Annals of Botany 85: 391±396, 2000
doi:10.1006/anbo.1999.1077, available online at http://www.idealibrary.com on

Seed Germination and Radicle Growth of a Halophyte, Kalidium caspicum(Chenopodiaceae)
K A Z U O TO B E * {, X I AO M IN G L I{ and K E N JI O MA S A {}{Environmental Biology Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba,
Ibaraki 305-0053, Japan and {Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences,
No. 40, South Beijing Road, Urumqi, Xinjiang 830011, People's Republic of China
Received: 23 August 1999

Returned for revision: 27 October 1999

Accepted: 19 November 1999

Eects of temperature, light, NaCl and polyethylene glycol (PEG)-6000 on seed germination and radicle growth in a
halophytic shrub, Kalidium caspicum (L.) Ung.-Sternb. were investigated. When seeds were incubated in deionizedwater at constant temperatures between 10 and 308C, the percentage germination in the dark exceeded 75%; light
suppressed seed germination at alternating temperatures. Incubating seeds with a hypersaline solution of NaCl for
30 d had no adverse eect on their germinability. The percentage germination of seeds incubated with a ÿ0.8 MPa
NaCl solution was 73, 80 and 54% at 10, 20 and 308C, respectively, but all radicles died before their length exceeded
5 mm. In contrast, when seeds were incubated with a ÿ0.8 MPa PEG solution at 208C, 68% of seeds germinated, and
95% of the emerging radicles survived beyond 5 mm. The high sensitivity of small radicles of this species to salinity
indicated that salt must be removed from the soil surface for seedling establishment.
# 2000 Annals of Botany Company
Key words: Chinese desert, radicle growth, germination, halophyte, Kalidium caspicum, salinity.

I N T RO D U C T I O N
In saline environments, adaptation of plants to salinity
during germination and early seedling stages is crucial forthe establishment of species (Ungar, 1978, 1991, 1995).
Seedlings are the most vulnerable stage in the life cycle ofplants, and germination determines when and whereseedling growth begins (Koller, 1964; Gutterman, 1993;
Kigel, 1995). Therefore, appropriate germination responsesof halophytic species to environmental parameters determine their distribution in saline environments.
Most seeds are located near the soil surface, where salt
accumulates in salinized locations (Ungar, 1978). The salt
concentration at the surface of salanized soil changes overtime: continuous evaporation of ground water graduallydeposits salt on the soil surface, but rainfall or melting
snow can quickly leach salt from the surface and supply
water to seeds. Thus, for the successful establishment ofplants in saline environments, seeds must remain viable athigh salinity and germinate when salinity decreases (Ungar,
1978, 1991, 1995).
Two Haloxylon species distributed mainly in non-saline
locations in the desert region of northwestern China were
found to show high adaptability to salinity in the germination and early seedling stages (Tobe et al., 2000).

* For correspondence. Fax 81 298 50 2586, e-mail tobe@nies.go.jp} Present address: Graduate School of Agriculture and Life
Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 1138657, Japan.

0305-7364/00/030391+06 $35.00/00

This suggested that adaptation to salinity in the initial life
stages does not determine geographical distribution of
species in this region. To further investigate this point, weinvestigated how a typical halophyte growing in saline
locations in this region adapts to salinity in the initial life
stages. We studied Kalidium caspicum (L.) Ung.-Sternb.
(Chenopodiaceae), a stem-succulent halophytic shrub(Lanzhou Institute of Desert Research, 1985), which is
restricted to highly salinized locations where the groundwater table is located about 0.5 m below the soil surface.
Older seedlings survive high salinity (Natori et al., 1992),
but little is known about the germination and early seedling
growth of this species.
We examined the eects of salinity on germination andradicle growth of K. caspicum. Because NaCl is the mostabundant salt in many salinized locations in the Chinese
desert (Fan et al., 1993), we used it in our experiments. We
investigated (1) whether seeds of K. caspicum remain viable
at high salinity, and (2) whether radicles could survive salt
concentrations that permit germination. To evaluate thespeci®c eects of NaCl on seeds and radicles, we compared
germination and early radicle growth in NaCl solutions
with those in isotonic solutions of polyethylene glycol(PEG)-6000, which cannot permeate plant cell walls(Carpita et al., 1979) and acts solely as an osmoticum(Sharma, 1973; Redmann, 1974; Hardegree and Emmerich,1990). In addition, because temperature and light aect
germination responses in many species and can alter
ecological behaviour (Koller, 1964; Gutterman, 1993;
Kigel, 1995), we also examined their in¯uence on seedgermination.
# 2000 Annals of Botany Company

