• TABLE OF CONTENTS
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 Historic note
 Title Page
 Credits
 Table of Contents
 Introduction
 Materials and methods
 Relation of temperature to...
 Relation of hydrogen-ion concentration...
 Relation of light to germinati...
 General discussion
 Summary and conclusions
 Literature cited














Group Title: Bulletin - University of Florida Agricultural Experiment Station ; 277
Title: Effects of certain environmental factors on germination of Florida cigar-wrapper tobacco seeds
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00026849/00001
 Material Information
Title: Effects of certain environmental factors on germination of Florida cigar-wrapper tobacco seeds
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 47 p. : charts ; 23 cm.
Language: English
Creator: Kincaid, Randall R ( Randall Rich ), 1903-
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1935
 Subjects
Subject: Tobacco -- Seeds   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 43-47.
Statement of Responsibility: Randall R. Kincaid.
General Note: Cover title.
General Note: Originally presented as: Thesis (Ph.D.)--University of Missouri.
Funding: Bulletin (University of Florida. Agricultural Experiment Station) ;
 Record Information
Bibliographic ID: UF00026849
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000924342
oclc - 18207286
notis - AEN4960

Table of Contents
    Historic note
        Unnumbered ( 1 )
    Title Page
        Title Page
    Credits
        Unnumbered ( 3 )
    Table of Contents
        Table of Contents
    Introduction
        Page 5
    Materials and methods
        Page 6
    Relation of temperature to germination
        Page 7
        Page 8
        Page 9
    Relation of hydrogen-ion concentration to germination
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Relation of light to germination
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
    General discussion
        Page 39
        Page 40
    Summary and conclusions
        Page 41
        Page 42
    Literature cited
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
Full Text





HISTORIC NOTE


The publications in this collection do
not reflect current scientific knowledge
or recommendations. These texts
represent the historic publishing
record of the Institute for Food and
Agricultural Sciences and should be
used only to trace the historic work of
the Institute and its staff. Current IFAS
research may be found on the
Electronic Data Information Source
(EDIS)

site maintained by the Florida
Cooperative Extension Service.






Copyright 2005, Board of Trustees, University
of Florida





Bulletin 277


UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA
WILMON NEWELL, Director





EFFECTS OF

CERTAIN ENVIRONMENTAL FACTORS

ON GERMINATION OF FLORIDA

CIGAR-WRAPPER TOBACCO SEEDS



RANDALL R. KINCAID






TECHNICAL BULLETIN









Bulletins will be sent free to Florida residents upon application to
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA


May, 1935









EXECUTIVE STAFF

John J. Tigert, M.A., LL.D., President of the
University
Wilmon Newell, D.Sc., Director
H. Harold Hume, M.S., Asst. Dir., Research
Harold Mowry, M.S.A., Asst. Dir., Adm.
J. Francis Cooper, M.S.A., Editor
Clyde Beale, A.B.J., Assistant Editor
Jefferson Thomas, Assistant Editor
Ida Keeling Cresap, Librarian
Ruby Newhall, Administrative Manager
K. H. Graham, Business Manager
Rachel McQuarrie, Accountant

MAIN STATION, GAINESVILLE

AGRONOMY
W. E. Stokes, M.S., Agronomist**
W. A. Leukel, Ph.D., Agronomist
G. E Ritchey, M.S.A., Associate*
Fred H. Hull, Ph.D., Associate
W. A. Carver, Ph.D., Associate
John P. Camp, M.S., Assistant

ANIMAL HUSBANDRY
A. L. Shealy, D.V.M., Animal Husbandman**
R. B. Becker, Ph.D., Dairy Husbandman
W. M. Neal, Ph.D., Associate in Animal
Nutrition
D. A. Sanders, D.V.M., Veterinarian
M. W. Emmel, D.V.M., Asst. Veterinarian
W. W. Henley, B.S.A., Assistant Animal
Husbandman
P. T. Dix Arnold, B.S.A., Assistant Dairy
Husbandman

CHEMISTRY AND SOILS
R. W. Ruprecht, Ph.D., Chemist**
R. M. Barnette, Ph.D., Chemist
C. E. Bell, Ph.D., Associate
B. B. French, Ph.D., Associate
H. W. Winsor, B.S.A., Assistant
H. W. Jones, M.S., Assistant

ECONOMICS, AGRICULTURAL
C. V. Noble. Ph.D., Agricultural Economist**
Bruce McKinley, A.B., B.S.A., Associate
Zach Savage, M.S.A., Associate
A. H. Spurlock. M.S.A., Assistant

ECONOMICS, HOME
Ouida Davis Abbott, Ph.D., Specialist**
L. W. Gaddum, Ph.D., Biochemist
C. F. Ahmann, Ph.D., Physiologist
J. T. Hall, Jr., B.S.Ch.E., Asst. Physiologist
ENTOMOLOGY
J. R. Watson, A.M., Entomologist**
A. N. Tissot, Ph.D., Associate
H. E. Bratley, M.S.A., Assistant
J. W. Kea, B.S.A., Assistant
HORTICULTURE
A. F. Camp, Ph.D., Horticulturist**
G. H. Blackmon, M.S.A., Horticulturist
A. L. Stahl, Ph.D., Associate
F. S. Jamison, Ph.D., Truck Horticulturist
R. J. Wilmot, M.S.A., Specialist, Fumigation
Research
R. D. Dickey, B.S.A., Assistant Horticulturist
PLANT PATHOLOGY
W. B. Tisdale. Ph.D., Plant Pathologist**
George F. Weber, Ph.D., Plant Pathologist
R. K. Voorhees, M.S., Assistant
Erdman West, M.S., Mycologist
Lillian E. Arnold, M.S., Assistant Botanist

In cooperation with U.S.D.A.
** Head of Department.


BOARD OF CONTROL

Geo. H Baldwin, Chairman, Jacksonville
A. H. Blanding, Bartow
A. H. Waeg, West Palm Beach
Oliver J. Semmes, Pensacola
Harry C. Duncan, Tavares
J. T. Diamond, Secretary, Tallahassee

BRANCH STATIONS

NORTH FLORIDA STATION, QUINCY
L. O. Gratz, Ph.D., Plant Pathologist in
Charge
R. R. Kincaid, Ph.D., Asso. Plant Pathologist
J. D. Warner, M.S., Agronomist
R. M. Crown, B.S.A., Asst. Agronomist
Jesse Reeves, Farm Superintendent

CITRUS STATION, LAKE ALFRED
John H. Jefferies, Superintendent
Geo. D. Ruehle, Ph.D., Associate Plant
Pathologist
W. A. Kuntz, A.M., Assoc. Plant Pathologist
B. R. Fudge, Ph.D., Associate Chemist
W. L. Thompson, B.S., Asat. Entomologist

EVERGLADES STATION, BELLE GLADE
A. Daane, Ph.D., Agronomist in Charge
R. N. Lobdell, M.S., Entomologist
F. D. Stevens, B.S., Sugarcane Agronomist
G. R. Townsend, Ph.D.. Assistant Plant
Pathologist
J. R. Neller, Ph.D., Biochemist
R. W. Kidder, B.S., Assistant Animal
Husbandman
Ross E. Robertson, B.S., Assistant Chemist

SUB-TROPICAL STATION, HOMESTEAD
H. S. Wolfe, Ph.D., Horticulturist in Charge
W. M. Fifield, M.S., Asst. Horticulturist
Stacy O. Hawkins, M.A., Assistant Plant
Pathologist

WEST CENTRAL FLORIDA STATION,
BROOKSVILLE
E. W. Sheets, D.Agri., Animal Husbandman
in Charge*
W. F. Ward, M.S.A., Asst. An. Husbandman*

FIELD STATIONS

Leesburg
M. N. Walker, Ph.D., Plant Pathologist in
Charge
W. B. Shippy, Ph.D,. Asso. Plant Pathologist
K. W. Loucks, M.S., Asst. Plant Pathologist
J. W. Wilson, Ph.D., Associate Entomologist
C. C. Goff, M.S., Assistant Entomologist
Plant City
A. N. Brooks, Ph.D., Plant Pathologist
R. E. Nolen, M.S.A., Asst. Plant Pathologist
Cocoa
A. S. Rhoads, Ph.D., Plant Pathologist
Hastings
A. H. Eddins, Ph.D., Plant Pathologist
Monticello
G. B. Fairchild, M.S., Assistant Entomologist
Bradenton
David G. Kelbert, Asst. Plant Pathologist
Sanford
E. R. Purvis, Ph.D., Assistant Chemist,
Celery Investigations













CONTENTS
PAGE
INTRODUCTION ... ---...------....-................ 5
EXPERIMENTAL WORK.................. ......... ....... ... .... ................ 5
Materials and methods .................. ......... ................. 6
Relation of temperature to germination....... ............. ............ 7
Summary of previous work................. ........ ............. 7
Cardinal temperatures ...-........ ...... ............... 7
Alternating temperatures ................... ........ .................-......... 8
Relation of hydrogen-ion concentration to germination-The apparent
isoelectric point ............................ .............. 10
Summary of previous work.................... ................... 10
Water absorption method..................................... 10
Ion adsorption method...... ........-...................... 15
Germination at various pH values.................................... 15
Relation of light to germination.......................... .......... 17
Summary of previous work .....-............ ............ ........ 17
Light requirement of Florida-grown tobacco seeds-.............-................ 20
Tests of various samples in light and in darkness....-..................-- .. 20
Effect of short exposure to direct sunlight........-..............-- ..... 21
Relation of amount of light to germination. .............. ................- 22
Water in relation to the light effect................. ................ 24
Effect of soaking on sensitiveness to light.............................. 24
Sensitiveness of air-dry seeds .......................... ............. 25
Influence of subsequent drying on the light effect.. ...................... 27
Wave-length and energy value of light...................................... 28
Amount of total and visible radiation required........................ 29
Effect of visible radiation only......................... ... ................ 29
Effect of wave-lengths strongly absorbed by seed coats-. ............... 29
Effect of moonlight on germination..................--- .......... 31
Temperature in relation to the light effect................. ........ 31
Effect of alternating temperatures and pressures..........................-. 31
Effect of light on the response to temperature....................... 32
Influence of temperature on the light effect-- -............................... 33
Depth of sowing and germination.................................. 37
GENERAL DISCUSSION ...... .... ..........-........... .-..... 39
SUMMARY AND CONCLUSIONS........... ........................... .. 41
LITERATURE CITED ..................................... .......43








THE EFFECTS OF CERTAIN ENVIRONMENTAL
FACTORS ON THE GERMINATION OF FLORIDA
CIGAR-WRAPPER TOBACCO SEEDS'
By RANDALL R. KINCAID

The study of the factors affecting the germination of tobacco
seeds is interesting from a physiological standpoint because the
seeds, being very small, contain only a small amount of reserve
material, and because they are generally classified among the
seeds which require light for germination. Literature on the
various phases of this subject is extensive, and reports concern-
ing the response of seeds of various types of tobacco to environ-
mental factors are often conflicting.
Therefore, it seemed desirable to study the effects of certain
environmental factors on the germination of Florida cigar-
wrapper tobacco seeds. These studies are intended to serve as
a basis for further work on the physiology and pathology of
cigar-wrapper tobacco varieties grown mainly in Gadsden County,
Florida.
Experimental work on the relation of temperature, hydrogen-
ion concentration and light to germination is reported in this
paper. The more important contributions from the literature
are summarized by way of introduction to the report of experi-
mental work on each of these factors.

