Optimization of Spore Germination, Gametophyte Liquid Culture, and Sporophyte Induction in the Endangered Medicinal Fern Cibotium barometz

Abstract

This study established a continuous in vitro propagation system for Cibotium barometz encompassing spore germination, liquid proliferation of gametophytes, air-lift bioreactor culture, and sporophyte induction. An L9(34) orthogonal design was used to screen combinations of NAA, TDZ, and GA3, and an L16(45) design was used to estimate the main effects of MS salt strength, sucrose, 6-BA, and IAA on gametophyte proliferation. Biomass, total flavonoids, total phenolics, soluble sugars, and electrical conductivity (EC) were monitored in a 5 L air-lift bioreactor. Treatment S-5 produced the highest 75 d germination rate (78.91%) and gametophyte longest-axis length (68.00 μm). M-6 yielded the greatest fresh biomass, whereas M-5 provided a more balanced dry-matter and metabolite yield. At 45 d, fresh weight, dry weight, and total flavonoid yield reached 35.02 g L−1, 2.79 g L−1, and 74.73 mg L−1, respectively. EC was negatively correlated with biomass and phenolic yields, and the first two principal components explained 97.48% of the variation. The best sporophyte-induction treatment achieved a conversion rate of 63.02% and an acclimatization survival rate of 91.77%. This system provides a methodological basis for ex situ conservation and scalable propagation of C. barometz.