Role of transgenic technology in vegetable improvement

WHY WE NEED NEW TECHNOLOGIES?

Global food demand is forecast to at least double by the year 2050. In India, the population has already exceeded 1.0 billion and our country is projected to be the most populous in the world with 1.5 billion by 2050.

The arable land is diminishing every year. Other resources like water, fertilizers and labour are also becoming scarce and costly.

About 1.2 billion people in the world are afflicted by severe poverty and suffer from malnutrition.

1.4 billion women (22% of world population of which 55% in the developing countries) suffer from iron deficiency anemia.

About 140 million children suffer from vitamin A deficiency.

More than 30% of our crop yields are lost to biotic factors.

Similarly, crop losses due to abiotic stresses.

What are transgenic plants?

TRANSGENIC PLANTS: Refers to plants in which functional foreign genes have been inserted.

Gene(s) for desirable traits from any organism can be functionally introduced into the crop of interest

BASIC STEPS INVOLVED IN THE DEVELOPMENT OF TRANSGENICS.

1. 2. 3. 4. 5.

Isolation of the desired gene Selection and isolation of a vector Construction of recombinant vectors Introduction of recombinant vector into the host cell Selection and multiplication of the host cells carrying the recombinant DNA molecule

Expression of the desired gene. PRE-REQUISITES OF THIS TECHNIQUE

6.

To genetically modify a plant, a genetic construct must be designed. The multiple copies of the gene of our interest. A vector for transmission. A reliable protocol for the regeneration of whole plants from tissue culture.

Plant Transformation Methods

Physical
Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Microinjection Silica/carbon fibers Lazer mediated SAT

Chemical
PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers

Biological In planta
A. Tumefaciens A. Rhizogenes Virus-mediated

INTERNATIONAL STATUS OF TRANSGENIC CROPS No. of countries 23 Area in the world=114.3MH Area in India = 6. 8 MH I transgenic plants 1983

Tobacco, Tomato ( Horsch et al., USA)

I commercial release 1994

Herbicide tolerant Soybean, USA

58

Status of GM Vegetables in India,2008

S.No Crop Lab stage Green Field house stage trial stage * * * * * * * * * * * * * * * * MLT

1 2 3 4 5 6

Brinjal Cabbage Cassava Cauliflower Okra Onion

7
8 9

Potato
Tomato Watermelon

*
* *

*
*

*
*
Renuswarup,2008 57

Why transgenics ?

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 Removal of certain specific defects in crops

60

TRADITIONAL PLANT BREEDING

Many genes are transferred

Desired Gene

X
Donor Plant

Commercial Plant Variety

New Plant Variety

Plant genetic engineering PLANT BIOTECHNOLOGY

Desired Gene Desired Gene

A single gene is transferred

Donor

Commercial Plant Variety

Improved Commercial Plant Variety

59

12

We need transgenic for

To meet the worlds need of food

To increase yield
To improve quality To cope better with climatic change Nutritional improvement Increased shelf life Improved taste and texture Stress resistance: drought, heat, cold, salt tolerance

To reduce loss during transportation and storage

Herbicide resistance Insect resistance

Virus resistance

AVENUES OF TRANSGENIC TRAITS

11

Development of GM foods
1950 First regeneration of entire plants from an in vitro culture

1973
1983 1985 1990 1994 1995

Researchers develop the ability to isolate genes

1st transgenic plant: antibiotic resistant tobacco engineered with a yeast gene GM plants resistant to insects, viruses, and bacteria are field tested for the first time USEFUL TRAITS First successful field trial of GM cotton crop Flavr-Savr tomato - 1st FDA approval for a food Monsanto's Roundup Ready soybeans approved for sale in the United States.

