Morphological and Physiological Indicators for Screening Cell Lines with High Potential for Somatic Embryo Maturation at an Early Stage of Somatic Embryogenesis in Pinus Koraiensis

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.

De Novo Sequencing and Transcriptome Analysis Reveal Genes’ Specific Expression in Chinese Fir (Cunninghamia lanceolata) Callus

“…On the other hand, NEC has high levels of MDA, POD, SOD, and CAT, so it is speculated that the observed high levels of phenolic substances and polyphenol oxidase may be the cause for the SE ability loss. In Pinus koraiensis, it was also found that the phenolic substances in NEC were significantly higher than those in EC, results consistent with our findings [5].…”

“…The development of an efficient in vitro regeneration system such as SE for Chinese fir is not only useful for elite germplasm mass propagation but also represents an essential enabling technology for future genetic transformation efforts. SE is considered to be one of the most promising strategies for bulking up elite conifers' verities (e.g., Picea pungens, Larix principis-rupprechtii, and Pinus koraiensis [3][4][5]. SE is a complex process of embryo development.…”

Plant peptide hormone phytosulfokine promotes embryogenic development of calli in Pinus massoniana

“…It has been shown that the embryogenic callus of Pyrus communis was high in solubility and protein, but low in starch (Ameri et al 2020). Peng et al (Peng et al 2022;Gao et al 2023) suggested that high starch content in the embryogenic callus was the basis for successful somatic embryogenesis. In this study,the contents of soluble sugar, protein, and starch in the Y7-20 cell line with higher embryonicity (better ESM structure development) were significantly higher than those in the cell lines with lower embryonicity.…”

Transcriptomic and Metabolomic Analysis of Korean Pine Cell Lines with Different Somatic Embryogenic Potential

“…However, there are still many problems that hinder the large-scale application of this technique. For example, there is a strong genotype dependence in the process of the SE of Korean pine, and the SE ability of callus is difficult to maintain for long periods of time [ 4 , 5 ]. Similar issues have been noted in other species.…”