standardization of protocol for genetic transformation of annexin gene in
TRANSCRIPT
STANDARDIZATION OF PROTOCOL FOR GENETIC TRANSFORMATION OF ANNEXIN
GENE IN Musa acuminata cv. Patakpura FOR DROUGHT RESISTANCE
A
THESIS SUBMITTED TO
THE ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY BHUBANESWAR
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
MASTER OF SCIENCE IN AGRICULTURE (AGRICULTURAL BIOTECHNOLOGY)
BY
DRAMADRI GERALD AFAYO
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY COLLEGE OF AGRICULTURE
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY BHUBANESWAR-751003, ODISHA
2014
THESIS ADVISOR: Dr. A.B.DAS
CONTENT
____________________________________________________________
CHAPTER PARTICULARS PAGE
______________________________________________________________________
I INTRODUCTION 1-17
II REVIEW OF LITERATURE 18-32
III MATERIALS AND METHODS 33-40
IV RESULTS 41-55
V DISCUSSION 56-62
VI SUMMARY AND CONCLUSION 63-64
VII REFERENCES i-xxv
VIII APPENDICES xxvi-xxviii
LIST OF TABLES
TABLE NO.
PARTICULARS PAGE NO.
1.1 Production of top 5 world producers of banana in 2012 4
1.2 Banana exports of top 5 world exporters in 2011 4
1.3 Production of top 5 world producers of plantains in 2012
5
1.4 Exports of top 5 world exporters of plantains in 2011 5
2.1 In vitro regeneration studies of banana 29
2.2 Genetic transformation of banana via Agrobacterium 30
4.1 Effects of surface sterilization on aseptic culture and survival of explants
41
4.2.1 Effect of growth regulators on callus induction from corm slices of banana cv. patakpura
43
4.2.2 Effect of various concentrations og growth regulators BAP and IAA along with Adenine sulphate on multiple shoot induction from corm slices of Musa acuminate cv. patakpura
44
4.3 Effect of various concentrations of cytokinin BAP and auxins IAA and NAA on in vitro shoot multiplication
45
4.4 Effect of various concentrations of IAA on root formation from multiple shoots of Musa acuminate cv. patakpura
46
4.6 Kanamycin based selection 47
4.7 Effect of cefotaxime on shoot induction in the medium containing different concentrations of cefotaxime
48
4.8 In vitro transformation studies using Annexine BJ2 gene
48
LIST OF FIGURES
FIG. PARTICULARS PAGE
1.1 Production of top 5 producers of banana in 2012 7
1.2 Banana export of top 5 exporters in 2011 7
1.3 Plantain production by top 5 producers of the world in 2012
8
1.4 Plaintain export of top 5 exporters of the world in 2011 8
2.1 An illustration of how Agrobacterium can be used to transform plant cells in order to regenerate transgenic plants
28
2.2 Mechanism of T-DNA transfer 28
3.1 Gene construct of AnnBj2 and AnnBj3 gene 35
3.2 Gene construct of GUS marker 35
3.3 Schematic map of binary vector pCAMBIA2301 35
4.1 Effect of growth regulators on callus induction 49
4.2 Effect of IAA concentration on root formation 49
4.3 Kanamycin based selection 50
4.4 Effect of cefotaxime concentration on shoot survival 50
LIST OF PLATES
PLATE PARTICULARS PAGE
Plate 1 A banana plant with a bunch of female flowers 6
Plate 2 1. Shoot initiation on MS +8mg/l BAP+1mg/l IAA after 3 weeks. 2. Shoot buds after 4 weeks. 3. Shoots after 4.5 weeks. 4. Elongated shoots after 5 weeks of culture. 5. Elongated shoots after 6 weeks. 6. Shoot multiplication on MS+4mg/l BAP+0.5mg/l IAA and
NAA. 7. Shoots after 1 month of culture on multiplication medium.
51
Plate 3 8. Root initiation and development on MS+1mg/l IAA+0.5mg/l activated charcoal.
9. Prehardening of rooted plants on 1:1:1(sand: soil: vermicompost) in culture room after 7days.
9a. Prehardened plants after 12 days. 10. Final hardening in poly house On 1:1:1 (sand: soil:
vermicompost) after7 days of transfer.
52
Plate 4 11. Final hardening after 12 days of transfer to poly house. 12. Hardened plants after 2 months in polyhouse. 13. Hardened plants after 3 months in polyhouse.
53
Plate 5 14. Callus initiation after 45 days of culture on MS +2mg/l BAP + 4mg/l 2, 4-D
15. Organogenesis from callus on MS + 4mg/l BAP + 1mg/l 2, 4-D
16. Somatic embryos scanned with S-3400N scanning Electron Microscope
16a. Somatic embryos scanned with S-3400N scanning Electron Microscope
54
Plate 6 17. Bacterial culture on selection medium containing kanamycin and rifampicin
18. Co-cultivation of embryogenic cell mass with bacteria
after infection 19. GUS stained embryogenic mass with distinctly visible
somatic embryos. 20. Shoot bud initiation on selection medium 21. Shoot regeneration on selection medium.
55
LIST OF ABBREVIATIONS USED
2, 4-D 2, 4-Dichlorophenoxy acetic acid Ads Adenine sulphate BAP Benzyl aminopurine CaMV Cauliflower Mosaic Virus cm centimeter d day DNA Deoxyribonucleic acid ECS Embryogenic cell suspension EDTA Ethylene diamine tetra acetic acid FAO Food and Agriculture Organization FAOSTA Online FAO Statistical Database containing statistics on agriculture, nutrition, fisheries, forestry, food aid, land use and population Fig. Figure GUS beta-glucuronidase HCl Hydrochloric acid Hrs Hours IAA Indole-3- acetic acid IBA Indole-3- butyric acid LB Left T-DNA border sequence LB Luria Bertani mg/l milligram per litre Min. Minute ml mililitre mm millimeter MS Murashige and Skoog NAA Napthalene acetic acid NaOH Sodium hydroxide nm Nanometer OD Optical density PCR polymerase chain reaction pH Hydrogen ion concentration RB Right T-DNA border sequence rpm Revolutions per minute TAE Tris acetic acid EDTA TBE Tris boric acid EDTA T-DNA Transferred DNA TDZ Thidiazuron v/v volume/volume w/v weight/volume X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronide µg Microgram µl Microlitre µM Micromolar
1
I. INTRODUCTION
1.1 Back ground
Bananas and plantains are monocotyledonous plants in the genus Musa
(Musaceae, Zingiberales). They are giant herbs, commonly up to 3 m in height, with
no lignifications or secondary thickening of stems that is characteristic of trees
(Tomlinson, 1969). The centre of origin of the group is in South-East Asia, where
they occur from India to Polynesia (Simmonds, 1962). The centre of diversity has
been placed in Malaysia or Indonesia (Daniells et al., 2001), although considerable
diversity is known throughout the range. The plants are distributed mainly on margins
of tropical rainforests (Wong et al., 2002).
Banana is one of the most important staple food crops in the tropics and a
source of income to millions of poor subsistence farmers. It is the fourth most
important food crop in developing world's after rice, wheat and maize (Bioversity
International, 2006). Banana is grown in over 120 countries worldwide (Thangavelu
and Mustaffa, 2012) covering about 10 million hectares, with an annual world
production estimated at 107 million tones. India is the largest producer in the world
(24.8 million tonnes) followed by China (10.8 million tonnes). Production share by
region indicated that Asia produced 56%, Americas 26.6%, Africa 15.6%,
Oceania1.5% and Europe 0.4%. In Africa, the total production of banana was
estimated at 15.8 million tonnes of which Uganda produced an estimated 5.7 thousand
tonnes (FAOSTAT, 2012). In Uganda, it is one of the most important staple crops
contributing about 30% of the total food consumption and 14% total crop value
(Kalyebara et al., 2005). About 24% of the agricultural households are engaged in
banana production. Banana being a year round fruiting crop ensures food security at
household level, providing food to more than 70% of Uganda’s population on a
regular basis. Banana is primarily grown for subsistence needs and any surplus for
sale to local markets.
Worldwide, well over a thousand banana cultivars or landraces are recognized.
The vast majority of the cultivated bananas (Pollefeys et al., 2004) are derived from
inter- and intraspecific crosses between two diploid (2n = 2x = 22) wild species, Musa
acuminata and Musa balbisiana (Simmonds and Shepherd, 1955). In terms of the
2
chromosome sets, these are designated as having the genome constitution AA (M.
acuminata) or BB (M. balbisiana). These diploid Musa species have seeded fruit with
little starch and only a small amount of fleshy pith, and are of not much value as a
crop. The cultivated bananas and plantains differ from their wild relatives by being
seedless and parthenocarpic – the fruit develops without seed development or
pollination and fertilization. The genetic basis of the mutation (or mutations) in the A
genome that gives rise to parthenocarpy has not been characterized, and no
parthenocarpy has been identified in B genome diploids, although hybrids of A and B
show the character. Most of the cultivars are wild collections made by farmers of
spontaneously occurring mutants with parthenocarpic fruit production, which were
brought into cultivation and then multiplied and distributed by vegetative propagation.
There is no straightforward botanical distinction between bananas and plantains but,
in general, bananas refer to the sweeter forms that are eaten uncooked, while starchy
fruits that are peeled with a knife when unripe and then cooked are referred to as
plantains and cooking bananas, while some cultivars are ‘beer bananas’ for
fermentation of the juice, or used for deep frying as banana chips.
Many of the domesticated bananas have proved to be triploid, 2n = 3x = 33,
with genome constitutions of AAA (mainly the sweet dessert bananas), AAB or ABB
(mainly but not exclusively starchy plantains eaten after cooking). There are also
seedless cultivated AA and AB diploids, and tetraploids (2n = 4x = 44) with genome
constitutions of AAAA, AAAB, AABB and ABBB. These various plants have been
collected from multiple, independent sources in the wild, so the hybridization events
and mutations giving rise to the seedless and parthenocarpic characters have occurred
many hundreds of times. Where fertile plants occur together, hybridization continues
to produce new diversity (Pollefeys et al., 2004) and parental combinations.
Simmonds (1962) considered five plant characteristics that lead to farmers picking
plants for cultivation: plant vigour, yield, seedlessness, hardiness and fruit quality, the
first four of which are related to polyploidy (triploidy).
1.2 Description of banana plant
Banana is a monoecious plant having male flowers at the tip of inflorescence and
female flowers behind (Fig. 1). The fruit of banana or plantain is a product of
parthenocarpy and characterised as berry with a leathery outer peel that contains much
3
collenchyma (Daniells et al., 2001). The fruits are formed in layers called combs or
hands, consisting of 10–20 bananas, and there are 6–15 combs per stalk. The latter
equals 40–50 kg per stalk or ten or more tons per acre. If commercially grown, the
large terminal bud and bracts are removed to redirect sugars to the developing fruits.
An unripened banana and the plantain have high starch and low sugar levels plus
copious amounts of bitter-tasting latex. Starch is converted to sugar as the fruit ripens,
so that bananas can eventually contain about 25% of total sugars. As the banana
ripens, the latex is also decomposed. Plantain has the stinging, bitter latex, so the peel
is removed with a knife and the pulp is soaked in salt water for 5–10 min prior to
cooking (http://www.crfg.org). Bananas are harvested unripe and green, because they
can ripen and spoil very rapidly. The fruits are cleaned of old floral parts, combs and
divided into smaller bunches. Poorly formed fruits are removed, and bunches are
thrown into a water bath, where latex is washed away. Then fruits are dried and
usually placed in a ripening room for several days before their transfer to market, or
exported after storing and packing with cushion (usually paper). Presence of naturally
formed ethylene gas, produced by ripe fruits, hastens considerably the ripening of
surrounding, greener fruits (http://www.botgard.ucla.edu)
1.3 Importance of Banana and Plantains
Banana is having great importance in the world due to their commercial and
high nutritional value. Bananas are multipurpose plants because most of their parts
can be used in various ways, depending on the species. The most important part is the
edible fruit, which can be eaten either ripe as a dessert, or unripe as boiled, fried or
roasted food (Smith et al., 2005). Nutritionally, the fruit is rich in carbohydrates,
vitamins A, B, and C, and potassium (Aurore et al., 2009). The unripe fruit can be
brewed to form beer and wine, or processed into sauce, flour, chips, crisps, smoked
products, and confectionary. Unripe fruit is also a source of amylase and starch (van
den Houwe et al., 2000). Male floral buds can be eaten as a boiled vegetable, whereas
pseudostems are a source of fiber for the manufacture of rope, paper, and textiles.
Banana leaves are used for thatching, in the production of fabric and cordage, and as
mulch and animal forage (Smith et al., 2005). Species such as M. ornata and M.
veluntina are popular ornamental plants (Heslop-Harrison and Schwarzacher 2007).
Banana has also been found effective against colorectal cancer (Deneo-Pellegrini et
4
al., 1996) breast cancer, (Zhang et al., 2009) and renal cell carcinoma (Rashidkhani et
al., 2005).
Bananas are popular as fresh fruit in temperate countries. In 2011, the world
export of bananas, consisting mainly of Cavendish-type dessert bananas, was
estimated to be 18 million tons (20% of world production), (FAOSTAT 2011). The
most important attributes that make the Cavendish subgroup the main bananas for
export are related to their reliability during transport and their shelf life, rather than
taste. In economic value, banana fruit ranked fifth in the world trade for agricultural
crops (Aurore et al., 2009).
Table 1.1: Production of top 5 world producers of banana in 2012
Country Production (× 1,000,000 tonnes) Percentage
India 24.8 24.38
China 10.8 10.63
China mainland 10.5 10.34
Philippines 9.0 9.04
Ecuador 7.0 6.87
Source: FAOSTAT 2012
Table 1.2: Banana exports of top 5 world exporters in 2011
Country Export (×1,000,000 tonnes) Percentage
Ecuador 5.7 30.86
Philippines 2.0 10.93
Costa Rica 1.9 10.22
Colombia 1.8 9.76
Guatemala 1.4 7.61
Source: FAOSTAT 2011
Uganda is the leading producer and consumer of plantains in the world
(FAOSTAT, 2012). East African Highland Bananas (EAHB) serves as the principle
staple food (‘matooke’) in Uganda with an average daily consumption of 0.6 kg/capita
(FAOSTAT, 2004). This is due to the continuous fruiting habit of EAHB varieties, an
5
ability that provides food to millions of families throughout the year without hunger-
gaps as opposite to cereal and root crop-based systems.
‘Matooke’ is the staple food for over 7 million people in Uganda (Karamura
and Karamura, 1994) with more than 66% of urban dwellers depending on it
(Rubaihayo, 1991). Besides providing a source of income through local sales in urban
centers, other uses of bananas in Uganda include livestock feeds, mulch, medicine and
fiber for thatching and making crafts (Rubaihayo and Gold, 1993).
