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Part 1. BETA AE‘D GAD’E'VLA iLADIJHTON SLUDTJLD IN
PISUH SAT TVUI-i
Part 11. T iii TY‘J;:F.RITAT'ICE OF ETSAPIUIH BOUT RO'
FUSARTUN; SULs‘iNI PISI, RESISTANCE IN PTSUM SA'PTl/Ui‘i

 

 

 

presented by

Dean E. Knavel

has been accepted towards fulfillment
of the requirements for

M degree in Horticulture

M(JZ/4L/é//Qj/éa74ug

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Date January 29, 1959

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LIBRARY

Michigan State
University

    

 

 

PART I. BETA AND GAMMA RADIATION STUDIES IN PISUM SATIVUM

PART II. THE INHERITANCE OF FUSARIUM ROOT-BOT, FUSARIUM
SOLANI PISI, RESISTANCE IN PISUM SATIVUM

By

Dean E: Knavel

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied ScienCe
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1959

«)6 WM mag
C//

‘ THES

 

 

ABSTRACT Dean E. Knavel

Beta and gamma radiation sources were evaluated for
producing mutations and resistance to Fusarium root-rot,

Fusarium solani f. pisi, in peas. Dry pea seeds and seeds

 

soaked in either water, solutions of colchicine, Endothal,

or uranyl nitrate of the variety Early Perfection were irra-
diated. The dry seeds were exposed to radiation dosages of
7,500r and 15,000r of the gamma source and 20,000 rep and
40,000 rep of the beta source. The soaked seeds were exposed
to dosages of 1,500r, 3,000r, 10,000 rep, and 20,000 rep.

Gamma irradiation was more effective than beta irradi—
ation of dry seeds for reducing germination and increasing
the numbers of plants showing dwarfing, leaf-distortion, and
leaf-variegations in the R1. Five per cent of the seeds
germinated and all of the surviving R1 plants showed visible
changes at a dosage level of 15,000r. In comparison, 16 per
cent of the seeds germinated, and only 58 per cent of the R1
plants showed similar visible changes at 40,000 rep.

Soaking seeds in the chemical solutions prior to either
radiation source did not cause any appreciable increase in
the variability in the R2 plants. However, soaking seeds in
water prior to gamma radiation was more effective than gamma
and beta irradiation of dry and chemically soaked seeds with
regards to the numbers of abnormal plants in the R2. Abnor—

mal R2 plants were characterized by chlorOphyll deficiencies,

 

sterility, lateness, earliness, tallness, dwarfness, and
plants that branched at the base.

Two gamma radiation-induced tall plants were studied
in detail. Plants from irradiated seeds in the R3 generation
flowered as early as Early Perfection plants from non-irradiated
seeds during long days, however, when the R4 was grown under
short day lengths and low light intensities, flowering was
delayed approximately one month longer than the non-irradiated
Early Perfection plants. Non-irradiated plants flowered in
52-56 days after seeding under long days.

All R5 plants grown from irradiated seed and tested
for Fusarium root-rot resistance were found susceptible to
the disease.

The variety Early Perfection was crossed to a Fusarium
root-rot resistant Foreign pea introduction, 140165, to study
the mode of inheritance for resistance to Fusarium root—rot,
to determine if resistance was associated with seed-shape
and seed coat color, and if resistant Perfection—type plants
could be obtained.

The Fusarium root~rot test of the F5 pOpulations, Early
Perfection X 140165 and 140165 X Early Perfection, indicated
that resistance to Fusarium root—rot was dominant. The curve
showing the distribution of the F5 progeny was not normal.

F segregates from wrinkled seeds and seeds with transparent

5

coats, which are characteristic of Early Perfection, were

E

 

 

 

 

similar in resistance as color coated and smooth seeds. The
latter seed-types are representative of the resistant parent,
140165. The short internode Perfection-type progeny of the
F5 populations were slightly more resistant than the long

internode 140165—type.

PART I. BETA AND GAMMA RADIATION STUDIES IN PISUM SATIVUM

PART II. THE INHERITANCE OF FUSARIUM ROOT—BOT, FUSARIUM
SOLANI PISI, RESISTANCE IN PISUM SATIVUM

By

Dean E. Knavel

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1959

 

 

a. 5/5.?—
&/ ,IIfIF/fiz/

DEDICATION

To my wife, Phyllis, in appreciation for her
encouragement, assistance, and many sacrifices.
To my mother-in-law, Margaret Seese, for her
continuing interest and loyal support during

the conduction of this investigation.

ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks to
Dr. J. C. Ballard for his generous help, valuable sugges-
tions, and direction throughout this investigation.

The author wishes to thank the other members of the
guidance committee, Drs. S. Honma, S. H. Wittwer, J. L.
Lockwood, and J. E. Grafius,for their helpful review of the
manuscript.

Grateful acknowledgement is expressed to Dr. J. L.
Lockwood for his generous help and advice on the pathologi-
cal techniques.

Sincere thanks are also extended to Dr. R. Nicholas
of the Department of Agriculture Engineering, Michigan State
University, and Dr. J. V. Nehemias of the Fission Products
Laboratory, University of Michigan, for their generous help
in performing the irradiation of seeds used in this study.

The author also wishes to thank Mrs. Mary Hillman

for typing the thesis.

 

 

TABLE OF CONTENTS

I° Introduction . . . . . . .

II. Literature Review. . . . .

III. Part

1.
2.
3.

4.
5.
6.
7.
I.

Sources of ionizing radiations
Irradiation of seeds

Irradiation of dry versus water—soaked

seeds. . . . . .

Mutation rates . .

Irradiation of chemically soaked

Beneficial mutations

Root-rot resistance.

Radiation Studies

0

0

Experimental materials and methods.

1. General methods. .

2. Irradiation of seeds and results

Experiment 1.
ditions . . . .
(1) Results . .
EXperiment 2.
house conditions
(1) Results . .

Experiment 5.

house conditions.

(1) Results .

Q

o

0

seeds

Beta irradiation of
soaked seeds under field and green—

Comparison of irradi-
ation of dry seeds under field con-

0

\ Gamma irradiation of
dry ani soaked seeds under green—

Page

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16
17

THE

 

 

Page

d. Experiment 4. Two dosage levels of
gamma and beta radiation of dry and
soaked seeds under greenhouse con~
ditions . . . . . . . . . . . . . . . 17

(1) Results . . . . . . . . . . . . . 19
e. Experiment 5. Three dosage levels of
gamma and beta radiation on dry and
soaked seeds under greenhouse con—
dit ions 0 0 O O O O O O O O O O O 0 O 19
(1) Results . . . . . . . . . . . . . 20
f. Experiment 6. Two dosage levels of
gamma and beta radiation on dry and
soaked seeds under field conditions . 20
(1) Results . . . . . . . . . . . . . 26
(a) Germination in the R1 . . . . 26
(b) Abnormalities in the R1 . . . 51
(c) Survival of R1 plants . . . . 52
(d) Variability in the R2 plants. 52
(e) Variability in the R5 plants. 57
(f) Test of R plants from irra—
diated seeds for Fusarium

root-rot resistance . . . . . 59

(g) Observation of two tall mu-

tants for three generations . 59
IV. Part III, Fusarium Root-rot Inheritance Studies 44
A. Experimental materials and methods . . . . 44

B. Experimental results . . . . . . . . . . . 46

1. Seed coat pigmentation of parental, Fl’
backcross, and F2 populations . . . . . 46

2. Test for resistance: backcross and F2
pOpulations . . . . . . . . . . . . . . 50

5. Test for resistance: F5 pOpulations.

4. Relationship between F5 progeny-types
and resistance to root—rot. . . . . .

V. DiscuSSion. C O O O O O O O O O O O O 0 O O O
A. Radiation studies. . . . . . . . . . . .
1. R1 data: germination . . . . . . . .

2. R1 data: dwarfing, leaf—variegation,
and leaf-diStortion o o o o o o o o o

5. R2 data 0 O O O 0 O O O O O O O O O O

4. R5 data 0 O O O O O O O O O O O O O O

5. Root-rot resistance test of R5 plants
from irradiated seeds . . . . . . . .

B. Inheritance studies. . . . . . . . . . .
VI. Summary and Conclusions . . . . . . . . . . .

VII. Literature Cited. . . . . . . . . . . . . . .

Page
51

51
62
62
62

65
66
69

71
72
74
77

ms

INTRODUCTION

Resistance to the various root-rots attacking pea,
Pisum sativum, has not been found. Commercial varieties have
been tested without success for resistance to Fusarium
solani f. Elfil- Six—hundred foreign pea assessions were
tested at Michigan State University. The tests included
seeds with pigmented and transparent coats. Resistance was
associated with seeds with a pigmented seed coat, and to
some extent, smooth seeds. Seeds with resistance varied
from red to various shades of brown, gray, and purple, usu—
ally with a brown mottling and/or purple dotting of the seed
coat.

Peas with a pigmented seed coat are not accepted for
the canned-pack. The canners demand peas with a transparent
seed coat, and moreover, a majority require a wrinkled or
sweet pea. A frozen pea pack should contain dark—green and
wrinkled seeds.

Therefore, it would be necessary either to separate
the resistance from seeds with the pigmented seed coat, or
induce resistance in a commercial variety by radiation
techniques.

