Effect of subzero temperature on seed viability and physiological characteristics of Quercus mongolica and Quercus acutissima

To investigate the differential low-temperature tolerance and the the underlying physiological mechanisms between Q. mongolica and Q. acutissima seeds, and to provide theoretical insights for improving oak seed storage.

Freshly harvested Q. mongolica and Q. acutissima seeds within one week were used as experimental materials. The experiment was conducted under simulated natural environmental conditions. After seed maturation, the seeds were stored sequentially at 0, -2, -4, -5, -6, -8, -10, -12, and -14 ℃ for cryopreservation, with each temperature gradient maintained for 7 days. Regular sampling was conducted to monitor germination percentage, comparing the seeds' low-temperature tolerance differences between the two oak species. Additionally, changes in the contents of small-molecule sugars, soluble proteins and starch were analyzed. The seeds were stored at -3 ℃ for one year, after which changes in seed viability indicators before and after storage were measured to investigate the subzero non-freezing storage strategy.

The low-temperature tolerance of Q. mongolica and Q. acutissima seeds differed significantly. As the temperature decreased, seed viability initially remained stable but then declined rapidly. Q. mongolica seeds could tolerate temperatures as low as approximately -8℃, whereas Q. acutissima seeds had a minimum tolerance threshold of around -5℃. Beyond these thresholds, seed viability sharply decreased. The contents of small-molecular sugars such as fructose, glucose, and sucrose were induced to accumulate with decreasing temperatures. However, raffinose, stachyose, lactose, galactose, xylose, ribose, and rhamnose were nearly undetectable. Soluble protein in both Q. mongolica and Q. acutissima seeds peaked at -2℃, while starch gradually declined with lower temperatures. In contrast, malondialdehyde (MDA) levels increased as temperatures decreased. After one year of cryopreservation at -3℃, the seeds of both species maintained a high viability, with a relative germination rate still exceeding 93.6%.

The seeds of Q. mongolica exhibited stronger low-temperature tolerance than those of Q. acutissima. Soluble proteins, starch, and sucrose played crucial roles in regulating seeds' cold tolerance, while the absence of non-reducing sugars such as raffinose and stachyose may be one of the primary factors contributing to the recalcitrant nature of both oak species. Both Q. mongolica and Q. acutissima seeds maintained high viability when stored at -3℃, demonstrating the feasibility of subzero non-freezing storage for recalcitrant seeds. These findings provide important insights for developing effective storage protocols for Quercus species with recalcitrant seeds.