agrobacterium, and production of transgenic plants in cotton, corn,soybean,mustard, recent status, problems in india and worldwide statistics.

1. THEROLE OF VECTOR MEDIATED GENETRANSFER AND
PRODUCTION OF TRANSGENIC PLANTS IN
COTTON,MAIZE,SOYBEAN AND OILSEED
Submitted To: Submitted By:
Dr. A. K. Sharma Smrutishree Sahoo
Professor M.Sc.(Ag.) PBG Previous
Department of PLANT BREEDING AND GENETICS
Collage of Agriculture
Swami Keshwanand Rajasthan Agricultural University, Bikaner

2. GENE TRANSFER IN PLANTS
• Transfer a gene from one DNA molecule to another DNA
molecule.
GENETIC TRANSFORMATION
• desirable gene transfer from one organism to another
• stable integration
• expression of foreign gene into the genome
• Transient transformation- DNA is not integrated into host genome
• Stable transformation - DNA is integrated into host genome and is
inherited in subsequent generations.
• The transferred gene is known as TRANSGENE and the organism
that develop after a successful gene transfer is known as
TRANSGENIC.

3. METHODS OF GENE TRANSFER
NATURAL METHODS
• 1. Conjugation
• 2. Bacterial transformation
• 3. Retroviral transduction
• 4. Agrobacterium mediated transfer( Ti vector)
ARTIFICIAL METHODS
Physical methods
• 1. Microinjection
• 2. Biolistics transformation/particle gun
• Chemical methods
• 1. DNA transfer by calcium phosphate method
• 2. Liposome mediated transfer
• Electrical methods
• 1. Electroporation
Ti vectors mostly used followed by
paricle gun and electroporation.

4. VECTORS FOR THE PRODUCTION OF
TRANSGENIC PLANTS
A vector is a DNA molecule capable of independent existence and replication.
Properties of a good vectors
1) replicate autonomously
2) easy to isolate and purify
3) easily introduced into the host cells.
4)suitable marker genes that allow easy detection of the transformed host cell.
Eg. Genes for amphicillin and Tetracycline resistance.
5) unique target sites for restriction enzymes
6) vector should contain suitable regulatory elements like promoter, operator,
ribosome binding sites.

5. Types of plant vectors
PLANT VECTORS
PLASMID VECTORS
1-pTi(tumour inducing
plasmid)
2-pRi (root inducing
plasmid)
PLANT VIRUS VECTORS
1-CaMV(cauliflower mosaic
virus)
2-Gemini virus
3-TMV(tobacco mosaic
virus)
4-BMV(brome mosaic virus)

6. Agrobacterium - mediated Gene
Transfer
• Most common method of engineering dicots, but
also used for monocots
• Pioneered by J. Schell (Max-Planck Inst.,
Cologne)
• Agrobacteria
– soil bacteria, gram-negative, related to
Rhizobia
species:
• tumefaciens- causes crown galls on many dicots
• rubi- causes small galls on a few dicots
• rhizogenes- hairy root disease
• radiobacter- avirulent

7. Infection and tumorigenesis
• Infection occurs at wound sites
• Involves recognition and chemotaxis of the
bacterium toward wounded cells
• galls are “real tumors”, can be removed and
will grow indefinitely without hormones
• genetic information must be transferred to
plant cells

8. Why Agrobacterium?
• Agrobacterium tumefaciens or Agrobacterium rhizogenes
mediated transformation is to date the most commonly used
method for obtaining transgenic plants.
• host plant species range A. tumefaciens include:
• Large number of dicots and some monocots and Gymnosperms.
• Agrobacteria are naturally occurring, ubiquitous soil borne
pathogens.
1- A. tumefaciens causes crown gall disease (tumors)
2- A. rhizogenes causes root hair disease (hairy root)
• Other bacterial groups also contain species capable of
interkingdom genetic exchange (Gelvin 2005).

11. Gene Transfer using Agrobacterium
Ti-plasmid(200kb)
Ti plasmid produce octopine and nopaline.
Ri plasmid produce agropine and
mannopine.
Regions in Ti plasmid-
1-T-DNA contains oncogene and opine
synthesis genes
2-vir region-regulates transfer of T-DNA to
plant cells
3-opine catabolism region
4-conjugative transfer region(oriT or tra )
region
5-origin of replication
Right and left border (RB, LB) sequences are the only parts of
T-DNA needed to enable transfer into plants.

