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Long-term subculture preservation significantly impacts somatic embryogenesis (SE) in various plant species. Studies on its effects in litchi (Litchi chinensis Sonn.) remain scarce. This study aimed to investigate the effects of subculture times on SE in litchi embryogenic callus (EC) and to uncover the metabolic basis for its decline, thereby providing a theoretical foundation for improving SE efficiency. Using 'Feizixiao' litchi EC materials from different subculture times (denoted as J11, J22, J48, J65 and J86). Somatic embryo induction rate, phenotypic characteristics, and dynamic changes in soluble sugars, organic acids, free amino acids, and energy-related substances were measured. The results showed that subcultures times significantly influenced SE in litchi EC. The embryo induction rate initially increased and then decreased with increasing subculture times, while the yield of normal somatic embryos (monocotyledonous, dicotyledonous, and polycotyledonous embryos) was also significantly affected. Among the five groups, J22 exhibited the highest induction rate (257.33%) and the greatest number of milky-white embryos. Sucrose content showed a significant positive correlation with SE, highlighting its role as the key carbon source for litchi SE. Fluctuations in soluble sugar content were primarily attributed to variations in sucrose. Low-subculture materials (J11 and J22) showed significant decreases in citric acid, succinic acid, and malic acid during the middle-late SE induction stages, indicating a stronger tricarboxylic acid (TCA) cycle flux compared to high-subculture materials (J48, J65, J86). The total amino acid content in EC initially increased and then stabilized during SE induction, with low-subculture materials maintaining higher overall amino acid levels. The contents of two major amino acids (glutamine and alanine) were significantly more abundant in low-subculture materials. Furthermore, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) initially increased and then decreased, with AMP being the most abundant. Low-subculture materials maintained significantly higher AMP levels during the first 49 days of SE induction, particularly in J22. The results demonstrate that subculture times is a critical factor influencing SE in litchi EC. Long-term subculture induces embryogenic decline by impacting energy supply and amino acid metabolism.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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