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Salt Tolerance Expressed as a Cellular Trait in Suspension Cultures Developed from the Halophytic Grass Distichlis spicata
Salt Tolerance Expressed as a Cellular Trait in Suspension Cultures Developed from the Halophytic Grass Distichlis spicata R. S. WARREN!) andA. R. GOULD 2 *) 1) Botany Department, Connecticut College, New London, CT 06320 U.S.A. 2) Plant Genetics Department, Pfizer Central Research, Eastern Point Road, Groton, CT 06340 U.S.A. Received March 22,1982· Accepted June 14, 1982
Summary Aberrant root tissue derived from primary in vitro explants of the halophytic grass Distichlis spicata, eventually gave rise to friable callus lines after prolonged manual selection for «calluslike» cultures. Rapidly dividing (cell doubling times - 40 h) cell suspension cultures initiated from friable callus, were exposed to NaCI concentra~ions up to 860 mM. Growth curves constructed from both packed cell volume and cell number estimations demonstrated no depression of growth at salinities as high as 170 mM and 200 mM NaC!. Even at much higher salt concentrations the Distichlis suspensions were still able to grow, although at reduced rates. In contrast, suspension cultures of Nicotiana sylvesiris and Zea mays were much less tolerant of salt. After 3 years of growth in non-selective (i.e. salt-fr~e) media, the Distichlis suspensions still expressed the salt tolerance trait. A comparison of previously published data on halophytes with similar data for D. spicata suggests that a considerable proportion of the salt tolerance expressed by this halophytic monocot is cellularly based and is not dependent upon anatomical and physiological specializations of whole plants.
Key words: Distichlis spicata, Nicotiana sylvestris, Zea mays, salt tolerance, plant tissue culture, cell-trait, halophyte.
Introduction The physiological basis of salt tolerance in halophytes has been studied extensively at the whole plant level. There is, however, very little direct information concerning the mechanisms of salt tolerance that must be operating at the cellular level (Flowers et aI., 1977 and reviews cited therein). One of the problems encountered in studying cellular processes and mechanisms has been the difficulty of functionally separating whole plant specializations such as succulence and salt glands from strictly cellular processes such as compartmentation or active exclusion of sodium, or the synthesis of osmotic protectants like glycine-betaine. The primary objective of this work was to develop a tissue culture system which would allow the cellular basis of salt tolerance *) To whom reprint requests should be sent. Z. Pf/anzenphysiol. Ed. 107. S. 347-356. 1982.
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in the halophytic, graminoid spike grass D. spicata L. to be studied free of whole plant anatomical and physiological specializations. D. spicata is a perennial grass common in Atlantic coastal tidal as well as inland saline marshes of North America (Chapman, 1974; Hansen et a!., 1976). In the field this species shows a very broad distribution with respect to soil salinity; it competes effectively in highly saline pannes (500-600 mM NaCI), as well as in areas of fresh water seepage (0-170 mM), and it is most commonly found in areas of moderate soil salinity (250-425 mM) (Taylor, 1939; Ungar, 1966; Niering and Warren, 1974, 1980; Hansen et a!., 1976). In laboratory growth studies of D. spicata from inland marshes Hansen et a!. (1976) report best growth at soil salinities of about 250 mM while Adams (1963) reported that optimal NaCI levels for greenhouse grown D. spicata from North Carolina marshes was about 170 mM. In the study by Taylor (1939) plants grew best in tap water but grew nearly as well in full sea water (600 mM). Recently Parrondo et a!. (1978) have reported that the dry weight increase of greenhouse grown D. spicata is unaffected at salinities up to 270 mM and is only slightly reduced at 540 mM. Kemp and Cunningham (1981) also report that growth rates of D. spicata, under high light intensity in controlled environment chambers, were not reduced significantly by hydroponic solutions containing up to 500 mM NaCI. It is apparent from these studies and from field observations (Niering and Warren, 1980) that D. spicata is likely to be the most salt tolerant grass in the Atlantic tidal marsh community and that it may well require some salt for optimal growth. A secondary objective of this study was to compare the salt tolerance of D. spicata callus cultures with cultured tissue from the dicotyledonous halophytes Suaeda mario tima and Salicornia europaea. Both these dicot species require salt for optimal growth in the field and their callus cultures have been tested for salt tolerance by von Hedenstrom and Breckle (1974). These authors reported that cultures of S. maritima and S. europaea grew more rapidly with some added NaC!, but optimal levels in culture were less than one half the optima for whole plants reported in the literature.
Material and Methods Plant Material Shoots of D. spicata were collected from two different marshes along the Connecticut shore of Long Island Sound during the last two weeks of July 1977. At that time the immature flowering heads were still enclosed and surrounded by several layers of immature leaves. Shoots were placed in plastic bags and returned to the laboratory within two hours. These were used immediately for explants or were refrigerated overnight and used the following morning.
Initiation a/Tissue Cultures The top eight to ten em of shoot was surface sterilized with 95 % ethanol for 60 s and then in 25 % Clorox for 4 min, followed by three rinses in sterile distilled water. The immature rachis and flowering head were dissected out and used as primary explants. The entire flowering stalk, and in some instances the head and rachis separately, were placed on a medium consisting of Gamborg's B5 salts (Gam borg et al., 1968) with the following organic constituents: inositol,
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100 mg I-I; thiamine-HCI, 10 mg I-I; nicotinic acid, 1 mg 1-\ pyridoxine-HCI, 1 mg I-I; 2,4dichlorophenoxyacetic acid (2,4-D), 2 mg I-I; sucrose, 2 % w/v; DifcoBacto agar, 0.8 % w/v. Aberrant root tissue, which produced abundant extracellular gel-like material (presumably root tip polysaccharide) was derived from primary explants. If the polysaccharide was removed at each subculture, the tissue would proliferate slowly. These cultures were maintained on the initiating medium with 2,4-D levels from 0.5 to 10 mg I-I and on a parallel series of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962) with organic supplements as for the B5 salts series. Cultures were transfered weekly and observed carefully at least once a week. Sectors of callus tissue which showed unusual appearance were picked out and maintained as separate lines. One line, derived by such selective passaging on MS + 4 mg I-I 2,4-D (MS4) produced much less gel-like material than other lines. After seven months of continuous subculture small callus-like sectors arose from this line, and by conserving and selecting calli for rapid growth, three rapidly growing lines of friable callus were developed. These friable callus lines show no potential for morphogenesis. During,the entire selection process the tissues were kept on MS4; all cultures were maintained at 25 ± 1 °C in 200-300 lux cool white VHO fluorescent light with a 16 h photoperiod.