EXPERIMENTAL WORK
Experiments on the effects of certain environmental factors on
the germination of Florida cigar-wrapper tobacco seeds were
begun at the University of Missouri during the academic year
1931-1932. The major portion of the work was conducted at the
North Florida Experiment Station during the period from Sep-
tember, 1932, to April, 1934.
The environmental factors investigated in relation to germina-
tion were temperature, hydrogen-ion concentration and light.
Water, wave-length and energy value of light, and temperature

1Thesis submitted to the faculty of the Graduate School of the University
of Missouri in partial fulfillment of the requirements for the degree of
Doctor of Philosophy. (Abridged for publication). The advice and en-
couragement given by Dr. W. J. Robbins and others of the University of
Missouri are hereby gratefully acknowledged. Thanks are due to Dr. L. 0.
Gratz and Dr. W. B. Tisdale of the University of Florida Agricultural
Experiment Station for helpful direction and for assistance in the prepara-
tion of the manuscript.





Florida Agricultural Experiment Station


were considered in relation to the light effect. Results of these
experiments are reported below.
MATERIALS AND METHODS
Seeds of tobacco, Nicotiana tabacum L., were used in all ex-
periments and, unless otherwise noted, were of variety No. 301,
developed at the North Florida Experiment Station for resistance
to the blackshank disease (63)2. Seed plants were self-pollinated
for several successive generations, and the seeds used were there-
fore probably a pure line.
The seed plants were grown under a cheesecloth shade, and
the seeds were produced under paper bags to prevent cross-
pollination. A sample of seeds harvested in August, 1931, was
used from November, 1931, to January, 1933, after which time
seeds of the same variety grown in 1932 were used. Both sam-
ples were cleaned with an air-blast apparatus, leaving very little
inert material, and stored in paper envelopes in the laboratory
until needed.
The average color of a mass of these seeds was Brussels brown
(58); many of the individual seeds were considerably lighter
or darker than this.
The seeds were generally counted by means of a mechanical
counter, which distributed them uniformly on the substrate.
Tests were made in three or more replications, 100 seeds per
replication. Petri dishes 90 millimeters in diameter were fitted
with two circular sheets of filter paper and the seeds were placed
on these. The paper was saturated with tap water, leaving only
a small excess, so that the water would not greatly disturb the
position of the seeds when the dishes were handled. Water was
added as necessary during incubation to keep the paper satu-
rated, and the dishes usually were wrapped in moist cloth or
enclosed in moist chambers to keep the seeds and filter paper
from drying out too rapidly.
Temperatures were taken in air near the dishes, using a
recording thermometer, or a laboratory thermometer which
generally rested in a small vessel of water. Room temperatures
between 18 and 2553 were used unless otherwise noted.
The seeds were counted and sowed in diffuse daylight or in
strong artificial light, and then placed in the incubator within
about an hour. For tests of germination in darkness, the seeds
2Reference is made by number (italic) in parenthesis to "Literature
Cited" in the back of this bulletin.
3All temperatures mentioned in this paper are stated in degrees Centi-
grade.





Environmental Factors Affecting Tobacco Germination 7

were incubated in light-proof wooden boxes or metal cans, which
were opened only in a photographic dark room or other situation
where light was practically excluded. The efficiency of the
method was checked repeatedly by means of controls not other-
wise exposed to light.
In experiments where final counts only were made, the seed-
lings were counted if the radicles had reached a length of about
2 millimeters. Whenever successive counts were made, the
seedlings with cotyledons free from the seed coats were counted
and removed from the dish. Observations on experiments in
light were continued at intervals of one or more days until the
percentage of germination had reached a maximum. Observa-
tions on experiments in darkness were made after from 10 to 14
days of incubation.
RELATION OF TEMPERATURE TO GERMINATION
SUMMARY OF PREVIOUS WORK
Considerable differences of opinion are expressed in the litera-
ture in regard to cardinal temperatures for the germination of
tobacco seeds. The minimum is 150 or lower, the optimum be-
tween 240 and 320, and the maximum between 320 and 400 (1,
11, 21, 29, 55). Differences in the temperature requirements
of various samples, associated with their light requirements,
have been reported (1, 26).
The influence of alternating temperatures has received little
attention, except in connection with attempts to make normally
light-requiring seeds germinate in darkness. For routine testing
of seeds in light, daily alternations, such as between 20 and
30, are commonly used (2, 55).
The experiments reported below served to establish the car-
dinal temperatures for the samples used, and also to determine
the effect of certain daily alternations of temperature on ger-
mination.
CARDINAL TEMPERATURES
Several experiments were conducted to determine the cardinal
temperatures for germination, which may be defined as follows:
Minimum, the lowest temperature at which any germination
occurs; optimum, the temperature at which the highest final
percentage of germination occurs within the shortest average
time; maximum, the highest temperature at which any germi-
nation occurs. The seeds were incubated at various controlled
temperatures, with occasional exposure to light. The tempera-
tures, which were observed twice daily, seldom fluctuated more






Florida Agricultural Experiment Station


than one degree above or below the average temperatures re-
ported. The averages of triplicate determinations and the mean
number of days required for germination are reported.
Table 1 shows the results of three consecutive trials, in which
the usual temperature intervals were from 3 to 4 degrees. These
results served to establish the cardinal temperatures with an
accuracy of perhaps 1 degree, as follows: Minimum (for about
50 days of incubation), 100; optimum, 240; and maximum, 34.
The temperature of incubation in subsequent experiments, on
the relation of other factors to germination, was optimal or
slightly lower. The effect of light on the response of seeds to
temperature will be considered in connection with the relation
of temperature to the light effect.
TABLE 1.-GERMINATION OF TOBACCO SEEDS IN LIGHT AT VARIOUS
CONSTANT TEMPERATURES.
Mean Total
Temperature Germination Incubation Period of
Trial No. Period Observation
C. % days days
1 242 73 7 17
28 38 8 17
S33 3 8 17
36% | 0 17
2 3 Oa 53
9 79 50 53
13 88 30 32
17 84 11 16
20 82 8 16
23 85 7 16
251/2 81 5 16
29 59 8 23
3 101 85b 28+ 28
16 70 13 18
20 77 8 15
24/2 84 6 15
28 46 7 18
31 8 7 15
33%1/ 3 8 15
_36 0 15
a Subsequently at room temperature, 90 percent.
'bMany had radicles only 1 millimeter long.

ALTERNATING TEMPERATURES
Experiments were conducted to determine the effect of certain
daily alternations of temperatures on germination in light. The
seeds were moved from one incubator to another of different
temperature at about 9 A. M. and back at about 5 P.M. each
day, and were thus exposed to light twice daily.






Environmental Factors Affecting Tobacco Germination 9

The results given in Table 2 show that alternations between
temperatures slightly below with temperatures slightly above
the optimum give a little more rapid germination than the
optimal constant temperature. Temperatures slightly above the
maximal constant temperature (34) in alternation with tem-
peratures at or below the optimum (240) give good germination
with little or no delay. Temperatures near or below the minimum
(100) for a part of each day increase the time required for
germination, but the final percentage is about the same as at
the optimum.
TABLE 2.-GERMINATION OF TOBACCO SEEDS IN LIGHT AT ALTERNATING
TEMPERATURES.
Temperatures
|I Mean
Trial No. Day Night Germination Incubation
8 hours 16 hours Period
C. CCC. % Days

1 24/% 241/ 73 7
2442 281/2 76 6
241 33 68 7
24% 361/2 68 9
24 40 52 17
2 24%1 2412 73 7
28 2412 75 6
33% 24 1/ 76 6
36% 24% I 79 7
40 24% 71 9
3 20 20 72 10
20 24 69 6
20 28 72 6
20 30% 66 6
20 34 69 7
4 20 20 72 10
24 20 76 8
28 20 74 7.
301/2 20 76 7
34 20 67 8
38 20 65 10
43 20 60 10
5 3 29 87 8
9 29 86 8
13 29 91 7
17 29 84 6
29 29 59 8
6 29 3 83 14
29 9 82 13
29 13 85 10
29 17 90 7
29 29 59 8
__________________________1____





Florida Agricultural Experiment Station


RELATION OF HYDROGEN-ION CONCENTRATION TO GERMINATION
-THE APPARENT ISOELECTRIC POINT
SUMMARY OF PREVIOUS WORK
Some samples of tobacco seeds germinate well in media having
pH values as low as 4.5 (11, 31), while for others pH 5.4 or more
acid has been found unfavorable (51). The alkaline limit is near
pH 9.0 (31).
There is considerable literature on the effect of hydrogen-ion
concentration on the growth of organisms, and on the absorption
of water and solutes by certain plant tissues. In many instances,
the workers obtained bimodal curves when they plotted these
functions against the pH of the solution. The reaction at the
minimum between two maxima has been interpreted by Robbins
(59) and others to represent the isoelectric point of the organism
or tissue.
Several investigators have reported results which indicate
that tobacco seeds and seedlings have an apparent isoelectric
point at a reaction near neutrality. Tobacco plants grown in a
neutral medium give a lower yield than those grown at pH 5.5,
6.1, 7.8, or 8.7 (38). Two maxima in the growth curves of small
seedlings have been observed, with a median minimum at about
pH 6.8 (31). It has been reported that neutral red, a basic dye,
stained the cells of tobacco seedlings only at reactions in the
neighborhood of neutrality, never at reactions more acid than
pH 5.5 (20).
Experiments were conducted by three methods to test this
relation. In the first method, the amount of water absorbed by
the seeds from solutions at different hydrogen-ion concentrations
was determined; the reaction at which a minimal value occurred
between two maxima was interpreted as the apparent isoelectric
point. In the second method, seeds were placed in acidified or
alkalized solutions of neutral salts; the reactions of both the
acidified and alkalized solutions shifted toward a certain pH
value, which was interpreted as the apparent isoelectric point.
In the third method, the seeds were sowed on soil, and the re-
action at which germination was slower than at reactions either
more acid or less acid was interpreted as the apparent isoelectric
point. The results of these three methods were found to be in
fair agreement, as shown by the experiments reported below.

