Plant Cell Reports
Plant Cell Reports (1987) 6: 476- 479
© Springer-Verlag1987
The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (Moench) Voss.* Faouzi Bekkaoui 1, Praveen K. Saxena 2, Stephen M. Attree 2, Larry C. Fowke 2, and David I. Dunstan 1 1 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, Saskatoon, SK, Canada, S7N 0W9 2 Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 0W0 Received September 8, 1987 / Revised version received October 15, 1987 - Communicated by F. Constabel
ABSTRACT Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea 91auca. A combination of 0.5% Cellulase R-tO, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with O.5M mannitol 6 and 5 mM CaCIg.2HgO produced an average of 4.5 X 10 protoplasts p~r g~am fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KMSP) media best supported mitotic divisions of protoplasts. A densitY of 10 protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique. ABBREVIATIONS BAP, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; FDA, fluorescein diacetate INTRODUCTION
Isolated protoplasts are viewed as one of the most important vehicles for genetic improvement of plants (Maheshwari e t a ] . , 1986). One such improvement would be the production of white spruce transformed for resistance to spruce budworm (Choristoneura fumifera) using the endotoxin gene isolated from Bacillus thuringiensis. However, research on protoplast isolation and regeneration of woody plants including conifers has been very limited (Dunstan and Thorpe, 1987). Protoplasts of conifers have been isolated from a variety of tissues such as cotyledons (Kirby and Cheng, 1979; David and David, 1979), roots (Faye and David, 1983), cell suspension cultures (Hakman and von Arnold, 1983), and needles (David et a l . , 1986). Such protoplasts showed differing degrees of divisional a c t i v i t y leading to cell cluster formation and occasionally to callus; no organogenesis was reported (David, 1987). The possibility of regenerating plants from protoplasts of recalcitrant species was f i r s t realized by Vasil and Vasil (1980) who successfully recovered adventive embryos from previously nonregenerable monocot (Pennisetum purpureum) protoplasts. This s u c c e ~ a - t T r i b u t e d to the source of protoplasts which was an embryogenic cell line derived from immature embryos. The use of such * NRCCNo. 27937
Offprintrequeststo: F. Bekkaoui
methods has recently led to successful regeneration of somatic embryos from protoplasts of Pinus taeda (Gupta and Durzan, 1987). The establishment of an embryogenic cell line of white spruce (Picea 9lauca) was described by Hakman and Fowke (1987). This communication reports the conditions for isolation, culture, and subculturable callus formation from protoplasts derived from that embryogenic cell suspension culture. MATERIAL AND METHODS Plant material. The isolation of embryogenic callus of white spruce (Picea 91auca (Moench) Voss.) and the establishment of cell suspension cultures have been described previously (Hakman and Fowke, 1987). Cells were maintained in LP medium (von Arnold and Erik~son, 1981) contai@ing 1% (w/v) sucrose, 2 mgl- 2,4-D and 1 mgl-" BAP. For subculturing, 15 ml aliquots from a 7-day-old culture were transferred to 50 ml of fresh medium dispensed into 250 ml DeLong flasks. Cell suspensions were agitated on a gyratory shaker (150 r~m) wnder continuous irradiance of 15-20 ~Em- sT M at 25°C. The cell suspension cultures used in this study, designated WSI, had been maintained for 14 months prior to commencing these experiments. They contained a heterogeneous population of cells including free cells, clusters of isodiametric cells and proembryos (Fig. 1). Isolation of Protoplasts. Cells from 6-day-old subcultures were collected on Miracloth (Chicopee M i l l , New York, U.S.A.) and transferred to Petri dishes containing a cell wall digesting solution (10 ml for 2 g cells). The enzyme solution consisted of 0.5% (w/v) CeIlulase R-IO, 0.25% each of Rhozyme HP-150, Macerozyme and Driselase, 5 mM CaCIg.2HgO and O.5M mannitol. In some experiments Maceroz~me End Rhozyme HP-150 were replaced by Hemicellulase and Pectinase at 0.25%. Prior to use the solution was centrifuged at 2500 g for 10 min at 4°C to remove impurities. The supernatant f l u i d was adjusted to pH 5.8 with KOH and f i l t e r - s t e r i l i z e d using disposable Nalgene filterware. Ceils were incubated in enzyme solution for 3-4 h on a shaker (50 rpm) at 25 ± 1°C in the dark. The resulting suspension was then passed through a 65 ~m nylon screen to remove debris and undigested cells and was centrifuged at 100 g for 5 min. The resulting pellet was gently resuspended in 5 ml of 20% (w/v) sucrose on top of which I ml of O.5M
477
~
. White spruce embryogenic cell suspension culture used for protoplast isolation. Note the proembryo free cells (FC) and isodiametric cells (IC). Bar = 0.2 mm. Fi 9. 2. Freshly isolated white spruce protoplasts. Bar = 30 ~m. Fig. 3. First division of cells derived from white spruce protoplasts. Bar = 30 um. Fig. 4. Cell cluster derived from white spruce protoplast after 2 weeks in LP medium containing 5 mM glutamine. Bar =_~0 ~m. Fig. 5. C~llus derived from white spruce protoplast after 2 subcultures on LP medium containing 2 mgl 2,4-D and 1 mgl- BAP. Bar = 2 mm.
