In Vitro Propagation and Gametophore Development of the Desiccation-Tolerant Moss Hypnum leptothallum

Hypnum leptothallum (Müll. Hal.) Paris, a pioneer moss species thriving in karst rocky desertification areas, exhibits remarkable desiccation tolerance and holds promise for ecological restoration initiatives. Despite its potential, the species faces limitations due to its slow growth rate. This study contributes significantly to the understanding of H. leptothallum by systematically documenting the developmental process from spore germination to young gametophore formation and establishing an efficient in vitro propagation system for widespread cultivation. Sequential developmental stages were identified through detailed morphological analysis, encompassing spore germination, protonemal differentiation, and gametophore formation. The culture system was optimized through singlefactor experiments and response surface methodology (RSM). Initial screening indicated that 0.2 mg L^-1 kinetin (KT) was the most effective plant growth regulator. Further RSM optimization determined that a medium composed of -1/2.67 Benecke + 0.2 mg L^-1 KT at pH 5.6 produced optimal growth. This medium yielded a protonemal patch diameter of 1.12 ± 0.047 mm, gametophore height of 1.97 ± 0.107 mm, gametophore yield of 9.75 ± 0.855 numbers, coverage of 19.32% ± 0.295%, and biomass of 0.17 ± 0.003 g. By following this established protocol, a sufficient amount of protonema material is ensured to facilitate the rapid cultivation of H. leptothallum using fragmented protonema and additional nutrients. This method provides a practical means for the mass production of H. leptothallum crusts, potentially contributing significantly to the mitigation of rocky desertification in karst regions.