SCARIFICATION OF SEEDS OF ACACIA ANGUSTISSIMA

Rincón-Rosales, R., Culebro-Espinosa, N.R., Gutierrez-Miceli, F.A. and Dendooven, L.

(2003), Seed Sci. & Technol., 31, 301-307

Scarification of seeds of Acacia angustissima (Mill.) Kuntze

and its effect on germination
R. RINCÓN-ROSALES1, N.R. CULEBRO-ESPINOSA1, F.A. GUTIERREZ-MICELI1
AND L. DENDOOVEN2*

1
Laboratory of Environmental Engineering, Instituto Tecnológico de Tuxtla-Gutierrez, Tuxtla-Gutierrez,
México
2
Laboratory of Soil Ecology Department Biotechnology and Bioengineering, Cinvestav, Av. Instituto
Politécnico Nacional 2508, C.P. 07000 México D. F., México

(Accepted June 2002)

Summary
Field germination of Acacia angustissima (Mill.) Kuntze, a N2-fixing tree whose bark is traditionally extracted
for tannins used in the leather industry, takes up to 55 days and the percentage of germination is generally less
than 20%. Abrasion, dry or wet heat, or immersing the seeds in different concentrations of sulphuric acid, chloric
acid or hydrogen peroxide for different lengths of time, were tested as germination enhancers. The application of
dry heat resulted in < 37% germination, less than the 44% found for untreated control seeds. The application of
wet heat for ≤ 30 s and abrasion for 1200 s resulted in 69% germination. Immersing seeds in concentrated H2SO4
for 900 s resulted in 77% germination while diluting H2SO4 or longer exposure to concentrated H2SO4 decreased
it. Immersing the seeds in concentrated or diluted H2O2 also increased germination, but to a lesser extent than
when immersed in H2SO4. We found that the exposure of seeds of A. angustissima to concentrated H2SO4 for
900 s appeared to be the best technique to stimulate their germination; the treatment gave the largest percentage
of germination in the shortest time, the technique is easy to apply and many seeds can be treated at once.

Introduction

Acacia angustissima (Mill.) Kuntze is a leguminous shrub with a distribution from the
southern United States to Costa Rica. The species can be found in arid and semiarid
regions of Mexico (Rzendowsky, 1978) and in Chiapas, mostly in the central depression
and in the valley of Comitan. A. angustissima is used as fire wood, the leaves as forage
for goats and sheep and its bark has traditionally been extracted for tannins used to tan
hide.
N2 fixing trees form fertility islands as they increase soil organic matter content,
prevent erosion, loss of valuable nutrients and run-off and form a refuge for fauna and
flora (e.g. Garner and Steinberger, 1989). Over-use, over-grazing, induced forest fires and
cultivation of land have greatly reduced the distribution and the density of these trees and
have led to a sharp decrease in soil fertility and loss of fertile topsoil.

* Author for correspondence

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The above characteristics make A. angustissima an ideal plant for restoration of

soil (SEDECO, 1999), so a reforestation program with this tree was started in the
state of Chiapas. Investigation in the field, however, has shown that germination of
A. angustissima takes up to 55 days and the percentage of germination is < 20% so
propagation of the species is low. A fast and easy technique that can be applied to a lot
of seeds at once and which results in a high percentage of germination would be useful
for a successful reforestation program. We investigated different physical and chemical
techniques to scarify seeds of A. angustissima and measured percentage of germination.

