In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications.

Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr) is a native conifer in North China with great economic and ecological values. Somatic embryogenesis (SE) is a powerful tool for the mass clonal propagation in plants. In this study, we described a high-efficiency SE system via indirect pathways and investigated the effect of genotype, culture conditions and phytohormones on SE. Immature zygotic embryos (IZEs) of L. principis-rupprechtii Mayr were used as explant materials. In the induction stage, embryogenic tissues (ETs) were induced on mLV medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzylaminopurine (6-BA). The initiation frequencies showed significant differences (p < 0.05) among 20 genotypes of open-pollinated mother trees with the highest induction frequency reaching 30%. For tissue proliferation, proliferation in liquid medium was more efficient compared with proliferation in semi-solid medium, providing a multiplication rate of 3.12 in an 8-day subculture period. As a necessary exogenous plant growth regulator (PGR) for somatic embryo maturation in conifers, abscisic acid (ABA) was optimized at 16 mg L⁻¹ in this system. Next, an orthogonal test on osmotic pressure factors showed 50 g L⁻¹ sucrose, 7 g L⁻¹ phytagel and 75 g L⁻¹ polyethylene glycol (PEG) was the optimal combination for somatic embryo maturation in L. principis-rupprechtii Mayr. Moreover, the dispersion culture method provided a more efficient somatic embryo maturation, up to 545 per gram of fresh weight (FW). Finally, 2 g L⁻¹ of active charcoal (AC) was found to increase the somatic embryo germination rate to 63.46%. The improved protocol of SE will serve as a foundation for establishing mass propagation and genetic transformation of L. principis-rupprechtii Mayr.

DNA methylation plays a crucial role in the development of somatic embryos (SEs) through the regulation of gene expression. To examine the impact of DNA methylation on gene expression during early SE development in Picea glauca, the demethylation reagent 5-aza-dC (5-aza-2′-deoxycytidine) was employed to modify DNA methylation regions and levels during the pre-maturation stage of somatic embryogenesis. The application of 2.0 µM 5-aza-dC did not induce toxicity to SEs in early development. Following treatment, the global DNA methylation level decreased significantly on the 7^th day of pre-maturation and the 1^st week of maturation. Methylated DNA immunoprecipitation (MeDIP) sequencing revealed that differentially methylated regions, as analyzed through Gene Ontology (GO), were related to plant development and reproduction and that they were hypomethylated on the 3^rd day but hypermethylated on the 7^th day in 5-aza-dC-treated embryogenic tissues. These findings indicate that 5-aza-dC treatment positively impacts early SE development, which was inhibited following 7 d of treatment. The expression of MSH7, JMJ14, and CalS10 was associated with DNA methylation, epigenetic regulation, and somatic embryogenesis. Further analysis of methylated regions revealed that the expression profiles of MSH7, JMJ14, and CalS10 were correlated with altered DNA methylation, suggesting DNA methylation at 5 mC may play a role in controlling the expression of these genes and regulating the early development of SEs in P. glauca. This study offers new insights into the regulation of somatic embryogenesis in conifers.

Pseudotsuga gaussenii is a forest species under protection in China. Propagation via somatic embryogenesis is efficient and needed for recovering this species. In this research, effects of medium supplements, i.e., methylglyoxal-bis (guanylhydrazone) (MGBG), polyethylene glycol (PEG), and maltose, on somatic embryo (SE) development were studied. More mature SEs developed with the pre-maturation treatment medium supplemented with 50 μM or 100 μM MGBG. During SE maturation, when the medium was supplemented with both PEG and maltose, the over-proliferation of embryogenic tissue was suppressed. When maltose was eliminated from the maturation medium, SEs were slightly smaller but developed better than other treatments due to the supply of lower carbon source, whereas when PEG was taken out from the maturation medium, no mature SE was obtained, only the heavily-proliferated plant tissue. Meanwhile, DNA methylation levels differed in these SE maturation cultures at the late culture stage of SE maturation. The level increased sharply with no maltose treatment (20.28%) while decreasing without PEG supplement (10.35%). DNA methyltransferase genes MET1-1, MET1-2, MET1-3, CMT3, DRM1, and DRM2 were partially cloned in this study to detect their expression level via qPCR. Expression of these genes, except MET1-3, in the culture of no PEG was higher significantly than the cultures of other treatments at weeks 1, 3, and 10. Our results suggested that MGBG and/or PEG play an important role in stimulating SE development and maturation. Furthermore, sugar supplements at a lower level benefited SE maturation in Pseudotsuga gaussenii.

Larix principis-rupprechtii Mayr, a coniferous species indigenous to Northern China, possesses significant ecological and economic value. Somatic embryogenesis offers a pathway with significant potential for large-scale propagation, long-term germplasm conservation, and genetic transformation in L. principis-rupprechtii Mayr. However, it remains unclear whether significant variations occur in embryogenic tissues during long-term successive subculturing, which could impact the productivity of somatic embryos. This is a pivotal concern that lacks comprehensive understanding. In this study, three embryogenic cell lines were used to explore the dynamics and relationships among proliferation rate, pre-treatment proliferation rate, and embryogenic capabilities across a series of 32 subculturing cycles. Proliferation rate, pre-treatment proliferation rate, and somatic embryo maturation rate showed no significant correlation with subculturing cycles. However, there was a positive correlation between subculturing cycles and pre-treatment proliferation rate and a negative correlation with somatic embryo maturation rate in the BFU1 cell line. In addition, we utilized ten SSR molecular markers to investigate the genetic stability in embryogenic tissues during long-term subculturing. No genomic variations were detected in any of the three embryogenic cell lines, which suggests that the observed phenotypic dynamics during subculturing may not be primarily driven by genomic alterations. This study provides novel insights into the dynamics of the long-term culture of embryogenic tissues, laying a foundation for the optimization and application of somatic embryogenesis techniques in L. principis-rupprechtii Mayr and potentially other coniferous species.