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How transgenic plants are made • Original approach used a natural plant system • Current plasmid based with various delivery systems • Currently many traits engineered or under development • Now augmented by newer high throughput genome sequencing techniques • Highly politicized, especially in EU Agrobacterium causes crown galls In the field …and in the lab A plasmid transfers DNA to the plant Used to make transgenic plants in 1983 Only small part of plasmid is transferred Transferred DNA ~30 KB Transfer region controls excision and integration of DNA into plant host Direct repeats 200 KB Growth factor stimulates gall growth in plant Transferring new genes to plant cells EXCISION SITES KanR KanR INTO PLANT GENOME KanR Antibiotic resistance genes included Two classes of plants require different methods MONOCOTS DICOTS Electroporation Agrobacterium Corn Rice Wheat Soy Cotton Aubergine Yams Tomato Potato SOME ORIGINAL TRANSGENIC CROPS Crop and release date Name Company Novel properties Tomato (1994) Flavr Savr Calgene Vine-ripened flavour, shelf life Zeneca Consistency of tomato paste Tomato (1995) Cotton Potato Maize (1996-97) Bollgard Monsanto Bacillus thuringiensis toxin for insect resistance Soybean Canola (rape seed) Cotton (1995-96) Corn Soy Beans Many others Roundup Ready Monsanto Glyphosate herbicide resistance New GMO crops are being approved Number of GMOs per crop Adoption in US is very high WHAT GENES ARE TRANSFERRED INTO PLANTS? • HERBICIDE RESISTANCE • DROUGHT RESISTANCE • SALT RESISTANCE • INHIBITORS OF RIPENING • NUTRITIONAL SUPPLEMENTS • PHARMACEUTICALS • INCREASED HEAVY METAL UPTAKE GMOs vary by trait Transferring bioluminescence gene Bacillus thuringenensis (Bt) - Insect Resistance • SPORE FORMING BACTERIA • SAME AS B. anthracis AND B. cereus PLASMIDS ONLY DISTINGUISH • PLASMIDS PRODUCE CRYSTALS OF CRY PROTEINS • MANY DIFFERENT KINDS OF CRY • SMALL PROTEIN = EASY MOLECULAR METHODS • MOST Bt HAVE SEVERAL TOXINS • TOXIC TO DIFFERENT INSECTS LONG HISTORY IN ORGANIC AGRICULTURE Microbial pesticides registered by EPA ONE Bt TOXIN STRUCTURE BINDS SPECIFICALLY TO A RECEPTOR INSERTS INTO GUT WALL PROTECTS THE PEPTIDE FROM DEGRADATION Bt proteins dissolve the insect gut lining TOXIN BINDS TO SPECIFIC RECEPTORS IN GUT WALL PROTEIN CLEAVED DISSOLVES GUT LINING NO TOXIN TOXIN Huge Diversity of Different Proteins SIMILAR MODES OF ACTION DIFFERENT INSECT SPECIFICITIES Bt CRYSTAL PROTEINS AND THEIR TARGET SPECIES Gene Insect Activity cryI; A(a), A(b), A(c), B, C, D, E, F,G Lepidopteran larvae (BUTTERFLIES) cryII; A, B, C Lepidopteran and Dipteran Larvae (FLIES) cryIII; A, B, Coleopteran larvae (BEETLES) cryIV; A, B,C,D Dipteran larvae cryV - cryIX Various MANY Bt-RICE VARIETIES HAVE BEEN MADE CHANGES IN GUT PROTEIN CAN MEDIATE RESISTANCE DIFFERENT TRANSGENIC GOAL – ADD NUTRIENTS • POLISHED RICE IS MAINLY CARBOHYDRATE • STAPLE FOOD IN AREAS WHERE VITAMIN DEFICIENCIES ARE COMMON • ADD VITAMIN A TO RICE ENDOSPERM? • RICE HAS ENZYMES TO MAKE VITAMIN A PRECURSOR CONVERT GGPP INTO CAROTENE WITH GENES FROM OTHER PLANTS LIMIT IN NORMAL RICE DAFFODIL ERWINIA DAFFODIL Herbicide Resistance - most common in US • Glyphosate = Roundup • Inhibits an enzyme needed to synthesize aromatic amino acids ( Phe, Tyr and Trp) • Roundup ready plants carry a bacterial version of the enzyme that is naturally resistant to Roundup • Animals do not have this system, so not toxic to humans • Resistance can evolve in a variety of ways in plants exposed to Roundup Diverse Methods & Goals, Many Controversies