agrobacterium tumefaciens mediated transformation of pigeonpea for
TRANSCRIPT
i
Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera
AND MOLECULAR ANALYSES OF SELECTED EVENTS
MAHALE BARKU MANOHAR
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005
JUNE, 2014
ii
Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera
AND MOLECULAR ANALYSES OF SELECTED EVENTS
Thesis submitted to the University of Agricultural Sciences, Dharwad
In partial fulfillment of the requirements for the
Degree of
Doctor of Philosophy
in
Molecular Biology and Biotechnology
By
MAHALE BARKU MANOHAR
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005
JUNE, 2014
iii
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD
CERTIFICATE
This is to certify that the thesis entitled “Agrobacterium tumefaciens MEDIATED
TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac,
cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera AND MOLECULAR
ANALYSES OF SELECTED EVENTS” submitted by Mr. MAHALE BARKU MANOHAR.,
for the degree of DOCTOR OF PHILOSOPHY in Molecular Biology and Biotechnology, to
the University of Agricultural Sciences, Dharwad, is a record of research work done by him
during the period of his study in this University under my guidance and the thesis has not
previously formed the basis for the award of any degree, diploma, associateship,
fellowship or other similar titles.
DHARWAD
JUNE, 2014 (B. FAKRUDIN)
CHAIRMAN
Approved by :
Chairman :
Members : 1.
2.
3.
(RAMESH BHAT)
(NARAYAN MOGER)
(B. M. KHADI)
(B. FAKRUDIN)
(S. S. UDIKERI)
4.
iv
Acknowledgement
One would not achieve whatever he is now, without all help, encouragement and
the well wishes of the near and dear ones. Teachers, parents, relatives, friends and all
well-wishers are an integral part of this. I owe them a lot and it is always a difficult task to
express the sense of gratitude for them in words.
First and foremost, I would like to express my deep sense of gratitude to
Dr. B. FAKRIDUN, Professor Dept. of Biotechnology, College of Agriculture, Dharwad
and the esteemed chairman of Advisory Committee for his inspiring and peerless
guidance, scholarly advice, thought provoking suggestions, sparing his time amidst busy
schedule and sustained encouragement throughout the course of investigation.
I record with sincerity my profound sense of gratitude to members of my Advisory
Committee Dr. B. M. KHADI, Directorate of Research, University of Agricultural
Sciences, Dharwad, , Dr. S. S. UDIKERI, Professor, Dept. of Agricultural Entomology,
College of Agriculture, Bijapur, UAS, Dharwad, Dr. RAMESH BHAT, Professor,
Department of Biotechnology, Dr. NARAYAN MOGER, Associate Professor, Department
of Biotechnology, for their encouragement and valuable suggestions during the course of
my investigation and evaluation of the manuscript.
I am grateful to my teachers Dr. P. U. Krishnaraj, Dr. Sumangla Bhat, Dr. S. K.
Prashanti and Dr. K. M. Sumesh, Department Biotechnology, for their cooperation
extended to me during the course of my study.
My untold heartfelt gratitude and profound indebtness to my beloved my
mother Smt. Suman and father Sri. Manohar whose prayers, unfailing love and affection
has upholded me at every moment of tension, difficulty and achievement. It is an
immense pleasure to express my sincere gratitude to my brother Mr. Mahesh, for their
sacrifice, co-operation, love and affection in every aspects of my studies and research
work. Without their support, I would never have come through this far.
I express my deep sense of gratitude to my seniors Shivu, Omkar, Siji, Rajkumar,
Abid, Suvarna, Dadakhalandar, Nagaraj and my friends Vaibhav, Sambhaji, for their
needful and timely help, moral support during my research period.
v
Life is not work all the time, one necessarily has to have some diversion once in
awhile, to be able to return to work with renewed enthusiasm and vigour. On a personal
note, I am very glad to mention sincere mental support, word of encouragement,
boundless love, selfless sacrifices of my friends Abhijit, Gourav, Bhabesh, Sandeep,
Neha, Diksha, Divya, Suhasini, Navya, Rose, Anil, Shrinvas, Dnyaneshwar,
Padmabhushan, Ramesh, Yogesh who have played an excellent role by showing friendly
attitude, love, generous hospitality right guidance and encouragement and all the trouble
they took for my sake, well association with them is truly of inestimable value. But, then
one does not need to thank the true friends.
I thank Lab maintainer Kallappa, Vijay, Santosh, Malik, Ravi, Basu and green
house maintainer Chanappa and Manju and other non-teaching faculty in the
Department of Biotechnology for their co-operation and help during the period of my
research work.
I sincerely acknowledge the necessary facilities provided by the concerned
authorities at U.A.S., Dharwad for smooth carrying out of my Ph. D. Programme. The
financial support of Indian Council for Agriculture Research (ICAR) under the framework
of National Fund for Basic Strategic Research in Agriculture (NFBSFARA) is gratefully
acknowledged.
Last but not the least, I express a special word of thanks to Mr. Kalmesh and Mr.
Arjun (Arjun Computers) for neat and timely printing of this manuscript.
Any omission in this acknowledgement does not mean lack of gratitude……….
DHARWAD
JUNE, 2014 (BARKU M. MAHALE)
vi
Affectionately Dedicated To
The Indian Farmers
vii
CONTENTS
Sl. No. Chapter Particulars
CERTIFICATE
ACKNOWLEDGEMENT
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
LIST OF APPENDICES
1. INTRODUCTION
2. REVIEW OF LITERATURE
2.1 Pigeonpea: Worlds important pulse crop
2.2 Major threats to pigeonpea production
2.3 Crop improvement strategies for insect pest resistance in pigeonpea
2.4 Pigeonpea pod borer, Helicoverpa armigera life cycle and damaging stages
2.5 Bacillus thuringiensis delta-endotoxin
2.6 Insecticidal proteins other than delta-endotoxin
2.7 Tissue culture studies in pigeonpea
2.8 Plant transformation studies
2.9 Selection marker
2.10 Southern Blotting
2.11 Immunoassay
2.12 Real-Time PCR
2.13 Insect bioassay
3. MATERIAL AND METHODS
3.1 Plant material and in vitro plantlet regeneration in pigeonpea
3.2 Maintenance of Agrobacterium tumefaciens strains and in vitro transformation of pigeonpea
3.3 Explant preparation and improvisation of in planta transformation protocol in pigeonpea
3.4 Identification of putative transformants
3.5 Transgene segregation analysis of T2 generation progenies
viii
Sl. No. Chapter Particulars
3.6 Transgene segregation analysis of T3 generation progenies
3.7 Insect culture and transgene bioefficacy analysis
3.8 Quantitative estimation of Cry protein using ELISA assay
3.9 Absolute real time qRT-PCR for cry transcript analysis
3.10 Genomic Southern blot analysis
3.11 Northern blot analysis
3.12 Recovering the site of transgene integration by TAIL PCR
3.13 The plant growth parameters
3.14 Statistical analysis
4. EXPERIMENTAL RESULTS
4.1
Effect of cytokinins and their concentration regimes on multiple shoot induction and plantlet regeneration in pigeonpea
4.2 Effect of different treatments on in planta transformation in pigeonpea
4.3 Generation of transgenic pigeonpea conferring expression of cry1Ac gene
4.4 Generation of transgenic pigeonpea conferring expression of cry2Aa gene
4.5 Generation of transgenic pigeonpea conferring expression of cry1F gene
4.6 Generation of transgenic pigeonpea conferring expression of cry1Acm gene
4.7 The plant growth parameters comparison between parental genotypes and transgenic lines developed
5 DISCUSSION
5.1 Improvisation of multiple shoot induction and plantlet regeneration in pigeonpea
5.2 Improvisation of in planta transformation protocol in pigeonpea
5.3 Generation of transgenic pigeonpea carrying different cry genes for pod borer resistance
5.4 Molecular characterization of pigeonpea transgenic lines expressing different cry genes
6 SUMMARY AND CONCLUSIONS
REFERENCES
APPENDICES
ix
LIST OF TABLES
Table No.
Title
1
Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from cotyledonary node with cotyledons explant of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture (50 explants)
2
Number of shoot bud induced in response to different concentration regimes of cytokinins (benzylamino purine, thidiazuron and zeatin) from cotyledonary node with cotyledons and embryo discs with half cotyledons explants.
3
Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from embryo discs with half cotyledon explants of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 12 days of culture (50 explants)
4
Effect of indole butyric acid (IBA) concentration regimes on root induction and number of root induced per shoot in pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture
5 The effect of targeting embryonic axis attached to single cotyledon for successful Agrobacterium tumefaciens infection and successful transformation.
6 The effect of targeting embryonic axis attached to single cotyledon with tobacco extract added in overnight grown Agrobacterium culture on plant transformation.
7 The effect of targeting embryonic axis attached to single cotyledon with air evacuation to increase the proximity between Agrobacterium tumefaciens and embryonic axis on transformation
8 Effect of different treatments used in in planta transformation of pigeonpea on explant survival, explants responded and transformation efficacy presented in per cent.
9 Summary of transformation carried out using cry1Ac gene in pigeonpea
10a Summary of transformation work being carried out using cry1Ac gene following in vitro kanamycin selection method in pigeonpea
10b Identification of T1 plants progenies of 10 putative transformants carrying cry1Ac gene. Testing for the presence of gene was done through gene specific PCR assay
11 The transgene segregation pattern in eighty eight transformants carrying cry1Ac in T2 generation revealed by gene specific PCR assay
x
12a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eighty eight putative transformants carrying cry1Ac gene in T2 generation
12b Comparison of H. armigera mortality in transgenic lines of
ICPL87119 and BSMR736 (by unpaired ‘t’-test at α=0.05)
12c Per cent corrected mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving in vitro generated putative transformants carrying cry1Ac gene in T1 generation
13a The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as
revealed by ELISA assay (α=0.05)
13b The Cry1Ac protein level detected in in vitro generated transgenic pigeonpea plants of T1 generation as revealed by ELISA assay in
leaf, flower and pod tissues (α=0.05).
13c The correlation analysis of insect mortality levels and estimated Cry1Ac protein in leaf, flower and pod tissues of T2 generation
plants (α=0.01)
14 Transgene segregation pattern in eight transgenic lines carrying cry1Ac gene in T3 generation
15a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eight putative transformants carrying cry1Ac gene in T3 generation.
15b The ‘t’-test analysis of Helicoverpa armigera mortality levels from T2
and T3 generation of eight cry1Ac transgenic lines (α=0.05)
16 The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay
17a The cry1Ac transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
17b The correlation analysis of insect mortality levels, estimated Cry1Ac protein and cry1Ac transcript in leaf, flower and pod tissues of T3
generation plants (α=0.01).
18 Juncture region analysis of cry1Ac cassette in AC20-2, AC20-3 and AC29-1 transgenic lines as revealed by TAIL-PCR analysis
19 Summary of transformation carried out using cry2Aa gene in pigeonpea
20 The transgene segregation pattern in sixty five transformants carrying cry2Aa in T2 generation revealed by gene specific PCR assay
21 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving sixty five putative transformants carrying cry2Aa gene in T2 generation
xi
22a The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as
revealed by ELISA assay (α=0.05)
22b The correlation analysis of insect mortality levels and estimated Cry2Aa protein in leaf, flower and pod tissues of T2 generation
plants (α=0.01)
23 Transgene segregation pattern in fifteen transgenic lines carrying cry2Aa gene in T3 generation
24a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fifteen putative transformants carrying cry2Aa gene in T3 generation
24b The ‘t’-test analysis of mortality levels from T2 and T3 generation of
fifteen cry2Aa transgenic lines (α=0.05)
25 The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay
26a The cry2Aa transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
26b The correlation analysis of insect mortality levels, estimated Cry2Aa protein and cry2Aa transcript in leaf, flower and pod tissues of T3
generation plants (α=0.01)
27 Juncture region analysis of cry2Aa cassette in selected transgenic lines as revealed by TAIL PCR analysis
28 Summary of transformation carried out using cry1F gene in pigeonpea
29 The transgene segregation pattern in fourteen transformants carrying cry1F in T2 generation revealed by gene specific PCR assay
30 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fourteen putative transformants carrying cry1F gene in T2 generation
31 The Cry1F protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay
32a The cry1F transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
32b The correlation analysis of insect mortality levels, estimated Cry1F protein and cry1F transcript in leaf, flower and pod tissues of T2
generation plants (α=0.05; 0.01)
33
Summary of transformation carried out using cry1Acm gene in pigeonpea
xii
34 The transgene segregation pattern in eleven transformants carrying cry1Acm in T2 generation revealed by gene specific PCR assay
35 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eleven putative transformants carrying cry1Acm gene in T2 generation
36 The Cry1Acm protein level detected in leaf, flower and pod tissues developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay
37a The cry1Acm transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
37b The correlation analysis of insect mortality levels, estimated Cry1Acm protein and cry1Acm transcript in leaf, flower and pod
tissues of T2 generation plants (α=0.01).
38 The per cent recovery of parental phenotypes for tested traits in transgenic plants carrying different cry genes
xiii
LIST OF FIGURES
Figure No.
Title
1a Construct map of the binary vector pBinBt3
1b Construct map of the binary vector pBinAR
1c Construct map of the binary vector pBinAR
1d Construct map of the binary vector pMKK1708. T-DNA carrying cry1Acm gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator
2 Response pattern of explants cultured on medium augmented with different cytokinins for multiple shoot induction in ICPL 87119 and BSMR 736 pigeonpea genotypes
3 Multiple shoot bud induced per explant cultured on medium supplemented with different cytokinins in ICPL 87119 and BSMR 736 pigeonpea genotypes
4 Effect of MS nutrient media strength and different IBA concentration regimes on rooting
5 Effect of different treatments on in planta transformation in pigeonpea.
6 Frequency distribution analyses of transgenic plants
7 Correlation analysis between insect mortality, Cry1Ac protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation
8. Correlation analysis between insect mortality, Cry1Ac protein and transcript levels in leaf tissues of developed transgenic pigeonpea plants in T3 generation
9. Frequency distribution analyses of transgenic plants.
10 Correlation analysis between insect mortality, Cry2Aa protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation.
11 Correlation analysis between insect mortality, Cry2Aa protein and transcript levels in leaf tissues of developed transgenic pigeonpea plants in T3 generation.
xiv
12 Frequency distribution analyses of transgenic plants
13 Correlation analysis between insect mortality, Cry1F protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation
14 Frequency distribution analyses of transgenic plants
15 Correlation analysis between insect mortality, Cry1Acm protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation.
16 The per cent deviation observed between transgenic plants and parental genotypes for selected morphological traits
xv
LIST OF PLATES
Plate No.
Title
1. Direct multiple shoot bud induction and plantlet regeneration from cotyledonary node with cotyledons, embryo discs with half cotyledon explants of pigeonpea
2 Establishment of primary and putative transformants carrying cry1Ac gene in transgenic containment facility
3 Gene specific PCR assay carried out for identification putative of transformants carrying cry1Ac gene and transgene segregation analysis in T2 and T3 generations.
4 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants.
5 Estimation of Cry1Ac protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay.
6 Absolut real-time quantification of cry1Ac gene transcript in transgenic pigeonpea plants
7 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 600bp cry1Ac gene fragment
8 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 600bp cry1Ac gene fragment
9 TAIL-PCR assays (tertiary reaction) conducted in T3 generation plants of AC20-2, AC20-3 and 29-1 transgenic lines carrying cry1Ac gene
10 Establishment of primary and putative transformants carrying cry2Aa gene in transgenic containment facility
11 Gene specific PCR assay carried out for identification putative transformants carrying cry2Aa gene and transgene segregation analysis in T2 and T3 generations
12 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants
13 Estimation of Cry2Aa protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay
xvi
14 Absolut real-time quantification of cry2Aa gene transcript in developed transgenic pigeonpea plants.
15 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 676bp cry2Aa gene fragment.
16 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 676bp cry2Aa gene fragment
17 TAIL-PCR assays (tertiary reaction) conducted for T3 generation plants of 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68 transgenic lines carrying cry2Aa gene.
18 Establishment of primary and putative transformants carrying cry1F gene in transgenic containment facility
19 Gene specific PCR assay carried out for identification putative transformants carrying cry1F gene and transgene segregation analysis in T2 generation
20 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants
21 Estimation of Cry1F protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay.
22 Absolut real time quantification of cry1F gene transcript in developed transgenic pigeonpea plants
23 Genomic Southern analysis: 15 µg of genomic DNA was digested with EcoRI and probed with Dig labelled 600bp cry1F gene fragment
24 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 600bp cry1F gene fragment
25 Establishment of primary and putative transformants carrying cry1Acm gene in transgenic containment facility
26 Gene specific PCR assay carried out for identification putative transformants carrying cry1Acm gene and transgene segregation analysis in T2 generations
27 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants
28 Estimation of Cry1Acm protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay
xvii
29 Absolut real-time quantification of cry1Acm gene transcript in developed transgenic pigeonpea plants
30 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 452 bp cry1Acm gene fragment
31 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 452 bp cry1Ac gene fragment
32 The comparison of plant growth parameters of developed transgenic plants and non-transgenic control plants.
xviii
LIST OF APPENDICES
Appendix No.
Title
I Appendix I
II Appendix II
III Appendix III
IV Appendix IV
V Appendix V
VI Appendix VI
1. INTRODUCTION
Pigeonpea [Cajanus cajan (L.) Millsp.] belongs to family Fabaceae, is one of the
major pulse crops grown in many countries of Asia, Africa and America. India ranks first
in the world accounting for 3.53 m. ha area with 2.51 million tons of pigeonpea
production (http://faostat3.fao.org). Pigeonpea is consumed as green peas, whole grain
or split peas and is a major source of protein (20 - 22%) to human population
particularly in developing countries (Singh and Eggum, 1984; Varshney et al., 2010).
The production and productivity of pigeonpea is threatened by many biotic and abiotic
stresses. The most important yield constraint in pigeonpea is from the polyphagous
lepidopteran pest, Helicoverpa armigera Hübner (Sharma et al., 2006). In India,
pigeonpea is prone to more than 200 species of insects, among which the gram pod
borer, H. armigera causes enormous losses (46.6 - 63.6%) which persists throughout
the year on one or other crops. In recent years other insect pests such as Maruca
(Maruca vitrata Fabricius), pod fly (Melanagromyza obtuse Malloch) and plume moth
(Exelastis atomosa Walsingham) are becoming economically important inflicting
significant yield losses (Choudhary et al., 2013).
An indiscriminate use of synthetic chemical insecticides has resulted in the
contamination of water and food sources, poisoning of non-target beneficial insects
leading to many serious environmental concerns (Kumar et al., 2008). Increased
understanding about the adverse environmental effects of indiscriminate use of
insecticides has promoted search of altermnative methods form pest control. In nature,
wild species of pigeonpea are rich source of resistance genes against many biotic and
abiotic stresses (Lal and Rathore, 2001). Poor crossing ability of cultivated pigeonpea to
species other than the closest ones, such as Cajanus cajanifolia and C. scaraboides
has limited the use of inter specific crosses in pigeonpea improvement (Varshney et al.,
2010). Conventional breeding approaches for pigeonpea improvement have been in use
for several decades, but have had limited success in overcoming different biotic and
abiotic challenges to sustainable crop production (Varshney et al., 2007; Saxena, 2008).
Hence, insect resistant transgenics expected to be an ideal solution in the interest of the
pigeonpea farmers and the productivity.
2
It has been reported that the global farm income gain due to use of genetically
modified insect resistance technology in maize and cotton have been $6.71 billion and
$5.3 billion respectively during 2012. The genetic engineering technology has been
adopted on large scale i.e. by 17.3 million farmers in 2012, demonstrating the important
economic benefits of this technology (Brookes and Barfoot, 2014). Cumulatively since
1996, the global farm income gain has been $32.3 billion for genetically modified insect
resistant maize and $36.3 billion for that of cotton. Considering the current statistics and
economic benefits from use of genetically modified insect resistant cultivars clearly
points at immense scope for use of genetic engineering technology in crop improvement
programme. Lawrence and Koundal (2001) have successfully transferred the cowpea
protease inhibitor gene (pCPI) in pigeonpea for biotic stress tolerance. Attempts have
been made to develop Spodoptera litura resistant pigeonpea by expressing synthetic
cryIE-C gene using transgenic technology (Surekha et al., 2005). Recently, Ramu et al.
(2012) have reported significant reduction in H. armigera feeding on genetically
modified pigeonpea lines expressing chimeric cry1AcF.
More than 500 different cry genes have been known and classified into 67
groups based on their primary amino acid sequence (Crickmore et al., 2010). Broadly
these cry genes have been divided in to four protein families such as, the family of three
domain Cry toxins (3D), mosquitocidal Cry toxins (Mtx), the binary-like (Bin) and the Cyt
family of toxins (Bravo et al., 2005). The Cry1Ac belong to 3D-Cry group proteins and
commercial Bt crops have been developed for expression of cry1Ac gene itself for the
control of many lepidopteran pests (Christou et al., 2006). Further, a second generation
Bt-cotton has been developed for Cry2Ab besides Cry1Ac as a resistance management
strategy. Bt-corn expressing Cry1Ac has been tested for their effectivity against
lepidopteran pests (Christou et al., 2006). Like any other Cry proteins, the mode of
action of Cry1F also involves the enzymatic cleavage of the protoxin to form core toxin
(Gao et al., 2006). Many studies have determined the activity of Cry1F against
lepidopteran species (Balog et al., 2011; Oppert et al., 2010). The native cry1Ac gene
sequence from Bacillus thuringiensis have been in silico modified and artificially
synthesized to overcome codon bias and other undesirable regulatory coding
sequences for its improved expression in transgenic plants. The study resulted in the
development of modified cry1Acm with increased GC content (Mohan, 2008).
3
There are very few reports on in vitro regeneration of pigeonpea through
organogenesis from unorganised callus (Kumar et al., 1983). Different explants such as,
cotyledons, embryonic axes, cotyledonary node from mature seeds and seedling
petioles have been studied for the multiple shoot production and plantlet regeneration
through direct organogenesis (Franklin et al., 1998; Srinivasan et al., 2004). The effect
of cytokinins such as, benzylamino purine (BAP) and furfurylamino purine (kinetin) and
their varied levels of concentrations on multiple shoot induction have been studied in
pigeonpea (Geetha et al., 1998). Similarly, Kashyap et al. (2011) has analysed the
frequency of multiple shoot bud induction using different cytokinins viz., BAP, kinetin
and TDZ in eleven Indian cultivars of pigeonpea. The type of explant, genotype and
concentrations of cytokinin can influence the frequency of shoot bud regeneration
(Geetha et al., 1998; Kashyap et al., 2011). A variable frequency (20-60 per cent) of
palntlet regeneration has been reported in pigeonpea (Srivastava et al., 2013).
However, there is a need to fine-tune the in vitro regeneration protocols to achieve high
frequency regeneration of plantlets.
The poor in vitro regeneration and low transformation frequency are the major
constrains for development of transgenics in pigeonpea. Efforts have been made for the
development of efficient protocols of Agrobacterium tumefaciens mediated and
microprojectile bombardment-based genetic transformations of pigeonpea (Rao et al.,
2008). Further, the in vitro regeneration conditions so far described are available for
only a few of the many cultivars/genotypes of pigeonpea (Rao et al., 2008). The in
planta plant transformation method has been developed and successfully applied in
wide range of crop plants such as, mulberry, soybean, rice, cotton, fieldbean, sunflower,
groundnut and safflower (Ping et al., 2003; Supartana et al., 2005; Keshamma et al.,
2008; Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b; 2001). In recent
efforts in planta transformation method has also been used for development of
transgenic pigeonpea for expression of a chimeric cry1AcF gene (Ramu et al., 2012).
Similarly, in case of alfalfa, this method has been employed for the development of
marker free transgenic plants (Weeks et al., 2008). Hence, the use of in planta methods
expected to be an important component for future transgenic development programs in
crops like pigeonpea.
4
Different characterization methods viz, insect bioassay, protein quantitation using
ELISA, mRNA quantitation using qRT-PCR assay, Sothern, western and northern blot,
and TAIL-PCR assay for juncture region analysis have been employed for transgenic
plants in many studies (Sharma et al., 2006; Ramu et al., 2012). Bhat and Srinivasan
(2002) has discussed genetic analysis of transgenic plants and indicated that handling
of primary transgenics to obtain progeny generation needs few careful considerations
such as, the breeding behaviour of the plant species, the performance of parent and
progeny etc. The selfing of the T0 plants helps to achieve homozygosity and resultant T1
progenies will have complex mixture of genotypes and their composition depends on
the genetic constitution of the T0 parents. Further, they have said that in case of multiple
copy integration of transgene, determination of exact genotypic constitution of individual
plants might be difficult due to difficulties in distinguishing hemizygotes from
homozygotes by Southern analysis (Bhat and Srinivasan, 2002).
Southern blotting methods have been assisted with gel electrophoresis. Southern
blotting involves a method for separating genomic DNA with exceptional resolving
power (Southern, 2006). Blotting enables the detection of specific molecules among the
mixture separated in the gel. DNA specific sequences are detected in the membrane by
molecular hybridization with labeled nucleic acid probes (Southern, 2006). The method
has been used in several key studies (Kan and Dozy, 1978; Jeffreys and Flavell, 1977).
The original proposal for the genetic mapping of the human genome has been based on
restriction fragment length polymorphisms (RFLPs) detected by blotting (Kan and Dozy,
1978). Introns have been first seen in blots of rabbit genomic DNA hybridized with
probes for the ß-globin gene (Jeffreys and Flavell, 1977). The first DNA fingerprints
have been produced by hybridizing restriction digests of human DNA with minisatellite
probes (Jeffreys et al., 1985). Acharjee et al. (2010) have used Southern blot technique
and showed the integration of cry2Aa gene in eight of the nine transgenic chickpea
lines, with integration of one, two or more copies of the transgene. The southern blot
technique has been used in many transgenic studies to check the presence and copy
number of transgene in transgenic plants (Mehrotra et al., 2011; Rao et al., 2008; Ramu
et al., 2012).
5
Bioassays have been used test the functionality of the transgene product (Bhat
and Srinivasan, 2002). The detached leaf bioassay tests have been undertaken for
transgenic pigeonpea plants against the pest S. litura, wherein study reported 80 per
cent insect mortality in developed transgenic plants (Surekha et al., 2005). Similarly,
many independent studies have performed bioefficacy tests against H. armigera
(Gopalaswamy et al., 2007; Ramu et al., 2012; Acharjee et al., 2010). The transgenic
plants of pigeonpea and chickpea, expressing different insecticidal cry genes have been
analysed for their bioefficacy levels against targeted insects by following bioassay
technique (Sanyal et al., 2005; Gopalaswamy et al., 2007; Ramu et al., 2012; Acharjee
et al., 2010).
An immunoassay technique based on antibodies has been a standard approach
for qualitative and quantitative detection of protein of a known target analyte (Brett et al.,
1999). Both monoclonal and polyclonal antibodies have been used depending on the
specificity of the detection system (Kamle and Ali, 2013). Sharma et al. (2006), using
this technique, have determined the synthetic Cry1Ab protein in developed transgenic
pigeonpea plants. Many parallel studies have reported the detection and estimation of
target protein in test samples using immunoassay technique (Gopalaswamy et al.,
2007; Ramu et al., 2012).
Another characterization method for genetically modified plants involves
transcript analysis of transgene using RT-PCR and northern blot techniques (Bhat and
Srinivasan, 2002). These methods have been used successfully in transgenic studies
for quantification of targeted cDNA molecules (Kamle and Ali, 2013). The real time PCR
has also been used for validating and estimating the number of copies of transgene into
the host genome in several genetically modified crop plants such as maize, cassava,
rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al., 2013; Beltrán et
al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007).
Considaring the severity of pod borer, H. armigera in pigeonpea, the Bt
transgenic approach has been felt essential. Hence, the present study on development
of Bt transgenic pigeonpea has been undertaken through all necessary precise
biotechnological and conventional tools with following objectives;
6
1. To improvise the in vitro plantlet regeneration and in planta transformation protocols
in pigeonpea
2. To develop genetically transformed pigeonpea lines using cry1Ac, cry2Aa, cry1F
and cry1Acm genes separately
3. To assess the bioefficacy and molecular characterization of selected putative
transgenic pigeonpea lines
2. REVIEW OF LITERATURE
Pigeonpea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) also known as red
gram, is one of the major grain legume (pulse) crops grown in the semi-arid tropics. It is
the second most important food legume in India, where more than 80% of the world
production occurs (FAO, 2008). It is cultivated in about 50 countries of Asia, Africa and
the America for a variety of uses like food, fodder, fuel wood, rearing lac insects,
hedges, wind breaks, soil conservation, green manure and roofing. It is often grown on
marginal soils and intercropped with cereals.
Pigeonpea is a major source of protein (21%) to human population mainly in
developing countries and several Latin American countries (Singh and Eggum, 1984).
Its production and productivity are constrained by several biotic and abiotic stresses,
whose levels of resistance in world germplasm accessions are low to moderate.
Breeding incompatibility problems associated with wild species warrant the exploration
of alternative approaches. Genetic engineering technology plays a significant role as an
additional tool for the introduction of agronomically useful traits in a high yield
background. The most important yield constraint in pigeonpea is from the lepidopteran
pest, Helicoverpa armigera. Insect resistant transgenics would be an ideal solution in
the interest of the pigeonpea farmers and the productivity. Among the many potential
genes, cry genes are generally effective against Lepidoptera, and are likely to be
species specific. Hence, genetic transformation of pigeonpea holds greater promise to
manage pod borer pest. A brief review on various aspects of present study is presented
below.
2.1 Pigeonpea: World’s important pulse crop
Pigeonpea is an important food legume crop, cultivation predominantly takes
place in tropical and subtropical regions of the world. It is a diploid crop with
2n = 22 and a genome size of 808 Mbp. Pigeonpea is a naturally drought tolerant crop
with large variation for days to maturity that ranges from 90 to 300 days duration.
Pigeonpea is cultivated as a sole crop as well as a mixed crop with cereals and
legumes. Globally pigeonpea is cultivated on 4.64 Mha, with an annual production of
3.43 million tons and productivity of 780 kg/ha. In pigeonpea growing countries, India
8
ranks first in the world accounting for 3.53 Mha area with 2.51 million tons of production
(Varshney et al., 2010). Pigeonpea consumed as green peas, whole grain or split peas
and contains 20-22 per cent protein. The seed, pod husks, dry branches and stems are
also used as a quality feed and also serve as domestic fuel. In field, pigeonpea plants
harbours nitrogen fixing bacteria, enriching soil fertility by symbiotic nitrogen fixation.
India alone occupies three-fourth of global harvested area and contributes almost
a similar share in production. After chickpea, pigeonpea is the second most important
pulse crop in India. Pigeonpea is mainly grown in Maharashtra, Karnataka and Andhra
Pradesh states (>60% area in India) with 60 per cent of production (1.4 million tons)
from these three states. Although there is increase in demand for pigeonpea, in last five
years, the area and production of pigeonpea has remained constant over a time period.
Despite its importance in subsistence and sustainable agriculture, the average global
productivity of pigeonpea has remained static over the last three decades. The yield gap
observed between the potential yield and on-farm yield is mainly due to prevalence of
various abiotic (Choudhary et al., 2011) and biotic factors together with the cultivation of
pigeonpea in marginal lands with low input supply and lack of efficient management
practices (Varshney et al., 2012). Wide yearly fluctuations have been observed in total
production mainly due to attack by insect pests including disease-causing pathogens.
2.2 Major threats to pigeonpea production
Pigeonpea plant growth and reproductive stage both are influenced by
environmental conditions. It has made crop breeding more complex in pigeonpea. In
nature, wild species of pigeonpea are rich source of resistance genes against many
biotic and abiotic stresses, the genes responsible for yield components such as, pods
per plant, length of fruiting branches, and number of primary branches per plant etc.
Due to the poor crossing ability of cultivated pigeonpea to species other than the closest
species, such as Cajanus cajanifolia and C. scaraboides has limited the use of inter
specifics in pigeonpea improvement. Use of biotechnological approaches viz., in vitro
rescue and propagation of wide cross hybrids, in conjunction with the use of bridge
crosses, has enabled the transfer of novel genes from a wider range of germplasm
within and outside the genus Cajanus (Varshney et al., 2010).
9
Although, pigeonpea is important crop in semi-arid regions of the world, less
concentration has been directed towards pigeonpea crop improvement. The important
factors responsible for poor productivity in pigeonpea are lack of improved cultivars,
poor crop husbandry, pests and diseases. Major disease and pests that have
threatened pigeonpea cultivation are fusarium wilt (Fusarium udum Butler), sterility
mosaic disease (Sterility mosaic virus) and phytophthora blight (Phytophthora
drechsleri), gram pod borer (Helicoverpa armigera), Maruca (Maruca vitrata), pod fly
(Melanagromyza obtusa), plume moth (Exelastis atomosa) and mite (Aceria cajani).
Besides, abiotic stresses such as water-logging, common in rain fed crop during early
stages, and stress from low water conditions in the later stages, and salinity has also
contributed significantly to reduce pigeonpea production (Varshney et al., 2010).
2.2.1 Major disease in pigeonpea
Among diseases, fusarium wilt (Fusarium udum) in Central and Southern states
followed by sterility mosaic (virus transmitted by eriophyid mite Aceria cajani), and SMD
and Phytophthora blight (Phytophthora drechsleri f.sp.cajani) diseases in the northeast
plain (Uttar Pradesh) cause substantial yield losses to the crop. Unchecked weeds also
cause 21-97% yield loss in pigeonpea. Fusarium wilt is one of the major diseases in
pigeonpea caused by Fusarium udum f. sp. cajani. This disease is characterized by
yellowing and slow withering of plant. The infected plants show symptoms of yellowing
followed by dropping and finally the whole plant dries up. Level of incidence is
significantly influenced by pathogen surviving saprophytically on the crop residue.
Phytopthora organisms are commonly referred to as water molds. The disease is
caused by the fungus P. drechsleri var. cajani and the disease is commonly known as
stem rot of pigeonpea. Pigeonpea seedlings are highly prone to infection and can
become infected as soon as they emerge (Sharma et al., 2010).
Bacterial Leaf Spot and Stem canker were first reported from India in 1950.
There is no specific period and the disease can occur anytime in the crop. Symptoms
on leaves are characterized by the appearance of minute, brown lesions surrounded by
a yellow halo. These lesions often coalesce and form larger ones. On the main stem
and branches, rough, cankerous dark brown lesions of various shapes and size appear.
The disease is caused by bacteria of Xanthomonas type belonging to the family of plant
10
pathogenic yellow pigmented pseudomonades. It is a species of Gram-negative rod-
shape flagellated bacteria known for being a common plant pathogen. Xanthomonas is
dependent on the type III protein secretion system, which relies on transport proteins, it
secrets hypersensitive and degradable exo-polysaccarides (Xanthan), causing an
interaction with the plant. X. campestris pv cajani is specific to pigeonpea and is seed
borne in nature (Sharma et al., 2010).
Sterility mosaic disease (SMD) was first described in 1931 from Pusa (Bihar) and
subsequently from rest of India and other countries. Patches of bushy pale and green
plants with smaller leaves, without flowers or pods are the common field symptoms and
is a serious disease in India. The disease is of a major concern in UP, Bihar, Gujarat,
Tamil Nadu and Karnataka. The disease is transmitted by mites (Aceria cajani).
Infection at an early stage (<45-day-old plants) results in a 95 to 100% loss in yield
while losses from late infection (>45-day-old plants) depend on the level of infection (i.e.
number of affected branches per plant) and range from 26 to 97% (Kannaiyan et al.,
1984).
Cercospora leaf spots is caused by a fungus Cercospora indica, causing several
types of spots on the leaves and petioles of affected plants. Initially small, circular,
necrotic spots forming typical concentric rings appear on the leaves during Alternaria
blight/leaf spot disease (Sharma et al., 2010). Collar rot /soft rot (Sclerotium rolfsii
Saccardo) disease appears within a month of sowing and is usually scattered over the
field. Seedlings turn slightly chlorotic before drying. Rotting in the collar region is often
covered with white mycelial growth accompanied with small mustard like bodies
(Sharma et al., 2010). Characteristic symptoms of Foliar Rust include dark brown raised
pustules (full of uredia) on the lower leaf surfaces. When the disease is severe, it
causes extensive defoliation. The disease is known to occur in Uttar Pradesh,
Maharashtra and Tamil Nadu (Gahukar and Peshney, 1985; Kannaiyan et al., 1984).
The powdery mildew symptoms appear mostly on older leaves, however, in severe
cases even young buds and pods also get infected (Sharma et al., 2010).
2.2.2 Major insect pests in pigeonpea
More than 200 species of insects have been identified, which feed on pigeonpea
from germination stage till harvest. Many of the insects attacking the crop are not
11
actually responsible for significant loss in production (Rangarao and Shanower, 1999).
Among the variety of insects feeding on pigeonpea, the pod borer (Helicoverpa
armigera Hubner) is the most damaging pest worldwide. Its frequent occurrence often
results in complete crop failure and cause losses of more than US $300 million annually
(Choudhary et al., 2013). The wide host range, high degree of migration, indiscriminate
pesticide application by farmers and innate ability of the insect to quickly develop
resistance to applied insecticides have made it attain the status of the key pest
(Vishwadhar et al., 2008). The pod fly infestation causes 2.5 to 86.8 per cent of grain
losses in different parts of the country. On other hand, the estimates of losses due to
pod borer have been recorded to be 30.2 per cent.
Nearly 30 species of Lepidoptera have been reported to feed on the reproductive
structures of pigeonpea. Most of these insects occur at low densities and are only
occasional pests or are of local importance. There are two most important species, viz.,
Helicoverpa armigera (Noctuidae) and Maruca vitrata (Dtestulalis) (Pyralidae). H.
armigera is the major biotic constraint to pigeonpea production. It is the key pest due to
the larval preference for feeding on nitrogen rich plant parts such as reproductive
structures and growing tips (Fitt, 1989). These plant parts are also the most suitable for
larval development. Besides its preference for feeding on reproductive structures, the
four important features of H. armigera life history that made it one of the most serious
and widespread insect pests are high fecundity, extensive polyphagy, strong flying
ability and a facultative diapause (Fitt, 1989). Other important pests of pigeonpea, M.
vitrata, is distributed throughout tropical and subtropical regions world-wide and has a
wide host range but is restricted to legumes (Atachi and Djihou, 1994). The M. vitrata
larvae are known to feed from inside a webbed mass of leaves, flowers and pods, and
are a serious pest of pigeonpea in India, Sri Lanka and Africa. Maruca is an important
pest of pigeonpea in early stage of growth and cause yield losses ranged up to 100 per
cent (Choudhary et al., 2013).
Another group of insects that belongs to Hemiptera involves mainly pests from
the families Alydidae, Coreidae, and Pentatomidae. They feed on pigeonpea and are
commonly referred to as pod-sucking bugs. The most important pests from this group
are the coreids Anoplecnemis spp., Clavigralla spp. (Acanthomia spp.) and Riptortus
spp. Both, adults and nymphs of Clavigralla spp. are known to feed on pigeonpea by
12
piercing the pod wall and extracting nutrients from the developing seeds (Bindra, 1965).
Damaged seeds appear dark and shriveled, and they are difficult to distinguish from
those damaged by drought.
Two Dipteran and one Hymenopteran insects feed on developing seeds of
pigeonpea within the pod. Among them the most important is Melanagromyza obtuse
Malloch (Diptera: Agromyzidae), the pigeonpea pod fly, which is observed mainly in
Asia. Pod fly damage has been reported from several countries. In India, the pod fly is a
more serious pest in northern and central areas than in other parts of the country
(Lateef and Reed, 1983). Damage levels in farmers’ fields range from 10 to 50 per cent
(Lal et al., 1992).
2.3 Crop improvement strategies for insect pest resistance in pigeonpea
The breeding strategies and methodologies for crop improvement have been
developed after understanding the important role of genetic inheritance of yield and
related traits. Relatively less effort has been made to understand the genetics of
important traits in pigeonpea, when compared with other economically important crops.
Saxena and Sharma (1990) have reported the importance of both additive effects and
dominant non-additive effects in determining yield, plant height and protein content. The
features like pleiotropic effects of genes, physiological changes and highly sensitive
nature of pigeonpea towards the environmental changes has made the interpretation of
yield and associated characters in pigeonpea difficult (Byth et al., 1981). Other than
yield related parameters, restoration of male fertility in cytoplasmic-genetic male-sterility
(CGMS) based hybrids has been critical and important trait in pigeonpea. Conventional
breeding approaches for pigeonpea improvement have been in use for several decades
but have had limited success in overcoming different biotic and abiotic challenges to
sustainable crop production (Varshney et al., 2007; Saxena, 2008).
2.3.1 Conventional breeding
Identification and utilization of resistant cultivars against these insects expected
to provide an environment friendly solution and sustainable management of these insect
pests. Many efforts have been made for the identification of sources of resistance to
various insects in pigeonpea by screening the pigeonpea genotypes for insect
13
resistance. The large variation in maturity period has been reported as a cause for
differential infestation specificity by insects. Wherein, the extra short-duration varieties,
harvested during October-November, and the medium-duration genotypes, harvested in
December-January, are more severely damaged (90 per cent) by pod borer. On other
hand, the long-duration genotypes maturing from February onwards shows lower pod
borer damage. Determinate types have heavier damage than the indeterminate types
(Choudhary et al., 2013). The pigeonpea genotype, UPAS 120, is fairly tolerant,
whereas ICPL 151 is highly susceptible. Lateef (1992) screened more than 14,000
pigeonpea germplasm accessions for identification of the sources of resistance to pod
borer. The study resulted in identification of only a few accessions with low levels of
resistance, wherein among the cultivated pigeonpea genotypes, ICPL 332, the resistant
check, was consistently less damaged than the susceptible check ICPL 87 (Lateef,
1992). The pigeonpea varieties ICPL87119 has been derived from a cross between C11
and a breeding line, and is medium duration variety with resistance to Fusarium wilt and
pigeonpea sterility mosaic virus (Dharmaraj and Lohithaswa, 2004).
Studies have reported that early maturing genotypes are more susceptible to pod
fly damage when compared with late maturing ones (Lal et al., 1998), and the
determinate types are less susceptible than the indeterminate types (Gupta et al.,
1991). Similarly, it has been revealed that long-duration varieties show heavier damage
than the early maturing types. More than 10,000 germplasm accessions of pigeonpea
have been screened for pod fly resistance (Lateef and Pimbert, 1990). Many
independent studies have identified the genotypes with a high degree of resistance
such as PDA 88-2E, PDA 92-1E, PDA 89-2E, PDA 92-2E, PDA 92-3E and PDA 93-1E
(Durairaj and Ganapathy, 1997; Srivastava et al., 1994; Lal and Rathore, 2001).
Although these lines can be used as the sources of resistances in the breeding
programmes, the field observations have indicated that these lines have small seed
size. Moudgal et al. (2008) have studied pigeonpea genotypes for physico-chemical pod
traits imparting resistance to pod fly under field conditions. The study reported that the
non-determinate genotype GP 75, extra early maturing, and GP 118, early maturing,
and determinate genotype GP 233, extra early maturing and GP 253, early maturing
genotypes recorded significantly lower pod and seed damage as compared to early
maturing checks Prabhat, determinate and Manak, non-determinate. The study also
14
indicated that resistance to pod fly has not been linked to plant growth habit and
maturity period of pigeonpea genotypes.
Furthermore, wild relatives of pigeonpea have been identified as useful sources
of resistance to pod borer (Romeis et al., 1999). Many studies have reported different
wild species especially C. scarabaeoides and C. platycarpus as the potential sources of
resistance to pod borer (Shanower et al., 1998; Mallikarjuna et al., 2011). Sharma et al.
(2009) have reported significant variation in egg laying, numbers of larvae and pod
damage among the wild relatives of pigeonpea under field conditions.
MacFoy et al. (1983) have recorded high concentrations of sugars and amino
acids in the cowpea cultivar Vita-1, which is susceptible to spotted pod borer/Maruca.
Similarly, in another stream of efforts, low amounts of polyphenols have been reported
to be associated with their high susceptibility to pod borer in the cultivated pigeonpea
(Sharma et al., 2009). It has been studied that the low amounts of phenols in pigeonpea
flowers are associated with susceptibility to spotted pod borer, M. testulalis. High
amount of soluble protein content have been reported in the pods of C. Scarabaeoides
when compared with those of ICPL 87, a cultivated variety of pigeonpea. High amounts
of polyphenols have been revealed in the resistant wild species as compared to the
cultivated pigeonpea (Sharma et al., 2009). Smith (1989) documented that the presence
of condensed tannins in plants act as insect growth inhibitors. The pod wall biochemical
traits have been studied for their role in pigeonpea resistance to insects (Moudgal et al.,
2008). Many studies have demonstrated that the pigeonpea pod walls with more wax,
total phenols and less reducing and non-reducing sugars and total amino acids suffers
less pod and grain damage by pod fly.
2.3.2 Molecular breeding
Studies have identified wild relatives of pigeonpea like C. scarabaeoides,
C. sericeus, C. acutifolius, C. albicans and Flemingia bracteata that possesses high
degree of resistance to pod borer, pod fly and pod wasp (Sharma et al., 2003;
Choudhary et al., 2013). Mallikarjuna et al. (2007) have developed the advanced
generation population from a cross utilizing C. acutifolius as the pollen parent and
reported introduction of resistance trait in progenies. Study also reported that some
15
lines having high level of resistance to pod borers, pod fly and bruchids under un-
protected field conditions (Mallikarjuna et al. 2007).
