Optimization Of Genetic Transformation System And Preliminary Verification Of TFL1 Gene Function In Watermelon

Watermelon(Citrullus lanatus),as one of the most popular horticultural crops,is favored by consumers for its juicy flesh and refreshing taste.Watermelon originates in southern Africa with a long history of cultivation and a wide range of regions.Since conventional genetic breeding methods of watermelon have long breeding cycle,difficulty and unstable genetic traits,it is particularly critical to obtain new germplasm through transgenic technology to improve or increase excellent traits.Although transgenic technology has been widely applied and achieved great success in many crops,so far,there has been no breakthrough in the application of watermelon,and most research is still focused on the core issue of improving the efficiency of genetic transformation.Therefore,exploring a stable and efficient genetic system is of great practical significance for improving or increasing the excellent traits of watermelon.In production,the excessive number of side branches of watermelon not only greatly increases the labor cost of pruning and branching,but also reduces the planting density,increases the risk of diseases and pests,and adversely affects the yield and quality of watermelon.Our research group previously mapped a gene Cl TFL1 controlling the number of lateral branches in watermelon through forward genetics.Therefore,on the basis of optimizing the genetic transformation system of watermelon,this study further uses CRISPR/Cas9 gene editing technology to knock out Cl TFL1 gene,further verifies the transformation efficiency,and further analyzes and verifies the gene function by using the obtained Cl TFL1-editing plants.The main conclusions of this study are as follows.1.In this study,watermelon germplasm resources existing in the laboratory with relatively high quality are used as experimental materials,and “YL” is finally selected as the acceptant material through statistical and analysis of germination rate,adventitious germination induction rate and germination state of watermelon seeds.After 2 days of sowing,YL seeds have basically germinated,the embryonic axis has begun to elongate and some have grown a small number of root hairs,and watermelon seeds in this state are most suitable for infection.The method of knife stroke to treat the injury of explants can significantly improve the fluorescence rate and fluorescence brightness in the differentiation stage of explants,and effectively improve the efficiency of explant differentiation into fluorescent buds.Negative pressure infection can assist Agrobacterium solution to enter deep tissue of explants,and its fluorescence rate is as high as 71.8% and callus induction rate is as high as 60.2%.The concentration of bacterial solution OD600=0.8 and the co-culture time of 3 days can make the explants have the highest fluorescence rate and the strongest fluorescence brightness,and the explants are significantly expanded and well differentiated in the differentiation stage.The addition of 0.5 mg/L of GA3 to the differentiation medium can induce emerald green callus from explants and accelerate the differentiation rate of explants,significantly improving the differentiation efficiency of adventitious buds.The addition of 0.5 mg/L of IBA to the rooting medium allows rootless seedlings to take root in a short period of time,and the root system grows vigorously and robustly.2.Bioinformatics analysis is conducted on the PEBP family genes of Cl TFL1,and HMMER model is used to predict that the PEBP family contains 7 genes,most of which contain 4 exons and 3 introns,predicting that the family genes are hydrophilic proteins.In addition,the phylogenetic tree is constructed with other gene families fits the classification of three subfamilies.The gene structure contains a stable PEBP conserved domain.The analysis on the promoter sequence of the members reveals that there are several elements related to light response,abscisic acid response,circadian rhythm regulation and auxin response on the promoter.3.In this study,we optimize a stable and efficient watermelon genetic transformation system,and further verify the transformation efficiency by using Cl TFL1 as the target gene.and find that the positive bud acquisition rate under this system is 17.5%,and the individual acquisition rate is 12.5%.The establishment of this system and the acquisition of edited plants lay a material foundation for us to further verify the function of Cl TFL1 gene and analyze its mechanism in regulating lateral branch differentiation.