Journal and proceedings of the Royal Society of New South Wales

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mayrAt SOCIETY
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2003
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Journal € Proceedings of the Royal Society of New South Wales, Vol. 135, p. 57-71, 2003

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The 33rd Liversidge Lecture for the Royal Society of NSW

Dietary Chemicals and Brain Function

GRAHAM A. R. JOHNSTON

Abstract

Phytochemicals in our diet may play a vital role in maintaining the brain’s chemical balance
by influencing the function of receptors for the major inhibitory neurotransmitter GABA.

The flavonoids apigenin and epigallocatechin gallate, found in chamomile and green tea
respectively, influence the way in which GABA receptors are modulated by drugs such as di-
azepam. Resveratrol, a flavonoid-like polyphenol found in red wine, acts on a subtype of GABA
receptors consistent with its action as a cognitive enhancer. Bilobalide from Ginkgo biloba, a
herb used in cognitive therapy, also influences GABA receptors. a-Thujone, a terpenoid in
the alcoholic beverage Absinthe, acts in a similar manner to bilobalide on GABA receptors.
(+)-Borneol and other terpenoids from Valerian, a herb used to promote sleep, enhance the
effects of GABA. The effects of these phytochemicals on GABA receptors are consistent with
the overall actions of the beverages and herbal preparations that contain them, thus providing
a rational basis for the use of these beverages and herbal preparations.

These studies provide evidence that chemicals in our diet may influence brain function in
a positive way. The chemical nature of these substances may lead to the development of new

therapeutic agents for the treatment of anxiety, epilepsy, memory disorders and insomnia.

Keywords: Brain function, chemicals, diet, balance, dosage

THE BRAIN’S CHEMICAL
BALANCE

Two simple chemicals, glutamic acid and
GABA (Figure 1), are responsible for most of
the communication between nerve cells in the
brain. Indeed, at a very simple level, brain func-
tion may be thought of as a balance between
excitation mediated by glutamic acid and inhi-
bition mediated by GABA.

All nerve cells in the brain have receptors
for glutamic acid and GABA. Some 40% of
nerve cells release glutamic acid as an excita-
tory neurotransmitter, while a different 40% re-
lease GABA as an inhibitory neurotransmitter.
The balance between these two chemical trans-
mitters is vital to normal brain function. An
excess of excitation over inhibition results in an

EMTHSONTA

JUN 1 7 2005

CIBKANiL-

over excited brain (as in Figure 1) that can be
manifested as anxiety, agitation, exhilaration,
convulsions and death. On the other hand, an
excess of inhibition over excitation can be man-
ifested by depression, anaesthesia, coma and
death. ‘he particular manifestations of such
imbalances in the brain depend on what neu-
ronal circuitry is involved.

Ethanol is an example of a chemical that
acts on both sides of the brain’s chemical bal-
ance. The CNS depression that results from in-
gestion of ethanol is due to a reduction in excita-
tion mediated by glutamic acid acting on a sub-
type of glutamate receptors known as NMDA
receptors and to an enhancement. of inhibition
mediated by GABA acting on GABAaj recep-

tors.

58 JOHNSTON

HoN COOH

COOH

Glutamic acid

Exitation

GABA

Inhibition
1)

Figure 1: The brain’s chemical balance between excitation mediated by the major excitatory
neurotransmitter, glutamic acid, and inhibition mediated by the major inhibitory neurotransmitter,

GABA.
GABA RECEPTORS

GABA (whose name is derived from the old
chemical name, y-aminobutyric acid) acts on
three main types of receptor to influence brain
function. GABA,g and GABAc receptors are
fast acting receptors that belong to the group
of receptors called ligand-gated ion channels
(LGICs) (Chebib and Johnston, 2000). GABA
acts as the ligand gating these receptors to
open channels specific for chloride ions, allowing
these ions to flow rapidly into nerve cells mak-
ing them more negative and thus harder to ex-
cite. GABAg receptors act more slowly, induc-
ing metabolic changes in nerve cells and belong
to the group of receptors called G-protein cou-
pled receptors (GPCRs) (Bowery et al. 1997).

The study of GABA receptors has been rev-
olutionised by the introduction of recombinant
receptor technology whereby receptors cloned
from human brain are expressed in cells that do
not normally express such receptors (Barnard et
al. 1998). The recombinant receptors so formed
may be studied in relative isolation using stan-
dard electrophysiological methodology. Since
all GABA receptors are made up of protein sub-
units, recombinant receptors of known subunit
composition may be studied.

The most common way to study recombi-
nant GABA receptors is to express them in

oocytes from the South African frog, Xenopus
laevis following injection of either DNA or RNA
cloned from human brain and coding for par-
ticular GABA receptor protein subunits. These
oocytes have the necessary cellular machinery to
make the human proteins and assemble them on
the surface of the oocytes as functional GABA
receptors. ‘The oocytes are approximately one
millimetre in diameter and readily penetrated
by glass microelectrodes. Using 2-electrode
voltage clamp methodology, the effects of chem-
icals on the function of these GABA receptors
may be assessed in a convenient quantitative
manner. For example, using recombinant recep-
tor technology, the effects of anti-anxiety agents
such as diazepam (Valium) on GABA recep-
tors can be easily shown to be restricted to a
specific sub-group of GABAg receptors. The
technology is not restricted to the study of pure
chemicals — relatively crude mixtures of chem-
icals can be studied, for example to follow the
purification of chemicals acting on GABA re-
ceptors from extracts of herbal products.

FLAVONOIDS AND TERPENOIDS

Flavonoids are polyphenolic chemicals widely
distributed in the plant kingdom particularly
in flowering plants. Flavonoids are responsi-
ble for many of the brilliant colours of fruits

DIETARY CHEMICALS AND BRAIN FUNCTION 59

and vegetables and are important constituents
of red wine, green tea and many herbal prepa-
rations (Aherne and O’Brien, 2002). Chemi-
cally, flavonoids are C15 compounds based on
the chromane ring structure. Flavonoids have
been studied extensively as anti-oxidants and
oestrogens (Collins-Burow et al. 2000). Many
of them show anti-cancer and anti-viral proper-
ties (Le Marchand, 2002).

There is an extensive literature on the effects
of flavonoids on GABA receptors (for a recent
review see Marder and Paladini (2002), dating
from the discovery of some plant derived isofla-
vans in bovine urine that inhibited the bind-
ing of diazepam to brain membranes (Luk et
al. 1983). In the present context of actions on
GABA receptors, the following flavonoids are
of interest: the flavone apigenin; the isoflavone
genistein; the flavanone naringenin; and the fla-

OH

OH O
Apigenin, a flavone found in

chamomile tea and related
beverages.

OH O

Naringenin, a flavone found in

grapefruit.

Figure 2: Some representative flavonoids

van, epigallocatechin gallate (Figure 2).

Terpenoids are also widespread in plants, es-
pecially in what are known as essential oils that
can be extracted from plants and have a wide
range of uses from perfume constituents to paint
thinners. Terpenoids are oxygenated products
formally derived from C5 isoprene units and are
classified by the number of C5 units in their
structure. ‘Thus monoterpenoids have 2xC5
units, sesquiterpenoids 3xC5 units, diterpenoids
4xC5 units and triterpenoids 6xC5 units. In
the present context of actions on GABA recep-
tors, the following terpenoids are of interest:
a-thujone, (+)-borneol, bilobalide and picrotox-
inin (Figure 3). Picrotoxinin is widely used ex-
perimentally as a non-competitive antagonist of
GABAjs and GABAcg receptors, however, its
convulsant action restricts its therapeutic use
(Chebib and Johnston, 2000).

OH
Genistein, an isoflavone found

in soy products, including tofu.

OH

OH
OH

(-)-Epigallocatechin Gallate,
a flavan found in green tea.

60

Hz. Es
_CHs

a-Thujone, a monoterpene
from Artemisia absinthium

JOHNSTON

H3C CH

CH3

OH

(+)-Borneol, a monoterpene
from Valerian officinalis

Bilobalide, a sesquiterpenoid
from Ginkgo biloba

Picrotoxinin, a sesquiterpenoid from
Anamirta cocculus

Figure 3: Some terpenoids that influence GABA receptors

DIETARY CHEMICALS AND BRAIN FUNCTION 61

APIGENIN FROM CHAMOMILE
TEA

The lead compound for our investigations was
apigenin (Figure 2), a flavonoid with a known
anti-anxiety action found in chamomile tea.
Chamomile tea is used widely to treat anxi-
ety and insomnia. Current therapeutic drugs
used for the treatment of anxiety and insom-
nia such as the benzodiazepines Valium and
Serepax act at GABAg receptors in the brain,
increasing chloride flow into neurones, result-
ing in decreased neural activity. There were
divergent reports of the effects of apigenin on
GABAag receptors. Viola et al. (1995) con-
cluded that apigenin is a benzodiazepine ago-
nist, like diazepam. However, Avallone et al.
(2000) concluded that apigenin was a benzodi-
azepine inverse agonist (the exact opposite of
diazepam).

Viola et al. (1995) based their conclusion
that apigenin is a benzodiazepine agonist on
the ability of apigenin to displace the bind-
ing of radiolabelled benzodiazepines from rat
brain membranes, coupled with benzodiazepine-
like effects in a rodent model of anxiety. How-
ever, binding studies do not reliably distinguish
between agonists, antagonists and inverse ag-
onists. Indeed, on the basis of similar bind-
ing studies, Dekermendjian et al. (1999) con-
cluded that apigenin was a benzodiazepine an-
tagonist (that is, it binds to the benzodiazepine
site, blocking the binding of benzodiazepine ag-
onists and inverse agonists, without having any
effect on GABA responses itself). Avallone et
al. (2000) used electrophysiological recordings
from rat neurones. This allowed a more di-
rect investigation of the activity of apigenin and
showed that apigenin inhibited currents due to
GABA, an effect which was blocked by the ben-
zodiazepine antagonist, flumazenil. This fits
the profile of a benzodiazepine inverse agonist.
However, Avallone et al. (2000) did find some
behavioural effects of apigenin which could be
consistent with an action as a benzodiazepine
agonist.

As part of her PhD studies, Erica Camp-

bell in our research group investigated the ac-
tion of apigenin on recombinant GABA recep-
tors. She used electrophysiological recordings
from Xenopus laevis oocytes injected with re-
combinant human RNA for the most common
subtype of GABAag receptor (a@)(272,) in the
brain. The actions of GABA at these receptors
are enhanced by a variety of modulators includ-
ing barbiturates, benzodiazepines, ethanol, and
neuroactive steroids.

She showed the action of apigenin on the
GABAag receptor is more complex than sug-
gested by earlier studies. The effects of api-
genin were biphasic dependent on the dose used.
Moderate doses of apigenin inhibited the ac-
tions of GABA, diazepam and the steroid al-
lopregnanalone, whereas low apigenin concen-
trations enhanced the effects of diazepam only
(Figure 4). These effects are unlikely to be due
to a simple action at the benzodiazepine site,
suggesting a new site on the GABAag receptor.

While the inhibitory actions of apigenin at
moderate doses were consistent with it acting
as a benzodiazepine inverse agonist, the abil-
ity of apigenin to enhance the enhancing action
of diazepam was novel. At low doses, apigenin
had no direct effect on the action of GABA on
these recombinant receptors. The presence of
diazepam was necessary in order to observe the
enhancing effects of apigenin. Thus apigenin
appeared to be modulating the action of a mod-
ulator, an action not previously described in
the pharmacological literature. Apigenin might
be described as a second order modulator that
influences the modulatory action of diazepam
as a first order modulator on the activation of
GABAag receptors.

The second order modulation of GABA,
receptors by apigenin requires the presence of
GABA and a first order modulator acting at
a benzodiazepine site. The sedative and anx-
iolytic actions of apigenin observed in rodents
(Avallone et al. 2000, Viola et al. 1995) can be
interpreted on the basis of apigenin potentiating
the action of endogenous benzodiazepine-like
agents in the brain. Evidence for the physiolog-
ically relevant presence of such agents, termed

62 JOHNSTON

endozepines, has come from the discovery of a
mutant GABAas receptor in childhood absence
epilepsy and febrile seizures that has diminished
sensitivity to benzodiazepines with little other
alteration in GABAg receptor function (Wal-
lace et al. 2001).

