Chemical mutagenesis of wheat for herbicide resistance.

Abstract

Weed infestation is one of the yield limiting factors in crop production. Weeds have negative effect on crop growth and productivity due to competition, allelopathy or hosting other harmful organisms. For large-scale wheat production, the use of wide spectrum pre-emergence or post-emergence herbicides remains the most valuable weed control tool. In South Africa, annual grass weeds are a major wheat production constraint, which is usually managed through application of pre-emergence herbicides. Due to limited water availability and low soil moisture content, these herbicides can often become ineffective and result into high weed infestations, which then have to be managed by manual cultivation or post-emergence herbicidal applications. However, there are no effective selective post-emergence herbicides available to control grass weeds in wheat. There is also limited option to use broad-spectrum post-emergent herbicides because they non-selectively kill the crop and weeds. Consequently, the use of herbicide resistant crops is a viable weed management system in wheat production. Breeding herbicide resistant crop varieties would allow farmers to safely use post-emergence herbicides without damaging the crop. Subsequently yield and quality losses will be reduced significantly. Thus, the development of herbicide resistant crop varieties through mutation breeding is a novel approach for effective weed management under both small-scale and commercial farmers. Mutagenesis has been recognized as one of the most efficient method to induce genetic variation in plants. Through induced mutations, development of new variants is possible that could be manipulated in plant breeding programs. Mutation leads to alteration of various traits in crop plants including plant height, improved nutritional quality, shorter growing period, increased tolerance or resistance to abiotic and biotic stresses. Ethylmethanesulphonate (EMS) is one of the most widely used chemical mutagens to induce mutagenesis in crop plants. The objectives of this study were to: 1) determine the optimum EMS concentration, treatment temperature and duration that would provide desired germination percentage and vigorous and healthy seedlings for effective mutagenesis in wheat, 2) investigate variations in agro-morphological traits in two selected wheat varieties (SST56 and SST875) after EMS mutagenesis and 3) select herbicide resistant wheat germplasm after inducing genetic variation using EMS using two selected wheat varieties (SST56 and SST875). The objectives were achieved through three independent studies as outlined below:

In the first study seeds of four selected wheat varieties (B936, B966, SST387 and SST875) were treated in two replicates with three EMS concentrations (0.3, 0.5, and 0.7%), three temperature regimes (30, 32.5 and 35 °C) at four time durations (0.5, 1, 1.5 and 2 hrs). Results showed highly significant interactions (P<0.01) among varieties, EMS concentrations, temperature and exposure time on seedling emergence, germination and seedling height. Seeds treated with the highest EMS dose (0.7%), temperature (35ºC) and long exposure time (2 hr) showed delayed emergence by 18 days. At 30ºC, 0.5hr and 0.3% EMS varieties B936, B966 and SST875 had early emergence (6 days). B936 and SST387 had 50% while B966 and SST875 had 53% and 57% germination, respectively. These results were observed at EMS level of 0.7%, 300C and 1.5 hr exposure time in B936 and EMS at 0.5%, 350C and 1.5 hr in B966. SST387 and SST875 required EMS dose at 0.5%, 32.50C and 2 hr treatment time. Other low or high treatment combinations were invariably ineffective comparedto untreated control. During the second study two selected varieties (SST56 and SST875) were subjected to EMS mutagenesis using 0.5% v/v EMS at 32.5oC for 1 hr. Field trials were carried out at Ukulinga research farm of the University of KwaZulu-Natal in the randomized complete block design with two replicates. Data on nine important agro-morphological traits were collected and analyzed using the analysis of variance (ANOVA), correlation and principal component analysis (PCA) procedures. Significant variations were found among the agro-morphological traits between M1 individuals compared to untreated checks. The mutagenesis significantly reduced seed germination in the field at 40% in both varieties. The treatment significantly delayed days to heading by 8 days and shortened days to maturity by 13 days in both varieties. EMS treatment also significantly reduced plant height at 18 cm in SST56 and 21 cm in SST875 and spike length reduced by ~2.5 cm in both varieties. Plant height had positive and significant correlation with number of tillers, number of seeds per spike, flag leaf length and 100 seed weight. However, it had negative correlation with the number of days to maturity. The PCA revealed that three principal components (PC1, PC2 and PC3) accounted to 57% of the total variations among the agro-morphological traits in both varieties. PC1 alone contributed to 27.7% of the variation which was well-correlated with plant height (0.767), tiller number (0.812), number of seeds per spike (0.599) and seed yield (0.720). PC2 explained 15.6% of the variation and well-correlated with germination percentage (0.784), spike length (0.554) and flag leaf length (0.772). PC3 accounted to 12.4% of the variation and had negative correlation with days to maturity (-0.730).

In the last study, seeds of two selected wheat varieties (SST56 and SST875) were treated with EMS at 0.5% concentration for 2 hr at 32.5ºC. Treated seeds and comparative controls were planted at the experimental farm of the University of KwaZulu-Natal using the randomized complete block design. Four weeks after planting M1 plants and untreated standard checks were sprayed with two herbicides, i.e. metsulfuron-methyl and bromoxynil at three different doses viz. 2x, 4x and 8x above the recommended rate of 4 g ha-1 and 2 kg ha-1, respectively. Two weeks after the treatment herbicide resistance were assessed. Results showed significant difference among varieties, tested herbicides and doses used. The EMS treated wheat lines showed variable degree of herbicide resistance compared to untreated controls. Overall, the study established the requirement of variety specific EMS dose and treatment temperature and duration that could be used for inducing large-scale mutation to select targeted mutant individuals in wheat. Further, the study found that EMS has the potential to increase agro-morphological variations in wheat to select useful and novel mutants with desired phenotypic traits and herbicide resistance which will be subjected for further selections to identify stable and herbicide resistance lines.