Medicinal and Aromatic Plants—Industrial Profiles

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who, in one year, measured a 62% decline between August and September followed by a
complete recovery in October.
Another experiment reported by Murtagh and Baker (1994) sampled plants of different
regrowth ages on the same day. The regrowth was aged between 145 and 354 days at one
site, and 82 and 292 days at another. The longest regrowth period included autumn—winter
when concentrations usually decline but the concentration per unit weight varied more with
the specific leaf area rather than the regrowth age. The specific leaf area is a measure of the
area per unit leaf weight. Young leaves tend to have a high specific leaf area because they
are thin and have less secondary thickening of cell walls. Thus the area, and perhaps the
number of oil glands, is greater per unit weight with young leaves leading to a higher oil
concentration per unit weight. When this effect was removed by expressing the oil
concentration per unit leaf area, there was little difference in oil concentration between
regrowth ages at one site, but it was more than double in the older regrowth at the other site.
These results illustrate the difficulty of finding a suitable expression of oil concentration
when contrasting leaves with very different specific leaf areas, as occurs between young
and medium to old leaves.
Figure 1 Seasonal changes in the oil concentration in (A) the humid subtropical environment of northern
NSW, and (B) the dry tropical environment of northern Queensland
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.

Daily Variation
Effects due to the leaf age distribution and specific leaf area can be removed from a
comparison by sampling over a short period when they would not change. When this was
done by sampling at about the same time each day over a sequence, the oil concentration
has been shown to vary. In one sequence over 8 days in November, the concentration halved
over 2 days and completely recovered by the next (Murtagh and Etherington 1990). There
was also a smaller 21% decline, followed by recovery, later in the sequence. These changes
were statistically significant and occurred on days that followed the warmest nights (minimum
temperatures of 17.7 and 15.9°C) during the sequence (Murtagh 1988). Subsequent sampling
during summer at daily intervals on 3 sites also showed significant changes in oil
concentration (Murtagh and Etherington 1990), but the changes were not related to
temperature (Etherington 1989).
The inverse correlation between night temperature and oil concentration prompted the
notion that the concentration declined because warmer nights increased the respiratory load
and the oil supplied at least some of the substrate for the process. Monoterpenes are the
major constituents of tea tree oil and are thought to be sometimes available for catabolism
(Croteau 1988). Warm nights reduced the oil concentration in peppermint (Mentha piperita),
especially during the flower initiation stage (Loomis and Croteau 1973). Curtis (1996) also
obtained evidence that a warm night can decrease the oil concentration in tea tree. That
result is discussed under the Environmental Effects heading.
Diurnal Variation
Studies that examined the diurnal pattern in oil concentration of tea tree have generally
found no significant changes. Etherington (1989) in three sequences found no variation.
Murtagh and Baker (1994) investigated 14 sequences of which three showed significant
variation. List et al. (1995) and Curtis (1996) examined one sequence each and found no
variation. The study by List et al. (1995) was done with potted plants in a polyhouse over 48
hours. All other studies were done in the field and a sequence occupied a day.
Another set of diurnal measurements, not included in the above, suggested that diurnal
fluctuations were related to the water vapour pressure deficit (VPD) of the atmosphere
(Murtagh 1991b). The samples were taken over three consecutive days from two watering
treatments; irrigated and rain watered. The soil on the second treatment was dry when
sampled. The tea trees were about 1.5m tall, and separate samples were taken from the
upper 40cm of the canopy (upper strata), and below 40cm (lower strata). The oil
concentration in the upper strata was significantly higher than the lower strata, and it
declined significantly within each of the first two days (
Figure 2
). There was a trend, not
always significant, for the concentration in the upper strata to be higher on the rain-
watered than the irrigated treatment, but the reverse applied in the lower strata. When
data from both strata were pooled, there was virtually no difference between the two
watering treatments. There was no change in the percent composition of the major
constituents in the oil over the three days.
The weather conditions differed between the three days. Day one was hot and dry and
the VPD continued to increase until sampling ceased. Day two was similar in the morning,
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.

