- Email: [email protected]
[60] The isolation of plant protoplasts
578
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
cotyledons, 33 castor bean endosperm, 34 cotyledons of legume seeds, as cotton seeds, onion bulbs, and cabbages. 36 nT. J. Jacks, L. Y. Yatsu, and A. M. Altschul, Plant Physiol. 42, 585 (1967). ~4R. L. Ory, L. Y. Yatsu, and H. W. Kircher, Arch. Biochem. Biophys. 123, 255 (1968). ~H. H. Mollenhauer and C. Totten, J. Cell Biol. 48, 533 (1971). ~L. Y. Yatsu, T. J. Jacks, and T. P. Hensarling, Plant Physiol. 48, 675 (1971).
[60] The Isolation of Plant Protoplasts B y EDWARD C. COCKING
It has recently been emphasized that isolated higher plant protoplasts are naked cells and that being cells they can under suitable conditions be induced to grow and divide. 1 These naked cells are in many respects ideal single-cell cultures. They have considerable potential for the cloning of plant cells generally. They are also well suited for studies on the fusion of plant cells, and the resultant heterokaryons serve as starting cultures for experimental investigation of the possibility of somatic hybridization of plants. 2 One remarkable attribute of these naked cells is that they can rebuild a cell wall. This resynthesis has been fully described and discussed previously2a; physiologically it results in the development of cells with the growth potential of cells as ordinarily cultured. Increasingly, plant biochemists and plant physiologists have become interested in such isolated protoplasts; moreover the removal of the cell wall exposes the plasmalemma directly to the influence of the culture medium. The presence of a cell wall is a complicating factor in studies on uptake generally, particularly so if the investigator is interested in the possible uptake of macromolecules or particles by the isolated protoplasts since the cell wall acts as a very efficient ultrafilter. Studies on the fusion and endocytotic activity of these naked cells have focused attention on the properties of the plasmalemma. Willison et al. 3 have postulated a mechanism for the pinocytosis (endocytosis) of latex spheres by tomato fruit protoplasts. Protoplasts were isolated from tomato fruit locule tissue obtained from tomato plants grown under controlled conditions. 4 Watering of plants should not be carried out for several hours 1E. C. Cocking, Annu. Rev. Plant Physiol. 23, 29 (1972). 2E. C. Cocking, Scienza Tecnica 74 (in press). 21E. C. Cocking, in "Dynamic Aspects of Plant Ultrastructure" (A. W. Robards, ed.), 1973 (in press). 3j. H. M. Willison, B. W. W. Grout, and E. C. Cocking, J. Bioenerg. 2, 371 (1971). ~J. W. Davies and E. C. Cocking, Planta 67, 242 (1965).
[60]
THE ISOLATION OF PLANT PROTOPLASTS
579
before the fruit is detached from the plant. The diced tissue was incubated with 0.5% Macerozyme (All Japan Biochemicals Ltd., Shingikancho, Nishinomiya, Japan) and 5% cellulase (Onozuka 1500, All Japan Biochemicals Ltd., Shingikancho, Nishinomiya, Japan) for 3 hours; the protoplasts released were then washed with 20% sucrose, in which they float? This enzymatic isolation of protoplasts makes use of cell wall degrading enzymes. As normally used these cell wall degrading enzymes are grossly impure, containing many enzymes other than those directly involved in the cell wall degradation. Toxic compounds can also be present, and although it is difficult to generalize, it would seem best always to treat the enzymes with Sephadex before use in order to remove certain enzymes, particularly nucleases, and low molecular weight toxic material. 6 Experience over several years and extending back to the early work of Tribe, ~ who used mechanical rather than enzymatic methods for protoplast isolation, has indicated that isolated protoplasts are far more resistant to such possible harmful effects when they are in the plasmolyzed condition. One reason for this has recently become clear from the work of Withers and Cocking? From studies using thorium dioxide as an electron dense electron microscopic marker, they showed that extensive uptake from the plasmolyticum took place during plasmolysis probably by largescale membrane invagination. In view of this extensive uptake, it would seem advantageous to carry out preplasmolysis in a suitable plasmolyticum before subjecting the tissue to digestion in a possibly harmful cell walldegrading enzyme mixture. As a result the procedures recommended for the isolation of tobacco leaf protoplasts for induced fusion studies with sodium nitrate involved preplasmolysis in 25 % sucrose. 