Seed Science Research (2004) 14, 345–353

DOI: 10.1079/SSR2004188

Effects of five different salts on seed germination and seedling
growth of Haloxylon ammodendron (Chenopodiaceae)
Kazuo Tobe1*, Xiaoming Li2 and Kenji Omasa3
1

Laboratory of Intellectual Fundamentals for Environmental Studies, National Institute for Environmental Studies,
16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan; 2School of Environmental Science and Engineering,
Shandong University, No. 27, South Shada Road, Jinan 250100, P.R. China; 3Graduate School of Agricultural andLife Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo, Tokyo 113–8657, Japan

Abstract

Introduction

Saline soils contain multiple types of salt, each of which
may exert a different effect on seed germination andseedling growth of plants. The effects of five types of salton the initial growth of Haloxylon ammodendron, a shrub
found on both saline and non-saline areas in deserts ofChina, were investigated. Seeds were incubated at 20°C
in the dark in a solution (0 to –5.1 MPa) of a salt (NaCl,
MgCl2, CaCl2, Na2SO4 or MgSO4) or polyethylene glycol(PEG)-6000, or in a salt (NaCl or MgCl2) or PEG solution
containing a low concentration of CaCl2. Seedgermination, seedling growth and cation (Na+, Mg2+, Ca2+
and K+) contents of seedlings were examined. Each salt
had a different effect on seed germination, seedling
growth and influx and outflux of cations in the seedlings.
In both NaCl and MgCl2 treatments, the addition of low
concentrations of CaCl2 favoured seed germination andseedling growth, and reduced K+ outflux from seedlings,
but caused no appreciable decrease in the influx of Na+or Mg2+ into seedlings. Marked abnormalities inseedlings were found only in treatments with Mg2+ salts,
but these effects were completely alleviated by a low
concentration of Ca2+ (Ca2+/Mg2+ = 0.012). The different
responses of the initial growth in H. ammodendron todifferent isotonic salt solutions were attributed todifferences among salt components in membrane
permeability, toxicity and effects on functions of theplasma membrane and/or the cell wall.

Saline soils contain multiple types of soluble salt
components, each of which has a different effect on
the initial growth of plants (Younis and Hatata, 1971;
Redmann, 1974; Hardegree and Emmerich, 1990; Tobe
et al., 2002, 2003), and the compositions of soluble
salts in saline soils differ greatly among locations(Tobe et al., 2002). Among these salt components, Ca2+
is noteworthy because it signiﬁcantly affects thesalinity responses of plants in both initial growth(Bliss et al., 1986; Marcar, 1986; Hamada, 1994; Tobe etal., 2002, 2003) and later developmental stages(LaHaye and Epstein, 1969; Cramer et al., 1986; Kurth
et al., 1986; Colmer et al., 1996).
One important effect of salts on the initial growthof plants is toxicity. At least some of the effects of salt
toxicity originate from the displacement of Ca2+
bound to the external surface of the plasma
membrane by metal cations, and the subsequent
impairment of membrane integrity and permeability.
Ca2+ in the external medium alleviates this type of salt
toxicity by re-displacing the cations on the membranewith Ca2+ (Cramer et al., 1985; Kent and Läuchli, 1985;
Lynch et al., 1987). This type of salt toxicity and its
alleviation by Ca2+ have been studied extensively formonovalent cations (especially Na+) in salts (e.g.
Cramer et al., 1985, 1986; Kurth et al., 1986; Rengel,
1992; Cramer, 2002); however, information on the
toxic effects of divalent cations (e.g. Mg2+) andalleviation of their effects by Ca2+ is limited (Marcar,
1986; Tobe et al., 2002, 2003).
Also, it is known from experiments, primarily
with NaCl, that salts affect the functions of themembranes and cell walls. First, NaCl affects thepermeability of the plasma membrane and increases
inﬂux of external ions and efﬂux of cytosolic solutes(Cramer et al., 1985; Kent and Läuchli, 1985; Zidan etal., 1991; Allen et al., 1995) in plant cells. Secondly,
NaCl causes hardening of the cell wall (Neumann,

