Mechanism analysis of glutamine and acid hydrolyzed casein enhancing the induction of callus in mature Pinus massoniana apical buds
摘要
Plant tissue culture facilitates efficient seedling propagation and, through indirect organogenesis in mature trees, enables conservation of elite traits and genetic transformation. However, organ regeneration from mature Pinus massoniana (Lamb.), an economically and ecologically important species, remains challenging. To overcome the challenge of organ regeneration in mature P. massoniana, this study utilized apical buds from mature P. massoniana to investigate callus induction, with a focus on the effects of exogenous glutamine (Gln) and acid hydrolyzed casein (CH) on endogenous phytohormones and differential gene expression during callus development. A modified quarter-strength Douglas-fir cotyledon medium (DCR), supplemented with 1.5 mg L−1 1-naphthaleneacetic acid (NAA), 0.5 mg L−1 6-benzylaminopurine (6-BA), and 1.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), yielded a 100% callus induction rate. Furthermore, exogenous supplementation with 500.0 mg L−1 Gln and 1000.0 mg L−1 CH significantly promoted cell division and morphological changes, thereby improving callus formation. Compared to CK (no additives), two treatments—A (Gln 500 mg L−1 with CH 1000 mg L−1) and B (CH 1000 mg L−1 alone)—led to the identification of 649 significantly differentially expressed genes (DEGs), which were annotated to 9 metabolic pathways. Among these, the cutin, suberin, and wax biosynthesis pathways were the most significantly enriched. This study has preliminarily identified optimal culture medium for callus induction via indirect organogenesis and has investigated the effects of Gln and CH on endogenous hormones and gene expression during callus optimization. These findings provide a theoretical foundation and key technical support for the large-scale asexual propagation of elite mature P. massoniana germplasm.