6.2.5 Effects of water availability and flooding
To determine the effects of relative water availability on the production of hypocotyl
hairs, 25 surface-sterilised seeds were placed onto each of four replicate Petri dishes
(i.e., 100 seeds per treatment) containing bacteriological-quality agar at
concentrations of 0.2 %, 0.5 %, 1 % or 10 % w/v. The Petri dishes were covered with
laboratory film and incubated as described above, and the development of hypocotyl
hairs was recorded for 21 days although Petri dishes were observed for 60 days. As
before, seedlings were classified into one of three development classes: fully
developed; partial or impaired development and absence of hypocotyl hairs. The
development of hypocotyl hairs was recorded also in relation to seedling
development; rates of germination, growth and geotropism (defined as radicle
touching the agar and the apical hypocotyl becoming vertical). To determine whether
seedlings that had not developed hypocotyl hairs could do so if they were exposed to
conditions of lower water availability, some seedlings were transferred after 14 days
of incubation from the 0.2 % to the 10 % w/v agar concentration.
Compound microscopy was used to document the zone of production of
hypocotyl hairs. Seedlings were taken from the 0.2 % and 10 % water-agar dishes
134
each day for 7 days, beginning one week after germination. Seedlings were cut from
agar and fixed in 2.5 % w/v glutaraldehyde (in pH 7.4 phosphate buffer), cleared in 10
% w/v KOH at 90
0
C for 3 hours and then stored in 70 % w/v ethanol until the last
samples were processed. Five seedlings were sampled from each treatment on each
day. Seedlings were then embedded in paraffin wax. Samples were arranged so that
longitudinal sections could be obtained. Sections were cut to 8
μ
m thickness and
stained with Mallory’s Triple Stain. Stained sections were photographed using a
Zeiss compound microscope with a Nikon Coolpix 4500 digital camera.
Magnifications were 16X, 25 X, 32 X and 45 X depending on parts to be illustrated
(scale is included on photographs (Fig. 2 a-d) to overcome alterations to magnification
upon printing).
To determine the effects of flooding, surface-sterilized seed was soaked in
sterilised distilled water for 2, 4, 8, 16, 32, 64, 128 or 256 hrs. From each treatment,
100 seeds were placed onto Whatmans #3 filter paper discs in four replicate Petri
dishes (i.e., 25 seeds per Petri dish) and covered with 8 mL of de-ionized water.
Seeds were incubated at 20
0
C under constant darkness; these conditions were chosen
because earlier trials had shown that hypocotyl hairs developed readily under this
temperature-light-salinity regime.
6.2.6 Effects of temperature, light and salinity
A factorial design, using a total of 36 treatments, was used to quantify the prime and
interactive effects of temperature, light and salinity on the development of hypocotyl
hairs. Because the seedlings developed also root hairs and secondary roots, this
135
experiment allowed us to examine also the chronology of hypocotyl hair development
in relation to the development of these other organs. The general protocol using seeds
germinated on filter discs (i.e., that used previously to examine the effects of flooding
on hypocotyl hair development) was re-used for these trials, except that two
contrasting light regimes, three temperatures and six salinities were used in a fully
factorial experimental design. Seeds were incubated under all the possible
combinations of these different sets of environmental variables, in order to
differentiate between prime effects and interactions among individual variables.
Surface-sterilised seed were incubated on Whatmans #3 filter paper discs in
Petri dishes (as before, 100 seeds per treatment, four Petri dishes and 25 seeds per
dish), covered with 8 mL of solution with the appropriate salinity. The light treatment
consisted of incubation under a) constant darkness or b) 12hr:12hr light:dark cycle,
with a light intensity of 40
μ
mol m
-2
s
-1
at the level of the dishes. Incubation at a
constant 10
o
C, 20
o
C or 30
o
C comprised the three temperature treatments. The six
salinities used to assess salinity effects spanned the range of surface-water salinities
reported for Dowd Morass over the past ~ 5 years; salinities used were 0, 1, 2, 4, 8 or
16 g L
-1
. Saline solutions were made up with a commercially available sea-salt
mixture (Red Sea brand, Heidelberg Aquarium Supplies).
