• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Review of literature
 Materials and methods
 Results and discussion
 Summary
 Conclusion
 Literature Cited
 Biographical sketch
 Copyright














Title: Some effects of ultraviolet light on barley and oat embryos
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
    List of Figures
        Page v
        Page vi
    Introduction
        Page 1
        Page 2
    Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Materials and methods
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Results and discussion
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
    Summary
        Page 69
        Page 70
    Conclusion
        Page 71
    Literature Cited
        Page 72
        Page 73
        Page 74
        Page 75
    Biographical sketch
        Page 76
        Page 77
    Copyright
        Copyright
Full Text












SOME EFFECTS OF ULTRAVIOLET LIGHT

ON BARLEY AND OAT EMBRYOS













By

ALLYN 0. LUNDEN


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
January, 1960
















ACKNOWLEDGEMENTS


The author wishes to express appreciation to Dr. A. T. Wallace,

Chairman of his Supervisory Committee, for cooperation and encourage-

ment in conducting this study and to Dr. Phares Decker, Dr. J. R.

Edwardson, Dr. Mildred Griffith, and Dr. Yoneo Sagawa, members of this

committee, for their encouragement and supervision. He wishes to ac-

knowledge assistance from Dr. F. H. Hull, Head of the Agronomy Depart-

ment, Florida Experiment Station, and Dr. P. H. Senn, Head of the

Agronomy Department, University of Florida, for provision of facilities

and equipment. A word of appreciation is also due his wife for assist-

ance in preparation of the manuscript and for moral support and under-

standing during this period of study.

























ii
















TABLE OF CONTENTS


Page

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

REVIEW OF LITERATURE . . . . . . . . ... . 3

MATERIALS AND METHODS. . . . . . . . .. . .. 17

RESULTS AND DISCUSSION . . . . . . . .... . .25

Cytological Studies . . . . ... . . . .25

Morphological Effects . . . . .. . . . 43

Genetic Study . . . . . . . .. . . . 51

General Discussion. . . . .. . . . . 64

SUMMARY. . . . . . . . . . . . . . 69

CONCLUSIONS. .... . . . . . . . . . .. . 71

LITERATURE CITED . . . . . . . .... .. . .72
















LIST OF TABLES


Page

Table 1. Summation of observed frequency of injury to ter-
minal apices of barley plumules following removal of
coleoptile and first leaf and exposure to dosages of 0,
30,000, 60,000, 120,000 and 240,000 ergs per mm.2 of
ultraviolet light . . . . . . . . ... 34

Table 2. Summation of frequency of injury to terminal apices
of barley plumules following removal of first leaf and
exposure to dosages of 15,000, 30,000 and 60,000 ergs
per mm.2 of ultraviolet light . . . . . . . 44

Table 3. Summation of frequency of injury to terminal apices
from barley plumules following removal of first leaf,
soaking for 24 hours and exposure to ultraviolet light, 46

Table 4. Average percentage of survival at 8 to 12 days of
growth of barley plants produced from plumules exposed
to ultraviolet light following removal of coleoptile and
first leaf -- 15 seeds per sample . . . . . .. 56

Table 5. Average height in centimeters at 8 to 12 days of
growth for barley seedlings produced from plumules ex-
posed to ultraviolet light following removal of coleoptile
and first leaf -- 15 seeds per sample -- 6 samples. . 57

Table 6. Average mature plant survival of barley plants pro-
duced from plumules exposed to ultraviolet light follow-
ing removal of coleoptile and first leaf -- 15 seeds per
sample . . . . . . . . . . . . 58

Table 7. Frequency of genetic segregation for chlorophyll de-
ficiencies observed in M2 progenies of barley plants
following removal of the first leaf and exposure of the
plumule to ultraviolet light. . . . . . . ... 61

Table 8. Summary of effects of ultraviolet light exposure to
vegetative apices of barley seeds as influenced by mois-
ture of seed. . . . . . . . .... ..... 65

Table 9. Summary of effects of dosage levels of ultraviolet
light exposure of vegetative apices of barley seeds . 67
















LIST OF FIGURES


Page

Figure 1. Dehulled barley seeds from which the following
tissues have been removed: left removal of seed coat
and coleoptile; center removal of seed coat, coleoptile
and first leaf; right removal of seed coat, coleoptile
and first and second leaves (9X). . . . . . ... 14

Figure 2. Barley seeds showing method of planting used for
study of seedling segregation in M2 populations .... 22

Figure 3. Barley seedlings following 7 days of growth in
rolled paper towels . . . . . . . . . 23

Figure 4, Normal seedling development from the seeds of a half
sector of a barley head at 7 days of growth ...... 24

Figure 5. The plumule of a dormant barley embryo from which
coleoptile and first leaf have been removed (150X). . 29

Figure 6. The plumule of a dormant barley embryo from which
coleoptile, first leaf and second leaf have been removed
(150X). . . . . . . . . . . . . 30

Figure 7. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf (645X) . . .. 33

Figure 8. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf and exposure to a
dosage of 60,000 ergs per mm.2 of ultraviolet light (645X) 35

Figure 9. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf and exposure to a
dosage of 240,000 ergs per mm.2 of ultraviolet light
(300X) . . . . . . . . . . . . . 36

Figure 10. Terminal apex of barley plumule after 8 days of
growth following removal of first leaf and exposure to a
dosage of 240,000 ergs per mm.2 of ultraviolet light
(300X) . . . . . . . . .. .. ... . 38

Figure 11. Terminal apex of oat plumule after 1 day of growth
following removal of first leaf (645X). . . . ... 40

Figure 12. Terminal apex of oat plumule after 2 days of growth
following removal of first leaf (645X). . . . ... 41













Page


Figure 13. Terminal apex of oat plumule after 4 days of
growth following removal of first leaf and exposure
to a dosage of 39,900 ergs per mm.2 of ultraviolet
light (645X). . . . . . . . ... .... 42

Figure 14. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf and exposure to
a dosage of 3,000 ergs per mm.2 of ultraviolet light
(300X) . . . . . . . . ... . . ... 48

Figure 15. Terminal apex of barley plumule after 4 days of
growth following removal of first leaf and exposure to
a dosage of 6,000 ergs per mm.2 of ultraviolet light
(645X). . . . . . . . . ... ...... 49

Figure 16. Terminal apex of barley plumule after 4 days of
growth following removal of first leaf and exposure to a
dosage of 12,000 ergs per mm.2 of ultraviolet light
(645X). . . . . . . . . . . . . 50

Figure 17. Segregation of 5 normal and 2 albino barley seed-
lings at 7 days of growth produced from a plant, the
embryo of which had been exposed to a dosage of 26,880
ergs per mm.2 of ultraviolet light. .. . . . . 63
















INTRODUCTION


The artificial induction of mutation is receiving much emphasis

in present-day genetics. Artificially induced genetic changes in many

types of cells and tissues have been produced as a result of treatment

by several different methods; these include chemical treatments, high

and low temperature, and exposure to ionizing and to nonionizing radia-

tions. The use of ultraviolet light, a nonionizing radiation, appears

to be more effective in the production of genetic changes because the

resultant changes appear to be less drastic than those produced by some

other mutagenic agents. Application of ultraviolet light has been

limited to pollen treatment in most higher plants because of its limited

penetration into tissue.

This study represents an attempt to expand the use of ultraviolet

light to include treatment of vegetative tissue of higher plants and

consisted of exposure of tissue at the tip of the terminal shoot of the

plumule of barley and oat caryopses to ultraviolet light. The feasi-

bility of inducing heritable genetic changes in plants by exposure of

vegetative tissue to ultraviolet light can be studied if suitable mate-

rial is chosen and effective techniques of exposure and growth are used.

The objectives of this study are to determine the extent of embryo

dissection necessary to provide exposure of the terminal shoot apices

of barley and oats, to determine the amount of injury caused by this

dissection, to measure depth and effectiveness of ultraviolet light

penetration into exposed tissue and type and degree of resultant in-

1














jury, to determine frequency of chlorophyll mutations induced by ir-

radiation treatment, and to study the modifying effects of moisture

content at time of treatment.

The relative effects of various dosages, of moisture content at

time of treatment, and of posttreatment exposure to visible light were

investigated and are described in regard to both resultant injury and

growth responses of tissue of the terminal apex. Resulting changes, in-

cluding both chlorophyll deficient types and morphological variants in

progenies from treated embryos, represent successful induction of herit-

able genetic changes from ultraviolet light exposure of vegetative tissue

in higher plants.
















REVIEW OF LITERATURE


The mutagenic effect of ultraviolet light has been demonstrated

on many plants, animals, and microorganisms since its first use on Dro-

sophila; the present status of this research being the result of exten-

sive work in each of these areas of study. A survey of the literature

pertaining to responses of living tissue to the effects of ultraviolet

light is based on information obtained in all of these areas, but

emphasis will be placed on research with higher plants wherever possible.

Studies of comparisons of physical and genetic effects of ionizing

and nonionizing radiations have been conducted by many workers on a number

of plant species. Notable is the work done by Stadler (1941), in which

both x-ray and ultraviolet light irradiated pollen were used to pollinate

a pure line of corn. The results of genetic studies of endosperm

characters and genetic and cytogenetic analyses of the progeny of these

crosses indicated a distinct difference between the two general types

of irradiation treatments. The ionizing type produced a preponderance

of changes involving relatively large sections of the chromosomes as

evidenced by presence of a high percentage of translocations; whereas

the nonionizing ultraviolet light treatment produced very few of these.

Steinitz-Sears and Sears (1957) also reported a difference between the

x-ray and ultraviolet light induced chromosomal abberations from pollen

treatment in wheat. They found ultraviolet light induced a higher fre-

quency of terminal deficiencies, whereas x-ray treatments more commonly

resulted in translocations and inversions.

3













Emmerling (1955) did not observe any gross qualitative differences

in his cytogenetic analysis of ultraviolet light and x-ray induced

genetic deficiencies in corn. Ultraviolet light produced a higher rela-

tive frequency of terminal deficiencies; however, it should be emphasized

that the only plants studied were those which carried genetically con-

trolled endosperm deficiencies so that no inversions or reciprocal trans-

locations were found. Barton (1954) obtained similar results with treat-

ment of tomato pollen. His data indicate an increase in the relative

number of chromosomal deficiencies and a decrease in translocations

with use of ultraviolet light as compared to x-ray irradiation.

