International Journal of Mediterranean Ecology - Ecologia ...

Material and methods
Seed collection sites
Seeds of the three Calligonum species were
obtained from wild plants which were collected
at El Borma (31 o 63’N, 09 o 27’E, 277 m
a.s.l.) for C. azel and C. arich and Tiert
(30 o 88’N, 10 o 16’E, 360 m a.s.l.) for C. comosum
in the Eastern Great Erg of Southern
Tunisia. The month and year of seed collection,
seed size and seed dry mass of the three
Calligonum species are shown in Table 1.
Seeds were cleaned and stored in the seed
bank at the Laboratory of the Arid Regions
Institute until further use (20 o C, 30% RH).
Both sites have an arid-type climate with dry
and hot summers and cold winters. The rains
are infrequent and irregular, sometimes with
no rain during long periods of several years.
The mean annual rainfall for 10 years is
52.3 mm and 61.6 mm, and the mean annual
temperature is 22 o C and 20 o C for El Borma
and Tiert, respectively (INM 1996). According
to Dhief et al. (2009), the mean maximum
temperature of the warmest month (August)
is 41.5 o C and 40.5 o C, and the minimum temperature
of the coldest month (January) is
3.8 o C and 2.2 o C for El Borma and Tiert sites,
respectively. Seed viability is variable, generally
between 22 and 57% (Dhief et al. unpubl.
data).
Germination experiments
Seeds were surface sterilized with Na
hypochlorite (12 o Chl) for one minute, subsequently
washed with deionized water and airdried
before being used in experiments to
avoid fungus attack. Seeds were sown on two
layers of filter paper (Whatman, No. 1) in
90-mm glass Petri dishes with 5 ml of deionized
water kept in a germination compartment
adjusted to day/night temperature of 25/10 o C
and 14 h of light (Luminincube II, analys,
Belgium; MLR-350, Sanyo, Japan). A completely
randomised design was used in the
germination tests. Seeds were subjected to six
pretreatments and one control. C: untreated
seeds (control); T1 : mechanical scarification
by grinding seeds in a mortar with a pinch of
clean silica sand; T2 : mechanical scarification
by rubbing seeds with sandpaper;
T3: immersion in boiling water for 10 min;
T4: immersion in concentrated sulphuric acid
(96%) for 10 min; T5 : immersion in concentrated
sulphuric acid for 20 min; T6 : immer-
ecologia mediterranea – Vol. 38 (1) – 2012
Effects of some seed-coat dormancy breaking treatments on germination
of three Calligonum species occurring in Southern desert of Tunisia
sion in concentrated sulphuric acid for
30 min. In T3, the seeds were immersed in
boiling water in a beaker for 10 min before
being removed and left to cool for 24 h. For
sulphuric acid treatments (T4-T6), seeds were
immersed in acid in a beaker for 10, 20 or
30 min, while stirring with a magnetic stirrer,
before being rinsed in running water for
45 min. For each treatment, four replicates of
25 seeds each were used. During 30 days, the
number of germinated seeds was counted and
removed every 2 days. A seed is considered
to have germinated when the emerging radical
elongated to 2 mm. Ungerminated seeds
were soaked in water at 30 o C for 24 h to test
their viability using tetrazolium chloride test.
Seeds were cut and the embryo soaked in 1%
tetrazolium chloride for 24 hours at 30 o C.
Pink embryos were scored as viable.
Germination calculation
From all germination data collected, the following
variables were determined according
to these formulas:
• germination percentage (%) = (number of
germinating seeds/number of seeds initiated)
× 100;
• relative germination percentage (%) = (number
of germinating seeds/number of viable
seeds initiated) × 100;
• germination index = G/t, where G is the relative
germination percentage at 2-day intervals
and t is the total germination period;
• dormancy percentage (%) = (number of
ungerminated but viable seeds/number of
seeds initiated) × 100;
• relative dormancy percentage (%) = (number
of ungerminated but viable seeds/number
of viable seeds initiated) × 100;
• mortality percentage (%) = (number of unviable
seeds/number of seeds initiated) × 100.
Statistical analysis
The germination data were transformed
before statistical analysis to ensure the homogeneity
of variance. The data were analysed
using SPSS for windows, version 11.5 (SPSS,
2002). A two-way analysis of variance
(ANOVA) was carried out to test the effects
of the main factors and their interaction on
germination characteristics. Duncan test was
used to estimate the least significant range
between means.
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