Abstract:

Objective: This study aims to elucidate the physiological and molecular regulatory effects of prolonged subculture on Picea abies embryogenic callus, and reveal the underlying mechanisms of its declining somatic embryogenic potential, so as to provide a theoretical basis for optimizing the somatic embryogenesis system and facilitating large-scale clonal propagation of conifers. Method: In this study, embryogenic callus from different P. abies cell lines with varying subculture generations was subjected to long-term subculture. Histocytological observations were performed, and the correlations between endogenous hormone content, gene expression patterns, and differentiation capacity were comprehensively analyzed. Result: 1) Analyses of histocytology and differentiation capacity indicated that as the number of subculture generations increased, the embryogenic callus of P. abies exhibited structural compaction and browning, accompanied by a progressive loss of embryogenicity. There were significant differences in the decline of differentiation capacity among the four cell lines, while the differentiation capacity of all four cell lines exhibited a unimodal pattern of first increasing and then decreasing. Among them, cell line 32-29 retained the highest potential for sustained differentiation. 2) Endogenous hormone analysis revealed that the dynamic changes of cytokinin (CTK) and gibberellin (GA₃) were consistent with the changes in differentiation rates of embryogenic callus, initially rising before falling. There was a significant positive correlation between the two, indicating that these hormones might play an important role in sustaining embryogenic potential. The content of indole-3-acetic acid (IAA) remained at a relatively low level with a transient increase followed by a slow decline, while the content of abscisic acid (ABA) exhibited a general decreasing trend. 3) An integrative analysis of gene expression level, somatic embryo number, and endogenous hormone content revealed that the expression levels of genes ARF6, BLH1, GH3.6, and SCL1 were significantly downregulated with increasing number of subcultures, and were significantly positively correlated with the number of differentiated somatic embryos, indicating positive regulators of somatic embryogenesis. On the other hand, the expression levels of genes ZAT9, WHY1, ZHD1, SCR, and AP2L2 were significantly upregulated with increasing number of subcultures, indicating potential negative regulatory factors. Furthermore, the expression levels of these key genes were closely related to the cytokinin (CTK) and gibberellin (GA₃) hormone signaling pathways. There was a significant positive correlation between ABA levels and WHY1, BLH1, SCR, SCL1, and ZHD1 expression, suggesting a co-regulatory network governing callus differentiation and development. Conclusion: In summary, the decline in somatic embryogenic potential of P. abies embryogenic callus is a biological process co-regulated by weakened cell division, an imbalance of endogenous hormone homeostasis, and the aberrant expression of key genes in somatic embryogenesis.

Key words: Picea abies, somatic embryogenesis, embryogenic callus, long-term subculture, endogenous hormones, gene expression