J.PlantPhysiol. Vol. 132.pp.

750-753 (1988)

The Germination Response of the Negatively Photoblastic Seeds

of Citrullus lanatus to Light of Different Spectral Compositions

FREDERIK c. BOTHA and J. G. CHRIS SMALL

Dept. of Botany, University of the Orange Free State, Bloemfontein 9300, South Africa

Received September 29, 1987 . Accepted November 25, 1987

Summary

The germination of Citrullus lanatus seeds is inhibited by far-red-, red-, blue-, green- and white light. The
inhibitory effect of light (with the exception of far-red) light is alleviated by a follow-up dark treatment.
In addition it was found that the inhibitory effect of far-red light could not only be alleviated by a follow
up red light treatment but also by short subsequent exposure to white- and blue light. The obtained re-
sults most probably indicate that germination is inhibited by red, blue, white, and, to a lesser extent, by
green light due to pigment cycling between Pr and Pfr- During periods of pigment cycling much of the
phytochrome probably accumulates in a form which can readily change to Pfr in a follow up dark treat-
ment. It appears that due to inherent differences in the Pfr-threshold value necessary for germination,
negatively- and positively photoblastic seeds cannot be made the same in their light requirements for ger-
mination merely by manipulating the phytochrome concentrations with light.

Key words: Germination, Citrullus lanatus, phytochrome.

Abbreviations: pto!) Total phytochrome; Pf) red light absorbing form of phytochrome; Pff) far-red light
absorbing form of phytochrome; LER, low energy reaction; HER, high irradiance reaction.

Introduction component of white light is the blue part of the spectrum

The germination behaviour at 27°C of the wild form of
Citrullus lanatus is negatively photoblastic (Botha et al.,
1982 a). The reaction of these seeds is typical of a low-energy
phytochrome system (LER) (Botha et al., 1982 a; Botha et al.,
1982 b).
One of the main differences between positively- and nega-
tively photoblastic seeds is that white light stimulates ger-
mination of the former but completely inhibits germination
of the latter. It would appear that positively photoblastic
seeds react to the red component and negatively photoblastic
seeds to the far-red component of white light (Smith, 1975).
The effect of white light could be explained by its effect on
several parameters of the pigment system namely, (1) the
photoequilibrium value, (2) the concentration of pto !) and (3)
the rate of pigment cycling between Pr and Pfr (Bewley and
Black, 1982). Normally inhibition in white light will only
occur when a photoequilibrium value of lower than 0.55 to
0.65 results (Bewley and Black, 1982). Another inhibitory
(Bewley and Black, 1982). The germination of both posi-
tively and negatively photoblastic seeds is usually inhibited
by blue light (Black and Wareing, 1957; Kendrick and
Franklin, 1969; Noronha et al., 1978; Wareing and Black,
1958). A feature of the germination of C. lanatus seeds is that
white light, almost devoid of a far-red component, also in-
hibits germination (Botha et al., 1982 a).
During seed ripening of some species a large part of the
phytochrome can be accumulated in the meta-Rb form (Ken-
drick, 1976). This phytochrome intermediate may revert to
Pfr during hydration of the seeds. When negatively photo-
blastic seeds are irradiated with far-red light after the conver-
sion of meta-Rb to Pfr is completed, the seeds will not ger-
minate unless exposed to red light (Kendrick and Franklin,
1969; Rollin et al., 1970; Yaniv et al., 1967). According to
Kendrick (1976) the only difference between a dark ger-
minating seed such as Amaranthus caudatus and a light-re-
quiring seed such as Lactuca sativa is the process by which
substantial amounts of Pfr appear in the seed during imbibi-

