SMITHSONIAN MISCELLANEOUS COLLECTIONS
VOLUME 94 NUMBER S
WAVE LENGTHS OF RADIATION IN THE VISIBLE
SPECTRUM INHIBITING THE GERMINATION
OF LIGHT-SENSITIVE LETTUCE SEED
BY
LEWIS H. FLINT
Division of Seed Investigations, Bureau of Plant Industry,
U. S. Department of Agriculture
AND
E. D. McALISTER
Division of Radiation and Organisms,
Smithsonian Institution
(PUBLI.CATION 3334)
CITY OF WASHINGTON
PUBLISHED BY THE SMITHSONIAN INSTITUTION
JUNE 24, 1935
€Be £orb at?atHrnorc (prtee
BALTIMORE, MD., C. 8. A.
WAVE LENGTHS OF RADIATION IN THE VISIBLE
SPECTRUM INHIBITING THE GERMINATION
OF LIGHT-SENSITIVE LETTUCE SEED
By lewis H. flint
Division of Seed Investigations, Bureau of Plant Industry,
U. S. Department of Agriculture
E. D. McALISTER
Division of Radiation and Organisms,
Smithsonian Institution
IXTRODUCTIOX
In studies of the light-sensitivity of " dormant " lettuce seed previ-
ously reported (3)' it was noted that radiation of the longer wave
lengths of visible light, characterizing the colors yellow, orange, and
red, promoted germination, whereas radiation of the shorter wave
lengths of visible light, characterizing the colors violet, blue, and green,
inhibited germination. The material appeared to be unusually well
adapted to the study of response to radiation, and steps were taken
to establish the relative effectiveness of radiation of various wave
lengths with respect to the germination of the seed.
While these studies were in progress, Johnston (6) at the Smith-
sonian Institution reported the results of a careful series of measure-
ments of phototropic response of the etiolated oat coleoptile, which
emphasized the fact that the shorter wave lengths of visible light were
responsible for such bending. He interpreted this activity as due to
an inhibitory effect of the shorter wave lengths upon the cells exposed
to such radiation.
On account of the obvious analogy between the results obtained
with the shorter wave lengths of light in respect to inhibition in
germination and in phototropism the facilities of the two research
divisions were combined in the furtherance of the germination study,
the cooperative investigation leading to the results here presented.
^ Numbers in parentheses refer to list of literature cited, at the end of this
paper.
Smithsonian Miscellaneous Collections, Vol. 94, No. 5
I
2 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 94
REVIEW OF LITERATURE
With respect to light and germination three classes of seeds have
been recognized for many years : ( i ) seeds germinating equally well
in light or darkness, (2) seeds whose germination is hindered by light,
and (3) seeds whose germination is favored by light. This classifica-
tion has emphasized the variety of the responses that may occur and
has proved satisfactorily descriptive for studies involving sunlight
or other white light.
In 1883 Cieslar (2) reported with respect to certain seeds that
yellow light favored germination, whereas violet light retarded it and
rendered them appreciably dormant. This varying response to differ-
ent wave lengths of light made it evident that in sunlight or other
white light certain components were acting to promote germination,
while certain other components were acting to retard it. Upon the rela-
tive effectiveness of these two groups of components, either through
the radiant energies involved or through the particular sensitivity of
the seed, one might presume to depend the gross efifect of the white
light with respect to germination. From the more technical standpoint,
therefore, there were but two classes of seeds with respect to germina-
tion : ( I ) those whose germination was not influenced by light, and
(2) those whose germination was influenced by light. Seeds of the
latter class were designated " light-sensitive seeds," as contrasted with
the widely occurring seeds of the class which germinate equally well
in light and in darkness. The present considerations are confined to
light-sensitive seeds.
Light-sensitivity as reported by Cieslar was limited for the most
part to small seeds without reserve food materials. In the seeds of
Poa nemoralis, Agrostis stolonifera, and Nicotiana nmcrophylla ger-
mination was reported as favored by white light, whereas the germina-
tion of seeds of Viscutn album was reported as hindered by white
light. In large measure the researches on light and germination by
various workers in subsequent years have been concerned with the
extension of these respective lists. In this respect it is to be noted
that sensitivity to light is now generally recognized as a widely oc-
curring characteristic of seeds.
The early distinction between the efifect of " yellow " light and
" violet " light gained precision through subsequent researches, and
one finds in Molisch (9) the statement that yellow to red light pro-
motes germination, whereas violet, blue, or green light inhibits ger-
mination. This information, however, has not been widely appreciated,
and the more recent studies of light in relation to germination, such
NO. 5 IRRADIATED LETTUCE SEED FLIXT AND McALISTER 3
as those of Gardner (4), Lehmann (7), Maier (8), Nathammer (10)
and Shuck (11), have concerned themselves for the most part with
white hght as a quantitative factor in germination.