Keywords:
calcium,
Haloxylon
ammodendron,magnesium, potassium, salinity, seed germination,
seedling growth, sodium*Correspondence
Fax: +81 29 850 2587
Email: tobe@nies.go.jp

346

K. Tobe et al.

1993; Neumann et al., 1994; Nabil and Coudret, 1995)
and a decrease in water conductance of the plasma
membrane (Azaizeh et al., 1992; Cramer, 1992). These
effects of NaCl on cellular functions are alleviated by
the addition of Ca2+ to the external medium (Cramer
et al., 1985; Kent and Läuchli, 1985; Kurth et al., 1986;
Zidan et al., 1990, 1991; Azaizeh et al., 1992; Cramer,
1992; Allen et al., 1995). These effects of salts on thefunctions of the cell membranes and the cell walls
may affect the water potential of the cytosol andcellular extensibility, and thus, may affect seedgermination and seedling growth. Although most ofthese reports are based on experiments with NaCl, it
is hypothesized that other salts have similar effects on
cellular functions, but to different degrees, dependingon the salt.
Studies to examine salinity effects on the initial
growth of plants have usually been carried out withindividual salts (especially NaCl); however, there
remains a question as to whether the results ofexperiments with individual salts simulate the actual
initial growth behaviour of plants in saline soils. To
clarify the responses of the initial growth of plants tosalinity, examination of the effects of various salts andthe interactive effects of various salts with Ca2+ on
seeds and seedlings is desirable. Furthermore,evaluation of ion movements into and out of
seedlings would aid in understanding the effects ofdifferent types of salt on seedlings.
In the present study, we compared the effects ofdifferent types of salts, with or without a low
concentration of Ca2+, on seed germination, seedling
growth and cation movement into and out of
seedlings of Haloxylon ammodendron (C.A. Mey.)
Bunge (Chenopodiaceae), a shrub found on both saline
and non-saline soils in desert regions of north-west
China (Fu and Jin, 1992). The seeds of this species
have no endosperm and have only a fully
differentiated spiral embryo coated by pericarp; thegermination process of this type of seed seems toconsist simply of water absorption by the dry spiralembryo and its subsequent elongation (Wallace et al.,
1968; Sharma and Sen, 1989). The seeds of H.
ammodendron germinate even in solutions having very
high NaCl concentrations, and the seedlings arehighly tolerant of NaCl (Tobe et al., 2000). Because themain salt components of saline soils are Na+, Mg2+
and Ca2+ cations and Cl and SO42 anions(Shainberg, 1975), we investigated the effects ofindividual salts (NaCl, Na2SO4, MgCl2, MgSO4 andCaCl2) and combinations of salts (NaCl and MgCl2)
with low concentrations of CaCl2 on the initial growthof H. ammodendron. Additionally, to evaluate theosmotic effects of the medium on seed germinationand seedling growth, we examined seed and seedling
responses to solutions of polyethylene glycol (PEG)6000. Furthermore, we measured the cation (Na+,Mg2+, Ca2+ and K+) contents in seedlings, to evaluate
their movements into and out of the seedlings.