8 Pieces of tobacco leaf taken from 50- to 60-day-old Nicotiana tabacum var. Xanthi plants, from which the lower epidermis had been removed (for practical details see Power and Cocking 9) were incubated for 2 hours in 25% sucrose. After this preplasmolysis the leaf pieces were incubated in a mixture of 5% w/v cellulase (Onozuka 1500) and 0.5% w/v Macerozyme in 25% sucrose, at 20 °, for periods of between 2 and 4 hours. The released protoplasts were washed by flotation in 25 % sucrose. The following two detailed examples may help the reader to appreciate more fully the procedures required for the isolation of protoplasts, first from cereal leaves, and second from suspension culture cells of rose. The
~E. Pojnar, J. H. M. Willison, and E. C. Cocking, Protoplasma 64, 460 (1967). P. K. Evans and E. C. Cocking, in "Plant Tissue and Cell Culture" (H. E. Street, ed.), pp. 100-120, 1972. 7H. T. Tribe, Ann. Bot. 19, 351 (1955). "L. A. Withers and E. C. Cocking, J. Cell Sci. 11, 59 (1972). 9j. B. Power and E. C. Coizking, J. Exp. Bot. 21, 64 (1970).
580
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
earlier report of Ruesink TM may also be useful as a further guide to the basic principles involved. Isolation of Cereal Leaf Protoplasts Mature leaves of Secale cereale (rye) or Triticum aestivum (wheat) or Hordeum vulgare (barley) or Avena sativa (oats) are employed. The cereal plants are between 21 and 28 days old, and the leaves are surface sterilized by treatment with 70% ethanol for 2 minutes followed by 30 minutes in 3% sodium hypochlorite (0.3-0.425% w / v available chlorine). It is necessary to add Teepol (0.5 % ) as a wetting agent ( B D H Chemicals Ltd., Poole, United Kingdom). It is important to remove thoroughly the hypochlorite which has been used as a surface sterilant. The hypochlorite is removed by three successive washes in sterile water. After this treatment the leaves are cut into narrow longitudinal threads using a scalpel and the leaf pieces immersed in a filter-sterilized enzyme mixture with 1 g fresh weight of pieces to every 7 ml of enzyme solution. The enzyme solution should be freshly prepared. A satisfactory enzyme solution was made up as follows: 4% Meicelase (Meiji Seika Kaisha Ltd., Tokyo) with either Pectinol R I 0 (Rohm and Haas, Philadelphia, Pennsylvania) or 1% Macerozyme (All Japan Biochemicals Ltd., Nishinomiya, Japan) and 1% potassium dextran sulfate with a sulfur content of 17.3% (Meito Sangyo Co. Ltd., Nagoya, Japan). These enzymes were dissolved in 0.7 M sorbitol or 0.7 M mannitol. Pectinol R I 0 is standardized with diatomaceous earth and a solution of the enzyme was prepared by suspending 20 g in 100 ml of distilled water for 2 hours. The insoluble material was removed by filtration and after the addition of the materials listed above the volume was made up to 100 ml. As only 6.5% of the Pectinol R I 0 is water soluble, this represents 1.3% solution of Pectinol R10. The pH was adjusted to 5.8 with normal HC1. The leaf shreds were incubated in the above medium at 25 ° for 5 hours. After this, the enzymes were removed and the leaf pieces were rinsed briefly with a sterile washing solution. The sterile washing solution was of the following composition: 0.7 M sorbitol, 1 mM KNO:~, 0.2 mM KHzPO4, 0.1 mM MgSo,, 1 mM CaC1,_,, 1 ~M KI, and 10 mM CuSO4. ~1 After this washing the leaf pieces were placed in a fresh enzyme solution. They were incubated in this fresh enzyme solution for a further 12 hours at 25 °, and after this the leaf shreds were teased apart in the washing solution; this resulted in the release of the protoplasts. The isolated cereal leaf protoplasts can be readily sedimented by cen:°A. W. Ruesink, this series, Vol. 23, p. 197. 11R. G. Jensen, R. I. B. Francki, and M. Zaitlin, Plant Physiol. 48, 9 (1971).
