Plant Cell Reports
Plant Cell Reports (1987) 6:31-34
© Springer-Verlag 1987
Tissue culture evaluation of NaCI tolerance in Cellular versus whole plant response
Medicagospecies:
T. J. McCoy Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA Received October 6, 1986 / Revised version received December 4, 1986 - Communicated by I. K. Vasil
ABSTRACT Tissue culture responses to three levels of NaCI (0, 85mM and 170 mM) were evaluated in several Medicago species including: M. dzhawakhetica, M. marina, M. rhodopea, M. rupestris , M. sativa (alfalfa) and M. suffruticosa. The whole plant responses of the same genotypes were evaluated in half-strength Hoagland's solution containing 0,51.5, and 103 mM NaCI. One or more genotypes of M. dzhawakhetica, M. rhodopea, M. rupestris, and M. sativa exhibited in vitro NaCI tolerance at 85 mM. In addition, one genotype each of M. dzhawakhetica, M. rhodopea, and M. sativa was tolerant of 170 mM NaCI. However, all of the genotypes that demonstrated NaCI tolerance in vitro were NaCI sensitive at the whole plant level. Conversely, M. marina the only species exhibiting whole plant NaCI tolerance, had the most NaCI sensitive genotypes at the in vitro level. Although an in vitro NaCI tolerance mechanism which confers whole plant NaCI tolerance was not observed, a potential NaCI tolerance germplasm source, M. marina, was identified. INTRODUCTION
and Rains 1984). However, in vitro selection has not resulted in the production of NaCI tolerant alfalfa plants. Although a halophytic type of cellular salt tolerance was obtained by Croughan et al. (1978), the plants regenerated from NaCI tolerant lines were so stunted and weak that whole plant tolerance was never evaluated (Stavarek and Rains, 1984). In a preliminary study, Smith and HcComb (1981b) found a correlation between in vitro and in vivo NaCI tolerance in alfalfa. However, after successful selection of a NaCI tolerant cell line capable of plant regeneration, Smith and McComb (1983) found the regenerated plants were as salt sensitive as the initial plants. This report describes the cellular and whole plant response to NaCI of several Medicago species. All of the species tested can be hybridized with alfalfa by using an embryo rescue method recently developed in our laboratory (McCoy and Smith 1986). One of the Species tested, M. marina L. is assumed to be salttolerant due to its exclusive habitat of seashore sands along the Mediterranean and the Atlantic coast of France, Portugal and Spain (Lesins andLesins 1979). The objective of this study was to determine if a cellular mechanism for whole plant NaCI tolerance could be identified either in cultivated or wild species of Medicago. If identified, the trait could be incorporated into cultivated germplasm using cellular evaluation of hybrids and backcross generations.
Research has demonstrated that the selection of NaCI tolerant cell lines is relatively straightforward and highly effective (Stavarek and Rains 1984). Almost all cases of cell culture selection of NaCI tolerant cell lines have relied on spontaneously occurring variation. Where mutagenesis was employed, e.g., selection of NaCI tolerant cell lines of Citrus (Kochba et al. 1982), it was found not to increase the recovery rate of NaCI tolerant cell lines. Unfortunately, although salt tolerant cell lines are easily selected, there have been only a few cases where in vitro selection resulted in heritable NaCI tolerance expressed at the whole plant level (Nabors et al. 1980, 1982). In tobacco, Nabors et al. (1980) identified heritable tolerance that resulted in plants tolerant of 3.3% NaCI. In most of the cases above either plants were not regenerated from NaCI tolerant cell lines, or if tested the plants have not demonstrated increased NaCI tolerance (Smith and McComb 1983). In one study, Chandler and Vasil (1984) demonstrated that NaCI tolerance was lost upon callus transfer to NaCl-free medium, and that plants regenerated from the selected callus were in fact more sensitive to NaCI than plants regenerated from unselected callus.
Callus cultures were initiated from immature ovaries of the following species (i0 to 51 genotypes were tested per species): M. dzhawakhetica Bordz. (2n=4x = 32), M. hybrida Traut. (2n=2x=16), M. marina L. (2n= 2x=16), M. papillosa Boiss. (2n=2x=16 and 2n=4x=32), M. rhodopea Velen. (2n=2x=16), M. rupestris M.B. (2n= 2x=16), M. sativa L. (2n=2x=16 and 2n=4x=32) and M. suffruticosa Ram. (2n=2x=16). The species were chosen based on the successful recovery of interspecific hybrids with alfalfa (M. sativa) either directly from seed (McCoy and Smith 1984) or by embryo rescue (McCoy 1985; McCoy and Smith 1986). All cultures were grown on the media of Schenk and Hildebrandt (1972), containing 2mg/l 2,4-D and 2mg/l kinetin (designated SH medium).
