Study on the regulation of Somatic Embryogenesis capacity in Chinese fir by Nicotinamide under Low-Temperature conditions

Liu Yifan, Wei Yuting, Lu Lu, Lin Zezhong, Zheng Weiwei, Zheng Renhua

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0

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南京林业大学学报（自然科学版）
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Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0 DOI: 10.12302/j.issn.1000-2006.202605008

Study on the regulation of Somatic Embryogenesis capacity in Chinese fir by Nicotinamide under Low-Temperature conditions

  • Liu Yifan1,2, Wei Yuting1,2, Lu Lu1, Lin Zezhong2, Zheng Weiwei3,*, Zheng Renhua1,2,*
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Abstract

【Objective】Somatic embryogenesis is an efficient technique for plant clonal propagation and is widely applied in large-scale propagation, elite germplasm breeding, and the establishment of genetic transformation systems. Embryogenic cell lines serve as the initial material for breeding and genetic transformation; however, their embryogenic potential is often gradually lost during frequent subculture. This study focused on the low-temperature preservation of Cunninghamia lanceolata embryogenic cell lines, aiming to establish and optimize an economical and practical low-temperature maintenance system for embryogenic cultures.【Method】The embryogenic cell line of the elite C. lanceolata genotype A4098-1-3 was used as experimental material. Cells were subcultured on proliferation medium supplemented with nicotinamide (NAM) and subsequently maintained in darkness at 4 °C, 8 °C, and 12 °C for 90 days. Embryogenic cultures maintained under normal subculture conditions served as the control. Cell viability, proembryogenic mass (PEM) structure, and somatic embryo induction efficiency were compared among treatments to evaluate the role of NAM in maintaining embryogenic potential under low-temperature conditions. Transcriptome analysis was further conducted to elucidate the molecular mechanisms by which NAM preserves embryogenicity during low-temperature culture.【Result】The results showed that after 90 days of culture at 4 °C, embryogenic cells treated with 4 mg/L NAM exhibited significantly higher viability than untreated cells cultured at the same temperature. The proembryogenic mass structure of cell aggregates treated with 4 °C + 4 mg/L NAM was consistent with that of cultures maintained under normal subculture conditions at 23 °C. Under all tested low-temperature conditions, untreated cultures displayed significantly lower somatic embryo induction efficiency than cultures maintained at 23 °C, whereas NAM-treated cultures showed significantly improved induction efficiency compared with untreated cultures. Notably, the somatic embryo induction efficiency of the 4 °C + 4 mg/L NAM treatment was significantly higher than that of the 23 °C control. Transcriptome analysis further revealed that NAM significantly upregulated genes involved in defense responses and auxin signal transduction pathways.【Conclusion】These findings suggest that NAM may maintain the embryogenic potential of C. lanceolata cells cultured at 4 °C by activating defense-related pathways, promoting auxin signaling, preserving cell viability, and maintaining the structural integrity of proembryogenic masses. This study preliminarily established a low-temperature preservation method for C. lanceolata embryogenic cultures, extending the subculture interval while maintaining embryogenic competence. The results provide a practical strategy and theoretical basis for addressing the gradual loss of embryogenicity in conifer embryogenic cell lines during repeated subculture at 23 °C, and offer important implications for advancing forest tree cell engineering and genetic improvement research.

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Cunninghamia lanceolata / Embryogenic cell line / Nicotinamide / Low-temperature preservation

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Liu Yifan, Wei Yuting, Lu Lu, Lin Zezhong, Zheng Weiwei, Zheng Renhua. Study on the regulation of Somatic Embryogenesis capacity in Chinese fir by Nicotinamide under Low-Temperature conditions[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 0 https://doi.org/10.12302/j.issn.1000-2006.202605008

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