Among available 21 wild species in the tertiary gene pool of pigeonpea,
C. platycarpus has been used for improving insect resistance through inter-specific
hybridization (Mallikarjuna et al., 2006). Sujana et al. (2008) has given the important
trait features of C. platycarpus as extra-early flowering and maturity, photoperiod
insensitivity, prolific flowering and podding, high harvest index, annuality and rapid
seedling growth including resistances to biotic stresses such as pod borer. These
characteristic features have become a major attraction for pigeonpea breeders.
Mallikarjuna et al. (2011) developed back cross progenies and reported a range of
morphological and resistance traits in the progeny lines for pod borer, bruchid, and pod
fly resistance. The study quantified that the presence of C. platycarpus genome in
advance generation progeny lines using Diversity Array Technology (DArT) markers.
The progeny screening for resistance to pod borer, pod fly and bruchids under
unprotected field conditions revealed observation of the damage ranged from 6.85 to
22.84 per cent in BC44F-A derivatives. Majority of the lines indicated less than 15 per
cent damage (Mallikarjuna et al., 2011).
Progeny lines derived from C. platycarpus have shown 0 to 7.44 per cent pod
borer damage when compared with compared with 14.5 per cent damage recorded in
the control lines. Study also reported that some of the lines showed less to no bruchid
damage with significant lower damage by pod borer. Three accessions of C.
platycarpus have been screened and identified for bruchid resistance (Mallikarjuna et
al., 2011). The studies also reported 82 to 91 per cent reduction in eggs hatch. The
minimum seed damage have been recorded on C. platycarpus accession ICPW 66
(14%), while the damage was moderate to medium in other C. platycarpus derivatives
compared with the susceptible check ICPL 85010 (75% damage). In advance
generation interspecific derivative lines, there have been 32.78 to 92 per cent eggs that
failed to hatch. Study also has reported low to moderate resistance for the pod fly in the
BC4F-A derivatives, which ranged between 4 to 22 per cent, with a single derivative
BC4F11-A, indicated a low damage of 3.73 per cent (Mallikarjuna et al., 2011).
16
Substantial progress has been made towards development of large-scale
genomic resources in pigeonpea especially during the last decade. These efforts have
resulted in the development of large-scale molecular markers, construction of
comprehensive genetic maps, identification of various marker-trait associations and
initiation of molecular breeding in this crop. The large numbers of SSR markers (Raju et
al., 2010), DArT markers (Mallikarjuna et al., 2011), SNPs (Dubey et al., 2011) and
ESTs (Raju et al., 2010) hold high promise for improvement of a number of economic
traits in pigeonpea. More than 3000 SSR markers have been developed that are useful
in development of inter as well as intra specific genetic maps using several F mapping
populations (Bohra et al., 2012). This represented the first instance of merging multiple
genetic maps in pigeonpea. Preliminary mapping efforts for trait mapping in pigeonpea
have resulted in mapping of Fusarium wilt resistance (Kotresh et al., 2006), SMD
(Ganapathy et al., 2010).
It has been reported that the presence of genetic variability for resistance to
insects particularly pod borer is not available in the primary gene pool of pigeonpea
(Choudhary et al., 2013). And hence, breeders need to opt wild relatives such as,
C. scarabaeoides, C. platycarpus that are considered reservoirs of superior alleles for
traits imparting resistance to insects that must have lost during domestication and
breeding. On other hand, the transfer of desirable alleles is not so simple because of
difficulty in efficient tracking for desired and non-desired alleles in breeding lines. This
problem can be overcome by advance backcross QTL based breeding. It is most
suitable for introducing novel alleles from wild relatives to the cultivated species
cultivars or varieties in a controlled manner (Tanksley and Nelson, 1996).
2.3.3 Genetic engineering
Few lines of pigeonpea have been identified for resistance to pod borer and pod
fly. Their resistance has been only partial and lines with absolute resistance are not
available in the cultivated germplasm (Grover and Pental, 2003). Attempts have been
made to produce insect resistant genotypes of pigeonpea using conventional breeding
methods with very less success. The reasons for limited success are limited resistance
sources in the crossable germplasm and incompatibility with wild species (Choudhary et
al., 2013). The advent of transgenic technology has provided the best alternative to
17
improve resistance to insect pests. A well defined reproducible regeneration and
transformation protocol have been developed for development of transgenics in
pigeonpea (Rao et al., 2008).
During 2001, transformation technique using Agrobacterium tumefaciens have
been used in pigeonpea (Cajanus cajan L. Millsp.) for expression of cowpea protease
inhibitor gene (pCPI), wherein pigeonpea embryonic axes excised from germinated
seeds on MS basal supplemented with BAP (2 mg/L) used as explants for
transformation (Lawrence and Koundal, 2001). Using embryonal segments as explants
the effective transformation and quick regeneration of transgenic plants of pigeonpea
has been documented by the expression of synthetic cryIE-C gene under a constitutive
35S promoter (Surekha et al., 2005). In their study selection of transformed shoots has
been performed on MS medium supplemented with 2.0 mg/L BAP, 250 mg/L cefotaxime
and 75 mg/L kanamycin and rooted on MS medium supplemented with 1.0 mg/L NAA.
In vitro insect bio-efficacy analysis of developed transgenic pigeonpea plants
expressing cryIE-C reported resistance against Spodoptera litura larvae.
Sharma et al. (2006) have reported novel tissue culture method involving the
direct regeneration of adventitious shoot buds from axillary bud region of in vitro
germinating seedlings through suppression of axillary and primary shoot buds on a
medium containing a high concentration of N 6 -benzyladenine (22.0 mM). Method has
been used for successful transfer and expression of synthetic cry1Ab in pigeonpea with
varied range of expression of gene in different tissues of the whole plant, showing the
highest expression in flowers (0.1 per cent of total soluble protein) and least in the
leaves (0.025 per cent of total soluble protein) (Sharma et al., 2006). It is also important
to develop efficient techniques for evaluation of transgenic plants for resistance to H.
armigera. Therefore, Gopalaswamy et al. (2007) evaluated the usefulness of detached
leaf assay to test the bioefficacy of transgenic pigeonpea carrying Bt-cry1Ab and SBTI
genes for resistance against H. armigera. The method could determine the levels of
Cry1Ab or SBTI (soybean trypsin inbhibitor) proteins in the transgenic pigeonpea plants
as not sufficient to cause significant deterrent effects on leaf feeding, larval survival and
larval weight of H. armigera and found to be quite useful for evaluation of transgenic
pigeonpea plants for resistance to H. armigera. Further, the field evaluation of
transgenic pigeonpea plants of ICPL 88039 and ICPL 87 carrying cry1Ab and soybean
18
trypsin inhibitor (SBTI) genes indicated lack of significant reduction in leaf feeding, larval
survival and larval weight indicated necessity to develop plants with high expression
levels of cry1Ab endotoxin or SBTI which would be sufficient enough to supress insect
attack (Gopalaswamy et al., 2007).
The major problems in development of transgenics in pigeonpea are poor in vitro
regeneration and low frequency transformation. Many efforts have been done to
develop efficient protocols of Agrobacterium and microprojectile bombardment based
genetic transformation of this crop and plant regeneration from in vitro cultured explants
via organogenesis as well as by somatic embryogenesis. Rao et al. (2008) have
proposed non-tissue culture-based method for transgenic pigeonpea using
Agrobacterium Ti plasmid mediated transformation system. The method involved raising
of whole plant transformants (T0 generation) directly from Agrobacterium infected young
seedlings. The plumular and intercotyledonary meristems of the seedling axes have
been targeted for transformation by pricking of the apical and inter-cotyledonary region
of the seedling axes of two day old germinating seedlings with a sewing needle and
infecting with Agrobacterium in Winans’ AB medium containing wounded tobacco leaf
extract. Co-cultivation is performed in the same medium for 1hr and seedlings are
transferred to soilrite for further growth and hardening and subsequent transfer of
seedlings to soil in pots in the greenhouse (Rao et al., 2008).
The merits associated with in planta transformation methods includes, ensured
generation of pigeonpea transgenic plants with considerable ease in a short time;
applicable across different genotypes/cultivars of the crop and offers immense potential
as a supplemental or an alternative protocol for generating transgenic plants of difficult-
to-regenerate pigeonpea. Besides, it also offers the option of doing away with a
selection step in the procedure and so facilitates transformation, which is free of marker
genes (Rao et al., 2008). Recently, this protocol has been used to express chimeric
cry1AcF (encoding cry1Ac and cry1F domains) gene in transgenic pigeonpea (Ramu et
al., 2012). Interestingly, chimeric Cry1AcF levels in developed transgenic plants ranged
3-15 µg/g of fresh tissue weight with the insect mortality ranged 0 to 100 per cent
indicating the potential effect of Cry1AcF against H. armigera (Ramu et al., 2012).
19
2.4 Pigeonpea pod borer, Helicoverpa armigera life cycle and damaging stages
The Helicoverpa armigera is also known with different synonyms such as,
Heliothis armigera (Hübner), Chloridea armigera (Hübner) Heliothis obsoleta Auctorum,
Chloridea obsoleta, Helicoverpa obsoleta Auctorum, Heliothis fusca Cockerell, Heliothis
rama Bhattacherjee and Gupta, Noctua armigera Hübner. It belongs to Kingdom
Animalia, Phylum Arthropoda, Class Insecta, Order Lepidoptera, Family Noctuidae and
Genus Helicoverpa. H. armigera completes its life cycles in 4-6 weeks starting from egg
to adult in summer, and 8-12 weeks in spring or autumn. The different stages in
Helicoverpa life cycle are egg, larva, pupa and adult (moth) (Begemann and Schoeman,
1999).
It has been studied that the adult moth wingspan in case of H. armigera is 30-45
mm; the forewings are brownish or reddish-brown (females) or dull greenish to yellow or
light brown (males); hindwings are pale with a broad, dark outer margin. The moths
have a pale patch near the centre of this dark region and feed on nectar (Shanower et
al., 1997). They live for around 10 days and females lay 1000 eggs in their life span.
The eggs are laid in clusters, on leaves, flower buds, flowers and developing fruits
(Begemann and Schoeman, 1999). Fertile eggs of H. armigera hatch in about three
days during warm weather (25 °C average) and 6 to 10 days in cooler conditions. With
their development the colour of eggs changes from white to brown to a black-head
stage before producing a hatchling. All laid eggs are fertile. At the same time different
physical factors such as temperature also dramatically affect egg survival and larval
establishment (Yin et al., 2009).
The hatched larva (neonate) of 1 to 1.5 mm long makes exit hole by eating
through the eggshell. Initially larvae feeds on tender young foliage, followed with buds,
flowers or young pods, bolls or fruits. There are six growth stages, also called as
instars, in larval development and it becomes fully grown in 2 to 3 weeks in summer or 4
to 6 weeks in spring or autumn. The larval development is noticed more rapid at higher
temperatures. Ninety per cent of all feeding by Helicoverpa is done by larva mainly from
the third instar, small medium larva of 8 to 13 mm long, onwards. Large Helicoverpa
larvae, more than 24 mm long, are the most damaging, because larvae consume about
80 per cent of their overall diet in the fifth and sixth instars (Yin et al., 2009). It indicates
20
the importance of controlling Helicoverpa larvae in early stages of development.
Helicoverpa larvae feed on different plant tissues such as, leaves, flower buds and
flowers, developing pods, fruits and seeds. Pupae are normally developed to produce a
moth in 10 to 16 days. The moth emerges, feeds, mates and the cycle continue.
2.5 Bacillus thuringiensis delta-endotoxin
The soil bacterium Bacillus thuringiensis (Bt) has been a source of insecticidal
toxins produced in commercial transgenic plants (Gatehouse, 2008). The Bt strains
constituted a large reservoir of genes encoding insecticidal proteins that has been
studied for their different specificities of insecticidal activity toward different pests. These
toxins accumulates in the crystalline inclusion bodies produced by the bacterium on
sporulation known as Cry proteins or Cyt proteins; or expressed during bacterial growth
known as Vip proteins (Gatehouse, 2008). The Cry proteins possessing three domains
have been extensively studied. The mechanism of action of Cry protein involved a
proteolytic activation step in insect gut after ingestion, followed by interaction of one or
both of domains II and III with surface cell receptors of the insect gut epithelium. This
interaction results in oligomerization of the protein and domain I forms an open channel
through the cell membrane resulting in breakdown of the gut due to ionic leakage in
cells leading to bacterial proliferation and insect death (Bravo et al., 2007).
More than 500 different cry genes have been classified into 67 groups based on
their primary amino acid sequence (Cry1-Cry67) (Crickmore et al., 2010). Further, these
cry genes have been divided in to four phylogentically non-related protein families such
as, the family of three domain Cry toxins (3D), the family of mosquitocidal Cry toxins
(Mtx), the family of the binary-like (Bin) and the Cyt family of toxins (Bravo et al., 2005).
Among these, the largest Cry family is the 3D-Cry group, which consisted of at least 40
different groups with more than 200 different gene sequences. The 3D-Cry group
proteins possess three domains, of which domain I is implicated in membrane insertion,
toxin oligomerization and pore formation. Domain II of 3D-Cry group proteins is involved
in receptor recognition. Both domain II and III are responsible for insect specificity by
mediating specific interactions with different insect gut proteins (Bravo et al., 2007).
21
Study has identified Cry1Ac binding proteins as V-ATPAse subunit A and actin of
brush border from M. sexta and H. virescens. It has indicated that the mode of action of
Cry toxins involve binding of the toxin with other components of the midgut cells (McNall
and Adang, 2003; Krishnamoorthy et al., 2007). Commercial Bt cotton has been
developed for expression of Cry1Ac protein for the control of Lepidopteran pests.
Further, a second generation Bt-cotton has been produced for Cry2Ab besides Cry1Ac
as a resistance managing mechanism. Bt-corn expressing Cry1Ac has been tested for
their effectively against lepidopteran pests (Christou et al., 2006).
The Cry2Aa belongs to an unusual subset of crystalline proteins. It possessed
broad insect species specificity against larvae from Lepidoptera and Diptera (Liang and
Dean, 1994). It has been reported that the Cry2Aa protoxin is significantly smaller (72
kDa) than those of the Cry1 proteins (~135 kDa). The Cry2Aa is a short protoxins and
are processed primarily at the N-terminal end. Such activated toxin follows complex
sequential binding events with different insect gut Cry-binding proteins and results in
membrane insertion and pore formation (Bravo et al., 2011).
Like any other Cry protein, the Cry1F protein mechanism of action involves the
enzymatic cleavage of the protoxin to form core toxin. During this process, a short
peptide, involved in the formation of crystalline inclusion bodies, are cleaved from both
N-terminal and C-terminal end (Gao et al., 2006). The remaining protein portion is
believed to be responsible for midgut membrane binding. After such a binding, a toxin
molecule forms oligomers and creates pores in the membrane, causing osmotic
destabilization and cell death (Jurat-Fuentes and Adang, 2001). Many studies have
determined the activity of Cry1F against lepidopteran species (Balog et al., 2011;
Oppert et al., 2010). The Cry1F toxin susceptible species include tobacco budworm,
beet armyworm, soybean looper, cotton bollworm, fall armyworm, lesser cornstalk
borer, wax moth and European corn borer (Adamczyk et al., 2008; Blanco et al., 2010;
Buntin, 2008; Tindall et al., 2009).
The native cry1Ac gene sequence from B. thuringiensis have been in silico
modified and artificially synthesized to overcome codon bias and other undesirable
regulatory coding sequences for its improved expression in transgenic plants. Mohan
(2008) targeted only toxic moiety (1.85 kb) for codon optimization and out of 620 codons
22
259 have been altered. The study resulted in the development of modified cry1Acm with
increased GC content to 42.60 per cent by removing the AT-rich regions that were
typical for cry1Ac genes. Study also involved modifications in specific undesirable
eukaryotic regulatory sequences.
Many novel Bt insecticidal proteins have been expressed in transgenic plants.
The Cry34/35 and Vip1/2 toxins have been known for activity against corn rootworm
(Diabrotica virgifera). Moellenbeck et al. (2001) have developed genetically modified
maize expressing the cry34/35 successfully. On other hand, the single chain Vip
proteins, like Vip3, have been studied for their activity against Lepidopteran larvae, with
a broader range of toxicity, which has been further extended by protein engineering
(Fang et al., 2007). Considerable efforts have been undertakes for production of
commercial transgenic plants expressing these proteins (Christou et al., 2006).
It has been hypothesized that combining domains from different insecticidal
proteins could generate active toxins with novel specificities. It has been reported that
the transfer of the carbohydrate binding domain III generated a Cry1Ab-Cry1C hybrid is
highly toxic to armyworm (S. exigua). Further, it has been shown that the presence of
the Cry1Ca domain III was sufficient to confer toxicity toward Spodoptera (de Maagd et
al., 2000). In similar study, Naimov et al. (2003) have reported that the transgenic potato
expressing a hybrid Cry protein, containing domains I and III from Cry1Ba and domain II
of Cry1Ia, conferred resistance to the lepidopteran pest potato tuber moth (Phthorimaea
operculella) and to the coleopteran Colorado potato beetle (Leptinotarsa decemlineata).
The parental Cry proteins in such hybrid proteins are Lepidopteran specific, with no
toxicity toward coleopterans such as the potato beetle, demonstrating the creation of a
novel specificity (Naimov et al., 2003).
The site-directed mutagenesis in Bt protein coding genes has been used as an
alternative to the domain swap approach to increase toxicity toward target pests. It has
been studied that the mutation of Cry1Ab has increased its toxicity toward larvae of
gypsy moth (Lymantria dispar) by 40 fold (Rajamohan et al., 1996). The similar
strategies have been used to increase the toxicity of Cry3A protein toward target
coleopteran pests (Wu et al., 2000). Furthermore, a directed evolution system based on
phage display technology have been used for producing toxins with improved binding to
23
a receptor and increased toxicity (Ishikawa et al., 2007). It has been reported that a
current commercial transgenic maize variety with resistance to corn rootworm,
MON863, expresses a modified version of Bt Cry3Bb1 toxin (Vaughn et al., 2005).
Many variants of the native Cry3Bb have been developed by incorporating a series of
specific mutations that aimed to improve the channel forming ability of the toxin and
have been screened for activity (English et al., 2003).
2.6 Insecticidal proteins other than delta-endotoxin
The α-amylase inhibitors from some legume seeds have been shown to be
effective in imparting resistance of specific varieties of legumes to coleopteran seed
weevils. The bean (Phaseolus vulgaris) α-amylase inhibitor gene has been expressed in
transgenic garden pea (Pisum sativum) and other grain legumes using seed-specific
promoter (Shade et al., 1994). It has been reported that the developed transgenic seeds
showed resistant against stored product pests, such as larvae of bruchid beetles and
field pests, such as larvae of the pea weevil Bruchus pisorum. Lectin genes have been
potentially explored to confer insect resistance in transgenic plants to target hemipteran
plant pests. The Man-specific snowdrop lectin (GNA) has been expressed in transgenic
rice plants using constitutive or phloem-specific promoters (Rao et al., 1998). The
studied reported that the transgenic plants were partially resistant to rice brown
planthopper (Nilaparvata lugens Stal) and other hemipteran pests, with reduction in
insect feeding, development and fertility of survivors up to 50 per cent (Foissac et al.,
2000).
A study incorporating a 90 days feeding trial reported no adverse effects resulting
from consumption of transgenic rice expressing GNA by rats (Poulsen et al., 2007). The
use of a Man-specific lectin from garlic has shown similar partial resistance to
hemipterans (Allium sativum) leaves (ASA-L) in transgenic rice (Saha et al., 2006b).
Further, the transgenic rice plants expressing ASA-L have shown decreased
transmission of Rice tungro virus by its insect vector, as a result of decreased feeding
by the pest (Saha et al., 2006a). Nematodes of Heterorhabditis species having
symbiotic enterobacteria have been widely used for small-scale biological control of
insect pests. It has been reported that the toxins secreted by the bacteria cause cell
death in the insect host, resulting in a lethal septicaemia (Gatehouse, 2008). The P.
24
luminescens, the well-investigated bacterial species have been reported for expressing
a large number of potentially insecticidal components (Ffrench-Constant et al., 2007).
The tcdA has been cloned and expressed with 5’ and 3’ untranslated region
sequences from a tobacco osmotin gene in transgenic Arabidopsis plants (Liu et al.,
2003). The developed transgenic plant has showed complete protection against larvae
of the lepidopteran tobacco hornworm (Manduca sexta). Bacterial cholesterol oxidase
has been studied for their insecticidal activity that is comparable to Bt toxins. Similarly,
avidin has been studied for their insecticidal effect on many insects. The transgenic
maize expressing avidin has been developed and it reported complete resistant to
larvae of three different coleopteran storage pests (Kramer et al., 2000).
2.7 Tissue culture studies in pigeonpea
Attempts have been failed to develop pigeonpea cultivars resistant to legume
pod borer (H. armigera) and fusarium wilt using conventional breeding methods, due to
narrow genetic variability and breeding incompatibility problems. The development of a
somaclonal variant have been reported in pigeonpea, which produces white seeds
exhibits 25 per cent increase in seed size and 30 per cent increase in yield (Saxena,
2005). The efficient plant regeneration protocols are a prerequisite in recombinant
technology. In vitro tissue culture methods have provided an opportunity to
micropropagate elite plant clones by organogenesis and somatic embryogenesis.
Through many studies, now it has become possible to regenerate pigeonpea plants
from differentiated and undifferentiated tissues in culture conditions. It has been
reported that in vitro regeneration of pigeonpea is often genotype-specific (Krishna et
al., 2010).
2.7.1 Organogenesis
The organogenesis has been the extensively used in pigeonpea due to its wider
adaptability among diverse genotypes. Many studies independently have reported
protocols for obtaining stable regenerants through organogenesis from apical meristem,
undifferentiated callus, differentiated nonmeristematic tissues like leaf, and various
seedling explants such as hypocotyls, cotyledons, cotyledonary nodes, epicotyls, and
embryonal axes (Krishna et al., 2010). Although, Villiers et al. (2008) have reported that
25
pigeonpea cultivars with differential crop maturity regenerate plants without any
abnormalities in growth, flowering and seed set, long-duration maturity cultivars of
pigeonpea respond better in tissue culture.
2.7.1.1 Callus mediated organogenesis
A method has been developed for regenerating pigeonpea [Cajanus cajan (L.)
Millsp.] plant through callus obtained from distal cotyledonary segments of mature
seeds (Mohan and Krishnamurthy, 1998). The study has reported the induction of large
number of shoot buds directly from explants of genotypes T-15-15 and GAUT-82-90
when cultured on basal media fortified with N6-benzylaminopurine, kinetin and adenine
sulfate. Seed and seedling explants of pigeonpea have been evaluated for
organogenesis and somatic embryogenesis (George and Eapen, 1994). Study has
involved de novo regeneration of plants through organogenesis using mature
cotyledons, primary leaves and roots of seedlings as explants. Production of multiple
shoots has been reported from the cotyledonary node in cultures of whole seeds on 6-
benzylaminopurine enriched medium. The somatic embryos have been induced from
immature cotyledons and embryonal axes (George and Eapen, 1994).
Three pigeonpea genotypes viz; ICPL 93086, Tanzania–7 and their hybrids have
been tested for whole plant regeneration in different combinations of growth regulators
(Tyagi et. al., 2001). Study has reported significant differences among explants from
leaf, shoot and roots in calli formation. The differential effect of genotype also has been
observed in callus formation (Tyagi et. al., 2001). Further, the largest percentage of calli
has been reported formed by leaf explants in all three genotypes. The calli formation
has been excellent in medium containing 2,4-dicholorophenoxy acetic acid (2,4-D),
Kinetin (KIN), Napthaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP). No
genotype or explant differences have been recorded in shoot and root regeneration
from calli formed from either type of explants in any three genotypes. The medium
containing indole - 3 acetic acid (IAA), 6-(yy-dimethylallylamino) Purine (2IP),
Gibberrellic acid (GA3) and BAP has been reported as the best culture medium for
rooting (Tyagi et al., 2001).
26
Effects on the developmental morphology of Cajanus seeds irradiated with
Cogamma rays have been investigated in vitro (Shamarao and Narayanaswamy, 1975).
Study has demonstrated that the exposure of seeds to 5 kR produced a cluster of
adventitious roots on a callusing medium. While the hypocotyl explants of germinated
seedlings has found stimulated for cell proliferation and abundant callusing on a
medium resulting in shoot buds and plantlets development. Further, it has been
reported that in controls and with higher doses growth have been limited to the
development of a spongy tissue to form a small callus mass from seeds and a restricted
callus proliferation from excised hypocotyls of irradiated seeds (Shamarao and
Narayanaswamy, 1975).
Kumar et al. (1983) have studied the efficiency of callus induction for four
different seedling explants namely leaf, epicotyl, root and cotyledons of pigeonpea with
different media. Study has reported that the Blaydes' medium supplemented with 2,4-
dichlorophenoxyacetic acid (2,4-D) and kinetin have been most effective in inducing
callus from various explants. Among the tested explants leaves have been found to be
most efficient in producing rapidly growing callus. Multiple shoots ranging 5-18 have
been induced from excised cotyledons on Blaydes' medium with 6-benzylaminopurine
(BAP) (Kumar et al., 1983). Shoot buds have been regenerated from cotyledonary
callus with 8-20 per cent frequency in Blaydes' medium with BAP and α-
naphthaleneacetic acid (NAA). The callus obtained from leaf cultures of ICP 7035 have
been regenerated in 14 per cent of the cultures on Blaydes' medium supplemented with
BAP, NAA and gibberellic acid (Kumar et al., 1983). The regenerated shoots in study
have been rooted on the same medium containing either NAA and KN or indole-3-acetic
acid and KN.
Sreenivasu et al. (1998) have reported the efficient plant regeneration via
somatic embryogenesis in pigeonpea. Cotyledon and leaf explants from 10 day old
seedlings have been subjected for development of embryogenic callus and somatic
embryos when cultured on Murashige and Skoog medium supplemented with
thidiazuron (TDZ). Further, subsequent withdrawal of TDZ from the induction medium
has showed induction of maturation and growth of the embryos into plantlets. In another
study, suspension cultures of calli derived from seedling leaf explants of Cajanus cajan
L. var. Vamban-1 have been used for production of somatic embryos (Anbazhagan and
27
Ganapathi, 1999). The highest embryogenic frequency have been recorded on
semisolid MS medium supplemented with 2,4-dichlorophenoxyacetic acid. Similarly, the
maximum frequency of somatic embryogenesis have been observed when this callus
was transferred to MS liquid medium supplemented with 2,4-D.
2.7.1.2 Direct organogenesis
The morphogenetic response of various explants of seven different cultivars of
pigeonpea has been studied. The stimulation and elongation of shoot buds into shoots
derived from the mature embryo axis and intact seed on Murashige and Skoog medium
supplemented with kinetin and benzyladenine have been found to be optimum in
Murashige and Skoog medium supplemented with kinetin, naphthalene acetic acid, and
gibberellic acid. The cotyledon and epicotyl explants of pigeonpea cultivars on the other
hand differentiated directly into four to eight and two to four shoots, respectively,
depending on the media composition and genotype (Naidu et al., 1995). On the other
hand, cotyledons excised from seedlings of Cajanus cajan have been grown on media
containing cytokinins (6-benzyladenine, zeatin, and zeatin riboside) and an allied
compound, thidiazuron (Chandra et al., 2003). It has been reported that with the
exception of zeatin riboside, initial response in terms of induction of organized
structures was very high. Subsequent regeneration of shoots from cotyledon explants
has been very poor. Anatomical studies on the regenerating explants have been
undertaken to study the pattern of morphogenesis. Cytokinins and thidiazuron induced
divisions in the epidermal and sub-epidermal cell layers have been led to the formation
of primary protrusions on the surface. This, further, has been followed by the
development of foci of high meristematic activity either on the surface or within the
primary protrusions. These foci differentiated into embryo like structures or shoot
meristem-like structures (Chandra et al., 2003).
Geetha et al. (1998) have demonstrated an efficient and direct shoot bud
differentiation and multiple shoot induction from seedling explants of pigeonpea. Study
has reported that the frequency of shoot bud regeneration has been influenced by the
type of explant, genotype and concentrations of cytokinin. Explants, such as, epitocotyl,
hypocotyl, leaf, cotyledon and cotyledonary nodal segments from 7 day old seedlings
have been cultured on MS medium augmented with different concentrations of
28
BAP/kinetin. Among the various concentrations tested, 2.0 mg/L BAP or kinetin has
been found to be the best for maximum shoot bud differentiation. Percentage as well as
the number of shoots per explant showing differentiation of shoot buds has been higher
on MS media supplemented with BAP compared to kinetin (Geetha et al., 1998).
Elongation of multiple shoots has been obtained on MS medium fortified with BAP in
combination with NAA and GA3. The combination of 1.0 mg/L BAP with 0.1 mg/L NAA
has resulted in increase number of multiple shoots and shoots elongation. Addition of
GA3 along with BAP and NAA combination has enhanced both multiple shoot
proliferation and shoot elongation in all the explants. Regenerated plants have been
successfully established in soil where 90–95 per cent of them developed into
morphologically normal and fertile plants (Geetha et al., 1998).
Villiers et al. (2008) has evaluated seven varieties of pigeonpea of varying growth
durations and adapted to a wide range of environments across eastern and southern
Africa for their shoot regeneration response in tissue culture. Study has reported that,
on a standardized shoot regeneration medium, the short duration varieties viz., ICPV
88091 and ICPV 86012, generally responded faster and better than the medium
duration viz., ICEAP 00554 and ICEAP 00557, and long duration viz., ICEAP 00020,
ICEAP 00040 and ICEAP 00053 varieties. All the tested varieties produced healthy
rooted plants in vitro that could be transferred to the greenhouse where they exhibited
normal growth, flowering and viable seed set (Villiers et al., 2008). Thidiazuron either
alone or in combination with IAA have been used for induction of high frequency shoot
regeneration from primary leaf segments of three pigeonpea cultivars (Eapen et al.,
1998). Study reported that that transfer of the cultures to medium with reduced
concentration of thidiazuron has been resulted in further development of the shoots.
The regenerated shoots have been subsequently transferred to medium supplemented
with BA, IAA and gibberellic acid where 5-10 per cent of the shoots elongated further.
Rooting of shoots has been obtained on half strength MS medium supplemented with
NAA (Eapen et al., 1998).
2.7.2 Somatic organogenesis
Somatic embryogenesis represents formation of embryos from somatic tissues
such as roots, cotyledons, leaves and stems on liquid and solid medium (Krishnaraj and
29
Vasil, 1995; Merkle et al., 1995; Sharma and Thorpe, 1995). The embryogenic calli
exhibit prolific multiplication and somatic embryo gives rise to plants by eliminating the
tissue culture steps. The regeneration approach through somatic embryogenesis has
been considered as the preferred pathway for transformation (Hansen and Wright,
1999). It has been studied that the meristematic tissues like immature zygotic embryos
and cotyledon explants are highly responsive for the induction of somatic
embryogenesis (Parrott et al., 1992; Sagare et al., 1995; Ahmed et al., 1996).
Patel et al. (1994) was the first to report somatic embryogenesis in pigeonpea.
Subsequently its pathway of regeneration has been reported in three cultivars (Patel et
al., 1994). Many independent studies has reported that the somatic embryos are
regenerated from diverse genotypes using various explant tissues such as mature
seeds, shoot apices, intact seedlings, leaves, petioles, hypocotyls, epicotyls,
cotyledonary nodes, cotyledons, internodes, roots, endosperm, and cell sus-pensions
(Sarangi et al., 1992; George and Eapen, 1994; Nalini et al., 1996; Sreenivasu et al.,
1998; Anbazhagan and Ganapathi, 1999; Mohan and Krishnamurthy, 2002; Singh et al.,
2003). Among all, cotyledon and leaf explants have been used for direct and callus-
mediated somatic embryogenesis, respectively (George and Eapen 1994; Nalini et al.
1996; Sreenivasu et al. 1998; Mohan and Krishnamurthy 2002). Further, the haploid
somatic embryoids have been derived from anthers of pigeonpea flowers when cultured
on MS medium added with KIN (2.0 mg/L) and IAA (4.0 mg/L) (Bajaj et al., 1980).
High somatic embryo regeneration up to 90-97.0 per cent has been observed
from pigeonpea cv. Gaut 82-90, Gaut 82-99 and T15-15 with cotyledon explants (Patel
et al., 1994; Mohan and Krishnamurthy, 2002). A low regeneration frequency of
embryos has been reported using leaf tissue as an explant from pigeonpea cv.
Vamban-1 (Anbazhagan and Ganapathi, 1999), followed by Pusa 852, H 86-5, Pusa
609, Pusa 856, and Pusa 855 (Sreenivasu et al., 1998). The effect of genotype on
somatic embryogenesis has been further evaluated by culturing the cotyledonary nodes
of various pigeon pea cv. Pusa 853, ICPH 8, Pusa 33, ICP 151, RWL 19, UPAS 120,
and ICP 8863 with a globular embryo formation of explant on the same medium (Singh
et al., 2003). It has been reported that the germination frequency from globular embryos
varies in relation to pigeonpea cultivars such as NP (WR) 15 (70%) (Patel et al., 1994)
and ICPL 87 (3%) (Nalini et al., 1996). These studies have suggested that less
30
responsive genotypes of pigeonpea for somatic embryogenesis can still be exploited for
organogenesis mediated regeneration.
Among the explants, better embryogenic frequency up to 74 per cent has been
observed from leaf explants, with 48 average somatic embryos, and a lower one (45 per
cent), with 34 embryos from each cotyledon explants (Sreenivasu et al., 1998).
Similarly, a higher embryo regeneration frequency has been achieved from immature
embryo axes (91 per cent) than with immature cotyledons (23 per cent). On the other
hand, mature cotyledons reported an embryogenesis frequency comparable to that from
immature embryonic axes (Patel et al., 1994). Moreover, Nalini et al. (1996) have
demonstrated somatic embryo formation from cotyledon, epicotyl, leaf and root
explants, of which only cotyledon-raised embryos converted into a plant. Explant tissues
of cotyledon from mature and immature seeds have been primarily used for achieving
somatic embryogenesis. While the leaf and hypocotyls have been less utilized for
somatic embryogenesis.
In many somatic embryo regeneration studies, TDZ has been most widely used
for pigeonpea (Sreenivasu et al., 1998). It is due to its combined activity of both auxin
and cytokinin (Thomas and Katterman, 1986; Saxena et al., 1992), and optimizing the
suitability of TDZ for somatic embryogenesis studies have indicated that the
concentrations of 1.0-2.0 mg/L realized 53-74 per cent frequency with about 38
embryos from each explant (Mohan and Krishnamurthy, 2002). Studies have also
reported that TDZ failed to regenerate somatic embryos and resulted in callus
proliferation or switched to organogenesis pathway directly, at extreme lower and higher
concentrations, with both cotyledons and leaf explants (Sreenivasu et al., 1998; Singh
et al., 2003). Interestingly some pigeonpea genotypes have responded better at higher
concentrations (4.0 mg/L TDZ) (Singh et al., 2003). The medium comprising BAP (1.0
mg/L) has promoted an average of 28 globular embryos from each explant, but
decreased the embryo formation at higher/lower concentrations (Mohan and
Krishnamurthy, 2002).
In pigeonpea, studies have been continued to exploit the function of cytokinins
(KIN) and auxins (IAA, IBA, NAA, and 2,4-D) either alone or in combination to improvise
the embryogenesis protocols. The study has found 2,4-D as most suitable to derive
31
callus mediated somatic embryos. Sucrose in combination with 2,4-D has found to
efficiently induce embryogenesis. On other hand, carbohydrate sources like glucose,
fructose and maltose have been found not optimal or ineffective (Anbazhagan and
Ganapathi, 1999). Further, comparison of the effect of 2,4-D, NAA and picloram on
somatic embryogenesis have revealed greater response of explant to 2,4-D with 91 per
cent frequency from embryonic axes explants. It was followed by picloram and NAA and
picloram showed 36 per cent response with cotyledon explants followed by a decrease
with 2,4-D and NAA (George and Eapen, 1994). These studies emphasize the need of
utilizing appropriate plant growth regulators for different explants to achieve maximum
response. The somatic embryo formation have been observed both on the adaxial and
abaxial surface of mature cotyledon segment on MS medium supplemented with BAP,
KIN and adenine sulphate (Patel et al., 1994). Nalini et al. (1996) have reported 3 per
cent of plant recovery from somatic embryos, regenerated from the surface of cotyledon
explants on MS medium fortified with NAA (50.0, 25.0, 10.0, and 5.0 mg/L) and BAP
(1.0 mg/L). Above studies clearly indicated that the less responsive leaf explants for the
somatic embryogenesis can be exploited by inoculating cotyledon explants on auxins
alone or on the medium augmented with auxin rich and cytokine-deficient medium
(Krishna et al., 2010).
2.8 Plant transformation studies
The plant transformation has started in the early 1980s. Researchers have
harnessed the causative agent of crown gall disease, Agrobacterium tumefaciens, for
introduction of defined fragments of DNA into plant cells (Newell, 2000). Plant
transformation refers to the introduction and integration of foreign DNA in plant cells and
the consequent regeneration of transgenic plants (Newell, 2000). Transfer of DNA into
plant cells results in transient or stable expression of the introduced DNA. Transient
expression, as name suggests, usually remains for few days only. However, it allows
the effects of experimental manipulations to be seen in a short time and hence occupies
a useful niche in such areas as development of transformation methodology or
metabolic studies. On other hand, stable transformation is often a time-consuming
process involving tissue culture techniques that facilitate the growth of whole plants
from treated cells or tissue explants. The introduced DNA through stable transformation
32
is integrated into the host cell DNA and is thereby eligible to be passed on to
succeeding generations.
2.8.1 Agrobacterium mediated method
The Agrobacterium tumefaciens, a Gram-negative soil bacterium, is soil
phytopathogen and has been identified as the causative agent of crown gall disease
(Smith and Towsend, 1907). Binns and Thomashaw (1988) have revealed that A.
tumefaciens is capable of transferring a particular DNA segment; transfer T-DNA of the
large tumor-inducing Ti plasmid into the nucleus of infected cells. Where, it is integrated
into the host genome subsequently resulting in the crown gall phenotype. The studies
have reported that the T-DNA contains oncogenic genes encoding enzymes involved in
synthesis of auxins and cytokinins, and the genes encoding for synthesis of opines
(octapine, nopaline, and agropine) (Zupan and Zambryski, 1995). Agrobacterium
rhizogenes is closely related to A. tumefaciens produces hairy root disease in
dicotyledonous plants. A. rhizogenes have been known to induce the formation of
proliferative multibranched adventitious hairy roots at the site of infection (Chilton et al.,
1982). The virulence plasmid of A. rhizogenes has been known as the Ri plasmid which
shares extensive functional homology with the Ti plasmid (Mehrotra and Goyal, 2012).
The process of Agrobacterium-mediated plant transformation is a highly complex
and depends on the genetic determinants of both the bacterium and the host plant cell
(Mehrotra and Goyal, 2012). Studies have identified the genetic components carried by
A. tumefaciens that are required for plant transformation, include the T-DNA, the Ti
plasmid virulence vir region, which is the master switch for transformation and three
chromosomal virulence loci, essential for transfer process, chvA, chvB, and pscA
(Statchel et al., 1985; Douglas et al., 1985; Thomashow et al., 1987). It has been
reported that the Ri and Ti plasmids share a functional similarity (White and Nester,
1980; Hooykaas et al., 1984; Sinkar et al., 1987). The gene of interest is placed
between the left and right border repeats of Agrobacterium-transferred T-DNA and the
T-DNA region is flanked by 25-bp border sequences in a directly repeated orientation
(Gelvin, 2003). Sheng and Citovsky (1996) have studied the ability of A. tumefaciens to
transfer a portion of its DNA, T-DNA, to the genome of the plant. The vir region includes
eight operons, virA, virB, virC, virD, virE, virF, virG, and virH encoding proteins, and has
33
been identified to regulate the processing and transfer of T-DNA. Study by Stachel et al.
(1986) has demonstrated that plant cells induce the expression of vir genes that are
essential for the process of plant transformation.
Stachel et al. (1986) has reported that Agrobacterium-infected cells excrete low
molecular weight compounds, which are recognized specifically by the Agrobacterium
as signal molecules that induce vir gene expression and thereby activate T-DNA
transfer. Plant proteins have been identified that contributes to Agrobacterium-mediated
transformation. Many proteins such as, BTI1, VIP1, Ku80, CAK2Ms, histones-H2A, H3-
11, H4, SGA1, UDP glucosyltransferase, and GALLS interacting proteins have been
reported to be involved in T-DNA and virulence protein transfer, cytoplasm trafficking,
nuclear targeting, T-DNA integration, stability and expression, and defense responses
(Gelvin, 2010; Magori and Citovsky, 2011; Tenea, 2012).
Agrobacterium-mediated transformation has been used for engineer plants to
produce a wide variety of useful high-value products and high-quality exogenous
proteins safely and effectively (Mehrotra and Goyal, 2012). Recombinant plant proteins
obtained from transgenic plants is an excellent potential source of recombinant
antibodies (Conrad and Friedler, 1994). A wide range of useful products have been
generated in genetically modified plant cell cultures (Marillonnet et al., 2005).
Biodegradable plastics, primary and secondary metabolites, biopharmaceuticals, and
commercially valuable plant traits have been engineered using Agrobacterium-mediated
transformation. Commercially important traits in trees like timber yield and decreased
generation time have also been a focus of Agrobacterium-mediated transformation
(Tzfira et al., 1998).
Recently, Thakur et al. (2012) have developed Agrobacterium-mediated
transformation in Populus and incorporated the silviculturally important traits like quality
paper production. Blanc et al. (2006) have developed efficient Agrobacterium-mediated
transformation method for Hevea brasiliensis. Similarly, Zombori et al. (2011) developed
highly efficient Agrobacterium-mediated transfer system in Brachypodium. On other
hand, Wang et al. (2012) developed an Agrobacterium-mediated method in the
medicinal herb, Bidens pilosa. Ribas et al. (2011) reported that Agrobacterium-mediated
transformation of embryogenic cultures as a viable and useful tool for coffee breeding
34
and functional analysis of agronomically important genes. A. rhizogenes-transformed
roots of coffee have been developed for functional genomic studies of coffee root genes
for coffee breeding and also have been used for the production of plant secondary
metabolites (Alpizar et al., 2008).
Agrobacterium-mediated transformation has been successfully used in
improvement of Brassica (Rafat et al., 2010). Singh et al. (2010) have reported efficient
transformation protocol in mustard using chickpea lectin gene. Bhuiyan et al. (2011)
have developed an efficient Agrobacterium mediated genetic transformation method for
Brassica juncea, wherein cotyledon explants were used as explants for successful gene
transfer. Similarly, Agrobacterium-mediated transformation has also been reported in
the biodiesel plant, Jatropha as well (Mazumdar et al., 2010; Zong et al., 2010).
Trick and Finer (1997) have described an efficient Agrobacterium based
transformation technology, sonication-assisted Agrobacterium-mediated transformation
(SAAT), in soybean. Many studies have demonstrated an efficient method of
transformation in legumes by regeneration of shoots from the cotyledonary node and
other meristematic explants after Agrobacterium infection (Trieu and Harrison, 1996;
Ohloft et al., 2003; Parmesha, et al., 2012). Efficient transformation of cotyledon
explants from Arachis hypogaea by Agrobacterium have been reported as a cost
effective, routine tool of peanut and chickpea transformation (Sharma and Anjaiah,
2000; Bhatnagar-Mathur et al., 2008). Transgenic soybean plants have been produced
by Agrobacterium-meditated T-DNA delivery (Trick and Finer, 1998). Recently, mature
seed embryos of Phaseolus vulgaris have been successfully transformed by
A. tumefaciens (Amugune et al., 2011). Similarly, Sathyanarayna et al. (2012) have
made a successful attempt for efficient and stable genetic transformation and
regeneration of legume Mucuna pruriens.
Agrobacterium-mediated transformation of commercially important monocots has
been initially achieved in rice and maize (Schlappi and Hohn, 1992; Chan et al., 1992).
Further, several monocots such as, barley, maize, rice, sorghum, triticale and wheat
have been transformed (Chan et al., 1993; Gurel et al., 2009; Harwood et al., 2009;
Raja et al., 2010). Enríquez-Obregón et al. (1998) have reported successful
Agrobacterium-mediated transformation in sugarcane meristems. Chan et al. (1993)
35
have reported the successful transfer and expression of a reporter gene driven by
alpha-amylase promoter in japonica rice using Agrobacterium-mediated gene transfer
system. Zhao et al. (2000) have made successful attempt of stably transformed
sorghum plants using Agrobacterium for production. Furthermore, a highly efficient and
reproducible Agrobacterium-mediated gene targeting transformation system has been
developed in rice (Ozawa and Takaiwa, 2010; Ozawa et al., 2012). Recently,
Ziemienowicz et al. (2012) developed a method of transgene delivery into Triticale
plants using the Agrobacterium-transferred DNA-derived nanocomplex. Many
independent studies have reported the Agrobacterium mediated transformation in
millets like Setaria italica, Panicum virgatum, Eleusine coracana and Pennisetum
glaucum (Liu et al., 2007; Wang et al., 2011; Li et al., 2010; Li et al., 2011; Sharma et
al., 2011).