Konzak and Singleton (1956) reported that treatment of male gametes

of barley with x-rays and ultraviolet light produced 50% and 3% of chromo-

somal interchanges, respectively. Treatment of corn pollen with thermal

neutrons, x-rays, gamma rays and ultraviolet light produced similar

effects but in different ratios for the various types of irradiation.

Work by Faberge' (1954) involving x-ray and ultraviolet light treatment

of corn pollen resulted in similar'chromosomal changes but in different

proportions.

Recent work by Nuffer (1957) substantiates the findings of these

workers by intensive study of very closely linked genes controlling en-

dosperm characters in corn. Greater differences between effects of the

two types of irradiation, however, were indicated. His treatment of

pollen showed that only by use of ultraviolet light was he able to pro-

duce genetic changes in a very small segment of the chromosome, while

x-rays appeared to produce changes involving larger chromosome segments.

The changes produced by ultraviolet light thus appear to be more similar













to "gene mutations" than are those produced by ionizing radiations as

they are of a much less severe physical or structural nature. These

changes seem to include a reduced frequency of chromosomal abberations

with a higher frequency of so-called "gene mutations." It is not known

whether these ultraviolet light induced mutations are deficiencies,

changes to the null level, or intragenic changes.

The effects of ultraviolet light upon bacteria have been studied

extensively by many workers. (Lea-1955, Spear-1955, Zelle and Hollaender-

1955). These microorganisms are especially suited to treatment as pene-

tration is not a problem and large populations can be tested in a short

period of time. These studies indicate effective induction of mutation-

al changes as well as effective germicidal or killing action. Ultra-

violet light exposures of protozoa (Kimball-1955) and Chlorella (Davis-

1952) have also been shown to result in various degrees of lethality and/

or induction of genetic changes.

The specific action of ionizing and nonionizing radiations might

well be discussed at this point as to the physical basis of their actions

and the genetic implications involved in resultant physical and chemical

changes within treated genes and chromosomes. Lea (1955) states that

ultraviolet light produces a change by producing an excitation in the

substance in which it is absorbed. This produces an increase in the

energy state of the recipient material which may result in a chemical

change without removal of electrons. X-rays and other ionizing radia-

tions refer to those types in which positively charged ions are produced

by ejection of electrons from atoms in the treated material. This probably

produces a chemical change in the molecule which contained the original













atom; and the end result generally is expected to be a more drastic

change than that of ultraviolet light absorption. Comparing these two

types on genetic, chemical, and physical bases indicates the probability

that ultraviolet light treatment may produce changes which are more

nearly comparable to those spontaneous mutations which are not associated

with detectable chromosomal changes.

A number of workers have used monochromatic irradiation to deter-

mine the most effective region of the ultraviolet light spectrum in re-

gard to induction of genetic changes. Swanson and Stadler (1955) report

production of maize endosperm deficiencies as the result of exposure of

corn pollen to light of 2350 A to 3020 X. The most effective of these

was 2650 A, and 2540 A light was about 85% as effective. The dosage levels

used were 1,000 to 132,000 ergs per mm.2 of tissue and a linear relation

did not exist between dosage and frequency of induced genetic changes

either at very low or at very high intensities. The absence of linearity

at low doses is the result of an excessive and disproportionate reduction

in effectiveness as measured by frequency of endosperm deficiencies.

Similar effects have been noted in other species. Absence of linearity

at high dosage levels is caused by internal filtration and by excessive

injury resulting in the killing of exposed tissue. It is interesting to

note that the maximum effectiveness dependence upon wave length is of the

same character as the absorption spectrum of nucleic acid.

Uber (1939) and Stadler and Uber (1942) have measured the ultra-

violet absorption of several types of material including the pollen wall

and the cellular components of corn pollen. They report that only about

22% of the incident light is transmitted through each 8-micron layer of













cytoplasm. This very poor penetration is in sharp contrast to the very

high transmission of ionizing x-rays through similar layers of tissue.

This factor of penetration has limited ultraviolet treatment to pollen

grains, pollen tubes, sperm, eggs, undeveloped zygotes and microorganisms.

The use of ultraviolet light in mutation production has been re-

stricted in the higher plants to those plants in which pollen can be

collected and treated and in which artificial pollination can be carried

out. It has been completely inapplicable in species restricted to asexual

reproduction and is not practical where hand pollination is extremely

difficult. Konzak (1957) states that the most useful plant mutants have

been obtained from treated seed, not from treated pollen.1 This reduced

apparent effectiveness of pollen treatment may be the result of more

frequent use of seed in irradiation treatment studies but may be also

due to the fact that treated pollen is in a stage of growth which is less

susceptible to genetic change. Lea (1955) states that the most effective

time for mutagenic treatment in any species is usually in late inter-

phase or early prophase of cell division and Sax (1957) indicates maximum

sensitivity at early prophase. Gunckel (1957) and Pratt (1959) conclude

that meristematic cells which have a high frequency of cell division

figures are most susceptible to injury. In view of these limitations an

efficient method of treating vegetative tissue with ultraviolet light

might be very desirable for use in many species.

Research indicates that the effectiveness of ionizing radiations

in inducing genetic changes in living tissue is dependent upon rate of



1. The term "seed" as used in this paper refers to grass caryopses
as well as to true seeds.













metabolism and water content of cells as well as on the stage of cell

division. A direct relationship between x-ray radiosensitivity and

moisture content of barley seeds was shown by Gelin (1956). Ehrenberg

(1955) noted that this held true only at 11% and above, and Caldecott

(1955) states that an inverse relationship existed at extremely low

moisture levels. Gustafsson (1940) has found that treatment of barley

embryos with x-rays is most effective in inducing genetic changes if

seeds have been soaked for 24 to 36 hours so that the cells to be treated

are no longer in a stage of dormancy and are near division. The impor-

tance of the stage of cell division and water content of cells indicates

the desirability of using material in which a relatively large propor-

tion of the cell population is in a receptive stage. This condition

can be approached by controlling the moisture and temperature conditions

of dormant embryos in such a way as to initiate growth, control water con-

tent, and obtain the desired rate of cell metabolism and nuclear activity

at time of treatment.

The effects of ultraviolet light exposure of vegetative tissue in

higher plants have not been analyzed genetically although physiological

effects have been described. Brumfield (1953) has reported a reduced

rate of growth and cell division in timothy roots as a result of exposure

to very low dosages. The reduced growth is evidenced by curvature of

roots which have been subjected to a specific dosage from one side of

the root. In this study, cell division and cell elongation in the epi-

dermal layer were delayed for approximately 24 hours after treatment.

Ultraviolet light exposure of apical cells in root tips of Scilla ob-

tusifolia produced much less chromosomal injury than was produced by x-













ray treatment (Martinoli-1954). He observed no telophase fragmentation

or micronuclei although inhibition of mitosis was evidenced by delay in

rate of root growth and cell division.

Bawden and Kleckowski (1952) have reported severe epidermal necro-

sis of bean leaves exposed to ultraviolet light. They also noted posttreat-

ment effects as the necrosis was much reduced in plants exposed to bright

light after ultraviolet light treatment, while necrosis remained severe

in plants kept in the dark. This characteristic of "photoreactivation"

whereby visible light repairs radiation injury was first recognized in

treatment of microorganisms. Brown (1951) has shown that posttreatment

with white light after ultraviolet light exposure of Neurospora will re-

duce the lethality and the relative frequency of induced reverse mutations,

but will increase the total number of reverse mutations. Altenberg and

Altenberg (1952) found that light exposure of Drosophila zygotes for 30

minutes after ultraviolet light treatment reduced the frequency of in-

duced mutations from about 7% to 1% which was not appreciably higher than

the control.

The tissue chosen for ultraviolet light exposure in the study being

reported here is the vegetative apex of monocotyledonous plants and per-

tinent literature will be discussed. A thorough knowledge of the organ-

ization and growth of the shoot apex is essential in this study and it is

fortunate that a considerable amount of information is available on this

subject. Modern emphasis on study of shoot apices was spearheaded by the

ideas set forth by Schmidt's tunica-corpus theory of apical organization.

Hanstein's histogen theory had been long accepted and the controversial

aspects of the two schools of thought served to provide renewed interest












which has continued until the present time. Comprehensive reviews

(Foster-1941) (Gifford-1954) (Popham-1951) have been presented which

provide a thorough discussion of the apical organization which exists

in various types of plants.

The usual type of Angiosperm apex was described by Popham (1951)

to contain four distinct regions which include mantle, subapical initial,

central meristem, and peripheral meristem. The surface mantle consists

of from one to several discrete layers of tissue in which cell division

is more or less limited to the anticlinal plane. The subapical initial

or central mother cell area consists of from one to several cells in a

terminal position immediately below the mantle layer. The central meri-

stem and peripheral meristem zones located below this area are responsible

for production of the pith and rib meristem cells and the cortex and pro-

cambium cells respectively. This zonation pattern is common in the Di-

cotyledons and is represented by Elodea spp. in the Monocotyledons

(Savelkoul-1957, Stant-1952).

The monocotyledonous Gramineae have a much simpler organization

pattern, consisting only of surface mantle and central mother cell zones.

Many common genera in this family including Triticum, Avena, and Zea have

been reported (Gifford-1954) to contain only a single tunica layer. In

contrast, Brown et al. (1957) conclude on the basis of study of eight

genera within five tribes of the Festucoideae that two tunica layers are

commonly present. This group includes the Avena, Hordeum, and Triticum

genera. In a recent paper Popham (1958) emphasizes that the tunica-

corpus concept is not fully acceptable by its definition, which restricts

the tunica to consist of those layers characterized by only anticlinal di-












visions. Periclinal divisions have been noted in a relatively high

frequency in the surface layer of tissue of apices of Triticum, Zea,

Avena, and Agropyron and less commonly in other genera. Stant (1952)

has found that young seedlings and young undeveloped buds of Triticum

and Avena contain only a single cell in the corpus initial area, while

the number is probably increased in shoot apices at a later stage of

development. Genetic studies of Hordeum indicate that a similar situa-

tion may exist in that species (Caldecott and Smith-1952, Ehrenberg and

Lundquist-1957) as a large proportion of induced genetic changes occur

in such a way as to produce spikes which consist entirely of mutant

tissue. MacKey (1954) states that the apex of Triticum contains no more

than 1 apical cell and that the apex of Zea contains 7 or 8 cells in the

apical initial zone.