In alfalfa, cellular selection for salt tolerance has resulted in NaCI tolerant cell lines (Croughan et al. 1978, 1981; Smith and McComb 1983; and Stavarek
After 28 days the best genotypes were visually selected on the basis of callus production. Poor callus producers were excluded at this time. Callus from
MATERIALS AND METHODS Cellular Response
32 the selected genotypes was subcultured (200-250 mg callus/lO0x20mm culture plate containing 50 ml. of media). Callus fresh weights were then determined after an additional 28 days, and the best genotypes with similar fresh weight increases were selected. Although this greatly limited the number of genotypes which could be tested, the genotypic response to NaCI should be due to actual differential tolerance of NaCI, and not to better callus growth rates per s e. The cellular response to NaCI was determined by transferring 200-250 mg of callus per 100x20mm culture plate containing 50 ml of SH media. NaCI concentrations tested were 0, 0.5% (85.6 mM) and 1.01 (171.1 mM), with five replicates per genotype. Final callus fresh and dry weights were determined after 28 days' growth in a growth chamber (25°C * 1 °, 24 h light and approximately 60% relative humidity). Whole Plant Response Vegetative propagules of each of the 36 gen0types were rooted in a mist bench and transferred to sand. Plants were grown in sand for eight weeks and then cut back and transferred to tanks containing 1 2 1 of half-strength Hoagland's solution. Each tank contained four propagules of each of eight genotypes for a total of 32 plants. All plants were grown in growth chambers at a constant temperature of 24°C ± 2° with a 16 h day (light intensity 125 pE.m-2.sec-l). Three days after transfer to the tanks NaCI was added. Preliminary experiments used the same NaCI concentrations that were used in the in vitro test: 0, 0.5% and 1.0% NaCI in half-strength Hoagland's solution. However, because the lowest concentration, 0.5%, was toxic to many of the genotypes subsequent experiments were conducted with 0.3% (51.4 mM) and 0.6% (102.7 raM) NaCI, with three replicates per genotype. As recommended by Greenway and Munns (1980), CaSO 4 was added so that the N a S a ratio (mM) did not exceed I7 (actual ratio was 15). Tanks were aerated for one h daily and all solutions were changed twice each week. Following three weeks growth, plants were harvested and fresh weight of shoots was determined. Table 1 Origin of all clones used in the experiments comparing whole plant and callus growth at different NaCI concentrations. Clone
Species
Origin
SAT-I,-2,-3,-4 M. sativa Regen-S (Bingham et al. 1975) SAT-5,-6,-14 M. sativa NMP 33 germplasm SAT-7,-8,-II,-13 M. sativa Florida 77 SAT-9,-12 M. sativa Moapa 69 SAT-10 M. sativa Narragansett MAR-I M. marina UAG 319 a MAR-2,-3 M. marina TM 1704 b MAR-4,-5 M. marina TM 1705 MAR-6,-7 M. marina UAG 320 MAR-8,-9 M. marina UAG 356 DZH-I,-2,-3,-4 M. dzhawakhetica UAG 98 RHO-I,-2 M. rhodopea UAG 493 RHO-3,-4 M. rhodopea UAG 494 RUP-I,-2,-3 M. rupestris UAG 1847 SUF-I,-2 M. suffruticosa UAG 1545 a)
UAG denotes Univ. of Alberta, Genetics Department. Seed of these accessions were obtained from the collection of Dr. K. Lesins.
b)
TM entries are maintained by the author. Original country of origin was Morocco. Accessions were collected in 1983 by Dr. Mel Rumbaugh, USDA/ARS. Utah State University, Logan, Utah.
RESULTS AND DISCUSSION The origin of the 36 genotypes selected for testing of NaCI tolerance at the callus and whole plant levels is given in Table i. None of the genotypes of M. hybrida or M. papillosa produced adequate callus ~or testing. One or more genotypes tolerant of 0..5% NaCI at the callus level were observed in all species exceptM. marina and M. suffruticosa (Table 2). In addition four genotypes: SAT-8, SAT-II, DHZ-2and RHe-4 were tolerant of 1.0% NaCI at the callus level without any selection for tolerance. However, none of these genotypes had a cellular tolerance mechanism that provided a high level of NaCI tolerance at the whole plant level (Table 3), although SAT-8 and S~T-II were two of the four M. sativa genotypes that survived the 0.6% NaCI treatment (shoot weight was 14.6% and 8.2% of control, respectively). Considering M. dzhawakhetica, although DZH-2 was the most NaCI tolerant at the callus level, this was the least tolerant of the four M. dzhawakhetica genotypes tested at the whole plant level. Based on the genotypes tested here, it appears there is an almost inverse relationship between the most NaCl.tolerant genotype at the whole plant level and cellular NaCI tolerance. This relationship is best exemplified by the M. marina genotypes. Results here confirm the assumed NaCI tolerance of M. marina plants (Table 3). All nine genotypes of M. marina (representing five accessions) were tolerant of 0.6% NaCI at the whole plant level. In fact, genotypes generally performed better in a 0.6% NaCI solution than in control solution, consistent with a halophytic growth habit. However, at the callus level, all M. marina genotypes were NaCI sensitive. In fact, M. marina genotypes were as NaCI sensitive at the cellular level as the most intolerant genotypes of M. sativa. Although M. marina does not have a cellular based mechanism for NaCI tolerance, perhaps efforts at incorporating the whole plant tolerance through interspecific hybridization will be fruitful. Embryo rescue experiments in our laboratory have demonstrated that interspecific hybrids between M. sativa and M. marina can be recovered (McCoy and Smith 1986). These hybrids will be evaluated for whole plant NaCI tolerance. Tests of the 36 genotypes of several Medicago species have not identified a cellular mechanism for whole plant NaCI tolerance. These results are similar to those of Hanson (1984) who found no differential NaCI tolerance between callus cultures of tomato (NaCI sensitive at the whole plant level) and its relative Solanum pennellii (NaCI tolerant at the whole plant level). Lack of a correlation between whole plant and callus cultures was also identified by Smith and McComb (1981a) who found the halophytic species Atriplex undulata and Suaeda australis to be as susceptible to NaCI as the glycophyte Phaseolus vulgaris. In contrast, other reports have identified cellular based mechanisms for NaCI tolerance in Hordeum (Orton 1980) Lycopersicon and Solanum (Tal et al. 1978) and Spartina (Warren et al. 1985). The inconsistency in the in vitro response to salt may be due to the multiplicity of mechanisms for whole plant salt tolerance. Levitt (1980) discusses several major avoidance and/or tolerance mechanisms for salt tolerance. Some of these may be operational at the cellular level only and thus expressed in vitro while other mechanisms may operate only at the organismal level. Although a cellular-based NaCI tolerance mechanism was not identified in Medicago, a high level of whole plant NaCI tolerance was demonstrated in M.
