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Symposium on 




Sponsored by the 

Committee on Plant Breeding and Genetics 

of the 
Agricultural Board 




Cornell University, Ithaca, N. Y. 
November 28 to December 2, 1960 

Publication 891 
National Academy Sciences— National Research Council 

Washington, D. C. 

Library of Congress 
Catalog Card Number 61-G0045 


J. HIS Symposium developed from the deliberations of the Com- 
mittee on Plant Breeding and Genetics which had been asked by the 
Agricultural Board of the Division of Biology and Agriculture, 
National Academy of Sciences — National Research Council, to make 
a realistic evaluation of the present status and future prospects of the 
use of induced mutations in the breeding of improved varieties of 
plants. A comprehensive symposium in this broad area of research 
had not been held for several years. In the meantime many labora- 
tories were active in both the theoretical and applied aspects of 
research in induced mutations. The deliberations of the Committee 
were directed primarily toward the research involving mutations 
produced by radiation. The scope of the program was broadened 
necessarily to include mutations regardless of origin, but with con- 
siderable emphasis remaining on those produced by radiation. The 
results of research in both the practical and theoretical investigations 
in genetics and breeding were emphasized. From the start of the Com- 
mittee's deliberations it was felt that the results of the Symposium 
should be of immediate, as well as long-time value, to geneticists and 
breeders, and should bring their research into closer juxtaposition. 

The meetings were held from the 28th of November through 
the 2nd of December, 1960. Afternoon and evening work sessions met 
during each of the first 4 days. Approximately 160 participants 
attended by invitation of the sponsoring Committee. The program 
consisted of 15 invitational papers, 5 formal discussions, and a formal 
resume. In addition, 34 volunteer contributions were given during 
the work sessions. These are not to be published by the Committee, 
but brief abstracts were multilithed and distributed to those in 

The sponsoring Committee gratefully acknowledges the financial 
support received from the United States Atomic Energy Commission, 
the National Institute of Health, the National Science Foundation, 
and the Agricultural Research Service of the United States Depart- 
ment of Agriculture. The encouragement of the Agricultural Board 
and the Agricultural Research Institute was helpful to the Committee 
in bringing the Symposium to fruition. 

I wish to acknowledge the help of all members of the Committee 
on Plant Breeding and Genetics, and especially that of H. F. Robin- 
son and G. F. Sprague who contributed greatly to the final 
development of the program. 

I especially wish to record my own as well as the Committee's 
appreciation and thanks to all of the formal participants Avhose papers 
are presented here; to the discussants, M. M. Rhoades, B. S. Strauss, 
W. M. Myers, I. J. Johnson, and H. H. Kramer; to the Editor, James 
D. Luckett; and, finally, to S. G. Stephens who undertook the major 
task of developing the resume, for their contributions to the 

R. P. Murphy, Chairman 

Committee on Plant Breeding and Genetics 

R. A. Brink H. F. Robinson 

W. M. Myers W. R. Singleton 

F. L. Patterson G. F. Sprague 

J. D. Luckett, Editor 
R. P. Murphy, Chairman 



Foreword V 

Session I: The Nature and Characteristics of Mutations 
R. P. Murphy, Chairman 

The nature of mutations in terms of gene and chromosome changes, 

John R. Laughnan 3 

Comparison of spontaneous and induced mutations, K. C. Atwood 

(By title only) 29 

Mutation, selection, and population fitness, C. C. Li 30 

Discussion of Session I, M.M. Rhoades 48 

Session II: Mutagenic Agents and Interpretation of Their Effects 
W. R. Singleton, Chairman 

Types of ionizing radiation and their cytogenetic effects, Arnold H. 

Sparrow 55 

Chemicals and their effects, Charlotte Auerbach 120 

Effects of preirradiation and postirradiation cellular synthetic events 

on mutation induction in bacteria, Felix L. Haas, Charles O. 

Doudney, and Tsuneo Kada 145 

Discussion of Session II, Bernard S. Strauss 173 

Session III: Evaluation of Mutations in Plant Breeding 
G. F. Sprague, Chairman 

Use of spontaneous mutations in sorghum, J. R. Quinby 183 

Use of induced mutants in seed-propagated species, Horst Gaul .... 206 
The use of induced mutations for the improvement of vegetatively 

propagated plants, Nils Nybom 252 

Discussion of Session III, W. M. Myers 295 

Session IV: Utilization of Induced Mutations 
F. L. Patterson, Chairman 

Screening methods in microbiology, Thomas C. Nelson 311 

Methods of utilizing induced mutation in crop improvement, James 
MacKey 336 


Factors modifying the radio-sensitivity ol seeds and the theoretical 
significance of the acute irradiation of successive generations, 
Richard S. Caldecott and D. T. North 365 

Discussion of Session IV, I. J. Johnson 405 

Session V: Possibilities for the Future 
H. F. Robinson, Cliairman 
Mutagenic specificity and directed mutation, Harold H. Smith . . . .413 
Increasing the efficiency of mutation induction, R. A. Nilan and C. F. 

Konzak 437 

The efficacy of mutation breeding, Walton C. Gregory 461 

Discussion of Session V, Herbert H. Kramer 487 

General Resume of Symposium 
Resume of symposium, S. G. Stephens 495 

List of those attending or participating in the Symposium 513 


Session I 

The Nature and Characteristics 
of Mutations 

R. P. Murphy, Chairman 
Cornell University, 
Ithaca, N. Y. 

The Nature of Mutations 
in Terms of Gene and Chromosome Changes 

University of Illinois, Urbana, Illinois 

For iHE breeder, variability of the biological entity, regardless of 
its source, is essential. But considered from an operational stand- 
point, his success in the selection of improved strains has depended 
to only an insignificant degree, or not at all, on an understanding of 
the ultimate basis for that variability. The fact that geneticists have 
so far been unable to provide a convincing demonstration of intra- 
genic mutation means that, for the breeder, the question of whether, 
in a particular instance, variability is attributable to intragenic or 
extragenic causes must for the time being remain academic. Mainly 
then, his approach to the problem has been to deal with the potential 
variability available in his source materials and to develop operation- 
al techniques aimed at manipulating this variability to the advantage 
of his program. Thus, the main impact which genetics has so far had 
on breeding programs has been to provide general techniques based 
on the principles of transmission genetics and, in my opinion, it is 
our lack of understanding of the physiological actions and inter- 
actions of genes, including their capacities to change, which is mainly 
responsible for differences of opinion and earnest debate over the 
breeding method to invoke in a particular instance. 

Nor is it always clear that a certain apparent degree of success in 
an improvement program is a function of the breeding technique 
employed. For one thing, superiority is often relative, difficult to 
assess, and sometimes embarrassingly short-lived. Then, too, nature 
is on the side of the breeder, and while he is therefore reasonably 
confident that he will finish with no worse than he started, he must, 
by the same token, ofttimes concede that his efforts may have been 
quite passive, alongside those of nature, in achieving a proved 
measure of success. 

'Currently Guggenheim Fellow on leave, Biology Division, California Institute of 
Technology, Pasadena. California. Work reported here was aided by a grant from the 
Xational Science Foundation. 


That there has been some success in the development of artificial 
methods to increase variability cannot be denied, and perhaps the 
introduction of various mutagenic agents is the outstanding example 
of this. However, I think it is safe to say that these agents have not 
had a significant impact on the development of improved techniques 
of selection, but are now viewed by the breeder primarily as a means 
of enhancing variability, with the added hope that an occasional 
spectacular, useful variant will appear. Rather, it should be antici- 
pated that the use of these agents, along with other techniques 
available to the geneticist and plant breeder, in the interests of a 
better understanding of the nature and action of the genetic elements, 
stands to add far more to the development of improved breeding 
techniques than their direct use to enhance, through increased 
variability, the prospects of success in a conventional selection 

But the traits of particular concern to the plant breeder are most 
often quantitative and are usually controlled by numbers of genes 
whose individual genetic analysis appears particularly unrewarding. 
Unless we hold with the idea that genes governing quantitative traits 
are unique in their actions, a premise that I consider indefensible, the 
reasonable alternative is to deal individually with genes having 
so-called qualitative effects. 

For some time it has been apparent that extragenic events make 
up a considerable portion of the occurrences we call mutations, 
and while we were at one time accustomed to think of variability in 
nature as due to the reshuffling of genetic entities whose differences 
were ascribable ultimately to qualitative changes that we preferred 
to think of as gene mutations, a great deal of painstaking work has 
led to increased emphasis on extragenic events as more immediate 
sources of variability in a population. As increasing numbers of muta- 
tions are resolved as extragenic events, the classical gene mutation 
appears more and more to be an elusive phenomenon and some, 
perhaps, would even doubt its validity as a biological concept. 

If, in fact, the propagation of coded genetic information from 
mother to daughter cells, from parent to offspring, is a far more 
accurate process than had earlier been considered, and we suppose 
that the intramolecular alteration we think of as gene mutation may 
range in frequency for individual loci from 10 -,! or 10 -7 to lower 

laugh nan: nature of mutations 5 

levels, it is obvious that, where technical difficulties place a limit on 
the size of population that may be analized effectively, our studies of 
the mutation phenomenon have unwittingly selected against the gene 
mutational event. We should be prepared to concede then that how- 
ever important may be the ultimate qualitative genetic change, the 
rarity of its occurrence in natural and even in experimentally manip- 
ulated laboratory populations may make it relatively impregnable to 
attack compared with other types of changes which make a greater 
immediate contribution to variability. 

I should like to consider here, in some detail, genetic analyses of 
certain derivatives of the A h complexes in maize which, on the basis 
of conventional criteria, appear not to be extragenic in origin and 
which, for this reason, are of particular interest in connection with 
the problem of gene mutation. 

The various forms of the A 1 gene in maize control the synthesis of 
varying amounts of anthocyanin pigment in the aleurone layer of the 
endosperm and in certain vegetative tissues. Under appropriate 
conditions they are also in control of the type of pigment deposited 
in cells of the pericarp. The phenotypes of aleurone and plant may 
range from deep purple, as in the presence of A, the type allele, 
through intermediate levels, to colorless aleurone and brown plant 
characteristic of individuals that are homozygous for the recessive a 
allele. In pericarp tissue the type allele A produces a red pigment and 
is dominant to recessive a, associated with brown pigmentation. The 
several alleles that go under the general designation A h , and their 
mutant derivatives, are unique in that they have divergent effects 
which will not allow their placement in consistent linear array witii 
sister alleles. Thus, A b , which was first described in a stock from 
Ecuador (5) 2 has a brown pericarp phenotype that is dominant to A, 
yet is weaker than the latter in its effect on plant pigmentation (6). 

Following the original finding (22) that A h mutates to an inter- 
mediate allele designated A d (dilute), with a frequency of about 5 X 
10~ 4 , detailed analyses (7, 8) employing suitable marker genes have 
established that A h consists of two closely linked, but separable, ele- 
ments, both concerned with anthocyanin pigmentation. 

This conclusion is based on an analysis of marker constitutions 
of A d strands derived from A b /a heterozygotes that carried various 

2 See References, page 25. 


combinations of markers. 

In summary, these experiments indicated that of 161 independ- 
ently occurring changes to A d , 148 were recombinants for the distal 
marker gene, while 13 were nonrecombinants. Thus, the member 
elements of A h consist of (a) the left-most A d element (hereafter 
designated alpha or a) which has intermediate effects on aleurone 
and plant pigmentation and carries the dominant brown pericarp 
effect of the parental complex, and (b) an adjacent element on the 
right (hereafter designated beta or /?) associated with purple plant and 
aleurone. Because in regard to aleurone and plant phenotypes the 
beta element is an isoallele of its parental A h complex, it is technically 
more difficult to isolate by crossing over than the alpha component; 
however, there have been 10 independent isolations of the beta 
element (13) and, as might be predicted from the strand constitutions 
of alpha isolations from the same complex, each of these was a 
recombinant for the proximal marker and each was shown to have a 
red pericarp effect. It may be concluded that the beta member of the 
A b complex is similar to the wild type A allele, and that the alpha 
element, which retains the dominance of its brown pericarp effect in 
spite of its intermediate plant and aleurone phenotypes, is the basis 
for the anomalous phenotypic behavior of A b . The genetic distance 
between alpha and beta is about 0.05 of a unit and the sequence of 
these elements in the long arm of chromosome 3 is centromere:alpha: 

More recent studies (9, 11) indicate that most of the alpha cases 
from A h homozygotes also occur in association with crossing over. 
This observation, along with evidence on the derivatives from certain 
special compounds involving A h , leads to the conclusion that alpha 
and beta, or the segments in which they reside, are members of an 
adjacent duplication in which the genetic materials are ordered in 
the same sequence. Thus A h , like bar in Drosophila, is a tandem, serial 
duplication Avhose members may engage in oblique synapsis. 

While these experiments indicate a strong association between 
the occurrence of the alpha derivative and crossing over in the A h 
segment, it is apparent, from the data cited above, that about 8 per 
cent of the alpha exceptions occur without an associated exchange 
in this region. The possibility was earlier considered (7) that these 
nonrecombinant alpha cases may represent double exchanges, one 

laughnan: nature of mutations 

crossover occurring between alpha and beta thus isolating the former, 
the second occurring between beta and the distal marker to reconsti- 
tute the parental combination. This explanation was not taken 
seriously, however, since it was found (8) that Ig A h ct /a sJi heterozy- 
gotes in which sJi (shrunken endosperm-2), the inside distal marker, 
is located 0.25 unit from A (14), gave rise to 43 alpha isolations, 4 of 
which were nonrecombinants for the sJi marker. It was discarded 
altogether on finding that alpha derivatives are obtained from hemizy- 
gous A b /a-X\ plants in which the X-ray induced deficiency u-Xl is 
substituted for recessive a. Since the deficient segment here includes 
the A locus as well as the SJi locus, crossing over between homologues 
can not be considered as a basis for these alpha occurrences. It should 
be noted that these experiments did not afford the opportunity to 
decide between several possible mechanisms to explain the anomalous 
nonrecombinant alpha derivatives, although gene mutation of 
the beta element of the A b complex was one of the possibilities 
suggested (8). 

A detailed analysis of the factors influencing the occurrence of 
the noncrossover alpha derivatives and, in particular, a test of the 
hypothesis that they occur as a result of gene mutation of beta 
are seriously hampered by the relatively low frequency of their occur- 
rence (ca. 5 X 10~ 5 ) among A b gametes of A 1 '/ a individuals. For this 
reason we have shifted the emphasis in these studies to other A b com- 
plexes whose yield of noncrossover alpha exceptions is greater than 
that of the original A b complex discussed above. 

We have carried out intensive analyses of two A b "alleles" of 
Peruvian extraction (the designation A b -V will be employed hereafter 
in general references to these) which share with the original A b of 
Ecuador the determination of purple plant and aleurone and a domi- 
nant brown pericarp. While it has been established (10) that these 
A b -F forms are likewise complexes consisting of separable alpha and 
beta elements, they differ from the original A b complex of Ecuador 
origin in several ways: 

(a) The alpha element of the A b -V complex is weaker in its effect 
on plant and aleurone pigmentation than that of A b . 

(b) The sequence of alpha and beta elements in A b -V is the 
reverse of that in A h . Using the C notation for the centromere, 
the order of the members of the A h complex is C : a : (3, 


whereas the sequence of members in the A h -V complex is 
C : p : (x. The evidence suggests that one complex is not sim- 
ply a gross inversion of the other, but rather that the indi- 
vidual members of the duplication have exchanged position 
while retaining the same serial order of the duplication as 
a whole. 
(c) There is a striking difference in the frequency of occurrence 
of the noncrossover alpha derivatives from the Ecuador A° 
complex (ca. 5 X 10 -5 ) as compared with that from the A h -P 
source (ca. 5 X 10 -4 ), whereas the crossover alpha derivatives 
occur with about the same frequency from both complexes. 

In attempting to test the hypothesis that the anomalous non- 
recombinant alpha derivatives are ascribable to gene mutation, the 
reduced phenotype of the alpha from A u -V and its enhanced fre- 
quency of occurrence are decided advantages. In what follows Ave 
have brought together the available evidence from several years' study 
in this laboratory bearing on the question of the gene mutation origin 
of the nonrecombinant alpha derivative. 

The markers employed in the experiments to be discussed are 
given in Figure 1 which provides a map of a portion of the long arm 
of chromosome 3, including the A locus with which we are concerned. 

7.1 0.25 12.8 

T A Sh Et 

1 ^ 1 

Figure 1. — Map of a portion of the long arm of chromosome 3 slwzo- 
ing position of A locus and of the marker loci employed in these studies. 
Centromere is located to the left of T. 

The symbol T refers to Translocation 2-3d (1) which has been used 
extensively in heterozygous condition to define a segment proximal 
to the A locus. Whenever it is used to designate chromosome constitu- 
tions, it refers specifically to an interchanged 2 :i chromosome carry the 
A locus. Similarly, in translocation heterozygotes the symbol A r refers 
to a normal (noninterchanged) chromosome 3. Since all individuals 
to be tested were crossed with homozygous normal testers, the pres- 
ence or absence of the interchanged chromosome in exceptional 
individuals among the offspring was easily determined by the pres- 

laughnan: nature of mutations [) 

ence or absence of aborted pollen, the former of which is typically 
associated with plants that are heterozygous for the translocation. 
This classification was confirmed by scoring the ears of exceptional 
individuals for the aborted condition. 

The sh 2 (shrunken-2) factor which produces a striking collapse 
of the mature endosperm is distal to A and, whenever possible, because 
of its close proximity to the latter, has been used as a marker in 
preference to et (etched endosperm, virescent seedling). 

Table 1 summarizes tests of over a million gametes from A h -F /a 
heterozygotes marked with T and sh. The A h -P alleles designated 
Lima and Cusco are extractions from two different plants of Peruvian 
origin and have been treated as separate entities throughout this 
presentation. Referring to the alpha derivatives in Table 1, we note 

Table 1 . — Constitutions of Alpha-bearing Strands from A h — P/a Individuals 

Marked with T and sh. 


A h -P 



Number and distr 
strands ai 



lg ol 

of alpha 
Tspring * 









N A h -Lima/T a sh 
jV^ b -Lima sh/T a 
T A b -Lima/jV a sh 




20 N a 

Sh T 



,V a sh 


7V/l b -Cusco/7 a sh 
7^yl b -Gusco sh/T a 
T A h -Cusco/N a sh 
T /l b -Cusco sh/N a 




















*As illustrated by the strand constitutions provided in the first row of this table, here and in 
similar tables to follow, nco-1 refers to a strand carrying the nonrecombinant parental markers of 
the parental A b -P chromosome; nco-2 refers to a strand carrying the parental markers of its homo- 
logue; co-1 refers to a recombinant strand that carries the distal marker of the parental A b -P chromo- 
some and the proximal marker of its homologue; and co-2 refers to a recombinant strand that 
carries the proximal marker of the parental A [ '-P chromosome and the distal marker of its homologue. 

that only two of the four possible marker combinations are repre- 
sented among these strands. One of these, the recombinant type 
designated co-1, carries the shrunken marker of the parental /J b -P 
chromosome and the T marker of the homologue. Since this recom- 


binant type constitutes over 20 per cent of the alpha strands from the 
/f 1 — Lima parent and almost half of those from the /J b -Cusco heterozy- 
gote, it is apparent that the crossover event is not independent of the 
event that isolates alpha from the complex. If these events were inde- 
pendent, the frequency of recombinant alpha-bearing strands, based 
on the genetic length of the T-sh segment, should be barely in excess 
of 7 per cent of the total alpha exceptions. This evidence, combined 
with the observation that the complementary recombinant type (co-2) 
is not represented among the alpha strands, confirms the original 
conclusion (10) that the order of elements in /4 b -Lima and ^4 b -Cusco 
is centromere:beta:alpha; accordingly, isolation of the alpha com- 
ponent from the parental A h -P complex by a crossover between the 
beta and alpha elements gives rise to recombination for the proximal 
marker, as illustrated: 

JV j8 a Sh T a S/i 

>- - (co-1) 

T a sh 

The alpha-bearing strand which is of particular interest, and 
which is predominant among those presented in Table 1 (nco-1), 
carries the nonrecombinant markers of the parental A h -V chromo- 
some. As \ve shall see, the alpha strand of nco-1 type is found among 
the progeny of various A h -V compounds. Its regular occurrence and 
the fact that it is not associated with recombination for marker loci 
suggest that it may be the result of gene mutation of the closely linked 
beta element. 

The possibility, that multiple exchanges within a short segment 
or a copy-choice phenomenon might account for the anomalous nco-1 
alpha derivatives should be considered. A detailed treatment of these 
possibilities will be given elsewhere, but it may be noted here that 
although the nco-1 alpha strands are expected on either or both of 
these schemes, they also call for the occurrence of alpha strands of 
nco-2 and co-2 constitutions, both of which are conspicuous by their 
absence in the data reported here. 

Tables 2 and 3 present the constitutions of alpha strands among 

laughnan: nature of mutations 


offspring from A h -V jA and A h -P/A h -Ec (original Ecuador source) 
heterozygotes. Again Ave note that the nco-1 alpha strands con- 
stitute a significant proportion of the total alpha occurrences 
and the prominence of co-1 and the absence of co-2 alpha strands 
are further evidence in support of the aforementioned order 
of components in the A h -V complexes. The absence of nco-2 deriv- 
atives (Table 2) from A h -V j A heterozygotes is expected since there is 

Table 2. — Constitutions of Alpha-bearing Strands from A h -Y*/A Individuals 
Marked with T and sh or with T and et. 


A h -Y* 




and distribution of c 
strands among offsp 







.V.4 b -Lima^/r.4 
T ^ b -Lima/,V A sh 




A" /l b -Cusco .s7*/"r /! 


X A h -Lima/T A et 



jV A h -Uma et/T A 



T A h -Uma/X A et 




A^l b -Cusco/T/l et 





*Here and in the following tables population is expressed in terms of ^l b -P gametes tested, though, 
of course, an equivalent number of gametes from the homologue were also scored. 

no evidence to indicate that the type A allele is a complex, let alone 
that it carries an alpha component. And the rare occurrence of nco-2 
alpha derivatives from the A h -P/A b -Ec heterozygotes (Table 3) is 
anticipated, since it is established independently that the A h of 
Ecuador extraction carries an alpha component that is infrequently 
isolated as a nonrecombinant. 

One further point may be mentioned here. The occurrence of 
crossover alpha derivatives from the A h -P/A b -Ec heterozygote indi- 
cates that these complexes have at least one homologous member in 
common. Evidence on crossover derivatives from A h -V homozygotes, 
omitted here because it does not bear on the order of the components, 
indicates that the members of this complex engage in oblique synap- 
sis from which it may be concluded that A h -V, like A h , is a duplication 


Table 3. — Constitutions of Alpha-bearing Strands from A h -P/A h (Ecuador) 
Individuals Marked with T and sh. 


A h -F 




and distribution of alpha 
strands among offspring 






N /l b -Lima/T A h sh 
T A h ~Uma sh/N A h 

j\ r A h -Cusco/TA h sh 













whose members have retained synaptic homology. 

Mention was made at an earlier point of the possibility that 
the nonrecombinant alpha cases might represent double exchanges 
in which one crossover occurs between the alpha and beta 
elements and another within the marked segment such as to 
reconstitute a parental marker combination. Critical evidence is 
provided on this point by an analysis of A h -V hemizygotes in which 
the homologue is deficient for a segment including the A locus. If 
both the recombinant (co-1) and nonrecombinant (nco-1) alpha 
derivatives from A h -V J a parents (Table 1) are dependent on a cross- 
over between the beta and alpha elements, it is expected that A h -P /Df 
u-X individuals in which the opportunity for synapsis of the A h -V 
complex and hence for exchanges between homologues in that region 
is removed, would yield no alpha offspring. 

The data summarized in Table 4 indicate that alpha occurrences 
are common among the offspring of such hemizygotes. The deficien- 
cies <7-Xl and a-XS are of X-ray origin (23) and are known to be 
deficient for a segment including the A locus and extending to the 
right beyond the 5/? locus. That the deficiency extends to the left 
beyond beta in the homologue is apparent from the absence of A T a SJ? 
recombinants (co-1) among the alpha strands from V'-marked hemi- 
zygotes. (See first four rows of Table 4.) Their complete absence is 
somewhat surprising since they might be expected as a result of 
coincidental exchange in the T — f3 segment. However, this coin- 
cidental event may be rarer than anticipated either as a result of an 
interfering effect of the event that gives rise to alpha, or because the 

laughnan: nature of mutations 13 

Table 4. — Constitutions of Alpha-bearing Strands from .4 b -P/Df a-X Hemizvgotes. * 

A h ~P Distribution of alpha-bearing 

Source gimetes strands among offspring 


T J b -Lima Sh/N a-Xl . . . .1 32,640 93 T a Sh 

T /t b -Cusco Sh/N a-Xl 400 1 T a Sh 

T A h -Uma Sh/N a-X3 20,580 12 T a Sh 

T 4 b -Cusco sh/N a-X3 2,350 3 T a sh 

No marking: 

JV A b -Uma Sh/N a-Xl . . . 1 28.280 83 A' « Sh 

N .4 b -Cuseo Sh/N a-Xl 86,355 44 N a Sh 

,V /l b -Lima Sh/N a-X3 4,555 4 X a Sh 

N i4 b -Cusco Sh/N a-X3 2,500 1 .V « Sh 

*The deficient segments in both Df a-Xl and Df a-X3 include the A locus and the Sh locus. 
See text for further details. 

deficiency may extend well to the left of the A locus. In any case, the 
regular occurrence of alpha strands from these deficiency heterozy- 
gotes indicates clearly that the phenomenon leading to the nonrecom- 
binant alpha occurrence can not be attributed to any mechanism 
requiring the direct participation of the homologue. 

One obvious explanation for the occurrence of the nonrecombi- 
nant alpha derivatives that does not conflict with the evidence so far 
presented would attribute them to mutation of the beta element. 
Since the beta element of the complex has a purple effect, it masks the 
pale or intermediate phenotype associated with the adjacent alpha 
component. A qualitative change in the beta element of the complex, 
rendering it ineffective in the production of pigment, would allow 
the expression of the adjacent alpha element under circumstances 
which would not involve the participation of the homologue and 
which would thus be independent of recombination for marker loci. 
However, since the crossover alpha derivatives (co-1) which have 
lost the beta element in the exchange event are phenotypically indis- 
tinguishable from the nonrecombinant alpha individuals (nco-1), 
the restriction is imposed that the presumed mutation of the beta 
element be to the null level, hereafter referred to as beta or /3 - 

The gene mutation hypothesis proposed here equates the occur- 
rence of the nonrecombinant alpha with a change in the A h -P com- 
plex from /? : a to /?o : a, where the former has the typical purple 
effect, the latter the typical alpha (pale) effect, and the hypothetical 


j3„ (imitated element) has a null effect which, so far as the aleurone 
phenotype is concerned, is equivalent to the colorless phenotype 
associated with recessive a. The validity of this hypothesis may be 
tested objectively by isolating the active beta element from the com- 
plex through a crossover event and subjecting it to appropriate 
mutational analysis to determine whether it has the predicted 
capacity to mutate to the null level, and if so, whether the frequency 
of this event corresponds to the frequency of occurrence of the 
nonrecombinant alpha derivatives from the fia complex. 

In order to provide a situation that permits the simultaneous 
comparison of mutation rates of the isolated beta element and the 
beta element in the beta:alpha complex, and that would eliminate 
the effect of modifiers, crosses were made to produce marked heterozy- 
gotes having the constitution T f3a Sh/X (3 sh, in which the isolated 
beta element is the same as that in the beta:alpha complex of the 
homologne. The steps involved in producing the desired heterozy- 
gotes are shown diagrammatically in Figure 2, which emphasizes the 

/? °C Sh T °C Sh j¥>^ Nonrecombinant (pale) 

—i 1 1 1 1 1 1 nco-l 

Nonrecombinant (colorless) 

A. B. C. 

Figure 2. — Diagrammatic presentation of the steps involved in testing 
the hypothesis of gene mntatoin of beta to beta,,. A, An exchange between 
beta and alpha isolates beta on A T f3 sh strand. B, Appropriate crosses 
are made to produce marked F x individuals of the type shown here, 
carrying the isolated beta in one chromosome and the bcta:alpfia com- 
plex from which it was isolated in the homologne. C, On the hypothe- 
sis, gene mutation of beta to beta in the F t parent should produce type J 
nonrecombinant strands (pale plienotype) and type 2 nonrecombinant 
strands (colorless phenotype), with equal frequencies. 

common origin of the two beta elements in the tested individual and 
also indicates the strand constitutions of exceptional offspring 
expected on the hypothesis that mutation of the beta element is 
responsible for the nonrecombinant alpha derivative in question. If 

laughnan: nature of mutations 15 

this hypothesis is valid, the frequency of nonrecombinant A T and sh, 
colorless derivatives, should equal the frequency of nonrecombinant 
T and Six, alpha derivatives. 

Since a number of independent isolations of beta derivatives 
were employed in these experiments, it will be helpful before pro- 
ceeding to the results to consider the criteria that were employed in 
establishing that an isolated beta was really dealt with. In all cases 
prospective beta isolates were tested for pericarp phenotype and only 
those which had lost the dominant brown effect and hence the alpha 
component in the crossover event were used in the present experi- 
ments. Incidentally, we have never obtained an alpha derivative from 
a crossover isolate whose pericarp test indicated a loss of the brown 
phenotype. A more serious problem is posed by the fact that a con- 
siderable number of beta isolates (lacking the alpha component) are 
new complexes of the /? : a type in which the a element from the 
homologue has taken the place of alpha in the complex. Detailed 
analyses of this phenomenon will be published elsewhere and it will 
suffice here to point out that this substitution is expected if a is paired 
to the right (see Figure 2A) of the crossover which takes place between 
beta and alpha. The j3 : a crossover derivative has a pericarp pheno- 
type indistinguishable from that of fi, and since it is established inde- 
pendently that homozygotes of the f3 : a complex yield both crossover 
and noncrossover a derivatives much as the /? : a complex yields 
crossover and noncrossover alpha derivatives, the unwitting use of 
/3 : a in the present experiment would lead to the isolation of non- 
crossover a derivatives which are not attributable to mutation of the 
beta component. Consequently, we have adopted the criterion that 
any beta isolate that has yielded crossover a derivatives among its 
progeny is a /? : a complex and is not legitimately included in the data 
to be presented. At the same time it is apparent that this runs the risk 
of mistakenly including data from those /? : a complexes whose prog- 
enies, speaking statistically, were too small to yield even a single 
crossover a case, even though they might produce occasional noncross- 
over a cases, which in that event would be mistakenly taken to repre- 
sent mutation of beta to beta . 

One particular beta extraction (designated isolate #1) has had 
extensive testing. The absence of the alpha component in isolate #1 
has been independently established by an analysis of over 50,000 



gametes from marked heterozygotes carrying beta and recessive a, 
among which no alpha exceptions were found. That this particular 
beta isolate does not carry an adjacent a element is suggested by the 
absence of a derivatives among over 15,000 offspring from marked 
compounds carrying the beta isolate in one chromosome and the 
original A h complex of Ecuador extraction in the other. 

The results of progeny analyses of heterozygotes carrying beta 
isolate #1 in one chromosome and the beta:alpha complex of A b - 
Lima, from which it was extracted, in the other, are presented in 
Table 5. We note that from T and sli marked heterozygotes of this 

Table 5. — Summary of Exceptional Cases from Heterozvgotes Involving Beta 

Isolate No. 1 and the Beta:alpha Complex of ^l b -LiMA from 

which it was Extracted. 


,i h -P 



Distribution of exceptional cases 
among the offspring 


nco-2 co-1 


T and sh marked: 

T A h -Umz/N /3-Lima sh 
T A h -Um&/N a sh 

No proximal marker: 
/J b -Lima/j3-Lima sh 


17 TaSh 

5 N a Sh 


17 T aSh 

9 A' a Sh 

37,905 34 a Sh; no colorless derivatives 

type five co-1 and 17 nco-1 alpha derivatives were obtained. The five 
recombinants are expected since they represent crossover isolations of 
the alpha element of the parental A h — Lima and carry the recombinant 
markers predicted from the knowledge that alpha is the distal element 
in this complex. Our special interest in these data concerns the 17 
nonrecombinant alpha cases since, according to the mutation 
hypothesis, they have the constitution /?„ : rx and are presumed to 
have resulted from the mutation of beta to beta,, in the /4 b -Lima com- 
plex. However, since the isolated beta element in the homologue is a 
replica of that carried in the A h — Lima complex, an equivalent num- 
ber of mutations ol the former to the /?<, level is expected. These 
would be scored phenotypically as a (colorless) derivatives, and since 
they should carry the N and sh parental marker combination, they 
would be expected to fall in the nco-2 registry of Table 5. Contrary to 

laughnan: nature of mutations 17 

this expectation, not a single a derivative was found. 

Data presented in the second row of Table 5, dealing with the 
number and distribution of alpha cases from T // h -Lima/A r a sli sibs, 
indicate a close agreement with regard to the frequencies of nco-1 
alpha strands from the two sources and may be taken as evidence 
that the frequency of this type of derivative from the sib (i :a//3 
heterozygote is not subject to any peculiarity having to do with its 
special genotype. 

In the third row of Table 5 are presented data from additional 
heterozygotes, involving beta isolate #1, which are without proximal 
marking. While it is thus not possible to distinguish between nco-1 
and co-l alpha derivatives, the data of Table 1 lead us to expect that 
at least half of these are noncrossovers; yet we note here the occur- 
rence of 34 alpha cases and again the absence of a derivatives. Thus, 
the data from beta isolate #1 pose a striking contradiction to the 
hypothesis that would attribute the noncrossover alpha to gene 
mutation of the adjacent beta element of the complex. 

Analyses similar to the foregoing have been carried out with 
nine other independently isolated beta derivatives from /(''-Lima and 
with eight such isolates from the /J''-Cusco complex. A summary of 
derivatives obtained from T and sJi marked heterozygotes involving 
these beta isolates and //''-Lima is given in Table 6. With the excep- 
tion of isolate #162-1, the data from individual beta isolates are too 
meagre to justify inferences concerning the mutation hypothesis but, 
taken as a whole, the occurrence of 41 nco-1 alpha cases as compared 
with two nco-2 a cases is in excellent agreement with the results 
obtained in the more extensive tests involving beta isolate #1. We 
conclude, therefore, that gene mutation of the beta element can not 
be the mechanism responsible for the vast majority of noncrossover 
alpha derivatives; nor is it certain that the two cases of nco-2 a 
derivatives shown in Table 6 are attributable to that phenomenon 
since some of these beta isolates may actually represent £ : a com- 
plexes, of the type discussed above, that were inadvertantly included 
here because, in the relatively small populations of tested gametes, 
they gave no crossover a derivatives to identify them as such. While 
further analyses of these questionable beta derivatives should resolve 
this question, their possible removal as nonvalid cases would only 
serve to emphasize the strong evidence, from the data as they stand. 


Table 6.- Summary of Exceptional Cases from Compounds Involving 17 
Independently Isolated Beta Elements and the /l b -Lima Complex. 

A h -P 


tribution of 

alpha and colorless 




derivatives among the 









7 ,4 b -Lima/N /3- 


















1 N 

a Sh 





































7 A h -\Ama/N /3- 



155 5 




a Sh 

1 N 





















1 N a 



























T ^ b -LimaAV a 







a Sh 

"These individuals occurred as sibs of the hetero/ygotcs listed above. Data presented in this row 
represent totals for all such sibs. 

against the gene mutation hypothesis. 

It is appropriate to consider at this point certain other experi- 
ments designed to test the mutation hypothesis for the origin of the 
noncrossover alpha cases. These studies, like those reported above, 
are based on the argument that if gene mutation of the beta element 
in the complex is responsible for the occurrence of the nonrecom- 
binant alpha, the latter, represented as /? : a (see Figure 2), should 
carry a null beta form which is susceptible to isolation by crossing 
over in advanced generation tests of these derivatives. These studies 
(20), carried out in this laboratory, not only fail to support the gene 
mutation hypothesis, but provide strong empirical support for the 
supposition that, if the event in question is not due to a qualitative 

laughnan: nature of mutations 10 

change in beta, it must be assigned to its removal altogether from the 

O o O 


One more line of evidence, less decisive perhaps than those present- 
ed above, may be brought to focus on the question of gene mutation of 
the beta element, and in this case the original A h complex of Ecuador 
origin is involved. As pointed out earlier, the elements of this com- 
plex are ordered in reverse (a : ft) of those of the A h -P complex. More- 
over, the alpha component of the former is isolated as a nonrecom- 
binant with a ten-fold lower frequency compared with the corre- 
sponding event from the A h -V complex. Among the crossover alpha 
isolations from the A h /a heterozygote, an occasional one is found (8) 
that is mutable (a-m) when the Dt gene is present in the genome. 
Kernels that carry this mutable alpha form are pale in phenotype, 
with dots (clusters of purple cells) distributed throughout the aleur- 
one. Since Dt is known to condition the mutation of recessive a to A 
(18, 19) and since the mutability of the a-m derivative is also con- 
trolled by Dt, it is inferred that a-m itself represents a complex of the 
type a : a, in which, as a result of a crossover in the A h /a heterozygote, 
the mutable a allele has been traded for, and taken the position of, 
beta in the alpharbeta complex. The mutability of this new complex 
is then considered to reside not in alpha itself but in the now adjacent 
a element. This argument is supported by the crossover extraction of 
the mutable a element from the a : a complex in experiments which 
also confirm that the order of the alpha and a elements in this 
complex, as expected, is centromere : alpha : a. 

Taking advantage of the influence of Dt on the mutation of a, 
we have obtained from a : a homozygotes a full-colored revertant 
whose phenotype is indistinguishable from A h . There is good reason 
to conclude that this revertant is a synthetic A h of the type a : A, in 
which A, originating from a as a result of mutation in situ, is substi- 
tuted for the original beta of the complex (Figure 3). This is sup- 
ported by tests which reveal that the revertant no longer yields a 
derivatives and that the mutant form, in heterozygotes with recessive 
a, yields alpha derivatives whose occurrence is associated with recom- 
bination for the distal marker. 

The substitution of A for beta in A h affords the opportunity to 
compare the frequencies of crossover and noncrossover alpha cases 
from the original and the synthetic complexes. This experiment 


A b 
^ Synthetic A b 

oC Q q. oC A 

_l — I — » 1 — ,_ 


Figure 3. — The steps involved in obtaining the synthetic A h . First step 
involves crossover isolation of the a:a complex. The second step involves 
Dt-induced imitation of a in this complex giving a'-A, the synthetic A h 
in which A replaces (j. 

would seem to he particularly appropriate, since, according to the 
mutation hypothesis which attributes the noncrossover alpha occur- 
rences to mutation ol the beta element, there is do reason to expect 
that the synthetic A h , which carries A instead ol beta, should yield 
alpha derivatives at the same rate that A h does. The data presented 
in the first two rows of Table 7 indicate that the original and the 

Table 7. — Yield of Alpha Derivatives from A h (alpha :beta) and from its Synthetic 
Counterpart (alpha^-1) in Heterozygotes with Recessive a. 

A h Alpha derivatives 

Source gametes 

tested nco co 


A h sh/aSh 9,180 \\ a Sh 

/l b -synthetic/a 12,340 Total of 15 a cases* 

-4 b -synthetic Sfi/a sh 36,700 3 a Sh Slash 

* These could not be stored for noncrossover or crossover origin since the parent lacked distal 

synthetic complexes yield alpha derivatives with similar frequencies. 
Moreover, it is apparent that the noncrossover alpha is a rare occur- 
rence among the progeny of the synthetic A h (Table 7, third row) 
just as it is among the progeny of the original A u . The 3 noncrossover 
cases among a total of 54 alpha derivatives reported here for the 
synthetic complex is in good agreement with the 10 noncrossovers 
among a total of 127 alpha cases reported for the original A b (8). From 
these results, indicating that the substitution of A for beta in the 
Ecuador complex has little if any effect on the frequency of noncross- 

laughnan: nature of mutations 21 

over alpha derivatives among the progeny, there is again no support 
for the gene-mutation hypothesis. 

On the basis of the evidence presented above, the contention 
that gene mutation of the beta element is responsible for the occur- 
rence of nonrecombinant alpha derivatives is indefensible. This 
should come as no surprise, however, since it fits a pattern that has 
by now become commonplace. The A alleles are among the most 
intensively studied in maize and, of the many "mutations" recorded 
at this locus, the noncrossover alpha derivatives analyzed here are 
among the select group that had survived previous tests designed to 
identify extragenic changes. It is difficult to avoid the conclusion 
that, if the currently favored hypothesis of gene mutation (25, 26) 
based on the alteration of structure at the molecular level is valid, we 
should not expect to encounter the phenomenon in maize in experi- 
ments which at best can deal statistically with events at the level of 
10" 5 . It is increasingly apparent that the classical gene mutation eludes 
the investigator, not so much because it defies characterization, but 
because it is of such infrequent occurrence that it is swamped by 
events that are extragenic. We can only conclude that nature is 
conservative in its display of gene mutations and that the predomi- 
nant and immediate contributions to variability in the population 
are functions of various extragenic events. 

In regard to the case discussed here we may summarize as follows: 

(a) The noncrossover event leading to the expression of alpha 
involves a loss of the beta (purple) phenotypic expression. 
This follows since the crossover and noncrossover alpha 
derivatives are identical in phenotype. 

(b) The noncrossover alpha derivative originates through a 
physical loss, not gene mutation, of the adjacent beta 

(c) The experiments dealing with heterozygous deficiencies 
indicate that the occurrence of the noncrossover alpha 
derivative (loss of beta) does not involve the participation 
of the homologue. 

(d) Loss of the beta element of the complex is conditioned by 
the presence of the adjacent alpha element since it has 
been shown that the isolated beta element "mutates" 
rarely, if at all. 


Mention should be made of a phenomenon at the II locus in 
maize which is a rather close parallel of that involving A b . It is now 
apparent (3, 4) that loss of the plant color effect of the (P) element in 
the (P) (S) Cornell complex, whether or not it is associated with 
recombination of marker genes, is attributable to physical loss rather 
than to gene mutation of the (P) element in the complex. Spontaneous 
deficiency of the (P) element or, alternatively, loss of this element in 
response to some type of activator, are two of the mechanisms pro- 
posed (4) to account for the event in question. However, neither of 
these explanations satisfactorily accounts for the noncrossover alpha 
derivatives from the beta: alpha complex since it has been shown 
that the isolated beta element, removed from its association with 
alpha, is surprisingly stable. 

Thus, an acceptable model on which to account for the loss of 
beta from the complex must satisfy the evidence that the event does 
not require the participation of the hoinologue and that it is uniquely 
dependent on the cis association with alpha. Moreover, the event must 
be highly restricted as to time of its occurrence since the vast majority 
of the derivatives occur as single kernels on ears of tested individuals, 
thus oivino: strono- indication that meiosis is involved. Several years 
aeo we sueeested (12) a mechanism which accommodated the evidence 

O uu \ 

available at that time and which appears not to be in conflict with 
that available now. It is based on the finding (9, 11) that the A b com- 
plexes are tandem, serial duplications whose members, at meiosis, 
engage in oblique synapsis with their complements in the homologue; 
and it assumes only that the adjacent members of the duplication 
have the alternative of pairing with each other, intrachromosomally, 
in a double-loop configuration at meiosis which will be referred to 
as A.A. (auto-association). As illustrated in Figure 4, rare exchanges 
between the beta and alpha segments comprising the double loop 
would result in the loss and/or gain of one complete member of the 
duplication, depending on the strands involved in the event. Occa- 
sional losses of the beta-carrying segment by this mechanism would 
yield apparent noncrossover alpha derivatives which, because of their 
origin through "deficiency" of beta, would be indistinguishable in 
phenotype from the crossover alpha derivatives. Moreover, since the 
event is dependent on a pairing phenomenon ordinarily restricted to 
meiosis, the observed individual occurrences of the alpha derivatives 

laughnan: nature of mutations 23 




Event ■ 1 

Event- 2 



T «c Sh 

/3?A Sh T fee Sh 

—i — i — i 1— ' i — i ' 


Figure 4. — Diagrammatic representation of proposed mechanism of auto- 
association. At the left, adjacent, homologous beta and alplia segments of 
a single chromosome are shown paired with each other at mciosis to form 
the double loop. The results of exchange event 1 (involving sister chroma- 
tids) and of exchange event 2 (involving a single chromatid) are shown at 
the right. Both events yield a "noncrossover" alpha strand, but the first 
event produces a complementary strand carrying tivo beta members 
plus an alpha, while the second event yields a parental-type strand 
carrying beta and alpha members plus an acentric ring representing the 
beta member. 

are explained. The observation that loss of beta is conditioned by 
the presence of alpha in the parental complex is also predicted since, 
on the model proposed here, the removal of the beta element is 
dependent on the adjacent alpha-carrying segment to provide the 
double loop. Finally, since the A.A. hypothesis involves a strictly 
intrachromosomal event, the occurrence of noncrossover alpha cases 
among the progeny of plants heterozygous for the a-X. deficiencies 
is also anticipated. 

In fact, assuming that the A.A. type of pairing at meiosis is in 
competition with conventional interhomologue pairing, the fre- 
quency of the former, and therefore of noncrossover alpha occur- 
rences, should be enhanced in the deficiency heterozygote, in which 
the opportunity for interhomologue pairing of members of the com- 
plex is removed. Results of experiments testing this prediction will 
be published elsewhere, but it may be mentioned here that studies 
of this type dealing with over 400 alpha cases and with tests of over a 
million gametes indicate a significant enhancement of the alpha rate 
in the hemizygote. 

The possibility that sister strand crossing over might account 
for the origin of the noncrossover alpha derivatives was treated in 
an earlier publication (8) but was not considered a likely explanation 
because (a) there was evidence against the occurrence of the phe- 


nomenon in Drosophila (15, 16), and (b) to explain the occurrence 
of noncrossover alpha cases, the sister strand exchanges would have 
to be unequal. More recently, evidence has been presented (21) in 
support of the occurrence of sister strand crossing over in maize, but 
it is apparent that the question of the existence of the phenomenon 
is still debated among geneticists. In any case, if it is argued that 
crossing over is a function of the reduplication process or that it 
occurs at the time of reduplication of strands, it is difficult to visualize 
how old and new member elements of the A h complex are, as 
required, in precise juxtaposition at the time of and immediately 
following replication, yet at the same time are in oblique association 
to facilitate the required unequal sister event. Moreover, according 
to the sister strand hypothesis, there is no apparent basis on which, 
to explain the enhancing effect of the deficient homologue on the 
frequency of occurrence of the noncrossover alpha derivative. 

We are now searching for independent cytological evidence 
bearing on the validity of the A. A. scheme proposed here and have 
chosen the bar locus in Drosophila for this purpose because it too 
represents a tandem, serial duplication and moreover, presents the 
opportunity for cytological analysis. To be sure, we cannot expect to 
observe the double loop in meiotic tissues of either Drosophila or 
maize, nor would such an observation be decisive. But Ave note 
(Figure 4) that the A. A. mechanism calls for the removal (or addition) 
of one complete member of the duplication, no more, no less. Thus, 
in the case of the bar duplication, the hypothesis calls for the occur- 
rence from bar of both noncrossover normal and noncrossover double- 
bar individuals, corresponding to the loss and gain, respectively, of 
one complete member of the duplication. Since the duplicated seg- 
ment in bar is clearly defined as carrying the seven bands of the 16A 
region of the salivary map, cytological analysis of noncrossover deriv- 
atives should constitute a critical test of the A. A. hypothesis. 

Studies carried on in this laboratory over the past year (17) indi- 
cate that noncrossover wild-type reversions from bar do occur, and 
with a sufficiently high frequency to permit their statistical analysis. 
Thus far, in addition to the expected crossover cases, reversions not 
associated with recombination for marker genes have been obtained 
from bar individuals heterozygous for the deficiency B-263-20, from 
homozygous bar individuals heterozygous for the C1B chromosome, 

laughnan: nature of mutations 25 

and from bar homozygotes carrying two normal X chromosomes. In 
addition, double bar has given rise to nonrecombinant bar offspring, 
and bar in turn, has produced noncrossover double-bar types. These 
derivatives are consistent with expectations on the A.A. hypothesis 
though their occurrence is not of itself decisive. It is of interest to 
note that Sturtevant, in his classical work on bar (24), recorded several 
cases of changes at the locus which were not associated with crossing 
over, though he considered their status as valid cases somewhat doubt- 
ful. Also, Braver (2), in the course of an experiment designed to test 
the effect of a nearby inversion on unequal crossing over at the bar 
locus, has obtained (personal communication) a nonrecombinant 
wild-type revertant from a homozygous-bar parent. 

Thus, it appears that the bar duplication has its share of anoma- 
lous derivatives, and cytological analysis of those now in hand may- 
be expected to shed light on a phenomenon which, though it is not 
attributable to gene mutation, is certainly a subtle substitute. On the 
basis of available evidence it may be anticipated that the phenomenon 
dealt with here is significant not only for the contribution it makes 
to variability through regressive changes, but for the progressive 
evolution of gene systems as well. 


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of the plant color component of the R r gene in Zea mays. Cold 
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Stephens: Strains of corn from Ecuador and Peru have the alpha and 
beta elements arranged in dilferent serial order. Do you have any simple 
mechanism which would derive one from the other? 

Lauhgnan: A scheme to derive one from the other was produced in con- 
nection with the original report (PNAS 41: 78—84) on the changed 
order of elements in the Peruvian complexes, ft is based on the find- 
ing that adjacent alpha and beta segments represent tandem serial dupli- 
cations whose members retain homology and may thus be obliquely 
synapsed in meiosis. Given the opportunity for crossovers within these 
members, and the occurrence of alpha-carrying chromosomes in the 
population, there are various ways of deriving the beta: alpha order 
from the alpha:beta order, and vice versa. For example, with exchanges 
in successive generations, we may go from alpha:beta to alpha:beta: 
beta to alpha:beta:alpha to beta:alpha. 

Peterson: Can the physical loss of beta be considered in the light of 
transposition events (simple drop out) associated with mutable gene 

Laughnan: It may be assumed that a Ds-like element resides at the 
beta locus and conditions an infrequent loss of the latter. Against this 
is the evidence indicating that the isolated beta element, removed from 
its association with alpha, is remarkably stable. Moreover, somatic alpha 
occurrences, expected as the ride on the insertion-transposition hypothe- 
sis, are all but absent, the vast majority of the alpha cases originating 
in connection with meiosis. And finally, progeny analysis of the alpha 
occurrences, a routine procedure in our studies, gives no hint of lowered 
gametophytic or sporophytic viability which would accompany the 
grosser aberrations predicted to occur at least occasionally on the trans- 
position hypothesis. 


Grobman: Examination of a large number of archeological ears of corn 
in Peru has not disclosed the presence of phenotypes with lower pigmen- 
tation intensity than those corresponding to the A b or a? levels (brown 
and brown-red pericarp, respectively), during at least the first 2,000 
years of a 2,900-year period represented by those ears. It is believed 
that A b , «p, or both represent the wild condition alleles at the A x locus 
in maize. The stability of A b phenotype seems to have been relatively 
high during a long period in the evolution of corn, suggesting stability 
of A h (a/3) allele complex, until such time as tripsacoid races of corn 
and lower level expression of A h down to a became prevalent. 

This apparent evolutionary stepping up of the changes at A, which 
we find coincides with the appearance of other conspicuous mutational 
events in other genes, suggests a direct or indirect effect of Tripsacum 
genetic material incorporated into corn. 

In the light of the information he has accumulated, would Doctor 
Laughnan care to comment on the possibility that the mutational events 
at A h were accelerated and are now being influenced by extragenic ele- 
ments originating from some kind of Zea-Tripsacum interaction? 

Laughnan: Experiments conducted by R. A. Emerson and E. G. Ander- 
son reported in 1932 established that A b and op, in the presence of the 
P factor, govern the synthesis of brown pigment in the pericarp of 
maize. However, since a number of other loci are known to affect pig- 
mentation of the pericarp, even in the absence of P, and since a variety 
of intergrading phenotypes is possible with respect to both quality and 
pattern of pigmentation, there is no way, other than by crossing and 
progeny test, to establish with certainty whether a particular pericarp 
phenotype is governed by the P locus. Under these circumstances, I 
consider it unsafe to make inferences about the //-locus constitution 
on the basis of pigmentation in archeological ears of corn, and even 
more hazardous to argue a shift in frequencies of alleles on this cri- 

So far as I can see, there is nothing about the A b events which can 
be taken to support or contradict the mentioned Zea-Tripsacum inter- 
action. I suppose some better understanding of this would come from 
genetic analysis of the presumed A elements of Andean Tripsacum. 

Auerbach: I wonder whether you think it is safe to generalize from 
maize to other organisms, in particular to animals. You said in your 
opening remarks that in maize intergenic changes are so much more fre- 
quent than gene mutations that, in over-all estimates, the bona fide 

laughnan: nature of mutations 29 

mutations are quite swamped. The opposite is true for Drosophila, 
where intergenic changes are much less frequent than mutations. It seems 
that plant chromosomes are much more easily broken than animal ones, 
at least those of Drosophila germ cells. 

If one assumes that most of the presumed Drosophila mutations are 
minute rearrangements, one would have to assume a very high frequency 
of rearrangements of undetectably small size. As far as I know, the size 
distribution of detectable rearrangements, in particular small deficien- 
cies, does not lead us to believe that this is so. 

Laughnan: I prefer not to generalize from maize to other organisms, 
including Drosophila, concerning the relative frequencies of gene muta- 
tions and extragenic events. But I should like to emphasize that, as 
with the alpha occurrences (beta losses) reported here, the change may 
be subtle to the point that only intensive genetic analyses of the changed 
form, or of the circumstances surrounding its origin, would be expected 
to reveal its extragenic nature; and upon this point I should not hesi- 
tate to generalize. 

It is clearly not established that most of the presumed Drosophila 
mutations are minute rearrangements, nor is it evident that, at the 
other extreme, they represent molecular alterations in the genetic mate- 
rial. But it seems to me, that to favor the latter interpretation on the 
basis of size distribution of detectable rearrangements, is to make the 
unwarranted assumption that opportunities for extragenic events are 
restricted to changes whose origin is similar to those we observe in 
gross rearrangements. 

Comparison of Spontaneous and Induced Mutations 


University of Illinois, Urbana, III. 

Paper presented, but no manuscript available. 

Mutation, Selection, and Population Fitness 

C. C. LI 

Graduate School of Public Health, 
University of Pittsburgh, Pittsburgh, Pa. 

The problems of imitation and selection in natural populations 
are varied and complicated. Probably each case merits a separate 
study, and it is doubtful if any attempt of abstract generalization is 
warranted. This situation, however, is in principle no different from 
that in physical sciences. A physical phenomenon may also be very 
complicated and yet physicists start out from very simple models and 
gradually build more and more realistic models as experimental 
evidence accumulates. It is in this sense that biologists may formulate 
mutation and selection models, granting and remembering that they 
are only first approximations of natural situations. Simple models do 
help us to understand certain biological phenomena and may serve 
as starting points for further research. 

Before proceeding, it may be well to define the scope of our 
discussion. The disciplines of population genetics, ecology, and evolu- 
tion are interlocked in such a way that it is impossible to disentangle 
one from the others. In a very general way, however, ecology deals 
with population growth in size and the joint distribution of more 
than one group of organisms under a variety of environmental condi- 
tions. Evolution is concerned with lono-term changes whatever the 
causes may be. In the present discussion, I will limit myself to the 
comparatively short-term changes in the genetic composition of a 
population in the face of recurring mutation and persistent selection. 

Probably all genes mutate. The history of mutation must be as 
old as the gene itself. Mutation is one of the most fundamental 
properties of a gene. It is also probable that each allele produces a 
different, however slight, effect on the organism in one respect or 
another which may or may not be detectable by our present tech- 
nique. As the title implies, the present discussion is further limited 
to mutations which affect the reproductive capacity of the organism. 



I. Definition of Selection and Fitness 

When a common word, in lieu of a new one, is drafted into 
science and used as a technical term, it is likely to introduce semantic 
difficulties if it is not precisely defined at the outset. Selection and 
fitness are such common words that have been drafted into biology 
and, regretfully, have caused a great deal of misunderstanding and 
unnecessary arguments, because different biologists take them to mean 
different things. The definition of these terms, given below, is for 
the purpose of the present discussion. This does not prevent other 
scientists defining them in some other way to mean something else, 
just so long as they are used consistently in the sense they are defined 
and not in any other sense. 

Consider two types, A and B, of organism (e.g. A = red wheat, 
and B = white wheat) that grow in the same environment and there 
is no intermixture. Suppose that in the initial period there are equal 
proportions of A and B, and that for every 100 living offspring pro- 
duced by the A type in the next generation, type B produces only 80. 
The relative numbers of the two types of organism in the entire 
population will be as follows: 

Generation Type A Type B Generation Type A Type B 

100 100 100 100 

1 100 80 1 125 100 

2 100 64 or 2 156.25 100 

It makes no difference which type is taken as the "standard"; 
the ratio of the relative numbers is the same in the two systems of 
presentation, viz., 100:04 = 156.25:100. In other words, the initial 
50%:50% distribution becomes, after two generations, approximately 
61%:39%. In a situation like this we say there is selection, meaning 
that there is differential reproduction, or differential contribution to 
the next generation, between the two types. Further, we say that type 
A is favored by selection, or that selection is against type B. Alterna- 
tively, we may say that type A has a greater fitness than B, or B has a 
lower fitness than A. All of these statements, inspite of the different 
wording, are equivalent and mean exactly the same thing. Selection 
and fitness are two words describing the same phenomenon of differ- 


ential reproduction within a given population under a given set of 
environmental conditions. 

Several observations may be made with respect to the definition 
of selection given above. First, if a population consists of only one 
type of organism (either all A or all B), there will be no selection to 
speak of and the statement that A or B is fit or unfit has no meaning. 
Second, selection or fitness is not something that can be determined 
by merely examining the organism itself as is the case in morphology, 
anatomy, and to a large extent, systematics. Rather, it is a description 
of the result of reproductive performance of one type relative to other 
types of the same population. The retrospective definition of fitness 
is quite analogous to that employed by Chinese historians: 
"The victorious is called a king; 
The defeated, a bandit." 

This leads to the third observation, viz., that selection and fitness 
are an overall verdict applying directly to the fact rather than the 
causes of differential reproduction. Thus Wright (15) 1 says: "Selec- 
tion is a wastebasket category that includes. . .such diverse phenome- 
na as differential viability at any stage, dispersal beyond the range 
of interbreeding, differential maturity, differences in mating tenden- 
cies, fecundity, and duration of reproductive capacity." In brief, selec- 
tion may operate through various mechanisms at any stage of the life 
cycle of the organism. Finally, a word of warning may well be 
injected here; in using the terms selection and fitness, we must get 
rid of the connotations of these words in common usage. Selective 
fitness has no necessary connection with physical appearance, vegeta- 
tive growth, market value, or social desirability. 

The definition of selection and fitness given for the two types of 
plants is equally applicable if we identify Type A and Type B as two 
alleles of a locus. "When one allele is reproduced, multiplied, repre- 
sented, or transmitted proportionally more frequently than another 
allele so that the relative frequency of the two alleles in the next 
generation changes, we say that there is selection and that the allele 
whose relative frequency has been increased has a greater fitness. The 
same definition may be extended to genotypes. 

II. Mutation-selection Balance 

Very broadly speaking, there are two kinds of equilibrium in 

'See References, page 4(3. 


natural populations. One is that the balanced condition is maintained 
by the opposing forces between selection and mutation. The other is 
that the equilibrium is the result of conflicting selection effects alone 
and that mutation plays a very little role. In this communication, only 
one or two examples of each kind will be given to illustrate the 
properties of equilibrium. A minimum bibliography is cited from 
which further discussions and other references may be found (1, 
8, 10, 16). 

A. Gametic Selection 

Consider the two alleles A and a which reproduce or transmit 
to the next generation in the ratio \iw, where w is called the (relative) 
fitness of gene a. If iv is smaller than unity, 'we may write w = 1 — s, 
where s is called the selection coefficient against gene a. Suppose that 
the initial frequencies of A and a in a population are p and q. Then, 
after one generation of selection, their frequency ratio will be 
p;q(\—s); that is, new p = pj{\—sq) and new q - q(\—s)/(\—sq). 
The decrease in frequency of a per generation is 

q(\s) -spq 

A q = new q — old q = — q — 

1 — sq 1 — sq 

Gene a will be eliminated from the population if the selection 
continues unopposed. Now, suppose that the rate (or probability) of 
mutation from A to a is u per generation, where u is a small number 
of the order of 10" 5 or 10" G . A mutation may occur at any time and 
at any stage of the life cycle; but for the sake of algebraic simplicity, 
let us assume that mutations occur after the operation of selection. 
Then, among the pj (l—sq) A genes, a small fraction it of them will 
mutate to a per generation. Selection tends to decrease q, but muta- 
tion tends to increase it. These two forces will balance each other so 
that there will be no change in gene frequency when 

spq p u 

1 — sq l—sq 

That is, sq = u, or q = n/s 

This is the simplest type of equilibrium between mutation and 
selection. The equilibrium is stable. That means, if q in any genera- 
tion is greater than u/s, it will decrease until the value of ujs is 
reached. If q is smaller than ujs, it will increase until the value of 


ii I s is readied. As long as the "rules of nature" (it and s) hold, the 
gene frequency will stabilize at the value ujs. Disturbances through 
artificial means can only increase or decrease the value of q temporari- 
ly. When the artificial agent is withdrawn and the population is left 
alone, the gene frequency will gradually restore to ujs. As long as 
there is a finite probability of mutation from A to a, there is really 
no permanent way of getting rid of gene a from a population for any 
appreciable length of time. The condition q = -ujs must be accepted 
as a fact of nature, and by all definitions of normality, it must be 
regarded as the normal condition. 

B. Genotypic Proportions 

Preparing for the discussion of genotypic selection, let us say a 
few words about the genotypic proportions in a population. Consider 
a locus with two alleles A and a, and let [) and q be their respective 
frequencies in the population (/; + q = 1). The proportion of the 
three genotypes A A, Aa, an (Table 1) depends upon the mating system 
being practiced in the population. Continued close inbreeding will 
result in complete homozygosis, and random mating yields the binom- 
ial proportions. The more general situation, especially in plants, is 
probably intermediate between the two extreme cases. The inbreed- 
ing coefficient F is formally defined as the coefficient of correlation 
between uniting gametes (Wright, 1922). 

Consider a plant population in which a fraction 6 is self- 
fertilized and the remaining fraction (1 — 6) is open-crossed (random 
mating) in each generation. It may be shown that after a few genera- 
tions the inbreeding coefficient for such a population is 


F = 

For instance, if 6 = 40 per cent of the plants is self-pollinated in 
each generation, the inbreeding coefficient F will be 0.40/(2.00 — 
0.40) = 0.25. The genotypic proportions in terms of F are given in the 
fourth column of Table 1. Note that such a population may be 
regarded as consisting of two components, viz., (1 — F) random and 
F inbred. 

Mating system determines how often the genes A and a are 
associated into the three types of pairs (genotypes) but does not 
change the frequency of the genes. Thus, in each of the middle three 


Table 1. — Genotypic Proportions in a Population. 

Geno- Random Continued Mating with General 

type mating inbreeding inbreeding coefficient F notation 

AA p 2 p (\-F)p 2 + Fp = p 2 + F pq D 

Aa 2pq {\-F)2p q = 2pq - 2Fpq H 

aa q 2 q (\~F)q 2 + F q = q 2 + Fpq R 

Total 1 1 (\-F) + F = 1 -0 1 

columns of Table 1, h~eq(A) = /; and heq(a) = q, although the geno- 
typic proportions for the three mating systems are quite different. 
If the three genotypic proportions are denoted by D, H, R, whatever 
the mating system, the gene frequencies can always be calculated 
accord i 112 to the relation 

p = D + l/,H, q = \/ 2 H + R 

where D + H + R = 1. Should D + H + R = W for some reason, the 
obvious modification is p = (D + i/ 2 H)/W, and q = (\/ 2 H + R)/W, 
so that /; + q = 1. 

C. Genotypic Selection 

Let W\, zuo, u's denote the selective fitness values of the three geno- 
types whose frequencies in the population are D, H, R, as shown in 
Table 2. The direct meaning of the id's is the probability with which 
the genotypes survive to reproduce; that is, we assume for the sake 
of simplicity that selection operates prior to reproduction. Then it is 
the selected group (that survives to reproduce) that determines the 
gene frequency of the next generation. The general procedure of 
calculating the new gene frequency after selection is given in Table 
2, and the selection effect on gene frequency is A<y = q' — q per gen- 
eration. In this simple model the w's are assumed to be constants and, 
as far as the selection effect on q is concerned, only their relative 
magnitude is relevant. Any other set of three fitness values propor- 
tional to ii'i'.iunitva will yield the same value of Aq. Hence, for prac- 
tical calculation, one of the three w's can always be taken as unity. 

To illustrate the balance between selection and mutation, I will 
mention only two examples: (a) When selection is against a dominant 
mutant gene (Table 2A) and (b) when selection is against the recessive 
genotype (Table 2B). In the former case, the frequency of the domi- 
nant deleterious gene is so low that we may assume homozygous 



Table 2. — General Procedure of Calculating Selection Effect. 





f IV 

New gene frequency 


Aa H 

aa R 

D wi 
H wi 
R Wi 

(Dwi + yiHwi) I w = p' 
(Rw s + l AHwi) I w = q ' 




A. Selection against dominant mutant gene A' 

D 1 D 

[D+ y 2 H(\-s)] I (1-Hs) = p' 
H \-s H(\-s) 

y 2 H(\s) I (l-ifr) = q' 


A A 
A A' 
A' A' 



w = 1 - Hs 


B. Selection against the recessive genotype 

AA D 1 D 

(D + l AH) / (1 - Rs) = p' 
Aa H 1 H 

(R-Rs + yH)/(\ -Rs) = q' 
aa R \-s R{\~s) 



w = 1 - Rs 


dominants are nonexistent in the population, so that D + H = 1 
and q = \/ 2 H is the frequency of the dominant mutant gene. The 
change in gene frequency due to selection is &q = q' — q = —s q 
(1 —H)l(\ —Hs) - —s q (\—H). Again, if u is the mutation rate from 
the type gene A to dominant mutant A', there will be up' = u 
(1 — H + i/ 2 H (1 +s)) new mutations. Equating the loss due to selection 
and the gain due to new mutation, we obtain the approximate equi- 
librium condition that could be written in various forms as follows: 
sq = u, q = l/ 2 H = u/s, H = 2nfs, u = \/ 2 Hs. 
When the mutant is dominant in its deleterious effect, the bal- 
anced condition q = u/s is of the same form as that of gametic 

Table 2B oives the result of selection against the recessives. 
The change in freq(?/) in one generation is 

q — Rs 

Af/ = q' — q = — q — — s [) R/zT> 

1 - Rs 


Equating this loss to the gain through mutation, viz., p u id, we 
obtain the equilibrium condition (in terms of the recessive propor- 
tion in population) 

sR = u, or R = u/s. 

In populations with inbreeding, 

-F + ^/F^+4(\-F)uTs 
R = (\-F)q 2 + F q = ujs, q = ' 

2(1 -F) 
In populations without inbreeding, 

R = q* = u/s, q = V^A* 
The equilibrium value of q with inbreeding is always lower than 
that without inbreeding. But, with mutation rate and selection 
coefficient fixed, the recessive proportion in the population is the 
same, with or without inbreeding. The equilibrium condition 
R = u/s is stable. 

III. Selectional Balance 
I). General Formula 

The second broad category of equilibrium is that maintained 
by selectional forces alone, without introducing new mutations into 
the population each generation. Referring to the general procedure 
outlined in Table 2, we see that equilibrium will be achieved when 
the new gene frequencies ({/ and q') after selection are equal to the 
old ones (p and q) before selection. Substituting D = p 2 + Fpq, etc. 
(Table 1) in the equation q' — q and simplifying, we obtain the 
equilibrium condition (11) 

(1 - F) (u'o - u'x) + F(w 3 - a',) 
q = 

(1 - F) [( Wa - Wi) + («' 2 - W B )] 
That is, when q assumes the value indicated above, the differen- 
tial selective fitness of the genotypes does not change the gene fre- 
quencies. In other words, the population remains the same inspite 
of the selective operation. Note that when Wx — iv%, the equilibrium 
condition is q = i/ 2 , with or without inbreeding. 

In order to make q a positive fraction in the expression above, the 
quantities iu 2 — u'i and w 2 — u'3 must be both positive (w 2 >ivi and iu 2 > 
w 3 ) or both negative and the inbreeding coefficient F not too high (see 



below). In other words, the equilibrium is possible only when the 
fitness of the heterozygote, w 2 , is greater or smaller than those of both 
homozygotes. One way of representing the former situation is as 

w»i = i-/,| 

( 10'j— It'i = t, j 
\ ?{'•»— Zt'3 = S, 

zv 3 = 1 —s> f 
Substitution of these values in the general expression for ecjiii- 
librium yields 

t—Fs s - F t 

q = - — , /> = - 

(\-F)(s+t) (\ -F)(s + t) 

where F must be smaller than t js and s/l, one of them being a frac- 
tion. To facilitate the appreciation of this type of selectional balance, 
a numerical illustration is provided in Table 3. Even without the 
details of mathematics, it is quite obvious that this equilibrium is 
stable. Since Aa has the greatest fitness, neither oene A nor gene a can 
be eliminated from the population. Furthermore, if freq(/l) or freq(fl) 
is too low, the greater fitness of Aa tends to raise it to a certain level 
and vice versa. The greater fitness of Aa and the loss of A A and aa are 
balanced in such a way that the gene frequency remains unchanged. 

It was mentioned previously that any set of three numbers pro- 
portional to w 1 :w 2 :w :i will yield the same result. Since 80:100:70 = 
100:125:87.5, the latter three numbers have been used in the lower 
portion of Table 3. The results are the same, of course. 

On the other hand, if the heterozygote fitness value is lower than 
that of either homozygote, the equilibrium is unstable. This, again, 
should be obvious. The elimination of a certain number of Aa from 
the population implies equal loss of the number of A and a genes. 
This inflicts proportionally a greater loss of the gene whose frequency 
is already lower than that required for equilibrium, and hence its 
frequency Avill decrease further. The frequency of the more common 
gene will, in turn, increase. Consequently, selection against heterozy- 
gotes will lead to near elimination of one of the alleles (to be kept at 
a very low level by new mutations). Unstable equilibria are not 
expected to exist in nature. 


Table 3. — Selectional Balance when the Heterozvgote has a Greater Fitness 
Value than Homozvgotes. * 

Genotype Frequency, / w j w New gene frequency 

AA 0.4225 + 0.0455 = 0.4680 0.80 0.3744 

Aa 0.4550 -0.0910=0.3640 1.00 0.3640 

aa 0.1225+0.0455=0.1680 0.70 0.1176 

0.5564/0.8560 = 0.65 
0.2996/0.8560 = 0.35 

Total 1.0000+0 =1.0000 w = 0.8560 1.00 

Another system of assigning fitness values 
A A p* + Fpj =0.4680 1.000 0.4680 

Aa 2pq-2Fpq =0.3640 1.250 0.4550 

aa q 2 + Fpq = 0.1580 0.875 0.1470 

0.6955/1.07 = 0.65 
0.3745/1.07 = 0.35 

Total 1+0 = 1.0000 iv = 1.0700 1.00 

*F = 0.20; t = 0.20; s = 0.30; p — 0.65; q = 0.35. 

E. Variable Fitness 

In all previous sections the value of the w's is assumed to be con- 
stant. This may be true for certain traits affecting reproduction and 
may not be so for others. It is quite possible that the fitness depends 
not only on the genotype itself, but also, possibly to a large extent, 
upon the genetic composition of the population as a whole. Some of 
the simplest examples of this nature are given in Table 4, in which 
the fitness value of a phenotype varies with the phenotypic frequency 
in the population. In part A it is assumed that selective advantage or 
disadvantage of one phenotype is proportional to the frequency of the 
other phenotype. Thus, in the first example, when recessives are rare 
in the population (R small and D + H = 1 — R large), the recessive 
fitness becomes large, being 1 + s(\—R), and hence the recessive pro- 
portion will increase. The same is true with the dominants. This type 
of selection will lead to a stable equilibrium. Conversely, if as R 
becomes small, the recessive fitness also becomes lower, 1 — s(D + H), 
the equilibrium is unstable and one of the alleles will be reduced to 
near extinction. The fitness of a phenotype in part B varies with fre- 
quency of its own phenotype. 

The equilibrium condition, when fitness varies with gene fre- 


Table 4.- Selectional Balance when Fitness Varies with Genotvpic Frequencies. 

Type Frequency, Advantage from Disadvantage from 

/ encounter, w encounter, iv 

A. Advantage or disadvantage proportional to/ of unlike phenotype 
Dominant D + H 1 +/ R 1 - / R 

Recessive R 1 + s(D + H) 1 - s(D + H) 

Equilibrium Stable Unstable 

B. Advantage or disadvantage proportional to/ of own phenotype 
Dominant D + H 1 + t(H + D) 1 - t(H + D) 

Recessive R \ -\- s R 1 — s R 

Equilibrium Unstable Stable 

quency, may be found either through the general procedure outlined 
previously (Table 2), or by the method of Wright (15) and Lewontin 
(9). In such simple cases as listed in Table 4, however, an expeditious 
short-cut may be used. The phenotypic frequency and the selective 
fitness should be so balanced that the population remains unchanged. 
This condition obtains when the fitness values of the two phenotypes 
are equal, thus 


for cases of Table 4A, / R = s(\-R), R = 


for cases of Table 4B, s R = t(\—R), R = 

where R = q 2 + Fpq with inbreeding, and R = q- without inbreeding. 
The stability of these equilibrium values has been indicated in Table 
4. The general principle which governs a large number of similar 
cases is 

selective advantage when abundant > unstable equilibrium 

selective advantage when rare > stable equilibrium 

This principle is not only important in studying selection within 
a population, but it is also important in ecological studies which deal 
with the equilibrium between different populations. 

F. Metrical Trait 

Consider a metrical trait (say, size) whose measurement is x. 


Probably in most cases, if not in all, natural selection favors not the 
extremely large or the extremely small, but some intermediate value. 
This value may then be called the optimum size with respect to fitness 
and be denoted by x . In any realistic situation in which the metrical 
trait is controlled by a number of loci, the optimum size x is prob- 
ably close to but does not necessarily coincide with the mean size 
x of the population. 

One way of representing the fitness that decreases with large as 
well as with small sizes is to let 

w = 1— c(x— X'o) 2 
where c is a constant. That is, the selection coefficient asrainst an 
organism of size x is proportional to the square of the deviation of x 
from optimum value. Suppose that the size of the three genotypes 
(A A, Aa, aa) are Xi = 11, .\*« =10, ,v 3 = 1, respectively, and that the 
optimum size is x = 8. Then the fitness of the three genotypes will be 
(taking c = 0.01) 

w 1 = 1 -c(ll -8) 2 = 0.91 
u , 2 = l — t (10 — 8) 2 = 0.96 
i Us = 1 - c ( 1 -8) 2 = 0.51 

Such a set of fitness values will lead to a stable equilibrium, as 
shown in section 4. If the optimum size is .v — 4, the three fitness 
values would be 0.51, 0.64, 0.91, leading to fixation of the small size. 

For a metrical trait determined by a number of loci, the situation 
is very complicated and selection for an optimum size does not always 
lead to stable equilibrium. Wright (14) found no stable equilibrium 
when there is no dominance or complete dominance with respect to 
size at all loci. 

Kojima (6, 7) recently showed that stable equilibrium points do 
exist when there is partial dominance or overdominance in size at all 
loci, and proposed a method of locating such stable points. In nature 
there must be a large number of metrical traits stabilized at certain 
optimum levels. It is unfortunate that the mathematics involved in 
this type of investigation should be so intricate that the most impor- 
tant type of selection in nature has been also the least understood. 
Kojima's finding must be hailed as a significant landmark in selection 

In all of the previous examples there is only one equilibrium 


point for a population under a given selection scheme. Tn more 
complicated situations, however, such as that involving multiple 
alleles, multiple loci, local differential selection scheme, differential 
selection in sexes, mutations of all directions, etc., there may be more 
than one stable equilibrium value at which the population may be 
stabilized. The selection scheme may change from time to time and 
this fluctuation may also keep a population in equilibrium (2). 

IV. Comparison of Population Fitness 

The selection effect that arises from the differential reproduction 
or transmission of different alleles or genotypes within a population, 
though quite involved at times, is at least in principle easy to com- 
prehend. It involves only the relative fitness of the genotypes con- 
cerned, and the problem of population growth, either in absolute 
size or in comparison with other similar populations, has not been 
taken into consideration. When we try to compare two or more popu- 
lations, difficulties arise because there does not exist a unique cri- 
terion (or scale) by which different populations may be judged. 

To illustrate the difficulty of comparing two populations, let us 
once more consider the mutation-selection equilibrium condition 
Rs = u, or R = ujs. The value of w = 1 — Rs at the bottom of Table 
2B is the total of the selected individuals, but it may also be viewed 
as the average fitness of the three genotypes in the population (average 
fitness of the population in short), with the understanding that Wi 
and w 2 are taken to be unity. Thus, Rs is the amount of recessive 
individuals to be eliminated by selection in each generation, although 
the same amount will be replaced through segregation of the old 
and new mutations so that there is no net change in the composition 
of the population. Mutation rate and selection intensity are param- 
eters of nature, and have been taken as constants in our models; that 
is, for short range genetical purposes. (They may vary widely in the 
history of evolution.) Some hypothetical equilibrium populations are 
listed in Table 5. The average fitness at equilibrium is 

W—]—sR=\ —s(u/s) = 1 — u, 
independent of selection intensity. This is also true for several other 
cases (5). 

Tn populations 1-3, mutation rate varies proportionally to 
selection intensity, so that the recessive proportion R remains the same 


Table 5. — Equilibrium Condition when Selection is Against the Recessives.* 
Population 1 Population 2 Population 3 

Parameters in nature u =0.000,018 u =0.000,045 u =0.000,090 

j = 0.02 s = 0.05 s = 0.10 

Recessive proportion R = 0.0009 R = 0.0009 R = 0.0009 

Average fitness w = 0.999,982 w = 0.999,955 w = 0.999,910 

Population 4 Population 5 Population 6 

Parameters in nature u = 0.000.050 u = 0.000,050 u = 0.000,050 

s = 0.02 5 = 0.05 j = 0.10 o 

Recessive proportion R = 0.0025 R = 0.0010 R = 0.0005 

Average fitness w = 0.999,950 w = 0.999,950 w = 0.999,950 

*wi = l — t; u>2 = I; a>8 = 1 — s. 

in all three populations. This means, the genetic composition of these 
three populations are the same at equilibrium. The average fitness of 
the populations, however, is decreasing from 1 to 3. In populations 
4-6, the mutation rate and consequently the average fitness of the 
population is the same, but the recessive proportion R, and thus the 
recessive gene frequency q, decreases from 4 to 6. 

How do we compare these populations? What criterion should 
be adopted? What is the value judgment involved here? In some 
discussions it is implied that a "good" population should have a low 
frequency of deleterious genes. This seems to suggest that the value 
of R should be used to compare different populations. If so, then 
populations 1, 2, 3 are considered to be equally good and 4 is worse 
than 5 which in turn is worse than 6. In other discussions the average 
fitness has been adopted as the criterion for judging populations on 
the theory that w shows the deviation of the actual population from 
an hypothetical one in which there is no deleterious mutation nor 
selection (all AA, vo x — w = 1). From this viewpoint, populations 
4-6 are equally good, but 1 is better than 2 which in turn is better 
than 3. 

The difference between these two criteria is essentially this: R is 
static in nature, describing the genetic composition of a population, 
while sR = \—w is dynamic, indicating the amount of selectional 
turnover in each generation. As an analogy, we may consider two 


gamblers A and B. The former likes to play big stakes and wins or 
loses a large sum in each bet but comes out even at the end of the 
game; the latter plays cautiously and wins or loses a small sum in 
each bet but also comes out exactly even at the end of the game. Who, 
then, is a better gambler? When they do lose, A loses a total of $50 
in two hands and B loses the same amount in ten hands, but both 
of them recover their losses before the end of the game so that there 
is no change in wealth distribution. Who shall Ave say is a better 

Comparing two populations in selectional balance involves more 
or le§s the same difficulty. For the sake of simplicity, let us assume 
F = 0, so that the equilibrium condition is, according to section 4, 

s t 

f) = and q = 

s+t s+t 

and the average fitness is 


w = 1 — tp 2 — sq 2 = 1 — 

s + t 

In Table 6 are listed some examples of equilibrium populations 
of this type. It is seen that populations 1 and 2 have the same genetic 
composition but different average fitness, while populations 3 and 4 
have different genetic composition but the same average fitness. Since 
in the case of selectional balance neither allele A nor allele a can be 
strictly considered deleterious, it may be easier to use w as an index 
which, as before, measures the amount of selection g-oinsf on in the 
population, although the selection produces no change on the genetic 
composition of the population at equilibrium. 

Finally, it should be pointed out that although w, based on rela- 
tive fitness of genotypes, measures the amount of selectional turnover 
per generation, it is not useful as an index for the absolute surviving 
ability of a population. If two populations, one with w = 0.94 and one 
with id — 0.64, are grown side by side in the same environmental con- 
ditions, there is no way to foretell which population will win out. 
The genetics of intrapopulation selection deal only with the genetic 
composition of a population and give no information as to inter- 
population competing abilities. The latter more properly belongs to 
the realm of ecology, to be studied by field naturalists and geneticists. 
In plants there are instances in which only heterozygotes survive, 


Table 6. — Equilibrium Condition when Selection Favors Heterozygotes. * 

Population 1 

Population 2 

Parameters in nature 

Gene frequency 
Genotype proportion 

wi = 0.90 

w 2 = 1.00 

w s = 0.85 

t = 0.10 

s = 0.15 

p = 0.60, q = 0.40 
0.36, 0.48, 0.16 

wi = 0.40 

/ = 0.60 

w 2 = 1.00 

} s = 0.90 
w 3 = 0.10 

p = 0.60, q = 0.40 
0.36, 0.48, 0.16 

Average fitness 

0.015 0.54 

0, = 1 = l - 0.06 w = 1 - - - = 1 - 0.36 

0.250 1.50 

Population 3 

Population 4 

Parameters in nature 

Gene frequency 
Genotype proportion 

wi = 0.92 

t = 0.08 

w 2 =1.00 

} s = 0.10 
iv 3 = 0.90 

p = 5/9, ? = 4/9 
25/81, 40/81, 16/81 

ivi = 0.95 

/ = 0.05 

w, = 1.00 

} s = 0.40 
w 3 = 0.60 

p = 8/9, 7 = 1/9 
64/81, 16/81, 1/81 

Average fitness 

0.008 0.02 

= 1 = 1 - 0.044 iv = 1 - = 1 - 0.044 

0.180 0.45 

*wi =1 — t; wz = 1; wa = 1 — 5. 

e.g., Oenothera; that is, w^ = w 3 = 0. The average fitness in such a 
case is very lo^v, meaning that there is a great deal of selection going 
on, but the plant population may nevertheless thrive. 

Summary and Conclusion 

Two major types of genetic equilibrium have been discussed. 

I. The balance between recurrent mutation and persistent 
selection. In this case, the gene frequency is always kept at a very low 
level, ranging from the order \/u to u, where u is the mutation rate 
to the gene under consideration. The small immediate harmful effect 
on the population as a whole must be viewed as the price the popula- 
tion has to pay for preserving the gene for possible further use in the 
course of evolution. Since mutations, the ultimate source of genetic 


variability, occur in nature, a population containing a small amount 
of mutants must be defined as the "normal" state of affairs. 

II. The balance maintained by selection scheme alone. Muta- 
tion plays a very little role in determining an equilibrium value of 
this type, because selection intensity is so much greater than mutation 
rate. The gene frequencies involved in this type of equilibrium are 
usually intermediate in value and thus result in genetic polymorph- 
ism. Although greater fitness of heterozygotes is the simplest and the 
most familiar mechanism by which selectional equilibrium may be 
attained, it is not a necessary condition in more complicated 
situations. A vast number of combinations of various factors may 
lead to stable selectional balance. Probably most of the common 
genetic characteristics, especially quantitative traits, are controlled 
by selection rather than by mutation. 

Only very simple examples have been chosen to illustrate the 
various points. The difficulty of comparing fitness of two different 
populations has been discussed. Populations with the same average 
fitness may have quite different selection schemes and different 
genetic compositions. More detailed discussions may be found else- 
where (3, 4, 12, 13, and 17). 


1. Crow, f. F. 1948. Alternative hypotheses of hybrid vigor. Genetics, 

33: 477-187. 

2. Dempster, E. R. 1955. Maintenance of genetic heterogeneity. Cold 

Spr. Harb. Symp. Quant. Biol., 20: 25-32. 

3. Dobzhansky, Th. 1957. Genetic loads in natural populations. Science, 

126: 191-194. 

4. , Krimbas, C, and Krimbas, M. G. 1960. Genetics 

of natural populations, XXIX: Is the genetic load in Drosopliila 
pseudoobscura a mutational or a balanced load? Genetics, 45: 741- 

5. Haldane, J. B. S. 1937. The effect of variation on fitness. Amer. Nat., 


6. Kojima, K. I. 1959 A. Role of epistasis and overdominance in sta- 

bility of equilibria with selection. Proc. Nat. Acad. Sci., 45: 984- 

7. . 1959. B. Stable equilibria for the optimum model. 

Proc. Nat. Acad. Sci., 45: 989-993. 


8. Levene, H. 1953. Genetic equilibrium when more than one eco- 

logical niche is available. Amer. Nat.j 87: 331-333. 

9. Lewontin, R. C. 1958. A general method for investigating the equi- 

librium of gene frequency in a population. Genetics, 43: 419-434. 

10. Li, C. C. 1955 A. Population Genetics. Univ. Chicago Press. 

11. . 1955 B. The stability of an equilibrium and the average 

fitness of a population. Amer. Nat., 89: 281-295. 

12. Wallace, B. 1959 A. The role of heterozygosity in Drosophila popu- 

lations. Proc. 10th Intern. Congr. Genet., 1: 408-119. 

13. . 1959. B. Studies of the relative fitness of experimental 

populations of Drosophila melanogaster. Amer. Nat., 93: 295- 

14. Wright, S. 1935. Evolution in populations in approximate equilib- 

rium. Jour. Genet., 30: 257-266. 

15. . 1955. Classification of the factors of evolution. Cold Spr. 

Harb. Symp. Quant. Biol., 20: 16-24. 

16. . 1956. Modes of selection. Amer. Nat., 90: 5-24. 

17. . 1959. Physiological genetics, ecology of populations, and 

natural selection. Perspectives in Biol. & Med., 3: 107-151. 


Auerbach: It seems to me that the examples discussed under I and II 
are not at all equivalent. In I, the equilibrium refers to a pair of alleles, 
in II to whole genomes, as in Oenothera, or whole chromosomes, as in 
Dobzhansky's Drosophila inversions. The balance in Oenothera is upheld 
by genes which are not allelic. This difference between equilibrium 
for alleles or for larger parts of the genome may not be important for 
the way a population achieves equilibrium in nature, but it is the 
important question in the assessment of genetical radiation damage. As 
far as I know, there are few examples for selectional equilibrium at 
the allele-level, the only level which has to be considered in this regard. 

Discussion of Session I 


Indiana University, liloomington, hid. 

At a symposium concerned with mutation and plant breeding it 
l would seem appropriate to consider briefly just what is meant 
by the term mutation and to describe some of the inherited changes 
in chromosomal organization which lead to modifications of the 
normal phenotype and hence are called mutations. 

