11

WELCOME

Ekatpure Sachin ChandrakantMSc. Agricultural Plant Biotechnology

2

WHICH

IS

MORE

HAZARDOUS?

2

Nuclear Crisis in Japan

3

Prospects and issues of Transgenics Prospects and issues of Transgenics in Agriculturein Agriculture

3

4

Outline

What is a transgenic? What is recombinant DNA technology? Requirements for transgenics Typical gene construct Steps in transgenic plant production Global status of trangenics General applications of transgenics Biosafety regulations for GM crops Possible consequences of transgenics Future prospects of transgenics Conclusion

4

5 5

The genetic material in living organism

Uniqueness of DNA

What is a transgenic?

Exotic gene added to a species through recombinant DNA technology - Transgene

The organism that develop after successful transformation - transgenic

6

BT COTTON GOLDEN RICE FLAVR SAVRROUNDUP READY

A TYPICAL GENE CONSTRUCT

Promoter

Marker gene

Transgene

Reporter gene

Terminating sequence

7

Recombinant DNA technology

Production of a unique DNA molecule

Joining together two or more DNA fragments not normally associated with each other

8

9

Requirements for transgenics

Gene of interest

Vector

Host

Gene delivery system

DNA modifying enzymes

Plant regeneration system (tissue culture) 9

(Agrobacterium)

(Gene gun)

Indirect

Direct

10

Steps in transgenic plant production

10

NATURAL

INDUCED

Kanamycin

11(James, 2011)

12(James, 2011)

1313

Approved for commercial cultivation

BT COTTON

Moratorium for environmental release

BT BRINJAL

Under various stages of field trialsCOTTON,BRINJAL,CABBAGE, GROUNDNUT, PIGEON PEA, MUSTARD, POTATO, SORGHUM, TOMATO, TOBACCO, RICE, OKRA and CAULIFLOWER

(Genes: Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, Cry1B, Cry2Ab)

IIndian status of transgenics

14

Applications of Transgenics in Agriculture

14

Directorate of Weed Science Research, 2010

Factors contributing to crop loss

15

16

Herbicide tolerence

Weed control - biggest challenge in crop production

Reduce crop yield and quality (37 per cent)

Transgenics offer tolerance to herbicides

eg. Roundup Ready soybean - glyphosate

T25 Maize - glufosinate

Roundup Ready Cotton - glyphosate

16

17

+ Glyphosate

X

Roundup Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

Plant EPSP synthase

Aromaticamino acids

Without amino acids, plant dies

X

18

BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

Roundup Resistant Plants

+ Glyphosate

With amino acids, plant lives

Roundup has no effect;enzyme insensitive to

herbicide

19

Soybean without Roundup spray Soybean with Roundup spray

Roundup Ready soybean

Cost/benefits of HTGM Soybean

(ISSAA, 2008) 20

YearCost

savings ($/ha)

Net cost saving/increase in

gross margins, inclusive of cost of technology ($/ha)

Increase in farm income at a national level

($ million)

Increase in national farm

income as a % of farm level value of

national production

1996 25.2 10.39 5.0 0.03

1997 25.2 10.39 33.2 0.19

1998 33.9 19.03 224.1 1.62

1999 33.9 19.03 311.9 2.5

2000 33.9 19.03 346.6 2.69

2001 73.4 58.56 1298.50 10.11

2002 73.4 58.56 1421.70 9.53

2003 78.5 61.19 1574.90 9.57

2004 60.1 40.33 1096.80 4.57

2005 69.4 44.71 1201.40 6.87

2006 81.7 56.96 1549.40 7.51

2007 82.7 57.96 1358.20 5.76

21

Insect resistance

“Bt” - Bacillus thuringiensis

- A soil bacterium with

crystalline (cry) protein

which transforms to δ

endotoxin in insect gut

In Bt crops, different cry genes incorporated for insect resistance

2121

Impact of Bt cotton in India

22

23

Cost/benefit analysis of Bt cotton

(ISAAA, 2009 )

24(Neilsen, 2003)

Cost/benefit analysis of Bt cotton contd….

