Establishment of a high-efficiency embryogenic callus-somatic embryogenesis regeneration system in the succulent plant Sinocrassula indica var. viridiflora

Abstract

The endangered succulent Sinocrassula indica var. viridiflora K. T. Fu faces severe threats from habitat fragmentation and overexploitation. To address its conservation and commercial propagation challenges, an in vitro regeneration system was developed using mature leaves as explants. Sterilized leaves were cultured on MS medium supplemented with optimized plant growth regulator (PGR) combinations screened through single-factor, full factorial, and L₉(3⁴) orthogonal experimental designs. Synergistic effects of 6-BA and 2,4-D significantly enhanced embryogenic callus induction, with 1.5 mg/L 6-BA + 1.0 mg/L 2,4-D yielding compact, bright-green callus (proliferation coefficient: 7.50). Orthogonal optimization further identified 1.5 mg/L 6-BA + 0.5 mg/L NAA + 1.0 mg/L 2,4-D as the optimal proliferation medium (proliferation coefficient: 11.65). White frost-like structures dynamically associated with somatic embryo differentiation were observed during callus proliferation. Plantlet regeneration was markedly improved by separating clustered shoots, increasing the regeneration coefficient from 3.1 to 12.5. Regenerated plants exhibited stable rosette morphology with red-tipped leaves. Recycling regenerated leaves and embryogenic callus achieved a total proliferation coefficient exceeding 70.0. Acclimatized plantlets showed 100 % survival and retained ornamental traits. Histological analysis confirmed indirect somatic embryogenesis as the regeneration pathway, emphasizing the critical role of PGR balance and proliferation-differentiation regulation. This system provides a scalable solution for germplasm conservation and commercial production of S. indica var. viridiflora, while offering insights into morphogenetic control in succulent micropropagation.