X-RA‘x' STUDIES
EN PHASEOLUS VULGARlS

Thesis for the, Degree of M. S.
MICHIGAN STATE COLLEGE

Clarence Fl Center

1939

 

X-RAY STUDIES IN PHASEOLUS VULGARIS

by

Clarence Frederick Genter

 

A THESIS
Submitted to the Graduate School of Vichigan State
College of Agriculture and Applied Science in
partial fulfilment of the requirements for
the degree of

YASTER OF SCIENCE

Department of Farm Crepe

1939

THEDL:

ACIQTOWLLSD GM 3N T

The writer is very grateful to Mr. H. M. Brown
for invaluable advice and assistance throughout
the course of this work.

Gratitude is also extended to Fr. E. E. Down
and to Dr. C. R. Megee for their willing co-oper-
ation and helpful advice and criticism.

The writer is indebted to Dr. J. C. Clark of
the Physics Department under whose direction all
of the x-ray treatments were made. The writer is
further indebted to Dr. Clark for technical advice
in writing this paper, and for the formula for the
calculation of the x-ray dosages used in these

experiments.

(V

CONTENTS

Introduction
Review of Literature
Physiological Effects of X-rays
Stimulative Effects
Deleterious Effects
Cytological Effects
Genetic Effects of X-rays
The First Mutations Induced by X-rays
Rate of Induced Mutation in Barley
Rate of Induced Mutation in Cats and Wheat
NacArthur's Work with Tomatoes
Experiments with Tobacco
Time of Irradiation
Types of Mutations Found
Ideas Concerning the Causes of Induced Mutations

Ideas Concerning the Nature of Induced Vutetions

Page

O'bvp-NNN

10
10
ll
12
12
13

Page

Experiment and Results 18
Object of the Experiment 18
Materials for the EXperiment 18
Basis for the Calculation of Dosages Given 19
Method of Treatment 20
The Treated Generation 22

Effects of the Treatments Shown by the Plants 22
The Method of Harvest 26
Production of Seed 26
The Second Generation 28
Description of Mutant Types 29
Distribution of Hutations 46
Discussion 49

Summary 52

1.

INTRODUCTION

Since the discovery of x-rays by Roentgen in 1895, many
investigations have been carried on to determine their effects
upon biological organisms, but there has been no reference in
the literature concerning the genetic effects of x-rays on
Phaseolus vulgaris. In 1938, an opportunity was presented to
begin a study of this type, and the work herein reported was
undertaken.

Before examining the data obtained in this study, the
nature of the x-ray and the findings of the earlier workers
will be discussed.

It is common knowledge that x-rays are produced when
very fast moving electrons experience collisions with the
orbital electrons of the stationary material of the x—ray
tube. X-rays, which are vibrations similar in nature to
visible light rays, vary greatly in length. The effective
wave length of the x-rays produced is dependent upon the
potential voltage difference between the source of electrons
(cathode) and the target (anode), and decreases as the
voltage increases. The long waves produced by low voltages
are often designated in the literature as "soft" x-rays.
Likewise, the short waves produced by high voltages are
called "hard" x-rays.

The third International Congress of Radiology which
met in Paris in 1951 adopted as a practical unit of x-ray

quantity the "Roentgen" or "r" unit. This is the quantity

of x-ray energy which, when fully utilized in the production
of ions, will produce one electrostatic unit of ions in one
cubic centimeter of air at standard conditions of temperature
and pressure.
For means of comparison, the dosage considered necessary
to redden human skin is approximately 600 r units (27).
REVIEW OF LITERATURE

No attempt will be made to present an extensive review
of literature. The material reviewed here will pertain
chiefly to plants, and only those references that seem to
bear directly on the discussion will be cited. A very
complete review of the literature has been made by Coodspeed
and Uber (7) and for further study the reader is referred to
that comprehensive review and bibliography.

It should be added that for the purposes of this paper,
the term "physiological effect" will be used to refer to the
discernible effect of the x-rays on the treated generation,
and the term "genetic effect" will be used to refer to the
transmissible effect of the x-rays on the treated or on
subsequent generations.

PHYSIOLOGICAL EFFECTS OF X-RAYS
Stimulative Effects
Although x-rays are now known to produce many genetic
effects in irradiated organisms, the earliest investigations
were entirely physiological in nature, and, if any significant
cytological or genetic effects were produced, they were over-

looked (7).

3.

In 1927, Science (26) published the report that potted
plants, grown from seeds that had been irradiated with "soft"
x-rays, grew more vigorously, flowered and fruited from 1 to 3
weeks earlier and yielded from 15% to 170% more than the
untreated material. Three lots of potatoes similarly treated
were reported to have given yield increases of 55, 107, and
170 percent over non-irradiated controls.

Shull and Mitchell (27) claimed stimulation of wheat, corn,
oats, and sunflower seedlings by treating with very low
dosages of x-rays in the early stages of germination. Treat-
ments of 30 to 120 r units were found to give best results
in their experiments. They think that the optimum is
probably specific for the various species and that the margin
between optimum dosage and over-dosage is small.

By irradiating dry soy bean seeds with small dosages of
soft x—rays, Long and Kersten (13) obtained slight stimulation
of plant growth over non-irradiated controls, using the
average green weight of the parts above ground as the basis
of comparison. Working with large numbers of plants, they
found that the average increase in weight of the irradiated
plants exceeded three times the probable error. This suggests,
but does not prove, a true stimulation as the result of x-ray
treatment.

On the other hand, Johnson (9) found that the irradiation

of sunflower seeds with a light dosage gave no increased rate

4.

of germination, no increased percentage of germination, no
increased growth during the seedling stage, and no increased
height nor weight at maturity. Working later (10) with
seedlings of tomato, sunberry, sunflower, and two species of
vetch which had all been given light treatments, she found
no stimulation of growth over controls as evidenced by
measurements of height and green and dry weight.
Deleterious Effects

Whereas little has been definitely proven concerning possible
stimulative effects of x-rays, much has beennwritten to show
conclusively that there may be very harmful effects of large
doses on the irradiated organisms. Soon after irradiation
experiments first began on living organisms, the fact that
the effects of the treatments varied directly with the dosage
in Roentgen units became evident. Many experiments have been
conducted that quite conclusively prove this point (2, ll, 14, 21).

One of the most convincing works was that of Packard (22)
in which he found that, if Drosophila eggs are exposed to
x-rays for a definite length of time, the quantitative
effects of the exposure, as measured by the number that fail
to hatch, could be used as a measure of the dosage. He found
that a carefully measured dose gave results which varied less
than 5%.

