Comparative salt responses of callus cultures of Vigna radiata L. Wilczek to various osmotic and ionic stresses

J Plant Physiol. Vol. 141. pp. 120-124 {1992}

Comparative Salt Responses of Callus Cultures of Vigna radiata (L.) Wilczek to Various Osmotic and Ionic Stresses ANJU GULATI

and PAWAN K. JAIWAL*

Department of Bio-Sciences, M. D. University, Rohtak-124001, India * To whom all correspondence should be addressed Received September 19, 1991 . Accepted June 25,1992

Summary

Leaf derived callus cultures of Vigna radiata (1.) Wilczek cv. K-851 were grown on modified PC-L2 medium salinized with various concentrations of NaCl, KCl, Na 2S04 or their mixture and mannitol to study the effect of osmotic and different types of salt stresses on callus growth and ion accumulation. The callus responded differently with stress and salt types. Callus growth was inhibited more severely with Na2S04, followed by KCl, salt mixture, NaCl and mannitol. A correlation between growth inhibition and the concentration of inorganic solutes in callus tissue was noted. Accumulation of Na+ in callus was accompanied by loss ofK+ at all levels of Na 2 S04, NaCl and salt mixture. Severe inhibition of cell growth with Na 2 S04 was due to more uptake of Na+ by cells under Na 2 S04 medium than any other salt and their mixture. S04 2 ion content in Na 2 S04 grown callus was 60-70 times higher than Na+. Similarly, K+ and Cl- contents of KCl callus were greater than Na+ and Cl- of NaCl grown callus. A comparison between salt mixture and its constituent different salts shows that some single salts (Na 2S04 and KCl) were more toxic than a salt mixture. An increase in the osmotic stress (by mannitol) inhibited callus growth less severely than any of the salts. This may be due to higher K + content of the cells under mannitol stress than iso-osmotic concentration of any of the salts. Key words: Callus culture, growth, ionic relations, salt and osmotic stress, Vigna radiata. Abbreviations: BAP thaleneacetic acid.

=

6-benzylaminopurine; 2,4-D

Introduction

The decline in. plant productivity due to salinity and drought in saline and arid lands necessitates the development of stress tolerant crops. Of various approaches, cell culture selection for mutants has received increasing attention in recent years (Maliga, 1984). Salt-tolerant cell lines have been isolated in a number of plant species (Rains et al., 1986; Chandler and Thrope, 1986) and tolerant plants have also been regenerated (Nabors et al., 1980, 1982; Tyagi et al., 1981; McHughen and Swartz, 1984; Nabors and Dykes, 1985). Moreover, cell cultures that lack the differentiation and structural integrity of higher plants can also be an ideal system to assess the physiological effects of salt and/or water stress at the cellular level (Lerner, 1985). In most of the salin© 1992 by Gustav Fischer Verlag, Stuttgart

=

2,4-dichlorophenoxyacetic acid; NAA

=

1-naph-

ity resistance studies in tissue culture, NaCl as a single salt has been used to induce salt stress (Sabbah and Tal, 1990). NaCl selection is likely to produce genotypes with resistance to Na+ or Cl- ions, but not to other ions contributing to salinity in certain agricultural situations (Rains et al., 1986). The results obtained with single different types of salt might differ significantly from those obtained when tissues are grown on salt mixtures to which plants may be exposed in nature. Therefore, in the present study, the response of callus cultures of Vigna radiata - a salt sensitive grain-legume- to NaCl, Na 2S04, KCl or their mixture and to mannitol (osmotic stress) with respect to growth and ion accumulation has been studied to acquire knowledge of the role and contribution of ions and osmotic stress to salinity.

