- CSIRO Publishing

DECAPITATED PEAS AND DIFFUSIBLE GIBBERELLINS
By D. COHEN,*t J. B. ROBINSON,*t and L. G. PALEG*
[Manuscript received November 26, 1965]
Summary
Growing and treatment conditions are described for a test in which decapitated
dark-grown pea seedlings manifest an elongation response proportional to the amount
of gibberellic acid supplied in 10 1'1 agar disks. This response removes the necessity of
treating tissues or extracts or both with extraction techniques which, it has been
shown, may cause structural changes in endogenous gibberellins.
Difficulties experienced by other authors in demonstrating a gibberellic acidinduced elongation response in decapitated pea seedlings have been shown to be due
to either the transitory nature of the potential of the internode to elongate, or to the
presence of light of greater intensity than about 10 f.c. before or during the treatment
period.
I. INTRODUCTION
The course of early advancement of knowledge concerning the physiological
effects of auxins was aided largely by the development of bioassays which were
relatively specific for auxins. Among these was one, the Avena curvature test (Went
and Thimann 1937), with which it was possible to measure auxin release from, or
transport through, various tissues without subjecting either the tissue or the active
compounds to grinding, drying, or solution in organic solvents. The results, though
restricted in scope, were clear-cut and enabled significant scientific progress.
In contrast to this, gibberellin technology has employed a wide range of bioassays, all of which, however, necessitate tissue extraction with pH adjustment and
organic solvents.
Jones and Phillips (1964) have recently shown that endogenous gibberellin-like
substances can be collected by classical agar diffusion techniques, although they then
extracted the agar with conventional organic solvent extraction methods. Jones
(1964), however, has indicated that such methods may cause changes in gibberellin
structure, and consequently, activity, thus clearly demonstrating the need for a test
which employs little or no potentially damaging treatment of tissue contents. The
following is a report of the development of a pea test for diffusible gibberellins based
on the observations of Lockhart (1957). Kentzer and Libbert (1961) mentioned the
use of a test in which gibberellin transport was measured by applying gibberellin
in agar to test plants but they furnished very few details about the technique.
* Department of Plant Physiology, Waite Agricultural Research Institute, University of
Adelaide.
t Present address:
t Present address:
Biology Department, Carleton University, Ottawa.
Botany School, Cambridge University.
Aust. J. Biol. Sci., 1966, 19, 535-43
D. COHEN, J. B ROBINSON, AND L. G. PALEG
536
II.
EXPERIMENTAL METHODS
(a) Planting and Growing Conditions
Dwarf pea seed (cv. W. F. Massey) was sorted and surface-sterilized for 10 min
in a mixture of 50 vol. H 202 and absolute ethanol (1 : 1 v/v). Following a thorough
rinsing in distilled water the seed was spread in a single layer on saturated autoclaved
vermiculite, thinly covered with more vermiculite, and allowed to imbibe at 20°0
for 24 hr. Selected seed was then planted in 9 by 4 by 2 in. plastic trays in two rows
25
t
I
+
T ___
1
20
THIRD INTERNODE
_
v
15
::E
~
z
0
,g~
w
t-+~l
~ +-i
11
~
10
I
5
o
. r~ ,r __
Xl
T
of
1
1
10-11
J
1
r-f/
/
~
1
SECOND INTERN ODE
!
o
Fig. i.-Elongation of GAa-treated
apical segments of etiolated dwarf peas
decapitated so that measured segments
were wholly located either in the second
or third internodes.
Vertical bars
indicate ± S.E.
10-10
I
10-9
1o-a
10- 7
GA3 (G(DISK)
of 10, with all radicles towards the near sides of the tray and pointing downward.
One litre of vermiculite was used with each tray and the seed was planted 1 in. deep.
Distilled water (450 ml) was added to each tray which was then incubated at 25°0
in the dark for 5-6 days. On the fourth day an additional 100 ml of distilled water
was added with the aid of a low intensity green safelight.
(b) Preparation of Agar Blocks
Agar (1·5%) containing either water or the test solutions was poured onto a
stainless steel plate 1 ·6 mm thick with 30 holes, 2·95 mm in diameter. The template
was held against plate glass which was, in turn, in contact with a chilled brass block.
Excess agar was removed from the agar disks by running a razor over the surface of
the template after the agar had solidified. The volume of the resulting blocks was 10 /1-1.
