Plant Cell Reports
Plant Cell Reports (1987) 6: 23-26
© Springer-Verlag 1987
Suspension and protoplast culture of hexaploid wheat ( Triticum aestivum L.) S. E. Maddock Biochemistry Department, Rothamsted Experimental Station, Harpenden, Hefts, AL5 2JQ, UK Received October 29, 1986 / Communicated by I. K. Vasil
ABSTRACT Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (TriticDm ~ L.). Most commonly, these "suspensions" are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions. ABBREVIATIONS 2,4-D = 2,4-dichlorophenoxyacetic acid; MS = Murashige and Skoog (1962) medium. INTRODUCTION A major barrier to the genetic manipulation of cereal plants by techniques such as transformation and protoplast fusion has been the inability to regenerate whole plants routinely from isolated protop±asts of graminaceous species (Dale 1983; Jones 1985). A significant advance was marked by the work of Vasil and Vasil (1980), in which plantlets of pearl millet (Pennisetum americanum) were obtained via somatic embryogenesis from callus derived from protoplasts of an embryogenic cell suspension. This was subsequently repeated with the species Panicum maximum (Lu et al. 1981) and Pennisetum DUrDureum (Vasil 9_~ A I ~ 1983). In none of these cases did the plants survive transfer from in vitro growth conditions, but in recent work with suspension-derived protoplasts of sugarcane (Saccharum officinarum), regenerated plants have been grown to maturity in the field (Srinivasan and Vasil 1986). This approach has been attempted in many laboratories with other members of the
Offprint requests to." S.E. Maddock
Gramineae, in particular the major crop species, and considerable success has recently been reported in the regeneration of rice plants from protoplasts from suspension or callus cultures (e.g., Fujimura et al. 1985; Toriyama and Hinata 1985; Yamada et al. 1986). However, the production of embryogenic cell suspensions is not an easy process, and in many species even the establishment of non-morphogenetic cell lines can be difficult. Hexaploid wheat (Triticum aestivum) has proved to be one of the more difficult cereal species to grow in suspension. This paper describes the production of suspension cultures of Triticum aestivum and the behaviour of protoplasts isolated from these cultures. MATERIALS AND METHODS Initiation Cultures
and
Maintenance
of Suspension
Cell suspensions were initiated either from embryogenic wheat callus, obtained by the culture of immature embryos and inflorescences as described previously (Maddock et al. 1983), or from individual wheat embryos. Embryogenic callus (20-80 days after initiation from 21 of the cultivars used in Maddock et al. 1983) and immature embryos (1-2 mm in length; cvs. Highbury, Marls Butler, Sicco and Timmo) were transferred to liquid medium and incubated at 25°C on a rotary shaker at 100 rpm. The cell lines MBE6 (cv. Marls Butler) and C82d (cv. Copain) were derived from embryogenic callus originating from immature embryos. Culture media used successfully for growth in suspension were based on MS salts and vitamins, and contained sucrose (30 g 1 -I) and 2,4-D (2.5 or 5.0 mg I-I), at pH 5.8. In the early stages of culture establishment, the medium was replaced at weekly intervals. As the material began to grow, extra medium was added and the cultures were subdivided. Once suspensions had become established (60 ml grown in 250 ml flasks), they were subcultured at weekly intervals at i:i dilution. The two cell lines MBE6 (cv. Marls Butler) and C82d (cv.
