Temperature and germination ...
97
TEMPERATURE AND GERMINATION OF THE
1,2
LEGUMINOSAE Enterolobium contortisiliquum
Consuelo Medeiros Rodrigues de Lima 3 , Fabian Borghetti 2 and
Marcelo Valle de Sousa 3 .
Laboratório de Termobiologia Luiz F. G. Labouriau, Instituto de Ciências
Biológicas, Universidade de Brasília, Brasília, DF. 4
ABSTRACT - Enterolobium contortisiliquum (Vell.)
Morong. (Leguminosae-Mimosoideae) is an arboreal
species of the Cerrado vegetation. The minimum
germination temperature lies between 10.9 and
11.9°C, and the maximum germination temperature is
located between 40.9 and 42.4°C. The germinabilities
are statistically not different from 100 % between 18.2
and 38.8 ºC. The germination rate becomes higher as
the incubation temperature increases. The plots of
relative frequency of germination are predominantly
platykurtic at lower temperatures and leptokurtic at
higher ones. The action of temperature as a regulating
factor in the germinative process is discussed, as well
as the relevance of certain seed morphology and
germination characteristics for the establishment of a
seed bank.
Additional index terms: dormancy, germination
frequency, germination rate, seed bank.
TEMPERATURA E GERMINAÇÃO DA
LEGUMINOSAE Enterolobium
contortisiliquum.
RESUMO- Enterolobium contortisiliquum (Vell.)
Morong. (Leguminosae-Mimosoideae) é uma árvore
de ocorrência na vegetação Cerrado. A temperatura
mínima de germinação das sementes localiza-se
entre 10,9 e 11,9°C, enquanto que a máxima está
entre 40,9 e 42,4°C. As germinabilidades não são
estatísticamente diferentes de 100% entre 18,2 e 38,8
ºC. A velocidade de germinação é crescente com o
aumento da temperatura. As curvas de frequência
relativa da germinação são predominantemente
1
Received 17/01/1997 and accepted 07/08/1997
2
Apolodoro Plausonio Foundation, Universidade de
Brasília and CNPq.
3
4
Lab. Bioquímica e Química de Proteínas, UnB.
email: [email protected] and
[email protected]
platicúrticas em baixas temperaturas e leptocúrticas
em temperaturas mais elevadas. Discute-se sobre a
atuação da temperatura como um fator regulador do
processo germinativo e sobre a relevância de certas
características da semente e de sua germinação para
o estabelecimento de um banco de sementes.
Termos adicionais para indexação: banco de
sementes, dormência, frequência de germinação,
velocidade de germinação.
INTRODUCTION
Enterolobium contortisiliquum (Vell.) Morong is a
Leguminosae-Mimosoideae tree, commonly known as
“orelha-de-negro”, “orelha-de-macaco”, “timbó” or
“tamboril”, among other names. It is found in many
regions of Brazil, especially in rain forest and
semi-deciduous forest (Lorenzi, 1992). Well adapted
to Cerrado, it can be used in foresting (Heringer,
1978), and its wood has a great economic value
(Lorenzi, 1992). Seeds of E. contortisiliquum present
tegument imposed dormancy, which can be overcome
by different scarification treatments (Eira et al., 1993).
Studies on the influence of temperature on the
germination of seeds is essential to understand the
ecological, physiological and biochemical aspects of
the process (Bewley & Black, 1982, 1994; Labouriau,
1983). In previous work, the germination of
Enterolobium seeds was studied in a limited
temperature range (Souza & Varela, 1989; Eira et al.,
1993), showing that this germination is slow and
irregular (Eira et al., 1993).
In the present work, the kinetic parameters and
cardinal points (Labouriau, 1972, 1983) for E.
contortisiliquum seed germination were established.
The importance of temperature in the kinetic control of
germination of E. contortisiliquum seeds and as a
climatic factor acting on a seed bank is discussed.
R. Bras. Fisiol. Veg., 9(2):97-102, 1997.
98
Lima et al.
MATERIAL AND METHODS
Legumes of E. contortisiliquum were collected in the
Campestral Club-House of the National Congress
(Brasília - DF) in 1995. The seeds were manually
removed from the legumes and stored at -20oC.
