Plant Cell Reports (1984) 3:45-47
Plant CeU Reports © Springer-Verlag 1984
Isolation of sodium chloride-tolerant callus line of Cicer arietinum L. cv. BG-203 Ruchira Pandey and P. S. Ganapathy Department of Botany, University of Delhi, Delhi 110007, India Received December 1, 1983 / Revised version received February 17, 1984 - Communicated by F. Constabel
ABSTRACT A N a C l - t o l e r a n t callus line of Cicer arietinum has been isolated, as a spontaneous variant, on agar-sotidified MS-medium supplemented with 100 mM NaCI. The growth of this line, in the presence of 100 mM NaCI, was comparable to that of the NaCl-sensitive callus line growing in the absence of NaCI. Regarding r e l a t i v e tolerance of the two callus lines towards NaCI (0 to 200 mM), the t o l e r a n t line performed poorly in the absence of NaCI and exhibited o p t i m a l growth at 50 mM NaCI. The tolerance persisted even a f t e r three passages of 4-wk each, tested so far, away from the selective agent. INTRODUCTION The application of plant tissue culture techniques for the selection of variant cell lines for crop improvement has been a topic of current interest (Maliga et el. 1982). The techniques involved in developing crop plants with improved tolerance to salinity could be instrumental in expanding agriculture to regions with higher salinity (Rains et el. 1980). Selection of a few cell lines exhibiting improved tolerance to salt stress has already been reported (Nabors et at. 1975; Croughan et al. I978; Rains et at. 1980; B e n - H a y y i m and Kochba 1982; Rangan and Vasi] 1983). In the present communication, we report the selection of a N a C l - t o t e r a n t callus line of Cicer arietinum (chickpea), a salt-sensitive crop.
fresh weight were inoculated on MS-medium supplemented with 25 to 200 mM NaCI. A t higher than 50 mM N a C l , c a l l i exhibited browning and arrested growth. Therefore, it was decided to select calli which would t o l e r a t e NaCI at /00 mM, and then increase the tolerance by successive cultures on increasing concentrations of NaCt. A t 100 mM,only /47% cultures showed retention of chlorophyll in defined sectors distinguishable from the remaining necrotic zones. These green sectors, presumably s a l t - t o l e r a n t , on transfer to a medium with the same concentration of NaCl (100 mM), exhibited an increase ( a p p r o x i m a t e l y 70-80%) in the cultures that survived, though their growth was still below optimal. Subsequently, during f i f t h and sixth passage, the surviving calli grew well and did not e x h i b i t discoloration at 100 mM NaCl. These variant calli have been designated R-100 to distinguish them from the NaCl-sensitive stock calli (C-0). To date, the NaCIt o l e r a n t calli have passed through 11 subcultures with a t o t a l culture period of about 11 months. To determine the s t a b i l i t y of the selected t r a i t , R-100 ealli were subcultured for three passages (4wk each) away from NaCl and, t h e r e a f t e r , grown again on the medium with 100 mM NaCl. Such a callus line has been designated R-100a. Fresh and dry weight measurements of C-0, R-100 and R-100a, and C-0 calli, grown at 100 mM NaCI, have been followed over a period of 40 days.
