J Plant Physiol. Vol. 148. pp.

677-683 (1996)
Germination of Chaenorrhinum minus Seeds in Response
to Gibberellin Treatments
ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTA
Department of Biology, Colgate University, Hamilton, NY 13346-1398, U.S.A.
Received June 2, 1995 . Accepted September 21, 1995
Summary
Seeds of Chaenorrhinum
l
minus (L.) Lange (Scrophulariaceae) exhibit embryo dormancy when released
from capsules of the parent plant, but can be induced to germinate when chilled or treated with gibberel-
lins (GAs). GAs found to have activity in breaking dormancy of C minus seeds were GA
3
, GAl, and
The degree of germination success, however, depended on pH, concentration, duration of incuba-
tion, and on the kind of GA applied. Newly mature seeds treated with GA
3
at a range of pH values had
optimum germination success in the pH range 8.5-10; by contrast, seeds that were stored for 10 months
at room temperature had optimum germination success in the pH range 5.5-8. GAl was the most active
of the GAs tested: in a 96 h incubation, the GAl concentration required for 50 % germination was approx-
imately 0.3 mmol L-I, compared to 2.5 mmol L-I for and 100 mmol L-I for GA
3
. In addition, the
maximum proportion (approx. 85 %) of seeds that germinated in GAl was greater than in the other GAs.
and produced almost identical responses, showing that the effects of and GAl in the mix-
ture were not additive. Two other GAs, GA
l3
and GArisolactone, were completely ineffective in breaking
dormancy. These results are discussed in terms of the affinity of the various GAs for possible receptor sites
in C minus seeds.
Key words: Chaenorrhinum minus (L.) Lange, dormancy, germination, gibberellin, seed.
Abbreviatiom: GA = gibberellin.
Introduction
Commonly, seeds are dormant when shed from the parent
plant. Such dormancy has, in general, one or more of several
causes (Mayer and Poljakoff-Mayber, 1989). These include
immaturity of the enclosed embryo; impermeability of the
seed coat to water and gases; mechanical prevention of em-
bryo elongation; and the presence of germination inhibitors,
which are often associated with the seed coat. The last three
are examples of the general phenomenon of coat-imposed
dormancy, which can be relieved by environmental phenom-
ena that soften the seed coat: these include microbial action,
abrasion by soil particles, and repeated freeze-thaw cycles.
These processes apparently do not involve active participation
by the embryo (Bewley and Black, 1985). Embryo immatu-
1 Spelled Chaenorhinum by some European authorities.
1996 by Gustav Fischer Verlag, Stuttgart
rity is, on the other hand, released by changes that take place
in the embryo itself and that have been termed afterripen-
ing when they occur during storage (Bewley and Black,
1985; Mayer and Poljakoff-Mayber, 1989). Such maturation
is often stimulated by temperature treatment or by applica-
tion of chemicals, including natural and synthetic plant
growth regulators and a miscellany of other compounds such
as respiratory inhibitors (e.g., cyanide, azide), nitrogenous
compounds (e.g., nitrate, thiourea), and even ethanol, hypo-
chlorite, and methylene blue (Bewley and Black, 1985).
Clearly, many of these compounds are unlikely to be encoun-
tered by seeds in their natural environment, but information
about dormancy and its release has been gained by laboratory
applications of them.
The most widely used and effective temperature treatment
for breaking embryo dormancy involves chilling seeds that
have been layered in moist sand or soil (a process called strati-
678 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTA
fication}. A temperature of 5C is most commonly employed,
but other temperature conditions, including fluctuating tem-
peratures, have also been successfully employed in some spe-
cies. The very extensive literature on this topic has been re-
viewed in, for example, Bewley and Black (1985) and Mayer
and Poljakoff-Mayber (1989).
A wealth of evidence (see, for example, Inoue, 1991; Len-
ton and Appleford, 1991) supports the involvement of gibbe-
rellins (GAs) in releasing seeds from dormancy. Much of this
evidence comes from experiments in which seed dormancy
has been broken by application of exogenous GAs, most
commonly gibberellic acid (GA
3
) or and GAl, either
separately or in mixture. These hormones have been shown to
be effective in breaking dormancy in seeds that otherwise re-
quire chilling (Bewley and Black, 1985) or exposure to light
(Derkx et al., 1994). Embryos of hazel (Corylus avellana) kept
at 5 C for 42 d and then transferred to 20 C for 8 d (Wil-
liams et al., 1974) had a much higher GAl and GA
9
content
than embryos kept at constant temperatures of 5 or 20 C,
suggesting that the chilling treatment somehow prepared the
embryo to synthesize the GAs in the subsequent exposure to
warmer temperatures. In contrast, recent work with Arabi-
dopsis thaliana by Derkx et al. (1994) indicates that the pro-
motion of seed germination by chilling (in association with
light) in this species is most likely not a consequence of in-
creased GA content. Indeed, Trewavas (1982, 1987) has pro-
posed that changes in the sensitivity of seeds to plant growth
regulators during maturation or post-dispersal events, rather
than changes in the levels of these regulators, may be the key
to understanding dormancy and germination. More studies
with other species, which address appropriate questions (see
Firn, 1986), are needed to clarify this situation.