Materials and methods

Location of the trees and collection of seeds

Seeds were collected from A. angustissima shrubs in the central depression of Chiapas
and the valley of Comitán (Mexico) where its bark has traditionally been extracted for
tannins used to tan hide. In the central depression in Piedra Blanca (Ocozocoautla, 16°45’
LN, 93°22’ LO), 20 mature plants with a canopy of ca 1.5 m2 located in an area 2180 m2
were sampled for seeds, 15 in la Cañada Suchiapa (16°37’ LN; 93°06’ LO) in an area
360 m2, 10 in Cruz Quemada Berriozabal (16°47’ LN; 93°16’ LO) in an area 400 m2 and
8 in Villaflores (16°13’ LN; 93°16’ LO) in an area 1230 m2. In the valley of Comitán in
Candelarias (16°15’ LN 92o07’ LO) 12 mature plants with a canopy of ca 1.5 m2 located
in an area 1040 m2 were sampled for seeds, 25 in Timbral (16°06’ LN 92°03’ LO) in
an area 1840 m2 and 20 in Las Margaritas (16°18’ LN 91°59’LO) in an area 5720 m2.
The seeds that were mature or with a black-red colour, complete, free of pathogens and
> 2 mm length were selected. Approximately 20000 seeds or 2 kg were obtained. They
were stored in the dark in sterile 1000 ml glass jars containing 2% ashes (< 2 mm). The
local farming community store leguminous seeds in ash as it prevents growth of fungi
and insects. Leguminous seeds can be stored for up ten years in this way (Hartmann and
Kester, 1987).

Characterization of the seeds

Four hundred seeds were selected at random and the polar and equatorial diameter,
thickness, total weight and volume were measured. Seed impermeability was measured
by submerging 400 seeds in H2O at 20°C for 10 days. Each day, the seeds that had taken
up water and were swollen were counted. The percentage non-swollen seeds defined the
impermeability (Quinlivan, 1971). Viability was measured by submerging 1000 seeds in
distilled H2O for 48 h. Hundred swollen seeds were selected at random and the softened
cuticula was removed under a stereoscopic microscope under sterile conditions so as not
to damage the embryo. The dissected embryos were placed on wet Whatman paper in
petri dishes in a illuminated incubator at 18-22°C (Hartmann and Kester, 1987). After 48
h, embryos were considered viable when they showed root and shoot development.

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Scarification of seeds
For each treatment, 100 seeds were used and the whole experiment was repeated four
times. Abrasion and dry and wet heat, in which time of exposure was changed, were
applied to stimulate germination (table 1). Seeds were placed on a heating plate at 144°C
for 30, 90, 150 or 270 s (considered the dry heat treatment) (Ruiz-Tovilla, 1987), in
95°C water for 10, 20, 30, 40, 50, 60, 70, 80, 120, 160 or 200 s (considered the wet
heat treatment), or the coat of the seeds was scratched mechanically between two metal
cylinders with an abrasive surface for 300, 600, 900 or 1200 s (considered the abrasion
treatment). Manual abrasion was not considered since the seeds are very small and
difficult to handle.
Additionally, seeds were immersed in 25% and 65% sulphuric acid (H2SO4), hydrogen
chloride (HCl) or hydrogen peroxide (H2O2) for 900 s or in 98% H2SO4, HCl or H2O2 for
900, 1800, 3600, 5400 or 7200 s (table 1). After submerging the seeds in acid, they were
washed five times in distilled water. Untreated seeds were used as control.
The treated and untreated seeds were planted at 0.5 cm depth in a seed raising mixture
locally purchased containing organic material (peat moss) and sand in a ratio of 3:1. Each
block of 100 planted seeds was placed at random in a greenhouse and watered daily. The
percentage of germination, defined as the complete formation of plumule and radicle, was
determined daily between day 4 and day 20. Kotowski’s velocity coefficient, which is a
measure of the distribution of germination regarding the number of seeds germinated in
time, was calculated after 20 days (Cervantes, Carabias and Vázquez-Yanes, 1996). All
statistical analyses were done using SAS (SAS Institute Inc., 1989).

Results and discussion

Characteristics of the seeds

The mean equatorial diameter of the seeds of A. angustissima was 2.7 mm, the polar
diameter 3.0 mm and the thickness 1.5 mm. The average weight of the seeds was 9.3
mg, the water content 2.5%, the water absorbed 0.4 mg and the impermeability 61%. The
viability of the seeds was > 95%.

