Plant Physiol. (1981) 68, 509-511
0032-0889/8 1/68/0509/03/$00.50/0
Short Communication
Reevaluation of the Cyanide Resistance of Seed Germination1
Received for publication May 20, 1980 and in revised form February 2, 1981
KENNETH S. YU', CARY A. MITCHELL', AND HENRY A. ROBITAILLE
Department of Horticulture, Purdue University, West Lafayette, Indiana 47907
ABSTRACT
Although high levels of KCN (53 micromoles per gram fresh weight of
seed, corresponding to 3.2 millimolar) failed to block germination of lettuce
seeds incubated in covered Petri dishes, the same levels totally blocked
germination in sealed dishes. Inhibition was reversed by removing the seal.
Placement of KCN remote from seeds also blocked germination in closed
systems. Cyanide effectiveness was enhanced by acidifying the KCN
solution but negated by the presence of a trap containing strong alkali.
Low levels of aqueous HCN (2.6 micromoles HCN per gram, corresponding
to 0.16 millimolar) injected into sealed dishes gave maximal inhibition of
germination, suggesting that the effectiveness of KCN was due to formation
of HCN in KCN solutions. Studies with nine additional crop species
generally supported the interpretation that cyanide inhibition of germination has been underestimated in the past due to escape of volatile HCN
from open systems.
The metabolic inhibitor cyanide has been reported to be ineffective in blocking seed germination (1, 3, 7). In fact, Roberts (3)
even found CN- salts to break dormancy in several species of
cereal grain, and Mayer et al. (2) found CN- to stimulate, mildly,
the dark germination of Grand Rapids lettuce seeds. Taylorson
and Hendricks (6) concluded that the promotive action of CNon lettuce-seed germination was nutritional, resulting from incorporation of cyanide into amino acids and protein. Yentur and
Leopold (8) showed that seed respiration of various species was
CN--resistant at early stages of germination but became CN-sensitive at later stages. Lettuce seed germination in the presence
of CN- has been found to proceed normally, even if seeds were
imbibed directly in solutions containing 10 mm KCN (9).
A common technique utilized in many previous germination
studies was to incubate seeds in covered Petri dishes. However, it
has been observed during experiments in our laboratory that, if
covered Petri dishes containing lettuce seeds imbibed in KCN
were sealed with thermoplastic film to prevent desiccation during
long incubations, germination was blocked even at very low levels
of KCN. Since preliminary observations suggested that inhibited
germination was not the result of sealing per se (ie. in the absence
of KCN), the apparent cyanide effect was investigated further.
The present communication presents evidence that small amounts
HCN formed in solutions of CN- salts inhibit germination in
' This research was supported in part by State and Hatch funds allocated
to the Purdue Agricultural Experiment Station. Journal Paper 7956 of the
Purdue Agricultural Experiment Station.
2 Present address: Austin Rogers Mesa Research Center, Austin, CO
81410.
'To whom correspondence should be addressed.
closed systems but are lost as a volatile emanation from open
systems.
MATERIALS AND METHODS
Seed Incubation Procedures. Lots of 50 lettuce (Lactuca sativa
L. cv. Waldmann's Green) seeds were imbibed in 6-cm-diameter
plastic Petri dishes on a double layer of Whatmann No. I filter
paper wetted either with 1.2 ml deionized-distilled H20 or with
aqueous KCN solution. All manipulations were performed in a
darkroom at 26 C, and all treatments were replicated three times.
Germination was potentiated by red-light treatment, as described
previously (9), and was scored as radicle emergence 24 h after
treatment.
Closed versus Open Systems. Strips of Parafilm were wrapped
tightly around the edges of covered Petri dishes containing seeds
and 1.2 ml test solution to achieve a closed system. KCN was
varied over a range of concentrations, up to 3.2 mm, in both closed
and open systems.
Alkali Traps in Closed Systems. A 1.2-cm-diameter plastic vial
cap was inverted in the center of each dish as a centerwell, and 0.1
ml of either H20 or 3.6 N KOH was added before sealing with
Parafilm. Seeds were imbibed directly in 1.2 ml test solution
consisting of either H20 or various concentrations of KCN. The
alkali trap served to scavenge volatile acidic substances that
otherwise would tend to accumulate within a sealed container.
