Long-term subculture of embryogenic callus leads to a decline in somatic embryogenesis and germination capacity, which may be associated with increased methylation levels. 5-Azacytidine (5-AzaC), a methylation inhibitor, modulates DNA methylation and is widely involved in regulating plant growth, development, and metabolism. In order to investigate the effect of 5-AzaC on somatic embryogenesis and germination in Larix olgensis, we supplemented the proliferation medium with different concentrations of 5-AzaC. The results showed that the addition of 5-AzaC inhibited the proliferation of embryogenic callus, with the proliferation of embryogenic line N2 completely inhibited at 100 μM, while that of embryogenic line N4 ceased at 20 μM. In contrast, treatment with 10 μM and 20 μM of 5-AzaC significantly increased the somatic embryo yield in both embryogenic lines, with the peak yield observed at 20 μM for embryogenic line N2 and at 10 μM for embryogenic line N4. Furthermore, the addition of 10 μM 5-AzaC effectively reduced the deformity rate during somatic embryo germination in embryogenic line N2 and N4, by 15.91% and 13.53%, respectively. These findings demonstrate that 5-AzaC can partially restore the somatic embryogenesis potential of embryogenic callus in L. olgensis under long-term subculture. Additionally, these results suggest that its effects may be both concentration-dependent and genotype-specific. The results provide a potential approach to mitigating the decline in embryogenic competence, while also demonstrating significant potential for large-scale propagation.

Larch is a cold-temperate tree species native to the northern hemisphere and tolerant to low temperatures. It is one of the most significant timber species in Northeast China. This study examined growth changes in hybrid larch seedlings from five lines to explore the physiological responses of these seedlings to low-temperature stress. Using 8-month-old hybrids of larch seedlings, we subjected the plants to cold stress at 4 °C and freezing stress at −20 °C over three periods of 6, 12, and 24 h, and treatment at 25 °C was used as a control. Results showed that significant correlations were found among the growth indicators, with larch line 1306 having the lowest incremental growth indicators, the largest root-to-crown ratio, and better cold tolerance than the other larch lines. The levels of soluble sugars (SSs), soluble proteins (SPs), malondialdehyde (MDA), and relative electrolyte leakage (REC) increased significantly in all lines under low-temperature stress. The activities of superoxide dismutase (SOD) and catalase (CAT) showed variation over time. Significant correlations were found between MDA and REL, SS, SR, Pro, CAT, and SOD in most of the lines; no significant correlation was found between MDA and the other indices in lines 1301 and 1309; and significant correlations were found between most of the physiological indices in line 1306.

Larix olgensis is a valuable timber species in northern China, typically propagated through somatic embryogenesis (SE). However, long-term subculture can lead to a loss of embryogenic potential. This study aimed to establish a simple and stable protocol for the cryopreservation and regeneration of L. olgensis embryogenic callus (EC) that preserves its SE potential and regenerative capacity. The slow-freezing method was employed for cryopreservation. A cryopreservation protocol for L. olgensis EC was developed by optimizing the preculture duration and conditions, cryoprotectant composition and thawing temperature. The results showed that optimal outcomes were achieved using a 24 h stepwise preculture on medium containing 0.2 and 0.4 mol∙L⁻¹ sucrose, followed by cryoprotectant treatment with 0.4 mol∙L⁻¹ sucrose, 2.5% (v/v) dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 6000 (PEG₆₀₀₀), and thawing at 37 °C. EC cryopreserved using this protocol achieved a 100% recovery rate. Moreover, the cryopreserved recoverable EC successfully underwent SE, progressing through germination and rooting. Cryopreservation duration (storage duration in liquid nitrogen) did not affect cell viability and proliferation rate, confirming the protocol’s suitability for long-term cryopreservation of L. olgensis EC. This study provides a valuable reference for the cryopreservation and regeneration of L. olgensis EC, with potential applications for other coniferous species. It establishes a robust foundation for the large-scale propagation of conifers.