Various workers have reported success in using radiation
to induce mutations in plant and animal material. Moreover,

irradiation of the organism is one method for increasing

the mutation rate. According to Muller (45), mutations can

arise through gene and chromosome abberations, and both oc-

cur naturally. A chromosome mutation may occur through one

of several ways, namely, translocation, inversion, deletion,

and duplication of chromosome parts.

The objectives are as follows:

1.
2.

Irradiation of pea seeds to induce mutations;
Determine the relationship between plant ab-
normalities in the first generation and those
in the second generation;

Correlation between Fusarium root—rot resist—
ance and various seed and plant characters;

Inheritance of resistance to Fusarium root—rot.

LITERATURE REVIEW

Irradiation of plants is not a new method for crOp
improvement. The value of using radiation as a tool for
increasing the number of mutants has been known since
Muller (41) and Stadler (55). Muller worked with the
Drosophila and Stadler with corn and barley. Since then
researchers in the various sciences have contributed to the
present knowledge of radiation and its effect on plants and

animals.

1. Sources of ionizing radiations
The sources of ionizing radiations are separated on the
basis of their penetrating power. According to Brock (6),

"The ionizing radiations either consist of,
or produce in the cell, charged particles of high
kinetic energy. Their biolOgical effect results
from the transfer of this energy to the cell either
by collision or by ionization of cellular compo-
nents--which results in a chain of chemical reac-
tions.

The radiations which consists of charged
particles have low penetration and their uses for
plant and animal material are limited. The charged
particles are either electrons, e. g. beta-rays
produced by radioactive phosphorous (P 52); or
atomic nuclei, e. g. alpha—rays produced by the de—
cay of radon or polonium. ‘

The radiations which produce charged parti-
cles in the cell are the electromagnetic radiations
(X-and gamma—rays) which produce electrons, and
neutrons which produce atomic nuclei (protons).
These have high penetrating Power and consequently
widespread use in biological experiments."

-3...

2. Irradiation of seeds

The majority of reports on induction of mutations by
radiation has been where the seeds have been irradiated dry
rather than irradiating the growing plants. The use of
seeds permits treating large numbers of progeny to obtain
beneficial mutations (55) (25). According to Gustafson (24),
irradiation of the seed is superior to treating either the

ovules or the pollen grains.

5. Irradiation of dry versus water-soaked seeds.
Smith (55), in reviewing the literature on radiation,

stated:

"It is of interest to note that radiation of dry

seeds was used, the method which today has been

most productive of beneficial mutations."

Wheat (1), tomatoes (55), and barley (8) (14) (25) (25)
(50) (55) (56) seeds with a high moisture content demonstrated
greater X—radiation sensitivity than dry seeds.

tadler (55) (56), Ehrenberg, et g1. (14), and Gustafsson
(25) soaked barley seeds prior to irradiation and increased
the mutation rate 7 to 15 times that of dry seeds. They also
found dry seeds survived dosages 15 to 20 times greater than
germinating seeds. Gustafsson (25) irradiated soaked barley

seeds which had moisture contents of 10 and 15 per cent.

The seeds with 10 per cent moisture content produced a larger

 

number of mutants. At the higher moisture level, more mu-
tants were obtained at a dosage of 5,000r than at 10,000r
units.
Gelin (19) found X-irradiated barley seeds with a
moisture content of 10 per cent produced plants of which
12 per cent had abnormal cell divisions, while at 15 per
cent moisture, 27 per cent showed abnormal cell divisions.
In comparing X-rays with neutrons, Key (50) and
Ehrenberg (15) found the effect of neutrons to be independ-

ent of the moisture content in the seeds.

4. Mutation rates.

Increase in the mutation rate has been reported by
Stadler (55), Muller (42), and Gustafsson (25) to be di-
rectly prOportional to the dosage of X—rays. However,
Gustafsson (25) found that the number of chromosome irregu-
larities was not directly prOportional to dosage. Never-
theless, as the number of chromosome rearrangements were
increased, there was an increase in the mutation rate (25)
(25).

Gustafsson (24) also reported that mutations in cere—
als were most frequently close to lethal dosages of X-rays.
At such dosages cell divisions contained nearly 100 per cent
fragments and bridges, and germination was reduced to about

10-20 per cent of normal.

 

6

Tedin (57) irradiated 5,528 seeds of yellow sweet lupine
and obtained 1.6 per cent mutations. From 8,616 X2 barley
plants, Akerberg (2) was able to select 6 plants which pro—
duced lines superior to the maternal strain in yield, weight,
straw strength, and earliness. Muller (42) found the rate
of desirable mutations in rye to be about 1 in 800 of those

plants surviving the radiation treatment.

5. Irradiation of chemically soaked seeds.

Chromosome re-arrangements and other mitotic disturbances
have been increased by pre-treating or soaking seed in metal-
lic salt solutions and other metabolic inhibitors prior to

irradiation.

Stadler (54) reported 0.5M solutions of Pb(N0 Ba(N0

5)2, 5)2,

and U02(N0 to be effective for increasing mutations in bar—

92

ley. A three-fold increase was obtained by Damato and

Gustafsson (10) with H202,

prior to X—irradiation of barley seeds.

KCN, and Butter Yellow when used

Colchicine, a mitotic poison, used at the concentration
of 0.1 per cent prior to X—irradiation of barley seeds increased
the mutation rate. Damato and Gustafsson (10) noted that at

0.005 per cent colchicine increased the number of "very rare

mutants."

 

 

6. Beneficial mutations.
Much of the radiation on plants in this country has
been to control the cat stem rust organism, Puccinia
raminis, var. avenae. Konzak (55) treated the cat variety
Mohawk and obtained plants which were resistant to race 7a
of stem rust. About the same time Frey (15) X—rayed oat
seeds of the variety Huron and produced three strains which
were resistant to race 7 of stem rust. Others who were
successful in producing resistance to stem rust in oats were
Meyers, gt gt. (40), Frey and Browning (l7), Singleton, gt
gt. (50), and MacKey (58). Resistance has been found for
other cereal diseases. Konzak, gt gt. (52) obtained wheat
mutants which were resistant to stripe rust, Puccinia
gluma um. Singleton, gt gt. (50) irradiated the cat vari-
ety Tamma and induced resistance to Helminthosporium Victoria
blight. Resistance to both diseases appeared to be recessive.
Irradiation of the cereals has resulted in beneficial

mutants which were earlier, higher yielding, and had greater
straw strength than non-irradiated (25) (25). Gustafsson
called these mutants erectoids. Similar types of mutants
have been reported in barley (2) (5) (18) (26) (51), cats
(16), and wheat (58). Froier (18) selected mutant barley
plants which yielded better at a medium level of nitrogen

than at a high level.

. Gregory (22) working with X—ray treated peanuts was
able to select plants resistant to stem rust and leaf spot.
He also selected five best-mutants and conducted yield
tests with the X5 generation in 1955 and 1955 and found an
increase in yields. This was one of the first works demon-
strating the use of radiation to improve a quantitative
character.

There are three X-radiation induced mutant crOp vari—
eties on the market. These are the Stralart pea, Primex
white mustard, and the Sanilac navy bean. The Stralart
pea is commercially important in Sweden because of its
ability to out—yield the mother strain by 6 per cent and
all commercial Swedish varieties by 10 per cent (20). Pri-
mex white mustard of Svalof is important for its high oil
yield. A mutation was found that had a 7 per cent increase
over the mother strain (5). Down and Anderson (12) made
use of a bush-type mutation which was produced by X-irradiation
of navy bean seeds of the variety Michelite. After 14 gen-
erations of crossing and back-crossing they improved its
quality and resistance to anthracnose. The improved bean
was released in 1957 as the variety Sanilac.

Progressive mutations have been induced in cotton by

X—ray treatment of dry seeds (27).

Mertens and Burdick (59) X-irradiated seeds of the
tomato variety Red Cherry and reported a mutation for early

fruit maturation.

7. Root-rot resistance.

A review of the literature revealed little information
on resistance to root-rot diseases of peas. Wade (59)
listed Fusarium root—rot as one in which the genetic basis
for resistance had not been worked out. Earlier, Jones (29)
reported that two varieties of peas, Rice 550 and Horal,
have some resistance to Aphanomyces euteiches. According
to Jones, resistance in these varieties was a "very small

factor in relation to total resistance."

 

 

PART I. RADIATION STUDIES
A. Experimental materials and methods

1. General methods.

Preliminary experiments were conducted in the field
during the summer of 1956 and the greenhouse during the
fall and winter of 1956-1957. The objective was to estab-
lish chemical and/or radiation treatments which would give
low germination and high numbers of plant—changes, as leaf—
variegation, leaf-distortion, and dwarfed pea—seedlings in
the R1 (first generation). Seeds of the variety Early Per-
fection were used in these studies.

