Genetic Engineering in Tomatoes

Improving Our Crop Plants

 Developing Modern Varieties of Crops
 Hybridization
 Crosses with Wild Relatives
 Hybrids
 Mutation
 Irradiation
 Chemicals
 Biotechnology
 Cell culture (Embryo Rescue, Somaclonal variation etc.)
 Genetic engineering
Traditional plant breeding Traditional donor Commercial variety New variety
DNA is a strand of genes, (many genes are transferred)
much like a strand of
pearls. Traditional plant
breeding combines many =
X
genes at once.
Desired Gene (crosses)
Desired gene

Plant biotechnology
Using plant biotechnology,
Desired gene Commercial variety New variety
(only desired gene is transferred)
a single gene may be
added to the strand. =
(transfers)
Desired gene
Basic Techniques in Plant Biotechnology
Plant Tissue Culture Genetic Engineering
Seed culture Gene Cloning
Embryo culture P.C.R.
Meristem culture invitro mutagenesis
Cell culture Transposable elements
Protoplast culture Molecular marker
Callus culture Transgenics
Bud culture Genomics
Organ culture
Biotechnology Can Add Value to Global
Agriculture

• Environmental Impact - Decreased Use of Pesticides

• Reduce Losses from Pests and Diseases

• Improve Nutrient Efficiency

• Improve Productivity
 Overview
• What is Genetic Engineering?
• How Genetic Engineering works?
• Characteristics of GM tomatoes.
• Disadvantages of GM tomatoes.
What is Genetic Engineering?

 Genetic engineering refers to a set of technologies that are

being used to change the genetic makeup of cells and move
genes across species boundaries to produce novel organisms.
. .

Definition
Genetic engineering can involve moving genes both
within or between species. Organisms modified in this way, are
referred to as being transgenic or genetically modified or
genetically engineered or simply as GMO’s. GM technology
thus gives the ability to add, subtract, alter or exchange an
individual gene or a group of genes, which are known to
influence an individual characteristic.

Source: http://www.advantageindia.com/english/gentical.htm
. .
 How Is It Done?
1. The scientist finds and isolates the
gene with the desired genetic
characteristics.

2. The scientist makes several copies of

the isolated gene. The copying process
is called PCR.

3. The scientist transfers the desired

genes to the plant's own genes.

4. The scientist creates a new plant from

the genetically modified plant tissue.

5. The scientist checks that the inserted

genes function as expected.

6. The scientist also checks that the

inserted gene appears in the plant’s
seeds.
 Introducing genes into plants
1) infecting plant cells with plasmids
as vectors carrying the desired gene;

2) shooting microscopic pellets

containing the gene directly into the cell.
Recombinant DNA techniques
 Genetic engineering of plants with
Agrobacterium tumefaciens

 A. tumefaciens:
used extensively for
genetic engineering
of plants.
 Contains T-DNA
(bacterial plasmid)
 Genes could be
integrated into the Tumor induced by
plant chromosomes A. tumefaciens

when the T-DNA is

transferred. http://courses.washington.edu/z490/gmo/natural.html
 Biology of A. tumefaciens
Well known to induce crown gall tumor

A.tumefaciens lives around

root surfaces (in rhizosphere)
where it using nutrients
that leak from the root tissues

infects only through wound sites

and actively chemotactic to them

www-genvagar.slu.se/teknik/ djup/plasm.htm

Bacterial T-plasmid produces Plant wound produces

receptors for acetosyringone acetosyringone
 Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumor-
producing
genes

T-DNA encoded
Opine catabolism
genes can be
Virulence region substituted by
target genes
ORI
 The basis of Agrobacterium-
mediated genetic engineering
 T-DNA of A. tumefaciens is excised and integrates into the
plant genome as part of the natural infection process.
 Any foreign DNA inserted into the T-DNA will also be
integrated.
.
Genetic Engineering Technology .

