Title: Post-irradiation development of chromosomal damage in seeds
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Title: Post-irradiation development of chromosomal damage in seeds
Physical Description: vi, 79 leaves : ill. ; 28 cm.
Language: English
Creator: Stevenson, Harlan Quinn, 1927-
Publication Date: 1963
Copyright Date: 1963
 Subjects
Subject: Plants -- Effect of radiation on   ( lcsh )
Seeds   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by Harlan Quinn Stevenson.
Thesis: Thesis (Ph. D.)--University of Florida, 1963.
Bibliography: Includes bibliographical references (74-78).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00097961
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000470641
notis - ACN5427
oclc - 37410790

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POST-IRRADIATION DEVELOPMENT OF

CHROMOSOMAL DAMAGE IN SEEDS












By

HARLAN QUINN STEVENSON


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA
August, 1963














SACKNOWLEDGEMENTS


The writer wishes to express his appreciation to Dr. Calvin Konzak,

Washington State University, for kindly supplying the seeds used in

this research; to the Department of Nuclear Engineering for use of the

cobalt-60 source and to Dr. Mendel Herzberg, Department of Bacteriology,

for providing office space during much of the cytological work and the

writing of the manuscript. He is deeply appreciative of the financial

aid provided by Graduate School and Nuclear Science Fellowships.

To the members of his committee, Dr. Yoneo Sagawa, Botany; Dr. H.

M. Wallbrunn, Biology; Drs. A. T. Wallace and J. R. Edwardson, Agronomy;

and especially to the chairman, Dr. Alan D. Conger, the writer owes an

immense debt of gratitude.

Many hands have helped to make the burden lighter. Thanks to my

laboratory companions who have been most willing to help when needed:

Doris Gennaro, Ram Prasad Sarda and William Blasky. Dr. R. G. Hoffman,

Statistical Laboratory, has been helpful in rendering statistical

advice, and Dianna Epperson, Department of Radiology, kindly confirmed a

number of the cytological observations. Marcie Norris has been of great

aid in typing from the manuscript, Joan Cheatham, in preparing the final

copy, and William Pettit, in drawing the figures.

No words can express the grateful heart I have for my wife, not

for her patience and understanding only, but for the very real support

she has given me throughout my work.

ii















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS . . . . . . . . ... . . ii

LIST OF TABLES . . . . . . . . ... ... . iv

LIST OF FIGURES . . . . . . . . ... . . v

INTRODUCTION . . . . . . . . ... ....... 1

REVIEW OF LITERATURE . . . . . . . . ... .. 2

MATERIALS AND METHODS . . . . . . . .... . 11

EXPERIMENTAL RESULTS . . . . . . . . ... . 18

Appropriateness of the Method . . . . . ..... . 18

Effect of Very Low Moisture Content . . . . ... 27

Effect of Higher Moisture Contents . . . . ... 44

Effects of Post-Irradiation Storage and Oxygen . . .. 53

General Cytological Observations . . . . . ... ..54

DISCUSSION . . . . . . . .... .. . . . 63

SUMMARY . . . . . . . . . . . . . 72

LITERATURE CITED . . . . . . . ... .. . . 74

VITA . . . . . . . . . . . . . . . 79














LIST OF TABLES


Table Page

1 Water Content, at Equilibrium, of Barley Grains
Stored at Different Constant Humidities . . .. 12

2 Comparisons Between Dishes of Mean Heights at 8
Days of Seedlings Which Either Had Two Root
Tips Excised or Were Left Intact . . .... 25

3 Comparisons Within Dishes of Mean Heights at 9
Days of Seedlings Which Either Had Two Root
Tips Excised or Were Left Intact . . . ... 26

4 Comparison of Per Cent Normal Metaphases as
Scored on Pairs of Roots, Each Pair From an
Individual Seedling . . .... . . . . 28

5 Mean Seedling Height for Treatments at Different
Doses and for Samples Examined Cytologically
for Per Cent Normal Metaphases . . . .... .43

6 Seedling Height and Per Cent Normal Metaphases
for'Seeds of Different Water Content, with
Summary of Correlation Analysis . ... ... . . 52

7 Seedling Height and Per Cent Normal Metaphases
for Seeds Given Various Post-Irradiation
Treatments, with Summary of Correlation
Analysis . . . . . .... . . . . 61















LIST OF FIGURES


Figure Page

la Frequency Distribution Histograms of Seedling
Heights for Barley Seeds With 2.8% Water
Content; Gamma-Irradiated in Air or in Oxygen
at Low Doses and Stored for 7 Days Post-
Irradiation . . . . . . . . ... .. 20

lb Frequency Distribution Histograms of Seedling
Heights for Barley Seeds With 2.8% Water
Content; Gamma-Irradiated at High Doses and
Stored Post-Irradiation . . . . . .... .22

2 Frequency Distribution Histograms of Seedling
Heights for Barley Seeds With 3.8% Water
Content; Gamma-Irradiated in Nitrogen and
Germinated Immediately . . . . . .... 24

3 Plot of Log Mean Seedling Height Versus Gamma-
Ray Dose for Barley Seeds With 2.8% Water
Content; Irradiated at High Doses and Stored
90 Days Post-Irradiation . . . . . .... .31

4 Plot of Mean Seedling Height Versus Gamma-Ray
Dose for Barley Seeds With 2.8% Water Content;
Irradiated at Low Doses and Stored 20 Days
Post-Irradiation . . . . . . . ... 33

5a Regression of Seedling Height on Per Cent Normal
Metaphases for Seeds With 2.8% Water Content;
Gamma-Irradiated at Low Doses and Stored 20
Days in Dry Air . . . . . . . . ... .35

5b Regression of Seedling Height on Per Cent Normal
Metaphases for Seeds With 2.8% Water Content;
Gamma-Irradiated at Low Doses and Stored 20
Days in Oxygen at One-Half Atmosphere Positive
Pressure . . . . . . . .. .. . .. .37

6 Regression of Seedling Height on Per Cent Normal
Metaphases for Seeds With 2.8% Water Content;
Gamma-Irradiated at High Doses and Stored 90
Days in Dry Air or in Oxygen at One-Half Atmos-
phere Positive Pressure . . . . . . ... 40










7 Plot of Log Mean Per Cent Normal Metaphases
and Log Mean Seedling Height at 8 Days Versus
Dose for Seeds With 2.8% Water Content; Gamma-
Irradiated and Stored 20 Days in Dry Air . . .. 42

8 Regression of Log Mean Seedling Height on Gamma-
Ray Dose for Seeds With Different Water Contents;
Composite Data From Three Experiments . . ... 47

9 Relative Sensitivity of Seeds of Different
Water Content Gamma-Irradiated and Stored in
Air, Nitrogen or Oxygen . . . . . . .. 49

10 Regression of Seedling Height on Per Cent Normal
Metaphases for Seeds of Different Water Content
Irradiated and Stored in Air . . . . .... .51

11 Plot of Log Mean Per Cent Normal Metaphases and
Log Mean Seedling Height Versus Dose for Barley
Seeds Gamma-Irradiated in Helium and Stored
at Different Oxygen Tensions . . . . ... 56

12 Plot of Log Mean Seedling Height Versus Dose for
Barley Seeds With 3.8% Water Content, Gamma-
Irradiated in Nitrogen and Stored at Different
Oxygen Tensions . . . . .... . . . 58

13 Regression of Seedling Height on Per Cent Normal
Metaphases for Seeds Irradiated in Nitrogen and
Stored at Different Oxygen Tensions . . . ... 60


Page


Figure














INTRODUCTION


A considerable body of data has accrued and a number of significant

hypotheses have been put forward in the last ten years concerning the

radiobiology of seeds. Much of this work has been based solely or

predominantly upon a single biological criterion, viz, seedling height.

Justification for extension of results obtained in such studies to the

consideration of other biological responses and to the mechanisms

possibly operative in effecting them rests on correlative studies of

the various criteria of radiation effect.

The present study was undertaken to investigate to what degree

seedling height reduction (length of first leaf) in barley is related

to the presence of chromosomal aberrations in the cells of the embryonic

root meristems of irradiated seeds. The effect of water content,

storage, and oxygen tension upon this correlation is examined on an

individual plant basis.














REVIEW OF LITERATURE


Plant seeds and such dry fruits as the grains of cereals (which are

commonly referred to as seeds) have been recognized as constituting

remarkably useful material for a broad spectrum of biological studies.

This review will be restricted to radiation effects and their modifica-

tion especially as studied in barley.


Early Work

The pioneer work of Stadler (1928a,b) establishing the mutagenic

effect of X rays and gamma rays on plants, and paralleling that of

Muller (1927) with Drosophila, was largely done with barley. Reading

these papers after thirty-five years one cannot help but be impressed

by the number of significant observations and hypotheses arrived at so

early. By 1930, Stadler (1930) was able to summarize his research and

draw a number of important, if tentative, conclusions, explicit or

implied, only some of which are cited below:

1. Induced mutations are similar to spontaneous ones.

2. Induced mutations are almost exclusively recessive.

3. Induced mutations are almost exclusively deleterious.

4. Induced mutations may in many cases be deletions.

5. Mutation frequency increases linearly with dose.

6. Mutation frequency is independent of X ray wave length.

7. A threshold effect may exist for mutation induction.

8. Radiosensitivity (mutation rate) is not affected by moisture

content.








9. Radiosensitivity is not affected by temperature.

10. Radiosensitivity (inviability) increases with moisture content.

11. Radiosensitivity (inviability) increases with post-irradiation

storage.

12. Radiation causes chromosomal disturbances.

13. Radiation imposed during early development is a useful tool

for studying ontogenesis and morphogenesis as well as gene

action.

A very impressive list of accomplishments indeed!

Sweden early became and has remained one of the major centers of

seed radiation research. Chief credit for this can be attributed to

the early and continued research of Ake Gustafsson. In a series of

papers (Gustafsson, 1937a, b, 1938) he summarized the results of his

early radiation experiments with barley. Most relevant to the work

considered here are his observations on frequency of "disturbed cells"

(i. e. those cells in division which show chromosomal aberrations)

following X irradiation and ancillary treatments.

He found a definite increase in frequency of disturbed cells with

increase in post-irradiation storage. This he termed "cytological

after-effect" (Gustafsson, 1937a, p. 326). None of his material was

germinated immediately after irradiation, but the proportion of dividing

cells showing aberrations when stored 18 days was almost double that

stored 4 days. In the same paper he reported that higher water content

is associated with a higher frequency of disturbances. It should be

noted, however, that his driest seeds were approximately 10% water and

that the comparison was made with seeds which were soaked for varying

periods of time toincrease their water content.








Gustafsson (1937b) maintained that following the X irradiation of

the resting barley seed, the prevalence of disturbed cells was greater

in certain regions of the roots upon germination. He attributed this

to increased cellular activity which in turn might be brought about by

higher water content in those cells at the time of irradiation. He also

found greater frequency of disturbances in irradiated aged seeds.

Gelin (1941), examining material from the same treatments employed

by Gustafsson, modified and extended Gustafsson's conclusions. He

found no real difference in the various histogens of the roots as

regards frequency of disturbed cells and therefore, combined the data

from these different layers. The summary table in which he compared

his cytological findings with the sterility and mutation data from

Gustafsson (1940) is reproduced here in simplified form.

Golden Barley 1939 Harvest

Irradiated 160 KV, 7.5 ma 4 mm Al 72 r/min Dose 10 kr

Water Content Disturbed Cells Sterility X1 Mutation X2

10% 12.66% 51.6% 7.9%

15% 27.97% 76.7% 13.4%

Soak 23 hours 53.80% 87.6% 26.2%

Gustafsson (1940, p. 7) had concluded that "Sterility and mutation

imply connected phenomena." The above results formed the basis for

Gelin's assertion that an even closer correlation exists between dis-

turbed cells and mutation rate in the X2.

Froier and Gustafsson (1944) demonstrated that the length of first

leaf and survival in the field increased directly with size of embryo

in irradiated wheat. They screened grain from each of two varieties

into four size classes and irradiated, at approximately 12% seed








water content, with 10 or 29 kr. They also showed a rather surprising

radiation protection effect provided by the presence of hulls in barley

and oats. The objection might be leveled that the barley used in their

study was of different varieties but in the case of oats, the hulless

condition was obtained by mechanical removal and there was no genetic

difference.