Initiation o/Suspension Cultures and Growth Measurements Six to eight pea sized pieces of rapidly growing callus were placed in each of five 125 ml flasks containing 50 ml of MS4 without agar. These cultures were incubated in a rotary shaker at 120 cycles min- 1 at 27 ± 1 °C in the dark. After three weeks 10 ml of suspension was transferred into 40 ml of fresh medium. Early growth measurements were made by allowing cells to settle out from a standard volume of culture in a graduated centrifuge tube. After one hour the total cell volume was measured (CV60). Cultures were transferred while in exponential growth and after five transfers two lines had CV60 doubling times averaging 2.3 days. These two lines were used to test growth as a function of NaCI concentration. In all growth experiments in which CV60 was the parameter, flasks were inoculated with 10 ml of cell suspension as described above. Final concentrations of NaCI tested were 20, 43, 85, 170,260,340,430,550,685 and 860 mM. CV 60 was determined for all cultures every two or three days. No differences could be seen between the two lines used and data from both were pooled. Cell lines were then maintained on salt free MS4 for a period of nine months after which a second set of CV60 growth trials were made with NaCl concentrations of 25,50, 100, 150, 200, 300 and 400 mM. Growth curves were replicated at least 5 times in both series of experiments and a NaCI free control culture was included in all tests. The response of D. spicata to NaCI was also compared to that of two glycophytes. N. sylvestris cultures (obtained from Dr. S. Flashman, North Carolina State University, July, 1977) were used in the first set of trials whilst in the second series, cultures of Z. mays var. Black Mexican (supplied by Prof. C. E. Green, University of Minnesota) were used. N. sylvestris cultures were grown in Murashige and Skoog (1962) tobacco medium supplemented with 0.4 mg I-I 2,4-D whilst cultures of Z. mays were grown in the same medium as D. spicata. After three years in culture without salt the D. spicata cell lines were again tested for expression of the salt tolerance trait. For the last two years, and in the experiments described below, the cultures were maintained on MS medium with 2 mg ml- I 2,4-D (MS 2), rather than the original MS 4 medium which contained 4 mg ml- I 2,4-D. In these trials rates of cell number increase were calculated by cell counting. 1 ml aliquots were removed from cultures and mixed with 5 ml of 10 % w/v aqueous chromium trioxide for subsequent determination of cell numbers per ml of culture as described by Gould (1977). Samples were counted in specially made, 1 mm deep chambers on an inverted microscope giving a field volume of 1.29 ILL Exponential rates of cell number increase were calculated by regression analysis. Cell number increase curves were determined for salt free control cultures, and for cells taken to salt containing medium in a single step from salt free cultures. These cells were referred to as «step up" cultures to dis-
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tinguish them from cells grown continuously (over a number of subcultures) in the presence of salt.
Results
Tissue Cultures /rom Primary Explants Cell enlargement occurred at the base of rachis explants within three days. After 7 to 10 days of culture, disorganised proliferation was apparent at the rachis base and in the axes of many florets. Excision and transfer of dividing tissue to fresh medium produced «aberrant root-like» cultures (Cure and Mott, 1978). Tissue grew in small dense spheres of 1 to 3 mm diameter. These produced a heavy coating of viscous gel into which cells were sloughed off. The gel could represent an uncontrolled production of root tip polysaccharide. Removal of the gel layer allowed tissue proliferation but the coating was rapidly reformed. Weekly removal of gel and transfer of callus to fresh medium maintained proliferation of the D. spicata tissues; if the coating was not removed, however, growth ceased within two weeks. Such arrested tissues remained viable and growth could be re-initiated (by removing the gel coating and transfering to fresh media) after as long as four months. Development of these aberrant root cultures was not influenced by excision of proliferating tissues from explants, or by the source of the explants; the same general response was observed in tissue derived from mesocotyl of germinating seed. Manipulation of hormone levels and ratios did not affect the development of explants to the aberrant root form on either MS or B5 basal salts. Although morphogenesis of initial tissues to roots was not influenced by exogenous hormones, the subsequent growth and development of the tissue could be modified by auxin. At 0.1 mg 1- 1 2,4-D, highly organized roots up to 6 mm long and 1 mm in diameter, with well developed root hairs, would grow out of the gel-coated tissue masses within two weeks of transfer. At 0.5 mg 1- 1 2,4-D, emerging roots elongated much more slowly and at 1.0 mg 1- 1 their development was limited to small bumps without root hairs. There were no apparent differences in growth or development with 2,4-D levels between 2 and 6 mg 1- 1; at 8 mg 1- 1 there was some reduction in growth and at 10 mg 1- 1 the inhibition was marked. Three other tidal marsh grasses were cultured in connection with this study: Spar· tina patens, Spartina pectinata and Panicum virgatum. Cultures from these species developed very similarly to D. spicata and also showed similar responses to varying levels of auxin.
Suspension Culture with Salt Growth rates of D. spicata suspension cultures as measured by packed cell volume (CV60) were unaffected by NaCl concentrations up to 170 mM. At this concentration a one day lag period was observed consistently, but the subsequent doubling time was the same as in salt free medium.· Above 170 mM, doubling times increased progresZ. Pjlanzenphysiol. Bd. 107. S.347-356. 1982.
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sively with increasing NaCllevels, and at 860 mM salt, only four of six inoculated flasks showed any growth even after two weeks of incubation. A set of CV60 growth curves from one representative trial is shown in Figure 1. In a second set of trials, after nine months of weekly subculturing without salt, D. spicata suspension cultures had the same pattern of salt tolerance as measured by CV60 growth curves, as they showed initially.
