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%.

Protoplast isolation and culture protocols were developed for ten cultivars of Hibiscus cannabinus L. (kenaf). Leaves from seedling lines maintained in vitro were used as donor tissues. Optimal cell wall digestions were achieved with a combination of cellulysin (1.0%) and macerase (0.5%). Average yields ranged from 0.9× 105 to 5.9× 106 protoplasts g fw -l leaf tissue with viability estimates ranging from 53% to 87%. This protocol was ineffective for leaf tissue taken from plants grown in vivo. Protoplasts harvested from plantlets maintained in vitro produced rapidly growing calluses when plated in semi-solid medium after an initial culture in liquid medium. First cell divisions were observed within four to six days after initial culture in medium containing plant growth regulators 2,4-dichlorophenoxyacetic acid (1.4/~M) and kinetin (13.8/~M). An electrofusion protocol which did not significantly reduce protoplast viabilities was developed for kenaf protoplasts. The maximum fusion frequency (4.6%) was obtained with an electrofusion voltage of 2.0 kV cm -I.