. .
 Tomato Genome Structure
• 12 chromosomes
telomere
euchromatin telomere

• 950MB of total DNA structure

pericentric
heterochromatin

• 220MB contiguous, 162 bp sub-

gene rich euchromatin centromere telomeric repeat

pericentric
heterochromatin

• Sequence only gene- euchromatin

7 bp telomeric
repeat

rich euchromatin
(>90% all genes)
Genetic Map
 The First Genetically Modified Tomato
• In 1994, the first “genetically modified” food was
approved by the FDA to go to market. The tomato, flavr
savr, was modified by Calgene (a biotechnology
company) using antisense technology resulting in altered
ripening.
 How To Flavr Savr was Made
• Plasmid transferred to E. coli host
• Hybrid gene (polygalacturonase) transferred to
A. tumefaciens plasmid (Ti plasmid)
• Tomatoes transformed w/Ti plasmid
• Kanamycin resistant cells identified in culture
• Southern & Northern blotting (confirmation)
• Regeneration & further testing
Flow chart for production of Flavr Savr
Isolation of PG gene

Cloning of gene to produce cDNA

Placing of PG gene between the Cauliflower Mosaic Virus (CaMV) 35S promoter & a termination
sequence

Introduction into plasmid in antisense direction

Transfer of plasmid in to bacterium E. coli to multiply

Transfer of DNA into bacterium Agrobcterium tumefaciens

Agrobcterium tumefaciens provide a vehicle for the transfer of the antisense PG gene
into tomato genome. Resulting in the development of late ripening tomato i.e. Flavr
Savr.
 Characteristics of GM Tomatoes

• Remain fresh and firm

longer
• Can tolerate lengthier
transport time
• Can be harvested
simultaneously
 Flavor Saver Traditional

The traditional
tomato must be
harvested while it
is still green and
firm so that it is
The Flavor Saver tomato not crushed on the
ripens on the vine – way to the
resulting in fuller supermarket.
flavour. It is modified so
that it remains firm after
harvesting.
The traditional
tomato is sprayed
with ethylene after
shipping to induce
ripening.

Ripe and Increased Flavor.

Ripe but decreased

Flavor.
Supermarket Supermarket
Why Antisense PG Gene?
Tomato Tomatine Content
(mg/100mg fresh wt)
Turning Stage Mature Stage
Anti-PG 1.7 0.4
Non-transgenic 1.4 0.5
Where We Are
 2005 is the 10th planting season for biotech
crops since their general use introduction in
1996
 Nearly 81 million hectares of biotech crops were
grown worldwide in 2004
 A 20% growth from 2003
 Despite the fact that the Flavr Savr Tomato was
the first approved transgenic food crop, virtually
all current production is confined to field crops:
soybeans, corn, cotton and canola
 Where We Are
 Papaya is the only commercially
available biotech fruit currently
available, only in the U.S, only from
Hawaii.

 Squash in the U.S. and Tomatoes and

Peppers in China are the only
commercially available vegetables
 What Happened and Why
FlavrSavr
• Calgene contracts directly with producers
attempts to control distribution and marketing

• Problems in shipping the delicate GE tomato

without damage

• Monsanto brings patent infringement lawsuit

• Calgene agrees to sell 54.6% of company to

Monsanto in 1995

• In 1997 Monsanto buys remaining shares of

of Calgene.
 The FlavrSavr Tomato
Late Ripening Variety

• 1991 Calgene request FDA Opinion

• 1992 FDA approves new Variety

• 1993 Tomatoes ready for test markets

• 1993 Public concerns prompt additional

FDA review, 1993 crop is not marketed

• 1994 Final FDA approval, and tomatoes

go to Chicago markets in mid-summer

• 1997 By March no more flavrsavr tomatoes

to be found
 What Happened and Why

Zeneca Seed Tomato Paste

• Public concerns over GE foods force Sainsbury

to pledge to withdraw product from market

• When supply was exhausted and product disappears

• No food approvals have been allowed since 1998

Demise of the Flavr Savr
 Consumer Issues
 Consumers did not perceive quality in tomato.
 Rising consumer backlash to GMO’s.

 Existence of Alternatives
 Israeli varieties (e.g., ‘Long Keeper’).
 Pioneer – ‘Super Life’ (commercial growers).
 DNA Plant Technology – other similar varieties.

 Patent infringement suit by Monsanto.

 Calgene “sells” technology.
Engineering of Tomato fruits for :-

Accumulation of polyamines, spermidine and Spermine.

