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Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase

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Abstract

Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying 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 substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the A19 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones 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 transformed with the mutated forms of the A19 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the A19 gene. In these transformation experiments the mutated A19 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, 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 R0 plants, and a single copy in one of the R0 plants transformed with Agrobacterium. As expected. progeny seedlings derived from outcrosses involving the R0 plant transformed with Agrobacterium segregated in a 1:1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 μg/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.

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Abbreviations

AHAS:

acetohydroxyacid synthase

CaMV:

cauliflower mosaic virus

ELISA:

enzyme linked immunosorbent assay

FW:

fresh weight

GUS:

β-glucuronidase

IC50 :

herbicide concentration that produces a 50% reduction in the fresh weight growth of cells

NAA:

α-naphthaleneacetic acid

NPT II:

neomycin phosphotransferase II

MS:

Murashige and Skoog (1962)

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Author notes

  1. Kanniah Rajasekaran

    Present address: ARS, SRRC, USDA, 1100 Robert E. Lee Blvd., 70124-4305, New Orleans, LA, USA

Authors and Affiliations

  1. Phytogen, J.G. Boswell Cotton Seed Company, 850 Plymouth Avenue, P.O. Box 787, 93212-0787, Corcoran, CA, USA

    Kanniah Rajasekaran, John W. Grula, Richard L. Hudspeth, Shoshana Pofelis & David M. Anderson

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  1. Kanniah Rajasekaran
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  2. John W. Grula
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  3. Richard L. Hudspeth
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  4. Shoshana Pofelis
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Rajasekaran, K., Grula, J.W., Hudspeth, R.L. et al. Herbicide-resistant Acala and Coker cottons transformed with a native gene encoding mutant forms of acetohydroxyacid synthase. Mol Breeding 2, 307–319 (1996). https://doi.org/10.1007/BF00437909

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