Optimization of protoplast methods suitable for transient CRISPR/Cas9 expression in Lepidium campestre

Sammanfattning: Lepidium campestre is a wild oil species with a number of traits that are beneficial from an agricultural point of view. CRISPR/Cas9 could be used transiently in protoplasts to accelerate domestication of L. campestre. In order for plants in Sweden to be classified as non-GMO they need to be modified without the addition of foreign DNA, therefore transient Cas9 expression is used. Since there are no protoplast methods optimized for L. campestre the aim of this project was to develop efficient methods for protoplast isolation, transfection and regeneration suitable for this species. Multiple isolation parameters were compared. When using higher enzyme concentrations significantly more protoplasts were obtained. No significant difference was found between gently shaking the leaves or keeping them stationary during the enzyme digestion. Cutting leaves with razorblades or scissors showed no significant difference in number of protoplasts isolated, and differences in regeneration capacity could not be evaluated due to infections. No significant difference was found when increasing the enzyme incubation time from 15 h to 18 h. Transfection was performed using the plasmid pEAQ-HT-GFP and the PEG incubation time was tested. Transfection was performed successfully using 25 % PEG4000 with incubation times 5 min and 10 min. Two regeneration methods were performed and differences in infection frequency and microcalli production were observed. Method 3.B suffered more infections, possibly due to a higher sensitivity or a contaminated solution. Microcalli were obtained from one plate, which was regenerated according to method 3.A. This shows that regeneration of the protoplasts is possible, and supports further optimization of method 3.A.