A Novel In Vitro Cyclic Micropropagation Protocol and Assessment of Genetic Fidelity in the Critically Endangered Woody Species Carpinus putoensis

Abstract

Carpinus putoensis is a critically endangered woody tree species with only a single known wild individual, facing severe reproductive barriers. To overcome the scarcity of germplasm, this study established a stable "in vitro–ex vivo–in vitro" cyclic micropropagation system. The protocol consists of three key stages. Initially (Primary Culture Establishment), semi-lignified stem segments were collected as initial explants from mature C. putoensis trees (derived from cuttings of the single wild individual). In this phase, the axillary bud induction rate was 28.6%, but the shoots exhibited slow elongation and severe rooting recalcitrance. To rescue this valuable germplasm, a modified heterologous micrografting technique was employed using in vitro C. putoensis shoots as scions and Glycine max (soybean) seedlings as rootstocks. Subsequently (Ex Vitro Acclimatization), the micrografted plantlets were successfully transferred to a greenhouse, serving as the source of explants for the next phase. Finally (In Vitro Culture Re-establishment), nodal segments from the acclimatized mother plants were re-introduced into culture. In this sustainable phase, shoot proliferation was significantly improved on WPM medium containing 0.3 mg·L⁻¹ 6-BA, 0.03 mg·L⁻¹ NAA, and 2 g·L⁻¹ activated charcoal, achieving a high axillary bud induction rate of 90.31%. A mean shoot elongation of 4.53 cm was obtained using 0.1 mg·L⁻¹ 6-BA and 0.8 mg·L⁻¹ GA₃, followed by a high rooting rate (89.3%) using a two-step IBA treatment. Genetic analysis using RAPD and ISSR markers confirmed the genetic fidelity of the micropropagated plantlets. This cyclic system provides a renewable and genetically stable method for the conservation of C. putoensis.