Harker and Maindonald, 1994; Inaba et al., 1995. The advantage of EIS is that electrical
circuit diagrams
can be
used to
interpret impedance spectra in terms of components of
resistance and capacitance associated with struc- tures at the cellular level Zhang et al., 1990;
Zhang and Willison, 1991.
Electrical impedance studies have provided a useful insight into ripening of nectarines Harker
and Dunlop, 1994; Harker and Maindonald, 1994, persimmon Harker and Forbes, 1997 and
tomato Varlan and Sansen, 1996. For example, the resistance of the apoplast declined by 60
when nectarine fruit were ripened immediately after harvest, and differences in cell wall resis-
tance were associated with the presence and ab- sence of woolly texture Harker and Maindonald,
1994. Furthermore, the development of chilling injury during storage of New Zealand-grown
‘Fuyu’ persimmon fruit was indicated by changes in the cytoplasmic resistance Harker and Forbes,
1997. In the present study, we have extended this research to kiwifruit; which has been used as a
model for studying fruit softening MacRae and Redgwell, 1992. The kiwifruit consists of four
distinct tissue zones: skin, outer pericarp, inner pericarp and seeds, and the core. Each of these
tissues differs in firmness Jackson and Harker, 1997, mineral and sugar contents Ferguson,
1980; MacRae et al., 1989, cell wall composition Redgwell et al., 1991, 1992 and cell characteris-
tics Hallett et al., 1992. The core is composed of a single population of sphericalellipsoidal cells
0.1 and 0.2 mm diameter, whereas the outer pericarp is composed of a population of large cells
0.5 – 0.8 mm diameter dispersed in a matrix of smaller cells 0.1 – 0.2 mm diameter Hallett et al.,
1992. The inner pericarp consists of the locules, which are enclosed within locule walls Hallett et
al., 1992. Each locule is composed of large radi- ally elongated thin-walled cells 0.2 – 0.4 mm × \
1 mm and seeds, while the locule wall is a narrow region
composed of
smaller thicker
walled cells Hallett et al., 1992. In this study, the
impedance characteristics of individual tissues, outer pericarp, inner pericarp and core, as well as
whole fruit, have been examined during fruit ripening.
2. Material and methods
2
.
1
. Plant material Kiwifruit Actinidia deliciosa C.F. Liang et
A.R. Ferguson cv. ‘Hayward’ weighing 130 – 150 g were harvested at commercial maturity from
orchards in the Bay of Plenty New Zealand in May 1995, 1996 and 1997, and stored at 0°C.
Fruit were stored for various lengths of time before they were ripened. Ripening was initiated
using a 12 h ethylene treatment 1 ml l
− 1
and then changes in firmness, soluble solids content
and electrical impedance were followed through- out the ripening process. All ripening treatments
and fruit evaluations were at 20°C, and a mini- mum of ten fruit per day was evaluated at each
assessment date. In 1995, fruit were stored for up to 10 weeks, and samples removed as required. In
1996, fruit were assessed on the day following harvest 15 May and again after 29 weeks storage
at 0°C. In 1997, fruit were ethylene-treated on 21 May, and ripening was followed over a 9-day
period.
2
.
2
. Fruit firmness and soluble solids content A patch of skin was removed from the fruit
equator and a 7.9 mm Effegi probe was driven into the flesh at 240 mm min
− 1
using an Instron materials testing machine Model 4301, Instron,
Canton, MA. The soluble solids content was determined for juice expressed from the blossom
and stem ends of each kiwifruit using a hand-held refractometer Model N-20E, Atago, Tokyo,
Japan.
2
.
3
. Preparation of tissue samples Electrical impedance was measured using a
range of different electrode arrangements and dif- ferent instruments discussion of the development
of these methods is covered in Section 3.1. In 1995, tissue samples consisted of cylindrical plugs
5 mm diameter, 25 mm length cut parallel to the fruit longitudinal axis from the outer pericarp,
inner pericarp and core. These tissue samples were connected to the measurement instrumentation
according to Repo 1994. The ends of a cylindri- cal tissue plug were inserted into insulating
tubes 30 mm long, 5 mm inner diameter filled with an electrode gel Sigma Gel, Parker Lab.
Inc., Orange, NJ. Silversilver chloride half cells AgAgCl; 2 mm diameter × 5 mm; World
Precision
Instruments, Sarasota,
FL were
inserted into the gel and connected to the measurement instrumentation. This method of
preparing the tissue samples and connecting them to instrumentation caused some prob-
lems see Section 3.1. Thus, a number of modifi- cations were made to the method in subsequent
years.