Cattell (1) found in his study of irradiated wheat

seedlings that, if the dosage remains the same, the physio-

logical effects remain the same.

5.

In a series of experiments with dry maize seeds, Collins
and Maxwell (2) found that a range of x—ray treatments existed
which caused all of the plants to die in the seedling stage
without reducing the percentage of germination. However, as
the dosages were increased, the rate of elongation of the
roots and shoots was increasingly reduced, with complete
"delayed killing" resulting from very high dosages (60,000 to
100,000 r units). They also found evidence that cell division
took place after treatments of 60,000 r units, and that not
all growth was due to elongation of the cells. They estimated
that dosages of approximately 2,000,000 r units would be re-
quired to completely inhibit germination.

Johnson (9) found that the plants recovered from
inhibitory effects of light doses in about three weeks and
differed very little thereafter in any respect from the
untreated controls.

Much variability of effect is shown by the plants that
survive, the effects are not immediately apparent, and plants
given lethal dosages have been found to live for several weeks
though little or no growth is made (11).

In an attempt to find the reason for the apparent inhibi-
tion of growth in the treated plants, Cattell (1) found, by
feeding x-rayed seedlings to mice, that the growth-promoting
Vitamin B in the embryo was destroyed by heavy doses of x-rays.

Skoog (28) later found that the "growth substance" was
inactivated by x-rays both in solution and in the intact

plant, and by comparable dosages. A water solution of growth

6.

substance, irradiated in an atmosphere of nitrogen, showed no
immediate inactivation, thus indicating that the reaction was
an oxidation. He also found that, if irradiated seedlings
were given a continuous supply of growth substance immediately
after irradiation, they were able to maintain a normal rate
of growth.compared to non-irradiated controls.
Cytological Effects

As far back as 1924, Komuro (12) made a rather thorough
study of the grosser cytological effects resulting from very
heavy doses of x-rays upon seedlings of Vicia faba. He found
that in 1.5 hours after irradiation, vacuolization of the
cytoplasm became apparent, chromatolysis was clearly evident,
and all observed mitoses were abnormal. Six hours after
irradiation, nuclear membranes were no longer visible due to
degeneration. Abnormal, binucleate cells were present. Nine
hours after irradiation, vacuolization of the cytoplasm was
manifest. Escaped nuclei were very often seen in the cyto-
plasm. Many abnormal binucleate cells, many giant nuclei,
and many multi-nucleolar cells were also found.

Stone (38) found that following x-ray treatment, mitosis
was soon stopped for a period, after which growth was slowly
resumed, with abnormal cell divisions generally occurring.
Cells about to divide at the time of treatment were found
to be prevented from further division, but those already in
mitosis were allowed to complete the division, owing to the

rapidity of the process.

7.

GENETIC EFFECTS OF X-RAYS

In spite of profound physiological and cytOIOgical
disturbances such as those already described, no genetic
effects were discovered until Muller demonstrated in 1928 (15)
that genetic variations could be induced in Drosophila by
x-ray treatment. Since that time, the use of x-rays in
genetic studies has been very extensive in both plants and
animals.

The First Plant Mutations Induced by X-Rays

Stadler (29), by irradiating germinating seeds of barley,
was the first to demonstrate that genetic effects, similar to
those produced by Muller in Drosophila, could also be produced
in plants. The cells from which the tillers will develOp are
already separated in the embryo of the dormant barley seed,
and a mutation in one of these cells will affect only one
tiller, unless an axillary bud subsequently develops above
the mutation. To be certain that a variant type in the
progeny was due to an induced mutation, the seed from each
tiller was planted separately. If the same variation appeared
in all of the head progenies of a single plant, the cause would
probably be due to a hybrid condition of the seed from which
the plant was grown. However, if a variation occurred in the
progeny of one head while the progenies of the other heads of
the same plant remained normal, the cause would probably be
due to a genetic change that occurred during the development
of the treated plant, presumably at the time of x-ray treatment.

In the first successful experiment, three mutant types were

8.

found in 70 head progenies from 26 plants grown from x—rayed
seeds. One mutation was a white seedling that was colorless
at the time of emergence and died in 2 or 5 weeks. One was
a virescent mutation that was colorless at time of emergence,
but became a pale green in a few days. One was green at
emergence, but later became yellow and died. Each of the
mutant types made up from one-fourth to one-eighth of a
single head progeny from a plant of which other head pro-
genies were entirely normal.

Rate of Induced Mutation in Barley

About 90% of the recognizable mutations induced by x-rays
may be detected in the seedling stage (30). Using this
assumption as an index upon which to base conclusions, several
genetic reactions of barley seeds under various conditions of
x-ray treatment have been noted.

The rate of mutation is not affected appreciably by changing
the percent moisture in the seeds by soaking, within the limits
of 15 and 40 percent moisture (35). The temperature of the
seeds during irradiation had no pronounced effect when
temperatures ranged from 10 to 50 degrees Centigrade (30).

No significant difference was shown by equal intensities
(r units) of irradiation when using 40,56,8l,98 and 116
kilovolts as the potential difference between the anode and
cathode of the x-ray tube (55).

The rate of mutation was found to be proportional to the
total intensity of the irradiation applied, within the limits

of the sampling error, for both dormant and germinating seeds (55).

9.

The average rate of mutation was about four to eight times
as high in germinating seeds as in dormant seeds in identical
treatments near the limiting intensities for germinating seeds (55).
However, dormant seeds were found to tolerate an x—ray dosage
of 15 to 20 times as great as did germinating seeds, and the
mutation rate possible with dormant seeds was about double
that possible with germinating seeds (55, 55).

Mutation was induced in dormant seeds whether they were
planted immediately after the treatment or not (50).

In some cases, two mutations occurred in the same head
progeny (55).

Rate of Induced Mutation in Cats and Wheat

Each of the genera Aygna,IHordeum and Triticum include
species of 7, 14, and 21 pairs of chromosomes, but the
cultivated barleys have only 7 pairs of chromosomes, whereas
the cultivated oat and wheat varieties contain 21 pairs of
chromosomes. The results of several experiments concerning
the rates of mutations of oats and wheat have been presented
by Stadler (52, as, 55).

Avena sativa and A. byzantine with 21 pairs of chromosomes
yielded no mutations with dosages sufficient to have produced
70 mutations in barley. ,Aygna'brevis and A. strigosa with 7
pairs of chromosomes gave 14 mutations at rates slightly
lower than, but not statistically different from, barley (52).