Comparative Salt Responses of Callus Cultures 160

Material and Methods

The callus cultures of Vigna radiata (L.) Wilczek var. radiata cv. K-851 were initiated from leaf explants of aseptically grown 7-dayold seedlings on modified PC-LZ (Phillips and Collins, 1979) medium containing 3 % sucrose, 0.7 % agar, 0.5 mgL -1 2,4-D, O.5mgL -1 NAA and 1 mgL -1 BAP. The callus cultures were grown under a 16h photoperiod of cool-white fluorescent light at 25 ± 2°C. After one subculture on the same medium, 250 ± 10 mg actively growing callus was divided into ten pieces (25 ± 2 mg) and cultured on modified PC-L2 medium containing increasing concentrations of NaCl (0, 100, 200, 300 and 400 mol m -3) in Petri dishes (100 mm x 17 mm) using five replicates. The Petri dishes were sealed with parafilm and incubated under the same photoperiod and temperature as for callus cultures. After 4 weeks of culture, callus growth was studied in terms of fresh and dry weights. The callus from Petri dishes was removed and its fresh and dry weights (oven dried at 80°C for 48 h) were determined for each treatment. The effects of Na 2S04 and KCI (equimolar to 100, 200, 300 and 400 mol m -3 NaCI) and mannitol (at concentrations iso-osmotic to 100,200,300 and 400 mol m -3 NaCl) were also investigated in a similar way. Three salts (NaCl, Na 2S04 and KCI) were used in the ratio of 8: 1 : 1 to prepare a salt mixture equimolar to 100, 200, 300 and 400molm- 3 NaCl. This salt mixture was added to modified PC-L2 medium to study its effect on callus growth.

121

140 0---0 NaCI

..-.. C>--O

120

~

Na2S04 KCI Salt mixture

~ c 0

u

100

'0 ;;-

-=

80

0'

'"~ '"

60

a

40

20

50

100

150

200

250

300

350

400

450

Salt concentration (mol.rii 3)

Fig. 1: Effect of NaCl, Na 2S04, KCI and their mixture on dry weight (% of control) of callus cultures of V. radiata cv. K-851.

Estimation ofNa+ and K+ ions

One hundred mg of dried well ground callus tissues was transferred to a 50cm 3 digestion flask to which 1mL 60% PCA, 5mL conc. HN0 3 and O.5mL conc. H 2S0 4 were added. The flasks were heated gently over a hot plate for 5 -10 min until the solution became colourless. The digest was cooled and diluted to 100 mL by addition of double distilled water. Na+ and K+ in the final acid digest extract were determined using an Elico flame photometer. Estimation ofCI- and SO;;2 ions

Chloride was extracted with concentrated HN0 3 and estimated by the mercuric nitrate titration method using diphenyl-carbazone and bromophenol blue mixed indicator (Krishnamurthy and Bhagwat, 1990). Sulphate was also extracted with concentrated HN0 3 and determined by turbidimetry (Tabatabai and Bremner, 1970) using blanks and standards prepared in concentrated HN0 3 • All the experiments were repeated at least twice. Chemicals


Results

1. Effect

0/ saline stress on growth

The dry weights (percent of control) of Vigna radiata callus cultures on modified PC-L2 medium containing increasing concentrations of NaCI, Na2S04, KCI and their mixtures are shown in Fig. 1. The dry weight of callus decreased with increasing concentration of any of the salinizing salts or their mixture in the culture medium. A 100 mol m- 3 salt resulted in a 62 to 94 % decrease in dry weight, depend-

ing on the salt, and at 300 mol m -3 dry weights were only 22 to 10 % of those of non-stressed callus (Fig. 1). Na 2S04 at 50 mol m- 3 stimulated callus growth in comparison to nonstressed callus, while both KCI and NaCI at this concentration had decreased it by 15 % (Fig. 1). Callus dry weight decreased sharply with further increase in concentration of any of the salts in the medium above 50molm- 3• Na2S04 at 200 mol m -3 or higher concentrations was more inhibitory than any of the salts and their mixture. KCI was less inhibitory for callus growth at 100molm- 3 than NaCI and salt mixture, while the reverse was true at higher concentrations (250 - 400 mol m -3). Callus growth was almost completely inhibited by Na 2S04 and KCI at 250 mol m- 3, and by NaCI at 350 mol m- 3• Thus, the inhibitory effect of the three individual salts on callus growth was not equal but increased according to the following order: Na2S04 < KCI < NaCl. Salt mixtures at 50 and 100 mol m -3 were more inhibitory than their constituent salts, while at higher concentrations (200-300 mol m- 3), they were more inhibitory than NaCI, and less inhibitory compared with Na 2S04 and KCl. Salt mixture at 350 mol m -3 had almost completely inhibited the growth of callus. Sodium levels in callus tissue increased with increasing concentrations of NaCI, Na 2S04 and salt mixture in the culture medium. Na+ accumulation was dependent upon the accompanying ions: in Na 2S04 grown callus Na+ was twice as great as in callus grown in NaCl and salt mixture (Fig. 2). However, Na+ accumulated in more or less similar amounts in callus grown on NaCI and salt mixture. In contrast, levels of K + in the callus declined continuously with increasing NaCI, Na2S04 and salt mixture (Fig. 2). On the contrary, K+ ions increased continuously with increase in KCI levels in the culture medium. K + ion accumulation from KCI was greater than that of N a + from N aCI and salt mixture.