© 1988 by Gustav Fischer Verlag, Stuttgart

 Effect of Light on Seed Germination 751

tion. Furthermore he states that negatively photoblastic Table 1: The effect of continuous irradiation with light from sowing
seeds, after a period of imbibition under continuous far-red of different spectral composition on the germination of Citrullus lao
light become qualitatively similar to positively photoblastic natus seeds.
seeds in that they then fail to germinate in the dark unless Percentage germination
given an exposure to red light. after:*)
This study was undertaken to determine which part of the Treatment 48 h 72h 96 h
electromagnetic spectrum of white light causes inhibition of White light 0 3±2 8± 1.5
germination of C. lanatus seeds and to ascertain whether the Red light 1± 1.5 6±5 33±10
seeds could qualitatively be made the same as positively Blue light 14±3 24±4 30± 2.4
photoblastic seeds by a far-red light treatment. From the re- Green light 4±2.7 22±7 73± 6
sults it was possible to formulate a hypothesis to explain the Far-red light 0 3±2 6± 1.5
Dark control 44±3 80±6 95± 4
mechanism of negative photoblastism as shown by C. lao
natus. *) periods include all pre-treatments.
± = standard deviation.

Material and Methods

Table 2: The effect of intermittent*) irradiation from sowing with
light of different spectral compositions on the germination of Cit·
Material rullus lanatus seeds.
Seeds of Citrullus lanatus Matsumura and Nakai and Lactuca sati· Percentage germination
va cv. Grand Rapids were obtained and stored as previously describ- after:
ed (Botha et al., 1982 a; Botha et al., 1982 b). Interference filter Ma7-1 Treatment 48 h 72h 96 h
(660 nm) with a half bandwith of 5 - 9 nm was obtained from White light 4±2 14±4 18± 6
SCHOTT. Red light 5±4 32±8 50±10
Red light** 8±3 28±7 44±12
Blue light 15±4 20±8 45± 11
Germination
Green light 20±8 38±9 79±13
Seeds were germinated in 9 cm Petri dishes containing a single Far-red light 0 0 8± 3
sheet of Whatman no 1 filter paper moistened with 5 m! of distilled Dark (control) 39±6 84±5 97± 6
water. In all cases 30 seeds were used per replicate. Germination *) 2 h of light in every 12 h cycle (see methods).
tests were conducted at 27°C in the light and in the dark. In trials **) monochromatic red light.
where seeds were exposed to intermittent light, a 2 h light treatment ± = standard deviation.
in every 12 h cycle was given. The cycle was started with the 2 h
light treatment immediately after sowing followed by 10 h darkness.
Germinated seeds were counted under a dim green safe light (Smith,
1975). A seed was considered germinated when the radicle had
Although part of the inhibition observed with C. lanatus
emerged. At least nine replicates per treatment were used through- seeds could be due to a high irradiance reaction (Kendrick,
out. 1976) the results obtained with intermittent light imply that
germination is controlled by a low energy reaction (Table 2).
Intermittent red, far-red, blue, green and white light are al-
Irradiation sources most as effective as continuous light of the same spectral
White light (16.6 /Lmol· m ~2. s~ I), red light (0.75 /Lmol· m ~2. composition (Table 1 and 2).
S~I) and far-red light (0.95/Lmol·m~2·s~l) were obtained as pre- The germination of Citrullus colosynthis seeds is also inhib-
viously described (Botha et al., 1982 b). Blue light was obtained ited by green light (Koller et al., 1963). These authors, how-
from «cool white» fluorescent tubes in combination with one layer ever, ascribe the inhibition to contamination of the green
no 20 Cinamoid filter which delivered a total quantum flux of light with other wavelengths of light. It is unlikely that the
0.80 /Lmo!· m ~ 2 . S ~ I at the position of the seeds. Green light was
same is true for the results obtained, especially with red light,
obtained by filtering the light form «cool white» fluorescent tubes
through two layers of no 39 Cinamoid filter which delivered a total in the present study with Citrullus lanatus seeds. Firstly, let-
quantum flux of 0.2 /Lmol· m ~ 2. S ~ I) at the position of the seeds. tuce seeds (Grand Rapids) attained a final germination of
Monochromatic red light (0.27 /Lmol· m ~ I . S ~ I) was obtained by 90 % and 86 % after a 48 h continuous white- and red light
filtering white light (Small et al., 1979) through the SCHOTT MA7-1 treatment, respectively, while in far-red light and in the dark
interference filter. only 8 % and 12 % of the seeds germinated. The high per-
centage germination of lettuce seeds in the red light indicate
that the inhibition of germination of Citrullus lanatus by red
Results and Discussion light is probably not due to contamination with other inhib-
itory wavelengths light. Secondly, it was found that intermit-
From the results presented in Table 1, it is evident that tent monochromatic red light also effectively inhibited the
continuous irradiation with blue, red, green and far-red light germination of Citrullus lanatus seeds (Table2).
is inhibitory for the germination of C. lanatus seeds. Red Germination is most probably inhibited by the different
and blue light are equally effective in inhibiting germination. light treatments in that the treatment results in a Pfr concen-
Although green light also inhibits germination of these tration lower than the critical threshold value required for
seeds, the inhibition is gradually alleviated with time. germination. The low Pfr concentration could be related to a
752 FREDERIK C. BOTHA and J. G. CHRIS SMALL

