Applications of Transgenic technology Transgenic technology n Breeding

Applications of Transgenic technology

Transgenic technology n Breeding method n Crop Improvement n The Potential traits: 1. controlling insects 2. manipulating petal color, 3. production of industrially important compounds 4. supporting plant growth in harsh conditions

The big six traits ü Herbicide Resistance ü Insect Resistance ü Virus Resistance ü Altered Oil Content ü Delayed Fruit Ripening ü Pollen Control

Herbicide Resistance ü Herbicides are a huge industry, with herbicide use quadrupling between 1966 and 1991, so plants that resist chemicals that kill them are a growing need. ü Critics claim that genetically engineered plants will lead to more chemical use, and possible development of weeds resistant to the chemicals.

Herbicide Resistance a) Glyphosate Resistance i. Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide ii. Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS (S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis), makes aromatic amino acids. iii. The gene encoding EPSPS has been transferred from glyphosate-resistant E. coli into plants, allowing plants to be resistant. Glufosinate Resistance i. Glufosinate (the active ingredient being phosphinothricin) mimics the structure of the amino acid glutamine, which blocks the enzyme glutamate synthase. ii. Plants receive a gene from the bacterium Streptomyces that produce a protein that inactivates the herbicide.

Herbicide Resistance c) Bromoxynil Resistance i. A gene encoding the enzyme bromoxynil nitrilase (BXN) is transferred from Klebsiella pneumoniae bacteria to plants. ii. Nitrilase inactivates the Bromoxynil before it kills the plant. d) Sulfonylurea. i. Kills plants by blocking an enzyme needed for synthesis of the amino acids valine, leucine, and isoleucine. ii. Resistance generated by mutating a gene in tobacco plants, and transferring the mutated gene into crop plants.

Roundup Ready™ Soybeans A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds. Herbicides such as Roundup. TM and Liberty Link. TM 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.

Insect resistance n Anti-Insect Strategy - Insecticides • From Bacillus thuringensis Toxic crystals found during sporulation n Alkaline protein degrades gut wall of lepidopteran larvae n • • n Corn borer catepillars Cotton bollworm catepillars Tobacco hornworm catepillars Gypsy moth larvae Sprayed onto plants – but will wash off

Insect resistance a) The Bt toxin isolated from Bacillus thuringiensis has been used in plants. The gene has been placed in corn, cotton, and potato, and has been marketed. b) Plant protease inhibitors have been explored since the 1990 s: i. Naturally produced by plants, are produced in response to wounding. ii. They inhibit insect digestive enzymes after insects ingest them, causing starvation. iii. Tobacco, potato, and peas have been engineered to resist insects such as weevils that damage crops while they are in storage iv. Results have not been as promising as with Bt toxin, because it is believed that insects evolved resistance to protease inhibitors.

Bt Corn 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

Virus resistance a) Chemicals are used to control the insect vectors of viruses, but controlling the disease itself is difficult because the disease spreads quickly. b) Plants may be engineered with genes for resistance to viruses, bacteria, and fungi. c) Virus-resistant plants have a viral protein coat gene that is overproduced, preventing the virus from reproducing in the host cell, because the plant shuts off the virus’ protein coat gene in response to the overproduction. d) Coat protein genes are involved in resistance to diseases such as cucumber mosaic virus, tobacco rattle virus, and potato virus X.

Virus resistance e) Resistance genes for diseases such as fungal rust disease and tobacco mosaic virus have been isolated from plants and may be transferred to crop plants. f) Yellow Squash and Zucchini Seeds are available that are resistant to watermelon mottle virus, zucchini yellow mosaic virus, and cucumber mosaic virus. g) Potato. a) Monsanto developed potatoes resistant to potato leaf roll virus and potato virus X, which also contained a Bt toxin gene as a pesticide. b) hain restaurants do not use genetically engineered potatoes due to public pressures. h) Papaya Varieties resistant to papaya ring spot virus have been developed.

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

Altered Oil Content Done in plants by modifying an enzyme in the fatty acid synthesis pathway (oils are lipids, which fatty acids are a part of). b) Varieties of canola and soybean plants have been genetically engineered to produce oils with better cooking and nutritional properties. c) Genetically engineered plants may also be able to produce oils that are used in detergents, soaps, cosmetics, lubricants, and paints. a)

Laurate canola oil n Canola plant modified with thioesterase gene obtained from California bay laurel tree • Enzyme produces lauric acid (up to 40% in oil from genetically modified (GM) canola seeds) Low saturated fat content n Heat tolerant n • Does not break down • Excellent for high temperature cooking processes

Delayed Fruit Ripening a) Allow for crops, such as tomatoes, to have a higher shelf life. b) Tomatoes generally ripen and become soft during shipment to a store. c) Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste.