Numerous authors have described the greater effectiveness
of GAs other than GA
3
in stimulating seed germination, es-
pecially in dicots: these GAs include (Loveys and Jusai-
tis, 1994; Derkx et al., 1994), (Karssen et al., 1989;
Derkx and Karssen, 1993), and GArisolactone (Derkx et al.,
1994).
This study reports work done with Chaenorrhinum minus,
an annual weed that grows in dry, gravelly soils, especially
along railways, throughout most of western Europe, northern
and central regions of the United States, and in southern
Canada. A previous study by Grime et al. (1981) has indi-
cated that C. minus seeds are released from dormancy when
chilled for 45 d at 5 C while buried in moist sand. In the
studies reported here, we investigated the germination re-
sponse of seeds of C. minus to exogenous treatment with sev-
eral different GAs.
Materials and Methods
Seeds of Chaenorrhinum minus were collected from local popula-
tions in July-September 1993 and 1994 in the vicinity of Hamil-
ton, NY. They were air-dried and stored at approximately 22 C
and 30 % relative humidity. Prior to all experiments, seeds were al-
lowed to imbibe deionized (milli-Q) water for 24 h. In these experi-
ments, each treatment group consisted of 6 replicates of 25 seeds
each.
Gibberellin treatments
Unless otherwise indicated, GA solutions were prepared by sus-
pending the crystalline GAs in phosphate-citrate buffer, pH 6.5,
containing 4.05 mmol L-1 K
1
HP0
4
and 1.0 mmol L-I citric acid
and adding 1mmol L-1 KOH one drop (aprox. 0.03 mL) at a time
until the GA dissolved. Then, 1.0 mmol L- citric acid was added to
adjust the pH to the required value (6.5, unless otherwise stated)
and more buffer was added to make the final solution.
a) Response to pH ofGA
3
solutions
Seeds were treated in 120 mmol L-1 solutions of GA
3
at a range
of pH values from 4 to 12 in 50 mmol L-1 phosphate buffer. The
pH treatments were conducted with seeds of the following ages:
seeds that matured and were collected in 1993 and then stored for
10 months under the conditions described above; seeds that
matured and were collected in 1994 and tested within 2 weeks of
maturity; and, seeds in the latter 1994 group that were stored under
the conditions described above for 10 months.
b) Dose responses to GA
3
, GA
4
, and GA
7
Seeds of C minus were immersed in solutions of gibberellic acid
(GA
3
, K-salt; Sigma Chemical Co., St. Louis, Missouri, USA) at pH
6.5 at concentrations in the range 0.03-480 mmol L-1 for 9Gh fol-
lowing the 1d imbibition with water. Other seeds were treated sim-
ilarly with or GA
7
(Sigma Chemical Co.) at concentrations in
the range 0.003-12mmol. L-1.
c) Response to duration oftreatment with GA
3
and GA
4
+
7
Seeds were incubated in 120 mmol . L-1 GA
3
or 3 mmol . L-I
for periods of time ranging from 24 to 144 h for GA
3
and 2 h
to 9Gh for all such seeds receiving less than 144h in GA
3
or
96 h in were returned to milli-Q water after the GA treat-
ment, and all seeds were transferred to germination dishes, as
described below when the longest GA treatment had been con-
cluded. To test the stability of the GA solutions at room temperature
over a 96 h period, some batches of seeds were treated with GA
3
or
for 9Gh beginning with 9Gh-old solutions.
d) Comparative effectiveness ofvarious gibbereLlins
Solutions at 3 mmol L-1 concentration of GA
7
, GA
7
isolac-
tone, (1.5 mmol L-1 of each), and GA
l3
were prepared by
dissolving the crystalline solids in 0.2 mL of 50 % ethanol and then
adding a relatively large volume (about 9 mL, depending on the
purity and molecular weight of the GA) of phosphate-citrate buffer,
pH 6.5, containing 4.05 mmol L-I K
1
HP0
4
and 1.0 mmol. L-
1
citric acid. Seeds were incubated in each GA solution for 36 hand
then returned to milli-Q water for 60 h. The 36 h GA incubation
time was chosen because it gave approximately 50 % of maximum
germination in GAl' thus making possible the detection of more
effective GAs while still providing an overall incubation time of 96 h
in bulk liquid prior to transfer of seeds to filter paper.
