Enhanced Somatic Embryogenesis and Plant Regeneration from Embryogenic Cultures Derived from Mature Loblolly Pine Zygotic Embryos by Suspension Culture and Medium Selection

Mature zygotic embryos of three families of loblolly pine (Pinus taeda L.)were cultured on callus induction medium containing 8 mg·L^-1 2, 4-dichlorophenoxyacetic acid (2, 4-D), 4 mg·L^-1 6-benzyladenine (BA), 4 mg·L^-1 kinetin (KT), 500 mg·L^-1 casein hydrolysate, and 500 mg·L^-1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub-cultured on the callus proliferation medium with 1.6 mg·L^-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.8 mg·L^-1 6-benzyladenine (BA), 0.8 mg·L^-1 kinetin (KT)for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM)and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16.9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole-butyric acid (IBA), gibberellic acid (GA3), BA, and activated charcoal and being lowered sucrose concentration for 4~12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy-one regenerated plantlets were transferred to a perlits:peatmoss:vermiculate (1:1:1)soil mixture, and 23 plantlets survived in the field.