ABSTRACT
An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1+/-2.1x10(6)/g FW) and viability (91.8+/-3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl(2).2H(2)O, and 0.011% (w/v) NaH(2)PO(4).2H(2)O. First divisions occurred 7-10 days following culture initiation. The highest division frequency (24.6+/-2.9%) and plating efficiency (6.88+/-0.8%) were obtained in liquid medium (MS) supplemented with 30 g l(-1) sucrose, 0.7M glucose, 0.1 mg l(-1) NAA, 1.0 mg l(-1) BA, and 1.0 mg l(-1) GA(3). After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l(-1) 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.