Triticum vulgare with 21 pairs of chromosomes, yielded
no mutations with dosages which would have yielded about 40

10.

in barley. 'T.'dg§gm and T, dicoccum with 14 pairs of chro-
mosomes mutated at a very low rate. 2, monococcum with 7
pairs of chromosomes, in a small test, yielded mutations at

a slightly higher, but not significantly different, rate than
barley (52).

Stadler (52) suggested as a probable explanation of these
results that in the formation of the polyploid species, a
certain amount of gene duplication took place. A single
recessive mutation induced in a cell containing two or three
dominant factors for the determination of a single plant
character would have no visible effect on the plant. Con-
sequently, a much lower rate of mutation could be detected
in the polyploid species. Stadler also noted that the
species with the higher numbers of chromosomes were injured
less by x-ray treatments.

MacArthur's Work with Tomatoes

In the progeny tests that MacArthur (14) made of tomato
plants grown from x-rayed seeds, he apparently disregarded
the possibility of there being chimeric tissue in the treated
material. Single fruit progenies were grown from the
irradiated material, and no attempts were made to determine
whether the mutant tissue was sectorial or not. Out of 546
progenies thus grown, 45 mutations were found, all of which
were recessive and monofactorial.

Experiments with Tobacco
Working with species of the genus Niggtigna, Goodspeed

11.

and Olson (6) experimented to discover the effects of
irradiation on sex cells. They x-rayed flower buds of all
ages up to anthesis. More than 1,000 plants came to
maturity, grown as seven populations from seven treated
capsules. More than 20 percent of variants appeared, one
capsule yielding 156 variants out of 168 plants. The
majority of variants exhibited some reduction in fertility,
but only rarely were they completely sterile, while a
number were completely fertile.

In experiments in which pollen was x-rayed and used to
fertilize untreated ovaries, variations appeared as readily
as when entire buds were treated (5, 6).

Further observations on tobacco have been reported. No
relation was found between the stage of maturity of the sex
cells and the effectiveness of irradiation (4). Sex cells
undergoing division yielded more mutations than those in the
resting condition (5). Mutations and chromosomal aberrations
may be produced with very low voltages by irradiation of the
sex cells (7). Mature pollen, like dry, dormant seeds, is
quite resistant to the effects of x-rays. (4, 7).

Time of Irradiation

Stadler (54) demonstrated that there was a great
advantage in x-raying the embryo at an early stage of
development. Corn treated after pollination showed affected
areas varying from one—half of the endosperm in seeds treated

28 hours after pollination to barely visible sectors in seeds

12.

treated 8 to 10 days after pollination. The entire embryo
seemed to be affected by changes induced by treatment 28
to 48 hours after pollination, whereas the chimeras grew less
and less in extent as more time elapsed between pollination
and irradiation. Obviously, the amount of differentiation
that had taken place in the embryo at the time a seed was
x-rayed greatly affected the size of the sector that arose
from any one disarranged cell.
Types of Mutations Found

0f the types of mutations that appear in the progenies
of treated materials, chlorophyll deficiencies make up
the vast majority of the mutants (14, 55). Numerous types
of modification of both vegetative and floral parts have
been reported (5, 6, 8, 14, 51, 55). Most of the mutant
seedling characters are lethal and almost all are unfavor-
able to growth (55). No case of a dominant mutation has
ever been reported from x-ray treated plant material, but
in all cases, the few mutants that reach maturity breed
true for their recessive characteristics (55).

IDEAS CONCERNING THE CAUSES OF INDUCED MUTATIONS

Stadler (55) defined a mutation as "a transmissible change
in the gene." Workers in the field are still not agreed as
to the real nature of mutations, nor are they sure that
induced mutations result in the same manner as the so-called
"Spontaneous" mutations found occurring naturally (55).

Muller (16) argued against the idea that the x-rays

produced a chemical within the cell and that the chemical then

13.

caused the mutation to occur. He reasoned that if this were
the case, other chemicals should accomplish the same effect.
All attempts by Patterson and Muller (25) to produce genic
mutations by the application of specific substances, without
irradiation, failed. Neither was there any case of "delayed
induction" as might be expected, if chemicals were the cause
of the induced mutations (16).

The theory that induced mutations were the direct result
of electronic hits which caused transmissible genic changes
was next advanced and generally accepted (25). A second
point was the fact that of two chemically identical allel-
omorphs present very near together in a treated cell, only
one becomes altered (25). Third, the degree of phenotypic
change (proportion of lethals to non-lethals) was obviously
independent of the dosage (25). Another argument was the
direct and simple proportionality that has been shown to
exist between the frequency of the induced mutation and
the amount of energy absorbed from the irradiation (7, 25).
Also, the number but not the degree or nature of the
individual mutations changed with the dosage (l5).

IDEAS CONCERNING THE NATURE OF INDUCED MUTATIONS
Although breakage and rearrangement of the chromosomes
was known to result from irradiation, the first mutations
were thought to be caused entirely by chemical changes in
the gene itself, apparently identical in gametic behavior
and, in many cases, in phenotypic effect with well-known

14.

mutations of spontaneous origin (7, 16, 17, 25, 24). In

the few cases tested by crossing the mutant type onto un-
treated individuals, the mutant characters behaved as would
be expected on the assumption that the mutation was a change
of a dominant gene to a correSponding recessive in the
somatic cell of a homozygous individual (55).

Cytological as well as genetic effects have been found
to increase linearly with dosage, within the limits of their
respective experimental errors (7, 56). Translocations
between non—homologous chromosomes arose in irradiated
Drosophila with nearly the frequency of detectable gene
mutations (18). Occasionally entire chromosomes were lost
from cells, but in the vast majority of cases, only a small
deletion occurred (25). Vorking with Cre is, Navashin (20)
concluded that there were no limitations on the size of
dislocated chromosome fragments, nor any regulation as to
the direction of the process.

Stadler (56) gives the types of chromosomal aberrations
that have been found by cytological investigations. There
may be the loss of either a terminal or internal segment of
a chromosome, the removal of a segment to a new position
in the same or in another chromosome, the inversion of a
segment in its original position, and the interchange of
segments between chromosomes. No case of simple trans-
location of a fragment to a whole chromosome has been found.

Although many deficiencies are associated with translocation,

15.

some deficiencies occur in cells which are otherwise normal.
The importance of these alterations is still not fully
appreciated and their presence and behavior is a subject
open for much further discussion.

