PlantCell Reports
Plant Cell Reports (1981) 1:48-51
© Springer-Verlag 1981
Isolation of Sodium Dependent Variants from Haploid Soybean Cell Culture Zhou Jia-Ping*, E. Jill Roth, William Terzaghi, and Karl G. Lark Department of Biology, University, Salt Lake City, UT 84112, USA Received August 6, 1981
ABSTRACT
i s o l a t i o n of Salt-Requiring Variants
Following u l t r a v i o l e t mutagenesis, we have isolated two Na+ dependent variants from haploid suspension cultures of soybean c e l l s . No selection was used in the i s o l a t i o n procedure. These cells cannot qrow in l mM Na+ but can grow in 17 mM Na+. The strains also grow in 33 or 65 mM Na+, concentrations in which the parent cell l i n e dies. [22Na] was used to measure the accumulation of Na+ by parent and variant cell l i n e s . Two- to t h r e e - f o l d less Na+ is retained by the variant cell lines.
Modified feeder plates using s t e r i l e paper f i l t e r s were prepared (Weber and Lark, 1979). One ml of SIN cells (concentration 0.8 x lO 5 to l x lO 6 c e l l s / m l ) was spread evenly on the paper f i l t e r . Ten plates were used in each experiment. Cells were then treated with 2000 erg/mm2 u l t r a v i o l e t l i g h t (Weber and Lark, 1980). A 15-W General E l e c t r i c germicidal lamp was used as a source of u l t r a v i o l e t l i g h t . The u l t r a v i o l e t f l u x at the surface of the cells was measured with an u l t r a v i o l e t i n t e n s i t y meter (BLAK-RAY). After i r r a d i a t i o n , surviving cells (usually 0.5% to 1.0%; Weber and Lark, 1980) were grown at 33oc ( e i t h e r in B5C or in B5C with 16 mM NAN03) for another six days in order to form microcolonies. To insure continuous growth, the paper f i l t e r s supporting the p l a t i n g cells were transferred to fresh medium and fresh feeder cells every two days. After three transfers (six days) the plated cells were grown on medium without feeder c e l l s . When colonies were larger (lO to 14 days l a t e r ) they were retested on B5C or B5C with 16 mM NaNO3. Later, they were tested on B5C with 32 mM NaNO3 or B5C with 64 mM NaNO3 .
INTRODUCTION The i s o l a t i o n of Na t o l e r a n t plants and plant c e l l s in tissue culture has been the subject of research in several laboratories (Abel and MacKenzie, 1964; Dix and Street, 1975; Nabors et a l . , 1975; Nabors et a l . , 1979). Such lines i f regenerated into s a l t - t o l e r a n t plants could prove of agronomic importance and also would be valuable tools in studies of the c e l l u l a r mechanism of s a l t tolerance. Preliminary results have indicated that s a l t - t o l e r a n t lines are also s a l t dependent. In the work described below, we have isolated Na dependent v a r i e t i e s of soybean using newly developed p l a t i n g and mutagenesis techniques (Weber and Lark, 1979; Weber and Lark, 1980). This was possible because the frequency of h e r i t a b l e variants produced by u l t r a v i o l e t mutagenesis is high and s a l t dependent variants can be found among clones which survive u l t r a v i o l e t treatment. An i n t e r e s t i n g aspect of the two Na-dependent variants described below is that both can t o l e r a t e Na concentrations in which the wild type dies. MATERIALS AND METHODS Haploid suspension cultures of soybean (SIN) described previously (SIN; Weber and Lark, 1980) were maintained at 33°C in the dark as 50 ml batch suspensions in 250 ml Delong flasks. They were grown on a gyratory shaker (120 rpm) in BBC medium [B5 medium (Gamborg et a l . , 1968) containing l ~g/ml 2,4D and 0.2% casein hydrolysate] with d i l u t i o n s to maintain the cell concentration between 5 x IO s and 2 x lO 6 c e l l s / m l . Cell numbers were counted in a Neubauer hemacytometer a f t e r converting c e l l suspensions into protoplasts. Cell v i a b i l i t y was determined cytol o g i c a l l y by use of the v i t a l s t a i n , trypan blue (Weber and Lark, 1979).