Table1.3: Production of top 5 world producers of plantains in 2012
Country Production (× 1,000,000 tonnes) Percentage
Uganda 9.2 24.75
Ghana 3.5 9.57
Cameroon 3.4 9.28
Colombia 3.3 8.95
Rwanda 3.2 8.66
Source: FAOSTAT 2012
Table1.4: Exports of top 5 world exporters of plantains in 2011
Country Export (10,000 tonnes) Percentage
Peru 10.8 25.88
Guatemala 10.2 24.47
Colombia 8.6 20.81
Nicaragua 3.9 9.49
Belgium 3.5 8.57
Source: FAOSTAT 2011
6
Plate 1: A banana plant with a bunch of female flower
7
Fig.1.1 Production of top 5 producers of banana in 2012
Fig.1.2 Banana export of 5 top exporters in 2011
0
5
10
15
20
25
30
India China China mainland Philippines Ecuador
Production (× 1,000,000 tonnes)
0
1
2
3
4
5
6
Ecuador Philippines Costa Rica Colombia Guatemala
Export (×1,000,000 tonnes)
8
Fig.1.3 Plantain production by top 5 producers of the world in 2012
Fig.1.4 Plantain export by top five exporters of the world in 2011
0
1
2
3
4
5
6
7
8
9
10
Production (× 1,000,000tonnes)
0
2
4
6
8
10
12
Export (10,000 tonnes)
9
1.4 Banana propagation.
The banana plant readily produces vegetative suckers next to the mother
pseudostem at the base of plant, with strong vascular connection to the mother. These
can be removed from the parent and planted separately, where they rapidly develop
new leaves and root systems, allowing rapid vegetative propagation and
multiplication. In cultivation, unwanted suckers are removed to avoid weakening the
parent plant. The suckers are the major source of planting material and normally
remain true-to-type. After planting, at a typical density of 1500 to 2500 plants per
hector, each plant produces a single pseudostem with one fruit bunch of 20–40 kg
harvested 9–14 months after planting. The plant is then cut to ground level, the leaves
removed and destroyed to control disease, and a side sucker allowed to grow up to
produce the next crop. In intensively managed plantations, the plants are replaced
with new, disease-free planting material after three-to-eight of these ratooning cycles.
Where plants are not replaced, a gradual and continuous yield decline is usually
observed, attributed to disease build-up.
During propagation, some somatic clonal variants have been observed and
selected, in particular for inflorescence, fruit and height characteristics (Krikorian et
al., 1993; Szymkowiak and Sussex, 1996). Good examples come from the
‘Cavendish’ group of dessert bananas, where there are several height variants such as
(in approximate descending order) ‘Lacatan’, ‘Robusta’, ‘Valery’, ‘Giant Cavendish’,
‘Grand Naine’, ‘Dwarf Cavendish’, ‘Petit Naine’ and ‘Dwarf Parfitt’, and other
variants, such as ‘Williams’ and ‘Zelig’. The changes giving rise to these
independently named varieties are considered to be genetic mutations, although
without the possibility of carrying out genetic segregation tests and without cloning
and sequencing the relevant genes this are not proven and they may be epigenetic
variants. However, there are some detectable changes between the ‘Cavendish’ groups
at the DNA level. The diversity of new forms derived through a combination of
accumulation of somatic mutations and human selection has led Ortiz (1997a) to
consider sub-Saharan Africa as a secondary centre of banana diversity.
In vitro tissue culture propagation systems are very efficient in Musa. These
can give high quality, uniform plants free of disease and nematodes, and much of the
planting material used in commercial plantations, and increasingly in smallholder
10
production, comes from mass micropropagation. Shoot tip cultures have been most
widely used (Strosse et al., 2004), but suspension cultures are also being developed
(Roux et al., 2001). In some tissue culture systems, high levels of chimerism are
found, where chromosome number and genotype vary (Roux et al., 2001) in the
resulting plants. The valued South Indian ‘Red’ sweet banana shows regular reversion
of the colour character to green, particularly in tissue-culture propagated plants but
also in the field (Stover and Simmonds, 1987), although the basis of this has not been
confirmed. A programme checking varietal characteristics of material grown up after
a decade of storage in vitro is showing that very few morphological or ploidy variants
have been induced (van den Houwe et al., 1995). Applications of molecular markers
do show some DNA changes (Ray et al., 2006) arising following tissue culture.
1.5 Banana production constraints
As with all other crop species, banana production faces major challenges from
biotic as well as abiotic stresses.
1.5.1 Biotic challenges
Banana production is limited due to several diseases and pests, such as
Fusarium wilt (Fusarium oxysporum f. sp. Cubense), black sigatoka (Mycospharella
fijiensisi), banana Xanthomonas wilt (Xanthomonas campestris pv. musacearum),
viruses (Banana bunchy-top virus, genus Nanovirus and Banana streak virus, genus
Badnavirus), weevils, and nematodes (Tripathi et al., 2008). Panama disease or
Fusarium wilt, caused by the fungus Fusarium oxysporum, has devastated banana
production and is widely regarded as one of the most destructive plant diseases
(Moore et al., 1995). Once established in an area, Fusarium cannot be controlled
chemically by fungicides or solid fumigants, or by cultural practices such as rotations
or soil amendment, so the only long-term option for continuing banana production is
replacement of a susceptible variety with a resistant variety (Hwang et al., 2004, Daly
et al., 2006). However, most commercial varieties are susceptible to ‘Tropical Race 4’
(Su et al., 1986). Although a number of varieties have been identified with resistance
genes that may be useful in breeding or gene transfer programs, these varieties have
weaknesses that makes them unsuitable as replacements for ‘Cavendish’ (Daly et al.,
2006). Another fungal disease, Black sigatoka leaf spot or Black Leaf Streak Disease
(BLSD, Mycosphaerella fijiensis) has been serious, with infection leading to around
11
50% crop losses (Ferreira et al., 2004). There is some genetic resistance in Musa with
potential for exploitation (Ortiz and Vuylsteke, 1995), and genomic studies of the
pathogen (BLSD, Mycosphaerella fijiensis), including complete sequencing, are
underway (Conde-Ferraez et al., 2007). A bacterial wilt caused by Xanthomonas is
spreading rapidly in East Africa; although control of disease spread by cultural
practices is being attempted, a long-term solution may again come through the genetic
resistance.
Viral diseases of banana include various diseases such as Banana Bunchy Top
and Banana Streak caused by BBTV and BSV respectively are controlled mainly by
eradication of infected plants. Harper et al. (1999) showed the BSV-related sequences
are integrated within the nuclear genome although integration is not an essential part
of the viral life cycle (Harper et al., 1999). Hull et al. (2000) and others have
speculated that the presence of integrated copies may confer virus resistance through
induction of transcriptional or post transcriptional gene silencing of homologous
sequences, and since then it has become clear that expression of these elements give
rise to a strong viral infection (Hull et al., 2000, Harper et al., 2002) .
Burrowing nematodes (Radopholus similis and Pratylenchus spp.) and weevil
(Cosmopolites sordidus) pests also constrain banana production, with little genetic
resistance in widely grown cultivars. Their infection often leads to conditions where
plantations becoming uneconomic and being abandoned. No known source of desired
level of resistance exists within the banana/plantain gene pool (Barekye et al., 2000;
Blomme, 2004; Fogain, 2001).
1.5.2 Abiotic challenges
Abiotic stresses caused by shortage or excess of water, salinity, wind or
temperature, affects the crop yield (Heslop-Harrison and Schwarzacher, 2007). Plants
can tolerate short periods of drought because of their water-filled reserves (Nelson et
al., 2006). Lack of water is associated with ‘maturity bronzing’ effect, manifested by
discoloration of mature bananas and cracking of the skin (Nelson et al., 2006). A soil
pH in the range 5.5-7.5 is suitable for growing bananas, with pH 5.5 considered as
optimal (Broadley et al., 2004). A low pH however solubilizes elements like iron,
aluminium and manganese; these can be toxic and have negative effects on the plant
growth. A low pH also reduces the availability of other nutrients such as calcium and
12
higher than 6.5, can reduce the availability of trace elements such as boron, zinc,
copper and iron (Broadley et al., 2004).
Despite high water requirements, water logging of the soil often results in
oxygen starvation of the roots (Daniells and Evans, 2005). Oxygen deficiency for
more than 6 hours results in root tip death, which in turn leads to branching of the
roots (Pattison and Lindsay, 2006). When sufficient water becomes available and
roots recommence growing, it may result in multiple branching, giving it appearance
of ‘witches broom’ (Pattison and Lindsay, 2006). Macronutrients required by banana
plants include nitrogen, potassium, phosphorus, calcium, magnesium and sulphur.
Deficiency of potassium results in reduced buoyancy, which interferes with the post
harvest production processes; the fruit sinks when dipped in hot water for the
treatment against certain diseases (Morton, 1987). The micronutrients required by
bananas include boron, iron, manganese, copper, zinc, molybdenum, chlorine and
cobalt. Deficiencies in these elements lead to morphological malformation of the
leaves, reduced growth and yield and poor fruit quality (Nelsonet al., 2006). Boron
deficiency alone can result in fruit that does not ‘fill’ (Broadley et al., 2004). Bananas
do not thrive in areas of high salinity, although some varieties show tolerance than
others. High levels of sodium result in reduced crop growth due to a reduction in
osmotic pressure of the soil, which leads to an increase in ions that are toxic to the
plant (Richards, 1992; Bohra and Doerffling, 1993; Gomes et al., 2002).
All Musa species grow best in the open sun provided that the moisture is not
limiting (Simmonds, 1962). Deep shade causes stools to die (Simmonds, 1962;
Nelson et al., 2006). Fire generally does not destroy the banana plant; they recover by
regrowing from the corm (Nelson et al., 2006). High humidity, >95%, during the final
stages of ripening can lead to ‘splitting’ of the fingers (Nelson et al., 2006). Bananas
are also susceptible to strong winds, which can twist and distort the crown. The leaves
can also be shredded by winds thus interfering with metabolism (Taylor and Sexton,
1972). Low temperatures retard growth although susceptibility to cold varies among
cultivars (Broadley et al., 2004). Impact of cold stress on plant growth includes; non-
emergence of bud from the stem at flowering time, cessation of root growth at
temperatures below 130C and destruction of the plant by frost (although the corm
normally remains viable) (Broadley et al., 2004). Choke Throat occurs when the
13
bunch gets mapped in the pseudostem at various stages of emergence. Less severe
cases result in bunches that only partially emerge from the pseudostem and are thus
susceptible to disease because they are difficult to cover (Daniells et al., 2004).
Keeping in mind the above stresses, it is emphasized that an ideal ideotype
cultivar is one which is disease and pest resistant, high yielding, photosynthetically
efficient, early maturing, display minimum delay between consecutive harvests, short
stature, strong roots for optimal nutrient uptake and greater resistance to wind
damage. Considering the global importance of banana, there is a great potential to
improve disease-free and high-yielding cultivars. As a step towards this, development
of efficient organogenesis and genetic manipulation techniques will come up with
new opportunity for the genetic improvement of banana.
1.6 Banana genetic improvement through conventional techniques
1.6.1 Sexual hybridization
Breeding of most cultivated bananas has relied upon conventional sexual
hybridization, involving the crossing of triploid cultivars with wild or cultivated
diploid parents. Generally, crossing triploid (3x) cultivars, which have residual
fertility with diploid (2x) parents, generates tetraploid (4x) hybrids (Pillay et al. 2002).
This strategy emphasized the need to cross improved diploids, which have good
agronomic qualities, with disease-resistant triploid accessions to generate diploid
hybrids with agronomic excellence, such as pest and disease resistances (Pedraza et
al., 2005).
However, diploid bananas generally have unacceptable low yields. The 3x/2x
procedure has generated triploid hybrids with low seed set (Smith et al., 2005).
Further crossing of these triploid hybrids with wild disease resistant diploids produced
tetraploid hybrids, but the latter were unsuitable for cultivation because of undesirable
features, such as premature senescence, fruit drop, short shelf-life, a weak
pseudostem, and production of seeds (Smith et al., 2005). The main factor hampering
progress in banana breeding using conventional genetic improvement methods is the
sterility of most edible varieties because of their triploidy (Assani et al., 2005).
However, the 3x/2x strategy enables the creation of AA diploid hybrids (Bakry et al.,
2009).
14
Banana breeding efforts have also focused on the improvement of selected wild, semi-
parthenocarpic and parthenocarpic diploid male parents. Intensive breeding of fertile
parthenocarpic edible diploids, which have large fruits of improved shape, resulted in
the development of hybrid M53 showing resistance to Sigatoka leaf spot and
Fusarium wilt (Bakry et al., 2009). A more recent strategy is the use of fertile diploid
AA hybrids, resulting from 3x/2x crosses, as parents or as starting material for
developing elite diploids, especially for plantains and East African highland bananas
that are resistant to Sigatoka leaf spot.
Another breeding strategy is the generation of secondary triploids by crossing
fertile tetraploid plants with diploid hybrids, a strategy that has been exploited to
genetically improve some cooking bananas. Using this breeding approach, some of
the banana cvs. obtained in this way have been AAB hybrids, such as IRFA909,
IRFA910, and IRFA914, and AAA hybrids, including FLHORBAN 918 and
FLHORBAN 920 (Bakry et al., 2009). A recent breeding strategy aimed at the
synthesis of triploid hybrids directly from diploid germplasm, which is based on the
specific combining ability between two diploids, one being the donor of diploid
gametes, was developed by CIRAD. The production of diploid gametes has been
achieved through chromosome doubling by treating selected mono-and inter-specific
diploids with colchicine to generate auto- or allotetraploids. Clearly, while sexual
hybridization will continue to be exploited for the genetic improvement of bananas,
this approach has limitations, emphasizing the relevance of tissue culture-based
technologies as important adjuncts to conventional breeding.
1.6.2 Induction of mutations
Induced mutation by treatment of in vitro material with physical (e.g., gamma
radiation) or chemical agents, such as ethyl methane-sulphonate, sodium azide, or
diethylsulphate, has been applied to banana breeding (Kulkarni et al., 2007) and has
been exploited in attempts to compensate for agronomic weaknesses in existing
cultivars (Heslop-Harrison and Schwarzacher, 2007). Although the production of
commercially interesting variants is possible by induced mutation, this approach has
been of limited success. However, Novaria and Klue Hom Tong KU1 are two of the
banana cultivars derived from gamma ray-induced mutation that have been released
15
commercially (Smith et al., 2005). The important agronomic traits of these mutants
include early flowering in Novaria and large bunches of fruit in Klue Hom Tong KU1
(Mak et al., 1996; Maluszynski, 2001; Roux, 2004; Smith et al., 2005). Most of the
banana mutants released commercially have been induced by gamma irradiation.
