SELECTION AND CHARACTERIZATION OF MUTANT COTTON (GOSSYPIUM-HIRSUTUM L) CELL-LINES RESISTANT TO SULFONYLUREA AND IMIDAZOLINONE HERBICIDES

Cell lines with spontaneous mutations conferring resistance to a sulfo nylurea herbicide, primsulfuron, were isolated from embryogenic cell s uspension cultures of cotton (Gossypium hirsutum L. Acala varieties SJ 2 and B1654) by direct selection at 2.13 mu M. The mutation frequency was approximately one in every two million cells. Growth inhibition st udies indicated that the IC50 values for the selected colonies were 10 0- to 1000-fold higher than the unselected control cells. Acetohydroxy acid synthase (the target enzyme of these herbicides) extracted from r esistant colonies was active in vitro at toxic levels of the herbicide . Leaf and petiole segments from plants derived from resistant colonie s readily produced callus in the presence of primsulfuron (0.06 to 0.2 1 mu M), whereas leaf and petiole segments from unselected control pla nts were bleached. The selected colonies were cross tolerant to two im idazolinone herbicides, imazerhapyr and imazaquin. Nucleotide sequence analysis of clones derived from two different colonies revealed the p resence of point mutations at the codon for amino acid Trp 563 in eith er one of two homeologous mutant genes, A5 (Trp --> Cys) or A19 (Trp - -> Ser). This is the first time the spontaneous occurrence of these pa rticular substitutions has been reported in a plant acetohydroxyacid s ynthase. Both substitutions yielded an active enzyme which was resista nt to toxic levels of sulfonylurea and imidazolinone herbicides.