Optimization of Protoplast Isolation and Transient\nExpression Systems for Lettuce (Lactuca sativa L.)

Plant\nprotoplasts are very useful in plant biotechnology, molecular\nbiology, and cell biology. However, an efficient method for protoplast\nproduction remains a challenge for many economically important dicotyledonous\nvegetables, including lettuce (Lactuca sativa L.). Herein, a protocol was optimized for efficient protoplast production\nfrom various tissues (leaf and shoot apex) of different lettuce subtypes\n(romaine, loose-leaf, and semiheading lettuces) by optimizing the\nmajor factors affecting protoplast yielding. The optimized protocol\nyields protoplasts up to 2.0 × 10<sup>7</sup>/g (fresh weight,\nFW) with viability more than 85%, which is 3–10 times higher\nthan those previously reported. This optimized protocol was also found\nto be applicable to other dicotyledonous plants (bok choy (Brassica parachinensis), celery cabbage (Brassica pekinensis), and Arabidopsis\nthaliana) for efficient protoplast production from\nleaves and shoot apexes. Moreover, an optimized poly(ethylene glycol)-mediated\ntransient expression system (TES), using lettuce shoot apex protoplasts\ngenerated via the aforementioned protocol, exhibited high transfection\nefficiency exceeding 80%. This was further evidenced by the elevated\nexpression levels and subcellular localization of four representative\nplasma membrane transporters: AAP2, ABCG22, ALMT10, and OATP. In conclusion,\nthe optimized protoplast production protocol along with TES developed\nin this study will be useful tools for the functional analyses of\ngenes in lettuce and other important dicotyledonous vegetables.