DECAPITATED PEAS AND DIFFUSIBLE GIBBERELLINS
537
(c) Treatment of the Plants
For the experiment illustrated in Figure 1, the method of marking used by
Lockhart (1957) was adopted, i.e. two marks were drawn 6 mm apart with a mixture
of lanolin and black marking ink. The upper mark was 2 mm below the vertex of
the hook, and the plant was decapitated at the vertex. In subsequent experiments
the upper mark was placed at the point on the third internode at which the hook
began to form and the second mark was 6 mm lower. The plants in these experiments
were decapitated by cutting through the upper mark.
1
TABLE
GA 3 ·INDUCED
ELONGATION RESPONSES
No. of
GA3
(gjdisk) Replicates
OF DIFFERENT PARTS OF PLANTS AT DIFFERENT AGES
Increase in Internode Length
(mm) in 3 Days ± S.E.
Elongation (mm) of 6-mm
Segment ± S.E.
Day 1
I
Day 2
I
Day 3
Second
Internode
Third
Internode
10-2±1·6
11-9±1-6
17-9±2-2
14-4±2'5
13·6±1-6
27-1±2-3
I
Third internode initially less than 6 mm
°10-
10
10- 8
IS
26
17
7'3±0'5
6-7±0-4
12-2±0-3
I
13·4±1·6·
13-7±1-3
29-1±2-3 I
15-S±2-6
16-2±2-1
32-1±2-6
I
Third internode initially greater than 6 mm
0
10-10
.10- 8
19
14
16
4·S±0-5
7'7±0-9
1l-1±0-5
I
7-1±1·O
1l·5±1-7
22-6±1-7
7-3±1-1
11-9±1-7
I 23-9±1-9
I
3-9±0-5
5-S±0-6
5-1±0-9
11-5±1·2
15'7±1-6
26-6±2-1
Agar blocks were placed on the decapitated stem tips within 5 min of apex
removal. The plants were then incubated at 25°0 for a further 24 hr_ No special
attempt was made to prevent the agar disks from drying out although the incubator
was maintained at a fairly high humidity level. The lengths of the marked segments
were measured to the nearest 0 -5 mm. In several experiments the length of the entire
third internode was also recorded and it was found to respond to gibberellic acid (GA3)
in the same way as the segment_ All manipulations were carried out under either a
low intensity green safelight or a 40-W white fluorescent tube (producing about
10 f.c_ of light at bench height)_
III.
RESULTS
In preliminary experiments plants were grown to a stage in which the third
internode was just beginning to elongate_ The marked section of these plants was
largely or wholly located on the second internode. The variation within each treatment
was high in these experiments but they indicated that the replacement of the apex of
dark-grown dwarf pea plants with an agar block containing GA3 resulted in stem
elongation proportional to the amount of GA3 applied.
An attempt was made both to reduce the variability mentioned above, and to
determine the distribution of the induced elongation_ Plants were used in which the
third internode was about 1 cm long and in which the measured segment was wholly
located in either the second or third internodes_ Figure 1 illustrates that the growth
D. COHEN, J. B. ROBINSON, AND L. G. PALEG
538
of such plants is largely a function of the elongation of the third internode, and that
by the time the third internode has reached a length of about 1 cm, the second internode has lost much, if not most of its ability to respond to GA 3.
The differential responses of the second and third internodes are more clearly
demonstrated in Table 1. In this experiment, plants were selected on the basis of the
length of the third internode at the time of decapitation. At this time the length of
the second internode was also recorded, and decapitation and the placing of the agar
blocks were all carried out under the green safelight. Table 1 indicates that the
growth potential of the second internode does not last indefinitely. If decapitation
and GA3 treatment are carried out when the third internode is less than 6 mm, the
second internode will elongate. If, on the other hand, treatment is postponed until
the third internode is greater than 6 mm, much of the potential response of the
second internode is lost.
TABLE 2
EFFECT OF LIGHT QUALITY DURING MANIPULATION ON SUBSEQUENT RESPONSE TO GAa
The number of replicates for each mean is shown in brackets
GAa
(gjdisk)
Elongation (mm) of 6·mm Segment in 24 hr
±
S.E.
Green Safelight
White Fluorescent Light
Daylight
3·5±0·4 (7)
2·0±0·1 (6)
2±0·3 (7)
10-10
4·0±0·4 (7)
4·0±0·2 (6)
2±0·3 (7)
10- 8
13·0±0·8 (7)
1l·5±0·1 (6)
2±0·4 (6)
°
The results in Table 1 also indicate that the elongation of the 6-mm marked
segment is more sensitive to exogenous GA3 if the third internode is initially greater
than 6 mm. The elongation of the 6-mm segment is an accurate indication of the
elongation of the third internode, and, as well, extension of the treatment time to
48 or 72 hr does not alter the relative response, although both the magnitude and the
variability of the results are increased.