A further breeding strategy is the triploid approach, which involves the
induction of tetraploids from diploids by colchicine treatment of parental tissues, the
subsequent selection of improved tetraploid lines, and hybridization of the selected
tetraploids with diploids to produce triploids suitable for final evaluation (Smith et al.,
2005). Both colchicine and oryzalin have been used as mutagens to induce tetraploids
and autotetraploids in banana (Hamill et al., 1992; van Duren et al., 1996), with the
manipulation of ploidy by in vitro mutation technology being integrated into several
Musa breeding programs. Escalant and Jain (2004) provided a useful discussion of the
relevance of induced mutations to banana breeding.
1.7 Banana improvement via genetic transformation.
Genetic transformation, involving the introduction and stable integration of
genes into the nuclear or plastid genomes with subsequent gene expression in
transgenic or transplastomic plants, offers an additional approach for the genetic
improvement of banana, particularly for those cultivars that are not amenable to
sexual hybridization, e.g., those from the Cavendish subgroup (Jones, 2000; Pillay
and Tripathi, 2007). Both particle bombardment (Becker et al., 2000) and
Agrobacterium-mediated gene-transfer techniques have been used to introduce
foreign genes into banana (Ganapathi et al., 2001; Khanna et al., 2004; Acereto-
Escoffie et al., 2005). Particle bombardment utilizes accelerated metal microparticles,
usually gold, coated with DNA to penetrate and deliver foreign genes into plant cells;
transformed cells, recovered by their ability to grow in the presence of a selective
agent, such as an antibiotic (e.g., kanamycin sulphate) or herbicide (e.g., glufosinate
ammonium), are selected and regenerated into plants. Both of these transformation
methodologies have been reviewed extensively (Davey et al., 2005a, b), while
Altpeter and Sandhu (2010) provided a detailed protocol for biolistics-mediated gene
transfer and listed earlier references relevant to this procedure.
16
In banana, embryogenic cell suspensions (Becker et al., 2000) and scalps (Sagi
et al., 1995) have been transformed by particle bombardment and meristems by
Agrobacterium-mediated gene delivery (May et al., 1995), with variable rates of
success. Agrobacterium-mediated DNA delivery resulted in low genetic
transformation rates with the induction of chimeras. As a consequence, this technique
is not commonly used for this crop (Smith et al., 2005) compared with biolistics-
mediated gene delivery. Foreign genes, such as those for reporter and selectable
markers, resistance to fungi (Sagi et al., 1998; Xin Wu et al., 2005), nematodes, and
viruses (Becker et al., 2000); delayed fruit ripening (Balint-Kurti et al., 2001),
tolerance to salt stress (Ismail et al., 2005), and the synthesis of therapeutic proteins
(e.g., hepatitis B surface antigen; Sunil et al., 2005), are some of the target genes for
banana transformation. The synthesis of vaccines, antibodies, and other therapeutic
proteins in transgenic bananas has several advantages because it eliminates costly and
time-consuming processing, such as extraction and purification. Most importantly,
this approach permits oral administration of vaccines to patients, including children,
because of palatability and digestibility without cooking, retaining heat-labile proteins
that would otherwise be destroyed (Pua, 2007). This is an important consideration in
the tropics and subtropics where economical vaccines are required to immunize large
human populations. Sunil et al. (2005) reported, for the first time, up to 38 ng per
gram fresh weight of leaf tissue of hepatitis B surface antigen (HBsAg) in the cv.
Rasthali (AAB). Although the expression level was low, this study demonstrated the
feasibility of expressing HBsAG and possibly other novel therapeutic proteins and
vaccines in banana (Pua, 2007).
The transformation of banana is influenced by several parameters, including
the plant genotype, the physiology of explants, and the totipotency of cells in culture
(Heslop-Harrison and Schwarzacher, 2007). However, transformation has the
potential to make a significant contribution to banana improvement.
An important aspect of banana transformation is the fact that there is little
chance of unintentional gene flow from transformed plants because of their sterility or
extremely low fertility, making them particularly environmentally safe (Smith et al.,
2005; Sunil et al., 2005; Pillay and Tripathi, 2007).
17
1.8 Research objectives:
1. Standardization of regeneration protocols of cv. Patakpura.
2. Standardization of transformation protocol with GUS marker gene.
3. Optimization of transformation with GUS and kanamycine markers.
4. Field transfer and hardening of control and transformed plants.
5. Histological and biochemical confirmation of regenerants.
18
REVIEW OF LITERATURE.
2.1 Tissue culture in banana improvement
Besides the production of healthy planting material, tissue culture has been
used for crop improvement since 1940s to create genetic variability and to increase
the number of desirable variants. Specially, in vitro techniques for the culture of
protoplasts, anthers, microspores, ovules and embryos have been used to create new
genetic variation in the breeding lines, often with haploid production with crop
improvement potential (Brown and Thorpe, 1995). The culture of single cells and
merited can be effectively used to eradicate pathogen from the planting material and
thereby dramatically improve the yield of established cultivars (Badoni and Chauhan,
2010). Tissue culture in combination with molecular techniques, have been
successfully used to incorporate specific trait through gene transfer.
2.1.1 Micro propagation
Traditionally, bananas and plantains are propagated by vegetative means
through suckers or corms. Tissue culture-based micropropagation systems are well
developed for bananas and, consequently, can be exploited to multiply elite
genotypes. Such procedures form a basis for germplasm conservation and genetic
improvement of this crop using somatic-cell techniques. Tissue culture was first
applied to shoot tips of M. acuminata AAA cv. Cavendish by Ma and Shii (1972), and
subsequently extended to other cultivars and tissue explants, including meristems,
rhizomes, inflorescences and immature male flowers, immature zygotic embryos, and
leaf bases (Cronauer and Krikorian 1985; Vuylsteke 1989, 1998). Interestingly, plant
material propagated in vitro has replaced completely the use of conventional
vegetative suckers in many regions where there is intensive cultivation of bananas. In
fact, bananas were one of the first fruit food crops to be micropropagated and are still
multiplied in vitro more than any other fruit crop, with annual production figures
estimated to exceed 2 million propagules (Swennen et al., 2004; Smith et al., 2005).
Certainly, micropropagation has become a standard practice for the production of
material for field planting of this seedless crop. Importantly, tissue culture enables
mass production of elite clones with desirable agronomic qualities, in preference to
the collection of more limited numbers of suckers from field-grown plants. Because
19
only quality material is selected for micropropagation, the growth and yield of such
propagules in the field are superior to traditionally produced plants. Thus, tissue
culture-derived banana plants generally outperform plants derived from conventional
planting materials with respect to their yield, finger size, cycle time, number of
suckers, efficiency of nutrient uptake, emergence, and crop uniformity, even in ratoon
crops.
Tissue culture enables plant material to be produced that is free of
contaminating microorganisms, pests, and diseases, because only axenic explants are
introduced into culture. Additionally, source materials may be virus-indexed prior to
introduction into culture (Hamill, 2000; Hwang and Su, 2000). The culture of shoot
tips, combined with virus indexing and quarantine procedures, guarantees the safe
dissemination and conservation of certified Musa germplasm and prevents
dissemination of serious diseases and pests from the native country (Vuylsteke, 1998).
Virus testing of germplasm is now recommended as a routine procedure to ensure safe
international distribution, because many viruses that affect Musa remain difficult to
eliminate even by the culture of meristems excised from stem apices. The use of in
vitro-derived planting materials can prolong the pest-free period of plants in the field,
providing access to new banana cultivars across quarantine zones on a global scale
and promoting the rapid introduction of elite selections (Vuylsteke, 1998). The use of
disease-free planting materials also ensures cost reduction and subsequent delay in the
necessity for pest and disease management. Environmental issues, such as tolerance to
drought, have been addressed using cultured shoot tips. For example, Ebrahim et al.
(2004) compared the drought tolerance of four Musa cultivars by exposing shoot tip-
derived plants to culture medium containing polyethylene glycol (PEG) to simulate
drought conditions. Similarly, Harb et al. (2005) included sea salt in the culture
medium to evaluate the salt tolerance of bananas.
Cell-culture technology is exploited extensively to multiply elite germplasms.
The ability to regenerate plants directly from cultured explants, explant-derived
callus, cell suspensions, and isolated protoplasts through organogenesis and/or
somatic embryogenesis also forms an essential basis for the generation of potential
new cultivars by the induction of mutations in cultured cells, exposure of somaclonal
variation, and genetic improvement through gene mobilization. The procedures
20
involved include somatic hybridization/cybridization involving protoplast fusion and
the introduction of specific genes by transformation. Cryopreservation to conserve
rare germplasms also depends on robust cell- and tissue-culture procedures, with
associated reproducible plant regeneration.
2.1.2 Plant regeneration from cultured cells by organogenesis and somatic embryogenesis
Plant regeneration in bananas can be achieved via organogenesis in the case of
cultured shoot tips, and by somatic embryogenesis from callus and cell suspensions.
In bananas, an efficient plant regeneration system via direct organogenesis and/or
somatic embryogenesis is vital as a basis for various biotechnological options. Shoot
apices containing meristems produce multiple new shoots following the inhibition of
apical dominance (Kulkarni et al., 2007). Suckers with sword-like leaves are normally
excised from parent plants to provide source material for micropropagation.
Micropropagated plants that originate from such “sword suckers” may act as a further
source of shoot tips for multiplication. Regenerated shoots provide material for
planting and research, whereas highly proliferating cauliflower (nodule)-like
meristems may also be established from cultured shoots to provide scalps with which
to establish embryogenic cell suspensions (Sadik et al., 2007). Scalps, the uppermost
parts of highly proliferating nodule like meristems, are rich in meristematic cells
(Panis and De Langhe,et al., 1990). Because plants can be regenerated from scalps,
the latter have been exploited as target material for genetic transformation (Acereto-
Escoffie et al., 2005) and cryopreservation (Strosse et al., 2006).
Effort has focused on the development of protocols to induce somatic
embryogenesis as a pathway of plant regeneration in genotypes of dessert and cooking
bananas, initially as a basis for micropropagation and, subsequently, as a basis for
genetic manipulation (Strosse et al., 2003). This procedure also underpins
cryopreservation. Somatic embryogenesis involves the formation of embryo-like
structures and their development into whole plants in a way analogous to zygotic
embryos (Strosse et al., 2006). Such somatic embryos are produced either directly
from somatic cells of cultured explants without an intervening callus stage or
indirectly from callus generated from somatic tissues and from cell suspensions
induced from callus. Cells develop into globular structures that progress to heart-
21
shaped embryos and, subsequently, to torpedo-shaped embryos with hypocotyls and
radicles in the case of dicotyledons, or globular, scutellar, and coleoptylar structures
in monocotyledons. Both embryo induction and development depend on the culture
conditions, including the composition of the culture medium, especially the
concentration and type of plant-growth regulators, the carbohydrate source, and the
osmotic potential of the medium (Jimenez, 2005). Plant regeneration via somatic
embryogenesis in bananas has been reported from embryogenic cell suspensions
established from embryogenic callus induced from apical meristems (Cronauer and
Krikorian 1985), corm-like tissues (Novak et al., 1989;), pseudostems, leaf bases and
rhizome fragments (Novak et al., 1989), highly proliferating scalps (Dhed’a et al.,
1991; Schoofs, 1997; Ganapathi et al., 2001b), immature zygotic embryos (Escalant
and Teisson, 1989; Marroquin et al., 1993), immature male flowers (Ma, 1991; Shii et
al., 1992; Grapin et al., 1998; Chung et al., 2006; Sidha et al., 2007; Jalil et al., 2008),
and immature female flowers (Grapin et al., 2000). In general, embryogenic cell
suspensions of banana are usually established from immature male flowers and scalps
(Strosse et al., 2003).
Somatic embryogenesis in banana is constrained, however, by several factors,
including the limited choice of explants, the restricted and often variable embryogenic
response of cells and tissues in vitro, labor-intensive and time-consuming
establishment of embryogenic cell suspensions, loss of embryogenic capability, and
high incidence of somaclonal variation associated with long-term culture (Strosse et
al., 2003, 2006). Dhed’a et al. (1991) observed 5%–10% abnormal somatic embryos
recovered from scalp derived cell suspensions of the banana cv. Bluggoe (ABB).
Morphological observations on plants regenerated from male flower-derived cell
suspensions of the cv. French Sombre (AAB) revealed 16%–22% somaclonal variants
, whereas Schoofs et al. (1999) reported an extremely high number (>90%) of ‘long
narrow leaf’ off-types for plants regenerated from scalp derived cell suspensions of
the cv. Williams (AAA). The same authors also noted that 9-year-old cell suspensions
of the cv. Bluggoe (ABB) were aneuploid and lacked four to five chromosomes, as
determined by flow cytometry. The latter technique is rapid for the quantification of
euploidy and aneuploidy in plants, particularly those regenerated from cell
suspensions, because only small numbers of cells are required for analysis (Schoofs et
al., 1999).
22
Secondary embryogenesis is frequent in banana cultures (Teisson and Cote, 1994;
Kosky et al., 2002), this process involving the induction of new somatic embryos
from similar pre-existing structures (Khalil et al., 2002). Consequently, secondary
somatic embryogenesis has the potential for plant multiplication across an extended
period of time, because new embryos are formed continuously from existing embryos.
Plant regeneration rates in bananas via this process varied between 1.5%–20% (Cote
et al. 1996) and 60%–89% (Kosky et al., 2002). The protocol of Kosky et al. (2002)
involved temporary immersion in liquid medium. Using such a procedure, Kosky et
al. (2002) reported an improvement in mass propagation of the banana AAAB cv.
FHIA-18 via somatic embryogenesis. Immature male flowers were induced to form
embryogenic tissue, the latter being used to generate embryogenic cell suspensions in
MS-based medium containing 1.0 mg l−1 biotin, 100 mg l−1 glutamine, 100 mg l−1
malt extract, 1.0 mg l−1 2,4-dichlorophenoxyacetic acid, and 45 g l−1 sucrose. A
temporary immersion system may not be available to all researchers and, indeed, may
not be essential in some cases, for example, a plant regeneration rate of 90% was
reported via somatic embryogenesis of the banana cv. Dwarf Brazilian (AAB) without
the need for a temporary immersion system, cell suspensions as source material, or
bioreactors as culture vessels (Khalil et al., 2002).
2.1.3 Generation of somaclonal variation
Variation may occur naturally during both conventional and in vitro
propagation of bananas (Vroh-Bi et al., 2010). While the incidence of somatic
mutations is low in bananas propagated conventionally, it is frequent in
micropropagated material (Stover, 1988; Robinson, 1996) and often constrains
regeneration by somatic embryogenesis (Strosse et al., 2006). Somatic mutations are
limited to non-reproductive cells, with somaclonal variation often being exhibited by
plants regenerated from cultured cells, particularly those regenerated via a callus
phase. Mutations have been associated with somaclonal variation, including point
mutations, gene duplication, chromosomal rearrangements, and changes in
chromosome complements.
