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Monitoring protoplast production from plant cells
Plant Science Letters, 4 (1975) 145--147 145 © ElsevierScientific PublishingCompany, Amsterdam-- Printed in The Netherlands
MONITORING PROTOPLAST PRODUCTION FROM PLANT CELLS
THOMAS E. FERRARI, J. ELIZABETHPALMER and JACK WIDHOLM Department of Agronomy, University of lllinois, Urbana, IlL 61801 (U.S.A.) (Received August 28th, 1974) (Revision receivedNovember 5th, 1974)
SUMMARY
A method is described for monitoring the progress of protoplast production from cultured carrot cells (Daucus carota) during enzymatic removal of cell walls. The procedure is based upon a differential response of protoplasts and cells still retaining a cell wall to a detergent, sodium dodecyl sulfate (SDS). The presence of a cell wall prevents release of cellular constituents when SDS dissolves the plasma membrane. Protoplasts, however, release many cellular constituents in a long, characteristic stream.
INTRODUCTION The most widely used method for production of protoplasts from plant tissues and cell cultures utilizes commercially available mixtures of walldegrading enzymes to remove cell wall material. While methods for this enzymatic procedure are established for many plant species and tissue cultures [1], the endpoint of wall degradation has remained for the most part subjective and seldom established by direct means. This is due to the absence of a simple, reliable technique for monitoring the appearance of protoplasts during the course of wall removal. The most commonly used method is staining with Calcofluor White, which forms a fluorescent complex with cellulose [2]. This method is inadequate as in advanced stages of wall degradation, but prior to protoplast production, fluorescence is difficult, if not impossible, to detect by eye. The presence of non-cellulosic cell wall constituents are also not detected, as Calcofluor White binds predominantly with cellulose. METHODS Carrot (Daucus carota) cell cultures were grown as described previously [3]. Prior to protoplast production, large clumps of cells were removed by filtering cells through a 177 ~ screen. Prior to use, the crude wall-degrading enzyme preparation (Cellulysin, 12 g; Macerozyme, 4 g - Calbiochem) was dissolved in Abbreviation: SDS, sodium dodecyl sulfate.
146
50 ml of 0.02 M sodium phosphate buffer (pH 6.0), clarified by centrifugation (37 000 x g, 10 min, 0--3 °) and the supernatant eluted from a Sephadex G-25 column with 0.02 M sodium phosphate buffer (pH 6.0). Material eluted in the void volume was freeze-dried. Cells (1 g fr.wt) were suspended in 20 ml protoplast production medium [4]. This medium is cell culture medium minus sucrose, glucose and 2,4-dichlorophenoxyacetic acid, but containing 0.57 M sorbitol and enzyme powder (25 mg/ml). Protoplasts are produced in this filter-sterilized medium at 28 ° within 6 to 24 h depending on the cell culture age and condition, and the activity of the enzyme preparation. Reagents used to monitor protoplast production were phenosafranine (0.1%) and phenosafranine (0.1%) plus SDS (5%). Each reagent was dissolved in protoplast production medium (minus wall-degrading enzymes) to prevent osmotic shock to protoplasts or cells with weakened cell walls. RESULTS AND DISCUSSION The progress of protoplast production was monitored by placing a drop of the cells suspended in protoplast production medium on a microscope slide at given time intervals. A drop of phenosafranine solution was mixed with the suspension, a cover slip applied and the cells viewed with a phase contrast microscope. The SDS-phenosafranine reagent was applied to one edge of the cover slip in sufficient quantity to cause a gentle but steady flow of medium across the slide. As the front of the SDS-phenosafranine solution moves across the slide, cytoplasmic membranes are dissolved by the SDS and proteinaceous material is stained by phenosafranine. Cells suspended in protoplast production medium lacking wall-degrading enzymes become plasmolyzed and cell wall is clearly visible surrounding the spherical cells. After addition of the SDS-phenosafranine reagent, the plasma membranes of the cells are dissolved, the cells become translucent, and many cellular inclusions are