WATER ABSORPTION METHOD
Lots of tobacco seeds weighing 1.000 gram were tied up in
bags of organdy cloth, and again weighed accurately. The bags






Environmental Factors Affecting Tobacco Germination 11

of seeds were soaked in dilute buffer mixtures, centrifuged to
remove excess free water, and then weighed accurately to de-
termine the increase in weight.
The weight of the cloth used in making each bag was about
0.2 gram. Rolls of cloth only, weighing about 1.2 grams each,
were used in one trial instead of bags of seeds. Each roll was
soaked and weighed three times, using buffers of three different
pH values. The three weights agreed closely among themselves
in all instances. Therefore, it was concluded that the differences
observed in the weights of the bags of seeds were not due to
any effect of the reaction on the amount of water retained by
the cloth.
Three bags of seeds were placed in a 40 cubic centimeter Erlen-
meyer flask, and a sufficient amount, about 25 cubic centimeters,
of the buffer mixture was added to cover the bags. The solution
was decanted and fresh solution was added about every hour,
except during the overnight period. After a certain period of
soaking, one bag of seeds from each buffer was removed, cen-
trifuged and placed in a moist chamber until the weighing were
made. After a second bag had been removed from each buffer,
the first was replaced and so on. This procedure was repeated
four times with each lot of seeds.
The buffers were mixtures of 0.02 molar NaH.PO4 and of 0.02
molar Na2HP04. A concentration of 0.02 molar osmoticc pres-
sure about 1.1 atmospheres) allowed good germination of the
seeds; 0.1 molar (about 5 atmospheres) was too concentrated
for germination. Mihailovici (47) reported that the maximal
osmotic pressure of solutions in which tobacco seeds would ger-
minate varied for different samples of seeds from 4.7 to about
12 atmospheres.
The final reaction of the buffers was determined frequently,
using samples of liquid drawn at about the time the seeds were
taken from the buffers. The average of the final readings on
a particular buffer mixture, which seldom varied more than 0.05
pH, was used in the graphs and calculations.
Two difficulties were encountered which caused considerable
error in the results. In the first place, there was some variation
in the amount of water lost by evaporation during the cen-
trifuging and weighing; however, the bags were taken at random
for each weighing. In the second place, the production of carbon
dioxide by the seeds made the reaction of the solution within
the bags more acid than the solution outside the bags, except






Florida Agricultural Experiment Station


possibly in the most acid solutions; this difficulty was partly
overcome in the second trial by occasional shaking of the flasks.
First Trial. Twenty-seven 1-gram samples of a mixed lot of
1932 seeds were used. The final pH values of the nine buffers
ranged from 6.02 to 7.20. The buffers were not stirred except
by the removing and replacing of the bags.
The first set of nine bags, one from each buffer, was weighed
after about 2, 4, 6 and 24 hours of soaking; the second set, after
about 21/2, 41/2, 61/2, and 241/2 hours; and the third set, after
about 3, 5, 7 and 25 hours. The weights for each set are reported
separately in Table 3. Each series of nine weights, if plotted
against the pH of the buffer, would produce a distinctly bimodal
curve. The weight in each series which is considered to represent
the first maximum is marked by a superscript "a"; the median
minimum, by a superscript "b"; and the second maximum, by
a superscript "c".
TABLE 3.-INCREASE IN WEIGHT OF 1-GRAM LOTS OF TOBACCO SEEDS
SOAKED IN BUFFER MIXTURES.
pH of Buffers
Initial......... ..... | 6.01 6.49 6.67 I 6.76 1 6.86 6.95 7.05 7.15 7.29
Final ................ 6.02 6.45 6.64 6.73 6.83 6.91 7.00 7.09 7.20
Time of
Soaking Increase in Weight (milligrams)
(hours)
2-...................... 405 410 407 411 424 426a 396b 411 420e
4....................... 467 471 464 465 476 477a 445b 473 488c
6.................... 467 477 481 481 490a 487 469b 477 484
24...... ................ 478 483 478 491 502a 494 478b 486 487c
2%2................... 425 427 425 428a 421 413b 428 426 424
41... .......... 461 467a[ 465 464 456bl 460 468e 468C[ 460
61/2................. 476 472 477a 467 468 466b 474 480c) 475
241/2 ............. 491 490 494 499al 492 I 489bl 499c 498 1 488
3- ...................... 461 464 476a 452b1 459 481c 463 465 457
5........- ........ 468 475 484a 475 | 469bl 485e 480 485el 464
7......................... 471 476 486a 473bl 475 ] 488 489Ce 489c 465
25... .......... ........... 509 508 532a( 502bi 5161 532e 526 526 505
aFirst maximum
bMedian minimum
c Second maximum
The first and second maxima and the median minima in Table
3 show a regression toward more acid points in each set of
weighing. For example, the average final pH value at which
the median minimum occurred was 7.00 in the first set of weigh-
ings, 6.89 in the second set, and 6.76 in the third. Since the
buffers were not stirred except by the removing and replacing
of the bags of seeds, the reactions prevailing among the seeds





Environmental Factors Affecting Tobacco Germination 13

were probably more acid than those on the outside of the bags,
as explained above. By the time when the third set of bags
was removed, the pH was probably about the same throughout.
The weights of each set of bags became somewhat greater at
each successive weighing, in spite of the loss by centrifuging and
drying while the bags were out of the buffers. However, it is
of interest to note that the differences between the maximal
and minimal weights were of about the same magnitude at each
weighing.
Second Trial. Thirty samples of seeds of variety No. 94 (63)
were used and a distilled water check was included with the
series of nine buffers. The solutions were changed about every
hour, and stirred once or twice between changes; a shaking
machine was not available. In one series of pH determinations,
samples of the buffers were centrifuged from the bags of seeds;
these showed about the same reactions as the samples taken
from outside the bags.


435


' 434


" 431

*1430
(D
1429
0
15 428


o.u0 ..3 .71 .79 .03 .*7 7.5 7."
Final pH of buffers
Fig. 1.-Average increase in weight of 1-gram lots of tobacco seeds
soaked in buffer mixtures.

The first set of 10 bags was weighed after about 1, 4, 6 and
10 hours of soaking; the second set, after about 11/4, 41/, 61/,
and 101 hours; and the third set, after about 11/2, 41/2, 61/
and 101/2 hours. The weights for each set are reported sepa-
rately in Table 4. The weights which are considered to represent
the first maximum, median minimum and second maximum, are
indicated by superscript letters as in the preceding table. Al-


0 a --
~z~z--^^
x __sz-




\ -r.l t ci^ rc T1






Florida Agricultural Experiment Station


though there was considerable variation in the pH values at
which the maxima and minima occurred, there was no regular
regression. The 12 weights for each pH value were averaged
as shown in the table, and plotted against the final reaction of
the buffers in Fig. 1.
The average results of the 12 series of weights show a distinct
minimum at final pH 6.79, between maxima at 6.43 and 7.18,
the latter pH value being the most alkaline of the series. At
still higher pH values the weights would probably have decreased,
as in the second and third weighing of the first trial. The
magnitude of the differences between maximal and minimal
average weights was rather small, because several of the aver-
ages included some weights which were at or near the maximum
and others which were at or near the minimum.
TABLE 4.-INCREASE IN WEIGHT OF 1-GRAM LOTS OF TOBACCO SEEDS SOAKED
IN BUFFER MIXTURES.
pH of Buffers
Initial........ 6.01 6.49 6.67 6.76 6.86 6.95 7.05 7.15 7.29 1HO
Final.......... 6.00 6.43 6.63 6.71 6.79 6.89 6.97 7.05 7.18 6.76
Time of
Soaking Increase in Weight (milligrams)
(hours)

1 327 334a 334a 325b 325b 332 334 327 335c 331
4 440 455a 443 442 441b 445 450 444 458C 444
6 444 459a 453 449b 449b 457 461 458 463c 453
10 447 461a 454 448b 451 455 460 456 465e 447
114 356 352 353 357a 353 357a 350b 358c 357 355
4% 458 452 454 457a 456 457a 450b 455 457c 446
61/4 481 471 474 475 476 477a 474b 476 480c 462
101% 466 467 465 466 470a 465 460b 462 4680 447
11 373 375 381a 376 375 368b 378 382c 378 379
4V2 449 447 448 451a 446 441b 444 457c 447 438
6% 450 454 455a 454 454b 454 457 469c 458 450
10% 449 453 454 462a 451b 456 455 469c 454 445

Averages 428.3 431.7 430.71 430.2 428.9 430.3 431.1 434.4 435.0 424.8

aFirst maximum
b Median minimum
c Second maximum

Summarizing the results in another way, the average reaction
at which the median minimum occurred in the 12 trials was
pH 6.85, and the maxima at 6.65 and at 7.13 or slightly more
alkaline.
It was concluded that, under the conditions of these experi-
ments, and within the range of reactions investigated, the





Environmental Factors Affecting Tobacco Germination 15

tobacco seeds show a minimum in water absorption at about
pH 6.8, an acid maximum at about 6.6, and an alkaline maximum
at 7.1 or slightly more alkaline.
The increase in weight of the seeds was attributed mainly to
the absorption of water. The amount and kind of ions absorbed
or adsorbed from the buffers were not investigated. It is inter-
esting to note that the increase in weight at the median minimum
in each series of weights was approximately equal to that in
distilled water, the final reaction of which was near the observed
isoelectric point of the seeds.
ION ADSORPTION METHOD
Attempts were made to determine the apparent isoelectric
point of seeds and seedlings by observing the change in hydrogen-
ion concentration when they were added to dilute salt solutions.
The method was generally as follows: One gram of air-dry
seeds or equivalent amount of seedlings was mixed with 10 cubic
centimeters of 1.0 normal or 0.1 normal NaC1 solution, the re-
action of which had been adjusted to pH 5 to 6 or to pH 7.5 to
8 by the addition of HCI or NaOH. After suitable intervals,
samples of the solutions were withdrawn for the pH determina-
tion, or quinhydrone was added and successive readings taken
directly on the entire volume of solution.
The pH of the acid solutions generally rose rapidly and reached
a value of from 6.6 to 6.8 within from 1 to 10 minutes, then
slowly declined to about 6.4. The pH of the alkaline solutions
fell rapidly to about 7.0 or 6.8 and then more slowly to about 6.4.
The gradual decrease in the pH value was attributed to the
production of carbon dioxide by the seeds. Seeds which had
been pressed to remove the oil, and thereby killed, showed a
slower change. When a solution of sodium sulfate was used,
and the samples withdrawn and heated in a boiling water bath
to expel the carbon dioxide, the pH was generally between 6.7
and 7.0.
The reaction toward which both the acidified and alkalized
solutions shifted at the beginning of each trial was slightly on
the acid side of neutrality.
GERMINATION AT VARIOUS pH VALUES
An experiment was conducted to determine whether the re-
action at which water absorption was at a minimum would also
give a slower rate of germination than at reactions more acid
or less acid. The seeds were germinated on soil, under approxi-





