Somatic embryogenesis and plantlet regeneration in Pinus armandii var. Amamiana

Abstract

ABSTRACT Embryogenic cell suspensions were induced from a mature seed of Pinus armandii var. amamiana known as an endangered species. MS liquid medium supplemented with 10 μ M 2,4-D and 3% sucrose was used for initiation of the suspensions. The suspensions were incubated in the dark at 24 °C. Obtained suspensions were transferred to NH4NO3-free MS liquid medium supplemented with 10 μ M 2,4-D, 3 mM L-glutamine and 3% sucrose and subcultured every 2 weeks. In order to develop somatic embryos, the suspensions were transferred into NH4NO3-free MS media supplemented with 3% sucrose, 100 μ M ABA, 0-20% PEG (MW4,000), 0-20 mM L-glutamine and 0.2% activated charcoal. After 1-2 weeks of incubation the suspension cultures were transferred onto NH4NO3-free media solidified with 0.4% gellan gum containing 100 μ M ABA, 0.2% activated charcoal, 0-10% PEG and 0-20 mM L-glutamine. The cultures were incubated under a 16 h light / 8 h dark photoperiod. After 1-2 momths of culture, differentiation of embryos progressed and cotyledonary embryos were obtained. These embryos were transferred on NH4NO3-free solid MS media containing 0-10 mM L-glutamine and incubated at 24 °C and under a 16 h light / dark photoperiod. After 2-3weeks some plantlets with roots and green cotyledons were obtained. Plantlets were transplanted into vermiculite containing modified MS liquid medium in 200 ml culture flasks. This is the first report of somatic embryogenesis and plantlet regeneration in P. armandii var. amamiana.