Genistein (Figure 2), an isoflavone found
in soy products, did not show the biphasic ef-
fects of apigenin. Genistein, a structural isomer

2
ess
5s 15
BOF
ag
aS
(eD)
eg I
S ga
ro 4
383
Sa
cure
Las 7
e335
Sp
cod
0
~0.5
0.01 0.1

of apigenin, showed only the GABA4g antago-
nist action of apigenin. In addition, a dihydro
derivative of apigenin, naringenin (Figure 2), a
flavanone found in grapefruit juice and other cit-
rus products, also showed only the GABA, an-
tagonist action of apigenin. Thus, the second
order modulatory action of apigenin is struc-
turally specific.

Enhancement

Inhibition

10 100

Apigen Concentration (uM)

Figure 4: Effects of apigenin on the enhancement by diazepam of the action of GABA on recom-

binant GABAg receptors (Campbell et al. 1999).

EPIGALLOCATECHIN GALLATE
FROM GREEN TEA

Epigallocatechin gallate (Figure 2, EGCG) is
the most abundant flavan in green tea (Camel-
lia sinensis). It is found in all teas made from
C’. sinensis but not in many other food products
(Arts et al. 2000b). Green tea is known to have
many beneficial effects, including prevention of
cancer, lowering of blood pressure and lipids,
and acting as an antioxidant. EGCG has been

shown to contribute to these effects and, in ad-
dition, has been shown to have neuroprotective
properties.

Erica Campbell investigated the actions
of EGCG on recombinant GABA receptors
(Campbell et al. 1999). She found that it shared
the same biphasic action of apigenin, enhancing
the action of diazepam at low concentrations
and inhibiting at higher concentrations. In both
the enhancement and inhibition phases, EGCG
was at least 10 times more potent than apigenin.

DIETARY CHEMICALS AND BRAIN FUNCTION 63

(+)-Catechin and (-)-epicatechin, the most
abundant flavans in nature, being found in
many foods (Arts et al. 2000a, Arts et al.
2000b), did not influence recombinant GABA
receptors, showing that the basic flavan ring
structure is not sufficient for either of the
actions of EGCG observed on recombinant
GABAag receptors.

The biphasic actions of apigenin and EGCG
emphasise the importance of dose in drug ac-
tion. Our experiments show that low doses of
apigenin and EGCG can enhance the activation
of GABA receptors under the right conditions
and thus could produce sedation and relief of
anxiety. On the other hand, higher doses have
the opposite effect and thus are likely to pro-
duce stimulation.

The second order modulatory action of api-
genin and EGCG might have therapeutic possi-
bilities. Low doses of these substances could
reduce the therapeutic dose of diazepam and
related benzodiazepines, while higher doses
might reduce the effectiveness of such benzo-

diazepines. The possibilities of interactions be-
tween benzodiazepine medication and the con-
sumption of chamomile and green tea need to be
considered, particularly as chamomile tea may
be used as a home remedy for those conditions
for which benzodiazepines are frequently pre-
scribed.

RESVERATROL FROM RED WINE

The relatively low incidence of coronary heart
diseases in France, despite intake of a high-
fat diet, — the “French Paradox” -— has been
attributed to the consumption of red wine
containing high levels of polyphenolic com-
pounds (Mojzisova and Kuchta 2001, Sun et al.
2002). Resveratrol (3,4’,5-trihydroxystilbene,

Figure 5) is one of the most interesting polyphe-
nolic compounds found in red wine. It has been
shown to have estrogenic (Turner et al. 1999)
and neuroprotective effects (Bastianetto et al.
2000).

OH

Resveratrol, a stilbene found
in red wine from Vitis vinifera

OH

Figure 5: Structure of Resveratrol

In view of the structural similarities be-
tween resveratrol and apigenin, we investigated
its effects on recombinant GABA receptors ex-
pressed in oocytes. To our surprise, resvera-
trol showed little action on GABAag receptors
but was a GABAc receptor antagonist (Camp-
bell and Johnston, 2003). Resveratrol non-

competitively inhibited the effects of GABA
(1uM) at GABAc receptors with an [C50 of
72 uM, while having no significant effect at
doses up to 100uM on the effects of GABA
(40 4M) at GABAg receptors. This is the first
report of a non-competitive antagonist show-
ing some selectivity for GABAc over GABAa

64 JOHNSTON

receptors, the widely used non-competitive an-
tagonist picrotoxinin being some 30 times more
potent at GABA,j than at GABAc receptors
(Chebib and Johnston, 2000).

We have a patent on the use of GABAg re-
ceptor antagonists to enhance cognitive activ-
ity and stimulate memory capacity (Johnston et
al. 1998). Thus it was interesting to note that
resveratrol has also been patented for the treat-
ment of mild cognitive impairment (Wurtman
and Lee, 2002) based on its ability to increase
the expression of soluble amyloid precursor pro-
tein.

Using resveratrol as a lead compound, we
are examining structural analogues to see if we
can develop more potent and selective com-
pounds acting on GABAc receptors. Recently
we discovered a range of very promising activ-
ities, including the ability to enhance GABAc
receptor activity, in a series of compounds syn-
thesized in the 1970s by David Collins and col-
leagues in Veterinary Physiology at ‘The Univer-
sity of Sydney as antiestrogenic and antifertility
agents (Collins et al. 1971).

There is great interest in drugs to treat
memory impairment in disorders such as
Alzheimer’s disease and schizophrenia. The use
of such “Smart Drugs” in healthy people to in-
crease their cognitive ability raises a variety of
ethical, legal and social issues (Rose 2002).

Resveratrol and related stilbenoids are
found in a variety of plants and herbs. Major di-
etary sources include grapes, wine, peanuts and
soy (Burns et al. 2002). These compounds are
also found in Itadori tea which has long been
used in Japan and China as a traditional rem-
edy for heart disease and stroke. For people
who do not wish to consume alcohol, Itadori
tea may be a substituent for red wine as a di-
etary source of resveratrol (Burns et al. 2002).
For those who prefer white wine to red, French
winemakers have created a Chardonnay, called
Paradoxe Blanc, that is enriched in polyphenols
and has been shown to be effective in reducing
oxidative stress in diabetic rats (Landrault et
al. 2003).

As noted above, ethanol itself enhances the

effectiveness of GABA acting on GABAg recep-
tors and there is evidence that moderate con-
sumption of alcoholic beverages is beneficial to
health. However, other substances in these bev-
erages, such as resveratrol, are likely to con-
tribute to the overall beneficial effects. Re-
cently it has been reported by Aoshima and col-
leagues (Hossain et al. 2002) that the fragrance
of whiskey is able to enhance the effectiveness
of GABA acting on GABAag receptors. Several
components of the fragrance showed this prop-
erty, the most potent being ethyl phenylpropi-
onate, which was shown to have an anticonvul-
sant action in mice on inhalation.

Enhancement of GABA action was also
found in extracts of other alcoholic drinks such
as wine, sake, brandy and sochu. Hossain
et al. (2002) noted “Although these fragrant
components are present in alcoholic drinks at
low concentrations (extremely small quantities
compared with ethanol), they may also modu-
late the mood or consciousness of the human
through the potentiation of the GABAag recep-
tor response”. Aoshima and colleagues have
shown that various perfume constituents and
aromatherapy agents potentiate GABAag recep-
tors (Aoshima and Hamamoto 1999, Aoshima
et al. 2001).

Clearly there are many interesting and inno-
vative ways to explore the possibilities of influ-
encing cognitive function.

BILOBALIDE FROM GINKGO
BILOBA

Extracts of Ginkgo biloba leaves are widely em-
ployed as herbal medicines to treat symptoms
associated with mild-to-moderate dementia, im-
pairment of other cognitive functions associated
with ageing and senility and related neurosen-
sory problems (Diamond et al. 2000). A study
has indicated that the cognition-enhancing ef-
fects of the Ginkgo leaf extracts may be partly
mediated by bilobalide via GABA receptors
(Sasaki et al. 1999b). Enhanced hippocampal
pyramidal neuronal excitability has been shown
to correlate with learning and memory (Power

DIETARY CHEMICALS AND BRAIN FUNCTION 65

et al. 1997). Bilobalide (Figure 3), a sesquiter-
penoid isolated from Ginkgo biloba leaves, has
been shown to enhance this excitability in rat
hippocampal slices, an action proposed to in-
volve blockade of GABAergic neurotransmis-
sion (Sasaki et al. 1999b).

In collaboration with Sasaki and colleagues,
Shelly Huang and Rujee Duke in our research
group have shown that bilobalide is a potent an-
tagonist of the action of GABA on recombinant
GABA,g and GABAc receptors (Huang et al.
2003). Bilobalide was only marginally less po-
tent than picrotoxinin in these actions but there
were subtle differences between the actions of
bilobalide and picrotoxinin. These findings
strongly support the proposal by Sasaki and col-
leagues (Sasaki et al. 1999b) that the observed
enhanced neuronal excitability in hippocampal
slices was due to its blockade of GABAergic neu-
rotransmission.

Bilobalide and picrotoxinin share common
structural features, including a hydrophilic cage
and lipophilic side chain. However, bilobalide
and picrotoxinin have opposite actions upon
systemic administration to animals. Bilobalide
is an anticonvulsant (Sasaki et al. 1999a,b)
whereas picrotoxinin is a potent convulsant
(Jarboe et al. 1968). There are, however, only
minor differences in their activities at recom-
binant GABAag and GABAc receptors. Bilob-
alide has been shown to increase GABA lev-
els in the hippocampus and cerebral cortex of
mice (Sasaki et al. 1999a). This increase may
override the GABA, antagonist action of bilob-
alide that would be expected to produce convul-
sions and result in the overall anticonvulsant
action. Nonetheless, the induction of epilep-
tic seizures by Ginkgo extracts has been noted
in rare cases (Granger, 2001). The anticonvul-
sant/convulsant actions of bilobalide need fur-
ther investigation and may provide vital clues as
to the safe use of Ginkgo extracts in the treat-
ment of mild cognitive deficits.

Ginkgo leaves were used traditionally in
Japan to protect books against harmful worms
and insects before the introduction of modern
insecticides. Like picrotoxinin, bilobalide is a

potent insecticide (Ahn et al. 1997), an action
likely to be due to blockade of insect GABA re-
ceptors.

THUJONE FROM ABSINTHE

Absinthe was the favoured drink of artists and
writers in Paris at the end of the 19th cen-
tury. ‘The emerald green liqueur made famous
by Van Gogh, Toulouse-Lautrec and their col-
leagues was banned in France and most other
countries by 1915 due to its ability to cause
convulsions, hallucinations and psychotic dis-
turbances.

The toxic component of absinthe has been
identified as the monoterpenoid a-thujone (Fig-
ure 3). It is also the active ingredient of worm-
wood oil and some other herbal medicines and
is reported to have antinociceptive, insecticidal,
and anthelmintic activity. Hold et al. (2000)
showed that a-thujone acted like picrotoxinin
as a GABAag receptor non-competitive antago-
nist. ‘They showed that a-thujone was rapidly
metabolised to less active metabolites and con-
cluded that “a-thujone in absinthe and herbal
medicines is a rapid-acting and readily detox-
ified modulator of the GABA-gated chloride
channel”.

Matthew Roper in our research group has
shown that a-thujone is a non-competitive in-
hibitor of the action of GABA on recombinant
a1 Goyer GABAag and p; GABAc receptors ex-
pressed in oocytes. Like picrotoxinin, a-thujone
was about 30 times more potent at GABA,
than at GABAc receptors. Furthermore, site-
directed mutagenesis studies showed that muta-
tions in the second membrane-spanning region
of the wildtype GABAc receptors influenced the
potency of a-thujone and picrotoxinin in a simi-
lar manner indicating that both convulsants in-
teract with the same amino acid residues on the
GABAcg receptor.

Many plant-derived essential oils, such as
wormwood, have been known for over a cen-
tury to have convulsant properties. Burkhard
et al. (1999) reported on case studies of plant-
related toxic seizures related to use of these oils

66 JOHNSTON

for therapeutic purposes. ‘They noted that “the
literature shows essential oils of 11 plants to be
powerful convulsants (eucalyptus, fennel, hys-
sop, pennyroyal, rosemary, sage, savin, tansy,
thuja, turpentine, and wormwood) due to their
content of highly reactive monoterpene ketones,
such as camphor, pinocamphone, thujone, cine-
ole, pulegone, sabinyl acetate, and fenchone.”
They went on to state “Nowadays the wide use
of these compounds in certain unconventional
medicines makes this severe complication again
possible” .

Absinthe is now available in a less potent
form that contains less than 10 parts per mil-
lion of a-thujone, whereas traditional absinthe
contained more than 250 parts per million.

