Effects of environmental factors on seed germination

Transcription

Plant Biosystems, Vol. 142, No. 2, July 2008, pp. 275 – 286
Effects of environmental factors on seed germination of
Anthyllis barba-jovis L.
MASSIMILIANO MORBIDONI1, ELENA ESTRELLES2, PILAR SORIANO2,
ISABEL MARTÍNEZ-SOLÍS3, & EDOARDO BIONDI2
1
Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Ancona, Italy,
ICBiBE - Jardı́ Botànic, Universitat de València, España and 3Universidad CEU – Cardenal Herrera, Departamento
de Fisiologı́a, Farmacologı́a y Toxicologı́a, Valencia, España
Downloaded By: [Biondi, Edoardo] At: 16:01 30 January 2009
2
Abstract
The influence of the main environmental factors on seed germination of Anthyllis barba-jovis L. were analysed. This work is
part of a broader investigation aimed at the reintroduction of this species on Mount Conero, Ancona (central Italy), where it
is at present extinct. The seeds were collected from the Gargano headland (southern Adriatic coast). Experimental analyses
were carried out to determine: (i) dormancy levels of seeds collected in successive years, and also collected from the soil seed
bank; (ii) effects of usual pre-treatments for overriding the physical dormancy of the seeds; (iii) optimal temperature range
for maximum germination; (iv) effects of fire on seed germination; and (v) effects of NaCl on germination and on early
stages of seedling development. Our results confirm that A. barba-jovis seeds have a physical dormancy due to their
teguments, which are water-impermeable. This barrier persists in naked seeds that remain in the soil. Regularly waterdrenched seeds show a high germinative ability. The optimal seed germination temperature is 208C, with germination
decreasing progressively at lower temperatures, and falling drastically over 208C. Fire and high temperatures positively
affected germination. The seeds were shown to be strongly resistant to salt stress, thus enabling the plants to colonize a
habitat suitable for halophytes.
Key words: Anthyllis barba-jovis, ecosystem restoration, fire species, germination, salt tolerance, seed dormancy
Introduction
Anthyllis barba-jovis L. is an evergreen shrub that is
found in different habitats along the rocky cliffs of
the western–central Mediterranean basin; in France
(Var, Bouches du Rhône, Hérault, Corsica), Italy
(Liguria, Tyrrhenian coast, Sardinia, Sicily, Adriatic
coast of Gargano, Tremiti Islands), Croatia, Algeria
and Tunisia (Cullen 1968; Greuter et al. 1989;
Pignatti 1992; Trinajstic 1994; Biondi et al. 1997;
Paradis 1997) (Figure 1).
Anthyllis barba-jovis is not included in the World
Conservation Union (IUCN) Red List, although it is
a protected species at a national level in France
(Danton & Baffray 1995) and Croatia (Trinajstic
1994). In Italy, it is not listed in the Protected Flora
(Conti et al. 1992), but it is considered to be in a risk
category in seven of the nine regions where it is
found (Conti et al. 1997). Although the areas
inhabited by this species are not generally directly
affected by anthropic modifications, due to their
inaccessibility, there have been quite recent cases
where A. barba-jovis has disappeared from its natural
habitats (Brilli-Cattarini 1965; Biondi 1986; Danton
& Baffray 1995; Paradis 1997; Benedi 1998). At
present, it has to be considered as extinct from the
Mount Conero promontory, Ancona (central Italy;
see Figure 2) (Brilli-Cattarini 1965; Biondi 1986),
although this territory was, for a long time, considered
to be its most northern known natural location along
the Adriatic coast of the Italian peninsula (Biondi
et al. 2002). Its presence in the past is testified by a
herbarium record, as it was collected in 1808 by the
naturalist Paolo Spadoni. In his work entitled
Xylologia Picena applicata alle arti, he claimed to have
collected it along the coast between Ancona and
Sirolo, including the coastal rocky marl and limestone
of Mount Conero (Spadoni 1826).
