Construction of Tissue Culture and Rapid Propagation System for Rhododendron hemsleyanum, a Plant Species with Extremely Small Populations

Purpose To establish a tissue culture system for Rhododendron hemsleyanum, a plant species with extremely small populations, and to investigate the optimal conditions for seed germination, seedling proliferation, root induction, and transplant substrate.

Methods The seeds with plump grains and similar size were selected as germination materials, the effects of light and dark conditions and different sterilization pretreatment on seed germination were studied. For the seedling proliferation and root induction experiments, the progeny seedlings were used as explants, and WPM was used as the basic culture medium, the optimal conditions for seedling proliferation and root induction were studied by a L₉(3³) orthogonal experimental design and Box-Behnken response surface methodology. For the hardening and transplant experiments, the successfully root-induced test seedlings were transplanted into five different substrates after nine days, and the survival rate of R. hemsleyanum in different transplant substrates was analyzed.

Results The best seed germination results were achieved with one minute treatment with 75% ethanol and ten minutes treatment with 1% NaClO solution, resulting in a germination rate of 66.67%. The best growth medium for seedlings was WPM+1.0 mg/L 6-BA; the optimal culture medium for seedling proliferation was WPM+0.5 mg/L NAA+1.5 mg/L ZT, and the pH value was 4.5, with a proliferation coefficient of 3.13. In the root induction culture medium, stem tips placed horizontally on the surface of the medium showed good rooting, while those inserted into the medium did not form adventitious roots, and after 40 days of culture, the basal part of the explants implanted in the culture medium decayed and turned black. The optimal rooting medium was found to be 1/2 WPM+0.5 mg/L NAA+1.0 mg/L IBA, resulting in a rooting rate of 88.00%. The most suitable transplant substrate for tissue-cultured seedlings was V_{humus soil}∶V_perlite∶V_vermiculite= 2∶1∶1, with a survival rate of 80.00%.

Conclusion This study successfully establishes a tissue culture rapid propagation system for R. hemsleyanum, providing a reference for tissue culture of other woody plants in the Ericaceae family. It also lays the foundation for future activities such as reintroduction, revitalization of wild populations, and sustainable development and utilization.