PROTOPLAST ISOLATION AND CULTURE OF KENAF (HIBISCUS CANNABINUS L.)

Abstract

Protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars [Everglades 41 (E41), E71, Guatemala 4 (G4), G45, G51, and Tainung 1]. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin (1% w/v; Calbiochem) plus macerase (0.5% w/v; Calbiochem), with a 24 hour digestion at 30°C in the dark. Yields reached 7.2 (10)6 protoplasts/g leaf tissue. Protoplast viabilities ranged from 65% to 96%. Minor cultivar differences were observed related to protoplast yield, but all viability estimates were in an acceptable range. Greatest cell division frequencies and plating efficiencies were obtained when protoplasts were initially cultured in liquid medium at a density of 1.0 (10)5 protoplasts/ml. Electrofusion protocols were developed for kenaf protoplasts testing the range from 1200 to 3000 V/cm. A fusion voltage of 2000 V/cm yielded the highest fusion frequency and retained viability above 80%.