The influence of high NaCl concentrations on seed germination in both light and darkness was examined in the species Centaurium pulchellum, C. erythraea, C. littorale, C. spicatum, and C. tenuiflorum. Salt tolerance was found to depend on the life history of the seeds. To be specific, seeds of all five species failed to complete germination when exposed to continuous white light if kept all the time in the presence of 100-200 mM and greater NaCl concentrations. However, when after two weeks NaCl was rinsed from the seeds and the seeds were left in distilled water under white light for an additional two weeks, all species completed germination to a certain extent. The percent of germination not only depended on NaCl concentration in the prior medium, but was also species specific. Thus, seeds of C. pulchellum, C. erythraea, and C. littorale completed germination well almost irrespective of the salt concentration previously experienced. On the other hand, seeds of C. tenuiflorum completed germination poorly if NaCl concentrations in the prior media were greater than 200 mM. When seeds after washing were transferred to darkness for an additional 14 days, they failed to complete germination if previously imbibed on media containing NaCl concentrations greater than 400 mM. However, the seeds of all species, even if previously imbibed at 800 mM NaCl, could be induced to complete germination in darkness by 1 mM gibberellic acid. .Uticaj visokih koncentracija NaCl na klijanje semena, kako na svetlosti tako i u tami, ispitivano je kod vrsta Centaurium pulchellum, C. erythraea, C. littorale, C. spicatum i C. tenuiflorum. Tolerantnost ovih biljaka na natrijum-hlorid zavisi od životnog ciklusa semena. Zapravo, semena svih pet ispitivanih vrsta nisu završavala klijanje kada su bila izložena kontinuelnoj beloj svetlosti u prisustvu 100-200 mM i pri većim koncentracijama. Pored toga, semena svih ovih vrsta, izložena imbibiciji na 800 mM NaCl, mogla su biti indukovana da završe klijanje u mraku uz dodatak 1 mM giberelinske kiseline. .nul

Dević, M.

Filipović, B.

Giba, Z.

Grubišić, D.

Živković, S.