BORNEOL FROM VALERIAN

Valerian (Valeriana officinalis) is a medicinal
plant used widely throughout the world. Ex-
tracts of the dried underground parts of the
plant are used to relieve anxiety, restlessness
and nervous sleep disorders. There is evidence
of its use by the ancient Greeks, Romans and
Chinese for healing purposes. Early herbalists
and physicians such as Hippocrates, Galen and
Culpeper noted the sedative and digestive prop-
erties of valerian, advocating its use as a mus-
cle relaxant, diuretic, expectorant and wound
healer (Plushner, 2000). Today there are over
400 commercially available products containing
valerian in Germany, more than 80 in the United
Kingdom and more than 30 available in Aus-
tralia (Houghton, 1999; Shohet et al. 2001).
Valerian extracts may be considered to be
a “herbal Valium”, given that they have a
benzodiazepine-like action reducing the latency
of sleep onset and increasing the depth of sleep
and the perception of well-being. These extracts
contain a large number of constituents, many of
which are thought to be active at GABA re-
ceptors. Compounds that have been identified
include acids (valerenic acid and isovalerenic
acid), ketones (valeranone), alcohols (valerenol,
maaliol), aldehydes (valerenal) and valepotri-
ates (valtrate, isovaltrate). Valerian extracts

also contain various flavonoids, alkaloids, tan-
nins and amino acids.

Renee Granger in our research group ob-
tained 2kg of the dried underground parts
of Valeriana officinalis from Newton’s Herbal
Pharmacy in Sydney. She extracted this
with hexane, ethyl acetate, methanol and
methanol:water (1:5), and fractionated the ex-
tracts using silica gel chromatography. ‘This
procedure produced more than 450 fractions,
which were assessed using thin layer chromatog-
raphy and functional studies on recombinant re-
ceptors many fractions influenced GABA action
on GABAag and GABAg receptors.

Dried valerian root normally contains 0.3-
0.8% volatile oil, including borneol, valerenic
acid, valeranone and kessy] glycol. These essen-
tial oil fractions proved very difficult to purify,
sO pure compounds were purchased and tested
on recombinant receptors. This produced a very
surprising result.

(+)-Borneol, the natural enantiomer found
in Valerian, produced a 12 fold enhancement
of the action of 104M GABA on recombinant
GABAag receptors under conditions whereby di-
azepam would give at best a 2 fold enhancement
(Figure 6; Granger et al. 2002). While relatively
high concentrations of (+)-borneol were needed
(EC50 400 uM), this degree of enhancement is
unprecedented.

Under these conditions, (-)-borneol pro-
duced a 4 fold enhancement (EC50 450 uM),
isoborneol a 7 fold enhancement (EC50
680uM), while the structurally related
monoterpenes camphor, bornyl acetate and p-
cymene each produced an approximately 3x
potentiation (with EC50’s around 300 uM).

While many general anaesthetics, barbitu-
rates and benzodiazepine are known to pro-
duce up to 2 fold enhancement of the re-
sponse of GABAag receptors to GABA (Belelli
et al. 1999b), the general anaesthetic etomidate
is known to cause much larger enhancements,
particularly at mutated GABA receptors, e.g.
etomidate produces a 10 fold enhancement of
GABA responses at GABAc receptors where
the wild type isoleucine residue at position 307

DIETARY CHEMICALS AND BRAIN FUNCTION 67

had been mutated to a serine (Belelli et al.
1999a).

(+)-Borneol represents an intriguing struc-
tural lead for the development of a new class of
therapeutic agents acting on GABA receptors.
Purified (+)-borneol has been used for medicinal
purposes in China and Japan (Hattori, 2000).

I)
oO (+)-Borneol
A (-)-Borneol
2
5 10
a
ox
eee faa)
oO <
5 0
2s
5 2
Bes
tH
A, 2
I see 3 ee DO oe Se ee eee
la a Ele tS =
0

Borneol is a common constituent of the essential
oil component of many plants and thus a com-
ponent of many herbal preparations. On the
basis of our studies, (+)-borneol would be ex-
pected to have antianxiety, anticonvulsant and
sedative properties.

"
na
a

Log Dose (uM)

Figure 6: Dose-response curve for the potentiation of the response to 10 uM GABA by (+)- and
(-)-borneol at recombinant GABAg receptors (Granger et al. 2002).

NATURAL VERSUS SYNTHETIC

Natural products derived from plants provided
the first medicines. These were complex mix-
tures of chemicals. ‘The active principles in
these mixtures were isolated and developed as
single chemical entities to use as drugs and
from which to develop new therapeutic agents.
The development of aspirin from the salicylates
found in Willow bark is a classic example of
this. Natural products continue to be an im-
portant source of new drugs. There are sophisti-
cated laboratories in many countries, including

Australia, using high throughput technology to
screen extracts of natural products for desired
biological activities.

Herbal preparations are by their nature mix-
tures of chemicals. It is a basic tenet of herbal
medicines that the whole is more than the sum
of the parts. The various chemicals in herbal
preparations are considered to act together in
a synergistic way to effect treatment of partic-
ular disorders. This is in direct contrast to the
“magic bullet” approach of single chemical en-
tity drugs designed to hit a particular target in a
highly selective manner. Both approaches have

68 JOHNSTON

their role in promoting health and well-being.

There is a widespread belief on the part of
the general public that natural substances are
inherently superior to synthetic substances with
regard to efficacy and safety in matters related
to human health. This question has been ad-
dressed recently by a task force of the Inter-
national Union of Pure and Applied Chemistry
(Topliss et al. 2002). A comparison of the char-
acteristics of natural and synthetic substances
used in a variety of therapeutic drugs, herbal
medicines, vitamins and nutrients shows a sim-
ilar range of favorable and unfavorable effects.
It was apparent that molecular structure and
dose determine the effect of chemicals on hu-
man health, not whether they are of natural or
synthetic origin.

Natural chemicals such as many flavonoids
have been consumed in our diet for countless
generations. This suggests that they would
be unlikely to have serious adverse effects se-
vere enough to prevent their use as therapeutic
agents. However, it is likely that the overall bal-
ance of flavonoids and related natural chemicals
in our diet is of vital importance, given the ex-
amples in this review of such chemicals having
opposing actions on GABA receptors. Recom-
binant receptor technology offers the means to
assess the overall effects of complex mixtures of
chemicals on the functioning of key receptors.
Such technology is expected to play an increas-
ingly important role in the quality control of
herbal preparations and “functional foods”.

Herbal preparations can have significant in-
teractions with therapeutic drugs, for example
by altering the metabolism of these drugs and
thus influencing their potency and duration of
action (Izzo and Ernst 2001). It is important
that health care professionals ask their patients
about their use of herbal products and consider
the possibility of herb-drug interactions. Food-
drug interactions are also important (Sorensen
2002) as many naturally occurring substances
influence the cytochrome p450 enzymes that
play such an important role in drug metabolism.
Grapefruit juice is a classic example. It con-
tains substances, including the flavonoid narin-

genin (Figure 2), that inhibit the p450 enzyme
CYP3A4 resulting in higher bioavailability of
drugs with a high firstpass metabolism (Fuhr
1998). While there may be a place for grape-
fruit juice as a drug-sparing agent, more re-
search is needed and drugs possibly influenced
by the consumption of grapefruit juice should
be appropriately labelled.

CONCLUSIONS

GABA receptors in our brain are susceptible to
a wide variety of chemicals consumed in the
diet. Our GABA receptors exist in a milieu
of substances that influences their function, of-
ten in opposing ways. ‘The balance between
the effects of these substances will determine
at any particular point in time how the recep-
tors respond to GABA, their natural neuro-
transmitter. This review has summarised the
effects of some substances found in four bever-
ages (chamomile and green tea, red wine, ab-
sinthe) and two herbal preparations (Gingko
and Valerian) that have significant actions on
recombinant GABA receptors consistent with
the overall actions of the beverages and herbal
preparations. The chemical nature of these sub-
stances may lead to the development of new
therapeutic agents for the treatment of anxi-
ety, epilepsy, memory disorders, and insomnia.
Does the concept of dietary substances influenc-
ing brain function indicate that we have entered
an era of neuraceuticals?

These studies provide a chemical basis for
some of the effects that these beverages and
herbal preparations have on brain function, and
may lead to rational improvements in their qual-
ity control and use, especially in combination
with other agents known to influence GABA re-
ceptors, such as alcohol, anaesthetics and ben-
zodiazepines.

ACKNOWLEDGEMENTS

It is a pleasure to gratefully acknowledge the
collaboration in these studies of Erica Camp-
bell, Mary Chebib, Rujee Duke, Renee Granger,

DIETARY CHEMICALS AND BRAIN FUNCTION 69

Belinda Hall, Jane Hanrahan, Shelly Huang,
Ken Mewett, Matthew Roper and Hue Tran.
Appreciation is also due to Gary Cutting,
George Uhl and Paul Whiting for the supply of
cloned GABA receptors and to the Australian
National Health and Medical Research Council
for financial support.

REFERENCES

Aherne, S.A. and O’Brien, N.M. 2002. Di-
etary flavonols: Chemistry, food content, and
metabolism. Nutrition, 18, 75-81.

Ahn, Y.J., Kwon, M., Park, H.M. and Han,
C.K. 1997. Potent insecticidal activity of
Ginkgo biloba derived trilactone terpenes
against Nilaparvata lugens. Phytochemicals
for pest control. American Chemical Society
Symposium Series, 658, 90-105.

Aoshima, H. and Hamamoto, K. 1999. Poten-
tiation of GABA(A) receptors expressed in
Xenopus oocytes by perfume and phytoncid.
Bioscience Biotechnology and Biochemistry,
63, 743-748.

Aoshima, H., Hossain, $.J., Hamamoto, K.,
Yokoyama, T., Yamada, M. and Shingai, R.
2001. Kinetic analyses of alcohol-induced po-
tentiation of the response of GABA(A) recep-
tors composed of alpha(1) and beta(1) sub-
units. Journal of Biochemistry, 130, 703-
709.

Arts, I.C., van de Putte, B. and Hollman, P.C.
2000a. Catechin contents of foods commonly
consumed in The Netherlands. 1. Fruits,
vegetables, staple foods, and processed foods.
Journal of Agricultural and Food Chemistry,
48, 1746-51.

Arts, I.C., van De Putte, B. and Hollman, P.C.
2000b. Catechin contents of foods commonly
consumed in The Netherlands. 2. Tea, wine,
fruit juices, and chocolate milk. Journal of
Agricultural and Food Chemistry, 48, 1752-7.

Avallone, R., Zanoli, P., Puia, G., Kleinschnitz,
M., Schreier, P. and Baraldi, M. 2000. Phar-
macological profile of apigenin, a flavonoid
isolated from Matricaria chamomilla. Bio-
chemical Pharmacology, 59, 1387-1394.

Barnard, E.A., Skolnick, P., Olsen, R.W.,
Mohler, H., Sieghart, W., Biggio, G.,
Braestrup, C., Bateson, A.N. and Langer,
S.Z. 1998. International Union of Pharmacol-
ogy - XV - Subtypes of y-aminobutyric acid,
receptors - classification on the basis of sub-
unit structure and receptor function. Phar-
macological Reviews, 50, 291-313.

Bastianetto, S., Zheng, W.H. and Quirion, R.
2000. Neuroprotective abilities of resveratrol
and other red wine constituents against nitric
oxide-related toxicity in cultured hippocam-
pal neurons. British Journal of Pharmacol-
Ogi, losis (11 (20,

Belelli, D., Pau, D., Cabras, G., Peters, J.A.
and Lambert, J.J. 1999a. A single amino
acid confers barbiturate sensitivity upon the
GABA p(1) receptor. British Journal of
Pharmacology, 127, 601-604.

Belelli, D., Pistis, M., Peters, J.A. and Lambert,
J.J. 1999b. The interaction of general anaes-
thetics and neurosteroids with GABA(A) and
glycine receptors. Neurochemistry Interna-
tional, 34, 447-452.

Bowery, N.G., Kerr, D.I.B. and Ong, J. (997.
GABA-B receptors - role and therapeutic im-
plications in CNS disorders. CNS Drugs, 7,
LO —1 75.

Burkhard, P.R., Burkhardt, K., Haenggeli, C.A.
and Landis, T. 1999. Plant-induced seizures:
reappearance of an old problem. Journal of
Neurology, 246, 667-670.

Burns, J., Yokota, T., Ashihara, H., Lean,
M.E.J. and Crozier, A. 2002. Plant foods and
herbal sources of resveratrol. Journal of Agri-
cultural and Food Chemistry, 50, 3337-3340.

