production of transgenics in oilseeds by kanak saxena
TRANSCRIPT
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