Many hold that mutations, either of spontaneous or induced 
origin, stem in large part from some kind of intragenic change at the 
molecular level, i.e., they are true or intragenic mutations. Inasmuch 
as the genetic information is determined by the combinations of the 
two pyrimidines and the two purines of DNA, it is believed that the 
substitution of one purine or of one pyrimidine for another, or of a 
purine for a pyrimidine and vice versa, would produce a new kind of 
genetic code which would lead in some cases to an altered phenotype. 
A change in gene action producing a mutant phenotype could also 
conceivably occur by an inversion in base order or by a deletion or 
duplication of one or more base pairs. That mutations of these kinds 
do in fact occur appears highly likely from the studies by Freese and 
Benzer on the mutagenic effect of base analogues and other chemical 
mutagens in phage. Some mutagens are thought to induce mutations 
in duplicating DNA and others in non-duplicating DNA. Comparable 
though less extensive results have been obtained with bacteria. 
Muller, Carlson, and Schalet suggest that a rotational substitution, 
whereby the two organic bases of a nucleotide pair become freed from 
the backbone and reversed in position upon reattachment, may be 
responsible for the whole body mutations in the non-replicating DNA 
of Drosophila sperm. 

Granting that intragenic mutations occur in phage and bacteria 
there is virtually no convincing evidence for intragenic changes in 
mutation studies of higher forms. This does not mean that intragenic 
changes do not occur in these organisms, and indeed on theoretical 
grounds it is difficult to deny that they do arise, but only that there are 
a number of extragenic events which simulate gene mutation. In prac- 
tice those mutations which cannot be ascribed to one or another 
extragenic mechanism are tentatively placed in the category of gene 



mutation. However, as Stadler points out, the mutations labeled as 
intragenic constitute a residual class at present unascribable to an 
extragenic event. As more sensitive and discriminating techniques 
have been developed the proportion of putative intragenic mutations 
becomes progressively smaller. The residual class remains suspect 
since no certain criteria exist which permit an unequivocal proof of 
true gene mutation. It has been suggested that the frequency of intra- 
genic changes is so low that they escape detection by the investigator 
who, at least in working with higher plants, usually restricts his 
attention to those cases where the mutation rate is high enough to 
afford an adequate number of changes for further analysis. A true or 
intragenic mutation rate of 1 X 10~ 5 for a single locus, which may be 
typical for many genes, would require a vast amount of effort to obtain 
an adequate sample of mutations. An exception would be the S alleles 
for incompatibility where the screening technique for mutations is as 
efficient as those employed for microorganisms. . . 

Different extragenic events which simulate gene mutation are 
as follows: 

1. Minute deficiencies. 

2. Position effects. 

3. Recombination between different mutant sites within a cis- 
tron or functional unit which yields a crossover strand with no 
affected sites and one with two mutant sites. 

4. The separation and isolation of the components of a complex 
locus by some kind of crossover mechanism. Mutations of this type 
have been described in Laughnan's paper at this symposium. Mecha- 
nisms 3 and 4 are associated with meiosis. 

5. Restoration of gene action through the loss of an adjacent 
inhibitor. Conversely, normal gene action may be modified when an 
inhibitor is placed next to a normal allele. One of the best examples 
comes from McClintock's studies on the Ds-Ac mutator system in 
maize where an apparent mutation of C— >c resulted from the inhibi- 
tory effect of Ds on the C allele when the two were brought into 
juxtaposition by transposition. Recovery of C action followed the loss 
of Ds. This occurs only when Ac is present in the nucleus. 

I am uncertain where to place the phenomenon called "gene 
conversion" because of the diverse opinions of the causal mechanisms 
and because it may consist of a heterogeneous class of changes. How- 


ever, there is no compelling reason to ascribe cases of gene conversion 
to intragenic changes since extragenic mechanisms are capable of 
producing the observed results. Whatever may be the cause of the 
intriguing cases of paramutation in maize described by Brink and his 
associates and by Coe, there is no evidence in these studies of 
intragenic modifications. 

It should be emphasized that in none of the many carefully con- 
ducted and extensive mutational investigations in maize is there 
unassailable proof of true gene mutation. In all well-analyzed cases an 
extragenic mechanism has either been demonstrated to be responsible 
for the mutant change or else one appears to be highly probable. 
Insofar as the mutation spectrum is concerned, it would be surprising 
if a comparable situation does not exist in other higher plants. Muta- 
tional studies are of concern not only to the geneticist but also to the 
plant breeder who utilizes mutations as a source of variation and who 
needs to be aware of the diverse mechanisms which simulate true 
oene mutation. 



Kramer: Since the waxy gene in corn produces pollen grains whose 
starch stains brown rather than blue with iodine, Dr. O. E. Nelson, 
by looking at stained pollen from Y 1 plants of crosses between tux stocks 
derived from independent reoccurrences of tux mutations, has been 
able to identify recombinants between different tux mutants by the rare 
occurrences of blue staining grains. A number of mutants have been 
placed in linear order within the waxy locus by this method. The large 
populations of pollen grains which can be screened, make this locus an 
ideal one and comparable to phage for genetic fine structure studies. 

Caspar: The latest chromosome theory, based on the information provided 
from electron microscope pictures of chromosomes and theoretical molecu- 
lar biology, states that there are 32 to 64 strands of DNA in the chromo- 
some of higher plants and animals. The theory holds that "gene" 
mutation is due to rearrangements in the base pair configurations of the 
DNA. A mutation is expressed when the chromosomes of a cell all carry 
the same mutated base pair configuration. It would seem that a chromo- 
some would have to go through at least 16 reduplications before this could 

How, then, does the investigator, testing for induced mutations in 
the higher plants and animals, score, let alone find, mutations which are 
due to induced changes in the base pair configurations? 


Rhoaoes: If chromosomes are multi-stranded it is, of course, difficult to 
understand how a mutational event consisting, for example, of a base 
substitution coidd occur simultaneously in all of the component strands 
and thus be immediately expressed. However, the genetic evidence for 
whole-body mutations occurring in gametes cannot be questioned. This 
has been interpreted as indicating that the chromosome of higher organ- 
isms consists of a single double helix of different DNA molecules which 
are linearly arranged and possibly connected by protein links. If the 
genetic data are deemed more reliable than the cytological studies, then 
one favors the latter concept of chromosome structure. This conflict has 
not been resolved. 

Caspar: With respect to the remark that corn does not respond in the 
same way as other organisms in radiation experiments, I should like to 
state that in some preliminary data of ours from an experiment with corn 
similar in design to the ones the Russells have done with mice in which 
the male gametes carrying dominant marker genes are radiated at different 
stages of gametogenesis and crossed on multiple recessive females, our 
results are quite similar. We find that the controls and material radiated 
after meiosis consist of losses of adjacent marker genes and most all the 
mutants also carry associated sterility effects, while the mutants from 
material radiated prior to meiosis consist of single gene losses and the 
mutants do not carry associated sterility effects. 

Session II 

Mutagenic Agents and 
Interpretation of 
Their Effects 

W. R. Singleton, Chairman 
University of Virginia, 
Charlottesville, Va. 

Types of Ionizing Radiation and Their 
Cytogenetic Effects 1 


llrooktiaven National Laboratory, Upton, Xew York 

Physicists now comprehend not only the structure of stars, the 
motion of our own and other galaxies, the curvature of space, 
the possible ways in which our universe has evolved, but also matter 
on a finer and finer scale: from familiar objects to molecules; then to 
the atoms of which the molecules are composed; the internal struc- 
ture of the atom with its electrons orbiting around nuclei; the nucleus 
itself, made up of protons and neutrons and the mesons which bind 
them together; and lately even something of a picture of the inside of 
the proton itself, complex and containing yet other particles. We are 
peeling an onion layer by layer, each layer uncovering in a sense 
another universe; unexpected, complicated, and — as we understand 
more — strangely beautiful." 

So begins a memorandum prepared for former President Eisen- 
hower by a Special Advisory Panel on High Energy Accelerator 
Physics (120). 3 In a similar manner, geneticists are probing at the ulti- 
mate secrets of life or the fine structure of the chromosome, the gene, 
and of their nucleoprotein components. It is of considerable interest 
that the use by geneticists of the knowledge and tools of the atomic 
and nuclear physicists is making a significant contribution to our new 
knowledge of genetic fine structure and also, we hope, to the useful 
application of radiobiological techniques in plant breeding. 

It is my assignment to try to outline the physical nature of the 
ionizing radiations of most interest to the geneticist, to explain some- 
thing of their interaction with matter, i.e., the process of energy 
transfer from the radiation into the atoms or molecules, and to survey 
briefly some of the biological effects produced by the complex series 

a Research carried out at Brookhaven National Laboratory, Upton, N. Y„ under the 
auspices of the U. S. Atomic Energy Commission. 

2 The author gratefully acknowledges the many helpful suggestions regarding the 
manuscript offered by Doctors H. J. Evans and Rae P. Mericle and by Miss Virginia 
Pond and Mrs. Rhoda Sparrow. 

3 See References, page 105. 



of changes initiated when ionizing radiations impinge upon living 
cells. Partly because of the space limitations and partly because of 
the large numbers of more detailed considerations of this topic already 
published elsewhere, this review will be brief and aimed primarily 
at readers looking for a fairly short and elementary survey of the 
subject. In the sections concerned with radiation effects, I shall 
emphasize chromosome breakage and derived phenomena rather than 
intragenic changes, partly because of my greater personal interest 
in this topic and partly because induced mutation in the more 
restricted sense is being covered in this Symposium by others who 
are better qualified to discuss it than I am. 

I. Ionizing Radiations and Their Characteristics 4 

By definition, ionizing radiations have the ability to produce 
ionization (ion pairs) when they interact with matter. With the 
removal of each electron a positively charged atom or molecule is 
left. In addition to the process of ionization, energy transfer also 
occurs by a process known as excitation. The major effect of the 
ionizing radiations is considered to result from their ability to ionize 
and to rupture chemical bonds. In contrast to the ionizing radiations, 
ultraviolet, except for the very shortest wave lengths, does not have 
the capacity to ionize but transfers energy primarily through the 
process of excitation. The fact that ultraviolet radiation does produce 
genetic effects (both mutation and chromosome breakage) is adequate 
evidence that excitation itself can produce a biological effect. 

Ionizing radiations include two different types: (a) electro- 
magnetic radiations which include X and gamma rays, and (b) the 
so-called particulate radiations (alpha, beta, protons, deuterons, etc.). 
Neutrons are generally classed as ionizing radiations, but it should 
be pointed out that they do not ionize directly but indirectly through 
the nuclear reactions which occur following their absorption by 
atomic nuclei. After absorption they may emit a, proton, electron, or 
7 rays, or disintegrate. 

Cosmic radiation is not of any serious interest to the plant 
breeder so far as artificially produced mutations are concerned but no 
doubt plays a part in the production of so-called spontaneous muta- 

4 For definitions of physical terms used, see Glossary of Terms in Nuclear Science (111). 

sparrow: cytogenetic effects of ionizing radiations 57 

tions. The cosmic rays are really a complex of radiations which are 
derived from interactions of very highly energetic particles. The less 
energetic particles interact with the atmospheric atoms and various 
reactions occur, including the production of mesons which in turn 
decay with the production of electrons, positrons, gamma rays, sec- 
ondary pair production, and the associated showers or avalanches of 
low-energy electrons. The amount of cosmic radiation varies with the 
altitude and with the position on earth with respect to the magnetic 
poles (112). 

With the rapid developments in space science, it is now possible 
to send seeds or spores into outer space and recover them. Since labo- 
ratory experiments using man-made radiations similar in nature to 
(but perhaps less energetic than) the particles from outer space are 
now under way, it will be possible to test the biological effects of both 
cosmic radiation and other ionizing; radiations in the Van Allen belt. 
Reports of such experiments will appear shortly in the literature and 
should show significant effects if the predicted doses (up to 3 X 10 4 r 
per hour) really exist (112). 

The characteristics of the various ionizing radiations are of con- 
siderable interest to physicists and some of their properties should be 
understood by radiobiologists. A partial list of the ionizing radiations 
and some of their more important properties are summarized in 
Table 1. This table may appear, at first sight, to be unnecessarily 
complicated. However, the minimum amount of information is given 
which, in my opinion, will allow the average biologist to use these 
radiations with some understanding of their practicality and the 
reason for differences in their relative biological effectiveness. 

It should be clearly understood that ionizing radiation generally 
produces an effect proportional to the energy absorbed in the tissue 
in question. The fraction of energy which passes right through a cell 
or tissue produces no effect nor does the energy absorbed in the air or 
medium surrounding the object in question (except for very small 
structures). Thus, in general terms, it is obvious that a radiation 
must have sufficient energy to penetrate to the position where its effect 
is sought and not sufficiently penetrating that most of it passes right 
through. The penetration of the different radiations is thus a major 
factor in deciding which radiations should be used in many experi- 
ments, and it also has an important bearing on their relative hazards. 



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The relative penetrations of several different radiations in various 
kinds of organic materials are indicated in Figure 1. Penetration into 
tissue depends upon density of the tissue, but is generally roughly 
comparable to that shown in Figure 1. 



12 3 

Energy, Mev 

Figure 1. — Range and half value thickness (H.V.T.) of various ionizing 
radiations or particles in (CH 2 ) n . Range or H.V.T. would be roughly 
the same in plant tissues of similar density. After Sun (182). 

In addition to penetration one should know the pattern of dis- 
tribution of absorbed energy in the irradiated object (131). The dose 

sparrow: cytogenetic effects of ionizing radiations 


at a particular position below the surface is called the depth dose 
and is determined by a large number of factors, including such things 
as kind and energy of radiation (Figure 2), size of the area irradiated, 
chemical composition and density of the specimen or tissue, collima- 






^ ¥0 


^ ^\ 

•/5-vMeV electi 

•'ons J I 

190 MeV deuterons 




5 10 

Depth in plastic 

Figure 2. — Comparison of the depth dose effect betzueen deuterons and 
electrons. Curve taken from Tobias, Anger, and Lawrence (1S9). These 
curves were determined experimentally, using a plastic having an absorp- 
tion coefficient similar to tissue. 

tion of the beam, etc. These factors are generally thoroughly discussed 
in books on radiological physics or radiation therapy (10, 51, 52, 76, 
113, for example). These should be consulted for further detail. 

Another important characteristic is the relative density of ioni- 
zations along the path of the ionizing particle. (See Figure 14.) As 
can be seen from Table 2, this varies widely for the different radi- 
ations, beginning at a minimum of approximately 8 ionizations per 


Table 2.- Ion Density Produced by Different Ionizing Radiations or Particles.* 


Mode of generation 

Rate of 
Mean linear transfer Ionizing 

ion density! of energy particle 

Very high energy j3 20-30 million volt betatron 
and 7 radiation radioactive elements 

X radiation 

"Supervoltage" 1,000 kv 
"Deep therapy" 200 kv 
From copper (K) 8 kev 
From Al (K) 1.5 kev 

J Cyclotrons at 12 Mev 
Neutron radiation J Cyclotrons at 8 Mev 
] 400 kv deuterium ions 
bombarding deuterium 

a radiation 

Atomic rays 

[Natural disintegration of 
i radon 
^Slow neutrons 

Uranium fission 






















a particle 



*After Gray (56). 

tlons per micron of tissue. 

micron and going up to 130,000 for very dense tracks made by urani- 
um fission fragments. Since each ionization is estimated to require 
32.5 ev, it is apparent that the denser the ionizations along the track 
the higher the rate of energy transfer from the ionizing particles to 
the atoms or molecules of the tissue. This concept known as linear 
energy transfer (LET) is defined as the energy released by the radi- 
ation per unit length of track in the absorbing material. It is usually 
expressed in units of kev/n and the LET varies directly with the 
square of the charge and inversely as the square of the velocity (or 
energy) of the particles. The charge on an ionizing particle does not 
change along its path, but as velocity (energy) remaining is gradually 
reduced, the frequency of ionization increases up to a maximum. The 
rate of energy loss for electrons in water is shown in Figure 3. The 
denser parts of the tracks are known as tails and are the most effective 






8 10 12 14 16 


i i i i 



1 2 3 4 5 6 7 


Figure 3. — Rate of energy loss for electrons in water. After Robertson 
and Hughes (129). 

portions for any radiobiological event requiring more than two 
ionizations. The radiobiological significance of differences in ioniza- 
tion density or LET is discussed in Section VIII. 

II. Units of Radioactivity and of Dosage Measurement 

Genetic effects can be produced by ionizing radiation regardless 
of the origin of the radiation. Today many geneticists use radiations 
from radioisotopes or nuclear reactors as well as from X-ray machines 
or accelerators. A list of isotopes used for biological experimentation 


is given in Table 3. These isotopes emit alpha, beta, or gamma rays, 
or a combination of two or more. 

It is imperative that careful dosage measurements be made if 
reproducible results are to be obtained in radiation experiments. 
Generally dosimetry should be done by someone well qualified to 

Table 3. — Radioisotopes Used in Biological Studies, Listed According to Half-life, 
with Radiations Emitted and, for Some Isotopes, Dose Rate per Curie at 1 Meter. 

Dose rate at 
Radioisotope Half-life* Radiation (s) 1 meter, r/hr 

emitted per curie f 

Manganese-56 2.56 h 0, 7 

Potassium-42 12.7 h 0, 7 

Sodium-24 15.00 h 0, 7 1-93 

Radon 3.82 d a 

Iodine-131 8.05 d 0, 7 0.231 

Phosphorus-32 14.3 d 

Rubidium-86 18.6 d 0, 7 

Iron-59 45.1 d 0, 7 0.65 

Strontium-89 53 d 

Sulfur-35 87.1 d 

Polonium-210 138.3 d a, 7 

Calcium-45 164 d 

Zinc-65 245 d 0+, 7 0.30 

Cobalt-60 5.27 y 0, 7 1-32 

Barium-133 7.2 y 7 

Hydrogen-3 (tritium) 1 2.46 y 

Strontium-90 28 y 

Cesium-1 37— barium-1 37 30 y 0, 7 0.356 

Radium-226 1,622 y a, 7 0.84 

Carbon-14 5,568 y 

Uranium-238 4.4xl0 9 y a 

*h = hour; d = day; y = year. 

tThese values taken from Kinsman (82, page 139). 

handle the appropriate apparatus. There are many pitfalls for the 
unwary, and, especially so, with internally deposited isotopes. A good 
idea of the complexities involved can be obtained from the book by 
Mine and Brownell (72). The details of dosimetry measurements are 
not appropriate to this article, but suffice it to say that the amount 
of radioactivity is determined by counting the number of particles 
emitted in a given time and that it is commonly designated in curies, 
millicuries, or microcuries. A curie (abbreviation: c) is defined as 

sparrow: cytogenetic effects of ionizing radiations 65 

3.7 X 10 ]0 disintegrations per second, without regard to the number 
of particles released per disintegration, their energies, or their prop- 
erties. It is possible to convert a known number of disintegrations 
into a standard unit of dosage measurement if both the number of 
particles emitted and their average energy is known. Dose rates 
obtained from several gamma emitting isotopes are shown in Table 3. 
Note that the dose rate per curie varies widely with different isotopes. 

There are several different units currently in use for the meas- 
urement of amounts of radiation produced in air or the amount of 
energy absorbed in tissue or similar material. The oldest unit still in 
general use is the roentgen (abbreviation: r). It is defined as "that 
quantity of X or gamma radiation sucJi tJiat the associated corpuscular 
emission per 0.001293 gram of air produces, in air, ions carrying 1 
e.s.u. of electricity of eitlier sign" . A roentgen is equivalent to the 
absorption of approximately 98 ergs/gm in water or tissue. One 
roentgen of X or gamma rays will produce 2.083 X 10 9 ion pairs per 
cc of air at standard temperature and pressure, 1.6 X 10 ]2 ion pairs 
per gram of tissue or about 1.8 ion pairs per [i 3 . For specific values of 
different radiations at various energies see Table 2 of Lea (93). 

A second unit, less commonly used, is known as the roentgen- 
equivalent-physical (abbreviation: rep) and can be used for any ioniz- 
ing radiation. A rep is defined as that quantity of corpuscular radi- 
ation which produces in tissue, per gram of tissue, an amount of 
ionization equivalent to that produced by 1 r of gamma radiation in 
air. 5 Both of these units have the disadvantage that they do not 
measure the energy absorbed but depend on the amount of ionization 
produced in air. In order to avoid this difficulty, a newer unit, the 
rad, has been adopted recently. It is a unit of absorbed dose and 1 rad 
equals 100 ergs/gm. For X-rays, one rad equals the amount of energy 
released by 1.08 r in water. 

More detailed information about the dosage units and measure- 
ments of ionizing- radiation are sfiven in raanv books and articles 
(43, 68, 72, 83, 98, 124, 193, for example). A summary titled "Recom- 
mendations of the International Commission on Radiological Protec- 
tion" has recently been published (74). This or a similar article 

B Unfortunately, the definition of the rep has undergone some revision and its value 
has appeared in the literature both as energy absorbed per unit mass or per unit volume. 
The rad unit avoids this difficulty. See glossary (111) for further comments. 


should be carefully read by anyone contemplating work with ionizing 

III. Methods of Exposure and Facilities Used 

Methods of exposing suitable material to radiation and the size 
and nature of the facilities used vary so widely that it is difficult to 
summarize them. For instance, experimental procedures used with 
plants vary from the treatment of a few milligrams of pollen or spores 
with an inexpensive ultraviolet source or small portable X-ray 
machine to the simultaneous treatment of hundreds or thousands of 
large plants or trees by gamma sources of many kilocuries in size 
(159, 167, 171). Space limitation does not allow a full description of 
all the methods used and facilities available, but representative radi- 
ation sources are listed in Table 4. A more complete description of a 
few types is given below. These have been selected largely on the 
basis of their actual usage or potential significance in cytogenetics or 
plant breeding. 

A. X-ray Machines 

X-ray machines are probably the most widely available source 
of ionizing radiation and have as their chief advantages availability, 
versatility, and, generally, ease of operation. They are also easier to 
shield than radioisotopes emitting highly energetic gamma rays such 
as Co 00 and Cs 137 and do not require such frequent calibration or cal- 
culations of dose as is necessary with isotopes, especially those with 
shorter half-lives. 

X-ray machines suitable for biological experiments are usually 
in the 50- to 300-kv range and their output may exceed 1,000 r per 
minute for small samples. Newer types may go much higher. As com- 
pared to Co G0 gamma sources, however, X-ray machines have as their 
chief limitations: (a) their requirement for considerable electric 
power; (b) that the maximum intensities and penetration of their 
radiation are in most cases much less than that of a Co co source; (c) 
that the size of the object to be irradiated is definitely limited with 
most installations, except for very low doses; (d) the fact that X-ray 
machines cannot be operated economically for continuous long 
exposures; and (e) maintenance costs are considerable for machines 
operated near full capacity. 

sparrow: cytogenetic effects of ionizing radiations 


B. Gamma Sources 

The availability of large amounts of long-lived isotopes emitting 
gamma radiation has given plant radiobiology an unequalled oppor- 
tunity for exposure of plants or plant parts to large amounts of ioniz- 
ing radiation and created new opportunities for comparing chronic 
exposure with the more usual acute methods of treatment. A list of 
common gamma-emitting isotopes and the intensity of gamma radi- 
ation per curie per hour at one meter is given in Table 3. The general 
relationship between the energy of gamma photons and their dose 
rate at 1 meter is given in Figure 4. 

0.001 0.002 0.005 0.01 0.02 0.05 0.1 0.2 0.5 1.0 2.0 5.0 I0.C 


Figure 4. — Dose rate in milUroentgens per millicurie per hour at 1 
meter in air (luithout absorption) from a point source. After Slack and 
Way (150). 

A large number of gamma ray sources (mostly Co G0 ) are now 
available for biological use. These vary from sources of less than a 
curie used for treatment of small samples in shielded containers up 
to those of many kilocuries. Some of these are located in large but 



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sparrow: cytogenetic effects of ionizing radiations 


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carefully protected open fields for chronic or acute treatment of 
plants of almost any desired size. Some uses of gamma sources in 
botanical research have been summarized by Sparrow (159). While 
much has been written about the value of these large gamma sources 
in plant breeding (146, 147, 167), their usefulness in this respect seems 
to be generally no greater than the more conventional radiation 
sources such as X-ray machines or shielded radioisotopes used under 
laboratory conditions. They are, however, very versatile and may be 
especially suited for certain specialized crops which are ordinarily 
reproduced by vegetative methods. The main advantage of these large 
oamma sources is the ease with which chronic treatments can be made 
at any desired dose rate and the very high intensities of radiation 
available at least in a restricted area near the source. Daily dose rates 
available at various distances from three Co 00 sources are given in 
Table 5. As shown in Figure 4, dose rates from a given size of source 

Table 5. — Dose-distance Relationship for Three Cobalt-60 Sources in Use in the 
Biology Department at Brookhaven National Laboratory. * 


from source, 



10-acre field 

0.21 curie (effective) 8.2 curies (effective) 2,040 curies (effective) 
r /20-hr day r /20-hr day r /20-hr day 




























After Sparrow (159). 

varies with the energy of the radiation. It is the opinion of the author 
that these sources should be regarded as versatile radiobiological tools 
and not primarily as facilities for the average plant breeder. 

While most installations to date have used Co 60 because of its 
availability, it would seem that Cs 137 would be more suitable for 
future installations. The main advantage of Cs m is its long half-life 
(30 years) compared to the 5.3 year half-life of Co 60 . Another advan- 

sparrow: cytogenetic effects of ionizing radiations 71 

tage of the Cs 1 * 7 is" its less energetic gamma which makes shielding 
easier. This is a potential disadvantage too, of course, wherever deep 
penetration into tissue is desired, and more curies are required to give 
the same output in r/hr. 

C. Accelerators and Cyclotrons 

The number of accelerators of various types available today is 
bewildering to the average biologist and no attempt will be made to 
enumerate all of them. The smaller accelerators for electrons cost 
only a few thousand dollars, but those more recently developed cost 
many millions of dollars. While the more expensive types can some- 
times be scheduled for biological experiments, one can hardly vis- 
ualize having such machines built at this time for our exclusive use. 

In addition to their great expense, accelerators are generally 
difficult to use for biological experiments and are in great demand 
by physicists. For these reasons they are used much less in biological 
experiments than other types of radiation sources. They are a major 
source now for biological experiments with fast electrons, protons, 
neutrons, and stripped nuclei (44). It is possible by appropriate design 
to obtain microbeams of a few microns in diameter from various 
accelerators (205). Protons or deuterons usually are used in such 
microbeams to reduce the amount of scatter which would occur with 
X-ray or electron beams. Mesons can be produced artificially by the 
acceleration of alpha particles or protons to energies in the Bev 
range. As far as the author is aware no genetic experiments with 
mesons have yet been performed owing to their elusive nature and 
the relatively low intensity available. With the development of the 
extremely high energy accelerators now under test or construction 
(45), it should soon be possible to obtain enough of these energetic 
particles to test them for their relative biological effectiveness in pro- 
ducing breakage and mutation. 6 However, since we know that the 
ionization density produced along their tracks varies from extremely 
low to extremely high values, it is predictable that their effectiveness 
will cover a broad range depending on their energy at the time they 
traverse a o-iven nucleus. 

"The Brookhaven Alternating Gradient Synchrotron will attain energies up to 30 
billion volts. 


D. Nuclear Reactors 

Unfiltered radiation from nuclear reactors is a complex mixture 
of radiations and is not suitable for well-defined biological experi- 
ments. Facilities with fairly complicated shielding are required to 
provide thermal or fast neutron beams with a small amount of gamma 
contamination (29). 

Some radioactivity is induced in the biological specimens treated 
with thermal neutrons, but the amount of radioactivity is usually 
small and of fairly short half-life. The radioactivity induced consti- 
tutes no serious hazard with reasonable precautions. For most plant 
material exposure times in the Brookhaven facility are in hours or 
days. A considerable amount of work has been done in the last decade 
in the treatment of seeds and other plant material with thermal 
neutrons for comparison with X- or gamma-ray treatment (35, 141, 
142, 160, 186, 190, 203). The main rationale behind this is that the 
relatively high ion density obtained with thermal neutron exposures 
may give results different from those one would get with the less 
densely ionizing radiation. 

E. Small Portable Irradiators 

In addition to the portable X-ray machines and ultraviolet 
sources, many other relatively inexpensive small sources have been 
designed and used for various purposes. Beta irradiators of various 
sizes and types can be used for localized exposures of suitable mate- 
rial either with small beams through minute pores or by surrounding 
the material to be irradiated with a radioisotope suitably bound to 
prevent corrosion or flaking (167). Maximum penetration of biologi- 
cal material with beta rays is usually only a few millimeters or less, 
depending upon the radioisotope used. Dosimetry of beta sources is 
difficult and complex (95). Similar irradiators can be made using 
alpha emitters and as these penetrate only one or a few cell layers, 
treatment of very localized areas is possible. Encapsulated radioactive 
materials in various sizes and shapes are now available commercially 
and can be used in a great variety of ways for experimental work. 

F. Internal Emitters 

Whenever radioisotopes are located inside cells or tissues, they 
are called internal emitters. Many internal isotopes have been studied 
with respect to their cytogenetic effect, but the four most commonly 

sparrow: cytogenetic effects of ionizing radiations 73 

used are probably phosphorus-32, sulfur-35, carbon-14, and tritium. 
These and others less frequently used are listed in Table 3 along with 
their respective half-lives and radiations emitted. Of special interest 
to geneticists are those radioactive elements or compounds which are 
selectively concentrated in chromosomes and nuclei. (See Caldecott 
(19) for references.) Tritiated thymidine is an example (94). Import- 
ant but less extensive use also has been made of various precursors 
labeled with C 14 , P 32 , S 35 , etc. (179, 181, 204). 

Tritiated thymidine was initially used by Taylor, Woods, and 
Hughes (187) because of its localization in the nuclei and high resolu- 
tion in autoradiography. Because of its concentration in the nucleus 
and the short, dense, ionization tracks produced, its energy is highly 
localized (Figure 5) and obviously can be expected to produce changes 
fairly efficiently in nuclei (129). A number of such studies have been 
made and some results obtained by Wimber (196) are shown in 
Figure 6. 

Other studies in which mutations have been induced by internal 
radioisotopes, including potentially useful ones, are too great to list 
here. The use of labeled chemical mutagens reported by Moutschen- 
Dahmen and co-workers (107) and by Smith (153) promises to be a 
very interesting and valuable new technique. The genetic effect will 
result from both chemical and physical activity. 

IV. General Survey of Cellular and Nuclear Changes 
Induced by Ionizing Radiation 

In the interests of completeness, we shall first list here briefly 
most of the known changes which occur in nuclei following exposure 
to ionizing radiations. (See also Section VI.) A few are secondary 
effects, e.g., polyploidy which results from a primary effect on the 
spindle. Many of these are not too well-understood and the signifi- 
cance of some with respect to genetic changes may be obscure or 

1. Gene mutation 

The author will not attempt to define gene mutation but uses 
it as a category of genetic change different from those involving some 
kind of chromosome aberration. Gene mutation almost without 
exception increases linearly with increasing dose. (See Muller (108) 
and other pertinent papers in this volume.) 



1000 R 


















0.01 — 

0.001 — 


V 2 I 1^2 2 2^2 3 3'/ 2 4 4'/ 2 5 


Figure 5. — Radiation doses about a point source of tritium. After Rob- 
ertson and HugJies (129). 

2. Chromosome, chromatid or subchromatid aberrations 

The type of aberration produced depends upon the number of 
strands present in the chromosome, on the length or density of the 

sparrow: cytogenetic effects of ionizing radiations 


0.5-1.0 1.0-2.0 1.5-3.0 2.0-4.0 2.5-5.0 3.0-6.0 3.5-7.0 40-8.0 



co 120 



t 100 



rr 80 



20 - 



3.6 RADS/HR. 
7.2 RADS/HR. 



30 40 50 60 70 











Figure 6. — Increase in chromosome fragmentation in Tradescantia palu- 
dosa exposed to 1 \\cjml H z -tliymidinc as a junction of autoradio- 
graphic grain number over dividing nuclei. 200 or more cells were scored 
for each point. Disintegrations per nucleus calculated on basis of 1 
grain per 143 disintegrations. After Wimber (196). 

ionizing track which traverses the strand or strands, and on a great 
number of other factors. (See sections on chromosome breakage and 
modifying factors.) 

3. Changes in chromosome number 

Changes in the basic chromosome number vary from the addi- 
tion or subtraction of one or a few chromosomes to the alteration of 
whole chromosome sets. Haploidy, triploidy, and tetraploidy have 


all been reported to result from treatment with ionizing radiation 
(158). Some cases of polyploidy apparently result from inhibition or 
destruction of spindles as in Trillium (156), others presumably from 
restitution caused by multiple bridge formation. Aneuploidy could 
result either from nondisjunction or from abnormal or multipolar 

4. Binucleate or multinucleate cells 

Either of these effects may result from the inhibition of the 
spindle. They are commonly seen after irradiation of meiotic or post- 
meiotic cells in many plants and less frequently in somatic cells. 

5. Effects on centromeres 

One frequently sees lagging chromosomes which behave as if the 
centromere were nonfunctional or at least delayed in functioning. 
A second effect of radiation on centromeres is misdivision of the 
centromere during which it splits at right angles to the longitudinal 
axis of the chromosome (70). Ideally, one should get two isochromo- 
somes each time misdivision occurs but both do not always survive. 
Nondisjunction frequently is reported in irradiated cells. It could also 
result from an effect on the centromere. 

6. Effect on chiasma frequency and crossing-over 

Radiation can increase or decrease the chiasma frequency, 
depending upon the time of exposure (92, 99). An effect on crossing- 
over has also been reported in Drosophila (108). 

7. Inhibition of cell division 

Retardation or inhibition of cell division occurs whenever suit- 
able doses of ionizing radiations are delivered to a tissue which would 
normally undergo further cell division. Such inhibition may result 
from a number of causes which may include genetic damage, inhibi- 
tion of DNA synthesis, or other unknown physiological disturbances. 
At low doses the effect may be transient but at higher doses recovery 
of the ability to undergo further division may be long delayed or 
may not occur. 

8. Induction of mitotic activity 

This sometimes occurs after irradiation in tissues or cells which 
would not normally undergo further cell division (62). 

sparrow: cytogenetic effects of ionizing radiations 77 

9. Death of nuclei or cells 

Death of a nucleus can lead to phenotypic change in binucleate 
cells or heterokaryons. It could also lead to the production of haploids 
if either a male or female nucleus had been sufficiently damaged prior 
to fertilization but survived long enough for pseudo-fertilization to 
occur and for development to begin. Such an effect, known as andro- 
genesis, has been reported in Habrobracon (192) and, as already men- 
tioned, some cases of haploidy resulting from exposure to ionizing 
radiation have been reported in plants (158, 160). More extensive 
cell death has been cited by Sagawa and Mehlquist (135) as the cause 
of the reversion of a periclinal chimera of Dianthus back to a non- 
chimera. Phenotypically such a reversion resembles a mutant but 
obviously it should not be so classified. Very high doses will also cause 
sufficient damage to kill whole meristems, organs, or even whole 

10. Sterility or partial sterility of various types may be produced 

Some of these are the result of genie or chromosomal change, but 
other types are not. (See Section V.) 

11. Miscellaneous nuclear or chromosomal changes 

There are a number of changes known to occur in irradiated 
cells whose significance with respect to genetic integrity is vague or 
unknown. These are listed below along with possible modes of effects 
in some cases. 

a. Cliromosome paling. — The production of pale regions (also 
called gaps) has been studied particularly in certain animal cells by- 
means of microbeam irradiation (205). An increase in the number of 
understained regions in plant chromosomes following exposure to 
ionizing radiation has also been reported (31). 

b. Chromosome stickiness and clumping. — These effects occur 
at late prophase, metaphase, and anaphase after sufficiently high doses 
shortly after irradiation and can lead to secondary consequences such 
as fragmentation and possibly polyploidy (183). 

c. Abnormal spiralization of chromonemata. — Abnormal spiral- 
ization has been reported in meiotic chromosomes of Trillium (169) 
and could lead to breakage due to entanglement of unspiralled 
chromosome arms or possibly to restitution at anaphase due to the 


long unspiralled arms behaving like bridges. Either of these effects 
would be more likely to occur when stickiness is also present. 

d. Persistent nucleoli. — These sometimes occur in irradiated 
cells and since nucleoli are known to modify chromosome mechanics 
(40, 101), it seems quite likely that cells with persistent nucleoli 
might behave differently with respect to terminalization of chiasmata 
and separation of chromosomes at anaphase. Irradiation of nucleoli 
by microbeams of ultraviolet for periods as short as 3 seconds can 
cause permanent stopping of cell division in Chortophaga neuroblasts 

e. Nuclear enlargement (without polyploidy). — This change 
sometimes occurs in irradiated cells and may have cytogenetic conse- 
quences if additional exposure occurs. (See Sparrow and Forro (164) 
for references.) 

f. Biochemical, cytocheiuical, or hislochemical changes. — Many 
changes falling in this category are known. Some may be the result 
of primary damage to the genetic system but others almost certainly 
are not. Many examples are known (7, 38, 53, 1 18, 125). 

V. Effects on Growth Rate, Growth Habit, Reproductive 
Capacity, and/or Phenotype 

1. Growth rate 

Growth rate may be retarded, completely stopped, or, in some 
cases, stimulated. Reduced growth, one of the commonest effects seen 
in plants, increases with increasing doses. However, different species 
differ greatly in the dose required to produce a given effect. The exact 
cause of the growth inhibition is difficult to pin down since the 
amount of inhibition can be correlated with both cytogenetic damage 
(18, 125, 163) and with a drop in level of auxin (53). In some cases 
buds may be inhibited for very long periods (161) without showing 
visible breakdown. Accelerated growth, growth stimulation, or pre- 
cocious maturation have also been reported in many plants (14, 62, 
125, 160). The nature of these growth-enhancing effects are not 
understood, but probably result from physiological disturbance rather 
than direct cytogenetic alterations. 

2. Abnormal growth habit including tumor induction 

This category is distinguished from the previous one by an 
abnormal growth pattern or growth habit rather than inhibition, and 

sparrow: cytogenetic effects of ionizing radiations 79 

may occur in a great variety of forms (59, 61, 62, 63). In the extreme, 
tumors may be formed as a result of the radiation treatment (166). 
It is not known whether the tumors originate as mutant cells or 
because of a general physiological disturbance. The latter seems most 
likely (151, 170). 

3. Reproductive capacity 

Reproductive capacity can be reduced to zero by gene or chromo- 
somal changes and also in several ways which may or may not result 
from direct cytogenetic damage, i.e., some may be due to physiological 
disturbances. Sterility may arise (a) by severe stunting or growth 
inhibition which prevents flowering; (b) flowers form but lack the 
necessary reproductive structures; (c) reproductive structures are 
present but pollen is aborted; (d) fertilization occurs but embryos 
are aborted before maturity; or (e) seeds form but fail to germinate 
properly or die after germination. 

4. Phenotypic appearance 

The phenotypic appearance of Xi plants may change for any of 
several reasons: (a) in haploid plants due to mutation; (b) in diploids 
due to dominant mutation, or if the plant is already heterozygous, 
due to recessive mutation or to deletion of the dominant locus; (c) 
reversion of a chimera as explained above (Section IV, 9); or (d) 
change of a characteristic presumably due to nongenetic physiological 
change, e.g., short shoots apparently devoid of chlorophyll may devel- 
op on chronically irradiated Tradescantia plants; however, they often 
regain normal color at later stages of development (155). 

VI. Radiation-induced Chromosome Aberrations, 
Abnormalties and Related Processes 

A. Terminology 

It would seem appropriate to explain briefly a number of proc- 
esses which occur during the production of or as a result of chromo- 
some breakage and a description of the various types of induced 
aberrations. (See also Section IV.) 

1. Fragmentation or breakage 

The process by which a chromosome, chromatid, or subchroma- 
tid is broken into two or more pieces. Less commonly used synonyms 
are fracture and shattering. 


2. Restitution 

The process in which broken ends unite back into the original 
configuration. The result leaves no cytologically detectable evidence 
of the break. 

3. Reunion or rejoining 

These words are used more or less interchangeably to describe 
any union of broken ends other than the type referred to above as 

4. Fragment 

Any portion of a chromosome separated from the main chromo- 
some by breakage. With the exception of a few organisms which have 
diffuse centromeres, fragments are commonly acentric. Terminal and 
interstitial fragments are frequently referred to as terminal and inter- 
stitial deletions or deficiencies. Small fragments or dot deletions which 
are the same in length as in width are also called isodiametric frag- 
ments and extremely small fragments are generally called minutes. 

5. Chromosome break 

The prerequisite to the production of fragments or other types 
of aberration. It occurs when a chromosome is irradiated before it 
has become effectively double (2-stranded). 

6. Chromatid break 

A break in one of the two chromatids of the duplicated chromo- 
some, usually seen at late prophase or metaphase. 

7. Subchromatid break 

A break in a subchromatid or half-chromatid at a time when the 
chromosome is 4-stranded. 

8. Isochromatid break 

A break in both chromatids of a chromosome at or near the same 
position. The broken ends of the chromatids may rejoin in such a 
fashion as to form a dicentric and a U-shaped fragment. The dicentric 
then usually forms a bridge at the first anaphase following its forma- 
tion. If both centromeres go to the same pole the dicentric will persist 
until the next anaphase or possibly longer. 

sparrow: cytogenetic effects of ionizing radiations 81 

9. Gaps or achromatic lesions 

Unstained or poorly stained short regions often seen in chroma- 
tids after irradiation. They may represent incipient breakage. 

10. Misdivision of the centromere 

A break in the centromere at right angles to the longitudinal 
axis of the chromosome. Such a division is in contrast to the usual 
lengthwise splitting. Misdivision of the centromere may result in the 
formation of isochromosomes, i.e., two chromosomes in each of which 
both arms are identical genetically and cytologically. 

11. Transposition 

The movement of a piece of chromosome or chromatid to a new 

12. Exchange 

Exchange occurs when broken ends produced by two separate 
breaks rejoin in a new combination. When two separate chromo- 
somes are involved it is called interchange, and when the two breaks 
involved are in the same chromosome an intrachange. A ring-shaped 
chromosome is usually such an intrachange. Isolocus chromatid 
exchanges usually result in a dicentric and a fragment. 

13. Simple translocation 

This results from the movement of a piece of one chromosome 
to another followed by union of the broken ends. 

14. Reciprocal translocation 

The structure arising when portions of two nonhomologous 
chromosomes are exchanged. These are much more common than 
simple translocations and are one type of exchange. 

15. Inversion 

An inversion occurs when a piece of a chromosome is inverted 
180° and inserted either in its original position or in a new position 
in the same chromosome. Inversions are of two types, depending upon 
the position of the centromere relative to the two breaks. Paracentric 
inversions are those confined to a single arm of the chromosome. If 
the centromere is included, it is a pericentric inversion. 


16. Duplication 

An abenation in which one or more segments are duplicated. 
They are also called repeats. In a triplication a segment is present 
three times. 

17. Deficiency or deletion 

Alternate names for fragments but usually restricted to mean 
small fragments which often are lost from the daughter nuclei by 
virtue of the fact that they are acentric. (See also 4 above.) 

18. Dicentric 

Any chromosome or chromatid which contains two centromeres. 
Tricentrics have three centromeres, quadri-centrics four. Those with 
more than two can be referred to as multi-centrics or poly-centrics. 
A chromosome with two or more centromeres may form a bridge 
(or bridges) at anaphase. 

19. Micronucleus 

A small nucleus which usually lies in the cytoplasm at some 
distance from the main nucleus and in irradiated cells usually results 
from one or more lagging acentric fragments. There may be one to 
several per cell. Micronuclei can also result from other causes, such 
as abnormal spindle behavior, and from the lagging of centric 

20. Spontaneous breakage 

This and resultant aberrations are know to occur in cells exposed 
only to background levels of radiation. Suitable experiments indicate 
that it is very doubtful if more than a small fraction of these spon- 
taneously appearing chromosome aberrations are the result of natural- 
ly occurring radiation (108). They presumably have their origin in 
the inherent instability of the nucleoprotein structural framework of 
the chromosome. There are wide fluctuations and variations in the 
rate of spontaneous breakage. 

The above description of cytological effects is necessarily brief. 
More detailed considerations of the aberrations are given in many 
places (21, 30, 39, 49, 78, 125, 137, 183, 198, 199). These sources 
should be consulted for descriptive diagrams and pertinent references. 
The "Bibliography on the Effects of Ionizing Radiations on Plants" 
(160) is also useful in finding original references. 

sparrow: cytogenetic effects of ionizing radiations 83 

B. Production of Aberrations 
1. General 

Although chromosome breakage normally appears in dividing 
cells shortly after exposure to ionizing radiation, in certain cases 
breakage does not occur for a relatively long period after treatment. 
For instance, the irradiation of meiotic first metaphase (or of later 
stages during meiosis) produces no apparent breakage at meiosis, 
but breakage and aberrations do appear during the subsequent micro- 
spore division (157). It is obvious that some kind of lesion must have 
been produced at the time of exposure, but that it did not develop 
to the point that it could be recognized as a break until a later stage 
or even until the nucleus had passed through an interphase. Such 
lesions or incipient damage are usually referred to as "potential 
breaks." Irradiation of certain stages, such as diplotene, may produce 
both immediate and delayed breakage. As one can readily imagine, 
this delayed expression of radiation damage has led to considerable 
confusion since chromosomes carrying potential breaks could be con- 
sidered normal if observations were not continued Ions enough to 
determine that delayed breakage also occurs in the next cell division. 

Chromosome or chromatid breaks may either restitute in the 
original position, rejoin in a new combination, or remain open. While 
much effort has been devoted to all aspects of this problem, it is diffi- 
cult to generalize concerning the time required for each of these 
events to occur or to define explicitly how and why restitution or 
reunion occurs. There is evidence that, in certain cases, breakage and 
reunion occur in a very few minutes, whereas in other cases, breakage 
does not occur for a very long period after exposure to radiation, 
and, under certain conditions, broken ends may remain open for long 
periods before union or reunion occurs (23). It is generally assumed 
that the initial pattern of chromosome breakage is completely random 
within a given karyotype, but there are several nonrandom cases 
reported (39, 183). These are generally thought to result from sec- 
ondary factors such as (a) differences in the freedom of movement of 
different regions of the chromosome, (b) the presence or absence of 
heterochromatin, (c) the position of heterochromatin within the 
karyotype, (d) the position of the nucleolus or nucleolar-organizing 
regions, and /or (e) the position of the centromere relative to any 
given locus. 


Although chromosome breakage predominates in the first divi- 
sion following exposure, the cytological consequences of breakage 
frequently also can be seen much later. In addition to chromatin 
bridges and micronuclei which may persist and be visible for more 
than one cell division, certain types of aberrations, such as inversions, 
translocations, and rings, may persist indefinitely and express them- 
selves by characteristic cytological configurations and by their con- 
comitant genetic or physiological effect. Small duplications in the 
form of centric fragments may also persist (15) and these, of course, 
can produce genetic effects. In certain cases a series of phenomena 
known as the breakage-fusion-bridge cycle can result whether the 
initial breakage is of spontaneous origin, produced by ionizing radi- 
ation or by a chemical mutagen. As reported by McClintock (100), 
this cycle may go on for many cell generations. Other examples of 
long-persisting effects are small dicentrics reported by Morrison (106) 
and certain conditions such as translocations found in hexaploid 
wheat by MacKey (96). For unknown reasons these are sufficiently 
unstable to lead to secondary changes such as deficiency-duplication 
or simple deficiencies. These, of course, can result in phenotypic 

2. The mechanisms of chromosome breakage 

Although much is known about the chemical changes produced 
by ionizing radiation (7, 8, 38, 73, 91, 113), our knowledge of the exact 
mechanisms by which chromosomes are broken is unfortunately 
rather meager. This lack of understanding is due partly to our inade- 
quate knowledge of the composition and structure of the chromo- 
some threads themselves, and partly to a lack of adequate definition 
and resolution which would enable us to see changes which are 
produced at the submicroscopic level in the interval between the 
initial chemical events and the final biological event recognized as a 
chromosome or chromatid break. 

A summary of the possible interrelationships of various events 
which occur preceding, during, and after chromosome breakage is 
given in Figure 7. Possible pathways of various modifying factors or 
processes are also given. The relationship of gene mutation to other 
types of chromosome damage is obscure. 

There has been much speculation concerning the manner in 
which damage to DNA protein or nucleoprotein molecules could lead 

sparrow: cytogenetic effects of ionizing radiations 85 






















-T OF 











Figure 7. — Suggested pathways of events concerned tuitJi the produc- 
tion of chromosome aberrations and jnutagenesis. After Thoday (188). 

to chromosome rupture and several possible hypotheses have been 
proposed (3, 4, 23, 24, 79, 80, 177, 178, 185, 197, 198). Regardless of 
the nature of the events which occur at the submicroscopic level, it is 
now fairly clear that not only can chromatids be broken independent- 
ly of each other but also that half chromatid or subchromatid break- 
age can occur (39, 195). Since the potentialities for recombination 
vary with the number of strands present and since the number of 
strands varies with the stage of meiosis and mitosis, it is apparent that 
the stage of cell division irradiated can have a great effect on the 
outcome of the initial breakage events which occur (194). The num- 


her of nucleoprotcin strands in a Tradescantia chromosome, accord- 
ing to recent work with the electron microscope, may be as high as 
32 before synthesis (177). One would expect that the number of 
strands or fibrils in a a chromatid, as well as their diameter, should 
have a bearing on the probability of breakage of that chromatid by a 
given ionization track or cluster. 

3. Dosage response curve for one-hit events 

Chromosome or chromatid breakage is considered to result from 
a cluster of 15 to 20 ionizations either within or very close to the 
chromatin strand in question (93). A sufficient density of ionization 
may occur at any position along the more dense tracks, such as those 
of alpha particles, or only near the end, or "tails", of less dense tracks. 
While each ionization is an independent event, in radiobiological 
parlance it is also possible to regard any large cluster or tail as a single 
event. When such a cluster or track passes through a chromosome 
(or chromatid), the event is called a "hit". Such clusters or tracks have 
a certain probability of producing a break (in contrast, it is often 
considered that gene mutation can be caused by as little as one ion 
pair). It lias been shown that various one-hit chromosomal events 
(one-hit interstitial deletions, terminal deletions) are independent of 
the dose rate, of the time of exposure, and of close fractionation. They, 
therefore, increase linearly with dose (Figure 8). In general, the 
kinetics of such events are similar to those of gene mutations, but the 
relative biological efficiency of different radiations in producing these 
end results would be expected to differ. (See also Section VIIT A.) 

4. Dosage response curves for two-hit events 

Certain types of chromosome or chromatid aberrations 
(exchanges, inversions, rings, etc.) are two-hit aberrations as far as 
X-rays and gamma rays are concerned and behave in a different 
fashion with respect to several of the variables mentioned above from 
one-hit chromosome aberrations. Whereas with one-hit aberrations 
the frequency is strictly proportional to dose, with two-hit aberrations 
the frequency increases disproportionately with increasing dose and 
with sufficiently high intensities becomes almost proportional to the 
square of the dose (Figure 8). The dose-squared relationship is best 
observed at the higher intensities when the exposure time is kept 
constant but the intensity varied (Figure 9). From these and similar 

sparrow: cytogenetic effects of ionizing radiations 

50 100 150 


Figure 8/ — Relation betiveen X-ray dosage and frequency of isochro- 
matid breaks (one-hit) and chromatid exchanges (two-hit). Time of 
exposure constant. After data of Sax modified by Giles (J9). 

observations, it can be concluded that the yield of two-hit exchange 
aberrations increases roughly as the square of the dose and that each 
of the two breaks is separately induced. Since the two breaks must be 
present simultaneously to permit the exchange to occur, it is obvious 
that such factors as intensity (Figure 9), exposure time, and dose frac- 
tionation should all have detectable effects on the frequency of two- 
hit events. Oxygen concentration also is a factor of considerable 
importance (39, 49, 80, 125). 














• 160 r/min 
A 20 r/min 
O 2.7 r/min 

-■■= , 1 1 1 1 

100 300 500 


Figure 9. — Effect of dosages of X-rays at different intensities upon tlie 
yield of chromosome exchanges. Data of Sax, after Giles (49). 

Certain types of chromosomal aberrations behave in a fashion 
intermediate between one-hit and two-hit events. For instance, the 
yield of interstitial deletions increases as the 1.5 power of the dose 
as shown by Rick (127), whereas isochromatid deletions have a value 
between 1.0 and 1.5. The exact value depends on the particular 
radiation used (84, 188). 

It has been known for some time that some types of two-hit 
aberrations fail to show the nonlinear response when cells are exposed 
to certain densely ionizing particles, such as neutrons, alpha rays, 
protons, etc. (Figure 10). The reason for this situation is that these 
particles have sufficient density of ionization and sufficient length of 
track to produce more than one break for each passage through a 
nucleus. Thus, what would for X- or gamma rays be a two-particle 
event (two separate ionization "tails") is, in actual fact, a one-particle 
event (one ionization track) for the densely ionizing particles men- 
tioned above. (See Swanson (183) for references.) 

sparrow: cytogenetic effects of ionizing radiations 




• XRAYS 190 kv 'k / 


/ / 
/ / 
/ / 

/ / 


/ / 
/ / 
/ / 

* / 


/ / 
/ / 
/ / 

/ -r/ 











400 r (XRAY) 
40 n (NEUTRONS) 

Figure 10.— Relation betioeen frequencies of chromosome intercliange 
(dicentric and centric rings) and dosages of fast neutrons and X-rays. 
1 n unit = ca. 2.5 r. After Giles (49). 

VII. Nature of Mutational Events Induced 
by Ionizing Radiation 

The relationship between the frequency of induced mutations 
and the amount of ionizing radiation depends in part upon the nature 
of the mutational event. Phenotypic change which results from 
chromosomal breakage, or the resultant aberrations, will have the 
same dosage response curves as the breakage or aberration event or 
events which caused it. For instance, simple one-hit deletions (of 
markers) will show a linear increase with increasing dose, whereas 
losses resulting from two-hit deletions will have a dosage response 
curve which is nonlinear and will approach the dose-squared 
relationship at high dosages or high intensities (163). 

It has been shown in a wide variety of organisms that point 
mutations increase linearly with increasing dose (Figure 11) and are 
generally independent of intensity (108). Occasional departures from 
linearity are found (see 110), but in this case it has been suggested 







io ; 




i i i i r 

T 1 1 1 1 GT 


34 68 

135 270 540 1080 2160 4320 


Figure 11. — Reverse mutations induced in met-2 auxotrophs by vari- 
ous doses of X-rays. After Demerec and Sa)us (34). 

t hat a selective mechanism was responsible for the nonlinearity. A 
recent case of nonlinearity has been found in mice in which mutation 
rate is dose-rate dependent (138). It was also reported in this paper, 

sparrow: cytogenetic effects of IONIZING RADIATION'S 91 

and confirmed later (134), that acute X- and gamma radiations are 
equally mutagenic to spermatagonia and that both are more effective 
than chronic gamma irradiation. No adequate explanation for this 
difference has been given, but it seems possible that such an effect 
would be expected if some of the "mutations" from the acute exposure 
were two-hit deletions and not true point mutations. Evidence of a 
dose-rate effect as well as a nonlinear response has been reported for 
marker losses in several different plant species. Such an effect for 
chronically irradiated Llliuni testaceum is shown in Figure 12. The 
explanation given is that some of the marker losses result from two-bit 
deletions and thus the dosage-squared component contributes sig- 
nificantly to nonlinear response (32, 162, 163). 

Classically, it has been considered that so-called point mutations 
or intragenic mutations are a valid category of genetic change. This 
may be true for Drosophila and for many extensively studied 
microorganisms. However, it was the view of Stadler (173, 174), who 
did early outstanding work on the induction of mutations in maize 
with ionizing radiations, that all the so-called point mutations in 
maize were actually minute chromosomal aberrations or deficiencies 
and that point mutations as defined above did not actually exist in 
this material. As far as this author is aware, it is questionable whether 
anyone has since analyzed mutations in higher plants with sufficient 
detail to disprove Stadler's conclusion. It is further of considerable 
interest that recent work of Demerec (33) has shown that most 
mutations induced by ionizing radiation in E. coli appear to be 
deficiencies, although point mutations apparently can be produced by 
ultraviolet radiation or arise spontaneously in this organism. In actual 
practise, the distinction between deletions and true mutations often 
becomes one of resolution, since only the most critical kind of test 
could reveal any distinction. Such tests are rarely applicable in higher 

If radiation-induced point mutations actually exist, one would 
expect that they could revert back to the original form by back 
mutation. For example, Giles (50) has shown in Neurospora that 
reverse mutation does occur at certain loci. These mutations would 
appear to meet the criterion for point mutation, but an equally 
clear case has not yet been found in higher plants. While the exact 
nature or even the actual existence of radiation-induced point muta- 












500 - 




TTI 1 1 1 I I I I I I 



i i i i i i 1 1 

i i i i i i i I 

.1 1.0 10.0 


Figure 12. — Somatic mutation rate in sepals and petals of chronically 
irradiated Lilium testaceum. After Sparrow , Cnany, Aliksche, and 
Scliairer (163). 

tions seems in doubt in higher plants, there is no corresponding doubt 
about mutational events derived from chromosomal breakage and 
rearrangement. The various kinds of such aberrations have been 
outlined above (Section VI) and almost all of these have been identi- 
fied with phenotypic changes of some type. For instance, duplications, 
deletions, translocations, inversions, isochromosomes, and breakage- 
fusion-bridge cycles have all been well documented elsewhere (16, 39. 

sparrow: cytogenetic effects of ionizing radiations 93 

64, 126, 158, 198, 199). In addition to these, position effect has been 
described by Catcheside (20) in Oenothera blandina. In spite of some 
of the practical difficulties sometimes associated with certain types of 
aberrations, e.g., reduced fertility, the record clearly shows that 
appropriately designed (or even accidental) cases of chromosome 
engineering can yield results of great potential value. An outstanding 
example is the work of Sears (140) in which leaf rust resistance was 
transferred from the wild glass, Aegilops umbellatum L., to common 
wheat, Triticum aestivum L., by means of a short interstitial trans- 
location. A similar transfer of stem rust resistance has since been 
reported by Elliott (36) from Agropyron elongatum to T. aestivum. 
A more extensive discussion of the usefulness of chromosomal aberra- 
tions in plant genetics and plant breeding is given in earlier publica- 
tions and elsewhere in this volume (46, 47, 64, 86, 97, 123, 158). 

VIII. Factors which Modify Cytogenetic or Other 
Radiobiological Responses 

One of the difficulties with the status of modern radiobiology is 
a plethora of facts and a deficiency of general principles. This regret- 
able state of affairs is well illustrated in the case of the literature 
on factors which modify radiation response. It is possible to catalogue 
these facts (Tables 6, 7, 8), but unfortunately the exact reason or 
mechanism by which modification comes about is often unknown. 

Since it is important that radiation experiments be as repro- 
ducible as possible, the maximum number of variables should be 
controlled carefully. Some things which may seem to be trivial details 
often turn out to be important modifying factors, i.e., moisture con- 
tent of seeds, exact age of seeds or seedlings, growth rate, and tem- 
perature. A diagram of some possible post-radiation events and of 
various stages at which modifying factors may act is given in Figure 13. 

In the interest of providing a brief summary of the known 
modifying factors and to provide a guide to investigators desiring 
uniformity of results, most of the major modifying factors are 
described or listed below. 

A. Physical Factors 
1. Miscellaneous factors 

Many physical factors other than total dose can modify the degree 






























<< 2 




a. — 





























t CO 




CO Ll) 









— v 5 



• 2 ° 


"5 ^ So 



z I 

< o 

Q -\A/V-* 



Q I 5 


< Q 

O co 

-=> ~ 

Pi C K 

§ - u 









Ph s 

sparrow: cytogenetic effects of ionizing radiations 95 

of the radiation response. Dose rate, dose fractionation (see Section 
VI, 4), linear ion density (see below), and temperature are probably 
the most important of these variables, but several others also can 
cause a significant modification under appropriate circumstances 
(Table 6). For instance, small doses of ultraviolet in combination 
with 1 rad of X-rays can give a yield of aberrations roughly equal to 
that normally obtained from 100 rads of X-rays alone (85). Similar 
but much less dramatic synergistic effects have been known for some 
time (see references in Table 6). It is difficult to make generalized 

Table 6. — Physical Factors Affecting the Radiosensitivity of Plants.* 
Factor References 

A. Dose fractionation 21,39,49,93,125,137,199,202 

B. Dose rate 21,39,86,93,125,137,183,199 

C. Linear ion density 21,26,57,88,93,125,202 

D. Previous exposure to ionizing radiation 202 

E. Exposure to other radiations 

1. Ultraviolet 183(page 382) 

2. Infrared 49(page 739),202(page 374) 

3. Visible light 160,202(page 358) 

F. Exposure to ultrasonic energy 125,1 37 (page 19) 

G. Bioelectrical potential 160 

H. Temperature 49(page 741),202(page 360) 

1. Centrifugation 137 (page 19) 

J. Pressure, hydrostatic 160 

K. Phase state 202(page 353) 

♦After Gunckel and Sparrow (62). The references in this table are held to a minimum and are in 
many cases to review papers rather than to the original research. References to many original papers 
can be found in the reviews cited or in the bibliography by Sparrow, Binnington, and Pond (160). 

statements concerning these factors. Other conditions of the experi- 
ment and the effect studied will determine, in part, the degree and 
direction of modification. The articles referred to in Table 6 contain 
extensive discussions of the various physical factors. 

2. Ionization density, LET and RBE 

It is well known that different radiations or the same radiation 
at different energies behave differently with respect to the average 
number of ionizations produced per micron of tissue traversed. (See 
Figure 14 and Table 2.) Since each ionization is estimated to require 
32.5 ev, it is apparent that the denser the ionizations along the track, 
the higher the rate of energy transfer from the ionizing particles to 









Figure 14. — Separation of ion clusters in relation to the size of virus 
particles 27 mji in diameter. After Gray (57 j. 

molecules of the tissue, i.e., the higher the linear energy transfer 
(LET). (See Section I.) 

The biological consequences of variations in LET have been 
studied for many years and are of great importance in radiogenetics. 
Certain qualitative differences and many quantitative differences in 
cytogenetic responses can be attributed to differences in the ioniza- 
tion density of the radiation used. The relative biological effect (RBE) 
for many responses increases with increasing LET but may go through 
a maximum (Figure 15) and then decrease. This saturation effect can 
be explained, in some cases at least, by the production of surplus 

sparrow: cytogenetic effects of ionizing radiations 




2.5 Mev 

I Mev 

10 15 20 30 39-40 48-50 


Figure 15. — The effect of ionizing radiation on different energies on 
their relative efficiency in inducing chromosomal aberrations in Tra- 
descantia microspores. After Sax (137). [See Conger, Randolph, and 
Johnston (25).] 

ionizations, e.g., if a sensitive target volume requires only x ionizations 
to produce an effect, then 2x ionizations in the same volume will not 
increase the detectable effect (Figure 14). Certain effects, such as those 
produced by single ionizations, are less efficiently produced with 
increasing LET (Figure 16). It is generally assumed that point muta- 
tion can be induced by one ion pair or one ion cluster (108, 199). For 
chromosome breakage, however, a different relationship apparently 

From early work, Lea concluded that chromosome breakage (in 
Tradescantia) required an estimated minimum of 15 to 20 ionizations 
to occur within a cross section of a chromatid thread not thicker than 
0.1 \i (93, page 280). This means that breakage of such chromosomes 
can only be produced by relatively dense ion clusters. The response 
of Tradescantia chromosomes to radiations of different ion density 
has recently been studied in detail by Conger, Randolph, Sheppard, 
and Luipold (26). By comparing the efficiency of various radiations 
to that of Co 60 gamma rays, they showed that the RBE climbs to a 



£ 1-0 





uj .8 





3 .* 




< .4 



1 l 


/ 1 

8 - 1 





^ / 

10,000 I 

1,0 00 

10 100 

I ONS / \i 

Figure 16. — The influence of ion density on chemical change and chro- 
mosome breakage. After Gray (55). 

peak of 8 to 11 between approximately 50 to 70 kev/j.i. It then falls 
considerably and flattens out in the LET range between roughly 120 
and 220 kev/[i. Less extensive but somewhat similar observations 
were made by Larter and Elliott (89) who found thermal neutrons 
more effective in producing translocations than X-rays, or beta rays 
from S 35 and P 32 . It also has been found that the dense ionization 
associated with thermal neutron treatment caused more breakage per 
unit inhibition of seedling growth than did X-rays (18). 

In addition to the LET effect on RBE's for chromosome break- 
age, there is evidence that breaks caused by high-ion density tracks, 
such as alphas, are less likely to rejoin than those caused by less dense 
tracks of X- or gamma rays (87). The RBE's of densely ionizing radi- 
ation with respect to various types of mutations also have been 
studied by several investigators. Theoretically, for point mutations 
low-ion density should be more efficient; however, the effect depends 
upon the kind of mutation produced (1, 13, 108, 109, 199). For 
example, Ehrenberg and Nybom (35) found a higher percentage of 
erectoides mutations in barley with thermal neutrons than with X 
rays. Other studies show that radiations of different ionization den- 
sities sometimes produce characteristically different spectrums of 

sparrow: cytogenetic effects of ionizing radiations 99 

mutations, at least in plants (46, 67, 86). An extensive bibliography 
(88) on RBE is available for those interested. 

B. Chemical Factors 

A very extensive literature exists relating to the modification of 
control of radiosensitivity by chemical substances. Variations in the 
amount of naturally occurring chemical constituents as well as in 
those which can be added experimentally to the internal or external 
cellular environment are all potentially suspect and many already 
are known to be effective (Table 7). While many of the results 
obtained could have been predicted, the reasons for some of the 
modifications are still obscure or unknown. However, the study of 
these factors is important since such studies may suggest some of the 
chemical steps involved in chromosome breakage and mutagenesis. 
Moreover, a better understanding should lead to more adequate 
control of the most important environmental variables and this, in 
turn, should yield more reproducible data. This also could have prac- 
tical applications, e.g., one could plan experiments in which the maxi- 
mum yield of a certain kind of cytogenetic effect could be obtained 
with the minimum amount of sterility or lethality. Such mutagenic 
specificity is summarized by Smith (153). 

Due to the complexity of the relationships involved and lack of 
space, it is impossible to explain here even the best known mecha- 
nisms by which protection or enhancement is brought about. How- 
ever, many authors have discussed at length chemical modification 
and the mechanisms of action involved (2, 5, 12, 22, 38, 71, 91, 125, 
153, 201). 

C. Biological Factors 

1. Cytological and genetic factors 

a. Chromosome number and polyploidy. — The chromosome 
number in diploids and the degree of polyploidy are both known to 
affect radiosensitivity (160, 163, 202). The degree of polyploidy and 
the kind of polyploidy are also known to affect the type of mutation 
and the rate (96). Likewise, the higher the degree of polyploidy, the 
more generations which must be grown to ensure the appearance of 
recessive mutations. 

b. Chromosome size or nuclear volume. — The size of the nucle- 


Table 7.- Chemical Factors Affecting the Radiosensitivitv of Plants.* 





A. Antioxidants (lipid) 69(pages 81,91), 


B. Auxins (indoleacctic acid) 53(page 10), 105 

C. Carcinogens 
1. Benzopyrenc 

157 (page 1522) 2. Urethane 

D. Chelating and complexing agents 

1. Cupferron 202(page 366) 3. 

2. Dowex-50 160 

E. Enzymes and cofactors 

tetraacetic acid 
(EDTA or Versene) 


1. Adenosinetriphosphate 





nucleotide (DPN) 

184 (page 242) 

2. Catalase 





3. Cytochrome C 


F. Metabolic inhibitors 

1. Carbon monoxide 



Triiodobenzoic acid 

137 (page 23) 



2. Cyanide 



Uranyl nitrate 

157 (page 1520) 

3. Maleic hydrazide 


G. Metabolites 

1. Carbon dioxide 

137 (page 23) 



157 (page 1523) 

2. Carboxylic acids: 




malonic and maleic acids 




3. Desoxypentose nucleic 



acid (DNA) 



(Bacteria, U.V.) 


a. Ascorbic acid 


4. Digitalis glycoside 


b. Synkavitc 


H. Miscellaneous 

1 . Casein hydrolysate 





2. "Kollidon" 

57 (page 389) 


Yeast extract 

202 (page 361) 

I. Mitotic poisons 

1 . Acenaphthene 





2. Colchicine 


J. Mutagens (chemical) 

1. Mustard oil 

130 (page 207) 


Nitrogen mustard 


*After Gunckel and Sparrow (62), Also, see footnote to Table fi. 

sparrow: cytogenetic effects of ionizing radiations 101 

Table 7. — Continued. 





K. Oxidizing substances 

1. Ferric sulfate 

2. Hydrogen peroxide 

3. Nitric oxide 

4. Oxygen 

L. Neutron absorbers 

1. Boron 

2. Cadmium 

M. Nutrients 

1. Boron 

2. Calcium 

3. Cobalt 

4. Magnesium 

157(page 1520) 5. Sodium 

91,125,180 peroxydisulfate 

2,122 6. Sodium pyrosulfate 
57(page 378), 

86 (page 38) 3. Lithium 
86 (page 38) 


149 6. Phosphorus 

175,176,177 7. Potassium 

60 8. Sulphur 

60 9. Zinc 


86(page 38) 

157(page 1521) 


157(page 1521) 


5. Nitrogen 
N. P H 

157(page 1521) 


O. Reducing substances (without -SH groups) 

1. Ascorbic acid 27,86(page 38) 4. Sodium hydrosulfite 

2. Hydrogen 57 (page 379) 5. Sodium hyposulfite 

3. Hydrogen sulfide 117 

P. Sulfhydryl compounds 

1. Cysteamine 73, 4. Glutathione 

202 (page 362) 

2. Cysteine 137 (page 24), 5. Thiourea 

202(page 362) 

3. 2,3-dimercaptopropanol 57(page 388), 
(BAD 1 25 

57 (page 388) 
57(page 388) 

57 (page 388), 

202(page 357) 

57 (page 388) 

us or chromosome is very important in determining the frequency 
of chromosome breakage per roentgen of exposure (116, 161, 163, 
165). Data collected from several species of plants with different 
nuclear volumes indicate that, under comparable conditions of 
chronic irradiation, somatic nuclei yield a more or less standard 
number of chromosome breaks per cubic micron of nuclear volume 
per roentgen of exposure (155). It is thus apparent that any nuclear 
volume difference between species, between stages in development, 
or between tissues in either plants or animals should have a great 


effect on the yield (per r of exposure) of breaks or on any of the 
known genetic changes derived from breakage. 

c. DNA content per nucleus. — The DNA content is related to 
average nuclear volume, and it appears that the larger the amount of 
DNA per complement, the higher the radiosensitivity (164, 168). 

d. Amount of lieterocliromatin. — The amount and distribution 
of heterochromatin lias been shown to affect the radiosensitivity of 
nuclei in various ways (39, 157). 

e. Number of nucleolar organizing regions.- — The number of 
such regions is often related to the amount of heterochromatin and 
may be related to the average amount of nucleolar material per 
nucleus. Both of these factors are known to affect the radiosensitivity 
of cells (41, 57). 

f. Number of nuclei. — It has been shown, for example in fungi, 
that the number of nuclei can affect the radiosensitivity (81). It 
would also be expected that multinucleate cells would be more resist- 
ant than the normal type of mononucleate cell. 

g. Stage of tlic nuclear cycle. — The stage of the nuclear cycle has 
been known for many years to affect the amount of chromosome 
breakage (14, 125, 138, 157) and the amount of mutation produced 
per roentgen of exposure. In some cases the extent of the differences 
is quite large. For instance, in Trillium erect urn there is about a 
60-fold difference in amount of fragmentation between the least sensi- 
tive and most sensitive stages of meiosis (39, 157). In Zea mays muta- 
tion rates also vary widely during microsporogenesis (148). Changes in 
length of chromonemata at different stages of meiosis in Trillium 
also has been correlated with sensitivity (169). 

h. Average lengtli of nuclear cycle or of intermitotic time. — It 
has been shown, especially for chronically irradiated material, that 
the longer the average nuclear cycle, the more radiation damage is 
accumulated under a given dose rate (121, 163). 

i. Position of a gene in the chromosome. — It has been clearly 
shown that the position of a gene with reference to the centromere 
has a significant effect on the frequency of deletion (42, 163). 

j. Position of the centromere and length of cliromosome arm. — 
Both of these factors are known to affect radiation response, although 
more work is required to establish clearly the degree of the effect 
(41, 42). 

sparrow: cytogenetic effects of ionizing radiations 103 

k. Portion of the cell irradiated. — Microbeam irradiation of cells 
has clearly illustrated that there are wide differences in sensitivity of 
different portions of cells (205). For instance, the nucleus is much 
more sensitive than the cytoplasm and for ultraviolet irradiation, at 
least, the nucleolus is an extremely sensitive spot in the nucleus (48). 

1. Genotype. — Genetic differences as small as single gene differ- 
ences can cause significant changes in sensitivity (2). Different varie- 
ties of a single species also may show different sensitivities (114, 152, 

2. Morphological organization and stage of development 

a. Type of cell or tissue. — In plants, as in animals, the type of 
cell or tissue in question may have a considerable influence on the 
radiosensitivity (8). For instance, meiotic cells are usually much 
more sensitive than somatic cells (138, 169). Shoot and scutellum are 
also less sensitive than roots in barley during early developmental 
stages (104). 

b. Stage of differentiation and age of plant. — There is a consid- 
erable amount of evidence which indicates that the tolerance of a 
plant, as well as the number of mutations or chromosome aberrations 
produced, may vary with the stage of differentiation of the seeds, 
seedling, or plant (11, 14, 103, 104, 136). 

c. Portion or amount of plant irradiated. — The radiosensitivity 
of a whole plant is greater than the sensitivity of a small area or 
volume of a plant (58, 102, 172). 

3. Physiological conditions 

The radiosensitivity is influenced by various physiological states 
of the cell, tissue, or plant. Space does not allow a detailed discussion 
of these, but they are summarized in Table 8. Of the various factors 
listed, ■water content of spores and seeds has been most extensively 
studied and is known to have a large effect on radiosensitivity and on 
yield of chromosome aberrations and mutations. Recent studies have 
shown that the water content during post-irradiation storage of seeds 
is also of great importance (28). It is therefore a factor which should 
be held constant whenever possible during radiation experiments. 

The other factors listed in Table 6 will not be mentioned, except 
to point out that it is becoming increasingly clear that the yield of 
mutations is dependent upon the time of exposure with regard to 



Table 8. Physiological Factors which Affect Sensitivity.* 







Age of cell, tissue or seed 

86(page 37), 
114(page 155) 


Diseased tissue 
Concentration in 



Synthetic activity 


cell suspensions 


(page 76) 

General (growth rate) 









of DNA 





of protein 


Water content 


(Bacteria, U.V.) 

65 (page 878), 


Post irradiation 


of auxin 

5 3 (page 10) 







Nutritional state 


'After Gunckel and Sparrow (62). Also see footnote to Table 6. 

certain physiological or biochemical steps involved in protein and 
nucleic acid synthesis (2, 66). Various factors listed above under 
chemical factors could perhaps have also been included in this section. 
Also, please refer to Table 7. 

Summary and Conclusions 

This paper attempts to review certain aspects of the physical 
nature of ionizing radiations and their effects upon nuclei, chromo- 
somes, and genes. The major physical characteristics of a number of 
the ionizing radiations most important to radiobiologists are pre- 
sented and some methods of treatment and facilities commonly used 
are described. The major biological effects, including nuclear and 
chromosomal changes known to be induced by ionizing radiation in 
plants or plant cells, are listed and explained briefly. Special attention 
is given to the problem of chromosome breakage and aberration. The 
kinetics of the dosage response curves obtained under different con- 
ditions for various types of aberrations and mutations are explained 
and discussed. The possible relationship between chromosome aber- 
ration and mutation, and the factors which influence radiosensitivity 
are considered in some detail. 

The review is mainly concerned with fundamental aspects of 
radiation cytogenetics and does not attempt to relate this knowledge 
to practical problems of plant breeding. It is concluded that further 
comparative studies of the effects of different physical and chemical 

sparrow: cytogenetic effects of ionizing radiations 105 

mutagens and of various techniques of exposure are urgently needed. 
In spite of rapid progress being made, our knowledge of radiobiology 
and radiation genetics leaves much to be desired with respect to our 
ability to define or predict the most efficient methods for the produc- 
tion of useful mutations in plants. Expansion of the research effort 
at both basic and applied levels is desirable. 


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Mehlquist: I am commenting with reference to your slide showing 
increased tolerance with increased size of the chromosomes and/or cell 
size. As you know, we tried some of our Delphinium hybrids along with 
carnations planted directly in the gamma field at Brookhaven and found 
that the Delphiniums were so sensitive that they were killed to the 
ground with the dosage at which the carnations showed little effect. 
Yet the Delphinium chromosomes are many times the size of carnation 

Chemicals and Their Effects 


Institute of Animal Genetics, 
Edinburgh, Scotland 

It is now almost exactly 20 years since the first highly effective muta- 
gens were detected. This symposium provides an opportunity for 
taking stock of developments in this new field of research. There can 
be no question of its expansion; an ever-increasing number of chemi- 
cals has been found to be mutagenic, and there is no reason to expect 
that this expansion Avill come to a standstill unless we halt it purposely 
because we feel that there is no good reason for testing more and more 
chemicals for mutagenic ability. This is one of the points which, I 
hope, we shall discuss at this meeting. But let us ask a different ques- 
tion: To what extent has work on chemical mutagens fulfilled the 
expectations with which it was started? 

Expectations are largely a personal matter, but I think that the 
main expectations of mutation workers at that time may be classed 
under three headings. Chemical mutagens were expected to help 
elucidate (a) the chemical nature of the genetic material and (b) the 
relationship between intragenic and intergenic changes, and (c) to 
open the way for the production of specific types of genetic change. 
How far have these expectations been fulfilled? 

The Nature of the Genetic Material 

The original, somewhat naive, idea was that by studying the chem- 
ical group or groups that produce mutations we would learn some- 
thing about the nature of the other partner in the reaction, the genet- 
ic material. This, I am afraid, has proved an illusion. We now know 
that there is not one magic group which confers mutagenic ability on 
a compound. Instead, vastly different compounds may have mutagenic 
abilities, and closely related ones may differ in this respect. On the 
other hand, during these 20 years our knowledge of the chemical 
nature of the genetic material has advanced spectacularly, but the 
advance did not come from mutation research. The situation now is 
almost exactly the reverse of what we expected 20 years ago. Instead 
of inferring the chemical nature of the gene from the nature of the 


auerbach: effects of chemicals 121 

substances which make it mutate, we tend to interpret the action of 
mutagenic compounds on the basis of what we know about the nature 
of the genetic material. In some cases, notably in mutation experi- 
ments on virus and bacteriophage, tins approach has already given 
highly interesting results, but as a general attitude to mutation 
research it narrows the area of investigation and is likely to result in 
misleading interpretations. Even if we accept it as highly probable 
that, in higher organisms as well as in bacteria and bacteriophages, 
the essential genetic specificity resides in the DNA of the chromo- 
somes, it still remains naive to imagine that a chemical — and often a 
highly reactive one — introduced into a cell will react with the DNA 
without at the same time producing a variety of chemical changes in 
the remaining constituents of the cell. Mutation, as we now know, 
is a complicated process of which we usually only see the beginning 
and the end: the introduction into the cell of a mutagenic aq.ent, 
and the emergence of an organism or a clone of cells with altered 
properties. In between are many steps. The chemical has to pass 
barriers of permeability; it may produce the actual mutagen by inter- 
action with the cytoplasm; the initial lesion in the genetic material 
may be restored or made permanent; chromosome breakage may be 
followed by restitution or rearrangement; the mutated gene has to 
create new biochemical pathways; the mutated cell has to multiply 
in the face of competition from non-mutated cells. The same chemical 
that produces a change in the DNA will often affect the course of these 
intermediary events by reactions outside the DNA. 

What we know at present about the action of most mutagens is 
little more than speculation. I think this is partly due to the concen- 
tration of effort towards the one question, how does the mutagen 
react with the DNA? I shall briefly discuss what is known about the 
action of some groups of mutagens. 

Alkylating Agents 
These include some of the most potent chemical mutagens — the 
"mustards", characterized by the presence of one or several chloro- 
ethyl groups, the epoxides, ethylene imines, methane sulphonates, 
and /3-propiolactone. A great number of alkylating agents have been 
synthesized for use in cancer therapy. The successful ones were sub- 
sequently tested on Drosophila, mainly by Fahmy and Fahmy (35) 1 in 

1 See References, page 136. 


London. Most of them were found to be mutagenic. The close cor- 
relation between carcinostatic and mutagenic abilities among the 
alkylating agents is therefore in large part due to a bias in the selec- 
tion of mutagens for testing. It is probable that chromosome breakage 
is the main mechanism by which alkylating agents kill dividing 
tumour cells. In view of the connection between mutation and chro- 
mosome breakage it would therefore be expected that alkylating 
agents with carcinostatic ability will be mutagens. The fact that many 
of them are also carcinogenic is less readily interpreted. It may be 
taken as support for the theory that the primary event in carcino- 
genesis may be a somatic mutation. 

Oncologists have found that all effective carcinostatic alkylating 
agents carry two or more active groups, e.g., ethylene inline or chloro- 
ethyl groups, and they put forward the theory that cross-linkage 
between biologically important molecules is a prerequisite for car- 
cinostatic as well as mutagenic ability. Yet monofunctional mustards 
have been found to be very effective in the production of mutations 
in Neurospora (88), and the monofunctional compounds ethylene 
oxide and ethylene inline are among the most effective mutagens for 
barley (31, 32), where they also produce many chromosome breaks. 
There still remained the possibility that polyfunctional compounds 
are relatively more effective than monofunctional ones in breaking 
chromosomes. If this were true, they should produce higher ratios of 
translocations to lethals in Drosophila. This was tested recently in 
our laboratory for the two compounds ethylene oxide and diepoxy- 
butane (65). The results suggested that cross-linkage plays no signifi- 
cant role in the production of chromosome breakage by alkylating 

Alkylating mutagens have been effective in all tested organisms 
from bacteria to mammals. Data on mammals are not easy to obtain. 
Two methods have been used successfully on mice. The first consists 
of injecting the chemical intraperitoneally and examining the Fi for 
heritable semisterility caused by induced translocations. By this 
method, translocations have been detected after treatment with 
nitrogen mustard (37), but toxicity was so strong that even with the 
highest tolerated dose the number of translocations was small. Possi- 
bly, treatment of semen for artificial insemination will be more 
successful and we intend to try this in our laboratory. Triethylene 

auerbach: effects of chemicals 123 

melamine (TEM) proved a much more suitable mutagen for mice 
(1G). Very high frequencies of dominant lethals and translocations 
were obtained alter intraperitoneal injection. One translocation is 
of particular interest because it produces a position effect on two 
recessive coat color genes in the neighborhood of the break. Both of 
these genes, when present in heterozygous combination with the 
translocation, give a flecked pattern (17). Position effects had pre- 
viously not been known in mice. Curiously enough, at the same time, 
several similar ones were found among X-ray-induced translocations 

A different method for testing mutagens on mammalian cells has 
been developed by Klein and Klein (52) and, independently, by 
Mitchison (61). It has been used in our Institute for testing TEM (27). 
Tumors are induced in hybrids between two inbred strains of mice 
which are isogenic apart from one allelic difference at the histo- 
compatibility locus. The hybrid tumors, possessing both antigens, 
will not take in either parental strain unless they have lost one of the 
antigens. The frequency with which this happens could be increased 
by TEM. Whether the underlying event is mutation proper, chromo- 
some loss, deficiency, or somatic crossing-over could not be decided. 

It is customary to interpret the effects of alkylating agents in 
terms of interaction with DNA. This may be correct, but it is not 
proved. The fact that the DNA, the transforming principle, is 
exceedingly sensitive to the destructive action of mustard gas (45) 
does not necessarily imply that the primary attack of mustard gas as a 
mutagen must be on DNA. Some evidence in favor of this assumption 
has recently been provided by the finding (58) that several alkylating 
agents, in particular ethyl methanesulphonate, can produce mutations 
in bacteriophage treated in vitro. 

Whatever its point of attack, mustard gas, like X-rays, act in a 
"hit"-wise fashion, that is, it produces mutations and chromosome 
breaks by independent reactions that affect separate points in a 
more or less random fashion. Eor X-rays this was established by stud- 
ies of dose-effect relations. Evidence of this type is not easy to obtain 
for the action of chemicals on a complex organism. For certain muta- 
gens a linear relationship between dose and effect has been shown 
to hold good within rather narrow ranges of cencentration (33, 42), 
but this finding is open to different interpretations. In particular, it 


suffers from the fact that the dose of a chemical as applied exter- 
nally bears only a remote relation to the amount that reaches the 
genes. We used a more indirect approach in experiments on Droso- 
phila (68). The effective dose of mustard gas was measured in terms 
of sex-linked lethals, and against this was plotted the frequency of 
translocations in the same tests. We found that translocation fre- 
quency increased almost exactly as the square of lethal frequency, 
and we took this to mean that mustard gas, like X-rays, acts by inde- 
pendent "hits" in or near the genetic material, one hit being suffi- 
cient to produce a lethal while two hits are required for a 

Corroborative evidence came from experiments in which treat- 
ment of Drosophila $ $ by mustard gas was immediately preceded or 
followed by X-rays (73). With both arrangements, the frequency 
of lethals after the combined treatment was the sum of the fre- 
quencies produced by either treatment separately. That of trans- 
locations was higher and calculation showed that it agreed well 
with what would be expected if rearrangements were formed indis- 
criminately from broken ends whether these had been produced by 
X-rays or mustard gas. It is interesting to note that Wolff and Luip- 
pold (93) obtained a different result when they exposed plant chro- 
mosomes to a combined treatment with neutrons and X-rays. In 
these experiments the frequency of interchanges was the sum of the 
frequencies that had been produced separately by either radiation. 
This was attributed to the fact that breaks produced by the closely 
ionized neutron track are not scattered at random throughout the 
nucleus, but lie close together and therefore tend to rejoin with 
each other rather than with independently produced X-ray breaks. 
It may be concluded that mustard gas, unlike neutrons, produces 
breaks which are scattered randomly. 


The ability of urethane to produce translocations in flower- 
ing plants was discovered by Oehlkers (71) at the beginning of the last 
war. Subsequently, the mutagenic action of urethane was con- 
firmed for Drosophila (92). Neurospora, on the other hand, has 
shown itself wholly refractory to its action (46). This may be con- 
nected with the fact that urethane shows an unusual degree of 
organism specificity also in its carcinogenic action. Rogers (77) found 

auerkach: effects of chemicals 125 

that the active principle in the production of lung tumors by 
urethane is a metabolite which is produced in mice but not in 
guinea-pigs. It is, therefore, possible that Neurospora is resistant 
to the mutagenic action of urethane because it cannot form the 
mutagenically effective metabolite. Oehlkers attributes the chro- 
mosome-breaking action of urethane in plant cells to general dis- 
turbances of metabolism. Rogers (78) has shown a close connec- 
tion between urethane and nucleic acid metabolism. In Droso- 
phila breaks produced by urethane and X-rays interact freely with 
each other to give the number of rearrangements expected from 
the overall number of breaks by both treatments (73). 


Oehlkers and his collaborators (72) have found that a num- 
ber of pharmacologically important alkaloids, such as morphine 
and scopolamine, produce chromosome rearrangements in plants. 
Quite recently, Clark in Australia (20) has established that certain 
alkaloids, e.g., heliotrin and related substances, are highly effec- 
tive mutagens for Drosophila. These substances occur in Senecio 
and some other plants and cause liver disease in sheep. 


This group forms a link between chemical and radiation muta- 
genesis, for peroxides have been shown to play the role of interme- 
diates in mutagenesis by ultraviolet light and, probably, X-rays. 
Hydrogen peroxide is a weak mutagen for micro-organisms (46), but 
is wholly ineffective in Drosophila where it is quickly destroyed 
by catalase. Certain organic peroxides, on the other hand, are effec- 
tive mutagens for both Drosophila and Neurospora (3, 28, 82). 


