Analysis On Recalcitrant Characteristics And Preservation Of Castanea Mollissima Bl.Seed

Chestnut(Castanea mollissima Bl.)is an important woody grain tree species,it is typically recalcitrant seed,which can not be preserved for a long time.The goal of this study was analyze the recalcitrant characteristics and put forward effective storage technology of the chestnut seeds.In this study,the‘Yanquan'chestnut seeds were used as experimental materials,the different dehydration and cryopreservation experiments of seeds and embryonic axis(embryo with partial cotyledons removed)were conducted,the germination rate of embryonic axis was determined with different moisture content.At the same time,differential scanning calorimetry(DSC)was used to measure the thermodynamic parameters of embryonic axis.The changes of microstructure,polysaccharides,proteins and ultrastructures in the cells of embryonic axis with different moisture contents were observed by anatomical study.The proteomic changes of embryonic axis with different moisture contents were analyzed by using the isobaric tags for relative and absolute quantitation(i TRAQ)technique.The results were as follows.(1)The moisture content of the cotyledons was lower than that of the embryonic axis of chestnut seed.The dehydration effect of silica gel was better than that of natural dehydration,the use of"dryer+fan"for embryonic axis had higher dehydration efficiency than other methods,the embryo moisture content was reduced to 11.8%at 16h.The moisture content of embryonic axis decreased from61%to 10%,and the germination rate decreased from 100%to 25.6%,indicated that chestnut seeds are sensitive to dehydration.(2)The results of differential scanning calorimetry(DSC)showed that with the decrease of embryo moisture content of chestnut seed,the onset temperature,peak temperature and enthalpy decreased regularly.When the moisture content decreased to 20%,no crystallization peak appeared.The critical moisture content of the embryonic axis of'Yanquan'chestnut was 23.9%.(3)The results of anatomical study showed that when the moisture content decreased to 30%,the cell wall separated,when the moisture content decreased to 10%,the cytoplasm shrunk seriously,and a large number of cells degraded.With the decrease of moisture content,the polysaccharides and proteins in the cells gathered first and then degraded.The serious shrinkage and degradation of the cytoplasm were important reasons for the dehydration injury of chestnut seed.(4)According to the isobaric tags for relative and absolute quantitation(i TRAQ),5201 credible proteins were identified in embryonic axis with different moisture contents,including 912 differential proteins.When the moisture content decreased to 30%,the“peroxisome”,“starch and sucrose metabolism”and“galactose metabolism”were significantly affected,and the"peroxisome"related genes were significantly up-regulated.When the moisture content decreased to 20%,“protein processing in endoplasmic reticulum”was significantly affected,"ribosome protein synthesis"related genes were significantly up-regulated,“protein processing in endoplasmic reticulum”and"metabolic pathway"related genes were significantly down-regulated.When the moisture content decreased to10%,there was a significant difference in"structural molecular activity"and"binding"of molecular function for the first time,the"metabolic pathway"related genes were significantly down-regulated,the"ribosome protein synthesis"related genes were significantly up-regulated.The endoplasmic reticulum protein processing pathway and ribosome protein synthesis pathway play an important role in the dehydration process of chestnut embryo.The lower moisture content will significantly affect the molecular function and metabolic pathway of the embryo.(5)The seeds of chestnut can be preserved for 12 months in a low temperature of 2?and sealed environment,and there was no significant difference in germination rate,root length and shoot length.In this study,the start germination time of chestnut seeds was 3d,and the mean germination time(MGT)was 6.3d.(6)The germination rate of vitrification method(20.0%)and programmed vitrification method(25.8%)was higher than that of other cryopreservation methods,which indicated that PVS₂ could improve the survival rate of embryo after cryopreservation.The optimized cryopreservation method can increased the germination rate of embryonic axis to 60%,which is suitable for cryopreservation of chestnut embryo.The thermodynamic characteristics of embryonic axis with different moisturecontent changed after PVS₂ treatment and cryopreservation.The basic medium types had the greatest influence on the differentiation rate,bud number and height of explant.In this study,the most suitable medium for tissue culture of embryonic axis seedlings of'yanquan'chestnut was DKW+2 mg/L 6-BA+0.2 mg/L NAA+0.04 mg/L IBA.In this study,a method for determining the critical dehydration water content of chestnut was developed based on DSC technology for the first time;It was found for the first time that endoplasmic reticulum protein processing pathway and ribosomal protein synthesis pathway had an important response to recalcitrant seed dehydration;The formation mechanism of chestnut embryo dehydration injury was proposed for the first time.The results provide a theoretical and technical reference for the study of recalcitrant mechanism and scientific storage of chestnut seeds.