Reserve substances and storage of Cyathea delgadii
ELIANA A. SIMABUKURO2, MARIA ANGELA M. DE CARVALHO3 and G. M.
(recebido em 03/06/97, aceito em 04/12/97)
ABSTRACT - (Reserve substances and storage of Cyathea delgadii Sternb. spores).
The spores used were collected from a population of trees of Cyathea delgadii Sternb.
growing in a gallery forest at the Reserva Biológica e Estação Experimental de Moji
Guaçu, São Paulo state, Brazil (22°18’S and 47°11’W). The germination of spores of
Cyathea delgadii decreases with time when kept in closed bottles under storage at 4°C
in darkness. Germination is still very high after storage for one year. Spores stored for
three years do not germinate. The results also show a decrease in soluble proteins and
an increase in starch after several months storage.
RESUMO - (Substâncias de reserva e armazenamento de esporos de Cyathea delgadii
Sternb.). Todos os esporos utilizados foram coletados de uma população de Cyathea
delgadii em uma mata ciliar na Reserva Biológica e Estação Experimental de Moji
Guaçu, SP (22° 18’S e 47°11’W). Com o passar do tempo, os esporos de Cyathea
delgadii deixam de germinar quando armazenados em vidros fechados mantidos no
escuro a 4°C. Germinação ainda é observada com esporos armazenados por um ano.
Não há germinação de esporos armazenados por três anos. Após vários meses de
armazenamento, foram observados uma diminuição do conteúdo de proteínas e um
aumento do nível de amido.
Key words - Tree fern, pteridophyta, lipids, proteins, sugars
Spores of tree ferns from the Cyatheaceae family lose viability in a few weeks of
storage at room temperature, with few exceptions (Page 1979).
There is a need to keep spores in storage. The literature recommends to keep spores
under low temperature and humidity (Dyer 1979).
Cyathea delgadii Sternb. is a tree fern which produces spores all the year round and one
frond can produce nearly 300 million spores (Simabukuro 1995). Spores of this species
were still viable after nine months of storage in closed bottles kept at 4°C and darkness
(Randi & Felippe 1988a). In this paper, the effect of storage at 4°C under darkness over
a period of four years was studied. The objective was to verify the ability of the spores
of C. delgadii to germinate during storage up to four years and to determine the level of
reserve substances during different storage periods of the spores.
Material and methods
Spores were collected from a population of Cyathea delgadii Sternb. growing in a
gallery forest in the Reserva Biológica e Estação Experimental de Moji Guaçu, São
Paulo state, Brazil (22º18' S and 47°11’W). After drying over absorbent paper at 25°C,
the spores from different harvests (different times from the same population) were
stored in closed bottles which were kept in darkness at 4°C. Some lots of spores were
kept for up to 48 months.
For germination the spores were sown in liquid medium (Marcondes-Ferreira & Felippe
1984) in 125 ml conical flasks (three per treatment) which were kept at 25°C under
white light; germination (protrusion of the rhizoid) was determined on day 7 from
sowing when maximum germination is attained in freshly collected spores (Randi &
Extractions - For the extraction of lipids, soluble proteins, phenolic compounds and
starch three samples of 100 mg of spores were used. Lipids (total lipids soluble in
hexane) were extracted and determined according to Gemmrich (1977). Soluble proteins
were determined according to Bradford (1976) using BSA as standard. The spores were
extracted with diethyl-ether. After centrifugation, the supernatant (diethyl-ether) was
discarded and the residue extracted with 0.1N NaOH and precipitated with 10% TCA.
The precipitated proteins were then re-suspended in NaOH. Phenolic compounds were
extracted in diethyl-ether and 0.1N NaOH (these two chemicals were used in the present
extraction of soluble proteins with the Bradford method mentioned above) and
determined according to Swain & Hillis (1959); total phenols are expressed as µg
equivalents of phenic acid. For the extraction of starch after the removal of soluble
carbohydrates the spores were first subjected to extraction in MCW
(methanol:chloroform:water - 12/5/3, v/v/v) followed by extraction in 10% ethanol,
according to Shannon (1968). The resulting residue was then treated with perchloric
acid to extract starch (McCready et al. 1950). Quantitative determination of starch was
done by the anthrone reagent using glucose as standard and applying the correction
factor 0.9 to convert glucose content to starch. For the extraction of soluble sugars 300
mg of spores were used. Extraction was done three times in 80% hot ethanol, according
to McCready et al. (1950). The pooled supernatants were concentrated in a rotary
evaporator and re-suspended in distilled water. Total sugars were determined by the
phenol-sulfuric acid procedure (Dubois et al. 1956) and reducing sugars according to
Somogyi (1945), using glucose as standard.
The number of globules of lipids in freshly-harvested and stored spores was determined
for six samples of 100 spores each.
