Preliminary Study On Tissue Culture In Pinus Densiflora Var. Zhangwuensis

Pinus densiflora var. zhangwuensis is a natural hybrid which was found by Sandfixation and Silviculture Institute of Liaoning province in1990in a man-made forest of P. sylvestris. It is one of the superordinary windbreak and sand afforestation species in northern China due to its fast growth, good shape, and high resistance to soil infertility and pest. Since its discovery, some researches on the origin, morphology, physiology, ecology and propagation of this species have been carried out. The species has been extended to Liaoning, Gansu, Shanxi, Shaanxi, Jilin, Heilongjiang and other provinces or autonomous regions in Three-North by using grafting. There is only small seed and cutting orchards which can’t supply enough propagation materials for silviculture of the hybrid. Grafting is the only method used in the forestry practice. However, grafting needs rootstock from3～5year-old P. sylvestris seedling and very strict technical requirement. Thus it is difficult to achieve rapid propagation. Therefore, it is of important practical significance for propagation of P. densiflora var. zhangwuensis to develope in vitro culture system.In this present study, dormant buds or new shoots were used as explants to in vitro propagate P. densiflora var. zhangwuensis. The influences of culture medium, PGR and other factors on the induction of axillary bud and callus, elongation and rooting of axillary shoot. This present study aimed at exploring the feasibility of propagating this new hybrid by tissue culture. The study results are as follows:(1) Different explants need different sterilization treatments. Treating with70%ethanol for30s following with0.1%HgCl2for5min is better for dormant bud explants. While new shoots need to be sterilized with0.1%HgCl2for7min.(2) Medium composition have a significant effect on the induction of Organogenesis. In the three basic media tested, WPM medium containing NH4NO3has the highest induction frequency (73.62%) of axillary bud. But the rates of induction on WPMG and MSG media lacking NH4NO3are only15.47%and0.98%, respectively.(3) BA, ZT and TDZ at low concentration induced organogenesis to some extent. While KT only induced axillary bud sprouting. The induction frequency of axillary bud on WPM medium containing2mg/L BA could reach57.5%, significantly higher than the frequency at1mg/L BA. But there was no significant difference on the induction frequency between2mg/L BA and3-5mg/L BA. Adding NAA at low concentration to the medium did not significantly improve the induction of organogenesis.(4) Sucrose, agar and other additives also influence the induction frequency of organogenesis to some extent. On WPM medium,30g/L sucrose,800mg/L NH4NO3,7g/L agar and1g/L inositol resulted in higher induction frequency than at other concentrations tested.(5) Collection time of explant has a significant influence on the induction frequency of organogenesis in P. densiflora var. zhangwuensis. Explants collected from October to next Aapril all generated axillary bud sprouting and axillary shoot induction to different extents. The best induction effect was obtained on dorminant buds collected from late October to December. New shoots collected in Apiral had significant lower induction rate of organogenesis than that of dorminant buds.(6) Axillary shoots obtained elongation and proliferation after subculture on WPM medium with or without PGR alternately. Adding1g/L activated carbon to the subculture medium is conducive to the elongation of the axillary shoots. While casein hydrolyzate and L-glutamine did not promote organogenesis significantly.(7) Combination of BA and NAA at suitable concentrations induced callus occuring at a rate of100%. Most of the calli are yellow-green and block or granular. But the current study failed to induce adventitious bud differentiation from the calli.