Gamma and beta radiation were used in these studies.
Gamma irradiation was accomplished by placing the bags of
dry seed in the center well of a colbalt-60 radiation
source where the dosage was approximately 250,000 rep/hr.
The dosage, measured in time of exposure and intensity, was
in roentgens and will be designated hereafter as "r." The
seeds were exposed to gamma radiation at the Fission Pro-
ducts Laboratory at the University of Michigan. Beta irra-
diation was accomplished by placing the dry seed, one layer
thick within the bag, on a metal tray which travelled be-

neath the electron beam at the rate of 12.2 cm/sec. The

- 10 _

ll

distance from the window of the electron tube to the base
of the tray was 47 cm. The dosage, measured by intensity,
is in roentgen-equivalen—physica1 and will hereafter be
designated as "rep." The seeds were exposed to high—speed
electron beta radiation of thecathode source at the Agri-
culture Engineering Department at Michigan State University.
Treatments prior to irradiation were performed by
soaking the seed in either water or the chemical solutions.
The seeds were placed in covered plastic dishes and soaked
in tap-water at room temperatures for six hours. At the end
of the six hours, the water was removed and either fresh
water or chemical solutions were added and the seeds allowed
to soak for an additional six hours. All seeds were covered
with the solution during the period. At the termination of
the l2-hours soaking period the seeds were washed in tap-
water and placed in polyethylene bags for irradiation. In
order not to introduce error because of the length of time-
lapse between the termination of the soaking period and the
time of irradiation, all irradiations were made approximately
six hours after the soaking treatments were completed. The

seeds were sown the same day they were soaked and irradiated.

2. Irradiation of seeds and results.
a. Experiment I. Comparison of gamma and beta irradi-

ation of dry seeds under field conditions.

12

One hundred dry seeds per treatment were irradi-
ated and planted in the field. The per cent germina-
tion and the percentage of the variegated, distorted,
and dwarfed seedlings which survived the radiation
treatments were determined.

(1) Results.

Gamma irradiation of dry seeds did not reduce
germination at dosage levels of 1,000r to 7,000r
as greatly as 10,000 rep and 20,000 rep of beta
irradiation (Figures 1 and 2). However, 21,000r
of gamma radiation was more lethal than 80,000 rep
of beta radiation. A dosage level of 10,000 rep
appeared to be comparable to a dosage level some-
where between 7,000r and 9,000r of gamma radiation
with regards to seed germination.

The numbers of deformed Rl seedlings were not
equivalent for both sources of radiation (Figures

1 and 2). The Rl seedlings showing the immediate

affects from radiation were slow growing and

greatly deformed. The color of the leaves on the
visibly affected plants were variegated and mot-
tled with large irregular areas devoid of chloro—
phyll (Figure 5). At 40,000 rep of beta radiation,

52 per cent of the R1 seedlings were deformed;

per cent

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Influence of beta radiation on pea

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15

 

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Figure 5. (top): normal plants. (bottom): dwarfed R
plants with variegation and distorted leaves as result
of gamma and beta radiation on dry and soaked seeds, and
seeds soaked in colchicine.

15

whereas, at a dosage level of 80,000 rep, only 21
per cent of the seedlings were visibly changed.
In comparison, all plants surviving dosage levels
of 15,000r or greater of gamma radiation were de-
formed.

b. Experiment 2. Beta irradiation of soaked seeds under

 

field and greenhouse conditions.

 

To determine if soaking seeds prior to irradiation
increased the effectiveness of beta radiation, 100 seeds
per treatment were irradiated following soaking in either
water, 0.01, or 0.05 per cent solutions of colchicine.

The seeds were sown in the greenhouse in flats of
non-sterilized soil, and a duplicated planting was made
in the field. The treatments were not replicated. The
per cent germination was determined.

(1) Results.

Seeds did not germinate in the field as well

as seeds in the greenhouse (Table l). The reduced

germination in the field was evident in all treat-

ments, regardless if beta-irradiated or not. A

dosage of 15,000 rep on soaked seeds appeared to

be too high for field studies. Colchicine solu—

tions of 0.01 and 0.05 per cent reduced germina—

tion and appeared to be too high.

16

Table 1. Influence of beta radiation with regards to per
cent germination of pea seeds under field and greenhouse
conditions as result of irradiation of seeds after soak-
ing in water or solutions of colchicine.

 

 

Radiation Germination
Soaking Treatment dosa e Field Greenhouse
(l X 10 rep) (per cent) (per cent)

 

Water—soaked

--- 70 98
15 0 4
25 0 4

Colchicine (0.01%)
—-- 61 68
15 0 4
25 0 0

(0.05%)
--- 4 l2
l5 0 0
25 0 0

 

--- without radiation.

0. Experiment 5. Gamma irradiation of dry and soaked
seeds under greenhouse conditions.
To compare the effect of gamma radiation on dry and
soaked seeds, seeds were soaked in 0.01 and 0.005 per
cent colchicine solutions and 0.001 and 0.005 per cent

Endothal solutions prior to irradiation. Wilson (60)

17

suggested that Endothal (disodium 5,6-endoxohexahydro-
phthalate) might be a mutagentic agent. A sample of
100 seeds were used in each treatment. The seeds were
sown in flats of non-sterilized soil in the greenhouse.
(1) Results.
Only 8 per cent of the dry seeds which were
exposed to a dosage of 15,000r germinated (Table 2).
Therefore, it appeared that 15,000r might be the
upper dosage level for satisfactory germination
under field conditions.

d. Experiment 4. Two dosage levels of gamma and beta

 

radiation on dry and soaked seeds under greenhouse

 

conditions.

 

To compare the effect of gamma and beta radiations
on dry seeds and seeds soaked in various chemical solu-
tions, seeds were soaked in either water, 0.002 per
cent colchicine, 0.002 per cent Endothal, or 0.25 per
cent uranyl nitrate solutions prior to irradiation. Dry
seeds were irradiated at either 10,000r, 15,000r, 20,000
rep, or 40,000 rep. Soaked seeds were irradiated at
either 2,000r, 5,000r, 10,000 rep, or 20,000 rep. A
sample of 100 seeds were used in each treatment. The

seeds were sown in flats of non-sterilized soil

18

Table 2. Influence of gamma radiation with regards to per
cent germination of pea seeds under greenhouse conditions as
result of irradiation of seeds dry or after soaking in water
or solutions of colchicine and Endothal.

 

 

 

Radiation dosage Germination

Treatment (1 X 102 r) (per cent)
Dry seed --- 96
5 76
8 80
10 64
l2 l2
l5 8
20 4
25 0
Water-soaked —-- 97
l 96
2 76
5 52
4 64
Colchicine (0.01%) --- 72
0.5 52
1 20
5 16
(0.005%) —-- 80
0.5 76
1 76
5 40
Endothal (0.001%) --- ‘ 92
0.5 88
l 88
5 48
(0.005%) -—— 72
0.5 68
l 64
5 48

 

--- without radiation.

 

19

in the greenhouse. The per cent germination was deter—
mined.
(1) Results.

Germination in all treatments appeared to be
satisfactory and, therefore, the treatments ap-
peared applicable for field studies (Table 5).

The concentrations of 0.002 per cent of Endothal
and colchicine, and 0.25 per cent uranyl nitrate
gave satisfactory germination. Each chemical
treatment resulted in germination similar to the
water-soaked control where 60 per cent of the seeds
germinated.

e. EXperiment 5. Three dosage levels of gamma and beta

radiation on dry and soaked seeds under greenhouse

conditions.

 

To determine the effect of gamma and beta radiation
in combination with treatment with chemicals, another
experiment was conducted using three dosage levels of
each radiation source. Dry seeds were exposed to
dosages of 7,500r, 15,000r, 22,500r, 20,000 rep, 40,000
rep, and 60,000 rep. Soaked seeds were exposed to
dosages of 1,500r, 5,000r, 4,500r, 10,000 rep, 20,000
rep, and 50,000 rep. The percentage germination and
the percentage of plants surviving the radiation treat—

ments, which exhibited dwarfing, leaf-variegation, and

2O

leaf-distortion, were determined. A sample of 100
seeds was used in each treatment. The seeds were sown
to flats in the greenhouse.

(1) Results.

Germination in this experiment was extremely
high for all treatments (Table 4). However, the
percentage of plants showing dwarfing, leaf—
variegation, and distortion appeared satisfactory.
Soaking seeds in Endothal and uranyl nitrate, not
followed with radiation, did not cause visible
changes in the R1 plants.

When the data on germination in Table 4 is
compared with that in Table 5, there is an indi-
cation that from experiment to experiment the same
treatments do not produce similar results under
greenhouse conditions. The experiment was not re—
peated because greenhouse space was limited.

f. Experiment 6. Two dosage levels of gamma and beta
radiation on dry and soaked seeds under field condi—
Eisne-

The results of the previous experiments conducted
in the greenhouse indicated that 7,500r, 15,000r,
20,000 rep, and 40,000 rep on dry seeds and 1,500r,
5,000r, 10,000 rep, and 20,000 rep on soaked seeds

might give satisfactory results in the field with

21

Table 5. Influence of gamma and beta radiation with regards
to per cent germination of pea seeds under greenhouse condi-
tions as result of irradiation of seeds dry or after soaking
in water or solutions of colchicine, Endothal, and uranyl
nitrate.

Radiation dosage Germination
Treatment (1 X 105) (per cent)

 

(Gamma radiation r)

Dry seeds --- 77
10 57

15 50

Water-soaked --- 60
2 52

5 l4

Colchicine (0.002%) --- 51
2 58

,5 15

Endothal (0.002%) '——- 65
2 56

5 1?

Uranyl nitrate (0.25%) --- 76
2 41

5 16

(Beta radiation rep)

Dry seeds 25 66
50 5O
Water-soaked 10 48
20 52
Colchicine (0.002%) 10 44
2O 29
Endothal (0.002%) 10 46
2O 58
Uranyl nitrate (0.25%) 10 47
2O 55

 

--- without radiation.

