Influence of (2-Chloroethyl) Trimethylammonium Chloride (Cycocel)

and Daylength on Gibberellin-like Activity in the Root Exudate of

the Poinsettia Euphorbia pulcherrima, Willd. cv. 'Paul Mikkelsen’1
Richard A. Criley2>3
University of California, Los Angeles

Abstract. Three substances with gibberellin-like activity in the dock and barley endosperm
bioassay systems were detected in the root exudate of poinsettia cultivar ‘Paul Mikkelsen’.
In the isopropyl ether:acetic acid (95:5, v/v) solvent system on 250 ft layers of silica gel G,
these substances had R£ values of 0-0.1, 0.4-0.6, and 0.7-0.8. The extracted exudate
material at R t 0-0.1 generally showed greater activity in plants grown under long day
conditions while the relative proportion of the other 2 fractions to the first increased
under short day conditions. Following a drench with (2-chloroethyl) trimethylammonium
chloride (Cycocel), the activity of all 3 fractions was reduced in comparison to the
control. The Rf 0-0.1 material evidenced lower activity in relation to the other 2
fractions in exudate from Cycocel spray-treated plants.

he occurrence of gibberellin-like substances in the
T bleeding sap has been demonstrated for a number of
different plant species (3, 8, 10, 13). The level of gibberel­
lin-like activity ranged from 0.007 to 30 pg gibberellic
acid (GA3) equivalents per plant per day, depending
upon the plant species. Such regular endogenous doses of
gibberellin-like substances were considered to exert a sig­
nificant effect upon the growth of the plant. The sites of
synthesis of these materials may include both the aerial
organs and roots.
Qualitative and quantitative changes in the levels of
gibberellin-like substances in plant exudates have been
reported. Reid and Carr (11) proposed that Cycocel
effected such changes through altered GA synthesis in
the roots. Kende and Sitton (8) reported a quantitative
change in the content of a gibberellin-like substance in
exudate from sunflower at the time of transition from
vegetative to reproductive status.
Unpublished data of Hackett and Kofranek with the
poinsettia cultivar ‘Paul Mikkelsen’ (a short day plant
with respect to flowering) have shown a promoting effect
of Cycocel upon flower initiation in plants growing under
long (16 hr) and marginal (131 4 hr) photoperiods. The
previously cited reports suggested that this could be re­
lated to an effect of Cycocel upon the gibberellins
involved in flower initiation.
Studies were undertaken to demonstrate that gibberel­
lin-like substances occur in poinsettia root exudate.
Additionally, an attempt was made to show that either
quantitative or qualitative changes in gibberellin-like
activity may be associated with transition to reproductive
status as induced by short photoperiods or treatment with
Cycocel.
M ater ia ls a n d M etho ds
Vegetative plants of ‘Paul Mikkelsen’ poinsettia were
grown in a 22 °C greenhouse with a 4-hr incandescent
light break (10 ft-c minimum) from 10 p m to 2 a m . Eight
hour photoperiods were achieved by covering the plants
with black cloth from 4 p m to 8 a m . Cycocel was applied
as a soil drench at the rate of 1 g per 6 inch pot or as a
0.25% foliar spray.
Root exudate was collected from 3-month-old plants.
deceived for publication July 12, 1969.
departm ent of Agricultural Sciences.
3Present address: Department of Horticulture, University of
Hawaii, Honolulu, Hawaii 96822.
120
The top of the plant was cut off to leave a stump about
3 inches tall. A short piece of latex rubber tubing was
affixed to the stump. An L-shaped glass spout was wedged
in a short collar of tygon tubing which was forced into
the rubber tube on the stump. The exudate was either
collected in separate containers attached to the spout or
drained into a central vessel through fine plastic tubing.
The accumulated exudate was bulked twice a day and
stored at —18° until used.
Frozen exudates were thawed and filtered through
Whatman No. 1 filter paper to remove debris, latex, and
precipitated substances. The aqueous exudate was meas­
ured and lyophilized to a powder.
