Effects of High Temperatures on Dry Seeds 1, 2
Nechama Ben-Zeev & Stephen Zamenhof
Department of Biochemistry, Columbia University, New York, N.Y.
It has recently been reporte(l that when bacterial
cells and spores are heated to high temperatures, a
high frequency of mutations is induced (19, 20).
The treatment is believed to affect the deoxyribonucleic aci(d (DNA) in that a loss of purines, and
concomitantly, a loss of genetic information occurs
(5). DNA is rather resistant to heat (18); thus,
special methods must be sought if one intends to inflict high frequency of genetic (lanlage to DNA without excessive lethality to the cell cause(d by high temperatures. It was found that survival at high temperatures and higher mutation frequencies can be obtained by subjecting vegetative cells or spores of bacteria to heat in the dried state under zvacumm (20).
This is partially due to the fact that without water
the proteins are less susceptible to hy(drolysis at
higher temperatures.
The purpose of this work is to extend these studies
to higher plants. Pollen and seeds can be conveniently subjected to drying; only the latter were used in
this study.
The effect of heat on seeds and pollen has been
the subject of several publications in the past (3. 4,
9-17). It was reportedl that the survival increases
with decreasing moisture content of the heated seeds
and that the heating producedl (lelay in germination
and other phenotypic as well as genotypic effects.
In the present work it was foun(d that. as in the
case of bacteria, on heating dry see(ds under vacuum
much higher tenlperatures can l)e usedl without letllal
effects than in the case of heating at atmosplleric
pressure. These higlh temperatures pro(lucedl pronounced phenotypic effects, both in the plhysiology
and morphology of the pl)ants. TIn addition, chromosome injuries were observed, so1lle of whichi imay
represent genetic (laillage; this u, ork -will be presente(d
at a later date.
Materials & Methods
Seeds. The dorniant seeds (except Hordeunt vdlgare, L.. var. Himalaya). uise(l in this work, were oh-
1 Received revised manuscript April 26, 1962.
2This investigation was supported by research grants
No. C-1760 from the National Institutes of Health of
the United States Public Health Service, No. G-4337
from the National Science Foundation, and No. E-52
from the American Cancer Society, and by a Senior Research Fellowship SF-241-R from the Public Health
Service. An abstract of this work has appeared (1).
8 The plants were grown in the greenhouse of the
Rockefeller Institute, New York. The authors are indebted t-o Dr. Armin C. Braun of the Rockefeller Institute
for extending these facilities to them.
3
tained from commercial sources. The species were
chosen for the highest survival at elevated temperatures and for smallness of seed size (Raphanuts satizuis,
L., Brassica napus, L., Brassica hirta, Moench., NTicotiana ruistica, L., Aniiaranthuis tricolor, L., LvcoPersiconI esculen turn1, Mill., & Nigella darniascenia L., var.
flor. plen. MIiss Jekyll.). Of these. B. niaopus alnd R.
satizuis Nere chosen for morplhological observationis;
N. da1tlascenIa lends itself for cytological stu(lies. InI
a(ldition, H. zuilgare, (lespite larger seedls, was studiecl
for comparison with renorted effects of ionizing radiation. The seedls of H. vilgare were obtained from
Dr. Seymnour Shapiro. Brookhaveni Natioinal Laboratory.
Chemlicals. Gibberellic acid and kinetin were obtained froml Dr. T. Stonier, the Rockefeller Tnstitute.
Thiourea was a commercial prodluct. (C.P.. Fisher
Scientific Co., New York).
Drying. The seedis were subjectedl to dirying for
12 to 72 hours at 35 C in a vacuum oven andl then
placed in 3 cim (liameter glass bulbs (as a layer one
see(l deep) which were evacuate(d to 10-1 to 10-4 mmII,
Hg (oil (liffusion pump) for 3 to 48 hours. TIn somlle
experiments (see figs) the bulbs, which were still
being evacuated, were immerse(d in a water bath at
55 to 60 C for 3 hours (pre-heatilig). to ilprove
(Irving.