33 Table 2 Final callus fresh weight of various genotypes containing 0%, 0.5%, or 1.0% NaCI.
following
Final Fresh Weight Clone
0% NaCI
four weeks growth on media
(g)a
0.5% NaCI
1.0% NaCI
SAT-I SAT-2 SAT-3 SAT-4 SAT-5 SAT-6 SAT-7 SAT-8 SAT-9 SAT-10 SAT-II SAT-12 SAT-13 SAT-14
3.67 2.91 2.28 2.56 1.78 4.55 4.62 2.84 3.16 3.61 2.15 2.64 5.03 2.12
± ± ± i t ± ± ± + ± ± ± ± ±
1.12 0.48 0.54 0.09 0.19 1.61 0.22 0.36 0.29 0.72 0.68 0.05 0.49 0.70
0.96 0.87 0.92 1.30 0.57 3.'23 1.48 2.30 0.71 0.50 3.19 0.63 2.65 0.38
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.52 0.29 0.18 0.19 0.41 0.39 0.52 0.63 0.41 0.08 1.21 0.31 0.83 0.22
(26.2) (29.9) (40.4) (50.8) (32.0) (71.0) (32.0) (81.0) (22.5) (13.9) (148.4) 23.9) 52.7) 17.9)
0.31 0.38 0.43 0.21 0.20 0.34 1.82 2.96 0.27 0.58 2.74 0.36 0.80 0.40
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24 0.27 0.22 0.05 0.09 0.06 0.85 1.21 0.ii 0.32 1.08 0.31 0.ii 0.08
( 8.4) (13.1) (18.9) ( 8.2) (11.2) ( 7.5) (39.4) (104.2) ( 8.5) (16.1) (127.4) 13.6) 15.9) 18.9)
MAR-I MAR-2 MAR-3 MAR-4 MAR-5 MAR-6 MAR-7 MAR-8 MAR-9
3.19 2.13 1.89 2.62 2.53 1.94 2.15 2.36 1.60
± ± ± ± ± ± ± ± ±
0.56 0.55 0.44 0.18 0.21 0.52 0.34 0.34 0.27
0.88 0.81 0.22 0.36 0.39 0.34 0.42 0.52 0.59
± ± ± ± ± t + ± ±
0.39 0.26 0.06 0.27 0.08 0.06 0.18 0.23 0.20
27.6) 38.0) ii.6) 13.7) 15.4) (17.5) (19.5) (22.0) (36.9)
0.51 0.30 0.46 0.52 0.18 0.28 0.37 0.31 0.34
± -+ ± i ± ± i ± ±
0.21 0.18 0.22 0.14 0.07 0.07 0.15 0.09 0.09
16.0)
DZH-I DZH-2 DZH-3 DZH-4
2.95 2.13 2.06 1.71
i ± ± ±
0.61 0.25 0.36 0.40
0.85 2.30 0.76 0.43
± ± ± ±
0.41 0.83 0~21 0.09
(28.8) (108.0) (36.9) (25.3)
0.29 2.55 0.65 0.33
± ± ± ±
0.ii 0.68 0.24 0.12
( 9.8) (119.7) 31.6) 19.3)
RHO-I RHO-2 RHO-3 RHO-4
3.85 1.98 4.06 3.19
± ± ± ±
0.88 0.45 1.44 0.68
4.28 3.67 2.81 4.68
± ± ± ±
1.16 0.8-3 0.97 1.33
(111.2) (185.4) ( 69.2) (146.7)
2.36 1.53 0.39 2.90
± ± ± ±
0.31 0.43 0.i0 0.81
61.3) ( 77.3) 9.6) 90.9)
RUP-I RUP-2 RUP-3
2.16 ± 0.46 5.81 ± 1.73 2.84 ± 0.99
2.08 ± 0.81 3.99 ± 0.95 3.18 ± 0.73
(96.3) (68.7) (111.8)
0.75 ± 0.48 0.46 ± 0.05 0.32 ± 0.12
34.7) 7.9) (11.3)
SUF-I SUF-2
2.28 i 1.01 2.97 ± 0.33
0.48 ± 0.36 0.51 ± 0.ii
(21.1) (17.2)
0.24 ± 0.13 0.13 ± 0.04
(10.5) ( 4.5)
a)
Mean ± standard deviation the percent control.
for five replicates.
marina, a species capable of interspecific hybridization with alfalfa (McCoy and Smith 1986). This opens one avenue of potentially improving the NaCI tolerance of alfalfa. An alternative approach is to identify a cellular NaCI tolerance mechanism by the direct selection of NaCI tolerant cell lines from originally NaCI sensitive genotypes which have the capacity to regenerate plants.
REFERENCES Bingham ET, Hurley LV, Kaatz DM and Saunders JW (1975) Crop Sci. 15:719-721 Chandler SF, Vasil IK (1984) Plant Sci. Lett. 37:157164
i4.1) 24.3) 19.8) 7.i) 14.4) 17.2) 13. i) 21.3)
Values in parenthesis
are
Croughan TP, Stavarek SJ, Rains DW (1978) Crop Sci. 18:959-963 Croughan TP, Stavarek SJ, Rains DW (1981) Env. Exp. Bet. 21:317-324 Greenway H, Munns R (1980) Ann, Rev. Plant Physiol. 31:149-190 Hanson MR (1984) In: Staples RC, Toenniessen GH (ed.) Salinity Tolerance in Plants. Wiley-Interscience, New York, pp 335-359 Kochba J, Ben-Hayyim G, Spiegel-Roy P, Saad S, Newmann H (1982) Z. Pflanzenphysiol 106:111-118 Lesins KA, Lesins I (1979) Genus Medicago (Leguminosae) A Taxogenetic Study. Dr. W. Junk by Publishers, The Hague, Berlin, Boston Levitt J (1980) Responses of Plants to Environmental Stresses Vol II. Water, Radiation, Salt and other Stresses (2nd edition) Academic Press, New'York
34 Table 3. Mean shoot fresh weight following three weeks growth in areated half-strength solution containing 0%, 0.3% or 0.6% NaCI.