Potential of GM crops in low input, sustainable agriculture

Traditional GM crop with pest resistance plus post-harvest qualities

4 tonnes/ha produced 25% losses post-harvest = 1 tonne/ha 3 tonnes/ha to eat

5 tonnes/ha 10% losses post-harvest = 0.5 tonne/ha 4.5 tonnes/ha to eat

CHNOLOGY OF TOMATO
Tomato was one of the first plants to be transformed

Agrobacterium tumefaciens and regenerated into fertile, productive plants (Fillaetttial., 1987).
The success of early work to obtain transgenic plants

allowed for the first commercial release of a transgenic food product, the Flavr Savr tomato, with extended shelf life of the ripe fruit.
A major goal of tomato genetic engineering has been to

manipulate the ripening process in order to delay fruit senescence and deterioration.

GE FOR CELL WALL MODIFICATION

Pectin: Cell rigidity

PG

Polygalacturonase

31

 The FlavrSavr tomato (introduced in 1994by

Calgene Inc.) is designed to ripen on the vine, with minimal softening.

Ripe tomatoes produces the enzyme

polyglacturonase(PG).
The PG enzyme is responsible for the breakdown

pectin, a building block in cell walls, which gives tomatoes their firmness.
To slow down the softening process, the Flavr

Savr employs antisense technology to block PG enzyme production.

ANTISENSE TECHNOLOGY
Antisense technology is a method of gene silencing. The first step in this process involved the isolation of

the PG gene from the tomato. Then clone the antisense DNA with the PG gene and insert this DNA into the plasmid of an agrobacterium. The bacterium is introduced to plant cells which transfers the gene of interest into plant cells. The cells with the plasmid are grown by adding specific hormones. The re-generated plants will express the antisense DNA and when the mRNA is made through the process of transcription the sense mRNA will bind to the anti-sense mRNA. This interferes with protein production (PG enzyme in tomato).

DNA

Summary of Antisense mechanism:

How enzyme is made?

PRODUCED

What Happens When A

Cloned Antisense DNA Is Added To The Original DNA?

When A Cloned Antisense DNA Is Added To The Original DNA:

Formation of antisense RNA blocks translation

Flavr Savr
The Flavr Savr tomato ripens on the vine resulting in fuller flavour. It is modified so that it remains firm after harvesting.

Traditional
The traditional tomato must be harvested while it is still green and firm so that it is not crushed on the way to the supermarket.

The traditional tomato is sprayed with ethylene after shipping to induce ripening.

Ripe and Increased Flavour.

Ripe but decreased Flavour.

Response to cracking after transport

PG antisense AC102 PG antisense AC105

Schuch et al., 1991

27

Wild type tomato

Transplastomic tomato Erwinia crtY

Transplastomic tomato Narcissus Lyc

Apel and Bock,2009

45

2.1 HPLC analysis of pigment accumulation in fruits

*P<0.05

Apel and Bock, 2009

44

2.2 HPLC analysis of pigment accumulation in leaves

*P<0.05

Apel and Bock, 2009

43

Transgenic tomato line 579HO

Fruit specific expression of yeast gene

ySAMdc

Tomatoes with 300% more Lycopene Increases total polyamines & shelf life
Mehta et al., 2002

42

3.1 Quality of transgenic tomato processed juice

Parameter Soluble solids (Brix) Acidity (% citric acid) pH

Precipitate weight ratio

Wild type 4.40.4 3.10.2 4.40.0 9.12.7 80.911.2

Azygous 3.70.1 3.00.2 4.40.0 9.82.0 94.94.8

Homozygous 566HO 4.1 0.2 3.0 0.1 4.4 0.1 13.0 1.5* 106 4.4* 579HO 4.3 0.0 3.3 0.2 4.3 0.0 14.5 0.3* 119.2 9.8*

Viscosity

*P<0.05
Mehta et al., 2002
41

3.2 Quality attributes of transgenic tomato (SAMdc)

**P<0.01

Mehta et al., 2002

40

3.3 Lycopene content transgenic tomato (SAMdc)

*P<0.05

Mehta et al., 2002

39

4.1 Fruit specific phenotypes of tomato

Wild type

Del/ Ros1C

Del/ Ros1N

Butelli et al., 2008

37

4.2 Anthocyanin levels in tomato lines

Butelli et al., 2008

36

7.1 Firmness of transgenic tomato fruit

control AC (white bar) AC-Exp1 (light stipple) AC-PG (heavystipple) H ACPG-EXP1(black bar) AC + Exp1 (hatched bar)

