Plant Science Letters, 20 (1981) 263--271 263

© Elsevier/North-Holland Scientific Publishers Ltd.

GERMINATION OF THE PHOTOBLASTIC SEEDS OF O C I M U M

T E N U I F L O R UM L.

DILIP AMRITPHALE and L.P. MALL

School of Studies in Botany, Vikram University, Ujjain, M.P. 456-010 (India)
(Received March 26th, 1980)
(Revision received August 12th, 1980)
(Accepted September 25th, 1980}

SUMMARY

Seeds of O c i m u m t e n u i f l o r u m L. are positively photoblastic. Photo-

sensitivity of seeds spreads t h r o u g h o u t the tested temperature range
(14---38°C). Treatment with suboptimal and relatively supraoptimal tem-
peratures before placing the seeds at optimal temperature led to an inter-
esting germination pattern. Gibberellic acid, although causing the seeds
to germinate in dark, failed to overcome the low temperature block to
germination both in light and dark. Prolonged darkness induced skoto-
d o r m a n c y in seeds. Low temperature and gibberellic acid alone or in con-
junction with red light failed to restore light sensitivity. Most interestingly
a prolonged continuous exposure to red light enabled the seeds to overcome
the skotodormancy. Significance of this adaptation in perpetuation of the
species in its natural environments is discussed.

INTRODUCTION

Exposure to light stimulates the germination of the seeds of many wild

species and of cultivated plants which have been subject to little selection.
In most species of light sensitive seeds there is sufficient evidence that light
sensitivity depends on the p h y t o c h r o m e system, the existence of which
was first detected physiologically in studies on light mediated seed germin-
ation.
Work done on light-sensitive seeds of tropical species is meagre com-
pared to the vast a m o u n t of work available on those o f sub-temperate
and temperate species. It is felt t h a t generalizations made, and inferences
drawn, from the studies of sub-temperate/temperate species may not re-
flect tropical species growing in a totally different climate. The present
work was u n d e r t a k e n in an a t t e m p t to understand the basic mechanism
264

underlying the p h o t o c o n t r o l of seed germination of Ocimum tenuiflorum L.

{Syn Ocimum sanctum L.) occurring at Port Blair and its environs {Andman
islands, India).

MATERIALS AND METHODS

Plant material. Seeds of Ocimum tenuiflorum L. were collected from

Port Blair and its environs {Andman islands 10°30'--13°41'N and 92°11 '-.-
93°7'E). The climate is tropical. Extremes of summer and winter are practic-
ally u n k n o w n , as also frost. Variations in temperature are slight (23--31°C).
These islands receive rainfall from both the monsoons. Average rainfall
at Port Blair is about 275 cm.
Seeds were collected in early part of February. They were brought to
laboratory at Ujjain {23°11'N and 75°43'E) in moisture free polythene
bags and then stored at r o o m temperature in dark. Experiments were started
with freshly collected seeds and were continued for about six months.
Their m a x i m u m germination in darkness at 26°C on water-saturated filter
paper was 0--2% in the beginning of the study and remained constant to
the end of the study.
Germination conditions. The standard experimental procedure is de-
scribed below. All departures from this procedure are noted in the descrip-
tions of individual experiments.
F i f t y seeds were sprinkled o n t o 5.5 cm disks of Whatmann No. 44 filter
paper which had been pressed into a 5 cm petridish and saturated with an
experimental solution or glass distilled water. The petri dishes were placed
into B.O.D. incubator at 26 + 0.5°C in light proof black polythene bags.
Germination was n o t e d on fourth day and the time thus allowed for germin-
ation was sufficient to permit m a x i m u m germination attainable under the
particular experimental conditions. All manipulations of seeds were carried
out in a dark room. Four replicates were employed per treatment. Criterion
of germination was the emergence of the radicle through the pericarp. All
the experiments were repeated at least twice and the average of the readings
are presented.
Light source. Red light was obtained by filtering the light from a bank of
cool white fluorescent tubes {4 tubes each of 1.2 m length giving a total
of 160 W) through 4 layers of red cellophane {for white and blue light 4
layers of transparent and blue cellophanes were used respectively). Far-red
light was obtained f r o m a bank of tungsten incandescent lamps (5 lamps
giving a total of 650 W) through 3 layers o f blue and 1 layer of red cello-
phanes. Transmission curves o f the filters were prepared and t h e y were
f o u n d quite satisfactory to produce red, blue and far-red light almost match-
ing the standard transmission spectra given by Smith [ 1].
Irradiation source to filter paper distance was kept 50 cm and in the case
of far-red irradiation, a 6-cm water filter was placed in between. Tern-
 265