Chromosome instability is among the most common causes of tissue culture
induced variations in bananas (Larkin, 2004; Msogoya et al., 2008).The movement of
23
transposable elements and changes in DNA methylation have also been implicated as
possible mechanisms associated with such variation.
During micropropagation of elite clones, somaclonal variation can result in
off-type plants of decreased commercial value. Indeed, the high incidence of off-types
resulting from the culture of banana meristems is of major concern to commercial
growers, with the incidence of morphological offtypes being more frequent when the
plants were propagated in vitro by meristem culture. Even a low percentage of off-
types is unacceptable in commercial production because the generation of off-types
can be extremely costly (Larkin, 2004). In contrast, several banana cultivars have
originated from spontaneous somatic mutations (Robinson, 1996; Heslop-Harrison &
Schwarzacher, 2007) and, in this respect, somaclonal variation is important for the
genetic improvement of banana (Khayat et al., 2004). Thus, exposure through culture
of naturally occurring genetic variation in somatic cells has the potential to generate
considerable novel and useful genetic variability not only in bananas but also for
crops in general. Mutant and somaclonal variant banana plants, exhibiting traits such
as tolerance to aluminum, dwarfism, and resistance to Panama and Sigatoka diseases,
have been released for commercial production, or are still being evaluated for their
growth potential and yield (Hwang, 2001; Hwang and Ko, 2004; Roux, 2004). Tai-Chiao
No.1, a variant from the banana cv. Pei-Chiao that resulted from multiplication of
material in vitro, showed improved agronomic characteristics, including resistance to
Tropical Race 4 of Fusarium wilt (Tang and Hwang, 1994). Likewise, ShiChuan and Ko
(2004) in Taiwan reported Cavendish banana cultivars resistant to Fusarium wilt.
Understanding natural and in vitro genomic variation and identifying such
changes at an early stage of plant development are vital for breeding, mutagenesis,
transgenic-plant characterization, and germplasm management (Vroh-Bi et al., 2010).
Banana off-types can be detected by their morphology and further characterized by
genomic fingerprinting techniques. Although potentially extremely useful, somaclonal
variation may be problematic in the genetic improvement of crops where individual
transgenic plants need to be tested exhaustively so that only proven elite plants are
selected for commercial release.
24
2.1.4 Somatic hybridization
Somatic hybridization, involving the reproducible isolation, fusion, and
culture of isolated protoplasts (Davey et al., 2010), is a procedure to circumvent
naturally occurring pre- and post-zygotic incompatibility barriers that normally
hamper sexual hybridization. Somatic hybridization can be exploited to manipulate
polygenic traits without the requirement to isolate DNA, or to have knowledge of
genes or their DNA-base sequences. Polygenic traits can be introgressed by nuclear
and/or organelle transfer through symmetric and asymmetric protoplast fusion. The
extensive genetic nuclear-cytoplasmic combinations generated by this procedure have
been reviewed (Davey et al., 2000a, b; 2005a, b, c; 2010) and far exceed those
combinations generated by sexual hybridization. The main constraint of somatic
hybridization is that it is labor-intensive and relies upon the development of robust
protoplast-to-plant systems. Consequently, to date, this method of gene introgression
has been applied to banana-breeding programs by only a limited number of workers
(Megia et al., 1993; Panis et al. 1993; Matsumoto and Oka, 1998; Assani et al., 2001,
2002, 2005). Although there exist a limited number of references relating to protoplast
technology in banana, it is recognized that protoplast-fusion technology is a potential
tool to overcome sterility and genetic variability in most edible banana varieties that
are triploid (Assani et al., 2005). Somatic hybridization is the only way to generate
banana hybrids between highly sterile cultivars, especially in the triploid Cavendish
group; protoplast fusion can accelerate and facilitate the crossing of bananas that is
difficult to achieve by conventional breeding methods (Bakry et al., 2009).
Procedures for protoplast fusion generally involve exposure of isolated
protoplasts to chemical fusion agents, such as polyethylene glycol (PEG), exposure of
mixtures of parental protoplasts to an alternating current, followed by high voltage
direct current pulses (electrofusion), or a combination of these procedures (Davey et
al., 2000b, 2005a). Generally, electrofusion is the preferred procedure to fuse banana
protoplasts (Matsumoto et al., 2002) and is the most efficient procedure to generate
somatic hybrid plants (Assani et al., 2005). Chen and Ku (1985) first attempted to
fuse isolated banana protoplasts using leaves as a source of protoplasts. Subsequently,
Matsumoto et al. (1992) isolated protoplasts from bracts. However, both research
groups were unable to culture the material resulting from protoplast fusion, until
Matsumoto et al. (2002) generated pentaploid somatic hybrid cells resulted from the
25
fusion of banana protoplasts. It is possible, by protoplast fusion, to generate somatic
hybrid tetraploid parents for use in interploid crosses with other diploid lines, or for
the direct release of triploid somatic hybrids by haploid/diploid protoplast fusion
(Assani et al., 2003). In extensive investigations, Matsumoto et al. (2002) reported the
generation of somatic hybrids following electrofusion of protoplasts of the cv. Maca
(ABB) with protoplasts of the cv. Lidi (AA), and the use of nurse cultures to stimulate
the growth of electrofusion-treated protoplasts. An interesting fact is that somatic-
hybrid plants were generated only after embryogenic cell suspensions were initiated
(Xu et al., 2005) and used as source material for the isolation of totipotent protoplasts
(Matsumoto et al. 2002; Assani et al., 2005), with 85% of the regenerated plants
being identified as somatic hybrids using random amplified polymorphic DNA
(RAPD) analysis (Matsumoto et al. 2002). In a study that compared the two most
frequently used fusion procedures, i.e., electrofusion and PEG, Assani et al. (2005)
found that the former technique was superior with respect to the subsequent mitotic
activity of treated protoplasts, somatic embryogenesis, and plant regeneration of
protoplast-derived tissues. However, PEG-induced fusion was optimal with respect to
the frequency of binary fusions. More recently, Matsumoto et al. (2010) summarized
the literature relating to the source of cells, enzyme mixtures, and media, which they
used to isolate and to culture banana protoplasts to plants. These workers also
provided detailed laboratory notes relating to all stages of the procedures involved to
develop a protoplast-to-plant system for banana. Interestingly, cells from suspension
cultures in liquid medium have featured as source material in most of these reports.
2.2 Genetic transformation of Musa species.
Genetic transformation is of great interest in banana because (i) the cultivated
varieties are triploid and sterile; (ii) some resistance sources are not available among
genetic resources (i.e. virus resistance) and (iii) the foreign gene within the genetically
modified plant cannot be transferred to another plant because the triploid plants will
not produce fertile pollen. Therefore, the risk of direct gene contamination is
minimized both for other plants and for the environment.
Two main methodologies are being used in genetic transformation. These are
particle bombardment and Agrobacterium-mediated transformation. The
Agrobacterium-mediated transformation method may be more widely applicable as it
26
is based on the use of differentiated tissue that can be routinely regenerated into whole
plants. In addition, it has been applied to a wide range of plantain and banana
cultivars and synthetic hybrids (Bosque-Perez, et al 1998). Moreover, Agrobacterium-
mediated transformation offers several advantages over direct gene transfer
methodologies like particle bombardment and electroporation (Shibata and Liu, 2000,
Hansen and Martha, 1999).
Various diseases caused by fungi and viruses and abiotic stress factors have
seriously endangered the production of banana and plantains. Through genetic
transformation technologies, disease and drought resistant varieties may be produced.
Transgenic plants have been produced for the cultivars Williams, Gros Michel,
Bluggoe and Three Hand planty, using gene constructs encoding for various
antifungal peptides which have previously proved to be highly active in vitro against
major pathogenic fungi of bananas (Remy et al., 2000). Agrobacterium-mediated
transformation of embryonic cell suspensions of the banana cultivars ‘Rasthali’,
Cavendish’, and ‘Ladyfinger’ has been achieved (Ganpathi et al., 2001).
Centrifugation assisted Agrobacterium-mediated transformation protocol developed
using banana cultivars from two economically important genomic groups (AAA and
AAB) of cultivated banana has been described (Buhariwalla et al., 2005). Relative
success in genetic engineering of bananas and plantains has been achieved recently to
enable the transfer of foreign genes into plant cells. In general, transformation
frequencies are reported to be cultivar dependent. Thus there is a need to develop
optimal transformation protocols for any particular type of banana.
2.2.1 Transformation via Agrobacterium tumefaciens
Many details of the key molecular events taking place in the bacterial cells
during T-DNA transfer have been elucidated, and some plant factors which were
elusive earlier have now been purified and characterized (Leelavathi et al., 2004). The
phytopathogenic soil bacterium Agrobacterium tumefaciens genetically transforms
plants by transferring a portion of the resident Ti plasmid, the T-DNA, to the plant.
Musa was generally regarded as recalcitrant to Agrobacterium-mediated
transformation. Agrobacterium tumefaciens is compatible with banana indicating the
potential for genetic transformation by this means (Hernandez et al., 1999).
Sreeramanan et al. (2006a) studied the chemotaxis of Agrobacterium tumefaciens
27
strains (EHA 101 and LBA 4404) towards wounded banana tissues, using swarm agar
plates (Sreeramanan et al., 2006a). Chemotaxis has a minor role in determining host
specificity and suggested that it could not be responsible for the absence of
tumorigenesis in banana under natural conditions as was observed. Agrobacterium-
mediated transformation offers several advantages over direct gene transfer
methodologies (particle bombardment, electroporation, etc.). For example the
possibility of transferring only one or few copies of DNA fragments carrying the
genes of interest at higher efficiencies at low cost and the transfer of very large DNA
fragments with minimal rearrangement ( 1998, Hansen and Wright , 1999, and
Shibata and Liu , 2000).
Since the success of Agrobacterium-mediated transformation of rice in the
early 1990s, transgenic plants have been regenerated in more than a dozen
monocotyledonous species, ranging from the most important cereal crops to
ornamental plant species. Many factors influencing Agrobacterium-mediated
transformation of monocot plants have been investigated and elucidated. The effect of
plant genotype (Carvalho et al., 2004), explants types (Carvalho et al., 2004) and their
transformation competence (Chateau et al., 2000), as well as the influence of
Agrobacterium strains and binary vectors have been reported (Cheng et al., 2004;
Khanna et al., 2004). In addition, a wide variety of inoculation and co-cultivation
conditions have been shown to be important for transformation of monocots.
These include antinecrotic treatments using antioxidants and bactericides,
osmotic treatments (Cheng et al., 2004), pre-culture with growth regulators (Chateau
et al., 2000), desiccation of explants before or after Agrobacterium infection, use of
surfactants like Pluronic F68 (Khanna et al., 2004), and composition of inoculation
and co-cultivation medium (Cheng et al., 2004). Transformation frequencies of wheat
inflorescence tissue were influenced by the duration of pre-culture, level of wounding,
and amount of bacterial cells infiltrated (Amoah et al., 2001). The effects of other
physical parameters like infection time and co-cultivation volume can also be
investigated. Dillen et al. (1997) and De Clercq et al. (2002) tested the influence of
co-cultivation temperature and 22°C was reported as the optimum. The effects of
Agrobacterium cell density during infection, medium, pH, age and size of calli,
density of calli during co-cultivation, and the concentration of acetosyringone on
28
transformation frequency were also studied (De Clercq et al., 2002). All these reports
highlight the importance of a complex and thorough optimisation of Agrobacterium-
mediated transformation procedures when dealing with new crops or plant species
2.2.2 Factors influencing Agrobacterium-mediated transformation
Fig. 2.1 An illustration of how Agrobacterium can be used to transform plant cells in order to regenerate transgenic plants (Adopted from http:// webschoolsolutions.com/biotech/transgen.htm)
Fig.2.2 Mechanism of T-DNA transfer; (adapted from Current Opinion in Biotechnology, 17:147–154, 2006)
29
Table 2.1: In vitro regeneration studies of Banana.
Name Genotype Basal Medium
Plantgrowth regulators and other supplements
Response References
Musa acuminata Cv. Mas
AA MS 2,4-D 1mg/l Biotin 1mg/l, Glutamine 100mg/l, Malt extract 100mg/l
Callus, embryogenic cell suspension
Huang et al. (2007)
Dwarf cavendish
AAA MS 2,4-D 4mg/l, IAA 1mg/l, NAA 1mg/l, Biotin 1mg/l.
Callus, embryogenic cell suspension, shoot regeneration, proliferation.
Perez et al. (2012)
Banana (Musa sp) Cv. “Sukali Ndiizi”
ABB MS 2,4-D 4.5 µl, Biotin 4.1 µl, Glutamine 680 µl, Malt extract 100mg/l, Ascorbic acid 20mg/l.
Callus, embryogenic cell suspension, shoot regeneration, proliferation.
Namuddu et al.(2013)
Cavendish banana Cv. Robusta
AAA MS 2,4-D 4mg/l, IAA 1mg/l NAA 1mg/l, Biotin 1mg/l.
Callus, embryogenic cell suspension, shoot regeneration, proliferation.
Ghosh et al.(2009)
Plantain Cv. “Gonja Manjaya”
AAB MS BAP 22mg/l, 2,4-D 1mg/l, zeatin 0.2mg/l, IBA 1mg/l, Ascorbic acid 100mg/l.
Multiple bud Induction, callus, Proliferation, Rooting.
Tripathi et al.(2012)
Banana Cv. Rasthali
AAB MS BA 6mg/l, NAA 0.2mg/l 5% coconut water, Ascorbic acid 100mg/l
Shoot induction, Shoot multiplication.
Subramanyam et al. (2011)
Banana Cv.Rastali
AAB MS BAP 5mg/l Shoot multiplication.
Miziah et al. (2007)
East African Highland Banana Cv. Mpologoma & Nakitembe.
AAA MS BAP 5mg/l, IAA 0.3mg/l, IBA 1mg/l, Ascorbic acid 100mg/l.
Proliferation, Elongation and maturation of shoots, rooting.
Tripathi et al. (2008)
Rastali ABB MS BAP 10mg/l Multiple bud clump induction.
Sreeremanan et al.(2009)
Furenzhi (Musa spp)
AA MS 2,4-D 1mg/l, Biotin 1mg/l, Malt extract 100mg/l, Glutamine 100mg/l.
Callus, initiation and maintenance of embryogenic cell suspension.
