ANALYSIS OF SUITABILITY OF DNA EXTRACTION PROTOCOLS FOR SOMACLONAL VARIATION ANALYSIS IN IN VITRO-CULTURED ORCHIDS

Orchid genetic transformation studies require the isolation of high-quality DNA from often small amounts of tissue. To date, not only are the number of successful genetic transformation studies of any orchid species limited, but also the DNA extraction method is in most cases (>90%) limited to the CTAB (or related) protocols. This study analyses the effectiveness of 12 previously published DNA extraction protocols (used for the extraction of DNA of both orchid and other species) in contrast to commercially available plant DNA extraction kits (3). In addition, an innovative, sonication-based, simple and efficient mini-prep to extract DNA from Cymbidium, Dendrobium, and Phalaenopsis is presented. DNA purity and quantification, EcoRI-restricted total genomic DNA, and RAPD analyses using three separate 10-mer primers show differences in the efficiency and applicability of the extraction method, even within the same clonal cultivar. The choice of explant on DNA analyses is also presented. These factors should be carefully considered when selecting a suitable DNA extraction protocol for somaconal variation or population genetic studies, or for transgene integration analyses.