Comparison of Growth Between Variants and SIN Wild Type Suspension cultures of salt-dependent variants, as well as SIN wild type, were grown in d i f f e r e n t concentrations of NaNO3. Every two days the packed volume was measured (Limberg et a l . , 1979). Cultures were d i l u t e d i f the packed volume was higher than 0.75 ml of c e l l s per 2.5 ml of cell suspension. Growth of d i f f e r e n t cell lines was compared. Determination of Na Uptake Cells were grown in B5 with 16 mM NaNO3. One experiment used SIN grown in B5. Two m i l l i l i t e r s of I0% (v/v) cultures were placed in p l a s t i c v i a l s (2.5 x 6 cm). For each c u l t u r e , samples were treated with 1.3, 17.5, 33.3, or 65.3 mM Na+ containing 0.2 ~Ci of [22Na]. These were shaken f o r l h at 33°C. At the end of l h, the cultures were centrifuged and the supernatant removed. The cells were then resuspended in 3 ml 500 ~M NaNO3 and again c e n t r i fuged. This was repeated f i v e times. After the l a s t wash, NaNO3 was added to give a t o t a l volume of l ml. This was transferred to 14 mls of hydrofluor s c i n t -
This work was supported by a grant from National Institute of Environmental Health Sciences (01498) to K.G.L. * Present address: Department of Botany, The State University of Rutgers, New Brunswick, NJ 08903, USA
0721-7714/81/0001/0048/$
01.00
49 i l l a t o r (purchased from New England Nuclear) and counted in a s c i n t i l l a t i o n counter. As seen in Figure l an i n s i g n i f i c a n t amount of [22Na] was removed from c e l l s a f t e r the f o u r t h c e n t r i f u g a t i o n . An experiment was also c a r r i e d out in which c e l l s were c e n t r i f u g e d once to remove the bulk o f [22Na] and then r a p i d l y washed on f i l t e r s . The r e s u l t (not shown) was i d e n t i c a l to the r e s u l t s o b t a i n e d by repeated c e n t r i f u g a t i o n .
3x1041i~104~)N 3x103 13.. 0 03 Z
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-
t r a n s f e r r e d to B5C or B5C + 16 mM NaNO3. From the number of c o l o n i e s screened, the estimated frequency o f the s a l t - d e p e n d e n t v a r i a n t s a f t e r u l t r a v i o l e t t r e a t m e n t was 2 x lO -3. The SIN-204 v a r i a n t was obtained by growing ultraviolet-irradiated c e l l s on B5C f o r two days, and then t r a n s f e r r i n g them to B5C + 16 mM NaNO3 f o r another s i x days. A f t e r lO to 14 days, c o l o n i e s were screened f o r i n a b i l i t y to grow on BSC. SIN-204 was one o f these. The e s t i m a t e d frequency o f Na dependent v a r i a n t s was 3 x lO: 3. On p l a t e s , the two v a r i a n t s grew w e l l in B5C + 16 mM NaNOs, B5C + 32 mM NAN03 and B5C + 64 mM NAN03. Upon repeated t r a n s f e r on feeder p l a t e s , 121 and 204 maintained t h e i r s a l t dependence. Both l i n e s died a t 33oc i f t r a n s f e r r e d to B5C medium. Both v a r i a n t s grew w e l l i f 16 or 32 mM NaCl replaced NAN03. The two v a r i a n t s were grown in suspension c u l t u r e to study the e f f e c t of added s a l t on growth. Cultures were grown in B5C w i t h or w i t h o u t added 16 mM NAN03. A f t e r lO days (lO g e n e r a t i o n s ) each c u l t u r e was washed by c e n t r i f u g a t i o n and d i v i d e d . One p o r t i o n was suspended in B5C, the o t h e r in BSC plus added NaNO3. Cell growth was measured as packed volume and c e l l suspensions were maintained by dilution at a d e n s i t y between 5 x IO s and 2 x lO ~. Figure 2 presents the r e s u l t s ( c o r r e c t e d f o r the cumulative d i l u t i o n s ) . 105
2
S121
102 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ':':':':~"
'"":':':':':':':':!:!:i:!:!:!:!