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

INTRODUCTION
The genus Centaurium Hill. (Gentianaceae) comprises
herbaceous plants, mostly annuals and biennials. The two
main regions of its distribution are the Mediterranean ar-
ea with Asia and the western part of North America and
Mexico. Natural habitats of C. spicatum are damp grassy
or sandy soils. In former Yugoslavia, it is widespread
along the Adriatic Coast. Centaurium erythraea is a com-
mon species distributed in forests, in light thickets, and
on field margins. The species C. tenuiflorum grows on
humid soils of marsh margins, on grasslands, and in
meadows, mostly in the Mediterranean area of the Balkan
Peninsula. In Serbia, this species is distributed only in
Vojvodina (Deliblato Sands). It is also found along the
Montenegrin Coast. Centaurium pulchellum is wide-
spread in Europe, but in Serbia its natural habitat is re-
stricted to a few regions on the territory of Vojvodina.
The species C. littorale grows on maritime grassland and
in sandy places near the sea. In Europe, it is distributed
southwards from Ireland to NW France, Austria, and SE
Russia. Thus some species of the genus inhabit coastal
regions and saline terrains. 
Germination is a critical part of plant life histories.
The ability of their seeds to germinate at high salt con-
centration in the soil is therefore of crucial importance for
the survival and perpetuation of these species. In saline
habitats, seed germination takes place after high precipi-
tation, i.e., under conditions of reduced soil salinity
(K h a n and R i z v i, 1994). The ability of the soil seed
bank to remain quiescent at a high salt level and to ger-
minate immediately after salinity reduction (B a j j i et
al., 2002) is very significant not only to halophytes, but
also to other species in colonizing their environment. Al-
though salinity stress mostly reduces the germination
percentage and delays the onset of germination, its ef-
fects are modified by interactions with other environmen-
EFFECT OF NaCl ON SEED GERMINATION IN SOME
CENTAURIUM HILL. SPECIES (GENTIANACEAE)
1S. ŽIVKOVIĆ,  1M. DEVIĆ,  1B. FILIPOVIĆ, 2Z. GIBA, and 1,2 D. GRUBIŠIĆ
1Siniša Stanković Institute for Biological Research, 11060 Belgrade, Serbia
2Institute of Botany, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
Abstract – The influence of high NaCl concentrations on seed germination in both light and darkness was examined in
the species Centaurium pulchellum, C. erythraea, C. littorale, C. spicatum, and C. tenuiflorum. Salt tolerance was found
to depend on the life history of the seeds. To be specific, seeds of all five species failed to complete germination when
exposed to continuous white light if kept all the time in the presence of 100-200 mM and greater NaCl concentrations.
However, when after two weeks NaCl was rinsed from the seeds and the seeds were left in distilled water under white
light for an additional two weeks, all species completed germination to a certain extent. The percent of germination not
only depended on NaCl concentration in the prior medium, but was also species specific. Thus, seeds of C. pulchellum,
C. erythraea, and C. littorale completed germination well almost irrespective of the salt concentration previously expe-
rienced. On the other hand, seeds of C. tenuiflorum completed germination poorly if NaCl concentrations in the prior
media were greater than 200 mM. When seeds after washing were transferred to darkness for an additional 14 days, they
failed to complete germination if previously imbibed on media containing NaCl concentrations greater than 400 mM.
However, the seeds of all species, even if previously imbibed at 800 mM NaCl, could be induced to complete germina-
tion in darkness by 1 mM gibberellic acid.
Key words: Germination, NaCl, light, gibberellin, Gentianaceae, Centaurium
UDC 582.923.1 : 581.142 : 581.133.1
227
Arch. Biol. Sci., Belgrade, 59 (3), 227-231, 2007. DOI:10.2298/ABS0703227Z
S. ŽIVKOVIĆ ET AL.228
Fig. 1. Effect of NaCl on light-induced seed germination in five  Centaurium species. Seeds of C. erythraea (A, B), C. littorale (C, D), C. pulchel-
lum (E, F), C. spicatum (G, H), and C. tenuiflorum (I, J) were imbibed  in distilled water (l) or NaCl in concentrations of 25 mM (m), 50 mM (ê),
100 mM (¶), 200 mM (p), 300 mM (r), 400 mM (q), 500 mM (s), 600 mM (u), 700 mM (¯) and 800 mM (Â)  and kept all a time in white
light on the medium containing salt (A, C, E, G, and I) or 14 days after the onset of imbibition were washed and transferred to distilled water and
incubated for an additional 12 days in white light (B, D, F, H, and J). Germination was scored each day either from the onset of imbibition (A, C, E,
