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Improved isolation and culture methods for cucumber mesophyll protoplasts
Plant Science Letters, 9 (1977) 45--51
45
© Elsevier/North-Holland Scientific Publishers, Ltd.
IMPROVED ISOLATION AND CULTURE METHODS FOR CUCUMBER MESOPHYLL PROTOPLASTS
R.H.A. COUTTS* and K.R. WOOD
Department of Microbiology, University of Birmingham, Birmingham B15 2TT (Great Britain) (Received October 6th, 1976) (Revision received November 18th, 1976) (Accepted November 23rd, 1976)
SUMMARY
Improved methods for the isolation of large numbers of viable protoplasts from cotyledons and first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China are described. Short-term isolation methods (STM), and long-term isolation methods (LTM), using a variety of commercial enzymes are outlined. Liquid culture and subsequent solid culture of isolated first leaf protoplasts resulted in sustained cell division and eventual compact callus formation, which has been induced to form roots. No shoot or whole plant regeneration has, as yet, been demonstrated.
INTRODUCTION
Aspects of plant protoplast production and culture have been continually improving since the original work of Cocking [1] and Takebe et al. [2]. Numerous'reports now exist on protoplast isolation, callus formation and whole plant regeneration (see reviews refs. 3, 4). However, tissue culture studies on the cucurbitaceae are relatively poorly advanced, with few reports on callus initiation, growth or organogenesis [ 5]. In this study we show that protoplasts isolated using a variety of methods, and enzymes, can be first induced to form micro-calli in liquid culture [6], then form cell clusters in solid media which undergo sustained cell division to produce compact callus, with eventual root formation. *Supported by a Leverhulme Fellowship; present address: The Dept. of Botany, Imperial College, Prince Consort Road, London, SW7 2BB. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid ; LTM, long-term isolation method; MES, 2-(N-morpholino)ethane sulphonic acid--sodium salt; MLS, Linsmaier--Skoog medium; STM, short-term isolation method.
46
M A T E R I A L S AND METHODS
Cucumber plants (Cucumis sativus L. cv. Ashley and China) were grown under conditions outlined previously [6,7]. Tissue surface sterilization was carried out as outlined before [6], using a 10-min immersion in 4% v/v "Chloros", plus a few drops of sterilized "Teepol" detergent as a wetting agent. Surface sterilant was removed by washing the tissue in sterile 10% w/v mannitol, and storage in fresh mannitol (petioles submerged) whilst peeling or slicing. The STM methods of isolation utilised two methods of leaf treatment, the normal epidermal peeling method, and a modification of the carborundum abrasion technique of Beier and Bruening [8], where eventually brushing the tissue alone with a stiff camel-hair brush, and slicing the material using a scalpel multi-blade arrangment, could be used to release protoplas£s. The LTM isolations followed the normal epidermal peeling method. All tissues were pre-plasmolysed in petri dishes [9] ; 1 h in the STM, and 2--3 h in the LTM at 25°C, in subdued light. Preplasmolytica were removed and replaced by a sterilized solution of enzymes, using one of the enzyme regimes outlined in Table I. Driselase and Sigma pectinase were purified by desalting on Sephadex 25 [3]. The peeled or stripped tissue pieces were then incubated in the dark for 18 h at 25°C in Parafilm-sealed petri dishes in the LTM, or for 3 h on a gyratory shaker (40 rpm) at 25°C, in the STM. After incubation protoplasts were released, and washed by centrifugation as before [6], using 10% w/v mannitol, plus 0.1 mM CaC12 • 2H2 O. The protoplasts were then cultured in 10 ml lots of liquid medium, incorporating antibiotics [7] and incubated as previously [6] at concentrations of 1 to 5. l 0 s/ml in 100 ml flasks. The media used were; half-strength Harada's (H) medium [10], containing the salts KNO3, MgSO4.7H2 O, CaCl2 • 2H2 O, KI, KH2 PO4, CuSO4 • 5H2 O, at 525.5,218.5, 159, 0.08, 48.6 and 0.0075 mg/l concentrations respectively, and full strength H medium both with 10% w/v mannitol. After 14--21 days in liquid culture developed cell clusters were embedded in 0.6% w/v agar solidified media using standard techniques [2,3]. Plating densities were from 5.104 to 1. l 0 s protoplasts/ml, in full strength H medium, with mannitol at 10% w/v concentration. After 2 weeks further incubation, agar strips were aseptically removed and overlaid onto fresh H medium with 6% w/v mannitol. Small green calli which had emerged, were sub-cultured onto fresh H medium with 3% w/v mannitol after 2--3 weeks culture. Callus subsequently developed was then subcultured onto either full-strength H medium minus mannitol, or modified MLS [11] containing nicotinic acid at 0.5 mg/1, pyridoxine HCI at 0.5 mg/1, glycine at 2.0 mg/1, L-glutamine at 140 mg/l, L-cysteine HC1 at 10 mg/1, sucrose at 40 g/l, 2,4-D at 1 mg]l, kinetin at 1 mg/ml and sterilized coconut milk extract at 10% v/v. Bacterial contamination of protoplasts often occurred, however, eradication was achieved by inclusion of chloramphenicol (P-L Biochemicals, Inc. Milwaukee, Wis. 53205, U.S.A.), in the mixed enzymes at 100/~g/ml. Agar-solidified long-term culture was only attempted with first leaf protoplasts.