M A T E R I A L A N D METHODS Calli of Cicer arietinum L. cv. BG-203 were i n i t i ated from embryos excised from imbibed seeds on B5 medium (Gamborg et al. 1968) with 0.8% agar, 2% sucrose, and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The calti were l a t e r maintained on MS-medium (Murashige and Skoog 1962) w i t h o u t adenine sulphate, but with 3% sucrose, I mg/l naphthaleneacetic acid (NAA), and 1 mg/l benzyl aminopurine (BAP). Stock cultures were thus obtained following subculture through 4-wk passages. When necessary, NaCI was incorporated into the MSmedium at 0, 25, 50, 100, 125, 150, and 200 mM. For all the combinations, pH of the media was adjusted to 5.8 before autoclaving. The cultures were maintained at 25-30°C under 10-11 h daily i l l u m i n a t i o n (500-560 lux). R e l a t i v e h u m i d i t y in the culture room varied from 5570%. The t o l e r a n t callus line was isolated following the direct selection procedure of biochemical variants (Maliga 1978). Calli pieces of a p p r o x i m a t e l y 100 mg
To compare NaCI tolerance of C-0 and R-100 callus lines, callus pieces of 100 _+ 10 mg were inoculated on media containing 0 to 200 mM NaCI. Fresh and dry weights of the two callus lines were determined a f t e r 20 days of culture. The data presented is an average of 12 replicates. RESULTS AND
DISCUSSION
A N a O l - t o l e r a n t callus line R-100 of C. arietinum has been isolated as a spontaneous variant, at 100 mM NaCl, a concentration normally inhibitory to the growth of NaOl-sensitive callus line C-0. Over a period of 40 days, C-0 caJlJ, in the absence of NaO], and R-100 calli at 100 mM NaO], exhibited a rapid increase in fresh weight (Fig.la) between 10 and 20 days which. continued up to 30 days, followed by a decline t i l l 40 days. This decline was higher in R-100 calli. C-0 callJ, when exposed to 100 mM NAG:], did not show any increase in fresh weight over the entire culture period (Fig.la). Similar changes in dry weight were
46 a'
60C
observations of Croughan et al. (1978) in Medicaqo sativa, Rains et al. (1980) in Qryza saliva, and BenH a y y i m and Kochba (1982) in Citrus sinensis. R-100a callus line exhibited a rapid increase in fresh (Fig.la) and dry (Fig.lb) weights between 10 and 20 days followed by a decline up to 30 days. The growth of R-100a calli
500i T
450
a
3O0
, "°IY .o/\ :o,,o./
150
\
;,oo: 0
I _ 0
10
_
20
~
I
30
40
Culture period (days)
i/ /
\
/
lOO 50
60t
I
I
25
50 75 loo 125 15o NclC[ concentrstion (mM)
I
t
i
1_
1
I
~75
200
5O cm
i >.
30
4(
c~
E 30
>" 20 f3 0 0
10
20
Culture period
30
40
(days) 10
Fig.1. Fresh weight (a) and dry weight (b) of callus as a function of time. 8--0 C-0 callus line growing on medium w i t h o u t NaCt, ~ R - 1 O 0 callus line growing at 100 mM NaCl, _A A_ R-100a callus line (grown for three passages in the absence of NaCI and again on a medium with 109 ram NaCt), ~ C-0 callus hne growing at 100 mM NaCl. Vertical bars represent standard error of the mean.
observed in the two callus lines (Fig.lb). The growth of R-1OO calli at 100 mM is comparable to that of C-0 in the absence o f NaCl. Our results confirm the
0 25
I 50
I 75
I 100
~ 125
I 150
I__ 175
I 200
Na£1 concentrQtion(mM)
Fig.2. C o m p a r a t i v e fresh weight (a) and dry weight (b) of C-O callus line (o---o) and R-1O0 callus line (e---O) as a function of NaCl concentration in the medium, Vertical bars represent standard error of the mean,