[60]
THE ISOLATION OF PLANT PROTOPLASTS
581
trifugation at 100 g. After centrifugation at 100 g for 5 minutes, the isolated protoplasts were resuspended in sterile 0.8 M sucrose. In 0.8 M sucrose the isolated protoplasts do not sediment, but because of their density they rise to the surface of the sucrose when the suspension is centrifuged; as a result further centrifugation at 200 g for 5 minutes caused intact protoplasts to rise to the surface, while cells, cell debris, and free chloroplasts sedimented to the bottom of the centrifuge tube. The layer of protoplasts was resuspended in a large excess of washing solution and centrifuged at 100 g for 5 minutes. The protoplasts sediment in this washing solution. The protoplasts were then suspended in a known volume of washing solution, and a small sample was taken for counting, using a modified Fuchs Rosenthai hemacytometer, 0.2 mm deep (Hawkesley Gelman, Lancing, England). High yields of protoplasts are obtained--of the order of 106 protoplasts per gram fresh weight of leaf. Such isolated cereal leaf protoplasts as these can be readily suspended at known cell densities in suitable culture media and their growth and development studied further. For further details, see Evans e t al. 1~ Isolation of Rose Suspension Culture Protoplasts Protoplasts isolated from "Paul's Scarlet" rose suspension culture cells have been extensively studied. 1~ The suspension culture cells themselves grow rapidly in batch culture on a fully defined medium originally devised by Davies. 13'~They are sterile, and therefore preliminary sterilization of the plant material is not required as in the case of cereal leaves. The fact that the starting material, i.e., the cells, is already sterile removes many of the uncertainties often present when dealing with material fresh from the plant. Indeed, the isolation of leaf protoplasts is beset with the variation that occurs even from variety to variety of the same species in relation to the efficacy of surface sterilization. The suspension culture of rose cells forms a pipettable suspension which is composed of single cells or small groups of cells. The fact that cells are largely separate from one another eliminates the need to use a pectinase enzyme. Cells from all phases of the growth cycle can be used (but see below). One gram fresh weight of cells filtered from the medium is added to every 10 ml of enzyme digestion mixture. The cell wall digestion mixture contains Meicelase (Meiji Seika Kaisha Ltd.) or Onozuka Cellulase (1500 or 3000). The concentration of enzyme which can be employed lies between 0.5% and 3%. The pH is adjusted to between 5.5 and 6.0 with NaOH or HCI. The incubation conditions are as follows: 20 ml of incuba1., p. K. Evans, A. G. Keates, and E. C. Cocking, Planta 104, 178 (1972). a3 R. Pearce, Ph.D. Thesis, Univ. of Nottingham, 1972. 1~. M. E. Davies, Phytochem. 10, 783 (1971).
