Plant Genetic Engineering

Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another Why do scientists want to change gene instructions? to produce needed chemicals to carry out useful processes to give an organism desired characteristics Genetic engineering is the process of manipulating and transferring instructions encoded by genes from one cell to another. This process involves copying of a gene from one living organism (e.g., bacteria, plant, or animal) and adding to another living organism using biotechnology techniques. Genetic engineering had opened up many possibilities: by adding a new gene into a living organism and thus producing new traits that may be beneficial to people. The examples of plant genetic engineering: - increasing yields and product quality - resistant plants to diseases and insects - medicines and vaccines: e.g., drugs used to treat diabetes (insulin), arthritis, heart disease and others - producing plastics or other materials traditionally synthesized artificially

THE SCIENCE OF GENETIC ENGINEERING Isolate desired gene for a new trait from any organism Isolate plasmid DNA Gene inserted into plasmid. Introduce modified plasmid into bacterium for replication. Grow in culture to replicate.

DNA of interest is EVERYWHERE The background is a picture showing a double stranded DNA molecule. The DNA molecule is found in every living organism: * ask students to come up with examples where they think they could find DNA The universal presence of DNA (deoxyribonucleic acid) in the cells of all living organisms makes the genetic engineering possible.

Plant Genetic Engineering Product Concepts and Technical Feasibility Building the Transformant Plant Transformation Selection Plant Breeding Seed Production and Marketing Detection of GMO Crops in the Commodity Chain

Requirement Plant transformation getting DNA into a cell getting it stably integrated getting a plant back from the cell Requirement a suitable transformation method a means of screening for transformants an efficient regeneration system genes/constructs Vectors Promoter/terminator reporter genes selectable marker genes ‘genes of interest’

Plant Transformation DNA Delivery to Target Cells Selection and Regeneration Event Selection

Transformation methods DNA must be introduced into plant cells Technique Methods Indirect Agrobacterium mediated gene transfer Viral vector Direct Lipid-mediated method, Calcium-phosphate mediated, Dextran-mediated, Electroporation, Biolistics, Polybrene, Laser transfection, Gene transfection enhanced by elevated temperature. Method depends on plant type, cost, application

Agrobacterium tumefaciens plasmid A large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid) Plasmid contains genes responsible for the crown gall disease Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Transfer DNA) There are 2 x 23bp direct repeat border

Agrobacterium tumefaciens 10

Ti plasmid 23 kb tra pTi ~200 kb vir genes T-DNA bacterial conjugation for transfer to the plant opine catabolism

Genetic engineering of the Ti plasmid Some considerations: Ti plasmid is 200 kb - too big for easy cloning need to remove tumor-inducing genes from T-DNA Cloning vector YFG T-DNA Ti plasmid Disarm Disarmed Ti plasmid VIR genes Agrobacterium Remember: Only VIR genes are required for T-DNA transfer Only LB and RB are required for T-DNA insertion into genome

The binary Ti plasmid system

Binary vector system

Binary vector system

Agrobacterium-mediated transformation Transformation by the help of agrobacterium Agrobacterium is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants

Agrobacterium tumefaciens Ti plasmid Agrobacterium Genomic DNA Plant cell Genomic DNA (carries the gene of interest) Gene of interest Empty plasmid Restriction enzyme A + Ti plasmid with the gene of interest Agrobaterium tumefaciens is a bacterium capable of inserting new DNA into the host plant cell upon infection. This ability makes Agrobacterium a natural genetic engineer. It contains a plasmid called the Ti plasmid that carries one or more genes. Once in the host cell, the gene(s) gets integrated into the plant cell’s DNA. Scientists alter the Ti plasmid to contain the gene they whish to incorporate into the plant cell. Next, the plasmid is reintroduced into the bacterial cell. When the bacterium with the altered Ti plasmid infects a plant cell, the new gene will be incorporated into the plant cell’s genome. Steps of the process: the Ti plasmid is removed from the bacterium using a restriction enzyme (biological scissors) the plasmid is cut at a specific manner 2) a gene of interest is cut out of the DNA of another organism (e.g. a plant cell) using the same restriction enzyme that will cut the DNA at the same manner as the Ti plasmid in the previous step 3) the gene is inserted into the plasmid (producing a recombinant DNA)

