Molecular characterization of Dendrobium ‘Earsakul’ mutants from in vitro selection for black rot resistance

Abstract: In vitro chemical mutagenesis of Dendrobium ‘Earsakul’ protocorm-like bodies (PLBs) followed by in vitro selection with Phytophthora palmivora culture filtrate (CF) generated several putative mutants potentially resistant to black rot. The objectives of this study were to evaluate the black rot resistance levels of these putative mutants, to estimate the genetic variability among them and non-mutagenized controls using inter-simple sequence repeat (ISSR) analysis, and to identify candidate markers with significant associations to black rot resistance. When 3 non-mutagenized control lines and 8 putative mutants, derived from ethyl methane sulfonate (EMS) or sodium azide (NaN₃) induced mutation, followed by in vitro selection were evaluated for black rot resistance using detached leaf assay with a virulent isolate NK-53-9, it was found that all controls were susceptible to black rot, but the resistance levels of putative mutants varied. Four of eight putative mutants were moderately resistant or resistant to the disease, suggesting their usefulness in the breeding program. ISSR analysis of these controls and putative mutants with 7 ISSR primers yielded 7 to 29 reproducible bands per primer, ranging in size from 170 to 2100 bp. A total of 114 amplified ISSR fragments were obtained, 53 of which were polymorphic (46.5%). All controls have the same DNA patterns, while all 8 putative mutants showed altered genetic profiles compared to controls and were identified as mutants. The mutant SUT13E18-A appears to have distinct genetic profile compared to others as well as high level of resistance to black rot. Moreover, five ISSR markers significantly associated with black rot resistance were identified. These results suggest that ISSR analysis is efficient for mutant identification and characterization, and in vitro chemical mutagenesis followed by in vitro selection with P. palmivora CF provides a useful tool for future improvement of black rot resistant Dendrobium.