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Tobe et al.ÐGermination and Radicle Growth of Kalidium caspicum
M AT E R I A L S A N D M E T H O D S

Seed collection
Seeds of K. caspicum (mean seed weight + s.d. 171 +45 mg, n  20) were collected in November 1996 from plants
growing on saline soil in Fukang, China (448180 N, 878550 E).
Annual precipitation and annual mean temperature in
Fukang are 172 mm and 6.18C (means from 1958 to 1980).
The monthly mean temperature is 508C from November
to March, 16±208C from May to September, and about
108C in April and October. The diurnal temperaturedierence is 10±208C. Precipitation from November
to March is about 36 mm (31% of annual precipitation)and usually falls as snow, which covers the ground during
winter and melts in early spring. The seeds of K. caspicum
ripen in November. Total salt content at a depth of0±10 cm in a typical habitat of K. caspicum ranged between
16 and 54 g per kg dry soil (measurements were made inlate-May, late-August, and mid-October 1992).
Seeds were initially stored at about 58C until they weretransported to Japan in March 1997, where they were stored
at about ÿ188C. The germination experiments were carriedout between April 1997 and March 1998.
Methods
In all experiments, 25 seeds were sown on three layers of®lter paper (Toyo, No. 1) in a 90 mm Petri dish. The ®lter
paper was moistened with about 10 ml of deionized wateror a solution of NaCl or PEG-6000, so that about half theseed was immersed in the solution. The Petri dishes werecovered with lids and kept in a controlled environment.
About two-thirds of the volume of the water or solution ineach Petri dish was replaced daily. Each experiment was
replicated four or ®ve times.
After each experiment, the ®nal percentage germination(GF) and the time taken to achieve 50% germination (TG50)
were calculated; TG50 was calculated only when the GF of
all replications exceeded 50%.
Eects of temperature and light on seed germination. To
examine the eects of temperature and light on seed germination, seeds were incubated in deionized water under
12 dierent constant or diurnally alternating temperature
regimes. For each temperature regime, germination wasstudied under conditions of both continuous dark and 12 h
dark/12 h light, but at 58C germination was examined in thedark only. When the temperature was alternated, seeds were
exposed to the lower temperature for 12 h (dark) and thehigher temperature for 12 h (light). For the light treatment,
seeds were illuminated with ¯uorescent lamps ( photon ¯uxdensity at the surface of the seeds: 80±90 mmol m ÿ2 s ÿ1over the wavelength 400±700 nm). Seeds were considered tohave germinated when the emerging radicles were over
2 mm long. The number of germinated seeds was counted
daily for 15 d.
Eects of NaCl pretreatment on seed viability. Seeds wereincubated with 3.0 mol kg-1 NaCl solution (49% saturation
at 208C) for 30 d at 208C in the dark. Seeds were then rinsed

with deionized water and transferred to Petri dishes
containing deionized water. The number of germinatedseeds was counted every day for 10 d after transfer.
Eects of NaCl or PEG solution on seed germination andradicle growth. Seeds were moistened with deionized wateror solutions of NaCl or PEG-6000 of known water
potential (CW). The NaCl and PEG-6000 solutions wereprepared according to the equations of Lang (1967) andMichel and Kaufmann (1973), but the CW of the prepared
PEG solutions was ®nally determined with an isopiestic
psychrometer (Boyer and Knipling, 1965) at 208C. The
numbers of both germinated seeds and seeds with emerging
radicles longer than 5 mm were counted every day for 20 d
(20 and 308C) or 30 d (108C). Seeds were considered to havegerminated when the emerging radicles were visible. We
calculated the ®nal percentage of seeds with emerging
radicles longer than 5 mm (SF) and the time taken until thepercentage of these seeds was 50% (TS50). TS50 was
calculated only when SF of all replications exceeded 50%.
DTS50 , which is a measure of the rate of radicle elongation,
was calculated as TS50 minus TG50 .
Statistical analysis
The statistical signi®cance of the dierence between twovalues was evaluated using Student's t-test. The values of
GF were arcsine transformed before analysis.
R E S U LT S
Eects of temperature and light on seed germination
GF was signi®cantly (P 5 0.01) lower in the light than in
the dark at 308C and at all alternating temperature regimes(Figs 1 and 2). In the dark, GF exceeded 75% at constanttemperatures between 10 and 308C and at all alternating
temperature regimes.