Luther Smith (1951), in his extensive review of the cytology and

genetics of barley, summarized en passant most of the early radiation

work. It will only be noted here that although search for fundamental

principles was not neglected, as evidenced by much of the foregoing,

the chief impetus behind many of the earlier investigations was the

desire to obtain a maximum yield of induced mutations for breeding work.

Stadler (1930) had clearly indicated that mutation induction by radiation

was not likely to prove a shortcut to success in this applied field

except in rare circumstances, but when no dramatic success was im-

mediately forthcoming, the whole field of radiation botany progressed

only slowly until after World War II.


The Nuclear Age

The atomic bombs which ended the war and their continued testing,

with the consequent increase in radioactive fallout, created a great

resurgent interest in radiobiology which has been paralleled by a

rapidly expanding technology. These have led to accelerated activity

not only in the applied field but in fundamental research as well:

witness the two International Conferences on the Peaceful Uses of Atomic

Energy (United Nations 1955, 1958).

Moisture effect

Among recent developments in the radiobiology of seeds has been








the discovery that seeds drier than the "normal" state, which is about

10 to 12% water, evince an inverse relationship between moisture content

and radiosensitivity based either on seedling height (Ehrenberg and

Nybom, 1954; Caldecott, 1954, 1955a) or on chromosomal aberrations in

the germinating shoot tip (Caldecott, 1955b). Concerning this latter

relationship, Caldecott showed that for seeds of different water con-

tents all receiving 20 kr of X irradiation there was a sharp rise in

per cent normal cells (those anaphases not showing bridges or fragments)

from embryos with 4% to those with 8% water, at which point the effect

of moisture on cytological damage leveled off. The actual values

obtained were 4.0% and 27.9% normal cells respectively.

More recently, Conger (1961) has reported that for "super dry"

seeds (less than 3% water) there is a decrease in radiosensitivity

(based on seedling height) which parallels the decrease in the number

of long-lived free radicals as determined by electron paramagnetic

resonance.


After-effect

The last paper cited above deals not only with water content but

with the influence this has during post-irradiation storage, i. e. the

"after-effect" phenomenon of Gustafsson as mentioned previously and

which recurs sporadically throughout the early literature (Tascher, 1929;

Wertz, 1940; Sax, 1941).

Kaplan (1951) and Caldecott and Smith (1952) observed that heat

treatments applied after irradiation was completed could alter the

response. The latter authors reported increased mutation rates and

seedling heights, reduced chromosomal aberrations both in root tips

and in microsporocytes, and no significant alteration of survival to








maturity with treatment of 75, 80, or 850 C for 30 minutes after ir-

radiation.

Adams et al. (1955) demonstrated a long term after-effect phenomenon

in irradiated barley in contrast to the short term responses cited above

which had also been reported for other organisms and for organic com-

pounds as well (cf. Mitchell and Holmes, 1954). Using seeds with

approximately 8% water content, an X ray dose of 7.5 kr and storage

times of 2, 4, 6 and 8 weeks, they found an approximate 80% increase in

bridges per cell, and a 60% decrease in seedling height and in germina-

tion at the longest storage time when in oxygen. The after-effects

were less when storage was in air or nitrogen. They were progressive

with storage time but showed a tendency toward leveling-off after

6 weeks.

This work confirmed and extended the observation of Ehrenberg

(1955a) that post-irradiation storage of'barley causes reduced growth.

Lawrence (1955) noted reduced germination and survival in stored

x-irradiated barley.

Curtis et al. (1958) in carefully controlled experiments carried

out in air, established that the after-effect increases as water content

decreases from 12% to 4% (the lowest value tested). They distinguished

two components, a brief one which is effective for only 4 hours post-

irradiation and is very moisture sensitive, and an extended one which

is much less sensitive to moisture content and continues to develop for

a month or longer. For seeds with 4% water content, storage for one

week post-irradiation increased damage by as much as a factor of twenty

over those which received the same dose that were immediately soaked

and germinated. This study was based solely on seedling height reduc-

tion as a measure of radiosensitivity.








Oxygen effect

Work on the effect of oxygen prior to the realization of the

significance of after-effects and of moisture content lacks the control

of variables necessary for a clear interpretation and must be considered

in this light.

Hayden and Smith (1949) had observed that when germinating barley

was irradiated in a partial vacuum, radiosensitivity as measured by

chromosomal aberration frequency and by seedling height reduction was

not as great as when irradiation was in air. Nilan (1954) found that

seedling height, survival, and mutation rate were unaffected by dif-

ferences in oxygen tension during irradiation but that cytological

effects were sensitive. Adams and Nilan (1958) found that although

post-irradiation storage under 100 pounds of oxygen increased radio-

sensitivity as determined by germination, chromosomal aberrations in

shoot tips, seedling height and survival in the field, it did not

alter mutation rate.

A recent review of the post-irradiation oxygen effect and summary

of their own work has been given by Nilan, Konzak, et al. (1961). Of

particular note are the following conclusions which they drew. Freezing

seed at dry-ice temperature (-780 C) suspends all after-effects until

thawing. Other things being equal, the magnitude of the oxygen effect

post-irradiation depends on the criterion of radiosensitivity which

is used. For reduction in seedling height this is eightfold, for

fragments per cell in M1 shoot tips this is sevenfold, and for chloro-

phyll mutations per 100 M2 seedlings this is sixfold. For seeds below

3% water content they find reduction in the after-effect. It becomes

negligible in some treatments at about 1% water content.








Caldecott (1961; cf. Bozzini, Caldecott and North, 1962) has

summarized results he has obtained with seeds of low moisture content

(about 4% water in the embryo). Of interest is his finding that post-

irradiation storage for 8 days in air resulted in skewed seedling height

distributions--even bimodality. Separating the seedlings into three

height classes and examining them for interchange frequency in X1

spikes and for mutant seedlings in the X2, he found a close correspon-

dence of these criteria with each other and with seedling height reduc-

tion. Wolff and Sicard (1961) reported data on "super dry" (about 2%

water) and "normal dry" (about 10% water) seeds at variance with the

results of Caldecott and of Curtis reported above. Their "super dry"

seed was obtained by two months' storage over calcium chloride and

their normal dry.seed was open-stored in the laboratory. When the super

dry seed was stored post-irradiation over the desiccant for varying

periods of time up to 32 days, there was'no increase in the radio-

sensitivity as determined by reduction in the mature length of the

first leaf. If, however, similar "super-dry" seed was stored under

room conditions, it grew even taller than "normal-dry" seed irradiated

and stored at room conditions. On the other hand, "normal dry" seed

if stored in the desiccator after irradiation showed progressively more

after-effect with time--giving a growth response essentially equal to

that of the "super-dry" desiccator-stored seed from 18 days onward.

Conger and Fairchild (1952) demonstrated that oxygen tensions

greater than that of a normal atmosphere can cause chromosomal aberra-

tions in dry pollen and microspores of Tradescantia which are identical

to those caused by ionizing radiation. Ehrenberg et al. (1957)

produced similar effects by exposing barley grains to 60 atmospheres








of oxygen for one and two week intervals. Adams and Nilan (1958) did

not observe any effect with oxygen at one atmosphere. More recently

several research groups have reported mutation induction by oxygen

treatment of barley grains (Kronstad et al., 1959; Moutschen-Dahmen

et al., 1959).

There is a great miscellany of observations published which touch

on the present work in one respect or another and which have not been

reviewed here. Some will be dealt with in the discussion section.

Two general works on seeds are those authored by Barton (1961) and

edited by Stefferud (1961). Numerous reviews and symposia have a bear-

ing on the present work. The most relevant are listed below:

Radiation Protection and Recovery, Hollaender, A. ed. (1960)

Symposium on the Effects of Ionizing Radiations on Seeds (1961)

Symposium on Mutation and Plant Breeding (1961)

Fundamental Aspects of Radiosensitivity, U. S. Brookhaven National

Laboratory (1961)

Radiation-induced Chromosome Aberrations, Wolff, S. ed. (1963)

Publications before 1955 are listed in Bibliography on The Effects

of Ionizing Radiations on Plants, Sparrow, A. H., J. P. Binnington and

V. Pond (1958).














MATERIALS AND METHODS


The seeds

The barley grains used in these experiments were of Hordeum vulgare

L. cultivar Himalaya (C. I. 620) from the selected strain maintained

by C. F. Konzak, Washington State University. All seeds were from the

1961 harvest.

Upon receipt the seeds were passed through a series of sieves.

The major fraction passed through U. S. Standard Sieve Series No. 6

(openings measuring 3360 microns or 0.132 inch) but not No. 7 (2830

microns or 0.111 inch). Only this portion was used. All apparently

defective seeds (broken, misshapen or discolored) were removed before

storage and the seeds were again checked before use.

The seeds were stored in desiccators over dry calcium chloride

from the time they were received until several months prior to use, at

which time they were transferred to desiccators maintained at the

appropriate relative humidity by dry chemicals or saturated solutions.

The various humidities, the means whereby they were obtained, and the

moisture content of the seeds are summarized in Table 1. When irradiated,

all seeds, regardless of treatments, were in a state of low physiological

activity ("resting") but not dormant (i. e. requiring "after-ripening"

before germinating).


Special seed treatments

In those experiments designed to study the "oxygen effect," seed

11







TABLE 1


WATER CONTENT, AT EQUILIBRIUM, OF BARLEY GRAINS STORED AT DIFFERENT
CONSTANT HUMIDITIES

Determinations from these experiments, and from the literature.

Relative Per Cent Weight Loss (Per Cent Water)
Storage Over: Humidity Whole Seeds* Embryo** Endosperm**
(at 200C) (at 200C) (1300C for 20 hrs.) (1050C, time unspecified)

P205 (evacuated) 0*** 2.8

Dry P205
(not evacuated) 0 3.8 4 5

Sat'd NaOH 6.5 6.7

Sat'd ZnC12 10 7.3

Sat'd KC2H302 20 9.2

Sat'd CaC12 32 10.1 6 8

Sat'd NaC103 75 15.5 11 14


*Determination following the method of Hart et al. (1959)

**Data from Caldecott (1955b)

-*'**A table of constant humidity at given temperatures over a saturated solu-
tion of various chemicals is given in Handbook of Chemistry and Physics,
44th Edition, 1962, pp. 2595-2596.








samples were placed in glass ampoules, attached to a glass manifold by

means of Tygon tubing and repeatedly evacuated and flushed with the

desired gas. In order to be reasonably sure of removing oxygen from

the seeds, initial evacuation was of several hours duration at a

pressure of 500 microns of mercury or less, as determined by a McLeod

gauge.

The gases used were: prepurified nitrogen with a tested concentra-

tion of oxygen of 0.5 + 0.2 ppm; oxygen; and air admitted via a column

packed with silica gel.

The ampoules were filled with the appropriate gas and the Tygon

tubing clamped. For nitrogen and oxygen an overpressure, either 40 or

80 mm Hg over atmospheric, was established as a precaution against

possible leaks. Without exception every ampoule retained positive

pressure during storage.


The irradiation source

The University of Florida Engineering Gamma Irradiator (UFEGI) was

used routinely as a source of radiation. This facility (Duncan et al.,

1960) consists of a distributed source of cobalt-60 wafers clad in

stainless steel and stacked vertically in each of a ring of twelve

aluminum tubes. Irradiation is carried out in a centrally situated

tube by lowering the specimen to a point at the center of the source.

Thus a very uniform field of high intensity gamma radiation is obtained.

External shielding is provided by immersion of the entire tube assembly

in a pool of water.

The total source originally had an activity of 835 curies and the

extrapolated dose rate over the period of these experiments varied

from 4.5 to 3.8 kr per minute at the central location which was employed.







All irradiations were carried out at ambient temperature which

because of the nature of the source fluctuated only approximately
0 o
10 C from 20 C.


Germination and growth

To initiate germination after irradiation the seeds were immersed

in water at room temperature for one-half to one hour and were then

sown on wet blotting paper in 90 mm petri dishes and covered. Extremely

dry seed lots showed impaired viability which was partially overcome

by "humidifying" them prior to soaking (except in those treatments

sown immediately after irradiation) by placing them over water in a

humidity chamber for several hours. Care was taken to ensure that the

embryos had free access to air by turning the seeds embryo side up

and keeping the water level very low.

Since it was desired that height reduction and chromosomal damage

be compared on an individual basis, it was necessary to provide a

means of ordering the seedlings in the dishes. This was accomplished

by making grids from glass rods of three or four millimeters diameter

that would fit inside the dishes and maintain the seedlings in a

precise order.