582
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[60]
tion mixture is placed in 100-ml Erlenmeyer flasks (too great a depth of mixture is deleterious to survival) and held at a constant temperature of 25 o for up to 24 hours. At the end of the incubation period the mixture is centrifuged for 1 minute at 300 g, and the protoplasts that collect at the surface of the liquid are transferred with a Pasteur pipette to a suitable concentration of sucrose. The protoplasts are then resuspended and centrifuged once more in the same molarity of sucrose. This procedure is then repeated. This centrifugation procedure results in the protoplasts forming a dense layer at the surface of the plasmolyticum while debris sinks to the bottom. This relatively simple procedure results in the isolation of rose protoplasts, largely entirely free of debris. The yield of protoplasts and the extent to which cells are absent from the protoplast suspension is related to the age of the suspension relative to the time interval between its current growth and the time when it was isolated and grown up from callus. Excellent preparations of isolated rose protoplasts, essentially completely free of debris, and from cells, were obtained when rose suspension cells were treated in this way when the rose suspension cells had been obtained from liquid cultures which had recently been grown up from callus on agar. Frequently almost all the suspension cells were converted to isolated protoplasts. When cells grown from subsequent passages of the suspension culture were used, it became necessary first to raise the incubation temperatures to 33 o and later to use 3 % purified cellulase (Cellulase 3000) to obtain satisfactory results. It was found, however, that if the suspension cells were returned to agar for several passages, then the original isolation conditions were once more satisfactory. It would thus seem likely that progressive changes either in wall composition or in the nature of enzyme inhibitors in the cells are responsible for these effects. It would also seem likely that there are progressive changes either in wall composition or in the level of inhibitors as the number of passages of the suspension cultures increases. As earlier mentioned, it may be advantageous with certain cell systems to preplasmolyze the tissues or cells from which it is desired to isolate protoplasts. It is difficult, however, to generalize in this respect; in the case of the isolation of rose protoplasts, preplasmolysis in sucrose for 0.5 or 24 hours reduced release of protoplasts without increasing total survival. Added Comments Although the enzymatic isolation of higher plant protoplasts has now become an acceptable laboratory procedure, each tissue must be investigated systematically in relation to the optimum conditions for protoplast release. It should be noted that protoplasts can also be isolated from tissues of higher plant cells by first separating the cells, under plasmolyzing condi-
[61]
ISOLATIONOF MICROSOMES, RIBOSOMES, AND POLYSOMES
583
tions, with Macerozyme and then isolating protoplasts using cellulase to degrade the walls of the separated cells. 14 This method is useful in that it enables the isolation of specifically palisade layer protoplasts from leaves. It is, however, more laborious than the mixed enzyme (Macerozyme plus Cellulase) procedure described for the isolation of cereal leaf protoplasts. Moreover, as discussed by Evans, Keates, and Cocking, 12 the sequential method of Takebe and his co-workers does not yield protoplasts from many cereal leaves. It is also possible to isolate protoplasts mechanically by means of the procedure first introduced by Klercker in 1892.1~ This method does not release protoplasts from meristematic cells since they do not plasmolyze sufficiently. The yield of protoplasts is usually low even from more mature cells. Nevertheless, such mechanically isolated protoplasts are very useful for comparisons between the metabolic activity of protoplasts isolated mechanically and enzymatically. Pilet and his co-workers 16 have recently pioneered the use of mechanically isolated protoplasts in this respect. a*T. Nagata and I. Takebe, Planta 92, 301 (1970). ~J. Klercker, Ofvers Vetensk. Akad. Forh., Stockholm, 49, 463 (1892). ~6p. E. Pilet, C. R. Acad. Sci. Ser. D 273, 2253 (1971).
[61] Isolation of Microsomes, Ribosomes, and Polysomes from Plant Tissues 1 B y JOE H. CHERRY
The first chemical indication that ribonucleoproteins played a direct role in the synthesis of proteins came in the early 1950's when animals were fed radioactive amino acids. When the labeled tissue was excised, homogenized, and then fractionated, the highest concentration of radioactive amino acids was found in the microsomal fraction. 2 This fraction has been shown to contain ribosome (and polyribosome) particles attached to membrane fragments. Careful kinetic studies on the rate of amino acid incorporation into different proteins support the likelihood that the newly synthesized polypeptides attached to ribosomes were precursors to the enzymes found in the cells. Furthermore, it was found that RNase stopped protein synthesis in isolated subcellular fractions. Experiments on whole 1Some of the research presented in this paper was supported by a contract (COO1313-31) from the U.S. Atomic Energy Commission. This is journal paper 4722 of the Purdue Agriculture Experiment Station. -~E. B. Keller, P. C. Zamecnik, and R. B. Loftfield, ]. Histochem. Cytochem. 2, 378 (1954).