Generation of HCN within Closed Systems. Seeds were imbibed
in 1.2 ml H20 instead of in KCN, but 0. l-ml aliquots of KCN at
various concentrations were added to a centerwell. Dishes were
immediately sealed with Parafilm, and 0.1 ml I N H2SO4 was
injected into the centerwell through a tape-sealed pinhole in the
cover of the dish. The hole was immediately resealed with tape.
Exogenous HCN. HCN gas was generated separately by injecting 0.6 ml 10 N H2S04 into a sealed 50-ml Erlenmeyer flask
containing 0.3 ml 1 N KCN. The generation flask was maintained
at 50 C with stirring. HCN gas distilled through a sidearm
connection to a collection flask containing 30 ml H20 held at ice
temperature. After 24 h of incubation, aliquots of the collecting
solution were withdrawn by syringe through a serum stopper, and
cyanide was determined colorimetrically, according to the method
of Robbie and Leinfelder (5). Once determined, precise volumes
of aqueous HCN were removed and injected into sealed Petri
dishes, either directly or after further dilution. This procedure
permitted control of the dosage of HCN delivered to, and potentially available for, interaction with the enclosed seeds.
Cyanide Reversibility. Seeds were incubated as described, either
in H20 or in 3 mm KCN, within sealed Petri dishes. Seals were
removed after 3 days, and seeds were maintained in test solutions
for an additional 4 days. Germination was evaluated daily under
green safelight without disturbing incubation conditions.
Species Survey of Cyanide Sensitivity. Seeds of nine additional
species were incubated in sealed or unsealed Petri dishes in the
absence or presence of KCN at two different levels. Seeds were
509
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YU ET AL.
510
Plant Physiol. Vol. 68, 1981
imbibed on filter papers moistened to glistening with test solutions
and incubated in darkness at 25 C until germination was near
maximum for the unsealed H20 controls of each species (from 24
h for lettuce to 96 h for pea). Each Petri dish contained from 10
(pea) to 50 (lettuce) seeds, and each treatment was replicated three
times. Results generally were reported as mean germination percentage ± SD of the mean.
Germination was evaluated as a function of dosage (i.e. concentration volume) of cyanide rather than of concentration alone,
because the volumes of KCN between experiments varied from
1.2 ml (in the case of direct imbibition) to 0.1 ml (in the case of
centerwell placement). Comparisons among experiments based
solely upon concentration would have little meaning, especially
since HCN was generated with high efficiency from a relatively
small volume of KCN in some cases (e.g. acidified centerwell) but
with lower efficiency from a relatively larger volume of KCN in
others (direct imbibition). Cyanide dosage was expressed in terms
of the initial dry weight of 50 seeds added to each dish.
x
RESULTS AND DISCUSSION
Seeds of Waldmann's Green lettuce incubated in covered Petri
dishes germinated readily in a range of concentrations of cyanide
up to 52.8 ,umolKCN g-1 fresh weight of seed, corresponding to
1.2 ml of 3.2 mm KCN per 6-cm-diameter Petri dish (Fig. IA).
We have previously reported near maximal germination by this
cultivar in 10 mm KCN (9). However, if covered Petri dishes were
sealed with thermoplastic film, seeds germinated poorly at KCN
dosages above 5.6t,mol g-' (0.32 mM), and they failed to germinate
at all above 16.7 ,umol g-1 (1 mM). These results suggest that a
volatile substance escaping from loosely covered Petri dishes was
responsible for blocking germination in the sealed system, especially since results with controls (zero CN) indicated that sealing
per se was not responsible for inhibited germination (Fig. IA).
Since HCN emanating from aqueous solutions of KCN appeared to be the most likely inhibitor of germination in closed
systems, strong alkali was placed in centerwells to scavenge HCN
(4) and, presumably, to prevent the inhibitory action of cyanide
in sealed Petri dishes. Lettuce seeds imbibed directly in aqueous
KCN germinated fully at 16.7,umol KCN g-' ( mM) if alkali
traps were present in sealed dishes, but no germination occurred
in their absence (Fig. IB). In fact, the germination profile from
sealed dishes containing alkali traps was identical with that from
unsealed dishes (cf. Figs. IA and IB).