Voucher branches with leaves, flowers and fruits were
deposited in the Herbarium of the Universidade de
Brasília (Brasília-DF) under the code “UnB - 001
Consuelo”.
Two scarification treatments were tested:
mechanical, in which a small opening was made on
the opposite side of the hilum and chemical, by
immersion of the seeds in concentrated sulphuric acid
P.A. (Reagen) for 15, 30 and 60 minutes, followed by
abundant washing, first in distilled and then in
deionized water. The germination tests were carried
out at 30oC with twenty five seeds per treatment. Seed
viability was estimated using 2,3,5-triphenyl
tetrazolium salt (Copeland, 1976).
kurtosis (G2) were also computed (Wilkinson, 1990).
All statistical tests considered an α = 0.05.
RESULTS AND DISCUSSION
The fresh and dry matter as well as the water
content of the seeds are shown in Table 1. The low
water content indicates that E. contortisiliquum is an
orthodox type seed (Bewley & Black, 1982). According
to the average fresh matter estimates, these seeds
can be considered large sized (Bewley & Black,
1978).
TABLE 1- Fresh and dry matter and water content of
seeds of Enterolobium contortisiliquum (Vell.)
Morong.a
Fresh matter
Dry matter b
Water content c
FM (g)
DM(g)
WC(%)
1
11.48
10.98
4.35
2
11.80
11.35
3.80
3
12.22
11.75
3.80
Seed water content was estimated according to
Justice (1972). Ten samples of 20 seeds each were
weighed before and after drying at 105oC for 16 hours.
4
11.48
10.91
4.96
5
11.69
11.15
4.60
6
12.60
12.15
3.60
The germination experiments were carried out in a
thermogradient block with 24 temperature stations. A
detailed description of the block can be found in
Labouriau & Cavalcanti (1996). Five replicates of 10
seeds were used for each temperature treatment
(each temperature corresponded to an experimental
station). The seeds were chemically scarified, washed
and placed in plexiglass plates with three layers of
qualitative filter paper. The plates were put inside pyrex
tubes, which were closed with hydrophobic cotton and
placed inside the stations. The observations were
made at different time intervals for each temperature
range, according to the seed germination rate (the
greater the rate, the smaller the time interval). At each
observation, the temperature was verified, the
germinated seeds were counted and removed, and
deionized water was added whenever necessary.
7
12.60
12.10
3.97
8
11.30
10.80
4.42
9
12.70
12.12
4.57
12.30
11.60
5.69
Average
12.02
11.49
4.38
Standard
deviation d
0.53
0.53
0.63
Sample
10
d
a
10 samples of 20 seeds each.
b
After drying at 105 o C for 16 h.
c
WC = [(FM - DM)/FM] x 100.
d
Sokal & Rohlf (1969)
Using the tetrazolium test, seed viability was
estimated in 100% for both chemically and
mechanically scarified seeds. For the germination
experiments, the chemical scarification method was
chosen because of its facility and greater
homogeneity.
The average germination time, variance of the
germination time, average germination rate and
variance of the germination rate were calculated
according to Labouriau (1972, 1983). Homogeneity of
Figure 1 shows the germinative behavior of E.
the average germination rate variances was verified
contortisiliquum seeds under different temperature
using the Kruskal-Wallis test, and multiple
treatments. The statistical analysis of the results
comparisons between the average germination rates
pointed out some germination parameters: the
were performed using the Mann-Whitney test (Sokal &
minimum germination temperature (Tmin) is located
Rohlf, 1969). The germinability confidence intervals
between 10.9 and 11.9 oC, and the maximum
were obtained from “Tablas Científicas” (Documenta
temperature (Tmax) lies between 40.9 and 42.4 oC. The
Geigy, 1965). The analysis of the seed germination
germinabilities are statistically not different from 100%
relative frequencies in all temperatures of incubation
between 18.2 and 38.8 ºC. The Mann-Whitney test (U
followed Labouriau & Pacheco (1978). The plots of
= 23) revealed more than one optimum germination
these relative frequencies were analysed by the
temperature range (Topt), the first one from 30.3 to
Kolmogorov-Smirnov test. The skewness (G1) and
R. Bras. Fisiol. Veg., 9(2):97-102, 1997.
Temperature and germination ...
