Plant Cell Reports
Plant Cell Reports (1988) 7:463-466
© Springer-Verlag 1988
In vitro selection for salt tolerant lines in
Lycopersiconperuvianum
Noaman S. Hassan* and Dennis A. Wilkins School of Biological Sciences, University of Birmingham, B152TT, UK Received January 2, 1988/Revised version received August 4, 1988 - Communicated by P. King
Tal
et al.
(1978)
compared
cell
cultures
of
ABSTRACT
Lycopersicon esculentum and L.peruvianum and found
A salt-tolerant callus line of Lycopersicon peruvianum has been obtained by exposing the cells, in suspension cultures and then in callus, to increasing concentrations of NaCI (50-350mM). This selected line grew better than the nonselected line at all levels of NaCI. Moreover, this selected line grew better in media containing salt than in those without it. It retained its tolerance after subculture for 3 passages (3 months) on salt-free medium. The growth of the selected line in mannitol was similar to that of the nonselected line, which suggested that the superiority of the selected line under salt stress was not due to osmotic stress tolerance. The ions SO 4 -" and K + were highly toxic to L. peruvianum root callus, while Na +, Mg ++ and CI- were less toxic.
the latter to be the more salt tolerant, but there are no reports of the selection of salt tolerant cells in tomato. The present study aims to isolate and study cell lines with an increased degree of salt tolerance from the already moderately tolerant wild tomato L. peruvianum.
INTRODUCTION One of the practical uses of cell culture techniques is for plant breeding (Rains et al. 1982). The use of such cultures for the selection of salt-tolerant lines may offer considerable potential, provided regenerative capacity is maintained and the tolerance character persists in regenerated plants and is inheritable. Selected cell lines of Nicotiana sylvestris and Capsicum annum (Dix and Street 1975), alfalfa (Croughan et al. 1978), Cicer arietinum (Pandey and Ganapathy 1984), Citrus aurantium (Kochba et al. 1982) and Ipomoea batatus (Salgado-Garciglia et al. 1985), retained their salt resistance when they were subcultured for 3 passages in the absence of NaCI. Embryogenic cell lines of Pennisetum americanum also retained this trait even after seven transfers on salt-free medium (Rangan and Vasil 1983). A salt resistant tobacco (N. tabacum) cell line showed stability through at least 24 passages in the absence of added NaCI (Watad et al. 1983). More interestingly, Nabors et al. (1980) were able to regenerate salt-resistant plants from a resistant cell line of tobacco which transmitted tolerance to two subsequent seed generations, and the level of NaCI resistance in regenerated plants was said to be higher than that of cell cultures.
MATERIALS
AND METHODS
Tissue and cell cultures Callus cultures were established from root explants of
Lycopersicon peruvianum. The culture medium was that of Murashige and Skoog (MS) (Murashige and Skoog 1962) supplemented with 4.0 mg/I naphthaleneacetic acid, 0.4 mg/I kinetin and 30 g/I sucrose. The medium was adjusted to pH 5.8, solidified with 0.8% agar and autoclaved at 121°C for 20 minutes. Cultures were maintained under 16 hours photoperiod at 26°C. After establishment the calli were subcultured at 4 weeks intervals on the same media. Suspension cultures were established by inoculating callus (5g fresh weight) into honey jars containing 50cm 3 of liquid MS medium kept on a rotary shaker in the dark at a temperature of 26°C. Selection Procedure At each subculturing 15cm 3 was transferred to a honey jar containing 50cm 3 fresh medium. The selected line was subcultured every 4 weeks in fresh medium with gradually increasing concentrations of NaCI (35,75,115,155 and 200mM). Samples were taken monthly before each subculture for a viability test using fluorescein di-acetate (Widholm 1972), and the number of living cells per one cm 3 was counted with a haemocytometer. The salt concentration was raised to 200mM over five four-week passages, after which the suspension cultures were allowed to settle for about 10 minutes, and most of the supernatant was decanted. Then these cells (or small cell aggregates) were inoculated on agar-solidified MS medium supplemented with 200mM NaCI. The resulting calli were subcultured every four weeks on fresh medium with a further increase
* Present address: Botany Department, Faculty of Science, Zagagig University, Egypt Offprint requests to. D. A. Wilkins
464 in NaCI concentration (250,300 and 350mM). 400mM NaCI was found to kill almost all the cells, so the selected line was grown and subcultured on fresh medium supplemented with 350mM NaCI. All soft and necrotic callus was discarded at each subculture. The whole selection process (in suspension culture and on agar-solidified medium) took 14 passages.
c) Proline content: We found that increasing the NaCI concentrations increased the proline content in all the three lines and that the selected lines accumulated more proline than the nonselected one (Fig.2).
