Medicinal and Aromatic Plants—Industrial Profiles


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BIOMASS AND OIL PRODUCTION
121
accumulation. Wiermann (1981) emphasised three aspects relating to the production of
secondary compounds of which tea tree oil is representative:
1.
Changes in the oil concentration represent a balance between gains from anabolism
and losses from catabolism. During periods with a constant concentration, turnover
might or might not be present.
2.
An increase in the rate of accumulation could result from direct synthesis, or trans
location from another organ.
3.
Changes in the accumulation rate may reflect interconversion rather than de novo
synthesis.
Using the definitions of Barz and Köster (1981), turnover occurs when secondary products
are further metabolised and even degraded. Catabolism is a form of turnover where there is
partial or complete degradation of the compound. When terpene turnover was measured in
intact plants, the rate of turnover was either slow (half-lives of 5–170 days) or undetectable
(Gershenzon et al. 1993, and references cited therein). This result contrasts with the rapid
turnover measured in many earlier studies that used detached plant parts (Mihaliak et al.
1991). The Gershenzon et al. (1993) study included tea tree and found no turnover over 14
days in a cohort of young leaves that were unfolding or just unfolded at the start of the
experiment.
Young leaves were sampled because their rapid rate of oil synthesis gave a measurable
incorporation of radiolabelled 
14
CO
2
 in the oil. The technique is not suited to older leaves,
and given the uncertainty regarding results obtained on detached plant parts, there is no
definitive picture regarding turnover in other than very young leaves.
Many essential oil plants synthesise the oil near, but not in, the storage organ. Symplastic
transport, probably through the many plasmodesmata in the walls of synthesising cells,
moves the oil to the storage organ (Cutter 1978). Under these conditions, factors that interfere
with short-distance transport could affect the rate of accumulation in secretory cells and
perhaps control synthesis by a feedback mechanism. Virtually all the relevant experiments
with tea tree have centered on the oil concentration, and only Gershenzon et al. (1993) have
provided a direct measurement of synthesis. They found a rapid rate of synthesis during
early leaf expansion, a result that agreed with the general situation with terpenoid producing
plants (Gershenzon and Croteau 1991).
When an increase in oil concentration is too rapid to be explained by an increasing
proportion of young leaves, it reflects either de now synthesis or conversion from bound
forms (Wiermann 1981). A third outside possibility is translocation from other organs, but
the storage glands for tea tree oil are virtually confined to leaves. Small numbers of glands
can be observed on fine stems, but produce no measurable quantities of oil when stems
alone are distilled.
Essential oil constituents are lipophilic compounds, and interconversion to more
water-soluble forms is essential if the oil is to move from the normally well sequestered
storage organ. Glycosides are common in plants and are thought to be important in
interconversion and transport (Stahl-Biskup 1987). Croteau (1988) provided evidence
that monoterpenyl glycosides are transport derivatives that can be found well away
from the site of synthesis.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