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Fig. 1: Growth curves from two representative trials comparing the influence of NaCI on the growth of suspension cultures of D. spicata and N. sylvestris. Salt concentration (mM NaCl) in medium given at the end of each curve. Three years after the initial explants were taken, and with over two years of subculture as a suspension in the absence of salt, the salt tolerance of one D. spicata line was tested again. However, in these trials, cell number increase rates were calculated from cell count data, and tolerance to various salt levels was expressed as hours per cell doubling, and final cell number per ml. Cells grown in salt-free medium were inoculated into MS2 giving final salt concentrations of 0, 50, 100, 200, 400 and 600 mM. Cell counting of these «step-up» cultures, at regular intervals generated the growth curves illustrated in Figures 2 A and B. Cells were also maintained in MS2 + 200 mM salt for 14 weekly subcultures and cell number increase curves were constructed on weeks 3, 12 and 14. Figure 2 B presents data for comparison of cell division rate and final cell number per ml for control (salt free), 200 mM stepup (salt free to 200 mM NaCl) and continuous subculture (14weeks of 7-day subcultures) in 200 mM NaCl. Cell doubling times for step-up experiments were 40.6 h ± 1.4 h for salt free controls (5 cultures); 43.9 h for 50 mM NaCI (1 culture); 42.4 h for 100 mM NaCI (1 culture); 54.4 h±2.0 h for 200 mM Z. Pjlanzenphysiol. Ed. 107. S. 347-356. 1982.
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Fig. 2 A: Representative cell number increase curves for D. spicata suspension cultures grown in salt-free (control), 50 mM, 100 mM, 400 mM and 600 mM salt containing medium. Fig. 2 B: Representative cell number increase curves for D. spicata suspension cultures grown continuously in medium containing 200 mM salt, or inoculated from salt free medium into 200 mM salt (step-up), compared with control.
NaCI (2 cultures); 103.7 h for 400 mM NaCI (1 culture); at 600 mM NaCl, cell numbers did not increase sufficiently to enable estimation of doubling time. The cell doubling time for cultures of D. spicata grown continuously in 200 mM salt was 40.5 h ± 2.0 hours (3 cultures) which is not significantly different from controls. Figure 2 B illustrates the different response of cultures stepped-up to 200 mM NaCI as compared with salt grown cultures at the same NaCI concentration. The reversion of such salt stressed cultures to doubling times similar to salt free controls had occurred within, at most, 2 subcultures in 200 mM NaCI after step-up to that salt level from salt free medium. It should be noted that cell number doubling times determined in these experiments are shorter than the CV60 doubling times obtained from initial measurements. A very common feature of suspension cultures during exponential cell division is a decline in average cell volume, and this is manifested as a discrepancy between packed cell volume doubling and cell number doubling (the latter being more rapid). Alternatively, the discrepancy could reflect a real difference due to the reduction in 2,4-D concentration in medium used in the cell counting experiments (4 to 2 mg 1-1) or a selection for more rapidly dividing cells over the years of weekly subculture. The essential point, however, is very clear; the salt tolerance trait is extremely stable in D. spicata cultures and is m;nntained without selection pressure. In contrast to D. spicata, the growth of N sylvestris and Z. mays suspension cultures was strongly inhibited by relatively low levels of NaCI. N sylvestris was the most Z. Pjl.anzenphysiol. Ed. 107. S. 347-356. 1982.
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sensltlve: growth was slowed considerably by a long lag period at concentrations above 43 mM and at 170 mM inhibition was complete. This is consistent with the observations of Nabors et al. (1975) with N. sylvestris suspensions. Only a few percent of the cells appeared to be alive when examined microscopically after one week in medium containing 170 mM salt. Growth curves from one representative trial are presented in Figure 1. Z. mays in suspension culture was somewhat more tolerant of NaCI than was N. sylvestris, but still dramatically less so than D. spicata. At 100 mM salt the doubling time (CV60) had increased by about 30 % in Z. mays suspensions (vs. about 400 % for N. sylvestris); at 200 mM there was still consistent although- very slow growth of the cultures. o Distichlis spicala o Zea mays 6.
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Fig. 3: Effect of salt on CV60 doubling times for suspension cultures of D. spicata, Z. mays and N. sylvestris. Data for S. maritima calculated from dry weight doubling times from von Hedenstriim and Breckle (1974).
The mean CV60 doubling times over eight separate trials for all three species are plotted against concentration of NaCl in the medium in Figure 3. Also included, is a plot of dry weight doubling time for S. maritima calculated from the data of von Hedenstr6m and Breckle (1974).