$204
~6--16,/'
16~16 e-
10 4
3o
I
I
I
I
I
1
2
3
4
5
Number
of
:'~10 3
Centrifugations
Figure I . Removal o f R a d i o a c t i v e [22Na] From SIN C e l l s by C e n t r i f u g a t i o n and Resuspension. SIN c e l l s were grown on B5 medium as described in the t e x t . Three ml samples (0.16 to 0.19 ml o f packed c e l l s ) were mixed w i t h [22Na] medium and incubated f o r l h as described in the t e x t . A f t e r t h i s they were washed by c e n t r i f u g a t i o n and a f t e r each resuspension 0.2 ml o f s u p e r n a t a n t was removed f o r c o u n t i n g . The f i n a l p e l l e t was also resuspended in a volume equal to the supernatant and 0.2 ml counted. (e) SIN i n cubated w i t h r a d i o a c t i v e [22Na]; (o) R a d i o a c t i v e medium w i t h o u t c e l l s ; (&) R a d i o a c t i v e medium plus h e a t - k i l l e d SIN c e l l s ; [ U B I ] Radioactivity o f l a b e l e d c e l l p e l l e t ; ::::::::::::::::::::::::::::::: R a d i o a c t i v i t y of heat-killed cell pellet. SIN heated c e l l s were t r e a t e d f o r 20 min a t 60°C. RESULTS Sodium S a l t Tolerance o f Soybean NAN03 and NaCl t o l e r a n c e o f SIN - - On p l a t e s c o l o n i e s arose w i t h equal e f f i c i e n c y of p l a t i n g ( e . o . p . ) a t Na c o n c e n t r a t i o n s equal t o , o r less than 64 mM. However, l o n g - t e r m growth as evidenced by s i z e and c o l o r became p r o g r e s s i v e l y worse above 16 mM NAN03. I s o l a t i o n o f the V a r i a n t s Using lO 7 c e l l s , two v a r i a n t s were s e l e c t e d which grew on BSC + 16 mM NaNO3 but not on B5C. One o f these was SIN-121. The SIN-121 v a r i a n t was obt a i n e d by growing u l t r a v i o l e t - t r e a t e d c e l l s on B5C f o r e i g h t days. A f t e r another nine days of ~rowth on 16 mM NAN03, p o r t i o n s o f the mature c o l o n i e s were
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Days grown Figure 2. Growth of Salt-Dependent V a r i a n t s (SIN121, SIN-204~ in Suspension Cultures in the Presence and Absence of Added NaNOm. The two suspension c u l t u r e s were grown in B5C ( [ [ ~ ] ] ) or B5C plus 16 mM NAN03 (-I~). Cultures grown in these media were washed (+) and resuspended in media w i t h or w i t h o u t added s a l t . Days o f growth p r i o r to the change in media are i n d i c a t e d by a negative s c a l e . Growth was measured as packed volume and c u l t u r e s d i l u t e d when necessary in o r d e r to maint a i n a c e l l d e n s i t y between 5 x IO s and 2 x lO 6 c e l l s per m i l l i l i t e r . The volumes on the o r d i n a t e have been c o r r e c t e d f o r d i l u t i o n . A lag in growth a f t e r c e n t r i f u g a t i o n (arrow) is o f t e n observed i f c e l l s are washed e x t e n s i v e l y to remove media. (A f o u r - to s i x - d a y recovery p e r i o d is not unusual.) I t can be seen t h a t SIN-121 or -204 c e l l s d i e in the absence of s a l t but can recover t h e i r normal growth r a t e i f NaNO3 is added a f t e r lO days. In c o n t r a s t to the w i l d + t y p e SIN c e l l s , both c u l t u r e s were t o l e r a n t of Na up to c o n c e n t r a t i o n s
50 of 32 or 64 mM NAN03. Figure 3 compares the growth of these three cell lines in B5C+ 16, 32 or 64 mM NAN03. As can be seen, neither SIN-121 or SIN-204 are i n h i b i t e d by the added 32 or 64 mM NAN03, whereas SIN eventually dies in the higher s a l t .
times that obtained under s i m i l a r mutagenic conditions for a phenotype a t t r i b u t e d to a single genetic locus (Weber and Lark, 1980). This suggests that i f mutation is involved, the phenotype is c o n t r o l l e d by a large mutable t a r g e t , possibly several l o c i . Table I.
