:.HE PRODUCTION OF HOMOZYGOUS DEFICIENT TISSUES     ,'
  WITH MUTAST CHARACTERISTICS BY ME-AX'S OF  i
                                                                \
    THE ABERRANT MITOTIC BEHAVIOR OF
         RING-SHAPED CHROMOSOIMES*

.TABLE OF CONTENTS                     P.\GE

I Introduction.                      315
II The mitotic behavior of ring-shaped chromosomes.                 3'8
I II The nature of the Bin 1-6~11 variegation               333
I\. Types of functional gametes produced by the two original plants.         336
\' Production of plants mosaic for homozygous deficiencies            346
\.I Simulation of the b>?t I phenotype through loss of the Bm I locus.            357
\`II The production and appearance of plants homozygous for Def2 R2.          360
\`I II The phenotypic effect of altered ring-chromosomes.          365
IS Discussion.. .               365
Summary. .                                         373
I .iterature cited.                                          375

                      I. INTRODUCTION

1 T IS the purpose of this paper to describe the method by which viable
tissues, homozygous deficient for a known region of a chromosome,
may be produced in maize. The chromosomal region involved includes
the locus of the gene BW 1 in chromosome V (allele of bm 1, brown mid-
rib, producing a brown color in the lignified cell walls). The lignified cell
walls of the homozygous deficient tissue exhibit the features characteristic
of the known recessive gene bm 1 although the locus of this gene is absent.
The method of obtaining the homozygous deficient tissue is related to
the unique behavior of ring-shaped chromosomes during somatic mitosis.
`I'his behavior has been briefly mentioned in previous publications (MC-
~LINTOCK 1932; RHOADES and MCCLINTOCK I 935). Ring-shaped chromo-
somes do not always maintain themselves unaltered through successive
nuclear cycles in the maize plant. They may (I) increase in size through
(luplication and reduplication of segments of the original ring, (2) decrease
in size by deletions of segrrents from the ring, (3) be totally lost from the
nuclei or (4) be present in increased numbers in the different nuclei. What-
ever the method by which a change in size occurs, only ring chromosomes
are produced from ring chromosomes:

In maize it has been found that deficiencies in certain regions of the

* `The cost of the half-tone illustrations in this paper is met by the Galton and Mendel
-\Iemorial Fund.

' Contribution from the Department of Botany in cooperation with the Department of Field
Crops, Genetics Research Proiect, Missouri Agricultural Experiment Station, Journal Series
TO. j70.


316                  BARBARA McCLINTOCK
chromosomes may be transmitted successfully through the egg but not
through the pollen (BURNHAM 1932; STADLER 1935). Pollen possessing a
deficient chromosome plus a ring-shaped fragment chromosome should bc
functional if the ring-shaped fragment completely compensates for the
deficiency. By utilizing a deficiency transmissible through the eggs ant]
rendered non-lethal in the pollen by the inclusion of a ring fragment
covering the deficiency, a zygote with two deficient chromosomes plus ;I
ring chromosome can be produced. This zygote is heterozygous for th,

FIGURE I.-TWO sides of a stalk of a variegated plant. The leaves at the two nodes
   have been removed. The dark bands are bnzl, the light bands: Bur 1.

deficiency. This heterozygosity in the resulting individual would be main-
tained as long as an unaltered ring chromosome was present. Should the
ring chromosome be lost in subsequent nuclear iivisions, or should it
change in size through loss of a segment within it, the tissues arising after
such 10~s or alteration would be homozygous deficient for the entire tic-
ficiency in the first case or for regions within the limits of the deficient!'
in the second case.

TWO cases of deficient rod chromosomes with complementary ring
chromosomes were available for this study. The two cases arose in the


              RING CHROMOSOMES IN MAIZE            3'7
progeny of X-rayed pollen containing a normal haploid complement with
the dominant gene Bm 1. This pollen, when placed upon silks of bm 1
plants with a normal chromosome complement, gave rise, among a progeny
of 466, to two individuals which were variegated for Bm 1 and bm 1
(figure I). Aberrant behavior of a ring chromosome produced by the X-ray
treatment and carrying the gene Bm 1 was suspected to be the cause of
the variegation.

II: r
     -
I '
I L

a

I
-

;
5
5 20,

b

II

5 1
4 0
3 2

FIGURE 2.-a. Diagram of a normal chromosome V. The slightly bulging section represents
the spindle fiber attachment region. The sets of arrows, I and II, point to the positions of breaks
which gave rise to the two deficient rod chromosomes and their compensating ring chromosomes
illustrated in I and II of b. The deficient rod and compensating ring chromosomes of I are referred
to in the text as Def 1 and R 1 respectively, those of II as Def 2 and R2 respectively

Examination of synaptic configurations in sporocytes revealed not only
the presence of a small ring-shaped chromosome in each plant but also a
deficiency in one chromosome V. In each case, the size of the ring-shaped
chromosome and the extent of the deficiency in the rod-chromosome were
comparable. The deficiency in both cases involved a section of the short
arm immediately adjacent to the spindle fiber attachment region. Since
the ring fragment in both cases possessed a small but definite spindle fiber
attachment region, these regions being clearly visible in meiotic prophases,


31%                 BARBARA McCLINTOCK
it was assumed that in each case the deficient rod and its compensating
ring chromosome arose as the result of two breaks in the normal chromo-
some V, one break passing through the spindle fiber attachment region,
the other breaking the chromosome at a distance from the spindle fiber
attachment region equal to approximately 1/20 (Case I) and 1/7 (Case
II) of the total length of chromosome V (figure 2). Fusions two by two
of the broken ends resulted in a deficient rod and a compensating ring
chromosome each with a section of the original spindle fiber attachment
region. Since both the deficient rod and the ring chromosome possessed a
section of the spindle fiber attachment region, both could be maintained
through nuclear cycles.

Proof that the ring chromosome represented the region for which the
rod chromosome was deficient was furnished by the synaptic configura-
tions produced by homologous associations of the three chromosomes : the
normal chromosome V contributed by the female parent, the deficient rod
chromosome V and the small ring chromosome contributed by the male
parent (figures 25 and 26 and photographs of the same, 17 and 18, Plate
II). Cytological examination of different portions of the tassel disclosed
the loss of the ring chromosome in several branches. Similarly, within a
single anther, groups of cells were found lacking the ring chromosome. It
was suspected, therefore, that the ring chromosome carried the locus of
Bm 1, its loss during somatic mitoses being responsible for the presence
of the bm 1 (brown) streaks in these plants. Conclusive proof for this was
derived from the progeny of these two plants when crossed to normal
bm 1 plants. The progeny included variegated (Bm 1 and bm 1) and
bm I plants. Of the variegated plants, microsporocytes of 148 individuals
were examined for the presence of the ring chromosome. The ring frag-
ment was found in 146 of these individuals although in many plants
several branches of the tassel lacked the ring fragment. In two plants
no ring chromosome was found in the several branches of the tassel
which were collected. Of the totally bm 1 plants, 47 were examined. In
no case was a ring chromo&me found. In a bm 1 tiller of a variegated
plant, a considerably reduced ring chromosome was found. It is probable
in this case that the Bm 1 locus had been deleted from the ring chromo-
some through somatic alterations to be described in the next section. In-
dividual collections were made on the two -sides of plants which were
approximately half bm 1 and half variegated. In these cases, the presence
of the ring chromosome could be established only on the variegated side.

II. THE MITOTIC BEHAVIOR OF RING-SHAPED CHROMOSOMES

The interpretation of the variegation and of the production of homozy-
gous deficient tissues has been based on a knowledge of the behavior of


              RING CHROMOSOMES IN MAIZE             319
ring-shaped chromosomes in somatic nuclear cycles. A description of what
has been observed regarding the appearance and behavior of the ring
chromosomes in meristematic regions is therefore necessary before the
individual cases can be considered. Although the primary cause of irregu-
larities in the nuclear cycles is undoubtedly the same for large and small
ring-shaped chromosomes, the subsequent behavior and the genetical con-
sequences vary in these two extremes. The behavior of large ring-shaped
chromosomes will be considered first; this willbe followed by an account
of the small ring-shaped chromosomes; finally, correlations and conclusions
will be drawn regarding ring-shaped chromosomes in general.

Mitolic behavior of large ring-shaped chromosomes

Since the two ring-shaped chromosomes of cases I and II, figure 2, are
both small, a large ring-shaped chromosome originally representing most
of chromosome II has been examined (MCCLINTOCK 1932). The observa-
tions were made on longitudinal sections of actively growing root tips.
Observations at meiotic prophase in this plant had clearly indicated that
changes in size and hence chromatin content of the ring chromosome were
occurring in the premeiotic nuclei. Groups of related cells usually had
similar ring chromosomes but the differences in unrelated cells were very
great. In a few cells the altered ring chromosome was larger than the
normal chromosome II. In some cells it had been reduced to only a few
chromomeres. All gradations between these two extremes were found in
different sporocytes of this same plant. The smallest ring chromosome has
obviously undergone a great loss of chromatin. The original ring chromo-
some possessed a single knob. Evidence for duplication of segments other
than the obvious increase in size of the ring chromosome was clearly
registered in some cells by the increase in the number of knobs. Rings
with two, three and four knobs were found.
It was suspected that the alteration in chromatin content of the ring
was related to the division cycle of the chromosome. Observations of
mitoses in root tip meristems suggested the manner in which the altera-
tions occur without, however, revealing the primary cause. If one assumes
that during the splitting process or after the split has occurred, a cross-
over took place between the two sister chromatids, a double-sized, con-
tinuous ring with two spindle fiber attachment regions would be produced.
-4 second crossover between the two sister chromatids could result in an
interlocking of the sister ring chromosomes provided the second crossover
did not counteract the first. The presence of double-sized rings with two
spindle fiber attachment regions at late anaphase and early telophase was
clearly evident in a number of cells (figures 5, 7, 15, 16; photographs 4,
3, 8, Plate I). Unfortunately the presence of interlocking rings could not


320                 BARBARA McCLINTOCK
be determined directly since the chromosomes of maize in somatic cells
are relatively small. Many anaphase figures were suggestive but none could
be definiteIy distinguished from double-sized rings with a twist at the
mid-region. From the point of view of the origin of such configurations it
would be important to know the relative percentage of each type. From
actual counts it is certain that the double-sized rings are present in at
least one-third of the aberrant figures. The actual number of late anaphase
and early telophase figures with chromatin bridges produced by double-
sized or interlocked rings amounted to approximately 8 percent of a total
of I 145 figures recorded in roots whose ring chromosome had not materialI!-
reduced in size in most of the cells (D, table I).

                      TABLE I
The frequency of normal and aberrant somatic anaphase and early telopkase configurations
           in plants wifh different ring chromosomes.

          RING CHROMOSOME        NORMAL     ABERRANT   70 ABERRXST

A       Rl             605          I        0.16
B       R2                    1195         14         1.1
C      R 2 plus enlarged R2       1169       76         6.1
D     Large ring chromosome II      1053        92        8.1

Since the fate of the double-sized or interlocked rings is not the same in
all late anaphase and telophase figures, a number of types of behavior
from anaphase to late telophase have been diagrammed in figure 3. Repre-
sentative drawings from different cells are given in figures 5 to 24 and
photographs of Plate I. In the diagrams, the behavior of double-sized
rings has been emphasized since this type could be clearly recognized in
many cells. They are either clearly open or show a twist at the mid-region.
Some of the interlocked rings should produce figures resembling those
shown in the diagram and would not be easily distinguished from them.

In most of the mitotic cycles the ring chromosome splits along a single
plane, separation of the two halves proceeding normally at anaphase,
figure 4. In the late anaphase figures the double-sized rings produce a
double bridge the chromatin of which is pulled taut (figure 5, photograph
I, Plate I). It is suspected that breakage of the chromatin bridges some-
times occurs during this period (photograph IO, Plate I). Since such
figures were not included in the counts mentioned above, the 8 percent
of anaphase and telophase figures with bridges represent the minimum
number of cells in which double-sized or interlocked rings occurred. Some
of the telophase figures suggest an early breakage of one or both strands
of the double bridge (figures 8 and 9 and photographs 5 and 6, Plate 1) I


RING CHROMOSOMES IN MAIZE             321

b

0
%I
h

8

i

FIGURE S.-Diagrams illustrating the behavior in somatic mitosis of double-sized ring chromo-
somes with two spindle fiber attachment regions produced from the two split halves of a single
ring chromosome.

a. Successive stages from mid-anaphase to mid-telophase of a medianly placed double-sized
ring chromosome. The cell plate determines the positions at which breaks will occur in the two
chromatin bridges,

b. Similar to a except that a twist is present in the bridge strands of the double-sized ring
chromosome.

c. L4ppearance in early and mid-telophase of a double-sized ring which was non-medianly
placed in the spindle figure. The components entering each daughter nucleus vary in chromosome
length and constitution.

d. Similar to c except that the upper portion of the double-sized ring chromosome is not in-
cluded in the reorganizing telophase nucleus. Such behavior results in the loss of a component of
the ring chromosome from one of the daughter nuclei.
e. i\ppearance at mid-telophase of a double-sized ring chromosome with one broken bridge
strand.

f. Appearance at mid-telophase of a double-sized ring chromosome with both strands broken.
g. Appearance at very early telophase suggesting an early breakage of bridge strands of a
double-sized ring chromosome (or two interlocked sister ring chromatids).
h. Comparable situation as illustrated in d except that the strands of the bridges are twisted
at the cell-plate region.

i. Fine bridge of chromatin between two resting nuclei suggesting that a breaking of the
strands had not occurred at telophase.