Campbell E.L., Chebib M. and Johnston
G.A.R. 1999, The flavonoid epigallocatechin
gallate blocks steroid, benzodiazepine and
GABA actions at the GABA-A receptor more
potently than apigenin. Proceedings of the
Australasian Society of Clinical and Experi-
mental Pharmacology and Toxicology, 6, 109

Campbell, E.L. and Johnston G.A.R. 2003.
Resveratrol is a GABAcg receptor antagonist.
Proceedings of the Australian Neuroscience
Society, 14, P156.

70 JOHNSTON

Chebib, M. and Johnston, G.A.R. 2000.
GABA-activated ligand gated ion channels:
Medicinal chemistry and molecular biology.
Journal of Medicinal Chemistry, 43, 1427-
1447.

Collins, D.J., Hobbs, J.J. and Emmens, C.W.
1971. Antiestrogenic and antifertility com-
pounds. 4. 1,1,2-Triarylalkan-l-ols and
1,1,2-Triarylalk-1-enes containing basic ether
groups. Journal of Medicinal Chemistry, 14,
O52 Tq

Collins-Burow, B.M., Burow, M.E., Duong,
B.N. and McLachlan, J.A. 2000. Estro-
genic and antiestrogenic activities of flavonoid
phytochemicals through estrogen receptor
binding-dependent and -independent mech-
anisms. Nutrition and Cancer-An Interna-
tional Journal, 38, 229-244.

Dekermendjian, K., Kahnberg, P., Witt, M.R.,
Sterner, O., Nielsen, M. and Liljefors, T.
1999. Structure-activity relationships and
molecular modeling analysis of flavonoids
binding to the benzodiazepine site of the rat
brain GABA(A) receptor complex. Journal
of Medicinal Chemistry, 42, 4343-4350.

Diamond, B.J., Shiflett, S.C., Feiwel, N., Math-
eis, R.J.; Noskin,':O., Richards, J.A. and
Schoenberger, N.E. 2000. Ginkgo biloba ex-
tract: Mechanisms and clinical indications.
Archives of Physical Medicine and Rehabili-
tation, 81, 668-678.

Fuhr, U. 1998. Drug interactions with grape-
fruit juice. Extent, probable mechanism and
clinical relevance. Drug Safety, 18, 251-72.

Granger, A.S. 2001. Ginkgo biloba precipitat-
ing epileptic seizures. Age and Ageing, 30,
023020;

Granger R.E., Campbell E.L., Duke R.K. and
Johnston G.A.R., 2002. Valerian and GABA
receptors, Proceedings of the Australian
Health and Medical Research Congress, p.
2029.

Hattori, A. 2000. Camphor in the Edo era -
camphor and borneol for medicines. Yakushi-
gaku Zasshi. The Journal of Japanese His-
tory of Pharmacy, 35, 49-54.

Hold, K.M., Sirisoma, N.S., Ikeda, T., Nara-

hashi, T. and Casida, J.E. 2000. a-thujone
(the active component of absinthe): 4-
aminobutyric acid type A receptor modula-
tion and metabolic detoxification. Proceed-
angs of the National Academy of Sciences of
the United States of America, 97, 3826-3831.

Hossain, S.J., Aoshima, H., Keda, iavand
Kiso, Y. 2002. Potentiation of the ionotropic
GABA receptor response by whiskey fra-
grance. Journal of Agricultural and Food
Chemistry, 50, 6828-6834.

Houghton, P.J. 1999. The scientific basis for
the reputed activity of Valerian. Journal of
Pharmacy and Pharmacology, 51, 505-12.

Huang, S.H., Duke, R.K., Chebib, M., Sasaki,
K., Wada, K. and Johnston, G.A.R. 2003.
Bilobalide, a sesquiterpene trilactone from
Ginkgo biloba, is an antagonist at recombi-
nant a,Go72, GABAg receptors. European
Journal of Pharmacology, 464, 1-8.

Izzo, A.A. and Ernst, E. 2001. Interactions be-
tween herbal medicines and prescribed drugs:
a systematic review. Drugs, 61, 2163-75.

Jarboe, C.H., Porter, L.A.-and Buckler, R.T.
1968. Structural aspects of picrotoxinin ac-
tion. Journal of Medicinal Chemistry, 2, 729-
(ak

Johnston, G.A.R., Burden, P.M., Chebib, M.
and Mewett, K.N. 1998. Neurologically ac-
tive compounds. PCT International. Appli-
cation, WO 98/58939.

Landrault, N., Poucheret, P., Azay, J., Kros-
niak, M., Gasc, F., Jenin, C., Cros, G. and
Teissedre, P.L. 2003. Effect of polyphenols-
enriched Chardonnay white wine in diabetic
rats. Journal of Agricultural and Food Chem-
istry, 15, 311-318.

Le Marchand, L. 2002. Cancer preventive ef-
fects of flavonoids - a review. Bzomedicine
and Pharmacotherapy, 56, 296-301.

Luk, K.C., Stern, L., Weigele, M., O’Brien,
R.A. and Spirst, N. 1983. Isolation and iden-
tification of “diazepam-like” compounds in
bovine brain. Journal of Natural Products,
46, 852-861.

Marder, M. and Paladini, A.C. 2002. GABA-
A receptor ligands of flavonoid structure.

DIETARY CHEMICALS AND BRAIN FUNCTION 71

Current Topics in Medicinal Chemistry, 2,
853-867.

Mojzisova, G. and Kuchta, M. 2001. Dietary
flavonoids and risk of coronary heart disease.
Physiological Research, 50, 529-535.

Plushner, S.L. 2000. Valerian: Valeriana offic-
inalis. American Journal of Health System
Pharmacy, 57, 335.

Power, J.M., Thompson, L.T., Moyer, J.R. and
Didsterhoft, J.F. 1997. Enhanced synaptic
transmission in CA1 hippocampus after eye-
blink conditioning. Journal of Neurophysiol-
ogy, 78, 1184-1197.

Rose, S.P.R. 2002. “Smart Drugs”: do they
work? Are they ethical? Will they be legal?
Nature Reviews Neuroscience, 3, 975-979.

Sasaki, K., Hatta, S., Haga, M. and Ohshika,
H. 1999a. Effects of bilobalide on y-
aminobutyric acid levels and glutamic acid
decarboxylase in mouse brain. Furopean
Journal of Pharmacology, 367, 165-173.

Sasaki, K., Oota, I., Wada, K., Inomata, K.,
Ohshika, H. and Haga, M. 1999b. Ef-
fects of bilobalide, a sesquiterpene in Ginkgo
biloba leaves, on population spikes in rat hip-
pocampal slices. Comparative Biochemistry
and Physiology C: Comparative Pharmacol-
ogy and Toxicology, 124, 315-321.

Shohet, D., Wills, R.B. and Stuart, D.L. 2001.
Valepotriates and valerenic acids in commer-
cial preparations of valerian available in Aus-
tralia. Pharmazie, 56, 860-3.

Sorensen, J.M. 2002. Herb-drug, food-drug,
nutrient-drug, and drug-drug interactions:
mechanisms involved and their medical im-
plications. Journal of Alternative and Com-
plementary Medicine, 8, 293-308.

Sun, A.Y., Simonyi, A. and Sun, G.Y. 2002.
The “French paradox” and beyond: Neuro-
protective effects of polyphenols. Free Radi-
cal Biology and Medicine, 32, 314-318.

Topliss, J.G., Clark, A.M., Ernst, E., Hufford,
C.D., Johnston, G.A.R., Rimoldi, J.M. and

Weimann, B.J. 2002. Natural and synthetic
substances related to human health - (Inter-
national Union of Pure and Applied Chem-
istry Technical Report). Pure and Applied
Chemistry, 74, 1957-1985.

Turner, R.T., Evans, G.L., Zhang, M.Z.,
Maran, A. and Sibonga, J.D. 1999. Is resvera-
trol an estrogen agonist in growing rats? E’n-
docrinology, 140, 50-54.

Viola, H., Wasowski, C., Levi de Stein, M.,
Wolfman, C., Silvera, R., Medina, A.E. and
Paladini, A.C. 1995. Apigenin, a component
of Matricaria recutita flowers, is a central
benzodiazepine receptors-ligand with anxi-
olytic effects. Planta Medica, 61, 213-216.

Wallace, R.H., Marini, C., Petrou, S., Harkin,
L.A., Bowser, D.N., Panchal, R.G., Williams,
D.A., Sutherland, G.R., Mulley, J.C., Schef-
fer, I.E. and Berkovic, $.F. 2001. Mutant
GABA(A) receptor y 2-subunit in childhood
absence epilepsy and febrile seizures. Nature
Genetics, 28, 49-52.

Wurtman, R.K. and Lee, R.K.K. 2002. Com-
positions and methods for treatment of mild
cognitive impairment. PCT International.
Application, 2002, WO 02/28141 A2.

Professor Graham Johnston AM

The Adrien Albert Laboratory of Medicinal
Chemistry, Department of Pharmacology

The University of Sydney DO6, NSW 2006
Australia

Phone: 612 9351 6117, Fax: 612 9351 2891
e-mail: grahamj@Qmail.usyd.edu.au

This paper is based on the 33°? Liversidge
Lecture presented before the Royal Society of
New South Wales at the Frencham School, Mit-
tagong, 22nd November 2002.

(Manuscript received 29.1.2003)

Journal & Proceedings of the Royal Society of New South Wales, Vol. 135, p. 73-83, 2003

ISSN 0035-9173/03/020073-11 $4.00/1

lonic Combating Mechanisms and their Comparative

Effects on Seed Hardening under Simulated
Supra-Optimal Environmental Conditions

M. A. KADER

Abstract

Heat extremes and limited moisture are two of the most dominant environmental factors
impacting stand establishment of rainfed sorghum (Sorghum bicolor L. Moench). Hardening
is the process of exposing plants to gradual levels of stress and acclimation to foster better re-
sponse to post-treatment stress factors. Three experiments were carried out under phytotron
and germination cabinet conditions to test the effects of osmotic soaking of sorghum seeds
with sodium chloride (NaCl) on germination and growth under simulated heat stress. The
hypothesis was that the NaCl treatment forms an acclimation to stress by inducing hormonal
responses to ionic toxicity caused by salt. This acclimation would lead to a lowered degree of
response when the seed is exposed to future stress; namely, heat and/or drought. Independent
variables included NaCl concentration, treatment duration, storage and genotype, whereas ger-
mination and growth were dependent variables. Further experiments tested various methods
of achieving the initial acclimation “signaling” whilst reducing ionic toxicity through combat-
ing ions. Longer soaking treatment durations (2-3 days) and higher NaCl concentrations (16 g
NaCl 1~*) were detrimental to germination in comparison to lower concentrations (8 g NaCl
1~') and shorter durations (1 day). An interaction between concentration and duration of
treatment existed where high concentrations performed better at lower treatment durations
and vice versa. Combining 10 g NaCl 17! with 5g calcium sulphate 1~’ to combat ionic toxicity
produced a greater advancement of germination than NaCl alone. Drying duration of seeds
did not affect subsequent germination nor did storage for 10 days to 1 month. The effects of
osmotic conditioning are discussed and could have potential for improving sorghum success
rates in harsh arid environments.

Keywords: ions, acclimation, stress, sorghum, germination

INTRODUCTION

The fate of sorghum (Sorghum bicolor L.
Moench) seeds sown into dry or gradually dry-
ing seedbeds that hamper emergence is not well
known. Even though some seeds may germi-
nate and give rise to seedlings, the majority
will fail to emerge (Al-Mudaris and Jutzi 1998).
This clearly sets back stand establishment and
subsequent yield due to thermo and other
forms of dormancy (Silvertown 1999). Treating
seeds with osmotic solutions before sowing, also
termed “osmoconditioning”, has been shown to
improve germination and seedling emergence in
a range of species (Heydecker and Gibbins 1978,
Brocklehurst and Dearman 1984). The use of

sodium chloride (NaCl) as the osmotic agent
in such treatments has also been investigated
and shown to yield enhanced germination pat-
terns in sorghum (Al-Mudaris 1998). Both the
concentration of NaCl and treatment duration
seem to play major roles in the response exhib-
ited by seeds. However, little is known of the
role of both factors or of the effect of various
salt mixtures on germination and early seedling
growth of treated seeds. This is especially the
case when ionic toxicity is taken into account
as this is the single most important factor af-
fecting NaCl usage as a priming agent. Ionic
combating by way of adding extra elements to
the NaCl solution may aid in reducing the neg-

74

ative effects of Nat and Cl~ ions (Al-Mudaris
and Jutzi 1999).