mannitol was layered. Following centrifugation (100 g for 5 min), viable protoplasts formed a band at the interface between sucrose and mannitol and were collected with a Pasteur pipette. The protoplasts were washed once with a O.5M mannitol solution and were f i n a l l y resuspended in culture medium. V i a b i l i t y of the protoplasts was tested with fluorescein diacetate (FDA) or Evans' Blue (Larkin, 1976; Constabel, 1982). The presence of cell walls was confirmed by calcofluor staining (Constabel, 1982). Culture of Protoplasts. Purified protoplasts were cultured~in 100 Rl ~rops at a density of either 10~, 2.5 X 10~ or 5 X 10 per ml on the lower halves of plastic Petri dishes (9 cm diameter), 5 drops per dish. A l t e r n a t i v e l y , in some experiments, 96 cell-well plates (Corning) were also used for protoplast culture (100 ~l per well). The media tested for culturing the protoplasts were B5 (Gamborg et a l . , 1968), MS (Murashige and Skoog, 1962), N6 -(-Chu-~, 1978), KMSP (Kao and Michayluk, 1975), LP (von Arnold and Eriksson, 1981) and LM (Litvay et a l . , 1981). All the culture media contained 0.37 M glucose as osmoticum and varying levels of BAP and 2,4-D. KM8Pmedium was f i l t e r - s t e r i l i z e d , a l l other media were s t e r i l i z e d by autoclaving. Some experiments involved the use of anti-browning compounds added to LP medium, these were polyvinylpyrrolidone (0.5%), spermidine (2.5 mM), spermine (2.5 mM) and n-propyl gallate (0.25 mM). These compounds were f i l t e r - s t e r i l i z e d . All cultures were maintained at 25°C i~ m~ist plastic boxes under diffuse l i g h t (7-10 ~E m- s- ). The percentage of
f i r s t division was estimated after 6 days in culture according to the formula: number of f i r s t divisions x 100 Percentage = i n i t i a l plating density At least 500 protoplasts per treatment were scored for the estimation of division frequency and 3-5 replicates were prepared for each treatment. All the experiments were repeated at least twice. Culture of microcalli. Microcalli are cultured using the following nurse culture method: 0.15g fresh weight of cells were harvested from the embryogenic cell suspension culture described e a r l i e r , plated onto agar-solidified (0.5%, Sigma) ~P medium containing 2 mgl- 2,4-D and 1 mgl- BAP and allowed to grow for four to seven days. After this time two layers of f i l t e r s were placed on top of each nurse callus. The lower f i l t e r was a Whatman no. 2 f i l t e r paper (7cm diameter) onto which was placed the second f i l t e r of black cellulose acetate (Millipore AA, 0.8 ~, 4.5 cm diameter). Microcalli, present in culture solution six weeks after protoplast isolation, were subsequently pipetted onto the black f i l t e r . The f i l t e r arrangement permitted observation of microcallus development while preventing mixing with cells of the nurse callus. After four weeks of nurse culture the developing c a l l i were transferred d i r e c t l y onto agar-solidified LP medium as described above. Chemicals. Cellulase R-IO and Macerozyme were obtained from Yakult Honsha Pharmaceutical Industry, Nishinomiya, Japan and Rhozyme HP-150 from Genencor,