To separate effects of alkali in scavenging volatile HCN from
possible confounding effects of scavenging C02, the KOH trap
was omitted in subsequent experiments with sealed systems. Instead, a small volume of KCN was placed in the centerwell, and
seeds were imbibed only in H20. Remote placement of KCN
proved to be just as effective as direct imbibition in KCN as long
as the system remained closed. However, since the pH of aqueous
KCN also is quite basic, a KCN solution would not only trap
some CO2 but would not liberate HCN as rapidly as would a more
acidic solution. Therefore, an excess of nonvolatile acid was added
to the KCN-containing centerwell to (a) eliminate C02-scavenging
by alkali as a confounding factor in the experiments and (b) drive
the volatilization of HCN closer to completion. Utilizing this
sytem in sealed dishes, small dosages of acidified KCN inhibited
germination more than did much larger dosages of nonacidified
KCN in direct contact with seeds (cf. Fig. IC with Figs. IA and
1B).
That the effectiveness of acidified KCN was actually due to
HCN was verified by experiments in which HCN was generated
separately and known amounts were injected into sealed Petri
dishes containing dry weeds. Increasing concentrations of aqueous
HCN gave a germination profile similar to that of acidified KCN,
although the HCN solution in direct contact with seeds was most
effective (cf. Figs. IC and ID). The threshold dosage of HCN for
D
40-
20L
L
-a 161
6 a
-4-
8
12
CN
Dosage
(p
moI./g
52
fw)
FIG. 1. Effect of various cyanide dosages on germination of Wald-
mann's Green lettuce seeds. A, Seeds were imbibed directly in aqueous
KCN test solutions. One set of covered Petri dishes was sealed (0), while
the other set remained unsealed (0). B, Seeds were imbibed directly in
KCN solutions within sealed Petri dishes. One set of dishes had centerwells
containing 0.1 ml H20(-), while the other set had centerwells containing
0.1 ml 3.6 N KOH (A). C, Seeds were imbibed in 1.2 ml H20, and 0.1-ml
aliquots of various concentrations of KCN plus I N H2SO4 were sequentially injected into the centerwells. D, Seeds were imbibed directly in
aqueous HCN solutions (generated and determined externally) and appropriately diluted, and 1.2-ml aliquots were injected into sealed dishes
containing dry seeds.
Table I. Reversibility of Inhibition of Lettuce-Seed Germination by
Cyanide
Seeds were imbibed within sealed Petri dishes either in H20 or in KCN,
illuminated with red light, and incubated in darkness at 26 C. Seals were
removed after 3 days, but seeds were maintained in the test solutions for
an additional 4 days.
Germination
Incubation Time
KCN (3 mM)
H20
days
0
2
3
4
5
6
7
%
0
0
98
98
98
98
98
98
0
0
0
0
96
98
99
4
inhibiting lettuce-seed germination proved to be 1.1,umol g-'
(0.066 mM). Inhibition was maximal at 2.6
,umol g-' (0.16 mM
HCN) (Fig.ID). A 20-fold higher dosage of KCN (52.8
,umol g-',
corresponding to 3.2 mM) failed to inhibit germination in covered
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Plant Physiol. Vol. 68, 1981 INHIBITION OF SEED GERMINATION BY HYDROCYANIC ACID
511
Table II. Effect of KCN at Two Levels on Seed Germination Percentage of 10 D!fferent Crop Species in Unsealed or Sealed Petri Dishes
Differences among treatments within a concentration for a given species were determined by analysis of variance and HSD at the 1% or 5% level of
significance.
Seed Germination
KCN (0 mM)
Unsealed
Sealed
KCN (I mM)
Unsealed
Sealed
Cucumber
Tomato
Oat
Wheat
Pea
Radish
Lettucea
Mungben
85
96
82
92
74
100
100
80
98
96
99
96
90
100
90
92
100
100
100
98
40
68
72
90
92
93
95
96
100
100
100
Soybean
Barley
Brly
Syean
ob
12b
70b
16b
oh
40b
16b
I0b
I,
KCN (10 mM)
7
2
34
Unsealed
oh
IC
0
Sealed
a Unlike other species, lettuce seeds received a red-light treatment.
b
Level of significance, 1%.
c Level of significance, 5%.
but unsealed dishes, suggesting that lettuce seeds are much more
sensitive to cyanide presented in the undissociated form than they
are to that in the dissociated form (cf. Figs. IA and ID). Although
aqueous solutions of KCN were basic (whereas those of HCN
were acidic), it is unlikely that the germination responses in this
study were due to pH alone; preliminary experiments with various
buffers indicated no significant effects of pH on germination over
a wide range of pH values, including those of the HCN and KCN
solutions used.
