全 文 ：Received 4 Nov. 2003 Accepted 26 Apr. 2004
Supported by the Significant Project of Introduction and Conservation of Tropical Plant Germplasm Resources and Research on Resource
Plants from The Chinese Academy of Sciences and Yunnan Province (WK2000-7), Postdoctoral Fellowship of National Research
Foundation of South Africa, the Knowledge Innovation Project of The Chinese Academy of Sciences (KSCX2-SW-117), Hundreds Talent
Program of The Chinese Academy of Sciences and Provincial Natural Science Foundation of Yunnan (2003C0068M), China.
* Author for correspondence. Tel: +86 (0)691 8715474; E-mail: .
Abbreviations: AFR, ascorbate free radical; APX, ascorbate peroxidase; AsA, ascorbic acid; BSA, bovine serum albumin; CAT, catalase; DHA,
dehydroascorbate; DHAR, dehydroascorbate reductase; EDTA, ethylenediaminetetra-acetic acid; GR, glutathione reductase; GSH, reduced
glutathione; GSSG, oxidized glutathione; MDA, malonylaldehyde; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NBT,
nitroblue tetrozulium; PVPP, polyvinylpolypyrrolidone; SOD, superoxide dismutase; TBA, thiobarbituric acid.
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Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 803-810
Desiccation Sensitivity of Trichilia dregeana Axes and
Antioxidant Role of Ascorbic Acid
SONG Song-Quan1, 2*, Patricia BERJAK2, Norman PAMMENTER2
(1. Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, China;
2. School of Life and Environmental Sciences, University of Natal, Durban 4041, Republic of South Africa)
Abstract: Recalcitrant seeds of Trichilia dregeana Sond. were used as experimental material, and
desiccation sensitivity of T. dregeana axes and antioxidant role of ascorbic acid were studied. Desiccation
tolerance of T. dregeana axes progressively declined with dehydration, water content at which 50% of axes
has been killed by dehydration (W50) was about 0.16 g H2O/g DW. During dehydration, electrolyte leakage
rate of axes gradually increased, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX),
catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) declined, and content
of thiobarbituric acid (TBA)-reactive products increased. Two point five-10 mmol/L ascorbic acid (AsA)
treatment could significantly increase desiccation tolerance and activities of SOD, APX, CAT and GR in
axes, and decrease electrolyte leakage rate and content of TBA-reactive products in axes. The results
showed that desiccation tolerance of T. dregeana axes was strongly correlated with the increase in the
activities of antioxidant enzymes and the decrease in lipid peroxidation.
Key words: ascorbic acid; ascorbate peroxidase; catalase; dehydroascorbate reductase; glutathione
reductase; lipid peroxidation; recalcitrant seed; superoxide dismutase; Trichilia dregeana
Recalcitrant seeds are shed at high water contents and
are intolerant of dehydration and often also of chilling. A
number of processes or mechanisms have been suggested
to confer, or contribute to desiccation tolerance. Different
processes may confer protection against the consequences
of loss of water at different hydration levels, and the ab-
sence or ineffective expression of one or more of these
could determine the relative degree of desiccation sensitiv-
ity of seeds of individual species (Pammenter and Berjak,
1999; Song et al., 2003a; 2003b). Free-radical generation as
a consequence of uncoordinated metabolism may well be a
major injurious factor during relatively slow dehydration of
recalcitrant seeds, underlying a spectrum of lethal lesions
(Smith and Berjak, 1995; Côme and Corbineau, 1996). Recal-
citrant seeds (or their embryos) do appear to possess anti-
oxidant mechanisms (Hendry et al., 1992; Finch-Savage et
al., 1994). However, these protective mechanisms may be-
come impaired under conditions of water stress (Smith and
Berjak, 1995; Walters et al., 2002); certainly, they are inef-
fectual in terms of protecting against desiccation damage.
A peculiar difference between orthodox and recalcitrant
seeds concerns the ascorbate recycling enzymes, AFR
redutase and DHA reductase. The DHA reduction capabil-
ity is low in recalcitrant seeds, but is high in the orthodox
ones. In contrast, AFR reductase activity is high in recalci-
trant seeds and low in the orthodox ones (Tommasi et al.,
1999).