Characteristics of the selected line
( ) Selected line (-----} SN line (------) Non-selecfed line
160 F For the measurement of salt tolerance, root callus pieces from the two callus lines were transferred to MS medium containing 0,100,200,250,300 or 350mM NaCI. In all experiments three calli per dish and six replicate dishes per treatment were used. One month later, callus fresh weight increase was determined and expressed as a tolerance index (T.l.=% of fresh weight increase without salt). The proline content of calli was also determined using the method described by Singh et al. (1973). The effects of mannitol and the salts KCI, MgCI 2 and Na2SO 4 were investigated in a similar way. Mannitol was used at concentrations iso-osmotic with those of NaCI (Harms and Potrykus 1978). The salts were used at the same molar concentrations as NaCI. With the two divalent salts the approximate concentration of NaCI which would have the same osmotic potential can be calculated from figures given by Harms and Oertli (1985). For 100mM MgCI 2 this would be 200mM, and in the case of Na2SO 4 it would be 170mM.
I-- lZ,0 X
120
¢-
100
U tm
},,
t=.
0
En
I"\,. \
60-
\\ .\. k\ ". T
-
k ' ~
e-
/+0L.
200
0
100
Stabilitv of salt tolerance of the selected line The selected line was allowed to grow on salt-free medium for three months, and the effect of NaCI was then studied as in the previous experiment. In all experiments 500mg fresh weight callus pieces were used as inocula.
\
80
¢1J
150
200
250
300
350
No.el concentration (mH) Figure 1 : Effect of NaCI on callus fresh weight tolerance index. The "SN line" was transferred from saline to non-saline medium for 3 months prior to testing. (Bars are s.d.)
RESULTS I.
Effect of NaCI.
.a-r-
o~ 60
a) Increase in callus fresh weight: 50 The selected line grew markedly better on NaCI than did the nonselected line (Fig.l). At 100mM NaCI, its tolerance index was almost twice that of the nonselected line. Doubling the NaCI concentration to 200mM reduced the T.I. of the selected line slightly and that of the nonselected line severely. At higher NaCI concentrations, the nonselected line stopped growing completely, while the selected line did not. It is notable that the selected line showed better growth at 100 and 200mM NaCI than that at 0 NaCI, thus giving it a T.I. of over 100%. b) Stability of the selected line: The stability of the selected line was tested after three months on salt-free medium by returning it to a medium containing salt. Some decline of tolerance was found, but at concentrations above 100mM NaCI (Fig.l) this callus line (designated SN) still showed better growth than the nonselected line.
i
( ~ ) SeLected line (--'--) SH line
C~,--'f ~
/_L
(-----) Non selected fine
~
9
T/.-~-~ T
"-" 4.0 E - -
30
% aJ
-~ 2o Q
==1o f
I
I
1
T
1
]
50
100
150
200
250
300
350
No.C[ concenfrafion (rnH}
Figure 2: Effect of NaCI on callus proline content per unit dry weight. (Bars are s.d.)
465
2. Effects of mannitol, MgCI. KCI and Na 2SO4 High concentrations of mannitol decreased the growth rate (Fig.3) and the difference between the two lines was not significant. Figure 4 shows the effect of 50, 100, 150 and 200mM MgCI 2 on the fresh weight increase of the two callus lines. Although the nonselected callus line showed little difference from the selected line at 50mM MgCI2, there were marked differences at 100mM and 150mM. Neither of the two lines could tolerate 200mM MgCI 2. lZ~0 -
I..-
120
--
_
.E 100
Na2SO4(mM) 50
100
Selected
66.7
0.0
Non/sel.
