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SOMATIC EMBRYOGENESIS IN PROTOPLAST CULTURES DERIVED FROM MESOPHYLL AND EMBRYOGENIC CALLUS OF SUGARCANE (SACCHARUM SPP. HYBRID CV. COL-54)

FAHEEM AFTAB AND JAVED IQBAL

Abstract: Protoplasts were isolated both from leaf mesophyll tissue as well as from compact globular white embryogenic callus of mesophyll origin in sugarcane (Saccharum spp. hybrid cv. COL 54). Totipotent protoplasts with an yield of 1x10⁶ and1x10⁵ ml^-1 with 72 and 68% viability were obtained respectively from mesophyll and embryogenic callus cultures. A heterogeneous population of protoplasts (30-60µm Ø) was obtained from both sources. Protoplasts isolated from compact globular white embryogenic callus were less heterogeneous (30-40µm Ø)and cytoplasmically dense. Of the several protoplast 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 differentiation of somatic embryos was not possible. Protocallus formation from protoplasts isolated from embryogenic callus cultures was achieved in dark using agarose-solidified KPR medium in 16 weeks. Shoot formation was achieved from such protocalluses by further transferring to MS basal medium containing 9µmol (2 mg^-1) 2,4D for 8 weeks under 16h photoperiod condition. Transfer of such proliferated callus masses to MS basal medium containing 9.29µmol (2 mg^-1) Kinetin + 5.37µmol (1.0 mg^-1) NAA and 200 mg^-1 activated charcoal for 10 weeks resulted in shoot formation.

Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.