TRANSGENIC TECHNOLOGY
Traits that plant breeders would like in plants n n n High primary productivity High crop yield High nutritional quality Adaptation to intercropping Nitrogen Fixation n n Drought resistance Pest resistance Adaptation to mechanised farming Insensitivity to photo-period Elimination of toxic compounds
Plant transformation ügetting DNA into a cell ügetting it stably integrated ügetting a plant back from the cell
Requirement 1. a suitable transformation method 2. a means of screening for transformants 3. an efficient regeneration system 4. genes/constructs ü Vectors ü Promoter/terminator ü reporter genes ü selectable marker genes ü ‘genes of interest’
Transformation methods DNA must be introduced into plant cells Indirect - Agrobacterium tumefaciens Direct - microprojectile bombardment - electroporation - Polyethylene glycol (PEG)-mediated - glass-beads - silicon carbide whiskers Method depends on plant type, cost, application
Agrobacterium-mediated transformation n n A natural genetic engineer 2 species • A. tumefaciens (produces a gall) • A. rhizogenes (produces roots) n Oncogenes (for auxin and cytokinin synthesis) + Opines n In the presence of exudates (e. g. acetosyringone) from wounded plants, Virulence (Vir) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.
BACTERIAL GALL DISEASES n Galls: overgrowth or proliferation of tissue, primarily due to increased cell division (hyperplasia) and increased cell size (hypertrophy). n Bacterial Galls: induced by bacteria in 3 different genera. • Agrobacterium • Pseudomonas • Clavibacter n Genes for plant hormone production found on bacterial plasmids!
Crown Gall Disease: Agrobacterium tumefaciens n n n Gram Dicots Worldwide
Disease Cycle
Agrobacterium tumefaciens n Characteristics • Plant parasite that causes Crown Gall Disease • Encodes a large (~250 kbp) plasmid called Tumor-inducing (Ti) plasmid n Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Tumor DNA)
Agrobacterium tumefaciens üT-DNA integrates stably into plant genome üSingle stranded T-DNA fragment is converted to ds. DNA fragment by plant cell Then integrated into plant genome n 2 x 23 bp direct repeats play an important role in the excision and integration process n
Agrobacterium tumefaciens n n n Tumor formation = hyperplasia Hormone imbalance Caused by A. tumefaciens • Lives in intercellular spaces of the plant • Plasmid contains genes responsible for the disease n Part of plasmid is inserted into plant DNA n Wound = entry point 10 -14 days later, tumor forms
Agrobacterium tumefaciens n What is naturally encoded in T-DNA? • Enzymes for auxin and cytokinin synthesis n n Causing hormone imbalance tumor formation/undifferentiated callus Mutants in enzymes have been characterized • Opine synthesis genes (e. g. octopine or nopaline) n n Carbon and nitrogen source for A. tumefaciens growth Insertion genes • Virulence (vir) genes • Allow excision and integration into plant genome
Ti plasmid of A. tumefaciens
1. Auxin, cytokinin, opine synthetic genes transferred to plant 2. Plant makes all 3 compounds 3. Auxins and cytokines cause gall formation 4. Opines provide unique carbon/nitrogen source only A. tumefaciens can use!
Agrobacterium tumefaciens n How is T-DNA modified to allow genes of interest to be inserted? • In vitro modification of Ti plasmid n n n n T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) Insertion genes are retained (vir genes) Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance geneticin (G 418) or hygromycin] Ti plasmid is reintroduced into A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) Antibacterial agents (e. g. chloramphenicol) added to kill A. tumefaciens G 418 or hygromycin added to kill non-transgenic plant cells Surviving cells = transgenic plant cells
Agrobacterium and genetic engineering: Engineering the Ti plasmid
Co-integrative and binary vectors LB RB Co-integrative Binary vector
Agrobacterium-mediated transformation Agrobacterium tumefaciens cause ‘Crown gall’ disease Agrobacterium is a ‘natural genetic engineer’ i. e. it transfers some of its DNA to plants
Expose wounded plant cells to transformed agro strain Electroporate TDNA vector into Agrobacterium and select for tetr Induce plant regeneration and select for Kanr cell growth
Microprojectile bombardment • uses a ‘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
n n In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot into the plant cell with a pulse of helium. Once inside the cell, the gene comes off the bead and integrates into the cell's genome.
n Model from Bio. Rad: Biorad's Helios Gene Gun
How do we get plants back from cells? We use tissue culture techniques to regenerate whole plants from single cells getting a plant back from a single cell is important so that every cell has the new DNA
Regeneration Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana
Somatic embryogenesis in peanut
Screening Technique
Not all cells take up DNA & not all cells can regenerate so Need an efficient regeneration system and transformation system i. e. lots of cells take up DNA and lots of cells regenerate into a plant to maximize chance of both happening regenerable cells Transformed cells Cells containing new DNA that are able to regenerate into a new plant
There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA therefore can get hundreds of plants back - maybe only 1% will be transformed How do we know which plants have taken up the DNA? Could test each plant - slow, costly Or use reporter genes & selectable marker genes
Selection n n Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by nontransformed. Usual to use a positive selective agent like antibiotic resistance. The Npt. II gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.
