A novel in vitro cyclic micropropagation protocol and assessment of genetic fidelity in the critically endangered woody species Carpinus putoensis

<i>Carpinus putoensis</i> 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 "<i>in vitro</i>-ex vivo-<i>in vitro</i>" 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 <i>C. putoensis</i> 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 <i>in vitro C. putoensis</i> shoots as scions and <i>Glycine max</i> (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 (<i>In Vitro</i> 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 <i>C. putoensis</i>.