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
cotyledons, 33 castor bean endosperm, 34 cotyledons of legume seeds, as cotton seeds, onion bulbs, and cabbages. 36 nT. J. Jacks, L. Y. Yatsu, and A. M. Altschul, Plant Physiol. 42, 585 (1967). ~4R. L. Ory, L. Y. Yatsu, and H. W. Kircher, Arch. Biochem. Biophys. 123, 255 (1968). ~H. H. Mollenhauer and C. Totten, J. Cell Biol. 48, 533 (1971). ~L. Y. Yatsu, T. J. Jacks, and T. P. Hensarling, Plant Physiol. 48, 675 (1971).
[60] The Isolation of Plant Protoplasts B y EDWARD C. COCKING
It has recently been emphasized that isolated higher plant protoplasts are naked cells and that being cells they can under suitable conditions be induced to grow and divide. 1 These naked cells are in many respects ideal single-cell cultures. They have considerable potential for the cloning of plant cells generally. They are also well suited for studies on the fusion of plant cells, and the resultant heterokaryons serve as starting cultures for experimental investigation of the possibility of somatic hybridization of plants. 2 One remarkable attribute of these naked cells is that they can rebuild a cell wall. This resynthesis has been fully described and discussed previously2a; physiologically it results in the development of cells with the growth potential of cells as ordinarily cultured. Increasingly, plant biochemists and plant physiologists have become interested in such isolated protoplasts; moreover the removal of the cell wall exposes the plasmalemma directly to the influence of the culture medium. The presence of a cell wall is a complicating factor in studies on uptake generally, particularly so if the investigator is interested in the possible uptake of macromolecules or particles by the isolated protoplasts since the cell wall acts as a very efficient ultrafilter. Studies on the fusion and endocytotic activity of these naked cells have focused attention on the properties of the plasmalemma. Willison et al. 3 have postulated a mechanism for the pinocytosis (endocytosis) of latex spheres by tomato fruit protoplasts. Protoplasts were isolated from tomato fruit locule tissue obtained from tomato plants grown under controlled conditions. 4 Watering of plants should not be carried out for several hours 1E. C. Cocking, Annu. Rev. Plant Physiol. 23, 29 (1972). 2E. C. Cocking, Scienza Tecnica 74 (in press). 21E. C. Cocking, in "Dynamic Aspects of Plant Ultrastructure" (A. W. Robards, ed.), 1973 (in press). 3j. H. M. Willison, B. W. W. Grout, and E. C. Cocking, J. Bioenerg. 2, 371 (1971). ~J. W. Davies and E. C. Cocking, Planta 67, 242 (1965).
[60]
THE ISOLATION OF PLANT PROTOPLASTS
579
before the fruit is detached from the plant. The diced tissue was incubated with 0.5% Macerozyme (All Japan Biochemicals Ltd., Shingikancho, Nishinomiya, Japan) and 5% cellulase (Onozuka 1500, All Japan Biochemicals Ltd., Shingikancho, Nishinomiya, Japan) for 3 hours; the protoplasts released were then washed with 20% sucrose, in which they float? This enzymatic isolation of protoplasts makes use of cell wall degrading enzymes. As normally used these cell wall degrading enzymes are grossly impure, containing many enzymes other than those directly involved in the cell wall degradation. Toxic compounds can also be present, and although it is difficult to generalize, it would seem best always to treat the enzymes with Sephadex before use in order to remove certain enzymes, particularly nucleases, and low molecular weight toxic material. 6 Experience over several years and extending back to the early work of Tribe, ~ who used mechanical rather than enzymatic methods for protoplast isolation, has indicated that isolated protoplasts are far more resistant to such possible harmful effects when they are in the plasmolyzed condition. One reason for this has recently become clear from the work of Withers and Cocking? From studies using thorium dioxide as an electron dense electron microscopic marker, they showed that extensive uptake from the plasmolyticum took place during plasmolysis probably by largescale membrane invagination. In view of this extensive uptake, it would seem advantageous to carry out preplasmolysis in a suitable plasmolyticum before subjecting the tissue to digestion in a possibly harmful cell walldegrading enzyme mixture. As a result the procedures recommended for the isolation of tobacco leaf protoplasts for induced fusion studies with sodium nitrate involved preplasmolysis in 25 % sucrose. 