478 New York, U.S.A. All other biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A.
concentration of 2,4-D was found to be 1 mg1-1 and was used in a l l subsequent experiments. In LP medium 2-3% of viable cells showed second division after 12 days. By 21 days clusters containing 5-6 cells were observed. After the addition of an equal volume of fresh medium most cells ceased to grow and eventually underwent necrosis. An early problem encountered in culturing the protoplasts of Picea 91auca was the occurrence of browning. The browning of protoplast-derived cells was apparent within 8-10 days. Similar observations have been recorded for protoplasts of Pinus coulter± (Patel et a l . , 1984), Pinus lambertiana ~upta and Durzan, 19863 and several angiosperms (see Saxena and G i l l , 1986). The phenomenon of browning in ceil cultures is usually attributed to the production of excess phenolics which causes cessation of cell proliferation. Inclusion of polyvinylpyrrolidone (0.5%) (Saxena and G i l l , 1986) or 2.5 mM each of sperm±dine and spermine postponed the occurrence of browning by 8-10 days but did not completely eliminate i t . The polyamines have been reported to be beneficial for improving the quality of cereal protoplasts (Galston et a l . , 1978). The use of n-propyl gallate did not r e s i g n the stabilisation of Pice,a protoplasts. This compound has been shown to delay the senescence of corn protoplasts (Saleem and Cutler, 1987). Glutamine had no significant effect on the induction of the f i r s t cell division over a concentration range of 0-10 mM, and at 20 ff~i or more was inhibitory to the f i r s t cell division. In
RESULTS AND DISCUSSION
Isolation of Protoplasts. The protoplasts (Fig. 2) were isolated from six-day-old cell suspension cultures. An effective enzyme combination for release of protoplasts comprised 0.5% Cellulase R-IO and 0.25% each of Macerozyme, Driselase and Rhozyme HP-150. Macerozymeand Rhozyme in the enzyme mixture given above could be replaced by Pectinase and Hemicellulase which were equally effective in releasing the protoplasts. The period of incubation required for the complete removal of the cell walls, confirmed with Calcofluor staining, wasA3-4 h. Under these conditions an average of 4.5 x 10~ protoplasts per gram fresh weight of suspension were isolated. The v i a b i l i t y of the protoplasts tested with FDA or Evans' Blue varied from 70-90%. Protoplast Culture and Influence of Nutrient Medium. A new cell wall was formed between 24-48h after protoplast isolation. The v i a b i l i t y of cells decreased to 30-40% after one week. The greatest number of protoplasts underwent f i r s t division (Fig. 3) in LP and KM8Pmedium (Table 1). This s i m i l a r i t y is not surprising as these media contain similar organic constituents. All other media produced a lower division frequency. We chose LP medium for further experiments for conveniencebecause i t was also being used for cell culture maintenance.
Table I . The influence of various nutrient media and growth regulators on the percentage of f i r s t division of protoplasts from an embryogenic cell suspensio~ of Picea 9lauca, after 6 days of culture, plating density 5 x 10 per ml. Concentration of 2,4-D (mg1-1)
Medi um 0
0.5
1.0
2.5
1.0+
2.0+
BAP 0.5
BAP 1.0
LP
1.2±0.4
4.9±1.4
7.3±1.1
4.3±1.2
3.3±0.4
5.1±1.0
LM
0.5±0.4
1.3±0.6
1.3±0.6
1.1±0.2
1.1±0.5
2.1±0.4
N6
0.7±0.6
3.1±1.0
3.7±0.3
3.0±0.5
3.4±1.3
3.0±1.0
KM8P
0.5±0.4
4.6±1.3
6.8±0.7
5.6±0.2
4.1±0.4
3.5±1.2
± standard error
As seen in Table 1, the presence of 2,4-D alone was sufficient to induce cell division, and BAP decreased the frequency of division. Kinetin also showed a similar effect (data not presented). A combination of an auxin and cytokinin is necessary for most protoplasts to develop cell walls and divide (see Maheshwari et a l . , 1986). However, in certain cases, usually with cell suspension cultures, auxin alone has been reported to trigger and sustain the mitosis (Kao et a l . , 1971; Grambow et a l . , 1972; Uchimiya and Murashige, 1976). Cell wall formation and division of Picea protoplasts in the absence of 2,4-D (Table I) can be attributed to endogenous levels of growth regulators. The optimal
contrast glutamine at 5mM sustained the growth of already dividing cells derived from Picea protoplasts, leading to the formation of clusters of 6-40 cells (Fig. 4) and microcalli (more than 40 cells in size); no microcalli were recovered in the absence of glutamine. The addition of glutamine to the culture medium has been reported to be beneficial for conifer protoplasts (Kirby and Cheng, 1979; David et a l . , 1984). David (1987) suggested that glutamine is beneficial because cells may be incapable of assimilating nitrogen in sufficient quantity to sustain growth. Even though i t was possible to obtain microcalli in the presence of 5mM glutamine, their number was
479 very low. The number of cell clusters and microcalli could be increased however (from very rare to about two clusters per culture drop) ~hen th~ plating density was lowered from 5 x i0 to 10- protoplasts per ml, using the drop culture method in Petri dishes. Browning was also ~ubstantially reduced. A plating density of 2.5 x i0 protoplasts per ml produced an intermediate result. When the same experiment was done using 96 c e l l - w e l l plates, approximately 15 clusters of c e l l s formed per drop (Table 2). The plating density was found to be c r i t i c a l with other species (Sticklen et a l . , 1985). The improved frequency of cell cluster formation in 96 c e l l - w e l l plates seemed to be due to reduced aggregation of protoplasts, which favoured c e l l d i v i s i o n in protoplast cultures of Picea. Table 2. Influence of the type of culture dish on the formation of clusters of 6-40 c e l l s from white spruce proto~lasts c u l t i v a t e d in LP medium with 2,4-D ( i m g l - ) and glutamine (5 mM) a f t e r 4 weeks. Protoplasts were cultured in 100 ~I drops at a plating density of i0 per ml.