That inhibition of germination by cyanide is a reversible physiological effect rather than an irreversible phytotoxicity was demonstrated by incubating lettuce seeds for 3 days in sealed dishes
containing 3 mm KCN, during which time no germination occurred (Table I). However, full germination occurred 3 days after
removing the seal from covered dishes, indicating not only that
the effect of HCN was nontoxic but that it was reversible.
Seeds of lettuce and nine additional crop species were incubated
in sealed or unsealed Petri dishes in the presence or absence of
KCN. Germination generally exhibited partial sensitivity to 1 mm
KCN in unsealed dishes (Table II). In sealed dishes, however,
germination response to 1 mm KCN ranged from complete suppression (in the case of tomato and barley) to full germination (in
the case of cucumber). At 10 mm KCN, differential sensitivity
remained in unsealed dishes, but germination of all species was
totally blocked in sealed dishes. Partial sensitivity to KCN in open
systems was probably due to uptake by seeds of some of the HCN
formed; this HCN otherwise would have escaped from the system.
The results of this study indicate that the apparent cyanide
resistance ofseed germination is largely an artifact ofexperimental
protocol, in that most previous studies have employed open systems from which volatile HCN formed from CN- salts can diffuse
out of the system. This finding does not mean that operation of
cyanide-resistant processes, such as the alternative electron transport pathway, are not important in the germination process (8, 9).
It merely means that HCN-sensitive processes also are required.
It is possible that the ability of HCN to block germination is
70
23
ob
ob
87
ob
80
ob
48
oh
40
oh
80
o
related to the greater permeability of cell membranes to uncharged
HCN molecules rather than to highly charged CN- anions. It also
is more likely that HCN entered seed tissue from aqueous solution
than from the gas phase. For instance, whenever HCN gas was
injected into, or generated within, a sealed system, subsequent
determination of cyanide in samples of the headspace atmosphere
indicated little or no gaseous HCN; analysis of the aqueous
imbibing solution always revealed an accumulation of dissolved
HCN. Nevertheless, a small proportion of HCN undoubtedly
volatilizes out of aqueous solution, and mass action drives its
continuous escape from open systems.
The findings of this investigation indicate that metabolic requirements for germination involve cyanide-sensitive as well as
cyanide-resistant processes. Identification of the key germinationlimiting steps will require further investigation at the level of cell
physiology and metabolism.
Acknowledgment-The authors wish to thank Cathy Nazimek for excellent technical assistance.
LITERATURE CITED
1. MAYER A, A POLJAKOFF-MAYBER 1962 Cytochrome oxidase in germinating
lettuce seeds. Plant Cell Physiol 3: 309
2. MAYER A, A PO01AKOFF-MAYBER, W APPLEMAN 1957 Studies on the oxidative
systems in germinating lettuce seeds. Physiol Plant 10: 1-13
3. ROBERTs E 1969 Seed dormancy and oxidation processes. Symp Soc Exp Biol 23:
161-192
4. ROBBIE W 1946 The quantitative control of cyanide in manometric experimentation. J Cell Comp Physiol 27: 181-209
5. ROBBIE W, P LEINFELDER 1945 Calcium cyanide solutions as constant sources of
HCN gas for animal experiments. J Ind Hyg Toxicol 27: 269
6. TAYLORSON R, S HENDRICKS 1973 Promotion of seed germination by cyanide.
Plant Physiol 52: 23-27
7. WILSON S, W BONNER 1971 Studies of electron transport in dry and imbibed
peanut embryos. Plant Physiol 48: 340-344
8. YENTUR S, AC LEOPOLD 1976 Respiratory transition during seed germination.
Plant Physiol 57: 274-276
9. Yu K, C MITCHELL, S. YENTUR, H ROBITAILLE 1979 Cyanide-insensitive, salicylhydroxamic acid-sensitive processes in potentiation of light-requiring lettuce
seeds. Plant Physiol 63: 121-125
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