AsA is a major primary antioxidant, reacting directly with
hydroxyl radicals, superoxide and singlet oxygen (Foyer,
1993; Buettner and Jurkiewicz, 1996). In addition to its im-
portance in photoprotection and the regulation of photo-
synthesis (Forti and Elli, 1995), AsA plays an important role
in preserving the activities of enzymes that contain pros-
thetic transition metal ions; and is also a powerful second-
ary antioxidant, reducing the oxidized form of a-tocopherol,
an important antioxidant in nonaqueous phases (reviewed
by Noctor and Foyer, 1998). Trichilia dregeana seed is a
recalcitrant seed, and is highly desiccation sensitive (Han
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004804
et al., 1997; Kioke et al., 1998; Drew et al., 2000). No
dehydrin-related polypeptides were detected in the mature
seeds, and seedlings of T. dregeana did not produce
dehydrins in the roots or cotyledons when exposed to ABA
or water-deficit-related stresses (Han et al., 1997). In the
present study, we have focused on the relationship be-
tween desiccation sensitivity and five main antioxidant
enzymes (SOD, APX, CAT, GR and DHAR) involved in
ascorbate-glutathione cycle and the antioxidant role of AsA
in desiccation sensitivity of T. dregeana axes.
1 Materials and Methods
1.1 Plant materials
Seeds of Trichilia dregeana Sond. were collected at
maturity from trees growing in Durban of South Africa
(rainfall 1 018 mm per year, mean temperature 20 °C with a
mean winter minimum about 10-12 °C and a mean summer
maximum about 30 °C) in March and April, 1999. And they
were soaked in water for 10-12 h to facilitate removal of the
waxy aril, cleaned in water, and then surface-sterilized in a
solution of 1% hypochlorite, and rinsed three times in ster-
ilized water. The axes were taken out from the seeds and
were treated.
Axes were first pre-dehydrated for 6 h, their water con-
tents were declined to about 0.40-0.45 g H2O/g DW (g/g),
and then axes were treated for 16 h at 25-28 °C in different
concentrations of AsA, and further dehydrated.
1.2 Desiccation treatments
Axis dehydration was achieved by placing axes taken
out of the fresh seeds in the small boat, and then the boat
was put over activated silica gel for different time within
closed plastic bucket at room temperature (25-28 °C).
1.3 Water content determinations
Water content of axes was determined gravimetrically
(80 °C for 48 h). Twenty axes were sampled each time for
these determinations. Water contents are expressed on a
dry mass basis (g/g).
1.4 Germination assessment
Batches of 20 axes were germinated on moist filter paper
in closed Petri dishes for 3 d in the dark at (24±1) °C. The
axes showing marked increase in length and volume and
appearing light green were counted as survived after dehy-
dration and subsequent re-imbibition or -germination. But
the axes showing no increase in length and volume and
appearing dark brown were counted as died.
1.5 Definition of W50
The W50 was used to describe the water content at
which 50% of T. dregeana axes have been killed by
dehydration.
1.6 Conductivity tests
Electrolyte leakage of individual axis was measured us-
ing a CM100 multicell conductivity meter (Reid and
associates, Durban, South Africa) for 6 h for seven
replicates. For directly dried axes, axes were placed in 1 mL
distilled water and conductivity of leakage was measured
immediately. For axes treated in AsA, axes were rinsed five
times in distilled water after desiccation, water of axes sur-
face was dried in paper towels, and then the axes were
placed in 1 mL distilled water and conductivity of leakage
was measured. Leakage rate was expressed in mS.cm-1.
mg-1 DW.h-1.
1.7 Assay of SOD
Forty axes of T. dregeana were homogenized to a fine
powder with a mortar and pestle under liquid nitrogen. Sub-
sequently soluble proteins were extracted by grinding the
powder in an extraction mixture composed of 50 mmol/L
phosphate buffer, pH 7.0, 1.0 mmol/L EDTA, 0.05% (V/V)
Triton X-100, 2% (W/V) PVPP and 1 mmol/L AsA. The ho-
mogenate was centrifuged at 16 000g for 15 min, after which
the supernatant was transferred to a new tube and keep at
-20 °C.
