Regeneration mechanisms in Swamp Paperbark (Melaleuca ericifolia Sm.) and their implications for wetland rehabilitation

Effects of water availability and flooding

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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 

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

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 
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
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 

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 
mol m
 at the level of the dishes.  Incubation at a 
constant 10
C, 20
C or 30
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
.  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 

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).  

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.   

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 

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 
m wide
 By contrast, 
root hairs were generally less than 5 mm long and 15 
m wide.  

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. 

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 
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 

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.  

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. 

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 

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
; at salinities of greater 
than about 4 g L
 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
C than at 10
C regardless of 
light regime, but differences in responses between 20
C and 30
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
 at 10
C under alternating light:dark 
conditions, but the proportion fell to around 10-15 % at 20
C and none produced fully 
functional hypocotyl hairs at this salinity if the temperature were increased to 30
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). 

Figure 6.5  Percentage of seedlings showing hypocotyl hair development at six 
salinities (0, 1, 2, 4, 8 or 16 g L
), three temperatures (10
C, 20
C, 30
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 

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
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
 under all treatments except for 30
C in dark conditions. 
A complete suppression of secondary roots occurred at 16 g L
 under all treatments 
except 20
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
, 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
 (Fig. 6 b) and there 
was a complete absence of hypocotyl hairs on seedlings grown at a salinity of 4 g L

(Fig 6 d).   At a salinity of 8 g L
, 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
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). 

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
: fully developed hypocotyl hairs, fully developed 
primary root with root hairs; b) salinity = 1 g L
: impaired development of hypocotyl 
hairs primary root with root hairs; c) salinity = 2 g L
:  impaired development of  
hypocotyl hairs no root hairs;  d) salinity = 4 g L
: no  hypocotyl hairs, no root hairs 
moderate development of primary root, moderate development of secondary root; e) 
salinity = 8 g L
: no hypocotyl hairs, no root hairs, stunted primary root, stunted 
secondary root; f) salinity = 16 g L
: no hypocotyl hairs, no root hairs, primary root 
prematurely desiccated, no secondary roots, cotyledons cupped and noticeably 

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 ArtemisiaMyrtus 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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