Development of highly tissue culture responsive lines of Lolium temulentum by anther culture

Most of the important forage and turf grasses are outbreeders, require vernalization to flower, and in some cases are polyploid. With the development of gene sequencing information in grasses, there is an urgent need for a model system to perform large-scale functional analysis of candidate genes. We propose to use Lolium temulentum L. (Darnel ryegrass) as a model system for genetic manipulation studies in forage and turf grasses because L. temulentum has the following advantages: self-fertile, short life cycle (11 weeks), diploid, easy to grow, and closely related with other major grass species. In order to improve tissue culture response of L. temulentum, two relatively responsive lines were crossed and putative hybrid seeds obtained. Analysis of the F1 plants by SSR markers confirmed that the F1 plants were true hybrids. Anthers were dissected from F2 plants of the cross, 3.0% of the cultured anthers responded with the formation of calluses. Green haploid plants were recovered from 48.9% of the anther-derived calluses. Seeds were harvested from doubled haploid plants and mature embryos were used as explants for comparing tissue culture responses with other lines. Besides anther culture, F2 seeds of the cross were subjected to a cycle of selection for callus formation and plant regeneration, and F4 seeds were obtained from the regenerated plants. Comparison of tissue culture response of different lines revealed that the doubled haploid plants had a much higher frequency of embryogenic callus formation than that of the parental lines and the F4 seeds. This is the first report on the generation of green haploid and doubled haploid plants in L. temulentum. The technique was successfully used for the rapid production of homozygous diploid plants that are highly tissue culture responsive. The anther culture-derived new L. temulentum lines could be valuable material for functional test of genes in grasses.