Selection and characterization of in vitro–induced mutants of Dendrobium ‘Earsakul’ resistant to black rot

The development of Dendrobium ‘Earsakul’ with improved resistance to black rot is essential for sustainable orchid production. In this study, in vitro mutagenesis and selection was used to breed D. ‘Earsakul’ for black rot resistance, evaluated black rot resistance levels in mutants and controls using detached leaf assay, and characterized putative resistant mutants using molecular markers, cytology, and morphological traits. Mutagenized protocorm-like bodies (PLBs) obtained by 1.4% (LD₃₀) and 1.8% (LD₅₀) ethyl methanesulfonate (EMS) concentrations and non-mutagenized PLBs were selected on a pea sucrose broth (PSB) medium supplemented with 0, 30, and 50% (first 2 cycles) and 0, 40, and 60% (third cycle) of Phytophthora palmivora culture filtrates (CFs). Fifty putative resistant mutants were obtained, and 42 of these were used for resistance level evaluation by detached leaf assay using P. palmivora isolate NK-53–9. The results revealed 13 resistant putative mutants, including 4 highly resistant putative mutants and 9 resistant putative mutants. Ten surviving resistant putative mutants were genetically different from the non-mutagenized controls and were confirmed as mutants after inter-simple sequence repeat (ISSR) analysis. Based on flow cytometry, the resistant mutants and non-mutagenized controls possessed the same chromosome number of 2n + 4n + 8n. Moreover, a mutant, SUT17E18316, exhibiting maximum DNA content and genome size was identified. Morphological characterization revealed that most of the black rot–resistant mutants were morphologically different on some characters from non-mutagenized lines, such as plant height and number of roots. Particularly, the highly resistant mutant SUT13E18305 which possessed outstanding characters may be useful for future commercialization.