Following the random distribution of seeds among the various treatments and
periodic shuffling of Petri dishes to randomise any within-cabinet variations in
environmental conditions, seedlings were checked for development of hypocotyl
hairs, root hairs and secondary roots every seven days. Seedlings were assigned to
one of the three development classes of hypocotyl hairs outlined previously, as well as
136
two development classes (i.e., present or absent) for root hairs and secondary roots.
The width and length of hypocotyl hairs were measured 6 hypocotyl hairs from five
randomly selected seedlings from each salinity treatment. These seedlings were
photographed using a Moticam 2000 2.0 pixel Digital Camera mounted on a Zeiss
compound microscope set at 40 X magnification and processed using a Motic Images
image-processing program. A representative seedling from each of the treatments is
presented in Figure 6.
During the course of the above studies casual observations were made
regarding the developmental progression of hypocotyl hairs and other aspects of the
post-germination processes of M. ericifolia seedlings. It was beyond the scope of this
paper to deal directly with the physiological aspects of hypocotyl hair development.
6.2.7 Data analysis
Data were analysed with Analysis of Variance (ANOVA) with the SPSS (version 12)
and Systat (version 11.5) computer packages. Where appropriate, percentage data
were arc-sine transformed before analysis. One-way and three-way orthogonal
ANOVA designs were used for analysis (Zar 1999). Post-hoc tests used Bonferonni-
corrected probability values. Estimates of variance were calculated using Microsoft
Office Excel (2003).
137
6.3 Results
6.3.1 Effects of surface sterilisation
Between 45 % and 65 % of seeds that were surface sterilized for less than 2 minutes
germinated. Soaking for periods longer than 2 minutes, however, markedly decreased
germination rates, regardless of whether seeds were soaked in sodium hypochlorite or
de-ionized water (Fig. 1). There was little difference in germination rates between
seeds that were surface sterilized and those treated with de-ionized water for 5
minutes, but surface sterilization for 30 minutes reduced germination rates to less that
10 %. Hypocotyl hairs were produced by those seeds that had been surface-sterilized
for 2 minutes, and about 30 % of these seedlings possessed hypocotyl hairs after 21
days of incubation with no additional hairs being produced after this time. Hypocotyl
hairs were not produced from seeds that had been surface-sterilized for 5 or 30
minutes, but were produced from a small proportion of seeds (< 10 %) that were
soaked in de-ionized water for these periods.
138
Figure 6.1 Percentage germination of M. ericifolia seeds and percentage of seedlings
that produced hypocotyl hairs after soaking seeds with sodium hypochlorite (0.5 to 30
minutes) and de-ionized water (5 or 30 minutes). The height of each bar indicates the
mean percentage germination and the black portion indicates the mean percentage of
seedlings that showed the presence of hypocotyl hairs.
6.3.2 Origin and characteristics of hypocotyl hairs
The seedlings produced in the surface-sterilisation treatment were examined with
microscopy for 60 days. Seedlings without developed hypocotyl hairs showed
enlarged hypocotyl cell cells in the region where hypocotyl hairs would normally
emerge (Fig. 2 a, b, c). The distinct zone where hypocotyl hairs develop is clearly
distinguishable from the radicle and the rest of the hypocotyl, and forms an enlarged
139
area at the base of the hypocotyl above the join with the radicle (Fig. 2b). No other
epidermal cells on the hypocotyl were observed to produce hairs. Hypocotyl hair
cells, while present on the embryo pre-germination, did not elongate until germination
took place. Elongation was synchronous and apparent within two to three days of
sowing. The single-cell nature of the hypocotyl hairs was evident from the stained
paraffin sections viewed under high-powered microscopy
(Fig. 2 c, d). Fully formed
hypocotyl hairs in M. ericifolia were about 20 mm long x 30
μ
m wide
.