A very interesting and important question was raised by Foster

(1939) as to the mechanism by which reproductive organs are initiated on

vegetative shoots. This problem was discussed a few years later by

Sharman (1945) in a study involving leaf and bud initiation in Agropyron

repens. His conclusion was that carpels were produced from cells derived

from the dermatogen layer but that the ovules within these carpels were

derived from the underlying residual cells of the hypodermis. Archesporial

tissue is derived from hypodermis of the anther which is assumed to orig-

inate in turn from hypodermis of the apex. The results of study of histo-

genesis in the cranberry by Dermen (1947) indicated that both pollen and

megaspore mother cells originate from tissues in the second tunica layer

in this multitunicate plant species. The significance of this is real-

ized when one considers the induction of heritable genetic changes which













must be produced in the tissue area responsible for production of re-

productive cells. Such changes must be produced in the apical initial

area in Agropyron repens which has a single tunica layer and in the

second tunica layer in the cranberry although these represent the second

layer of cells in either of these two diverse species. This indicates

that a similar situation may exist in other plant genera.

The comparative morphology of dormant embryos and seedlings of

certain species in the Gramineae has been studied by Avery (1930), Bonnet

(1935, 1936, 1937, 1953) and Kiessellback (1949). Anatomical features

are essentially similar for oats, wheat, and barley in which the plumule

consists of well-developed coleoptile and first leaf, a moderately well-

developed second leaf, and a somewhat rudimentary third leaf surrounding

the terminal apex. The primordium of a lateral bud is found in the axil

of the coleoptile, and subsequent lateral bud primordia are formed in leaf

axils as the plant develops.

The species Hordeum vulgare has been used quite extensively for

genetic studies of radiation induced mutations. Nybom (1954, 1955) has

described various chlorophyll deficient types which include albinism,

"xantha" or yellow, viridiss" or light green, and various types of de-

ficiencies resulting in mosaic color patterns on seedling leaves. Each

of these types has been produced by various mutagenic agents and has

generally resulted in the characteristic M2 segregation ratio of 3 normal

seedlings to 1 seedling expressing a mutant phenotype.

A considerable amount of preliminary experimentation was conducted

by Wallace and Lunden over a period of several months to develop desir-

able techniques of treatment involving ultraviolet light exposure of













vegetative tissues.2 These experiments included: (1) germination and

seed treatment methods for barley and oat seeds, (2) dissection of plant

tissues overlying the shoot apex in plumules of dormant embryos, (3) ex-

posure of dissected shoot apices to various dosages and intensities of

ultraviolet light and (4) methods for obtaining optimum growth from treat-

ment until maturity. The results of these experiments have been utilized

in formulating the procedures described in the materials and methods

section of this paper.

The first experimental work involved a general study of the ana-

tomical characteristics of embryos in barley kernels after removal of the

adherent lemmas. The overlying fused pericarp and seed coat, coleoptile

and initial foliage leaves were removed with a very small blade with the

aid of a dissection microscope. The blade first used was fashioned by

grinding the sharp end of a needle to a delicate edge but later a surgical

knife which was of finer, more durable steel was used. This study indi-

cated that removal of either the first leaf or both first and second

leaves was possible. Figure 1 shows three dormant seeds from which

various tissues have been removed.

The second leaf is in the form of a closed cylinder and a barely

perceptible opening can be seen at its tip. Tissue which is observed

after removal of the second leaf is the primordium of the third leaf

with the terminal apex forming a small bulge at its center. This indi-

cates that removal of the second leaf would guarantee ultraviolet light



2. A. T. Wallace and A. O. Lunden. Progress Report of Florida
Agricultural Experiment Station. Project 848. July, 1959.



























































Figure 1. Dehulled barley seeds from which the following tissues
have been removed: left--removal of seed coat and coleoptile; center--
removal of seed coat, coleoptile and first leaf; right--removal of seed
coat, coleoptile and first and second leaves (9X).













exposure of treated apices but that removal of only the coleoptile and

first leaf would produce little or no exposure of the apex. Growth

tests of dissected seeds without any ultraviolet light exposure resulted

in survival at 10 days after planting of 60% after removal of the first

leaf and only 32% after removal of both first and second leaves. Growth

following ultraviolet light exposure after dissection produced 61% and

16% survival respectively. The result of tests involving moisture pre-

treatments before ultraviolet light exposure was that survival was ap-

preciably reduced only for seeds from which both leaves had been removed.

This served to indicate that exposure was not effective following removal

of only a single leaf from the dormant embryo in barley.

The results obtained from treatment of oats were similar to those

described for barley. The nonlinearity of dosage-survival curves was

again interpreted as being due to ineffective exposure from removal of

only the first leaf from the plumule.

The surviving plants from each of these preliminary experiments were

transplanted and grown to maturity so that each treated plant produced M2

seeds upon selling. A single mutant line was observed in the M2 popula-

tions of exposed barley seeds from which the first two leaves had been

removed. This line was characterized by presence of 10 xantha (yellow)

seedlings and 15 normal green seedlings which represented the bulked seed

of several spikes of an embryo exposed to 24,000 ergs per mm.2 of ultra-

violet light. This may have been either a genetic change to a dominant

gene or a cytoplasmically inherited chlorophyll deficiency. The actual

condition existing cannot be tested as none of the xantha type seedlings

survived to maturity and the green plants produced no segregating seedlings.







16




Plants from exposed embryos from which only the first leaf had

been removed were not mature at the time of initiation of major experi-

mentation. These plants were also grown to maturity and were later

tested for frequency of induction of mutations for chlorophyll deficiencies.
















MATERIALS AND METHODS


Treatment of vegetative tissue involved shoot apices in embryos

from which overlying tissues had been removed to afford direct ex-

posure of surface cells in an attempt to overcome the problem of

limited penetration of ultraviolet light. Two species, Hordeum vulgare

(common barley) and Avena byzantina (red oats), were used with primary

emphasis on the first of these. Barley was selected for the following

reasons: (1) it is a diploid; (2) extensive genetic literature exists;

(3) it has been widely used in irradiation studies; (4) its seeds pro-

vide material which is convenient to treat; (5) normal self-pollination

occurs; and (6) induced chlorophyll deficient mutants can be readily

recognized by segregating M2 seedlings. Gustafsson (1940) and Nybom

(1954) have studied barley and its chlorophyll deficiency symptoms

and causes and have described several classes of specific mutant types

along with the spontaneous and ionizing irradiation induced frequency

of each. The oat plant is very similar although it is an allopolyploid

of hexaploid constitution and a lower frequency of mutagenically in-

duced chlorophyll deficiencies would be expected in the presence of

genome replication.

The tissue type selected for treatment is contained in the dor-

mant embryo. This material was selected because it is possible to ob-

tain the desired stage of cell division in a majority of the cells to

be treated and because it is relatively easy to expose the apex. Bonnet

17













(1935) has studied the anatomy of the vegetative shoot of barley as

well as that of other grains, His study shows that the embryo con-

tains a very well-developed first leaf, partly developed second leaf,

and primordium of the third leaf at the base of the terminal apex.

These embryos also contain primordia of lateral apices in the axil of

each leaf as well as in the axil of the coleoptile.

Shoot apices of barley embryos were described by Nybom (1954)

to have only a single corpus initial which functions as the progenitor

of succeeding growth in the interior of the culm. Tunica initials are

responsible for production of epidermal tissue throughout the culm,

If this situation exists, any heritable change induced in the corpus

initial cell will be passed on to its daughter cells and the resultant

culm will consist of a normal epidermis surrounding a core of cells

carrying the changed genotype. If more than one corpus initial cell

is contained in any treated apex, the question of cell competition

during growth will be very important and the resultant culm might be

variously sectored depending upon the number of corpus initials, genetic

changes in these initials, and degree of competitive power of the

changed cells.

The barley, which was used for treatment, was seed of Florida

Experimental No. 1079 which represents the F9 generation of a cross

of Manchuria (MS)2 X Robat barley and was produced at the Florida Ex-

periment Station Agronomy Farm in 1957-58. This specific line,

selected in the F5 generation, had been bulked without further selec-

tion since that time. The red oats seed which was used for treatment

was of the Floriland variety and was produced in 1957-58 at the Agronomy













Farm. These seeds were stored at a temperature of just above freezing

until their moisture content was about 10%, at which time the various

treatments were initiated.

In this study the light source used was an instant starting

General Electric G36T6 germicidal type bulb which delivered a measured

energy of 112 ergs/mm.2/sec. at a distance of 7-3/4 inches from the

center of the bulb. This bulb, 34 inches long and 3/4 inch in diameter,

was placed in the upper center of an 18 x 20 x 36 inch cabinet, the

front side of which consisted of a hinged glass door. The actual me-

chanics of each treatment consisted of orienting the individual kernels

under the light source after removal of the lemma, pericarp and seed

coat, coleoptile, and the oldest one or two leaves. Various dosage

levels were obtained by exposure for specific periods of time and the

effect of various moisture contents in the treated cells was determined

by use of seeds which had been presoaked before treatment. Effect of

rate of metabolic activity and stage of growth upon the susceptibility

to induced genetic changes was determined by control and variation of

temperature during the presoaking period.

Treatment after exposure was very important, as it was necessary

to maintain optimum growth conditions to prevent excessive injury of

exposed apices. Maintenance of maximum relative humidity in the vicin-

ity of the treated seeds was of primary concern as appreciable decrease

in moisture for an extended period of time caused severe desiccation

of tissue in the apex. Desirable conditions were maintained by placing

treated seeds on moistened blotters in covered plastic boxes which

were 35 x 31 x 4 inches in size. Fifteen seeds were placed in each













box and moisture was maintained by daily watering.

The young seedlings remained in these boxes until time to trans-

plant (about 10 days) and complete freedom of observation was possible

during this period. It was desirable to remove all lateral buds which

appeared during the entire period of growth as the position of develop-

ment of lateral bud primordia indicated that tissue in that area could

not possibly have received any exposure of ultraviolet light. Trans-

planting involved transfer of the entire blotter with all roots intact

to field plots or to greenhouse pots. The injury resulting from trans-

planting was negligible.