16mM
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I
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Days grown Figure 3. Growth of Salt-Dependent Variants, SIN-121, SIN-204 and Wild Type SIN Cells in SusPension in the Presence of Added 16 mM, 32 mM and 64 mM
NaNO~. SIN-121 ( - - o - - ) , SIN-204 ( - . . A . - . ) and SIN ( - - e - - ) were grown in B5C plus 16 mM NAN%, 32 mM NaNO3 or 64 mM NaNO3. Cultures had been pregrown in these media for 18 days before the period shown in the f i g u r e . Growth was measured as packed volume and cultures d i l u t e d to maintain a cell density between 5 x lO 5 and 2 x lO 6 cells per ml. The volumes on the ordinate have been corrected for d i l u t i o n . Mechanisms of Na Dependence-Na Tolerance In an e f f o r t to characterize the Na dependence of the SIN-121 and -204 variants, we compared the uptake of [22Na] with that of wild type. Cells were suspended in media containing a constant amount of radioactive Na+ (see Materials and Methods) and a f t e r one hour washed free of radioactive Na (as described in Materials and Methods). Both variants retained much less Na (between I / 4 and I / 3 than the wild type,parental c e l l l i n e (Table l ) . I t can be seen that NaT is excluded from w i l d type and variant c e l l l i n e s , but that the variants exclude Na+ more e f f e c t i v e l y than the parental cell l i n e . Since Na+ was not retained by h e a t - k i l l e d cells (Figure l ) , the retention of Na+ during washing cannot be the r e s u l t of passive absorption to the c e l l surface or to cell membranes. DISCUSSION The a b i l i t y to obtain high frequencies of variant clones using new p l a t i n g and u l t r a v i o l e t mutagenesis techniques (Weber and Lark, 1980) has allowed us to i s o l a t e Na-dependent variants nons e l e c t i v e l y . These variants have been maintained and passaged under non-selective conditions, i . e . , conditions under which the wild type also can grow. The fact that both variants also are s a l t t o l e r a n t suggests that the two phenotypes, s a l t dependence and s a l t tolerance, could be the r e s u l t of a single genetic change (or mutation). The frequency at which the variants occurred (2 to 3 x lO -3) is about lO
Sodium Uptake by SIN and Salt-Dependent Variants
Cell Type
(a)
(b)
(c)
(d)
SIN SIN-121 SIN-204
1.3 mM
2.16
5,900 2,100 2,200
0.036 mM O . O l 3 1mM O . O l 3 1mM
SIN SIN-121 SIN-204
17.3 mM
2.10
9,920 3,345 2,380
SIN SIN-121 SIN-204
33.3 mM
2.00
7,700 2,450 2,250
1.28 .41 .37
mM mM mM
SIN SIN-121 SIN-204
65.3 mM
1.75
13,130 4,560 4,145
4.90 1.70 1.55
mM mM mM
.817 .276 .196
mM mM mM
(a) (b)
Sodium concentration in the medium. Radioactivity in the medium, lO 5 counts per minute per m i l l i l i t e r of medium. (c) Radioactivity per cell as counts per m i l l i l i t e r of packed c e l l s . (d) Sodium concentration in washed c e l l s . Cells were incubated for one hour in B5 media containing radioactive [22Na] and d i f f e r e n t concent r a t i o n s of NAN03. B5 medium contains ca. 1.3 mM NaNO3. In other media 16, 32 or 64 mM NaNO3 was added. Cells were incubated and washed as described in Materials and Methods. We have been unsuccessful in i s o l a t i n g spontaneous revertants to the parental phenotype (thus cells able to grow in low Na medium do not appear to arise with a frequency of I / l O 6 or higher). However, we have observed such revertants a f t e r u l t r a v i o l e t treatment (unpublished data). These observations suggest that the variants are the r e s u l t of a stable h e r i t a b l e change and could be the r e s u l t of genetic mutation. An i n t e r e s t i n g phenotype common to both variants is that they are osmotically f r a g i l e in the presence of high s a l t . As a consequence, i t is d i f f i c u l t or impossible to p u r i f y the variants by conversion to isolated protoplasts, regenerating these into c e l l clones. The simultaneous appearance of two phenotypes, i . e . , s a l t dependence and s a l t tolerance, could be explained in part by the observation that both variants exclude Na more e f f i c i e n t l y than wild type c e l l s . Na dependence could be explained i f we assume that soybean cells require a certain i n t r a c e l l u l a r concentration of Na+ for growth. Below this concent r a t i o n they cannot survive and Na dependent variants only achieve this concentration at higher external Na concentrations. Dependent variants may also e x h i b i t tolerance to Na, i f higher concentrations do not increase the i n t r a c e l l u l a r concentration to the same level as wild type (although the resistance to 65.3 mM Na suggests that they do t o l e r a t e higher i n t r a c e l l u l a r Na concentrations). REFERENCES Abel A, MacKenzie AJ (1964) Crop Science 4:157-161 Dix PJ, Street HE (1975) Plant Science Letters 5: 231-237
51 Gamborg OL, M i l l e r RA, Ojima K (1968) Exp Cell Res 50:151-158 Limberg M, Cress D, Lark KG (1979) Plant Physiol 63: 718-721 Nabors MW, Daniels A, Nadolny L, Brown C (1975) Plant Science Letters 4:155-159
Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1979) Z Pflanzenphysiol 97:13-17 Weber G, Lark KG (1979) Theor Appl Genet 55:81-86 Weber G, Lark KG (1980) Genetics 96:213-222
Plant Cell Reports (1981) 1:48-51
© Springer-Verlag 1981
Isolation of Sodium Dependent Variants from Haploid Soybean Cell Culture Zhou Jia-Ping*, E. Jill Roth, William Terzaghi, and Karl G. Lark Department of Biology, University, Salt Lake City, UT 84112, USA Received August 6, 1981
ABSTRACT
i s o l a t i o n of Salt-Requiring Variants
Following u l t r a v i o l e t mutagenesis, we have isolated two Na+ dependent variants from haploid suspension cultures of soybean c e l l s . No selection was used in the i s o l a t i o n procedure. These cells cannot qrow in l mM Na+ but can grow in 17 mM Na+. The strains also grow in 33 or 65 mM Na+, concentrations in which the parent cell l i n e dies. [22Na] was used to measure the accumulation of Na+ by parent and variant cell l i n e s . Two- to t h r e e - f o l d less Na+ is retained by the variant cell lines.
