EFFECT OF NaCl ON SEED GERMINATION IN SOME CENTAURIUM HILL . SPECIES ( GENTIANACEAE ) 1

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.


INTRODUCTION
The genus Centaurium Hill.(Gentianaceae) comprises herbaceous plants, mostly annuals and biennials.The two main regions of its distribution are the Mediterranean area 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 common 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 widespread in Europe, but in Serbia its natural habitat is restricted 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 concentration 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 precipitation, i.e., under conditions of reduced soil salinity (Khanand Rizvi, 1994).The ability of the soil seed bank to remain quiescent at a high salt level and to germinate immediately after salinity reduction (Bajjiet al., 2002) is very significant not only to halophytes, but also to other species in colonizing their environment.Although salinity stress mostly reduces the germination percentage and delays the onset of germination, its effects are modified by interactions with other environmen-  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 (Malcolm, 1964) and light (Kahn, 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 germination in relation to light requirements are rather scarce.To our best knowledge, apart from data on the germination of seeds of C. erythraea (Mijajlović et al., 2005) and the species C. spicatum and C. tenuiflorum (Živković et al., 2002) [reported in the survey of Nikolaevaet al. (1985)] and information about ecological aspects of germination (Silvertown, 1980), no other 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.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 ± 2 0 C 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 µmol 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 Instrument, vacuum receiver 3021).Gibberellic acid (GA 3 ) 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 exceeded 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 germinate, even if kept in continuous white light.The extent of germination at lower NaCl concentrations varied for different 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,  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 concentrations in the medium than the other four species (Fig. 1, C and D).Their germination could be ensured only 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 experienced.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 transferred to darkness in the presence of 1 mM GA 3 , 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 (Ungar, 1995).Salinity tolerance of many perennial halophytes differs greatly (Khan, 2002) and is frequently dependant on a variety of abiotic factors such as light and temperature (Baskinand Baskin, 1998).Generally, plants display 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 conditions.Most seeds are located near the soil surface.Salt concentration in the soil surface changes over time.Continuous water evaporation causes surface salt deposition (Ungar, 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 (Khanand Ungar, 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 continous red light.The germination of C. spicatum seeds can also be induced, apart from red light, by continuous far red light (Živković et al., 2002).Although Cen-taurium species are not typical halophytes, their seeds germinated well at a moderately elevated salt concentration.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 osmotic (Kahn, 1960;Loercher, 1974;Reynolds, 1975), but the nature of its effect is unknown.It is improbable that high NaCl concentrations affect the photoconversion of Pr to Pfr (Scoreret al., 1985).It is more likely that low water potentials in seeds affect phytochrome transduction chain (Grubišić and Konjević, 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 action 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 phytochrome induces a decrease of water potential in excised lettuce embryos, thus increasing their growth potential (Scheibeand Lang, 1965;Carpitaet 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 effect of gibberellic acid, which under the same conditions was able to stimulate germination in darkness (Fig. 3).

Fig. 1 .
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. pulchellum (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).
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 Biological Research (Belgrade) in 2004.

Fig. 2 .
Fig. 2. Effect of NaCl on Centaurium seed germination in darkness.Seeds of C. erythraea (l), C. littorale (p), C. pulchellum (m), C. spicatum (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 experimental period.

Fig. 3 .
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 GA 3 (hatched bars) and kept in darkness for an additional 14 days.Germination was scored at the end of the experimental period.