4? was comparable to that of R-100 t i l l 20 days, and then it was almost 50% of that of R-IO0. R-100a calli resumed growth in subsequent passages. This showed the persistence of the tolerance t r a i t in the absence of NaCI (up to three passages tested so far) - as reported earlier in other species (see Dix and Street I975; Ben-Hayyim and Kochba 1982; Rangan and Vasil 1983).On the contrary, Hasegawa et al. (1980), in Nicotiana tabacum, reported the loss of NaCl-toleranee upon growing the cells for five generations away from the selective agent. Fig.2a represents relative tolerance of C-0 and R-100 callus lines towards NaCI, in increase in fresh weight. The C-0 calli had a slightly larger fresh weight at 0 salinity, at 25 mM both lines had almost a similar fresh weight~ and at 50 and I00 mM R-100 catli had significantly higher fresh weight. At still higher concentrations (I25; 150, and 200 mM) of NaCI while the two lines exhibited a decrease, R-100 ca]It still had higher fresh weigh[. A similar trend in changes in dry weight of the two callus lines was also observed (Fig.2b). These results are in agreement with those of Croughan et al. (1978) in Medicaqo saliva and Rains et al. (1980) in Oryza saliva. A r e l a t i v e l y poor growth in the absence of NaCI~ and growth stimulation by the addition of NaCI in NaCltolerant calli, possibly indicate a shift towards halophytic mode of salt tolerance in chickpea as also suggested by Croughan et at. (1978) in s a l t - t o l e r a n t cell line of Medica9o sativa. Another feature of halophytes is their capacity to accumulate and tolerate high internal concentrations of certain ions (Yea 1983). Preliminary studies on t h e estimation of Na +, K + C I - a n d PO43- in the two callus lines grown at 0 to 200 mM NaCI indicate that up to 50 mM; there was no significant difference in the levels of Na+ and C I - whereas at 100 mM, R-100 ealli accumulated =more Na + and C I - than C-0 catli. While K + and P O / 2 - levels in both callus lines decreased with increasing NaCl, R-100 calli still maintained higher levels of these two ions despite high Na+ in the system. The u l t i m a t e proof of a true genetic variant lies in the regeneration of s a l t - t o l e r a n t plants (see Mathur et at. 1980; Nabors et al. 1980; Smith and McComb 1983) and genetic analysis of their F1 progenies as shown by Nabors et al. (1980) in Nicotiana tabacum. Attempts to obtain regeneration of plants from N a C l - t o l e r a n t calli of Cicer are in progress. ACKNOWLEDGEMENTS We are grateful to Professor B.M. 3ohri for c r i t i c a l l y going through the manuscript. One of us (R.P.) thanks Council of Scientific and Industrial Research, New Delhi, for financial assistance.
REFERENCES Ben-Hayyim 87-9/4
G,
Croughan TP, 18:959-963 Dix
PJ,
Street
Kochba
Left
27:
8tavarek SJ, Rains DW (1978) Crop
Sci
HE
3 (1982)
(1975)
Gamborg OL, M i l l e r RA, 50:151-158
Plant
Plant
Sci
Sci
Lett
5: 231-237
Ojima K (1958) Exp Cell Res
Hasegawa PM, Bressan RA, Handa AK (1980) Plant Cell Physiol 21:1347-1355 Maliga P 41978) In: Thorpe TA (ed) Frontiers of plant tissue culture. Proc 4th ]ntl Congr Plant Tissue and Cell Culture, Univ Calgary, Calgary, pp 381-392 Maliga P, Menczet L, Sidorov V~ Marten L~ Cseplo A, Medgyesy P~ Dung TM~ Lazar G, Nagy F (1982) In: Vasil [K, Scowcroft WR, Fcey KJ (eds) Plant improvement and somatic cell genetics. Academic Press, New York, pp 221-237 Mathur AK, Ganapathy PS, John BM (1980) Z Pflanzenphysiol 99:287-294 Murashige T, Skoog F (1962) Physiol Plant 15 : 473-/497 Nabors MW, Daniels A, Plant Sei L e t t 4 : I 5 5 - 1 5 9
Nadotny
L,
Brown
C (1975)