322                BARBARA McCLINTOCK
In many cases, breakage of the strands composing the bridge does not
occur at anaphase; compare photographs 9 and IO, Plate I. The moving
apart of the spindle fiber attachment regions in the double-sized rings is
retarded by the tension of the chromatin bridges. The subsequent behavior
is conditioned by the position in the spindle figure of this retarded ring
or of two retarded interlocked rings. As the telophase sets in there is an

2

   5

+is

                      9                     IO

a

6

FIGURE 4.-Normal separation of a ring chromosome in somatic anaphase.
  FIGURE 5.--A double-sized ring chromosome in early telophase.
FIGURE 6.-A double-sized ring chromosome at a slightly later stage than
           that shown in figure 5.

FIGURE `].-Double-sized ring chromosome at mid-telophase. The bridge strands close to the
cell-plate have become very thin. The shape of the chromosome within the nuclei has become
discernible.
FIGURE 8.-Mid-telophase. Early breakage at the cell-plate region of two bridge strands of the
      double-sized ring. See comparable figure, photograph 6, Plate I.
   FIGURE o.-Mid-telophase. Early breakage at the cell-plate region of one bridge
        strand of a double-sized ring. See photograph 5, Plate I.
FIGURE ro.-Late telophase. Breakage of bridge strands of a double-sized ring chromosonrc
at the cell-plate region and withdrawal of the chromatin into the nucleus at the lower part of the
figure.

immediate release of tension on the chromatin bridges produced through
the swelling of the forming nuclei (photograph 2, Plate I). As the nuclei
continue to swell and approach the cell-plate, the chromatin of the ring
within the nuclei is relaxed, allowing the form of the ring chromosome to
be clearly defined (figures 8, 9, II, 16; photographs 3, 4, 5 and 8, Plate 11.
At this stage the tension on the chromatin threads from the nuclear men)-


RING CHROMOSOMES IN MAIZE            323

0
8
0

14

IS              16

Q
  0

0

17

    FIGURE rr.-Double-sized ring at mid-telophase. See photograph 3, Plate I.
FIGURE rz.-Similar stage to that shown in figure IO resulting from a previous double-sized ring
with a twist at the mid-region or from two sister ring chromatids which were interlocked.
FIGURE q--Sister nuclei at late telophase. The positions of the ring chromosomes suggest a
           previous bridge formation which has broken.

  FIGURE r4.-Resting stage. Sister nuclei with a fine connecting chromatin bridge.
FIGURE r3.-Non-median position of a double-sized ring chromsome at late anaphase. See
photograph 7, Plate I. In the photograph there is a twist in the ring chromosome.
FIGURE r6.-Mid-telophase. The result of a non-median placement of a double-sized
    ring chromosome at anaphase. See photograph of the same, 8, Plate I.
FIGURE r7.-Mid-telophase. The result of a non-median placement of a double-sized ring
    chromosome with a twist, or of two interlocked sister ring chromatids.

brane to the cell-plate again increases. The threads become thin and taut
as if being pulled into the nuclei (figures 7, 16, 17; photograph 8, Plate I).
In a few cases, these fine chromatin threads are seen in relatively late telo-
phase nuclei (figure 14). Since they usually do not persist into late stages,
breakage must usually occur during the earlier telophase period. There
were many sister telophase nuclei observed in which the ring chromosome
in each nucleus was close to the region of the nuclear membrane lying
nearest the cell-plate (figures 12 and 13). Such figures probably represent
the last stage in the progress of thepreviously double-sized or interlocked
rings. It should be emphasized that fusions of broken ends must occur
after such breakage, since only ring chromosomes have been found to
arise from ring chromosomes although rod chromosomes might be ex-
pected.

It sometimes happens that the passage of one spindle fiber attachment


324                 BARBARA McCLINTOCK
region of a double-sized ring proceeds toward its pole in advance of the
opposing spindle fiber region. Consequently, the double-sized ring is not
medially placed in the spindle figure. The cell-plate then intercepts the
chromatin bridges in a non-median position (figure 15, photograph 7,
Plate I). As a result, the components of the double-sized ring entering
sister telophase nuclei will be unequal in size and chromatin constitution.
One segment of the double-sized ring is sometimes not included in the
telophase nucleus on its side of the cell-plate (figures 16 and 17, photo-
graph 8, Plate I).

If the chromosome is not split at anaphase, fusions of broken ends could
give rise in the next division to normally disjoining sister ring chromo-
somes, or if twists are present in the chromonema before fusion, to a con-
tinuous double-sized ring or interlocked sister ring chromosomes when

EXPLANATION OF PLATE I

All magnifications are approximately X I roe.

Plate I.-Individual cells from longitudinal sections of the growing points of roots. Photo-
graphs I to ID are of the large chromosome II ring. Photographs II to 14 show an enlarged R2
chromosome. Photographs 15 and 16 are of the normal R2 chromosome.
Photograph I. Late anaphase. Bridge produced by separation of the split halves of a ring-
shaped chromosome which is in the form of a double-sized continuous ring. There is a twist of
the strands at the mid-region.

Photograph a. Early telophase. Beginning of relaxation of tension on the strands of the double
bridge.

Photograph 3. Mid-telophase. Complete relaxation of tension on strands of bridge.
Photograph 4. Mid-telophase. Double-sized ring chromosome.
Photograph 5. Mid-telophase. A double-sized ring chromosome. The strand to the right ap-
pears to be broken.

Photograph 6. Mid-telophase. A double-sized ring chromsome. Both strands appear to be
broken at the cell-plate region.

Photograph 7. Late anaphase. xon-median placement in the spindle figure of a double-sized
ring chromosome.

Photograph 8. Mid-telophase. The result of a non-median placement in the spindle figure of a
double-sized ring chromosome. The strands adjacent to the cell-plate have become attenuated.
The lower segment of the ring chromosome was excluded from the forming nucleus.
Photograph 9. Very early telophase. Chromatin bridge produced by a double-sized ring chro-
mosome with twisted strands, or possibly two interlocked sister ring chromatids.
Photograph IO. Very early telophase. Figures such as this suggest an early breakage of the
strands of a double-sized ring chromosome or of interlocked sister ring chromatids.
Photograph I I. Typical late anaphase position of a small ring-shaped chromosome which xvi11
be excluded from the reforming telophase nuclei.
Photograph 12. Early telophase. Excluded ring chromosome which was previously non-
medianly placed in the spindle figure. The cell-plate has passed below it.
Photograph 13. Late anaphase. Stage in the process of exclusion of two closely associated
ring chromosomes.
Photograph 14. Similar to photograph 13.
Photograph 13. Typical late anaphase position of a small ring-shaped chromosome which Jvill
be excluded from the telophase nuclei.

Photograph 16. Mid-telopha'se. The result of a previously excluded ring-shaped chromosonn'
The cell-plate has passed below the ring.


RING CHROMOSOMES IN MAIZE             325

   - P . .
. ,

PLATE J


326                 BARBAR. McCLINTOCK

,
  --                  21

PLATE 11


               RING CHROMOSOMES IN MAIZE            327
simple assumptions are made regarding the method of splitting or re-
duplication of a chromonema along a single plane. If the chromosomes are
split at anaphase, two by two fusions of the two adjacent broken ends of
sister chromatids could result immediately in a continuous double-sized
ring. If fusions two by two took place between the non-adjacent broken
ends, continuous double-sized rings or interlocked sister ring chromatids
could result. If the single (no anaphase split) threads were very much
twisted or the double (split present at anaphase) threads coiled about one
another, complex configurations would appear in the next anaphase. Only
rarely was a figure found suggesting any complexity. If such behavior
were the secondary cause of double-sized or interlocked ring chromatids
(it cannot be the primary cause, see discussion), adjacent cells in the
longitudinal rows could be expected to show chromatin bridges in an
appreciable percent of the cases. They were present in a number of longi-
tudinally adjacent cells. However, a very large number of such figures
would be necessary to allow a satisfactory statistical study to be made.
Although a large number of anaphase and telophase figures with aberrant
configurations have been observed, the numbers of these in adjacent cells
were insufficient for such a study.

    The mitotic behavior of small ring-shaped chromosomes
The mitotic behavior of small ring-shaped chromosomes differs from
that of large ring-shaped chromosomes in (I) the reduced frequency with

EXPLANATION OF PLATE II

PLATE II.--All photographs are of pachytene configurations in microsporocytes. XI 100.
Photograph I 7, Synaptic association of a normal chromosome V, a Def 2 chromosome V and
an R2 chromosome. See text figure 25.

Photograph 18. Similar to photograph 17. See text figure 26.
Photograph 19. For description, see text figure 27.
Photograph 20. Synaptic association of a normal chromosome V and a Def 2 chromosome V.
See text figure 28.

Photograph z I. From a sporocyte of a plant with a normal chromosome \`, a Def 2 chromo-
some V and two R2 chromosomes. The two rod-chromosomes have associated with one another
(note buckle at spindle fiber attachment region). The two ring chromosomes have associated with
one another.

Photograph 22. Synaptic association of two R2 chromosomes.
Photograph 23. Late pachytene. The arrow points to the tiny Rl chromosome.
Photograph 24. Collapsed Rl (upper) and R2 (lower) chromosomes associated at their
spindle fiber attachment regions.

Photograph 25. Collapsed R 1 (left) and R2 (right) chromosomes associated at their spindle
fiber attachment regions.

Photograph 26. Rl chromosome (arrow). Its spindle fiber attachment region is stuck to that
of bivalent chromosome VIII.

Photograph 27. Collapsed R 1 chromosome (arrow) whose spindle fiber attachment region is
associated with that of a normal chromosome V bivalent.
Photograph 28. Synaptic association of a normal chromosome V and a Def 1 chromosome V.
See text figure 30.


328                 BARBARA McCLINTOCK
which double-sized or interlocked rings arise; (2) the more frequent loss
of the ring chromosomes from the nuclei; (3) the considerably less frequent
occurrence of changes in size of the ring chromosomes and (4) the occa-
sional increase in the number of rings in a nucleus.
The two small rings in cases I and II, figure I, have been used to study
the behavior of small ring-shaped chromosomes in mitosis. In the sub-
sequent discussions these two ring chromosomes will be referred to as
R 1 and R 2 respectively. Cytological examination of the sporocytes in
different branches of the tassel in plants with either of these rings had
indicated that loss of the.ring chromosome was occurring far more fre-
quently than changes in size of the ring. This is in direct contrast to the
behavior of large ring chromosomes, where changes in size are more fre-
quent than loss. To obtain evidence on the method of loss, examinations
of the meristematic regions of the roots of such plants were made, The
tiny R 1 ring chromosome is clearly visible in the prophase nuclei of these
cells. However, the description will confine itself to the behavior of the
larger of these two rings, R 2, and one of its enlarged derivatives, since
anaphases showing aberrant configurations of the R 1 chromosome are
found only very rarely.
The aberrant anaphase and telophase configurations are characterized
by the median or nearly median position of the double-sized (or inter-
locked) ring chromosome in the spindle figure (photograph 15 for the
normal R 2 and photograph II for the enlarged R 2, Plate I). However,
they occasionally lie some distance from this position (figure 22 and photo-
graph 12, Plate I). The ring chromosome in these configurations, as with
the large ring chromosomes, frequently appears to be double-sized. In roots
in which most of the nuclei contained the normal R 2 chromosome, 14 of
the 1209 anaphase and telophase figures counted, or 1.1 percent showed
these aberrant configurations (B, table I). They were observed many times
in roots where counts were not made.
The fate of the delayed double-sized ring depends upon its position in
the spindle figure as the cell-plate appears. If it is in the .middle, the cell
plate passes through it, dividing it into relatively equal or decidedly un
equal segments (figures 19 and 20). If it is not medially placed, the cell-
plate passes to one side and the double-sized ring remains in the cytoplasm
of one of the daughter cells (figures 21 and 22, and photographs of same,
12 and 16, Plate I). If it lies rather far away from the mid-region, it may
be included in one of the nuclei. If this occurs and if normal splitting of
this double-sized ring with two spindle fiber attachment regions folloJvs
in the next division, two double-sized rings, each with two spindle fiber
regions should then be found lying close together in the spindle figure
when the two spindle fiber regions on the same chromatid pass to opposite


            RING CHROMOSOMES IN MAIZE             329
poles. Several configurations have been observed in which two rings were
lying very close together (figures 23 and 24, and photographs of the same,
13 and 14, Plate I). The exact contours of the individual rings could not
be accurately followed and therefore have not been shown in the drawings.
Since the contours of the two ring chromosomes could not be accurately

cl
0

0
0

18              I9                20

22                  23                    24

II

FIGURE r8.-Simultaneous loss at late anaphase of two enlarged R2 chromosomes.
FIGURE rg.-Late telophase appearance after loss of an R2 chromosome. The double-sized
      ring chromosome has been intercepted by the cell-plate.
        FIGURE zo.-Similar to figure 19 but a later stage.
FIGURE zr.-Telophase. The excluded R2 chromosome has not been intercepted by
         the cell-plate. See photograph 16, Plate I.