Conditioning whole plants to stress by grad-
ual exposure to limited and increasing levels of
the particular stress factor has been found to
alleviate part of the stress at later stages due to
an early peaking of abscisic acid (ABA) produc-
tion and a better hormonal balance in favour
of Kinetin (Al-Mudaris 1998a). The concept
of conditioning seeds to heat stress by expos-
ing them to pre-germination salinity stress may
aid in “hardening” seeds and improving subse-
quent germination and seedling growth under
heat stress.

This paper investigates the effects of NaCl
concentration, treatment duration, salt mix-
tures and storage on germination and growth
parameters in four sorghum genotypes. It also
evaluates the effect of combining NaCl with
other salts in advancing the “hardening” ef-
fect to improve germinative performance under
drought and/or heat stress by way of ionic com-
bating.

MATERIALS AND METHODS

Effect of Salt Concentration,
Osmoconditioning Treatment
Duration and Drying

Seed lots of the sorghum variety IRAT 204 were
used in this set of experiments. Seed tests
revealed a 1000 seed weight of 34g, a mois-

Time (hh.mm) Temperature (°C)

24.00 — 05.00 15
06.00 — 08.00 Le
09.00 — 12:00 29
13.00 — 19.00 43
20.00 — 21.00 20
22.00 — 23.00 23

Relative Humidity (%)

65
62
50
38
D0
58

KADER

ture content of 15%, viability of 100% and ger-
minability of 98.0% following International Seed
Testing Association (ISTA) rules (ISTA 1993).

Previous work (Al-Mudaris and Jutzi 1998
and 1998a) showed the upper range of posi-
tive responses to NaCl to lie between 10 and
20g NaCl 1~!. Therefore, two concentrations
around this range were used. These were 8 and
16g NaCl 1~!. A water-soaked (distilled water)
wet control was also evaluated. Sodium chloride
solutions were prepared at the concentrations
mentioned, and seeds soaked in them at 10°C
in the dark inside glass beakers for one of three
durations; namely 1, 2 or 3 days (d). The wet
control was also soaked in water for the same
periods of time. The 10°C incubation temper-
ature proved low enough to prevent premature
germination (visible signs of radicle emergence
after imbibition) during soaking in water.

After treatment, seeds were either sown
fresh without drying or surface dried by expos-
ing to an airflow of 25°C for 3 hours (h) inside
an incubator and sown dry. Seeds were sown
in batches of 100 in 1000mL polystyrene trays
filled with equal volumes of sieved sand (weight
basis) and irrigated to weight with 200mL of
water, initially, and as they lost one third of
their weight thereafter. Trays were weighed
daily at 7:00am. Experiments were conducted
in a 18m? walk-in phytotron (Heraeus-Voetsch,
Germany) set at the environmental conditions
shown in Table 1. ‘These conditions were an at-
tempt to simulate likely heat stress during the
course of a day (Al-Mudaris 1998a).

Light (33 klux)

Absent
Activated
Activated
Activated
Activated
Absent

Table 1. The course of temperature, relative humidity and light activation in the phytotron during

a 24 hour cycle.

IONIC COMBATING MECHANISMS IN SEED 79

Treatment combinations were replicated
four times each at 100 seeds per replicate (3
treatments x 3 durations x 2 drying treatments
x 4 replicates = 72 experimental units). Trays
were arranged in a Randomized Complete Block
Design (RCBD). Emergence counts were taken
daily for 12d and from them the final germina-
tion percentage (FGP), mean germination time
(MGT) and germination index (GI) calculated.
MGT and GI were calculated following Orchard
(1977) and Benech Arnold et al. (1991), respec-
tively. The MGT is a measure of the mean time
taken for a seed lot to germinate, while the GI
assigns maximum weight to a higher number
of seeds germinating earlier (Al-Mudaris 1998).
Data were arcsin transformed (Yang et al. 1999
and Houle et al. 2001) and analysed in ANOVA
using the General Linear Model (GLM PROC)

of the SAS® statistical package for Windows®.

At 12 days of age seedlings were collectively
harvested from each tray, separated into shoots
and roots, and, after washing roots under run-
ning tap water, dried at 80°C for 4d in a reverse
cycle oven (Conviron Industries, Canada). Av-
erage dry weights of shoots (DWS) and roots
(DWR) and the shoot: root ratio (SRR) were
obtained and averaged. Also, after harvest,
the contents of each tray were emptied into a
sieve with 2mm openings, and germinated but
unemerged seeds retrieved and counted (here-
after termed GUE). Those that had neither ger-
minated nor emerged were classified as non-
germinated seeds and represented the difference
between emerged and germinated unemerged
seeds (Munir et al. 2001). These were sub-
jected to a tetrazolium test of viability following
ISTA (ISTA 1993) to verify their viability sta-
tus, but data was not analysed. Individual seeds
were also studied under a dissecting microscope
for further viability and anatomy notes follow-
ing Hidayati et al. (2001). Germination and
growth data were exposed to one-way and two-
way analysis of variance (ANOVA) procedures
for single factors and interactive factors of seed
treatment x duration (Weber and D’Antonio
1999).

Effect of Salt Concentration,
Osmoconditioning Treatment
Duration and Storage

The same NaCl treatments mentioned above
(0, 8 and 16g NaCl 1~') were used in combi-
nation with the same treatment durations (1,
2 or 3d). After treatment, IRAT 204 seeds
were dried back at 25°C for 24h and stored at
22°C and 50 to 52% relative humidity for 30d
in the dark. Thereafter, seeds were retrieved
from storage and sown in batches of 100 in
1000 mL polystyrene trays lined with creased fil-
ter paper. Each tray was moistened with 40 mL
of a polyethylene glycol solution (PEG) (Fluka
Chemie, Germany) and covered with a lid to
minimise evaporation. The solution had an os-
motic potential (w,) of -10 bar (circa -1.0 MPa)
simulating drought (Marschner 1995, Dodd and
Donovan 1999). The PEG molecular weight
(m.w.) was 10,000. Trays were placed in an in-
cubator set at 42/25°C (day/night, 12h/12h).
Germination counts were taken daily for 12d
and from them the FGP, MGT and GI calcu-
lated. Statistical arrangements were similar to
the first experiment with experimental factors
being treatment and soaking duration applied
in RCBD (Dodd and Donovan, 1999).

Effect of Salt-Based Mixtures and
Drying Duration

Three sorghum varieties were tested in this trial.
‘These were IGSV 112, ICSV 7¢45-and Me5=-1.
Seed lot tests revealed germinability, moisture
content and viability levels comparable to those
of IRAT 204 sorghum used in the previous two
experiments.

The hypothesis to be tested here was that
mixing NaCl with other minerals might pro-
vide both the stress acclimation (hardening) ef-
fect and a mineral uptake effect, thus improving
seed performance under stress. The “softening”
effect of other elements in the mixture might,
thus, lead to a balance and a combating of Na*
and Cl~ ions. A basic 10g NaCl 17! (wv, of
-7.7 bar) (Knauer Osmometer, Germany) treat-

76 KADER

ment was mixed with calcium sulphate or one
of three fertilizers as follows:

T,: Dry Control (dry, untreated seeds)

Do 10s NaGitie: 4 og CaSO,4.2H2O ie
(calcium sulphate)

10g NaCl 1~! + 7.5¢ Urea fertilizer 17!
(water-soluble nitrogen-source fertilizer)
10g NaCl 1—! + 10g NPK fertilizer 1~*
(slow release compound fertilizer with
6% N, 12% P2Os and 18% K20O)

10g NaCl 1~! + 15g DAP fertilizer 1~!
(di-ammonium phosphate,

soluble phosphorous source)

10g NaCl 17?

Ts:

dare

Te:

Ee:

Seeds were soaked in the above mentioned
solutions for $d’ at 18°C: in ‘thedark. “The
concentrations were based on earlier work with
sorghum (Al-Mudaris 1998). After treatment,
seeds were dried back at 25°C for 7, 14 or 21h
and stored for 1 month at 24°C in the dark.
Subsequently, seeds were sown in batches of
100 in polystyrene trays at 40/20°C (day/night
temperatures, 12h/12h) under a PEG-induced
(m.w. 10,000) drought of -3.3bar (0.3 MPa).
The change in temperature down to 40/20°C
was based on observation of partial fungal infec-
tion at the 42/25°C level in the previous trial.
Again, the FGP and MGT were calculated in
addition to the coefficient of velocity of germi-
nation (CVG) (Jones and Sanders 1987). The

CVG increases when the number of germinated

Seed FGP MGT GI
Treatment (%) (day)

Wet Control 78.8 a BS a 278.6 a
8g NaCl 17! Vora 3 OM 262.2 a
16g NaCl 17! 62.1 a 335. b 228.0 b

seed increases and the time required for ger-
mination decreases. Theoretically, the highest
CVG possible is 100 and would occur if all seeds
germinated on the first day (Jones and Sanders
1987). Statistical procedures were the same as
those for the second experiment.

RESULTS AND DISCUSSION

Effect of Salt Concentration,
Osmoconditioning Treatment
Duration and Drying

The 16g NaCl 17! treatment reduced the FGP
and GI in comparison to the wet control and the
8g NaCl1l7! treatment. Both salt soaks reduced
the time needed to germinate as seen from lower
MGT values (Table 2). Neither growth param-
eters (DWS, DWR, and SRR) nor the number
of GUE seeds differed between treatments. The
longer the duration of soaking in NaCl solutions,
the lower the final germination percentage and
germination speed, and the higher the number
of GUE seeds (Table 3). Drying seeds, on the
other hand, did not affect germination charac-
teristics (FGP, MGT and GI) but reduced the
DWR and, thus, increased the SRR (Table 4).

The interactive effects of NaCl concentra-
tion, soaking duration and drying on germina-
tion and growth characteristics of IRAT 204 re-
vealed no significant interactions between treat-
ment combinations regarding the parameters
studied (data not shown)

DWS DWR SRR GUE
(mg) (mg) (seeds)
6.6 a 94a 0. 75sa; A a
6.6 a Gilea 0.75 a A Dua,
6.3 a 8.6 a 0.84 a 6.0 a

Table 2. Effect of NaCl treatments on germination and growth characteristics of sorghum IRAT

204 under phytotron conditions.

Means in columns followed by similar letters are not significantly different (a < 0.05). FGP:
Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS:
Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated

Unemerged Seeds.

IONIC COMBATING MECHANISMS IN SEED 77

Treatment FGP(%) MGT GI DWS DWR SRR GUE
Duration (day) (mg) (mg) : (seeds)
(days)

i 79.4 a S020 296.8 a 6.7 a 8.6 a 0.84 a 2010
2 70.0 b 4.0a 242.5-b 6.3 a 98a 0.69 a 5.0: a
3 64.7 b 3.7 ab 229.5 b 6.5 a 8.7 a 0.80 a 5.9 a

Table 3. Effect of duration of soaking in NaCl solutions on germination and growth characteristics
of sorghum IRAT 204 under phytotron conditions.

Means in columns followed by similar letters are not significantly different (a < 0.05). FGP:
Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS:
Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated
Unemerged Seeds.

Drying Treatment FGP MGT GI DWS DWR SRR GUE
(%) (day) (mg) (mg) (seeds)

No Drying (Fresh Sown) 72.7 a 3.6 a 263.0a 6.54 10.0 a 0.69 b 46a

Drying (25°C, 3h) 70.1 a 3.9 a 249.5a 65a 8.1 b 0.86 a Orca)

Table 4. Effect of drying after seed treatment on germination and growth characteristics of sorghum
IRAT 204 under phytotron conditions.

Means in columns followed by similar letters are not significantly different (a < 0.05). FGP:
Final Germination Percentage, MGT: Mean Germination Time, GI: Germination Index, DWS:
Dry Weight of Shoot, DWR: Dry Weight of Root, SRR: Shoot : Root Ratio and GUE: Germinated

Unemerged Seeds.

Effect of Salt Concentration,
Osmoconditioning Treatment
Duration and Storage

The germination pattern of treated seeds after
storage revealed that in both the Wet Control
and 8g NaCl 1~! treatment, a soaking dura-
tion of 2 days yielded better FGP values than
3 or 1 day soaking treatments, respectively. At
the apparently high 16 g NaCl 17! level, soaking
seeds for 1 day only was superior to soaking for
2 or 3 days (Table 5). Neither the MGT nor
the GI were clearly affected by treatment and
duration interactions. However, the highest GI
value (best germination percentage and germi-
nation speed relationship) was observed in seeds
treated with 8g NaCl 17! for 2 days.