47
Enzyme mixtures were adjusted to pH 5.8 with 2 N HCI, and centrifuged at 500 g for 15 min before Millipore sterilization. MES was omitted from the STM isolations. R I 0 enzymes were supplied by the Kinki Yakult Manuf. Co., Tokyo, Japan. Driselase, by the Kyowa Hakko Co., Ltd., Tokyo, Japan. Pectinase was supplied by the Sigma Chemical Corp., St. Louis, U.S.A. RESULTS
Protoplast isolations STM. Protoplasts were successfully isolated from both first leaves and cotyledons using either the epidermal peeling or carborundum dusting/slicing methods. However, the STM m e t h o d possibly has more application for cotyledon tissue, since first leaf protoplasts isolated by this method were
TABLE I CONDITIONS F OR THE ISOLATION OF CUCUMIS S A T I V U S L. FI RS T LEAF AND COTYLEDON PROTOPLASTS Leaf treatment
STM
Carborundum dusted and sliced Epidermis peeled
Enzyme mixture composition (percentage w/v) for isolation from Cotyledon
First leaf
1.5% Driselase (D) 0.5% Sigma pectinase (P)
2.0% 0.5% 2.0% 0.5%
LTM a
D P D P
0.7% D 0.7% P
0.2% Purified driselase (PD) or
Epidermis peeled
0.2% Purified sigma pectinase (PP)
0.2% PD 0.2% PP
or 0.7% Macerozyme R10 0.7% Cellulase R10 aAll LTM enzymes were dissolved in a simple salts solution containing : KH 2 PO 4 KNO~ CaCI 2 , 2H 2 O KI CuSO 4 - 5H~ O MgSO4.7H 2 0
rag/1 27.2 101 148 0.16 0.0025 247
Mannitol at 10% w/v concentration. Potassium dextran sulphate 0.5% w/v (molecular wt. source Dextran 560, S content 17.3%, Meito Sangyo Co. Ltd., Tokyo, Japan) and MES 3 raM. STM enzymes were dissolved in 10% w/v mannitol alone.
48 smaller and sub-protoplast in appearance when compared to LTM isolated material (Fig. 1). Cotyledon protoplasts were similar morphologically to LTM first leaf protoplasts, though slightly larger (40--60 ~ m compared to 30--50/~m), possibly due to a plasmolysis effect. Yields of protoplasts were on average 6. l 0 s/cotyledon. LTM. Several modifications to the original overnight isolation procedure have been introduced; the use of mannitol to remove excess surface sterilant maintains the tissue in a plasmolysed state, when large numbers of leaves or cotyledons have to be peeled. Several combinations of mixed enzymes can be used to isolate protoplasts from first leaf tissue (Table I). Protoplasts from either preparation were morphologically indistinct (Fig. 1). Yields varied from 1 to 4.104 protoplasts/leaf. Leaf material of the correct age and texture (14--21-day-old plants, fully expanded leaves) gave Viable protoplasts, younger leaves gave rise to large numbers of fusion bodies [12], whilst older material was often refractive to e n z y m e digestion except by the purified enzymes. The inclusion of MES (Table I, ref. 13), together with the simple salts medium [6,9] assisted in protoplast stability as seen previously [13, 14].
