Effect of storage of achenes of Bidens gardneri Baker on light sensitivity
during germination
REGINA M. SASSAKI1, LILIAN B.P. ZAIDAN1 and G.M. FELIPPE1
(recebido em 10/06/98; aceito em 13/11/98)
ABSTRACT - (Effect of storage of achenes of Bidens gardneri Baker on light
sensitivity during germination). Bidens gardneri is a very common herbaceous species
in the cerrados of the state of São Paulo, whose seeds become light sensitive at 25°C
only. Achenes of this species were stored in refrigerator at 4°C and in cerrado soil and
in forest soil. The field experiments were carried out in the cerrado at the Reserva
Biológica e Estação Experimental de Moji Guaçu, in Moji Guaçu and in the forest of
the Instituto de Botânica, in São Paulo, Brazil. Achenes of B. gardneri vary in size and
achenes from 7 to 12 mm long were used. Achenes stored for up to 6 months at 4°C
showed light sensitivity; after 9 months storage, the difference in germination between
light and darkness had disappeared for the smallest and the largest achenes used. Seeds
of B. gardneri germinated during the period of storage in soil; the number of germinated
seeds increased over the storage time, while the number of intact achenes decreased for
the same period, no matter if the experiment was being carried out in the cerrado or in
the forest. Therefore, the achenes germinated in soil in darkness. Light sensitivity was
lost in intact achenes that had been stored in soil for three months.
RESUMO - (Efeito do armazenamento dos aquênios de Bidens gardneri Baker na
sensibilidade à luz durante a germinação). Bidens gardneri é uma planta herbácea muito
freqüente nos cerrados do estado de São Paulo, cujas sementes só germinam em luz na
temperatura de 25°C. Aquênios foram armazenados a 4°C e enterrados em solo de
cerrado e solo de mata. Os experimentos de campo foram realizados no cerrado na
Reserva Biológica e Estação Experimental de Moji Guaçu, em Moji Guaçu e na floresta
do Instituto de Botânica de São Paulo, em São Paulo, Brasil. Os aquênios de B. gardneri
variam em tamanho e aquênios de 7 a 12 mm de comprimento foram usados. Os
aquênios armazenados até 6 meses a 4°C apresentaram sensibilidade à luz, que foi
perdida depois de 9 meses de armazenamento, tanto para os menores como para os
maiores aquênios usados nos experimentos. Aquênios da espécie germinaram durante o
período de armazenamento no solo; o número de aquênios germinados aumentou com o
tempo de armazenamento e o número de aquênios intactos diminuiu com o período de
armazenamento; estes resultados foram independentes do fato de o experimento ter sido
feito em cerrado ou na floresta. Isto mostra que aquênios germinaram no escuro quando
armazenados em solo. Os aquênios intactos perderam a sensibilidade à luz após
armazenamento no solo por três meses.
Key words - Bidens gardneri, longevity, light sensitivity
Introduction
The natural cover of about 25% of the land area of Brazil was savannah vegetation
given the generic name of cerrado. The cerrado vegetation is being quickly destroyed
for the use of the land for short term agriculture. As the cerrado soil is characteristically
poor in mineral nutrients (Alvim et al. 1968) frequently the land, after the removal of
the original vegetation, is then left to regenerate. Thus seed bank and its features, like
longevity of seeds, are very important.
Dormancy can be a determining factor for seed longevity in soil. Two types of longevity
can be distinguished, ecological and potential longevity. The ecological longevity refers
to the duration of the dormancy in natural conditions, the interval between maturation
and dispersal of the seeds and germination or death in the soil; the potential longevity
(viability) is the maximum duration of capacity to germinate of the dormant seeds
stored in optimal conditions (Bewley & Black 1985). The study of the longevity of
seeds is very important when considering species that can form a seed bank.
