Optimization of Protoplast Isolation and Transient Expression Systems for Lettuce (<i>Lactuca sativa</i> L.)

Plant protoplasts are very useful in plant biotechnology, molecular biology, and cell biology. However, an efficient method for protoplast production remains a challenge for many economically important dicotyledonous vegetables, including lettuce (Lactuca sativa L.). Herein, a protocol was optimized for efficient protoplast production from various tissues (leaf and shoot apex) of different lettuce subtypes (romaine, loose-leaf, and semiheading lettuces) by optimizing the major factors affecting protoplast yielding. The optimized protocol yields protoplasts up to 2.0 × 107/g (fresh weight, FW) with viability more than 85%, which is 3–10 times higher than those previously reported. This optimized protocol was also found to be applicable to other dicotyledonous plants (bok choy (Brassica parachinensis), celery cabbage (Brassica pekinensis), and Arabidopsis thaliana) for efficient protoplast production from leaves and shoot apexes. Moreover, an optimized poly(ethylene glycol)-mediated transient expression system (TES), using lettuce shoot apex protoplasts generated via the aforementioned protocol, exhibited high transfection efficiency exceeding 80%. This was further evidenced by the elevated expression levels and subcellular localization of four representative plasma membrane transporters: AAP2, ABCG22, ALMT10, and OATP. In conclusion, the optimized protoplast production protocol along with TES developed in this study will be useful tools for the functional analyses of genes in lettuce and other important dicotyledonous vegetables.