24 Copain) were subcultured intervals at 1:5 dilution. Testina for Morphogenetic Suspension Cultures
at
weekly
Capacity
of
Morphogenetic capacity of cell suspension material was tested on a wide range of agar or agarose media, based on MS, B5 (Gamborg et ~/_~ 1968) and N6 (Chu et ~61~ 1975) salts and vitamins, containing various concentrations and combinations of auxins, cytokinins, gibberellic acid, abscisic acid, coconut milk and casein hydrolysate. Cultures (7-21 days old) were first passed through 500 ~ m stainless steel sieves. The cell aggregates retained on the sieve were spread onto the surface of the solid media and the filtered liquid was plated separately. Material was then incubated at 25°C (16 h light/8 h dark cycle, 120 ~Em-2s-l). ProtoDlast
Isolation
A standard protocol was developed for the isolation of protoplasts from cell lines MBE6 and C82d. Cell aggregates from 6-12 day-old cultures were resuspended in an enzyme mixture containing 2% Cellulase R-10 and 0.3% (w/v) Pectolyase Y-23 (750~i0 mOsM, pH 5.6). Mannitol was used as osmoticum throughout the isolation procedure. After incubation for 3-6 hrs in petri dishes on a rotary shaker (30 rpm; at 25°C in the dark), material was passed through 50 ~ m and 38 ~ m stainless steel sieves and the protoplasts pelleted by centrifugation at 100 g for 10 min. The pellets were washed a total of three times by resuspension either in MS medium without hormones or in mannitol alone (750~i0 mOsM, pH 5.8), followed by centrifugation at 100 g for 5 min. With protoplasts from line C82d, an additional flotation step was included after the first wash. 2.5 ml aliquots of protoplasts suspended in washing medium were layered over 5 ml cushions of 25% sucrose (w/v) and centrifuged at 700 g for 30 min. The protoplast layers on top of the sucrose cushions were removed and washed two further times. After the final wash, protoplasts were resuspended in a culture medium consisting of MS salts and vitamins, sucrose (30 g I-i), and 2,4-D (i mg 1 -I) (750±10 mOsM, pH 5.8) and passed through 38 ~m stainless steel sieves. Protoplast density was counted and adjusted to 1-5 x 105 protoplasts ml -I with additional culture medium. Protoplast viability was determined by staining with fluorescein diacetate (Larkin 1976). 5 ml aliquots of protoplasts were incubated in 5 cm plastic dishes (Nunc, Denmark) at 25°C in the dark. MS-based media were used for the culture of protoplasts of cell line C82d, but in establishing a standard procedure, a range of more complex media was compared, including KMSP (Kao and Michyaluk 1975), Schenk and Hildebrandt (1972) Medium Vl and P2 (Potrykus 9_t al. 1977), each containing various combinations and concentrations of sugars, amino acids, and phytohormones.
Culture of Dividing Protoplasms Colonies which developed from protoplasts of cell line C82d were either grown in liquid medium, plated over agar or agarose media, or cultured using the "beadtype" technique (Shillito et al. 1983). For growth in liquid culture or on solid media, colonies were transferred at 3-4 weeks after isolation. For liquid culture, the contents of each culture dish were transferred to 50 ml flasks, together with 5 ml fresh medium, and incubated on a rotary shaker (50-100 rpm) at 25°C. These secondary suspensions were subsequently transferred to 250 ml flasks, diluted with suspension culture m e d i u m (2.5 or 5.0 mg 1 -I 2,4-D) and maintained according to the standard procedure outlined above. For growth on solid media, the contents of each culture dish were diluted with 5 ml fresh incubation medium and repeatedly drawn gently in and out of a Pasteur pipette, to dislodge the colonies from the bottom of the dish. 2.5 ml aliquots were then distributed over agar or agarose plates containing MS salts and vitamins, sucrose (30 g 1 -I) and 2,4-D (0.2 or 1.0 mg i-i). After a further 2-4 weeks of culture, when individual calluses had reached ~ 1 mm in diameter, they were picked off and cultured separately on fresh agar plates. Successful culture using the "beadtype" technique was only possible if the protoplasts had started to divide (~6 days after isolation) before they were embedded in agarose. RESULTS AND DISCUSSION Initiation and Growth of S~Hpension Culture More than 400 suspensions were initiated from embryogenic wheat callus, and the establishment of relatively homogeneous cultures took at least 