Dormancy and germination in two Australian native species (Acacia aneura and Rhodanthe floribunda)

Abstract

In the laboratory of plant physiology of the University of Queensland (Gatton Campus), a seed germination experiment was undertaken on seeds of two Australian native plant species – Rhodanthe floribundato and Acacia aneura. Most Acacia, including A. aneura exhibit a physical dormancy due to the waxy coat covering the seed. Comparably, just a few species of Rhodanthe are studied as to their dormancy. However, they are also known to present different forms of dormancy. To understand and describe these dormancy mechanisms, a seed germination experiment was conducted on Acacia aneura and Rhodanthe floribunda. This experiment will either add to the existing knowledge regarding these species’ dormancy or corroborate them. It is expected that both species display some form(s) of dormancy.

Full text

1
DORMANCY AND GERMINATION IN TWO
AUSTRALIAN NATIVE SPECIES
(ACACIA ANEURA AND RHODANTHE FLORIBUNDA)
Paul Theophile EPEE MISSE
CONTENTS
ABSTRACT ..................................................................................................................................................... 2
INTRODUCTION ............................................................................................................................................ 2
MATERIALS AND METHODS.......................................................................................................................... 2
RESULTS ........................................................................................................................................................ 3
ACACIA DORMANCY ................................................................................................................................. 3
Effect of light on dormancy .................................................................................................................. 3
Effect of scarification on dormancy ...................................................................................................... 4
Effect of GA
3
on dormancy ................................................................................................................... 4
RHODANTHE DORMANCY ........................................................................................................................ 4
Effect of light on dormancy .................................................................................................................. 4
Effect of GA
3
on dormancy ................................................................................................................... 5
SEED CONTAMINATION ............................................................................................................................ 5
DISCUSSION .................................................................................................................................................. 5
Acacia aneura dormancy .......................................................................................................................... 5
Rhodanthe floribunda dormancy ............................................................................................................. 6
Seed contamination ................................................................................................................................. 6
CONCLUSION ................................................................................................................................................ 7
REFERENCES ................................................................................................................................................. 7

2
ABSTRACT
In the laboratory of plant physiology of the University of Queensland (Gatton Campus),
a seed germination experiment was undertaken on seeds of two Australian native plant
species – Rhodanthe floribundato and Acacia aneura. Most Acacia, including A. aneura
exhibit a physical dormancy due to the waxy coat covering the seed. Comparably, just a
few species of Rhodanthe are studied as to their dormancy. However, they are also known
to present different forms of dormancy. To understand and describe these dormancy
mechanisms, a seed germination experiment was conducted on Acacia aneura and
Rhodanthe floribunda. This experiment will either add to the existing knowledge
regarding these species’ dormancy or corroborate them. It is expected that both species
display some form(s) of dormancy.
INTRODUCTION
Germination constitutes an early stage of development for most naturally growing plants.
A wide array of plant species, however, exhibit germination blocks termed dormancy.
Five types of dormancy are identified: physiological, morphological, morpho-
physiological, physical and combinational dormancy (Baskin & Baskin 2004). To
determine what type of dormancy a particular seed species exhibit, a specific set of
experiments are performed. In the laboratory of plant physiology of the University of
Queensland (Gatton Campus), a seed germination experiment was undertaken on seeds
of two Australian native plant species – Rhodanthe floribundato and Acacia aneura.
Dormancy across the Acacia genera and ways to overcome it are well documented
through a number of studies. Some of these include Acacia caven (Escobar et al. 2010),
Acacia tortilis, Acacia oerfota (Abari et al. 2012), Acacia aroma, A. cavenand and A.
Furcatispina (Funes & Venier 2006). Most Acacia, including A. aneura exhibit a physical
dormancy due to the waxy coat covering the seed (Auld 1986; Al-Mudaris et al. 1999).
Comparably, just a few species of Rhodanthe are studied as to their dormancy (Hoyle et
al. 2008). However, they are also known to present different forms of dormancy (Bunker
1994). To understand and describe these dormancy mechanisms, a seed germination
experiment was conducted on Acacia aneura and Rhodanthe floribunda. This experiment
will either add to the existing knowledge regarding these species’ dormancy or
corroborate them. It is expected that both species display some form(s) of dormancy.
MATERIALS
AND
METHODS
Rhodanthe floribunda seeds used for this experiment were provided by the Centre for
Native Floriculture of the University of Queensland, while those of Acacia aneura were
collected from the Nindethana seed service.
The seed germination trial was conducted in the plant physiology laboratory of the
University of Queensland, Gatton campus. Seeds were first sterilized in 2% sodium