Protoplast culture STM cotyledon protoplasts. Cotyledon protoplasts have only so far been examined in the simple salts m e d i u m of Otsuki et al. [15], and in this medium, protoplasts showed characteristic expansion, cystrophy of chloroplasts, and cytoplasmic streaming [4,6]. However, since this simple medium contains no metabolisable carbon source or growth factors, no divisions would have been expected. First leafprotoplasts. Protoplasts isolated by either the LTM or STM using any of the e n z y m e mixtures outlined in Table I, all showed characteristic cultural changes first in liquid and then agar-solidified culture. Morphologically, freshly prepared protoplasts had a distinctive appearance as shown before (Fig. 1, ref. 6). Both half or full-strength H medium gave cell divisions within 3--4 days (Fig. 2), while cell clusters appeared in 5--7 days in full strength H or 4--5 days in half-strength H m e d i u m (Fig. 3). The formation of micro-calli proceeded in liquid culture as before {Fig. 3, ref. 6). After 14--21 days in liquid culture visible masses of dividing cells were embedded in full strength agar-solidified H medium, and after 8 days of solid culture as m a n y as 60% of the cell clusters entered further rounds of division (Fig. 4). Subsequent passage of clusters in agar strips (Fig. 5), onto fresh H medium every 2 weeks, reducing the osmoticum level, resulted in the eventual formation of a green/white compact callus (Fig. 6). Transfer of this callus onto MLS gave root formation within 6 weeks (Fig. 7), and callus could also be sub-cultured onto either fresh MLS or H m e d i u m minus mannitol, during this time.
49
Figs. 1--7. The bar line represents 25 um in Figs. 1--3. Fig. 1. Cucumber first leaf mesophyll protoplasts freshly prepared after LTM isolation. Fig. 2. Fluorescence micrograph of regenerated cell undergoing the first divisions after 4 days in liquid H medium (full strength), note fluorescence of division wall and newly formed cell-wall when stained with Tinopal B.O.P.T. [6 ]. Fig. 3. Cell clusters obtained within 6 days of culture, stained with fluorescent brightener. Fig. 4. Cell clusters embedded in full strength agar solidified H medium in a petri plate, 14 days after original isolation. Fig. 5. Agar strips plated on H medium with mannitol at 6% w/v concentration, 14 days after original solid culture. Fig. 6. Callus formed on H medium with mannitol at 3% w/v concentration, 4 weeks after Fig. 5. Fig. 7. Root formation on cucumber callus grown on MLS, 6 weeks after transfer from Fig. 6 situation. Photographs are of cultured protoplasts from C. sativus L.cv. China, cultured protoplasts of the Ashley variety appeared identical, with a similar time course of events.
50 DISCUSSION
Either the STM or LTM could be used to successfully isolate protoplasts from both cotyledons or first leaf tissue of two cucumber varieties, and first leaf protoplasts isolated by either method behave similarly in culture. The use of mannitol dUring tissue sterilization, and MES as a buffer in the LTM enhanced protoplast stability [13]. Several workers have used different combinations of mixed enzymes for protoplast isolation [3,4], and similar mixtures can be used for cucumber mesophyll tissue. Antibiotics used either in the enzyme isolation mixture or the liquid culture medium, or both together had no effect on cell division or callus proliferation and reduced chance contamination. Callus of first leaf protoplasts obtained within 6--10 weeks of culture in either cucumber variety could be induced to form roots though shoot formation has not yet been demonstrated. Other workers have experienced difficulty in organogenesis of protoplast derived callus [ 16--20], and the possibility that the addition of an essential undefined plant extract to the growth media may be required cannot be excluded, since the only report of whole plant regeneration from cucumber callus used such an extract [5]. Such extracts have also been used recently in organogenesis experiments with bean callus [21]. However, subtle alteration of hormone levels may offer more reproducible possibilities [16], and with this view, work is currently in progress to obtain complete organogenesis in both healthy cucumber protoplast-derived callus, and cucumber mosaic virus-infected protoplasts [22], to produce virus-free plants. ACKNOWLEDGEMENTS