Hoagland's
Shoot Fresh Weight (g)a Clone
0% NaCI
0.3% Nacl
0.6% Nacl
SAT-I SAT-2 SAT-3 SAT-4 SAT-5 SAT-6 SAT-7 SAT-8 SAT-9 SAT-10 SAT-II SAT-12 SAT-13 SAT-14
6.72 6.70 8.07 i0.04 6.01 12.10 7.91 ii.89 11.50 9.50 18.01 5.86 18.56 ii.44
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.27 2.07 2.23 1.05 0.93 3.30 2.30 2.20 3.08 3.36 2.57 1.90 2.87 2.19
1.25 1.87 1.58 1.86 0.73 4.47 1.89 5.88 2.29 1.31 5.69 2.23 9.05 2.04
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.31 0.71 0.73 0.48 0.20 1.26 0.38 0.98 1.02 0.36 1.16 0.92 2.64 1.40
(18.6) (27.9) (19.6) (18.5) (12.1) (36.9) (23.9) (49.5) (19.9) (13.8) (31.6) (38.1) (48.8) (17.8)
Died Died Died Died Died 1.49 Died 1.73 Died Died 1.48 Died 2.25 Died
MAR-I MAR-2 MAR-3 MAR-4 MAR-5 MAR-6 MAR-7 MAR-8 MAR-9
1.66 2.05 1.70 1.21 1.90 1.83 1.57 1.14 1.42
± ± ± ± ± ± ± ± ±
0.64 0.74 0.55 0.44 0.45 0.50 0.37 0.34 0.38
1.70 3.79 1.60 1.70 2.39 3.14 1.37 2.58 1.75
± ± ± ± ± ± ± ± ±
0.30 0.98 0.32 0.66 0.31 0.29 0.39 0.62 0.15
(102.4) (184.9) (94.1) (140.5) (125.8) (171.6) (87.3) (226.3) (123.2)
2.16 3.28 2.06 2.47 3.44 3.18 2.01 2.35 1.73
DZH-I DZH-2 DZH-3 DZH-4
2.76 3.56 1.28 2.75
_+ 1.22 ± 1.89 ± 0.29 ± 0.91
2.17 1.42 1.66 3.33
± ± ± ±
0.63 0.43 0.40 0.72
(78.6) (39.9) (129.6) (121.1)
Died Died Died Died
RHO-I RHO-2 RHO-3 RHO-4
4.37 3.71 2.54 2.86
± 1.76 ± 1.45 ± 1.13 _+ 0.66
1.95 1.06 1.86 0.94
± ± ± ±
0.30 0.21 0.93 0.51
(44.6) (28.6) (73.2) (32.9)
Died Died 0.81 ± 0.43 Died
RUP-I RUP-2 RUP-3
1.97 ± 0.73 1.24 ± 0.69 3.45 ± 1.61
0.93 ± 0.41 1.00 ± 0.29 0.78 ± 0.36
47.2) 80.6) 22.6)
Died Died Died
SUF-I SUF-2
5.61 ± 1.73 3.26 ± 0.88
1.07 ± 0.53 0.93 ± 0.21
19.1) 28.5)
Died Died
a)
± 0.36
(12.3)
± 0.63
(14.6)
± 0.81
(8.2)
+_ 0.67
(12.1)
± ± ± ± ± ± ± ± ±
(130.1) (160.0) (121.1) (204.1) (181.1) (173.8) (128.0) (206.1) (121.8)
0.33 0.99 0.81 0.87 0.82 0.65 0.22 0.38 0.64
(31.9)
Mean ± standard deviation for three replicates; each replicate consisted of four vegetative propagules. Values in parenthesis are the percent control.
