biotechnology in the vegetable crop, transgenics,

1. Transgenics in Vegetable Crop
Name –Vivek Yadav
Department of Horticulture
Sikkim University
Course – 523 Breeding in Vegetable Crop

2. Why transgenic ?
Traits can be combined beyond species border( viruses,
• bacteria, fungi, insects, animals, human beings and
• genes synthesized in the laboratory)
Quicker & more targeted development of new varieties
• with desired traits
Gene pyramiding
• Increase the efficiency of plant breeding for genetic stock . Elite lines from
multiple parents.
Removal of certain specific defects in crops
Serve as bioreactors for molecular farming
A plant in which a
foreign gene has been
transferred through
genetic engineering is
called a transgenic
plant and the gene so
transferred is called
transgene (Singh
2001).

3. Sl no Particulars Gene technology Traditional breeding
01 Sexual process No Involved
02 Direct single gene transfer Possible Not possible
03 Gene transfer b/w unrelated organism Possible Not possible
04 Transgene Involved Not involved
05 Technical skill Very high Moderate
06 Accuracy of method Very high Moderate
07 Bioethical measures Required Not required
08 Bio safty measures Required Nor required
Difference between traditional and gene technology
Singh P. introduction to biotechnology2005

4. TRADITIONAL PLANT BREEDING
Desired Gene
X
Many genes are
transferred
Donor
Plant
Commercial
Plant Variety
New Plant
Variety
PLANT BIOTECHNOLOGY
+
A single gene is
transferred
Desired Gene
Commercial
Plant
Variety
Improved
Commercial
Plant
Variety
Desired
Gene
Donor

5. Status of GM Vegetables in India,2008
S.No Crop Lab stage Green house stage Field trial stage MLT
1 Brinjal * * * *
2 Cabbage * * *
3 Cassava *
4 Cauliflower * * * *
5 Okra * * * *
6 Onion *
7 Potato * * *
8 Tomato * * *
9 Watermelon *
Renuswarup,2008

6. Steps in gene technology variety release
1:Isolate gene of interest
2: Clone into a Ti-plasmid: Gene
of interest + herbicide
resistance gene
3:Transform the plant with the
altered Ti-plasmid
4: Regenerate entire plant from
transformed cells, callus
or seeds
5: Large field trials followed by
small scale trials.

7. Tomato
• Constitutive expression of the phytoene
desaturase (crtI) gene from the bacterium
Erwinia uredovora resulted in elevated β-
carotene in tomatoes, but also led to reduction
in total carotenoid levels (Romer et al., 2000)
• The reduction in total carotenoids is believed to
be an effect of feedback regulation from
b-carotene (Bramley, 2002)
• Fruit-specific over expression of the native
lycopene b-cyclase resulted in increased b
carotene accumulation, without decrease in
total carotenoids (Rosati et al., 2000)
β-Carotene
Lycopene
Xanthophyll
Anthocyanins
zinc

8. • Fruit specific expression of yeast gene ySAMdc
• Tomatoes with 300% more Lycopene
• Increases total polyamines & shelf life
Transgenic tomato line 579HO
Mehta et al., 2002

9. Produce new breeds of food
Cancer susceptible Trp53-/- mice fed a diet
supplemented with the high anthocyanin
tomatoes showed a significant extension of
life span.
Butelli et al., 2008

10. Tomatoes with high Zn
Mouse Metallothionein mt-1 a cysteine-rich metal binding protein
was transferred to tomato
The average Zn content in transgenic tomato leaves was 32.7
mg/100 g (1.6 times higher than wild type.
Sheng et al.,2007

11. Flavr Savr Tomato
Tomato plants with antisense PG
gene showed 95% reduction in PG
enzyme.
Flavr-Savr stayed firm for a longer
period of time.
The first GM crop approved for marketing was the Flavr-Savr
tomato by Calgene, Inc. (US) in 1994.
The consultative synthases of PG antisence
RNA in transgenic plant resulted in a
substantial reduction in the levels of PG
mRna ans enzymatic activity

13. GENETIC MODIFICATION FOR TASTE & FLAVOUR

14. Tomatoes that smell like a
Rose, geranium and lemon grass
Scientists in Israel and at Rutgers and the University
of Michigan, USA, transferred a gene geraniol
synthase (GES) from Ocimum basilicum into
tomato plants which converts lycopene to aroma
molecules.
Transgenic tomato plants produced fruits enriched in
several acyclic flavour components such as
geraniol, citral, citronellol, linalool, etc.,

15. Aromatic Tomato
 Tomato plants transgenic for S-linalool synthase
(LIS) gene, accumulate S-linalool & 8-hydroxylinalool
in ripening fruits
Shimon et al .,2006
Linalool is a major component of the
scent of many flowers & present in many
edible fruits( guava, peach, plum,
pineapple, passionfruit)

16. Rikanati et al., 2007
Enrichment of tomato flavor

17. virus Transgenic product Tranformed plant Origin of transgene Ref.
Potato virus X Viral coat protein Potato PV X Homenway et al.
1988
Potato virus Y Viral coat protein Potato
Kufri jyothi
PV Y CPRI, Shimla
TYLCV Viral coat protein Tomato TYLCV Kunik et al. 1994
Cl- gene Tomato TYLCV Brunetti et al. 1997
TSWV N-gene Tomato TSWV Pang et al. 1992
Tomato Mosaic
Virus
Anti sense RNA Tomato ToMV Beachy et al. 1997
Cucumber Mosaic
Virus
Satellite RNA Tomato CMV Sotmmel et al. 1998
Viral resistance
vegetables engineered against viruses (Singh et al., 2001).