Discussion The appearance and the response to auxin of D. spicata tissue cultures initiated from primary explants are consistent with the observations of Cure and Mott (1978) and Mott and Cure (1978) on seven different grass species. This supports the interpretation that the tissues are organized and root-like. The growth of the initial cultures of D.spicata (and the other grasses used in this study) at 0.1 and 0.5 mg 1-1 2,4-D fits Cure and Mott's Class 1 category of aberrant root development; at
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1.0 mg 1-1 the pattern is similar to their Class 2 and above that level of auxin the growth can be described as their Class 3. True parenchymous callus was derived from the D. spicata primary aberrant root culture. The transformation was gradual and was accomplished by severe selection pressure for desirable culture phenotypes. This was possible due to the stable inheritance of changes that were expressed as different patterns of cell growth and tissue organization. In the selection process a number of phenotypically distinct and stable tissue lines were developed and could be maintained, as could the aberrant root tissue and the friable callus lines. The growth of D. spicata suspensions in media with added NaCl demonstrates that a significant and critical portion of this species' tolerance to salt, functions at a strictly cellular level; it is not dependent upon tissue organization or anatomical specializations such as salt glands, the endodermis or succulence. This conclusion is consistent with the results of von Hedenstrom and Breckle (1974) from the marsh dicots S. maritima and S. europaea, and also with the results of Tal et al. (1978) comparing salt tolerant and sensitive species of Lycopersicon and Solanum. These results, however, are all in contrast with those of Strogonov (1973) who failed to find a difference in salt tolerance between callus from Salicornia species and a number of glycophytes. A comparison of growth performance under salt stress between S. maritima and S. europea cultures (von Hedenstrom and Breckle, 1974) and D. spicata cultures is interesting in respect of the expression of whole plant characteristics in tissue culture. Optimal levels of salt for growth of S. maritima and S. europea suspension cultures (about 85 mM and 170 mM respectively; see Figure 3 for S. maritima) were about half the optima for whole plants (Flowers et al., 1977). At salt levels corresponding to field conditions (about 340 mM) growth of S. maritima suspension cultures was very strongly inhibited (no detailed data were presented for S. europea). Thus, whilst salt tolerance of the Suaeda and Salicornia species has a strong cellular basis, anatomical and physiological specializations such as succulence and salt glands also playa large role at the whole plant level. In contrast to these two dicotyledonous species, the growth rates of cell cultures of D. spicata are not stimulated by NaCl. Growth as measured by a cell volume parameter (CV60) is not inhibited until NaCl concentrations exceed 170 mM. In terms of cell number increase, 200 mM NaCl is insufficient to depress the generation time of D. spicata suspension cultures if they are continuously subcultured at this level of salt stress, and cell doubling time is only very slightly influenced if cultures are taken from 0 to 200 mM NaCl in a single step. CV60 doubling times increase steadily above 170 mM NaCl but growth is rapid at salinities typical of field conditions and can still occur at 860 mM salt. In suspension culture, therefore, D. spicata can deal more effectively with NaCl than can S. maritima, although in the field they appear to be approximately the same in that respect. D. spicata suspensions are also at least as salt tolerant as those of S. europea, yet as a whole plant the latter can survive at molar or greater soil NaCl, whilst D. spicata is very rarely found with soil salinities much
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above 680 mM (Niering and Warren, 1980). These comparisons suggest that salt tolerance in D. spicata has a relatively more important cellular component, and is less dependent upon whole plant organization than is the tolerance of S. europea of S. maritima. In a recent publication, Smith and McComb (1981) report that two dicot halophytes, Atriplex undulata and Suaeda australis, exhibit no cellularly based salt tolerance in callus cultures, and the authors conclude that in these species, salt tolerance depends entirely upon anatomical and physiological specializations at the whole plant level. Clearly, the data presented here do not support such a conclusion for D. spicata, and there are at least two possible explanations for this disagreement. Smith and McComb (1981) used fresh weight determinations to calculate growth in their callus cultures, and they emphasized that such measurements are open to criticism. Certainly, the different osmotic environments suffered by callus cells under different degrees of salt stress, could alter the relative contribution of cell associated water to changes in fresh weight. The results reported here may provide the first evidence to support Jagels' (1973) hypothesis that the salt tolerance of monocots has, in general, a stronger cellular basis than that of dicots. They also suggest, as do the results of Orton (1980), that selection for salt tolerance in tissue culture, especially with monocots, should be a realistic strategy for improving the salt tolerance of crop speCles.
References ADAMS, D. c.: Factors influencing vascular plant zonation in North Carolina salt marshes. Ecology 44, 445-446 (1963). CHAPMAN, B. J.: Salt marshes and salt deserts of the world. 1st Edition. Interscience Publishers, New York, pp. 392, 1974. CURE, W. W. and R. L. MOTI: A comparative anatomical study of organogenesis in cultured tissues of maize, wheat and oats. Physio!. Plant. 42, 91-96 (1978). FLOWERS, T. J., P. F. TROKE, and A. R. YEO: The mechanism of salt tolerance in halophytes. Ann. Rev. Plant. Physio!. 28,89-121 (1977). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Exptl. Cell Res. 50,151-158 (1968). GOULD, A. R.: Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G 1 transition probability mode!. Planta 137,29-36 (1977). HANSEN, D. J., P. DAYANANDAN, P. B. KAUFMAN, and J. D. BROTHERSON: Ecological adaptations of salt marsh grass Distichlis spicata (Gramineae), and environmental factors affecting its growth and distribution. Am. J. Bot. 63, 635-650 (1976). HEDENSnOM, H. VON and S. W. BRECKLE: Obligate halophytes? A test with tissue cultllre methods. Z. Pflanzenphysiol. 74,183-185 (1974). JAGELS, R.: Studies of a marine grass, Thalassia testudinum. I. Ultrastructure of the osmoregulatory leaf cells. Am. J. Bot. 60, 1003-1009 (1973). KEMP, P. R. and G. L. CUNNINGHAM: Light, temperature and salinity effects on growth, leaf anatomy and photosynthesis of Distichlis spicata (L.) Greene. Am. J. Bot. 68, 507-516 (1981). MOTI, R. L. and W. W. CURE: Anatomy of maize tissue cultures. Physio!. Plant. 43, 139-145 (1978).
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MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio!. Plant. 15, 473-497 (1962). NABORS, M. W., A. DANIELS, L. NADOLNY, and C. BROWN: Sodium chloride tolerance lines of tobacco cells. Plant Sci. Let. 4,155-159 (1975). NIERING, W. A. and R. S. WABREN: Tidal wetlands of Connecticut I: Vegetation and associated animal populations. Dept. of Env. Protection, State of Conn., Hartford, 1974. - - Vegetation patterns and processes in New England salt marshes. BioSci. 30, 301-307 (1980). ORTON, T. J.: Comparison of salt tolerance between Hordeum vulgare and R jubatum in whole plants and callus cultures. Z. Pflanzenphysio!. 98, 105-118 (1980). PARRONDO, R. T., J. G. GOSSELINK, and C. S. HOPKINSON: Effects of salinity and drainage on the growth of three salt marsh grasses. Bot. Gaz. 139,102-107 (1978). SMITH, M. K. and J. A. MCCOMB: Effect of NaCl on the growth of whole plants and their corresponding callus cultures. Aust. J. Plant Physio!. 8, 267-275 (1981). STROGONOV, B. P.: Salt tolerance in isolated tissues and cells. In: Structure and Function of Plant Cells in Saline Habitats. New Trends in the Study of Salt Tolerance. Trans!. from Russian by Israel Program for Scientific Translations; Jerusalem, pp. 1-33, 1973. TAL, M., H. HEIKIN, and K. DEHAN: Salt tolerance in the wild relatives of the cultivated tomato: Response of callus tissue of Lycopersicon esculentum, L. peruvianum and Solanum pennellii to high salinity. Z. Pflanzenphysio!. 86, 231-240 (1978). TAYLOR, N.: Salt tolerance of Long Island salt marsh plants. N. Y. State Mus. Circ. 23, pp 42, 1939. UNGAR, I. A.: Salt tolerance of plants growing in saline areas of Kansas and Oklahoma. Ecology 47, 154-155 (1966).