Wholly unaware of the foregoing background of researches by-
German workers in this field, the senior author discovered that so-
called " dormant " lettuce seed would germinate readily in white light,
and further, that yellow, orange, or red light promoted this germina-
tion, whereas violet, blue, or green light inhibited it. It now appears
that the germination response to radiation of specific wave lengths
noted for dormant lettuce seed represents types of reactions of wide
occurrence among seeds. This fact suggests that the further study
of such responses may be warranted as promising results of both prac-
tical and theoretical significance in relation to germination and pos-
sibly also in relation to other aspects of growth. The results obtained
by Flint (3) with green Wratten filters indicated that color alone was
not a safe criterion to use in the interpretation of results obtained with
filters, thus directing attention to their wave-length transmission.
METHODS
The principal line of attack in this investigation involved the use
of a spectrum, and to a large extent the work comprised successive im-
provements in the technique of utilizing the spectrum to the greatest
Fig. I.—Schematic drawing of the apparatus used in the study of spectral ligl
in relation to germination.
advantage in relation to germination. The set-up as finally elaborated
(see fig. i) consisted of a fixed light source, a condenser lens con-
centrating light upon an adjustable slit, an achromatic lens, a prism,
and a silvered mirror. A light-proof house surrounded the set-up,
with a partition at the slit (not shown in the figure). With this set-up,
using a single filament 1,000 lumen 6.6 ampere Mazda street-lighting
bulb as a light source, a spectrum was obtained which was about i foot
long in the visible range.
4 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 94
For exposures of the material in the spectrum special boxes lo x
4x1 inches were made of brass and provided with parallel center
plates of monel metal about 4x1 inches, spaced at 0.4 inch. Two such
boxes placed end to end thus more than filled the visible spectrum, and
each of the 48 compartments was subjected to a band of radiation
ranging in width from the order of 50 A in the low violet to that of
200 A in the high red. The spectrum and boxes were provided with
a secondary light-proof housing, in which, at an elevation of about
1 foot, were installed two 20-inch milk-glass lumiline lights wrapped
in red cellophane having no appreciable transmission for wave lengths
shorter than 6000 A. These lights were so arranged that in conjunc-
tion with end-mirrors no shadows were cast in any compartment of
the boxes. The intensity of illumination was regulated by a rheostat.
The focal plane of the spectrum obtained was located by inserting
a plate of didymium glass between the condenser lens and the slit.
The sharp absorption lines of didymium also provided a convenient
means of establishing the wave lengths of all regions of the visible
spectrum. Wave lengths in the near ultraviolet region were established
by substituting a mercury arc as a light source and using uranium glass
to pick up through fluorescence the lines characteristic of mercury.
Wave lengths in the infrared region were established by following the
absorption characteristics of water vapor with a thermopile. The
radiation energies throughout the entire spectrum were established
by means of a thermocouple.
The procedure in each experiment was as follows. Two boxes
were placed in the spectrum and half filled with tap water, which
served as the medium of germination. Dormant or light-sensitive
lettuce seeds were then scattered into the compartments, surface ten-
sion bringing about a fairly uniform distribution of the seeds over
the available water surface. About 100 seeds could be conveniently
accommodated in each compartment.
After 2| hours presoaking the seeds were given exposures of spec-
tral light, of red lumiline light, or of both lights, depending upon the
particular objective. The red lumiline light, by suitable modification
of the duration or intensity of the exposure, was used for the most
part to effect a 50 percent germination of the seeds independent of the
spectral light—a feature ordinarily offering some difficulty, but en-
tirely feasible with the material at hand, as had been attested by tables
2 and 3 of Flint's paper (3). Upon this base the nature and extent
of any promoting or inhibiting influence of the spectral light was
registered as a departure. After 24 hours the boxes were removed.
NO. 5 IRRADIATED LETTUCE SEED FLINT AND McALISTER 5
and the seeds in each compartment were transferred immediately to
numbered petri dishes, placed in a refrigerator at 3° C, and exposed
to blue light (to prevent further germination), where they were kept
until germination counts could be made.
In plotting the germination percentages against radiation the wave
length falling on the median line of each compartment was taken as
the wave length for the seeds of that compartment.
In plotting the inhibitory influence in the violet-blue-green region
as corrected for energies involved, the curves were inverted to facili-
tate subsequent comparison with other data.
The transmission curves of the Wratten filters and of the ether
extracts of lettuce seeds were made in the conventional manner with
a double monochromator and a thermocouple.