Dried residues were handled according to a procedure
adapted from Aung and de Hertogh (1). The dry exudate
was taken up in equal parts (v/v) of 0.33 M KH2P 0 4
buffer at pH 8.0 and petroleum ether. Following shaking
and separation from the petroleum ether, the aqueous
phase was extracted twice with ethyl acetate. The ethyl
acetate was discarded, and the aqueous phase acidified
to pH 2.5 with HC1 and partitioned 3 times against ethyl
acetate. The aqueous phase was discarded and the excess
water present in the ethyl acetate was frozen out. The
acidic ethyl acetate fraction was evaporated to a yellow
gummy residue. This residue was taken up in a small
portion of acetone or redistilled ethyl acetate for streak­
ing on a 250 \i layer of silica gel G. The thin layer plate
was developed at room temperature for 15 cm by ascend­
ing chromatography with isopropyl ether: acetic acid
(95:5, v/v).
Fractions of the chromatograms representing the origin
and 10 zones 1.5 cm wide were scraped from the plate
and eluted with water-saturated ethyl acetate. The ethyl
acetate was evaporated and the residue taken up in dis­
tilled water for bioassay.
Dock leaf senescence bioassay. The procedures of
Whyte and Luckwill (16) were followed using leaves
collected from clonally propagated broadleaved dock,
Rumex obtusifolius, L., plants grown in the greenhouse.
In this assay gibberellins prevent the degradation of
chlorophyll in leaf discs kept in the dark. The amount of
chlorophyll remaining is proportional to the gibberellin
concentration. Chlorophyll remaining in the leaf discs
after 5 days was extracted with methanol and the ab­
sorbency (optical density) read at 665 mp, on a Bausch and
Lomb Spectronic 20. The sensitivity of the assay was such
J. Amer. Soc. Hort. Sci. 95(1): 120-124. 1970.
as to give an almost linear reading (Fig. 1) in proportion
to the negative logarithm of the gibberellic acid concen­
tration between 10"4 and 5 X 10"3 ^ig GA3 /ml. Standard
GA3 concentrations were run with each test.
The raw optical density readings from the replicated
bioassay samples were used to find the standard deviation
of the mean difference (sa) which was used to find the
Least Significant Difference value from the equation
L.S.D. = sa*t 05. The t value was selected for the one-tail
test as the only differences for which this assay would be
expected to be significant were those greater than the
control. Since chlorophyll content was expressed as a per
cent of the control, the LSD value was transformed into a
percentage figure by dividing by the mean optical density
reading for the control. The planned comparison for the
LSD value was for the given treatment peaks with the
control.
Barley endosperm bioassay. The technique used was as
described by Jones and Varner (7). In this assay the re­
lease of a-amylase by embryoless half seeds of Hordeum
vulgare ‘Himalaya’ is reported as being proportional to
the logarithm of the gibberellic acid concentration be­
tween 0.0005 and 0.05 fig/ml. Standard GA3 concentra­
tions were run with each test.
The data are given as optical density units according
to the formula,
O.D. units = A O.D. X T v where
t X v
T v = volume of supernatant
A O.D. = O.D. of zero time control minus O.D. of
sample
t = time of incubation with starch
v = volume of supernatant taken for incubation.
When 2 O.D. values were obtained for each sample
which differed by no more than 4 on a scale of 1 to 100,
their mean was taken and entered into the above
formula. There was no need for statistical treatment
because of the close internal agreement of the readings.
>ig GA -3 /m l
Fig. 1. Example of standard
curve for gibberellic acid (GAS)
in the dock leaf assay. Each
point represents the mean of
10 bioassay standards for a
given concentration. Two sep­
arate series of 10 bioassays
each are shown as one curve
as agreement was very close.
J. Amer. Soc. Hort. Sci. 95(1): 120-124. 1970.
R esults
Presumably, the collected sap was translocated in the
xylem, as injury to the phloem when the rubber tube
was fitted on the stump did not appear to affect exudate
flow. In the woody basal area of the stem, the exuding
of latex from the laticifers in the bark was not a problem.