Initially the in planta transformation protocol has been developed in the model
plant, A. thaliana (Feldman and Marks, 1987). Further, the floral dip method developed
by Clough and Bent (1998), which eliminated the step of vaccum infilteration. The
Camelina sativa have been transformed by floral dipping along with vacuum infiltration
(Lu and Kang, 2008). Trieu et al. (2000) have developed floral infilteration method and
seedling infilteration methods in Medicago. Similarly, Weeks et al. (2008) have
developed in planta transformation method in alfalfa for development of marker free
transgenic plants. In this method the seedlings have been cut at the apical node and
vortexed with suspension of Agrobacterium in sterile sand and then seeds were
subsequently grown to maturity. Many studies have reported in planta techniques
successfully applied to obtain transformants in mulberry, soybean, rice, and cotton
wherein Agrobacterium has been targeted to wounded apical meristem of differentiated
seed embryo in which various organs and primodial had been formed (Ping et al., 2003;
Supartana et al., 2005; Keshamma et al., 2008).
Similar strategy was also applied to pigeonpea, fieldbean, sunflower, and
safflower (Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b). The in planta
approach has been effectively used to introduce agronomically important traits in
groundnut (Rohini and Rao, 2001). In planta transformation has also been
demonstrated in other species like maize, rice, and wheat (Mehrotra and Goyal, 2012).
Razzaq et al. (2011) have developed in planta transformation protocol in wheat through
36
apical meristem. Ramu et al. (2012) reported the expression of a chimeric cry1AcF
(encoding cry1Ac and cry1F domains) gene in transgenic pigeonpea developed used in
planta method and studied their resistance towards H. armigera.
The effect of air evacuation has been studied in Arabidopsis thaliana by Clough
and Bent (1998), it has indicated two folds increase in transformation rate when flower
tissues have been air evacuated with Agrobacterium culture in place of flower dip
method. Dehestani et al. (2010) using in planta transformation method in Arabidopsis
has showed that plants infected with Agrobacterium strain GV3850 only reported
highest transformation frequency of 1.54 per cent. Whereas, using of vacuum infiltration
during Agrobacterium infection, transformation efficiency has been improved by 3.0 per
cent (Dehestani et al., 2010). Similarly, Habashi et al. (2012) have studied the effect of
using vacuum infiltration during Agro-inoculation in two pear (Pyrus communis L.)
cultivars viz., Bartlett and Harrow Delight and reported significant increase in
transformation efficiency (10.63 per cent) that that of common Agro-inoculation method
(4.06 per cent).
2.8.2 Direct methods
Through many studies, protocols for the electroporation of cell suspensions have
been worked out for many species such as, tobacco, rice and wheat (Rafsanjani et al.,
2012). Using this method so far the best results have been obtained for maize (Lurquin,
1997). To et al. (1996) reported that the efficiency of electroporation have been found to
be relatively high and 90 transgenic maize plants have been regenerated from 1440
embryos (6.25 per cent) and 31 plants from 55 calli (54.6 per cent). The efficacy of this
method has been fully comparable with the best results obtained for maize after micro
bombardment (Chowrira et al., 1996). Modifications in electroporation such as post
pulse addition of ascorbic acid or ascorbate could significantly increase the efficiency of
process without having any negative influence on cell viability (Lazzeri, 1995). The
electroporation is a simple and effective method, but its application is limited to only few
species. In addition, application of electric current damages the gene leading to
misleading codons and wrong translational end product (Gaertig et al., 1994; Rakoczy-
trojanowska, 2002).
37
Microinjection is another method wherein, DNA is directly injected into plant
protoplasts or cells, specifically into the nucleus or cytoplasm, using fine needle or tip of
0.5-1.0 micrometre diameter made up of glass needle or micropipette (Rafsanjani et al.,
2012). This method of gene transfer has been used to introduce DNA into large cells
such as oocytes and the early embryonic stage (Oard, 1991; Casas et al., 1995;
Neuhaus et al., 1987). In yet another method, the silicon carbide fibres are simply
added to a suspension, containing both plant tissue and the plasmid DNA, and
rigorously mixed so that DNA-coated fibres penetrate the cell wall (Rakoczy-
trojanowska, 2002). Although this approach is easy, fast and economical method that is
applicable to various plants but this methodology have disadvantages like low
transformation efficiency, damage to cells that negatively influence their further
regeneration capability, and the necessity of taking precautions during lab work as
breathing the fibres in, especially asbestos ones, can lead to serious sicknesses (Asad
et al., 2008; Sailaja et al., 2008).
It is well studied that elevated temperature regimes enhances the gene transfer
that is successfully adopted and conformed to animal cell transfection process (Asad et
al., 2008). It has been documented that at temperature above 37 °C, the gene transfer
tendency found getting increased and further rise in ambient temperature to 43 °C with
certain period of time provided greater transient transfection (Rafsanjani et al., 2012).
Many studies have confirmed this concept through the work on interleukin-2 and swine
growth hormone expressions using indirect ELISA (Baron et al., 2001; Kenel et al.,
2010). Dillen et al. (1997) worked on the effect of temperature on Agrobacterium
tumefaciens-mediated gene transfer in plant. They reported the effect of temperature
(15 °C to 29 °C) in biotransformation process with A. tumefaciens involved in co-
cultivation of Phaseolus acutifolius and further in Nicotiana tabacum biotransformation
(Dillen et al., 1997). In both the situations, irrespective of the type of helper plasmid, the
level of transient uidA expression decreased notably when the temperature was raised
above 22 °C, further lowered down in temperature at 27 °C and was undetectable at 29
°C (Iba, 2002).
38
2.9 Selection marker
Genetic engineering involves the delivery, integration and expression of
defined genes into plant cells, which can be grown to generate transformed plants. The
system to select the transformed cells, tissues or organisms from the non-transformed
is important and hence, marker genes are vital to the plant transformation process.
From the development of first transgenic plants during early 1980s and subsequently
after its commercialization worldwide over a decade, antibiotic and herbicide resistance
selectable marker genes have become one among the integral feature of plant genetic
modification (Ramessar et al., 2007).
Many studies have revealed that antibiotics such as, kanamycin or hygromycin
and herbicide such as, phosphinothricin, PPT have been widely used as selection agent
since the early days of plant transformation (Sundar and Sakthivel, 2008). Positive
selection systems are those that allow the growth of transformed plant cells. Based on
their functionality the positive selection systems can be classified into conditional and
non-conditional positive selection system (Sundar and Sakthivel, 2008). A conditional-
positive selection system involves a gene encoding for a protein, usually an enzyme
that confers resistance to a specific substrate that may be toxic to the untransformed
plant cells or that facilitates the growth as well as differentiation of the transformed cells
alone.
Bacterial aminoglycoside 3’-phosphotransferase II (APH [3’] II, E.C 2.7.1.95),
also known as neomycin phosphotransferase II (NPTII) has been used as an effective
selectable marker in mammalian and yeast cells, it was the first to be tested in plants
(Miki and McHugh, 2004). The NPTII catalyses the ATP-dependent phosphorylation of
the 3’-hydroxyl group of the amino-hexose portion of certain amino-glycosides including
neomycin, kanamycin, geneticin (G418), and paramomycin (Miki and McHugh, 2004).
Hygromycin B is an aminocyclitol antibiotic inhibitor of protein synthesis. It has a broad
spectrum activity against prokaryotes and eukaryotes including in plants. The E. coli
gene aphIV (hph, hpt), coding for hygromycin B phosphotransferase (HPT, E.C.
2.7.1.119), has been reported for conferring resistance against bacteria, fungi, animal
cells and plant cells by detoxifying hygromycin B via an ATP-dependent phosphorylation
of a 7’’-hydroxyl group (Waldron et al., 1985; van den Elzen et al., 1985). Chimeric
39
genes have been studied for their effectiveness in diverse plant species, including
dicots, monocots and gymnosperms, and have been used as a selectable marker when
nptII was not found to be effective (Tian et al., 2000; Twyman et al., 2002).
The gene coding for streptomycin phosphotransferase (SPT, APH [3’], E.C.
2.7.1.87) have been derived from the bacterial transposon, Tn5 (Mazodier et al., 1985).
A mutant form of SPT, containing a two amino acid deletion near the carboxy terminus
of the protein, have been placed under the control of the T-DNA transcript promoter and
introduced into N. tabacum (Miki and McHugh, 2004). This marker system has not been
adopted for general use. Another marker gene, the aminoglycoside-N-acetyl
transferases (AAC), is another class of aminoglycoside-modifying enzyme. It potentially
acts as plant selectable marker genes (Nap et al., 1992). Two of such enzymes viz.,
AAC(3)-III and AAC(3)-IV, have been examined in petunia and Arabidopsis under the
control of the 35S promoter and nos 3 sequences (Hayford et al., 1988). The study
reported that the gene was effective in a variety of plants including Brassica napus,
Nicotiana tabacum and tomato. According to Nap et al. (1992) aminoglycoside-O-
nucleotidyl transferases, bacterial aadA gene, codes for the enzyme aminoglycoside-3’-
adenyltransferase represent the third class of enzymes that modify the aminoglycoside
antibiotics and have been used as plant selectable marker genes.
2.10 Southern Blotting
Southern blotting methods are an assisted to gel electrophoresis. It involves a
method for separating DNA with exceptional resolving power. Blotting enables the
detection of specific molecules among the mixture separated in the gel. The major steps
involve transfer of molecules from the gel to a porous membrane, which is achieved by
soaking solution through the gel and the membrane using absorbent paper. For DNA,
specific sequences are detected in the membrane by molecular hybridization with
labeled nucleic acid probes (Southern, 2006). The method has been used in several key
studies. The original proposal for the genetic mapping of the human genome has been
based on restriction fragment length polymorphisms (RFLPs) detected by blotting (Kan
and Dozy, 1978). Introns have been first seen in blots of rabbit genomic DNA hybridized
with probes for the ß-globin gene (Jeffreys and Flavell, 1977). The first DNA fingerprints
have been produced by hybridizing restriction digests of human DNA with minisatellite
40
probes (Jeffreys et al., 1985). Many of the early applications of the method have been
replaced by Sanger sequencing, which gives more information, or methods based on
the polymerase chain reaction (PCR), which is simpler to perform (Southern, 2006).
The transgenic status of regenerated plants has been performed employing PCR
amplification of the marker gene or transgene. Southern analysis is an important
component of transgene analysis to prove the integration of the foreign gene into host
genome (Bhat and Srinivasan, 2002). Southern hybridization helps to assess the
number of independent transgene insertions, which is important to find transformants
with single, unaltered transgene insertions and considered ideal for analysis (Bhat and
Srinivasan, 2002). It is also necessary to check the presence of multicopy tandem
insertions and other rearrangements at the given locus. Surekha et al. (2005) used
Southern blotting analysis for the confermation of 18 pigeonpea transgenic samples,
which showed clear amplification in PCR. The method confirmed the presence of
transgene and number of the inserts, size of the inserts in different plants confirming the
independent origin of the transgenic lines.
Southern blot analysis has been carried out for the genomic DNA from the T1
generation pigeonpea transgenic plants in order to determine the integration pattern and
the copy number (Sharma et al., 2006). The PCR amplicon of cry1Ab fragment have
been used as probe for the Southern hybridization analysis and hybridization signals
detected in eight of the 12 plants analyzed for the integration of Bt gene in the genome
of these plants. The blotting analysis revealed that four of six transgenic plants
possessed single-copy integrations (Sharma et al., 2006). Eleven of the plants, positive
in the dot blot hybridization; have been tested for genomic Southern analysis of nptII
gene (Rao et al., 2008). The results indicated hybridization signal in all the test eleven
plants except in the non-transgenic control plant sample confirming integration of the
transgene in the pigeonpea genome. Further, in similar king of study, Ramu et al.
(2012) studied the integration pattern using genomic Southern analysis, which was
carried out with restrict digested DNA from three selected plants. A strong signal and
the difference in the hybridization pattern in the selected transgenic plants revealed the
single copy integration in these plants, whereas no signal was observed with the DNA of
untransformed plants.
41
The PCR and Southern analysis of the genomic DNA from T1 progenies of
selected transgenic chickpea plants have showed amplification of expected amplicons
of 800 and 995 bp for cry1Ab and cry1Ac genes respectively similar to the positive
controls (Mehrotra et al., 2011). The result of Southern blot hybridization have shown
single DNA fragments of size ranging from 4.16 to 6.57 kb hybridizing with 1.845 kb
radiolabelled cry1Ab/ c probe in T1 transgenic plants. Results of Southern hybridization
also indicated that the insertion of cry1Ab and cry1Ac genes as single copy integrations
(Mehrotra et al., 2011). In similar study, Acharjee et al. (2010) Southern blotting of DNA
from pooled progeny of nine independent transgenic chickpea lines have been
performed and showed the presence and integration of the cry2Aa gene in eight of the
nine lines. Among all, few lines showed integration of one copy of the transgene into the
genome, few contained two or more copies of the transgene (Acharjee et al., 2010).
2.11 Immunoassay
An immunoassay technique based on antibodies is a standard approach for
qualitative and quantitative detection of protein of a known target analyte (Brett et al.,
1999). Both monoclonal, which is highly specific, and polyclonal, which is often more
sensitive, antibodies have been used depending on the specificity of the detection
system (Kamle and Ali, 2013). On the basis of typical concentrations of a transgenic
material in plant tissues, the limit of detection (LOD) of a protein immunoassay can
predict the presence of recombinant protein in genetically modified plant tissue (Stave,
2002).
The ELISA has shown a significant advantage for protein analysis in transgenic
plants. A sandwich ELISA is the preferable immunoassay used for the detection of Bt
protein, where an analyte is sandwiched in between the two antibodies; a capture
antibody and the detector antibody. In sandwich ELISA protein concentration is directly
proportional to the colour intensity (Kamle and Ali, 2013). ELISA has been successfully
used for the detection of protein encoded by cp4-epsps gene in a RR soybean (Rogan,
1999). Similarly, monoclonal antibodies have been used for the development of
sensitive and single epitope specific immunoassays for the detection of Bt proteins like
Cry1Ac and Cry1Ab (Vázquez-Padrón, 2000). For the detection of Cry1Ab, a capillary
electrophoresis competitive immunoassay and a highly sensitive quanti-dot based
42
fluorescence linked emmuno sorbant assay have been developed (Giovannoli et al.,
2008; Zhu et al., 2011). Similarly, a monoclonal antibody based sandwich immunoassay
having a 100 ng/g LOD for Cry1Ac and a 1 pg/g LOD for Cry2Ab in cotton seed/leaf
samples have been reported (Kamle et al., 2011, 2013; Shan et al., 2007).
A nitrocellulose-strip rather than microtiter wells have been developed and used,
which has resulted in the development of lateral flow strip/ dipstick/immuno-strip
technology (Kamle and Ali, 2013). Immobilized double antibodies, specific to recognize
expressed protein have been conjugated to a colour reactant (gold nano-particles) and
incorporated into a nitrocellulose strip. This nitrocellulose strip when dipped in the
protein extract of plant tissue harboring a GM protein, leads to an antibody reaction
releasing colour. This red coloured gold conjugated complex flows to the other end of
the strip through capillary movement to a porous membrane that has two captured
antibody zones. The immuno-strips can give results as either ‘Yes’ or ‘No’ within 5 to 10
min. The immuno-strip is an economical, easy and field tractable detection method.
These immuno-strips are commercially available to detect Cry1Ab, Cry1Ac, Cry2Ab and
CP4-EPSPS (Lipton et al., 2000; Fagan et al., 2001). The method has been used and
revealed that chimeric Cry1AcF levels in developed transgenic pigeonpea plants ranged
3-15 µg/g of fresh tissue weight (Ramu et al., 2012).
An immunoassay technique has been used to estimate the Cry1Ac protein levels
in developed transgenic chickpea plants and it ranged from 14.5 to 23.5 mg 1-1
extractable protein with high levels of toxicity in insect feeding bioassay with larvae of
pod borer insect H. armigera (Sanyal et al., 2005). Mehrotra et al. (2011) has used
modified cry1Ab and cry1Ac insecticidal genes to introduce into chickpea by
Agrobacterium-mediated transformation of pre-conditioned cotyledonary nodes.
Interestingly, the pyramided transgenic plants having moderate expression levels (15-20
ng/mg) showed high-level of resistance against pod borer larvae of H. armigera when
compared with high level expression of a single individual Cry toxin.
2.12 Real-Time PCR
Many studies have used real-time PCR to quantify a targeted cDNA molecules
(Kamle and Ali, 2013). Real-time PCR has shown great value in validating and
estimating the number of copies of inserted genes into the host genome (Zhang et al.,
43
2003). The method has been reported for several genetically modified crops such as
maize, cassava, rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al.,
2013; Beltrán et al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007).
Furthermore, a sensitive loop mediated isothermal amplication method employed for the
detection of three GM rice events has been reported (Chen et al., 2012; Kiddle et al.,
2012).
The 5′ event-specific/hmg-taxon gene real-time polymerase chain reaction (PCR)
protocol coupled to 2−∆∆CT analysis have been used to determine the MON 810 insert
copy number per haploid genome across 26 genetically modified commercial maize
varieties (Aguilera et al., 2008). The end-point and real-time polymerase chain reaction
methods have been used to detect EE-1 brinjal (Ballari et al., 2013). The study reported
the limits of detection and quantification for SYBR-based real-time PCR assay, which
were 10 and 100 copies respectively. Beltrán et al. (2009) developed real-time
polymerase chain reaction-based methods for the primary scrutiny of putative
transgenic plants. They tested for the presence of transgenes, estimated copy number,
and quantified messenger RNA (mRNA) levels of genes introduced through
Agrobacterium. Copy numbers for the genes ß-glucuronidase and hygromycin
phosphortransferase have been estimated in 15 transgenic lines. The study indicated
that although real-time PCR has been efficient for classifying transgenic lines with one
or more transgenes inserted, for conclusive analysis of gene copy number, i.e., in a
potential breeding line, the Southern blot may still be required (Beltrán et al., 2009).
Further, high, medium and low levels of mRNA expression have been detected but
study revealed that no direct relationship between copy number and expression level of
transgenes was obvious, suggesting that factors like position effects or DNA
rearrangements led to differential expression.
The quantitative real-time PCR have been used to determine transgene copy
number in transgenic wheat (Li et al., 2004). A conserved wheat housekeeping gene,
puroindoline-b, have been used as an internal control to calculate transgene copy
number. Estimated copy number in transgenic lines using real-time quantitative PCR
has been correlated with actual copy number based on Southern blot analysis in
transgenic wheat lines and it has been reported that the real-time PCR is an efficient
method for estimating copy number in transgenic wheat (Li et al., 2004).
44
2.13 Insect bioassay
Surekha et al. (2005) performed detached leaf feeding tests on T1 and T2
generation pigeonpea plants for insect resistance using the 1st and 2nd instar larvae of
the pest S. litura. Study reported significant variation between transgenic samples and
the control wild-type plants. The highest mortality of the larvae found in the transgenic
plants was 80 per cent. Further, the plant that showed high mortality rate or larval
damage was more than another plant. The effect of transgenic pigeonpea carrying
cry1Ab and SBTI genes on the growth and development of H. armigera have been
studied for three successive generations using detached leaf bioassay (Gopalaswamy
et al., 2007). Plant numbers Bt 6.1 (1.7%), Bt 1.2 (2.0%), SBTI 2.2 (2.0%), Bt 6.2
(2.2%), Bt 3.6 (2.3%), Bt 9.2 (2.3%), SBTI 1.4 (2.3%), Bt 3.2 (2.7%), SBTI 4.3 (2.7%) Bt
2.1 (3.0%), Bt 6.6 (3.0%), and SBTI 2.5 (3.0%) showed lower leaf feeding compared to
the non-transgenic plants of ICPL 88039 (4.5). whereas, plants Bt 6.1 (10.0%), SBTI 1.4
(13.3%), Bt 3.2 (16.7%), and Bt 6.2 (16.7%) also showed significantly less larval
survival than the non-transgenic control, ICPL 88039 (30.0%). Further, they have
reported that the larval weights in test samples were lower on Bt 2.1 (0.517 mg), Bt 8.1
(0.542 mg), Bt 3.2 (0.567 mg), Bt 7.2 (0.597 mg), Bt 1.2 (0.600 mg), Bt 6.2 (0.622 mg),
SBTI 4.3 (0.628 mg), SBTI 2.5 (0.633 mg), SBTI 1.2 (0.650 mg), and SBTI 7.5 (0.733
mg) as compared to the non-transgenic plants of the respective genotypes.
Ramu et al. (2012) tested the bioefficacy of the plants against first and second
instar Helicoverpa revealed significant variability in larval mortality and damage in both
the pods and leaves. Study indicated that the both the damage and the mortality in
insects varied from 0 to 100 per cent among the putative transformants. The effect of
the cry1AcF gene has been seen on the larva as there was a considerable difference in
the size of the larva that fed on the transgenics and wild type. The transgenic plants that
showed high mortality exhibited less damage in leaves and pods (Ramu et al., 2012).
Selected transgenic chickpea lines, BS2A, BS5A and BS6H have been tested in
insect bioassays using neonate H. armigera and compared to the non-transgenic cv
ICCV 89314 and cv. Semsen (Acharjee et al., 2010). Study reported significantly
greater larval mortality among the larvae receiving transgenic leaves than those
receiving leaves from the controls. Larval mortality was highest on the BS6H transgenic
45
line where almost all larvae died during the assays, wherein larval death on leaves from
BS6H was 100 per cent (Acharjee, et al., 2010).
The toxicity of T0 and T1 plants expressing Cry1Ac protein have been tested
using either isolated or whole plants in feeding assays with second or third instar
neonate larvae of H. armigera (Sanyal et al., 2005). The larvae ceased feeding after 2
days on leaves of A3, A4, B5, B8, B10, C6, C7, D2 and D3 chickpea plants expressing
high level of toxin and exhibited significant reduction in their weight decreased to 40 to
90 per cent and finally showed high mortality. Similarly, the high correlation between
feeding damage caused by H. armigera larvae on the transgenic chickpea leaves has
been reported (Lawo et al., 2008). For the susceptible H. armigera strain, leaf damage
has been significantly higher for control leaves than for Bt chickpea leaves after 24 hr of
feeding.
46
3. MATERIAL AND METHODS
The present study was undertaken at Department of Biotechnology, UAS,
Dharwad for development and molecular characterization of transgenic pigeonpea
plants expressing different cry genes viz., cry1Ac, cry2Aa, cry1F and cry1Acm
separately. The in vitro plantlet regeneration and in planta transformation protocols
were improvised and used for transformation of pigeonpea cultivars viz., ICPL 87119
(Asha) and BSMR 736. The details on material used and methods adopted are as
follows:
3.1. Plant material and in vitro plantlet regeneration in pigeonpea
Two pigeonpea varieties viz., ICPL 87119 (Asha), a moderately resistant variety
for fusarium wilt, sterility mosaic disease and BSMR 736, a sterility mosaic disease
resistant variety were used for present experiment as a plant material. Seeds of these
cultivars were obtained from Agriculture Research Station (ARS), Gulburga, which
were maintained by the breeders with purity. Healthy seeds with uniform size, shape
and colour were surface sterilized with 70% ethanol treatment for one min followed by
two rinses with sterile distilled water for two min each. Further, seeds were subjected to
0.1% (W/V) aqueous mercuric chloride solution treatment for 5 min followed by two
rinses with sterile distilled water for 2 min each. The surface sterilized seeds were used
for inoculation on Murashige and Skoog’s (MS) medium having different cytokinins and
their consecration regimes.
3.1.1 Culture media and conditions
The culture medium was that of MS with 3% (W/V) sucrose. For multiple shoot
bud induction, the culture medium was augmented with 1, 2, 3, 4, 6, 8 and 10 mg/L
BAP, TDZ and zeatin, separately. The pH of the medium was adjusted to 5.8 prior to
adding 8 g/L agar-agar. Initially, MS medium was sterilized at 121 °C for 15 min and
filter sterilized growth hormones was added. Cultures were maintained at 25±2 °C
under light intensity of 1000 lux with 16 hr/day photoperiod.
47
3.1.2 The effect of cytokinins on shoot bud induction
The experiment was designed to compare the effect of three cytokinins viz.,
BAP, TDZ and zeatin with their concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) on
shoot bud induction. The explants used for study were cotyledonary node with
cotyledons and embryo discs with half cotyledon. For cotyledonary node with
cotyledons as explants, the surface sterilized seeds were inoculated on basal MS
supplemented with increasing concentrations of growth hormones. In case of embryo
discs with half cotyledon as explants, the surface sterilized seeds were soaked
overnight in sterile distilled water; seed coat along with half cotyledon was detached
using sterile forceps retaining embryo discs with other half part of cotyledon. After 8-10
days of culture, the number of explants forming shoots buds and shoot buds per
explant were counted. The cotyledonary node with induced multiple shoot buds were
sub-cultured on the MS medium with respective growth hormone augmentations for
shoot bud elongation up to 10-12 days. Experiment was repeated twice using a total of
50 explants for each treatment. The growth hormone solutions were prepared viz., BAP
in 1N NaOH; TDZ in DMSO and Zeatin in 1N NaOH, and filter sterilized before using in
culture medium. The explants cultured on MS medium devoid of growth hormone
fortification were maintained as experimental controls and the effect of different
cytokinins and their concentration regimes was analyzed.
3.1.3 Rooting of elongated shoots
The shoots (~3 cm) devoid of roots were transferred to MS basal media fortified
with (0-2 mg/L) IBA. The treatments maintained for rooting were culture medium MS +
(0-2 mg/L) IBA; ½ MS and ½ MS + 0.50 mg/L IBA. A set of fifty shoots per rooting
medium treatment was maintained for this experiment and the experiment was
repeated twice. After 10-15 days of incubation in rooting media, individual shoots were
observed for root induction and the number of roots induced per shoot.
3.2 Maintenance of Agrobacterium tumefaciens strains and in vitro transformation of
pigeonpea
The A. tumefaciens strains carrying constructs with different cry genes were
grown and maintained on YEMA media (Appendix I) supplemented with respective
48
selection pressures. Such actively growing A. tumefaciens cultures were used in in
vitro transformation process.
3.2.1 Culture and maintenance of A. tumefaciens strains
The freshly prepared YEMA media was sterilized by autoclaving. The media
was allow to cool up to 50 to 60 °C and respective filter sterilized selection
pressures/antibiotics were added and thoroughly mixed before pouring it in UV
sterilized polystyrene petri dishes (Fisher Scientific Pvt. Ltd.). The antibiotics used
were, for A. tumefaciens strain with cry1Ac: Kan100; A. tumefaciens strain with cry2Aa:
Kan50 + Rif10; A. tumefaciens strain with cry1F: Kan50 and A. tumefaciens strain with
cry1Acm: Kan50 + Rif25 (Fig. 1a to 1d). Freshly inoculated culture was grown over night
at room temperature and stored at 4 °C. The cultures were revived after every 20 to 25
days. The cry1Ac, cry2Aa and cry1F gene constructs were provided by Dr. P. Ananda
Kumar, NRC on Plant Biotechnology, IARI, New Delhi as a part of ICAR-NFBSFARA
funded pulse project.
3.2.2 In vitro transformation of pigeonpea
The binary plant transformation vector carrying cry gene was used for
pigeonpea transformation. The pigeonpea seeds were surface sterilized with 70%
ethanol and allowed to germinate overnight in distilled water. The seed coat from
germinated seeds were removed and embryonic axis attached to single cotyledon were
surface sterilized using 1.2% (w/v) aqueous sodium hypochlorite solution for 2 min.
The embryonic axis of germinating seed was injured and infected with Agrobacterium
tumefaciens harbouring binary vector for 30 min. The explants were co-cultivated on
basal MS media for two days at 28 °C in dark. Co-cultivated explants were washed
repeatedly in sterile distilled water containing 300 mg/L cefotaxime and transferred to
selection medium containing MS basal + 2 mg/L BAP + 200 mg/L Kanamycin + 300
mg/L Cefotaxime media in plant growth chamber maintained at 25 ± 2 °C, light intensity
of 1000 lux with 16/8 hr light-dark duration. The survived explants were again sub-
cultured onto Kanamycin containing media for an additional round of testing. Well-
developed shoots were excised and transferred to half MS containing 0.5 mg/L IAA for
rooting. The rooted plants were hardened to establish in transgenic containment
facility.
49
Fig. 1a: Construct map of the binary vector pBinBt3. A. Construct map of the binary vector pBinBt3 carrying cry1Ac and nptII marker gene. B. T-DNA carrying cry1Ac gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator.
A
B
50
A
B
Fig. 1b: Construct map of the binary vector pBinAR. A. Construct map of the binary vector pBinAR carrying cry2Aa and nptII marker gene. B. T-DNA carrying cry2Aa gene with 35S promoter and ocsA terminator; nptII marker genes with nos promoter and terminator.
51
Fig. 1c: Construct map of the binary vector pBinAR. A. Construct map of the
binary vector pBinAR carrying cry1F and nptII marker gene. B. T-DNA carrying cry1F gene with 35S promoter and ocsA terminator; nptII marker genes with nos promoter and terminator.
A
B
52
nos
P
nptII
nos
T
35S
AMV
cry1Acm
uidA
nptII
nos
T
Xba I Bam HI
Fig. 1d: Construct map of the binary vector pMKK1708. T-DNA carrying cry1Acm gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator.
53
3.3 Explant preparation and improvisation of in planta transformation protocol in
pigeonpea
The in planta transformation method reported by Ramu et al. (2012) for pigeonpea
using seeds (embryonic axis) as a source of explants was further optimized to increase
the transformation efficiency. The seeds were surface sterilized with 70% ethanol and
allowed to germinate overnight in sterile distilled water. The seed coat from germinated
seeds were removed and embryonic axis attached to single cotyledon were injured using
fine needle and infected with Agrobacterium tumefaciens culture harbouring binary
plasmid for 30 min. Different treatments such as, (a) A. tumefaciens infection alone, (b) A.
tumefaciens infection using A. tumefaciens culture with tobacco extract added in
overnight grown culture (1.0 g of tobacco leaf tissue per 100 ml of A. tumefaciens culture)
and (c) A. tumefaciens infection with air evacuation to increase the proximity between A.
tumefaciens and embryonic axis (vacuum infiltration for 10 min at 450 mm Hg). A total of
200 explants in four batches (50 explants per batch) were subjected for transformation
using above mentioned treatments. The explants were kept in dark for two days at 25 ± 2
°C for co-cultivation on moist germination paper to avoid the drying of explants. After two
days of co-cultivation explants were allowed to grow at 16/8 hr light-dark period for seven
days on moist germination paper. Well responding seedlings, of seven days old, were
transferred to plastic trays containing sterile coco peat and hardened in green house
conditions for one week period. The well grown healthy seedlings, also called as primary
transformants (T0 generation), were transplanted in earthen pots carrying media mixture
of soil and FYM, and allowed to grow in transgenic containment facility till harvesting. The
per cent explants survived, per cent explants responded and per cent transformation
efficiency were calculated using following formulae;
Explants responded (%) = No. of explants responded
x 100 No. of explants infected
,
Explants survived (%) = No. of explants survived
x 100 No. of explants responded
Transformation efficiency (%) = No. of PCR +ve plants identified in T1
x 100 No. of explants infected
54
3.4 Identification of putative transformants
The putative transformants were identified by sowing the harvested seeds from
each primary transformant (T0) in plant to row progeny manner (all available seeds from
each T0 plants, if more seeds were available, up to 60 seeds). The progenies were tested
using cry and nptII gene specific PCR assay and putative transgenic plants (T1
generation) were identified.
3.4.1 Collection of plant samples and genomic DNA preparation
DNA was extracted from individual plants of primary or putative transformants and
non-transgenic plants. Leaves from 2-3 weeks old plants were collected, surface sterilized
with 70 per cent alcohol and frozen in liquid nitrogen. The leaf samples were stored at -80
°C until further use. The genomic DNA was prepared following the method of Krishna and
Jawali (1997) with a few minor modifications. Frozen tissue sample (2.0 g) was ground
into fine powder in liquid nitrogen, using autoclaved mortar and pestle and immediately
transferred to 2.0 ml Eppendorf tube containing 900 µl of extraction buffer and 90 µl of 20
per cent sodium dodesyl sulphate (SDS). The grounded tissue was not allowed to thaw;
the contents were mixed well and incubated at 65 °C for 10 min with intermittent shaking
for proper mixing of contents. The contents were then cooled on ice for 10 min and
potassium acetate 3 M (300 µl) was added and mixed thoroughly. Further, the contents
were spun for 20 min at 13000 rpm at 4 °C and about 600 µl of supernatant was
transferred to fresh tube and the remaining was discarded along with the tube. About 600
µl of isopropanol-ammonium acetate mixture was added to supernatant to precipitate
nucleic acids. The contents were mixed thoroughly and centrifuged for 20 min at 13000
rpm to pellet the nucleic acids. The supernatant was discarded and the pellet was washed
with 70 per cent alcohol, tubes were inverted on blotting paper to dry the pellet and the
supernatant was carefully drained to avoid damage/slippage of nucleic acid pellet at this
step. Finally, pellet was dissolved in 200 µl of T10E1.
The RNAase (5 µl) (10 mg/ml) was added to each tube. DNA was re-dissolved by
tapping the pellet suspended in RNAase and incubated at room temperature (37 °C) for
30 min. The DNA was precipitated by adding 1/10th volume of 3 M sodium acetate and
2.5 volumes of absolute ethanol. The contents were mixed gently and incubated at 4 °C
for 30 min. The contents were centrifuged for one min at 3000 rpm. The DNA pellet was
55
rinsed with 70 per cent ethanol twice and centrifuged at 3000 rpm for one min.
Supernatant was discarded and the tubes were put upside down on paper towel to
eliminate excess ethanol. Care was taken not to allow the DNA to over dry. Pellet was
dissolved in 100 µl of T10E1 and stored in -20 °C untill further use.
3.4.2 Purification and quantification of extracted genomic DNA
Equal volume (100 µl) of phenol: chloroform: isoamylalcohol (25:24:1) mixture was
added to each tube and the contents were mixed by inverting. The contents were spun at
2500 rpm for 10 min and supernatant was transferred to fresh tubes, 10 µl of 3 M sodium
acetate (1/10 volume of aqueous layer) and 2.5 volumes of chilled absolute ethanol was
added to supernatant and mixed gently and incubated at -20 °C for 15-20 min. The DNA
was spooled in 1.5 ml eppendorf tubes using a glass hook, washed with 70 per cent
alcohol and dried. The DNA was dissolved in 100 µl T10E1 and kept at -20 °C till its further
use. The amount of DNA in each sample was quantified by taking the readings at 260 nm
and 280 nm in the Nano Drop (ND1000-UV/Vis Spectrophotometer, METUS, USA).
Initialization of the instrument was done with nanopure water. The instrument was set
blank with the help of 3 µl T10E1. The quantity of DNA was measured by loading 1-2 µl
DNA sample on Nano-Drop spectrophotometer pedestal. The DNA quantity in ng/µl and
OD value for each sample was noted. The ratio between the readings at 260 and 280 nm
(OD 260/OD 280) was used as an estimate of the purity of the DNA samples. Pure
preparations of DNA have 260 nm/ 280 nm OD ratio between 1.7 and 1.8 (Sambrook and
Russel, 2001). Computed OD values were used to dilute the DNA samples to the working
concentrations of 100 ng/µl for PCR. The DNA working solutions were stored at -20 °C till
further use.
3.4.3 PCR analysis of transformants
Total genomic DNA isolated from young leaves of putative transformants and non-
transgenic plant was used for PCR analysis. The cry and nptII gene fragments were
amplified using PCR assay with gene specific primers. The PCR reaction mixture (20 µl)
contained 0.3 µl (1 U) Taq DNA polymerase (New England Biolabs Pvt. Ltd., UK), 1µl
(1X) assay buffer, 0.5 µl (200 µM) of each dNTP, 1µl (5 µM) of each forward and reverse
primer (Sigma Aldrich Pvt. Ltd., USA) and 1µl (100 ng) template DNA. The DNA extracted
from non-transgenic plants was used as a negative control and the construct carrying cry
56
gene as a positive control. The PCR reaction profile comprised of 38 cycles, with initial
denaturation at 94.0 °C for 3 min followed by cycle denaturation at 94 °C for one min,
annealing at 55.0-64.0 °C (for cry gene specific primers) and 60.0 °C (for nptII gene
specific primers) for 30 sec and extension at 72.0 °C for one min with final extension for
10 min at 72.0 °C. The amplified products were electrophoresed on a 1.2% agarose gel
and visualized under ultraviolet light (Appendix II) (Sambrook and Russel, 2001).
List of gene specific primer pairs used for transgene analysis.
Primer Nucleotide Sequence (5’-3’) Annealing
temperature
cry1Ac Forward: 5’-ACCCAAACATGAAGGAATGC-3’
55.0 °C Revers: 5’-CGGATAGGGTAGGTTCTGGAG-3’
cry2Aa Forward: 5’-GTGGATGGAGTGGAAGAG-3’
64.0 °C Revers: 5’-GAAGAGGGACCAGATGG-3’
cry1F Forward: 5’-CTGCCAATTTGCATCTCTC -3’
56.0 °C Revers: 5’-CCCAGACAGTTTGAGACC -3’
cry1Acm Forward: 5’-TACGACTCAAGGCGATACCC-3’
58.0 °C Revers: 5’-GTGCTGGGAAGATTGGTTGT-3’
nptII Forward: 5’-GAGGCTATTCGGCTATGACTG-3’
60.0 °C Revers: 5’-ATCGGGAGCGGCGATACCGTA-3’
3.5 Transgene segregation analysis of T2 generation progenies
The putative transgenic plants (T1) were maintained in greenhouse condition and
harvested seeds, from each putative transformants, were sown in plant to row progeny
manner. The progenies (T2 generation) were subjected to gene specific PCR assay using
cry and nptII gene specific primer pairs and gene segregation pattern was analysed
based on number of PCR positive and negative plants. The chi-square test was used to
57
study the significance of observed gene segregation pattern with expected 3:1 monogenic
gene segregation pattern.
3.6 Transgene segregation analysis of T3 generation progenies
The putative T2 generation transgenic plants were maintained in greenhouse
condition and harvested seeds, from each putative transformants, were sown in plant to
row progeny manner. The progenies (T3 generation) were subjected to gene specific PCR
assay using cry and nptII gene specific primer pairs and gene segregation pattern was
analysed based on number of PCR positive and negative plants. The chi-square test was
used to study the significance of observed gene segregation pattern with expected 3:1
monogenic gene segregation pattern.
3.7 Insect culture and transgene bioefficacy analysis
The Helicoverpa armigera insect culture was maintained at 26-28 °C and 60%
relative humidity in insect culture room. The chickpea artificial diet was used to feed the
different growth stages of H. armigera. The insect bioassay was carried out in insect
culture room for five days.
3.7.1 Maintenance of insect culture
The oviposition jars for adult moths were prepared by placing moth food petri
plates inside the oviposition jars. Soaked absorbent cotton wad in 50% honey solution
was placed as adult food. Fifteen to twenty moths were released per oviposition jar and it
was covered with black cotton cloth, secured with rubber bands. The eggs were collected
from 2nd day and it was ended on 5th day from each jar. After 2-3 days of egg collection,
hatched neonates were reared on artificial medium. The artificial medium for neonate
larvae was consisted of Part A that was blended with 200 ml of distilled water for 3-5 min,
retaining 200 ml of water (Appendix III). Agar-agar was boiled (Part B) in 400 ml of water
till the agar dissolves completely by intermittent shaking and allowed to cool for 4-5 min
(Appendix III). The molten agar was poured into the blender containing Part A. The steam
was allowed to escape and another 150 ml of water was added in to it. The mixture was
blended for five min and allowed to cool down to 60 °C for 2-3 min. The mixture of sorbic
acid, streptomycin, bavistin, Wesson’s salt mixture and other micro-nutrients (Part C) was
58
mixed completely by rinsing it with 50 ml of water and blended for 2-3 min (Appendix III).
The prepared diet was poured into trays when it is still hot. The diet was cooled at room
temperature for 30 min and used to feed the larvae.
3.7.2 Insect bioassay
The transgenic plants confirmed for the integration of transgene were subjected to
insect bioassay to assess their resistance to H. armigera. Fully expanded leaves from 30-
35 days old plants and 5 days old flowers and pods were excised and leaf petiole/ flower
stalk was inserted into water agar gel (2%) block in the disposable plastic petri plates.
Different plant tissues such as tender pods, flowers and leaves from untransformed plants
served as negative control. On each tissue sample ten first instar neonate larvae (one day
old) were released. Larval mortality and per cent leaf damage was recorded at 24, 48, 72,
96 and 120 hr. The larvae, which failed to show any movement, were considered as
dead. The mortality in transformed plant tissues was corrected with Abbott’s formula
(Abbott, 1925). Two biological and two technical replications were maintained throughout
the experiment and standard deviation was calculated. The experimental design used for
statistical analysis was completely randomized block design and means were compared
at five per cent level of significance using Duncan’s multiple range and Tukey’s test.
3.8 Quantitative estimation of Cry protein using ELISA assay
The quantitative estimation of Cry protein in putative transgenic lines was carried
out using commercially available Quanti-ELISA plates pre-coated with specific Cry
antibody from Envirologix Pvt. Ltd., Hyderabad, India. The sandwich ELISA was carried
out according to the manufacturer’s instructions.
Different tissues samples viz., leaf, flower and pod tissues from transgenic and
non-transgenic plants were collected in disposable 1.5 ml tube. The weight of samples
were taken and used in estimating the Cry protein concentration in µg/g of fresh leaf
tissue. The collected tissues samples were ground by rotating the pestle against the sides
of tube. The process was continued for 20-30 sec and 0.5 ml of 1X extraction buffer
provided with kit was added in to tube along with ground samples. The grinding step was
repeated to mix the tissue samples in extraction buffer. All reagents were allowed to
reach room temperature prior to use. The 100 µl of negative control, 100 µl of each
59
calibrator and 100 µl of each sample extract were added to in two replicates in their
respective places. The content of the well was mixed well by moving the strip holder in
rapid circular motion on the bench top for a full 20-30 sec. Care was taken to avoid cross-
contamination.
Further, the wells in plate were covered with aluminium foil to prevent evaporation
and incubated at ambient temperature for 15 min in shaking condition at 200 rpm on
orbital shaker. The 100 µl of Cry protein specific Cry-enzyme conjugate was added to
each well and mixed thoroughly. Care was taken to avoid cross-contamination. The plate
was covered with aluminium foil to prevent evaporation and incubated at room
temperature for one hour on orbital shaker at 200 rpm. After incubation, the covering was
removed carefully, the content of the well were vigorously shaken and decanted into sink.
The wells were flooded with wash buffer. The washing step was repeated three times.
The plate was inverted and slapped on paper towel to remove as much water as possible.
The 100 µl of substrate was added to each well, the content of well was thoroughly mixed
and covered with aluminium foil. The plate was incubated for 30 min at ambient
temperature in dark condition. The plate was observed for colour development and the
reaction was stopped by adding 100 µl stop solution give with kit.
The spectrophotometric measurements were taken by setting microtiter plate
reader to 450 nm. Based on the slope obtained from slandered graph generated from
calibrators the Cry protein concentrations were calculated by using following formula
given below.
Cry protein conc. (g/mg)
=
(Abs. in test sample – Abs. in negative sample x
Slop x 5
Weight of sample (mg)
3.9 Absolute real time qRT-PCR for cry transcript analysis
The absolute quantification of cry mRNA levels was carried out using real time
qRT-PCR assay. The cry transcript levels were analyzed in three different tissue type viz.,
leaf, flower and pod tissues from selected transgenic events carrying different cry genes
and non-transgenic plant tissue samples.
60
3.9.1 Tissue collection
The selected transgenic pigeonpea plants/events were used to collect leaf, flower
and pod tissues. Normal non-transgenic pigeonpea plants of same genotype were used
as controls. The leaf, flower and pod tissues were harvested from each transgenic plant
and non-transgenic control, separately and carefully labelled. The tissue samples were
collected in such a way to have two biological and two technical replications and stored
immediately in liquid nitrogen, shifted to -80 °C.
3.9.2 Preparation of RNase free water
The RNasa free water was prepared by treatment of diethyl pyrocarbonate (DEPC)
(Sigma Aldrich, USA) to the millipure water and used for preparation of all the solutions
required for RNA isolation. The 0.1% DEPC (one ml) was added to required quantity of
water (999 ml) and kept overnight with gentle shaking. Overnight DEPC treated water
was autoclaved next day in order to degrade the DEPC and this DEPC free water was
used for the preparation of reagents. The utensils and materials used for RNA isolation
was treated with DEPC treated un-autoclaved water for overnight and autoclaved before
used to make them RNase free.
3.9.3 Isolation and purification of total RNA
Total RNA was isolated from leaf, flower and pod tissues samples from selected
transgenic events expressing different cry genes and non-transgenic control pigeonpea
plants using TRIzol reagent (Sigma-Aldrich Pvt. Ltd. USA) as following protocol.
Frozen tissue sample approximately 1.0 g of tissue was grounded into fine powder
in a pre-chilled mortar and pestle and the ground tissue was transferred into a pre-chilled
50 ml conical tube and 5-10 ml of TRIzol reagent (one ml of TRIzol/100 mg of tissue) was
added. Contents were mixed well with vortex and one ml of the mixture was transferred
into labelled, RNase-free 1.5 ml tubes to incubate for 5 min at room temperature.
