Mesophyll cell protoplasts were isolated from potato (Solanum tuberosum L. cv. Russet Burbank) leaves (e.g. 2, 6, 8, 9) have been induced to reform their cell walls, undergo sustained proliferation, and ultimately redifferentiate whole plants. This feature of protoplast totipotency among the experimental plant species studied thus far suggests a viable new approach toward crop and varietal improvement (3) once similar success is achieved for species of major economic consequence.The potato, which ranks fourth among world food crops (19), has been recalcitrant in tissue culture. In only a few cases (5, 10, 14, 20) has plant regeneration been achieved from excised tissues other than shoot tips. Recently, however, there has been definite progress toward the development of an in vitro regeneration system from single cells. Upadhya (18) cultured protoplasts from potato leaves and obtained calli which differentiated roots but not shoots. Behnke (1) plated cells from dihaploid suspension cultures and reported that "small shoots" emerged in a high percentage of individual calli, although mention was not made of whether whole plants were obtained. In the present study, calli raised from single mesophyll protoplasts of potato were induced to undergo shoot formation and eventually regenerate whole plants. The developmental sequence, while dependent upon the proper balance of phytohormones, was also sensitive to numerous other constituents of the culture medium.

No 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...

Enzyme profiles during synchronous development This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol19/iss1/2 To++en,R. E. and ti.g Howe, Jr. Enzyme profiler The following enzymes have been investigated previously during during synchronous development of conidiophorer development of conidio: alcohol dehydrcgenare and glucore-5-phosphate dehydrogenare (Weiss and Turion 1966 J. Gen. Micrcbiol.44:407), inand conidio in N. crassa.-vertare and trehalore (