In 1996, rectangular blocks of tissue 5 × 5 × 35 mm were excised from the outer pericarp so that
one surface consisted of the fruit skin, and thus was undamaged. The tissue block was im-
paled by a linear array of four silver wire 0.5 mm diameter; World Precision Instruments, Sarasota,
FL electrodes, spaced at 10 mm intervals, as described previously Harker and Dunlop,
1994; Harker and Maindonald, 1994. In turn, silver wire electrodes 1 and 2, then 1 and 3,
and finally 1 and 4, were connected to the measurement
instrumentation. In
1997, the
impedance was measured in whole fruit and ex- cised blocks of tissue 5 × 5 × 35 mm from the
outer pericarp, inner pericarp and core. Measure- ment of excised tissues was carried out as de-
scribed for the 1996 studies. However, for whole fruit impedance measurements, two AgAgCl
half-cells were impaled 20 mm apart into fruit. Measurements were made on opposite sides of
each fruit.
2
.
4
. Electrical impedance measurements Initially in 1995, the tissue impedance compo-
nents were measured according to Zhang and Willison 1991 and Harker and Dunlop 1994,
using a function generator Thurlby Thandar Model TG230, Huntingdon, UK and an oscillo-
scope Iwatsuo Model SS-7610, Tokyo, Japan. Alternating current at frequencies between 50 Hz
and 1 MHz was passed through the tissue sam- ples. Impedance characteristics were determined
from the dimensions of an ellipse traced on the oscilloscope. In later work, impedance character-
istics were determined using either a Hewlett- Packard Model HP4194A impedance analyser
Hewlett-Packard, Hyogo, Japan, or a Hewlett- Packard Model HP4284A Precision LCR meter
Hewlett-Packard, Hyogo, Japan. The HP4194A was operated at 100 mV generator voltage, and
scanned 101 spot frequencies between 100 Hz and 10 MHz. The HP4284A was operated at genera-
tor voltages of 300 mV for excised tissues and 500 mV for whole fruit, and scanned 36 spot frequen-
cies between 50 Hz and 1 MHz.
2
.
5
. Calculations of tissue resistance and reactance
When electrical contact with the tissue sample was made via AgAgCl half cells placed into an
electrode gel or directly into whole fruit, the electrode resistance the resistance associated
with electrochemical interactions at the electrode surface was minimal, and the impedance data
could be directly used in electrical models see below. However, when an array of silver wire
electrodes was impaled into the tissue sample, it was necessary to account for the electrode resis-
tance before the data could be modelled. A linear plot of resistance and reactance against interelec-
trode distance did this. The intercept on the y axis reflects the electrode resistance or reactance
and the slope reflects the tissue resistance or reactance Zhang and Willison, 1991. Only tis-
sue resistance and reactance were considered fur- ther.
2
.
6
. Modelling of impedance in fruit tissue For each tissue sample, tissue resistance and
reactance was fitted to an electrical circuit dia- gram Fig. 1 using S-PLUS functions described
by Harker and Maindonald 1994. The model is based on the structure of plant cells, as proposed
by Zhang et al. 1990.
2
.
7
. Electrolyte leakage and mineral analysis For electrolyte leakage, fruit unripe and
ripened for 9 days at 20°C were transversely cut
into 4 mm thick slices, and 5 mm diameter discs of outer pericarp or core tissue excised using
a cork borer. It was not possible to use discs of inner pericarp tissue, due to poor tissue
cohesion during incubation in solutions. The discs were rinsed for 1 min in 0.6 osmol mannitol
corresponding to the isotonic concentration of the fruit, Harker and Hallett, 1994 and
blotted dry. For each ripening treatment, four lots of discs, about 2 g of tissue, were incubated
in 20 ml 0.6 osmol mannitol or 20 ml of water for 4 h at 25°C in a shaking water bath.
Electrolyte leakage was estimated at regular intervals throughout the experiment using a con-
ductivity meter Konductimeter CG875, Schott Geraete, Germany. The total electrolyte leakage
was measured after the discs had been freeze- thawed.
Upon completion of the experiment, the min- eral content of the tissue and an aliquot of the
leakage solution was determined. Samples were dried
at 60°C,
digested in
HNO
3
– HClO
4
, and analysed for calcium, magnesium and potas-
sium content by atomic absorption spectrophoto- metry.
3. Results and discussion
