Department of Plant Breeding and Genetics

CREDIT SEMINARON

Jawaharlal Nehru Krishi Vishwa Vidyalaya Jabalpur (M.P.)

(2015-16)

“PRODUCTION OF TRANSGENICS IN

OILSEED CROPS”

Guided by Speaker

Dr.(Mrs.) Rajani Bisen

Kanak SaxenaRoll no. 234

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Introduction Why transgenic production Need of transgenics in oilseeds Steps of transgenic Production Methods of Transformation Transgenic Oilseed crops (Case studies) Application Limitations Conclusion

CONTENT

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• India is the fourth largest oilseed producing country in the world• India has the largest area and production of oilseeds in the world• Oilseeds are rich sources of energy and nutrition.• Oilseeds also contain carbohydrates, vitamins and minerals• Oilseeds and oilseed meals have an important role in relieving the

malnutrition and calorie nutrition of human and animal population. In addition, the vegetable oils are useful as lubricants, surface coatings, cosmetics and as raw material for various industrial products like, paints, varnishes, hydrogenated oil, soaps, perfumery, lubricants, etc

• Oil-cake which is the residue after the oil is extracted from the oilseeds, forms an important cattle-feed and manure

INTRODUCTION

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• The most important annual oilseed crops are

1. Groundnut 2. Rapeseed-Mustard 3. Sesame 4. Sunflower 5. Safflower 6. Soybean 7. Niger 8.Castor and 9. Linseed.

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Area, Production and Productivity

Source --Department of Agriculture and cooperation DAC (2013-14)

AREA(lakh ha)

285.25

PRODUCTION(million tonne)

328.77

PRODUCTIVITY(Kg/ha)

1094

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Oilseeds productivity (kg/ha) in India and World (2012)

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Why transgenic production ?

Rapid method of crop improvementOvercome problems related to wide hybridizationEvolution of New GenotypesEffectivenessGenerates useful genetic variation

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Survey says-

• Reasons for growing transgenics:–Weed control–Better yield, more return, profit–Reduce costs–Less time

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Survey says-

• Reasons for not growing transgenics:– Overall costs– No need to change– Market access

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Why we need Transgenic technology?

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Need of transgenics in oilseeds?

• India is a leading importer of vegetable oil so we have to develop some improved varieties so we can able to break all constraints in oilseed production

• Although we have wide variability in oilseeds ; wild as well as cultivated but we are not able to mitigate the requirement of vegetable oil in our country

• So there is a need to create variability in the existing population in the existing crop and for this creation of variability transformation method is the only milestone in the improvement of oil production

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Sr. No. Crop Trait Countries where approved

1Sunflowe

r Herbicide tolerance Canada

2Argentine

canola

Herbicide tolerance and improved protection against

weedsCanada, USA,

3 Soybean Improved weed control and

herbicide tolerance, increased cooking quality

USA, Argentina, Japan, Canada, Uruguay, Mexico,

Brazil and South Africa, Czec Republic, European Union, Korea, Russia, Switzerland,

Taiwan, U.K., Philippines and Australia

4Flax,

Linseed

Herbicide tolerance, antibiotic resistance and improved weed

protectionCanada, USA

Worldwide Transgenic crops approved for commercial use

Source ; the Indian Society of Oilseeds Research,DOR, Hyderabad (2013)13

Institute Plants/crops

Transgene(s) inserted

Aim of the project

Indian Agricultural Research, Institute, New Delhi

Mustard/ rapeseed

Arabidopsis annexin gene

To generate stress-tolerant plants

Indian Agricultural Research, Institute, New Delhi

Mustard/

rapeseed

Choline dehydrogenase

To generate abiotic stress-tolerant plants

Delhi University, South Campus, New Delhi

Mustard/ rapeseed

Bar, Barnase, baraster

To generate herbicide-tolerant plants, male sterile and restorer lines for hybrid seed production