but a sea breeze arrived at noon and lowered the VPD. On this day, the decline in the oil
concentration was arrested even before the instruments detected the change in the weather.
Day three was overcast with a low VPD throughout. The relation between the oil
concentration in the upper strata and VPD was summarised by two intersecting straight
lines, that showed a significant decline in the concentration once a threshold VPD was
exceeded (
Figure 3
). Separate relations were fitted to the irrigated and rain-watered
The threshold VPD were 1.5kPa on the irrigated treatment, and 1.0kPa if rainwatered.
Whereas the threshold occurred at a lower VPD on the rain-watered treatment, the decline
was greater on the irrigated treatment and equalled 22% at the highest VPD that was
measured. The slower rate of decline on the rain-watered treatment suggested that these
plants had acclimated to dry conditions.
However subsequent data obtained by Murtagh and Baker (1994) indicated the VPD
effect was not the sole factor involved in diurnal fluctuations in oil concentration. In nine of
the 14 sequences mentioned above, the VPD exceeded 2kPa at sometime during the day,
but only one of the nine sequences showed a significant decline in concentration. It is
relevant that the sequence with the decline had the highest oil concentration of the nine
sequences, starting the day at 55mg/g. The declines discussed earlier occurred at higher
concentrations, suggesting that diurnal fluctuation is more likely at high concentrations.
One sequence in the 14 showed a significant increase of 26% in the oil concentration during
a day that was warm and particularly humid, with the VPD being less than 0.9kPa for most
of the day.
Figure 2 Diurnal variation in oil concentration over three consecutive days, in the upper ( ) and
lower ( ) strata of the canopy, on irrigated (solid symbol) and rain-watered (open symbols) treatments.
The water vapour pressure deficit (VPD) is also shown
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.

Post-harvest Oil Concentration
Two studies have shown that the oil concentration remains constant for a considerable period
after harvest in treatments that are handled the same whilst fresh. Murtagh and Curtis (1991)
exposed samples taken from a common batch of twig material to a range of drying treatments
that were designed to provide varying rates of drying, respiration, and opportunities for
volatilisation. They found no effect on the oil concentration over 13 days in one experiment,
and seven days in another. Twig samples were distilled, and although not presented in their
paper, repeated sampling showed that the proportion of leaf in the twig sample was constant
throughout each experiment, thus eliminating variation in the leaf to twig ratio as a possible
source of error.
Whish and Williams (1996) also tested for post-harvest losses of oil. They distilled leaf
samples, and when the leaf was stripped from the fine stems whilst fresh, there was no
change in the oil concentration between distillations done immediately or 20 days later.
However, when the twigs were air dried before the leaf was removed, the oil concentration
was 28% greater. They suggested that oil movement from the stem gave the higher
concentration in the latter result, but an alternative explanation is that the striping of green
leaves from stems caused some oil loss. As discussed later, detaching branches or rough
handling can increase volatilisation losses in other species. Zrira and Benjilali (1991) observed
a 62% increase in oil concentration of Eucalyptus camaldulensis after shade drying, but it is
not certain if the results were corrected for the varying water contents in the leaves. This
issue must be clarified because if oil is lost when leaves are stripped while fresh, there is a
Figure 3 The pattern of change in oil concentration in the upper strata shown in 
Figure 2
 with increasing
vapour pressure deficit (VPD) on irrigated (solid symbol) and rain-watered (open symbols) treatments
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.

strong argument to either distill twigs and adjust the concentration to a leaf basis by
separately determining the leaf to twig ratio, or air dry the twigs before stripping leaves.
Oil losses after harvest vary between species, and those with a high water content tend to
lose more (Guenther 1948). Fresh tea tree leaves are relatively dry with 26–47% dry matter
(Murtagh and Smith 1996), and this combined with the subepidermal position of the oil
glands would tend to reduce the loss of tea tree oil after harvest (Flück 1963; Murtagh and
Curtis 1991). However, the relatively thin epidermal cap cell above oil glands could offset
the positional protection in both Eucalyptus (Welch 1920) and Melaleuca species (List et
al. 1995).
Factors Related to Oil Concentration
Environmental Effects and Water Content
Curtis (1996) studied the effect of temperature on the oil concentration in plants. When tea
trees were grown in controlled environment chambers, the oil concentration increased from
13mg/g at 15°/10°C (day/night temperatures) to 32mg/g at 30°/25°C; a rate of increase of
1.27mg/g/°C. The concentration was less at 35°/30°C. In a field study, Murtagh and Smith
(1996) estimated that an increase in the mean temperature over the 3 months preceding
harvest increased the oil concentration at a rate of 1.02mg/g/°C. Although the trees carried
a mixture of leaves of 0–12 months age, the temperature over the final 3 months was a
better predictor of the temperature effect than the temperature over the complete regrowth
When Curtis (1996) measured the oil concentration in leaves under a range of
environmental conditions in the field, the concentration was principally related to leaf age
and the minimum temperature during the morning preceding harvest. In 100 day old leaves,
the predicted oil concentration was 50mg/g with a 10°C minimum, 46mg/g with 15°C, and
42mg/g with 20°C. The temperature effect was absent at 300 days when the predicted
concentration was 35mg/g. Curtis (1996) also found that the proportion of terpinen-4-ol in
oil in leaves aged more than 100 days increased from 32% when the daily mean air
temperature was 10°C, to 45% at 25°C.
Several studies have noted a positive correlation between oil concentration and water
content in fresh tea tree leaves, and this was interpreted as an indication of the effect of
water supply on oil concentration (Murtagh 1988). However, subsequent tests showed
that irrigation increased the water content without a corresponding change in oil
concentration (Murtagh 1992). Consequently, the effect of water stress on oil concentration
is an open issue at this time. Gershenzon et al. (1978) noted a negative relation between
the monoterpene concentration in field populations of Satureja douglasii and moisture
stress. Many species of herbs and shrubs, but not trees, respond to water stress by slowing
growth while continuing to produce secondary metabolites, leading to higher
concentrations (Gershenzon 1984).
The relation between the concentration of oil (dependent variable) in growing tissue and
water (independent variable) is difficult to interpret for two reasons. The water concentration
is included in the dependent variable as it is used to adjust the oil concentration to a dry
weight basis, and also as the independent variable in the relation. This commonality to both
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.