FIGURE zz.-Telophase. The excluded enlarged R2 chromosome was not medianly pIaced in the
       spindle figure. See photograph of the same, IZ, Plate I.
FIGURE z3.-Late anaphase. Two enlarged R2 chromosomes lying close together at the cell-
  plate region. For description, see text. See photograph of the same, 13, Plate I.
   FIGURE z4.-Similar to figure 23. See photograph of the same, 14, Plate I.

followed, such figures could represent two interlocked ring chromosomes
from a similar condition to that described above, if instead of a double-
sized ring, two interlocked sister ring chromosomes had been included in
the preceding telophase nucleus.

Since anaphases and telophases with the ring chromosome lying in the
middle of the spindle figure are the most frequent types of aberrant con-
figurations, and since these rings are subsequently excluded from the


330                BARBARA McCLINTOCK
telophase nuclei, the frequently observed absence of the ring chromosome
from branches of the tassel or from groups of cells in an anther can be ex-
plained.
In connection with the problem of the mechanism of movement of
chromosomes in the spindle it would be of interest to explain why these
small double-sized rings do not appear to be under greater tension as their
spindle fiber regions pass toward opposite poles. It is possible that move-
ment toward opposite poles is initiated at the spindle fiber region of the
chromosome at early anaphase but that continued movement is made
possible by other forces exerted on the chromosomes when they have
reached a region in the spindle which is some distance away from the
equatorial plate. These double-sized ring chromosomes may be too small
to reach this region by the pull exerted at the spindle fiber region. The
behavior of intermediate sized rings lends support to this assumption since
these sometimes remain in the equatorial plate either with or without
evidence of tension.
From cytological examination of sporocytes it was known that changes
in size of the small ring chromosomes do occur though with relatively low
frequency. The R 2 chromosome has been observed to decrease to several
chromomeres and also to increase to seven or eight times its original size.
Photograph 15, Plate I, represents a normal R 2 chromosome, photographs
II and 12, an enlarged R 2 chromosome. It has also been seen that an
increase in the number of these rings in a nucleus, usually with alterations
in size, sometimes occurs. As many as six ring chromosomes in the nuclei
of a sector of a plant which very probably possessed but one ring in the
zygote have been observed in a single instance.
If a double-sized ring is included in one of the daughter nuclei, as
described above, a start in the direction of increase in number of rings
has been made. The chance of loss of this ring chromosome in subsequent
divisions is great. However, as already shown, the ring chromosome may
be directly broken in two by opposed poleward forces at anaphase, or the
poles of the spindle may lie so close together that the spindle fiber regions
of the ring are readily included in the two nuclei, after which the resulting
chromatin bridges are cut through by the cell-plate and drawn into the
nuclei. When two double-sized sister ring chromosomes each with two
spindle fiber regions are present in an anaphase figure, and when each of
these is subsequently broken and the broken ends drawn into the telophase
nuclei, the initial event in the production of a sector of tissue with two
altered ring chromosomes has occurred. It is apparent, on this basis, why
increase in number of these small ring chromosomes is relatively rare: one
infrequent event must be followed by another.


              RING CHROMOSOMES IN MAIZE             331
     Conclusions regarding ring chromosome behavior
The foregoing account has indicated the probable method by which
rings are altered in size and genie constitution or are lost from the nuclei
altogether. From both cytological and genetical observations it has been
concluded that the rate at which this occurs is dependent upon the length
of the chromonema composing the ring: the longer the chromonema, the
more frequent the occurrence of aberrant mitoses involving the ring.
Counts of the aberrant ring chromosome configurations in late anaphase
and very early telophase are given in table I. The counts are from roots
in which the ring chromosome was present in most of the cells. In A, the
R 1 chromosome of case I, figure I, is represented. One aberrant configura-
tion of the ring chromosome was observed in these roots which were
counted. Judging from the relatively small amount of bm 1 tissue shown
by plants with this R 1 chromosome, loss of the ring chromosome must be
relatively infrequent. In B, the small R2 chromosome of case II is repre-
sented. The observed aberrant configurations of the ring chromosome in
these roots amounted to 1.1 percent of all the figures recorded. As stated
above, these aberrant configurations result mainly in loss of the ring
chromosome from both nuclei. Variegation, expressed by the bm 1 tissue
in these plants, is considerably greater than in the plants from which the
counts of table I, A, were obtained. Table I, C, represents the counts
from a plant which possessed two ring chromosomes, a normal R 2 and an
R 2 enlarged approximately three times. In the roots from which the
counts were made, both rings were present in many of the nuclei. Aberrant
configurations involving the enlarged R 2 chromosome were more frequent
than those involving the normal R 2 chromosome. The size of the enlarged
R2 chromosome lies at the border line between those rings whose aberrant
configurations lead mainly to exclusion from the telophase nuclei (small
rings) and those whose aberrant configurations lead mainly to changes in
size of the ring chromosomes (large ring chromosomes). For the enlarged
R 2, both types of configurations were frequently encountered. In table I,
D, from a plant with a ring chromosome approximately twice the size of
the enlarged R 2 chromosome, the aberrant configurations amounted to
8 percent of the total number recorded. In this case, as described above,
the figure is possibly too low. The telophase figures here were characterized
mainly by changes in size of the ring chromosome rather than loss from
the nuclei.
If a ring chromosome in a given individual carried a dominant gene and
the two normal rod chromosomes carried a recessive gene, the expression
of variegation produced by losses or changes in constitution of the ring
chromosome would depend upon (I) the size of the ring chromosome and


332                 BARBARA McCLINTOCK
(2) the position of the gene with respect to the spindle fiber attachment
region. In relatively small ring chromosomes, which are mainly lost from
the nuclei, the expression of variegation is directly dependent upon the
actual size of the ring chromosome: the larger the ring chromosome the
greater the amount of variegation exhibited. This is strikingly illustrated
by the two ring chromosomes, R 1 and R 2. The variegation produced by
Rl is very much less than that produbed by R 2. TO check this conclusion
without prejudice, cultures were obtained in which either R 1 or R2 or both
R 1 and R 2 chromosomes were expected to be present in individual plants

TABLE 2

Comparisons of the predicted and observed ring chromosome constitz~tions in cultwes segregating
           plants z&h one, two and three ring chromosomes.

PREDICTION:         PRED-CTION :
  1Rl             lR2

PREDICTION:
2 rings

PREDICTION:
3 rings

Correct Deviation   Correct Deviation   Correct Deviation   Correct Deviation
                           5 (I ring)*
14   1(Rl+R2)     15    3(Rl)      24   1(3 rings) t     3       0

* Three showed one R 1; one showed one R 2 in an estimated two R 2 plant; one showed one
R2 in an estimated R 1 plus R2 plant. Complete agreement in all cases could not be expected in
two- and three-ring plants from sporocyte examinations since loss of one of the ring chromosomes
in the developmental stages of the tassel is expected in some cases. This particularly applies to
the R2 chromosome.

t The estimate for this plant was two Rl. Some of the two Rl plants have practically no
bml tissue. A two R 1 plant could be difficult to distinguish from a three R2 plant.

of the culture. In some of these cultures plants with three ring chromo-
somes were expected. From the expression of the variegation exhibited
by each plant a prediction was made as to the ring chromosome constitu-
tion of the plant. Cytological observations were subsequently made to
determine the correctness of these predictions. Table 2 shows the correla-
tion of these observations with the predictions. Cultures of plants with
the R 1 chromosome can readily be separated from cultures whose individ-
uals possess the R 2 chromosome through observations of the variegation
alone (see following section for more complete discussion).

With relatively large ring chromosomes, which are characterized mainly
by changes in size of the ring chromosomes, the expression of variegation
would depend upon the nearness of the locus of the gene to the spindle
fiber region. The farther away the locus, the greater is the amount of varie-
gation that should be expressed.

The method of alteration of the ring chromosomes as suggested by the
somatic anaphase and telophase configurations should produce rod-shaped
chromosomes. Although thousands of microsporocytes have been exam-
ined in many of which an alteration of the ring chromosome has been ob-


               RING CHROMOSOMES IN MAIZE             333
served, no rod-fragments have been recognized. It can not be stated that
they do not occasionally occur, but certainly their frequency must be
exceedingly 10~. If the method by which ring chromosomes change in size
has been correctly interpreted from the study of somatic anaphase and
telophase figures, one is forced to conclude that the broken ends of the
chromosomes unite, thus regstablishing a ring.

It might be stated here that when two ring chromosomes are present in
the nuclei of a plant, it is rare that both rings show aberrant configurations
in the same cell (figure 18). Each ring chromosome apparently acts inde-
pendently with regard to the formation of double-sized or interlocked
rings.

That the behavior of ring chromosomes in maize is a consequence of
their form and not of their genie constitution can be definitely stated,
since a number of different ring chromosomes, each involving segments of
chromosomes not strictly comparable, have been found so far. These in-
clude segments from chromosomes II, V, VI, VIII and IX. Most of them
were detected by the variegation which they produced but three were
isolated independently of any visible effect.

III. THE NATURE OF THE Bm I-bm 1 VARIEGATION

The gene bm 1 when homo- or hemizygous produces a brown color of
the cell walls. The color appears in the walls as soon as lignification sets in.
It is not present before this period. The depth of color, on external exam-
ination of bm 1 plants, is greatest in those tissues which are composed
largely of thickened cell walls, such as the midrib of the leaf, the veins in
the leaf sheath and the stalk tissue. The brown color is not easily detected
in the leaf tissues other than the midrib since the cell walls arelthin and
the color is masked by the chlorophyll.

As the plant matures in the field, the brown color has been noted to
fade considerably in exposed regions of the plant but remains deep in
those regions which are well protected from light. It was suspected that
direct sunlight was causing a change in the structure of the brown pigment
which resulted in loss of color. To determine if this was correct, black paper
was placed about exposed parts of several bm 1 plants when the brown
color was intense. The bands of black paper remained about these parts
for a period of three weeks. When the paper was removed, the tissues
protected from light had retained their original deep brown; the brown
color in the tissues above and below the protected region had faded con-
siderably.

In plants possessing two normal chromosomes V with bm 1 (or one
normal chromosome V with bm 1 and one of the deficient chromosomes
V) and a ring chromosome with Bm 1, streaks of bm 1 tissues are pro-


334                 BARBARA McCLINTOCK
duced and can be seen by external examination of the plants (figure I).
Over 7000 variegated plants have been examined in the progeny of the
two original variegated plants. Cytological observations have indicated
that loss of the ring chromosome carrying Bm I is the primary cause for
the appearance of the bm 1 tissues. *Losses can occur anywhere in the
ontogeny of the plant. The patterns of the bm 1 tissues should give some
indication of where and when these losses occurred. Although wide or
narrow bands on the stalk (figure I) indicate an early or late loss of the
ring chromosome, respectively, cross sections of the stem, where most
of the cell walls are heavily lignified, give even a better indication of the
time of loss. If loss occurred early in ontogeny, the whole plant would be
bm 1. If the first loss occurred in one of the cells which is to give rise to
the part of the plant above the ground, a wide sector of bm 1 would
be produced. Still later losses would produce sectors of various widths in
the stem. Very late losses would produce streaks or patches composed of
a few cells only. All of these types of variegation patterns have been ob-
served.
When a stalk with a relatively wide external band of bm 1 tissue is
cross-sectioned and examined with low magnification, the brown-walled
tissue is seen to be composed of a V-shaped sector with the tip of the V
pointing toward the center of the stalk. Many narrow surface streaks are
produced by similar sectors but the V is smaller and the tip considerably
removed from the center of the stalk. Very narrow streaks may be com-
posed of only a few cells. Such streaks are visible on external examination
of the stalk only if they lie at or close to the surface. Patches of bm 1
cells not close to the surface cannot be seen by external examination.
Dilution of color in the brown (bm 1) cell walls on the side of the wall
adjacent to the white (Bm 1) cell walls was a striking feature of the
variegation in all plants. That it is a dilution produced by the adjacent
Bm 1 cells and not a spreading of the brown color from the bm 1 cell
walls is suggested by the considerable reduction in intensity of color in
the brown walls of the very small patches composed of only a few cells,
and by the dilution of color of a row of bm 1 epidermal cells on the side
adjacent to inner Bm 1 cells.
The variegation in plants possessing an R 2 chromosome is expressed
by a few totally bm 1 plants where the ring chromosome has been lost
before the cells which are to produce the stem meristem have been differ-
entiated, to plants which are composed of many bm 1 streaks of different
widths. Cross sections of the stems of the average variegated plant show
wide V-shaped sectors, smaller V-shaped sectors and many irregular
patches of bm 1 composed of few to many cells.
The variegation patterns in plants with the R 1 chromosome were similar


