establishment of efficient transformation system in cicer arietinum with gus gene

Establishment Of Efficient Transformation System In Cicer

arietinum WITH gus GENE

PRIYA GOSWAMI4th Semester

Plant Physiology Biochemistry & Plant Molecular biology Special Paper , 2013

Why Chickpea?

Cicer arietinum (Common Name :- Chickpea) is an important food crop in much of the developing countries.

Ranks third in production among food legumes.

It is good as a source of carbohydrate (48.2 - 67.6%), protein (12.4 - 31.5%), fat (6%) and nutritionally important minerals.

This pulse crop is an important source of dietary protein for human consumption, it is also important for the management of soil fertility due to its nitrogen-fixing ability

Provides excellent quality of protein at affordable prices to the poor and average income families.

Fig 1 : Chickpea plant

*SYSTEMATIC POSITION* Kingdom : Plantae Order : Fabales Family : Fabaceae Sub-family: Faboideae Genus: Cicer Species: C. arietinum

•Binomial name: Cicer arietinum L.

Short Description Of Chickpea Plants

Fig 2 : Different Parts Of Chickpea Plant

Leaves

Flower

Pod

Chickpea is an annual plant with plant height ranging between 30-70cm.

The plant has a deep root system with nodules & thus very efficient in fixing atmospheric nitrogen.

They have compound leaves which are arranged in alternate phyllotaxy and are generally imparipinnate with 11-13 leaflets and covered with glandular hairs.

Flowers are solitary axillary and are generally arranged in inflorescence of two to three. They are white, pink, purplish, or blue in colour.

Chickpeas are a type of pulse with one seedpod containing two to three peas.

Fig 3 : Different Coloured Flowers Of Chickpea

Fig 4: Chickpea PodFig 5: Chickpea Pod Containg Peas

Origin & Cultivation

ORIGIN & CULTIVATION : Chickpea is probably originated in an area of south-eastern Turkey and adjoining Syria, but is now cultivated throughout the semi-arid regions of the world.

Fig 6: Cultivation Of Chickpea Throughout The World

Primary Zone Of Chickpea Production

Secondary Zone Of Chickpea Production

Tertiary Zone Of Chickpea Production

CHICKPEA PRODUCTION IN INDIA : India is the largest producer and consumer of chickpea in the world, and accounts for more than 60% of the area harvested and of the total worldwide production.

Fig 7 : Different Zones Of Chickpea Production In India

GROWING CONDITIONS :

TYPES :

TEMPERATURE 18-26°C @ day and 21-29°C @ night

RAIN FALL 600-1000 mm

Fig 8: Desi Fig 9 : Kabuli

Small, angular seeds that are

variously pigmented and

are grown principally on the

Indian subcontinent and

in East Africa

Relatively large, round seeds of

white or pale cream colour and are predominantly grown in the

Mediterranean region and in Central and South America.

Growing Conditions & Types

Nutritional Content & Uses

Fig 10 : Pie Chart For The Nutritional Content Of Chickpea

Carbohydrate (with 47% starch)

Protein (with 8% crude fibre)

Fat

P,Fe,ash &other water soluble vitamins

NUTRITIONAL CONTENT :

USES :

AS FOOD AS MEDICINE OTHER USES

Due to high protein, high unsaturated fatty acid, cellulose, calcium, zinc, potassium, high amount of vitamin- B content chickpeas are used as food.

• Chickpeas contain isoflavone which has a big influence on the health of women, their estrogenic activity & also prevents the proliferation of cancer cells causing cancer cell death.• They also contain chromium which plays an important role in glucose & lipid metabolism.

• Animal feed.• Preparation of adhesive.• Preparation of dye.• Preparation of vinegar.• Cooked chickpea-milk mixture is good for feeding infants, effectively controlling diarrhoea.• Used for textile sizing.