Chloroform 200 l per one ml of TRIzol was added and vortexed for 20 sec and
incubated the content for 10 min at room temperature and centrifuged at 13,000 rpm for
10 min at 4 °C. The aqueous phase was carefully transferred into a new RNase-free 1.5
61
ml tube without disturbing the other phases and the tube was placed on ice as soon as
transferred. Equal volume of isopropanol was added and mixed gently by inverting 2-3
times and incubated it on ice for 30 min and centrifuged at 13,000 rpm for 20 min at 4 °C.
A very small pellet was visible at the bottom of the tube that was total RNA. The
supernatant was decanted and allowed to stand upside down on kimwipes (Kim tech
science, Canada) for 5 min. The pellet was washed with 75 per cent ethanol (500 l). The
liquid was decanted and the inside of the tube was wiped to dry with a clean Kimwipe,
without touching the pellet. The pellet was re-suspended in 500 l of RNase-free water
and was incubated on ice for at least an hour and pipetted occasionally for dissolving the
pellet. The content was centrifuged at 13,000 rpm for 20 min at 4 °C and the supernatant
was transferred into a new RNase-free 1.5 ml tube. It was precipitated with 10 per cent
3M sodium acetate and equal volume of isopropanol. Contents were incubated on ice for
one hr or overnight at -80 °C and centrifuged at 13,000 rpm for 20 min at 4 °C. The final
pellet was re-suspended into 10 l of RNase-free water and was stored at -80 °C.
The total RNA was treated to remove DNA using Turbo DNA-freeTMkit (cat#
AM1907 Ambion, USA) as per the manufacturer’s instruction. In order to eliminate the
genomic DNA, 20 g of total RNA was digested with RNase-free DNase I and finally the
total RNA was precipitated into desired volume of water. The quantity and quality of total
RNA was checked using NanoDrop ND-1000 spectrophotometer (NanoDrop
Technologies, USA). The total RNA was subjected to Nanodrop ND-1000 using RNase-
free water as blank: absorbance was recorded at 260/280 and 260/230. Further, the
sample was fractionated over a formaldehyde agarose gel (Appendix IV). Absence of
genomic contamination was subsequently confirmed by PCR with total RNA as template
(Caldana et al., 2007).
3.9.4 Preparation of cDNA
Single stranded cDNA was prepared by using High Capacity cDNA Reverse
Transcription kit (cat# 4368814, Ambion, USA) as per the manufacturer’s protocol. All
reagents were thawed and stored on ice in RNase and DNase free work environment. All
individual reagents were mixed thoroughly and spin down and pipetted. About two g of
total RNA in a single 20 l reaction was quantitatively converted to single-stranded cDNA
using standard thermal condition mentioned below.
62
Composition of components used for the cDNA synthesis reaction
Sl. No. Components Volume/reaction (ml)
1 10X RT buffer 2.00
2 25X dNTP (100mM) 0.80
3 10X RT random primers 2.00
4 MultiScribeTM Reverse Transcriptase 1.00
5 RNase inhibitor 1.00
6 Nuclease free water 3.20
7 Total RNA (2 mg) 10.00
Total volume 20.00
Thermal condition for single-stranded cDNA conversion
Step 1 Step 2 Step3 Step 4
Temperature 25.0 °C 37.0 °C 85.0 °C 4.0 °C
Time 10 min 120 min 5 sec Hold
3.9.5 Standardization of real-time PCR condition
The primer concentration is one of the key factors in real-time quantitation of any
gene expression. Primer concentrations ranging from 150 nM, 200 nM, 250 nM, 300 nM,
350 nM, and 400 nM were used to optimize the amplification. Primer at 200 nM
concentration gave single melting curve, low Ct value, high fluorescence value and no
primer dimer when loaded on gel. Thus, this concentration of primer was used for rest of
the experiment. Similarly, it is also essential to determine optimal annealing temperature
63
for each primer pair before their use. All primers designed for cry genes were tittered for
optimal annealing temperature ranges from 55 to 60 °C.
3.9.6 qRT-PCR reaction
The master mix of different components of real-time PCR was prepared fresh to
avoid handling errors. The reaction mixture of 10 l containing 1.0 ng cDNA, 200 nM of
each gene-specific primer and 5 l of 2X SYBR green reagents (Cat. #4367659, Ambion,
USA) were used in the experiment. Individual components of reaction mixture were
standardized for 10 l reaction volume. An Eppendorf mastercycler ep realplex
instrument (Eppendorf Pvt. Ltd., Germany) was used for all real-time PCR amplifications.
Two biological and two technical replications were maintained for each treatment. An
optimal PCR conditions used for amplification are given as follows.
Real-time PCR amplification conditions
Stage Step Temperature
(°C) Duration No. of cycles
1 Initial denaturation 95.0 °C 10 min 1
2 Denaturation 95.0 °C 15 sec
40 3 Annealing (vary with
primer)
55.0-58.0 °C 20 sec
4 Extension 60.0 °C 20 sec.
5 Melting curve 95.0 °C 10 min 1
64
List of primer used for real time qPCR analysis.
Primer Nucleotide Sequence (5’-3’) Annealing
temperature
rt1Ac Forward: 5’-ACGAAATCCCACCACAGAAC-3’
58.0 °C Revers: 5’-ACGGAACTGTTGCTGAATCC-3’
rt2Aa Forward: 5’-CCGCTCCATTACAACCAGAT-3’
56.5 °C Revers: 5’-ATGGTGAAGCCGGTGTAGTC-3’
rt1F Forward: 5’-TACTGGGGCTTAGGGGAGT -3’
55.0 °C Revers: 5’-GCGGACAAAGGTAACGTGAT-3’
rt1Acm Forward: 5’- TGCCTCCCTTACAACCAATC-3’
56.0 °C Revers: 5’- CGCTATTATCTTGGGGTGGA-3’
3.9.7 Absolute quantification of mRNA levels
Absolute quantification of target cry mRNA was performed based on the standard
curve. The cry gene fragments were PCR amplified, eluted and purified. The purified PCR
products were quantified using Nanodrop ND-1000 and absorbance was recorded at
260/280 and 260/230. The purified product was diluted to prepare standards of 1.0 ng/µl,
10.0 ng/µl and 100.0 ng/µl and used as calibrators during real time PCR reaction. The
observed absorbance for prepared standards was plotted against the known
concentration of template and slop was calculated. Based on the slop, the cry mRNA
levels in test samples was quantified in ng/µl.
3.10 Genomic Southern blot analysis
Genomic Southern blot analysis was carried out using the DIG-High Prime DNA
Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany, cat. no.
11585614910). Genomic DNA was isolated from the young leaves of T2/T3 generation
plants and blotting was carried out following standard protocol (Sambrook et al., 1989).
The 15 µg of high molecular weight genomic DNA was digested with restriction
65
endonuclease viz., EcoRI, HindIII and BamHI separately overnight at 37 °C. The total
volume of the digestion reaction was set to 20 µl. One µl of each sample was checked for
digestion in 0.8% (w/v) agarose gel stained with ethidium bromide and visualized under
UV light. The digested DNA was heated in boiling water for 10 min and quickly chilled on
ice. Such DNA samples were electrophoresed at 40 V 6-8 hr on 0.8% (w/v) agarose gel
followed by denaturation solution treatment (Appendix V) for 30 min and neutralization
solution treatment (Appendix V) for 30 min.
The DNA fragments were transferred from the gel to the Biodyne® B, 0.45 µm
Positively-charged Nylon 6,6 Transfer Membrane (PALL Life Sciences) by capillary
movement of transfer solution (10X Sodium-sodium citrate; SSC) through agarose gel in
to transfer membrane kept overnight. Further, the membrane was exposed to UV light for
UV crosslinking for 10 min. The membrane was pre-hybridized for at least two hr in 10 ml
of hybridization buffer (Appendix V) without probe. The cry gene specific PCR-amplified
fragments from cry gene construct were random primed labelled with DIG-11-dUTP and
were used as hybridization probes. Probe hybridization was performed overnight at 48 °C
in hybridization chamber and the membranes were washed three times each in wash
buffer I (Appendix V) for 10 min, followed by wash buffer II (Appendix V) for 10 min at 68
°C, and wash buffer III (Annexure V) for 10 min. The membrane was treated with blocking
solution (Appendix V) for 30 min followed with antibody treatment (Appendix V) for 30 min
and washed twice with wash buffer III, 15 min each.
Next, the membrane was incubated in freshly prepared substrate solution
(Appendix V) in an appropriate container in the dark without disturbing. Reaction was
stopped by washing the membrane for five minutes with 20 ml sterile water. The
membrane was dried and results were documented by scanning the blot.
3.11 Northern blot analysis
Northern blot analysis was carried out using the DIG-High Prime DNA labelling and
Detection Kit (Roche Diagnostics, Mannheim, Germany, cat. no. 11585614910). Total
RNA was isolated from the young leaves, flower and pod tissues of transgenic and non-
transgenic control plants and blotted following standard protocol (Sambrook et al., 1989).
The 10 µg of total RNA was mixed with 10 µl of 5X sample buffer (one ml glycerol, one ml
10X MOPS and one pinch of bromophenol blue), heated at 65 °C for 15 min, put on ice,
66
then loaded into gel wells and electrophoresed at 40 V for 2 hr on 1.0% (w/v)
formaldehyde agarose gel (Appendix IV) followed by denaturation solution treatment for
30 min and neutralization solution treatment for 30 min.
The RNA strands were transferred from the gel to the Biodyne® B, 0.45 µm
Positively-charged Nylon 6,6 Transfer Membrane (PALL Life Sciences) by capillary
movement of transfer solution (10X SSC) from agarose gel in to transfer membrane, kept
overnight. Further the membrane was exposed to UV light for UV crosslinking for 10 min.
The membrane was pre-hybridized for 2 hr in 10 ml of hybridization buffer (Appendix V)
without probe. Specific PCR-amplified fragments from respective cry construct were
random primed labeled with DIG-11-dUTP and were used as hybridization probes. Probe
hybridization was performed overnight at 48 °C in hybridization chamber and the
membranes were washed three times each in wash buffer I (Appendix V) for 10 min,
followed by wash buffer II (Appendix V) for 10 min at 68 °C, and wash buffer III (Appendix
V) for 10 min. The membrane was treated with blocking solution (Appendix V) for 30 min
followed with antibody treatment (Appendix V) for 30 min and washed twice with wash
buffer III, 15 min each. Then the membrane was incubated in freshly prepared substrate
solution (Appendix V) in an appropriate container in the dark without disturbing. Reaction
was stopped by washing the membrane for 5 minutes with 20 ml sterile water. The
membrane was dried and results were documented by scanning the blot.
3.12 Recovering the site of transgene integration by TAIL PCR
Selected transgenic (T3 generation) plants expressing cry gene were analyzed for
site of integration of T-DNA using TAIL-PCR. T-DNA specific primers, 35S Reverse (5’-
GATAGTGGGATTGTGCGTCA-3’) and arbitrary degenerate AD1 (5’- NTCGAS
TWTSGW-3’), AD2 (5’-NGTCGASWGANAWGAA-3’) and AD3 (5’-AGWGNAGW ANCA-
3’) primers were used to recover the flanking genomic region. The TAIL-PCR included
primary, secondary and tertiary amplification reactions. The primary PCR product was
diluted to 1:29 ratio using nano-pure water and used as template for secondary reaction.
The product of secondary reaction was diluted to 1:9 ratio using nano-pure water and
proceed to tertiary reaction. The primary, secondary and tertiary products were run in
adjacent lanes on a 1.0% low melting agarose gel. The specificity of the products was
confirmed by the expected size change between the secondary and tertiary products.
67
Higher most band from tertiary PCR was eluted and taken for transformation. The eluted
product was ligated into the pTZ vector by using InsTAcloneTMPCR Cloning Kit as per
manufactures instruction.
Components of primary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction
Sl. No. Reaction components Quantity (µl)
1 Water 12.8
2 Buffer 2.0
3 dNTP’s 2.0
4 Specific primer 0.6
5 Arbitrary degenerate primer 0.6
6 Taq polymerase 1.0
7 DNA 1.0
Total 20
Components of secondary and tertiary Thermal Asymmetric Inter Laced - Polymerase
Chain Reaction
Sl. No. Reaction components Quantity (µl)
1 Water 16.7
2 Buffer 2.5
3 dNTP’s 2.0
4 Specific primer 1.0
5 Arbitrary degenerate primer 1.0
6 Taq polymerase 0.8
7 DNA 1.0
Total 25.0
68
Primary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction programme
Particulars No. of cycles
95.0 °C, 3 min; 95.0 °C, 1 min
94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 5
94.0 °C, 30 sec; 25.0 °C, 2 min; 72.0 °C, 3 min 1
94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 15 each
94.0 °C, 30 sec; 41.6 °C, 1 min; 72.0 °C, 2 min
72.0 °C, 15 min
Secondary and tertiary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction
programme
Particulars No. of cycles
94.0 °C, 1 min
94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 2 cycles
94.0 °C, 30 sec; 41.6 °C, 1 min; 72.0 °C, 2 min 20 cycles
72.0 °C, 30 min
3.12.1 Preparation of competent cells
The competent cells were prepared using E. coli DH5α culture by following the
protocol given by Sambrook et al. (1989) with minor modification as described below. An
isolated colony from E. coli DH5α plate was inoculated into 5 ml Luria broth (LB)
(Appendix VI) and incubated at 37 °C overnight on shaking condition at 200 rpm. The
culture was diluted to 1:100 using LB i.e., 0.5 ml of culture was added to 50 ml of LB. The
69
culture was incubated for 2 to 3 hr till it attained an OD of 0.3 to 0.4 at 600 nm. The
culture was chilled in ice for 30 min and 25 ml of culture was dispensed into two
centrifuge tubes of capacity 50 ml. The cells were pelleted at 6000 rpm for 5 min, the
supernatant was discarded and pellet was suspended in 12.5 ml of ice-cold 0.1M calcium
chloride. The centrifuge tubes were again incubated in ice for 45 min and later centrifuged
at 4000 rpm for 10 min. The pellet was dispensed in one ml of 0.1M CaCl2 and to this 88
µl of dimethyl sulfoxide (DMSO) was added. About 200 µl of cells were distributed to each
chilled 1.5 ml micro centrifuge tubes and immediately used.
3.12.2 Transformation of E. coli DH 5α
A freshly prepared competent cell stock (about 100 µl) was taken in a chilled
centrifuge tube and of ligated mixture (10 µl) was added into the tube and was mixed
gently. The mixture was chilled in ice bath for 45 min. Heat shock was given by shifting
the chilled mixture to preheated 42 °C water bath for exactly two min and then shifted to
ice to chill for 5 min. About 800 µl of Luria broth was added to the mixture and incubated
at 37 °C at 200 rpm for 45 min. The culture was centrifuged at 13,000 rpm for one min
and about 700 µl of supernatant was discarded. The pellet was dissolved in remaining
supernatant and spread on the plates having Luria agar with Amp100, X-gal and IPTG,
and incubated overnight at 37 °C. The plates were observed for growth of colonies and
the white colonies were selected and re-streaked on Luria agar plate with Amp100, X-gal
and IPTG.
3.12.3 Colony PCR
The PCR was performed by using selected colonies: loop of colony was taken and
inoculated in the reaction mixture. The reactions were carried out using a master thermal
cycler. The amplified products were assayed by electrophoresis on 1.2% agarose gels.
The colonies, which showed the expected size of the amplified fragment by M13 forward
(5’-GTTGTAAAACGACGGCCAGT-3’) and M13 reverse (5’-CAGGAAACAGCTATGACC-
3’) primers were selected for further confirmation of the transformed colonies.
70
Components used in colony polymerase chain reaction
Sl. No. Components Volume/reaction (µl)
1 Standard Taq reaction buffer (10X) 2.50
2 dNTP’s (1mM) 0.50
3 Forward primers (5 pmol/µl) 1.00
4 Reverse primers (5 pmol/µl) 1.00
5 Template (colony) 1.00
6 Taq DNA polymerase 0.50
7 Water 18.5
Total volume 25.00
Colony polymerase chain reaction programme
Particulars Duration No. of cycles
94.0 °C 4 min 1
94.0 °C 1 min
35 54.0 °C 1 min
72.0 °C 1 min
72.0 °C 10 min 1
4.0 °C ∞ 1
3.12.4 Plasmid isolation
White colonies were inoculated to 10 ml LB carrying ampicillin (100 µg/ml) as a
selection pressure and incubated overnight at 37 °C on shaker at 200 rpm. Overnight
grown culture was centrifuged at 5000 rpm for 2 min in 2.0 ml micro centrifuge tubes. The
71
supernatant was removed and pellet was washed with STET buffer (0.25 volume of
original culture) (Appendix VI), then it was centrifuged at 5000 rpm for two min. The pellet
was re-suspended in 200 µl of ice-cold alkaline-lysis solution-I (Appendix VI) by vigorous
vortexing. The 400 µl of freshly prepared alkaline-lysis solution-II (Appendix VI) was
added to each tube and the contents were mixed by inverting the tubes for 4 to 5 times
and kept in ice for about 5 min. To this suspension, 300 µl of alkaline-lysis solution-III
(Appendix VI) was added and again mixed thoroughly by gently inverting the tubes 4-5
times. The tubes were stored on ice for 5 min and centrifuged at 13,000 for 8 min at 40
°C.
The supernatant was transferred to fresh tubes and equal volume of phenol:
chloroform: Isoamyl alcohol (25:24:1) was added to precipitate proteins and mixed well.
The tube was centrifuged at 13,000 rpm for 10 min at 4 °C. The aqueous layer was
transferred to a fresh tube and two volumes of isopropanol were added. The contents
were mixed and allowed to stand for 2 minutes at room temperature. The solution was
later centrifuged at 13,000 rpm for 5 min. The supernatant was discarded and pellet was
washed with 70 per cent ethanol and spun for 1 min at 13,000 rpm to recover the plasmid.
The supernatant was discarded, pellet was dried completely and dispensed into 30 µl of
T10E1 (pH 8.0) containing 3 µl of RNase A (10 mg/ml). The solution was kept at 50 °C for
15 min and then stored at -20 °C. The confirmation of the presence of cloned fragment
was done by PCR amplification.
3.12.5 Sequencing of clones and sequence analysis
The independent clones were sequenced using M13 primers at Sci-Genom Pvt.
Ltd., Kochi. The obtained sequences (forward and reverse) were multiple aligned and
contig was formed. Such contigs were processed to remove vector backbone by vector
backbone trim algorithm available with BioEdit software (Hall, 2011). Such processed
contigs were subjected for blast analysis against pigeonpea draft genome sequence
database by establishing local blast search using BioEdit software. The T-DNA flanking
genomic region was recovered based on local blast results.
72
3.13 The plant growth parameters
The selected plant growth parameters were observed in developed transgenic
plants and compared with the respective parental genotypes used during transformation
processes (ICPL 87119 and BSMR 736). The observations for different plant growth
parameters were taken in greenhouse conditions.
1. Stem colour: The stem colour of developed transgenic plants was observed and
compared with the typical characteristic green stem colour of ICPL 87119 and BSMR
736 non-transgenic plants.
2. Flower colour: The transgenic plants were observed for flower colour and compared
with that of ICPL 87119 (Yellow with pink streaks) or BSMR 736 (Yellow) non-
transgenic parental genotypes.
3. Growth habit: The growth habit of transgenic plants was analysed and categorised into
spread, semi spread and compact types and compared with that of ICPL 87119 (semi
spread) or BSMR 736 (semi spread) non-transgenic plants.
4. Leaf colour and shape: The observation of leaf colour and shape were taken for
identified transgenic plants and compared with that of ICPL 87119 (light green and
tapering) or BSMR 736 (dark green tapering) non-transgenic parental genotypes.
5. Branching pattern: The branching pattern in developed transgenic plants was observed
and categorised into more, medium and few (ICPL 87119 and BSMR 736; medium).
6. Fresh pod colour: The transgenic plants were observed for fresh pod colour (light
green and green with purple streaks) and compared with that of ICPL 87119 and
BSMR 736 non-transgenic plants.
7. Seed colour: The seed colour development was noticed in transgenic plants and
parental ICPL 87119, BSMR 736 non-transgenic parental genotypes.
8. Plant height: The trait, plant height, in case of transgenic plants was observed and
compared with that of non-transgenic control plants (ICPL 87119 and BSMR 736).
73
3.14 Statistical analysis
Required number of replications (technical and biological) was maintained for all
experiments and standard deviation was calculated. The experimental design used for
statistical analysis was completely randomized block design and means were evaluated
at five per cent level of significance using Duncan’s multiple range tests and Tuke’s test
(MSTAT-C program). For statistical analysis of per cent data, arcsine transformation was
performed for per cent data before subjecting it for any statistical test. Statistical
independent and unpaired ‘t’-test was performed to compare the performance of two
groups in different experimental treatments. The correlation analysis was performed using
SPSS statistical program for insect mortality, Cry protein and cry gene transcript levels
from different transgenic lines. The chi-square analysis was done to test the transgene
segregation pattern using MS-Excel program.
74
4. EXPERIMENTAL RESULTS
The present investigation was carried out to improvise the pigeonpea
transformation protocol using in vitro and in planta transformation methods. The
transformation protocol with good transformation efficiency was employed to develop
transgenic pigeonpea expressing different cry genes viz., cry1Ac, cry2Aa, cry1F and
cry1Acm separately. The transgenic lines were characterised for their bio-efficacy
against H. armigera. The investigation also encompassed the molecular characterisation
of selected transgenic lines of pigeonpea. The experimental results are presented below.
4.1 Effect of cytokinins and their concentration regimes on multiple shoot induction
and plantlet regeneration in pigeonpea
The surface sterilized seeds, cultured on MS basal and MS medium
supplemented with different cytokinins and their concentration regimes indicated 50-60
per cent germination after 7 days in both the genotypes viz., ICPL 87119 and BSMR
736. The different concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) of three cytokinins
viz., BAP, TDZ and zeatin were assessed to study their effect on shoot bud induction in
pigeonpea.
4.1.1 Multiple shoot bud induction in response to BAP concentration regimes
The number of cotyledonary node with cotyledon explants responded to different
concentration regimes of BAP for shoot bud induction ranged from 0.0 to 36.0 in case of
ICPL 87119 and from 0.0 to 36.5 in case of BSMR 736 (Table 1). The maximum number
of explants responded for shoot bud induction was noticed at 2.0 mg/L BAP
concentration level, whereas it was list at 10 mg/L BAP, in both genotypes. The number
of shoot bud induced per explant ranged from 0 to 52.6 for ICPL 87119 and from 0 to
53.7 for BSMR 736 (Table 2). The maximum number of shoot buds induced was
recorded in MS medium supplemented with 2.0 mg/L BAP. For embryo disc with half
cotyledon explants, the explants response ranged from 0 to 26.5 (ICPL 87119) and 0 to
27.5 (BSMR 736) in pigeonpea genotypes (Table 3). The number of explants responded
to BAP fortification was at its maximum when medium was fortified with 2.0 mg/L BAP.
Interestingly, there was a concomitant decrease in explants response with increase in
BAP concentration. Similar kind of explants response to multiple shoot bud induction was
75
Table 1: Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from cotyledonary node with cotyledons explant of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture (50 explants)
Growth hormone
concentrations
Number of explants responded (mean ±SD)
Grand mean
Benzylamino purine Thidiazuron Zeatin
ICPL 87119
BSMR 736
ICPL 87119
BSMR 736
ICPL 87119
BSMR 736
0 mg/l 0.0±0.0 c 0.0±0.0d 0.0±0.0e 0.0±0.0d 0.0±0.0c 0.0±0.0c 0.0±0.0d
1 mg/l 23.5±2.1b 21.0±1.4b 22.5±3.5c 23.5±2.1bc 29.5±2.1a 30.5±3.5a 25.1±3.9b
2 mg/l 36.0±1.4a 36.5±2.1a 23.5±2.1c 23.5±3.5bc 33.0±1.4a 32.0±1.4a 30.8±5.8a
3 mg/l 32.0±2.8a 32.5±2.1a 29.5±2.1ab 29.0±1.4ab 22.5±3.5b 22.0±1.4b 27.9±4.6a
4 mg/l 26.0±2.8b 24.5±2.4b 33.5±3.5a 33.5±2.1a 21.5±3.5b 21.5±2.1b 26.8±5.5b
6 mg/l 24.0±1.4 b 24.5±0.7b 25.5±2.1bc 24.5±3.5bc 21.5±2.1b 20.5±1.2b 23.4±1.9b
8 mg/l 22.5±2.1 b 21.5±1.4b 23.5±2.2c 22.5±3.5bc 19.0±2.8b 19.0±1.8b 21.3±1.9bc
10 mg/l 21.0±1.4 b 16.0±2.8c 17.5±3.5d 18.5±2.9c 18.5±2.1b 17.5±3.5b 18.2±1.7c
Grand mean 23.1±10.6a 22.1±11.1a 21.9±10.1a 21.9±9.9a 20.7±9.7a 20.4±9.8a 24.8±9.7
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
76
Table 2: Number of shoot bud induced in response to different concentration regimes of cytokinins (benzylamino purine, thidiazuron and zeatin) from cotyledonary node with cotyledons and embryo discs with half cotyledons explants.
Growth hormone conc-
entrations
Number of shoot bud induced per explant (mean ±SD)
Cotyledonary node with cotyledons
Grand mean
Embryo discs with half cotyledon
Grand mean
Benzylamino purine Thidiazuron Zeatin Benzylamino purine Thidiazuron Zeatin
ICPL 87119 BSMR 736 ICPL 87119
BSMR 736 ICPL 87119
BSMR 736
ICPL 87119
BSMR 736
ICPL 87119
BSMR 736 ICPL 87119
BSMR 736
0 mg/l 0.0±0.0f 0.0±0.0
f 0.0±0.0
e 0.0±0.0
e 0.0±0.0
e 0.0±0.0
e 0.0±0.0
e 0.0±0.0
e 0.0±0.0
e 0.0±0.0
d 0.0±0.0
d 0.0±0.0
d 0.0±0.0
d 0.0±0.0
d
1 mg/l 29.9±5.6c 30.2±6.2
c 22.8±4.6
d 26.0±3.9
d 35.9±4.9
b 35.3±6.0
b 30.2±5.1
c 2.8±0.6
cd 3.1±0.6
bc 2.5±0.5
c 2.8±0.6
c 4.2±1.0
a 4.2±0.8
a 3.2±0.7
b
2 mg/l 52.6±5.9a 53.7±4.9
a 30.5±3.9
bc 32.2±3.3
bc 40.4±4.5
a 40.9±5.8
a 41.7±9.8
a 4.8±0.8
a 4.4±0.5
a 3.1±0.7
c 3.3±0.7
bc 3.5±0.5
b 3.6±0.5
b 3.7±0.6
a
3 mg/l 40.4±5.8b 41.2±4.9
b 31.9±4.3
b 34.9±2.6
b 30.8±3.0
c 29.3±3.9
c 34.7±5.0
b 3.5±0.5
b 3.2±0.6
b 4.0±0.9
b 3.9±1.1
ab 2.8±0.8
c 2.9±0.7
c 3.3±0.5
b
4 mg/l 32.9±5.2c 33.8±4.2
c 41.1±5.7
a 46.1±5.3
a 27.3±4.4
c 25.3±3.8
d 34.4±7.9
b 3.0±.0.7
bc 2.7±0.7
cd 4.7±1.0
a 4.5±0.8
a 2.7±0.7
c 2.6±0.5
c 3.3±0.9
b
6 mg/l 23.9±4.2d 25.9±1.7
d 31.4±2.9
bc 33.4±3.0
b 22.4±3.3
d 23.5±3.3
d 26.7±4.5
c 2.5±0.5
cd 2.6±0.5
d 2.7±0.7
c 2.9±0.6
c 2.5±0.5
c 2.4±0.5
c 2.6±0.2
c
8 mg/l 19.7±4.3de
21.2±3.7e 27.6±3.7
c 29.6±4.5
c 20.0±3.8
d 21.7±3.3
d 23.3±4.2
d 2.4±0.5
d 2.6±0.5
d 2.7±0.7
c 2.7±0.5
c 2.4±0.5
c 2.5±0.5
c 2.5±0.1
c
10 mg/l 18.7±2.8e 20.1±3.4
e 22.8±2.7
d 25.8±3.1
d 21.0±3.0
d 22.0±2.5
d 21.7±2.4
d 2.3±0.5
d 2.5±0.5
d 2.5±0.5
c 2.6±0.5
c 2.6±0.5
c 2.4±0.5
c 2.4±0.1
c
Grand mean 27.2±15.7a 28.2±15.9
a 26.0±12.0
a 28.5±13.1
a 24.7±12.3
a 24.7±12.0
a 26.5±13.0 2.6±1.4
a 2.6±1.2
a 2.7±1.4
a 2.8±1.3
a 2.5±1.2
a 2.5±1.2
a 3.1±1.2
Note: The means followed with same letters are within student ‘t’ range at α =0.05.
77
Table 3: Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from embryo discs with half cotyledon explants of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 12 days of culture (50 explants)
Growth hormone concentrations
Number of explants responded (mean ±SD)
Grand mean Benzylamino purine Thidiazuron Zeatin
ICPL 87119 BSMR 736 ICPL 87119 BSMR 736 ICPL 87119 BSMR 736
0 mg/l 0.0±0.0e 0.0±0.0e 0.0±0.0c 0.0±0.0c 0.0±0.0c 0.0±0.0d 0.0±0.0d
1 mg/l 19.0±1.4bc 18.5±3.5bc 16.5±2.1b 18.0±2.8b 21.0±3.5a 21.0±1.4ab 19.0±1.8b
2 mg/l 26.5±2.1a 27.5±2.1a 19.0±1.4b 19.0±1.4b 21.5±4.9a 22.0±2.8a 22.5±3.6a
3 mg/l 23.0±1.4ab 23.0±2.8ab 22.0±2.8a 23.5±2.1a 16.0±2.8b 17.5±3.5abc 20.8±3.2ab
4 mg/l 16.5±2.1cd 18.0±1.4bc 22.0±5.6a 25.0±1.4a 16.5±2.1b 17.0±1.4bc 19.1±3.5b
6 mg/l 13.5±2.1d 15.0±1.4cd 17.5±3.5b 16.5±2.1b 15.0±4.1b 16.0±1.4c 15.5±1.3c
8 mg/l 12.0±1.4d 12.5±0.7d 16.5±2.1b 15.5±0.7b 16.0±1.4b 15.5±1.2c 14.6±1.9c
10 mg/l 12.0±2.8d 11.0±1.4d 17.5±0.7b 16.0±2.4b 16.5±2.1b 16.0±1.4c 14.8±2.6c
Grand mean 15.3±8.1a 15.6±8.3a 16.3±6.9a 16.6±7.6a 15.3±6.7a 15.6±6.8a 18.1±7.1
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
78
noticed in both pigeonpea genotypes. The number of shoot bud induced from embryo
discs with half cotyledon explants was ranged from 0 to 4.8 in ICPL 87119, whereas it
was from 0 to 4.4 in BSMR 736 (Table 2). In MS medium supplemented with 2.0 mg/L
BAP recorded highest number shoot buds (4.8; ICPL 87119) when embryo discs with
half cotyledon was used as explant. Interestingly, there was a concomitant decrease in
resonance of explants for shoot bud induction and number of shoot bud induced with
further increase in BAP levels in growth medium (Fig. 1; 2).
The behavioral response of two genotypes to same BAP level was non-significant
(p > 0.72) as revealed by ‘t’-test statistical analysis. At the same time, there was a
significant difference between cotyledonary node with cotyledons and embryo discs with
half cotyledon explants for their response to same BAP concentration regime (p < 0.05).
Similarly, there was no significant difference for number of shoot bud induced per
explants in response to same BAP level between two genotypes (p > 0.82). Whereas,
the effect of same BAP level on shoot bud induction from cotyledonary node with
cotyledons and embryo discs with half cotyledon was found be significant (p < 0.001).
4.1.2 Multiple shoot bud induction in response to TDZ concentration regimes
The number of cotyledonary node with cotyledon explants responded to different
TDZ concentration regimes ranged from 0 to 33.5 in case of both genotypes (Table 1).
Further, in both genotypes, maximum number of explants responded for shoot bud
induction was noticed at 4.0 mg/L TDZ concentration level (Plate 1). It was observed that
the number of shoot bud induced per explant ranged from 0 to 41.1 (ICPL 87119) and
from 0 to 46.1 (BSMR 736) in respective genotypes (Table 2). The embryo disc with half
cotyledon explants response to increasing TDZ concentration regimes ranged from 0 to
22.0 in ICPL 87119 and 0 to 25.0 in BSMR 736. The maximum number of explants
responding was recorded in BSMR 736 at 4.0 mg/L TDZ concentration regime (Table 3).
The number of shoot bud induced from embryo discs with half cotyledon explant was
ranged from 0 to 4.7 (ICPL 87119) and from 0 to 4.5 (BSMR 736) (Table 2). At TDZ
concentration regime of 4.0 mg/L, the highest number
shoot buds (4.7) was recorded in ICPL 87119 when embryo discs with half cotyledon
was used as explant. Similarly, it was noticed that any further increase in TDZ
concentration levels imparts reduction in multiple shoot bud induction in both explants
types of pigeonpea (Fig. 1 and 2).
79
80
81
Plate 1: Direct multiple shoot bud induction and plantlet regeneration from cotyledonary node with
cotyledons, embryo dises with half cotyledon explants of pigeonpea A. Multiple shoot buds
induced from cotyledonary node with cotyledons explants cultured on MS +2mg/L BAP after 10
days of inoculation. B.shoot growth after first round of subculture on MS + 4 mg/L TDZ. C.
Development of sugary white callus around induced shoot buds when explants were cultured on
MS +10 mg/L.TDZ.D. Multiple shoot buds induced from embryo discs with half cotyledon explants
cultured on MS + 1 mg/L zeatin after 15 days of inoculation.E. Shoots with profuse rooting on MS
+0.5 mg/LIBA. F. Multiple shoot buds induced (up to 52 shoot buds) from cotyledonary node with
cotyledons explants cultured on MS + 2mg/LBAP after 14days of inoculation.
82
There was no significant difference between the responses of genotypes to
different levels of TDZ concentrations (p > 0.43). On other hand, the effect of TDZ
concentration regime on explants response was found significant (p < 0.001). The
statistical analysis revealed that there was no significant difference between genotypes
for number of shoot bud induced per explant in response to same TDZ level (p > 0.83).
At the same time, the two explants types showed significant difference for their response
to multiple shoot bud induction at same TDZ concentration regimes in both genotypes (p
< 0.02). The analysis of means revealed that the treatment 4.0 mg/L of TDZ was most
effective for induction of multiple shoot buds per explants from both explants and
genotypes.
4.1.3 Multiple shoot bud induction in response to zeatin concentration regimes
The augmentation of MS with different zeatin concentration regimes resulted in
the multiple shoot bud induction in cotyledonary node with cotyledon explants, which
ranged from 0 to 33.0 in ICPL 87119 and from 0 to 32.0 in BSMR 736 (Table 1). In both
genotypes, the maximum number of explants responded for shoot bud induction was
recorded at 2.0 mg/L zeatin concentration regime. Further, the maximum number of
shoot bud induced per explant was recorded at 2.0 mg/L zeatin concentration level,
which was 41.1 for ICPL 87119 and 46.1 for BSMR 736 (Table 2). The maximum
number of explants responding was recorded at 2.0 mg/L zeatin concentration regime,
which was 21.5 in ICPL 87119 and 22.0 in BSMR 736 (Table 3). The number of shoot
bud induced from embryo discs with half cotyledon explant was ranged from 0 to 4.2 in
both genotypes (Table 2). From embryo discs with half cotyledon explants, at 1.0 mg/L
zeatin concentration regime the highest number shoot buds was recorded. There was a
concomitant decrease in resonance of explants for shoot bud induction and number of
shoot bud induced with further increase in zeatin levels in growth medium
(Fig. 1 and 2).
The ‘t’-test statistical analysis indicated that the response of two genotypes at
same level of zeatin was non-significant (p > 0.84). At the same time, there was a
significant difference between the two explants response to same level of zeatin
concentration regimes for multiple shoot bud induction (p < 0.03). It was observed that
there was no significant difference between two genotypes response for number of shoot
83
bud induced per explants at same zeatin concentration regime (p > 0.96). Whereas, the
two explants (cotyledonary node with cotyledons and embryo discs with half cotyledon)
noticed differ significantly for their response to shoot bud induction at same zeatin
concentration regime in both genotypes (p < 0.001). The analysis of means showed that
the two treatments, 1.0 mg/L and 2.0 mg/L zeatin, were on par for number of explants
responded to zeatin fortification. On other hand, the treatment with 2.0 mg/L zeatin level
was superior over other treatments in cotyledonary node with cotyledons explants and
the treatment with 1.0 mg/L zeatin level was superior in embryo discs with half cotyledon
explants for number of shoot bud induced per explant.
4.1.4 Rooting and establishment of plantlets
Elongated and well developed shoots (~ 3 cm long) were excised from shoot
clumps on basal MS medium and cultured on MS medium augmented with increasing
IBA concentration regimes. The frequency of rooting varied with different IBA
concentration regimes ranging from 20 to 80 per cent in both genotypes (Table 4). The
highest root induction was noticed in MS media with 0.5 mg/L IBA. The observed number
of roots per shoot ranged from 1.4 to 4.8 per shoot. The maximum number of roots
induced per shoot was recorded in MS fortified with 0.5 mg/L IBA, in both genotypes.
The induced roots were thick, white in color with less fine root hair developed on it (Plate
1). The analysis of means revealed that the response to root induction and number of
roots induced was maximum in MS supplemented with 0.5 mg/L IBA for both genotypes
(Table 4) (Fig. 3).
4.2 Effect of different treatments on in planta transformation in pigeonpea
The improvisation of in planta transformation protocol to increased transformation
efficiency was performed by using different treatment such as,
A. tumefaciens infection alone, A. tumefaciens infection using A. tumefaciens culture
with tobacco extract added in overnight grown culture and A. tumefaciens infection using
air evacuation to increase the proximity between A. tumefaciens and embryonic axis. For
each treatment, a set of 200 explants in four batches of 50 explants each were used for
transformation.
84
Table 4: Effect of indole butyric acid (IBA) concentration regimes on root induction and number of root induced per shoot in pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture
Growth hormone concentrations
Per cent rooting
(mean ±SD) Grand mean
Number of roots per shoot (mean ±SD) Grand
mean ICPL 87119 BSMR 736 ICPL 87119 BSMR 736
MS 25.0±7.0c 20.0±8.0c 22.5±3.5c 1.4±0.5e 1.5±0.4e 1.4±0.1e
½ MS 25.0±8.0c 15.0±8.0c 20.0±7.1c 1.8±0.4e 1.8±0.4e 1.8±0.2e
MS + 0.25 mg/l IBA 50.0±6.0b 55.0±5.0b 52.5±3.5b 2.6±0.5d 2.7±0.5d 2.6±0.1d
MS + 0.50 mg/l IBA 75.0±7.0a 80.0±4.0a 77.5±3.5a 4.6±0.8a 4.8±0.7a 4.7±0.1a
MS + 0.75 mg/l IBA 55.0±5.0ab 60.0±6.0b 57.5±3.5ab 4.0±0.7b 3.7±0.5b 3.8±0.2b
MS + 1.0 mg/l IBA 50.0±7.0b 55.0±5.0b 52.5±3.5b 3.4±0.6c 3.4±0.4bc 3.4±0.2bc
MS + 1.5 mg/l IBA 45.0±5.0bc 45.0±7.0b 45.0±2.0b 3.4±0.7c 3.3±0.7bc 3.4±0.3bc
MS + 2.0 mg/l IBA 50.0±4.0b 45.0±6.0b 47.5±3.5b 3.3±0.6c 3.1±0.5cd 3.2±0.4c
½ MS + 0.50 mg/l IBA 50.0±6.0b 50.0±7.0b 50.0±6.5b 3.3±0.7c 3.2±0.4c 3.2±0.4c
Grand mean 47.22±15.2a 47.22±19.8a 47.22±17.1a 3.1±1.1a 3.1±0.9a 3.0±0.9
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
85
86
The use of A. tumefaciens infection alone to injured embryonic axis attached to
single cotyledon resulted in observation of 160 explants responding after the co-
cultivation period (Table 5). Out of responded 160 explants, a total of 86 explants could
survive and developed into seedlings, also called as primary transformants (T0
generation). The seeds from these set of 86 transformants were harvested and plant to
row progeny test employing gene specific PCR assay resulted in the identification of six
putative transgenic plants in T1 generation. In case of embryonic axis attached to single
cotyledon infected with A. tumefaciens culture with tobacco extract, out of infected 200
explants, 170 noticed responding well (Table 6). Among 170 well responded explants,
153 could survive and developed into seedlings. The plant to progeny screening of these
primary transformants resulted in identification of a set of 13 putative transformants in T1
generation as revealed by cry gene specific PCR assay. On other hand, A. tumefaciens
infection using air evacuation to increase the proximity between A. tumefaciens and
embryonic axis resulted in the observation of comparatively lesser number of explants
responding (133 explants) to this treatment (Table 7). The seeds were harvested from
121 survived primary transformants and plant to row progeny screening of them
identified a set 24 putative transformants in T1 generation, based on PCR analysis.
The statistical analysis revealed 80.00 per cent explant response, 53.75 per cent
explant survival and 3.0 per cent transformation efficiency in case of A. tumefaciens
infection alone (Table 8) (Fig. 4). Whereas, in case of A. tumefaciens infection using A.
tumefaciens culture along with tobacco extract recorded 85.00 per cent explant
response, 90.00 per cent explant survival and 6.5 per cent transformation efficiency.
Similarly, 66.50 per cent explant response, 90.98 per cent explant survival and 12.0 per
cent increased transformation efficiency was recorded for the treatment involving air
evacuation to increase the proximity between A. tumefaciens and embryonic axis.
87
Table 5: The effect of targeting embryonic axis attached to single cotyledon for successful Agrobacterium tumefaciens infection and successful transformation.
Sl. No.
Number of explants
infected/ batch
Number of explants
responded
Number of
explants survived
Plant to row screening in next
generation (T1)
Number of PCR +ve plants
identified in T1
1 50 36 20 20 0
2 50 39 18 18 3
3 50 40 27 27 1
4 50 45 21 21 2
Total 200 160 86 86 6
Table 6: The effect of targeting embryonic axis attached to single cotyledon with tobacco extract added in overnight grown Agrobacterium culture on plant transformation.
Sl. No.
Number of explants
infected / batch
Number of explants responde
d
Number of explants survived
Plant to row screening in next
generation (T1)
Number of PCR +ve plants
identified in T1
1 50 45 40 40 2
2 50 43 38 38 3
3 50 42 38 38 4
4 50 40 37 37 4
Total 200 170 153 153 13
Table 7: The effect of targeting embryonic axis attached to single cotyledon with air evacuation to increase the proximity between Agrobacterium tumefaciens and embryonic axis on transformation
Sl. No.
Number of explants
infected/ batch
Number of explants responde
d
Number of explants survived
Plant to row screening in next
generation (T1)
Number of PCR +ve plants
identified in T1
1 50 37 35 35 6
2 50 33 31 31 7
3 50 31 30 30 5
4 50 32 25 25 6
Total 200 133 121 121 24
88
Table 8: Effect of different treatments used in in planta transformation of pigeonpea on explant survival, explants responded and transformation efficacy presented in per cent.
Sl. No.
Treatments Explant
responded (%)
Explant survived
(%)
Transformation efficiency (%)
1 Agrobacterium infection alone
80.00a 53.75b 3.0c
2 Tobacco extract was added in overnight grown Agrobacterium culture
85.00a 90.00a 6.5b
3 Air evacuation to increase the proximity between Agrobacterium and embryonic axis
66.50b 90.98a 12.0a
LSD value (α=0.05) 0.1518 0.1554 0.1558
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
89
90
4.3 Generation of transgenic pigeonpea conferring expression of cry1Ac gene
Transgenic plants expressing cry1Ac gene were developed using both improvised
in vitro and in planta transformation methods. The in planta method, modified in present
study, was found to be the most effective and less laborious when compared with in vitro
transformation and plantlet regeneration method. The transgenic pigeonpea plants were
developed and forwarded to advanced generations (up to T3 generation). The analyses
of developed transgenic lines was done by performing their bioefficacy analysis against
H. armigera, quantitative Cry1Ac protein analysis using ELISA assay and cry1Ac gene
segregation analysis using PCR assay. Furthermore, the molecular analyses of selected
good transgenic lines were assessed by RT-PCR analysis of cry1Ac transcripts,
Southern and northern blot, and juncture region analysis using TAIL-PCR assay.
4.3.1 Development of transgenic pigeonpea carrying cry1Ac gene
A total of 1400 explants were infected with A. tumefaciens strain carrying construct
with cry1Ac gene and nptII as marker gene using in planta transformation protocol (Fig.
5). A set of 741 primary transformants were established, of which 728 could grow well till
plant maturity (Table 9) (Plate 2). The T1 seeds were harvested form well-established
primary transformants after reaching to maturity stage and T1 generation was raised. The
T1 plant progeny screening using cry1Ac gene specific PCR assay identified a set of
eighty eight putative transgenic plants for integration of cry1Ac gene (Plate 3). The
identified putative transformants were grown till maturity and T2 seeds were harvested. In
the embryonic axis infection followed by in vitro screen effort, a set of 2800 explants
were infected and co-cultivated. Upon kanamycin selection and rooting, a set of ten
plants could be established (Table 10a). The plantlets surviving on Kan selection
medium were confirmed for presence of cry1Ac gene using gene specific PCR assay
(Plate 3). The in vitro generated transformants (T0 generation) were hardened and grown
in transgenic containment facility, T1 seeds were harvested. The T1 seeds were sown in
plant to row progeny manner and T1 plants carrying cry1Ac gene were identified based in
PCR assay (Table 10b).