McCoy TJ (1985) Can. J. Genet. Cytol. 27:238-245 McCoy TJ, Smith LY (1984) Can. J. Genet. Cytol. 26: 511-518 McCoy TJ, Smith LY (1986) Theor. Appl. Genet. 71: 772-783 Nabors MW, Gibb SE, Bernstein CS, Meis ME (1980) Z. Pflanzenphysiol 97:13-17 Nabors MW, Kroskey CS, McHugh DM (1982) Z. Pflanzenphysiol. 105:341-349 Orton TJ (1980) Z. Pflanzenphysiol. 98:105-118 Schenk RU, Hildebrandt AC (1972) Can. J. Bot. 50: 199-204 Smith MK, McComb JA (1981a) Aust. J. Plant Physiol.8: 267-275
Smith MK, McComb JA (1981b) Aust. J. Plant Physiol.8: 437-442 Smith MK, McComb JA (1983) Plant Cell Reports 2:126128 Stavarek SJ, Rains DW (1984) In: Staples RC, Toenniessen GH (ed.) Salinity Tolerance in Plants, Wiley-Interscience, New York pp 321-334 Tal M, He±ken H, Dehan K (1978) Z. Pflanzenphysiol. 86:231-240 W~rren RS, Baird LM, Thompson AK (1985) Plant Cell Reports 4:84-87
Plant Cell Reports (1987) 6:31-34
© Springer-Verlag 1987
Tissue culture evaluation of NaCI tolerance in Cellular versus whole plant response
Medicagospecies:
T. J. McCoy Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA Received October 6, 1986 / Revised version received December 4, 1986 - Communicated by I. K. Vasil
ABSTRACT Tissue culture responses to three levels of NaCI (0, 85mM and 170 mM) were evaluated in several Medicago species including: M. dzhawakhetica, M. marina, M. rhodopea, M. rupestris , M. sativa (alfalfa) and M. suffruticosa. The whole plant responses of the same genotypes were evaluated in half-strength Hoagland's solution containing 0,51.5, and 103 mM NaCI. One or more genotypes of M. dzhawakhetica, M. rhodopea, M. rupestris, and M. sativa exhibited in vitro NaCI tolerance at 85 mM. In addition, one genotype each of M. dzhawakhetica, M. rhodopea, and M. sativa was tolerant of 170 mM NaCI. However, all of the genotypes that demonstrated NaCI tolerance in vitro were NaCI sensitive at the whole plant level. Conversely, M. marina the only species exhibiting whole plant NaCI tolerance, had the most NaCI sensitive genotypes at the in vitro level. Although an in vitro NaCI tolerance mechanism which confers whole plant NaCI tolerance was not observed, a potential NaCI tolerance germplasm source, M. marina, was identified. INTRODUCTION
and Rains 1984). However, in vitro selection has not resulted in the production of NaCI tolerant alfalfa plants. Although a halophytic type of cellular salt tolerance was obtained by Croughan et al. (1978), the plants regenerated from NaCI tolerant lines were so stunted and weak that whole plant tolerance was never evaluated (Stavarek and Rains, 1984). In a preliminary study, Smith and HcComb (1981b) found a correlation between in vitro and in vivo NaCI tolerance in alfalfa. However, after successful selection of a NaCI tolerant cell line capable of plant regeneration, Smith and McComb (1983) found the regenerated plants were as salt sensitive as the initial plants. This report describes the cellular and whole plant response to NaCI of several Medicago species. All of the species tested can be hybridized with alfalfa by using an embryo rescue method recently developed in our laboratory (McCoy and Smith 1986). One of the Species tested, M. marina L. is assumed to be salttolerant due to its exclusive habitat of seashore sands along the Mediterranean and the Atlantic coast of France, Portugal and Spain (Lesins andLesins 1979). The objective of this study was to determine if a cellular mechanism for whole plant NaCI tolerance could be identified either in cultivated or wild species of Medicago. If identified, the trait could be incorporated into cultivated germplasm using cellular evaluation of hybrids and backcross generations.
Research has demonstrated that the selection of NaCI tolerant cell lines is relatively straightforward and highly effective (Stavarek and Rains 1984). Almost all cases of cell culture selection of NaCI tolerant cell lines have relied on spontaneously occurring variation. Where mutagenesis was employed, e.g., selection of NaCI tolerant cell lines of Citrus (Kochba et al. 1982), it was found not to increase the recovery rate of NaCI tolerant cell lines. Unfortunately, although salt tolerant cell lines are easily selected, there have been only a few cases where in vitro selection resulted in heritable NaCI tolerance expressed at the whole plant level (Nabors et al. 1980, 1982). In tobacco, Nabors et al. (1980) identified heritable tolerance that resulted in plants tolerant of 3.3% NaCI. In most of the cases above either plants were not regenerated from NaCI tolerant cell lines, or if tested the plants have not demonstrated increased NaCI tolerance (Smith and McComb 1983). In one study, Chandler and Vasil (1984) demonstrated that NaCI tolerance was lost upon callus transfer to NaCl-free medium, and that plants regenerated from the selected callus were in fact more sensitive to NaCI than plants regenerated from unselected callus.
Callus cultures were initiated from immature ovaries of the following species (i0 to 51 genotypes were tested per species): M. dzhawakhetica Bordz. (2n=4x = 32), M. hybrida Traut. (2n=2x=16), M. marina L. (2n= 2x=16), M. papillosa Boiss. (2n=2x=16 and 2n=4x=32), M. rhodopea Velen. (2n=2x=16), M. rupestris M.B. (2n= 2x=16), M. sativa L. (2n=2x=16 and 2n=4x=32) and M. suffruticosa Ram. (2n=2x=16). The species were chosen based on the successful recovery of interspecific hybrids with alfalfa (M. sativa) either directly from seed (McCoy and Smith 1984) or by embryo rescue (McCoy 1985; McCoy and Smith 1986). All cultures were grown on the media of Schenk and Hildebrandt (1972), containing 2mg/l 2,4-D and 2mg/l kinetin (designated SH medium).