FIG. 1. Time courses of percentage germination of seeds incubated with
deionized water at 208C in the dark (d, j) or in 12 h dark/12 h light(s). Before the experiment, seeds were either not pretreated (d, s) or
incubated with deionized water at 58C for 15 d (j). Each point
represents the mean of four replications; bars indicate s.e.

Tobe et al.ÐGermination and Radicle Growth of Kalidium caspicum

FIG. 2. Eects of temperature and light on ®nal percentage germination(GF). Seeds were incubated with deionized water for 15 d either in thedark (d) or in 12 h dark/12 h light (s). Each point represents the meanof four replications; bars indicate s.e.

393

FIG. 3. Eects of temperature and light on TG50 . Seeds were moistenedwith deionized water for 15 d either in the dark (d) or in 12 h dark/
12 h light (s). Each point represents the mean of four replications; bars
indicate s.e. TG50 was calculated only when all four replications of GF
were over 50%.

FIG. 4. Time courses of percentage germination (d, j, m) and percentage of seeds with radicles over 5 mm (s, h, n). Seeds were moistened with
NaCl or PEG solutions at dierent temperatures. Each point represents the mean of ®ve replications. (d, s) 0 MPa; (j, h) ÿ0.2 MPa;(m, n) ÿ0.4 MPa.

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Tobe et al.ÐGermination and Radicle Growth of Kalidium caspicum

FIG. 5. Eects of NaCl or PEG treatments on ®nal percentage germination (GF) (d) and ®nal percentage of seeds with radicles over 5 mm (SF) (s)at dierent temperatures. Each point represents the mean of ®ve replications; bars indicate s.e.

TG50 in the dark was 10.2 d at 108C and 5.5 d at 158C,
and between 2.0 and 4.7 d at other temperature regimes(Fig. 3). TG50 was signi®cantly (P 5 0.01) lower in thelight than in the dark at all temperature regimes except 15
and 308C.
GF was zero at 58C (Fig. 2), but when the ungerminatedseeds that had been incubated at 58C for 15 d were transferred to a constant temperature of 208C they germinatedrapidly (Fig. 1) [TG50 after transfer was 0.58 + 0.09 d(mean + s.e.) compared with 2.99 + 0.08 d for nonpretreated seeds].

Eects of NaCl pretreatment on seed viability
During pretreatment with 3.0 mol kg ÿ1 NaCl solution
for 30 d, no seed germination was observed. The seeds
germinated rapidly when transferred to deionized water.
TG50 after transfer was 1.69 + 0.09 d (mean + s.e., n  5),compared with 2.99 + 0.08 d for non-pretreated seeds.

Eects of NaCl and PEG on seed germination and radicle
growthWhen seeds were incubated with NaCl solution of CW
lower than ÿ0.2 MPa (108C) or ÿ0.3 MPa (20 and 308C),
or PEG solution of ÿ1.1 or ÿ1.6 MPa, some or mostradicles were necrotic and stopped elongating before they
were over 0.5 mm long; these seedlings died eventually
without further elongation (Figs 4 and 5). Consequently, in
the NaCl treatment, SF was much smaller than GF in mostof the CW and temperature conditions tested. SF in the PEG
treatment was slightly smaller than the corresponding GF atmost values of CW (Fig. 5).
Both germination percentage and radicle growth at 108C
were considerably slower than those at higher temperatures(Fig. 4). TG50 was signi®cantly higher (P 5 0.01) at 108C
than at 20 and 308C at all CW (Fig. 6). DTS50 was
signi®cantly higher (P 5 0.01) at 108C than at 20 and
308C at 0 and ÿ0.1 MPa. Larger GF and smaller TG50 at
108C, 0 MPa (Figs 5 and 6) compared with those in theTobe et al.ÐGermination and Radicle Growth of Kalidium caspicum

395

FIG. 6. Eects of NaCl or PEG treatments on TG50 (d) and DTS50 (s) at dierent temperatures. Each point represents the mean of ®veandindicate that values are signi®cantly dierent from those at 0 MPa at P 5 0.05 and P 5 0.001,
replications; bars indicate s.e.
respectively. TG50 was calculated only when all ®ve replications of GF were over 50%, and DTS50 was calculated only when all ®ve replications of
SF were over 50%.