A convenient form for the grid, and one which was used quite

successfully, consisted of three parallel rods approximately 15 mm

apart fused at right angles to one rod crossing at their midpoint. As

many as five seeds can then be placed in each sector of the grid,

giving a total of forty seedlings; when arranged "broadside" to each

other there is extremely slight danger of displacement during the

growth period.

Throughout germination and growth the seedlings were in a room








maintained at 210 C + 20 C and under continuous illumination by day-

light white fluorescent lamps at an intensity of approximately 3200

lux.

It is generally observed that, even for radiation doses which

result in 100% lethality, the coleoptile and first seedling leaf

usually elongate one to two centimeters when germination is attempted

(Moutschen et al., 1956). This "growth" can be attributed to the

swelling of the existing cells as a result of the water imbibed, and

to a limited amount of metabolic activity (Haber et al., 1961). Conger

(unpublished) has determined that under general experimental conditions

the mean value of this expansion or "elongation height" is 16 mm for

the first leaf in barley and suggests that for purposes of comparing

heights of seedling plants relative to controls this base line value

logically ought to be subtracted from all measured means. This procedure

was followed in all the original data presented here and is referred

to as "corrected" mean height.


Root tip collection

Since individual comparisons of growth response and chromosomal

damage on the same seed were to be made, it was necessary to collect

mitotic material from the seedlings but not to injure the seedling it-

self. It proved possible to remove several root tips when they were

three to five millimeters long without appreciable injury to the

seedlings (Tables 2 and 3). The roots attain this length about 24

to 36 hours after sowing, depending on the treatment. This represents

the peak of the first division cycle of mitotic activity (Caldecott

and Smith, 1952).

At the time of collection some seeds have failed to develop roots








and others have roots too short to collect without danger of excessive

damage to the embryo, hence, no roots were collected from such seed-

lings. Since these seedlings also tend to be shorter than average the

mean height of those seedlings examined cytologically is greater than

the mean height for the whole population. In an attempt to reduce this

bias the "harvesting" of root tips in most of the experiments was timed

to coincide with the time at which a maximum number of seedlings had

roots of the proper length. Thus, some plants were excluded because

their roots were too long and a somewhat larger proportion because their

roots were too short. In some experiments root tips were collected

from every seedling which developed them. This was accomplished by

successive harvests.

The excised roots were put into vials containing a few drops of

0.2% w/v colchicine in aqueous solution and allowed to continue growth

for four to five hours and then fixed by adding approximately 5 ml of

Carnoy's fixative (6 ethanol: 3 chloroform: 1 acetic acid).

Since it was impractical to attempt to examine all root tips, the

vials were stored until after seedling height determination had been

made. At that time a sample was selected from the irradiated material.

This sample was representative of the full range of heights except for

those in the very lowest classes which in Figures 1 and 2 have the

following designations and meanings: X, those seeds with no roots or

shoots; 0, those seedlings with roots but with first leaf less than

5 mm long; 1, those seedlings with leaves from 5 to 15 mm long; 2,

those seedlings with leaves from 15 to 25 mm long. In general, when

attempts were made to examine root tips from these very short seedlings,

the few division figures that could be found were very aberrant.








In the case of controls, cytological examination was deliberately

carried out on a sample which contained a high proportion of those

seedlings which were shorter than the mean. This was done in order to

enhance the likelihood of detecting any possible "spontaneous" chromo-

somal aberrations, since the rate is known to be extremely low.


Cytological method

For cytological examinations the fixed root tips were prepared by

the Feulgen method (9 minute hydrolysis in IN HC1 at 600 C followed by

staining with leucobasic fuchsin). The root tips were then softened by

fifteen to thirty minutes digestion with 5% w/v aqueous pectinase, a

slight modification of the procedure suggested by Wolff (1956). The

prepared squashes were preserved either as temporary mounts by sealing

with dentist's sticky wax (Conger, 1960) or 25 permanent slides by the

freeze dry method of Conger and Fairchild (1952) using liquid nitrogen

for freezing and diaphane for mounting.

Each root tip was squashed separately and the slides coded. All

scoring were made by one observer (the writer) solely on the basis of

'whether or not any aberration was present in the colchicine-arrested

metaphases. In one series of observations scored independently by a

second cytologist, the differences in per cent normal metaphases

estimated did not exceed 5.














EXPERIMENTAL RESULTS


Appropriateness of the method

In order to investigate the relationship between growth response

and chromosomal damage in barley seedling populations arising from

grain irradiated and stored under very dry conditions, the usual

procedure of subsampling is not appropriate. This.is because of the

highly heterogeneous, even bimodal,growth response of such seedlings

(Figure la and lb) which contrasts sharply with the fairly normally

distributed heights of populations obtained in the usual radiation

experiments with more moist seeds or those not stored (Figure 2).

Consequently, the method used in these studies consisted of

individual sampling of two root tips from seedlings which were then

permitted to continue growth until measured for height. (See methods

section for details of the procedure.)

First it was necessary to determine whether the removal of several

root tips per se resulted in any impairment of subsequent growth. Data

gathered in the course of experiments designed primarily for other

purposes demonstrate rather conclusively (Tables 2 and 3) that under

the differing conditions of these experiments there is no effect upon

growth during the time period which is involved, namely, seven to nine

days.

Secondly, it was important to determine the consistency of aber-

ration frequency between roots from the same seedling.

Barley grains upon germination usually develop a total of seven

18















FIGURE la


FREQUENCY DISTRIBUTION HISTOGRAMS OF SEEDLING HEIGHTS FOR BARLEY SEEDS
WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED IN AIR OR IN OXYGEN AT LOW
DOSES AND STORED FOR 7 DAYS POST-IRRADIATION


The data illustrate the bimodal distribution of seedling heights which
occurs with very dry seed stored after low doses of gamma-irradiation.

Upper figures: seeds irradiated and stored in air over P205
Lower figures: seeds irradiated and stored in oxygen at one atmosphere
positive pressure

Doses were 0, 2, 4, 6, 8 kr Co60 gamma-irradiation at 4.25 kr/min, with
approximately 75 seeds per treatment. Seedlings were measured after
nine days growth.

Height class interval 1 cm centered upon integral centimeters. Zero
class indicates seeds which developed roots, but had a shoot less than
5 mm long. The class below this (to the left of zero class) showed no
evidence at all of germination and was usually excluded from calculation
of means since it showed no relation to dose.

The mean height for each treatment is indicated on each histogram with
the conventional symbol 3.











o z

4 "LU + o0
o

y (0 _
110 II















4 ax 0 IX

N N
0

z
aO =0 >(O-
Ix





a 0 IX




ix x OI
N














0
















0
0
N
Z

















O- WO
S0 IX 1X
N














0 UIJ



I I l I I n


SONn033S JO


0 0
v) N\j
839fNnN















FIGURE Ib


FREQUENCY DISTRIBUTION HISTOGRAMS OF SEEDLING HEIGHTS FOR BARLEY SEEDS
WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED AT HIGH DOSES AND STORED
POST-IRRADIATION


The data illustrate that the bimodal distribution of seedling heights
which occurs with very dry seed after gamma-irradiation is clearly
manifest with a dose of 18 kr but is not present with a dose of 36 kr
due to the severity of damage.

Combined data from two experiments which differed only in length of
post-irradiation storage in dry air, viz., 3 days and 160 days. The
means were nearly the same and the modes were practically identical.

Doses were 0, 9, 18, 36 kr Co60 gamma-irradiation at approximately
4.5 kr/min. The total number of seedlings per dose is 241, 238, 228,
and 209, respectively. Seedlings were measured after eight days growth.

Height classes are as in Figure la. That class designated x showed no
signs of germination.












II I I
























O






0
x

I.










U
,y, -











0-
4 N0




en
-j

















I I I I I

0 0 0 0 0 0 0
(M 0 CD to It N
SoNi-1033 S .JO U38InN
N
















StNI1O33 Jf N














FIGURE 2


FREQUENCY DISTRIBUTION HISTOGRAMS OF SEEDLING HEIGHTS FOR BARLEY SEEDS
WITH 3.8% WATER CONTENT; GAMMA-IRRADIATED IN NITROGEN AND
GERMINATED IMMEDIATELY


The data illustrate the essentially normal distribution of seedling
heights which is usually found in radiation experiments other than those
involving very dry seeds subjected to post-irradiation storage.

Doses were 0, 20, 30 and 50 kr at 3.83 kr/min. The number of seeds per
dose were respectively 78, 78, 73 and 77. Seedlings were measured after
nine days growth.





24















L0) --- *

Oil











IIx
'a
0


4













11 ?! L
X -













Ix w
Oi I
I


















N -

S n N
0



















-- --I-1-- I

SONI'1033S J0 a39filN








TABLE 2


COMPARISONS BETWEEN DISHES OF MEAN HEIGHTS AT 8 DAYS OF SEEDLINGS
WHICH EITHER HAD TWO ROOT TIPS EXCISED OR WERE LEFT INTACT


Seeds
air 8


with 2.8% water content gamma-irradiated as specified and stored in
days post-irradiation.


Dose
(kr)


0


CUT
No. of
Seed-
Dish lings

1 24
2 25
3 26
4 26


Treatment Mean


9 1 25
2 26

Treatment Mean


18 1
2
3
4 .


Treatment Mean


36 1 19
2 19
3 21

Treatment Mean


Mean
Height
(mm)

117.9
106.4
110.0
111.9

111.5


55.6
54.2

54.9


47.1
56.0
50.0
50.9

51.0


27.9
31.1
35.7

31.7


UNCUT
No. of
Seed-
Dish lings


Control Mean


Control Mean


Control Mean


Control Mean


Mean
Height
(mm)

114.0
108.0


111.0


48.5
60.0

54.3


41.5
60.5


51.0


28.9
32.6


30.8


Difference (mm)
Cut Uncut


0.5


0.6


0.9


Mean 0.5


No statistical test is required to demonstrate the lack of effect of ex-
cision of two roots on seedling height.








TABLE 3

COMPARISONS WITHIN DISHES OF MEAN HEIGHTS AT 9 DAYS OF SEEDLINGS
WHICH EITHER HAD TWO ROOT TIPS EXCISED OR WERE LEFT INTACT

Seeds with 3.8% water content gamma-irradiated in nitrogen at different
doses and either germinated immediately (NI) or stored in nitrogen (NSN),
air (NSA), or oxygen (NSO) for 5 days before germination.


Treatment


Dose (kr)


Mean Height (mm)
Cut Uncut


132.5

115.4

74.4

60.3


127.9

113.6

128.1

66.9


NSN


NSA


1.75

3.5

8


NSO


1.5

3

6


Total

Mean


135.9

130.2

114.2

136.9


141.4

125.5

119.1

81.4
1803.7

112.7


134.9

131.1

104.9

65.4


131.4

137.6

132.8

98.8


126.0

124.6

130.3

90.1


133.9


137.4

97.9

83.8
1860.9

116.3


Difference (mm)
Cut Uncut

2.4

-15.7

-30.5

5.1


3.5

-24.0

4.7

-31.9


9.9


5.6


-16.1

46.8


-11.9

21.2

- 2.4
-57.2

- 3.58


n = 16


s = 19.77


s_ = 4.94
x


Each pair of mean heights was obtained from 32
same dish, 16 of which had two roots excised.


t = 0.724


seedlings grown in the


P 0.4








roots. The radical emerges as a primary root soon accompanied by two

lateral seminal roots. These are in turn followed by another pair

laterally disposed, and then finally by two more. In the experiments

recorded here, routinely, two roots were excised. These usually con-

sisted of the first two laterals, or, less frequently, the primary

root and one lateral. No attempt was made to distinguish between these

roots in the cytological examinations which followed.

The squash preparations of the individual root tips were coded

before scoring and the members of a pair were not scored consecutively--

indeed, in most instances several days intervened. Inspection of the

data from a number of paired estimates of per cent normal metaphases

(Table 4) shows typical results. The difference in per cent normal

metaphase estimates for the two roots seldom exceeded 10%. Additional

comparisons of paired estimates can be made for the data illustrated in

Figures 5b, 6, and 13.


Effect of very low moisture content

To obtain a very dry state, seeds which had been stored several

months over phosphorous pentoxide were pumped several hours and held

under vacuum for 24 hours. Before irradiation, the seeds were placed

in ampoules which were then evacuated and refilled with either dry air

or oxygen at one-half atmosphere positive pressure. This procedure

resulted in a moisture content which was determined to be 2.8% (see

Table 1).

