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.

This work aimed to explore the effects of three combined bacterial inoculants of Bacillus spp. on the growth of Quercus mongolica seedlings, and clarify the growth-promoting effects of different bacterial inoculants, so as to provide a theoretical basis for the application of Bacillus in the cultivation of Q. mongolica seedlings.

The container seedlings of the one-year-old Q. mongolica seedlings were inoculated with Bacillus subtilis inoculants (B1), B. subtilis and Bacillus licheniformis mixed inoculants (B2) as well as B. subtilis, B. licheniformis and Bacillus amyloliquefaciens mixed inoculants (B3). Each bacterial inoculant had two concentrations of the stock solution (C0) and 20-fold diluent (C2), and water was used as the control (CK). Subsequently, the effects of different types and concentrations of bacterial inoculants on the growth indexes, photosynthetic parameters and non-structural carbohydrates (NSC) of Q. mongolica seedlings were analyzed.

Bacillus increased the seedling height, ground diameter, leaf number and biomass accumulation of Q. mongolica seedlings, enhanced the photosynthesis of seedlings, and also changed the morphology and NSC distribution pattern of roots. Bacterial inoculants B1, B2 and B3 increased the biomass ratio of leaves, stems and roots, reduced the NSC content of seedlings, elevated the ratio of soluble sugar to starch in leaves, and promoted the distribution of NSC to roots. B1 was better than B2 in promoting seedling biomass and photosynthesis. However, the B2 inoculant showed greater effects on the aboveground part, root morphology and NSC of seedlings than the B1 inoculant.

Bacillus inoculants can regulate the growth of Q. mongolica seedlings and improve their growth potential. Among the three kinds of bacterial inoculants, the mixed inoculant of B. subtilis, B. licheniformis and B. amyloliticus has the best effect. This study can provide theoretical support for the application of Bacillus in the cultivation of high-quality seedlings of Q. mongolica.

The Quercus mongolica seeds are recalcitrant and not resistant to storage. In order to provided scientific basis and technical reference for the reasonable storage of Q. mongolica seeds, the changes of endogenous hormones and cellular structure characteristics of seeds under different initial moisture content and storage temperatures were explored.

The silica gel drying was used to control the initial seed moisture content to 50% (initial moisture content after harvesting), 40%, 30% and 20%, and stored at low-temperature (4 ℃) and room-temperature (20 ℃) respectively. During the storage, the seed vigor, endogenous hormones, and cell microstructure of Q. mongolica seeds were detected.

1) The germination of newly harvested Q. mongolica seeds could reach 100%, and the seed vigor was not affected after rapid drying by silica gel. 2) During seed storage, the rates of seeds vigor loss were different at different initial moisture content and storage temperatures. The optimal moisture content for storing Q. mongolica seeds was 30%-40%, and low-temperature storage was superior to room-temperature storage. 3) Under room-temperature storage, with the increase of seed initial moisture content, the mold increased seriously, while reducing seed initial moisture content or under low-temperature storage, the seed deterioration was mainly browned or softened. 4) During the storage, the GA₃ content of Q. mongolica seeds was decreased with the decrease of the seed initial moisture content, and it decreased sharply within the first 14 days of storage. The ABA content responded differently to the storage temperature. ABA content was stable under low-temperature storage, while under room-temperature storage conditions, ABA content changed differently with different initial seed moisture contents. 5) Microstructure observation showed that the cotyledon cells of newly harvested Q. mongolica seeds were closely arranged and regular, and had no inclusions, while plumule cells were small and loosely arranged. After 14 days of storage, seed cells produce inclusions. The seed deterioration was accompanied by strong cell shrinkage or damage, unclear cell boundaries, and loss of internal substances.

Reducing the initial moisture content and storage temperature of Q. mongolica seeds is beneficial to improve seed storage tolerance, but seed water content less than 20% is not conducive to seed storage.