RESULTS
In the experiments of Flint (3) two green Wratten filters were
found to transmit light that promoted germination, and 10 green
Wratten filters were found to transmit light that inhibited germina-
tion. The spectral transmission of all the green Wratten filters was
studied, and the energy transmission curves were obtained by mul-
tiplying the percentage transmission by the energy radiated from a
Mazda lamp at each wave length. The energy radiated by the lamp at
each wave length was obtained from its known spectral energy curve.
These energy values were used in conjunction with the inverse square
law and the distances at which the respective filters (when used with
the Mazda lamp) gave equal response with a Weston photronic cell.
The energies transmitted by representative green Wratten filters are
shown in figure 2.
It is to be noted from the curves of figure 2 that the two green
filters which had been found to transmit light promoting germination
(64 and 67) transmitted more of the ultraviolet and less of the long
visible red than the green filters which had been found to transmit
light inhibiting germination (56 and 60). This fact suggested that
the promotion was associated either with a promoting influence in the
ultraviolet or with an inhibiting influence in the long red or near
infrared.
A substantial series of exposures of moist dormant lettuce seed
to various wave lengths in the ultraviolet ranging from the lower
limits of the visible spectrum to below the ultraviolet characterizing
solar radiation gave uniformly negative results, whereupon attention
was directed to the infrared regions. Earlier studies with a spectrum
SMITHSONIAN MISCELLANEOUS COLLECTIONS 94
by the senior author had given an approximate range of 5200 to
7000 A for the promoting effect, with a sharp faUing off in germina-
tion in the long red. No inhibitory effect had been suspected as as-
sociated with this falHng off, however, until the effort was made to
Fig. 2.—Energy transmission curves of green Wratten filters. The ordinates
are relative energies transmitted at each wave length (indicated as abscissae).
Numbers 56 and 60 are typical of the green filters transmitting light that in-
hibits germination. Numbers 64 and 67 transmit light that promotes germination.
explain the physical basis for tha promoting effect of the light trans-
mitted by the two aberrant green filters. The spectrum set-up previ-
ously used by the senior author had been considerably modified in these
cooperative studies to permit a more precise measurement of the wave
NO. 5 IRRADIATED LETTUCE SEED FLINT AND McALISTER /
lengths to which seeds were exposed. With the apparatus and pro-
cedure described in the foregoing section such results as those given
in figure 3 were obtained in the long red region.
Such results as those presented in figure 3 established the presence
of a strong inhibitory influence in the region of 7600 A.
By applying the same methods to the violet, blue, and green regions
of the spectrum, such results as those presented in figure 4 were
obtained.
In conjunction with the foregoing experiments a study of ether
extracts of lettuce seed was carried, out. These extracts contained
oil and pigments. Definite absorption in the region 4200 A to 5200 A
was evidenced by the transmission curves. These remained bimodal,
even after appreciable oxidation had taken place, and thus appeared
suggestive in relation to the bimodal curve of inhibition given in
figure 4. Definite absorption in the region 5200 A to 7000 A was also
evidenced by the transmission curves, suggesting the presence of some
precursors of chlorophyll and allied pigments. No appreciable ab-
sorption in the region 7600 A was noted.
DISCUSSION
The discovery of a strong inhibitory influence in the region of
7600 A, although made in an effort to explain the difference in the
response to the light transmitted by certain green Wratten filters, and
quite incidental to the study of the precise nature of the curve of
inhibition in the regions characterizing violet, blue, and green light,
may well transcend in importance the original objective of the coopera-
tive studies. Since this discovery appears to offer a clearer approach
to biological problems involving light, it has been given precedence in
these considerations.
All the green Wratten filters used by Flint (3) were found to trans-
mit in the 7600 A region, but the two filters transmitting light which
promoted germination had such a low transmission in this region that
the eft'ects of the promoting regions—the yellow, orange, and red
—
predominated over the effects of the inhibiting regions—the long red,
the violet, the blue, and the green. Since many blue and violet glass,
liquid, or gelatin filters transmit in the region 7600 A, it follows that
the newly discovered inhibitory band becomes a potential source of
confusion as to the effectiveness of radiation in the more visible spec-
trum with respect to the germination of light-sensitive lettuce seed.
Moreover, since the same type of light-sensitivity has been recognized
as characterizing other seeds, this factor may well be of some general
significance with respect to light-sensitivity in seeds.
SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 94
100
NO. 5 IRRADIATED LETTUCE SEED FLINT AND McALISTER 9
In connection with the later consideration of inhibitory influences
associated with wave lengths characterizing violet, .blue, and green
light, it is to be noted that with both solar and Mazda radiation the
energy at 7600 A is much greater than at the shorter wave lengths of
the visible spectrum. In solar radiation there is a sharp absorption
band in the 7600 A region interpreted as due to oxygen in the sun
and water vapor in the earth's atmosphere. Notwithstanding this ab-
sorption, however, the energy of solar radiation at this wave length
region is large. In consequence it would appear that under natural
outdoor conditions and under customary indoor experimental con-
ditions radiation of a wave length in the long red exerts a relatively
powerful inhibitory influence upon the germination of dormant lettuce
seed, although this influence is ordinarily more than counteracted by
the promoting influence in the yellow-orange-red region. The extent
to which the 7600 A region has an analogous effect upon other seeds
and upon other phases of light-sensitivity is not known at this time,
but because of the high energy and universal occurrence of the radia-
tion, its potential significance becomes one of the most intriguing
results of its discovery. Further studies of the possible effectiveness
of this region in respect to the germination of other seeds and in
respect to other phases of light-sensitivity are now in progress.
An examination of the germination responses to light of the wave
lengths indicated in figure 4 reveals that there are two maxima of
inhibition in the violet-blue-green region—a major one at 4400 A, and
a somewhat subordinate one at 4800 A. It may now be noted that
Bachmann and Bergann (i) and Johnston (6), studying the etiolated
coleoptiles of Avena sativa Culberson, obtained curves of phototropic
sensitivity having two maxima at about these same regions. The two
types of data, the one indicating an inhibitory influence of light on the
germination of seeds, the other an influence of light on the direction
of growth of young etiolated shoots, have been brought together to
facilitate comparison in figure 5.
An examination of figure 5 reveals that within the range of experi-
mental error the two types of plant response to light show identical
critical wave lengths. Johnston (6, p. 14) and others interpret photo-
tropic response as an index of growth retardation on the theory that
the light on the exposed side of the shoot inhibits elongation, while
on the opposite unexposed side elongation is relatively uninfluenced.
The results here reported obviously tend to sustain the correctness of
this interpretation. Both of the foregoing types of plant response to
light involved etiolated structures, and further studies are in progress
relating to the types of plant response characterizing green tissues.
10 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 94
In resume, the results obtained with the Wratten filters, taken in
conjunction with the curve of violet-blue-green inhibition given by
the spectral data and with the newly discovered inhibitory influence in
the 7600 A region, make it more than ever obvious that the effects ob-
tained through the use of any color filter may not safely be interpreted
without an analysis of its spectral transmission.
The results presented in this paper indicate the general relative
effectiveness of radiation of various wave lengths in the visible spec-
trum found to inhibit the germination of light-sensitive lettuce seed.
00
NO. 5 IRRADIATED LETTUCE SEED FLIXT AND McALISTER II
effectiveness in bringing about phototropic response in etiolated col-
eoptiles of oats. Both phenomena may be represented by bimodal
curves showing critical wave lengths in the regions 4400 A and 4800 A.
LITERATURE CITED
1. Bachmann, Fr., and Bergann, Fr.
1930. Uber die Werkigkeit von Strahlen verschiedener Wellenlange fur
die phototropische Reizung von Avcna sativa. Planta, Arch.
Wiss. Bot., vol. 10, pp. 744-755.
2. Cieslae, Adolph
1883. Untersuchungen iiber den Einfluss des Lichtes auf die Keimung
der Samen. Pp. 270-295.
3. Flint, Lewis H.
1934. Light in relation to dormancy and germination in lettuce seed.
Science, vol. 80, pp. 38-40.
4. Gardner, W. A.
1921. Effect of light on germination of light-sensitive seeds. Bot. Gaz.,
vol. 71, pp. 249-288.
5. Gassner, G.
1915. Beitrage zur frage der Lichtkeimung. Zeitschr. Bot., vol. 7,
pp. 609-661.
6. Johnston, Earl S.
1934. Phototropic sensitivity in relation to wave length. Smithsonian
Misc. Coll., vol. 92, no. 11, pp. 1-17.
7. Lehmann, E.
1918. Uber die minimale Belichtungzeit welche die Keimung der Samen
von Lythrum salicaria auslost. Ber. Deutsch. Bot. Ges., vol.
36, pp. 157-163.
8. Maier, Willi
1933. Das keimungsphysiologische Verhalten von Phleum pratense, L.,
dem Timotheegras. Jahrb. Botanik, vol. 78, pt. i, pp. 1-42.
9. MoLiscH, Hans
1930. Pflanzenphysiologie als Theorie der Gartnerei. P. 323.
10. Nathammer, Ann.
1927. Keimungphysiologische Studien unter Hervorhebung des Licht-
keimungsproblems. Biochem. Zeitschr., vol. 185, pp. 205-215.
11. Shuck, A. L.
1935. Light as a factor influencing the dormancy of lettuce seeds. Plant
Phys., vol. 10, pp. 193-196.