Profiling of black rot resistant Dendrobium 'Earsakul' induced by in vitro sodium azide mutagenesis

In Thailand, Dendrobium 'Earsakul' is one of the most important commercial orchids. However, its production is negatively impacted especially by black rot disease caused by Phytophthora palmivora. Therefore, new orchid varieties with high quality and resistance to the disease are urgently needed. The objectives of this research were to assess the resistance levels in sodium azide (NaN3) induced black rot resistant D. 'Earsakul' mutants by detached leaf assay and by characterizing the putative mutants using morphological characters, molecular markers, cytology, and protein profiles of black rot resistant mutants. The results revealed that two D. 'Earsakul' mutants, namely SUT17N05304 and SUT17N05308, were highly resistant and SUT17N05305 and SUT17N05501 were resistant. Their morphological characters including number of nodes, number of leaves, and number of roots differed from non-mutagenized controls showing susceptibility to the disease. Genetic variability by means of inter-simple sequence repeat (ISSR) analysis further demonstrated that these resistant mutants were genetically different from non-mutagenized controls (0.186 genetic dissimilarity; GD). Using flow cytometry, all of the resistant mutants were identified to be mixoploid (2n +4n+ 8n) and their DNA content and genome size, ranging from 3.77 to 3.90 pg 2C(-1) and 3,640 to 3,764 Mbp, respectively, were higher than all the non-mutagenized controls. Moreover, SDS-PAGE showed up-regulation of a protein with a molecular weight of 39 kDa at 3 to 5 days after inoculation in four black rot resistant mutants, while that of non-mutagenized controls appeared at 4 to 5 days after inoculation. In addition, a 13 kDa protein was uniquely induced only in two of the black rot resistant mutants (SUT17N05304 and SUT17N05305). These results suggest the usefulness of these resistant mutants for future development of new black rot resistant Dendrobium varieties.