G.J.MURTAGH
122
A possible explanation for the rapid decline followed by a recovery to the original oil
concentration is a process of conversion to a water soluble form, then a reversion to the
original form. The water soluble form need not move within the plant for the distillation
process to record a drop in oil concentration, as water soluble compounds such as glucosides
do not distill (Banthorpe et al. 1972). Even if the water soluble forms did distill they would
not be recovered during the oil/water separation phase unless breakdown occurred during
distillation.
Loss Pathways
Of the three potential loss pathways, volatilisation, interconversion and catabolism,
information with tea tree is only available for volatilisation. Using intact tea trees with
16 week old regrowth placed in a darkened chamber at 25°C, Murtagh et al. (1993),
measured a volatilisation rate of 1.3µg/g DW leaf/h, a rate that is towards the lower end
of the general range of 0.1 to 10µg/g/h for monoterpene emissions (Tingey et al. 1991).
If the measured rate held constant it would take 3.6 days to volatilise 1% of the oil,
which is too slow to explain the negative movement in oil concentration discussed
earlier.
Kawakami et al. (1990) used a stream of nitrogen to collect the emissions from detached
tea tree twigs that were cut into 10cm lengths and held at 15°C. They measured an emission
rate of 0.5–2.5mg/100 g fresh leaf/h, that translates to approximately 5.6µg/g DW leaf/h; a
much higher rate than the first study. The difference could arise from the use of twig sections
in the second study. Removing a branch from Salvia mellifera more than doubled the short-
term volatilisation (Dement et al. 1975), and rough handling was observed to increase the
volatilisation from Pinus radiata (Juuti et al. 1990).
Murtagh et al. (1993) measured the volatilisation from tea tree at 15, 25 and 35°C. A
Q
10
 value was used to quantify the temperature effect and equalled 3.1 with the olefins
and 5.6 with the oxygenated compounds. Both values exceeded the effect of temperature
on the saturated vapour pressures of the major compounds in emissions. Tingey et al.
(1991) also noted the same difference across a number of species, and attributed it to
either changes in pathway conductance or differences in pool size. The Q
10
 of 3.1 with
the olefins is close to the expected value of 2–3 for enzymatically controlled processes
(Salisbury and Ross 1992), and values in this range have been presented as evidence
that pool size regulates the emission flux (Tingey 1981). Pool size had the greatest
effect on the emission rate in the Murtagh et al. (1993) study. The even higher Q
10
 with
the oxygenated compounds is well above the expected value for metabolic processes,
and suggests that diffusion is controlled by the cuticular membrane (Schönherr and
Bukovac 1978).
The volatilisation of individual compounds was also measured in a study by Murtagh
et al. (1993) (
Table 2
). This was done with the 35°C treatment because the discrimination
was best at a high temperature. The leaf oil concentration, given in Table 2, was measured
on a solvent extract to avoid converting precursor compounds to terpinen-4-ol (Southwell
and Stiff 1989). In agreement with studies with other species (Tingey et al. 1991), the
tea tree emissions had a different chemical composition to the leaf oil. At 35°C, the
emissions contained 40% olefins whereas the leaf oil had 32% olefins. The mean cineole
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

BIOMASS AND OIL PRODUCTION
123
concentration shown in Table 2 is higher than the concentration in commercial tea tree
oil because a tree with an exceptionally high cineole concentration was included in the
study.
The leaf oil concentration, sometimes referred to as the pool size, had the greatest
effect on the emission rates of the individual compounds (Table 2). The effect was
non-linear, meaning that the compounds present at the larger concentrations did not
volatilise at an equivalent rate to those with a smaller concentration. A standardised
emission rate that accounted for much of the concentration effect was obtained by
dividing the rate by the concentration raised to the power of 0.9. The method of
estimation used a statistical procedure to balance out the uneven weighting of results
when some compounds were not detected in all runs. The high standardised emission
rate for 
α-pinene, ß-pinene and p-cymene relative to the other olefins can be attributed
to their relatively high SVP, but the reason for the high standardised rate with trans-
sabinene hydrate is not clear. Both sabinene hydrate isomers are evident in flush
growth but are converted to end products and little or none remains as leaves mature
(Southwell and Stiff 1990). With ongoing synthesis, the recently formed juvenile oil
with both sabinene hydrate isomers could be held at a location that is more susceptible
to volatilisation loss.
Comparisons of the relative volatilisation rates of various compounds are invariably
confounded by their different physical properties. As these differences are less with 
α- and
ß-pinene these compounds were used for comparison. In both the Murtagh et al. (1993) and
Kawakami et al. (1990) studies, the ratio of 
α- to ß-pinene was about 2:1 in the plant oil,
and 1:1 in the emissions. In other words, ß-pinene was favoured in emissions relative to the
Table 2 The composition of leaf emissions at 35°C, and leaf oil, from regrowth of M.
alternifolia. Also listed are the standard emission rates (E/C
0.9
) after correction for pool size
effects, and saturated vapour pressures (SVP) at 35°C
The rate of emission (e’miss.) of each compound is expressed as the weight per unit doublesided
leaf area per hour. The concentration (conc.) is given as the weight per double-sided leaf area
(mg/m
2
).
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