Nabors MW~ Gibbs SE, Becnstein CS, Meis ME (1980) Z Pflanzenphysiol 97:13-17 Rains DW, Croughan TP, Stavarek S3 (1980) In: Rains DW, Valentine RC, Ho]laender A (eds) Genetic engineering of osmoregulation: impact on plant productivity for food, chemicals and energy. Plenum Press, New York, pp 279-292 Rangan TS, Vasil IK (1983) Ann Bat 52:59-64 Smith MK, 126-128
McComb
JA
(1983) Plant
Yea AR (1983) Physio] Plant 58:214-222
Cell
Reports
2:
Plant CeU Reports © Springer-Verlag 1984
Isolation of sodium chloride-tolerant callus line of Cicer arietinum L. cv. BG-203 Ruchira Pandey and P. S. Ganapathy Department of Botany, University of Delhi, Delhi 110007, India Received December 1, 1983 / Revised version received February 17, 1984 - Communicated by F. Constabel
ABSTRACT A N a C l - t o l e r a n t callus line of Cicer arietinum has been isolated, as a spontaneous variant, on agar-sotidified MS-medium supplemented with 100 mM NaCI. The growth of this line, in the presence of 100 mM NaCI, was comparable to that of the NaCl-sensitive callus line growing in the absence of NaCI. Regarding r e l a t i v e tolerance of the two callus lines towards NaCI (0 to 200 mM), the t o l e r a n t line performed poorly in the absence of NaCI and exhibited o p t i m a l growth at 50 mM NaCI. The tolerance persisted even a f t e r three passages of 4-wk each, tested so far, away from the selective agent. INTRODUCTION The application of plant tissue culture techniques for the selection of variant cell lines for crop improvement has been a topic of current interest (Maliga et el. 1982). The techniques involved in developing crop plants with improved tolerance to salinity could be instrumental in expanding agriculture to regions with higher salinity (Rains et el. 1980). Selection of a few cell lines exhibiting improved tolerance to salt stress has already been reported (Nabors et at. 1975; Croughan et al. I978; Rains et at. 1980; B e n - H a y y i m and Kochba 1982; Rangan and Vasi] 1983). In the present communication, we report the selection of a N a C l - t o t e r a n t callus line of Cicer arietinum (chickpea), a salt-sensitive crop.
fresh weight were inoculated on MS-medium supplemented with 25 to 200 mM NaCI. A t higher than 50 mM N a C l , c a l l i exhibited browning and arrested growth. Therefore, it was decided to select calli which would t o l e r a t e NaCI at /00 mM, and then increase the tolerance by successive cultures on increasing concentrations of NaCt. A t 100 mM,only /47% cultures showed retention of chlorophyll in defined sectors distinguishable from the remaining necrotic zones. These green sectors, presumably s a l t - t o l e r a n t , on transfer to a medium with the same concentration of NaCl (100 mM), exhibited an increase ( a p p r o x i m a t e l y 70-80%) in the cultures that survived, though their growth was still below optimal. Subsequently, during f i f t h and sixth passage, the surviving calli grew well and did not e x h i b i t discoloration at 100 mM NaCl. These variant calli have been designated R-100 to distinguish them from the NaCl-sensitive stock calli (C-0). To date, the NaCIt o l e r a n t calli have passed through 11 subcultures with a t o t a l culture period of about 11 months. To determine the s t a b i l i t y of the selected t r a i t , R-100 ealli were subcultured for three passages (4wk each) away from NaCl and, t h e r e a f t e r , grown again on the medium with 100 mM NaCl. Such a callus line has been designated R-100a. Fresh and dry weight measurements of C-0, R-100 and R-100a, and C-0 calli, grown at 100 mM NaCI, have been followed over a period of 40 days.