State

Pesticide reduction Yield increase Net profit

/ha % Q/ha % /ha %

Andhra Pradesh 4,594 58 4.9 24 12,717 92

Karnataka 2,930 51 3.3 31 6,222 120

Maharashtra 2,591 71 3.6 26 5,910 66

Gujarat 3,445 70 2.9 18 8,564 164

Madhya Pradesh 2,200 52 5.4 40 9,594 68

Average 3,202 60 4.2 29 7,737 78

Virus resistance

No valid control measure for viral diseases in plants

Various approaches used

in transgenics

Antisense mediated Satellite RNA Ribozyme mediated Coat protein mediated

25

Papaya ringspot resistance

GM Papaya in Thailand

Virus resistant transgenic plants

CMV infected CMV resistant (GM)

27

Crop Virus

Tobacco Tobacco mosaic virus

Potato Potato virus x

Tomato Tomato yellow leaf curl virus

Maize Maize dwarf mosaic virus

Rice Rice strip virus

Citrus Citrus tristeza virus

28

Disease resistance

A large number of plant defense genes encoding

antimicrobial proteins have been cloned

eg. Chitinase and glucanase

Breaks chitin and glucan in the cells of fungal pathogen

Other antimicrobial proteins - Thionin, Lysozyme, Polygalacturonase inhibitor

28

29

Disease resistant transgenic plants

Crop Gene transferred Controlled pathogen

Tobacco Bacterial chitinase Alternaria longipes

Chitinase Rhizoctonia solani

Potato 1,3,-beta glucanase Phytophthora infestans

Tomato Chitinase Fusarium oxysporum

Tobacco Ribosome inactivating protein

Rhizoctonia solani

GM Grapes

31

Quality improvement

Long term storage for tomato

Antisense technology

(polygalacturonase)

Golden Rice - enhanced vitamin A and Fe content

Golden rice 1 - 1.6 μg/g β carotene Golden rice 2 - 37 μg/g β carotene Golden rice 3 - + 7 fold iron content

32

The Golden Rice Production

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

β -carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

β-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

Transgenic flowers with longer vaselife

Post harvest life of flowers -ethylene based

Transgenic carnations expressing antisense ACC oxidase

Dianthus caryophyllus- approved in Australia

(Tanaka et al., 1998)

33

Transgenic plants as bioreactors

Act as living bioreactors - Molecular pharming

Inexpensive production of chemicals and pharmaceuticals

Carbohydrates, fatty acids, proteins,

vaccins (edible)

Transgenic banana, tobacco and potatoes for hepatitis B virus vaccine 34(Giddings et al., 2001)

35

Overview of Molecular Pharming

35

MOLECULAR PHARMING

Biosafety of GM Crops

Policies and procedures adopted to ensure environmental safety

36

Genetic manipulations - against nature !!!

37

Safety assessment approach for GM crops

3737

38

Elements of assessment

3838

39

Regulatory framework in India

3939

Possible consequences of transgenics

40

41

Risk of toxicity/allerginicity

Addition of new genetic material may activate toxic pathways

Appearance of novel metabolites

Introduction of new protein

Non-immunogenic protein could become immunogenic

41

(Kaeppler, 2000)

Brazil nut allergy in soybean

A well-known case of GM soya allergy

2S albumin gene from Brazil nut to soybean for enhanced methionine

(Julie et al.,1996)

Allergen transferred unintentionally from Brazil nut to GM soya

Investigations with GM soya revealed immune reactions in people with Brazil nut allergies

42

43

Impact of marker genes

Marker gene could induce antibiotic resistance Would reduce effectiveness of antibiotics to fight diseases

Possibilities for HGT (horizontal gene transfer) : DNA must be free from the cells Bacterial recipient must be competent Sequence homology for integration Acid environment of human stomach is not congenial HGT of nptII gene can occur 1 in 10 billion Specificity of promoter

43(Redenbaugh et al.,2008)

Use of promoters of virus origin

Concerns expressed regarding promoter sequences

eg. 35S promoter of CaMV

If it invades human cells and turns on certain genes !!