Bidens gardneri (Asteraceae) is a common herb of the cerrados of the state of São
Paulo, producing ornamental flowers and a great number of achenes. The species has
been the subject of a few papers, relating to the nutrient conditions (Felippe & Dale
1990), flowering (Klein et al. 1992) and nutrient conditions and photoperiod (Klein et
al. 1996). Felippe (1990) showed that the species presents light sensitivity when
germination is studied at 25°C; with a long storage period in dry conditions at 4°C (up
to 14 months) the achenes lose their light sensitivity and can easily germinate in dark
conditions. But is this also true when the seeds are stored in moist soil? Felippe (1990)
also showed that germination in light was high when long achenes were used and very
low with short achenes. The objective of this paper was to try to find out if light
dormancy is maintained when achenes of B. gardneri are artificially stored in the soil.
Material and methods
The achenes (seeds) of Bidens gardneri Baker were collected in 1996 in the cerrado of
Itirapina, state of São Paulo, Brazil (22°51'S and 47°52'W). A capitulum of Bidens
gardneri carries 39 achenes of different sizes (measured in 15 capitula). The distribution
of achenes according to their lengths is shown in table 1, most being between 8 and 12
mm long. The relative mass is smaller the longer the achene as can be seen in the ratio
mass/length shown in table 1. Changes in viability and light sensitivity during storage
were followed in achenes of different sizes in length, four groups of achenes were used:
7 - 8 (1st group); 9 (2nd group); 10 (3rd group) and 11- 12 mm long (4th group).
The achenes were stored in three conditions:
1. in closed glass bottles, at 4°C (± 2C) in darkness (refrigerator);
2. mixed with soil and buried in the cerrado at the Reserva Biológica e Estação
Experimental de Moji Guaçu, in Moji Guaçu, state of São Paulo, Brazil (22°18'S and
47°11'W). A description of this area and its vegetation is given in Mantovani & Martins
(1993);
3. mixed with soil and buried in the forest at the Instituto de Botânica, in São Paulo,
state of São Paulo, Brazil (23°39'S and 46°37'W). Description of the area and its
vegetation can be seen in Struffaldi-De Vuono (1985).
The chemical and physical analyses of the soils used and the rainfall and the
temperature of the two regions are presented in Simabukuro et al. (1998) and Sassaki et
al. (1999).
The achenes mixed with soil were placed in small bags (10 x 15 cm) made of very fine
black nylon gauze ("Sombrite"). In general 500 achenes were used in each bag. The
bags were buried at 10 cm deep in the cerrado or in the forest at the beginning of April
1996. The bags were removed from the soil 1, 3, 6 and 9 months afterwards. At
removal, the number of intact and germinated achenes was checked and the moisture
content (%) of the intact achenes was determined (three replicates of 20 achenes):
MC % = [(FM - DM).FM-1)] . 100,
where FM is fresh mass and DM is dry mass obtained after drying the achenes for 48
hours at 80°C (ISTA 1976).
Germination of intact achenes was analysed after 1 and 3 months storage in the soil but
with longer storage periods there were not enough intact achenes for the tests.
Germination was carried out at 25°C in 5 cm diameter Petri dishes on moistened filter
paper in darkness or in white fluorescent light at 437µW.cm-2.s-1 at Petri dish level
(Ruggiero & Zaidan 1997); three replicates of 30 seeds were used. Darkness was
obtained by enclosing the Petri dishes in two black plastic bags and seeds were
examined under a green safe light (Labouriau & Costa 1976).
Imbibition was determined using five replicates of 20 achenes, according to: %
imbibition = [(Mt - Mi) . Mi-1)] . 100, where Mt is the mass after imbibition and Mi the
initial mass (before imbibition started).
Germination and moisture content were also measured for achenes stored at 4°C for 1,
3, 6, 9 and 12 months. Germination rate (t) was calculated as the period of time in days
when 50% germination was reached (Labouriau 1967).
The germination percentage was subjected to angular transformation and different
treatments were compared by analysis of variance (Snedecor 1962).
