[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

[Tissue cultivation of tiller buds of Epimedium wushanense].

OBJECTIVE: To established the rapid tissue propagation system of Epimedium wushanense, in order to provide theoretical basis for industrialized seed cultivation. METHOD: Tiller buds E. wushanense were used as explants, with MS, B5, WPM as basic media, and added with different concentrations of plant growth regulators such as 6-BA, NAA and GA3, in order to conduct a systematic study on induction and propagation conditions for tiller buds. RESULT: The suitable method for sterilizing bud was to disinfect with 75% ethanol for 30 s, and then treated with 0.1% HgCl2 for (4 + 2) min for consecutively twice, which could control the pollution rate below 5% and the survival rate above 75%. The optimal medium for bud induction was WPM + 6-BA 2.0 mg x L(-1) + NAA 0.1 mg x L(-1) + GA3 0.5 mg x L(-1), with the induction rate of 75.5%; meanwhile, the basic medium and 6-BA showed significant effect on the induction rate. The propagation medium suitable for buds was MS +6-BA 2.0 mg x L(-1) + NAA 0.5 mg x L(-1), with the propagation rate of 3.3. The optimal growth of rooting medium was 1/2 WPM + IBA 0.5 mg x L(-1) + activated carbon which (0.05%), with the rooting rate of 90%, three to six strong seedlings in each plant. CONCLUSION: The disinfection method suitable for tiller buds and the medium combination suitable for induction, propagation and rooting of adventitious buds are screened out to establish the rapid cultivation system for tiller buds of E. wushanense.