Materials and methods
Seeds of H. ammodendron [seed weight: 3.48 ± 0.48 mg(mean ± SE; n = 20)] were collected soon after
maturation in November of 2001, from plants
growing on a non-saline soil in the Tulufan Botanical
Garden of the Xinjiang Institute of Pedology, Biology
and Desert Research, Tulufan, China (42°56N,
89°12E). The collected seeds were initially stored atroom temperature until transport to Japan thefollowing January. After transport to Japan, the seeds
were stored at c. 18°C to avoid germination loss,
and germination experiments were carried out within
12 months after the maturation of the seeds.
Replicates of 25 seeds were sown on three layers ofﬁlter paper (Toyo, No. 1) in 90-mm plastic Petridishes. About 15 ml of deionized water, a salt solution
or a PEG-6000 solution was added to each Petri dish,so that about half the volume of each seed was
immersed. The PEG solution of known water
potential (ΨW) was prepared according to acalibration curve that was determined from isopiestic
psychrometry(BoyerandKnipling,
1965)
measurements at 20°C. The ΨW of each salt solution
was calculated from van’t Hoff ’s law, assuming thatall salts in solution were dissociated into ions. In
addition to ΨW, salt concentrations in solutions wereexpressed on a molal basis (mMolal; millimole perkilogram water). The Petri dishes were covered withlids, and the seeds were incubated at 20°C in the dark,
because the seeds germinate favourably under these
conditions (Tobe et al., 2000). About two-thirds of thevolume of the solution in each Petri dish was replaced
daily with fresh treatment solution, to avoid changes
in solute concentration; because no decrease in thevolume of solution in each dish caused byevaporation or water uptake by the seeds was
detectable, the daily solution replacement is believed
to have been sufﬁcient to maintain a constant soluteconcentration in the dish.
Experiment 1: Effects of individual salts and PEG on
seed germination and seedling growth

Seeds were incubated in deionized water, a salt
(NaCl, Na2SO4, MgCl2, MgSO4 or CaCl2) solution (ΨW:
1.0 to 5.1 MPa) or a PEG solution (ΨW: 1.0 to4.0 MPa). The seeds were observed daily for 20 d in
dim light through a magnifying glass with a scale; thenumbers of both germinated seeds (seeds withemerging seedlings longer than 3 mm) and seeds withemerging seedlings longer than 20 mm were counted,
and seeds with emerging seedlings longer thanEffects of different salts on initial plant growth

20 mm were discarded. After 20 d of incubation, theﬁnal germination percentage (GF) and the ﬁnal
percentage of seeds with emerging seedlings longer
than 20 mm (SF) were determined. Each treatment
was replicated four times.
Experiment 2: Effects of salt and PEG solutions withdifferent CaCl2 concentrations on seed germinationand seedling growth

Seeds were incubated in a salt (NaCl or MgCl2) or
PEG solution (ΨW: 2.0 MPa) with different CaCl2
concentrations (0, 1.0, 5.0 and 25 mMolal). Seeds wereobserved as described in Experiment 1, and after 20 dof incubation, GF and SF were determined as
described in Experiment 1. Each treatment was
replicated four times.

347

solution was analysed for cation (Na+, Mg2+, Ca2+ andK+) content by ICP spectrometry (ICAP-750, Nippon
Jarrell-Ash, Japan). From the results, cation content
per seedling and cation concentrations in seedlingwater (the water contained in the seedlings) were
calculated. Each treatment was replicated ﬁve times.
Statistical analysis

For comparison of multiple means, one-way ANOVA
and Tukey’s test were used. Percentage values werearcsine transformed prior to statistical analysis. All
statistical tests were conducted at P < 0.05.

Results
Effects of individual salts and PEG on GF and SFExperiment 3: Evaluation of water uptake by
seedlings

Seeds were incubated in either a salt (NaCl, Na2SO4,
MgCl2, MgSO4 or CaCl2) solution (ΨW: 2.0 or 3.1
MPa) or a PEG solution (ΨW: 2.0 or 3.0 MPa), or ina salt (NaCl or MgCl2; ΨW: 2.0 or 3.1 MPa) or PEG(ΨW: 2.0 or 3.0 MPa) solution containing
5 mMolal CaCl2. After 4 d (2.0 MPa treatments) or
6 d (3.0 or 3.1 MPa treatments) of incubation, thepercentage of seeds with an emerging seedling
longer than 10 mm (S10) was determined. For seeds
with emerging seedlings longer than 10 mm,
seedlings were detached from the pericarp and were
blotted dry with tissue paper, and the fresh weight ofthe seedlings from each dish was measured. Afterdrying the seedlings at 80°C for more than 3 d, thedry weight of the seedlings was determined. From
fresh and dry weights of the seedlings, the watercontent per seedling was calculated. Each treatment
was replicated four times.
Experiment 4: Measurement of cation content inseedlings