Delayed Fruit Ripening Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. e) What happened to the Flavr Savr tomato? i. Produced by Calgene 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. ii. Plants were transformed with the anti-sense PG gene, which is m. RNA that base pair with m. RNA that the plant produces, essentially blocking the gene from translation. iii. First genetically modified organism to be approved by the FDA, in 1994. iv. Tomatoes were delicate, did not grow well in Florida, and cost much more than regular tomatoes. v. Calgene was sold to Monsanto after Monsanto filed a patentinfringement lawsuit against Calgene, and the Flavr Savr tomato left the market. d)

The Flavr Savr. TM Tomato (First transgenic Plant Product)

DNA Summary of Antisense mechanism: How enzyme is made? PRODUCED

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

First biotech plant product – Flav’r Sav’r tomato

Flavr-Savr tomato n n n “Rot-Resistant Tomato” Calgen, Inc. Anti-sense gene complementary to polygalacturonase (PG) • PG = pectinase accelerates plant decay/rotting

Pollen Control a) Hybrid crops are created by crossing two distantly related varieties of the same crop plant. b) The method may generate plants with favorable traits, such as tall soybean plants that make more seeds and are resistant to environmental pressures. c) For success, plant pollination must be controlled. This is usually done by removing the male flower parts by hand before pollen is released. Also, sterilized plants have been genetically engineered with a gene from the bacteria Bacillus amyloliqueifaciens (barnase gene). This gene is dominant gene for male sterility

Genetic laser approach n n n n Targetting the expression of a gene encoding a cytotoxin by placing it under the control of an ather specific promoter (Promoter of TA 29 gene) Expression of gene encoding ribonuclease (chemical synthesized RNAse-T 1 from Aspergillus oryzae and natural gene barnase from Bacillus amyloliquefaciens) RNAse production leads to precocious degeneration of tapetum cells, the arrest of microspore development and male sterility. It is a dominant nuclear encoded or genetic male sterile (GMS), although the majority of endogenous GMS is recessive Success in oilseed rape, maize and several vegetative species Used antisense or cosuppression of endogenous gene that are essential for pollen formation or function Reproducing a specific phenotype-premature callose wall dissolution around the microsporogenous cells Reproducing mitocondrial dysfunction, a general phenotype observed in many CMS

Fertility restoration ü Restorer gene (RF) must be devised that can suppress the action of the male sterility gene (Barstar) 1. 2. 3. a specific inhibitor of barnase Also derived from B. amyloliquefaciens Served to protect the bacterium from its own RNAse activity by forming a diffusion-dependent, extremely one to one complex which is devoid of residual RNase activity ü The use of similar promoter to ensure that it would be activated in tapetal cells at the same time and to maximize the chance that barstar molecule would accumulate in amounts at least equal to barnase ü Inhibiting the male sterility gene by antisense. But in the cases where the male sterility gene is itself antisense, designing a restorer counterpart is more problematic

Production of 100% male sterile population ü ü When using a dominant GMS gene, a means to produce 100% male sterile population is required in order to produce a practical pollination control system Linkage to a selectable marker Use of a dominant selectable marker gene (bar) that confers tolerance to glufosinate herbicide Treatment at an early stage with glufosinate during female parent increase and hybrid seed production phases eliminates 50% sensitive plants ü Pollen lethality add a second locus to female parent lines consisting of an RF gene linked to a pollen lethality gene (expressing with a pollen specific promoter)

Plant Biotechnology Revolution Cold and Drought Tolerance and Weather. Gard Genes. 1. Plants such fruits are subject to frost damage at low temperatures, as well as from loss of water. They can be genetically engineered to resist these conditions, and increase crop yields as a result. 2. To resist cold weather, cold-regulated (COR) genes are also called “antifreeze genes, ”, which encode proteins that protect plant cells from frost damage. 3. A transcription factor for a group of COR genes called “CBF” was patented as Weather. Gard in 1997 by a group at Michigan State University. The genes also provide drought tolerance and tolerance to high-salt soils. 4. All major crop species, including corn, soybean, and rice contain CBF genes. 5. Genetically engineering plants with CBF genes survive temperatures as much as 4 to 50 C lower than non-engineered plants.

Plant Biotechnology Revolution Genetically Engineered Foods. n n Foods that contain an added gene sequence Foods that have a deleted gene sequence Animal products from animals fed GM feed Products produced by GM organisms

Plant Biotechnology Revolution Genetically Engineered Foods. 1. More than 60% of processed foods in the United States contain ingredients from genetically engineered organisms. 2. 12 different genetically engineered plants have been approved in the United States, with many variations of each plant, some approved and some not. 3. Soybeans. a) Soybean has been modified to be resistant to broad-spectrum herbicides. b) Scientists in 2003 removed an antigen from soybean called P 34 that can cause a severe allergic response. 4. Corn a) Bt insect resistance is the most common use of engineered corn, but herbicide resistance is also a desired trait.

