J.Japan.Soc.Hort.Sci.63(2) : 453-459.1994.

A Simple Method for Vacuum Extraction

Internal

Ethylene

and Quantitative

of Excised

Determination

of

Apple Tissue

Tomonori Kawano* and Keishi Shimokawa

FacultyofAgriculture,
Miyazaki
University,
Miyazaki
889-21
Summary
A rapid and simplemethodfor extractinginternalethylenefromexcisedappletissue
and its quantitativedetermination
are described.The methodis suitablefor correlating
the ethyleneconcentration
withthe physiological
statusof the planttissues.Oneadvantage was that the timerequiredfor the tissueexcision,vacuumextractionof ethylene,
and injectionof a gas sampleintoa gas chromatograph
wasabouta minute.Themethod
providesaccurateand reproducible
results,i.e. ca. 80%or moreof the internalethylene
in appletissuecan be extractedand determined.Anotheradvantageof this methodis
that the tissuefromwhichethylenewasextractedis still availableforfurtheranalyses.
Themethodwasappliedto the studyethyleneregeneration
in appletissueafterthe removalof the internalethylene.Theformationof ethylenein appletissueincreasedafter
the existingethylenewas removedled us to proposethat ethylenesynthesisis governed
by a feed-back
inhibition.Theinhibition
(ca.20%)of ethyleneformationwassignificantly
relatedto the levelof internalethylenein appletissue.

and not internal ethylene.

It is difficult to estimate the level of ethylene existing in plant tissues from the amount of ethylene
diffused from tissue. Staby and De Hertogh (1970)
detected ethylene in the internal atmosphere
of 5
bulb species. Vacuum extraction
of ethylene from

Introduction
Ethylene is the simplest olefin which regulates
various aspects of plant growth, development, and
senescence. Under normal physiological
conditions
it exists in plant as a gaseous hormone. Even in
the earlier stages of ethylene research, gas chromatography
has been used for its determination
because the instruments
can accurately
detect minute quantities of the gas. Ethylene diffusing from

plant tissues has been conducted

by Beyer and
Morgan
(1970).
Blanpied
(1971)
reported
a
method of extracting ethylene from fresh plant tissue, but the apparatus consisted of a lot of rubber
materials.
We have divised a new, simple and
sensitive method for withdrawing internal ethylene
from tissues to determine the concentration
with a
high degree of sensitivity.
A method like this has
been needed to concurrently
associate the occurring phenomena inside plants and the quantity of
internal ethylene. We have applied this method to
the study
of the biofeed
back regulation
of
ethylene synthesis in apple tissues.

plant tissues when placed in a desiccator can be

detected and assayed through gas chromatography,
using a gastight syringe for sampling. The syringe
and all the other instruments
must be made of
materials
which
do not produce
or adsorb
ethylene; hence, rubber stoppers must be avoided.
When the concentration
of ethylene is extremely
low, mercuric
perchloride
(MP) solution can be
used to trap the ethylene; the gas is liberated by
treating the MP/ethylene
complex with LiCI or
HCl (Young et al., 1952). Whether MP is used or
not, the ethylene detected is that which diffused
Received for publication 20 September 1993.
* Present address : School of Biological Sciences

Materials

and Methods

Mature
apple fruit (Malus pumila Mill. var.
domestica Schreid L. Borkh. cv. Fuji) was obtained
from a local market and stored at 4 C. The fruits
was incubated at 25 C for 24 hr before the ex-

, Uni-

versity of East Anglia, Norwich, England

453

454

T. Kawano

periments
port

and
were

all

plication,

apple

mm

thick)

the

equator

processes

conducted
tissues

were

that
mm

from

each

fruit,

in

temperature.

(10

excised

of

kitchen

described

at

in

with

re-

and

For

re-

vacuum

10

out

and

cortex
a

just

cork

K. Shimokawa

this

diameter

the

and

below

borer

close

until

Hg,

and

knife.

stopcock
the

hold

pressure

apparatus

apparatus

tissue

ml

and

and

(GL

50-l

1-3),

the

cm)
mesh,

GL

2.

and
1.

sampling
(Fig.

the

a
Fig.

column

into

of

ethylene;

2)

apple

tissue

while

Insert

the

cone

resin

rated

to

to

mm

negative

from

TR ~ (60

steps;

the

apple

the

gas

sam-

sampled

the

piston

piston

rod

rod
into

is
the

the

gas

To

removed

tle

cylinder

ure

(A

excision

bottle

bottle

2-7).