Another source of variation in both published reports and bioassay procedure
is the alteration in the GA3-induced response due to the presence or absence of light.
This aspect was investigated by marking and decapitating plants in three different
types of light, i.e. low intensity green safelight, white fluorescent light (lO f.c.), or
daylight. The results (Table 2) indicate that the GA 3-induced elongation was almost
as great when the plants were manipulated in fluorescent light as under the green
safelight although daylight completely eliminates the response.
The response of decapitated and intact seedlings to temperature was determined
by growing plants at 25°C up to the time of decapitation and treatment. Following
treatment, the trays were enclosed in plastic sleeves and incubated for an additional
24 hr at 15, 20, 25, and 30°C. Maximum elongation of the 6-mm segment of intact
plants occurred at 25°C with significant reduction in elongation evident at 30°C
(Fig. 2). The results also indicate that the GA 3-induced elongation can take place
DECAPITATED PEAS AND DIFFUSIBLE GIBBERELLINS
539
over at least a lO-degree range in temperature. When the apex was removed and
replaced with GA3 in agar, a temperature of 30°0 was no longer inhibitory, but, in
I
15
i
L.S.D.
z
0
f:
<:
~
z
o
W
Fig. 2.-Effect of different temperatures
on segment elongation of intact (D) or
decapitated dwarf peas. Decapitated
peas treated with GA3 as follows:
/':, 5 X 10- 7 g per disk;
o 5 X lO- 9 g per disk;
+ 5 X 10- 11 g per disk;
X No GA3.
//B __:
~
0
5%
/0/0
10
.J
=
5
~
~~~o
::::::--::-::t=- =-~~~
OLI--~1~5------------~2~0~----------~2~5------------~30
TEMPERATURE (DC)
fact, possibly stimulatory. Also at 30°0 the elongation increment induced by 5 X 10-7 g
GA3 per disk was greater than that of intact plants.
15
15
(b)
(a)
/t
.,
~
10
10
:E
Z
0
1=
<:
~
z
0
/
.J
W
5
~~
o
o
i"'6
I-~
t
---::; ¢
¥-l
Q
I I
5XtO- lt
J:
5
I
5Xl0 9
5Xl0-7
GAs
oL..L.j
0
(1
f-Q ____ 9IAA
I
I
5Xl0- 11
-9
I
I
5Xl0 9
5X10""7
(GjDISK)
Fig. 3.-Comparison of segment elongation induced by various concentrations of IAA
or GA3 in white fluorescent (a) or green (b) light. Vertical bars indicate ± S.E.
The effects and interaction with GA3 of several naturally occurring substances
which might influence the response was also examined. 3-Indolylacetic acid (IAA)
was without appreciable effect over a wide concentration range (Fig. 3) when
D. COHEN, J. B. ROBINSON, AND L. G. PALEG
540
manipulations were carried out under either green or low intensity white fluorescent
light. Glucose, fructose, and sucrose at two concentrations were also unable to
duplicate the effects of GA3 (Table 3). When either IAA (Table 4) or glucose (Table 5)
were added in combination with GA 3, there was a somewhat decreased response.
In addition, IAA produced a characteristic response (noted earlier by Lockhart 1957)
in that all of the plants treated with the higher concentration, and many at the
lower, became swollen for up to 5 mm below the cut surface.
TABLE
EF]'ECT OF SUGARS AND
GAa
3
ON SEG~'!ENT LENG'rHS
The number of replicates for each mean is shown in brackets
Sugar
Concentration
(gjdisk)
Glucose
°
2 X 10- 5
2 X 10- 4
Fructose 2 X 10- 5
2 X 10- 4
Sucrose 2 X 10- 5
2 X 10- 4
Elongation (mm)
of 6-mm Segment
in 24 hr ± S.E.
I'2±0'I
0·9±0·2
0·8±0·I
0·9±0·I
0·9±0·2
I·I±O·I
I·I±0·2
IV.
(21)
(15)
(15)
(15)
(15)
(15)
(15)
GAa
Concentration
(gjdisk)
Elongation (mm)
of 6-mm Segment
in 24 hr ± S.E.
5 X 10-11
5 X 10- 9
5 X 10-7
I·3±0·I (15)
5·4±I·6 (15)
7·4±2·1 (15)
DISOUSSION AND CONOLUSIONS
The ability of the decapitated pea seedling to respond to GA3 in agar has been
clearly established, and several parameters of the response have been determined.