Cytological studies which were conducted involved collection of

embryos from barley and oat seeds as well as collection of seedlings.

These represented embryos which had been allowed to develop for various

periods of time following treatments which included: (1) tissue re-

moval, (2) soaking of seeds, (3) ultraviolet light exposure, and (4)

posttreatment exposure to photoreactivating light. The staining pro-

cedure which was used was the warm safranine-fast green method (Shapiro-

1947). This type of stain was shown by Sagawa and Mehlquist (1957) to

be very desirable for use in analysis of irradiation induced injury of

plant tissue. The resultant staining pattern is very characteristic

and consists of a deep red color of lignified tissues, nuclei, nucleoli,

chromosomes, and injured cells, while normal cytoplasm and cellulose

are stained a light blue-green color.

Genetic study involved analysis of segregation of seedlings grown

from seed produced by treated plants of barley. This study consisted

of growth of more than 50,000 seeds representing over 2,000 treated













plants. Due to limited available space and time a rapid method of

germination and screening was devised. The germination process was

as follows: (1) separate planting of the seeds from half sectors of

each head between the folds of a moistened paper blotter which was then

placed upon a piece of waxed paper, (2) rolling into loose cylinders

which were placed on end in plastic boxes, (3) covering of these boxes

with thin plastic to prevent excess moisture loss, (4) storage at low

temperature of 5 t 20C. for 5 days to break dormancy of fresh seeds, and

(5) growth for 7 to 1C days at 20 + 2C. to produce nearly maximum de-

velopment of the first leaf. Figures 2, 3, and 4 represent the method

of planting and growth from seeds treated in this manner. Chlorophyll

deficient types were readily recognizable at the stage of growth repre-

sented by Figure 4.











































Figure 2. Barley seeds showing method of planting used for
study of seedling segregation in M2 populations.











































Figure 3. Barley seedlings following 7 days of growth in
rolled paper towels.













































Figure 4. Normal seedling development from the seeds of a half
sector of a barley head at 7 days of growth.
















RESULTS AND DISCUSSION


This study has involved a continuous series of experiments. The

results obtained in each of these have been needed and utilized in de-

velopment of each subsequent step in the over-all pattern of research.

It is for this reason that the author has found it desirable to report

the results of each experiment in the order of completion, along with

a discussion of these results.


Cytological Studies

Cytological studies were initiated to obtain a better under-

standing of the physical effects of exposure to ultraviolet light ir-

radiation. These studies involved collection and sectioning of barley

and oat kernels, embryos and seedlings at various stages from dormancy

until 16 days of growth. The general schedule for cytological study

included killing and fixing of the plant material, removal of fixing

solution, infiltration with paraffin, sectioning from paraffin blocks,

mounting the sections on slides, and staining the material on the slides

with a suitable stain. The first schedule which was attempted involved

use of Randolph's solution as a killing and fixing fluid with iron alum-

hematoxylin in the stain procedure. This stain produces very dark

staining of nuclei and chromosomes in dividing cells but does not give

the necessary differentiation of normal and injured cells and cytoplasm

and in addition is a very slow method requiring 6 to 8 hours for the

entire staining process. The staining procedure which was finally













adopted was the warm safranine-fast green method, which involves killing

and fixing in Craf solution, infiltrating and mounting as above, and

staining with warm safranine followed by transfer to a fast green-clove

oil solution in which fast green replaces safranine. The relative in-

tensity of each color is dependent upon the degree to which the original

red is replaced by green stain and this is directly controlled by the

amount of time the material remains in fast green-clove oil solution.

The method is very rapid as the entire staining process can be completed

in less than an hour. The resultant stain is ideal because it allows

rapid identification of injured tissues, which stain deep red, as do

lignified tissues, nuclei, and chromosomes, while cytoplasm and cellu-

lose stain light blue to green.

The first objective of the study was to obtain a clear understand-

ing of the anatomical features of the plumule in the seed used; Experi-

ment I, designed for this purpose, involved sectioning and staining of

dormant seeds and embryos within seeds of barley. Dormant seeds contain

a thin pericarp and seed coat over the entire embryo and the plumule

consists of a well-developed, somewhat fleshy, coleoptile, a well-

developed first leaf producing a double layer of tissue over the apex

and a partly developed second leaf in the form of a nearly closed coni-

cal cylinder with a small aperture at its tip. Within the cylinder

formed by the second leaf are found the bluntly conical terminal apex

and the third leaf which is a ridge of tissue at the base of the apex.

A partly formed lateral bud is deeply buried at the 'juncture of the

coleoptile base and the outer edges of the first leaf. The form of the

second leaf and its orientation to the axis of the grain indicate that













ultraviolet light may be able to penetrate directly to the apex with-

out removal of this second leaf. Should this be possible the material

would be ideal for treatment as only a minimum amount of injury would

result and both the second and third leaves would be in a healthy

state of growth for rapid development of protection for the treatment

injured apex. It should be emphasized, however, that the above-mentioned

opening is not at a right angle to the long axis. This means that only

a fraction of the incident surface light energy will be available to

the terminal apex for induction of genetic changes. The terminal apex

tunica layer cells are generally 6 to 10 microns thick, the underlying

cells in the corpus initial area are about 8 to 12 microns thick and

the cells walls in these meristematic tissues are very thin. The actual

penetration into tissue which is directly exposed can be estimated if

it is assumed that ultraviolet light penetration into barley cytoplasm

is similar to that of the corn cytoplasm studied by Stadler and Uber

(1942). One could then estimate that less than 20% of the light in-

cident at the surface of the apex actually penetrates into underlying

cells and that about 10% is available to the nuclei of these underlying

cells. These figures refer to apices of embryos from which both leaves

have been removed. Ultraviolet light exposure following removal of

only the first leaf will provide only a small fraction of the total

surface energy available to nuclei of corpus initials as the result of

the limited size and unfavorable angle of the aperture at the tip of

the second leaf.

The next sectioning work (Experiment II) involved study of barley

embryos from which only the first leaf, as well as those from which












leaves one and two, had been removed. Treatments also involved ultra-

violet light exposure. Results of this study showed that in general a

great deal of injury was caused by removal of only the first leaf.

Figures 5 and 6 represent the plumules of dormant barley embryos from

which one and two leaves have been removed, respectively. Note the

poor stain differentiation caused by resistance of dry dormant tissues

to absorption of the various dyes. Several very important points are

suggested by observation of these pictures. The first point is the

obvious difficulty of removing the second leaf as in Figure 6 without

physical injury to the apex or the third leaf. This was shown by de-

termination of frequency of injury to the apex in embryos which were

collected after dissection. Embryos from which only one leaf had been

removed contained less than 8% of injured apices immediately after dis-

section while removal of the second leaf resulted in about 77% of in-

jury to apices. Injury increased to 100% if seeds were grown for 2 to

5 days following removal of both leaves while injury remained infre-

quent after removal of only the first leaf.

The fact that removal of the second leaf did provide excellent

exposure was very evident but it was also evident that nearly all the

growing plant tissue at the apex was removed, and that the plant would

surely have great difficulty in attempting to overcome this injury even

in the absence of physical injury to the apex proper. It should be

noted that ultraviolet light exposure as in Figure 5 would cause injury

to much of the second leaf, to almost none of the third leaf, and only

to the tip of the apex, thus leaving enough leaf tissue to produce rapid

recovery. Exposure of the apical area in Figure 6 would, however, cause











































Figure 5. The plumule of a dormant barley embryo from which
coleoptile and first leaf have been removed (150X).












































Figure 6. The plumule of a dormant barley embryo from which
coleoptile, first leaf and second leaf have been removed (150X).













injury to nearly all the apex as well as to the third leaf, and almost

no intact tissue would remain to aid in recovery following treatment.

The first part of this experiment involved use of embryos from

which two leaves had been removed followed by germination for 1, 2, 3

and 4 days after dissection. Cell division first occurs between 24 and

48 hours after planting at room temperature, indicating that the optimum

time for irradiation exposure would be soon after planting. The best

time would probably approach 24 hours of growth to provide the early

prophase stage of cell division which is considered to produce the

maximum frequency of induced mutations from treated cells. The last

part of this experiment involved use of various dosages of ultraviolet

light exposure after removal of both leaves. Embryos were exposed to

total dosages of 0, 39,900, 79,800, 159,600 and 319,200 ergs per mm.2

and were allowed to grow at room temperature for 24 hours in petri

dishes after which time they were collected and processed through stain-

ing. The results of these treatments showed no apical injury at the

lowest dosage, slight injury produced by the 79,800 ergs per mm.2 dosage,

and severe injury caused by treatment at the highest levels. Injuries

in the form of severe necrosis (breakdown of cells and severely injured

or killed tissue areas) were clearly evident by the characteristic red

staining of injured tissue.

It should also be noted that exposure to ultraviolet light appeared

to cause delayed cell division. This delay was evidenced by scarcity of

dividing cells after 48 hours of growth in material exposed to 319,200

ergs per mm.2 compared to normal frequent cell division in unexposed

growing plants collected after the same period of growth. Delayed cell













division in addition to severe injury of a very large area of the grow-

ing plant resulted in disruption of the organization of the terminal

apex and its protective leaves. Thus, the removal of both leaves would

be expected to result in a very low survival rate, largely due to physi-

cal injury of the protective tissue at or near the shoot apex. Damage

will also cause growth competition between the terminal bud and lateral

buds which are out of range of ultraviolet light penetration.

It seemed desirable to carry out experiments in which only the

first leaf was removed to determine if exposure of the corpus initial

area was possible without removal of the second leaf. A treatment with

only one leaf removed would be highly desirable (Experiment III) if

proper apical penetration could be obtained, because it would provide a

maximum dosage to the corpus initial area while preventing any appreci-

able injury to the third leaf and retaining the second leaf to insure

maximum possible protection to the exposed injured terminal area. Pene-

tration was very effective as evidenced by ultraviolet light treatment

induced necrosis. Epidermal and subepidermal injury were evident and

study of frequency of injury for each growth period served to provide a

general idea of the time period required for this injury to appear as

well as the time required for recovery.

The observed frequencies of apical injury for all treatments of

Experiment III are shown in Table I.

The data in this table show that maximum frequency of injury

appears by 4 days of growth and that recovery from injury occurs soon

after. Conclusive evidence is also provided that exposure is effective

with removal of only the coleoptile and first leaf. Figure 7 represents
















Figure 7. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf (645X).