56.2
0.0
\\
KCL(mM)
~~.\\
( ~ ) Selected line (----)Non selected line
8O
o
~
Table 1" Effects of Na2SO4 and KCI on callus fresh weight tolerance index.
T \
e-
~
Table 1 shows that the two callus lines were equally sensitive to Na2SO 4 and to KCI. Comparison with Fig.1 shows that even taking into account the differences of osmotic potential the cells are more sensitive to these two salts than to NaCI.
60
100
200
250
Selected
37.2
6.9
0.0
Non/sel.
43.5
0.0
0.0
40 #u~
D I S C U S S I O N
,2.
20±
o
100
200
300
/+00
500
600
HQnnifo[ concentration (rnH)
Figure 3: Effect of mannitol on callus fresh weight tolerance index. (Bars are s.d.)
( } Selected line I (----) Non selected line
For a selected trait to be agriculturally useful, the desirable characteristic selected in vitro must maintain stability even in the absence of the stress (Kochba et al. 1982; Salgado-Garciglia et al. 1985). In the present work, when the selected callus line was tested on salt-containing medium after subculturing on salt-free medium for three months, it showed a reduced level of tolerance, but it still showed a significantly better growth rate than the nonselected line. This result may imply that the selected line consists of a mixture of (a) adapted cells which lost their tolerance when transferred to salt-free medium, and (b) true genetic variant cells which retained their tolerance in salt-free medium.
IO0 ~-
80
x
\
T
~- 60
\\\\ NNN~ ~
2O
50
100
150
We succeeded in selecting a salt tolerant callus line of the wild tomato Lycopersicon peruvianum which grew better than the nonselected callus line at all levels of NaCI. Moreover, this selected line displayed a shift toward a halophytic behaviour as it was stimulated by concentrations of NaCI up to 200mM. Similar observations were reported in alfalfa (Croughan et al. 1978), rice (Rains et al. 1980), orange (Ben-Hayyim and Kochba 1983), and sweet potato (Salgado-Garciglia et al. 1985). For comparison Tal et a1.(1978) found that little growth occurred in their L.peruvianum calli, to which our unselected line corresponds, at salt concentrations above about 200mM.
200
HgCt2 concenfrofion (mM)
Figure 4: Effect of MgCI 2 on callus fresh weight tolerance index. (Bars are s.d.)
However, the ultimate proof of a true genetic variant lies in the regeneration of salt tolerant plants and then testing the inheritance of tolerance at whole plant level. Unfortunately our attempts to regenerate plants from the selected cells failed.
466 T h e accumulation of proline by the two lines was similar in the absence of salt, but at 200mM NaCI or more the proline content of the selected line was much higher than that of the nonselected one. Similar results were obtained by Watad et al. (1983) with Nicotiana and by Pandy and Ganapathy (1985) with Cicer. The opposite result was reported by Tal et al. (1979) in comparisons between callus of relatively salt resistant and sensitive species of Lycopersicon., and by Dix and Pearce (1981) with cell lines of Nicotiana. These divergent results imply that the part played by proline accumulation in salt tolerance still remains to be clarified. It has frequently been assumed to be involved in osmotic adjustment, but much clearly depends on the conditions of the experiment. Growth of the nonselected callus line was less severely inhibited by mannitol than by NaCI, suggesting that in the latter case it was affected by ion toxicity. Growth of the selected line, on the other hand, was better in NaCI than in mannitol. This may be a further reflection of the stimulating effect of salt on halophytic plants. Replacing CI- with SO4-- inhibited growth completely in both callus lines at concentrations higher than 50mM. When NaCI was replaced by KCI both cell lines were inhibited almost completely, while when NaCI was replaced by MgCI 2 there was little change of growth rate in either callus line. These results suggest that SO4-- and K + are both highly toxic to L. peruvianum root callus, while Na +, Mg ++ and CI" are less toxic, and are in general agreement with those of Kochba et a1.(1982) on Citrus.
ACKNOWLEDGEMENT
The authors wish to thank the Egyptian Education Bureau for the financial support of this work.