Like the induced gene mutations, translocations are
commonly accompanied by lethal or deleterious character
changes (18, 19). Partial sterility is also common as a
result of chromosomal aberration because the alteration is
often lethal in the haploid condition to he gametOphyte
generation (54).

Because the early mutations were all changes from the
dominant to the recessive, some workers believed that they
were due to destruction of the gene or parts of the gene
by the x-rays (56). If such were the case, no forward steps
could ever be taken. Considerable research was undertaken to
attempt to induce dominant mutations. Muller (15), Patterson
and Muller (25), and Timefeeff-Ressovsky (59), have claimed
progressive mutations in Drosophila, produced by causing
mutant types that originally resulted from irradiation to
return to the original type by further irradiation.

The fact that mutations can be induced either of two
different ways, and that a cycle of mutational change can
be completed is considered by Patterson and Muller (25) as
proof that not all mutational changes caused by x-rays
consist of losses. They also believe the "progressive"

mutations can probably be produced by irradiation in cases

16.

where there is a possibility of their occurring at all.
Muller (15) further believes that the production of mutations
by x-rays in each of two ways proves that the x-rays have a
reconstructive rather than a destructive action on the genes.
The changes in the chemical composition of the genes is
thought by Muller (16) to be "endless in their eventual
possibilities." Muller also suggested that, if the "spon-
taneous" gene mutations serve as the basis of evolution, as
is apparently the case, hen the artificially produced muta-
tions must also include amongst them changes as good as those
which occurred naturally (l6).

Stadler (57) questioned the assumption of Muller that the
induced mutations are simple chemical changes of the genes.
No one has proven the nature of change in the chromosome of
a natural mutation, let alone of the induced ones. He says:
"In plants it is only on the assumption that deficiencies are
invariably lethal to the gametophyte that it is possible to
Justify the description of viable mendelian variations in
general as gene mutations. This assumption is obviously
invalid in the polyploid series, and is of doubtful validity
in the other species." He believes (56) that mutations are
not a single homogeneous class of germinal variations, but
may include variations in the arrangement of the chromosomes
as well as variations entirely within the gene itself. He
stated that there are many induced mutations that are known
by cytological investigation to be the result of chromosomal

aberrations, and that it is very possible that the rest are

17.

due to chromosomal aberrations that are too small to be
detected cytologically. Although he grants that the reverse
mutations are a convincing argument in favor of simple genie
changes, he argues that cases are known in corn, in which
mutant endosperm tissue, known to be due to a deficiency

that had been induced by x-raying the pollen, occasionally
grew small areas that showed the recovery of the lost dominant
gene. He further believes that too little is yet known about
the causes of natural mutations even to be able to prove

that the types of mutations involved in gene evolution are
affected by x—irradiation.

Muller and Altenburg (18) made the following statement
regarding the evolutionary value of x-ray induced variations:
"The lethal and other deleterious effects of most transloca-
tions, when homozygous, do not rule out translocations as
factors in evolutionary change any more than the lethal or
deleterious character of the vast majority of detectable
gene mutations rules out gene mutations as the main building
blocks of evolution. It is not to be expected that the
majority of any changes occurring at random will have survival

value."

18.

EXPERIMENT AND RESULTS
OBJECT OF THE EXPERIMENT

The primary objects of the experiment with x-rays herein
reported were to determine the types of mutations that might
be produced, and the rates of these mutations in both dormant
and germinating seeds of white pea beans. Of secondary
importance was to be the study of any physiological and
genetical variations that might present themselves.

MATERIALS FOR THE EXPERIMENT

For this study, the Michelite variety of Phaseolus
vulgaris which was recently introduced by Michigan State
College was used. The beans were a representative sample
of an increase lot grown on the college farm in 1957.

Several factors entered into the choice of the white
I pea bean as the subject for this experiment. The treatments
were to be given in the spring, and a second generation
would necessarily have to be grown in order to allow any
recessive mutations to segregate out. It was important,
therefore, that the plants used be annuals. In order to
have the segregation of recessive factors in the shortest
possible time, it was necessary to self-fertilize the
hybrid material, and a normally self—fertilized plant would
be highly desirable. A plant that had not yet been studied
for genetic effects of irradiation, and one that was of

considerable importance in Michigan was further desired.

19.

BASIS FOR THE CALCULATION OF DOSAGES GIVEN

Since intensity of electromagnetic energy, such as x-rays,
is expressed in the units of energy per square centimeter per
second, and since the inverse-square-law applies to x-rays as
well as to visual rays, by making use of the work of Ulrey (40),
on the spectrum of x-rays produced by a thick tungsten target
bombarded by electrons of various energies, it may be shown
that the intensity of the x—ray beam varies inversely as
the square of the distance from the target, and directly
as the first power of the tube current, and as the square
of the tube voltage.

A Coolidge cathode x-ray tube with a thick tungsten target,
such as was used in the experiments herein reported, when
operating at 100 K.V. and 10 m.a. produces an x-ray beam of
intensity equal to 0.54 r per second per square centimeter
at a distance of one meter from the target (5).

In these experiments, the tube voltage and current was
maintained at 60 K.V. and 10 m.a., respectively. The material
for irradiation was placed as close to the x—ray tube target
as was practical, both from the standpoint of electrical
insulation and heating effect. This distance was 15 centi-
meters from the center of the target to the center of the
irradiated material.

Remembering that the total energy given off from the
target is proportional to the square of the tube voltage,
and inversely proportional to the square of the distance

from the target, and using the constant of 0.54 r per second

20.

per square centimeter with the conditions of 100 K.V. and
ten m.a. at 1 meter, the intensity of the x—ray beam 15
centimeters from the target of a tube operated at 60 K.V.
and 10 m.a. may be computed by the equation

100 )2

H 2 60
I 0.04 X (1'5“) X (160

I = 7.2 r per second per square centimeter
METHOD OF TREATMENT
In an attempt to reduce experimental error, the beans
were graded over a size 15 round hole screen to remove the
smaller sizes, and then all checked, weathered and distinctly
odd-shaped beans were removed.

The beans were then divided into three groups for treat—
ment. One group was left dormant, a second was germinated
for 18 hours before treatment, and the third was germinated
for 56 hours before treatment.