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Summary Aberrant root tissue derived from primary in vitro explants of the halophytic grass Distichlis spicata, eventually gave rise to friable callus lines after prolonged manual selection for «calluslike» cultures. Rapidly dividing (cell doubling times - 40 h) cell suspension cultures initiated from friable callus, were exposed to NaCI concentra~ions up to 860 mM. Growth curves constructed from both packed cell volume and cell number estimations demonstrated no depression of growth at salinities as high as 170 mM and 200 mM NaC!. Even at much higher salt concentrations the Distichlis suspensions were still able to grow, although at reduced rates. In contrast, suspension cultures of Nicotiana sylvesiris and Zea mays were much less tolerant of salt. After 3 years of growth in non-selective (i.e. salt-fr~e) media, the Distichlis suspensions still expressed the salt tolerance trait. A comparison of previously published data on halophytes with similar data for D. spicata suggests that a considerable proportion of the salt tolerance expressed by this halophytic monocot is cellularly based and is not dependent upon anatomical and physiological specializations of whole plants.
Key words: Distichlis spicata, Nicotiana sylvestris, Zea mays, salt tolerance, plant tissue culture, cell-trait, halophyte.
Introduction The physiological basis of salt tolerance in halophytes has been studied extensively at the whole plant level. There is, however, very little direct information concerning the mechanisms of salt tolerance that must be operating at the cellular level (Flowers et aI., 1977 and reviews cited therein). One of the problems encountered in studying cellular processes and mechanisms has been the difficulty of functionally separating whole plant specializations such as succulence and salt glands from strictly cellular processes such as compartmentation or active exclusion of sodium, or the synthesis of osmotic protectants like glycine-betaine. The primary objective of this work was to develop a tissue culture system which would allow the cellular basis of salt tolerance *) To whom reprint requests should be sent. Z. Pf/anzenphysiol. Ed. 107. S. 347-356. 1982.
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in the halophytic, graminoid spike grass D. spicata L. to be studied free of whole plant anatomical and physiological specializations. D. spicata is a perennial grass common in Atlantic coastal tidal as well as inland saline marshes of North America (Chapman, 1974; Hansen et a!., 1976). In the field this species shows a very broad distribution with respect to soil salinity; it competes effectively in highly saline pannes (500-600 mM NaCI), as well as in areas of fresh water seepage (0-170 mM), and it is most commonly found in areas of moderate soil salinity (250-425 mM) (Taylor, 1939; Ungar, 1966; Niering and Warren, 1974, 1980; Hansen et a!., 1976). In laboratory growth studies of D. spicata from inland marshes Hansen et a!. (1976) report best growth at soil salinities of about 250 mM while Adams (1963) reported that optimal NaCI levels for greenhouse grown D. spicata from North Carolina marshes was about 170 mM. In the study by Taylor (1939) plants grew best in tap water but grew nearly as well in full sea water (600 mM). Recently Parrondo et a!. (1978) have reported that the dry weight increase of greenhouse grown D. spicata is unaffected at salinities up to 270 mM and is only slightly reduced at 540 mM. Kemp and Cunningham (1981) also report that growth rates of D. spicata, under high light intensity in controlled environment chambers, were not reduced significantly by hydroponic solutions containing up to 500 mM NaCI. It is apparent from these studies and from field observations (Niering and Warren, 1980) that D. spicata is likely to be the most salt tolerant grass in the Atlantic tidal marsh community and that it may well require some salt for optimal growth. A secondary objective of this study was to compare the salt tolerance of D. spicata callus cultures with cultured tissue from the dicotyledonous halophytes Suaeda mario tima and Salicornia europaea. Both these dicot species require salt for optimal growth in the field and their callus cultures have been tested for salt tolerance by von Hedenstrom and Breckle (1974). These authors reported that cultures of S. maritima and S. europaea grew more rapidly with some added NaC!, but optimal levels in culture were less than one half the optima for whole plants reported in the literature.
Material and Methods Plant Material Shoots of D. spicata were collected from two different marshes along the Connecticut shore of Long Island Sound during the last two weeks of July 1977. At that time the immature flowering heads were still enclosed and surrounded by several layers of immature leaves. Shoots were placed in plastic bags and returned to the laboratory within two hours. These were used immediately for explants or were refrigerated overnight and used the following morning.
Initiation a/Tissue Cultures The top eight to ten em of shoot was surface sterilized with 95 % ethanol for 60 s and then in 25 % Clorox for 4 min, followed by three rinses in sterile distilled water. The immature rachis and flowering head were dissected out and used as primary explants. The entire flowering stalk, and in some instances the head and rachis separately, were placed on a medium consisting of Gamborg's B5 salts (Gam borg et al., 1968) with the following organic constituents: inositol,
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100 mg I-I; thiamine-HCI, 10 mg I-I; nicotinic acid, 1 mg 1-\ pyridoxine-HCI, 1 mg I-I; 2,4dichlorophenoxyacetic acid (2,4-D), 2 mg I-I; sucrose, 2 % w/v; DifcoBacto agar, 0.8 % w/v. Aberrant root tissue, which produced abundant extracellular gel-like material (presumably root tip polysaccharide) was derived from primary explants. If the polysaccharide was removed at each subculture, the tissue would proliferate slowly. These cultures were maintained on the initiating medium with 2,4-D levels from 0.5 to 10 mg I-I and on a parallel series of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962) with organic supplements as for the B5 salts series. Cultures were transfered weekly and observed carefully at least once a week. Sectors of callus tissue which showed unusual appearance were picked out and maintained as separate lines. One line, derived by such selective passaging on MS + 4 mg I-I 2,4-D (MS4) produced much less gel-like material than other lines. After seven months of continuous subculture small callus-like sectors arose from this line, and by conserving and selecting calli for rapid growth, three rapidly growing lines of friable callus were developed. These friable callus lines show no potential for morphogenesis. During,the entire selection process the tissues were kept on MS4; all cultures were maintained at 25 ± 1 °C in 200-300 lux cool white VHO fluorescent light with a 16 h photoperiod.