The present study is the first part of a broader
programme focused on acquiring further knowledge
Correspondence: Edoardo Biondi, Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, via Brecce Bianche
s.n., 60131 Ancona, Italy. E-mail: [email protected]
ISSN 1126-3504 print/ISSN 1724-5575 online ª 2008 Società Botanica Italiana
DOI: 10.1080/11263500802150514
276
M. Morbidoni et al.
Figure 1. Present-day distribution of Anthyllis barba-jovis.
Figure 2. Geographic location of Mount Conero and the seed harvesting zone in the Gargano headland.
of the autoecology of A. barba-jovis, its ex-situ
conservation, and its reintroduction in the Regional
Natural Park of Mount Conero. Restoration programmes are a priority in the management of natural
populations, and thus our study is aimed at providing
protocols that cover the first steps of plant culture.
Present results relate to various aspects of the
germination physiology of A. barba-jovis seeds. In
particular, the following issues were addressed:
(i) the dormancy level in seeds collected in successive years and taken from the soil seed bank;
(ii) the effects of standard seed pre-treatments to
override their physical dormancy, with verification of the results at a histological level;
(iii) the optimal temperature range for maximum
germination;
(iv) the effects of fire temperatures on seed
germination; and
Germination in Anthyllis barba-jovis
(v) the effects of NaCl on the germination phase,
the first stages of seedling development, and
the relationships between light conditions, the
thermal optimum and salt concentrations.
Materials and methods
Plant material
Anthyllis barba-jovis seeds were collected from San
Menaio, in the Gargano headland territory of the
Italian peninsula (southern Adriatic basin; Figure 2)
in September 2003 and July 2004. In this region, the
A. barba-jovis population is large, and although seed
harvesting was abundant, it was not enough to
damage the natural rate of reproduction. The
monosperm legumes are generally retained inside
the dry flower remains; they were collected at
maturity directly from the plants and desiccated
under laboratory conditions. The fruits were cleaned
by rubbing them between two rubber sheets, and
then separating them through differently sized
metallic sieves; the small pods were then opened
using a scalpel. The best procedure was to cut the tip
and then open the pod longitudinally with a blunt
blade. This had to be done with special care to avoid
damage to the seed coat, which could destroy seed
impermeability. The cleaned seeds were kept in
airtight bags at room temperature until used.
Germination protocol
For germination, the seeds were placed on 55 mm
diameter Petri dishes containing 0.6% agar, which
were kept in climate-controlled rooms under various
temperature and light conditions, as specified below.
The observation period was generally one month,
and for each treatment 4 replicates of 25 seeds were
used. A seed was considered to have germinated
when the radicle was longer than 1 mm.
Mechanical and chemical scarification
The study of physical dormancy was performed
through the application of different treatments,
including mechanical and chemical scarification,
pre-heating and the alternation of freeze/thaw temperatures using liquid nitrogen. Since similar experiments have not been performed previously on A.
barba-jovis, the various parameters were selected
following the studies on Anthyllis cytisoides and
Anthyllis lagascana (Ibanez & Passera 1997; Prieto
et al. 2004). These species are considered to be
phylogenetically and ecologically similar to A. barbajovis (Nanni et al. 2004).
Two different scarification methods were used:
one mechanical, with sand paper, and the other
277
chemical, with 96% sulphuric acid for 5, 10 or
15 min. For the pre-heating treatments, two different
types of heat were used: dry heat and humid heat,
with a factorial experimental design. The dry heat
was applied using a Selecta thermo-block: the seeds
were exposed to at 808C or 1008C for 5 or 10 min.
The seeds were subjected to humid heat by immersion in water at 808C for 5 s, 10 s and 5 min. The
freeze/thaw protocol was for 20 min at –1968C in
liquid nitrogen, followed by 10 min thawing in a
waterbath at 408C, for 10, 20 and 30 successive
cycles.
All of the basic germination tests for the dormancy
of seeds were carried out in the dark at 208C. The
experiments were carried out in December 2003,
with seeds gathered in September 2003.