By contrast,
root hairs were generally less than 5 mm long and 15
μ
m wide.
140
Figure 6.2 Microscopy images of M. ericifolia seedling without (a, b) and with (c, d)
hypocotyl hairs. Unexpanded hypocotyl hair cells (image b) and hypocotyl hairs
(image d) are evident as enlarged or elongated cells surrounding the base of
hypocotyls. Unexpanded hypocotyl hair cells and formed hypocotyl hairs are marked
with arrows.
141
6.3.3 Hypocotyl hairs and seedling development
A large proportion of seedlings (65 %) emerged from the seed coat with hypocotyl
hairs that were impaired to well-developed. In other seedlings, however, hypocotyl
hair growth was delayed by several days. Radicle elongation began shortly after
germination in seedlings without hypocotyl hairs, and occurred sooner and more
rapidly than in those with hypocotyl hairs. Those seedlings with hypocotyl hairs were
more likely to produce root hairs on the radicle than those without. After 6-8 weeks
the hypocotyl hairs began to change in appearance, becoming wrinkled and had most
likely ceased to function. In general this event coincided with establishment of root
hairs.
6.3.4 Effects of water availability and flooding
A range of experiments showed that the relative dryness of the environment had a
strong effect on the development of hypocotyl hairs. Variations in relative water
availability, produced by incubating seeds on media made up with various
concentrations of agar, did not significantly affect germination rate, which remained
relatively constant around 20 % (data not shown). Water availability, however, did
influence strongly the development of hypocotyl hairs in those seeds that did
germinate (Fig. 3). Over 98 % of seedlings on the driest substrate (10 % w/v agar)
developed complete hypocotyl hairs and the 2 % of remaining seedlings showed
partially developed or impaired hypocotyl hairs. In this driest treatment, hypocotyl
hairs developed rapidly and reached full elongation with three days of emergence of
the hypocotyl from the testa. About 80 % of seedlings developed hypocotyl hairs on
142
the 1 % agar treatment, consisting of about 50 % with fully developed hairs and the
remaining 30 % showing impaired hair development. Seedlings on the substrates with
highest water availability (0.2 % and 0.5 % agar) did not develop any hypocotyl hairs
at all. No systematic data were collected on survival of seedling without hypocotyl
hairs but it was noted that most of these seedlings had perished at the end of the 60-
day observation period compared with almost complete survival of seedlings that had
produced hypocotyl hairs for the same period.
To determine whether seedlings that had not developed hypocotyl hairs in the
wetter environments could do so if exposed to conditions of lower water availability,
we moved some seedlings from the 0.2 % to the 10 % w/v agar-concentration
treatment. Seedlings that were moved to the drier conditions still failed to produce
hypocotyl hairs. Within days of transfer, the stem and to a lesser extent the radicle of
the surviving seedlings became highly pigmented (pink), possibly indicating a stress
response, and produced a mass of short root hairs but no hypocotyl hairs. Most of the
transferred seedlings died within three weeks.
To determine the effects of flooding, surface-sterilized seed was soaked in
sterilised distilled water for 2, 4, 8, 16, 32, 64, 128 or 256 hrs. Flooding had a
strongly negative effect on hair development, and hypocotyl hairs were not produced
under any of the flooding treatments (data not shown). This result is consistent with
the results shown in Fig. 1, which showed that surface sterilization for periods of
more than 2 minutes resulted in the failure of hypocotyl hairs to develop and that only
few seeds that were soaked in de-ionized water for 5 or 30 minutes developed hairs.
143
From these experiments we conclude that seeds soaked for more than about one hour
are unlikely to develop hypocotyl hairs.