Plant Biotechnology Revolution Genetically Engineered Foods 4. Corn a) Bt insect resistance is the most common use of engineered corn, but herbicide resistance is also a desired trait. b) Products include corn oil, corn syrup, corn flour, baking powder, and alcohol. c) By 2002 about 32% of field corn in the United States was engineered. 5. Canola. a) More than 60% of the crop in 2002 was genetically engineered; it is found in many processed foods, and is also a common cooking oil. 6. Cotton. a) More than 71% of the cotton crop in 2002 was engineered. b) Engineered cottonseed oil is found in pastries, snack foods, fried foods, and peanut butter. 7. Other Crops Other engineered plants include papaya, rice, tomato, sugar beet, and red heart chicory.

Plant Biotechnology Revolution Nutritionally Enhanced Plants Golden Rice 1. More than one third of the world’s population relies on rice as a food staple, so rice is an attractive target for enhancement. 2. Golden Rice was genetically engineered to produce high levels of beta-carotene, which is a precursor to vitamin A. Vitamin A is needed for proper eyesight. 3. Biotechnology company Syngenta, who owns the rights to Golden Rice, is exploring commercial opportunities in the United States and Japan. Monsanto will provide licenses to Golden Rice technology royalty-free. 4. Other enhanced crops include iron-enriched rice and tomatoes with three times the normal amount of betacarotene

Plant Biotechnology Revolution Nutritionally Enhanced Plants 6. Cause for Concern? The Case of Star. Link Corn. a) Star. Link corn had been approved for animal consumption, but in 2000 ended up in Taco Bell taco shells. The shells were immediately recalled. b) Aventis Crop. Science believed that precautions regarding the corn were in place, but some farmers did not know the corn was not for humans. c) Engineered and non-engineered corn was mixed in mills, contaminating food. d) Star. Link contained two new genes: i. Resistance to butterfly and moth caterpillars by a modified Bt toxin gene called Cry 9 c. ii. Resistance to herbicides such as Basta and Liberty. e) Star. Link was approved for animals because the Cry 9 c protein could be an allergen in humans because it was more stable to heat and in the stomach.

Plant Biotechnology Revolution Nutritionally Enhanced Plants Currently, no cases of allergic reactions have been reported, and the EPA ruled in 2001 that Star. Link was not safe for humans. 7. Cause for Concern? Genetically Engineered Foods and Public Concerns. a) The release of the Flavr Savr tomato generated much discussion over the potential risks of genetically engineered food: i. The primary public fear was that genetically engineering a plant may produce unexpected results, such as allergic reactions or even shock. ii. Genetically engineered food may also raise concerns about the selection of food if, for example, an apple has a gene from an animal. iii. The use of antibiotic resistance markers may possibly inactivate antibiotics, leading to scientists trying to find ways to remove markers from plants. f)

Plant Biotechnology Revolution Nutritionally Enhanced Plants iv. Another concern is that deleting genes may bring about side effects when ingested, such as secondary metabolites that may protect people from compounds that would normally be broken down by the plant. v. Uncharacterized DNA included along with the gene of interest may produce unexpected, harmful side effects in the plant. vi. Crops may spread the trait to other plants through pollination, which may damage ecosystems. Male-sterile plants may deal with this problem.

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

Plant Biotechnology Revolution Molecular Farming 1. A new field where plants and animals are genetically engineered to produce important pharmaceuticals, vaccines, and other valuable compounds. 2. Plants may possibly be used as bioreactors to mass-produce chemicals that can accumulate within the cells until they are harvested. 3. Soybeans have been used to produce monoclonal antibodies with therapeutic value for the treatment of colon cancer. Drugs can also be produced in rice, corn, and tobacco plants.

Plant Biotechnology Revolution 4. 5. Molecular Farming Plants have been engineered to produce human antibodies against HIV and Epicyte Pharmaceuticals has begun clinical trials with herpes antibodies produced in plants. The reasons that using plants may be more costeffective than bacteria: a) Scale-up involves just planting seeds. b) Proteins are produced in high quantity. c) Foreign proteins will be biologically active. d) Foreign proteins stored in seeds are very stable. e) Contaminating pathogens are not likely to be present (no animal contaminations).

Plant Biotechnology Revolution Molecular Farming 6. Edible Vaccines a) People in developing countries have limited access to many vaccines. b) Making plants that produce vaccines may be useful for places where refrigeration is limited. c) Potatoes have been studied using a portion of the E. coli enterotoxin in mice and humans. d) Other candidates for edible vaccines include banana and tomato, and alfalfa, corn, and wheat are possible candidates for use in livestock. e) Edible vaccines may lead to the eradication of diseases such as hepatitis B and polio.