***

former experiment (Figs 2 and 3) were the result of a longer
incubation time (30 d) and dierent germination criteria(see Materials and Methods).
GF was signi®cantly lower (P 5 0.01) at 208C in PEG
than in NaCl at ÿ0.8 MPa (Fig. 5). TG50 was signi®cantly
higher (P 5 0.01) at 208C in PEG than in NaCl at ÿ0.4
and ÿ0.8 MPa (Fig. 6). TG50 at ÿ0.2 MPa in NaCl tended
to be slightly smaller than that at 0 MPa, but TG50 at lower
CW tended to be larger than that at 0 MPa in both NaCl
and PEG treatments. DTS50 tended to be larger at lower
CW (Fig. 6).
DISCUSSION
Seed germination of K. caspicum was relatively insensitive
to temperature (Fig. 2), and we found no indication of seed
dormancy. These results indicate that K. caspicum seeds arepotentially germinable in the species' habitat at any time
between April and October. The CW of the NaCl solutionthat reduced GF of K. caspicum was not largely dierent
from that which reduced germination of other halophytic
species (Macke and Ungar, 1971; Williams and Ungar,
1972; Khan and Ungar, 1984; Myers and Morgan, 1989;
Khan and Rizvi, 1994; Ungar, 1996).
Moistening seeds with 3.0 mol kg-1 NaCl solution for
30 d had no adverse eect on their germinability in deionized water.This indicates that K. caspicum seeds can remain
ungerminated and viable on the salt-rich soil surface. In
many other halophytes, seeds remain viable after moistening with hypersaline solution (Macke and Ungar, 1971;
Okusanya, 1977; Ungar, 1996; Katembe, Ungar and
Mitchell, 1998).
Many radicles died in NaCl solutions with CW lower
than ÿ0.2 MPa (108C) or ÿ0.4 MPa (20 and 308C),
although most seeds germinated (Fig. 5). This indicatesthat germination, even at relatively low salinity, does not
ensure establishment of seedlings. In many other halophytes, most small seedlings survived and radicles

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Tobe et al.ÐGermination and Radicle Growth of Kalidium caspicum

continued to elongate at salinities that allowed seeds togerminate (Khan and Ungar, 1984; Stumpf et al., 1986;
Katembe et al., 1998). The radicles of K. caspicum are very
sensitive to salinity compared to those of many other
halophytic and non-halophytic species (Stumpf et al., 1986;
Lombardi et al., 1998; Tobe et al., 1999). In contrast, mostradicles continued to elongate in PEG solutions of CW
higher than ÿ1.1 MPa until their length exceeded 5 mm.
The marked dierence in the eect of radicles of isotonic
NaCl and PEG indicates that the deleterious eect of NaClis salt-speci®c toxicity rather than osmotic inhibition.
Although K. caspicum is distributed on highly saline soil
and 60-d-old seedlings can survive at a high level of NaCl
(Natori et al., 1992), the small radicles were sensitive to
NaCl. This is in marked contrast to the salinity responses ofHaloxylon persicum, a non-halophytic shrub distributed in
the same region as K. caspicum. H. persicum showed 64%
germination in ÿ3.0 MPa NaCl solution, and the small
seedlings continued to elongate in ÿ3.0 MPa NaCl withoutsuering from salt toxicity, but older seedling of H. persicum
were sensitive to salinity (Tobe et al., 2000). These results
indicate that salinity tolerance in the early seedling stage is
not correlated with salinity tolerance in later developmental
stages, and that salinity adaptability in the initial developmental stages does not determine the geographical distribution of species in this region.
Our results indicate that, although high salinity does notaect the viability of seeds, the relatively low salinity(ÿ0.4 to ÿ1.2 MPa) that causes seed germination results inhigh mortality of the radicles. For the establishment of
K. caspicum in saline environments salt must be completely
removed at the soil surface.
The establishment of K. caspicum in saline environments
may depend on the leaching of salt from the soil surface bysnow-melt in early spring. This study showed that although
seeds do not germinate at 58C, an increase in temperatureinduces very rapid germination. This indicates that seeds
imbibe water at low temperatures in early spring and, assoon as the temperature rises, seeds germinate rapidly.
Although radicle growth is reduced by low temperatures,
low temperatures also reduce the accumulation of salt on
the soil surface and thus favour seedling establishment.
Precipitation in the following warmer season would alsoleach salt from the soil surface and allow germination;
however, high temperatures would result in more evaporation and faster accumulation of salt on the soil surface,which would kill small radicles. To ascertain whether thisprediction is correct, ®eld observations will be needed.
L I T E R AT U R E C I T E D
Boyer JS, Knipling EB. 1965. Isopiestic technique for measuring leaf
water potentials with a thermocouple psychrometer. Proceedingsof the National Academy of Sciences, USA 54: 1044±1051.
Carpita N, Sabularse D, Montezinos D, Delmer DH. 1979. Determination of the pore size of cell walls of living plant cells. Science 205:
1144±1147.
Fan Z, Chang Q, Tian C, Yabuki S, Okada A, Liu C. 1993. Genesis andcharacteristics of salt-aected soils in Tarim Basin. In: Ichikuni