Hu et al.(2013)
30
Table 2.2: Genetic transformation of banana via Agrobacterium
Name Genotype Explant Gene Efficiency Detection method/Analysis
References
Musa acuminata Cv. Mas
AA Embryogenic Cell Suspension (ECS)
Gus A and npt II
490 transgenic plants per 0.5 PCV of ECS
Geneticin resistance, Gus expression, PCR, Southern blotting
Huang et al.(2007)
Dwarf cavendish
AAA ECS from immature male flowers.
Uid A intron cassette and npt II
89.7% PCR analysis, Southern blot hybridization, Histochemical Gus assay
Perez et al. (2012)
Banana (Musa sp) Cv. “Sukali Ndiizi”
ABB ECSs from male flowers.
Modified carica papaya cystatin (CPCYS)
96.4% Hygromycin resistance, Histochemical Gus assay, PCR analysis, Southern blotting.
Namuddu et al.(2013)
Cavendish banana Cv. Robusta
AAA ECS from immature male flowers.
Uid A 30 transgenic plants/50mg settled cell mass
Hygromycin resistance, Gus histochemical asay, PCR,southern analysis.
Ghosh et al.(2009)
Plantain Cv. “Gonja Manjaya”
AAB ECS from Apical shoot tips.
Uid A and npt II
95-96% Kamamycin resistance, histochemical Gus analysis, southern blot analysis.
Tripathi et al.(2012)
Banana Cv. Rasthali
AAB Suckers. Hpt II and Gus genes
39.4% Hygromycin resistance, Gus histochemical assay, PCR amplification and southern blot analysis.
Subramanyam et al. (2011)
Banana Cv.Rastali
AAB Multiple bud clumps from Corm slices of in vitro plantlets.
Rice chitinase (RCC2)
5-20% Hygromycin resistance, PCR, southern blot analysis.
Miziah et al. (2007)
East African Highland Banana Cv. Mpologoma & Nakitembe
AAA Fine cross-sections of intercalary merismatic tissues from in vitro shoots.
Gus A and npt II
10% Kanamycin resistance, Gus histochemical asay, PCR analysis, southern hybridization.
Tripathi et al. (2008)
Rastali ABB Multiple bud clumps from Corm slices.
Rice chitinase(RCC2) and hpt II
5-20% Hygromycin resistance, histochemical Gus staining, PCR analysis.
Sreeremanan et al.(2009)
Furenzhi (Musa spp)
AA ECS from immature male flowers.
Chitinase (Chit 42)
20% Hygromycin resistanmce, histochemical Gus assay, PCR analysis, southern blotting.
Hu et al.(2013
31
2.4 Annexin gene for abiotic stress tolerance
Plants are continuously exposed to various abiotic stresses such as salinity,
cold, drought, chemical toxicity, which are the primary causes of crop losses
worldwide ( Bray et al., 2000). They adapt to these unfavorable conditions by
perceiving and transducing the stress signal(s) through a cascade of molecular
networks eventually leading to the expression of stress-related genes (Zhu, 2001). In
plant cells, calcium ions (Ca2+) serve as a second messenger during abiotic stress
signaling (Sanders et al., 2002). The increase in calcium levels during abiotic stress is
perceived and transduced by certain calcium-binding proteins such as calmodulin,
calcium-dependent protein kinases (CDPKs) and calcineurin-B-like proteins (CBL
proteins) (Knight and Knight, 2001). There is increasing evidence that another class
of proteins, annexins also bind calcium and play important roles in abiotic stress
responses in plants (Cantero et al., 2006, Jami et al., 2008, Kovacs et al., 1998, Lee et
al., 2004).
Annexins are calcium-dependent phospholipid-binding proteins. They are
ubiquitous in animal and plant kingdoms. Plant annexins were first identified in
tomato (Boustead et al., 1998) and subsequently, isolated and characterized in a wide
range of plant species (Mortimer et al., 2008). In vertebrates, annexins are represented
by at least thirteen distinct members (Raynal and Pollard, 1994). DNA blot analyses
in Arabidopsis (Gidrol et al., 1996), maize (Battey et al., 1996), bell pepper (Proust et
al., 1996) and tobacco (Proust et al., 1999) have indicated that the annexin gene
family in plants is relatively simple and possesses at least two different annexins.
However, with the availability of complete genomic sequence data in Arabidopsis and
rice, there appears to be eight and ten different annexin cDNA sequences, respectively
(Cantero et al., 2006, Clark et al., 2001). The primary structure of mammalian and
non-vertebrate metazoan annexins is characterized by a tetrad repeat of 70 amino
acids containing calcium-binding endonexin sequence usually referred as G-X-G-T-
{38}-(D/E) motif that binds calcium. Within the family of plant annexins, the
endonexin sequence is only conserved within the first and fourth repeats with the
presence of type-II calcium-binding sites (acidic residues). Recent studies on crystal
structural analysis showed that calcium binds to cotton annexin in repeats first and
fourth in the presence of acidic phospholipid vesicles (Hu et al., 2008).
32
Plant annexin gene expression is influenced by tissue/cell specific
developmental controls and environmental signals. They were reported to possess
phosphodiesterase activity, F-actin binding activity, calcium channel activity, and
participate in Golgi-mediated secretion (Mortimer et al., 2008). Annexins might also
participate in the regulation of callose and cellulose synthase activity (Hofmann,
2004). The annexin AnnAt1 from Arabidopsis thaliana has been shown to possess
peroxidase like activity (Gorecka et al., 2005) which enabled it to protect both E. coli
and Arabidopsis thaliana from oxidative stress. The transgenic tobacco plants
constitutively expressing AnnBj1 from Brassica juncea showed enhanced stress
tolerance (Jami, 2008).
Interestingly, antioxidative property of this annexin has also enabled it to
protect human tumour cell line from TNF (Tumor necrosis factor) induced apoptosis
(Kush and Sabapathy, 2001). Jami et al., (2009) reported that the highly contrasting
expression patterns of AnnBj2 and AnnBj3 in different treatments indicate that they
are a good combination of genes for deployment together in transgenic plants for
deriving abiotic stress tolerance. Therefore attempts were made in present study to
transform Musa acuminate cv. Patakpura with annexin (AnnBj2), a member of
annexin gene family of Brassica juncea and to probe its possible role, if any, in
plants’ defense, particularly, tolerance against drought.
33
MATERIALS AND METHODS
The present study on transgenic research in banana was carried out at the Department
of Agricultural Biotechnology, College of Agriculture, Orissa University of
Agriculture and Technology, Bhubaneswar-751003 Odisha, India. The details of
materials used and the experimental techniques adopted during the course of
investigation are presented in this chapter.
3.1 MATERIALS
3.1.1 Plant materials
The suckers of Musa acuminate cv. Patakpura were collected from the
backyard of Department of Agricultural Biotechnology, College of Agriculture,
OUAT, Bhubaneswar and were used for the organogenesis and Agrobacterium
tumefaciens- mediated genetic transformation studies.
3.1.2 Apparatus required
� Laminar airflow cabinet
� Precision scale digital balance
� pH meter
� Autoclave
� Environmental shaker
� Centrifuge machine
� Incubator
� UV spectrophotometer
� Scanning Electron Microscope ( S-3400N)
3.1.3 Plant growth regulators (Source-MP Biomedical)
The following plant growth regulators were used in different experiments at
different concentrations.
34
Auxins:
• Indole Acetic Acid (IAA)
• Naphthalene Acetic Acid (NAA)
• 2, 4-Dichlorophenoxy Acetic Acid (2,4-D)
Cytokinin:
• Benzylaminopurine (BAP)
3.1.4 Plant nutrient medium
Murashige and Skoog (1962) basal salt mix (Source- MP Biomedical) were used.
3.1.5 Antibiotics (Source-MP Biomedical)
The following antibiotics were used in different experiments at different
concentrations.
• Kanamycin
• Rifampicin
• Cefotaxime
3.1.6 Agrobacterium strain and plasmid vector
The Agrobacterium tumefaciens strain EHA105 is an L, L-succinamopine
strain with a C58 chromosomal background. It contains pEHA105 as virulence helper
plasmid derived from supervirulent pTiBo542 (Hood et al. 1986, 1993) harboring
binary vector, pCAMBIA2301 having the neomycin phosphotransferase gene (nptII)
as selection marker and betaglucuronidase gene (gusA) with a catalase intron as a
reporter was obtained from CAMBIA for the transformations (Fig.3.3). Both the
genes were under the regulation of CaMV35S promoter.
Gene construct of AnnBj2 gene
The gene construct AnnBj2/Bj3 has CaMV35S promoter in the upstreme
region and next to that nptII (neomycin phosphotransferase II) gene in the left border
is present. In the downstream Kanamycine gene is present attached to the AnnBj2/Bj3
gene.
35
Fig.3.1 Gene construct of AnnBj2 and AnnBj3 gene
Fig.3.2 Gene construct of GUS marker
Fig.3.3 Schematic map of binary vector pCAMBIA2301
36
3.1.7 Histochemical Gus assay.
X-Gluc solution (Jefferson 1987):
� 2mM X-Gluc
� 100 mM NaH2 PO4 (pH 7.0)
� 50 mM Potassium ferricyanide
� 50 mM Potassium ferrocyanide
3.2 METHODOLOGY
3.2.1 Collection of plant materials
The suckers were uprooted, trimmed with the help of a knife to remove the
adventitious roots and part of the pseudo stem. They were then washed thoroughly
under running tape water and then placed in a beaker of water and brought to the
laboratory.
3.2.2 Sterilization of explants for plant tissue culture
Since suckers are present below the rhizosphere region, contain many bacteria
and viruses, it is necessary to follow an effective surface sterilization method. Shoot
tips were prepared by trimming corm and outer leaf sheaths from the suckers of Musa
acuminate cv. Patakpura. The shoot tips were treated with 0.1% HgCl2 solution for 2-
16 min and then rinsed with sterile distilled water 3 times under aseptic conditions in
a laminar airflow cabinet.
3.2.3 Direct plant regeneration
3.2.3.1 Multiple shoot induction
Corm slices were placed on autoclaved Murashige and Skoog (MS medium,
1962) medium with different concentrations of cytokinin (BAP; 1-10mg/l) and auxin
(IAA; 0.5-3mg/l) along with 100mg/l Adenine Sulphate. The pH of medium was
adjusted to 5.6 prior to autoclaving with the help of 0.1N NaOH and 0.1N HCl. The
cultures were incubated in the culture room at 25-27°C under 16h photoperiod using
cool white fluorescent bulbs (Philips fluorescent light tubes) of 150µ mol m-2s-1. After
4-6 weeks, multiple shoots were initiated and developed in the culture and were
transferred to fresh medium for further growth.
37
3.2.3.2 Elongation and multiplication of shoots
The shoots obtained from the above experiment were subcultured on MS
medium supplemented with different concentrations of cytokinin BAP (2.0 -8.0 mg/l)
in combination with auxins IAA and NAA (0.5-3.0 mg/l) along with Adenine sulphate
100 mg/l and number of elongated shoots per culture and days taken for elongation
were recorded.
3.2.4 Callus mediated regeneration
3.2.4.1 Callus induction
Apical shoot tips were cultured on MS medium supplemented with BAP (1.0-
2.0 mg/l) and 2, 4-D (1.0-5.0 mg/l) along with ascorbic acid (10mg/g), 5% coconut
milk and malt extract (100mg/l) and incubated in dark for callus initiation.
Observations were recorded on days to callus initiation, number of explants
responding, quantity and type of callus induced.
3.2.4.2 Regeneration through callus
Calli induced on the different media combinations were subcultured for
regeneration on graded doses of BAP (1.0-6.0mg/l) and 2, 4-D (1.0-4.0mg/l). Types
of response, days to shoot bud iniation, number of shoots regenerated from callus
were recorded.
3.2.4.3 Somatic embryogenesis
Somatic embryos were produced directly from somatic cells of cultured
explants without an intervening callus stage. Cells developed into globular, scutellar,
and coleoptylar structures. The somatic embryos were treated with different
concentrations of alcohol (30%, 50%, 70% and 100%) and scanned with S-3400N
Scanning Electron Microscope (Hitachi Company) (plate 5:16 and 16a ).
3.2.5 Preparation of Agrobacterium culture
The Agrobacterium tumefaciens strain carrying plasmid binary vector,
pCAMBIA2301 containing Annexin Bj2 gene construct was maintained on the solid
L. B. agar medium (HiMedia) containing 30µl/100ml kanamycin and rifampicin
(Plate 6: 17). Sub-culturing was done every month on fresh medium in order to
38
refresh and maintain the cultures for long term preservation. For transformation
experiment, a single colony of Agrobacterium was taken from the stock plate and
streaked on fresh L. B. agar plate containing the same antibiotics concentration and
the plates were kept in the incubator at 26-280C for overnight colony growth. The
next day, a single colony was transferred into 2ml of L. B. broth having the above
concentration of antibiotics and was kept at 26-280C for overnight. 200µl of
Agrobacterium culture was transferred into 100ml of L. B. broth and kept in the same
condition as above. The optical density (O.D) at 600 nm of the culture was then taken
at regular intervals of 1 hr in UV spectrophotometer until the culture reached up to the
required value (0.6 to 0.8). The Agrobacterium culture was then centrifuged in sterile
centrifuge tube at 5000rpm for 10 min. The supernatant was discarded and the pellet
gently dissolved in cold distilled water with the help of sterile brush under laminar
airflow. The dissolved pellet was again centrifuged at 3000rpm at 40C for 5 min and
the supernatant discarded under laminar air flow. The pellet was then mixed with
10ml of MS liquid medium and then centrifuged at 3000rpm for 5min and supernatant
discarded as above. Finally, the pellet was mixed in 10ml of liquid plant growth
media (LPGM) with optimized acetosyringone concentration of 100µM and used for
co-cultivation with somatic embryos.
3.2.6 Transformation of somatic embryos
Seven days post sub cultured embryogenic cells were used for cocultivation.
The somatic embryos were cocultivated with Agrobacterium in liquid basal MS
medium supplemented with 100 µM Acetosyringone for 3days. Post-coculture
embryogenic cells were transferred to semi solidified MS medium supplemented with
2, 4-D (1 mg/ml), biotin (1 mg/ml), malt extract (100 mg/l), glutamine (100 mg/ml)
and 4.5% sucrose along with cefotaxime (200 mg/l) for germination of embryos.
3.2.7 Co-cultivation
The somatic embryogenic cell mass was pre-cultured for 7 days prior to
Agrobacterium infection. The embryos were mildly injured using a sterile needle and
then immersed into a centrifuge tube containing LPGM (liquid plant growth medium)
along with Agrobacterium suspension for different periods of time (10, 15, 20, 25, 30
min) with an optimized acetosyringone concentration of 100µM. Acetosyringone is
known to activate the virulence genes of the Ti plasmid and to initiate the transfer of
39
the T-DNA. The explants were then blotted dry on sterile filter paper and co-
cultivated for three days in the dark at 260C on petridish containing LPGM (liquid
plant growth medium) along with 100µM acetosyringone. The petridishes were
wrapped with aluminum foil and kept in the dark for 3 days.