Florida Agricultural Experiment Station


mately normal conditions of moisture and aeration. The amount
of light was sufficient for germination, but much less than that
under field conditions.
Finely sifted soil, having a pH value of 5.4, was acidified with
sulfuric and phosphoric acids to pH 3.9. A similar lot of soil
was alkalized with potassium, calcium and magnesium hydrox-
ides to pH 8.0. These two lots of soil were mixed together in
various proportions to give soils of intermediate pH value.
About 20 cubic centimeters of dry soil were placed in each petri
dish, and 100 seeds were sowed on the top of the soil. When
water was added, some of the seeds were covered with soil to
a depth of perhaps 1 millimeter. The cultures were incubated
at room temperature for 9 days, with diffuse light admitted to
the seeds from one side only. The pH value of the soil was
determined on dried samples by the quinhydrone method 2 days
before and 4 days after the period of incubation. One part by
weight of soil was mixed with two parts of water, and readings
were made with the soil in suspension. However, the suspension
effect was probably quite small in the sandy soil used. The
initial, final and average pH values of the soil, and the average
germination of three trials at each reaction, are given in Table 5.
TABLE 5.-GERMINATION OF TOBACCO SEEDS ON SOIL AT VARIOUS
PH VALUES.
pH Value of Soil Percentage
__ of Remarks
Initial Final Average Germination
3.95 4.2 4.1 73 Seedlings normal
4.15 4.45 4.3 76
4.65 5.1 4.9 70
5.1 5.5 5.3 68
5.55 6.05 5.8 54
6.05 6.45 i 6.25 49 "
6.4 6.75 6.6 60
6.5 6.75 6.65 53
6.65 6.95 6.8 42 "
6.85 7.2 7.05 47
7.35 7.6 7.5 50 Few root hairs
7.7 7.8 7.75 63 "
7.95 8.0 8.0 68 "

The percentage of germination was lowest at reactions aver-
aging pH 6.8. Germination was fair throughout the range of
reactions used, pH 4 to 8. At reactions from pH 7.5 to 8.0 the
roots were abnormal, with few root hairs or none. Evidently
the seeds were able to absorb water and germinate, but the
medium was unfavorable for the development of root hairs.
The method of pH adjustment used produced an abnormally
high concentration of electrolytes in the soil solution. In order






Environmental Factors Affecting Tobacco Germination 17

to avoid this, it would have been better to adjust the reaction
of the soil by additions of hydrogen-clay and of calcium-clay or
potassium-clay.
From the experiments reported above, and others mentioned
in the review of literature, it was concluded in agreement with
the suggestion of Katchioni-Walther (31), that the tobacco
seeds and seedlings investigated show an apparent isoelectric
point at about pH 6.8. This reaction is more alkaline than the
isoelectric point reported by Vickery and coworkers (65) for the
globulin isolated from tobacco seeds, about pH 5.4.
The tap water used to moisten the seeds and filter paper in
all other germination experiments had a pH value of about 7.3.
This medium was evidently satisfactory, because high percent-
ages of germination and normal seedlings were obtained in the
controls of all experiments.
RELATION OF LIGHT TO GERMINATION
SUMMARY OF PREVIOUS WORK
There is considerable disagreement in the literature as to
whether tobacco seeds require light for germination. Many
workers have found samples of tobacco seeds which germinated
to much higher percentages in light than in darkness (1, 8, 15,
23, 24, 27, 29, 33, 34, 37, 56, 66). On the other hand, other
workers have reported samples which germinated about as well
in darkness as in light (7, 16, 18, 19): however, some of these
results, at least, can be attributed to short periods of illumination
during examination of the seeds (42, 66). Since workers with
large numbers of samples (1, 23, 24) have observed all grada-
tions of germination in darkness from excellent to none, there
must exist considerable differences in the light requirements
of tobacco seeds.
It has been reported that certain samples of tobacco seeds
pass through a stage of after-ripening, during which light
and sharp daily alternations of temperature are required for
germination (53); afterwards, they actually germinate better
in darkness (54).
Seeds must imbibe water in order to be made sensitive to light.
The period of soaking4 required varies with different samples
and with the strength of the light to which the seeds are ex-
posed, from 1 hour or less to 72 hours or more (66). Sensitive-
ness increases to a maximum after a certain period of soaking
4Inaccurately called "presoaking" by various authors.






Florida Agricultural Experiment Station


and afterwards decreases, so that more light is required to in-
duce germination (3). Maximal sensitiveness to light is reached
at about the same time as that at which the maximal amount
of water is absorbed (34).
The period of exposure may be as short as 1 second (66),
and the effect of light depends on the product of the intensity
and the duration of exposure, through a considerable range of
intensities and periods of exposure (3). The effectiveness of
light of a given energy value varies directly with the wave-
length, except for a slight deviation in the green region, which
is most strongly absorbed by the seed coats (35).
A photochemical theory of the light effect has been proposed
(35), according to which the resulting germination is propor-
tional to the number of quanta which pass through the seed
coats and act upon the seed contents. The conclusion, that light
acts directly on the contents of the seeds and not on the seed
coats, is supported also by the results of genetical studies (24-
26), and of chemical treatments applied to the seed coats (15,
26). On the other hand, the germination of normally light-
requiring seeds in darkness after the removal of the seed coats
(3), or after treatment of the seeds with solutions of potassium
nitrate (1), has been reported; these observations indicate that
the seed coats may possibly have some part in the light effect.
The effect of light on seeds is conditioned not only by the
supply of water and the characteristics of the light used, as
mentioned above, but also by the supply of oxygen. Seeds incu-
bated for a few days in a vacuum are not sensitive to light while
they remain in the vacuum, and the effect of exposure to light
under favorable conditions can possibly be reversed by subse-
quent storage in a vacuum (66). Increased partial pressure of
oxygen may also inhibit the effect of the usual amount of light
required to induce germination, but this inhibition can be over-
come by increasing the amount of light (5).
When seeds are incubated in darkness, the increase and sub-
sequent decrease in sensitiveness to light closely parallel the
rise and fall in the rate of respiration, suggesting a possible
close relationship between these two functions (34). When
fully imbibed seeds are exposed to light, the decrease in respira-
tion is immediately checked (34, 61).
Several theories of the nature of the light effect are based on
a supposed reducing effect of light, as follows: (a) The effect
of light and the effect of oxygen are opposed to each other,
because the inhibiting effect of prolonged soaking or of incuba-






Environmental Factors Affecting Tobacco Germination 19

tion in high concentrations of oxygen can be overcome by in-
creasing the amount of light (5). (b) There is an oxidation-
reduction equilibrium within the seeds, which is displaced by
light in one direction, and by oxygen in the other; sensitiveness
to light may be closely associated with the oxidation-reduction
potential, and with the rate of respiratory activity (34).
(c) Light activates specific substances, perhaps enzymes, by a
process of reduction (34); in this connection, the rate of respi-
ration has been observed to increase within about 2 hours after
exposure to light (34, 61), but attempts to demonstrate an im-
mediate effect of light on the activity of catalase and peroxidase
were not successful (61).
Genetical studies have shown that light-requirement is domi-
nant and light-indifference is recessive in some crosses, while
in others there is a maternal preponderance (24-26). Some
samples of light-requiring tobacco seeds gradually lose this re-
quirement during several years in storage (24-26), while others
do not (56). Tobacco seeds are generally sowed on the top of
the soil (62), but they may germinate under a 1-centimeter
covering of soil (7). They may remain viable when buried in
moist, fallow soil for 8 years or more (28).
Tobacco seeds which require light for germination at constant
temperature may be made to germinate in darkness by suitable
daily alternations of temperature (1, 26); this observation may
explain the germination of seeds under a thin covering of soil.
Seeds treated in water at 600 for a few seconds may germinate
better in darkness than untreated seeds (15). Incubation at
temperatures near the maximum has been found to improve
the germination of the light-requiring seeds of certain other
species (40), but not of tobacco seeds (1, 15).
Various other methods have been tried in attempts to increase
the germination of tobacco seeds in darkness. Negative results
have been reported for the use of various substrates, such as
soil, sand and peat (15, 56). Positive results have been reported
for the treatment of seeds with weak acids (14, 15), or with
solutions of ether or potassium nitrate (1); negative or variable
results have also been reported for the use of potassium nitrate,
ether, and various other substances (15, 26, 53). The effect
of light on the seeds of some species may be replaced by the
action of proteolytic enzymes (41).
The fact that the effect of light on seeds may be at least
partially replaced in many instances by various other agents
has led some to the opinion that there remains very little basis






Florida Agricultural Experiment Station
I


for the notion of a light requirement for germination (30, p.
131). Nevertheless, a brief exposure to light certainly does
initiate a remarkable response in imbibed seeds of tobacco and
several other species. Many of the conflicting and variable
results which have been reported in the literature are due,
without doubt, to the failure of the workers to appreciate the
vary small amount of light which may be effective, and there-
fore uncontrolled exposure to light may have been a factor in
the results.
The factors which condition the effect of light apparently
have not been clearly distinguished from those which condition
the subsequent stages of germination. Among the factors which
are known to condition the effect of light are the supply of water,
wave-length and energy value of light, and the supply of oxygen;
alternating temperatures and solutions of certain chemicals may
replace the effect of light in some instances. Temperature as
a conditioning factor deserves more attention.
Studies on the relation of light to the germination of Florida
cigar-wrapper tobacco seeds are reported below. Water, wave-
length and energy value of light, and temperature were consid-
ered in relation to the light effect.
LIGHT REQUIREMENT OF FLORIDA-GROWN TOBACCO SEEDS
The necessity of light for the germination of the seeds of
variety No. 301 was demonstrated in an early experiment, in
which only 1 percent of the seeds germinated at 23 in darkness
while 84 percent germinated in diffuse daylight. The seeds
which had been incubated in darkness had not lost their viability,
because they subsequently germinated in light 81 percent.
To determine the effect of longer periods of incubation on
germination in darkness and on subsequent germination in light,
seeds were incubated in darkness at 220 for 11, 20 and 30 days,
then transferred to light. The percentage of germination in
darkness was 1 percent or less. The same seeds germinated
subsequently in light 76 percent or more, showing that they
were still viable after 30 days of soaking in darkness.
Tests of Various Samples in Light and in Darkness. It was
hoped to find seeds which would germinate well in darkness,
for use as controls in the experimental work. Eighteen lots of
seeds, including most of the cigar-wrapper varieties and strains
grown commercially in Florida, were tested in light and in dark-
ness at 220. The results of tests on 100 seeds in light and 100
seeds in darkness are given in Table 6.






Environmental Factors Affecting Tobacco Germination 21

TABLE 6.-GERMINATION OF FLORIDA CIGAR-WRAPPER TOBACCO SEEDS IN
LIGHT AND IN DARKNESS.


