Cui Ying, Gao Ying, Zhao Ruirui, Zhao Jian, Li Yixuan, Qi Shuaizheng, Zhang Jinfeng, Kong Lisheng
Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, College of Biological Science and Biotechnology, Beijing Forestry University, Beijing, China.
Department of Biology, Centre for Forest Biology, University of Victoria, Victoria, BC, Canada.
Front Plant Sci. 2021 Sep 1;12:694229. doi: 10.3389/fpls.2021.694229. eCollection 2021.
Cryopreservation is one of the key technologies for the mass propagation of conifers via somatic embryogenesis. Cryotolerance and embryogenecity of conifer somatic embryos (SEs) could be affected by different temperature treatments, for which the underlying mechanisms were unknown. In this study, the developing SEs of obtained their cryotolerance with a survival rate of 100% when cultured on maturation medium at either 23°C for 4 weeks or 4°C for 10 weeks. However, only the embryos that underwent 4°C acclimation remained high embryogenicity, i.e., 91.7% based on cryovials or 29.3% on the plant tissue. Analysis of differentially expressed genes (DEGs) revealed that both 23 and 4°C treatments led to drastic changes in the gene expression, i.e., 21,621 and 14,906 genes, respectively, and the general increase in many oligosaccharides and flavonoids, in addition to the content change of proline (1.9- and 2.3-fold at 23 or 4°C) and gallic acid (6,963- and 22,053-fold). There were 249 significantly different metabolites between the samples of 23 and 4°C treatments and the changing trend of the sorbitol, fatty acids, and monosaccharides differed between these samples. During 4°C-acclimation, the metabolites of the arginine biosynthesis pathway increased between 2.4- and 8.1-fold, and the expression of antioxidant genes was up-regulated significantly. At 4°C, the up-regulated genes were for germ-like proteins, instead of seed storage proteins at 23°C. Concentrations of abscisic acid and jasmonic acid increased up to 2- and 1.5-fold, respectively, in the cold-acclimated embryos. After 10 weeks at 4°C, the embryos stayed at pre-cotyledonary stage with 17.1% less DNA methylation and fewer storage substances than those at 23°C for 4 weeks, which developed cotyledons. This research provides new insights into mechanisms underlying the response of SEs to different culture temperatures and benefits method development for germplasm conservation in conifers.
超低温保存是通过体细胞胚胎发生实现针叶树大规模繁殖的关键技术之一。针叶树体细胞胚（SEs）的耐冻性和胚胎发生能力可能受到不同温度处理的影响，但其潜在机制尚不清楚。在本研究中，获得的发育中的SEs在23°C培养4周或4°C培养10周的成熟培养基上培养时，其耐冻性达到100%的存活率。然而，只有经过4°C驯化的胚仍保持较高的胚胎发生能力，即基于冻存管的为91.7%，基于植物组织的为29.3%。差异表达基因（DEGs）分析表明，23°C和4°C处理均导致基因表达的剧烈变化，分别为21,621个和14,906个基因，并且许多寡糖和黄酮类化合物普遍增加，此外脯氨酸含量变化（23°C或4°C时分别为1.9倍和2.3倍）和没食子酸含量变化（6,963倍和22,053倍）。23°C和4°C处理的样品之间有249种显著不同的代谢物，这些样品中山梨醇、脂肪酸和单糖的变化趋势不同。在4°C驯化期间，精氨酸生物合成途径的代谢物增加了2.4至8.1倍，抗氧化基因的表达显著上调。在4°C时，上调的基因是类胚蛋白基因，而不是23°C时的种子贮藏蛋白基因。在冷驯化的胚中，脱落酸和茉莉酸的浓度分别增加了2倍和1.5倍。在4°C下培养10周后，胚停留在子叶前期，其DNA甲基化比在23°C下培养4周并发育出子叶的胚少17.1%，贮藏物质也更少。本研究为SEs对不同培养温度的响应机制提供了新的见解，并有助于针叶树种质保存方法的开发。