3.2.8 Washing of explants
After 3 days of incubation, the infected embryogenic cell mass were
thoroughly washed with sterile distilled water having cefotaxime (200mg/l), blot dried
on sterile filter paper and inoculated on semisolid MS + 2, 4-D (1 mg/ml), biotin (1
mg/ml), malt extract (100 mg/l), glutamine (100 mg/ml) and 4.5% sucrose along with
cefotaxime (200 mg/l) + 100mg/l kanamycin medium for germination of embryos..
The washing of the explants and transfer to fresh medium with the above composition
was continued with gradual increase in the concentration of the antibiotics until the
Agrobacterium stopped growing in the selection medium.
3.2.9. Cefotaxime sensitivity test
To find out the suitable concentration of cefotaxime to avoid bacterial
contamination during the regeneration period and to know the minimal level of
cefotaxime which would completely eliminate the excess bacteria after co-cultivation,
this test was conducted at 100, 200, 300, 400, 500, 600, 700, 800 and 900 mg/l
cefotaxime along with control.
3.2.10 Optimization of lethal dose for the kanamycin based selection
For the determination of lethal dose of kanamycin on plant regeneration, the
normal, untransformed somatic embryos were transferred to fresh regeneration
medium supplemented with different concentrations of kanamycin (50, 100, 150, 200,
250, 300mg/l) to design the medium for selection of transformed plants.
3.2.11 GUS histochemical assay
Transformed somatic embryos were analyzed for β-glucuronidase expression
by using X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) as the substrate .The
histochemical reaction was allowed to proceed at 370C for overnight. Subsequently,
embryos were cleared in 70% (v/v) ethanol. To serve as control, un-transformed
embryos were included at all staining occasions.
40
3.2.12 Biometrical observations
Various biometrical observations on the following parameters were recorded
as given below.
3.2.12.1 Assessment of percent shoot induction
Shoot induction at the end of 35 days of culture period was assessed by
calculating number of explants responded for multiple shoot induction and was
expressed in percentage.
Percent shoot induction= No. of explants with multiple shoots x 100% Total no. of explants cultured
3.2.12.2 Assessment of percent callus initiation
Response of callus to callus initiation at the end of 45 days of culture was
assesssed by calculating number of explants responded for callus initiation and
expressed in percentage.
Percent callus initiation= No. of explants with callus initiation x 100% Total no. of explants cultured
3.2.12.3 Assessment of percent rooting
Response of regenerated shoots to rooting at the end of 30 days of culture was
assessed by calculating number of shoots responded for rooting and expressed in
percentage.
Percent rooting = No. of shoots with rooting x 100 Total no. of explants cultured
3.2.12.4 Assessment of percent kanamycin resistance (Putative transformants)
All the co-cultivated explants were cultured on shooting medium containing
200mg/l kanamycin to obtain putative transformants and then the percentage of
putative transformants was calculated.
Percent putative transformants= Total no. of explants survived on 100mg/l kanamycin x 100% Total no. of shoots obtained from co-cultivated explants
41
RESULTS 4.1 Standardization of surface sterilization
Suckers are present below the rhizosphere region and contain many bacteria,
fungi and viruses. It is therefore necessary to follow an effective surface sterilization
method. The results of surface sterilization of banana (Musa accuminata cv.
Patakpura) suckers using different chemicals were presented in the table 4.1. The
suckers were excised from old banana plants and the 3–4 outer layers were removed.
They were soaked in 2% commercial fungicide ‘‘Bavistin’’ (BASF, India) and 0.2%
streptocyclin (Bayers India limited) for 2 h. The suckers were surface sterilized with
70% ethanol for 2 min, 0.1% mercuric chloride for different time intervals to obtain
sterile explants. Finally, the suckers were rinsed several times with sterile double-
distilled water. Treatment of the apical shoot tips from suckers with 0.1% mercuric
chloride was found suitable as it resulted in 88% aseptic culture and 88.75%
survivability. Treatment with 0.1% HgCl2 for 12 min resulted in 90% aseptic culture
with lower (76.25%) percent survivability. The untreated explants (control) had 100%
survival but all the cultures were found to be infected with microbes.
Table 4.1 Effects of surface sterilization on aseptic culture and survival of explants
Treatment Chemical concentration Time (Min)
In vitro culture
Survival % Aseptic culture % Death %
T1 No treatment (control) 0 0.0 0.00 100.00
T2 0.1% HgCl2 +4drops of
Tween-20 2 40 12.20 27.80
T3 0.1% HgCl2 +4drops of
Tween-20 4 45 15.40 29.60
T4 0.1% HgCl2 +4drops of
Tween-20 6 80 20.50 59.50
T5 0.1% HgCl2 +4drops of
Tween-20 8 92 22.20 70.20
T6 0.1% HgCl2 +4drops of
Tween-20 10 88 11.25 88.75
T7 0.1% HgCl2 +4drops of
Tween-20 12 90 23.75 76.25
T8 0.1% HgCl2 +4drops of
Tween-20 14 85 33.50 51.50
T9 0.1% HgCl2 +4drops of
Tween-20 16 75 30.70 44.30
42
4.2 In vitro regeneration.
A successful application of in vitro techniques for crop improvement rests on a
reproducible plant regeneration protocol and it is also helpful for genetic
transformation. There are two methods of in vitro regeneration i.e. direct and indirect
regeneration. Corm slices obtained from suckers were tried in MS medium
supplemented with various combinations of growth hormones and the regeneration
via callus culture and direct multiple shoot regeneration via apical meristems was
obtained. The rate of success mostly depends upon the combinations of different
growth regulators like BAP, IAA and NAA as well as IBA and Kn.
4.2.1 Indirect regeneration via callus induction and organogenesis
Callus is unorganized mass of plant cells and its formation is controlled by
growth regulating substances present in the medium. Hormones, especially auxins
were absolutely necessary for callus induction. Morphology of the callus varied with
different plant growth regulators used in the medium. Various combinations of growth
hormones like BAP (1.0-2.0 mg/l) and 2,4-D (1.0-5.0 mg/l) presented in the table
4.2.1 were tried with MS medium supplemented with 5% coconut water, 10mg/l
Ascorbic acid and malt extract (100mg/l) for callus induction from corm slices of
Musa accuminata cv. Patakpura. Initiation of callus was observed on the explants
after about 25-30 d when MS medium with BAP alone or in combination with auxin
2, 4-D, was used. Among these combinations, highest response of callusing observed
was in 2mg/l BAP and 4mg/l of 2, 4-D on MS Medium supplemented with 5%
coconut water, 10mg/l ascorbic acid and 100mg/l malt extract. Light yellow friable
callus was obtained and the calli were sub-cultured on to regeneration media
containing different concentrations of BAP in combination with 2, 4-D in four weeks
intervals to provide adequate nutrients. Best response on organogenesis from callus
was obtained with 4mg/l BAP and 1mg/l 2, 4-D along with adenine sulphate 100mg/l.
43
Table 4.2.1 Effect of growth regulators on callus induction from corm slices of banana cv. patakpura (Mean data#SE)
MS+ Growth regulators (mg/l)
Callus induction mean ±SE
Culture period (weeks)
No. of explants cultured
Average No. of explants
responded in percentage
(%)*
Types of response
MS +0 0 12 10 0 - MS +BAP (1.0) +2, 4 –D (1.0)
34.5±2.23 12 10 4 (40.0) Low
MS +BAP (1.0) +2, 4 –D (2.0)
39.2±3.30 12 10 6 (60.0) Moderate
MS +BAP (1.0) +2, 4 –D (3.0)
42.5±5.22 12 10 7.5 (75.0) Moderate
MS +BAP (1.0) +2, 4 –D (4.0)
44.05±3.03 12 10 6 (60.0) Moderate
MS +BAP (1.0) +2, 4 –D (5.0)
48.30±3.55 12 10 7 (70.0) Moderate
MS +BAP (2.0) +2, 4 –D (1.0)
55.45±2.23 12 10 6 (60.0) Moderate
MS +BAP (2.0) +2, 4 –D (2.0)
67.22±6.33 12 10 7 (70.0) Moderate
MS +BAP (2.0) +2, 4 –D (3.0)
80.50±4.55 12 10 10 (100.0) High
MS +BAP (2.0) +2, 4 –D (4.0)
95.35±5.20 12 10 10 (100.0) High
MS +BAP (2.0) +2, 4 –D (5.0)
70.52±6.40 12 10 6 (60.0) Moderate
*10 no. of explants were cultured per treatment in 3 replication
4.2.2 Direct regeneration via multiple shoot induction from apical meristem
Most often direct regeneration occurs through shoot proliferation from pre-
existing meristems instead of de novo formation of a meristem. Direct in vitro
regeneration can be from different explants like shoot tip, apical meristem, leaves,
nodal explants etc. Corm slices were inoculated on MS with different concentration
and combinations of cytokinin (2.0-10mg/l BAP) and auxin (1.0-3.0 mg/l IAA)
supplemented with 100 mg/l Adenine sulphate as presented in Table 4.2.2. The
highest multiple shoot induction (40.45%) was observed from MS medium with BAP
8mg/land IAA 1mg/l. The multiple shoots were sub-cultured in different
concentrations of hormones on MS medium at 3-4 weeks interval for providing
adequate nutrients.
44
Table 4.2.2 Effect of various concentrations of growth regulators BAP and IAA along with Adenine sulphate on multiple shoot induction from corm slices of Musa acuminata cv. Patakpura
MS +BAP (mg/l)
IAA (mg/l) Adenine sulphate (mg/l)
No. of explants
inoculated
Culture period (weeks)
Mean number of shoots per plant (%)*
0 0 100 10 6 5.20±1.02 0 0.5 100 10 6 6.20±2.00
2.0 0.5 100 10 6 8.20±2.52 4.0 0.5 100 10 6 12.20±4.88 6.0 0.5 100 10 6 24.20±2.54 8.0 0.5 100 10 6 30.20±8.65 10 0.5 100 10 6 22.45±6.22 0 1.0 100 10 6 28.22±3.22
2.0 1.0 100 10 6 25.54±4.20 4.0 1.0 100 10 6 35.20±3.22 6.0 1.0 100 10 6 40.20±4.55 8.0 1.0 100 10 6 40.45±5.20 10 1.0 100 10 6 35.00±1.00 0 1.5 100 10 6 20.40±2.64
2.0 1.5 100 10 6 18.23±4.56 4.0 1.5 100 10 6 10.60±1.54 6.0 1.5 100 10 6 10.00±1.22 8.0 1.5 100 10 6 28.22±2.32 10 1.5 100 10 6 17.16±3.55 0 2.0 100 10 6 2.20±0.02
2.0 2.0 100 10 6 15.00±5.00 4.0 2.0 100 10 6 14.25±3.55 6.0 2.0 100 10 6 28.02±2.50 8.0 2.0 100 10 6 30.00±6.45 10 2.0 100 10 6 9.20±4.33 0 2.5 100 10 6 1.20±0.32
2.0 2.5 100 10 6 10.50±2.54 4.0 2.5 100 10 6 15.20±3.20 6.0 2.5 100 10 6 16.5±3.02 8.0 2.5 100 10 6 20.20±8.54 10 2.5 100 10 6 16.55±3.80 0 3.0 100 10 6 2.20±0.75
2.0 3.0 100 10 6 14.30±5.44 4.0 3.0 100 10 6 15.20±2.22 6.0 3.0 100 10 6 20.20±8.54 8.0 3.0 100 10 6 25.40±8.54 10 3.0 100 10 6 15.20±5.54
(*10 no. of explants were cultured per treatment in 3 replications.)
45
4.3. Shoot multiplication
The multiple shoots obtained after inoculation of apical shoot meristems were
separated into individual plantlets and multiplied on MS medium supplemented with
different concentrations of cytokinin BAP (2.0 -8.0 mg/l) in combination with auxins
IAA and NAA (0.5-3.0 mg/l) along with Adenine sulphate 100 mg/l as in the Table
4.3.The highest rate of shoot multiplication (35.30%) was achieved on MS medium
having 4mg/l BAP and 0.5 mg IAA and NAA along with 100mg/l Adenine sulphate.
Table 4.3 Effect of various concentrations of cytokinin BAP and auxins IAA and NAA on in vitro shoot multiplication
MS+BAP (mg/l)
IAA + NAA (mg/l)
Adenine sulphate (mg/l)
No.of explants
inoculated
Culture period
(Weeks)
Mean number of shoots per plant (%.)*.
0 0 100 1 4 2.24±0.24 0 0.5 100 1 4 3.47±1.44
2.0 0.5 100 1 4 7.24±2.24 4.0 0.5 100 1 4 35.30±2.54 6.0 0.5 100 1 4 20.22±3.40 8.0 0.5 100 1 4 15.20±2.00 0 1.0 100 1 4 3.20±2.54
2.0 1.0 100 1 4 15.22±5.50 4.0 1.0 100 1 4 30.33±5.54 6.0 1.0 100 1 4 20.50±3.00 8.0 1.0 100 1 4 15.50±5.20 0 2.0 100 1 4 5.20±1.40
2.0 2.0 100 1 4 10.30±2.54 4.0 2.0 100 1 4 15.20±2.40 6.0 2.0 100 1 4 10.01±5.54 8.0 2.0 100 1 4 15.22±3.22 0 2.5 100 1 4 5.3±2.54
2.0 2.5 100 1 4 6.20±3.54 4.0 2.5 100 1 4 10.30±2.44 6.0 2.5 100 1 4 24.30±2.54 8.0 2.5 100 1 4 12.45±3.25 0 3.0 100 1 4 08.45±4.88
2.0 3.0 100 1 4 5.34±2.05 4.0 3.0 100 1 4 5.20±1.55 6.0 3.0 100 1 4 3.20±0.54 8.0 3.0 100 1 4 4.39±0.40
*1 no. of explants was cultured per treatment in 3 replications.
46
4.4. Root initiation from in vitro regenerated plants
For better survival of plantlets in the field, well developed rooting system is
necessary. Root induction was observed when regenerated shoots were cultured on
medium with low concentrations of auxins, whereas at higher concentrations shoots
formed callus. The multiple shoots were transferred to MS supplemented with IAA
(0.1-1.0 mg/l) for root differentiation along with 0.5 mg/l of activated charcoal. The
maximum root differentiation i.e. profuse rooting from multiple shoots was achieved
on MS supplemented with 1.0mg/l IAA along with 0.5 mg/l of activated charcoal
after 30 d of transferring into rooting medium as presented in table 4.4.
