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A method for obtaining callus cultures from mesophyll protoplasts of melon (cucumis melo L.)
Plant Science Letters, 34 (1984) 195--201
195
Elsevier Scientific Publishers Ireland Ltd.
A METHOD FOR OBTAINING CALLUS C U L T U R E S FROM MESOPHYLL P R O T O P L A S T S O F MELON (CUCUMIS MELO L.)
V. MORENO a, L. ZUBELDIA a and L.A. ROIG b
aDepartment of Genetics and bDepartment of Microbiology, E.T.S.I.A. Universidad Politdcnica de Valencia, Camino de Vera 14, Valencia (Spain) (Received June 6th, 1983) (Revision received October 18th, 1983) (Accepted October 18th, 1983)
SUMMARY
Calli were obtained from protoplasts o f plants of three cultivars of Cu. cumis melo L. grown under axenic conditions. The composition of the culture medium in which the axenic plants were grown greatly influenced the rate of recovery of protoplasts capable of undergoing sustained divisions. Under the best conditions, cell wall regeneration was accomplished by up to 90% of the protoplasts and the first divisions were observed within 3--4 days of culture. After 20 days, the percentage of dividing cells was 26% in the cultivar 'Valenciano Pinyonet', 21% in the cv. 'Pinyonet Piel de Sapo' and 52% in the cv. 'Cantaloup Charentais'. T w o subsequent cultures on media with lower molarity led to calli with elevated growth rates. When plants of the first t w o cultivars were cultivated in a suboptimal medium, preculture of the leaves was essential in order to obtain protoplasts with mitotic activity.
Key words: Melon -- Cucumis melo L. -- Protoplast culture -- Axenic plants
INTRODUCTION
Although the techniques o f isolation, culture and regeneration of plant protoplasts are being applied to a great n u m b e r of species, the family Cucurbitaceae has received little attention with the exception of cucumber [1--3]. We previously described [4] the isolation of protoplasts from melon cell suspension cultures and the induction of sustained cell divisions in mesophyU protoplasts from plants grown under greenhouse conditions. The Abbreviation: MS, Murashige and Skoog mineral solution. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
196
problems associated with greenhouse-grown plant material, such as the low mitotic activity of the protoplast-derived cells, frequent culture contaminations, poor reproducibility and impossibility of maintaining the uniformity of the plant material throughout the year, were partially overcome by use of the technique of preculturing the leaves as proposed by Gatenby and Cocking [5]. We present here an alternative method based on the axenic culture of shoot apices, with or without preculture of the leaves in a medium which induces divisions. Our ultimate goal was to obtain protoplasts with a high rate of successive divisions and rapid development into calli. MATERIALS AND METHODS Plan t material Leaves of Cucumis melo L. cvs. 'Cantaloup Charentais', 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' from axenic shoot cultures were used for protoplast isolation either directly or after preculturing in C medium [4], consisting of Murashige and Skoog [7] mineral solution (MS), 4% sucrose, 2.5 rag/1 naphthalene acetic acid, 1 mg/l 6-benzylaminopurine, 100 rag/1 myo-inositol, 1 mg/l thiamine--HC1 and 0.8% agar (Technical No. 3, Oxoid), for 2 days. Axenic plants were initiated as follows: surface sterilized seeds were germinated in MG medium consisting of MS salts, 1% sucrose and 1% agar. After 11--13 days, apical buds (3--5 mm length) were aseptically planted into 250-ml bottles containing 50 ml of either MG medium supplemented with 4% sucrose and 0.8% agar (MS-4 medium) or MS-4 medium supplemented with 0.1% yeast extract (MEL medium). Plants were cultured for 30--35 days in both media. The incubation was carried out in a culture room at 27 + 2°C during the light period (16 h under 2000 lux provided by cool white fluorescent tubes, Gro-lux, Sylvania) and 24 + 2°C during the dark. Protoplast isolation Fresh as well as precultured leaves from axenic plants were cut into 1--2 mm wide strips, and these strips were then placed in 50-ml Erienmeyer flasks containing 4 ml of a filter-sterilized enzyme solution consisting of 2% cenulase Onozuka R-10 (Yakult Pharmaceutical Ind. Ltd.), 0.6 M-mannitol in MS mineral solution, at pH 5.7. One milliliter of enzyme solution was used for each 0.1 g of leaf tissue. The incubation was performed in the dark at 28°C for 5 h in a reciprocal shaker at 100 strokes/min (amplitude 20 ram). After incubation, the protoplasts were poured through stairAess steel filters of 1 0 0 ~ m and 75~am mesh, centrifuged three times at 75 × g and resuspended in 0.6 M-mannitol at pH 5.7. Four successive cell counts were carried out in a Howard mould-counting chamber to estimate the yield.