by

insert-

the

and

gas

sampling

(GL

Science

cylinder

silicone

then

inject
for

is

the
analy-

with

stop

sampling

gauge

(GL

type

bot-

inlet

kgEcm

press-

-2,

GL

sci-

used.

preparation

of

must

be

ethylene

to
is

1 `

3)

tissue

done

prior

quickly

before

to

measurement.

in

this

extracting

estimate

experiment

tissue.

efficiency

the

impossible

be

we

In

ethylene
method

from
by

The

piston
the

rod

according
until

CoCl2

solution.

to
the

the

tissue
The

infil-

in

ml

the

of

became

into

the

cylin-

was

described
saturated

of

gas

10 M

Ethylene

previously

of

vacuum

inserted

plugs

ethylene

tissue
10

closed.

estimate

procedure

the
the

was

stopcock

to

test

tissue,

containing

ethylene

internal

this

immersing

cylinder

all

attempted
of

extracted.

efficien-

extract

percentage

could

extraction

to

Therfore

approximate

cedure

min

expro-

with

apple

tissue

the
was

into
after

vials
the

and

gas

incubated

extraction.

at

25

Ethylene

for

that

120

diffused

Science)

(Hamilton,
(A

sampling
an

cock

Tokyo)

bottle

syringe

was

of ethylene

it

and

gas

seconds.

difficult

the

the

60 -760`3

(step

20

since

placed
Ltd

cylinder,

of

Test

tracted

1. The apparatus for extracting internal ethylene

of apple tissue and an inlet pressure gauge.

in

preparation
only

der

science)

the

(Fig.

through

pressure

and

loss

CoCl2.

GL

containing

chromatograph

ethylene

sampling

pressure

Open

air

sampling

the

sampling

1-4)

extracting

tration

Inlet

siliaspi-

2-6).

inside

2-8)

gas

the

bottle.
gas

syringe
(Fig.

type

Fig.

extracting

kgcm-2,

the

sampling

bottle

sampling
of

inside

the

gas

Ltd.,

that

gastight

into

the

gauge

the

gas

of

(Fig.

gas

the

pressure

bottle

determine

and

from

50-,u1

(Fig.

gas

gastight
the

gas

It

50-m1

ml

sis.
in

cy

3.

20

through

sampling

let

cock

atmosphere

of

Exp. 1.

2.

in

the

it

vacuum

the
the

50-l

stopper

took

needle

let

immediately

of

gas

and

into

the

ing
1)

the

Hg

Open

tip
insert

atmospheric

Sample

Each

100-m1

to
it

the

of

mm

flow
3.

minimize

4.

a
rod

90

The

out

cock

and

stopcock

The

1.

2-3).

close

on

2-5)

stopper

restore

to

and

stop

and

needle

80

Step

`8

divided

a
(Fig.

ethylene

(200

alumina

Fit

ence

Fig.

the

create
piston

reaches

(Fig.

ethylene

air

cylinder's

Nevada,

glass

be

weighed

cylinder
2-1).

1-1),

determination.

Place

2-4).

100-

Ltd.).

can
extraction

3)

Step

cylinder
sec

the

Open

cylinder

(Shimadzu

activated

Science

procedure

vacuum

pling;

with

Ltd.,

(Hamilton,

with

ap-

stopcock

Fig.

Science

chromatograph

fitted

packed

Tokyo,

(GL

syringe
gas

of

with

Ltd.

bottle

Tokyo)

0.32

The

Science

from

consists
equipped

gastight
and

ethylene

determination

sampling

GC-8AIF,

15

To
the

procedure

extracting

syringe

needle
gas

Fig.

its

sampling

50-m1
1-2),

for

and

gas

the

for

forces

ethylene-free

ple

in
it

2-2).
slide

tissue.
of

The

(Fig.
cylinder,

vacuum

then

Step
Description

the
inside

Nevada)
60

0-760 E3

from
30-min

the

tissue
intervals.

in

the

vials

was

sampled

at

15-and

455

J. Japan. Soc. Hort. Sci. 63 (2) : 453-459. 1994.

Fig. 2. Step-wise
termination.