Water availability had a strong effect also on the development of geotropism
in M. ericifolia seedlings (Fig. 4). Over 80 % of seedlings growing on the driest
media (10 % w/v agar) showed positive geotropism, and this percentage fell
uniformly until only about 20 % of seedlings that were sown on 0.05 % w/v agar
showed a positive response. Seedlings growing on 0.02 % w/v agar did not show any
positive geotropism at all.
Figure 6.3 Percentage of M. ericifolia seedlings that produced hypocotyl hairs after
germination and growth on substrata of different agar concentrations ranging from 0.2
% to 10 % w/v. The black portion of each bar indicates the mean percentage of
seedlings showing full hypocotyl hair development; the grey portion indicates the
mean percentage of seedlings with impaired development of hypocotyl hairs; the
white portion indicates the mean percentage of seedlings showing no development of
hypocotyl hairs.
144
Figure 6.4 Percentage of seedlings exhibiting positive geotropism after germination
and growth on substrata of different agar concentrations ranging from 0.2 % to 10 %
w/v. Error bars show standard deviations. N = 4
6.3.5 Effects of temperature, light and salinity
The individual effects of salinity, light and temperature were highly significant (P <
0.001) terms of both the percentage of seeds germinating and the formation of
hypocotyl hairs on those seeds that did germinate (Fig. 5). In addition to the effects of
these three prime variables, there were also significant interaction (P < 0.05) terms in
the ANOVA and these precluded making many generalizations as to any overall
impacts of temperature, light or salinity. Nevertheless, the strongest individual
inhibitory effect on hypocotyl hair formation was salinity, which accounted for 65 %
of the variance in response. Across all temperature and light-regime treatments, there
was a consistent decrease with increasing salinity of the proportion of seedlings
145
having fully developed hypocotyl hairs. Although there was an important interaction
between salinity and temperature (see below), seedlings showed fully developed
hypocotyl hairs only at the lowest salinities of 0 and 1 g L
-1
; at salinities of greater
than about 4 g L
-1
there was either no, or very poor, development of hypocotyl hairs.
This pattern occurred regardless of temperature or light regime.
Temperature exerted a smaller individual influence than salinity (10 % of
variance) but still returned a highly significant prime effect ( P< 0.001) on hypocotyl
hair formation (Fig. 5). Germination was better at 20
o
C than at 10
o
C regardless of
light regime, but differences in responses between 20
o
C and 30
o
C were conditional on
whether seedlings were incubated under constant darkness or under an alternating
light:dark cycle. Temperature also modulated the effect of salinity on hypocotyl hair
formation, and higher temperatures exacerbated the inhibitory effects of salinity on
the formation of these structures. For example, almost all seedlings developed fully
functional hypocotyl hairs at a salinity of 1 g L
-1
at 10
o
C under alternating light:dark
conditions, but the proportion fell to around 10-15 % at 20
o
C and none produced fully
functional hypocotyl hairs at this salinity if the temperature were increased to 30
o
C.
The alternating light:dark treatment, although having the smallest inhibitory
influence on hypocotyl hair production (<0.2 % of variance), nevertheless also
returned a highly significant prime effect (P < 0.001). The formation of hypocotyl
hairs was generally better in the dark than under the alternating light:dark cycle. As
noted before, however, it is difficult to draw general conclusions because of the
significance of the many interaction terms. Moreover, the interactive effect of all
three factors - salinity, light and temperature - was also highly significant ( P< 0.001).
146
Figure 6.5 Percentage of seedlings showing hypocotyl hair development at six
salinities (0, 1, 2, 4, 8 or 16 g L
-1
), three temperatures (10
o
C, 20
o
C, 30
o
C) and under
contrasting light regimes of a) alternating 12hr:12hr light:dark cycle or b) complete
darkness. Means are shown; error bars have been excluded for clarity. The black
portion of each bar indicates the mean percentage of seedlings showing full hypocotyl
hair development; the grey portion indicates the man percentage of seedlings with
147
impaired development of hypocotyl hairs; the white portion indicates the mean
percentage of seedlings showing no development of hypocotyl hairs.