variables introduces autocorrelation and the possibility of creating a false correlation. It can
be avoided by using the direct measurement of concentration per unit wet weight, but if so
the results will be confounded by changes in the water concentration between samples.
Also the positive correlation between oil and water content of leaves will dampen the true
variation in oil concentration if expressed on a wet weight basis (Murtagh and Smith 1996).
This is a problem for commercial growers who typically obtain a wet weight of their biomass
and use this to calculate the oil concentration.
The second problem with interpretation relates to the difference between water content
and water availability in leaf tissue (Waring and Schlesinger 1985). Factors such as changes
in cell wall elasticity, membrane permeability, and the concentration of solutes in cells
can all counter the effect of changing water concentration. Another factor could be the
ratio of apoplastic to symplastic water in leaves. E. globulus has a relatively high proportion
of cell wall water that is associated with the relatively high proportion (41%) of dry
matter in fresh leaves (Gaff and Carr 1961). Tea tree has a similar proportion of dry
matter, and it could be that the amount of apoplastic or symplastic water, rather than total
water content, is required to interpret the effect of water content on the synthesis and loss
of tea tree oil.
The effect of humidity is also not clear cut. In a controlled environment experiment
(Lowe and Murtagh 1995), the oil concentration was 38% lower in plants grown at a
continuous daytime VPD of 1.9kPa (40% relative humidity at 25°C) than at 0.6kPa (81%).
Also, as described earlier, a high VPD (drier air) was implicated in the diurnal decline in
concentration when the concentration was at a high level. Both experiments had a large
difference or change in the VPD, but from a regional perspective where the differences in
the mean VPD are less, there is no obvious effect of humidity on oil concentration. For
instance, the mean daytime VPD is almost always higher at Mareeba in northern
Queensland than at Lismore in northern NSW, but there is no obvious difference in the
general level of oil concentrations that can be attributed to humidity effects (
Figure 1
The mean daytime VPD in January is 1.43kPa at Mareeba, and 1.39kPa at Lismore.
Corresponding values in July are 0.98kPa and 0.80kPa. Both districts also have the same
seasonal trend in oil concentration but because of the high correlation between monthly
temperature and VPD, the possible effect of temperature or VPD cannot be distinguished.
Overall, it appears that large changes in humidity are required to affect the oil concentration,
and while such changes can occur over short periods they are not present in long term
Oil Gland Density
As detailed in 
Chapter 7
 the oil concentration is under strong genetic control, and this
control could be expressed through the density of oil glands in a leaf. List et al. (1995)
examined the number of oil glands in recently fully expanded leaves, and found slight
variation within a plant, but a large variation between plants from a common seed source. In
addition, the oil concentration was not correlated with oil gland density.
Hojmark-Andersen (1995) studied the frequency distribution of differently sized oil glands
throughout several tea trees. He found the distribution varied between trees, between the
north/east and south/west aspects, and between the two surfaces of a leaf but not within a
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.