high Pr concentration or to a high concentration of phy- Table 4: The effect of a short light treatment after 22 h of incubation
tochrome intermediates. In an Amaranthus sp., depending on on the germination of Citrullus lanatus seeds.
the fluence rate, up to 30 % of Ptot can be in the intermediate Pre-treatment Treatment between Percentage germination
phytochrome forms after a white light treatment (Kendrick for 22 h 22 hand 25 h*) after:**)
and Spruit, 1972). Even after irradiation with monchromatic 48 h 72h 96 h
red light a substantial amount of phytochrome intermediates Dark 1 h far-red light 4±3 8±4 9±5
can exist (Kendrick and Spruit, 1972). 2 h dark
During any light treatment of which the quality falls in the
Dark 1 h far-red light 80±7 97±3 100±0
spectral region where the absorption spectra of Pr and Prr 1 h red light
overlap, a substancial amount of phytochrome intermediates 1 h dark
will exist at the photostationary state (Kendrick and Spruit,
Dark 1 h far-red light 85±5 89±6 93±4
1976). As the reaction step meta-Rb to P rr is the slowest reac-
1 h white light
tion in the Pr .... Prr .... Pr-cycle (Kendrick and Franklin, 1969) 1 h dark
any light treatment which causes pigment cycling between Pr
and Prr will probably result in a high meta-Rb concentration Dark 1 h far-red light 15±6 30±5 40±7
2 h blue light
(Kendrick and Spruit, 1974; Kendrick and Spruit, 1976). It is
only during treatment of seeds with monochromatic far-red Dark dark 81±6 93±3 97±2
light that no accumulation of meta-Rb occurs as Pr does not *) thereafter all seeds where kept in the dark.
absorb much light with a wavelength longer than 720 nm **) periods include all pre-treatments.
(Smith, 1975). ± = standard deviation.
The inhibitory effect of white, blue, green and red light is
alleviated when seeds of C lanatus are transferred to the dark
after a 48-h light treatment (Table 3). In contrast the inhibi- P" but a large portion of the Ptot will probably still be in the
tion caused by far-red light is not alleviated by a follow-up intermediate form which could revert to Prr . If the far-red
dark treatment. The alleviation of the inhibitory effect of light treatment is therefore followed by a dark period, suffi-
white light during a follow- up dark treatment seems to be a cient intermediates will revert to Prr to render a P rr concen-
general phenomenon in negatively photo blastic seeds tration high enough for germination (Black and Wareing,
(Gorski and Gorska, 1979; Kendrick and Franklin, 1969; 1957). (b). Secondly, if the seeds are exposed to far-red light
Negbi and Koller, 1964). after 22 h but before 30 h of dark incubation the larger part
Based on these results and previous work (Botha et al., of meta-Rb would already be converted to Prr and therefore
1982 b) in which it was shown that the germination response not enough would be left to revert to Prr in the dark after far-
of C lanatus seeds is typical of a LER-phytochrome me- red light treatment to enable the seeds to germinate [(Botha