The frequency distribution of seedling heights for such dry seeds

irradiated and stored in dry air or oxygen tends to be bimodal (Figure

la and Ib) for doses up to about 25kr; beyond this, damage is so great

that the population becomes skewed severely toward very low growth.








TABLE 4


COMPARISON OF PER CENT NORMAL METAPHASES AS SCORED ON PAIRS OF ROOTS,
EACH PAIR FROM AN INDIVIDUAL SEEDLING

Seeds with 2.8% water content gamma-irradiated and stored as specified.


Atmos- Height Root A
phere (mm) % Normal


Root B Differ- Difference x 100
% Normal ence Mean % Normal


Pairs With 100 Metaphases Scored Per Root Tip


Air
Oxygen
Air
Air
Air
Oxygen
Oxygen
Oxygen
Air
Air
Oxygen
Oxygen


154
43
128
128
37
166
54
50
120
96
122
48


Mean Difference


7.33


Pairs With at Least 75 Metaphases Scored Per Root Tip


Air
Air
Oxygen
Oxygen
Air
Air
Air
Oxygen
Oxygen
Air
Air
Air
Air


152
49
142
138
36
138
59
112
26
130
130
60
50


90.0
51.7
79.2
79.0
7.8
80.0
17.0
56.7
18.0
97.4
89.0
62.8
81.4


87.0
68.5
68.0
77.8
8.0
85.3
20.9
77.0
23.9
98.7
88.7
54.0
73.3


3.0
16.8
11.2
0.2
0.2
5.3
3.9
20.3
5.9
1.3
0.3
8.8
8.1


Mean Difference


Dose
(kr)


8.2
61.1
9.7
6.9
3.6
3.1
0.0
3.2
17.1
5.1
22.6
25.8


3.4
28.0
15.2
0.3
2.5
6.4
20.5
30.4
28.1
1.3
0.3
15.1
10.5


6.56







At 5 kr the corrected mean height is approximately one-fourth that of

the unirradiated control. At this point there is a sharp break in the

log per cent height-dose curve (Figure 3). At lower doses there is

marked decrease in height as dose increases; beyond this point the

decrease is much more gradual.

The height-dose curve of an experiment restricted to the sensitive

logarithmic region is presented in Figure 4. It can be observed that

an approximate eightfold increase in oxygen tension, during irradiation

and storage, over that in the normal atmosphere had only a slight sen-

sitizing effect upon seedling height reduction.

Practically no.seedlings from the controls in any of the irradia-

tion experiments had metaphase abnormalities, but, as can be seen in

Figures 1 and 2, there is a wide range in the control seedling heights,

hence, there is no correlation between seedling height and per cent

normal metaphases for the unirradiated treatments. Despite this, a

high positive correlation on an individual basis does exist between

these two criteria for the seeds which were gamma-irradiated with 1, 2,

3, 4 or 5 kr. Statistical analysis gave a value for the correlation

coefficient, r = 0.885 with a probability that the data are not cor-

related, P < 0.001. A distribution plot for seedling height versus

per cent normal metaphases for seeds irradiated and stored in air is

given in Figure 5a. Each point represents the mean value for the in-

dependent estimates of per cent normal metaphases from each of two

root tips from a seedling of the height indicated. The regression line

for seedling height on per cent normal metaphases is shown.

In Figure 5b, similar data for seeds irradiated and stored in

oxygen at one-half atmosphere positive pressure are presented. Here















FIGURE 3


PLOT OF LOG MEAN SEEDLING HEIGHT VERSUS GAMMA-RAY DOSE FOR BARLEY SEEDS
WITH 2.8% WATER CONTENT; IRRADIATED AT HIGH DOSES AND STORED 90 DAYS
POST-IRRADIATION


Irradiation and storage was either in dry air or in oxygen a 0one-half
atmosphere positive pressure. Doses were 0, 9, 18, 36 kr Co gamma-
irradiation at 4.25 kr/min with approximately 100 seeds per treatment.
Seedlings were measured after eight days growth.

The mean height for each treatment, minus the elongation height (16 mm),
is plotted as log per cent of the corrected, pooled control height,
98.9 mm.

The data indicate no appreciable difference between the two treatments
at these doses. Note the sharp break in the curve which occurs at or
below 5 kr. With this dose damage is so severe that further'increments
of dose have only slight additional effect.












l O--- I I I I


801-


*1


* I



I
* I









1
I


40F


LEGEND:
* AIR
0 OXYGEN


301-


701-


60 -


50F


\ 0



~ - _ _^0


GAMMA-RAY DOSE (Kr)















FIGURE 4


PLOT OF MEAN SEEDLING HEIGHT VERSUS GAMMA-RAY DOSE FOR BARLEY SEEDS WITH
2.8% WATER CONTENT; IRRADIATED AT LOW DOSES AND STORED 20 DAYS
POST-IRRADIATION


Irradiation and storage were either in dry air or in oxygen at one-half
atmosphere positive pressure. Doses were 0, 1, 2, 3, 4, and 5 kr at
4.05 kr/min with 100 seeds per treatment. Seedlings were measured after
eight days growth.

The mean height for each treatment, minus the elongation height (16 mm),
is plotted as per cent of the corrected specific control height, 92.1 mm
for air and 87.9 mm for oxygen.

The data suggest slight differences between the two atmosphere treat-
ments at the doses used. The points for oxygen in all cases but one
(2 kr) fall below those for air even though they are relative to a
slightly smaller control value. The general sigmoidal form of the curve
as drawn by inspection is typical of response over this dose range.



















0







75-

O
0
T
I-
Z
0
U

U \
u

U
w

V)

0
50-



a


I-
O

w 2


u
3-


I0


w 25 -LEGEND:
(/ AIR
0 OXYGEN









0 2 3 4
GAMMA RAY DOSE (Kr)















FIGURE 5a


REGRESSION OF SEEDLING HEIGHT ON PER CENT NORMAL METAPHASES FOR SEEDS
WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED AT LOW DOSES AND STORED
20 DAYS IN DRY AIR


Treatment mean heights and other data for this experiment are given in
Figure 4.

Two root tips were excised from each seedling during the second day of
growth and analyzed separately for per cent normal metaphases. An
average of 50 metaphases was scored per slide resulting in a mean dif-
ference of approximately 7% normal metaphases between the members of a
pair. The mean value of each pair (in a few cases, the value for a
single root tip where the partner was unanalyzable) is plotted as a
point on the distribution. The regression equation for seedling height
on per cent normal metaphases is Y = 24.1 + 1.153X and the standard
error of estimate, se = 17.3 mm. The controls are not included in this
regression since in all those examined there were no aberrations re-
gardless of height, even though most of them were shorter than the mean
height for controls.






















0 0
0


* ,V


0

08


* *
*


0/ / 9


e


LEGEND:
0 I Kr
G 2Kr
* 3Kr
o 4Kr
9 5Kr
D CONTROL


10 20 30 40 50 60 70 80 90
SEEDLING HEIGHT (mm.)


100 110 120


60
u,

I

w

.J
50


4

040
z

z
I-
2


y 30


%


* / 0















FIGURE 5b


REGRESSION OF SEEDLING HEIGHT ON PER CENT NORMAL METAPHASES FOR SEEDS
WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED AT LOW DOSES AND STORED
20 DAYS IN OXYGEN AT ONE-HALF ATMOSPHERE POSITIVE PRESSURE


Treatment mean heights and other data for this experiment are given in
Figure 4 and general procedural details accompany Figure 5a.

Each root tip was analyzed separately. The pair observations are plotted
separately and connected by vertical lines. Observations were made only
on 0, 2 and 5 kr doses as indicated by the appropriate symbols on the
figure. With the exception of two seedlings all values are based on a
minimum of 50 analyzed metaphases. The regression equation for seedling
height on per cent normal metaphases is Y = 4.32 + 1.683X and the
standard error of estimate, se = 29.0 mm. As in 5a the controls are not
included in this regression. In the shorter-than-average controls
examined a single abnormal metaphase was observed.




























75-












w
V)
I

w






25
0 50 1

SEEDLING HEIGHT (mm.)
I-


w
a 0
25-






/LEGEND:
Q 2 Kr
6) 5 Kr
0 I) CONTROL





0 50 100
SEEDLING HEIGHT (rmm.)








the value for each root tip is plotted, the pairs being connected by

vertical lines. Data was obtained only for the 2 and 5 kr doses. For

these data r = 0.955 with probability P < 0.001 that this is a chance

association.

These data further indicate that within the dose range of this

experiment the length attained by individual seedlings is a function

of per cent normal cells, irrespective of the dose received. Similar

results were obtained in other experiments with doses up to 36 kr,

though fewer observations were made at these higher levels. Figure 6

shows the distribution of data from one such experiment. Here, the

value for each root tip is indicated and the pairs are connected by

vertical lines as in Figure 5b. Data for air and oxygen treatments

are combined in this analysis of seeds given 5, 15, or 25 kr gamma

irradiation. The correlation coefficient, r = 0.913 with P< 0.001.

A logarithmic plot of per cent corrected specific mean control

height, and of per cent normal metaphases versus dose for the experi-

*ment illustrated in Figure 4 is present in Figure 7, and a summary of

the data is given in Table 5. The curve for per cent normal metaphases

was determined from subsamples of those individuals which were examined

cytologically. These subsamples were selected to have mean heights

equal to those of the whole population at each dose level. Since, for

every dose level the total cytological sample had a higher mean height

than that of the whole treatment, for reasons previously stated, the

representative subsample was obtained by exclusion of a sufficient

number of the highest plants to reduce the mean height to that of the

whole treatment. This procedure was used to prevent subjective bias

in selection.















FIGURE 6


REGRESSION OF SEEDLING HEIGHT ON PER CENT NORMAL METAPHASES FOR SEEDS
WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED AT HIGH DOSES AND STORED
90 DAYS IN DRY AIR OR IN OXYGEN AT ONE-HALF ATMOSPHERE POSITIVE
PRESSURE


Treatment mean heights and other data for this experiment are given
in Figure 3; procedural details are the same as those accompanying
Figure 5b.

No distinction is made, here, between the air and oxygen treatment.
Only root tips of seedlings from 5, 15 and 25 kr doses were analyzed
in this experiment. The three seedlings represented by the points in
the upper left of the figure were excluded from the calculations since
their growth was reduced by causes extraneous to this experiment (fungal
infection).

The regression equation for seedling height on per cent normal meta-
phases is Y = -10.7 + 1.827X and the standard error of estimate,
se = 33.4 mm. Note that the horizontal axis has its origin at 20 mm.





40


100 -lI I I I
I.



90




80 -




70
o I







C,/

W 60-
V)
I


7- 50-
_1

a
40 -
40-
Z
w
U

a


LEGEND:
5 Kr
20 0 15 Kr
(D 25 Kr




10




o0 II
20 30 40 50 60 70 80 90 100 110 120 130 140 150
SEEDLING HEIGHT (mm)















FIGURE 7


PLOT OF LOG MEAN PER CENT NORMAL METAPHASES AND LOG MEAN SEEDLING HEIGHT
AT 8 DAYS VERSUS DOSE FOR SEEDS WITH 2.8% WATER CONTENT; GAMMA-IRRADIATED
AND STORED 20 DAYS IN DRY AIR


The cytological data are a portion of that presented in different form
in Figure 5a. The method for selection of these data is discussed in the
text and the data are summarized in Table 5.

The treatment mean heights for the seedlings contributing to the cyto-
logical data are identical with those for the total population which
are plotted here. Thus direct comparisons can be made between the two
curves.

The regression equation for log seedling height on dose is log = 150.7
- 1.517X and that for per cent normal metaphases on dose is .log Y = 114.8
- 1.551X.

The HD50 (that dose which reduces the height to one-half that of control)
is 2.65 kr; the CD50 (that dose which reduces per cent normal metaphases
to one-half) is 1.90 kr.





42


100 - |-..|---


90



80-



70-
\\





60 -

60
O \



\ o
0 0 \
50\


WU
VZ

\LL
O \

Zo 40-







Jcr
4w










w LEGEND:

n0 SEEDLING HEIGHT




20
z\









'5\
U \
























IIIII

0 I 2 3 4 5
GAMMA-RAY DOSE (Kr)








TABLE 5


MEAN SEEDLING HEIGHT FOR TREATMENTS AT DIFFERENT DOSES AND FOR SAMPLES
EXAMINED CYTOLOGICALLY FOR PER CENT NORMAL METAPHASES

Seeds with 2.8% water content gamma-irradiated in air and stored 20
days in dry air post-irradiation. The representative cytological sub-
samples were selected to have mean heights corresponding to those of
the entire treatment. Seedlings measured at 8 days.