12. LB RB
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
T-DNA
These genes have typical eukaryotic expression signals!
1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by wounded plant cells
activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation groups), span about 30 kb of Ti
plasmid.
auxA auxB cyt ocs
Vir (virulent) genes

13. Overview of the Infection Process

14. A. tumefaciens & Crown Gall Disease

15. Agrobacterium tumefaciens for TRANGENIC PLANTS
DRAWBACKS:
1) Auxine/Cytochine made by T-DNA do not allow proper
plant regeneration
2) Opine is not usefull for plant
3) Ti plasmids are big (200-800Kb)
4) Monocots don't produce AS in response to wounding.
5) couldn't regenerate plants from tumors

16. 1) BINARY VECTOR SYSTEM
Strategy:
1. Move T-DNA onto a separate, small plasmid.(mi-Ti)
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in T-DNA.
4. Vir genes are retained on a separate plasmid.(helper Ti)
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
2) CO-INTEGRATEpTi VECTORS
• Produced by integrating the modified E. coli plasmid(pBR322)in to disarmed pTi.
VECTORS DERIVED FROM pTi-
Disarmed pTi- the deletion of genes governing auxin and cytokinin production
from T-DNA of a Ti-plasmid.

17. Tearless Onion
Colorful Cauliflowers
Purple tomatoes
Blue Roses

18. TRANSGENIC PLANTS
• “Transgenics” or GMOs are defined as those
organisms with a gene or genetic construct of
interest that has been introduced by molecular
or recombinant DNA techniques.
• The power of this technique lies in its ability to
move genes from one organism to crop plants
to impart novel characteristics.
• It is possible to transfer genetic material from
algae, bacteria, viruses or animals to plants or
to move genes between sexually incompatible
species.

19. TARGET TRAITS
•Disease resistance
•Improving the quantity of the protein
•Increasing vitamin content
•Stress tolerance
•Herbicide resistance
•Delayed ripening
•Edible vaccine

20. VEGETABLES
Tomato, Potato, Eggplant
Lettuce, Celery, Cauliflower
Cabbage, Sugarbeet, Carrot,
Cucumbers, Sweetpotato,
Cassava
FRUITS
Apple, Strawberry,
Walnut, Muskmelon,
Papaya, Grape
Transgenic Crops
for Food
EDIBLE OILS
Mustard
Oilseed rape
Canola
Sunflower
CEREALS
Wheat, Rice
Maize, Rye
LEGUMES
Soybean, Pigeon pea,
Chick pea

21. TRANSGENIC COTTON
• HERBICIDE RESISTANCE
 Glyphosate Resistance
i. Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide
ii. Marketed under the name Roundup, glyphosate inhibits the enzyme
EPSPS (S-enolpyruvlshikimate-3 phosphate – involved in
chloroplast amino acid synthesis), makes aromatic amino acids.
iii. The gene encoding EPSPS has been transferred from glyphosate-
resistant E. coli into plants, allowing plants to be resistant.

22. EPSP SYNTHASE
Phosphoenol
pyruvate
Tryptophan
Tyrosine
Phenylalaline
ROUNDUP
(Glyphosate )
Glucose
3 phosphoglycerate
Glycolysis
EPSP SYNTHASE
ROUNDUP
(Glyphosate )
In transgenic plant, herbicide cannot bind the mutant of EPSPS
(Roundup resistant cotton and soybean)
ROUNDUP (Glyphosate) RESISTANCE
( HERBICIDE TOLERANCE IN CROPS)

23. Bt COTTON-INSECT RESISTANCE
• Genetically modified to produce insecticidal toxins derived from
the bacterium Bacillus thuringiensis.
• Toxins are crystalline proteins (Cry-proteins) that target specific
pests.
• DISCOVERY OF Bt- COTTON
• Ernst Berliner isolated a bacteria that had killed a Mediterranean
flour moth in 1911, and rediscovered Bt. He named it Bacillus
thuringiensis, after the German town Thuringia where the moth
was found.
• INTRODUCTION TO INDIA?
• In 2002, a joint venture between Monsanto and Mahyco
introduced Bt cotton to India. In 2011, India grew the largest GM
cotton crop at 10.6 million hectares.