Longer vine
Increased lycopene

Mattoo et al. 1987

 OTHER GE TOMATOES
Resistance to insect Source Resistance develped
against
Tomato Bt Heliothis armigera (Fischoff
et al., 1987)
Resistance to virus due to
coat protein gene
Tomato CP-TMV TMV
CP-AMV AMV
CP-TYLCV TYLCV (Powell et al., 1986)
Tomato Ribozyme CEVd (Atkins et al., 1995)
Resistance to fungal and
bacterial diseases
Tomato Chitinase and 1,3-β Fusarium oxysporum
glucanase lycopersici

Anti-microbial protein from Trichoderma hamatum

Hevea brassiliensis
Pseudomonas syringae
Pto gene (Martin et al., 1993)
 Tomato plants infected with tobacco mosaic virus (which attacks tomato
plants as well as tobacco). The plants in the back row carry an introduced gene
conferring resistance to the virus. The resistant plants produced three times as
much fruit as the sensitive plants (front row) and the same as control plants.
 Product Company Altered Trait Purpose Sources Agency
of New Action 1

Genes
Tomato Calgene Delayed Ripening Enhance fresh Tomato, USDA
market value Bacteria, approved 2

Virus
FDA approved 3

Tomato DNA Plant Delayed Ripening Enhance fresh Tomato, USDA approved
Technology market value Bacteria, FDA approved
Virus

Tomato Monsanto Delayed Ripening Enhance fresh USDA approved

market value Bacteria FDA approved

Tomato Zeneca / PetoSeed Thicker Skin, Enhance Tomato, USDA approved

altered pectin processing value Bacteria, FDA approved
Virus

1. Agency action may respond to wither voluntary or required submissions from companies
•2. USDA approval means that the product has been approved to enter market by the Food Safety and
Inspection Service (FSIS) division of the United States Department of Agriculture.
•3. FDA approval means that the Food and Drug Agency has completed consultations with a company and
will allow the product to enter the market once regulatory requirements are met at other agencies. Except for
the Calgene tomato approved in 1994, FDA consultations are abbreviated reviews of company safety assessments
 In Summary
Barriers to Commercialization of Horticultural GE Crops

• Biological Diversity
Too Many Varieties Too Few Concentrated Large Markets
• Intellectual Property
Patent right infringement, transactions cost in obtaining Freedom to Operate
Multiple Patented Genetic Technology in single cultivar ($100,000 * x)
Who owns what and Who owes what to whom
• Post-Commercialization Stewardship
Identity Preservation and Segregation in Distribution
• Regulatory Requirements
Each transgenic event must be separately tested and approved for each variety
• Lack of Compelling Benefits to Consumers
Benefits that consumers demand not just producers/processors benefit from
Food Safety (Consumer) Concerns
• Possibility of toxins in food.
• Possibility of new pathogens or pests.
• Reduced nutritional value.
• Introduction of human allergens.
• Transfer of antibiotic resistance to
humans.
• Unexpected immune system and genetic
effects from novel compounds.
. .

Loss of biodiversity

Due to the pest resistant transgenic crops the insect will die, in the
lack of food so the farm birds and other components of food chain
will face the problem of survival.

. .
 Public Research Concerns
• Research is supported to a larger degree
by private research dollars.
• Transgenic research is now a private
industry agenda.
• Intellectual property rights.
– Farmer restrictions (cannot save seed).
– Regional and local needs left unaddressed.
 Disadvantages
• Effect on food chain
• Possible allergic reaction
• Potential danger to environment
• Decline in number of farms
GE crops will not address small farmers’ needs
Farmers will be dependent; seed diversity will be lost
Environmental risks - gene flow, insect resistance
Insufficiency of biosafety regulations
Only large agrochemical companies will benefit
 Achievements in GE Tomatoes
 Salt Tolerance
 Cold Resistance
 Improved Nutritional Quality
 Insect Resistance
 Disease Resistance
 Herbicide Resistance (Roundup Tomato)
Some Limitations on Biotechnology Applications

in DEVELOPED Countries

Intellectual property rights

Regulatory costs

Economic incentives

Limited ability of public sector to

participate effectively
Some Limitations on Biotechnology Applications

in DEVELOPING Countries

Legal issues

Scientific and infrastructure insufficiencies

Unique political and economic hurdles

Societal inequalities

Lack of funding for public sector to

participate effectively
CONCLUSION