              RING CHROMOSOMES IN MAIZE            335
to those produced by plants with the R2 chromosome but the total amount
of bm 1 tissue was very much less. There were feper sectors of all types
in these plants, making cultures of the two types of variegated plants
readily distinguishable. This is expected from the cytological examinations
since the smaller ring chromosome is lost less frequently in somatic divi-
sions than the larger ring chromosome. The extent of variegation is a
direct expression of the rate of loss of the ring chromosome.
Plants with two ring chromosomes show considerably less variegation
than plants with one ring chromosome. Loss of one ring chromosome
followed later by loss of the second ring chromosome or simultaneous
losses of both ring chromosomes must occur before the bm 1 tissue could
be produced. The patterns of the bm 1 tissues in cross-sections of the
stem clearly show this relationship. These fall into three main types of
sectors: (I) solid V-shaped sectors, (2) spotted V-shaped sectors, and (3)
small patches of bm 1 tissue.
The solid V-shaped sectors are interpreted as relatively early losses of
one ring followed slightly later by loss of the second ring chromosome or
by occasional simultaneous losses of both rings. The spotted V-shaped
sectors reveal more closely the relationship between loss of one ring fol-
lowed considerably later by losses of the second ring. They are detected
as a cluster of bm 1 patches in an isolated region of a stem which other-
wise shows very few bm 1 patches. When each of the brown patches in
such a cluster is traced with a camera lucida and lines drawn joining the
outer boundaries of the outermost patches, the lines converge in the direc-
tion of the center of the stem. They clearly define a V-shaped sector.
Such spotted V-shaped sectors would be expected if loss of one ring carry-
.ing Bm 1 is followed later in development by losses in different cells of the
the second ring chromosome carrying Bm 1.
The small patches of brown walled tissue, usually composed of only a
few cells, can be interpreted as relatively late, successive, or occasionally
simultaneous, Iosses of the two rings.
There are three types of plants with two ring chromosomes: (I) those
with two Rl, (2) those with one Rl and one R 2 chromosome, and (3)
those with two R 2 chromosomes. Since somatic loss of the R 2 chromosome
is considerably more frequent than the Rl chromosome, the amount of
bm 1 tissue produced in each of these plants is progressively greater.
Plants with two R 2 chromosomes have considerable amounts of bm 1
tissue; those with one Rl plus one R2, very much less, and those with
two Rl chromosomes exceedingly-little bm 1 tissue. In this latter type
of plant it is often necessary to examine cross-sections of the stem to
determine if any bm 1 tissue is present. Such tissue, when not close to
the surface, cannot be detected from field examinations of the plants.


336                 BARBARA McCLINTOCK
Plants with three ring chromosomes of the constitution two R 2 plus
one R 1 chromosome or one R 2 plus two R 1 chromosomes, have been
obtained. These plants frequently show no external evidence of bm 1
tissues. In all cases, however, careful examinations of the stalks have
revealed small patches of bm 1 cells. The bm 1 cells could arise only
after loss (mainly successive) of all three ring chromosomes from the
nuclei.

For the sake of comparison, the stalks of a number of plants with a
normal chromosome constitution carrying Bm 1 in one chromosome V
and bm 1 in its homologue were examined. In no case was there any
evidence of bm 1 tissue.

In conclusion it can be emphasized that the genetic expression of varie-
gation is in full agreement with expectation on the basis of the cytological
observations given in the previous section. In these plants with small ring
chromosomes whose aberrant mitotic configurations are followed mainly
by loss of the ring chromosome from the nucleus, the extent of variegation
is a direct indication of the length of the chromonema composing the ring
chromosome, the larger the ring chromosome the higher the rate of loss
and thus, the greater the amount of exhibited variegation. Loss of the
ring chromosome can occur at any stage in the development of the plant,
early loss giving rise to a totally bm 1 plant, later loss to wide sectors
of bm 1 and very late losses to small patches of bm 1 cells. The patterns
exhibited by two and three ring chromosome plants are those expected
from the cytological observations where it has been shown that simultane-
ous loss of the several ring chromosomes from a nucleus is rare. The cause
of the aberrant mitotic configuration arises independently in each ring
chromosome.

Knowledge gained from a study of variegation in these plants has been
utilized in the analysis of tissues of plants mosaic for homozygous de-
ficiencies (section V).

IV. TYPES OF FUNCTIONAL GAMETES PRODUCED BY THE

TWO ORIGINAL VARIEGATED PLANTS

Each of the two original variegated plants possessed one normal chromo-
some V with bm 1, one deficient chromosome V and a ring-shaped frag-
ment chromosome corresponding in size to the deficiency in the rod
chromosome (figure 2). In case II (Def 2, R2) prophase meiotic associa-
tions had indicated the homology of the ring chromosome with the region
in the rod chromosome which had been deleted (figures 25 and 26; photo-
graphs of the same, 17 and 18, Plate II). Most frequently, the ring chromo-
some did not associate with its homologous section in the normal rod


              RING CHROMOSOMES IN MAIZE             337
chromosome but remained separate and collapsed (for meiotic prophase
behavior of ring-shaped chromosomes, see MCCLINTOCK 1933). In all cells
the deficient rod chromosome V and the normal chromosome V were asso-
ciated. The normal V had to buckle to compensate for the deletion in the
deficient V. Figures 27 and 28, and photographs of the same, 19 and 20,
I late II, illustrate this association. In the plant from which figure 27

FIGURE zs.-Pachytene association of a normal chromosome V, a Def 2 chromosome V and
its compensating R2 chromosome. The ring chromosome has been drawn with a finer line. The
spindle fiber attachment region has been drawn as a slight bulge. The dark bodies toward the
end to the right are the knobs. See photograph of the same, I 7, Plate II.

was drawn, two ring chromosomes were present. They are separate and
collapsed. Another figure from the same plant, photograph 21, Plate II,
shows the not infrequent association of the two R2 chromosomes to form
a true ring-shaped configuration and also the association of the normal
and Def 2 chromosomes.

FIGURE s6.-Similar situation to that shoan in figure 25. See photograph of the same, 18, Plate II.

In case I (Def 1, Rl), no figures were observed showing the association
of the ring chromosome with its homologous section in the normal chromo-
some. It is probable that it occurred in a small percentage of the cases but


338                BARBARA McCLINTOCK
would be difficult to detect except in the most favorable figures because
of the smallness of the deficiency and the ring chromosome. Figures 29
and 30 illustrate the pachytene association of the Def 1 chromosome with

FIGURE Pg.-Pachytene association in a microsporocyte of a plant with one normal chromo-
some V, one Def 2 chromosome V and two R2 chromosomes. Note the buckle in the normal
chromosome V at the spindle fiber attachment region and the two unassociated, collapsed ring
chromosomes. The ring chromosomes have a similar chromatin constitution to that of the buckle.
See photograph of the same, rg, Plate II.

a normal chromosome V. The small ring chromosome (Rl) lies free and
is collapsed. In figure 30 and photograph of the same, 28, Plate II, non-
homologous associations about the deficient region have resulted in a

FIGURE s8.-Pachytene association of a normal chromsome V and a Def2 chromosome 1`.
          See photograph of the same, 20, Plate II.

separation of the spindle fiber attachment regions of the two chromosomes
(for expected non-homologous associations, see M~CLINTOCK 1933)~ The
small buckle below the lower spindle fiber region or the distance between


              RING CHROMOSOMES IN MAIZE             339
the two spindle fiber regions represents the extent of the deficiency.
Photographs 23, 26 and 27, Plate II, illustrate the appearance of the Rl
ring at meiotic prophase. In photograph 23, very early diplotene, the

FIGURE pg.--Pachytene association in a microsporocyte of a plant with one normal chromo-
some V, one Def 1 chromosome V and an R 1 chromosome, The ring chromosome is collapsed and
is not associated with its homologous region (buckle) in the normal chromosome V.

ring shape of the chromosome is clear. In photograph 26 the spindle fiber
region of the Rl chromosome is stuck to that of chromosome VIII. In
photograph 27, the spindle fiber region of the collapsed R 1 chromosome is
adjacent to that of a normal chromosome V bivalent. Photographs 24

FIGURE so.-Pachytene association of a normal chromosome V and a Def 1 chromosome V.
Through non-homologous associations, the buckle which compensates for the deficiency, has
shifted into the long arm of the normal chromosome V. Note the displacement of the spindle fiber
attachment regions. See photograph of same, 63, Plate II.

and 25, Plate II, illustrate the relative sizes of Rl and R2 when both
are present in the same sporocyte. In both photographs the collapsed ring
chromosomes are associated by their spindle fiber attachment regions. In
photograph 24 the Rl chromosome is above, the R2, below. In photo-
graph 25 the Rl chromosome is to the left, the R2 to the right.

In plants heterozygous for either deficiency and its compensating ring


340                  BARBARA McCLINTOCK
chromosome, the deficient rod chromosome and its normal homologue
proceed quite normally during the meiotic mitoses, two spores of a quartet
receiving the deficient chromosome, two the normal chromosome. The
behavior of the ring chromosome, on the other hand is irregular. In the
case of RI chromosome the split halves separate and pass to opposite poles
at anaphase I along with the disjoining bivalents (except where double-
sized or interlocked ring chromosomes are formed). The split halves of
the R2 chromosome likewise separate at I, either at the same time that
the bivalents disjoin or slightly later. They are nearly always included
in the first division telophase nuclei.
In the second meiotic mitosis, the behavior of the ring chromosome is
variable. They do not divide again but either pass to one of the poles
along with the other chromosomes of the complement or remain in the
spindle figure and are excluded from the telophase nuclei. The behavior
of the rings in the two sister cells is not always the same.
As a result of meiosis, four types of spores are to be expected. They
carry the following chromosomes :

I. Normal chromosome V.
2. Deficient chromosome V.
3. Normal chromosome V plus the ring fragment.
4. Deficient chromosome V plus the ring fragment.
The percentage of each type in an anther would depend upon (I) the
proportion of the sporocytes which lacked a ring chromosome, giving only
types I and 2 above, and (2) the percentage of cases in which the ring
chromosome, when present, was included in the second meiotic telophase
nucleus.
Examination of the pollen has given some indication of the percentage
of each of these four types which are present in an anther. Pollen from a
bml sector of a plant known to have a normal chromosome V (carrying
bml), a deficient chromosome V (Def l), and an R 1 chromosome, showed
three types of grains: (I) large well filled grains, (2) small partially filled
grains and (3) small totally empty grains. The proportions of each type
are shown in table 3, A. In these bml sectors it is assumed on good evi-
dence (see sections I and II) that the ring chromosome has been lost.
Equal proportions of type I and type 2 grains should be present. If the
small partially filled grains represent those with the Def 1 chromosome,
the large filled grains, those with the normal chromosome V, they should
be present in equal proportions. A total of 5535 normal pollen grains to
5547 small partially filled grains clearly indicates this association. The 347
small empty grains represent 3.3 percent of the total. In all samples of


               RING CHROMOSOMES IN MAIZE             341
pollen from normal maize plants there is a small percentage of these empty
grains. They probably represent the products of abnormalities in meiosis
which are not infrequently observed in normal plants.
Anthers from Bml regions of the plant, in which the ring chromosome
is present, give a higher proportion of normal well-filled grains (table 3, B).
The difference is interpreted as due to the presence of the ring chromosome
in some of the grains which have a deficient rod chromosome (type 4,
above). Since the ring chromosome, if unaltered, covers the deficiency,
a normal appearing pollen grain is expected. On this interpretation, the
number of each type in a particular anther can be estimated. Pollen types
I and 2 should be present in equal numbers. Likewise, types 3 and 4
should be present in equal numbers. Type 2 grains can be directly re-
corded. An equal number of the normal appearing grains should belong
to type I. When this number is subtracted from the total number of
normal appearing grains, the remainder can be equally distributed to
types 3 and 4. The estimates of each type of grain from the Bml anthers
in table 3 B, are: type I, 1994; type 2, 1994; type 3, 743 ; type 4, 743, or
36.5 percent each of types I and 2 and 13.5 percent each of types 3 and 4.
In one plant, heterozygous for Def 1 RI, four types of pollen grains
were present, 619 large well-filled grains, 170 small but well-filled grains,
426 small partially-filled grains (type 2) and 49 small empty grains. If
it is assumed that the small well-filled grains represent type 4 with an
altered ring chromosome which does not completely cover the deficiency,
pollen types 2 and 4 can be directly recorded. If, on the other hand, these
grains are included in the normal appearing class, and calculations made
as above, the proportion of types are: type I, 426; type 2, 426; type 3,
184; type 4, 184. It is obvious that there is a close agreement in the cal-
culated number of 184 for type 4 grains and the 170 grains which have
been assumed to represent this type.
In plants heterozygous for Def 2 and R2, the type 2 pollen grains are
large but almost completely empty. These grains cannot be distinguished
from the few empty grains produced by other causes than the presence of
the deficiency in chromosome V. However, if these latter grains are
assumed to represent two percent of all the grains, an approximate esti-
mate can be made of the number of grains with each of the four chromo-
somal constitutions. The counts from the bml anthers are given in table
3 C, and similar counts from the Bml anthers, with estimates of propor-
tions of types, in table 3 D.
The functional capacity of each of the four types of gametes can best
be illustrated by reference to the types and numbers of individuals result-
ing from the crosses given in table 4. Section A in the table represents the


342                BARBARA McCLINTOCK
                   TABLE 3
A. Pollen counts from bml anthers of plants will; the conslilution Def l/bml/Rl.

