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

…and in the lab Agrobacterium causes crown galls In the field

A plasmid transfers DNA to the plant

Used to make transgenic plants in 1983

Transferred DNA ~30 KB Transfer region controls excision and integration of DNA into plant host Growth factor stimulates gall growth in plant 200 KB Only small part of plasmid is transferred Direct repeats

EXCISION SITES INTO PLANT GENOME Transferring new genes to plant cells Kan R Antibiotic resistance genes included

Two classes of plants require different methods MONOCOTS Electroporation DICOTS Agrobacterium Corn Rice Wheat Soy Cotton Aubergine Yams Tomato Potato

Crop and release dateNameCompanyNovel properties Tomato (1994)Flavr SavrCalgeneVine-ripened flavour, shelf life Tomato (1995)ZenecaConsistency of tomato paste Cotton Potato Maize ( ) BollgardMonsantoBacillus thuringiensis toxin for insect resistance Soybean Canola (rape seed) Cotton ( ) Corn Soy Beans Many others Roundup Ready MonsantoGlyphosate herbicide resistance SOME ORIGINAL TRANSGENIC CROPS

WHAT GENES ARE TRANSFERRED INTO PLANTS? HERBICIDE RESISTANCE DROUGHT RESISTANCE SALT RESISTANCE INHIBITORS OF RIPENING NUTRITIONAL SUPPLEMENTS PHARMACEUTICALS INCREASED HEAVY METAL UPTAKE

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 Bacillus thuringenensis (Bt) - Insect Resistance

LONG HISTORY IN ORGANIC AGRICULTURE Microbial pesticides registered by EPA

INSERTS INTO GUT WALL BINDS SPECIFICALLY TO A RECEPTOR PROTECTS THE PEPTIDE FROM DEGRADATION ONE Bt TOXIN STRUCTURE

TOXIN BINDS TO SPECIFIC RECEPTORS IN GUT WALL PROTEIN CLEAVED DISSOLVES GUT LINING NO TOXIN TOXIN Bt proteins dissolve the insect gut lining

SIMILAR MODES OF ACTION DIFFERENT INSECT SPECIFICITIES Huge Diversity of Different Proteins

Bt CRYSTAL PROTEINS AND THEIR TARGET SPECIES GeneInsect Activity cryI; A(a), A(b), A(c), B, C, D, E, F,G Lepidopteran larvae ( BUTTERFLIES) cryII; A, B, CLepidopteran and Dipteran Larvae (FLIES) cryIII; A, B,Coleopteran larvae (BEETLES) cryIV; A, B,C,DDipteran larvae cryV - cryIXVarious

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

LIMIT IN NORMAL RICE DAFFODIL ERWINIA DAFFODIL CONVERT GGPP INTO CAROTENE WITH GENES FROM OTHER PLANTS

SIMPLER TRANSFORMATION SURVEY OF PSY GENES SHOWED MAIZE GENE MOST ACTIVE PROTOTYPE OF PRODUCING OTHER COMPLEX MOLECULES IN EASY-TO-CULTURE PLANTS RECENT DEVELOPMENTS

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