Modified feeder plates using s t e r i l e paper f i l t e r s were prepared (Weber and Lark, 1979). One ml of SIN cells (concentration 0.8 x lO 5 to l x lO 6 c e l l s / m l ) was spread evenly on the paper f i l t e r . Ten plates were used in each experiment. Cells were then treated with 2000 erg/mm2 u l t r a v i o l e t l i g h t (Weber and Lark, 1980). A 15-W General E l e c t r i c germicidal lamp was used as a source of u l t r a v i o l e t l i g h t . The u l t r a v i o l e t f l u x at the surface of the cells was measured with an u l t r a v i o l e t i n t e n s i t y meter (BLAK-RAY). After i r r a d i a t i o n , surviving cells (usually 0.5% to 1.0%; Weber and Lark, 1980) were grown at 33oc ( e i t h e r in B5C or in B5C with 16 mM NAN03) for another six days in order to form microcolonies. To insure continuous growth, the paper f i l t e r s supporting the p l a t i n g cells were transferred to fresh medium and fresh feeder cells every two days. After three transfers (six days) the plated cells were grown on medium without feeder c e l l s . When colonies were larger (lO to 14 days l a t e r ) they were retested on B5C or B5C with 16 mM NaNO3. Later, they were tested on B5C with 32 mM NaNO3 or B5C with 64 mM NaNO3 .
INTRODUCTION The i s o l a t i o n of Na t o l e r a n t plants and plant c e l l s in tissue culture has been the subject of research in several laboratories (Abel and MacKenzie, 1964; Dix and Street, 1975; Nabors et a l . , 1975; Nabors et a l . , 1979). Such lines i f regenerated into s a l t - t o l e r a n t plants could prove of agronomic importance and also would be valuable tools in studies of the c e l l u l a r mechanism of s a l t tolerance. Preliminary results have indicated that s a l t - t o l e r a n t lines are also s a l t dependent. In the work described below, we have isolated Na dependent v a r i e t i e s of soybean using newly developed p l a t i n g and mutagenesis techniques (Weber and Lark, 1979; Weber and Lark, 1980). This was possible because the frequency of h e r i t a b l e variants produced by u l t r a v i o l e t mutagenesis is high and s a l t dependent variants can be found among clones which survive u l t r a v i o l e t treatment. An i n t e r e s t i n g aspect of the two Na-dependent variants described below is that both can t o l e r a t e Na concentrations in which the wild type dies. MATERIALS AND METHODS Haploid suspension cultures of soybean (SIN) described previously (SIN; Weber and Lark, 1980) were maintained at 33°C in the dark as 50 ml batch suspensions in 250 ml Delong flasks. They were grown on a gyratory shaker (120 rpm) in BBC medium [B5 medium (Gamborg et a l . , 1968) containing l ~g/ml 2,4D and 0.2% casein hydrolysate] with d i l u t i o n s to maintain the cell concentration between 5 x IO s and 2 x lO 6 c e l l s / m l . Cell numbers were counted in a Neubauer hemacytometer a f t e r converting c e l l suspensions into protoplasts. Cell v i a b i l i t y was determined cytol o g i c a l l y by use of the v i t a l s t a i n , trypan blue (Weber and Lark, 1979).