G.J.MURTAGH
124
composition of the plant oil. This contrasts with the likely effect of the relative physical
properties that would favour the volatilisation of a-pinene, as it has the higher SVP (727 Pa
and 524 Pa at 25°C for the a and ß isomers respectively) and a lower viscosity than ß-pinene
(Drew et al. 1971). As the ratio of 
α- to ß-pinene is 1:1 in the juvenile oil found in flush
growth (Southwell and Stiff 1990), a better explanation is that the emissions were sourced
from juvenile oil. This would explain the 1:1 ratio in the absolute volatilisation rates of the
pinene isomers. Furthermore, for those compounds that were sourced from the juvenile
pools of unknown concentration, the absolute emission rate given in 
Table 2
 is a better
measure than the standardised rate.
The presence of cis- and trans-sabinene hydrate in the emissions provided further evidence
that they were sourced from juvenile oil. Southwell and Stiff (1990) found that both isomers
were well represented in flush leaves but declined to trace or zero levels as the leaves
developed to leaf node 13 and beyond. However, the observed lack of turnover in young
leaves aged to 15 days (Gershenzon et al. 1993) complicates the issue. With typical leaf
emergence rates of 0.5–1.0/d (Curtis 1996), leaf node 13 corresponds to a leaf age of 13–26
days. The majority if not all of this period coincides with the measured period of no turnover
and hence no volatilisation from the young leaves. This suggests that the emissions were
sourced from juvenile oil in mature leaves. The presence of juvenile oil in nonflush growth
is consistent with ongoing synthesis in mature leaves, a process that could give the positive
changes in oil concentration described earlier. The quantity of juvenile oil could be quite
small and difficult to detect in a leafoil analysis because of the diluting effect of the bulk oil.
Also, its presence could be transient according to the extent of replacement synthesis of oil
as exhibited in the changing oil concentrations.
Double Pool Conceptual Model
A number of authors including Barz and Köster (1981) have noted that ongoing synthesis
or interconversion is required to provide positive changes in oil concentration. In contrast
with this view is the concept that oil stored in glands is well sequestered and isolated
from the normal functioning of the plant (McKey 1979). The isolation need not be
absolute and solubilising reactions can promote translocation and catabolism (Croteau
1988).
With tea tree, the fluctuations in oil concentration are more marked in mature, but
not aged leaves, and in leaves with a high oil concentration. Also, emissions appear to
be sourced from pools with a more juvenile oil than the bulk leaf oil. These observations
can be accommodated in a double pool model of oil accumulation and loss. One pool
represents a stable storage, with some seasonal decline in oil concentration, especially
during winter. The second pool has a more variable concentration, and is subject to
both additions and loss even in the short term. This is not a new idea. Banthorpe et al.
(1972) suggested that plants may have two distinct pools of terpenes; one of which is
affected by outside influences and the other is more inert. Loomis and Croteau (1973)
used the concept of metabolic and storage pools to explain some of the periodic changes
in oil concentration. Janson (1993) and a number of other authors proposed the equivalent
of the double pool model for emissions from conifers, with the standing pool in resin
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

