HERBICIDE-RESISTANT ACALA AND COKER COTTONS TRANSFORMED WITH A NATIVEGENE ENCODING MUTANT FORMS OF ACETOHYDROXYACID SYNTHASE

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants c arrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitut ions at residue 563 or residue 642 of the precursor polypeptide. Trans formation with the mutated forms of the A19 gene produced resistance t o imidazolinone and sulfonylurea herbicides (563 substitution), or imi dazolinones only (642 substitution). The herbicide-resistant phenotype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were trans formed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained wit h the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coke r and Acala plants were regenerated from a subset of transformation ev ents. Embryonic cell suspension cultures of both Coker and Acala varie ties were biolistically transformed at high frequencies using cloned c otton DNA fragments carrying the mutated forms of the A19 gene. In the se transformation experiments the mutated A19 gene served as the selec table marker, and the efficiency of selection was comparable to that o btained with the NPT II gene marker of vector Bin 19. Using this metho d, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A19 transgene in one of the biolistically transformed R (0) plants, and a single copy in one of the R(0) plants transformed wi th Agrobacterium. As expected, progeny seedlings derived from outcross es involving the Ro(0) plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant pr ogeny grew normally after irrigation with 175 mu g/l of the imidazolin one herbicide imazaquin, which is five times the field application rat e. In contrast, untransformed sibling plants were severely stunted.