Effect of Salt-Based Mixtures and
Drying Duration

The interactive analysis of seed treatment,
genotype and drying duration did not reveal
significantly different germination percentages.
From Table 6 it can be seen that the low-
est FGP was observed in the NaCl + DAP
fertilizer treatment. All seed osmocondition-
ing treatments reduced the MGT (seeds ger-
minated faster) and increased the CVG (bet-
ter germination percentage and rate) over un-
treated seeds. This means that seed osmocon-
ditioning increased overall germination speed.
Genotypes differed in their response to osmo-
conditioning with M35-1 performing in an in-

78 KADER

ferior manner in comparison to ICSV 112 and the germination parameters studied (data not
ICSV 745 as far as the FGP, MGT and CVG _ shown) pointing to the possibility of drying
were concerned (Table 7). The duration of dry- seeds after treatment in order to improve stor-
ing, on the other hand, did not affect any of age life.

Seed Treatment Duration (days) FGP (%) MGT (day) GI

Wet Control 1 78.6 be 4.2 ab 255.0 b-d
ya 86.6 ab O.2' a Z210rored
3 t2.6.C 4.7 ab 223.6 cd
8g NaCl 17} 1 bard 34 b 201.0 cd
94.6 a 3.6 b 348.0 a
3 86.6 ab 4.0 ab 298.3 ab
16g NaCl 17! 1 84.0 be 4.4 ab 275.3 be
58.0 d 4.1 ab 200.0 cd
3 54.0 d 4.1 ab 185:3 a

Table 5. Interactive effects of NaCl concentration and soaking duration on the germination of
sorghum IRAT 204 seeds after storage for 10d under ambient conditions.

Means in columns followed by similar letters are not significantly different (a < 0.05). FGP: Final
Germination Percentage, MGT: Mean Germination Time and GI: Germination Index.

Seed Treatment FGP (%) MGT (day) CVG
Dry Control (4.2) 3.7 a PASTE
NaCl (10g/1) + Calcium Sulphate (5g/l) 81.2 a 29K 36.1 ab
NaCl (10g/1) + Urea Fertilizer (7.5 g/1) 66.4 c 2,9) be 33.9 ab
NaCl (10 g/l) + NPK Fertilizer (10 g/1) 7120) o¢ Dae 36.8 a
NaCl (10g/1) + DAP Fertilizer (15 g/1) 56.8 d Sw 6) 32) (ib
NaCl (10 g/1) 68.7% be Zales 36.4 ab

Table 6. Effect of NaCl and NaCl-based seed osmoconditioning treatments on germination char-
acteristics of sorghum varieties ICSV 112, ICSV 745 and M35-1 after storage for 1 month at 30°C.
Means in columns followed by similar letters are not significantly different (a < 0.05). FGP:
Final Germination Percentage, MGT: Mean Germination Time and CVG: Coefficient of Velocity
of Germination.

Genotype FGP (%) MGT (day) CVG
1eSV 112) 2b 2.9 b Son hoe

ICSV 745 84.4a 2.9 b 34.9 a
M35-1 53.6 ¢ 3.2 a BIE)

Table 7. Effect of genotype on germination characteristics of sorghum after storage for 1 month at
5 O-

Means in columns followed by similar letters are not significantly different (a < 0.05). FGP:
Final Germination Percentage, MGT: Mean Germination Time and CVG: Coefficient of Velocity
of Germination.

IONIC COMBATING MECHANISMS IN SEED 79

From the results of the first experiment,
which was carried out under simulated condi-
tions in the phytotron, it is clear that NaCl
concentration is decisive in the post-treatment
germinative response seeds exhibit. While 8g
NaCl 1~! improved germination, 16g NaCl 17!
retarded it in terms of FGP and GI. Both con-
centrations of salt, however, increased germina-
tion speed over water-soaked controls, but nei-
ther affected seedling growth or the fate of seeds
which did not germinate. The longer the dura-
tion of soaking, the less positive effects were ob-
served. Additionally, longer soaking durations
(2 or 3 d in comparison to 1 d) almost doubled
the number of germinated but unemerged seeds
(Table 3). This response observed in fresh sown
seeds was also observed in the second experi-
ment where seeds were dried back and stored.
The 8g NaCl 17! and water-soaking treatments
were better than 16g NaCl 17! except that a
2-day treatment duration was observed to be
better than 1d for water and 8g treatments,
whereas | d was more suited for 16 g treatments.
Again, 8g NaCl 1~! combined with a 2d treat-
ment duration advanced germination as seen
from higher GI values. This shows that stor-
age of treated seeds at ambient temperatures
for a duration of 30d does not negatively affect
performance of pre-storage-hardened seed.

The fact that longer treatment durations in-
creased the number of germinated unemerged
seeds (first experiment) and reduced overall ger-
mination (16g NaCl for 2 or 3d in the second
experiment) may be a direct effect of NaCl tox-
icity to seeds. That seeds germinate but do not
emerge reflects a reduced vigor and/or abnor-
mality (microscopic evidence, data not shown)
since all seeds were planted at the same depth
of 2cm. Moisture supply was also regulated by
weight and so the effect of external factors seems
unlikely. A replication of the same treatments
was conducted in a separate experiment and
seeds/seedlings retrieved at 1, 3, 5 and 7 days
after sowing. These were analysed for Nat and
Cl~ content and showed levels up to five times
as high as non-treated seeds/seedlings. The re-
sults of this experiment will be reported in a

subsequent paper, but would indicate that ionic
toxicity occurred. An analysis of hormonal lev-
els would point to a sharp increase in ABA lev-
els during soaking treatments and a mild in-
crease during and after heat shock (Kader, un-
published data). This is where the hardening
effect is likely to have taken effect, but at ex-
cessive NaC] levels a germination block would
have occurred due to the shortage in Kinetin
and GA3 as a direct result of this physiologi-
cal shock (Al-Mudaris 1998, Kader and Jutzi
2002). This points to the possibility that hard-
ening can only provide alleviation from stress at
moderate stress levels and becomes a stress it-
self at higher degrees of heat, drought or salinity
(Noe and Zedler 2000).

The observation that 1d was better for 16g
NaCl 1~! treatments tends to confirm the toxi-
city hypothesis, since 1d would not be enough
to inflict such stress due to the uptake of water
by the seed at high rates during the first day
of treatment (Al-Mudaris and Jutzi 1998ab).
Bussell and Gray (1976), in their work on os-
moconditioning tomato seeds at -5 to -15 bars,
observed that the length of soaking period had
little effect at low osmotic potentials but not at
high potentials. The 8 and 16g NaCl 1~! treat-
ments measured -6.02 and -11.1 bar on the os-
mometer, respectively. This means that longer
durations would expose the seeds to more os-
motic stress.

Soaking sorghum in water alone, on the
other hand, has been reported to increase germi-
nation speed as soaking period increased (Har-
ris 1996). This has also been reported for pep-
per seed treated with the non toxic, inert man-
itol (Georghiou et al. 1987). The problem,
then, lies within the nature of the osmoticum
used. Seeds coming into contact with NaCl
solutions have been found to have higher Cl7
concentrations in their different components,
the accumulation of which, in the embryo (Es-
echie 1995) and endosperm (Al-Mudaris, 1998),
was associated with germination failure. Ful-
bright (1988) observed reduced germination in
Indiangrass (Sorghastrum nutans cv. Cheyenne,
Lometa) at 0.12mol NaCl 1~! (6.9g NaCl 17?),

80 KADER

whereas Shanmugasundaram and Kannaiyan
(1989) found 1.0% NaCl (10g NaCl 1~*) to give
the highest germination percentage in pear! mil-
let (Pennisetum glaucum L. R. Br.) in compar-
ison to 2.5% (25g NaCl 1~*).

Increasing the salt concentration of a so-
lution in contact with sorghum seeds reduces
a-amylase and protease activity, reducing and
non-reducing sugar contents and the rate of re-
serve protein mobilization (Khan et al. 1989).
Additionally, if a seed takes up solutes or mobi-
lizes its reserves in an osmotically active form,
its own water potential will be reduced although
physiological processes may be inhibited both
by the low water potential and the toxicity of
ions (Bannister 1978). Ells (1963) reported that
the priming effect of seed treatment with nutri-
ent solutions (including 2% NaCl) is not due to
the salts, nor to the amount of water retained
by the seed from the treatment, but rather to
certain enzymatic activities which take place
within the seed while it is being held in a moist
condition.

Reduced emergence, after seed germination
(germination in the seedbed, but lack of emer-
gence above the soil surface) was observed in
the phytotron trial. If the radicle emerges from
the testa and the soil water content is reduced
below the initial soil water content due to dry-
ing, emergence is reduced (Helms et al. 1996).
This could have been the case where trays were
re-irrigated by weight only when they lost one
third of their initial moisture content. This
would have been more deleterious to seeds os-
moconditioned for longer periods due to their
higher initial moisture contents and would have
led to a loss of the capacity to emerge (Peske
1983).

Combining NaCl with calcium sulphate im-
proved seed response as seen from the third ex-
periment, whereas other mixtures did not raise
the final germination percentage but improved
germination speed over controls. This may be
due to the fact that the fertilizers used were,
in themselves, salts and as such contributed
to the ionic stress discussed above. A notable
point is that of solubility. Calcium sulphate was

not totally soluble at the rates used, whereas
sodium chloride was. The NPK fertilizer was
also only partly soluble, whereas urea and DAP
were more soluble.

Mixing sodium chloride with calcium prob-
ably increased Cat? content of seeds (Bharati
and Vaidehi 1989, Al-Mudaris 1998, Kader, un-
published data). Calcium has been found to
play a major role in tolerance to NaCl where tol-
erant genotypes of vegetables contained higher
Cat? reserves in their seeds (Guerrier 1983).
It has also been noted as modifying seed re-
sponse to heat shock in maize (Gong et al. 1997)
due, probably, to increasing membrane stabil-
ity (Marschner 1995). Amazallag et al. (1997)
also reported that leaf malformations in plants
exposed to high NaCl concentrations were pre-
vented by the addition of Cat? to the nutrient
solution.

Drying duration did not affect seed response
to osmoconditioning. ‘This agrees with the re-
sults of Emmerich and Hardegree (1996) who
found that the germination of four warm-season
grasses was not affected by length of the de-
hydration period. It is also in line with the
work of Brocklehurst and Dearman (1984) who
found that drying vegetable seeds after treat-
ment did not interact with either the priming
chemical used or the species tested and those of
Al-Mudaris and Jutzi (1998ab) and Al-Mudaris
(1998a). Drying the seed slowly by control-
ling humidity may impact germination rates
achieved through conditioning (Mueller 1996).
The loss of cell membrane integrity during dry-
ing is repaired when seeds are allowed to imbibe
water, albeit after a certain period (Knypl and
Khan 1981). It follows that the rate of dry-
ing (Dell Aquila and Trito 1990) and its timing
after initial imbibition (Kutschera 1995) play
the major roles (Al-Mudaris and Jutzi 1999 and
1999a). Both the degree of drying and its timing
used here seem to fall within the range which
does not alter osmoconditioning effects. ‘This
ranged between 14 and 19.5% moisture content
following treatment and after drying prior to
storage.

IONIC COMBATING MECHANISMS IN SEED 81

CONCLUSIONS

The three experiments were carried out under
varying temperature and drought stress situ-
ations and after storage at ambient tempera-
tures. The response of sorghum seeds to os-
moconditioning in all three cases was gener-
ally more affected by the priming agent itself,
its concentration and the duration of treatment
than by drying or storage. ‘This drying and stor-
age is of great practical significance in the field,
for if hardening is to be practiced, convenience
in handling seeds must be fostered. This is dif-
ficult to achieve in moist batches of seed, which
would be prey to fungal infection and render
sowing a difficult task.

In conclusion, it would appear that seed
hardening of sorghum via an NaCl-based soak
has some potential to improve performance un-
der post-treatment supra-optimal environmen-
tal conditions like drought or heat stress. The
decisive factor in this is the thin line between
this hardening actually inducing germination
and it being an additional plant stress factor
itself.

REFERENCES

Al-Mudaris, M. 1998. Notes on various param-
eters recording the speed of seed germina-
tion. Journal of Agriculture in the Tropics
and Subtropics, 98, 147-154.

Al-Mudaris, M. 1998a. Studies on osmotic con-
ditioning seed treatments to enhance germi-
nation and early seedling growth of sorghum
and pearl millet under drought, heat and
combined stress conditions, Tectum Verlag
Publishers, Marburg, Germany, 225p.

Al-Mudaris, M. and Jutzi, S. 1998. Emergence
and growth characteristics of sorghum and
pearl millet intercropped with French beans
after PEG-based seed priming under green-
house and phytotron conditions. Journal of
Agriculture in the Tropics and Subtropics, 98,
31-42.