Culture dish
Petri dish Cell-well
Number Numberof Numberof of 100 Drops with clusters ~l Drops clusters
MeanNumber of clusters per drop
78
21 (26%)
133
1.7
100
100 (100%)
1520
15.2
Culture of Microcalli. At the time of inoculation to nurse culture the microcalli varied in size from 0.1 - 0.3 mm diameter. After 4 weeks of nurse culture, c a l l i increased in size to approximately 3-5 mm diameter. The recovery of transferrable c a l l i from microcalli was 60-80% e f f i c i e n t . Most of the transferred c a l l i were green and f r i a b l e (Fig. 5), and consisted of groups of small isodiametric cells. Other c a l l i were white and f r i a b l e , consisting of groups of small isodiametric cells and occasional elongated cells. Such c a l l i have been maintained for up to nine weeks (3 passages) after removal from nurse culture. In this time green c a l l i increased about 2-3 fold each passage and have been subcultured, whereas the white c a l l i exhibited negligible growth. I t remains to be seen i f either type of c a l l i have competence for embryogenesis or organogenesis.
The present study has resulted in the development of an i s o l a t i o n and culture technique which permits the reproducible recovery of c a l l i from protoplasts of embryogenic suspensions of Picea 91auca. This has been achieved by studies on the effects of enzyme solution, base medium formulation, glutamine concentration and plating density. Modifications of this basic technique have also been applied to other embryogenic suspension lines of Picea 91auca. One of these l i n e s , WS3, has subsequently been shown to be capable of regeneration into somatic embryos (Attree et a l . , in press).
ACKNOWLEDGEMENTS This work was supported by a strategic research grant (G-1868) from Natural Sciences and Engineering Research Council of Canada to L.C. Fowke and D.I. Dunstan.
REFERENCES von Arnold S, Eriksson T (1981) Can. J. Bot. 59: 870-874. Chu CC (1978) I n : Proceedings of Symposium on Plant Tissue Culture, Science Press, Peking, pp 43-50. Constabel F (1982) In: Wetter L.R., Constabel, F. (eds.) Plant t~sue culture methods. NRC of Canada Prairie Regional Laboratory, Saskatoon, Saskatchewan, pp 38-48. David A (1987) I n : Bonga, J.M., Durzan, D.J. (eds.) Cell and Tissue culture in f o r e s t r y , vol. 2: specific principles and methods, pp 2-15. David A, David H (1979) Z. Pflanzenphysiol. 94: 173-177. David H, Jaret E, David A (1984) Physiol. Plant 61: 477-482. David H, Deboucaud MT, Gaultier JM, David A (1986) Tree Physiol. i : 21-30. Dunstan DI, Thorpe TA (1987) In: Vasil, I.K. (ed.) Cell culture and somatic cell genetics of plants, Academic Press, pp 223-241. Faye M, David A (1983) Physiol. Plant. 59: 359-362. Galston AW, Altman A, Kaur-Sawhney R (1978) Plant Sci. Lett. 11: 69-79. Gamborg OL, M i l l e r RA, Ojima K (1968) Exp. Cell Res. 50: 151-158. Grambow HJ, Kao KN, M i l l e r RA, Gamborg OL (1972) Planta 103: 348-355. Gupta PK, Durzan DJ (1987) Bio/technology 5: 710-712. Gupta PK, Durzan DJ (1986) Plant Cell Reports 5: 346-348. Hakman I , Fowke LC (1987) Plant Cell Reports 6: 20-22. Hakman I , Arnold S. Von (1983) Plant Cell Reports 2: 92-94. Kao KN, Gamborg OL, M i l l e r RA, Keller WA (1971) Nature New Biol. 232: 124. Kao KN, Michayluk MR (1975) Planta 126: 105-110. Kirby EG, Cheng T (1979) Plant Sc. Lett. 14: 145-154. Larkin PJ (1976) Planta 128: 213-216. Litvay JD, Johnson MA, Verma DL, Einspahr DW, Weyrauch K (1981) Inst. Paper Chem. Pap. Ser. 115: 1-17. Maheshwari SC, G i l l R, Maheshwari N, Gharyal PK (1986) In: Reinert J, Binding H (eds.) D i f f e r e n t i a t i o n of protoplasts and of transformed c e l l s , Springer Verlag, Berlin. pp 3-36. Murashige T, Skoo9 F (1962) Physiol. Plant. 115: 473-479. Patel KR, Shekhawat RS, Berlyn AP, Thorpe TA (1984) Plant cell tissue and organ culture 3: 85-90. Saleem M, Cutler AJ (1987) J. Plant Physiol. 128: 479-484. Saxena PK, G i l l R (1986) Biol. Plant. 28: 313-315. Sticklen MB, Lineberger D, Domir SC (1985) Plant Sc. 41: 117-120. Uchimiya H, Murashige T (1976) Plant Physiol. 57: 424-429. Vasil V, Vasil K (1980) Theor. Appl. Genet. 56: 97-99.
Plant Cell Reports (1987) 6: 476- 479
© Springer-Verlag1987