perature during irradiations was n o t controlled since both the red and
far-red light reactions have temperature coefficients near unity [ 2].
Aqueous solutions of gibberellic acid {GA~ --- Phylaxia, Budapest, Hun-
gary} of different molarities were prepared by continuously agitating the
crystals, for several hours in warm glass distilled water. Solutions were used
on the day of preparation or were held at 5°C and used within 1 or 2 days
after preparation.
Data were analysed statistically for the S.E. and variance ratio, F. Arcsin
transformation of the percentage germination data was performed prior to
their treatment with analysis of variance.

RESULTS

Seeds o f Ocimum tenuiflorum were f o u n d to germinate in light just after

harvest and thus required no after-maturation period. Seeds exposed to
short-time irradiations showed the standard red/far-red reversal of germin-
ation thus indicating the operation of the low-energy p h y t o c h r o m e system
(Table I}.
Effect o f light and temperature interaction. Germination of Ocimum
seeds was tested at different temperatures ranging from 14°C to 38°C in the
light as well as in the dark {Table II). Seed germination was inhibited or
prevented by low temperature b u t higher temperature had relatively little
effect. Photosensitivity was, however, found t h r o u g h o u t the temperature
range.
In another set of experiments on the interaction of light and temperature
on germination, seeds of Ocimum were allowed to imbibe in the dark before
or after exposure to 10 min red light at 14°C and 38°C before transferring
them on fifth day to 26°C. While transferring to 26°C in dark, seeds which
had not been pretreated with red light were exposed to it for 10 min. A
peculiar response to 14°C and 38°C in darkness and light is evident from

TABLE I
C O N T R O L O F O C I M U M S E E D G E R M I N A T I O N BY R E D A N D F A R - R E D L I G H T
( I R R A D I A T I O N T I M E : 10 MIN; T E M P . 26 -+ 0.5°C)

Irradiation programme % germination

Dark 2 ~ 0.50
W h i t e light 55 ± 2.43
Red 50 + 4.87
Far-red 18 ± 2.55
Red/Far-red 12 ± 0.91
Red/Far-red/Red 48 ± 3.85
Red/Far-red/Red/Far-red 11 -~ 1 . 9 6
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TABLE II
CO NTR OL OF OCiMUM SEED G E R M I N A T I O N BY LI G H T- TEMPERA TU RE
INTERACTION
Analysis of Variance: F-value: 142.78 for temperature significant at 1% level. In working
out F-value, germination data under different temperatures in light only considered.

Light treatment % germination of seeds at indicated temperature (°C)

14 20 26 32 38

Dark 0 0 0 0 0
Light a 0 12 56 56 51

aRed light: 10 min.

Table III. Seeds allowed to imbibe in dark at 14°C w i t h o u t a pretreatment

with red light showed higher germination (55%) at 26°C than seeds ,~hat
were pretreated with red light (34%). A just opposite trend was obtained
for seeds that were given 38°C temperature treatment {].4% and 52% without
light pretreatment and with light pretreatment respectively).
Effect ofgibberellic acid treatment. Ocimum seeds have a hard seed coat
and also become mucilagenous on wetting. O p t i m u m concentration of
gibberellic acid for germination of Lepidium virginicum seeds having similar
characteristics was found to be 10 -3 M b y Toole and Cathey [3]. Evenari
et al. [4] obtained 10 -4 M concentration of gibberellic acid as o p t i m u m
for lettuce seed germination. A number of gibberellic acid concentrations

TABLE III
I N T E R A C T I O N OF LIGHT AND T E M P E R A T U R E (PRE- AND POSTTREATMENTS)
ON G E R M I N A T I O N OF OCIMUM SEEDS
Analysis of Variance: F-values: 9.14, 5.45, 50.78 for temperature, light and temperature
× light respectively. F-values for temperature and light significant at 5% level, whereas
for temperature x light significant at 1% level. Values in parentheses indicate percentage
germination attained at 14°C and 38°C on fifth day.