The seeds of the first two groups were irradiated for
periods of 5, 15, 50 and 60 minutes. This is equivalent to
dosages of approximately 2,160, 6,500, 15,000 and 26,000 r
units respectively. The seeds of the third group were given
a single dosage of twelve minutes which was equivalent to
approximately 5,200 r units.

Care was exercised while the beans were being x—rayed to
keep the seeds reasonably cool. A blast of air from an
electric fan was kept on the apparatus to insure circulation
of air over the beans.

The treatments are summarized in Table 1.

Table l.

ment in minutes and dosage in r units.

 

Condition of

Number of

Number of seeds treated and the length of treat-

 

Treatment

Dosage in

 

 

 

 

 

 

 

 

 

i

beans at time seeds in
Of treatment irradiated minutes r units
500 0 0
400 5 2,160
Dormant 400 15 6,500
400 50 15,000
400 60 26,000
200 O 0
400 5 2,160
Germinated
400 15‘ 6,500
lShmms
400 50 15,000
400 60 26,000
Germinated 200 , 0 l O
56 hours 400 12 s ! 5,200

 

I

of treatments at this point.

a The x—ray tUbe burned out

and terminated this series

-—

 

22.

THE TREATED GENERATION
The seeds were all planted within an hour or two after
the treatment was made. Plantings were made on the following
dates in 1958:
All dormant seeds, treated and untreated-~-—June 8.
All seeds germinated 18 hours —————————————— —June 9.
All seeds germinated 56 hours -------------- ~June 10.
Complications arose immediately after the beans were planted.
Heavy rains fell either during the actual planting or within a
very few hours afterwards in every case. The soil was fairly
heavy and it baked more or less before the beans had germin-
ated and broken through the surface of the ground. The fact
that there was a crust on the ground undoubtedly affected the
number of seedlings that broke the crust and survived.
Effects of the X-ray Treatments Shown by the Plants
Soon after the plants began to come up, it was noted that
many of the seedlings had pushed through the ground, but had
died without producing any leaves whatsoever. The stems
would straighten up in most cases, and the cotyledons would
open, but the shoot buds would produce no apparent growth.
The seedlings might remain thus for a week or ten days and
then die. The roots of these plants were found to be about
one-half an inch long, thick and swollen. The root tips
also failed to grow actively and finally rotted.
These dying seedlings, tOgether with the fact that, due
to the crust on the ground, beans still kept coming up after

three weeks from the time of planting, made accurate germina—

25.

tion and survival counts rather difficult. However, on

June 27, the number of plants that were above ground and were
apparently healthy were counted. The number that had come up
and had died soon afterwards were also counted. The number
that had failed to come up at all was obtained by subtracting
the total number of plants that had come up from the number
of seeds planted. This was done for each plot, and the
results are given in Table 2.

When germinated seed was treated, the number that failed
to come up increased with the length of the x-ray treatment.
When dormant seed was treated, the number that failed to
come up increased only in the 50 and 60 minute treatments,
and only the 60 minute treatment showed a large increase in
the number.

The germinating seeds were injured more than the dormant
seeds by identical treatments except by the lightest treat-
ment, in which little difference could be found between the
two lots. The seeds that had been germinated for 56 hours
before the treatment was given were severely injured by the
12 minute exposure, even more so than the seed that germin-
ated 18 hours was injured by the 15 minute exposure.
Apparently, the susceptibility to injury by x—rays increases
very rapidly after the seeds begin to germinate.

It is not known how many of the beans that came up, after
the first count was made on June 27, died without further

grOVJth o

24.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Germination and survival counts of treated beans.
_*Dose NUmber Not up Dead, Living, Living+Living, Total
in by har-
mino treated June 27 June 27 June 27 July 8 July 22 vested
Hfifii Seeds dormant when treated
o 500 7 25 260 265 264 25s;
5 400 28 55 557 546 544 540
15 400 22 45 525 550 550 522
so 400 59 f 95 266 256 242 168
60 400 170 i 118 112 82 75 62
Seeds germinated 18 hours when tteated ‘fi—l
o 200 9 14 177 177 176 0-4
5 400 28 51 541 552 550 556 l
15 400 61 52 267 271 260 22s
50 400 165 70 147 156 121 95
60 400 282 54 64 54 52 49
_seeds‘gernin5ted 56 hours when treated 2‘;1
l o 200 e 20 171 175 175 160
12 400 150 50 220 '220 212 200

 

 

 

 

 

 

 

’—

25.

The inhibitory effects of the x-rays on the plants that
survived remained in.evidence by the stunted growth of the
treated material. The 5-minute treatments gave only a slight
stunting effect, but the effects became progressively more
severe as the length of the treatment increased. As in the
seedling observations, the seeds that were germinated at
the time of treatment continued to show more severe injury
than the seeds treated in the dormant stage by identical
treatments.

Many of the plants, especially in the more lightly treated
plots, apparently recovered from the effects of the irradi-
ation in three or four weeks and grew normally. However,
even these never quite overtook the early lead gained by the
controls. The more heavily treated plants were severely
retarded and grew very slowly in the early part of the
summer.

No great difference was noted in the shape or structure
of the leaves or plants grown from the treated material.

The only differences noted seemed to be due entirely to the
slow growth of the plants. .

Late in the summer when a period of wet weather set in,
many of the heavily treated plants began to grow vigorously
and many set a heavy crop of seed. Late in the fall, the
treated plots were rather characteristically spotted by the
dark green foliage of late-maturing plants that had been
retarded earlier by the irradiation. The untreated controls

matured much more evenly and a majority of the plants

26.

matured much earlier than did the treated ones.
The Method of Harvest

Because there was so much variation in the time of
maturity of the treated material, and since the most retarded
plants seemed logically to be the most affected by the x-ray
treatment, knowledge as to whether there was a correlation
between the time of maturity and the percentage of mutations
was desired. Accordingly, sacks were numbered consecutively,
and as the plants matured, the pods were picked, placed in
the proper Sack, dried and stored. The results of this
method of harvesting are given in Table 3.

It will be noted that in nearly every case, the heavier
the treatment, the later was the date that the greatest
number of plants were harvested. This seems to bear out
very well the earlier conclusions made concerning the ap-
parently retarding effects of the x-rays.

Production of Seed
During the winter months the seeds were threshed from
the pods. Later, in getting the material ready for planting,
the number of seeds from all of the treated material and from
one dormant sample of untreated material was counted. These
findings are given in Table 3.

‘With only one exception, the average number of seeds
per plant dropped sharply on the final date of harvest.
Because of this, the plants that matured late in the fall
tended to reduce the average number of seeds produced

'within a given treatment. The only large differences in

Table 3.