M A T E R I A L A N D METHODS Calli of Cicer arietinum L. cv. BG-203 were i n i t i ated from embryos excised from imbibed seeds on B5 medium (Gamborg et al. 1968) with 0.8% agar, 2% sucrose, and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The calti were l a t e r maintained on MS-medium (Murashige and Skoog 1962) w i t h o u t adenine sulphate, but with 3% sucrose, I mg/l naphthaleneacetic acid (NAA), and 1 mg/l benzyl aminopurine (BAP). Stock cultures were thus obtained following subculture through 4-wk passages. When necessary, NaCI was incorporated into the MSmedium at 0, 25, 50, 100, 125, 150, and 200 mM. For all the combinations, pH of the media was adjusted to 5.8 before autoclaving. The cultures were maintained at 25-30°C under 10-11 h daily i l l u m i n a t i o n (500-560 lux). R e l a t i v e h u m i d i t y in the culture room varied from 5570%. The t o l e r a n t callus line was isolated following the direct selection procedure of biochemical variants (Maliga 1978). Calli pieces of a p p r o x i m a t e l y 100 mg
To compare NaCI tolerance of C-0 and R-100 callus lines, callus pieces of 100 _+ 10 mg were inoculated on media containing 0 to 200 mM NaCI. Fresh and dry weights of the two callus lines were determined a f t e r 20 days of culture. The data presented is an average of 12 replicates. RESULTS AND
DISCUSSION
A N a O l - t o l e r a n t callus line R-100 of C. arietinum has been isolated as a spontaneous variant, at 100 mM NaCl, a concentration normally inhibitory to the growth of NaOl-sensitive callus line C-0. Over a period of 40 days, C-0 caJlJ, in the absence of NaO], and R-100 calli at 100 mM NaO], exhibited a rapid increase in fresh weight (Fig.la) between 10 and 20 days which. continued up to 30 days, followed by a decline t i l l 40 days. This decline was higher in R-100 calli. C-0 callJ, when exposed to 100 mM NAG:], did not show any increase in fresh weight over the entire culture period (Fig.la). Similar changes in dry weight were
46 a'
60C
observations of Croughan et al. (1978) in Medicaqo sativa, Rains et al. (1980) in Qryza saliva, and BenH a y y i m and Kochba (1982) in Citrus sinensis. R-100a callus line exhibited a rapid increase in fresh (Fig.la) and dry (Fig.lb) weights between 10 and 20 days followed by a decline up to 30 days. The growth of R-100a calli
500i T
450
a
3O0
, "°IY .o/\ :o,,o./
150
\
;,oo: 0
I _ 0
10
_
20
~
I
30
40
Culture period (days)
i/ /
\
/
lOO 50
60t
I
I
25
50 75 loo 125 15o NclC[ concentrstion (mM)
I
t
i
1_
1
I
~75
200
5O cm
i >.
30
4(
c~
E 30
>" 20 f3 0 0
10
20
Culture period
30
40
(days) 10
Fig.1. Fresh weight (a) and dry weight (b) of callus as a function of time. 8--0 C-0 callus line growing on medium w i t h o u t NaCt, ~ R - 1 O 0 callus line growing at 100 mM NaCl, _A A_ R-100a callus line (grown for three passages in the absence of NaCI and again on a medium with 109 ram NaCt), ~ C-0 callus hne growing at 100 mM NaCl. Vertical bars represent standard error of the mean.
observed in the two callus lines (Fig.lb). The growth of R-1OO calli at 100 mM is comparable to that of C-0 in the absence o f NaCl. Our results confirm the
0 25
I 50
I 75
I 100
~ 125
I 150
I__ 175
I 200
Na£1 concentrQtion(mM)
Fig.2. C o m p a r a t i v e fresh weight (a) and dry weight (b) of C-O callus line (o---o) and R-1O0 callus line (e---O) as a function of NaCl concentration in the medium, Vertical bars represent standard error of the mean,