To obtain a reproducible system which will manifest a proportional elongation
response to concentrations of GA3 between 10-10 and about 10- 7 g per 10 p.l agar
block, the following techniques may be helpful:
(1) Dwarf pea seed should be sterilized and allowed to imbibe for 24 hr before
planting.
(2) The seed should be planted under conditions providing for maximum
uniformity (constant depth, constant seed orientation, etc.), and grown in
the dark at 25°C for about 5-6 days.
(3) When the plants have attained the proper physiological age, i.e. third
internode about 10-20 mm long, two marks 6 mm apart should be placed
on the stem so that the upper one is at the point of formation of the hook.
The plants can then be decapitated by cutting through the upper mark.
(4) Manipulations, including replacing the apex with the desired size agar disk
or block, may be carried out with a green safelight or weak white fluorescent
light (about 10 f.c.), and the test plants should be incubated at 25-30°C for
a further 24-48 hr before measuring the marked section.
Plants treated in this way do not respond comparably with IAA, glucose, fructose,
and sucrose, and relatively high concentrations of these substances are required
before they markedly interfere with the GA 3-induced response.
ELLINS
DECAP ITATED PEAS AND DIFFUS IBLE GIBBER
541
gibbere llin
Several attemp ts to employ the assay to measur e endoge nous
"stame n
ted
designa
stage
the
[at
concen trations were made. Barley main stem apices
the test,
in
tion
stimula
ant
signific
a
initials " by Aspinal l and Paleg (1963)] produc ed
and
initials
leaf
three
d
include
apex
equival ent to about 10- 9 g GAg per apex, if each
ty.
humidi
high
of
ons
conditi
under
was allowed to diffuse into an agar disk for 12 hr
TABLE 4
INTERACTION OF IAA AND GAa ON SEGMENT EXTENSION
Each value is the mean of 9 replicate s. Least significa nt
differenc e at 5% level = 1·1, at 1 % level = 1· 5
Elongat ion (mm) of 6·mm Segmen t in 24 hr
IAA
(gjdisk)
0
No GAa
15 X 10- 11 g GAa
5x 10- 9 g GAa
per Disk
per Disk
1·8
2·6
6·1
5X
lO-7
1·7
2·3
4·4
5X
lO-5
3·2
3·7
4·4
was not signific ant.
If only two leaves were include d, the stimula tion observe d
of Nicholl s and May
These results lend confirm ation, in a general way, to the findings
they found that
(1964). Using the barley endosp erm test (Nicholls and Paleg 1963)
TABLE 5
AND GAa ON SEGMENT EXTENSION
GLUCOSE
OF
INTERACTION
8 replicate s. Least significa nt
of
mean
the
is
Each value
differenc e at 5 % level = 1·0, at 1 % level = 1·4
Elongat ion (mm) of 6·mm Segmen t in 24 hr
Glucose
(gjdisk)
No GAa
i 5x 10- 11 g GAa
5 X lO-9 g GAa
per Disk
per Disk
o
1·8
1·8
5·2
5 X lO-5
1·4
1·8
5·2
5 X 10- 4
1·1
1·2
3·2
detecta ble amoun ts
barley apices at a similar stage of develop ment contain ed easily
attache d when the
of endoge nous gibbere llin-like activity if the smalles t leaf was still
more sensitiv e than
tissue was extract ed. Since the endosp erm test is conside rably
leaf tissue is require d
the test used here, it is not too surprisi ng to find that even more
to produc e a signific ant response.
just below the
In anothe r test, 6-mm section s of pea stems were remove d from
grown in the
and
old
days
6
were
third node of W. F. Massey seedlings. The plants
6
was placed
GA3
g
10ing
contain
same way as the test plants. A disk of 1·5% agar
542
D. COHEN, J. B. ROBINSON, AND L. G. PALEG
on the morphological top of each section which was, in turn, placed on an agar disk
to which no GA3 had been added. The sections were left to transport GA3 for 12 hr
at 20°C, and at the end of this period the lower blocks were tested for the presence
of gibberellin. The results are shown in Figure 4, and indicate that gibberellin activity
can be transported through such sections, substantiating the work of Kato (1958),
and that this transport can be measured with the bioassay.
Mention should also be made of negative results obtained with pea apices.
Both dwarf (W. F. Massey) and tall (Telephone) dark-grown, 6-day-old seedlings
were decapitated in green light just above the vertex of the hook. The apices were
placed on agar disks for 12 hr at 20°C, after which the agar disks were bioassayed.