3
hypochlorite for 2 to 3 minutes, rinsed in sterile distilled water and placed on 2 sheets of
moistened filter paper in a Petri dish. The petri dishes were then carefully sealed with
parafilm and incubated at 25°C.
The trial was a completely randomized design of six treatments with eight replicates:
intact seeds moistened with water (the control); scarified moistened seeds; intact seeds
soaked in GA
3
at 100 mg L
-1
for 24 hours; and two light levels (light and dark). For the
dark treatments, dishes were covered with an aluminum foil. Acacia seeds were scarified
by making incisions on the seed coat at the opposite side of the embryo using N°10 scalpel
blade. Seeds of Rhodanthe were too small to be scared and therefore, this treatment was
not considered for this species. The dishes were then incubated at 25°C.
A day eight following incubation, the petri dishes were inspected for signs of germination
or contamination. A Seed was considered to have germinated when the radicle emerged
from the seed coat. Contaminated seeds with fungi or bacteria were discarded. For each
treatment, the number of contaminated and germinated seeds was recorded. With dark
treatments, the aluminum foil was carefully and quickly peeled off to inspect for
contamination, removal of contaminated seeds and germination count and then
immediately reinstate. Petri dishes were all resealed and returned to the incubation room
(25
o
C). The week after, all Petri dishes were removed from their covers, checked for
contamination and germination count.
The recorded data were statistically analyzed using the statistical software package R.
The effects of the various treatments were assessed by the ANOVA followed by the Least
Significant Difference test for means comparison at the probability level of P
< 0.05.
RESULTS
ACACIA
DORMANCY
E
FFECT OF LIGHT ON DORMANCY
Acacia aneura displayed a positive response to dark eight days following imbibition. The
germination rate of intact and GA
3
treated seeds in the dark was twice as much as in the
light (Table 1). Scarified seeds incubated in the dark recorded a higher germination rate
(80%) compare to light incubated seeds (57.5%). Fifteen days later, there was no
significant difference for both light levels except for the GA
3
treatment (57.5% in dark
and 35% in light).

4
Table 1: Effect of scarification, GA
3
and light/dark on percentage germination at 8 days and
15 days from imbibition on
Acacia aneura
Periods of
observation from
imbibitions
Germination (%)
Intact (control) Scarified GA
3
Dark Light Dark Light Dark Light
8 days
from
imbibition
37.5bc 17.5c 80a 57.5ab 40bc 17.5c
15 days
from
imbibition
80ab 65ab 92.5a 87.5a 57.5bc 35c
NB: Treatment means within the same row followed by the same letter(s) are not significantly
different at P< 0.05
E
FFECT OF SCARIFICATION ON DORMANCY
Scarified A. aneura seeds germinated faster and more than others. At the first seven days
of the trial, their germination rate in both light levels was at least twice higher than the
control and the GA
3
treatment (Table 1). Seven days afterward, scarified seeds still
recorded the highest germination rates (92.5%), particularly in the dark.
E
FFECT OF
GA
3
ON DORMANCY
The GA
3
treatment had a negative effect on A. aneura germination especially at the
second period of the experiment. The germination rate of this treatment was about half
the rate of intact seeds (control) independent of the light level.
RHODANTHE
DORMANCY
E
FFECT OF LIGHT ON DORMANCY
R. floribunda seeds responded positively to light. Eight days and 15 days from incubation,
the number of intact seeds (control) germinated in the light was nearly six times as much
as in the dark (Table 2). Irrespective of the treatment and the period, germinated seeds in
the light were at about twice or higher as much as in the dark. Furthermore, the highest
germination rate (100%) was recorded on seeds incubated in the light.
Table 2: Effect of GA
3
and light/dark on percentage germination at 8 days and 15 days from
imbibition on
Rhodanthe floribunda
Periods of observation
from imbibitions Germination (%)
Intact (control) GA
3
Dark Light Dark Light
8 days from
Imbibition
5c 27.5b 32.5b 80a
15 days
from
Imbibition
12.5c 52.5b 57.5b 100a
NB: Treatment means within the same row followed by the same letter(s) are not significantly
different at P< 0.05

