Objective To create sexual polyploid resources of medicinal Dendrobium species via the 2n gamete pathway.

Method Using D. officinale, D. aphyllum, and D. loddigesii as materials, we systematically observed the development of microspores in D. officinale through pollen pressing and DAPI staining techniques. The correlation between microspore development and flower bud morphology was analyzed, and the frequency of 2n male gamete occurrence in the three medicinal Dendrobium species was compared. Hybrid populations ('D. officinale × D. aphyllum' 'D. loddigesii × D. aphyllum') were constructed via distant hybridization, and the 2n male gamete formation rates of the parental and F₁ generations were recorded. High-frequency 2n male gametes were screened for secondary distant hybridization, followed by phenotypic observation and flow cytometry to identify sexual polyploids.

Result The microspore development process in D. officinale was positively correlated with flower bud size. When the flower bud length was between 0.92~1.03 cm and the diameter was 0.30~0.37 cm, the microspores were in the late stage of meiosis Ⅱ (MⅡ). The asynchrony of meiosis led to the formation of 2n male gametes, primarily as dyads and triads in D. officinale. The occurrence of 2n male gametes was observed in all three medicinal D. species, but the frequency varied among species D. aphyllum (0.55%±0.07%) > D. loddigesii (0.22%±0.03%) > D. officinale (0.16%±0.04%). Distant hybridization significantly enhanced the formation of 2n male gametes in the F1 generation, with 46.9% of progeny in the D. officinale × D. aphyllum cross having≥1% (13 plants reaching 5%~24%), and 20.4% of progeny in the D. loddigesii × D. aphyllum cross having ≥0.9%. Using F₁ generation individuals with high-frequency 2n male gametes, secondary distant hybridization was conducted. After tissue culture, 106 potential polyploid progeny were obtained, with one sexual triploid identified via flow cytometry.

Conclusion Distant hybridization can effectively induce 2n male gamete formation in medicinal Dendrobium species, and the 2n gamete pathway successfully creates sexually derived polyploid resources in medicinal Dendrobium, providing a new strategy for genetic improvement of Dendrobium species.