PLANT

LARGE FILLED   SMALL PARTIALLY      EMPTY      70 EMPTY
  GRAINS      FILLED GRAINS      GRAINS         GRAINS

sgSA-2          II81        1148           49            2.0
SgSA-z        832       91;        3S            2.1
597A-2          775           751           66            4.1
597A-2          610           611           21           1.6
59SA-16        692         678           42            2.9
sgSA-3           523         526           70            6.2
597I3-6           922        916           61            3.2

Totals         5535         5547         347           3.3

B. Pollen counls from Bml anthers of a plant with the coditutiox Def l/bml)R 1.

PLANT

         SYALL
LARGE                              ESTIMATES OF FOUR TYPES
       PARTIALLY    EMPTY
FILLED                70 EMPTY   OF GRAINS IN PERCENT
        FILLED     GRAINS
GRAINS                       GRAINS
        GRAINS                       I     2    3 4

59SA-2      `43'       so2       47       2.0     36   36    1.1    14
SgSA-z      1094      561       26       I.5      34    34    16    16
59SA-2     956     631       28       I.7      40    40    IO    IO

Totals     3481      1994       101       1.8

C. Pollen counts from bml anthers of a plant with the constituiiox Def2,lbml/R2.

PLANT

NORMAL GRAINS            EMPTY GRAINS

9X9-6                  681                  733
9s3B-6                  943              918
953B-6              864              962
953B-6              761                  779
953B-6             1036                  1079

Totals             4285                4471

D. Pollen counls from Bm 1 anthers of plants with the cons&&ion Def 2/bml jR2.

PLANT

                        ESTIMATE OF FOUR TYPES OF
                              GRAINS IN PERCENT
NORXAL GRAINS    EMFTY GRAINS
                             I     2    3 4

1009-10        906         864        4S   4S    *    2
1009-S           95s         623       38   38    12    12
1009-8          1281         847       38   38    12    12
roog-8          I 109           822          41    41    9    9

Totals         4251        3156


               RING CHROMOSOMES IN MAIZE             343
progeny from crosses of the two original variegated plants. Section B rep-
:c+ents crosses of plants from A of this table, which were heterozygous
/,,r the deficiency and compensating ring, with normal bm 1 plants. It
can be seen that all f our gametes produced by plants heterozygous for
Dei 1 Rl can be transmitted through the eggs. Through the pollen,
zametes of type I and 3 are readily transmitted but gamete type 2 is
not transmitted and gamete type 4 only rarely in competition with
gametes I and 3.

TABLE 4

  TYPEOFCROSS
(Female parent to left)

Def l,`bml/RlXbml

CONSTITUTION OF PLANTS RESULTING FROM CROSSES
   bml PLANTS          VARIEGATED PLANTS

bml/bml    Deflbml   bml/bml/R   Def/bml/R

218          9         16          I2

.\.
  bmlXDefZ/bml/RZ       38          0          0          8

____~~~~
  DefZ bml/R2Xbml        352         I*        13          II
   reciprocal           696       4*       353        IS.4
13.
  Defl, bml/RlXbml        3*          13         29         =7
   reciprocal              74          0         =7          I

C.  Def 1,`bml Xbml           IS5          6          0          0

* These bml plants probably arose through very early loss of the ring chromosome. The
Def 2 chromosome is not transmitted without the R 2 chromosome.

In plants heterozygous for Def 2 R2, gametes I, 3 and 4 are trans-
mitted through the eggs but gamete 2 is not transmitted. Through the
pollen, gametes I and 4 are readily transmitted. The frequent transmis-
sion of the Def 2 R 2 combination through the pollen contrast with the
\-erv infrequent transmission of the Def 1 R 1 combination through the
pollen. Both cases are in agreement in the lack of transmission of the
deficient chromosome minus its compensating ring chromosome through
the pollen.

To obtain high counts on transmission of the deficiency carrying gam-
etes, the gene bt (brittle endosperm) was introduced into the normal
chromosome. The bt gene is located in the long arm of chromosome V
(RHOADES 1936) and gives very little crossing over with bml. No posi-
tive case of crossing over between bt and the two deficiencies of chromo-
some V has been found. Thus, plants with a deficient chromosome carry-
ing Bt and a normal chromosome with bt, when crossed to normal bt should
give the percentage of deficiency-carrying gametes (Bt kernels) directly,
without the necessity of growing large progenies and testing each indi-


344                 BARBARA McCLINTOCK
vidual plant for the presence of the deficient chromosome. The results
of such crosses are given in table 5. In many of the crosses bml was like-
wise involved. The deficiency carrying plants were Def Bt/Bml bt with
or without a ring chromosome carrying Bml. These were crossed with
normal bml bt individuals. The Bt kernels should give rise to variegated
(BmI-bm1) plants when the ring chromosome is present or totally bml
plants when the ring chromosome is absent; the bt kernels should give
rise to totally Bm1 plants. The results of a test of the Bt and bt kernels
are summarized in table 6. If no crossing over occurred between the de-
ficiency and Bt, bml could appear in these crosses only when a deficient
chromosome was present. 177 of the 358 individuals showing bml were
tested for the presence of the deficient chromosome. It was present in
every case. Thus, the data in table 5 can be used as a direct means of
determining the functioning of the gametes which carry a deficient chro-

mosome.

           TABLE 5
Bt kernel lest of transmissions of Def 1 and Def 2 chromosomes.

CROSS*                  Bl

bt

I. +Bt/+btXbt              2893              2746
2. reciprocal                    797              889

3. Def 1 Bt/+bt/R I X bt             1217             6%
4. reciprocal                       4                 5544

5. Def 1 Bt/+bt (no ring)Xbl          927                 I 2682
6. reciprocal                       0                  323

7. Def2 Bt/+bt/R2Xbt
8. reciprocal

g. Def2 Bt/+bf (no ring)Xbl
10. reciprocal

187                5135
363                 19s=

0                13x9
0             4065

* A non-deficient chromosome V is represented by +. The pollen parent is placed at the right
in each cross.

The results given in lines 3 and 5 of table 5 are particularly interesting
with respect to the functioning of the Def 1 chromosome through the
eggs. If there had been no megaspore selection in favor of the spore carry-
ing. the normal chromosome and if all the eggs (or zygotes) which carried
the Def 1 chromosome functioned, the ratio of Bt to bt should be equal.
In all cases the number of bt kernels was greater than Bt and in all cases
the ears were incompletely filled. The presence of the abortive grains on
the ear indicate that there has been little if any selection of normal chro-
mosome carrying megaspores. Therefore, the percentage of Bt kernels,


               RING CHROMOSOMES IN MAIZE            345
when the bt kernels are taken as the standard of expectancy, indicates
the extent of functioning of the deficiency carrying eggs (or zygotes).
The 927 Bt kernels in line 5, table 5, represent 7.2 percent of the deficiency
carrying eggs (or zygotes) which functioned. In the 74 ears which contrib-
uted to this count, the percentages ranged from o to 23.2 with half of
the ears falling within the range of 2 to 8 percent. When the ring chro-
mosome (Rl) was present (line 3, table 5) the Bt kernels on the 30 ears
contributing to this count ranged from I to 40 percent of expectancy on
the individual ears alld averaged 20.1 percent. That .this increase can be
attributed to the presence of the ring chromosome covering the deficiency
in many of the eggs can be seen from line I, table 6.

TABLE 6

CROSS

PLANTSPROM Bt KERNELS

Bml-bml      bml
variegated

PLANTSFROM bt KERNELS

Bml        bml

Defl Bf/Bml bt/RlXbml bt      40          23      not grown
Def 1 Bt/Bml btXbm1 bt          0        85       328          0
Def2 BtjBml bt/R2Xbm1 bt      106          7         342          0
bml btXDef2 Bt/Bml btfR2      95          2          24         0

That pollen containing the Def 1 chromosome without its compensat-
ing ring chromosome does not function in competition with normal pollen
can be concluded from line 6, table 5. By use of a 170 wire mesh screen
these grains, which are small and partially filled with starch, can be segre-
gated from the normal grains and the possible factor of competition with
the grains carrying the normal chromosome eliminated. Some of the
ears pollinated with sifted pollen gave no kernels at all, others a few bt
kernels through passage of a few small but normal chromosome pollen
grains through the wire mesh. It can be definitely stated, therefore, that
these grains are incapable of producing an effective pollination when placed
upon normal silks.

Line 4, table 5, suggests that the grains with a Def 1 and an R 1 chro-
mosome normally do not effect a pollination in competition with normal
chromosome carrying grains. The four Bt kernels were grown to deter-
mine the chromosome constitution of' the resulting individuals. TWO of
these Bt kernels were definitely produced through contamination,  one
through functioning of a Def 1 R 1 grain and one could have been a cross-
over between Bt and the deficiency although contamination could not be
excluded definitely. If this kernel represents a crossover, it is the only
evidence so far obtained of crossing over between the deficiency and bt.


346                 BARBARA McCLINTOCK
From this evidence it could be concluded that (I) the ring-shaped chro-
mosome, R 1, does not completely cover the deficiency due to a loss of a
small section either at the time of irradiation or during the development
of the original plant or that (2) a mutation affecting pollen tube growth
appeared at the time of irradiation or (3) the particular chromosomal
modification (position effect) is responsible for the reduced pollen tube
activity. If (I) above is correct, .the deficiency, when homozygous, does
not produce a visible effect in the tissues of the mature plant (see section
v>.

That the gametes with Def 2 function only when the ring chromosome,
R2, is present is evident from tables 5 and 6. The Def2 gametes with a
complete R2 chromosome have an equal chance in competition with
normal chromosome carrying gametes. The discrepancy in the percent-
ages of Bt kernels in the reciprocal crosses, lines 7 and 8, table 5, can be
understood when it is realized that the ear arises from a definite sector
of tissue which originally may or may not have had the ring chromosome
in its nuclei (6 of the 36 ears had no Bt kernels) whereas the pollen is
shed from all parts of the tassel which is usually a mosaic of sectors with
and without the ring chromosome. The percentage "expected" Bt kernels
on the 36 individual ears in the cross summarized in line 7, table 5, ranged
from o to 8.9 percent, those on the six ears summarized in line 8, from 4.5 to
2 I .6 percent.

As stated in section II, changes in size and genie constitution of the
ring.chromosomes occasionally occur during ontogenesis of a plant. This
being so, it could be objected that the two original ring chromosomes could
not be kept constant through successive generations. Small duplications
within a ring chromosome are not phenotypically detectable. They must
be determined through cytological examination. In contrast, the deficien-
cies within the ring chromosome can be detected through phenotypic
appearances of certain plants (see section V) and through pollen trans-
missions which tend to eliminate gametes with the deficient ring chromo-
some. However, change in size of the ring chromosome is not frequent and
with proper care, it is not difficult to maintain stocks with unaltered ring
chromosomes.

V. PRODUCTION OF PLANTS MOSAIC FOR HOMOZYGOUS DEFICIENCIES

As shown in the previous section, the progeny of the crosses of the two
original variegated plants by normal bml included a number of individuals
with chromosomal constitutions similar to the two original plans. Plants
heterozygous for Def 1 and R 1 when crossed by plants heterozygous for
Def 2 and R2 should produce twelve types of plants, each with a different
chromosomal constitution :


               RING CHROMOSOMES IN MAIZE             347
I. bml/bml     5. Def l/bmf/R2    9. Def 2/bml/Rl/R2
2. bml/bml/R2  6. Def l/Def 2/R2   IO. Def l/bm l/R 1
3. Def2/bml/R2 7. bml/bml/Rl    II. Defl/bml/Rl/R2
4. Def l/bm1    8. bml/bml/Rl/R2  12. Def l/Def 2/Rl/R2   '
In the cross of heterozygous Def 1 R 1 by normal bm 1 plants, a number
of individuals with the constitution of plant type 4, above, were obtained.
When these, in turn, are crossed by plants heterozygous for Def 2 R2,
the first six types of plants listed above should be produced.
All of the plants except 6 and 12 from the first cross, and all the plants
except 6 from the second cross can be distinguished by the presence or
absence of variegation, the type of variegation exhibited and the type of
pollen shown by each plant. Cytological examinations of a number of
these plants were in agreement with the field determinations.
The appearance of plants of type 6 and 12 could not be predicted. In
actual experience it proved very simple to identify them. Both types of
plants possess two deficient chromosomes, Def 1 and Def 2. Plant 6 has
one ring chromosome, R 2, plant I 2, two ring chromosomes, R 1 and R 2.
These two types of plants will be designated R 2 double-deficient and R 1
R 2 double-deficient.
In the R2 double-deficient plants, the ring chromosome covers both
deficiencies. Its loss in somatic nuclear divisions should result in cells
homozygous deficient for the extent of the deficiency in the Def 1 chromo-
some. If these cells were viable and continued to multiply at the same rate
as the surrounding heterozygous deficient cells, which are close to normal
in growth rate, both wide and narrow sectors of homozygous deficient
tissues should be produced through early and late losses, respectively, of
the ring chromosome during ontogeny. In the Rl R2 double-deficient
plants, both ring chromosomes cover the homozygous deficient segment
in the rod chromosomes. Simultaneous loss of both ring chromosomes or
loss of one ring chromosome followed later by loss of the second ring chro-
mosome must occur in order that homozygous deficient tissue can be
produced. The total amount of homozygous deficient tissues produced
in the R 2 double-deficient plants should be considerably greater than that
produced by the R 1 R 2 double-deficient plants. If tissues homozygous
deficient for the extent of the deficiency in Def 1 were visibly modified,
the two types of plants should be readily distinguishable. However, no
prediction as to the nature of the homozygous deficient tissue was possible
before the appearance of these plants.
In addition to the bml and variegated plants resulting from the cross of
Defl/bmlXDef2/bml R2, one plant appeared (table 7) which was not
bml and did not show the ordinary Bml-bmi variation. This plant was
stunted in growth habit, the leaves and leaf sheaths were uniformly


348                 BARBARA McCLINTOCK
                   TABLE 7

Def l/bm 1 (no ring)XDef 2/bm l/R 2.