Initiation o/Suspension Cultures and Growth Measurements Six to eight pea sized pieces of rapidly growing callus were placed in each of five 125 ml flasks containing 50 ml of MS4 without agar. These cultures were incubated in a rotary shaker at 120 cycles min- 1 at 27 ± 1 °C in the dark. After three weeks 10 ml of suspension was transferred into 40 ml of fresh medium. Early growth measurements were made by allowing cells to settle out from a standard volume of culture in a graduated centrifuge tube. After one hour the total cell volume was measured (CV60). Cultures were transferred while in exponential growth and after five transfers two lines had CV60 doubling times averaging 2.3 days. These two lines were used to test growth as a function of NaCI concentration. In all growth experiments in which CV60 was the parameter, flasks were inoculated with 10 ml of cell suspension as described above. Final concentrations of NaCI tested were 20, 43, 85, 170,260,340,430,550,685 and 860 mM. CV 60 was determined for all cultures every two or three days. No differences could be seen between the two lines used and data from both were pooled. Cell lines were then maintained on salt free MS4 for a period of nine months after which a second set of CV60 growth trials were made with NaCl concentrations of 25,50, 100, 150, 200, 300 and 400 mM. Growth curves were replicated at least 5 times in both series of experiments and a NaCI free control culture was included in all tests. The response of D. spicata to NaCI was also compared to that of two glycophytes. N. sylvestris cultures (obtained from Dr. S. Flashman, North Carolina State University, July, 1977) were used in the first set of trials whilst in the second series, cultures of Z. mays var. Black Mexican (supplied by Prof. C. E. Green, University of Minnesota) were used. N. sylvestris cultures were grown in Murashige and Skoog (1962) tobacco medium supplemented with 0.4 mg I-I 2,4-D whilst cultures of Z. mays were grown in the same medium as D. spicata. After three years in culture without salt the D. spicata cell lines were again tested for expression of the salt tolerance trait. For the last two years, and in the experiments described below, the cultures were maintained on MS medium with 2 mg ml- I 2,4-D (MS 2), rather than the original MS 4 medium which contained 4 mg ml- I 2,4-D. In these trials rates of cell number increase were calculated by cell counting. 1 ml aliquots were removed from cultures and mixed with 5 ml of 10 % w/v aqueous chromium trioxide for subsequent determination of cell numbers per ml of culture as described by Gould (1977). Samples were counted in specially made, 1 mm deep chambers on an inverted microscope giving a field volume of 1.29 ILL Exponential rates of cell number increase were calculated by regression analysis. Cell number increase curves were determined for salt free control cultures, and for cells taken to salt containing medium in a single step from salt free cultures. These cells were referred to as «step up" cultures to dis-
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tinguish them from cells grown continuously (over a number of subcultures) in the presence of salt.
Results
Tissue Cultures /rom Primary Explants Cell enlargement occurred at the base of rachis explants within three days. After 7 to 10 days of culture, disorganised proliferation was apparent at the rachis base and in the axes of many florets. Excision and transfer of dividing tissue to fresh medium produced «aberrant root-like» cultures (Cure and Mott, 1978). Tissue grew in small dense spheres of 1 to 3 mm diameter. These produced a heavy coating of viscous gel into which cells were sloughed off. The gel could represent an uncontrolled production of root tip polysaccharide. Removal of the gel layer allowed tissue proliferation but the coating was rapidly reformed. Weekly removal of gel and transfer of callus to fresh medium maintained proliferation of the D. spicata tissues; if the coating was not removed, however, growth ceased within two weeks. Such arrested tissues remained viable and growth could be re-initiated (by removing the gel coating and transfering to fresh media) after as long as four months. Development of these aberrant root cultures was not influenced by excision of proliferating tissues from explants, or by the source of the explants; the same general response was observed in tissue derived from mesocotyl of germinating seed. Manipulation of hormone levels and ratios did not affect the development of explants to the aberrant root form on either MS or B5 basal salts. Although morphogenesis of initial tissues to roots was not influenced by exogenous hormones, the subsequent growth and development of the tissue could be modified by auxin. At 0.1 mg 1- 1 2,4-D, highly organized roots up to 6 mm long and 1 mm in diameter, with well developed root hairs, would grow out of the gel-coated tissue masses within two weeks of transfer. At 0.5 mg 1- 1 2,4-D, emerging roots elongated much more slowly and at 1.0 mg 1- 1 their development was limited to small bumps without root hairs. There were no apparent differences in growth or development with 2,4-D levels between 2 and 6 mg 1- 1; at 8 mg 1- 1 there was some reduction in growth and at 10 mg 1- 1 the inhibition was marked. Three other tidal marsh grasses were cultured in connection with this study: Spar· tina patens, Spartina pectinata and Panicum virgatum. Cultures from these species developed very similarly to D. spicata and also showed similar responses to varying levels of auxin.
Suspension Culture with Salt Growth rates of D. spicata suspension cultures as measured by packed cell volume (CV60) were unaffected by NaCl concentrations up to 170 mM. At this concentration a one day lag period was observed consistently, but the subsequent doubling time was the same as in salt free medium.· Above 170 mM, doubling times increased progresZ. Pjlanzenphysiol. Bd. 107. S.347-356. 1982.
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sively with increasing NaCllevels, and at 860 mM salt, only four of six inoculated flasks showed any growth even after two weeks of incubation. A set of CV60 growth curves from one representative trial is shown in Figure 1. In a second set of trials, after nine months of weekly subculturing without salt, D. spicata suspension cultures had the same pattern of salt tolerance as measured by CV60 growth curves, as they showed initially.