stained but are not released. In contrast, treatment of protoplasts, that is cells lacking complete walls, with SDS-phenosafranine results in the release of the cellular contents into the medium. The protoplast's plasma membrane is first dissolved by the SDS and then the cytoplasmic constituents are released in a long stream of protoplasm and cellular particles. After 10 to 15 sec no trace of the original protoplast remains. It has been noted in an earlier report [5] that detergent releases chloroplasts from protoplasts of immature tomato fruits. These developments were in contrast to cells still possessing some enclosing cell wall materials. Partial protoplasts, which apparently retain some residual cell wall material, can also be detected by detergent treatment. Such treatment releases a small stream of material devoid of visible cellular particles from the cells. The residual wall material is apparently acting as a screen and retaining larger cellular materials. Unlike protoplasts, cells with significant wall material present remain visible long after SDS had dissolved the plasma membrane. At this stage of wall degradation, no fluorescence can be detected using the Calcofluor White
147 m e t h o d for detection of wall material (observations from unpublished experiments). Phenosafranine stains dead cells [6] in addition to SDS-released proteins. Thus, using this new cell dissolution procedure, one can first measure the percent mortality in a cell preparation using phenosafranine and then after addition of the SDS-phenosafranine reagent, monitor the progress of protoplast production. ACKNOWLEDGEMENTS This research was supported b y funds from the Illinois Agricultural Experiment Station and a grant from the National Science Foundation (GB 34716). REFERENCES
1 E.C. Cocking, Ann. Rev. Plant Physiol., 23 (1972) 29. 2 B.J. Harrington and K.B. Raper, Appl. Microbiol., 16 (1968) 106. 3 T.E. Ferrari and J.M. Widholm, Analyt. Biochem., 56 (1973) 346. 4 H. Grambow, K.N. Rao, R.A. Miller and O.L. Gamborg, Planta (Berl.), 103 (1972) 348. 5 M.A. Boulware and N.D. Camper, Physiol. Plant., 26 (1972) 313. 6 J.M. Widholm, Stain Tech., 47 (1972) 189.
MONITORING PROTOPLAST PRODUCTION FROM PLANT CELLS
THOMAS E. FERRARI, J. ELIZABETHPALMER and JACK WIDHOLM Department of Agronomy, University of lllinois, Urbana, IlL 61801 (U.S.A.) (Received August 28th, 1974) (Revision receivedNovember 5th, 1974)
SUMMARY
A method is described for monitoring the progress of protoplast production from cultured carrot cells (Daucus carota) during enzymatic removal of cell walls. The procedure is based upon a differential response of protoplasts and cells still retaining a cell wall to a detergent, sodium dodecyl sulfate (SDS). The presence of a cell wall prevents release of cellular constituents when SDS dissolves the plasma membrane. Protoplasts, however, release many cellular constituents in a long, characteristic stream.
INTRODUCTION The most widely used method for production of protoplasts from plant tissues and cell cultures utilizes commercially available mixtures of walldegrading enzymes to remove cell wall material. While methods for this enzymatic procedure are established for many plant species and tissue cultures [1], the endpoint of wall degradation has remained for the most part subjective and seldom established by direct means. This is due to the absence of a simple, reliable technique for monitoring the appearance of protoplasts during the course of wall removal. The most commonly used method is staining with Calcofluor White, which forms a fluorescent complex with cellulose [2]. This method is inadequate as in advanced stages of wall degradation, but prior to protoplast production, fluorescence is difficult, if not impossible, to detect by eye. The presence of non-cellulosic cell wall constituents are also not detected, as Calcofluor White binds predominantly with cellulose. METHODS Carrot (Daucus carota) cell cultures were grown as described previously [3]. Prior to protoplast production, large clumps of cells were removed by filtering cells through a 177 ~ screen. Prior to use, the crude wall-degrading enzyme preparation (Cellulysin, 12 g; Macerozyme, 4 g - Calbiochem) was dissolved in Abbreviation: SDS, sodium dodecyl sulfate.