8 Pieces of tobacco leaf taken from 50- to 60-day-old Nicotiana tabacum var. Xanthi plants, from which the lower epidermis had been removed (for practical details see Power and Cocking 9) were incubated for 2 hours in 25% sucrose. After this preplasmolysis the leaf pieces were incubated in a mixture of 5% w/v cellulase (Onozuka 1500) and 0.5% w/v Macerozyme in 25% sucrose, at 20 °, for periods of between 2 and 4 hours. The released protoplasts were washed by flotation in 25 % sucrose. The following two detailed examples may help the reader to appreciate more fully the procedures required for the isolation of protoplasts, first from cereal leaves, and second from suspension culture cells of rose. The
~E. Pojnar, J. H. M. Willison, and E. C. Cocking, Protoplasma 64, 460 (1967). P. K. Evans and E. C. Cocking, in "Plant Tissue and Cell Culture" (H. E. Street, ed.), pp. 100-120, 1972. 7H. T. Tribe, Ann. Bot. 19, 351 (1955). "L. A. Withers and E. C. Cocking, J. Cell Sci. 11, 59 (1972). 9j. B. Power and E. C. Coizking, J. Exp. Bot. 21, 64 (1970).
580
SUBCELLULARFRACTIONS DERIVED FROM PLANT TISSUE
[60]
earlier report of Ruesink TM may also be useful as a further guide to the basic principles involved. Isolation of Cereal Leaf Protoplasts Mature leaves of Secale cereale (rye) or Triticum aestivum (wheat) or Hordeum vulgare (barley) or Avena sativa (oats) are employed. The cereal plants are between 21 and 28 days old, and the leaves are surface sterilized by treatment with 70% ethanol for 2 minutes followed by 30 minutes in 3% sodium hypochlorite (0.3-0.425% w / v available chlorine). It is necessary to add Teepol (0.5 % ) as a wetting agent ( B D H Chemicals Ltd., Poole, United Kingdom). It is important to remove thoroughly the hypochlorite which has been used as a surface sterilant. The hypochlorite is removed by three successive washes in sterile water. After this treatment the leaves are cut into narrow longitudinal threads using a scalpel and the leaf pieces immersed in a filter-sterilized enzyme mixture with 1 g fresh weight of pieces to every 7 ml of enzyme solution. The enzyme solution should be freshly prepared. A satisfactory enzyme solution was made up as follows: 4% Meicelase (Meiji Seika Kaisha Ltd., Tokyo) with either Pectinol R I 0 (Rohm and Haas, Philadelphia, Pennsylvania) or 1% Macerozyme (All Japan Biochemicals Ltd., Nishinomiya, Japan) and 1% potassium dextran sulfate with a sulfur content of 17.3% (Meito Sangyo Co. Ltd., Nagoya, Japan). These enzymes were dissolved in 0.7 M sorbitol or 0.7 M mannitol. Pectinol R I 0 is standardized with diatomaceous earth and a solution of the enzyme was prepared by suspending 20 g in 100 ml of distilled water for 2 hours. The insoluble material was removed by filtration and after the addition of the materials listed above the volume was made up to 100 ml. As only 6.5% of the Pectinol R I 0 is water soluble, this represents 1.3% solution of Pectinol R10. The pH was adjusted to 5.8 with normal HC1. The leaf shreds were incubated in the above medium at 25 ° for 5 hours. After this, the enzymes were removed and the leaf pieces were rinsed briefly with a sterile washing solution. The sterile washing solution was of the following composition: 0.7 M sorbitol, 1 mM KNO:~, 0.2 mM KHzPO4, 0.1 mM MgSo,, 1 mM CaC1,_,, 1 ~M KI, and 10 mM CuSO4. ~1 After this washing the leaf pieces were placed in a fresh enzyme solution. They were incubated in this fresh enzyme solution for a further 12 hours at 25 °, and after this the leaf shreds were teased apart in the washing solution; this resulted in the release of the protoplasts. The isolated cereal leaf protoplasts can be readily sedimented by cen:°A. W. Ruesink, this series, Vol. 23, p. 197. 11R. G. Jensen, R. I. B. Francki, and M. Zaitlin, Plant Physiol. 48, 9 (1971).