The authors are grateful to the Royal Society for supplying the environmental cabinet, and to the Leverhulme Trust for financial assistance. REFERENCES 1 E.C. Cocking, Nature, 187 (1960) 927. 2 I. Takebe, G. Labib and G. Melchers, Naturwissenschaften, 58 (1971) 318. 3 O.L. Gamborg, F. Constable, L. Fowke, K.N. Kao, K. Ohyama, K.K. Kartha and L. Pelcher, Can. J. Genet. Cytol., 16 (1974) 737. 4 E.C. Cocking, Ann. Rev. Plant Physiol., 23 (1972) 29. 5 W. Maciejewska-Potapezykova, A. Rennert and E. Milewska, Acta Soc. Bot. Polon., 61 (1972) 329. 6 R.H.A. Coutts and K.R. Wood, Plant Sci. Lett., 4 (1975) 189. 7 R.H.A. Coutts, Ann Barnett and K.R. Wood, Nucl. Acid Res., 2 (1975) 1111. 8 H. Beier and G. Bruening, Virology, 64 (1975) 272. 9 B.W.W. Grout and R.H.A. Coutts, Plant Sci. Lett., 3 (1974) 397. 10 H. Harada, Z. Pflanzenphysiol., 69, 1 (1973) 77. 11 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 12 R.H.A. Coutts, Linn. Soc. (in press). 13 K.N. Kao and M.R. Michayluk, Planta, 126 (1975) 105. 14 L.E. Pelcher, O.L. Garnborg and K.N. Kao, Plant Sci. Lett., 3 (1974) 107.
51 15 16 17 18 19 20 21 22
Y. Otsuki, T. Shimomura and I. Takebe, Virology, 50 (1972) 45. C.R. Landgren, Am. J.Bot., 63 (1976) 473. O.L. Gamborg, J. Shyluk and K.K. Kartha, Plant Sci. Lett., 4 (1975) 285. S. Poirier-Hamon, P.S. Rao and H. Harada, J. Exp. Bot., 25 (1974) 752. Aliza Vardi, P. Spiegel-Roy and E. Galun, Plant Sci. Lett., 4 (1975) 231. M.D. Upadhya, Potato Res., 18 (1975) 438. O.J. Crocomo, W.R. Sharp and J.E. Peters, Z. Pflanzenphysiol., 785 (1976) 456. R.H.A. Courts and K.R. Wood, Arch. Virol., (in press).
45
© Elsevier/North-Holland Scientific Publishers, Ltd.
IMPROVED ISOLATION AND CULTURE METHODS FOR CUCUMBER MESOPHYLL PROTOPLASTS
R.H.A. COUTTS* and K.R. WOOD
Department of Microbiology, University of Birmingham, Birmingham B15 2TT (Great Britain) (Received October 6th, 1976) (Revision received November 18th, 1976) (Accepted November 23rd, 1976)
SUMMARY
Improved methods for the isolation of large numbers of viable protoplasts from cotyledons and first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China are described. Short-term isolation methods (STM), and long-term isolation methods (LTM), using a variety of commercial enzymes are outlined. Liquid culture and subsequent solid culture of isolated first leaf protoplasts resulted in sustained cell division and eventual compact callus formation, which has been induced to form roots. No shoot or whole plant regeneration has, as yet, been demonstrated.
INTRODUCTION
Aspects of plant protoplast production and culture have been continually improving since the original work of Cocking [1] and Takebe et al. [2]. Numerous'reports now exist on protoplast isolation, callus formation and whole plant regeneration (see reviews refs. 3, 4). However, tissue culture studies on the cucurbitaceae are relatively poorly advanced, with few reports on callus initiation, growth or organogenesis [ 5]. In this study we show that protoplasts isolated using a variety of methods, and enzymes, can be first induced to form micro-calli in liquid culture [6], then form cell clusters in solid media which undergo sustained cell division to produce compact callus, with eventual root formation. *Supported by a Leverhulme Fellowship; present address: The Dept. of Botany, Imperial College, Prince Consort Road, London, SW7 2BB. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid ; LTM, long-term isolation method; MES, 2-(N-morpholino)ethane sulphonic acid--sodium salt; MLS, Linsmaier--Skoog medium; STM, short-term isolation method.
