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 regioncontrols

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

EXCISIONSITES

INTO PLANT GENOME

Transferring new genes to plant cells

KanR

KanR

KanR

Antibiotic resistance genes included

Two classes of plants require different methods

MONOCOTS

Electroporation

DICOTS

Agrobacterium

CornRice

Wheat

SoyCotton

AubergineYams

TomatoPotato

Crop and release date Name Company Novel properties

Tomato (1994) Flavr Savr Calgene Vine-ripened flavour, shelf life

Tomato (1995) Zeneca Consistency of tomato paste

CottonPotatoMaize (1996-97)

Bollgard Monsanto Bacillus thuringiensis toxin for insect resistance

Soybean Canola (rape seed) Cotton (1995-96)CornSoy BeansMany others

Roundup Ready

Monsanto Glyphosate 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 PEPTIDEFROM DEGRADATION

ONE Bt TOXIN STRUCTURE

TOXIN BINDS TOSPECIFIC RECEPTORS

IN GUT WALL

PROTEIN CLEAVEDDISSOLVESGUT LINING

NO TOXIN TOXIN

Bt proteins dissolve the insect gut lining

SIMILAR MODESOF ACTION

DIFFERENTINSECT

SPECIFICITIES

Huge Diversity of Different Proteins

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 VITAMINDEFICIENCIES 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