Regeneration of Ceratozamia euryphyllidia (Cycadales, Gymnospermae) plants from embryogenic leaf cultures derived from mature-phase trees.

Embryogenic cultures were induced from pinnae removed from young leaf flushes of mature-phase trees of the endangered cycad species, Ceratozamia euryphyllidia. Induction media consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l^-1 glutamine, 100 mg l^-1 asparagine, 100 mg l^-1 arginine, 60 g l^-1 sucrose, 2 g l^-1 gellan gum, 4.65-13.94 μM kinetin and 4.52-9.05 μM 2,4-dichlorophenoxyacetic acid. Cultures were maintained in darkness. Embryogenic cultures were comprised of precotyledonary somatic embryos that proliferated by somatic polyembryogenesis following subculture onto medium without plant growth regulators. Somatic embryo development and maturation occurred spontaneously from proliferating cultures on medium without plant growth regulators. Somatic embryos were monocotyledonous and mature somatic embryos germinated on semisolid medium without growth regulators. Subsequent development, which included the elongation of the first leaves, occurred only after subculture onto semisolid medium without plant growth regulators containing 0.5% (wt/vol) activated charcoal and under low light intensity. The time period from explanting to plant recovery was approximately 3 years.