Establishment of an effective protocol for cultivation of tissue cultured seedlings in Pinus massoniana superior provenance

ObjectivePinus massoniana is a main tree species for ecological construction and timber in southern areas of China. However, the productivity of plantation is generally low resulting from the limitation of improved varieties, which leads to the slow development of industrialization in P. massoniana. It is necessary to develop an effective propagation system of elite germplasm for P. massoniana in order to promote the use of improved varieties, accelerate the industrial development, and enhance the competitiveness of industry.

MethodIn this study, ‘Tongmiansong’ (TM), the backbone of P. massoniana breeding resources was used as the research object, zygotic embryos excised from immature cones were applied to explants, and mature somatic embryos (SE) obtained via somatic embryogenesis were used for testing materials. Concerning the technical bottlenecks of P. massoniana tissue culture, including low germination rate, poor shoot growth, and recalcitrance to rooting, effects of active charcoal (AC), basal media, and plant hormones on SE germination as well as reinvigoration and adventitious rooting of shoots were investigated in the present study.

Result(1) AC significantly improved germination of TM mature SE, while a high level of AC was able to weaken their germination effects, and the best effect was observed at the 0.83 mol/L AC treatment. Based on the application of AC in the medium, the basal medium composed of high N, low ratio of NH₄⁺/NO₃⁻ , and moderate K and Ca furtherly enhanced the germination of SE, reaching 94.1% of germinating rate. (2) 0.42 mol/L AC effectively promoted the elongation of germinated SE. Under the treatment of 4 μmol/L TDZ, induction of axillary buds was better, achieving effective bud proliferation coefficient of 5.6/35 d, and shoot height of 9.2 cm/50 d. (3) After 60 days of 1.2 μmol/L NAA + 2 μmol/L PBZ application in the rooting medium, rooting rate was 94.3%, root number was 6.4, and survival rate was 95.8% after 3-month transplanting.

ConclusionThe effective breeding system by tissue culture for TM was firstly established via a combined approach of somatic embryogenesis and organogeneis in this study, which would be used for the rapid propagation of elite germplasm for P. massoniana as well as for the research on genetic transformation, providing solid foundation for industrialization of improved varieties and molecular breeding in P. massoniana.