REFERENCES
Ben-Hayyim G, Kochba J (1983) Plant Physiol. 72: 685-690 Croughan TR, Stavarek SJ, Rains DW (1978) Crop. Sci. 18:959-963 Dix PJ, Pearce RS (1981) Z. Pflanzenphysiol. 102S: 243-248 Dix PJ, Street HE (1975) Plant Sci. Lett. 5:231-237 Harms CT, Potrykus I (1978)Theor.Appl.Genet. 53:57-63 Harms CT, Oertli JJ (1985) J. Plant Physiol. 120:29-38 Kochba J, Ben-Hayyim G, Spiegel-Roy P, Saad S, Neumann H (1982) Z.Pflanzenphysiol. 106S: 111-118 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1980) Z.Pflanzenphysiol. 97:13-17 Pandey R, Ganapathy PS (1984) Plant Cell Rep. 3:45-47 Pandey R, Ganapathy PS (1985) Plant Sci. Lett.40:13-17 Rains DW, Croughan TP, Stavarek SJ (1980) In: Rains DW,Valentine RC, Hollaender A, eds., Genetic Engineering of Osmoregulation, Plenum Press, New York, pp. 279-292 Rains DW, Csonka L, Le Rudulier D, Croughan TP, Yang SS, Stavarek SJ, Valentine RC (1982) In: San Pietro A, ed., Biosatine Research: A Look to the Future, Plenum Press, New York, pp. 283-302 Rangan TS, Vasil IK (1983) Ann. Bot. 5 2 : 5 9 - 6 4 Salgado-Garciglia R, Lopez-Gutierrez F, Ochoa- Alejo N (1985) Plant Cell Tis.Org.Cult. 5:3-12 Singh TN, Paleg LG, Aspinall D (1973) Aust. J. Biol. Sci. 26:45-56 Tal M, Heikin H, Dahan K (1978) Z.Pflanzenphysiol. 86:231-240 Tal M, Katz A, Heikn H, Dehan K (1979) New Phytol. 82: 349-355 Watad AEA, Reinhold L, Lerner HR (1983) Plant Physiol. 73:624-629 Widholm J M (1972) Stain Techn. 47:189-194
Plant Cell Reports (1988) 7:463-466
© Springer-Verlag 1988
In vitro selection for salt tolerant lines in
Lycopersiconperuvianum
Noaman S. Hassan* and Dennis A. Wilkins School of Biological Sciences, University of Birmingham, B152TT, UK Received January 2, 1988/Revised version received August 4, 1988 - Communicated by P. King
Tal
et al.
(1978)
compared
cell
cultures
of
ABSTRACT
Lycopersicon esculentum and L.peruvianum and found
A salt-tolerant callus line of Lycopersicon peruvianum has been obtained by exposing the cells, in suspension cultures and then in callus, to increasing concentrations of NaCI (50-350mM). This selected line grew better than the nonselected line at all levels of NaCI. Moreover, this selected line grew better in media containing salt than in those without it. It retained its tolerance after subculture for 3 passages (3 months) on salt-free medium. The growth of the selected line in mannitol was similar to that of the nonselected line, which suggested that the superiority of the selected line under salt stress was not due to osmotic stress tolerance. The ions SO 4 -" and K + were highly toxic to L. peruvianum root callus, while Na +, Mg ++ and CI- were less toxic.
the latter to be the more salt tolerant, but there are no reports of the selection of salt tolerant cells in tomato. The present study aims to isolate and study cell lines with an increased degree of salt tolerance from the already moderately tolerant wild tomato L. peruvianum.
INTRODUCTION One of the practical uses of cell culture techniques is for plant breeding (Rains et al. 1982). The use of such cultures for the selection of salt-tolerant lines may offer considerable potential, provided regenerative capacity is maintained and the tolerance character persists in regenerated plants and is inheritable. Selected cell lines of Nicotiana sylvestris and Capsicum annum (Dix and Street 1975), alfalfa (Croughan et al. 1978), Cicer arietinum (Pandey and Ganapathy 1984), Citrus aurantium (Kochba et al. 1982) and Ipomoea batatus (Salgado-Garciglia et al. 1985), retained their salt resistance when they were subcultured for 3 passages in the absence of NaCI. Embryogenic cell lines of Pennisetum americanum also retained this trait even after seven transfers on salt-free medium (Rangan and Vasil 1983). A salt resistant tobacco (N. tabacum) cell line showed stability through at least 24 passages in the absence of added NaCI (Watad et al. 1983). More interestingly, Nabors et al. (1980) were able to regenerate salt-resistant plants from a resistant cell line of tobacco which transmitted tolerance to two subsequent seed generations, and the level of NaCI resistance in regenerated plants was said to be higher than that of cell cultures.
