Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables

Abstract

Plant protoplasts-based platform shows high totipotency, sensitivity, and versatility, and thus promotes botanical advances. However, an efficient protoplasts-based platform remains a challenge for many economically significant dicotyledon vegetables such as lettuce (Lactuca sativa L.). In the present study, a highly efficient protocol was optimized and developed for isolation of protoplasts from various tissues (leaf, shoot apex, and root tips) of different lettuce subtypes (romaine, loose-leaf, and head lettuces) by optimizing the major factors affecting protoplast yield. It provided the protoplasts yield up to 1.95× 107 /g (fw) and viable cells > 85%, which are 3–10 times higher than that of previous report. This protocol was also successfully applied to other dicotyledon plants for efficient isolation of protoplasts from their leaves and shoot apices, including Arabidopsis thaliana, celery cabbage (brassica pekinensis), and bok choy (Brassica parachinensis). Furthermore, the optimized polyethylene glycol-mediated transient expression system (TES) based on the lettuce shoot apex showed a high transfection efficiency (> 80%), achieving protein subcellular localization of four typical plasma membrane genes and their induced expression function investigations in lettuce. The optimized protoplast isolation protocol and TES provide a versatile technique for functional analyses of genes in lettuces and other important dicotyledon vegetables.