Germination trials
After GA treatments, seeds were washed with 3 changes of milli-
Q water and transferred in a small known volume of milli-Q water
to plastic disposable Petri dishes containing two layers of filter paper
(Whatman # 3). Sufficient milli-Q water was added to each dish to
bring the total volume added to 6.5 mL. All dishes were weighed
and then incubated in a plant growth chamber (Model PG-77, Per-
cival Manufacturing Co., Boone, Iowa), equipped with a combina-
tion of fluorescent (160 W Gro-Lux, Osram Sylvania, Inc., Dan-
vers, Massachusetts, USA) and 60 W incandescent bulbs. Incubation
conditions were a I6L18D photoperiod, with temperatures 25 C
and 15C, respectively, and relative humidity 70 2 %. Dishes were
examined daily, germinated seeds were scored and removed, and suf-
ficient milli-Q water was added to each dish to restore its mass to its
original value. Since the mean mass of seeds was only 49 /lg (range =
14-77/lg, N= 150 seeds) it was considered unnecessary to compen-
sate for the mass of germinated seeds that had been removed. A seed
was considered to have germinated when the radicle produced any
degree of outgrowth visible at a lOx magnification of a dissecting
microscope; a mere swelling of the embryo, even when accompanied
by rupture of the seed coat, was not considered successful germina-
tion. Experiments were terminated when there was no further ger-
mination over 3 successive d.
Statistical analysis
In all experiments resulting in percentage data, statistical tests and
function fitting operations were carried out after arcsine transforma-
tion of the percentages (as arcsin [proportion seeds germinated] 'h) in
order to satisfY the assumptions of analysis of variance and regres-
sion that the error terms are homoscedastic and normally distributed
(Sokal and Rohlf, 1995). Where indicated, Tukey tests for multiple
comparisons among means (Zar, 1984) were carried out after analy-
sis of variance.
Results
Seed germination in Chaenorrhinum minus 679
60
-I<)!....
'0
(J)
1U
50
c:
'E
iii
Ol 40
(/J
'0
(J)
(J)
(/J
30
c:
0
'
0
c.
20
e
.e:
(J)
c:
Ow
10

0
2 4 6 8 10 12 14
pH of incubation solution
Fig. 1: Germination of newly matured and stored seeds of Chaenor-
rhinum minus in response to the pH of the 120 mmol L-I gibberel-
lic acid (GA
3
) solution in which they were incubated for 96 h fol-
lowing a 24 h imbibition in water. Newly mature seeds (0) were
collected in 1994 and tested within 2 weeks of capsule dehiscence;
stored seeds (e) wete collected in 1993 and 1994 and stored at
room temperature for 10 months prior to testing. Vertical bars
around the means are SE. Curves were fitted by a robust locally
weighted regression (LOWESS) method (Cleveland, 1979, 1981).
Table 1: Germination response of seeds of Chaenorrhinum minus to
various commercially-available gibberellins (GAs). Seeds were in-
cubated in 3 mmol L-I solutions of each gibberellin, or mixtute, for
36 h following a 24 h imbibition in water, returned to water for
60 h, and then incubated in a growth chamber as described in Mate-
rials and Methods.
Experiments with gibberellins
Dormancy of C minus seeds was broken by pretreatment
with solutions of the gibberellins GA
3
, GA
7
, and
but the effectiveness depended on pH (Fig. 1), con-
centration (Fig. 2), and duration of incubation (Fig. 3) in GA
solutions and on the kind ofGA applied (Fig. 2 and Table 1).
Nature ofdormancy in Chaenorrhinum minus
In all treatments reported here, including controls in-
cubated in water, seeds became swollen and soft, indicating
they had imbibed water and that the seed coat was no barrier
to the uptake of water. In some GA treatments, seeds that did
not reach the point of successful germination, by our criteria,
did undergo embryo enlargement sufficient to rupture the
seed coat at one end. This typically did not occur in control
seeds.