Strain


73-3-3-1-1-1-.......-- .................
94-26...... ---.............................
135-2-3-2-2.............................
220-3-2-2.........................
226-2-2-1........ ............ ... .
261-4-1.......................... ...
271--1.................................-
942-3........................-...... .
942-20...............................-...
942-24 ................................ ..
944-12 .................... ............ .
944-18......... ............ ........
P 30-3-1............... ..... ............
R 29-2........ ...... .......- .-. ...
R 30-3...................................
Connecticut Round Tip............
Big Cuba.........................
Little Cuba........... ............ ... ..-


Germination
in Light

%
79
99
95
90
86
45
82
94
86
93
92
93
83
80
74
64
80
78


Germination


In
Darkness
%
0
0
0
2
1
0
6
0
0
0
0
0
2
1
2
0
2
1


Subsequently
in Light
%
85
96
91
89
84-
50
78
93
73
83
93
85
83



73


Every lot germinated poorly in darkness, and therefore the
further experiments had to be conducted without the benefit of
seeds which naturally germinated well in darkness.
Effect of Short Exposure to Direct Sunlight. As was demon-
strated by Wieser (66), the effective time of exposure to light
may be as short as 1 second. It was therefore of interest to
determine the shortest exposure to intense light (direct sun-
light at midday) which would induce germination. The seeds
were incubated in darkness for 8 days, exposed to light, and
returned to darkness for 14 days.
The exposures were made in a camera with lenses removed;
the aperture was 3.5 centimeters in diameter. The moist filter
papers bearing the seeds were removed from the petri dishes
and placed in plate holders, the transfers being made in a pho-
tographic dark room. Four plate holders at a time were prepared
for the camera and the seeds were exposed to direct sunlight
with the usual photographic precautions. Then the plate hold-
ers were taken back to the dark room, and the seeds returned
to the petri dishes. This procedure was followed four times, a
different plate holder being used each time for the exposure of
a given duration.
Controls were placed in the camera and the slide of the plate
holder was removed for 10 seconds; this served as a check on
the light leaking through the closed shutter. Four other dishes





Florida Agricultural Experiment Station


of seeds were exposed to full sunlight for 5 minutes, and four
were left in the light-proof box.
Exposures of 0.01 and 0.1 second were timed by means of the
automatic shutter of the camera; the exposures of 1.0 second
were corrected by comparison with a metronome marking sec-
onds. Since the seeds were distributed over a circular area 7.1
centimeters in diameter, and the aperture was 3.5 centimeters
in diameter, about one-fourth of the seeds were exposed to direct
sunlight.
The germination of the four trials for each length of exposure,
and of the controls in full sunlight and in the light-proof box,
are shown in Table 7.
TABLE 7.-GERMINATION OF TOBACCO SEEDS EXPOSED TO DIRECT SUNLIGHT.
Plate- Duration of Exposure (seconds)
holder
No. Controls 0.01 I 0.1 1.0 300
Percentage of Germination
1......................... 35 51 57 74 68
2............................. 29 62 56 69 83
3............................. 9 67 67 77 84
4............................ 20 63 62 73 86
Av......................... 23a 61 61 73 80
aApparently due to a slight leak in the shutter; controls in light-proof
box did not germinate.

The controls placed in the camera evidently received a con-
siderable amount of light by leakage. An additional exposure
to direct sunlight for 0.01 second was sufficient to induce a
significant increase in germination over that of the controls.
Relation of Amount of Light to Germination. It was consid-
ered of importance in the interpretation of experimental results
to determine whether equal increments in the amount of light
of given wave-lengths produce equal increments in the percent-
age of germination. In order to investigate this question, seeds
were incubated in darkness for 4 days, exposed to light for
various periods of time, and returned to darkness for 10 days
more. A 60-watt lamp and Wratten filter No. 00 were used to
produce a total radiation of about 5 x 10-4 gram-calorie per
square centimeter per second, or visible radiation of about 4 x
10-6 gram-calorie per square centimeter per second5. The periods
of exposure varied from 5 to 1,800 seconds. Controls in dark-
ness were left in the light-proof box. The average germination

5The measurement of the radiation is discussed later in this paper.






Environmental Factors Affecting Tobacco Germination 23

of six trials for each period of exposure is given in Table 9.
The average germination is plotted against the logarithm of
the time in Fig. 2.


5 15 60 10s 600 1800
Exposure in seconds (logarithmic scale)
Fig. 2.-Germination of tobacco seeds exposed to light for various periods
of time.

TABLE 9.-GERMINATION OF TOBACCO SEEDS EXPOSED TO A TOTAL RADIATION
OF 5 x 10-4 GRAM-CALORIE PER SQUARE CENTIMETER PER SECOND FOR
VARIOUS PERIODS OF TIME.
Duration of Exposure Germination
seconds logarithm %
0 ........ 0.0
5 0.70 11.5
15 1.18 17.2
60 1.78 24.2
180 2.26 30.2
600 2.78 40.0
1,800 3.26 42.2


These results indicate that the amount of light must be suc-
cessively increased by approximately constant multiples of the
preceding amount, rather than by equal amounts, in order to
produce equal increments in the percentage of germination,
through the range of percentages investigated. This generaliza-
tion applies also to data reported by Wieser (66, p. 564).
If the entire range of possible percentages were investigated,
it might be determined whether the distribution curve of seeds
in a certain sample according to their light requirement, or to





Florida Agricultural Experiment Station


its logarithm, approaches a normal curve. If so, the method
of statistical analysis proposed by Bliss (4), which makes allow-
ance for normal variation, might prove useful in the interpreta-
tion of such data. The cause of this variation in the amount
of light required might lie in differences in the color of the seed
coat, or in its resistance to the absorption of water or oxygen.
WATER IN RELATION TO THE LIGHT EFFECT
The factors which condition the light effect should be dis-
tinguished from those which condition germination after ex-
posure to light. Among the factors which condition the light
effect are water content of the seeds, wave-length and energy
value of light, and temperature. Experiments on the relation
of these factors are reported in the following pages.
The effect of soaking seeds in water on their sensitiveness
to light has received considerable attention. Since differences
have been observed in the optimal period of soaking for various
samples of seeds (66), it was necessary to determine this period
for the seeds used in these experiments.
Effect of Soaking on Sensitiveness to Light. In preliminary
experiments concerning the effect of soaking seeds on their
sensitiveness to light, they were incubated in darkness at 220,
Seeds soaked for about 4 days became completely sensitive to
an exposure of from 3 to 10 minutes to diffuse daylight. They
did not show any loss of sensitiveness to the light used with
further soaking for periods as long as 7 days.
In another experiment, the seeds were soaked at room tem-
perature, varying from 19 to 23, for periods of from 1 to 12
days. Then they were exposed for 100 seconds to a total radia-
tion of 1.3 x 10-5 gram calorie per square centimeter per second,
of which only about 1 percent was in the visible region between
497 and 700 millimicrons. The average germination of quintupli-
cate trials is shown in Table 9.
The percentage of the seeds sensitive to the light used in-
creases with soaking for 4 days, and afterward declines with
further soaking until the 10th day, when an equilibrium is
reached. This loss of sensitiveness can be overcome by exposure
to more light, as shown by the writer's observation reported
above, that seeds soaked in darkness for 20 or 30 days subse-
quently germinated well in diffuse daylight.
In further experiments on the effect of light on germination,
the period of soaking was generally 4 or 7 days, as indicated
for each experiment reported.






Environmental Factors Affecting Tobacco Germination 25

TABLE 9.-GERMINATION OF TOBACCO SEEDS INCUBATED IN DARKNESS FOR
VARIOUS PERIODS AND EXPOSED FOR 100 SECONDS TO A TOTAL RADIATION
OF 1.3 x 10-5 GRAM-CALORIE PER SQUARE CENTIMETER PER SECOND.
Period of Soaking Germination
days %0
0 ........... ---- .. .... 0 ........... ----- --------- o
S.......................... .......... ................ ................... 0
2 ........2... .................... ..... ... .... .... .... .................. ........ 2
3 .... .................................... ............................................ 11
43 ................................................................................... 22
5 ............................... .... ......................... ......................... 226
65 .................. ........ ............... ............................ 12
76 ...... .................. ........... .... ....... .....15.............. ..... 12
8 ............ .......... ........ .. ...... ... ........ ...... ... 14
9 ................................ .....-.. ............ .............. .......-- 8
10 ................... .. ................... ........................--- ... 4
11 .............. ............ .............. ............................ ..... 4
12 ......... ....... ..... .......................... .............. 4


This optimal period of soaking is much longer than the period
of 32 hours or less, mentioned by Wieser (66), Bihlmeier (3),
and Kipp (34), for most of the samples which they investigated.
There was evidently considerable difference in their various
samples, because Wieser reported one which, when exposed to
light after 72 hours of soaking, germinated to a percentage far
below the maximum for that sample.
Temperature may also be a factor in the period of soaking
required. Bihlmeier (3) and Wieser (66) soaked the seeds at
28 and 300, respectively, at which temperatures water absorp-
tion probably occurred more rapidly than at 19 to 23', the
temperature used by the writer. Indeed, this relation of tem-
perature to water absorption was demonstrated by Poptzoff (52).
Sensitiveness of Air-Dry Seeds. The sensitiveness of seeds
to light is conditioned by the imbibition of water, as demonstrat-
ed by the writer and several others. According to the hypothesis
of Kipp (34), water makes the reserve substances in the seed
labile. It may also allow the light to pass through the seed
coats more directly, and it was considered possible that light
might be effective on the contents of air-dry seeds if the seed
coats were soaked with a colorless liquid other than water.
Glycerin was found unsuitable, because it promoted the growth
of fungi. Paraffin oil was found suitable, because it neither
promoted the growth of fungi nor impaired the germination of
seeds when water was added directly to oil-soaked seeds and
filter paper.
Air-dry seeds on dry filter papers were soaked in paraffin oil
for several days, after which they were exposed to direct sun-





Florida Agricultural Experiment Station


light for 15 minutes. Then water was added and the seeds
were tested, some in light and some in darkness. Those in light
germinated promptly, but those in darkness failed to germinate.
Since soaking air-dry seeds in paraffin oil does not make
them sensitive to light, it is inferred that the imbibition of water
by the seed coats only would not be sufficient to make the seeds
sensitive. This further supports the opinion of Kipp (34) and
others that the seed contents must be imbibed. On the other
hand, Bihlmeier (3) reported that seeds shelled dry germinated
to a considerable percentage in darkness, but the cause of this
behavior was not explained.
Air-dry seeds are not sensitive to ordinary sunlight, and there-
fore it was of interest to determine whether they are sensitive
to a more intense radiation. Seeds were exposed for 1 hour to
noonday sunlight, which was concentrated four times by means
of a reading glass, adjusted so that the diameter of the area
irradiated was half that of the lens. Other seeds were exposed
to direct sunlight, and untreated controls were kept in the lab-
oratory. The treated and untreated seeds germinated well in
light, but failed to germinate in darkness, thus showing that the
treatment of the dry seeds had no effect.
Experiments were conducted to determine whether seeds
might become sensitive to light by absorption of water vapor
from a moist atmosphere. Small vials containing 100 air-dry
seeds each were placed in glass moist chambers over water or a
solution of sulfuric acid. After a few days of storage in light,
the seeds were sowed on dry filter paper, moistened and taken
immediately to the dark room.
In one experiment, seeds were stored for 21 days over sulfuric
acid at relative humidities of from 75 to 95 percent; the con-
centrations of sulfuric acid required were calculated from data
of Olsen (49). The moisture content of the seeds on the basis
of the dry weight after 6 hours at 1050, and the average of du-
plicate germination tests in light and in darkness, are given in
Table 10.
Seeds stored in light for 21 days at relative humidities of
from 75 to 95 percent absorbed a certain amount of water. How-
ever, they did not germinate when tested in darkness, indicating
that they are not made sensitive to diffuse daylight by this
treatment. According to Poptzoff (52), the maximal water con-
tent of seeds soaked in water is about 31 percent.
In another experiment, nine lots of seeds were stored in light
for 6 days over water, and then tested in light and in darkness.