L


""I













a normal apex of barley from an embryo which had been soaked for 48

hours. The general structure is similar for any stage from dormancy

to 16 days after planting. The apex of barley in Figure 7, bluntly

conical with a diameter of 98 microns, contains a single tunica layer

of tissue. The corpus initial area appears to contain more than one

cell and the third foliage leaf is well developed.


Table 1. Summation of observed frequency of injury to terminal
apices of barley plumules following removal of coleoptile and first
leaf and exposure to dosages of 0, 30,000, 60,000, 120,000 and 240,000
ergs per mm.2 of ultraviolet light.


Control Samples-No Ultra- Summation of All the Ultra-
violet Light Exposure violet Light of Exposures
No. of Total no. Per cent No. of Total no. Per cent
Days of apices of apices of apices of apices of
Growth injured studied injury injured studied injury

1 0 9 5 23 22

2 0 10 11 19 58

4 0 10 21 21 100

8 0 0 15 25 60

16 0 5 2 14 14


Figures 8 and 9 are photomicrographs of apices of plumules which

have been exposed to dosages of 60,000 and 240,000 ergs per mm.2 fol-

lowed by two days of germination. Surface cells of the apex and a single

cell at the tip of the third leaf were killed, as shown in Figure 8 by

deeply stained cytoplasm in these cells, and the resultant discontinuity

of the surface layer is evident. The more severe dosage represented in

Figure 9 has resulted in injury extending into the second layer of

cells at the tip of the apex and in the death of a number of the epi-
























Figure 8. Terminal apex of barley plumule after 2 days of
growth following removal of first leaf and exposure to a dosage of
60,000 ergs per mm.2 of ultraviolet light (645X).


FO-












































Figure 9.
growth following
240,000 ergs per


Terminal apex of barley plumule after 2 days of
removal of first leaf and exposure to a dosage of
mm.2 of ultraviolet light (300X).













dermal cells on both sides of the second leaf. The injury evidenced

at 2 days of growth is generally in the form of deeply staining cell

contents in injured cells with no other abnormal cell development.

The conditions existing at 4 days are generally cell enlargement,

cell vacuolation, discontinuity of the surface layer, and abnormal

growth of cells near necrotic areas..

Figure 10 is a photomicrograph of a barley apex which received

a dosage of 240,000 ergs per mm.2 and had been allowed to grow for a

period of 8 days. Partial recovery of normal organization is indicated

by the condition of the apex, and the unusual plane of division of sur-

face cells should be noted. Original injury extended to the second

layer of cells and cells below this area of injured tissue have nearly

repaired the resulting discontinuous epidermal layer by division in an

anticlinal plane. Cell enlargement and vacuolation are common at 8

days of growth and recovery is generally complete or very nearly complete

by 16 days of growth.

A study of oats (Experiment IV) involved sectioning of intact

dormant embryos as well as those from which the pericarp and seed coat,

the coleoptile, and the first leaf or the first two leaves had been

removed. Results indicated that it should be possible to obtain effec-

tive exposure by removal of the first leaf, as with barley. Groups of

embryos from which the first leaf had been removed were exposed to

ultraviolet light dosages of 0, 39,900, 79,800 and 159,600 ergs per mm.2

with collections of growing embryos being made 1, 2, 4 and 8 days after

exposure. Ten seeds were treated dry for each of the 16 treatment

combinations. Sectioning studies revealed that effective penetration,










































Figure 10. Terminal apex of barley plumule after 8 days of
growth following removal of first leaf and exposure to a dosage of
240,000 ergs per mm.2 of ultraviolet light (300X).













as indicated by injured cells in the apex, was possible following re-

moval of only the first leaf. Total injury of the terminal apex

observed at 4 days of growth amounted to 75% for all the ultraviolet

light treatments and only 10% for seeds which received no exposure.

The procedure used prevented actual determination of survival rates

inasmuch as treated seeds were removed for sectioning before it was

possible to determine if recovery would be complete.

Figure 11 represents a normal apex from the plumule of an oat

embryo which had been germinated for only 1 day and Figure 12 represents

one which had been germinated for 2 days. The slightly tapered conical

form of the apex is quite different from that of barley but the apex

becomes less tapered with continued growth.

Figure 13 represents the apex from a plumule exposed to a dosage

of 39,900 ergs per mm.2 and grown for 4 days. The injury appearing

here is essentially similar to that observed from treatment of barley;

however, oat apices appear to be more resistant as higher dosages appear

necessary to produce similar effects.

The use of plastic boxes for germination chambers, blotters

moistened with distilled water, and controlled germination temperatures

of 20 t 20C. for these experiments served to provide excellent con-

ditions for growth and for optimum treatment recovery. This growth

method was used in all further tests as it provides space for plant

elongation, allows maximum observation during growth, provides maxi-

mum humidity near the plant and allows removal of disease infection

areas as soon as they develop.











































Figure 11. Terminal apex of oat plumule after 1 day of growth
following removal of first leaf (645X).










































Figure 12. Terminal apex of oat plumule after 2 days of growth
following removal of first leaf (645X).










































Figure 13. Terminal apex of oat plumule after 4 days of growth
following removal of first leaf and exposure to a dosage of 39,900 ergs
per mm.2 of ultraviolet light (645X).













Morphological Effects

The second group of experiments was designed to determine

actual growth responses of barley following ultraviolet light ex-

posure of plant tissue in exposed embryos and to obtain additional

information by continuation of the cytological studies. Experiment

V was designed to measure moisture effects and utilized dosages of 0,

15,000, 30,000 and 60,000 ergs per mm.2. Twenty-five barley seeds

were planted for each treatment and 24 samples were treated. The

effect of moisture was measured by soaking half the seeds on moist

blotters for 30 minutes and the remainder for a period of 4 hours ber

fore treatment. Collections for cytological study were made at 2, 4

and 8 days of growth for 12 of the seeds in each plastic box and the

remaining 13 were transplanted to pots of soil after 10 days of growth.

The method of transplanting involved transfer of each entire blotter

along with remaining surviving roots and plants held intact into pots

of soil. All surviving plants were then grown to maturity but a very

low rate of mature plant survival and seed set were obtained due to

undesirable environmental conditions existing after transplanting.

Observation of these plants showed that early growth, as indicated by

appearance of green tissue, was slightly retarded in those samples re-

ceiving ultraviolet light exposure. The delayed growth effect was

evident at 6 days after planting but was no longer recognizable by 8

days of growth. The survival rate at 10 days of growth for non-

irradiated samples was 71%. The over-all average for all treatments

was 62% for seeds soaked 30 minutes, and 46% for seeds soaked 4 hours.

These results indicate that the greatest amount of injury occurred in













seeds containing higher moisture levels.

The frequency of injury, observed by sectioning studies, did

not appear to substantiate data of survival rates although injury ap-

peared more rapidly in seeds treated at the higher moisture content.

The frequency of injury to the terminal apex for each moisture level

is shown in Table 2. These data suggest a delay in expression of in-

jury following treatment of seeds which contained the lower moisture

content. This characteristic is very evident at 2 days of growth, but

data collected at 4 and 8 days after exposure do not indicate that any

differences exist in regard to total sensitivity over this range of

moisture.

Lateral buds appeared as early as 4 days after exposure at which

time some were nearly as large as the terminal bud. Unusually rapid

development of lateral buds is probably based upon injury of the terminal

apex as the apical dominance of this tissue might be lost as the result

of injury.


Table 2. Summation of frequency of injury to terminal apices of
barley plumules following removal of first leaf and exposure to dosages
of 15,000, 30,000 and 60,000 ergs per mm.2 of ultraviolet light.


Seeds Exposed to Moisture on Seeds Exposed to Moisture on
Blotters for 30 Minutes Blotters for 4 Hours
No. of Total no. Per cent No. of Total no. Per cent
Days of apices of apices of apices of apices of
Growth injured studied injury injured studied injury

2 5 21 24 9 14 64

4 13 14 93 13 14 93

8 11 17 65 6 9 67













Since a limited supply of the F9 population of Florida Experi-

mental No. 1079 barley was available early in the spring of 1958, a

seed sample was sent to South Dakota for increase. The seed produced,

representing the FIO generation, was returned in August and was stored

at low temperature until its moisture content was about 10%. Since

these seeds were to be used in the experiments designed to determine

genetic effects of treatment, it seemed desirable to study the physical

structure and specific effects of treatment. It is possible that seed

produced in a completely different environment might be characterized

by anatomical features which render it less desirable for ultraviolet

light exposure treatments. Experiment VI was designed to determine the

relative ease of apical exposure in the new seed as well as to obtain

additional information on the effect of moisture content. This experi-

ment involved use of 36 lots of 10 seeds each, which represented treat-

ment of seeds from which the first leaf had been removed. The seeds

were placed in inverted plastic box covers and ultraviolet light was

applied at dosage levels of 0, 15,960 and 39,900 ergs per mm.2 for dry

seeds and at dosages of 0, 39,900 and 319,200 ergs per mm.2 for seeds

soaked 24 hours. The experiment involved sectioning of these treated

seeds which were collected following 2, 4, 6, 8, 10 and 12 days of growth

after treatment. The data from this experiment showed that ultraviolet

light exposure produced similar results to those obtained with the orig-

inal seed.

Experiment VII involved ultraviolet light exposure of 24 samples

and was designed to determine the importance of photoreactivation. Each

sample contained 15 barley seeds which had been moistened at room tempera-













ture for 24 hours after removal of the first leaf from each embryo.

Dosage levels were 1,500, 3,000, 6,000, 12,000, 24,000 and 48,000 ergs

per mm. and posttreatments were immediate exposure of seeds to light

versus 48 hours of complete darkness. The results observed by section-

ing of plants which had been grown for 2 days and 4 days after treat-

ment indicated that exposure of seeds to visible light from several in-

candescent and fluorescent bulbs after exposure to ultraviolet light

had some photoreactivating effect. The frequency of injury resulting

from these treatments is summarized in Table 3.


Table 3. Summation of frequency of injury to terminal apices from
barley plumules following removal of first leaf, soaking for 24 hours and
exposure to ultraviolet light.