The mutagenic action of formaldehyde was discovered by 
Rapoport (74) soon after the last war. He obtained high mutation 
frequencies by mixing formaldehyde with the food of Drosophila 
larvae. Mutations can also be produced by injecting aqueous solu- 
tions of formaldehyde into the abdomen of adult flies (6). This is 
a less effective method but will be discussed first because there are 
reasons to believe that it acts via the formation of organic peroxides 
from formaldehyde and metabolically produced hydrogen perox- 


ide. Mutation frequency is enhanced in Drosophila $ $ whose cata- 
lase has been poisoned by KCN (83, 8-1). Under these conditions, 
formaldehyde injection even produces some mutations in 9 9 , which 
otherwise are quite refractory to it. Solutions of formaldehyde are 
but weakly mutagenic lor conidia of Neurospora; so are solutions 
of hydrogen peroxide (46). A mixture of both compounds in solution 
is strongly mutagenic (28), and addition of dihydroxydimethyl 
peroxide compound is a good mutagen for Drosophila (82). 

When formaldehyde is applied as an admixture to the food 
of Drosophila, it probably acts in quite a different way. While 
injection produces mutations mainly in mature spermatozoa, form- 
aldehyde food acts preferentially or exclusively on the long- 
growth stage of the primary spermatocyte that, in the larvae, pre- 
cedes meiosis (9). Female larvae are wholly resistant to formaldehyde 
food; so are adults. Experiments with isotopically labelled formal- 
dehyde (47) have shown that lack of penetration into certain cells 
cannot be responsible for these results; for large amounts of labelled 
formaldehyde were found inside the germ cells of adults, of female 
larvae, and of spermatogonia. The fact that bunches of identical 
or complementary cross-overs were obtained from treated $ larvae 
(85) shows that the effective substance enters the nucleus of sperma- 
togonia; yet it hardly, if ever, produces mutations at this stage, 
for several hundred cross-tests between autosomal lethals that 
appeared as clusters in the progeny of individual $ 6 yielded no 
clusters of identical lethals (9). 

It is tempting to assume that the preferential mutagenic action 
of formaldehyde food on the growth stage of the primary sperma- 
tocyte is somehow connected with the synthetic processes going on 
during this stage. This assumption is supported by various observa- 
tions. Thus, any condition that slows down larval development 
reduces the mutagenic effectiveness of formaldehyde food (8). More 
recently, Alderson found that, in a synthetic and sterile medium, 
formaldehyde requires adenosine riboside for its mutagenic action 

Substances Related to Nucleic Acid 

Even before DNA acquired its predominant role in our model 
of the genetic material, substances related to nucleic acid metab- 
olism were tested for mutagenic activity, many of them with success. 

auert-ach: effects of chemicals 127 

Acridines, which react with nucleic acid, have produced mutations 
in at least one series of experiments with barley (22). One of them, 
proflavine, is a very effective mutagen for bacteriophage (15). Pyronin, 
which reacts with ribonucleic acid, is mutagenic for Drosophila 
(18). Its overall effect is weak, but certain treated individuals 
yield very high mutation frequencies. It seems that very special 
conditions have to be fulfilled for its effective action (7, 19). 

Many purines produce mutations in micro-organisms (41, 70), 
and mutations and chromosome breaks in plants (49). For many 
years, no mutagenic pyrimidines were found in spite of attempts 
to detect them. This has changed since it was discovered that it 
is possible to force bacterial cells to take in analogues of the 
normal pyrimidine bases by closing the normal pathway leading 
to pyrimidine synthesis. Bacteria that are unable to synthesize 
thymine may replace it quantitatively with offered bromouracil. 
Mutations occur in such bacteria and in bacteriophages grown on 
them (13, 57, 79). 

The fact that bromouracil is so readily incorporated into 
bacterial DNA makes it tempting to assume that it is mutagenic 
through incorporation, and ingenious schemes of mutagenesis 
through base changes in the genetic DNA have been based on this 
assumption (40). Some mutagenic purines, such as caffeine, are 
either not incorporated at all or only in indetectably small amounts 
(69) and proflavine probably does not act by incorporation. For 
purines, interference with enzymes concerned with nucleic acid 
metabolism has been suggested as an alternative cause of muta- 
genic ability (69). Particularly interesting, though still unexplained, 
is the fact that in bacteria mutagenesis by purines like caffeine and 
theophylline can be prevented by "antimutagens" in particular 
adenosine riboside (69). Radiation-induced mutation frequency is 
not depressed by these antimutagens, and a certain proportion of 
spontaneous mutations is likewise refractory to them. A search for 
similar systems of mutagen-antimutagen balance seems a promis- 
ing approach to a study of the complex biochemical interactions in 

Nitrous Acid 

In 1939, Thorn and Steinberg (90) produced variants in Asper- 
gillus grown on food with an admixture of mannitol-nitrite. Ten 


year later, admixture of sodium nitrite to the food of Drosophila 
produced mutations in one experiment, but repetitions gave 
negative results (76). These tests had been undertaken with the idea 
that nitrite, like formaldehyde, might produce mutations by acting 
on the proteins, which then were thought to be carriers of genetical 
specificity. Quite recently, nitrite has been used again and with spec- 
tacular success. Starting with very precise chemical predictions 
about desamination of the bases in nucleic acid, a group of Ger- 
man workers (63) found a method for the production of mutations 
in tobacco mosaic virus treated hi vitro. In this case, there can be 
little, if any, doubt that mutations were produced by direct chemical 
action on the genetical material, for treatment was equally effective 
when applied to the naked RNA of the virus. Subsequently, sim- 
ilar results have been obtained with the RNA of polio virus (14) 
and the DNA of the pneumococcus-transforming principle (56). 
Nitrite has also been used successfully for the production of muta- 
tions in bacteriophage (91) and bacteria (48). Treatment of bac- 
teriophage produced many sectored mutants, as would be expected 
from chemical reaction with one of the two strands of DNA. When 
an exceptional phage strain with single-stranded DNA was treated, 
there were no sectored mutants (89). Although it seems plausible 
to assume that nitrite also acts directly on DNA in bacteria, proof is 
still lacking. 

In conclusion of this section I would like to say this. Work with 
chemical mutagens has not thrown new light on the nature of the 
gene, except insofar as the effects of certain mutagens can be under- 
stood best from our present model of gene structure. In my opinion, 
it would be regrettable if further research were directed wholly 
towards the study of this particular class of mutagens. The very 
variety of mutagenic chemicals shows that the stability of the hered- 
itary mechanism is under complex control. Analysis of many 
different types of mutagen may help us unravel some of these 
complexities. It may also teach us how to loosen the control in ways 
that produce desirable mutations for applied purposes. 

The Relation Between Intergenic and Intragenic Changes 

The question whether there is an essential difference between 
intergenic and intragenic changes has been often discussed (62), 

auerbach: effects of chemicals 129 

sometimes heatedly. There are good reasons, theoretical as well as 
observational, for answering; it in the affirmative, but decisive 
experimental evidence would be desirable. One possibility of obtain- 
ing such evidence was to look for a chemical that would produce 
gene mutations but no chromosome breakage. No clear case of 
this kind has been encountered. Although mutagens differ widely 
in their chromosome-breaking ability, most of them can be shown 
to possess this ability to some extent and none can be shown not 
to possess it. The opposite is not true. Several compounds that 
break the chromosomes of plants have failed to produce mutations 
in Drosophila. This, however, cannot be taken as evidence that 
these substances act on intergenic bonds rather than on genes, unless 
it can be shown that they can also break the chromosomes of Droso- 
phila. A priori, it is probable that substances that can do this will 
also produce small deficiencies that appear as recessive lethals, and 
position effect rearrangements that appear as visible mutations. 
There are good reasons for supposing that a substance that breaks 
plant chromosomes may not have the same effect on Drosophila 
chromosomes. Not only are the metabolic differences between these 
organisms likely to affect the action of introduced chemicals. It 
should also be kept in mind that plant chromosomes, in contrast 
to the very stable chromosomes in the germ cells of Drosophila, 
are easily broken, e.g., by low doses of X-rays, excessive oxygen 
pressure (21) or, conversely, anoxia (60). Thus, only a substance 
that fails to produce mutations in the same organism in which it 
produces chromosome breaks can be used as evidence in favor 
of distinct intergenic bonds. No clear case of such a substance has 
so far been found. 

Again chemical mutation research has failed to provide a 
decisive answer to a definite question. An answer, albeit still a 
partial one, has come from a different field of genetics. Analysis of 
the fine structure of the genetic material in bacteriophage has shown 
that a mutation may affect only one or at most a few nucleotides 
(12), surely a change that must be called intragenic. Since, however, 
the chromosomes of higher organisms are much more highly struc- 
tured than those of bacteriophage and, in particular, have various 
types of protein intimately associated with their DNA, the rela- 
tion between intergenic and intragenic changes remains an impor- 


t an t subject of investigation. Crystal lographic, electronmicroscopic, 
biochemical, and biophysical investigations are likely to play a 
major role in this research. Analysis of the effects of chemical muta- 
gens will, I think, be mainly important by showing the complex- 
ity of the processes that result in chromosome breakage and by 
separating out some of the relevant factors. 

One very important factor has been discovered in radiation 
studies. Wolff and Luipold (93), found that the reunion of broken 
chromosome ends is an energy-requiring process which proceeds 
only under conditions that permit protein synthesis. This makes it 
necessary to distinguish between the effects of mutagens on the 
cohesion of the chromosomes and on the mechanism of reunion. 
A chemical may affect these processes separately, and the effects 
may differ in dependence on the cellular environment. Thus, chro- 
mosomes that have been broken by 8-ethoxycaffeine readily form 
rearrangements in Vicia, but tend to remain as free fragments in 
Allium (50). Conversely, urethane produces mainly fragments in 
Vicia and mainly rearrangements in Oenothera (26). Discrepan- 
cies between the reports of cytologists on the effects of a given 
mutagen will often be due to differences in the choice of organism 
or cell stage. 

Only barley and Drosophila have been used for systematic 
attempts at comparing the intergenic and intragenic effects of chem- 
icals. In Drosophila, most chemicals produce fewer rearrangements 
for a given frequency of mutations (sex-linked lethals) than do 
X-rays. That this is not due to a shortage of breaks but to failures 
of reunion has been shown for two substances, mustard gas (68) and 
TEM (34). Compared with a dose of X-rays giving the same fre- 
quency of sex-linked lethals, both these substances produce too lew 
translocations but at least as many chromosome fragmentations 
resulting in zygotic lethals. The main cause for the relative shortage 
of reunions is apparently the delayed effect of these compounds; 
for breaks that open in different cell cycles cannot form rearrange- 
ments. Indeed, when mustard gas-treated spermatozoa were stored 
in the seminal receptacles of the 9 , there was an increase in the 
frequency of large rearrangements (5). 

The tendency of chemical mutagens to produce intrachromo- 
somal rather than interchromosomal changes mav be likewise a 

aueruach: effects of chemicals 131 

consequence of their delayed action, if it is assumed that latent 
breaks in the same chromosome have a greater chance than latent 
breaks in different chromosomes to open in the same cell cycle (81). 

Storing of mustard gas-treated spermatozoa in the seminal 
receptacles of 5 5 did not affect the frequency oi sex-linked lethals, 
although calculations showed that the increase should have been 
perceptible if delayed mutation had occurred at the same rate as 
delayed breakage (10). Yet it is known that mutation is often delayed 
after mustard gas treatment (4). It appears, therefore, that delayed 
gene mutation requires replication of the chromosome, while 
delayed breakage does not. If this can be firmly established, it will 
form a distinguishing feature between chemically induced intergenic 
and intragenic changes in Drosophila. 

Even if the mechanisms, by which mustard gas produces gene 
mutations and chromosome breaks should differ in their final 
stages, they must have a common initial step. This follows already 
from the previously mentioned experiment in which it was shown 
that the frequency of translocations increases as the square of the 
frequency of lethals (68). It is further substantiated by the find- 
ing that during the late spermatogonial stage, which is the most 
highly sensitive one to the mutagenic effects of mustard gas, lethals 
and breaks leading to translocations increase proportionally in 
frequency (87). 

Minute deficiencies and other minute rearrangements occupy 
a somewhat intermediate position between gene mutations and 
large chromosome rearrangements. This is so in radiation muta- 
genesis, where the frequency of minute rearrangements increases 
linearly with dose, that of large rearrangements more nearly as 
the square of dose. It is also so in chemical mutagenesis, where 
compounds that are inferior to X-rays in the production of large 
rearrangements may yet be superior in the production of minute 
ones (36, 81). It is possible that a proportion of minute deficien- 
cies is produced not by breakage and reunion but by some other 
mechanism such as unequal crossing-over, or errors in chromosome 
replication; but this is certainly not true for all of them, and probably 
only for a small minority. Reverse repeats, e.g., cannot arise without 
previous chromosome breakage, and they have been found repeatedly 
after exposure of Drosophila to chemical mutagens (59, 67, 81). The 


tendency of chemical mutagens to produce minute rather than 
large rearrangements may be due to some spreading effects of the 
chemical damage. 

Of particular interest is the ability of a few — and possibly of 
more — chemical mutagens to produce high frequencies of small 
duplications. Many duplications were found in Vicia chromosomes 
that had been treated with nitrogen mustard (38), and formalde- 
hyde food produced many duplications in Drosophila (81). 

While there is good evidence that a common mechanism is 
responsible for the production of gene mutations, chromosome 
breaks, and minute rearrangements by chemicals, crossing-over 
seems to be induced in some different way. Formaldehyde food pro- 
duces crossing-over but not mutations in spermatogonia (83, 86). For 
a given frequency of mutations, mustard gas produces considerably 
fewer cross-overs than does formaldehyde (85). The relative fre- 
quencies of lethals and induced cross-overs were markedly changed 
when mustard gas was given to male pupae rather than imagines 
(10). These findings agree with observations on plants which show 
that there is no correlation between the ability of a chemical to 
produce translocations and its effect on chiasma frequency (55). 
Further, chemical treatment of plants can produce translocations 
in pachytene when crossing-over is completed (54). 

In barley, gene mutations are scored as segregants for visible 
abnormalities, mainly chlorophyll defects, in F 2 , while chromo- 
somal changes are recognized by their effect on the fertility of the F t . 
Judged by these criteria, chemical mutagens differ widely in their 
relative abilities to produce intergenic and intragenic changes (30). 
Curiously enough, the extremes are formed by two purines — 8- 
ethoxycafFeine, which produces almost exclusively chromosome 
changes, and nebularine (purine-9-riboside) which produces almost 
exclusively mutations. Unfortuntately, it is not possible to deter- 
mine how many of the segregating chlorophyll mutations are caused 
by small deficiencies or rearrangements. Even so, the striking dif- 
ference between the effects of these two substances, and similar 
differences between other chemicals, lend support to the model of 
a chromosome in which the genes are connected by links of non- 
gen ic material. 

auerbach: effects of chemicals 133 

The Production of Specific Genetical Changes 

From its early stages, work with chemical mutagens brought 
evidence for the possibility that these substances might be more 
specific than X-rays in their effects on the genetic material. Dur- 
ing many years, however, the detectable specificity was regional 
rather than genie. The distribution of chemically induced chromo- 
some breaks in plants was found not to be random (38, 51, 72, 75). 
After treatment with certain chemicals, it was highly localized (23). 
Most chemicals appear to produce more breaks in heterochromatic 
than dichromatic regions. In Drosophila, too, the distribution of 
sex-linked lethals over the X-chromosomes was found to differ 
between X-rays and certain chemicals (11, 35). 

The discovery of specific effects of mutagens on individual 
loci meant a great step forward in mutation research. Many cases 
of "mutagen specificity" are now known in a variety of organisms, 
but it is very doubtful whether all of them have a similar basis. 
The most striking example is that of one particular region in the 
chromosome of bacteriophage T4 (39), where mutations induced 
by 5-bromouracil or 2-aminopurine are crowded together into pre- 
ferred sites. While it is at least probable that these "hot spots" arise 
from specific interactions between mutagen and genetic site, this is 
not necessarily — and not even plausibly — so in many other cases of 
mutagen specificity. In bacteria (24) and fungi (53), different loci and 
different alleles of the same locus show different mutational reponses 
to a variety of physical and chemical mutagens. In extreme cases, 
a gene may be "mutagen stable", i.e., it may fail to respond to some 
or all tested mutagens, while yet being able to mutate spontan- 
eously (25). These "mutation spectra" can be profoundly changed 
by introduction of an additional mutant gene into the same nu- 
cleus (43). We do not yet know how this is brought about, but vari- 
ous explanations can be envisaged and tested. An influence of the 
residual genotype on the reaction between gene and mutagen is 
the least likely one. We have come to realize that mutation is a 
complicated process of which the reaction between mutagen and 
gene is only one step, although it is the essential one. Interactions 
between mutagen and cytoplasm, conditions of the cell preceding 
and following treatment, degree and type of competition between 
the newly mutated cell and the remaining non-mutated ones, as 


well as other circumstances which it would lead too far to dis- 
cuss here, all these decide whether the essential step will take place 
and whether it will result in a detectable mutation. Specificity may 
occur at any one of these levels; at which one, cannot be decided 
without special analysis of each individual case. 

In higher organisms, apparent mutagen specificity at individ- 
ual loci may be an expression of regional specificity along the chro- 
mosome. In Drosophila the distribution of visible mutations over 
the X-chromosome depends to some extent on the mutagenic treat- 
ment (36). In the silkworm, two linked genes determining the color 
of the embryonic membranes appear to mutate in different ratios 
after treatment with X-rays and several chemical mutagens (66). 
Most or all of these apparent mutations are due, however, to defi- 
ciencies, and the specific responses observed are therefore properties 
of the chromosome regions in which the genes are located. Also, spec- 
ificity may occur at some later step in the mutagenic process, such 
as the rejoining of broken chromosome ends. This is suggested by 
the observation that the differential response of the two loci to muta- 
gens varies with the sex of the treated animal (64). 

The mutagen specificities that have been observed in barley 
give rise to similar ambiguities of interpretation. The mutation 
spectra of both chlorophyll and erectoides mutations have been 
shown to vary according to the mutagen used (44). Some of these spec- 
ificities have been correlated with detectable chromosomal aberra- 
tions. How many may be due to differences in the frequencies and 
types of undetected chromosome rearrangements cannot at present 
be decided. Moreover, in barley as in the silkworm, the mutation 
spectrum is under the influence of physiological conditions (29), 
such as hydration or degree of pregermination, and thus is unlikely 
to reflect specificities of chemical interaction at the level of the 
gene itself. 

These doubts concerning the origin of specific responses to 
mutagens do in no way detract from the great theoretical and prac- 
tical importance of the observed phenomenon of mutagen specificity. 
Theoretically, the recognition that mutagen specificity need not 
always result from specific chemical reactions between mutagens and 
nucleotides should make the geneticist more rather than less inter- 
ested in chemical mutagens. It should make him realize that chemical 

auerbach: effects of chemicals 135 

imitation research, in addition to and often instead of supplying the 
chemist with food for speculation, is one of the best tools for ana- 
lyzing the fascinating series of events that culminate in induced 
mutation. The practical value of mutagen specificity depends on 
economical considerations and not on theories about the origin of 
specificity. If a treatment can be found that profitably produces 
a desired type of mutation, this will be a tremendous practical 
success, whatever its theoretical basis. Indeed, it seems to me that 
specificity of the reaction between mutagen and DNA is least 
likely to lead to such a success. Research with micro-organisms 
strongly suggests that this type of specificity differs between alleles 
and sites of the same locus; in general, it will therefore not appear 
as locus specificity. The aim of mutation breeding, on the other 
hand, is phenotypical specificity, that is the specific production of 
mutations that yield a desired phenotypical effect. If such specificity 
exists at all, it is more likely to occur at a late step in mutagenesis, 
when mutations leading to similar final effects may have a common 
biochemical pathway. A judicious combination of mutagenic and 
anti-mutagenic treatment may be one approach to its detection. 

If we now look back at the questions with which we started 
this stocktaking of progress in chemical mutagenesis, it turns out, 
surprisingly, that the greatest progress has been made along the path 
that at first seemed by far the steepest, the path directed towards the 
detection of mutagen specificity. Progress along the two other paths, 
directed towards analysis of the chemical nature of the gene and of 
the relationship between intergenic and intragenic changes, has been 
somewhat disappointing. I believe this has been due mainly to two 
opposing tendencies on the part of investigators. One is the tendency 
to disperse efforts by hunting for more and more mutagens. The 
other, which comes into play when a new mutagen has been detected, 
is the tendency to channel all efforts at analysis into the field of 
nucleic acid chemistry. Outside these two methods of approach lie 
many neglected possibilities of biological analysis, whose exploration 
almost certainly Avill contribute to our understanding of the structure 
of higher chromosomes and of the complexities of the mutagenic 



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Ross: Is there any evidence for chemical specificity either for chromo- 
some breaks or gene mutations at the same locus of both homologues 
of a chromosome in one cell? 

Auerbacii: This is a possibility that interests me very much. I hope that 
it will be tried this year in our Institute by Doctor Oster who has 
worked out a scheme for testing it in Drosophila. 

Milan: Results obtained at 1'ullman with diethyl sulfate have some 
bearing on Doctor Auerbach's discussion of the delayed effect of cer- 
tain chemical mutagens on chromosomes. In barley, very high fre- 
quencies of chlorophyll-deficient seedling mutations were induced by 
this chemical. However, only a very few chromosome aberrations were 
found. Such aberrations, resulting from chromosome interchanges, are 
common following irradiation. On further study, some evidence for 
an appreciable frequency of inversions has been obtained. Thus, it 
appeal's that more intrachromosomal than interchromosomal rearrange- 
ments have occurred, indicating a delayed effect of this chemical on 

Caldecott: In regard to the relation between chemical mutagens and 
ionizing radiations, I would like to re-emphasize the importance of the 
physiological state of the cell and the kinds and frequencies of genetic 
damage that can be detected. Clearly, it would be quite impossible to 
design an experiment where there coidd be a precise comparison between 

Auerbach: I quite agree; it is too often overlooked that any mutagenic 
treatment — in particular with a chemical — allects the cell, and some- 
times the organism as a whole, in addition to acting on the chromo- 

auerbach: effects of chemicals 143 

MacKfy: I greatly appreciate the cautiousness shown by Dr. Auer- 
bach in discussing results from different organisms in relation to muta- 
genic treatment. I believe there is great danger in trying to explain 
different radiobiological phenomena observed in different research 
objects according to one pattern. For example, the radiosensitivity in 
relation to polyploidy goes a completely different way in a ploidy series 
of wheat or any higher plant and yeast. From diploid level onwards, 
radioresistance gradually increases with genome number in the first 
case but decreases in the latter. The results cannot be explained only 
by substitution of cells in the multicellular organism. It seems likely that 
a factor not yet discussed here intervenes, viz., the mutual interaction of 
cells in a multicellular tissue. Work by Dr. A. M. Clark with Habro- 
bacon favors such an interpretation. He found haploid embryos more 
resistant to X-rays during cleavage. No significant difference in radio- 
sensitivity existed at blastema and earl) larval stages, and at late larval, 
prepupal, and pupal stages, the diploids shoAved the higher resistance. 

Kramer: There is perhaps some precedent for this approach in the 
use of such chelating agents as ethylene diamine tetra acetic acid on 
Chlamydomonas and Drosophila. Some preliminary work by Nuffer, I 
believe, has indicated less success with corn, but this may be due to 
inadequate methods of treatment. Surely this approach has value both 
in plant breeding and in learning more about recombination. 

Auerbach: The importance of induced recombination for plant breed- 
ing deserves, I think, more consideration than it has received so far. 
It seems that the efficiencies of mutagens in affecting crossing over, on 
the one hand, and producing chromosome rearrangements, on the other, 
are only loosely correlated. It might, therefore, be promising to under- 
take a screening program — first, cytologically, then genetically — in a suit- 
able organism like maize for chemicals that enhance recombination 
with little concomitant sterility or other undesirable effects resulting 
from rearrangements. 

Vallentyne: I would like to comment briefly on the first of your three 
initial hopes, viz., the possibility of learning something about the chem- 
ical nature of the hereditary unit from a knowledge of the chemistry of 
mutagenic substances. As you were talking about the action of formalde- 
hyde and nitrous acid, it occurred to me that there is a reaction that 
should be considered in relation to the natural process of mutation. 
This is the reaction between keto and amino compounds first studied 


by L. C. Maillard in 1912, and subsequently known as the Maillard 
reaction (or the browning reaction in food chemistry). It is of interest 
that the reaction proceeds at measurable rates in the temperature range 
to which living matter is normally subjected; secondly, that the reac- 
tive groups are not only present in DNA, but present in juxtaposition; 
and thirdly, that the groups involved in the reaction are those that give 
specificity to the bases of DNA which in turn give specificity to the 
DNA itself. The juxtaposition of the keto and amino groups in 
the Watson-Crick model of DNA seems rather unusual in terms 
of the possibility of a Maillard-type reaction. Perhaps the groups 
are protected by the very existence of hydrogen-bonding in the double 
helical arrangement. This I do not know. It is conceivable, however, 
that the groups would be reactive on rupture of the double helix, either 
with each other or with keto and amino compounds in the surrounding 
nuclear plasm of cytoplasm. I wonder if the Maillard reaction could be 
operating in nature to produce mutation in a manner analogous to 
that of nitrous acid. Would you care to comment on this? 

Auerbach: I am afraid my knowledge of chemistry is not good enough 
for an answer to this interesting question. 

Effects of Preirradiation and Postirradiation Cellular 
Synthetic Events on Mutation Induction in Bacteria 1 


The University of Texas M. D. Anderson Hospital and Tumor Institute, 
Houston, Texas 

During the past few years several investigations have made it 
evident that the processes leading to mutation induction by 
ultraviolet light (UV) are intimately related to gene replication, 
which is then followed by the influence of the modified gene in 
enzyme synthesis. Since these events are primarily biochemical, 
involving interrelations in the syntheses of deoxyribonucleic acid 
(DNA) ribonucleic acid (RNA), and protein, it seems reasonable 
that one can no longer examine such biological phenomena as muta- 
tion induction and expression without simultaneously studying their 
biochemical basis. Conversely, mutation induction and mutation 
expression are endpoints of the biochemical events of DNA replica- 
tion and genetic control of enzyme synthesis respectively, and they 
may serve as useful tools in working out the biochemistry of these 

This paper is concerned with several theoretical aspects of muta- 
tion induction which have been investigated by such combined bio- 
chemical and biological studies. The experimental results also have 
some implications for modern theories of genetic DNA replication. 
At the very least, it is hoped that the value and relative ease of 
performing such integrated studies will be demonstrated. 

The importance of the physiological state of the biological mate- 
rial used in such investigations, and probably in all biological investi- 
gations, cannot be overemphasized. It is obvious that the same 
biochemical and physiological processes are not operating at all 
times during the life cycle of any cell. Important events may be 
entirely obscured when working with random populations composed 
of members of all ages and stages of physiological development. 

x This investigaton is supported in part by research grant C-3323 from the National 
Institutes of Health and by Atomic Energy Commission contract AT-(40-l)-2139. 

Postdoctoral Research Fellow; Research Training Grant CRT-5047 from the 
National Institutes of Health. 



The experiments presented here were carried out with an 
appreciation both for the simultaneous study of biochemistry and 
biology, and for uniformity of the biological material. Various strains 
of the bacterium Escherichia coli were employed, and in all experi- 
ments the bacteria used were synchronized as to cell division and 
development by a cold treatment method (I7). 3 In most of the experi- 
ments the syntheses of RNA, DNA, and protein were followed in the 
cultures simultaneously with mutation induction and expression. 

Materials and Methods 
Description of bacterial strains and the mutation followed in each 

E. coli strain B. — Originally obtained from Oak Ridge National 
Laboratory stock of Dr. A. Hollaender. Mutation — aberrant colonial 
color response on Difco eosin-methylene blue (EMB) agar after 2 days 
incubation at 37° C. 

E. coli strain WP2. — Originally isolated by Witkin (26) as a 
tryptophan-requiring mutant of E. coli strain B/r. Mutation — rever- 
sion of the tryptophan requirement to the non-requiring state. 

E. coli strain 1 5 T - ML ,- T .vr- — A triple auxotroph of E. coli strain B/r 
requiring thymine, methionine, and tyrosine isolated in our labora- 
tory from thymine-requiring E. coli strain I5 T - following ultraviolet 
light exposure. Strain 15 T - was obtained from Dr. S. Zamenhoff of 
Columbia University. Mutation — reversion of tyrosine requirement. 

Description of growth media 

M medium. — A salts-glucose basal growth medium to which 
various supplements under test, or various metabolic inhibitors, were 
added. The composition of the medium has previously been described 
(7). In experiments using auxotrophs appropriate amounts of the 
required growth factors were added. All preirradiation and post- 
irradiation incubation was carried out in liquid M medium supple- 
mented as indicated in the various experiments. 

Agar plating medium 

1. For color mutants. — Difco EMB agar was used as the final 
plating medium. Survivors and mutations were scored on the same 

3 See References, page 169. 


2. For prototrophic reversion. — M medium supplemented with 
2.5 per cent Difco nutrient broth and solidified with 3 per cent Bacto 
agar was used for final platings. The low amino acid concentrations 
in this medium allows unreverted cells to make sufficient divisions to 
develop small visible colonies (26). It also enables reverted cells to 
become phenotypically expressed. Unreverted survivors are counted 
as small colonies on high-dilution platings, while revertants appear 
as large colonies against a background of auxotrophic growth at low 

3. For mutation expression. — M medium solidified with 3 per 
cent Bacto agar was used. No nutrient broth was added to this 

Radiation sources 

Ultraviolet irradiation (W). — A model 30600 Hanovia mercury- 
vapor lamp. The UY output at the position of the cells was 92.5 
ergs/mm 2 /sec at wave lengths below 2800 A (determined by a Han- 
ovia model AY-971 ultraviolet meter). 

X-ray. — A General Electric "Maxitron 250" unit set at 200 KVP 
and 30 MA, with 1-imn aluminum filtration added. X-ray output at 
the locus of cell suspensions was approximately 2860 r/min. 

Preirradiation and postirradiation treatments 

The techniques employed have been previously described (3, 4, 
7, 9). Briefly they are as follows: A 24-hour-old slant culture was used 
to inoculate 50 ml sterile M medium (plus growth factors in the case 
of auxotrophic strains). This culture was grown for 15.5 hours at 
37° C with aeration, and then held at 6 C for 1 hour to synchronize 
cell division. The cells were centrifuged down in the cold and 
resuspended in 50-ml fresh M medium (plus supplements if neces- 
sary). In the preirradiation experiments supplements under test were 
added to this medium. The culture was grown for an additional 
period, aliquots taken for test at various intervals, and chilled to halt 
further growth. For postirradiation experiments the cells were incu- 
bated for 50 minutes before chilling. Chilled cells were washed with 
cold 0.9 per cent saline and M medium, then resuspended in cold 
M medium so as to titer approximately 6 X 10 8 colony-forming organ- 
isms per ml in the UY studies and 2 X 10 10 organisms per ml in the 
X-ray studies. Aliquots of this suspension at proper dilution were 


plated on appropriate medium for determination of the number of 
organisms subjected to the radiation. Other aliquots were irradiated 
with UV or X-ray depending on the experiment. 

In preirradiation supplementation experiments, immediately 
following irradiation, appropriate dilutions were plated on agar plat- 
ing media by the glass rod spreader technique. 

For postirradiation experiments aliquots of the irradiated culture 
were diluted 1 :4 into appropriately supplemented growth medium 
and incubated at 37° C on a reciprocal shaker for time intervals 
indicated in the various experiments prior to plating on agar media. 
All plates were incubated at 37° C for 2 days in the case of color 
mutants and for 3 days in the cases of prototrophic reversions before 
scoring: for survivors and mutants. 

Biochemical determinations 

Culture samples were taken at indicated intervals during pre- 
irradiation and postirradiation incubation for analysis of RNA, 
DNA, and protein. The samples were precipitated and washed with 
0.5N perchloric acid, and the nucleic acids then hydrolyzed by incu- 
bation in perchloric acid for 50 minutes at 70° C (20). They were 
then analyzed for DNA content by the Burton (1) method. For RNA 
determination, the UV absorption at 260 mil and 290 rati were deter- 
mined (25), and the amount of DNA, as determined by the Burton 
analysis, subtracted with correction for extinction coefficients. Protein 
was determined by the Folin (15) method. 

Experimental Results and Discussion 

I. Effect of Preirradiation Growth Factor Supplementation on 
Radiation-Induced Mutations 

Early experiments were carried out using E. coli strain B, and 
consisted of attempts to identify possible extragenic factors which 
might be affected by UV so as to produce mutations. These experi- 
ments have been previously reported (7). The induced mutation- 
radiation dose curves previously obtained by many investigators 
suggested that radiation-sensitive material present in the cell was 
activated or altered by radiation as a prerequisite to mutation induc- 
tion. The material also appeared to be limited in amount since the 
induced mutation frequency leveled off at high doses of radiation 
(18, 19, 29). 


The mutations followed were those giving aberrant colonial 
color response on EMB agar (color mutants). Synchronized cell cul- 
tures were incubated for 1 hour in M medium supplemented with 
either casein hydrolysate (2 mg/ml), purines and pyrimidines (0.01 
mg/ml each of adenine, guanine, cytosine, and uracil), or with a 
mixture of B vitamins (1 (.ig/inl of each). Following incubation, ali- 
quots were irradiated with increasing doses of UV and then dilutions 
were plated on EMB agar. The results indicated that cells incubated 
with purines and pyrimidines yielded somewhat higher induced 
mutation frequencies following UV exposure. Other supplements 
produced no changes in the subsequent UV-induced mutation fre- 
quency from that obtained with the unsupplemented control except 
for the growth factors, riboflavin and p-amino benzoic acid. Further 
investigation, using various combinations of purines and pyrimidines 
and a UV dose giving maximum mutation frequency, demonstrated 
that maximum increase in mutation frequency was obtained when 
uracil, cytosine, and either adenine or guanine were present. Substi- 
tution of thymine for uracil resulted in considerable reduction in 
the mutation frequency obtained (7). 

Experiments were next conducted to determine the duration of 
incubation in purines and pyrimidines necessary for attaining the 
maximum increase in UV-induced mutation. It was found that an 
initial incubation period of 20 minutes in purine-pyrimidine medium 
is necessary for initiation of the increase in subsequent radiation- 
induced mutation frequency. Following this lag a rapid increase is 
observed and the maximum frequency is attained at 30 to 35 minutes 
incubation. On the other hand, when cells are incubated in yeast 
extract there is no lag, and increase in susceptibility to subsequent 
mutation induction starts immediately with incubation. This sug- 
gested the possibility that the lag period observed with purine- 
pyrimidine incubation represents the time necessary for synthesizing 
radiation-reactive substances from the purines and pyrimidines. 
Further experiments, using ribosides (adenosine, guanosine, uridine, 
and cytidine) supported this concept since these supplements led to 
reduction of the lag period to less than 5 minutes (7). 

Similar experiments using X-ray suggest that a large fraction of 
X-ray-induced mutations are mediated through similar mechanisms 
(8). Incubation of cells in yeast extract or purine-pyrimidine medium 


prior to irradiating increases the induced mutation frequency some- 
what, although the effect is not as marked as with UV. 

II. Effect of Postirradiation Treatments on the Fate of Potent ial 
UV-induced Mutations 

Witkin (26) lias suggested that the immediate postirradiation 
synthesis of protein is necessary for expression of induced prototrophs 
with certain auxotrophic strains of E. coli and Salmonella typliimuri- 
um. In her experiments, expression of induced prototrophic mutants 
was directly related to availability of a complex supply of amino acids 
during the first hour of postirradiation growth. Chloramphenicol 
(CMP), which specifically interferes with protein synthesis, prevented 
mutation expression if the cells were treated during the first post- 
irradiation hour. We have confirmed the existence of similar relation- 
ships for the color mutations of E. coli strain P>, and have shown that 
the mutation frequency increase due to preirradiation incubation in 
purines and pyrimidines is also dependent on postirradiation amino 
acid supply. Involvement of postirradiation macromolecular syn- 
theses (protein, RNA, DNA) in the mutagenic process has been 
studied for both color mutants of strain B and for prototrophic muta- 
tions of various auxotrophic strains. While the results were essentially 
comparable, for technical reasons most of these postirradiation studies 
were accomplished with the latter system. 

Mutation frequency decline 

Involvement of metabolic processes in mutation induction has 
been studied by observing the effects of various conditions limiting 
to amino acid or protein synthesis on UV-induced mutation (3, 4, 8). 
Immediately following irradiation the cells were incubated in 
nitrogen-free medium for periods of time varying from to 90 
minutes, prior to plating on M plus 2.5 per cent nutrient broth agar. 
Using this technique, it was found that when nitrogen-dependent 
synthetic activities are limited there is a rapid decline in induced 
mutation frequency obtained at plating on nutrient broth-containing 
medium. The decline in mutation frequency is observed almost 
immediately and proceeds to a level unaffected by further incubation 
within 20 to 30 minutes at 37° C. The decline rate is similar in cul- 
tures incubated in M medium and in purine-pyrimidine-supple- 
mented medium prior to irradiation. Evidence that the processes 


involved in decline are enzymatic was obtained by studying the 
decline rate at different incubation temperatures ranging from 6° to 
37° C. This established that the mutation frequency decline (MFD) 
process has a Q 10 of approximately 2. The hypothesis was suggested 
that when amino acids or a nitrogen source were absent, some enzy- 
matic process removes the potential mutation before it is "fixed" in 
the genetic structure. Amino acids might then serve in "stabilization" 
of the pre-mutation or mutagen, removing it from susceptibility to 
MFD until those processes leading to incorporation in the genetic 
structure could be completed. 

The role of CMP in interfering with mutation induction was 
investigated and found to promote MFD in a manner completely 
analogous with that caused by nitrogen or amino acid deprivation 
(4). It was thus considered that CMP acts to prevent some process of 
"mutation stabilization" (MS) involving amino acids, thus allowing 
MFD to proceed. Figure 1 compares the time course and relationships 
of MFD and MS in regard to tryptophan-requirement reversions in 
E. coli strain WP2. When the cells are plated immediately after irradi- 
ation onto broth-supplemented M agar the mutation frequency 
attained at the UV dose used was about 40 prototrophs per 10° sur- 
vivors. However, this level of nutrient broth supplementation affords 
amino acid concentrations much lower than optimum for maximum 
mutation response as is apparent from the Min + A A curve in Figure 
1. Here the cells were incubated following irradiation in M medium 
supplemented with higher levels of amino acids for increasing time 
intervals before plating onto the limiting broth-supplemented M 
agar. It is apparent that when the cells are incubated for 30 to 40 
minutes with excess amino acids, the induced mutation frequency 
obtained on subsequent plating almost doubles. Therefore, the amino 
acids have been taken into the cell and are involved in processes 
leading to mutation induction. On the other hand, when the cells 
are incubated in medium containing no amino acids or other nitrogen 
source (Min — N curve in Figure I), or if CMP is added to amino 
acid-supplemented medium (Min + AA + CHL curve in Figure 1), 
MFD leads to a low mutation frequency within 20 minutes. Oxidative 
phosphorylation is probably required for MS since, if the irradiated 
cells are incubated in complete medium to which dinitrophenol 
(DNP) has been added, MS does not take place (Min + AA + DNP 




^miiiiiiiiiaiii ■"■•iiiiiiii„ ii-iiiiiii „„„» 

MIN. * AA. 

40 60 


Figure 1. — Comparison of "mutation stabilization" (MS), 'imitation 
frequency decline" (MFD), and tJic effect of dinitrophenol on UV-induced 
reversion of tryptophan requirement in Escherichia coii strai?i WP2 
The UV-irradiated cells were incubated at 37°C in the folloxving media 
for the indicated time periods and then plated on M + 2.5 per cent nutri- 
ent broth agar: (1) Min + AA = M medium + casein hydrolysate (NBC, 
vitamin free, enzymatic, 2 ?ng/?nl) and dl-tryptophan, 0.2 mgjml; (2) 
Min + AA + DNP — medium (1) plus dinitrophenol (5 X 10' 3 molar); 
(3) Min + AA + Chi = medium (1) plus chloramphenicol (20 micro- 
gramsjml); (4) Min — N = M medium xoith ammonium sulfate deleted. 
The plates were incubated 3 days then scored for mutation and survival. 
A T o significant changes in survival level xoere produced by the above 

curve in Figure 1). DNP specifically uncouples oxidative phosphoryla- 
tion and also prevents MFD when it is added to nitrogen-free medi- 


urn. When incubation in complete medium or M minus nitrogen 
medium is allowed for short intervals before adding; DNP intermedi- 
ate levels of MS or MFD are obtained. Experiments in which high 
levels of amino acids were added to the incubation medium subse- 
quent to or during MFD indicate that the process is not reversible. 
Once MFD has proceeded to any level, addition of amino acids will 
not increase the mutation frequency obtained with subsequent 
incubation beyond that level. 

Witkin (26, 27) has suggested that delay in DNA synthesis could 
increase mutation since more time would be available for protein 
synthetic processes involved in mutation induction prior to DNA 
synthesis. On the other hand, Kimball, et al (13) propose that delay 
in DNA synthesis would decrease mutation since more time would 
be available for reversion of an unstable "pre-mutational" state to a 
stable state not inducing a mutational change. 

It has previously been demonstrated that UV will cause a delay 
in DNA synthesis (12), and several investigators (2, 6, 10) have since 
shown that resumption of DNA synthesis following UV requires 
prior synthesis of RNA and protein. In our experiments several inhib- 
itors which block RNA or protein synthesis prior to initiation of 
DNA synthesis in a UY-irradiated culture were found not only to 
inhibit DNA synthesis, but also to promote a decided decrease in the 
induced mutation frequency (5). If a quantitative correlation could 
be established between the effects of these agents on mutation fre- 
quency and their effect in delaying DNA synthesis, then the hypothe- 
sis advanced by Kimball, et al (13), would be supported. But if MFD 
occurs independently of the effect on DNA synthesis, then the 
hypothesis that MFD is an active enzymatically controlled process (3) 
would be more likely. Several experiments designed to give evidence 
as to the most likely of these alternatives were performed. These 
experiments measured the effect of increasing periods of postirradi- 
ation treatments blocking RNA or protein synthesis on the subse- 
quent DNA synthesis and mutation frequency. Absence of nitrogen 
source, absence of tryptophan for the tryptophan-requiring auxo- 
troph, presence of 6 aza uracil, and presence of CMP were used as 
inhibitors. With all experiments the results demonstrated that condi- 
tions sufficient in duration to lead to lower mutation frequency 
through blockage of RNA or protein synthesis produce no significant 


effect on subsequent DNA synthesis. Only with treatment of at least 
20 minutes is maximum MFD obtained and this decline is not asso- 
ciated with any change in the pattern of DNA synthesis. The same 
holds true for RNA and protein synthesis in the culture. Net RNA 
and protein syntheses begin only after 25 to 30 minutes postirradiation 
incubation, and treatments during the first 20 minutes causing maxi- 
mum MFD do not appear to appreciably change the subsequent 
pattern of RNA and protein synthesis. It is apparent from these 
experiments that delay in DNA synthesis cannot be held responsible 
for MFD following UV irradiation. 

Mutation fixation 

The potential mutation eventually becomes established in the 
cell to the extent that it is no longer susceptible to conditions pro- 
moting MFD. This process has been termed "Mutation Fixation" 
(MF) (4). It should not be confused with the final process of mutation 
induction involving DNA synthesis (see below). CMP challenge (see 
Figure 2 for outline of technique) is most frequently used for demon- 
starting MF. The CMP challenge technique is based on the principal 
that, when irradiated cells are incubated in complete medium, all 
processes involved in mutation induction will proceed. However, 
when CMP is added after a given incubation interval, all potential 
mutations remaining "unfixed" at the time of CMP addition will be 
eliminated by the MFD processes. In Figure 2 comparison of the 
MS curve with that for MF (determined by CMP challenge) shows 
that the potential mutations are stabilized in the cell for a consider- 
able period prior to initiation of the MF process. As late as 30 minutes 
after irradiation all potential mutations remain subject to processes 
promoting MFD, but after this time decreasing numbers are affected 
by CMP challenge and after 75 minutes none are susceptible. Neither 
MS nor MF is correlated with gross protein synthesis since this process 
is not initiated until 10 minutes after MF has begun. 

Mutation fixation and mutation expression in relation to 
macromolecular syntheses 

When the progress of RNA, DNA, and protein synthesis are 
simultaneously followed with MF in a UV-irradiated culture, an 
interesting correlation is apparent (Figure 3). Progression of MF is 
closely correlated with RNA synthesis. Both processes are initiated at 


20 40 60 



Figure 2. — Comparison of "imitation stabilization" (MS) and "mutation 
fixation" (MF) in Escherichia coli strain WP2. Mutation folloived zuas 
reversion of the tryptophan requirement. Following UV irradiation 
cells xocre incubated at 37°C in M medium plus casein hydrolysate 
(2 mg/ml) and dl-tryptoplian (0.2 mg/ml). Culture aliquots were plated 
at the indicated times (MS) on brotli-supple?nented M agar. Also, after 
these intervals of incubation cJdorampJienicol (CMP) (20 micrograms / '?nl) 
was added to samples which were incubated for an additional 45 min- 
utes before plating. This latter procedure is termed "CMP cliallenge" 
and measures MF since all mutations remaining subject to the CMP- 
promoted MFD process at the time of CMP addition are eliminated 
during the subsequent 45 minutes incubation. Relative protein content 
of the culture at the times of CMP addition is also given. 

about the same time and MF attains its maximum when the RNA 
has doubled in amount. It thus appears that DNA synthesis per se 
may be segregated from the MF process. 

The uridine analogue, 5-hydroxyuridine (5-HU), when present 
during postirradiation incubation, promotes marked decline in muta- 
tion frequency (Figure 4). However, this is not the MFD process 


co 16 



W 12 








o 6 


H 4 


„ 2 

RNA / 













I 5 •- 





10 20 30 40 50 60 70 80 90 


Figure 3. — Relation of "mutation fixation" (MF), as indicated by CMP- 
challenge, to synU)csis of RNA, DNA, and protein in a UV-irradiated 
culture of EscJicrichia coli strain ]VP2. Folloxoing irradiation the bac- 
terial suspension xvas diluted 1:4 into M medium phis casein hydrolysate 
(2 mg/ml) and dl-tryptophan (0.2 mg/ml) and incubated at 37°C. 
At the indicated intervals aliquots were taken and CMP (20 micro- 
grams/ml) added to them. They were then incubated for an additional 
45 minutes at 37°C to alloxv CMP-promoted MFD to take place and 
plated on M + 2.5 per cent broth agar. At the time tliat samples xuere 
taken for CMP treatment other samples xoere taken for RNA, DNA, 
and protein determinations. 

revealed by incubation in CMP or amino acid-deficient media. With 
5-HU treatment decline in mutation frequency does not start 
immediately but only after some 20 minutes incubation in its pres- 
ence. Moreover, 5-HU promoted decline appears inversely correlated 
with MF and with RNA synthesis. This evidence, which suggests that 
5-HU is exerting its effect through RNA synthesis, would account 
for the lag in initiation of the decline in mutation frequency. One 
possible explanation for the 5-HU promoted decline in mutation 
frequency is that the analogue is incorporated into new RNA, thus 



10 20 30 40 50 60 


Figure 4. — Comparison of the effects of 6-aza uracil (6AU) and 5- 
hydroxy uridine (5HU) on macromolecular syntheses and the freqcuncy 
of induced prototrophs in UV-irradiated Escherichia coli strain 1VP2. 
Folloioing irradiation the bacterial suspension was diluted 1:4 i)ito M 
medium plus casein hydrolysate (2 rngfinl) and dl-tryptopha?i (0.2 
mg/ml). This zoas immediately divided into tioo portions and 6AU 
(0.05 micrograms/ ml) was added to one portion and 5HU (0.05 micro- 
grams/ml) to the other. Both cultures ivere tJien incubated at 37°C, 
and after the indicated incubation intervals samples icere taken from 
each tube and plated on M plus 2.5 per cent nutrient broth agar. At 
the same tiynes samples were taken for RNA, DNA, and protein analyses. 

producing nonfunctional RNA and using up the radiation-produced 
mutagenic precursors. 

MFD is also obtained in the presence of the analogue 6-aza 
uracil (6-AU) (Figure 4), and is similar with this analogue to that 
obtained with CMP. Like CMP, 6-AU blocks protein synthesis, but 
unlike CMP, this is brought about through direct blockage of RNA 
synthesis. Addition of uridine will reverse the blockage to RNA and 
protein syntheses and also prevent 6-AU promoted MFD. 


Schwartz and Strauss (21) have shown that incubation of UV- 
irradiated E. coli strain WP2 in the presence of tryptazan (a trypto- 
phan analogue which is incorporated into protein) will result in a 
decline in mutation frequency. They suggest that this decline is 
caused by protein synthesis utilizing tryptazan. Their results consti- 
tute significant biochemical evidence implicating protein synthesis 
in mutation induction. If protein formed in the presence of trypta- 
zan and subsequent to mutation fixation were intimately involved 
with RNA in the replication of genetic DNA, then "nonfunctional" 
protein would probably prevent utilization of the corresponding 
RNA in DNA replication and in so doing use up the mutagenic 

All these experiments indicate that MF is closely correlated with 
the RNA synthesis, and that the mutations are established in some 
structure or form before any measurable DNA synthesis takes place. 
They support our previously presented hypothesis (4) that RNA and 
protein syntheses are intimately involved in replication of genetic 
DNA; and that the RNA, modified by incorporation of a radiation- 
altered precursor, in some manner leads to a corresponding modifi- 
cation in newly formed DNA. This implies that induced mutations 
first appear in the daughter DNA initially synthesized after irradi- 
ation, and that a period of time is thus necessary between UV treat- 
ment and establishment of the mutation in the genome. During this 
interval manipulations interfering with RNA and protein syntheses 
can prevent the potential mutation from being induced. 

It is of considerable importance to determine at what point in 
the sequence of postirradiation events the induced mutation is first 
capable of being phenotypically expressed. Establishment of this 
point would differentiate processes involved in gene synthesis from 
those of enzyme synthesis involved in phenotypic expression. Dur- 
ing the course of experiments with reversion of the tryptophan 
requirement of E. coli strain WP2, it became apparent that, dur- 
ing the early stages of postirradiation incubation, if the bacteria 
were plated on M agar rather than on broth-supplemented M agar, 
much lower induced mutation frequencies were obtained. After 90 
minutes postirradiation incubation in complete medium, however, 
the mutation frequency obtained was the same on both M agar and 
broth-supplemented M agar. It seemed probable that the lower level 


of mutation obtained on M agar might be due to inability of the 
induced reversions in these tryptophan-deficient strains to be pheno- 
typically expressed without some initial supplementation. If true, this 
behavior would indicate that mutation expression requires macro- 
molecular synthetic events following those involved in gene repli- 
cation and separable from the former when studying prototrophic 
mutations in an auxotrophic strain. The results of experiments to 
test this hypothesis have been previously reported (9). 

A typical CMP challenge experiment was performed; also at the 
same time, samples were plated on M agar as well as on broth- 
supplemented M agar (Figure 5). As usual MF closely follows RNA 
synthesis. However, when identical aliquots are plated at the same 
incubation times on M agar, few mutations appear until after 40 
minutes of incubation. At this point MF has practically been com- 
pleted. After 40 minutes incubation, and closely following DNA 
synthesis, the mutation frequency obtained on the M agar plates rises 
sharply, and after 90 minutes the frequency of induced mutation 
measured is the same on both plating media. The results indicate 
that a period of protein synthesis subsequent to DNA replication is 
necessary for expression in the case of auxotrophic reversions. This 
hypothesis is susceptible to further testing. If the irradiated cells are 
incubated in complete medium for a time sufficient to allow initiation 
of DNA synthesis and then CMP is added to the incubating culture, 
mutation expression should be prevented in that, portion of mutant 
cells which have not yet synthesized the requisite protein. This late 
CMP treatment should not promote MFD, however, since mutation 
fixation would already have been completed (see Figure 5). When 
such an experiment was performed, it was found that if CMP is added 
after 60 minutes incubation in complete medium, DNA synthesis 
continues in its presence; but further mutation expression is pre- 
vented when the cells are plated on M agar. When the culture is 
treated with CMP after 100 minutes of incubation and then plated 
on M medium, the mutation frequency is the same as that obtained 
in an untreated twin culture plated on broth-supplemented M agar. 
Other experiments were performed with an auxotroph requiring 
thymine, methionine, and tyrosine (E. coli strain 15 T -Me-Tyr-)- The 
mutation followed was reversion of the tyrosine requirement. When 
this strain is deprived of thymine but not amino acids during post- 




i r i r 

20 30 40 50 60 70 


Figure 5. — Relation of "mutation fixation" (MF) and "mutation expres- 
sion" to macromolecular synthesis in a UV-irradiated culture of Escheri- 
chia coli strain WP2. Following irradiation the bacterial suspension was 
diluted 1:4 into M medium plus casein hydrolysate (2 nig/ml) and dl- 
tryptophan (0.2 mg/?ul) and incubated at 37°C. MF zoas determined 
by the "CMP-challcnge" method (see Figure 2) followed by plating on 
M plus 2.5 per cent nutrient broth agar. "Mutation expression" was 
determined by plating directly onto M agar after the indicated periods 
of incubation in M medium plus casein liyrdolysate and tryptophan. 
Prototrophic mutations are expressed as the percentage of maximum 
mutation frequency response (SO phototrophs/10 6 UV survivors xohen 
plating was on broth-supplemented M agar, and 60 prototrophs/ 10 G UV 
survivors when plating zvas on M agar). At the same times that samples 
were taken for CMP challenge and for platiiig on M agar, samples were 
also analyzed for UNA, DNA, and protein. 

irradiation incubation, no DNA synthesis takes place and mutation 
expression does not occur with subsequent plating on M plus 


thymine plus methionine medium. However, when thymine is 
restored to the incubating culture, DNA synthesis resumes and 
mutation expression is observed on the latter plating medium. 

While processes leading to mutation induction occur prior to 
DNA synthesis and involve macromolecules other than the DNA, the 
mutation apparently comes finally to reside in new DNA but is not 
expressed until this DNA has functioned to establish the requisite 

III. Involvement of Postirradiation Macromolecular Syntheses 
in X-ray-Induced Mutation 

Considerable time has been devoted recently to examining the 
relation of postirradiation macromolecular synthesis to X-ray-induced 
mutation. Earlier it was stated that preirradiation supplementation 
experiments indicated that a part of X-ray-induced mutations are 
due to incorporation of radiation-altered precursors during post- 
irradiation synthetic processes (7, 8). Because of this we have been 
examined the postirradiation synthetic events of X-irradiated bacteria 
for the same processes involved in UV-induced mutation. The tech- 
niques and methods elaborated for the UY work were used in the 
X-ray studies, except that the bacteria were at a higher concentration 
during exposure. The total dose in most experiments was 10 Kr given 
at a rate of 2860 r per minute. The cells were held in an ice bath 
during exposure. 

Figure 6 presents results obtained when X-irradiated cells are 
plated on M agar and on broth-supplemented M agar after various 
intervals of postirradiation incubation in complete medium. It is evi- 
dent from the M agar plates that an appreciable portion of the total 
mutational yield is expressed after only 10 minutes incubation in 
complete medium. With UV irradiation a much lower proportion of 
prototrophs were expressed prior to initiation of DNA synthesis (see 
Figure 5). In the case of X-ray exposed cells, during the first 10 
minutes of postirradiation incubation approximately 40 per cent of 
the prototrophs are expressed in the absence of measurable DNA 
synthesis. DNA synthesis is initiated after 10 minutes and the M agar 
platings indicate that additional mutations are expressed following 
initiation of DNA synthesis. 

When protein synthesis is inhibited by adding CMP immediately 
after irradiation, there is no appreciable effect on mutation frequency 



- 2.0 


20 30 40 


Figure 6. — Comparison of mutation frequency expressed on M agar wit!) 
that expressed on brotJi-suppIetneuted M agar, and the relation of DNA 
synthesis to mutation expression in an X-irradiated culture of Escher- 
ichia coli strain WP2. Folloiving X-ray irradiation (10 kr) the bacterial 
suspeusioti ivas diluted 1:4 into M medium plus casein hydrolysate 
(2 mg/ml) and dl-tryptophan (0.2 mg/ml) and incubated at 37°C. At 
the indicated times aliquots were withdrawn and plated on M agar 
and on M plus 2.5 per cent nutrient broti) agar. Identical samples were 
also analyzed, for DNA content at each time. 

observed with plating the cells on broth-supplemented M agar after 
various intervals of incubation. However, when plated on unsupple- 
mented M agar, the mutation frequency is quite low (Figure 7). While 
a large fraction of the potential mutations are not susceptible to CMP 
(are not unstable and subject to MFD as in UV irradiation), it is 
obvious that their expression is prevented by CMP. Therefore, all 
X-ray-induced mutations, as wll as UV-induced mutations, evidently 
require a period of protein synthesis following establishment in 
the genome if they are to attain expression. When CMP is added after 
10 minutes incubation in complete medium (Figure 7) it is found 
that a large part of the mutations appearing on broth-supplemented 



(O 150- 

» 100 - 

10 20 30 40 


Figure 7.— Effect of CMP in an X-irradiated culture of Escherichia coli 
strain 1YP2. Following X-ray irradiation (10 kr) the bacterial suspen- 
sion was diluted 1:4 into M medium plus casein hydrolysate (2 mg/ml) 
and dl-tryptophan (0.2 mg/ml). This culture was then divided into 
two portions. To one portion chloramphenicol (20 micrograms/ ml) 
ivas added immediately and incubation at 37° C started. The other por- 
tion was incubated for 10 minutes at 37°C before adding chloramphen- 
icol (20 ?nicrograms/?nl) and then incubation was continued. At the 
indicated incubation times samples were withdranm from each culture 
and plated on M agar and on M plus 2.5 per cent nutrient broth agar. 

plates have become susceptible to CMP. Within 10 minutes incuba- 
tion potential mutants can be divided into two classes, viz., those 
completely expressed and not sensitive to CMP, and those which are 
not capable of being expressed and are sensitive to CMP. 

If we assume that X-ray has the capacity to mutate genetic mate- 
rial at two different levels, viz., (a) in the parental DNA, presumably 
by direct physical action, and (b) in the daughter DNA, by some 
copy-error mechanism, these results can be readily accounted for. 


Those mutations induced in the parental DNA should require only 
a short period of protein synthesis following induction. With muta- 
tions to prototrophy, expression on M agar would occur as soon as 
protein synthesis for new enzyme production takes place. Treatment 
with CMP after this time would not interfere with their expres- 
sion. On the other hand, mutations induced in daughter DNA, 
presumably during the process of replication, might be dependent 
on mutagenic precursors or on chemical mutagens produced by the 
X-ray and acting at some stage in the implicative process. These 
would correspond to the so-called "delayed" or "indirect" muta- 
tions produced by X-ray. In Figure 7 these correspond to that frac- 
tion susceptible to MFD after CMP addition at 10 minutes and 
revealed by the broth-supplemented M agar platings. 

This hypothesis is readily testable when CMP challenge is 
run on X-irradiated cells, and the cells then plated on both broth- 
supplemented M agar and on M agar (Figure 8). From the supple- 
mented M agar plates it is evident that during the first 10 minutes 
of postirradiation incubation in complete medium about half of 
the mutations become susceptible to CMP challenge. Tins sensi- 
tivity remains constant for approximately 20 minutes and then is 
lost with further incubation. All mutations are insensitive to CMP 
after 30 to 40 minutes incubation. Similar results have been obtained 
usins the uracil analogue 6-AU, and are additional evidence that the 
CMP-sensitive fraction of X-ray-induced mutants is induced in daugh- 
ter DNA during the implicative process and that RNA and protein 
are involved in replication. 

Other experiments designed to determine whether the CMP- 
resistant fraction of mutants can be expressed in the absence of 
DNA synthesis were carried out using E. coll strain 1 5 T - Me - Tj i-, follow- 
ing reversion of the tyrosine requirement in this strain. It was found 
that immediately after irradiation few mutants were expressed on 
M plus thymine plus methionine agar but large numbers were 
expressed on broth-supplemented M agar. In further experiments 
with this strain thymine was withheld from tyrosine and methionine- 
supplemented postirradiation incubation medium for increasing 
periods ol time and then added. All platings were on thymine and 
methionine supplemented M medium after a total postirradiation 
incubation period of 80 minutes. In these experiments approximately 







20 30 40 50 60 70 80 


Figure 8. — Separation of X-ray-induced mutations in Esclicrichia coli 
strain WP2 into two classes by CMP challenge followed by plating or 
M agar and broth-supplemented M agar. Folloiving X-ray irradiation 
(10 kr), the bacterial suspension was diluted 1:4 into M medium plus case- 
in hydrolysatc (2 mgjml) and dl-tryptophan (0.2 mg/ml) and incubated 
on a reciprocal shaker at 37° C. At the indicated times CMP (20 micro- 
grams/ml) xeas added to aliquots of the culture and incubation continued 
in the presence of CMP for 40 additional minutes. All samples zocre then 
plated on M agar and on M plus 2.5 per cent nutrient broth agar. 

half of the tyro 1 mutations were able to be expressed on this medium 
after 20 minutes incubation. During the 20 to 40 minute incubation 
period no more mutations were expressed. At this time thymine was 
added to the incubation tubes and the incubation continued, and 
during the next 20 minutes the remaining half of the mutations were 
expressed. Therefore, after 60 minutes the mutation frequency 
obtained was the same as that of the control, which had been 
incubated in complete medium. No DNA synthesis was observed in 
the test culture until thymine addition, and then DNA synthesis 


started immediately and was correlated with expression of the second 
mutant fraction. 

Summary and Conclusions 

Several years ago Witkin (26) expressed the opinion that "the 
time interval between absorption of radiant energy and produc- 
tion of stable genetic changes can no longer be regarded as infini- 
tesimal." She pointed out that many postirradiation treatments 
altered the ultimate fate of a potential genetic change. Our experi- 
ments have indicated that the basis for the delay between appli- 
cation of inducing agents and production of stable genetic changes 
is due to the necessity for replication of the genetic DNA. 

Our experiments with X-ray-induced mutations indicate that 
certainly with this inducing agent mutations are produced through 
at least two mechanisms, or at two different levels of genetic structure 
development. Recent studies by Witkin and Thiel (28) and by 
Kada, Brim, and Marxovich (11) indicate that the same is probably 
true for UV-induced mutations. In one type of mutation induc- 
tion with either agent the initial damage is unstable and capable 
of modification by postirradiation treatments. The second type is 
stable, and is not influenced significantly by postirradiation condi- 
tions. Both types are analyzed in our X-ray experiments, but we 
have dealt only with the former type in the UV experiments. 

In regard to the unstable type of UV-induced mutation, while 
our studies suggest that the potential mutation is initially in the 
form of a mutagenic nucleic acid precursor formed by action of the 
radiation on a purine- or pyrimidine-containing monomer (4), we 
have no conclusive evidence for this; and the possibility remains 
that the initial irradiation damage is to the cellular DNA. Sins- 
heimer (22, 23) has shown that exposure of uridylic or cytidylic 
acids to UV results in stable and unstable byproducts, and our 
experiments indicate that cytidine and uridine are effective com- 
pounds in increasing UV-induced mutation frequency. 

Studies on the postirradiation events involved in mutation 
induction by UV have led to delineation of several biochemical 
interrelations influencing mutation induction and probably involved 
in DNA replication. Those biochemical interactions occurring prior 
to any detectable macromolecular syntheses and tending to increase 


the mutation frequency are said to be involved in mutation stabili- 
zation. The frequency of mutation is therefore determined by the 
effectiveness of the MS processes. The nature of this process is not 
known; however, the fact that 5-HU will lower the induced muta- 
tion frequency (presumably through incorporation into RNA) in 
a process always correlated with MS suggests that stabilization 
involves "templating" preparatory to macromolecular synthesis. 

If MS does not take place an antagonistic process occurs which 
removes the potential mutation from pathways leading to muta- 
tion induction. This process, which we have termed mutation fre- 
quency decline, is enzymatically mediated (27, 5) and requires 
energy, but not RNA or protein synthesis. Also DNA synthesis 
is not required since the process occurs in the absence of thymine 
with a thymine-requiring auxotrophic strain (5). Moreover, MFD 
can be carried to completion without influencing either the timing 
or the rate of subsequent syntheses definitely involved in mutation 
induction. Therefore, the processes involved in either MFD or MS 
must take place prior to the RNA and protein synthesis made 
necessary by UV irradiation to further DNA replication (2, 5, 6, 
10). These results would seem to eliminate hypotheses for MFD 
based on modification of timing of DNA synthesis so as to give more 
time for "decay of a premutational state" at a constant rate (13), or 
disappearance of "mutagens" at a rate independent of the treat- 
ment (14). 

Ultimately, the potential mutation is fixed in the cell and 
no longer subject to conditions causing MFD. The striking correla- 
tion between RNA synthesis and MF (measured by challenge pro- 
cedures specifically blocking RNA or protein synthesis) leads us to 
believe that MF is mediated by RNA synthesis. There appears to be a 
relation between the amount of postirradiation RNA synthesized at 
the time of CMP addition and the rate of DNA synthesis in the pres- 
ence of the inhibitor, as well as the level of induced mutation 
obtained. If the RNA and protein necessary for genetic replication 
have not been formed prior to CMP addition, there is no DNA 
produced and the potential mutation is lost through MFD. It is 
probable that the RNA and protein involved in MF are identical 
with that involved in postirradiation recovery of the DNA-synthesiz- 
ing system. The experiments make quite clear that acquisition of 


capacity to synthesize DNA rather than DNA synthesis per sc is 
necessary for mutation fixation, since DNA synthesis during CMP- 
challenge is not necessary. 

A number of hypotheses have been advanced in regard to the 
mechanism of mutation induction by UV. However, it is quite 
difficult to explain most of the postirradiation data on the basis of 
"direct-induction" theories. In our opinion, the hypothesis for the 
unstable portion of UV-induced mutation which best accommodates 
the experimental data is that which we have previously advanced 
(4). According to this hypothesis, DNA synthesis involves transfer of 
information from the parental DNA to the daughter DNA through 
an RNA-protein intermediate. Incorporation of a UV-modified pre- 
cursor into the RNA leads to a "copy error" involving substitution of 
a different nucleotide pair or perhaps deletion of the usual nucleotide 
pair in the subsequently formed daughter DNA. While little experi- 
mental support at the molecular level exists for this indirect mecha- 
nism of DNA replication, the same situation also exists for theories 
of a more direct mechanism. There are also several indications that, 
at least during phage DNA replication, the genetic information is at 
certain stages carried in some structure or molecule other than the 
DNA (16, 24). 

Our findings thus far for X-ray-induced mutations can be 
briefly summarized as follows: Approximately half of the X-ray- 
induced reversions of certain amino acid-requiring auxotrophs of 
E. Coli are sensitive to CMP, or to 6-AU. A short period of incu- 
bation involving protein synthesis is required for this sensitivity to 
become manifested. These mutations require RNA, DNA, and 
protein syntheses for induction and are apparently induced in a 
manner comparable to that for the major portion of UV-induced 
reversions. It is probable that these mutations are induced dur- 
ing replication of the daughter DNA. The other fraction of X-ray- 
induced mutations are not lost by MFD processes when the cells 
are incubated in the presence of CMP or 6-AU. These mutations 
are expressed when the culture is plated on M agar after a short 
period of incubation in complete medium, and during this incu- 
bation no detectable DNA synthesis occurs. This suggests that the 
second fraction of mutations is induced by X-ray in the gene, and 
that DNA replication is not necessary for induction. However, RNA 


and protein syntheses subsequent to their induction are required 
for phenotypie expression of the reverted character. 


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Auerbach: Is the separation into mutation stabilization and mutation 
frequency decline not an artifact, caused by your having used for base- 
line mutation frequency on plates with an intermediate amount of 
broth? If you had used mutation frequency on minimal medium as base 
line, would not both phenomena have turned out to be the same — or 
rather reverse sides of the same phenomenon? 

Haas: It may very well be that mutation stabilization and mutation 
frequency decline are reverse sides of the same phenomenon, but they 
most certainly are not the same thing; nor is the separation an artifact 
caused by using mutation frequencies obtained on plates supplemented 
with an intermediate amount of broth for the base line. We use the 
intermediate level of broth supplementation so that we can demonstrate 
and study both processes at the same time on identical samples, but 
the phenomena of mutation stabilization and of mutation fixation, 
as well as the relation between the two, can be demonstrated just as 
well with plating on minimal medium. Similarly, the phenomena of 
mutation stabilization and mutation frequency decline can be demon- 
strated on broth-agar plates containing a much higher degree of broth 

It is quite probable that both mutation stabilization and mutation 
frequency decline are affecting the same biosynthetic step in DNA 
replication, but one process (stabilization) leads to a maximum fre- 
quency of mutations (provided mutation fixation takes place before any 
decline process occurs), while the other (mutation frequency decline) 
leads to a less number of mutations. Therefore, the processes certainly 
must be different. When one incubates irradiated cells in buffer con- 
taining a high level of amino acids, no increase in the mutation fre- 
quency will be obtained with incubation, and neither will there be any 
mutation frequency decline. However, these incubated cells are still 
fully susceptible to either phenomenon and if, after a period of say 
30 minutes buffer-amino acid incubation, conditions suitable for meas- 
uring mutation fixation (addition of minimal medium to the culture) 
or conditions suitable for mutation frequency decline (addition of 
chloramphenicol) are initiated, we find that both phenomena will take 


place to the same extent as when they are initiated immediately after 
irradiation. Thus the potential mutations have been stabilized by the 
amino acids and they have neither been fixed nor have they disappeared. 
Furthermore, if one interrupts the process of mutation fixation at some 
intermediate level and then subjects the cells to conditions causing 
mutation frequency decline, no decline is obtained but neither is any 
further mutation fixation. Similarly, if at some intermediate level of 
mutation frequency decline one interrupts this process and introduces 
conditions favorable for mutation fixation, no increase in the frequency 
of mutants fixed above this level is obtained and neither is there any fur- 
ther mutation frequency decline. It is quite apparent that in the former 
case, where mutation fixation was being measured, the mutations already 
fixed were not subject to processes causing mutation frequency decline, 
while the fraction remaining unfixed was equally susceptible to either 
mutation fixation or mutation frequency decline. In the latter case, 
when mutation frequency decline was being measured, the potential 
mutants that have disappeared cannot be made to reappear by intro- 
ducing conditions favorable to fixation, and the fraction which has 
not disappeared is as susceptible to mutation frequency decline as to 
mutation fixation. 

Discussion of Session II 

Tlie University of Chicago, Chicago, III. 

It is evident now that mutation is a complex process; the inter- 
mediate steps depending on the particular mutagen used to 
initiate the process. As Doctor Auerbach points out, we interpret 
the action of mutagenic agents in terms of what we know about the 
structure of the genetic material rather than learning about the 
genetic material as a result of studies of mutagenic agents. This 
process of interpretation may lead to useful results even though 
its presuppositions are incorrect. 

In 1939, Thorn and Steinberg (16) 1 reported that nitrous acid 
induced genetic changes in fungi. Their experiments were based on 
the idea that the genetic material was protein in nature and their 
interpretation was based on the well-known reaction of nitrous 
acid with the amino groups — of the amino acids. Nitrous acid 
is one of the more useful of the mutagens and its action is interpreted 
today on the basis of its reaction with the amino groups — of the 
purines and pyrimidines — since we now know, or feel sure that we 
know, that genetic material is polynucleotide in nature. I think 
Thorn and Steinberg should be given more credit for having- first 
studied the mutagenic action of nitrous acid and I think also that 
it is instructive to note the pitfalls a favorite hypothesis can create. 

Nitrous acid-induced mutation represents a direct type of 
mutagenesis. The agent produces a base analogue in situ which 
can then yield a slight increase in the rare frequency of pairing 
mistakes during DNA duplication. Ultraviolet light-induced muta- 
tion as described by Doctor Haas probably represents the other 
extreme, a complex of photochemical and biochemical events being 
required for the complete mutation process. (In the discussion, 
Doctor Atwood suggested that ultraviolet light-induced mutation 
might be due to an iv situ photocatalytic hydroxy lation of a pyrimi- 
dine DNA constituent. Since ultraviolet light does not cause muta- 

^ee References, page 178. 



tion of an isolated transforming principle (6) in contrast to the 
action of nitrous acid, I believe ultraviolet-induced mutation must 
be a more complex process.) Doctor Haas, and others (17), have 
demonstrated that synthetic processes intervene between the pri- 
mary ultraviolet absorption product and the final mutant state. 

The process of ultraviolet-induced mutation is in fact a very 
special case. A portion of the ultraviolet-induced changes can be 
reversed by visible light and this photoreactivation is unique to 
ultraviolet-induced mutation. (The photoreactivation process is 
itself only partially understood. It is known to be enzymatically 
mediated.) While it is established that protein synthesis, or some 
closely related process, is required for the fixation of ultraviolet- 
induced mutations, it is equally evident, both from the work report- 
ed by Doctor Haas at this symposium and from other work, that 
protein synthesis is not a general requirement for the fixation of 
mutations. Bromouracil is mutagenic for bacteriophage even when 
introduced along with chloramphenicol as an inhibitor of protein 
synthesis (7). Protein synthetic processes do not play an important 
role in the fixation of those mutations induced by the alkylating 
agent ethyl sulfate, although they may be required for the expres- 
sion of the mutations (14). The frequency of ethyl sulfate-induced 
mutation is not reduced when E. coli is treated in the presence of 
any one of the inhibitors methyltryptophan, ethionine, or 6-mer- 
captopurine nor does post-treatment incubation in buffer, or in buffer 
containing these inhibitors, seem to alter the mutation frequency 

According to the ideas presented by Doctor Atwood earlier 
in this symposium, the genetic material is a linear sequence of 
deoxyribonucleotides. The sequence forms a code which is deci- 
phered by the cell and the process of deciphering represents gene 
action. Base analogues incorporated into the DNA lead to muta- 
tion only by increasing the probability that a mistake will be made 
on duplication leading to the substitution of one nucleotide pair 
for another. DNA containing a base analogue is not of itself mutant — 
bacteriophage with bromouracil completely substituted for thymine 
can produce normal progeny (8). The mutation process can only be 
considered complete when a changed DNA (itself able to replicate) 
containing only normal bases is Formed as a result of a replication. 


Consider the behavior of bacteriophage treated with ethyl 
methanesulfonate. Although phage treated in vitro produce mutants 
when used to infect bacteria, such treated phage are not yet mutant. 
Single bursts yield groups with a few mutants and others with many, 
indicating that the "mutation" may occur at any time during the 
replication of the treated DNA (9). 

Our present ideas require that a mutation represent a change 
in base order within the genetic DNA. All mutagens must lead to 
such a changed base order. If this hypothesis is accepted, the prob- 
lem becomes a determination of how each particular mutagen may 
bring about a change. It is not required, for example, that a chem- 
ical mutagen react directly with DNA. As Szybalski (15) has shown, 
the alkylating agent triethylene melamine may react with pyri- 
midines and pyrimidine derivatives to give products which are 
in turn incorporated into the DNA where they behave as analogues 
leading to an increased mutation frequency. It must not be sup- 
posed that mutations need represent changes in only single nucle- 
otide pairs. Freese (1) points out that the class of mutations he 
calls transversions may represent merely the change of a purine 
for a pyrimidine base. A transversion may also result in a more 
extensive change in the genetic material since incorporation of a 
purine at a pyrimidine site could result in sufficient deformation of 
the double helix of DNA to allow for the substitution of other 
bases. Fresco and Alberts (2) have shown how noncomplementary 
bases can be accomodated in a helix by the formation of loops com- 
ing out of the helix. Such loops might, on DNA duplication, result 
in the addition or deletion of portions of the nucleotide chain. It 
is important to realize that current ideas of mutation as a result of 
base substitution are not so restrictive that they require all muta- 
tions to be simple changes in single base pairs. 

It is also essential to emphasize that the mutagenic agents, 
particularly the reactive mutagenic alkylating agents, may have 
several different reactive sites within the organism and that their 
reaction, even at a single site, may have both mutagenic and non- 
mutagenic consequences. The alkylating agent diethyl sulfate (ethyl 
sulfate) not only is mutagenic but also produces a permeability 
change in the bacterial cell which makes magnesium ion more 
available to chelation and removal by citrate (13). Many alkylating 


agents combine with bacteriophage in a two stage process; the first 
step is a true combination, while the second is a decay event lead- 
ing to phage inactivation (10). Only certain of these alkylating 
agents produce bacteriophage mutations in vitro (9). 

There is a post-treatment decay reaction in phage plaque- 
forming ability and in the activity of transforming principle from 
B. subtilis following treatment with either the mutagenic ethyl 
methanesulfonate or the nonmutagenic (for virus) methyl methane- 
sulfonate. Incubation of bacteriophage T2 following treatment with 
ethyl methanesulfonate does not greatly alter the frequency of r 
mutants recovered. It seems likely that there is more than one site 
of reaction of these alkylating agents with genetic material and it is 
still difficult to determine the chemical difference between the muta- 
genic and nonmutagenic reactions. Certainly it is established that 
the reaction of alkylating agents with cellular constituents is a 
complex process. 

The hypothesis of mutation due to base substitution is logically 
satisfying but there are cases in which the mechanism by which 
this substitution occurs is not yet apparent. Thymine starvation 
is mutagenic and Kanazir (3) has shown that this mutagenic action 
occurs during the first 30 minutes of thymine starvation and before 
there is any lethality due to the thymine starvation. DNA duplica- 
tion does not occur. To my knowledge there is no way at present 
to account for this mutagenicity. Caffeine and other methylated 
purines are mutagenic (11), but these compounds are not incorpo- 
rated or metabolized (5) and presumably act by inhibiting some 
enzyme involved in DNA metabolism. We do not know how this 
inhibition results in a change in base order. 

Breaking a single one of the chains of the DNA double helix 
can be mutagenic. Radioactive decay of P-32 leads to a transmu- 
tation event in which the phosphorus changes to S-32. In most of 
the decay events the daughter nucleus recoils from the molecule 
leaving a "hole". Kaudewitz, et al. (4) have shown that this decay 
event is mutagenic and that the production of mutation is inde- 
pendent of the (3 radiation accompanying the radioactive decay. 
Kaudewitz reports (personal communication) that about 30 to 40 
per cent of the mutants obtained were able to revert to the wild- 
type, indicating that a proportion (at least) of the strains obtained 
were true mutants as distinguished from deletions. The removal 


of phosphorus from the DNA chain constitutes a definite break in 
that chain. How then does a break in the backbone chain cause 
a change in the base order? Kaudewitz (personal communication) 
suggests that a break in one of the two (replicating) chains of the 
DNA molecule leaves the distance between these chains unfixed 
permitting a mispairing, the actual mutation (change in base order) 
in this case being a secondary effect. Somewhat similar is the obser- 
vation of Freese that treatment of bacteriophage at 45° C and pH 
5 results in the production of mutants. Freese (1) supposes that 
these mutations are due to the formation of apurinic acid at lower 
pH. The initial reaction at pH 5 is different from those mentioned 
above, but once again the DNA is unstabilized in a way that permits 
unusual base pairings. 

In such cases the mutation is not "fixed" and in fact by defi- 
nition can not be considered established until a new DNA strand 
has been synthesized with the new base order. In order to have a 
mutation which is functional before DNA duplication it is neces- 
sary to produce, as a result of the action of the mutagen, a strand 
which is read differently by the deciphering mechanism. It is this 
requirement of the basic hypothesis that makes Doctor Haas's 
report of X-ray-induced mutation in the absence of DNA synthe- 
sis so interesting. Spontaneous mutation can occur in the absence 
of DNA synthesis, but an energy source is required before such 
mutations can occur (12). Doctor Haas reports that reversions can 
be induced in the absence of DNA synthesis but that these muta- 
tions require protein and RNA synthesis for their expression. 
Unfortunately, it is not possible to exclude the possibility of DNA 
synthesis from some of the data as presented. The frequency of 
prototrophs induced is of the order of 100 per 10'' cells, or 0.01 per 
cent. Doubling of the DNA of these particular cells would give 
a change of only 0.01 per cent, three cell divisions of this selected 
population would still give less than a 0.1 per cent increase. Con- 
sidering the analytical methods which must be employed, it is 
doubtful if the results are good to within 1 per cent. But 1 per cent 
could represent over five divisions of a selected cell population. If 
one accepts the fact that the thymine-requiring organisms do not 
make DNA, then the results using these mutants do seem to indi- 
cate mutation and mutation expression in the absence of DNA 


We assume that mutation represents a functional change in 
base order. These X-ray-induced mutations must therefore have a 
base order which looks different to the "reading mechanism" of the 
cell. Perhaps the X-rays produce a truly different base (base pair?) 
within the DNA or perhaps there is some sort of "transnucleotida- 
tion" corresponding to the known transpeptidation induced by 
agents such as X-rays which cause breaks in DNA chains. It is almost 
necessary to suppose a mechanism for rare base substitution within 
intact DNA molecules to account for mutation in the absence of 
DNA duplication. 


1. Freese, E. 1959. On the molecular explanation of spontaneous and 

induced mutations. Brookhaven Sy?np. in Biol., 12: 63-75. 

2. Fresco, J. R., and Alberts, B. 1960. The accomodation of noncoin- 

plementary bases in helical polyribonucleotides and deoxy- 
ribonucleic acids. Proc. Nat. Acad. Sci., 46: 311-321. 

3. Kanazir, D. 1958. The apparent mutagenicity of thymine deficiency. 

Biocliem. Biophyv. Acta, 30: 20-33. 

4. Kandewitz, F., Vielmetter, W., and Friedrich-Freksa, H. 1958. Muta- 

gene Wirkung ties Zerfalles von radioaktiven Phosphor nach 
Einbau in Zellen von Escherichia coli. Zcit. Natnrjorsch., I3b: 

5. Koch, A. L. 1956. The metabolism of methylpurines by Escherichia 

coli: I. Tracer studies. Jour. Biol. Chem., 219: 181-1SS. 

6. Litman, R. M., and Ephrnssi-Taylor, H. 1959. Inactivation et muta- 

tion des facteurs genetiques de l'acide desoxyribonucleique du 
pneumocoqne par l'ultraviolet et par l'acide nitreux. Compt. 
Rend., 249: 83S-S40. 

7. and Pardee, A. B. 1959. Mutations of bacteriophage T2 

induced by broinouracil in the presence of chloramphenicol. Virol- 
ogy, 8: 125-127. 

8. , . 1960. The induction of mutants of bacterio- 

phage T2 by 5-bromouracil: III. Nutritional and structural evi- 
dence regarding mutagenic action. Biocliem. Biophys. Acta, 42: 
9. Loveless. A. 1959. The influence of radiomimetic substances on 
deoxyribonucleic acid synthesis and function studied in Escheri- 
chia coli phage systems: III. Mutation of T2 bacteriophage as 


a consequence of alkylation in vitro: the uniqueness of ethyla- 
tion. Proc. Roy. Soc. London, Ser. B 150: 497-508. 

10. and Stock, J. 1959. The influence of racliomimetic sub- 
stances on deoxyribonucleic acid synthesis and function studied 
in Escherichia coli phage systems: 1. The nature of the inactiva- 
tion of T2 phage in vitro by certain alkylating agents. Proc. Roy. 
Soc. London, Ser. B 150: 423-145. 

11. Novick, A., and Szilard, L. 1951. Experiments on spontaneous and 

chemically induced mutations of bacteria growing in the chemo- 
stat. Cold Spring Harbor Symp. Quant. Biol, 16: 337-343. 

12. Ryan, F. J. 1959. Bacterial mutation in a stationary phase and the 

question of cell turnover. Jour. Gen. Microbiol., 21: 530-549. 

13. Strauss, B. 1961. Production of a permeability defect in Escherichia 

coli by the mutagenic alkylating agent, ethyl sulfate. Jour. Bad., 
81: 573-580. 

14. and Okubo, S. 1960. Protein synthesis and the induction 

of mutations in Escherichia coli by alkylating agents. Jour. Bad., 
79: 464-473. 

15. Szybalski, W. 1960. The mechanism of chemical mutagenesis with 

special reference to triethylene melamine action. In Developments 
in Industrial Microbiology (Miller, ed.), A T ciu York: Plenum 
Press, 231-241. 

16. Thorn, C, and Steinberg, R. A. 1939. Chemical inductions of gene- 

tic changes in fungi. Proc. Nat. Acad. Sci., 25: 329-335. 

17. Witkin, E. M. 1959. Post-irradiation metabolism and the timing of 

ultraviolet-induced mutations in bacteria. Proc. 10th Intern. 
Congr. Genetics, 1: 280-299. 


Auerbach: If UV produced mutations only at gene replication, then 
all lactose mutants on EMB agar should be sectored. As far as I remem- 
ber Witkin's experiments, she obtained mainly whole-colony mutants 
when nuclear number in the treated cells had been reduced to one. 

Strauss: The operational definition of mutation is that it is an inherited 
change. It is therefore required that any change be subjected to the 
test of reproduction. One might conceive of an alteration in DNA that 
produced a change in heterocatalytic activity but which could not be 
reproduced. We could not call this a gene mutation. At this level the 
discussion is one of semantics. 

Doctor Auerbach has pointed out a very surprising experiment and her 


memory is correct. In addition, Kaudewitz has shown that nitrite-treated 
bacteria produce nonsectored mutants. Mutants arising as a result of 
P-32 decay tend to be sectored. If one assumes that the Watson-Crick 
structure as confirmed by the experiment of Meselson and Stahl repre- 
sents the genetic material at all times, and further assumes that a muta- 
genic agent affects only one of the two DNA strands, then all mutants 
should be sectored. With chemical mutagens such as alkylating agents, 
the sectoring should be more complex for reasons indicated in my dis- 

Now Witkin's experiment (for lactose nonfermenters) is quite clear 
and, yet, Witkin herself at the Montreal Congress reported experiments 
indicating that certain UV-induced mutations were "fixed" only at the 
time of gene replication. There appears to be a paradox and essentially 
the same point raised here by Doctor Auerbach was raised by Marshak 
in the discussion of Witkin's paper on sectoring. If UV acted on a "chro- 
mosomal" level and affected both strands, there would be no sectoring. 
Perhaps the paradox is due to our assumption that the genetic material 
is always double-stranded. 

Witkin irradiated her material to a survival of 10~ 3 . At this survival 
one expects about seven lethal hits per organism. Since this Symposium, 
it has been shown by Marmur and Grossman that one of the effects 
of UV is to tie the two strands of DNA together by covalent bonds. 
Nitrous acid treatment has a similar effect. DNA treated with either 
of these two mutagens does not consist of two strands which can sep- 
arate merely by breaking hydrogen bonds. I think these findings will 
lead to an explanation for the lack of sectoring. 

Session III 

Evaluation of Mutations 
in Plant Breeding 

G. F. Sprague, Cluiirman 

U. S. Department of Agriculture, 

Beltsville, Mel. 

Use of Spontaneous Mutations in Sorghum 


Texas Agricultural Experiment Station, Substation No. 12, Chillicothe, Texas 

Sorghum in the United States is separated by usage into grain 
sorghum, dual-purpose sorghum, sweet sorghum, sudangrass, 
and broomcorn. Statistics on world production of sorghum are 
inadequate, but the total area devoted to the crop for grain pro- 
duction is thought to be more than 80 million acres. Sorghum is 
grown on all the continents below latitudes of 45 degrees and 
on many of the islands of the East and West Indies. Sorghum is 
the chief food grain in parts of Africa, India, and China. As a world 
food grain, sorghum ranks third, being exceeded only by rice and 

In the United States, sorghum is used for human food only as 
sorghum sirup, dextrose, starch, and oil. As a grain crop, sorghum 
is exceeded in production only by wheat and corn and has been 
grown on over 20 million acres in recent years. The sorghum crop 
is now about one fifth as large as the corn crop, whereas 10 years 
ago it was about one tenth as large. Prior to 1942, the acreage har- 
vested for forage exceeded that harvested for grain, but since that 
time the reverse has been true. 

