Plant& CtU Physiol. 15: 341-349 (1974)
The effect of growth regulators on the growth and
pigmentation of "Baccara" rose flowers
N. Zieslin, I. Biran and A. H. Halevy
Department of Ornamental Horticulture, The Hebrew
University of Jerusalem, Rehovot, Israel
"Baccara" rose buds were treated with various growth regulators during late
stages of bud development. The effect of these substances on growth and pigmentation
were determined. Growth regulators were applied by spray or injection or as a lanolin
paste, also in the nutrient media on which petals were cultured in vitro. Injection
of GA into the base of the receptacle caused elongation of the bud whereas IAA, K,
ABA, AMO-1618, CCC, and SADH had little or no effect. CCC and MeCl-F did
not reduce the elongation caused by GA. GA treatments also enhanced flower weight
and petal pigmentation and MeGl-F decreased the gibberellin effect on pigmentation.
GA treatments of intact flowers and excised petals cultured in vitro, were only effective
at low temperatures.
Gibberellin treatments increased the size of petals, the receptacle and the pedicel
only if applied directly to the receptacle. Treatments at lower positions on the flowering shoot either had no effect at all, or caused elongation of only the receptacle.
Endogenous gibberellin levels are higher in the receptacle than in petals or in
the pedicel. Injection of GA into the receptacle significantly increased gibbereUin
activity in all flower parts whereas injection into the flowering-shoot base increased
gibberellin activity only in the receptacle.
The possibility is discussed that GA, which is exogenously supplied directly to the
receptacle, enhances flower dimensions and pigmentation by drawing photosynthates
to the flower as a consequence of intensification of the sink.
Flowering may be divided into four main stages: induction, differentiation,
development of the bud and blooming (anthesis). Environmental conditions,
especially daylength and temperature, are known to affect the first two stages in
many plants. Changes in levels of endogenous growth regulators connected with
induction and initiation of flowers, as well as the effects of exogenous hormone
treatments have recently been fully summarized in books (1, 2). Less is known
about effects of environmental conditions on hormonal balance during the later
stages of flowering (3). Information about the physiology of flowering in autonomously induced plants, e.g. roses, is especially scanty (4). Environmental
Abbreviations: GA, gibberellic acid; IAA, indole 3 acetic acid; K, kinetin; SADH, succinic
acid-2,2-dimethyl hydrazide AMO-1618, ammonium (5-hydroxycarvacryl) trimethyl chloride
piperidine carboxylate; CCC, (2-chloroethyl) trimethylammonium chloride; ABA, abscisic acid;
MeCl-F, 2-chloro-9-hydroxyfluorene-9-carboxylic acid; A, absorbancy.
341
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(Received August 17, 1973)
342
N. Zieslin, I. Biran and A. H. Halcvy
conditions such as temperature, light intensity, mineral nutrition and COa concentration affect flower size and also the pigmentation of roses {5-10). Changes
in endogenous levels of growth substances occurring during the development of
photoperiodically induced flowers, have recently been described {11, 12), as have
changes in the levels of phytohormones occurring during the early stages of flower
development in roses {4, 13). The objective of the present study was to investigate
the effect of various growth regulators on the final stages of bud development and
on early stages of flowering in "Baccara" roses.
Materials and methods
Growth regulator treatments
Growth regulators were applied in four different ways: 1) Injection of the
indicated amount of regulator as 5 /x\ solution into the receptacle by a Hamilton
microsyringe (Fig. 1). 2) Spraying 0.5 ml solution on the upper or basal leaf
(Fig. 1). 3) Application of 50 mg of lanolin paste containing the substance to be
tested, to the base of the receptacle. 4) Sterile culture of excised petals on nutrient
media. The five outermost petals were removed before formation of anthocyanin,
from buds which had reached a diameter of 12 mm. Forty-five petals were incubated for 6 days in 500 ml Erlenmeyer flasks containing 250 ml of Jacobs et al.
{14) liquid medium to which 3 % sucrose and the substance to be tested had been
added. The medium was aerated constantly with sterile air and treatments were
replicated twice.