2-3 months. A total of 200 immature embryos were used to start cultures, but only 80 of these showed sustained growth, and cultures took at least 4 months to become established. The morphology commonly attained by wheat material in shaken liquid culture cannot be described as that of a true cell line (King 1980). These "suspensions" consist of mixtures of small, callus-like or root-type (King 9£h al. 1978) aggregates (up to 2 mm in diameter) and many dead or elongate single cells, which give the medium a cloudy appearance. No capacity for embryogenesis or shoot formation was shown by such suspensions, either in liquid culture or on a wide range of solid media, although rooting occurred readily if 2,4-D levels were lowered. Good preparations of protoplasts could not be obtained from these suspensions. However, three lines that were more finely d i v i d e d in appearance did become established, and two of these have been maintained for more than 4 years. These were originally derived from embryogenic callus of the cvs. Maris Butler (line MBE6) and Copain (line C82d), and were not distinguishable from other types of suspensions until 15-18 months after
25 initiation. They were then carefully cultured to select for smaller aggregates of cytoplasmic cells, and by increasing the frequency and dilution of subculture, more rapidly-dividing lines were obtained. MorDhoioav and MorDhoaenetic CaDacitv of SusDension Cultures MBE6 and C82~ Both of these lines consist predominantly of small groups (< 500 ~ m diameter, ~8 cells) of cytoplasmic cells (Figures la and 2a), although larger aggregates also develop. Cells of line C82d are more densely cytoplasmic than those of line MBE6. Both cell lines contain relatively few dead or empty cells. They have doubling times of 3 days (MBE6) and 5 days (C82d) in the standard MS-based medium, as measured by increases in packed cell volume, fresh weight, and dry weight (data not shown). No improvement in growth rate or culture morphology was obtained by changing to other growth media. No embryoid-like structures develop in liquid culture of these lines, nor do they show the rooting capacity of the more typical "suspensions". Suspension cells were plated onto a wide range of agar or agarose media in attempts to induce morphogenesis, but these lines have only very limited morphogenetic capacity. Cell groups smaller than 500 ~ m , as well as larger aggregates, were able to grow as callus. MS-based media supported good callus growth, which was most rapid at low levels of 2,4-D (0.2 mg i-i). Cytokinins tended to inhibit growth and caused browning of the callus. Some evidence of morphogenetic capacity was found in the form of localized areas of chlorophyll or anthocyanin production, occasional root morphogenesis, development of more compact, nodular tissue, and more rarely the formation of leaf-like structures. A single green plantlet was obtained from culture MBE6, which was rooted on medium lacking hormones (Figure Ib), but it did not develop sufficiently for transfer to the glasshouse. Isolation and Culture _of P r Q ~ Z P ~ ~ Lines MBE6 and C82d Good yields of protoplasts (up to 7.5 x 106 protoplasts from 1 ml cell volume) were obtained from lines MBE6 and C82d at 6-12 days after subculture, after which
both the yield and survival of protoplasts were reduced. Best yields were obtained at about 750 mOsM, although the protoplasts were able to tolerate a wider range of osmolarities. Clean preparations of intact, cytoplasmic protoplasts were obtained consistently (Figures ic and 2b). Multi-nucleate protoplasts were sometimes observed. These probably resulted from spontaneous cell fusions during or after isolation. Occasionally, individual single cells were present in the final preparation, but these lacked cytoplasm and did not undergo division. Filtration through 38 ~ m sieves removed any small groups of cells. Viability of the protoplasts immediately after isolation was almost 100% as measured by FDA staining. General observations on the behaviour of protoplasts from lines MBE6 and C82d were made from many separate isolations. A standard MS-based medium containing sucrose (30 g 1 -I) and 2,4-D (I mg 1 -I) was found to be most satisfactory for culture. Protoplasts from both lines were cytoplasmic and relatively dense, so that shortly after isolation they settled to the bottom of the culture dish and within 