46
M A T E R I A L S AND METHODS
Cucumber plants (Cucumis sativus L. cv. Ashley and China) were grown under conditions outlined previously [6,7]. Tissue surface sterilization was carried out as outlined before [6], using a 10-min immersion in 4% v/v "Chloros", plus a few drops of sterilized "Teepol" detergent as a wetting agent. Surface sterilant was removed by washing the tissue in sterile 10% w/v mannitol, and storage in fresh mannitol (petioles submerged) whilst peeling or slicing. The STM methods of isolation utilised two methods of leaf treatment, the normal epidermal peeling method, and a modification of the carborundum abrasion technique of Beier and Bruening [8], where eventually brushing the tissue alone with a stiff camel-hair brush, and slicing the material using a scalpel multi-blade arrangment, could be used to release protoplas£s. The LTM isolations followed the normal epidermal peeling method. All tissues were pre-plasmolysed in petri dishes [9] ; 1 h in the STM, and 2--3 h in the LTM at 25°C, in subdued light. Preplasmolytica were removed and replaced by a sterilized solution of enzymes, using one of the enzyme regimes outlined in Table I. Driselase and Sigma pectinase were purified by desalting on Sephadex 25 [3]. The peeled or stripped tissue pieces were then incubated in the dark for 18 h at 25°C in Parafilm-sealed petri dishes in the LTM, or for 3 h on a gyratory shaker (40 rpm) at 25°C, in the STM. After incubation protoplasts were released, and washed by centrifugation as before [6], using 10% w/v mannitol, plus 0.1 mM CaC12 • 2H2 O. The protoplasts were then cultured in 10 ml lots of liquid medium, incorporating antibiotics [7] and incubated as previously [6] at concentrations of 1 to 5. l 0 s/ml in 100 ml flasks. The media used were; half-strength Harada's (H) medium [10], containing the salts KNO3, MgSO4.7H2 O, CaCl2 • 2H2 O, KI, KH2 PO4, CuSO4 • 5H2 O, at 525.5,218.5, 159, 0.08, 48.6 and 0.0075 mg/l concentrations respectively, and full strength H medium both with 10% w/v mannitol. After 14--21 days in liquid culture developed cell clusters were embedded in 0.6% w/v agar solidified media using standard techniques [2,3]. Plating densities were from 5.104 to 1. l 0 s protoplasts/ml, in full strength H medium, with mannitol at 10% w/v concentration. After 2 weeks further incubation, agar strips were aseptically removed and overlaid onto fresh H medium with 6% w/v mannitol. Small green calli which had emerged, were sub-cultured onto fresh H medium with 3% w/v mannitol after 2--3 weeks culture. Callus subsequently developed was then subcultured onto either full-strength H medium minus mannitol, or modified MLS [11] containing nicotinic acid at 0.5 mg/1, pyridoxine HCI at 0.5 mg/1, glycine at 2.0 mg/1, L-glutamine at 140 mg/l, L-cysteine HC1 at 10 mg/1, sucrose at 40 g/l, 2,4-D at 1 mg]l, kinetin at 1 mg/ml and sterilized coconut milk extract at 10% v/v. Bacterial contamination of protoplasts often occurred, however, eradication was achieved by inclusion of chloramphenicol (P-L Biochemicals, Inc. Milwaukee, Wis. 53205, U.S.A.), in the mixed enzymes at 100/~g/ml. Agar-solidified long-term culture was only attempted with first leaf protoplasts.
47
Enzyme mixtures were adjusted to pH 5.8 with 2 N HCI, and centrifuged at 500 g for 15 min before Millipore sterilization. MES was omitted from the STM isolations. R I 0 enzymes were supplied by the Kinki Yakult Manuf. Co., Tokyo, Japan. Driselase, by the Kyowa Hakko Co., Ltd., Tokyo, Japan. Pectinase was supplied by the Sigma Chemical Corp., St. Louis, U.S.A. RESULTS
Protoplast isolations STM. Protoplasts were successfully isolated from both first leaves and cotyledons using either the epidermal peeling or carborundum dusting/slicing methods. However, the STM m e t h o d possibly has more application for cotyledon tissue, since first leaf protoplasts isolated by this method were
TABLE I CONDITIONS F OR THE ISOLATION OF CUCUMIS S A T I V U S L. FI RS T LEAF AND COTYLEDON PROTOPLASTS Leaf treatment
STM
Carborundum dusted and sliced Epidermis peeled
Enzyme mixture composition (percentage w/v) for isolation from Cotyledon
First leaf
1.5% Driselase (D) 0.5% Sigma pectinase (P)
2.0% 0.5% 2.0% 0.5%
LTM a
D P D P
0.7% D 0.7% P
0.2% Purified driselase (PD) or
Epidermis peeled
0.2% Purified sigma pectinase (PP)
0.2% PD 0.2% PP
or 0.7% Macerozyme R10 0.7% Cellulase R10 aAll LTM enzymes were dissolved in a simple salts solution containing : KH 2 PO 4 KNO~ CaCI 2 , 2H 2 O KI CuSO 4 - 5H~ O MgSO4.7H 2 0
rag/1 27.2 101 148 0.16 0.0025 247
Mannitol at 10% w/v concentration. Potassium dextran sulphate 0.5% w/v (molecular wt. source Dextran 560, S content 17.3%, Meito Sangyo Co. Ltd., Tokyo, Japan) and MES 3 raM. STM enzymes were dissolved in 10% w/v mannitol alone.