diated reaction we propose the following mechanism for the et al., 1982 b) and Tables 3 and 4].
germination response of these seeds to light: (2) During white, blue, green and red light treatment of
(1) The reaction of these seeds to an inhibitory far-red the seeds pigment cycling between Pr .... Prr .... Pr will prob-
light treatment can be divided into two phases. (a). Firstly, ably take place (Kendrick and Spruit, 1972). Any of these
the period before 20 h, during which the conversion of treatments will therefore result in the formation of sub-
phytochrome intermediates (presumably meta-Rb ) to Prr is stantial amounts of phytochrome intermediates. In the case
not completed (Botha et al., 1982 b). If seeds are exposed to of C lanatus seeds the Prr-concentration during all these light
far-red light at this stage most of the P rr will be converted to treatments is probably lower than the threshold value re-
quired for germination. However, if the seeds are transfered
to the dark after any of these light treatments, dark-reversion
Table 3: The effect of irradiation with light of different spectral of intermediates to P rr will result in a Prr concentration
composition for different periods from sowing on the germination higher than the threshold value required for germination
of Citrullus lanatus seeds.
(Table3). If this postulate is valid, then any light treatment
Percentage germination resulting in pigment cycling should alleviate the inhibitory
after:*) effect of far-red light. Tables4 and 5 show that this is indeed
Tratment 72h 96 h 120 h the case, provided that the light treatment is followed by a
Continuous red light 6±5 33±10 50±8 dark period. Red, blue, and white light are effective to cause
48 h Red light followed by dark 84±4.7 98± 1.4 98± 1.4 a significant alleviation of the inhibitory effect of far-red
Continuous blue light 24±4 30± 2.4 38±6 light. The inhibitory effect of far-red light, however, cannot
48 h Blue light followed by dark 62±7 83± 2.9 96±3
be alleviated by continuous white or red light (Table 5).
Continuous far-red light 0 0 0
3±2 3±2 The statement by Kendrick (1976) that positively and
48 h Far-red light followed by dark 0
Continuous green light 22±7 73± 6 86±4 negatively photoblastic seeds will qualitatively be the same
48 h Green light followed by dark 81± 1.4 94± 1.9 94±1.9 in their light requirements after a far-red light treatment does
Continuous white light 3±2 6± 1.5 10±3 not appear to hold for C lanatus. Positvely photo blastic
48 h White light followed by dark 46±4 72± 6 96±2 seeds are able to reach a Prr concentration high enough for
Continuous dark 80±6 94± 3 98± 1.5 germination during treatment of the seeds with continuous
*) periods include all pre-treatments. white and red light. Negatively photoblastic seeds, however,
± = standard deviation. cannot attain a Prr concentration high enough for germina-
 Effect of Light on Seed Germination 753