24. (Agrobacterium mediated)

25. • Bacillus thuringiensis (or Bt)
• a Gram-positive, soil-dwelling bacterium,
commonly used as a biological pesticide
• Cry gene- Upon sporulation, B. thuringiensis forms
crystals of proteinaceous insecticidal δ-endotoxins
(called crystal proteins or Cry proteins), which are
encoded by cry genes.
• How Bt works?
• 1. Insect eats Bt crystals and spores.
• 2. The toxin binds to specific receptors in the gut
and the insects stops eating.
• 3. The crystals cause the gut wall to break down,
allowing spores and normal gut bacteria to enter
the body.
• 4. The insect dies as spores and gut bacteria
proliferate in the body.
alkaline digestive tracts denature the
insoluble crystals soluble and being
cut with proteases found in the insect
gut, which liberate the toxin from the
crystal. The Cry toxin paralyzing the
digestive tract and forming a pore.The
insect stops eating and starves to
death.

28. ADVANTAGE AND DISADVANTAGE
• Advantages
– High insect specificity
• Control crop damage and disease vectors
– Nontoxic to non-target species
– Biodegradable
– Reduction of other insecticides
• 94.5 million kg (19.4%) from 1996 to 2005 for cotton
– Yield increases
• Limitations
– Susceptible to resistance
– High seed cost

29. Bt-COTTON INDIA
• At present, 96% of India
cotton cultivation area is
under Bt cotton crops
but it wasn’t always so.
• Cotton production rose
from 14 million bales in
the pre-Bt year of 2001-
'02 to 39 million bales in
2014-'15, a rise of
almost 180%. India’s
cotton imports fell,
exports grew and as of
2015-16 India is
expected to have
overtaken China as the
biggest cotton producer
it the world.

31. PROBLEMS FACED
1. the adoption of Bt cotton is that the seeds are more expensive than local,
non-genetically modified varieties.
2. the seeds cannot be reused and farmers need to buy new stock for every
growing season. This, along with licencing agreements with local seed
companies, has given Monsanto a near monopoly on cotton seeds in
India that has been the biggest worry for activists.
3. the diffusion of illegal Bt hybrids that hadn't been cleared for biosafety
standards, leading to fears of environmental toxicity.
4. Bt hybrids were unsuitable for rain-fed cotton lands so yield stagnation of
more than 1000 varieties cause farmer suicide.
5. Bt provides protection only against one type of cotton pest, use of
insecticides has risen again close to the levels of the pre-Bt years.

32. PRESENT ISSUES
1. Despite Monsanto's warning the government cut royalty by
more than 70% on March 9, 2017and followed that up by
capping the price of seeds at Rs 800. They were earlier sold
at between Rs 830 and Rs 1,000.
2. Monsanto faces a big challenge from the Central Institute of
Cotton Research, which has introduced Bt genes into 21
cotton seed varieties and is offering to provide these seeds
to farmers at 10% the cost of Monsanto’s products.
3. if Monsanto leaves then India will lose access to the new
iterations of its Bollgard seed that farmers might need in the
next three to five years

33. TRANSGENIC CORN(MAIZE)
HERBICIDE RESISTANT MAIZE
• Corn varieties resistant to glyphosate herbicides
were first commercialized in 1996 by Monsanto, and
are known as "Roundup Ready Corn".They tolerate
the use of Roundup.
• Bayer CropScience developed "Liberty Link Corn"
that is resistant to glufosinate.
• Pioneer Hi-Bred has developed and markets corn
hybrids with tolerance to imidazoline herbicides
under the trademark "Clearfield"

34. INSECTICIDE-PRODUCING CORN
Bt-CORN
• The European corn borer, Ostrinia nubilalis, destroys
corn crops by burrowing into the stem, causing the
plant to fall over.
• The European corn borer causes about a billion
dollars in damage to corn crops each year.
• In recent years, traits have been added to ward
off corn ear worms and root worms, the latter of
which annually causes about a billion dollars in
damages.
• Bt-corn is poisonous to these insects.

