Development of an asymbiotic reference line for pea cv. Bohatyr by de novo mutagenesis

Comparison of asymbiotic and symbiotic pairs of lines (difference technique) is a reliable and accessible method for determining the contribution of symbiotic nitrogen fixation to the yield of legume crops. To obtain an isogenic reference line for a commercial pea (Pisum sativum L.) a mutagenesis program was initiated with cv. Bohatyr. Ethyl methanesulfonate (EMS) treatment of large seeds was optimized in a closed system to scale-up the process and to improve safety. The 18-h exposure of non-presoaked seeds to 0.15% (w/w) EMS solution resulted in 30% field emergence in the M, generation and in 1.04% of chlorophyll mutants and 3.64% of mutants classified as symbiotic in the M-2. However, only 48.4% of the growth chamber-detected and 21.6% of the field-detected presumable symbiotic mutants were found to be stable genetic deviations in the M-3 to M-5. Twenty-two preliminary characterized stable mutants comprised one nonnodulating, one with reduced nodulation, one with markedly delayed nodulation, and 19 lines with affected nodule development and function. Further evaluation under controlled conditions and in field trials identified the nonfixing line Budfix2 as a suitable reference line for field estimates of symbiotic nitrogen fixation. This line was shown to be physiologically equivalent to the original cultivar when grown under asymbiotic conditions, i.e., when the symbiotic fault was compensated for by exogenous mineral nitrogen. The availability of Budfix2 allows for direct application of the difference method in the particular pea cultivar Bohatyr, as well as in further varieties after trait introgression. The additional symbiotic mutants generated in the course of the search for a reference line are expected to enhance the variability available for the genetic dissection of rhizobial nodulation.