Gamma-Ray Dose (kr)
0 1 2 3 4 5


Entire Treatment


Number


Mean Height (mm)
(corrected)*

% Control Height
(mm) (corrected)*


97 97 84 82 81 83


92.1 88.6 58.3 43.7 27.4 16.1


100


96.2 63.3 47.4 29.8 17.5


Entire Cytological Sample


Number


Mean Height (mm)
(corrected)*

% Normal Meta-
phases


12 21 19 20 17 19


95.6 96.6 69.1 63.9 32.4 42.4


91.4 50.5 41.2 21.9 31.4


Representative Cytological Sub-Sample


Number


Mean Height (mm)
(corrected)*

% Normal Meta-
phases


15 16 13 15 10


89.0 59.1 44.0 27.5 16.9


76.1 44.7 31.2 20.5 12.5


*Mean height (corrected) equals actual mean height minus
due to elongation.


16 mm, height








The fact that the curve for cytological effects falls below that

for gross effects indicates that the cytological effects are more

radiosensitive. There is approximately 700 r difference for equal

response of cytological and gross effects relative to controls through-

out the range tested. The 50% level of effect for height reduction

(HD50), after correction for cell elongation (see methods section) was

approximately 2.7 kr in this experiment, while that for chromosomal

damage (CD50) was 1.9 kr, giving a relative sensitivity of 1.4 for

these two criteria of radiation damage.

The regression lines do not intercept the response axis at zero

dose but instead indicate the presence of a threshold around 300 r for

chromosomes and 980 r for leaf length. This phenomenon was not investi-

gated further in these experiments, but it is a common observation in

many radiation experiments, i. e. a "multi-hit" response. It is inter-

preted as implying that several independent unitary events of the type

causing damage are required before damage is made manifest. It is

known, furthermore, that very low doses may actually stimulate growth

(e. g. Suess, 1961).

The results of these experiments with very dry seeds irradiated

and stored in dry air or oxygen at room temperature suggest that the

heterogeneous growth response (Figure 1) is a consequence of chromosomal

damage which differs greatly in extent among the individual irradiated

seeds. The cause for this difference among seeds receiving the same

treatment is not known.


Effect of higher moisture contents

For these experiments seeds were stored at a wide range of dif-

ferent humidities until their moisture content was stabilized. Samples








were withdrawn, placed in glass tubes fitted with rubber stoppers,

irradiated in air and returned to their respective humidities for post-

irradiation storage.

Height-dose regression lines for the composite data of three

experiments are given in Figure 8. The results indicate the inverse

relationship between moisture content of the seeds and radiation damage

to growth which occurs over this range of humidities. There is scatter

in the data, much of which results from difficulty in obtaining the

same degree of response from experiment to experiment. This difficulty

is encountered frequently in this material and will be referred to in

the discussion.

In Figure 9 the interpolated values for doses giving a corrected

mean seedling height 50% that of the related control (HD50) are plotted

against water content. For comparison similar data from Ehrenberg

(1955b) are included. There is indication of a maximum effect around

13% water with a decrease at higher as well as at lower values, but

this particular point was studied in only one experiment and not

confirmed.

Cytological observations were made on individuals from the various

treatments within one such experiment. Particular effort was made to

collect data from the dose at each moisture level in the midrange of

the "log phase" of response to dose, i. e. 40% of corrected specific

control mean height. This was done in order to determine whether

reduction in seedling height is directly related to observable chromo-

somal damage over a wide range of seed water content. The results are

presented graphically in Figure 10 and are summarized in Table 6. The

regressions of seedling height upon per cent normal metaphases as















FIGURE 8


REGRESSION OF LOG MEAN SEEDLING HEIGHT ON GAMMA-RAY DOSE FOR SEEDS WITH
DIFFERENT WATER CONTENTS; COMPOSITE DATA FROM THREE EXPERIMENTS


All seeds were stored over appropriate desiccant or solution as specified
in Table 1 for several months prior to use, with the exception of the
wettest treatment which was stored for only several weeks to prevent
impaired germination. Following irradiation in air all treatments were
returned to their respective desiccators for post-irradiation storage.

Experiment
a b c

Days stored post-irradiation 8 6 38
Days growth before measurement 9 8 9

Points from individual experiments are indicated by appropriate letter.
Data at 2.8% water from Figure 7 are included.

Straight lines were fitted to the data by linear regression and that
dose which gives 50% height reduction relative to specific control (HD50
was determined.

Relative
Per Cent Water Regression Equation HD50 Sensitivity
A
2.8 Y = 150.7 1.517X 2.65 15.1
3.8 = 86.9 1.263X 2.6 15.4
6.7 Y = 120.5 1.252X 4.0 10.0
7.3 Y = 85.9 1.119X 5.0 8.0
9.2 Y = 110.0 1.113X 7.3 5.5
10.1 Y = 133.7 1.079X 13 3.1
12.7 = 124.7 1.023X 40 1
15.5 Y = 225.7 1.063X 25 1.6
























LEGEND:
7o H20 HD50

2.8 2.65
3.8 0 2.6
6.7 (D 4.0
7.3 e 5.0
9.2 0 7.3
10.1 13.0
12.7 e 40.0
\ 15.5 9 25.0


20 30
GAMMA-RAY DOSE (Kr)


U30
IL
U
w
CL
a
i.)














FIGURE 9


RELATIVE SENSITIVITY OF SEEDS OF DIFFERENT WATER CONTENT GAMMA-IRRADIATED
AND STORED IN AIR, NITROGEN OR OXYGEN


The HD50 (that dose reducing seedling growth to 50% that of unirradiated
controls) values obtained from the data in Figure 8 are plotted against
seed water content (heavy line). For comparison the data from Ehrenberg
(1955b) for x-rayed barley seed treatments in nitrogen and oxygen are
included.

The point for 12.7% water content was determined from a single experi-
ment and not confirmed.












I j I I I I I I


40


36


24-


20-


0// I -- "-
:






P/ LEGEND:
0 AIR
/ 0 NITROGEN
e OXYGEN


I I I I I I I I


0 2 4 6 8 10 12 14
SEED WATER CONTENT


16 18 20 2
(PERCENT)














FIGURE 10


REGRESSION OF SEEDLING HEIGHT ON PER CENT NORMAL METAPHASES FOR SEEDS
OF DIFFERENT WATER CONTENT IRRADIATED AND STORED IN AIR


Details for this experiment are those of Experiment a, Figure 8. The
cytological data and statistical computations are presented in Table 6.
The cytological analysis presented here was made on seedlings from that
dose at each moisture level closest to 40% control height. The actual
values and the regression equations from Table 6 are summarized here.


Seed Moisture
Content, in
Per Cent

3.8
6.7
9.2
12.7
15.5


Mean Height
Dose as Per Cent
(kr) of Control


Regression Equation


A
Y.=

9=


38.4
40.6
37.7
48.5
37.1


7.37
-10.98
1.20
9.69
5.05


1.178X
1.538X
1.681X
1.702X
1.131X


The analysis indicates that the regression of seedling height on cyto-
logical damage is not affected by differences in water content.










































0 0


LEGEND:
07o H20

(D 3.8
0 6.7
9 9.2
12.7
0 15.5


20


SEEDLING HEIGHT (mm.)














- I
-~ N

oLN 0 '0 4) %D
C/) Cd -
1. . 0 O o0




T3 X0or CM >-
) E c i t o c )o 4 U
n^- N-'.0-.Z -.ZL( (nc N-^ u- N- Ci 0
0'- ) + co r- > *-H
LI-I + uj) 00 Wd4-1
H 0 0 0 O cN



4 -4 4 0 C C 4 Lo







H Cd '' 0
N 0 r C4
w- 00 o- o o -ooo C) r^ 0C C) U) r Lr) r- 11 11 0










0 p L
H > ;4 O0 m- Cd4

C 0 Cs ) <-Iot00 C r O cy0 U 0 -









0 *0
0 t oo m 0 n r L N- 4- i
H 0f 0'-' + + O
w ND C <1 N 0 0
l 01 It -4 V) 10
N <1 'cil0 '.0 4^o ^ -r o l o m o I 1 -J
Cd -4 4-) -4 LC1 0 -4 0<


0 1 4 C cI
c r-0'.0-l-N-0 c) C) LC) 00 1 -4 1




















M~o cyJ0 ^0 + + N 0y -.
Z CO NC Z Co cU> -
H 0 . q rC
[JZ -H 8- tNNo0Lt (o uoo -t o|o "i r4 0' 0- n 4
cu 01 ce C) r^r r- -c F*g r-4 c^ c e) -4f


C:)ca 0 N Zo P













pH iI O^~lCOO ^O CM* O OO r
) Co . . . .N
OHi o 4-i ON-s 0ojm o -~ ^ ^0_
CD*0.) LC1










0 c CO cOOi 0 m U N II c- II
C ULr 0 q0 LN- L y co CC o' I-l
0%-" + + C)c' Ci











*O 4- -.z-
So oi 0- | l 0I
4 41 crO 0 N -4 .











m~4 r, C)u a r a u
HO p c0.a C ON m* 0 cn 4.



02j + -4- P-
*" S'- Cd 0's-' .*:t +- N C0 cS (M 4->
N- N-i '! -0 N- >
MC 0 N CO O 0











o a c' '. 0a 0 -0 4C
O-) u-i CN- 0, r, -4 C14 c cn .1




S Cd 'o c -' N N . o .0










rf, jj o ^- e m o~ o 00clo ^ r^11 01^
on 1 C a no C O4-

P- 0'- N + + N CO c^ r
2; CO M -.zt C ~ 1
4 -4-1 CO C) u 0 0 -0
-n ocf 2 . .- i C
H- CU T b dC Coo oc'i ^ -NC C C' r CO II II
p0 4da- Ci'.. M- 0' "- 0- a%
ffi ~ ~ ~ ~ ~ 1 4j 'tgo^o~M co^ n I Ic
> 4 cfl r- r, cq 1I c i i l H
.w 0 '% r 1% )
!rD 00l c
0 :; %D C~) C-4 -4< )-IN








0 4-i


Z )C Cd 1 -
-4 r-4 00s -It4








4C C 4 4 -4
Cr1 F >n Ci Cc4 Cd-'-4



MCCC1 4-I 4-i Ci) OZ0 Ci14-4
-a fl -aCC Ci CC cd i- -4- ZCC Ci c
CiCC 00 CCaC)C C i~ 0~










C) C4 'D 0 00 g c -l bO S 0 0 !
r=: -H QJU 3 H 0 5~ O0
r. M C YA Q o S Y K







determined for each moisture level do not differ significantly. The

regression lines for 3.8 and 15.5% water content appear somewhat

divergent from the others, probably because the seedlings at these

two moisture levels displayed impaired growth in the unirradiated con-

trols when compared'to those of the other moisture levels. Such reduced

vigor can reasonably be expected in the irradiated individuals as well.

Supplementary data from other treatments within the same experi-

ment as well as the general experience of the writer confirm the direct

relationship of reduction in seedling height and observable chromosomal

damage at the individual level within the wide range of water contents

and consequent radiosensitivities which were examined.


Effects of post-irradiation storage and oxygen

The seeds used in these experiments were stored for several months

over phosphorus pentoxide prior to use. Their initial water content

was not measured for every experiment but was assumed to be about 3.8%

as previously determined for these conditions. After vigorous pumping

and flushing (see methods section), all seed samples were sealed in

glass ampoules in an anoxic atmosphere of nitrogen for at least two

days before irradiation.

After irradiation some seed samples were soaked immediately (NI)

within 30 seconds post-irradiation. Other ampoules were opened and

placed, within 45 seconds, in a pressure bomb which was held at 200

pounds over atmospheric in dry oxygen (NSO) giving an oxygen concentra-

tion approximately 65 times that of air. Still others were opened to

air via a drying column packed with silica gel and then transferred to

storage in air over phosphorus pentoxide (NSA). Finally, one group

was left sealed in the atmosphere of nitrogen in which it was ir-

radiated (NSN). Post-irradiation storage was four to five days.








