Studies On Establishment Of Rapid Propagation System In Vitro Of Medicinal Plant Pithecellobium Clypearia

Pithecellobium clyperria (Jack) Benth. named as weixianshu, jiaolongmu in Chinese is a plant of Leguminosae, grown in Zhejiang, Guangdong, Fujian, Yunnan, Taiwan and other regions in China. Its young shoots and leaves are usually used for medicinal purposes in the southern china, have functions of heat-clearing, detoxifying, eliminating dampness, healing sore. It is the major raw material of some famous clinical medicines, such as Pithecellobium clypearia antichloristic tablet, granule and capsule. The main collection way of herbs is from wild resources. Because of the excessive development and collection, the wild resources have been destructed seriously, and now the market demand cannot be satisfied in long term. Therefore, it is imperative to carry out artificial cultivation to offer medicinal materials, protect wild resources and keep sustainable utilization.Through seed breeding is the main propagation path of Pithecellobium clyperria. According to the seed biological characteristics and the seed physiology, the seeds of Pithecellobium clyperria are hard to store, while the germination rate drop sharply after cryopreserved 3 months. The other serious problems are the low purity of seeds, the high infesting rate. Consequently, the supplies of seeds are difficult to met production need. In addition, cutting propagation of wildlife resources do not has practice effect because of the low rooting and survival rate. Therefore, it is significant to establish the rapid propagation system in vitro of medicinal plant Pithecellobium Clypearia.1 The review of domestic and foreign literaturesAfter reviewing a large number of relative literatures, the research status of Pithecellobium Clypearia was summarized, including the progress of the history, pharmacognosy, chemical composition, pharmacologic action, clinical application, quality standard and so on. The overview of tissue culture about Mimosoideae woody plants was summarized from the following several aspects:the establishment of sterilizing system, cluster buds induction and proliferation, callus induction and differentiation, rooting induction, acclimatization and transplantation.2 The establishment of sterilizing systemThe experimental materials were collected from mature plant, and studied the effect of different explants, sterilization procedure, and anti-browning methods on the survival rate. The results showed it was hard to overcome the browning and infected bacteria problems. After the seeds of Pithecellobium Clypearia were collected and used as explants, through compared of the pre-treatment ways, sterilization procedure and culture condition, the optimal method was found. The treatment with 0.1% HgCl2 for 12 minutes was suitable to sterilize seed embryos, and the medium for culture was 1/2MS+AC 1.0 g/L, with contamination rate of 16.10% and the sprout rate of 78.66%. The seedlings sprouted from the seeds in the 1-2 months were acted as explants, and optimum sterilization time was 30 s with 70% ethyl alcohol and 12 min 0.1% HgCl2, cultured in the medium 1/2MS with 1.0 g/L PVP, the survival rate up to 49.17%.3 Cluster buds induction and proliferationEffectiveness of different kinds, concentrations and combinations of plant growth regulators on the cluster buds initial induction were compared. Results showed that the optimal medium for cluster buds induction was MS medium containing 0.50 mg/L 6-BA and 0.05 mg/L NAA, with the induction rate of 100%, proliferation coefficient of 3.78, and the growth vigor was better.In the proliferation culture, the effect of plant growth regulators, sugar sources and concentrations, hydrolytic lactalbumin, hyponex 5 and illumination time were researched and compared. The suitable medium was MS+6-BA 0.70 mg/L+NAA 0.05 mg/L+IBA 0.30 mg/L+ Hyponex 50.50mg/L～0.80mg/L + sucrose 20 g/L. The light intensity was 2500～3000 lx, with irradiating 10～ 12 h/d. After 45 d, the proliferation coefficient reached 4.20～4.94, and the results remained stable in sub cultured 3 times.4 the rooting induction, and plantlets transplantationIn the studies of strong seedlings and rooting induction, comparing with basic medium, plant auxins, sucrose concentrations and activated carbon, the optimum medium for direct rooting method was screened out. Furthermore, the different ways of rooting induction were studied, including direct way, dipped method, two-step treatment, and the use of new rooting agent.1/2 MS+IBA 0.50 mg/L+AC 0.10～0.30 g/L+ sucrose 20 g/L was the optimum medium for plantlet rooting and strong plant regeneration occurred 60d later with the rooting rate up to 100% and developed root system.After being acclimated with 3 to 5 days, the survival rate can reach 91.11% when the plantlets were transplanted on the substratum composed of vermiculite and Coconut chaff in equal volume proportion.