M, ed. Proceedings of the Japan-China international symposium on
the study of the mechanism of deserti®cation. Tokyo: Science andTechnology Agency, 219±226.
Gutterman Y. 1993. Seed germination in desert plants. Berlin: SpringerVerlag, 169±206.
Hardegree SP, Emmerich WE. 1990. Partitioning water potential andspeci®c salt eect on seed germination of four grasses. Annals of
Botany 66: 587±585.
Katembe WJ, Ungar IA, Mitchell JP. 1998. Eect of salinity on
germination and seedling growth of two Atriplex species (Chenopodiaceae). Annals of Botany 82: 167±175.
Khan MA, Rizvi Y. 1994. Eect of salinity, temperature, and growth
regulators on the germination and early seedling growth ofAtriplex grithii var. stocksii. Canadian Journal of Botany 72:475±479.
Khan MA, Ungar IA. 1984. The eect of salinity and temperature on
the germination of polymorphic seeds and growth of Atriplex
triangularis Willd. American Journal of Botany 71: 481±489.
Kigel J. 1995. Seed germination in arid and semiarid regions. In: Kigel
J, Galili G, eds. Seed development and germination. New York:
Marcel Dekker, 645±697.
Koller D. 1964. The survival value of germination-regulating mechanisms in the ®eld. Herbage Abstracts 34: 1±7.
Lang AGR. 1967. Osmotic coecients and water potentials of sodium
chloride solutions from 0 to 408C. Australian Journal of Chemistry
20: 2017±2023.
Lanzhou Institute of Desert Research, Chinese Academy of Sciences.
1985. Desert ¯ora of China, vol. 1. Beijing: Science Press, 381±382.
Lombardi T, Fochetti T, Onnis A. 1998. Germination of Briza maxima
L. seeds: eects of temperature, light, salinity and seed harvestingtime. Seed Science and Technology 26: 463±470.
Macke AJ, Ungar IA. 1971. The eects of salinity on germination andearly growth of Puccinellia nuttalliana. Canadian Journal of
Botany 49: 515±520.
Michel BE, Kaufmann MR. 1973. The osmotic potential of polyethylene glycol 6000. Plant Physiology 51: 914±916.
Myers BA, Morgan WC. 1989. Germination of the salt-tolerant grass
Diplachne fusca. II. Salinity responses. Australian Journal of
Botany 37: 239±251.
Natori T, Mo W, Li X, Jiang S, Omasa K. 1992. The culture of Chinese
desert plants in a greenhouse. Acta Horticulturae 319: 453±458.
Okusanya OT. 1977. The eect of sea water and temperature on thegermination behaviour of Crithmum maritimum. Physiologia
Plantarum 41: 265±267.
Redmann RE. 1974. Osmotic and speci®c ion eects on the germinationof alfalfa. Canadian Journal of Botany 52: 803±808.
Sharma ML. 1973. Simulation of drought and its eect on germinationof ®ve pasture species. Agronomy Journal 65: 982±987.
Stumpf DK, Prisco JT, Weeks JR, Lindley VA, O'Leary JW. 1986.
Salinity and Salicornia bigelovii Torr. seedling establishment.
Water relations. Journal of Experimental Botany 37: 160±169.
Tobe K, Li X, Omasa K. 2000. Eects of NaCl on seed germination andgrowth of two Chinese desert shrubs, Haloxylon ammodendronand H. persicum (Chenopodiaceae). Australian Journal of Botany(in press).
Tobe K, Zhang L, Omasa K. 1999. Eects of NaCl on seed germinationof ®ve nonhalophytic species from a Chinese desert environment.
Seed Science and Technology (in press).
Ungar IA. 1978. Halophyte seed germination. Botanical Review 44:
233±264.
Ungar IA. 1991. Ecophysiology of vascular halophytes. Boca Raton:
CRC Press, 9±48.
Ungar IA. 1995. Seed germination and seed-bank ecology in halophytes. In: Kigel J, Galili G, eds. Seed development and germination. New York: Marcel Dekker, 599±628.
Ungar IA. 1996. Eect of salinity on seed germination, growth, and ion
accumulation of Atriplex patula (Chenopodiaceae). American
Journal of Botany 83: 604±607.
Williams MD, Ungar IA. 1972. The eect of environmental parameterson the germination, growth, and development of Suaeda depressa(Pursh) Wats. American Journal of Botany 59: 912±918.