22

Table 4. Influence of gamma and beta radiation with regards
to per cent germination of pea seeds and the percentage of
surviving R1 plants showing dwarfing, leaf-variegation, and
leaf-distortion under greenhouse conditions as result of
irradiation of seeds dry or after soaking in water or solu-
tions of colchicine, Endothal, and uranyl nitrate.

J:_

 

Dwarfing, leaf-
Radiation dosage Germination variegating, and
Treatment (1 X 102) (per cent) leaf-distortion
(per cent)

 

(Gamma radiation r)

Dry seeds --- 99 0
7.5 95 100
15.0 77 100
22.5 70 100
Water-soaked ~-- 96 0
1.5 100 9
5.0 87 60
4.5 65 100
Colchicine (0.002%) -—— 97 25
1.5 91 55
5.0 95 48
4.5 74 100
Endothal (0.002%) -—- 95 0
1.5 98 18
5.0 86 25
4.5 45 51
Uranyl nitrate -—- 97 0
(0.25%) 1.5 99 12
5.0 88 56
4.5 71 68

(Beta radiation rep)
Dry seeds 20.0 99 26
40.0 86 42
60.0 52 69

(Continued on page 25)

25

Table 4. (Continued)

 

 

Dwarfing, leaf—

Radiation dosage Germination variegation, and

Treatment (1 X 105) (per cent) leaf-distortion
(per cent)

 

(Beta radiation rep)

Water-soaked 10.0 76 15
20.0 55 9

50.0 46 5

Colchicine (0.002%) 10.0 89 59
20.0 48 46

50.0 46 17

Endothal (0.002%) 10.0 75 7
20.0 75 6

50.0 65 5

Uranyl nitrate 10.0 81 9
(0.25%) 20.0 56 7
50.0 50 4

 

—-— without radiation.

regards to germination and variability. Hence these
radiation dosages were used in the 1957 field experi—
ment. The seeds were exposed to gamma radiation on

May 29 and beta on May 51. A sample of 500 seeds were
used in each treatment. The treatments were randomized
in a split-plot design. The main plots were the sources
of radiation, while the sub—plots were the soaking treat—
ment and/or radiation treatments. The rows in each re-
plication were 56 feet long. Each treatment was re-

plicated four times.

 

 

24

The Rl seedlings which showed visible leaf and
plant changes were tagged and harvested separately
from the normal-appearing plants to observe if any
relationship existed between the variability in the

R1 plants and the variability obtained in the R (second

2
generation) plants. The per cent germination, percent—
age of abnormal plants, and the percentage of plants
which survived were determined in the R1. The near-
dry plants were harvested by hand. Further drying was
accomplished in a forced warm—air drying oven at 80'0 F.
The pods were threshed by machine.

The R2 generation was grown in the field in the
summer of 1958. A sample of 500 seeds, where seed was
available, from the separated Rl plants was planted
for each treatment. Seeds of the R1 plants which
showed visible differences were sown adjacent to the
seeds from plants of the same treatment which showed no
visible effects of radiation in the R1. The treatments
were randomized in a paired split-plot design. Four
replications were used.

The R2 plants were observed for visible differ-
ences. Seeds of the plants differing from the normal

perfection-type plant were harvested separately. Five

seeds from each plant were sown in pots in the greenhouse

 

 

25

for additional observation in the R5 (third generation),
and an additional five seeds from the same plants were
saved and tested for Fusarium root—rot resistance.

One pod was harvested from each of the remaining
R2 plants for each treatment. The seeds were bulked
and saved to test for root—rot resistance. Since test-
ing large numbers of seeds is desirable and space was
limited in the greenhouse, four-200 seed samples from

each of the following treatments were tested for resist-

ance to Fusarium root-rot:

Treatment Radiation dosage (l X 105)

 

(Gamma r)

Dry seeds 7.5
15.0

Water—soaked 1.5
5.0

Colchicine 1.5
5.0

Endothal 1.5
5.0

Uranyl nitrate 1.5
5.0

(Beta rep)
Dry seeds 20.0

40.0

 

 

26

The seeds were sown in quartz sand in wooden flats in
the greenhouse. Each flat contained a row of non—
irradiated Early Perfection as a control.

The plants were inoculated with isolate 48 of
Fusarium solani f. ptgt. The technique of Lockwood and
Ballard (57) was used. Spore suspensions were cali-
brated to 70 per cent light transmittance with a photo-
electric colorimiter. When the suspensions were diluted
to 1:4, there was approximately 200,000 spores per m1.
Ten ml. of the diluted suspension was pipetted to each
row with the inoculum being directed to the base of
the seedlings when all seedlings had emerged. Some
seedlings were larger than others at the time of inocu-
lation because of the difference in rate of emergence.

(1) Results.

(a) Germination in the R1.

As the radiation dosage levels were in—
creased, there was a reduction in germinmion
(Figures 4, 5, and 6). The effect of beta radi-
ation on germination was more pronounced at the
lower dosages of gamma radiation in both dry and
soaked seeds. Eighty-two per cent of the seeds
germinated in the dry control. At a dosage of

20,000 rep, 28 per cent of the seeds germinated.

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28

 

 

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50

When the dosage was doubled to 40,000 rep, 16 per
cent of the seeds germinated. In comparison, a
dosage of 7,500r gave 59 per cent germination.
However, when the dosage was doubled to 15,000r,
only 5 per cent of the seeds germinated.

A similar reduction in germination was
found in irradiated soaked seeds; however, dosages
of 1,500r, 5,000r, 10,000 rep, and 20,000 rep on
soaked seeds did not cause as great a reduction as
7,500r, 15,000r, 20,000 rep, and 40,000 rep on dry
seeds. Seeds exposed to 10,000 rep of beta radi—
ation after soakingin either water, Endothal,
colchicine, or uranyl nitrate gave less germina-
tion than seeds which received the same soaking
treatments and exposed to l,500r of gamma radi—
ation. A similar decrease was obtained in both
sources of radiation for the water and chemical
treatments at dosages of 5,000r and 20,000 rep.
Soaking seeds in the chemical solutions and sub-
jecting them to either source of radiation did not
appear to increase germination over water-soaking
and irradiation.

Treatment with colchicine and Endothal
without subsequent radiation reduced germination,

when compared with the water—soaked control. On

51

the other hand, soaking seeds in uranyl nitrate
appeared to stimulate germination over the water-
soaked control. Ninety and 92 per cent of the
uranyl nitrate soaked seeds germinated, whereas
only 79 and 87 per cent of the water—soaked seeds
germinated (Figure 4).
(b) Abnormalities in the R1.

Dry and soaked seeds exposed to gamma
radiation resulted in more leaf and plant abnor-
malities than exposing seeds to beta radiation
(Figures 4, 5, and 6). The exposure of seeds to
dosage levels of 7,500r and 15,000r of gamma radi-
ation resulted in visible changes in 100 per cent
of the plants, while beta radiation produced simi-
lar changes in only 62 per cent of the plants at
20,000 rep and 58 per cent at 40,000 rep (Figure 6).

Soaking seeds prior to gammaradiation
slightly increased the number of visibly changed
plants (Figure 4). In comparison, beta treatment
did not produce any changes in the R1 plants from
soaked seeds (Figure 5).

Colchicine alone produced greater changes
than either radiation treatment on pre—soaked seeds

for the production of leaf—variegation and distor-

tion in the R1 plants (Figures 5 and 6). In both

52

sub-plots, colchicine-treatment of seeds resulted
in 85 per cent of the R1 seedlings exhibiting leaf
changes. No changes were noted where Endothal and
uranyl nitrate were used alone.

(c) Survival of R1 plants.

Plant survival was not appreciably in-
creased or decreased by the action of gamma and
beta radiation on dry and soaked seeds.

(d) Variability in the 32 plants.

Abnormal plants Observed in the R2 were
distinguished as chlorOphyll deficiencies, ster-
ility, lateness, earliness, tallness, dwarfness,
and branching at the base (Table 5). Seventy—two
per cent of the changes observed were the result
of gamma irradiation, while only 16 per cent can
be associated with beta irradiation.

The chlorophyll deficiencies, which com—
prised 19 per cent of observed off—type plants,
included viridis, chlorina, xantha, and albino
types (Table 5). Seventy—eight per cent of the
chlorOphyll deficient plants were viridis and al-
binos.

Forty-four per cent of the abnormal
plants exhibited degrees of fertility (Table 5).

Nine per cent of these plants were sterile,

55

 

 

 

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55

vegetative, and flowered continuously without
setting pods. One per cent of the sterile

plants had petaloidic flowers with as many as 15
petals per flower. These flowers, which were
without pistils and stamens, were usually in clus-
ters. Types of sterile plants are shown in Figure
7.

The differences observed in the R2 plants
were not associated with the visibly changed R1
plants, except for the following treatments:
7,500r and l5,000r on dry seeds, and l,500r on
water-soaked seeds (Table 5). Nine off—type
plants were found in the 7,500r, and l? in the
l5,000r treatments. However, the water—soaked—
l,500r treatment, had 15 off-type plants which
originated from the changed Rl plants, as compared
to only three from the non—changed R1 plants.