Sorghum, Sorghum vulgar e Pers., is a large grass of many vari- 
eties that probably originated in Africa and has been cultivated 
since ancient times. An Assyrian ruin dating from 700 B. C. con- 
tains a carving depicting sorghum. In the United States, where 
the species was introduced, as many as 400 varieties have existed, 
many of which are sweet sorghums or sorgos. The number of varie- 
ties grown on farms has shrunk rapidly as horses and mules have 
disappeared from farms. It has become a cash grain crop and hybrids 
have appeared. In Africa and Asia, numerous other varieties exist. 

These varieties, even including sudangrass [var. sudanesis 
(Piper) (Hitch.)], cross-pollinate readily and produce fertile off- 
spring. Vinall, et al. (43) 1 have considered all varieties as belong- 
ing to the same species, but Snowden (35) has classified some 3,000 

'See References, page 202. 



forms into 31 species. Snowden's collection was worldwide and his 
classification is useful even though his giving races specific rank may 
he questionable. Characteristics such as plant color, juicy or pithy 
stem, pericarp color, presence or absence of testa, presence or absence 
of awns, starchy and waxy endosperm, white or yellow endosperm, 
dehiscent or indehiscent spikelets, height and duration of growth, 
are known to be genetic. But the inheritance of the complex of 
characters that makes up the groups of varieties or races such as the 
broomcorns, kafirs, feteritas, milos. kaoliangs, hegaris, shallus, sumacs, 
etc., is not understood. Laubscher (16) has considered modifying 
complexes to be important in separating the races of sorghum. 
Reviews of sorghum genetics literature have been made by Martin 
(20), Myers (23), and Quinby and Martin (31). 

Introduction Into the United States 

Sorghum has been grown in the United States for a little over 
a century. Accounts of the early introductions and early work with 
sorghum have been presented by Vinall, et al. (43), Martin (20), 
Quinby and Martin (31), and Quinby, et al. (30). 

The first sweet sorghum that reached the United States came 
from China by way of France in 1853 and is now known as Black 
Amber. Before the distinction between sorghum and sugarcane, 
Saccharum officinarutn , was well understood, the Indian Service 
sent seed of this sorghum to the Comanche and Brazos Reserves in 
Throckmorton and Young Counties in Texas as Chinese Sugar- 
cane. To this day, farmers in West Texas call sweet sorghum 
"cane". Fifteen sorgo varieties, several of which survive, were intro- 
duced in 1857 from South Africa. Two other sweet sorghums were 
introduced in 1881 and 1891, the first from Natal, South Africa, and 
the second from Australia where the variety was undoubtedly intro- 
duced. The present American sorgo varieties originated from these 
17 introductions and a 1951 introduction from Ethiopia. 

The first grain sorghums grown in the United States were 
White and Brown Durras which reached California from Egypt in 
1874. These two varieties were never widely grown outside Cali- 
fornia and have been unimportant as parents of present-day vari- 
eties and hybrids. The progenitor of the milo varieties was intro- 
duced into South America and reached South Carolina from Colum- 


bia in 1879. The variety was called "Millo Maize" (14). How Black- 
hull Kafir reached die United States is unknown (43), but the varie- 
ty was growing on farms in Kansas about 1890. A late-maturing 
kafir was introduced from Columbia in 1880 (14) and was distributed 
in South Carolina as "White Millo Maize". Perhaps Blackhull 
Kafir originated as an early-maturing mutation in this late-matur- 
ing kafir. White Kafir and Red Kafir from Natal were shown at the 
Centennial Exposition at Philadelphia in 1876 and seed from the 
exhibit was later distributed. Pink Kafir, Feterita, Hegari, and 
Sundangrass were introduced in 1904, 1906, 1908, and 1909, respec- 
tively. Broomcorn has been grown in the United States since Ben- 
jamin Franklin began its culture with seeds plucked from an 
imported broom. 

Transformation of Sorghum in the United States 

The mechanization of agriculture and changes in sorghum 
that began about 30 years ago have changed sorghum from a feed 
crop for use on the farm into a cash grain crop. The chief changes 
in sorghum on the farm since 1930 have been a reduction of about 
40 centimeters in height and a shortening of the duration of growth 
by about a week. The reduction in height and dry-headedness asso- 
ciated with early maturity have made combine harvesting possible. 
Sorghum production has expanded into higher latitudes and higher 
elevations as extremely early-maturing varieties have become avail- 
able. Recently, a method of hybrid sorghum seed production using 
cytoplasmic male-sterility has been devised and hybrids have resulted 
in an expansion of acreage in areas outside the "sorghum belt" as 
well as within it because of higher yields. 

These changes have come about from making use of alleles 
discovered in the species. The transformation of sorghum in the 
United States within the last half century is actually a further domes- 
tication of the species to make it fit the needs of mechanized farm- 
ing. Most improvement in sorghum has resulted from accumulating 
desirable alleles in a single variety. 

Genes Used in Sorghum Improvement 

Many of the contrasting characters present in sorghum were 
in one or another of the varieties introduced into the United States, 


but a few alleles resulted from mutations after sorghum was intro- 
duced about 100 years ago. All of the alleles must have arisen as 
spontaneous mutations originally and no effort will be made to 
distinguish between recent and ancient mutations. 

Nevertheless, eight mutations have occurred in milo and have 
been preserved during 80 years in the United States. Four of these 
eight that control response to photoperiod occurred at three 
loci. Two were alleles that reduce height, one affects pericarp color, 
and one causes resistance to a root-rotting fungus. The Y gene that 
causes colored pericarp mutated to recessive y in milo before 1910 
and White Milo resulted. The recessive mutation causing resistance 
to periconia root-rot must have occurred many times because a few 
resistant plants could be found in almost any badly diseased field 
of any of several milo varieties when the disease first became seri- 
ous. Probably mutations at some loci occur again and again. Con- 
versely, many other genes have not been seen to mutate in the 50 
years that sorghum has been worked with by plant breeders in the 
United States. For instance, the yellow endosperm character was 
never seen in the United States until O. J. Webster returned with it 
from Africa in 1951. 

Maturity Genes 

The history and evaluation of milo in the United States has 
been presented by Karper and Quinby (13, 14) and by Vinall, et al. 
(43). Another early-maturity genotype has appeared in more recent 
years. When "Millo Maize" reached the United States in 1879 it 
was a tall, late-maturing variety. When tested at the Louisiana 
and Kansas Experiment Stations in 1888, the variety matured in 
about 120 days, indicating a time of blooming about 90 days from 
planting. By 1900, the variety in use in western Texas was Standard 
Milo that bloomed in about 65 days and matured in 95 days. Before 
1910, farmers in western Texas were growing Early White Milo 
that bloomed in 50 days and matured in 80 days. In 1938, a farm- 
er in California found a still earlier maturing milo that bloomed 
in 44 days and matured in 75 days. Commercial production of this 
early-maturing milo, called Ryer Milo, began about 1948. 

The inheritance of duration of growth in milo has been reported 
by Quinby and Karper (25, 29). Three genes influence plant 
response to photoperiod and control the time of floral initiation. 

quinby: mutations in sorghums 187 

The genes have been named Max, Mrt 2 , and Ma 3 . Only one recessive 
allele is known at each of the Mai and Ma» loci, but two recessive 
alleles are known at the Ma s locus. Recessive max and ma\ act 
like amorphic alleles since they cause early floral initiation when 
homozygous. Recessive ma 2 and ma 3 modify the expression of the 
dominants and are not amorphic. The various milo maturity geno- 
types (but not the infrequent cross-over genotypes) that emerge 
from crosses of Early White Milo with Yellow Milo and with 
Ryer Milo are shown in Table I. The "Millo Maize" introduced 

Table 1. — Milo Maturity and Pericarp Color Genotypes and Phenotypes, 
Excluding Crossover Classes, at Chillicothe, Texas, from 
Early June Plantings. 


Ma\ Ma 2 (Mazy) . . 
Mai Ma* (mail") . . 
Mai Ma 2 (waV) • 
Mai Ms (Maty) . . 
mai Ma 2 (Ma 3 y) . . . 
Mai ma* (mazl") . . 
Mai ma* (ma 3I). ■ 
max Ma-i (mazY) . . . 
ma\ Mai (wa R 3 T). . 
mai ma2 (Many). ■ ■ - 
mai mai (mazl') ... 
mai mc2 (ma 3T) . . . 

Days to bloom 

Pericarp color 

























in 1879 was evidently dominant for all three maturity genes and 
has been reconstituted by selecting the late-maturing genotype 
from the F 2 generation of appropriate crosses. A genetic linkage 
exists between the white pericarp color gene y and Ma 3 and between 
the maturity gene Max and the dwarfing gene diu^. 

Of the many varieties grown in the United States and tested 
for photoperiod response, only Yellow Milo (60-day), Hegari, and 
Early Hegari, and a few of their derivatives, are dominant Max. The 
remainder, including the parents of the present sorghum hybrids, 
are recessive ma x and have a critical photoperiod high enough to 
allow relatively early floral initiation and maturity. The dominant 
or recessive condition of the genes at the Ma 2 and Ma 3 loci is 
unknown for most varieties other than the milos, but most are 



apparently dominant for either Ma 2 or Ma 3 or both. Yellow milo 
(60-day) is genetically Ma! ma 2 ma 3 . Hybrids of 60-day Milo and 
Texas Blackball Kafir, Hegari, Early Hegari, and California White 
Durra are extremely late in maturity. Hybrids of 60-day Milo and 
Early Kalo, Kalo, Bonar Durra, Feterita F. C. 81 1, Manko, and Fargo 
are later in maturity than either parent but not extremely late. The 
latter group is probably dominant for either Ma 2 or Ma s and the 
former for both Ma 2 and Ma 3 . Obviously, more than three major 
maturity genes may exist in sorghum and all the milos might be 
dominant for a fourth gene. The late-maturing kafir introduced into 
the United States in 1880, since it was so late in maturing, must have 
been dominant for all three genes. If Blackhull Kafir came from this 
late-maturing kafir, it arose as a recessive mutation at the M«i locus. 
Several varieties are sensitive to photoperiod at the tempera- 
tures that prevail in the summer months at Chillicothe, Texas, but 
many other varieties are insensitive or show varying degrees of 
sensitivity. Data in Table 2, taken from Quinby and Karper (26), 
show the response of 12 varieties to photoperiod at Chillicothe, 

Table 2. — Effect of 10-hour Photoperiod on Time of Floral Initiation, Leaf 

Number, and Time of Anthesis of Sorghum Varieties Planted July 13, 1941, 

at Chillicothe, Texas. 

Number of days 

from planting to 

TVTi tT-*-.K***~ 

of leaves on 
ire plant 

Head differentiation 

















Sooner Milo 







Texas Milo 





















Calif. White Durra 







Spur Feterita 



































Blackhull Kafir 







Dwf. Broomcorn 







•Short day exposure to sunlight was 10 hours. In July at Chillicothe, Texas, the sun is abo\e 
horizon for about 14 hours. 


Texas, in 1941. Several varieties are sensitive to photoperiod, sev- 
eral are less sensitive, and one, Dwarf Brooincorn, is quite insensi- 
tive. Although this point has not been established experimentally, 
it can be inferred from work with other species that the thermal 
requirements have not been met if a sorghum variety is not sensi- 
tive to photoperiod. Likewise, it can be inferred that varying degrees 
of sensitivity indicate that the thermal requirements have been 
partially met. Insensitivity to photoperiod is undoubtedly the 
mechanism that allows late maturity of varieties in the tropics. 
In some tropical areas, sorghum is planted before a rainy season 
of 3 or 4 months duration and the crop heads and matures after 
the rainy season. Such a long duration of growth under 12-hour 
days would not be possible with varieties sensitive to photoperiod. 
Three or four maturity genes, allelic series at those loci, and vary- 
ing degrees of insensitivity to photoperiod might well account for 
all the maturities found in sorghum. 

Early floral initiation, low leaf number, and small plant size 
are associated, as shown by data presented by Quinby and Karper 
(25) and reproduced in Table 3. At the time these data were collected, 

Table 3. — Data Showing the Size of Four Milo Maturity Genotypes Grown 

Under Normal Photoperiods from a Planting at Chillicothe, Texas, 

Made on June 20, 1 944. 


Criteria 50-day 60-day 80-day 90-day 

ma\Ma<> maz Ma\ ma^ maz Ma\ ma* Maz Ma\ AIa-> maz 

No. of days to anthesis 48.6 ± 0.3 68.6 ± 0.4 82.6 ± 0.7 102.4 ± 0.04 

No. of leaves 16.4 ± 0.1 22.0 ± 0.1 23.5 ± 0.3 31.5 ± 0.2 

Height of plant, cm 85.8 ± 1.2 87.7 ±1.3 121.6 ± 3.4 151.8 ± 2.7 

Length of leaf, cm 54.1 ± 1.5 65.1 ± 0.5 65.3 ± 0.8 78.1 ± 0.5 

Diameter of stalk, cm 1.42 ± 0.02 2.30 ± 0.04 2.22 ± 0.05 2.53 ± 0.01 

Weight of head, lbs. 0.24 ± 0.02 0.36 ± 0.02 0.33 ± 0.03 0.27 ± 0.02 

Weight of plant, lbs. 0.43 ± 0.03 0.62 ± 0.04 0.64 ± 0.04 0.97 ± 0.06 

Ryer milo maturity was not known -which accounts for the fact 
that a Ryer population was not measured. The data in Table 3 
show that the duration of the vegetative period is positively cor- 
related with plant size, the best measure of which is total dry weight 
per plant. The figures show that a two-fold difference in size result- 


ed from a difference of 54 days in blooming. This difference in size 
is apparently the result of the time element alone and not a differ- 
ence in rate of growth because no appreciable difference occurred 
in the time that corresponding successive leaves appeared on the 
four genotypes. The fact that larger leaves appeared on the later- 
maturing genotypes indicates only that the growing point con- 
tinued to grow in circumference from day to day in the early life 
of the plant, and a leaf arising from a higher node would be expected 
to be larger than one arising from a lower node. Some additional 
data are presented in Table 3 to show the morphological relation- 
ships of the various genotypes. As would be expected, there is a 
positive correlation between leaf number and number of days to 
anthesis, height, stalk diameter, and leaf size. The four genotypes 
measured are similar to one another in appearance, except that 
each genotype is a larger counterpart of the one immediately pre- 
ceding it in earliness of maturity. Early maturing varieties are used 
by farmers because the later maturing varieties exhaust the soil 
moisture before maturity or need additional irrigations. 

A logical explanation of the nature of the maturity genes in 
milo would be that Ma 1} Ma 2 , and Ma s are dominant inhibitors 
that block some essential reaction leading to floral initiation. This 
assumption would mean that the "wild type" was small and very 
early in maturing because of early head initiation. Sorghum must 
have been domesticated by the preservation in the tropics of larg- 
er and later maturing plants that carried inhibitors that prevented 
early floral initiation. When tropical varieties came to the United 
States and were grown at latitudes above 30 degrees, they were 
too late in maturity to be of greatest use and man again preserved 
mutations, but this time those that inactivated the inhibitors 
to some degree. 

Height Genes 

In most of the sorghum-producing areas of the world, tall 
stature is preferred; but, in the United States, farmers have used 
shorter and shorter varieties as they became available. The dwarf- 
ing genes that have been useful in sorghum improvement shorten 
only the internodes. Four such dwarfing genes are known in sor- 
ghum and are inherited independently (28). 

Recessive chu 4 was in Standard and Early White Milos and 

ouimiy: mutations in sorghums 191 

in Blackhull Kafir that farmers were growing by 1900. Recessive 
chi'i was found before 1905 and recessive dw 2 about 1910, both in 
milo. The combine height varieties that originated in the 1920's 
and later were recessive for three height genes were obtained by 
selecting 3-dwarf segregates from crosses between milo (dwi Dw-> 
Diux dw 4 ) and kafir (Du^ Div 2 dw 3 dw 4 ). The dominant gene in all 
of the 3-dwarf varieties of milo-kafir parentage is Diu 2 and in the 
3-dwarf milos Dru s . Four-dwarf strains have been obtained by cross- 
ing these two 3-dwarf genotypes (diui Dw 2 dw 3 dw± and dtOi div 2 
Div?, du'i) and selecting 4-dwarf segregates. The genetic height 
constitution of many varieties and parents of sorghum hybrids 
and broomcorn is known. The genetic identity of many strains is 
given in Table 4. At Chillicothe, Texas, on dryland 2-dwarfs were 

Table 4. — Classification of Sorghum Varieties According to Height. 

Genotype Variety 

Dw\ Dw-i Dwz D1V4 None identified 

Dw\ Dw-2 D1V3 dw i Tall White Sooner Milo SA 1170, Spur Feterita, Manchu Brown 

Kaoliang, Shallu SA 401, Sumac 
Dw\ Dwi dw% Da<4 Standard Broomcorn 
Dwi dwz Dwz Dwi None identified 
dw\ D1V2 Dwz D1V4 None identified 
Dw\ Dw< dwz did Texas Blackhull Kafir, Kalo, Early Kalo, Chiltex, Shantung Dwarf 

Dw\ diL'2 D1V3 dim Bonita, Early Hegari, Hcgari 
dw\ D1V2 D1V3 du>4 Dwarf Yellow and White Milos, Sooner Milo 
Dw\ dwt dw% D1U4 Acme Broomcorn 
dw\ D1V2 dwz Dwi Japanese Dwarf Broomcorn 
dw\ dwz Dw% Dwi None identified 
Dwi dwi dw% dwi None identified 

dw\ D1V2 divz dw4 Combine Kafir 60, Combine 7078, Martin, Plainsman, Wheatland, 

Caprock, Combine White Feterita SA 396, the Redbines, Day 
dw\ div* Dws diet Double Dwarf Yellow Milo, Double Dwarf Yellow and White 

Sooner Milos, Ryer Milo 
dw\ dwz dw% D1V4 None identified 

dwi du)2 du>3 dwi SA 403, 4-dwarf Martin, 4-dwarf Kafir 

usually about 50 cm shorter than 1 -dwarfs; 3-dwarfs about 40 cm 
shorter than 2-dwarfs; and 4-dwarfs about 10 cm shorter than 3- 
dwarfs. As more recessive ^enes accumulate in a strain, the dwarfing 
effect is obviously lessened. 

Two of four height genes are unstable. Mutations to tallness in 


kafir were reported by Karper (11) at a ratio of 1 mutation to 604 
zygotes. Such a mutation rate produces almost 100 tall plants per 
acre when plant population per acre is above 50,000. Tall plants 
make fields unsightly and farmers are suspicious of seed production 
practices when such large numbers of off-type appear in their fields. 

All varieties unstable for height are recessive dr«» 3 , except for 
Early Hegari. Some varieties recessive for dw s , such as Shantung 
Dwarf Kaoliang and Acme and Japanese Dwarf Broomcorns, are 
stable. Hegari and Early Hegari have the same height constitution, 
DiVi dwo Du's du> 4 , but Hegari is stable for height and Early Hegari 
is unstable. The unstable gene in Early Hegari has not been identi- 
fied. The cause of the instability of height genes in sorghum is not 

Sorghum hybrids of the future will probably be heterozygous 
3-dwarf or homozygous 4-dwarf. The seed-parents will be 4-dwarfs. 
The pollinators will exist in two versions: the 4-dwarf and the 
stable 3-dwarf, drui dw 2 Du> 3 diu^. If the 4-dwarf version of the 
hybrid is too short in stature in some areas, the heterozygous 3- 
dwarf hybrid would be used. In the original height inheritance 
paper (28), the possibility of using stable 3-dwarfs was pointed out. 
Stephens (38) then suggested the conversion of unstable 3-dwarfs 
to 4-dwarfs by a backcrossing process followed by a recovery of 
stable 3-dwarfs following mutation to dominant Dzu s . In producing 
hybrids using 4-dwarf seed-parents and stable 3-dwarf pollinators, 
mutations to 3-dwarf would still occur in the 4-dwarf female. The 
mutated gamete would be dxox dw» Div 3 dw± and the plant pro- 
duced from it would be homozygous 3-dwarf. The rest of the hybrid 
population would be heterozygous 3-dwarf and only a few centi- 
meters shorter than the plants that carry the mutated gene. In the 
field, the few homozygous 3-dwarfs would be inconspicuous. 

These dwarf genotypes in sorghum are brachytic. Brachysm, by 
definition, is dwarfness characterized by shortening of the inter- 
nodes only. Some unpublished data bearing on this point and col- 
lected in 1947 are presented in Table 5. Plants of two Sooner milo 
populations segregating for height were studied. The data from 
plants heterozygous for height were discarded, leaving only the 
data from homozygous height genotypes to be summarized. 

The mean difference in height between 1 -dwarf plants in pop- 

quinby: mutations in sorghums 193 

Table 5. — Comparison of Plant Characters in Sooner Milo Genotypes that 
Differ by One Height Allele in Populations Grown at Chillicothe, Texas, in 1947. 

Plant character 





SA 1295 population 

1 -dwarf 


Height to flag leaf, cm 

136.4 ± 1.65 

95.8 ± 1.11 



No. of leaves 

21.1 ± 0.17 

21.1 ± 0.17 



Days to bloom 

62.6 ± 0.31 

62.0 ± 0.23 



Width of 15th leaf, mm 

72.0 ± 1.20 

69.8 ± 1.06 



No. of stalks per plant 

2.3 ± 0.18 

2.5 ± 0.12 



Weight of heads, gms. 

186 ± 16.2 

181 ± 13.1 



SA 5535 population 

2— dwarf 


Height to flag leaf, cm 

102.1 ± 1.37 

55.4 ± 0.73 



No. of leaves 

19.4 ± 0.21 

19.0 ± 0.17 



Days to bloom 

59.0 ± 0.44 

58.7 ± 0.45 



Width of 15th leaf, mm 

68.6 ± 0.73 

68.6 ± 0.77 


No. of stalks per plant 

3.2 ± 0.14 

3.2 ± 0.13 


Weight of heads, gms. 

209 ± 10.3 

172 ± 10.0 



ulation SA 1295 was 41 cm. The difference between 2-dwarf and 3- 
dwarf plants in population SA 5535 was 47 cm. These differences were 
highly significant. No significant differences occurred between the 
height genotypes in number of leaves, days to bloom, width of leaf, 
or number of stalks per plant. The 5-gram difference in weight of 
heads in the height classes of 1 -dwarf and 2-dwarf plants in popu- 
lation SA 1295 was not significant. However, the 37-gram differ- 
ence in favor of the 2-dwarf over the 3-dwarf class of population 
SA 5535 was significant statistically. Why the 3-dwarf plants in 
population SA 5535 produced lighter heads than 2-dwarf plants 
is not apparent. Perhaps the smaller head yield of the 3-dwarf class 
could be due to shading from the taller 2-dwarf plants as the two 
heights occurred at random in the rows. Regardless of this excep- 
tion in head weight, the dwarfing genes in sorghum appear to be 
truly brachytic. 

This brachysm, true to its definition, does not influence length 
of the sheath of the upper leaf, peduncle length, or head length. 
Fortunately, the length of the peduncle is not shortened, otherwise 
the head of short plants would not emerge from the boot. Also, 
if the rachis and seed branches of the head were shortened as much 
as the internodes, short-statured sorghums would have heads as 
compact as those of Pig-nosed durras. Data to show the lack of 


influence of dwarfing genes on leaf sheath, peduncle length, and 
head length are shown in Table (5. Kafir plants of these height geno- 
types growing together at Chillicothe, Texas, in 1960 were tagged 

Table 6. — Days from Planting to Blooming and Size of Various Plant Parts of 
Three Height Genotypes of Kafir at Chillicothe, Texas, in 1960. 

Height genotype 


2-dwarf 3— dwarf 4-dwarf 

Days from planting to blooming 55.5 ± 0.6 55.6 ± 0.4 56.8 ± 0.4 

Flag leaf sheath length, cm 33.4 ± 0.4 32.5 ± 0.6 30.6 ± 0.4 

Height to upper node, cm 79.4 ± 1.0 34.2 ± 0.5 20.9 ± 0.5 

Peduncle length, cm 32.5 ± 0.9 31.7 ± 0.8 35.6 ± 0.8 

Head length, cm 20.2 ± 0.4 23.1 ± 0.4 21.6 ± 0.3 

Total height, cm 132.2 ± 1.3 89.0 ± 1.1 78.1 ± 1.0 

Stalk diameter, cm 1.65 ± 0.04 1.83 ± 0.03 1.58 ± 0.04 

for days from planting to blooming and were measured for height 
from the base of the culm to the upper node for peduncle length, 
head length, and flag leaf sheath length. The 2- and 3-dwarf classes 
were isogenic lines, but the 4-dwarf class was a derived line that 
was similar in maturity but not isogenic with the other two. The 
data show that even the 4-dw-arf genotype has a peduncle and head 
as long as those of taller 3-dwarf and 2-dwarf genotypes. 

Rapid internode elongation in sorghum follows floral initia- 
tion, indicating some connection between the presence of a floral 
bud and internode elongation. The mechanism involved in the 
shortening of the internodes without a shortening of the peduncle 
and head is as yet unknown. Far-red radiation is now known to induce 
floral initiation and influence internode elongation (8). Probably 
dwarfing genes in sorghum slow down the synthesis of the sub- 
stance that causes elongation. 

Genes For Resistance to Disease and Insects 

The diseases of sorghum and the organisms that cause them 
have been described by Leukel, et al. (19). Resistance to some of the 
diseases is present in one or more varieties and resistance to some 
diseases has been bred into some varieties. 

All of the different strains of milo grown on farms in the United 
States prior to 1937 were susceptible to milo disease, later identified 


as periconia root rot (18) and caused by the fungus Periconia cir- 
cinata. This disease suddenly became widespread during the drought 
years beginning in 1934. A number of varieties of milo parentage, 
including Day, Colby, Wheatland, and Beaver, were also suscep- 
tible as was Darso and one strain of Sumac, a sorg;o variety. Resistant 
plants in Dwarf Yellow milo were quickly found in Kansas (44) and 
in Texas (27). Bowman, et al. (4) reported susceptibility as being 
partially dominant and controlled by a single major gene. In Texas 
(27), resistant plants were invariably found to be homozygous resist- 
ant. Resistant plants that occurred occasionally in susceptible 
strains must have occurred due to mutation. In pure milo, resist- 
ant selections were found in one strain in Kansas, two in Texas, 
and three in California (31). Resistant plants of Beaver were found 
at both Dalhart, Texas, and Garden City, Kansas. Resistant plants 
of Day and Colby were found in Kansas. Resistant Darso selections 
were found at Temple and Chillicothe, Texas, and at Stillwater, 
Oklahoma. Resistant plants of exact "Wheatland type were never 
found in diseased fields of Wheatland, but resistant plants found 
in such fields were distributed as Martin, Resistant "Wheatland No. 
288, and Dalhart Resistant Wheatland. 

Yield trials over long periods of years at the Texas Substations 
at Chillicothe and Lubbock (27) have shown that losses of 50 to 
60 per cent can be expected whenever a susceptible variety is grown 
on infested soil. Fortunately, all varieties grown to any extent and 
most parents of hybrids now in production are resistant to periconia 
root rot. 

The three smuts of sorghum in the United States are covered 
kernel smut, SpJiacelotJieca sorglii, loose kernel smut, S. cruenta, and 
head smut, S. relliana. Both kernel smuts can be controlled with chem- 
ical seed treatments, but head smut is soil-borne. Resistance to the 
four races of covered kernel smut is present in Spur Feterita. Casady 
(5) has recently reported on the inheritance of resistance to ,S. sorglii. 

Head smut has recently become a problem in Texas, probably 
because of the widespread planting of a susceptible variety, Com- 
bine 7078, and several susceptible hybrids. The inheritance of 
resistance has not yet been determined, but heritable resistance 
exists since selections from resistant strains in a nursery in Refurio 
County on the Texas Gulf Coast in 1959 were resistant again in 


1960. Resistance occurs in a number of varieties, including milo, 
hegari, and feterita. Among the parents of sorghum hybrids, Com- 
bine White Feterita is highly resistant but not immune. Other 
resistant parents are Wheatland, Plainsman, Caprock, and several 
of the Redbines. Most of the kafirs are moderately susceptible and 
Martin slightly so. The head smut resistance of a hybrid cannot 
be predicted if one parent is resistant and one susceptible to the 
fungus. Some parents, such as Caprock, are quite resistant them- 
selves, but their hybrids with susceptible parents, such as Combine 
Kafir Tx 3197, are susceptible. Combine White Feterita, Tx 09, 
is also resistant, but its hybrid with Tx 3197 is quite resistant. 
Most hybrids with two resistant parents are resistant. 

A destructive stalk rot of sorghum, caused by the fungus Macro- 
pliomina pliaseoli (Maubl.) Ashley, is called charcoal rot. Severe 
lodging results from the presence of the disease because the culms 
break over close to the ground level when the pith of the lower 
internodes rots away. The fungus, Fusarhim inoniliforme Sheldon, 
produces symptoms that are sometimes quite similar to those pro- 
duced by M. pliaseoli, and there is a tendency to attribute the dam- 
age to charcoal rot regardless of which fungus is responsible. Tullis 
(42) reported on work done with moniliforme and cited the perti- 
nent literature. Resistance to both these diseases exists in sorghum, 
but the expression of susceptibility is variable from year to year 
and it has been impossible to determine the mode of inheritance 
of resistance to these stalk-rotting fungi. Breeding for resistance to 
charcoal rot has not been very effective in sorghum up to the 

Anthracnose of sorghum caused by Colletotriclrum gramini- 
colum (17) severely damages sorgo varieties grown in Mississippi. 
Leaves are injured sometimes to the point of defoliation and rot- 
ting causes a break-down of the tissues of the stalk. LeBeau and 
Coleman (17) found resistance to the leaf phase of this disease in 
recently introduced varieties from Africa. They found resistance to 
the disease to be a simple dominant. 

The chinch bug, Blissus leiicopterus (Say.), is a serious pest 
on sorghum in some areas in certain years. In Texas and Oklahoma, 
the insect migrates from glass pastures or barley fields to sorghum 
by flying and the usual measures of building barriers to stop the 
crawling insects is not effective. An effective breeding program to 


produce adapted chinch bug-resistant varieties was carried on at 
Lawton, Oklahoma. This breeding work, and studies of resistance 
that went on at the same time, gave some information on the inher- 
itance of resistance. Snelling, et al. (34) reported on work at Man- 
hattan, Kansas, and Lawton, Oklahoma. Resistance was found to 
be dominant and transgressive segregation for resistance indicated 
that more than one gene was involved. Chinch bug resistance was 
not put on a definite basis by this work, but there was ample evi- 
dence of its inheritance. Varieties of sorghum have been classified 
for susceptibility to damage from chinch bugs by a number of 
workers in several states and this information has been summarized 
in a book by Painter (24). Dahms (6), working in Oklahoma, found 
that resistant varieties showed the greatest tolerance to a uniform 
infestation of chinch bugs, that the insects preferred susceptible 
varieties as hosts, that female chinch bugs lived longer on suscep- 
tible varieties than on resistant ones, that more eggs were laid on 
susceptible varieties, and that nymphs developed faster on suscep- 
tible varieties. This work is significant and shows that there is in 
plants actual resistance to insects produced by influencing the 
biology of the insect. 

Male-sterile Genes 

Hybrid vigor was known to exist in sorghum long before the 
difficulties inherent in producing crossed seed in a species with per- 
fect flowers -were overcome. The story of the advent of sorghum 
hybrids has been told at least twice (31, 30). Cytoplasmic male- 
sterility was the final answer to the problem of hybrid seed produc- 
tion, but several genetic male-steriles that were apparently mutations 
were worked with before cytoplasmic male-sterility was discovered. 

Antherless -was the first genetic male-sterile worked with and 
was found in 1929 (15). Male-steriles 1 and 2 were found about 
1935, one in India and the other in Texas. It was proposed by Ste- 
phens (36) in 1937 and ms 2 might be used for the production of 
hybrid sorghum seed. By 1946, -work had progressed with ms 2 to 
a point where it appeared that hybrid sorghum might be put into 
production. However, a still better male-sterile had been found 
and work with ms 2 was abandoned. 

In 1943, Glen H. Kuykendall found in the Day variety a male- 
sterile that must have arisen as a mutation. This abnormality proved 


to be a genetic male-sterile that gave male-sterile offspring when 
crossed to some varieties and fertile offspring when crossed to others. 
The proposed procedure for the production of hybrid seed by a 
8-way cross was published by Stephens, el al. in 1952 (40). The Day 
male-sterile was distributed to plant breeders by the Texas Agri- 
cultural Experiment Station in 1951. Because cytoplasmic male- 
sterility was found, the Texas Station did not put any hybrids into 
production using the Day male-sterile. However, one seed company 
did produce hybrids for a year or two using the Day male-sterile. 

Beginning in 1949, work was done that established the exist- 
ence of cytoplasmic male-sterility. In 1950, F 2 populations of recip- 
rocal crosses involving Sooner Milo and Texas Blackhull Kafir 
were grown and partial male-sterility was found in the popula- 
tions whose female parent was milo. Holland (10) reported the 
early data from this study in a thesis. It soon became apparent that 
male-sterility in this case was due to the interaction between milo 
cytoplasm and kafir nuclear factors as reported by Stephens and 
Holland in 1954 (39). The degree of male-sterility was increased 
as the proportion of kafir chromosomes in milo cytoplasm was 
increased. The number of genes involved was not determined by 
Stephens because of the pressure of work to get sorghum hybrids into 
production and because unfavorable weather in 1952 and 1953 at 
Chillicothe confused the expression of genetic sterility. Maunder 
and Pickett (21) reported cytoplasmic male-sterility to be depend- 
ent on a single pair of recessive genes, ms c ms c , interacting with 
sterile cytoplasm. Plants in their fertile classes in segregating pop- 
ulations varied in seed set from 5 per cent upward. Obviously, 
important modifying genes must exist. Finding fertility restorers 
was no problem in sorghum. All milos and milo derivatives with 
sterile cytoplasm carry the dominant allele, Ms e ; otherwise they 
would be male-sterile. The dominant restorer also exists in many 
varieties with normal cytoplasm. 

The genetic male-steriles that arose as mutations are not impor- 
tant today in the production of hybrid sorghum seed, but work 
with them contributed to the discovery of cytoplasmic male-ste- 
rility. J. C. Stephens had been working with mutant male-steriles 
in sorghum for more than 20 years when he and R. F. Holland 
announced, in 1954, the attainment of cytoplasmic male-sterility. 


Other Useful Genes 

Seed color is an important economic characteristic of sorghum. 
The colors are varied and occur in different layers of the caropsis. 
(7, 41, 32, 37). The contrasting colors, such as white, yellow, red, 
and brown, occur as a result of the color content of the- epicarp, 
the thickness of the mesocarp, the presence or absence of a brown 
testa, and the dominant or recessive condition of a gene designated 
as S that controls the presence or absence of brown pigment in the 
epicarp when a brown testa is present. Six genes, exclusive of the 5 
gene, have been reported to cause color in the epicarp. The genetics 
of seed color in sorghum has been summarized by Quinby and 
Martin (31). 

All of the possible pericarp colors are not represented by 
varieties in the United States. Most of the grain produced in the 
United States is genetically red or pink. Small amounts of white 
grain are produced. White grain frequently is used as poultry feed 
as chickens seem to prefer white grain for some reason. True yellow 
is not represented by a commercial variety in the United States 
but may soon be as the yellowest yellow endosperm varieties or 
hybrids have a yellow pericarp. 

Brown seeds result when a brown testa or undercoat is pres- 
ent along with a dominant spreader gene that allows the presence 
of brown in the epicarp. When the seeds are dominant red or pink 
also, the seeds are dark reddish brown. The brown color is caused 
by the presence of tannin and the astringency of developing brown 
seeds is some protection from birds. In Africa, brown-seeded 
varieties are used to make beer. In the United States brown-seeded 
varieties are grown only in the humid areas or where birds are a 

Most soroo varieties have brown seeds and the brown color 
is associated with high tannin content. Atlas and Tracy, two widely 
grown sorgo varieties that have no brown testa in their seeds, orig- 
inated as selections from crosses involving brown-seeded and non- 
brown-seeded varieties. 

Most discolored spots on sorghum grain result from injury 
caused usually by sucking insects or diseases. When sorghum plants 
are damaged, any dead tissue becomes pigmented. The spots become 
blackish brown, reddish brown, or tan in color, depending upon 


the genetic constitution of the plant with respect to two plant 
color genes, Q and P. Plants with black glumes (QP) have blackish 
brown spots, those with red glumes (qP) have reddish black spots, 
and those with sienna (qp) or mahogany glumes (Qp) have tan 
spots. The pigment in the spots is water-soluble and tan spots are 
the least objectionable in starch manufacture by the wet milling 
method. The beautiful sienna glume color of Sweet sudangrass 
is the result of the variety being recessive for both q and p. 

Two kinds of starch are found in varieties of many cereals, 
including rice, maize, millet, coix, barley, and sorghum. Waxy 
starch, which stains red with iodine, is amylopectin, a branched 
glucose polymer. Common starch that stains blue with iodine is a 
mixture of amylopectin along with an unbranched or linear poly- 
mer, amylose. The properties of waxy starch make it of particular 
interest to industry as it forms a clear paste which has little tendency 
to gel on standing. Extensive studies of the chemical properties of 
sorghum grain and of the properties of sorghum starch have been 
made by Barnaul, et al. (3). 

Waxy endosperm of sorghum was first reported by Meyer 
(22). Karper (12) found waxy to be a simple recessive to starchy 
in segregating populations. Waxy varieties and hybrids are in com- 
mercial production. The waxy allele is present in Black Amber, 
the first sorghum to become established after being- introduced into 
the United States. The waxy allele bred into waxy grain sorghums 
came from the Batad variety that came from the Philippines. Seeds 
of a number of varieties from India and several kaoliangs are also 

A sugary endosperm character in sorghum resembles sugary 
in maize. The character occurs in a number of Indian varieties 
and the sugary allele was found as a mutation at Chillicothe (31). 
Ayyangar, et al. (1) found sugary to be a simple recessive. Sugary- 
varieties in India have "dimpled" grains. An analysis of sugary 
grains in India showed the amount of reducing sugars to be three 
times the quantity found in nondimpled grains, the nonreducing 
sugars being equal in both. No commercial use has been made of this 
character in the United States. In India, sugary grain sorghum 
is parched by placing green heads in beds of coals. The grain is 
then knocked from the heads and eaten. 

quinby: mutations in sorghums 201 

A yellow endosperm character was introduced into the United 
States from Nigeria by O. J. Webster in 1951. The yellow color is 
caused by carotene and xanthophyll. The yellow endosperm character 
is being bred into adapted varieties. The amounts of carotene and 
xanthophyll in the yellowest sorghum varieties obtained thus far 
are one-quarter to one-half those contained in yellow corn. 

Sorghum stalks are pithy or juicy and sweet or nonsweet. 
According to Hilson (9), pithy is a simple dominant to juicy. Accord- 
ing to Ayyangar, et al. (2), 17 to 20 per cent of juice can be extracted 
from pithy-stalked varieties and 33 to 48 per cent from juicy vari- 
eties. Pithy-stalked plants have white leaf midribs and juicy-stalked 
plants opaque midrids. Sorgos are quite juicy and grain sorghums less 
juicy or quite dry. Kafirs have juicy stems and feteritas and broomcorn 
dry stems. 

The inheritance of sweet and nonsweet stalks was studied by 
Ayyangar, et al. (2) and they found nonsweet to be a simple dom- 
inant to sweet. These authors also reported genes for sweetness 
and juiciness to be independent in inheritance. Sorgos have sweet 
stems, but most grain sorghums have little sweetness. However, 
feterita, which has dry stems, tastes sweet if the dry pith is chewed. 
Sweet sudangrass is both juicy and sweet, whereas the introduced 
variety is pithy and nonsweet. 


Sorghum is an introduced species in the United States and is 
grown on about 20 million acres. The mechanization of agricul- 
ture and changes in sorghum that began about 30 years ago have 
changed sorghum from a feed crop for use on the farm into a cash 
grain crop. 

The species has been transformed by producing early matur- 
ing: and shorter statured varieties. Alleles for resistance to several 
plant diseases and to one insect have been found. Cytoplasmic 
male-sterility has been found and methods of hybrid sorghum seed 
production devised. Several other desirable characters have been 
bred into improved varieties, including improved seed and glume 
colors, waxy endosperm, yellow endosperm, and either pithy or juicy 



I. Ayyangar. G. N. R., Ayyar, M. A. S., Rao, V. P., and Nambiar, A. K. 
1936. Inheritance of characters in sorghum — the great millet, 9, 
dimpled grains. Indian Jour. Agr. Set., 6: 938-9-15. 

1936. Mendelian segrega- 

tions for juiciness and sweetness in sorghum stalks. (Research note.) 
Madras Agr. Jour., 24: 247-24 S. 

3. Barham, H. N., Wagoner, J. A., Campbell, C. L., and Harclerode, E. 

H. 1946. The chemical composition of some sorghum grains and 
the properties of their starches. Kansas Agr. Exp. Sta. TcrJi. Bui. 

4. Bowman, D. H., Martin, J. H., Melchers, L. E., and Parker, }. H. 

1937. Inheritance of resistance to Pythium root-rot in sorghum. 
Jour. Agr. Res., 55: 105-115. 

5. Casady, A. J. 1961. Inheritance of resistance to psychotic races 1, 

2, and 3 of Spltacelotlieca sorglii in sorghum. Crop. Sci., 
1: 63-6S. 

6. Dahms, R. G. 1948. Effect of different varieties and ages of sorghum 

on the biology of the chinch bug. Jour. Agr. Res., 76: 271-2SS. 

7. Graham, R. J. D. 1916. Pollination and cross-fertilization in the juar 

plant (Andropagon sorglium Brotj. India Dept. Agr. Mem. Bot., 
Ser. 8: 201-216. 

8. Hendricks, S. B., and Borthwick, H. A. 1959. Photocontrol of plant 

development by simultaneous excitations of two interconvertible 
pigments. Proe. Nat. Aead. Sei., 45: 344-349. 

9. Hilson, G. R. 1916. A note on the inheritance of certain stem char- 

acters in sorghum. Agr. Jour. India, 11: 150-155. 

10. Holland, R. F. 1952. Tiiesis, Texas Agricultural and Mechanical 

College, College Station, Texas. 

11. Karper, R. E. 1932. A dominant mutation of frequent recurrence 

in sorghum. Amer. Nat., 66: 511-529. 

12. ■. 1933. Inheritance of waxy endosperm in sorghum, Jour. 

Hered., 24: 257-262. 

13. and Quinby, J. R. 1946. The history and evolution of 

milo in the United States. Jour. Amer. Soc. Agron., 38: 441- 

14. , -. 1947. Additional information concerning the 

introduction of milo into the United States. Jour. Amer. Soc. 
Agron., 39: 937-93S. 

15. and Stephens, J. C. 1936. Floral abnormalities in sorghum. 

Jour. Hered., 27: 1S3-194. 


16. Laubscher, F. X. 1945. A genetic study of sorghum relationships. 

Union South Afr. Dept. of Agr. and For., Sci. Bui 242. 

17. LeBeau, F. J., and Coleman, O. H. 1950. The inheritance of resist- 

ance in sorghum to leaf anthracnose. Agron. Jour., 42: 33-34. 

18. Leukel, R. W., and Martin, J. H. 1953. Four enemies of sorghum 

crops. U. S. Dept. Agr. Yearbook, 368-377. 

19. , , and Lefebvre, C. L. 1951. Sorghum diseases 

and their control. U. S. Dept. Agr. Farmers Bui. 1959. 

20. Martin, J. H. 1936. Sorghum improvement. U. S. Dept. Agr. Year- 

book, 523-560. 

21. Maunder, A. B., and Pickett, R. C. 1959. The genetic inheritance 

of cytoplasmic-genetic male-sterility in sorghum. Agron. Jour., 51: 

22. Meyer, Arthur. 1886. Ueber Starke Korner, Wekhe sich mit Jod 

roth Farben. Bericht der Deutsch. Bot. Gesell., 4: 337-363. 

23. Myers, W. M. 1947. Cytology and genetics of forage grasses. Bot. 

Rev., 13: 319-421. 

24. Painter, R. H. 1951. Insect Resistance in Crop Plants. New York: 

The MacMillan Co. 

25. Quinby, J. R., and Karper, R. E. 1945. The inheritance of three 

genes that influence time of floral initiation and maturity date 
in milo. Jour. Amer. Soc. Agron., 37: 916-936. 

26. , . 1947. The effect of short photoperiod on sor- 
ghum varieties and first-generation hybrids. Jour. Agr. Res., 75: 

27. , . 1949. The effect of milo disease on grain and 

forage yields of sorghum. Agron. Jour., 41: 118-122. 

28. , . 1954. Inheritance of height in sorghum. Agron. 

Jour., 46: 211-216. 

29. , . 1961. Inheritance of duration of growth in the 

milo group of sorghum. Crops Sci., 1: 8-10. 

30. , Kramer, N. W., Stephens, J. C, Lahr, K. A., and Karper, 

R. E. 1958. Grain sorghum production in Texas. Texas Agr. Exp. 

31. and Martin, J. H. 1954. Sorghum improvement. Advances 

in Agronomy, 6: 305-359. New York: Academic Press, Inc. 

32. Sieglinger, J. B. 1933. Inheritance of seed color in crosses of brown- 

seeded and white-seeded sorghums. Jour. Agr. Res., 47: 663-667. 

33. . 1940. A sorghum seed color chimera. Jour. Hered., 31: 


34. Snelling, R. O., Painter, R. H., Parker, J. H., and Osborn, W. M. 


1937. Resistance of sorghums to the chinch bug. U. S. Dept. Agr. 
Tech. Bui. 5S5. 

35. Snowden, J. D. 1936. The Cultivated Races of Sorghum. London: 

Adlard and Sons. 

36. Stephens, J. C. 1937. Male-sterility in sorghum: Its possible utiliza- 

tion in production of hybrid seed. Jour. Amer. Soc. Agron., 29: 

37. . 1946. A second factor for subcoat in sorghum seed. Jour. 

Amer. Soc. Agron., 38: 340-342. 

38. . 1956. A breeding method to eliminate tall mutations 

in combine grain sorghum. Texas Agr. Exp. Sta. Tech. Article 

39. and Holland, R. F. 1954. Cytoplasmic male-sterility for 

hybrid sorghum seed production. Agron. Jour., 46: 20-23. 

40. , Kuykendall, G. H., and George, W. W. 1952. Experi- 
mental production of hybrid sorghum seed with a three-way cross. 
Agron. Jour., 44: 369-373. 

41. , and Quinby, J. R. 1938. Linkage of the Q B Gs group 

in sorghum. Jour. Agr. Res., 57: 747-757. 

42. Tidlis, E. C. 1951. Fusarium monilijorme, the cause of a stalk-rot 

of sorghum in Texas. Pliytopatlwlogy , 61: 529-535. 

43. Vinall, H. N., Stephens, J. C, and Martin, J. H. 1936. Identifica- 

tion, history, and distribution of common sorghum varieties. 
U. S. Dept. Agr. Tech. Bui. 506. 

44. Wagner, F. A. 1936. Reaction of sorghums to the root, crown, and 

shoot rot of milo. Jour. Amer. Soc. Agron., 28: 643-654. 


Gabelman: You assumed the wild-type genes for maturity were prob- 
ably all recessive and that the selection in the tropics was in favor of 
the dominant genes. Under what photoperiods would this natural selec- 
tion have taken place? Do you assume that this is selection brought 
about by domestication, or was it natural selection? 

Quinby: It seems reasonable to suppose that late maturity and large 
size resulted from domestication. Photoperiodic response is influenced 
by temperature and floral initiation would be delayed even with 12-hour 
days at some temperatures. Snowden, in his book "The Races of Sor- 
ghum", presents evidence that leads him to believe that present-day 
varieties evolved from grassy types. However, my opinion in regard to 


the "wild type" being early maturing grows out of the fact that the 
alleles for late maturity act like dominant inhibitors. 

Singleton: The dwarf 3 (clw 3 ) appears like the effect of pseudo alleles 
similar to the case of Star-asteroid eye characters in Drosophila analyzed 
by E. B. Lewis who found crossing over within the locus. When two 
wild-type alleles were located on a single chromosomal strand (cis posi- 
tion), the phenotype was wild. If a similar situation exists for dwarf 3 
in sorghum, a tall plant would be expected following a rare crossover 
within the locus. If this is the proper interpretation, it might be cor- 
rected by radiating the recovered tall plants, deleting the + allele so that 
crossing over would not give rise to a tall (+) plant. 

Nuffer: I would like to suggest a method of solving the problem of 
instability of the dio 3 locus. This situation seems especially well-suited 
for the application of ionizing radiation. Since the reversion of div 3 to 
Dw 3 prevents the production of true breeding dxu^ dw 2 dw 3 dzv 4 lines, a 
likely procedure would be to induce a deficiency at the Div 3 locus. This 
could best be done by subjecting seed of a reverted dw x dw 2 Dw 3 dw± 
line to X-radiation. Plants from the treated seed would then be selfed 
and a search made in the progenies for 4-dwarf plants. Since X-ray- 
induced changes are usually irreversible, the new dw 3 should be stable. 

Quinby: Dr. W. R. Singleton has also suggested this same procedure 
and I shall send him seed of several reverted dn) x dw 2 Dw 3 dw A strains 
to be X-rayed and returned to me. 

Mehlquist: Relative to your suggestion that the mutation for disease 
resistance has occurred many times, is it not reasonable to assume that 
this gene has existed for a long time but did not show until the nonre- 
sistant plants were exposed to the disease? 

Quinby: Perhaps the genes for resistance might have been carried along 
in the populations, but resistance was not always found in a variety 
the first time it was grown in a diseased field. Also, resistance was found 
in many different varieties, or the same variety at different locations, 
and the incidence of resistant plants was quite low, sometimes as low 
as two or three plants to 5 or 10 acres. 

Use of Induced Mutants in Seed-propagated Species 


Max-1'huuk-lnstitut fiir YJlchtungsfoyscliung 
Koln-1'ogelsmtg, (lermahy 

Plant breeding is controlled evolution. Two of the major factors 
of evolution, recombination and selection, are extensively used 
by breeders and refined methods were developed during the first 
half of this century to exploit them. In the last 30 years research 
has shown that mutations, the third major factor in evolution, 
offer an additional tool, which is potentially able to modify and 
improve cultivated plants in a way similar to the conventional 
breeding methods. These results, however, do not imply that the 
efficiency of the new method is equal to or greater than that of 
the older. If the efficiency can be increased, the mutation technique 
could be used more often in conjunction with the other breed- 
ing methods. These questions will be discussed throughout this 
paper, particularly in the last part. 

The interest in the induction of mutations for plant breeding 
has increased considerably all over the world in the last 10 years. 
However, the enhanced interest is not only a consequence of pure 
objectivity, but also results from the fact that funds for mutation 
research are often easily obtained. Although theoretical work on 
the action of mutagenic agents, on the nature of mutations, etc., 
has a long history, strict and intensive investigations concerning the 
practical application in breeding has not been conducted until 
recently. This should be kept in mind when considering the objec- 
tion often made to the new method, viz., that up to date so few 
varieties which have their origin in induced mutants have been 

In the last few years a series of meetings and symposia has 
taken place dealing with general radiobiology and with physi- 
cally and chemically induced mutations from both a theoretical 
and practical point of view. Among the reviews of the last 5 to 
6 years relevant to the present paper, the following may be men- 

x Thc author is grateful to Doctors R. S. Caldecott and R. A. Nilan for critical reading 
of the manuscript and for linguistic corrections. 


caul: induced mutants in seed-propagated speciks 207 

tinned (9, 14, 25. 28, 34, 48, .59, 65, 66, 71, 74, 75, 80, 82, 83, 94, 
99, 100, 101, 104, 115, 123).- In the review by Prakken (83), there 
is an appendix with a bibliography covering 789 titles and the 
symposia, proceedings, handbooks, etc., are quoted separately. In 
this connection the bibliography of Sparrow, et al. (102) also deserves 
mention, since it covers nearly 2,600 titles, published between 1896 
and 1955, on the effects of ionizing radiations on plants. 

In these numerous reviews on mutations and plant breeding 
there is naturally much repetition. There is, therefore, no need to 
present again a detailed picture and to quote every relevant publi- 
cation. Instead, I shall try to give a critical evaluation of the pres- 
ent stage and future possibilities of breeding with mutations. Cer- 
tainly this review will sometimes be colored by my personal point 
of view. 

First, the nature of induced mutations will be discussed, includ- 
ing genetics and types of mutants. Then the methods of breeding 
with mutations will be outlined. Thereafter, the problem of 
induction and selection will be reviewed and some special hints 
will be given. Finally, an attempt will be made to evaluate the 
use of mutations in plant breeding. 

Nature of Induced Mutants 

In plants, the most comprehensive information about charac- 
ters which can be modified by induced mutations has been obtained 
in barley and snapdragon. Recently, however, the number of vital 
mutations is rapidly increasing in many other species, e.g., toma- 
toes (113, 114, 116), flax (60), soybeans (129), peanuts (41), peas 
(35, 38, 68, 69, 126), bush beans (85), potatoes (52), and millet 
(107, 108). There are also numerous reports on wheat and rice 
(cf. symposium on the effects of ionizing radiations on seeds, 
Karlsruhe, Germany, 1960, in press). Concerning barley there are 
large collections of induced mutants in Sweden, Germany (Gaters- 
leben, Halle, Koln-Vogelsang), and Belgium (Gembleaux), each 
of them consisting of several hundred forms. 

The induced variability in the species mentioned is striking. 
Practically all morphological and physiological characters can be 

2 See References, page 240. 


chano-ed by means of induced mutations within the framework 
of the species or even beyond it. 

Genetics of Mutants 

The great majority of these mutations are recessive and segre- 
gate in a 3:1 ratio; sometimes, however, with a deficiency of reces- 
sives and occasionally with an excess. In diploid organisms completely 
dominant mutations have scarcely been found, except some types 
which are lethal or semilethal in the homozygous condition (cf. 79, 
110). Therefore, whenever a dominant deviation is met with in 
mutation experiments with diploids, extreme caution is advisable. 
For instance, a great deal of the fungi-resistant variants, claimed to be 
dominant mutants, may probably be a consequence of contamination 

There are, however, a few reports of true dominant mutations. 
Notzel (78), for instance, investigated 40 different barley muta- 
tions, including types of erectoides, intermedium, macrolepis, and 
earliness. One of the earliness mutants was dominant, while the 
other 39 were recessive. Another dominant mutation of earliness 
has been reported by Scholz (93). Out of 70 erectoides mutants 
investigated by Hagberg (50), 2 were almost completely dom- 
inant for ear density and 1 was partially dominant for the same 
character. However, other characters affected by the same muta- 
tion may behave as recessive or intermediate. For example, erectoides 
mutants may be recessive for ear density, but dominant for length of 
the upper internode of the culm (48, 49). 

Along with the pronounced pleiotropic action of all (or nearly 
all) mutations, the manifestation of the relative degree of reces- 
sivity in the different characters concerned can vary considerably. 
Moreover, superdominance and superrecessivity have been repeat- 
edly observed for various characters in lethal, sublethal, and vital 
mutations (47, 49, 112). The degree of dominance may also be 
greatly influenced by the genetic background (e.g. 45, page 624; 51, 
72) and/or the environment. The dominant tomato mutant sub- 
sistens, for instance, which was studied by Fndlich (21), is lethal in 
the homozygous condition. In the heterozygot the mutant allel 
showed a varying degree of expressivity in different environments. 
Under low light intensity or high temperature, the mutated char- 
acter appeared to be recessive or nearly recessive. 

gaul: induced mutants in seed-propagated species 209 

In contrast to diploids, dominance or semidominance seems to 
be a fairly common phenomenon for many characters of vital muta- 
tions in polyploids like wheat (70). Here, the phenotypic change is 
probably more often caused by chromosome mutations instead of 
point mutations and polyploids are more tolerant to chromosomal 
aberrations as compared with diploids. 

There is no essential difference between the alleles available 
from the world collections of natural forms and those from induced 
mutations. This has been adequately demonstrated for some char- 
acters in barley. Thus, some erectoides mutants (50, 78), macrolepis 
mutants (78), naked-kernel mutants (92), smooth-awned mutants, and 
a mutant with waxy stalks and leaves (Scholz as cited by Stubbe, 
117), have been shown in crosses to be allelic with corresponding 
forms of the world collections. In tomatoes several loci of induced 
mutants of Lycopersicoii pirn pinelh folium have been proved identi- 
cal with those of the corresponding esculentum mutants (117). 

Identity of the locus does not mean that the alleles are iden- 
tical. The occurrence of multiple alleles or of pseudoalleles can 
usually be studied only with mutants which do not differ other- 
wise in their gene content. From a careful morphological study 
of 70 erectoides mutants in barley, including the various pleiotropic 
effects, Hagberg (50) arrived at the conclusion that it seems to be 
impossible to copy an individual mutation. These 70 mutants were 
scattered over 22 separate loci. Concerning the phenotype belong- 
ing to an individual erectoides allele there are important differ- 
ences between the loci, but there is also a remarkable variation 
within a locus. This finding is in general accord with the evidence 
accumulated from microorganisms and Drosophila in the last 10 to 
15 years which has shown that genes have a complex nature. So 
far as plant breeding is concerned, these results imply that the 
induction of mutations does not simply reproduce the natural 
variability, but may expand it to a large extent. Through muta- 
tions of a given character, not only may loci which are not yet 
known be discovered, but also new alleles within the loci may be 

Phenotypic Classification of Mutants 

The alteration of characters induced by mutations may be 
large or small. There are transitions all the way from macro-muta- 


dons to niicro-niiitations. In Table 1 vital mutations are classified 
in lour types according to degree of change of the phenotype. 

Table 1. — Phenotypic Classification of Vital-mutations. 

1. Macro-mutations Large mutations, drastic mutations (Grossmutationen) 

Detectable in a single plant 

a. Transspecific Systemic mutations, organization mutations 

b. Intraspecific 

2. Micro-mutations Small mutations (Kleinmutationen) 

Detectable in a group of plants 

a. Manifest 

b. Cryptic Detectable if the environment and/or genetic back- 

ground is changed 

Those deviations, which are easy to recognize in a single plant, 
may be called macro-, or large, mutations. Macro-mutations usually 
affect characters which are already known in the species, but some- 
times the induced character is unknown in the species, in the genus, 
or even in the family. (Particularly in cultivated plants the previous 
separation into different species is sometimes not justified, e.g., 
in wheat (61).) 

In contrast to these are micro-, or small, mutations (Klein- 
mutationen) which cannot be detected with certainty in a single 
plant but only in a group of at least, say, 30 or more individuals. 
These micro-mutations may be classified into manifest and cryptic 
micro-mutations, depending upon their degree of detection. Mani- 
fest micro-mutations are detectable if, in the unchanged environ- 
ment and genetic background, appropriate methods of screening 
and observations are applied. Cryptic micro-mutations cannot be 
recognized even in a large group of plants under "normal" grow- 
ing conditions. A drastic change of the environment and/or the 
genetic background may enable them to become manifest, where- 
as under normal conditions they behave in a neutral manner. The 
existence of cryptic mutations is more or less hypothetical and there 
is scarcely any experimental evidence for them until now (see, how- 
ever, 72). Nevertheless, they may have significance in evolution and 
they could be of value for certain breeding purposes. The existence 
of the other types of mutations has been shown repeatedly. Further 
use of the term micro-mutation in this paper refers to manifest ones 

gaul: induced mutants in seed-propagated species 211 

Naturally, the grouping as suggested in Table 1, is arbitrary 
and has no meaning; relative to the nature of changes of the genetic 
material. Whether in individual cases the phenotypic change is 
considered to be small or large will often depend on the methods 
and personal view of the observer. Though it is not possible to 
construct sharply distinguished categories of mutations, the classi- 
fications suggested may be of value for the communication of evolu- 
tionary and plant breeding problems. 

Transspecific Macro-mutations 

The occurrence of transspecific mutations is Aery rare. Never- 
theless, a number of more or less clear cases of both induced and 
spontaneous mutations have been reported (see reviews in 44, 45, 79, 
111, 117, 119). Recently, in barley, another induced macro-mutation 
(57) has been described in more detail (91). The culms of that mutant 
have no differentiated nodes. All the nodes are located in close 
succession immediately above the root, and the culm is formed only 
by one internode. Such a character is common in the Cyperaceae. 
Another interesting example of a spontaneous macro-mutation was 
recently described by Staudt (105) in Fragaria. 

Macro-mutations that are transspecific usually have a stronger 
reduced vitality and/or fertility. They are of no immediate value 
for plant breeding. However, if a definite character has to be trans- 
ferred from a foreign species, it might be easier to use transspe- 
cific mutants instead of interspecific hybridization. In agricultur- 
al plants transspecific mutations which could serve for practical 
breeding are scarcely known. Yet, it might be possible that they 
will be found in the future, owing to the increased production of 
mutations. Such mutations might also be of considerable interest in 

Sometimes the manifestation of the mutant characters is not 
constant but variable. This holds true, e.g. for some mutants of 
Antirrhinum majus which were investigated by Stubbe, (111, 
117). Thus, the mutant transcendens has a tendency to reduce the 
number of stamens per flower from four to two. Flowers with two 
stamens are characteristic in adjacent genera of the Scrophul- 
ariaceae to which Antirrhinum belongs. However, in the mutant, 
only 40 per cent of the flowers possess two stamens and the others 
form three and four. Selection of stable types with two stamens 



proved impossible. When crossing this mutant with wild species 
of the genus Antirrhinum, Stubbe (111, 117) had success in the 
selection of forms which possess two stamens in every flower. From 
Figure 1 it can be seen that already in the F 5 generation, 98 per 




A majus gelblich 

ssp. majus van slri alum 

George de ffalamus (Corbierel 

A. majus S.50 mul. Irons cendens 2 

1949 1350 1351 1952 1953 1954 

1 2 '3 In 1 5 1 6 17 



Figure 1. — Curve of selection of F 2 to F g for two stamens, after crosses 
of the mutant trancendens 2 of Antirrhinum majus to A. majus gelblich 
ssp. majus var. s trie turn. From Stubbe (117). 

cent of the flowers had two stamens. The introduction of numerous 
other genes evidently increased the penetration of the mutated 
character and led to its stabilization in the new "genetic environ- 
ment". With other mutants forming variable numbers of stamens 
up to eight, similar success in stabilizing was possible (111, 117). 
These examples have been broached here because they offer 
an interesting model for plant breeding. Macro-mutations may not 
only be stabilized in a new genetic background, but it also seems to 
be possible to modify their action and particularly to eliminate 
undesired pleiotropic effects, as discussed later in this paper. 

caul: induced mutants in seed-propagated species 213 

Intraspecific Macro-mutations 

Intraspecific macro-mutations are the most common type of 
all mutations that have been selected. Numerous characters of 
value for plant breeding have been found, but usually these mutants 
have a reduced vitality. Reduced general performance may, how- 
ever, not be unexpected on a priori reasoning. A gross change in 
the genetic system, though it may mean the induction of a valu- 
able character, will usually result in a disturbance of the delicate 
gene balance, which had been built up by the breeder with great 
efforts over a long time. Perhaps the frequency of productive large 
mutants might be higher in those plants in which greater breed- 
ing work has not yet been done (Cf. 25). However, a small portion of 
these large mutations represent a valuable source for further 
recombination work as discussed later. 

Micro-mu tations 

Small mutations generally deserve more attention from plant 
breeders than macro-mutations. Like the large mutations, the small 
ones may affect all morphological and physiological characters. The 
sionificance of small mutations in evolution was first recognized 
and emphasized by Baur (3). Since then it has been repeatedly 
discussed (119). From the fact that most character differences in 
race and species hybrids show complex segregation, the majority 
of research workers (see, e.g., 106) agree today with East (18, page 
450) that "the deviations forming the fundamental material of 
evolution are the small variations of Darwin". Though small muta- 
tions were early described in Antirrhinum (109) and plenty are known 
in Drosophila (63, 120), it is surprising that relatively little atten- 
tion has been paid to them. Also, in the extensive work with barley, 
small deviations of various characters, such as kernel size, leaf size, 
straw height, protein content, etc., have been known for a long time 
(43, 57, 73, 79, 93, 95, 96). Yet, the suggestion to put the emphasis in 
breeding programs on small mutations was not made until recently 
by Nybom (79) and Gregory (39, 40), and meets perhaps with 
even stronger emphasis by myself (25, 28). 

Most plant attributes of interest to the breeder are quantitative 
characters which are controlled by many genes. These are called 
polymeric genes, multiple genes (or factors), or polygenes. When 


compared with genes controlling so-called qualitative characters, 
there are no convincing reasons to assume that polymeric genes are a 
different sort of genes so far as their nature is concerned. A mutation 
step in one of these multiple genes may affect a quantitative char- 
acter in a measurable way. For instance, increasing yielding poten- 
tial of, say, 5 per cent in a top variety of barley, wheat, or oats is 
usually considered to be a desired goal of breeding. It seems possi- 
ble that such progress can be obtained by mutation of only one 
or a few polymeric genes without a pronounced morphological 
effect. However, the experimental evidence for such a speculation 
is as yet meagre, except for the extensive evidence with peanuts 
from the careful investigations of Gregory (39, 40, 41, 42). This 
author found a striking increase in the genetic variance for yield 
in progenies of normal-appearing M 2 plants which were selected 
at random. He also succeeded in the selection of mutants with higher 
yielding capacity. 

In addition, Cooper and Gregory (15) presented evidence 
of small mutations exerting a quantitative effect on leaf-spot resist- 
ance. Starting with progenies of normal-appearing M 4 plants in 
rice, Oka, et al. (81) found a considerable increase in the genetic 
variance for plant height and heading date. For both these char- 
acters, the authors succeeded in selecting plus and minus mutants. 
In barley, a study of induced variability for quantitative char- 
acters has recently been presented by Moes (73). He describes posi- 
tive and negative alterations of a great number of characters, such 
as number of tillers per plant, number of seeds per spike, kernel 
size, lodging-resistance, leaf size, straw height, etc. Also at our 
laboratory in Koln-Vogelsang, following a program to develop 
selection methods for small mutations in barley, we succeeded in 
obtaining a greater number of small variants in spring barley and 
winter bailey, and some of the results will be presented later in 
this paper. 

Despite these studies, the field of small mutations is largely 
unexplored as compared with that of large mutations. More infor- 
mation is needed about their features and their frequency for an 
evaluation of their significance in plant breeding. A priori it may 
be expected, that the more genes that are involved in a character, 
the higher the probability of obtaining an alteration by a mutation 

gaul: induced mutants in seed-propagated species 215 

of one of the multiple genes concerned. Indeed the results obtained 
in Drosophila (63, 97, 120) and peanuts (39, 41, 42) suggest that 
the frequency of small mutations is considerably higher than that of 
large mutations. 

Methods of Breeding with Mutations 

Mutants can be utilized in various ways in plant breeding 
and the most important methods are reviewed in Table 2. The term 
"mutation breeding" is often loosely used. In the classification of 
Table 2, it is suggested that the term mutation breeding be used 
only when a new variety results from the direct propagation of 
the mutant. Extensive use of mutants can be made in the various 
methods of cross-breeding. Besides, there are a number of special 
aims for which mutagens may prove valuable, and these are outlined 
in Table 2. 

Table 2. — Methods of Breeding with Mutations and Use of Mutagenic Agent 
in Seed-propagated Species. 

A. Self-pollinating species mainly 

1. Immediate use of mutants: Mutation breeding proper 

2. Use of mutants in cross-breeding 

a. Within the same variety 

b. With foreign varieties 

c. In hybrid populations, as an additional source of variability 

d. In heterosis-breeding (including cross-pollinating species) 

B. Cross-pollinating species 

3. Induction of mutations in cross-pollinating populations 

C. Self- and cross-pollinating species 

4. Mutagenic agents as a tool for special purposes 

a. Induction of translocations for interspecific or intergeneric transfer of desired char- 

b. Diploidization of artificially produced polyploids 

c. Use of translocations (with localized breakage points) for "directed" duplications 

d. Use of radiation to induce transitory sexuality in apomicts 

e. Use of radiation to produce haploids 

f. Use of radiation to break incompatibility in distant crosses 

Mutation Breeding 

The direct use of mutants offers theoretically the greatest 
advantage in utilizing mutations in plant breeding. As compared 
with cross-breeding, it may save nearly half of the time necessary 
to create a new variety. Evidently here lies the field in which the 


significance of small mutations should be tested. The experience 
with large mutations has shown that, besides their relative rarity, 
only a very small fraction exerts a positive influence on yield. 

The chance of producing more or less large mutations with 
higher yield is, however, a matter of fact which cannot be over- 
looked. In Table 3 some examples are given in barley. There is no 

Table 3. — Some Examples of Barley Mutants with Well-established 
Higher Yielding Capacity. * 

Character and Serial No. years Relative Author 

Early, M s 90 4 108 HOFFMANN (57) 

Large-kernelled, 44/7 10 107 FROIER (24) 

Early, less straw-stiff, 44/4 9 109 FROIER (24) 

Large-kernelled, early, 3978 6 115 SCHOLZ, pers. com., also (93, 96) 

Erectoides, 2660 5 105 SCHOLZ, pers. com., also (93, 96) 

Erecloides, 2654 8 103 SCHOLZ, pers. com., also (93, 96) 

Smooth-awned, 4033 7 107 SCHOLZ, pers. com., also (93, 96) 

Semi-smooth-awned, 3945 6 113 SCHOLZ, pers. com., also (93, 96) 

Early, W 3 4 107 GAUL 

Winter Barley 
Early, 506 5 114 SCHOLZ pers. com., also (93) 

Early, 481 8 107 SCHOLZ pers. com., also (93) 

'Kernel yield of the mother line is taken as 100. 

doubt that the higher yield is well established because these figures 
are based on drill tests with several replicates over a period of at 
least 4 years. It should be noticed, however, that in these examples, 
the higher yielding power is proved only for one location. 

The objection often raised as to why such mutants have not 
been released as varieties is not valid. Under the conditions in 
which they were produced, the total expenditure in cross-breeding 
as compared with "mutation-breeding", may have been of the order 
of 50:1, or even 500:1. It is not surprising that by the time the supe- 
riority of these mutants was established, through conventional breed- 
ing methods, new lines were developed which were superior even 
to these "yield-mutants". Moreover, a great deal of those progres- 
sive mutants reported in the past were just derived as a by-product 

Number of 





Spring Barley 

















gaul: induced mutants in seed-propagated species 217 

of theoretical mutation experiments. This also holds true for our 
mutant of Haisa II (Table 3), which was selected from a theoreti- 
cal experiment described elsewhere (26). Haisa II was marketed 
in 1950 and used to be one of the most extensively grown varie- 
ties in Western Germany. However, in recent years, it has con- 
tinuously lost acreage. The mutants investigated by Scholz (93 and 
personal communication) belong to "fairly old" varieties, and most 
of those recorded in Table 3 have now reached the same level of 
yield as the present top varieties in that area of Germany. 

Scholz (96) also gave an example showing that the yield of 
mutants can be raised in a second radiation cycle. A barley mutant 
with naked kernels (2) proved to possess a yielding capacity of 
90 to 91 per cent as compared with the mother variety Haisa, which 
has been established in yield trials over a period of 7 years. Because 
the glumes contribute about 10 per cent of the total kernel weight, 
the mutant reached the level of the mother variety in terms of "net 
yield". Irradiation of this mutant residted in a new mutant which 
yielded 98 per cent of Haisa on an average of 4 years of testing and 
which, in addition, is 7 to 8 days earlier. Thus, the net yield of the 
new mutant has been raised beyond that of Haisa. 

A few years ago the first varieties derived by propagation of 
a mutant were marketed. In Middle Germany the winter barley 
Jutta was released, which is derived from an induced mutant in the 
variety Kleinwanzlebener Mittelfriihe. In this mutant winterhardi- 
ness, strawstiffness, and yield are improved. At present this mutant 
covers about 10 per cent of the total acreage of winter barley (125 
and personal communication). In Sweden a barley erectoides 
mutant, named Pallas, and a pea mutant, called Weibulls original 
Stralart, are grown (8). Earlier a new variety of oil rape and another 
of white mustard were marketed, which were selected from X-rayed 
material. However, both these species are cross-pollinating and there 
is no clear evidence that the new varieties originated by mutations. 

Use of Mutants in Cross-breeding 

Information about the use of mutants in cross-breeding is still 

more limited than that of mutation breeding proper. If characters 

of two mutants belonging to the same variety are to be combined, 

a small number of F 2 plants is sufficient. The breeding procedure 


is very simple, because the two mutants differ (practically) only by 
two genes. This is in contrast to the great expenditure necessary 
in combination breeding on the basis of variety crosses. Occasion- 
ally, two or even more genes may be changed in one mutant at the 
same time, but this does not complicate seriously the breeding pro- 
cedure. Usually, however, crossing of two mutants results in normal 
bifactoriel segregation (78, 93). 

In extensive crosses among different erectoides mutants of 
barley, Hagberg- (50) studied the additive effect of the combined 
loci as expressed by the internode length in the ear. He succeeded 
in the addition of up to four homozygous mutations in one plant. 
By this, the average internode length was reduced from 33 mm of 
the mother line (no mutation) to 24 mm in those containing a single 
mutation, to 16 mm for those containing two mutations, to 13 mm 
where three o-enes were involved, and to 12 mm in the line con- 
taining all four mutated genes. This result indicates that the addi- 
tion of each further gene had a smaller effect than the previous 
one. Hoffmann (discussion of 50) reported that in another polyfac- 
torial character, namely, earliness, the combination of two mutations 
sometimes results in a multiplication effect. Thus, crossing a 3-day 
earlier with a 6-day earlier mutant resulted in a double recessive 
being about 18 days earlier. Crosses between various intermedium 
mutants of barley (Hoffmann, loc. cit.) led sometimes to double reces- 
sives which were dwarfs. These examples indicate that certainly not 
all mutations can be combined without reducing the vitality. On the 
other hand, crossing of small mutations inter se has led in peanuts to 
heterosis (41, 42) and the use of micro-mutations should also be 
explored in combination breeding. 

Also crosses of mutants with foreign varieties offer the chance to 
obtain rapid and simple results. Their advantage is particularly 
to be expected when a "rare" character, which is known only in 
primitive or non-adapted forms, is to be transferred. If that char- 
acter is available from a mutant collection of an adapted variety, it 
may be much easier to use the mutant instead of the non-adapted 
variety for a cross-breeding program. Characters from mutant collec- 
tions which may be useful are for instance: resistance against fungi, 
variability in relation to different soil and climatic conditions, 
strawstiffness, high protein content of seeds (95, 95), earliness, and 

gaul: induced mutants in seed-propagated species 219 

numerous other properties. In barley, for example, the smooth- 
awned and naked-kernel mutants of Bandlow (2) are certainly a 
valuable source for an easy production of top varieties (96). 

An example along this line has already been given by Down 
and Anderson (17) in bush beans. These authors crossed an earli- 
ness mutant, induced by X-rays from the variety Michelite, with 
other strains resistant to Colletotrichum lindemuthianum. The 
new variety Sanilac, derived from these crosses, combines earliness 
with resistance and is higher yielding than Michelite. 

A similar case which theoretically deserves still more interest 
is reported by Stubbe (118) in Antirrhinum. In breeding this orna- 
mental plant, an erect growth has been desired for a long time. 
The mutant eramosa approaches that aim and forms usually one 
culm only. It has almost completely lost the ability of branching 
and, in addition, it possesses a number of other pathological charac- 
ters, like inhibition and deformation of the flowers. By crossing 
eramosa with other varieties, Vogel (according to Stubbe, 118) suc- 
ceeded in breeding a new nice-looking and vigorous snapdragon 
which forms only one culm and has normal flowers. Evidently, 
the undesired pleiotropic by-effects of the mutation have been 
"dissolved" in a new genetic background. 

This model demonstrates how macro-mutations may be of 
use in cross-breeding agricultural plants. Little has been done in 
this field. There are some reports from practical breeders; and 
though the evidence is not conclusive, two examples may be men- 
tioned. In barley breeding, the erectoides mutants deserve great 
interest because of their strawstiffness. However, the dense spike 
of the mutants is often considered a disadvantage because generally 
the seed quality is lowered. Thus, the German breeder v. Rosenstiel 
(84 and personal communication) crossed one of the Swedish erec- 
toides mutants (ert 12) with other strains of his material carry- 
ing the mildew-resistance of Hordeum spontaneum, H 204 (86). 
He selected lines with extremely stiff straw and, in some of them, 
the dense spike had disappeared. Because these high-yielding lines 
with a "normal" spike carry also the mildew-resistance of H. spon- 
taneum, the procedure of v. Rosenstiel is an example of how a char- 
acter from a wild species and a mutant may be combined. Two of 
these lines are now in the stage of official yield trials of the Federal 


Variety-Board in Germany. From the experience he has in his cross- 
material, v. Rosenstiel is fairly sure that the unusual s traws tiff n ess 
obtained was derived from the erectoides parent. 

Another example that may be given is the mildew-resistant 
mutant of barley found by Freisleben and Lein (22). This mutant 
is resistant against all race groups isolated in Germany. However, 
the older plants have strongly chlorotic spots and, in turn, a reduced 
yield. Using this mutant as cross parent, Vettel (personal communi- 
cation) was able to "separate" the resistance from the leaf spots. He 
selected high-yielding lines carrying the mutant resistance. 

There are further possibilities for a combination of cross- 
breeding with mutations. Thus, induction of mutations in F 2 seeds 
of a hybrid population may be useful because it means an additional 
increase of variability. Up to date there is little experimental evi- 
dence of such an approach. Likewise, the possibilities of using mutants 
for heterosis breeding are almost completely unexplored. In the 
latter case there is, however, the interesting instance of the com- 
mercial use of a tobacco mutant (121, 122) which has been over- 
looked in the older literature concerning mutations and plant breed- 
ing. This (lightgreen) chlorina mutant of Vorstenland tobacco was 
produced in Java. The leaves had an attractive color and quality. 
Since the homozygous chlorina mutant produced too few leaves, its 
Fi hybrid, with the ancestral type, was used, and had to be produced 
again every year. In the second half of the 1930's these hybrid plants 
were grown extensively in the Netherlands East India, but the 
mutant was lost during the war as far as is known. This seems to be 
the first case of an induced mutant that has ever been used in prac- 
tice (8, 83). 

Induction of Mutations in Cross-pollinating Populations 

There is no experiment known to me which proves crucially 
the use of mutations in cross-pollinating species. However, the new 
varieties of oil rape and white mustard in Sweden already mentioned 
were selected from X-rayed material. There are also a number of 
other reports where variants with valuable characters have been 
selected after application of mutagens, either with or without self- 
ing of the Mi generation. This is, for example, the case in Tri folium 
pratense (10, 89), in Plialaris aritndinacea (53), in Alope curus. pra- 
tensis (128), and in Meli lotus alb us (90). It may be expected that 

gaul: induced mutants in seed-propagated species 


mutagenic treatment of cross-pollinating populations will result 
in additional and possibly new genetic variability. 

A conclusive experiment of recurrent irradiation with success- 
ful selection for small mutations was reported in Drosopliila inel- 
anogaster (11, 97). This may serve here as a model for plant popu- 
lations. The subject of selection was a quantitative character, the 
sternopleural bristle number of the flies. In this experiment, two 
strains were treated with X-rays at every second generation. Two 
further strains, serving as controls, were nonirradiated but sub- 
jected to the same selection procedure as the irradiated lines. As 
illustrated in Figure 2, the two irradiated strains (Ap and Bp) 
responded strikingly to the selection. The hair number could be 

i s 


Figure 2. — Progress of artificial selection for high number of sterno- 
pleural liairs of Drosopliila melanogaster in two X-ray-treated lines 
(Ap and Bp) and in two non treated lines (Cp and Dp). From Buzzati- 
Traverso and Scossiroli (11)- 


raised from 26 to 41 after 10 selection cycles. In contrast, the con- 
trol strains (Cp and Dp) did not undergo any appreciable change. 

Mutagenic Agents as a Tool for Special Purposes 

Physical and chemical mutagens may be exploited in plant 
breeding in various other ways than those discussed above. A num- 
ber of other possibilities are outlined in Table 2 and will be brief- 
ly discussed here. The use of translocations in transferring a valu- 
able character from one species or genus to another has recently 
been reviewed (28), including the typical and fully analysed 
example of Sears (98). In the same review (28), the problem of 
diploidization of artificially produced tetraploids is outlined. 

Use of duplications to produce "directed mutations" has been 
suggested by Hagberg (50, see there also older references). For this 
project, it is necessary to have a very large set of translocations in the - 
genotype in which the breeding work shall be done and to have 
these translocations localized. If a greater gene content of the chro- 
mosomes is also mapped, then it should be fairly easy to produce 
definitely localized duplications (and deletions) of short segments 
carrying desired genes through crosses of adequate translocation 

Irradiation may also be useful in apomictic species to produce 
transitory sexuality for one or a few generations, as has been shown 
by Julen (62) for Poa pratensis. This enables the breeder to make 
crosses and to select in later generations new apomictic forms with 
improved characters. 

That irradiation can be exploited for the production of hap- 
loids has been known for a long time and is outside the scope of 
the present paper. Attempts have also been made to overcome the 
interspecific incompatibility by exposing male or female gametes 
or tissues to gamma radiation prior to crossing. In one instance, 
that of the cross Brassica olerocea X B. nigra, the technique appeared 
to be successful and many interspecific hybrids were produced 

Methods of Induction and Selection of Mutants 

The significance of mutations in plant breeding will largely 
depend on the progress of their production methods. It is impor- 
tant to obtain a greater yield of total mutations and particularly of 

gaul: induced mutants in seed-propagated species 223 

progressive mutants. The final output of mutants for plant breed- 
ing depends on both the methods of original induction and of selec- 
tion. According to the arrangement of Table 4, I have recently 

Table 4. — Possibilities of Obtaining a Higher Yield of Mutants for use in 

Plant Breeding. 

A. Control of induction 

1 . Through raising of mutation rate per surviving Mi plants 

2. Through alteration of the proportion of chromosome mutations vs. factor mutations 

3. Through alteration of the mutation spectrum 

B. Control of selection 

1 . Through knowledge of chimera formation and diplontic selection in Mi plants 

2. Through improved screening methods of mutants 

reviewed the progress in obtaining more efficient production meth- 
ods of mutants (28). The arrangement of Table 4 will also be the 
basis of the following considerations. 

Induction of Mutations 

There is no need to review the subject of a control of mutation 
induction a°;ain in detail, though in the meantime some remarkable 
results have been obtained (13, 46, 67, 75, 76, 77). Such a limited 
control appears to be possible through the different actions of the 
great number of known physical and chemical mutagens that can 
be used either alone or in combination with various secondary 
factors. Also the stage of the plant development and the parts of the 
plant being treated are important. 

Mutation research is in a stage of rapid development, and the 
theoretical progress is fascinating in the fields outlined under Al 
to A3 of Table 4. Yet, its practical application relative to "recipes" 
for useful techniques in plant breeding is still limited. With regard 
to the theoretical progress, I am, however, inclined to suppose that 
methods to obtain a greater total mutation frequency (Table 4, Al) 
will be available for everybody in the near future. I also would like 
to speculate that techniques for a certain control of the relative fre- 
quency of chromosome mutations vs. factor mutations (Table 4, 
A2) will be developed in, say, the next 5 to 15 years. Alteration of 
the mutation spectrum (Table 4, A3) is most apart from being 
used in practical plant breeding. Up to date, conclusive evidence in 
this field has only been obtained with chlorophyll mutations (cf. 28). 


Exciting progress has been made with chemical mutagens. At 
present, two of the most powerful substances for higher plants are 
ethylene inline (20) and especially ethyl methane sulfonate or 
EMS (54, 55, 56). The high mutagenic efficiency of EMS is demon- 
strated in Table 5 for chlorophyll mutations in barley. After X-irra- 
diation of barley seeds the highest mutation rates obtained are of 

Table 5. — Some Results of the Mutagenic Efficiency of EMS (CH 3 S0 2 OC2H 5 ) in 
Barley Seeds Obtained by Heslot. * 

Treatment Number of Number of Mutants, 

Concentration duration Temperature spikes M 2 plants %, 


24 hours 

24° C 





24 hours 

24° C 





3 days 

3° C 




*Personal communication. LD-50 is approximately for 24 hours, 24° C:V3oo, and for 3 davs, 
3° C:Vioo. 

the order of 3 to 4 per cent mutants per 100 M 2 plants (12, 33), 
while in Table 5 with EMS 14 per cent are recorded. According 
to Heslot (Table 5), around 12 per cent were obtained with an 
EMS treatment that led to approximately 50 per cent Mi surviv- 
als. The values mentioned for X-ray treatment were only reached 
with doses exerting a greater Mi lethality, except in one experiment 
(33, treatment 30,000 rp). Consequently, the mutagenic efficiency 
of EMS appears to be somewhere between 3 to 8 times higher than 
that of X-rays, if these results are reproducible. Similar high muta- 
tion rates Avith EMS have now also been obtained by Ehrenberg 
(19). Apparently EMS has a relatively low toxic effect and a high 
genetic effect as compared with X-rays. Moreover, Heslot (56) found 
many more morphological and physiological mutations per chloro- 
phyll mutation after treatment with EMS than after X-raying. 
EMS treatment results in much lower chromosome breakage (Hes- 
lot, personal communication, also 67), however, it causes high Mi 
sterility. This discrepancy needs to be further investigated. The 
mutagenic activity of EMS has already been proved in other spe- 
cies (56, 87). Apparently, EMS is the most efficient mutagen for plant 
breeding known at present and it deserves greatest interest in further 
theoretical and practical applications. 

Most of the applied mutation work has been done with radia- 

gaul: induced mutants in seed-tropagated species 225 

tion of seeds. Certainly seed treatment offers a great number of 
advantages (77), but this does not necessarily mean that it is the 
best method for the induction of mutations in plant breeding. In 
Table 6 some other stages of plant development are shown which 

Table 6. — Plant Stages for Mutagenic Treatment. 

1. Seeds, dormant, presoaked. germinating 

2. Pollen 

3. Flowers, prior to, during, and after meiosis 

4. Zygotes, immediately after fertilization 

5. Buds in various stages 

6. Any other stage 

can be used. Particularly, flowers which have passed meiosis and 
also zygotes deserve attention. In the last case, the whole organism 
is represented by one cell only; consequently, no chimera forma- 
tion will take place and high mutation rates might be expected 
because there is no intercellular competition. After treatment of 
seeds or buds diplontic selection results in a great loss of muta- 
tions (34). With gamma-radiation sources, like cobalt 60 and caesium 
137, acute irradiation of various plant-stages can easily be done, and 
there are also useful methods for the application of chemical 
mutagens (88). 

The experience with chronic irradiation has shown that this 
treatment apparently offers no advantage as compared with acute 
irradiation. The "maximal" mutation rate seems to be rather low- 
er with chronic irradiation which may be easily explained by the 
phenomenon of diplontic selection (27, 28, 30, 33). However, these 
results are based mainly on chlorophyll mutations and it is unknown 
if the same holds true for vital mutations. Theoretically, diplontic 
selection could eventually be utilized as a screening procedure 
for progressive mutations, but experimental evidence is lacking for 
this speculation. 

In relation to radiation of seeds, we have recently presented a 
number of detailed suggestions on how a breeder may best proceed 
(34). These suggestions were based mainly on our experience with 
barley, and they include the description of an early test to find the 
most efficient dose on the base of the seedling length (31). Further- 
more, suggestions are given as to the most suitable sowing technique, 


the procedure in recurrent irradiation programs, and various other 
topics. Previously it was suggested to use a dose which leads to about 
50 per cent surviving Mj plants (23, 43). In contrast to these earlier 
findings we have recommended applying higher doses which result 
in 10 to 20 per cent (or even less) survival. At 90 per cent lethality of 
Mi plants the mutation rate may be nearly doubled as compared 
with 50 per cent lethality. The discrepancy with the older results 
is mainly a consequence of the fact that the mutation frequency 
was not measured correctly (27, 32). 