The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (Moench) Voss.* Faouzi Bekkaoui 1, Praveen K. Saxena 2, Stephen M. Attree 2, Larry C. Fowke 2, and David I. Dunstan 1 1 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, Saskatoon, SK, Canada, S7N 0W9 2 Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 0W0 Received September 8, 1987 / Revised version received October 15, 1987 - Communicated by F. Constabel
ABSTRACT Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea 91auca. A combination of 0.5% Cellulase R-tO, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with O.5M mannitol 6 and 5 mM CaCIg.2HgO produced an average of 4.5 X 10 protoplasts p~r g~am fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KMSP) media best supported mitotic divisions of protoplasts. A densitY of 10 protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique. ABBREVIATIONS BAP, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; FDA, fluorescein diacetate INTRODUCTION
Isolated protoplasts are viewed as one of the most important vehicles for genetic improvement of plants (Maheshwari e t a ] . , 1986). One such improvement would be the production of white spruce transformed for resistance to spruce budworm (Choristoneura fumifera) using the endotoxin gene isolated from Bacillus thuringiensis. However, research on protoplast isolation and regeneration of woody plants including conifers has been very limited (Dunstan and Thorpe, 1987). Protoplasts of conifers have been isolated from a variety of tissues such as cotyledons (Kirby and Cheng, 1979; David and David, 1979), roots (Faye and David, 1983), cell suspension cultures (Hakman and von Arnold, 1983), and needles (David et a l . , 1986). Such protoplasts showed differing degrees of divisional a c t i v i t y leading to cell cluster formation and occasionally to callus; no organogenesis was reported (David, 1987). The possibility of regenerating plants from protoplasts of recalcitrant species was f i r s t realized by Vasil and Vasil (1980) who successfully recovered adventive embryos from previously nonregenerable monocot (Pennisetum purpureum) protoplasts. This s u c c e ~ a - t T r i b u t e d to the source of protoplasts which was an embryogenic cell line derived from immature embryos. The use of such * NRCCNo. 27937
Offprintrequeststo: F. Bekkaoui
methods has recently led to successful regeneration of somatic embryos from protoplasts of Pinus taeda (Gupta and Durzan, 1987). The establishment of an embryogenic cell line of white spruce (Picea 9lauca) was described by Hakman and Fowke (1987). This communication reports the conditions for isolation, culture, and subculturable callus formation from protoplasts derived from that embryogenic cell suspension culture. MATERIAL AND METHODS Plant material. The isolation of embryogenic callus of white spruce (Picea 91auca (Moench) Voss.) and the establishment of cell suspension cultures have been described previously (Hakman and Fowke, 1987). Cells were maintained in LP medium (von Arnold and Erik~son, 1981) contai@ing 1% (w/v) sucrose, 2 mgl- 2,4-D and 1 mgl-" BAP. For subculturing, 15 ml aliquots from a 7-day-old culture were transferred to 50 ml of fresh medium dispensed into 250 ml DeLong flasks. Cell suspensions were agitated on a gyratory shaker (150 r~m) wnder continuous irradiance of 15-20 ~Em- sT M at 25°C. The cell suspension cultures used in this study, designated WSI, had been maintained for 14 months prior to commencing these experiments. They contained a heterogeneous population of cells including free cells, clusters of isodiametric cells and proembryos (Fig. 1). Isolation of Protoplasts. Cells from 6-day-old subcultures were collected on Miracloth (Chicopee M i l l , New York, U.S.A.) and transferred to Petri dishes containing a cell wall digesting solution (10 ml for 2 g cells). The enzyme solution consisted of 0.5% (w/v) CeIlulase R-IO, 0.25% each of Rhozyme HP-150, Macerozyme and Driselase, 5 mM CaCIg.2HgO and O.5M mannitol. In some experiments Maceroz~me End Rhozyme HP-150 were replaced by Hemicellulase and Pectinase at 0.25%. Prior to use the solution was centrifuged at 2500 g for 10 min at 4°C to remove impurities. The supernatant f l u i d was adjusted to pH 5.8 with KOH and f i l t e r - s t e r i l i z e d using disposable Nalgene filterware. Ceils were incubated in enzyme solution for 3-4 h on a shaker (50 rpm) at 25 ± 1°C in the dark. The resulting suspension was then passed through a 65 ~m nylon screen to remove debris and undigested cells and was centrifuged at 100 g for 5 min. The resulting pellet was gently resuspended in 5 ml of 20% (w/v) sucrose on top of which I ml of O.5M