Probability is very low and no such report so far

Current process – tissue specific/plant based promoters44

Eating of foreign DNA

DNA present in all living things and eaten by humans with every meal

Broken down during digestion process

Small amount absorbed in blood stream or excreted

According to WHO, amount of DNA ingested- 0.1 to 1 g/day (novel DNA represents 0.0001%)

(Chawla, 2009) 45

Changes in nutritional elements

Accidental changes in nutritional component of GM crop

Roundup Ready soybean produces large quantities of phytoestrogen (causes breast cancer) after glyphosate spray ??

Investigation revealed no such increase

46

(SAG report, 2000)

47

Gene Flow

Accidental cross between GM plant and traditional varieties

Contaminate local variety with transgene

35S promoter sequence found in non GM Maize in Mexico (Chapela, 2001)

CIMMYT amplified 28 accessions from Oaxacan landraces with CaMV 35S promoter primer

Samples did not show presence of 35S promoter

47 (Gaur et al., 2010)

Creation of superweed

GM crop may hybridize with weedy relatives

Transgene may be transferred

eg. Ryegrass highly resistant to glyphosate found in Australia

48

Strategies to prevent gene flow

Isolation zone

Trap crop

Male sterility

Chloroplast transformation

Clean gene technology

- Devoid of vegetation

- Use of non-transgenic variety

- Inactive pollens

- Gene construct introduced into chloroplast genome

- Markerless GM49(Chawla, 2009)

Resistance of target species

Insect/ virus population rapidly adapt to environmental pressure

Development of new strains

Gene pyramiding - Best solution

50

Impact on biodiversity

Reduction in genetic diversity by development and global spread of improved varieties

Sustainable agriculture depends on mixed cropping and crop rotation

Not unique to GM but is relevant to

all improved varieties

Bt protein is highly unstable & rapidly degraded in stomach juices of vertebrates and in soil 51

Monarch butterfly incidence

Monarch butterfly caterpillars died when fed on Bt maize pollen

It was a lab experiment Butterfly fed only on Bt maize For toxic effect of Bt protein it should meet specific dose

requirement In nature it is not possible to meet that dose level

52

(Losey et al,1999)

Warangal Attempt Cattle and sheep dying on consuming Bt

cotton plants in Warangal Dist. of Andhra Pradesh??

No one has so far conclusively proved that Bt protein in the Bt cotton plants was the real culprit

Why do the cattle die eating Bt cotton only in the Warangal region of Andhra Pradesh ?

Clearly a mischievous and cheap attempt (Rao, 2007) 53

Ethical issues

Unacceptable intervention in “God’s creation” violating barriers in natural world!!

Objections to consuming animal genes in plants and vice-versa

Most species already share a very significant amount of common genetic material. Man & chimpanzee - 99.4%

54

Future prospects

From 2011 to 2015 about 12 new countries projected to adapt GM crpos

Golden rice expected in 2013

In north America herbicide tolerant wheat expected to be ready by about 2017

GM potato for late blight resistance

New events ready for playing role against climate change55

Still confused?

Transgenic Cisgenic

Choice is yours

Wild relativeNo risk of gene flowNo risk of foreign DNANo risk of allergyNo ethical issueFewer biosafety testing

Unrelated organismRisk of gene flowRisk of foreign DNARisk of allergyEthical issuesBiosafety testing

56

57

Conclusion

Transgenic crops have potential to solve world’s hunger and malnutrition problems

Safety testing and regulations can ensure its superiority

The society should be enlightened about the scientific technology

Newer and faster techniques required to evaluate transgenic products

57

Thank you…