Simple and Efficient Protoplast Culture Procedure of Lettuce, Lactuca sativa L.

Abstract

Simple and efficient procedure of protoplast culture was developed in lettuce. For protoplast isolation, cotyledons from aseptically grown 7 to 10 days old seedlings of lettuce cv. Kaiser were sliced into ca. 1mm strips, and incubated in 0.04% Macerozyme R-10, 0.2% Meicelase, containing O.029M sucrose, 0.5M mannitol and l/2 inorganic salts and vitamins of MS except NH₄NO₃ (200mg/l), for 16h at 25°C without shaking. Protoplasts liberated with gentle shaking by hand were passed through nylon mesh (50μm), and washed three times with 0.5M mannitol containing CPW salts or the modified MS used for enzyme treatment. Isolated protoplasts were cultured at a density of 2.5×l0⁴/ml in the medium with 1/2 inorganic salts and vitamins of MS except NH₄NO₃ (80mg/l), 5mM sodium succinate, 0.5g/l casamino acid. 0.3mg/l BA, 0.3M sucrose, 0.3% Gelrite and an auxin (2, 4-D or NAA) under dim light (ca. 100lux) at 25°C. Colonies were transferred to the liquid medium with the same composition except sucrose (O.15M) after 7 to 10 days, and onto the Gelrite-solidified medium with the same composition as the initial culture medium except sucrose (0.088M) after further 7 to 10 days. Calli larger than 1mm in diameter were transferred onto MS agar medium with BA to induce shoots. Additions of Gelrite to the initial culture medium gave a rapid colony growth, and efficient shoot regeneration. Frequency of shoot regeneration was more influenced by auxin of initial culture medium than by cytokinin of the shoot regeneration medium. The shoots were obtained most frequently when the initial culture medium contained 1mg/l NAA. Shoot primordia differentiated at an early stage of colony growth. By selecting the calli differentiating green shoot primordia, most of calli regenerated plants on the MS agar medium with 0.3mg/l BA. The frequency of calli with green shoot primordia was also influenced by the density of calli in a plate. The higher the density of calli, the lower the frequency of shoot regeneration. Shoot regenerated from isolated protoplasts within 2 months. Only three changes of media were enough to regenerate shoots. The number of colonies was ca. 300per 7.5×10⁴ protoplasts cultured, and most of the colonies regenerated plants. Protoplasts of 7 cultivars of lettuce and 1 of endive were cultured using this procedure, and regenerated plants were obtained from all the cultivars of lettuce and endive.