Regeneration Of Potato Protoplasts And Transient Transformation Using CRISPR/Cas9

Potato is an important food crop in China.Complex genetic background and self-incompatibility lead to a very slowly process of potato breeding.Lack of high-quality potato varieties for fresh market and industry processing has been a big issue for long time.Biotechnology is a powerful and effective tool to shorten the breeding process.Transient expression in protoplast can avoid the integration of foreign gene on the plant genome,and create new germplasm materials with target traits.The purpose of this study is to establish a technology system of transient transformation by potato protoplast with CRISPR/Cas9 construct,and provides a useful tool for potato genetic improvement and new germplasm resources creation.In this study,the potato cultivar Atlantic was used as the material.To establish and optimize the two key segments of protoplast isolation,transformation and regeneration,which includes such as the concentration of enzymatic hydrolysate,time,osmotic pressure,exogenous hormone and initial density were studied.The pCAMBIA2300-GBSS vector was constructed by the 20 bp sequence on exon 9 of StGBSS gene reported in the literature as the target site(GBSS9).Using CRISPR/Cas9editing technology,it was transformed into mesophyll protoplasts instantaneously.The results as follow:1.The separation conditions were optimized and the optimal conditions for protoplast separation were cellulase concentration of 0.7%,osmotic pressure of 0.4 M mannitol concentration,and the enzymatic time of 8-9 h.2.By optimizing the culture conditions,it was found that the concentration of exogenous hormone 2,4-D was 2 mg/L and the initial density of protoplast culture was 0.5×10~4-1×10~4 cells/mL,which was the most favorable condition for protoplast culture.The protoplast culture was studied from cell division stage to differentiation into callus,and finally dedifferentiation into seedlings.A rapid and stable protoplast regeneration system was established.In this study,it took only about 40 days from callus stage to bud induction,which shortened the time of formation and regeneration.3.Using PEG-mediated transient transformation in potato protoplasts,45regenerated plants were obtained.Ten of the regenerated plants were identified by PCR and sequencing.In the later stage,in order to obtain sufficient transformed seedlings,secondary transformation has been carried out,and knockout detection is also carried out simultaneously.