Electron microscopy
Observations were carried out on the morphology of
the seed coat of three different seed samples: (i) nontreated seeds collected from the plant; (ii) seeds
treated with 96% sulphuric acid for 15 min; and (iii)
non-treated seeds collected from the soil seed bank.
The seeds, are, in general, found in a dehydrated
condition, so that they were analysed directly without
the need for any dehydration process. The samples
for the ultramorphological analysis were mounted on
an aluminium base, and kept in place using doublesided carbon tape (STR tape, 8 mm, Sinto Paint Co.
Ltd). They were then metallized with SC 500 Sputter
Coater (Bio-Rad) for a cover of gold–palladium of
*200 Å, and examined by scanning electron
microscopy (FE HITACHI 4100), which included
a system for the collection of digital images. The
voltages used were 5 kV and 10 kV.
Germination temperature
The optimum germination temperature was established by testing a range of temperatures from 58C to
358C, at intervals of 58C. A second test was carried
out to determine the percentages of seeds that
germinated under conditions of alternating temperature, using 108C and 188C, with a period of 10 h and
14 h, respectively. In all cases, the seeds were pretreated with chemical scarification using 96% sulphuric acid for 15 min, followed by profuse washing
with sterilized water; they were kept in the dark for
germination. The experiments were carried out in
October 2004 with seeds collected in September
2003.
Dormant seed collection from the seed bank
For testing the degree of dormancy, three seed
samples were collected. Seeds were collected directly
278
M. Morbidoni et al.
from the plants, in July 2003 and in July 2004, and
extracted from the fruits as described above. Naturally aged seeds from the soil seed bank were gathered
in September 2004, by taking soil samples at a 3–
4 cm depth at the base of some adult A. barba-jovis
individuals. Samples consisted of marly detritus, with
some humus and decomposing organic matter, and
were in contact with the compact rock. The seeds
were partly contained in the legumes and partly
exposed; they were separated out using metal forceps.
They were germinated in the dark, either under the
alternating temperature conditions indicated above
(108C/188C for 10 h/12 h), or after chemical scarification with 96% sulphuric acid for 15 min. These
tests were performed in summer 2005.
Pre-treatment with heat or fire
Figure 3. Germination of seeds subjected to different scarification
pre-treatments. Ctr, control; abr, scarification with sandpaper;
S.A.5, S.A.10, S.A.15, scarification with 96% sulphuric acid for 5,
10 and 15 min. The different letters indicate means that show
significant differences (P 5 0.05).
To simulate the effects of fire, different experimental
conditions were examined: (i) a seed sample underwent thermal shock pre-treatment at temperatures of
508C, 1208C and 1508C, and with different exposure
times of 30, 60 and 120 min, 1, 5 and 10 min, and
1 min, respectively. To do this, the seeds were placed
in glass test tubes containing sterilized sand, which
were then placed in a stove; for the short treatment
periods (1208C and 1508C), the sand was pre-heated
to the required temperatures; (ii) a seed sample was
directly exposed to the naked flame of a Bunsen
burner for a few seconds (Vuillemin & Bulard 1981);
and (iii) a seed sample was left inside the fruits and
the dried remains of the flowers (the conditions in
which they were gathered from the soil) and ignited
over a Bunsen burner flame and allowed to burn until
the flame extinguished spontaneously. The seeds
were then recovered from the ashes and immediately
prepared for germination. All seeds were germinated
in the dark, under the alternating temperature
Figure 4. Germination of seeds pre-treated with heat (A) and liquid nitrogen (B), compared with control and mechanical scarification. Ctr,
control; abr, scarification with sandpaper; UM.5s, 10s, 5 min, humid heat 5 s, 10 s, 5 min; 80–5 min/10 min, 100–5 min/10 min, 808C for
5 min/10 min, 1008C for 5 min/10 min; 10-N, 20-N, 30-N, 10, 20, 30 freeze (–1968C)/thaw cycles. The different letters indicate means that
show significant differences (P 5 0.05).
conditions indicated above (108C/188C for 10 h/
12 h). The experiments were carried out in June 2005
with seeds that had been gathered in July 2004.