In alfalfa, cellular selection for salt tolerance has resulted in NaCI tolerant cell lines (Croughan et al. 1978, 1981; Smith and McComb 1983; and Stavarek
After 28 days the best genotypes were visually selected on the basis of callus production. Poor callus producers were excluded at this time. Callus from
MATERIALS AND METHODS Cellular Response
32 the selected genotypes was subcultured (200-250 mg callus/lO0x20mm culture plate containing 50 ml. of media). Callus fresh weights were then determined after an additional 28 days, and the best genotypes with similar fresh weight increases were selected. Although this greatly limited the number of genotypes which could be tested, the genotypic response to NaCI should be due to actual differential tolerance of NaCI, and not to better callus growth rates per s e. The cellular response to NaCI was determined by transferring 200-250 mg of callus per 100x20mm culture plate containing 50 ml of SH media. NaCI concentrations tested were 0, 0.5% (85.6 mM) and 1.01 (171.1 mM), with five replicates per genotype. Final callus fresh and dry weights were determined after 28 days' growth in a growth chamber (25°C * 1 °, 24 h light and approximately 60% relative humidity). Whole Plant Response Vegetative propagules of each of the 36 gen0types were rooted in a mist bench and transferred to sand. Plants were grown in sand for eight weeks and then cut back and transferred to tanks containing 1 2 1 of half-strength Hoagland's solution. Each tank contained four propagules of each of eight genotypes for a total of 32 plants. All plants were grown in growth chambers at a constant temperature of 24°C ± 2° with a 16 h day (light intensity 125 pE.m-2.sec-l). Three days after transfer to the tanks NaCI was added. Preliminary experiments used the same NaCI concentrations that were used in the in vitro test: 0, 0.5% and 1.0% NaCI in half-strength Hoagland's solution. However, because the lowest concentration, 0.5%, was toxic to many of the genotypes subsequent experiments were conducted with 0.3% (51.4 mM) and 0.6% (102.7 raM) NaCI, with three replicates per genotype. As recommended by Greenway and Munns (1980), CaSO 4 was added so that the N a S a ratio (mM) did not exceed I7 (actual ratio was 15). Tanks were aerated for one h daily and all solutions were changed twice each week. Following three weeks growth, plants were harvested and fresh weight of shoots was determined. Table 1 Origin of all clones used in the experiments comparing whole plant and callus growth at different NaCI concentrations. Clone
Species
Origin
SAT-I,-2,-3,-4 M. sativa Regen-S (Bingham et al. 1975) SAT-5,-6,-14 M. sativa NMP 33 germplasm SAT-7,-8,-II,-13 M. sativa Florida 77 SAT-9,-12 M. sativa Moapa 69 SAT-10 M. sativa Narragansett MAR-I M. marina UAG 319 a MAR-2,-3 M. marina TM 1704 b MAR-4,-5 M. marina TM 1705 MAR-6,-7 M. marina UAG 320 MAR-8,-9 M. marina UAG 356 DZH-I,-2,-3,-4 M. dzhawakhetica UAG 98 RHO-I,-2 M. rhodopea UAG 493 RHO-3,-4 M. rhodopea UAG 494 RUP-I,-2,-3 M. rupestris UAG 1847 SUF-I,-2 M. suffruticosa UAG 1545 a)
UAG denotes Univ. of Alberta, Genetics Department. Seed of these accessions were obtained from the collection of Dr. K. Lesins.
b)
TM entries are maintained by the author. Original country of origin was Morocco. Accessions were collected in 1983 by Dr. Mel Rumbaugh, USDA/ARS. Utah State University, Logan, Utah.
RESULTS AND DISCUSSION The origin of the 36 genotypes selected for testing of NaCI tolerance at the callus and whole plant levels is given in Table i. None of the genotypes of M. hybrida or M. papillosa produced adequate callus ~or testing. One or more genotypes tolerant of 0..5% NaCI at the callus level were observed in all species exceptM. marina and M. suffruticosa (Table 2). In addition four genotypes: SAT-8, SAT-II, DHZ-2and RHe-4 were tolerant of 1.0% NaCI at the callus level without any selection for tolerance. However, none of these genotypes had a cellular tolerance mechanism that provided a high level of NaCI tolerance at the whole plant level (Table 3), although SAT-8 and S~T-II were two of the four M. sativa genotypes that survived the 0.6% NaCI treatment (shoot weight was 14.6% and 8.2% of control, respectively). Considering M. dzhawakhetica, although DZH-2 was the most NaCI tolerant at the callus level, this was the least tolerant of the four M. dzhawakhetica genotypes tested at the whole plant level. Based on the genotypes tested here, it appears there is an almost inverse relationship between the most NaCl.tolerant genotype at the whole plant level and cellular NaCI tolerance. This relationship is best exemplified by the M. marina genotypes. Results here confirm the assumed NaCI tolerance of M. marina plants (Table 3). All nine genotypes of M. marina (representing five accessions) were tolerant of 0.6% NaCI at the whole plant level. In fact, genotypes generally performed better in a 0.6% NaCI solution than in control solution, consistent with a halophytic growth habit. However, at the callus level, all M. marina genotypes were NaCI sensitive. In fact, M. marina genotypes were as NaCI sensitive at the cellular level as the most intolerant genotypes of M. sativa. Although M. marina does not have a cellular based mechanism for NaCI tolerance, perhaps efforts at incorporating the whole plant tolerance through interspecific hybridization will be fruitful. Embryo rescue experiments in our laboratory have demonstrated that interspecific hybrids between M. sativa and M. marina can be recovered (McCoy and Smith 1986). These hybrids will be evaluated for whole plant NaCI tolerance. Tests of the 36 genotypes of several Medicago species have not identified a cellular mechanism for whole plant NaCI tolerance. These results are similar to those of Hanson (1984) who found no differential NaCI tolerance between callus cultures of tomato (NaCI sensitive at the whole plant level) and its relative Solanum pennellii (NaCI tolerant at the whole plant level). Lack of a correlation between whole plant and callus cultures was also identified by Smith and McComb (1981a) who found the halophytic species Atriplex undulata and Suaeda australis to be as susceptible to NaCI as the glycophyte Phaseolus vulgaris. In contrast, other reports have identified cellular based mechanisms for NaCI tolerance in Hordeum (Orton 1980) Lycopersicon and Solanum (Tal et al. 1978) and Spartina (Warren et al. 1985). The inconsistency in the in vitro response to salt may be due to the multiplicity of mechanisms for whole plant salt tolerance. Levitt (1980) discusses several major avoidance and/or tolerance mechanisms for salt tolerance. Some of these may be operational at the cellular level only and thus expressed in vitro while other mechanisms may operate only at the organismal level. Although a cellular-based NaCI tolerance mechanism was not identified in Medicago, a high level of whole plant NaCI tolerance was demonstrated in M.