There are large differences in the radiosensitivity between 
various species, necessitating quite different doses. In an extensive 
study, Sparrow, et al. (103) considered nuclear volume (of mer- 
istematic cells), polyploidy, and chromosome number as the major 
factors determining radiosensitivity. A table of doses leading to 
approximately 50 per cent survival in various agricultural plants 
can be found in Gustafsson and v. Wettstein (48). 

Diplontic Selection 

Mutagenic treatment of seeds or other parts of the plant results 
in the formation of chimeras. The efficiency of mutant selection 
depends, therefore, on the problem as to which parts of the plant the 
progenies should be grown and whether the elimination of mutations 
within the plant (diplontic selection) can be inhibited. Unfortunate- 
ly, little is known in this field. After treatment of barley seeds, we Avere 
able to show that about the first five tillers per plant possess a 
considerably higher mutation frequency than tillers formed later 
(27, 30, 33). It has therefore been recommended to space the M x seeds 
extremely close in order to obtain high mutation rates (34). We also 
have demonstrated that the size of chimeras varies with the dose. With 
very low X-ray dose the average size of a mutated sector may comprise 
about one quarter of the generative tissue of a single spike. With 
increasing dose this size increases correspondingly and finally all the 
florets of a spike contain the same mutation. With greater tillering, 
the mutated sector may then even include several spikes of a plant 
(30, 33). The decrease of mutation frequencies in later formed tillers 
has recently also been demonstrated in rice by Bekendam (4, 5). In 
addition, the same author was able to show that in rice, as in barley, 
after irradiation of seeds the generative tissue of a single head may 
derive from one to four embryo cells. The chimeric structure has also 

gaul: induced mutants in seed-propagated species 227 

been investigated in several other plants, e.g. in maize (1), sorghum 
(64), pea (6), and blue lupin (36). 

In practical breeding work, selection for mutations may perhaps 
alreardy be started with seedlings of the Mi generation. "We have 
shown that raising X-rayed barley seeds in the greenhouse before 
transplanting them to the field may result in 4 to 5 times better 
survival than when seeds are sown directly in the field (33). The 
mutation frequency of the "greenhouse seedlings" was not essentially 
lower than that of the "field seedlings". This finding complements 
interesting results of Caldecott (13), who demonstrated that heavily 
damaged seedlings (shortest height class of the length of the first leaf) 
have a 2 to 4 times higher mutation rate than those which belong 
to the tallest height class. Thus, with the technique of raising seed- 
lings in the greenhouse, it might be expected (a) that higher doses 
can be applied, resulting in higher mutation rates, and (b) that the 
selection of seedlings with short leaves (but which are still viable) 
will result in an additional increase in mutation rates. 

Similarly, Blixt, et al. (7) found recently in peas, after treatment 
with ethylen inline, a correlation between the M 2 mutation rate and 
the frequency of leaf-spots on the first leaves of Mi plants. In order to 
obtain a high rate of M 2 mutations, they suggest, therefore, taking 
offsprings from those plants only which show the highest frequency of 
leaf-spots at the Mi seedling stage. 

Selection of Mutants — General Results 

In practical breeding work it will often be advisable to com- 
mence with the selection of mutants only after mutagenic treatments 
have been applied for several years or generations to the same mate- 
rial (23, 34). Recurrent induction of mutations leads to an accumula- 
tion of mutants in the treated population and increases the efficiency 
in breeding with mutations. 

Selection for fertility can already start in the Mi generation or 
in case of recurrent induction of mutations, it may be repeated in 
each succeeding generation. After treatment of barley seeds, we have 
shown repeatedly that the mutation frequency of fertile Mi spikes is 
no smaller than that of the Mi spikes with partial sterility if the tiller- 
ing of the Mi plants is reduced (25, 28, 29, 34). Recent results in rice 
(4, 5) agree basically with barley. Selection of fertile (or nearly fertile) 


Mi spikes results therefore in elimination of chromosome mutations 
without reducing the frequency of factor mutations. We have also 
shown in barley that the M 2 sterility in progenies of partially sterile 
Mi spikes is actually several times greater than in the progenies of 
fertile Mi spikes. However, in one experiment, even a twofold selec- 
tion for fertility, namely, in the Mi and M 2 generation, still resulted 
in the M 3 generation in 3 per cent M 2 spike progenies with partial 
sterility (34). Usually the breeder is interested in point mutations 
only and not in chromosomal aberrations. This is valid at least for 
diploid plants. Selection of fertile M x spikes means, therefore, an 
increase of efficiency in breeding with mutations. 

Selection of mutants has commonly been done in the M 2 genera- 
tion. In that generation only macro-mutations can be recognized. 
Moreover, owing to the chimeric structure of the Mi plants and their 
sterility, a great part of mutations are only represented in the hetero- 
zygous condition in the M 2 generation. These become manifest for 
the first time in the M 3 generation. Thus, in peas, Gottschalk (37) 
recognized only 60 per cent of the mutants in the M 2 generation and 
40 per cent in the M 3 . Similar results have been obtained also in 
barley (73). If selection of mutants is started in the M 2 generation, it 
should be continued in the M 3 and following generations. 

The procedure of selection depends on the intention of the 
breeding program. The selection may be directed towards a special 
goal, like yield, fungi resistance, baking quality in wheat (58), high 
protein content in barley (95, 96), germination under low tempera- 
ture in soybeans (129), strawstiffness, earliness, etc., without any 
interest for other mutants. Or selection may be nondirected and every 
mutant which might be of value for plant breeding and which 
becomes recognized is picked up for further investigations. Of course, 
directed and nondirected selection may be combined. 

Screen ins: for small mutations can start for the first time in 
progenies of normal-appearing M 2 plants, and it should be com- 
menced on a lame scale. Selection has to be continued in the follow- 
ing generations, and the material will become continuously smaller 
by the elimination of nonmutant and undesired lines. As compared 
with the pedigree method, for example, the breeder needs a different 
approach. Breeders using the pedigree method are used to observe 
large differences among the various progenies and desired types are 

gaul: induced mutants in seed-propagated species 229 

selected, at least in the beginning, by eye inspection. With small muta- 
tions, the basic phenotype is often not changed; and under these 
conditions it is hard, if not impossible, to detect, for instance, an 
increase in the yield potential of 5 to 10 per cent by simple eye 
inspection. There is a need to develop mass selection methods, and 
these will be different for different purposes and plants. This demand 
is shared with conventional breeding methods, and indeed many of 
the known selection methods can be applied to mutants in a more 
or less modified way. There are, however, some basic differences 
between conventional breeding and breeding; with mutations, and 
these will often require a different approach and special selection 
techniques. It is not possible to go into more detail here on this 

For the selection of useful mutants, particularly of higher yield- 
ing mutants, it might be possible to utilize the pleiotropic gene action 
which seems to be connected with every (or nearly every) mutation. 
I have suggested that one looks for "indicator characters" (25, 28). 
These should be mutations which are relatively easy to detect, either 
in a "normal" environment or under extremely changed (laboratory) 
conditions. They are characterized by morphological or physiological 
deviations which do not necessarily have breeding value. As a conse- 
quence of the pleiotropy, a certain proportion of these mutants may 
possess progressive features, i.e., they may have a greater yielding 

Selection of Mutants — Special Results 

We have started preliminary studies along this line in our labo- 
ratory. Among various such indicator mutations we have considered, 
the earliness character appears to be suggestive. Because earliness is a 
polyfactorial character, the probability of a mutation-event is relative- 
ly great; and earliness mutations have been frequently met with in all 
species. Moreover, it is possible to recognize even small differences 
with regard to the beginning of flowering. In cereals, the date of head- 
ing is a character that can be recorded fairly precisely and small 
differences become evident. In order to recognize small genetic differ- 
ences, it is however necessary (a) to have a great number of plants, 
(b) to work with replications, (c) to observe the material at least once 
a day during the heading time, and (d) to observe the material for 
several years. 



Table 7. — Heading Data of 56 Earliness Mutants of Spring Barley as Compared 

with the Mother Line Haisa II and Tentative Results About 

the Yield Potential. * 



% kernel 



than Haisa II 

Number of 

of Haisa II 

Number of 





(fr. No) 































































































































































































































































































gaul: induced mutants ln seed-propagated species 231 

Table 7. — Continued. 



% kernel 



than Haisa II 

Number of 

of Haisa II 

Number of 






(fr. No) 























































































































*Where no data are recorded the yield is lower than 80 per cent of Haisa II. All mutants 
originated by X-rays and were selected in Ma and Ms between 1954 and 195& from various experiments. 

In Table 7, headins: data of 56 earliness mutants are recorded 
which are all derived from the barley variety Haisa II. These data are 
based on observations of at least 2 years, but in most cases the results 
from 3 to 7 years are recorded. Occasionally, the mutants were grown 
in the same year in two different trials and therefore the number of 
observations recorded in Table 7 is sometimes greater than the num- 
ber of years tested. The most extreme mutant we have is 10.1 days 
earlier than the mother line on the average of eight observations in 7 
years, and there are all transitions to mutants only 0.2 day earlier. 
In each mutant the degree of earliness varies somewhat from year to 
year, indicating the influence of environmental factors on the expres- 
sivity of that character. It seems that the variability is usually greater 
with more extreme earliness. 

The mutant character of 28 of these earliness mutants could be 
firmly recognized in the M 2 generation. They may be considered, 
therefore, as large mutations. The other 28 were either questionable 
in Mo or were found for the first time in the M 3 generation. Most of 


these last-mentioned 28 may be considered as small mutations, and 
generally they are not earlier than 2 days (on the average of several 
observations). The results reported in Table 7 are not representative 
with regard to the relative frequencies of small and large earliness 
mutations because they are derived from various experiments and the 
selection methods have been inconsistent. There is no doubt that 
the frequency of small earliness mutants is considerably higher than 
indicated. These mutants have not been considered enough in the 
earlier experiments and perhaps not in some later ones too. 

The distribution of these earliness mutants on yield classes is 
indicated in Table 8. The various yield data are derived from differ- 
ent experimental arrangements. Most results are based on drilled 

Table 8. — Distribution of the Earliness Mutants of Barley on Yield Classes.* 

Yield class 

< 80 90 ■ 100 

- 110 


Number of mutants 

15 11 18 



Mean value of days of earlier heading 

than Haisa II 

4.8 2.5 1.4 



♦Mutants from Table 7. Kernel yield of Haisa II is taken as 100. Tentative results. 

trials, some on plots sown by hand, and some on hills. The lack of 
yield data in Table 7 indicates poor-looking mutants which have not 
been tested for yield. These mutants yield definitely less than 80 per 
cent of the control. Particularly, the results of those mutants having 
more than 95 per cent yield of Haisa II were based mainly on drilled 
trials, which had often a plot size of about 10 m 2 and three replica- 
tions. The yield of two of the three mutants recorded in the class 
> 1 10 per cent of Table 8 is based on one year only (1960), and the 
plot size was smaller. 

The results shown in Table 8 seem to indicate that 12 out of the 
56 earliness mutants possess a greater yielding potential than the 
mother line. These tentative investigations will be continued to 
obtain more reliable results. However, even if the proportion of 
high-yielding earliness mutants goes down to 1 / 10 , earliness seems to 
be an indicator character of practical interest. Yet it has to be con- 
sidered that these results are valid only for one variety (Haisa II) 
and for one location (Koln-Vogelsang). It may be expected that with 
other varieties and growing conditions, the proportion of high- 
yielding earliness mutants will be lower or higher. 


From Tables 7 and 8 it is further evident that, on the average, 
more extreme earliness mutants have lower yields. The higher 
yielding mutants are all 1/9 to 4 days earlier than Haisa II. 

Another possible indicator character under investigation in our 
laboratory is seed size. As with earliness, kernel size is certainly con- 
trolled by polymeric genes and the great heritability of 1,000-kernel 
weight is well known. In one of these investigations winter barley, 
variety Breustedts Atlas, was used. The procedure of selection is out- 
lined in Table 9. We started with nearly 1,500 normal-appearing M 2 

Table 9. — Procedure of Recurrent Selection for 1,000-kernel Weight in X-rayed 
Progenies of Winter Barley, Variety Breustedts Atlas. 

Number of 
plants or 1,000-kernel weight 

Year Generation progenies Grown as determined with 


1957 M 3 1,494 Single plants 200 and 300 kernels 

(drilled bulk) 

1958 M 3 135 Hills 2 X 300 kernels (from 2 field 


1959 M 4 31 Micro-drill test 2 X 1 ,000 kernels (from 2 field 


1960 M 5 31 Micro-drill test 2 X 1 ,000 kernels (from 2 field 


plants which were taken at random from an M 2 bulk. From these, 135 
with the highest 1,000-kernel weight were grown further. By a two- 
fold selection Ave ended with 31 M 2 progeny lines which were 
investigated more carefully in micro-drill tests for 2 years. 

The results of the first drill trial in 1959 are shown graphically 
in Figure 3. From that histogram it is evident that only 4 out of 31 
lines had a lower 1,000-kernel weight than the mother variety. Most 
of the differences are only in the range of 6 per cent as compared with 
the mother variety, but they go up to around 16 per cent. Thus, it 
appears that the relatively simple selection procedure has been sur- 
prisingly effective. According to an analysis of variance, differences 
between the lines are highly significant (F value lines/error: 17.80, F 
value of the table at 0.1 per cent level: 2.97). Also in 1960, differences 
between the lines were highly significant which is not demonstrated 
here (simple lattice, F value lines/intra-block error: 6.73, F value of 
the table at 0.1 per cent level: 3.58). 











t, - 



1 - 











Figure 3. — Effect of recurrent selection for higher 1 ,000 -kernel weight 
in progenies of X-rayed winter barley (variety Breustedts Atlas), accord- 
ing to the procedure of Table 9, results of 1959. Control is taken as 100. 
Only 4 lines out of 31 have a lower 1 ,000-kernel weight than the control. 

In Figure 4 a scatter diagram of the results in 1958 and 1959 is 
represented. There is a highly significant correlation between both 
years (r = 0.745, P > 0.1 per cent). A similar correlation was obtained 
between 1959 and 1960, as is shown in Figure 5 (r = 0.797, P > 0.1 
per cent) and also between 1958 and 1960 (r = 0.790, P > 0.1 per cent). 
Because of severe lodging in 1960, only marginal plants could be sam- 
pled from the drilled plots. Thus, the sampling has been done in 
1958 from hills, in 1959 from drilled plots, and in 1960 from marginal 
plants of drilled plots. Because of the high correlation between the 
different years and growing conditions, there can be no doubt that 
the differences between most of the lines are genetically controlled. 

At present we are not completely sure, however, whether all these 
variants of kernel size are true mutants though this is very probable 
for the majority at least. The seeds, which were irradiated in 1955 
were "Zuchtgarten-Elitegemisch" and not a pure line obtained by 
propagation of a single plant for only a few generations. According to 
the breeding history, the propagation methods, and his experience, 
the breeder of this barley believes it is not possible that the variants 
selected in the irradiated material were already present in the starting 
material (Breustedt, personal communication). 











Figure 4. — Correlation of 1,000-kernel weight between 195 S and 1959 
in 31 selected lines from X-rayed winter barley with regression lines. 
One barley line, marked by a dot with arrow, lies outside of the dia- 
gram (195S, 55.2; 1959, 50.2). r = 0.745. 

We also investigated this question in more detail by statistical 
means. The irradiated seeds were in the F 2 i generation and practical- 
ly all genes were, therefore, in a homozygous condition. The irradi- 
ated material was a mixture of 15 "nursery-lines", which were pheno- 
typically Aery similar, if not alike. In 1960, progenies of eight of these 
(untreated) lines were available for us to determine the 1,000-kernel 
weight. In the meantime, 23 lines had been developed from the eight 
progenies. The variance of the 1,000-kernel weight of the 23 lines was 
s 2 = 0.571 and the corresponding variance of our 31 selection lines 
was s 2 = 4.92 (determined as mean square deviation in relation to the 
mean value of our 31 selection lines). According to that, the variance 
of the selection lines is 8 to 9 times greater. The difference is highly 
significant (P > 0.1 per cent). 

A similar selection program as with winter barley was conducted 
with spring barley, variety Haisa II (Hochzucht). The results of the 
first drill test in 1959 are demonstrated in Figure 6. It is obvious 
that a oreat number of small variants of kernel size have been 



44 45 46 47 



Figure 5. — Correlation of 1 ,000-kerncl weiglit between 1959 and 1960 
in 31 selected Ivies front X-rayed winter barley ivitli regression lines. 
r = 0.797. 

screened. Again, differences between the lines are highly significant 
(simple lattice, F value lines/intrablock error: 16.90; F value of the 
table at 0.1 per cent level:2.79). Also, the correlation between 1959 
and 1960 is highly significant (r = 0.652, P > 0.1 per cent). The breed- 
ers of Haisa II consider this variety to be a pure line within reason- 
able limits (Vettel and Lein, personal communication). 

The selection experiments described are of a preliminary char- 
acter. They were conducted to determine the feasibility and value of 
initiating more exact and intensive experiments, and to gather tech- 
nical experience. The results have been broached here because they 
indicate at least that screening for increased kernel size is very simple, 
if there is a corresponding genetic variability in the starting popula- 
tion. Experiments are underway in our laboratory to obtain conclu- 
sive information on the nature of these variants, as well as on their 
general significance in breeding. 

Besides the experiments described, we started, several years ago, 
a program of recurrent selection for kernel yield in irradiated proge- 

gaul: induced mutants in seed-propagated species 








16 -- 

14 - 

11 - 
10 — 









Figure 6. — Effect of selection for higher 1 ,000 -kernel weight in progenies 
of X-rayed spring barley. Control is taken as 100. Results of 1959. Only 
9 lines out of 44 liave a lower 1,000-kernel xveight than the control. 

nies of spring barley. This program includes already an untreated 
control. It seems that it is relatively easy to screen directly for small 
mutations affecting yield and the results obtained so far will be 
published elsewhere. 



It has been shown that the induction of mutations offers a new 
tool which is potentially able to make progress in plant breeding- 
similar to that obtained with conventional methods. Mutants can be 
used directly to establish a new variety ("mutation breeding"). They 
can also be used in cross-breeding and for special aims. Generally, the 
use of mutants is time-saving as compared with the traditional 

However, the rate of progressive mutations and, hence, the 
efficiency of mutation breeding has been low until now. At present, I 
doubt, therefore, if generally this method is as useful and more eco- 
nomical than cross-breeding when a given aim is to be reached. 

At present, the uses of mutants in cross-breeding seems to be 
more promising than mutation breeding. In cross-breeding, mutants 
may complement the natural gene resources. Particular interest 
attaches to the possibility of removing the undesired pleiotropic 
effects of a mutation in a new genetic background. 

To summarize, the significance and importance of mutations in 
practical breeding now lies in their complementary use to the well- 
established breeding methods. Increasing the efficiency of the muta- 
tion technique can lead to a greater use of mutations in conjunction 
with the conventional methods. 

It should be emphasized that any evaluation of mutation breed- 
ing is speculative at the present time. Crucial evidence on compara- 
tive efficiency of traditional cross-breeding versus breeding with muta- 
tions is almost completely lacking. Strict utilization of mutations in 
practical breeding programs has scarcely been attempted until very 

In appraising the efficiency of and in defense of breeding with 
mutations one could challenge the followers of the traditional meth- 
ods with the view that the efficacy of cross-breeding is not extremely 
great either. Thus, Vettel (125), one of the most successful practical 
breeders in Germany, recently reviewed 30 years of his experience in 
cross-breeding with cereals. He gave a statistic of all crosses he has 
made in wheat, barley, and oats, analyzing 5,045 different cross- 
progenies. Only 0.3 per cent from these 5,045 F 2 populations resulted 
in commercially used varieties. In an evaluation of this figure, one 
has to realize that very much work of further selection and breeding 


has to be done in the generations succeeding the F 2 . The structure of 
total work to be done by a breeder may be compared with a pyramid 
having the top in the Fi generation and becoming broader with suc- 
ceeding generations. Another example of efficiency in cross-breeding 
was recently given by Williams (127) in tomatoes. The frequency of 
successful hybrid combinations appears to be around 2 per cent in 
that crop. Two such Fx hybrids which are used commercially in 
England were analysed in order to fix heterosis and Williams isolated 
1 desirable recombinant in every 1,000 to 1,500 F 2 individuals. 

Gregory (42), on the other hand, in his extensive work with 
small mutations in X-rayed peanuts, indicates that the frequency of 
mutants which are superior in yield may be of the order of 1 among 
500 to 5,000 Mo population plants. Gustafsson (45, cf. also 43, 44, 48) 
stated that a higher productive mutant is formed "once in 500 to 
1,000 genotypical changes". It should be emphasized that this estimate 
is based mainly on the selection of superior large mutations. From 
our preliminary studies in barley it appears that out of 5 to 10 small 
or fairly small earliness mutations, 1 may outyield the mother line. 
Earliness mutations are frequent and are easy to detect; the small 
ones, however, only in a group of plants. 

Consequently, if one critically tries to compare both methods, 
breeding with mutations and traditional cross-breeding, there is no 
evidence that the first is inferior. Much more experimental evidence 
and practical experience are necessary for a sound evaluation of the 
significance of breeding with mutations. 

The significance of mutations in the future depends largely on 
the question of whether a higher total mutation rate can be obtained, 
and particularly, whether the output of useful mutants can be 
increased. The final output of mutants depends on the technique of 
original induction as well as of selection. In both these fields theoreti- 
cal progress has been made, and there is hope that the efficiency of 
mutation production will be considerably increased. 

It would be useful, if, in various parts of the world, mutant 
collections of the main crops could be established which are based 
on adapted varieties with high performance. Collections of large 
mutations are easy to establish even with present methods. The 
expenditure is not too great as compared, e.g., with collection trips to 
centers of genetic diversity. These large mutations imply a valuable 


source in cross-breeding programs. Selection of small mutations 
involves more work than that of large mutations. Yet, mutation 
breeding proper might offer the greatest advantage as compared 
with conventional cross-breeding. Further theoretical and practical 
work will serve a final evaluation of mutation breeding. 


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Dermen: I am still unclear about the difference between macronut tat ions 
and micromutations. 

Gaul: The grouping has practical purposes only and may serve for 
the communication of evolutionary and breeding problems. It con- 
siders the phenotype only and has nothing to do with the change of the 
genetic structure. The classification is simply based on the method 
of detection, i.e., whether the mutation can be recognized with cer- 
tainty in a single plant or in a large group of plants only. It is arbitrary 
because there are all transitions from large alterations to small ones. How- 
ever, in plant breeding, the selection procedure entirely depends on 
whether one is dealing with macro- or micromutations. 

Strauss: It seems that the terms micromutation and macromutation 
have been used by you in an operational or field sense only. Should not 
these terms be defined more carefully in terms of actual gene changes? 

Gaul: This would be good if it is possible. A priori, I would not expect 
that a "small change of the gene structure" would necessarily correspond 
to a small change of the phenotype, and vice versa. 

MacKey: As to Doctor Gaul's definition of macromutations versus micro- 
mutations, I must say that the plant breeder urgently needs a separation 
of two such groups in his discussion, since they relate to the selection 
methods applied. Personally, I do not hesitate to enlarge the earlier 
concept of macromutation, since such shifts in meanings are common 
in biological denomination rules. Doctor Gaul's "transpacific macro- 
mutation" will be the old concept now in a strict sense. 

Frey: You have suggested improving the efficacy of mutation by (a) 
better control of mutation induction, and (b) better control of selection. 
I would like to see you add another category of great importance, name- 
ly, analysis of the elements that constitute the characteristic to be modi- 
fied and then an intelligent and logical selection of parent material 
(the genotype) to be irradiated. 


Gall: I agree completely with you. For instance, it might be expected 
that it will be easier to find high-yielding earliness mutants in a late- 
ripening variety than in an early one. 1 think however, that a thorough 
selection of the genotype of the starting material is an objective not only 
valid for the use of mutations but is common for all breeding methods. 

Sarvella: Are the first five tillers referred to in the theory of diplontic 
selection formed from the five buds present in the seed after irradia- 
tion (primary axillary spikes), or from the first five tillers emerging 
from the ground? Would you expect the mutation rate at a node to be 
lower in the tillers which are formed after the primal) axillary tiller 
is formed? 

There is evidence that the mutation rate per spike and per seedling 
in the plant appear to be the same for the whole plant, for the primary 
axillaries, and for the whole plant excluding the apical spikes and the 
primary axillary spikes (secondaries, etc.) at some nodes. The primary 
axillary spikes were tagged when the X-l seedlings were 3 weeks old. 

Caul: In reference to your first question. The hypothesis of diplontic 
selection considers the ontogenetically first five tillers, which do not 
always correspond with the first five tillers emerged from the ground. 
In reference to your second question. The first second-order tillers 
derived from axillary bud 1, 2, and perhaps also 3 presumably contain 
fewer mutations. With excessive tillering, the mutation rate of the later 
second-order tillers depends, I suppose, on the number of surviving 
L Il-initial cells of the corresponding axillary bud. If there are several 
surviving initial cells in these buds or primordias, the mutation rate 
of the later second-order tillers will usually be smaller than that of the 
tillers derived from primary axillary buds. This is a consequence of inter- 
cellular competition. But if there is only one surviving L Il-initial cell 
and this carries a mutation, no drop of the mutation frequency is 
expected because the generative tissue of the later secondary tillers is 
supposed to originate from this initial cell. This question is fully dis- 
cussed in the paper "Studies on diplontic selection" which is now in 
press (Symposium on the Effects of Ionizing Radiation on Seeds and 
their Significance for Crop Improvement, Karlsruhe, 1960). 

We have now evidence from four different experiments that the muta- 
tion rate of the later formed tillers is smaller than that of the first by 
about five. 

Nilan: From the literature and some discussions at this symposium, I 
find that frequently the induced mutation technique and the cross 

gaul: induced mutants in seed-propagated species 251 

breeding method are compared as methods in plant breeding. I feel 
that this comparison is not fair since for the most part induced muta- 
tions will be just a complement but not a supplement to a cross-breeding 
program. As plant breeders, we realize that genetic diversity is the basic 
ingredient of any plant improvement program. This diversity, which has 
arisen by mutation, can be introduced into a cross-breeding program 
through varieties and related species and genera. This diversity can also 
be obtained through induced mutations. Thus, induced mutations com- 
prise an important source of genetic diversity for a cross-breeding pro- 

The Use of Induced Mutations for the 
Improvement of Vegetatively Propagated Plants 


Jiahgard Fruit Breeding Institute, 
Fjalkestad, Sweden 

If we look at lists of new fruit varieties, a great many will be found 
to constitute spontaneous mutations. A glance at a rose nursery 
catalog will tell us the same thing, and we also know that much of 
the variation in form and color among flower bulbs and other orna- 
mental plants goes back to spontaneous mutations that have been 
taken care of and have been cultivated as new and more attractive 
varieties. In orange trees, potatoes, strawberries, and many other 
clonal plants there are often said to occur different "races", some of 
which may be claimed to be better keeping, better yielding, or 
deviating in other respects from the original types (65, 72, 77, 92, 
100). 1 

For several reasons, these sports seem to have been of special 
importance among the vegetatively propagated plants. Their greater 
constancy, preserved through clonal propagation, permits the detec- 
tion of even slight phenotypical changes. Thanks to the vegetative 
mode of reproduction, practically all such changes may be propa- 
gated, even such that would lead to complications and perhaps rapid 
elimination in a seed-propagated plant. 

The vegetatively propagated plants often have a long period of 
sexual reproduction. They usually turn out to be highly heterozy- 
gous, and our knowledge of their genetical relationships is still very 
imperfect. All this has made improvements by means of the classical 
methods, involving crossing, recombination and selection, time- 
consuming and dearly bought. To a considerable extent this may 
also have favored the use of spontaneous mutations in these plants. 

Towards the end of the twenties, means were found for the arti- 
ficial induction of mutations, as it seemed of the most varying kinds 
and in unlimited amounts. No wonder that plant breeders tried to 
apply this possibility in their work. I am not going to relate this story 
in detail here as it has been dealt with elsewhere (46, 49, 50, 86, 104). 

1 See References, page 285. 


nybom: vegetahvely propagated species 253 

We all know that the experiences and the opinions based on these 
studies in many cases turned out to be quite negative. Either one did 
not find any greater amounts of mutations at all, or those obtained 
did not appear very promising as a basis for the production of new 
and better varieties. 

The interest in induced mutations was revived when it indeed 
turned out to be possible to produce undeniable, progressive muta- 
tions of practical interest for the breeder (17, 39, 46). The increasing 
interest in the application of atomic sciences also has helped to focus 
the interest of the plant breeder on this "new" path or tool in plant 
breeding, the mutation method (47, 107). 

Although the inclination of many plant breeders has changed 
"from fairly sceptic to moderately optimistic" (80), one still meets 
fairly conflicting statements concerning the prospects of the mutation 
method. It is one of the chief aims of the present conference to collect 
our experiences, to focus the interest on the most promising areas, 
and, as far as possible, to present a realistic evaluation of the mutation 
method in plant breeding. 

A Survey of Mutation Experiments in 
Vegetatively Propagated Plants 

In order to give an idea of the achievements arrived at in various 
plants and to provide a basis for the discussion to follow, I shall begin 
by summarizing experiences from the more important mutation 
experiments in the vegetatively propagated plants that have been 


Some of the more extensive and most promising work on induced 
mutations has been reported with the potato by my compatriot, A. 
Heiken. As I happen to be rather familiar with his results, I shall 
take the liberty to deal with them at some length. This may be the 
more justified as his work is published in a journal that may not be 
generally available. 

However, for the sake of justice, I should like to begin by refer- 
ring to the classical work of Asseyeva. She published during the 
twenties and early thirties a series of papers on the chimaeric structure 
of different spontaneous potato mutants (2). She found that most of 
the spontaneous mutants were periclinal chimaeras; and with the 


eye excision method, she was able to unveil the genotypical constitu- 
tion of the deeper lying tissues. She also published some work on 
radiation-induced changes (3, 4). X-ray treatment was found to be 
"a powerful and reliable means" for bringing about tissue recom- 
binations similar to those obtained by eye excision. Not all changes 
were such tissue recombinations, however. Some behaved like origi- 
nal mutations. Unfortunately, her promising experiments were dis- 
continued. It was 20 years before they were taken up again (51, 54, 
55, 58). 

Heiken's own results were supplemented by a survey of the 
reported spontaneous mutations in the potato. His list of "tuber skin 
color aberrations" contains more than 50 cases, and to this list may 
be added changes in skin structure, shape and color of tuber, stem, 
foliage and flowers, as also the very characteristic potato mutations 
"bolters" and "wildings". 

The author also presents the results of his own extensive studies 
on the occurrence of similar aberrations in untreated material. In 
addition to 1 per cent bolters, he found 25 cases of such spontaneous 
aberrations in a material of several million plants, i.e., somewhat 
more than 1 in 200,000 plants. 

For irradiation, Heiken divided each tuber into two parts. One 
half was irradiated and the other was sown as a control. In this way 
it was possible to detect and remove all cases of contamination and 
tuber-carried diseases. The irradiations were carried out at different 
times. With regard both to the lower lethality and the considerably 
higher mutation frequency, the best results were obtained just at the 
beginning of germination, during February to March. A certain 
number of primary tubers then yielded six times more mutations 
than if irradiated during dormancy, i.e., during November to Decem- 
ber. After germination had started, April to May, a drop in mutation 
frequency was noted. The most suitable X-ray dose was around 
4,000 r. 

All tubers from the surviving Xi plants (plants growing from the 
irradiated tubers) were harvested and sown the next year to form 
the X 2 families, the plants in which then carried the X^ tubers. 

In the first year, irradiation gave rise to typical "primary effects", 
viz., increased fleshiness of leaves, disturbances in vein branching, 
deformation of leaves, etc. However, only a very small portion of 

nybom: vegetatively propagated species 255 

these effects persisted into the next generation. The frequency of 
transmitted aberrations was very much the same in those families that 
had passed the X 2 germination without primary effects. Observations 
on the X-? plants, on the other hand, showed that most of the induced 
changes had already been found in X 2 . 

As far as distinct, easily observed changes are concerned, selec- 
tion work may therefore be concentrated in the second year only. 
However, a breeder looking for less drastic changes might prefer to 
postpone selection to X 3 , when it can be based on more or less pure 
tuber lines. 

In the second year, changes turned up in certain families. Only 
changes in the above-ground plant parts were looked for. The dose 
response was evidently linear. Thus, 2,000 r gave 4.9 per cent mutated 
X 2 families, 4,000 r 10.5 per cent, and 8,000 r 20.8 per cent. However, 
due to the differences in lethality, the absolute number of mutated 
families always was higher after 4,000 r. The 640 tuber-halves irradi- 
ated with 4,000 r gave 409 surviving X! plants and the same number 
of X 2 families. Of these, 364 were normal while 45 contained muta- 
tions. The "segregation" ratio in X 2 was about 2 aberrant plants to 8 
normal plants per family. 

By means of eye excisions Heiken demonstrated that most, 
though evidently not all, e.g., none of the "wildings", of the isolated 
changes were periclinal chimaeras, having normal tissue below the 
mutated. All isolated types were also grafted on virus-free stocks in 
order to test the presence of any sap-transmissible principles, but no 
such indications were found. 

Even though these studies were intended as a "preliminary exer- 
cise" for mutation breeding work in the potato, like most other 
studies on induced mutation hitherto they were not planned as a 
mutation breeding project. The author wanted, in the first round, 
to determine the range of variability induced and concentrated mainly 
on distinct and easily identifiable mutations. Thus not less than 55 
of the isolated 109 aberrations consisted of various malformations, 
17 of them were dwarf types, and 27 were different flower color vari- 
ations. Stranoe enough, no bolters were found after irradiation. 

The average yield of these isolated, drastic changes was, as one 
could expect, considerably reduced. Most of them gave between 70 
to 80 per cent of the original clone, whereas some were rather 
similar to the mother variety in vigor. 


The dormancy period of the tubers was also studied in special 
experiments. Some of the isolated mutants had increased periods of 
dormancy while others started germination earlier than the normal 

Alter the last, main irradiation experiment a series of less marked 
and probably more interesting changes were isolated, but as they 
required another year for verification and testing, they were not 
included in the report. Summarizing his results, Heiken states that, 
"the somatic aberration frequencies obtained have been high enough 
to make continued research in this field very desirable". He suggests 
that a systematic selection of induced mutations could be combined 
with the present virus-testing routines. 

One of the keys to Heiken's success was the fact that he had the 
privilege of working at an institute specializing in virus diseases of 
potatoes and in growing virus-controlled stocks of the varieties used. 
I should like to stress this fact, as it is ol general importance for muta- 
tion studies in vegetatively propagated crops. Virus diseases often 
simulate o-enetical changes and may, therebv, cause serious trouble. 


A very interesting and promising piece ol work on induced muta- 
tions in Chrysanthemum indicum was published some years ago by 
Jank (59) in Germany. Chrysanthemum is also a plant where spon- 
taneous mutations, especially concerning flower color, have often 
been observed and also have been of great practical importance in 
the breeding work. 

]ank also combined his work with an extensive survey of the 
reported spontaneous color variations. Not less than 318 such "sports" 
have been described in 170 different varieties. It turned out that 
rose-colored varieties especially often give rise to mutations with 
varying new colors. White varieties usually are somewhat less mutable, 
and the same is true of the bronze-colored ones. There are also violet, 
red, orange, yellow, brown, and other colors which seem to be more 

Three varieties were selected for the main experiment. Day 
Dream, Vogue, and Berta Talbot, all with different shades of rose. 
Cuttings were taken on February 2. On March 18 the rooted and 
potted plants were decapitated in order to give rise to side shoots, and 
a few weeks later they were X-rayed. The suitable doses had been 


found to lie between 1,000 and 2,000 r. The roots were also irradiated 
in order to avoid excessive root shoot formation. 

In addition to reduced growth rate, the first shoots to appear 
showed the expected primary effects, thick succulent leaves with 
irregular margine and uneven surface. The author was well aware 
that the mutations should be expected to form aberrant sectors in the 
shoots formed, and that repeated bud and branch formation would be 
necessary in order to isolate the changes in a pure condition. 

Therefore, the shoots formed were again decapitated and the 
tips so obtained planted in order to form new roots. This process was 
repeated as often as possible; in all, seven times between April and 
June. The original 144 irradiated plants thus gave rise to 1,144 new 
plants, which were allowed to grow undisturbed and which flowered 
during October. Some changes in foliage were then noticed, as also 
some types with possibly deviating flowering periods, late flowering- 
being a desirable character. 

In addition, a great many variations in flower color were 
recorded, in all 281 different cases. Practically all of the originally 
irradiated plants yielded such color changes, most of them a whole 
series. The following new nuances were found: intensive rose, flesh- 
colored, copper-rose, copper-red, cream-yellow to cream-rose, cream, 
yellow, bronze, brown, red, and violet. White was obviously not 

The new colors were either found as sectors in the flowers or as 
single deviating flowers on a plant. Whole, changed plants were also 
observed, mostly after the later decapitations; the early ones usually 
giving narrow sectors. 

The percentage of recorded changes was highest alter the highest 
dose, 2,000 r. The author points out that it is important to use doses 
that permit an extensive clonal propagation of the irradiated indi- 
vidual in order to isolate the numerous changes induced at the 
moment of irradiation. 

On the whole, Jank's results appear very impressive and promis- 
ing, and he also summarized his work by stating that, "the experi- 
mental induction of mutations by means of X-rays may be regarded 
as an effective way of creating new color variations in Chrysanthem inn 
indicum" . 


Flower Bulbs 

Another category of vegetatively propagated plants where spon- 
taneous mutations have had, and indeed still have, a great connner- 
cial importance is the ornamental bulb plants, particularly tulips and 
hyacinths, but also iris, daffodils, lilies, freesias, and others. The 
variety collections are continuously enriched by new, and at least 
sometimes improved variations of the old standard varieties. Let us 
take one example ol such a "mutation family", that of the Bartigon 
tulip. This old and popular variety is now found in the catalogs as 
a double-flowered form, "Double Bartigon"; as a parrot form, "Red 
Champion"; as a giant or "maximum" mutation, "Bartigon Max"; 
as a white-variegated type, "Cordell Hull"; and as a series of new 
color sports, including salmon red "Queen of the Bartigons", scarlet 
red, "All Bright", deep rose "Philip Snowden", etc. 

The flower bulbs have also been the object of some relatively 
successful experiments with induced mutations, primarily by de Mol 
in Holland who described his results in a long series of papers. He 
summarizes the results of the first 13 years of X-ray experiments with 
tulips and hyacinths (74, 75). More than 70 different cases of induced 
changes are described, found in about 30 different varieties. The 
mutations mostly concerned flower color, but flower shape was also 
involved, ranging from highly irregular monstrosities to rather slight 
changes, e.g., from rounded to more angular flowers. 

During 1933-34 he irradiated in all 1,472 bulbs belonging to 
60 different varieties, using 1,200 r units of X-rays. In this material 
he later isolated 41 different mutations out of 25 of the varieties. Most 
of these mutations seem to have been cultivated further on in pure 

De Mol was aware that the older spontaneous sports may be 
periclinal chimaeras, e.g., the parrot-tulip Gemma, and that a 
radiation-induced reversion to the mother type, La Reine, was not a 
true mutation from the genetical point of view but as a "modifica- 
tion", or rather as a tissue recombination. But he also induced 
changed color in parrot tulips without loss of the parrot character, 
and also new parrot types from normal varieties. Except for a few 
possible tissue recombinations, most of de Mol's changes obviously 
were original mutations. 

In a special chapter, at the end of his 1944 paper (74), de Mol 


presents practical hints for the mutation breeder. The best time for 
irradiation seems to be September because the bulbs are then easier 
to handle than earlier in the summer when the new side-bulbs 
are formed. A suitable dose is said to be 800 r units. Like Tank, 
de Mol advocates the use of doses low enough to permit a rich forma- 
tion of new buds and shoots. The material should be propagated by 
side-bulbs for 4 years in order to obtain all changes in a pure state. 
The growings conditions should also be modified so as to stimulate the 
production of new side-bulbs. 

There was a pronounced similarity between the induced muta- 
tions and those known to occur spontaneously. There was also, in the 
main, parallel variations in induced and spontaneous mutability 
among the varieties. Many of the induced changes occurred, however, 
in varieties in which they were not known before. 

More recently a Swedish plant breeder, Carlsson, at the plant 
breeding station at Gullaker, Haininenhog, has taken up irradiation 
experiments with flower bulbs on a comparatively large scale. I 
believe we have irradiated some 25,000 flower bulbs for him at our 
cobalt 60-source at Balsgard since 1955. The doses used have been 
considerably higher than those mentioned by de Mol, namely, 
between 2,000 and 5,000 r. This difference may to some extent depend 
on the deviating irradiation conditions, acute X-rays compared with 
semi-chronic gamma during 5 to 6 days in the open air. 

Carlsson (22) has not yet published any results from his works, 
most of his material still being in an early stage. He has, however, 
already isolated a series of different changes and seems convinced 
that he shall be able to select types among them that ought to become 
of future value. In addition to the more common color changes, he 
has found several cases of white-variegated flowers, similar to the 
"Cordell Hull" sport of Bartigon. He also believes he has obtained 
giant sports in several varieties, corresponding to "Bartigon Max" 
out of Bartigon. 

Other Ornamental Plants 

Preliminary results have also been reported from mutation 
experiments with some other ornamental plants, the best known 
examples being those with carnations described by Richter and 
Singleton (91) and by Sagawa and Mehlquist (94, 95). 

Some of these changes, such as reversions from the spontaneous 


sports White Sim and Pink Sim back to the original red William 
Sim, obviously are tissue recombinations. However, some other new 
colors were also obtained as well as changes in flower morphology. 
These studies are being continued by Mehlquist (71). 

In Saiutpaulia, Sparrow and his co-workers have reported very 
interesting irradiation experiments (107 and 108). In the white- 
flowered and semi-double variety D wight's White mutations with 
violet and lavender-colored flowers were induced as also types with 
single and double flowers and a series of leaf changes. 

After irradiating leaf-petioles with 2,000 and 3,000 r X-rays, 14.2 
and 25.2 per cent, respectively, mutated plants were recorded, in all 
154 cases. A feature of special interest concerning these mutants is 
that they are mostly homogeneous, i.e., not chimaeric. This is also 
to be expected as the new plants are derived from single cells of the 
primary, irradiated leaf-petiole. 

In this connection there are some unpublished Swedish results 
with roses that I have been allowed to mention here. Doctor Gelin 
(39) at Weibullsholm Plant Breeding Institute irradiated 1957 
summer buds of five varieties of roses with 2,500 to 10,000 r gamma- 
rays from cobalt 60. After irradiation these were budded into com- 
mon rootstocks. About half of them took and developed into new 
shoots next year. In order to isolate the possible induced changes, 
buds were again taken from these new shoots in 1958. 

In addition to the primary effects the first year, the isolation the 
next year also gave rise to persistent changes. Besides changes in 
thorniness (increased thorniness), leaf color (increased anthocyanin 
content), and leaf shape, there was also found, after 5,000 r, a darker 
colored mutation in the variety Peace, somewhat similar to the 
spontaneous "Pink Peace," which is now under propagation for 
further tests (40). 

Thus, these first experiments with roses did give at least one 
mutation that might become of direct commercial importance if 
further trials show it to deviate in a positive way from the other types. 

It might be added here that similar-looking mutations should 
always be worth further trial and comparison, as varying pleiotropic 
changes seem to be a rule rather than an exception. Some of the red 
Delicious mutations in apples, for example, at the same time show a 
distinctly different and, from some points of view, an improved mode 
of growth and fruit production (1, 64). 


Fruit Trees and Small Fruits 

Most of the imitation experiments with vegetatively propagated 
plants fall within this group. For the sake of surveyability, the various 
mutation projects known through publications have been collected 
in Table 1. 

A special kind of mutation of great practical interest is repre- 
sented by the self-fertility mutations of Lewis, who has given detailed 
descriptions of their induction (66, 67). Irradiation was done during 
the resting stage before meiosis. Irradiation of mature pollen grains 
had no effect as the substances responsible for the incompatibility 
reaction are obviously by then already formed. In cherries, 800 r on 
an average gave one seed after self-pollination (or incompatible cross- 
pollination) of 130 flowers. Ten per cent of the seedlings so formed 
turned out to be completely self-fertile and to give rise to self-fertile 
offspring in their turn. 

The artificial induction of self-fertile types must be said to be of 
very great importance, not only from the point of view of fruit pro- 
duction but also with regard to the possibility of obtaining individu- 
als homozygous for rare recessive genes, leading, e.g., to extreme 
earliness (67). 

The method most commonly used for the induction of somatic 
mutations in fruit trees has been to irradiate dormant scion wood 
during winter or early spring and then to graft these scions into other 
trees. In order to ensure a better union between stock and scion, 
Grober (45) and Zwintzscher (112) recommend that only the upper 
part of the scions be irradiated. Granhall (42) and Zwintzscher (112) 
also overcame the same difficulty by irradiating complete young trees 
already growing on an understock. Granhall used 1 -year-old "year- 
linos", while Zwinzscher used somewhat older trees, the crowns of 
which had been pruned back in order to form new shoots. 

Other methods of irradiation described, e.g., by Hough and 
Weaver (57), involve continuous exposure of growing trees at a 
stationary cobalt 60-source. This method of irradiation has also been 
practiced in Sweden, although we are not prepared to judge whether 
it is more efficient than acute irradiation. We have also, like Grober 
(45), irradiated bud sticks of apples and pears during August and 
inserted the irradiated buds into suitable rootstocks. As far as we 
can judge, this method seems to offer certain advantages. 



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nvbom: vegetativelv propagated species 263 

On the branches formed from the irradiated scions various effects 
may be observed, primary changes like shoot bifurcations, disturb- 
ances in the leaf spiral, deformed leaves, and so on, but also fruit 
changes that may be genetical in nature, like sectors with deviating 
over-color. Such sectors may be rather common at first, but it is a 
general experience that if the shoots are allowed to grow undisturbed 
for several years these sectors eventually disappear. They may get 
broader later on but at the same time more rare, and only in a very 
few cases thus far have "stable" or "pure" changes been observed 
affecting whole branches (10, 12, 45). 

German workers especially have paid much attention to the 
problem of recovery and isolation of induced changes. A piece of 
work of great interest in this connection is that of Bauer (6) on black 
currants. According to his experiences, the first primary shoots com- 
ing from the irradiated buds only Aery rarely reveal any mutational 
changes. But if these shoots are pruned back to the originally irradi- 
ated stock and new shoots thereby forced to develop, a certain propor- 
tion of these will show such changes. These second-year shoots may 
then be removed, transplanted, and new shoots forced to develop 
again. This was repeated for several years, and after five years of 
selection in this way not less than 324 aberrant plants had been 
isolated from the originally irradiated 343 shoots. 

The reason why the unpruned primary shoot (like the Xj potato 
plant) only exceptionally gives rise to any changes may be explained 
by intrasomatic elimination which is supposed to take place in an 
irradiated multicellular organism and leads to an elimination of the 
mutated cells and a "normalization" of the plant (34, 63, 83). In 
barley, e.g., the primary roots may contain up to 100 per cent cyto- 
logically disturbed cells immediately after irradiation; but still, after 
completed ontogenesis, most of the cells in the spikes and the roots 
may look quite normal again. When mature buds are irradiated, the 
changes will form narrow sectors along the primary shoot. When the 
shoots grow, these sectors run a great risk of being eliminated due to 
intrasomatic competition. This must be part of the reason for the 
meagre results of many mutation breeding projects in fruit trees. 

Zwintzscher (112, 113) has adopted a special system in order to 
unravel the irradiated tissue and to isolate the induced changes in 
pure condition. The shoot developed from an irradiated bud is cut 


back close to the originally irradiated scion so that adventitious buds 
are forced to develop. The basal buds on the detached shoot may be 
expected to contain induced, sectorial changes and are, therefore, 
budded over on new rootstocks, either a certain number beginning 
from the base, e.g., five, or only those that sit in the axis of a leaf 
showing primary effects. The primary effects can certainly not be 
called "mutations" but may perhaps be taken as an indication that 
the tissue in question was in a sensitive condition during irradiation 
and that it has not had the chance of getting rid of the changes to 
the same extent as the buds sitting with normal leaves further to the 
tip of the growing shoot. The originally irradiated wood may be kept 
for several seasons as a reservoir of induced changes. 

Grober (45) is using a similar pruning method. After the end of 
the first growing season, all shoots coming from irradiated buds are 
cut back on three or four buds, which are then allowed to break next 

The types of mutations induced are also briefly indicated in 
Table 1. On the whole, these mutations are very similar to those 
known to occur spontaneously in fruit trees, as listed, e.g., by Shamel 
and Pomeroy (102). From the genetically better explored organisms 
Ave know that most mutations, whether induced or spontaneous, con- 
sist of destructive changes, deformations with more or less reduced 
vigor, etc. The same is. of course, true of mutations found among the 
fruit trees. 

From almost all mutation projects, however, there are also 
reported mutations that may be of practical importance, e.g., the solid 
red sports of Cortland apple (11); the high-quality, late-ripening 
Elberta peach (57); mutations with later dowering period in the black 
currant (6); with earlier ripening in grapes (18); the "self-thinning" 
changes in the seedless Perlette Grape (85); and certainly the self- 
fertile cherry mutations (67). 

Most of the mutation breeding experiments in these time- 
consuming plants have been going on for a relatively short time. We 
are undoubtedly still in the beginning of the exploration of the muta- 
tion method, and even if the positive achievements are far from 
definite, this is of course still less true of failures. Preliminary results 
are also reported from other mutation experiments with vegetatively 
propagated plants (16, 23, 69, 70, 73, 88). Of special interest is the 

nybom: yegetatively propagated species 


still unpublished work of Shapiro (103) carried out at the Brookhaven 
National Laboratory with roses, chrysanthemums, geraniums, and 

Mutation Experiments at Balsgard 

As briefly as possible, I should like to account for the mutation 
experiments on fruit trees and small fruits carried out at my own 
station at Balsgard. In cooperation with The Swedish group for 
Theoretical and Applied Mutation Research, we have now mutation 
material under way of most of the plants Ave work with at the Station. 

Since 1952, we have had stationary cobalt 60-sources for irradi- 
ation purposes. The old one, shown in Figure 1, was enclosed in a 
metal tube inserted into the ground. This source was removed last 
year and we are now installing another one, shown in Figures 2 and 3. 
As Ave have not found any advantage in the chronic irradiation of 

Figure 1. — Fruit trees being irradiated at the old cobalt source. Arrow 
itidicates the position of the source. 



The Gamma Station at Bals ga'rd ' v "" 

Figure 2. — Plan of the neiu gamma irradiation field at Balsgdrd, Sweden. 

plants, we arc going to concentrate on acute or semi-chronic expo- 
sures. Therefore, we have made the field as small as possible in order 
to save space. The strength of the source is now 40 curies, which seems 
suitable for our purposes. It is enclosed in a lead container sur- 
rounded by a ring-shaped soil wall. The source may be lifted up out 
of the container, which may also be used as a transport container, by 
means of an oil piston. A small hand-driven oil pump is placed, 
together with control lamps, in a box at the gate. The plexiglass tube 
above the container houses the oil piston. 



Figure 3. — Close-up of the cobalt pot. A now indicates upper position 
of the source. 

The method mostly used for the irradiation of fruit trees is 
shown in Figure 1. Young, 1 -year-old trees are taken from the nursery 
in the spring the second year after budding and placed at the cobalt 
source so that the source is closer to the tip than to the root. By adjust- 
ing the inclination of the trees we may get the killing dose of 10,000 r 
at the tips and the physiologically rather harmless dose of 2.000 r at 


the roots. Somewhere between, we then have the optimal dose, which 
may vary from time to time, depending on kind of fruit, chromosome 
number, condition of the trees, and so on. Through repeated pruning, 
first in order to remove all growth from the lower portion of the tree 
leaving only the uppermost three living buds, and then again the 
next year back to the basal buds of the surviving shoots, we try to 
"dissolve" the tissue having got the most suitable irradiation dose. 

The technique described might perhaps be improved by iso- 
lating the basal buds of the first year's growth over onto clonal root- 
stocks. The trees that have been exposed to irradiation or into which 
a lot of irradiated scions have been grafted usually will be rather irreg- 
ularly developed, and it is extremely difficult to identify these minor 
changes that may be of the greatest practical interest, such as changes 
in color, period of ripening, size or form of fruit, etc. When raising 
new trees on clonal rootstocks by taking buds from previously irradi- 
ated material, one might expect more uniform tree material among 
which it should be easier to trace slighter changes. 

We have also irradiated summer buds of apples and pears which 
have then been budded onto suitable rootstocks. Figure 4 shows such 
a shoot comino from an irradiated bud. About the same result would 
be obtained by irradiating dormant winter wood and grafting it into 
a rootstock just above soil level. After pruning the next year, such a 
mutated bud would result in a more or less completely changed tree 
without primary effects and other disturbances in growth habit. We 
have some such material with irradiated Williams pears among which 
numerous minor changes have been noted. However, the deviating 
trees must be tested further, budded over onto new rootstocks, togeth- 
er with control material, before we can be sure of the true nature of 
the changes. Some drastic aberrations have been recorded, however, 
like the irregularly corrugated mutants in Figure 5 and the "seedless" 
mutant in Figures 6 and 7. 

The irregularly furrowed fruits obviously are homologous to the 
spontaneous sport Corrugated Bartlett (101) and probably also to the 
irregular apple types described by Gilmer and Einset (41). A mutant 
in Cortland, quite similar to the neutron-induced one of Einset 
and Pratt (37), has been found in the same variety after gamma- 
irradiation, Figure 8. 

The seedless Williams type also deviates by its more elongated 

nybom: vegetatively propagated species 


Figure 4. — X-ray -induced tissue recombination leading to chlorophyll- 
deficient slioot in the chimaerical sport Striped Williams. 

fruits, Figure 6, a common effect of reduced seed formation in pears 
(9). In the seedless fruits, shown on both sides of a normal pear in 
Figure 7, only the empty seed coats are to be found in the carpels. 
The fruit-set of this seedless type did not look markedly influenced. 
Another change, probably also without practical interest, is the 
small-flowered mutant shown in Figure 9, Cox's Pomona gamma- 

Increased russeting, i.e., a rougher fruit skin, obviously is a 
rather common change both spontaneously and after treatment. It has 
been induced in the pear varieties Seigneur d'Esperen, Graf Moltke, 
and Williams, as also in the apple variety Belle de Boskoop. 

Of more commercial interest are the red-colored changes of 



Figure 5. — X-ray -induced mutations in Williams pear with irregularly 
furrowed fruits. Three normal pears above. 

which two are shown in Figures 10 and 11. In Cox's Orange, Figure 

10, (gamma-irradiation), a whole branch carrying apples with dis- 
tinctly increased color has originated. In Cox's Pomona (after pile 
neutrons), one branch, represented by the first two rows in Figure 

11, is still sectorial in constitution, practically all fruits being sectored 
with deep red. The other two branches formed on the same irradiated 
tree, represented by the apples of the other two rows of Figure 1 1, are 
quite normal. 

The chlorophyll-deficient shoot shown in Figure 4 (X-rays on 
summer buds) is induced in the pear clone Striped Williams (43). 
This Striped Williams, itself a spontaneous mutant very similar to 
the one of Gardner el al. (38), is most probably an endochimaera 
(Figure 12) that, besides "mutating" back to normal Williams, also 
oives rise to numerous such white shoots when irradiated. Thus, these 
changes are probably not true, original mutations, only tissue 



Normal Williams pears 

"Seedless" mutant 

Figure 6. — X-ray-induced mutation in Williams pear with "seedless" 
elongated fruits. 

recombinations similar to those found in other plants (4, 58, 74, 87, 
91, 94). 

These tissue recombinations may still be of considerable interest, 
however, and a deliberate production of such changes is also on our 
irradiation program. As should be clear, most somatic mutants are 

Figure 7. — Cross-sections of "seedless" fruits at both sides of a normal pear. 



Figure 8. — Gamma-ray-induced mutation in Cortland apple with fur 
roxued fruits. Four normal apples in the upper row. 

Figure 9. — Gamma-ray-induced mutation in Cox's Pomona apple with 
small Powers. Mutated branch to the left, normal branch to the right. 



Figure 10. — Gamma-ray-induced mutation in Cox's Orange apple zuitli 
increased red over-color. Normal apple to the left. 

built up as more or less stable periclinal chimaeras. Some of them 
make trouble by being too labile, like the reverting Starking Deli- 
cious (20), while others may be very constant. Such "ectochimaeras" 
(Figure 12), as for example thornless blackberry sports (26) or types 
with tetraploid epidermis (36), have normally not been accessible for 
further breeding work, because, as Darrow (26) says, "unfortunately 

Figure 11.— Neutron-induced sectorial mutation witli increased red color 
in Cox's Pomona apple. The first tivo rows represent the fruits from an 
affected branch, still sectorial and mutable. The other two roivs from 
normal brandies. 



Growth of the 
mutated tissue. 


primary sectors, unstable. stable. / <-c n f the 

leading to: sectorial or periclinal or mu tat ion 



Ty pes of 

periclinal mutations: 










to normal 

Figure 12. — Schematic presentation of mode of origin and chimaerical 
structure of somatic mutations. 

no way has been found to use these thorn less sports in breeding for 


As pointed out already by Asseyeva and de Mol and also verified 
in later experiments, especially those of Howard (58) and Pratt (87), 
irradiation seems to be a potent means of producing such tissue 
recombinations, not only "stripping off" external mutated tissue, but 
also giving rise to solid mutants (Figure 12). Therefore, we have this 
year irradiated some ectochimaeric mutations in order to make them 
available for future breeding work, namely, the blackberry sport 
Thornless Evergreen and a colchicine-induced ecto-chimaera in 
Prunus cerasijera. The latter was irradiated as slimmer buds, whereas 
the blackberry was irradiated as leaf-bud cuttings propagated under 

Part of the anatomical background for such tissue recombina- 
tions has been treated by Crockett (24). 

Finally, I should like to mention another case when such tissue 
recombinations might be the true purpose of an irradiation treat- 
ment, and that is in order to make a single layer chimaera more stable 
by transforming it into a diectochimaera or into a solid mutant. We 
have irradiated material of a spontaneous, but unfortunately not very 
stable nor very intensively, red sport of the James Grieve apple. If we 
could make it both more stable and more intensely colored, that 
should be a great advantage. I believe that the secondary, stable types 
of Delicious, like Starkrimson which has originated from the unstable 
Starking, very well might be examples of such tissue recombinations 
(1, 28). It is not clear, however, whether Starking is an ecto- or a 

Reduced fertility is another kind of genetical change that may 
turn out to be an undesirable by-product in some cases but that might 
even be desired in other cases. We have been noting that the fruit-set 
among: the irradiated trees sometimes seems to be reduced. In fact, 
some branches with aberrant foliage never have set any fruit. We 
have also found that there is a rather high frequency of reduced 
pollen fertility among apple trees that were irradiated 5 years earlier. 

Buds from fertile and partially sterile branches have been 
brought over onto new rootstocks, and in time it will be possible to 
see whether this reduction in fertility is also reflected in reduced fruit- 
set and perhaps in increased fruit size. In some fruit varieties of 
apples, plums, and grapes, too many fruits are normally formed with 
reduced size and quality as a consequence. In such varieties a slight 


reduction in fertility could be a great advantage. This already seems 
to have been successfully attacked by Olmo (85). We have also had 
our attention directed towards the same aim and have irradiated 
plum varieties, like Victoria and Reine Claude d'Oullins, with the 
intention of inducing some degree of sterility. It may be mentioned 
in this connection that in many ornamental plants even complete 
sterility, for several reasons, might be a highly desirable change, and 
it should be a rather easy one to produce. 

Finally, I should like to touch upon two other projects that we 
have also just started and therefore cannot say much about as yet. 
The first concerns the induction of thornless sports in blackberries. 
To that end we have irradiated seeds of an English variety, Merton 
Thornless, which is said to be apomictic (53). Irradiation of apomicts 
is a very interesting possibility, both because some of the induced 
changes might then be constantly reproduced by seeds, but also 
because the irradiation is known to be able to break down the 
apomictic mechanism itself which might be desirable in order to 
release the variation of apomictic species (52, 61, 62). 

In addition to mutagenic treatments with various radiations, we 
have also performed experiments with injected radio-isotopes (33), 
and also more recently with mutagenic chemicals. From the point of 
view of mutation induction the treatments with P 32 were not very 
successful, but this hardly permits us to say that this kind of treat- 
ment would generally be so (111). 

The treatments with chemicals involved ethyleneoxide, ethylene- 
imine, and ethylmethanesulphonate. Usually young trees, like those 
shown in Figure 1, taken just when the buds begin to swell, have been 
dipped in water solutions of the chemicals for 24 hours. 

Various chemicals that are active mutagenes in microorganisms 
have turned out to be rather ineffective on higher plants as for exam- 
ple the mustard gas substances (48). Chemical mutagenesis in these 
plants has, therefore, until recently not been very promising. Ehren- 
berg, et al. (35) found, however, that ethyleneimine (EI) in barley was 
even more effective than X-rays and neutrons, 20 per cent compared 
with 4 to 5 per cent mutations per spike progeny. EI did produce 
some of the typical primary effects of radiations, e.g., yellowish 
patches on the leaves of young apple seedlings, but not to the same 
degree as X-rays. This year we made the treatments with ethylmeth- 


anesulphonate (EMS), which had been shown by Heslot, et al. (56) to 
give up to 40 to 50 per cent mutations per spike progeny. The pri- 
mary effects on fruit trees were more pronounced with EMS than 
with the other chemicals. Even white sectors were found in Striped 
Williams, so this chemical seems to offer definite possibilities and 
should be tested further. Treatments with 0.5 to 1 per cent gave the 
best results; 2 per cent killed most of the buds. 

Mode of Origin of Somatic Mutations 

If we look back at this survey of mutation experiments we shall 
find certain features that are common to vegetatively propagated 
plants. In the first place, I think of the mode of origin and the dis- 
tribution of mutated tissue. When a mutation originates at a growing 
point, it will be localized in a single cell. When the tissue grows, the 
mutation will normally be delimited to a certain one of the histogenic 
layers at the growing point. There are usually supposed to be three 
(or four) such histogenic layers, or germ layers, the dermatogen, the 
periblem, and the plerome, the innermost one (98). These layers are 
also called L I, L II, and L III, respectively (29, 30, 36). 

As the mutation is only very rarely induced exactly in the central 
part of the growing point but more often at the side of it, it will form 
a more or less narrow sector at the base, or at the side, of the growing 
shoot (Figure 12). The shoot will become a mericlinal chimaera, and 
when side buds are to be formed, these may get several alternative 
constitutions, as shown in Figure 12. They may become completely 
normal again, they may retain a sectorial constitution, or they may 
also become complete, periclinal chimaeras. Only in the last case do 
we get a stable mutant more or less relieved of the competition, i.e., 
the intrasomatic elimination, taking place in the growing plant. In 
the side shoots, the mutated sectors usually will be broader, but also 
more rare than in the original, central shoot. Many of the induced 
mutants will probably be lost right in the beginning of the shoot 
growth and never be included in any side buds. 

In some plants there is normally provision for the formation of 
enough basal buds in a way that effectively conserves the mutations. 
In the potato plant, for example, the X 2 eyes on the tubers formed 
by the irradiated Xi tubers have gone through three such bud forma- 
tions rather rapidly, first when the stolon is formed in the axis of a 


basal leal, then when the tuber is formed on the stolon, and finally 
when the eyes are formed on the X 2 tubers. 

However, in other plants, like fruit trees, the irradiated bud 
may grow vigorously, several feet long into a shoot where the mutated 
sectors are probably found only at the basal buds. These buds are 
usually sitting close together, and normally they never take up growth 
again due to the apical dominance. These conditions, which have 
already been briefly touched upon in connection with the survey 
of the fruit tree experiments, are no doubt the main reason 
for the differences in mutation yield between different mutation 

Genetic Background of Somatic Mutations 

With few exceptions, the genetic background of stable somatic 
mutations have not been the object of closer studies. This certainly 
is due to the fact that most of the plants concerned are very incom- 
pletely investigated genetically. An exception is formed by the studies 
of Blakeslee and Avery (14) on spontaneous changes in Datura. These 
were practically all found to be due to chromosome changes, not to 
gene mutations. Also in chrysanthemum spontaneous mutations have 
been found often to be associated with changes in chromosome 
number (32, 96). 

The studies on radiation induced somatic aberrations in the 
endosperm of maize carried out by Dollinger (31) showed that most 
of these were losses from a genetical point of view, and that they were 
mostly associated with chromosome structural changes and deletion 
of certain chromosome segments. Some of them, however, were not 
cytologically detectable. This information on the cyto-genetic nature 
of these changes is also consistent with the repeated finding that most 
of them are eliminated at meiosis (97). 

Although we know very little from other plants, it seems reason- 
able to assume that conditions would be about the same, namely, 
that most, though not all, of the induced somatic changes are made 
up of gross cytological changes with phenotypical effects, most of 
them, perhaps, simply being losses or duplications of gene material. 

By experience we know that most of the radiation-induced muta- 
tions studied in seed-propagated plants are recessive, only quite few 
clear-cut dominant or semi-dominant induced changes having been 


found (80). Although most of the endosperm changes studied by 
Dollinger turned out to be losses, changes in recessive direction, he 
also found indications of dominant changes. In apples we know that 
gain in color as well as loss of color both do occur (9, 12). It is, how- 
ever, not necessarily so that the former changes should be dominant. 
They might as well be caused by losses of suppressor factors. Cases of 
obvious dominant mutations are known, however. The deep-red 
mutant of the Williams pear, Max-Red Bartlett, for example, seems 
to be a really dominant subepidermal factor mutation as it gives rise 
to 50 per cent seedlings with the increased anthocyanin content in 
the shoot, typical also for the Max-Red Bartlett. 

The Technique of Mutagenic Treatment 

As is evident from Table 2, most of the various types of diaspores 
used in vegetative propagation also have been exposed to mutagenic 
treatment, including scions, buds, cuttings, bulbs, tubers, etc. The 

7 Q 7 O 7 3 7 

range of doses that have been used is, however, narrow compared with 
the wide range of doses tolerated by seeds (50, 82) or by continuously 
growing plants (81, 105, 106). The most common doses lie between 
2,000 and 4,000 r units of X-rays or gamma rays. For especially sensi- 
tive, herbaceous material these doses may be halved, while for many 
lignified or dormant structures they may be doubled. 

Table 2. — Examples of Radiation Doses that Have Been Used in Order to Induce 
Mutations in Various Kinds of Vegetatively Propagated Plants. 

Apples and Pears: 

Dormant scions, 3,000 to 6,000 r, X-rays and gamma rays, Bishop, 1955; Granhall, 

et. al. 1949; Grober, 1959; Zwintzscher, 1959; 4-6x1 12 Nth/cm 2 , Bishop, 1958 
Summer buds, 2,000 to 4,000 r, Grober, 1959 
Growing trees, 25 to 50 r/day, several years; 50-100 r/day, 5 months, Granhall and 

Nybom, unpublished 
Flower buds before meiosis, 1,000 to 3,000 r, Lewis and Crane, 1954 


Dormant scions, 2,000 to 4,000 r, Zwintzscher, 1955 
Flower buds before meiosis, 800 r, Lewis and Crane, 1954 


Dormant scions, 2,000 to 6,000 r, Zwintzscher, 1955 

Growing trees, 25 to 50 r/day, several years, Granhall and Nybom, unpublished 


Growing trees, 10 to 60 r/day, 8 to 20 months, Hough and Weaver, 1959 


Table 2. — Continued. 


Dormant buds, 2,000 to 3,000 r, Breider, 1956; Olmo, 1960 

Summer buds, 5,000 to 10,000 r, Gelin and Gustafsson, unpublished 
Black currants: 

Dormant wood cuttings, 3,000 r, Bauer, 1957 

Leaf bud cuttings, 4,000 to 6,000 r; dormant seeds of apomictic species, 10,000 to 20,000 
r, Koch and Nybom, unpublished 

Spring suckers, 10,000 to 12,000 r, Nybom, unpublished 

Resting tubers, 3,000 to 4,000 r, Asseyeva, et al., 1935; Hagberg and Nybom, 1954; 
Heiken, 1960; Howard, 1958 
Sweet potato: 

Leaf cuttings, 3,000 to 4,000 r, Nishiyama, et al, 1959; root-tubers, 2,500 to 15,000 r, 
Matsumura and Fujii, 1957 
Tulips and Hyacinths: 

Dormant bulbs, summer or early fall, 800 r, X-rays, de Mol, 1944; 2,000 to 5,000 r, 
gamma, semi-chronic, Carlsson, unpublished 

Stem cuttings, 1,000 to 2,000 r, Jank, 1957 

Stem cuttings, 5,000 r, Sagawa and Mehlquist, 1957 

Growing plants, 200 r/day, 105 days, Richter and Singleton, 1955 
Camphor tree: 

Seedlings and cuttings, 2,000 to 6,000 r, Matsumura and Fujii, 1957 

Growing plants, 20 to 100 r/day, several months; seeds, 10,000 to 20,000 r, Moh and 
Orbegoso, 1960 

Leaf cuttings, 2,000 to 3,000 r, Sparrow and Konzak, 1958 
Various Bulbs and Corms: 

5,200 r, Spencer, 1955 

Rooted cuttings, 3,000 to 4,000 r, Murray, 1956 

Shoots, up to 250 r/day; Cuany, et al, 1958 

Rooted cuttings, 50 to 200 r/day, several months, Sand, el al., 1960 
Poa pratensis: 

Apomictically produced seeds, 20,000 to 30,000 r, Julc'n, 1954 

In most cases slight modifications of the propagation methods 
commonly used for the plants in question seem easily applied for 
irradiation of the material. That large variations in the frequency 


of isolated mutations might be anticipated after irradiation at differ- 
ent stages of development is shown by Heiken's results (55), but still 
Ave know far too little for making general recommendations. The 
method of decapitating rooted cuttings sometime before irradiation 
seem to be worth attention (59, 94). As mentioned before, we have 
been irradiating leaf-bud cuttings of blackberries rooted under mist. 
It might be added here that the modern mist-propagation methods 
seem to offer excellent possibilities for the management of irradiated 
material of vegetatively propagated plants, including the procedure 
of repeated tipping for the isolation of the induced changes (93). 

The intrasomatic elimination is a process that should be coun- 
teracted by all means. In barley we know that this loss of mutated 
cells is more pronounced under some conditions than under other. 
It is, for example, influenced by the hydration of the seeds, by tem- 
perature, oxygen pressure, and dose, being more severe after high 
doses (34). It plays a much smaller role after neutron irradiation 
than after irradiation by X-rays or gamma rays (68, 83), which may be 
one of the reasons why neutrons have been considered to be a more 
effective mutagen than X-rays in fruit trees (10). In fact, this elimina- 
tion is of great importance also for the yield of induced mutations in 
seed-propagated plants. In peas, for example, it will tend to reduce 
the part of the plant which is heterozygous for the induced mutation, 
and according to Blixt and Gelin (15), this part is smaller, i.e., elimi- 
nated to a larger extent after X-rays than after neutrons. The chemi- 
cal mutagens tested during later years seem to follow the neutrons 
more than the X-rays in this respect, which is, of course, much to 
their credit (15). 

We have reasons to believe that this intrasomatic selection is 
more pronounced in an object where there is great variation in radio- 
sensitivity between the cells or between the primordia. The irradi- 
ation should, therefore, be carried out at a staoe when the tissue that 
afterwards is to give rise to the new plant consists of as few and 
undifferentiated cells as possible. 

By introducing chronic irradiation Ave originally thought we 
might be able to differentiate between mutation induction, showing: 
no dose-rate dependence, and the probable physiological and dose- 
rate dependent damage leading to intrasomatic elimination. Thereby, 
Ave should o-et an accumulation of mutations above the maximal 


frequency obtainable by acute irradiation. For some plants, e.g., 
barley, we know now that this does not seem to be possible (84). Also, 
the results of Cuany, et al. (25) do not point to any conspicuous 
advantage of chronic irradiation over acute. 

Obviously even growing plants are able to eliminate a certain 
fraction of the induced changes. Therefore, as pointed out by Sand, 
et al. (97), the "pertinent dose", i.e., the dose corresponding to the 
number of recovered mutations, is "considerably less than that admin- 
istered to the whole plant as chronic irradiation during its develop- 
ment". Even though further studies on the possible differences in 
efficiency between chronic and acute exposures are required, there 
are no results yet to indicate any disadvantage of the more convenient 
method of acute or semi-chronic irradiation. 

Consequences of Chimaeric Structure of Somatic Mutations 

There are certain consequences of this fact that most of the 
induced and, probably to a still higher extent, the spontaneous 
somatic mutations are propagated as periclinal chimaeras (7). 

One practical consequence is that some of the induced muta- 
tions will be more or less unstable, more so perhaps if the new geno- 
type has a reduced competitive ability compared with the original 
one. Another consequence, also mentioned earlier in this paper, is 
that most of these mutations when used in crosses will turn out not 
to transmit the changes to the offspring unless they also comprise 
the subepidermal cell layer giving rise to the gametes. 

However, irradiation seems to constitute a very efficient tool for 
bringing about tissue recombinations so that di-ectochimaeras or 
tissue-homogeneous plants are produced. If this succeeds, we may 
then expect these new types to be both more stable and perhaps 
more pronounced in phenotypical expression. As far as the geno- 
typical change itself does not impair the fertility or viability of the 
subepidermal mutants, they should then also become accessible for 
further breeding work by means of crossing. The spontaneous muta- 
tions isolated hitherto present in this way a very attractive and com- 
pletely unexplored raw material for further irradiation experiments. 

Still another consequence of this chimaeric structure is that it 
permits the use of such changes that, when tissue-homogeneous, they 
would lead to complete sterility or complete inviability of the plant. 


The genotypical background might in such cases consist of losses of 
large chromosome sections or other gross cytological changes, leading 
to profound changes of skin properties, for example. This is probably 
not a very rare example of variety improvement that would not easily, 
or not at all, be attainable by ordinary cross breeding but only by 
means of mutations, delimited to a special tissue of the plant. There 
are thornless blackberry varieties, lor example, which seem to give 
rise to crippled dwarfs when tissue-homogeneous (26), and there are 
variegated Pelargonium types which, under the same conditions, 
either are practically lethal or not able to form any flowers (8). 

One could also well imagine that many ectochimaeric mutations 
with retained acceptable characteristics of the deeper lying tissue 
might not be usable as tissue-homogeneous clones because they would 
impair fruit quality, flesh color, and other properties. Again, the use 
of somatic mutations offers quite unique possibilities perhaps some- 
what hypothetical but indeed not unlikely. I might refer, for exam- 
ple, to the negative correlation often found between quality and 
disease resistance (21, 60). 

Use of Mutations in Relation to Other Breeding Methods 

After his review on radiation in the production of useful muta- 
tions, Smith (104) arrives at the conclusion, "The crux of the problem 
is — is it economically feasible to use radiations in plant improve- 
ment? There is no one answer. . . the answers will be different depend- 
ing on the material, objectives, and circumstances". 

I agree with this statement in all its details. The sometimes con- 
tradictory and sweeping statements about the value of the mutation 
method, or the irradiation method, must come from those who have 
not understood that the mutation method, like any other single 
method or tool in plant breeding, cannot possibly alone give us all 
the completely new and improved varieties that we need and that 
we are aiming at. The mutation method should be regarded as a 
complement not an alternative to other plant breeding methods, 
varietal crosses, species crosses, inbreeding, polyploidi/ation, etc. 

We may observe that in some cases the mutation method has 
indeed been "economically feasible". It has led to the production of 
varieties that are paying for themselves. That it has indeed been an 
"economical failure" in other cases is also evident. In addition to 


methodological shortcomings, the limited profit of the mutation 
experiments on vegetatively propagated plants reported so far may 
be explained by the fact that practically none of these mutation 
experiments were planned or carried out as real plant breeding- 
projects. We have been lacking, and are still lacking, most of the 
knowledge that would permit us to do so. We still know much too 
little about the possibilities of the mutation method and still less 
about its limitations. As long as the chief aim in plant breeding is 
to produce basically new and, from many points of view, improved 
varieties, I believe the cross breeding method will still be the back- 
bone of the breeding program. At least this appears to be the situation 
in fruit breeding. 

But it may well turn out that after 20 years' expensive and hard 
work we are left with a variety that is nearly perfect in nine of ten 
important characteristics. In order to get the fully acceptable variety 
we might have needed 100,000 seedling trees instead of the 10,000 
we could afford. Then, the mutation method might be the only way 
of coming closer to our aim. 

At Alnarp, Sweden, they have had a new seedling clone of apple 
under test and consideration for quite a few years. It has good quality 
and good handling properties but lacks distinct appeal in appearance. 
This year we were shown a beautiful, red spontaneous mutation of 
this variety. Obviously the basis for releasing this new variety on 
the market has changed completely with the detection of this red 

The organizers of this conference on mutation and plant breed- 
ing have raised some questions: How fully has natural variation been 
used? Should more emphasis be placed on its utilization? Will muta- 
tion induction be the one method when this variation is exhausted? 
Again these questions can not be given one answer. If I may limit 
myself to fruit again, I think one can say that we have, in general, an 
overwhelming natural variation, a variation so rich that I think it 
could not possibly be exhausted within reasonable time. The difficulty 
is to bring all the desired characters together into one and the same 
variety; so generally, we would not have to irradiate material only in 
order to o;et variation. 

But if we »o to single fruit kinds or to single characters, the 
variation is not always so rich nor so easily available. In black currants 


(6), the variation among available varieties with regard to size of the 
trusses (ease of picking) or length of the dormancy period (resistance 
against late frosts) is not as wide as we might wish. What do we do 
then, start crossing or irradiating? 

In blackberries we have some relatively acceptable varieties, 
which are, however, all terribly spiny. There are extremely few thorn- 
less types available for breeding, like the diploid weak-growing and 
winter-sensitive Rubus ulmifolius inermis. It might take us 15 years 
to bring its recessive inermis gene over into acceptable polyploid 
varieties, if we do succeed. Which will be cheapest, crossing or 
irradiation? If given resources, we should of course try both ways, 
well knowing that not all breeding projects started so far have resulted 
in improved varieties. 

Even after critical consideration there are applications like these 
and others where the mutation method indeed seems worth trying 
and where it no doubt in many cases will turn out to be economically 
feasible. With regard to the chimaeric nature of somatic mutations 
there are also occasions when the mutation method offers quite 
unique possibilities, or even turns out to be the only practicable way. 

We should not forget those plants that lack regular sexual repro- 
duction, e.g., the apomictic types, or where this can not be utilized 
because of self-sterility or incompatibility or the absence of llower 
formation. In the imperfect microorganisms, e.g., the antibiotic pro- 
ducing ones, deliberate mutation breeding has turned out to be highly 
successful (5). Also, the possibility of using pollen irradiation in order 
to realize difficult species crosses certainly deserves more attention 
among vegetatively propagated plants (27, 78, 90). 

Especially if we extend the concept to include all applications 
of mutagenic agents, it seems beyond doubt that the mutation method 
will become not only a valuable but even an indispensable tool for 
the plant breeder. But we shall have to learn much more before we 
can say when and how to apply it. 


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90. Reusch, J. D. H. 1956. Influence of gamma irradiation on the breed- 

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91. Richter, A., and Singleton, R. W. 1955. The effect of chronic gamma 

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92. Rogers, W. S., and Fromow, M. G. 1958. Royal Sovereign strawberry 

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94. Sagawa, Y., and Mehlquist, G. A. L. 1957. The mechanism respon- 

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98. Satina, S., Blakeslee, A. F., and Avery, A. G. 1940. Demonstration 

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der Obstzuchtung: I. Die Isolierung von Mutanten in Anlehnung 
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Caluecott: You have indicated that you obtain as many mutants from 
acute irradiation as from chronic irradiation. If this is true, does the 
gamma field still serve a useful purpose in a plant breeding program — 
note a plant breeding program, not a basic research program? 

Nybom: I think we know still too little to distinguish between a muta- 
tion breeding program and a basic research program. Certainly more 
studies are required to elucidate the different effects of chronic and acute 
irradiations, irradiations at different stages, etc. Also, for the irradia- 
tion of bulky objects, like fruit trees and others, a gamma source seems 
to be a most suitable tool, so 1 do not think such a source can be said 
to be out-of-date. 

nybom: vegetatively propagated species 293 

MacKey: In the discussion as to preference of chronic or acute radia- 
tion, I think it is very dangerous to generalize. Species with different 
speeds in cell division and different patterns of diplontic selection are 
bound to respond very differently to the two kinds of treatment. 

Hough: In reference to the use of chronic irradiation in gamma field 
versus acute irradiation, we have obtained useful mutations in peaches 
from both chronic irradiation of whole trees and acute irradiation of 
buds. We think that acute irradiation of buds and acute and semi- 
chronic irradiation of dormant or potted trees will be most useful in 
the future. 

Gamma irradiation appears to be quite valuable in irradiating some 
Prunus species in which microsporogenesis occurs in midwinter and in 
which the buds can be maintained only on the tree for the remainder 
of the dormant season. Thus, for irradiation during microsporogenesis 
in these Prunus species, whole-tree gamma irradiation is required. 

Singleton: Concerning chronic versus acute radiation it has also been 
our experience that a Co 00 source in a gamma field is useful in provid- 
ing gamma radiation to a wide variety of plants brought near the 
source for short periods of time similar to the way Nybom uses his 
gamma field. The gamma field at the Blandy Experimental Farm has 
been used since 1957 to give semi-acute doses of radiation in a 24-hour 
period. This machine is an economical radiation source. The machine 
is on loan to us by the Atomic Energy Commission. Total cost of the 
gamma field, 1 acre in extent, exclusive of the radiation machine, was 

Strauss: It seems that the terms micromutation and macromutation 
have been used also by you in an operational field sense only. Should not 
these terms be defined more carefully in terms of actual gene changes? 

Nybom: I have used the terms in the same sense as Doctor Gaul, just 
for the sake of convenience only. In higher plants we still know very 
little about the actual genetical changes underlying the phenotypical 
effects observed. 

Dermen: In the research of the African Violet are the mutations in 
the control or irradiated plants from adventitious buds? 

Nybom: According to the paper of Doctor Sparrow, quoted in the list 
of references, the new plantlets are derived from single cells of the leaf 


petiole. They are, therefore, not chimaeric in structure but represent 
homogeneous changes. 

Dermen: What is the chimeral make-up of yellow variegation in the 

Nybom: I believe the pear clone in question has normal epidermis 
and second layer and a chlorophyll deficiency in the third cell layer. 
It remains to be investigated further, however. 

Discussion of Session III 1 


University of Minnesota, St. Paul, Minnesota 

IN the early papers of this symposium, and particularly in Doctor 
Rhoades' discussion, reference has been made to different kinds of 
eYents which collectiYely may be referred to as mutation. These may 
include true changes in the molecular structure of the gene as well as 
minor chromosome changes such as deletion, duplication, or position 
effects resulting from iiiYersions. They may also include major chro- 
mosomal derangements, especially in the polyploids and in vegeta- 
tively propagated organisms, including losses or duplications of large 
chromosome segments or entire chromosomes. Finally, recombination 
by normal crossing-over may result in effects which can be distin- 
guished from true mutation only by refined genetic and cytological 
analysis, if at all. These may include sudden "large" changes due to 
crossing-over between members of a compound locus or, on the other 
hand, merely increases in genetic variability in characters controlled 
by polygenes. 

In the experiments on the role of mutations in plant breeding, 
it is evident that the very old and very broad definition of mutation 
as a sudden heritable change has been applied. The necessary cyto- 
eenetic tests have not been used in most studies to classify in a more 
specific fashion than this the kinds of mutations observed. Indeed, it 
is evident that with presently available knowledge sufficiently critical 
tests are not possible, in most instances, to distinguish between 
minute duplications or deficiencies on the one hand and true changes 
in the physical-chemical structure of the gene on the other. Further- 
more, for the purely practical assessment of the value of mutations in 
plant breeding, it is of little or no consequence whether the heritable 
changes observed were the result of changes in the DNA molecule 
itself or of some chromosomal aberration. To be sure, there are good 
reasons why the plant breeder wants to know the basic nature of the 
genetic change. But it is not inappropriate that in many of the plant 
breeding studies, no effort is expended in determining it. 

iPaper No. 1062, Miscellaneous Journal Series, Minnesota Agricultural Experiment 



The title of this section of the program is '"The Evaluation of 
Mutations in Plant Breeding". During the discussion so far, there 
has been consideration of the role of so-called "mutation breeding" 
and some concern regarding the relative efficacy of "mutation breed- 
ing" versus conventional cross breeding. Perhaps a consideration of 
the essential phases involved in plant breeding will aid in answering 
these questions. The three essential phases are: 

1. Finding or developing populations in which there is adequate 
and appropriate genetic variation, 

2. Effective selection, including; critical evaluation of the selections, 

3. Appropriate use of the selections, either directly as the varieties 
of commerce or indirectly in building such varieties. 

In connection with the discussions in this symposium the third 
phase is not a matter of concern, but the first two are and therefore 
warrant some further consideration. 

Consideration of the first phase is based on the premise that all 
genetic variability is dependent, basically, on differences in function 
at corresponding loci of homologous chromosomes; in other words, 
in the classical genetic terminology, that two or more alleles exist 
of one or more genes. The premise is also held that these differences 
at various gene loci have resulted from mutation. Therefore, all ge- 
netic variation must have had its origin in mutation. If these premises 
are valid, and this discussant can see no obvious alternative, the 
question posed by the title of this program can be given a definitive 
answer — mutation is a necessary prerequisite to successful plant 
breeding for, without mutation, there could be no genetic variation 
and without genetic variation plant breeding would be impossible. 

The words adequate and appropriate were used to describe the 
required genetic variation. By appropriate genetic variation, it is 
meant that for success in the plant breeding endeavor there must be 
in the population with which the breeder is working the genes which 
will, in proper combination, condition the expression of the charac- 
teristics sought in the improved variety. By selection, the breeder can 
do no more than extract from the population lines or new popula- 
tions which have combinations of the alleles that existed in the popu- 
lation. Genetic advance, that is the success of the breeding effort, is a 
function of the selection differential times the heritability. Both of 
these factors are determined in part by the extent of genetic vari- 


ability in the population. Therefore, within limits, the greater the 
appropriate genetic variability, the better the average chance and 
magnitude of success. 

Before considering the tools available to the plant breeder for 
developing populations within which he practices selection, it will 
be desirable to consider the kinds of genetic variation, from the stand- 
point of source, available to him. For purposes of consideration, these 
may be divided into the following four classes: 

1. Spontaneous mutations that were in the wild ancestor of the 
species or that have occurred and been preserved since its 

2. Spontaneous mutations that have occurred and been preserved in 
related species, 

3. Spontaneous mutations that arise in the breeding cultures, 

4. Induced mutations produced by any of the mutagenic agents. 

From the considerations given above, it becomes clear that the 
only basic difference between so-called "mutation breeding" and 
"conventional breeding" is in the source of genetic variability used 
by the breeder. It would seem preferable, therefore, not to speak of 
"mutation breeding" as a distinct method, but simply to recognize 
induction of mutations as a tool for obtaining genetic variability. 
For the population with which he works, the plant breeder may accept 
those available, either from natural sources or from mutagen-treated 
materials. On the other hand, he may build populations by cross- 
breeding, using parents that differ either because of natural muta- 
tions, induced mutations, or both. Finally, he may produce new 
populations with more desirable frequencies and combinations of 
genes by such tools as backcrossing or recurrent selection. Here again, 
he will be using mutations as the sources of genetic variability and 
these mutations may be spontaneous in origin, induced by mutagenes, 
or both. Induction of mutations as a breeding tool must be considered 
for efficiency or value, therefore, not in comparison with "conven- 
tional" breeding methods, but rather in terms of induced versus 
spontaneous mutations. It is appropriate, therefore, to consider in 
more detail the kinds of variation outlined above. 