91
Table 9: Summary of transformation carried out using cry1Ac gene in pigeonpea
Sl. No. Number of
explants co-cultivated/set
Number of explants
responded
Number of primary
transformants (T0) established in greenhouse
Number of primary transformants
tested in plant-to-row progeny for
identifying putative transformants
Number of putative
transformants identified (T1)
1 50 20 10 10 00
2 50 22 08 08 00
3 50 45 27 27 05
4 50 40 21 21 03
5 50 16 12 12 00
6 50 00 00 00 00
7 50 45 40 40 03
8 50 46 37 37 04
9 50 42 30 30 03
10 50 45 29 29 03
11 50 30 32 32 00
12 50 42 40 40 00
13 50 45 40 40 21
14 50 30 00 00 00
15 50 43 38 38 09
16 50 16 10 10 00
17 50 17 10 10 00
18 50 42 38 38 13
19 50 40 37 37 16
20 50 25 00 00 00
21 50 45 40 27 08
22 50 41 35 35 00
23 50 46 42 42 00
24 50 44 40 40 00
25 50 38 30 30 00
26 50 36 32 32 00
27 50 34 31 31 00
28 50 35 32 32 00
Total 1400 970 741 728 88
92
93
94
Table 10a: Summary of transformation work being carried out using cry1Ac gene following in vitro kanamycin selection method in pigeonpea
No. of sets
Number of explants co-
cultivated/set
Number of explants survived and established on Kan selection media
Number of plantlets rooted and established
in greenhouse
1 100 0 0
2 100 0 0
3 100 0 0
4 100 4 2
5 100 3 2
6 100 0 0
7 100 0 0
8 100 0 0
9 100 0 0
10 100 0 0
11 100 0 0
12 100 0 0
13 100 2 2
14 100 0 0
15 100 0 0
16 100 0 0
17 100 0 0
18 100 4 1
19 100 1 1
20 100 0 0
21 100 0 0
22 100 0 0
23 100 0 0
24 100 0 0
25 100 2 2
26 100 0 0
27 100 0 0
28 100 0 0
Total 2800 16 10
95
Table 10b: Identification of T1 plants progenies of 10 putative transformants carrying cry1Ac gene. Testing for the presence of gene was done through gene specific PCR assay
Transgenic plants
Number of plants
screened
Number of PCR positive plants
Number of PCR negative plants
AC1 09 03 06
AC2 07 04 03
AC3 07 06 01
AC4 21 14 07
AC5 09 06 03
AC6 09 04 05
AC7 08 05 03
AC8 16 05 11
AC9 10 05 05
AC10 12 05 07
96
4.3.2 The cry1Ac gene segregation analysis in T2 generation progenies
The obtained T2 seeds were sown in plant to row progeny manner to get T2
generation plants. From each parental T1 generation plants up to 40 seeds were sown
and obtained T2 plants were subjected for gene specific PCR assay (Plate 3). The gene
segregation pattern was assayed using chi-square test. Among developed eighty eight,
for forty eight transgenic plant progenies of T1 generations plants recorded chi-square
calculated value less than table chi-square value (3.84) (Table 11). For rest forty
transgenic plants the chi-square value was observed to be more than the table value,
clearly indicating that the cry1Ac gene segregation in those transgenic plant progenies
did not follow 3:1 gene segregation pattern in T2 generation.
4.3.3 Characterization of developed transgenic lines in T2 generation
Bioefficacy of the transgenic plants expressing cry1Ac against first instar
Helicoverpa larvae revealed significant variability in larval mortality in all targeted tissue
types viz., leaf, flower and pod. The larval mortality in case of leaf tissues ranged from
25.0% to 70.0% (Table 12a). Whereas, it was 22.5% to 52.5% in case of flower tissues
and 17.5% to 47.5% in case of pod tissue. It was noticed that the majority of putative
transformants were having insect mortality ranging from 40.0% to 60.0%, with the
highest mortality recorded in Ac140-13 (70.0%) and Ac272-18 (70.0%) transgenic line
(Fig. 6). It was noticed that the larval feeding was highest in transgenic lines Ac279-8
(30.0%) and Ac30-12 (25.0%). Few transgenic plants like, Ac16-7, Ac30-12, Ac46-11
showed consistency in the insect mortality levels across different tissue types, though
mortality was low. The larval feeding pattern on different tissue types of transgenic and
non-transgenic control plants is shown in Plate 4. The statistical analysis of variance
(ANOVA), indicated that the transgenic lines differ significantly (p < 0.001) from each
other for larval mortality in all tissue types. The ‘t’-test analysis of larval mortality in
different tissue types indicated significant difference between mortality levels in leaf and
flower (p ≤ 0.001), and in leaf and pod tissues (p ≤ 0.001). Interestingly, the larval
mortality levels were comparable between flower and pod tissue (p ≥ 0.41) types as
indicated by ‘t’-test analysis. The un-paired ‘t’-test analysis of bioefficacy levels noticed in
different transgenic plants with two different genomic backgrounds (ICPL87119 and
BSMR736) indicated that the effect of Cry1Ac through thransgenic plant tissues of
97
Table 11: The transgene segregation pattern in eighty eight transformants carrying cry1Ac in T2 generation revealed by gene specific PCR assay
Plant ID Number of plants tested using gene
specific PCR
Number of plants positive using gene
specific PCR
Number of plants negative using gene
specific PCR
Expected number of
positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
Ac5-2 24 14 10 18.00 6.00 3.56 Yes Ac8-19 22 14 08 16.50 5.50 1.52 Yes Ac8-15 23 13 10 17.25 5.75 4.19 No Ac12-11 21 12 09 15.75 5.25 3.57 Yes Ac12-15 24 13 11 18.00 6.00 5.56 No Ac16-1 29 17 12 21.75 7.25 2.50 Yes Ac16-7 25 14 11 18.75 6.25 4.81 No Ac16-11 21 11 10 15.75 5.25 5.73 No Ac18-12 26 15 11 19.50 6.50 4.15 No Ac20-2 21 13 08 15.75 5.25 1.92 Yes Ac20-3 29 21 08 21.75 7.25 0.10 Yes Ac21-4 23 14 09 17.25 5.75 2.45 Yes Ac25-1 20 10 10 15.00 5.00 6.67 No
Ac29-1 30 19 11 22.50 7.50 2.18 Yes Ac29-3 30 23 07 22.50 7.50 0.02 Yes Ac30-12 24 14 10 18.00 6.00 3.56 Yes Ac31-1 24 18 06 18.00 6.00 0.00 Yes Ac31-2 32 19 13 24.00 8.00 4.17 No Ac37-8 20 12 08 15.00 5.00 2.40 Yes Ac39-2 22 12 10 16.50 5.50 4.91 No Ac41-7 18 11 07 13.50 4.50 1.85 Yes Ac46-11 24 13 11 18.00 6.00 5.56 No Ac48-3 25 15 10 18.75 6.25 3.00 Yes
(Table chi-square = 3.84) Cont…
98
Plant ID Number of plants tested using gene
specific PCR
Number of plants positive using gene
specific PCR
Number of plants negative using
gene specific PCR
Expected number of
positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
Ac49-5 20 11 09 15.00 5.00 4.27 No
Ac50-1 32 20 12 24.00 8.00 2.66 Yes
Ac50-3 22 12 10 16.50 5.50 4.91 No
Ac51-1 22 13 09 16.50 5.50 2.97 Yes
Ac53-23 21 11 10 15.75 5.25 5.73 No
Ac55-16 19 11 08 14.25 4.75 2.96 Yes
Ac56-3 23 13 10 17.25 5.75 4.19 No
Ac57-10 24 15 09 18.00 6.00 2.00 Yes
Ac58-23 20 12 08 15.00 5.00 2.40 Yes
Ac61-4 20 11 09 15.00 5.00 4.27 No
Ac63-20 24 15 09 18.00 6.00 2.00 Yes
Ac66-12 25 13 12 18.75 6.25 7.05 No
Ac73-3 26 14 12 19.50 6.50 6.21 No
Ac75-3 20 14 06 15.00 5.00 0.27 Yes
Ac88-2 24 16 08 18.00 6.00 0.89 Yes
Ac110-10 25 16 09 18.75 6.25 1.61 Yes
Ac140-13 22 14 08 16.50 5.50 1.52 Yes
Ac150-6 24 13 11 18.00 6.00 5.56 No
Ac152-15 28 17 11 21.00 7.00 3.05 Yes
Ac161-4 20 14 06 15.00 5.00 0.27 Yes
Ac162-8 20 11 09 15.00 5.00 4.27 No
Ac167-4 16 09 07 12.00 4.00 3.00 Yes
Ac168-7 26 15 11 19.50 6.50 4.15 No
Ac170-2 24 10 14 18.00 6.00 14.22 No
(Table chi-square = 3.84) Cont…
99
Plant ID Number of plants tested using gene
specific PCR
Number of plants positive using gene
specific PCR
Number of plants negative using
gene specific PCR
Expected number of
positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
Ac201-3 25 18 07 18.75 6.25 0.12 Yes
Ac202-3 24 15 09 18.00 6.00 2.00 Yes
Ac203-5 20 14 06 15.00 5.00 0.27 Yes
Ac204-2 22 13 09 16.50 5.50 2.97 Yes
Ac205-2 23 16 07 17.25 5.75 0.36 Yes
Ac206-6 24 13 11 18.00 6.00 5.56 No
Ac207-11 21 12 09 15.75 5.25 3.57 Yes
Ac208-19 19 10 09 14.25 4.75 5.07 No
Ac251-3 23 12 11 17.25 5.75 6.39 No
Ac252-3 21 11 10 15.75 5.25 5.73 No
Ac253-8 20 12 08 15.00 5.00 2.40 Yes
Ac254-12 21 11 10 15.75 5.25 5.73 No
Ac255-4 27 15 12 20.25 6.75 5.44 No
Ac256-9 26 14 12 19.50 6.50 6.21 No
Ac257-5 22 14 08 16.50 5.50 1.52 Yes
Ac258-12 24 13 11 18.00 6.00 5.56 No
Ac259-3 27 15 12 20.25 6.75 5.44 No
Ac260-9 24 14 10 18.00 6.00 3.56 Yes
Ac261-3 25 13 12 18.75 6.25 7.05 No
Ac262-16 24 15 09 18.00 6.00 2.00 Yes
Ac263-4 24 14 10 18.00 6.00 3.56 Yes
Ac264-15 26 16 10 19.50 6.50 2.51 Yes
Ac265-5 20 14 06 15.00 5.00 0.27 Yes
Ac266-13 23 12 11 17.25 5.75 6.39 No
(Table chi-square = 3.84) Cont…
100
Plant ID Number of plants tested using gene
specific PCR
Number of plants positive using gene
specific PCR
Number of plants negative using
gene specific PCR
Expected number of
positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
Ac267-21 22 12 10 16.50 5.50 4.91 No
Ac268-18 24 14 10 18.00 6.00 3.56 Yes
Ac269-7 25 15 10 18.75 6.25 3.00 Yes
Ac270-6 24 12 12 18.00 6.00 8.00 No
Ac271-14 26 15 11 19.50 6.50 4.15 No
Ac272-18 24 16 08 18.00 6.00 0.89 Yes
Ac273-11 26 17 09 19.50 6.50 1.28 Yes
Ac274-3 21 12 09 15.75 5.25 3.57 Yes
Ac275-6 20 11 09 15.00 5.00 4.27 No
Ac276-2 24 13 11 18.00 6.00 5.56 No
Ac277-11 22 14 08 16.50 5.50 1.52 Yes
Ac278-9 23 15 08 17.25 5.75 1.17 Yes
Ac279-8 22 11 11 16.50 5.50 7.33 No
Ac280-3 24 14 10 18.00 6.00 3.56 Yes
Ac281-5 26 13 13 19.50 6.50 8.67 No
Ac282-1 24 13 11 18.00 6.00 5.56 No
Ac362-21 20 10 10 15.00 5.00 6.67 No
(Table chi-square = 3.84)
101
Table 12a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eighty eight putative transformants carrying cry1Ac gene in T2 generation
Plant ID Per cent corrected mortality
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
Ac5-2 45.00abcd 32.50abc 37.50abc Ac48-3 50.00abcd 37.50abc 37.50abc
Ac8-19 60.00abc 47.50ab 42.50ab Ac49-5 35.00bcd 27.50bc 32.50abc
Ac8-15 45.00abcd 37.50abc 42.50ab Ac50-1 47.50abcd 37.00abc 35.00abc
Ac12-11 50.00abcd 42.50abc 42.50ab Ac50-3 50.00abcd 37.50abc 42.50ab
Ac12-15 40.00abcd 32.50abc 32.50abc Ac51-1 55.00abcd 37.50abc 42.50ab
Ac16-1 42.50abcd 37.00abc 35.00abc Ac53-23 45.00abcd 37.50abc 37.50abc
Ac16-7 35.00bcd 27.50bc 32.50abc Ac55-16 50.00abcd 32.50abc 32.50abc
Ac16-11 50.00abcd 42.50abc 42.50ab Ac56-3 40.00abcd 27.50bc 27.50abc
Ac18-12 40.00abcd 32.50abc 32.50abc Ac57-10 60.00abc 42.50abc 42.50ab
Ac20-2 65.00ab 47.00ab 45.50a Ac58-23 60.00abc 42.50abc 37.50abc
Ac20-3 65.50ab 50.50ab 47.00a Ac61-4 40.00abcd 32.50abc 32.50abc
Ac21-4 55.00abcd 42.50abc 37.50abc Ac63-20 60.00abc 47.50ab 42.50ab
Ac25-1 35.00bcd 22.50c 22.50bc Ac66-12 30.00cd 22.50c 22.50bc
Ac29-1 52.00abcd 42.50abc 40.00ab Ac73-3 40.00abcd 32.50abc 27.50abc
Ac29-3 47.50abcd 40.50abc 37.00abc Ac75-3 25.00d 22.50c 27.50abc
Ac30-12 25.00d 22.50c 22.50bc Ac88-2 55.00abcd 47.50ab 47.50a
Ac31-1 55.50abcd 42.50abc 40.00ab Ac110-10 45.00abcd 37.50abc 42.50ab
Ac31-2 52.50abcd 40.00abc 42.50ab Ac140-13 70.00a 52.50a 47.50a
Ac37-8 60.00abc 47.50ab 47.50a Ac150-6 50.00abcd 37.50abc 37.50abc
Ac39-2 45.00abcd 32.50abc 37.50abc Ac152-15 50.00abcd 37.50abc 37.50abc
Ac41-7 55.00abcd 42.50abc 42.50ab Ac161-4 60.00abc 47.50ab 47.50a
Ac46-11 35.00bcd 32.50abc 32.50abc Ac162-8 45.00abcd 37.50abc 37.50abc
Note: The means followed with same letters are within student ‘t’ range at α=0.05. Cont…
102
Plant ID Per cent corrected mortality
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
Ac167-4 60.00abc 42.50abc 42.50ab Ac262-16 60.00abc 47.50ab 42.50ab
Ac168-7 40.00abcd 32.50abc 37.50abc Ac263-4 65.00ab 42.50abc 42.50ab
Ac170-2 30.00cd 22.50c 27.50abc Ac264-15 50.00abcd 37.50abc 32.50abc
Ac201-3 60.00abc 42.50abc 42.50ab Ac265-5 55.00abcd 42.50abc 42.50ab
Ac202-3 50.00abcd 42.50abc 37.50abc Ac266-13 45.00abcd 37.50abc 37.50abc
Ac203-5 60.00abc 47.50ab 47.50a Ac267-21 35.00bcd 27.50bc 27.50abc
Ac204-2 60.00abc 47.50ab 47.50a Ac268-18 60.00abc 47.50ab 47.50a
Ac205-2 55.00abcd 42.50abc 42.50ab Ac269-7 50.00abcd 37.50abc 37.50abc
Ac206-6 40.00abcd 27.50bc 27.50abc Ac270-6 45.00abcd 37.50abc 32.50abc
Ac207-11 60.00abc 42.50abc 42.50ab Ac271-14 50.00abcd 37.50abc 37.50abc
Ac208-19 30.00cd 22.50c 27.50abc Ac272-18 70.00a 47.50ab 47.50a
Ac251-3 45.00abcd 37.50abc 37.50abc Ac273-11 65.00ab 47.50ab 42.50ab
Ac252-3 40.00abcd 32.50abc 37.50abc Ac274-3 45.00abcd 32.50abc 32.50abc
Ac253-8 55.00abcd 42.50abc 47.50a Ac275-6 45.00abcd 37.50abc 32.50abc
Ac254-12 45.00abcd 37.50abc 37.50abc Ac276-2 40.00abcd 32.50abc 32.50abc
Ac255-4 30.00cd 27.50bc 27.50abc Ac277-11 55.00abcd 37.50abc 32.50abc
Ac256-9 45.00abcd 37.50abc 37.50abc Ac278-9 35.00bcd 22.50c 22.50bc
Ac257-5 60.00abc 47.50ab 47.50a Ac279-8 30.00cd 22.50c 17.50c
Ac258-12 55.00abcd 42.50abc 42.50ab Ac280-3 55.00abcd 37.50abc 32.50abc
Ac259-3 30.00cd 27.50bc 27.50abc Ac281-5 40.00abcd 32.50abc 32.50abc
Ac260-9 60.00abc 42.50abc 42.50ab Ac282-1 40.00abcd 27.50bc 27.50abc
Ac261-3 50.00abcd 37.50abc 37.50abc Ac362-21 40.00abcd 32.50abc 32.50abc
SD (±) 0.055 0.039 0.038
CD 0.002 0.001 0.001
CV (%) 6.04 4.70 4.35
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
103
104
105
pigeonpea was independent of genotypes used with respect to their bioassay against H.
armigera (p > 0.1) (Table 12b).
There was a significant variation in Cry1Ac protein levels of different transgenic
lines irrespective of tissue type viz., leaf, flower and pod as revealed through ELISA
assay (Plate 5). The Cry protein level ranged from 0.305 to 0.847 µg/g of fresh leaf
tissue (Table 13a). Whereas, it was 0.221 to 0.671 µg/g of fresh flower tissues and 0.250
to 0.685 µg/g of fresh pod tissue. Interestingly, it was noticed that the majority of putative
transformants were accumulating the Cry1Ac protein from 0.6 to 0.8 µg/g of fresh tissue,
wherein the highest Cry1Ac proteins was detected in Ac140-13 (0.847 µg/g in leaf)
transgenic line (Fig. 6). Similarly, in case of flower and pod tissues, the majority of
transgenic plants were accumulating Cry1Ac protein from 0.3 to 0.5 µg/g of fresh tissue.
The transgenic line Ac140-13 (0.671 µg/g) showed highest accumulation of Cry protein
in flower and Ac201-3 (0.685 µg/g) in pod tissues, as indicated by ELISA assay. On
other hand, the lower level of Cry protein was recorded in transgenic line Ac73-3 (0.250
µg/g) for leaf tissues and in Ac75-3 (0.305 µg/g in flower and 0.221 µg/g in pod) for
flower and pod tissues. The ‘t’-test analysis of Cry protein accumulation in different tissue
types indicated significant difference in leaf and flower (p ≤ 0.001) and in lead and pod
tissues (p ≤ 0.001), and the Cry protein accumulation was non-significant for flower and
pod tissue (p > 0.44) types. The positive correlation was noticed between insect morality
and Cry1Ac protein accumulation (r2=0.8303) (Fig. 7). Similarly, the frequency
distribution analysis of transgenic plants following 3:1 transgene segregation pattern and
other than 3:1 segregation pattern revealed that majority of high Cry1Ac protein
accumulating transgenic plants were following 3:1 transgene segregation pattern (Fig. 7).
4.3.4 Characterization of in vitro generated transgenic plants in T1 generation
The bioefficacy analysis of in vitro developed transgenic plants showed the larval
mortality ranged from 40.00% to 55.50% in case of leaf tissues. Similarly, it was 27.50%
to 42.50% in case of flower and pod tissues (Table 12c). The highest larval mortality
recorded in case of transgenic plant AC4 in leaf and flower tissues, whereas in case pod
tissues, it was maximum in AC2, AC5 and AC7. On other hand, least larval mortality was
noticed in transgenic line AC10 for all tissue types. In leaf tissues, the Cry1Ac protein
level ranged from 0.344 to 0.716 µg/g of fresh tissue weight. Similarly, the Cry protein
accumulation in flower tissues was noticed from 0.273 to 0.604 µg/g and from 0.291 to
106
Table 12b: Comparison of H. armigera mortality in transgenic lines of ICPL87119 and
BSMR736 (by unpaired ‘t’-test at α=0.05)
Genotypes ICPL87119 BSMR736
Mean 47.84 53.19
Variance 115.62 73.14
Observations 80 8
Hypothesized Mean Difference 0
Df 9
t Stat -1.64
P(T<=t) two-tail 0.13
t Critical two-tail 2.26
Table 12c: Per cent corrected mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving in vitro generated putative transformants carrying cry1Ac gene in T1 generation
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues
AC1 45.00c 37.50b 32.50c
AC2 50.00b 42.50a 42.50a
AC3 40.00d 32.50c 32.50c
AC4 55.50a 42.50a 40.00ab
AC5 52.50ab 40.00ab 42.50a
AC6 45.00c 32.50c 37.50b
AC7 55.00a 42.50a 42.50a
AC8 45.00c 37.50b 37.50b
AC9 50.00b 32.50c 32.50c
AC10 40.00d 27.50d 27.50d
SD (±) 0.043 0.039 0.051
CD 0.002 0.001 0.003
CV (%) 5.12 4.69 5.26
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
107
Plate 5: Estimation of Cry 1Ac protein in different test tissues of developed
transgenic pigeonpea plants using ELISA assay. TR: Technical
Replications; NC: Negative control; NTC: Non-Transgenic Control
108
Table 13a: The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2
generation as revealed by ELISA assay (α=0.05)
Plant ID Cry1Ac protein level (µg/g FW)
Plant ID Cry1Ac protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
Ac5-2 0.592 0.460 0.474 Ac48-3 0.637 0.542 0.550
Ac8-19 0.721 0.599 0.554 Ac49-5 0.332 0.267 0.301
Ac8-15 0.640 0.513 0.522 Ac50-1 0.345 0.287 0.291
Ac12-11 0.656 0.534 0.557 Ac50-3 0.612 0.528 0.538
Ac12-15 0.344 0.273 0.291 Ac51-1 0.708 0.536 0.572
Ac16-1 0.398 0.339 0.342 Ac53-23 0.627 0.508 0.523
Ac16-7 0.332 0.299 0.316 Ac55-16 0.626 0.450 0.464
Ac16-11 0.558 0.489 0.504 Ac56-3 0.423 0.383 0.398
Ac18-12 0.410 0.321 0.356 Ac57-10 0.690 0.584 0.594
Ac20-2 0.769 0.702 0.710 Ac58-23 0.780 0.636 0.586
Ac20-3 0.756 0.646 0.669 Ac61-4 0.378 0.300 0.318
Ac21-4 0.691 0.594 0.545 Ac63-20 0.719 0.638 0.638
Ac25-1 0.423 0.284 0.304 Ac66-12 0.310 0.292 0.301
Ac29-1 0.605 0.567 0.561 Ac73-3 0.404 0.363 0.250
Ac29-3 0.405 0.325 0.342 Ac75-3 0.305 0.221 0.266
Ac30-12 0.315 0.267 0.289 Ac88-2 0.682 0.584 0.572
Ac31-1 0.454 0.378 0.399 Ac110-10 0.529 0.454 0.489
Ac31-2 0.587 0.502 0.519 Ac140-13 0.847 0.671 0.599
Ac37-8 0.721 0.561 0.562 Ac150-6 0.557 0.461 0.486
Ac39-2 0.632 0.505 0.522 Ac152-15 0.640 0.575 0.574
Ac41-7 0.716 0.604 0.601 Ac161-4 0.700 0.626 0.645
Ac46-11 0.459 0.397 0.400 Ac162-8 0.636 0.505 0.520
Cont…
109
Plant ID Cry1Ac protein level (µg/g FW)
Plant ID Cry1Ac protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
Ac167-4 0.712 0.616 0.612 Ac262-16 0.744 0.611 0.587
Ac168-7 0.426 0.319 0.339 Ac263-4 0.806 0.584 0.604
Ac170-2 0.340 0.262 0.299 Ac264-15 0.560 0.490 0.452
Ac201-3 0.725 0.615 0.685 Ac265-5 0.685 0.500 0.500
Ac202-3 0.629 0.530 0.470 Ac266-13 0.605 0.473 0.355
Ac203-5 0.760 0.604 0.602 Ac267-21 0.539 0.438 0.442
Ac204-2 0.688 0.603 0.610 Ac268-18 0.799 0.619 0.619
Ac205-2 0.683 0.517 0.532 Ac269-7 0.673 0.486 0.493
Ac206-6 0.447 0.322 0.344 Ac270-6 0.567 0.439 0.365
Ac207-11 0.768 0.616 0.637 Ac271-14 0.620 0.421 0.434
Ac208-19 0.354 0.278 0.290 Ac272-18 0.807 0.632 0.642
Ac251-3 0.627 0.504 0.528 Ac273-11 0.835 0.536 0.504
Ac252-3 0.515 0.414 0.439 Ac274-3 0.543 0.405 0.417
Ac253-8 0.648 0.545 0.558 Ac275-6 0.587 0.426 0.397
Ac254-12 0.547 0.434 0.447 Ac276-2 0.406 0.360 0.362
Ac255-4 0.342 0.265 0.274 Ac277-11 0.708 0.447 0.410
Ac256-9 0.527 0.467 0.471 Ac278-9 0.457 0.311 0.338
Ac257-5 0.749 0.618 0.615 Ac279-8 0.337 0.259 0.223
Ac258-12 0.698 0.598 0.612 Ac280-3 0.701 0.467 0.488
Ac259-3 0.325 0.250 0.261 Ac281-5 0.453 0.403 0.403
Ac260-9 0.701 0.497 0.512 Ac282-1 0.467 0.358 0.363
Ac261-3 0.552 0.411 0.410 Ac362-21 0.406 0.325 0.334
SD (±) 0.016 0.019 0.018
CD 0.006 0.010 0.012
CV (%) 3.35 4.13 4.06
110
111
0.601 µg/g of fresh pod tissue (Table 13b). The higher level of Cry1Ac proteins was
detected in AC4 transgenic line irrespective of tissue type. The statistical analysis
revealed that the transgenic plants differ significantly for both insect mortality levels and
Cry1Ac protein accumulation (p < 0.01). There was a strong positive correlation noticed
between the different tissue types, and insect mortality and Cry1Ac protein in transgenic
lines (Table 13c).
4.3.5 The cry1Ac gene segregation analysis in T3 generation progenies
In initial efforts of plant transformation a set of eight transgenic plants viz., Ac16-1,
Ac20-2, Ac20-3, Ac29-1, Ac29-3, Ac31-1, Ac31-2 and Ac50-1 carrying cry1Ac gene were
identified and forwarded to T3 generation. The T3 seeds were harvested from T2
generation plants and were sown in plant to row progeny manner to get T3 generation
plants. From each parental T2 generation plant up to 40 seeds were sown and obtained
plants were subjected for gene specific PCR assay (Plate 3). Using chi-square test, the
cry1Ac gene segregation pattern was assayed. Among eight transgenic lines, in five
transgenic lines we identified homozygosity. The homozygosity in T2 parental plants viz.,
20-2-7, 20-3-2, 29-1-10, 31-2-9, 31-2-12, 31-1-2 and 31-1-3 belonging to five transgenic
lines were confirmed using cry1Ac gene specific PCR assay (Plate 3) (Table 14). Many
of other plant progenies was found to heterozygous as revealed by chi-square analysis
(table chi-square value is 3.84).
4.3.6 Characterization of eight transgenic lines carrying cry1Ac gene in T3 generation
The insect bioassay of the transgenic pigeonpea plants recorded significant
variability in larval mortality for eight transgenic lines. It was noticed that the larval
mortality was ranged from 41.25 to 61.25 per cent in case of leaf tissues whereas; it was
32.5 to 47.5 per cent in case of flower tissues and 35.0 to 52.5 per cent in case of pod
(Table 15a). The highest larval mortality recorded in case of transgenic line Ac20-2 for all
tissue types (Plate 4). On other hand, least larval mortality was noticed in transgenic line
Ac16-1. The analysis of variance indicated that the transgenic lines differ significantly (p
< 0.001) for larval mortality in all tissue types. The ‘t’-test analysis of larval mortality in
112
Table 13b: The Cry1Ac protein level detected in in vitro generated transgenic pigeonpea plants of T1 generation as revealed by ELISA assay in leaf, flower and pod
tissues (α=0.05).
Plant ID Cry1Ac protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues
AC1 0.454 0.378 0.399
AC2 0.587 0.502 0.519
AC3 0.423 0.383 0.398
AC4 0.716 0.604 0.601
AC5 0.637 0.542 0.550
AC6 0.540 0.413 0.422
AC7 0.656 0.534 0.557
AC8 0.527 0.408 0.423
AC9 0.526 0.450 0.464
AC10 0.344 0.273 0.291
SD (±) 0.011 0.016 0.021
CD 0.012 0.011 0.014
CV (%) 4.12 4.89 5.04
113
Table 13c: The correlation analysis of insect mortality levels and estimated Cry1Ac
protein in leaf, flower and pod tissues of T2 generation plants (αααα=0.01)
Insect mortality
in leaf tissues
Insect mortality in
flower tissues
Insect mortality
in pod tissues
Cry1Ac protein in leaf
tissues
Cry1Ac protein in
flower tissues
Cry1Ac protein in
pod tissues
Insect mortality in leaf tissues
1 0.936** 0.861** 0.911** 0.893** 0.867**
Insect mortality in flower tissues
1 0.921** 0.846** 0.875** 0.834**
Insect mortality in pod tissues
1 0.798** 0.839** 0.855**
Cry1Ac protein in leaf tissues
1 0.946** 0.915**
Cry1Ac protein in flower tissues
1 0.970**
Cry1Ac protein in pod tissues
1
**Correlation is significant at the 0.01 level (two tailed).
114
Table 14: Transgene segregation pattern in eight transgenic lines carrying cry1Ac gene in
T3 generation
Plant ID
Total number of
plant progenies
tested
Number of PCR positive plants
Number of PCR
negative plants
Expected number
of positive plants
Expected number
of negative plants
Calculated chi-square
Estimated plant
zygosity
20-2-1 15 14 01 11.25 03.75 02.69 Hetero 20-2-2 16 12 04 12.00 04.00 00.00 Hetero 20-2-3 14 07 07 10.50 03.50 04.67 Hetero. 20-2-4 16 11 05 12.00 04.00 00.33 Hetero. 20-2-6 16 12 04 12.00 04.00 00.00 Hetero. 20-2-7 15 15 00 11.25 03.75 05.00 Homo.
29-3-2 05 05 00 03.75 01.25 01.67 Homo. 29-3-3 19 10 09 14.25 04.75 05.07 Hetero. 29-3-6 10 08 02 07.50 02.50 00.13 Hetero. 29-3-8 08 05 03 06.00 02.00 00.67 Hetero.
29-3-10 09 05 04 06.75 02.25 01.81 Hetero. 29-3-11 15 08 09 11.25 03.75 08.29 Hetero.
20-3-1 14 09 05 10.50 03.50 00.86 Hetero. 20-3-2 11 11 00 08.25 02.75 03.67 Homo. 20-3-4 13 12 01 09.75 03.25 02.08 Hetero. 20-3-5 14 07 07 10.50 03.50 04.67 Hetero. 20-3-6 11 04 07 08.25 02.75 08.76 Hetero. 20-3-9 14 08 06 10.50 03.50 02.38 Hetero.
29-1-6 12 08 04 09.00 03.00 00.44 Hetero. 29-1-7 11 05 06 08.25 02.75 05.12 Hetero. 29-1-8 13 07 06 09.75 03.25 03.10 Hetero. 29-1-9 15 14 01 11.25 03.75 02.69 Hetero.
29-1-10 10 10 00 07.50 02.50 03.33 Homo. 29-1-15 11 10 01 08.25 02.75 01.48 Hetero.
16-1-1 24 16 08 18.00 06.00 00.89 Hetero. 16-1-2 23 19 04 17.25 05.75 00.71 Hetero. 16-1-3 10 10 00 07.50 02.50 03.33 Homo. 16-1-4 11 05 06 08.25 02.75 05.12 Hetero. 16-1-5 07 07 00 05.25 01.75 02.33 Hetero. 16-1-6 10 09 01 07.50 02.50 01.20 Hetero.
31-2-6 16 11 06 12.00 04.00 01.08 Hetero. 31-2-7 15 14 01 11.25 03.75 02.69 Hetero. 31-2-9 16 16 00 12.00 04.00 05.33 Homo.
31-2-10 21 12 09 15.75 05.25 03.57 Hetero. 31-2-11 23 15 08 17.25 05.75 01.17 Hetero. 31-2-12 16 16 00 12.00 04.00 05.33 Homo.
31-1-1 13 12 01 09.75 03.25 02.08 Hetero. 31-1-2 11 11 00 08.25 02.75 03.67 Homo. 31-1-3 10 10 00 07.50 02.50 03.33 Homo. 31-1-5 18 10 08 13.50 04.50 03.63 Hetero. 31-1-6 11 07 04 08.25 02.75 00.76 Hetero. 31-1-7 20 10 10 15.00 05.00 06.67 Hetero.
Contd…
115
Plant ID
Total number of
plant progenies
tested
Number of PCR positive plants
Number of PCR
negative plants
Expected number
of positive plants
Expected number
of negative plants
Calculated chi-square
Estimated plant
zygosity
50-1-1 18 13 05 13.50 04.50 00.07 Hetero.
50-1-2 20 12 08 15.00 05.00 02.40 Hetero.
50-1-3 14 05 09 10.50 03.50 11.52 Hetero.
50-1-4 17 10 07 12.75 04.25 02.37 Hetero.
50-1-5 21 14 07 15.75 05.25 00.78 Hetero.
50-1-6 17 12 05 12.75 04.25 00.18 Hetero.
Where; Hetero.: Heterozygous; Homo.: Homozygous
116
Table 15a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eight putative transformants carrying cry1Ac gene in T3 generation.
Event I D
Corrected per cent larval mortality
Leaf tissues Flower tissues Pod tissues
Ac29-3 42.50d 35.00d 37.50d
Ac31-2 47.50c 37.50c 40.00c
Ac29-1 51.25b 42.50d 45.00b
Ac16-1 41.25e 32.50e 35.00e
Ac20-2 61.25a 47.50a 52.50a
Ac20-3 53.75a 45.00a 47.50a
Ac31-1 50.00b 37.50b 37.50d
Ac50-1 45.00d 35.00d 37.50d
SD (±) 0.078 0.033 0.030
CD 0.019 0.010 0.017
CV (%) 4.91 5.00 6.24
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
Table 15b: The ‘t’-test analysis of Helicoverpa armigera mortality levels from T2 and T3
generation of eight cry1Ac transgenic lines (α=0.05)
Leaf tissues Flower tissues Pod tissues
T2
generation T3
generation T2
generation T3
generation T2
generation T3
generation
Mean 53.50 49.06 42.13 39.06 40.25 41.56
Variance 68.07 42.75 21.98 28.46 20.57 37.39
Observations 8 8 8 8 8 8
Pooled Variance 55.41 25.22 28.98
Hypothesized Mean Difference
0 0 0
Df 14 14 14
t Stat 1.19 1.22 -0.49
P(T<=t) two-tail 0.25 0.24 0.63
t Critical two-tail 2.14 2.14 2.14
117
different tissue types indicated significant difference between mortality levels transgenic
lines in leaf and flower (p ≤ 0.003), and in leaf and pod tissues (p ≤ 0.02). The larval
mortality between two tissue types viz., flower and pod were on par with each other with
p ≥ 0.19, by ‘t’-test analysis. Further, ‘t’-test analysis revealed that there was no
significant difference between the mortality levels observed in T2 and T3 generation in all
tissue types of eight transgenic lines (Table 15b).
It was interesting to notice the significant variation in Cry1Ac protein levels of eight
transgenic lines irrespective of tissue types as revealed through ELISA assay. In leaf
tissues, the Cry1Ac protein level ranged from 0.322 to 0.736 µg/g of fresh tissue weight
(Table 16). Similarly, the Cry protein accumulation in flower tissues was noticed from
0.254 to 0.646 µg/g and from 0.269 to 0.691 µg/g of fresh pod tissue. The higher level of
Cry1Ac protein was detected in Ac20-2 transgenic line irrespective of tissue type. On
other hand, the lower level of Cry1Ac protein was recorded in transgenic line Ac50-1.
The statistical analysis indicated that the transgenic lines were differencing significantly
for Cry protein accumulation with p < 0.001 in case of leaf, flower and pod tissue types.
Interestingly, the ‘t’-test analysis of Cry protein accumulation in different tissue types
indicated that there was no significant difference in leaf and flower
(p > 0.19), flower and pod tissues (p > 0.38) and between leaf and pod tissue types (p
>0.29). The correlation analysis between insect morality and Cry1Ac protein
accumulation revealed positive correlation between them with r2=0.8262 (Fig. 8).
4.3.7 The cry1Ac transcript analysis using qRT-PCR assay
Absolute real time analysis of cry1Ac transcript levels in different tissue types of
eight transgenic lines indicated significant variation in cry1Ac transcript levels as
revealed by standard graph (Plate 6). The cry1Ac transcript level was ranged from 24.6
to 165.1 ng/µl in fresh leaf tissue (Table 17a). Similarly, the cry1Ac transcript level in
flower was ranged from 15.6 to 149.5 ng/µl and in pod from 18.3 to 152.4 ng/µl.
Interestingly, the highest transcript level was detected in all tissue types of Ac20-2
transgenic lines. On other hand, the lower level of cry1Ac transcripts was recorded in
case of transgenic line Ac50-1. The ANOVA indicated the observation of significant
difference between transgene transcript levels of eight transgenic lines (p < 0.01). The
‘t’-test analysis of transcript levels between tissue types revealed non-significant
difference in the transcripts levels of cry1Ac gene in different tissue types within
118
Table 16: The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay
Event ID
Cry1Ac protein level (µg/g FW)
Leaf tissue Flower tissue Pod tissue
Ac29-3 0.375d 0.304e 0.324e
Ac31-2 0.526bc 0.469cd 0.498cd
Ac29-1 0.566b 0.512bc 0.529bc
Ac16-1 0.340d 0.298e 0.312e
Ac20-2 0.736a 0.646a 0.691a
Ac20-3 0.709a 0.617ab 0.634ab
Ac31-1 0.437cd 0.361de 0.388de
Ac50-1 0.322d 0.254e 0.269e
SD (±) 0.030 0.028 0.008
CD 0.021 0.061 0.050
CV (%) 5.99 5.53 2.93
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
119
120
121
Table 17a: The cry1Ac transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
Event ID cry1Ac transcript level in leaf tissue
(ng/µl)
cry1Ac transcript level in flower tissue
(ng/µl)
cry1Ac transcript level in pod tissue
(ng/µl)
Ac29-3 056.3f 049.6f 052.1f
Ac31-2 102.5d 098.5d 102.1d
Ac29-1 120.6c 105.2c 110.4c
Ac16-1 045.2g 023.9g 025.8g
Ac20-2 165.1a 149.5a 152.4a
Ac20-3 156.9b 145.3b 149.3b
Ac31-1 075.6e 057.4e 061.0e
Ac50-1 024.6h 015.6h 018.3h
SD (±) 3.358 3.005 2.916
CD 0.943 0.973 0.941
CV (%) 3.59 3.72 3.47
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
122
developed transgenic plants (p ≥ 0.31). The correlation analysis between Cry1Ac protein
accumulation and transgene transcript levels showed positive correlation (r2=0.988) (Fig.
8). The correlation analysis of larval mortality, Cry1Ac protein and cry1Ac transcript
revealed strong positive in leaf, flower and pod tissues for these traits of T3 generation
transgenic lines (Table 17b).
4.3.8 Southern blot analysis
The Southern blotting was performed to study the integration pattern of T-DNA in
to plant genome, for which from three selected transgenic plants genomic DNA was
digested. It was performed with HindIII as the enzyme that cuts in the T-DNA region and
probed with 600 bp cry1Ac PCR product. An observation of strong signal and the
difference in the hybridization pattern in the three selected transgenic plants viz., Ac29-1,
Ac20-2 and Ac20-3, revealed the single copy integration in these plants (Plate 7).
Whereas, there was no hybridization signal noticed with the DNA of non-transformed
plants. Interestingly, in case of Ac20-2 and Ac20-3 the hybridization signals revealed
similar kind of banding pattern in both transgenic lines.
4.3.9 Northern blot analysis
The presence of cry1Ac transcript in transgenic pigeonpea plants was also
confirmed with northern blotting using 600 bp cry1Ac PCR product as a probe (Plate 8).
The hybridization signals were noticed in all selected transgenic plant samples viz.,
Ac31-2, Ac29-1, Ac31-1, Ac29-3, Ac20-2 and Ac20-3. The observation of strong signals
revealed the production of transcripts in respective transgenic plants.
4.3.10 Juncture region analysis of T-DNA integration
The site of insertion of T-DNA/cry1Ac in the pigeonpea genome was identified by
recovering the genomic sequence flanking the left border (LB) of T-DNA by TAIL-PCR in
transgenic plants. Primary TAIL-PCR with 35S revers and arbitrary primer AD-3
produced a smear in PCR product. The multiple bands were noticed in secondary TAIL-
PCR when amplified product was electrophoresed on one per cent agarose gel.
However, there was a reduction in number of multiple bands produced with development
of one to more thick bands in the tertiary TAIL-PCR (Plate 9). The bright bands with
more than 1.5 kb amplicon size were eluted and cloned into pTZ57R/T and sequenced.
123
Table 17b: The correlation analysis of insect mortality levels, estimated Cry1Ac protein and cry1Ac transcript in leaf, flower and pod
tissues of T3 generation plants (αααα=0.01).
Insect mortality
in leaf tissues
Insect mortality in flower tissues
Insect mortality
in pod tissues
Cry1Ac protein in
leaf tissues
Cry1Ac protein in
flower tissues
Cry1Ac protein in
pod tissues
cry1Ac transcript
level in leaf tissue
cry1Ac transcript
level in flower tissue
cry1Ac transcript
level in pod tissue
Insect mortality in leaf tissues
1 0.957** 0.935** 0.909** 0.890** 0.904** 0.887** 0.868** 0.867**
Insect mortality in flower tissues
1 0.982** 0.965** 0.952** 0.953** 0.950** 0.941** 0.941**
Insect mortality in pod tissues
1 0.950** 0.941** 0.944** 0.930** 0.925** 0.923**
Cry1Ac protein in leaf tissues
1 0.996** 0.996** 0.994** 0.991** 0.990**
Cry1Ac protein in flower tissues
1 0.998** 0.995** 0.991** 0.990**
Cry1Ac protein in pod tissues
1 0.995** 0.990** 0.989**
cry1Ac transcript level in leaf tissue
1 0.993** 0.993**
cry1Ac transcript level in flower tissue
1 1.000**
cry1Ac transcript level in pod tissue
1
**Correlation is significant at the 0.01 level (two tailed)
124
125
Sequence of all the three lines viz., Ac20-2, Ac20-3 and Ac29-1 were analysed by using
BioEdit bioinformatics algorithm.
The obtained sequences (forward and reverse) were assembled and contigues
were formed. Prior to that the contigues were assembled with available T-DNA left
border sequences and T-DNA backbone was removed. The local blast was set for these
processed contigue sequences and it was noticed that flaking genomic region recovered
from Ac20-2 showed 99 per cent homology (268 bp of 270 bp) with Scaffold130851 of
pigeonpea genome (Table 18). It was interesting to notice that flaking genomic region
recovered from Ac20-3 also showed 100 per cent homology (311 bp of 311 bp) with
Scaffold130851 of pigeonpea genome. On other hand, in case of Ac29-1, the recovered
flaking genomic region indicated 100 per cent homology (85 bp of 85 bp) with
Scaffold137204 of pigeonpea draft genome.
4.4 Generation of transgenic pigeonpea conferring expression of cry2Aa gene
The in planta transformation method was employed for development of transgenic
plants carrying cry2Aa gene. The developed pigeonpea transformants were forwarded to
advanced generations (up to T3 generation). The characterization of transgenic lines
carrying cry2Aa gene was performed by their bioefficacy analysis against H. armigera
larvae and quantitative Cry2Aa protein analysis using ELISA. The transgene segregation
pattern was analysed using cry2Aa gene specific PCR assay. The selected transgenic
lines were subjected for molecular characterization using qRT-PCR for cry2Aa transcript
level, Southern and northern blot, and juncture region analysis using TAIL-PCR assay.