24 h began to adhere to the surface. Many of the protoplasts collapsed during the first few days after isolation, but in the surviving protoplasts cytoplasmic rearrangements occurred within 24 h and cell wall formation began within 2-3 days. Sustained cell divisions did not occur in protoplasts from line MBE6, although budding was commonly observed and sometimes protoplasts remained intact for 2 weeks or more. In contrast, protoplasts from line C82d consistently underwent repeated cell divisions when cultured (Figures 2c-h). Division of C82d protoplasts occurred only when they were cultured at an initial density of at least 1 x 105 protoplasts ml -I. Clear cell divisions were usually seen at 7-8 days after isolation (Figures 2c and d), but occasionally dividing cells were present as early as 5 days after isolation. The proportion of protoplasts undergoing division was low (
Plant Cell Reports (1987) 6: 23-26
© Springer-Verlag 1987
Suspension and protoplast culture of hexaploid wheat ( Triticum aestivum L.) S. E. Maddock Biochemistry Department, Rothamsted Experimental Station, Harpenden, Hefts, AL5 2JQ, UK Received October 29, 1986 / Communicated by I. K. Vasil
ABSTRACT Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (TriticDm ~ L.). Most commonly, these "suspensions" are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions. ABBREVIATIONS 2,4-D = 2,4-dichlorophenoxyacetic acid; MS = Murashige and Skoog (1962) medium. INTRODUCTION A major barrier to the genetic manipulation of cereal plants by techniques such as transformation and protoplast fusion has been the inability to regenerate whole plants routinely from isolated protop±asts of graminaceous species (Dale 1983; Jones 1985). A significant advance was marked by the work of Vasil and Vasil (1980), in which plantlets of pearl millet (Pennisetum americanum) were obtained via somatic embryogenesis from callus derived from protoplasts of an embryogenic cell suspension. This was subsequently repeated with the species Panicum maximum (Lu et al. 1981) and Pennisetum DUrDureum (Vasil 9_~ A I ~ 1983). In none of these cases did the plants survive transfer from in vitro growth conditions, but in recent work with suspension-derived protoplasts of sugarcane (Saccharum officinarum), regenerated plants have been grown to maturity in the field (Srinivasan and Vasil 1986). This approach has been attempted in many laboratories with other members of the
Offprint requests to." S.E. Maddock
Gramineae, in particular the major crop species, and considerable success has recently been reported in the regeneration of rice plants from protoplasts from suspension or callus cultures (e.g., Fujimura et al. 1985; Toriyama and Hinata 1985; Yamada et al. 1986). However, the production of embryogenic cell suspensions is not an easy process, and in many species even the establishment of non-morphogenetic cell lines can be difficult. Hexaploid wheat (Triticum aestivum) has proved to be one of the more difficult cereal species to grow in suspension. This paper describes the production of suspension cultures of Triticum aestivum and the behaviour of protoplasts isolated from these cultures. MATERIALS AND METHODS Initiation Cultures
and
Maintenance
of Suspension
Cell suspensions were initiated either from embryogenic wheat callus, obtained by the culture of immature embryos and inflorescences as described previously (Maddock et al. 1983), or from individual wheat embryos. Embryogenic callus (20-80 days after initiation from 21 of the cultivars used in Maddock et al. 1983) and immature embryos (1-2 mm in length; cvs. Highbury, Marls Butler, Sicco and Timmo) were transferred to liquid medium and incubated at 25°C on a rotary shaker at 100 rpm. The cell lines MBE6 (cv. Marls Butler) and C82d (cv. Copain) were derived from embryogenic callus originating from immature embryos. Culture media used successfully for growth in suspension were based on MS salts and vitamins, and contained sucrose (30 g 1 -I) and 2,4-D (2.5 or 5.0 mg I-I), at pH 5.8. In the early stages of culture establishment, the medium was replaced at weekly intervals. As the material began to grow, extra medium was added and the cultures were subdivided. Once suspensions had become established (60 ml grown in 250 ml flasks), they were subcultured at weekly intervals at i:i dilution. The two cell lines MBE6 (cv. Marls Butler) and C82d (cv.