33 Table 2 Final callus fresh weight of various genotypes containing 0%, 0.5%, or 1.0% NaCI.
following
Final Fresh Weight Clone
0% NaCI
four weeks growth on media
(g)a
0.5% NaCI
1.0% NaCI
SAT-I SAT-2 SAT-3 SAT-4 SAT-5 SAT-6 SAT-7 SAT-8 SAT-9 SAT-10 SAT-II SAT-12 SAT-13 SAT-14
3.67 2.91 2.28 2.56 1.78 4.55 4.62 2.84 3.16 3.61 2.15 2.64 5.03 2.12
± ± ± i t ± ± ± + ± ± ± ± ±
1.12 0.48 0.54 0.09 0.19 1.61 0.22 0.36 0.29 0.72 0.68 0.05 0.49 0.70
0.96 0.87 0.92 1.30 0.57 3.'23 1.48 2.30 0.71 0.50 3.19 0.63 2.65 0.38
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.52 0.29 0.18 0.19 0.41 0.39 0.52 0.63 0.41 0.08 1.21 0.31 0.83 0.22
(26.2) (29.9) (40.4) (50.8) (32.0) (71.0) (32.0) (81.0) (22.5) (13.9) (148.4) 23.9) 52.7) 17.9)
0.31 0.38 0.43 0.21 0.20 0.34 1.82 2.96 0.27 0.58 2.74 0.36 0.80 0.40
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.24 0.27 0.22 0.05 0.09 0.06 0.85 1.21 0.ii 0.32 1.08 0.31 0.ii 0.08
( 8.4) (13.1) (18.9) ( 8.2) (11.2) ( 7.5) (39.4) (104.2) ( 8.5) (16.1) (127.4) 13.6) 15.9) 18.9)
MAR-I MAR-2 MAR-3 MAR-4 MAR-5 MAR-6 MAR-7 MAR-8 MAR-9
3.19 2.13 1.89 2.62 2.53 1.94 2.15 2.36 1.60
± ± ± ± ± ± ± ± ±
0.56 0.55 0.44 0.18 0.21 0.52 0.34 0.34 0.27
0.88 0.81 0.22 0.36 0.39 0.34 0.42 0.52 0.59
± ± ± ± ± t + ± ±
0.39 0.26 0.06 0.27 0.08 0.06 0.18 0.23 0.20
27.6) 38.0) ii.6) 13.7) 15.4) (17.5) (19.5) (22.0) (36.9)
0.51 0.30 0.46 0.52 0.18 0.28 0.37 0.31 0.34
± -+ ± i ± ± i ± ±
0.21 0.18 0.22 0.14 0.07 0.07 0.15 0.09 0.09
16.0)
DZH-I DZH-2 DZH-3 DZH-4
2.95 2.13 2.06 1.71
i ± ± ±
0.61 0.25 0.36 0.40
0.85 2.30 0.76 0.43
± ± ± ±
0.41 0.83 0~21 0.09
(28.8) (108.0) (36.9) (25.3)
0.29 2.55 0.65 0.33
± ± ± ±
0.ii 0.68 0.24 0.12
( 9.8) (119.7) 31.6) 19.3)
RHO-I RHO-2 RHO-3 RHO-4
3.85 1.98 4.06 3.19
± ± ± ±
0.88 0.45 1.44 0.68
4.28 3.67 2.81 4.68
± ± ± ±
1.16 0.8-3 0.97 1.33
(111.2) (185.4) ( 69.2) (146.7)
2.36 1.53 0.39 2.90
± ± ± ±
0.31 0.43 0.i0 0.81
61.3) ( 77.3) 9.6) 90.9)
RUP-I RUP-2 RUP-3
2.16 ± 0.46 5.81 ± 1.73 2.84 ± 0.99
2.08 ± 0.81 3.99 ± 0.95 3.18 ± 0.73
(96.3) (68.7) (111.8)
0.75 ± 0.48 0.46 ± 0.05 0.32 ± 0.12
34.7) 7.9) (11.3)
SUF-I SUF-2
2.28 i 1.01 2.97 ± 0.33
0.48 ± 0.36 0.51 ± 0.ii
(21.1) (17.2)
0.24 ± 0.13 0.13 ± 0.04
(10.5) ( 4.5)
a)
Mean ± standard deviation the percent control.
for five replicates.
marina, a species capable of interspecific hybridization with alfalfa (McCoy and Smith 1986). This opens one avenue of potentially improving the NaCI tolerance of alfalfa. An alternative approach is to identify a cellular NaCI tolerance mechanism by the direct selection of NaCI tolerant cell lines from originally NaCI sensitive genotypes which have the capacity to regenerate plants.
REFERENCES Bingham ET, Hurley LV, Kaatz DM and Saunders JW (1975) Crop Sci. 15:719-721 Chandler SF, Vasil IK (1984) Plant Sci. Lett. 37:157164
i4.1) 24.3) 19.8) 7.i) 14.4) 17.2) 13. i) 21.3)
Values in parenthesis
are
Croughan TP, Stavarek SJ, Rains DW (1978) Crop Sci. 18:959-963 Croughan TP, Stavarek SJ, Rains DW (1981) Env. Exp. Bet. 21:317-324 Greenway H, Munns R (1980) Ann, Rev. Plant Physiol. 31:149-190 Hanson MR (1984) In: Staples RC, Toenniessen GH (ed.) Salinity Tolerance in Plants. Wiley-Interscience, New York, pp 335-359 Kochba J, Ben-Hayyim G, Spiegel-Roy P, Saad S, Newmann H (1982) Z. Pflanzenphysiol 106:111-118 Lesins KA, Lesins I (1979) Genus Medicago (Leguminosae) A Taxogenetic Study. Dr. W. Junk by Publishers, The Hague, Berlin, Boston Levitt J (1980) Responses of Plants to Environmental Stresses Vol II. Water, Radiation, Salt and other Stresses (2nd edition) Academic Press, New'York
34 Table 3. Mean shoot fresh weight following three weeks growth in areated half-strength solution containing 0%, 0.3% or 0.6% NaCI.