Germination of seeds
The application of wet heat for 10, 20 or 30 s resulted in 69% germination after 20
days (table 1). Increasing the time of exposure to wet heat reduced the percentage of
germination (table 1), but it still remains remained larger than in the untreated control
seeds (table 1). The application of wet heat for longer time reduced the germination
percentage. Application of wet heat generally increases germination of seeds (e.g.
Rolston, 1978), although it is sometimes lethal (Teketay, 1996). Cervantes et al. (1996)
found that boiling seeds of three woody Acacia species from the mountainous tropical
subhumid region of southern Mexico (A. cochliacantha, A. farnesiana or A. pennatula)
for 60 s increased germination, but boiling for 300 or 600 s reduced the germination
capacity. Isikawa (1965) found that boiling seeds of Acacia mollisima for 300 s yielded
germination percentages of 75 to 80% and the time of boiling, ranging between 15 s and

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Table 1. Percentage of germination after 4, 8 and 20 days and Kotowski’s velocity coefficient after 20 days for
seeds of Acacia angustissima.

Time of Percentage of germination Kotowski’s

application velocity coefficient
Treatment (sec) 4 days 8 days 20 days after 20 days

Dry heat (144°C) 30 0 15 30 7.03

Dry heat (144°C) 90 0 13 33 7.84
Dry heat (144°C) 150 0 17 36 8.05
Dry heat (144°C) 270 0 3 5 7.86
Wet heat (95°C) 10 0 45 69 8.03
Wet heat (95°C) 20 4 44 69 8.05
Wet heat (95°C) 30 5 48 69 6.70
Wet heat (95°C) 40 1 31 49 6.94
Wet heat (95°C) 50 4 43 61 7.90
Wet heat (95°C) 60 1 33 51 7.90
Wet heat (95°C) 70 3 39 60 6.84
Wet heat (95°C) 80 1 28 43 6.89
Wet heat (95°C) 120 2 38 58 6.98
Wet heat (95°C) 160 2 18 24 7.07
Wet heat (95°C) 200 2 43 61 7.15
Abrasion 300 16 56 63 6.96
Abrasion 600 22 59 68 6.84
Abrasion 900 21 60 67 7.17
Abrasion 1200 31 62 69 6.88
Untreated – 2 27 46 6.98
H2SO4 25% 900 1 29 51 7.03
H2SO4 65% 900 26 60 69 7.84
H2SO4 98% 900 46 69 77 8.05
H2SO4 98% 1800 30 60 70 7.86
H2SO4 98% 3600 41 63 71 8.03
H2SO4 98% 5400 35 56 59 8.05
HCl 25% 900 1 16 50 6.70
HCl 65% 900 1 19 45 6.94
HCl 98% 900 19 43 49 7.90
HCl 98% 1800 12 26 28 7.90
HCl 98% 3600 4 7 26 6.84
HCl 98% 5400 2 5 17 6.89
H2O2 25% 900 0 24 47 6.98
H2O2 65% 900 0 32 51 7.07
H2O2 98% 900 1 31 52 7.15
H2O2 98% 1800 2 29 68 6.96
H2O2 98% 3600 0 15 42 6.84
H2O2 98% 5400 1 34 51 7.17
H2O2 98% 7200 1 25 61 6.88