Chemical soaking of seeds prior to irra-
diation did not increase the variability in the R2
as compared to irradiation of dry seeds. On the
other hand, soaking with water prior to gamma ir—
radiation increased the number of off—type plants.
At a dosage of l,500r, 18 plants or ll per cent of
the abnormal plants came from seeds which had been

soaked in water prior to irradiation.

 

Figure 7. Degrees of sterility in R2 pea plants as result

of gamma and beta irradiation of seeds.

Left: petaloidic flowers; no pods.

Middle: normal-appearing flowers; pods later drop.

Right: normal-appearing flowers, pods with rudimentary ovules.

57

(e) Variability in the R plants.

5

Plants of Lines 2-B, 5, 6-A, 6HB, 7-A,
7-B, 8, and 9 bred true for tallness in the R5 and
were significantly taller than the non-irradhflmd.
plants (Table 6). Plants of Line 5 bred true for
dwarfness, and were significantly smaller. Plants
of Line 1 were homozygous for dark green seed—coat.
Plants of Line 2-0 were homozygous for small pods
and small seeds. The fifth internode of the
plants of Line 2-C were non—measurable without
histological observation, and instead of the usual
type of double flowering where the peduncle branches
dichotomously, these plants had two peduncles from
the leaf-axila, each producing a single flower.

The 9-l5th internodes of plants in Line
5 averaged 0.5 inches in length as compared to 1.5
inches for the non-irradiated Early Perfection plants.

Plants of Line 4, from seeds treated with
colchicine, had thick stems and leaves and heavy
short pods and low fertility. The plants had the
appearances of polyploids.

The plants of Line 7—B were tall, averag—
ing 45 inches as compared to 17 inches for the non-
irradiated plants. The foliage was gray rather

than the normal green. The leaflets were narrower

~than the Perfection-type.

58

 

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59

Double branching at the base of the main
stem was characteristic in the plants of Line 10.
(f) Test of R5 plants from irradiated seeds
for Fusarium root-rot resistance.
None of the R5 plants exhibited resist-
ance to Fusarium root-rot.
(g) Observation of two tall mutants for three
generations.
Two tall plants were found in the prelim-
inary studies. One in the R and the other in the

2

R These plants originated from seeds which were

5.
exposed to 20,000r of gamma radiation. These plants
were increased and observed in subsequent generations.
Means and standard deviations are presented in

Table 7 for height, number of nodes to the first
flower, and internode length. The comparative

height of plants of Line A and Early Perfection
plants from non—irradiated seeds are shown in Figures

8 and 9. The R of Line A which was grown from

5
January to March flowered a month later than the

non—irradiated plants and attained a height of 64
inches, as compared to 28 inches for the control.
The R4 of the same line, grown in April, May, and

June, flowered with non-irradiated Early Perfection

and averaged 41 inches in height. The control plants

40

Table 7. Comparison of non-irradiated pea plants and plants
of Lines A and B of Early Perfection which resulted from

gamma irradiation of dry seeds with regards to plant heights,
numbers of nodes to first flower, and the length of internodes

Height Nodes to Internode

 

Pepulation Generation (in.) flower length (in.)
Non—irradiated** 28 i 4.3 15 i 1.4 1.5 i 0.85
Line A 2* 46 -—- 21 --- 2.0 ---
R5** 64 i 5.0 22 i 2.4 2.5 i 0.48
R4*** 41 i 2.4 18 i 0.5 2.5 i 0.22
Line B R5** 45 —-- 18 --- 1.8 -—-
R4*** 42 i 5.8 20 i 1.5 1.8 i 0.81
Non-irradiated*** 29 i 5.1 16 i 1.0 1.5 i 0.24

 

--- only one plant observed.
* plants grown from Feb.-Mar.
** plants grown from Jan.-Mar.
*** plants grown from Apr.-June.
averaged 29 inches when grown from April to June.
The R4 of Line B was similar to the R4 of Line A
when grown under the same conditions. The plants
can be considered homozygous for tallness because
the lines were no more variable than the non-
irradiated control.

Cytological examination of mother—megaspores

in the R

4 plants showed meiosis to be normal.“

 

* Verification by G. B. Wilson, Professor of Botany,
M.S.U.

41

Measurements of pollen grains, stomata, and cell-
sizes of epidermis tissue indicated no significant

differences between Line A and the control.

42

I.

1

 

 

 

 

Figure 8. Early Perfection (three plants on the left) and R5
plants of the Line A tall mutant of Early Perfection which
resulted from gamma irradiation of dry seeds.

45

 

 

 

 

 

 

Figure 9. Early Perfection (two plants on the left) and R
plants of the Line A tall mutant of Early Perfection which
resulted from gamma irradiation of dry seeds.

PART II. FUSARIUM ROOT-HOT INHERITANCE STUDIES
A. Experimental materials and methods

Plants of an Afghanistan pea-introduction, 140165, which
exhibited resistance to Fusarium solani f pig; in a screen-
ing test, were crossed to the root-rot susceptible commercial
parent, Early Perfection. Parental crosses and backcrosses
were made in the following ways:

(1) 140165 X Early Perfection
BC (a) Fl X 140165
BC (b) Fl X Early Perfection
(2) Early Perfection X 140165
BC (c) Fl X Early Perfection
30 (d) Fl X 140165
Hybridizations were made without bagging the flowers. However,
since the stigmas were exposed, frequent spray applications
were made to control greenhouse insects. A11 crosses were
marked with tags. Naturally selfed seeds were saved from
both parents and the F1 populations.

The F2 pOpulations were grown in an air-cooled green-
house room during the summer of 1958 to facilitate a larger
pod~set and higher seed-yields. Sixty seeds of each Fl pOpu—
lation were distributed among 10 pots. Each F2 pOpulation
represented a random sample of seed harvested from selfed Fl
plants.

_ 44 _

45

The seeds of parental, Fl’ backcross, and F2 pOpulations
were classified according to various seed characteristics to
determine whether the resistance as found in the parent was
associated with any specific seed character. The seed char—
acters were wrinkled and smooth, either with or without
purple dotting of the seed coats.

The testing of seeds for resistance was conducted in the
greenhouse. The seeds were mechanically scarified and sur—
face sterilized for 15 minutes in a chlorox solution, diluted
1:10. The seed was sown approximately one and one-half
inches deep in washed quartz sand in metal pans. Twenty
seeds, from the larger lots were sown in each row. Each pan
included a check row of Early Perfection. The pans were
placed in tanks in which the water was maintained at 82° F.
The test contained a sample of the parents and all seed-
both F

classes of both E and the four backcross popula-

l,
tions. The test of the F

29
5 progenies were from both F2 pOpu—
lations. The seedlings were inoculated by the previously
described technique of Lockwood and Ballard (57).

The plants were carefully lifted approximately three
weeks after inoculation and the roots examined for Fusarium
root-rot symptoms. Plants of the parental, backcross, and

F2 pOpulations were divided into four classes depending on

their severity of symptom expression. These classes were

46

none-O, slight-l, moderate—2, and severe-5. To determine
whether resistance was either qualitative or quantitative,

plants of the F test were classified according to the

5
severity of the disease on three areas of the plant, tOp,
collar (point of seed attachment), and the roots. One to
three points were allowed for each area of the plant de-
pending upon the severity of the disease symptoms. The top
of the plant was classified by the degree of wilting. The
collar was classified by the amount of brown discoloration.
The roots were classified by the amount of discoloration and
the number of rootlets. The total number of points, summa-
tion of points for the three areas, served as the disease
rating. A rating system of 0 to 9 was used with O repre—
senting disease—free plants. Rated plants are shown in

Figure 10.
B. Experimental Results

1. Seed coat pigmentation of parental, F], backcross, and Fp.

 

The seed of the cross, Early Perfection X 140165, had
a transparent seed coat. The reciprocal cross, 140165 X
Early Perfection, produced seeds with pigmented coats, simi-
lar to seeds of the resistant parent. Seeds in each F2 and
each backcross to Early Perfection were separated into eight
classes (Table 8). Backcrosses to 140165, as well as the F1’

140165 X Early Perfection, had only two classes. No

Figure 10. F5 pea
plants rated ac-
cording to Fusarium
root-rot incidence
on top of plant
(left number),
collar-point of
seed-attachment
(middle number),
and roots (right
number). 0 repre-
sents disease-free;
5 represents a
badly diseased area.
Left-right:

(TOp) O—O-O;
0-0—2;
O-l—O.

(Middle) O—l-l;

0-2-1;
O—5-l.
(Bottom) 0—5-2;
1—5-2;
5*5r5.