197
Protoplast culture Protoplasts were cultured in 35 m m diameter Falcon Petri dishes containing 2 ml of the liquid medium, at a final concentration of 1--2 X 10 s protoplasts/ml. The Zapata et at. medium (ZEPC) medium was routinely used for all experiments. It contained B5 mineral solution [9], 1% sucrose, 0 5 % glucose, 0.5 mg/1 naphthalene acetic acid, I mg/1 2,4-dichlorophenoxyacetic acid, 0.5 rag/1 6-benzylaminopurine, 100 mg/l myo°inositol, 10 mg/l thiamine--HC1, 1 rag/1 nicotinic acid, 1 mg/l pyridoxine--HC1 and 0.6 Mmannitol. The pH was adjusted to 5.7 before autoclaving. After 20 days of incubation at 27°C in the dark, cells and small cellclusters originating from protoplasts were subcultured in the same liquid medium or plated on ZEPC solidified with 0.8% agar at a reduced osmotic pressure (0.3 M-mannitol). The final concentration was kept at 2.5--5.0 × 104 cells/ml. After 20 days more of culture, the small calli were grown on the surface of either the same medium or the C medium without osmotic stabilizer, under the light regime used for the axenic plants. RESULTS
Protoplast isolation Although initially different kinds and concentrations of enzymes were assayed, a simple maceration mixture consisting of MS salts, 2% cellulase and 0.6 M-mannitol was found to be satisfactory for the isolation of mesophyll protoplasts from axenic plants of melon. Table I shows the yields obtained for each of the three cultivars from mother-plants grown on the t w o media. High yields were achieved in all cases, ranging from approx. 7 × 106 to 12 × 104 protoplasts/g of leaf tissue. Significantly poorer results were obtained when precultured leaves were used as source o f protoplasts. Yields did not" exceed 106 protoplasts/g of tissue in any of the cultivars. Addition of other enzymes (pectinase and/or hemicellulase) did n o t increase the protoplast release. Neither an initial preplasmolysis of the leaves (either precultured or not) TABLE
I
PROTOPLAST
Y I E L D (× 10 6) P E R G R A M
OF LEAF TISSUE
Values expressed as the m e a n value + S.D. of four cell counts per each replicate, at least 10 replicates for each experiment.
Plants grown on MS-4 m e d i u m
cv. 'Valenciano Pinyonet' cv. 'Pinyonet Piel de Sapo' cv. 'Cantaloup Charentais'
7.2±1.3 7.8±3.6 11.8±3.9
MEL m e d i u m
10.8±5.3 10.2±5.1 12.9±3.9
198
F~g."1. Isolation and culture of melon protoplasts. (A) Axenic plant grown on M E L m e d i u m used as source of protoplasts. (B) Freshly isolated mesophyll protoplasts after 5 h in the enzyme mixture. (C--G) Development of protoplasts cultured in Z E P C m e d i u m with 0.6 M-mannitol: (C) after 3--4 days, (D) after 5--7 days, (E,F) after 10 days and (G) cell-clusterat 20th day of culture. (H) Microcalli obtained after plating in solid Z E P C m e d i u m 0.3 M-mannitol for 20 days. (I) Calli developed on mannitol-free C mediu m after 20 days of culture. in 0.6 M-mannitol, nor the removal of mineral salts from the enzyme mixture affected the release or viability of the protoplasts. MesophyU protoplasts from axenic plants (Fig. 1B) were morphologically similar to those from greenhouse-grown plants [4], but slightly smaller
199
(21 p m in diameter, mean o f 100 values) and with the chloroplasts more irregularly distributed. Protoplasts derived from precultured leaves were similar to those from leaves that were n o t precultured, even though some of them had fewer vacuoles and mature chloroplasts.