CULTURE

b??Zl AND
VARIEGATED

Def l/Def2/R2

692                     220                    I
693                     23                    0
36-30                   97                    0

Totals                 340                    I

streaked with fine bands of colorless tissue, as shown in figure 32 (com-
pare with figure 31, a normal leaf). In the cross of Def l/bml/R 1 XDef 2/
bml/R2, besides the bml and variegated plants, two new types of
plants appeared (table 8). One type was similar to the plant just described.
The other type was considerably larger, approaching normal in growth
habit, but was not a typical bml or variegated plant, and presented the
same fine streaks of colorless tissues in the leaves and leaf sheaths as the
first new type. However, the total amount of such tissue was markedly
less and the pattern of this tissue was not uniform, figures 35 and 36.

      TABLE 8
Def l/bml/RIXDef 2jbmljRZ.

CULTURE

bml Am
VARIEGATED

Def l/Def 2/R2   Def l/Def2/Rl/R2

694               40                I               I
6%               41                I               2

35-9             126               0               0

35-10             243                I               0

35-I 1             =?A               5               I
36-24             172                2               2

Totals         856               IO             6

It was suspected that the two new types of plants represented the two
expected types of double deficients, and that the colorless streaks rep-
resented the homozygous deficient tissues. Since these streaks of colorless
tissue were not wide, as some of them theoretically should be from homol-
ogies tiith the bml streaks in normal variegated plants, it was suspected
that the cells of the homozygous deficient tissue were unable to multiply
at the same rate as the surrounding heterozygous deficient cells. As stated
in section II, loss of the R2 chromosome occurs much more frequently
than loss of the RI chromosome. If the rate of loss for each of these two
chromosomes is uniform throughout development, a uniform distribution
of colorless streaks should be present in the R2 double-deficient plants.


              RING CHROMOSOMES IN MAIZE            349
In the case of the R 1 R2 double-deficient plants, early loss of the R 1
chromosome should give a sector of tissue with the pattern of colorless
streaks characteristic of the R 2 double-deficient plants, since the chromo-
some constitution within the sector is the same. If the R2 chromosome
were lost early in ontogeny, a sector of tissue with a different pattern of
colorless streaks should result. These colorless streaks should be consider-
ably less frequent since the Rl chromosome is lost from the nuclei less
frequently. Nevertheless, the distribution of such streaks should be uni-
form. If this hypothesis were correct, the small, uniformly but heavily
streaked plants should be the R2 double-deficient plants, the larger, non-
uniformly streaked plants, Rl R2 double-deficient plants. That these
two types represented the expected R2 and R 1 R 2 double-deficient plants
was established through cytological observations and confirmed by pollen
examinations and appropriate crosses.

In the intercrosses of plants heterozygous for Def 2 and R2, the union
of a Def2 R2 gamete with a similar gamete results in a plant with two
Def 2 chromosomes plus two R2 chromosomes. (It is of theoretical inter-
est to point out that the chromosome number has been increased in these
plants without changing the genome complement, that is, these as-chro-
mosome plants are genomically equivalent to normal go-chromosome
plants.) Since these plants are markedly different from the double-de-
ficient plants, a description will be postponed until section VII. The
functional gametes produced by these plants contain the Def 2 chromo-
some plus one or two R2 chromosomes. The gamete most frequently
transmitted through the pollen contains but one R2 chromosome. In the
crosses of Def 1/6mI and Def l/bm1/R 1 by plants homozygous for Def 2
and R2, all the eggs which carry the normal chromosome with 6ml
should give rise to normal variegated plants, all those that carry a deficient
chromosome to double-deficient plants. The results of these two types of
crosses are given in tables 9 and IO. The test for the functioning of
deficiency-carrying eggs is similar to the Bt and bt tests described in the
previous section. The correlation between the proportions of functional

         TABLE 9
Def I/bm 1 (no ring) XNomozygoru Def 2, R2.

CULTURE

NQRYAL Bm 1 - bm 1
VARIEGATED*

Def l/DefZ/R2

     814               76                    0
    36-28                   61                   8
    36-29                  13.5                   20
    37-56                   I3                    0

      37-57                64                    0

     Totals                349                2s
* A few plants were bm 1, see page 364.


BARBARA McCLINTOCK

CULTURE

36-25
36-26
37-52
37-53
37-54
37-55

         TABLE IO
Def l/bml/RIXHomozygous Def2, R2.
NORMAL Bm I - bm 1
VARIEGATED'     Def l/Def 2/R2   Defl/Def2/Rl/R2

    106              18              I5
    56               9               4
     154              17               I2

     I75               22              31
     107                2               5
     162               5              18

350

   Totals         760
* A few plants were bm 1. See page 364.

63           85

eggs with a normal chromosome V, a deficient chromosome V, and a
deficient chromosome V plus its ring chromosome, respectively, is similar
in the two tests.

It remains to be shown that the colorless streaks in the double-deficient
plants represent the homozygous deficient tissues produced after loss of
the ring chromosomes during somatic mitosis. Adequate confirmation of
this relationship is obtained from the patterns of such tissues in double-
deficient plants with the following ring chromosomes: one R 1, one R2,
two R2, one Rl plus one R2, two Rl plus one R2, one Rl plus two R2.
Double-deficient plants with different combinations of ring chromosomes
can be obtained from crosses of R2 and R 1 R2 double-deficient plants
by plants homozygous for Def 2 R2 and from intercrosses of the double-
deficient plants. The results of the respective crosses are given in tables
II, 12, and 13. It should be noted that only plants homozygous for Def 2
R2 and double-deficient plants result from these crosses.

Classification of these plants into the two categories of homozygous

                       TABLE II

CULTURE

823
987
988
989
990
991
992
37-8s
37-86
37-87
37 88
37-89
37790
37-91

Def l/Def 2/R LXHomozygous Def 2, R 2.
HOMOZYGOUS      Def I /Def 2
Def 2, R2          one R2
     0                I
     0                2
     2                3
     9                II

     4              18

     7                4
     3                20
     I               7
     3               17
     0               I5
      I                9
     4                20

     4               8

     4               18

Def l/Def 2
two R2
   0

   5

I3
a

4
6

5

Totals            42           I53             51


RING CHROMOSOMES 1N MAIZE             351

               TABLE 12
   Def i/Def 2/R l/R2XHomozygo~s Def 2, R2.
HOYOZYGOUS  Def 1 /Def 2   Def 1 /Def 2   Def l/Def 2
Def 2, R2*    ONE R2    TWO R2 ONE Rl, ONER2
   II          30          9          I4
   6          10          II         16

    .5          8          2          4
    I          0          0          8
    I          II          II          0

    3          14          3         30
    3          14          0         33
    5          5          0          4
    0          7          0          8

    4          II           2         `3
    0          2          1          6
    I          6          I          I2

    3          I           I          6
    2          8          6          14
   14         16          3          20
   12          16          5          27
    5          8          5          `9
    6         IS           I          20

   82        152         61        254

Def l/Def 2
THREE RINGS

   3
    2
   0

0

0
2

4
3
2
8
2

5
5
2
6

17
3

Totals
* Some of these plants had, in addition, an R 1 chromosome.

67

CULTURE

37-58
37-59
37-b
37-61
37-62
37-63
37-64
37-6s
37-66
37-67
37-68
37-69
37-70
37-7'
37-72
37-73
37-74
37-7s

Def 2 R2 and double-deficient was simple since the former type of plant
has a peculiar growth habit (see section VII) and does not show the par-
ticular streaks which are always present in the double-deficient plants.
The double-deficient plants, in turn, were classified as to their ring chro-
mosome constitution on the basis of the patterns of the colorless streaks.
If the colorless streaks represent the homozygous deficient tissue, then
the pattern exhibited by the double-deficient plants with the R 1 or R 2
chromosome or various combinations of two or three of these rings should

                  TABLE 13
      Plants obtained from sib CroxSeS o,f Def l/Def 2/R l/R 2.
           HOMOZYGOUS
CULTURE              Def l/Def2    Def l/Def 2    Def l/Def 2
        Def2, R2*       I RING         2 RINGS     3 RINGSt
37-76           4           IS            15            9
37-77            r            14            27            II
37-78           3             0            `9            5
37-79           5            6            20            8
37-h           0            3             4            6
37-81            2             0            9             I
37-82           6            17            ?I            =7
1000            0       8      27            II
1001            9             I            14             9
1003            2            7            22            IO

Totals          32           71        178        87

* Several of these plants had, in addition, an R 1 chromosome.
t Several of these plants were suspected to have four rings.


3.52               BARBARA McCLINTOCK
be predictable from the knowledge of their behavior in somatic mitosis
(section II) and from the knowledge gained from a study of the patterns
of bml tissues in normal variegated plants with these same combinations
of ring chromosomes (section III). A number of these plants were examined
cytologically to establish the value of the prediction. The results are sum-
marized in table 14. The agreement between predicted and observed is
obvious from the table.

             TABLE I4
Comparisons qf predicted and observed chromosomnl constitutions.

PREDICTED CONSTITUTION           NO. PLANTS         DEVIATION FROM
l?ltOM APPEARANCE OF PLANT           EXAMINED           EXPECTATION

Homozygous Def 2, R 2
Def I /Def 2+one R2
Def l/Def2+two R2
Def l/Def2+one Rl and one R2
Def l/Def2+two Rl and one R2
Def l/Def 2+one R 1 and two R2

17         0
18          0
IO       I (I Rl)
24       I (I Rl)
5       2 (2 R2; I RI+2 R2)
6       I (Rl+R2)

Totals
* See footnote *. table 2.

80        5*

The patterns of colorless tissue exhibited by the various double-deficient
plants will be briefly described. Photographs of parts of leaves of double-
deficient plants with various ring chromosome combinations are given in
figures 31 to 38. To conserve space only a small part of a leaf is shown,
which considerably limits the effectiveness of the demonstration.

The colorless streaks in the one R2 double-deficient plants are uniformly
distributed throughout the leaf area and are relatively closely spaced
(figure 32). Double-deficient plants with the Rl chromosome have been
produced only by very early loss of the R 2 chromosome in plants originally
possessing both the R 1 and R 2 ring chromosomes. There is considerably
less streaking but the distribution of these streaks is uniform (figure 34).
Plants with two R2 chromosomes are clearly distinguishable from those
with an R 1 and R 2 chromosome. In both types of plants the streaking is
not uniformly distributed. The two R2 chromosome plants have a con-
siderably greater total amount of colorless tissue. There are numerous
sectors of various widths with a pattern similar to that shown by the one
R2 plants (figure 33, sector to right). This is to be expected since loss of
either ring chromosome would give rise to cells with the same chromosome
constitution as the one R2 plants. Sectors with the R 1 pattern, figure 34
are not found. The Rl R2 double-deficient plants have fewer streaks
than the two R2 plants. The sectors in these plants are either of the one
R2 type (figure 36, sector to left), or of the Rl type (figure 35, sector to
right of mid-rib), through early loss of the R 1 or R 2 chromosome, respec-


              RING CHROMOSOMES IN MAIZE            353
tively. In the three ring double-deficient plants, very few colorless streaks
were observed. This is particularly evident in the two R 1 plus one R 2
plants. Many of the leaves in these plants have no well defined sectors
butIonly:scattered streaks here and there. Sectors, when present, are

FIGURE 3r.-(upper) Surface view of a small region of a leaf of a plant with a normal chro-
mosome constitution. The wide clear band is the midrib. The finer parallel bands are the veins.
FIGURE 3z.-(middle) Surface view of a small region of a leaf of a plant with Def 1, Def 2 and
an R2 chromosome. The wide clear band is the midrib. Note the many fine streaks of colorless
homozygous deficient tissue.

FIGURE As.-(lower) Surface view of a small region of a leaf of a plant with Def I, Def 2 and
two R2 chromosomes. Note the sector to the right of the midrib composed of many fine streaks
(one ring sector) and the sector immediately to the left of the midrib with comparatively few
streaks (two-ring sector).

usually narrow (figures 37,38). The two R2 plus one R 1 plants have more
streaking and more well defined sectors.
That the colorless streaks represen; the homozygous deficient tissues
resulting from loss of the ring chromosome from the nuclei seems certain
from the correlations of the patterns of this tissue in the six types of
plants.