MATERIALS
AND METHODS
Tissue and cell cultures Callus cultures were established from root explants of
Lycopersicon peruvianum. The culture medium was that of Murashige and Skoog (MS) (Murashige and Skoog 1962) supplemented with 4.0 mg/I naphthaleneacetic acid, 0.4 mg/I kinetin and 30 g/I sucrose. The medium was adjusted to pH 5.8, solidified with 0.8% agar and autoclaved at 121°C for 20 minutes. Cultures were maintained under 16 hours photoperiod at 26°C. After establishment the calli were subcultured at 4 weeks intervals on the same media. Suspension cultures were established by inoculating callus (5g fresh weight) into honey jars containing 50cm 3 of liquid MS medium kept on a rotary shaker in the dark at a temperature of 26°C. Selection Procedure At each subculturing 15cm 3 was transferred to a honey jar containing 50cm 3 fresh medium. The selected line was subcultured every 4 weeks in fresh medium with gradually increasing concentrations of NaCI (35,75,115,155 and 200mM). Samples were taken monthly before each subculture for a viability test using fluorescein di-acetate (Widholm 1972), and the number of living cells per one cm 3 was counted with a haemocytometer. The salt concentration was raised to 200mM over five four-week passages, after which the suspension cultures were allowed to settle for about 10 minutes, and most of the supernatant was decanted. Then these cells (or small cell aggregates) were inoculated on agar-solidified MS medium supplemented with 200mM NaCI. The resulting calli were subcultured every four weeks on fresh medium with a further increase
* Present address: Botany Department, Faculty of Science, Zagagig University, Egypt Offprint requests to. D. A. Wilkins
464 in NaCI concentration (250,300 and 350mM). 400mM NaCI was found to kill almost all the cells, so the selected line was grown and subcultured on fresh medium supplemented with 350mM NaCI. All soft and necrotic callus was discarded at each subculture. The whole selection process (in suspension culture and on agar-solidified medium) took 14 passages.
c) Proline content: We found that increasing the NaCI concentrations increased the proline content in all the three lines and that the selected lines accumulated more proline than the nonselected one (Fig.2).
Characteristics of the selected line
( ) Selected line (-----} SN line (------) Non-selecfed line
160 F For the measurement of salt tolerance, root callus pieces from the two callus lines were transferred to MS medium containing 0,100,200,250,300 or 350mM NaCI. In all experiments three calli per dish and six replicate dishes per treatment were used. One month later, callus fresh weight increase was determined and expressed as a tolerance index (T.l.=% of fresh weight increase without salt). The proline content of calli was also determined using the method described by Singh et al. (1973). The effects of mannitol and the salts KCI, MgCI 2 and Na2SO 4 were investigated in a similar way. Mannitol was used at concentrations iso-osmotic with those of NaCI (Harms and Potrykus 1978). The salts were used at the same molar concentrations as NaCI. With the two divalent salts the approximate concentration of NaCI which would have the same osmotic potential can be calculated from figures given by Harms and Oertli (1985). For 100mM MgCI 2 this would be 200mM, and in the case of Na2SO 4 it would be 170mM.
I-- lZ,0 X
120
¢-
100
U tm
},,
t=.
0
En
I"\,. \
60-
\\ .\. k\ ". T
-
k ' ~
e-
/+0L.
200
0
100
Stabilitv of salt tolerance of the selected line The selected line was allowed to grow on salt-free medium for three months, and the effect of NaCI was then studied as in the previous experiment. In all experiments 500mg fresh weight callus pieces were used as inocula.
