Selection and characterization of mutant cotton (Gossypium hirsutum L.) cell lines resistant to sulfonylurea and imidazolinone herbicides

Abstract Cell lines with spontaneous mutations conferring resistance to a sulfonylurea herbicide, primsulfuron, were isolated from embryogenic cell suspension cultures of cotton ( Gossypium hirsutum L. Acala varieties SJ2 and B1654) by direct selection at 2.13 μM. The mutation frequency was approximately one in every two million cells. Growth inhibition studies indicated that the IC 50 values for the selected colonies were 100- to 1000-fold higher than the unselected control cells. Acetohydroxyacid synthase (the target enzyme of these herbicides) extracted from resistant colonies was active in vitro at toxic levels of the herbicide. Leaf and petiole segments from plants derived from resistant colonies readily produced callus in the presence of primsulfuron (0.06 to 0.21 μM), whereas leaf and petiole segments from unselected control plants were bleached. The selected colonies were cross tolerant to two imidazolinone herbicides, imazethapyr and imazaquin. Nucleotide sequence analysis of clones derived from two different colonies revealed the presence of point mutations at the codon for amino acid Trp 563 in either one of two homeologous mutant genes, A5 (Trp → Cys) or A19 (Trp → Ser). This is the first time the spontaneous occurrence of these particular substitutions has been reported in a plant acetohydroxyacid synthase. Both substitutions yielded an active enzyme which was resistant to toxic levels of sulfonylurea and imidazolinone herbicides.