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300 s, did not affect their viability. The seeds of A. angustissima were more susceptible
to wet heat than those of A. mollisima, A. cochliacantha, A. farnesiana or A. pennatula
confirming the fact that the time of boiling required to obtain maximum germination is
different among species (Vázquez-Yanes, 1983; Cervantes et al., 1996). Percentages of
germination after the application of wet heat is unequal even among seeds from different
crops within a single species (Karssen, 1981; Cervantes et al., 1996).
The application of dry heat at 144°C for 30, 90, 150 or 270 s was detrimental to
germination when compared to the control treatment (table 1). The temperature of 144°C
might have been too high. However, Mott, Cook and Williams (1982) found that exposing
seeds of 13 legumes to high temperatures increased germination. A. angustissima was thus
very sensitive to heat and the temperature of 144°C even when applied for only 30 s was
too high. Although forest fires might reduce the overall germination of A. angustissima,
they are often required in the wild to break the seed coat and, after rainfall the Acacia
seedlings sprout. The effect of dry heat on the germination of seeds is known to vary
widely between species. Teketay (1996) reported that seeds of Acacia senegal were very
sensitive to dry heat while those of Acacia oerfata were not. Most of the seeds of A.
senegal died after being exposed to a temperature of 80°C for 900 s while most of the A.
oerfata seeds were intact even after being treated for 3600 s at 100°C.
Mechanical abrasion increased the percentage of germination compared to the
untreated control seeds, but gave similar values as the wet heat treatment. Percentage of
germination after 20 days was 63% for seeds treated for 300 s and between 67 and 69%
for those treated longer (table 1). Teketay (1996) reported that mechanical scarification
resulted mostly in larger cumulative germination for most species than boiling, but not
always. Airi, Rawal, Samant and Dhar (1998) while working with four multipurpose trees
of central sub Himalaya, found that mechanical scarification did not improve germination.
Conversely, MacKay, Davis and Sankhla (1995) obtained almost 100% germination for
seeds of Lupinus havardii (Big Bend bluebonnet) after mechanical scarification. Tigabu
and Odén (2001) also obtained 100% germination for seeds of Albizia gummifera and
80% for seeds of Albizia grandibracteata, two multipurpose species from Ethiopia,
compared to < 10% germination for unscarified seeds.
Immersing seeds in 65% H2SO4 for 900 s increased the percentage of germination, but
immersion in 25% H2SO4 did not (table 1). The largest percentage of germination (77%)
and Kotowski’s velocity coefficient (8.05) was found for seeds submerged for 900 s in
concentrated H2SO4. Immersing the seeds for more than 900 s reduced the percentage
of germination, but germination remained larger than in the untreated control seeds.
Concentrated H2SO4 is known to be consistently effective, resulting in rapid, uniform and
high germination (e.g. Clemens, Jones and Gilbert, 1977; Cavanagh, 1987, Cruz, Perez-
Urria, Martin, Avalos and Vicente 1995; MacKay et al., 1995, Teketay 1996). Teketay
(1996) found that germination of all 20 different species he studied increased when
exposed to concentrated H2SO4. He reported that in some species, germination increased
when exposure time increased from 900 to 3600 s, that in some species the percentage
germination decreased as found in our experiment, while in other species the germination
reached a peak and then decreased again.

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Submerging seeds in diluted or concentrated H2O2 increased germination compared to

the untreated control seeds (table 1). The largest percentage of germination, i.e. 68%, was
found when seeds were submerged for 1800 s in concentrated H2O2. Submerging seeds in
diluted or concentrated HCl for 900 s increased the percentage of germination compared
to the control treatment (table 1). The increase, however, was small and ≤ 50% of the
seeds germinated. Immersing the seeds in concentrated HCl for > 900 s strongly reduced
the percentage of germination.
We found that the exposure of seeds of A. angustissima to concentrated H2SO4 for 900
s appeared to be the best technique to stimulate their germination. This treatment resulted
in the largest percentage of germination in the shortest time. The technique is easy to
apply and can be used on a lot of seeds at once.

Acknowledgements

We thank the Instituto Nacional de Investigaciones Forestales y Agropecuarios (INIFAP)

for technical assistance. The research was funded by Consejo Nacional de Ciencia y
Tecnología (CONACyT, México) project No 3966N-9402 and the Laboratorio de Plantas
Piloto, Instituto Tecnológico de Tuxtla-Gutierrez, México. L.D. received grant aided
support from FOSIBEJ Project 20000506030.

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