The effect of plant growth regulators on centaury (Centaurium erythraea Rafn) seeds germination

Abstract

Centaury seeds are light-requiring. Long-term red light irradiation caused more than 80% of seeds to germinate. Seeds did not germinate in darkness. Gibberellic acid and GA7 can replace light, but N-substituted phtalimide AC 94,377 was ineffective. Light-induced germination was inhibited by abscisic acid and growth retardants such as ancymidol, tetcyclacis, and paclobutrazole. Growth retardant-caused inhibition can be overcome by the addition of gibberellic acid.

Full text

Arch. Biol. Sci., Belgrade, 57 (1), 25-28, 2005.
THE EFFECT OF PLANT GROWTH REGULATORS ON CENTAURY
(CENTAURIUM ERYTHRAEA RAFN.) SEED GERMINATION
NADA MIJAJLOVIĆ
1
, D. GRUBIŠIĆ
2,3
, Z. GIBA
3
andR. KONJEVIĆ
3
1
"Eko-lab" Quality Management Section Ltd.;
2
Siniša Stanković Institute for Biological Research, University of Bel-
grade, BulevarDespota Stevana 142, 11060 Belgrade;
3
Institute of Botany, Faculty of Science, University of Belgrade,
Takovska 43, 11000 Belgrade, Serbia and Montenegro
Abstract - Centaury seeds are light-requiring. Long-term red light irradiation caused more than 80% of seeds to germi-
nate. Seeds did not germinate in darkness. Gibberellic acid and GA
7
can replace light, but N-substituted phtalimide AC
94,377 was ineffective. Light-induced germination was inhibited by abscisic acid and growth retardants such as ancymi-
dol, tetcyclacis, and paclobutrazole. Growth retardant-caused inhibition can be overcome by the addition of gibberellic
acid.
UDC 582.923.1 : 581.142
INTRODUCTION
Centaurium erythraea Rafn. (centaury), like some other
species from the Gentianaceae family, is a medicinal
plant whose aerial parts are used in folk medicine as a
drug. The crude drug "Centaurii herba" has been
described in pharmacopoeias of many countries in
Europe. The whole herb is appetite-stimulating, aromat-
ic, bitter, cholagogic, diaphoretic, digestive, emetic,
weakly febrifugal, hepatic, stomachic, and tonic. The
plant is also used for preparation of commercial bever-
ages. It grows in open woods, meadows, and dry grass-
lands, often on chalky soils. It is in flower from June to
October, and the seeds ripen from August to October. The
scented flowers are hermaphroditic (have both male and
female organs) and are pollinated by bees, flies, and bee-
tles. Centaury is widely spread in Europe. However, due
to heavy harvesting by tearing out, centaury has become
a rather threatened plant species. Although it is not criti-
cally endangered, some measures should be applied to
protect centaury. For example, areas severely devastated
by previous harvests should be repopulated. Production
of "Centaurii herba" on plantations is another way of pro-
tection. The simplest and easiest way of repopulating or
planting would be by means of seed sowing. However,
knowledge about centaury seed germination is rather
scarce. Germination of these seeds is treated from the
ecological point of view in the paper of Silvertown
(1980). It is also mentioned in a survey of basic require-
ments for germination of dormant seeds (Nikolaeva
et al. 1985). The present paper describes the effect of
some plant growth regulators on the germination of Cen-
taurium erythraea seeds.
MATERIAL AND METHODS
Seeds and seed manipulation
Seeds collected from different localities in Western
Serbia (neighborhood of the town of Šabac) were used
throughout these studies. Lots of a hundred seeds each
were placed in Petri dishes 6 cm in diameter with 1 ml of
distilled water or test solution. The fungicide Nistatin
was supplied at a concentration of 500 mg L
-1
in order to
prevent fungal infections. The seeds were kept in dark-
ness until germination score, or after 24 h of imbibition
in darkness were continuously irradiated with red light
until the end of experiment. All experiments were repeat-
ed at least two times, each with four replicates, at a con-
stant temperature of t = 25 ± 0.2
0
C. Germination was
25
Key words: Centaurium erythraea, centaury, germination, gibberellins, growth retardants, light
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NADA MIJAJLOVIĆ et al.
scored 7 days after the onset of imbibition.