GA
3
4
7
4+7
7-isolactone
13
Mean percent
germination SE
o
37.25.93
85.34.08
37.43.22
o
o
a) Respome to pHofGA
3
solution
Figure 1 shows the dependence of C minus seed germina-
tion on the pH of the GA
3
solutions in which seeds were in-
cubated. Seeds that matured in 1993 and were stored for 10
months had a broad optimum germination response in the
pH range 5-8; newly mature seeds collected in 1994 had a
sharper optimum in the pH range 8.5-10. Seeds from this
latter batch that were stored for 10 months, however, showed
a pH response indistinguishable from the lO-month-old seeds
from the previous year (F= 1.37, P>0.05). The responses of
newly-mature and stored seeds were significantly different
from one another (F for age as a factor = 158.32, P <0.001).
In addition, the response of seeds of both age groups ap-
peared to be quadratic (F for quadratic term for age X pH in-
teraction = 7.14, P<.05) but reaching similar maxima (at ap-
prox. 65 % and 67 % germination success for newly mature
and stored seeds, respectively) but at significantly different
pH values (Ffor pH x age interaction = 65.86, P<O.OOI).
b) Dose responses to GA
3
, GA4' and GAl
The responses of seeds to different concentrations of GA
3
,
and GA
7
is shown in Figure 2. For the and GA
7
treatments, the arcsine transformed percentage values met the
assumptions of normality and homoscedasticity of the error
terms (residuals) in the regressions; these assumptions were
not met by the original percentage data.
An increase in germination success with increasing GA
concentration was seen in all three treatments. In the case of
GA
3
, the effective range of concentration in which increasing
germination was observed was 3-120 mmol L-1: further in-
680 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTA
60
96
144
24 48 72
Hours of treatment with
24 48 72 96 120
Hours of treatment with GA
3
A
.....
60..,-----------------,
50 B
Q>
Cl
rJ) 40
1
c 30
o
'E
8. 20

10
.! 0 +-I__
<{ 0
.....

60
'E
Q> 50
Cl
rJ)
i 40
3l
c 30
o
.-E
8. 20
e
.9: 10
CD
.5 0
0
nated = sin
2
64.87 = 0.82). It is noteworthy that the propor-
tion of seeds that germinated at each GA
7
concentration (in-
cluding those that gave maximum germination success) was
markedly greater than that in the corresponding treat-
ments.
The regression functions may also be used to calculate the
GA concentration required for 50 % germination as a direct
way of comparing GA effectiveness. The resulting values are
2.5 mmol . L-1 for and 0.29 mmol L-1 for GA
7
, again
showing the much greater effectiveness of the latter GA. The
corresponding concentration for GA
3
can be estimated by in-
terpolation in Figure 2, which gives a value in the vicinity of
100 mmol . L-1. Thus, GA
7
is more effective than GA
3
by a
factor of almost 350 in breaking dormancy of C minus seeds.
In addition, seeds germinated much sooner following
treatment with and GA
7
than with GA
3
. This is shown
by comparison of tso (time for 50 % germination) values for
the three treatments: seeds treated with or GA
7
at
3 mmol L-1 (the lowest concentration to give maximum ger-
mination) had a tso of approximately 7d (from initial imbibi-
tion with water; 6 d after beginning GA treatment); in con-
trast, seeds treated with GA
3
at 120 mmol . L-1 (correspon-
dingly, the lowest concentration giving maximum germina-
tion) had a tso of approximately 17 d (i.e., 16 d after begin-
ning GA treatment).
Fig. 3: Germination success of seeds of Chaenorrhinum minus
treated with solutions of the gibberellins GA
3
and +7 fot diffet-
ent periods of time following a 1d imbibition in water. Vertical bars
around the means are SE. Regression equations are:
A. GA
3
: Y= 50.77/(1 + 10.73e-o.067X). R
2
=0.80).
B. Y= 54.64/(1 + 2.23e-O.129X); R
2
= 0.89).
