Abstract:

Objective: In this study the critical moisture content of embryo axis of recalcitrant seed Castanea mollissima was studied,the seed cooling preservation technique and embryo axis cryopreservation technique were explored in order to provide scientific basis for the preservation of recalcitrant seeds in seed bank of chestnut and other recalcitrant forest trees. Method: Chestnut embryo axes were cut from the seed and subjected to rapid dehydration with silica gel to target moisture content,after which the germination rate of embryo axes at different moisture content was determined. Differential scanning calorimetry (DSC) was used to determine the thermodynamic characteristic parameters of embryo axis at different moisture content. According to the relationship between embryo axis moisture content and corresponding enthalpy,the critical moisture content of Chinese chestnut was calculated. Finally,the chestnut seeds were preserved at low temperature with the method of low temperature sealing preservation. The embryo axis cryopreservation of Chinese chestnut with different moisture content was determined by direct freezing,programmed freezing,vitrification freezing and programmed vitrification freezing. Result: 1) When the embryo axis moisture content decreased to 20%,the germination rate began to decline. When the embryo axis moisture content decreased to 10%,the germination rate decreased to 25.6%. 2) The DSC results showed that the onset temperature of crystallization,peak temperature and enthalpy were all decreasing with the decrease of embryo axis moisture content,and there was a significant positive correlation between them.When the embryo axis moisture content dropped to 25%,the onset temperature of crystallization drops to -37.2℃,the peak temperature dropped to -44.1℃,and the enthalpy value was 5.0 J·g^-1 dw.When the embryo axis moisture content decreased to 20%,no peak appeared in cooling crystallization and heating melting stages. 3) Base on the embryo moisture content and corresponding enthalpy,the critical moisture content of this chestnut variety was 23.9%. 4) Chestnut seeds were sealed and stored in a low temperature environment of 2℃,there was no significant change (P>0.9) in germination rate chestnut seeds and shoot lengths and root lengths of chestnut seedlings after 6 months storage. 5)Among the four cryopreservation methods,vitrification and programmed vitrification had higher embryo axis germination rate than direct freezing and programmed freezing,and the best method was programmed vitrification freezing. Conclusion: Chinese chestnut is a typical recalcitrance seed,and the embryo axis moisture content decreased to 20% or less will affect the germination activity of the embryo axis. Based on differential scanning calorimetry technique combined with embryo axis germination test of different water contents can effectively determine the critical water content of this chestnut variety of 23.9%. Chestnut seeds can be preserved more than 6 months under low temperature (2℃) with no significant change in germination rate and shoot lengths and root lengths. The embryo axis germination rate of chestnut can be improved after cryopreservation by adding PVS₂.

Key words: Chinese chestnut, recalcitrant seed, thermodynamic analysis, desiccation sensitivity, differential scanning calorimetry