477
~
. White spruce embryogenic cell suspension culture used for protoplast isolation. Note the proembryo free cells (FC) and isodiametric cells (IC). Bar = 0.2 mm. Fi 9. 2. Freshly isolated white spruce protoplasts. Bar = 30 ~m. Fig. 3. First division of cells derived from white spruce protoplasts. Bar = 30 um. Fig. 4. Cell cluster derived from white spruce protoplast after 2 weeks in LP medium containing 5 mM glutamine. Bar =_~0 ~m. Fig. 5. C~llus derived from white spruce protoplast after 2 subcultures on LP medium containing 2 mgl 2,4-D and 1 mgl- BAP. Bar = 2 mm.
mannitol was layered. Following centrifugation (100 g for 5 min), viable protoplasts formed a band at the interface between sucrose and mannitol and were collected with a Pasteur pipette. The protoplasts were washed once with a O.5M mannitol solution and were f i n a l l y resuspended in culture medium. V i a b i l i t y of the protoplasts was tested with fluorescein diacetate (FDA) or Evans' Blue (Larkin, 1976; Constabel, 1982). The presence of cell walls was confirmed by calcofluor staining (Constabel, 1982). Culture of Protoplasts. Purified protoplasts were cultured~in 100 Rl ~rops at a density of either 10~, 2.5 X 10~ or 5 X 10 per ml on the lower halves of plastic Petri dishes (9 cm diameter), 5 drops per dish. A l t e r n a t i v e l y , in some experiments, 96 cell-well plates (Corning) were also used for protoplast culture (100 ~l per well). The media tested for culturing the protoplasts were B5 (Gamborg et a l . , 1968), MS (Murashige and Skoog, 1962), N6 -(-Chu-~, 1978), KMSP (Kao and Michayluk, 1975), LP (von Arnold and Eriksson, 1981) and LM (Litvay et a l . , 1981). All the culture media contained 0.37 M glucose as osmoticum and varying levels of BAP and 2,4-D. KM8Pmedium was f i l t e r - s t e r i l i z e d , a l l other media were s t e r i l i z e d by autoclaving. Some experiments involved the use of anti-browning compounds added to LP medium, these were polyvinylpyrrolidone (0.5%), spermidine (2.5 mM), spermine (2.5 mM) and n-propyl gallate (0.25 mM). These compounds were f i l t e r - s t e r i l i z e d . All cultures were maintained at 25°C i~ m~ist plastic boxes under diffuse l i g h t (7-10 ~E m- s- ). The percentage of
f i r s t division was estimated after 6 days in culture according to the formula: number of f i r s t divisions x 100 Percentage = i n i t i a l plating density At least 500 protoplasts per treatment were scored for the estimation of division frequency and 3-5 replicates were prepared for each treatment. All the experiments were repeated at least twice. Culture of microcalli. Microcalli are cultured using the following nurse culture method: 0.15g fresh weight of cells were harvested from the embryogenic cell suspension culture described e a r l i e r , plated onto agar-solidified (0.5%, Sigma) ~P medium containing 2 mgl- 2,4-D and 1 mgl- BAP and allowed to grow for four to seven days. After this time two layers of f i l t e r s were placed on top of each nurse callus. The lower f i l t e r was a Whatman no. 2 f i l t e r paper (7cm diameter) onto which was placed the second f i l t e r of black cellulose acetate (Millipore AA, 0.8 ~, 4.5 cm diameter). Microcalli, present in culture solution six weeks after protoplast isolation, were subsequently pipetted onto the black f i l t e r . The f i l t e r arrangement permitted observation of microcallus development while preventing mixing with cells of the nurse callus. After four weeks of nurse culture the developing c a l l i were transferred d i r e c t l y onto agar-solidified LP medium as described above. Chemicals. Cellulase R-IO and Macerozyme were obtained from Yakult Honsha Pharmaceutical Industry, Nishinomiya, Japan and Rhozyme HP-150 from Genencor,
