Optimization Of Rapid Propagation For Cremastra Appendiculata And Construction For Cdna Pcr Library Of Dendrobium Candidum Leaves

Shancigu is commonly used for traditional Chinese medicine and used as anticancer medicine clinically because of its effectiveness for heat-clearing and detoxicating, swelling-reducing and detumescence. According to Pharmacopoeia of People’s Republic of China (Pharmacopoeia Commission of People’s Republic of China,1995,2000,2005), the resources of Shancigu are the drying pseudobulbs of Cremastra appendiculata (D. Don) Makino, Pleione bulbocodioides (Franch) Rolfe and Pleione yunnanensis Rolfe. These three plants mainly grow in Sichuan, Guizhou, Yunnan, and other provinces and regions of the south of Yangtze River in China. But the amount is steadily declining due to low rate of propagation in nature and huge demand for Shancigu in pharmaceutical applications. Therefore, it’s very important and significant for Shancigu to be multipropagated in vitro. In this study, the green protuberances of Shancigu cultures (C. appendiculata) on solid medium were cutted as explants, and then the explants were cultured on different media for finding out the optimal basal medium, optimal type and concentration of plant growth regulators and organic supplements. The results shown that1/2MS was best for basal medium,1.0mg/L NAA optimal for plant growth regulator,100g/L PE optimal for organic supplement, and through the orthogonal test, the optimal medium for growth and development of C. appendiculata was that of1/2MS basal medium supplemented with50g/L BE,50g/L PE,0.75mg/L6-BA,0.75mg/L NAA,30.0g/L sucrose, and3.0g/L activated charcoal. The medium was solidified with5.5g/L agar and pH was adjusted to5.8～6.2. Lastly, with the traditional paraffin section method, cultures of Shancigu at different developmental stages on the optimal medium were chosen for mo rp ho genetic research of C. appendiculata by optical microscope.Based on the RACE, the total RNA of Dendrobium candidum leaves was subjected to reverse transcription and terminal deoxynucleotidyl transfer resulting in the cDNA which contained the specific sequences in two ends. The obtained cDNA was subjected to PCR using primers which contained the specific sequences. Then the cDNA was amplified and the cDNA PCR library was constructed. The cDNA PCR library constructed through this method magnified the cDNA number more than one hundred times. By comparison, the PCR effect of the cDNA PCR library obviously outstripped that of reverse transcription products. The cDNA PCR library constructed using5pg total RNA as starting material still had satisfactory PCR amplification effect. This construction method of the cDNA PCR library, with the low cost and easy manipulation, provided a facility for cloning and expression analysis of low abundant genes.