Ligbt treatment Percentage germination

Temperature Treatment a
14/26°C 38/26°C

Dark b 55 (0) 14 ( 1 )
Light c 34 (0) 52 (46)

aSeeds a l l o w e d t o i m b i b e in dark b e f o r e or after e x p o s u r e to 10 rain red light at 14°C

and 38°C for 4 days and t h e n transferred to 26°C.
bRed light for 10 min on fifth day only when transferred to 26°C
CRed light for 10 rain after 12 h dark imbibition only.
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TABLE IV
INTERACTION OF GIBBERELLIC ACID, RED LIGHT AND TEMPERATURE ON
G E R M I N A T I O N O F OCIMUM S E E D S
A n a l y s i s o f V a r i a n c e : F-values: g i b b e r e l l i c acid -~ 3 4 0 . 8 1 , gibberellic acid x light
3 6 0 . 4 6 ; light -* 5 5 3 . 4 0 , g i b b e r e l l i c acid x t e m p e r a t u r e -~ 8 4 . 1 2 ; t e m p e r a t u r e -~ 1 1 7 7 . 0 4 ,
light × t e m p e r a t u r e ~ 1 3 2 . 8 1 . G i b b e r e l l i c acid x light x t e m p e r a t u r e ~ 1 1 1 . 5 3 . All t h e
values o f F significant at 1% level.

S o l u t i o n a n d light t r e a t m e n t % g e r m i n a t i o n o f seeds at i n d i c a t e d
temperature (C)

14 26 38

Water
Dark 0 2 0
Light a 0 58 55
G A ( 1 • 10 - 4 M )
Dark 0 46 49
Light a 0 55 54

a R e d light: 10 min.

(10 -2 M, 10 -3 M, 10 -4 M, 10 -s M) were tried and 1 • 10 -4 M was found as

o p t i m u m concentration.
Gibberellic acid caused Ocimum seeds to germinate in darkness at optimal
and at relatively supraoptimal temperatures (46% and 49%). It however
failed to cause the seeds to germinate at suboptimal temperature in dark-
ness or even after p r o m o t i o n with red light {Table IV).
Effect o f imbibition time in dark. Seeds of Ocimum were allowed to
imbibe in dark at 26°C for varying time periods. Ten-minute red light in-
duced germination in 54% of the seeds up to 5 days of dark imbibition.
After that time, induction of germination b y short exposure to red light
decreased gradually for some time and then rapidly. Seeds ultimately became
completely insensitive to short-term irradiation with red light after approx.
20 days o f dark imbibition (Fig. 1). Secondary d o r m a n c y thus induced by
prolonged darkness was called s k o t o d o r m a n c y by Evenari [ 5 ].
In order to break the s k o t o d o r m a n c y the seeds were subjected to low
temperature (14°C) and high temperature (38°C) for 24 h before light
treatment. None of the treatments was found to restore the light sensitivity
of the seeds (Table V).
As gibbereUic acid was f o u n d to cause the seeds of Ocimum to germinate
in dark, it was thought that it can restore the light sensitivity of dark im-
bibed seeds. However, gibberellic acid either alone or in conjunction with
10 min red light failed to overcome the induced dormancy (Table V).
With an idea to overcome the d o r m a n c y caused by prolonged dark im-
bibition b y prolonged continuous irradiation the s k o t o d o r m a n t seeds were
268

60

40

~G

20

0 10 20 30

Days of dark imbibition

Fig. 1. Effect of dark imbibition on germination of O c i m u m seeds.

TABLE V
E F F E C T O F R E D LIGHT, G I B B E R E L L I C A C I D A N D T E M P E R A T U R E ON G E R M I N -
A T I O N O F OCIMUM S E E D S I M B I B I N G IN D A R K A T 26°C F O R A P P R O X . 20 D A Y S

Treatment Programme % germination

R e d light 10 m i n 2 ± 0.58
Gibberellic acid 1 • 10 -4 M G A for 0 ± 0.00
24h
Gibberellic acid 1 • 1 0 - ' M G A for 3 ± 0.50
+ 24 h a n d t h e n
R e d light 10 m i n r e d light
14°C + R e d light 24 h e x p o s u r e t o
14°C a n d t h e n red I -+ 0.41
light for 10 rain
38°C + R e d light 24 h e x p o s u r e t o 0 • 0.00
38°C and t h e n red
light f o r 10 min
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TABLE VI
HIGH IRRADIANCE RESPONSE OF OCIMUM SEEDS ALLOWED TO IMBIBE IN
DARK AT 26°C APPROX. 20 DAYS (IRRADIATION TIME: 300 MIN)

Irradiation quality % germination

White 47
Red 44
Far-Red 12
Blue 0

irradiated with red, far-red and blue light for 300 min. Most interestingly
continuous irradiation with red light enabled the seeds to overcome the
skotodormancy. Far-red light could break it only to a limited extent while
blue light failed completely {Table VI).