Examination of the flowers
Injected flowers were examined when 3-5 petals burst open from the bud
Print of In)«c1loi
or application of
lanolin?"'*
Uppar Msf
Fig. 1.
"Baccara"
rose flower parts
nuasured, and
points where growth regulator treatments were applitd.
Point o4 inaction
Into baaa of
tha flowanng
•hoot
Baial laaf
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Rose plants of the cv "Baccara", grafted on Rosa indica major were grown in
10 liter-containers in a green-house covered with fibreglass. Plants were grown
under two different temperature regimes: 14°C night and 20°C day temperature
referred to hereafter as "low temperature", and, 20°C night and 30cC day temperature referred to as "high temperature".
343
Growth and pigmentation of rose flowers
("blooming stage"). The lengths of the flower, receptacle and pedicel were measured (see Fig. 1). The 3 outermost petals were removed for pigmentation analyses.
These were dried at 55°C and ground. Pigments were extracted by shaking petals
for 20 hr at 4°C with methanol + 1% HC1. Anthocyanin content was estimated
by measuring the absorbance at 515 nm, (0.4 mg dry weight in 1 ml methanol).
This wavelength had previously been found to be the wavelength of the maximum
absorbance by the mixture of the anthocyanins cyanidin 3,5 diglucoside and
pelargonidin 3,5 diglucoside which are found in "Baccara" petals (9). Each treatment included at least 10 flowers. Results were analysed by the Multiple Range
Test.
Gibberellin activity assays
Results
1.
Effect of growth regulators injecUd into rose buds on the length and weight of the flowers
Receptacles with at least 10 flower buds growing under "low temperature"
conditions were injected with various growth regulators when the buds had a
diameter of 8-10 mm. Two separate experiments were set up. In one experiment
D
I E NGTH
WEIGHT
£25
Fig. 2. Effect of injecting 5 fig of growth
inhibitors or GAs into "Baccara" rosi buds grown
at "low temperatures" on the weight and length of
the bud.
I
«»
;
1
20
-
CONTROL
GA
SADH
AMO
III!
CCC
ABA
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GA activity was determined in flower parts harvested at anthesis. Parts were
frozen in liquid air, then were lyophilised and stored at —20°C until use. 1 g of
dry matter was used for each determination. The extraction, fractionation and
chromatography procedures, as well as the barley endosperm bioassay for gibberellins, were conducted according to Halevy and Shilo (75). Since in preliminary tests most of the GA activity was found in the acidic ethyl acetate fraction
at Rf values 0.4-0.8, only these sections were assayed for GA activity.
344
N. Zieslin, I. Biran and A. H. Halevy
Fig. 3. Effect of injecting 5 fig of GA, (GA) or water
(C) into "Baccara" rose buds, growing at "high" (20j
30°C) and "low" (1412O°C) tempaatares, on bud wtight
and pigmentation. Columns with different letters
are significantly different at the 5% level.
2.
Effect of GA injected into the receptacle or included in the medium in sterile cultures,
on flower size and pigmentation, under "high" and "low" temperature conditions
a) Receptacles of at least ten, 8-10 mm diameter flower buds growing under
either "high" or "low" temperature conditions were injected with 5 fig GA3.
GA had no effect under "high" temperature conditions whereas under "low"
Table 1 Effect of 2.5mgjliter GA in the nutrient medium and incubation temperaturt on the growth and pigmentation of excised "Baccara" rose petals cultured in vitro
Temp.
(°C)
Treatment
Fresh wt of
1 petal
( m g)
Parameter
Dry weight of
1 petal
()
Relative
pegmentation
of 1 petal
27
Control I '
Control II '
GA
23.9
42.5
45.3
6.8
7.2
7.8
100
103
104
18
Control I
Control II
GA
25.9
48.6
61.5
6.3
12.1
12.0
100
207
264
14.5
Control I
Control II
GA
22.8
67.3
89.0
5.4
8.1
12.0
100
268
335
' Control I, petals before incubation.
* Control II, petals incubated at the indicated temperature on nutrient medium alone.