48 smaller and sub-protoplast in appearance when compared to LTM isolated material (Fig. 1). Cotyledon protoplasts were similar morphologically to LTM first leaf protoplasts, though slightly larger (40--60 ~ m compared to 30--50/~m), possibly due to a plasmolysis effect. Yields of protoplasts were on average 6. l 0 s/cotyledon. LTM. Several modifications to the original overnight isolation procedure have been introduced; the use of mannitol to remove excess surface sterilant maintains the tissue in a plasmolysed state, when large numbers of leaves or cotyledons have to be peeled. Several combinations of mixed enzymes can be used to isolate protoplasts from first leaf tissue (Table I). Protoplasts from either preparation were morphologically indistinct (Fig. 1). Yields varied from 1 to 4.104 protoplasts/leaf. Leaf material of the correct age and texture (14--21-day-old plants, fully expanded leaves) gave Viable protoplasts, younger leaves gave rise to large numbers of fusion bodies [12], whilst older material was often refractive to e n z y m e digestion except by the purified enzymes. The inclusion of MES (Table I, ref. 13), together with the simple salts medium [6,9] assisted in protoplast stability as seen previously [13, 14].
Protoplast culture STM cotyledon protoplasts. Cotyledon protoplasts have only so far been examined in the simple salts m e d i u m of Otsuki et al. [15], and in this medium, protoplasts showed characteristic expansion, cystrophy of chloroplasts, and cytoplasmic streaming [4,6]. However, since this simple medium contains no metabolisable carbon source or growth factors, no divisions would have been expected. First leafprotoplasts. Protoplasts isolated by either the LTM or STM using any of the e n z y m e mixtures outlined in Table I, all showed characteristic cultural changes first in liquid and then agar-solidified culture. Morphologically, freshly prepared protoplasts had a distinctive appearance as shown before (Fig. 1, ref. 6). Both half or full-strength H medium gave cell divisions within 3--4 days (Fig. 2), while cell clusters appeared in 5--7 days in full strength H or 4--5 days in half-strength H m e d i u m (Fig. 3). The formation of micro-calli proceeded in liquid culture as before {Fig. 3, ref. 6). After 14--21 days in liquid culture visible masses of dividing cells were embedded in full strength agar-solidified H medium, and after 8 days of solid culture as m a n y as 60% of the cell clusters entered further rounds of division (Fig. 4). Subsequent passage of clusters in agar strips (Fig. 5), onto fresh H medium every 2 weeks, reducing the osmoticum level, resulted in the eventual formation of a green/white compact callus (Fig. 6). Transfer of this callus onto MLS gave root formation within 6 weeks (Fig. 7), and callus could also be sub-cultured onto either fresh MLS or H m e d i u m minus mannitol, during this time.
49
Figs. 1--7. The bar line represents 25 um in Figs. 1--3. Fig. 1. Cucumber first leaf mesophyll protoplasts freshly prepared after LTM isolation. Fig. 2. Fluorescence micrograph of regenerated cell undergoing the first divisions after 4 days in liquid H medium (full strength), note fluorescence of division wall and newly formed cell-wall when stained with Tinopal B.O.P.T. [6 ]. Fig. 3. Cell clusters obtained within 6 days of culture, stained with fluorescent brightener. Fig. 4. Cell clusters embedded in full strength agar solidified H medium in a petri plate, 14 days after original isolation. Fig. 5. Agar strips plated on H medium with mannitol at 6% w/v concentration, 14 days after original solid culture. Fig. 6. Callus formed on H medium with mannitol at 3% w/v concentration, 4 weeks after Fig. 5. Fig. 7. Root formation on cucumber callus grown on MLS, 6 weeks after transfer from Fig. 6 situation. Photographs are of cultured protoplasts from C. sativus L.cv. China, cultured protoplasts of the Ashley variety appeared identical, with a similar time course of events.
