O. VARELA AND G.

LIZARDO

Varela, O. and Lizardo, G. (2010), Seed Sci. & Technol., 38, 528-531

Research Note

Seed viability and effect of scarification with sulphuric acid on

germination of Enterolobium contortisiliquum (Fabaceae) seeds
O. VARELA1 AND G. LIZARDO2

1
Instituto de Ecologia, Fundación Miguel Lillo, Miguel Lillo 251, San Miguel de Tucumán, CP 4000, Argentina
- Instituto de Ambientes de Montañas y Regiones Áridas, Universidad Nacional de Chilecito, La Rioja,
Argentina (E-mail: varelao@uolsinectis.com.ar)
2
Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, Tucumán,
Argentina

(Accepted April 2010)

Summary

Enterolobium contortisiliquum (Vell) Morong., a large leguminous tree from South America, produces dormant
seeds with low germination capacity. Laboratory experiments were conducted to evaluate seed viability of
E. contortisiliquum and to test the effect of different times of seed scarification in concentrated sulphuric acid
(30, 60, 90, 120 minutes) on germination capacity. The results show that 98% of the seeds (n = 120) were
viable. Germination of control seeds reached 2%. All pretreatments with sulphuric acid broke seed dormancy
earlier and increased germination percentage and rate significantly as compared with control seeds. Scarification
for 90 minutes promoted the highest germination percentage (94%). Our findings will be particularly useful to
the requirements of a rapid mass production of seedlings of E. contortisiliquum.

Experimental and discussion

Understanding seed viability, dormancy and requirements for germination is of ecological

and economic importance (Khurana and Singh, 2001). Enterolobium contortisiliquum
(Vell) Morong. (Fabaceae), popularly known as “timbó”, “pacará” and “oreja de negro”,
is a majestic tree of big size that occurs in Brazil, Bolivia, Paraguay, Uruguay, and
Argentina. It is widely cultivated as an ornamental and shade tree, and is considered a
promising tree for mixed reforestation in degraded lands, mainly due to its rapid initial
growth (Lorenzi, 1992; Montagnini et al., 2006).
As many other hard-seed legume species, seeds of E. contortisiliquum requires
pretreatments of scarification, for improve germination (Eira et al., 1993; Malavasi and
Malavasi, 2004). Previous studies have reported varying degrees of dormancy in seeds
of E. contortisiliquum at different localities in Brazil in responses to pregerminative
treatments (Eira et al., 1993). Relatively little information is available concerning seed
viability and dormancy in pacará trees that occur in northwestern Argentina. The aims
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SEED VIABILITY AND GERMINATION IN ENTEROLOBIUM CONTORTISILIQUUM

of this study were to assess seed viability of E. contortisiliquum and test the effect of
different times of scarification in concentrated sulphuric acid (30, 60, 90, 120 minutes)
on germination capacity (germination percentage, minimal latency, germination rate).
Understanding these factors is relevant to improve reforestation plans and the commercial
production.
Mature seeds of E. contortisiliquum were collected during August 2008 from 10
randomly selected plants that grow naturally at Reserva Experimental Horco Molle
Department Yerba Buena, Province of Tucumán, Argentina. Seeds were carefully
examined under a stereoscopic microscope to exclude aborted seeds or seeds parasitized
by bruchid beetles (seed-eating insects).
To assess viability of E. contortisiliquum seeds we used a standard bioindicator
(TTC, 2,3,5 triphenil tetrazolium chloride) that detects seed viability by staining the
embryo tissue pink/red (Moore, 1973). Ten replicates of 12 seeds were used for each
treatment. Seeds were classified as viable (if the embryo stained pink/red) or dead (if
embryo remained white). To study the effect of chemical scarification on germination
seed samples were immersed in sulphuric acid (H2SO4) concentrated at 98% for 30, 60,
90, and 120 minutes. Untreated seeds were used as control group. Acid scarification was
performed in Erlenmeyer flasks covered with a cork; the flasks were regularly agitated to
ensure a homogeneous acid action. Following acid treatment seeds were washed during
10 minutes with abundant running water until pH was neutral, and then rinsed in sterile
water. Ten replicates of 10 seeds were used for each treatment.
Germination assays were carried out in a germination chamber set at 32°C. Seeds
were sown in plastic trays contained a mixture of sand (50%) and soil (50%) collected in
the study area, previously sterilized in a stove (100°C) for 8 hours. The substrate was kept
moist with running water applied by mist spraying. The number of germinated seeds was
recorded three times a week during two months. Seeds were considered germinated when
the tip of the radicle emerged from the seed coat. At end of the experiment, seeds that did
not germinate were subjected to tetrazolium reagent to assess viability.
Results indicate that 98.3 ± 5.3% (Mean ± SD) of E. contortisiliquum seeds were
viable. Germination percentage of unscarified seeds (control) was of 2% (figure 1).
Germination percentage differed significantly among treatments (One-way ANOVA, P
< 0.001) with significantly higher values in seeds treated with sulphuric acid than in
control (unscarified) seeds (figure 1). Seed scarification in acid for 90 minutes promoted
the highest germination percentage (figure 1). Likewise, acid scarification was equally
efficient in shortening germination time and increasing germination rate. Seeds scarified
with sulphuric acid began to germinate at day 2 after sowing and reached a maximum
germination rate between days 21 and 24. Significant differences among treatments were
found in relation to minimal latency (One-way ANOVA, P < 0.001) and germination
rate (One-way ANOVA, P < 0.001). Post-hoc comparisons showed that all scarification
treatments with sulphuric acid had a significantly lower minimal latency and a higher
germination rate than control (table 1). Finishing the experiment, non-germinated seeds
remained dormant, except a non-viable 18% of the seeds treated with sulphuric acid
during 120 minutes.