Powell et al.,2003

24

Parthenocarpy

Control

F1

transgenic

DefH9-RI-iaaM
Rotino et al., 2005
7

13.1 Performance of transgenic tomato lines

Genotype Marketable yield Unmarketable yield Green fruits Rotten fruits Yield Pl-1 No.of Yield Pl-1 (g) fruits (g)
3.40.5 6.21.9 5.60.6 6.71.5 3.80.6 5.21.2 6.91.4 5.20.9

Yield Pl-1 (g)

Allflesh(F1)
UC82(control)

No.of fruits

Fr wt (g)

1906362 32.36.1 1380301 21.64.3 1538159 43.25.0 1227147 33.73.6

60.53.4 9822 65.52.5 19665 37.01.1 12315 38.01.5 14944

Ri4(trans) Ri5(trans)

Rotino et al., 2005

6

13.2 Performance of transgenic tomato lines

Genotype Shape index seed (%) No. of seeds fruit-1 Brix (o) -Carotene (g g-1 dw)
491.119.7 566.68.6 698.910.1 643.615.1

Allflesh UC82 Ri4 Ri5

1.270.02 86.75.4 68.86.2 1.250.02 85.08.8 36.56.5 1.240.03 26.75.4 18.43.8 1.270.03 20.04.7 11.43.8

5.30.18 3.80.14 4.50.12 4.20.23

Rotino et al., 2005

5

Purple Tomatoes, Rich In Health-Protecting Anthocyanins, Developed With Help Of Snapdragons

Cancer susceptible Trp53-/- mice fed a diet supplemented with the high anthocyanin tomatoes showed a significant extension of life span

Butelli

et al., 2008

GENETIC MANIPULATION OF ETHYLENE BIOSYNTHESIS:

Second transgenic approach have been used to reduce

endogenous ethylene production in ripening fruit in order to delay the onset and rate of fruit ripening. The pathway of ethylene biosynthesis is now well known and the final two steps in the pathway, conversion of Sadenosyl methionine (SAM) to 1-aminocyclopropane-1carboxylic acid (ACC) and its oxidization to ethylene have been targeted for modification in transgenic plants. One approach has been to metabolize either SAM or ACC to an inactive product and the second approach has been to specifically suppress the expression of the two ethylene biosynthetic enzymes required to catalyze the final steps in the pathway.

TRANSGENICS FOR PARTHENOCARPY

Parthenocarpic mutants have been identified in

several plant species, but their use for generating parthenocarpic varieties is limited by the reduction of fruit set and fruit size. The parthenocarpic trait is polygenic and it proves cumbersome in breeding programmes. For these reasons biotechnology may prove to be an interesting alternative method. Transgenic eggplants expressing the coding region of the iaaM gene from Pseudomonas syringae driven by an ovule-specific promoter show parthenocarpic development.

 The iaaM gene codes for an indolacetamide

monoxygenase that converts tryptophan to indolacetamide, a precursor of the plant hormone auxin.
Transgenic plants produced seedless fruit of

marketable size when the flowers were emasculated or in adverse conditions when untransformed lines where unable to set fruit.
iaaM gene is a powerful biotechnological tool for

generating parthenocarpic eggplants that proves to be superior to the use of both agricultural practices and traditional genetic methods

POTATO
Potato breeding started around the beginning of the 19th

century but its complex tetraploid genetics means that targeted breeding is a time-consuming exercise.
Comparatively speaking, potato has a narrow genetic base,

which, at least in part, contributes to slower progress in crop improvement than with other major crop species. Attempts to introgress genes from wild relatives has met with some success for disease resistance traits but undesirable side effects from hybridization minimizes the crop improvement through conventional methods. Since back-crossing to remove undesirable effects is not an easy option for potato, the approach of improving existing cultivars using gene transfer or genetic engineering technology has been an attractive proposition.