AFFECTED CHICKPEA

YIELD

BIOTIC STRESS

ABIOTIC

STRESSTHESE ARE MAINLY DISEASES eg: DRY ROOT ROT BOTRYTIS GREY MOLD COLLAR ROT FUSARIUM WILT

eg:DROUGHTSALINITYLOW TEMPERATURE

Main Problems In Chickpea Production

AFFECTED

CHICKPEA

YIELD

BIOTIC

STRESS

ABIOTIC STRESS

THESE ARE MAINLY DISEASES eg: DRY ROOT ROT BOTRYTIS GREY MOLD COLLAR ROT FUSARIUM WILT

eg:DROUGHTSALINITYLOW TEMPERATURE

Main Problems In Chickpea Production

Diseases are the most serious constraints of chickpea production

LEAD UPTO 100% LOSS OF YIELD

Some Common Diseases In Chickpea

Aims & Objective Of My Project

Tissue culture and transformation in chickpea using gus gene governed by i. Constitutive Actin1 Promoter, ii Pod specific Promoter.

•In vitro germination of surface sterilised chickpea seeds for embryo axis development using MSO media.

•Agrobacterium mediated transformation of the developed explants.

•Selection of the plants in hygromycin.

•Hardening of the selected transformants in the soil.

•PCR analysis of the transgenics ..

Fig 11 : Chickpea Seeds Of Variety DCP-92-3

Fig 12 : Imbibition Of Chickpea Seeds

Fig 13: Surface Sterilized Seeds Were Plated In MSO* Plates After Removal Of The Seed Coat

Fig 14 : 2 Day Grown Seeds In MSO* Media

Fig 15 : 3 Day Grown Seeds In MSO* Media

Some Seeds Were Further Used For Transformation

Fig 16 : Control Plants Were Grown In The Greenhouse Condition*

in vitro germination of surface sterilised chickpea seeds for embryo axis development using MSO media.

MSO*= Murashige & Skoog Basal Media (1962)Greenhouse Condition*= with 16/8 (light/dark) photoperiod temp. 20°C under high humidity with frequent watering.



•in vitro germination of surface sterilised chickpea seeds for embryo axis development using MSO media.





Fig 15 : 3 Day Grown Seeds In MSO Media Are Used As Explants For Transformation

Agrobacterium LBA4404 with vector containing Pod Specific Promoter

Agrobacterium LBA4404 with vector containing Actin1 Constitutive Promoter

Fig 17: 20 ml Agrobacterium Culture In LB Media With Rifampicin & Kanamycin Antibiotic & kept In the Incubator Shaker @ 28°C & 120 rpm For 2 Days

O.D. Value Of 2day Old Agrobacterium Culture Was Measured (i.e. approximately around 3.0)

The Cuture Was Pellet Down @ 3500 rpm for 20 mins At The Bottom Of The Oakridge Tube

Pellet was dispersed in 40ml Infiltration Media (MS20*)+ 40µl Acetosyringone & poured in the petriplates

Explants Were Pricked With Injection Syringe Then Transferred To The Petriplate For

Agrobacterial Infection.

The Petriplate Was Kept In The Vacuum Desiccator For 20 minutes. The Pressure Is Maintained at 600

mm of Hg

Agrobacterium mediated transformation of the developed explants.

After 20 mins The Explants Were Blotted Dry & Transferred To Petriplates Containing

MSO-As* Media & Covered With Aluminium Foil & Kept In The Incubator @ 28°C For 2

days.

MS20*=MS basal media with 20g/l sucroseMSO-As*=MS basal media with acetosyringoneFig 18: Vacuum Desiccator

Vector Construct Used For Transformation

Fig 20: pCAMBIA 1301 (-) GUS Vector -Fig a: Containing Actin1 Constitutive Promoter; Fig b: Containing Pod Specific Promoter

LB = Left BorderRB = Right BorderActin1 = Constitutive Promoterpds= Pod Specific Promoternos = Nopaline synthasehpt = Hygromycin phosphotransferase.gus = Reporter Gene nptII= neomycinphosphotransferase II

Fig 19 : Vector map of pCAMBIA 1301

Fig: a

Fig: b

nptII

nptII

Selection Of The Plants In Hygromycin.

After 2 days from the Agrobacterial infection,the explants were transferred to Magenta boxes containing selection media with hygromycin (50 mg/l) for plant selection.

The transformed explants can be easily distinguished from the non transformed ones on the basis of their viability in the medium.

Fig 21: Transformed Plants In Hygromycin Selection Media

Fig a:-With Constitutive Actin1 PromoterFig b:-With Pod Specific Promoter

b.a.