4.4.1 Development of transgenic pigeonpea carrying cry2Aa gene
A set of 600 explants were subjected for A. tumefaciens infection using
A. tumefaciens strain carrying cry2Aa gene construct with nptII as marker gene (Fig. 9).
A total of 348 primary transformants were established in transgenic containment facility
(Plate 10). All the primary transformants were allowed to grow till plant maturity and T1
126
Table 18: Juncture region analysis of cry1Ac cassette in AC20-2, AC20-3 and AC29-1 transgenic lines as revealed by TAIL-PCR analysis
Transgenic lines
Pigeonpea contig Position (bp) Query coverage E value
Ac20-2 Scaffold130851 37339-37340 99% (268/270 bp) e-143
Ac20-3 Scaffold130851 37339-37340 100% (311/311 bp) e-175
Ac29-1 Scaffold137204 398597-398598 100% (85/85bp) 1e-040
127
seeds were collected. The plant to row progeny screening in T1 generation, using cry2Aa
gene specific PCR assay identified a set of sixty five putative transformants carrying
cry2Aa gene (Table 19) (Plate 11). The identified putative transformants were grown till
maturity and T2 seeds were harvested.
4.4.2 The cry2Aa gene segregation analysis in T2 generation progenies
The collected T2 seeds were sown in plant to row progeny manner and T2
generation progenies were raised (Plate 10). The chi-square analysis of observed
number of plants with and without cry2Aa gene indicated that among sixty five transgenic
lines, sixteen were following 3:1 (positive: negative) ration for transgene segregation
(Table 20) (Plate 11). Whereas, in case of rest forty nine transgenic lines the chi-square
value was observed to be more than the table value (3.84), clearly indicating that the
cry2Aa gene segregation in those transgenic plant progenies did not follow 3:1
segregation pattern for transgene in T2 generation.
4.4.3 Characterization of developed transgenic lines in T2 generation
The insect bioassay in case of cry2Aa transgenic plants showed significant
variability in larval mortality in different tissues viz., leaf, flower and pod. It was noticed
that the larval mortality in case of leaf tissues ranged from 5.25% to 65.75% (Table 21).
On other hand, larval mortality was between 5.25% to 40.5% in case of flower tissues
and 10.25% to 50.50% in case of pod tissue. It was interesting to notice that the majority
of transformants were having insect mortality between 30.0% - 40.0% (Fig. 10). The
highest larval mortality was recorded in 21A12-24 (65.75%) transgenic line for leaf
tissues, in 21A3-4 and 21A6-12 transgenic line (40.50%) for flower and in 1A2-1
(50.50%) for pod tissues. It was noticed that the larval feeding was highest in transgenic
line 21A5-11, which was comparable with non-transgenic plant. The larval feeding
pattern on different tissue types of transgenic and non-transgenic control plants is shown
in Plate 12. The transgenic lines, 21A4-21, 21A12-26, 1A2-30, 1A2-42 were found
consistent in their performance across different tissue types. The statistical analysis of
variance/ANOVA indicated that there was significant difference in observed insect
mortality levels of different transgenic lines. The ‘t’-test analysis of larval mortality in
different tissue types indicated significant difference between mortality levels in leaf and
flower, and leaf and pod, and between flower and pod tissues types (p < 0.01).
128
129
Table 19: Summary of transformation carried out using cry2Aa gene in pigeonpea
Sl. No.
Number of explants co-
cultivated/set
Number of explants
responded
Number of primary
transformants (T0)
established in greenhouse
Number of primary
transformants tested in plant-to-row progeny for
identifying putative
transformants
Number of putative
transformants identified (T1)
1 50 38 30 30 6
2 50 35 20 20 3
3 50 31 30 30 0
4 50 35 32 32 7
5 50 33 31 31 0
6 50 39 25 25 5
7 50 36 29 29 12
8 50 35 33 33 6
9 50 37 35 35 0
10 50 34 27 27 12
11 50 34 31 31 11
12 50 32 25 25 3
Total 600 419 348 348 65
130
131
Table 20: The transgene segregation pattern in sixty five transformants carrying cry2Aa in T2 generation revealed by gene specific PCR assay
Plant ID
Number of plants tested using gene specific
PCR
Number of plants positive using gene specific
PCR
Number of plants negative using gene specific
PCR
Expected number of
positive plants
Expected number of negative
plants
Calculated chi-square
Gene segregation in
3:1 ration
1A2-1 36 24 12 27.00 09.00 01.33 Yes 21A2-2 32 22 10 24.00 08.00 00.67 Yes 21A2-3 35 17 18 26.25 08.75 13.04 No 21A3-4 40 25 15 30.00 10.00 03.33 Yes 21A3-5 38 18 20 28.50 09.50 15.47 No 21A6-6 34 20 14 25.50 08.50 04.75 No 21A6-7 37 20 17 27.75 09.25 08.66 No 21A6-8 34 15 19 25.50 08.50 17.29 No 21A6-9 29 20 09 21.75 07.25 00.56 Yes 21A5-10 30 21 09 22.50 07.50 00.40 Yes 21A5-11 38 20 18 28.50 09.50 10.14 No 21A6-12 40 25 15 30.00 10.00 03.33 Yes 21A6-13 40 17 23 30.00 10.00 22.53 No 21A5-14 34 23 11 25.50 08.50 00.98 Yes 21A5-15 39 20 19 29.25 09.75 11.70 No 21A5-16 36 22 14 27.00 09.00 03.70 Yes 21A5-17 40 16 24 30.00 10.00 26.13 No 21A5-18 33 19 14 24.75 08.25 05.34 No 21A5-19 36 22 14 27.00 09.00 03.70 Yes 21A4-20 35 17 18 26.25 08.75 13.04 No 21A4-21 40 24 16 30.00 10.00 04.80 No 21A12-24 33 21 12 24.75 08.25 02.27 Yes 21A12-25 40 28 12 30.00 10.00 00.53 Yes
(Table chi-square = 3.84) Cont…
132
Plant ID
Number of plants tested using gene specific
PCR
Number of plants positive using gene specific
PCR
Number of plants negative using gene specific
PCR
Expected number of
positive plants
Expected number of negative
plants
Calculated chi-square
Gene segregation in
3:1 ration
21A12-26 37 20 17 27.75 09.25 08.66 No 21A12-27 36 20 16 27.00 09.00 07.26 No 1A22-28 34 16 18 25.50 08.50 14.16 No 1A22-29 39 18 21 29.25 09.75 17.31 No 1A2-30 40 17 23 30.00 10.00 22.53 No 21A6-31 38 20 18 28.50 09.50 10.14 No 21A6-32 36 19 17 27.00 09.00 09.48 No 21A6-33 37 16 21 27.75 09.25 19.90 No
2A22-39 33 16 17 24.75 08.25 12.37 No
1A2-40 39 19 20 29.25 09.75 14.37 No 1A2-41 40 24 16 30.00 10.00 04.80 No 1A2-42 36 21 15 27.00 09.00 05.33 No 21A6-43 37 20 17 27.75 09.25 08.66 No
21A4-50 35 19 16 26.25 08.75 08.01 No 21A4-51 37 19 18 27.75 09.25 11.04 No 21A4-52 38 18 20 28.50 09.50 15.47 No 21A5-53 39 20 19 29.25 09.75 11.70 No 21A5-57 39 22 17 29.25 09.75 07.19 No 21A5-58 34 11 23 25.50 08.50 32.98 No 21A5-59 34 24 10 25.50 08.50 00.35 Yes
21A5-60 33 21 12 24.75 08.25 02.27 Yes 21A4-61 36 20 16 27.00 09.00 07.26 No 21A4-62 40 28 12 30.00 10.00 00.53 Yes 21A4-63 40 20 20 30.00 10.00 13.33 No
(Table chi-square = 3.84) Cont…
133
Plant ID
Number of plants tested using gene specific
PCR
Number of plants positive using gene specific
PCR
Number of plants negative using gene specific
PCR
Expected number of positive plants
Expected number of negative
plants
Calculated chi-square
Gene segregation in
3:1 ration
21A4-64 38 19 19 28.50 09.50 12.67 No
21A4-65 40 22 18 30.00 10.00 08.53 No
21A4-66 31 14 17 23.25 07.75 14.72 No
21A4-67 34 06 28 25.50 08.50 59.65 No
21A6-68 26 19 07 19.50 06.50 00.05 Yes
21A6-69 40 21 19 30.00 10.00 10.80 No
21A6-70 27 04 23 20.25 06.75 52.16 No
21A6-71 33 09 24 24.75 08.25 40.09 No
21A4-72 39 20 19 29.25 09.75 11.70 No
21A4-73 36 22 14 27.00 09.00 03.70 Yes
21A4-74 40 17 23 30.00 10.00 22.53 No
21A4-75 40 21 19 30.00 10.00 10.80 No
21A4-76 36 13 23 27.00 09.00 29.04 No
21A4-77 39 20 19 29.25 09.75 11.70 No
21A4-78 34 09 25 25.50 08.50 42.71 No
21A12-79 40 21 19 30.00 10.00 10.80 No
21A12-80 38 21 17 28.50 09.50 07.89 No
21A12-81 35 18 17 26.25 08.75 10.37 No
(Table chi-square = 3.84)
134
Table 21: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving sixty five putative transformants carrying cry2Aa gene in T2 generation
Plant ID Per cent corrected mortality
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
1A2-1 55.25bc 35.50ab 50.50a 21A12-25 60.25ab 30.25bc 45.25b
21A2-2 60.50ab 35.25ab 45.50b 21A12-26 30.75de 25.75cd 30.75e
21A2-3 25.50ef 20.25de 25.50f 21A12-27 20.25fg 10.25fgh 20.25g
21A3-4 60.25ab 40.50a 50.25a 1A22-28 25.50ef 10.75fg 20.75g
21A3-5 25.25ef 20.50de 25.75f 1A22-29 30.50de 15.25ef 25.25f
21A6-6 35.50d 25.50cd 30.25e 1A2-30 25.25ef 20.50de 25.50f
21A6-7 25.50ef 15.25ef 25.75f 21A6-31 35.25d 20.50de 30.50e
21A6-8 30.50de 30.75bc 35.25d 21A6-32 25.50ef 20.25de 25.25f
21A6-9 65.25a 35.25ab 50.50a 21A6-33 30.50de 15.25ef 25.25f
21A5-10 55.75bc 30.75bc 40.50c 2A22-39 25.50ef 15.50ef 25.50f
21A5-11 05.25h 05.25i 10.25i 1A2-40 35.25d 10.50fg 20.50g
21A6-12 60.75ab 40.50a 50.25a 1A2-41 25.75ef 10.25fgh 20.25g
21A6-13 25.25ef 15.50ef 20.50g 1A2-42 30.25de 25.25cd 30.25e
21A5-14 55.50bc 30.25bc 40.50c 21A6-43 25.75ef 15.50ef 30.50e
21A5-15 15.50g 05.25hi 10.25i 21A4-50 30.25de 20.50de 35.50d
21A5-16 55.25bc 20.50de 35.25d 21A4-51 30.50de 15.50ef 25.50f
21A5-17 30.25de 15.50ef 30.50e 21A4-52 35.50d 10.25egh 20.50g
21A5-18 20.50fg 15.25ef 20.50g 21A5-53 30.25de 15.75ef 25.50f
21A5-19 60.50ab 30.25bc 40.50c 21A5-57 30.25de 15.25ef 30.50e
21A4-20 25.50ef 20.50de 30.50e 21A5-58 25.50ef 10.75fg 15.25h
21A4-21 30.25de 25.50cd 30.50e 21A5-59 55.50bc 30.25bc 45.75b
21A12-24 65.75a 25.50cd 40.50c 21A5-60 50.50c 35.50ab 45.25b
Note: The means followed with same letters are within student ‘t’ range at α=0.05. Cont…
135
Plant ID Per cent corrected mortality
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
21A4-61 30.25de 15.50ef 25.75f 21A4-72 35.50d 15.25ef 25.50f
21A4-62 55.75bc 35.25ab 45.25b 21A4-73 60.25ab 30.25bc 40.50c
21A4-63 35.25d 20.25de 30.50e 21A4-74 05.75h 05.50ghi 15.25h
21A4-64 35.75d 15.50ef 25.50f 21A4-75 35.25d 10.50fg 20.75g
21A4-65 25.25ef 10.25fgh 20.25g 21A4-76 30.75de 15.25ef 25.25f
21A4-66 30.50de 15.75ghi 20.25g 21A4-77 25.25ef 15.25ef 20.50g
21A4-67 20.50fg 10.25fgh 20.50g 21A4-78 35.50d 20.50de 30.50e
21A6-68 65.25a 35.75ab 45.50b 21A12-79 35.50d 20.50de 25.25f
21A6-69 35.25d 15.25ef 25.25f 21A12-80 35.25d 10.25fgh 20.25g
21A6-70 35.50d 15.50ef 30.25e 21A12-81 30.25de 15.50ef 25.50f
21A6-71 25.50ef 15.50ef 25.50f
SD (±) 0.021 0.026 0.012
CD 0.005 0.007 0.003
CV (%) 3.43 4.74 2.11
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
136
137
138
The significant variation in Cry2Aa protein levels of different transgenic lines
irrespective of tissue types was revealed through ELISA assay (Plate 13). It was
observed that the Cry2Aa protein level in leaf tissue ranged from 0.013 to 3.231 µg/g of
fresh leaf tissue (Table 22a). Whereas, it was 0.010 to 2.854 µg/g in case of fresh flower
tissues and 0.012 to 2.811 µg/g in case of fresh pod tissue. Further, it was noticed that
the majority of putative transformants were accumulating the Cry2Aa protein from 0.0 to
0.5 µg/g of fresh tissue (Fig. 10). The highest Cry2Aa protein accumulation was recorded
in 21A12-24 transgenic line in both leaf and flower tissues, whereas in case of pod tissue
it was more in 21A4-73. On other hand, the lower level of Cry2Aa protein was recorded
in transgenic line 21A5-11 for all tissues types. The analysis of variance indicated
significant difference between Cry2Aa protein levels of different transgenic lines (p <
0.01). Interestingly, the ‘t’-test analysis of Cry2Aa protein accumulation in different tissue
types indicated non-significant difference between Cry protein levels among them (p >
0.45). There was a positive correlation between insect morality and Cry2Aa protein
accumulation with r2=0.8171 (Fig. 11). Similarly, the frequency distribution analysis of
transgenic plants following 3:1 transgene segregation pattern and other than 3:1
segregation pattern showed that majority of high Cry2Aa protein accumulating transgenic
plants were following 3:1 transgene segregation pattern (Fig. 11). The correlation
analysis of insect mortality and Cry2Aa protein levels in different tissues types of
developed transgenic plants indicated the positive correlation among them at α =0.01
(Table 22b).
4.4.4 The cry2Aa gene segregation analysis in T3 generation progenies
Based on the observation of transgene segregation analysis, bioefficacy test and
Cry2Aa protein accumulation in T2 generation progenies, a set of fifteen transgenic lines
were selected and advanced in to T3 generation. The transgenic lines advanced to T3
generation were 21A2-2, 21A3-4, 21A6-9, 21A5-10, 21A6-12, 21A5-14, 21A5-16, 21A5-
19, 21A12-24, 21A12-25, 21A5-59, 21A5-60, 21A4-62, 21A6-68 and 21A4-73, wherein
transgene reported 3:1 segregation pattern, comparatively high larval mortality and
Cry2Aa protein accumulation. The T3 seeds collected from T2 plants were sown in plant
139
140
Table 22a: The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2
generation as revealed by ELISA assay (α=0.05)
Plant ID Cry2Aa protein level (µg/g FW)
Plant ID Cry2Aa protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
1A2-1 1.320 0.969 1.001 21A12-25 2.387 1.859 1.958
21A2-2 2.704 1.981 2.014 21A12-26 0.315 0.224 0.234
21A2-3 0.180 0.121 0.145 21A12-27 0.140 0.096 0.091
21A3-4 2.708 2.341 2.258 1A22-28 0.023 0.025 0.024
21A3-5 0.557 0.485 0.491 1A22-29 0.258 0.159 0.210
21A6-6 0.446 0.315 0.325 1A2-30 0.038 0.029 0.031
21A6-7 0.777 0.419 0.108 21A6-31 0.338 0.289 0.275
21A6-8 0.835 0.589 0.612 21A6-32 0.098 0.089 0.075
21A6-9 2.913 2.124 2.302 21A6-33 0.173 0.124 1.350
21A5-10 2.550 2.152 2.189 2A22-39 0.159 0.128 0.124
21A5-11 0.013 0.010 0.012 1A2-40 0.214 0.189 0.191
21A6-12 2.003 1.485 1.541 1A2-41 0.104 0.095 0.099
21A6-13 0.048 0.051 0.045 1A2-42 0.192 0.124 0.141
21A5-14 1.950 1.428 1.521 21A6-43 0.140 0.115 0.121
21A5-15 0.085 0.059 0.069 21A4-50 0.088 0.074 0.079
21A5-16 1.833 1.305 1.285 21A4-51 0.133 0.110 0.101
21A5-17 0.491 0.298 0.324 21A4-52 0.476 0.296 0.301
21A5-18 0.124 0.099 0.102 21A5-53 0.136 0.110 0.128
21A5-19 2.204 1.897 1.793 21A5-57 0.186 0.128 0.134
21A4-20 0.138 0.990 0.105 21A5-58 0.124 0.112 0.108
21A4-21 0.305 0.199 0.201 21A5-59 2.076 1.856 1.812
21A12-24 3.231 2.854 2.795 21A5-60 1.782 1.412 1.329
Cont…
141
Plant ID Cry2Aa protein level (µg/g FW)
Plant ID Cry2Aa protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues
21A4-61 0.157 0.124 0.119 21A4-72 0.062 0.051 0.058
21A4-62 2.161 1.854 1.799 21A4-73 3.080 2.749 2.811
21A4-63 0.126 0.110 0.109 21A4-74 0.090 0.074 0.081
21A4-64 0.189 0.125 0.154 21A4-75 0.087 0.078 0.064
21A4-65 0.131 0.110 0.124 21A4-76 0.042 0.041 0.038
21A4-66 0.121 0.102 0.113 21A4-77 0.120 0.110 0.118
21A4-67 0.143 0.121 0.119 21A4-78 0.077 0.061 0.068
21A6-68 3.112 2.458 2.621 21A12-79 0.170 0.149 0.157
21A6-69 0.198 0.154 0.161 21A12-80 0.133 0.124 0.119
21A6-70 0.191 0.148 0.156 21A12-81 0.103 0.098 0.100
21A6-71 0.095 0.068 0.071
SD (±) 0.016 0.016 0.017
CD 0.002 0.001 0.001
CV (%) 2.04 2.63 2.52
142
143
Table 22b: The correlation analysis of insect mortality levels and estimated Cry2Aa protein in leaf, flower and pod tissues of T2
generation plants (αααα=0.01)
Insect mortality in leaf tissues
Insect mortality in flower tissues
Insect mortality in pod tissues
Cry2Aa protein in leaf
tissues
Cry2Aa protein in flower tissues
Cry2Aa protein in pod
tissues
Insect mortality in leaf tissues
1 0.821** 0.890** 0.904** 0.884** 0.886**
Insect mortality in flower tissues
1 0.945** 0.795** 0.781** 0.767**
Insect mortality in pod tissues
1 0.834** 0.820** 0.806**
Cry2Aa protein in leaf tissues
1 0.985** 0.977**
Cry2Aa protein in flower tissues
1 0.971**
Cry2Aa protein in pod tissues
1
**Correlation is significant at the 0.01 level (two trials).
144
to row progeny manner. The gene specific PCR assay identified T3 plant progenies
carrying cry2Aa gene. The chi-square test analysis of observed number of plants with
and without transgene indicated that majority of plants were heterozygous in nature for
cry2Aa locus. Among fifteen transgenic lines, in four transgenic lines homozygous nature
of cry2Aa locus was detected. The T2 parental plants with homozygous nature were
21A2-2-1, 21A3-4-7, 21A5-16-1 and 21A4-62-6 (Plate 11) (Table 23).
4.4.5 Characterization of fifteen transgenic lines carrying cry2Aa gene in T3 generation
The bioefficacy analysis of T3 generation transgenic lines revealed consistent
nature of their efficacy against neonate larvae of Helicoverpa armigera. The bioassay
results reported that the larval mortality in case of leaf tissue, which ranged from 43.33 to
68.33 per cent. The larval mortality ranges in case of flower tissue were 23.5 to 48.5 per
cent and in case of pod it ranged from 28.5 to 53.25 per cent (Table 24a). The highest
larval mortality recorded in case of transgenic line 21A6-68 for all tissue types. On other
hand, least larval mortality was noticed in transgenic line 21A5-16. The analysis of
variance showed that the transgenic lines differ significantly (p < 0.01) for larval mortality
in all tissue types. The ‘t’-test analysis of larval mortality levels in different tissue types
indicated significant difference between mortality levels in leaf and flower (p ≤ 0.001) and
in leaf and pod tissues (p ≤ 0.001). It was interesting to notice that the larval mortality
levels between flower and pod were on par with each other with p ≥ 0.06. The ‘t’-test
analysis of mortality levels in T2 and T3 generations for developed transgenic plants
carrying cry2Aa gene reported non-significant difference in insect mortalities between
two generations for all tissue types (p ≥ 0.08) (Table 24b).
The similar pattern of consistent Cry2Aa protein accumulation was noticed in
selected fifteen transgenic lines in T3 generation. The Cry2Aa protein levels in leaf
tissues was recorded and ranged from 0.370 to 1.417 µg/g of fresh tissue weight (Table
25). The Cry2Aa protein accumulation in flower tissues was noticed ranged from 0.312 to
1.021 µg/g and in pod it ranged from 0.309 to 0.989 µg/g. The higher level of Cry2Aa
proteins was recorded in 21A6-68 transgenic line irrespective of tissue type. On other
hand, the Cry2Aa accumulation was lowest in transgenic line 21A4-62. The statistical
analysis indicated that the transgenic lines were differencing significantly for Cry2Aa
145
Table 23: Transgene segregation pattern in fifteen transgenic lines carrying cry2Aa gene in T3 generation
Plant ID
Total number of progenies
tested
Number of PCR positive plants
Number of PCR
negative plants
Expected number of
positive plants
Expected number of negative plants
Calculated chi-square
Estimated plant
zygosity
A2-2-1 16 16 00 11.25 3.75 05.00 Homo.
A2-2-5 16 06 10 12.00 4.00 12.00 Hetero.
A2-2-7 15 10 05 11.25 3.75 00.56 Hetero.
A2-2-8 15 11 04 11.25 3.75 00.02 Hetero.
A2-2-9 19 14 05 14.25 4.75 00.02 Hetero.
A2-2-12 12 09 03 09.00 3.00 00.00 Hetero.
A3-4-2 16 15 01 12.00 4.00 03.00 Hetero.
A3-4-4 12 08 04 09.00 3.00 00.44 Hetero.
A3-4-5 19 13 06 14.25 4.75 00.44 Hetero.
A3-4-7 17 17 00 12.75 4.25 05.67 Homo.
A3-4-8 17 10 07 12.75 4.25 02.37 Hetero.
A3-4-9 15 11 04 11.25 3.75 00.02 Hetero.
A6-9-3 17 11 06 12.75 4.25 00.96 Hetero.
A6-9-4 21 14 07 15.75 5.25 00.78 Hetero.
A6-9-6 19 11 08 14.25 4.75 02.96 Hetero.
A6-9-9 20 08 12 15.00 5.00 13.07 Hetero.
A6-9-10 10 02 08 07.50 2.50 16.13 Hetero.
A6-9-12 13 08 05 09.75 3.25 01.26 Hetero.
A5-10-1 11 07 04 08.25 2.75 00.76 Hetero.
A5-10-4 13 08 05 09.75 3.25 01.26 Hetero.
A5-10-5 14 06 08 10.50 3.50 07.71 Hetero.
A5-10-8 12 07 05 09.00 3.00 01.78 Hetero.
A5-10-13 10 06 04 07.50 2.50 01.20 Hetero.
A5-10-14 14 08 06 10.50 3.50 02.38 Hetero.
A6-12-1 15 11 04 11.25 3.75 00.02 Hetero.
A6-12-2 14 08 06 10.50 3.50 02.38 Hetero.
A6-12-3 15 09 06 11.25 3.75 01.80 Hetero.
A6-12-4 15 10 05 11.25 3.75 00.56 Hetero.
A6-12-8 16 07 09 12.00 4.00 08.33 Hetero.
A6-12-9 14 09 05 10.50 3.50 00.86 Hetero.
A5-14-1 18 14 04 13.50 4.50 00.07 Hetero.
A5-14-2 15 12 03 11.25 3.75 00.20 Hetero.
A5-14-4 17 12 05 12.75 4.25 00.18 Hetero.
A5-14-5 14 10 04 10.50 3.50 00.10 Hetero.
A5-14-6 16 12 04 12.00 4.00 00.00 Hetero.
A5-14-10 14 08 06 10.50 3.50 02.38 Hetero.
Contd…
146
Plant ID
Total number
of progenies
tested
Number of PCR positive plants
Number of PCR
negative plants
Expected number
of positive plants
Expected number
of negative plants
Calculated chi-square
Estimated plant
zygosity
A5-16-1 15 15 00 11.25 3.75 05.00 Homo.
A5-16-2 18 16 02 13.50 4.50 01.85 Hetero.
A5-16-3 12 08 04 09.00 3.00 00.44 Hetero.
A5-16-4 14 10 04 10.50 3.50 00.10 Hetero.
A5-16-11 16 11 05 12.00 4.00 00.33 Hetero.
A5-16-12 14 08 06 10.50 3.50 02.38 Hetero.
A5-19-4 12 08 04 09.00 3.00 00.44 Hetero.
A5-19-5 14 08 06 10.50 3.50 02.38 Hetero.
A5-19-7 10 06 04 07.50 2.50 01.20 Hetero.
A5-19-8 14 09 05 10.50 3.50 00.86 Hetero.
A5-19-9 13 10 03 09.75 3.25 00.03 Hetero.
A5-19-10 14 08 06 10.50 3.50 02.38 Hetero.
A12-24-1 14 07 07 10.50 3.50 04.67 Hetero.
A12-24-2 15 08 07 11.25 3.75 03.76 Hetero.
A12-24-3 13 06 07 09.75 3.25 05.77 Hetero.
A12-24-4 14 09 05 10.50 3.50 00.86 Hetero.
A12-24-6 15 10 05 11.25 3.75 00.56 Hetero.
A12-24-8 10 06 04 07.50 2.50 01.20 Hetero.
A12-25-3 16 10 06 12.00 4.00 01.33 Hetero.
A12-25-4 15 10 05 11.25 3.75 00.56 Hetero.
A12-25-6 20 13 07 15.00 5.00 01.07 Hetero.
A12-25-7 15 10 05 11.25 3.75 00.56 Hetero.
A12-25-8 14 08 06 10.50 3.50 02.38 Hetero.
A12-25-10 12 08 04 09.00 3.00 00.44 Hetero.
A5-59-7 12 08 04 09.00 3.00 00.44 Hetero.
A5-59-5 12 09 03 09.00 3.00 00.00 Hetero.
A5-59-1 14 09 05 10.50 3.50 00.86 Hetero.
A5-59-6 13 08 05 09.75 3.25 01.26 Hetero.
A5-59-3 12 05 07 09.00 3.00 07.11 Hetero.
A5-59-4 10 05 05 07.50 2.50 03.33 Hetero.
A5-60-2 12 08 04 09.00 3.00 00.44 Hetero.
A5-60-3 17 11 06 12.75 4.25 00.96 Hetero.
A5-60-9 11 09 02 08.25 2.75 00.27 Hetero.
A5-60-10 13 08 05 09.75 3.25 01.26 Hetero.
A5-60-11 06 04 02 04.50 1.50 00.22 Hetero.
A5-60-1 10 07 03 07.50 2.50 00.13 Hetero.
A4-62-10 09 05 04 06.75 2.25 01.81 Hetero.
A4-62-3 12 11 01 09.00 3.00 01.78 Hetero.
A4-62-2 14 08 06 10.50 3.50 02.38 Hetero.
A4-62-7 12 08 04 09.00 3.00 00.44 Hetero.
A4-62-6 11 11 00 08.25 2.75 03.67 Homo.
A4-62-8 09 06 03 06.75 2.25 00.33 Hetero.
Cont…
147
Plant ID
Total number
of progenies
tested
Number of PCR positive plants
Number of PCR
negative plants
Expected number
of positive plants
Expected number
of negative plants
Calculated chi-square
Estimated plant
zygosity
A6-68-4 12 08 04 09.00 3.00 00.44 Hetero.
A6-68-1 14 09 05 10.50 3.50 00.86 Hetero.
A6-68-10 12 08 04 09.00 3.00 00.44 Hetero.
A6-68-4 15 11 04 11.25 3.75 00.02 Hetero.
A6-68-11 14 10 04 10.50 3.50 00.10 Hetero.
A6-68-2 13 09 04 09.75 3.25 00.23 Hetero.
A4-73-7 15 10 05 11.25 3.75 00.56 Hetero.
A4-73-3 10 08 02 07.50 2.50 00.13 Hetero.
A4-73-5 12 08 04 09.00 3.00 00.44 Hetero.
A4-73-4 12 07 05 09.00 3.00 01.78 Hetero.
A4-73-1 11 07 04 08.25 2.75 00.76 Hetero.
A4-73-2 14 08 06 10.50 3.50 02.38 Hetero.
Where; Hetero.: Heterozygous; Homo.: Homozygous
148
Table 24a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fifteen putative transformants carrying cry2Aa gene in T3 generation
Event ID Corrected per cent larval mortality
Leaf tissues Flower tissues Pod tissues
21A2-2 58.33abc 43.50ab 50.50a
21A3-4 58.33abc 43.00ab 49.00a
21A6-9 63.33ab 43.00ab 45.00ab
21A5-10 48.33cd 38.00abc 43.00abc
21A6-12 53.33bcd 33.50bcd 36.50bcd
21A5-14 48.33cd 28.00de 32.00d
21A5-16 48.33cd 23.50e 28.50d
21A5-19 58.33abc 48.50a 52.50a
21A12-24 68.33a 43.25ab 47.25a
21A12-25 58.33abc 38.50abc 44.50ab
21A5-59 48.33cd 30.50cde 34.50cd
21A5-60 43.33d 28.00de 32.00d
21A4-62 43.33d 28.25de 33.25cd
21A6-68 68.33a 48.25a 53.25a
21A4-73 53.33bcd 28.50de 34.50cd
SD (±) 0.024 0.063 0.063
CD 0.004 0.012 0.012
CV (%) 3.22 6.75 5.84
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
149
Table 24b: The ‘t’-test analysis of mortality levels from T2 and T3 generation of fifteen
cry2Aa transgenic lines (α=0.05)
Leaf tissues Flower tissues Pod tissues
T2
generation T3
generation T2
generation T3
generation T2
generation T3
generation
Mean 57.15 54.66 31.75 36.42 42.73 41.08
Variance 52.12 65.95 30.33 68.42 28.08 70.77
Observations 15 15 15 15 15 15
Pooled Variance 59.04 49.37 49.43
Hypothesized Mean Difference 0 0 0
Df 28 28 28
t Stat 0.89 -1.82 0.64
P(T<=t) two-tail 0.38 0.08 0.53
t Critical two-tail 2.05 2.05 2.05
150
Table 25: The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay
Event ID Cry2Aa protein level (µg/g FW)
Leaf tissue Flower tissue Pod tissue
21A2-2 1.046d 0.910b 0.915c
21A3-4 0.975e 0.825c 0.812d
21A6-9 1.170b 0.914b 0.931b
21A5-10 0.667i 0.589h 0.605i
21A6-12 0.829g 0.712f 0.728g
21A5-14 0.609j 0.548i 0.543j
21A5-16 0.485k 0.401j 0.398k
21A5-19 0.892f 0.759d 0.782e
21A12-24 1.127c 0.912b 0.939b
21A12-25 0.889f 0.729e 0.742f
21A5-59 0.457l 0.356k 0.371l
21A5-60 0.424m 0.387j 0.392k
21A4-62 0.370n 0.312l 0.309m
21A6-68 1.417a 1.021a 0.989a
21A4-73 0.798h 0.685g 0.691h
SD (±) 0.017 0.019 0.018
CD 0.005 0.005 0.004
CV (%) 2.19 2.78 2.65
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
151
protein accumulation with p < 0.001 in case of leaf, flower and pod tissue types.
Interestingly, the ‘t’-test analysis of Cry protein accumulation in different tissue types
indicated that there was significant difference in Cry2Aa protein levels in leaf and flower
(p < 0.08), whereas it was on par between flower and pod tissues (p > 0.07) and
between leaf and pod tissue types (p >0.09). The correlation analysis between insect
morality and Cry2Aa protein accumulation revealed positive correlation between them
with r2=0.8434 (Fig. 12).
4.4.6 The cry2Aa transcript analysis using qRT-PCR assay
The observation of absolute real time analysis of cry2Aa transcripts in different
tissue types of selected fifteen transgenic lines indicated significant variation in cry2Aa
transcript levels (Plate 14). It was noticed that the cry2Aa transcript level ranged from
48.5 to 134.5 ng/µl in fresh leaf tissue of transgenic plants (Table 26a). Similar
observation was noticed in flower tissues where the cry2Aa transcript level ranged from
41.2 to 110.2 ng/µl and in pod from 42.8 to 100.5 ng/µl. The highest transcript level was
recorded in 21A6-68 for leaf and flower tissues and in 21A2-2 for pod. The lower level of
cry2Aa transcripts was recorded in case of transgenic line 21A4-62. The ANOVA
statistical analysis indicated the observation of significant difference between transgene
transcript levels of transgenic lines (p < 0.01). The ‘t’-test analysis of transcript levels
between tissue types revealed non-significant difference in the transcripts levels of
cry2Aa gene in different tissue types within developed transgenic plants (p ≥ 0.11). The
correlation analysis between Cry2Aa protein accumulation and transgene transcript
levels revealed presence of positive correlation (r2=0.9469)
(Fig. 12). The correlation analysis of larval mortality, Cry2Aa protein and cry2Aa
transcript reported positive in leaf, flower and pod tissues for these traits of T3 generation
transgenic lines (Table 26b).
4.4.7 Southern blot analysis
The genomic DNA from four selected transgenic plants was digested using HindIII
restriction endonuclease and Southern blotting was performed to analyse the T-DNA
integration pattern in to plant genome. The blotted digested genomic DNA was probed
with 676 bp labelled cry2Aa PCR product. A single but weak hybridization signals were
noticed for two samples belonging to 21A2-2 and 21A3-4 transgenic lines. The Southern
152
153
154
Table 26a: The cry2Aa transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
Event ID cry2Aa transcript level in leaf tissue
(ng/µl)
cry2Aa transcript level in flower tissue
(ng/µl)
cry2Aa transcript level in pod tissue
(ng/µl)
21A2-2 124.2c 105.1b 100.5a
21A3-4 100.4e 095.6d 094.6b
21A6-9 115.2d 099.8c 095.4b
21A5-10 084.6h 081.5g 078.9d
21A6-12 091.2g 085.4ef 081.6c
21A5-14 078.1i 071.5h 068.9e
21A5-16 054.9j 050.6j 048.6g
21A5-19 098.4e 084.5f 078.2d
21A12-24 128.1b 104.3b 094.6b
21A12-25 095.2f 087.3e 082.4c
21A5-59 054.8j 049.5j 047.2g
21A5-60 052.6k 045.7k 042.9h
21A4-62 048.5l 041.2l 042.8h
21A6-68 134.5a 110.2a 099.8a
21A4-73 086.2h 061.5i 062.9f
SD (±) 2.421 2.734 2.639
CD 0.779 0.774 0.823
CV (%) 2.73 3.49 3.54
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
155
Table 26b: The correlation analysis of insect mortality levels, estimated Cry2Aa protein and cry2Aa transcript in leaf, flower and pod
tissues of T3 generation plants (αααα=0.01)
Insect mortality
in leaf tissues
Insect mortality in flower tissues
Insect mortality
in pod tissues
Cry2Aa protein in
leaf tissues
Cry2Aa protein in
flower tissues
Cry2Aa protein in
pod tissues
cry2Aa transcript
level in leaf tissue
cry2Aa transcript
level in flower tissue
cry2Aa transcript
level in pod tissue
Insect mortality in leaf tissues
1 0.829** 0.811** 0.960** 0.934** 0.935** 0.936** 0.891** 0.863**
Insect mortality in flower tissues
1 0.988** 0.848** 0.852** 0.856** 0.857** 0.873** 0.855**
Insect mortality in pod tissues
1 0.838** 0.848** 0.848** 0.854** 0.866** 0.853**
Cry2Aa protein in leaf tissues
1 0.984** 0.976** 0.973** 0.939** 0.925**
Cry2Aa protein in flower tissues
1 0.998** 0.986** 0.962** 0.959**
Cry2Aa protein in pod tissues
1 0.986** 0.962** 0.958**
cry2Aa transcript level in leaf tissue
1 0.970** 0.958**
cry2Aa transcript level in flower tissue
1 0.994**
cry2Aa transcript level in pod tissue
1
**Correlation is significant at the 0.01 level (two tailed).
156
hybridization pattern was different for both samples. On other hand, there were strong
hybridization signals noticed in other two other samples viz., 21A6-9 and 21A6-68.
Interestingly, the hybridization pattern was same in both the samples, indicated the
possibility of same position of T-DNA integration in plant genome. An observation of
single hybridization signal in all tested transgenic plants indicated the single copy
integration of transgene (Plate 15).
4.4.8 Northern blot analysis
The transcription of cry2Aa gene was further confirmed using northern blot
analysis using 676 bp labelled cry2Aa PCR product as a probe (Plate 16). The
hybridization signals were noticed in all selected transgenic plant samples viz., 21A2-2,
21A3-4, 21A6-9, 21A12-24 and 21A6-68. The observation of strong signals revealed the
transcription of cry2Aa gene in transgenic lines.
4.4.9 Juncture region analysis of T-DNA integration
The site of insertion of T-DNA carrying cry2Aa gene in the pigeonpea genome
was identified by recovering the genomic sequence flanking the left border (LB) of
T-DNA. The smear was noticed in the primary TAIL-PCR product obtained from 35S
revers and arbitrary primer AD-2. The secondary TAIL-PCR reported development of
multiple bands when diluted primary product was used as template. However, there was
a reduction in number of multiple bands produced with development of single thick bands
in the tertiary TAIL-PCR (Plate 17). The bright bands with more than one kb amplicon
size were eluted and cloned into pTZ57R/T and sequenced. Sequence of all the five
lines viz., 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68 were analysed by using
BioEdit bioinformatics algorithm.
The sequences were analysed and detected vector backbone sequences were
trimmed. Such processed forward and revers sequences were assembled and contigues
were formed. The local blast was set using BioEdit tool for processed contigue
sequences and it was observed that flaking genomic region recovered from 21A2-2
showed 100 per cent homology for 89 nucleotide bases of 144 bp with Scaffold137204 of
pigeonpea genome (Table 27). Similarly, the flaking genomic region recovered from
21A3-4 showed 100 per cent homology for 58 bp out of 186 bp and genomic region
157
158
Table 27: Juncture region analysis of cry2Aa cassette in selected transgenic lines as revealed by TAIL PCR analysis
Transgenic lines Pigeonpea contig Position (bp) Query coverage E value
21A5-14 Scaffold137204 398597-398598 89/144 (100%) 4e-043
21A6-7 Scaffold134438 5542-5543 58/186 (100%) 2e-024
21A5-60 Scaffold127179 23885-23886 89/150 (100%) 4e-043
21A3-4 Scaffold127179 23885-23886 85/195 (100%) 8e-036
21A5-10 Scaffold134438 5481-5482 119/243 (99%) 2e-058
recovered from 21A12-24 showed 99 per cent homology for 119 bp out of 243 bp with
Scaffold134438. On other hand, in case of 21A6-9 and 21A6-68, the recovered flaking
159
genomic region indicated 100 per cent homology (89 bp of 150 bp and 85 bp of 195 bp
respectively) with Scaffold127179 of pigeonpea draft genome.
4.5 Generation of transgenic pigeonpea conferring expression of cry1F gene
The transgenic pigeonpea expressing cry1F was developed using improvised in
planta transformation method (Fig. 13). The developed transgenic pigeonpea plants
were characterized for their bioefficacy against H. armigera larvae and quantitative
Cry1F protein analysis using ELISA. The transgene segregation pattern was assessed
using cry1F gene specific PCR assay. The developed transgenic lines were subjected
for cry1F transcript analysis using qRT-PCR, Southern and northern blot analysis.
4.5.1 Development of transgenic pigeonpea carrying cry1F gene
A total of 350 explants were infected with Agrobacterium tumefaciens strain
carrying cry1F gene carrying construct and set of 177 primary transformants were
established, which was considered as T0 generation (Table 28). The T1 seeds were
harvested form well-established primary transformants and T1 generation was raised.
The T1 progeny plant screening using cry1F gene specific PCR assay identified a set of
fourteen putative transgenic plants for integration of cry1F gene (Plate 18). The identified
putative transformants were grown till maturity and T2 seeds were harvested.
4.5.2 The cry1F gene segregation analysis in T2 generation progenies
The obtained T2 seeds were sown in plant to row progeny manner to get T2
generation plants. From each parental T1 generation plant up to 40 seeds were sown
and obtained plants were subjected for gene specific PCR assay. The gene segregation
pattern was assayed using chi-square test. Among developed fourteen, for seven
transgenic plants the chi-square calculated value was recorded less than table chi-
square value (3.84) (Table 29) (Plate 19). The observation of chi-square analysis results
160
Table 28: Summary of transformation carried out using cry1F gene in pigeonpea
Sl. No Number of
explants co-cultivated/set
Number of explants
responded
Number of primary
transformants (T0)
established in greenhouse
Number of primary transformants tested
in plant-to-row progeny for
identifying putative transformants
Number of putative
transformants identified (T1)
1 50 38 25 25 4
2 50 34 20 20 0
3 50 32 26 26 0
4 50 37 30 30 0
5 50 35 28 28 6
6 50 31 25 25 0
7 50 30 23 23 4
Total 350 237 177 177 14
161
162
Table 29: The transgene segregation pattern in fourteen transformants carrying cry1F in T2 generation revealed by gene specific PCR assay
Plant ID
Number of plants tested using gene specific
PCR
Number of plants positive using gene specific
PCR
Number of plants negative using gene specific
PCR
Expected number of positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
1F-22 28 17 11 21.00 7.00 03.05 Yes
1F-32 25 15 10 18.75 6.25 03.00 Yes
1F-38 24 16 08 18.00 6.00 00.89 Yes
1F-19 28 15 13 21.00 7.00 06.86 No
1F-10 24 15 09 18.00 6.00 02.00 Yes
1F-20 27 16 11 20.25 6.75 03.57 Yes
1F-25 27 14 13 20.25 6.75 07.72 No
1F-21 28 12 16 21.00 7.00 15.43 No
1F-37 20 12 08 15.00 5.00 02.40 Yes
1F-9 28 15 13 21.00 7.00 06.86 No
1F-36 25 12 13 18.75 6.25 09.72 No
1F-35 25 12 13 18.75 6.25 09.72 No
1F-33 35 22 13 26.25 8.75 02.75 Yes
1F-45 24 12 12 18.00 6.00 08.00 No
(Table chi-square = 3.84)
163
revealed the 3:1 (positive: negative) cry1F gene segregation pattern in transgenic plant
progenies of 1F-22, 1F-32, 1F-38, 1F-10, 1F-20, 1F-33 and 1F-37 in T2 generation. For
rest seven transgenic plants the chi-square value was noticed more than the table value,
clearly indicating that the cry1F gene segregation in those transgenic plant progenies
was not following 3:1 gene segregation pattern in T2 generation.
4.5.3 Characterization of developed transgenic lines in T2 generation
The bioefficacy of the plants against first instar H. armigera larvae revealed
significant variability in larval mortality and damage in all targeted tissue types viz., leaf,
flower and pod. The larval mortality in case of leaf tissues ranged from 22.5 to 62.5 per
cent (Table 30). It was 20.0 to 57.5 per cent in case of flower tissues and 10.0 to 47.5
per cent in case of pod. It also was noticed that the majority of putative transformants
were having insect mortality from 40.0 to 60.0 per cent in both leaf and flower tissue, with
the highest mortality recorded in 1F-22 transgenic line for leaf and in 1F-33 for flower
(Fig. 14). On other hand, in case of pod, the majority of putative transformants showed
insect mortality from 30.0 to 50.0 per cent with the highest mortality recorded in 1F-22
and 1F-33. The larval feeding was highest in transgenic line 1F-25. The larval feeding
pattern on different tissue types of transgenic and non-transgenic control plants is shown
in Plate 20. The statistical analysis, analysis of variance (ANOVA), indicated that the
transgenic lines differ significantly (p < 0.001) from each other for larval mortality in all
tissue types and for leaf damage. The ‘t’-test analysis of larval mortality in different tissue
types indicated significant difference between mortality levels in leaf and flower (p ≤
0.01), and in flower and pod tissues (p ≤ 0.01). Interestingly, the larval mortality levels
were comparable between leaf and pod tissue (p ≥ 0.9) types as indicated by ‘t’-test
analysis.
There was a significant variation in Cry1F protein levels of different transgenic
lines irrespective of tissue types viz., leaf, flower and pod as revealed through ELISA
assay (Plate 21). The Cry protein level ranged from 0.170 to 1.032 µg/g of fresh leaf
tissue (Table 31). Whereas, it was 0.113 to 0.870 µg/g of fresh flower tissues and 0.117
to 0.782 µg/g of fresh pod tissue. Interestingly, it was noticed that the majority of putative
transformants were accumulating the Cry1F protein from 0.4 to 0.6 µg/g of fresh leaf
tissue, wherein the highest Cry1F proteins was detected in 1F-22 transgenic line (Fig.