99
FIGURE 1- Temperature
dependence
of
the
germinability ( ) and the
germination rate ( ) of
E n t e r o l o b i u m
seeds.
contortisiliquum
The bars represent 95%
confidence intervals of
germinability (Documenta
Geigy, 1965), but are
absent at the points where
the germinabilites are not
statistically different from
100%.
®
33.0 ºC and the other lying between 34.1 and 40.9 ºC.
The existence of a germination rate plot with multiple
temperature points or ranges which statistically do not
differ from each other has been described earlier
(Labouriau & Pacheco, 1979). The variances of the
average germination times decrease as incubation
temperatures increase, and the variances of the
germination rates are predominantly heterogeneous
(data not shown).
Figure 2 presents the relative frequencies of seed
germination in some representative temperatures as
well as skewness and kurtosis. By means of the
Kolmogorov-Smirnov test, it was found that none of
the frequency distributions of germination follow a
gaussian distribution (P<0.05 in all temperatures). The
estimated skewness was positive for all temperatures
but the values decreased with increasing
temperatures (the higher the temperatures, the
smaller the right tail). Most of the germination
distributions were platykurtic but the kurtosis
approaches zero and even becomes negative as the
incubation temperatures approach the optimum
temperature range. These results indicate a large
physiological
heterogeneity
concerning
the
germinative process of E. contortisiliquum seeds in
relation to this environmental factor. At lower
temperatures, there is a larger temporal distribution of
seed germination, contrasting with temperatures near
or at the optimum germination range, where the
germination variances are smaller (hence, a
leptokurtic distribution becomes more evident).
Furthermore, as the incubation temperature
increases, the average germination rate rises and
stablishes a high average germination rate plateau
near Tmax, even when germinability begins to decrease
(figure 1).
The role of temperature in regulating the kinetic
parameters of the seed germinative process has been
discussed in previous reports (Labouriau, 1972, 1978;
m
Labouriau & Pacheco, 1979; Labouriau & Osborn,
1984; Labouriau & Agudo, 1987). It has been found
that, in certain temperatures, seed germination is
heterogenous (polymodal), showing a large variance,
thus resulting in the spread of the propagule
germination in a large time interval. For example, a
wider dispersion of germination in time at
temperatures closer to Tmin and Tmax was found in the
germination of seeds of Salvia hispanica (Labouriau &
Agudo, 1987). E. contortisiliquum seeds show this
platykurtic pattern of germination at the infra-optimum
temperature range. On the other hand, the existence
of a temperature “signal” has been suggested which,
in certain situations, can overcome the aleatory
thermic noise in germination control (Labouriau, 1978,
1983; Labouriau & Osborn, 1984). In such a case, the
germination plots acquire a leptokurtic distribution and
seed germination becomes more homogeneous
(unimodal).
In the current case, this kind of frequency
distribution predominates at temperatures close to or
at the optimum temperature ranges. While in some
incubation temperatures seed germination shows a
polymodal pattern, in others (usually near or in the
optimum range of germination) the unimodal
distribution of germination predominates. Besides, it
has been verified that in temperatures near Tmax,
where germinability is decreasing, germination rate is
still high. These results had already been detected in
germination studies with Dolichos biflorus (Labouriau
& Pacheco, 1979), Lycopersicom esculentum
(Labouriau & Osborn, 1984) and Sesbania virgata
seeds (Zayat, 1996).
The physiological meaning of the above behavior, in
which the few seeds that germinate do it with high
velocity, requires further exploration, as well as the
factor(s) involved in the regulation of the degree of
synchronism of seed germination in relation to
temperature (Labouriau, 1978). A biochemical
R. Bras. Fisiol. Veg., 9(2):97-102, 1997.
100
Lima et al.
FIGURE 2- Frequency polygons
of the times of isothermal
germination of Enterolobium
contortisiliquum seeds at some
temperatures.