Environmental Factors Affecting Tobacco Germination 27

Some lots failed to germinate; others germinated well, but this
result was obviously due to water condensed in the vials and not
to water vapor.
TABLE 10.-MOISTURE CONTENT AND PERCENTAGE OF GERMINATION OF
TOBACCO SEEDS STORED FOR 21 DAYS OVER SOLUTIONS OF SULFURIC ACID.
Moisture Germination
Relative in
H1SO, Humidity Seeds In Light In Darkness
% %0 % /%0 %

30.4 75 10.3 89 0
27.0 80 11.5 89 0
23.1 85 13.2 91 0
18.3 90 15.2 86 0
11.7 95 20.1 86 1

From the results of these experiments, it was concluded that
seeds must be soaked in water in order to make them sensitive
to light.
Influence of Subsequent Drying on the Light Effect. Experi-
ments were conducted to determine whether the effect of light
on imbibed seeds persisted after the seeds were dried. Seeds
were incubated on moist filter paper for a certain number of
days, then the lids were removed from the petri dishes, and the
seeds and paper were allowed to dry in darkness or in light.
The germination of the dried seeds was tested in darkness and
in light, and the averages of triplicate germination tests are
shown in Table 11.
TABLE 11.-GERMINATION OF TOBACCO SEEDS SOAKED AND THEN DRIED IN
DARKNESS OR IN LIGHT.


Period of Soaking
SLight
Length I Conditions
days _

0 Light
1 "
2 "
4
6
0 Light
3
6
6 Darkness
20

10 Darkness
5 "$


Period of

Length
days

5
5
5
5

3
3
3
3
3
140
3


Drying
Light
Conditions


Light




Light


Light

Darkness
"


Germination
In I In
Light Darkness


73 13
73 22
68 45
48a 25a
4a 3a
85 2
88 81
la 2a
69 65
76 65
68 0
86 0


aRemainder germinated during soaking, and were killed by drying.





Florida Agricultural Experiment Station


The results show that seeds which are exposed to light in an
imbibed condition and then dried do not lose their ability to
germinate subsequently in darkness. A similar observation on
seeds of Ranunculus sceleratus was reported by Gassner (17).
Seeds soaked and dried in darkness did not germinate in dark-
ness, showing that the effect of drying in darkness is not suffi-
cient to offset the light requirement.
The light effect is conditioned by the presence of a considerable
amount of water in the seed contents, but it is not reversed
when the water is removed by drying.
Gassner (17) reported that the effect of light on imbibed seeds
of Ranunculus sceleratus persisted after the seeds were dried.
On the basis of this observation, Wieser (66) proposed a theory
to account for the occurrence of light-indifferent samples of
seeds of Lythrum salicaria, which generally require light for
germination. Wieser considered it possible that the seeds might
be moistened with dew or rain while they were still in the
capsule, and that the effect of light on the moist seeds would
persist after they again became dry. If this theory should prove
applicable to tobacco seeds, some of the differences in the light
requirement of tobacco seeds might be explained.

WAVE-LENGTH AND ENERGY VALUE OF LIGHT
Several experiments were conducted to determine the amount
of radiant energy of certain wave-lengths required to induce
germination.
The source of radiation was a 10-watt lamp, mounted in one
end of a metal can; the opposite end was closed by means of a
No. 00 or a No. 3 Wratten filter. The total radiation from this
lamp was measured in gram-calories by means of a thermopile,
and compared with that from a total-radiation standard prepared
by the Bureau of Standards, Washington, D. C. It had been
previously proved that the deflection of the galvanometer was
proportional to the amount of radiant energy received by the
thermopile. Radiations of similar color were compared at vari-
ous distances from the lamp by means of a photoelectric cell.
Both of the filters used transmitted freely wave-lengths of'
about 700 millimicrons and longer. Measurements of the visible
radiation only were made, using a water cell containing copper
sulfate solution, equivalent to a 1-centimeter layer of 1 percent
solution. This cell absorbed the infra-red and some red radiation,
and therefore the visible radiation was a little stronger than






Environmental Factors Affecting Tobacco Germination 29

that reported. Unless otherwise noted, the copper sulfate cell
was not used when the seeds were exposed to light.6
Amount of Total and Visible Radiation Required. Several
trials were conducted with exposures at various distances from
the lamp to determine the amount of total and visible radiation
required to induce the germination of seeds which had been
previously incubated in darkness. The average germination of
three replications in each trial with filter No. 00, and of six
replications in each trial with filter No. 3, are shown in Table
12. In each trial, germination of controls not exposed to light
was 1 percent or less.
In the first trial, a significant response was produced by a 1-
second exposure to a total radiation of 1.3 x 10-6 gram-calorie
per square centimeter, or to visible radiation of 1.1 x 10-8 gram-
calorie per square centimeter. A seed of average size and typical
shape, presenting an area of 0.0036 square centimeter, received
about 5 x 10-9 gram-calorie of total radiation, or about 4 x 10-11
calorie of visible radiation. Calculated from a wave-length of
600 millimicrons, the number of quanta of visible radiation
striking a seed was about 5 x 108.
In the second trial, 100 times as much radiation produced a
somewhat smaller percentage of germination. Therefore, these
calculated values probably represent little more than the order
of magnitude of the radiation required in each trial.
Effect of Visible Radiation Only. To determine whether the
infra-red and red radiation absorbed by the copper sulfate solu-
tion was important in inducing germination, exposures were
made with and without the copper sulfate cell. The results,
given in Table 12, Trials 3 and 4, show that germination was
slightly reduced by the use of the cell. This may be attributed
to the absorption by the cell of a certain amount of radiation
in the red region.
Effect of Wave-Lengths Strongly Absorbed by Seed Coats. An
attempt was made to find a light of such wave-length and energy
value that seeds being incubated in darkness might be examined
without affecting their germination. The visible radiation trans-
mitted by filter No. 3 was from 483 to 580 millimicrons, strongest
at 544 millimicrons. Kommerell (35) found that radiation of
510 millimicrons was most strongly absorbed by the seed coats
6The writer is indebted to Dr. R. T. Dufford of the Department of Physics,
University of Missouri, for direction and assistance in measuring the radia-
tion; also to Mr. Lloyd A. Jones of the Eastman Kodak Company, for
furnishing the transmission curves of the Wratten filters.











TABLE 12.-GERMINATION OF TOBACCO SEEDS EXPOSED TO RADIATION OF KNOWN WAVE-LENGTH AND INTENSITY.


Trial
No.


1






2



3

4

5


Filter
No.


00






00



00

00

3


a Copper sulfate cell used


Visible
Region
Transmitted
m4

497-700






497-700



497-700

497-700

483-580


Period
of
Soaking
days

8






5



7

4


4


Period I
of Radiation per Sq. Cm.
Exposure Total Visible
sec. gram-calories gram-calories

1 1.3 x 10-6 1.1 x 10-8
10 1.3 x 10-5 1.1 x 10-7
100 1.3 x 10 4 1.1 x 10 6
1,000 1.3 x 10- 1.1 x 10-5
1 1.3 x 10-5 1.1 x 10-7
10 1.3 x 10-4 1.1 x 10-6
100 1.3 x 10 3 1.1 x 10-5

10 1.3 x 10-4 1.1 x 10-6
100 1.3 x 10-3 1.1 x 10-5
1,000 1.3 x 10-2 1.1 x 10-4
10,000 1.3 x 10-1 1.1 x 10-3

100 1.3 x 10 3 1.1 x 10-5
100 .......... a 1.1 x 10-5

100 1.3 x 10-3 1.1 x 10-5
100 .............a 1.1 x 10-5

300 6 x 10-2 3 x 10-4


Germination


39
52
63
67
62
58
63

29
43
55
55

14
12

15
12

10






Environmental Factors Affecting Tobacco Germination 31

and least effective in inducing germination. The eye is most
sensitive to radiation of low intensity at about 540 millimicrons,
according to the Eastman Kodak Company (12), or at 535 milli-
microns, according to Hodgman (22, p. 483).
Seeds were exposed to light through filter No. 00. The esti-
mated energy value of the radiation, and the average results of
sextuplicate germination tests, are reported in Table 12, Trial 5.
The light used was too faint to permit the counting of seedlings,
but an exposure of only 300 seconds induced considerable ger-
mination. It may be noted that the effect of this light on germi-
nation was less than that of light of the same energy value in
the region transmitted by filter No. 00.
These results show that very sensitive seeds such as those
used cannot safely be examined during incubation in darkness.
However, if another sample had been available which required
more light, it might have been possible to make successive
counts of germination in darkness without affecting the results.
Effect of Moonlight on Germination. It may be of some interest
to note that exposure of imbibed seeds for 15 minutes to strong
moonlight (bright enough to permit the reading of a typewritten
page) induced a high percentage of germination. The energy
value of the light was not measured, but the intensity to the
eye appeared to be comparable to that of the artificial light used
in some of the trials reported above.
TEMPERATURE IN RELATION TO THE LIGHT EFFECT
The effects of temperature and light on the germination of
tobacco seeds are closely related. Studies on the interrelation-
ship of these two factors are presented below.
Effect of Alternating Temperatures and Pressures. To deter-
mine whether a sharp daily alternation of temperatures would
increase the percentage of seeds which germinate in darkness,
several experiments were conducted. Tests at constant tempera-
ture were incubated at 230, and those at alternating tempera-
tures were moved to the refrigerator at 50 for 8 hours of each
day. The results of the germination tests at constant and
alternating temperatures are shown in Table 13.
From these results, it was concluded that seeds which germi-
nate poorly or not at all in darkness at constant temperature
may be caused to germinate well in darkness by suitable alter-
nation of temperatures. This result is in agreement with those
of Arnaudov (1) and Honing (26).