Indian Agricultural Research, Institute, New Delhi

Brassica Chitinase, glucanase and RIP

To generate plants resistant to fungal attack

Major Developments in Transgenic Research and its Applications in Public Sector

Source ; the Indian Society of Oilseeds Research,DOR, Hyderabad (2013)14

Institute Plants/crops

Transgene(s) inserted

Aim of the project

Proagro PGS (India) Ltd, Gurgaon

Brassica/

mustard

Bar, barnase, barstar

To develop superior hybrid

cultivars

Tata Energy Research Institute, New Delhi

Mustard

Ssu-maize Psy and Ssu-tpCrt1 gene

To generate plant containing high level of b-carotene

Major Developments in Transgenic Research and its Applications in Private Sector

Source ; the Indian Society of Oilseeds Research,DOR, Hyderabad (2013)15

Steps for developing transgenic crops

o Identification of geneo Gene transfero Regeneration from callus/tissue/protoplasto Gene expression to the desired levelo Backcross to produce varietieso Field testo Approval for commercialization

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Methods for gene transfer

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Vector Based Method:- Infecting plant cells with plasmid as vector carrying the desired gene

1) Agrobacterium tumefaciens 2) Agrobacterium rhizogenes

Direct Gene Transfer Method :- Shooting microscopic particle containing gene

directly into the Cell 1. Particle Bombardment (Gene Gun) 2. Electroporation of protoplast 3. Microinjection 4. Liposome mediated gene transfer

Direct & Indirect Methods of Gene Transfer

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A.tumifaciens

GENETIC TRANSFORMATIO

N

Agrobacterium gene Transformation method

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Genetic Transformation

disarmed T-DNA (contains transgene)

gene transfer (Ti) plasmid

Agrobacterium tumefaciens

bacterialchromosome

Transformed plant cell with geneplant

chromosome

inserted gene

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To identify cells/tissues in which new genes are incorporated into plant’s DNA, grow in media containing antibiotics or herbicides.

Successful transformant

Identification of Transformed cell

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Whole plants with inserted genes are regenerated through tissue culture.

Regeneration of Transformed cells

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1. Electroporation – Electrical impulses are used to increase membrane and cell wall

permeability to DNA contained in the surrounding solution.

Direct Methods

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2. Microinjection - injection of DNA directly into the cell Nucleus using an ultrafine needle.

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3.Biolistic / Gene Gun

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4.Polyethelyne glycol –

Plant cell protoplasts treated with PEG are momentarily permeable, allowing uptake of DNA from the surrounding solution.

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• Resistant to herbicide – eg. Soybean, Linseed• Resistant to virus – eg. Sunflower• Production of male sterile lines – eg. Brassica spc.• Improved oil quality and quantity – eg. Canola

• Improved nutritional quality – eg. Canola

Applications Of Transgenic In Oilseeds

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SOYBEAN 28

Herbicide Resistant Soybean

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A mutant aro gene from bacteria Salmonella typhimurium has been used for developing tolerance to glyphosate.

The target site of glyphosate is a chloroplast enzyme 5-enol pyruvylshikimic acid 3-phosphate synthase (EPSPS)

Introduction to mutant aro gene produces modified EPSPS, not recongnizable to glyphosate.

Development of herbicide resistant crops allows the elimination of surrounding weeds without harm to the crops.

Roundup Ready Soybeans; The first variety was also known as GTS 40-3-2 are a series of genetically engineered varieties of glyphosate resistant soybeans produced by Monsanto.

Steinrucken, H.C et al., 198030

SESAME 31

Genetic transformation of cultivated sesame through particle bombardment using 5-day-old apical,meristematic tissues of germinating seedlings

• An in vitro plant generation and genetic transformation protocol was established in sesame (sesamum indicum l. cv rama) through biolistic particle gun bombardment.

• 5-day-old apical, meristematic tissues of in vitro-germinating seedlings were used as explants.

• A synthetically designed bialaphos resistance gene (bar)was used for transformation.

Bhattacharyya, et al., 201532

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SUNFLOWER 34

Generation of white mold disease-resistant sunflower plants expressing human lysozyme gene

• Gene used - human lysozyme gene• Method used - Agrobacterium mediated transformation

• The plasmid pNGL was used for transformation of Agrobaterium tumefaciens. It contains the structural gene for neomycin phosphotransferase-II npt II, which encodes resistance to the antibiotic kanamycin sulphate, the β-glucuronidase gus reporter gene and the human lysozyme gene.