Protoplast culture Several media were assayed in preliminary experiments. As a result, ZEPC medium was selected as the most suitable for the culture of melon protoplasts from plants grown under axenic conditions, although it had been developed originally for t o m a t o protoplasts [8]. In this medium, the protoplasts were able to regenerate a new cell wall and actively divide. The behaviour of the protoplasts was influenced by the plant growth media employed (Table II). The use of plant growth medium without yeast extract led to protoplasts which were unable to divide in two of the three cultivars. Medium with this organic addendum (at 0.1% w/v) permitted the growth of protoplasts from all three cultivars. Plants of cv. 'Cantaloup Charentais' grown on medium with yeast extract gave protoplasts with 80--90% of cell wall regeneration within 2--3 days of culture. First divisions could be seen after 3--4 days; 2% of the cells were dividing on the 5th day of culture, and a b o u t 50% of them after 20 days (Fig. 1 C--G}. Cell wall regeneration and first divisions were similar in the other t w o cultivars b u t the proportion of cells with mitotic activity were lower: 15% in cv. 'Valenciano Pinyonet' and 20% in cv. 'Pinyonet Piel de Sapo' after 20 days of culture. Preculture of the leaves overcame the inability of protoplasts from the cultivars 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' to divide when T A B L E II C U L T U R E O F M E L O N P R O T O P L A S T S IN ZEPC M E D I U M Percentage o f cells undergoing divisions (D) and forming colonies (C) after 20 days of incubation. Percentage expressed as the m e a n value ± S.D. o f at least 10 replicates for each experiment. P: precultured leaves. NP: n o t precultured leaves. Plants grown on MS-4 m e d i u m NP
MEL m e d i u m P
NP
P
cv. ' V a l e n c i a n o P i n y o n e t '
D C
0 0
6.4±0.8 1.2±0.3
15.2±2.9 5.5±2.1
26.5 ± 6.2 10.3 ± 2.4
cv. ' P i n y o n e t Piel de S a p o '
D C
0 0
18.9±2.2 5.9±2.4
19.8±4.2 5.1±1.3
21.2 Jr 4.5 6.0 +- 2.1
cv. 'Cantaloup Charentais'
D C
12.6±1.6 3.8±0.9
48.2±6.6 15.8±4.1
52.1 + 7.5 20.1 4"-6.3
10.0±2.5 2.1±0.5
200 the mother-plants had been grown on yeast extract-free medium. However, preculture of the leaves did not induce a significant increase in the percentage of dividing cells when mother-plants were grown on medium supplemented with yeast extract. Subculturing the small cell-clusters for 20 days in the same medium (either liquid or solidified with 0.8% agar) at a lower osmotic pressure facilitated the growth of hundreds of microcalli (Fig. 1H). The transfer of these microcalli onto solid C medium without mannitol 20 days later, induced an elevated growth rate of calli (Fig. lI). DISCUSSION The protoplast yields of more than 5 × 106/g of leaf tissue obtained in the present study, are comparable to those reported for axenic tobacco plants [10] and were higher than those found in other species [11--13] or in greenhouse-grown melon plants [4]. Preculture of the leaves from axenic plants strongly diminished the protoplast yield, as already observed by other authors [5,14], indicating that quantitative and/or qualitative changes in the cell wall occurred as a result of the culture in vitro [5]. Although preplasmolysis of plant material and presence of metallic ions in the maceration mixture have been reported to increase the protoplast viability in cucumber [ 1,2], we found no such requirements in melon. Plant growth conditions turned out to have a striking effect on the behaviour of the protoplasts from the three cultivars tested. This fact is in agreement with the results reported by several workers who used greenhousecultured plants [15--19] or axenic cultures [10,11,13,20]. So far, the most intensively studied factor has been the influence of light fiuence during the growth of the axenic plants upon the further response of the protoplasts isolated from them. In addition, Durand's work [13] demonstrated the influence of the composition of the plant culture medium on the development of the protoplasts. Our work presents new evidence in this regard. Preculture of the leaves from axenic plants of cvs. 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' cultivated under suboptimal conditions, gave rise to protoplasts capable of sustained divisions at a moderate rate, as previously reported for melon protoplasts obtained from greenhouse-grown plants [4]. The successful application of this technique to systems of where protoplasts divide poorly had been already demonstrated in other plant species [9,14]. In conclusion, the axenic culture of plants, with or without preculture of their leaves is an improvement over our previously reported method [4] because of its simplicity, plating efficiency, absence of contamination problems, uniformity of plant material throughout the year and total reproducibility of results. Morphogenetic responses of the protoplast-derived calli of melon are being investigated at present, stimulated by the recent observation that somatic embryos were obtained under certain conditions (in preparation).
