Somatic embryogenesis in protoplast cultures derived from mesophyll and embryogenic callus of sugarcane (Saccharum spp. hybrid cv.COL-54)

Protoplasts were isolated both from leaf mesophyll tissue as well as from c ompact globular white embryogenic callus of mesophyll origin in sugarcane ( Saccharum spp. hybrid cv. COL 54). Totpotent protoplasts with an yield of 1 x10(6) and 1x10(5) ml(-1) with 72 and 68% viability were obtained respectiv ely from mesophyll and embryogenic callus cultures. A heterogeneous populat ion of protoplasts (30-60 mu m phi) was obtained from both sources. Protopl asts isolated from compact globular white embryogenic callus were less hete rogeneous (30-40 mu m phi) and cytoplasmically dense. Of the several protop last culture methods tried, the best results were achieved using protoplast embedding in agarose-solidified medium. Medium P9 proved good for somatic embryogenesis from mesophyll-derived protoplasts in which further different iation of somatic embryos was not possible. Protocallus formation from prot oplasts isolated from embryogenic callus cultures was, achieved in dark usi ng agarose-solidified KPR medium in 16 weeks. Shoot formation was achieved from such protocalluses by further transferring to MS basal medium containi ng 9 mu mol (2 mgl(-1)) 2,4-D for 8 weeks under 16h photoperiod condition. Transfer of such proliferated callus masses to MS basal medium containing 9 .29 mu mol (2 mgl(-1)) Kinetin + 5.37 mu mol (1.0 mgl(-1)) NAA and 200 mgl( -1) activated charcoal for 10 weeks resulted in shoot formation.