surface. The oil concentration was correlated with the number of large oil glands, and the
total oil gland area on the abaxial, but not the adaxial surface of leaves. These results highlight
the need for thorough sampling to obtain a representative sample to measure either the
gland density or oil concentration in a complete tree. Dawkins (1915) also commented on
the difficulty of obtaining a representative sample when relating the oil content of several
tree species to the number and size of oil glands.
Plant Vigour and Juvenile Effect
One experiment showed a positive relation between the biomass yield at one harvest and
the oil concentration 12 months later at the next (Murtagh and Smith 1996). The effect
was most marked when the regrowth age at the first harvest was extended beyond 12
months giving a corresponding increase in biomass. The following harvest had some of
the highest oil concentrations, but also some of the lowest biomass yields in the 4 years of
the experiment (Murtagh 1996). The low biomass yields were thought to reflect
environmental conditions during the growing season, and not the increased yield at the
first harvest. These results, but not all, fit Gershenzon’s (1984) observation of a negative
correlation between growth and production of secondary compounds in many species.
They could reflect an adaptive response or a preferential allocation of resources when
stress reduced growth. However the result does not fit the average seasonal trend when
both growth rate and oil concentration are highest during summer. A likely explanation is
that the low growth/high oil concentration years are an occasional event that is masked in
average trends.
The same experiment differed from most field experiments in that there was a significant
variation in oil composition. In 31% of samples, there was a significant decline in the
proportion of 
γ-terpinene, α-terpinene and terpinolene, with the proportion of each being
strongly, negatively correlated with the proportion of p-cymene in the oil. This is consistent
with the oxidation of the former compounds to p-cymene (Guenther and Althausen 1949).
There was no relation between the formation of p-cymene and the oil concentration, and
it was only observed when 400 g samples were distilled in laboratory flasks and not when
5–10kg samples were distilled in a larger vessel. Thus the formation of p-cymene was
viewed as a distillation artifact, as did Koedam et al. (1979) with a number of other
The remaining 69% of samples had a normal proportion (0.3–1.7%) of p-cymene, but
compositional changes affected the other four major monoterpene olefins, 
γ-terpinene, α-
terpinene, terpinolene and 
α-pinene. The changes differed from those discussed above in
that they were related to the oil concentration. When the concentration exceeded a threshold
of 63–67mg/g, there was virtually no further increase in the weight per leaf of the compounds
Figure 4
). Whilst the effect was small for the minor compounds, it was statistically significant
for all four. At the lower, unaffected oil concentrations, these compounds represented 39%
of the total oil, and 55% of the samples in this analysis exceeded the threshold of 63–67mg/
g oil concentration.
The other constituents in the oil were not affected. While the decreasing proportion of
olefins must have caused a corresponding increase in the proportion of the other compounds,
the change was not statistically significant and the weight of the other compounds per leaf
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.

changed at a constant rate over the full range of oil concentrations. Murtagh and Smith
(1996) concluded that selective volatilisation, perhaps promoted by the pressure of oil in
cells or glands at high concentration (Dussourd and Denno 1991), was the most likely
explanation of observed changes in oil composition. Olefins have higher octanol/water
partition coefficients, and hence a higher permeance through cuticular membranes (Schönherr
and Riederer 1989), than the other major compounds in the oil.
In a series of experiments that were conducted in a controlled environment glasshouse,
the oil concentration was low across all treatments even when some treatments provided
very favourable growing conditions (Lowe et al. 1996). Most treatments had oil
concentrations of less than 35mg/g, and all were less than 50mg/g. By growing plants under
similar conditions in the open, it was shown that the low concentration was not related to
the environment in the glasshouse. Most experiments were done in 5 L pots, and while
there was little response to increases in pot volume, the concentration increased from 36mg/
g in the first, to 47mg/g in the third growth cycle, with each cycle growing over 5 months
(Lowe and Murtagh 1997). In other words, the generally low oil concentrations reflected a
juvenile plant effect. This result, together with the observation that fluctuating oil
concentrations are generally restricted to the higher concentrations, suggests that mature
plants should be used in experiments.
Controlling Processes
The oil concentration is a state variable that represents the balanced outcome from a number
of processes that can include biosynthesis, catabolism, transport, interconversion and
Figure 4 Changes in the weight per leaf of four olefins with an increasing oil concentration per leaf;
γT, γ-terpinene; αT, α-terpinene; TP, terpinolene; αP, α-pinene. The symbol marks the threshold
concentration at which the normal rate of accumulation changed. Taken from Murtagh and Smith
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