| Dendrobium sinenese is an endemic plant species inhabiting the tropical cloud forest or montane rainforest with altitudes above 1000m in Hainan Island, China. Dendrobium is used as a tranditional medicinal plant by Li minority people, and numerious compounds,such as stilbenoids and bibenzyls with cytotoxic, antioxidant and antibacterial bioactivities have been extracting from its whole plant. Reasons include habitat destruction, scarce resources, low acclimatization and poor growth vigor , which make the introduced domesticated and artificial cultivation difficult of Dsinenese. Polyploid has the potential advantages in increasing plant vegetative organs,increasing resistance and improving nutrient content of plant, and it is possible to solve the above problems.In this study, the polyploidy of D.sinense induction and development from embryo protocorms in vitro by chemical inducer, different concentration and duration were studied and analyzed. Then, the tetraploid seedlings were identified and selected by morphology,stomatal microscopy, karyotype characterization and flow cytometry analysis. The identification process was optimized. Finally,the quality evaluation of tetraploid D.sinenese was carried out from three aspects: the stability of proliferation, the heat resistance and the polysaccharide content.The aim of this study was to establish a complete and efficient platform for the induction and identification of tetraploid D. sinenese, and to provide scientific basis for the new germplasm cultivation, utilization and cultivation of D.sinenese.The results are as follows:(1) Colchicine and oryzalin are successful in obtaining autotetraploid D.sinenese.The best treatment of colchicines was 0.10% in 8 days; the inducing ratio was 22.0%;seedlings immersed in 20mg·L-1 oryzalin solution for 4 days reached to the highest inducing ratio 35.0%.(2) Firstly, the mutant plants with dwarf and stout stems and thick dark green leaves were screened out by morphological identification to improve the efficiency of identification.Then using flow cytometry for identification of ploidy was simpler, rapid and accurate, the relative DNA content of tetraploid and diploid D. sinense nearly 2:1 in ratio.Finally, the stomatal size and chromosome number were observed and further verified.The epidermal stomata of the tetraploid plants increased by 1.59 times, and the number of stomata in the unit field of view were halved. Stoma guard cell chloroplast count increase. The chromosome number of tetraploid cells was 2n=4x=76, while that of diploid cells was 2n=2x=38.(3) The results shows tetraploid and diploid have the same proliferation effect,regeneration plants are grows healthy.(4) D.sinense seedlings easily injury under high temperature, RDA demonstrated that relative conductivity, MDA content and SPAD value could be used as heat tolerance index of D.sinense seedlings.Relatively smooth changes in SPAD value and relative conductivity of tetraploid D.sinense, and get heat injury delay.Inferred that tetraploid has higher heat resistance.(5) The content of polysaccharides in the stems of diploid D.sinense was slightly higher than that of tetraploid D.sinense.Chemical inducer colchicine and oryzalin are efficient and effective in polyploid induction of medicinal plant D.sinenese. This study has developed a toolbox to rapidly obtain and confirm the polyploid of Dendrobium. These results could have practical implications for the optimal utilization of D.sinense in horticultural industry. |
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