Embryogenic Cell Culture, Protoplast Regeneration, Cryopreservation, Biolistic Gene Transfer and Plant Regeneration in Japanese Cedar (Cryptomeria japonica D. Don)

Somatic embryogenesis in Cryptomeria japonica was initiated at a relatively high frequency from immature seeds collected from the end of June to mid-July. Induction of embryogenic cultures was possible on media with or without plant growth regulators, and the initiation frequency varied from 5 to 16%. Embryogenic cell lines have been maintained and proliferated for more than 2 years in solid and liquid media. For long-term storage embryogenic cells were cryopreserved using a simple freezing method. Cotyledonary embryos were obtained mostly on maturation media containing abscisic acid (ABA) and polyethylene glycol (PEG) as osmotic agent, however, the plant conversion rate was still low. Plants regenerated from somatic embryos continued growing in a greenhouse. Furthermore, a procedure for the individual culture of protoplasts isolates from embryonal masses, and an approach for microprojectile bombardment-mediated transformation using pIPT and pMAT vectors was also described.