478 New York, U.S.A. All other biochemicals were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A.
concentration of 2,4-D was found to be 1 mg1-1 and was used in a l l subsequent experiments. In LP medium 2-3% of viable cells showed second division after 12 days. By 21 days clusters containing 5-6 cells were observed. After the addition of an equal volume of fresh medium most cells ceased to grow and eventually underwent necrosis. An early problem encountered in culturing the protoplasts of Picea 91auca was the occurrence of browning. The browning of protoplast-derived cells was apparent within 8-10 days. Similar observations have been recorded for protoplasts of Pinus coulter± (Patel et a l . , 1984), Pinus lambertiana ~upta and Durzan, 19863 and several angiosperms (see Saxena and G i l l , 1986). The phenomenon of browning in ceil cultures is usually attributed to the production of excess phenolics which causes cessation of cell proliferation. Inclusion of polyvinylpyrrolidone (0.5%) (Saxena and G i l l , 1986) or 2.5 mM each of sperm±dine and spermine postponed the occurrence of browning by 8-10 days but did not completely eliminate i t . The polyamines have been reported to be beneficial for improving the quality of cereal protoplasts (Galston et a l . , 1978). The use of n-propyl gallate did not r e s i g n the stabilisation of Pice,a protoplasts. This compound has been shown to delay the senescence of corn protoplasts (Saleem and Cutler, 1987). Glutamine had no significant effect on the induction of the f i r s t cell division over a concentration range of 0-10 mM, and at 20 ff~i or more was inhibitory to the f i r s t cell division. In
RESULTS AND DISCUSSION
Isolation of Protoplasts. The protoplasts (Fig. 2) were isolated from six-day-old cell suspension cultures. An effective enzyme combination for release of protoplasts comprised 0.5% Cellulase R-IO and 0.25% each of Macerozyme, Driselase and Rhozyme HP-150. Macerozymeand Rhozyme in the enzyme mixture given above could be replaced by Pectinase and Hemicellulase which were equally effective in releasing the protoplasts. The period of incubation required for the complete removal of the cell walls, confirmed with Calcofluor staining, wasA3-4 h. Under these conditions an average of 4.5 x 10~ protoplasts per gram fresh weight of suspension were isolated. The v i a b i l i t y of the protoplasts tested with FDA or Evans' Blue varied from 70-90%. Protoplast Culture and Influence of Nutrient Medium. A new cell wall was formed between 24-48h after protoplast isolation. The v i a b i l i t y of cells decreased to 30-40% after one week. The greatest number of protoplasts underwent f i r s t division (Fig. 3) in LP and KM8Pmedium (Table 1). This s i m i l a r i t y is not surprising as these media contain similar organic constituents. All other media produced a lower division frequency. We chose LP medium for further experiments for conveniencebecause i t was also being used for cell culture maintenance.
