Establishment of in vitro efficient regeneration system for leaves and hypocotyls of Toona sinensis, a multifunctional woody plants

Abstract

Establishing an efficient in vitro regeneration of Toona sinensis (A.Juss.) Roem. (T.sinensis) is critical for advancing industrial applications. However, current regeneration efforts are constrained by low efficiency and challenges in redifferentiation. In this study, hypocotyls and leaves of T.sinensis were utilized as explants for the establishment of a regeneration system and optimization of age, distribution site of explants, and orientation. Hypocotyls of 21-day-old seedlings were placed horizontally on a medium containing plant growth regulators (PGRs): 0.75 mg/L Zeatin (ZT), 0.15 mg/L Indole acetic acid (IAA), and 0.05 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D). The callus induction rate of hypocotyls was 73.03%, with an average of 14.80 adventitious buds per callus. The proximal ends of leaves in the second developmental stage were placed upright on a medium with 1.0 mg/L ZT, 0.05 mg/L IAA, and 0.20 mg/L 2,4-D. The callus induction rate of leaves was 77.77%, with an average of 15.78 adventitious buds per callus. Additionally, the proliferation and rooting of regenerating buds were investigated. When regenerated shoots were transferred to a proliferation medium containing 1.5 mg/L 6-Benzylaminopurine (6-BA) and 0.1 mg/L Indole-3-butyric Acid (IBA), shoot proliferation coefficients peaked at 4.18 for leaves and 3.17 for hypocotyls. Transferred these proliferating buds to the rooting medium, reached a 100 % rooting rate with PGRs at 1.0 mg/L, and the survival rate of regenerated plants after transplanting reached 98 %. This study establishes a more efficient in vitro regeneration system for T.sinensis, offering a robust foundation for molecular breeding efforts. The results have the potential to support molecular improvement breeding and the innovation of germplasm resources of T.sinensis.