18. Disease Casual organism Gene Transferred
Downy mildew Perenospora parasitica Chitinase
Wire stem Rhizoctonia solani Glucanase
Leaf spot Alternaria brassicae Thionin
Cabbage yellows Fusarium oxysporum RIP
Damping off Phythium spp. Permatins
White rust Albugo candida Osmotin
Black rot Xanthomonas compestris Chitinase
Soft rot Erwinia caratovora Chitinase
Fungal resistance
resistance genes against various fungal diseases
(Singh et al 2001).
Mechanism :Degradation of fungal cell wall and proteins

19. Resistance against Transgenic product Origin of transgene Crop
Erwinia caratovora Lysozyme T4 bacteriophase Potato
Erwinia caratovora Tachypelsin Horseshoe crab Potato
Erwinia caratovora Pestate lyase Erwinia caratovora Potato
Erwinia caratovora Glucose oxidase Aspergillus niger Potato
Pseudomonas syringae var
phaseocola
Phaselotoxin
insensitive OCTase
Pseudomonas syringae
var phaseocola
Bean
Bacterial resistance (Singh et al 2001).
Mechanism: Detoxification of Bacteriotoxins

20. Bt denotes Bacillus thuringiensis, a soil borne gram positive
bacteria, whose genes produce delta endotoxins that are toxic
to insect pest. These toxins interfere with ion uptake in
alkaline insect midgut.
They are not toxic to humans and animals because it works in
the alkaline digestive track of insects.
Bt- insect toxin

21. Transgenic
product
Organism of
transgene
Target insect Transformed
plant
Ref.
Cry 1 AB Bacillus
thuringiensis
Helicoverpa
armigera
Tomato Kumar et al. 1998
Cry 1 H Bacillus
thuringiensis
Helicoverpa
armigera
Corn Duck et al. 1997
Cry 3 A Bacillus
thuringiensis
Leptinotarsa
decimlineata
Brinjal Jelenkovic et al.
1998
Cry 1 A Bacillus
thuringiensis
DBM
Plutella xylostella
Cabbage Sharma 1999
Cry 1 A Bacillus
thuringiensis
Butterfly
Pieries brassicae
Cabbage Sharma 1999
Bt genes transferred for insect resistance in
vegetables

22. Synthetic Bt (NRC Pl. Bio Tech.)
GENE CROP / PEST VARIETY REMARK
Gy 1Ab Brinjal
Leucinodes arbonaslis
Pusa Purple Long 70% resistance
Gy 1AC Tomato
Helicoverpa armigera
Pusa Ruby 100% rsst in Lab
Gy 1Ac, Gy 113 Cabbage
Plutella xylostella
Under trial

23. • Acidic stomach
• Very low pH (∽1.5 in humans)
• Absence of required receptors
Bt is safe to non-target organisms-
HUMAN
Epithelium
Dissolving of crystals and
activation of toxins
Receptors
Toxins bind to receptors
Perforation of
gut membrane
Crystals
Hilbeck and Schmidt (2006)

24. Transgene Source Resistant to Transferred plant Basis of resistance
Glutathione-S-
transferase
Atrazine Tomato Detoxification of
herbicide
bxn (nitrilase) Klebsiella
pneumoniae
Bromoxynil Tomato ,,
bar (phosphinothricin
acetyl transferase)
Streptomyces spp. L- phosphinothricin Tomato, Sugarbeet ,,
Engineering for herbicide tolerance (Rai
&Rai 2006)

25. • Heat shock protein encoding gene HSP17.7 is found in carrot (Malik et al. 1989)
which can be transferred to other species for inducing thermal stress.
• Anti freezing protein gene has been transferred from winter flounder fish to tomato
and soyabean (Hightower et al. 1999).
Engineering for Abiotic stress resistance

27. Parthenocarpy
Rotino et al., 2005
DefH9-RI-iaaM

28. Purpose Transgene
product
Origin of
transgene
Transformed
plant
Ref.
Improved
shelf life
Antisense
polygalacturon
ase
Tomato Tomato Sheehy et al.
1988
Inhibition of
ripening
Antisense ACC
synthase
Tomato Tomato Oeller et al.
1991
Antisense ACC
oxidase
Tomato Tomato Hamilton et al.
1990
Fruit
pigmentation
Phtoene
synthase gene
Tomato Tomato Bird et al.
1991
Engineering for post harvest traits

