Studies On The Anatomical,Physiological And Genetic Characteristics Of Hydroxyproline Tolerant (HYP) Cauliflower Mutants

Broccoli（scientific name:Brassica oleracea var.botrytis Linnaeus）is a variety of wild cabbage in the mustard family and Brassica genus.It contains vitamin A,vitamin C,protein,fat,calcium,andβ-Carotene,etc.,has the effect of preventing cancer and various coronary artery diseases.Broccoli is not resistant to long-term frost,and the suitable temperature for growth is 17 to 20℃.At 25℃,the seeds germinate fastest,and the suitable temperature for seedling growth is 20 to 25℃.However,broccoli is deficient in germplasm resources in China,especially in cold resistant germplasm resources.In this study,the loose callus of cauliflower was used as the experimental material to obtain mutant cell lines through EMS somatic cell mutation and hydroxyproline screening,and then somatic embryogenesis was used to obtain cauliflower mutant plants.The purpose was to cultivate cold resistant cauliflower varieties;In order to further verify the cold tolerance characteristics of cauliflower mutants,the mutant plants were transplanted after elongation and rooting culture.After one month of survival,their leaf anatomy（stomatal density,etc.）and photosynthetic characteristics were studied.At the same time,physiological indicators and transcriptome measurements were conducted on their leaves after 12 hours of low-temperature treatment to find out the changes in phenotypes and genetic expression of cold resistance of the mutant plants,To further verify the feasibility of using somatic cell in vitro mutagenesis technology to screen cold resistant mutant plants in cauliflower,with a view to laying a certain foundation for the cultivation of cold resistant varieties of cauliflower.The main research results are as follows:（1）The leaves of cauliflower mutant lines were dissected with bare hands and sliced for observation.It was found that there were significant differences in leaf thickness among cauliflower mutant lines.Among the 7 mutant lines,6 had leaf thickness greater than CK,and the leaf thickness of the 7 cauliflower mutant lines and CK was ranked as follows:Y232>Y308>Y266>Y7>Y140>Y8>CK>Y149;In terms of epidermal cell thickness,the mutant plants showed no significant changes;The changes in stomatal characteristics of the lower epidermis of mutant leaves are extremely significant,and the stomatal density of all 7mutant lines is less than CK,and the difference is extremely significant.The order of pore density size is:CK>Y140>Y232>Y149>Y8>Y7>Y266>Y308.（2）The photosynthesis of cauliflower mutant strains was measured using a photosynthetic apparatus,and the results showed significant differences in the photosynthetic characteristics of cauliflower mutants.From the perspective of net photosynthetic rate,the net photosynthetic rate of cauliflower mutant lines is higher than that of CK,and overall,the net photosynthetic rate of cauliflower mutant lines has increased by 1.080 compared to CKμmol/m²/s-16.832μmol/m²/s;From the perspective of intercellular CO₂ concentration,except for Y149,the intercellular CO₂concentration of the 7 mutant lines was significantly higher than that of CK,all other lines were lower than that of CK;From the analysis of stomatal conductance,the stomatal conductance of strain Y149 was significantly lower than that of CK,at409.227mmol/m²/s,which was 0.883 times that of CK.The stomatal conductance of the other six strains was significantly higher than that of CK;From the perspective of transpiration rate,there is little variation between cauliflower mutants and normal cauliflower plants.Among them,the transpiration rates of Y8,Y266,and Y308 are significantly higher than those of CK,at 4.588mmol/m²/s,1.976 mmol/m²/s,and 2.232 mmol/m²/s,respectively,which are 3.872,1.668,and1.884 times higher than those of CK.The transpiration rates and CK of other plant lines are not significantly different.（3）Physiological indicators were measured on the leaves of 7 cauliflower mutant lines after low-temperature treatment.The analysis results showed that the free Pro content,SOD activity,soluble sugar content,POD activity,soluble protein content,and CAT activity of 70%-90%of the hydroxyproline tolerant cauliflower mutant lines showed an upward trend,while the malondialdehyde content showed a downward trend.On the basis of the above,this article conducted a membership function analysis on eleven indicators,including anatomical indicators,physiological indicators,and stomatal density,to comprehensively evaluate the cold resistance between hydroxyproline tolerant cauliflower mutants and non mutagenic cauliflower.The results showed that the comprehensive evaluation values were Y140（0.699）>Y266（0.682）>Y308（0.617）>Y232（0.587）>Y7（0.563）>Y8（0.482）>CK（0.328）>Y149（0）.Therefore,compared with CK,the hydroxyproline tolerant cauliflower mutant has stronger cold resistance.（4）Through high-throughput sequencing technology,the transcriptome of mutant plants and CK after low temperature treatment was studied and analyzed.The results showed that after low temperature stress,9580 differential genes were identified between the hydroxyproline tolerant cauliflower mutant（Y308）and the non mutagenic cauliflower（CK）,of which 5273were up-regulated and 4307 were down regulated.A total of 4211 genes from the 9580differentially expressed genes identified were successfully enriched on GO Term,and were annotated on 10,11,and 2 GO categories of molecular function,biological process,and cellular component,respectively.The most annotated differentially expressed genes were in biological processes,followed by molecular function and cellular components.Differentially expressed genes mainly participate in biological regulation,cellular processes,response to stimuli,metabolic processes,and localization related to biological processes.Differently expressed genes in molecular functional items mainly affect binding and catalytic activity,The differentially expressed genes related to cell component entries are mainly involved in protein containing complexes and cellular anatomical entities.Among the 9580 differentially expressed genes identified,a total of 8341 genes were successfully enriched into 5 pathway branches and19 KEGG pathways.Among them,the metabolic pathway branch had the highest expression of differentially expressed genes,including 11 KEGG pathways and a total of 5574 genes,including the global overview map pathway and the carbohydrate pathway.Other KEGG pathways include transport and catabolic pathways,membrane transport,signaling pathways,folding sorting and degradation,replication and repair,transcription,translation,and environmental adaptation pathways.The first six KEGG Pathways in the significantly enriched KEGG Pathway belong to metabolic pathways,namely carbon fixation in photosynthetic organisms,photosynthesis antenna proteins,porphyrin metabolism,MAPK signaling pathway plant Carbon metabolism and biosynthesis of amino acids.Further analysis of the MAPK signaling pathway revealed that the significantly enriched KEGG Pathway is:MAPK signaling pathway-plant,plant pathogen interactions,and plant hormone signaling.The above comprehensive explanation indicates that differentially expressed genes are mainly enriched in the pathways of photosynthesis and carbohydrate metabolism.The MAPK pathway is a widely present signal transduction pathway in eukaryotes,with significantly enriched gene expression levels,indicating that mutant Y308 has a stronger role in regulating protein activity and signal transduction compared to CK.Among them,differential genes in plant hormone signal transduction pathways are significantly enriched,and endogenous hormones in plants can quickly perceive external environmental changes,regulate stress,and enhance plant stress resistance.Therefore,the above pathways have improved the cold tolerance of hydroxyproline tolerant cauliflower mutants,effectively protecting their growth and development under adverse conditions,and improving their survival rate.