A Rapid and Highly Effective Protocol for Sporophyte Proliferation of Drynaria roosii and the Potential Regulation Mechanism by Plant Growth Regulators.

Sporophyte propagation in medicinal ferns is influenced by explant type, owing to their distinctive two-phase life cycle: haloid gametophyte and sporophyte. However, few studies have systematically investigated sporophyte proliferation using different explants. This study established an efficient in vitro propagation system for the medicinal fern Drynaria roosii using two types of explants: with green globules (A) and without them (B). An L9(34) orthogonal design tested 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on direct sporophyte regeneration. Results showed that sporophytes regenerated directly without a callus phase. For explant A, 6-BA was the dominant factor, with optimal proliferation at 3.00 mg/L 6-BA + 1.50 mg/L NAA + 0.05 mg/L 2,4-D. Explant B was less hormone-responsive but achieved a higher proliferation coefficient (116.50) under lower hormone levels. Endogenous hormone profiling revealed that increasing 6-BA triggered a biphasic cytokinin response, suppressed auxins, and elevated GA9 in explant A, while most hormones in explant B were significantly lower. Transcriptomic GO enrichment highlighted differentially expressed genes involved in the chloroplast envelope, thylakoid membrane, and photosynthesis, indicating that membrane remodeling and photosynthetic reorganization underpin sporophyte dedifferentiation and redifferentiation. We propose that 6-BA modulates proliferation through a biphasic cytokinin response, auxin suppression, and GA9 induction, coupled with transcriptomic reprogramming of the chloroplast envelope and thylakoid membrane components.