ANJU GUl......T! and PAW.....N K, J. . .IWAL

122

aE

4 00

JO 0

SE

,

[

5.'

N,

, I

K'

Na'

0

5.'

, I

I

K'

[[Th

5.'

I

... o

Na+

5.'

5.'

J

, J K' Na

, I, 1 5.'

Na'

K'

SE , I N,' K'

5.'

, I

-20 0 ;;

. E

c

•

c

."

,

15 0

o

u

+1'11

Z

100

0

AaCDEFGHI

ABCD E FGHI

KCl

NaCI

I

A B CDEFGHI

Salt mixturQo

abcde f g

Mannitol

Fig. 2: Effect of NaCI, Na2S0 .., KCI and t heir mixture and mannitol on Na+ and K+ contents of V. radiata callus cultures, A, 0; B, SO; C, 100: D, 200: E, 250: F, 300: G, 350: H ,400: I, 450 mol m- J salt and a, 0; h, 180; c, 360; d, 450; e, 540; f, 630; g, 720 mol m- 3 mannitol. Vertical lines.are the minimum and maximum standard error observed with the data in this figure.

Mannitol (mot. m- l ) 360 44g3 540

180

Table 1: Effect of different salts and their mixture on 0- content

62g

(mmol (kg-' dry weight)} of Vigna radiata caJlus.

no

160 • Salt mixtu re • Mannitol a NaCl Fresh weight ----O r y wl'igllt

//\\

"

~ 120 o

-

\\

- ._

~ 100r-~~~~~~~'--T~~---

.

225±14.1 225± 14.1 497±28.2 564±27.8 535±28.2 564±28.6 564±40.0 535±84.6 620± 16.0 733±21.0

Salt N,Cl 225±14.1 564±29.0 592±84.6 592±84.6 620±20.0

Salt mixture 225±14.1 592±28.2 620±29.6 648±28.2 789±56.4

T he C l- levels of callus tissue i ncreased with increasing concentrations of NaCl, KCI and salt mixture in the medium. CI- accumulation was greater relative to Na + + K + in callus grown in salt mixture t han in NaCI or KCl grown

40

~

ones (Table 1).

20

o

0 50 100 200 400

KCL

Values are the mean of five replicates ± standard error.

<

•

Concentration of salt mmol- J Na2S0..

50

100

200

250

300

350

!tOO

!t50

NaC l jSalt mixturE' (rnol ni 3 )

Fig. 3: Effect of NaCI, salt mixture and mannitol on fresh and dry weiglm (% of control) on callus cultures of V. radiata.

Sulphate in callus tissue increased with increasing concentration of N a2 SO .. in the culture medium. The concentration of 504"2 accumulation in Na 2SO .. grown callus was 10-12 fold g reater than the concentration of CI- in callus grown on approximately equimolar amounts of NaCl. Na 2S04 grown callus contained 60 to 70 times more sulphate than Na+. T he amount of 50.;-2 in callus grown on salinity

Comparative Salt Responses of Callus Cultures

with mixed cation salts remained almost similar to increased salinity as if the media had contained only NaCI. II Osmotic stress

Mannitol at 180 and 360 mol m -3 stimulated both the fresh and dry weights of the callus over those of the control. These parameters decreased with further increase in the concentration of mannitol (Fig. 3). Callus incubated at higher concentrations of mannitol remained green and was less necrotic than callus on iso-osmotic concentrations of NaCl and salt mixture. The reduction in dry weight was also less with mannitol than with all of the iso-osmotic concentrations of the saline media (Fig. 3). Mannitol grown callus accumulated less monovalent ions than any of the salt treatments. Accumulation of Na+ and Cl- remained uneffected but the K + content of the callus decreased with increase in the concentration of mannitol. However, K+ content was greater in tissue grown on mannitol than NaCl, Na 2 S04 and salt micture containing media (Fig. 2). Discussion