279
experiments were concluded after the first true leaves
appeared on the control plants (approximately one
month from germination), and the lengths of the
stems and roots were measured.
Salt stress
The germination tolerances to 10 increasing concentrations of NaCl were determined: 0, 50, 100,
150, 200, 250, 300, 350, 400 and 500 mM. The
seeds were scarified before use with 96% sulphuric
acid for 15 min, and they were germinated at the two
best temperatures (158C and 208C) in the dark. Salt
effects on seedling development led to the hypothesis
that germination behaviour varied according to
salinity and illumination. Therefore, the salinity
effects were also investigated under a 12 h/12 h
photoperiod at 158C. The experiments were carried
out in October 2004 with seeds that had been
gathered in September 2003. These germination
experiments were extended for up to two months, as,
by the end of the first month, there were seeds that
were still in active germination, and in particular at
the higher NaCl concentrations.
Further development of plantlets under salt stress
was carried out under a 12 h/12 h photoperiod at
208C. Small bottles (height 35 mm, diameter
65 mm) were used that contained 30 ml 1% agar
as substrate, supplemented with Murashige and
Skoog salts (MS; 4.3 g/l) to satisfy the nutritional
demands of the seedlings. NaCl was added at the
concentrations specified above. The seeds were
scarified with 96% sulphuric acid for 15 min. The
Table I. Germination after different scarification pre-treatments,
expressed as percentages + standard error.
Chemical scarification with 96%
sulphuric acid
Days
Control
Mechanical
scarification
5 min
10 min
15 min
9
12
19
29
32
0 + 0.0
0 + 0.0
1 + 1.0
3 + 1.9
3 + 1.9
78 + 9.6
78 + 9.6
78 + 9.6
78 + 9.6
78 + 9.6
4 + 2.3
4 + 2.3
15 + 1.9
21 + 1.0
22 + 1.2
11 + 3.0
16 + 4.3
29 + 1.9
32 + 3.3
33 + 4.1
16 + 6.3
45 + 5.4
80 + 3.5
84 + 2.4
84 + 2.4
Statistical analysis
The data obtained were analysed statistically by
ANOVA for different factors, with a Tukey post-hoc
test to identify homogeneous groups, and a significance level of 0.05 to confirm the differences
between the arithmetic means. The percentages
obtained had a normal behaviour, and therefore an
arcsin transformation was not necessary. In the
histograms, the different letters indicate data that
are significantly different.
Results
Physical dormancy
The non-treated seeds showed very low levels of
germination (*3%), which was also particularly slow
(Figures 3 and 4; Table I). The pre-treatments were
all more effective. The seeds treated with abrasive
paper showed germination levels that were significantly greater than control levels (78 + 9.6%);
germination was particularly fast, with maximum
germination already achieved on the first day of
sampling (Figure 3; Table I). Seeds subjected to
chemical scarification using sulfuric acid showed the
highest germination rate (84 + 2.4%); germination
levels were proportional to treatment times (Figure 3;
Table I).
Immersion in hot water (humid heat at 808C) gave
modest results (Figure 4A; Table II); the longest
treatment (5 min) yielded the highest germination
levels (34 + 6.2%). This 5-min treatment, which was
considerably longer than most of the others, did not
appear to compromise seed vitality, although, in
most seeds, germination was slowed down by about
one week.
The same slow germination was seen following dry
heat treatment of the seeds, at both 808C and 1008C
(Figure 4A). The highest levels of germination were
observed with an exposure to 1008C for 5 min
Table II. Germination after seed exposure to thermal shock, expressed as percentages + standard error.