BIOMASS AND OIL PRODUCTION
125
ducts providing a relatively stable source of emissions and synthesising cells providing
a variable source.
The issue of quantitative limits within a pool is uncertain, but it is interesting that the
composition of leaf oil changed when the concentration exceeded about 65mg/g in the
Murtagh and Smith (1996) study. As a first step for the genetic lines currently used by the
industry, 65mg/g could be taken as the upper limit for the stable pool. With the highest
observed oil concentrations near 100mg/g, the difference of 35mg/g would be the capacity
of the variable pool. While the more variable oil concentrations tend to occur at the higher
concentrations, this is not an absolute effect. Consequently, the model would not require
the stable pool to be full before some accumulation could start in the variable pool.
BIOMASS YIELD
The leaf yield is the second major component of the yield of tea tree oil. As with the oil
concentration it varies, but for different reasons and in a different pattern to the oil
concentration (Murtagh 1991 a). In plantation production, the tea trees are grown as a row
crop and are influenced by many of the agronomic factors that affect cropping in general.
These are reviewed in 
Chapter 3
 and the current chapter concentrates on the physiology of
biomass production.
The leaf yield is strongly correlated (r=0.94) with the total yield of biomass, and the two
vary only through changes in the proportion of leaf in twigs, and the proportion of twig in
total biomass (Murtagh 1996). As explained earlier, twigs consist of fine stems and leaves,
and contain virtually all the leaves on a tree. Working with twigs that were not subject to
leaf shedding, the proportion of leaf in twig on a dry weight basis was usually between 0.60
and 0.73, and cool conditions appeared to restrict the growth of leaves more than fine stems,
giving a lower ratio (Murtagh 1996). In the same experiment, the proportion of twig in total
biomass decreased from 0.69 to 0.43 as trees increased in size and the main stems occupied
an increasing proportion of the total biomass.
Environmental Effects
Tea trees grow best at high temperatures. In a controlled environment experiment, Curtis
(1996) measured a near linear increase in biomass weight between day/night temperatures
of 15/10°C and 35/30°C. The leaf emergence rate also increased with temperature, but
the rate of increase began to slow at the higher temperatures. The emergence rate was 0.1/
d at 15/10°C, 1.9/d at 30/25°C, and 2.1/d at 35/30°C. In a field calibration of the temperature
response, Murtagh (1996) found that a one degree increase in temperature above a threshold
for growth of 16°C, increased the biomass yield by 105 g/plant/yr. As discussed later
however, the temperature response was greater at some stages of growth than others.
When tea trees are grown at the higher temperatures quoted above, the young leaves are
limp, or nearly so, depending on the evaporative demand for moisture. Richards, quoted
by McKey (1979) has suggested that in such leaves expansion precedes differentiation. If
so the early differentiation and filling of oil glands, discussed above, may be delayed in
high temperature growth.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

G.J.MURTAGH
126
The effect of water stress on biomass production depends heavily on the availability of
subsoil moisture. When tea tree was grown on a site with a permanently damp subsoil at
about 600mm depth, the response to irrigation was largely restricted to one growth stage
(Murtagh 1992). The situation is different when the subsoil dries out. While severe water
stress may not kill trees, it will cause extensive defoliation and yield loss. Hence irrigation
is essential on such soils (Drinnan 1997).
Murtagh’s (1992) experiment measured the response to irrigation during the typically
dry spring-early summer period on the north coast of NSW. The period can be divided
into a pre-flush stage that extends until early October, flushing until early December, and
a post-flush stage. The pre-flush stage was characterised by low soil temperatures and a
period when the dominant resistance to water flow through a plant was in the liquid
rather than the vapour phase (Figure 5). This differed from the more usual situation where
the dominant resistance is in the vapour phase and reflects stomatal movements. When
the mean air temperature was less than about 19°C, the liquid-flow resistance was dominant
and was not altered by irrigation. Consequently the resistance probably arose within the
plant rather than in the soil. Many factors can increase the liquid-flow resistance within a
plant including temperature effects on root hydraulic resistance (Jones et al. 1985),
cavitation or plugging within the xylem pathway (Zimmermann and Milburn 1982), and
the effectiveness of mycorrhizas in water absorption (Jones et al. 1985). Regardless of
the mechanism, the effect was temperature driven but exhibited as a water stress. The
results in Figure 5 were measured on mature shoots, whereas all fresh shoots had a low
Figure 5 The ratio of the resistance to water flow in the liquid to the vapour phase at different
temperatures on irrigated ( ) and rain-watered (
•) treatments. All readings were taken at least 3.5
hours after sunrise
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