35. Bt-corn
Non
Bt-corn
Bt-corn reduces the need for
conventional pesticides:

36. • DROUGHT RESISTANCE
• In 2013 Monsanto launched the first transgenic drought
tolerance trait in a line of corn hybrids called DroughtGard.
• The MON 87460 trait is provided by the insertion of the cspB
gene from the soil microbe Bacillus subtilis;
• it was approved by the USDA in 2011and by China in 2013
• SWEET CORN
• GM sweet corn varieties include "Attribute", the brand name
for insect-resistant sweet corn developed by Syngentaand
Performance Series™ insect-resistant sweet corn developed
by Monsanto

37. PRESENT SCENARIO(INDIA)
• India does not currently allow the growing of GM food crops
but the government of Prime Minister Narendra Modi, keen
to improve farms' productivity, has encouraged open field
trials after a five-year de facto ban.
• Monsanto's (MON.N) Indian subsidiary expects to submit final
trial results for its genetically modified (GM) corn to
lawmakers within a year for the government to then decide
on a commercial launch.
• the resulting corn will be insect- and herbicide-tolerant,
helping raise yields by 15-20 percent.
• The current average corn yield in India is 2-2.25 tonnes per
hectare, compared with 10 tonnes in the top producer United
States

38. TRANSGENIC SOYBEAN
• In 1994 the first genetically
modified soybean was introduced
to the U.S. market, by Monsanto.
• In 2014, 90.7 million hectares of
GM soy were planted worldwide,
82% of the total soy cultivation
area
• ROUNDUP READY SOYBEAN
• Roundup Ready Soybeans (The first
variety was also known as GTS 40-
3-2 (OECD UI: MON-04032-6)) are a
series of genetically engineered
varieties of glyphosate-
resistant soybeans produced
by Monsanto.

40. TRANSGENIC OILSEEDS

41. ß-carotene rich Mustard oil

42. GM HYBRIDISED MUSTARD,
• As it is claimed, gives up to 30% more yield than the present best
varieties.
• Researchers have used “barnase / barstar” technology for genetic
modification. A barnase gene is isolated from a soil bacterium
called Bacillus amyloliquefaciens.
• The gene can code for a protein that impairs the pollen production
in a plant making it male-sterile.
• This male-sterile variety is crossed with a parent variety having a
gene called ‘barstar’ to block the action of barnase gene.
• The resulting variety, having both foreign genes, is a fertile plant
and it can increase yield of the crop.
• This canola crop is cultivated 10% in Canada.

44. STATISTICS
Global overview Values
Acreage of genetically modified crops worldwide 179.7m ha
Acreage of genetically modified soybean crops worldwide 92m ha
Leading GMO crops producing country, based on acreage United States
Acreage of genetically modified crops in the U.S. 70.9m ha
Adoption of GMO technology for soybeans worldwide 83%
Adoption of GMO technology for cotton worldwide 75%

45. Global Adoption of Biotech
Soybean, Maize, Cotton, and Canola
• The most planted biotech crops in 2016 were soybean, maize, cotton, and canola. Although there was only 1% increase in the planting of
biotech soybean, it maintained its high adoption rate of 50% of the global biotech crops or 91.4 million hectares. This area is 78% of the
total soybean production worldwide.

46. Global Area of Biotech Crops in 2015 and 2016: by
Country (million hectares)
rank Country 2015 2016
1 USA* 70.9 72.9
2 Brazil* 44.2 49.1
3 Argentina* 24.5 23.8
4 Canada* 11.0 11.6
5 India* 11.6 10.8

47. Canada overview Values
Genetically modified soybeans seeded in Ontario 718.3k ha
Genetically modified corn for grain seeded in Ontario 679.9k ha
Genetically modified soybeans seeded in Quebec 210k ha
Genetically modified corn for grain seeded in Ontario 303k ha
U.S. overview Values
Share of GMO corn in the U.S., based on acreage 92%
Share of GMO cotton in the U.S., based on acreage 80%

48. Attitudes of farmers and consumers Values
U.S. Millennials who think that GMOs should never be used in food and beverages 34%
U.S. Millennials who think that GMOs need more testing 32%
U.S. consumers who drink less coffee because of GMO concerns 22%
GMO is the most important food safety issue for U.S. consumers 24%
U.S. consumers strongly agree that GMOs should be labeled as such 48%

49. crop variety
Maize MON 810, MON 863, StarLink
Soybean Roundup ready soybean, Vistive gold
mustard DMH-11,Varuna ,the east European EH-2
Cotton PAU Bt 1, F1861 and RS 2013