Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp hybrid cv CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp. hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mu mol (0.5 mg l(-1))picloram under dark conditions at 27+/-1 degrees C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mu mol (2 mg l(-1)) 2,4-D and 500 mg l(-1) CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l(-1) sucrose, 500 mg l(-1) CH and 2.26 mu mol (0.5 mg l(-1)) 2,4-D after 4-6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l(-1)) and sucrose (60 g l(-1)). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium. Totipotent protoplasts with an average yield of 2.0x10(7) to 1.0x10(8) ml(-1) were obtained from embryogenic cell suspension cultures at log phase, i.e., 4-5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0x10(5) m l(-1). Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-D (2 mg l(-1)) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium + 9.29 mu mol kinetin (2 mg l(-1) + 5.37 mu mol NAA (1.0 mg l(-1)) + activated charcoal (200 mg l(-1)) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2-4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.