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Fig. 1: Growth curves from two representative trials comparing the influence of NaCI on the growth of suspension cultures of D. spicata and N. sylvestris. Salt concentration (mM NaCl) in medium given at the end of each curve. Three years after the initial explants were taken, and with over two years of subculture as a suspension in the absence of salt, the salt tolerance of one D. spicata line was tested again. However, in these trials, cell number increase rates were calculated from cell count data, and tolerance to various salt levels was expressed as hours per cell doubling, and final cell number per ml. Cells grown in salt-free medium were inoculated into MS2 giving final salt concentrations of 0, 50, 100, 200, 400 and 600 mM. Cell counting of these «step-up» cultures, at regular intervals generated the growth curves illustrated in Figures 2 A and B. Cells were also maintained in MS2 + 200 mM salt for 14 weekly subcultures and cell number increase curves were constructed on weeks 3, 12 and 14. Figure 2 B presents data for comparison of cell division rate and final cell number per ml for control (salt free), 200 mM stepup (salt free to 200 mM NaCl) and continuous subculture (14weeks of 7-day subcultures) in 200 mM NaCl. Cell doubling times for step-up experiments were 40.6 h ± 1.4 h for salt free controls (5 cultures); 43.9 h for 50 mM NaCI (1 culture); 42.4 h for 100 mM NaCI (1 culture); 54.4 h±2.0 h for 200 mM Z. Pjlanzenphysiol. Ed. 107. S. 347-356. 1982.
R. S. WARREN and A. R. GOULD
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Fig. 2 A: Representative cell number increase curves for D. spicata suspension cultures grown in salt-free (control), 50 mM, 100 mM, 400 mM and 600 mM salt containing medium. Fig. 2 B: Representative cell number increase curves for D. spicata suspension cultures grown continuously in medium containing 200 mM salt, or inoculated from salt free medium into 200 mM salt (step-up), compared with control.
NaCI (2 cultures); 103.7 h for 400 mM NaCI (1 culture); at 600 mM NaCl, cell numbers did not increase sufficiently to enable estimation of doubling time. The cell doubling time for cultures of D. spicata grown continuously in 200 mM salt was 40.5 h ± 2.0 hours (3 cultures) which is not significantly different from controls. Figure 2 B illustrates the different response of cultures stepped-up to 200 mM NaCI as compared with salt grown cultures at the same NaCI concentration. The reversion of such salt stressed cultures to doubling times similar to salt free controls had occurred within, at most, 2 subcultures in 200 mM NaCI after step-up to that salt level from salt free medium. It should be noted that cell number doubling times determined in these experiments are shorter than the CV60 doubling times obtained from initial measurements. A very common feature of suspension cultures during exponential cell division is a decline in average cell volume, and this is manifested as a discrepancy between packed cell volume doubling and cell number doubling (the latter being more rapid). Alternatively, the discrepancy could reflect a real difference due to the reduction in 2,4-D concentration in medium used in the cell counting experiments (4 to 2 mg 1-1) or a selection for more rapidly dividing cells over the years of weekly subculture. The essential point, however, is very clear; the salt tolerance trait is extremely stable in D. spicata cultures and is m;nntained without selection pressure. In contrast to D. spicata, the growth of N sylvestris and Z. mays suspension cultures was strongly inhibited by relatively low levels of NaCI. N sylvestris was the most Z. Pjl.anzenphysiol. Ed. 107. S. 347-356. 1982.
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sensltlve: growth was slowed considerably by a long lag period at concentrations above 43 mM and at 170 mM inhibition was complete. This is consistent with the observations of Nabors et al. (1975) with N. sylvestris suspensions. Only a few percent of the cells appeared to be alive when examined microscopically after one week in medium containing 170 mM salt. Growth curves from one representative trial are presented in Figure 1. Z. mays in suspension culture was somewhat more tolerant of NaCI than was N. sylvestris, but still dramatically less so than D. spicata. At 100 mM salt the doubling time (CV60) had increased by about 30 % in Z. mays suspensions (vs. about 400 % for N. sylvestris); at 200 mM there was still consistent although- very slow growth of the cultures. o Distichlis spicala o Zea mays 6.
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The mean CV60 doubling times over eight separate trials for all three species are plotted against concentration of NaCl in the medium in Figure 3. Also included, is a plot of dry weight doubling time for S. maritima calculated from the data of von Hedenstr6m and Breckle (1974).
Discussion The appearance and the response to auxin of D. spicata tissue cultures initiated from primary explants are consistent with the observations of Cure and Mott (1978) and Mott and Cure (1978) on seven different grass species. This supports the interpretation that the tissues are organized and root-like. The growth of the initial cultures of D.spicata (and the other grasses used in this study) at 0.1 and 0.5 mg 1-1 2,4-D fits Cure and Mott's Class 1 category of aberrant root development; at
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1.0 mg 1-1 the pattern is similar to their Class 2 and above that level of auxin the growth can be described as their Class 3. True parenchymous callus was derived from the D. spicata primary aberrant root culture. The transformation was gradual and was accomplished by severe selection pressure for desirable culture phenotypes. This was possible due to the stable inheritance of changes that were expressed as different patterns of cell growth and tissue organization. In the selection process a number of phenotypically distinct and stable tissue lines were developed and could be maintained, as could the aberrant root tissue and the friable callus lines. The growth of D. spicata suspensions in media with added NaCl demonstrates that a significant and critical portion of this species' tolerance to salt, functions at a strictly cellular level; it is not dependent upon tissue organization or anatomical specializations such as salt glands, the endodermis or succulence. This conclusion is consistent with the results of von Hedenstrom and Breckle (1974) from the marsh dicots S. maritima and S. europaea, and also with the results of Tal et al. (1978) comparing salt tolerant and sensitive species of Lycopersicon and Solanum. These results, however, are all in contrast with those of Strogonov (1973) who failed to find a difference in salt tolerance between callus from Salicornia species and a number of glycophytes. A