Optimization of nitrogen source ratios for somatic embryo maturation in Pinus elliottii

Somatic embryogenesis represents a critical biotechnology platform for large-scale propagation and germplasm conservation of Pinus elliottii . However, low maturation efficiency remains a major limitation for commercial application. This study systematically investigated the regulatory effects of nitrogen source ratios on somatic embryo maturation in slash pine. Embryogenic callus [Ca(NO₃)₂, Asn, CH, Gln, and Pro], followed by optimization of nitrogen combinations based on an L₁₈(3⁵) orthogonal experimental design. Results indicated that Medium No. 14 was most conducive to the formation of total embryos of both S7-5 and S24-1, and to S7-2 cotyledonary embryo formation; Medium No. 11 proved optimal for total embryos of S7-2 and differentiation of S24-1 cotyledonary embryos; whereas Medium No. 13 was most favorable for S7-5 cotyledonary embryo formation. Correlation analysis revealed significant promoting effects of Ca(NO₃)₂ and Gln on embryo maturation. Enzymatic activity assays indicated that the optimized nitrogen ratio significantly upregulated GS activity while downregulating NR and NiR activities, facilitating the assimilation of nitrogen into organic forms. This study established an nitrogen regulatory system for slash pine somatic embryo maturation and elucidated the regulatory mechanisms of key nitrogen metabolic enzymes in conifer embryogenesis, thereby providing theoretical foundations and technical support for large-scale breeding and genetic improvement of superior slash pine varieties.