In a preliminary experiment of this nature using helium as an

anoxic atmosphere, other workers in this laboratory (Gennaro and Harrer,

unpublished) obtained the growth and cytological response to dose shown

in Figure 11. (Coding of the treatments is the same as that given

above except that He was used instead of N.) The cytological data

were collected from root tips of an unmeasured subsample. A good cor-

respondence was obtained between damage to the chromosomes and reduced

height. There was considerable scatter in the data; and it is inter-

esting to note that especially for the least sensitive treatment (Hel)

the cytological response was more precise than that of seedling height,

although these observations were based on many fewer individuals.

Their use of oxygen at 1750 pounds pressure resulted in chromosomal

aberrations in 17% of the metaphases examined in unirradiated controls.

In a recent experiment (Figures 12 and 13) individuals were

examined cytologically from that dose level, for each type of post-

irradiation treatment, which resulted in a mean seedling height between

40 and 50% of control height. Correlations between height and per cent

normal metaphases for these groups of individuals did not differ sig-

nificantly (Table 7) despite doses as disparate as 6 and 50 kr. Agree-

ment would probably have been even greater had the range of heights

sampled been more nearly the same in all treatments.


General cytological observations

All observations were made on root tip cells arrested by colchicine

at metaphase of the first division cycle upon sprouting of the seeds.

The criterion employed in these experiments for scoring chromosomal

effects was the presence or absence of any detectable chromosomal aber-

ration. Records of type and number of aberrations with a given cell

were made only on cells of unusual interest.














FIGURE 11


PLOT OF LOG MEAN PER CENT NORMAL METAPHASES AND LOG MEAN SEEDLING HEIGHT
VERSUS DOSE FOR BARLEY SEEDS GAMMA-IRRADIATED IN HELIUM AND STORED
AT DIFFERENT OXYGEN TENSIONS


Doses were 0, 0.67, 1.33, 2.65, 5.3, 10.6, 21.2 and 31.8 kr at 5.3 kr/min.
Seedlings were measured after seven days growth.

The treatments were as follows: The seeds had a water content of ap-
proximately 3.8%; all seed ampoules were evacuated and flushed repeatedly
and then stored at a slight positive pressure of helium for one day
prior to irradiation.

Hel immediately after irradiation seeds were soaked in anoxic
water and germinated.

HeSA immediately after irradiation seed ampoules were evacuated and
dry air admitted for 3 days storage prior to germination.

HeSO immediately after irradiation seed ampoules were opened and
placed in a pressure bomb and stored 3 days in oxygen at 1750
pounds pressure prior to germination. This is approximately
585 times the concentration of oxygen in air.

The doses (in kr) giving 50% reduction for height and for normal meta-
phases as well as the associated relative sensitivities estimated from
this plot are:

Relative Relative
HD Sensitivity CD Sensitivity
50 50
Hel 50 1 20 1
HeSA 5.3 9.4 3.2 6.2
HeSO 1.7 29.4 1.0 20

This data is from an experiment by Gennaro and Harrer (unpublished) and
seedling heights were not corrected for elongation. Such "correction"
steepens the slope of the seedling height lines making the comparison
with per cent normal even closer.






















N


\
\


N
N
N
N


\
E3\\


LEGEND:
07o NORMAL METAPHASES
----------- HSO
------ ----- HeSA
---------- Hel


07o CONTROL HEIGHT



A.
----- ----
----- -l--


GAMMA-RAY DOSE (Kr)














FIGURE 12


PLOT OF LOG MEAN SEEDLING HEIGHT VERSUS DOSE FOR BARLEY
3.8% WATER CONTENT, GAMMA-IRRADIATED IN NITROGEN AND
DIFFERENT OXYGEN TENSIONS


SEEDS WITH
STORED AT


Doses were 0, 1.5, 1.75, 2.5, 3, 3.5, 6, 8, 30 and 50 kr at 3.83 kr/min.
Seedlings were measured after nine days growth.


The treatments
placed in each
repeatedly and
nitrogen for 5


NSN


NSO


were as follows: Approximately seventy-five seeds were
ampoule; all seed ampoules were evacuated and flushed
then stored at 60 cm Hg positive pressure with prepurified
days prior to irradiation.


immediately after irradiation seeds were soaked in distilled
water and germinated.

after irradiation seed ampoules were stored unopened for 5
days prior to germination.

immediately after irradiation seed ampoules were opened and
placed in a pressure bomb and stored 5 days in oxygen at 200
pounds pressure prior to germination. This is approximately
65 times the concentration of oxygen in air.


The doses (in kr) giving 50% reduction in height relative to specific
controls as estimated from this plot are:

Relative
HD50 Sensitivity


NI
NSN
NSO


1
1.4
5.9





58




00 4-


90



80-
O


70 -

- I
O
a:
I-
z
O 60
U \
S\
S\ \

L50- 5


z
U \
u o\





I
0



z

bJ
030 -





LEGEND:
0 NSO
NSA
D NSN
9 NI


20-








15
0 10 20 30 40 50
GAMMA- RAY DOSE (Kr)















FIGURE 13


REGRESSION OF SEEDLING HEIGHT ON PER CENT NORMAL METAPHASES FOR SEEDS
IRRADIATED IN NITROGEN AND STORED AT DIFFERENT OXYGEN TENSIONS


Details for this experiment are given with Figure 12. Cytological data
and statistical computations are presented in Table 7. The data pre-
sented here were obtained from seedlings from that dose level for each
post-irradiation treatment which gave a mean height between 40 and 50%
of that of the pooled controls. Each point represents the per cent
normal metaphases for a single root tip. The data representing pairs
of roots from a single seedling are connected by a vertical line. The
data from Table 7 are summarized below:

Mean Height
Dose as Per Cent
Treatment (kr) of Control Regression Equation
A
NI 50 40.2 Y = 31.16 + 1.53X
NSN 30 49.3 Y = 30.03 + 1.21X
NSO 6 49.1 f = 31.62 + 1.09X






















OMITTED FROM CALCULATIONS I


I e


LEGEND:
0 NSO
D NSN
e NI


100
SEEDLING HEIGHT (mm.)


(V)
w
()

.50

w

-j

0

L-
z
w
U
2r
0-25





61


TABLE 7


SEEDLING HEIGHT AND PER CENT NORMAL METAPHASES FOR SEEDS GIVEN VARIOUS
POST-IRRADIATION TREATMENTS, WITH SUMMARY OF CORRELATION ANALYSIS

Seeds with 3.8% water content gamma-irradiated in nitrogen and stored 5
days post-irradiation in oxygen at 200 pounds pressure (NSO) or nitrogen
(NSN) or germinated immediately (NI). Seedlings measured at 9 days.


Treatment


Dose


NSO

6 kr


NSN


30 kr


50 kr


Y
Height
(mm)

134
132
106
104
83
74
62
57
57
52
47
47
32


Sum


Mean


Treatment
Mean


Regressions
Height on
% Normal
% Normal
on Height

Correlation
Coefficient


75.9


49.1


X
R Per Cent
Normal

95*
60
35
85
44
41
44
24
25
30
22
26
0

531

40.8


Y
Height
(mm)

138
134
108
98
88
86
80
77
76
70
57
44
14

1070

82.3


X
x Per Cent
Normal

76
74
52
39
80
40
45*
29
23
26
28*
39
0

561

43.2


49.3


Y = 31.62 + 1.09X

X = -9.85 + 0.6673Y


r = 0.8513


A
Y = 30.03 + 1.21X

X = -2.14 + 0.5509Y


r2 = 0.8154


Y
Height
(mm)

97
89
88
82
68
62
58
58
56
50
46
40
34
30
858

61.3


X
1 Per Cent
Normal

49
28
22
30
15
20*
20
18
5
16
17
9
14
13
276

19.7


40.2


A
Y = 31.16 + 1.53X
A
X = -4.3 + 0.3916Y


r3 = 0.7732


z = 1.262

s- = 0.447
1,2
s- = 0.4369
2,3
s- = 0.4369
1,3,


z = 1.143

t = 0.266

t = 0.262

t = 0.533


z3 = 1.028

d.f. = co

d.f. = oo

d.f. = co


*Single root examined cytologically


P>0.8

P>0.8

P>0.5







It became evident during the course of the observations, however,

that there exists a definite inverse relationship between per cent

normal metaphases and average number of aberrations per cell. Tall

plants had few abnormal metaphases and these usually consisted of only

a single fragment. They rarely had aberrations resulting from multiple

breaks. Conversely, many multiple break aberrations (e. g. rings,

dicentrics, occasional tricentrics, and translocations) as well as a

high number of fragments were present in short individuals which had

few or no normal metaphases regardless of dose.

One abnormality observed only rarely was an apparent lack of

synchrony indicated by the presence of one pair of daughter chromosomes

in an extended state (2 to 4 times the length expected by comparison

with the other chromosomes) and with little matrix material. These

"extended" chromosomes characteristically are separated by some distance

from the others in the squash preparations, but this may be an artifact

resulting from pressing the cells. Whether these chromosomes represent

a precocious loss of matrix and uncoiling, or the failure to attain

normal metaphase development is unknown; however, kinetochore separa-

tion had occurred in all the cases observed. This would tend to support

the former hypothesis. Records were not kept initially, but later data

indicate a frequency of this abnormality in irradiated cells of 0.00015.

None was observed in controls.

It should be noted that the barley used in these experiments is

remarkably free from chromosomal abnormalities for all the treatments

used in these investigations except irradiation. Only two aberrations

were observed in approximately seven thousand cells examined from

control plants.















DISCUSSION


It was recognized at the meeting held in Karlsruhe, on the effects

of ionizing radiations on seeds, (Sparrow, 1961, p. 646) that one of

the pressing needs in the area of seed irradiation investigations is, if

not a standardization of method, at least an itemization of the numerous

variables involved in each experiment so that results can be evaluated

properly and comparisons drawn. As yet the bill of particulars promised

has not been published. It is with this in mind that the following

comments are made.


Methodology

The barley seed supply, at least for most workers in this country,

is fairly uniform. Konzak, Nilan, Caldecott, Curtis, Conger, Wolff,

and others all use the same selected strain of the variety Himalaya.

However, even within this carefully selected and hand-harvested material

there is considerable variation. An occasional harvest results in

poor germinability (Konzak, 1963). Seed stored less than eight months

or more than three or four years is somewhat unreliable and the condi-

tions of storage (temperature and humidity) can influence both germina-

tion and radiosensitivity (cf. Davidson, 1960).

Even after careful screening and the removal of obviously defective

seeds the weight of individual seeds used in these experiments ranged

from about 0.025 g to 0.055 g, a twofold difference, when stored over

P205. Wetter seeds should vary even more. Much of this admittedly

63








reflects variation in endosperm rather than embryo size since the embryo

represents only about 3% of total weight, but it is one source of

variability.

If the caryopses are not soaked thoroughly in water, preferably

by submersion and mild aspiration prior to sowing on wet blotting

paper, their germination is slower and more erratic.

If the seed is sown with the embryo turned down and if there is

more than a thin film of moisture on the blotting paper (i. e. if the

embryo is actually submerged) the seedling may be delayed a day in

germinating.

In the very dry seeds, particularly, any rough handling can readily

fracture the embryo which is very brittle. An occasional seed, approxi-

mately one in two or three hundred, encounters mechanical difficulty in

rupturing the pericarp and the shoot is delayed or prevented from

emerging or else carries the endosperm aloft with it, thus depriving

itself of nutrition.

These are all minor variables but do contribute to the total

variability.

One difficulty in relating experimental results from different

laboratories which the writer has encountered, is the lack of any

standardized method of determining water content. Some workers grind

the seeds; others leave them intact. All, so far as the writer is

aware, use some method of drying and estimating original water content

on the basis of weight loss. The drying, however, may be in a vacuum

oven, or an air oven. The temperature and duration of treatment varies

widely (cf. Nilan et al., 1962).







The writer has based his estimates on the procedure of Hart et al.

(1959). These workers have developed a simple method of oven drying

samples which differs from others such as those of the Association of

Official Agricultural Chemists (Horwitz, 1960, p. 169, 158, 124) prin-

cipally in that the seeds are not ground but are heated whole in roughly

10-gram samples for 20 hours at 1300 C. This avoids any unknown or

uncontrolled gain or loss of moisture in the grinding process and the

time has been set by testing against the presumed highly precise chemical

method known as the Karl Fischer method (Hart et al., 1957) from which

it gave a mean deviation of -0.01% and standard deviation of 0.29%. The

procedure as outlined above was tested on grain of normal moisture.

How well it applies to extremes of moisture content, as often used in

experiments, is not known. Conger (unpublished) has demonstrated the

feasibility of estimating water content of seeds by nuclear magnetic

resonance techniques. In any event scientists owe it to their colleagues

to specify what method they have used, and yet many do not.