4? was comparable to that of R-100 t i l l 20 days, and then it was almost 50% of that of R-IO0. R-100a calli resumed growth in subsequent passages. This showed the persistence of the tolerance t r a i t in the absence of NaCI (up to three passages tested so far) - as reported earlier in other species (see Dix and Street I975; Ben-Hayyim and Kochba 1982; Rangan and Vasil 1983).On the contrary, Hasegawa et al. (1980), in Nicotiana tabacum, reported the loss of NaCl-toleranee upon growing the cells for five generations away from the selective agent. Fig.2a represents relative tolerance of C-0 and R-100 callus lines towards NaCI, in increase in fresh weight. The C-0 calli had a slightly larger fresh weight at 0 salinity, at 25 mM both lines had almost a similar fresh weight~ and at 50 and I00 mM R-100 catli had significantly higher fresh weight. At still higher concentrations (I25; 150, and 200 mM) of NaCI while the two lines exhibited a decrease, R-100 ca]It still had higher fresh weigh[. A similar trend in changes in dry weight of the two callus lines was also observed (Fig.2b). These results are in agreement with those of Croughan et al. (1978) in Medicaqo saliva and Rains et al. (1980) in Oryza saliva. A r e l a t i v e l y poor growth in the absence of NaCI~ and growth stimulation by the addition of NaCI in NaCltolerant calli, possibly indicate a shift towards halophytic mode of salt tolerance in chickpea as also suggested by Croughan et at. (1978) in s a l t - t o l e r a n t cell line of Medica9o sativa. Another feature of halophytes is their capacity to accumulate and tolerate high internal concentrations of certain ions (Yea 1983). Preliminary studies on t h e estimation of Na +, K + C I - a n d PO43- in the two callus lines grown at 0 to 200 mM NaCI indicate that up to 50 mM; there was no significant difference in the levels of Na+ and C I - whereas at 100 mM, R-100 ealli accumulated =more Na + and C I - than C-0 catli. While K + and P O / 2 - levels in both callus lines decreased with increasing NaCl, R-100 calli still maintained higher levels of these two ions despite high Na+ in the system. The u l t i m a t e proof of a true genetic variant lies in the regeneration of s a l t - t o l e r a n t plants (see Mathur et at. 1980; Nabors et al. 1980; Smith and McComb 1983) and genetic analysis of their F1 progenies as shown by Nabors et al. (1980) in Nicotiana tabacum. Attempts to obtain regeneration of plants from N a C l - t o l e r a n t calli of Cicer are in progress. ACKNOWLEDGEMENTS We are grateful to Professor B.M. 3ohri for c r i t i c a l l y going through the manuscript. One of us (R.P.) thanks Council of Scientific and Industrial Research, New Delhi, for financial assistance.
REFERENCES Ben-Hayyim 87-9/4
G,
Croughan TP, 18:959-963 Dix
PJ,
Street
Kochba
Left
27:
8tavarek SJ, Rains DW (1978) Crop
Sci
HE
3 (1982)
(1975)
Gamborg OL, M i l l e r RA, 50:151-158
Plant
Plant
Sci
Sci
Lett
5: 231-237
Ojima K (1958) Exp Cell Res
Hasegawa PM, Bressan RA, Handa AK (1980) Plant Cell Physiol 21:1347-1355 Maliga P 41978) In: Thorpe TA (ed) Frontiers of plant tissue culture. Proc 4th ]ntl Congr Plant Tissue and Cell Culture, Univ Calgary, Calgary, pp 381-392 Maliga P, Menczet L, Sidorov V~ Marten L~ Cseplo A, Medgyesy P~ Dung TM~ Lazar G, Nagy F (1982) In: Vasil [K, Scowcroft WR, Fcey KJ (eds) Plant improvement and somatic cell genetics. Academic Press, New York, pp 221-237 Mathur AK, Ganapathy PS, John BM (1980) Z Pflanzenphysiol 99:287-294 Murashige T, Skoog F (1962) Physiol Plant 15 : 473-/497 Nabors MW, Daniels A, Plant Sei L e t t 4 : I 5 5 - 1 5 9
Nadotny
L,
Brown
C (1975)
Nabors MW~ Gibbs SE, Becnstein CS, Meis ME (1980) Z Pflanzenphysiol 97:13-17 Rains DW, Croughan TP, Stavarek S3 (1980) In: Rains DW, Valentine RC, Ho]laender A (eds) Genetic engineering of osmoregulation: impact on plant productivity for food, chemicals and energy. Plenum Press, New York, pp 279-292 Rangan TS, Vasil IK (1983) Ann Bat 52:59-64 Smith MK, 126-128
McComb
JA
(1983) Plant
Yea AR (1983) Physio] Plant 58:214-222
Cell
Reports
2:
