Characterization of sethoxydim and haloxyfop tolerant mutants selected from tissue culture

--W.B. Parker, F.A. Keith, J.D. Burton, D.A. Somers, D.L. Wyse, J.W. Gronwald1 and B.G. Gengenbach
1USDA-ARS

Sethoxydim is a postemergence herbicide that is toxic to most grass species, including corn. Haloxyfop is a herbicide from a different family with the same mechanism of herbicide action as sethoxydim. It also is toxic to grass species. Both herbicides have recently been shown to inhibit monocot Acetyl- Coenzyme A carboxylase (EC 6.4.1.2) (Burton et al., Biochem. Biophys. Res. Comm. 148:1039-1049, 1987). Corn hybrids tolerant to these herbicides would increase the herbicide options available for control of annual and perennial grasses in corn. The objectives of this research were to select sethoxydim- and haloxyfop-tolerant corn tissue culture lines, characterize the level and basis of tolerance, and determine if tolerance could be transmitted to regenerated plants and their progeny.

Initial experiments were conducted using "Black Mexican Sweet" (BMS) corn tissue cultures as a model system. Sethoxydim and haloxyfop were toxic to unselected BMS corn tissue cultures at 5 and 0.1µM concentrations, respectively. Three sethoxydim- tolerant BMS culture lines (B10S, B50S, and B100S) were selected. Sethoxydim concentrations reducing growth by 50% were 11-, 88-, and 40-fold higher in B10S, B50S, and B100S, respectively, than in BMS. Haloxyfop concentrations reducing growth by 50% were 9-, 64-, and 51-fold higher in B10S, B50S, and B100S, respectively, than in BMS. Tolerance was stable for one year when B10S was grown in the absence of sethoxydim indicating a genetic or epigenetic basis for tolerance. ACCase activity was 79, 167, and 88% higher in extracts from B10S, B50S and B100S, respectively, compared to BMS. Herbicide concentrations reducing ACCase activity by 50% in total protein extracts were not significantly different in selected lines compared to BMS. SDS-PAGE of total protein extracts probed with avidin-conjugated horseradish peroxidase indicated that levels of a biotin-containing protein, presumably ACCase, were increased in selected lines corresponding to their increase in ACCase activity. These results indicated that sethoxydim tolerance in selected BMS culture lines was conferred by increased ACCase activity likely due to elevated levels of the enzyme. Further investigations to determine the mechanism of increased enzyme expression are in progress.

Regenerable, friable, embryogenic callus cultures (A188 x B73) were selected for sethoxydim and haloxyfop tolerance. Two sethoxydim-tolerant (S1 and S2) and two haloxyfop-tolerant (H1 and H2) corn callus culture lines were obtained. S1 and S2 callus exhibited 100- and >100-fold increases in sethoxydim tolerance, respectively, compared to the unselected control callus. S1 and S2 also exhibited 4-and 30-fold cross-tolerance to haloxyfop, respectively. H1 and H2 callus exhibited no sethoxydim tolerance and 60- and 4-fold increases in haloxyfop tolerance compared to control callus, respectively. ACCase activity levels of the unselected control, S1, S2, and H1 were similar in the absence of herbicide. ACCase activity from H2 was 5-fold higher than the unselected control in the absence of herbicide. ACCase activity from S1 and S2 was inhibited 50% by sethoxydim concentrations that were 4- and 40-fold higher and haloxyfop concentrations 3- and 7-fold higher, respectively, than concentrations required for 50% inhibition of wildtype ACCase activity. ACCase activity from H1, H2, and the unselected control was inhibited 50% by similar sethoxydim concentrations. ACCase activity from H1 was inhibited 50% by a 6-fold greater haloxyfop concentration than required for the unselected control; whereas, ACCase activities of wildtype and H2 were inhibited by similar haloxyfop concentrations. These results indicated that herbicide tolerance was conferred either by elevated ACCase activity as in line H2 or by an altered ACCase activity that was less sensitive to herbicide inhibition.

Plants regenerated from unselected wildtype callus were killed by greenhouse applications of 0.05kg/ha sethoxydim and 0.01kg/ha haloxyfop. Plants regenerated from S1 and S2 callus lines were injured by 0.11 and 0.22kg/ha sethoxydim, respectively, but plants from both lines survived 0.44kg/ha sethoxydim, which is twice the field application rate of sethoxydim. S1, S2, and H1 plants were injured, but survived a 0.01kg/ha haloxyfop treatment; whereas, they were killed by 0.10kg/ha haloxyfop.

Progeny from reciprocal crosses of S1 and S2 plants x wildtype inbreds segregated 1:1 for tolerant:susceptible plants when treated with 0.44kg/ha sethoxydim. Progeny from self- pollinated S1 and S2 plants segregated 1:2:1 for uninjured:tolerant:susceptible plants when treated with 0.44kg/ha sethoxydim. Progeny from self-pollinated H1 regenerated plants segregated 1:2:1 for uninjured:tolerant:susceptible plants when treated with 0.01kg/ha haloxyfop. These results indicated that the S1, S2, and H1 plants regenerated from tissue culture were heterozygous for single dominant allele(s) which conferred sethoxydim tolerance. Putative homozygous plants that survived sethoxydim treatment have been grown to maturity and selfed. Seed set appears normal and selfed progeny will be evaluated for herbicide tolerance to determine if the trait can be maintained in a homozygous condition.


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