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 O. VARELA AND G. LIZARDO

100 a
a
a
a
Germination (%) 80

60

40

20

b
0
Control H2SO4 H2SO4 H2SO4 H2SO4
30 min 60 min 90 min 120 min

Treatments
Figure 1. Mean germination (%) of E. contortisiliquum seeds immersed in concentrated sulphuric acid for
different periods compared with an untreated control. Vertical bars denote standard error. Columns labelled with
different letters indicate significant differences (Tukey HSD, P ≤ 0.05).

Table 1. Minimal Latency and germination rate of seeds of Enterolobium contortisiliquum subjected to different
periods of scarification in concentrated sulphuric acid compared with an untreated control. Values are mean ± SE
(Standard error). Means values followed by different letters in the same row are significantly different (Tukey
HSD, P ≤ 0.05). Minimal latency is the mean number of days from sowing until the first seed germinated.
Germination rate is the sum of the quotients of the number of seeds germinating each day divided by the time
(number of days) from the start of the germination test. It was calculated according to Maguire equation (Naylor,
1981).

Seed treatments

Germination H2SO4 H2SO4 H2SO4 H2SO4

Control
parameters 30 min 60 min 90 min 120 min

a b b b b
Minimal latency 33 ± 4 3.3 ± 0.8 2.2 ± 0.1 2 ± 0.0 2.3 ± 0.2

c a,b b a a,b
Germination rate 0.01 ± 0.005 2.5 ± 0.5 2.3 ± 0.4 4.0 ± 0.2 2.9 ± 0.6

Our results are consistent with previous studies (Eira et al., 1993; Malavasi and
Malavasi, 2004) in that the seed coat is the only factor restricting germination of
E. contortisiliquum, since the alteration of seed coat structure by the effect of sulphuric
acid promotes rapid germination and a high germination percentage.
Results also reveal no significant differences in germination between treatments of 30-
120 min of scarification in acid, in agreement with the findings of Malavasi and Malavasi
(2004) for Brazilian conspecific seeds. Likewise, the germination percentages obtained
for the control and chemical treatments range within the values reported by those authors.
In our study, scarification in concentrated sulphuric acid for 90 minutes was the most
efficient treatment in promoting maximum germination (figure 1). Malavasi and Malavasi
(2004) reported the highest germination percentage (93%) with sulphuric acid treatment
for 180 minutes.
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SEED VIABILITY AND GERMINATION IN ENTEROLOBIUM CONTORTISILIQUUM

Sulphuric acid breaks physical (seed coat) dormancy by corroding the outermost layers
of the seed coat, improving seed permeability (Baskin and Baskin, 1998). When the seed
is soaked in acid for a longer period it becomes more permeable to water (Baskin and
Baskin, 1998). However, a prolonged seed exposure to sulphuric acid can cause seed death.
In our study, 18% of the seeds exposed to acid during 120 minutes appeared hydrated,
soft, and mucilaginous, indicating that they were damaged by the acid treatment.
Among the different treatments employed to break physical dormancy of pacará
seeds, mechanical scarification has been suggested as the best procedure (Malavasi and
Malavasi, 2004). Mechanical scarification utilizes abrasion, particularly by rough surfaces.
This technique is tedious, labour intensive and time consuming if done manually, and is
not practical for large seed lots.
Our study provides further evidence that sulphuric acid is a highly effective scarifying
agent to overcome dormancy of pacará seeds and is particularly appropriate for treating
large numbers of seeds (hundreds to thousands). Such procedure is simple, of easy
application and inexpensive. Our findings will be particularly useful to the requirements of
a rapid mass production of seedlings of E. contortisiliquum for commercial or restoration
purposes.

Acknowledgements

We are thankful to E.H. Bucher for their comments during preparation of the manuscript,
and to J. Brasca for improving the English style. This research was partially funded by
Fundación Miguel Lillo.

References

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seeds. San Diego Academic Press, California, USA.
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contortisiliquum (Vell.) Morong.-Leguminosae. Revista Brasileira de Sementes, 15, 177-181.
Khurana, E. and Singh, J.S. (2001). Ecology of tree seed and seedlings: Implications for tropical forest
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Lorenzi, H. (1992). Árvores Brasileireas: manual de indentificacao e cultivo de plantas arbóreqas nativas do
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Moore, R.P. (1973). Tetrazolium staining for assessing seed quality. In Seed Ecology (ed. W. Heydecker),
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Naylor, R.E.L. (1981). An evaluation of various germination indices for predicting differences in seed vigour in
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