An advanced protocol for the establishment of plantlets originating from somatic embryos in Pinus massoniana

This study describes critical factors affecting germination of somatic embryos and plantlet regeneration in Pinus massoniana. Somatic embryos from the same embryogenic line 27 of P. massoniana were used as test materials. The supplementation of activated charcoal (AC) in the medium was essential for the germination of mature somatic embryos, while the addition of excessive AC to the medium was prohibitive for somatic embryo germination. The highest germination rate was found on the medium containing 10 g/l AC, and the addition of 5 g/l AC to the medium was optimal to the growth of germinating somatic embryos. Thidiazuron (TDZ) was linearly related to the number of sprouting axillary buds. However, the growth of sprouting buds was retarded when > 4 µmol/l TDZ was added into culture medium. Exogenous plant growth regulators added to the medium significantly improved the root regeneration capacity of shoots. The highest root regeneration rate was observed under the treatment of 1.2 µmol/l ɑ-naphthaleneacetic acid (NAA) plus 2 µmol/l paclobutrazol (PBZ), reaching 96.3%. One year after the field transfer, the growth performance of plant height, caliper, and survival rate for rooted shoots was significantly better than that of plantlets directly developed via somatic embryogenesis. The presented results provide useful instruction for the establishment of plantlets originating from somatic embryos, and would be able to make a great contribution to the clonal forestry of P. massoniana.