Growth inhibition by saline stress is commonly accepted to be due to a lowering of the water potential of growth media caused non-specifically by dissolved excess ions (Flowers et aI., 1977; Greenway and Munns, 1980). Most studies concerning the effects of salinity on callus growth have dealt with the effect of one salt, NaCI. Studies restricted to NaCl stress can not describe the relationship of inorganic solute accumulation to growth. Callus did not respond equally to stress nor to the compounds used for the stressing salt. Callus growth was inhibited more severly with Na 2 S04, followed by KC1, salt mixture and NaCI. Such severe inhibition of cell growth with Na2S04 compared with NaCl has also been reported in Beta vulgaris (Pua and Thorpe, 1986). This is due to more uptake of Na+ by cells growing on Na 2S04 medium than NaCl and salt mixture containing media. Moreover, Na 2S04 grown cells accumulated 60-70 times more S04 2 than Na+. Thus, results show that the effect of Na+ is highly dependent on the nature of the accompanying anion, for reasons yet not clearly understood. Stimulation of callus growth at low levels (50 mol m- 3) of Na 2S04 may be due to sub-optimal osmotic strength of the medium. Callus growth at low concentrations of KCl was higher compared with NaCl and salt mixture. Subsequently , growth at higher concentrations of KCI decreased and became less rapid than on NaCl and salt mixture. One difference between callus grown on KCI and NaCl was greater K+ and CI- contents of the KCI callus than Na+ and Cl- accumulation in NaCl grown callus. It is suggested that the results can be explained by the different degree of solute accumulation, which at lower levels can be beneficial to callus, allowing osmotic adjustment to occur, and at higher levels can become toxic. The difference in ionic uptake from KCl and NaCl media implies greater permeability of the cells in KCl medium.

123

A comparison between salt mixture and NaCl systems shows that at the same salinity level the Cl- accumulation in callus grown on salt mixture was higher than on NaCl, resulting in a greater reduction in growth. S04 2 ions of salt mixture seem to be inhibitory to K+ uptake as reported earlier by Rains (1972). Plant cells show variable response in their performance under salt and water stresses. Several halophytic plants responded similarly when subjected to sea water or PEG, indicating that most of the results can be ascribed to low osmotic potential and not salinity per se Qefferies et al., 1979). In the present study, callus growth was less severely inhibited by mannitol than by any of the salt treatments, suggesting that in the latter cases it was affected by ion toxicity. Similar results were also obtained by Tal and Katz (1980). They reported that tomato cell cultures showed no correlation between salt stress and PEG induced water stress. The results that we obtained using mannitol were similar to those obtained by other workers with PEG (Handa et aI., 1982; Sabbah and Tal, 1990). The dry weight of cells growing in mannitol was much higher than in cells growing in the presence of iso-osmotic concentrations of any of the salts and their mixture. Similar results were. also reported by Heyser and Nabors (1981), Binzel et al. (1985) and Ben-Hayyim (1987). The capability of the cells for good growth on mannitol rather than on any salt/salt mixture may be due to osmotic adjustment of the tissue by accumulation of more K + ions under mannitol induced osmotic stress than at iso-osmotic concentrations of any of the salts and their mixture. The present study shows that the response of callus varies with stress and salt types. SO 42 and K + are highly toxic to Vigna radiata leaf callus while Na+ and Cl- are less toxic. The results also suggest that single salts (Na 2S04 and KC1) are more toxic to the tissue than a salt mixture. Acknowledgement

The authors are thankful to CSIR and UGC (3-22/89 SR II), New Delhi, for financial assistance.