X-axes
correspond to time (h) and
Y-axes
refer
to
relative
frequencies. Skewness (G1)>0
long right tail, Kurtosis (G2)>0
platykurtic
distribution
(Wilkinson, 1990). 12.9oC G1 =
5.2, G2 = 36.2; 18.2oC G1 = 2.5,
G2 = 5.5; 29.7oC G1 = 1.1, G2 =
-0.04; 37.9oC G1 = 1.2, G2 = 0.6;
38.8oC G1 = 0.98, G2 = 0.45;
40.9oC G1 = 2.1, G2 = 4.4.
approach could lead us to speculate on the
involvement of proteins in the physiological control of
this process (Labouriau & Labouriau, in press).
On the other hand, this temperature-dependent
physiological behavior is ecologically important to the
species by contributing, e. g., for the formation of a
heterogeneous seed bank (Ross & Harper, 1972;
Metzger, 1992). Eira et al. (1993) verified that E.
contortisiliquum seed germination was irregular,
which has been corroborated in the present work. This
could be an adaptive characteristic, since an irregular
germination, distributed along a larger period of time,
increases the probability that at least some of the
seeds will germinate and establish themselves in more
favorable environmental conditions (Labouriau &
Agudo, 1987; Bewley & Black, 1994).
Hopkins & Graham (1987) reported that seeds of
the majority of the pioneer or late secondary species
remain viable but dormant in the soil. This agrees with
ecological characteristics of E. contortisiliquum is a
pioneer species, but more frequent in secondary
successions (Lorenzi, 1992). The role of the
germinative behavior of the seed bank in regulating
this pattern of distribution remains to be further
explored.
It has been reported that seeds with water
impermeable tegument usually require treatments
with low and/or high temperatures, alternating
temperatures or even fire in order to break this
physical barrier thus allowing imbibition and,
subsequently, germination (Baskin & Baskin, 1989). In
the specific case of seeds of some Mimosoideae
species, it has been determined that high
temperatures play a role in breaking physical
dormancy by rupturing the strophiolar plug (Dell, 1980;
Hanna, 1984). Seeds of Dimorphandra mollis, a
typical Cerrado species, which require scarification in
order to germinate (Lorenzi, 1992), do so after a high
temperature treatment, e.g., 90ºC for 20 minutes (F.
Borghetti, unpublished results). The effect of this
thermal stress in overcoming dormancy of E.
contortisiliquum seeds remains to be studied. These
facts indicate the importance of verifying the effect of
fire on the germinative pattern (see Thanos &
Georghiou, 1988) and in breaking dormancy of
Cerrado seeds, where fire occasionally occurs. This
R. Bras. Fisiol. Veg., 9(2):97-102, 1997.
Temperature and germination ...
question was considered by Labouriau (1966), but is
still unexplored.
The presence of seeds in the soil on the one hand
is adaptive in establishing a potential genetic reserve
(Simpson et al., 1989), on the other makes the
diaspores susceptible to pathogens and predators
which can compromise seed viability, thus negatively
influencing the seed bank (Foster, 1986; Louda, 1989).
It has been verified that large seeds tend to attract
more predators than small ones (Foster, 1986). This
could be the case for E. contortisiliquum seeds. It
would be interesting to verify whether enterolobin, a
protein found in these seeds (Sousa & Morhy, 1989),
contributes to the reduction of seed predation rate in
soil by both insects and rodents, since this protein is
cytolytic (Sousa & Morhy, 1989; Sousa, 1991), lethal
to Callosobruchus maculatus coleopteran larvae
(Sousa et al., 1993) and inflammatory to rat tissues
(Cordeiro et al., 1991).
An eco-physiological study on the importance of
proteins as an adaptive mechanism of dormancy
maintenance and regulation of the kinetic parameters
of germination (Labouriau & Labouriau, in press), as
well as a selected mechanism of protection of seeds
in the soil against natural predators, requires further
investigation.
ACKNOWLEDGEMENTS
To Fábio N. Noda for technical help, Pedro J.P.
Zanotta for computational programming and Patrícia
G.B. de Carvalho for competent assistance in writing
the manuscript. This paper is dedicated in memorian
to Prof. Dr. Luiz F.G. Labouriau, ti whom we are
extremely indebted.
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