Results and Discussion
As shown previously by Felippe (1990) the newly collected achenes of B. gardneri
showed light sensitivity during germination (figure 1). The results of germination for
achenes stored at 4°C up to 12 months are also shown in figure 1. The achenes showed
light sensitivity, germination being statistically higher in light than in darkness up to 6
months storage at 4°C. By 9 months storage in this condition the difference in
germination between light and darkness had disappeared for the smallest and the largest
achenes (7 and 8 mm long; 11 and 12mm long). Thus it seems that light sensitivity in B.
gardneri decreases with storage for a long period at 4°C. This is clearly observed when
achenes of all sizes were used (figure 2). There was a positive correlation between
period of storage and germination in darkness. Bidens odorata (Corkidi et al. 1991) and
Bidens pilosa (Forsyth & Brown 1982) show light sensitivity and also showed an
increase in germination in dark conditions with long period of storage. Both
germination in light and in darkness were affected by the size of the achenes (figure 1).
There was a very high positive correlation between size and germination for achenes
stored for 12 months (r = 0.9972 for germination in light and r = 0.9758 for germination
in darkness). The size of the achenes and the period of storage at 4°C also had an effect
on the rate of germination as measured by the medium time of germination, t (table 2).
In general, the higher the size of the achene and the longer the period of storage the
faster was the germination (lower values of t). Germination in light conditions was
higher in the longer achenes of Bidens odorata than in the shorter ones (Corkidi et al.
1991).
Figure 1. Germination in light (open symbols) and in darkness (closed symbols) of
Bidens gardneri achenes (7-8; 9; 10; 11-12 mm long) stored at 4°C for 0, 1, 3, 6, 9 and
12 months. The results of analysis of variance are shown by letters. Small letters (a, b)
compare germination in light and darkness in each period of storage and for each size of
achene. Capital letters (A,B,C) compare germination in light for each period of storage.
Capital letters with apostrophe (A', B', C') compare germination in darkness for each
period of storage. Results shown up to day 20 (values were the same afterwards, up to
day 30).
Figure 2. Correlation between period of storage at 4°C and maximum germination in
darkness (closed symbols) or in light (open symbols) in achenes of Bidens gardneri.
When stored in soil some seeds of B. gardneri germinated during the period of storage;
the number of germinated seeds increased and number of intact achenes decreased with
period of storage in the soil. Thus, there was a negative correlation between number of
intact achenes collected from the soil and period of storage and a positive one between
germinated achenes in the soil and period of storage, as can be seen in figure 3. When
storage was longer than 3 months very few intact achenes were recovered from burial in
the soil; because of this it was impossible to carry germination experiments. Achenes of
Bidens polylepis behave in a different way when stored in soil: only 2% of them
germinated while buried in soil in a non heated greenhouse (Baskin et al. 1995). The
moisture content (%) was much higher in the intact achenes removed from the soil than
for achenes stored at 4°C (table 3); this is not surprising as the achenes over the storage
period in the soil imbibed water from the rain (see rain data in Simabukuro et al. 1998
and Sassaki et al. 1999).
Figure 3. Correlation between period of storage in the soil in the cerrado of Moji Guaçu
and in the forest of the Instituto de Botânica and number of intact achenes (open
symbols) or germinated achenes of Bidens gardneri (closed symbols) when the
containers were removed from the soil.
The germination of the intact achenes stored in the cerrado for 1 and 3 months is shown
in figure 4. After 1 month storage in the soil the higher germination values were
observed in the longer achenes; this was also shown for achenes stored for 1 month at
4°C but the difference in germination values in darkness and in light was reduced,
compared with achenes stored for 1 month at 4°C. The statistical analysis (small letters)
showed differences between germination in light against darkness. In light there was no
difference in germination values when the length of the achenes was considered. In
darkness germination was higher in the 11-12 mm long achenes and the lowest
germination value was detected in the 7-8mm long achenes (see A' and C'). By 3
months storage in the soil the achenes had lost their sensitivity to light. Germination of
the achenes stored in forest for 1 and 3 months is shown in figure 4. With achenes
stored for 1 month germination was higher in light for the longer achenes and the lowest
values were found in the 7-8 mm long achenes; by the third month the achenes had lost
their sensitivity to light. Thus, light sensitivity is lost after storage for 9 to 12 months at
4°C while with storage in the soil (cerrado or forest) this happens with storage for one
month. Thus, soil storage radically changes the behaviour of the seeds of this species to
their sensitivity to light.