G, and I) or from the transfer to distilled water (B, D, F, H, and J).
tal factors such as temperature (M a l c o l m, 1964) and
light (K a h n, 1960). Salinity can affect germination by
affecting the osmotic component, which influences water
uptake, and by interfering with the ionic component,  i.e.,
Na and Cl accumulation. Data on centaury seed germina-
tion in relation to light requirements are rather scarce. To
our best knowledge, apart from data on the germination
of seeds of C. erythraea (M i j a j l o v i ć et al., 2005)
and the species C. spicatum and C. tenuiflorum (Ž i v -
k o v i ć et al., 2002) [reported in the survey of N i k o -
l a e v a et al. (1985)] and information about ecological
aspects of germination  (S i l v e r t o w n, 1980), no oth-
er data on centaury seed germination are available in the
literature. The present study deals with germination of
NaCl-stressed seeds in five species from this genus.
MATERIALS AND METHODS
Seeds of C. erythraea were collected around Lake
Vlasina (Serbia) in 2001 and those of C. spicatum in
2004 in the vicinity of Herceg Novi (Montenegro). The
seeds of other species (C.littorale, C. pulchellum, and C.
tenuiflorum) were harvested from plants grown in the
greenhouse of the Siniša Stanković Institute for Biologi-
cal Research (Belgrade) in 2004. Lots of 30 seeds each
were placed in Petri dishes 6 cm in diameter with 2 ml of
distilled water or NaCl solution. The fungicide Nistatin
was supplied at a concentration of 500 mg L-1 in order to
prevent fungal infections. Seeds were germinated at 25 ±
20C in continuous white light or induced to germinate by
white light for 14 days and then transferred to darkness.
White light was provided by white fluorescent tubes (65
W, 4500 K, “Tesla”, Pančevo, Serbia) giving a fluence
rate of 17.46 mmol m-2 s-1 at seed level. Each solution
change was done by washing the seeds with copious
amounts of distilled  water using a Buchner funnel and
side-arm flask attached to a vacuum pump (Gilford In-
strument, vacuum receiver 3021). Gibberellic acid (GA3)
was purchased from Sigma (St. Louis, USA), and 1 mM
were solutions prepared. Germination was scored for 12
days after the onset of imbibition or four weeks after seed
pretreatment. All experiments were repeated three times.
Standard errors are not shown because they never ex-
ceeded 5 %.
RESULTS
Figure 1 shows the effect of NaCl in the medium on
the germination of all five Centaurium species. As evi-
dent, seeds kept all the time in a medium containing 200
mM and higher concentrations of NaCl did not germi-
nate, even if kept in continuous white light. The extent of
germination at lower NaCl concentrations varied for dif-
ferent species (A, C, E, G, and I). However, if after two
weeks NaCl was washed away and the seeds were left in
distilled water in white light for an additional two weeks,
SEED GERMINATION IN CENTAURIUM HILL. SPECIES 229
Fig. 2. Effect of NaCl on Centaurium seed germination in darkness.
Seeds of  C. erythraea (l), C. littorale (p), C. pulchellum (m), C. spi-
catum (r), and C. tenuiflorum (n) were incubated at different NaCl
concentrations (indicated on the abscissas) in white light for 14 days,
washed and transferred to distilled water and kept in darkness for an
additional 14 days. Germination was scored at the end of the experimen-
tal period. 
Fig. 3. Effect of gibberellic acid on Centaurium seed germination in
darkness. Centaurium seeds were incubated in 800 mM NaCl in white
light for 14 days, washed, and transferred to distilled water (open bars)
or 10-3 M GA3 (hatched bars) and kept in darkness for an additional 14
days. Germination was scored at the end of the experimental period.
all species germinated to a certain extent (Fig. 1, B, D, F,
H, and J). Seeds of some of them germinated up to 100%
even if pre-treated with the highest NaCl concentration. It
seems that C. littorale is better adapted to higher salt con-
centrations in the medium than the other four species
(Fig. 1, C and D). Their germination could be ensured on-
ly if the washed seeds were kept in white light. If after
rinsing they were incubated in distilled water in darkness
for an additional 14 days, they either did not germinate
(C. pulchellum and C. spicatum) or germinated poorly,
depending on the NaCl concentration that they experi-
enced. However, seeds of none of the species germinated
in darkness if previously kept at 500 mM of NaCl (Fig.
2). On the other hand, when seeds were rinsed and trans-
ferred to darkness in the presence of 1 mM GA3, more
than 70% seeds of all species germinated (Fig. 3).
DISCUSSION
The group of plants that are well adapted to saline
habitats are called halophytes. Their seeds germinate well
in freshwater and the germination is similar to that of