No one who has worked with the crop plants can help but be 
impressed with the enormous natural variation that exists in most 
species. Doctor Quinby has pointed out the variation in sorghum. The 


3,000 forms studied by Snowden were classified into 31 species on 
morphological grounds; yet all of these may be freely interbred and, 
on a genetic basis, can be considered as one species. Doctor Quinby 
has reviewed for this symposium the accomplishments in remodeling 
this species from a too tall, too late, tropical forage grass into short, 
early maturing, grain-producing varieties adapted throughout most 
of the Great Plains of the United States. In this biological engineer- 
ing program, the basic materials were mutations, particularly at three 
loci controlling floral initiation, four loci controlling plant height, 
and several loci which condition grain color, grain type, and disease 
and insect reaction. Finally, a gene which reacts in the appropriate 
cytoplasm to produce male sterility has provided the mechanics for 
hybrid seed production, thus enabling the plant breeder to reap the 
benefits of heterosis on a practical scale. In connection with this story 
of the use of "macro" mutations in remodeling the sorghum crop, it is 
important to be aware also of the innumerable minor genes of which 
suitable combinations have provided for varietal superiority and 
heterosis within the larger groups determined by the major genes. 

Variability of the magnitude found in sorghum is also encoun- 
tered in other crops. Corn, wheat, and alfalfa are notable examples. 
All students of plant breeding are familiar with the writings of 
Vavilov based on most extensive plant explorations and collections 
by him and other Russian scientists. A recent, posthumous publica- 
tion (8) 2 dealing with world resources of cereals, grain, leguminous 
crops, and flax appeared in 1957. Vavilov, as noted by Hayes, Immer, 
and Smith (5), for example, defined eight principal regions of origin 
of crop plants. It is in these regions that the greatest genetic diversity 
in a crop is found. 

Many others, including Harlan (4), have also written about the 
enormous natural variation found in most crop plants. It has been 
estimated that domestication of most of the present-day crops began 
50 to 70 centuries ago and the species from which domestication 
started may well have arisen many centuries or millennia before that. 
One could assume in the thousands of generations and vast popula- 
tions of a crop that have existed that every possible genetic change 
has occurred, not once but probably many times. To be sure, it is 
probable that most of these spontaneous changes have been lost by 

2 See References, page 305. 


natural elimination or by chance. Even so, it is not surprising that 
so much variability exists. Even with the greatly accelerated incidence 
of mutations induced by mutagenic agents, the plant breeder may, 
with his limited time and facilities, be unable by controlled mutagene- 
sis to exceed or even to equal the work of nature. 

Faced with this fact and realizing that mutations provide the 
"life blood" of plant breeding, it seems appropriate to sound again 
the alarm regarding the danger of losing our natural sources of germ 
plasm. In a sense, the plant breeder is his own worst enemy. Improved 
varieties developed by him replace the natural stands, farmer's seed 
lots, and land races that are the reservoirs of the accumulated spon- 
taneous mutations. It is fortunate for the future of plant breeding 
that the eight regions of origin, as defined by Vavilov, are in the 
less-developed areas of the world. But this situation is changing 
rapidly. Some of the great gene centers may have already been 
destroyed by the advancing science of plant breeding (4). So-called 
"World Collections", valuable as they are, represent only a fraction 
of the world's germ plasm. The "Germ Plasm Bank" at Fort Collins, 
Colorado, is only a beginning of the facilities required if the best of 
the world's germ plasm is to be preserved. Plant breeders throughout 
the world must increase their efforts to assemble and save the store- 
house of variability which has accumulated over so many centuries. 

This discussant believes that in many crop plants, natural vari- 
ation can be found for almost any characteristic that might be sought 
in the crop. Obviously, there are limitations. One would probably 
not expect to find a genotype in wheat capable of symbiosis with 
Rhizobium bacteria. One of the interesting stories of extensive search 
for and the eventual finding of mutations required is that of the work 
done to convert the Lupinus spp. from wild plants to cultivated. This 
may be found in the publications of Von Sengbusch and his German 
associates which are reviewed by Hackbarth and Troll (3). As one 
example, Von Sengbusch and his staff examined more than one and 
one-half million plants searching for low alkaloid content. A total of 
six such plants were found. 

In search for genetic variability in one crop, the plant breeder 
can use as a guide the range of variability found in related species 
and genera. This is in accordance with the Law of Homologous Series, 
proposed by Vavilov (5). It is interesting that Doctor Quinby has 


pointed out the parallelism between mutant types found in sorghum 
and corn, i.e., endosperm color, waxy endosperm, sugary endosperm, 

Added to the genes of the cultivated species as possible sources 
of variability are the vast array of genes in related species. Classical 
examples of transfer from a related wild species of genes not known 
to be available in the cultivated species are found in wheat, potatoes, 
tobacco, and other crops. Modern advances in embryo culture, poly- 
ploidy, use of bridging species, alien chromosome and gene substitu- 
tion, and other techniques are expanding the range of species from 
which desired genes can be obtained (6). 

The third class of variation, as outlined above, consists of the 
spontaneous mutations that occur in the breeding cultures. These 
probably occur more frequently than was once thought. An early 
recognition of the frequency of spontaneous mutations was by East. 
From studies of maternal diploids of Nicotian a rustica, obtained in 
attempts at interspecific hybridization, he found that each progeny 
row was astonishingly alike, more so than "any ordinary inbred 
population that I have ever examined". When these lines were con- 
tinued by self-fertilization, they were, within three or four genera- 
tions, as variable as ordinary inbred populations (5). Despite their 
frequency, however, spontaneous mutations do not occur often 
enough in the breeding cultures to provide the amount and kinds of 
variation required in plant breeding programs. 

The papers of Doctors Nybom and Gaul have dealt in consider- 
able detail with the potentialities of using induced mutations. Doctor 
Nybom has emphasized particularly the use of induced mutations in 
improving varieties of fruits, bulb flowers, and other perennial orna- 
mentals that are vegetatively propagated. In these crops, in the 
absence of a sexual cycle, the only genetic variation that is found in 
a variety must arise from mutation. When a sexual cycle is used to 
provide for variation, the extreme heterozygosity of the parent variety 
or varieties results in populations within which the favorable gene 
recombinations can rarely be found in the size of sample with which 
the breeder can work. Furthermore, the long life cycle of many of 
these species limits the number of generations which can be grown. 
It is not surprising, therefore, that in these crops so many of the new 
varieties are mutant types from older varieties. Obviously, in such a 


crop, agents that will increase the frequency of mutation can be a 
powerful tool. This is particularly true with the ornamentals in which 
deviations from existing varieties have immediate appeal and in 
which vigor and reproductive capacity are not characters of prime 

Two points made by Doctor Nybom, in addition to the docu- 
mentation of varietal improvements resulting from induced muta- 
tions, seem to deserve special mention. One of these is his suggestion 
that mutation induction may be expected in the vegetatively repro- 
duced crops to pave the way for production of new commercially 
important varieties only if generally "good" varieties are used in 
mutation induction. The other point is that what appear to be 
mutations can be produced in periclinal chimeras by tissue 

Doctor Gaul has cited cases of favorable mutations induced in 
seed-propagated crops and of varieties released which owed their 
improvements apparently to induced mutations. From these reports 
one could conclude that induced mutations are a valuable addition 
to the natural variability that exists in the crop species. In fact, one 
is tempted to say that these reports are "too good to be true". This is 
not a valid reason for failure to accept the documented results of 
research. It does seem appropriate, however, to point out some pre- 
cautions that must be used both in conducting mutation experiments 
and in evaluating reported successes. 

The first precaution is the need for adequate controls. The 
varieties of self-pollinated crops are not, contrary to a commonly held 
opinion, pure lines. Rather they are mixtures of pure lines and, 
although they may appear to be uniform, they exhibit variability in 
physiological character and often, under new environments, in mor- 
phological characters as well. It is not sufficient proof of mutation- 
induced variability merely to have selections from a treated variety 
which differ from the variety in physiological characteristics. The 
treated population must be, in fact, a pure line or an untreated por- 
tion of the population must be subjected to selection pressure 
comparable to that used in the treated material. 

The nature of varieties of self-pollinated crops, namely, that 
they are mixtures of pure lines, is probably in some cases responsible 
for their broad adaptation in years and locations. When single lines 


are selected from such a variety following mutagen treatment, there 
is real danger that the derived lines, being more nearly "pure lines", 
will have a narrower range of adaptation than the original variety. 

A second precaution required is the prevention of contamination 
due to cross-pollination. Some natural cross-pollination occurs in 
almost every self-pollinated crop and the amount of such contamina- 
tion is sharply increased by the sterility in the first generation follow- 
ing treatment with the mutagene. 

Caldecott, et al. (2) have suggested that many of the variant types 
found in populations following radiation may actually have been of 
this origin. Subsequent studies by Ausemus (unpublished) have, in 
fact, shown that the rust-resistant segregates found in wheat popula- 
tions following radiation (7) were probably the result of cross- 
pollination with other rust-resistant varieties, even though the X x 
generation had been spatially isolated in the breeding nursery. 

A third precaution has to do with the validity of plot trials at one 
or two locations in f or 2 years. Such preliminary data provide 
a very uncertain basis for concluding that true increases have been 
obtained in yield or other characters having low heritability. 

An example of efficacy of "mutation breeding" that might be 
viewed with some reservation because of the precautions stated is the 
case reported by Doctor Gaul of Jutta winter barley obtained as an 
induced mutant from Kleinwanzlebener. The new variety is said to 
have improved winterhardiness, straw-stiffness, and yielding ability. 
This seems to be a large order of favorable effects to have occurred in 
a single line from treated material. 

Doctor Gaul has suggested that particular emphasis be given to 
"micro" mutations involving the genes of polygenic systems. In this 
connection it should be noted that so-called micro mutations might 
result either from a change in a minor gene (polygene) or from a 
small change in a gene having ordinarily major effect. As Doctor Gaul 
has pointed out, most of the characters of major importance in plant 
breeding are quantitative in nature and dependent upon polygenes. 
Whether or not there is more likelihood of favorable mutations and 
less of strongly deleterious mutations in these numbers of polygenic 
systems and whether or not, because of the number of polygenes 
involved, the chances of mutations significantly affecting quantitative 
characters are greater, are questions which must be settled by further 


experimentation. Nevertheless, as Doctor Gaul has suggested, more 
attention in mutation experiments needs to be given to changes in 
the polygenic systems. 

In connection with the studies of "micro" mutations, Doctor 
Gaul has pointed out the need lor better selection procedures that 
will permit accurate mass screening of populations for heritable vari- 
ations in the quantitive characters. Returning for a moment to our 
three phases of plant breeding, it is evident that this suggestion 
applies to the second phase, i.e., selection. Efficient selection methods 
are, of course, just as important in populations where the variability 
is due to accumulated spontaneous mutations as in those where it is 
due to induced mutations. 

The suggestion that efficiency of selection for yield and other 
less readily evaluated characters might be increased by selection in 
mutagene-treated materials for more readily observable changes, 
should be viewed with some reservation. When the more readily 
observable character is a component of the less readily observable 
one, the method seems to have potentialities. Selection for increased 
seed size with the objective of getting increased yield, as illustrated 
by Doctor Gaul, is a good example. On the other hand, there seems 
in many cases to be no a priori reason to assume that the pleiotropic 
effect of a macromutation on a quantitative trait such as yield will 
always be favorable. It might just as logically be expected to be 

As part of a discussion of the papers presented in this program, it 
seems appropriate to list some of the questions which are pertinent 
to the problem of the efficacy of induction of mutations to provide 
genetic variability for use in plant breeding. Some of these questions, 
as yet unanswered, are: 

1. Are the induced mutations the same as spontaneous ones? Related 
to this are the questions of whether mutations can be induced at 
loci where such mutations are not available in natural popula- 
tions or whether new kinds of mutant alleles can be induced at 
loci at which alleles from mutation are already known. It is evi- 
dent that a definitive answer to this question is not yet available. 
Also related is the question of whether true changes in molecular 
gene structure (true gene mutations) actually can be induced in 
higher plants by mutagenic agents. There seems to be fairly gen- 
eral acceptance of the fact that they can be. Yet of the induced 


imitations in corn which have been subjected to sufficiently 
critical genetic analysis none has been of this type. 

2. What is the frequency of total mutations? What proportion of the 
total mutants are favorable? Doctor Gaul has reported some of 
the information now available in answer to these questions, but 
it is evident that much more information is required before a 
definite answer can be given for any crop species. 

3. Are more efficient methods of treatment possible so that the total 
percentage of mutations can be increased? Doctor Gaul has dealt 
with this problem, as have others in this symposium. It is obvious 
that this is still a fertile field of research. 

4. Can we look forward to directed mutations? There is already 
some evidence that, by appropriate treatments with different muta- 
genes, the relative frequencies of "mutations" versus gross chro- 
mosomal changes and of various kinds of "mutations" can be 
altered. Even if some day true directed mutations become possible, 
■\ve might then ask how useful this will be to the plant breeder. 

5. Are the results of mutation, recombinations, etc., in viruses and 
bacteria completely applicable to the higher plants? There have 
been suggestions at this symposium and elsewhere (9) that differ- 
ences in chromosomal organization and gene action might exist 
which would limit the validity of extrapolation of results from 
the lower to higher organisms. 

Another consideration, not directly related to mutations but 
certainly involved in the extent of genetic variability, is that of link- 
age and recombination. Numerous experiments have shown some- 
thing of the magnitude of the genetic variability, particularly for 
characters determined by polygenes, that is "locked up" in chromo- 
some blocks by linkage and that is slowly released in succeeding 
generations by recombination. Anderson (1) has written about the 
hinderance to recombination imposed by linkage and refers to the 
cohesiveness of the germplasm. It has already been suggested in this 
discussion that sudden (or gradual) changes that appear to result from 
mutation may actually be caused by recombination. One might 
indeed question whether the results of the selection_experiment in 
Drosophila, referred to by Doctor Gaul, might have been due to 
increased genetic recombination rather than to mutations as was 


It is known that treatment with gamma- or X-rays or with high 
temperature results in small increases in frequency of crossing-over at 
least in some regions of the chromosome, notably in regions proximal 
to the centromere (10). Most chemical mutagenes that have been 
tested cause some crossing-over in the male Drosophila as also does 
radiation, according to Doctor Auerbach in personal conversation. 
The possibility, as yet virtually unexplored, exists that certain chemi- 
cals or other treatments (not necessarily mutagenic) have the capacity 
greatly to increase crossing-over. The natural variability that could 
be "unlocked" by radiation, chemical, or other treatment might equal 
or exceed any new variability that could be induced by mutagenic 

In conclusion the following points might be made: 

1. There is a growing body of information pointing to the possibili- 
ties of inducing favorable mutations in crop plants. 

2. Most plant breeders, working with crops in which an enormous 
amount of genetic variability exists, should concentrate at present 
on more effective use of that natural variability. 

3. Plant breeders should not, with available information and tech- 
niques, use mutagenic agents in their breeding programs with 
only the vague objective of increasing variability. 

4. Plant breeders who have specific reasons for increasing genetic 
variability may find mutagenic agents are useful tools. An exam- 
ple might be the case where resistance to a specific race of a disease 
organism is not known and is needed. Another example would 
be with crops which seem to have limited natural genetic variation 
such as peanuts. (See Gregory, this symposium, for example.) 

5. Research on the questions listed above and others relating to the 
efficacy of induced mutation versus use of naturally occurring 
genetic variation should be carried out intensively by plant 
breeders, geneticists, and cytogeneticists who have the facilities 
and opportunities to do a competent job of such research. 


1. Anderson, Edgar. 1949. Introgressive Hybridization. A T ew York: John 

Wiley ir Sons, Inc. 

2. Caldecott, Richard S., Stevens, Harland, and Roberts, Bill J. 1959. 

Stem rust resistant variants in irradiated populations — mutations 
or field hybrids. Agron. Jour., 51: 401-^103. 


?). Hackbarth, Jaochim, and Troll, Hans-Jiirgen. 1959. Lupinen als 
Kornerleguniinosen nnd Futtcrpflanzen. Handbuch der Pflauzeu- 
ziiclttung, 4: 1-51 . Berlin: J'erlag Paul Parey. 

4. Harlan, Jack R. 1956. Distribution and utilization of natural vari- 

ability in cultivated plants. In Genetics in Plant Breeding. Brook- 
haven Symp. in Biology, No. 9, 191-206. 

5. Hayes, H. K., Iinmer, F. R., and Smith, D. C. 1955. Methods of Plant 

Breeding. Nexv York: McGraiu-Hill Book Co., Inc. Ed. 2. 

6. Myers, W. M. 1960. Some limitations of radiation genetics and plant 

breeding. Indian Jour. Gen. if Plant Breed., 20: 89-92. 

7. , Ausemus, E. R., Koo, F. K. S., and Hsu, K. J. 1950. Resist- 
ance to rust induced by ionizing radiations in wheat and oats. 
Proc. of the Intern. Conf. on the Peaceful Uses of Atomic Energy, 
Geneva. Next) York: United Nations, Vol. 12: 60-62. 

8. Vavilov, N. 1. 1957. World resources of cereals, grain, leguminous 

crops, and llax and their utilization in plant breeding. In Agroc- 
cological Su)~uey of the Principal Field Crops. Izdatel stvo Akademii 
Nauk. SSSR Moskva-Leningrad. (Plant Breed. Abs., 28: 3576. 1958.) 

9. Westergaard, M. 1960. A discussion of mutagenic specificity: 1. 

Specificity on the "geographical" level. Chem. Mutagenese Erwin- 
Baur-Geddclitnisvorlesungcn. 1. Abhandlungen der Deutschen 
Akademie der Wissenschaften zu Berlin, 116-121. 
10. Whittinghill, Maurice. 1955. Crossover variability and induced cross- 
ing-over. Symp. on Gen. Recombination, Jour. Cellular and Com- 
parative Physiol,, 45:Suppl. 2, 189-211. 


Gaul: If I understood it correctly, you have the feeling that the example 
of Professor Vettel which I gave might not be representative. Vettel 
indicated that only 0.3 per cent of his 5,045 F 2 populations resulted in 
new varieties. You mentioned that professor Hayes needed fewer F 2 popu- 
lations for the development of new varieties. Such a comparison is, 
however, very limited because it depends on the breeding procedure. I 
would like to repeat that Vettel is one of the most successful German 
breeders. In Europe many breeders prefer to have many F 2 popula- 
tions and analyze only a few very carefully. If I understand, you at 
Minnesota did the contrary. You had very few hybrid populations which 
you analyzed very carefully. Thus, I think that a comparison of Vettel's 
results and yours is very difficult, if not impossible. 

Myers: I quite agree with Doctor Gaid that a comparison of Doctor 


Vettel's results and Doctor Hayes' results is of limited validity, since 
one has the choice of growing many F 2 populations with few plants in 
each or few F 2 populations with many plants in each. Perhaps the 
most important single factor in success is the total number of plants 
that can be grown. Nevertheless, the plant breeder who selects care- 
fully the parents he uses, as did Doctor Hayes, will make fewer crosses 
and have fewer F 2 populations. If, however, he was successful in selec- 
tion of the parents, the average value of his F 2 populations would exceed 
that of the F 2 populations of the breeder who did not select the parents 
so intensively. 

The real point I was trying to make, however, is that the low per- 
centage of Vettel's crosses from which new varieties were obtained is 
not, in itself, a valid reason for condemning the pedigree method. If, 
to take an extreme example, one grew only one F 2 plant of each cross, 
there would be little likelihood that any superior varieties would result 
from 5,045 F 2 populations. 

MacKey: The spontaneous mutations occurring in the breeding cul- 
tures mentioned by Doctor Myers as one cause of variability should 
definitely not be overlooked. In wheat, using a marker gene on chromo- 
some IX, 1 found 0.7 per cent mutations (speltoids) in a homozygous 
stable variety, close to 20 per cent after optimal X-irradiation. close 
to 40 per cent after optimal neutron irradiation, but more than 50 
per cent in heterozygous Fj material, all calculated on a per plant prog- 
eny basis. 

Davies: In relation to Doctor Myers' comment on artificially stimulating 
crossing over, we have already some results in this field. If we expose 
certain early meiotic stages to low doses of gamma radiation, a definite 
increase in the number of true chiasmata is observed in the bivalents 
at metaphase. 

Langham: There seems to be a tendency of new breeders to ignore par- 
tially the natural genetic variability in a species (such as sesame) and 
go directly to radiation equipment to look for induced mutations. I 
believe emphasis should be placed in (1) obtaining a world collection 
of genetic variability and (2) looking for new methods of screening this 
material for useful genes. In this connection, the High-Low method 
of breeding, as used in our sesame program, has given positive results 
and offers a useful tool in screening available breeding material. The 
High-Low method is as follows: 


A. It assumes that genes with strong potentials: 

— exist in the population under study 

— are not showing their maximum expression due to specific buffers 

— are maintained in a buffered condition in High X High or Low X 
Low crosses (such crosses give some transgressive segregation) 

— segregate free of their specific buffers as a result of High X Low 
crosses (such crosses give strong transgressive segregation) 

— receive an enhancing effect from specific buffers of the other 

]}. It consists of: 

— crossing the best parent with the worst parent for the character 
under study (High X Loav) 

— growing large F 2 populations from such crosses 

— selecting at both ends of the curve for plants showing extreme 
transgressions (H' and L'); H' is H minus its buffers, and L' is 
L minus its buffers 

— intercrossing favorable segregates (H' X H', or L' X L') to accu- 
mulate genes affecting same character or complementary char- 

— crossing H' with L and selecting in ¥ 2 for H" (H" = H' plus 
enhancers from L) 

— crossing L' with H and selecting in F 2 for L" (L" = L' plus 
enhancers from H) 

— crossing H" X H" and L" X L" for further advance in respective 

C. It works as demonstrated by results of sesame breeding programs 
involving the following characters: 

— length of pods 

— gland number 

— disease resistance (Alternaria and Cercospora) 

— insect resistance (aphid s) 

— number of branches 

— dehiscence 

Session IV 

Utilization of Induced 

F. L. Patterson, Chairman 
Purdue University, 
Lafayette, Indiana 

Screening Methods in Microbiology 


Eli Lilly and Company, Indianapolis, Indiana 

Screening methods as used in both basic and applied microbiology 
are designed to uncover organisms possessing specific physiologi- 
cal characteristics. The infrequent occurrence of these organisms, 
existing as only a small fraction of the population, requires that an 
effective screen be devised. Efficient screening techniques have been 
developed for various types of organisms of interest in basic research. 
Problems arise in adapting such techniques for industrial use, since 
the aims of the two types of investigation are different. In basic 
research the process and not the result is often of sole interest, the 
yield of variant types being used as a measure of effectiveness of vari- 
ables, as in studies on mutagenesis. When it is the variant organism 
that is of interest, as in studies on the pathways of biosynthesis using 
nutritionally deficient mutants, the property sought is a lack rather 
than a gain of physiological function. Industrial screening searches 
for a gain of function, either qualitative, in the production of a new 
antibiotic, or quantitative, in yield improvement. Screens to detect 
such changes, especially quantitative variations, are difficult to design. 
It is however possible that some screening methods for readily 
obtained variants can be adapted to industrial problems. This article 
will discuss modifications of some screening methods of basic research 
and the applicability of the resulting readily obtained variants to anti- 
biotic production. Other industrial fermentations and aspects of 
production are reviewed yearly (26). x 

Variants with Increased Yield 

The bulk of developmental microbiology in the antibiotics indus- 
try consists in screening for increased yield, "potency" in industrial 
jargon. More effort is expended in this phase of development than in 
experimentation with media composition, conditions of fermenta- 
tion, and purification. The reasons for this are, first, increase in yield 
through culture selection has been continuous and usually larger in 

'See References, page 327. 



magnitude than improvements by other modifications and, secondly, 
chemical purification can only remove what is already present and 
now approaches complete recovery. With culture development 
responsible for yield improvement from the 1 to 10 gamma /ml to 
the 1,000 gamma/ml range, further increase is expected. 

Practically no experimental methods and results are published 
from industrial laboratories on this topic, since these are trade 
secrets (59). A complete and detailed account of strain selection for 
increased penicillin yield has been published (8). The absolute yields 
are, however, below present industrial production levels. References 
to published papers concerning strain selection of commercially 
important antibiotics have been published (2); other papers appear 
in symposia (14, 41) and review volumes (62). 

No published work clearly separates the effects of culture selec- 
tion from changes in media, methods of growing inocula, and condi- 
tions of fermentation on yield of commercially important antibiotics 
during industrial production. An interesting account of a strain selec- 
tion program is given by Kashii, et al. (67) using successive mutagenic 
treatments, isolation, and application of statistics. 

When a decision is made to produce pilot plant quantities of a 
new antibiotic for pharmacological and clinical tests and determina- 
tion of structure, a program of strain selection and media develop- 
ment is usually begun on a laboratory scale. This program then 
expands to include conditions of inoculum development and fer- 
mentor tank operation in the pilot plant. Strain selection programs 
in the laboratory follow a sequence of steps, regardless of the specific 
organism and antibiotic. These steps are routine and have been in 
use throughout the period of industrial antibiotic production. These 
will be described with some speculations on different approaches 
suggested by recent genetic findings. 

A word of caution concerning measuring quantitative differences 
in antibiotic yield needs introduction here. The only proof of the 
worth of an antibiotic-producing culture is the yield obtained under 
actual production conditions. Since strains with an improved yield 
from a production level culture are rare, a less expensive system than 
tank fermentations returning results rapidly on a large scale is neces- 
sary. The "shake flask" system is commonly used. The antibiotic 
yield of various isolates is determined in a small volume of medium 

nelson: screening methods in microbiology 313 

in "fennentor flasks", inoculated from a previously grown "vegeta- 
tive flask", stirred and aerated on rotary shakers at constant tempera- 
ture. The misnomer "fermentation" is industrial jargon for a highly 
aerobic process. The activity of the antibiotic is measured by bioassay 
which has a readily determinable error, usually less than 5 per cent. 
Problems of bio-assay are discussed in a recent article (68). 

An apparent increase in yield may be due to the production of 
related antibiotics, hence all new strains must be tested for the pres- 
ence of other components. The major problem in shake flask testing 
has been the large variation between separate runs under apparently 
identical conditions. Attention to many details is necessary to reduce 
this variation below 30 per cent when determined by an analysis of 
variance between and within runs. While different antibiotic fer- 
mentations will each be found to have their own peculiarities, the 
major factor causing variation between runs has been the different 
states of the vegetative growth. The age and extent of vegetative 
growth can be determined and controlled on pilot plant and produc- 
tion scales but not in shake flasks, where smaller volumes of media 
and larger numbers of tests are required. Several inoculations of 
vegetative growth at different times may be used to compensate for 
different ages of growth. 

Any claims of increased antibiotic yield should be accompanied 
by a measure of reliability based on repetitive determinations in 
separate experiments, preferentially using pilot plant scale equip- 
ment. Use of sequential analysis as an aid in making decisions has 
been described (91). An increase in yield may occur on "scaling-up" 
the fermentation from the smaller laboratory equipment through 
the pilot plant. Since the same culture and media are used in both 
shake flasks and fermentor tanks, the fortuitous increase in yield is an 
indication that the medium can support more antibiotic production 
than is found in shake flasks. The limiting factor in laboratory scale 
fermentations may be the physical characteristics of the equipment, 
often giving insufficient aeration and agitation. Strains selected for 
higher yield in shake flasks may possess a different response to these 
physical limitations and not scale-up to larger equipment. 

Purification of Culture 

A new antibiotic-producing culture is usually "unstable", pro- 
ducing colonies with different morphology and variegated sectors 


within individual colonies. These different morphological types may 
be "purified" by any of several methods for obtaining single cultures. 
A spectrum of stable but different morphological types will be 
obtained along with the unstable prototype. The yield of different 
types will often be different and a pattern of production associated 
with morphology may be found. The variability may be due to hetero- 
karyosis (57). An isolate with stable morphology and high yield is 
established to supply inocula. The original culture should be retained 
since some derivatives are found to "degenerate", that is, decrease in 
yield, to be susceptible to actinophages, or fail to survive mainte- 
nance techniques such as lyophilization. 

Isolation of Colonies with Different Morphology 

Usually, some variation in morphology can be found in cultures 
that have been repeatedly "purified". New morphological types can 
be uncovered by plating on different media. An increase in morpho- 
logical variation follows mutagenic treatment and mav be used as a 
measure of effectiveness. Increased variation may not be due to muta- 
tion, however. Dissociation of homokaryotic sectors from hetero- 
karyotic growth, induction and lysis by temperate phage or bacterio- 
cins, changes in cytoplasmic determinants of morphology, if such 
exist, and changes of balance of nuclear types may produce variants 
that are not mutants. Thus, the use of morphological variation as a 
measure of induced mutation is not valid unless proved by genetic 
analysis. Such variation may be a partial explanation of pleomorph- 
ism found among the actinomycetes as well as the higher fungi (13). 

Experience with morphological variation in antibiotic-producing 
cultures can be used to predict what types do not produce desirable 
yields, but not what types will produce higher yields than the cultures 
established as the norm or "controls". Two types common to both 
the actinomycetes and higher fungi and producing no or very little 
yield are aconidial and colorless forms. 

Random Isolation 

Isolation of large numbers of colonies derived from individual 
spores surviving various mutagenic treatments is the next phase of a 
strain selection program. It is essential that these isolates be pure 
clones and not mixtures of mutant and nonmutant genomes. Muta- 
genic treatments applied to suspensions of nongerminating conidia 

nelson: screening methods in microbiology 315 

followed by dilution and plating to obtain well separated colonies 
will usually give pure colonies since the conidia probably contain a 
single haploid genome. Mutagenic treatments applied 10 germinating 
conidia and to mycelial growth will give genetically mixed colonies 
derived from several oenomes. Testing; such isolates is fruitless. An 
intermediate growth step with sporulation and replating is necessary 
to eliminate this error when the mutagenic treatment is applied to 
growing cultures. It is convenient to use this step to measure the yield 
of several classes of easily recovered variants (resistance to antibiotics 
and phage) as a function of the treatment method. These "muta- 
genicity indices" may be used to maximalize production and recovery 
of mutants. While a generalization of effective conditions to muta- 
tions affecting yield may not be valid, it is possible to eliminate 
inefficient methods which would prevent the recovery of any isolates 
with improved yield. 

Small yield improvements may sometimes be obtained by ran- 
dom selection of colonies plated out from production fermentor 
tanks. Such isolates may be returned to production fermentor 
tanks without more than one shake flask test showing yields at or 
above production levels. This program can often be coupled with 
continuous changes in operation of fermentor tanks and minor 
changes in media constitution in a form of evolutionary operations. 
Unfortunately, a statistical interpretation of results on either a labo- 
ratory or a production scale is not often possible since projected 
demands for material lor processing and the cost of operating non- 
productive equipment as "controls" may prevent carrying out valid 

Random isolation methods are distinguished from other tech- 
niques by the fact that the culture isolated is not known to be a 
variant. So many tests of different isolates may have to be made as to 
make random isolation an inefficient program. It is advisable to set 
a limit to the number of isolates to be run with various treatments 
to avoid an unrewarding search. 

Isolation of Variants with Known Physiological Differences 

The possibility of determining the potential yield of isolates 
without an initial quantitative test in shake flasks has occupied most 
industrial workers, btit no adequate method has yet been reported. 
A technique commonly used is the "streak plate", on which several 


isolates of an antibiotic-producing culture are grown using a suitable 
solidified medium, and their antibiotic production measured by 
streaking a sensitive indicator organism on the uninoculated portion 
of the plate. Various refinements of this method have been devised, 
the most elaborate is a "top-layering" method in which the well 
developed colonies of the producing organism are covered with an 
inoculum of a sensitive organism. The size of the zone of inhibition 
of the sensitive organism around a colony of the antibiotic producer, 
or the ratio of zone to colony size, is used as a measure of production. 
A positive growth response may be substituted for inhibition by using 
an antibiotic-requiring organism, such as streptomycin-dependent 
Escherichia coli (36) or macrolide-dependent Micrococcus (64). 

While logical in theory, these rapid test methods are difficult in 
practice. The antibiotic-producing organism must be plated in con- 
venient number, develop at a constant rate to the same extent, posi- 
tion of colonies on the test plates must not influence results, a medium 
adequate for growth of both the antibiotic-producing and sensitive 
organisms chosen, and the producing organism must be isolated free 
of the test organism. This method has been disappointing with the 
exception of hyphal tip isolations of Penicillia (44). Nonproducing 
isolates can be recovered. There is usually little correlation between 
increased inhibition zone diameter and the yield in submerged fer- 
mentation (14). Again, as with correlations of yield and morphology, 
one can go backwards but not forwards. Possible explanations for an 
absence of correlation are that the test organism is inhibited by 
factors other than the antibiotic, and that antibiotic yields obtained 
by colonial growth on the surface of media are not responsive to the 
same factors controlling yield in submerged fermentation. 

A popular approach to isolates with increased yield has been 
the selection of antibiotic-resistant variants (113). An isolate produc- 
ing a specific level of antibiotic is assumed to be resistant to this 
level, regardless of whether the antibiotic is being produced. Isolates 
of greater resistance are assumed to be immune by virtue of increased 
yield. As an initial screening method for new cultures or for "degen- 
erated" cultures, this technique is effective but fails for variants 
resistant to greatly increased amounts of antibiotic. These highly 
resistant isolates produce no antibiotic and are often asporogenous. 

A method based on a different rationale consists in selecting vari- 

nelson: screening methods in microbiology 317 

ants resistant to related antibiotics. Mutants resistant to amino acid 
analogs have been Pound to excrete small quantities of the homolo- 
gous amino acid due to a lack of repression of enzyme synthesis 
(1, 31, 32). Assuming that similar enzyme repression mechanisms 
control antibiotic synthesis, it should be possible to select variants 
lacking suppression, and therefore yielding more antibiotic. The 
problem of producing analogs of antibiotics is solved for those anti- 
biotics existing in families, such as the inositol amines streptomycin, 
neomycin, kanamycin, paromomycin, and the hygromycins, and the 
macrolide family. Streptomycin- and hygromycin-producing actino- 
mycetes were found to be sensitive to other inositol amine antibiotics 
and resistant variants easily obtained on gradient plates, but no 
increase in production occurred. Erythromycin-producing actino- 
mycetes proved to be resistant to other macrolides so that no test was 

A general repressor effect of excess glucose on synthesis of 
enzymes in bacteria may explain the delay in antibiotic synthesis 
that occurs until growth is nearly complete (34). Occasional increases 
in antibiotic yield have been obtained by feeding carbohydrate slow- 
ly, maintaining the measurable external level low. However, the 
effect of sugar feeds may be due to better pH stabilization rather than 
to absence of glucose repression. Mutants of bacteria have been found 
that are not suppressed by glucose (46, 78, 82). Similar variants have 
not been described in actinomycetes. No increases in yield were found 
by substituting carbon sources that do not produce glucose on 
hydrolysis, however. 

A recently described group of mutants lacks permease activity 
for inorganic ions (76) or amino acids (77, 92). It is difficult to devise 
a method increasing antibiotic synthesis by interfering with the 
utilization of amino acids, the principal nitrogenous components of 
fermentor media. An inability to concentrate phosphate ion might 
shift metabolism to antibiotic synthesis earlier with a subsequent 
yield improvement (39, 61). The ability of certain Penicillia strains 
to accumulate sulfate favors penicillin synthesis (105). "Boostable 
enzymes" have been described, but restricted growth is necessary for 
their occurrence (5, 55, 103). 

Variants resistant to actinophages as well as the phage are readily 
obtained. Various results have been obtained with phage resistant 


strains, high yields or no yield (85, 90). Complex patterns of resistance 
to virulent phage, production of temperate phage, and growth 
inhibition by bacteriocins occur among actinomycetes (17, 23, 98, 
1 12). The occurrence ol lysogeny raises the possibility of transferring 
characteristics between strains by transduction or conversion with 
temperate phage (7, 101). Survivors of a phage exposed population 
have a varied cobalamin vield (65). The usefulness of transduction is 
unexplored in strain selection. The success of such a program would 
depend upon the efficiency of the screening technique used to detect 
change. As an example, consider a series of strains resistant to high 
levels of an antibiotic that is produced by most of the strains at low 
levels, and at a high level by at least one strain. Production by the 
low level strains may be inhibited by genetic factors replaceable with 
different alleles from the production level strain. With a different 
genetic constellation, these strains may produce antibiotic at yields 
above the level of the former production strain. 

Various correlations between physiological properties during 
fermentation and yield have been noticed but are usually specific to 
the strain and conditions used and cannot be generalized or adapted 
for use as a screening method. Correlations between catalase activity 
and streptomycin production (72) and fluorescence and tetracycline 
production (88) have been described. 

Nutritionally exacting variants, auxotrophs, of antibiotic- 
producing microorganisms can be obtained by modifications of 
screening methods applied to Neurospora and Escherichia. Auxo- 
trophs described in the literature have been obtained for use as selec- 
tive and differential genetic markers in recombination studies, and 
groups of mutants blocked in the production of known metabolites 
have been used to determine pathways of biosynthesis. Two differ- 
ences exist between these studies and possible applications to anti- 
biotic production. Biosynthetic pathways blocked in auxotrophs lead 
to metabolites that are completely utilized during growth for further 
synthesis of more complex substances, there being little or no over- 
production and excretion of the metabolite into the medium by non- 
mutants, as occurs with antibiotics, and none of these metabolites 
serve as necessary intermediates in the biosynthesis of antibiotics of 
major commercial importance with the exception of penicillin. Tims, 
the selection of auxotrophs of a given biochemical class cannot be 
predicted to affect yield. 

nelson: screening methods in microbiology 319 

Excluding a direct link between the specific block in the auxo- 
troph and the antibiotic synthesized, is there any reason to suspect a 
change in yield? Continued improvements in yield have been con- 
sidered to result from mutations preventing diversion of energy and 
substrate into side reactions (41). Thus, the more nutritional defi- 
ciencies introduced into a strain, the higher the expected yield, 
providing the fermentation medium is supplied with a sufficiency or 
the required nutrients. A corallary of this hypothesis is the prediction 
that strains with high yields are auxotrophs. No significantly 
improved yields have been found among auxotrophs. However, the 
levels of loss of biochemical function which affect antibiotic synthesis 
may be more subtle than those existing in all-or-none auxotrophs. 

Another line of reasoning predicting yield increases in auxo- 
trophs follows from the suggestion that a medium with a surplus of 
energy sources but limiting in nutrients essential to growth, but not 
antibiotic synthesis, results in antibiotic production (61). Thus, anti- 
biotic production can be viewed as a consequence of unbalanced 
growth (33). While phosphate is usually the growth limiting factor, a 
lack of phosphate may depress the rate of production of high energy 
intermediates and limit antibiotic production as well as the level of 
growth. Furthermore, continuing ribose nucleic acid synthesis may 
be a prerequisite for enzyme synthesis and maintenance. Limiting 
phosphate would adversely affect both energy generation and enzyme 
synthesis. A specific method for blocking deoxyribose nucleic acid 
synthesis is required. Physical (ultraviolet irradiation) or chemical 
(addition of pyrimidine analogs or transmethylation inhibitors) 
methods are too cumbersome for industrial use. A thymine-requiring 
auxotroph would have a built-in method for growth level adjust- 
ment by control of the thymine concentration of the medium. 

Mutants blocked in alternate pathways of dissimilation and 
energy generation, such as the "poky" strains of Neurospora (58), 
would be expected to have changed rates of synthesis. Strains resistant 
to acridine dyes, which may clear the cell of plasmid-like portions of 
the cytochrome system (47), proved to have lower growth rates and 
antibiotic synthesis. 

Mutagenic Techniques 

There are no clues to suggest that one mutagen is more effective 
than another in producing variants with improved yield. The action 


of mutagens lias been shown to be "geographically" specific but not 
functionally specific (6). When a mutagen is found more effective in 
producing one rather than another physiological property, and when 
the effect is not due to differential sensitivity of one locus primarily 
affecting synthesis or to plasmid clearing, a selective effect of the 
system for recovering variants should be suspected. 

Although no specific mutagenic treatment can be singled out as 
most effective, certain pitfalls of inefficient use must be avoided. Some 
mutagens require special conditions for maximal effectiveness. Thus, 
ultraviolet irradiation is most effective when applied to growing cells 
containing large pools of nucleic acid intermediates, followed by 
incubation in a medium deficient in these compounds but enriched 
with amino acids. Conditions may be allele as well as mutagen 
specific (40, 99, 102, 114, 115). Certainly a mutagen will be ineffective 
if it or the products of its action cannot penetrate the cell. Pretreat- 
ment of bacteria is necessary to obtain maximum effectiveness of 
manganous ion due to its low permeability (38). 

The discovery of "hot spots" within a locus more sensitive to 
one than another mutagen, as well as the locus specificity of mutagens 
(probably due to such regions), favors a cyclical application of differ- 
ent mutagens (9, 37, 52). Continued application of the same mutagen 
may be fruitless if the most probable mutations inducible by the 
mutagen have already been recovered. Besides the use of a variety of 
mutagens and careful design of the techniques of application optimal 
to induction, detection, and recovery of mutants, the order of appli- 
cation is important. Thus, X-ray and ultraviolet irradiation may be 
initially used to obtain radio-kinetic data on the genetic constitution 
(30, 89), and to obtain auxotrophs for mutagenicity indices and 
incorporation of mutagenic purine and pyrimidine base analogs. 

Recombination Between Antibiotic-Producing Strains 
An approach to higher yields lies in the use of existing variation 
among different antibiotic-producing strains. A combination of 
desirable characteristics and elimination of unwanted properties 
mio-ht occur during joint cultivation of two different strains (80), but 
such a "mixed fermentation" would be difficult to stabilize. A single 
strain possessing only the desirable properties should be obtained 
through some form of genetic recombination (73) between differing 
isolates, the form of recombination depending upon the plasticity of 

nelson: screening methods in microbiology 321 

the organism and the technical competence of the worker. The possi- 
bility of improved yield through recombination has been recognized 
in a patent (84) but no examples of increased yield have been 
reported. An attempt to improve yields of organic acids by the use 
of heterokaryons did not succeed (29). Parasexual recombination in 
penicillin-producing strains demonstrated the genetic control of 
penicillin synthesis but did not result in yield improvement (28). 
A diploid recombinant between variants of the Wisconsin family of 
Penicillia separated by several mutational steps was reported to give 
a 50 per cent yield improvement (93, 94). 

The discovery of recombination between actinomycetes leading 
to heterokaryons and stable recombinants (22, 96) opened the way 
to possible industrial application of such techniques to antibiotic- 
producing microorganisms. A symposium on the genetics of actinomy- 
cetes contains further papers (104). The formation of antibiotic- 
producing recombinants from nonproducing mutants has been 
described (21). In summary, these and other papers (4, 15, 16, 18, 19, 
20, 25, 63, 89, 97) report the possibility of forming heterokaryons 
within but not between "species" of streptomycetes and the occasional 
strain-specific formation of stable recombinants, but no recovery of 
industrially useful derivatives. 

This work on genetic interaction among actinomycetes provides 
the basis for the possible production of isolates with increased anti- 
biotic yield obtained as recombinants between strains of diverse 
origin. Antibiotic production is probably dependent upon a complex 
of genetically separable factors, affecting an estimated 30 different 
biosynthetic steps through their enzymes and control systems. Recom- 
binants obtained between genetically different strains may give 
higher yields than either parental strain by reassortment of factors 
limiting and enhancing these individual biosynthetic steps. An essen- 
tial character of the strains selected for this analysis, other than the 
necessity for the production of the same antibiotic, is divergent 
geographic origin, ecology, and physiology. Recombinants derived 
from mutants selected from the same line, of similar "pedigree", would 
not be expected to have an enhanced yield and no increases with 
measures of significance have been reported for such recombinants 
(4, 93). Changes in ploidy within the same strain could affect yield 
without the necessity of "outcrossing" (95). 


A strain improvement program following this principle has been 
carried out by the author and associates. The highest yielding strain 
derived through a linear sequence of mutagenic treatments from the 
original erythromycin-prodncing isolate of Streptomyces erythreus 
(81) was selected as one parent. Other erythromycin-prodncing strains 
of differing geographic origin, morphology, physiology, and levels of 
synthesis have occasionally been found by the new antibiotics screen- 
ing program. A number of single and double auxotrophs were 
selected in these strains. In addition, it was necessary to prove the 
different strains were not mutually inhibitory due to cross-sensitivity 
and production of temperate phage, bacteriocins, or other antibiotics. 
Various genetic markers other than nutritional deficiency were used 
as aids to the recovery of infrequent recombinant classes or for proof 
of recombination. Interaction between strains, probably due to 
heterokaryosis, but no formation of stable recombinants has been 

Screening for New Antibiotics 

Lack of knowledge of the role of antibiotics in ecology, paths of 
biosynthesis, and mode ol action prevent a rational as opposed to an 
accidental approach to uncovering new antibiotics. Resumes of 
screening methods for soil microorganisms are given by Waksman 
(110) in several of his books with extensive literature references. The 
approach of present day screening programs is not very different from 
that described eight years ago (87). An outline of a generalized screen 
ing method will acquaint the reader with the procedure: 

(a) Soil samples are obtained from diverse sources and individual 
organisms selected by plating suspensions on solid media. A pre- 
liminary growth step, in selective liquid media, usually to sup- 
press molds and bacteria and favor actinomycetes, is often used. 

(b) Individual organisms are grown in a variety of media favoring 
antibiotic synthesis. 

(c) The spent culture media, sterilized, concentrated, or extracted, 
are tested for antibiotic activity against a variety of bacteria and 
molds, and possibly viruses and tumors (43, 108, 116). 

(d) Organisms producing activity in this initial screen are regrown 
under a larger variety of conditions and the antibiotic categorized 
by paper chromatography to determine whether it is known or 
new. A disappointing feature of this method is not the rarity but 

nelson: screening methods in microbiology 323 

the prevalence of organisms showing activity, most of them too 
weak to identify further or consisting of known antibiotic com- 
plexes (100). It is difficult to specify the exact number of known 
antibiotics due to incomplete chemical characterization, over- 
lapping families, and use of different names for the same 
substance (100). 

Either an extensive or an intensive approach may be used to 
increase the effectiveness of the screen. A more extensive program 
would increase the combination of isolates, media, conditions, and 
tests, usually by an increase at the input end, and the number of new 
cultures tested. This number is already large, 20,000 per year in one 
company's program, and the recovery of new clinically useful anti- 
biotics is low — 3 in 10 years (74). More extensive testing of new 
isolates may not be effective if the same organisms are merely being 
encountered more often. This suspicion is supported by the increas- 
ingly frequent recovery of the same antibiotic types. 

An increase in the variety of media used for fermentation may 
yield more active isolates. The effectiveness of a given medium may 
not be due to specific precursors but to the lack of inhibiting sub- 
stances. However, the effect of a medium is usually quantitative and 
the variety of media is probably not as great as the variety of organ- 
isms. Detection of activity depends upon inhibition or killing of 
sensitive bacteria, but more subtle effects on cell metabolism may be 
used to screen for useful metabolites that no longer fulfill the strict 
definition of antibiotic. No changes in media, conditions of fermenta- 
tion, or methods of detection can be expected to replace extensive 
testing of many different microorganisms. 

The intensive approach depends upon concentrating effort on 
different groups of microorganisms. Elective culture methods are 
often proposed to isolate one group of microorganisms from a mixed 
collection in their natural habitat. Difficulties encountered here are, 
first, elective culture methods are designed to recover a single type 
of organism rather than all members of its physiological group, and, 
second, such methods depend upon the selective use and not produc- 
tion of a metabolite. Thus, it is possible to isolate one or a few 
actinomycetes but not all actinomycetes by repetitive passage through 
appropriate media favoring actinomycete growth, without a pref- 
erential selection of antibiotic-producing types, however. 


Elective culture methods, operating by reduction of populational 
variability through selective growth, diminish the number of differ- 
ent types ol organisms originally present in a soil sample. Various 
methods have been proposed to retain and separate physiological 
and taxonomic groups in toto. Besides the physical difficulty in sepa- 
rating all organisms from their micro-environments in a heterogene- 
ous soil suspension, the microorganisms will be in different physio- 
logical states and growth phases from active metabolism and multi- 
plication to dormancy. Separation of the various groups assumes a 
greater difference between groups than between members within the 
same group. Thus, various physical separation techniques may be 
effective in resolving artificial mixtures of different organisms during 
"reconstruction experiments" but fail when applied to soil samples. 

Resides methods favoring the growth of one group of micro- 
organisms, there is the reverse technique of inhibiting all but one 
group. Addition of antibiotics to plating media is common (42). This 
method may be adequate for the recovery of new strains producing 
known antibiotics but could not be expected to uncover organisms 
producing new antibiotics. A variation could be used to isolate strains 
producing unknown antibiotics in such low yield as to make their 
initial identification impossible. Spent broths showing activity in 
step (c) but too dilute to classify in step (d) could be concentrated 
and added to plating media in step (a) to recover, by a system of 
positive feedback, strains producing higher yields or similar anti- 
biotics of greater specific activity. 

A separate intensive approach concentrates upon different 
groups of microorganisms isolated without an initial regard to their 
ability to produce antibiotics. The most intensively studied group 
has been the aerobic sporulating actinomycetes since success breeds 
success. Related groups, either less differentiated, such as the nocar- 
dias, corynebacters, and propionic acid bacteria, or more complex 
forms, such as the actinoplanes and streptosporangia, would be logical 
departures. Other groups might be chosen on the basis of similar 
habitat, growth forms, physiology, production of organic compounds, 
or possible unbalanced growth. 

It is impossible to predict what group o! organisms or set of 
conditions will yield new antibiotics. Even macroorganisms may pro- 
duce useful substances (70, 106). This is due to a lack of connection 

nelson: screening methods in microbiology 325 

between antibiotic production as an industrial process and produc- 
tion of similar substances in a natural habitat. Even though no con- 
vincing demonstration of production with chemical isolation of a 
major commercially important antibiotic under natural conditions 
has been reported, the suspicion remains that antibiotic production 
is but a magnified and variant expression of a normal biochemical 
function (27). Experiments to determine whether antibiotic produc- 
tion occurs in the soil or to determine the effects of antibiotics in soil 
have been performed, but none have suggested methods for the ready 
recovery of new antibiotic-producing organisms. 

The premise that microorganisms produce antibiotics in the 
soil as a mechanism conferring adaptive value in competition with 
other soil organisms does not have to be accepted before speculating 
on the function of antibiotic production. Antibiotic activity may not 
be the prime property of these substances conferring an adaptive 
advantage upon the producing organism. The principal groups of 
antibiotic-producing microorganisms, the actinomycetes, possesses a 
little but not a lot of morphological differentiation during the growth 
cycle, both during colony formation on solid media and during sub- 
merged growth in liquid media. Antibiotic production may represent 
a sloughing off of structures in passing through stages of differenti- 
ation (12), such as discarded or abnormal cell wall fragments or other 
components of the vegetative cell or spore forming cells. Production 
of bacitracin by Bacillus species, a group of microorganisms that may 
lie on the evolutionary path of actinomycetes, occurs at sporulation 
(10, 11). Antibiotic activity may be a secondary and accidentally 
derived property of these substances. If this view is correct, then sub- 
stances chemically similar to antibiotics but inactive should occur. 
None have been found, but the present biological screen would not 
be expected to uncover them. Chemical tests for antibiotics do not 
depend upon biological activity but are too insensitive and non- 
specific. Although antibiotics can be classed with major biochemical 
groups, none have known roles in biochemistry even when related 
structurally to normal metabolites. 

An approach to new antibiotics, alternative to random screening 
of neAvly isolated cultures, is induction of antibiotic synthesis in 
nonproducing organisms. Such proposals take one of two forms, viz., 
(a) the potential to produce an antibiotic may be considered already 


present but dormant and the correct conditions, usually a substrate 
or "precursor", or a "challenge" with a competing organism or dele- 
terious environment must be chosen to "induce" production; or (b) 
a culture may be considered to lack the immediate potential to pro- 
duce but may be "forced" to evolve antibiotic production in order to 
survive by subjecting it to an artificial selection system with 
competing microorganisms. 

The second proposal can be disposed of by some calculations on 
the time necessary for the evolution of an estimated 30 enzymes in 
the biosynthesis scheme of an antibiotic. The first proposal, restated 
in operational terminology and stripped of unpalatable and uncritical 
nonmechanistic allusions, is more attractive on our limited time scale. 
The difficulty arises in devising a method for "inducing" and recover- 
ing such microorganisms from a complex soil population or in deter- 
mining which, of a large number of pure cultures, is capable of 

Attempts to isolate new antibiotic-producing microorganisms 
from soil seeded with pathogenic bacteria have failed (111). Various 
mutagenic methods that have been proposed are probably selective 
rather than inductive in mechanism (69). Examples of gain of func- 
tion are cited as supporting evidence, but critical evaluation of these 
weigh against such proposals (45, 49, 50, 51, 56, 79). 

Any effective change in screening methods in the near future 
will probably come by way of increased efficiency of the screen 
coupled with a wider range of sources of organisms. Specific changes 
in screening methods can be tested by "reconstruction" experiments. 
Soil samples can be salted with a known number of spores of an 
antibiotic-producing actinomycete, productive at the same low levels 
typical of new isolates, and the efficiency of recovery of the added 
organism determined. This form of proofing of a new method would 
be most effective when the introduced strain is genetically marked 
to allow its identification and recovery at various stages of the 
screen (54). 

Success in uncovering new antibiotics and strains with improved 
yields has been dependent upon empirical methods. When continued 
application of routine and standardized methods does not produce 
results, the usual solution has been to increase the scope of the opera- 
tion. Any approach but this empirical one has been closed by lack 

nelson: screening methods in microbiology 327 

of knowledge of the biosynthetic paths and mode of action of anti- 
biotics at the molecular level. A beginning to the solution of these 
problems is now being made (35, 48, 53, GO, 71 , 83, 8(3, 1 1 7). It is possi- 
ble that the empirical approach has been over extended, and an 
amount of effort comparable to that expended on repetitive screening 
but directed towards these two problems would provide methods for 
desisTiino rather than discovering new antibiotics. 


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strains of Streptomyces griseus. Bact. Proc, Abs. 51st Gen. Meet., 
Soc. Anier. Bact., 15. 

114. Witkin, E. 1956. Time, temperature, and protein synthesis: A study 

ol ultraviolet-induced mutation in bacteria. Cold Spr. Harb. 
Sym p. Quant. Biol., 21: 123-140. 

115. and Theil, E. C. I960. The effect of post-treatment with 

chloramphenicol on various ultraviolet-induced mutations in 
Escherichia coli. Proc. Nat. Acad. Sci., 46: 226-231. 

116. Yamazaki, S., et al. 1956. Cylinder plate method of testing the anti- 

cell effect. Jour. Antibiotics (Japanese), Ser. B, 9: 135-140. 

117. Yarmolinsky, M. B., and de la Haba, G. L. 1959. Inhibition by 

puromycin of amino acid incorporation into protein. Proc. Nat. 
Acad. Sci., 45: 1721-1729. 


Auerbach: Did you carry out cytological observations? When 1 was in 
Moscow this year, 1 saw very beautiful preparations made by Prokof- 
jewa-Belgowskaja, which showed a close correlation between the stages 
of growth and antibiotic production in Streptomyces and Actinomyces 
with the appearance of the structure of nuclear bodies. On the basis of 
these correlations she has been able to advise on the use of culture 
methods for antibiotic production. 

Nelson: Inspection of stained or unstained mycelia is often used to 
determine when a pilot plant or production scale vegetative growth 
tank is ready for use as an inoculum. It has not been used as a method 
of strain selection since we don't know what changes to look for nor 

nelson: screening methods in microbiology 335 

how to avoid confusion of hereditary changes with physiologically 
induced morphological changes as the culture ages. 

Laxgham: You defined potency as yield. If you consider potency for 
potency's sake, it is known that the potency of insecticides, such as 
pyrethrum, can be increased about 100 per cent by the use of synergids. 
This was discovered during the last Avar when sesame oil was used in 
aerosol bombs. Chemical tests showed that both sesamin and sesamolin 
were responsible for the observed synergism, and further thnt the meth- 
ylenedioxyphenyl group of these compounds was the ellective one. Later, 
other compounds with this same group proved effective and are now 
used in commercial insecticides. (These are piperonyl butoxide, sulf- 
oxide, etc.) Have these chemicals been tested for a possible synergistic 
effect on antibiotics? 

Nelson: Those specific compounds have not been tested and I suspect 
that they would be too toxic for intravenous administration. However, 
a large part of clinical research concerns synergisms and antagonisms 
between cl liferent antibiotics and potentiating effects of derivatives upon 
antibiotic effectiveness. 

Methods of Utilizing Induced Mutation in 
Crop Improvement 


Swedisli Seed Association, Svulnf, Sweden 

Thirty years of applied mutation work have gradually sapped 
the first pessimistic idea that induced mutations represent only 
the deleterious types of spontaneous mutation. More and more, genet- 
icists have become convinced that the fundamental evolutionary 
force, the creation of variability, can now be controlled by man. 
From our present knowledge, it seems theoretically not too incon- 
siderate to accept ideas like the one held by Gaul (26, p. 276). 1 when 
he says, "Today there is little doubt that all the genes involved in the 
world collections of our cutlivated plants can be reproduced by 
induced mutations". But theory is one tiling and practical plant 
breeding another. For that reason, it is still a debate, but it is now 
less concerned with whether the imitation is possible or not than 
with whether it is economical or not. 

Proper economical evaluations need more evidence and experi- 
ence than does a statement of purely academic significance. However, 
since plant breeding is nothing more than controlled and directed 
evolution, I think it is easier for us to make the correct evaluation if 
we always keep the theoretical aspects in mind. If we compare with 
nature, I have no feeling that we use the two evolutionary forces, 
recombination and selection, in a different way in our plant breeding. 

Can we today make the same statement in connection with the 
third force, mutation? I do not think so, unless we accept such an 
extreme idea that macromutations are of overwhelming importance 
and are independent of background genotype. Most of our evidence 
that mutation breeding is a reality is based on distinct off-types either 
from a morphological or from a physiological standpoint, and these 
off-types have mostly been evaluated as raw mutants. It is logical first 
to build up proof in this way, since a macromntation in a self- 
fertilized crop plant can be made to constitute absolute evidence of 

1 See References, page 3M. 


mac key: induced mutation in crop improvement 337 

the progressive value of induced mutation if proper precautions are 
taken. If we now believe in a more balanced, more subtly differenti- 
ated, and a more dynamic evolution, such proofs will, however, be 
insufficient to rate mutation experiments as an ingredient in plant 
breeding. The meagre data available on induced mutation in quanti- 
tative traits and on the combining ability of induced mutations make 
it very difficult completely to understand the evolutionary implica- 
tion of artificial mutation. The very promising results obtained in 
the sparse experiments along these two lines (2, 3, 17, 33, 34, 37, 76, 
81, 91, 92, 104, 106), however, seen: to justify more optimism. 

Our deficient knowledge of how to utilize mutation in crop 
improvement must be borne in mind when we try to evaluate muta- 
tion experiments as a technique in plant breeding. It seems very 
likely that mutation work in the future will be more completely in- 
terwoven with the so-called conventional methods, with which it now 
too often is set in opposition. We have also to recall that most of the 
mutation experiments done so far have been biased by theoretical 
considerations and not planned as strict breeding projects. 

In addition, our knowledge of the relation between genotype 
and the possibility of inducing progressive mutations is most meagre. 
Nearly all our efforts have been concentrated on methods to increase 
mutation rate and to find mutagens with a specific and effective mode 
of action. For a plant breeder, it seems just as important to explore 
the specific response of genotype to mutation, since the careful choice 
of parental material is perhaps the most decisive part of the program. 
Not even the most clever and observant handling can compensate 
for unsuitable material. 

Table 1 lists some of the more important factors influencing the 
genotypic response to progressive mutation. Available information is 
too scant on many points to allow me to present more than self- 
evident items of knowledge in this paper, and some of the problems 
will be taken up by other participants in this symposium. For these 
reasons, I will discuss only some factors related to the choice of 
parental material which may be important for progressive mutation. 

From this special point of view, inherent mutagenic resistance 
with respect to survival is only indirectly important as long as it influ- 
ences the total yield of mutations. Drastic interspecific differences in 
radio-resistance have been demonstrated in connection with both 


Table 1. — Factors Influencing the Genotvpic Response to Progressive Mutation 

in Cultivated Plants. 

Nutrimental and industrial or ornamental use 

Age from standpoint of evolution, cultivation, regional adaptation, and scientific breeding 

Extent and availability of natural variation, including possibilities for interspecific gene 

Anatomy and differentiation of meristematic tissue 
Annual and biannual or perennial habit of growth 
Autogamy, allogamy, or clonal propagation 

Genie duplication and tolerance to chromosomal rearrangements 
Inherent mutagenic resistance and environmental possibilities to its modifications 
Degree of heterozygosity 

Phenotypic buffering in relation to characteristics 
Presence of desirable precursor genes 
Combining ability with genotypic background 
Goal of the specific breeding program and availability of effective selection techniques 

acute and chronic radiation with chromosome size and level of ploidy 
as the most decisive characteristics (39, 52, 72, 73, 80, 100). It is inter- 
esting, though by no means conclusive, that practical results in muta- 
tion breeding were early reported for rape and mustard, two extremes 
in high seed radio-resistance among agricultural crops (2, 3). 

Clear differences in primary radio-resistance are also found 
between closely related genotypes. The degree, and thus the chance 
to obtain maximum yield of mutation, may be controlled by inherent 
differences in simple metabolic activities (51). It may depend on indi- 
vidual chromosomes (114) or even genes (30, 61, 62, 99). It has been 
proved to vary witth degree of genotypic stability (8), can easily be 
recombined, and is dependent on type of radiation (34, 35). From 
Drosophila experiments, Ave can expect varietal differences in the 
response to chromosome breaks (16, 18). Gregory (34, 35) has even 
shown that it is possible to induce mutants with improved resistance 
to primary radiation injury. 

An example on induced decrease in radio-resistance is given in 
Table 2. Such a phenomenon has implication also in connection with 
recurrent radiation experiments in the same or successive generations. 
In the latter case, Shestakov, et al. (cited by Hoffmann, 48) found an 
increased sensitivity to radio-phosphorus in the second generation of 
treatment, and the same trend was found by Hoffmann (48) in acute 
X-irradiation of successive generations of wheat. 

With respect to the degree of heterozygosity, it appears that not 

mac key: induced mutation in crop improvement 339 

Table 2. — Artificially Induced Decrease in Radio-resistance. 

Control 10,000 r 15,000 r 20,000 r 

No. of seeds sown 150 300 300 300 

No. of seedlings, % 

Skandia III winter wheat 86.0 86.3 84.0 80.7 

X-ray-induced deficiency-speltoid of 

Skandia III 92.0 79.7 78.3 52.7 

Difference against difference in control —12.6 —11.7 —34.0 

iridiff. ±4.72 xx ±4.80 x ±5.14 xxx 

Seed moisture: 14.7 per cent Seed size: 2.50 - 2.75 mm 

only radio-resistance but also width of mutation spectrum may be 
influenced. Unfortunately, no definite information is yet available on 
this aspect on genotypic response to mutability. At present, there are 
thus only some rather vague statements on the advantage of using 
heterozygous parental material in mutation work (36, 40, 69), and the 
problem can therefore only be pointed out here. Heterozygosity 
implies, however, more unlike genes that can be changed, rearranged 
in blocks, or split up. The fact that heterozygosity in itself implies an 
enhanced pressure and chance of mutation (69) may also increase the 
efficiency of the applied mutagen. Such an idea is in a way supported 
by the fact that rare and new mutations become gradually more 
frequent in experiments including radiation of successive generations 
(48, 103). 

Phenotypic buffering, i.e., the ability to resist and absorb muta- 
tion without severe deviations from type, is another genotypic factor 
controlling rate and spectrum of progressive mutation. Duplication, 
epistasis, polymery, and other cumulative or complementary gene 
effects are added to allelism to form a very complex system of inter- 
ference and interaction within the germ plasm. The extent, type, and 
direction of this interdependence of genes, as well as their position 
and stability, will vary with genera, species, and biotype and also 
with character to form different patterns of buffering. 

The example I present is somewhat special, since it deals with 
macromutations in a ploidy series, but I am sure of its wider applica- 
tion. In Table 3, the relative prevalence of chlorophyll mutations 
per plant progeny in relation to all other phenotypically distinct 
mutations in M 2 of 2x monococcum, 4x dicoccum, and 6x vulgare 



Table 3. — The Relative Prevalence of Chlorophyll and Other Phenotypically 
Distinct Mutations in M 2 of X-ray- and Neutron-treated 2x, 4x, and 6x Wheat. 

The relative prevalence in M> of 
Species N 

Chlorophyll Other phenotypically 

mutations, % distinct mutations, % 

2x monococcum wheat 282 66.3 33.7 

4x dicoccum wheat 389 43.7 56.3 

6x vidgare wheat 983 0.7 99.3 

Test of heterogeneity 

X 2 d.f. P 

2x - 4x wheat 32.7 1 <0.001 

4x - 6x wheat 687.7 1 <0.001 

2x-6x wheat 721.2 1 <0.001 

wheat is calculated from a set of X-ray and neutron experiments 
(72, 73). 

A sharp decrease in the relative rate of chlorophyll mutations 
with increasing level of ploidy is associated with the fact that the 
total mutation rate increased twofold in the X-ray experiments and 
threefold in the neutron experiments from diploid to hexaploid 
wheat. What is interesting from our present approach is that the data 
demonstrate that the ability to produce chlorophyll, and presumably 
also other characteristics essential for the ability to complete life 
and to reproduce, are better buffered in the polyploid wheats. The 
induced macromutations in these wheats center more in characteris- 
tics which are not decisive for life or death but rather interfere with 
ecological adaptation, etc. 

As evident from Table 4, the same trend can be observed even 
within the category of mutations influencing the genetic control of 
the chlorophyll apparatus. Mutations harmful to the carrier already 
at the young seedling stage, like albina, xaiitha-types and viridoal- 
bina, decrease in relative frequency with increasing level of ploidy in 
contrast to types like tigrina and viridis where the production of 
chlorophyll is less completely blocked (38). 

This observation that the majority of mutations in one species 
can be centered on more harmful changes than in another is not only 
observed in ploidy series. If we still keep to the cholorophyll niuta- 

mac key: induced mutation in crop improvement 


Table 4. — Types of Chlorophyll Mutations and Their Relative Prevalence in 
Mi of X-ray- and Neutron-treated 2x, 4x. and 6x Wheat. 

The relative prevalence in M> of 

Species N xanlha 

albina xan.alb. virido- tigrina vindis All 

alb.xan. albina other 

2x monacoccum wheat .. . 295 42.4 6.4 9.8 2.7 34.6 4.1 

4x dkoccum wheat 191 27.8 4.2 6.3 18.3 40.S 2.6 

6x vulgare wheat 15 6.7 26.7 53.3 13.3 

Test of heterogeneity 

X 2 d.f. P 

2x - 4x wheat 43.4 5 <0.001 

4x - 6x wheat 3.9 1 * <0.05 

2x - 6x wheat 12.5 1 * <0.001 

*Albinu, xtmtlia-X^pes and viridoalbina is one group; tigrina and viridis is another. "All other' 1 

tions, which generally though not always (10, 43, 61, 74, 111) are 
detrimental, and refer only to diploids, we will find great differences 
in the relative commonness especially of alb bias between cereals, on 
the one hand (72), and large-seeded leguminous plants, such as peas 
(89), lupines (110), soybeans (117), and beans (Sjodin, unpublished), 
on the other. It is also symptomatic that Steuckhardt (103) found the 
albina mutations gradually increasing in relative frequency when 
irradiation of millet was repeated over successive generations and the 
buffering in this way gradually broken down. 

The varying degree of progress in the use of mutation experi- 
ments in crop improvement may partly be due to differences like 
those discussed here. The relatively high frequency of lethal or sub- 
vital mutations and a very strict, morphological architecture of, e.g.,- 
the barley plant, may partly account for the comparatively meagre 
results in contrast to those with peas, beans, and peanuts. Similar 
differences but of different magnitude can also be found on an inter- 
varietal level within species. To take only one example, Hagberg 
(46) found different erectoides loci more or less stable with a different 
pattern for different varieties. In the variety Gull 4 out of 8 
induced erectoides mutations involved locus b which never has been 
found to mutate in the varieties Maja and Bonus, where 7 and 87 


erectoidcs imitations, respectively, have been analyzed. As a general 
rule for all characteristics, it seems more difficult to induce and 
observe an improvement in a direction to which the parental variety 
has already been intensively bred (70). 

It is thus evident that the possibility to be able to induce, 
observe, and properly evaluate a mutation is highly dependent on 
parental genotype, and for that reason the breeder should not restrict 
his basic materal too drastically (15) and only to his very best strain. 
The choice of material especially suitable for the detection of the 
desired mutations can sometimes greatly improve the experiment. 
Thus, Melchers (75) demonstrated the use of haploid plants in 
Antirrhinum for detection of recessive mutations. Monosomies or 
other types with known deficiencies could be used in a similar man- 
ner. Mertens and Burdick (76) used the technique of irradiating 
autodiploids (2n ex-haploids) and then backcrossing and comparing 
them with an untreated control in order to detect mutations in quan- 
titative traits that could exhibit a dominance type of heterosis in 
tomato to Fi's. Genotypes with appropriate marker genes or trans- 
locations can greatly improve the detection of desirable chromosomal 
rearrangements in an irradiated material (19, 46, 93). 

The choice of the most suitable genotype in accordance with 
the intention of the mutation experiment is, however, only one side 
of the problem of how to render mutation breeding as efficient as 
possible. The chance to induce the desired mutations also depends 
much on the metabolic stage of the treated cells, the mutagen, and 
the dose applied as well as on the handling of the material before, 
during, and after treatment. These problems are, however, taken up 
by other speakers on this symposium, and we will here continue to 
center our interest in pure breeding techniques. 

In the treated Mi generation, the breeder has certain possibili- 
ties to influence the final yield of induced mutations. As in a hybrid 
bulk population, competition between genotypes will occur, but this 
competition will start already on the cellular level if plants or seeds 
are treated. Such an intrasomatic or diplontic selection is desirable 
as long as only deleterious mutations are screened away as in the very 
first cell divisions after treatment. Later on, the risk increases that 
cells carrying valuable mutations may also be suppressed and lost, 
and it is, therefore, important to overcome this phenomenon of 
intraplant selection. 

mac key: induced mutation in crop improvement 343 

In principle, this can be achieved in two different ways. One is to 
hinder or eliminate competition between primordia or shoots already 
differentiated before the mutagenic treatment. In radiation experi- 
ments with seed of cereals, Freisleben and Lein (24, 25) suggested and 
Gaul (28, 29) more definitely proved that the highest relative yield 
of mutation will be recorded if the Mi plants are permitted to develop 
a maximum of one tiller. In this way only one of the three to five 
differentiated ear primordia of the treated embryo can develop. The 
simplest method to suppress tillering is dense and late sowing, prefer- 
ably with an ordinary drill and with proper calculation with respect 
to lowered seed vitality. High temperature and light deficiency, as in 
ordinary greenhouse conditions, are also efficient. In vegetative!)' 
propagated plants, Bauer (5) showed that the detection of mutations 
greatly improved if bud competition was reduced. This can be 
done by cutting back the treated shoot successively or dividing it into 
small cuttings which are allowed to root and develop separately. 

The other method of overcoming the disadvantages of diplontic 
selection is to adjust the dose and its time of application. If only one 
initial is allowed to develop, a stronger dose will increase the fre- 
quency of mutated over normal meristematic cells. The relative rate 
of mutation will thus increase much longer with dose than if com- 
petition between affected and unaffected initials is allowed to counter- 
select (29). A treatment in a late ontogenetic phase of plant develop- 
ment will decrease both inter- and intrameristematic competition, 
since the floral shoots gradually lose their interdependence and the 
time for the diplontic selection to work, will be shortened. The most 
radical solution along this line will be to treat meiotic stages or even 
gametes and then, preferably, pollen (13, 21, 29). In connection with 
ionizing radiation and treatment with radiomimetic chemicals, such 
extreme efficiency may, however, at least in certain species, be coun- 
teracted by the exceptional response of very condensed chromosomes 
resulting nearly entirely in chromosome aberrations (98, 101, 102). 
The delay in treatment should, under such circumstances, be 
restricted preferably to the somatic stages of the plant ontogeny. 

As Mericle (unpublished) has shown, treatment in early embry- 
onal stages offers similar advantages without the undesirable effects 
of treating gametes. A higher mutation frequency and a higher ratio 
of mutant to normal offsprings can thus, in this way, be combined 
with a much lower demand on dose. 


A chronic or recurrent treatment will act similar to a delayed 
application, but offers, in addition, chance lor a continuous selection 
against very harmful mutations and a successive accumulation of 
more vital changes. Pursuant to this reasoning, Mikaelsen (77) found 
chronic radiation of barley definitely more effective than the com- 
parative, acute dose already given to the seed. By applying a recurrent 
treatment, gross deleterious effects may be eliminated before new 
valuable mutations are added step by step. In vegetatively propagated 
plants, where a shift in generation over a sexual phase may break 
down a complex but well-balanced, heterozygous genotype, such a 
recurrent treatment may have great possibilities even to inactivate a 
dominant gene in homozygous position. As will be discussed by 
Caldecott (11) in this symposium, recurrent treatment of successive 
generations of seed-propagated plants has special merit, since a muta- 
tion can here be removed from competition with the parental geno- 
type before new mutations are induced. 

In certain situations, imitation rate can be enhanced by proper 
selection of M : individuals. Selection can directly be made for domi- 
nant mutations or recessive changes in heterozygous position. Induced 
mottling or streaking of primary leaves or other signs of injury are 
generally correlated with mutagenic effect (55, 68, 117) and can 
preferably be used as a basis for selection when treatment is not 
uniform among individuals. Thus, Blixt, et al. (6) were able greatly 
to increase mutation rate by selecting mottled pea seedlings treated 
with ethyleneimine. A similar improvement is reported by Gregory 
(33) after selection for Xi seedling injury in peanuts. Since there does 
not appear to exist any correlation between chromosome and point 
mutations if competition between shoot initials is eliminated (27, 29), 
a shift to either category can be obtained by selecting semifertile or 
fertile Mi plants, respectively. 

Even with the best choice of parental material, the highest 
efficiency in mutagenic application, and the most appropriate han- 
dling of the treated generation, the vast majority of scorable muta- 
tions will be harmful or practically uninteresting. The economical 
success of mutation breeding will, therefore, depend greatly on the 
efficiency of selection in the segregating generations. It is also symp- 
tomatic that the most rapid and elegant completions in mutation 
breeding are generally directly associated with a specific screening 

mac key: induced mutation in CROT IMPROVEMENT 345 

technique by which a most rare mutant can easily be detected in a 
large population. 

Screening for disease resistance by means of artificial infection 
with known races (58, 59) or extracted toxin (116) allows enormous 
amounts of individuals to be handled. Rapid chemical selection 
methods like those elaborated for detection of sweet lupines (97) or 
coumarin-free sweet clover (88, 90) will also greatly improve efficiency 
in mutation breeding. The same is true for micro-methods for quality 
analyses of individual plants like the new Svalof method for oil 
determination which has radically changed the possibilities in breed- 
ing oil crops for higher yield of fat (82, 112, 113). 

Properties like frost, heat, drought, and sprouting resistance, 
where artificial selection methods are elaborated (57, 65), have prefer- 
ence in mutation breeding. An interesting work along these lines is 
reported by Zacharias (117). By simply germinating X_. material of 
soybeans at low temperatures, he was able to select mutants with 
the ability to develop and start growth earlier, which is of greatest 
importance for extending soybean cultivation to cool climates. In 
crops with distinct response to clay length or vernalization, mass 
screening may also be used at low cost. By spring sowing of X-material 
of Skandia III winter wheat, off-types were easily selected which were 
able to shoot and ripen before the end of the vegetation period (69). 
This technique is now used by Konzak (personal communication) in 
large-scale breeding projects for better spring wheats. 

In all situations where aim and detection technique are precise, 
selection can very well have set in already in M 2 , the first segregating 
generation. If the inventory is laborious or otherwise expensive, selec- 
tion in M 2 will even be preferable, since it will imply the highest 
chance to find the desired mutation among the smallest number of 
plants (103). If the screening is easily done and the treatment given 
to seeds or seedlings, it may, however, be advisable to select first in 
M 3 , where every mutation is no longer represented by single plants 
but rather by a whole group of plants. By testing only a part of each 
M 2 progeny, screening can also work efficiently even if the tested 
material has to be destroyed during the analysis. All M 2 lines proved 
to include the desired off-type can in this way be picked out and 
propagated. In most situations, where the desired mutations are 
difficult to detect due to imperfect selection methods or too vague a 


phenotypic penetration, or the aim of the imitation experiment is 
indeterminate, the advantage of screening; groups instead of single 
plants will often be decisive. For that reason, more and more muta- 
tion experts recommend selection first in M 3 (24, 25, 26, 29, 49, 50, 
103), a trend definitely stimulated by the promising experiments on 
induced mutations in quantitative traits. 

The rapid increase in size of population from Mi to Ma can 
partly be eliminated by proper sampling. Since mutation in a uniform 
parental material is a chance event but seeds traced back to one and 
the same germ line more or less interrelated, the sampling should 
be based on as many independent inflorescence units and as few seeds 
from each such unit as possible (24, 25, 56). The more seeds taken 
per independent unit, the lower relative frequency of mutant indi- 
viduals will appear in the next generation, but the higher will be 
the chance to detect all mutations induced in the given material. The 
last statement, however, has a limit set by the segregation ratio 
between normal and mutant types. In barley, for example, the aver- 
age chlorophyll mutant frequency per segregating Xj head progeny 
has been found to vary between 13 to 18 per cent, depending on 
tillering (28, 68). The corresponding figure for X 2 is about 20 per 
cent (78). Within the 99 per cent probability limit, therefore, not 
more than about 30 vital seeds per unit should be tested. The simple 
system of sampling by means of dense sowing under unfavourable 
conditions, resulting in few seeds per plant, can be used also in M_>, 
but this has to be weighed against the risk of interplant competition 
(107). Negative or positive mass selection in M- is another method to 
keep down size of population. Natural selection under more or less 
extreme conditions as to overwintering, disease infection, day length, 
vegetation period, etc., can also be of great help. Highly sterile plants 
may be discarded if only point mutations are desired. Selection on the 
basis of morphological properties may generally be effective for physi- 
ological features as well as due to the high chance for character 

Unless the goal of the breeding project is not very determinate, 
one of the greatest difficulties with practical mutation experiments is 
to decide which mutants should be selected and incorporated in 
future work. Induction of mutation as an independent method of 
plant breeding is only restricted to cases where the plant is vegetative- 

mac key: induced mutation in crop improvement 347 

ly propagated and where a imitation is able to change a highly valu- 
able autogamous genotype in a positive direction. In all other cases, 
the new mutant will be utilized through recombination, and com- 
bining ability will be about as difficult to see "from outside" as it is 
between entries in a world collection. This limitation of mutation 
breeding as an independent method is often overlooked, or perhaps 
it is better to say that there has so far been a tendency to restrict, 
mutation breeding to such specific uses where it is sufficient by itself. 
It is true that one of the great promises in mutation breeding is the 
possibility to add a single characteristic to a delicate system of genie 
balance in which recombination may cause a breakdown. It is, how- 
ever, just as true that a mutation will seldom be induced in its own 
optimal genie environment. The extensive work at Svalof on stiff- 
strawed crectoides mutants in barley may be a good example in this 

We now know that it was a fairly gross simplification, when 
Gustafsson and MacKey in 1948 (43) stated that "strength of straw 
can be produced at will" in barley radiation experiments. The prom- 
ised stiffness of straw was no mistake, but from hitherto 166 crectoides 
mutations analysed extremely few have given a direct practical result. 
Only one, the Pallas barley, has been released (7). The overwhelming 
majority was induced in inferior germ plasm or had pleiotropic 
by-effects which made them either lower in yield, more sensitive to 
drought, more specialized in soil and nutritional demands, more 
liable to stay in boot, etc. It is also important to observe that, the 
erectoides factor in Pallas shows a different pleiotropic pattern, both 
in relation to straw and head, if transmitted from the original Bonus 
genotype. Thus, it interferes generally not as Avell with the brittle 
straw of Carlsberg II but even better with the elastic straw of Rika 
(Hagberg, personal communication). 

The erectoides mutations, as well as other categories of induced 
mutations, have shown us that it is often much more efficient to 
transfer a successful mutation than to try to induce it anew in another 
genie background. It is further just as likely to make progress by- 
trying to transfer interesting mutations into new genotypes with the 
hope for improved combining ability than to start searching in new 
Mo material. The recent introduction of growth chambers to speed 
up the backcross technique has added further arguments along this 


line, since this method is especially suitable for the transfer of the 
distinct types of mutation here discussed. We need, however, a more 
detailed understanding about the combining ability of induced 
macromutations, since there are many apparent differences between 
the genetic behavior of natural and induced factors. Thus, we have 
produced a great many mutations resistant to diseases. Almost all of 
these mutations are recessive, while dominant mode of inheritance is 
more common among natural genes of the same kind. If Fisher (23) 
is right, this difference may depend on genie environment and 

The combining ability of a mutation has been proved to vary 
greatly with environment and background genotype (4, 10, 41, 42, 
105). Only one example, cited by Stubbe (106), will be given here. 
In the snapdragon, a mutation called erumosa was characterized by a 
nearly complete loss in the ability to branch and by a very erect 
growth, desirable properties for an ornamental plant. Unfortunately, 
however, less pleasant features were also associated with the eramosa 
mutation, viz., considerable inhibition and deformation of the flow- 
ers. By recombining the pathological mutant with other genotypes, it 
was possible to neutralize parts of the pleiotropic complex and to 
find a genie background where the desirable but not the undesirable 
characteristics of the eramosa mutation could be established (Vogel, 
unpublished). Induction of new neutralizing mutations can be con- 
sidered another variation on the same thema (34, 37, 44). 

In principle, there is no difference in the behavior of macro- and 
micromutations. They gradually pass into each other, and their 
effects may be cooperant or opposed. Quantitative variation can 
seldom definitely be proved to depend on multigenic differences of 
small magnitude only. The smaller phenotypic contribution of indi- 
vidual micromutations necessitates, however, recombination or recur- 
rent induction, since only their additive effects can be phenotypically 
scored. Recombination, which allows accumulation of valuable and 
omission of negative modifiers, occurs to great advantage automati- 
cally in allogamous plants. In autogamous plants, it must, however, 
be artificially stimulated if the evolutionary so important micro- 
mutations are to be fully utilized. For this reason and also for the 
chance to evaluate the induced macromutations in different oenic 
environments, mutation experiments with self-fertilizers should pref- 

mac key: induced mutation in crop improvement 3-19 

erably be combined with heterozygosity and /or outcrossing. Instead 
of carefully purified parental stocks and rigorous isolation in order to 
meet high demands on scientific exactness (12), the purely practical 
mutation breeder should stimulate outcrossing and recombination 
above what decreased fertility can give. It would thus be interesting 
to try artificial induction of mutations in connection with composite 
crosses or multi-cross bulks where male sterility was added to augment 
genie exchange. 

In evolution, the mutation processes are not only restricted to 
interior changes of the genes. Their deletion, duplication, and posi- 
tion, alone or in blocks, interfere with evolutionary fitness. The loss 
of genie material is definitely the most common type of chromosome 
mutation, and the rare evolutionary advantage of such a process is 
largely responsible for the discredit of this whole group of gross 
mutations. A loss may have a positive effect as a sequence of duplica- 
tion (70, 72, 94). Duplications are, however, definitely more interest- 
ing, since they may imply a cumulative effect and also a buffering 
which allows vital genes to mutate in directions that might have been 
impossible otherwise. In genetically well-studied objects like maize 
and barley, systematic production of duplications starts to become a 
reality (I, 31, 45, 46). 

In barley, the breeder is interested in duplicating a segment of 
chromosome 6 carrying the gene "orange lemma". This gene is strong- 
ly associated with high a-amylase activity and a duplication may thus 
offer a chance to improve malting properties. Another idea is to 
duplicate parts of the 40 centimorgans long segment of chromosome 5, 
where 13 of the 14 known loci for mildew resistance are incorporated 
(22). Such a procedure may augment the degree of resistance of some 
of the weaker genes, and it may offer a possibility to overcome difficul- 
ties with very close linkage. It would also allow more than one allele 
to be present simultaneously in the homozygous condition necessary 
for a stable barley variety. The last-mentioned possibility to fix a 
heterozygous condition would give a chance to utilize superdom- 
inance also in autogamous plants. Such a relation is found by Wiebe 
(cited by Hag-berg, 46) to exist between the alleles V for 2-row and v 
for 6-row barley. By a proper duplication in chromosome 2, a geno- 
type of the constitution VV ' , vv would allow the advantageous inter- 
action between V and v without the risk of segregation and 


The above-mentioned examples are taken from the work of my 
Svalof colleague Hagberg- (46). Since duplication followed by differ- 
entiation must be considered as one of the most constructive phases 
in evolution, works of this kind may soon drastically change our ideas 
of induced mutation in plant breeding. The principle of producing 
well-defined duplications will have to go via the induction of trans- 
locations involving the same two chromosomes but with different 
positions of the breakage points. The combination of two such trans- 
locations will imply a duplication of the segment between the breaks. 
For the proper use of this technique in plant breeding, a large set of 
well-defined translocation lines and a detailed gene map are essential. 

Induced chromosomal rearrangements may be valuable in many 
situations. They may be useful in splitting up gene blocks and char- 
acter associations which had an evolutionary advantage in the wild 
plant but are undesirable from the demand of man. In the opposite 
way, valuable gene blocks may be inverted to prevent crossing-over 
or moved inside localized chiasmata. A fascinating way of using 
radiation experiments for incorporation of small segments from non- 
pairing chromosomes is well demonstrated by Sears' (9.°>) already 
classical transfer of leaf-rust resistance from Aegilops umhellulata to 
common wheat. A similar approach is successfully accomplished by 
Elliott (19, 20) in transferring stem rust resistance from Agropyron 
elongatum to wheat, and the attempts to transfer bunt resistance 
from the same donor species are progressing (60, 63). Also, in Europe, 
this technique is now applied (unpublished by McKelvie, and 

The great merits of induced chromosome mutation are definitely 
in a well-defined chromosome engineering work. Repeatedly, how- 
ever, suggestions have been put forward to use recurrent radiation 
experiments for the diploidization of artificial polyploids, where 
differentiation of identical or too closely related chromosomes was 
thought to improve disomic pairing (26, 48, 71). No definite report of 
success along these lines is yet available, if we except the observation 
by Vettel (1 15) in Triticale that morphological X-mutants with long, 
dense heads had an improved seed setting. The Svalof work along this 
line, including 4x barley, flax, and rye, does not indicate any succes- 
sive improvement with recurrent radiation, but there is some hope 
that suddenly types with improved fertility may appear. In the light 

mac key: induced mutation in crop improvement 351 

of the recent finding of a genetic control of the disomic behavior of 
hexaploid wheat chromosomes (86, 87, 95, 96), it seems jnst as possible 
that one mutational step would be enough to improve fertility as a 
successive accumulation of many small changes of the chromosome 

All through the above presentation, the importance of an inter- 
fusion between mutation experiments and conventional breeding 
methods is stressed. Mutation is the first step in a creative process 
followed by efforts to bring it in progressive interaction with the back- 
ground genotype. This dependence on other breeding methods, how- 
ever, can also be reversed. Mutations may open possibilities to apply 
new approaches in breeding a specific crop. Thus, Lewis (6(5, 67) and 
Pandey (83) were able to increase the mutation rate of sell- 
incompatibility alleles in Oenothera organensis and Tri folium 
species, respectively, by the use of X-irradiation. Similar phenome- 
non can also be produced as primary and thus nonheri table effects of 
radiation (9). It may also be of interest to mention that gamete 
irradiation is a possibility to overcome certain interspecific barriers 
(14, 84, 85, 108, 109). Primary irradiation is also known to induce 
intersexuality in hemp (79). 