Comparison of Growth Between Variants and SIN Wild Type Suspension cultures of salt-dependent variants, as well as SIN wild type, were grown in d i f f e r e n t concentrations of NaNO3. Every two days the packed volume was measured (Limberg et a l . , 1979). Cultures were d i l u t e d i f the packed volume was higher than 0.75 ml of c e l l s per 2.5 ml of cell suspension. Growth of d i f f e r e n t cell lines was compared. Determination of Na Uptake Cells were grown in B5 with 16 mM NaNO3. One experiment used SIN grown in B5. Two m i l l i l i t e r s of I0% (v/v) cultures were placed in p l a s t i c v i a l s (2.5 x 6 cm). For each c u l t u r e , samples were treated with 1.3, 17.5, 33.3, or 65.3 mM Na+ containing 0.2 ~Ci of [22Na]. These were shaken f o r l h at 33°C. At the end of l h, the cultures were centrifuged and the supernatant removed. The cells were then resuspended in 3 ml 500 ~M NaNO3 and again c e n t r i fuged. This was repeated f i v e times. After the l a s t wash, NaNO3 was added to give a t o t a l volume of l ml. This was transferred to 14 mls of hydrofluor s c i n t -
This work was supported by a grant from National Institute of Environmental Health Sciences (01498) to K.G.L. * Present address: Department of Botany, The State University of Rutgers, New Brunswick, NJ 08903, USA
0721-7714/81/0001/0048/$
01.00
49 i l l a t o r (purchased from New England Nuclear) and counted in a s c i n t i l l a t i o n counter. As seen in Figure l an i n s i g n i f i c a n t amount of [22Na] was removed from c e l l s a f t e r the f o u r t h c e n t r i f u g a t i o n . An experiment was also c a r r i e d out in which c e l l s were c e n t r i f u g e d once to remove the bulk o f [22Na] and then r a p i d l y washed on f i l t e r s . The r e s u l t (not shown) was i d e n t i c a l to the r e s u l t s o b t a i n e d by repeated c e n t r i f u g a t i o n .
3x1041i~104~)N 3x103 13.. 0 03 Z
103
--
-- \~L~
o,I
3
x
-
t r a n s f e r r e d to B5C or B5C + 16 mM NaNO3. From the number of c o l o n i e s screened, the estimated frequency o f the s a l t - d e p e n d e n t v a r i a n t s a f t e r u l t r a v i o l e t t r e a t m e n t was 2 x lO -3. The SIN-204 v a r i a n t was obtained by growing ultraviolet-irradiated c e l l s on B5C f o r two days, and then t r a n s f e r r i n g them to B5C + 16 mM NaNO3 f o r another s i x days. A f t e r lO to 14 days, c o l o n i e s were screened f o r i n a b i l i t y to grow on BSC. SIN-204 was one o f these. The e s t i m a t e d frequency o f Na dependent v a r i a n t s was 3 x lO: 3. On p l a t e s , the two v a r i a n t s grew w e l l in B5C + 16 mM NaNOs, B5C + 32 mM NAN03 and B5C + 64 mM NAN03. Upon repeated t r a n s f e r on feeder p l a t e s , 121 and 204 maintained t h e i r s a l t dependence. Both l i n e s died a t 33oc i f t r a n s f e r r e d to B5C medium. Both v a r i a n t s grew w e l l i f 16 or 32 mM NaCl replaced NAN03. The two v a r i a n t s were grown in suspension c u l t u r e to study the e f f e c t of added s a l t on growth. Cultures were grown in B5C w i t h or w i t h o u t added 16 mM NAN03. A f t e r lO days (lO g e n e r a t i o n s ) each c u l t u r e was washed by c e n t r i f u g a t i o n and d i v i d e d . One p o r t i o n was suspended in B5C, the o t h e r in BSC plus added NaNO3. Cell growth was measured as packed volume and c e l l suspensions were maintained by dilution at a d e n s i t y between 5 x IO s and 2 x lO ~. Figure 2 presents the r e s u l t s ( c o r r e c t e d f o r the cumulative d i l u t i o n s ) . 105
2
S121
102 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ':':':':~"
'"":':':':':':':':!:!:i:!:!:!:!