Figure 4. Germination in light (open symbols) and in darkness (closed symbols) of
Bidens gardneri achenes (7-8; 9; 10; 11-12 mm long) stored in cerrado soil in the
cerrado of Moji Guaçu for 1 and 3 months and in forest soil in the forest of the Instituto
de Botânica for 1 and 3 months. The results of analysis of variance are shown by letters.
Small letters (a, b) compare germination in light and darkness in each period of storage
and for each size of achene. Capital letters (A,B,C) compare germination in light for
each period of storage. Capital letters with apostrophe (A', B', C') compare germination
in darkness for each period of storage.
Felippe (1980) has shown for Rumex obtusifolius that for seeds which only germinate in
the light at 25°C, alternating temperatures could have the same promotive effect as
light. Bidens gardneri, another light sensitive seed, may behave in the same way as
Rumex. Ortolani & Pinto (1972) have shown that in Ribeirão Preto, state of São Paulo
(21°10'S and 47°50'W) in summer the median air temperature was 26°C. In our
experiments, in April, the air max temperature was around 30°C and the mininum
around 20°C for Moji Guaçu and Instituto de Botânica (Simabukuro et al. 1998, Sassaki
et al. 1999). According to Ortolani & Pinto (1972) alternating temperatures occurred in
the soil at 5 cm deep (22°C at 8.00 h and 35°C at 16.00 h) and at 10 cm deep (23°C at
8.00 h and 34°C at 16.00 h, but the temperatures were nearly constant at 20 cm deep
(25°C and 27°C). As the seeds in this work were buried at 10 cm deep, some alternating
temperatures occurred. Although the achenes were stored in the soil in a relatively dry
April, some rain occurred soon after the beginning of storage (see Simabukuro et al.
1998, Sassaki et al. 1999) and the alternating temperatures could have affected the
imbibed seeds. This could explain the fact that seeds germinated when buried, but does
not explain the lack of light sensitivity of the intact achenes removed from the soil.
The soil of the cerrado of Moji Guaçu is very porous presenting a high content of sand
(Simabukuro et al. 1998) and thus the water from the rain can easily reach the buried
seeds. The soil of the forest of the Instituto de Botânica contains about half the sand of
the cerrado soil but a higher level of clay and in this case can retain the water (Sassaki et
al. 1999). The rainfall occurring by the beginning of the storage could have been enough
for the seeds to imbibe, as they take up water very fast: in an experiment to check
imbibition the percentage of imbibition of the achenes of B. gardneri (stored for 12
months at 4°C or newly collected) was around 40% after 5 hours from the beginning of
the test (data not shown). The differences in germination (see figure 1) between longer
and shorter achenes is not a result of imbibition rates as imbibition was the same for the
achenes of different sizes used here (data not shown). Thus, the differences in
germination can not be explained by differences in relation to water permeability.
However, shorter achenes have higher mass/length ratio than longer ones (table 1). This
could be due to a thicker coat which is permeable to water, although the thickness of the
achenes coat was not measured in the present work. One possibility is that this thicker
seed coat could present some difficulty for the radicle to go through thus reducing
germination. This seems to be the case as Felippe (1990) has shown that coat removal
promoted germination of the shorter achenes of Bidens gardneri. Short achenes of
Bidens bipinnata also show dormancy (Dakshini & Aggarwall 1974). Buried short
achenes of Bidens gardneri may have slowly degraded their coats to allow seed
germination only later in the season compared to the faster germination of the long
achenes. Thus the requirements of the short achenes for germination will be responsible
for the formation of a transitory seed bank. In contrast, the long achenes that will
germinate soon after dispersal do not contribute to a seed bank.
Acknowledgements - Are due to CNPq for financial support and research grants for the
authors. Thanks are also due to Josimara N. Rondon for technical assistance. Thanks are
also due to Prof. John E. Dale (University of Edinburgh) for correcting our mistakes in
English and his comments on the manuscript.
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4005, 01016-970 São Paulo, SP, Brazil. Research Fellow, CNPq.