The ability of radiation to disrupt a biochemical sequence and 
to induce a heritable shift in the same metabolic pathway is also 
demonstrated by the interesting transfer from apomixis to sexuality in 
Poa pratensis (32, 53, 54) and Potentilla species (Asker, unpublished). 
Apomixis is evidently dependent on a rather complex gene balance 
where many different genetic events may interfere and induce a high- 
er or lower degree of sexuality. Due to the high tolerance to chromo- 
some disturbances in the multiploid Poa, even losses of whole chro- 
mosomes were ultimately found to result in sexuality. The artificial 
induction of such a shift enables the breeder to utilize very strictly 
apomictic clones as parental components in crosses or merely to 
explore the variability inherent to an extreme heterozygote. In both 
cases he may select further among more or less sexual segregates or 
isolate fixed, apomictic types, where the balance is restored (Figure 1). 

The above review is an attempt to understand mutation experi- 
ments and their possibilities in plant breeding. Just as the induced 
mutation can be evaluated only in relation to its background geno- 
type, mutation breeding can be evaluated only as an integral part 





morphologically and physiologically different 

Figure 1. — X-ray-induced shift to sexuality in Poa pratensis. After Grazi, 
el ft I. (32). 

in our efforts to improve our cultivated plants. An evolutionary sys- 
tem based on mere addition of mutations under different selective 
pressure would be a rigid and uneconomical mechanism of adjust- 
ment to environment. Mutation breeding must be considered as one 
method of approach, sometimes practically impossible, sometimes 
inferior, sometimes insufficient but definitely promoting, and some- 
times superior or the only solution. Its ability to furnish basic vari- 
ability should not be considered as a substitute, but rather as a 
complement to our world collections and other sources of natural 
variation (47). 

Our skepticism and hesitation today can partly be referred to 
the novelty of the method and to the feeling that nature should first 
be exhausted of its resources, but partly also to our incomplete genet- 
ic knowledge in relation to our specific breeding objects. The work 
on maize and barley shows doubtless that a more profound knowledge 
and a more systematic accumulation of gene and chromosome mark- 
ers will greatly improve our chance to use mutation experiments in 
plant breeding. In the collection of all these data and marker types, 

mac key: induced mutation in CROP IMPROVEMENT 353 

mutation induction will be one of our best helps. This indirect con- 
tribution of mutation genetics to crop improvement is a large and 
fascinating subject worth another story. It is evident, however, that 
we have to possess ourselves in patience before we will be able fully 
to understand the merits and drawbacks of induced mutation as a 
method of augmenting the food production of the world. 


Besides the metabolic staere at time of treatment, the mutagen 
and dose applied, and the method of handling before, during, and 
after treatment, the genotypic constitution of the material subjected 
to mutation experiments plays an important role. The total yield of 
mutations depends on inherent resistance to the mutagen applied, 
probably to degree of heterozygosity, to extent and direction of pheno- 
typic buffering, and to the presence of necessary precursor genes. 
From all these aspects, inter- as well as intraspecific differences occur, 
which greatly influence the yield of desirable mutations from case to 
case. Material especially suitable for the detection of desired 
mutations may improve the chance for success. 

Maximum yield of interesting mutations is also dependent on 
handling of Mj. A diplontic selection over the first cell divisions after 
seed or plant treatment should be prohibited by special arrangements. 
Different kinds of positive or negative mass selection may also increase 
the mutation rate in desired directions. 

Due to the very low frequency of valuable mutations, the success 
in mutation breeding depends largely on efficient screening. Selection 
in Ab is preferred for very easily detectable mutations. In other cases, 
selection in Mg may be better, since every mutation will here be 
represented by a whole group of off-type individuals. Proper sampling 
in Mo can keep down population size. 

Valuable mutations seem often so rare that it may be easier to 
transfer them to other genotypes than to try to induce them anew. 
Greater emphasis should be laid on micromutations. The combining 
ability of induced mutations varies drastically with background 

Amonsr chromosome mutations, translocations may be very use- 

o J J 

ful for a systematic production of duplications, for splitting up or 
building up gene blocks, and for interspecific gene transfers. Their 


vole in diploidizing artificial polyploids and inducing disomic pairing 
and better fertility is not yet well understood. 

Induced mutations may open possibilities to apply new breeding 
methods to specific crops. Induced self-compatibiiity in self-sterile 
plants and induced sexuality in apomictic plants are given as 

It is essential not to set mutation breeding in opposition to more 
conventional breeding methods but rather to evaluate this novelty as 
an integral and interdependent part. In this way, and with more 
complete knowledge in relation to the specific breeding objects, the 
value of induced mutation in crop improvement will certainly 


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some species of the section Alatae. Jap. Jour. Breed., 7: 39-44. 

110. Tedin, O., and Hagberg, A. 1952. Studies on X-ray-induced muta- 

tion in Lupinns Intens L. Hereditas, 38: 267-296. 

111. Tollenaar, D. 1938. Untersuchungen uber Mutation bei Tabak: II. 

Einige kiinstlich erzeugte Chromosom-Mutantcn. Genetica, 20: 
2 S 5-294. 


112. Troeng, S. 1955. Oil determination of oilseed: Gravimetric routine 

method. Jour. Amer. Oil Chem. Soc, 32: 124-126. 

113. . 1955. Oljehaltsbestamning i svenskt oljevaxtiro. Kungl. 

Lantbr. akad. tidskr., 94: 125-140. 

114. Tsunewaki, K., and Heyne, E. G. 1959. Radiological study of wheat 

monosomies. Genetics. 44: 933-954. 

115. Vettel, F. K. 1959. Mutationsversuche an Weizen-Roggenbastarden 

(Triticale): 1. Mutationsausldsung bei Triticale Rimpau. Zuchter, 
29: 293-317. 

116. Wheeler, H. E., and Luke, H. H. 1955. Mass screening for disease- 

resistant oats. Science, 122: 1229. 

117. Zacharias, M. 1956. Mutationsversuche an Kuliurpllan/en: VI. Rout- 

genbestrahlungen der Sojabohne (Glycine soja (L.) Sieb. et Zucc). 
Zuchter, 26: 321-338. 

Bkewraker: May 1 comment first that there are two misconceptions 
that appear in the discussions of mutations involving incompatibility 
alleles. First, that a curious sort of revertible mutation can occur in 
which the gene mutates to an inactive form to permit fertilization and 
then reverts in the X x embryo to its original form. The evidence sup- 
porting this double mutation event is extremely tenuous, and gains no 
support in our Petunia work. Second, there is no evidence whatsoever 
of "point mutations" resulting in the formation of a new S allele (our 
studies alone exceeding 30 million pollen grains) and reports of mutation 
by inactivation of subunits in the S gene (as in the cherries) can be 
suggested to result from chromosomal aberrations, such as transloca- 
tion or centric fragments. 

Now to my question — having screened a great number of such self- 
fertile mutants in Petunia, in which S locus duplication is an elegant 
theoretical way to produce "mutants", we have no evidence at all for 
such duplications, although irradiations have been applied premeio- 
tically. Unless one has a particularly effective screen for duplications, 
how optimistic do you feel we can be regarding such duplications which 
might be, as you suggest, of considerable value in mutation breeding? 

MacKkv: 1 share your skepticism on the correct interpretation 
of the mechanism behind the induced compatibility in some cases. This 
will, however, not interfere with the fact that the plant breeder can 
make use of this shift in his breeding procedure, even if a chromosome 
mutation may be a little more complicated to work with than a point 


As to your second problem, the possibility is certainly extremely remote 
of being able to induce a very specific type of duplication directly by 
prophase irradiation. Thus, the success will depend more or less entirely 
on the efficiency of the available screening technique. For thai reason, 
the method must have a limited, practical value. However, this does not 
necessarily mean that it must always be preferable to work via trans- 
location lines. I would definitely prefer more data to be compiled before 
any clear answer can be given to your question. 

Caldecott: The need for attention being directed to the production 
of duplications is most important. Today we are relying almost entirely 
on translocation between the same arms of homologous chromosomes 
that were broken at dissimilar points. 

What we need is a good method of obtaining cells in which the chro- 
mosomes are uniformly in the bipartite condition before irradiation. 
This would enchance sister strand reunions and in instances where the 
breaks were at dissimilar points should prove very effective. 

Dr. B. H. Heard is studying the problem using the 2— row vs. G-row 
gene in barley. His procedure is to irradiate seeds in which the genes 
are in the hetero/ygous state and screen in the X ;! for "nonsegregating 
permanent heterozygotes". 

Auerbach: The two-step process of the production of duplications, 
which Doctor Caldecott outlined for X-rays, occurs normally at a high 
frequency after treatment with chemical mutagens whose breakage 
often is delayed until after the treated chromosome has divided into 
chromatids. Ford found many small duplications after treatment of 
Vicia with nitrogen mustard, and Slizynska found many after formalde- 
hyde treatment on Drosophila. 

Lewis: In cotton many introductions cannot be evaluated in the lati- 
tude of the United States because of a short-day photoperiod require- 
ment. Do you think irradiation to break this reaction might be feasible? 

MagKey: The answer to this question greatly depends on the genetic 
complexity of the photoperiodic response. A complex genetic background 
greatly impairs the chance to break the short-day reaction by induced 
mutation, while a simple genetic control woidd greatly favor a pro- 
gram along this line. A shift in photoperiodic response can easily be 
detected in a huge material and a simple mutation is likely sometimes 
to hit just right. The chance that many mutational events should happen 
simultaneously according to a fixed pattern is, however, practically nil. 

mac key: induced mutation in crop improvement 363 

Judging from other crops, the genetic control of photoperiodism is 
likely to be rather simple. Thus, it would seem worth trying this experi- 
ment in cotton. 

Patterson: The pleiotropic effects of the crcctoides mutations on straw 
strength are of special value for use in cross breeding for straw strength 
in barley. Selection for the crcctoid type results in selection for straw 
strength, a character usually difficult to obtain. 

MacKey: 1 think that the Swedish barley breeding along conventional 
lines, with results like Rika, Herta, and Ingrid, proves that straw strength 
can be achieved also in other ways than through inducing crcctoides 
mutations. The important thing is that the two ways are different with 
possibilities for transgression when combined. 

Konzak: Since terminology in this new field has an important bearing 
on the understanding of methods and treatments, I wish to recom- 
mend that we utilize a standard designation system for describing the 
generation following mutagen treatment as the M l5 M 2 , etc., designa- 
tion system. In addition, f would suggest that we consider an idea 
expressed to me by Doctor Wellensiek recently — this is to use a super- 
script to denote the mutagen and the treatment used, as Mj EMS 4hr02 
for the first generation following ethyl methane sulfonate treatment 
or to designate the first generation from a 15 Kr gamma radiation treat- 
ment as Mj ir * Kr G . 

In a case where repeated treatments are studied, the designation might 
be modified thus: 

M x i_- first generation, x = kind of treatment and dose, if desired 

second cycle M = mutagen treatment 

M\>_ 2 second generation, 1 = 1st generation 

second cycle 2 = 2nd generation treatment 

MacKey: I think it is highly desirable to agree upon a standard desig- 
nation system, but the abbreviations should not be so exclusively used 
that people outside our group will not be able to understand our pub- 

Konzak: It should be recorded here for the benefit of those interested 
that we put into use two additional microanalyses techniques in our 
mutation and plant breeding programs with wheat. 

One of these involves a rapid microtest for protein analyses. The 
equipment used is an Udy protein analyzer which, using a dye bind- 


ing method, can give protein analyses with accuracy comparable to or 
better than, Kjeldhals in about %i/ 2 to 5 minutes time per sample. We 
now consider it more accurate than the Kjeldahls because it measures 
oid) amino nitrogen and does not measure nitrate. This is important, 
especially for the analysis of plots fertilized with nitrogen. This machine 
was developed at the Regional Wheat Quality Laboratory at Pullman, 
Washington, and is now being manufactured and further developed 
by Dr. Doyle C. Udy, its inventor at Pullman. 

Using this equipment, we have sorted out induced variation in wheat 
and will be able to evaluate material on a large scale. 

Another machine for rapid analyses also was developed in the same 
laboratory. This machine, the Micromill, permits isolation of better 
milling selections using visual identification of selections according to 
bran cleanup. Two operators can stud)' about 1,000 samples per clay. In 
breeding, we use 1.5 to 5 grams of seed, but we hope that we can identify 
lines carrying promising material by recognizing one or few clean nearly 
endosperm-free bran (lakes among a bulk sample from an M 2 progeny. 

Olmo: In retrospect, would it have been more economical to produce 
a Pallas-type barley by conventional breeding methods than by the radia- 
tion breeding technique? 

MacKey: A strict answer to your question cannot be given, since the 
two kinds of approaches have not been tried. I have the feeling that 
erect oides types became of interest to the barley breeders first when 
induced in a highbred genie environment. As with many other things 
hidden in our world collections, they did not attract the breeders when 
combined with otherwise undesirable genes and this the more so when 
their mode of inheritance was less well explored. Now that the informa- 
tion is available, transfers by backcross are likely to be more effic ient than 
trying to induce just the right type of ereetoides characters anew in 
another variety. 

Factors Modifying the Radio-Sensitivity of Seeds 

and the Theoretical Significance of the Acute 

Irradiation of Successive Generations 1 


University of Minnesota, St. Paul, Minn. 

The purpose of this report is twofold, namely, to present data 
which demonstrate the use of dormant seeds in biophysical 
studies and to discuss the possible genetic and applied significance of 
the irradiation of successive seed generations of the small-drained 

Part I. Modification of the Radiosensitivity of Seeds 

For years it has been obvious to investigators working with seeds 
that these biological structures apparently represent an unique system 
in that they can be subjected to extremes of environment without 
impairment of function when restored to the conditions required for 
normal growth and development. Using seeds, it has been possible 
to study the biological consequences of treatments that were other- 
wise only possible in vitro. This was perhaps best shown in studies 
where, by decreasing the water content well below levels at which 
physiological activity was possible, seeds tolerated temperature 
extremes ranging from those of liquid nitrogen to 112° C. 

It also was shown that under conditions where physiological 
activity was not detectable, the expression of damage to X-rayed seeds 
could progressively increase for weeks after initial photon absorption 
(1,2, 5, 12). 3 Furthermore, the rate and degree of this injury enhance- 

^This work was conducted under Contract No. AT (1 1—1 )— 332 between the University 
of Minnesota and the U. S. Atomic Energy Commission. The report is a compilation o£ 
two reports previously presented at symposia sponsored by the IAEA and FAO in 
Karlsruhe, Germany, August, 1960, and the AAAS in Chicago, 111., in December, 1959. 
Contribution from the U. S. Department of Agriculture, Field Corps Research Branch, 

2 The writers are pleased to acknowledge the help of Dr. Alessandro Bozzini on the 
studies relating to seedling height and genetic injury, Miss Victoria L. Bergbush on all 
phases of the temperature investigations, and Miss Louise Heine and Mr. Fa-ten Kao 
for assistance with the cytological investigations. 

3 See References, page 398. 



ment was shown to be dependent on temperature. Thus, the post- 
irradiation increase in injury to seeds was negligible over a period of 
96 hours at the temperature of solid carbon dioxide. 

Studies complementary to those on the relation of time and tem- 
perature to the manifestation of post-irradiation injury have shown 
that the availability of oxygen to the seed following X-irradiation 
also has a profound effect on the expression of injury. Thus, seeds that 
are stored or hydrated in the presence of oxygen immediately after 
X-irradiation are more severely injured than are seeds that are stored 
or hydrated anaerobically (5, 10). Significantly, sensitivity to oxygen 
following irradiation diminishes as a function of time and can be 
completely eliminated by a brief post-irradiation temperature treat- 
ment administered at 75° C. 

In a recent series of comprehensive and refined experiments, 
using bacterial spores which will tolerate desiccation to levels com- 
parable to those commonly used in the seed work, Powers, et al. (21) 
have corroborated and expanded upon much of the work that has 
been done with seeds. This parallel response, at least in the more basic 
considerations, of a multicellular and a unicellular organism is a 
fortunate circumstance in that it gives credence to the wealth of data 
accumulated in biophysical studies with seeds that has often been 
considered exceptional and has, accordingly, too often been ignored. 
Furthermore, analyses of these two organisms supplement one 
another. The bacterial spore techniques are amenable to rapid 
analysis of large populations to determine the relative lethality of 
specific treatments; whereas the seed material is ideal for the most 
refined cytogenetic and genetic studies, which have resulted in pin- 
pointing the lethal consequences of the treatments as originating in 
the chromosome. 

The work with seeds was given further significance in an origi- 
nal experiment by Zimmer, et al. (31) in which it was shown that the 
paremagnetic resonance spectra obtained in X-rayed barley seeds 
was similar to that obtained from free radicals. Equally relevant was 
the demonstration that irradiation in air gave rise to more magnetic 
centers than irradiation in the presence of nitrogen. Later studies 
have confirmed and extended this work (11, 13). 

It is of interest that much higher dosages were necessary to obtain 
clear electron spin resonance signals (ESR) than were necessary to 


demonstrate the storage phenomenon and other after-effects of irradi- 
ation with seeds. If the ESR methods enable the detection of the 
same kinds of events that initiate detectable biological injury, it 
seems apparent that the biological system has a greater resolving 
power than the physical system by several orders of magnitude. In 
this connection, because the production of some reactive entities may 
be dose-dependent, there is a distinct need for the physicist and the 
biologist to maintain the closest liaison in all of their work on pre- 
and post-irradiation effects. Only in this way can it be certain that 
the biological significance of a particular event observed with elec- 
tron spin resonance methods will be placed in proper perspective. 

Material and Methods 

In the entire series of experiments reported, dormant seeds of 
Himalaya barley were used as the test material. In all instances they 
were of uniform size from the same harvest year and had an embryo 
water content of about 4 per cent before being used experimentally. 

The X-rays used in the studies were unfiltered and were gen- 
erated with a constant potential machine operated at 100 KV and 7 
ma. For irradiation, the seeds were placed on a turntable 9.7 inches 
from the target, with the embryo oriented toward the beam. Under 
these conditions the seeds received a dose of approximately 3,000 r 
per minute. 

In different experiments the seeds were subjected to a variety of 
pre- and post-irradiation treatments before germination. To elimi- 
nate confusion, these particular conditions are indicated in the experi- 
mental results. However, in all experiments, hydration immediately 
before the initiation of germination was accomplished by steeping 
the seeds for 45 minutes in boiled distilled water through which 
either oxygen or nitrogen was continuously flushed. This was done 
to eliminate a variable that had previously been demonstrated to be 
of profound significance (5, 10). Furthermore, on all occasions where 
seedling height determinations were made, the seeds were grown in 
petri dishes in a controlled environment room. Measurements were 
made to the nearest millimeter after either 6 or 7 days of growth, 
depending on the objectives of the particular experiment. 

To determine the frequency of interchanges in the irradiated 
generation, cytological analyses were made at the first meiotic meta- 



phase of Xi plants that were grown to maturity (7). This laborious 
procedure was used because it avoided the controversy about the 
accuracy of using root tip cells for determining interchange frequen- 
cies (4, 30). Seeding chlorophyll mutation data were obtained on Xo 
populations using the method of Stadler (27). 

Experimental Results 
Relation of seedling height to genetic injury 

After treatment with 5,000 r of X-rays, a large sample of seeds 
was divided into four lots. Two lots were immediately hydrated for 
45 minutes, one in the presence of oxygen and the other in the pres- 
ence of nitrogen. The remaining two lots were stored over phosphorus 
pentoxide for 8 days at room temperature. After this period one lot 
was hydrated aerobically and the other lot anaerobically. 



£ 50 



i: 30- 




2 20- 


Hydroted in presence of / | 




oxygen ( — ) / 1 



nitrogen ( — ) / ' 







/ / 
/ / 

/ / 


/ / 


/ / 



/ / 
/ / 


/ / 

k \ 

/ / 

\ \ 

y / 

\ \ 

^*^^ ^ 
^^*-*~^ *" 

\ \ 

— t — i-—-t i i i i I 


i \ i\ i l 

5,000 r 

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 

Seedling Height-cm. 

1. — Distribution of seedling lieights at 7 days from seeds given 
of X-rays and tJien immediately hydrated in the presence of either 
or nitrogen. 



Immediately after hydration the seeds were plated out in petri 
dishes and grown for 7 days. At that time individual seedling heights 
were determined (compare the frequency distributions in Figures 1 
and 2) and the seedlings were placed in one of three height classes, 
viz., to 5 cm, 5.1 to 9 cm, and taller than 9.1 cm. The seedlings in 
each height class were than transplanted and grown to maturity to 
obtain interchange and mutation data (Figures 3 and 4, Table 1). 

350 r 

300 - 

250 - 

^200 - 




Hydroted in presence of 

oxygen ( ) 

nitrogen ( — ) 

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 

Seedling Height -cm. 

Figure 2. — Distribution of seedling heights at 7 days from seeds given 
5,000 r of X-rays and then stored for 8 days before hydration in tlie 
presence of either oxygen or nitrogen. 

It is significant that for those seeds hydrated immediately after 
X-irradiation (Table 1) the data are not presented in terms of genetic 
damage in different height classes but rather for the population as a 
whole. The reason for this is that the distribution of seedling heights 



Table 1.— Height of Seedlings at 7 days After 5,000 r Irradiation in Relation to 
Survival, Interchange Frequency, and Mutation Frequency. * 









x 2 













ity, analyzed, 










Hydrated Immediately After 


o 2 







1 92 









Hydrated 8 Days 

After Ir 































N 2 





























*Nonirradiated control populations evidenced no interchanges in 383 Xi spikes and an X? 
chlorophyll mutation frequency of 0.001 mutant seedling per 100 X2 plants. 

about the mean was close to normal (Figure 1). Accordingly, too few 
seedlings fell in either the tall or short height classes to gjive a reliable 
estimate of the injury in those classes in comparison with those which 
fell within plus or minus one standard deviation of the mean. 

From the data presented it is apparent that there is a good 
correlation between the extent of injury, as measured by seedling 
height, and the degree of genetic injury as measured both in terms 
of mutations and interchanges. It is also apparent (Table 1) that 
populations of individuals can be selected from seeds that have been 
X-rayed and stored which evidence no increase in genetic injury 
resulting: from storage. 

Relation of oxygen to manifestation of genetic injury 

The demonstration that post-irradiation hydration in the pres- 
ence of oxygen resulted in greater seedling injury to dormant seeds 
than post-irradiation hydration in the presence of nitrogen (5, 10) 
necessitated a critical analysis of the relation between dose of X-rays 











\ ^r~ 

- Seedling Height Distribution 


- V 


Hydrated in presence of 


~ 200 





- / 

i i 


oxygen ( — ) 
nitrogen ( — ) 

o 150 


- [ 

1 1 
1 ' 



I ' 

\ /^ 

2 100 

1 1 
1 1 
1 i 


I I 
I i 
i i 
1 1 


1 1 
1 1 
1 1 
1 1 
1 1 . 

1 . \ \ 

- 40 


- 30 

20 «u 

- 10 

5 9 14 

Seedling Height-cm. 
Figure 3. — Relation of seedling height at 7 days to interchange frequency 
at microsporogenesis. (Plants grown from dormant barley seeds subjected 
to 5,000 r of X-rays and stored for S days before hydration in the presence 
of either oxygen or nitrogen.) 

and gnenetic injury under the two conditions of hydration. In this 
connection, a study was set up wherein seeds were subjected to one 
of a wide range of doses of X-rays and then immediately hydrated, 
either aerobically or anaerobically, and planted in the field. Immature 
inflorescences were collected from the X x plants and microsporocytes 
were examined to determine the frequency of interchanges induced 
in the material (Figure 5). At maturity up to five heads were removed 
from each plant and the seeds grown to determine the X 2 chlorophyll 
mutation frequency (Figure 6). 

These genetic data demonstrate that when seeds are hydrated 
aerobically after X-irradiation there is an exponential relation 






•p 200- 




100 - 

5 9 

Seedling Height -cm. 

Figure 4. — Relation of X t seedling height at 7 days to X, seedling muta- 
tion frequency. (X x plants grown from dormant barley seeds subjected to 
5,000 r of X-rays and stored for S days before hydration in the presence 
of either oxygen or nitrogen.) 

between dose and interchange frequency, but that when they are 
hydrated anaerobically the relation is linear. Furthermore, they show 
that the mutation frequency is linear under both conditions of 

The data suggest a relation between ion density and the role of 
oxygen in the production of both one- and two-hit events that will be 
presented in the discussion. Also, they indicate the likely reason for 
the conflicting views that have been expressed by different cytogenet- 
icists relevant to the nature of the regression of interchanges on dose 
(4, 30). 



Y» 1.9444 X 



DOSE OF X-RAYS (10 r units) 

Figure 5. — Relation of interchange frequency to close when seeds are 
hydrateel in the presence of eitlier oxygen or nitrogen immediately follow- 
ing X-irradiation. 

g 3.0 




1 1 1 
/ /- 

Y = 


* / / - 


/ / 

W 2 5 

/ "/ 



/ / 



^ Y ' 0. 158 X - 




b b> 








x _ 










/ o 




1 1 1 

5 10 

DOSE OF X RAYS (10* r units) 



Figure 6. — Relation of imitation frequency to dose when seeds are 
hydrateel in the presence of either oxygen or nitrogen immediately follow- 
ing X-irradiation. 


Relation of pre- and post-irradiation temperature to manifestation 
of injury 

Pre-irradiation temperature and the oxygen effect. — In previous 
investigations, prior to the demonstration that, following irradiation, 
seeds were injured by aerobic hydration and protected by anaerobic 
hydration, it was shown that protection from X-rays was also afforded 
to barley seeds when they were treated with a barely sub-lethal tem- 
perature immediately before irradiation (6, 24, 26). Present studies 
have proved that a pre-irradiation heat treatment of 75° C or 85° C 
gives effective protection when the treatment is for a duration of 24 
hours. Furthermore, the protective effect is not necessarily associated 
with a detectable water loss and cannot be attributed to this factor. 
It has also been demonstrated that two months can elapse between 
the temperature treatment and irradiation without loss of protection, 
provided the seeds are not hydrated in the interim. 

Because protection from X-irradiation was obtained by both pre- 
irradiation temperature treatment and by anaerobic post-irradiation 
hydration, it was deemed essential to determine whether or not the 
two types of protection were complementary. To determine this point 
seeds that had been heated for 24 hours at a temperature of 75° C and 
then X-rayed with a wide range of doses were immediately hydrated, 
either aerobically or anaerobically, and compared with seeds that 
were not subjected to the temperature treatment but otherwise 
handled in an identical manner (Figure 7). 

These data show that the protective effect of the heat treatment 
was obtained under both conditions of hydration. Because other data 
(Figure 3) suggest a casual relation between genetic damage and 
seedling injury, and because it has been demonstrated that a pre- 
irradiation treatment with heat reduces the frequency of interchanges 
in X-irradiated seed (26), it is apparent that the protective effect 
afforded by post-irradiation hydration in the presence of nitrogen and 
pre-irradiation treatment with heat must be complementary; in the 
sense, that is, that they are both influencing the manifestation of the 
same kinds of genetic injury. 

Pre-irradiation temperature and storage effect. — As previously 
indicated (5, 12), earlier work has shown that the injury to barley 
seeds from a given dose of X-rays can be enhanced by post-irradiation 
storage. In addition, data have been presented which show that a pre- 



25 35 45 55 

DOSE OF RAYS (r Units) x I0 3 

Figure 7. — Protection jrom X-irradiation by pre-heat treatment. 

irradiation heat treatment results in a reduction in the radiosensitiv- 
ity of seeds. The logical next step was to determine whether or not 
pre-irradiation heat had any influence on post-irradiation sensitivity 
to storage. To determine this the following study was conducted. 
Seeds were subjected to a temperature of 75° C for 24 hours and then 
irradiated with a dose of 15,000 r. After irradiation, different segments 
of the population were stored at room temperature for periods rang- 
ing from to 48 hours before being hydrated either aerobically or 
anaerobically. Six days after hydration seedling height data were 
obtained and compared with data obtained from populations treated 
in precisely the same way except that they were not subjected to the 
pre-irradiation temperature treatment. The data presented compare 
only the two extremes of the post-irradiation storage period, and 
48 hours, because they illustrate the points of concern (Figures 
8,9, 10). 

The following conclusions can be drawn from the data in this 
experiment. First, that under conditions of either aerobic hydration 





co 20 


10 - 

10 ■ 

1 5,000r + aerobic hydration 
x = 4.5 cm. 




Heat + I5,000r ♦ aerobic hydration 
k = 7.6 cm. 





I 5,000 r + 48 hrs. storage 
+ aerobic hydration 
x = 5.3 cm. 


Heat + I5,000r + 48 hrs. storage 
+ aerobic hydration 
x = 8.5 cm. 




Figure 8 

injury to 

I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 

— The effect of a 75° C pre-irradialion heat treatment on the 
seeds that are hydrated aerobically before and after storage. 

(Figure 8) or anaerobic hydration (Figure 9) the distribution of seed- 
ling heights was more nearly normal in the populations that received 
heat treatment than in the population that did not. Furthermore, the 
most skewed distribution occurred in the populations that were not 







£ 30 


i 20 



I5,000r + anaerobic hydration 
x = 9.6 cm. 

fin,-, ^n^„„r.n„nnr.n 



Heat + I5,000r + anaerobic hydration 
x = 1 1.4 cm. 


n n nri 


I5,000r + 48 hrs. storoge 

+ anaerobic hydration 

x = 6.2 cm. 

II.-, n n n 

.a nnfinn 


n rt i-i 

Heat+I5,000r +48 hrs. storoge 

+ anaerobic hydration 

x = 11.0 cm. 


n nn 


I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 

Figure 9. — The effect of a 75°C prc-irradiatiou heat treatment on the 
injury to seeds tliat are hydrated anaerobically before and after storage. 

heated but in which germination was delayed for 48 hours (Figures 
8 and 9, part C). Second, pre-irradiation heat treatment completely 
eliminated the increase in injury that usually occurs when seeds are 
stored after irradiation (Figures 8 and 9, compare parts A and C with 






v> 10 



fj Heot * I5,000r + oerobic hydrotion (x = 7. 6 cm.) 
Heot * I5,000r ♦ onoerobic hydrotion (x = 11.4 cm.) 




n. 5 J 

Kl Ri ra 


c 25- 


2 20 J 




Q Heot + I5,000r t 48hrs. storage * aerobic hydrotion (x=8.5cm.) 

Heot + I5,000r + 48 hrs. storage + onoerobic hydrotion (x = 1 1.0 cm.) 

^ q EL 







6 7 8 9 10 


12 13 

14 15 

Figure 10. — Elimination of sensitivity to storage but not to oxygen result- 
ing from a prc-irradiation temperature treatment at 75°C. 

parts B and D). Third, while pre-irradiation heat treatment elimi- 
nated storage injury, it did not modify the sensitivity of the seeds to 
aerobic hydration, even when they were stored for 48 hours before the 
initiation of hydration (Figure 10). This particular observation is 
considered to be of especial significance and will be dealt with in the 

Post-irradiation temperature extreme and oxygen and storage 
effects. — The demonstration that the injury to seeds could be 
increased by storing them at room temperature after X-irradiation 
emphasized the need to determine to what extent the injury progres- 
sion, as a function of time, was temperature dependent. To elucidate 
this problem, an extensive two phase study was set up. In the first 
phase seeds were irradiated and then immediately stored at one of 
three temperatures (—78° C, 20° C, and 85° C) for different periods of 
time before they were hydrated either aerobically or anaerobically 
(Figure 1 1A). In the second phase, after X-irradiation, the seeds were 



stored for 120 hours at —78° C. They were then removed from this 
temperature and subjected to additional storage periods, at either 
20° C or 85° C, before being hydrated (Figure 1 IB). 

O 40 

Stared immediotely offer 7500r of x-roys ot either 20*C. 
or 85°C before hydrotion 

'85*C. •» oerobic hydrotion 

;20*C. * onoerobic hydrotion 

T20*C. ♦ 

oerobic hydrotion 

Stored 120 hrs. ot -78*C. immediotely offer 7500r of x-roy», 
followed by storoge ot either 20*C. or 85*C before hydrotion 

-85*C. ♦ onoerobic hydrotion 

20*C. ♦ onoerobic hydrotion 

20'C. ♦ oerobic hydration 

24 30 


Figure 11. — The effect of post-irradiation temperature on X-ray -induced 
storage injury and tJie suppression of injury progression by loiu tempera- 

These data clearly show that, for at least the first few hours, 
at both 20° C and 85° C injury increases progressively as a function 
of time stored, with the most rapid increase occurring at the highest 
temperature. Furthermore, they show that sensitivity to hydration 
in the presence of oxygen is eliminated first by the highest temper- 
ature. In addition, they demonstrate that after about 48 hours storage 
at 85° C there is a partial recovery from injury. Essentially the same 
result was obtained when seeds were stored at 75° C for longer periods 
of time (Figure 12). 

The data also demonstrate (16) that storing seeds at —78° C for 
120 hours essentially prevents the progression of post-irradiation 



Stored immediately after 3000r of x-rayt 

ot either -78*C, 20*C. or 75*C. before onoerobic hydrotion 


Figure 12. — Initial thermal enhancement of and ultimate thermal re- 
covery from X-ray -induced storage injury. 

injury. Apparently the radiation-induced damage is maintained in 
relatively labile state by the low temperature because, on removal 
from this temperature to temperatures of either 20° C or 85° C, the 
progression of injury, as a function of time, is similar to that obtained 
when seeds are stored at these temperatures immediately after 
irradiation (Figure 11A and B). 


Seedling injury (growth retardation during a finite period) fol- 
lowing germination of irradiated seeds has been used commonly as 
an indication of the genetic injury that has been induced in the cells 
of the meristem. The validity of using this estimate is reasonable on 
a priori grounds. In addition, it has been repeatedly demonstrated 
that there is a good correlation between seedling injury and genetic 
injury over a wide range of doses with X-rays and other irradiations. 
Despite this, the problem of the relation between seedling height 
and genetic injury has long been of concern because the distribution 
of seedling heights about the mean was often more skewed in X-rayed 
material than in neutron-irradiated seed (9). The problem took on 
added significance when studies were undertaken on the effects of 
post-irradiation storage on seedling injury because the variability in 
seedling heights in the stored populations became extreme; actually, 
it often approached the bi modal. 

Clearly, there was a need to determine if the suspected relation- 


ship between seedling height and genetic injury held true when seed 
lings in populations of X-rayed seed, which had been subjected to 
post-irradiation storage, were subdivided into different height classes 
and analzed for frequencies of interchanges and mutations. Such a 
study was conducted and the data presented show beyond any reason- 
able doubt that there is a good correlation between seedling injury 
and genetic injury (Figures 3 and 4, Table f). Indeed, they warrant 
the conclusion that the relationship is one of cause and effect. 

At the present time there is no obvious explanation as to why 
seeds in a population undergo different degrees of storage injury 
(Figure 2), although it seems likely that it is associated with either 
an environmental variable that has not been elucidated, to perme- 
ability of the cell, or to a different physical state of radiosensitive sites 
within the nuclei of different seeds. These possibilities will be 
explored later. 

Basing his arguments on the first root tip cell divisions in the 
germinating seed, Wolff (29) came to the opposite conclusion to that 
presented here, viz., that changes in seedling height during storage 
were not correlated with radiation-induced chromosomal damage. He 
suggested that, "the pattern of damage can be correlated to the 
behavior under similar regimes of long-lived radiation-induced radi- 
cals". Presumably the injury resulting from such radicals was con- 
sidered to be nonoenetic in nature because it did not become mani- 
fest in the form of chromosome breaks. It is unlikely that the differ- 
ence between Wolff's results and those presented here is due to the 
method of analyzing for genetic injury, because root-tip studies similar 
to Wolff's that have been conducted in this laboratory confirm the 
interchange and mutation data that are presented. There must be 
some other explanation for the difference and this problem should 
be re-examined. 

Early work demonstrated that the relation between interchange 
frequency and dose was exponential when Tradescantia microspores 
were subjected to X-irradiation (22) and linear when they were bom- 
barded with neutrons (16). From these data it was concluded that, 
when biological material is treated with neutrons, the dense clusters 
of ionization that occur usually break two chromosomes simultane- 
ously which then have a good probability of exchange; thus, the one 
to one relation between interchange frequency and dose. In contrast 


with this, it was postulated that the free electrons resulting from 
absorption of X-rays, because they can produce a range of ion densi- 
ties within a sensitive volume, often only break one chromosome at a 
time. Accordingly, the probability of having two breaks simultane- 
ously present for exchange would increase at a greater rate than the 
first power of the dose, with the result that the dose interchange 
relationship would be exponential. It has since been demonstrated 
(8) that, with barley seeds, the relation between dose and interchange 
frequency is linear when the seeds are subjected to both X-rays and 
thermal neutrons. It is now known that the linear relation obtained 
with X-rays only occurs in seeds with a moisture content above 10 
per cent at the time of irradiation or in seeds with water contents 
below that level when they are hydrated anaerobically immediately 
after irradiation (see following discussion). From these earlier results 
(8) it was concluded that only the densely ionizing tails of the X-ray- 
induced free electrons cause immediate chromosome breakage and 
that then two chromosomes are usually broken "simultaneously". 

This conclusion was disputed by Wolff and Luippold (30) on 
the grounds that the scoring of dicentric bridges in root-tip cells was 
a poor means of determining interchange frequencies. They pointed 
out that in their analyses there was actually an exponential relation 
between interchange frequency and dose when decentrics were scored 
at metaphase and a linear relation when they were scored at ana- 
phase. This particular consideration ignored the fact that the inter- 
change frequency as detected at the first metaphase division of micro- 
sporogenesis was also linear (8). A more pertinent and convincing 
argument should have included data on neutron bombardment, the 
water content of the seeds at the time of irradiation, the conditions 
under which the seeds were hydrated after irradiation, and the 
frequencies of interchanges at microsporogenesis. 

What appears to be a more likely answer to the problem is that 
the two workers were probably using test materials that had non- 
uniform water contents and, therefore, differential sensitivities to 
post-irradiation modification. To develop the significance of this 
consideration, it will be necessary to diverge briefly to examine one 
possible explanation as to why post-irradiation effects were observed 
with X-rays and not with neutrons (8, 12). 

It has been postulated that post-irradiation sensitivity to oxygen 


might depend upon the distribution of energy within critical sites. 
As stated earlier, with X-rays there would be some sensitive volumes 
that were sparsely ionized and some that were densely ionized. If 
post-irradiation oxygen treatment influenced only sites that received 
one or a few ionizations, effecting a single chromosome break, the 
frequency of such sites, and their corresponding breaks, would 
increase linearly with the dose while the frequencies of interchanges 
between such breaks would increase at a power greater than unity. 

To test this consideration seeds were treated with one of a wide 
range of doses of X-rays and then immediately hydrated either aerobi- 
cally or anaerobically. The seeds were then permitted to develop into 
mature plants and the frequency of interchanges was determined at 

The data obtained (Figure 5) are in agreement with the hypothe- 
sis presented, for the frequency of interchanges in this material 
increased exponentially when the seeds were hydrated in the presence 
of oxygen and linearly when they were hydrated anaerobically. 

These data have greater significance than merely in apparently 
resolving the confusion that has heretofore existed concerning the 
interchange-dose relationship. They suggest that many radiation- 
induced breaks are in fact "potential" breaks or "lesions", that either 
become manifest as actual breaks or are eliminated, depending upon 
the presence of oxygen in the system. 

In regard to these lesions, it is worth considering" whether or not 
it would be correct to use the term "restitution" in referring to the 
elimination of breaks under conditions of anaerobic hydration. Wolff 
and I have discussed this matter in personal communications and are 
agreed that, if the relationship between dose and interchange fre- 
quency is exponential for seeds hydrated in the presence of oxygen, 
which it is, then it is reasonable to conclude that "restitution" (as the 
term is defined in radiobiological literature) occurred in the material 
hydrated ill the presence of nitrogen. 

The effect of temperature treatments before, during, and after 
irradiation on X-ray-induced injury in seeds has been studied over a 
period of 30 years (25). Unfortunately, in the early work, the pro- 
found effects of slight changes in the water content of seeds on their 
senstitvity to pre- and post-irradiation treatments was not appreciated. 
Consequently, there was often little correspondence between differ- 


ent workers' findings and the data were very largely ignored. Added 
to this was the tact that, at the times these studies were conducted, 
the test materials used in most radiobiological studies were not suit- 
able for demonstrating the influence of environment on radiosensi- 
tivity and there was general skepticism concerning reports that the 
damage to biological systems could continue after the cessation of 
irradiation. Indeed, this particular opinion was expressed very 
recently, as far as a post-irradiation effect of oxygen is concerned (3). 

These two reasons undoubtedly account for the fact that little 
consideration was given to the work of Kempton and Maxwell (17), 
who demonstrated that maize seeds X-rayed at either the temperature 
of liquid air or at 61° C evidenced less radiosensitivity than seeds 
X-rayed at room temperature. 

Now that some of the environmental parameters which affect 
radiosensitivity can be controlled, and it is established beyond any 
reasonable doubt that this applies to damage to the chromosome, 
speculation is warranted concerning the biophyiscal mechanisms that 
are involved. 

Of the possibilities that might be examined, three seem worthy 
of mention. First, that all damage to the chromosome results from the 
direct absorption of energy. This does not impugn the possible sig- 
nificance of intramolecular energy transfer mechanisms. Second, that 
all damage to the chromosome results from absorption of energy by 
the ambient layer, the effect on the chromosome being initiated by 
active radicals or some other mechanism for transferring energy. The 
following discussion would appear to rule out the need for further 
consideration of this possibility. Third, that damage to the chromo- 
some results from a combination of the first two kinds of events. 

For the reasons indicated (loc. cit.), it seems reasonable to con- 
clude that the energy released in regions of high specific ionization 
is more than sufficient to break a chromosome at the initial center of 
absorption. This particular type of event would not likely be modi- 
fiable by the changes in the physical structure of the chromosome that 
cotdd conceivably result from high temperature treatment or the 
other conditions that have been imposed on seeds during the course 
of this work. Data supporting this statement have been obtained in 
the study relating to the nature of the interchange-dose relationship 
(Figure 5) when seeds were hydrated in the presence of oxygen and 


nitrogen. They show that the linear components in both curves are 
indistinguishable from one another. This is rather good evidence that 
oxygen, at least, has no discernible effect on breaks resulting from 
dense clusters of ionization. 

From these considerations, it seems reasonable to suggest that 
most, if not all, environmental factors that effect radiosensitivity 
either mitigate or enhance the probability of sparsely ionized sites 
becoming biologically detectable. On the assumption that the detect- 
able events are a manifestation of genetic injury, one could conceive 
of both metastable (reversible) states within the chromosome and in 
the ambient layer as initial stages. Distinguishing between these two 
possibilities would be difficult, if not impossible, in studies with cells. 
Possibly studies with virus particles should be expanded in an 
attempt to resolve the problem. 

At this point it should be emphasized that, because of the sig- 
moidal nature of the curve expressing seedling injury as a function 
of dose (Figure 7), it is often necessary to choose specific doses to 
demonstrate the significance of particular kinds of pre- and post- 
irradiation treatments. This accounts for the fact that 15,000 r was 
used to show the protective effect of a pre-irradiation temperature 
of 75° C (Figures 8, 9, 10), whereas 7,500 r was used to show the effect 
of post-irradiation temperature (Figure 11) on the progression of 
injury from storage. 

It is now appropriate to look at the interrelations between 
hydration, storage, and oxygen availability in connection with the 
manner in which they influence the manifestation of radiation-incited 
changes as seedling injury. It can be stated with certainty that the 
addition of water to the seed after irradiation, irrespective of other 
pre- and post-irradiation treatment conditions, has a quenching influ- 
ence on those radiation-induced changes which might otherwise 
progress for a considerable period of time. Furthermore, absorption 
of water is equally effective in eliminating injury progression under 
both anaerobic and aerobic conditions, although more total injury 
may occur in the material subjected to aerobic hydration under 
specific treatment regimes. 

Most previous investigators have suggested that the increase in 
injury which occurs during storage includes the oxygen-sensitive 
component (5, 12). It was demonstrated (5) that as post-irradiation 


storage time increased, sensitivity to oxygen decreased. This particu- 
lar relation appears to hold under all but one set of conditions. When 
seeds are subjected to a temperature of 75° C for 24 hours before they 
are irradiated, they subsequently show no sensitivity to storage. How- 
ever, their sensitivity to oxygen immediately after irirradiation is as 
great as in non-heat treated seeds and, furthermore, it persists for at 
least 48 hours (Figure 10). These last data suggest that there is no 
relation between the effects of oxygen and storage on seeds. This 
problem will be discussed later. 

There appear to be two reasonable explanations as to why a pre- 
irradiation treatment with heat prevents the dissipation of sensitivity 
to oxygen. First, that the cell membranes are modfied by the tempera- 
ture treatment so that they become impermeable to oxygen. Second, 
that radiosensitive molecules within the nucleus are so re-oriented by 
high temperature that the reactive sites are enfolded within them 
and are not exposed to oxygen until hydration occurs. Both of these 
explanations suggest that heat-treated seeds would retain a radiation- 
induced labile state more or less indefinitely under appropriate condi- 
tions. The data presently available support this suggestion. 

In their recent report Powers, et al. (21) have demonstrated that, 
in bacteria, maximum protection from X-irradiation by high temper- 
ature (80° C) only occurs when the temperature treatment is admin- 
istered during or immediately after irradiation. Furthermore, maxi- 
mal thermorestoration or thermal annealment, as they have chosen 
to call this phenomenon, takes place only in the absence of oxygen. 
In contrast with the spore work, for the first few hours following 
irradiation there is a more rapid increase in the injury to X-rayed 
seeds when they are maintained at 85° C (Figures 11 and 12). Inter- 
estingly enough, however, seeds maintained at the higher temper- 
atures for periods of 48 hours or more showed some thermorestoration 
of injury. One other significant fact is that sensitivity to oxygen was 
completely eliminated after a 15-minute post-irradiation heat treat- 
ment at either 75° C or 85° C, whereas it persisted for considerably 
longer periods of time at 20° C. 

Speculation seems warranted concerning the mode of action of 
high temperatures on critical molecules that results in the elimination 
of injury from storage, but not from oxygen, when the temperature 
treatment is given before irradiation and the reverse when it is given 


after irradiation. Let tis assume that the injury occurring during 
storage results from the transfer of energy from the site of initial 
absorption (primary event) to a second more sensitive site (secondary 
event), whereas injury from oxygen results directly from oxidation 
of the primary event. Let us further assume that it is a matter of 
chance whether or not oxidation occurs at the primary site before 
the energy associated with it is transmitted to a secondary site. If such 
was the case, it is conceivable that pre-treatment with heat could 
modify the structure of the critical molecule in such a way that the 
energy associated with the primary event could not be transferred or 
otherwise dissipated. Under such a circumstance the oxygen-sensitive 
primary site should persist indefinitely, which is precisely what the 
data indicate (Figure 10). 

LIsing the same model, the effect of a post-irradiation heat treat- 
ment, which rapidly eliminates sensitivity to oxygen, could be 
explained by assuming that the thermal energies involved were 
sufficient to enhance the rate at which energy was transferred away 
from the site of primary absorption. 


Dormant seeds of barley with an embryo water content of 4 per 
cent were used for biophysical studies concerned with the influence 
of pre- and post-irradiation treatment conditions on the manifestation 
of seedling and genetic injury. In the course of the investigations, 
designed to examine some of the environmental parameters that were 
known to affect radiosensitivity, the following observations were 

1 . Seeds germinated immediately after X-irradiation and then grown 
for 8 days showed a near normal distribution of seedling heights 
about the mean. However, when seeds were stored at room tem- 
perature before germination, the distribution of seedling heights 
about the mean became progressively skewed, as a function of 
time, for a period of at least 8 days. Determination of the extent 
of genetic injury in different seedling height classes in material 
that was stored for 8 days showed that there was an inverse relation 
between seedling height and genetic injury. On the average, seeds 
that fell into the shortest height class evidenced between two and 
four times as many interchanges and mutations as seeds that fell 
into the tallest height class. 


2. The interchange frequency increases exponentially with dose 
when seeds are hydrated aerobically immediately after X-irradi- 
ation but linearly with dose when they are hydrated anaerobically. 
For both conditions of hydration the mutation frequency 
increases linearly with dose. However, aerobic hydration results 
in the production of more mutations per roentgen than does 
anaerobic hydration. 

3. Pre-irradiation temperature treatments for 24 hours at 75° C, 
showed a protective effect over a wide range of doses of X-rays. 
The protective effect of the treatment was manifest when the seeds 
were hydrated either aerobically or anaerobically and was not asso- 
ciated with a detectable change in water content. Furthermore, 
the percentage protection obtained, per unit of injury, under 
both conditions of hydration was similar. It was also demonstrated 
that the protective effect of the heat treatment against a subse- 
quent dose of X-rays persisted for at least 2 months between heat- 
ing and irradiation provided the humidity of the storage atmos- 
phere was not changed. In addition, a pre-irradiation temperature 
of 75° C eliminated the increase in injury that usually accom- 
panies post-irradiation storage. However, such a pre-treatment 
did not modify the sensitivity of the seeds to post-irradiation 
hydration in the presence of oxygen. 

4. In contrast with the effects of a pre-irradiation heat treatment, 
a short post-irradiation storage temperature of either 75° C or 
85° C generally enhanced the rate of increase in injury that was 
observed during post-irradiation storage at room temperature. 
However, 48 hours storage at both of these temperatures resulted 
in a distinct thermorestoration of injury. Of particular significance 
was the demonstration that treatment of the seeds at either 75° C 
or 85° C for as little as 15 minutes after they were irradiated 
eliminated their sensitivity to post-irradiation hydration in the 
presence of oxygen. 

Part II. Theoretical Considerations Relating to Acute 
Irradiation of Successive Generations 

Actually since 1930, but particularly since the conclusion of the 
Second World War, ionizing radiations have been used by plant 
geneticists in efforts to induce heritable changes that will be of eco- 


nomic significance. By and large these studies have involved attempts 
to induce so-called point mutations. The organisms most commonly 
studied have been the small-grained cereals, peanuts, and corn. Data 
obtained from studies with these species have been discussed in other 
sections of this Symposium. The intent of the present report is to 
mention only theoretical considerations that may be of relevance to 
the plant breeder and which have received very little attention by 
most workers. 

In using ionizing radiations in plant improvement, one must 
take into consideration the degree of ploidy and the breeding 
behavior or mode of reproduction of the species with which he 
intends to work. In addition, one must be cognizant of the fact that 
ionizing radiations not only induce point mutations but also all 
manner of structural chromosomal anomalies, such as reciprocal 
translocations, inversions, duplications, and deletions. Furthermore, 
he must be aware that the frequency with which these types of events 
are produced, and their ease of detection, will be dependent upon 
the species with which he is working and the ontogenetic stage of the 
plant to which the irradiation is given. 

To reduce this complex of variables in the present report, unless 
otherwise stated, we shall consider only what may possibly be achieved 
through seed irradiation of the naturally self-fertilizing cereals which 
occur in nature as diploids, tetraploids and hexaploids. 

Mutations From a Single Dose of Radiation 

On a priori grounds, in the X 2 from populations receiving one 
dose of radiation it would be expected that plants expressing mutant 
phenotypes for qualitative characters would occur commonly in the 
diploid species, with a lower frequency in tetraploid species, and only 
rarely in hexaploid species. The reason for this is simply that most 
radiation-induced mutations are recessives and the simultaneous 
mutation of genes which influence the expression of the same charac- 
ter, but are located on homeologous chromosomes, would be a rela- 
tively rare event, even under optimal conditions of treatment. The 
essential validity of this consideration has been borne out by chloro- 
phyll mutation studies on tetraploid and hexaploid oats and wheat. 

In addition to the above consideration, there is another important 
factor which makes it difficult to recover a large number of mutant 



phenotypes following administration of a single dose of radiation to 
an organism. This is because ionizing radiations induce chromosomal 
aberrations as well as mutations. Such aberrations are often respon- 
sible for cell death and it is apparently a simple matter of chance 
whether or not either a mutational event, a chromosomal anomaly, 
or both, are induced in a cell following the absorption of photons or 
ionizing particles. Accordingly, as the dose to which the population 
is subjected is increased, the number of cells in the population which 
contain both aberrations and mutations also increases. Because only 
one or a very few aberrations can result in cell death, it is apparent 
that the probability is very low of inducing mutations at more than 
a few loci before a lethal aberration is induced. 

From these considerations, it should be apparent that similar 
complications prevail when attempts are made to obtain mutations 
for quantitative traits. As many of the most important characters with 
which the breeder works are under polygenic control, the desirability 
of using ionizing radiations in other than a "one-shot" approach in 
breeding programs should be obvious. 

Mutation From a Dose Administered to a Sequence of Generations 

In diploids 

It seems obvious that a method that would increase the relative 
frequency of mutations to lethal or semi-lethal aberrations in a pop- 
ulation, before it is subjected to the expensive process of screening 
for economic mutations, would be of distinct value to the geneticist. 
At this time there appears to be only one simple way in which this 
can be achieved. That is to irradiate successive seed generations of 
the material under test, always selecting for re-irradiation seed from 
those spikes or panicles which do not evidence structural chromo- 
somal anomalies. This is a particularly simple procedure in diploid 
species, such as barley, because during the development of the plant 
from irradiated seed all cells which are cytologically deficient in their 
chromosome complement are incapable of competing with more 
normal cells. The end result is that, effectively, only reciprocal inter- 
changes and inversions are detected in the analysis of sporocytes aris- 
ing from irradiated diploid seed. Reciprocal interchanges invariably 
induce 25 to 50 per cent sterility. Thus, when selecting seed for 
re-irradiation one can select it only from fertile heads. Because muta- 


tions and chromosomal aberrations tend to be independently induced 
events (15), seed from fertile heads will carry as many mutations as 
seed from semi-sterile heads. While seed selected in this way may 
incidentally carry an inversion it would likely be of little consequence 
to the breeder and could be easily eliminated if the need was 

In suggesting the use of recurrent irradiation in diploid species 
for inducing maximum genetic variability for both quantitative and 
qualitative traits, it is recognized that gene mutations of both a dele- 
terious and beneficial nature will be induced in the same cells and 
that the method does not provide for their separation between gen- 
erations. From this it follows that the immediate products from 
recurrent irradiation of a diploid could not likely be used as com- 
mercial varieties. However, after screening for mutants of agronomic 
value in a population that had been subjected to recurrent irradi- 
ation, it would be easy to produce hybrids between the original 
progenitor and the irradiated material to place the character, or char- 
acters, of value in what 'was an otherwise desirable genetic back- 
ground. In so far as the author is aware, this particular approach 
has received little consideration by plant breeders. 

In polyploids 

When attempting the induction of detectable mutations in the 
self-fertilizing polyploids among the small grains, a special set of 
circumstances confronts the geneticist. For every pair of genes that 
influence the expression of a character in the diploid, both quantita- 
tive and qualitative, there presumably usually exist two and three 
times as many genes in the tetraploid and hexaploid, respectively. 

If considering mutation only for qualitative characters, theoreti- 
cal assumptions suggest that continued re-irradiation of the seed 
generation of the polyploids, in which the genes governing the same 
order of biochemical function are in the homozygous dominant con- 
dition on homeologous chromosomes, will ultimately result in the 
appearance of a high frequency of chlorophyll mutations in the popu- 
lation. When such mutations appear, F 2 segregation for the mutant 
trait in crosses with the original parental type should be 15:1 in the 
case of the tetraploid and 63:1 in the case of the hexaploid. Among 
the normal F 2 phenotypes from these crosses there should exist geno- 


types which contain the normal gene in the homozygous dominant 
condition on one pair of homologs and are homozygous recessive on 
the other pair or pairs of homeolgous chromosomes. In plants of this 
genetic constitution crosses with the recessive phenotype should give 
a 3:1 segregation for the mutant character. In effect, then, it can be 
said that the polyploid has been "diploidized". It follows that the 
recurrent re-irradiation of any polyploid that shows bivalent pairing 
should eventually result in the material used in such studies 
being indistinguishable, by simple Mendelian tests, from naturally 
occurring diploids. 

It might well be asked if there exist any factual data to support 
these considerations. The answer is simply that the incidence of 
chlorophyll mutations in hexaploid oats has increased each genera- 
tion through six successive generations of re-irradiation. Mutant types 
have been perpetuated, but crosses with the parental type have not 
been made. 

In addition to the hexaploid oat work, which will be mentioned 
again in the next section, recurrent irradiation studies are also under- 
way at the University of Minnesota with diploid and tetraploid oats 
and tetraploid and hexaploid wheat. The incidence of chlorophyll 
mutations in the wheat material after irradiation of two successive 
seed generations supports the general considerations. 

It seems pertinent to ask the following cpiestion: If diploidiza- 
tion of a polyploid is indeed possible, is it likely that it 'will have any- 
practical significance? The answer, based on theory and existing 
recurrently irradiated plant material, is yes. To give substance to this 
assertation it is again necessary to resort to theoretical considerations 
and the findings of other workers. Heterosis in corn is commonly 
attributed to differences in alleles which govern expression of the 
same characters, i.e., heterozygosity at several loci, or to epistasis. 
Similarly, it has been demonstrated by Wallace and Vetukhiv (28) in 
Drosophila that genes which are recessive and deleterious when in 
the homozygous condition may benefit the survival and growth of a 
fly in which they are in the heterozygous state. 

What has this to do with diploidization of a self-fertilizing poly- 
ploid, specifically, for ease of presentation, hexaploid wheat? As has 
been indicated, in the hexaploid often there are presumably six genes, 
one on each of three pairs of homeologous chromosomes, which 


influence the expression of a given character. Furthermore, apparent- 
ly these genes are often in the homozygous dominant condition on 
more than one pair of the homeologous chromosomes. Now, if in 
diploids a heterotic effect is obtained when differences exist between 
alleles on a pair of homologs, it seems possible that in a polyploid 
differences between genes which govern the expression of the same 
character, but are on homeologous chromosomes, may also confer 
increased vigor to plants in which the condition exists. If such is the 
case, the self-fertilizing polyploid would have an advantage over the 
diploid in that the "heterozygous" state (between genes on homeolo- 
gous chromosomes) could be perpetuated by selfing and thus provide 
permanent heterosis by merely maintaining the alleles on one pair 
of homologous chromosomes in the homozygous dominant condition. 

In regard to the possible usefulness of this approach in plant 
breeding, it seems relevant to mention a promising study that is in 
progress, using recurrent irradiation on hexaploid oats. In this pro- 
gram the following procedure has been used. Seed from a single plant 
from three different oat varieties, Park, Missouri 0-205, and Clint- 
land, was increased and irradiated. In the X 1; 1,000 fertile panicles 
were chosen from different plants in each of the three varieties. The 
seed from these panicles was re-irradiated and planted in panicle 
rows. From these panicle rows one fertile panicle was chosen from 
one agronomically suitable-looking plant to form the seed source for 
the next cycle of radiation. This procedure has been continued 
throughout the entire program, which has now run through six cycles 
and is presently in the seventh. 

Some in teres tin sf sfeneral observations on this material are worth 
reporting, although at this time there are no grounds for assuming 
that a state of permanent heterosis exists in any of the material. First, 
and most significant, is the fact that variability in the material for 
height, panicle type, and maturity date was not in evidence until 
after the second cycle of radiation. Subsequent to this there has been 
a continued increase in variability for these characters. At the end 
of the fifth cycle of radiation the variability was so extreme in all of 
the material, with a considerable number of types that evidenced 
characteristics of agronomic interest, that a number of plant selections 
were made for testing with the parental type. These selections were 
increased in 1959 and the promising ones were turned over to experi- 


enced plant breeders who will compare them in yield tests with the 
parental types in 1960. It is important to emphasize that after this 
lono series of recurrent irradiation, where selection was made each 
generation for desirable plant types, most of the material in the 
nursery is agronomical)' undesirable. This is particularly true of the 
material derived from Clintland and to a lesser extent to that derived 
from Missouri 0-205 and Park, in that order. 

Another point that warrants consideration is the fact no attempt 
was made to isolate the irradiated material from outcrossing. How- 
ever, as indicated, each year seed for re-irradiation was taken only 
from completely fertile panicles. Accordingly, there should have been 
no more outcrossing in the material than the breeder usually finds 
in his nursery plots. As the incidence of outcrossing should be quite 
low, it seems unlikely that this can account for the extreme variability 
that has been observed or for the apparently desirable types that have 
been selected lor further study. However, this possibility cannot be 
ignored and studies are presently under way with polyploid series in 
oats and wheat, where isolation is being practiced, to resolve this 

The Use of Intraspecific Chromosome Structural Changes 

Among the complex types of chromosomal aberrations induced 
by ionizing radiations is a class known as the "duplication". As the 
name implies, this means that a duplication of genetic material gov- 
erning specific gene functions exists in the chromosome complement. 
Duplications may arise in two simple ways which do not simultane- 
ously result in a loss of genetic material in the cell. The first involves 
translocations between corresponding arms of homologous chromo- 
somes that were broken at dissimilar points. The second involves 
breakage and reunion of sister chromatids in which the break points 
were not at corresponding loci. 

From the preceding discussion on polyploids it should be evident 
that they have built into them a system of duplications of what can 
amount to whole sets of genes in the case of a true allopolyploid. It 
would appear, then, that radiation-induced duplications would likely 
be of greatest practical use in diploid species. Accordingly, the discus- 
sion will center around their possible use in self-fertilizing diploids. 


The previously mentioned work of Wallace and Vetukhiv (28) 
with Drosophila, which has demonstrated that flies heterozygous for 
one or a lew loci may be better adapted to their environment than 
when they are either homozygous dominant or recessive for these 
loci, is information that could be of import to the plant breeder 
using diploid species if he could devise some method of obtaining 
lines that would breed true for the heterozygous condition. There 
seems to be only one possible method for achieving this end, within 
the confines of the diploid, and that is to obtain a duplication of the 
first type mentioned so that the normal and mutant alleles are located 
on the same chromosome. This would necessitate irradiating F x seeds 
that were heterozygous for the allele under consideration. 

One might well ask three questions concerning the feasibility of 
using the method: (A) What is the probability of obtaining a trans- 
location at dissimilar points on homologous chromosomes? (B) What 
is the probability that the duplicated segment of the interchange will 
involve the gene in question? (C) What is the likelihood of recog- 
nizing the duplication once it is produced? 

The answer to the first question is that translocations between 
opposite arms of homologous chromosomes occur with a frequency 
that is apparently due to chance (19). It can be assumed, therefore, 
that translocations between corresponding arms would occur with 
the same frequency. From this it follows that the lower the number of 
chromosomes in the species under study, the more readily will such 
interchanges arise. Actually, in diploid barley and maize, cytogenetic 
studies have shown that translocations between opposite arms of 
homeologous chromosomes are a relatively common occurrence 
(4, 6, 20)". 

The second question cannot be answered except to point out 
that, based on purely physical assumptions, a gene in the median 
position of an arm should be involved in duplications more commonly 
than one close to the centromere or on the distal end of an arm. 

The answer to the third question is simply that it depends on 
whether or not the heterozygous state produces a recognizable pheno- 
type. If it does, plants carrying a duplication involving a dominant 
and recessive allele should be readily detected because they will 
either not segregate for the character in question or give aberrant 
segregation ratios. 


Duplications arising in the second-mentioned manner, viz., by 
joining of sister chromatids that were broken at dissimilar points, 
are exceedingly common, as based on cytological observations. How- 
ever, they maye be extremely difficult to detect phenotypically and 
would appear to have little practical value except in cases where 
additive gene action prevailed in the production of a particular trait, 
such as disease resistance, pigmentation, etc. 

Production of fertile tetraploids 

Since the discovery that colchicine was an effective polyploidiz- 
ing agent, plant breeders have doubled the chromosome complement 
of numerous species with which they work. In only a few instances 
have the induced polyploids been useful economically. One immedi- 
ate reason for this is that meiosis in an autopolyploid is disturbed by 
multivalent formation which, along with some undetermined physio- 
logical imbalance, apparently results in sterility and aneuploid types 
arising in the progenies. If these problems could be overcome, ade- 
quate tests of polyploids, originating from common genetic back- 
grounds, could be undertaken and the genetic diversity within a 
species that was available to the breeder coidd be increased 

One possible method of overcoming multivalent associations 
would be to reorganize structurally the chromosomes of a species to 
the point that very little or no homology remained between the 
structurally modified lines and the progenitor. At this stage F/s 
between the modified lines and the progenitor could be doubled to 
produce a fertile tetraploid that would be similar to artificially and 
naturally occurring amphidiploids. 

At attempt to produce such structural modification experimen- 
tally in barley is underway, using ionizing radiations to induce trans- 
locations, inversions, duplications, and deficiencies. For the past six 
generations, the procedure has been to use irradiation to induce at 
least one interchange per generation in each of four lines originally 
selected from four different varieties. Incidental to the addition of 
the interchanges, but equally effective in inducing structural differ- 
entiation, are the addition of the other anomalies mentioned. 

Hybrids between some of the material, in which structural differ- 
entiation of the chromosomes is being attempted, and the parental 


genotype have just been achieved. They will be examined cytologi- 
cally in the spring of 1961 and it is to be hoped that some of the F x 's 
will show the kind of reduced pairing that is common in two of the 
asynaptic mutants that have arisen from the material. 

Use of Interspecific and Intergeneric Chromosome Interchanges 
With few exceptions, cereal breeders are limited in their 
improvement programs to using the phenotypes they can obtain by 
vecombining the genes that exist in the species with which they are 
working. The reason for this is simply that when wide crosses can be 
effected, there is frequently little homology between chromosomes 
from the two parents and correspondingly little recombination takes 
place. The consequence is that there is often either divergence toward 
parental types or the progenies from such crosses contain additions 
of whole chromosomes to the basic complement. The lines in which 
whole chromosomes have been added to a basic complement have 
invariably proved too inferior for commercial usage. 

In most instances, what the breeder is seeking from intergeneric 
and inter-specific crosses is one or a few characters, such as disease or 
insect resistance, that he can incorporate in a commercial strain 
without impairment to that strain. Recently ionizing radiations have 
been effectively used to achieve this where conventional methods 
failed. In this regard, Sears (23) has reported the successful transloca- 
tion of a segment of chromosome carrying a gene for leaf rust resist- 
ance from Aeiglops umbellulata to Tritiairn vulgare, and Elliott (14) 
has had comparable success in transposing a piece of chromosome 
carrying a gene for stem rust resistance from Agropyron elongation 
to T. vulgare. 

Because of these successes, there would appear to be considerable 
merit to the sug-grestion that ionizing radiations should be used exten- 
sively in hybrids involving Triticum, Avena, and their related genera, 
to obtain recombinant types that cannot be obtained by conventional 
methods. Using the method outlined, it should be possible to derive 
lines that contained all the 42 chromosomes of T. vulgare plus one 
pair from a related genus. Theoretically, where there is no homology 
between chromosomes involved in an intergeneric cross, it should be 
possible to derive as many strains containing a different pair of 
chromosomes from the related genus as in the "n" number of chro- 
mosomes in that genus. By screening the derived strains, it could be 


determined whether or not the added pair of chromosomes carried 
a factor or factors of economic significance. In cases where some eco- 
nomic worth was apparent, irradiation studies could be undertaken 
to translocate a region of the chromosome carrying the gene or genes 
of significance into the T. vulgare background of chromosomes. Once 
this had been achieved it would be a simple matter to eliminate the 
extra pair of chromosomes from the related genus by backcrossing 
to T. vulgare and selecting only those progenies with a complement 
of 42 chromosomes. 

Concluding Remarks 

In recent Years a great deal of attention Iras been given to the use 
of one acute dose of ionizing radiation for inducing mutations of a 
beneficial nature. At the same time, relatively little consideration has 
been directed to other possible approaches, involving both mutations 
and chromosome structural modifications, which would appear to 
have great potential promise. The present report is an attempt to 
emphasize proved and theoretical considerations which suggest that 
effectively utilized ionizing radiations will likely become of increasing 
significance to the applied geneticist. 


1. Adams, J. D., and Nilan, R. A. 1958. After effects of ionizing radia- 
tion in barley: II. Modification by storage of X-irradiated seeds 
in different concentrations of oxygen. Radiation Res., 8: 11 1-122. 

2 , , and Gunthardt, H. 1955. Alter effects of ionizing 

radiation in barley: I. Modification by storage of X-rayed seeds in 
oxygen and nitrogen. Northwest Sri., 29: 101-10S. 

3. Alper, T. 1956. The modification of damage caused by primary 

ionization of biological targets. Radiation Res., 5: 573-5S6. 

4. Caldecott, R. S. 1955. The effects of X-rays, 2 Mev electrons, thermal 

neutrons, and fast neutrons on dormant seeds of barley. Ann. New 

York Acad. Sri., 59: 514-535. 
5. . 1958. Post-irradiation modification of injury in bailey — its 

basic and applied significance. Proc. 2nd Intern. Conf. Peaceful 

Uses of Atomic Energy. New York: United Nations, 27: 260-269. 
6. and Smith, L. 1952. The influence of heat treatments on 

the injury and cytogenetic effects of X-rays on barley. Genetics, 37: 



7. , . 1952. A stud) of X-ray-induced chromosomal 

aberrations in barley. Cytologic, 17: 224-242. 

8. , Beard, B. H., and Gardner, C. O. 1954. Cytogenetic effects 

of X-ray and thermal neutron irradiation on dormant seeds of 
barley. Genetics, 39: 240-259. 

9. , Frolik, E. F., and Morris, R. 1952. A comparison of the 

effects of X-rays and thermal neutrons on dormant seeds of barley. 
Proc. Nat. Acad. Set., 38: 804-809. 

10. , Johnson, E. F., North, D. T., and Konzak, C. F. 1957. 

Modification of radiation-induced injury by post-treatment with 
oxygen. Proc. Nat. Acad. Sri., 43: 975-983. 

11. Conger, A. D., and Randolph, M. L. 1959. Magnetic centers (free 

radicals) produced in cereal embryos by ionizing radiation. Radia- 
tion Res., 11: 54-66. 

12. Curtis, H. J., Delhias, N., Caldecott, R. S., and Konzak, C. F. 1958. 

Modification of radiation damage in dormant seeds by storage. 
Radiation Res., 8: 526-534. 

13. Ehrenberg, A., and Ehrenberg, L. 1958. The decay of X-ray-induced 

free radicals in plant seeds and starch. Arkiv Fysik, 14: 133-141. 

14. Elliott, F. C. 1957. X-ray-induced translocation of Agropyron stem 

rust resistance to common wheat. Jour. Hcred., 48: 77-81 . 

15. Gaul, H. 1958. Present aspects of induced mutations in plant breed- 

ing. EupJiytica, 7: 275-289. 

16. Giles, N. H. 1940. Induction of chromosome aberrations by neutrons 

in Tradescantia microspores. Proc. Nat. Acad. Sci., 26: 567-575. 

17. Kempton, J. H., and Maxwell, L. R. 1941. Effect of temperature dur- 

ing irradiation on the X-ray sensitivity of maize seeds. Jour. Agr. 
Res., 62: 603-618. 

18. Konzak, C. F., Caldecott, R. S., Delhias, N., and Curtis, H. J. 1957. 

The modification of radiation damage in dormant seeds. Radiation 
Res., 7: 326. 

19. Koo, F. K. S. 1959. Expectations on random occurrence of structural 

interchanges between homologous and between non-homologous 
chromosomes. Amer. Nat., 43: 193—199. 

20. Morris, R. 1955. Induced reciprocal translocations involving homo- 

louous chromosomes in maize. Amcr. Jour. Bot., 42: 546-550. 

21. Powers, R. L., Webb, R. B., and Ehret, C. F. 1961. Storage transfer 

and utilization of energy from X-rays in dry bacterial spores. 
Biocuergetics symposium. Radiation Res., Suppl. 2. In press. 

22. Sax, K. 1941. Types and frequencies of chromosal aberrations induced 

by X-rays. Cold Spr. Harb. Symp. Quant. Biol., 9: 93-101. 


23. Sears, E. R. 1956. The transfer of leal-rust resistance from Aegilops 

umbellulata to wheat. Brookhaven Symp. in Biol., 9: 1-22. 

24. Smith, L. 1946. A comparison of the eilects of heat and X-rays on 

dormant seeds of cereals with special reference to polyploidy. Jour. 

Agr. Res., 73: 131-158. 

25. . 1951. Cytology and genetics of barley. Bat. Rev., 17: 1-355. 

26. and Caldecott, R. S. 1948. Modification of X-ray effects on 

barley seeds by pre- and post-treatment with heat. Jour. Hexed., 34: 


27. Stadler, J,. J. 1930. Some genetic eilects of X-rays in plants. Jour. 

HerecL, 21:3-19. 

28. Wallace, B., and Vetukhiv, M. 1955. Adaptive organization of the 

gene pools of Drosophila populations. Cold Spr. Harb. Symp. 
Quant. Biol., 20: 303-310. 

29. Wolff, S. I960. Post-irradiation storage and the growth of barley 

seedlings. Radiation Res., 12: 4SJ. 

30. and Luippold, H. E. 1957. inaccuracy of anaphase bridges 

as a measure ol radiation-induced nuclear damage. Nature, 179: 

31. Zimmer, K. G., Ehrenberg, L., and Ehrenberg, A. 1957. Xachweis 

langlebiger magnetischer Zentren in bestrahlten biologischen Med- 
ien und deren Bedeutung fur die Strahlenbiologic. Strahlenthera- 
pie, 103: 1-15. 


Robinson: I question here, and in Doctor MacKey's presentation, the 
assumption of the superiority of the hetero/ygote, i.e., the importance 
of overdominance, particularly in quantitative characters. Reference has 
been made to work in Drosophila for the importance of epistasis. 1 
cite the work of Vethukiv (Evol., 1954) and by Yethukiv and Beardmore 
(Genetics, 1959) where the later results do not support the conclusion 
of important epistasis given in 1954. 

The importance of super- or overdominance in quantitative char- 
acters has, to my knowledge, still to be convincingly demonstrated. 

Heterosis occurs in variety crosses of maize, but neither overdomin- 
ance nor epistasis may be necessary for an adequate explanation of the 
heterosis. Genetic diversity resulting from different alleles in different 
parents could account for the heterosis observed. 

Caldecott: 1 woidd make three points because 1 believe they reflect 


our differing views on the motivation for doing the research reported 
and the nature of the genetic systems involved. 

1. To test the hypothesis, or "assumption" to use your word, that the 
heterozygote is superior to the homozygote is not, in itself, bad. One 
could have set up the opposite hypothesis which would have been sub- 
ject to the same kind of criticism that you render but which, 1 sense, 
you would condone. 

2. The fact that overdominance has not been "convincingly" demon- 
strated is the crux of the whole issue. If it had been clearly demonstrated, 
or proved not to exist, both in diploids and polyploids (and 1 empha- 
size the latter), 1 would have no interest in conducting the studies out- 
lined as they would contribute nothing new to our knowledge. In this 
regard, in my opinion, it is dangerous to generalize from what are admit- 
tedly inadequate studies with two species, if you accept the negative 
data in Drosophila, both of which are diploids, and neither of which 
is naturally inbred or exists in nature at the polyploid level. 

3. You make a distinction between genetic diversity and epistasis. 
These two concepts need not be, indeed they cannot realistically be, 
treated independently. Therefore, it seems inconsistent to me that, on the 
one hand, you postidate that heterosis may result from genetic diversity 
but, on the other hand, not from epistasis. If you recall, in the text it 
is specifically stated that the procedures being used should be of sig- 
nificance if heterosis is due either to the differences between genes 
governing the same order of biochemical function or to epistasis. 

Mac Key: Wheat is neither strictly autoploid nor alloploid. It varies with 
the characteristic. My own investigations indicate this rather clearly. 
Characteristics essential for the ability to complete life and to repro- 
duce show a high degree of autoploidy, while other characteristics not 
decisive to life or death but determining morphology, adaptation, etc., 
may follow a more alloploid pattern. The reason for this difference is 
that the former category will have a more conservative differentiation 
on the diploid level, while the latter may evolve rather freely as evident 
from the morphology of Triticutii monococcum, Acgilops speltoides, 
and A. squarrosa, the parental genomes of fix wheat. 

First, when diploids are added to form polyploids, the essential genes 
can mutate more readily without severe deleterious effects, but also 
here, at least, one pair has to be kept intact. The others are, however, 
now free to mutate in directions that may have been impossible on the 
diploid level. The degree of diploidization revealed in my experiments 
with dicoccum favors the idea that this process has selective advantage. 


It may be a kind of genomic heterosis ellect. In addition, the dosage 
of every gene is very unlikely always optimal at the level reached by 
the addition of whole genomes. For phylogenetical reasons, 1 am just 
now studying the degree of diploidization in different 4x wheats, since 
it is still not settled whether they should be traced to one or more 
processes of tetraploidi/ation. 

Caldecott: What you have stated is precisely the basis on which the 
studies outlined here were conducted. 

Grun: There are two aspects on which I have questions to ask. The first 
concerns the plan described to see whether you can establish perma- 
nent heterosis in self-pollinated hexaploid cereals. As I understand it, 
you plan to irradiate plants repeatedly over a number of generations, 
then cross back onto the original parental lines and attempt to select 
vigorous lines from the progeny. While you may well increase vigor by 
this procedure, I wonder whether you would be safe in assuming that 
such vigorous derivatives are a result of a heterotic interaction between 
homozygous dominant and homozygous recessive alleles of the same 
gene on homologous chromosomes. Might not such vigorous deriva- 
tives result instead from new epistatic interactions resulting from the 
induced mutations? 

The second question concerns your use of the word "diploidization". 
I am assuming that you are implying by this a progressive change from 
autoploidy toward alloploidy. I wonder whether mutations from dom- 
inant to recessive alleles of polyploid plants, as you have described, 
woidd necessarily lead the plant more towards alloploidy. Alight not such 
changes be better termed as simple gene mutations than adding the 
implications involved in calling them diploidizations? 

Caldecott: In reference to your first question, the answer is yes, and 
this fact is mentioned in the text. 

Regarding the second question, the term diploidization was chosen 
simply because after recurrent irradiation one should be able to make 
crosses that would give a wide range of genes that would segregate in a 
3:1 ratio. (See text.) 

Dkrmen: If mutation was induced in the genotype AA AA AA, in a 
hexaploid wheat, and it was changed successively to Aa Aa Aa, would 
all three mutations be alike? 


Caldecott: They could be, but it is very unlikely. 

Derm en: Does AA AA AA constitution represent an autoploid or an 
amphiploid condition? 

Caldecott: It could represent either, providing one was dealing with 
bivalent pairing. 

Garber: On what basis are allopolyploids and allopolyploids to be 
distinguished when individual loci are involved? When the term "diploid- 
ization" is used, is it restricted to converting replicate loci (autoploids) 
to only two loci for alleles or may it be used for converting any poly- 
ploid to a strictly bivalent type? 

Caldecott: As I am working with allopolyploids I have not concerned 
myself with tin's problem. The distinction will, of course, be related to 
whether or not there is bivalent or quadrivalent pairing. The use of 
the term diploidization should be generally taken to imply the conver- 
sion of replicate genes, those which govern the same order of biochem- 
ical function, to one of their allelic forms so that, after selecting appro- 
priate genotypes out of F 2 hybrids between the mutated stock and the 
progenitor, genotypes can be obtained which, when crossed with one 
another, will give 3:1 ratios. 

Gabelman: Seedlings produced from seeds irradiated with low levels of 
X-radiation (less than 1,500 r) occasionally show stimulation in growth. 
What is the nature of this stimulation? How do you rationalize this 
potential stimulatory effect in evaluating and interpreting the response 
of seedlings to higher levels of irradiation as used in your experiments 
on radiosensitivity? 

Caldecott: I am not aware of any reproducible studies with barley 
seeds that show that low doses of ionizing radiations cause a stimulation 
in growth, albeit this may be true in other species. I do not know what 
the nature of the stimulation is in other species and, because it does 
not exist in barley, have not had the problem of rationalizing the effect 
with the data presented. 

Osborne: As Doctor Caldecott has mentioned, the Brookhaven coop- 
erative Program has for some years included preradiation stabilization 
of seeds at controlled relative humidity which, I believe, is 65 per cent. 


I would merely add that, since the beginning of the UT-AEC Coopera- 
tive Program in 1956, we have also brought all seeds to constant moisture 
at 65 per cent relative humidity. They are then sealed in plastic, irradi- 
ated, and returned to the cooperator. There seems little doubt that 
this procedure keeps sensitivity (in terms of toxicity), storage effects, and 
oxygen ellects at a minimum and the experiments are highly repeat- 

Discussion of Session IV 


Caladino Farm Seeds, Inc., Wlieaton, Illinois 

Progress in plant improvement may be dependent upon the 
incorporation into a new genotype of either one or many gene 
pairs. Several examples can be given in which but a single gene with 
major effects has meant the difference between success or failure in 
the economic culture of a crop plant. The best examples of the 
importance of single (or few) gene effects are the addition of disease 
resistance lacking in otherwise agronomical!}' desirable genotypes in 
wheat, oats, barley, Hax, and a number of other crops. Often, also, a 
simple gene mechanism may control plant height, as in sorghum, and 
a relatively few genes may control photoperiodic response or time of 
maturity and thereby adaptation to a new environment. All favorable 
simple gene affects need not be due to the action of dominant genes. 
Many cases can be cited where the recessive is the "desirable" type 
and the dominant the "undesirable" type. Hence, in the total realm 
of plant improvement, one must not confine his thinking only to 
quantitative characters for which expression usually is conditioned 
by many genes, each with small effects and with action varying from 
additive to epistatic. 

Throughout the long evolutionary processes in the development 
of plants there has been accumulated a large number of mutant types. 
These have been partially catalogued in several crops and maintained 
as world collections. These mutant types provide a "gene pool" and 
are the basic working" stocks in many of our economic food plants. 

The mere existence of a known source of supply of genes to meet 
specific needs does not imply that all of the genes useful in plant 
breeding are now in existence. When a search for desired genes fails 
to uncover sources of new characters desired in plant improvement, 
speeding the process of natural mutation through artificial means 
surely becomes an important part of plant breeding. During the 
progress of this symposium, several examples (dwarf cotton, early- 
maturing Hybiscus, and rust-resistant Merion bluegrass) of new 



genes useful in plant improvement and arising through the use of 
mutagenic agents have been reported. 

Many studies reported in the literature have shown that irrespec- 
tive of the mechanism involved or the kind of mutagenic asent 
employed there is no problem in producing mutant types. MacKey in 
his paper strongly emphasized the importance of devising more ade- 
quate screening procedures to isolate those kinds of mutants from the 
many arising from irradiation that may be useful in higher plants. 
Nelson also has shown the complexity of screening for useful mutants 
(as well as natural variants) in microorganisms. 

Nearly every study in mutation breeding has shown that newly 
formed mutants generally are expected to be associated with chromo- 
some aberrations that may be deleterious to the organism as a whole. 
This is not surprising. Present techniques to produce mutations are 
not selective. For these reasons it would be indeed surprising if a 
single gene mutant in higher plants would produce a line useful 
per se. This does not limit the usefulness of mutation breeding, but 
only serves to emphasize the necessity of transferring a newly formed 
desirable mutant gene into a more usable gene background through 
hybridization. The example given by Langham for nondehiscent 
sesame is an example of the difficulty that can occur in separating the 
undesirable plieotropic "side affects" from the major gene or genes. 

The discussion to this point largely has been concerned with 
single or simple gene effects in naturally self-pollinated crops. Perhaps 
it would be appropriate to discuss next the more complex prob- 
lems associated with mutation breeding for modification of char- 
acteristics in a desired direction when character expression is depend- 
ent upon multi-gene effects. In seeking to improve quantitative char- 
acters by irradiation the problem of identification becomes most, 
difficult. General experience has shown that the heritability of quan- 
titative characters is low and hence only limited progress can be 
expected from selection on a single plant basis. Evaluation of mutant 
progenies in replicated trials (as in the normal procedure of testing 
variability derived by hybridization) consequently becomes a 

In any approach to plant improvement the breeder must com- 
pare the probability of obtaining improvement by alternative 
approaches. The long history of plant improvement has clearly shown 


the advantages to be gained by first defining the needs for improve- 
ment, then carefully surveying the parental material available which 
together may contain the desired genes for recombination, followed 
by hybridization to create the pool of variability upon which subse- 
quent selection and evaluation can be practiced. To me, one of the 
most serious shortcomings of mutation breeding for characters whose 
expression is conditioned by many genes is the transfer from the 
philosophy of "creating planned variability" to creating "chance 
variability". At best, the desired recombination of many genes has a 
relatively low probability, but it is advantageous to know something 
about the odds as based on previous experiments. 

During the progress of this symposium examples have been cited 
of gains made by following the mutation breeding route. Other 
examples have been given by Burton and Caldecott showing lack of 
significant progress. These demonstrations through experimentation 
that gains or lack of gains have been made neither prove nor disprove 
the value of mutation breeding. The critical test is the comparative 
progress that could have been made with similar expenditure of time 
and resources to achieve a particular goal. Modern plant breeding 
approaches with the customary limitations of resources at research 
institutions require that progress be equated in terms of gains per 
unit of investment. 

Throughout the reports in the literature on mutation breeding 
for improvement in quantitative characters there seems to be an urge 
on the part of the breeder to achieve progress faster than he has a 
right to expect. As previously stated, presently used mutagenic agents 
are not selective. It would be expected that as many (or more) undesir- 
able chromosome alterations would be derived as favorable ones. 
The evolutionary development of favorable internal and relational 
balance in our present economic plants has been a slow one. Evidence 
presented in the literature 1 has shown that after many generations 
following hybridization in barley important measurable changes 
have occurred in such hybrid-derived populations. Is it not likely that 
much more potential gains could be achieved if populations derived 
from induced mutation were allowed to be subjected to a consider- 
able period of time for "restoration" to chromosome balance before 

^lal, 11. S., Snneson, C. A., and Romage, R. T. Genetic shift during 30 generations 
of natural selection in barley. Agron. Jour., 51: 555-557. 1959. 


attempts were made to select individuals from them for progeny 
evaluation? Selection of plants from a heterogeneous population with- 
in the limits of sampling that are imposed at best represent only a 
limited portion of the potential population range. It would seem to 
me that the probability of excluding the most unproductive end of 
the population distribution would be greatly enhanced if selection 
pressure for survival of individuals were allowed to operate on such 
mutation-derived variants. 

During the progress of this session of the symposium major 
emphasis has been directed to the use of mutagenic agents to produce 
new variants in self-pollinated plants. The literature does not record 
many irradiation experiments with the large group of economic 
plants in which cross pollination is the rule. Obviously, it would be 
difficult to distinguish between natural and induced mutations in 
populations that predominantly are heterozygous as a consequence 
of random cross pollination. The real question, however, is whether 
or not there is need to produce new variability within a crop in which 
abundant natural variability already exists. It is a common belief of 
plant breeders that we have sampled and evaluated only a small por- 
tion of the total gene supply in such species and hence adding to this 
reserve, for the most part, is not likely to be an important breeding 
procedure. It should not be inferred, however, that among established 
inbred lines (as in maize) all desired characters have been fixed by 
inbreeding and selection. The use of mutagenic agents to produce 
desired variants among existing inbred lines is in reality a problem 
comparable to their use in naturally self-pollinated crops. 

In summary, to this discussant, one should view the usefulness of 
induced mutations not as a sole means to attain a desired objective, 
but rather as a supplement to our present well-established procedures 
that have adequately demonstrated their value in plant breeding. 
This is not a "negative" point of view. If a desired character cannot 
be found within the existing pool of germ plasm in a species or in its 
related species, there is ample justification to create new variability 
through induced mutations even though our present techniques are 
comparatively crude in respect to gene selectivity. Finally, the use of 
mutation breeding techniques rarely can be expected to immediately 


produce agronomical]}' desirable variants. The undesirable and 
uncontrolled side effects due to chromosome rearrangements may 
upset the balance of gene complexes that characterize the total genetic 
complex needed in our present-day highly specialized crop varieties. 

Session V 

Possibilities for the 

H. F. Robinson, Chairman 
North Carolina State College, 
Raleigh, N. C. 

Mutagenic Specificity and Directed Mutation 1 


Brookliaven National Laboratory, 
Upton, L. I., N. Y. 