comparison of growth performance under salt stress between S. maritima and S. europea cultures (von Hedenstrom and Breckle, 1974) and D. spicata cultures is interesting in respect of the expression of whole plant characteristics in tissue culture. Optimal levels of salt for growth of S. maritima and S. europea suspension cultures (about 85 mM and 170 mM respectively; see Figure 3 for S. maritima) were about half the optima for whole plants (Flowers et al., 1977). At salt levels corresponding to field conditions (about 340 mM) growth of S. maritima suspension cultures was very strongly inhibited (no detailed data were presented for S. europea). Thus, whilst salt tolerance of the Suaeda and Salicornia species has a strong cellular basis, anatomical and physiological specializations such as succulence and salt glands also playa large role at the whole plant level. In contrast to these two dicotyledonous species, the growth rates of cell cultures of D. spicata are not stimulated by NaCl. Growth as measured by a cell volume parameter (CV60) is not inhibited until NaCl concentrations exceed 170 mM. In terms of cell number increase, 200 mM NaCl is insufficient to depress the generation time of D. spicata suspension cultures if they are continuously subcultured at this level of salt stress, and cell doubling time is only very slightly influenced if cultures are taken from 0 to 200 mM NaCl in a single step. CV60 doubling times increase steadily above 170 mM NaCl but growth is rapid at salinities typical of field conditions and can still occur at 860 mM salt. In suspension culture, therefore, D. spicata can deal more effectively with NaCl than can S. maritima, although in the field they appear to be approximately the same in that respect. D. spicata suspensions are also at least as salt tolerant as those of S. europea, yet as a whole plant the latter can survive at molar or greater soil NaCl, whilst D. spicata is very rarely found with soil salinities much
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above 680 mM (Niering and Warren, 1980). These comparisons suggest that salt tolerance in D. spicata has a relatively more important cellular component, and is less dependent upon whole plant organization than is the tolerance of S. europea of S. maritima. In a recent publication, Smith and McComb (1981) report that two dicot halophytes, Atriplex undulata and Suaeda australis, exhibit no cellularly based salt tolerance in callus cultures, and the authors conclude that in these species, salt tolerance depends entirely upon anatomical and physiological specializations at the whole plant level. Clearly, the data presented here do not support such a conclusion for D. spicata, and there are at least two possible explanations for this disagreement. Smith and McComb (1981) used fresh weight determinations to calculate growth in their callus cultures, and they emphasized that such measurements are open to criticism. Certainly, the different osmotic environments suffered by callus cells under different degrees of salt stress, could alter the relative contribution of cell associated water to changes in fresh weight. The results reported here may provide the first evidence to support Jagels' (1973) hypothesis that the salt tolerance of monocots has, in general, a stronger cellular basis than that of dicots. They also suggest, as do the results of Orton (1980), that selection for salt tolerance in tissue culture, especially with monocots, should be a realistic strategy for improving the salt tolerance of crop speCles.
References ADAMS, D. c.: Factors influencing vascular plant zonation in North Carolina salt marshes. Ecology 44, 445-446 (1963). CHAPMAN, B. J.: Salt marshes and salt deserts of the world. 1st Edition. Interscience Publishers, New York, pp. 392, 1974. CURE, W. W. and R. L. MOTI: A comparative anatomical study of organogenesis in cultured tissues of maize, wheat and oats. Physio!. Plant. 42, 91-96 (1978). FLOWERS, T. J., P. F. TROKE, and A. R. YEO: The mechanism of salt tolerance in halophytes. Ann. Rev. Plant. Physio!. 28,89-121 (1977). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Exptl. Cell Res. 50,151-158 (1968). GOULD, A. R.: Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G 1 transition probability mode!. Planta 137,29-36 (1977). HANSEN, D. J., P. DAYANANDAN, P. B. KAUFMAN, and J. D. BROTHERSON: Ecological adaptations of salt marsh grass Distichlis spicata (Gramineae), and environmental factors affecting its growth and distribution. Am. J. Bot. 63, 635-650 (1976). HEDENSnOM, H. VON and S. W. BRECKLE: Obligate halophytes? A test with tissue cultllre methods. Z. Pflanzenphysiol. 74,183-185 (1974). JAGELS, R.: Studies of a marine grass, Thalassia testudinum. I. Ultrastructure of the osmoregulatory leaf cells. Am. J. Bot. 60, 1003-1009 (1973). KEMP, P. R. and G. L. CUNNINGHAM: Light, temperature and salinity effects on growth, leaf anatomy and photosynthesis of Distichlis spicata (L.) Greene. Am. J. Bot. 68, 507-516 (1981). MOTI, R. L. and W. W. CURE: Anatomy of maize tissue cultures. Physio!. Plant. 43, 139-145 (1978).
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MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio!. Plant. 15, 473-497 (1962). NABORS, M. W., A. DANIELS, L. NADOLNY, and C. BROWN: Sodium chloride tolerance lines of tobacco cells. Plant Sci. Let. 4,155-159 (1975). NIERING, W. A. and R. S. WABREN: Tidal wetlands of Connecticut I: Vegetation and associated animal populations. Dept. of Env. Protection, State of Conn., Hartford, 1974. - - Vegetation patterns and processes in New England salt marshes. BioSci. 30, 301-307 (1980). ORTON, T. J.: Comparison of salt tolerance between Hordeum vulgare and R jubatum in whole plants and callus cultures. Z. Pflanzenphysio!. 98, 105-118 (1980). PARRONDO, R. T., J. G. GOSSELINK, and C. S. HOPKINSON: Effects of salinity and drainage on the growth of three salt marsh grasses. Bot. Gaz. 139,102-107 (1978). SMITH, M. K. and J. A. MCCOMB: Effect of NaCl on the growth of whole plants and their corresponding callus cultures. Aust. J. Plant Physio!. 8, 267-275 (1981). STROGONOV, B. P.: Salt tolerance in isolated tissues and cells. In: Structure and Function of Plant Cells in Saline Habitats. New Trends in the Study of Salt Tolerance. Trans!. from Russian by Israel Program for Scientific Translations; Jerusalem, pp. 1-33, 1973. TAL, M., H. HEIKIN, and K. DEHAN: Salt tolerance in the wild relatives of the cultivated tomato: Response of callus tissue of Lycopersicon esculentum, L. peruvianum and Solanum pennellii to high salinity. Z. Pflanzenphysio!. 86, 231-240 (1978). TAYLOR, N.: Salt tolerance of Long Island salt marsh plants. N. Y. State Mus. Circ. 23, pp 42, 1939. UNGAR, I. A.: Salt tolerance of plants growing in saline areas of Kansas and Oklahoma. Ecology 47, 154-155 (1966).
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