Research On Establishment Of High Efficient Regeneration System In Vitro And Its Endogenous Hormones Changes Of Larch

Larch is one of main afforestation tree species in north China. There are some limitations with the conventional breeding methods, such as breeding period is long, response is slow efficiency, offspring traits can’t be orientation improvement and so on, so it is necessary to establish rapid propagation system for the purpose of shorten the breeding period and orientation improving the offspring traits. The research based on the mature zygotic embryos of Larch, studying on vitro regeneration of hybrid larch and Larix olgensis and endogenous hormones changing, and the vitro regeneration systems of Larch were established. It established foundation for further research on larch genetic transformation. The main results were as follows:1. Establishment of highly efficient in vitro regeneration system of Hybrid larchThe research based on the mature zygotic embryos of Hybrid Larch(L.gmelinii 10×L.kaempferi 13), Study on the effect of various factors of regeneration from explants mature embryos of hybrid larch and efficient regeneration system was firstly developed.The results showed that BM medium was suitable for adventitious bud induction of Hybrid Larch and induction rate was 60.7%. Under the condition of combination BM and 1.5 mg·L-1 TDZ, induction rate was the highest,67.73%.BM medium could promote the proliferation of adventitious buds and the value-added coefficient is 4.48, when it was cultured on BM+1.0 mg·L-1 TDZ, the coefficient reached 5.23. The active carbon at the level of 2 g·L-1 can also elongation. When the adventitious buds were cultured on the condition of 1/2BM+0.05 mg·L-1 6-BA+0.2 mg·L-1 NAA, the pumping stems rate was the highest, up to 73.96% and 29.85% was the highest rooting rate when meet with the condition of 1/2BM+0.5 mg-L"1 IBA+1.0 mg·L-1 NAA. Survival rate of regeneration plant reached 62.5% after being transplanted.2. Establishment of highly efficient in vitro regeneration system of Larix olgensisThe establishment and optimization of regeneration system of Larix olgensis of Jixi provenance using mature zygotic embryos were studied.Explants sterilization, callus induction, adventitious bud induction and proliferation were studied by different disinfection methods, culture medium and hormone combination. Effects on elongation of adventitious buds were studied by different concentrations of GA3 and activated carbon. The rooting was studied by inner tubes and outer tubes two methods. Peeled seeds disinfected 15 minutes by 4% NaClO and embryos disinfected 2 minutes by 0.5% NaClO was the best method; The induction rate of callus was the highest in BM medium with 2.0 mg·L-1 TDZ, which was 88.9%; The induction rate of adventitious buds was the highest BM medium with 1.5 mg·L-1 TDZ, which was 80.0%; When it was cultured on BM with 1.0 mg·L-1 TDZ, the proliferation coefficient reached the highest at 4.98; GA3 had a toxic effect on elongation of adventitious buds, but 2.0 g·L-1 activated carbon had a good effect on it and the elongation rate reached 261.76%; When it was cultured on 1/2BM medium with 0.5 mg·L-1 IBA and 1.0 mg·L-1 NAA for the in vitro rooting, the rooting rate was 26.09%, but tube external rooting by dipping 25 seconds with 750 mg·L-1 NAA was the highest,37.14%.3. Study on Endogenous Hormones Changes During Vitro Culture of LarchUsing high performance liquid chromatography and mass spectrometry method(HPLC-MS/MS) the content changes of endogenous hormones in Hybrid Larch and Larix olgensis plantlet regeneration process has been studied. Results showed that the hybrid larch and Larix olgensis endogenous ABA and IAA content were higher and amplitude of variation was bigger, and IAA content overall showed high-low-high change rule; while the endogenous ZT and GA3 content was relatively low, but the changes of content in the different stages in the process of adventitious bud growth changes was obviously. Changes of endogenous hormone content differences may be related to its genetic characteristics in Hybrid Larch and Larix olgensis. IAA is one of indispensable endogenous hormones in the process of larch plantlet regeneration; ZT, GA3 can accelerate larch growth and differentiation.