Al-Mudaris, M. and Jutzi, S. 1998a. The influ-

ence of genotype, priming material, tempera-

ture and osmotic potential of priming solution
on imbibition and subsequent germination of
sorghum and pear] millet seeds during and af-
ter treatment. Journal of Agriculture in the
Tropics and Subtropics, 98, 133-145.

Al-Mudaris, M. and Jutzi, S. 1998b. The effect
of incubation temperature during presowing
seed treatments on the subsequent germina-
tion behavior in Sorghum bicolor and Pen-
nisetum glaucum. German Journal of Agron-
omy, 2, 131-134

Al-Mudaris, M. and Jutzi, S. 1999. The in-
fluence of fertilizer-based seed priming treat-
ments on emergence and seedling growth of
Sorghum bicolor and Pennisetum glaucum in
pot trials under greenhouse conditions. Jour-
nal of Agronomy and Crop Science 182, 135-
142

Al-Mudaris, M. and Jutzi, S. 1999a. Osmot-
ically primed seed and seedling reactions to
variations in day/night temperature. Jour-
nal of Agronomy and Crop Science, 182, 217-
22\e

Amzallag, G., Seligmann, H. and Lerner, H.
1997. Leaf malformation during early devel-
opment in Sorghum. Evidence for an em-
bryonic developmental window. Physiologia
Plantarum, 99, 470-476.

Bannister, P. 1978. INTRODUCTION TO PHYS-
IOLOGICAL PLANT ECOLOGY, Second Print-
ing, Oxford, Blackwell Scientific, UK.

Benech Arnold, R., Fenner, M. and Edwards,
P. 1991. Changes in geminability, ABA con-
tent and ABA embryonic sensitivity in devel-
oping seeds of Sorghum bicolor (L.) Monech
induced by water stress during grain filling.
New Phytologist, 118, 339-347.

Bharati, P. and Vaidehi, M. 1989. ‘Treatment
of sorghum grains with calcium hydroxide for
calcium enrichment. Food and Nutrition Bul-
letin, 11, 53-56.

Brocklehurst, P. and Dearman, J. 1984. A
comparison of different chemicals for osmotic
treatment of vegetable seed. Annals of Ap-
plied Biology, 105, 391-398.

Bussell, W. and Gray, D. 1976. Effects of pre-
sowing seed treatments and temperatures on

82 KADER

tomato seed germination and seedling emer-
gence. Scientia Horticulturae, 5, 101-109.

Dell Aquila, A. and Trito, V., 1990. Ageing and
osmotic priming in wheat seeds: effects upon
certain components of seed quality. Annals of
Botany, 65, 21-26.

Dodd, G. and Donovan, L. 1999. Water po-
tential and ionic effects on germination and
seedling growth of two cold desert shrubs.
American Journal of Botany, 86, 1146-1153.

Ells, J. 1963. The influence of treating tomato
seed with nutrient solutions on emergence
rate and seedling growth. Proceedings of the
American Society for Horticultural Science,
83, 684-687.

Emmerich, W. and Hardegree, S. 1996. Partial
and full dehydration impact on germination
of 4 warm-season grasses. Journal of Range
Management, 49, 355-360.

Esechie, H. 1995. Partitioning of chloride
ion in the germinating seed of two forage
legumes under varied salinity and tempera-
ture regimes. Communications in Soil Sci-
ence and Plant Analysis, 26, 19-20.

Fulbright, T. 1988. Effects of temperature, wa-
ter potential, and sodium chloride on Indian-
grass germination. Journal of Range Man-
agement, 41, 207-210.

Georghiou, K., Thanos, C. and Passam;’ H.
1987. Osmoconditioning as a means of coun-
teracting the ageing of pepper seeds during
high-temperature storage. Annals of Botany,
60, 279-285.

Gong,”'M., ‘Yong=Jun, L., ‘Dai,’ XxX.) Tians iM.
and Li, Z. 1997. Involvement of calcium and
calmodulin in the acquisition of heat-shock
induced thermotolerance in maize seedlings.
Journal of Plant Physiology, 150, 615-621.

Guerrier, G. 1983. Germination in vegetables
and oil plants in the presence of NaCl. Seed
Science and Technology, 11, 281-292.

Harris, D. 1996. The effects of manure, geno-
type, seed priming, depth and date of sow-
ing on the emergence and early growth of
Sorghum bicolor (L.) Moench in semi-arid
Botswana. Soil and Tillage Research, 40,
73-88.

Helms, T., Deckard, E., Goos, R. and Enz, J.
1996. Soil moisture, temperature, and drying
influence on soybean emergence. Agronomy
Journal, 88, 662-667.

Heydecker, W. and Gibbins, B. 1978. The
“Priming” of seeds. Acta Horticulturae, 83,
213-215.

Hidayati, S., Baskin, J. and Baskin, C. 2001.
Dormancy-breaking and germination require-
ments for seeds of Symphoricarpos orbicula-
tus (Caprifoliaceae). American Journal of
Botany, 88, 1441-1451.

Houle, G., Morel, L., Reynolds, C. and Siegel,
J. 2001. The effect of salinity on different
developmental stages of an endemic annual
plant, Aster laurentianus (Asteracea). Amer-
ican Journal of Botany, 88, 62-67.

ISTA, 1993. International Rules for Seed Test-
ing. Seed Science and Technology, 21. |

Jones, K. and Sanders, D. 1987. The influence
of soaking pepper seed in water or potassium
salt solutions on germination at three tem-
peratures. Journal of Seed Technology, 11,
O7- 102.

Kader, M. and Jutzi, S. 2002. Temperature, os-
motic pressure and seed treatments influence
imbibition rates in sorghum seeds. Journal of
Agronomy and Crop Science, 188, 286-290.

Khan, A., Azmi, A. and Ashraf. M. 1989. In-
fluence of NaCl on some biochemical aspects
of two sorghum varieties. Pakistan Journal of
Botany, 21, 74-80.

Knypl, J. and Khan, A. 1981. Osmocondi-
tioning of soybean seeds to improve perfor-
mance at suboptimal temperatures. Agron-
omy Journal, 73, 112-116.

Kutschera, U. 1995. KURZES LEHRBUCH DER
PFLANZENPHYSIOLOGIE. Wiesbaden, Quelle
und Meyer Verlag.

Marschner, H. 1995. MINERAL NUTRITION OF
HIGHER PLANTS (Second Edition), London,
Academic Press.

Mueller, D. 1996. Germination and root growth
of 4 osmoconditioned cool-season grasses.
Journal of Range Management, 49, 117-120.

Munir, J., Dorn, L., Donohue, K. and Schmitt,
J. 2001. The effect of maternal photoperiod

IONIC COMBATING MECHANISMS IN SEED 83

on seasonal dormancy in Arabidopsis thaliana
(Brassicaceae). American Journal of Botany,
88, 1240-1249.

Noe, G. and Zedler, J. 2000. Differential ef-
fects of four abiotic factors on the germina-
tion of salt marsh annuals. American Journal
of Botany, 87, 1679-1692.

Orchard, T. 1977. Estimating the parameters of
plant seedling emergence. Seed Science and
Technology, 5, 61-69.

Peske, S. 1983. Germination and emergence
of soybean seeds as related to moisture
stress. Dissertation Abstracts International,
44, 668 B.

Shanmugasundaram, V. and Kannaiyan, M.
1989. Effect of concentration of seed hard-
ening chemicals on physiological characters of
pearl millet (Pennisetum typhoides Stapf and
Hubb). Journal of Agronomy and Crop Sci-
ence, 163, 174-176.

Silvertown, J. 1999. Seed ecology, dormancy
and germination: A modern synthesis from

Baskin and Baskin. American Journal of
Botany, 86, 903-905.

Weber, E. and D’Antonio, C. 1999. Germi-
nation and growth responses of hybridising
Carpoprotus species (Azzoaceae) from coastal
California to soil salinity. American Journal
of Botany, 86, 1257-1263.

Yang, J., Lovett-Doust, J. and Lovett-Doust,
L. 1999. Seed germination patterns in green
dragon (Arisaema dracontium, Araceae).
American Journal of Botany, 86, 1160-1167.

M. A. Kader
Director, Consultica Worldwide, PO Box 3089

Tamarama NSW 2026
Australia
m.kader@mbox.com.au

(Manuscript received 5.11.2002)
(Manuscript received in final form 04.02.2003)

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iD

Journal & Proceedings of the Royal Society of New South Wales, Vol. 135, p. 85-86, 2003

ISSN 0035-9173/03/020085-2 $4.00/1

Edgeworth David Medal 2002

PROFESSOR MARCELA BILEK

Professor Marcela Bilek was appointed Pro-
fessor of Applied Physics at Sydney University
in 2000. She graduated from Sydney University
with the University Medal in Physics in 1991
and has since worked in a number laboratories
on projects that include atomic scale computer
simulation, plasma processing, thin film mate-
rials and surface modification. In 1993 she was
awarded the Minerals, Metals and Materials So-
ciety Reduction Technology Prize for her suc-
cessful computer simulation of bubble stirring
effects in aluminium cells, an achievement of
considerable economic importance to the alu-
minium smelting industry.

A Peterhouse College and Cambridge Com-
monwealth Trust scholarship enabled her to
obtain a PhD from Cambridge University in
plasma technology for the fabrication of thin
solid films. She continued similar work at Cam-
bridge under an Emmanuel College Research
Fellowship. Specialised materials, increasingly
needed in microelectronics, biomaterials and op-
tics, are enhanced by these methods. Much of
this work has been done in collaboration with
research groups in Australia.

She was the first to accurately model the
transport of cathodic arc plasmas through mag-

netic filters, enabling the removal of microparti-
cles from beams and produce beams with homo-
geneous cross sections for uniformly processing
large wafers. Her models for the structure of
both hydrogenated amorphous carbon and hy-
drogenated silicon carbide, based on quantum
mechanical treatment of the bonding electrons,
has been confirmed by numerous experimental
observations. One socially significant example
of her current research is the surface coating of
materials to be implanted in the human body as
prosthetic devices. Biocompatibility, adhesion
and corrosion resistance is always a problem in
the body which sees such devices as foreign and
attempts to remove them. Her plasma implan-
tation methods promise to significantly extend
the useful life of such devices.

She has raised more than a million dollars
for research since her Sydney appointment, con-
vened a conference on biological effects of mi-
crowave radiation, delivered the 14th Pollock
Lecture, been awarded a Young Tall Poppy
Award 2001 by the Australian Institute of Po-
litical Science and the 2002 Malcolm McIntosh
Prize for Physical Scientist of the Year.

JCK

86 CLARKE MEDAL

The Clarke Medal for 2002

PROFESSOR ROBERT HILL

Professor Robert Hill is a Senior Research
Fellow in the School of Earth and Environmen-
tal Sciences at the University of Adelaide and
is Head of Science at the South Australian Mu-
seum. He is a graduate of the University of
Adelaide. He completed his Ph.D. on Tertiary
plant macrofossils in 1981, and his D.Sc. on
the interaction between climate change and the
evolution of the living Australian vegetation in
1997. In 1979 he accepted a position as Tutor in
Botany at James Cook University, and in 1980
was offered a lecturing position in the Depart-
ment of Botany at the University of Tasmania
where he remained until 1999, being promoted
to Professor in 1993. He was Head of the School
of Plant Science for 6 years prior to his depar-
ture, and was awarded Professor Emeritus sta-
tus by the University of Tasmania Council in
2000. In 1999 he returned to the University of
Adelaide to take up his current position.

Professor Hill has had a lifetime interest in
the evolution of the vegetation in Australia and
Antarctica. He has published more than 125
refereed journal papers, 35 book chapters, sev-
eral symposium papers and has edited or co-

edited four books, including The History of the
Australian Vegetation (Cambridge University
Press), Ecology of the Southern Conifers (Mel-
bourne University Press), The Ecology and Bio-
geography of Nothofagus Forests (Yale Univer-
sity Press), and Vegetation of Tasmania (Aus-
tralian Biological Resources Study).

Professor Hill is President and a Fellow of

the Australian Institute of Biology and a Fel-

low of the Linnean Society of London. His cur-
rent research interest is the adaptation of the
Australian vegetation to increasing aridity dur-
ing the last 30 million years. He is developing
a research program on the impact of fire on the
Australian vegetation during this time period.
He is best known for his research on the fossil
history of the southern beech, Nothofagus, and
the southern conifers. His work on the fossil his-
tory of Nothofagus has been critical in refining
our understanding of its evolution and has led
to a major revision of our understanding of the
biogeography of this critical southern genus.

