Application Of The Protoplasts Electrofusion Technique And Ultraviolet Mutagenesis In Germplasm Improvement Of Rhodiola Yunnanenisis, Aloe Vera And Penicillium Oxalicum

With the development of cell electrofusion and UV-mutagenesis technologies, they not only provide the efficient methods for the research of the nucleo-cytoplasmic relation and gene regulation, but also become the key technology for cross-breeding and germplasm enhancement. In this paper, cell electrofusion and UV-mutagenesis technologies were applied in germplasm improvement for Rhodiola yunnanenisis L., Aloe vera L. and Penicillium oxalicum.Using the sterilized seedings of Aloe vera L. and the callus of Rhodiola yunnanenisis L. cultured in our laboratory as samples, we developed the methods of protoplasts isolation and purification and studied the optimum conditions of electrofusion to provide theoretical and experimental fundamention for the germplasm improvement of Chinese medicinal plants. The parameters of protoplasts isolation were studied firstly. The CPW solution contained 2.0% cellulase, 0.5% pectolyase, 0.5% bovine serum albumin, 0.1% MES and 0.6 mol/L manmitol under the condition of 25±1℃, pH 5.6, and dark-incubated for 3 h. The highest yield of the A. vera L. was obtained as 7.03×105 /mL. The material for the isolation of R. yunnanenisis L. protoplasts was the callus cultivated for 10 d in dark. The optimized method for protoplast separation was that the callus were dissolved for 3 h in the CPW solution containing 2.0% cellulase, 1% pectolyase, 0.3% macerating enzyme, 0.5% bovine serum albumin, 0.1% MES and 0.7 mol/L manmitol. After acquiring the protoplasts, the electrofusion of A. vera L. protoplast and R. yunnanenisis L. protoplast was estimated. The results showed that the appropriate parameters for the highest binary fusion frequency (17.8%) were 160 V/cm, 1 MHz, 20 s for alternating-electric field (AC) and 0.9 kV/cm, 30μs, 1 times for direct current field (DC). Under the basis of the intraspecific fusion parameters, the electrofusion conditions between A. vera L. and R. yunnanenisis L. were futher investigated. The optimal conditions were 180 V/cm, 1 MHz, 30 s for AC field and 0.9 kV/cm , 30μs,1 times for DC field.The original strain of Penicillium oxalicum with cellulase activity was an endophytic fungi screened from Ginkgo biloba by our laboratory. High-cellulase-yield mutant PO1M was selected by UV-irradiation and the cellulase activity of the PO1M remained stable after several generations. The mutant exhibited distinct differences in morphology, cellulase activity and gene compared to the original strain. Random amplified polymorphic DNA (RAPD) was employed to study the genetic variation between the mutant and the parent strain. We used 10 random primers to examine the change of genetic background. The primer G02 was efficient and obtained specific bands in the mutant. This result demonstrated that the DNA of the UV mutants had been altered by the UV treatment. Single factor experiment on liquid fermentation of mutant PO1M was carried out to show the optimal medium compositions: (w/v) 20% potato, 2% microcrystalline cellulase, 0.5% peptone, 0.3%KH2PO4, 0.15% MgSO4, 0.001% vitamineB1, natural pH. The appropriate parameters were as follows: the shake speed 150 r/min, temperature 28℃. The cellulase activities were examined after fermention for 5 d. The activities of filter paper(FPAse), endoglucanase(EG),β-glucosidase(BGL) and cellobiohydrolase(CBH) were 0.48, 3.95, 0.27 and 0.26 U/mL, respectively. This research for resolving the problem of low cellulase production and callulase activity is effective and promising.