Day
9
12
19
29
32
Humid 808C
5s
Humid 808C
10 s
Humid 808C
5 min
Dry 808C
5 min
Dry 808C
10 min
Dry 1008C
5 min
Dry 1008C
10 min
9 + 2.5
14 + 2.6
16 + 2.3
16 + 2.3
16 + 2.3
7 + 3.0
9 + 3.0
14 + 2.0
17 + 1.0
17 + 1.0
5 + 1.9
10 + 2.0
23 + 5.0
30 + 5.3
34 + 6.2
2 + 1.2
5 + 1.0
18 + 3.5
22 + 2.6
24 + 1.6
3 + 1.9
7 + 3.4
26 + 6.2
32 + 5.4
33 + 5.3
5 + 1.0
7 + 1.0
31 + 3.4
33 + 4.1
34 + 5.0
1 + 1.0
3 + 1.9
15 + 3.0
21 + 6.6
22 + 6.2
280
M. Morbidoni et al.
Electron microscopy
The quality of the external tegument and the
alterations that were caused by the artificial scarification produced by sulphuric acid were examined by
electron microscopy. These seeds were compared
(34 + 5.0%); twice this treatment time resulted in
the death of some of the seeds.
The highest germination rates were obtained with
seeds treated for 20 or 30 freeze/thaw cycles through
repeated immersion in liquid nitrogen: ca. 85% of
these seeds germinated (Figure 4B).
Figure 5. General ventral views of seeds. Left panels: magnification, 47 6. Right panels: magnification, 1500 6. A. Seed collected from the
plant that can be seen to be completely intact, with the characteristic cavities recognizable under the higher magnification: dépressions
plissées, sensu Saint-Martin (1986). B. Seed following scarification, where the modifications to the hilum area can be seen; the characteristic
cavities in the non-scarified seed are no longer seen at the higher magnification. Instead, more or less deep hollows are visible, with cracks. C.
Seed collected from the soil, with the hilum area showing similar modifications to those seen in the seed following artificial scarification;
moreover, the surface of the seed is cracked and not so smooth. Under the higher magnification, the tegument is seen to be altered, even
though to a lesser extent than in B.
281
Table III. Germination under different thermal regimes, expressed as percentages + standard error.
Days
7
13
20
26
31
58C
108C
158C
208C
258C
308C
10/188C
0
0
2.5 + 2.5
11.3 + 5.5
26.3 + 7.5
0
17.5 + 3.2
30.0 + 5.4
37.5 + 2.5
55.0 + 3.5
37.5 + 2.5
51.3 + 2.4
57.5 + 2.5
57.5 + 2.5
58.8 + 1.3
0
45.0 + 5.4
78.8 + 3.1
82.5 + 1.4
83.8 + 2.4
0
10.0 + 3.5
22.5 + 5.2
36.3 + 3.1
53.8 + 3.8
0
0
1.3 + 1.3
5.0 + 2.0
5.0 + 2.0
52.0 + 6.5
63.0 + 3.4
69.0 + 1.9
70.0 + 1.1
75.0 + 1.0
Anthyllis. They have been described as ‘‘dépressions
plissées’’. Pitting, with cracking, was seen following
artificial scarification (Figure 5B), but also in seeds
collected from the soil (Figure 5C).
Optimal germination temperature
Over the range of temperatures tested, A. barba-jovis
showed significantly variable germination responses
(Table III; Figure 6). The temperature that can be
considered to be optimal for seed germination was
208C. At lower temperatures, germination decreased
progressively, whereas at temperatures above 208C
they fell drastically (e.g. 5% at 308C; 0.0% at 358C).
Figure 6. Germination under the different thermal regimes (as
indicated), in the dark. The different letters indicate means that
Degree of dormancy in seeds of different ages
The germination capacity of seeds of different ages
did not show any striking differences (Figure 7), with
more than one year needed to show significant aging.
In our samples, the physical dormancy was not
reduced following one year under laboratory conditions. Whether or not the seeds were scarified, those
that had aged in the soil showed a tendency to
germinate quicker and in greater percentages. The
seeds from the soil seed bank remained dormant, and
scarification was still needed to break this dormancy.
