| Rapeseed is the major oil crop in China.As the main producing areas of rapeseed,the main cropping system of Yangtze River basin is rice-rape multiple cropping systems.Recent years,farmers prefer to choose the simplified cultivation and direct seeding method for rape cultivation,which resulting in a large area delayed of rape sowing date.And low temperature of late sowing have become one of the obstacle factors of rape cultivation by affecting seed germination and emergence.In this study,germination test were carried through at low temperature with 30 varieties(materials)of rapeseed,and different resistant materials were selected for comparative studies.We established an evaluation system of low-temperature-resistant of rapeseed germination,estimate the base temperature of germination,and discussed the response pathway of seed germination and seedling growth to low temperature stress.Through the above work,we intend to provide some theoretical and technical approach to mitigate the restrictions of low temperature to seed germination and seedling morphogenesis,achieve uniform and strong seedling of direct-sowing rape,and promote cultivation acreage of rapeseed.The main results are as follows:1 Low temperature tolerance evaluation of rapeseed during germination and emergenceThrough principal component analysis,we screened out six indexes effectively as the evaluation index,emergence percentage,seedling vigor index,root length,total length,shoot fresh weight,total fresh weight.According to these six index we use the membership function to calculate,and found that in these 30 materials:Fengyou 823,Deheyou 999 and Qinyou No.7 have the robust germination ability under low temperature;Shengguang 127,HN4,Xiangzayou No.2 and Huayouza No.14 also have a strong ability.A35,Huazao 291 and Deyou No.8 have poor performance which should be avoided to sowing late.2 Base temperature of germination and emergence may at 0.24 ℃ and 0.80 ℃When average temperature is equal,the variable temperature is more conducive to promote germination and emergence of low temperature sensitive varieties under the low temperature stress.The base temperature variation of eight varieties is 0.24℃～4.97 ℃and 0.80℃～5.55 ℃,respectively(based on GI),when germination and emergence occur at constant temperature.In the natural variable temperature environment,the estimate of emergence base temperature is 1.23℃～4.19℃.The base temperature of resistant varieties(materials),such as Qinyou No.7,Wenyou 99,HN3,Ganyouza No.5,are much lower than sensitive ones,and they are all below 2.5 ℃.Comparing different estimation methods,we found that reciprocal time of median germination performed well in the low temperature threshold estimation,which is more suitable for the parameter estimation of the relatively independent stage.And the germination index and mean daily germination is simple and easy to calculate,the result is comparatively reliable,these can be used for fast estimation.The correlation coefficient of fitting result of germination rate index was larger,but the estimation error was also the largest.Repeated probit analysis includes all the data points in the whole process of germination,the amount of data is large,the estimation error is small,and the results were more statistical significant.3 Effect of low temperature stress on seed germination and seedling growthLow temperature significant inhibition of the rape seed germination and seedling morphogenesis,reduce the germination percentage,emergence percentage and seedling vigor index,extend the mean germination time.Seedling fresh weight,seedling length and root length were significantly reduced under low temperature.Low temperature at different periods have a different inhibition degree to root and shoot,root inhibition degree is higher than shoot in the germination stage,but in the seedling stage shoots performed more sensitive.4 Physiological responses of rape seedlings to low temperature stressThe strategy of low temperature resistance of different varieties may be differences.Under low temperature stress,osmotic regulation system priority calls against low temperature in resistant varieties(Qinyou No.7),subsequently the antioxidant enzyme system.While in Sensitive varieties(Fengyou 730),the osmotic regulation system and antioxidant enzyme system were stimulated simultaneously.Fengyou 730 is more sensitive to low temperature stress,which start physiological defense much earlier than Qinyou No.7,while Qinyou No.7 need a lower temperature or longer stress duration to trigger cold response.5 Transcription response of rape seedlings to low temperature stressTranscriptome sequencing and analysis utilized on samples collected from resistance material Qinyou No.7 after cold treatment about 0 h,24 h,48 h and 96 h.Compared with the control,the amount of differentially expressed genes including un-regulated gene 1077,1090,1167,and down-regulated gene 1249,1244,1244,respectively.Among them,there were 428 up-regulated genes and 381 down-regulated genes common expression in three samples.GO analysis shows that the main molecular function of these differentially expressed genes involved in metabolism process,cellular process and single-organism process.KEGG pathway analysis also shows that the main enrichment of differentially expressed genes is in metabolic pathways and genetic information processed terms,and include the signal transduction in environmental information process,adaptability of organisms system to environment.Through the STEM clustering analysis 7 patterns of expression was significantly associated,in which continues up-regular pattern,we found include the protein processing in endoplasmic reticulum,protein export,amino acid metabolism,starch and sucrose metabolism,glutathione S-transferase and peroxidase related genes.In addition,transcription factors like zinc finger,MYB family and MADS-box family,and the key enzyme and calmodulin of IP3/Ca2+ and ABA signal transduction pathway were all observed differentially expressed under low temperature stress,showed the corresponding pathways had participated in the response of rape seedlings to low temperature stress.The results of qRT-PCR was consistent with the results from sequencing.Differentially expressed genes provides a valuable candidate genes for function analysis and genetic transformation of rapeseed cold resistance related gene. |