Table 5: The germination of Citrullus lanatus seeds after different BOTHA, F. c., J. G. C. SMALL, and N. GROBBELAAR: Seed germination
light treatments. in Citrullus lanatus. 2. The involvement of phytochrome and
ethylene in controlling light sensitivity. S. Afr. J. Bot. 1,
Pre-treatment Treatment between Percentage germination 131-133 (1982 b).
for 22 h 22 hand 96 h after 96 h*)
ELLER, B. M., F. C. BOTHA, N. GROBBELAAR, andJ. G. C. SMALL: Seed
Far-red light 74 h red light 10±6.0 germination in Citrullus lanatus. 3. The possibility of light as an
Far-red light 74 h white light 8±4.3 inhibitory factor for germination of seeds within fruits based on
Far-red light 1 h red light 96±3.2 light measurement studies. S. Afr. J. Bot. 1, 133 -134 (1982).
73 h dark GORSKI, T. and K. GORSKA: Inhibitory effects of full daylight on the
Far-red light 1 h white light 93±6.0 germination of Lactuca sativa. Plant Physio!. 144, 121-124
73 h dark (1979).
Far-red light 74 h dark 8±4.5 JONES, B. M. and L. F. BAILY: Light effects on the germination of
*) period includes all pre-treatments. seeds of Henbit (Lamium amplexicaule L.) Plant Physio!. 31,
± = standard deviation. 347-349 (1956).
KENDRICK, R. E.: Photocontrol of seed germination. Sci. Prog. 63,
347 -369 (1976).
tion in continuous white light, and in the case of C. lanatus KENDRICK, R. E. and B. FRANKLIN: Photocontrol of germination of
also in red, blue and green light. This difference between the Amaranthus caudatus. Planta 85, 326-339 (1969).
two types of seeds remains even after treatment of the seeds KENDRICK, R. E. and C. J. P. SPRUIT: Light maintains high levels of
with far-red light. phytochrome intermediates. Nature New Bio!. 237, 281- 282
(1972).
A possible explanation for this phenomenon could be that
- - Inverse dark reversion of phytochrome: an explanation.
negatively photoblastic seeds exhibit a higher Pfr threshold Planta 120, 265-272 (1974).
value requirement for germination than positively photo- - - Intermediates in the photoconversion of phytochrome. In:
blastic seeds. If this is true then it would not be possible to H. SMITH, ed. Light and plant development Butterworths,
make positively and negatively photo blastic seeds the same London (1976).
in their light requirements merely by manipulating the - - Phototransformations of phytochrome. Photochem. Photo-
phytochrome concentration in the seeds with different light bio!. 26, 201-214 (1977).
treatments. KOLLER, D., A. POL]AKOFF, A. BERG, and T. DISKIN: Germination-
In addition it appears possible that dark-germinating seeds, regulation in Citrullus colocynthis. Amer. J. Bot. 50, 598 - 603
as exemplified by C. lanatus, might exhibit higher rates of (1963).
pigment cycling when irradiated and/or longer intermediate NEGBI, M. and D. KOLLER: Dual action of white light in the photo-
control of germination of Oryzopis miliacea Plant Physio!. 39,
lifetimes than positively photo blastic seeds.
247-253 (1964).
NORONHA, A., M. VINCENTE, and G. M. FELIPPE: Photocontrol of
Acknowledgements germination in Cucumis anguria. Bio!. Plant. 20, 281-286 (1978).
ROLLIN, P., R. MALCOSTE, and D. EUDE: Le role du phytochrome
This project was financially supported by the University of the dans la germination des graines de Nemophila insignis L. Planta
Orange Free State and the Foundation for Research Development. 91,227 -234 (1970).
SMALL, J. G. c., c. J. P. SPRUIT, G. BLAAUW-JANSEN, and o. H.
BLAAUW: Action spectra for light-induced germination in dor-
References mant lettuce seeds. Planta 144, 125-131 (1979).
SMITH, H.: Phytochrome and photomorphogenesis. McGraw-Hill,
BEWLEY, J. D. and M. BLACK: Physiology and Biochemistry of Seeds. London (1975).
2. Viability, dormancy and environmental contro!' Springer- WAREING, P. F. and M. BLACK: Similar effects of blue and infra-red
Verlag, New York (1982). radiation on light sensitive seeds. Nature (Lond.) 181,
BLACK, M. and P. F. WAREING: Sensitivity of light-inhibited seeds to 1420-1421 (1958).
certain spectral regions. Nature (London) 180, 398 (1957). YANIV, Z., A. L. MANCINELLI, and P. SMITH: Phytochrome and seed
BOTHA, F. c., N. GROBBELAAR, andJ. G. C. SMALL: Seed germination germination. 3. Action of prolonged far-red irradiation on the
in Citrullus lanatus. 1. Effect of white light and growth sub- germination of tomato and cucumber seeds. Plant Physio!. 42,
stances of germination. S. Afr. J. Bot. 1, 10-13 (1982 a). 1479 -1482 (1967).