SOD (EC 1.15.1.1) activity assay was based on the
method of Beauchamp and Fridovich (1971), who measured
inhibition of the photochemical reduction of NBT at 560
nm, modified as follows. The 3 mL reaction mixture con-
tained 50 mmol/L phosphate buffer, pH 7.8, 0.1 mmol/L
EDTA, 13 mmol/L methionine, 75 mmol/L NBT, 16.7 mmol/L
riboflavin and enzyme extract (ca. 50 mg protein). Ribofla-
vin was added at last and the reaction was initiated by
placing the tubes under two 9-W fluorescent lamps. The
reaction was terminated after 15 min by removal from the
light source. An illuminated blank without protein gave the
maximum reduction of NBT, therefore, the maximum absor-
bance at 560 nm. SOD activity (mean of five replicates) is
presented as absorbance of sample divided by absorbance
of blank, giving the percentage of inhibition. In this assay,
1 unit of SOD is defined as the amount required to inhibit
the photoreduction of NBT by 50%. The specific activity
of SOD was expressed as unit/mg protein.
1.8 Assays of APX, CAT, GR and DHAR
A fine powder of 40 axes homogenized in a mortar under
liquid nitrogen was extracted by grinding in 5 mL of 50
mmol/L Tris-HCl, pH 7.0, containing 20% (V/V) glycerol, 1
mmol/L AsA, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 1
mmol/L GSH, 5 mmol/L MgCl2 and 1% (W/V) PVPP. After
two centrifugation steps (6 min at 12 000g and 16 min at
26 900g, respectively), the supernatant was stored at
-20 °C for later determinations of enzyme activities of APX,
SONG Song-Quan et al.: Desiccation Sensitivity of Trichilia dregeana Axes and Antioxidant Role of Ascorbic Acid 805
CAT, GR and DHAR.
APX (EC 1.11.1.7) was assayed as the decrease in ab-
sorbance at 290 nm due to AsA oxidation, by using the
method of Nakano and Asada (1981). The reaction mixture
contained 50 mmol/L potassium phosphate, pH 7.0, 1 mmol/
L sodium ascorbate, 2.5 mmol/L H2O2 and enzyme source
(ca. 50 mg protein) in a final volume of 3 mL at 25 °C.
CAT (EC 1.11.1.6) activity was determined by directly
measuring the decomposition of H2O2 at 240 nm as de-
scribed by Aebi (1983), in 50 mmol/L potassium phosphate,
pH 7.0, containing 10 mmol/L H2O2 and enzyme source (ca.
50 mg protein) in a final volume of 3 mL at 25 °C.
GR (EC 1.6.4.2) was determined as the decrease in ab-
sorbance at 340 nm due to the oxidation of NADPH, ac-
cording to Halliwell and Foyer (1978) in 50 mmol/L Tris-HCl
buffer, pH 7.5, containing 5 mmol/L MgCl2, 0.5 mmol/L GSSG,
0.2 mmol/L NADPH and enzyme extract (ca. 100 mg protein)
in a final volume of 3 mL at 25 °C.
DHAR (EC 1.8.5.1) was assayed directly by following
the formation of AsA at 265 nm, according to Hossain and
Asada (1984). The reaction mixture contained 50 mmol/L
potassium phosphate, pH 7.0, 0.5 mmol/L DHA, 2.5 mmol/L
GSH and enzyme extract (ca. 100 mg protein) in a final vol-
ume of 3 mL at 25 °C.
1.9 Lipid peroxidation product
Lipid peroxidation was determined as the concentration
of TBA-reactive products, equated with MDA, as origi-
nally described by Heath and Packer (1986), but modified
as in Hendry et al. (1993), where the products were quanti-
fied from the second derivative spectrum against standards
prepared from 1,1,3,3-tetraethoxypropane. All determina-
tions are means of five replications, and content of TBA-
reactive products (MDA) was expressed as nmol/mg protein.
1.10 Protein assay
Protein was measured following the procedure of
Bradford (1976), using BSA as a standard.
1.11 Statistical analysis
All data were analyzed using a one-way ANOVA model
from the SPSS 12.0 package for Windows (SPSS Inc.).