 

O I. ..
.. ’ .9 o
.30 leg; . v.
'9 fix. "
i d. i, 5‘ [*9
a .3 l‘ ‘1'
‘ \
a.
106?.-
«- 'er
"dL(.b " ‘ip ‘1
r! “‘7‘ - ‘Bf—l- ' ‘

a
4.2; 1ft
33“
.1
‘5,
3’
I
*5

‘
'4‘
J
' . é“

 

48

 

 

 

 

mcHoeH H HH
6 s m mm c: 4 H m .HHmH HHHmH
o H s we 4m H H. m H moHosH Homv
moHosH H HH
0 0 HH mm mm H H m mcHosH H
o o H mm mu H H m .HHoH HHHmH Homv
a o o m OH H w H
m OH o o mH H w a
o o m s n H w m
o m 0 9H MH H w m
m m o # 0H 4 H a H
o H m MH 4H H H H .HHoH HHHMH H H
o o m d n H H m meo¢H N
e m 0 mm 4m H H m .HHmH HHHMH Homv
m 6 HH am we 4 H m .HHmH HHHmH
c c H mm an H H m H mcHosH AHHV
mm m: mm ma OOH H H m meosH N
.HHom mHHmm AHmV
m d w om #0 H H m
o w m m¢ mm H H m meosH
mHm mHH H m 00H H o a .HHmH HHHwH
Ampqum Ho .oZv ApHmonHv AHv Hammad mwv nome Agv doHMnHHB HOHHMHSHOH
mHmbmm mpwsmcoz pHmHHm mnoz HOprHsmom mmv pammmHm AMV BOHHmM Amv_npooam
mdepwm thHmbmm mmwmmHm comm Ho mmflppod HOHoo mowwadm
proe mHHHsm nodehpoo IUmcm

 

HOHHHOHHHmmmHo poem

.meo¢H .pqumm pumpmHmmH poHIpooH adHHmmsm oflp.op
HOHpomHHmm hHHmm Ho mommoHc aoHH dopquwHHo HOHQH machstmom mm dam .mmoHoMomQ .Hm How
QOHHHOHHHmmmHo 60mm op ccpmeH mm mHHpmH thHm>cm mmmmde Hem mpqum Ho mHmQafiz .w oHQwe

49

 

 

 

m m o mH m H w H
w o H m H H w H
o o 0 OH H H o m
m H mH mm HH H u m
m m m mm m H H H
H m m no HH H H H
H H H Hm mH H H m .HHmH HHHHH
H 8 MH mmH Hm H H m H mcHoHH HmHv
H m 0 HH m H o H
m H H om H H w H
o o H mm m H u m
OH NH m mo mH H w m
c H 6 mm m H H H
um um mH mm 6H H H H
mH mH HH mm mm H H m mmHOHH H
H m om mmH om H H m .HHmH HHHHH HNHV
o H o m m H u H
o o o H cH H w H
o o o m o H u m
o m o 6 NH H w m
o c o H MH H H H H
m o o H mH H H H .HHoH HHHHH H H
o o o m OH H H m .HHmH HHHHH
o H 0 6H AH H H m H mcHOHH Homv
mmpquH 90 .02v ApflmeHmmv AHV pmompd mwv Hmme Aav @mHMdHHH HOHpdemom
mp»pwmnwpwnwepalpanHmnman HOHHHHsHoH HHV HammmHH HHV HOHHmH Hmv Hpooam
mWQHPwm hHHH®>mw mmfimmflm Ummm Ho MQprod HOHOO . mommhdm
Hmpoa onHsm nochhpoo Idmmm

 

QOHHHOHHHmmmHo doom

I!

Avmdfiwpdoov .m mewB

50

transparent Perfection-type seed was obtained in the F and

2
backcross pOpulations. Seeds with transparent coats were

obtained from F2 plants irrespective of flower color. This
can be seen where data in Table 10, which lists the classi-
fied F5 seeds, is compared with data in Table 9. No white-

flowered plants produced seeds with either colored and/or

purple dotted seed coats.

2. Test for resistance: backcross and F2 populations.

 

Disease ratings of the backcross and F2 pOpulations
were grouped on the basis of seed-type to determine if wrinkled
seeds and non-purple—dotted seeds were as resistant as smooth
seeds and purple—dotted seeds (Table 8). Plants in the rat-
ings of 0 and 1 were considered as resistant. The ratios,
based on the number of resistant plants to the total number
of plants tested for each type of seed, are presented in
Table 11. The smooth—seeded plants had considerably more re-
sistance than those from wrinkled seeds within each popula-
tion. Plants from purple-dotted and non-dotted seeds were
equal in resistance (Table 11).

Seed coats were removed from seeds of 140165 to determine
whether the seed coat itself, other than color, was influenc-
ing resistance to Fusarium root—rot. It was found that seed-
coat removal did not influence resistance. Cuttings made of

disease—free plants of the resistant parent were inoculated

51

to further test the role of the seed coat in resistance.
Plants from cuttings were as resistant as those plants grown

from seed.

5. Test for resistance: F5 pOpulations.

 

Both F5 pOpulations were approximately equal with regard
to resistance (Figure 11). The 140165 parent had slightly
more resistance than either F5, while Early Perfection was
highly susceptible to the disease. The data were based on
the total number of plants for the combined classes of seed
in each pOpulation. Resistance was not associated with the
color of the seed coat, nor with the seed-shape. The data
showing this relationship is shown in Figures 12, 15, 14,
and 15. Seeds with transparent coats were as resistant as

pigmented seeds.

4. Relationship between F5_progeny-types and resistance to
root-rot.

The intermediate length internode (0.8cm) F5 plants ap-
peared to be more resistant than the long internode (2.0 cm)
140165-type plants. Ratios were obtained by rating each
plant on a point system in order to establish relative degrees
of resistance. The total number of points was obtained by
multiplying the number of short plants in each disease rating
in Table 10 by their disease rating. The total number of

points was divided by the total number of plants to give a

52

ratio. The following ratios were obtained: 0.55 for
140165—type plants as compared to 0.45 for the Perfection—
type plants in the cross, 140165 X Early Perfection; 0.50
for the 140165—type plants as compared to 0.44 for the
Perfection—type plants in the reciprocal cross, Early Per-

fection X 140165.

55
Table 9. Description of F2 seeds and F2 plants of the popula-

tions, Early Perfection X 140165 and 140165 X Early Perfection,
used in the Fusarium root-rot inheritance study.

 

 

 

ngseeds ‘ Eggplants
Seed- Cotyledon Purple Flower Node
surface color dotting color color
Seed Smooth (S) Yellow (Y) Present (P) Pigmented (P) Pigmented (P)
lot Wrinkled (W) Green (G) Absent (A) White (W) Colorless (C)

 

140165 X Early Perfection

(a
<5:

101
102
105
104
105
106
107
108
109
110
111
112
115
114
115
116
117 w
118 W
119 W
120 W
121 W
122. S
123 S
124 S

S

S

S

smmmméssssmmmmm
'WWWWWWWEWWWEWWWWéWfiW

2‘:

125
126
127

Early Perfection X 140165

‘WWWWW

é.
OOOWWWOWWOwOOWOOOfiOWWWWOWWE

HHHHHHHHHHHHHHHHHHHHammmmmm
HtwthHtttHHHHHHHHHHHHHHHtH

128
129
150
151
152
155
154
155

HHESHHmE
memmmmm
wamtwwb
wmtwtwtt
OfiOOWWOW

 

 

54

Table 9. (Continued)

 

 

F2 seeds Fp plants
Seed- Cotyledon Purple Flower Node
surface color dotting color color
Seed Smooth (S) Yellow (Y) Present (P) Pigmented (P) Pigmented (P)
lot Wrinkled (W) Green (G) Absent (A) White (W) Colorless (C)

 

W
W

156
157
158
159
140
141
142
145
144
145
146
147
148
149
150
151
152
155
154
155
156

mma‘séséammmmmmmmmmmmé
NMMKF<F<F<F<F<F<I~<QQ¢QG3F<F<F<F<M
*dtdtdtdtdtdtdtdtdtdtdtdpbbtdbbbbb
*dtdwtdtdtdWFUW-tdtdtd-‘éi'fitdtdtdtdtd

*UFUOHJFUFUO’UO’UFUOOOFUWFUOFUOO

 

 

 

 

 

H H H N NH mH ON HH mH HH 9 H OH om OH 00 HNH
O H N O N H H m m m O H H mH H OH ONH
O H O O O H H HH H O O H m NN O mNH
H O H H H O m m O O O H m mN H HN HNH
N O O O m m O m HH HH O H O OH O OH mNH
o o o H H m Hm OH OH OH H H Honv HH HH mm NO mmH
H H H O O O O O O O a H H H H HNH
O O H O O N H H m N O H H HH 2 ONH
O O O O O H O O N O O H m m H OHH
o H o o o H H m H H o H H HH H OHH
O N O O O O O O O O a H H O HHH
O H O O O O m m N H O H m H H OHH
H H O O H O NH O OH H O H N mH O mm mHH
H H H N O MH OH HH OH H O H AOHHV OH Hm mH OO HHH
O H O O O N H H O O H H H H OH mHH
O O H H H N O m O H H H H HH O NHH
H H H O O O N O O O O H N H H HHH
O O O O m m m H m O O H H ON OHH
N O O O m O OH O NH m e H 5 HH 3 OOH
O H O O O N O H N m O H O O H OOH
O O O O O O O m N H a H O O HOH
O O O H N O OH O H MH O H O m HH OOH
O H O H m N m O O O H H O OH OOH
O O O O O m H m H O H H O O HOH
O O O O O O N m N O a H O mH mOH
O O O H N O N O O O O H O m NOH
O O O O H H O H H H O H O H NH HOH
HOHeoeHHeH HHssH H mOHoHH

HammoHH Ho .ozu Aav pachqumHe Amcmom Ho .oZv 90H

m m H O m H m m H 6 H6O eeseHoo .so .HoH .HHH .oam seem

quHpmH HHHHm>om ommcmHH HOHoo AHV pammnH mOwammHO HBO OOHHQHHH
sees eeem HHO HammesH HHO HOHHmH HmO epooam
quppoO HOHoo oomHHSm
oHHHSH qumHHpoo Ivomm
wOmow mm

