Establishment And Applications Of Efficient Protoplast Transient Transfection System In Brassica Crops

As a new generation of breeding technology,genome editing technology has been widely used in the breeding improvement of many major crops.In the application or development of plant genome editing,it is necessary to verify the activity of gene editing vector and iterate testing of gene editing tools.Protoplast transient transfection system(PTTS)is the most widely used in plant genome editing due to its advantages of simple operation,short transformation period and high transfection efficiency.However,in Brassica crops,there are only a few reports on the establishment of PTTS,and the isolated protoplasts viability and transfection efficiency are lower than those in other species.Therefore,how to establish an efficient and universal PTTS in Brassica vegetable crops is one of the important problems to promote the application of genome editing technology in Brassica vegetable crop breeding.Based on the above challenges,this study took Brassica rapa and Brassica oleracea as experimental objects,and systematically explored the most commonly used enzymatic hydrolysis methods for protoplast isolation and transient transfection mediated by PEG-Ca2+.The key factors affecting protoplast isolation and transfection efficiency were optimized,and several key conditions affecting the transfection efficiency were explored for the first time,such as osmotic pressure concentration,heat shock method,calcium ion concentration,etc.An efficient and universal PTTS was successfully established in B.rapa and B.oleracea.This PTTS was used to verify the editing activity of B.rapa genome by CRISPR/Cas9 system,and realized the editing of B.oleracea genome by CRISPR/Cas12a system for the first time The main research results are as follows:First,based on the PTTS established in Arabidopsis thaliana,the key factors of protoplast isolation of B.rapa and B.oleracea were optimized,such as the material size,enzymatic hydrolysis time and mannitol concentration in the enzymolysis solution.The true leaves of B.rapa and B.oleracea grown to 2 cm were used,the concentration of mannitol was 0.6 M in the enzymolysis solution,and after enzymolysis for 6 h,the protoplast yield was 4.7×105/g FW and 4.9×105/g FW,respectively.The cell viability of protoplasts was 97%and 95%respectively.These results indicated that a large number of protoplasts with high viability could be obtained by this established isolation system.Second,the PTTS was optimized,and it was found that the transfected protoplasts cultured with W5 solution could maintain the cell vitality of protoplasts to the greatest extent.It was found for the first time that the concentration of mannitol in MMG solution was the key factor affecting the transfection efficiency.When mannitol concentration was 0.3 M,the transfection efficiency of B.rapa and B.oleracea was increased by about 2.5-fold and 3.9fold,respectively.In B.rapa,using 0.2 M calcium chloride,20 μg plasmid,heat treatment at 37℃ for 6 min and transfected for 15 min,the transfection efficiency reached 86%.In B.oleracea,using 0.3 M calcium chloride,20 μg plasmid,and transfected for 15 min at room temperature,the transfection efficiency reached 69%.The above results indicated that an efficient transient protoplast transformation system was successfully established in B.rapa and B.oleracea.Third,The established PTTS of B.rapa protoplast was applied to different varieties of B.rapa.It was found that the transfection efficiency of wild type A03 and inbred line LG reached 73%and 74%respectively.The results showed that the transient transfection system was not limited by the material genotype.In B.rapa protoplasts,the CRISPR/Cas9 system was used to simultaneously edit three genes of BrAOP2,with an efficiency of 22.2%.In addition,by exploring the influence of culture temperature on transfection efficiency of protoplasts after transfection,it was found that when protoplasts were cultured at 29℃ after transformation,the transformation efficiency was similar to that of 25℃,indicating that this PTTS could be applied to some temperature-sensitive editing vector activity verification.When the established PTTS was applied to other crops in Brassica,it was found that the high transfection efficiency was also achieved in B.chinensis,B.oleracea var.alboglabra and B.rapa var.rapa L.Fourth,CRISPR/Cas12a-mediated gene editing was first performed in B.oleracea.In B.oleracea protoplasts,CRISPR/Cas12a was used to edit two BoPDS genes of B.oleracea.The results of Sanger sequencing showed that 3-16 bp was mainly deleted at the four target sites of two BoPDS genes,which expanded the application range of gene editing technology in B.oleracea.In conclusion,this study successfully established an efficient and universal PTTS by systematically exploring the isolation and transient transfection conditions of B.rapa and B.oleracea protoplasts.In addition,this PTTS can be successfully applied to different varieties of B.rapa,and B.chinensis,B.oleracea var.alboglabra and B.rapa var.rapa L.in Brassica,indicating that this PTTS has the potential to be widely applied to other Brassica vegetable crops.Furthermore,this study established CRISPR/Cas12a-mediated gene editing in B.oleracea for the first time,extending the application range of gene editing technology in B.oleracea,and providing important technical support for promoting Brassica vegetable crop breeding with genome editing technology.