seeds of nonadapted species (glycophytes). However,
they differ from glycophytes in their ability to germinate
at higher salt concentrations in the soil (U n g a r, 1995).
Salinity tolerance of many perennial halophytes differs
greatly (K h a n, 2002) and is frequently dependant on a
variety of abiotic factors such as light and temperature
(B a s k i n and B a s k i n, 1998). Generally, plants dis-
play great diversity with regard to soil salinity tolerance.
Species distribution and survival mainly depend on the
seed ability to complete germination and the seedling
ability to develop successfully under unfavorable condi-
tions. Most seeds are located near the soil surface. Salt
concentration in the soil surface changes over time. Con-
tinuous water evaporation causes surface salt deposition
(U n g a r, 1991) while rain dissolves and washes away
salt deposits and provides enough water for germination.
In the course of evolution, seeds have adapted to such
changes, staying viable at high soil salinity and being
able to germinate under appropriate external conditions
(K h a n and U n g a r, 1997).
Our previous and preliminary results showed that
seeds of all five Centaurium species examined by us are
light-requiring. Centaurium erythraea, C. tenuiflorum,
and C. spicatum seeds are induced to germinate by con-
tinous red light. The germination of C. spicatum seeds
can also be induced, apart from red light, by continuous
far red light (Ž i v k o v i ć et al., 2002). Although Cen-
taurium species are not typical halophytes, their seeds
germinated well at a moderately elevated salt concentra-
tion. However, none of the seeds germinated if kept all
the time at relatively high concentrations of NaCl in the
medium. Yet they remained viable and germinated in
light when washed and transferred to a medium free of
NaCl (Fig. 1 B, D, F, H, and J). Published data show that
the effect of NaCl on seed germination is mostly osmot-
ic (K a h n, 1960; L o e r c h e r, 1974; R e y n o l d s,
1975), but the nature of its effect is unknown. It is im-
probable that high NaCl concentrations affect the photo-
conversion of Pr to Pfr (S c o r e r et al., 1985). It is more
likely that low water potentials in seeds affect phyto-
chrome transduction chain (G r u b i š i ć and K o n -
j e v i ć, 1990) because Centaurium seeds incubated at
high NaCl concentrations could not germinate when
transferred to darkness, even if previously washed. This
fact indicates that  phytochrome did not complete its ac-
tion in seeds at a low seed water potential. Germination
requires the embryonic axis to develop a growth potential
high enough to counteract mechanical restraints imposed
by the surrounding tissues. It has been shown that phyto-
chrome induces a decrease of water potential in excised
lettuce embryos, thus increasing  their growth potential
(S c h e i b e and L a n g, 1965; C a r p i t a et al., 1979).
Germination of NaCl-stressed Centaurium seeds under
continous white light may therefore be attributable to the
fact that phytochrome is needed for a longer period of
time for the embryo to develop of the appropriate growth
potential. This view seems to be corroborated by the ef-
fect of gibberellic acid, which under the same conditions
was able to stimulate germination in darkness (Fig. 3).
Acknowledgments – The present work was supported by the Ministry of
Science and Environment Protection of Serbia (Grant No. 143031).
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SEED GERMINATION IN CENTAURIUM HILL. SPECIES 231
ЕФЕКАТ НАТРИЈУМ-ХЛОРИДА НА КЛИЈАЊЕ СЕМЕНА КОД НЕКИХ ВРСТА 
РОДА CENTAURIUM HILL. (GENTIANACEAE)
1С. ЖИВКОВИЋ, 1М. ДЕВИЋ, 2З. ГИБА и 1,2Д. ГРУБИШИЋ
1Институт за биолошка истраживања “Синиша Станковић”, 11060 Београд, Србија
2Институт за ботанику, Биолошки факултет, Универзитет у Београду, 11000 Београд, Србија
Утицај високих концентрација NaCl на клијање семе-
на, како на светлости тако и у тами, испитивано је код
врста Centaurium pulchellum, C. erythraea, C. littorale,
C. spicatum и C. tenuiflorum. Толерантност ових биља-
ка на натријум-хлорид зависи од животног циклуса
семена. Заправо, семена свих пет испитиваних врста
нису завршавала клијање када су била изложена кон-
тинуелној белој светлости у присуству 100-200 mM и
при већим концентрацијама. Поред тога, семена свих
ових врста, изложена имбибицији на 800 mM NaCl,
могла су бити индукована да заврше клијање у мраку
уз додатак 1 mM гиберелинске киселине.


Effect of NaCl on seed germination in some Centaurium Hill. Species (Gentianaceae)

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Effect of NaCl on seed germination in some Centaurium Hill. Species (Gentianaceae)

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