INSECT-RESISTANT POTATO
Nature Mark potato lines named NewLeaf confer

resistance to Colorado potato beetle.

The potatoes were transformed with a gene, which

encodes for the Cry3A protein from the bacteria Bacillus thuringiensis (var. tenebrionis) using the constitutive 35S CaMV promoter.
The product has been endorsed by the World Health

Organization (WHO) and other regulatory agencies throughout the world. The protein affects directly only the target pest, Colorado potato beetle (CPB) and has no effect on other insects, mammals or wildlife. Mammalian toxicology and digestive fate studies, which have been conducted and confirmed that these Cry proteins are non-toxic to humans and pose no significant concern for allergicity.

GENETIC ENGINEERING OF CUCURBITS FOR DISEASE RESISTANCE

PEPPER

Transgenic hot peppers showing resistance to bialaphos, a non-selective Herbicide have been generated by transfer of the bar gene via A. tumefaciens. Transgenic plants were capable of withstanding 5000 mg/L of bialaphos applied to the leaves. Sweet peppers expressing (phosphinothricin N acetyl transferase) gene introduced via A.tumefaciens exhibited tolerance to applications of 0.44% of the commercial preparation of Basta containing 20% phosphinothricin.

LEGUMES

BIOTECHNOLOGY OF BULKY ORGANS ( CARROTS, SWEET POTATOES, ALLIUM SPS)

Roots, bulbs and other bulky organs used as vegetables

belong to a wide range of horticultural species. Except for the potato, very few of them have received attention in terms of improvement via biotechnology. Resistance to insects and fungi The most important pests of sweet potatoes are insects, mainly the sweet potato weevil (Cyclas formicarius, Fab). Losses dues to insect attacks may reach 60 to 100%. The genetics of sweet potatoes is complex due to the hexaploid genome and self-incompatibility. Transfer of foreign genes via biotechnology is therefore of great interest. Moran et al. obtained several clones of the Jewel sweet potato cultivar carrying the CryIIIA gene that exhibited some resistance to sweet potato weevil infestation under greenhouse and field conditions as compared to control plants although the level of expression of the gene was

Quality traits

The sugar content of carrots represents an important

quality trait.

Transgenic carrots in which sucrose synthase, vacuolar

and cell wall invertase and tonoplast H+ATPase have been repressed were generated.
The transgenic plants had an altered phenotype with

smaller roots and the strategy for improving the quality of the carrot (e.g. in terms of sugar content) remains to be established.

Resistance to abiotic stresses

The generation of plants resistant to environmental

stress is of great practical interest.

Modifications of heat tolerance in carrots have been

achieved by constitutively expressing or downregulating a small heat shock protein gene, Hsp Constitutive expression resulted in an increase of heat tolerance, while down-regulation resulted in lower tolerance.

BIOTECHNOLOGY OF LEAFY VEGETABLES (CABBAGE, BROCCOLI, CAULIFLOWER, LETTUCE, SPINACH) AND ASPARAGUS

Resistance to viruses

Cauliflower Transgenic cauliflower carrying the capsid gene and antisense VI of gene the cauliflower mosaic virus have been generated through A.tumefaciens-mediate transformation.

Resistance to insects and fungi Cauliflower Insect pests represent a serious problem for cauliflower cultivation. A trypsin inhibitor from the sweet potato has been transferred to Taiwan cauliflower cultivars that gave transgenic primary transformants substantial resistance to local insects.
Cabbage & Broccoli Metz et al. have generated a large number of transgenic broccoli lines carrying the Bt Cry1A(c) gene, most of them causing 100% mortality of first instar larvae of the diamond moth. More recently, a synthetic Bt Cry1C gene was introduced. In addition, theCry1C-transgenic broccoli were also resistant to other lepidopteran pests of crucifers such as cabbage looper and imported cabbageworm.