Seeds Receiving Dosages of
1,500 and 3,000 Ergs per
mm.2 of Ultraviolet Light
No. of Total no. Per cent
apices of apices of
injured studied injury

1 8 12

3 6 50

11 15 73

13 15 87


Seeds Receiving Dosages of
6,000, 12,000, 24,000 or
48,000 Ergs per mm.2
of Ultraviolet Light
No. of Total no. Per cent
apices of apices of
injured studied injury

2 4 50

5 7 71

12 13 92

12 13 92


Seeds which were exposed to visible light after treatment developed

injury at a lower frequency and at a much slower rate than those which

were immediately transferred to a dark chamber, thus indicating that

light served to reduce ultraviolet light induced injury. It is signifi-

cant that photoreactivation reduction of injury represented only a small


Post-
treat-
ment
Light

Normal

Dark

Normal

Dark


Days of
Growth

2

2

4

4













part of the total amount of induced injury, for considerable injury

occurred even in those samples placed in visible light after treat-

ment. This relatively slight reduction of injury may be due to use of

a source of visible light which was not of sufficient intensity to pro-

duce maximum effectiveness of recovery from induced injury. The experi-

ment also showed that the lowest dosages of 1,500 and 3,000 ergs per

mm.2 resulted in appreciably less injury than the higher dosages.

The pattern of recovery from injury was indicated in several of

the embryos which were sectioned, three of which are reproduced in

Figures 14, 15 and 16. Figure 14 shows injury to the terminal apex of

an embryo which received a dosage of only 3,000 ergs per mm.2 followed

by 4 days of growth. The effect of this relatively small dosage is

evident although resultant injury is not severe.

Figure 15 represents an embryo which received a dosage of only

6,000 ergs per mm.2 followed by a germination period of 4 days. The

pattern of recovery, which ultimately results in production of a normal

intact and continuous tunica layer over injured areas, is quite clearly

indicated in this apex. Immediately below the injured area at the tip

of the apex are two pair of cells which have recently divided and a

third cell which is beginning to divide. The presence of these division

figures in the terminal apical area is strong evidence of recovery being

the result of enlargement and periclinal division of underlying cells.

The terminal apex represented by Figure 16 received a dosage of

12,000 ergs per mm.2 followed by a growth period of 4 days. The cell

enlargement in cells immediately below the injured area at its apex was

a typical result of treatment. The dividing cell just to the right of












































Figure 14. Terminal apex of barley plumule after 2 days of growth
following removal of first leaf and exposure to a dosage of 3,000 ergs
per mm.2 of ultraviolet light (300X).












































Figure 15. Terminal apex of barley plumule after 4 days of growth
following removal of first leaf and exposure to a dosage of 6,000 ergs
per mm.2 of ultraviolet light (645X).














r


Figure 16. Terminal apex of barley plumule after 4 days of growth
following removal of first leaf and exposure to a dosage of 12,000 ergs
per mm.2 of ultraviolet light (645X).


I*#j













the two enlarged cells contains that which appears to be an anaphase

bridge. This was the only apparent abnormality observed in study of

over 600 apices which were sectioned. This probably represents delayed

separation of a dicentric chromosome or is the result of chromosome

stickiness which has often been observed following ultraviolet light ex-

posure.

The results of these experiments in sectioning studies indicate

several points which should be considered in designing procedures to

measure genetic results of treatment. These considerations include

choice of a wide range of dosage levels and especially inclusion of

dosages as low as 6,000 ergs per mm.2. Exposure of the apex can apparent-

ly be obtained by removal of only the first leaf, a method which will

provide protection to aid in recovery from exposure. The importance of

testing for the effects of moisture, for preventing photoreactivation,

for removing lateral buds during growth and development, and for main-

taining maximum humidity in the area of the treated apices to prevent

excessive loss of moisture from treated tissue are indicated.


Genetic Study

Experiment VIII, designed on the basis of the previous studies,

will be described in detail. It is the first of two major experiments

designed to determine and analyze the presence and frequency of induced

genetic changes in vegetative apices as the result of ultraviolet light

exposure. The seed used was Florida Experimental No. 1079 barley which

had been stored in a refrigerator at 5 t 2C. Moisture content of the

seed was about 10% at time of selection for treatment. Methods of dis-

section and methods of ultraviolet light exposure were identical to













those described before and dissection involved removal of only the

first leaf. The four levels of dosage exposure of 0, 6,000, 12,000

and 24,000 ergs per mm.2 were obtained by exposure of seeds in open

plastic boxes for 0, 1, 2, and 4 minutes at a distance of 61 inches

from the center of the bulb. The use of high dosage levels was con-

sidered necessary since only a very small percentage of the incident

light is actually available to the corpus initial area, as was pre-

viously noted. Posttreatment light intensity was varied in such a way

that half of the above samples were treated in darkness and remained

in the dark for a 24-hour period after treatment, whereas the remain-

ing samples were immediately placed in contact with a continuous effec-

tive light intensity of 150 candle power and at a constant temperature

of 20 + 2C. Control of moisture level included use of seeds which

had been either moistened immediately before treatment or placed on

moist blotters one hour before treatment. The first of these had a

moisture content of slightly over 10%, while those seeds soaked for

an hour had a moisture content of about 18% at time of treatment, This

experiment contained 32 lots of seed, 15 seeds in each lot, which repre-

sented two replications, four dosage levels, two moisture level pretreat-

ments and two light intensity posttreatments.

The kernels were planted upon moist blotters in covered plastic

containers to maintain optimum growing conditions for recovery from

treatment injury. This method provided an atmosphere of very high

humidity which prevented excessive drying of the exposed apical tissue

and also provided a means for close observation measurement of treatment

survival. Close observation was necessary to obtain an accurate measure













of survival rates of the terminal apex due to the presence of lateral

buds in dormant embryos. The plants in which growth developed from

rapid elongation of lateral apices would give neither a measure of

survival nor of frequency of mutation induction as they would be de-

veloped from underlying tissue which had not been exposed. These

characteristics of lateral bud formation, coupled with the observation

that cell division and growth of the terminal apex were retarded as a

result of exposure to ultraviolet light, necessitated removal of all

lateral buds as soon as possible after their emergence. All samples

were watered daily and growth notes were taken every second day until

transplanting into pots of soil which was done at ten days after treat-

ment. Results of these treatments showed 84% survival at ten days of

growth with no significant differences between irradiated and nonirradi-

ated samples. The resulting plants were transplanted to soil when

normal development had occurred to the extent that sufficient growth

had been produced to provide protection over the developing terminal

apices.

Transplanting was achieved by transfer of the entire section of

blotter containing the growing roots into a separate pot of soil for

each sample. Pots were then placed in the growth chamber at a tempera-

ture of 21 t 3C. with light intensity of 1000-candle power until matu-

rity. The average stand at 22 days of growth was 72% of the treated

seeds and the number of plants which grew to maturity and actually pro-

duced seed was only 72% of these surviving plants. The resulting sur-

vival to maturity involved only 52% of the treated seeds; no significant

differences in survival rates were found for any treatment, including













comparisons between irradiated and nonirradiated samples. A relatively

small average number of seeds produced on each mature spike indicated

that the conditions existing in the growth chamber were not ideal for

induction of a normal reproduction cycle in this material. Many infer-

tile florets were noted in most of the heads and many heads were complete-

ly unproductive. Two specific environmental conditions which were known

to be other than optimum for reproduction of this plant were the light

intensity which should have been increased to about 1500-candle power

and the dark period temperature which should have been considerably

lower than the constant temperature used.

Experiment IX, the largest experiment conducted, was designed on

the basis of results of all previous work. The seeds used were the F10

population of Florida Experimental No. 1079 barley grown for increase in

South Dakota which had been dried and stored in a refrigerator until the

moisture content was about 10%. The experiment contained 274 lots of

seed, each lot containing 15 seeds, a total of 4,110 seeds for all treat-

ments. The entire experiment utilized 6 levels of moisture and tempera-

ture pretreatments which included: dry seeds; seeds soaked 1, 2, 3 and

4 days at 5 1 2C.; and seeds soaked 24 hours at room temperature of

20-30*C. The seed lots contained 10%, 30%, 35%, 38%, 40% and 42% mois-

ture, respectively, and represented various levels of growth and metabo-

lism. Exposure was given from the same lamp, at a distance of 7-3/4

inches from the center of the bulb, to produce an intensity of 112 ergs

per mm.2 per second. The treatment intensity here was equalized for

all seeds by placing the material to be treated in the inverted lids of

plastic boxes. Exposure in these lids eliminated unequal exposure caused













by obstruction of light rays through side panels of the plastic boxes.

Treatment durations used were 0, 1, 1, 2, 4 and 8 minutes giving total

dosages of 0, 3,360, 6,720, 13,440, 26,880 and 53,760 ergs per mm.2.

Each set of samples contained all dosages, a single level of moisture

content and three replications of each treatment. Three replicated

sets were planted in the case of both dry seeds and seeds soaked for

24 hours at either low temperature or room temperature, while only two

replicated sets were used in case of seed soaked 2, 3 and 4 days at low

temperature. Posttreatment methods were also varied somewhat from the

previous experiment as all samples were exposed to an initial period of

24 hours of darkness. The growing plants were given an effective light

intensity of 900-candle power from 24 hours after ultraviolet light ex-

posure until transplanting. This intensity was maintained for each 18-

hour light period followed by a 6-hour period of darkness at constant

temperature of 21 30C. The actual exposures were made during the

period from October 24 to November 17, 1958.

Daily observations were made on seedlings from time of treatment

until time of transplanting and all lateral buds were removed as soon as

possible after they became evident. It was noted that in all cases ex-

cept for dry seeds, green tissues appeared more rapidly for each decreas-

ing dosage of ultraviolet light. Measurements of height were taken at

time of transplanting at 8 to 12 days after ultraviolet light exposure.

Results of Experiment IX in regard to survival rates for all

treatments have been determined. Comparisons between dry seeds, seeds

soaked 24 hours at room temperature, those soaked 24 hours at a low tem-

perature and those soaked for longer periods at this low temperature are













reported in Table 4 on the basis of percentage survival from treated

seeds. These figures represent the survival 8 to 12 days after treat-

ment at which time the plants were transplanted.


Table 4. Average percentage of survival at 8 to 12 days of growth
of barley plants produced from plumules exposed to ultraviolet light fol-
lowing removal of coleoptile and first leaf -- 15 seeds per sample.


