Isolation, culture, and plant regeneration from Echinaceapurpurea protoplasts.pdf

Echinacea purpurea Protoplasts 211 211 From: Methods in Molecular Biology, vol. 318: Plant Cell Culture Protocols, Second Edition Edited by: V. M. Loyola-Vargas and F. Vázquez-Flota ? Humana Press Inc., Totowa, NJ 18 Isolation, Culture, and Plant Regeneration From Echinacea purpurea Protoplasts Zeng-guang Pan, Chun-zhao Liu, Susan J. Murch, and Praveen K. Saxena Summary 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 purifica- tion, 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 μmol/L benzylaminopurine (BA), and 5.0 μmol/L 2,4-dichlorophenoxy- acetic 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 μmol/L BA and 2.0 μmol/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 μmol/L BA and 2.0 μmol/L IBA. Further subculture onto basal medium resulted in the regeneration of complete plantlets. Key Words: Callus; Echinacea purpurea; medicinal plant; protoplast isolation. 1. Introduction Echinacea purpurea L. belongs to the family Asteraceae, and has been used extensively in medicinal preparations for the treatments of many dis- eases including colds, toothaches, snake bites, rabies, and wound infections (1). Echinacea preparations are among the best-selling herbs in North America, making up about 10–12% of the natural health product market with an annual value of about $33