Light sources
Red light (λ = 660 nm) was provided by Philips TL
20/15 red-light fluorescent tubes with a 3-mm plastic
Rohm and Haas (Darmstadt, Germany) filter No. 501
producing a fluence rate 5.76 µmol m
-2
s
-1
at the seed
level. Light was measured with a Li-Cor (Lincoln, Nebr.,
U.S.A.) LI-1905A quantum sensor and a 660/730 SKR
110 sensor (SKYE Instruments Ltd., Liandrindod Wells,
Powys, Wales, U.K.).
Chemicals
Gibberellic acid (GA
3
) was purchased from the
Sigma Company, U.S.A; GA
7
from Serva, Germany;
ancymidol (α-cyclopropyl-α-(4-methoxy-phenyl)5
pyrimidine methanol) from Eli Lily and Co., Indianapo-
lis, Ind., U.S.A.); tetcyclacis [(5-(4-chloro-phenyl)-
3,4,5,9,10 -pentaaza-tetra-cyclo-5,4,1,0
2,6
,0
8,11
-dodeca-
3,9-diene)] from BASF, Germany; paclobutrazol [(2RS,
3RS)-1-(4-chloro-phenyl)-4,4-dimethyl-2-(1H-1,2,4-tria-
zol-1-yl) pentan-3-ol] from ICI, Bracknell, Berks., Eng-
land. N-substituted phtalimide, AC 94,377
[1-(chlorophtalimido)cyclohexanecarboximide] was
obtained from American Cyanamide Co., USA.
RESULTS
Seeds of centaury did not germinate in darkness.
Continuous irradiation with red light induced germina-
tion reaching a maximum at about 80 h after the start of
irradiation (Fig. 1). Germination in darkness can be
induced by exogenously applied gibberellins. If the seeds
were treated with gibberellins from the onset of imbibi-
tion, they germinated up to 95%, depending on the
applied concentration and the type of gibberellin used. To
be specific, GA
7
proved to be more effective than GA
3
,
inducing the same percent of germination at concentra-
tions 10 times lower a those of GA
3
. However, a physio-
logical analog of gibberellins, the N-substituted phtalim-
ide AC 94,377, was completely ineffective (Fig. 2). On
the other hand, light-induced germination can be com-
pletely inhibited by the application of abscisic acid (Fig.
3). The complete arrest of germination was achieved
26
Fig. 1. Light-induced germination of Centaurium erythraea seeds. Seeds were
imbibed for 24 h in distilled water in darkness at 25
0
C and then irradiated with
continuous red light as indicated on the x axis.
Fig. 2. The effect of GA
3
(˜), GA
7
(š), and AC 94,377 (n) on the centaury seed
germination in darkness. Seeds were imbibed at indicated concentrations of
growth regulators from the onset of imbibition. Germination was scored seven
days after the onset of imbibition.
Fig. 3. The effect of abscisic acid on the germination of red light irradiated cen-
taury seeds. Seeds were imbibed in different concentration of abscisic acid for 24
h in darkness and then irradiated with continuous red light. Germination was
scored 7 days after the onset of imbibition.
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27
GROWTH REGULATORS ON CENTAURY (CENTAURIUM ERYTHRAEA RAFN.) SEED GERMINATION
when ABA was present in the incubation medium in a
concentration of 3 x 10
-4
M.
Fig. 4 shows the effect of growth retardants on light-
induced germination of centaury seeds. As can be seen,
all of the applied retardants inhibited germination. The
most effective was tetcyclacis. It prevented germination
completely even at a concentration as low as 3 x 10
-6
M.
The inhibition of germination of centaury seeds caused
by growth retardants can be overcome by gibberellic
acid. Fig. 5 shows that the application of 3 x 10
-3
M gib-
berellic acid to paclobutrazole (3 x 10
-5
M)-inhibited
seeds reverted germination to close to 100%.
DISCUSSION
Information about centaury seed germination is
rather limited. To our best knowledge, apart from data on
seed germination, in the survey of Nikolaeva et al.
