Host: The Japanese Forestry Society
Somatic Embryogenesis and plant regeneration from immature seeds of Pinus armandii var. amamiana, a species threatened with extinctionEmilio Maruyama, Yoshihisa Hosoi, and Katsuaki Ishii (Forestry and Forest Products Research Institute)Pinus armandii var. amamiana (Yakutanegoyou) is a five-needle pine species endemic to Japan. The natural population of Yakutanegoyou is restricted to the Tanegashima and Yaku Islands (Kagoshima Prefecture). Because, it is believed that the natural population is only 1,500 to 2,000 individuals, this species has been registered as endangered species in the Red Data Book of Japan Wildlife1). In situ and ex situ conservation of this species has being considered as a prior importance. So, we try to reproduce this species by tissue culture techniques through somatic embryogenesis as an alternative of ex situ conservation.Somatic embryogenesis in Pinus armandii var. amamiana was initiated from immature seeds collected from early July. Collected seeds were washed with 100% ethanol before disinfection in 2.5% (w/v available chlorine) sodium hypochlorite solution for 30 min. Then, seeds were rinsed with sterile distilled water. After seed coats had been removed, the megagametophytes that containing immature zygotic embryos were cultured on EM1-2) medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Embryogenic cells were transferred to fresh medium and subcultured at three-week intervals in the same medium. After proliferation, embryogenic cells were transferred to EM media containing maltose and abscisic acid (ABA), and supplemented with and without polyethylene glycol (PEG) and activated charcoal. After two months of culture, the best result regarding to the number of somatic embryos per Petri dish was obtained on medium containing maltose, ABA, PEG, and activated charcoal. Cotyledonary somatic embryos were transferred to EM medium without plant growth regulators. About 50% germination frequency was achieved. Germinated embryos that showed epicotyl growth were transferred into vessels containing vermiculite or fluorialite substrate fertilized with modified Nagao's plant food solution1-2) and kept in culture room before ex vitro acclimatization.1) Environment Agency of Japan (2000)Threatened wildlife of Japan -Red Data Book 2nd ed.-, Japan Wildlife Research Center2) Maruyama, E. et al. (2000) Plant Biotech. 17: 281-2963) Maruyama, E. et al. (2002) J. For. Res. 7:23-34