Significant gibberellin-like activity was detected in the
0-0.1 Rf zone on the chromatogram of the first day’s
collection, but not in the second. Only the first day’s col­
lection was used unless otherwise noted. Fig. 1-4 are
representative of the several trials which were analyzed.
The rate of flow from plants maintained on long days
was somewhat greater than for plants subjected to short
days or treated with Cycocel as a drench of spray (Table
1) . The effect of a Cycocel treatment, either as a drench
or spray, was also to decrease the volume of exudate col­
lected. There was, however, no apparent injury to the
root system of the Cycocel-treated plants which might
account for the decrease in volume.
Three zones of gibberellin-like activity appeared in
chromatograms of extracts of poinsettia exudate. The
peak which appeared at Rf 0-0.1 (sometimes into 0.1-0.2)
was designated Fraction A (a peak which occasionally
appeared in the range 0.2-0.4 was designated A'); that in
mid-range which appeared from 0.4-0.6 , Fraction B; and
that at Rf 0.7-0.9, Fraction C.
The data in Fig. 2 show relatively higher gibberellin-
like activity in plants receiving 7 or 14 short days (SD)
in comparison to their long day controls. In the barley
assay under long days, however, the presence of Fraction
A is not evident to the same extent as in the dock assay.
The B and C components appear greater under short
days than long. The gibberellin equivalent values when
corrected for assay method and number of plants (Table
2 ) bear out this observation.
The application of 1.0 g Cycocel per 6 inch pot as a
soil drench reduced the level of gibberellin-like substances
in root exudate as evidenced by the dock assay (Fig. 3).
Table 1. Mean volume and dry weight of collected poinsettia
exudates expressed on a per plant per day basis.
First day collections Second day collections
Treatments
Volume (ml) Dry wt (mg) Volume (ml) Dry wt (mg)
Long days... 8.64 ± 4.65 30.5 ± 16.1 7.69 ± 5.60 13.6 ± 8.2
Short days. . 6.03 ± 3.93 41.9 ± 31.3 5.44 ± 1.73 14.4 ± 13.9
Long days +
Cycocel
spray........ 6.97 ± 1.22 16.3 =fc 0.2 6.03 ± 3.77 ------
Long days +
Cycocel
drench.. . . 4.86 ± 3.43 ------ 3.40 ± 3.40 ------
Table 2. Gibberellin A3 equivalents per plant for root exudate of
long and short day plants as assayed in the dock and barley endo­
sperm assays. Values derived from Fig. 2 following adjustments
for number of plants and conditions of assay.
Fraction
(GA3 equivalents in fig X 10-5)
Treatment
A A' B C Total
Long days
Barley. . . . ............. 2.7 4.06 5.4 6.1 18.26
D ock........ ............... 5 4 3 12.0
Short days
Barley. . . . ............... 6.7 32.6 13.3 52.6
D ock........ ............... 11.4 8.6 3.6 23.6
121

Fig. 2. Acidic ethyl acetate extracts of exudates from long day and
short day poinsettia plants. A and B assayed in the dock leaf
system. C and D assayed in the barley endosperm system. A. 665
ml (2.52 g dry wt) exudate from 60 long day (LD) plants. B. 730
ml (2.30 g dry wt) from 84 plants which received 7 short days
(SD). C. 875 ml (2.68 g dry wt) exudate from 59 LD plants. D. 370
ml (1.25 g dry wt) exudate from 60 plants which received 14 SD.
All extracts chromatographed on TLC. Solvent, isopropyl ether:
acetic acid (95:5, v/v). In A and B, the LSD value indicates a
significant difference from the control at the 5% level of risk.
In C and D each peak represents the average of two optical
density unit values derived from separate replicates, but no test
of significance was attempted. Control blanks contained barley
half-seeds and distilled water instead of TLC eluant.
The active zones in the Cycocel assay, while still appear­
ing at the expected R /s were below the reliable extra­
polation of the gibberellin standards. The GA3 equiva­
lent activities of the long day peaks A, B, and C were 2
X 10"4, 1.6 X 10"4, and 4 X 10"4 ug/plant/day, respec­
tively.