\
80
¢1J
150
200
250
300
350
No.el concentration (mH) Figure 1 : Effect of NaCI on callus fresh weight tolerance index. The "SN line" was transferred from saline to non-saline medium for 3 months prior to testing. (Bars are s.d.)
RESULTS I.
Effect of NaCI.
.a-r-
o~ 60
a) Increase in callus fresh weight: 50 The selected line grew markedly better on NaCI than did the nonselected line (Fig.l). At 100mM NaCI, its tolerance index was almost twice that of the nonselected line. Doubling the NaCI concentration to 200mM reduced the T.I. of the selected line slightly and that of the nonselected line severely. At higher NaCI concentrations, the nonselected line stopped growing completely, while the selected line did not. It is notable that the selected line showed better growth at 100 and 200mM NaCI than that at 0 NaCI, thus giving it a T.I. of over 100%. b) Stability of the selected line: The stability of the selected line was tested after three months on salt-free medium by returning it to a medium containing salt. Some decline of tolerance was found, but at concentrations above 100mM NaCI (Fig.l) this callus line (designated SN) still showed better growth than the nonselected line.
i
( ~ ) SeLected line (--'--) SH line
C~,--'f ~
/_L
(-----) Non selected fine
~
9
T/.-~-~ T
"-" 4.0 E - -
30
% aJ
-~ 2o Q
==1o f
I
I
1
T
1
]
50
100
150
200
250
300
350
No.C[ concenfrafion (rnH}
Figure 2: Effect of NaCI on callus proline content per unit dry weight. (Bars are s.d.)
465
2. Effects of mannitol, MgCI. KCI and Na 2SO4 High concentrations of mannitol decreased the growth rate (Fig.3) and the difference between the two lines was not significant. Figure 4 shows the effect of 50, 100, 150 and 200mM MgCI 2 on the fresh weight increase of the two callus lines. Although the nonselected callus line showed little difference from the selected line at 50mM MgCI2, there were marked differences at 100mM and 150mM. Neither of the two lines could tolerate 200mM MgCI 2. lZ~0 -
I..-
120
--
_
.E 100
Na2SO4(mM) 50
100
Selected
66.7
0.0
Non/sel.
56.2
0.0
\\
KCL(mM)
~~.\\
( ~ ) Selected line (----)Non selected line
8O
o
~
Table 1" Effects of Na2SO4 and KCI on callus fresh weight tolerance index.
T \
e-
~
Table 1 shows that the two callus lines were equally sensitive to Na2SO 4 and to KCI. Comparison with Fig.1 shows that even taking into account the differences of osmotic potential the cells are more sensitive to these two salts than to NaCI.
60
100
200
250
Selected
37.2
6.9
0.0
Non/sel.
43.5
0.0
0.0
40 #u~
D I S C U S S I O N
,2.
20±
o
100
200
300
/+00
500
600
HQnnifo[ concentration (rnH)
Figure 3: Effect of mannitol on callus fresh weight tolerance index. (Bars are s.d.)
( } Selected line I (----) Non selected line
For a selected trait to be agriculturally useful, the desirable characteristic selected in vitro must maintain stability even in the absence of the stress (Kochba et al. 1982; Salgado-Garciglia et al. 1985). In the present work, when the selected callus line was tested on salt-containing medium after subculturing on salt-free medium for three months, it showed a reduced level of tolerance, but it still showed a significantly better growth rate than the nonselected line. This result may imply that the selected line consists of a mixture of (a) adapted cells which lost their tolerance when transferred to salt-free medium, and (b) true genetic variant cells which retained their tolerance in salt-free medium.
IO0 ~-
80
x
\
T
~- 60
\\\\ NNN~ ~
2O
50
100
150
We succeeded in selecting a salt tolerant callus line of the wild tomato Lycopersicon peruvianum which grew better than the nonselected callus line at all levels of NaCI. Moreover, this selected line displayed a shift toward a halophytic behaviour as it was stimulated by concentrations of NaCI up to 200mM. Similar observations were reported in alfalfa (Croughan et al. 1978), rice (Rains et al. 1980), orange (Ben-Hayyim and Kochba 1983), and sweet potato (Salgado-Garciglia et al. 1985). For comparison Tal et a1.(1978) found that little growth occurred in their L.peruvianum calli, to which our unselected line corresponds, at salt concentrations above about 200mM.