Florida Agricultural Experiment Station


TABLE 13.-GERMINATION OF TOBACCO SEEDS IN LIGHT AND IN DARKNESS
AT CONSTANT AND AT ALTERNATING TEMPERATURES. (The number of
tests averaged is given in parentheses.)

Trial Constant Temperature Alternating Temperature
No. Light Darkness I Light I Darkness
S % % 1 % 1 %
1 51 (3) 2 (9) 60 (3) 54 (9)
2 80 (3) 2 (6) 77 (3) 73 (6)
3 84 (3) 0 (6) | 85 (3) 75 (6)


It is most surprising that alternation of temperatures should
replace the effect of light. Vafiha (64) suggested that alterna-
tion of temperatures might favor the movement of gases within
the seeds, and thus promote the removal of carbon dioxide and
the renewal of oxygen, conditions which are favorable to in-
creased respiration. To determine whether alternation of pres-
sures in the air surrounding the seeds would accomplish the
same purpose as alternation of temperature, two trials were
conducted with pressures varying from normal to about 19
centimeters of mercury below normal.
A glass culture vessel was attached to a motor-driven pump,
automatically controlled by means of a mercury manometer and
a relay. At normal pressure the pump was started, and after
a few seconds' operation about one-fourth of the air was exhaust-
ed and the pump was stopped. A period of from 1 to 2 hours
was required for the pressure to return to normal, after which
the pump was started again. The trials were conducted at room
temperature, in light and in darkness, each in quadruplicate.
Controls were incubated under similar conditions, except at
normal pressure.
In the two trials, the seeds germinated in light at either
normal or alternating pressure 84 percent or more in light, 1
percent or less in darkness. These results show that an alter-
nation between normal pressure and about three-fourths normal
pressure every 1 to 2 hours does not induce germination in
darkness. It would be interesting to determine the effect of
a greater reduction in pressure, and also of pressures above
normal.
Effect of Light on the Response to Temperature. In order to
determine whether light affects the response of seeds to tem-
perature, seeds which had been made rather indifferent to light






Environmental Factors Affecting Tobacco Germination 33

by soaking in darkness and drying in light were tested at various
temperatures in light and in darkness in comparison with un-
treated seeds of the same lot. The tests were made in triplicate,
each set of three petri dishes being enclosed in a metal can.
Tests in light were exposed for a few minutes daily to direct
sunlight in a greenhouse. The seeds tested in darkness were
incubated until the corresponding tests in light had reached their
maximal percentage of germination, and were removed from
the cans in a dark room for aeration and watering every third
day. The averages of triplicate germination tests of untreated
and treated seeds in light and in darkness at six different tem-
peratures are shown in Table 14.
TABLE 14.-GERMINATION OF UNTREATED (LIGHT-REQUIRING) AND TREATED
(LIGHT-INDIFFERENT) TOBACCO SEEDS IN LIGHT AND IN DARKNESS AT
VARIOUS TEMPERATURES.
Mean Incubation
Tempera- Total Germination Period
ture In Darkness 1 In Light In Light
Untr. Tr. | Untr. Tr. Untr. Tr.
C % _% ,-__ % days days
16 0.3 68 94 89 14.2 10.7
201/2 1.7 47 93 91 8.9 7.4
25 0.3 54 93 89 7.5 6.0
29 0.3 55 67 84 8.8 4.8
332 0.0 17 3 78 10.6 8.1
38 0.0 0 0 0 ...... --


These results show that the treated (light-indifferent) seeds
germinate much better at 331/20 in light than in darkness, and
also that the treated seeds germinate much better than untreat-
ed (light-requiring) seeds when tested at 331/20 in light. Treat-
ed seeds germinate faster at all temperatures than untreated
seeds. The difference between untreated and treated seeds may
have been due, at least partly, to the effects of soaking and
drying, although, as shown above, seeds soaked and dried in
darkness still require light for germination.
Influence of Temperature on the Light Effect. The work of
Kommerell (35) indicated that the effect of light on tobacco
seeds is a photochemical reaction. Dhar (10, p. 320) stated,
"According to the law of photochemical equivalence, temperature
should not have any influence on the velocity of a photochemical
reaction." Therefore light should be equally effective through-
out a considerable range of temperatures. Experiments were
conducted to test this relationship.





Florida Agricultural Experiment Station


Seeds which had been incubated in darkness at fairly constant
room temperatures for 7 or 10 days were exposed to a light of
constant intensity for 100 seconds. The temperature of exposure
was controlled by floating six petri dishes at a time in a pan of
water, which was left covered for from 2 to 7 minutes to allow
the temperature of the seeds to approach that of the water
bath. The cover was removed and three of the dishes were
removed from the water bath just before the light was turned
on; these served as checks on the effect of the temperature
treatment in the absence of light. However, the treatment was
about 2 minutes shorter, and possibly less extreme, than that
of the seeds exposed to light. Both the initial and final tem-
peratures of the bath were recorded; the latter probably repre-
sented the approximate temperature of the seeds at the time of
exposure, except for the higher temperatures of the second trial,
which rose gradually during the period of the treatment. After
exposure to light, the seeds were incubated in darkness at room
temperature for from 9 to 14 days. The average results of tripli-
cate determinations in each of six trials are shown in Table 15.
The germination of seeds exposed to light at from 0 to 20
was greater in every trial than the germination of seeds exposed
to light at from 19 to 220. The significance of the increases
in the six trials is indicated by odds of more than 10,000 to 1
when calculated by Love's modification of Student's method (45).
It did not seem probable that light was actually more effective
at 00 than at 20', because the treatment at low temperature
might have exerted a favorable effect on some of the seeds, but
not enough to induce germination in darkness. A favorable
effect of low temperature treatments had already been observed,
since a daily alternation between 50 and 23 induces germination
in darkness, as mentioned above.
Among the seeds exposed to light, a minimal percentage of
germination was observed in the seeds treated at about 20. The
germination of seeds exposed to light, as well as of the controls
in darkness, began to increase at about 300 and reached a maxi-
mum at about 400. The germination of seeds treated at about
500 was reduced, and most of the seeds treated at 600 were
killed. Gardner (15) reported that seeds treated in water at 60
for only 15 or 30 seconds germinated much better in darkness
than untreated seeds.
Thus it appears that the effect of light is similar to the effect
of temperature. In an attempt to separate these effects, three
more trials were made, using seeds which had been incubated






Environmental Factors Affecting Tobacco Germination 35

in darkness for 7 days. The duration and intensity of the ex-
posure to light was the same as in previous trials. The time
allowed for the seeds to come to equilibrium with the tempera-
ture of the water bath was 5 minutes. Some of the seeds were
exposed to light at 20 before the temperature treatment at 0
or 40. Others were given the temperature treatment, brought
back to 200, and then exposed to light. Controls were given
similar temperature treatments at 0, 20' and 40, with and
without exposure to light during the treatment. Table 16 shows
the average germination of the seeds in three trials. Each
treatment consisted of six replications.
TABLE 15.-GERMINATION OF TOBACCO SEEDS EXPOSED TO LIGHT AT
VARIOUS TEMPERATURES.
Temperature of Total Germination
Trial Exposure to Germination After Subsequent
No. Light Incubation in Light
Initial Final Light Darkness | Light I Darkness
C. C. % % I % %

1 0 0 58 0
10 10 49 0
20 20 37 0
31 28 77 1
S 41 36 90 68
54 42 87 50
2 0 0 78 0 92 93
5 8 70 0 88 93
14 14 70 0 90 92
22 21 61 0 84 90
28 30 92 25 92 84
S 37 42 93 68 94 92
44 52 87 33 89 90
50 64 0 0 23 33
3 0 0 49 5
9 12 54 6
20 20 38 4
30 31 89 36
41 41 91 76
50 44 85 63
4 0 2 31 0
20 20 25 0
40 44 91 37
5 0 2 8 0
20 20 6 0
40 46 81 7
6 0 2 21 0
19 20 19 0
27 26 19 0
33 31 77 4
40 36 87 24















TABLE 17.-GERMINATION OF TOBACCO SEEDS AFTER EXPOSURE TO LIGHT AT VARIOUS TEMPERATURES, AND AFTER
TEMPERATURE TREATMENTS.

Percentage of Germina


Treatment Applied to the Seeds


Exposed to light at 200 before temperature treatment at 0....................... ......
during 0.............................
200 after 0............................


20 during
200 before
400 during
20 after

Controls not exposed to light-
i -


Trial
1


c" 20 ............... .......................

" "C 40o.............. -.... ...........
C cc 400............ ..... ...............
CC 40 .................... ....................

" '' 0
c" 200...... .... ..................
" 40o.........................................
,, 4 0 ".- -------------................................


Trial
2


aAfter 2 treatments, 2 hours apart: 1 per cent
bAfter 2 treatments, 1 hour apart: 26 per cent


VARIOUS
0

tion
Trial
3





6
ti






82
81


Oa
0a


7b

8:
;t'
7b






Environmental Factors Affecting Tobacco Germination 37

Seeds exposed to light at 20 before or after the temperature
treatment at 0 gave practically the same germination as seeds
exposed to light during the temperature treatment at 0. Simi-
larly, light at 20' and light at 400 had the same effect on seeds
which, in addition, received the temperature treatment at 40.
From these results it was concluded that, under the conditions
of these experiments, the effect of light alone on imbibed seeds
is independent of the temperature of the seeds at the time of
exposure.
The combined effect of light and high temperature treatment
was greater in several instances than the sum of the results
when light (at 200 or 210) and high temperature treatment were
applied to different lots of seeds. Either the light or the temper-
ature treatment, or both, may have had an effect on some of the
seeds, but neither of these effects was sufficient to induce ger-
mination of those seeds in the absence of the other. Two tem-
perature treatments at 400 induced a greater percentage of
germination in darkness than one treatment.
Lehmann (43) found that the minimal period of illumination
required to induce the germination of seeds of Lythrum salicaria
was much shorter at 30 than at 20'. Since no trials with still
higher temperatures were reported, it is not possible to decide
whether this result is in agreement with the results on tobacco
seeds reported above.
The fact that the effect of light is independent of the tempera-
ture of the seeds at the time of exposure to light supports
Kommerell's photochemical theory of the light effect, because
the rate of photochemical reactions in general is not directly
affected by the temperature.
This instance of the absence of a temperature effect is in
striking contrast to the case of many biological reactions. To
cite a well-known example, the work of Matthaei (46) showed
that the rate of carbon dioxide assimilation in leaves of Prunus
sp. was approximately doubled for each increase of 100, thus
agreeing with the Van't Hoff rule.