1000
. , I
0.01 0.1 1 10 100
Gibberellin concentration (mmolr
1
)
I
0.001
10
70..,-------------------
creases in GA
3
concentration to 240 and 480 mmol L-1 pro-
duced no significant change in germination success (Tukey
test: q for each comparison <4.96, P >0.05). No seeds ger-
minated at GA
3
concentrations less than 3 mmol . L-1. At-
tempts to fit regression functions to the GA
3
data were not
successful: the If of the best-fitting function (a logistic one)
was only 0.47 and the resultant curve did not converge with
the plotted means. Treatment of seeds with resulted in a
similar relationship between germination success and concen-
tration but at much lower GA concentrations (Fig. 2). Here,
concentrations in the range 0.03-3 mmol L-1 resulted
in a progressive increase in the proportion of seeds that ger-
minated, but further increases in concentration to 6 and
12 mmol L-1 produced no further significant change in ger-
mination success (Tukey test: qfor each comparison <4.90, P
>0.05), and there was no germination at concentrations
of 0.03 mmol L-lor less. The significant regression function
for treatment implies a maximum germination success
of approximately 69 % (proportion seeds germinated =sin
2
56.14 = 0.69). Finally, still lower concentrations (0.006
mmol L-1) of GA
7
were effective in breaking seed dormancy:
as GA
7
concentration increased, an increase in germination
success, similar to that for was observed, reaching a
maximum at 3 mmol- L-1 GA
7
Again, further increases in
GA
7
concentration to 6 and 12mmol L-1 produced no signif-
icant change in response from the 3 mmolL-1 value (Tukey
test: q for each comparison <4.60, P >0.05). In the case of
GA
7
, the regression function implies a maximum germina-
tion success of approximately 82 % (proportion seeds germi-
Fig. 2: Gibberellin dose responses of seeds of Chaenorrhinum minus.
Seeds were incubated with GA
3
(0), (e), or GA
7
(0) fot 96h
following a 24 h incubation in water. Vertical bars around the
means are SE. Regression e9nuations are:
y= 56.14/(1 +0.5ge- .95X); =0.88;
GA
7
: y= 64.87/(1 +0. 16e-
O
.
83
x); =0.92.
The GA
3
data did not adequately fit a logistic function.
c) Response to duration oftreatment with GA
3
or GA4+7
Germination success of C. minus also depended on the du-
ration of treatment with GA solurions following the 1d imbi-
bition in water (Fig. 3). Figure 3 A shows the response to
GA
3
: there was a progressive increase in germination success
with increasing lengths of exposure from 1 to 4 d, but appar-
ently.no further increase at 5 and 6 d of treatment. A logistic
function gave the best fit to these data, which is consistent
with germination success reaching a maximum (approx. 60 %
germination) following about 4 d incubation in GA
3
and
then remaining constant. There were no significant differ-
ences among the 96, 120, and 144h percent germination val-
ues (Tukey test: q>2.5 for each comparison, P>0.5), show-
ing that maximum germination success (approx. 60 % germi-
nation) resulted from approximately 96 h of incubation in
GA
3
and remained constant thereafter.
Figure 3 B shows the response to As in the case of
the response to GA
3
, a logistic function provides the best fit
to these data. There were no significant differences among
the 48, 72, and 96 h percent germination values (Tukey test:
q >2.5 for each comparison, P >0.5), showing that max-
imum germination success (approx. 66 % germination) re-
sulted from approximately 48 h of incubation in +7 and
remained constant thereafter.
The stability test results showed that there was no signifi-
cant difference in the proportions of seeds that germinated
when seeds were incubated for 96 h beginning with freshly-
prepared versus 96h-old GA
3
and (F= 0.76 and 0.88,
P@0.05 for GA
3
and respectively).
d) Comparative effectiveness ofvarious gibberellins
Data in 'fable 1 show that, at a 3 mmol L-I concentration
and 36 h treatment duration, GA
7
was the most effective GA
of. tested in breaking dormancy in C. minus (85 % ger-
success); and the mixture were equally
effective (Tukey test: q = 0.133, P >0.9), producing approx.
37% germination success, but these two GA treatments pro-
duced significantly less germination success than GA
7
alone
(Tukey test: q for GA
7
versus = 10.28 P <0.01 and q
for GA
7
versus = 10.03, P <0.01). Finally, GA
3
, GA
13
and GArisolactone induced no germination, although GA
3
at this same concentration (3 mmol L-I) had a slight stimu-
latory effect (approx. 8 % germination success - see Fig. 2)
when seeds were incubated in this hormone for 96, rather
than 36 h. The seed coats in approximately half of the seeds
in this 36 h GA
3
treatment ruptured during the incubation,
although they did not develop beyond that point. By con-
trast, no such rupture of the seed coat in the GArisolactone
nor GA
I3
treatments was seen, even in additional seeds main-
tained in these GAs for 96 h.
Discussion
The significance, if any, of the difference in pH optimum
of newly-matured and stored seeds with respect to GA
3
-sti-
mulated germination is not clear to us. The broad pH opti-
mum (approx. 5-8) of stored seeds corresponds almost ex-
actly to the reported pH range for soils in which C. minus
Seed germinarion in Chamo"hinum minuJ 681
grows in nature (Grime et al., 1988); the species has not been
reported in soils of pH in the corresponding, somewhat nar-
rower, pH optimum range (approx. 8.5-10) for newly- ma-
ture seeds. It is thus possible that the pH requirement for ger-
mination of newly mature seeds is part of a mechanism that
prevents premature germination in the late summer/early fall,
when seedlings would be killed by forthcoming winter condi-
tions. We have not examined the pH response of other GAs.