Seeds were incubated in a salt (NaCl, Na2SO4, MgCl2,
MgSO4 or CaCl2) solution (ΨW: –3.1 MPa), or in a salt
(NaCl or MgCl2) solution (ΨW: –3.1 MPa) containing
5 mMolal CaCl2. After 5 d of incubation, seedlings
with emerging lengths longer than 10 mm werecollected, detached from the pericarp, blotted drywith tissue paper and the fresh weight of theseedlings from each dish was measured. Then, theseedlings were dipped in water and quickly blotted
dry again to remove any surface salts, and theseedlings were dried at 80°C for more than 3 d. After
determining the dry weights of the seedlings, they
were ground, digested with HNO3 on a hot plate, andﬁnally dissolved in c. 100 ml water and the aqueous

In all treatments, both seed germination percentage
and seedling elongation progressively increased as
ΨW in the medium increased [see Tobe et al. (2000) for
daily changes in germination percentage of NaCl- or
PEG-treated seeds], but in most treatments, thepercentages of germinated seeds and of seeds withemerging seedlings longer than 20 mm substantiallyplateaued before the end of 20 d of incubation.
Figure 1 shows GF and SF for each treatment. At a
ΨW of –3.0 MPa or less, GF of PEG-treated seeds was
lower than that of seeds treated with any salt solutionof the same calculated ΨW. At 4.1 and 5.1 MPa, GF
was higher in treatments with SO42 salts than intreatments with Cl salts. At these values of ΨW, GF
tended to decrease as SO42 concentration in thesolutions of the same calculated ΨW increased, and to
decrease as Cl concentration increased; for example,at 4.1 MPa, GF decreased in the order MgSO4 (SO42
concentration:
840 mMolal),
Na2SO4(SO42

concentration: 560 mMolal), NaCl (Cl concentration:
840 mMolal), MgCl2 (Cl concentration: 1120 mMolal)
and CaCl2 (Cl concentration: 1120 mMolal)
treatments (the values of mean GF were 97%, 76%,
65%, 49% and 46%, respectively).
The ratio SF/GF at a ΨW of 2.0 MPa or less waslowest in the MgCl2 treatment (0.39 at 2.0 MPa), andhighest in treatments with Na+ salts (at 4.1 MPa,0.83 and 0.84 for NaCl and Na2SO4 treatments,
respectively). For CaCl2 and PEG treatments, SF/GF
was high, except for 4.1 MPa CaCl2 (0.22) and3.0 MPa PEG (0.11).
In treatments with Mg2+ salts, all the seedlings,
including those of lengths exceeding 20 mm, wereabnormal in colour (yellowish or blackish) and shape(bent and/or coiled). For treatments with other salts
and PEG, although some seedlings showed slightly
abnormal colour and/or shape, many seedlings
appeared normal (white and straight).

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K. Tobe et al.

Figure 1. Effects of individual salts and polyethylene glycol (PEG)-6000 on ﬁnal germination percentage (GF) () and ﬁnal
percentage of seeds with emerging seedlings longer than 20 mm (SF) (). Water potentials of salts were calculated from van’t
Hoff ’s law. Each point represents the mean of four replications; bars indicating SE are shown only where SE is larger than thesize of the point.

Effects of the addition of CaCl2 to salt or PEG
solutions on GF and SF

Effects of the addition of CaCl2 to a salt (NaCl or
MgCl2) or PEG solution of 3.0 MPa on GF and SF
were examined (Table 1). In the MgCl2 treatments,
both GF and SF were signiﬁcantly increased byincreasing CaCl2 concentration, while in the NaCl
treatments, both GF and SF were high irrespective of
CaCl2 concentration. In the PEG treatments, GF was
signiﬁcantly lower at the highest CaCl2 concentration.
At 0 mMolal CaCl2 in the MgCl2 treatment, allseedlings showed abnormalities as described above,
but at 1.0 mMolal CaCl2 (Ca2+/Mg2+ = 0.0024),
although many seedlings showed abnormalities, thedegrees of the abnormalities were less conspicuousthan those at 0 mMolal CaCl2. At 5.0 and 25 mMolal
CaCl2 in the MgCl2 treatments (Ca2+/Mg2+ = 0.012 and0.056, respectively), most seedlings appeared normal(white and straight). For NaCl and PEG treatments,

many seedlings appeared normal irrespective of
CaCl2 concentration.
Effects of salts and PEG with or without 5 mMolal
CaCl2 on seedling water uptake

