| Gymnocarpos przewalskii is a relic species of the ancient Mediterranean xerozoic flora,which has the characteristics of drought resistance,salinity tolerance,resistance to wind erosion and sand burial.It is one of the important woody species in rocky desert areas.In order to explore the key factors for the establishment of tissue culture and regeneration system of Gymnocarpos przewalskii,and to determine the origin of regenerative adventitious shoots,the stem segments of the healthy and strong branches from Gymnocarpos przewalskii were used as the explants,and the combination of different concentration from 6-BA and IBA for tissue culture were taken in this experiments.The optimal combination of hormone concentrations for callus proliferation and the key influencing factors for adventitious shoot differentiation and its rooting induction were confirmed,then the regeneration system was established.To confirm the shoot origin from the tissue,the process of adventitious shoot differentiation was analyzed.The main findings are as follows:(1)The best disinfection time for Gymnocarpos przewalskii stems was 3% sodium hypochlorite for 24 min in 2 times,then the 72%±2.6 probability of sterile stems was gained successfully.Callus induction rate can get to 90% after initial culture.(2)During the callus proliferation culture of Gymnocarpos przewalskii,there were three differentiation directions appeared,which were the dark yellow-green callus,half-translucent yellow-white callus with the differentiated root on the surface,and yellow-green callus with the differentiated shoots in it.(3)The best callus proliferation medium was MS+1 mg/L IBA+1 mg/L 6-BA+ 30g/L sucrose+ 7 g/L agar.After 4 weeks’ culture for proliferation,the yellow-green callus formed and grew vigorously with a diameter of 2.72 cm.The effect of IBA was more critical than that of 6-BA,and the callus size increased and then decreased with the increasment of IBA concentration.(4)The best adventitious shoot induction medium was MS+0.5 mg/L 6-BA+30 g/L sucrose+7 g/L agar.After 21 days culture,the adventitious shoots height increased to 0.5cm,and the number of induced adventitious shoots was 4.4 ±0.5/block,6-BA and IBA were the key influencing factors.(5)The best rooting medium was SH+0～10 g/L sucrose+7 g/L agar.The stem growth height got to 5 cm and milky white well-developed adventitious roots emerged with the 91.3%±0.5 rooting rate.The basic medium and sucrose concentration are the key factors in this process.Lesser callus were induced from SH medium during rooting,which is beneficial to rooting.Low concentration or no addition of sucrose can effectively reduce the callus regeneration during rooting.Low concentrations of IBA and NAA can promote rooting.(6)The most suitable substrate for seedling cultivation was 55% humus and 45%perlite,and the survival rate got to 78%.The plant grows with the well-developed condition,and there are rarely the roots rot in this process.The healthy plant can be obtained.(7)Anatomical structure analysis of adventitious shoots regeneration process from callus showed that adventitious shoots originated from meristematic cells in the surface layer of vascular nodules,which were extraneous origins.In this study,the regeneration system of Gymnocarpos przewalskii was established through the organogenesis pathway,and the extrinsic origin of adventitious shoots was confirmed.This thesis is of great value for the protection of the biological resources of Gymnocarpos przewalskii,and provides a research basis for its rapid reproduction and large-scale utilization,and also provides a new way for the protection and reproduction of this rare and endangered plant. |
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