29. Antisense technology - Gene
silencing
POLY
GALACTURONASE
SILENCING OF POLY
GALACTURONASE GENE
EXPRESSION STOPS THE
SOFTENING OF CELL WALL
&PREVENTS BRUISING
Attacks the cell
walls of mature fruit
& softens the skin of
the fruits by
degrading the cell
wall pectin

30. Pectin: Cell rigidity
PG
Polygalacturonase
GE FOR CELL WALL MODIFICATION

31. • Male sterility is a tool for economic hybrid seed production
• In crops like Okra, where male sterility hasn’t been found in
cultivated as well as wild relatives we can transfer it from micro
organisms by using R-DNA technology
• Already used in Brassicas and Tomato
Engineering for male sterility

32. • Grain Amaranthus - A. hypochondriacus contain higher storage
grain protein content.
• This protein contain 2-4 folds more essential amino acids –
Methionine, Lysine, Leucine & Threonine than normal ones
• The gene responsible for this protein AmA1 has been transferred
successfully into Potato (Chakraborty et al. 2000 JNU, ND)
Engineering for Quality & Nutrition

33. • Potato var. with high sugar content leads to charring of
sugar while chips making
• Potato varieties with high starch content are needed by
Food industry.
• ADPGPP- ADP Glucose Pyro Phosphorylase, the first
enzyme in the pathway of starch biosynthesis.
• This gene has been transferred to potato cultivars from
E.coli, which resulted in dramatic increase in starch content
in potato tubers. (Monsanto, USA)

34. TRAIT CROP NAME COMPANY PRODUCT STATUS
QUALITY (vine ripen flavour-
shelflife)
Tomato Flavr-savr Calgene Released 1994
QUALITY (vine ripen flavour-
shelflife)
Tomato Endless summer DNA plant
technology
Blocked by patent
cliams
QUALITY
(paste consistancy)
Tomato - Zenca Released 1995
Viral resistance Tomato
Capsicum
Released in china
1993-94
CPB resistance Potato New leaf Monsanto Released 1996
CPB resistance (cry 3B) Potato Black jacket Monsanto Expression of gene is
not sufficient
Viral resistance Squash Freedom II Asgrow Released in 1995
COMMERCIALLY RELEASED TRANSGENIC VEGETABLE
VARIETIES
IN PRIVATE SECTOR (Rai & Rai 2006)

35. • In second season sowing of harvested transgenic seeds, the plant growth is
abnormal as it is hooked by the RFP (rat fat protein gene). Until and unless
spraying of specific chemicals the plant growth will not be normal.
• This was developed by “Giant Geneca”, UK based Transgenic company
Verminator Gene Technology

36. • In this technology the transgene expression in the plant is tied to a
specific chemical.
• To get the expression of desirable transgene the plants are to be sprayed
with that particular chemical or the seeds are to be soaked in that
chemical before sowing.
• In these Verminator & Triator cases the chemical to be used are known
only by that particular company and the farmer has to purchase from
them.
Triator Gene Technology

37. Tieman et al., 2000
Fruit ripening in LeETR and NR antisense lines

38. Bao et al.,2007
Fruit firmness of tomato lines
Silencing of the
LeEIN2 gene
suppressed fruit
ripening
Fu et al., 2005
Positive regulators of
Ethylene synthesis
Silencing of the
LeEILs gene
suppressed fruit
ripening

39. Singh et al. 2013,
Mortality of fruit and shoot borer neonate larvae on fruit on fruit slice of different Brinjal

40. • The improvement of more efficient transformation protocols.
• The search and development of new genes of agronomical
interest.
• The development of strategies to better meet with public
acceptability of transgenic plants.
• Improvement of secondary or under-exploited species through
Transgenic.
• Multi disciplinary approach to develop multiple resistant
varieties with agronomical interest.
• Discouraging the suicidal / devil seed technologies.
Future thrust

41. Hamilton, A.J., Lycett, G.W. and Grierson, D. 1990. Antisense
gene that inhibits systhesis of the hormone ethylene in transgenic
plants. Nature. 336: 284-287
Nakajima ,Y., Yamamoto, T., Muranaka, T., and Oeda, K. 1999.
Genetic variation of petalord male-sterile cytoplasm of carrots
revealed by sequence-tagged sites (STSs). Theory and
Application. Genetics. 99: 837-843.
Rai, N. and Rai, M.2006. Heterosis Breeding in Vegetable Crops.
New India Publishing Agency. pp. 530.
Singh, M., Chakraborty, S., Srivastava, Kiriti and Kaloo, G. 2001.
Biotechnological advances in vegetable crops. In: Emerging
scenario in vegetable research and development. Kaloo, G. and
Singh, K. (Eds.) Research periodical and book publising house.
pp: 58-84.
Yin, Y. Wang, Z.Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T.
and Chory ,J. 2002. BES1 accumulates in the nucleus in response
to brassinosteroids to regulate gene expression and promote stem
elongation. Cell 109 :181–191.

42. Thank youIngo Potrykus