Effects of different solutions with or without
5 mMolal CaCl2 on seedling water uptake were
examined (Table 2a, b). For either ΨW, seedling watercontent was signiﬁcantly less in the PEG treatments
than in any treatment with salt. Among salt
treatments without 5 mMolal CaCl2, no signiﬁcantdifference in water content was detected. At
2.0 MPa, the addition of CaCl2 caused signiﬁcantly
higher S10 and higher water content in both NaCl and
MgCl2 treatments, while at 3.1 MPa, the addition of
CaCl2 caused signiﬁcantly higher water content onlyin the NaCl treatment. In the PEG treatments, theaddition of CaCl2 resulted in no signiﬁcant changes in
S10 or seedling water content at either ΨW. S10 tendedEffects of different salts on initial plant growth349

Table 1. Effects of NaCl and MgCl2 solutions (water potential: 3.1 MPa) and apolyethylene glycol (PEG) solution (water potential: 3.0 MPa) with different CaCl2
concentrations on ﬁnal germination percentage (GF) and ﬁnal percentage of seeds withemerging seedlings longer than 20 mm (SF). In each column, the values with the same
superscript letter are not signiﬁcantly different from each other (P < 0.05; Tukey’s test)
CaCl2
concentration(mMolal)

NaCl (630 mMolal)

MgCl2 (420 mMolal)

PEG-6000

GF

SF

GF

SF

GF

SF

0
1.0
5.0
25

91 a
97 a
97 a
95 a

85 a
95 a
96 a
92 a

79 b
97 a
99 a
95 a

14 c
43 b
96 a
93 a

63 a
51 ab
47 ab
31 bc

7a
9a
3a
3a

Table 2. Effects of NaCl and MgCl2 solutions [water potential: 2.0 (a) or 3.1
MPa (b)] and polyethylene glycol (PEG) solutions [water potential: 2.0 (a) or3.0 MPa (b)] with (+Ca) or without (Ca) 5 mMolal CaCl2 on ﬁnal percentage ofseeds with emerging seedlings longer than 10 mm (S10) and seedling watercontent. In each column, the values with the same superscript letter are notsigniﬁcantly different from each other (P < 0.05; Tukey’s test)
Treatment solution(a) PEG or salt (2.0 MPa)
PEG (2.0 MPa)Ca
+Ca
NaCl (420 mMolal)
Ca+Ca
MgCl2 (280 mMolal)Ca+Ca
Na2SO4 (280 mMolal)
Ca
MgSO4 (420 mMolal)
Ca
CaCl2 (280 mMolal)(b) PEG (3.0 MPa) or salt (3.1 MPa)
PEG (3.0 MPa)Ca
+Ca
NaCl (630 mMolal)
Ca+Ca
MgCl2 (420 mMolal)Ca+Ca
Na2SO4 (420 mMolal)
Ca
MgSO4 (630 mMolal)
Ca
CaCl2 (420 mMolal)

to be similar in treatments with SO42 salts and Cl
salts of the same ΨW; differences in seedling water
uptake were not conspicuous between the treatmentswith SO42 and Cl salts of the same ΨW.
Effects of salts with or without CaCl2 on cation
content in seedlings

A marked increase in Na+, Mg2+ or Ca2+ concentrationin seedling water was detected when seedlings weretreated with a salt containing each cation (Table 3).
Na+ concentrations, in treatments with Na+ salts, andS10 (%)