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1 ng of the growth promotors IAA, K and GA3 were tested, while in the second,
5 fig of GA and the growth inhibitors SADH, AMO-1618, CCC and ABA were
used. In the first experiment, GA significantly (P 5%) elongated flower buds by
approximately 20% whereas IAA and K had no effect. In the second experiment,
growth inhibitors had little or no effect on the length and weight offlowers,whereas
GA, again significantly increased flower length as well as weight (Fig. 2).
Growth and pigmentation of rose flowers
Fig. 4.
TOO
Effect of injecting 5 ug o/GAs into rose buds {8-10 mm), growing at low ttmt>eraha-ts, onflowerdimen-
sions (see Fig. 1) at the blooming stage. Q, length of receptacle; £ , fresh wt. of flower; A. fresh weight
of petal; Q, length of petiole; A. length of flower.
temperatures both the weight offlowersand pigmentation of the petals were significantly enhanced (Fig. 3). Low temperatures alone, also significantly enhanced
the weight and pigmentation of flowers.
b) Excised petals were cultured at a constant temperature of 27°, 18° or 14.5°C
in aerated liquid nutrient media to which 2.5 mg/liter GA had been added. GA
had no effect on petals cultured in vitro under high temperatures (27°C) whereas
under low temperature conditions (18° and 14.5°C) both petal weight and pigmentation increased (Table 1), similar to the increase found following the injection
of intact buds with GA.
c) The effect of different concentrations of GA on flower size was determined by
injection of buds which were growing at "low" temperatures, when at the 8—10 mm
stage. Increasing concentrations of GA increased the length of the flower and the
receptacle and also increased flower weight. Optimum concentration of GA was
generally 5 fig per bud (Fig. 4).
3.
Effects of the position of application and method used to apply GA on flower dimensions
Flowering shoots growing at low temperature and bearing 8-10 mm buds
were treated with GA by various methods (Table 2): injection, lanolin paste
application and spraying the green leaves (see Materials and methods and
Fig. 1). Each treatment contained 10 replications. Results presented in Table 2
show that both the injection of GA and application of lanolin paste containing GA
to the bud, increased all the flower dimensions measured. Spraying the upper
leaf increased flower weight and the lengths of the receptacle and pedicel, however,
it had no effect on flower length. Spraying the basal leaf had no effect on any of
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iO
345
N. Zieslin, I. Biran and A. H. Halevy
346
Table 2 Effect of various mtthods of application of GA at different positions on the plant onfiowtr weight and
the length of various flower parts
Position and method of
application of GA
Flower
length
Injection into receptacle (5 ,ug per bud)
Application of lanolin paste into receptacle
(0.1%)
Spraying of upp«r leaf (100 mg/liter)
Injection of shoot base (5 /ig per stem)
Spraying of basal leaf (100 mg/liter)
Parameter
Flower
Receptacle
weight
length
•
Pedicel
length
124
132
137
135
162
158
121
101
100
98
126
104
103
133
133
105
129
106
101
150
Data are expressed as % of the control.
uor 6-
0 06
PEDICEL
RECEPTACLE
Fig. 5.
PETALS
0.5
p o / bud
Fig. 6.
Fig. 5. Effect of tht position oftht injation ofGA% on gibbtnUin actimty in differentfloralorgans as dettrrmntd
by the barley endosperm bioassaj. (Activity at Rf values 0.4—0.8 of acidic ethyl acetate fraction, solvent
system : Isopropanol : ammonia: water, 8 : 1 : 1). Horizontal line marks GA activity without
exogenous GA treatment. Activity is expressed as the increase in reducing sugars (measured a' A
at 550 nm) over water.
Fig. 6.
Effect of the injection of various amounts of CCC and MeCl-F together with 5 ftg GA onflowerlength
and pigmentation. O> weight; A> length; Q, pigmentation. Points along a curve not followed by
the same letter are significantly different at the 5% level.
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the flower dimensions measured. Injection at the base of the flower shoot had
only one effect, the flower receptacle elongated, all other floral dimensions were
unaffected.
Growth and pigmentation of rose
flowers
347
4.