201 ACKNOWLEDGEMENTS
T h e a u t h o r s are g r a t e f u l t o Dr. J. H a n s e n f o r r e v i e w i n g a n d c o r r e c t i n g t h e English m a n u s c r i p t . T h e r e s e a r c h was p a r t i a l l y s u p p o r t e d b y t h e Ministry o f E d u c a t i o n a n d Science o f t h e S p a n i s h G o v e r n m e n t g r a n t t o Dr. V. Moreno. REFERENCES
1 2 3 4
R.H.A. Coutts and K.R. Wood, Plant Sci. Lett., 4 (1975) 189. R.H.A. Coutts and K.R. Wood, Plant Sci. Lett., 9 (1977) 45. R.H.A. Coutts, A. Barnett and K.R. Wood, Nucl. Acid Res., 2 (1975) 1111. V. Moreno, L.A. Roig and B. Garcfa, Proceedings Eucarpia Meeting "In Vitro Culture Applied to Vegetable Breeding", Versailles,France, 1980, 166. 5 A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 8 (1977) 275. 6 H. Binding, Plant Sci. Lett., 2 (1974) 185. 7 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 8 F.J. Zapata, P.K. Evans, J.B. Power and E.C. Cocking, Plant Sci. Lett., 8 (1977) 114. 9 0 . L . Gamborg, R.A. Miller and K. Ojima, Exp. Cell Res., 50 (1968) 151. 10 H. Binding, Physiol. Plant., 35 (1975) 225. 11 R. Nehls, Plant Sci. Lett., 12 (1978) 183. 12 R. Gill, A. Rashid and S.C. Maheshwari, Physiol. Plant., 47 (1979) 7. 13 J. Durand, Z. Pflanzenphysiol., 93 (1979) 283. 14 G. Donn, Z. Pflanzenphysiol., 86 (1978) 65. 15 F. Constabel, J.W. Kirkpatrick and O.L. Gamborg, Can. J. Bot., 51 (1973) 2105. 16 J.F. Shepard and R.E. Torten, Plant Physiol., 55 (1975) 689. 17 S. von Arnold and T. Eriksson, Physiol. Plant., 36 (1976) 193. 18 A.C. Cassells and M. Barlass, Physiol. Plant., 42 (1978) 236. 19 M. Tal and J.W. Watts, Z. Pflanzenphysiol., 92 (1979) 207. 20 H. LSrz, W. Wemicke and I. Potrykus, Planta Medica, 36 (1979) 21.
195
Elsevier Scientific Publishers Ireland Ltd.
A METHOD FOR OBTAINING CALLUS C U L T U R E S FROM MESOPHYLL P R O T O P L A S T S O F MELON (CUCUMIS MELO L.)
V. MORENO a, L. ZUBELDIA a and L.A. ROIG b
aDepartment of Genetics and bDepartment of Microbiology, E.T.S.I.A. Universidad Politdcnica de Valencia, Camino de Vera 14, Valencia (Spain) (Received June 6th, 1983) (Revision received October 18th, 1983) (Accepted October 18th, 1983)
SUMMARY
Calli were obtained from protoplasts o f plants of three cultivars of Cu. cumis melo L. grown under axenic conditions. The composition of the culture medium in which the axenic plants were grown greatly influenced the rate of recovery of protoplasts capable of undergoing sustained divisions. Under the best conditions, cell wall regeneration was accomplished by up to 90% of the protoplasts and the first divisions were observed within 3--4 days of culture. After 20 days, the percentage of dividing cells was 26% in the cultivar 'Valenciano Pinyonet', 21% in the cv. 'Pinyonet Piel de Sapo' and 52% in the cv. 'Cantaloup Charentais'. T w o subsequent cultures on media with lower molarity led to calli with elevated growth rates. When plants of the first t w o cultivars were cultivated in a suboptimal medium, preculture of the leaves was essential in order to obtain protoplasts with mitotic activity.