2 Results
2.1 Changes of water content and germination percent-
age during dehydration of T. dregeana
Water contents of whole seed and axis of T. dregeana
were about 1.4 and 2.0 g/g. Water content and survival of T.
dregeana axes progressively declined with dehydration
(Fig.1). Survival of axes, however, remained 100% at water
content of 0.3 g/g; but decreased to 35% at 0.11 g/g (Fig.
1b). The W50 for survival of T. dregeana axes was about
0.16 g/g (Fig.1). These results showed that axes of T.
dregeana were desiccation sensitivity, and gradually lost
desiccation tolerance with dehydration.
2.2 Effects of AsA treatment on desiccation tolerance of
T. dregeana axes
The desiccation tolerance of T. dregeana axes was sig-
nificantly increased by 2.5-15 mmol/L AsA, and decreased
by higher than 15 mmol/L AsA (P = 0). Survival of axes
increased from 24.7% in control to peak in 5 mmol/L AsA,
and then decreased from peak with increasing AsA
concentration; the optimum concentration of AsA appeared
to be 5 mmol/L for increase in desiccation tolerance of axes
(Fig.2a).
Figure 2b also indicates that desiccation tolerance of T.
dregeana axes gradually lost with dehydration as describe
in Fig.1, and that 2.5 and 5.0 mmol/L AsA could increase
desiccation tolerance of axes and decrease the W50 for
survival of axes. W50 for survival of axes treated with
0, 2.5 and 5.0 mmol/L AsA were about (0.18 ± 0.010), (0.15 ±
0.008) and (0.13 ± 0.008) g/g, respectively shown in Fig. 2b
(P = 0.001).
Fig.1. The changes of water content (a) and survival (b) during
dehydration of Trichilia dregeana axes. The axes were immedi-
ately dehydrated over the activated silica gel at 25-28 °C after
taking out of fresh seeds. The axes showed marked increase in
length and volume and appeared light green were counted as survived.
All values are means ± SD of three replicates of 20 axes each.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004806
2.3 Changes in the leakage rate of electrolyte during
dehydration of T. dregeana axes and effects of AsA treat-
ment on leakage rate of electrolyte
Electrolyte leakage rate of T. dregeana axes dramati-
cally increased with dehydration (P = 0, Fig.3a). For example,
when water content of axes declined from 0.81 to 0.25 and
0.08 g/g; electrolyte leakage rate of axes increased from
0.96 to 2.28 and 3.83 mS.cm-1.mg-1 DW.h-1, increased by
about 138% and 300% than that of control, respectively (Fig.3a).
AsA treatment could decline electrolyte leakage rate of
T. dregeana axes, the optimum concentration of AsA was
about 5 mmol/L (P = 0, Fig. 3b).
2.4 Changes in the activities of antioxidant enzymes
during dehydration of T. dregeana axes
Activities of five antioxidant enzymes, SOD, APX, CAT,
GR and DHAR, were monitored during dehydration of T.
dregeana axes (Fig.4). Activities of SOD and APX were
significantly decreased with dehydration of axes (P =
0). Water content of axes was dehydrated from 1.78 to 0.12
g/g; Activities of SOD and APX decreased by about 30%
and 20.7%, respectively (Fig.4a).
Activities of CAT and GR in axes firstly increased, and
then decreased with increasing dehydration (P = 0), activ-
ity peak of CAT and GR was about at (0.120 ± 0.006) g/g
water content of axes (Fig.4b). DHAR activity of axes,
however, showed a little increase at the initial phase of
dehydration and then a rapid decrease (P = 0), activity peak
of DHAR was about at (0.711 ± 0.013) g/g (Fig.4c).
2.5 Effects of AsA treatment on activities of antioxidant
enzymes in T. dregeana axes
Activities of SOD (P = 0), APX (P = 0.028), CAT (P =
0.001), GR (P = 0.003) and DHAR (P = 0) in T. dregeana axes
were significantly affected by AsA treatments (Fig.5). Ac-
tivities of SOD, APX, CAT and GR in axes were increased
by 2.5-10 mmol/L AsA treatments, and decreased by AsA
treatment higher than 15 mmol/L AsA treatments (Fig.5a,
b). Two point five-20 mmol/L AsA treatments, however,
markedly declined DHAR activity of axes (Fig.5c).