One persistent source of methodological diversity is the time at

which seedling height (length of first leaf) is determined. Moes (1961)

measured his plants when the controls were about 75 mm. Wolff (1961)

measured his when the first leaf was fully grown. Caldecott and also

Konzak at one time measured their seedlings at 14 days (Caldecott

et al., 1952; Konzak, 1955) but more recently Caldecott (1961) measured

7 day old seedlings, and Konzak et al. (1960) varied the time from 5 to

11 days depending on the temperature at which the seedlings were grown.

Most workers seem to favor 6 to 8 days or a control mean height around

10 to 12 cm. In a test of growth with time, the writer found that for

the growth conditions specified in the present work the first leaf on








control plants continued to grow for fourteen days to a maximum of

20-21 cm but growth was exponential only from day two through day eight

at which time the maximum length was 16 cm. Plants at dose levels

giving about 50% height reduction had essentially completed growth by

the seventh day. One must conclude that data based on height relative

to control under these diverse conditions can be compared in a quanti-

tative manner only with great difficulty and considerable uncertainty,

and not at all in a surprising number of cases where no actual heights

are given.

Another misleading practice is the citing of mean heights with no

indication of the distribution of the population about the mean. In

the principal study dealt with here, that of very dry seeds gamma-

irradiated and stored in air, the mean quite regularly falls between

two modes and is often one of the least frequently represented portions

of the distribution (Figures 1 and 2). Yet most of the data in the

literature treats this exceptional material in the same way as that

which is more normally distributed. In fact, for cytological purposes,

random samples, often quite small, are drawn from these populations

and assumed to be truly representative.

Until a few years ago almost all of the cytological data on first

division cycle mitoses in irradiated barley grains was on anaphase

figures, either in root tips or shoot tips. It was not until Wolff

and Luippold (1956) published their method for obtaining numerous well-

spread metaphases that metaphase analysis became practical. The dis-

crepancy between metaphase analysis and anaphase analysis (Wolff, 1957)

indicates that as dose increases an increasing proportion of aberrations

visible at metaphase is no longer discernible at anaphase; hence meta-

phase analysis is preferable.








Comparison of data

Turning now to results comparable to those of the present work,

let us consider first the results with very dry seeds. Caldecott et al.

(1952) observed that barley seed "which had been kept in a humidity

controlled desiccator for at least three weeks prior to treatment" gave

rise to a great range in individual seedling heights when sown after

receiving 20 kr of X rays, in contrast to the much more uniform effect

of thermal neutrons. Lower doses of X rays gave more normal distribu-

tion. Neither the precise water content nor how much time may have

intervened between irradiation and germination was stated but the

phenomenon of heterogeneity was observed. The cytological data were

reported only on an average basis. Apparently an attempt was made

(Caldecott and Smith, 1952, p. 141) to obtain cytological data from

root tips of plants which were then permitted to grow for seedling

observation but this was abandoned as impractical and random sampling

was continued as the preferred method.

Curtis et al. (1958) present a photograph (their Figure 1) which

clearly shows the great heterogeneity of seedling height response in

barley seeds of 4% water content which were x-irradiated and stored for

24 hours in air before soaking and sowing. They do not mention this

phenomenon in the text and treat all the data solely as means.

The first approach to considering these widely distributed popula-

tions as anything other than normal populations occurs in the paper

Caldecott (1961) presented at Karlsruhe. Here he subdivided the

population into three height classes: 0 5 cm, 5.1 9 cm, and 9.1 cm

and taller. There was a good inverse relationship between height class

and frequency of interchanges in microsporocytes, a lesser one with








mutant X2 seedling frequency. The results are not strictly comparable

to those presented here because the seeds were soaked and sown immedi-

ately after irradiation.

The question arises, why should there be so much variation in the

response of different individuals to the same treatment? Gustafsson

(1937a,b) struggled with this problem many years ago. His hypothesis

was that the embryonic nuclei within the seed are not really resting

but that vital processes are under way preparatory to their reproduction.

A locally high concentration of water would promote greater activity

and concomitantly greater radiosensitivity.

His evidence for locally active regions lay in the fact that serial

fixations of hydrating seeds resulted in ever increasing proportions of

dividing cells. There was no general synchrony to the.initiation of

division within the root. This he interpreted as indicating that in

the "resting" embryo some nuclei are more advanced in their preparations

for division and are more radiosensitive.

It is rather difficult to imagine that seeds which have been stored

over strong desiccants for as long as a year or which have been evacuated

vigorously for a number of hours can have such dissimilar water content

that their differential radiosensitivity can be attributed to this cause

alone. Ehrenberg (1955b, p. 208) has presented a statistical argument

which implicates unequal water content in this differential response,

but he also demonstrated a temperature sensitive period in early germina-

tion when low temperatures are most injurious.

Bozzini, Caldecott and North (1962) put forward the hypothesis that

the variation in response within these treatments of dry seeds, irradiated

and stored dry, may be associated with the manner in which specific







critical radiosensitive molecules within the seed give up bound water

during dehydration. Although they know of no way to test this hypothesis

unequivocally, they do have some supporting evidence. Post-irradiation

heat treatments of one to twenty-four hours duration at 750 C completely

eliminates the heterogeneity which otherwise occurs, without altering

sensitivity to aerobic hydration. This suggests that the heat treatment

has induced the same physical state in all radiosensitive sites. This

they suggest, may possibly be due to molecular reorientation with the

loss of bound water.

In the present study, irradiation of the seeds, at the center of

a relatively large distributed-type source of cobalt-60, ensured a

uniform dose to the individual embryos. In the context of the reactive

site hypothesis, the heterogeneous response observed here with respect

to per cent normal metaphases for different individuals within the

same radiation treatment must either be ascribed to differences in the

number of such sites or to differences in sensitivity of such sites or

to both.

Wolff (1963) has obtained data on chromosome exchange frequencies

at various moisture levels which he interprets as indicating that in

very dry seeds the number of radiosensitive sites does not increase

but that sensitivity does. This, he holds, is consistent with the

concept that free radicals are produced which have a longer life in the

dry system and thus have an increased probability of inducing biological

damage.

Osborne et al. (1963), in a survey of the effect of different

water contents on radiosensitivity of the seeds of diverse species,

conclude that it is characteristic for seeds to show a minimum








radiosensitivity at some intermediate moisture content and to increase

in sensitivity at either extreme. In barley this effect has been re-

ported by some workers (Ehrenberg, 1955b) and not by others (Caldecott,

1955a,b). In the present work there was a suggestion that there is

such a minimum but the humidity at which this was achieved was not one

of the routine ones being studied so the data are from one experiment

only. Figure 9 compares the present observation with those of Ehrenberg

(loc. cit.). Ohba (1961) found Japanese red pine seed least sensitive at

13% water content.

An explanation for this effect, if it should prove consistent, can

be based on the free radical hypothesis as reviewed by Osborne in the

aforementioned paper. At low water concentrations radicals have a high

probability of interaction with biologically significant molecules

since relatively few competing water molecules are present. As water

concentration increases more harmless, radical-radical and radical-

water "nullifying reactions" occur until a point is reached where, al-

though radical decay is rapid, the increase in radicals formed, their

greater proximity to critical molecules, andtheir possibly greater

mobility more than offsets the harmless "nullifying reactions" and

sensitivity once more increases.

In the case of post-irradiation treatment with oxygen immediately

following irradiation in nitrogen (NSO) the HD50 (dose required to reduce

height by 50%) obtained in the experiment illustrated by Figure 11 was

5 kr while the HD50 for the seeds left in nitrogen (NSN) was 30 kr.

This sixfold increase is comparable to that reported in the literature

(e. g. Nilan et al., 1961). In two other experiments the respective

values were 2.5 to 17.5 and 2.5 to 8.5 giving a 7-fold and a 3.4-fold








"oxygen effect" during storage. This type of fluctuation from experi-

ment to experiment is typical of that encountered and suggests that

there may be several variables which are not being controlled.

Additional confirmation that there is a direct relationship between

the frequency of aberrations in root tips and various other criteria of

radiosensitivity has come from widely separated laboratories and with

other varieties of barley. Ivanov and Kalikov (1960) tested a number

of varieties of both wheat and barley and found the relationship to hold.

Yanushkevich (1961), while finding a similar relationship, could not

relate radiosensitivity to age, maturity, storage or water content, but,

rather, associated it with locality of cultivation. Avanzi (1960) in

studies of both shoot and root from the same plants found that with

irradiation there was close agreement in growth inhibition, but that

plants averaging 10% growth inhibition in both organs had four times as

many chromosome breaks in the shoot as in the root.

It becomes clear that full understanding of the interrelations

between the many observable consequences of radiation treatments of such

a complex biological system as that of barley grain must await the

identification of and carefully controlled experimentation with each

and every significant variable. That day is fast approaching!














SUMMARY


Barley grains were irradiated at a central location within a

distributed-type cobalt-60 gamma source at a dose rate of approximately

4,000 r per minute for total doses of one to 75 kr. The effects of

moisture content from 2.8 to 15.5% water (by weight) and different

oxygen tensions, during and after irradiation, upon radiosensitivity

were determined. The criteria of radiation damage were: 1, reduction

in growth of the first seedling leaf with respect to controls and 2,

the per cent of cells showing no detectable chromosomal aberrations

when arrested, by colchicine, at metaphase of the first division cycle

in root tips. These observations were made on the same individuals.

Results demonstrate that excision of two root tips from day old

seedlings has no significant effect on subsequent growth. Comparison

of the cytological data obtained from the two related roots shows that

a close correspondence usually exists, the difference in per cent normal

metaphases being about seven on the average.

There is a high positive correlation (r> 0.85) between seedling

height and per cent normal metaphases for all conditions that were

examined. These were as follows:

1. Seeds of 2.8% water content irradiated in air or oxygen with

doses from one to 36 kr and stored at least five days;

2. Seeds with 3.8, 6.7, 7.3, 9.2, 10.1,. 12.8 and 15.5% water con-

tent irradiated in air with doses-up to 50 kr and stored at the

same water content in air at least six days;

72








3. Seeds with 3.8% water content evacuated and stored at least

one day under slight positive pressure of nitrogen; irradiated

in nitrogen with doses up to 75 kr and either germinated im-

mediately, or stored for at least four days in nitrogen, or

air, or in oxygen under moderate positive pressure.

The high positive correlation between seedling height and per cent

normal metaphases holds true for individuals within a treatment as well

as for mean values between treatments. This response of individuals

within treatments was the subject for special study with very dry seeds

(less than 3% water content) where great diversity in leaf length within

treatments has been generally observed.

The writer concludes that the variable extent of chromosomal damage

among individuals within treatments is a sufficient cause for the hetero-

geneous growth response. This great diversity can not be attributed

to variation in the dose absorbed. The "direct effect" must therefore

be relatively uniform. Evidence for "indirect effect" has been amply

demonstrated by other workers and is apparent from post-irradiation

modification experiments reported here. The present study suggests that

indirect effect on seedling height is mediated through the chromosomes,

and that, for it to be modified, there must be either protection or

repair of the chromosomes themselves.















LITERATURE CITED


Adams, V. D., R. A. Nilan, and H. M. Gunthardt. After-effects of ioniz-
ing radiation in barley. I. Modification by storage of x-rayed
seeds. A preliminary report. Northwest Sci. 29:101-108. 1955.

Adams, V. D., and R. A. Nilan. After-effects of ionizing radiation in
barley. II. Modification by storage of x-irradiated seeds in
different concentrations of oxygen. Radiation Research 8:111-122.
1958.

Avanzi, S. (Preliminary data on relative radioresistance of the apex
of the shoot and the root apex of barley seeds.) in Italian. Genet.
Agrar. 13:113-122. 1960.

Barton, L. V. Seed preservation and longevity. New York, Interscience.
1961.

Bozzini, A., R. S. Caldecott and D. T. North. The relation of seedling
height to genetic injury in x-irradiated barley seeds. Radiation
Research 16:764-772. 1962.

Caldecott, R. S. Inverse relationship between the water content of
seeds and their sensitivity to X rays. Science 120:809-810. 1954.

Caldecott, R. S. Effects of ionizing radiations on seeds of barley.
Radiation Research 2:339-350. 1955a.

Caldecott, R. S. Effects of hydration of X ray sensitivity in Hordeum.
Radiation Research 3:316-330. 1955b.