0 Storage. In somle experiments, the glass, bulbs
were seale(d under vacuulim after the seedIs lha(d beeni
(irie(i as ill(licate(l above; the see(ls were thieni store(l
at roomil temiiperature for 4 to 70 dlays before lbeatinig
to highi temiperatures as explaine(d below. Note that
in the case of bacterial cells this storage before hleating greativ imiproved the survival (20), undoubte(dly
because of additional w\vater remiioval. TlI other experiments the seedIs Nere stored for 14 (lays (unider
vacuumi or un(ler atmospheric lpressure) after hieating to highi temperatures, before germinatioll.
Heating to High Temperatures. The bulbs \with
seeds at atmosphier-ic pressure (contr-ol) or) uind(1er
vacuum and(l continint11g evacuation as (escrib)e(l abov-e
(experimental) w\ith or without pire-lheatillg, w\ere
heated to 100 to 138 C for 16 miillnutes, by totally im11milersing inlto an oil bath pre-heate(l to these temilperatures. The actual temiperature reachie(d by the see(ds
while heatinig was measured 1y a thermocouple inselrted into the glass bulb so as to touclh the see(ds.
and conlnecte(d to ain electroniic temli)erature in(licator
outside of the vacuuml system (21). This miieasurl-emlent is considlere(l essential as the data in the ollelliterature often refer to the temperature of the oven
and do not permit estimation of the actual temiiper-ature of the seeds.
-
-
-
696
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
697
BEN-ZEEV & ZAMENHOF-HIGH TEMPERATURE EFFECTS ON SEEDS
Additional measurements have shown that in the
system used in the present experiments the surface
of the seeds (thermocouple) reaches the temperature
of the bath within 1 minute after immersion of the
bulb. The smallness of the seeds assures that the
embryo in the interior of the seed reaches this temperature shortly aftervard.
- Deternmination of Moisture Content.
The moisture content of heated and non-heated seeds was determined in small aliquots of seeds, as the difference
between the weight of seeds before and after drying
in vacuum for 46 hours at 100 C in a vacuum oven.
Germination. The main aliquots of heated and
non-heated seeds were allowed to germinate on a
wxet filter paper in covered petri dishes at 23 C or 30 C,
either in the dark or under light (two 15 xv fluorescent tubes and one 40 watt incandescent bulb, 25
cm fronm the seeds, 15 hr per 24 hr). In some experiments the filter paper was soaked in 2.5 X
10-3 Ai aqueous solution of gibberellic acid or 10-s M
aqueous solution of kinetin or 3 X 10-2 M solution
of thiourea, as described by Haber et al. (8).
For each species and each heating condition 50
to 180 seeds were used. The germination was scored
as successful if the emergence of rootlets and cotyledons alone occurred. One week after germination
had started the seedlings were transferred to pots
in the green house whenever further study was de-
sirable.
Results & Discussion
The loss of moisture (typical examples) of dormant seeds heated in vacuum as described below is
represented in table I.
The results concerning the survival and the delay
in germination are represented in figures 1 to 5
'40
Table I
Moisture Content of Dormant Seeds Before
(Control) & After Heating in Vacuum*
After heating
Before heating
moisture, % Final heating, Moisture %
R. sativus
B. napus
5.3
4.8
137
133
1.4
1.8
1.75
N. aramascena
4.6
120
H. vulgare
7.2
127
5.2
* Pre-heating 3 hours, 60 C; final heating 16 minutes
at temperatures indicated.
which also indicate the particular treatments in each
experiment.
As can be seen from figures 1 and 2, the heating
in the dry state in vacuum yields surviving seeds at
temperatures 20 to 35 C higher than without vacuum.
Thus, seeds heated under vacuum survive temperatures as high as 138 C, and such drastic treatment
allows one to produce abnormalities not obtainable
at lower temperatures.
Dormant seeds of other species tested that survive 120 C (16 min in vacuum) were N. rustica (3 %
survival), A. tricolor (33 %), B. hirta (10-32 %),
and L. escutlentunti (11 %). Note that high survival
temperatures reported in the older literature actually
may refer to the ambient temperature and not the
seed itself.