146
50 ml of 0.02 M sodium phosphate buffer (pH 6.0), clarified by centrifugation (37 000 x g, 10 min, 0--3 °) and the supernatant eluted from a Sephadex G-25 column with 0.02 M sodium phosphate buffer (pH 6.0). Material eluted in the void volume was freeze-dried. Cells (1 g fr.wt) were suspended in 20 ml protoplast production medium [4]. This medium is cell culture medium minus sucrose, glucose and 2,4-dichlorophenoxyacetic acid, but containing 0.57 M sorbitol and enzyme powder (25 mg/ml). Protoplasts are produced in this filter-sterilized medium at 28 ° within 6 to 24 h depending on the cell culture age and condition, and the activity of the enzyme preparation. Reagents used to monitor protoplast production were phenosafranine (0.1%) and phenosafranine (0.1%) plus SDS (5%). Each reagent was dissolved in protoplast production medium (minus wall-degrading enzymes) to prevent osmotic shock to protoplasts or cells with weakened cell walls. RESULTS AND DISCUSSION The progress of protoplast production was monitored by placing a drop of the cells suspended in protoplast production medium on a microscope slide at given time intervals. A drop of phenosafranine solution was mixed with the suspension, a cover slip applied and the cells viewed with a phase contrast microscope. The SDS-phenosafranine reagent was applied to one edge of the cover slip in sufficient quantity to cause a gentle but steady flow of medium across the slide. As the front of the SDS-phenosafranine solution moves across the slide, cytoplasmic membranes are dissolved by the SDS and proteinaceous material is stained by phenosafranine. Cells suspended in protoplast production medium lacking wall-degrading enzymes become plasmolyzed and cell wall is clearly visible surrounding the spherical cells. After addition of the SDS-phenosafranine reagent, the plasma membranes of the cells are dissolved, the cells become translucent, and many cellular inclusions are stained but are not released. In contrast, treatment of protoplasts, that is cells lacking complete walls, with SDS-phenosafranine results in the release of the cellular contents into the medium. The protoplast's plasma membrane is first dissolved by the SDS and then the cytoplasmic constituents are released in a long stream of protoplasm and cellular particles. After 10 to 15 sec no trace of the original protoplast remains. It has been noted in an earlier report [5] that detergent releases chloroplasts from protoplasts of immature tomato fruits. These developments were in contrast to cells still possessing some enclosing cell wall materials. Partial protoplasts, which apparently retain some residual cell wall material, can also be detected by detergent treatment. Such treatment releases a small stream of material devoid of visible cellular particles from the cells. The residual wall material is apparently acting as a screen and retaining larger cellular materials. Unlike protoplasts, cells with significant wall material present remain visible long after SDS had dissolved the plasma membrane. At this stage of wall degradation, no fluorescence can be detected using the Calcofluor White
147 m e t h o d for detection of wall material (observations from unpublished experiments). Phenosafranine stains dead cells [6] in addition to SDS-released proteins. Thus, using this new cell dissolution procedure, one can first measure the percent mortality in a cell preparation using phenosafranine and then after addition of the SDS-phenosafranine reagent, monitor the progress of protoplast production. ACKNOWLEDGEMENTS This research was supported b y funds from the Illinois Agricultural Experiment Station and a grant from the National Science Foundation (GB 34716). REFERENCES
1 E.C. Cocking, Ann. Rev. Plant Physiol., 23 (1972) 29. 2 B.J. Harrington and K.B. Raper, Appl. Microbiol., 16 (1968) 106. 3 T.E. Ferrari and J.M. Widholm, Analyt. Biochem., 56 (1973) 346. 4 H. Grambow, K.N. Rao, R.A. Miller and O.L. Gamborg, Planta (Berl.), 103 (1972) 348. 5 M.A. Boulware and N.D. Camper, Physiol. Plant., 26 (1972) 313. 6 J.M. Widholm, Stain Tech., 47 (1972) 189.