The amount of homozygous tissue in a double-deficient plant bears an


354                BARBARA McCLINTOCK
inverse relation to the size of the plant, the greater the total amount of
homozygous deficient tissue present, the smaller the plant. The one R2
double-deficient plants are smaller than the two R2 plants, which in
turn, are smaller than the Rl R2 plants. The three ring plants are prac-
tically equal in size and vigor to. plants with a normal chromosome con-
stitution.

FIGURE 34.-(upper) Surface view of a small region of a leaf of a plant with Def 1, Def 2 and
one Rl chromosome. Note the distribution of streak of homozygous deficient tissue. Compare
with figures 32 and 33.

FIGURE 35.-(middle) Surface view of a small region of a leaf of a plant with Def 1, Def 2 an
Rl and an R2 chromosome. Note the Rl sector in the middle of the region to the right of the
midrib and the sectors to either side of it which are comparatively free of streaks of homozygoua
deficient tissue.

FIGURE 36.-(lower) Surface view of a leaf of a plant with the same constitution as that in
figure 3.5. Note the R2 sector (left) and the comparatively small number of homozygous deficient
tissue streaks in the tissue to the right.

From homologies of the bml sectors in normal variegated plants (sec-
tion III), wide sectors of homozygous deficient tissues in the double-
deficient plants would be expected to be found if the cells of such tissues
could grow and multiply at the same rate as the surrounding cells. It is


              RING CHROMOSOMES IN MAIZE            355
reasonable to assume that these cells, with a deficient chromosome com-
plement, would be incapable of an equal growth rate. The juxtaposition
of two tissues with unequal growth rates should cause considerable dis-
tortion of the cells about the boundaries of the two tissues. This is obvious
from the microscopic observations of the two types of tissues in the double-
deficient plants. The normal tissues appear to be pulling in the direction
of the homozygous deficient tissues. The more rapid growth of the normal

FIGURE 37.-(upper) Surface view of a small region of a leaf of a plant with Def 1, Def 2,
two R2 and one R 1 chromosomes. Note the two narrow sectors with streaks of homozygous de-
ficient tissue.

FIGURE 38.-(lower) Surface view of a small region of a leaf of a plant with Def 1, Def 2, two
R 1 and one R2 chromosomes. Note the narrow sector to the left of the midrib with a few streaks
of homozygous deficient tissue.

tissues may exert enough pull upon the homozygous deficient cells to
cause separation at cell boundaries and the production of a hole in the
midst of a patch of homozygous deficient tissue. In the one R2 double-
deficient plants, which have the most homozygous deficient tissue, the
unequal growth rates of the two types of tissue is reflected in a roughened,
finely corrugated surface of the considerably reduced and narrowed leaf.

The evidence for considering the colorless streaks as tissues homozygous
deficient for the extent of the deficiency in the Def 1 chromosome can be
briefly summarized :

I. Plants which show these colorless' streaks must contain two deficient
rod chromosomes, one of which must be the Defl chromosome. This has
been proven by cytological examination, pollen examination and appropri-
ate crosses. Plants homozygous for Def2 R2 do not show the colorless
streaks characteristic of the double-deficient plants. It will be shown in


356                 BARBARA McCLINTOCK
section VII that cells homozygous for the full deficiency in the Def2
chromosome (which is more than twice as long as that in the Def 1 chromo-
some) are incapable of surviving.
2. Plants with these colorless streaks can arise only in crosses where
double-deficient plants are expected. For confirmation, see tables 7 to 13.
3. When streaked plants are crossed to normal plants, no streaked
plants should appear in the progeny. This has been fully confirmed.
4. The amount of streaked tissue present in a plant and the pattern
exhibited should be correlated with the number and kinds of ring chromo-
somes present. (See table 14 and previous discussion.)
5. A reduced growth rate in the cells homozygous deficient for such a
relatively large section bf the chromosome is to be expected. This is re-
flected in the small size of the colorless streaks and the distortion of tissues
produced by the juxtaposition of tissues with unequal growth rates.
In mentioning the crosses given in tables II, 12 and 13, little was stated
concerning the functional gametes produced by the double-deficient plants.
The one R2 double-deficient plants produce four types of gametes (I)
Def 1, (2) Def2, (3) Def 1 R2, (4) Def2 R2. As shown previously, the
gamete with Def 2 (2 above) will not function in pollen or ovule; (I) above
will function in some but not all of the eggs in which it is present but will
not function through the pollen. Gamete (4) is equally viable through the
eggs and pollen (tables II to 13). Gamete (3) is a new type the functioning
of which had to be tested. It was found to function readily through the
eggs. That it does not function through the pollen in an appreciable
amount in competition with (4) is shown by the following test. Pollen of
R2 double-deficient plants was placed upon silks of normal brnl plants.
The resulting plants should be variegated (Bm I and bm 1) and heterozy-
gous for either Def 1 or Def 2. Since both types of plants can be distin-
guished through pollen examinations (see section IV), the pollen of 171
plants resulting from this cross was examined. All showed the presence of
the Def2 chromosome. Cytological verification was obtained from 28 of
these plants. From this evidence, it can be concluded that type (4) pollen
grain is the only normally functioning grain produced by these plants.
The functional gametes of the R 1 R2 double-deficient plants have been
determined. Of the eight possible gametes: (I) Defl, (2) Defl Rl, (3)
Defl R2, (4) Defl Rl R2, (5) Def2, (6) Def2 Rl, (7) Def2 R2, (8) Def2
Rl R2, the egg can transmit all except (5) and possibly (6). The pollen
transmits only (7) and (8).
The dissimilarity in functional capacity of the different types of gametes
in pollen and ovules is reflected in the Pr and pr ratios (Pr, purple aleur-
one; pr, red aleurone) in reciprocal crosses (table IS). Pr is located in the
long arm of chromosome V, 18-24 units from Bmf (EMERSON, BEADLE and


               RING CHROMOSOMES IN MAIZE            357
FRASER, 1935; the percentage of crossing over varies within this range in
different strains). As is obvious from the discussion in this paper, Bml
is very close to the spindle fiber region in the short arm of chromosome V.
.A measure of the crossing over between the deficiency (or spindle fiber
region) and Pr can be obtained directly from the crosses shown in table

CROSS

     TABLE Is
L?issimiladies in reciprocal crosses.

    Pr       %        PI       %

Def 1 pr/Def 2 Pr/R 2 X pr         60        31.5        130      68.5
reciprocal               5192      81.3      1198      18.7

Def 1 Pr/Def 2 $w/R2 XQv       85        61.2         54      38.8
reciprocal                495        19.0        2106        81.0

Defl pr/Def2 Pr/Rl/R2X$7      I77      27.8       458        72.2
reciprocal            4964      83.7        973      16.3

Def 1 Ps/Def2 pf/Rl/R2XQr     `65        66.6       83       33.4
reciprocal                 3'3       18.0        1422        82 .o

16. Since no ring chromosome is present, only the pollen grains carrying
the normal chromosome function. Crossing over is not altered in plants
heterozygous for Def 1 and very little in plants heterozygous for Def 2.

                      TABLE 16

    CROSS             Pr           fi      y. CROSSING OVER
@XDef 1 Pr/+pr       27%           9'7.5            23.3
prXDef 1 pr/+Pr         1286             425          24.8
pXDef2 Pr/+fw        562             2201            20.3
pIXDef 2 #r/+Pr       823         I84           18.2

In double-deficient plants, with one Def 1 and one Def2 chromosome,
crossing over is similar to that in plants heterozygous for the Def 2 chro-
mosome, "reciprocal" crosses (table I 5).

VI. SIMULATION OF THE bml PHENOTYPE THROUGH
       LOSS OF THE &?Zl LOCUS

When one-ring and two-ring doubleTdeficient plants were closely ex-
amined, fine streaks of brown tissue were seen in the leaf sheath, the mid-
rib and the veins of the leaf. These fine streaks were many times more fre-
quent in the one-ring plants than in the two-ring p1ant.s. The shade of
color and its association with cells having thickened walls were strikingly
similar to the effect produced by brown midrib (bml). In some of the
streaks it was obvious that the brown color of the vein was associated


35%                 BARBARA McCLINTOCR
with adjacent parenchyma cells lacking chlorophyll. Since it was known
that normal bml produces a brown color in the lignified cell walls, which
can be seen in sections of any lignified tissue, the leaf sheaths with brown
streaks were removed, sectioned fresh and examined microscopically. It
was immediately observed that the brown color was in the cell walls. It
was similar in its range of color and in its deposition in the cell wall to that
of ordinary bml. When brow0 patches appeared in regions where plastids
are normally lacking, that is, heavy-walled schlerenchyma cells, the ad-
jacent parenchyma cells, when too thin-walled to show the brown clearly,
frequently lacked plastids. When theadjacent plastid deficient parenchyma
cells possessed thickened walls, a brown color could be seen in these walls.
The brown-walled and plastid deficient cells formed a definite unit of
tissue. It was suspected that these small sectors represented homozygous
deficient tissues and that a brown pigmented cell wall would accompany
all such tissues. However, the walls of the cells in the colorless streaks in
the leaf, except about the veins, are too thin, and the concentration of
brown pigment insufficient for a visible effect. If the homozygous deficient
cells, produced by loss of the ring chromosome from the nuclei, have brown
cell walls, cross-sections of the stem of the single-ring double-deficient
plants should show many small, uniformly distributed patches of cells
with brown cell walls, just as the leaf is uniformly streaked with fine
stripes of colorless tissue. This proved to be true for each of the many R 2
double-deficient plants examined. The sections of the stem were advan-
tageous in relating the brown-walled cells to those which lacked plastids
since the normally plastid-carrying parenchyma in the outer region of the
stem is thick walled and has a sufficient concentration of brown to bc
readily seen. Considerable distortion of the bundles and cells was present,
particularly in the regions about relatively large patches of brown-walled
cells. The types of distortion suggested that the brown-walled cells had
grown at a slower rate than the surrounding white-walled cells.
In contrast to the single-ring plants, cross-sections of stems of the two-
ring plants showed fewer brown patches. Such would be expected if the
brown color were limited to cells which were homozygous deficient; loss
of one ring followed by loss of the second ring must occur before the homo-
zygous deficient cells could be produced. Frequently a cluster of brown
patches was present in a restricted region of the stem. When the extent
and position df these brown patches were traced with a camera lucida and
a boundary drawn about the cluster, it was clear that they formed one
continuous sector. In the two R2 double-deficient plants, the number and
distribution of the brown patches in such a sector were similar to those
of the one R2 plants. In the R 1 R 2 plants, some of such sectors were
similar to the above and some had considerably fewer patches in a cluster,


               RING CHROMOSOMES IN MAIZE             3.59
In the former plant, the sectorial clusters of brown patches correspond
to the one-ring sectors seen in the leaf through early loss of one of the R2
chromosomes. In the latter plant, the two types of sectorials correspond
to early losses of the R 1 or R2 chromosome, respectively.
If the brown-walled, plastid-deficient patches of cells represent the
homozygous deficient tissue, the three-ring double-deficient plants should
show very few such patches. When present, their distribution should cor-
respond to that observed in the leaves of these plants. The results obtained
were in complete agreement. There were very few such patches in these
three-ring plants. This is especially true of the two R 1 plus one R 2 plants.
It might be stated here that plants homozygous for Def 2 R2 do not
show these patches of brown-walled, plastid-deficient cells. The homozy-
gous deficient cells in these plants, as will be shown in the next section, are
inviable.
(I) The correlation of the brown cell walls with the cells lacking plas-
tids, (2) their presence in narrow streaks, (3) the restricted growth capac-
ity of these cells, (4) their uniform distribution in one-ring double-deficient
plants, (3) their reduced frequency in two R2, Rl R2, and three-ring
plants, respectively, and (6) the distribution patterns in sectorials in these
two- and three-ring plants summarize the homologies of the brown-walled
cells with the streaks of colorless cells in the leaf which were shown to be
homozygous deficient in the previous section.
The brown-walled cells are homozygous deficient for the full extent of
the deficiency in Def 1. At this point it should be emphasized that the locus
of Bml is carried by the ring chromosome and that there is no locus for
Bml or bm I in the deficient rod chromosomes. If the brown color in the
walls of these homozygous deficient cells is similar to bml, it should ap-
pear in the development of the wall at the time lignification sets in, as
has been shown for normal bml (section III). Furthermore, the brown
color of the walls in such cells, when adjacent to white-walled cells, should
be diluted on the side adjacent to the white-walled cells, as observed in
normal variegated plants. Thirdly, when exposed to intense light, the
brown color should fade just as normal bml fades on exposure to light.
Immature cells are present at the base of each leaf sheath. These cells
rapidly merge into fully mature cells just above this region. If a leaf
showing a prominent brown streak is removed and serial sections made to
trace the brown streak as it emerges from the immature cells, it becomes
obvious that the brown color appears as the cell walls become lignified in
a manner fully comparable to normal bml. Thus, the time of appearance
of the color in the development of the wall is similar to that in normal
bm 1 plants.
When examining the brown patches in sections of the stem it was ob-


360                BARBARA McCLINTOCK
served that the color of the brown in the walls of the plastid deficient
cells was considerably diluted on the side adjacent to the white-walled,
plastid containing cells. This, in turn, is comparable to the observations
in normal variegated plants.