5
E
FFECT OF
GA
3
ON DORMANCY
R. floribunda seeds reacted positively to GA
3
treatment. At the first period of the trial, the
germination rate under this treatment was threefold that of the control for light incubated
seeds, and up to sixfold for dark incubated seeds. At the second period of observation, the
rate of GA
3
treated seeds in light was almost twice as much as in the control.
SEED
CONTAMINATION
Seed contamination was monitored during the whole trial. Eight days from incubation
micro-organisms began infesting seeds in petri dishes. At the second period,
contamination kept progressing mostly on Acacia (Table 3). The Rhodanthe
contamination remained quite stable over both periods with a little increase of the
contamination rate of dark incubated intact seeds. However, these are just tendencies
since no major significant difference was noticed over both periods and across treatments
except between the Rhodanthe intact dark incubated seeds and those treated with GA
3
in
the light, 12.5% and 0% respectively.
Table 3: Seed contamination rate at 8 and 15 days from imbibition of two Australian native
species
Species Periods of
observation
from
imbibitions
Contamination (%)
Intact Scarified GA
3
Dark Light Dark Light Dark Light
Acacia
aneura
8 days
2.5a 2.5a 0a 5a 2.5a 12.5a
15 days
7.5a 7.5a 2.5a 2.5a 2.5a 17.5a
Rhodanthe
floribunda
8 days
7.5a 2.5a 5a 0a
15 days
12.5a 2.5ab 5ab 0b
NB: Treatment means within the same row followed by the same letter(s) are not significantly
different at P< 0.05
DISCUSSION
A
CACIA ANEURA DORMANCY
Scarified seeds of A. aneura germinated faster than the intact seeds. This result is
consistent with a number of studies on Acacia dating back in the 1980’s (Auld 1986).
Pound et al. (2014) report that mechanical scarification enhances Acacia germination,
with a higher percentage being achieved between 10 to 15 days. Acacia seeds are
generally recovered with a seed coat.To germinate, seeds need water and the seed coat
recovering the Acacia seed is a barrier to water absorption. Scarification provides
openings through which water and oxygen are absorbed triggering imbibition, the first
germination stage. It is also likely that some other compounds inhibiting germination are
released through these openings (Pound et al. 2014). However, not all Acacia exhibit

6
physical dormancy due to seed coat for Schelin (2004) found that Acacia macrostachya
lacked physical dormancy and could germinate properly under favorable conditions
despite its seed coat.
The darkness promoted Acacia germination. Although it is established that seeds with
physical dormancy do not exhibit any sensitivity to light (Baskin et al. 2004), our
observations contradict this assumption, particularly during the first week following
incubation.
The germination percentage of Acacia seeds was lower when treated with GA
3
. GA
3
appears to be an inhibitor of Acacia germination. Although the control and the GA
3
treatment were quite similar a week after incubation, a significant difference was
noticeable a week later, with GA
3
treated seeds displaying a lower germination rate.This
indicates that GA
3
slows down or blocks Acacia germination.
R
HODANTHE FLORIBUNDA DORMANCY
Rhodanthe floribunda seeds responded to light as well as GA
3
, an indication that
dormancy mechanisms may be controlled by both factors. This corroborates other
findings on Asteracea to which the Rhodanthe genera belongs to. For example, Merritt
(2006) noted that Gibberellic acid (or GA
3
) and light stimulated the germination of some
Australian Asteracea seeds. Similarly, Bunker (1994) reported the germination
stimulating effects of GA
3
on Rhodanthe moschata and Rhodanthe polygalifolia as well
as that of light on Rhodanthe humboldtiana and Rhodanthe stricta. In some dormant
seeds, germination is triggered by light which activates phytochromes. Phytochromes
seem to control the synthesis of Gibberellins (Bewley 2013). Gibberellins concentrations
usually increase during germination to support active cell enlargement by controlling the
transcription of genes encoding hydrolic enzymes. Upon activation of these genes by
Gibberellins, enzymes are released into the endosperm to decompose proteins and starch
into nutrients assimilated by the developing embryo (Hopkins and
Hüner, 2009). Similar
biochemical processes might have occurred in GA
3
treated Rhodanthe seeds incubated in
light, suggesting that the concentration levels of Gibberellins were lower to spark
germination.
S
EED CONTAMINATION
The number of contaminated seeds tended to increase over time. Rhodanthe seeds were
less subject to infestation than Acacia. The highest contamination occurred on Acacia
GA
3
treated seeds followed by the Rhodanthe intact seeds (control). This suggests that
the sterilization with 2% sodium hypochlorite was not strong enough to destroy
completely the microbes off the seeds or that the growth media were not completely
sterile. Therefore, sterrilising seeds with 4% sodium hypochlorite with the adjonction of
pesticides application could prevent contamination. For instance, Bunker (1994) added
fungicide on the petri dishes at the start of his experiment although other aseptic measures
might have been taken to keep the medium sterile.

7
CONCLUSION
This experiment revealed that Rhodanthe floribunda and Acacia aneura present different
forms of dormancy. The former exhibited a physiological dormancy while the later
displayed a physical dormancy. Mechanical scarification proved effective to break the
physical dormancy of A. aneura. However, this method can be time-consuming and risky
for the embryo, particularly if done manually on a large number of seeds. Consequently,
further investigation on chemical scarification could provide an alternative. To break R.
floribunda dormancy, seeds should be soaked in a solution of GA
3
at the concentration of
100 mg L
-1
for 24 hours and incubated in the light. Finally, to limit seed microbial
contamination strict aseptic measures should be combined with the use of
environmentally friendly pesticides.
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Australian Acacia Species through Seed Treatments Overcoming Coat-Imposed
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combinations of gibberellin, scarification, light and dark', Scientia Horticulturae, vol. 59,
no. 3, pp. 243-52.
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& Sons, Hoboken, NJ.

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Dixon, KW 2006, 'Effects of a butenolide present in smoke on light-mediated germination
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