In Vitro Culture And Genetic Diversity Evaluation Of Chunlan (Cymbidium Goeringii Rchb.f.) In GuiZhou

Chunlan (Cymbidium goeringii Rchb.f.) is a kind of highly ornamental Orchidaceae plant. Conventionally, it was propagated by separation of bulbs. With the development of modern biotechnology, tissue culture may be effectively employed to the propagation. The elucidation of the genetic diversity of wild Chunlan germplasm can provide essential information for the conservation, phylogeny, identification, genetic improvement and reasonable exploitation of this orchid. To satisfy the increasing demand of its germplasm utilization, in vitro culture and genetic diversity evaluation were carried out in the present study. The main results are as follows:1 Establishment and optimization of aseptic culturesThe seeds of Cymbidium goeringii Rchb were germinated in asymbiotic culture. Collecting-time, sterilization methods, gibberellin pre-treatment, basal media, carbon source and regulators significantly affected the germination rate. The results showed that the best medium to induce germination was MS + NAA(1.0 mg/L) + 6-BA(1.0 mg/L) + GA(1.5mg/L) + Sucrose(3.0%) + agar(0.7%). With the discussion of the factors which affected the germination rate, the optimized in vitro germination system was established.2 The optimization of RAPD-PCR protocol in CymbidiumTo establish the reproducible RAPD-RAPD protocol in Cymbidium, the concentrations of Mg²⁺, dNTPs, Taq DNA polymerase, and primers were also optimized in the present work. With the total volume of reaction was 25μL, the optimal system using 75 ng genomic DNA, 2.5 mmol/L Mg²⁺, 0.15 mmol/L dNTPs, 0.75 U Taq DNA polymerase, and 0.4 μmmol/L primers, were employed to carry out PCR in 20 accessions from 11 species of Cymbidium.3 Genetic diversity evaluation among Cymbidium goeringii and its relativesMorphological traits and RAPD markers were used to evaluate the relationship of Cymbidium goeringii Rchb and its relatives. With 35 morphological traits, considerably close relationships were found between Cymbidium sinense Willd and Cymbidium kanran Makino. In addition, a significant relatedness was also obtained between Cymbidium nanulum Y.S.Wu and Cymbidium lancifolium Hook. Based on the RAPD data yielded from the 23 screened primers, five subclusters were obtained from the given accessions.4 Genetic diversity in wild accessions as revealed by RAPD analysisTo satisfy its genetic improvement and phylogenetic information, genetic variation of 65 wild accessions of Cymbidium goeringii Rchb from nine provinces of China was investigated at the DNA level using the 48 screened arbitrary primers. With these primers, 324 discernible RAPD bands were scored, and 201 (62.04%) were polymorphic.The results showed that it has considerable correlation between genetic distance and geographical distance.