Fire and temperature effects on germination
Figure 7. Germination of seeds of different ages gathered directly
from the plant in the years 2003 and 2004 and stored in the
laboratory, and of seeds from the soil bank gathered either with
their legume or ‘‘naked’’. The different letters indicate means that
with those that were collected directly from the plant
or the soil. The seeds collected from the plant
(Figure 5A) had clearly evident radial depressions
and cavities; these were described by Saint-Martin
(1986) as being characteristic of many Leguminosae
from the Loteae tribe, and in particular of the genus
At temperatures that simulated the effects of fire, the
best results were obtained by exposing the seeds to
1208C for 1 min (44 + 6.3%), with longer exposure
times resulting in lower germination rates (5 min,
25 + 4.1%; 10 min, 9 + 2.5%). One minute of
exposure at a temperature of 1508C resulted in very
low levels of germination (4 + 1.6%). Whereas
modest levels of germination were obtained after
exposure of the seeds to flame (20 + 2.8%), burning
of the seeds inside the fruits produced improved
germination levels (43 + 4.1%) (Table IV; Figure 8).
Salt and light effects on germination
As expected, germination rates decreased as the salt
concentrations increased. Germination also occurred
282
M. Morbidoni et al.
Table IV. Effects of high temperatures on germination, expressed as percentages + standard error.
Control
508C
30 min
508C
60 min
508C
120 min
1208C
1 min
1208C
5 min
1208C
10 min
1508C
1 min
Naked
flame
Burnt
8 + 4.3
23 + 3.4
32 + 3.3
14 + 2.0
44 + 6.3
25 + 4.1
9 + 2.5
4 + 1.6
20 + 2.8
43 + 4.1
Discussion and conclusions
Figure 8. Germination of pre-treated seeds following different
exposures to high temperatures. The different letters indicate
means that show significant differences (P 5 0.05).
at 400 mM NaCl, a salt concentration that would
usually block seed germination. Furthermore, under
salt stress conditions, different effects were seen
regarding light and temperature conditions. At high
salt concentrations in the dark there was a greater
level of germination at 158C than at 208C (Figure 9;
Table V). Under different light regimes and salt
concentrations at 158C slightly superior levels of
germination were observed in the dark than with a
photoperiod of 12 h / 12 h (Figure 10; Table V).
These effects became more evident with the extension of the experiments to a total of 60 days, with
increased germination levels at 300 mM NaCl (38%)
and higher.
Salt effects on seedling development
Increasing the salt concentration in the substrate
led to a progressive slowing down of growth that
was equally evident in roots and stems, although
these inhibitory effects on growth became more
evident with the stems at the higher NaCl
concentrations (Figure 11). Indeed, a concentration of 300 mM NaCl appeared to constitute a
limit to stem development, although radicle
growth continued very slowly up to 400 mM NaCl
(Figure 11).
This study has confirmed that A. barba-jovis seeds
have a physical dormancy, with the germination
barrier only consisting of the tegument, due to its
water resistance, as usually occurs in most Fabaceae
(Baskin & Baskin 1989). The results obtained with
the more efficient scarification techniques demonstrate the high vitality of the seeds.
A high level of physical dormancy was also found
in the naturally aged seeds present in the soil; only
after being pre-treated with chemical scarification
did these seeds show a high germination rate. Thus,
it can be seen that dormancy was not reduced (or
only very slightly) by age. The seed longevity also
remained high.
The histological observations on the external
surface of the seed tegument showed that this was
fully continuous in the intact seeds, and was changed
in various ways and fractured by the action of the
acid. For the seeds collected in the soil, of those that
were examined microscopically, none was found that
had manifest signs of breakage or degradation of the
tegument. This condition was in agreement with the
germination trials with the same seeds. Here, as
indicated above, there was a slight, but non-significant, increase in germination levels compared with
those obtained for seeds collected from the plants.
The temperatures to which A. barba-jovis seeds
were subjected in the present study were very similar
to those that can be generated during a fire of shrub
vegetation at the most superficial layer of the ground
(0–5 cm), where most of the dormant seeds (the soil
seed-bank) are found. These temperatures have also
given positive results in other studies on Leguminosae (Auld & O’Connell 1991; Herranz et al. 1997).