Hoagland's
Shoot Fresh Weight (g)a Clone
0% NaCI
0.3% Nacl
0.6% Nacl
SAT-I SAT-2 SAT-3 SAT-4 SAT-5 SAT-6 SAT-7 SAT-8 SAT-9 SAT-10 SAT-II SAT-12 SAT-13 SAT-14
6.72 6.70 8.07 i0.04 6.01 12.10 7.91 ii.89 11.50 9.50 18.01 5.86 18.56 ii.44
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.27 2.07 2.23 1.05 0.93 3.30 2.30 2.20 3.08 3.36 2.57 1.90 2.87 2.19
1.25 1.87 1.58 1.86 0.73 4.47 1.89 5.88 2.29 1.31 5.69 2.23 9.05 2.04
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.31 0.71 0.73 0.48 0.20 1.26 0.38 0.98 1.02 0.36 1.16 0.92 2.64 1.40
(18.6) (27.9) (19.6) (18.5) (12.1) (36.9) (23.9) (49.5) (19.9) (13.8) (31.6) (38.1) (48.8) (17.8)
Died Died Died Died Died 1.49 Died 1.73 Died Died 1.48 Died 2.25 Died
MAR-I MAR-2 MAR-3 MAR-4 MAR-5 MAR-6 MAR-7 MAR-8 MAR-9
1.66 2.05 1.70 1.21 1.90 1.83 1.57 1.14 1.42
± ± ± ± ± ± ± ± ±
0.64 0.74 0.55 0.44 0.45 0.50 0.37 0.34 0.38
1.70 3.79 1.60 1.70 2.39 3.14 1.37 2.58 1.75
± ± ± ± ± ± ± ± ±
0.30 0.98 0.32 0.66 0.31 0.29 0.39 0.62 0.15
(102.4) (184.9) (94.1) (140.5) (125.8) (171.6) (87.3) (226.3) (123.2)
2.16 3.28 2.06 2.47 3.44 3.18 2.01 2.35 1.73
DZH-I DZH-2 DZH-3 DZH-4
2.76 3.56 1.28 2.75
_+ 1.22 ± 1.89 ± 0.29 ± 0.91
2.17 1.42 1.66 3.33
± ± ± ±
0.63 0.43 0.40 0.72
(78.6) (39.9) (129.6) (121.1)
Died Died Died Died
RHO-I RHO-2 RHO-3 RHO-4
4.37 3.71 2.54 2.86
± 1.76 ± 1.45 ± 1.13 _+ 0.66
1.95 1.06 1.86 0.94
± ± ± ±
0.30 0.21 0.93 0.51
(44.6) (28.6) (73.2) (32.9)
Died Died 0.81 ± 0.43 Died
RUP-I RUP-2 RUP-3
1.97 ± 0.73 1.24 ± 0.69 3.45 ± 1.61
0.93 ± 0.41 1.00 ± 0.29 0.78 ± 0.36
47.2) 80.6) 22.6)
Died Died Died
SUF-I SUF-2
5.61 ± 1.73 3.26 ± 0.88
1.07 ± 0.53 0.93 ± 0.21
19.1) 28.5)
Died Died
a)
± 0.36
(12.3)
± 0.63
(14.6)
± 0.81
(8.2)
+_ 0.67
(12.1)
± ± ± ± ± ± ± ± ±
(130.1) (160.0) (121.1) (204.1) (181.1) (173.8) (128.0) (206.1) (121.8)
0.33 0.99 0.81 0.87 0.82 0.65 0.22 0.38 0.64
(31.9)
Mean ± standard deviation for three replicates; each replicate consisted of four vegetative propagules. Values in parenthesis are the percent control.
McCoy TJ (1985) Can. J. Genet. Cytol. 27:238-245 McCoy TJ, Smith LY (1984) Can. J. Genet. Cytol. 26: 511-518 McCoy TJ, Smith LY (1986) Theor. Appl. Genet. 71: 772-783 Nabors MW, Gibb SE, Bernstein CS, Meis ME (1980) Z. Pflanzenphysiol 97:13-17 Nabors MW, Kroskey CS, McHugh DM (1982) Z. Pflanzenphysiol. 105:341-349 Orton TJ (1980) Z. Pflanzenphysiol. 98:105-118 Schenk RU, Hildebrandt AC (1972) Can. J. Bot. 50: 199-204 Smith MK, McComb JA (1981a) Aust. J. Plant Physiol.8: 267-275
Smith MK, McComb JA (1981b) Aust. J. Plant Physiol.8: 437-442 Smith MK, McComb JA (1983) Plant Cell Reports 2:126128 Stavarek SJ, Rains DW (1984) In: Staples RC, Toenniessen GH (ed.) Salinity Tolerance in Plants, Wiley-Interscience, New York pp 321-334 Tal M, He±ken H, Dehan K (1978) Z. Pflanzenphysiol. 86:231-240 W~rren RS, Baird LM, Thompson AK (1985) Plant Cell Reports 4:84-87
