Development of a microspore culture method to produce haploid and doubled-haploid asparagus (Asparagus officinalis L.) plants

The effects of genotype, cold pretreatment of flower buds and microspore isolation method were investigated for asparagus microspore culture. Binucleate microspores resulting from symmetric rather than asymmetric pollen mitosis I were observed after seven days cold treatment of flower buds and divided further in vitro after being shed from anthers. Significant correlations were found among the percentage of late-uninucleate microspores in the anther before cold treatment, the frequency of symmetric pollen mitosis after seven days of cold treatment and yield of microspore derived calli. Approximately 30% viable microspores were produced from two mechanical isolation methods, homogenization and microblending with viable microspore enrichment on Percoll gradients, while shed microspore culture generated more than 70% viable microspores. Calli were obtained only from shed microspore culture. Genotypes responded differently to shed microspore culture; responses for six genotypes were 144, 60, 56, 41, 4 and 0 calli produced per 100 anthers cultured.