(upper figure) and number of seeds

Dates of harvest,

27.

number of plants harvested

51 ant (lo1er fig

‘

per

 

 

 

 

ure).

-—~---—‘___.———.-——s-‘——

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

pgsefNumber Date harvested
in I H---....,-...__- -,. _..____ _- -
min. treated d/ioIo/isis/isis/saqo/ olio/egio/ia tr. otal
Seeds dormant when treated
‘ .
0 g 500 0 96’ 62 52! 19 28! 21 ' 0 258
; 0 62. 62.7 65. 8 54 8 45.01 11.5 0 56.0
5 3 400 213 56 56 87i 71 21! 28 0 540
i 95.2 82.5 75.7 66.6!EO.8 56.2; 22.8 0 65.4
n l
15 ’ 400 2 55 25! 79! 65 453 48 0 522
74.4 66.5 65.6'72.li68.8 45.7 28.7 1 0 60.5
50 400 0 1 5 5? 25; 25 109 2 168
l l
60 400 0 0 0; 0‘ 12 8 29 15 62'
, 0 0 0; 0 75.5 77.8 56.7 4.4 45.1
‘ Seeds germinated 18 hours when treated
' 5 f 400 26 45 59: 78 55 9 85 g 1 556
64.1 78.9 70.6 57.5 48.7 51.2 55.4 38.0 ‘54.5
15 400 14 17 20 59 24 41 68 g 0 225
84.9 82.8 92.0 71.0 67.5 55.4 26.5, 0 57.0
1
50 400 1 7 6 15 12 41 1 5 95
45.0 27.4‘80.0'60.0 49.1 59. i 29.5; 4.0 40.1
60 400 0 1 2 0 10 8’ 22 l 6 49
I 0 91.0 99.0 0 80.9 90.6; 56.5. 7.0 54.5
.4..L - L L.._._. L-.. 1 -
Seeds germinated 56 hours when treated 4
3 0 200 49' 51 7 9‘ 20? 0 180;
‘ 6il 810 safd counts \.ere Jade ; j
! I
E 12 E 400 18:02516g 54 451 5:; 50: 5 200;
l 1 .69.5|5 . 551.5,65.8 69.0;5 5.7; 22.7; 1.5 55'5i

__.-_-—.

 

 

 

 

 

 

 

 

 

 

 

 

 

The plants in this column were taken from the field

Sept. 1 and matured in the greenhouse.

 

28.

the average number of seeds produced per plant that were
evident between the various treatments were in the 50-minute
treatments of the dormant and the germinated seeds. In each
of these treatments, a large percentage of the plants were
harvested on the last harvest day.

About the middle of the flowering period a rather
severe and prolonged drought occurred that may have influ-
enced the seed set to quite an extent. The earlier plants
would have set the most seed and the later ones would set
under the handicap of dry weather. This drought, however,
was not important to many of the more retarded plants be-
cause early in August a period of wet weather occurred and
those plants, still growing steadily because they had not
yet set any seed, flowered profusely and set a late crop.
The fall was late and many of these late plants fully
matured. Many, however, matured a very small number of
seeds, the importance of which will be discussed.

THE SECOND GENERATION

In the spring of 1959, the material to be planted was
sorted according to the number of seeds per plant in order
that plots of the same length could be planted in a block.
The seeds were planted about 5 inches apart in the row. Most
of the material was planted June 7 just before a rain, but
several of the longer plots were planted under rather unfavor-
able conditions two days later.

Several of the larger progenies of the more lightly

treated material were not planted for lack of space.

29.

Observations were made every few days after the beans
first came up, and at longer intervals thereafter. Only
well-defined variations were classed as mutant types.

Progenies showing mutant types were thinned to give the
variants more favorable growing conditions.

Description of Mutant Types

A total of 152 mutants were found up to August 15, and
a large variety of forms occurred. The mutations that were
apparently the same in phenotypic appearance were grouped.
The following classification of mutant types, with the

number of progenies producing each type, resulted:

No. of
progenies Description of Mutant Type
29 Yellow seedlings that died soon after emergence.
13 White seedlings, entirely lacking in pigment, that
died soon after emergence.

9 Light green seedlings that died soon after emergence.

8 Light green seedlings that lived and formed light ‘
green secondary leaves; some of these plants have
died.

6 Green primary leaves; light green secondary leaves;
some plants nearly normal in size.

6 Light green primary leaves; secondary leaves light
green when first formed, but turn darker green with
age.

4 Yellow primary leaves; secondary leaves light green.

3 Yellow primary leaves; secondary leaves dark green.

30.

No. of
progenies Description of Mutant Type
2 Light green primary leaves; several very bright yellow
secondary leaves produced; died in a few weeks.
2 Yellow primary leaves; produced a few secondary
leaves; then died.
1 Light green seedling; many very small secondary
leaves. Fig. 1.
15 Dark, glossy green; veins distinctly indented;
chiefly dwarfs. Fig. 2.
1 Yellow edges and veins on secondary leaves; very
small plants.
1 Secondary leaves light green along veins; plant
viny; leaves somewhat smaller than normal. Fig. 3.
2 White chimeras; plants nearly normal in siZe. Fig. 4.
ll Leaves long and narrow, "willow leaf" type; plants
rather small. Fig. 5.
5 hick leathery leaves; slow growth. Figs. 6 and 7.
4 Small bushy plants with small, rough, irregular
leaves. Fig. 8.
1 Narrow primary leaves, dwarf plant.
3 Appearance of leaves resembles mosaic; plants
normal height, but slender. Fig. 9.
l Mottled primary leaves; dwarf plant.
3 Dark green, small, slender vine; no branching.
1 Small plants with small narrow leaves.
2 Seedlings very erect and tall; very erect later

growth.

51.

No. of
progenies Description of Mutant Type
4 Long narrow leaves, but wider than "willow leaf"
type.
7 Dwarf in size and structure; leaves proportional
in size to the size of the plant; plants 4 to 7
inches high.
4 A great many very slender branches; plants of
good size. Fig. 10.
l Rounded leaves; number of leaflets varies from
one to three. Fig. ll.
1 Leaves have 5 leaflets instead of 5; small plant.
Fig. 12.
1 Center leaflet football shape; attachment to petiole
flat. Fig. 15.
l Dwarf, squat plants, rosette in form; died.
1 Erect, dense, cylindrical in form; dark glossy
green color; plants 5 to 10 inches high. Fig. 14.
l Stems appear very susceptible to rust; tips of vines

green with the rust becoming progressively worse
down the stem.

