APPLICATIONS OF GENETIC ENGINEERING TECHNIQUES IN

AGRICULTURESUBMITTED BY

B.DEVADATHA

M.SC BMB

DEPT. OF BMB

PONDICHEERY UNIVERSITY

Why genetically engineer plants?

• To improve the agricultural, horticultural or ornamental value of a crop plant• To serve as a bioreactor for the production of economically important proteins or

metabolites• To provide a powerful means for studying the action of genes (and gene

products) during development and other biological processes

Genetic engineering techniques applied to plants

METHOD SALIENT FEATURES

1.VECTOR MEDIATED GENE TRANSFERa. Agrobacterium mediated

gene transferb. Plant viral vectors

Very efficient but limited to a selected group of plants

Ineffective, hence not widely used

2.DIRECT OR VECTORLESS DNA TRANSFERa. Electroporation

b. Microprojectile

c. Liposome fusion

d. Silicon carbide fibres

Mostly confined to protoplasts that can be regenerated to viable plants

Limited use only one cell can be microinjected at a time

Confined to protoplasts that can be regenerated into viable whole plants

Requires regenerable cell suspensions

3 CHEMICAL METHODSa. Polyethylene glycol mediated

b.Diethylaminoethyl(DEAE)dextran- mediated

Confined to protoplasts. Regeneration of fertile plants is frequently problematical

Does not

Agrobacterium tumefaciens as a vector for transferring foreign genes into plant chromosome

Characteristics

Plant parasite that causes Crown Gall Disease

Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid

Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Tumor DNA)

T-DNT-DNA integrates stably into plant genome

A ss DNA fragment is converted to dsDNA fragment by plant cell

Then integrated into plant genome

2 x 23bp direct repeats play an important role in the excision and integration process

Infection Process

Vir genes copy T-DNA

Open channel in bacterial cell membrane for T-DNA to pass through

T-DNA enters plant through wound, integrates itself into plant chromosome

Applications of Plant Genetic Engineering

A.Crop Improvement B.Genetically Engineered Traits: The Big Six

1.Herbicide Resistance 2.Insect Resistance 3.Virus Resistance 4.Altered Oil Content 5.Delayed Fruit Ripening 6.Pollen Control

C.Biotech Revolution: Cold and Drought Tolerance and Weather-Gard Genes

D.Genetically Engineered Foods 1.Soybeans 2.Corn 3.Cotton 4.Other Crops

Herbicide-resistant plants: Reducing the ability of the herbicide-sensitive target to bind to the herbicide

• Herbicide: Glyphosate (better known as Roundup)• Resistance to Roundup (an inhibitor of the enzyme EPSP

involved in aromatic amino acid biosynthesis) was obtained by finding a mutant version of EPSP from E. coli that does not bind Roundup and expressing it in plants (soybean, tobacco, petunia, tomato, potato, and cotton)

• 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a chloroplast enzyme in the shikimate pathway and plays a key role in the synthesis of aromatic amino acids such as tyrosine and phenylalanine

• This is a big money maker for Monsanto!

insect-resistance plant's• bt toxin• cowpea trypsin inhibitor• proteinase inhibitor ll• alfa amylase inhibitor• bacterial chloesterol oxidase• combination of the above

Anti-Insect Strategy – Insecticides From Bacillus thuringensis

Toxic crystals found during sporulation Alkaline protein degrades gut wall of

lepidopteran larvae Corn borer catepillars Cotton bollworm catepillars Tobacco hornworm catepillars Gypsy moth larvae

Sprayed onto plants – but will wash off

Transgenic plants for crop improvements

1. Virus resistant transgenic plants:

a. Pathogen-derived resistance (PDR b. non-pathogen-derived resistance (non-

PDR)

2. Stress resistant transgenic plants.

Anti-Viral Strategy• TMV-coat protein inserted into tobacco and

tomato plant cells using Ti plasmid • Viral capsids inhibit viral replication of TMV

when infected Cucumber mosaic virus (CMV)

• the most important viruses • more than 800 host plants • absent the resistance genes in the

germplasm of most crops Other trials using capsid proteins: potato

leaf-roll virus, cantaloupe mosaic virus, rice strip virus

Development of stress- and senescence-tolerant plants: genetic engineering of flavorful tomatoes Fruit ripening is a natural aging or senescence process that involves

two independent pathways, flavor development and fruit softening.

Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow for safe shipping of the fruit.

Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and contribute to fruit softening.

In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping of the fruit, polygalacturonase gene expression was inhibited by introduction of an antisense polygalacturonase gene and created the first commercial genetically engineered plant called the FLAVR SAVR tomato. Flavor development pathway

Fruit softening pathway

Green Red

Hard Softpolygalacturonase

antisense polygalacturonase

Modification of plant nutritional content: increasing the vitamin A content of plants

• 124 million children worldwide are deficient in vitamin A, which leads to death and blindness

• Mammals make vitamin A from b-carotene, a common carotenoid pigment normally found in plant photosynthetic membranes

• Here, the idea was to engineer the b-carotene pathway into rice

• The transgenic rice is yellow or golden in color and is called “golden rice”

GGPP

Phytoene

Lycopene

b-carotene

Vitamin A

Daffodil phytoene synthase gene

Bacterial phytoene desaturase gene

Daffodil lycopene b-cyclase gene

Endogenous human gene

Antisense technology

• Used to produce the Flavr-Savr tomato in 1994.

• Enzyme polygalacturonase breaks down structural polysaccharide pectin in wall of a plant.

• This is part of the natural decay process in a plant

• Monsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme.

Edible Vaccines – Ongoing Research Areas

Hepatitis BDental caries - Anti-tooth decay Ab (CaroRxTM) (anti-Streptococcus mutans)Autoimmune diabetesCholeraRabiesHIVRhinovirusFoot and MouthEnteritis virusMalariaInfluenzaCancer

Frost Resistance

• Ice-minus bacteria• Ice nucleation on plant surfaces caused by bacteria that

aid in protein-water coalescence forming ice crystals @ 0oC (320F)

• Ice-minus Pseudomonas syringae• Modified by removing genes responsible for crystal

formation• Sprayed onto plants

• Displaces wild type strains• Protected to 23oF

• Dew freezes beyond this point• Extends growth season• First deliberate release experiment – Steven Lindow – 1987-

sprayed potatoes

• Frost Ban • Different strain of bacteria – Julie Lindemann led different

project – 1987• Strawberries in California

Potentially Harmful Effects

• Contamination by pesticides• Co-purification of plant chemicals (e.g. nicotine)• Different glycosylation in plants versus animals

• Interference with normal function of protein in animals• Stimulation of hypersensitivity reactions in animals (allergies)• Research is underway to engineer tobacco to synthesize “human-

compatible” glycans

Environmental Risks• Pharmaceutical products may inadvertently

be introduced into the general food supply• Cross-pollination

• Pollen from a drug-containing crop fertilizes a neighboring related crop (or wild relatives) used for animal consumption

• Wind

• InsectsConsumption of GM plant by insects Food chain

• Accumulation in birds – extinction? (e.g. DDT and bald eagle)

• Deleterious effects on non-target organisms (NTO’s)• NTO’s = organisms in the environment that are

affected by the product unintentionally• Insects, arthropods• Risk to NTO’s

• Depends on recombinant protein involved• Risk assessment carried out case-by-case