The barley endosperm assay was used to measure gib-
berellin-like activity in exudate of plants receiving a
series of 0.25 per cent Cycocel sprays at weekly intervals
for 5 weeks (Fig. 4). The activity of the A fraction was
greater in the long day plants which were not treated
with Cycocel, but the B and C fractions were greater in
the treated plants (Table 3). The effect of a spray on
gibberellin-like activity in root exudate was less marked
than the effect of a soil drench. The levels of gibberellin-
like activity indicated in the Cycocel spray treatments
w ere g rea ter th a n th e h ig h e s t g ib b e r e llin sta n d a r d o f 1 X
10'2 jig GA3/ml. The fraction represented by the peak
designated C appeared at a higher Rf than was usually
found to be the case and may have been an artifact of the
extraction solvent.
D iscussion
Bioassays in repeated samplings of root exudate have
revealed the presence of three fractions active as gib-
122
berellins. The first, designated Fraction A, was commonly
observed in the Rf 0-0.1 zone but also in the origin and
in the 0.1-0.2 Rf region of the isopropyl ether:acetic acid
system. Gibberellins A1? A2, A3, A8, and A10 have been
reported to occur in the 0-0.1 Rf zone with several others,
A4, A5, A6, A7, and A14 occurring in the 0.1-0.2 region in
this solvent system (4). All of the latter have been placed
at higher Rf’s with A6 at 0.2-0.3 and A4, A5, and A7 in the
region of 0.3-0.4 (9). The B fraction of the exudate
appeared variously from Rf 0.4-0.6 when it was evident
at all. The known gibberellins for these regions (4) may
include A9, An and A15, although A9 was also reported
to have an Rf of 0.75 (9) and of 0.84 (Dr. B. O. Phinney,
personal communication). The C fraction of gibberellin-
like activity was regularly placed at 0.7-0.8 with occa­
sional appearance in the 0.8-0.9 region and could cor­
respond to A9.
Whyte and Luckwill (16) reported the dock bioassay
to be most sensitive to GA3 while GA4 was only a tenth
as effective with other known gibberellins still less effec­
tive (GA5, one-hundredth; GA9, one-thousandth). How­
ever, as their data have received no separate confirma­
tion and as the substances extracted in these studies have
not been positively identified as gibberellins, the assay
data should be interpreted cautiously. Similarly, Jones
and Varner (7) reported that GA5 induced less response
in a 24-hr period in the barley assay than GA4, GA3,
GA4 or GA7 while Stoddart and Lang (15) reported
GA9 to be only 0.1% as active as GA3 in this system.
Thus, suspected GA5-like and GA9-like components may
be underestimated by this assay, and interpretations as
to their presence or absence should be made cautiously.
Assuming the flow rates of Table 1 and the GA equiva­
lent activity of Table 3, a calculation was made estimat­
ing the production at 0.0012 jig GA3 equivalents/plant/
day or approximately 0.14 pg/1. Such a figure is compa­
rable to other reports (10, 13).
The presence of gibberellin-like activity in the sap does
not necessarily confirm its origin as a product of the root.
Jones and Phillips (6) recognized this and provided evi­
dence for considering the root as one organ involved in
gibberellin synthesis. Their use of the agar block diffu­
sion technique with roots, coupled with extraction of root
tissue, confirmed that the apical 3-4 mm of the root ac­
tively synthesized gibberellin. Butcher (2) had previously
isolated gibberellin-like substances in excised tomato
roots in vitro, and Sitton et al. (12) demonstrated that
isolated root apices incorporated C14-mevalonate into gib­
berellin intermediates. A preliminary experiment was
conducted using the agar block diffusion technique to
check the possibility that the root apex may be involved
in the synthesis of gibberellin-like substances in poinset­
tia. The level of biological activity evidenced in the
barley endosperm assay (Fig. 5) broadens the basis for
considering the root apex as a source of gibberellin-like
substances.