 

 

.mOomm mH Ho HOHHHHHommO Haw mHmQasd op HmpmHmH

mm OquwH HPHHm>om mmmmmHO poHIpoos adHmeSH Home HH HchoHH mH Ho mHmQasz .OH OHQOB

56

 

 

 

 

 

O O O O N H O H O O O H H OH OOH
H O O O H H N O O O O H O OH O OOH
O O O O O O H O H N H H H H HOH
H O H H H H HH O O H O H O HN a OOH
O O O O O O O O H H O H H H a NOH
o o o o o o m H o o H H m H H HOH
O O O O O H N H N H O H H H OOH
O N O O H H N H OH N a H H OH H OHH
O O O O O N N O O O O H H O O OHH
O O O O O H N O O O O H H OH N OH HHH
O O O O O O H N N N H H H OH OHH
o o o o o o O H m m o H o H OH OHH
O H O O H H O N O H H H O HH HHH
O O O H H H O O O O O H O O NH OHH
O H O O H H H O O O O H O N O NHH
O O O H N H O H H H O H O HH HHH
O H O O H OH HH NH H H O H OH HH H HH OHH
O N O N H O O O H O O H H NO OOH
O O O O N N O H H O H H H O O OOH
O NH H O OH HN OH HO ON O a H AOHOV HO ONH HO ONH HOH
O O O O O O O HH N O H H O OH 3 OOH
O O O O O O O H O H O H O O a OOH
O O O O N O O O H O O H O HH a HOH
O O O O O O O O O O H H O O OOH
O O O O O O O H N O O H O O NOH
H H O N HOH
O O O O O N H H N O O H O O OOH
O O O O O O N N H H a H O O ONH
O O O O O O H N O H O H O O ONH
OOHOHH H HOHpomemm HHHHH
NHQmOoHH Ho .ozv Nev pmonmqumpe Amdmmm Ho .ozv HOH
m m H O O H m m H 0 H0O omHOHoo .aw .HmH OHHH .oam Hmmm
mqupmm HpHHmbmm omwmmHO HOHoo AHV pammnH NOV qmmHO HBO OoHHnHHB
pwoo doom HHO pmommam HHO HBHHmH HmO Hpooam
OmepoO HOHoo mommhdm
mHHHSH. nodeHpoo IOmoO
mdmmm wH

 

 

.AcmqupqooO .OH memH

Table 11.

total number of plants tested on basis of seed-shape and
purple dotting of the seed coats for F1, backcross, and

F2 populations.

 

57

Ratios of Fusarium root-rot resistant plants to

 

Population

Seed-shape

Purple dotting
Smooth Wrinkled Present Absent

 

(F ) 140165 X
Early Perfection

(BC) Early Perfection
X 140165, F X
Early Perfection

(BC) Early Perfection
X 140165, Fl X 140165

(BC) 140165 X Early
Perfection, Fl X
140165

'(BC) 140165 X Early
Perfection, F X
Early Perfectlon

(F2) Early Perfection
X 140165

(F ) 140165 X
Eagly Perfection

0.906
0.866

0.911

0.484
0.670

0.784

0.885

0.748

1.000

0.981

0.556
0.789

0.865

0.855

0.589

1.000

0.952

0.741
0.826

0.878

 

----- all seeds were smooth and round.

58

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61

 

 

 

1:)

isease severity

Jisjribution
.Df tile F

Figure 1;) .
wrinfiloi
regards

'I'.'\.--- ' C‘
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to performance in

. ’ .4-:-. —"
HS (Imitin; 01 g;
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severely d5.

of the progeny from smoo R
14019; I Early Lerfection, with
rusarium root-rot test.

 

DISCUSSION

A. Radiation Studies

1. R1 data: germination.

 

Gamma radiation dosages of l,500r and 5,000r on soaked
seeds were too low for maximum reduction in germination
(Figure 4). An interaction of genotype and environment was
evident in both field and greenhouse tests. Therefore, the
results obtained at any one time are dependent upon the ac-
tion of radiation on genes and chromosomes and the inter-
action of gene products with the existing environmental
conditions. Results of radiation treatment cannot be pre—
dicted, since mutations and chromosome irregularities occur
.at random and the same changes are infrequently repeated (42).
Higher radiation dosages could have been used in these stu—
dies on seeds soaked in water or chemical solutions, but
since irregularities and mutations are more frequent at high
dosage levels (42), there are also great detrimental changes
with most favorable mutations (55). Therefore, it was rea-
soned that low dosage levels might produce beneficial changes
with fewer deleterious ones.

The dosages of 7,500r and l5,000r of gamma radiation and

20,000 rep and 40,000 rep on dry seeds, and 10,000 rep and

-62-

65

20,000 rep of beta irradiation on soaked seeds gave satis-
factory results in the Rl’ as measured by the low germina—
tion (Figure 5). The concentrations of Endothal and uranyl
nitrate used in this study were low, 0.002 per cent and

0.25 per cent, respectively. As a result, these chemicals
appeared to have no visible radiomimetic effects on Rl
plants. However, the fact that the chemicals did not pro—
duce visible effects does not eliminate the possibility that
Endothal and uranyl nitrate may be radiomimetic at higher
concentrations, since the concentrations used were adjusted
to give germination comparable to that of the water-soaking
treatments. On the other hand, colchicine appeared to have
radiomimetic prOperties at a concentration as low as 0.002
per cent. This is less than that which is effective in pro—

ducing chromosome doubling and induction of mutations (lO).

2. R1 data: dwarfing, leaf-distortion, and leaf variegation.
The abnormal distortion and variegation observed in the
early stages of growth in the R1 plants did not continue with
growth and eventually disappeared with the withering and
dropping of the lower leaves. Similar plant and leaf abnor-
malities were obtained from X-irradiated maize seeds by
Anderson, et gl. (4) and Randolph (47). They observed the

typical plant dwarfing and mottling of the lower leaves.

64

Similar leaf changes were found when tomato seeds (28) and
Fodder pea seeds (21) were X-rayed. Since these changes
were not carried over in the later generations as heredi-
tary characters, there is an indication that the abnormali-
ties might have been the result of either the effect of radi—
ation on cell differentiation, effecting only the older,
outer layer of cells of the embryo, or to a temporarily
disturbed metabolism and the inhibition of plant auxins. It
has been postulated that the first compound formed in irra-
diated cells is hydrOgen peroxide (58). According to
Quastler and Baer (46) and Skoog (52), the delay in growth
of irradiated plants is the result of auxin inactivation by
the peroxide. Clark (9) attributes the reduced growth-
response in irradiated plants to a delay in mitosis, while
death of irradiated seeds, and seedlings from irradiated
seeds is caused by the inactivation of enzymes (55).
Histological examination of mottled leaves of plants
from X—rayed seeds of sunflower by Noyucki (45) revealed
that the chlorOplasts were in clusters and that the chloro-
phyll was diffused throughout the leaf-cells. It was con-
cluded that the distortion was the result of wavy, irregular—
shaped epidermal cells with no distinction between the pali—
sade and the parenchyma tissue. The mottled leaves were the
result of a differential concentration of chlorOplasts and

intercellular spaces. Dark areas were composed of great

 

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65

concentrations of chlorOplasts with little or no intercellu—
lar spaces, while the light areas contained few chlorOplasts
with large intercellular spaces.

Beta irradiation of dry seeds resulted in 62 per cent
plant—abnormalities in the R1 at a dosage of 20,000 rep
(Figure 6). At the higher dosage of 40,000 rep there was a
decrease in the percentage of plants showing dwarfing, leaf-
distortion, and leaf—variegation. The data presented in
Figure 2 support the results with regards to the action of
beta radiation on seeds. With regards to the number of
plant abnormalities in the R1, 52 per cent of the plants
showed leaf—changes at 40,000 rep, while 21 per cent showed
changes at 80,000 rep.

The inability of electrons to penetrate deeply into
living cells, may explain the large number of plants surviv-
ing the high dosages of beta radiation and the small per-
centage of plants exhibiting leaf—changes in the R1. By
comparison, seeds which were irradiated with high dosages,
7,500r and lB,OOOr, of gamma rays produced plants which were
all visibly changed (Figure 6). Gamma is a high ion pene-
trating radiation source (5). If the seeds receiving the
beta radiation had been oriented so that the young embryos
were toward the radiation source, equal penetration might
have occurred and the effect might have been equal to that

of gamma radiation. However, the cotyledons of the seeds

66

probably absorbed the rays and protected the embryo, since
the electrons have a low penetrating power. Seeds which
were exposed to 80,000 rep (Figure l) and germinated and
showed visible changes in the R1 mighthave originated frmn
seeds that had their embryos oriented to the side, per-
pendicular to the electron beam, and within the penetration
range of the electrons. The depth-dose function relative

to the per cent penetrance of the ions per unit density of
material is shown in Figure 16. The specific gravity of dry
Early Perfection seed is 1.4 and the seeds average 0.70 X
1.0 cm. through the short and long axis. The unit density
of pea seed was determined by multiplying specific gravity
by unit density (centimeters). At a depth of 0.1 cm., which
is the depth of the embryo in Perfection seeds, the unit
density is 0.14. This gives approximately 85 per cent pene-
trance of the ions from its source. Virtually no ions can
penetrate the embryo to a depth of 0.5 cm. An embryo at

the base of the seed, 0.7 cm. from the point of 100 per

cent penetrance, would be unaffected by ionization.