DISCUSSION

A low-energy phytochrome system operates in seed germination of

Ocimum tenuiflorum. Similar to Lepidium virginicum [6] photosensitivity
in Ocimum seeds spreads throughout the temperature range and thus the
block to germination by the photocontrol mechanism is neither overcome
nor bypassed at any temperature. However, in contrast to Lepidium, it is
the lower temperature that prevents seed germination of Ocimum indicating
a different underlying mechanism.
Inhibition of germination of the seeds of Ocimum that imbibed in dark
at 14°C with a light pretreatment m a y result from phase transitions of m e m -
brane lipids, the phase transitions being promoted by Pfr form of phyto-
chrome. Germination inhibition of Ocimum seeds imbibing in dark at 38°C
without a light pretreatment m a y be explained on the basis of partial anaero-
biosis. It is presumed that such partial anaerobiosis m a y not occur in seeds
that were given a pre-light treatment, due to the presence of a threshold
concentration of Pfr, the later being regulatory in membrane permeability
changes [7].
Gibberellins can cause many photoblastic seeds to germinate in dark
and also over a broader temperature range [3,8]. Gibberellic acid, although
allowing the germination process in Ocimum seeds to bypass the light
requirement, failed to overcome the low temperature block to germination
alone or even in conjuction with light.
Ocimum tenuiflorurn seeds became insensitive to light after approx. 20
days of dark imbibition in contrast to a period of 3--4 days for lettuce
[9], and 10 days for Ocirnum arnericanum [10]. Low temperature treat-
ment that could release skotodormancy [5] of dark imbibed seeds of
Ocirnurn arnericanum [10] was found as ineffective for Ocimum tenuiflorum
seeds.
270

In accordance with the results of Vidaver and Hsiao [ 11] and Speer et al.
[12] for lettuce seeds, neither red light nor gibberellic acid alone could
break the s k o t o d o r m a n c y in Ocimum seeds. However, the red light and
gibberellic acid which in conjuction released the s k o t o d o r m a n c y of lettuce
seeds [11,12] failed to cause the s k o t o d o r m a n t Ocimum seeds to germinate.
On this basis it is assumed t h a t the two metabolic pathways described for
s k o t o d o r m a n t lettuce seeds by Vidaver and Hsiao [11] are irreversibly
blocked by extended dark imbibition in Ocimum.
Failure of short-term irradiation with red light in restoring light sensitivity
may be due to the low levels of Pfr f o r m e d by it in skotodormant seeds,
insufficient to establish an adequate product level for germination. Pro-
longed continuous irradiation with red light probably maintains high
threshold levels o f Pfr, thereby inducing s k o t o d o r m a n t seeds to germinate.
Alternatively, a possibility of the existence of other excited species of Pfr
(besides the Pfr of Mohr [13]) as an effector molecule of high irradiance
response to red light m a y be considered.
The aforesaid mechanism of breaking s k o t o d o r m a n c y may be of special
significance to Ocimum tenuiflorum occurring at Port Blair where seed
dispersal (January-February) is followed by dry months (March-April).
Heavy rains commencing from the m o n t h of May thicken the already exist-
ing green canopy thus altering the R / F R ratio in a way to throw the seeds
to unfavourable light conditions as the canopy light is relatively very rich
in far-red light compared to unfiltered light [14].
Temperature at Port Blair and its environs does not drop to an appreciable
level so as to break the s k o t o d o r m a n c y and also the seeds do not possess
such an adaptation {Table V). Further gibberellic acid neither alone nor in
conjunction with red light is potent to overcome the induced dormancy.
In face o f these findings, the positive response of s k o t o d o r m a n t seeds to
prolonged continuous exposure to red light, becoming available when
the canopy thins out, m a y help the species, at least in part, to survive and
spread.

ACKNOWLEDGEMENTS

Thanks are due to Dr. N.P. Balakrishnan, Regional Botanist and Dr.
M.K. Vasudeo Rao, Systematic Botanist of Botanical Survey o f India,
A n d m a n and Nicobar circle, for identification of plant species and to Dr.
V.P. Singh, School o f Studies in Botany, Vikram University for technical
cooperation. Financial assistance from University Grants Commission as
a special grant for a field trip to A n d m a n Islands is acknowledged.

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205.
 271

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