Tissue Culture And Evolutionary System Analysis Of Endangered Plant Species-Betula Halophila In Xingjiang

Betula halophila, which distributed only in Xinjiang, faced to the dangerous situation of extinction. For this reason, it is very necessary for us to protect and prevent it from extinction. Regeneration and reproduction by establishing tissue culture system of Betula halophila may be a promising way. In this thesis, tissue culture technique of Betula halophila and all various factors which affect this system were studied, and finally we established Betula halophila regeneration system and the optimal combination of various factors in this system. We used stems and leaves as explants, investigated the effects of different components, such as basic media (MS,WPM,LS ,1/2MS ) , hormones (BA,KT,TDZ,NAA,2,4-D,IAA,IBA) , culture conditions , and explants on shoot propagation, callus induction, adventitious shoot differentiation and root inducing of Betula halophila. The system was established and optimized as follows: 1. The segments of shoot with bud and leaves of Betula halophila were taken as explants and cultured in vitro to induce the plant regeneration. Results showed that the immature stems are good explants of shoot multiplication; the multiplication medium is MS+TDZ1.0mg/L+IBA0.2mg/L; the medium for rooting is 1/2MS +IBA 0.5mg/L + sucrose 30g/L, after three or seven days in dark; then transferred to callus inducing medium LS+BA0.5 mg/L +NAA0.4 mg/L. 2. The segments of shoot with bud and leaves were used to induce high efficient callus differentiation and adventitious shoot regeneration. Results showed that the immature stems are good explants of callus induction and adventitious bud differentiation; callus differentiation medium is LS+BA0.5 mg/L +NAA0.02 mg/L; the best medium for high efficient callus induction and adventitious bud differentiation is LS+ BA0.5 mg/L + NAA0.2 mg/L; the rate of callus induction is 82%, and adventitious bud differentiation is 93.6%. 3. Four factors:type of culture medium, different concentrations of hormones, content of sucrose and illumination condition, were investigated for their effects on rooting of regeneration plantlets of Betula halophila in vitro by L27(313) orthogonal design experiment. The optimization media for rooting were as follows: MS +IAA 0.2mg/L + sucrose 30g/L,1/2MS +IBA 0.5mg/L + sucrose 30g/L + three or seven days in dark, MS + NAA 0.2mg/L + sucrose 10g/L . The sequences of nrDNA in ITS area (including ITS -1, 5. 8SrDNA and ITS -2) of Betula halophila were analyzed. Results showed that the difference of the sequence in ITS area between Betula halophila and other 14 Betula L. plants was not significant, the homology was from 100.0% to 96.7%. With DNAMAN analysis, the phylogenetic trees were constructed based on Betula halophila ITS sequences. The data set of ITS sequences from 15 Betula L. plants was divided into two classes, Betula halophila was belong to the same class with Betula alnoides,Betula pendula,Betula populifolia,Betula alba,Betula nana, and moreover, Betula halophila formed an independent sub-class within this class, it demonstrated that Betula halophila had its own distinctive classification status, and this study no doubt had significance in the evolution system research of Betula L..