Table 3. GA3 equivalents per plant for root exudates from long day
p lan ts an d long day plan ts w hich received five weekly sprays of
0.25% Cycocel. Values derived from Fig. 4 following adjustment
for number of plants and condition of assay.
Fraction
(GA3 equivalents in Mg X 10- 3)
Treatment
A B C Total
Long d ay..................... 0.48 0.4 0.32 1.28
Long day + Cycocel. 0.16 1.6 + 1.2 2.96
J. Amer. Soc. Hort. Sci. 95(1): 120-124. 1970.
 >igGA-3/ml *
Fig. 3. Acidic ethyl acetate extracts of two-day exudate collections from LD and LD + Cycocel drench poinsettia
plants. A. 465 ml (1.5 g dry wt) exudate from 30 LD plants. B. 465 ml (1.61 g dry wt) exudate from 34 LD plants
which received 1 g Cycocel per 16 inch pot one week previously. Chromatographed on TLC. Solvent, isopropyl
ether: acetic acid (95:5, v/v). Bioassay with dock leaf. LSD values indicate significant difference from the
control at the 5% level of risk.

Fig. 4. Acidic ethyl acetate extracts of exudate from LD and LD + Cycocel sprayed poinsettia plants. A. 335 ml
(1.22 g dry weight) from 50 LD plants. B. 290 ml (0.93 g dry weight) from 50 LD plants which received 5 weekly
sprays of 0.25% Cycocel. Chromatographed on TLC. Solvent, isopropyl ether:acetic acid (95:5, v/v). Bioassay
with barley endosperm. Peaks represent mean of two replicates.

J. Amer. Soc. Hort. Sci. 95(1): 120-124. 1970. 123


Fig. 5. Diffusate of 138 root tips diffused for 24 hr in darkness.
Chromatographed on TLC. Solvent isopropyl ether: acetic acid
(95.5, v/v). Bioassay with barley endosperm system. Peaks repre­
sent mean of two replications.
The evidence of Fig. 3 for a Cycocel drench and of Fig.
4 for a Cycocel spray confirmed the observation (11) that
repeated applications of Cycocel reduce the gibberellin
production of the root. The sprays applied to the top of
the plant did not reduce the content of gibberellin-like
substances in root exudate as much as might have been
expected if the shoot apex were the sole source of gib-
berellins.
The effect of Cycocel in reducing the level of gibberel­
lin-like activity in the poinsettia is of interest because of
the relation of Cycocel to bud initiation under marginal
and non-inductive photoperiods as previously discussed.
Cycocel is thought (5) to affect one or more of the steps
involved in conversion of the C-20 gibberellins to C-19
gibberellins. In Fig 4, for example, the A fraction (Rf 0-
0.1), which may represent the terminal product of inter­
conversions, is less active in the exudate of treated plants.
124
Cycocel may lower the level of some gibberellin or gib­
berellin precursor which is inhibitory to flower bud initi­
ation in the poinsettia. Such a phenomenon may be
restricted to conditions in which natural production is
just barely sufficient to effect the inhibition, i.e., under
marginal photoperiods.
Similarly, under short daylengths, the A fraction was
lower in relation to the other fractions than in long day
exudates. Stoddart (14) and Stoddart and Lang (15) ad­
vanced the concept of daylength-mediated steps in gib­
berellin metabolism. The alteration in gibberellin bal­
ance in root exudate during the transition from the
vegetative to the floral state also has been documented
(8). The possibility of conversion between gibberellins
arising in the root and in the shoot complicates this con­
cept but injects the thought that Fractions B and C may
have their origin in the shoot apex and Fraction A in the
root tip. These may interact or function independently.
Under short days flower initiation in the poinsettia may
result as a response to a decreased ratio of Fraction A to
other gibberellin-like substances or as a response to a
higher level of the other gibberellin-like substances, no
matter what the level of Fraction A.
L i t e r a t u r e C it e d
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and Cell Physiol. 8:201-205.
2. B u t c h e r , D. N. 1963. The presence o f gibberellin in excised
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