Table 4.4 Effect of various concentrations of IAA on root formation from multiple shoots of Musa acuminata cv. Patakpura
Treatment Concentrations of IAA (mg/l)
Activated charcoal
(mg/l)
No. of explants
inoculated
Root (%) ( Mean # S.E) Type of root
1 0 0.5 10 0 No rooting 2 0.1 0.5 10 0 No rooting 3 0.2 0.5 10 4.20±2.20 Slow rooting 4 0.3 0.5 10 14.35±4.65 Slow rooting
5 0.4 0.5 10 24.50±5.20 Very less no.
of roots
6 0.5 0.5 10 44.54±7.32 Less no. of
roots
7 0.6 0.5 10 54.20±4.20 Long ,
multiple 8 0.7 0.5 10 85.20±7.32 Long multiple
9 0.8 0.5 10 89.20±6.52 Long
,multiple
10 0.9 0.5 10 90.80±5.20 Long
,multiple, branching
11 1.0 0.5 10 94.20±4.20
Long, multiple,
hairy branching,
thick. *10 no. of explants were cultured per treatment in 3 replications.
4.5. Pre-hardening and hardening of regenerated plants
The regenerated rooted plantlets were first prehardened by removing them
from the medium and roots washed thoroughly under tap water to remove traces of
agar then placed back in a beaker of tap water for 24 hours so as to acclimatize them.
47
Then these plantlets were transferred to plastic cups containing soil: sand:
vermicompost (1: 1: 1) and kept in the culture room for 7 days. After 7 days, these
plants were transferred to clay pots containing soil: sand: vermicompost (1: 1: 1) and
kept in the poly-house for final hardening and establishment of plants. All the plants
transferred to pots survived under field conditions (plate 4:13).
4.6. Kanamycin based selection of putative transformants
As per the gene construct, kanamycin is employed for selection of transformed plants.
Therefore it is essential to find out the lethal dose of kanamycin for the selection of
transformed plants. The growth inhibiting dose of kanamycin was determined by
transferring the transformed and non- transformed somatic embryos on MS medium
supplemented with various concentrations of kanamycin (50-300mg/l) (table 4.6). It
was observed that the lethal dose for control explants was 200mg/l kanamycin used
for primary screening of putative transformants. The optimized selection method
eliminates the regeneration of non-transformed plants.
Table 4.6 Kanamycin based selection
Treatment Kanamycin concentrations (mg/l)
Survival (%) in control plants after 30 days.
Survival (%) in transformed plants after 30 days.
1 50 80.44±5.50 60.04±3.60 2 100 75.54±3.40 65.84±5.30 3 150 75.67±4.50 75.57±7.40 4 200 65.85±9.50 85.80±5.54
5 250 85.69±8.26 95.68±9.66 6 300 55.45±6.28 45.44±8.88
4.7. Cefotaxime sensitivity test.
Influence of cefotaxime on shoot induction and subsequent development was
checked by culturing somatic embryos on shoot induction and shoot multiplication
media containing different concentrations of cefotaxime (0, 50, 100, 200, 300, 400,
500, 600, 700, and 800 mg/l). Of the different concentrations analyzed, 300 mg/l
cefotaxime had less negative effect on shoot induction and multiplication, but
effective concentration against the growth of the Agrobacterium strain used. Above
500 mg/l cefotaxime, the induction of shoots was severely affected.
48
Table 4.7 Effect of cefotaxime on shoot induction in the medium containing
different concentrations of cefotaxime
Treatment Concentration of cefotaxime (mg/l)
No. of explants
Shoot number
Agrobacterium growth.
1 0 10 33 +++ 2 50 10 31 +++ 3 100 10 29 +++ 4 200 10 27 ++ 5 300 10 25 + 6 400 10 23 + 7 500 10 17 + 8 600 10 7 - 9 700 10 5 - 10 800 10 2 -
*10 no. of explants were cultured per treatment in 3 replications. +++: Prominent growth of Agrobacterium. ++: Moderate growth of Agrobacterium. +: Slow growth of Agrobacterium. -: complete inhibition of Agrobacterium growth.
Table 4.8 In vitro transformation studies using Annexine Bj2 gene
No. of shoots obtained after co-cultivation
No. of shoots obtained on
kanamycin 20 days after inoculation
No. of plantlets transferred to
growth chamber.
Transformation frequency (%) ( based on
kanamycin selection)
136 69 62 50.7
4.8 Histochemical GUS assay of transformed somatic embryos.
Embryogenic cell mass was tested and intense blue staining was readily observed in
all the positive cells and no staining in control tissues as shown in plate 6:18.
49
Fig. 4.1 Effect of growth regulators on callus induction
Fig. 4.2 Effect of IAA concentration on root formation
0
20
40
60
80
100
120
MS +0 BAP (1.0) +2, 4 –D
(1.0)
BAP (1.0) +2, 4 –D
(2.0)
BAP (1.0) +2, 4 –D
(3.0)
BAP (1.0) +2, 4 –D
(4.0)
BAP (1.0) +2, 4 –D
(5.0)
BAP (2.0) +2, 4 –D
(1.0)
BAP (2.0) +2, 4 –D
(2.0)
BAP (2.0) +2, 4 –D
(3.0)
BAP (2.0) +2, 4 –D
(4.0)
BAP (2.0) +2, 4 –D
(5.0)
Average No. of explants responded in percentage (%)Callus induction mean ± SE
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11
Effect of IAA concentration on root formation
Concentrations of IAA (mg/l) Root (%)
50
Fig.4.3 Kanamycin based selection
Fig. 4.4 Effect of cefotaxime concentration on shoot survival
0
50
100
150
200
250
300
350
1 2 3 4 5 6
Kanamycin concentrations (mg/l)
Survival (%) in control plants after 30 days.
Survival (%) in transformed plants after 30 days.
0
100
200
300
400
500
600
700
800
900
1 2 3 4 5 6 7 8 9 10
Concentration of cefotaxime (mg/l) Shoot number
51
Plate 2:
1. Shoot initiation on MS +8mg/l BAP+1mg/l IAA after 4 weeks.
2. Shoot buds after 5 weeks.
3. Shoots after 6 weeks.
4. Elongated shoots after 7 weeks of culture.
5. Elongated shoots after 8 weeks.
6. Shoot multiplication on MS+4mg/l BAP+0.5mg/l IAA and NAA.
7. Shoots after 1 month of culture on multiplication medium.
52
Plate 3:
8. Root initiation and development on MS+1mg/l IAA+0.5mg/l activated charcoal.
9. Prehardening of rooted plants on 1:1:1(sand: soil: vermicompost) in culture room
after 7days.
9a. Prehardened plants after 12 days.
10. Final hardening in poly house on 1:1:1 (sand: soil: vermicompost) after 7 days of
transfer.
53
Plate 4:
11. Final hardening after 12 days of transfer to poly house.
12. Hardened plants after 2 months in polyhouse.
13. Hardened plants after 3 months in polyhouse.
54
Plate 5:
14. Callus initiation after 45 days of culture on MS +2mg/l BAP + 4mg/l 2, 4-D 15. Shoot proliferation from callus on MS + 4mg/l BAP + 1mg/l 2, 4-D
16. Somatic embryos (arrows) scanned with S-3400N scanning Electron Microscope
16a. Somatic embryos scanned with S-3400N scanning Electron Microscope
55
Plate 6:
17. Bacterial culture 18. Co-cultivation of embryogenic cell mass with bacteria after infection 19. GUS stained embryogenic mass with distinctly visible somatic embryos 20. Shoot bud initiation on selection medium 21. Shoot regeneration on selection medium 22. Shoot proliferation on selection mediu
56
V. DISCUSSION
5.1 General discussions
Banana (Musa spp.) constitutes the core food for hundred millions of people
and has an important role in the economy of several countries of the developing
world. More than three quarters of its production is consumed as a staple food in the
country of production, whereas the export market is dominated by the cultivar
subgroup Cavendish (Musa AAA) which is highly susceptible to a wide range of
biotic and abiotic stresses (Roux et al., 2008; van Asten et al., 2011; Figueroa-
Ya´n˜ez et al., 2012 ). Traditional plant breeding in this cultivar subgroup is difficult
because of the long life cycle, triploidy and sterility. Genetic transformation is an
attractive alternative way to introduce agronomically important genes in these
cultivars. Moreover, it can be used as a tool to elucidate gene function and regulation
using technologies like gene and promoter trapping, activation tagging or RNA
interference (Peraza-Echeverria et al., 2007; Roux et al., 2008; Santos et al., 2009).
Thus, a high throughput transformation pipeline is necessary for reliable functional
analysis of sequence data that are becoming available with the ongoing Musa genome
sequencing project (Roux et al., 2008).
Transgenic banana plants have been obtained in different cultivars using
biolistics (Sa´gi et al., 1995; Becker et al., 2000; Arinaitwe et al., 2004; Vishnevetsky
et al., 2011), Agrobacterium-mediated transformation (May et al., 1995; Ganapathi et
al., 2001; Arinaitwe et al., 2004; Khanna et al., 2004; Remy et al., 2005; Pe´rez-
Herna´ndez et al., 2006a; Ghosh et al., 2009), and protoplast electroporation (Sa´gi et
al., 1994). At present, most Agrobacterium tumefaciens mediated banana
transformation protocols use embryogenic cell suspensions (ECSs) (Sa´gi et al., 1995;
Ganapathi et al., 2001; Arinaitwe et al., 2004; Khanna et al., 2004; Remy et al., 2005;
Pe´rez-Herna´ndez et al., 2006a; Ghosh et al., 2009). To increase transformation
efficiency, several parameters have been optimized, for example Agrobacterium strain
(Khanna et al., 2004; Pe´rez-Herna´ndez et al., 2006a), age of the ECSs (Ganapathi et
al., 2001; Arinaitwe et al., 2004), time of infection (Arinaitwe et al., 2004), time of
co-culture and bacterial cell density (Khanna et al., 2004; Pe´rez-Herna´ndez et al.,
2006a), co-centrifugation of embryogenic cells and Agrobacterium (Khanna et al.,
2004; Ghosh et al., 2009), heat-shock pretreatment of ECSs (Khanna et al., 2004), the
use of semisolid or liquid medium for co-cultivation (Ghosh et al., 2009), use of
57
surfactants like Pluronic F68 (Khanna et al., 2004) or Agrobacterium vir gene
inducing compounds like acetosyringone (Khanna et al., 2004; Pe´rez-Herna´ndez et
al., 2006a).
Notwithstanding, these optimizations, reported transformation efficiencies do
not exceed 100 independent transgenic plants per 50 mg of ECS, and are generally
genotype and cell line dependent.In addition establishing cell suspensions is,
however, a lengthy process and is cultivar dependent. Developing resistant varieties
through genetic engineering potentially is the most cost-effective and sustainable
method of averting the impact of abiotic and biotic stresses. Such improvement
initiatives demand efficient transformation frequencies and a standard, rapid, and
reproducible protocol that can be used to transform all banana genomic groups. A
transformation system using apical meristems from various cultivars of Musa has also
been established (May et al., 1995; Tripathi et al., 2005). To date, there has been no
report describing the regeneration, establishment and transformation of M. acuminata
cv. Patakpura despite its popularity in all parts of Orissa for its high productivity. For
these reasons, the present investigation was undertaken to establish a rapid,
reproducible protocol for regeneration of M. acuminata cv. patakpura for genetic
transformation with Annexin Bj2 gene for drought resistance through Agrobacterium
mediated transformation.
5.2. In vitro regeneration
5.2.1. Surface sterilants
The success of in vitro regeneration basically depends on aseptic conditions
and microbes free sterile explants/cultures. The explants were sterilized with different
sterilizing agents (Table 4.1). It was observed that increasing the time of treatment
increased the percentage of aseptic cultures but decreased the survival of the explants.
The highest percentage (88.75) of aseptic culture was obtained when explants were
treated for 10 min. Jaisy and Ghai (2011) who worked on in vitro propagation of
banana also found treatment of explants with HgCl2 (0.1%) for 6 minutes most
effective surface sterilization procedure registering maximum culture establishment
with minimum contamination.
58
5.2.2 Direct regeneration via shoot multiplication from apical meristem
The highest multiple shoot induction (40.45%) was observed from MS
medium with BAP 8mg/l and IAA 1mg/l along with 100mg/l Adenine sulphate after
six weeks of culture (Plate 2: 1 and 2). Shoot tips being easy to culture are extensively
preferred as starting material for micropropagation for a wide range of banana
cultivars (Cronauer and Krikorian, 1984a; Banerjee and De-Langhe, 1985; Vuylsteke,
1989; Israeli et al., 1996). Ganapathi et al. (1998) reported regeneration of banana,
Lal Kela (AAA genotype) from shoot tips and obtained 5-6 shoots per explants.
Priyono (2001) reported that micro-propagation of Musa paradisiaca through cormlet
initiation by in vitro culture of apical meristem slices. Josekutty et al. (2003)
established the efficient micropropagation of Apat regular and Apat fissuse (cooking
bananas) using shoot meristem. Hamide and Pekmeze (2004) used shoot tips to
multiply banana cultivars dwarf Cavendish. Diro and Staden (2005) also re-ported
rapid in vitro protocol for multiplication of Enset vetricosum from shoot tips.
Kanchanapoom and korapatchaikul (2012) reported induction of yellow compact
calluses from in vitro-grown shoot tips of diploid bananas (Musa acuminata, AA
group) ‘Kluai Sa’ and ‘Kluai Leb Mu Nang’. Unlike other methods that need for field
access and seasonal dependence, main advantage of this procedure is that it by passes
this procedure.
Proliferation rate of shoot and elongation are affected by the type and
concentration of plant growth regulators. Cytokinins and auxins are used as growth
regulators for in vitro propagation of Musa spp. As concentration of exogenous
cytokinin appears to be the main factor affecting shoot multiplication, most widely
used and most effective cytokinin for this purpose is adenine based cytokinin; N6-
benzylaminopurine (BAP) (Cronauer and Krikorian, 1984a;Vuylsteke 1989; Hamide
and Pekmeze, 2004; Rabbani et al., 1996).The same hormone was also used in this
studies. Others include isopentyladenine (2-ip) (Cronauer and Krikorian, 1984a),
zeatin (Vuylsteke and De Langhe 1985) and kinetin (Cronauer and Krikorian, 1984b).
Cronauer and Krikorian (1984a) obtained 9.1 shoots per explants during in vitro
multiplication of Phillippine lacatan and Grand naine, on a modified MS medium
supplemented with 10µM BAP (Cronauer and Krikorian 1984a), while Rahman et al.