Key words: Melon -- Cucumis melo L. -- Protoplast culture -- Axenic plants
INTRODUCTION
Although the techniques o f isolation, culture and regeneration of plant protoplasts are being applied to a great n u m b e r of species, the family Cucurbitaceae has received little attention with the exception of cucumber [1--3]. We previously described [4] the isolation of protoplasts from melon cell suspension cultures and the induction of sustained cell divisions in mesophyU protoplasts from plants grown under greenhouse conditions. The Abbreviation: MS, Murashige and Skoog mineral solution. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
196
problems associated with greenhouse-grown plant material, such as the low mitotic activity of the protoplast-derived cells, frequent culture contaminations, poor reproducibility and impossibility of maintaining the uniformity of the plant material throughout the year, were partially overcome by use of the technique of preculturing the leaves as proposed by Gatenby and Cocking [5]. We present here an alternative method based on the axenic culture of shoot apices, with or without preculture of the leaves in a medium which induces divisions. Our ultimate goal was to obtain protoplasts with a high rate of successive divisions and rapid development into calli. MATERIALS AND METHODS Plan t material Leaves of Cucumis melo L. cvs. 'Cantaloup Charentais', 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' from axenic shoot cultures were used for protoplast isolation either directly or after preculturing in C medium [4], consisting of Murashige and Skoog [7] mineral solution (MS), 4% sucrose, 2.5 rag/1 naphthalene acetic acid, 1 mg/l 6-benzylaminopurine, 100 rag/1 myo-inositol, 1 mg/l thiamine--HC1 and 0.8% agar (Technical No. 3, Oxoid), for 2 days. Axenic plants were initiated as follows: surface sterilized seeds were germinated in MG medium consisting of MS salts, 1% sucrose and 1% agar. After 11--13 days, apical buds (3--5 mm length) were aseptically planted into 250-ml bottles containing 50 ml of either MG medium supplemented with 4% sucrose and 0.8% agar (MS-4 medium) or MS-4 medium supplemented with 0.1% yeast extract (MEL medium). Plants were cultured for 30--35 days in both media. The incubation was carried out in a culture room at 27 + 2°C during the light period (16 h under 2000 lux provided by cool white fluorescent tubes, Gro-lux, Sylvania) and 24 + 2°C during the dark. Protoplast isolation Fresh as well as precultured leaves from axenic plants were cut into 1--2 mm wide strips, and these strips were then placed in 50-ml Erienmeyer flasks containing 4 ml of a filter-sterilized enzyme solution consisting of 2% cenulase Onozuka R-10 (Yakult Pharmaceutical Ind. Ltd.), 0.6 M-mannitol in MS mineral solution, at pH 5.7. One milliliter of enzyme solution was used for each 0.1 g of leaf tissue. The incubation was performed in the dark at 28°C for 5 h in a reciprocal shaker at 100 strokes/min (amplitude 20 ram). After incubation, the protoplasts were poured through stairAess steel filters of 1 0 0 ~ m and 75~am mesh, centrifuged three times at 75 × g and resuspended in 0.6 M-mannitol at pH 5.7. Four successive cell counts were carried out in a Howard mould-counting chamber to estimate the yield.