There is no reason, however, to expect major differences be-
cause all the GAs we tested and that showed activity have in
common a single carboxyl group attached to carbon-6 of the
B ring and no other ionizable functional group. With regard
to the overall response of seeds to pH, we do not know
whether the variation in germination success of seeds within
each age group in our study is the result of pH effects on
metabolic enzymes or, more specifically, on the molecular
forms of added (and indigenous) GAs.
In comparing the effectiveness of different gibberellins (Ta-
ble 1), especially the components and GA
7
with the mix-
ture +7, a treatment duration of 36 h was chosen because,
as shown in Figure 3 B, this duration was expected to yield
substantial bur less than maximum germination (approx.
45 %) in thus allowing for the possibility of detecting
GAs more active than as well as those equally or less
active. It is of particular interest that GArisolactone had no
activity in breaking dormancy in C. minus. Derkx et al.
(1994) found this GA, along with to be the most active
in stimulating germination in Arabidopsis thaliana seeds, and
they also reported that GA
7
was among the least active of
those tested. There can be little doubt that sensitivity to the
various GAs is highly species-specific. For example, GAl ap-
pears to be the main biologically active compound in wheat
(Lenton and Appleford, 1991); and GA
9
are particularly
abundant in conifers Junttila, 1991); and GA
l9
and GA
20
seem to be the dominant kinds in woody dicotyledons Junt-
tila, 1993). Moreover, GArisolactone has also been shown to
have low activity in other plant systems in which other GAs,
especially and GA
7
, are effective. For example, GAriso-
lactone has little stimulatory effect on the growth of seedlings
of dwarf cultivars of cucumbers, lettuce, and peas (Serebrya-
kov et al., 1984) and is ineffective in controlling the fruit rus-
set disorder of apples (Looney et al., 1992). Thus, the lack of
sensitivity of C. minus seeds to GArisolactone is not, in and
of itself, surprising. Furthermore, examination of the struc-
ture of this GA in light of one of the proposed characteristics
of GAs (see Derkx et al., 1994), namely that the pos-
sessIOn of a C
lO
-C
I9
lactone bridge below the plane of the
A-ring of the molecule is important to activity, suggests a
possible explanation: GArisolactone has a C
2
-C
19
(instead
of a C
lO
-C
I9
) lactone bridge. This suggested characteristic
also explains the inactivity of GA\3' which has no lactone
bridge at all. Additionally, GA\3 has three carboxyl groups
plus a hydroxyl group, which will make it significantly more
polar than the other GAs tested and perhaps further reduce
its affinity for a presumed GA receptor to which GAs may
bind as a prerequisite for cellular activity.
A further difference between GA
7
and GArisolactone lies
in the position of the double bond in the A-ring adjacent to
the lactone bridge: it is between C
I
and C
2
in GA
7
and be-
tween C
1
and C
lO
in GArisolactone. Derkx et al. (1994) also
682 ROBERT M. ARNOLD, JENNIFER A. SLYKER, and TARA H. GUPTA
report that GArisolactone is converted to GA
7
by KOH,
which is commonly used to initially dissolve GAs prior to di-
lution with water or buffer. These workers also report in-
creased activity of GAs dissolved in ethanol compared to
those dissolved in KOH. In the experiments to compare the
different gibberellins reported here, small volumes of ethanol,
rather than KOH, were used to initially dissolve GAs for the
experiments to compare activity of the various GAs tested.
This treatment seems to have not caused conversion among
GA
7
isomers; given the high reactivity of C. minus seeds to
GA
7
, any conversion of GArisolactone to GA
7
would likely
have been detected as a greater-than-zero germination success
in the former. In regard to our use of KOH to assist the dis-
solving of GAs in other experiments reported here, the sim-
ilar germination maxima produced by GA
7
when dissolved by
the KOH (Fig. 2) and ethanol (Table 1) methods suggests
that our KOH method (in which GAs were dissolved in buf-
fer and then KOH was added dropwise to dissolve the GAs)
is less destructive to GAs than when GAs are initially dis-
solved in KOH followed by dilution with buffer. This latter
method would, of necessity, expose the GAs to more strongly
basic conditions (pH close to 14) than would the former
method, in which the pH of the GA solutions did not exceed
8.5.