Reporter genes most common - easy to visualise or assay - ß-glucuronidase (GUS) (E. coli) - green fluorescent protein (GFP) (jellyfish) - luciferase (firefly, bacterial, jellyfish etc)
GUS Cells that are transformed with GUS will form a blue precipitate when tissue is soaked in the GUS substrate and incubated at 37 o. C this is a destructive assay (cells die) The Uid. A gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.
GUS Bombardment of GUS gene - transient expression Stable expression of GUS in moss Phloem-limited expression of GUS
HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic plant expressing a HAESA: : GUS fusion. It is expressed in the floral abscission zone at the base of an Arabidopsis flower. Transgenic plants that harbor the AGL 12: : GUS fusions show rootspecific expression.
GFP (Green Fluorescent Protein) ü Fluoresces green under UV illumination ü Problems with a cryptic intron now resolved. ü Has been used for selection on its own. GFP glows bright green when irradiated by blue or UV light This is a nondestructive assay so the same cells can be monitored all the way through
GFP protoplast colony derived from protoplast mass of callus regenerated plant
Selectable marker genes - let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance Most common: antibiotic resistance - e. g. kanamycin, hygromycin [ only phytotoxic antibiotics can be used] herbicide resistance - e. g. phosphinothricin (PPT); glyphosate
Only those cells that have taken up the DNA can grow on media containing the selection agent
APPLICATIONS transfer of exogenous genes Pathogen resistance Herbicide resistance Bioreactors/molecular farming Delivery systems Plant improvement manipulation of endogenous genes Gene silencing/ downregulation
Gene silencing/ downregulation of endogenous genes Antisense RNA – delayed ripening; Flav. R Sav. R tomatoes - modified flower colour (paler flowers) Post-transcriptional gene silencing induces cytoplasmic RNA degradation system induced by ds. RNA highly sequence specific
Applications of Plant Biotechnology A. B. Crop Improvement 1. The following traits are potentially useful to plant genetic engineering: controlling insects, manipulating petal color, production of industrially important compounds, and plant growth in harsh conditions. Genetically Engineered Traits: The Big Six. 1. Herbicide Resistance a) 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. b) Critics claim that genetically engineered plants will lead to more chemical use, and possible development of weeds resistant to the chemicals.
Applications of Plant Biotechnology Glyphosate Resistance i. Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS, makes aromatic amino acids. ii. The gene encoding EPSPS has been transferred from glyphosate-resistant E. coli into plants, allowing plants to be resistant. d) 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. c)
Applications of Plant Biotechnology e) f) 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. 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.
Applications of Plant Biotechnology 2. 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.
Applications of Plant Biotechnology 3. 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.
Applications of Plant Biotechnology e) f) g) h) 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. Yellow Squash and Zucchini i. Seeds are available that are resistant to watermelon mottle virus, zucchini yellow mosaic virus, and cucumber mosaic virus. 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. Papaya. a)Varieties resistant to papaya ring spot virus have been developed.
Applications of Plant Biotechnology 4. Altered Oil Content a) 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. 5. 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.
Applications of Plant Biotechnology 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
What Happens When A Cloned Antisense DNA Is Added To The Original DNA?
When A Cloned Antisense DNA Is Added To The Original DNA:
Applications of Plant Biotechnology 6. 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.
Applications of Plant Biotechnology C. Biotech 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.
Applications of Plant Biotechnology D. 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 broadspectrum 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.
Applications of Plant Biotechnology 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 a) Other engineered plants include papaya, rice, tomato, sugar beet, and red heart chicory. b)
Applications of Plant Biotechnology E. Nutritionally Enhanced Plants—Golden Rice: An International Effort. 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. Golden Rice was developed by Ingo Potrykus and Peter Beyer, and several agencies are attempting to distribute the rice worldwide. 4. 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. 5. Other enhanced crops include iron-enriched rice and tomatoes with three times the normal amount of betacarotene
Applications of Plant Biotechnology 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.
Applications of Plant Biotechnology 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)
Applications of Plant Biotechnology 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.
Applications of Plant Biotechnology F. 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. Clot-busting drugs can also be produced in rice, corn, and tobacco plants.
Applications of Plant Biotechnology 4. Plants have been engineered to produce human antibodies against HIV and Epicyte Pharmaceuticals has begun clinical trials with herpes antibodies produced in plants. 5. 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.
Applications of Plant Biotechnology 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.
Applications of Plant Biotechnology 7. 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.
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