Pre-heating to 55 to 60 C and resulting additional
drying markedly improves the survival (figs 1 & 2).
It is of interest to note that the heat treatment prior
to X-ray treatment also reduced final injury (2).
Storage for 24 to 48 hours in a desiccator (vacuum
l00 105 110 *C 115 120 125 130 35 40
Fig. 1. Germination of dormant seeds of R. sativus (radish) in 7 days after heating in vacuum for 16 minutes
at temperatures indicated on the abscissa. Curve 1, heating at atmospheric pressure; 2, the same but preceded by
pre-heating at 55 to 60 C for 3 hours; 3, heating in vacuum (10-3-10-4 -mm Hg); 4, the same but preceded by
pre-heating as in curve 2. Control seeds, 100 % germination. See text for details.
Fig. 2. Germination of dormant seeds of B. napus (rape) in 8 days after heating. The conditions and the symbols
as on figure 1. Control seeds, 98 % germination.
85
90
95
tO
105
110 115
*C
120
125
130
135
85
90
95
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
698
PLANT PHYSIOLOGY
8 miiiii Hg) p11ior to ligh vacuunii resulte(d in a 15 to
18 (( imlprovement in survival. However, the same
effect cani be obtainedl by replacing the desiccator
treatmiietnt by- a longer (up to 48 hr) evacuation (high
vacuuIll ). On the other hand, prolonged storage
unIder vacuum (sealedl bulbs') for 2 to 3 weeks before
heatilng increased the delay in germination and decreasedl the survival (A. d(umiiascc;ia). Storage under
vacuum (sealed bulbs after fincal heatilng, for 20
d(las. had the ol)posite effect.
The increase(l resistance to heat upoIn desiccation
uni1der vacuum is probal)ly (lue to the protection of
cell constituent against heat denaturation (hydrolysis)
bv the removal of water (20) and, possibly, also of
oxYgenl ( 17).
A\miiong tle plhenotylpe chainges in the surviving
see(ls l)ro(luce(l by heat the most conspicuous one is
the delav in germination (see also 3. 9). In the
preselit workl delay anl inllibitioll of germlination of
up to 4 mointlhs was stu(die(d (N. dainascena, fig 3).
rhe addition of gibberellic aci(d (8) pronmptly relieved
the delav alld inhiibitioni (figs 3 & 4) anid resulted in
a mltuclh higlher final survival. Kinetin (but not
thiourea) (8) also has an effect but to a smaller degree. These studIies on a convenient material (N.
d(a1ilasccila) may eventually lea(l to elucidation of the
possil)le sites of injury. For instance, gibberellic acid
may stimulate the pro(luction of a hormlonie necessary
to overcome the injury or serve as a hormone itself.
However, niote that the treatment with these substances did lnot give higlher surxival temlperatures.
It is of interest that all seeds still capable of
germination. ith or x\ithout addition of gibberellic
acid or kinetin, completely inhibited the growth of
mol,( even if germlination xvas (lelayed up to 4 months.
hI ad(lition to delay in germlination, the flowering
of B. no puts xvas also completely inhibited for at least
90rF
2 months. This inhibition could also be relieved by
the addition of gibberellic acid.
Figure 5 represents the effect of heatinig on survival in H. ulWgare. Considerable survival can be
obtained even at 127 C. The physiological anid
miiorphological changes induced by heat and not observed in any of the non-heated controls, included:
A. Germination process: Emiiergence of thle
cotyledlon and epicotyl before the hypocotyl (B. llapits,
R. sativtus). Elongatioin of the hypocotvl before thle
groxvth of the root [B. niapufs in 100 (4 of the seeds;
R. sativuts in 80 %( of the seeds; H. qUlg(irc in 5 e 1.
The enmergence of the coleoptile before tlle grox-th
of the root (H. vidgarc). Doubling of the stem
(H. zdgaorc). Shorteninig of the stemii (1f. vidy(irc).
It is to be note(d that tlle latter resuilts are analogous
to the effects of ra(liatioin (7).