Pharmaceutical Production in Plants Genetically modified plants have been used as “bioreactors” to produce therapeutic proteins for more than a decade. A recent contribution by transgenic plants is the generation of 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. Edible vaccines produced by transgenic plants are attractive for many reasons. The cost associated with the production of the vaccine is low, especially since the vaccine can be ingested directly, and vaccine production can be rapidly up scaled should the need arises. Edible vaccine is likely to reach more individuals in developing countries. 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.

Edible Vaccines One focus of current vaccine effort is on hepatitis B, a virus responsible for causing chromic liver disease. Transgenic tobacco and potatoes were engineered to express hepatitis B virus vaccine. During the past two years, vaccines against a E. coli toxin, the respiratory syncytial virus, measles virus, and the Norwalk virus have been successfully expressed in plants and delivered orally. These studies have supported the potential of edible vaccines as preventive agents of many diseases. There is hope to produce edible vaccines in bananas, which are grown extensively throughout the developing world. Vol. 19, No. 3 Feb. 1, 1999

Examples of Biopharmaceuticals n n n Hepatitis B and other subunit vaccines Urokinase (clot dissolving drug) Human serum albumin (liver cirrhosis treatment) Hemoglobin Human erythropoietin Glucocerebrosidase (Gaucher’s disease)

Examples of Biopharmaceuticals n n n Blood coagulants Proteases (e. g. trypsin) Protease inhibitors (e. g. aprotinin - used by surgeons) Growth promoters HIV viral coat protein (HIV therapy) Nutraceuticals (Vitamin A and E, amino acids)

Examples of Biopharmaceuticals n n n n Neurologically active agents (human enkephalins) Protein based sweetener (Brazzein) Avidin Beta-glucoronidase Indirect thrombin inhibitor (Hirudin – anticoagulant originally isolated from the leech Hirudo medicinalis) Human epidermal growth factor Human interferon-alpha (Hepatitis B and C treatment)

Examples of Biopharmaceuticals n n n Bacterial enterotoxins Human insulin Norwalk virus capsid protein “Natural” plastic (plastic-like polymers) (Biopol) Human alpha-1 antitrypsin (cystic fibrosis/liver treatment) Angiotensin-1 -converting enzyme (hypertension)

Edible Vaccines Ongoing Research Areas ü Hepatitis B ü Dental caries - Anti-tooth decay Ab (Caro. Rx. TM) (anti. Streptococcus mutans) ü Autoimmune diabetes ü Cholera ü Rabies ü HIV ü Rhinovirus ü Foot and Mouth ü Enteritis virus ü Malaria ü Influenza ü Cancer

Edible Vaccine n n n Foodborne Pathogen Vaccine exists, cost prohibitive delivery Plant-based vaccination can be cost effective Improved safety of the food supply Safety evaluation of the vaccine protein

Plant Biotechnology Revolution Biopolymers and Plants a) Plant seeds may be a potential source for plastics that could be produced and easily extracted. b) A type of PHA (polyhydroxylalkanoate) polymer called “poly-beta-hydroxybutyrate”, or PHB, is produced in Arabidopsis, or mustard plant. c) PHB can be made in canola seeds by the transfer of three genes from the bacterium Alicaligenes eutrophus, which codes for enzymes in the PHB synthesis pathway. d) Monsanto produces a polymer called PHBV through Alicaligenes fermentation, which is sold under the name Biopol.

Areas of ongoing debate n n Environment Human Health Food security Socio-economic concerns

Environment n n Anti-GM Loss of biodiversity Cross-pollination Emergence of superweeds and superbugs Potential increase in use of herbicides *Opinions are generalized, and not all opponents or proponents may hold all of these views. n n n Pro-GM Need to increase yields to feed growing population Possibility of reducing need for pesticides, fertilizers Grow more food on same amount of land

Human Health Anti-GM n n n Fear of unknown allergens Spread of anti-biotic resistance Inadequate regulation of new products Pro-GM n n Greater regulations than other foods Potential benefits to nutrition • golden rice • enhanced protein content in corn • soybean oil with less saturated fat

Food Security n n n Anti-GM Need redistribution, not just more Farmers will not be able to afford expensive seed Developing countries should not have to eat the food others reject n n Pro-GM Modified seeds will allow farmers to grow more to feed their family and to sell, reducing the need for food aid Public-private cooperation can transfer technology

Socio-economic concerns n n n Corporations benefit, not those in need Products needed in developing countries are not being developed because the market is not profitable It is wrong to patent life n n Patents needed because new strains are intellectual property Publicly funded research can benefit the public good