5. R2 data.

 

Only a few "possible—mutations" were found in this study
(Table 5). The frequency of abnormal R2 plants increased

with an increase in the dosage rate on dry seeds. However,

 

 

 

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68

there appeared to be little change when the dosage was in-
creased on soaked seeds, except where water was used. In
contrast, Freisleben and Lein reported the actual mutation

6

frequency falls continuously from 18.4 X 10- at 4,000r to

5.6 X 10‘6

at 14,000r in X—irradiated dry barley seeds (25).
This frequency is considerably lower than a frequency of
14.2 X 10"5 obtained in this study where the dry seeds were
exposed to 15,000r. However, the data presented do not in-
clude the lethals which may or may not be considered as
mutations. Gustafsson (25) suggested that Freisleben and
Lein may not have considered the non-germinated seeds as
lethals. He postulated that the mutation rate might have
been greater at the dosage of 14,000r if lethals had been
included.

A conclusion might be made that the visible changes
observed in the R1 plants were not hereditary, since there
appeared to be no association between the R1 plants and the
number and kind of abnormal R2 plants. Therefore, seeds
from Rl plants could be used without regard to their ap-
pearance in the R1 when radiation is used for the production
of mutations. I

The infertility observed in some plants, as measured by

the occurrence of pods with rudimentary ovules, indicated

that fertilization may have occurred, but as result of

69

possible differences in the chromosomes, the ovules aborted.
Radiation induced sterility has been reported in tomatoes
(7), Fodder peas (21), and corn (4). Anderson and co-workers
explained sterility on the basis of chromosome irregulari-
ties which gave rise to pollen grains deficient for some por—
tion of the chromosome. Lamprecht (54) obtained partially
sterile plants from X—irradiated pea seeds which were char-
acterized by an interchange in the X1, and segregation in

the X2 into 50 per cent fertile and 50 per cent semi-sterile
plants. He also observed plants with a chromosome dupli-
cation which resulted in segregation into 75 per cent fertile
and 25 per cent semi-sterile in the X2 plants.

Forty-four per cent of the chlorOphyll deficient plants
found in the R2 were virides, and 54 per cent albino types.
Rosen (49) did not find any albinos in plants from X—irradiated
pea seed, but frequently found xantha, lutescens, chlorina,
and albo-virides types. Gustafsson (26) classified chlorOphyll
mutations in X—irradiated pea material as xantha and virides

‘types.

4. R5 data.

 

Gamma radiation appeared to be more effective than beta
radiation for producing mutations (Table 6). Gamma irradia-
tion of dry seeds was responsible for the production of the
less common mutations, for example: dark—green seeds in Line

1 and small seeds and pods in Line 2-C.

70

Plants of Line 1 were tall in the R2 generation and
produced dark-green seeds. There was no change in the R3
plants. This indicates that a mutation had occurred for
these characters.

The mutation in plants of Line 2-C, which had small
pods and seeds, might be explained on the basis of either a
"position effect," or some other chromosome change. Since
plants of Line 7-B were tall and had normal type foliage
in the R2, while the plants in the succeeding generation
were all tall and narrow-leafed. Thus, a recessive gene
mutation might have been responsible for the different leaf—
type. Recessive mutations occur in the R5 of self-pollinated
plants which originated from seeds which were exposed to
radiation (55).

Tall R3 plants, as illustrated in Figures 8 and 9, ap-
peared to be the most frequent type of mutation found. This
mutation appears to be a dominant character. According to
the review of pea-genetics by Wade (59), a series of genes
or modifying genes control plant—height which is determined
by the number of internodes prior to flowering, and the
length of the internodes. However, other growth-inhibiting
genes are present which condition the action of the genes
for long internodes° According to DeHann (10) at least one
of the inhibiting genes must be in the recessive state with

the gene which controls long internodes in order to have a

71

tall plant. A mutation of the genes for tallness and of one
or several of the growth-inhibiting genes might have occurred
for an intermediate height Early Perfection plant to mutate

to tall. Plants of Lines A and B had longer internodes and

a greater number of nodes prior to flower emergence (Table 7).

Plants of Line A responded differently at different
times of the year, either the length of day or the light
intensity may have influenced the action of the genes which
determine vine-height. Investigations by Heath and Wittwer
(48) indicated that the pea variety Early Perfection responded
to day lengths.

The work by Lewis (56) on heterosis in tomato may help
support the supposition that light intensity is an influ-
encing factor. He found the growth rate of the cultivated
and the wild tomato to be similar at low light intensities.
0n the otherhand, at a high light intensity, the wild species
had a greater growth rate than the cultivated one. Neverthe-
less, even though the reaction in tomato is in reverse to
that of the supposition presented, there is an indication that
light intensities may induce differential growth-response in

plants.

5. Root—rot resistance test of R5 plants from irradiated seeds.

 

No resistance was obtained to Fusarium root-rot in the
R5 plants from seeds which were exposed to radiation. Fur-

thermore, no R plant rated better than any non—irradiated

5

 

72

Early Perfection control. However, it must be considered
that only a small pOpulation was tested, because of inade-

quate space and facilities in the greenhouse.
B. Inheritance Studies

Resistance appeared to be highly associated with smooth
seeds where the plants from the smooth-seeded classes in
each F2 pOpulation were combined into the following two
groups: plants of ratings 0 and 1 as resistant plants;
plants of ratings 2 and 2 as susceptible plants (Table 11).
However the F1 Early Perfection X 140165, had a distribution
intermediate to the parents, even though the seeds were
smooth. The reciprocal cross, where the seeds were also
smooth, produced an F1 in which the plants had resistance
similar to 140165. A comparison of the two F5 pOpulations
(Figures 14 and 15) indicated that plants from wrinkled seeds
were as resistant as plants from smooth seeds. Similarly,
plants from seeds with transparent coats were as resistant
as plants from pigmented—coated seeds (Figures 12 and 15).
Therefore, on the basis of F5 data, a conclusion can be made
that resistance is not associated with either the seed-shape
nor with the seed coat color.

Resistance to Fusarium root—rot appeared to be a quan-

titative character. However, the distribution of F5 plants

75

was skewed (Figure 11) in favor of the resistant parent.
Two possible explanations for the skewness of the curve are
as follows:

1. It may be possible that resistance is linked or
associated with a character or characters other
than seed coat color, seed—shape, and purple dot-
ting of the seed coat.

2. A series of allelic genes with modifiers may
determine the degree of resistance to Fusarium

root-rot.

SUMMARY AND CONCLUSIONS

Soaking seeds with either Endothal, uranyl nitrate, or
colchicine, prior to either source of radiation, did not
appreciably increase the variability. However, colchicine
was similar to radiation in producing dwarfing, leaf-distortion,
and leaf-variegation in the R1 plants. Soaking seeds in water
prior to gamma irradiation was as effective as any treatment
for producing abnormal plants in the R2. These abnormal
R2 plants were characterized by chlorOphyll deficiencies,
sterility, lateness, earliness, tallness, dwarfness, and
branching.

The exposure of dry seeds to gamma, as compared to ex-
posure to beta irradiation, resulted in a greater reduction
in germination and a greater increase in the numbers of plants
showing leaf and plant abnormalities in the R1. Data are
presented to support the theory that the low penetrating
power of beta ions is responsible for the ineffectiveness
of the cathode radiation source.

No association was noted between the variability in the
R2 plants with that in the R1 plants. Therefore, there would
be no advantage in saving seed from abnormal Rl plants rather
than from the normal-appearing plants.

All R plants from the radiation treatments which were

5

tested for Fusarium root-rot resistance were found to be susceptible.

_ 74 _

 

'75

The study revealed that precise results from radiation
treatments cannot be predicted and that environmental con-
ditions, such as temperature and soil moisture, exert a
profound influence on plant-growth and tend to confound the
reaction to radiation. Optimum specifications with regards
to dosage levels, as established under greenhouse conditions,
are not directly transferable to the field.

Reciprocal crosses were made between the variety Early
Perfection and a Fusarium root—rot resistant plant intro—
duction, 140165, to determine the mode of inheritance of
resistance to this disease. The backcross and F2 popula—
tions produced only colored seeds. Therefore, no definite
conclusions could be made, on the basis of F2 data, concern-
ing the mode of inheritance. Segregation for seed coat
color occurred in the F5 pOpulations. On the basis of F3
data, transparent coated and wrinkled seeds were as resist-
ant as pigmented and smooth seeds.

Resistance to Fusarium root~rot appeared dorminant. The
distribution of the Fusarium root-rot tested F5 progeny was
not normal. Two suppositions were presented to explain the
skewed distribution.

Clear-cut results were not possible in the inheritance
studies, primarily because the resistant parent, 140165 was

not immune.

76

Resistance was obtained in seeds with transparent coats
and in desirable plants. One should be able to initiate a
breeding program with reasonable assurance that Fusarium

root-rot resistant commercial-type plants could be produced.

10.

ll.

12.

LITERATURE CITED

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between the Office of Naval Research and MSU.

 

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