BIOMASS AND OIL PRODUCTION
127
ratio. No units are given for the ratio of the resistances in the liquid and vapour phases as
two phases are physically dissimilar (Cowan and Milthorpe 1967), and the ratio was used
as an index only.
The growth rates were related to the water potential on the various treatments (Figure 6).
During the pre-flush stage, both shoot water potentials and growth rates were low, and the
absolute response to irrigation was only moderate. Growth rates were much higher, even on
dry soils, during the flushing stage but the response to irrigation remained moderate. In
contrast there was a marked response to irrigation and increases in the water potential in the
post-flush stage. Regardless of the stage, wetting the soil increased the shoot water potential
by about 0.5MPa.
Two factors are particularly important in the interpretation of these results. The
experiment was done at a site with a permanently damp or wet subsoil, and the tea tree
stand had a strong spring flush that typically follows winter frosts. Under these
conditions, the only appreciable response to irrigation can be expected during the post-
flush phase. In the Lismore district where the experiment was done, the post-flush
period just precedes the summer rains and irrigation responses are small over a full
regrowth cycle.
Murtagh (1996) used a growth model to describe the effect of temperature and water
stress on the field growth of tea tree. The temperature effects are described above. Water
stress began when the top soil had dried to less than 69% of the total available water content,
and declined in a linear manner with further drying of the soil.
Other Effects
Tea trees are harvested by cutting the main stem near ground level, leaving a bare
stump that will produce the coppice regrowth. However it takes time to produce a new
canopy and during the first 3 months after harvest, the growth rate was 46% of that
Figure 6 The relation between the leaf growth rate and shoot water potential (
ψ) during three growth
stages. The results were collected over two years on irrigated ( ) and rain-watered (
•,ο) treatments,
with closed and open symbols indicating predominantly wet or dry topsoil respectively
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.

G.J.MURTAGH
128
during months 4–6, the most efficient stage (Murtagh 1996). After 6 months, new shoots
were less vigorous than before and the growth rate declined to 71% of the stage two
rate. The relative rates were calculated with all other conditions remaining constant.
The best yields of biomass were obtained by timing harvests so that the second and
third regrowth stages coincided with the best environmental conditions for growth. The
analysis of the effect of water stress on yield provided a good example of the gains
from following this approach. Across all harvest times, water stress reduced biomass
yields by an average of 24%. However, an October harvest that matched the first stage
of regrowth to the driest period, had only a 7% reduction in yield due to water stress.
This result reflected in part the assumption, supported by the functional analyses, that
there is no water stress soon after harvesting because of the large root to shoot ratio
(Blake and Tschaplinski 1986).
Melaleuca species are reputed to be very tolerant of water logging. Gomes and Kozlowski
(1980) found no effect of 30 days flooding in stagnant water on the growth of M.
quinquenervia, but longer periods did reduce growth. Bolton and Greenway (1996) obtained
good growth from M. alternifolia growing in 100–150mm deep, flowing sewage effluent
over 20 months, but it is unlikely if the same tolerance would be present in stagnant water
with a higher oxygen tension. Colton and Murtagh (1990) noted that growth was depressed
on waterlogged soil.
OIL YIELD
Because the two are not closely related, the combined effect of oil concentration and
leaf yield gives a wide range in potential oil yield. Colton and Murtagh (1990) indicated
that the oil yield could range from a low of 43kg/ha/yr to a high of 392kg/ha/yr, with a
yield of 150–200kg/ha/yr representing a realistic target for most plantations in northern
NSW.
Since these projections were recorded in 1990, there has been no confirmed advance
in the potential oil concentration on a plantation scale, but the situation could change in
the near future. Doran et al. (1996) recorded a 60% increase in oil concentration in
plants grown from seed from a selected provenance, over the concentration in plants
from selected lines used in commercial nurseries. Williams (1995) has developed clones
that produced more than twice the oil yield of unselected trees. Efforts are proceeding
to confirm that these gains can be achieved in commercial plantations, but at this stage
it would be premature to use either set of work to adjust the projected oil yields. Apart
from the use of genetic improvement to increase the oil concentration, it might be
possible to use preharvest treatments to increase the concentration, but little work has
been done in this regard.
Some progress has been made since 1990 towards producing higher leaf yields by fine-
tuning the agronomic procedures in growing a crop (Murtagh 1998). The expected yields in
Table 3
 were obtained by increasing the previous estimates of leaf yield (Colton and Murtagh
1990) by 5%, and using the leaf: twig and twig: biomass ratios in Murtagh (1996), and a
mean dry matter content of 40% to calculate the other plant yields. In addition to the above
gain, more growers are producing crops in the higher yielding categories, so the industry
average has increased by more than 5%. A realistic yield target would be 170–220kg oil/ha.
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint,

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