B. Leaves of see(llings: Clhange inl color alnd
the appearance of (lark spots (R. satizvus) ; thickenillg
a.nd elongation of the cotyledon, fusioni of txvo leaves,
leaves xvith dissections (nlotches), abnormal contouirs,
snmooth edges, cup-like slhape, or larger cotyledons
(B.
niapuis).
C. Root: The groxvth of the secon(lary roots
insteadl of the p)rimary root (R. satiZvts, B. llaptus).
D. Stenm: Fasciniatioln (B. 1apulS ), formation
of additional vegetative buds, and thickeninig of tlle
stem (R. satizvts).
E. Inflorescence: No flowxers in 2 miionitlhs xithout gibberellic acid; slhorteningF, of floxering stalk
aInd re(luction in nunmber anl size (R. sativits), xxhen
gibberellic aci(d wX-as usedl.
Some of the possible biochemical illju-ries by heat
have been reported (17).
As miientione(l before, somlle of the morphological
0/0
80
C
70
60
50
40
G
30
20
10
2
4
6
8
10
12
WEEKS
i4
16
18
20
22
Fig. 3. Delay and inhibition of germination of dormant seeds of N. dainiascena after heating in conditions
as on curve 4 (fig 1), and the effects of 2.5 X 10-3 M
gibberellic acid (G) or 10-5 A kinetin (K), added at a
time indicated by an arrow (for curve G, 121 C, added
immllediately after heating). C, control (not heated).
The temperatures indicated refer to heatinig in vacuum.
See text for details.
0
KG
120
121
K
122
Fig. 4. The increase in final percentage of germination of heated dormant seeds of N. damiiascena upon treatment with gibberellic acid (G) and kinetin (K), over the
final percentage of germination of seeds heated but nlot
treated with these substances. The values at 118 C represent mean of three experiments. Other concditionis as on
figure 3.
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
699
BEN-ZEEV & ZAMENHOF-HIGH TEMPERATURE EFFECTS ON SEEDS
Literature Cited
1. BEN-ZEEV, NTECHAMA & S. ZAMENHOF. 1961. Ef-
0/1
70
60
60
2.
2.
-;vV v
50
3.
0*s
2
40
%
0
4.
30
a
5.
/
20
6.
10i
7.
120
121
'
122
'
124
123
'
125
'
126
127
C
8.
Fig. 5. Germination of dormant seeds of H. vulgare
(barley) in 10 days at 23 C after pre-heating (60 C, 3
hr) and final heating in vacuum for 16 minutes at temperatures indicated on the abscissa. Curve 1, germination; curve 2, heights of shoots (in % of control). Control germination 100 %; control average height 14 mm.
9.
fects of high temperatures on dry seeds. Records
Genetics Soc. Am. 30: 61.
CALDECOTT, R. S. & L. SMITH. 1952. The influence
of heat treatments on the injury & cytogenetic
effects of X-rays on barley. Genetics 37: 136-157.
CARTLEDGE, J. L., L. V. BARTON, & A. F. BLAKESLEE.
1936. Heat & moisture as factors in the increased
mutation rate from datura seeds. Proc. Am.
Philos. Soc. 76: 663-685.
DAVIs, B. M. 1947. The appearance of a balanced
lethal situation in the c nothera heterozygote
Gaudens. h franciscana following severe heat treatment of seeds. Genetics 32 185-199.
GREER, S. & S. ZAMENHOF. 962. Studies on deJ. Molec. Biol. 4:
purination of DNA by he,
123-141.
GREGORY, W. C. 1941. Phylogenetic & cytological
studies in the Ranunculac'-ae. Tras. Am. Phil.
Soc. NS 31: 443-521.
GUNCKEL, J. E. & A. H. SPARROW. 1954. Aberrant
growth in plants induced by ionizing radiation.
Brookhaven Symp. in Biol. 6: 252-279.
HABER, A. H. & HELEN J. LUIPPOLD. 1959. Dormancy resulting from gamma-irradiation of lettuce
seed. Intern. J. Radiation Biol. 1: 317-327.
HIORTH, G. 1930. Ein Versuch uber den Einfluss
der Erwarmung des Pollens auf die Nachkommen-
shaft. Z. Induktive Abstammungsu. Verebungslehre 56: 39-50.