Fig.2. Effects of different concentrations of AsA on desiccation
tolerance of Trichilia dregeana axes. After pre-dehydration, axes
were treated with indicated concentration of AsA, and dehydrated
to single water content of (0.169 ± 0.030) g H2O/g DW (a) and to
different water contents (b) over activated silica gel, and then
measured the survival as described in Material and Methods. All
values are means ± SD of three replicates of 20 axes each.
Fig.3. Changes of electrolyte leakage rate during dehydration of
Trichilia dregeana axes (a) and effects of AsA on leakage rate of
electrolyte (b). For directly dried axes, dehydration of axes was
conducted as described in Fig.1. For axes treated with AsA, pre-
dehydration, AsA treatment and subsequent dehydration of axes
were conduced as described in Fig.2, but water content of axes is
(0.131 ± 0.018) g H2O/g DW. Individual axis was placed in 1 mL
distilled water and then the conductivity of leakage was immedi-
ately measured. All values are means ± SD of seven replicates.
SONG Song-Quan et al.: Desiccation Sensitivity of Trichilia dregeana Axes and Antioxidant Role of Ascorbic Acid 807
2.6 The accumulation of lipid peroxidation products dur-
ing dehydration of T. dregeana axes and the effects of AsA
treatment on lipid peroxidation in axes
The content of TBA-reactive products in T. dregeana
axes slowly increase at the initial phase of dehydration,
and then rapidly increased (Fig.4c). The increase in TBA-
reactive products in axes is strongly correlated with the
decline of axis survival (Fig.1) and with the activities of
antioxidant enzymes (Fig.4).
The content of TBA-reactive products in T. dregeana
axes was declined by 2.5-10 mmol/L AsA treatment, and
increased by 15 and 20 mmol/L AsA treatments (Fig.5c) (P
= 0.004). After treatment of AsA, the decrease in TBA-reac-
tive products in axes is strongly correlated with the in-
crease in the activities of SOD, APX, CAT and GR, the
increase in TBA-reactive products, on the contrary, and
the decrease in the activities of SOD, APX, CAT and GR
(Fig.5a, b).
3 Discussion
The dry weight of axis and whole seed in T. dregeana
seeds were about 0.007 5g and 1.253g respectively; the
ratio of axis to whole seed was about 0.60%. Whole seeds
of T. dregeana lost viability progressively with increasing
dehydration, no seeds remained viable by the axis water
content at 0.55 g/g. Flash-dried, excised axes could main-
tain a high level of viability by the 0.16 g/g (Kioko et al.,
Fig.4. Changes in the activities of SOD, APX, CAT, GR and
DHAR and in content of TBA-reactive products during dehydra-
tion of Trichilia dregeana axes. Axes were dehydrated to indicate
water content. Activities of enzymes and content of TBA-reac-
tive products were assayed as described in Material and Methods.
All values are means ± SD of five replicates.
Fig.5. Effects of AsA treatment on activities of SOD, APX,
CAT, GR and DHAR and on content of TBA-reactive products
in Trichilia. dregeana axes. Pre-dehydration, AsA treatment and
subsequent dehydration of axes were conduced as described in
Fig.2, and axes were dehydrated to (0.131 ± 0.018) g H2O/g DW.
Activities of enzymes and content of TBA-reactive products
were assayed as Material and Methods. All values are means ±
SD of five replicates.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004808
1998). The results by Kioko et al. (1998) indicated that des-
iccation tolerance of T. dregeana axes was higher than that
of whole seed. Survival of T. dregeana axes gradually lost
with increasing dehydration, and W50 for axes survival was
about 0.16 g/g (Fig.1). These results showed that axes of T.
dregeana were very sensitive to desiccation, and that re-
calcitrance of T. dregeana axis is not an all-or-nothing situ-
ation (Berjak and Pammenter, 1994) and is a quantitative
feature (Vertucci and Farrant, 1995; Walters, 1999).
The desiccation tolerance of T. dregeana axes is signifi-
cantly changed by the treatment of 2.5-20.0 mmol/L AsA
(Fig.2). Two point five and 5.0 mmol/L AsA could increase
desiccation tolerance of axes and decrease the W50 for sur-
vival of axes (Fig.2).