197
Protoplast culture Protoplasts were cultured in 35 m m diameter Falcon Petri dishes containing 2 ml of the liquid medium, at a final concentration of 1--2 X 10 s protoplasts/ml. The Zapata et at. medium (ZEPC) medium was routinely used for all experiments. It contained B5 mineral solution [9], 1% sucrose, 0 5 % glucose, 0.5 mg/1 naphthalene acetic acid, I mg/1 2,4-dichlorophenoxyacetic acid, 0.5 rag/1 6-benzylaminopurine, 100 mg/l myo°inositol, 10 mg/l thiamine--HC1, 1 rag/1 nicotinic acid, 1 mg/l pyridoxine--HC1 and 0.6 Mmannitol. The pH was adjusted to 5.7 before autoclaving. After 20 days of incubation at 27°C in the dark, cells and small cellclusters originating from protoplasts were subcultured in the same liquid medium or plated on ZEPC solidified with 0.8% agar at a reduced osmotic pressure (0.3 M-mannitol). The final concentration was kept at 2.5--5.0 × 104 cells/ml. After 20 days more of culture, the small calli were grown on the surface of either the same medium or the C medium without osmotic stabilizer, under the light regime used for the axenic plants. RESULTS
Protoplast isolation Although initially different kinds and concentrations of enzymes were assayed, a simple maceration mixture consisting of MS salts, 2% cellulase and 0.6 M-mannitol was found to be satisfactory for the isolation of mesophyll protoplasts from axenic plants of melon. Table I shows the yields obtained for each of the three cultivars from mother-plants grown on the t w o media. High yields were achieved in all cases, ranging from approx. 7 × 106 to 12 × 104 protoplasts/g of leaf tissue. Significantly poorer results were obtained when precultured leaves were used as source o f protoplasts. Yields did not" exceed 106 protoplasts/g of tissue in any of the cultivars. Addition of other enzymes (pectinase and/or hemicellulase) did n o t increase the protoplast release. Neither an initial preplasmolysis of the leaves (either precultured or not) TABLE
I
PROTOPLAST
Y I E L D (× 10 6) P E R G R A M
OF LEAF TISSUE
Values expressed as the m e a n value + S.D. of four cell counts per each replicate, at least 10 replicates for each experiment.
Plants grown on MS-4 m e d i u m
cv. 'Valenciano Pinyonet' cv. 'Pinyonet Piel de Sapo' cv. 'Cantaloup Charentais'
7.2±1.3 7.8±3.6 11.8±3.9
MEL m e d i u m
10.8±5.3 10.2±5.1 12.9±3.9
198
F~g."1. Isolation and culture of melon protoplasts. (A) Axenic plant grown on M E L m e d i u m used as source of protoplasts. (B) Freshly isolated mesophyll protoplasts after 5 h in the enzyme mixture. (C--G) Development of protoplasts cultured in Z E P C m e d i u m with 0.6 M-mannitol: (C) after 3--4 days, (D) after 5--7 days, (E,F) after 10 days and (G) cell-clusterat 20th day of culture. (H) Microcalli obtained after plating in solid Z E P C m e d i u m 0.3 M-mannitol for 20 days. (I) Calli developed on mannitol-free C mediu m after 20 days of culture. in 0.6 M-mannitol, nor the removal of mineral salts from the enzyme mixture affected the release or viability of the protoplasts. MesophyU protoplasts from axenic plants (Fig. 1B) were morphologically similar to those from greenhouse-grown plants [4], but slightly smaller
199
(21 p m in diameter, mean o f 100 values) and with the chloroplasts more irregularly distributed. Protoplasts derived from precultured leaves were similar to those from leaves that were n o t precultured, even though some of them had fewer vacuoles and mature chloroplasts.
