Establishment Of Vitro Culture System Of Oplopanax Elatus Nakai

Oplopanax elatus Nakai (O. elatus), a member of Oplopanax genus Araliaceae family, is used as a folk medicine in Russia and China for its excellent pharmacological activities in preventing and curing many diseases. Due to sharing some similar pharmacological functions to Panax ginseng, it is also known as the "woody ginseng". Besides, it has now been listed as a national second-grade protection plant, owing to a shortage of its resources. Fortunately, with the rapid development of the plant tissue culture technology, the callus, suspension cells and plant regeneration become the effective way to protect the endangered medicinal plants. Therefore, studies on tissue culture technology of O. elatus are of important theoretical and practical value for resources protection and rational utilization. To solve these urgent questions mentioned above, we successfully established a culture system of O. elatus in vitro.Based on different explants, we inducted callus that had different differentiation abilities. The morphology and cytology of different types of callus were compared in this study. Moreover, the culture conditions of callus were optimized by response surface method, which was prepared for suspension culture system of O. elatus. And, the growth dynamics of callus and suspension cells were also studied. Finally, we explored the methods of embryonic callus regeneration and cryopreservation. Our main conclusions are as follows:1. Callus induction and optimization of callus culture conditionsIn our study, we find the optimum explant was five-day-old leaf for callus induction of O. elatus and the induction rate reached 83%. Besides, the optimum culture medium was MS medium supplemented with 1 mg/L NAA and 1 mg/L 6-BA. In this culture, the callus induction rate was up to 95%. In addition, the callus growth cycle was an s-shaped curve type and biomass peaked at 32 d. And, the total sugar content of callus was higher than the reducing sugar content. Meanwhile, the sugar content in growth cycle of callus was raised at beginning and fell into balance at last.The best subculture conditions of O. elatus callus were white light,24 ℃. pH 6. 40 g/L sucrose,1 mg/L NAA,1 mg/L 6-BA. After using the response surface method, the best culture conditions were 1.07 mg/L NAA,0.94 mg/L 6-BA,39.44 g/L sucrose. And the predicted value is 7.93199 g, while the actual value was 8.2 g.2. Establishment of suspension culture systemWe successfully established the suspension cell system of O. elatus. The growth curves of suspension cell were all s-shaped, when the inoculation amount of the cells were 2 g,1 g.0.5 g, respectively. However, the cell biomass achieved the highest after 20 d cultivation when the inoculation amount of the cells was 2 g. In this culture. pH showed a trend of increase and then decrease after the first drop, and nitrate nitrogen content was continued to decline. Meanwhile, the results showed that the most appropriate condition for biomass and proliferation of suspension cell were pH 6,40 g/L sucrose,2-2 mg/L NAA-6-BA, without yeast extract and hydrolysis casein.3. Induction, regeneration and cryopreservation of embryonic callusThe optimum explant for embryonic callus induction was seed and the induction rate reached 50%. Moreover, the optimum induction medium was MS medium supplemented with 1 g/L glutamine,1 mg/L 2,4-D,0.5 mg/L 6-BA,30 g/L sugar, and 7 g/L AGAR, while the optimum regeneration medium was MS medium supplemented with 1 g/L glutamine,1 mg/L GA,0.5 mg/L 6-BA,30 g/L sucrose and 7 g/L AGAR.From the perspective of cytology, embryonic callus cells were very small, arranged closely and were rich with nucleus. Moreover, embryonic callus cells exhibited high cytoactive and differentiation ability, and some of them differentiated into tracheid. However, callus cells had less nucleus, big vacuole, less cytoplasm and low differentiation capacity, which had different degree of crushing.The optimum time pretreatment 0.4 mol/L sorbitol was 24 h, and the best cryopreservation agent was 5% DMSO+5% glycerin+0.4 mol/L sorbitol. Then, after being cryopreserved for 1d,7 d and 30 d, the cell activity of callus was 50.333%, 33% and 50.333%, respectively.