DEPTH OF SOWING AND GERMINATION
Tobacco seeds are generally sowed on the top of the soil, where
they are exposed to light, but sometimes, according to Busse
(7), they are sowed under a covering of fine soil 1 centimeter
thick. In order to determine whether alternating temperature
might offset the light requirement of seeds planted under a
covering of soil, the seeds were sowed on soil of pH 5.4 in petri






Florida Agricultural Experiment Station


dishes and covered with soil to various depths. The total depth
of the soil in all dishes was 10 millimeters. Tap water was
added until the soil was nearly saturated. The cultures were
incubated in glass moist chambers, at a constant temperature
of 24, or at alternating temperatures of 24 for 18 hours and
5 for 6 hours of each day. On the 27th day of incubation, the
tests at constant temperature were transferred to alternating
temperature for a period of 14 days. The average of triplicate
germination tests at each depth, at constant and alternating
temperatures, are shown in Table 17.
TABLE 17.-GERMINATION OF TOBACCO SEEDS SOWED AT DIFFERENT DEPTHS
IN SOIL, AT CONSTANT AND ALTERNATING TEMPERATURES.
CONSTANT TEMPERATURE ALTERNATING TEMPERATURES
Days of Incubation Days of Incubation
Depth 11 131 I18 21 27 ] 41a 11 i 13 | 18 23 j 30 1 35 [ 41
nmm. Percentage of Germination Percentage of Germination
0 86 88 89 90 90 90 68 81 84 85 86 86 86
1 62 70 76 77 77 88 43 66 86 87 88 89 89
2 21 25 28 32 32 43 18 29 67 80 81 82 82
3 14 17 20 20 20 28 6 12 57 67 73 74 75
4 10 12 13 14 14 23 4 9 46 62 67 69 69
5 11 13 17 18 18 21 5 12 48 64 67 67 67

aAfter 14 days at alternating temperatures.

Germination at a depth of from 2 to 5 millimeters was much
better at alternating temperatures than at constant temperature,
indicating that alternating temperatures offset the light require-
ment of the seeds, permitting them to germinate in darkness.
At constant temperature, the highest percentage of germination
was obtained when the seeds were sowed on top of the soil. At
alternating temperatures, fair germination was obtained at 5
millimeters, the greatest depth tried; the time required for the
emergence of the seedlings varied directly with the depth of
sowing.
After 41 days of incubation, the soil was dried for 7 days to
kill any seedlings, pulverized and remoistened; thus the remain-
ing seeds were mixed throughout the soil. After 17 days of
incubation at alternating temperatures, there was some germi-
nation in all except one of the dishes in which seeds had origin-
ally been sowed at 2 millimeters or deeper. This indicated that
some seeds had remained ungerminated and viable in moist soil
for a period of 41 days.






Environmental Factors Affecting Tobacco Germination 39

GENERAL DISCUSSION
The experimental work reported in this paper shows conclu-
sively that the tobacco seeds used require light for germination
at constant temperature. Exposure of imbibed seeds to a visible
radiation of the order of 10-8 gram-calorie per square centimeter,
or 10-11 gram-calorie per seed, is sufficient to induce considerable
germination.
This observation suggests for consideration the question, "Is
the effect of light on the seeds a photochemical reaction?" Two
observations support the photochemical theory proposed by
Kommerell (35). First, the results of Kommerell show that the
germination resulting from exposure of imbibed seeds to light
is proportional to the number of quanta which act upon the seed
contents. Second, data presented by the writer in this paper
show that the effect of light on imbibed seeds is independent of
the temperature of the seeds at the time of exposure to light.
The fact that the exposure to light may be replaced by a
temperature treatment at 400 is not in serious contradiction
to the photochemical theory of the effect of light, because some
other reactions, which proceed very slowly at a given tempera-
ture unless light is applied, may proceed quite rapidly in darkness
at higher temperatures; e.g., photographic plates may be fogged
in darkness at high temperatures. The fact that the exposure
to light may be replaced by a sharp daily alternation of tem-
perature is not readily explained on the basis of the photochemi-
,cal theory.
The reaction which occurs when imbibed tobacco seeds are
exposed to light and the coagulation of proteins are similar in
respect to the effect of certain factors on these processes. Some
points of similarity are as follows:
(1) The amount of light required to produce equal increments
in the percentage of germination must be successively increased
by constant multiples of the preceding amount, within the range
of percentages investigated by the writer. Therefore, a loga-
rithmic curve is obtained when the percentage of germination
is plotted against the period of exposure. Likewise, Lepeschkin
(44) found a logarithmic relation between the temperature and
the time required to kill living plant tissue, e.g., root of beet.
(2) The coagulation of a protein by ultra-violet light, accord-
ing to Bovie (6), has a negligible temperature coefficient, the
rate of coagulation being nearly as great at 0' as at higher






Florida Agricultural Experiment Station


temperatures. The same relation holds for the effect of light
on tobacco seeds, as shown by the findings of the writer.
(3) Tobacco seeds in an air-dry condition are not sensitive
to light. They must be soaked in water for a certain length
of time before they become sensitive. Likewise, dry seeds of
wheat are much more resistant to the killing effect of heat than
moist seeds, as shown by Robbins and Petsch (60).
Thus the reaction which occurs when imbibed tobacco seeds
are exposed to light resembles in certain respects the coagula-
tion of proteins. However, the evidence at hand does not justify
a conclusion concerning the type of reaction involved.
The germination resulting from exposure to light is separated
from the exposure by a considerable period of time, and by
many complex processes of metabolism. This suggests the ques-
tion, "What is the immediate effect of light on tobacco seeds?"
Kipp (34) proposed to explain the effect of light by the activation
of certain substances, perhaps enzymes, by a process of reduc-
tion. The first measurable change in the seeds after exposure
to light, observed by Kipp (34) and Schr6ppel (61), was an
increase in the rate of respiration, which took place within about
2 hours, but Schroppel was not able to demonstrate an immediate
effect of light on the activity of the respiratory enzymes, catalase
and peroxidase.
Navez and Rubenstein (48) reported the acceleration of the
activity of diastase by light, especially in the presence of a
fluorescent dye, and they attributed this result to the removal
of a diastasic inhibitor, possibly by oxidation. Since tobacco
seeds contain no starch, this observation on the activation of
diastase does not appear to be applicable to the present problem.
Since the most abundant reserve substance in tobacco seeds
is fat, a study of the activity of lipase in relation to light and
other environmental factors should prove interesting. In fact,
several observations suggest the possibility of a close relation
between light and the activity of lipase in seeds. Coe and
Leclerc (9) found that the development of rancidity in fatty
food materials by oxidation and hydrolysis is hastened by light,
especially in certain regions of the spectrum, the region from
490 to 560 millimicrons being least active. This is the region
reported by Kommerell (35) to be least effective in inducing the
germination of tobacco seeds. Oosthuizen and Shedd (50) re-
ported that lipase is absent or of doubtful activity in ungermi-
nated tobacco seeds. Unpublished data by the writer are in
agreement with this observation, and show also that the activity





Environmental Factors Affecting Tobacco Germination 41

of lipase increases rapidly during the first few days of ger-
mination.
Ralski (57) and Kummer (39) found that graminaceous seeds
which contain nearly neutral fat require light for germination,
while those in which the fat has an appreciable acid number
germinate in darkness. Kandilis and Karnis (32) reported that
the acid value of tobacco seed oil varies from 2.25 to 16.93. It
would be interesting to determine whether these wide differences
in acid value are associated with the activity of lipase or with
the requirement of light for germination.
The respiration of fat, according to Kostychev (36, p. 121),
is associated with a comparatively low value of the ratio of
carbon dioxide to oxygen, and is undoubtedly effected by way
of the intermediate stage of sugar. However Ermakoff and
Iwanoff (13) reported that the ratio of carbon dioxide to oxygen
in the fatty seeds of flax, castor bean and almond in the early
stages of germination is near unity, the same as that of seeds
rich in carbohydrates. The quality and quantity of the oil in
flax seeds remains unchanged during the first hours of germi-
nation. After about 2 days, the respiration quotient falls to
0.78, indicating the respiration of the fatty reserves.
On the contrary, Kipp (34) and Schrippel (61) reported that
the ratio of carbon dioxide to oxygen in germinating tobacco
seeds starts at about 0.7 and thereafter declines. The low value
of this ratio could represent the incomplete oxidation of the
carbohydrate materials present in small amounts in tobacco seeds.
The similarity between the effect of light on imbibed tobacco
seeds and the coagulation of proteins, and the suggestions con-
cerning the relation of light to the activity of lipase, are pre-
sented as a basis for further work on this very interesting
phenomenon.
SUMMARY AND CONCLUSIONS
Experimental work on the relation of certain environmental
factors to the germination of Florida cigar-wrapper tobacco
seeds is reported in this paper. The more important conclusions
drawn from these studies are as follows:
The cardinal temperatures for germination are approximately
10, 240 and 34. Germination at certain daily alternations of
temperature is a little more rapid than at the optimum.
The apparent isoelectric point of the seeds, as indicated by
three types of experiments, is about pH 6.8.





Florida Agricultural Experiment Station


Light was required for the germination of all 19 samples of
seeds tested.
Exposure of imbibed seeds to direct sunlight for 0.01 second
induces a significant increase in germination.
The amount of light must be successively increased by ap-
proximately constant multiples of the preceding amount to pro-
duce equal increments in the percentage of germination.
Sensitiveness of the seeds to a comparatively weak light
reaches a maximum after about 4 days of soaking at a room
temperature of from 190 to 230, and declines with further
soaking until an equilibrium is reached after about 10 days.
Air-dry seeds are not sensitive to light.
The light effect in imbibed seeds is not reversed by subsequent
drying.
Seeds soaked and dried in darkness still require light for
germination.
Considerable germination is produced by exposure of imbibed
seeds to a total radiation of about 10-6 gram-calorie per square
centimeter, of which about 10-8 gram-calorie is in the visible
region from 497 to about 700 millimicrons. The visible radiation
per seed is of the order of 1011 gram-calorie, and the number
of quanta is about 108.
Green light, of a wave-length to which the eye is most sensi-
tive, and to which the seeds are least sensitive, induces consid-
erable germination, although the light is not strong enough to
be used in counting seedlings.
Exposure of imbibed seeds to moonlight for 15 minutes induces
a high percentage of germination.
Seeds which germinate poorly or not at all in darkness at
constant temperature germinate well in darkness at tempera-
tures alternating daily between 230 and 5. Attempts to induce
germination in darkness with alternating pressure were not
successful.
Seeds made indifferent to light by soaking in darkness and
drying in light germinate much better at 331/2o in light than
in darkness.
The effect of light on imbibed seeds is independent of the
temperature of the seeds at the time of exposure, from 0 to
about 400. A short temperature treatment at 00 in darkness
does not induce germination in darkness but appears to exert
a favorable influence on seeds which are exposed to light. A
short temperature treatment at 40 in darkness induces consid-






Environmental Factors Affecting Tobacco Germination 43

erable germination in darkness, and also exerts a favorable
influence on seeds which are exposed to light.
Seeds germinate in soil at a depth of 5 millimeters much
better at alternating temperatures than at constant temperature.
A general discussion of the relation of light to germination
is given.

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