• The expression of the human lysozyme gene is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and is terminated by the nopaline synthase Nos gene terminator.

• Southern blot analysis, Western blotting ,Northern blot analysisSawahel, et al.,(2006)

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BRASSICA 36

 Jagannath, et al.,2012

The barnase/barstar gene system in the GM mustard

• Barnase/barstar gene system, in which herbicide resistance is linked

with male sterility, so that the herbicide will kill the male fertile lines,

leaving the seed producing male sterile plants unharmed.

• Barnase, a ribonuclease (an enzyme) from bacillus amyloliquefaciens,

inhibits pollen formation and results in male sterility in the transgenic

plants.

• The bar gene from streptomyces hygroscopicus encodes for the

enzyme phosphinothricin acetyltransferase, that restores male fertility

in the transgenic lines.

• A phsphonithricin resistance-coding (pat) gene is used to eliminate

undesirable segregates, by spraying an herbicide.

•

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Combined expression of a barley class II chitinase and type I ribosome

inactivating protein in transgenic Brassica juncea provides protection

against Alternaria brassicae

Gene used - barley antifungal genes class II chitinase (AAA56786) and type I ribosome inactivating protein (RIP; AAA32951) Method used - Agrobacterium mediated transformation

The plasmid GJ42 contains two antifungal genes, barley chitinase (AAA56786) and RIP (AAA32951) under the control of CaMV35S promoter and a selectable marker gene, neomycin phosphotransferase (nptII)The stable integration and expression of transgenes in plants were confirmed by Southern blot and Western analysis.

Result - The transgenic plants showed up to 44% reduction in A. brassicae hyphal growth in in vitro antifungal assays.

 Chhikara, et al.,(2011)38

CANOLA

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Improved rapeseed

• Improved rapeseed cultivars were free of erucic acid and glucosinolates. Erucic acid tastes bitter and had prevented the use of rapeseed oil in food.

• Gluconsinolates, which were found in rapeseed meal leftover from pressing, are toxic and had prevented the use of the meal in animal feed. These new cultivars are known as "double-zero" rapeseed. In Canada, where "double-zero" rapeseed was developed, the crop was renamed "canola" (Canadian oil) to differentiate it from non-edible rapeseed.

Bin jhu, et al.,(2011)40

Laurate canola oil• GM rapeseed enriched with lauric acid can be used for producing

fat-based coatings in food processing• Canola plant modified with thioesterase gene obtained from

California bay laurel tree• Enzyme produces lauric acid from genetically modified (GM)

canola seeds• Low saturated fat content• Heat tolerant• Does not break down• Excellent for high temperature cooking processes•

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Limitations Unstable performance Difficult to transfer polygenic traits Costly method High Technical Skill Effect on natural Evolution Undesirable combination

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Various varieties of oilseeds have been developed

through Transgenic for different traits and released for

commercial cultivation.

Genetic engineering offers considerable potential for

genetic improvement of crop plants, especially for

disease and pest resistance, and improved quality

characteristics.

The Transgenic technology has great potential to

generate new varieties along with the conventional

breeding.

CONCLUSION

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References• Ag-West Biotech, “Canola- Biotechnology’s Powerhouse

Crop,” AgBiotech Infosource Issue 21, May , 1996 http://www.agwest.sk.ca/event_inf_may96.shtml>, April, 2002.

• Department of Agriculture and cooperation DAC (2011-12)• FAOSTAT 2012• Jagannath, Arun ; Arumugam, N. ; Gupta, Vibha ; Pradhan,

Akshay ; Burma, Pradeep Kumar ; Pental, Deepak (2002)Development of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea) Current Science, 82 (1). pp. 46-52

• Steinrücken, H.C.; Amrhein, N. (1980). "The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase". Biochemical and Biophysical Research Communications 94 (4): 1207–12.

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Thank You45