(1985) and on ecological aspects of germination (Sil-
vertown, 1980), no other data on centaury seed germi-
nation can be found in the literature. As can be seen from
our results, seeds of Centaurium erythrea are light-
requiring. They did not germinate in darkness. However,
gibberellins can replace light and induce maximum ger-
mination in darkness. Some 50 years ago, Lona(1956)
established the role of gibberellins in control of seed ger-
mination by showing that the application of exogenous
gibberellins promoted lettuce seed germination. In many
other light-requiring seeds, gibberellins also stimulate
germination in the absence of light (Borthwick et al.
1964; Grubišić, 1985; Grubišić et al. 1988, 1995).
On the other hand, our results (Fig. 4), as well as the
results of other authors (Gardner, 1983; Grubišić
and Konjević, 1987; Grubišić et al. 1988), show
that light-induced germination can be prevented by tetcy-
clacis, ancymidol, and paclobutrazol (inhibitors of gib-
berellin biosynthesis). All three retardants block gib-
berellin biosynthesis by interfering with oxidation steps
in the conversion of ent-kaurene to ent-kaurenoic acid.
These steps are catalyzed by so-called "mixed oxidases"
(Coolbaughand Hamilton, 1976; Dalzieland
Lawrence, 1984; Rademacher et al. 1984;
Rademacher, 2000).
Not all retardants were equally effective. Tetcyclacis
arrested germination at a concentration 10 times lower
than those of ancymidol and paclobutrazol. The higher
level of inhibition obtained with tetcyclacis points to the
possibility that this growth retardant may have a different
site of action or some side effects (Grossman etal.
1985). However, the inhibition of centaury seed germina-
tion caused by growth retardants can be overcome by the
application of exogenous gibberellins (Fig. 5). This was
previously demonstrated in the case of Paulowniatomen-
tosa seed germination (Grubišić etal. 1988). The
findings presented here, together with those reported in
the literature, suggest the possibility that light controls
seed germination by regulating the endogenous gib-
berellin level (Toyomasu etal. 1998; Yamaguchi
etal. 2002). However, these are only indirect data as far
as germination of centaury seeds is concerned. A clear-
Fig. 4. Growth retardants inhibited germination of centaury seeds. Centaury seeds
were imbibed in different concentration of tetcyclacis (š), ancymidol (˜), and
paclobutrazol (£) for 24 h in darkness and then irradiated with continuous red
light. Germination was scored 7 days after the onset of imbibition.
Fig. 5. The effect of gibberellic acid on paclobutrazol-inhibited germination of
centaury seeds. Seeds were simultaneously imbibed in inhibitori concentration of
paclobutrazol (3 x 10
-5
M) and indicated concentration of GA
3
. They were then
irradiated with continuous red light. Germination was scored 7 days after the
onset of imbibition.
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NADA MIJAJLOVIĆ et al.
28
cut conclusion on this matter should be based on more
detailed studies and in particular on analyses of the
endogenous gibberellin level in light-stimulated and
growth retardant-inhibited seeds.
Ackowledgements - The present work was supported by the Ministry of Science
and Enviroment Protection of Serbia (Grant # 1696).
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ЕФЕКАТ РЕГУЛАТОРА РАСТЕЊА НА КЛИЈАЊЕ СЕМЕНА КИЧИЦЕ
(CENTAURIUM ERYTHRAEA RAFN.)
Клијање семена кичице је зависно од светлости.
Дуготрајно осветљавање црвеном светлошћу
доводи до клијања преко 80% семена. Семена
не клијају у мраку. Гиберeлна киселина и GA
7
замењују потребу за светлошћу док је
N субституисани фталимид AC 94,377 неефикасан.
Клијање индуковано светлошћу инхибирају
абсцисинска киселина и ретaрданти растења
као што су тетциклацис, анцимидол и пакло-
бутразол. Инхибиција клијања изазвана рета-
ндантима може да се превазиђе додавањем
гиберелина.
НАДА МИЈАЈЛОВИЋ
1
, Д. ГРУБИШИЋ
2,3
, З. ГИБА
3
и Р. КОЊЕВИЋ
3
1
"Еко-лаб" Оделење за контролу квалитета;
2
Институтза биолошка истраживања ”Синиша Станковић”, Универзитет
уБеограду, БулеварДеспота Стефана 142,11060 Београд,
3
Институт за ботанику и ботаничка башта, Биолошки
факултет, Универзитет у Београду, Таковска 43, 11000 Београд, Србија и Црна Гора
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