Hardening of the selected transformants in the soil

Rooted plantlets with varying shoot lengths were carefully taken out of the magenta boxes and roots were washed thoroughly in water to remove the agar and placed in the soil and then they were kept in the greenhouse with containment facility, maintained with 16/8 (light/dark) photoperiod,20°C temperature,under high humidity with frequent watering.

Fig 22 : Transformed plants with GUS gene a. Governed by Constitutive promoter b. Governed by Pod Specific Promoter -were grown successfully in soil.

a. b.

PCR analysis of the transgenics.

Fig 23 :Gel Showing PCR Products Of The Genomic DNA Of Chickpea

L1,L2,L3: Transformed Plants with Pod Specific PromoterL4,L5: Transformed Plants with Actin1 PromoterL6:Negative Control (Non Transformed Plant)L7:Positive Control Plasmid

Result: Distinct bands were observed in L2,L3,L4,L5,L6.Thus the PCR analysis showed the presence of gus gene.

567 bp(Amplified gus)(Approx.)

L1 L2 L3 L4 L5 L6 L7

TABLE 1: SHOWING THE TRANSFORMATION FREQUENCY OF CHICKPEA

CULTIVAR OF CHICKPEA

PROMOTER USED FOR TRANSFORMATION

NO. OF SEEDS PLATED

NO. OF SEEDS GERMINATED

NO. OF PLANTS TRANSFORMED

% OF TRANSFORMED PLANTS WITH

RESPECT TO TOTAL NO. OF SEEDS

% OF TRANSFORMED PLANTS WITH RESPECT TO

GERMINATED PLANTS

DCP-92-3 Actin1 47 41 4 8.51% 9.75%

DCP-92-3 Pod Specific 156 139 25 16.02% 17.99%

% Of Germination % Of Transformation Of@ Total Seeds

% Of Transformation Of@ Germination

020406080

100120

Graph Showing Germination & transformation Efficiency

DCP-92-3 (With Actin1 Promoter) DCP-92-32 (With Pod Specific Promoter)

Histochemical Staining

• From the regenerated explants, the section of the seeds were excised using a sterile scalpel and were immersed in GUS staining solution.

• Then the excised explants in the staining solution were incubated at 37°C for 24 hours.

Fig 24 : gus expression a: For Transformed Plants (with Actin1 promoter)B: For Wild Type Plants

a. b.

Thus in this study ,the following have been accomplished-

i. Development of efficient regeneration system has been studied and experimentally standardized.

ii. Agrobacterium mediated gene transformation protocol for chickpea variety DCP-92-3 has been developed with gus gene &

iii. Transient gus expression has been confirmed by the presence of specific bands in PCR & through histochemical staining.

So transformation of pCAMBIA 1301 containing the gus gene into the chickpea variety DCP-92-3 was successfully done in my project.

Conclusion

Acknowledgement

At the very beginning, I feel highly privileged to extend my sincere gratitude to my respected supervisor Dr. Karabi Datta for suggesting me this interesting project work. It is my privilege that I got the opportunity to work under her benevolent guidance.

Moreover, I would like to thank Prof A.K Biswas who has always helped and motivated me during my project work.

I would also like to convey my deepest gratitude and sincere regards to Dr Sailendra Nath Sarkar for his continuous support, useful advice and encouragement in completing my project work. I would also wish to acknowledge with sincere appreciation and deepest gratitude to my co guide Dr. Moumita Ganguly who has guided me at every step and has helped me immensely throughout this project. I am also thankful to Ms. Sayani Majumder for her constant help and support.

The content of the text, pictures, figures and protocols are incorporated in this review based on the literature available in the text books, E-survey and various Research papers. Last but not the least; I am ever thankful to all the scholars of Plant Molecular Biology and Biotechnology Laboratory for their constant suggestions and kind guidance during the course of my project work.

Overall it was a great learning experience and I am indebted to my friends and family for their support and to everyone with whomever I have had the opportunity to work with.

THANK YOU

establishment of efficient transformation system in cicer arietinum with gus gene

Education

embryo axis development

pod specific promoter

constitutive actin1 promoter

project tissue culture

gus gene governed

agrobacterium mediated transformation

chickpea variety dcp

actin1 promoter