24 Copain) were subcultured intervals at 1:5 dilution. Testina for Morphogenetic Suspension Cultures
at
weekly
Capacity
of
Morphogenetic capacity of cell suspension material was tested on a wide range of agar or agarose media, based on MS, B5 (Gamborg et ~/_~ 1968) and N6 (Chu et ~61~ 1975) salts and vitamins, containing various concentrations and combinations of auxins, cytokinins, gibberellic acid, abscisic acid, coconut milk and casein hydrolysate. Cultures (7-21 days old) were first passed through 500 ~ m stainless steel sieves. The cell aggregates retained on the sieve were spread onto the surface of the solid media and the filtered liquid was plated separately. Material was then incubated at 25°C (16 h light/8 h dark cycle, 120 ~Em-2s-l). ProtoDlast
Isolation
A standard protocol was developed for the isolation of protoplasts from cell lines MBE6 and C82d. Cell aggregates from 6-12 day-old cultures were resuspended in an enzyme mixture containing 2% Cellulase R-10 and 0.3% (w/v) Pectolyase Y-23 (750~i0 mOsM, pH 5.6). Mannitol was used as osmoticum throughout the isolation procedure. After incubation for 3-6 hrs in petri dishes on a rotary shaker (30 rpm; at 25°C in the dark), material was passed through 50 ~ m and 38 ~ m stainless steel sieves and the protoplasts pelleted by centrifugation at 100 g for 10 min. The pellets were washed a total of three times by resuspension either in MS medium without hormones or in mannitol alone (750~i0 mOsM, pH 5.8), followed by centrifugation at 100 g for 5 min. With protoplasts from line C82d, an additional flotation step was included after the first wash. 2.5 ml aliquots of protoplasts suspended in washing medium were layered over 5 ml cushions of 25% sucrose (w/v) and centrifuged at 700 g for 30 min. The protoplast layers on top of the sucrose cushions were removed and washed two further times. After the final wash, protoplasts were resuspended in a culture medium consisting of MS salts and vitamins, sucrose (30 g I-i), and 2,4-D (i mg 1 -I) (750±10 mOsM, pH 5.8) and passed through 38 ~m stainless steel sieves. Protoplast density was counted and adjusted to 1-5 x 105 protoplasts ml -I with additional culture medium. Protoplast viability was determined by staining with fluorescein diacetate (Larkin 1976). 5 ml aliquots of protoplasts were incubated in 5 cm plastic dishes (Nunc, Denmark) at 25°C in the dark. MS-based media were used for the culture of protoplasts of cell line C82d, but in establishing a standard procedure, a range of more complex media was compared, including KMSP (Kao and Michyaluk 1975), Schenk and Hildebrandt (1972) Medium Vl and P2 (Potrykus 9_t al. 1977), each containing various combinations and concentrations of sugars, amino acids, and phytohormones.
Culture of Dividing Protoplasms Colonies which developed from protoplasts of cell line C82d were either grown in liquid medium, plated over agar or agarose media, or cultured using the "beadtype" technique (Shillito et al. 1983). For growth in liquid culture or on solid media, colonies were transferred at 3-4 weeks after isolation. For liquid culture, the contents of each culture dish were transferred to 50 ml flasks, together with 5 ml fresh medium, and incubated on a rotary shaker (50-100 rpm) at 25°C. These secondary suspensions were subsequently transferred to 250 ml flasks, diluted with suspension culture m e d i u m (2.5 or 5.0 mg 1 -I 2,4-D) and maintained according to the standard procedure outlined above. For growth on solid media, the contents of each culture dish were diluted with 5 ml fresh incubation medium and repeatedly drawn gently in and out of a Pasteur pipette, to dislodge the colonies from the bottom of the dish. 2.5 ml aliquots were then distributed over agar or agarose plates containing MS salts and vitamins, sucrose (30 g 1 -I) and 2,4-D (0.2 or 1.0 mg i-i). After a further 2-4 weeks of culture, when individual calluses had reached ~ 1 mm in diameter, they were picked off and cultured separately on fresh agar plates. Successful culture using the "beadtype" technique was only possible if the protoplasts had started to divide (~6 days after isolation) before they were embedded in agarose. RESULTS AND DISCUSSION Initiation and Growth of S~Hpension Culture More than 400 suspensions were initiated from embryogenic wheat callus, and the establishment of relatively homogeneous cultures took at least 2-3 months. A total of 200 immature embryos were used to start cultures, but only 80 of these showed sustained growth, and cultures took at least 4 months to become established. The morphology commonly attained by wheat material in shaken liquid culture cannot be described as that of a true cell line (King 1980). These "suspensions" consist of mixtures of small, callus-like or root-type (King 9£h al. 1978) aggregates (up to 2 mm in diameter) and many dead or elongate single cells, which give the medium a cloudy appearance. No capacity for embryogenesis or shoot formation was shown by such suspensions, either in liquid culture or on a wide range of solid media, although rooting occurred readily if 2,4-D levels were lowered. Good preparations of protoplasts could not be obtained from these suspensions. However, three lines that were more finely d i v i d e d in appearance did become established, and two of these have been maintained for more than 4 years. These were originally derived from embryogenic callus of the cvs. Maris Butler (line MBE6) and Copain (line C82d), and were not distinguishable from other types of suspensions until 15-18 months after