58 cd

Water content(mg seedling1)
3.52 d

54 d
88 ab
96 a
77 bc
91 a
64 cd
87 ab
90 ab

3.49 d
5.06 bc
5.91 a4.51 c5.71 ab4.62 c4.91 bc
4.84 c

26 c
9c
70 b
72 b
66 b
65 b
76 b
92 a
60 b

2.86 d2.71 d
4.64 bc
6.84 a3.98 c
4.19 bc
4.65 bc
4.87 b
3.96 cMg2+ concentrations, in treatments with Mg2+ salts, inseedling water were, respectively, 52–66% of Na+concentrations and 41–58% of Mg2+ concentrations in
the treatment solutions. For 420 mMolal CaCl2
treatment, the Ca2+ concentration in seedling water
was rather low (23% of the Ca2+ concentration in thetreatment solution).
Potassium (K+) content per seedling was
signiﬁcantly higher in 420 mMolal CaCl2 than in any
other treatment, and was signiﬁcantly lower in NaCl(Ca) and Na2SO4 treatments than in any othertreatment. In NaCl and MgCl2 treatments, theK. Tobe et al.

350

Table 3. Cation content per seedling and cation concentration in seedling water (in parentheses) for seedlings
treated with different salt solutions (water potential: 3.1 MPa) with (+Ca) or without (Ca) 5 mMolal CaCl2.
In each column, the values with the same superscript letter are not signiﬁcantly different from each other (P <0.05; Tukey’s test)
Cation content per seedling (nmol seedling1) and cation concentrationin seedling water (mMolal) in parentheses
Treatment solution
Ca+Ca
MgCl2 (420 mMolal) Ca+Ca
Na2SO4 (420 mMolal) –Ca
MgSO4 (630 mMolal) Ca
CaCl2 (420 mMolal)
NaCl (630 mMolal)

Na+

Mg2+

Ca2+

K+

1899 b (414 b)
2370 a (418 b)
32 c (8 d)47 c (11 cd)
1958 b (439 a)
16 c (4 d)
108 c (28 c)

114 c (25 e)
106 c (19 e)
927 b (245 b)
914 b (216 c)
109 c (25 e)1116 a (255 a)
138 c (36 d)

13 bc (3 b)
27 b (5 b)
13 bc (3 b)
18 bc (4 b)
9 c (3 b)
9 c (2 b)
377 a (97 a)

371 d (81 ef)
472 c (83 e)
478 c (127 c)
663 b (157 b)
290 e ( 65 f)
465 c (106 d)
832 a (215 a)addition of 5 mMolal CaCl2 resulted in signiﬁcant
increases in K+ content per seedling.
No signiﬁcant differences were detected in Na+,
Mg2+ or Ca2+ contents per seedling among treatments
in which the treatment solutions did not contain thatcation (Table 3). It seemed that because the cation
content in seedlings treated with a solution
containing that cation was so much larger, statisticaltests were not effective in determining differences incation contents among treatments in which thetreatment solutions did not contain that cation. Thus,
we carried out statistical comparisons among cation
contents in seedlings treated with solutions that did
not contain that cation. The results (not presented)
indicated that Na+ or Mg2+ content was signiﬁcantly(P < 0.05) higher in seedlings treated with 420 mMolal
CaCl2 than in those treated with any other solutionthat did not contain Na+ or Mg2+. On the other hand,the addition of 5 mMolal CaCl2 to the NaCl or MgCl2
solution did not have any signiﬁcant effect on Mg2+ orCa2+ content in the NaCl treatments, or on Na+ or Ca2+
content in the MgCl2 treatments (Table 3). Moreover,comparison of Na+, Mg2+ or Ca2+ content among
treatments in which treatment solutions containedthat cation (results not presented) did not indicate
signiﬁcant effects (P > 0.05) of 5 mMolal CaCl2 on any
cation content.
The addition of 5 mMolal CaCl2 to NaCl caused asigniﬁcant increase in Na+ content per seedling,whereas it caused no signiﬁcant change in Na+
concentration in seedling water; this was interpreted
to have resulted from an increase in seedling water
uptake due to Ca2+ and an eventual increase in Na+
inﬂux into the seedlings. The addition of 5 mMolal
CaCl2 to MgCl2 caused no signiﬁcant change in Mg2+
content per seedling, but caused a signiﬁcant (butsmall) decrease in Mg2+ concentration in seedlingwater.

