Transient Transformation System Of Cotton Protoplasts Establishment And Optimization

Cotton has enormous economic value worldwide.Due to the nature of allopolyploid and time-consuming genetic transformation steps,the development of cotton functional genomics is slow.The most commonly cultivated cotton is allotetraploid cotton,and the large and complex genome makes it difficult to study the functional genome of cotton.The prerequisite for identifying the genetic function of cotton and selecting new varieties is to obtain mutants.CRISPR/Cas9 genome editing technology can accurately edit the genome,effectively generating mutant materials for target gene mutations,providing strong technical support for achieving cotton molecular breeding.However,to apply genome editing technology to cotton,a crop that is difficult to transform or has a long transformation time,it is necessary not only to optimize the efficiency of genome editing,plant cell delivery and regeneration systems,but also to develop efficient transient transformation methods to quickly verify the activity of CRISPR/Cas mediated genome editing vectors.The transient transformation system of protoplasts can achieve rapid expression of foreign genes in cells.By detecting the expression of target genes in protoplasts alone,gene expression in cells can be studied.This method has been widely applied in related research fields such as protein subcellular localization and protein interaction.However,successfully isolating a large number of live protoplasts suitable for genome editing in cotton remains a challenge.This study selected the entire root of cotton as the source of protoplasts,and explored the optimal conditions for the preparation of cotton root protoplasts and PEG-Ca2+ mediated instantaneous transformation by referring to the preparation and transformation conditions of Arabidopsis leaf protoplasts.An efficient cotton root protoplast instantaneous transformation system was established,and its feasibility and applicability were verified.The main results are as follows:1.Optimization of extraction conditions for cotton root protoplasts.Using cotton whole roots cultured in water for different times(60h,72h,84h)as research materials,the protoplasts of cotton whole root tissues were extracted using enzymatic hydrolysis.Statistics showed that the protoplast extraction effect of cotton roots cultured in water for 72 hours was the best,with a yield of up to 3.55 × 105 protoplasts/g FW(fresh weight),FDA staining showed that the vitality of protoplasts was greater than 92%.2.Optimization of instantaneous transformation conditions for cotton root protoplasts.Based on the optimal preparation conditions of cotton root protoplasts,the transient transformation conditions of PEG-Ca2-mediated protoplasts were optimized.By comparing the factors of PEG-mediated protoplast transfection(such as Ca2+ concentration in PEG solution,incubation time,and plasmid concentration),it was found that the concentration of cotton root protoplasts was 1.5 X 106 pieces/mL,Ca2+ concentration of 200 mmol·L-1,total plasmid DNA of 20 μg.When the transformation time is 20 minutes,the instantaneous transformation efficiency of protoplasts reaches 84%.3.Application of instantaneous transformation system for cotton root protoplasts(1)Subcellular localization detection.The Golgi localization protein Man49(Man49 mCherry)labeled with mCherry and an empty control vector(pEASY-35S:GFP)were combined to instantaneously transform cotton root protoplasts.The results showed that the fluorescent protein could accurately determine the subcellular structure located in the Golgi,confirming the application of cotton root protoplasts in subcellular localization.(2)Double molecule fluorescence complementary(BiFC)analysis.We used GhBIN2(GhA09G0713)and GhBZR3(Gh_A10G0312)as candidate targets for cotton protoplast BiFC detection.The results showed that BZR3 and BIN2 interacted,demonstrating their application in protein interactions in cotton root protoplasts.(3)The activity of genome editing vector was detected.The successfully constructed pKSE401-GFP,pKSE401-CLA,pKSE401-PDS,and pKSE401-DMR6 vectors were transformed into cotton root protoplasts.After 48 h of culture,the protoplast genomic DNA was extracted,and the editing ability of genome editing vectors to target sites was tested by PCR/RE combined with sequencing.The results showed that the genome editing vector produced a small number of base deletions and insertions at the gene target sites,indicating that the established transient protoplast transformation system can be used for the rapid verification of genome editing vectors,providing a reliable reference for stable genetic transformation.(4)CRISPR/Cas9 extended base editing range variant efficiency.Through this transient transformation system,the editing efficiency of up to 27.46%was achieved at the target site of cotton A genome pKSE401-SpG-GhCLA1 gene,and different variants had different editing efficiency at the same target site,all of which realized genome editing in cotton by using extended base editing range vector.To sum up,the whole root protoplast somatic cell cells of 72h hydroponic cotton obtained by enzymolysis separation have a yield of 3.55×105 protoplasts/g fresh weight[FW])。According to FDA staining statistics,92%of cells have intact structures and high vitality;By optimizing the PEG-Ca2+ mediated transient transformation method,the specific experimental parameters were determined when the PEG concentration was 40%and the plasmid amount increased to 20 μg.The conversion time is 20 minutes,and the conversion efficiency reaches a maximum of 84%at 200 mM Ca2+.The established efficient transient transformation system of cotton protoplasts can be used for subcellular localization,BiFC analysis and activity verification of genome editing vectors,which provides a fast and efficient platform for cotton genome editing research and is conducive to accelerating cotton functional genomics research.