Abstract
Slash pine (Pinus elliottii Engelm.) is an economically significant tree species valued for its timber and resin production. Establishing a somatic embryogenesis system is essential for the effective utilization of slash pine. This study aims to develop an optimized cultivation system for the long-term proliferation of embryogenic callus (EC) in slash pine. The study used the EC induced from immature zygotic embryos as materials to investigated the effects of plant growth regulators (PGRs), subculture cycles, and subculture durations on the EC proliferation coefficient and the number of somatic embryos (SE). Additionally, we monitored the changes in the proliferation coefficient, the number of SE, and physiological and biochemical indicators of EC during long-term proliferation. The EC proliferation coefficient reached its highest value (7.65) when the DCR medium was supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (6-BA). Refreshing the culture medium every 14 days, EC maintaining proliferation coefficient (4.05) and embryogenic potential for approximately three year (980 d), but the number of somatic embryos was low and significantly declined after 140 days.The additional supplementation of 1.0 mg/L 2- (1-naphthyl) acetic acid (NAA) to the original proliferation medium further enhanced SE induction, achieving a maximum of 143.25 SE per gram of EC. Under the optimal proliferation culture conditions, EC was subcultured for 392 days, with no significant differences observed in the proliferation coefficient and the number of SE. The results of correlation and path analyses between physiological indicators and proliferation coefficient indicate that the proliferation coefficient of EC is mainly related to the content of storage substances (starch and soluble sugars) and the activities of redox-related enzymes (POD and CAT). The optimized long-term proliferation culture system for EC in slash pine provides an efficient approach to enhance the production of EC and SE.