Ultraviolet
Light Dosage
in Ergs per
mm.


0

3,360

6,720

13,440

26,880

53,760


Dry Seeds
(9 Samples)

67%

73%

68%

62%

73%

71%


Seeds Soaked
24 Hrs. at
Room Temp.
(9 Samples)

76%

67%

68%

66%

50%

54%


Seeds Soaked
24 Hrs. at
Low Temp.
(9 Samples)

79%

72%

65%

57%

65%

42%


Seeds Soaked
2, 3 and 4 Days
at Low Temp.
(18 Samples)

73%

61%

62%

61%

49%

44%


The data summarized here show no significant effect of variation

in dosages for treatment of dry seeds. This does not hold true, however,

for seeds which have been soaked, as they undergo a great deal more in-

jury. A positive correlation exists between amount of injury and in-

crease in the levels of dosages for all soaked seeds.

Measurements of height of these seedlings are reported in Table 5.

Comparison in seedling development indicates results of treatment

which are very similar to those obtained in regard to comparison of sur-

vival. The effect of dosage levels on dry seeds is not correlated with

rate of seedling development although reduced growth is evident at

higher dosages of seeds containing increased levels of moisture. It













should be emphasized that comparisons between moisture treatments should

not be made because of differences in duration of growth periods until

time of collection of seedling data.


Table 5. Average height in centimeters at 8 to 12 days of growth
for barley seedlings produced from plumules exposed to ultraviolet light
following removal of coleoptile and first leaf -- 15 seeds per sample --
6 samples.


Ultraviolet Light Seeds Soaked
Dosage in Ergs Dry 24 Hrs. at Seeds Soaked at 5 2C.
per mm.2 Seeds Room Temp. 24 hr. 48 hr. 72 hr. 96 hr.

0 6.0 6.1 6.4 6.9 7.6 7.3

3,360 5.9 6.2 5.4 6.4 5.7 6.2

6,720 6.1 6.4 6.0 5.6 6.3 6.5

13,440 5.6 6.1 4.5 5.9 5.4 5.6

26,880 5.9 5.6 4.6 5.0 5.1 4.8

53,760 5.3 5.1 5.2 5.3 4.4 4.7


The transplanting technique for these seedlings

used in the previous work, in which the entire blotter


system was transferred to soil.


was similar

with intact


In this case, however, the plants were


planted in the field rather than in pots in the growth chamber. The

field planting received normal care, including protection-from frost in

the winter of 1958-59 by covering the area with polyethylene during several

limited periods of low temperature. To maintain identity of terminal buds

it was frequently necessary to remove all lateral buds that developed

during this period. Tiller buds or lateral buds would be produced from

tissue which had not been treated; therefore, calculation of frequency of

induced genetic changes or mature plant survival would be meaningless in


to that

root













the presence of secondary growth of this type. Treated plants were

harvested during the period of March 29 to April 21, 1959. A total

of 1,825 plants were harvested from the field planting in this group

of treatments. Table 6 represents the mature plant survival on the

basis of percentage of seeds treated.


Table 6. Average mature plant survival of barley plants produced
from plumules exposed to ultraviolet light following removal of coleoptile
and first leaf -- 15 seeds per sample.


Ultraviolet
Light Dosage
in Ergs per
mm.2


0

3,360

6,720

13,440

26,880

53,760


Dry Seeds
(9 Samples)

48%

56%

59%

59%

48%

50%


Seeds Soaked
24 Hrs. at
Room Temp.
(9 Samples)

56%

50%

56%

49%

42%

44%


Seeds Soaked
24 Hrs. at
Low Temp.
(9 Samples)

54%

41%

49%

38%

47%

34%


Seeds Soaked
2, 3 and 4 Days
at Low Temp.
(18 Samples)

47%

40%

49%

38%

30%

32%


These results indicate that no relationship exists between dosage

and survival rates in treatment of dry seeds but that a correlation does

exist in those groups of samples in which seeds were exposed to moisture

before treatment.

Genetic studies involved observation of growth of the treated

plants until maturity, as well as observation of growth of M2 seedlings.

Morphological' changes in treated Ml plants were observed only in Experi-

ment IX and consisted of two plants which produced longitudinal yellow

stripes on the leaves and one in which leaf color was of a lighter hue













than the normal dark green. These three variant types apparently repre-

sented changes induced by treatment. The specific cause could be a

dominant mutagenic change, a change in chloroplasts, or a type of virus

attack or other change resulting in abnormal leaf coloration.

Recessive changes would be discovered only upon observation of

segregation in the M2 generation produced from self-fertilization of

plants carrying induced heterozygosity in their germinal tissue. The

expected phenotypic ratio in this diploid species would be of 3 normal

plants to 1 mutant plant. The mutants which were studied represented

chlorophyll deficient changes and were of 3 major types which correspond

to types previously recognized and described as ionizing irradiation in-

duced changes. These include albinism, which is complete absence of

chlorophyll; xantha seedlings, which are of a distinct yellow color; and

viridis, which are characterized by light green color of seedling leaves.

The method used to determine mutation frequency was designed to

study chlorophyll deficiencies in the seedling stage of the M2 generation

and consequently to recognize chimeral sectoring in the treated plant

which may have resulted from the ultraviolet light exposure. The process

involved drying the mature spikes for two weeks after harvest followed

by careful removal of the dry kernels from each spike. The kernels from

each side of the rachis were collected and labeled separately and were

planted as soon as possible. Growth study involved use of a 5-day period

of low temperature in moist paper towels to overcome dormancy followed

by space planting in the field or in upended rolled towels in plastic

boxes at 21 30C. Genetic changes were observed in the seedlings of

preliminary experiments as well as in the 2 major experiments.













Three chlorophyll deficient types were discovered on growth of

seedlings from seed produced by plants which were exposed to ultra-

violet light in the preliminary studies of Wallace and Lunden.3 These

consisted of a longitudinally striped type giving segregation values

of 8 normal and 3 striped seedlings, xantha type deficiency resulting

in 15:2 segregation and an albino type giving a segregation ratio of 2

normal to 3 mutant type seedlings. The xantha type appeared to repre-

sent a chimeral type of mutagenic change with 8 normal seedlings being

produced from seed produced on one side of the rachis and 7 normal and

2 xantha on the other side. The striped seedlings were produced from

both head segments of the treated plant in apparent 3:1 ratios. Plant-

ings of M3 seedlings from normal M2 plants produced continued similar

segregation ratios in all but the xantha type. M3 segregation of the

albino line was for 18 albino and 16 normal seedlings. Further genetic

analysis of the conditions existing in these lines is being carried out.

These mutant types were obtained from treatment dosages in excess of

24,000 ergs per mm.2 and represent a total mutation rate of 2.7%.

Only 2 chlorophyll deficient lines were observed as the result

of genetic study of Experiment VIII. These were produced following

dosages of 6,000 and 24,000 ergs per mm.2 of tissue and posttreatment

in darkness. The mutant types segregated in ratios of 16:2 for the

xantha character and 14:4 for albinism.

The results of genetic studies of Experiment IX are presented in

Table 7.



3. Wallace and Lunden, loc. cit.












Table 7. Frequency of genetic segregation for chlorophyll de-
ficiencies observed in M2 progenies of barley plants following removal
of the first leaf and exposure of the plumule to ultraviolet light.


Ultraviolet Light Moisture Treatment of Seeds
Dosage in Ergs 24 hr. at Time at low temp.
per mm.2 Dry room temp. 24 hr. 48 hr. 72 hr. 96 hr.

0 0 0 0 0 0 0

3,360 0 0 0 0 0 0

6,720 0 1 0 0 0 0

13,440 0 0 0 0 0 0

26,880 0 2 2 0 0 0

53,760 0 0 2 0 0 1


Each moisture and dosage combination reported in Table 7 represents

135 treated seeds for the first 3 moisture levels and 90 seeds for low

temperature soaking of 48 hours or longer. These represent surviving M2

progeny tests of about 90 and 60 plants of each treatment combination.

Eight chlorophyll deficient mutants were recognized in Experiment

IX among the total of 1,485 treated plants which were tested. None were

observed in 340 which had received no ultraviolet light exposure. The

mutant types were viridis (light green), xantha (yellow) and albino. One

progeny produced M2 segregation of 15 normal and 5 viridis seedlings on

one segment of the head and 17 normal plants on the other segment. The

3 xantha mutants and the 4 albino mutants segregated on only one side of

each head with 6 to 17 normal seedlings being produced from the other half

of each segment. Segregating xantha segments produced normal: yellow

types of 6:1, 9:2 and 0:1 while normal: albino segregations were 5:2, 8:1,

3:1 and 19:1. Seven of these 8 mutant phenotypes appear to be controlled












by recessive genes in the homozygous condition. The numbers involved

are in each case representative of normal 3:1 ratios on the basis of chi-

square tests. The only mutation which does not appear to be of this type

was obtained from exposure to a dosage of 53,760 ergs per mm.2 of ultra-

violet light following moisture exposure for 96 hours at low temperature.

This was the only mutant type produced in Experiment IX from any treat-

ment other than the 24 hour period of soaking. The observed segregation

for this plant progeny of 19 normal plants to one albino may have been

the result of a single spontaneous mutation. Figurez-'.l has been included

to represent a typical example of the growth produced as the result of a

mutation for albinism.

The screening procedures used were designed to study seedling

chlorophyll deficiencies and for that reason other morphological changes

were generally not noted. A single morphological mutant was also recog-

nized in Experiment IX as a type which can best be described by the term

"split coleoptile." This was characterized by M2 segregation of 13 normal

and 6 split coleoptile type seedlings. The coleoptile of these abnormal

seedlings was not in the form of a closed cylinder as isinormanl, but was

expanded into a flat organ from its base. This variant type was produced

from exposure to 13,440 ergs per mm.2 of ultraviolet light following pre-

treatment by soaking the seeds for 24 hours at room temperature.

Analysis of the results of this experiment provides two important

conclusions regarding the conditions necessary to produce genetic changes.

The first and most important factor appears to be moisture treatments of

samples. Mutations were produced most commonly in samples which had been

soaked for not more than 24 hours before ultraviolet light exposure. The













































Figure 17. Segregation of 5 normal and 2 albino barley seedlings
at 7 days of growth produced from a plant, the embryo of which had been
exposed to a dosage of 26,880 ergs per mm.2 of ultraviolet light.













effect of dosage is also important, as 7 of the 8 observed chlorophyll

deficiencies were found from treatments at the two highest dosages used.

