ABSTRACT
The embryogenesis capacity of conifer callus is not only highly genotype-dependent, but also gradually lost after long-term proliferation. These problems have seriously limited the commercialization of conifer somatic embryogenesis (SE) technology. In this study, the responsive SE cell line (R-EC), the blocked SE cell line (B-EC), and the loss of SE cell line (L-EC) were studied. The morphological, physiological, transcriptomic, and metabolomic profiles of these three types of cells were analyzed. We found that R-EC had higher water content, total sugar content, and putrescine (Put) content, as well as lower superoxide dismutase (SOD) activity and H2O2 content compared to B-EC and L-EC. A total of 2566, 13,768, and 13,900 differentially expressed genes (DEGs) and 219, 253, and 341 differentially expressed metabolites (DEMs) were found in the comparisons of R-EC versus B-EC, R-EC versus B-EC, and B-EC versus L-EC, respectively. These DEGs and DEMs were mainly found to be involved in plant signal transduction, starch and sugar metabolism, phenylpropane metabolism, and flavonoid metabolism. We found that the AUX1 and AUX/IAA families of genes were significantly up-regulated after the long-term proliferation of callus, resulting in higher auxin content. Most phenylpropane and flavonoid metabolites, which act as antioxidants to protect cells from damage, were found to be significantly up-regulated in R-EC.
Subject(s)
Pinus , Tracheophyta , Transcriptome , Hydrogen Peroxide , Pinus/genetics , Cell Line , Embryonic Development , Flavonoids , Republic of Korea , Sugars , Gene Expression Regulation, Plant , Plant Somatic Embryogenesis TechniquesABSTRACT
Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine (BSO, the inhibitor of glutathione synthase) on the EC proliferation rate in Korean pine (Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.
ABSTRACT
Many cell lines in the embryogenic callus cannot produce somatic embryos (SEs) even if they meet the optimal SE maturation culture conditions during conifer somatic embryogenesis. This phenomenon hinders the progress of the industrial-scale reproduction of conifers. Therefore, there is an urgent need to obtain morphological and physiological markers to screen embryogenic calli in response to SE maturation conditions. To detect cell lines with high somatic embryogenesis potential during the proliferation process, we counted the number of pro-embryos and early SEs (ESEs) in different cell lines and storage substances, endogenous hormones, and polyamine contents. The results showed that the yield of P. koraiensis SEs was heavily dependent on genotype (p = 0.001). There were high levels of PE III (pro-embryo III) number, ESE number, and soluble protein content, in the response cell lines (R cell lines), which were 1.6-, 3-, and 1.1-fold those of the obstructive cell lines (B cell lines), respectively. The B cell line had high levels of starch, auxin (IAA), Put, Spd, and putrescine: spermine (Put: Spm) compared to the R cell line. In addition, the numbers of PE III, ESEs, and soluble protein content were significantly positively correlated with SE yield. In contrast, the contents of starch, abscisic acid (ABA), Put, Spm, and Spd were significantly negatively correlated with SE yield. To ensure the accuracy of the results, we used nine cell lines to test the results. The PE III and ESE numbers and the Spm and Spd contents were positively correlated with SE yield, while the levels of starch, ABA, IAA, Put: Spd, and Put: Spm were negatively correlated with SE yield. Thus, we recommend using high PE III and ESEs as morphological indicators and low levels of starch, IAA, ABA, and Put: Spm as physiological markers to screen cell lines with a high somatic embryogenesis potential. In addition, we also found that the relationship between Spd, Spm, and SE yield was opposite in the two experimental results. Therefore, we speculate that the differences in Spd and Spm content are mainly affected by genotype. In conclusion, this study obtained the morphological and physiological markers of some high-somatic embryogenic cell lines by comparing the differences between nine somatic embryogenic cell lines. Our results can guide the improvement of conifer somatic embryogenesis technology and can provide a theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.