50 DISCUSSION
Either the STM or LTM could be used to successfully isolate protoplasts from both cotyledons or first leaf tissue of two cucumber varieties, and first leaf protoplasts isolated by either method behave similarly in culture. The use of mannitol dUring tissue sterilization, and MES as a buffer in the LTM enhanced protoplast stability [13]. Several workers have used different combinations of mixed enzymes for protoplast isolation [3,4], and similar mixtures can be used for cucumber mesophyll tissue. Antibiotics used either in the enzyme isolation mixture or the liquid culture medium, or both together had no effect on cell division or callus proliferation and reduced chance contamination. Callus of first leaf protoplasts obtained within 6--10 weeks of culture in either cucumber variety could be induced to form roots though shoot formation has not yet been demonstrated. Other workers have experienced difficulty in organogenesis of protoplast derived callus [ 16--20], and the possibility that the addition of an essential undefined plant extract to the growth media may be required cannot be excluded, since the only report of whole plant regeneration from cucumber callus used such an extract [5]. Such extracts have also been used recently in organogenesis experiments with bean callus [21]. However, subtle alteration of hormone levels may offer more reproducible possibilities [16], and with this view, work is currently in progress to obtain complete organogenesis in both healthy cucumber protoplast-derived callus, and cucumber mosaic virus-infected protoplasts [22], to produce virus-free plants. ACKNOWLEDGEMENTS
The authors are grateful to the Royal Society for supplying the environmental cabinet, and to the Leverhulme Trust for financial assistance. REFERENCES 1 E.C. Cocking, Nature, 187 (1960) 927. 2 I. Takebe, G. Labib and G. Melchers, Naturwissenschaften, 58 (1971) 318. 3 O.L. Gamborg, F. Constable, L. Fowke, K.N. Kao, K. Ohyama, K.K. Kartha and L. Pelcher, Can. J. Genet. Cytol., 16 (1974) 737. 4 E.C. Cocking, Ann. Rev. Plant Physiol., 23 (1972) 29. 5 W. Maciejewska-Potapezykova, A. Rennert and E. Milewska, Acta Soc. Bot. Polon., 61 (1972) 329. 6 R.H.A. Coutts and K.R. Wood, Plant Sci. Lett., 4 (1975) 189. 7 R.H.A. Coutts, Ann Barnett and K.R. Wood, Nucl. Acid Res., 2 (1975) 1111. 8 H. Beier and G. Bruening, Virology, 64 (1975) 272. 9 B.W.W. Grout and R.H.A. Coutts, Plant Sci. Lett., 3 (1974) 397. 10 H. Harada, Z. Pflanzenphysiol., 69, 1 (1973) 77. 11 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 12 R.H.A. Coutts, Linn. Soc. (in press). 13 K.N. Kao and M.R. Michayluk, Planta, 126 (1975) 105. 14 L.E. Pelcher, O.L. Garnborg and K.N. Kao, Plant Sci. Lett., 3 (1974) 107.
51 15 16 17 18 19 20 21 22
Y. Otsuki, T. Shimomura and I. Takebe, Virology, 50 (1972) 45. C.R. Landgren, Am. J.Bot., 63 (1976) 473. O.L. Gamborg, J. Shyluk and K.K. Kartha, Plant Sci. Lett., 4 (1975) 285. S. Poirier-Hamon, P.S. Rao and H. Harada, J. Exp. Bot., 25 (1974) 752. Aliza Vardi, P. Spiegel-Roy and E. Galun, Plant Sci. Lett., 4 (1975) 231. M.D. Upadhya, Potato Res., 18 (1975) 438. O.J. Crocomo, W.R. Sharp and J.E. Peters, Z. Pflanzenphysiol., 785 (1976) 456. R.H.A. Courts and K.R. Wood, Arch. Virol., (in press).