Distribution of gibberellin in the flower following application at different sites
The reason for the non-elongation of petals following application of GA to the
shoot base was sought in the following experiment. Flowering shoots grown at
low temperature and bearing buds at the 8-10 mm stage were treated by a) injecting 5 /ig of GA into the base of the shoot or b) injecting 5 fig of GA into the
receptacle. GA-like activity was determined in the pedicel, the receptacle and the
petals 7 days after treatment. Results summarized in Fig. 5 show that when the
receptacle was injected, GA activity increased in all three flower parts tested,
whereas, when the substance was injected into the shoot, activity increased only
in the receptacle. GA levels before exogenous treatment were relatively higher
in the receptacle as compared to the other floral parts.
Effect of growth inhibitors on the GA induced increase in flower weight and pigmentation
8—10 mm flower buds growing in "low" temperatures were injected with
GA and various amounts of MeCl-F and CCC (Fig. 6). As in previous experiments,
GA alone increased weight, length and pigmentation of the flowers. CCC did
not affect the increase in weight or length caused by GA, but significantly decreased
pigmentation. MeCl-F did not affect the elongation caused by GA but significantly
decreased both weight and pigmentation.
Discussion
In most research work concerning flowering, the effects of environmental and
hormonal factors have been studied on early stages of flower development. In
this study, the effects which growth regulators have on the later stages of flower
development were investigated. Most growth and pigmentation occurs during
these later stages (16). The effect of growth promoters and inhibitors on elongation
of flower parts, flower weight and pigmentation were examined. Application of
gibberellin to the bud itself, caused elongation of the petals, whereas application
of other growth regulators, both those considered promoters (IAA, K), and those
considered inhibitors (ABA, SADH, AMO-1618 and CCC), had no effect (Fig. 2,
6). In combined treatments of GA with CCC or MeCl-F, the inhibitors did not
reduce the promotion of elongation caused by gibberellin. The elongating effect
of gibberellin is known today in many plant organs (17). Gibberellin enhances
the elongation of stems and also the leaves of certain species. In contrast, the
inhibitors used — especially those of the growth retardant type only, inhibit stem
growth and occasionally that of the petioles but not the growth of leaves or petals
(18). In this study, the part of the flower measured for length was actually only
the length of the petals (see Fig. 1). Gibberellin increased the fresh and dry
weights of flower buds, enhanced pigmentation and also increased petal length.
The effects only occurred under low temperatures both in the experiments with
flowers in vivo and in excised petals cultured in vitro. The increase in flower
weight caused by GA may be explained by the greater movement of photosynthates
into the growing upper parts of the plant (19, 20). It seems that GA enhances
movement not only in the intact plant but also in excised petals which absorb more
metabolites from the nutrient medium.
Two aspects of GA activity revealed in this study are worth special mention.
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5.
348
N. Zieslin, I. Biran and A. H. Halevy
References
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The first is that the effects of GA were only expressed at low temperatures. We
have no explanation for this phenomenon and have found no examples of similar
behaviour in other plants. The second interesting point is that GA caused elongation of the petals, only when the substance was applied directly to the receptacle,
i.e. to the base of the petals (Table 2). Applications of GA to other parts of the
flowering shoot below the pedicel, caused elongation and an increase in the weight
of the pedicel or the receptacle but not of the petals. It seems that the receptacle,
which has a high endogenous level of gibberellin (Fig. 5), is a strong sink and is
capable of drawing gibberellin to itself even when the exogenous application is
relatively far away. In contrast, the petals and pedicel receive gibberellin only when
it is applied in their vicinity. When we examined the distribution of GA activity
in different flower parts following injection into the receptacle or the shoot base,
we found that GA accumulated in the receptacle in both cases, whereas in petals
or the pedicel the level increased only when the substance was applied to the receptacle; not when it was applied lower on the shoot (Fig. 5). The flower parts
where GA effects were observed following application of the substance at different
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is antagonistic to GA in its effect on flower weight and also pigmentation. CCC
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emphasize the wide range of activities which these regulators have and also the
possibility that they act differently in different tissues and different processes (4).
GA seems to affect the flower by forming a sink in the position where it accumulates, thus drawing photosynthates to this site. This causes the increase in
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The point which needs further clarification is whether environmental factors
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similar to the effects of growth regulators, act via the endogenous hormonal balance
or act by other means.
Growth and pigmentation of rose
flowers
349
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