Evaluation Of Cold-resistant Germplasm And Study On Cold-resistant Physiology And Molecular Mechanism Of Pinus Massoniana Lamb.

Pinus massoniana Lamb.has become the most important native timber species in China with its excellent characteristics of rapid growth,high yield,strong adaptability,high comprehensive utilization and wide range of uses.The natural distribution areas of improved provenances of P.massoniana are mostly in the North tropical and south subtropical regions.The improved provenances of P.massoniana in Guangxi have the advantages of rapid growth and high yield.However,due to the great differences in the tolerance range of temperature among different provenances and families of P.massoniana,low temperature has become the primary limiting factor for the promotion of improved varieties of P.massoniana in the south to the north.Therefore,it is crucial to improve the cold resistance of P.massoniana and to select and breed excellent cold resistant families for the promotion of improved varieties of P.massoniana in China.In this study,254 P.massoniana family seedlings from different seed orchards were screened for cold resistance based on the method of the field cold resistance identification.Then one cold resistant family and one non-cold resistant family from the same seed orchard were selected based on the field cold resistance screening results to investigate the changes of chlorophyll fluorescence characteristics,physiological indexes and microstructure of different cold resistant families under different cold stress so as to explore their response to cold stress.The transcriptomics and fluorescence quantitative PCR techniques were also used to investigate the cold resistance regulatory network,key gene screening and gene expression patterns.The main findings were as follows.（1）A total of 254 seedlings of P.Massoniana families from different seed orchards were naturally screened in the field,and 12 cold-resistant families were selected by combining seedling height growth,survival rate and frost damage index.（2）According to the field screening results,cold resistant families and non-cold resistant families in the same seed orchard were selected to study the chlorophyll fluorescence characteristics,physiology and biochemistry under different low temperature treatments.The results showed that the actual photochemical efficiency（ФPSⅡ）,electron transfer rate（ETR）,photochemical quenching coefficient（q P）,soluble sugar content and peroxidase activity of cold resistant families under low temperature stress were significantly higher than those of non-cold resistant families,while the content of malondialdehyde was significantly lower than that of non-cold resistant families,showing a stronger resistance to cold.（3）There was no significant difference in cell morphology between the two families under low temperature treatment.The accumulation of starch grains in stems and leaves and lignin in roots,stems and leaves of cold resistant families under low temperature was higher than that of non-cold resistant families,indicating that cold-resistant families can accumulate more nutrients and metabolites to improve their cold resistance under low temperature.（4）The gene expression of P.massoniana families in response to freezing stress was analyzed at the transcriptional level by transcriptome sequencing.In the 0 h and 24 h comparison groups,there were 7148 significantly differentially expressed genes,of which4401 were up-regulated and 2747 were down-regulated;There were 1991 differentially expressed genes in the 0 h and 48 h comparison groups,including 1123 up-regulated genes and 868 down regulated genes.Transcriptome analysis revealed that the differentially expressed genes in P.massoniana seedlings under low temperature stress were mainly enriched in metabolic pathway,plant pathogen interaction,MAPK signaling pathway,galactose metabolism,starch sucrose,plant hormone transduction and phenylpropanoid biosynthesis.（5）Seven functional genes and six transcription factors from the major cold resistance gene enrichment pathways were selected as cold resistance candidate genes and their expression patterns were investigated in different cold resistance families of P.massoniana.All of them were found to be actively involved in the response of P.massoniana seedlings to low temperature.Among them,peroxidase genes related to lignin synthesis were significantly up-regulated under low temperature stress.We also found that peroxidase activity and lignin content increased in all tissues of P.massoniana seedlings under low temperature stress in our previous study.Therefore,it is hypothesized that under low temperature stress,P.massoniana seedlings regulate peroxidase activity by regulating peroxidase gene to increase lignin content in plants and thus improve its cold resistance.