Objective To establish an efficient and stable in vitro cluster bud rapid propagation system for fragrant Oncidium Twinkle 'Chian-Tzy Deilight', fill the technical gap in industrial seedling raising of this cultivar, and break through the two core bottlenecks of rapid propagation: proliferation and rooting.

Method Multiple shoots induced from flower stalk axillary buds were used as experimental materials. L₁₆(4⁵) and L₉(3⁴) orthogonal designs were adopted to systematically investigate the effects of five factors (basic medium, 6-BA, KT, NAA and sucrose) on multiple shoot proliferation, as well as four factors (basic medium, NAA, IBA and organic additives) on multiple shoot proliferation. so as to screen the optimal combination each factor.

Result The priority order of factors multiple shoot proliferation was 6-BA > basic medium > NAA > KT > sucrose. The optimal medium for proliferation was 1/2MS + Hyponex No.1 1.5 g/L + 6-BA 1.2 mg/L + KT 0.3 mg/L + NAA 0.15 mg/L + sucrose 30 g/L + agar 6.5 g/L, with an average proliferation coefficient of 5.77 after 45 days of culture. The optimal rooting medium was Hyponex No.1 3.0 g/L + NAA 0.1 mg/L + IBA 0.2 mg/L + potato powder 10 g/L + sucrose 30 g/L + agar 7 g/L + activated carbon 0.5 g/L. The rooting rate reached 100% after 75 days, with an average of 9.5 roots per plantlet and an average root length of 4.0 cm.

Conclusion The cluster bud-based micropropagation technique enhances the asexual propagation efficiency of Oncidium Twinkle 'Chian-Tzy Deilight', offering theoretical basis and technical models for culture medium optimization of fragrant congeners in the same hybrid series.