Bt BRINJAL

What is Bt Brinjal
Bt Brinjal is a transgenic brinjal created out of inserting a gene (Cry 1Ac) from the soil bacterium Bacillus thuringiensis into Brinjal. The insertion of the gene into the brinjal cell in young cotyledons through an Agrobacterium-mediated vector. This gives to the brinjal plant resistance against lepidopteran insects like the brinjal fruit and shoot borer (L.orbonalis) and fruit borer (Helicoverpa armigera). when ingestion of the Bt toxin by the insect, there would be disruption of digestive processes, ultimately resulting in the death of the insect.

Primary damage

Shoot damage

Brinjal Shoot and Fruit Borer

Pesticide Use in Vegetables

Chillies: 5.13 kg of active ingredient /ha Brinjal: 4.6 kg of active ingredient /ha =Rs 12,000 ha Okra: 3.71 kg of active ingredient/ha
Indian Chemical Industry, 2007

MHB-4 Bt, MHB-9 Bt, MHB-10 Bt, MHB-11 Bt, MHB-39 Bt, MHB-80 Bt and MHBJ-99 Bt

Mode of action

Considerable reduction in cost of production by saving on cost of insecticides and labour labor cost as a result of reduced spraying. Manifold increase in yield per unit area by saving fruits from damage caused by FSB. Significant improvement in marketable fruits thereby increasing income per unit area. Reduction in direct exposure to insecticides leading to lesser health problems.

Reduction in pesticide residues in soil and water in brinjal fields. Lesser pollution of air and local environment due to decreased use of insecticides.
Protection of naturally occurring predators and parasitoids and other beneficial organisms due to reduced use of insecticides. Reduction in soil and ground-water contamination. Safeguarding soil micro flora and invertebrates from damage caused by unintended and excessive use of insecticides.

Effect of Bt on non-target Pests or beneficial insects

No direct effect was found on sucking pests and beneficial insects. (IGMORIS: Chapter 5, p 72 of Vol 1.)

Capability to reduce Pesticidal Application

The number of pesticides sprays in the field is considerably less for transgenic compared to nontransgenic brinjal. The number of sprays were reduced from 8-10 to 3-5 sprays.

Yield Advantage

Under high level of FSB infestation in the crop, there

was 80-100 per cent yield advantage in transgenic brinjal over non-transgenic counterpart
(Chapter 1 p69 of Vol 1)

Effect of Bt on soil micro flora

No negative

effect on soil micro flora was seen.

No significant cry protein was detected in soil samples of Bt brinjal grown field and there was no harmful effect on the microbial population.
(Chapter 6 p89 of Vol 1. Details are given in Vol 5.)

Information on gene transfer to cultivated or related species

No gene flow has been detected in cultivated or related species of brinjal Pollen flow studies were conducted by Indian Institute of Vegetable Research (IVRI ICAR), Varanasi. (Chapter 5.7, p80 of Vol 1)

Information on food safety, Allergenicity and toxicity

No toxic effect has been found (Chapter 7.2 p111-129 of Vol1).

The skin allergy test was conducted on white rabbits by the accredited lab INTOX Pvt Ltd., Pune.
No skin irritations were seen.

Detailed Skin irritation test results are published (vol 2)

Allergenicity was studied in rats in Rallis India Ltd., Bangalore and no adverse effect was seen.

Information is available on the level of Bt protein present in Bt brinjal (raw) vs., processed or cooked brinjal

Information is available on the level of Bt protein present in Bt brinjal (raw) and cooked samples (Desigen Diagnostics, Jalna). There is no Bt protein detectable in cooked or processed brinjal in any form as it is completely degraded on cooking. Cry protein was absent in the cooked fruit (roasted, shallow fried, deep fried and steamed) suggesting that the protein was completely degraded on cooking. (Chapter 7 p105 of Vol 1).

Long-term effect on human or animal health

It needs to be ascertained Monitoring of Bt brinjal over a long time period will only be able to answer this issue.

Risks associated with Bt brinjal

1. Safety

Potential human health implications.

Out-crossing

Inevitable out-crossing of transgenic plants with naturally occurring ones.

