Field assessment of agronomic traits and in vitro acetolactate synthase characterisation of imazapyr herbicide tolerant sugarcane.

Weed control is a major cost for growers in the sugarcane industry, especially for monocotyledonous species such as Cynodon and Rottboellia spp. The introduction of imazapyr-tolerant sugarcane would be advantageous as this herbicide has shown to be effective against the above-mentioned weeds but it also kills sugarcane. In a previous study in our laboratory, several sugarcane putative-mutant lines of variety N12 were generated by in vitro exposure of embryogenic callus to 16 mM ethyl methanesulfonate (EMS), followed by selection on imazapyr-containing medium. Tolerance to a low dose of imazapyr was confirmed in seven of those lines when the herbicide was applied (182 g a.i. ha-1) to 3 month-old plants in pots. The aim of the present study was to identify which of the seven herbicide mutant lines had agronomic characteristics at least equivalent to un-mutated N12. The objectives were to: 1) confirm tolerance to increased rate (312 and 625 g a.i. ha-1) of imazapyr in field plants; 2) measure the agronomic characteristics of these lines; 3) determine the effect of residual soil herbicide activity on germination of sugarcane setts. The seven mutant lines (Mut1-Mut7) and un-mutated N12 were clonally propagated in vitro by shoot multiplication followed by rooting and planted in three plots (untreated, sprayed with 312 or 625 g a.i. ha-1 imazapyr), in the field, in a randomized complete block design. In the untreated control plot there were no significant differences between the control and the mutant plants for agronomic traits (tiller number/plot, stalk height and stalk diameter) or estimated yield (kg/plot) after 10 months, indicating that the mutation process had no effect on general plant phenotype. In the sprayed (312 and 625 g a.i. ha-1) plots, Mut1, Mut4, Mut5, Mut6 and Mut7 plants showed tolerance to imazapyr as the leaves remained green compared with Mut2, Mut3 and N12 control plants, which displayed chlorotic leaves and eventually died in the plot sprayed with 625 g a.i. ha-1. Post-herbicide application, the yields of Mut5, Mut6 and Mut7 (52.33, 43.43 and 41.43 kg/plot, respectively) from the 312 g a.i. ha-1 plot were not significantly different from that of N12 control (53. 61 kg/plot) in the untreated plot. However, in the 312 g a.i. ha-1 plot, the yield and agronomic trait measurements of the untreated N12 control were significantly higher than those of the herbicide-susceptible plants Mut2 and Mut3. Similarly, in the 625 g a.i. ha-1 plot, the recorded yields for Mut4, Mut6 and Mut7 were 41.60, 43.44 and 36.30 kg/plot, respectively, indicating that their imazapyr tolerance and yield characteristics were comparable to the untreated N12 control. Imazapyr is conventionally applied to a fallow field 3-4 months prior to planting sugarcane as there is residual herbicide activity in the soil that suppresses sugarcane germination and growth. Therefore, in order to establish if the herbicide-tolerant mutants could germinate in iii an imazapyr-treated field, 3-budded setts of the mutant lines (Mut1-Mut7) and N12 control were planted in two plots, one unsprayed and one sprayed with 1254 g a.i. ha-1 imazapyr, 2 weeks previously. Germination was calculated after 3 weeks as the number of germinated setts in each plot/no. germinated setts in unsprayed plot x100. In the sprayed plot, the setts from Mut1, Mut4 and Mut6 displayed the highest germination percentages (60, 71 and 74%, respectively) compared with Mut2 (24%), Mut3 (46%), Mut5 (34%), Mut7 (40%) and the N12 control (12%). The in vitro acetolactate synthase (ALS) enzyme activity of 10 month-old plants from the untreated plot was assessed in the presence of 0-30 μM imazapyr to determine the herbicide concentration that inhibited ALS activity by 50% (IC50). The IC50 values for the mutated lines were between 3 and 30 μM, i.e. 1.5-8.8 times more tolerant to imazapyr than the N12 control plants, with Mut6 displaying the highest IC50 value (30 μM). On the basis of the results, it was concluded that Mut1, Mut6 and Mut7 lines were more tolerant to imazapyr than N12 and the other tested lines. Future work includes phenotypically assessing these lines for traits including sucrose content, fibre content, actual yield (tons cane ha-1) and altered pest and disease resistance. Once isolated and sequenced, the ALS gene conferring imazapyr tolerance can be used in genetic bombardment in the genetic modification approach as the gene of interest or as a selectable marker. In addition, the imazapyr-tolerant line can be used for commercial purposes in the field and as the parent plant in the breeding programme.