Exp. 2.

procedure

Further ethylene formation

of internal ethylene

Two apple

plugs

were

excised

for extracting

after the removal

from the cortex

ethylene

from excised

apple tissue

for ethylene

de-

just below the equator of a fruit and the ethylene

in one plug was removed and the sample assayed
as above. The extracted plug was transferred
to a
10-ml vial with a silicone cap. The second plug

456

T. Kawano

which

had

placed

in

were

undergone

in

at

both

gastight
for

as

25

vials
at

and

cubation

period

estimate

of

min.

ethylene

The

atmoswith

intervals

amounts

.two

vials

separating

of

diffused

the

was

Both

30-min

which
by

the

120

sampled

The

that

extraction
control.

15-and

ethylene.

ethylene

for

were

syringe

assayed

ethylene

vial

incubated

phere
a

not
another

plugs

produced

and

and

the

initial

during

the

in-

should

give

an

after

the

initial

extraction.
Results

Reproducible

accuracy

Twelve
4

tracting

were

plugs

per

ethylene.

each

plug

and

injected

mate

accuracy.
of

imately

one

series

of

amounts

each

of

ethylene

After
ment,
cubated

for

were

120

sampled
in

at

Table

2.

maining

in

traction

diffused

presented

the

are

took

com-

approx-

Data

from

each

almost

the

same

from

each

extracted

from

the

method

of

apple

tissue

is

efficiency

of

ethylene

plugs

were

min

during

15-

or

and

placed

all

apple

tissue

averages

in

which

30-min

Almost

out
the

esti-

the

accurate.

extraction
apple

to

that

extracting

the
the

syringe

to

it

been

and

exfrom

tissue

that

ethylene

reproducible

Test

of

demonstrating

internal

highly

with

replicate.

had

1),

measuring

times

showed

ethylene

(Table

fruits
for

chromatography

extraction,

for

apple
used

extracted

excision

replicates
of

tissue

gas

ethylene

min

were

ethylene
four

the
From

the

from

which

sampled
into

samples

excised
fruit

The

was

pletion

Discussion

of replicate

tissues

yielding

are

and

the

intervals.

120
of

the

internal
after

within

CoCl2

quadruple

treat-

vials

the
min.

and

gas

in

invials

The

A small percentage
of diffused ethylene in the
vials was considered
to be new ethylene produced
after the extraction
operation
because ethylene
synthesis
was not inhibited
perfectly
despite
CoCl2 treatment (ca. 30% inhibition of control for
120 min). The amount of diffused in the vials gradually
ceased increasing
within the 120
min
(Table 2). Further
apparent
increase in ethylene
concentration
in the vials could not be observed
throughout
the few hours
after the 120
min
measurement
(data not shown). The mean of the
extracted
ethylene was ca. 75 nl and that of the
diffused ethylene sampled at the end of the incubation period was ca. 18 nl. Assuming
all the
ethylene that diffused out in the vials had already
existed before extraction
and that all the internal
ethylene in apple tissue which had not been extracted
diffused out within 120 min period, the
tests indicate that more than ca. 80% of the internal ethylene
was extracted
from apple tissue
through
this procedure.
This efficiency
is high
enough to be useful in many plant biological and
horticultural
studies.
Further ethylene formation
nal ethylene

after the removal of inter-

The results are from a typical experiment which

was replicated
18 times. Though a larger amount
of ethylene diffused from the control tissue than
from the ethylene-extracted
tissue during the incubation
period,
incremental
rates of ethylene
diffusion after the first 15-min measurement
was

data

ethylene

re-

internal

ex-

The

K. Shimokawa

data

injections.

Table 1. Reproducibility of extracting ethylene from 4 plugs

of apple tissue excised from 3 different fruits.

Table 2. Time-course of cumulative ethylene evolved and evolution

rate by 'Fuji' apple tissues after vacuum extraction of internal
ethylene during a 2-hr incubation.

J. Japan. Soc. Hort. Sci. 63(2) : 453-459. 1994.

incubation
period was considered
to be total
ethylene production for the ethylene-extracted
tissue. The summation of the ethylene which was diffused during incubation
and that extracted
after
the incubation period was considered to be the total ethylene production
for the control tissue (Fig.
3). Apparently
more ethylene was produced by the
ethylene-extracted
plugs than the control plugs indicates that ethylene synthesis
in excised apple
tissue was enhanced by the removal of internal
ethylene. However in a few replicates,
such enhancement of ethylene production
was not detectable when the volume of internal
ethylene
extracted
was very small. The enhancement
of
ethylene
production
was proportional
to the
amount of extractable
ethylene in the tissue (Fig.
4). We attribute
the difference
in the rate of
ethylene production between two tissues to the difference in residual concentrations
of the internal
ethylene because the amounts of ethylene at the
end of the incubation
periods were very similar