12). Similarly, in case of flower and pod tissues, the majority of transgenic plants were
164
Table 30: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fourteen putative transformants carrying cry1F gene in T2 generation.
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues
1F-22 62.5a 55.0a 47.5a
1F-32 57.5ab 50.0ab 42.5abc
1F-38 60.0a 47.5ab 42.5abc
1F-19 47.5c 40.0bc 37.5bc
1F-10 50.0bc 50.0ab 45.0ab
1F-20 47.5c 42.5b 37.5bc
1F-25 22.5e 20.0d 10.0d
1F-21 47.5c 35.0c 40.0abc
1F-37 57.5ab 55.0a 40.0abc
1F-9 30.0d 25.0d 12.5d
1F-36 47.5c 47.5ab 40.0abc
1F-35 50.0bc 47.5ab 37.5bc
1F-33 60.0a 57.5a 47.5a
1F-45 47.5c 47.5ab 35.0c
SD (±) 0.057 0.060 0.058
CD 0.043 0.047 0.044
CV (%) 5.15 5.42 5.25
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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Table 31: The Cry1F protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay
Plant ID Cry1F protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues
1F-22 1.032a 0.870a 0.782a
1F-32 0.834b 0.557b 0.523b
1F-38 0.683d 0.511c 0.499c
1F-19 0.489g 0.327h 0.301g
1F-10 0.570e 0.412f 0.412d
1F-20 0.552e 0.462d 0.425d
1F-25 0.170j 0.113k 0.117i
1F-21 0.457h 0.301i 0.309g
1F-37 0.556e 0.433e 0.422d
1F-9 0.286i 0.194j 0.181h
1F-36 0.522f 0.394g 0.342f
1F-35 0.470h 0.386g 0.383e
1F-33 0.714c 0.547b 0.513bc
1F-45 0.502g 0.430e 0.410d
SD (±) 0.025 0.027 0.026
CD 0.007 0.006 0.007
CV (%) 4.60 4.47 4.69
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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accumulating Cry1F protein from 0.3 to 0.6 µg/g of fresh tissue. The transgenic line 1F-
22 showed highest accumulation of Cry protein in both flower and pod tissues as well, as
indicated by ELISA assay. On other hand, the lower level of Cry protein was recorded in
transgenic line 1F-25 and 1F-9. The statistical analysis of variance clearly indicated the
significant difference in Cry1F protein levels among developed transgenic lines (p <
0.001). The ‘t’-test analysis of Cry protein accumulation in different tissue types indicated
significant difference in leaf and flower (p ≤ 0.01), in flower and pod tissues (p < 0.03)
and between leaf and pod tissue (p ≤ 0.03) types as indicated by ‘t’-test analysis. There
was a positive correlation between insect morality and Cry1F protein accumulation with
r2=0.781 (Fig. 15). Further, the frequency distribution analysis of transgenic plants
following 3:1 transgene segregation pattern and other than 3:1 segregation pattern
showed that majority of high Cry1F protein accumulating transgenic plants were
following 3:1 transgene segregation pattern (Fig. 15).
4.5.4 The cry1F transcript analysis using qRT-PCR assay
Absolute real time analysis of cry1F transcript levels in different tissue types
indicated significant variation in cry1F transcript levels in developed transgenic lines as
revealed by standard graph (Plate 22). The cry1F transcript level ranged from 47.6 ng/µl
to 105.3 ng/µl in fresh leaf tissue (Table 32). Similarly, the cry1F transcript level in flower
was ranged from 45.2 ng/µl to 101.0 ng/µl and in pod from 48.5 ng/µl to 103.2 ng/µl.
Interestingly, the highest transcript level was detected in all tissue types of 1F-25 and 1F-
9 transgenic line. On other hand, the lower level of cry1F transcripts was recorded in
transgenic line 1F-35. The statistical analysis of variance clearly indicated the significant
difference in cry1F transcript levels among developed transgenic lines (p < 0.001). The
‘t’-test analysis revealed non-significant difference in the transcripts levels of cry1F gene
in different tissue types within developed transgenic plants (p ≥ 0.94). There was a
positive correlation between Cry1F protein accumulation and cry1F transcript levels
(r2=0.0431) (Fig. 15). The correlation analysis of larval mortality, Cry1F protein and cry1F
transcript reported positive in leaf, flower and pod tissues for these traits of T2 generation
transgenic lines (Table 32b).
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Table 32a: The cry1F transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
Event ID cry1F transcript level in leaf tissue (ng/µl)
cry1F transcript level in flower tissue (ng/µl)
cry1F transcript level in pod tissue (ng/µl)
1F-22 105.3c 101.0c 103.2c
1F-32 080.5d 081.2d 079.5d
1F-38 075.1e 073.4e 072.5e
1F-19 060.2h 061.9h 061.9g
1F-10 071.6f 070.8f 070.4e
1F-20 070.5fg 068.1g 072.1e
1F-25 140.2a 137.6a 142.3a
1F-21 059.4h 060.2h 060.1g
1F-37 068.7g 066.9g 068.1f
1F-9 128.2b 130.8b 129.3b
1F-36 054.3i 053.6i 052.0h
1F-35 047.6j 045.2k 048.5i
1F-33 078.5d 079.1d 078.0d
1F-45 049.2j 050.1j 048.6i
SD (±) 1.414 1.212 1.313
CD 0.905 0.889 0.843
CV (%) 1.82 1.52 1.69
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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Table 32b: The correlation analysis of insect mortality levels, estimated Cry1F protein and cry1F transcript in leaf, flower and pod
tissues of T2 generation plants (αααα=0.05; 0.01)
Insect mortality
in leaf tissues
Insect mortality in flower tissues
Insect mortality
in pod tissues
Cry1F protein in
leaf tissues
Cry1F protein in
flower tissues
Cry1F protein in
pod tissues
cry1F transcript
level in leaf tissue
cry1F transcript
level in flower tissue
cry1F transcript
level in pod tissue
Insect mortality in leaf tissues
1 0.625* 0.750** 0.827** 0.780** 0.817** 0.796** 0.791** 0.771**
Insect mortality in flower tissues
1 0.497 0.556 0.647* 0.647* 0.539 0.511 0.524
Insect mortality in pod tissues
1 0.747** 0.664* 0.687* 0.811** 0.821** 0.788**
Cry1F protein in leaf tissues
1 0.954** 0.952** 0.924** 0.921** 0.906**
Cry1F protein in flower tissues
1 0.992** 0.876** 0.851** 0.858**
Cry1F protein in pod tissues
1 0.882** 0.858** 0.864**
cry1F transcript level in leaf tissue
1 0.995** 0.996**
cry1F transcript level in flower tissue
1 0.992**
cry1F transcript level in pod tissue
1
*Correlation is significant at the 0.05 level (two tailed). **Correlation is significant at the 0.01 level (two tailed).
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4.5.5 Southern blot analysis
The Southern blotting was performed to study the integration pattern of T-DNA in
to plant genome, for which from six selected transgenic plants the genomic DNA was
digested (Plate 23). It was performed with EcoRI as the enzyme that cuts in the T-DNA
region and probed with 600 bp cry1F PCR product. An observation of strong signal and
the difference in the hybridization pattern in the four selected transgenic plants viz., 1F-
22, 1F-32, 1F-38 and 1F-33, revealed the single copy integration in these plants.
Whereas, there was no hybridization signal noticed with the DNA of non-transformed
plants. On other hand, in two plant samples viz., 1F-25 and 1F-9, hybridization signals
were noticed in two places indicating integration of two T-DNA copies in plant genome.
4.5.6 Northern blot analysis
The presence of cry1F transcript in transgenic pigeonpea plants was also
confirmed with northern blotting using 600 bp cry1F PCR product as a probe (Plate 24).
The hybridization signals were noticed in all selected transgenic plant samples viz., 1F-
25, 1F-32, 1F-33, 1F-9, 1F-22 and 1F-38. The observation of strong signals revealed the
production of transcripts in respective transgenic plants.
4.6 Generation of transgenic pigeonpea conferring expression of cry1Acm gene
The in planta transformation method was employed and transgenic pigeonpea
expressing cry1Acm was developed. The characterization of developed transgenic
plants was done by performing insect bioassay against H. armigera larvae and
quantitative Cry1Acm protein analysis using ELISA. The segregation pattern of cry1Acm
was assessed using gene specific PCR assay. Further, the developed transgenic lines
were also subjected for cry1Acm transcript analysis using qRT-PCR, Southern and
northern blot analysis.
4.6.1 Development of transgenic pigeonpea carrying cry1Acm gene
The A. tumefaciens infection was made to a total of 250 explants using
Agrobacterium tumefaciens strain carrying cry1Acm gene carrying construct .The
transformation efforts resulted in the development of total of 120 primary transformants,
which were established in green house facility (Table 33). The T1 seeds were collected
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form well-established primary transformants and T1 generation was raised. The T1
progeny plant screening identified a set of eleven putative transgenic plants carrying
cry1Acm gene as revealed by gene specific (cry1Acm) PCR assay (Plate 25). The
identified putative transformants were grown till maturity and T2 seeds were harvested.
4.6.2 The cry1Acm gene segregation analysis in T2 generation progenies
The collected T2 seeds were sown in plant to row progeny manner and T2
generation progenies were raised. The chi-square analysis of observed number of plants
with and without cry1Acm gene indicated that among eleven transgenic lines, seven
were following 3:1 (positive: negative) ration of transgene segregation (Table 34) (Plate
26). Whereas, in case of rest four transgenic lines the chi-square value was observed to
be more than the table value (3.84), clearly indicating that the cry1Acm gene segregation
in those transgenic plant progenies did not following 3:1 segregation pattern for
transgene in T2 generation.
4.6.3 Characterization of developed transgenic lines in T2 generation
The insect bioassay against first instar Helicoverpa armigera larvae reported
significant variability in larval mortality targeted tissue types. It was noticed that, the
larval mortality in case of leaf tissues ranged from 40.0% to 62.5% (Table 35). Similarly,
in case of flower bioassay it ranged 37.5% to 57.5% and 35.0% to 52.5% in case of pod.
The majority of putative transformants were having insect mortality from 50.0% to 62.5%
in leaf bioassay, with the highest mortality recorded in M135-4 transgenic line (Fig. 17).
The larval feeding was highest in transgenic line M33-12. The larval feeding pattern on
different tissue types of transgenic and non-transgenic control plants is shown in Plate
27. The statistical analysis (ANOVA) indicated that the transgenic lines differ significantly
(p < 0.001) from each other for larval mortality in all tissue types. The ‘t’-test analysis of
larval mortality in different tissue types indicated non-significant difference between
mortality levels in leaf, flower and pod tissues with p > 0.15.
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Table 33: Summary of transformation carried out using cry1Acm gene in pigeonpea
Sl. No Number of explants
co-cultivated/set
Number of explants
responded
Number of primary transformants (T0)
established in greenhouse
Number of primary transformants tested in plant-to-row progeny for identifying
putative transformants
Number of putative transformants identified (T1)
1 50 45 20 20 2
2 50 46 27 27 2
3 50 42 20 20 3
4 50 45 29 29 3
5 50 46 24 24 1
Total 250 224 120 120 11
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Table 34: The transgene segregation pattern in eleven transformants carrying cry1Acm in T2 generation revealed by gene specific PCR assay
Plant ID
Number of plants tested using gene specific
PCR
Number of plants positive using gene specific
PCR
Number of plants negative using gene specific
PCR
Expected number of positive plants
Expected number of negative plants
Calculated chi-square
Gene segregation in
3:1 ration
M2 24 16 08 18.00 6.00 0.89 Yes
M3 24 13 11 18.00 6.00 5.56 No
M5 22 14 08 16.50 5.50 1.52 Yes
M7 23 12 11 17.25 5.75 6.39 No
M10 22 15 07 16.50 5.50 0.55 Yes
M12 21 13 08 15.75 5.25 1.92 Yes
M16 24 15 09 18.00 6.00 2.00 Yes
M33 25 14 11 18.75 6.25 4.81 No
M55 24 14 10 18.00 6.00 3.56 Yes
M133 25 13 12 18.75 6.25 7.05 No
M135 22 13 09 16.50 5.50 2.97 Yes
(Table chi-square = 3.84)
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Table 35: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eleven putative transformants carrying cry1Acm gene in T2 generation
Plant ID Per cent corrected mortality
Leaf tissues Flower tissues Pod tissues
M2-14 60.0ab 50.0abc 50.0ab
M3-4 42.5cd 40.0bc 35.0c
M5-1 47.5cd 50.0abc 52.5a
M7-5 45.0cd 42.5bc 40.0bc
M10-3 62.5a 57.5a 52.5a
M12-10 50.0cd 50.0abc 47.5ab
M16-1 60.0ab 52.5ab 50.0ab
M33-12 45.0cd 37.5c 40.0bc
M55-6 52.5bc 50.0abc 47.5ab
M133-10 40.0d 42.5bc 42.5abc
M135-4 62.5a 57.5a 52.5a
SD (±) 0.060 0.061 0.066
CD 0.054 0.066 0.056
CV (%) 5.85 6.40 5.74
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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Analysis of Cry protein in different tissue types indicated significant variation in
Cry1Acm protein levels of different transgenic lines irrespective of tissue types (Plate
28). The Cry protein level in leaf tissue ranged from 0.322 to 0.907 µg/g of fresh leaf
tissue; 0.202 to 0.503 µg/g of fresh flower tissue and 0.186 to 0.405 µg/g of fresh pod
tissue (Table 36). The highest Cry1Acm protein was detected in M135-4 transgenic line,
as indicated by ELISA assay (Fig. 17). On other hand, the lower level of Cry protein was
recorded in transgenic line M133-10. The statistical analysis of variance clearly indicated
the significant difference in Cry1Acm protein levels among developed transgenic lines (p
< 0.001). The ‘t’-test analysis of Cry protein accumulation in different tissue types
indicated non-significant difference for Cry1Acm protein accumulation in flower and pod
tissues (p > 0.08). Whereas, there it was significant between leaf and flower; and leaf
and pod tissues (p < 0.001) as indicated by ‘t’-test analysis. There was a positive
correlation between insect morality and Cry1Acm protein accumulation with r2=0.6542
(Fig. 18). Further, the frequency distribution analysis of transgenic plants following 3:1
transgene segregation pattern and other than 3:1 segregation pattern showed that
majority of high Cry1Acm protein accumulating transgenic plants were following 3:1
transgene segregation pattern (Fig. 18).
4.6.4 The cry1Acm transcript analysis using qRT-PCR assay
The absolute real time analysis of cry1Acm transcripts varied among different
transgenic lines but was constitutive between different tissue types as indicated by
standard graph (Plate 29). The cry1Acm transcript level was ranged from 40.15 to 105.5
ng/µl in fresh leaf tissue; in flower it ranged from 39.80 to 103.0 ng/µl and in pod from
39.83 to 102.9 ng/µl (Table 37a). Interestingly, the highest transcript level was detected
in all tissue types of M135-4 transgenic line. On other hand, the lower level of cry1Acm
transcripts was recorded in transgenic line M3-4. The statistical analysis of variance
showed the significant difference in cry1Acm transcript levels among developed
transgenic lines (p < 0.001).The ‘t’-test analysis revealed non-significant difference in the
transcripts levels of cry1Acm gene in different tissue types within developed transgenic
plants (p ≥ 0.45). Further, positive correlation was noticed between Cry1Acm protein
accumulation and cry1Acm transcript levels with r2=0.4886 (Fig. 15). The correlation
analysis of larval mortality, Cry1Acm protein and cry1Acm transcript showed positive in
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Table 36: The Cry1Acm protein level detected in leaf, flower and pod tissues developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay
Plant ID Cry1Acm protein level (µg/g FW)
Leaf tissues Flower tissues Pod tissues
M2-14 0.519e 0.394d 0.323e
M3-4 0.328gh 0.230h 0.204f
M5-1 0.519e 0.363e 0.335d
M7-5 0.359f 0.282f 0.204f
M10-3 0.542de 0.392d 0.322e
M12-10 0.568d 0.426c 0.363c
M16-1 0.734b 0.482b 0.392b
M33-12 0.352fg 0.253g 0.186g
M55-6 0.621c 0.427c 0.384b
M133-10 0.322h 0.202i 0.188g
M135-4 0.907a 0.503a 0.405a
SD (±) 0.023 0.014 0.023
CD 0.011 0.013 0.014
CV (%) 4.48 4.07 2.03
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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Table 37a: The cry1Acm transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay
Event ID cry1Acm transcript level in leaf tissue
(ng/µl)
cry1Acm transcript level in flower tissue (ng/µl)
cry1Acm transcript level in pod tissue
(ng/µl)
M2-14 72.10d 71.60e 70.82e
M3-4 40.15g 39.80i 39.83h
M5-1 75.75c 73.75d 75.00d
M7-5 59.50e 60.00g 60.00f
M10-3 85.40b 87.10b 84.20c
M12-10 86.65b 85.00c 87.00b
M16-1 87.45b 87.00b 85.90bc
M33-12 50.00f 51.75h 51.20g
M55-6 87.65b 86.05bc 86.05bc
M133-10 60.00e 62.30f 60.60f
M135-4 105.5a 103.0a 102.9a
SD (±) 3.599 3.342 2.185
CD 0.923 0.677 0.923
CV (%) 4.88 4.55 2.99
Note: The means followed with same letters are within student ‘t’ range at α=0.05.
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leaf, flower and pod tissues for these traits of T2 generation transgenic lines (Table 37b).
4.6.5 Southern blot analysis
The genomic DNA from four selected transgenic plants was digested using
BamHI restriction endonuclease and Southern blotting was performed to analyse the T-
DNA integration pattern in to plant genome. The blotted digested genomic DNA was
probed with 452 bp labelled cry1Acm PCR product. A single but weak hybridization
signals were noticed for all samples viz., M2, M10, M16 and M135 transgenic lines. The
Southern hybridization pattern was different for all samples, indicated different position of
T-DNA insertion in plant genome. Further, an observation of single hybridization signal in
all tested transgenic plants indicated the single copy integration of transgene (Plate 30).
4.6.6 Northern blot analysis
The presence of cry1Acm transcript in transgenic pigeonpea plants was also
confirmed with northern blotting using 452 bp cry1Acm PCR product as a probe (Plate
31). The hybridization signals were noticed in all selected transgenic plant samples viz.,
M135, M55, M2, M5 and M7. The observation of strong signals revealed the production
of transcripts in respective transgenic plants.
4.7 The plant growth parameters comparison between parental genotypes and
transgenic lines developed
The different plant growth parameters such as, stem colour, flower colour, growth
habit, leaf colour and shape, branching pattern, fresh pod colour, seed colour, and plant
height were phenotypically observed in transgenic and non-transgenic control plants. It
was interesting to notice the change in flower colour development, slight pinkish yellow,
in few transgenic lines such as Ac63-20, Ac61-4, with BSMR 736 genomic background.
Whereas, it was observed that typical yellow flower colour developed in BSMR 736 non-
transgenic plants (Plate 32). Similarly, it was also noticed that another plant parameter
altered in transgenic plants i.e. fresh pod colour. The typical fresh pod colour developed
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Table 37b: The correlation analysis of insect mortality levels, estimated Cry1Acm protein and cry1Acm transcript in leaf, flower and
pod tissues of T2 generation plants (αααα=0.01).
Insect mortality
in leaf tissues
Insect mortality in flower tissues
Insect mortality
in pod tissues
Cry1Acm protein in
leaf tissues
Cry1Acm protein in
flower tissues
Cry1Acm protein in
pod tissues
cry1Acm transcript
level in leaf tissue
cry1Acm transcript
level in flower tissue
cry1Acm transcript
level in pod tissue
Insect mortality in leaf tissues
1 0.878** 0.789** 0.809** 0.857** 0.781** 0.781** 0.789** 0.766**
Insect mortality in flower tissues
1 0.912** 0.843** 0.871** 0.874** 0.919** 0.921** 0.909**
Insect mortality in pod tissues
1 0.762** 0.815** 0.838** 0.874** 0.877** 0.870**
Cry1Acm protein in leaf tissues
1 0.946** 0.914** 0.921** 0.906** 0.910**
Cry1Acm protein in flower tissues
1 0.971** 0.926** 0.910** 0.918**
Cry1Acm protein in pod tissues
1 0.922** 0.901** 0.913**
cry1Acm transcript level in leaf tissue
1 0.997** 0.999**
cry1Acm transcript level in flower tissue
1 0.997**
cry1Acm transcript level in pod tissue
1
**Correlation is significant at the 0.01 level (two tailed).
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in case of ICPL 87119 was green with purple strips, whereas in case of few transgenic
plants (1F-22) with ICPL 87119 as genomic background recorded dark purple colour
development in freshly developing pods. Further the other important plant growth
parameter, wherein the changes were noticed was plant height. Apart from theses, there
were no phenotypically visible changes noticed in other parameters of transgenic and
non-transgenic pigeonpea plants. The per cent recovery of parental phenotypes for
selected traits in transgenic plants carrying different cry genes is presented in Table 38
(Fig. 19). It was noticed that the non-parental phenotypes were observed in transgenic
plants expressing cry1Ac and cry1F gene.
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Table 38: The per cent deviation observed between transgenic plants and parental genotypes for selected morphological traits
Plant growth parameters Transformants
carrying cry1Ac
Transformants carrying cry2Aa
Transformants carrying cry1F
Transformants carrying cry1Acm
Stem colour 0.00 0.00 0.00 0.00
Flower colour 4.55 0.00 7.14 0.00
Growth habit 2.27 0.00 0.00 0.00
Leaf colour and shape 0.00 0.00 0.00 0.00
Branching pattern 2.27 0.00 0.00 0.00
Fresh pod colour 3.41 0.00 7.14 0.00
Seed colour 0.00 0.00 0.00 0.00
Plant height 2.27 0.00 0.00 0.00
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5. DISCUSSION
Pigeonpea belonging to family Fabaceae is cultivated in more than 25 countries of
the world including Indian subcontinent, Africa and Central America. It ranks sixth among
grain legumes in production and grown over 4.7 million hectares of agriculture land in
world wide with the production of around 3.69 million tons annually
(http://faostat.fao.org). India, alone contributes more than 80 per cent of total world
pigeonpea production (Parde et al., 2012). Pigeonpea production and productivity are
constrained by several biotic and abiotic stresses, whose levels of resistance in world
germplasm accessions are low to moderate. Attempts to develop pigeonpea cultivars
resistant to biotic stresses, such as resistance to legume pod borer (Helicoverpa
armigera) and fusarium wilt by conventional breeding methods have shown limited
success due to narrow genetic variability among the germplasm accessions. Breeding
incompatibility problems associated with wild species warrant the exploration of
alternative approaches. Due to high level of resistance to different insecticides in H.
armigera, pigeonpea cultivation also has been difficult. Such problem has been resorted
by Bt transgenic technology adopted in cotton and other crops world wide. Genetic
engineering technology plays a significant role as an additional tool for the introduction of
agronomically useful traits in a high yielding background.
There are very few reports on in vitro regeneration of pigeonpea through
organogenesis from unorganised callus (Kumar et al., 1983). Many independent studies
have reported the multiple shoot production and plantlet regeneration through
organogenesis from different explants viz., cotyledons, embryonic axes, cotyledonary
node from mature seeds and seedling petioles (Franklin et al., 1998; Srinivasan et al.,
2004). The attempts have been made to initiate in vitro culture from different tissue
sources in pigeonpea (Geetha et al., 1998). However, the in vitro regeneration protocols
further need to be fine-tuned and improvised to achieve high frequency regeneration of
plantlets. In this contest, the enhancement in multiple shoot bud induction by use of
cytokinins in nutrient medium expected to be one of the potential approaches.
Cytokinins, as a hormone, are associated with cell division, modification of apical
dominance, shoot differentiation etc and incorporated in tissue culture media for cell
197
division and differentiation of adventitious shoots from callus and organs. The commonly
used cytokinins for plant tissue culture includes benzylamino purine (BAP), isopentenyl-
adenine (2-ip), furfurylamino purine (kinetin), thidiazuron (TDZ) and zeatin. The effect of
cytokinins such as, BAP, kinetin and their varied levels of concentrations on multiple
shoot induction has been studied in pigeonpea (Geetha et al., 1998). In another study,
frequency of multiple shoot bud induction has been titrated using different cytokinins viz.,
BAP, kinetin, TDZ in eleven Indian cultivars of pigeonpea using leaf as explant (Kashyap
et al., 2011).
Independent studies have reported that the type of explant, genotype and
concentrations of cytokinin usually influence the frequency of shoot bud regeneration
(Geetha et al., 1998; Kashyap et al., 2011). The present study aims at improvisation of
multiple shoot induction frequency and plantlet regeneration in pigeonpea using
cotyledonary node with cotyledons and embryo discs with half cotyledon explants. Two
pigeonpea genotypes viz., ICPL 87119 (Asha) and BSMR 736 were tested for their
response to different cytokinins such as, BAP, TDZ and Zeatin, most commonly used
cytokinins for organogenesis. The study identified the best concentration level of
cytokinin in medium for highest number of shoot bud inductions. The medium fortified
with identified concentration levels of respective cytokinins can be used for crop
improvement programmes involving production of transgenic pigeonpea with improved
transformation efficiency by formation of more number of shoots per explant co-
cultivated.
5.1 Improvisation of multiple shoot induction and plantlet regeneration in pigeonpea
In present study, two genotypes viz., ICPL 87119 and BSMR 736 reported their
behavioral response to same BAP level, which was comparable in both genotypes. On
other hand it was interesting to notice that the two explants i.e. cotyledonary node with
cotyledons and embryo discs with half cotyledon reported different behavioral response
to same BAP concentration regime. As far as the number of shoot bud induced per
explants was concern both genotypes showed similar kind of response to same BAP
level. There was a significant difference in number of shoot bud induced from two
explants in response to same BAP fortification level. The analysis of means indicated
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that the two treatments, 2.0 mg/L and 3.0 mg/L BAP, were on par for number of explants
responded to BAP fortification. On the other hand, the treatment with 2.0 mg/L BAP level
was superior over other treatments for number of shoot bud induced per explant.
Interestingly, there was a concomitant decrease in resonance of explants for shoot bud
induction and number of shoot bud induced with further increase in BAP levels in growth
medium. The similar kind of response by explants to increasing concentrations of
cytokinins has been documented by Geetha et al. (1998) in pigeonpea. The effect of two
cytokinins viz., BAP and Kinetin, and their concentration regimes have been studied for
shoot bud induction from cotyledonary node, epicotyl, hypocotyl, cotyledon and leaf
explants; reported that the 2.0 mg/L BAP level could potentially induced maximum
number of shoot buds from all explants (Geetha et al., 1998).
The results of present study reported parallel response of two pigeonpea
genotypes to different TDZ concentration levels. The effect of different TDZ
concentration regime on two explants response was significant. At the same time, both
genotypes responded well and equally for induction of multiple shoot bud in response to
same TDZ level. Further, there was a distinct response of two explants for multiple shoot
bud induction at same TDZ concentration regimes. The TDZ concentration regime 4.0
mg/L was the most effective for induction of multiple shoot buds per explants from both
explants and genotypes. Recently, Shekhar et al. (2012) have titrated different TDZ
concentration levels (1 to 6 mg/L) for shoot bud induction from mature zygotic embryo
explants of pigeonpea. They identified 4.0 mg/L TDZ as the most effective for multiple
shoot induction from mature zygotic embryo explants in pigeonpea (Shekhar et al.,
2012).
Similarly, the results of present study also revealed that any further increase in
TDZ concentration levels imparts reduction in multiple shoot bud induction in both
explants of pigeonpea. The same kind of explant response to TDZ levels in growth
medium has been reported in cotyledonary node of Cassia sophera and mature zygotic
embryo explants of pigeonpea (Shekhar et al., 2012; Parveen and Shahzad, 2010).
Furthermore, TDZ (1.0 mg/L) in combination to 2,4-D (1 mg/L) have been found effective
for shoot generation from cotyledon and hypocotyl explants of L. campestre as well
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(Ivarson et al., 2013). Studies have reported that embryonic axis with cotyledons are the
best for producing maximum of 55.02 shoots per explant when cultured on MSB5
medium with 2 mg/L 2-isopentenyladenine (2-iP), 1 mg/L thidiazuron (TDZ) and 0.4 mg/L
kinetin (KIN) in chickpea (Cicer arietinum L.) (Pawar et al., 2012). Although, in present
study, the best TDZ concentration observed for more number of shoot induction seems
to be high (4.0 mg/L), in previous reports same TDZ concentration level has been
reported for more number of shoot induction from mature zygotic embryo in pigeonpea
(Shekhar et al., 2012). Further, any artefacts such as deformed shoots, when explants
were cultured on MS augmented with different TDZ concentration regimes, were not
observed in the present study.
The behavioral response of both genotypes and explants to zeatin fortification
was same as that of BAP and TDZ. Results indicated that ICPL 87119 and BSMR 736
response to zeatin augmentation was on par with each other. Whereas, two explants
(cotyledonary node with cotyledons and embryo discs with half cotyledon) responded
distinctly for the number of multiple shoot buds induced at same level of zeatin
concentration regimes. Further, results indicated absence of merit difference for number
of induced multiple shoot buds in two genotypes experiencing same zeatin concentration
regime: whereas, the two explants recorded merit difference for shoot bud induction at
same zeatin concentration regime in both genotypes. The mean analysis reported that
the two treatments, 1.0 mg/L and 2.0 mg/L zeatin, were on par and superior over other
treatments for number of shoot bud induction per explant. As that of BAP and TDZ, the
similar kind of response curve was also noticed for shoot bud induction at different zeatin
levels. The effect of change of zeatin concentration on shoot bud induction was similar to
that of effect of change in BAP or TDZ concentration levels. The observation of
development of white sugary callus might be the reason for reduction in multiple shoots,
when medium is supplemented with higher concentrations of cytokinins. Similar zeatin
levels (2.0 mg/L) in growth medium have been documented as most effective for shoot
regeneration from cotyledonary node in pigeonpea (Srivastava et al., 2013). In another
study, the zeatin (2.0 mg/L) in combination to 2,4-D (1.0 mg/L) has been reported for
shoot generation from cotyledon and hypocotyl explants of L. campestre (Ivarson et al.,
2013).
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Present study reported induction of more number of shoot buds in cotyledonary
node with cotyledons explants when compared with that of embryo discs with half
cotyledon explants. The cotyledonary node with cotyledons explants has immense
potential for multiple shoot bud induction. It is interesting to notice such difference, and
might be it was due to removal of half cotyledon retaining embryo discs with other half
part of it. Such detachment of half cotyledon might have resulted in reduction in
cotyledonary node compactness, which might be essential for more number of shoot bud
induction. Although there are no previous reports indicating this phenomenon, based on
present experimental results it seems obvious. Many individual studies have reported the
response of different explants to multiple shoot bud induction in response to cytokinin
fortification (Geetha et al., 1998; Kashyap et al., 2011). As per previous literature or
information until this time the first report indicating such huge number of multiple shoot
bud induction from cotyledonary node with cotyledons explants in pigeonpea has been
emerged through present investigation.
Furthermore, results of present study clearly indicated that the MS with 0.5 mg/L
IBA was most suitable for healthy root induction with comparatively more number of
roots. Further increase in IBA levels in rooting medium did not affect the root induction
significantly. Many studies have documented the effectiveness of lower concentrations of
IBA on root induction in pigeonpea (Geetha et al., 1998; Guruprasad et al., 2011).
Pigeonpea suffers from many biotic and abiotic stresses, and the level of
resistance in world germplasm accessions is low to moderate. Hence, there is a great
need and opportunity to use tissue culture based methods to improve pigeonpea crop
resistance to different stresses. The results of present study reported the efficient
multiple shoot bud induction and plantlet regeneration method for pigeonpea. As
pigeonpea genotypes used in present study, ICPL 87119 and BSMR 736, are moderate
to resistant to fusarium wilt and sterility mosaic disease, in vitro plantlet regeneration
methods in such genotypes can be exploded for genetic engineering programs to
develop multiple stress tolerance in pigeonpea.
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5.2 Improvisation of in planta transformation protocol in pigeonpea
The development of transgenics in pigeonpea has remains dogged due to poor in
vitro regeneration and low transformation frequency. Although numerous efforts have
been made for the development of efficient protocols of Agrobacterium tumefaciens and
microprojectile bombardment-based genetic transformation of pigeonpea, the in vitro
regeneration conditions so far described are available for only a few of the many
cultivars/genotypes of pigeonpea (Rao et al., 2008). It has also been reported that the
morphogenetic response of pigeonpea crop is known to be a genotype-specific
phenomenon (Mohan and Krishnamurthy, 1998).
An in planta transformation method have been successfully applied in wide range
of crop plants such as, mulberry, soybean, rice and cotton (Ping et al., 2003; Supartana
et al., 2005; Keshamma et al., 2008). This method of transformation also has been
documented in other crop plants like, pigeonpea, fieldbean, sunflower, groundnut and
safflower (Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b; 2001). In
resent efforts in planta transformation method has been successful in maize, rice and
wheat transformation (Mehrotra and Goyal, 2012). Further, very recently in planta
method has also been used for development of transgenic pigeonpea for expression of a
chimeric cry1AcF gene encoding Cry1Ac and Cry1F domains (Ramu et al., 2012). In
case of alfalfa, the marker free transgenic plants have been developed employing in
planta transformation method (Weeks et al., 2008).
In present study, for improvisation of in planta transformation protocol in
pigeonpea different treatment such as, A. tumefaciens infection alone, A. tumefaciens
infection using A. tumefaciens culture with tobacco extract added in overnight grown
culture and A. tumefaciens infection using air evacuation to increase the proximity
between A. tumefaciens and embryonic axis were employed. Results of present
investigation indicated merit differences between explants response to different in planta
transformation treatments with p < 0.03. The two treatments viz., A. tumefaciens
infection alone and A. tumefaciens infection using A. tumefaciens culture with tobacco
extract were comparable with each other with respect to explants response to
treatments. Whereas, there was a significant reduction in explants response to A.
tumefaciens infection using air evacuation treatment with 18.5 per cent.
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Similarly, the number of primary transformants developed during test treatments
was significant at p < 0.001. The two treatments viz., A. tumefaciens culture with tobacco
extract and A. tumefaciens infection using air evacuation were on par and superior for
development of more number of primary transformants when compared with treatment
involving A. tumefaciens infection alone. Further, study reported that the in planta
transformation treatment, A. tumefaciens infection using air evacuation, was most
effective in developing more number of transformants in pigeonpea with 12.0 per cent
transformation efficiency. Other two treatments were found to be statistically on par with
each in case of transformation efficiency.
Clough and Bent (1998) have reported two folds increase in transformation rate
when flower tissues were air evacuated with A. tumefaciens culture in place of flower dip
method in Arabidopsis thaliana. Dehestani et al. (2010) employed in planta
transformation method in Arabidopsis and showed that plants infected with A.
tumefaciens strain GV3850 only reported highest transformation frequency of 1.54 per
cent. The use of vacuum infiltration during A. tumefaciens infection has improved
transformation efficiency to 3.0 per cent through their study. Further, Habashi et al.
(2012) has studied the effect of vacuuming during Agroinoculation in two pear (Pyrus
communis L.) cultivars, Bartlett and Harrow Delight and showed significant increase in
transformation efficiency (10.63 per cent) when compared with the common
Agroinoculation method (4.06 per cent).
According to Rao et al. (2008) the use of in planta transformation method has
provided many advantageous like, ensured generation of pigeonpea transgenic plants
with considerable ease in a short time, applicable across different genotypes/cultivars of
the crop and offers immense potential as a supplemental or an alternative protocol for
generating transgenic plants of difficult-to-regenerate pigeonpea. Present study reported
identification of effective method involving air evacuation during A. tumefaciens infection
for pigeonpea transformation. Such high transformation efficiency might be due to the
removal of air from injured parts of actively developing embryo, which further might have
allowed the A. tumefaciens to reach and infect damaged tissues parts more efficiently.
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As the targeted explants involved developing embryo, the injured tissues may be healed
faster and grow normal due to actively dividing and growing cells after A. tumefaciens
infection and co-cultivation period. As the air evacuation during A. tumefaciens infection
improved in planta transformation efficiency to greater extent, can be employed for
genetic engineering programmes from crop improvement in pigeonpea.
5.3 Generation of transgenic pigeonpea carrying different cry genes for pod borer
resistance
The pigeonpea variety ICPL87119 have been derived from a cross between C11
and a breeding line, and is medium duration variety with resistance to Fusarium wilt and
pigeonpea sterility mosaic virus (Dharmaraj and Lohithaswa, 2004). The extra short-
duration varieties and the medium-duration genotypes have been found to be severely
damaged (90 per cent) by pod borer (Choudhary et al., 2013). Many independent studies
have reported that pigeonpea pod walls with more wax, total phenols, less reducing and
non-reducing sugars, total amino acids and high amount of soluble protein content have
been associated with plants resistance reaction in pigeonpea (Sharma et al., 2009;
MacFoy et al., 1983; Moudgal et al., 2008). The back cross progenies have been
developed and reported a range of resistance traits in the progeny lines for pod borer,
bruchid and pod fly resistance ranged from 6.85 to 22.84 per cent (Mallikarjuna et al.,
2011).
Substantial progress has been made towards development of large-scale
genomic resources in pigeonpea especially during the last decade, these efforts have
resulted in the development of large-scale molecular markers, construction of
comprehensive genetic maps, identification of various marker-trait associations and
initiation of molecular breeding in this crop (Raju et al., 2010; Mallikarjuna et al., 2011;
Dubey et al., 2011). Although inter as well as intra specific genetic maps in pigeonpea
have been developed, preliminary mapping efforts have resulted in mapping of Fusarium
wilt resistance and SMD only (Kotresh et al., 2006; Ganapathy et al., 2010). The
presence of genetic variability for resistance to insects particularly pod borer is not
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available in the primary gene pool of pigeonpea (Choudhary et al., 2013). Although the
advance back cross QTL based breeding is most suitable for introducing novel alleles
from wild relatives, the transfer of desirable alleles from wild relatives is not so simple
because of difficulty in efficient tracking for desired and non-desired alleles in breeding
lines (Tanksley and Nelson, 1996).
Hence, many plant transformation studies using genetic engineering have been
undertaken for development of transgenic in pigeonpea expressing different alien genes
such as, cowpea protease inhibitor gene (pCPI) (Lawrence and Koundal, 2001),
synthetic cryIE-C (Surekha et al., 2005), synthetic cry1Ab (Sharma et al., 2006), etc.
Recently, in planta transformation method has been successfully used to express
chimeric cry1AcF in pigeonpea (Ramu et al., 2012). It has been reported that Cry1Ac
belongs to 3D-Cry group and binds to V-ATPAse subunit A and actin of brush border in
case of M. sexta and H. virescens. Further the mode of action of Cry1Ac toxins involve
binding of the toxin with other components of the midgut cells (McNall and Adang, 2003;
Krishnamoorthy et al., 2007). Cry2Aa belongs to an unusual subset of crystalline
proteins and possessed broad insect species specificity against Lepidoptera and Diptera
(Liang and Dean, 1994). The Cry2Aa protoxins are significantly smaller (72 kDa) and are
processed primarily at the N-terminal end. Such activated toxin follows complex
sequential binding lines with different insect gut Cry-binding proteins and results in
membrane insertion and pore formation (Bravo et al., 2011).
The Cry1F protein mechanism of action involves the enzymatic cleavage of the
protoxin to form core toxin (Gao et al., 2006). Many studies have reported the activity of
Cry1F against lepidopteran species (Balog et al., 2011; Oppert et al., 2010). The Cry1F
toxin susceptible species also includes tobacco budworm, beet armyworm, soybean
looper, cotton bollworm, fall armyworm, lesser cornstalk borer, wax moth and European
corn borer (Adamczyk et al., 2008; Blanco et al., 2010; Buntin, 2008; Tindall et al., 2009).
The native cry1Ac gene sequence from B. thuringiensis have been in silico modified and
artificially synthesized to overcome codon bias and other undesirable regulatory coding
sequences for its improved expression in transgenic plants (Mohan, 2008). The study
resulted in the development of modified cry1Acm with increased GC content and
modifications in specific undesirable eukaryotic regulatory sequences (Mohan, 2008).
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In present study, eighty eight transgenic plants were developed carrying cry1Ac
gene using improvised in planta transformation protocol. The transgene segregation
analysis in T2 generation plants revealed 3:1 (positive : negative) cry1Ac gene
segregation pattern in forty eight transgenic lines. According to Mendel’s law of
inheritance, the characters govern by single gene segregate in 3:1 (dominant: recessive)
ratio in F2 generation (Bowler, 1989). The observation of 3:1 (positive : negative) ratio for
cry1Ac gene in T2 generation plants indicated the possibility of singly copy transgene
integration in pigeonpea genome. Although, in case of A. tumefaciens mediated
transformation the possibility of multiple copy insertion in very less to rare, the
observation of absence of 3:1 transgene segregation pattern in large number of
transgenic lines i.e. forty, was interesting. One of the possible reasons for observation of
such behaviour might be due to the presence of more than single copy transgene
insertion. Another possibility could be the number of plants tested to study transgene
segregation pattern might be less, as the 3:1 monogenic segregation ratio is population
phenomenon (Bowler, 1989). The in vitro transformation efforts resulted in generation of
ten transgenic plants. The transformation frequency observed in case of in vitro
transformation method was very less i.e. 0.36 per cent, whereas it was 6.29 per cent in
case of in planta plant transformation method for cry1Ac gene.
In case of cry2Aa, sixty five putative transformants were developed using
improvised in planta transformation protocol. The assessment of transgene segregation
pattern identified sixteen transgenic plants following 3:1 (positive : negative) cry2Aa gene
segregation pattern in T2 generation progenies. Such observation of 3:1 (positive :
negative) ratio for transgene segregation clearly indicated the possibility of singly copy
transgene integration in pigeonpea genome in those sixteen lines. Similar to that of
cry1Ac gene carrying transgenic plants, in case of cry2Aa transformants, the observation
of absence of 3:1 transgene segregation pattern in many transgenic lines i.e. forty nine,
was interesting. And it was probably either due to the presence of more than single copy
transgene insertion or the less number of plants tested to study transgene segregation
pattern. In order to achieve multiple generation per year, the transgenic plants were not
allowed to reach their full harvestable/ maturity stage and only initial pod yield was
harvested. Hence, due to availability of limited seed material, 30-40 seeds per transgenic
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line were sown and obtained next generation plants were used to study transgene
segregation analysis.
Further, fourteen transgenic plants were developed carrying cry1F gene using
improvised in planta transformation protocol. The transgene segregation analysis in T2
generation plants revealed 3:1 (positive : negative) cry1F gene segregation pattern in
seven transgenic lines. The observation of 3:1 (positive : negative) for cry1F gene in T2
generation plants indicated the possibility of singly copy transgene integration in
pigeonpea genome. The possible reasons for observation of absence of 3:1 transgene
segregation pattern in remaining seven transgenic lines was might be due to the
presence of more than single copy transgene insertion or could be due to the number of
plants tested to study transgene segregation pattern might be less.
In case of cry1Acm, eleven putative transformants were developed. The
assessment of transgene segregation pattern identified, among eleven, seven transgenic
plants were following 3:1 (positive : negative) cry1Acm gene segregation pattern in T2
generation progenies. Such observation of 3:1 (positive : negative) for transgene
segregation revealed the possibility of singly copy transgene integration in pigeonpea
genome. Similar to that of other cry gene carrying transgenic plants, in cry1Acm
transformants the observation of absence of 3:1 transgene segregation pattern in four
transgenic lines might due to the presence of more than single copy transgene insertion
or the less number of plants tested to study transgene segregation pattern.
Bhat and Srinivasan (2002) discussed genetic analyses of transgenic plants and
indicated that handling of primary transgenics to obtain progeny generation needs few
careful considerations, wherein the breeding behaviour of the plant species is the
deciding factor. It is important to take into account the performance of parent and
progeny. To obtain homozygosity researchers resort to selfing of the T0 plants. It results
in T1 progenies with the complex mixture of genotypes and their composition depends on
the genetic constitution of the T0 parents. In case of multiple copy integration of
transgene, determination of exact genotypic constitution of individual plants may be
difficult due to difficulties in distinguishing hemizygotes from homozygotes by Southern
analysis (Bhat and Srinivasan, 2002).
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5.4 Molecular characterization of pigeonpea transgenic lines expressing different cry
genes
Different transgene characterization methods viz., insect bioassay, protein
quantitation using ELISA, mRNA quantitation using qRT-PCR assay, Sothern, western
and northern blot, and TAIL-PCR assay for juncture region analysis have been employed
in many studies (Sharma et al., 2006; Ramu et al., 2012). Sharma et al. (2006) have
developed transgenic plants expressing synthetic cry1Ab in pigeonpea, which showed
varied range of expression of gene in different tissues of the whole plant, highest
expression in flowers (0.1 per cent of total soluble protein) and least in the leaves (0.025
per cent of total soluble protein). Gopalaswamy et al. (2007) evaluated the usefulness of
detached leaf assay to test the bioefficacy of transgenic pigeonpea and determined
levels of Cry1Ab or SBTI (soybean trypsin inbhibitor) proteins in the transgenic
pigeonpea plants as not sufficient to cause significant deterrent effects on leaf feeding,
larval survival and larval weight of H. armigera and found to be quite useful for evaluation
of transgenic pigeonpea plants for resistance to H. armigera (Gopalaswamy et al., 2007).
