Tissue culture of Juniperus chinensis: effects of sex and leaf morphology on in vitro regeneration

Background and Aims In the tissue culture of Juniperus chinensis, issues such as inadequate sterilization, severe browning, and poor rooting have significantly hindered the widespread application of its asexual propagation techniques. Meanwhile, J. chinensis presents three sexes: female (FP), male (MP) and monoecious (MOP), with leaf morphological differentiation into scale leaves (SL) and spiny leaves (SPL). Variations in sexes and leaf morphogenesis may affect the establishment of tissue culture systems. However, there are currently no studies that have established tissue culture systems by integrating both sex and leaf morphogenesis and the differences in responses of explants with different sexes and leaf morphogenesis during culture remain unclear. Methods Using SL and SPL from FP, MP and MOP as explants, we conducted optimization of explant disinfection protocols, screening of culture medium, selection of anti-browning agents, determination of antioxidant enzyme activities, proliferation and rooting culture, plants hardening and transplantation. Comprehensively explore the differences in the process of establishing tissue culture among J. chinensis with different sexes and leaf morphogenesis. Key Results Disinfection of explants with two leaf morphogenesis from three sexes can be achieved by treating them with sodium hypochlorite (NaClO) for 18 minutes. The basal medium suitable for the early growth of explants with three sexes and two leaf morphogenesis is Douglas-fir Cotyledon Revised medium (DCR). Polyvinylpyrrolidone (PVP) can inhibit browning by reducing the activity of oxidases. During the proliferation stage, explants derived from MOP demonstrate consistent hormone requirements. The adventitious bud proliferation efficiency of SPL is notably higher than that of SL, and all proliferated adventitious buds are SPL. At the rooting stage, the rooting ability of adventitious buds from SPL is inferior to that of SL. Conversely, MOP still maintain consistent hormone demands during the rooting stage. Post-transplantation, sterile plants display vigorous growth, with stem segments undergoing progressive lignification, the survival rate shows variability influenced by both sexes and leaf morphogenesis. Conclusions This study established a multi-dimensional tissue culture system for Juniperus chinensis based on three sexes (FP, MP, and MOP) and their corresponding SL and SPL. It analyzed the differences and preferences among different sexes and leaf forms at various stages of tissue culture, providing technical support for the targeted breeding, germplasm resource innovation and industrial development of J. chinensis. Meanwhile, it offers novel perspectives for research on sex differentiation, mechanisms of leaf form transformation and other related areas in polygamous or heterophyllous tree species.