JCK

Journal €& Proceedings of the Royal Society of New South Wales, Vol. 135, p. 87-88, 2003

ISSN 0035-9173/03/020087-2 $4.00/1

Biographical Memoir

Te hi

SIR ARTHUR RODEN CUTLER, V.C., AK,
GMEG., K.C.V.0., C.B.E., K: St.Ji,
B.Ec (Syd.), L1.D (Hon.) (Syd.},

D.Sc. (Hon.) (NSW & Newcastle)
1916-2002

Arthur Roden Cutler, known as Sir Roden,
was born on 24 May, 1916, at Manly, son of
Arthur William Cutler and Ruby Daphne (née
Pope) of Bathurst.

The Cutler family arrived in New South
Wales as free settlers in 1833. The Roden fam-
ily, of which his mother was a direct descendent,
arrived even earlier (1827) with the army.

Roden Cutler was educated at Sydney Boys’
High School and Sydney University, graduat-
ing B.Ec in 1935. At the University he excelled
in sports, gaining Blues from both Sydney and
the Australian Universities in swimming, and

reprinted with permission of the Australian War Memorial

participating in water polo and shooting. As a
teenager he made an heroic surf rescue, risking
his life against a large shark.

He joined the Public Trust Office of NSW
on graduating, taking leave in 1940 to join the
A.LF. (2/5 Field Regiment). Roden Cutler’s
war service was relatively short, but spectacu-
lar, and became legendary. In Syria on 19 June,
1941 he showed “exceptional courage” in driving
the enemy back and establishing outposts which
were important factors in capturing Merdijay-
oun. Three weeks later (6 July), at Damour, he
went forward against heavy machine-gun fire,
was severely wounded, and subsequently had his
leg amputated. He was awarded the Victoria
Cross on 28 November, 1941.

Between 1942-43 he was a member of the
Commonwealth Aliens Classification and Advi-
sory Committee, Assistant Deputy Director of

88 BIOGRAPHICAL MEMOIR

the Security Service of NSW (1943), and from
1943 to 1946 Assistant Commissioner of the
Repatriation Department. He was also NSW
State Secretary for the R.S.L, 1942-43.

In 1946 he married Helen Gray Annetta
Morris (d. 1990) and a new career path opened
with his appointment as High Commissioner
to New Zealand (to 1952), to Ceylon (now Sri
Lanka), and the equivalent position (Australian
Minister) to Egypt between 1955-56. Then fol-
lowed two years in Canberra where he was Chief
of Protocol in the Department of External Af-
fairs, Secretary-General of a S.E.A.T.O. Meet-
ing, and ACT President of the RSL.

In 1959 he moved overseas again as High
Commissioner to Pakistan, with a brief inter-
lude in the Somali Republic, and then to New
York, where he was Australian Consul-General
from 1961-65, and a delegate to the United Na-
tions. His final overseas posting was as Ambas-
sador to the Netherlands, 1965-66.

His association with this Society began in
1966, when he was appointed Governor of New
South Wales, a post he held until 1981, the
longest-serving Governor of the State. During
this time he was also Administrator of Australia
(acting Governor-General) on six occasions. He
accepted the Council’s invitation to joint Pa-
tronage (with the Governor-General), and was
a strong supporter (with his wife) of the Soci-
ety, during a period of turmoil, with the move
from Science House and a considerable drain of
the Society’s finances.

At the Centenary Dinner in 1966 Sir Ro-
den stressed the importance of scientific work

in modern times, and the contribution made by
Australian scientists.

Sir Roden’s speech at the 1974 Dinner, held
at the Sydney Opera House, was memorable,
marked as it was by some humorous compar-
isons with former Governors who had been asso-
ciated with the Society (and its ancestors), but
with discussion of more serious Society matters.
He said “The Royal Society of New South Wales
may not be as widely known as it deserves, nor
may its functions be fully understood”. While
there was encouragement that membership had
increased in the previous year, “the real value
is in the learned qualifications of your member-
ship, not in the total number”, and the medals
were held in high regard.

“The Society’s task is to bring a balance into
people’s assessment of the advantages and lim-
itations of scientific progress. You need to en-
courage research and investigation, and occa-
sionally express a word of warning”. He com-
mended a “most valuable function” of the So-
ciety, the “keeping of a library,” which he saw
in its final open arrangement when opening the
the Science Centre in 1977.

Sir Roden displayed in his speeches a good
knowledge of the workings of the Society, de-
spite the many other calls on his time, and his
widespread interests. He died on 21 February,
2002 after a short illness, and is survived by his
second wife, Lady Jane C. Cutler and four sons
from his first marriage. The Society was repre-
sented at the State Funeral.

DFB

Journal € Proceedings of the Royal Society of New South Wales, Vol. 135, p. 89-90, 2003
ISSN 0035-9173/03/020089-2 $4.00/1

Index to Volume 135

A

Aeronautica Antipodean. D.A. CRADDOCK, Presidential Address 2002 1

Aerial and Below Ground Biomass Production of Acacia as Influenced by Organic Waste
Substrates During Nursery-Stage Seedling Growth. KADER, H.A., OMARI, M.A.

and HATTAR, B.I. 1
Awards, Citations for 2001 37-43
Awards, Citations for 2002 85-86
Annual Report of Council for the year ended 31 March 2002 49
B
BILEK, Professor Marcela, Edgeworth David Medal for 2002 85
Biomass Production Dy,
Biographical Memoirs

Samuel Warren CAREY AO 45

Andrew John CORBYN 47

Sir Roden CUTLER 87
C
CRADDOCK, David A. Aeronautica Antipodean. Presidential Address 2002 1
Clarke Medal 2001 43
Clarke Medal 2002 86
CAREY AO, Samuel Warren, Obit 45
D
Deposition of Trace Elements from the Atmosphere in the Sydney Region. D.J. SWAINE 20
E
Edgeworth David Medal for 2001 39
Edgeworth David Medal for 2002 85
H

HATTAR, B.I., KADER, M.A. and OMARI, M.A. Aerial and Below Ground Biomass

Production of Acacia as Influenced by Organic Waste Substrates During

Nursery-Stage Seedling Growth. 17
HILL, Professor Robert, Clarke Medal for 2002 86

K
KADER, M.A., OMARI, M.A. and HATTAR, B.I. Aerial and Below Ground Biomass
Production of Acacia as Influenced by Organic Waste Substrates During
Nursery-Stage Seedling Growth. ii

continued on next page

90 INDEX

O

OMARI, M.A., HATTAR, B.I. and KADER, M.A., Aerial and Below Ground Biomass
Production of Acacia as Influenced by Organic Waste Substrates During
Nursery-Stage Seedling Growth.

Obituary: Andrew John CORBYN

Obituary: Sir Roden CUTLER

P

Presidential Address 2002

PARKER, Michael William, Walter Burfitt Prize for 2001
PACKHAM, Gordon Howard, Clarke Medal for 2001

R
RICHARDSON, Samantha Jane, Edgeworth David Medal for 2001

S

SWAINE, D.J. Deposition of Trace Elements from the Atmosphere in the Sydney Region.

Society’s Medal 2001

T
Trace Elements in Sydney Region

W
Walter Burfitt Prize for 2001
WILLIAMS, P.A., Society’s Medal for 2001

Al
37

JOURNAL AND PROCEEDINGS
OF THE

ROYAL SOCIETY
O F
NEW SOUTH WALES

Volume 135 Parts 1 to 4
(Nos 403 — 406)

2003
ISSN 0035-9173

PUBLISHED BY THE SOCIETY
PO BOX 1525, MACQUARIE CENTRE, NSW 2113
Issued June 2002 (Parts 1 and 2), April 2003 (Parts 3 and 4)

THE ROYAL SOCIETY OF NEW SOUTH WALES

OFFICE BEARERS FOR 2002-2003

Patrons His Excellency the Right Reverend Dr Peter Hollingworth AC, OBE,
Governor General of the Commonwealth of Australia.
Her Excellency Professor Marie Bashir, AC, Governor of New South Wales.
President Mr D.A. Craddock, BSc(Eng) NSW, Grad.Cert. Management UWS.
Vice Presidents Prof. P.A. Williams, BA (Hons), PhD Macq.
Dr W.E. Smith, MSc Syd, MSc Oxon, PhD NSW, MInstP, MAIP.
Mr C.F. Wilmot

Hon. Secretary (Gen.) vacant (acting Hon. Sec. Prof. P.A. Williams)

Hon. Secretary (Ed.) Mrs M. Krysko von Tryst, BSc, Grad.Dip.Min.Tech NSW, MAusIMM.
Hon. Treasurer Prof R.A. Creelman, BA, MSc, PhD

Hon. Librarian Dr E.V. Lassak, MSc, PhD NSW, ASTC; FRACI

Councillors Mr J.R. Hardie, BSc Syd, FGS, MACE.

Prof. J. Kelly, BS¢ Syd, PhD Reading, DSc NSW

Ms K. F. Kelly, BSc(Hons)

Mr M.F. Wilmot, BSc

Prof M.A. Wilson, PhD, DSc. Auck, FRACI, C.Chem.
Southern Highlands Rep. Mr C.M. Wilmot

The Society originated in the year 1821 as the Philosophical Society of Australasia. Its main function is
the promotion of Science by: publishing results of scientific investigations in its Journal and Proceedings;
conducting monthly meetings; organising summer science schools for senior secondary school students;
awarding prizes and medals; and by liason with other scientific societies. Special meetings are held for:
the Pollock Memorial Lecture in Physics and Mathematics, the Liversidge Research Lecture in Chemistry,
the Clarke Memorial Lecture in Geology, Zoology and Botany, and the Poggendorf Lecture in Agricultural
Science.

Membership, as an Ordinary, Associate or Absentee Member, is open to any person whose application is
acceptable to the Society. An application must be supported by two members of the Society. Subscriptions
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the Journal and Proceedings. Manuscripts from non-members must be communicated through a member.

ISSN 0035-9173

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and data published on behalf of authors rests with the relevant authors, not with the Royal Society of New
South Wales.

CONTENTS
Vol. 135 Parts 1 and 2

CRADDOCK, DAVID, A.
Antipodean Aeronautica, Presidential Address 2002

KADER, M.A., OMARI, M.A. & HATTAR, B.I.
Aerial and Below Ground Biomass Production of Acacia as Influenced by

Organic Waste Substrates During Nursery-Stage Seedling Growth

SWAINE
Deposition of Trace Elements from the Atmosphere in the Sydney Region

AWARDS
The Society’s Medal 2001
Edgeworth David Medal 2001
The Walter Burfitt Prize for 2001
The Clarke Medal for 2001

BIOGRAPHICAL MEMOIRS
Samuel Warren Carey AO
Andrew John Corbyn

ANNUAL REPORT OF COUNCIL FOR THE YEAR ENDED 31%* MARCH 2002

Vol. 135 Parts 3 and 4

JOHNSTON, GRAHAM A. R.

Dietary Chemicals and Brain Function

KADER, M. A.

Jonic Combating Mechanisms and their Comparative Effects on Seed Hardening

under Simulated Supra-Optimal Environmental Conditions
AWARDS

Edgeworth David Medal 2002

The Clarke Medal for 2002

BIOGRAPHICAL MEMOIR

Sir Arthur Roden Cutler, V.C., AK, K.C.M.G., K.C.V.O., C.B.E., K. St.J.,

INDEX TO VOLUME 135

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73

85
86

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89

NOTES

NOTES

32

os

NOTICE TO AUTHORS

Manuscripts should be addressed to The Hon-
orary Secretary, Royal Society of New South Wales,
PO Box 1525, Macquarie Centre, NSW 2113.
Manuscripts submitted by a non-member (through
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Manuscripts are subjected to peer review by an in-
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Papers, other than those specially invited by the
Editorial Board on behalf of Council, will only be
considered if the content is substantially new ma-
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iN

CONTENTS 3 9088 01308 49

Vol. 135 Parts 3 and 4

JOHNSTON, GRAHAM A. R.

Dietary Chemicals and Brain Function

KADER, M. A.
Ionic Combating Mechanisms and their Comparative Effects on Seed Hardening

under Simulated Supra-Optimal Environmental Conditions

AWARDS
Edgeworth David Medal 2002
The Clarke Medal for 2002

BIOGRAPHICAL MEMOIR
Sir Arthur Roden Cutler, V.C., AK, K.C.M.G., K.C.V.O., C.B.E., K. St.J.,

INDEX TO VOLUME 135

ADDRESS Royal Society of New South Wales,
PO Box 1525, Macquarie Centre, NSW 2113, Australia
http://nsw.royalsoc.org.au

DATE OF PUBLICATION April 2003

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