No gibberellin-like activity was found. These experiments, although not definitive
in any way, provide indirect substantiation of the difficulty experienced by Lockhart
10
J,
z
o
i=
""oz
--'
w
;(
T/f-t------'
:>
~
5
~-I
o~
o
./f
~i./
Fig. 4.-GA3-induced segment elongation ( X ) and segment elongation produced by agar disks containing 12-hr
diffusate from base of 6-mm pea epicotyl
sections to the apical ends of which agar
blocks containing 10- 6 g GAe were
applied (D) . Vertical bars indicate
± S.E.
I
I
10-
10
GA3
10-9
(G/DISKI
I 8
10-
10-7
(1957) in inducing elongation of decapitated pea seedlings by grafting the apex back
into position. He suggested, as one possible explanation for his lack of success, that
the endogenous gibberellin-like substance(s) could not be transported through the
cut surface. In the present work, however, GA3 appears to have been transported
through two cut surfaces of epicotyl sections. One possibility not discussed by
Lockhart is that the endogenous gibberellin-like substances in dark-grown peas are
transported upward (possibly from the cotyledons) rather than downwards from
the apex.
Certain features of the response are of interest in other respects, as well as in
connection with the bioassay. As pointed out by Paleg (1965) there is conflicting
evidence in the literature pertaining to the question of whether or not gibberellin
can replace the pea stem apex in its ability to stimulate elongation of the sub tending
internodes. Lockhart (1957) has suggested that GA3 can, and Vlitos and Meudt (1957)
and Kuraishi and Muir (1963, 1964) have concluded that GA3 cannot replace the apical
control of internode elongation in decapitated peas. The results of the present study
make several conclusions possible. The sensitivity of any given section or part of the
pea stem to GA3 will vary depending on its physiological age. As indicated in Figure 1
and Table 1, the second internode begins to lose its ability to respond as the third
internode begins to extend. It can be anticipated that decapitation of plants of this
DEOAPITATED PEAS AND DIFFUSIBLE GIBBERELLINS
543
age or older, at any point below the third internode, will produce an unresponsive
system. This point explains the lack of response found by Vlitos and Meudt (1957),
since they decapitated their plants below the third internode and used the unreactive
remainder.
Table 2 illustrates the fact that the system loses the potential to respond to GA3
when manipulations are carried out in the light. This conclusion explains the results
of Kuraishi and Muir who used greenhouse. grown plants.
Another aspect of the response is also of interest. It is obvious from previous
reports of Kuraishi and Muir (1963, 1964) that IAA can stimulate the elongation
of decapitated peas under the proper circumstances. In this work, and in that of
Lockhart (1957), no pronounced effect of IAA on elongation was recorded, though
an induction of swelling towards the tip of the decapitated seedling was observed.
Lockhart suggested that this might be interpreted as evidence for the fact that IAA
was penetrating the stem. Another interpretation which might be put forward is that
the induction of swelling is due to the destruction of IAA at the cut surface of the stem.
Pronounced 1M· oxidase activity at cut surfaces is well established and it is possible
that, by some aspect of their technique, Kuraishi and Muir were able to prevent the
oxidation, and consequently increase the elongation.promoting effects of the applied
1M.
V. ACKNOWLEDGMENTS
This work was carried out while D. Cohen held a Barley Improvement Trust
Fund Studentship and J. B. Robinson held a Deciduous Tree Fruits Studentship.
VI.
REFERENCES
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JONES, D. F. (1964).-Examination of the gibberellins of Zea mays and Phaseolus multijlorus
using thin.layer chromatography. Nature, Lond. 202: 1309-10.
JONES, R. L., and PHILLIPS, 1. D. J. (1964).-Agar·diffusion technique for estimating gibberellin
production by plant organs. Nature, Lond. 204: 497-9.
KATO, J. (1958).-Non.polar transport of gibberellin through pea stem and a method for its
determination. Science 128: 1008.
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gibberellin treatment. Plant Physiol. 39: xliv.
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LOCKHART, J. (1957).-Studies on the organ of production of the natural gibberellin factor in
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initiation of internode elongation in the inflorescence. Aust. J. BioI. Sci. 17: 619-30.
NICHOLLS, P. B., and PALEG, L. G. (1963).-A barley endosperm bioassay for gibberellins. Nature,
Lond. 199: 823-4.
PALEG, L. G. (1965).-Physiological effects of gibberellins. Annu. Rev. Pl. Physiol. 16: 291-322.
VLITOS, A. J., and MEUDT, W. (1957).-The effect of light and of the shoot apex on the action
of gibberellic acid. Contrib. Boyce Thompson Inst. 19: 55-62.
WENT, F. W., and THIMANN, K. V. (1937).-"Phytohormones." (The Macmillan 00.: New York.)