It is still possible, however, that our use ofKOH to prepare
GA solutions for the dose response experiments may explain
a striking difference between our results and those of Derkx
et al. (1994). The most effective of the GAs (GA
7
) tested in
the present" study needed to be at a 1mmol . L-1 concentra-
tion or higher to produce maximum germination success; in
contrast, Derkx et al. (1994) obtained maximum germination
success in seeds of A. thaliana using GA concentrations in the
range 0.01-0.1 mmol L-1. Alternatively, this apparent incon-
sistency may be due to characteristics inherent in the differ-
ent plant species used in the two studies.
A second proposed characteristic of active GAs (Stoddart,
1986) is substitution at the C
13
position. Interestingly, of the
GAs tested here, only GA
3
has substitution (by a hydroxyl
group) at this position, yet this GA was much less active than
or GA
7
. Clearly, other factors besides C
13
substi-
tution playa role here.
Comparison among the GAs also shows that the effects of
the mixture, can not be predicted from knowledge of
the effectiveness of the separate components. Based on the
much greater effectiveness of GA
7
than one would pre-
dict that would have intermediate effectiveness. This
was not the case, however, since and were almost
identical in effectiveness. This observation also provides evi-
dence that there may be only one GA receptor for and
GA
7
in C. minus, since the effects of the two growth regula-
tors are not additive. Derkx et al. (1994) observed a similar
lack of additivity in experiments with GA
7
and GArisolac-
tone in A. thaliana and, accordingly, proposed that the two
GAs compete for the same binding site. We suggest that the
same kind of competition for a single type of receptor site,
here involving and GA
7
, may occur in C. minus.
Stronger evidence in favor of such competition would come,
however, from studies of dose responses to GA
7
in the pres-
ence of varying quantities of A measure of the relative
strength of binding of and GA
7
to the receptor(s) is
given by the [GA] 50 values (the GA concentration giving half
the maximum response). These quantities can be determined
from the logistic function for each GA, and have values
0.57 mmol L-1 and 0.11 mmolL-1 for and GA
7
, respec-
tively. The logistic curves at [GA] 50 for the two GAs are al-
most identical (the slopes, obtained by differentiation of each
function at an abscissa value of [GAJso, were 13.3 and 13.5
for and GA
7
, respectively). By analogy with the Michae-
lis constant for an enzyme-catalyzed reaction, the very differ-
ent [GA] 50 values suggest that GA
7
has a greater affinity for
its receptor than has for the same (or separate) receptor,
especially as the two logistic functions are essentially parallel
at this concentration (Firn, 1986; Weyers et al., 1987).
The greater effectiveness of GA
7
in breaking dormancy of
C. minus seeds is also evident from the significantly greater
maximum response (approx. 82 % germination success) than
was observed with (approx. 65 %) and GA
3
(approx.
55 %). This is in contrast to work by Derkx et al. (1994),
who found that the maximum response of A. thaliana seeds
was similar (and close to 100%) in all GAs tested over a suffi-
ciently broad range of concentrations to produce a maximum
response. This difference between and GA
7
again is con-
sistent with competition between the two GAs for the same
receptor sites, to which GA
7
has greater affinity, and hence
stimulates a greater response.

The authors thank Colgate students Kerri Blum, Tamara Mer-
chant, John Mulhall, and Allison Wayman for conducting prelimi-
nary experiments for this study; Dr. T. Sparks of the Institute ofTer-
restrial Ecology, Monks Wood, Cambridgeshire, U.K. for statistical
analysis of the pH data; Dr. K. Valente of the Department of Mat-
hematics, Colgate University for help in estimating the mid-point
slopes of logistic functions; and Dr. V. McMillan of the Department
of Biology, Colgate University for critically reading the manuscript.
The research was supported by a grant from the Colgate Research
Council.
References
BEWLEY, J. D. and M. BLACK: Seeds: Physiology of Development
and Germination. Plenum Press, NY. (1985).
CLEVELAND, W. S.: Robust locally weight regression and smoothing
scatterplots. J. Amer. Statist. Assn. 74,829-836 (1979).
- LOWESS: A program for smoothing scanerplots by robust lo-
cally weighted regression. Amer. Statist. 38, 54 (1981).
DERKX, M. P. M. and C. M. KARSSEN: Variability in light-, gibberel-
lin-, and nitrate requirement of Arabidopsis thaliana seeds due to
harvest time and conditions of dry storage. J. Plant Physiol. 141,
574-582 (1993).
DERKX, M. P. M., E. VERMEER, and C. M. KARSSEN: Gibberellins in
seeds of Arabidopsis thaliana: biological activities, identification
and effects of light and chilling on endogenous levels. Plant
Growth Regul. 15,223-234 (1994).