(2002) obtained 4.52 shoots per explants on the same media in variety Bari-1. It
59
indicates different genotypic response towards the cytokinin BAP. Aziah and Khalid
(2002) used higher concentration of BAP for regeneration, using whole meristems and
scalps as explants. Scalps were induced on MS medium supplemented with coconut
water and BAP (75µM). The average number of shoots produced from scalps was six
times more than that produced from a single shoot tip. Venkatachalam et al. (2006)
achieved direct regeneration from leaf sheaths of silk banana (AAB) on MS medium
supplemented with BAP (22.4 µM). Thiadiazuran (TDZ) is a urea based cytokinin,
which is frequently used in banana micro-propagation. Hamide and Pekmeze (2004)
tested the effects of BAP (5, 10, 20 and 30 µM) and TDZ (0.4, 1, 2 and 3 µM), either
alone or in combination with 1 µM indole acetic acid (IAA) for shoot multiplication
in three banana types. They observed that in all the three type, shoot proliferation and
elongation were significantly greater with TDZ as compared with BAP. Also each
cytokinin, in combination with 1 µM IAA increased shoot proliferation and
elongation more than when used alone. Strosse et al. (2008) cultured shoot tip
explants to determine the influence of five cytokinins [BAP, kinetin,
isopentenyladenine (2iP), zeatin, and thidiazuron (TDZ)] each at three concentrations
(1, 10 and 100 µM) added to the basal corn shoot multiplication (CSM) medium, on
multiple shoot formation . When shoot tips of banana variety Williams (AAA) were
cultured on basal CSM medium devoid of plant growth regulators, all explants grew
into elongated single shoots. The highest number of explants developed into elongated
shoots. The highest number of explants developing into multiple shoots was observed
with TDZ (up to 100%) followed by BAP (up to 92%). These studies suggest that a
combination of cytokinins, with or without auxin have been used for enhancing shoot
proliferation in banana regeneration.
5.2.3 In vitro shoot multiplication
The highest rate of shoot multiplication (35.30%) was achieved on MS
medium having 4mg/l BAP and 0.5 mg IAA and NAA along with 100mg/l Adenine
sulphate (Plate 2: 6 and 7). Cytokinins are known to reduce the dominance of apical
meristem and induce axillary as well as adventitious shoot formation from
meristematic explants (Pandey and Jaiswal, 2002). Amongst the cytokinins, BAP is
the widely used, most effective and affordable cytokinin for the proliferation of
multiple shots (Johnson and Manickam, 2003). Even though, cytokinins have been
known to induce shoot formation, there exist differences in the relative strength of the
60
different types of cytokinins in shoot induction of diploid and triploid Musa cultivars.
Banana micropropagation protocols via shoot tip culture invariably use BAP (Kacar et
al., 2010). Multiple shoot production from MS medium supplemented with BAP and
IAA in lower concentration was reported in two diploid cultivars of South India
(Mukunthakumar and Seeni, 2005). BAP at 22.2µM was considered optimal for shoot
proliferation as well as shoot elongation from excised scalps of banana cultivars
(Shirani et al., 2010). Cronauer and Krikorian (1985 a; 1985b) obtained multiple
shoot clusters from the terminal floral apices of Musa acuminate cv. Dwarf Cavendish
(AAA), inoculated on modified MS medium supplemented with 22.2µM BAP and
10% (v/v) coconut water. Shoot apices explants also produced similar results
(Cronauer and Krikorian, 1984).Thus the findings in the current investigation were
similar to the earlier investigations. The high performance of BAP over the other
cytokinins in inducing multiplication in shoot tip cultures has been reported in
different cultivars of banana (Ikrak-ul-Haq and Dahot, 2007). In other plants such as
Oryza sativa, Bacopa monerria and Penthorun chinense important role of BAP for
stimulation and proliferation of multiple shoot growth were reported (Medina et al.,
2004; Mohapatra and Rath, 2005; Yang and Peng, 2009). The marked effects of BAP
on shoot formation compared to kinetin and 2ip may be attributed to its high stability
in in vitro cultures which is in agreement with Buah et al. (2010). BAP is not easily
broken down and therefore persists in the medium. It is also possible that the amount
of BAP that gets conjugated in the medium was smaller than what happened to other
plant hormones. Therefore, larger amount of BAP exiting in free or ionized forms in
the medium are readily available to plant tissues.
5.2.4 Root initiation from in vitro regenerated plants
The maximum root differentiation i.e. long, multiple, hairy, branching and
thick roots from multiple shoots was achieved on MS supplemented with 1.0 mg/l
IAA along with 0.5 mg/l of activated charcoal after 30 d of transferring into rooting
medium (Plate 3:8). Rooting can be stimulated when individual shoots are transferred
to basal medium alone (Cronauer and Krikorian 1984; Jarret et al., 1985). However,
auxins may induce further root initiation (Vuylsteke, 1989). Cronauer and Krikorian
(1984) reported no differences in the root-inducing effects of NAA, IAA or IBA in
presence of 0.025% (w/v) activated charcoal. Hwang et al. (1984) recommended 0.1-
61
0.25% activated charcoal. Therefore, the results of the present study are in total
agreement with these above earlier investigations.
5.2.5 Pre-hardening and hardening regenerated plants
All the rooted plantlets (100%) survived on hardening in sand, soil and
vermicompost (1:1:1) mixture (Plate 3: 9-10, plate 4:11-13). This indicates that the
sequential hardening enabled the plants to acclimatize to field conditions and hence
the plants grew normally after transferring to the polyhouse and then field conditions
with 100% survival. Sharma and Thorpe (1990) showed that complete plantlets of
Morusalba were successfully established (100%) in the field condition.
5.3 Genetic transformation
Breeding for drought-resistant banana cultivars using classical methods
remains a tedious endeavour because of high sterility, polyploidy, and long generation
times of most of edible cultivars. Biotechnology involving modern tissue culture, cell
biology and molecular biology provides an opportunity to develop new germplasm
better adapted to changing demands (May et al., 1995). Agrobacterium mediated
transformation is a major DNA delivery system for novel transgenic technologies.
However, low transformation efficiency has become the greatest challenge in the
application of this technology in recalcitrant crops, especially monocotyledonous
plants, like banana which are not naturally susceptible to Agrobacterium spp.
(Philippe Vain, 2007).
A number of genes have been isolated and used in genetic transformation of
plants including banana (Rout et al., 2000). Several of these genes have been cloned,
and their expression regulated by CaMV 35S promoter. The CaMV 35S promoter,
which is also used here in the current study, was preferred above other potential
promoters because it is a more powerful promoter than others and is not greatly
influenced by environmental conditions or tissue types (Sagi et al., 1997).
5.4 Kanamycin based selection of putative transformants
The antibiotic effect of kanamycin is normally attributed to its ability to inhibit
translation in prokaryotes, plastids and mitochondria of eukaryotes, by binding to 30S
subunit (Misumi et al., 1978). As the gene construct used here carried npt-II as a
62
selectable marker, its expression detoxifies the kanamycin. It was important to find
out the toxic level of kanamycin which can completely inhibit the growth of the
normal plants so that putative transformants can be isolated. It was observed that
lethal dose for control plant was 200mg/l kanamycin and this was used for primary
screening of putative transformants. The optimized selection method eliminates the
regeneration of nontransformed plants.
5.5 Characterization of regenerated transgenic plants
Transgenic plant production has been intimately connected to the β-
glucuronidase (UidA or GUS) gene used as a reporter or marker gene. The enzyme
stability and the high sensitivity and amenability of the Gus assay to qualitative
(histochemical assay) and to quantitative (fluorometric or spectrophotometric assay)
detection are some of the reasons that explain the extensive use of uidA gene in plant
genetic transformation. Methods for UidA (GUS) gene detection have been
thoroughly described in the literature (Cervera, 2005).
Histochemical Gus assay results, used as a marker for transformation efficiency,
showed that embryogenic cells from cultivar “patakpura” were competent and
susceptible to Agrobacterium tumefaciens infection and hence transformable. This
was due to observation of blue colour in the stained cells and tissues. The GUS gene
inserted in the transformed cells produced a protein which has enzymatic activity, β-
galactosidase, which turned the colorless substrate (x-gluc in the stain) into blue.
Transient GUS expression assay three days after cocultivation of explants showed
uniform blue coloration confirming transient expression of the reporter gene in all the
surface cells. Expression of β-glucuronidase in banana somatic embryo proves the
efficacy of this system for expression of any other useful foreign protein in cv.
Patakpura.
63
VI. SUMMARY AND CONCLUSION
Bananas are important staple foods, which are difficult to breed due to the
high sterility of commercial cultivars. Sustainable banana production is vital to ensure
a constant supply of fruit to meet world food demand. However, fruit production faces
challenges from changing economic, social, and environmental conditions.
The genetic improvement of banana is one of the strategies to ensure sustained
production. Consequently, strategies that exploit both conventional and
biotechnological approaches, particularly genomic analyses and transformation, have
considerable potential to play a role in achieving sustainable fruit production. Genetic
and physical mapping of the Musa genome will facilitate the isolation of genes that
are potentially useful in genetic transformation, with significant progress being
achieved in this area in recent years. Improved understanding of genomes will
facilitate targeted breeding and more efficient use of existing Musa biodiversity.
In vitro-based technologies, particularly genetic transformation, offer
excellent opportunities to create novel cultivars with targeted traits through the
manipulation of nuclear and cytoplasmic genomes. Exposure to somaclonal variation
through basic tissue-culture procedures will continue to generate new cultivars,
whereas somatic hybridization and cybridization by protoplast fusion will also enable
the mobilization of genetic material without the requirement to isolate and
characterize DNA. Overall, the genetic improvement of bananas is crucial to generate
new cultivars that are productive as well as adapted to different environmental
conditions. It requires the availability of suitable germplasm combined with
experimental procedures and the practical expertise and theoretical knowledge of
biotechnologists and breeders to manipulate nuclear and cytoplasmic genomes using
both conventional and biotechnological approaches. In the long term, genetically
improved banana cultivars could ensure sustained fruit production for food security,
with the additional advantage of guaranteed income for farmers in producing
countries.
The results obtained from this study demonstrate that “patakpura” cells are
highly competent and transformable by Agrobacterium mediated transformation
64
system. Importantly, an efficiency of more than 50% was demonstrated under this
study with “patakpura” embryogenic cells, which suggests that selectable markers
could be unnecessary in the selection of transgenic plants.
In this study it is also evident as high GUS specific activity was observed in
embryogenic cell mass after cocultivation. Transformation efficiency could have been
further enhanced if embryo germination was done in non-selective medium. There are
reports suggesting that antibiotics allow formation of transgenic embryos but interfere
with embryo germination (Yao et al. 1995; Bretagnesagnard and Chupeau 1996).
Hence, the use of subsequent germination medium with reduced levels or removal of
kanamycin promises to enhance the regeneration frequency of transgenic plants.
This dissertation contributes to the current information about improvement of
transformation and regeneration efficiency of bananas. Studies in the recommended
areas will add useful information on the long term integration and stability as well as
heritability of transgenes in these transgenic “patakpura” plants.
RECOMMENDATIONS
The following recommendations can be made about the transformation of
“patakpura” cells with annexin BJ2genes.
1. There is need to perform further molecular analyses like PCR, Southern blotting,
RT-PCR and Western blotting on screen house samples of transgenic “patakpura” to
ascertain the gene integration pattern as well as gene stability in these plants.
2. Morphological characteristics, of the regenerated plants, like leaf emergence rates,
pseudostem vigor and girth width also need to be evaluated to establish the expression
status as well as effect of BJ2 genes to the growth rate of the transgenic plants in vivo.
i
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APPENDICES
Appendix I: Composition of MS medium
MS major salt
Mg/l medium 500 ml stock (20x)
NH4NO3
1650 mg 16.5 gm
KNO3
1900 mg 19 g
CaCl2.2H20
440 mg 4.4 g
MgSO4.7H2O
370 mg 3.7 g
KH2PO4
170 mg 1.7 g
MS minor salt
Mg/l medium 500 ml stock (200x)
H3BO3
6.2 mg 620 mg
MnSO4.4H2O
22.3 mg 2230 mg
ZnSO4.7H2O
8.6 mg 860 mg
KI
0.83 mg 83 mg
Na2MoO4.2H2O
0.25 mg 25 mg
CoCl2.6H2O
0.025 mg 2.5 mg
CuSO4.5H2O
0.025 mg 2.5 mg
MS Vitamins
Mg/l medium 500ml stock (200x)
Thiamine (HCl)
0.1 mg 10 mg
Niacin
0.5 mg 50 mg
Pyridoxine (HCl)
0.5 mg 50 mg
xxvii
Iron-EDTA stock (500 ml, 200x)
Dissolve 3.725 g of Na-EDTA (Ethylene diamine tetra acetic acid, disodium salt) in
250 ml double distilled water. Dissolve 2.785g of FeSO4.7H2O in 250 ml double
distilled water. Boil Na2-EDTA solution and add it to FeSO4 solution gently by
stirring.
• Major 50 ml
• Minor 5 ml
• Iron 5 ml
• Vitamin 5 ml
• Myo-inositol 100 mg
• Glycine 2.0 mg
• Sucrose 30 mg
Make final volume to 1 litre by adding double distilled water, set pH 5.6-5.8. Add
agar 7 g/l, autoclave at 15 psi/ 121oC for 20 min.
Appendix II: LB medium (1 litre)
10gm tryptone, 5gm yeast extract,10 gm NaCl, 15gm agar, pH 7.0.
Appendix III: Preparation of the phytohormone stock
1mM IAA stock solution 100 ml (MW 175.2gm)
17.52 mg of IAA was dissolved in 1N NaOH (1-1.5 ml) and sterile double distilled
water was added slowly with constant stirring, followed by makeup of volume to 100
ml with sterile water. Store in the freezer.
1mM NAA stock solution 100 ml (MW 186.2 gm)
18.62 mg of NAA was dissolved in 0.5 ml Dimethyl sulfoxide and added sterile
double distilled water, stirred up and volume made up to 100 ml and stored at 4oC.
1M 2, 4-D stock solution 100 ml (MW 221gm)
xxviii
22.1 mg of 2, 4-D dissolved in 0.5 ml 1N NaOH and 10 ml water by vortexing and
make up the volume to 100 ml by adding sterile double distilled water, store it at 4oC.
1mM BAP stock solution 100 ml (MW 224.2gm)
22.5 mg of BAP was dissolved in 1N NaOH (0.3-0.5M) and add double distilled
water slowly with stir, volume make up to 100 ml and store at 4oC.
Appendix IV: substrate preparation for Gus assay: Reagent
Volume taken for 25 ml in ml
Substrate A 0.1 M Na2HPO4.2H2O or 0.5KH2PO4
5
Sodium EDTA
2.5
Potassium fericyanide 50mM
2.5
Potassium ferrocyanide 50mM
2.5
0.1% Triton-X
2.5
Distilled water
10
Substrate B X-gluc (5-bromo-4- chloro-3-indolylβ-D-glucuronide) dissolved in Dimethyl formamide
25 mg of X-gal (MP Biomedicals) substrate in 250µl Dimethyl formamide