Establishment Of Rapid Propagation Protocol In The Culture Of Dendrobium Shixingense And In Vitro Flowering

In2010Dendrobium shixing has been found in shixing county, northern of Guangdong, and now it had been found only a few areas of Guangdong and Jiangxi border distribution, appropriate elevation of about400～600m. In recent years, the resources of the wild Dendrobium was predatory acquisition, already has been facing endangerment and extinction situation.Used the Dendrobium shixingense seeds as explants. we select the optimal medium for germination of seeds, multiplication and differentiation of protocorms, multiplication of adventitious bud, protocorms induction and rooting of the plant in vitro. The based medium was MS and B5supplemented with different concentration of6-BA, KT, TDZ, IBA,NAA and AC. Then compared the different treatment on survival rate of plantlets. And determinate all kinds of kinetic parameters changes of suspension cultures of protocorms. At the same time, in vitro flowering by using plantlets produced with about4-month old tissue culture to study the effects of6-BA, TDZ, sucrose, nitrogen, photoperiod, temperature and light quality.The results showed that the optimal medium for germination of seeds was MS containing1.5mg·L-16-BA,0.6mg·L-1NAA and0.1%active carbon, in which germination rate was100%. For multiplication of protocorms it was MS containing0.8mg·L-16-BA,0.3mg·L-1NAA and4.5%Sucrose, the propagation coefficient was7.75. Differentiation rate was the best on MS medium containing2.5mg·L-16-BA,0.5mg·L-1NAA and0.03%active carbon, in which differential rate reached92%. As for protocorms induction,1/2MS Supplemented with0.1mg·L-1KT and0.1mg·L-1NAA, in which induction rate reached41.67%.For multiplication of adventitious bud it was1/2MS containing0.7mg·L-16-BA,0.8mg·L-1NAA and1.0mg·L-1KT, the propagation coefficient was5.25.As for rooting,1/2MS Supplemented with0.5mg·L-1IBA and0.05active carbon, in which rooting rate reached95.3%. Used the moss, bark and volcanic rock for ground, then transplanted for the rooting plantlets, the survival rate was more than93%.As the weight-culture of protocorms basic, research the process of protocorms culture about its growth status, dynamic change of the medium such as carbon source, nitrogen, phosphate and pH. The results of protocorms showed that:stagnant period for0～4d, growth for4～18d, stabilization period for18～22d,dieth for22～24d. During the process of cultivation, sucrose content decreases rapidly to9.43g·L-1on the6th day. On the contrary, the content of fructose rises gradually in the same time. We know that fructose consume fast at the last stage of cultivation, and total sugar present slow decline in the whole growth process. Phosphate decreases firstly before the6th day, and then rise again; however, the content of phosphate declines to stable slowly. Ammonium nitrate nitrogen declines sharp to112mg·L-1on the12day, and remains only87mg·L-1. However, the changes of nitrate are relatively slow, since its content decreases slowly during the whole cultivation. The pH from5.5to3.6at first, while there is a slowly arise process at the late stage. the pH maintains4.5on the18th day.The results showed that floral bud induction rate was not obvious at6-BA,20.00%at0.10mg L-1TDZ, and16.67%at4.5%sucrose supplemented in MS. The best medium for floral bud induction was MS (1/2N,3P) containing0.10mg L-1TDZ and3.5%sucrose,14h d-1photoperiod,25℃temperature and1500lx intensity of illumination,the floral bud induction rate was25.00%.In photoperiod experiments, when the temperature is20℃, photoperiod for12h d-1, flower bud induction rate was35.00%; in temperature experiments, when the photoperiod for12h d-1, and the temperature is20℃, the flower bud induction rate was33.33%.