Caldecott, R. S. Seedling height, oxygen availability, storage and
temperature: Their relation to radiation-induced genetic and seed-
ling injury in barley. In Symposium on the effects of ionizing
radiations on seeds, Karlsruhe, 1960. Proceedings Series. pp. 3-24.
Vienna, International Atomic Energy Agency. 1961.

Caldecott, R. S. and L. Smith. A study of x-ray-induced chromosomal
aberrations in barley. Cytologia 17:224-242. 1952.

Caldecott, R. S., E. F. Frolik and R. Morris. A comparison of the
effects of X rays and thermal neutrons on dormant seeds of barley.
Proc. Natl. Acad. Sci. U. S. 38:804-809. 1952.

Conger, A. D. Dentists stickywax: A cover-sealing compound for tem-
porary slides. Stain Technol. 35:225. 1960.








Conger, A. D. Biological after-effects and long-lived free radicals in
irradiated seeds. J. Cellular Comp. Physiol. 58(3) Suppl. 27-32.
1961.

Conger, A. D. and L. M. Fairchild. Breakage of chromosomes by oxygen.
Proc. Natl. Acad. Sci., U. S. 38:289-299. 1952.

Curtis, H. J., N. Delihas, R. S. Caldecott and C. F. Konzak. Modifica-
tions of radiation damage in dormant seeds by storage. Radiation
Research 8:526-534. 1958.

Davidson, D. Protection and recovery from ionizing radiations: Mechan-
isms in seeds and roots. In Hollaender, A. ed. Radiation Protection
and Recovery pp. 175-211. New York, Pergamon. 1960.

Duncan, J. M., R. K. Stock, J. A. Wethington, Jr., and H. J. Teas.
Versatile gamma facilities at the University of Florida. Nucleonics
18:126-129. 1960.

Ehrenberg, L. The influence of post-irradiation factors on effects
produced in barley. Radiobiol. Symposium, Proc., Liege (1954).
,pp. 285-289. 1955a.

Ehrenberg, L. The radiation induced growth inhibition in seedlings.
Botan. Notiser 108:184-215. 1955b.

Ehrenberg, L. and N. Nybom. Ion density and biological effectiveness
of radiations. Acta Agr. Scand. 4:396-418. 1954.

Ehrenberg, L., J. Moutschen-Dahmen and M. Moutschen-Dahmen. Aberrations
chromosomiques produites dans des graines par de hautes pressions
d' oxygen. Acta. Chem. Scand. 11:1428-1429. 1957.

Froier, K. and A. Gustafsson. The influence of seed size and hulls on
X ray susceptibility in cereals. Hereditas 30:583-589. 1944.

Gelin, 0. E. V. The cytological effect of different seed treatments in
x-rayed barley. Hereditas 27:209-219. 1941.

Gelin, 0. E. V. Mitotische Storungsfrequenzen in rontgenbestrahlter
Gerste. Agr. Hort. Genetica 11:66-81. 1953.

Gustafsson, A. The different stability of chromosomes and the nature
of mitosis. Hereditas 22:281-335. 1937a.

Gustafsson, A. Der Tod als ein Nuklear Prozess. Hereditas 23:1-37.
1937b.

Gustafsson, A. Studies on the genetic basis of chlorophyll formation
and the mechanism of induced mutating. Hereditas 24:33-93. 1938.

Gustafsson, A. The mutation system of the chlorophyll apparatus.
Lunds. Univ. Arssk. N. F. And. 2:36:1-40. 1940.








Haber, A. H., W. L. Carrier and D. E. Foard. Metabolic studies of
gamma-irradiated wheat growing without cell division. Am. J. Botany
48:431-438. 1961.

Hart, J. R. and M. H. Neustadt. Application of the Karl Fischer method
to grain moisture determinations. Cereal Chem. 34:26-37. 1957.

Hart, J. R., L. Feinstein and C. Columbic. Oven methods for precise
measurement of moisture content of seeds. Marketing Research Report
304. U. S. Dept. Agri. Washington, D. C. 16 pp. 1959.

Hayden, B. and L. Smith. The relation of atmosphere to biological
effects of X rays. Genetics 34:26-43. 1949.


Horwitz, W. ed. Official Methods of Analysis. 9th ed.
Official Agricultural Chemists. Washington, D. C.


Association of
832 pp. 1960.


Ivanov, Y. A. and B. N. Kalikov. (Response of varieties and
wheat and barley to irradiation with radioactive cobalt.)
Tsitologiia 2:736-739. 1960. Referat Zhur, Biol. 1961
(Transl.)


strains of
in Russian.
No. 15G304


Kaplan, R. W. Chromosomen und Faktormutationsraten in Gerstenkornern
bei verschiedenartigen Quellungsbehandlungen oder Kalte wahrend oder
nach der Rontgenbestrahlung sowie bei Dosisfraktionierung. Z. induk-
tive Abstammungs-u. Vererbungslehre 83:347-382. 1951.

Konzak, C. F. Radiation sensitivity of dormant and germinating barley
seeds. Science 122:197. 1955.

Konzak, C. F. 1963 (Personal communication).

Konzak, C. F., H. J. Curtis, N. Delihas and R. A. Nilan. Modification
of radiation-induced damage in barley seeds by thermal energy. Can.
J. Genet. Cytol. 2:129-141. 1960..

Kronstad, W. E., R. A. Nilan and C. F. Konzak. Mutagenic effects of
oxygen on barley seeds. Science 129-1618. 1959.

Lawrence, T. The production of mutations by the irradiation of Montcalm
barley. Can. J. Botany 33:515-530. 1955.

Mitchell, J. S. and B. Holmes eds. The chemistry of biological after-
effects of ultraviolet and ionizing radiations (Symposium). Brit.
Jour. Radio. 27:36-144. 1954.

Moes, A. Water content, wave length and sensitivity to X rays in
barley. In Symposium on the effects of ionizing radiations on seeds,
Karlsruhe, 1960. Proceedings Series. pp. 631-641. Vienna, Inter-
national Atomic Energy Agency. 1961.

Moutschen, J., Z. M. Bacq and A. Herve. Action du rayonnement X sur la
croissance de la plantule d'orge. Experientia 12:314-315. 1956.








Moutschen-Dahmen, M., J. Moutschen and L. Ehrenberg. Chromosomal dis-
turbances and mutations produced in plant seeds by oxygen at high
pressures. Hereditas 45:230-244. 1959.

Muller, H. V. Artificial transmutation of the gene. Science 66:84-87.
1927.

Nilan, R. A. Relation of carbon dioxide, oxygen and low temperature to
the injury and cytogenetic effects of X rays in barley. Genetics
39:943-954. 1954.

Nilan, R. A., C. F. Konzak, R. R. Legault and J. R. Harle. The oxygen
effect in barley seeds. In Symposium on the effects of ionizing
radiations on seeds, Karlsruhe, 1960. Proceedings Series. pp. 139-
154. Vienna, International Atomic Energy Agency. 1961.

Nilan, R. A., C. F. Konzak, J. R. Harle and R. E. Heiner. Interrelation
of oxygen, water and temperature in the production of radiation-
induced genetic effects in plants. In Strahlenwirkung und Milieu
pp. 171-181. Suppl. to Strahlentherapie 51. 1962.

Ohba, K. Radiation sensitivity of pine seeds of different water content.
Hereditas 47:283-294. 1961.

Osborne, T. S., A. 0. Lunden and M. J. Constantin. Radio-sensitivity
of seeds. III. Effects of pre-irradiation humidity and gamma-ray
dose on seeds from five botanical families. Radiation Botany 3:19-28.
1963.

Sax, K. The behavior of X ray induced chromosomal aberrations in
Allium root tip cells. Genetics 26:418-425. 1941.

Smith, L. Genetics and cytology of barley. Botan. Rev. 17:1-51, 133-
202, 285-355. 1951.

Sparrow, A. H. (Closing discussion) In Symposium on the effects of
ionizing radiations on seeds, Karlsruhe, 1960. Proceedings Series.
pp. 643-646. Vienna, International Atomic Energy Agency. 1961.

Sparrow, A. H., J. P. Binnington and V. Pond. Bibliography on the
effects of ionizing radiations on plants. Brookhaven National
Laboratory. 1958.

Stadler, L. J. Mutations in barley induced by X rays and radium.
Science 68:186-187. 1928a.

Stadler, L. J. The rate of induced mutation in relation to dormancy,
temperature, and dosage. Abstract. Anat. Record 41:97. 1928b.

Stadler, L. J. Some genetic effects of X rays in plants. J. Heredity
21:3-19. 1930.

Suess, A. Uber die Wirkung kleiner Strahlendosen auf das Pflanzen-
wachstum. Naturwiss. 48:650. 1961.








Symposium on the effects of ionizing radiations on seeds, Karlsruhe,
1960. Proceedings Series. Vienna, International Atomic Energy
Agency. 1961.

Symposium on mutation and plant breeding, Cornell University, 1960.
NAS-NRC Publication 891. Washington, D. C., National Research
Council. 1961.

Tascher, W. R. Experiments with X ray treatments on the seeds of cer-
tain crop plants. PhD Thesis University of Missouri. 37 pp. 1929.

United Nations. International Conference on the Peaceful Uses of Atomic
Energy, Geneva. 1955.

United Nations. Second International Conference on the Peaceful Uses of
Atomic Energy, Geneva. 1958.

U. S. Brookhaven National Laboratory. Fundamental aspects of radio-
sensitivity. Brookhaven Symposia in Biology, No. 14. Upton, N. Y.,
Brookhaven National Laboratory. 1961.

Wertz, E. Uber die Abhangigkeit der Rontgenstrahlen-wirkung vom
Quellungszustand der Gewebe, nach Untersuchungen an Gersten-Kornern.
Strahlentherapie 67:309-321, 536-550, 700-711; 68:136-164, 287-302.
1940.

Wolff, S. Inaccuracy of anaphase bridges as a measure of radiation-
induced nuclear damage. Nature 139:208-209. 1957.

Wolff, S. and H. E. Luippold. Obtaining large numbers of metaphases
in barley root tips. Stain Technology 31:201-205. 1956.

Wolff, S. and H. E. Luippold. Studies on the sensitivity of chromosomes
to radiation. (Abstract) Genetics 48:916-917. 1963.

Wolff, S. and A. M. Sicard. Post-irradiation storage and the growth
of barley seedlings. In Symposium on the effects of ionizing
radiations on seeds, Karlsruhe, 1960. Proceedings Series. pp. 171-
179. Vienna, International Atomic Energy Agency. 1961.

Yanushkevich, S. I. (Resistance of the seeds of barley and wheat to
T-rays in relation to the conditions of cultivation before ir-
radiation. Agrobiologiia 1:95-102. 1961. Referat Zhur. Biol.
1961 No. 19G293 (Transl.)














VITA


Harlan Quinn Stevenson, son of Wilbur Harlan and Roberta Quinn

Stevenson, was born April 1, 1927, and raised on a farm at Midvale,

Pennsylvania. He attended public elementary school at Rouzerville and

high school at Waynesboro, Pennsylvania. He enlisted in the United

States Naval Reserve March, 1945, and served seventeen months active

duty principally in the Philippines.

He attended college at Pennsylvania State University, receiving

the degree of Bachelor of Science in Science, February, 1950, and im-

mediately commenced graduate study in Botany at that University, holding

a graduate teaching assistantship for three semesters. He transferred

September, 1951, to Cornell University where he again held a graduate

teaching assistantship in Botany while majoring in Cytogenetics.

September, 1955, he took employment at Brookhaven National

Laboratory as an Associate in Biology. Several papers were published

in collaboration with Dr. Harold H. Smith as a result of work done

there. In September, 1960, he entered the University of Florida as a

graduate student in Botany with a major in Radiation Biology. He was

recipient of a Graduate School Fellowship 1960-61 and Nuclear Science

Fellowship 1961-63.

He married the former Katharine L. Gebhard August, 1960, and has a

daughter, Pamela Jean, born March 1, 1962.

He is a member of Gamma Alpha, graduate scientific fraternity

(Cornell) and Phi Sigma, biological honor society (University of Florida).








This dissertation was prepared under the direction of the chair-

man of the candidate's supervisory committee and has been approved by

all members of that committee. It was submitted to the Dean of the

College of Agriculture and to the Graduate Council, and was approved

as partial fulfillment of the requirements for the degree of Doctor

of Philosophy.

August, 1963






Dean, College of Agriculture


Dean, Graduate School


SUPERVISORY COMMITTEE:



Chairman



i i ,








1




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