Recently, transgenic pigeonpea plants carrying chimeric cry1AcF have been developed
and showed that chimeric Cry1AcF levels in developed transgenic plants ranged 3-15
µg/g of fresh tissue weight with the insect mortality ranged from 0 to 100 per cent
indicating the potential effect of Cry1AcF against H. armigera (Ramu et al., 2012).
Bioassays can be used to test the functionality of the transgene product. However,
in primary transgenics, somaclonal and transgene effects are expected to be confounded
and hence bioassay analysis of transgenic plant progenies is recommended (Bhat and
Srinivasan, 2002). Considering the same, in present study, transgenic plants developed
were analysed through bioassay in T2 and T3 generations. The bioefficacy of developed
transgenic plants expressing cry1Ac in terms of larval mortality in three tissue viz., leaf,
flower and pod reported distinct response of transgenic plants toward insect resistance.
The larval mortality in case of leaf tissues ranged from 25.00 to 70.00 per cent, in case of
flower from 22.50 to 52.50 per cent and in case of pod from 17.50 to 52.50 per cent over
non-transgenic control. The expression of Cry1Ac protein resulted in reduction in leaf
tissues damage over non-transgenic plants. There was a concomitant decrease in leaf
damage with increase in larval mortality.
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Amoungst transgenic plants expressing cry2Aa gene, the larval mortality in case
of leaf tissues ranged from 5.25 to 68.33 per cent, between 5.25 to 48.5 per cent in case
of flower tissues and 10.25 to 53.25 per cent in case of pod tissue. When compared with
cry1Ac gene carrying transgenic plants, cry2Aa transformants showed wide range of
insect mortality starting from very low, 5.25 per cent, to almost comparable with the
highest mortality level as that of cry1Ac transgenic lines. In case of flower tissues cry1Ac
transgenic plants were more effective when compared with cry2Aa transformants.
Whereas, both the transgenic plants carrying cry1Ac and cry2Aa, separately, were
equally effective in leaf and pod bioassay. Similar to that of cry1Ac transgenic plants,
there was considerable reduction in leaf tissue damage in case of cry2Aa transgenic
plants as well. The expression of Cry2Aa protein reported significant reduction in leaf
tissues damage over non-transgenic plants.
Further, the transgenic plants carrying cry1F gene reported varied response in
case of larval mortality in tissue types. The larval mortality in case of leaf tissues ranged
from 22.5 to 62.5 per cent, in case of flower from 20.0 to 55.0 per cent and in case of
pod from 10.0 to 47.5 per cent over non-transgenic control. There was considerable
reduction in leaf tissue damage in case of transgenic plants. The larval mortality levels of
cry1F transgenic lines were comparable with both cry1Ac and cry2Aa transgenic lines in
case of flower bioassay. Whereas, the bioefficacy levels in leaf and pod tissues were
relatively low in case of cry1F transgenic plants when compared with other two cry gene
carrying transformants. There was a concomitant decrease in leaf damage with increase
in larval mortality probably due to reduced larval feeding on transgenic plant tissues
expressing cry1F gene.
The transgenic plants carrying cry1Acm gene also showed varied response in
case of larval mortality and leaf tissue damage in tissue types. The larval mortality in
case of leaf tissues ranged from 40.0 to 62.5 per cent, in case of flower from 37.5 to 57.5
per cent and in case of pod from 35.0 to 52.5 per cent over non-transgenic control. The
results indicated that there was considerable reduction in leaf tissue damage in case of
transgenic plants in response to Cry1Acm protein accumulation. When compared with
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other transgenic plants carrying different cry genes. The larval mortality levels of
cry1Acm transgenic lines were comparable with other transgenic lines. There was a
concomitant decrease in leaf damage with increase in larval mortality.
Surekha et al. (2005) has performed detached leaf feeding tests on transgenic
pigeonpea plants expressing cry1Ac for the pest S. litura. Study has reported significant
variation between transgenic samples and the control wild-type plants with highest
mortality of 80 per cent (Surekha et al., 2005). The effect of transgenic pigeonpea
carrying cry1Ab and SBTI genes on the growth and development of H. armigera have
been reported using detached leaf bioassay (Gopalaswamy et al., 2007). Ramu et al.
(2012) has indicated that the both the damage and the mortality in insects varied from 0
to 100 per cent among the putative transformants carrying cry1AcF. The effect of the
cry1AcF gene has been seen on the larva as there was a considerable difference in the
size of the larva that fed on the transgenics and wild type (Ramu et al., 2012). Selected
transgenic chickpea lines viz., BS2A, BS5A and BS6H have been tested in insect
bioassays using neonate H. armigera and compared to the non-transgenic cv ICCV
89314 and cv Semsen (Acharjee et al., 2010). Study reported significantly greater larval
mortality among the larvae consumed transgenic leaves, with highest mortality on leaves
of BS6H, up to100 per cent (Acharjee et al., 2010). Further the toxicity of T0 and T1
plants expressing Cry1Ac protein have been tested using either isolated or whole plants
in feeding assays with second or third instar neonate larvae of H. armigera (Sanyal et al.,
2005). There was inscrease in feeding of H. armigera larvae within two days of exposure
to transgenic chickpea expressing high levels of Bt toxins. The effective larval weight
reduction was 40.0 to 90.0 per cent with high mortality (Sanyal et al., 2005).
The ELISA has shown a significant advantage for protein analysis in transgenic
plants. A sandwich ELISA is the preferable immunoassay used for the detection of Bt
protein, where an analyte is sandwiched in between the two antibodies; a capture
antibody and the detector antibody (Kamle and Ali, 2013). ELISA has been successfully
used for the detection of protein encoded by cp4-epsps gene in a RR soybean (Rogan,
1999). In present study, the analysis of Cry1Ac protein in different tissues of developed
transgenic plants reported significant variation in Cry1Ac protein levels in leaf, flower and
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pod tissues of different transgenic lines. The ELISA results indicated that the majority of
putative transformants were having 0.4 to 0.6 µg/g of Cry1Ac protein in different tissue
types. There was a positive correlation between the Cry1Ac protein level and insect
mortality. With increase in Cry protein level, there was a concomitant increase in insect
mortality in transgenic plants. It was noticed that transgenic plants expressing higher
level of Cry1Ac protein showed significant reduction in tissue damage as well. The
similar kind of correlations has been reported in transgenic pigeonpea plants expressing
cry1AcF gene (Ramu et al., 2012).
There was a significant variation in Cry2Aa protein levels of different tissue types
as noticed in case of transgenic plants expressing cry2Aa gene. The majority of cry2Aa
transformants were having 0.0 to 0.5 µg/g of Cry2Aa protein in different tissue types.
There was a positive correlation between the Cry2Aa protein level and insect mortality
and with increase in Cry2Aa protein level, there was a concomitant increase in insect
mortality in transgenic plants. In few transgenic lines such as, 21A12-24, although there
was a very high accumulation of Cry2Aa protein, up to 3.231 µg/g, the insect mortality
levels were as comparable with transgenic plants expressing cry1Ac gene (0.847 µg/g of
Cry1Ac). This observation gives a probable indication of more effective activity of Cry1Ac
protein when compared to that of Cry2Aa against Helicoverpa armigera attack in
pigeonpea.
The analysis of Cry1F protein in different tissues of developed transgenic plants
reported significant variation in Cry1F protein levels in leaf, flower and pod tissues of
different transgenic lines. The ELISA results indicated that the majority of putative
transformants were having 0.4 to 0.6 µg/g of Cry1F protein in leaf, 0.3 to 0.6 µg/g of
Cry1F protein in both flower and pod tissue. With increase in Cry protein level, there was
a concomitant increase in insect mortality in transgenic plants. On other hand, transgenic
plants expressing higher level of Cry1F protein showed significant reduction in tissue
damage. The cry1F protein accumulation in developed transgenic plants were
comparable and slightly above (1.032 µg/g in leaf tissue; 0.870 µg/g in flower and 0.782
µg/g in pod) to that of cry1Ac protein accumulation (0.847 µg/g in leaf tissue; 0.671 µg/g
in flower and 0.685 µg/g in pod) in cry1Ac transgenic plants.
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Further, Cry1Acm protein analysis in different tissues of developed transgenic
plants indicated accumulation of Cry1Acm protein, 0.3 to 0.9 µg/g of Cry1F protein in
leaf, 0.2 to 0.5 µg/g of Cry1F protein in both flower and pod tissue. With increase in
Cry1Acm protein level, there was a concomitant increase in insect mortality in transgenic
plants. On other hand, transgenic plants expressing higher level of Cry1Acm protein
showed remarkable reduction in tissue damage when compared with non-transgenic
control plant tissue. The cry1Acm protein accumulation in developed transgenic plants
were comparable the cry1Ac protein accumulation in cry1Ac transgenic plants. Based on
comparative analysis of different transgenic events expressing cry1Ac, cry2Aa, cry1F
and cry1Acm genes independently, it was indicative that the transgenic events
expressing differential levels of cry genes were varying for their insecticidal activity
against H. armigera.
In previous studies, Sanyal et al. (2005) has estimated the Cry1Ac protein levels
in developed transgenic chickpea plants, which ranged from 14.5 to 23.5 ng/mg
extractable protein with high levels of toxicity against pod borer. Similarly, Mehrotra et al.
(2011) has reported that the Cry protein accumulation in cry1Ab and cry1Ac insecticidal
genes pyramided transgenic plants ranged between 15-20 ng/mg and showed high-level
of resistance against pod borer larvae of H. armigera.
RT-PCR and northern hybridization techniques are employed to assess the
expression of the introduced gene (Bhat and Srinivasan, 2002). Studies have reported
the use of real time PCR for quantification of targeted cDNA molecules (Kamle and Ali,
2013). The real time PCR has also been undertaken for validating and estimating the
number of copies of inserted genes into the host genome (Zhang et al., 2003). The
method has been reported for several genetically modified crops such as maize,
cassava, rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al., 2013;
Beltrán et al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007). Beltrán et al. (2009)
developed real-time polymerase chain reaction-based methods for the primary scrutiny
of putative transgenic plants. They tested for the presence of transgenes, estimated copy
number and quantified messenger RNA (mRNA) levels of genes introduced through A.
tumefaciens.
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In present study, real-time PCR assay was employed to quantify the cry gene
transcript levels in transgenic plants. The real time transcript analysis of cry1Ac transcript
level reported that transgene transcripts ranged from 24.6 to 165.1 ng/µl in leaf, 15.6 to
149.5 ng/µl in flower and from 18.3 to 152.4 ng/µl in pod tissues of transgenic plants.
Interestingly, Ac20-2 transgenic lines indicated the highest cry1Ac transcript level in all
tissue types. Similarly, the real time transcript analysis of cry2Aa reported that transgene
transcripts ranged from 48.5 to 134.5 ng/µl in leaf, 41.2 to 110.2 ng/µl in flower and from
42.8 to 100.5 ng/µl in pod tissues of transgenic plants. In terms of transcription of cry
genes, cry1Ac transgenic plants were more effective than that of transformants
expressing cry2Aa gene. Considering the lower levels of Cry1Ac protein levels, although
the transcript levels of cry1Ac were high when compared with cry2Aa transgenic plants,
the possible reason for such behaviour may be the less stability of cry1Ac transcript in
plant cell. On other hand, cry2Aa transcripts might be more stable leading to more
accumulation of Cry2Aa protein in transgenic plant cell. It has been reported that the
high levels of expression of cry1Ac gene cannot be routinely achieved and this has been
attributed to the instability of the transcript (Rawat et al., 2011).
The real time transcript analysis of cry1F transcript level reported that transgene
transcripts ranged from 47.6 to 105.3 ng/µl in leaf, 45.2 to 101.0 ng/µl in flower and from
48.5 to 103.2 ng/µl in pod tissue of transgenic plants. Interestingly, 1F-25 and 1F-9
transgenic lines indicated the highest cry1F transcript level in all tissue types. On other
hand, the observed insect mortality and Cry1F protein was significantly less in 1F-25 and
1F-9 transgenic lines, contradicting the positive correlation of it with cry gene transcript
level. The transcript levels of cry1F gene in developed transgenic plants were
comparatively lower than that of cry1Ac and cry2Aa gene transcripts in their respective
transgenic plants. Further, in case of cry1Acm transgenic plants, M135-4 transgenic line
showed highest cry1Acm transcript accumulation in all tissue types. The transcript levels
of cry1Acm gene in developed transgenic plants were comparatively lower among all
other cry gene carrying transgenic plants. The northern blot analysis further confirmed
the presence of cry transcripts in selected transgenic pigeonpea plants and validated the
results obtained from qRT-PCR based transcript analysis.
213
Southern analysis is an important component of transgene analysis to prove the
integration of the foreign gene into host genome (Bhat and Srinivasan, 2002). Southern
hybridization helps to assess the number of independent transgene insertions, which is
important to find transformants with single, unaltered transgene insertions and
considered ideal for analysis (Bhat and Srinivasan, 2002). Southern analysis of selected
three transgenic lines indicated presence of single cry1Ac carrying T-DNA copy
insertions in pigeonpea genome. The hybridization pattern was similar for two lines
Ac20-2 and Ac20-3, whereas Ac29-1 produced distinct band with higher molecular
weight. It was interesting to notice the same position of T-DNA insertion in pigeonpea
genome for transgenic line Ac20-2 and Ac20-3 as revealed through juncture region
analysis. The recovered genomic flanking region from these two lines identified
sequence homology with pigeonpea genome sequence Scaffold130851. In case of
transgenic line Ac29-1, the position of T-DNA integration was in the genomic region
represented in Scaffold137204. Although, the pigeonpea draft genome is available, as it
is not processed completely, we could not retrieve more information about identified
genomic region for its gene content and other genomic features. The identification of
same position of T-DNA insertion and similar Southern hybridization pattern gave the
possibility of Ac20-2 and Ac20-3 as same transgenic line.
For cry2Aa transgenic plants, southern analysis of selected four transgenic lines
indicated integration of single cry2Aa carrying T-DNA in pigeonpea genome. The
hybridization pattern was similar for two lines 21A6-9 and 21A6-68, whereas 21A2-2 and
21A3-4 produced distinct banding pattern with higher molecular weight. It was interesting
notice the same position of T-DNA insertion in pigeonpea genome in case of transgenic
line 21A6-9 and 21A6-68 as revealed through juncture region analysis. The recovered
genomic flanking region from 21A6-9 and 21A6-68 transgenic lines identified sequence
homology with Scaffold127179. Further, in case of transgenic line 21A2-2 the position of
T-DNA integration was in the genomic region represented in Scaffold137204. In the
same Scaffold, the recovered flanking genomic region from cry1Ac transgenic line Ac29-
1 also reported sequence homology.
Similarly, in case of transgenic line 21A3-4 the position of T-DNA integration was
in the genomic region represented in Scaffold134438. The identification of same position
of T-DNA insertion and similar Southern hybridization pattern observed in case of 21A6-
214
9 and 21A6-68 given the possibility of these two transgenic lines as same transgenic
lines. The junction region analysis of 21A3-4 and 21A12-24 transgenic line reported
recovery of genomic region homologous to Scaffold134438. The Southern analysis of
21A12-24 expected to give interesting insights regarding the two transgenic lines 21A3-4
and 21A12-24 expressing cry2Aa gene.
The two transgenic lines carrying cry1Ac gene (Ac20-2 and Ac20-3) reported
same position of transgene insertion in pigeonpea genome. As these lines were
generated from same primary transformant i.e. Ac20 (T0 generation) might be the reason
for such observation. Similar kind of result was also recorded in case of two transgenic
lines carrying cry2Aa gene i.e. 21A6-9 and 21A6-68, having generated from same
primary transformant i.e. 21A6. Contradictory to above notion, although identified from
the different primary transformats viz., 21A3 and 21A12, the observation of same
position of insertion in case of 21A3-4 and 21A12-24 transgenic lines was interesting.
Southern analysis of 1F-25 and 1F-9 indicated presence of two T-DNA copy
insertions in pigeonpea genome, the probable reason for the reduced Cry protein and
insect mortality. The presence of two transgene copy might have resulted in post
transcriptional or post-translational silencing of the transgene expression. There are
many factors such as, DNA methylation, trans-inactivation, co-suppression may reduce
the gene expression (Li et al., 2002). The similar type transgene silencing phenomenon
has been reported in transgenic tobacco expressing uidA gene (Li et al., 2002). Study
reported the multiple copy insertion could result in the reduced to no GUS activity in
transgenic tobacco plants. The other transgenic plants, 1F-22, 1F-32, 1F-38 and 1F-33,
tested for Southern analysis indicated single copy T-DNA insertion in pigeonpea genome
and also found superior in insect mortality, cry1F gene expression in terms of both
protein and transcript level.
The genomic Southern blotting in case of cry1Acm gene carrying transformants
confirmed the integration of T-DNA in to plant genome. A weak hybridization signals
were recorded for all four samples viz., M2, M10, M16 and M135. The observation of
different Southern hybridization banding pattern clearly indicated different position of T-
DNA insertion in plant genome. Furthermore, an observation of single hybridization
215
signal in all tested transgenic plants unambiguously reported the single copy integration
of transgene.
Surekha et al. (2005) have used Southern blotting analysis for confirmation of 18
pigeonpea transgenic samples. Based on Southern blotting they studied the presence of
transgene and number of the inserts, size of the inserts in different plants confirming the
independent origin of the transgenic lines (Surekha et al., 2005). The PCR amplicon of
cry1Ab fragment have been used as probe for the Southern hybridization analysis and
hybridization signals detected in eight of the 12 plants analyzed for the integration of Bt
gene in the genome of these plants. The blotting analysis revealed that four of six
transgenic plants possessed single-copy integrations (Sharma et al., 2006).
Rao et al. (2008) performed Southern blot and confirmed the integration of the
transgene in the pigeonpea genome. Further, in similar kind of study, Ramu et al. (2012)
studied the transgene integration pattern using genomic Southern analysis, where strong
signal and the difference in the hybridization pattern in the selected transgenic plants
revealed the single copy integration in these plants. The Southern analysis of the
genomic DNA from T1 progenies of selected transgenic chickpea plants have shown
single DNA fragments of size ranging from 4.16 to 6.57 kb hybridizing with 1.845 kb
radiolabelled cry1Ab/ c probe (Mehrotra et al., 2011). Acharjee et al. (2010) have
Southern blotted the genomic DNA from pooled progeny of nine independent transgenic
chickpea lines and showed the integration of cry2Aa gene in eight of the nine lines, with
integration of one, two or more copies of the transgene.
Further, in present study, the observation of transgenic plants for selected plant
growth parameters indicated the appearance of non-parental phenotypes for five traits
viz., flower colour, growth habit, branching pattern, fresh pod colour and plant height.
The change in these traits was noticed in transgenic plants carrying cry1Ac and cry1F
genes. The appearance of such non-parental types was either may be due to integration
of cry gene or the somatic changes that might have happed during the transformation
and regeneration process. Rawat et al. (2011) have reported the development of
abnormal phenotypes such as extreme retardation in the growth of the plant, reduced
flowering, abscission of the flowers following crossing and no setting of bolls in
transgenic cotton plant.
216
As in case of developing countries, conventional methods of pest control have
been least effective and the greatest improvement in yields due to use of genetically
modified insect resistance technology has occurred there. It has been reported that at
the aggregate level, the global farm income gain due to use of genetically modified insect
resistance technology in maize and cotton have been $6.71 billion and $5.3 billion
respectively during 2012 (Brookes and Barfoot, 2014). Cumulatively since from 1996, the
global farm income gain has been $32.3 billion for genetically modified insect resistant
maize and $36.3 billion for that of cotton. Furthermore, from the past 17 years, the
adoption of crop biotechnology (by 17.3 million farmers in 2012) has clearly
demonstrated the important economic benefits of genetic engineering technology.
Reports also showed that many of farmers, especially from developed countries, have
benefited due to lower costs of production (Brookes and Barfoot, 2014). Current statistics
and understanding of economic benefits from use of genetically modified insect resistant
cultivars clearly points at immense scope for use of genetic engineering technology in
crop improvement programme.
The gene pyramiding in transgenic plants expected to be potentially more viable
strategy for supressing insect evolution leading to resistance against single cry genes
(Cao et al., 2002). Although transgenic lines carrying different cry gene (cry1Ac, cry2Aa,
cry1F and cry1Acm) developed in present study showed insect resistance up to 60.0 to
70.0 per cent, they can be further potentially used for gene pyramiding of different cry
genes for development of broad spectrum and durable resistance. Based on the
experience of Bt transgenic cotton performance world wide and in India as well, pyramid
products expressing cry1Ac anlong with any other gene would be ideal for commercial
products amoung the lines developed in present study. It would ideal in terms of
resistance management also. The pigeonpea transgenic expressing cry1F, cry1Acm etc.
would also be ideal in terms of bio-activity against H. armigera, if second generation Bt
cottons are grown around. Some lines showed uniform expression (as per bioassay) in
different tissue types which is principally appreciable. Although, the insect mortalities are
low in such lines, this lot will make separate study material for enhancement.
217
Furthermore, as the transformation method followed in present study found efficient and
easy for pigeonpea transformation, can be explored to developed more number of
transgenic plants with new cry gene sources that can be characterised for identification
of superior transgenic line with complete resistance to H. armigera.
218
6. SUMMARY AND CONCLUSIONS
Pigeonpea is an important pulse crop, particularly in India. Pigeonpea
production is limited by wide range of insects in which pigeonpea pod borer,
Helicoverpa armigera is major. Although, conventional breeding approaches have
been attempted in pigeonpea crop improvement, development of genetically modified
crops for insect resistance would be an ideal solution in the interest of the pigeonpea
farmers and crop productivity. Many Cry proteins are well known for their insecticidal
activity, and expression of them could improve insect resistance in many crop plants
such as, cotton, maize and soybean. The improvisation in in vitro regeneration
methods through organogenesis is important for future transgenic programs in crops
like pigeonpea. The in planta transformation method have shown immense potential in
transgenic development in many crop plants. Development of transgenic pigeonpea
expressing insecticidal cry genes and understanding their bioefficacy against
pigeonpea pod borer attack is important. Present study involved improvisation of in
vitro and in planta transformation and plantlet regeneration methods, development and
characterization of transgenic pigeonpea carrying four cry genes viz., cry1Ac, cry2Aa,
cry1F and cry1Acm separately. The summary of present investigation is presented
here;
1. The increasing concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) of three
cytokinins viz., BAP, TDZ and zeatin were assessed to study their effect on shoot
bud induction in two pigeonpea genotypes, ICPL 87119 and BSMR 736.
2. The explants response to different concentration regimes of BAP for shoot bud
induction ranged from 0.0 to 72.0 per cent in case of ICPL 87119 and from 0.0 to
73.0 per cent in case of BSMR 736. The number of shoot bud induced per
explant ranged from 0 to 52.6 for ICPL 87119 and from 0 to 53.7 for BSMR 736.
The maximum number of explants responded and number of shoot induced
noticed at 2.0 mg/L BAP concentration regime.
3. In case of embryo disc with half cotyledon explants, the explants response
ranged from 0 to 53.0 per cent (ICPL 87119) and 0 to 55.0 per cent (BSMR 736)
in pigeonpea genotypes. The number of shoot bud induced ranged from 0 to 4.8
219
in ICPL 87119 and from 0 to 4.4 in BSMR 736. In MS medium supplemented
with 2.0 mg/L BAP recorded highest number shoot buds (4.8 in ICPL 87119).
4. The cotyledonary node with cotyledon explants responded to different TDZ
concentration regimes ranged from 0 to 67.0 per cent in case of both test
genotypes. Maximum number of explants responded for shoot bud induction was
at 4.0 mg/L TDZ concentration level. The number of shoot bud induced per
explant ranged from 0 to 41.1 (ICPL 87119) and from 0 to 46.1 (BSMR 736) in
test genotypes.
5. The embryo disc with half cotyledon explants response to increasing TDZ
concentration regimes ranged from 0 to 44.0 per cent in ICPL 87119 and 0 to
50.0 per cent in BSMR 736. The number of shoot bud induced from embryo
discs with half cotyledon explant ranged from 0 to 4.7 (ICPL 87119) and from 0
to 4.5 (BSMR 736). At TDZ concentration regime of 4.0 mg/L, the highest
number shoot buds (4.7) was induced in ICPL 87119.
6. To the effect of zeatin concentration regimes in MS, the cotyledonary node with
cotyledon explants response ranged from 0 to 66.0 per cent in ICPL 87119 and
from 0 to 64.0 per cent in BSMR 736. The maximum number of shoot bud
induced per explant found at 2.0 mg/L zeatin concentration level. The number of
shoot bud induced from embryo discs with half cotyledon explant ranged from 0
to 4.2 in both genotypes.
7. There was an induction of more number of shoot buds in cotyledonary node with
cotyledons explants when compared with that of embryo discs with half
cotyledon explants. The cotyledonary node with cotyledons explants reported
immense potential for multiple shoot bud induction.
8. There was a concomitant decrease in resonance of explants for shoot bud
induction and number of shoot bud induced at higher concentration regimes of
tested cytokinins in growth medium. There was an absence of any artefacts such
as deformed shoots when explants were cultured on MS augmented with
different TDZ concentration regimes.
220
9. Well developed shoots of more than 3 cm long were excised and cultured on MS
medium augmented with increasing IBA concentration regimes. The frequency of
rooting varied with different IBA concentration regimes ranging from 20 to 80% in
both genotypes. The highest root induction was noticed in MS media with 0.5
mg/L IBA.
10. The improvisation of in planta transformation protocol to increased
transformation efficiency was performed using following treatment such as,
Agrobacterium tumefaciens infection alone, A. tumefaciens infection using A.
tumefaciens culture with tobacco extract added in overnight grown culture and A.
tumefaciens infection using air evacuation to increase the proximity between A.
tumefaciens and embryonic axis.
11. There was 80.00 per cent explant response, 53.75 per cent explant survival and
3.0 per cent transformation efficiency recorded in case of A. tumefaciens
infection alone. In case of A. tumefaciens infection using A. tumefaciens culture
along with tobacco extract, recorded 85.00 per cent explant response, 90.00 per
cent explant survival and 6.5 per cent transformation efficiency. The 66.50 per
cent explant response, 90.98 per cent explant survival and 12.0 per cent
increase in transformation efficiency was recorded for the treatment involving air
evacuation to increase the proximity between A. tumefaciens and embryonic
axis.
12. By following improvised in planta transformation protocol, the T1 plant progeny
screening of developed 741 primary transformants identified a set of eighty eight
putative transgenic plants for integration of cry1Ac gene. The in vitro generated
ten transformants (T0 generation) were hardened and grown in transgenic
containment facility.
13. The obtained T2 seeds were sown in plant to row progeny manner to get T2
generation plants. From each parental T1 generation plants up to 40 seeds were
sown and obtained T2 plants were subjected for gene specific PCR assay.
Among developed eighty eight, for forty eight transgenic plant progenies of T1
generations plants recorded chi-square calculated value less than table chi-
square value.
221
14. The larval mortality in case of leaf tissues ranged from 25.0 to 70.0 per cent.
Whereas, it was 22.5 to 52.5 per cent in case of flower tissues and 17.5 to 47.5
per cent in case of pod tissue. The Cry protein level ranged from 0.305 to 0.847
µg/g of fresh leaf tissue, from 0.221 to 0.671 µg/g of fresh flower tissue and
0.250 to 0.685 µg/g of fresh pod tissue.
15. Set of eight transgenic plants viz., Ac16-1, Ac20-2, Ac20-3, Ac29-1, Ac29-3,
Ac31-1, Ac31-2 and Ac50-1 carrying cry1Ac gene were identified and forwarded
to T3 generation. The homozygosity in T2 parental plants viz., 20-2-7, 20-3-2, 29-
1-10, 31-2-9, 31-2-12, 31-1-2 and 31-1-3 belonging to five transgenic lines were
confirmed using cry1Ac gene specific PCR assay.
16. The larval mortality in T3 generation plants ranged from 41.25 to 61.25 per cent
in case of leaf tissue, 32.5 to 47.5 per cent in case of flower tissue and 35.0 to
52.5 per cent in case of pod. In leaf tissues, the Cry1Ac protein level ranged from
0.322 to 0.736 µg/g of fresh tissue weight. Similarly, the Cry protein
accumulation in flower tissues was noticed from 0.254 to 0.646 µg/g and from
0.269 to 0.691 µg/g of fresh pod tissue. The cry1Ac transcript level ranged from
24.6 to 165.1 ng/µl in fresh leaf tissue, from 15.6 to 149.5 ng/µl in flower and
from 18.3 to 152.4 ng/µl in pod tissue.
17. In Sothern blot analysis, an observation of strong signal and the difference in the
hybridization pattern in three selected transgenic plants viz., Ac29-1, Ac20-2 and
Ac20-3, revealed the single copy integration in these plants. Northern blot
analysis indicated hybridization signals in all selected transgenic plant samples
viz., Ac31-2, Ac29-1, Ac31-1, Ac29-3, Ac20-2 and Ac20-3.
18. Juncture region analysis revealed that flanking genomic region recovered from
Ac20-2 and Ac20-3 showed homology with Scaffold130851 of pigeonpea
genome. In case of Ac29-1, the recovered flaking genomic region indicated
homology with Scaffold137204.
19. A set of sixty five putative transformants carrying cry2Aa gene was identified
from 348 primary transformants. The chi-square analysis of observed number of
plants with and without cry2Aa gene indicated that among sixty five transgenic
222
lines, sixteen were following 3:1 (positive: negative) ration for transgene
segregation in T2 generation.
20. The larval mortality in case of leaf tissues ranged from 5.25 to 65.75 per cent,
between 5.25 to 40.5 per cent in case of flower tissue and from 10.25 to 50.25
per cent in case of pod tissue. The transgenic lines, 21A4-21, 21A12-26, 1A2-30,
1A2-42 found consistent in their performance across different tested tissue types.
The Cry2Aa protein level in leaf tissue ranged from 0.013 to 3.231 µg/g of fresh
leaf tissue. Whereas, it was 0.010 to 2.854 µg/g in case of fresh flower tissues
and 0.012 to 2.811 µg/g in case of fresh pod tissue.
21. The transgenic lines viz., 21A2-2, 21A3-4, 21A6-9, 21A5-10, 21A6-12, 21A5-14,
21A5-16, 21A5-19, 21A12-24, 21A12-25, 21A5-59, 21A5-60, 21A4-62, 21A6-68
and 21A4-73 were advanced to T3 generation. Among these fifteen, in four
transgenic lines homozygous nature of cry2Aa locus was detected. The T2
parental plants with homozygous nature were 21A2-2-1, 21A3-4-7, 21A5-16-1
and 21A4-62-6.
22. The bioassay results reported that the larval mortality in case of leaf tissue
ranged from 43.33 to 68.33 per cent, in case of flower tissue from 23.5 to 48.5
per cent and in case of pod from 28.5 to 53.25 per cent. The Cry2Aa protein
levels in leaf tissues ranged from 0.370 to 1.417 µg/g of fresh tissue weight, from
0.312 to 1.021 µg/g in flower tissue and from 0.309 to 0.989 µg/g in in pod.
Further, the cry2Aa transcript level ranged from 48.5 to 134.5 ng/µl in fresh leaf
tissue, from 41.2 to 110.2 ng/µl in flower tissue and from 42.8 to 100.5 ng/µl in
pod.
23. There was a positive correlation between insect morality and Cry protein
accumulation. The frequency distribution analysis of transgenic plants following
3:1 transgene segregation pattern and other than 3:1 segregation pattern
revealed that majority of high Cry protein accumulating transgenic plants were
following 3:1 transgene segregation pattern. The correlation analysis of larval
mortality, Cry protein and cry transcript revealed strong positive in leaf, flower
and pod tissues for these traits of T3 generation transgenic lines.
223
24. The southern bolt analysis revealed a single but weak hybridization signals for
two test samples belonging to 21A2-2 and 21A3-4 transgenic lines with different
prob hybridization pattern. There were strong hybridization signals in other two
other test samples viz., 21A6-9 and 21A6-68 with similar hybridization pattern,
indicating the possibility of same position of T-DNA integration in plant genome.
The northern blot analysis showed hybridization signals in all selected transgenic
plant samples viz., 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68.
25. The local blast was set using BioEdit tool for processed contigue sequences and
it was observed that flaking genomic region recovered from 21A2-2 showed
homology with Scaffold137204 of pigeonpea genome. Similarly, the flaking
genomic region recovered from 21A3-4 and 21A12-24 showed homology with
Scaffold134438. In case of 21A6-9 and 21A6-68, the recovered flaking genomic
region indicated with Scaffold127179 of pigeonpea draft genome.
26. The T1 seeds were harvested from well-established 177 primary transformants
and T1 generation was raised. The T1 progeny plant screening using cry1F gene
specific PCR assay identified a set of fourteen putative transgenic plants for
integration of cry1F gene. Among developed fourteen, for seven transgenic
plants the chi-square calculated value was recorded less than table chi-square
value.
27. The larval mortality in case of leaf tissues ranged from 22.5 to 62.5 per cent, it
was 20.0 to 57.5 per cent in case of flower and 12.5 to 47.5 per cent in case of
pod. The Cry protein level ranged from 0.170 to 1.032 µg/g of fresh leaf tissue,
0.113 to 0.870 µg/g of fresh flower tissue and 0.117 to 0.782 µg/g of fresh pod
tissue. The cry1F transcript level ranged from 47.6 to 105.3 ng/µl in fresh leaf
tissue, 45.2 to 101.0 ng/µl in flower tissue and from 48.5 to 103.2 ng/µl in pod
tissue.
28. The correlation analysis of larval mortality, Cry1F protein and cry1F transcript
reported positive in leaf, flower and pod tissues for these traits of T2 generation
transgenic lines.
224
29. An observation of strong Southern hybridization signals and the difference in the
hybridization pattern in the four selected transgenic plants viz., 1F-22, 1F-32, 1F-
38 and 1F-33, revealed the single copy integration in these plants. Whereas, in
two plant samples viz., 1F-25 and 1F-9, two copies of T-DNA integration in plant
genome was noticed. The northern blot analysis indicated hybridization signals in
all selected transgenic plant samples viz., 1F-25, 1F-32, 1F-33, 1F-9, 1F-22 and
1F-38.
30. The T1 progeny plant screening from 120 primary transformants identified a set
of eleven putative transgenic plants carrying cry1Acm gene as revealed by gene
specific PCR assay. The chi-square analysis of observed number of plants with
and without cry1Acm gene indicated that among eleven transgenic lines, seven
were following 3:1 (positive: negative) ration of transgene segregation.
31. The larval mortality in case of leaf tissues ranged from 40.0 to 62.5 per cent, in
case of flower it ranged from 37.5 to 57.5 per cent and in pod from 35.0 to 52.5
per cent. The Cry protein level in leaf tissue ranged from 0.322 to 0.907 µg/g of
fresh leaf tissue; 0.202 to 0.503 µg/g of fresh flower tissue and 0.186 to 0.405
µg/g of fresh pod tissue. The cry1Acm transcript level was ranged from 40.15 to
105.5 ng/µl of fresh leaf tissue; in flower it ranged from 39.80 to 103.0 ng/µl and
in pod from 39.83 to 102.9 ng/µl.
32. A single but weak Southern hybridization signals were noticed for all test
samples M2, M10, M16 and M135 transgenic lines. The Southern hybridization
pattern was different for all samples, indicated different position of T-DNA
insertion in plant genome with single copy integration of transgene. The northern
hybridization signals confirmed production of transgene transcripts in all selected
transgenic plant samples viz., M135, M55, M2, M5 and M7.
33. Among tested plant eight growth parameters, development of non-parental type
phenotypes were noticed for plant height, flower colour and pod colour traits.
34. Based on comparative analysis of different transgenic events expressing cry1Ac,
cry2Aa, cry1F and cry1Acm genes independently, it was indicative that the
225
transgenic events expressing differential levels of cry genes were varying for
their insecticidal activity against H. armigera.
35. Although transgenic lines carrying different cry gene (cry1Ac, cry2Aa, cry1F and
cry1Acm) developed in present study showed insect resistance up to 60.0 to
70.0 per cent, they can be further potentially used for gene pyramiding of
different cry genes for development of broad spectrum and durable resistance.
36. Some lines showed uniform expression (as per bioassay) in different tissue types
which is principally appreciable. Although, the insect mortalities are low in such
lines, this lot will make separate study material for enhancement. The
transformation method followed in present study can be explored to developed
more number of transgenic plants with new cry gene sources that can be
characterised for identification of superior transgenic line with complete
resistance to H. armigera.
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APPENDIX I
a. Yeast Mannitol Agar (YEMA) broth (100 ml)
Mannitol 1.0 g
Orthophosphate (KH2PO4) 20.0 mg
Dipotassium hydrogen phosphate (K2HPO4) 20.0 mg
Yeast extract 100.0 mg
Magnesium sulphate (MgSO4) (1M) 80.0 µl
Calcium chloride (CaCl2) (1M) 40.0 µl
b. Yeast Mannitol Agar (YEMA) (100 ml)
Mannitol 1.0 g
Orthophosphate (KH2PO4) 20.0 mg
Dipotassium hydrogen phosphate (K2HPO4) 20.0 mg
Yeast extract 100.0 mg
Magnesium sulphate (MgSO4) (1M) 80.0 µl
Calcium chloride (CaCl2) (1M) 40.0 µl
Agar agar 1.8 g
259
APPENDIX II
a. Loading dye composition
Loading dye (6X) 0.25% bromophenol blue
40% (w/v) sucrose in water
b. Ethidium bromide
Dissolve 10 mg/ml in distilled water and store at 4 °C in a dark bottle.
c. Recipe for 1% agarose gel (40 ml)
Agarose 400.0 mg
1X Tris-acetate-EDTA (TAE) 40.0 ml
Ethidium bromide (10 mg/ml) 2.0 µl
d. 50X Tris-acetate-EDTA (TAE) composition
Tris base 242.0 g
Glacial acetic acid 57.1 ml
0.5 M Ethylenediamine tetraacetic acid (EDTA) (pH 8.0) 100.0 ml
Make up the volume to 1000 ml with double distilled water
260
APPENDIX III
a. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part A)
Kabuli gram flour 100.0 g
Yeast 30.0 g
Sterile water 400.0 ml
b. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part B)
Agar agar 14.0 g
Sterile water 400.0 ml
c. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part C)
Wesson’s salt 7.0 g
Casein 5.0 g
Ascorbic acid 3.0 g
Multivitamin mix (Polybion SF) 4.0 ml
Sorbic acid 1.0 g
Methyl parahydroxy benzoate (MPHB)* 2.0 g
Streptomycin sulphate 0.25 g
Bavistin 2.0 g
* Dissolve in 5.0 ml of 95.0% Ethanol and added to the diet
261
APPENDIX IV
a. RNA loading dye composition (5X) (10 ml)
Saturated aqueous bromophenol blue solution 16.0 µl
EDTA (0.5M; pH: 8.0) 80.00 µl
Formaldehyde 720.0 µl
Glycerol (100 %) 2000.0 µl
Formamide 3048.0 µl
10X FA gel buffer 4000.0 µl
RNase free water 100.0 µl
b. Ethidium bromide
10 mg/ml in RNase free water. Store at 4°C in a dark bottle.
c. Recipe for 1.2% formaldehyde agarose gel (60 ml)
Agarose 720.0 mg
Formaldehyde 1.0 ml
Ethidium bromide (10 mg/ml) 3.0 µl
1X 3-(N Morpholino) propanesulphonic acid (MOPS) buffer 60.0 ml
d. 10X 3-(N Morpholino) propanesulphonic acid (MOPS) electrophoresis buffer
3-(N Morpholino) propanesulphonic acid (MOPS) 41.8 g
Sodium acetate (DEPC-treated) 20.0 ml
EDTA (DEPC-treated; pH: 8.0) 20.0 ml
Total volume 1000 ml with DEPC treated water
262
APPENDIX V
a. Denaturation Buffer (500 ml)
Sodium hydroxide (0.4M) 8.0 g
Sodium chloride (3M) 87.7g
Sterile water Make up the vol. 500.0 ml
b. Neutralization Buffer (500 ml)
Tris (1M) pH 7.4 78.5 g
Sodium chloride (1.5M) 43.9 g
Sterile water Make up the vol. 500.0 ml
c. Pre-hybridization buffer (100 ml)
20X Saline Sodium Citrate (SSC) 25.0 ml
Formamid 50.0 ml
Sterile water 25.0 ml
Casein 0.5 g
SDS 0.1 g
d. Marker preparation (10 µl)
Dig-labelled Marker 3.0 µl
Bromo phenol blue 3.0 µl
Sterile water 4.0 µl
e. Hybridization buffer
Pre-hybridization buffer 10.0 ml
Dig-labelled DNA probe 10.0 µl
263
f. Wash buffer I (200 ml)
20X Saline sodium citrate (SSC) 20.0 ml
10% Sodium dodecyl sulfate (SDS) 2.0 ml
Sterile water 178.0 ml
g. Wash buffer II (200 ml)
20X Saline sodium citrate (SSC) 5.0 ml
10% Sodium dodecyl sulfate (SDS) 2.0 ml
Sterile water 193.0 ml
h. Wash buffer III (500 ml) pH 7.5
Malic acid (2.0M) 25.0 ml
Sodium chloride (4.0M) 16.15 ml
Twin 20 1.5 ml
Sterile water Vol. made 500 ml
i. Malic acid buffer (500 ml) pH 7.5
Malic acid (2.0M) 25.0 ml
Sodium chloride (4.0M) 16.15 ml
Sterile water Vol. made 500 ml
j. Blocking solution (30 ml)
Malic acid buffer pH 7.5 27.0 ml
Blocking reagent 3.0 ml
264
k. Antibody solution (15 ml)
Blocking solution 15.0 ml
Antibody 3.0 µl
l. Detection buffer (100 ml) pH 9.8
Tris-Cl (1M) 10.0 ml
Sodium chloride (4M) 2.5 ml
Sterile water Vol. made 500 ml
m. Substrate solution (10 ml)
Detection buffer 10.0 ml
NBT/BCIP 200.0 µl
265
APPENDIX VI
a. Luria agar (LA)
Tryptone 10.0 g/l
Yeast extract 5.0 g/l
Sodium chloride 5.0 g/l
Agar 18.0 g/l
b. Sodium Chloride-Tris-EDTA (STE) buffer
Tris-HCl (pH 8.0) 10 mM
NaCl 0.1 M
EDTA (pH 8.0) 1.0 mM
Autoclaved and stored at 4 °C
c. Alkaline-lysis solution I
Glucose 50 mM
Tris-HCl (pH 8.0) 25 mM
EDTA (pH 8.0) 10 mM
Autoclaved and stored at 4 °C
d. Alkaline-lysis solution II
Sodium hydroxide (NaOH) 0.2 N
Sodium dodecyl sulfate (SDS) 1% (w/v)
(Prepared fresh and used at room temperature)
e. Alkaline-lysis solution III
5 M Potassium acetate 60.0 ml
Glacial acetic acid 11.5 ml
Double distilled water 28.5 ml
Autoclaved and stored at 4 °C
Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST
Helicoverpa armigera AND MOLECULAR ANALYSES OF SELECTED EVENTS
2014 MAHALE BARKU M. Dr. B. FAKRUDIN
Major Advisor ABSTRACT
In ICPL87119 and BSMR736, MS medium supplemented with 2.0 mg/l BAP,
4.0 mg/l TDZ and 2.0 mg/l zeatin, separately, induced maximum shoot buds, 53.7,
46.1 and 40.9 respectively; any further increase in cytokinins levels resulted in
reduced shoot buds. The MS basal with 0.5 mg/l IBA induced maximum and healthier
roots (4.8±0.7). In planta transformation revealed 80.00, 85.00, 66.50% explant
response, 53.75, 90.00, 90.98% explant survival and 3.0, 6.5, 12.0% transformation
efficiency in Agrobacterium tumefaciens infection alone, A. tumefaciens culture with
tobacco leaf extract and air evacuation, respectively. The 88 putative transformants
carrying cry1Ac were developed, of which 48 showed 3:1 transgene segregation
pattern in T2. Insect mortality ranged from 25.0 to 70.0% whereas, Cry1Ac protein
level from 0.31 to 0.85 µg/g and cry1Ac transcript level from 15.6 to 165.1 ng/µl,
validated through northern blotting in different tissues (leaf, flower and pod). In case
of cry2Aa, 65 transformants developed, of which 16 showed 3:1 transgene
segregation in T2. Insect mortality ranged from 5.25 to 65.75% whereas, Cry2Aa
protein and transcripts ranged from 0.01 to 3.23 µg/g and 41.2 to 134.5 ng/µl,
respectively. Southern and juncture analyses of selected three cry1Ac and five
cry2Aa transformants confirmed T-DNA integration in plant genome. Fourteen
transformants carrying cry1F were developed, of which seven showed 3:1 transgene
segregation pattern in T2, wherein insect mortality ranged from 10.0 to 62.5%, Cry1F
protein level from 0.113 to 1.032 µg/g and transcripts ranged from 45.2 to 105.3
ng/µl. Similarly, eleven cry1Acm transformants were developed, of which seven
showed 3:1 transgene segregation in T2. Insect mortality ranged from 35.0 to 62.5%
whereas, protein level and transcripts ranged from 0.19 to 0.91 µg/g and 41.2 to
134.5 ng/µl respectively, in tested tissues. Pigeonpea transformation procedures and
generated events of present study could be prospected for their further use.