Results from planting head segments showed that mutational changes

had probably occurred only in a portion of each of these heads, as mutant

types were generally not observed in both head segments of any sample.

This observation appears to be in direct contrast to the results of Ehren-

berg and Lundquist (1957) who did not find any sectoring in treated bar-

ley. The factor involved here which appears to cause this difference is

probably stage of growth. The treatments used here which produced muta-

genic changes consisted of exposure of apices which had been soaked for

24 hours and the material treated consisted only of terminal apices and

no lateral buds. Treatments of lateral buds would involve apices which

were much less developed and would be expected to produce fewer chimeral

changes due to the unicellular embryonic condition existing in bud

primordia. Chimeral sectoring would be expected if each apex had con-

tained more than one corpus initial cell at time of exposure treatment.

Results indicate that a complex apex probably exists in barley.


General Discussion

The structure of apices of barley and oats has been studied exten-

sively in this investigation. Embryos of each of these species contain

a single tunica layer over the terminal apex. Axillary or lateral buds

are in an undeveloped condition although the terminal apex is well de-

veloped which accounts for the low percentage of sectoring within culms

from ionizing radiation treatments. Many or all of the undeveloped

lateral buds would be in the embryonic single celled condition at time

of treatment. Chimeral sectoring occurred very commonly among the in-













duced chlorophyll deficiencies in this study following treatment of ter-

minal apices, which suggests that a multicellular condition exists in

the corpus initial area of the terminal apex.

It is evident on the basis of results of these studies that genetic

changes can be induced by treatment of vegetative tissue in apices of

barley. Cells in the treated tissue which carried these induced changes

were able to compete successfully with normal unchanged cells throughout

the growth cycle from time of treatment in undeveloped terminal apices

until maturity of the plant following flowering and reproduction.

Tables 8 and 9 are included to represent a final summary of tabu-

lation of all chlorophyll deficiency mutants to provide an over-all com-

parison of moisture levels and dosage rates.


Table 8. Summary of effects of ultraviolet light exposure to
vegetative apices of barley seeds as influenced by moisture of seed.


Condition of Seed
- Soaking Period


Dry

Wetted

30 Min.

1 Hr.

2 Hr.

4 Hr.

24 Hr.

48 Hr.

72 Hr.

96 Hr.


Total Number of
Progenies Tested


396

108

20

117

18

15

606

159

179

173


Chlorophyll Deficiency Mutants
Total number Mutation rate

1 0.3%

0 0

1 5.0

2 1.7

1 5.6

0 0

7 1.2

0 0

0 0

1 0.6












The variation in numbers of progenies tested and durations of ex-

posure make comparison of moisture effects difficult but study of the

general trend of mutation frequency is of value. The trend is that mois-

ture content attained by soaking seeds for periods of not more than 24

hours is the most desirable. This probably corresponds to a receptive

stage of cell division in the exposed apex as well as to desirable mois-

ture content levels. The extremely low frequency of genetic changes ob-

served following treatment of seeds soaked at low temperature for extended

periods is difficult to explain. The sensitivity to induction of genetic

change appears to be controlled by factors other than the simple inter-

action between cell moisture and cell division as both of these conditions

should have been optimum for seeds soaked 48 to 96 hours at that tempera-

ture.

The dosage results must be interpreted with caution because the

dosages listed include variable moisture treatments and variations in

numbers of progeny tested. The general trend of treatments is of value,

as it indicates that relatively high dosages in excess of 20,000 ergs

per mm.2 are necessary to obtain maximum effectiveness of treatment. The

relatively low frequency of mutations produced at dosages of under 20,000

ergs per mm.2 was unexpected on the basis of cytological observations of

maximum injury as low as 6,000 ergs per mm.2 and severe injury as low as

1,500 ergs per mm.2. This can probably be best explained by noting that

heritable mutations must be produced in the second layer of tissue and

assuming that low dosages provide insufficient exposure at that depth.

Choice of optimum dosage and moisture levels for this type of treatment

should produce a mutation frequency of 5% to 10% on the basis of results













reported in Tables 8 and 9.


Table 9. Summary of effects of dosage levels of ultraviolet light
exposure of vegetative apices of barley seeds.


Dosage in
Ergs per nrm.2


0

3,360

6,000 and 6,720

12,000 and 13,440

18,000

24,000 and 26,880

30,000

36,000

42,000

48,000 and 53,760


Total Number of
Progenies Tested


381

307

432

364

16

336

10

14

14

278


Chlorophyll Deficiency Mutants
Total number Mutation rate

0 0%

0 0

2 0.5

0 0

0 0

6 1.8

0 0


Cytological observations of method of recovery from treatment in-

jury indicate that the tunica layer is very rapidly killed, followed by

enlargement and growth of the corpus initial cells immediately beneath

this layer. These cells then divide in a plane opposite to the surface

of the apex, producing new tunica cells.while maintaining their original

role as corpus initial cells. This type of recovery causes the first

two surface layers to consist of daughter cells of the original under-

lying cell. An effective dosage is one which is high enough to pass

through the tunica layer with enough penetration to produce a change in

the underlying tissue.












Heritable genetic changes can be produced by exposing vegetative

apices of plants to ultraviolet light. The over-all frequency of observed

mutations was too low to make the technique of practical value, but with

proper choice of dosage and time of exposure its use could prove very

practical. Use of this treatment method over a wide range of species

should be possible with development of methods to expose vegetative

apices of various parts of each type of plant. Although this study was

conducted on barley apices which contain a single tunica layer, ultra-

violet light should not be limited to monotunicate plants, since repro-

ductive cells arise from the second layer of tissue in other plant types

as well.

The use of ultraviolet light on vegetative tissues provides a

method of studying the specific physical and chemical effects of this

type of irradiation on plant cells. Successful analysis and study of

these effects should provide a better insight into the changes involved

in "gene mutation" and into the structure of "genes."
















SUMMARY


The purpose of this study was to determine the effects of ultra-

violet light on the apices and apical tissue of barley and oats. Treat-

ment involved exposure of the terminal apices within embryos to various

dosages of ultraviolet light of wave length 2537 A following removal of

overlying tissues and subjection of caryopses to various moisture con-

ditions.

Preliminary experimentation involved anatomical study of shoot

apices by sectioning of intact embryos to determine the extent of tissue

removal necessary to provide direct exposure of the apex. This indicated

need for removal of the fused pericarp and seed coat, coleoptile, and

either one or two foliage leaves, which was accomplished with aid of a

dissection microscope. Effects were studied on the basis of results of

cytological analysis and survival of plants exposed to ultraviolet light

following removal of only one or of both of these leaves. These studies

showed that excessive injury resulted from exposure following removal of

both leaves and that exposure was effective and plant survival was sat-

isfactory after removal of only the first leaf. The effects were similar

on both oats and barley although the former was more resistant at a

similar dosage.

Cytological analysis of the recovery process indicated that exposed

tunica cells at the tip of the apex are rapidly killed, followed by en-

largement and growth of underlying corpus initial cells into the open

area remaining when these cells are sloughed off. These enlarged cells

69












then divide in a plane parallel to the surface of the apex producing

new tunica cells while retaining their original role as corpus initial

cells.

Frequencies of ultraviolet light induced genetic changes were de-

termined by analyses of observed M2 seedling segregation for chlorophyll

deficiencies in progenies produced from exposed apices. Treatments re-

sulted in a total of thirteen mutations for chlorophyll deficiencies.

These included six progenies producing segregation for albinism, one

segregating for a deficiency resulting in formation of narrow longitu-

dinal green and white stripes on leaves, five segregating for the xantha

(yellow) type of seedling, and one segregating for light green leaf

color viridiss). The majority of these mutant types appear to be reces-

sive, which in turn most commonly are evidenced by chimeral sectoring in

the treated plant. The most effective treatments consisted of exposure

to dosages in excess of 20,000 ergs per mm.2 of ultraviolet light and in

exposure of terminal apices from which only the first leaf had been re-

moved. The treatment of dry caryopses was not as effective as exposure

of those soaked for periods of 30 minutes to 24 hours, while soaking

for 48 to 96 hours at low temperature resulted in very few mutations.















CONCLUSIONS


Hereditary genetic changes can be produced in barley by ultra-

violet light exposure of vegetative apices in dissected barley embryos.

The most effective treatment in this study consisted of dosages

in excess of 20,000 ergs per mm.2 delivered to seeds soaked for periods

of 30 minutes to 24 hours.
















LITERATURE CITED


Altenberg, Luolin S. and Edgar Altenberg. The Lowering of the Mutagenic
Effectiveness of Ultraviolet by Photoreactivating Light in Drosophila.
Genetics 37:545-553. 1952.

Avery, George S. Jr. Comparative Anatomy and Morphology of Embryos and
Seedlings of Maize, Oats, and Wheat. Bot. Gaz. 89:1-39. 1930.

Barton, Donald W. Comparative Effects of X-ray and Ultraviolet Radiation
on the Differentiated Chromosome of the Tomato. Cytologia 19:157-
175. 1954.

Bawden, F. C., F.R.S. and A. Kleckowski. Ultra-Violet Injury to Higher
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BIOGRAPHICAL SKETCH


The author, Allyn Oscar Lunden, was born at Toronto, South

Dakota on February 5, 1931.

He attended High School at Walsh County Agricultural School,

Park River, North Dakota. His undergraduate work consisted of a major

in Agronomy with minors in Chemistry and Botany at South Dakota State

College, Brookings, South Dakota. He attended this institution from

September, 1948 until June, 1952, at which time he received the degree

of Bachelor of .Scente in:Agriculture.

His graduate work consisted of research in the field of seed tech-

nology and formal course work in the field of Agronomy. This work was

completed in June of 1956 at which time he received the degree of Master

of Science, with a thesis entitled "The Effect of High Temperature Con-

tact Treatment on Hard Seeds in Alfalfa."

He is a member of Gamma Sigma Delta and Alpha Zeta.














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.


January, 1960.




Dean, College of Agriculture



Dean, Graduate School


SUPERVISORY COMMITTEE:



Chairman












---------------- --










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TITLE: Some effects of ultraviolet light on barley and oat embryos / (record
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