Discussion
For H. ammodendron seedlings treated with salts, bothinﬂux of external cations and efﬂux of internal cations
were found, and the degree of cation movement intoand out of seedlings differed among treatments withdifferent salts (Table 3). Efﬂux of K+ was prominent intreatments with Na+ salts without 5 mMolal CaCl2,
suggesting that Na+ enhances outward movement of
K+ through ion channels in the plasma membrane. Ion
movements into and out of seedlings would haveaffected the ΨW of seedling water, and thus seedgermination and seedling growth of H. ammodendron.
In CaCl2 treatments, although Ca2+ movement into theseedlings was more limited than inward movement of
Na+ or Mg2+ in treatments with Na+ or Mg2+ salts, theGF of seeds treated with CaCl2 was similar to that ofseeds treated with MgCl2 of the same ΨW (Fig. 1). This
would, at least partly, be attributable to the action of
Ca2+ in decreasing the permeability of many ion
channels (Tyerman and Skerrett, 1999), thus reducing
efﬂux of intracellular solutes and maintaining a low
ΨW of seedling water. The fact that seed germination
was favoured more in treatments with either salt thanin treatments with PEG of similar ΨW values may be
because movement of external ions into seedlings
favoured seedling water uptake. However, it should
be stressed that the ΨW values for salt solutions
presented in this study are calculated values and may
deviate from the actual ΨW values of the solutions.
For example, 1050 mMolal NaCl was calculated tohave a ΨW of 5.1 MPa at 20°C, while its actual ΨW at
20°C is 4.8 MPa according to the report of Lang
(1967). More strict discussion of the osmotic effects ofsalts on seed germination needs to be based on actual
ΨW values. In addition to differences in thecharacteristics of inward and outward movements ofions, differences in the effects of different salts on cellwall extensibility and/or water conductance ofEffects of different salts on initial plant growth

membranes may have caused the different responses
of GF and SF of H. ammodendron to different salts.
Low concentrations of Ca2+ favoured seedgermination, seedling water uptake and seedling
growth of H. ammodendron in both NaCl and MgCl2
treatments. Because these effects of Ca2+ were notdetected in the PEG treatments, they are believed to
be attributable to an interaction of Ca2+ and salt
components. For both NaCl and MgCl2 treatments,
the addition of low concentrations of Ca2+ reduced K+
leakage from H. ammodendron seedlings. Similar
results have been reported for crop plants (Cramer etal., 1985; Kent and Läuchli, 1985; Colmer et al., 1996)
and charocean macroalgae (Tufariello et al., 1988;
Whittington and Smith, 1992), for both of which K+
leakage from plant cells treated with NaCl was
reduced by adding Ca2+ to the medium. On the other
hand, the addition of low concentrations of Ca2+ did
not cause any appreciable reduction in thepermeation of Na+ or Mg2+ into H. ammodendron
seedlings in NaCl or MgCl2 treatments. This is incontrast to the results with crop plants (Zidan et al.,
1991; Allen et al., 1995; Roberts and Tester, 1997;
Tyerman et al., 1997) and charocean algae (Tufariello etal., 1988; Hoffmann et al., 1989; Whittington andSmith, 1992; Davenport et al., 1996), for which Na+permeation into the plant cells treated with NaCl was
reduced by adding Ca2+ to the medium. The
promotive effects of Ca2+ on seed germination andseedling growth of H. ammodendron in salt treatments
would, at least partly, have resulted from reduced
efﬂux of K+ (and possibly other solutes) from theseedlings, and eventual maintenance of a low ΨW ofseedling water. Additionally, it is possible that Ca2+
alleviated the reduction in cell wall extensibility
caused by salts (Kurth et al., 1986; Zidan et al., 1990)
and/or water conductance of the plasma membrane(Azaizeh et al., 1992; Cramer, 1992) in H. ammodendron
seedlings, thus favouring water uptake and extension
growth.
Marked abnormalities in seedlings were foundonly in treatments with Mg2+ salts, but noabnormalities
wereobserved
whenlowconcentrations of Ca2+ (Ca2+/Mg2+ = 0.012) wereadded to the medium. The most probable explanationof the interactive effects of Mg2+ and Ca2+ on H.
ammodendron seedlings would be that thedisplacement of plasma membrane-bound Ca2+ with
Mg2+ in the medium impaired normal growth, andthat restoration by re-displacement with Ca2+ in themedium allowed normal seedling development(Cramer et al., 1985; Kent and Läuchli, 1985; Lynch etal., 1987). Because Mg2+ inﬂux into H. ammodendron
seedlings was not markedly decreased by Ca2+, theabnormalities of Mg2+-treated seedlings would nothave resulted from cytosolic Mg2+ accumulation. This351

