Isolation, Culture, and Plant Regeneration From Echinacea purpurea Protoplasts

A plant regeneration system from the isolated protoplasts of Echinacea purpurea L. using an alginate solid/liquid culture is described in the chapter. Viable protoplasts were isolated from 100 mg of young leaves of 4-wk-old seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase, and 0.3 mol/L mannitol. After isolation and purification, the mesophyll protoplasts were embedded into 0.6% Na-alginate at the density 1 � 10⁻⁵ mL and cultured in modified Murashige and Skoog (MS) culture medium supplemented with 0.3 mol/L sucrose, 2.5 �mol/L benzylaminopurine (BA), and 5.0 �mol/L 2,4-dichlorophenoxyacetic acid (2,4-D ). The visible colonies were present after 4 wk of culture. The protoplast-derived clones were transferred onto gellan gum-solidified basal medium supplemented with 1.0 �mol/L BA and 2.0 �mol/L indole-3-butyric acid (IBA) and formed compact and green calli. Shoot development was achieved by subculturing the calli onto the same basal medium supplemented with 5.0 �mol/L BA and 2.0 �mol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets.