Development of soybean (Glycine max L.) mutants resistant to chlorsulfuron (su group) herbicide through EMS mutagen and ALS gene sequence analyses

Soybean (Glycine max L.) is a legume plant that has the highest production among oilseed plants. It uses as a source of both oil and protein in the world. As in other crop plants, weeds are one of the most important factors reducing yield in soybean. The number of herbicides registered for soybean is very limited for the non transgenic production in the world as in Turkey. Consecutive applications of herbicides with the same mode of action in the same field may cause herbicide resistance in weeds and thus struggling with weeds are getting difficult gradually. Applying only one herbicide to control broad-leaf weeds along with narrow-leaf ones without damaging soybean is an important issue. From this perspective, it is aimed to develop soybean mutants resistant to sulfonylurea (SU) herbicides. For this purpose, 0.1% EMS was applied to 10.000 seeds of the Arısoy soybean variety and 4150 M1 plants were grown. 300.000 M2 plants obtained from the M1 population were tested for resistance to the SU group herbicide. In order to accelerate the process and ensure reliability in the tests, M2 plants were also screened under the hydroponic system beside the field conditions. 7 plants from open field conditions and 3 plants from hydroponic system were selected as potential herbicide resistant mutants. These were furher evaluated for confirmation in hydroponic system and 1 mutant come from open field condition confirmed as a herbicide resistant. This resistant source were also exposed to 60 ppm and 90 ppm doses which were 2 and 3 times more than the optimum dose of herbicide to see the resistance level and the mode. The resistant mutant showed no symptoms again in this higher dosess. Analyses at molecular level were conducted to determine changes in the ALS gene of the resistant source. For this purpose, the resistant plant was selfed and 8 progenies were grown. 2 out of 8 progenies were susceptible to the herbicide while the remains were resistant to the herbicide. DNA sequence analysis indicated that 8 point mutations were detected and 7 sense amino acid changes were found in these six resistant progenies. According to the ALS gene protein sequence of the Arabidopsis thaliana model plant, tyrosine-154 serine (Tyr154-Ser), alanine-155 valine (Ala155-Val), valine-163 aspartate (Val163-Asp), proline-197 serine (Pro197-Ser), glycine-609 valine (Gly609-Val), threonine-616 methionine (Thr616-Met) and glutamine-644 histidine (Gln644-His) amino acid changes were founded in these resistant plants. Only one common mutation in six mutant progenies is the proline-197 serine alteration at point 197 according to the Arabidopsis thaliana ALS gene sequence. By these data, herbicide resistance was confirmed by both molecularly and phenotypically. It has been determined that the resistance obtained by EMS mutation provided target site resistance.