Agrobacterium tumefaciens Ti plasmid with the new gene + Transformation Plant cell cell’s DNA The new gene Transgenic plant 4) the plasmid is reinserted into Agrobacterium and incubated with plant cells 5) Agrobacterium transfers the new gene into the plant cell’s nucleus and the gene gets incorporated into the plant cell’s genome (DNA) 7) as the plant cell divides subsequent cells receive the new gene 8) a transgenic plant is produced where every cell contains the new gene A. tumefaciens infects broadleaf plants like tomatoes and soybean. Cell division

Biolistics ‘gene gun’ DNA is coated onto gold (or tungsten) particles (inert) Gold is propelled by helium into plant cells if DNA goes into the nucleus it can be integrated into the plant chromosomes Cells can be regenerated to whole plants

DNA coated golden particles “Gene Gun” Technique DNA coated golden particles Plant cell Cell’s DNA Gene gun A plant cell with the new gene Transgenic plant The other methods have been developed for the plant species that Agrobacterium does not infect (e.g., corn, rice, wheat, etc.). The gene gun techniques is known as the biolistic particle delivery system. Gold particles (1mm in diameter) are coated with DNA containing the gene of interest, and are propelled using the gene gun into the host plant tissue. The particles are able to penetrate the plant cell walls and deliver the DNA inside the cell. Only those cells that incorporate the new gene into their genomic DNA are selected and grown into the full transgenic plants. Cell division

Tearless Onion

Colorful Cauliflowers

Purple tomatoes

Blue Roses

Herbicide Resistance A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds. Herbicides such as RoundupTM and Liberty LinkTM are able to kill a wide range of weeds and have the advantage of breaking down easily. Development of herbicide resistant crops allows the elimination of surrounding weeds without harm to the crops.

ROUNDUP (Glyphosate) TOLERANCE ( HERBICIDE TOLERANCE IN CROPS) Glucose ROUNDUP (Glyphosate ) 3 phosphoglycerate Tryptophan Glycolysis EPSP SYNTHASE Phosphoenol pyruvate EPSP SYNTHASE Tyrosine Phenylalaline ROUNDUP (Glyphosate ) In transgenic plant, herbicide can not bind the mutant of EPSP synthase (Example: RR-Cotton, RR-Soybean)

Insect Resistance Various insect resistant crops have been produced. Most of these make use of the Cry gene in the bacteria Bacillus thuringiensis (Bt); this gene directs the production of a protein that causes paralysis and death to many insects. Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis GENE FOR Bt TOXIN WAS TRANSFERRED TO OBTAIN BT TRANSGENIC PLANTS PLANT SYNTHESIZES INACTIVE PROTOXIN PROTEINASE DIGESTION IN INSECT GUT MAKES THE ACTIVE TOXIN INSECT FEEDS ON TRANSGENIC PLANT Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death

Papaya infected with the papaya ringspot virus Virus Resistant Crops Papaya infected with the papaya ringspot virus Virus resistance gene introduced The Freedom II squash has a modified coat protein that confer resistance to zucchini yellows mosaic virus and watermelon mosaic virus II. Scientists are now trying to develop crops with as many as five virus resistance genes

Delayed Fruit Ripening Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. It is produced by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found in plant cell walls. Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA that the plant produces, essentially blocking the gene from translation.

Flav’r Sav’r tomato “Rot-Resistant Tomato” Anti-sense gene  complementary to polygalacturonase (PG) PG = pectinase  accelerates plant decay/rotting

Golden Rice Normal rice Transgenic technology produced a type of rice that accumulates beta-carotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. “Normal” rice “Golden” rice

Gernayl Gernayl diphosphate (GGPP) Phytoene synthase Phytoene Phytoene desaturase Lycopene Lycopene cyclase Beta carotene Complete biochemical pathway in the rice for production of beta-carotene, a precursor for vitamin A.

Edible Vaccines Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

Approved Transgenic plants Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco Carnations Potato Flax Papaya Chicory Rice Melon