The environmental conditions that encouraged the formation of hypocotyl
hairs also encouraged the formation of root hairs: increasing salinity, temperature and
exposure to light inhibited the production of root hairs (data not shown). Conversely,
the production of secondary roots, with concurrent inhibition of primary radicles, was
stimulated by increases in salinity and temperature and with exposure to light.
Complete suppression or premature death of both the primary radicles and secondary
roots took place at the highest salinities, as well as at 30
o
C and with exposure to light.
Thus there was an inverse relationship between the formation of hypocotyl hairs/root
hairs and the formation of secondary roots. Indeed, secondary roots were initiated
only at salinities above 2 g L
-1
under all treatments except for 30
0
C in dark conditions.
A complete suppression of secondary roots occurred at 16 g L
-1
under all treatments
except 20
0
C under constant darkness.
Fig. 6 shows the development of hypocotyl hairs on seedlings incubated under
contrasting salinity regimes. At the lowest salinity of 0 g L
-1
, the hypocotyl hairs
were fully developed, as was the primary root and root hairs (Fig. 6a). Hypocotyl hair
development was visibly impaired at a salinity as low as 1 g L
-1
(Fig. 6 b) and there
was a complete absence of hypocotyl hairs on seedlings grown at a salinity of 4 g L
-1
:
(Fig 6 d). At a salinity of 8 g L
-1
, there were no hypocotyl hairs, no root hairs, only a
stunted primary root and a stunted secondary root. The highest salinity of 16 g L
-1
generated seedlings with no hypocotyl hairs, no root hairs, the primary root
prematurely desiccated, no secondary roots, and the cotyledons cupped and noticeably
thickened (Fig 6 f).
148
Figure 6.6 Microscopy images of the development of hypocotyl hairs and root hairs
in M. ericifolia seedling grown under six contrasting salinity regimes. Photographed
at day 14. a) salinity = 0 g L
-1
: fully developed hypocotyl hairs, fully developed
primary root with root hairs; b) salinity = 1 g L
-1
: impaired development of hypocotyl
hairs primary root with root hairs; c) salinity = 2 g L
-1
: impaired development of
hypocotyl hairs no root hairs; d) salinity = 4 g L
-1
: no hypocotyl hairs, no root hairs
moderate development of primary root, moderate development of secondary root; e)
salinity = 8 g L
-1
: no hypocotyl hairs, no root hairs, stunted primary root, stunted
secondary root; f) salinity = 16 g L
-1
: no hypocotyl hairs, no root hairs, primary root
prematurely desiccated, no secondary roots, cotyledons cupped and noticeably
thickened.
149
6.4 Discussion
The location of hypocotyl hairs on the base of the hypocotyl of M. ericifolia
seedlings, and not on the radical, clearly indicates that these structures are not part of
the normal root system of young Swamp Paperbark plants. They are transitory in
nature and, although produced soon after germination, begin to desist within a few
weeks. In most cases, hypocotyl hairs were present as the seedling emerged from the
seed coat or else they developed very shortly afterwards. A similar developmental
pattern has been reported for other woody plant species by Young and Martens
(1991), Arrone and de Micco (2004) and Matsuo and Shibayama (2002) for the
genera Artemisia, Myrtus and Monochoria.
Arrone and de Micco (2004) and Young and Martens (1991) proposed that
hypocotyl hairs, which radiate out along the substrata, were important in providing
anchorage and in facilitating the development of geotropism in young seedlings.
Certainly these results show a strong correlation between the development of
hypocotyl hairs and the development of positive geotropism (cf. Figs. 3 and 4). The
ability of seedlings to produce hypocotyl hairs is likely to be particularly important for
wetland plants such as M. ericifolia, which occurs in habitats where inundation is
common, making it critical for young plants to be able to orientate themselves
appropriately when establishing in mobile, possibly disturbed, substrata.
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