The data were submitted to analysis of variance and LSD 5% (Tukey test) was
determined when necessary (Snedecor 1962). Percentage of germination was
transformed to angular values for the analyses.
Results and Discussion
The germination of C. delgadii spores under white light is very high in freshly harvested
spores sown in liquid medium, being 86.0% on day 7 after sowing (table 1).
Germination of the spores decreases with storage and is near zero after 32 months of
storage. The values shown in table 1 are very similar to those obtained by Randi &
Felippe (1988a) for spores kept under 4°C and darkness, during two and nine months.
Although there is no decrease in germination between two and at least 10 months, there
is a decrease between freshly collected spores and spores stored for two months. Spores
of C. delgadii do not germinate under darkness (Marcondes-Ferreira & Felippe 1984).
The content of lipids in the spores of C. delgadii is very high in all periods of storage
analysed (table 1). This agrees with data from Marcondes-Ferreira & Felippe (1984) and
Randi & Felippe (1988b), and high content of lipids has been shown in several papers
for different species (Randi & Felippe 1988b). It is also very high in another member of
the Cyatheaceae also collected in São Paulo state, Trichipteris corcovadensis (Felippe et
al. 1989). A single large lipid globule is present in the majority of spores independent of
the period that they were kept stored (table 2). Most of the whole grain is a lipid
globule, which confirms the high content of lipids shown in table 1. In Psilotum sp.
there was a relation between the number of globules of lipids and germination: only
spores with a large number of globules germinate (Whittier 1990) and spores of
Equisetum fluviale var. verticillatum presenting only one small central globule of lipid
did not germinate (Gullvåg 1968). In C. delgadii, lipid content does not seem to be
related to germination in stored spores.
Table 1. Germination and reserves substances in stored spores (0 to 48 months) of C. delgadii.
Germination on day 7 from imbibition. Different lots of spores (spores collected in different dates) were
used in the extraction. Different letters indicate significant differences.
Table 2. Percentage of spores of C. delgadii with different number of globules of lipids. Letters compare
each column; different letters indicate significant differences.
According to Cohen & DeMaggio (1986) and Mattoo et al. (1987), the Bradford method
is questionable as co-extracted phenolic compounds that can interfere in the method and
thus give a spurious high value for soluble proteins; on the other hand, in the case of
proteins the method only measures larger peptides. In a preliminary experiment, the
results for phenolic compounds in equivalents of phenic acid were 6.18 µg per mg of
spore for the diethyl-ether extract compared with 0.18 µg per mg of spore for 0.1N
NaOH extract; these results show that diethyl-ether extracts most of the phenolic
compounds and very little is extracted by NaOH; thus in the present case there seem to
be little interference of phenolic compounds in soluble protein content. Soluble protein
(at least the larger peptides) content does not change during the first year of storage, but
drops strongly thereafter (table 1). Synthesis of proteins occurs during germination of
spores of Cyathea delgadii (Randi & Felippe 1988c), Pteridium aquilinum (Raghavan
1970) and Pteris vittata (Gemmrich 1979).
Between three and seven months storage, there is an increase in the level of starch in
spores (table 1); the level remained high during the rest of the experiment. There was
great fluctuation in values for soluble and reducing sugars (table 1). This could reflect
the low amount of material used here; thus it is difficult to reach some conclusions with
the present results. During germination of Cyathea delgadii, there was an increase in the
level of reducing sugars and starch and a decrease in soluble sugars (Randi & Felippe
1988c). A reduction of soluble and reducing sugars was observed during the
germination of Adiantum capillus-veneris (Minamikawa et al.1984).
The values for soluble protein, soluble sugars, reducing sugars and starch for freshly
collected spores (table 1) are much higher than the values presented by Randi & Felippe
(1988c) for the same species. This can be expected as the spores represent genic pools
from two distinct populations. The spores used in the present paper are from the
Reserva Biológica e Estação Experimental de Moji Guaçu and the ones used by Randi
& Felippe were collected at the Reserva Biológica do Parque Estadual das Fontes do
Ipiranga, in the city of São Paulo, Brazil (23°29'S and 46°37’W). Whittier (1990) has
shown differences in spores of Psilotum sp. collected in different regions of the United
States and these differences were maintained even with the same treatments of light,
temperature or nutrition.
In conclusion, spores of Cyathea delgadii lose their ability to germinate with time when
kept in closed bottles under storage at 4°C in darkness; however germination of about
60% still occurs after one year storage, falling to circa 25% after two year storage; by
three years, percentage germination is zero. The results for the first year storage for
germination confirm the data shown by Randi & Felippe (1988a).
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1. Financial support by International Scientific Cooperation EC-Brazil (CI1/0620) and CAPES (Brazil).
2. Universidade Federal de Pernambuco, Departamento de Botânica, 50670-901 Recife, PE.
3. Instituto de Botânica, Caixa Postal 4005, 01061-970 São Paulo, SP, Brazil. Research Fellow, CNPq.