[60]
THE ISOLATION OF PLANT PROTOPLASTS
581
trifugation at 100 g. After centrifugation at 100 g for 5 minutes, the isolated protoplasts were resuspended in sterile 0.8 M sucrose. In 0.8 M sucrose the isolated protoplasts do not sediment, but because of their density they rise to the surface of the sucrose when the suspension is centrifuged; as a result further centrifugation at 200 g for 5 minutes caused intact protoplasts to rise to the surface, while cells, cell debris, and free chloroplasts sedimented to the bottom of the centrifuge tube. The layer of protoplasts was resuspended in a large excess of washing solution and centrifuged at 100 g for 5 minutes. The protoplasts sediment in this washing solution. The protoplasts were then suspended in a known volume of washing solution, and a small sample was taken for counting, using a modified Fuchs Rosenthai hemacytometer, 0.2 mm deep (Hawkesley Gelman, Lancing, England). High yields of protoplasts are obtained--of the order of 106 protoplasts per gram fresh weight of leaf. Such isolated cereal leaf protoplasts as these can be readily suspended at known cell densities in suitable culture media and their growth and development studied further. For further details, see Evans e t al. 1~ Isolation of Rose Suspension Culture Protoplasts Protoplasts isolated from "Paul's Scarlet" rose suspension culture cells have been extensively studied. 1~ The suspension culture cells themselves grow rapidly in batch culture on a fully defined medium originally devised by Davies. 13'~They are sterile, and therefore preliminary sterilization of the plant material is not required as in the case of cereal leaves. The fact that the starting material, i.e., the cells, is already sterile removes many of the uncertainties often present when dealing with material fresh from the plant. Indeed, the isolation of leaf protoplasts is beset with the variation that occurs even from variety to variety of the same species in relation to the efficacy of surface sterilization. The suspension culture of rose cells forms a pipettable suspension which is composed of single cells or small groups of cells. The fact that cells are largely separate from one another eliminates the need to use a pectinase enzyme. Cells from all phases of the growth cycle can be used (but see below). One gram fresh weight of cells filtered from the medium is added to every 10 ml of enzyme digestion mixture. The cell wall digestion mixture contains Meicelase (Meiji Seika Kaisha Ltd.) or Onozuka Cellulase (1500 or 3000). The concentration of enzyme which can be employed lies between 0.5% and 3%. The pH is adjusted to between 5.5 and 6.0 with NaOH or HCI. The incubation conditions are as follows: 20 ml of incuba1., p. K. Evans, A. G. Keates, and E. C. Cocking, Planta 104, 178 (1972). a3 R. Pearce, Ph.D. Thesis, Univ. of Nottingham, 1972. 1~. M. E. Davies, Phytochem. 10, 783 (1971).