is in contrast to the situation in NaCl-treatedcharocean algae, in which Ca2+ alleviated salt toxicity
by decreasing Na+ inﬂux into the cells (Tufariello etal., 1988; Whittington and Smith, 1992).
Alleviatory effects of Ca2+ on the toxicity of Na+
and Mg2+ on radicles have been found in our previous
studies with a halophyte Kalidium capsicum (Tobe etal., 2002) and three non-halophytes (Tobe et al., 2003);
the proportion of Ca2+ needed to alleviate salt toxicity
differed among these species; the Ca2+/Mg2+ ratio
needed to alleviate Mg2+ toxicity on radicles ranged
between 0.025 and 0.16, depending on the species.
Compared to these species, H. ammodendron required
a lower proportion of Ca2+ (Ca2+/Mg2+ = 0.012) toalleviate Mg2+ toxicity, showing higher salt tolerance
in seedlings of H. ammodendron. The reason for theabsence of abnormalities in NaCl-treated H.
ammodendron seedlings is believed to be because Na+,
being much less toxic than Mg2+ (Tobe et al., 2002,
2003), did not cause appreciable injurious effects on
the salt-tolerant H. ammodendron seedlings. The resultof this study indicates that seedlings of H.
ammodendron can survive in saline areas over a wider
range of salt compositions, and reinforces theconclusions of previous studies (Tobe et al., 2002,
2003) that salt composition is a factor in determining
which species is dominant.
In addition to cations, anions seem to also affectseed germination of H. ammodendron, although they
did not cause any appreciable effect on seedling
growth. The promotive effect of SO42 andsuppressive effect of Cl on seed germination were
found in K. capsicum in a previous study (Tobe et al.,2002) as well as in H. ammodendron in this study.
However, because the ΨW values presented in thisstudy may deviate from the actual ΨW values of thesalt solutions, the possibility cannot be ruled out that
this result was caused by larger deviations ofcalculated values of ΨW in SO42 salts than in Cl
salts. Further research is needed to clarify the effects
of anions on seed germination.
Different salts caused different responses in seedgermination and seedling growth of H. ammodendron.
This result is in agreement with the results of studies
on other species (Younis and Hatata, 1971; Redmann,
1974; Myers and Morgan, 1989; Hardegree andEmmerich, 1990). The different responses of seeds andseedlings of H. ammodendron to different salt solutions
are believed to have resulted from differences among
different salt components in membrane permeability,
toxicity and effects on functions of the plasma
membrane and/or the cell wall. These results suggestthat examination of the initial growth responses ofplants to different salt components is needed to
predict whether or not they will establish in saline
areas.

352

K. Tobe et al.

Acknowledgements
We thank Dr Nishikawa and Ms Kumada for
analysing the cation content of H. ammodendron
seedlings by ICP spectrometry.

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Received 11 May 2004
accepted after revision 19 August 2004
© CAB International 2004