25 initiation. They were then carefully cultured to select for smaller aggregates of cytoplasmic cells, and by increasing the frequency and dilution of subculture, more rapidly-dividing lines were obtained. MorDhoioav and MorDhoaenetic CaDacitv of SusDension Cultures MBE6 and C82~ Both of these lines consist predominantly of small groups (< 500 ~ m diameter, ~8 cells) of cytoplasmic cells (Figures la and 2a), although larger aggregates also develop. Cells of line C82d are more densely cytoplasmic than those of line MBE6. Both cell lines contain relatively few dead or empty cells. They have doubling times of 3 days (MBE6) and 5 days (C82d) in the standard MS-based medium, as measured by increases in packed cell volume, fresh weight, and dry weight (data not shown). No improvement in growth rate or culture morphology was obtained by changing to other growth media. No embryoid-like structures develop in liquid culture of these lines, nor do they show the rooting capacity of the more typical "suspensions". Suspension cells were plated onto a wide range of agar or agarose media in attempts to induce morphogenesis, but these lines have only very limited morphogenetic capacity. Cell groups smaller than 500 ~ m , as well as larger aggregates, were able to grow as callus. MS-based media supported good callus growth, which was most rapid at low levels of 2,4-D (0.2 mg i-i). Cytokinins tended to inhibit growth and caused browning of the callus. Some evidence of morphogenetic capacity was found in the form of localized areas of chlorophyll or anthocyanin production, occasional root morphogenesis, development of more compact, nodular tissue, and more rarely the formation of leaf-like structures. A single green plantlet was obtained from culture MBE6, which was rooted on medium lacking hormones (Figure Ib), but it did not develop sufficiently for transfer to the glasshouse. Isolation and Culture _of P r Q ~ Z P ~ ~ Lines MBE6 and C82d Good yields of protoplasts (up to 7.5 x 106 protoplasts from 1 ml cell volume) were obtained from lines MBE6 and C82d at 6-12 days after subculture, after which
both the yield and survival of protoplasts were reduced. Best yields were obtained at about 750 mOsM, although the protoplasts were able to tolerate a wider range of osmolarities. Clean preparations of intact, cytoplasmic protoplasts were obtained consistently (Figures ic and 2b). Multi-nucleate protoplasts were sometimes observed. These probably resulted from spontaneous cell fusions during or after isolation. Occasionally, individual single cells were present in the final preparation, but these lacked cytoplasm and did not undergo division. Filtration through 38 ~ m sieves removed any small groups of cells. Viability of the protoplasts immediately after isolation was almost 100% as measured by FDA staining. General observations on the behaviour of protoplasts from lines MBE6 and C82d were made from many separate isolations. A standard MS-based medium containing sucrose (30 g 1 -I) and 2,4-D (I mg 1 -I) was found to be most satisfactory for culture. Protoplasts from both lines were cytoplasmic and relatively dense, so that shortly after isolation they settled to the bottom of the culture dish and within 24 h began to adhere to the surface. Many of the protoplasts collapsed during the first few days after isolation, but in the surviving protoplasts cytoplasmic rearrangements occurred within 24 h and cell wall formation began within 2-3 days. Sustained cell divisions did not occur in protoplasts from line MBE6, although budding was commonly observed and sometimes protoplasts remained intact for 2 weeks or more. In contrast, protoplasts from line C82d consistently underwent repeated cell divisions when cultured (Figures 2c-h). Division of C82d protoplasts occurred only when they were cultured at an initial density of at least 1 x 105 protoplasts ml -I. Clear cell divisions were usually seen at 7-8 days after isolation (Figures 2c and d), but occasionally dividing cells were present as early as 5 days after isolation. The proportion of protoplasts undergoing division was low (