Therefore, it is clear that in relation to the highest
temperatures registered at a few centimetres above
ground level (about 5008C), temperatures of only
around 408C to 508C are found at a depth of 5 cm,
and above 608C in the most superficial layer (0.5 cm)
(Auld & Bradstock 1996). These temperatures can be
higher when natural organic matter is present on the
ground, in which case they can reach 1408C at a 2-cm
depth (Bradstock et al. 1992).
Exposure of the seeds to a direct naked flame or
to the burning of the dry remains of the flowers
were based on the consideration that, due to the
pedological characteristics of the zone in which
283
Figure 9. Germination of seeds under increasing saline concentrations, at 208C (left) and 158C (right) in the dark. Results shown one month
from sowing. The different letters indicate means that show significant differences (P 5 0.05).
Table V. Germination of seeds under increasing salt concentrations at one and two months after sowing, expressed as
percentages + standard error. The different letters indicate means that show significant differences (P 5 0.05).
1 month
208C in the dark
158C photoperiod 12 h/12 h
158C in the dark
2 months
158C photoperiod 12 h/12 h
158C in the dark
0
50
100
83.8 ab
91.3a b
86.3 ab
47.5 ch
85.0 ab
90.0 ab
25.0 g
81.3 af
80.0 ab
0
50
100
92.5 a
88.8 a
87.5 a
90.0 ab
86.3 a
80.8 ab
A. barba-jovis grows (rocks that are more or less
degraded, and very shallow or completely absent
soil), the action of fire on buried seeds would appear
less likely than a more direct effect on the seed
surface. The effects of the flame on the seed is a test
that no doubt has a highly random effect that is
difficult to reproduce. However, the germination
percentages were greater than those obtained with
the maximum times of exposure to temperatures of
1508C, 1208C and 508C, and significantly greater
than the controls. Exposure to a temperature of 508C
was assumed to simulate the thermal shock produced
not only by the action of fire, but also by the direct
effects of solar irradiation on the seeds when the
protection of the plant cover is eliminated. The
action of high temperatures on seed germination in
the Leguminosae growing in Mediterranean climates
shows strong interspecific variations (Herranz et al.
1997; Hanley et al. 2001). In almost all of the cases
NaCl concentration (mM)
150
200
250
33.8 g
63.8 c
93.8 b
45.0 cg
17.5 d
71.3 cf
1.3 e
0e
20.0 d
NaCl concentration (mM)
150
200
250
75.0 b
95.0 a
30.0 c
88.8 a
8.8 d
83.8 ab
300
350
400
500
0e
0e
5.0 e
0e
0e
0e
0e
0e
0e
0e
0e
0e
300
350
400
500
1.3 e
38.8 c
0e
15.0 d
0e
3.8 de
0e
0e
studied, an increase in germination response has
been seen with exposure to temperatures between
708C and 1508C for more or less prolonged periods
of time. However, with many species from environments that are exposed to frequent fires, an exposure
to temperatures of 1108C to 1208C would lead to
embryo death, even if it is only for 4 min (Auld &
O’Connell 1991). The seeds of A. barba-jovis appear
to have an intermediate behaviour among the
Leguminosae, which can show evident positive
effects when exposed to high temperatures. These
effects can produce germination levels of 75% with
moderately prolonged exposures (10 min) to 1208C
(e.g. Cytisus scoparius, Psoralea bituminosa and Ulex
europaeus), with others that apparently do not obtain
any benefit (e.g. Scorpiurus muricatus) (Herranz et al.
1997). In the present study, treatment at 1208C for
10 min was detrimental (or at least did not produce
any advantage), and a temperature of 1508C was
284
M. Morbidoni et al.
Figure 10. Germination of seeds under increasing saline concentrations at 158C, one and two months after sowing. The different letters
indicate means that show significant differences (P 5 0.05).
clearly lethal for many embryos, as the germination
levels were lower than those of the control. The
very important role of the Leguminosae (either
grasses or shrubs) in the recolonization of burnt
environments is well known (Doussi & Thanos
1994; Arianoutsou & Thanos 1996; MartinezSanchez & Herranz 1999). Indeed, many of them
appear to obtain indirect benefits from the passage
of fire, in addition to those mainly deriving from the
greater availability of space due of the lack of
competing plants.