Approximately 67% of the mutations were chlorophyll
abnormalities, almost all of which (i.e. of those that
survived) were less than normal size despite the fact that
'the rows had been thinned to give the mutant types more

:favorable growing conditions.

32.

 

 

Fig. 1. Small bushy plant with very small leaves.

The primary leaves were light green.

33.

 

 

Dark, glossy green plant; veins distinctly

Fig. 2.

indented; small in size.

34.

 

 

 

Fig. 3. Plant showing light green areas along the

Veins of upper leaves; viny habit of growth.

35.

 

 

Fig. 4. Plant showing a white chimera.

36.

 

 

 

Fig. 5. Bean plant showing long, narrow, "willow leaf"

type of leaves.

37.

 

 

leathery leaves; slow

Plant showing thick,

Fig. 6.

clusters of very small leaves at the growing

growth;

points.

38.

 

 

 

Fig. 7. Glossy green plant showing thick lower leaves
and clusters of very small leaves at the growing points.

Normal plants on each side of the mutant in the center.

39.

 

 

 

Fig. 8. Small plant in the center showing small, rough,

irregular leaves. TFormal plants surround the mutant.

40.

 

 

 

Fig. 9. Plant in center showing small, dull, rolling

leaves; Normal plant at the left.

41.

 

 

 

Fig. 10.
Mutant in center showing a large number of very slender

branches.

42.

 

 

 

Fig. 11. Bean plant showing large rounded leaves and

the number of leaflets varying from 1 to 5.

43.

 

Small plant showing the mutation from 5 leaflets

Fig. 12.

to 5 leaflets per leaf.

44.

 

 

 

Fig. 13. Small, glossy plant showing the football
shaped center leaflet, and the flat attachment to the

petiole of the leaf.

45.

 

 

 

Fig. 14. Small center plants showing the mutant type

with glossy green leaves, and erect cylindrical form.

46.

In the plots segregating out the mutant types, the ratio
of mutants to total progeny population varied greatly. At one
extreme were segregations of 1:1 and 3:5 in the smallest plots,
and 8:54, 7:50, and 15:42 in the larger plots. At the other
extreme were ratios of 1:65, 2:105, 1:52, and 2:60. Ratios
ranging from one extreme to the other were found.

Distribution of Mutations

Except for the large percentage of mutants in the 5-minute
treatment, the percent of mutants appearing from the various
treatments of dormant seeds was approximately proportional
to the dosage given.

A similar relationship did not hold for the germinated
seeds (Table 4). The percent of mutations that appeared as
a result of the 15—minute treatment in this group was about
three times that resulting from the 5-minute treatment, but
the percent of mutations resulting from the heavier treat-
ments was far below that expected on the assumption that the
rate of mutation is proportional to the dosage.

The rate of mutation in the germinating seeds exceeded
that in the dormant-treated seeds in identical treatments in
only the 15-minute treatments.

Table 4 also shows that the rate of mutation tended to
increase with the later dates of maturity.

Table 5 shows that the average percent of mutations
appearing in the progenies increased with the size of the

progeny in every case.

Table 4

Number of progenies planted in 1959 (upper figure)

vest in 1958, and percent of mutation.

and number of mutations (lower figure) for each date of har—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treat- L_‘ Date harvested in 1958 %of muta-
ment. : ~ ions per
in min.E/12 h/15J9/19 9/23 9/30J10/e 10/13 Gr. Total treatment
_ - Seeds dormant when treated -
5 7 42 27 52 27 15 26 O 196
1 2 l 5 0 2 1 0 10 5.1
15 17 24 16 48 45 58 47 O 255
O 2 4 8 2 0 4 O 20 8.6
50 O 1 5 5 25 25 109 2 168
0 0 2 1 8 7 12 0 50 17.9
60 0 O O O 12 8 29 15 62
O O O O 6 5 7 1 19 50.7
Seeds germinated 18 hours when treated
5 2O 25 25 55 51 9 78 1 262
1 0 l 5 5 2 5 O 15 5.0
15 9 9 8 28 18 50 67 O 169
4 5 2 5 2 5 10 O 27 16.0
50 1 7 6 15 12 8 41 5 95
0 1 O 0 O 2 l 1 5 5.5
60 O 1 2 O 10 8 22 6 49
O l O 0 5 2 6 O 12 24.5
Seeds germinated 56 hours when treated
12 18 25 16 54 45 51 5O 5 200
0 1 O l 5 2 7 0 16 8.0
Totals 72 134 103 235 239 172 449 30 1434
6 10 10 19 28 25 51 2 152 10.6
% of mut-8.5 7.5 9.7 8.1 11.7 14.5 11.4 6.7 10.6
ations per
date of
m harvest

 

 

 

Table 5.

Number of progenies of the various sizes (upper

figure), numher of mutations appearing in each size (lower

figure), and average percent of mutations in each size.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Number of seeds per progeny Net * Total
in *Over
minutes 1-10 11-20 21-50 51-45 46-70 71 planted planted
1 Seeds dormant when treated
5 13 26 54 4o 83 o 152 196
O 0 0 2 8 O 10
15 12 52 29 54 86 2O 89 255
0 2 5 5 9 1 20
5O 40 27 20 51 55 15 168
O 2 5 5 12 8 50
60 19 2 8 9 11 15 62
l O O 5 7 8 19
Seeds germinated 18 hours when treated
5 22 25 47 65 91 14 74 262
0 2 5 5 5 O 15
15 15 22 2 55 56 19 54 169
1 5 5 6 7 5 27
5O 11 16 16 25 12 15 95
l 0 1 5 O 0 5
60 7 8 4 7 5 18 49
O 0 l 4‘ O 7 12
Seeds germinated 56 hours when treated
12 14 22 17 45 42 60 200
O 5 1 5 5 2 16
Totals 155 178 197 511 421 174 569 1454
5 12 19 54 55 51 152
Ave. % of
mutations l.7 6.7 9.6 10.9 12.6 17.8 10.6
* Net planted for lack of room. All progenies not planted

contained more than 71 seeds.

 

49.

Two types of mutations were found in each of 11 different
progenies.

Only one segregating progeny was found among 160 controls,
which tends to indicate that the material was rather uniform
when the experiment began.