References BEN-HAYYIM, G.: Relationship between salt tolerance and resistance to polyethylene glycol-induced water stress in cultured citrus cells. Plant Physiol. 85, 430-433 (1987). BINZEL, M. L., P. M. HASEGAWA, A. K. HANDA, and R. A. BRESSAN: Adaptation of tobacco cells to NaC!. Plant Physiol. 78, 118 -125 (1985). CHANDLER, S. F. and T. A. THORPE: Variation from plant tissue cultures: Biotechnological application to improving salinity tolerance. Biotech Adv. 4, 117 -135 (1986). FLOWERS, T. J., P. F. TROKE, and A. R. YEO: The mechanism of salt tolerance in halophytes. Annu. Rev. Plant Physiol. 28, 89 -121 (1977). GREENWAY, H. and R. MUNNS: Mechanisms of salt tolerance in nonhalophytes. Annu. Rev. Plant Physiol. 31, 149-190 (1980). HANDA, A. K., R. A. BRESSAN, S. HANDA, and P. M. HASEGAWA: Tolerance to water and salt stress in cultured cells. In: A. FUJIWARA (ed.): Proc. 5th Int. Congr. Plant Tissue and Cell Culture, 471-474. Abe Photo Printing Co., Tokyo, 1982.

124

ANJU GULATI and PAWAN K. JAIWAL

HEYSER, J. W. and M. W. NABORS: Growth, water content and solute accumulation of two tobacco cell lines cultured on NaCl, dextran and polyethylene glycol. Plant Physiol. 68, 1454 -1459 (1981). JEFFERIES, R. L., T. RUDMIK, and E. M. DILLON: Responses of halophytes to high salinities and low water potentials. Plant Physiol. 68, 989-994 (1979). KRISHNAMURTHY, R. and K. A. BHAGWAT: A rapid, simplified and sensitive method for the determination of chloride, calcium and magnesium in plant material. Indian J. Exp. BioI. 29, 444-447 (1991). LERNER, H. R.: Adaptation to salinity at plant cell level. Plant and Soil 89, 3-14 (1985). MALIGA, P.: Isolation and characterization of mutants in plant cell culture. Ann. Rev. Plant Physiol. 35,519-542 (1984). McHuGHEN, A. and M. SWARTZ: A tissue culture derived salt tolerant line of flax (Linum usitatissimum). J. Plant Physiol. 117, 109-117 (1984). NABORS, M. W., S. E. GIBBS, C. S. BERNSTEIN, and M. E. MEIs: NaCltolerant tobacco plants from cultured cells. Z. Pflanzenphysiol. 97, 13 -17 (1980). NABORS, M. W., C. S. KROSKEY, and D. M. McHUGH: Green spots are predictors of high callus growth rates and shoot formation in normal and in salt stressed tissue cultures of Oat (A vena sativa L.). Z. Pflanzenphysiol. 105,341-349 (1982). NABORS, M. W. and T. A. DYKES: Tissue culture of cereal cultivars with increased salt, drought and acid tolerance. In: Biotechno-

logy International Rice Research Institute, 121-138. Manila, Philippines (1985). PHILLIPS, G. C. and G. B. COLLINS: In vitro tissue culture of selected legumes and plant regeneration from callus cultures of red clover. Crop. Sci. 19, 59-64 (1979). PUA, E. C. and T. A. THORPE: Differential response of non-selected and N azSO 4 selected callus cultures of Beta vulgaris L. to salt stress. J. Plant Physiol. 123,241-248 (1986). RAINS, D. W.: Salt tolerance by plants in relation to salinity. Annu. Rev. Plant Physiol. 23, 367 -388 (1972). RAINs, D. W., S. S. CROUGHAN, and T. P. CROUGHAN: Isolation and Characterization of mutant cell lines and plants: Salt Tolerance. In: I. K. VASIL (ed.): Cell Culture and Somatic Cell Genetics of Plants, 537 -544. Academic Press Inc. Orlando, Florida (1986). SABBAH, S. and M. TAL: Development of callus and suspension cultures of potato resistant to NaCI and mannitol and their response to stress. Plant Cell Tissue Org. Cult. 21,119-128 (1990). TABATABAI, M. A. and J. M. BREMNER: A simple tubidimetric method for determining total sulphur in plant materials. Agron J. 62, 865-866 (1970). TAL, M. and A. KATZ: Salt Tolerance in the wild relatives of the cultivated tomato: the effect of proline on the growth of callus tissue of Lycopersicon esculentum and L. peruvianum under salt and water stress. Z. Pflanzenphysiol. 98, 283 - 288 (1980). TYAGI, A. K., A. RASHID, and S. C. MAHESHWARI: Sodium chloride resistant cell line from haploid Datura innoxia Mill: A resistance trait carried from cell to plantlet and vice versa in vitro. Protoplasma 105, 327 -332 (1981).