$204
~6--16,/'
16~16 e-
10 4
3o
I
I
I
I
I
1
2
3
4
5
Number
of
:'~10 3
Centrifugations
Figure I . Removal o f R a d i o a c t i v e [22Na] From SIN C e l l s by C e n t r i f u g a t i o n and Resuspension. SIN c e l l s were grown on B5 medium as described in the t e x t . Three ml samples (0.16 to 0.19 ml o f packed c e l l s ) were mixed w i t h [22Na] medium and incubated f o r l h as described in the t e x t . A f t e r t h i s they were washed by c e n t r i f u g a t i o n and a f t e r each resuspension 0.2 ml o f s u p e r n a t a n t was removed f o r c o u n t i n g . The f i n a l p e l l e t was also resuspended in a volume equal to the supernatant and 0.2 ml counted. (e) SIN i n cubated w i t h r a d i o a c t i v e [22Na]; (o) R a d i o a c t i v e medium w i t h o u t c e l l s ; (&) R a d i o a c t i v e medium plus h e a t - k i l l e d SIN c e l l s ; [ U B I ] Radioactivity o f l a b e l e d c e l l p e l l e t ; ::::::::::::::::::::::::::::::: R a d i o a c t i v i t y of heat-killed cell pellet. SIN heated c e l l s were t r e a t e d f o r 20 min a t 60°C. RESULTS Sodium S a l t Tolerance o f Soybean NAN03 and NaCl t o l e r a n c e o f SIN - - On p l a t e s c o l o n i e s arose w i t h equal e f f i c i e n c y of p l a t i n g ( e . o . p . ) a t Na c o n c e n t r a t i o n s equal t o , o r less than 64 mM. However, l o n g - t e r m growth as evidenced by s i z e and c o l o r became p r o g r e s s i v e l y worse above 16 mM NAN03. I s o l a t i o n o f the V a r i a n t s Using lO 7 c e l l s , two v a r i a n t s were s e l e c t e d which grew on BSC + 16 mM NaNO3 but not on B5C. One o f these was SIN-121. The SIN-121 v a r i a n t was obt a i n e d by growing u l t r a v i o l e t - t r e a t e d c e l l s on B5C f o r e i g h t days. A f t e r another nine days of ~rowth on 16 mM NAN03, p o r t i o n s o f the mature c o l o n i e s were
~
10 2
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10
--
0
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"6
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I I I -8
0
I I I ] I I 8
16
24
I 32
"
I I I -8
0
I q I I I 8
16
24
I I 32
Days grown Figure 2. Growth of Salt-Dependent V a r i a n t s (SIN121, SIN-204~ in Suspension Cultures in the Presence and Absence of Added NaNOm. The two suspension c u l t u r e s were grown in B5C ( [ [ ~ ] ] ) or B5C plus 16 mM NAN03 (-I~). Cultures grown in these media were washed (+) and resuspended in media w i t h or w i t h o u t added s a l t . Days o f growth p r i o r to the change in media are i n d i c a t e d by a negative s c a l e . Growth was measured as packed volume and c u l t u r e s d i l u t e d when necessary in o r d e r to maint a i n a c e l l d e n s i t y between 5 x IO s and 2 x lO 6 c e l l s per m i l l i l i t e r . The volumes on the o r d i n a t e have been c o r r e c t e d f o r d i l u t i o n . A lag in growth a f t e r c e n t r i f u g a t i o n (arrow) is o f t e n observed i f c e l l s are washed e x t e n s i v e l y to remove media. (A f o u r - to s i x - d a y recovery p e r i o d is not unusual.) I t can be seen t h a t SIN-121 or -204 c e l l s d i e in the absence of s a l t but can recover t h e i r normal growth r a t e i f NaNO3 is added a f t e r lO days. In c o n t r a s t to the w i l d + t y p e SIN c e l l s , both c u l t u r e s were t o l e r a n t of Na up to c o n c e n t r a t i o n s
50 of 32 or 64 mM NAN03. Figure 3 compares the growth of these three cell lines in B5C+ 16, 32 or 64 mM NAN03. As can be seen, neither SIN-121 or SIN-204 are i n h i b i t e d by the added 32 or 64 mM NAN03, whereas SIN eventually dies in the higher s a l t .
times that obtained under s i m i l a r mutagenic conditions for a phenotype a t t r i b u t e d to a single genetic locus (Weber and Lark, 1980). This suggests that i f mutation is involved, the phenotype is c o n t r o l l e d by a large mutable t a r g e t , possibly several l o c i . Table I.
16mM
..32rnMz //~/~t f'. ....
I
64m M
.•103 -
/ ....