DISCUSSION

Several factors may have contributed in making the obServed
rate of mutation quite different from the actual mutation rate,
especially in the germinated seeds. The crust that formed on
the ground the first year and killed many of the weaker seed-
lings may have been a factor, because the data shows that the
plants that were injured and retarded by the x-rays tended to
produce the greatest percentage of mutations.

The large number that failed to mature may have been a
factor also, because of the small progenies of many of the
plants harvested late in the fall. The size of the progeny
seems rather important to the observed results because the
rate of mutation increases with the size of the progeny.

Several of the treated plants which were moved into the
greenhouse September 1 either died without producing any seed
whatsoever or matured a very small number of seed as shown in
Table 5. These plants were the most retarded of those sur—
viving the treatments, and they came chiefly from the heavily
treated and the germinated plots.

Much more accurate results could undoubtedly be obtained,
especially in the seeds most severely injured by the x-rays,
if the treated material all fully matured, and if favorable

50.

soil conditions were present to afford maximum survival of
seedlings.

The regular increase in the rate of mutation as the size of
the progeny increases is probably due to the fact that only a
small sector of the plant may be affected by the induced
mutation in the seedling. Consequently, the tissue is
chimeric and the number of seeds that will be homozygous
for the mutant character will depend upon the number of
seeds produced and the chance that the character will be
segregated out. If the mutant character is caused by a
recessive factor, the expected ratio will be 5:1 for the
number of seeds produced by the affected area. The larger
the number of seeds produced, therefore, the greater the
likelihood that the seeds produced by the affected sector
will contain one or more seeds homozygous for the mutant
factor.

The germinating seeds failed to exceed the dormant seeds
in the rate of mutation in identical treatments except in the
one treatment already mentioned (lS-minute). This may have
been due to the apparently high mutation rate in the 5-minute
treatment of the dormant seeds, and to the unfavorable
environmental factors to which the weakened heavily treated
germinating seeds were subjected. By far the majority of the
germinated seeds that were treated for 50 minutes matured
plants very late in the fall and had a low average yield per
plant. Many of the progenies of the heavily treated plants

were grown under unfavorable conditions. It is also

51.

apparent that the lethal dosage for germinating seeds is
much smaller than for dormant seeds. There was a great
difference in the effect of the x-rays on plants with a
given treatment, and it is probable that only the least
injured of the germinated seeds survived. More carefully
controlled growing conditions would be necessary to accu-
rately determine the true effects of the x-rays on the germr
inating seeds, but the environmental factors were so unfavor-
able to the injured plants that the data must have been
greatly influenced.

No cytological studies were made to attempt to explain
the causes of the mutations. However, the fact that certain
types of mutations occurred several times, whereas many of the
types occurred only in one progeny, promotes speculation as to
probable cause. Are certain genes more easily changed than
others or are there several complementary genes working
together to produce the readily mutant characters? Surely
translocations and inversions would not occur regularly in
the specific manner necessary to affect any given gene.
The field seems necessarily limited, therefore, to either
deletions or simple chemical changes of the gene itself.
And are the various chlorophyll abnormalities, leaf varia-
tions, etc., allelomorphic or is a different gene affected
in each? The only means of determining the number of genes
affected in the many progenies producing the same or similar

mutant characters is by crossing plants, either of the type

52.

or carrying the mutant factor, in one plot with affected
plants from other plots.

Some question arose concerning the segregation for suscept~
ibility for disease in one control plot. The ratio was 22:8
or nearly 5:1 for normal:disease. Since the ratio is so close
to the expected ratio for a monohybrid, the author believes
that it is due to a chance cross with a susceptible plant in
1957.

SUMMARY

Both dormant and germinated bean seeds were x-rayed to
study the effects of the treatments on the treated generation,
and to observe the types and rates of mutations that might
show up in the progenies of the treated material.

The physiological effects of the x-rays varied with the
intensity of the treatment. Germinated seeds were found to
be more severely injured than dormant seeds by identical
treatments.

The x-ray treatments tended to retard the growth and
development of the plants of the treated generation.

Considerable differential effect was evidenced by the
treated plants, but no great difference was noted in the
shape or structure of the leaves or plants except that common
to very slow'growing bean plants.

Mutations were readily produced in the white pea bean and
many different types of mutants were found.

The rate of mutation was roughly proportional to the

intensity of the dosage for dormant seeds, but the propor-

55.

tionality did not hold for germinated seeds.

The ate of mutation in the germinated seeds exceeded
the rate in the dormant seeds in only the l5—minute treatments.

The rate of mutation tended to increase with the later
harvest dates in the fall.

There was a steady increase in thex¢ate of mutation as the
size of the progeny increased.

A mutation produced in a treated plant is undoubtedly
sectorial because of the wide ratios found in the progeny

tests.

10.

54.

LITERATURE CITED

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wheat seedlings. Science. 75: 551-553. 195 .

Collins, G.N. and Maxwell, L.R. Delayed killing of

maize seedlings with x—rays. Science. 85: 575-576.

1956.

Compton and Allison. X—rays in Theory and Experiment

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Goodspeed, T.H. The effects of x-rays and radium on
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-------------- . Cytological and other features of

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Nicotiana tabacum. The Bot. Gaz. 87: 565-582. 1929.

Goodspeed, T.H. and Olson, A.R. The production of

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Horlacher, WQR. and Killough, D.T. Somatic changes

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Johnson, E.L. Growth and germination of sunflowers

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———————————— . On the alleged stimulating effects of

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11.

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‘15.

16.

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18.

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20.

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Johnson, E.L. The relation of x-ray dosage to degree
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Komuro, H. Cytological and physiological changes in
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Long, T.P. and Kersten, H. Stimulation of growth of
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Muller, H.J. The production of mutations by x—rays.
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----------- . Radiation and genetics. Am. Nat. 64: 220—251.

----------- . Types of visible variations induced by
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----------------------------- . The frequency of trans-
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15: 285-511. 1950.

Navashin, M.. A preliminary report on some chromosome
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Packard, C. The biological effects of short radiations.

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Painter, T.S. and Muller, H.J. Parallel cytology and
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Patterson, J.T. Proof that the entire chromosome is not
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----------- . Genetic effects of x-rays in.Maize.

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----------- . Chromosome number and the mutation rate
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--- ---------- . Some genetic effects of x—rays in plants.

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3.55.?

C )1
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0

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------------ . The experimental modification of heredity
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47: 815-826. 1955.

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Physical Review. 11: 401-410. 1918.

tags ROOM USE ONLY

, II” 71955

 

 

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