582
SUBCELLULAR FRACTIONS DERIVED FROM PLANT TISSUE
[60]
tion mixture is placed in 100-ml Erlenmeyer flasks (too great a depth of mixture is deleterious to survival) and held at a constant temperature of 25 o for up to 24 hours. At the end of the incubation period the mixture is centrifuged for 1 minute at 300 g, and the protoplasts that collect at the surface of the liquid are transferred with a Pasteur pipette to a suitable concentration of sucrose. The protoplasts are then resuspended and centrifuged once more in the same molarity of sucrose. This procedure is then repeated. This centrifugation procedure results in the protoplasts forming a dense layer at the surface of the plasmolyticum while debris sinks to the bottom. This relatively simple procedure results in the isolation of rose protoplasts, largely entirely free of debris. The yield of protoplasts and the extent to which cells are absent from the protoplast suspension is related to the age of the suspension relative to the time interval between its current growth and the time when it was isolated and grown up from callus. Excellent preparations of isolated rose protoplasts, essentially completely free of debris, and from cells, were obtained when rose suspension cells were treated in this way when the rose suspension cells had been obtained from liquid cultures which had recently been grown up from callus on agar. Frequently almost all the suspension cells were converted to isolated protoplasts. When cells grown from subsequent passages of the suspension culture were used, it became necessary first to raise the incubation temperatures to 33 o and later to use 3 % purified cellulase (Cellulase 3000) to obtain satisfactory results. It was found, however, that if the suspension cells were returned to agar for several passages, then the original isolation conditions were once more satisfactory. It would thus seem likely that progressive changes either in wall composition or in the nature of enzyme inhibitors in the cells are responsible for these effects. It would also seem likely that there are progressive changes either in wall composition or in the level of inhibitors as the number of passages of the suspension cultures increases. As earlier mentioned, it may be advantageous with certain cell systems to preplasmolyze the tissues or cells from which it is desired to isolate protoplasts. It is difficult, however, to generalize in this respect; in the case of the isolation of rose protoplasts, preplasmolysis in sucrose for 0.5 or 24 hours reduced release of protoplasts without increasing total survival. Added Comments Although the enzymatic isolation of higher plant protoplasts has now become an acceptable laboratory procedure, each tissue must be investigated systematically in relation to the optimum conditions for protoplast release. It should be noted that protoplasts can also be isolated from tissues of higher plant cells by first separating the cells, under plasmolyzing condi-
[61]
ISOLATIONOF MICROSOMES, RIBOSOMES, AND POLYSOMES
583
tions, with Macerozyme and then isolating protoplasts using cellulase to degrade the walls of the separated cells. 14 This method is useful in that it enables the isolation of specifically palisade layer protoplasts from leaves. It is, however, more laborious than the mixed enzyme (Macerozyme plus Cellulase) procedure described for the isolation of cereal leaf protoplasts. Moreover, as discussed by Evans, Keates, and Cocking, 12 the sequential method of Takebe and his co-workers does not yield protoplasts from many cereal leaves. It is also possible to isolate protoplasts mechanically by means of the procedure first introduced by Klercker in 1892.1~ This method does not release protoplasts from meristematic cells since they do not plasmolyze sufficiently. The yield of protoplasts is usually low even from more mature cells. Nevertheless, such mechanically isolated protoplasts are very useful for comparisons between the metabolic activity of protoplasts isolated mechanically and enzymatically. Pilet and his co-workers 16 have recently pioneered the use of mechanically isolated protoplasts in this respect. a*T. Nagata and I. Takebe, Planta 92, 301 (1970). ~J. Klercker, Ofvers Vetensk. Akad. Forh., Stockholm, 49, 463 (1892). ~6p. E. Pilet, C. R. Acad. Sci. Ser. D 273, 2253 (1971).
[61] Isolation of Microsomes, Ribosomes, and Polysomes from Plant Tissues 1 B y JOE H. CHERRY
The first chemical indication that ribonucleoproteins played a direct role in the synthesis of proteins came in the early 1950's when animals were fed radioactive amino acids. When the labeled tissue was excised, homogenized, and then fractionated, the highest concentration of radioactive amino acids was found in the microsomal fraction. 2 This fraction has been shown to contain ribosome (and polyribosome) particles attached to membrane fragments. Careful kinetic studies on the rate of amino acid incorporation into different proteins support the likelihood that the newly synthesized polypeptides attached to ribosomes were precursors to the enzymes found in the cells. Furthermore, it was found that RNase stopped protein synthesis in isolated subcellular fractions. Experiments on whole 1Some of the research presented in this paper was supported by a contract (COO1313-31) from the U.S. Atomic Energy Commission. This is journal paper 4722 of the Purdue Agriculture Experiment Station. -~E. B. Keller, P. C. Zamecnik, and R. B. Loftfield, ]. Histochem. Cytochem. 2, 378 (1954).