FIRN, R. D.: Growth substance sensitivity: the need for clearer ideas,
precise terms, and purposeful experiments. Physiol. Plant. 67,
267-272 (1986).
GRIME, J. P., G. MASON, A. V. CURTIS, J. RODMAN, S. R. BAND, M.
A. G. MOWFORTH, A. M. NEAL, and S. SHAW: A comparative
study of germination characteristics in a local flora. J. Ecol. 69,
1017-1059 (1981).
GRIME, J. P., J. G. HODGSON, and R. HUNT: Comparative Planr
Ecology. Unwin-Hyman, London (1988).
INOUE, Y.: Role of gibberellins in phytochrome-mediated lettuce
seed germination. In: TAKAHASHI, N., B. O. PHINNEY, and J.
MAcMILLAN (eds.): Gibberellins, pp. 289-295. Springer-Verlag,
New York (1991).
JUNTTILA, 0.: Gibberellins and the regulation of shoot elongation in
woooy plants. In: TAKAHASHI, N., B. O. PHINNEY, and J. MAc-
MILLAN (eds.): Gibberellins, pp. 199-210. Springer-Verlag, New
York (199l).
- lneeraction of growth retardants, daylength, and gibberellins A
19
,
A
2o
, and Al on shoot elongation in birch and alder. J. Plane
Growth Regul. 12, 123-127 (1993).
KARSSEN, C. M., S. ZAG6RSKI, J. KEpCZYNSKI, and S. P. C. GROOT:
Key role for endogenous gibberellins in the control of seed germi-
nation. Ann. Bot. 63,71-80 (1989).
LENTON, J. R. and N. E. J. APPLEFORD: Gibberellin production and
action during germination of wheat. In: TAKAHASHI, N., B. O.
PHINNEY, and J. MAcMILLAN (eds.): Gibberellins, pp. 125-135.
Springer-Verlag, New York (1991).
LOONEY, N. E., R. 1. GRANGER, C. 1. CHU, S. J. McARTNEY, 1. N.
MANDER, and R. P. PHARIS: Influences of gibbereJlins At, At.?,
and At +iso-A? on apple fruit qualiry and tree productiviry. I. Ef-
fects on fruit russet and tree yield componenes. J. Hort. Sci. 67,
613-618 (1992).
LoVEYs, B. R. and M. JUSAITIS: Stimulation of germination of quan-
dong (Santa/um acuminatum) and other Australian native plane
seeds. Aust. J. Bot. 42, 565-574 (1994).
Seed germination in Chamorrhinum minUJ 683
MAYER, A. M. and A. POLJAKOKFF-MAYBER: The germination of
seeds, 4th Ed. Pergamon Press, Oxford (1989).
SEREBRYAKOV, E. P., V. N. AGNISTIKOVA, and 1. M. SUSLOVA:
Growth promoting activiry of some selectively modified gibber-
ellins. Phytochemistry 23, 1847-1854 (1984).
SOKAL, R. R. and F. J. ROHLF: Biometry: the principles and practice
of statistics in biological research, 3rd Ed. W. H. Freeman and
Co., New York (1995).
STODDART, J. 1.: Gibberellin receptors. In: CHADWICK, C. M. and
D. R. GARROD (eds.): lneercellular and ineracellular communica-
tion, 1. Hormones, receptors and cellular ineeractions in plants,
pp. 91-114. Cambridge Universiry Press, New York (1986).
TREWAVAS, A. J.: Growth substance sensitiviry: the limiting factor in
plant development. PhysioJ. Plane. 55, 60-72 (1982).
- Sensitiviry and sensory adaptation in growth substance responses.
In: HOAD, G. v., M. B. JACKSON, J. R. LENTON, and R. K. ATKIN
(eds.): Hormone Action in Plane Developmene, pp. 19-38. But-
terworths, London (1987).
WEYERS, J. D. B., N. W. PATERSON, and R. A'BROOK: Towards a
quaneitative definition of plane hormone sensitiviry. Plane Cell
Environ. 10, 1-10 (1987).
WILLIAMS, P. M., J. W. BRADBEER, P. GASKIN, and J. MAcMILLAN:
Studies in seed dormancy. VIII. The identification and determi-
nation of gibberellins AI and A
9
in seeds of Cory/us ave/lana 1.
Planta 117, 101-108 (1974).
2M, J. H.: Biostatistical Analysis, 2nd Ed. Prentice-Hall, Inc., Eng-
lewood Cliffs USA, 1984.