Isolation and Regeneration of Tobacco Mesophyll Cell Protoplasts under Low Osmotic Conditions

Abstract

A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 M sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.Protoplasts have been isolated from tobacco mesophyll cells by a variety of enzymatic techniques (5, 16) and have also been induced to regenerate normal plants (4, 10, 11). In each of these studies, protoplasts were prepared and subsequently plated for regeneration studies in the presence of a concentrated osmoticum. It would seem desirable, nonetheless, to have available a similar technique in which protoplasts, at least at the outset, are exposed to lesser degrees of plasmolysis. Plasmolysis is known to influence a variety of cell functions (9) and a more direct determination of additional effects should be possible if protoplasts could be maintained within a broad range of osmoticum concentrations.In this paper, we report a highly efficient and consistent technique for the isolation and regeneration of tobacco mesophyll cell protoplasts in solutions containing relatively low concentrations of a primary osmoticum. During this investigation, it was found that a considerable number of modifications over those previously reported (10) Protoplast Isolation. Tobacco leaves were collected soon after they had reached 25-to 26-cm length provided that plants were less than 0.762 m tall and had not begun to flower. The leaves were surface-sterilized for 15 min in 0.525% sodium hypochlorite (10% Clorox), rinsed in sterile H20 with free moisture, then removed in a Microvoid II-C sterile air chamber. Leaves were finally placed in sterile paper towels in the refrigerator overnight. They were then rinsed in 70% ethanol for 3 min, removed, and dried in a sterile air chamber. All ensuing steps were conducted aseptically. Usually, the lower epidermis was removed, but leaves could also be shredded into narrow strips (about 1 mm) to facilitate enzyme penetration. Approximately 2 g of leaf tissue were placed in a 250 ml evacuation flask containing 100 ml of the following filter sterilized solution: 0.5% macerozyme (Yakult Biochemicals Ltd.), 4% cellulase (Onozuka SS, Yakult Biochemicals), 2% PVP (mol wt 10,000, Sigma Chemical Co.), and 0.2 M sucrose, pH 5.4. The enzyme preparation at the outset always contained significant amounts of insoluble material. This w...

Mesophyll Cell Protoplasts of Potato

“…Well expanded leaflets (9-13 cm in length) were collected from plants conditioned under short photoperiods and surface-sterilized with 0.53% sodium hypochlorite and 70% ethanol (16). Lower leaflet surfaces were gently stroked with a soft nylon brush until they appeared light green (15) and were then cut into squares approximately 2 cm in diameter.…”

Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles

“…The filtered solution was centrifuged in Babcock bottles (Kimble No. 1000) (14) in a one-speed Jalco centrifuge (model 58) for 5 min at approximately 500g. During centrifugation, a vacuole and protoplast mixture floated to the surface and collected in the neck of the Babcock bottle.…”

Promotive effect of polyvinylpolypyrrolidone on pollen embryogenesis in Datura innoxia

“…1978) and addition of exogenous phenolic compounds to tbe ctjlture medium completely inhibits plantlet production in anther culture of Nicotiana tabacum (Weatherhead et al 1979). Polyvinylpyrrolidone has also been used to maintain stability of protoplasts during isolation (Shepard and Totten 1975). Therefore, it seems that polyvinylpolypyrrolidone is enhancing the response in anther cultures of Datura innoxia by removing phenolic compounds produced by cultured anthers and which inhibit the development of pollen grains into embryos.…”