Protoplast culture Several media were assayed in preliminary experiments. As a result, ZEPC medium was selected as the most suitable for the culture of melon protoplasts from plants grown under axenic conditions, although it had been developed originally for t o m a t o protoplasts [8]. In this medium, the protoplasts were able to regenerate a new cell wall and actively divide. The behaviour of the protoplasts was influenced by the plant growth media employed (Table II). The use of plant growth medium without yeast extract led to protoplasts which were unable to divide in two of the three cultivars. Medium with this organic addendum (at 0.1% w/v) permitted the growth of protoplasts from all three cultivars. Plants of cv. 'Cantaloup Charentais' grown on medium with yeast extract gave protoplasts with 80--90% of cell wall regeneration within 2--3 days of culture. First divisions could be seen after 3--4 days; 2% of the cells were dividing on the 5th day of culture, and a b o u t 50% of them after 20 days (Fig. 1 C--G}. Cell wall regeneration and first divisions were similar in the other t w o cultivars b u t the proportion of cells with mitotic activity were lower: 15% in cv. 'Valenciano Pinyonet' and 20% in cv. 'Pinyonet Piel de Sapo' after 20 days of culture. Preculture of the leaves overcame the inability of protoplasts from the cultivars 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' to divide when T A B L E II C U L T U R E O F M E L O N P R O T O P L A S T S IN ZEPC M E D I U M Percentage o f cells undergoing divisions (D) and forming colonies (C) after 20 days of incubation. Percentage expressed as the m e a n value ± S.D. o f at least 10 replicates for each experiment. P: precultured leaves. NP: n o t precultured leaves. Plants grown on MS-4 m e d i u m NP
MEL m e d i u m P
NP
P
cv. ' V a l e n c i a n o P i n y o n e t '
D C
0 0
6.4±0.8 1.2±0.3
15.2±2.9 5.5±2.1
26.5 ± 6.2 10.3 ± 2.4
cv. ' P i n y o n e t Piel de S a p o '
D C
0 0
18.9±2.2 5.9±2.4
19.8±4.2 5.1±1.3
21.2 Jr 4.5 6.0 +- 2.1
cv. 'Cantaloup Charentais'
D C
12.6±1.6 3.8±0.9
48.2±6.6 15.8±4.1
52.1 + 7.5 20.1 4"-6.3
10.0±2.5 2.1±0.5
200 the mother-plants had been grown on yeast extract-free medium. However, preculture of the leaves did not induce a significant increase in the percentage of dividing cells when mother-plants were grown on medium supplemented with yeast extract. Subculturing the small cell-clusters for 20 days in the same medium (either liquid or solidified with 0.8% agar) at a lower osmotic pressure facilitated the growth of hundreds of microcalli (Fig. 1H). The transfer of these microcalli onto solid C medium without mannitol 20 days later, induced an elevated growth rate of calli (Fig. lI). DISCUSSION The protoplast yields of more than 5 × 106/g of leaf tissue obtained in the present study, are comparable to those reported for axenic tobacco plants [10] and were higher than those found in other species [11--13] or in greenhouse-grown melon plants [4]. Preculture of the leaves from axenic plants strongly diminished the protoplast yield, as already observed by other authors [5,14], indicating that quantitative and/or qualitative changes in the cell wall occurred as a result of the culture in vitro [5]. Although preplasmolysis of plant material and presence of metallic ions in the maceration mixture have been reported to increase the protoplast viability in cucumber [ 1,2], we found no such requirements in melon. Plant growth conditions turned out to have a striking effect on the behaviour of the protoplasts from the three cultivars tested. This fact is in agreement with the results reported by several workers who used greenhousecultured plants [15--19] or axenic cultures [10,11,13,20]. So far, the most intensively studied factor has been the influence of light fiuence during the growth of the axenic plants upon the further response of the protoplasts isolated from them. In addition, Durand's work [13] demonstrated the influence of the composition of the plant culture medium on the development of the protoplasts. Our work presents new evidence in this regard. Preculture of the leaves from axenic plants of cvs. 'Valenciano Pinyonet' and 'Pinyonet Piel de Sapo' cultivated under suboptimal conditions, gave rise to protoplasts capable of sustained divisions at a moderate rate, as previously reported for melon protoplasts obtained from greenhouse-grown plants [4]. The successful application of this technique to systems of where protoplasts divide poorly had been already demonstrated in other plant species [9,14]. In conclusion, the axenic culture of plants, with or without preculture of their leaves is an improvement over our previously reported method [4] because of its simplicity, plating efficiency, absence of contamination problems, uniformity of plant material throughout the year and total reproducibility of results. Morphogenetic responses of the protoplast-derived calli of melon are being investigated at present, stimulated by the recent observation that somatic embryos were obtained under certain conditions (in preparation).
201 ACKNOWLEDGEMENTS
T h e a u t h o r s are g r a t e f u l t o Dr. J. H a n s e n f o r r e v i e w i n g a n d c o r r e c t i n g t h e English m a n u s c r i p t . T h e r e s e a r c h was p a r t i a l l y s u p p o r t e d b y t h e Ministry o f E d u c a t i o n a n d Science o f t h e S p a n i s h G o v e r n m e n t g r a n t t o Dr. V. Moreno. REFERENCES
1 2 3 4
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