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27
9
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J
18 20 22 24 26
Irllll[ll [rltllllllil 18 20 22 24 26 18 20 22 24 26
Days grown Figure 3. Growth of Salt-Dependent Variants, SIN-121, SIN-204 and Wild Type SIN Cells in SusPension in the Presence of Added 16 mM, 32 mM and 64 mM
NaNO~. SIN-121 ( - - o - - ) , SIN-204 ( - . . A . - . ) and SIN ( - - e - - ) were grown in B5C plus 16 mM NAN%, 32 mM NaNO3 or 64 mM NaNO3. Cultures had been pregrown in these media for 18 days before the period shown in the f i g u r e . Growth was measured as packed volume and cultures d i l u t e d to maintain a cell density between 5 x lO 5 and 2 x lO 6 cells per ml. The volumes on the ordinate have been corrected for d i l u t i o n . Mechanisms of Na Dependence-Na Tolerance In an e f f o r t to characterize the Na dependence of the SIN-121 and -204 variants, we compared the uptake of [22Na] with that of wild type. Cells were suspended in media containing a constant amount of radioactive Na+ (see Materials and Methods) and a f t e r one hour washed free of radioactive Na (as described in Materials and Methods). Both variants retained much less Na (between I / 4 and I / 3 than the wild type,parental c e l l l i n e (Table l ) . I t can be seen that NaT is excluded from w i l d type and variant c e l l l i n e s , but that the variants exclude Na+ more e f f e c t i v e l y than the parental cell l i n e . Since Na+ was not retained by h e a t - k i l l e d cells (Figure l ) , the retention of Na+ during washing cannot be the r e s u l t of passive absorption to the c e l l surface or to cell membranes. DISCUSSION The a b i l i t y to obtain high frequencies of variant clones using new p l a t i n g and u l t r a v i o l e t mutagenesis techniques (Weber and Lark, 1980) has allowed us to i s o l a t e Na-dependent variants nons e l e c t i v e l y . These variants have been maintained and passaged under non-selective conditions, i . e . , conditions under which the wild type also can grow. The fact that both variants also are s a l t t o l e r a n t suggests that the two phenotypes, s a l t dependence and s a l t tolerance, could be the r e s u l t of a single genetic change (or mutation). The frequency at which the variants occurred (2 to 3 x lO -3) is about lO
Sodium Uptake by SIN and Salt-Dependent Variants
Cell Type
(a)
(b)
(c)
(d)
SIN SIN-121 SIN-204
1.3 mM
2.16
5,900 2,100 2,200
0.036 mM O . O l 3 1mM O . O l 3 1mM
SIN SIN-121 SIN-204
17.3 mM
2.10
9,920 3,345 2,380
SIN SIN-121 SIN-204
33.3 mM
2.00
7,700 2,450 2,250
1.28 .41 .37
mM mM mM
SIN SIN-121 SIN-204
65.3 mM
1.75
13,130 4,560 4,145
4.90 1.70 1.55
mM mM mM
.817 .276 .196
mM mM mM
(a) (b)
Sodium concentration in the medium. Radioactivity in the medium, lO 5 counts per minute per m i l l i l i t e r of medium. (c) Radioactivity per cell as counts per m i l l i l i t e r of packed c e l l s . (d) Sodium concentration in washed c e l l s . Cells were incubated for one hour in B5 media containing radioactive [22Na] and d i f f e r e n t concent r a t i o n s of NAN03. B5 medium contains ca. 1.3 mM NaNO3. In other media 16, 32 or 64 mM NaNO3 was added. Cells were incubated and washed as described in Materials and Methods. We have been unsuccessful in i s o l a t i n g spontaneous revertants to the parental phenotype (thus cells able to grow in low Na medium do not appear to arise with a frequency of I / l O 6 or higher). However, we have observed such revertants a f t e r u l t r a v i o l e t treatment (unpublished data). These observations suggest that the variants are the r e s u l t of a stable h e r i t a b l e change and could be the r e s u l t of genetic mutation. An i n t e r e s t i n g phenotype common to both variants is that they are osmotically f r a g i l e in the presence of high s a l t . As a consequence, i t is d i f f i c u l t or impossible to p u r i f y the variants by conversion to isolated protoplasts, regenerating these into c e l l clones. The simultaneous appearance of two phenotypes, i . e . , s a l t dependence and s a l t tolerance, could be explained in part by the observation that both variants exclude Na more e f f i c i e n t l y than wild type c e l l s . Na dependence could be explained i f we assume that soybean cells require a certain i n t r a c e l l u l a r concentration of Na+ for growth. Below this concent r a t i o n they cannot survive and Na dependent variants only achieve this concentration at higher external Na concentrations. Dependent variants may also e x h i b i t tolerance to Na, i f higher concentrations do not increase the i n t r a c e l l u l a r concentration to the same level as wild type (although the resistance to 65.3 mM Na suggests that they do t o l e r a t e higher i n t r a c e l l u l a r Na concentrations). REFERENCES Abel A, MacKenzie AJ (1964) Crop Science 4:157-161 Dix PJ, Street HE (1975) Plant Science Letters 5: 231-237
51 Gamborg OL, M i l l e r RA, Ojima K (1968) Exp Cell Res 50:151-158 Limberg M, Cress D, Lark KG (1979) Plant Physiol 63: 718-721 Nabors MW, Daniels A, Nadolny L, Brown C (1975) Plant Science Letters 4:155-159
Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1979) Z Pflanzenphysiol 97:13-17 Weber G, Lark KG (1979) Theor Appl Genet 55:81-86 Weber G, Lark KG (1980) Genetics 96:213-222