Directed control of induced mutation is an important objective 
in both theoretical genetics and its practical application. The 
means of achieving and improving this control is through better 
understanding of mutagenic specificity. Evidence of specificity has 
been reported for mutations among genes (inter-locus) and among 
alleles (inter-allelic and/or intra-locus), for localization of chromo- 
somal breakpoints, and for relative frequencies of gene mutations vs. 
chromosome breaks. 

In the first part of this paper, experimental evidence will be sum- 
marized for a nonrandomness, differential effect or specificity of 
induced mutation in organisms ranging from higher plants and 
animals to bacteriophage. The selectivity may be expressed in broad 
spectrum experiments of "forward" mutations and chromosome 
breakage, or, more specifically, in differences of back mutation 
response of particular alleles to a series of mutagenic agents. This 
discussion will be followed by considerations relating interpreta- 
tion of mutagenic specificity to mechanisms of action and structure of 
the genetic material. 

Evidence for Mutagenic Specificity 

Higher Plants 

The evidence for mutagenic specificity in higher plants is con- 
fined for the most part to "forward" (impaired function) mutations 
between loci (inter-locus specificity) and gene mutations vs. chromo- 
some breaks. The Swedish group of investigators have been most 
active in this field and have reported results on different spectra of 
mutations produced in barley following treatment with different 
mutagens (28, 29, 71).- Chlorophyll mutations of the vir'tdis type are 

1 Research carried out at Brookhaven National Laboratory under the auspices of the 
U. S. Atomic Energy Commission. 
2 See References, page 430. 



induced with increasing frequency and of the albino type with com- 
parative decreasing frequency through the series of mutagenic agents: 
neutrons, X-rays, ethylene oxide, myleran, nitrogen mustard, ethylene 
imine, di(/?-chloroethyl) phenyl alanine, and nebularine (purine-9- 
d-riboside). The spectrum of spontaneous chlorophyll mutations lies 
between that of myleran (di-methane-sulfonyloxy-butane) and nitro- 
gen mustard. There is also an indication of different frequencies in 
mutations for mildew resistance and susceptibility compared to chlo- 
rophyll mutations when the spectrum of spontaneous. X-ray-induced, 
and chemically induced types are compared (22). 

Differential inter-locus response following exposure to ionizing 
radiations of different ion densities has been reported for 69 analyzed 
radiation-induced erectoid mutants located at 22 different loci in bar- 
ley (29). Comparisons among mutants at five of the loci are particu- 
larly informative. Locus a (11 to 12 mutants) has not given mutations 
with neutrons, locus b (4 mutants) gave mutations in only one variety 
(Golden) of the three tested, locus c (17 mutants) gave mutations from 
exposure to sparsely as well as densely ionizing radiations and these 
were commonly associated with detectable chromosome breakage, and 
loci d (10 mutants) and m (7 mutants) also gave mutations from both 
types of irradiation but in only one case was a detectable chromosome 
break involved. These results are indicative of differential mutagenic 
effects of different radiation sources, though there is still a resrretable 
paucity in numbers. 

Localization or nonrandomness of chromosome breakage is an- 
other manifestation of differential control of the mutation process in 
its broader sense. It has been known for about a decade that radio- 
mimetic compounds have the ability to break chromosomes prefer- 
entially. In contrast, most evidence indicates that radiations are non- 
specific, or at least less specific, in effecting breakage between and 
within chromosomes. The highly reactive compounds, namely, nitro- 
gen mustards, di (2:3-epoxypropyl) ether, and beta-propiolactone, 
induce breaks selectively in heterochromatic regions in the middle 
of the short chromosomes of the Vicia jaba complement. The more 
weakly reactive radiomimetic compounds that have been tested exten- 
sively, namely, maleic hydrazide and 8-ethoxycaffeine, produce a con- 
centration of breaks in the long satellited chromosome of Vicia jaba. 

smith: directed mutation 415 

With the former, a structural isomer of the pyrimidiue base uracil, 
breaks are concentrated near the centromere; with the latter, a purine 
derivative, breaks are mainly in the region of the nucleolar constric- 
tion (47, 64, 65). The effects of maleic hydrazide and 8-ethoxycaffeine 
may conceivably be related to their structural resemblance to nucleic 
acid bases. KCN treatments produce an apparent random distribu- 
tion of breaks among Yicia chromosomes which are, however, concen- 
trated within chromosomes in heterochromatic regions (36). Analysis 
of exchanges between localized and randomized chromatid breaks 
may be used to gain information on chromosome structure in specific 
regions (46). 

Specificity of the mutagenic agent itself is clearly shown by the 
wide variation in the ratio of gene mutations to chromosomal aberra- 
tions. For some chemicals, as maleic hydrazide, the ratio is essentially 
zero, for others, as nebularine. it is large if not infinite; while certain 
alkylating agents and ionizing radiations fall in between these 
extremes (17). Diethyl sulphate has been found to combine the quali- 
ties of inducing a high frequency of mutations in barley with few 
accompanying chromosomal structural aberrations (31). There is, 
also, some indication of a different spectrum of chlorophyll mutations 
produced by diethyl sulphate compared to gamma radiation. The 
highly efficient mutagenic alkanesulphonic esters, as ethyl methane 
sulphonate (33) appear to cause little chromosome breakage. On the 
other hand, myleran is efficient in producing localized chromosome 
breaks in Hordeum and Vicia (48). We have found that ethyl methane 
sulphonate is ineffective at 0.1 M in producing loss of endosperm 
marker genes in the short arm of chromosome 9 in treated pollen of 
maize. By this same test (39) diepoxybutane was found to induce 
breaks approximately at random along this chromosomal arm, a 
region in which there is no conspicuous localization of heterochro- 
matin. The number of anaphase bridges induced by maleic hydra- 
zide and certain of its derivatives was observed by Graf (27) to be 
correlated with the number of heterochromatic knobs in the variety 
of maize treated. 

The clearest evidence of specificity in induced chromosome 
breakage is, then, the preference shown by certain chemicals for affect- 
ing particular heterochromatic regions. No definitive explanation is 
yet available; but the phenomenon may be due to different spacial 


relations of chromosomal strands in en- vs. hetero-chromatic regions, 
or to different placement with reference to the nuclear membrane. 
Significant information may be forthcoming from experiments under- 
way utilizing radioactive-labeled chemical mutagens. Such experi- 
ments should provide answers to questions regarding incorporation 
and possible localization of the mutagen in the chromosome and 
whether the mutagenic agent acts directly or indirectly with the 
chromosomal constituents. Callaghan and Grun (10), using C 14 
labeled maleic hydrazide, observed that this compound is incorpo- 
rated into chromatin material, is localized in the nucleoli, and is 
uniformly distributed in eu- and hetero-chromatin. 

An example of induced mutation leading to directional genetic 
change in populations is found in investigations on radiation-induced 
polygenic variation. Experiments with Drosophila will serve as an 
illustration. Utilizing lines that no longer responded to selection for 
number of sternopleural hairs, Scossiroli (59, 60) and Clayton and 
Robertson (11) were able to induce, with radiation, new genetic vari- 
ability. Selection was practiced for both higher and lower number oi 
hairs, but response was realized only in the high lines. In our labora- 
tory, Daly (12) induced genetic variability for flowering time in 
Arabidopsis thaliiuia with y-radiation and selected for early and late 
flowering. In the R^ generation, 80 lines were developed from selected 
R 2 plants. Up to 50 per cent of the lines selected for earliness were 
found to be significantly earlier than nonirradiated selected control 
populations. None of the late-flowering lines differed significantly 
from the corresponding controls. We have had similar experiences 
with a radiation-induced quantitative character in Nicotiana tab ile- 
um. Although it cannot be stated unequivocally that the mutations 
per se are biased in one direction, it is nevertheless clear that the 
ultimate result of induced variability in quantitative characters may 
frequently, and perhaps characteristically, be a directed change in the 
population. Further information on this phenomenon is urgently 
needed for clarification of its genetic basis as well as its evolutionary 
and practical significance. It is not evident that the "easy direction" 
is necessarily a detrimental one, as e.g., earliness in Arabidopsis; but 
rather it appears to be characteristic of the genotype to respond to a 
mutagen by a greater yield of genetic variability in one direction from 
the parental mean compared to another. 

smith: directed mutation 417 


The most extensive series of experiments on differential muta- 
genicity reported with metazoan material are those of O. G. and M. J. 
Fahmy on Drosophila melanogaster. As in the experiments on higher 
plants, these involve selectivity of mutagenic agents among loci, and 
are concerned with "forward" mutations which are frequently asso- 
ciated with deficiencies. In earlier publications the Fahmy's reported 
the following differences in mutants recovered after treatment with 
certain alkylating agents compared to X-rays: a higher proportion of 
visible to lethal mutations, new visible mutations not previously pro- 
duced spontaneously or after irradiation, and a different distribution 
of breakpoints and lethals in the X chromosome. These claims have 
been critically reviewed recently by Auerbach (4) and Altenburg and 
Altenburg (3) and will not be discussed further here. 

Subsequently, additional papers by Fahmy and Fahmy (19, 20, 
21) have been published giving further experimental evidence of 
differentia] induction of mutations in Drosophila. In one of these (20) 
was reported differential response of different regions of the X 
chromosome in Drosophila to the same mutagenic compound (phenyl- 
alanine mustard). In the region proximal to / they found an excess 
of visibles in the chromosome segment f-car and an excess of lethals 
in the segment from car to the centromere. The Fahmy's contend that 
their data indicate a qualitative difference in the physical nature of 
visibles and lethals. They suggest that visibles may result from 
molecular re-orientation which alters the transmission of the genetic 
code, whereas lethals can be attributed to elimination or inactivation 
of the gene. Obviously, it is not presently possible to test this 
hypothesis. The Fahmy's have recently shown (21) that S-2-chloro- 
ethylcysteine is virtually specific to a particular cell stage of the male 
germ line in Drosophila melanogaster. This compound, they reported, 
is ineffective on mature sperm, but is very effective on the early 

In the silkworm Xakao, Tazima, and Sakurai (51) found differ- 
ences in the relative frequencies of two egg color mutants (pe and re) 
following treatment of the males with two structurally different muta- 
genic mustards, nitromine and alanine "mustard". ^Vith the former 
the ratio of pe to re was over 1 (1.06-1.58), with the latter it was 0.6. 
Most of the mutations were clue to deficiencies so that tentatively 


there appears to be a specilic relation between the chemical agent 
and the position of breakage of the chromosome. 


The most productive and illuminating of recent studies on muta- 
genic specificity are those carried out with microorganisms: fungi, 
bacteria, and bacteriophages. The outstanding advantage of the 
experimental methods used with these materials is the extraordinarily 
high resolving power which permits localization of genetic events with 
sufficient accuracy to approach a correlation with macromolecular 
substructure. These investigations differ from those on higher plants 
and animals in that inter-allelic or iutra-locus rather than inter-locus 
specificity is tested, and also, back-mutation analysis is used almost 

In Neurospora mutagenic specificity has been reported for two 
particular alleles at two loci, one adenineless, the other inositolless, 
which have been combined in a double mutant. These alleles have 
mutated at different relative rates under the action of eioht different 
agents tested, i.e., six chemical mutagens of the alkylating type, ultra- 
violet light, and X-rays (38, 69, 70). The proportion of ad* : inos + 
mutants recovered ranged from 1.5 (ethyl methane sulphonate) to 
3,800 (bromoethyl methane sulphonate) among the chemical muta- 
gens. With ultraviolet light the proportion was 0.5, for X-rays 16, and 
for spontaneous mutations 10. The widely different results with the 
two methane sulphonates, ethyl and bromoethyl, demonstrate 
the pronounced specificity which related chemicals may have for the 
genetic material. A further indication of differential mutagenic effect 
of these two alleles is that, although ultraviolet induced only about 
half as many reversions to ad* as i))Os + , if the UV exposure is preceded 
by a 20-minute treatment with formaldehyde then about four times as 
many ad + as itws* mutants are recovered (45). 

Preliminary evidence of gene-specific effects, i.e., inter-locus 
specificity, for 8-ethoxycaffeine, dimethyl sulfate, and ultraviolet 
light, has been obtained recently in the ascomycete OpJiiostoma mid- 
tifUDiulatui)) (75) and in Scliizosaccharomyces pom be (32). 

In bacteria early evidence indicating that not only different loci 
but also different alleles of the same locus may react differently to the 
same mutagen was reported by Demerec (13) in 1953. Thirty-five 

smith: directed mutation 419 

nutritional deficiencies in Escherichia coli involving 11 amino acids 
were tested for frequency of spontaneous reversions and those induced 
by MnGIo, ultraviolet, and beta-propiolactone. The material included 
a number of alleles governing deficiencies for tryptophane, methio- 
nine, histidine, and leucine. These studies were extended by Glover 
(26) to additional alleles and mutagens. He showed that in E. coli the 
frequency of induced mutations at one locus can be influenced by the 
type of allele present at another locus. 

Extensive evidence of inter-allelic specificity of induced muta- 
tion has now been demonstrated in Salmonella typhimurium. About 
60 per cent of auxotrophic and fermentation alleles in this species are 
"mutagen stable", i.e., although they mutate spontaneously, their 
mutation rates are not increased by treatment with any of a large 
number of mutagens which are effective on other alleles (14). Back- 
mutation tests have revealed that different "mutagen-labile" alleles 
at a locus may differ widely in rates of induced mutability. The most 
extensive analysis reported to date is that of Kirchner (37) with a 
series of alleles at the histidine locus in two strains of S. typhi murium. 
The histidine mutants were either of spontaneous origin or induced 
by either ultraviolet irradiation or 2-aminopurine. The mutagenic 
agents used to revert the mutant alleles to prototrophy were 2-amino- 
purine (2AP), 5-bromodeoxyuridine (BDU), sodium nitrite (N0 2 ), 
t-butyl hydroperoxide (TBP), and beta-propionactone (BPL). Of 54 
mutants tested in one strain 23 were mutagen-stable. Data on the 31 
mutaaen-labile mutants are summarized in condensed form from 
Kirchner' s results in Table 1. 

In the table + signifies an increased back-mutation frequency, — 
means that there was no increase. The highly reactive polymerizing 
agents, BPL and TBP, are grouped, as are the base analogues, BDU 
and 2AP. The mutant alleles show considerable specificity of response 
to the mutagens, and this is further emphasized if the relative amount 
of increased mutation is also taken into consideration. Seventeen of 
the alleles responded to the mutagenic action only of the highly 
reactive compounds, nine to both groups, four only to the analogue 
2AP, and only one to all three categories of mutagenic agents. Eleven 
of the 1 2 auxotrophs that were obtained originally after treatment 
with 2AP showed an increased reversion frequency with BDU, 
whereas the ultraviolet-induced auxotrophs showed no particular 



Table 1. — Induction of Reverse Mutations with Chemical Mutagens in 31 
Mutagen-labile Alleles at the Histidine Locus of Salmonella lyphimurium. * 

Highly reactive 




No. of 























































*After Kirchner (37). See text for meaning of symbols. 

mutational pattern. The interpreted significance of these results will 
be discussed below under mechanisms of mutagenic action. 

Patterns of reversion of 22 mutants at two tryptophan loci (try C 
and try D) in Salmonella typhimurium have been investigated follow- 
ing treatment with four chemical mutagens (15, 56). The compounds 
used were the base analogues 5-bromouracil (5BU) and 2— amino- 
purine (2AP), and the alkylating agents diethyl sulphate (DES) and 
beta-propiolactone (BPL). Many of the alleles responded differently 
to the treatments, as shown in Table 2, where + indicates that rever- 
sions were induced and — that there was no increase in frequency of 
reversions. As with alleles at the histidine locus, those tested at the 
tryptophan loci also showed marked specificity to chemical mutagens. 
DES induced reversions in all 18 of the mutants that reverted, of 

Table 2. — Induction of Reverse Mutations with Chemical Mutagens in 22 Alleles 
at Two Tryptophan Loci of Salmonella typhimurium. * 


ig age 





of alleles 





. + 






























*After Balbinder in Denierec, et at. (15). See text for meaning of symbols. 

smith: directed mutation 421 

which 15 also responded to the base analogues. Mutants that were 
affected by one base analogue were also, with one exception, affected 
by the other. 

Zamenhof (73) has reported that heating (1 35° to 155° C) of vege- 
tative cells (E. coli) or spores (B. subtilis) in the dried state in vacuum 
produced mutations at frequencies of 1 to 10 per cent, certain mutants 
appearing more frequently than others. It was suggested that this 
difference may be attributed to a differential susceptibility to altera- 
tion by heat of different loci. 

The first evidence that the base analogue 5-bromouracil induces 
mutations more frequently at specific sites, distinctly different from 
those most mutable spontaneously ("hot spots"), was afforded by the 
experimental results of Benzer and Freese (8) with rll alleles of bac- 
teriophage T4. These experiments were carried out consequent to 
evidence that 5-bromouracil is incorporated in place of thymine in 
the DXA of phage and bacteria (16, 74) and that this incorporation 
causes mutation (42). Subsequently, Brenner, Benzer, and Barnett 
(9) demonstrated the induction by proflavine of another series of 
mutations which occur preponderantly at sites of the rll locus dis- 
tinctly different from those characteristic of the spontaneous and 
5-bromouracil spectra. The mutant alleles can be mapped by tech- 
niques of high resolution which permit the positioning of mutant 
sites in regions of the order of magnitude of a few nucleotide pairs 
in a DXA molecule. 

Freese (23, 24, 25) has extended these investigations with rll 
alleles to include mutagenic patterns of additional base analogues to 
test the reversion frequencies of induced and spontaneous mutations, 
and particularly to provide a theoretical basis for better understand- 
ing of the mutation process and mutagenic specificity. Of the addi- 
tional analogues investigated, 2— aminopurine, 2,6-diaminopurine, 
and 5-bromodeoxyuridine were found to be mutagenic. The muta- 
bility spectrum of 5-bromodeoxyuridine was essentially the same as 
that of 5-bromouracil but different from that of 2-aminopurine. Each 
differed from the spectrum of spontaneous mutants. A striking differ- 
ence was shown in the pattern of reversion of rl I alleles that had been 
induced originally by different mutagens. Of the mutants induced by 
base analogues, 95 to 98 per cent were reversible by base analogues; 
whereas only 14 per cent of spontaneous mutants and 2 per cent of the 


proflavine mutants were reversible by treatment with base analogues. 
Furthermore, mutants induced by 5-bromouracil are more readily 
reverted by 2-aminopurine than by 5-bromouracil and, vice versa, 
for those originally induced by 2-aminopurine. In summary, muta- 
genic specificity has been demonstrated in ill alleles both for forward 
mutations, i.e. different spectra of "hot spots", and reverse mutations. 
The latter show different reversion rates both in senera] . according 
to the origin of the mutant, as well as individually, according to the 
specific site. 

Mechanisms for Mutagenic Specificity 

Most of the evidence for mutagenic specificity, as can be seen 
from the preceding review, comes from experiments in which nucleic 
acid base analogues or alkylating agents were used as the mutagenic 
agents. It is unlikely that mutations are induced by a common 
mechanism in all organisms or for all mutagens. However, the best 
working hypothesis so far proposed is that hereditary information is 
determined by the base pair sequence in DNA and that true intra- 
genic mutations involve fundamentally an alteration in this sequence. 

Freese (23, 24, 25) postulated that certain base analogues through 
"mistakes" in incorporation and replication of DNA would cause 
errors in base pairing and an ultimate change in sequence of the 
normal bases. Mutations are considered to result from these "transi- 
tions", i.e., replacements of purine by purine or pyrimidine by pyrim- 
idine. For example, diagrammatical ly: 

1. Substitution of a pyrimidine analogue, 5-bromouracil (5BU) 
A-T (5BU) -* A-5BU -* G-5BU ^» G-C 

G-C 5(BU) -> G-5BU -> A-5BU -* A-T 

2. Substitution of a purine analogue, 2-aminopurine (2AP) 
T-A (2AP) -» T-2AP -> C-2AP -* C-G 

C-G (2AP) -> C-2AP -> T-2AP -* T-A 
A and G represent the natural DNA purine bases adenine and 
guanine, respectively; T and C, the pyrimidine bases thymine and 
cytosine. The new stable base pair, i.e., transition from one normal 
purine-pyrimidine pair to another, at the mutant site would be 
expected to be established after two replications of DNA, subsequent 
to the initial incorporation of the analogue. Rudner (55) confirmed 
that mutations induced by 5-bromodeoxyuridine and 2-aminopurine 

smith: directed mutation 423 

in Salmonella typhimurium become established as mutant clones only 
after two DNA replications (50). 

The importance to the problem of mutagenic specificity of these 
recent concepts and experiments is that mutagenic activity can be 
related to genetic molecular structure. The mapping of sites with 
different mutation frequencies under the influence of different muta- 
genic agents can be localized by use of Benzer's high-resolving power 
technique (6, 7), with an accuracy that approaches the nucleotide 
level. If it is assumed that only two kinds of nucleotide pairs are pres- 
ent in DNA, the appearance of different "hot spots" would seem to 
indicate that the mutability of a particular nucleotide pair is depend- 
ent upon its position in the genome, perhaps the specific sequence in a 
particular area of the macromolecule. 

The reversion of base analogue-induced mutants in both phage 
(24, 25) and bacteria (37) by subsequent treatment with base ana- 
logues is confirmatorv evidence that the original mutations were 
caused by transitions for which change in both directions would be 
expected. Reversion by a particular mutagen of specific mutants, 
which involve a restoration of function owing (in theory) to re-estab- 
lishment of a nucleotide sequence, can be considered as specificity in 
the strictest sense. This is the basis of the back-mutation test used in 
Neurospora and bacteria and for reverting rll-type mutants in phage. 

The inirequency of base analogue inducible reversions with 
mutants of spontaneous or proflavine origin led Freese to postulate 
that most of these arose by a process different from "transition", name- 
ly, "transversion", which involves a replacement of a purine by a 
pyrimidine, or vice versa in a given DNA chain. 

Nitrous acid will produce mutations in tobacco mosaic virus 
after hi vitro treatment of isolated RNA (49, 57). Since de-amination 
with nitrous acid converts cytosine to uracil, adenine to hypoxan- 
thine, and guanine to xanthine (58), it is considered that these new 
bases produced /'/) situ will have altered pairing properties and cause 
the change of a nucleotide pair in subsequent replication of DNA 
(25). The mutagenic effect would depend upon "transitions", as with 
the base analogues. Nitrous acid has been found to be highly muta- 
genic for E. coli (34, 35) and to produce a spectrum of mutations 
which differs significantly in some respects from the spontaneous and 
ultraviolet-induced spectra, but not from that caused by disintegra- 


tion of incorporated phosphorus-32. Nitrous-acid-induced ill mu- 
tants in T4 phage are, for the most part (87 per cent), reverted by base 
analogues (25). 

In contrast to the sound theoretical basis (actually as yet unproved 
by experiment) for the mutagenicity, and by plausible extension the 
mutagenic specificity, of base analogues and nitrous acid, there is little 
known at present which would explain mutagenic specificity of alky- 
lating agents. These compounds react with proteins, nucleic acids, 
and nucleoproteins; chiefly with sulphhydryl (— SH), amino (— NH 2 ), 
and acid (-COOH) groups (54, 61). Reactions with the phosphate 
groups, amino and imino groups, and ring nitrogens ol the purines 
and pyrimidines of DNA or precursors are, in all probability, of 
importance in mutagenicity. It is not known which of these reactions 
is primarily responsible for the ultimate genetic changes. Based on 
in vitro reactions some investigators have been inclined to emphasize 
esterification of the phosphate groups (1, 2), which results in the 
formation of unstable triesters; others, the direct alkylation of the 
purine or pyrimidine ring (40, 41). Ethylation in phage DNA may 
be particularly significant and may lead to faulty replication (43). 
Reiner and Zamenhof (53) reported differential sensitivity of DNA 
bases of call thymus when treated in vitro with the alkylating agents 
dimethyl sulphate, diethyl sulphate, and nitrogen mustard. These 
agents attach their alky] group to the purines, but not the pyrimi- 
dines, of DNA on the nitrogen in position 7 (72). 

The alkylated DNA cannot itself be the mutant hereditary deter- 
minant since such a structure could not be replicated by metabolic 
cell processes. A preferred interpretation is that alkylation of a DNA 
group interferes with synthesis so as to increase the chance of errors 
in copying (44). 

Szybalski (66) treated Escherichia coli cells with the alkylating 
agent triethylenemelamine (TEA!) and investigated the mechanism 
by which mutants for streptomycin independence are produced. 
TEA! reacted extensively with pyrimidines and with their nucleo- 
sides and deoxynucleosides, but not with the purines adenine and 
guanine. Szybalski postulated several sequential reaction steps to 
explain the effect of TEAI: (a) semireversible absorption of TEM by 
resting cells; (b) chemical reaction between the mutagen and pyrimi- 
dine deoxynucleotides with the production of fraudulent DNA pre- 

smith: directed mutation 425 

cursors; (c) incorporation of these unnatural analogues into DNA 
during synthesis of the latter; and (d) occurrence of permanent but 
nonlethal errors (mutations) in the base sequence during subsequent 
replication of the modified DNA. It is conceivable that if nitrogen 
mustards alkylate purines (Zamenhof) and ethylene imines pyrimi- 
dines (Szybalski), a basis may be provided for mutagenic specificity 
between these two classes of alkylating agents. 

An ultimate step in demonstrating the mechanism of mutagenic 
specificity would be to show that a hereditary change induced by 
altering the nucleic acid has, in turn, produced a change in amino 
acid sequence of associated protein. Tsugita and Fracnkel-Conrat 
(67) have reported that the protein of a differential host mutant 
isolated after nitrous acid treatment and reconstitution of tobacco 
mosaic virus (TAR') RNA differs from that of the parent strain in 
that three amino acid residues are replaced by three others (proline, 
aspartic acid, and threonine by leucine, alanine, and serine). 

Mutagenic Specificity and 
Directed Mutation in Higher Organisms 

Experiments on microorganisms, fungi, bacteria, and bacterio- 
phage, have provided the unequivocal evidence for mutagenic specific- 
ity. These analyses have, for the most part, made use of techniques 
developed to explore the genetic fine structure of complex loci (14). 
It has been shown that specificity is characteristic of alleles at numer- 
ous sites in the gene locus rather than of the locus in general. This 
intra-locus or inter-allelic specificity has been demonstrated in Xeuro- 
spora and bacteria only by the back-mutation test; in phage by both 
forward ("hot spot" spectra) and reverse mutations. Aueibach and 
Westergaard (5) suggest that forward mutations involve damage or 
malfunction by diverse mechanisms and are therefore less strictly 
specific; whereas, repair of a damaged allele would require a specific 
mechanism. Two mutagens which show an allele-specific effect in a 
back-mutation test may not show any striking specificity in a broad 
spectrum forward-mutation experiment. 

A question of primary interest is, to what extent can the results 
with microorganisms be related or applied to higher organisms 
There are differences in the organization of the heritable material 
from phage to the chromosomes of higher forms in strand composi- 


tion, chemical composition, and possibly in the organization of genet- 
ic fine structure. In higher plants and animals there is now evidence 
that the chromosome consists in cross section of 32 DNA strands 
before synthesis and 64 after DNA synthesis in prophase through 
metaphase (63). In contrast, from T2 phage to bacteria the number 
of DNA strands is considered to be usually 2, with exceptions. Fur- 
thermore, in phage, and apparently in bacteria, there is no close 
association of protein with the DNA as in the nucleoprotein composi- 
tion of chromosomes in higher forms. It is not yet clear that gene loci 
of higher forms are subdivisible into units of mutation, recombina- 
tion, and function in the same way as complex loci in microorganisms; 
but the presence of pseudo-alleles in Drosophila and maize suggests 
that a basic similarity of organization may exist. Westergaard (70) has 
discussed further differences between genetic mechanisms in micro- 
organisms and higher forms -which may be significant in specificity. 

The evidence for mutagenic specificity in higher plants and ani- 
mals rests primarily on (a) the different spectra of gene mutants pro- 
duced by different mutagens in barley and in Drosophila; and (b) 
the differential effect of certain mutagens on chromosome breakage 
both with respect to localization of breaks (usually in heterochromatic 
regions) and in the ratio of gene imitations to chromosomal aberra- 
tions. A common cause for many of these results may be that there is 
regional (particularly heterochromatic) sensitivity of the chromo- 
some to damage by different mutagens and that the expressed change 
may vary in degree with the mutagen to give gross chromosomal 
breaks, small deficiencies (lethals), or "visible" gene mutations. (For 
more detailed discussion, see 4, 5, 17). It has been suggested by West- 
ergaard that the higher proportion of visibles over , lethals in Droso- 
phila and the higher proportion of less drastic viridis over more 
extreme albino chlorophyll mutations in barley may reflect in each 
case a less drastic effect in the mutation event of certain chemical 
mutagens compared to ionizing radiations. 

A large gap remains to be bridged between the intra-locus or site 
specificity observed in microorganisms and the inter-locus or chromo- 
somal region specificity observed in higher plants and animals. The 
former implies a correlation between hereditary function and struc- 
ture within DNA molecules; the latter may depend more on the chem- 
ical bonds that hold the DNA molecules together and also conceiv- 

smith: directed mutation 427 

ably the binding with protein. Attachment of DNA molecules longi- 
tudinally as well as DNA strands laterally may be through bonding of 
diester phosphates with a divalent metal (62). These sites, as well as 
attachment points of DNA through diester phosphate groups to 
amino groups of proteins, may be susceptible to attack by alkylating 
agents which are known specifically to react with phosphate groups 
of DNA (54). What relation may exist between such reactions and 
DNA genetic coding is unknown, but conceivably alteration of base 
sequence between DNA "species" could be as significant as that within 
a single DNA molecule. 

In order to formulate a direct analogy between current explana- 
tions for inter-allelic specificity for reverse (restoration of function) 
mutations in microorganisms in terms of DNA base sequence and 
inter-locus specificity for forward (loss of function) mutations in 
higher forms, it would appear necessary to postulate a qualitative or 
quantitative difference in reaction sites affecting DNA code within 
the functional gene locus involved. That is, either that the gene loci 
differ qualitatively in susceptibility to change into viable or lethal 
mutant sequences by the action of one mutagen compared to another, 
or that there are different numbers of ways (numerically more or 
fewer susceptible sites) among gene loci to change to heritable loss of 
function with one mutagen compared to another. Parenthetically, to 
those who work with cultivated plants forward and reverse mutations 
may often lack evident distinction. 

Close analogues of normal DNA bases have not, to date, been 
reported as mutagenic in higher plants and animals (nebularine may, 
however, be considered in this category). Owing to interest in the use 
of these analogues in cancer chemotherapy much is known about their 
incorporation and antimetabolic activity in mammalian cells (30). 
Less is known about their effects on plants (52). We have 
recently obtained preliminary evidence from experiments with 
tritiated 2-aminopurine that this analogue is incorporated 
in the DNA of nuclei of root tip cells of Vicia faba (Figure 
1). Root tips were placed in a solution of tritiated 2-amino- 
purine (31 X 10~ c M, 3.7 [.ic/ml specific activity) for 8 hours 
at 23° C. They were then grown in Hoagland's solution at 20° C for 
24- and 48-hour periods, respectively; placed for 2 hours in 0.05 per 
cent colchicine; fixed; and stained by the standard Feulgen smear 




* % 

** it**"- ** « 

% « 



Figure 1. — Nuclei of root tip cells of Vicia fab a that have been grown in 
tritiated 2-ami nop urine for S hours. A and B slioiv labeled metaphase 
chromosomes 24 hours after removal from the base analogue solution. 
C siioivs labeled (left) and unlabeled (right) resting nuclei after 4S hours 
of recovery. 

technique. The slides were then dipped in photographic emulsion 
and exposed for 12 days. The 24-hour recovery period slides showed 
evidence of tritium decay in a number of metaphase chromosomes 
(Figure 1, A and B) and some resting nuclei. After 48 hours of recov- 
ery few metaphase chromosomes appeared labeled, but many resting 
nuclei were (Figure 1, C). Some of the label is observed in the cyto- 
plasm. These observations are tentatively interpreted to indicate 

smith: directed mutation 429 

that 2-aminopurine, or at least that part with which the tritium 
label remains, is incorporated into the chromosomes and cytoplasm. 
It has been reported recently (68) that only a small quantity of 
unchanged 2-aminopurine is incorporated into DNA and RNA of 
E. coli, and that a large proportion is transformed into adenine and 

Striking differences in growth of Arabidopsis t Indiana can be 
induced by incorporation of 2-aminopurine and /or 5-iododeoxyu- 
ridine in the medium on which this plant can be cultured. Threshold 
concentrations which barely permit the plants to mature are 0.0008 
M 2-aminopurine, 0.00005 M 5-iododeoxyuridine, and a combina- 
tion of 0.0004 M of 2-aminopurine with 0.00005 M of 5-iododeoxy- 
uridine. There has not yet been time to test for induced mutations 
in the progeny of plants so treated. 

In a symposium on "Mutation and Plant Breeding" it is appro- 
priate to make reference to the use of directed mutation. This has 
been an objective of plant breeders since artificial induction of muta- 
tions became possible. Today we are able, by a choice of mutagenic 
agents, to exercise some selectivity in differentially inducing chromo- 
some breaks compared to gene mutations. This accomplishment is 
recent and there are many ramifications to explore in theory and to 
exploit in practice. Applications can be made in spite of our lack of 
understanding of the phenomena involved. 

An ultimate objective would seem to be able to induce one 
particular mutation to the exclusion of all others. However, a more 
legitimate objective on the basis of present working hypotheses about 
the structures that store and transmit genetic information, i.e., se- 
quences of a four (or fewer) word code, is limited control of the 
spectra of mutations rather than a complete "all-or-none" direction 
of specific mutations. As we have seen, there is limited evidence that 
with higher plants this can be accomplished in broad spectra experi- 
ments by using a variety of mutagens. The theoretical basis for 
specificity in forward mutations among gene loci remains obscure, 
and it will be difficult to devise definite experiments of sufficient 
resolving power with higher plants. The best material lor experi- 
mentation would appear at present to be Neurospora where refined 
tests can be applied to a plant with a hereditary apparatus of appar- 
ently the same level of organization as green plants. Experiments 


using base analogues as mutagens should be particularly informative, 
if indeed, these compounds are mutagenic in higher forms. 


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smith: directed mutation 435 

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Auerbach: I should like to throw in a word of caution about the so- 
called "true specificity" of Doctor Smith. I think one has to distinguish 
between the result, the mutation, and the process by which it is 
obtained. If one accepts the DNA model of the gene and the base-change 
model of mutation, then the result — a change of one allele into another — 
certainly is specific; but this does not mean that the action of the chem- 
ical on the DNA is specific. In fact, there is so far no proof that this 
is so in any case, although there is strong presumptive evidence for it 
in the case of bacteriophage. In other cases, reported for bacteria and 
fungi, mutagen specificity might reside at any one of the steps by which 
the primary chemical event is transformed into a detectable mutant. 
Doctor Haas has dealt with some of these steps, and there may be more 
of them. In some cases it could, in fact, be shown that the detected 
mutagen specificity depended on residual genotype, plating medium, 
or different kinds of pretreatment. This skeptical attitude to the theo- 
retical interpretations of mutagen specificity does not imply a corre- 
sponding skeptical attitude to the possibility of obtaining specific types 
of desirable mutants by chemical treatments. I think, however, that the 
best hope for success in this field lies in attempts to find specificity at 
some of the later steps in mutagenesis. I should recommend strongly to 
investigate the influence of treated stage and experimental conditions 
on the mutation spectrum produced by a given chemical mutagen. 


Peterson: In terms of specificity (and, although it may be a special 
case or class of cases), one might consider the "microspecificity" that is 
involved in mutable gene systems in corn. The specificity involves the 
mutable systems concerned with a specific locus — example a x . Among 
the different strains of maize there are present at this locus five or six 
different systems which have been analyzed with their own controllers 
of mutability. Each of these controllers has their own specific mutator. 
There is no interaction between members of different systems. Here is 
a case where the controller is responsive to its own specific activator. 
Note that the specificity does not only involve the locus, but the specific 
controlling elements. 

Coe: Although I do not work with mutability factors, I feel compelled 
to point out some aspects of these systems in relation to mutational 
specificity. First, these are broad-spectrum mutagens. In the work by 
McClintock originally, and since then in studies by Kramer, Nuffer, 
and others, these systems have been used to induce a variety of changes. 
Still, there is an interesting quantitative specificity involving the chro- 
mosome on which the factor is located, according to Brink. Second, 
these are highly specific mutagens, as shown in the original case reported 
by Rhoades for Dt and A in maize, in further study of A by Nuffer, 
and in the studies of wx by Sprague. In these cases, a mutable system, 
once induced, permits many variations in the expression of the affected 
factor. There is little doubt that employment of variants induced orig- 
inally by mutability factors and further affected by subsequent 
mutations can provide specificity at least as great as that provided by 
present mutagens known to affect higher plants. 

Increasing the Efficiency of Mutation Induction 1 


Washington State University, Pullman, Washington 

Great strides have been made in the study and use of induced 
mutations for the improvement of agricultural crops. This 
success has spurred researchers to accelerate their pace toward obtain- 
ing greater efficiency of mutation induction for plant improvement 
programs of the future. 

A principal approach to this endeavor is through investigations 
of the induced mutation process. The specific objectives of these 
investigations are (a) to increase the total induced mutation yield 
through increasing dose tolerance of tissues and through altering the 
ratios ol mutations to chromosome aberrations, and (b) to control and 
direct the induced mutation process for the production of desired 

These objectives are being pursued through the control and 
manipulation of secondary factors which alter the response of tissues 
to radiation through the use of certain chemical mutagens that 
induce effects different from radiation and through the transfer of 
plant-cell compounds from radio-resistant to radio-sensitive species. 

Several other approaches to increasing mutation yield and to 
increasing the economic feasibility of artificial mutagenesis in future 
plant breeding programs have been investigated. These include (a) 
the use of pollen and embryos in mutation induction, (b) diploidiza- 
tion of loci in polyploids by mutagen treatment, (c) alteration of 
reproductive mechanisms (especially self-incompatibility and apom- 
ixis) by mutagen treatment, and (d) development of efficient recur- 
rent irradiation techniques. Such approaches are concerned with the 
appearance, detection, and selection of induced mutations rather than 
with the induced mutation process per se. However, they are so 
intimately related to the problem of increasing the efficiency of muta- 
tion induction that they must be considered in this discussion. 

Scientific Taper No. 2065 Washington Agricultural Experiment Stations, Pullman, 
Wash. Research supported by U. S. Atomic Energy Commission Contract AT(45-l)-353 
and U. S. Public Health Service Grant A-2184. 



The results already obtained from these investigations concerned 
with increasing the efficiency of mutation induction as well as indica- 
tions of necessary future research will be the subject of this paper. 

Investigations Related to the Induced Mutation Process 

The manipulation of the induced mutation process appears to 
be a promising way for increasing the efficiency of mutation induction 
in plant improvement programs. Here the prime objective is to 
increase total mutation yield. Eventually we may obtain sufficient 
control and direction over the induced mutation process so that 
specific mutations may be produced. 

Some progress toward both objectives has been achieved through 
the control and manipulation of secondary factors that influence the 
response of plant tissues to ionizing radiation. Progress has been 
achieved also through the use of certain chemical mutaoens, such as 
diethyl sulfate and ethyl methane sulfonate, which induce a spectrum 
of effects different from radiation. 

Plant radiobiological studies have shown that there are many 
factors that alter the response of tissues to the sparsely ionizing radi- 
ations such as X and gamma rays. Of these, the factors that have been 
more intensively investigated (21, 29) 2 are genotype, age, stage of 
cellular development, chromosome number and size, nuclear volume, 
moisture, temperature, atmosphere (oxygen, nitrogen, carbon di- 
oxide, hydrogen sulfide, etc.), infra red radiation, and chemicals 
(colchicine, cysteamine, etc.) (21, 29). 

Most modifying factors, however, do not seem to change the 
response of plant tissues to neutrons and other densely ionizing types 
of radiation (21, 29). Therefore, in mutation breeding, neutrons give 
repeated and predictable results regardless of the physiological condi- 
tion and environment of the plant tissue. In spite of this advantage, 
neutron treatments cannot be experimentally modified for increasing 
total mutation yield and altering the induced mutation process. In 
this section, therefore, reference will be made only to the investiga- 
tions involving X and gamma radiation. 

In the investigations of the influence of secondary factors in irrad- 
iated tissues, the seed has been the chief experimental material. This 

-Sec References, page 455. 


is because the seed has several unique properties not possessed by the 
more actively metabolizing tissues, such as the vegetative bud or fresh 

j O O 

pollen. One important characteristic of the seed in these radiobiologi- 
cal studies is its adaptability. It can be irradiated under a range of 
conditions that greatly alter the cellular environment. For instance, 
the seed can be desiccated, soaked, or frozen. It can be maintained 
under a vacuum, almost free of oxygen, or under high pressures of 
oxygen or other gases for extended periods. When dry it is resting, 
almost biologically inert, and the severe environmental treatments 
apparently cause little or no biological damage. After such treatments 
and controlled rehydration, the irradiated seed can be measured for 
damage using several biological criteria. 

Rigid controls of environmental conditions before, during, and 
after exposure to radiation provide a means for learning about the 
specific modifying factors that influence the degree ot radiation 
damage. Controls are also necessary for revealing the basic physical 
and chemical processes responsible for the cell damage incited by 
radiation (23, 32). 

For other reasons, such as ease of handling during irradiation, 
chemical treatment, and culture, the seed has been the most widely 
used plant organ for the induction of mutations in plant breeding. 
Thus, the fundamental studies of the induced mutation process in the 
seed can provide pertinent information which may have an immedi- 
ate and direct application for increasing the efficiency of mutation 
induction in crop plants. 

Among the seeds used for radiobiological and chemical mutagen 
studies, the barley seed (Hordeum vulgare or Hordeum distichum) 
has been by far the most useful (29). The chief advantage in using this 
seed in these studies is that the effects caused by radiation and by 
chemical mutagens can be measured in terms of several criteria. These 
include the linear rate of Mi seedling growth over a finite period; 
survival of Mi plants following treatment and frequencies of seedling 
leaf flecking and cholorophyll-deficient chimeras in the Mi plants; the 
number of spikes per Mi plant and number of seedlings produced 
in the M x plant and spike; frequencies of chromosome bridges and 
fragments in the shoot-tips of mutagen-treated seeds; chromosome 
translocation and inversions at meiosis in the Mi plants; and the fre- 
quency and proportion of chlorophyll-deficient seedling mutations 


among the M 2 progenies. These criteria present a broad base for inter- 
preting the effects of the radiation and the interaction between radi- 
ation effects and secondary factors and for comparative analysis of the 
action of chemical mutagens on plant cells. By measuring both chro- 
mosomal and genetic damage induced by the same treatment, one 
may obtain a greater understanding of the nature and control of the 
induced mutation process. 

Increasing Induced Mutation Yield 

An increased total of induced mutations has been sought 
through techniques that increase the radiation dose tolerance of tis- 
sues and reduce the amount of chromosome damage without an associ- 
ated reduction of induced mutation frequencies in the treated cells. 
These techniques have involved the control and manipulation of 
secondary factors in irradiated seeds, the transfer of plant-cell com- 
pounds from radio-resistant to radio-sensitive species, and the use of 
the chemical mutagens diethyl sulfate and ethyl methane sulfonate 
which induce high mutation frequencies but relatively few chromo- 
some aberrations. 

Increasing the radiation dose tolerance 

Under normal conditions, plant tissues have distinct levels of 
tolerance to doses of ionizing radiation. Doses of radiation above these 
levels lead to such low cell survival that most mutations are lost with- 
in the plant. These levels, which vary from species to species, are 
governed to a large extent by numerous secondary factors, only a few 
of which are understood. Nevertheless, through proper manipula- 
tion of a few of them in irradiated seeds, it has been possible to 
increase the radiation dose tolerance, and, hence, mutation yield. 

Extensive studies have shown that after-effects, as they are related 
to time, oxygen, moisture, and temperature, are a most important 
influence on the degree of damage in X- and gamma-rayed seeds 
(3, 5, 6, 9, 10, 21, 23, 29, 30, 31, 32). Through proper control and 
manipulation of oxygen, moisture, and temperature before, during, 
and after irradiation, the dose tolerance of the seed has been greatly 
altered. Because of the several recent and extensive reviews on this 
subject, only a brief summary will be presented here. 

Oxygen-effects and after-effects in irradiated barley seeds increase 
radiation-induced damage, which in turn causes low cell and plant 


x - Planted immediately 
O- Stored in O2 planted 

I week later 
Q - Stored in N2 planted 

I week later 

Seeds 4% moisture 

Gamma rays 

Figure 1. — Seedling Jieight response of barley seeds irradiated frorii 
10 to 60 Kr and then stored in N 2 arid 2 for 1 week before planting. 



survival. Typical oxygen- and after-effect data are presented in Figure 
1. Seeds at 4 per cent moisture were gamma-rayed from 10 Kr to 60 Kr 
and then either planted immediately or stored in oxygen or nitrogen 
for 1 week before planting. Severe after-effects occurred in the oxygen- 
stored seeds, while considerably fewer after-effects occurred in the 
nitrogen-stored seeds. It is also interesting to note that a typical dose 
response was exhibited by the seeds planted immediately after radi- 
ation, but no dose response occurred in the irradiated, stored seeds. 

These oxygen- and after-effects now can be controlled (32). They 
occur only slightly in seeds with over 12 per cent moisture, and are 
even more reduced if these moist seeds are partially evacuated of 
oxygen before irradiation and then irradiated at dry ice (—78° C) 
temperature. Controlling both the oxygen- and after-effects also 
depends on the rehydration of the irradiated seeds in oxygen-free 
water. The length of time necessary for rehydration depends upon 
temperature of the water. Usually 1 1/ 2 to 2 hours at 30° C is adequate. 
Longer rehydration may be required for very dry seeds. Typical 
results of these environmental controls on irradiated barley seeds are 
shown in Figure 2. 

By manipulating the oxygen and moisture content and control- 
line - the after-effects, seeds can be made to tolerate and survive high 


"^-^.-a-.V 13% EMBRYO MOISTURE 


HELIUM BUBBLED x "~""' s **** 1 4, l 


■..■■■a — 


■ — .t 

10 20 30 40 50 60 70 80 90 100 

Figure 2. — The interrelation of moisture content and oxygen effect 
when seeds are frozen in dry ice (S0°C) prior to and during irradia- 


radiation dosages. For instance, air-dried barley seeds, about 9 per 
cent moisture, given no proper pre- or post-treatment to control the 
oxygen- and after-effects can not survive 20 Kr of X-rays (even less if 
stored), whereas seeds given proper treatments to control after-effects 
can survive dosages of 80 to 100 Kr (23, 24). Mutation yields from 
these latter dosages are the highest yet recorded for irradiated 
barley seeds. Furthermore, with these treatments, more predict- 
able and reproducible results have been obtained in seed irradiation 

Another important means to alter the dose tolerance of plant tis- 
sues is the application of certain compounds that affect plant response 
to radiation. Only recently has this possibility been investigated 
(1, 2). Soaking pine seeds in extracts of mustard seeds increased their 
LD.-,o, where soaking mustard seeds in extracts of pine seeds reduced 
their LD.-.o- Protective effects were obtained after treatment with both 
acidic and basic fractions from mustard seeds. Furthermore, the phen- 
olic and neutral fractions of the mustard seed extracts contained ger- 
mination inhibitors. A study of the chemical fractions of pine seed 
extract revealed that the sensitizing action was centered in the 
organic acid fraction and was possibly a result of the presence of oleic 
and linoleic acids. It was suggested (1) that peroxides produced by 
irradiation of the unsaturated fatty acids may be the primary cause of 
the sensitization. 

To date, the LD 50 has been measured only in terms of seed ger- 
mination. The influence of these radio-resistant or radio-sensitive 
fractions on the induced-mutation process has not been investigated. 
It appears, however, that this line of research may open up a whole 
new approach to the manipulation and possible control of the radi- 
ation tolerance of plants for increased mutation induction. 

Increasing the ratio of induced mututations to chromosome 

Possibly one of the chief causes of cell death in tissues irradiated 
at high dosages are chromosome aberration. Caldecott (3) has shown 
that chromosome aberrations in barley increase exponentially, where- 
as the mutations increase linearly with dose. This means that at high 
doses, chromosome aberrations concurrently induced limit the 
mutation yield. Therefore, for radiation to become more efficient 
for inducing mutations in plant breeding, the frequency of chromo- 


some aberrations in irradiated cells must be decreased. Such a decrease 
will lead to a more favorable ratio of mutations to chromosome 

Some progress lias already been achieved in decreasing frequen- 
cies of chromosome aberration in irradiated cells. Several reports 
(7, 12, 13, 17, 28) have indicated that manipulation of various factors 
of seed environment may decrease X-ray-induced chromosome aber- 
ration frequencies and maintain or increase the Mi plant survival 
and Mo mutation frequency. 

A convincing demonstration of a possible control over the ratio 
of radiation-induced mutations to aberrations has been heat-shock 
post-treatments in our laboratory (23, 24). 3 Seeds were frozen in dry 
ice at about —78° C, exposed to 80 Kr and 100 Kr, then immediately 
plunged into water at 60° C for 1 minute, and hydrated in distilled 
oxygen-free water at 32° C for \\/ 2 hours. Compared with the non- 
shocked treatments, there was an appreciable increase in survival of 
the Mi plants from the heat-shocked seeds. Mutation yield from these 
treatments was higher than any previously recorded for irradiated 
barley seeds. It was determined that the improved survival rate after 
heat-shock was in part due to a reduction of chromosome aberrations 
(Table 1). We have more recently found that certain growth- 
reo-ulator treatments after X-radiation also reduce chromosome 
aberration frequencies without decreasing mutation frequencies. 

The mechanism conditioning this more favorable ratio of muta- 
tions to chromosome aberrations is yet unknown. Apparently it must 
affect processes that either cause mutations and gross chromosome 
aberrations at different frequencies or that result in restitution or 
repair of chromosome damage. 

Considerable support for the possibility of obtaining more favor- 
able ratios of mutations to chromosome aberrations in irradiated 
seeds has come from experiments involving certain chemical muta- 
gens. It must be recalled here that most chemical mutagens are con- 
sidered to be radiomimetic since their effects and even possibly their 
basic mechanisms of action are similar to those of ionizing radiations. 
Diethyl sulfate and ethyl methane sulfonate have produced excep- 
tionally high frequencies of mutations in barley (11, 15, 18, 19, 20, 

3 More recent studies have established that the heat shock response, at least in part, 
depends on oxygen and moisture in the pre-radiation storage environment of the seeds. 



Table 1. — Response of Irradiated Barley Seeds to Post-Radiation 
Heat-shock Treatment. 

80 Kr. 

100 Kr. 

Aberrations Dry seeds 

Moist seeds 

Dry seeds 

Moist seeds 

No No No No 

Shock shock Shock shock Shock shock Shock shock 

Cells scored 



























Total frag- 





























Survival * 










Per cent/ 

Mi plant 









Per cent/ 

Mi spike 









Per cent/ 

Mo seedling 









'Percentage of control. 
tNot recorded. 

24). (See Figure 3.) The important new finding, however, is that these 
chemicals produce very few gross chromosome aberrations of the 
interchange type. (18, 24). (See Table 2.) 

Recently, it has been found that chromosome bridges and, less 
frequently, fragments and bridges occur at anaphase I in M, plants 

Table 2. — Frequencies of Chromosome Interchanges in Shoot-tip Cells of Treated 

Seeds and in Pollen Mother Cells of Mi Plants Following Treatment of 

Barley Seeds with Diethyl Sulfate and Gamma Rays. 











vival * 


per cell 

per cell 



Gamma radiation 60 Kr. 







Diethyl sulfate (1) 1 ]4 hr. 








Diethyl sulfate (2) 1 ]/ 2 hr. 30°Cf 







•Percentage of control. 

tMore severe treatment with new, more purified chemical. 


90|- • • Diethyl Sulfate 

O O 

A A Gamma Radiation 









Mutated Plants 

Reduction of 

Mutated Plants 

/ Reduction of *V f 

/ y Survivol o I 

20 40 60 80 100 Kr gamma rays 

I 11/2 2 Hrs., Diethyl Sulfate 

Mutagen Treatment 
Figure 3. — Comparison of effects of diethyl sulfate and gamma rays 
on sii)~uival of M 1 plants and frequencies of chlorophyll-deficient muta- 
tions in M 2 barley seedlings. 


from the chemical treatments. These aberrations indicate that inver- 
sions have been induced by the chemicals. This is of considerable 
interest since inversions are relatively rare in plants from irradiated 
seed. Furthermore, the finding that these chemicals induce a higher 
frequency of intra-chromosomal compared to inter-chromosomal type 
of aberration than radiation suggests a more subtle and delayed effect 
of the chemicals on the chromosomes. 

It is already apparent that the effectiveness and efficiency of the 
chemical agents may be greatly influenced by modifying factors. For 
example, the uniformity in the growth of seedlings from seeds treated 
with diethyl sulfate seems to be measurably increased by the addi- 
tion of certain divalent cations to the treatment solution. These 
results suggest that the hydrolysis product, ethyl sulfuric acid, in the 
treatment solution may chelate divalent cations in the cell membranes 
and facilitate the penetration of the active agent into the cells of the 

It is also interesting to speculate that the hydrolysis products 
from both diethyl sulfate and ethyl methane sulfonate may have 
important roles in the mutagenic and cytogenetic activity of these 
agents. Alcohol might be expected to act as a surface-active agent 
aiding penetration of the alkylating agents. The ethyl sulfuric and 
methane sulfonic acids, weak chelating agents, may affect cell mem- 
brane permeability. Also, by their chelating action on the divalent 
cations bonding the DNA, they may cause an opening-up of the multi- 
stranded chromosome, permitting the alkylation of additional as 
well as internal sites, as suggested by the Steffensen model (38). It is 
now already indicated, however, from the results of Strauss (39) that 
in bacteriophage at least (which seems to have a double or single- 
stranded chromosome), the chelating effect does not appreciably influ- 
ence the frequency of induced mutations but reduces survival. Thus, 
the effect hypothesized may relate only to higher organisms. 

It is also possible that chromosome aberrations of the type 
observed may result from the temporary chelation of the divalent 
ions bonding the DNA rather than from the alkylation of bases or 
phosphate groups. However, what is more important is that these 
possibilities can be subjected to experimental test, and future studies 
may provide even more exciting information on the nature of 
chemical reactions leading to chromosome aberrations and mutations. 


It is obvious that more intensive studies on the nature and origin 
of chromosome aberrations and repair of chromosome damage are 
required before we can more effectively reduce or eliminate chromo- 
some aberrations and increase mutations in irradiated cells. Investi- 
gations pursued by Wolff and his collaborators (42) have shed some 
light on the factors that govern the rejoining and restitution of 
chromosome breaks. However, recent papers by Revell (35, 36) sug- 
gest that radiation-induced chromosome reunions and exchanges or 
incomplete reunions and exchanges are derived from possible chemi- 
cal bonding initiated by the ionizing radiation. If this suggested 
mechanism proves to be real, it could mean that radiation-induced 
changes in the chromosome may not result in aberrations until the 
late prophase of the irradiated cells. This suggests that a relatively 
long period during mitosis would be available for altering the course 
of radiation damage to the chromosomes. Experiments designed to 
test and then use this new hypothesis for the reduction or elimination 
of qtoss chromosome aberrations and for increasing the efficiencv of 
mutation induction are now underway in our laboratory. 

Directing the Induced Mutation Process 

The greatest concentration of effort in directing the induced 
mutation process has been through the manipulation of secondary 
factors in irradiated seeds and the use of chemical mutagens. Here 
again, these studies are only possible because of the wide variety 
of conditions and controls that can be applied to seeds. 

Indications that the spectrum of chlorophyll-deficient muta- 
tions in barley can be altered have been published by the Swedish 
group (41) in the past several years. Small shifts of the mutation 
spectrum have been caused by certain variations of the experimental 
conditions during X-irradiation. It also has been reported that the 
chlorophyll-deficient mutations produced by neutrons may show a 
different spectrum from those induced by X-rays. However, these 
results have not always been reproducible. It has not been determined 
whether the results are due to alterations in the sensitivities of indi- 
vidual genes or to unequal selection of these mutations during the 
ontogeny of the plant. 

Probably some of the most convincing evidence that different 
mutagenic treatments can produce differential mutability of loci is 


from studies of the erectoid mutations of barley. Treatment with 
densely ionizing neutrons will induce more erectoid mutants than 
treatments with sparsely ionizing X and gamma rays. Furthermore, 
neutron treatments will affect only some individual erectoid loci; 
with X-ray, other loci will be affected (16). Whether these results are 
due to differential sensitivities of specific loci, to changes in modify- 
ing genes at other loci, to environmental or other modifying factors, 
or to differential selection of certain mutations, the practical impor- 
tance of these findings still remains — a specific changed type can be 
more readily selected from one kind of treatment than from another. 

Some of our investigations on the influence of moisture and 
heat shock on radiation damage also show a shift in the mutation 
spectrum among M 2 chlorophyll-deficient seedlings. From dry seeds 
but not from moist, the proportions of albino and viridis to total 
mutants decreased with increasing doses. On the other hand, the 
albo-viridis and xantlia mutations from the dry seeds increased with 
increasing radiation dose. The mutation spectrum in the heat-shock 
experiments more closely resembled the spectrum obtained from 
seeds exposed to lower doses (24). There is some indication that 
these alterations in the proportion of mutation types are associated 
with the selective influence of induced chromosome aberrations. 
A previous report (10) has suggested that the proportion of viridis 
mutants was smaller and that of the xantJia type larger at higher 
radiation doses. This alteration appeared to be associated with spike 
sterility which, in turn, may be a result of chromosome aberra- 

After treatments of barley seeds with several chemical muta- 
gens, considerable variation in the proportion of M 2 chlorophyll- 
deficient seedling mutants has been observed (11, 15, 41). In our 
studies, it has been found that the gamma-radiation spectrum of 
induced-mutation types differs from that of diethyl sulfate (Table 3). 
This difference might be related to the fact that very few chromo- 
some aberrations are produced by the chemical. In the plant from 
irradiated seed, aberrations would influence the selection and recov- 
ery of induced mutations. On the other hand, it is possible that in the 
DNA molecule some of these chemicals can bring about a specific 
change which is expressed as a specific type of mutation. Stronger 
evidence that there is a differential sensitivity of individual genes 



Table 3. — Comparison of Mutation Spectra Induced by Diethyl Sulfate 
and Gamma Radiation. 


cent of 

99% confidence 

Number of mutations 

total mutations 




































2.2- 6.7 







3.4- 9.0 

3.6- 9.2 






1.8- 6.1 





to chemical treatment has been obtained from the experiments of 
Westergaard and his associates with back mutations with Xenro- 
spora (40). 

Investigations Related to Appearance, Detection, 
and Selection of Induced Mutations 

Several other approaches to greater efficiency and to increased 
economic feasibility of artificial mutagenesis in plant breeding 
should be explored more intensely. These include (a) the use 6f 
pollen, zygotes, and embryos in mutation induction; (b) diploid- 
ization of loci in polyploids by mutagen treatment; (c) alteration 
of reproductive mechanisms, especially self-incompatibility and 
apomixis, by mutagen treatment; and (d) development of efficient 
recurrent mutagen treatment techniques. Most of these are con- 
cerned with the appearance, detection, and selection of mutations 
rather than with the induction of mutations per se. However, they 
will be considered briefly here since progress along these lines can 
extend the usefulness of mutagen techniques. 

Irradiation of Pollen, Zygotes, and Embryos 
Most mutation breeding experiments have been initiated either 
with seeds of sexually propagated crops or with buds of cuttings in 
vegetatively propagated plants. However, both seeds and buds are 
multi-cellular, complex tissues presenting certain difficulties for 
induced mutation detection and selection. These difficulties relate 


to (a) the chimeral nature of mutated sectors; (b) a rapid loss of 
induced cytogenetic changes during subsequent mitoses and, in 
sexual crops, during meiosis of the Mi plant; and (3) overgrowth 
of mutated cells by nonmutated cells (8, 33). 

Greater efficiency might be obtained by irradiating a plant when 
half or all of its o-enes are located in an individual cell. This imme- 
diately suggests the application of mutagens to pollen or zygotes. 
However, pollen, zygotes, and embryos have been used for inducing 
mutations in very few physical or chemical mutagen experiments. 
Conger (8) has discussed in some detail a possible improved method 
for inducing mutation by using pollen instead of seeds, and some 
results have been obtained. 

Much more work needs to be done on this technique, since 
its efficiency does not approach modern seed irradiation technics. 
From exploratory studies, Konzak (21) has noted that pollens of 
diploid maize and barley suffered such pronounced damage from 
radiation that high seedling mutation rates were not obtained. In 
one study (22), barley pollen was exposed to doses of X-rays, thermal 
neutrons, and ultraviolet radiation. Records were kept of the num- 
ber of seeds set, number of aborted and shriveled seeds produced, the 
number and condition of the plants grown from the Mi seed, and the 
number of second generation progenies showing mutations. Mi plants 
carrying chromosome translocations or inversions and the frequencies 
of semi-sterility in M 2 and M 3 progenies were also recorded. Although 
the numbers studied, totalling approximately 1,200 seeds from 
irradiated pollen, were not large enough for firm conclusions, a dis- 
appointingly low number of mutations was recovered for the effort 
expended. Moreover, the ratio of mutations to chromosome trans- 
locations induced by X-rays and neutrons in pollen was considerably 
less than observed in seed. Several chromosome translocations were 
induced by the ultraviolet radiation treatments, but the frequency 
of mutations observed also was relatively low compared with that 
obtained from X-ray treatment of seeds. 

A renewal of extensive work with pollen would seem to be war- 
ranted, however, if for no other reason than to take advantage of the 
fact that the mathematical analysis of induced mutation rates can 
be more critically estimated. Also, with irradiated pollen, an Mi 
plant carrying a mutation is almost always completely heterozygous 


for the mutations rather than a sectorial chimera as is the case with 
irradiated seeds. Moreover, better comparisons would be possible 
with chemical and ultraviolet treatments of pollen. Before these 
studies can be practical, however, certain major difficulties must be 
surmounted. These include maintaining pollen viability in some 
species and adequate control over modifying factors. 

As early as 1930, Stadler (37) compared the probable advantages 
of inducing mutations in zygotes or pro-embryos over those of seeds. 
However, because of several difficulties inherent in these materials, 
including the low dose tolerance, no further studies were conducted 
to explore the merit of zygotes or even immature embryos in higher 
plants as organs tor the induction of mutations by use of radiation. 

In the past two years, the use of these organs for mutation 
induction in barley has been investigated with some promising 
results (26, 27). The irradiation of pro-embryos has resulted in a sig- 
nificant increase in the size of the M 2 mutant population and in 
the frequencies of tillers of a single plant with the same mutation 
compared with that obtained from treated seed. These advantages 
indicate that irradiated zygotes or pro-embryos may have definite 
practical significance lor the induction of mutations in certain self- 
fertilizing crops. However, the efficiency of this technique will need 
considerable improvement to approach the results now possible 
with seed irradiation. 

Diploidization of Polyploids 

Another means of facilitating the detection and selection of 
mutations is by the diploidization of loci in polyploids through 
mutagen treatment. Many of our crop species are polyploids; thus 
the duplication, triplication, etc., of loci, restrict the segregation 
and appearance of induced mutations. It may be possible to alter 
these loci through mutagen treatment so that they behave as in a 
diploid. Their alleles would then segregate in normal Mendelian 
ratios, and induced changes at these loci would be easier to detect. 
Some success with this technique has already been achieved and 
reported at this symposium (6) and elsewhere (4). 

Alteration of Reproductive Mechanisms 

The reproductive system in cross-fertilized species influences 
and limits the segregation and recovery of induced mutations. For 


this reason, very little research has been conducted to determine 
the possibilities of mutation induction in these species. In the 
self-incompatible species, this limitation might be overcome by 
producing self-compatible or self-fertile strains of these plants through 
physical or chemical mutagen-induced changes of the self-incom- 
patible (S) alleles. A precedent for this approach may be found in 
a paper by Lewis (25). 

Recurrent Mutagen Treatments 

Another means to produce higher mutation yields is mutagen 
treatment of successive seed generations prior to selection of muta- 
tions. In a self-fertile crop, such as barley, the procedure would be 
to treat the seeds from fertile inflorescences at each generation. As 
shown by Gaul (14), the frequency of point mutations or muta- 
tions not associated with gross aberrations is independent of fer- 
tility. Thus, through successive mutagen treatments of seeds from 
only fertile inflorescences, it should be possible to accumulate high 
frequencies of mutations in a line. 

This approach, as outlined by Caldecott (4) in more detail for 
irradiation treatments, is essentially a mechanical means for remov- 
ing induced chromosome aberrations while increasing the total 
accumulated radiation dose that can be applied. It is probably far 
less efficient than the controlled seed irradiation procedures described 
earlier, but since it can be applied independently from other con- 
trols or treatments, it has special merit. 

Summary and Conclusions 

Considerable progress has been made in recent years in increas- 
ing the efficiency of mutation induction by radiation in seeds. 
Greater precision, repeatability, and prediction in radiation experi- 
ments have been achieved and higher frequencies of induced muta- 
tions have been obtained through rigid control of after-effects, 
moisture, oxygen, and temperature. Furthermore, through con- 
trol and manipulation of these secondary factors, certain deleterious 
effects of the radiation, such as low survival and high chromosome 
aberration frequencies can be reduced; and some control and 
direction of the induced mutation process may be realized. 

Certain chemical mutagens, such as diethyl sulfate and ethyl 


methane sulfonate, may be more efficient than radiation in certain 
aspects of mutation induction. In barley seeds, these chemicals 
induce very high mutation frequencies without an appreciable fre- 
quency of gross chromosome aberrations, and apparently produce 
a slightly different mutation spectra from X or gamma rays. 

It is obvious, however, that the required information for still 
greater progress in increased efficiency of mutation induction in this 
area is at present only fragmentary. Much more work should be 
conducted in radiobiology, biochemistry, genetics, and cytology to 
obtain more adequate information on induced mutations for plant 
breeders of the future. 

In the final analysis, it is apparent that the greatest advances 
toward increasing the efficiency of mutation induction in plant 
tissues will come only when the basic mechanisms of radiation and 
chemical mutagen effects in cells are completely understood. Studies 
of the influence of secondary factors on radiation damage in seeds 
have extended our concept of the physical and chemical pathways 
of effects of ionizing radiation in cells. They have shown that much 
of the radiation damage in the chromosomes or genes is due not to 
the direct ionizing event but to intermediate agents (10, 32). Cer- 
tain of these agents, free radicals, have been detected in seeds 
following irradiation. Furthermore, they seem to be influenced by 
the various secondary environmental factors to the same degree as 
the radiobiological effects. The apparent association of these radia- 
tion-induced radicals with biological damage was first demonstrated 
with barley seeds and now has been confirmed and extended with 
bacterial spores (34). Further studies on the nature of these inter- 
mediate agents are necessary to increase our knowledge of mech- 
anisms that produce radiation damage in seeds. 

It is unfortunate that much useful information obtained from 
the seed radiobiological research has not been generally applied 
by plant scientists, particularly plant breeders, in seed radiation 
studies for practical or theoretical purposes. The techniques for 
controlling secondary factors that have been described here and in 
more detail in other publications have been ignored to a large 
extent. And yet it should now be apparent to all who use seeds in 
radiation experiments that the biological effects of a given dose of 
radiation are meaningless unless the moisture content of the seed 


is known and unless oxygen, temperature, and after-effects have 
been adequately controlled. Certainly, information is now avail- 
able that will provide greater precision, repetition, and prediction 
in seed radiation experiments. A discussion of the techniques and 
methods that should be followed in all seed radiation experiments 
will be summarized by the authors in an early issue of Radiation 

As we consider the needs of future plant breeders in terms of 
genetic \ariability and of techniques for manipulating this vari- 
ability, it is imperative that we keep abreast of modern advances in 
genetics and biochemistry. Many of these advances may well pro- 
vide means of inducing mutations in plants with greater efficiency 
than ever before possible with physical or chemical mutagens. The 
oene mutation controlling elements of McClintock and Brink, and 
gene transduction through plant viruses may well be a future means 
for inducing high frequencies of directed mutations in crop plants. 
These and other similar possibilities, however, may be completely 
overshadowed when we consider that deoxyribonucleic acid (DNA), 
the basic genetic material, may soon be synthesized artificially. When 
this is accomplished and when methods for its incorporation into 
reduplicating chromosomes are found, man may have his most 
powerful means for controlling and directing the heredity of the 
plants and animals upon which he depends for his survival. 


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The oxygen effect in X-rayed barley seeds. In Effects of Ionizing 
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ruhe, Germany, Aug. S-12. 

33. Osborne, Thomas, S. 1957. Symposium on Radiation genetics: 

Mutation production by ionizing radiation. Proc. Soil and Crop 
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34. Powers, E. L., Webb, R. B., and Ehret, C. F. I960. Storage, trans- 

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age and its relevance to a new interpretation of chromatid aber- 
rations induced by ionizing radiations. Proc. Roy. Soc. (Loudon) 
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ical agents. In Chcmischc Mutagenese. Abhandl. dtsch. Akad. 
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39. Strauss, B. Unpublished communication. 

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Davies: I wish to comment on acute irradiation. We have compared 
somatic mutation rates at the V b y locus in Trifolium repens with dose 
rates from 25 to 80,000 rads per hour, and find that we obtain about 
10 times as many with the latter as the former. 

Lewis: If I understood the use of terms mutation yield and mutation 
spectrum correctly, would you not finally be more interested in spectrum 


than yield? In other words, the emphasis would be on different rmitants 
rather than repeats of the same mutant. Possibly yield and spectrum 
are closely correlated. 

Nilan: Yes, as we have discussed in the paper we are very much inter- 
ested in finding ways of altering the mutation spectrum and of directing 
the induced mutation process toward specific desired changes. How- 
ever, we think that increasing yield of many mutations in a genotype 
is also very important. I am afraid we do not have enough information 
yet to determine the relationship, if any, of yield and spectrum. 

Brawn: Albinos may be due to a variety of causes ranging from point 
mutations to deletions of some size. Beneficial mutations are not likely 
the result of deletions and so may differ in environment-treatment 
response. What is the likelihood that the treatments you have used 
may be specific for inducing only those mutants (albinos, etc.) which 
you used as the criteria for measuring mutation rate? Are beneficial 
mutants induced to the same degree and in the same way? 

Grobman: Doctor Brawn's question has relevance in regard to resolution 
limits in the evaluation of different mutagens as to their effects in pro- 
ducing variable mutation spectra. I am particularly disturbed with the 
presentation in this paper and the preceding ones of chlorophyll muta- 
tion data to substantiate differences in mutation spectra due to different 
mutagens. The synthesis of chlorophylls in chloroplast grana is a Aery 
complex biochemical process, conditioned by at least 20 environmental 
factors, and in barley by at least 200 to 300 different genes, as it appears 
to be evidenced by Swedish mutation work (and Doctor MacKey may 
check me on this point). Scoring of chlorophyll mutations would, there- 
fore, be a gross and inefficient manner of defining the limits of particular 
mutagen-induced mutation spectra. Characters conditioned in their 
phenotypic experssion by few genes, if selected, would give a much 
finer resolution of mutation spectra, in studies where differences between 
different mutagens were of primary interest. 

Nilan: On the basis of the high chlorophyll mutation frequencies from 
diethyl sulfate, we have looked for morphological mutants in mature M 2 
barley plants. Last summer high frequencies of these mutants were found. 
This indicates, I believe, that there is a good correlation between the 
chlorophyll and morphological mutants in barley. 


Mfriclk, R. P.: One factor which has not been given much consideration 
to date at this meeting is that of dose-rate. By this, I do not mean chronic 
versus acute, but rather, within the framework of acute or semiacute. 
In our proembryo irradiations of barley, for example, we have achieved 
more than a 6-fold increase in mutation rate to albinism by a 4-fold 
decrease in dose-rate, when total dose and irradiation conditions were 
identical. These mutations were in all cases ones in which the mutant- 
carrying sector encompassed two or more heads in the X-l plants, and 
are probably of a similar nature since segregation ratios in the X-2 
were the same. With the continued interest in increasing mutation 
rates, while at the same time decreasing the frequency of gross chromo- 
somal aberrations, we suggest that, especially in instances when it is 
desirable to use large radiation doses, the usual dose-rates be reduced 
by 1 / 4 , 1 /w, or so. An optimum balance between total dose and rate 
of delivery should be sought, since lowering the dose-rate too much may 
result in loss of effective mutation frequencies. 

The Efficacy of Mutation Breeding 1 


Xortli Carolina Agricultural Experiment Station, Raleigh, N. C. 

It is the purpose of this paper to discuss some of the factors 
expected to affect the efficacy of mutation breeding and to bring 
forward data from which comparisons of efficacy may be drawn 
between hybridization and mutation as methods of generating 
genetic variance for purposes of selection. The paper will not be 
concerned with the efficacy of various selection and screening pro- 
cedures, it being considered that these, important as they are, are 
by no means restricted to mutation breeding and, if improved, 
would benefit any method of breeding. The discussion has mean- 
ing in man's effort to create plant breeding capital in contrast to 
the accepted methods of liquidating natural mutational assets 
accumulated over evolutionary time. 

The basic questions are (a) whether the power of present known 
mutagens is such as to produce the kinds of changes which natural 
mutation and selection have provided us, and (b) given this power, 
with what efficacy can it be used with plants of various breeding 

The Power of Mutagens 

There is reason to believe that despite the different frequen- 
cies of similar mutations obtained with different mutagens (15, 
14, 54), 2 the chief limiting factor in mutation production and 
mutant recovery is the genie constitution of the experimental organ- 
ism and not the type of mutagen used. Thus, for the plant breeder, 
a knowledge of what might be called mutant expectation in his 
material may be more important than a resolution of the mechanism 
of mutational change at the submicroscopic level. 

Contribution from the Field Crops Department, North Carolina Agr. Exp. Sta., 
Raleigh, N. C. Published with the approval of the Director of Research as Paper No. 
1253 of the Journal Series. This work was supported by the U. S. Atomic Energy Com- 
mission as part of Contract AT-(40-l)-1747. 

"See References, page 482. 



Useful mutant occurrences in many kinds of plants have been 
reported and a number of reviews have been published concerning 
their usefulness in plant breeding (2, 17, 31, 38, 40, 46, 48, 51, 55). 
Numerous characteristics, such as grain yield, straw stiffness, chloro- 
phyll type, maturity date, grain weight, plant height, disease resist- 
ance, alkaloid content, etc., have been observed to vary significantly 
under the influence of mutagens. MacKey (31) concluded his paper 
on mutation breeding in Europe by saying that the evidence attests 
that any agronomic characteristic can be improved by induced 
mutations. The more recent reviews of Borg, et al. (2), Gaul (17), 
Prakken (40), Scholz (46), and Stubbe (51) have given generally 
optimistic accounts of the possibilities of mutation breeding. 

In interpreting the results obtained with useful individual 
mutants it should be remembered that the statistical characteristics 
and therefore interpretations are considerably different in situa- 
tions where useful deviates are recognized among a great number 
of variants in an artificially mutated population and in situations 
where a character is pre-chosen for study. Few bona fide cases of the 
latter can be identified with certainty in the literature on mutation 
plant breeding. Among these cases, Brock and Latter (6) pre-chose 
flowering date in their studies on subterranean clover, Oka, et al. 
(39) pre-chose heading date and plant height in studies with rice, 
and Gregory and Gregory (unpublished) pre-chose flowering response 
to day length in their study of Hibiscus. 

Some other pre-chosen characteristics have not proved so suc- 
cessful as breeding ventures. For example, Gregory (unpublished) 
pre-chose the reversal of geotropism in the young peanut fruit. 
Following; an individual observation of hundreds of thousands of 
X 2 and X 3 plants he has not, to date, observed what could be estab- 
lished as a diminution of the geotropic response in peanut fruits. 
Apple (unpublished) found in three different flue-cured varieties 
of tobacco, that two of the varieties were naturally very highly 
susceptible to "black shank", Pliytophthora parasitica nicotianae; 
the third had a measurable tolerance to the disease. By making use 
of artificial inoculation in a combination of seedling flat and field 
techniques, Apple was able to screen extremely large numbers of 
individual X 2 plants. After several years of work he has been unable 
to discover any increase in tolerance to black shank in the two 


highly susceptible varieties. In the variety bearing some tolerance 
to the disease significant increases in tolerance have been selected 
among its progenies. Cooper and Gregory (9) observed an increase 
in the variability of tolerance to Cercospora leaf spot in peanuts, 
but this also was in a variety where tolerance was already at a rela- 
tively high level. 

The conclusion appears inescapable that despite the large num- 
ber of desirable mutations reported in the literature there have been 
sought highly desirable characteristics which have not been attained 
by artificial mutation after extensive trials. Furthermore, since 
many investigators do not report the negative results of breeding 
trials, there may be many more instances of inefficacy of mutation 
breeding than we suspect. It has to be admitted also that with a new 
character no a priori decision can be made as to (a) whether a certain 
desired change can be had through mutation, (b) how much mutagen 
should be applied, or (c) how large a population would be required. 
One might ask, for example, what it would require of mutation 
breeding to produce a peanut with tendrils. Miracles of mutation 
or even the more optimistic predictions of breeders simply cannot 
be realized because of points of no return attained already in the 
evolution of species. 

These considerations appear to contradict the thesis that muta- 
genic agents simply increase the rate of natural mutation and the 
logical conclusion therefrom that since nearly every conceivable 
change has occurred in plants and animals, nearly every conceiv- 
able change should occur if sufficient number of mutations were 
produced. The fallacy lies in the failure to recognize the evolved 
relationships between variation at the gene level and the acceptance 
of that variation by the genome and by the organism. 

Under the various conditions of nature various organisms 
have found the balances which worked for them in particular environ- 
ments. They have opposed the conservatism of linkage to the oppor- 
tunities of crossing over, the possession of a few large chromosomes to 
having a lame number of small ones, self-fertilization to cross- 
fertilization, and have evolved many of the possible compromises of 
different levels of these characteristics. 

Mutation, sometimes thought of erroneouslv as an extra-organis- 
mal force, is a characteristic of organisms related particularly to 


their breeding structures. If the hazard of mutation has been met by 
different breeding structures in different ways these adaptations 
affect the expected efficacy of mutation breeding. 

Mutation and Breeding Structure 

By breeding structure I refer to the manner of reproduction 
of an organism and the consequent organization of its genome. It 
is the purpose of this section to point out that efficacy of mutation 
breeding cannot be divorced from the role of mutation in the evolu- 
tion of breeding structure. That breeding structure is a result of 
natural selection was an early contribution of genetics and received 
its first general summary in Darlington's Evolution of Genetic Sys- 
tems (11). This was followed by more explicit association of mating 
system with the polygenetic organization of the genome as brought 
out especially in a series of papers by Mather and given a general 
summary by him (32). The developments in population genetics 
during the last two decades (13), together with the indicated genomic 
control of mutation in plants (3, 4, 5, 16, 33, 34, 35, 42, 43), have 
suggested that the kind and abundance of mutation itself have evolved 
in conjunction with the evolution of breeding structure. 

The concept of the crossbred population as a system of het- 
erozygous genotypes maintained in frequencies optimal to the 
demands of environment has been generally established (8, 13, 23, 
24, 28, 29, among others). Much work has been done that demon- 
strates the advantages of deleterious recessives in higher than base 
mutation-rate frequencies in crossbreds (28). The maintenance 
of heterozygosity by selection in crossbreds was further supported 
when Dempster (12) showed that mutation to deleterious recessive 
was inadequate to account for the variance observed. Recent tests 
of specific situations in known genetic material show that individual 
heterozygotes may be at a disadvantage in uniform environment 
(18, 26) with a consequent return to homozygosity (30). These facts 
suggest that a price in excellency may be required in a chosen 
environment in return for the opportunity to change in a chang- 
ing environment. How generally these conclusions apply is still 
subject to experimental confirmation since the numerical relation- 


ships of ]ethals, sublethals, vitals, supervitals, etc., are only imper- 
fectly known even in Drosophila (7, 37). 

Numbers of loci which are carrying deleterious alleles are 
thought to be maintained in various degrees and frequencies in 
some sensitive equilibrium by breeding system as well as bv external 
environment. The fact that such a genetically variable population 
presents, in the wild at least, a uniformity of wild type has been 
discussed by Lerner (28). The widely accepted explanation of the 
coincident maintenance in the crossbred of genetic diversity and 
phenotypic uniformity lies in the phenomenon of heterozygosis. 
Thoday (52, 53) was able to show that heterozygosity was essential 
for the preservation of bilateral symmetry in the fruit fly. 

The conclusion that mutation itself is in equilibrium with 
requirements of the mating system derives from the simple genetic 
situation that given a mutant A, advantageous in heterozygous 
state, every other mutation which occurs affecting breeding sys- 
tem will be selected in terms of As conferred benefits. Furthermore, 
that particular organization of the genome which permitted the 
mutability of A will be favored in the sense that not only will those 
forms possessing the A-mutating quality be favored in selection but 
also those forms that possessed qualities controlling the A-mutating 
frequency. The genome itself and its intrinsic characteristics are 
conceived to evolve with breeding system to give the breeding 
structure of the population. The cross-pollinator is thought to have 
a genotype suited not only to meeting the contingencies of its 
environment but to the controlling of the variability in its own 
heterozygous organization. A measurable supply of new mutation 
is steadily furnished the crossbred (47, 49). It is thought that the 
rate of supply is determined by natural selection in keeping with 
the demands of breeding system and environment. Such a dom- 
inance-dependent organization would tend to neglect the evolu- 
tion of high thresholds to expressivity for individual alleles. Any 
radical change in breeding system, external environment, or muta- 
tion rate would result in immediate shifts in the genetic composi- 
tion of the population. 

The above considerations concern the efficacy of mutation 
breeding with normally outbred organisms where the chief protec- 
tion against deleterious mutation resides in heterozygosity. If selec- 


tion has already increased the load of mutations to the optimum 
for the exigencies of the environment for population size, for loss 
of mutant alleles through homozygosis, and for balanced mutation 
rate through modulators, any marked increase in mutation would 
likely result in a diminution of fitness. Hutchinson (27) and Muller 
(37) especially have maintained that little is to be expected of muta- 
tion breeding with this system of mating. These strictures do not 
preclude positive results from mutation breeding of crossbreds (38) 
in instances of small breeding populations or where the crossbred 
population is near or at the limit of its range, either case necessitat- 
ing a past history of inbreeding. They do suggest that mutation 
breeding, in a panmictic population of large size and great com- 
plexity, unaccompanied by a radical departure from customary 
breeding procedure, would probably be ineffectual. The somewhat 
negative conclusion here should take into account the selection work 
reported on irradiated populations of Drosophila (45). 

Lacking the necessary experimental data to prove or to disprove 
the above conclusion and unable to review the enormous litera- 
ture allied to this subject, I would like to stop with the suggestion 
that the effectiveness of selection following mutagenic treatment 
vs. no treatment, with and without radical change of breeding pro- 
cedure, be investigated in a panmictic crop plant — shall we say rye 
or corn . 


Mather has emphasized the contrasting organizations of the 
genetic systems in self- and cross-pollinators. In an extension of 
Mather's thesis, Gregory (19, 22) postulated that self-pollinators, 
faced with the alternatives of elimination or fixation of every muta- 
tion, have evolved genomic systems capable of absorbing relatively 
large numbers of mutations of small effect without their necessarily 
reaching the thresholds for phenotypic expression. According to this 
hypothesis genetic organization favoring resiliency of the pheno- 
type of the homozygote in the self-pollinator would tend to become 
established by selection. The genetic factors controlling the ten- 
dency of mutations to occur in the direction of resiliency would 
likewise become ensconced in the genome. The genome would 
become laced with supporting modifier gene complexes with little 


other obvious or major effects than to provide variation in expres- 
sivity consistent with a changing environment. Each chromosome 
would come into a form of internal balance, as Mather has postu- 
lated, with respect to the amount of modulating material optimum 
for the genome to maintain in relation to external environment. 

The relations of self-pollination, mutation, and expressivity 
permits a genetic explanation for Stebbins' (50) observation that 
self-pollinators possess, "a relatively high degree of phenotypic 
plasticity so that the individual is susceptible of tremendous modi- 
fication in the face of adverse or extreme conditions". 

The homozygote's capacity to adapt to great environmental 
change combined with the preservation of phenotype suggests that 
protection against expression of newly acquired mutation may 
be high (22). It is the possibility that the height of this barrier may 
permit a sufficient number of simultaneous mutations that makes 
the mutation breeding of selfbreds attractive. It should be said 
that not every breeder of self-fertilized crops shares this view (1, 

Comparative effects of Irradiation of 
Pure Lines and Their Hybrids 

Notwithstanding the theoretical possibility that normally self- 
fertilizing species may utilize mutation as a resource of genetic 
adaptability in a manner comparable to the use of heterozygosity 
by cross-fertilizing species, it is still uncertain whether self-ferti- 
lizers are capable of absorbing to advantage very much larger muta- 
tion rates than those established in the species by natural selection. 
The solution of this problem requires knowledge of the effects 
of mutation on characters equatable to fitness in a number of dif- 
ferent species. Interest in the effects of mutagenic treatment on 
quantitative characters, some of which are the most likely indi- 
cators of fitness, has been slow in its development. As a consequence 
of this it will be difficult to give a satisfactory assessment of the sit- 
uation at the present time. 

Papers which have laid the foundation for further experi- 
ments on quantitative characters have been published by Brock 
and Latter (6), Daly (10), Gregory (19), Mertens and Burdick (36), 
Oka (39), and Rawlings, et al. (41). For an assessment of the efficacy 


of mutation breeding, experiments designed to make comparisons 
between conventional breeding procedure and mutation breeding 
are required. Gregory (20) suggested that radiation-induced variance 
should be cumulative with that induced by hybridization. Pre- 
liminary results have been reported (21) on expressions of F 2 dom- 
inance in hybrids of mutant selections from the same pure line. In 
the absence of such work from other laboratories it is my purpose 
to devote the remainder of this paper to the description of such an 
experiment conducted with peanuts. (See, however, Krull, C. F., 
Agronomy Abstracts, 1960, page 50.) 

In 1953 all possible hybrid combinations were made among six 
lines of peanuts. All of these lines had been reproduced from initial 
single plant selections followed by sufficient automatic self-fertiliza- 
tion to assure their relative homozygosity. Three of them were 
selections in X r , generation of high-yielding mutants from the same 
pure line. The other three were selections in F n generation from 
the hybridization and progeny testing program which, at that time, 
was conducted separately from the radiation experiments. A suffi- 
cient number of cross-pollinations was made to produce at least 
100 Fi seeds of each of the 15 possible Fi hybrids. In each cross 
the 100 Fj seeds were divided into two lots of 50 each. One 50-seed 
lot of each hybrid and a 125-seed sample of each parent were 
given a treatment of 15 Kr of X-rays. 

The packages of peanuts were arranged all at a time upon a 
curved target surface in random order. At half time the packages 
were all removed, turned over, re-randomized, and placed upon 
the target surface for the remainder of the treatment. The radia- 
tion was delivered at the rate of 62 to 63r per minute at a distance 
of 1 meter from a 1,000-Kv tube equipped with beryllium window. 
It is thought that all received equal and uniform doses of X-rays. 
The seed had been stored dry for 6 months prior to irradiation and 
moisture content was uniform. (The author is indebted to Dr. 
William T. Ham, Jr. (25) for dosimetry measurements and the 
use of the X-ray tube, Biophysics, Medical College of Virginia, 
Richmond.) The treated seeds and their controls were planted in 
individual plots the day following X-ray treatment. In the fall of 
1954 the plants were harvested individually. The treated hybrids 
were designated as FiXj. The F 2 and F 2 X 2 generations were grown 


in 1955. At the end of the F-> and F>X-> year, 12 of the 15 original