Table I . The influence of various nutrient media and growth regulators on the percentage of f i r s t division of protoplasts from an embryogenic cell suspensio~ of Picea 9lauca, after 6 days of culture, plating density 5 x 10 per ml. Concentration of 2,4-D (mg1-1)
Medi um 0
0.5
1.0
2.5
1.0+
2.0+
BAP 0.5
BAP 1.0
LP
1.2±0.4
4.9±1.4
7.3±1.1
4.3±1.2
3.3±0.4
5.1±1.0
LM
0.5±0.4
1.3±0.6
1.3±0.6
1.1±0.2
1.1±0.5
2.1±0.4
N6
0.7±0.6
3.1±1.0
3.7±0.3
3.0±0.5
3.4±1.3
3.0±1.0
KM8P
0.5±0.4
4.6±1.3
6.8±0.7
5.6±0.2
4.1±0.4
3.5±1.2
± standard error
As seen in Table 1, the presence of 2,4-D alone was sufficient to induce cell division, and BAP decreased the frequency of division. Kinetin also showed a similar effect (data not presented). A combination of an auxin and cytokinin is necessary for most protoplasts to develop cell walls and divide (see Maheshwari et a l . , 1986). However, in certain cases, usually with cell suspension cultures, auxin alone has been reported to trigger and sustain the mitosis (Kao et a l . , 1971; Grambow et a l . , 1972; Uchimiya and Murashige, 1976). Cell wall formation and division of Picea protoplasts in the absence of 2,4-D (Table I) can be attributed to endogenous levels of growth regulators. The optimal
contrast glutamine at 5mM sustained the growth of already dividing cells derived from Picea protoplasts, leading to the formation of clusters of 6-40 cells (Fig. 4) and microcalli (more than 40 cells in size); no microcalli were recovered in the absence of glutamine. The addition of glutamine to the culture medium has been reported to be beneficial for conifer protoplasts (Kirby and Cheng, 1979; David et a l . , 1984). David (1987) suggested that glutamine is beneficial because cells may be incapable of assimilating nitrogen in sufficient quantity to sustain growth. Even though i t was possible to obtain microcalli in the presence of 5mM glutamine, their number was
479 very low. The number of cell clusters and microcalli could be increased however (from very rare to about two clusters per culture drop) ~hen th~ plating density was lowered from 5 x i0 to 10- protoplasts per ml, using the drop culture method in Petri dishes. Browning was also ~ubstantially reduced. A plating density of 2.5 x i0 protoplasts per ml produced an intermediate result. When the same experiment was done using 96 c e l l - w e l l plates, approximately 15 clusters of c e l l s formed per drop (Table 2). The plating density was found to be c r i t i c a l with other species (Sticklen et a l . , 1985). The improved frequency of cell cluster formation in 96 c e l l - w e l l plates seemed to be due to reduced aggregation of protoplasts, which favoured c e l l d i v i s i o n in protoplast cultures of Picea. Table 2. Influence of the type of culture dish on the formation of clusters of 6-40 c e l l s from white spruce proto~lasts c u l t i v a t e d in LP medium with 2,4-D ( i m g l - ) and glutamine (5 mM) a f t e r 4 weeks. Protoplasts were cultured in 100 ~I drops at a plating density of i0 per ml.
Culture dish
Petri dish Cell-well
Number Numberof Numberof of 100 Drops with clusters ~l Drops clusters
MeanNumber of clusters per drop
78
21 (26%)
133
1.7
100
100 (100%)
1520
15.2
Culture of Microcalli. At the time of inoculation to nurse culture the microcalli varied in size from 0.1 - 0.3 mm diameter. After 4 weeks of nurse culture, c a l l i increased in size to approximately 3-5 mm diameter. The recovery of transferrable c a l l i from microcalli was 60-80% e f f i c i e n t . Most of the transferred c a l l i were green and f r i a b l e (Fig. 5), and consisted of groups of small isodiametric cells. Other c a l l i were white and f r i a b l e , consisting of groups of small isodiametric cells and occasional elongated cells. Such c a l l i have been maintained for up to nine weeks (3 passages) after removal from nurse culture. In this time green c a l l i increased about 2-3 fold each passage and have been subcultured, whereas the white c a l l i exhibited negligible growth. I t remains to be seen i f either type of c a l l i have competence for embryogenesis or organogenesis.
The present study has resulted in the development of an i s o l a t i o n and culture technique which permits the reproducible recovery of c a l l i from protoplasts of embryogenic suspensions of Picea 91auca. This has been achieved by studies on the effects of enzyme solution, base medium formulation, glutamine concentration and plating density. Modifications of this basic technique have also been applied to other embryogenic suspension lines of Picea 91auca. One of these l i n e s , WS3, has subsequently been shown to be capable of regeneration into somatic embryos (Attree et a l . , in press).
ACKNOWLEDGEMENTS This work was supported by a strategic research grant (G-1868) from Natural Sciences and Engineering Research Council of Canada to L.C. Fowke and D.I. Dunstan.
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