Sensitivity to desiccation in orthodox and recalcitrant
seed tissues can be expressed in electrolyte leakage rate
(Berjak et al., 1993; Vertucci et al., 1993; Leprince et al.,
1995). Electrolyte leakage rate of T. dregeana axis dramati-
cally increase with dehydration (Fig.3a), showing that the
membrane function of T. dregeana axis is damaged with
dehydration. AsA could significantly decrease electrolyte
leakage rate of axes (Fig.3b), indicating that AsA could im-
prove the membrane function during dehydration of T.
dregeana axis.
O2
-. and H2O2 are synthesized at very high rates in the
cells even under optimal conditions. They are involved in
virtually all major areas of aerobic biochemistry and are
produced in copious quantities by several enzymes sys-
tems (e.g. plasmalemma-bound NADPH-dependent super-
oxide synthase and SOD). The chief toxicity of O2
-. and
H2O2 is thought to reside in their ability to initiate cascade
reactions that result in the production of the hydroxyl radi-
cal and other destructive species such as lipid peroxides
(reviewed by Noctor and Foyer, 1998). Efficient destruc-
tion of O2
-. and H2O2 requires the action of several antioxi-
dant enzymes acting in synchrony. Superoxide produced
in the different compartments of plant cells is rapidly con-
verted to H2O2 by the action of SOD. CAT converts H2O2
to water and O2. An alternative mode of H2O2 destruction
is via peroxidase, which is found throughout the cell and
which has a much higher affinity for H2O2 than CAT. In
plant cells, the most important substrate for H2O2 detoxifi-
cation is AsA. APX uses two molecules of AsA to reduce
H2O2 to H2O and to form DHA. DHA is reduced to ascor-
bate by DHAR, using GSH as the reducing substrate. This
reaction generates GSSG, which is in turn re-reduced to
GSH by NADPH, a reaction catalyzed by GR (Smith and
Berjak, 1995; Côme and Corbineau, 1996; Noctor and Foyer,
1998; McDonald, 1999). During dehydration of T. dregeana
axes, activities of SOD and APX significantly decreased;
and those of CAT, GR and DHAR showed marked increase
at the initial phase of dehydration, and then rapid decrease
(Fig.4). These results indicated that changes in activities of
antioxidant enzymes were closely related to desiccation tol-
erance of T. dregeana axes; and are similar to those of Li
and Sun (1999), who found that activities of SOD, APX and
peroxidase significantly decreased with dehydration of im-
mature and mature Theobroma cacao axes. The treatment
that prolongs the seed storage life increases SOD activity
and decreases the O2
-. level (Chaitanya and Naithani, 1998).
Lipid peroxidation has considerable potential to dam-
age membranes and may be a principal cause of seed dete-
rioration (Smith and Berjak, 1995; McDonald, 1999). Loss
of viability and declining vigor were associated with in-
crease in lipid peroxidation in rapidly aged soybean seeds
(Khan et al., 1996). Viability loss during desiccation in the
recalcitrant seeds of Quercus robur (Hendry et al., 1992;
Finch-Savage et al., 1994), Castanea sativa and Aesculus
hippocastanum (Finch-Savage et al., 1994), and Clausena
lansium (Wu et al., 2002) was accompanied by increased
lipid peroxidation. During dehydration of T. dregeana axes,
the content of lipid peroxidation (TBA-reactive products)
markedly increased and is also strongly correlated with the
decline in the activities of SOD, APX, CAT, GR and DHAR
(Fig.4). The reason why 2.5-10.0 mmol/L AsA treatment
declined the content of TBA-reactive products in T.
dregeana axes could be that they increased activities of
SOD, APX, CAT and GR (Fig.5). C. lansium seeds treated
by 5 mmol/L AsA showed increasing desiccation tolerance
and SOD activity and decreasing content of TBA-reactive
products (Song and Fu, 1997). AsA is a water soluble com-
pound capable of reacting with free radicals (R×) and O2
-.
and ×OH (reviewed by McDonald, 1999).
Acknowledgements: We are grateful to Mr. D Erdey, Mr.
T Ntuli, Mr. A Schrueder, Mr. J Kioko, Mrs C Calistru and
Mrs M Norris for providing some experimental help.
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(Managing editor: HE Ping)