Table 3. Cumulative ethylene evolved (C) and evolution

rates (R) from vacuum extracted 'Fuji' apple plugs
vs untreated control plugs.

higher in the vial of tissue which had undergone

ethylene extraction (Table 3). The concentration
of
CO2 in both vials at the end of the incubation
period was less than 0.3% which is statistically
negligible
(Bufler,
1986).
Quantitatively,
very
similar amounts
of internal
ethylene
were extracted from the paired plugs after the incubation

(Fig. 3) and the rate of ethylene production

gradually became closer (Table. 3).
Ethylene is known to regulate its own biosynthesis, both positively
and negatively.
A negative

period. Comparison of the total ethylene produced

from the test and the control tissues reveals that
the summation of the extracted ethylene before incubation, the diffused ethylene from the incubation
period,

and the ethylene-extracted

Fig.

3.

again

after

Comparison
from

the

column

represent

tracted

before

which

diffused

tracted

at
). The

the

control

phenomenon
biosynthesis

the

of total

excised

ethylene

opposite
the

end

right
apple

of

in

the

column
tissues.

of

summation

(middle, );

the

produced

sides

incubation

457

102

vial

between

the

same

of

internal

(upper, );
the

min

incubation

symbols

internal

the
are

two

apple.

ethylene

the

ethylene

ex-

ex(lower,

ethylene
same

left

ethylene

period

total

plugs

The

the

and

represents
The

in which
ethylene
inhibits
its
is known as autoinhibition.
This auto-

as

from
above.

458

T. Kawano

and K. Shimokawa

Fig. '4. Relationship between the amount of initial

enhanced ethylene production in excised apple
The graph represents
the relationship between
hibition of ethylene production and the content
the tissue.

inhibition
has been recognized
in a number of
fruits and vegetative
tissues (Saltveit and Dilly,
1978; Vendrell and IvIeGlasson, 1971; Yang and
Hoffman,
1984; Zauberman
and Fuchs,
1973).
Vendrell
and McGlasson
(1971)
showed
that
ethylene treatment
significantly
inhibited
wound
ethylene production
in banana pulp slices. Likewise, slicing of flavedo tissue of citrus fruit enhanced
wound
ethylene
production,
but this
ethylene production
was greatly inhibited by exogenous ethylene (Riov and Yang, 1982). Saltveit
and Dilly (1978) demonstrated
that autoinhibition
of ethylene production
occurred
in pea segments
by exposing them to exogenous ethylene or propylene. They found that the inhibition was rapidly
reversed following the removal of ethylene.
In our experiments,
ethylene biosynthesis
in the
evacuated tissue was promoted, whereas it was inhibited in the control plugs. We conclude from
these results that autoinhibition
of ethylene production naturally
occurs in apple fruit when the
concentration
of internal ethylene rises.
We believe that the ethylene produced
by the
excision of apple tissue during the sampling period
is minimal because it requires
only about a mi-

ethylene extracted and

tissue within 120 min .
the strength of autoinof internal ethylene in

nute. In the efficiency test, ethylene formation by

wounding/excision
was inhibited
by CoCl2, a
known inhibitor of ethylene biosynthesis.
Howev-er , Smith et al. (1992) reported that Co2 appears
to stimulate the process at concentrations
above
ca. 20 MM. Ethylene
formation
via ACC (1aminocyclopropane-l-carboxylic
acid) oxidase by
excised plugs was inhibited by 10 MM Co2+ compared to the non-treated plugs (data not shown). In
the last experiment,
in order
to study
how
ethylene is formed in tissue after the removal of
internal ethylene, we did not use any inhibitor of
ethylene biosynthesis.
Thus, the control tissue and
the treated tissue continued
to produce ethylene
during
the
incubation
period.
If significant
amounts of ethylene were produced as a result of
wounding,
ethylene production
should have been
promoted in all samples, irrespective
of internal ethylene (Fig. 4).

of the level

Acknowledgement
We would like to show our gratitude
to Dr.
David Wildon, School of Biological Sciences, University of East Anglia, England, for critical reading of the manuscript
and thank Mr. K. Nakachi

459

J. Japan. Soc. Hort. Sci. 63(2) : 453-459. 1994.

for his cooperation

in this

investigation.

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ometric
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