10. HUTCHINSON, J. B. 1944. The drying of wheat.
of temperature
germination
*
* temperatutesII.Te
~~~~~~III.The effect
raue onngriain
fecoftm
to high
changes observed after heating
104-107.
Ind.
Trans.
63:
Soc.
Chem.
J.
capacity.
by
changes
resemble
ay
.
. .
.
11. JOZEFOWICZ, MARY. 1930. The effects of certain
oind
hion tn .ration
(7); however, the mechanism of injury in these
two cases is probably entirely different. The action
of radiation may involve the production of free radicals; the action of heat may include coagulation of
proteins, breaking of hydrogen bonds, and depurination of nucleic acids (5).
Summary
treatments on the germination of tomato seeds.
Ann. Appl. Biol. 12: 504-513.
12. JOZEFOWICZ, MARY. 1930. Some observations on
tomato plants from seed submitted to high temperatures. Ann. Appl. Biol. 12: 514-521.
13. NAVASHIN, M. & P. SHKVARNIKOV. 1933. Process
of mutation in resting seeds accelerated by in14.
482.
creased temperature. Nature 132:
PETO, F. H. 1933. The effect of aging & heat on
the chromosomal mutation rates in maize & barley.
Previous reports demonstrated that heating of dry
Can. J. Res. Sec. C. 9: 261-264.
bacterial cells and spores to high temperatures in
15. PETO, F. H. 1937. Genetical studies on mutants
vacuum induced a high frequency of mutations. The
in the progeny of heat-treated barley. Can. J.
treatment was designed to inflict genetic damage to
Res., Sec. C. 15: 217-229.
DNA without excessive lethality to the cell (proand
of Triticum
F. H. 1938.
16. PETO,
Hybridization
number
of chromosome
V. Doubling
Agropyron.
tection of protein by drying in vacuum). To study
in T. vulgare & F1 of T. vulgare X A. glaucunt
these effects in higher plants, dormant seeds of R.
satits, B. naputs, N. damascena, and H. zulgare were
by temperature treatments. Can. J. Res., Sec. C.
dried and heated (16 min) in vacuum. Survival was
16: 516-519.
17. SIEGEL, S. M. 1953. Effects of exposures of seeds
obtained at 122 C to 138 C which was up to 35 C
to various physical agents. II. Physiological &
higher than without vacuum. Such treatment prochemical aspects of heat injury in the red kidney
duced delay and inhibition of germination for more
bean embryo. Botan. Gaz. 114: 297-312.
than 4 months (N. damascena); both effects were
18. ZAMENHOF, S., HATTIE E. ALEXANDER, & GRACE
relieved by gibbellic acid and, to a lesser degree,
LEIDY. 1953. Studies on the chemistry of the
o
rphonkenun
btoe
Pronounced
by
kineti
I. Resistance to physical
butnotythiure.
transforming
activity.J. Exptl.
in the resulting plants were also prological changes
& chemical agents.
Med. 98: 373-397.
duced. Some of these changes may resemble the
19. ZAMENHOF, S. & S. GREER. 1958. Heat as an agent
effects of ionizing radiation.
producing high frequency -of mutations & unstable
-genes in Escherichia coli. Nature 182: 611-613.
20. ZAMENHOF, S. 1960. Effects of heating dry bacAcknowledgments
teria & spores on their phenotype & genotype
The authors are indebted to Dr. J. Herbert Taylor,
Proc. Natl. Acad. Sci. 46:101-105.
RockeThe
C.
Columbia University, Dr. Armin
Braun,
46: 101-105.
Z P by
Natl.
Downloaded from on June 14, 2017 -21Published
www.plantphysiol.org
1961.
New York
5. rights
1961.reserved.
Gene unstabilization induced
feller Institute, and Dr. Richard
Klein,American
CopyrightM.
© 1962
Society of Plant AMENHOF,
Biologists. All
by heat & by nitrous acid. J. Bacteriol. 81: 111Botanical Garden, for advice and help in the course of
117.
experimentation.