Anther culture and haploid plant regeneration in purple coneflower (Echinacea purpurea L.)

Anthers containing microspores at the uninucleate stage were excised from capitula of field grown plants of purple coneflower, Echinacea purpurea, and cultured on medium conducive to callus growth. In callus induction cultures, N6 basal medium was more effective than Murashige and Skoog (MS), and a combination of benzyladenine (BA) at 2.22 mu M with naphthaleneacetic acid (NAA) at 0.054 mu M was more effective than 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.52, 9.05 and 13.57 mu M. Callus induction rate as high as 85.8% was achieved, and no statistically significant differences were found among cultures with various densities of 20, 40, 60, 80 and 100 anthers per bottle each filled with 40-ml medium. Shoots were regenerated from calluses on MS medium containing 2.22 mu M BA and various concentrations of NAA, with the highest regeneration rate of 95.24% obtained when 0.27 mu M NAA was applied. Although a large portion of the regenerated shoots had prominent symptom of vitrification, some normal shoots could be easily rooted on MS medium containing 0.054 mu M NAA. Thirty regenerated plants were randomly selected and 19 of them were confirmed to be haploid by observation of chromosome number of root-tip cells.