For the species studied here, positive effects of
high temperatures were evident on seed germination,
although they were not greatly significant. This may
be one of the numerous possible ways of breaching
the impermeability of the seed coat, although it may
not be the main one. Due to the high sensitivity to
high temperatures shown by the embryo, it appears
that this plant is not a pyrophyte; the ecological
benefit that might be derived from the passage of fire
will be determined by the indirect factors described
above. There is a joint action of legume indehiscence
and secondary dormancy, which persists with time in
the seeds in the ground, and which can be eliminated
by drastic scarification. These, therefore, constitute
control mechanisms for germination, preventing
seeds from germinating under unfavourable conditions, and also generally retarding the germination
process.
This presence of such germination barriers, along
with the prolonged vitality of the seeds, allows us to
hypothesize that in nature there is an accumulation
of a considerable reserve of seeds that settle in the
ground with time (Bewley & Black 1985; Guardia
et al. 2000). This is an important adaptive aspect, as
it is evident that in a difficult environment like these
285
Figure 11. Effects of increasing the saline concentrations of the substratum on the lengthening of the stem and radicle. Data collected 40
days after germination.
rocky cliffs, the availability of an important seed
reserve in itself constitutes an important factor for
species survival.
Anthyllis barba-jovis seeds displayed a high resistance to salt stress. After two months of observation,
germination in up to 400 mM NaCl was recorded, a
concentration that would cause a total block of
germination in most plants. The ability to germinate
at such high or more salt concentrations is typical of
halophytic species. Even higher NaCl concentrations
(800 mM NaCl) can be tolerated only by the
hyperspecialized halophytes; for example, species of
the genera Arthrocnemum, Salicornia and Salsola
(Khan & Gul 1998; Khan 2002).
In the species under investigation here, germination under salt stress was modulated by temperature
and light conditions. In the absence of salt, seed
germination was indifferent to light, and had a
precise temperature optimum. Under high salt
concentrations, any variation in temperature relative
to the thermal optimum resulted in decreased
germination, while light also behaves as a fundamental factor (Naidoo & Naicker 1992; Khan &
Ungar 1999; Khan & Gulzar 2003; Zia & Khan
2004). Some ecologists have interpreted such behaviour as the result of an adaptative process. The salt
concentrations in the ground are very variable, also
depending on the time of the year; for instance, a
part of the salt is washed away during rainy periods,
and, on the contrary, rises to the surface in dry
periods and during drought. For A. barba-jovis, as
shown here, the constant temperature that was the
optimum under conditions of low soil salinity (208C)
was no longer the optimal in the presence of salt,
while light definitely works against germination. It is
clear, therefore, that A. barba-jovis can germinate in
soil that is rich in salt, and can compete with the
halophytes that are directly exposed to the action of
the sea. Indeed, in the environment where this
species grows, high salt concentrations have been
measured in the waters that bathe the seeds in the
summer following occasional rainy periods, or on the
surface of the soil as a tide effect, or due to the sea
breeze; these are all unfavourable situations that can be
overcome by the temperature–salinity–light interactions. Germination could be inhibited by high
temperatures in the first case, and by light in the
second. The intolerance to light in the presence of salt
means that a seed is more likely to germinate when it is
buried, thereby activating a mechanism based on this
photoinhibition that inhibits the superficial development of the seedling (Thanos et al. 1989), at least
during the most unfavourable times of the year.
For A. barba-jovis, although germination is the
most critical phase of its development, adaptations to
living in environments that are characterized by high
salt concentrations in the soil are also undoubtedly
seen in the early stages of seedling growth which
appears to be almost indifferent to 50 mM NaCl,
while little damage appears to be caused by
concentrations up to 100–150 mM.
Acknowledgements
We are very grateful to the staff of Servicio Central de
Soporte a la Investigación Experimental (SCSIE) –
Universidad de Valencia, for help in preparing the
samples investigated and in subsequent observations.
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Effects of environmental factors on seed germination

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