Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.)

A procedure is described to regenerate plants from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Somatic embryogenic calli were induced from ginger shoot tips on solid MS medium with half the concentration of NH4NO3 and supplemented with 1.0 mg l−1 2,4-Dichloroacetic acid (2, 4-D) and 0.2 mg l−1 Kin. Rapid-growing and well-dispersed suspension cultures were established by subculturing the embryogenic calli in the same liquid medium. Protoplasts were isolated from embryogenic suspensions with an enzyme solution composed of 4.0 mg l−1 cellulase, 1.0 mg l−1 macerozyme, 0.1 mg l−1 pectolyase, 11% mannitol, 0.5% CaCl2 and 0.1% 2-(N-morpholino) ethane sulphonic acid (MES) for 12–14 h at 27°C with a yield of 6.27 × 106 protoplasts g−1 fresh weight. The protoplasts were cultured initially in liquid MS medium with 1.0 mg l−1 2, 4-D and 0.2 mg l−1 Kin. Then the protoplast-derived calli (1.5 cm2) were transferred to a basal MS medium containing 0.2 mg l−1 2, 4-D, 5.0 mg l−1 benzyladenine (BA), 3% sucrose and 0.7% agar. The white somatic embryos were transferred to MS medium lacking growth regulators for shoot development. Shoots developed into complete plantlets on a solid MS medium supplemented with 2.0 mg l−1 BA and 0.6 mg l−1 α-Naphthaleneacetic acid (NAA). In addition, the effects of AgNO3, activated charcoal (AC) and ascorbic acid (AA) on browning of protoplast-derived calli are discussed.