200
HgCt2 concenfrofion (mM)
Figure 4: Effect of MgCI 2 on callus fresh weight tolerance index. (Bars are s.d.)
However, the ultimate proof of a true genetic variant lies in the regeneration of salt tolerant plants and then testing the inheritance of tolerance at whole plant level. Unfortunately our attempts to regenerate plants from the selected cells failed.
466 T h e accumulation of proline by the two lines was similar in the absence of salt, but at 200mM NaCI or more the proline content of the selected line was much higher than that of the nonselected one. Similar results were obtained by Watad et al. (1983) with Nicotiana and by Pandy and Ganapathy (1985) with Cicer. The opposite result was reported by Tal et al. (1979) in comparisons between callus of relatively salt resistant and sensitive species of Lycopersicon., and by Dix and Pearce (1981) with cell lines of Nicotiana. These divergent results imply that the part played by proline accumulation in salt tolerance still remains to be clarified. It has frequently been assumed to be involved in osmotic adjustment, but much clearly depends on the conditions of the experiment. Growth of the nonselected callus line was less severely inhibited by mannitol than by NaCI, suggesting that in the latter case it was affected by ion toxicity. Growth of the selected line, on the other hand, was better in NaCI than in mannitol. This may be a further reflection of the stimulating effect of salt on halophytic plants. Replacing CI- with SO4-- inhibited growth completely in both callus lines at concentrations higher than 50mM. When NaCI was replaced by KCI both cell lines were inhibited almost completely, while when NaCI was replaced by MgCI 2 there was little change of growth rate in either callus line. These results suggest that SO4-- and K + are both highly toxic to L. peruvianum root callus, while Na +, Mg ++ and CI" are less toxic, and are in general agreement with those of Kochba et a1.(1982) on Citrus.
ACKNOWLEDGEMENT
The authors wish to thank the Egyptian Education Bureau for the financial support of this work.
REFERENCES
Ben-Hayyim G, Kochba J (1983) Plant Physiol. 72: 685-690 Croughan TR, Stavarek SJ, Rains DW (1978) Crop. Sci. 18:959-963 Dix PJ, Pearce RS (1981) Z. Pflanzenphysiol. 102S: 243-248 Dix PJ, Street HE (1975) Plant Sci. Lett. 5:231-237 Harms CT, Potrykus I (1978)Theor.Appl.Genet. 53:57-63 Harms CT, Oertli JJ (1985) J. Plant Physiol. 120:29-38 Kochba J, Ben-Hayyim G, Spiegel-Roy P, Saad S, Neumann H (1982) Z.Pflanzenphysiol. 106S: 111-118 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1980) Z.Pflanzenphysiol. 97:13-17 Pandey R, Ganapathy PS (1984) Plant Cell Rep. 3:45-47 Pandey R, Ganapathy PS (1985) Plant Sci. Lett.40:13-17 Rains DW, Croughan TP, Stavarek SJ (1980) In: Rains DW,Valentine RC, Hollaender A, eds., Genetic Engineering of Osmoregulation, Plenum Press, New York, pp. 279-292 Rains DW, Csonka L, Le Rudulier D, Croughan TP, Yang SS, Stavarek SJ, Valentine RC (1982) In: San Pietro A, ed., Biosatine Research: A Look to the Future, Plenum Press, New York, pp. 283-302 Rangan TS, Vasil IK (1983) Ann. Bot. 5 2 : 5 9 - 6 4 Salgado-Garciglia R, Lopez-Gutierrez F, Ochoa- Alejo N (1985) Plant Cell Tis.Org.Cult. 5:3-12 Singh TN, Paleg LG, Aspinall D (1973) Aust. J. Biol. Sci. 26:45-56 Tal M, Heikin H, Dahan K (1978) Z.Pflanzenphysiol. 86:231-240 Tal M, Katz A, Heikn H, Dehan K (1979) New Phytol. 82: 349-355 Watad AEA, Reinhold L, Lerner HR (1983) Plant Physiol. 73:624-629 Widholm J M (1972) Stain Techn. 47:189-194