2. Monophagous pest

3. Farmers do not plant refuges to manage resistance.

4. Resistance development
Defects in protease production
Elevated immune response Enhanced esterase production

5.Labeling
Mixing GM crops with non-GM confounds labeling attempts.

(Human Genome Project Information (2003),

http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood. shtml)

6. POLLEN FLOW -Isolation distance

-India is considered a centre of diversity.

9.Minor pest become major pest Sucking pest 10. seed price regulation Bt seed high cost

Comparative figures from experiences with IPM packages applied in some locations.

Bt brinjal Non Bt2005-06 Brinjal

Two checks

IPM by GAU 2001

IPM by ANGRA U 2000-02

17.72 %

Non chemical IPM by OUAT Summer 2004

Fruit damage

13.5 % 16.02 %

28.7% 27.72 % 157.08 q/ha 190.26 q/ha

29.4% 27.69 % 182.15 q/ha 192.86 q/ha

10.64%

13.07+ 7.54 %

Marketabl 231.69 le yield q/ha 223.39 q/ha

266.25 q/ha

203.98 q/ha

214.5+ 16.3

Transgenic tomato cv. Pusa Uphar expressing a bacterial mannitol-1phosphate dehydrogenase gene confers abiotic stress tolerance.
Neeraj Khare Danswrang Goyary Singh, N. K. Pramila Shah Meenal Rathore Sivalingam Anandhan Dinesh Sharma Mohomad Arif Zakwan Ahmed Plant Cell, Tissue and Organ Culture. 2010. 103: 2, 267-277. many ref.

A bacterial mannitol-1-phosphate dehydrogenase (mtlD)

gene driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter was transferred into tomato plants using an Agrobacterium tumefaciens-mediated transformation protocol in an attempt to improve abiotic stress tolerance in the transformed plants Upon exposure to low temperature stress (4 degrees C) in a cold chamber, transgenic plants survived up to 48 h, while non-transformed plants were unable to survive and gradually died.Drought (polyethylene glycol in medium) and salinity (sodium chloride in medium) tolerance tests revealed that transgenic lines exhibited a higher tolerance for abiotic stresses than non-transformed plants. These findings indicate that the introduction of a bacterial mtlD gene into tomato conferred tolerance to abiotic stresses to the transformed tomato plants.

Potato R1 resistance gene confers resistance against Phytophthora infestans in transgenic tomato plants.
Faino, L. Carli, P. Testa, A. Cristinzio, G. Frusciante, L. Ercolano, M. R. European Journal of Plant Pathology. 2010. 128: 2, 233-241. 36 ref.

R1 potato gene was transferred into tomato

lines.All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.

Fruit quality of transgenic tomatoes with suppressed expression of LeETR1 and LeETR2 genes.
Bao Bili Ke LeQin Jiang JianMei Ying TieJin Asia Pacific Journal of Clinical Nutrition. 2007. 16: supp 1, 122-126. 22 ref.

The effect of antisense suppression of ethylene

receptor genes LeETR1 and LeETR2 over the quality of tomato fruit was investigated in this paper.

. The data suggest that fruit with suppressed LeETR1

and LeETR2 genes expression have stronger ethylene response, which accelerate fruit ripening and greatly altered tomato variety characteristics

Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle.
Arpaia et al.,

The presence of the CryIIIB toxin in leaf extracts

was demonstrated in 57 out of 93transgenic plants tested by DAS-ELISA assay. High Bt-expressing plants contained a 74-kDa protein cross-reacting with serum anti-CryIIIB toxin.Twenty three out of 44 S. melongena plants tested by insect bioassay showed significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus,transgenic Bt eggplants represent a very effective means of CPB pest control.

CONCLUSION Growth

in agriculture is less than 2%. If we have to achieve our ambitions of growing at a rapid pace of over 8%, we must aim at an agricultural growth rate of over 4%. Hope that progress of agriculture through use of modern science & technology will spread to every nook and corner of the country.
Dr. Manmohan Singh Honble Prime Minister

83