To test the third correlation, fading of the color when exposed to light,
black paper was placed over part of a conspicuous brown streak in a leaf
sheath or midrib of a leaf. Upon removal, several weeks later, the brown
color under the paper had retained its intensity, that above and below
had lost much of its intensity. In this respect, the brown of the homozy-
gous deficient tissues is similar to normal bml.
The range in color of normal bml varies in some plants from a deep
wine red to a light orange, the deep red color being present in the stem
toward the basal nodes, the light orange in the regions toward the top of
the plant. This same gradation of color in comparable regions was found
in the brown patches of the double-deficient plants.
To summarize: The two browns, the normal bml and the brown pro-
duced in cells possessing no locus for this gene, are comparable in (I) time
of appearance of the color in the development of the cell walls, (2) in
dilution of the color in regions adjacent to white-walled (Bml) cells, (3)
in loss of intensity of color when exposed to light, and (4) in range of color
variations in specific regions of the plant. No differences could be detected
in the expression and behavior of the brown color in the two cases. Al-
though it has not been proven that bml is due to a deficiency in chromo-
some V, it can be stated that absence of the locus of Bml will duplicate
the phenotypic expression of bml.

VII.THE PRODUCTION AND APPEARANCE OF PLAN-k
      HOMOZYGOUS FOR Def2 R2

Mention of plants homozygous for Def2 R2 has been made in the
previous section. The first of these plants appeared in the progeny of sib
crosses of Def 2/bml/R 2 through the union of two gametes each contain-
ing Def 2 and R2. Such plants were to be expected. However, as in the case
of the double-deficient plants, no prediction could be made as to appear-
ance other than that they should not be typical bml or variegated plants.
Nevertheless, they are readily recognizable. They are short, usually deep
green in color, with, thickened, erect leaves and do not show normal Bm l-
bm I variegation. The streaks of colorless tissue, so characteristic of double-
deficient plants are absent from the leaves.

In later generations many plants homozygous for Def 2 R2 were ob-
tained. Cytological examination of microsporocytes at pachytene has con-
firmed the accuracy of the phenotypic classification (table 14). The two
deficient chromosomes V synapse homologously throughout their length


               RING CHROMOSOMES IN MAJZE          361
The changed arm ratio produced by the deficiency is clearly evident. In
the normal chromosome V, the chromomeres adjacent to the spindle fiber
region on the short arm are relatively large and deep-staining. With the
removal of this section in the production of the ring chromosome, small,
light-staining chromomeres are brought adjacent to the spindle fiber re-
gion, making this deficient chromosome V readily recognizable at pachy-
tene. The two ring chromosomes either synapse to form a ring-shaped
bivalent, similar to the rings in photographs 21, 22, Plate II, or remain
unsynapsed and appear as two collapsed rings, similar to the rings in
photograph, 19, Plate II. There is no tendency for the ring chromosomes
to synapse with any part of the deficient rod chromosomes V.
The pollen of these plants is always highly abortive. Only those grains
possessing a ring chromosome are normal in appearance and capable of
functioning.
The loss of one ring chromosome followed by loss of the second ring
chromosome should give rise to cells homozygous deficient for the full
extent of the deficiency in the rod chromosomes. If these cells were viable
and capable of multiplying, evidence of such tissue would be expected in
the leaves of these plants from homologies with the double-deficient plants
described in the previous two sections. Since evidence of such tissue did
not appear in the leaf, it was suspected that the cells whose nuclei were
homozygous deficient for this relatively long section were inviable or in-
capable of further multiplication. Longitudinal sections of growing points
of roots of these plants were made with the view of finding evidence of the
fate of these cells since such cells necessarily are formed.
In all root meristems of plants homozygous for Def 2 R2, the following
peculiar cell type was found. It was confined to roots of these plants, not
being present in normal or double-deficient plants. Very much enlarged,
heavily vacuolate groups of two or more cells in positions indicating rela-
tion in descent, were observed in regions of the root where enlarged cells
are not normally encountered (figures 39, 40, 41), When a mitotic figure
was observed in one of these cells, the chromosomes were short, thickened
and sometimes irregularly placed in the spindle. Daughter telophase nu-
clei were sometimes joined by a connecting nuclear bridge. In older regions
of the root, degeneration processes in these cells, depicted first by an aber-
rant staining reaction of the nucleolus followed by a pycnotic condition
of the cytoplasm (figure 42), and finally by a collapse of these cells due to
the pressure of the normal surrounding cells (figure 43). Should the space
formerly occupied by these cells be extensive, the surrounding cells divide
in planes other than normal, filling in this space which might otherwise
have remained as a hole in the tissue (figure 43), The numbers of such
patches of enlarged cells varied considerably in different roots. Some had
many, others relatively few.


362                   BARBARA McCLINTOCR

FIGURE 39.-(left) Longitudinal section of a root tip of a plant homozygous for Def2 and R2.
Note the two adjacent, enlarged (homozygous deficient) cells near the tip of the growing point
and the row of enlarged, degenerating (homozygous deficient) cells in the root cap directly above.
Mag. X 160.

FIGURE 4o.-(right) Similar to figure 39. Note the transverse row of four enlarged
         (homozygous deficient) cells. Mag. X 160.

The conditions depicted conform strictly to expectancy if these cells
represent the homozygous deficient cells resulting from loss of the ring
chromosomes from their nuclei. Loss of one ring chromosome would cause
no obvious tissue alteration since tissues heterozgyous for Def 2 are normal
in appearance. Loss of the second ring chromosome, such loss taking place
at anaphase by being left at the equatorial plate of a mitotic figure, would
result in two adjacent cells whose nuclei would be homozygous deficient
for the extent of the deficiency in the Def 2 rod chromosomes. The occur-
rence of pairs of enlarged cells has been mentioned. In many cases, it was
possible to see the cast-out ring chromosome in the cytoplasm of one of
these cells but also in many cases, degeneration processes in the cytoplasm
had advanced too far for such a determination. No ring chromosome was
observed in the few cells which had mitotic figures but the small ring chro-
mosome could have been obscured by one of the rod chromosomes so that
evidence of the homozygous deficient conditions of these cells from direct
observations of the chromosome constitution was not considered satis-
factory.

The numbers of such patches of cells and their distribution in the differ-
ent roots lend strong supporting evidence for the homozygous deficient


RISG CHROMOSOMES IN MAIZE

363

I;ICURIC qr.-Longitudinal section of a root tip of a plant homozygous for Def2 and R2. Note
    the row of very much enlarged (homozygous deficient) cells. &fag. X 160.

FIGURE .+a.-(left) Longitudinal section immediately below the actively meristematic region
of a root of a plant homozygous for Def 2 and R2. Sote the row of four enlarged, degenerating
(homozygous deficient) cells. Nag. X 160.

FIGURE 4g.-(right) Longitudinal section of a root tip of a plant homozygous for Def 2 and R 2.
Note that there has been a proliferation of cells about the degenerated streak. Msg. X160.

nature of these cells. They were present in all roots but the frequency was
variable. In a root in which only one ring chromosome is present in the
nuclei of the normal cells, a high frequency of such patches should be


344                  BARBAR,% JlcCLTSTOCK
observed. In this latter case, every loss of the ring chromosome would be
reflected in a patch composed of t&o or more such cells. In roots where
two ring chromosomes are present in many of the nuclei, loss of one ring
chromosome followed by loss of the second ring chromosome or the very
occasional simultaneous loss of both ring chromosomes would have to
occur to produce such a patch. Thus, the frequency of such patches in
these roots would be considerably less than in the former type of root.
Since a root could contain but one ring chromosome in its normal cells
or be a mosaic of one- and two-ring sectors of various sizes, variations in
the numbers of patches of abnormal, enlarged (homozygous deficient) cells
in the different roots of thehomozygous Def 2 R2 plants is to be expected.
(I) The presence of these patches of abnormal cells only in plants homo-
zygous for Def 2 R2 and their absence in double-deficient and normal
plants, (2) the observed presence of the R2 ring in the cytoplasm of one
of these cells in many cases, (3) the frequency and distribution of these
patches in different roots, (4) their arrangement in descent, that is, rows
of two or more, (5) the rapid distintegration of these cells, and finally (6)
the absence of evidence of homozygous deficient tissues in the mature
cells of the stalk and leaves of these plants as contrasted with double-
deficient plants, strongly support the view that they represent the homo-
zygous deficient cells since such cells must be produced in these plants.
Since early death is the fate of these cells? it is clear why the leaves of these
plants are not streaked with modified tissues which could be interpreted
as representing the homozygous deficient tissues.
At this point it might be mentioned that a third deficiency (Def3), not
previously considered in this paper! which is outside the limits of Defl but
within the limits of Def2 and therefore covered by the R2 chromosome,
produces the same pattern of homozygous deficient tissues as that ex-
hibited by the R2 double-deficient plants when the constitution of the
plant is Def2/Def3/R2. However, in this case, the homozygous deficient
tissue has an even poorer growth capacity than tissues homozygous for
the deficiency in the Defl chromosome. To return to the Def2 chromo-
some, the deficiency is apparently too long, the loci deleted too important
in cell physiology for survival of cells which are homozygous deficient for
this region.
When pollen of plants homozygous for Def 2 R 2 is placed upon silks of
normal bml plants, the progeny should all be variegated for Bm 1 and
bm I except in those cases where the ring chromosome has been lost suf-
ficiently early in development to be absent from that part of the embryonic
tissue which produces the visible part of the plant.* In this latter case.
  ? Functional male gametes without a ring chromosome, which would give rise to bv11 plants.
might be produced as the result of a loss of the ring chromosome in the second microspore mitosis-


               RING CHROMOSOMES IN MAJZE          365
the plant would be bm 1. The progeny of 13 such crosses totalled 1,829
variegated to 68 bml plants. To exclude the possibility that this 3.5
percent of bml plants represented contaminations, 42 of them were ex-
amined for the presence of the deficient chromosome. In 41 of these plants
the deficient chromosome was present; one represented a contamination.

The number of kernels which develop on the ear of a plant homozygous
for Def 2 R2 is always very small, ranging from o to 30 kernels with
the average about IO. This would be expected since only those gametes
which possess a ring chromosome are functional. Many of the ears should
arise from one-ring sectors and others should be composed of both one-
and two-ring sectors. Since, in the two-ring sectors, the two small ring
chromosomes frequently do not synapse, of if so, do not remain together
at the first meiotic mitosis, their elimination in the two meiotic mitoses
is frequent. Relatively few ovules with eggs containing a ring chromosome
would be expected and thus only a few kernels should be expected on an
ear. Since the exsertion of anthers and shedding of pollen is dependent
upon the presence of a number of well-formed grains in the anther sac,
pollen collected from such plants usually contains enough functional grains
(those with a ring chromosome) to produce a complete set of seed when
placed on silks of normal plants. Since both deficient chromosomes are
similar, there is no selection in favor of one or the other chromosome either
through the pollen or eggs. Reciprocal backcrosses of Pr/pr plants give
I : I ratios.

VIII. PHENOTYPIC EFFECTS OF ALTERED RING CHROMOSOMES

As stated in section II, ring-shaped chromosomes not only are lost from
nuclei during somatic mitoses but also change in size. They may increase
in size through duplications of segments composing the ring or decrease
in size through losses of segments. The frequency of such changes depends
upon the size of the ring chromosome. In the case of the small R 2 chromo-
some, such changes are relatively infrequent. Cytological examinations of
large numbers of plants and many nuclei within each plant have given
abundant evidence, however, of such changes. The R2 chromosome has
been seen to increase to approximately seven times its original size and to
have decreased to several chromomeres. The duplicated segments do not
result in tissues showing extensive modification. Slight modifications are
visible with higher multiples resulting in smaller plants with thickened,
erect leaves.

In the case of the double-deficient plants, loss of the ring chromosome

In this case, the tube nucleus would contain the ring chromosome and could be expected to func-
tion normally in pollen germination. Thus, sperm nuclei lacking an R2 chromosome, could be
introduced into the embryo sac.


366                BARBARA McCLINTOCK
results in viable tissues which are homozygous deficient for the extent of
Def 1, as shown in the previous sections. This tissue is poor in growth
capacity, has brown cell walls similar to normal bml, possesses no plastids
and, on continuous exposure to sunlight this tissue in the leaves dries and
shrivels. In Def 1 the four chromomeres. adjacent to the spindle fiber at-
tachment region on the short arm of chromosome V have been removed.
The R2 chromosome includes these four chromomeres plus the next five
chromomeres.3 Should changes in size of the ring chromosome delete one
or more of the four chromomeres covering the deficiency in Def 1 or frac-
tions of them, tissues homozygous deficient for sections within the limits
of the deficiency in Def 1 should result. Since tissues homozygous for the
full extent of the deficiency in Def 1 are viable, fractional deficiencies
within this region might be expected to have even better viabilities and
should reveal themselves by wider sectors of homozygous deficient tissue
having a specific modification, this modification should be repeatedly en-
countered as a sectorial in large populations of such plants. In other words,
the nature of these sectorials should vary depending upon the fraction of
the four chromomeres which has been deleted from the ring chromosome.
The same region should be independently deleted in a number of instances
and therefore the same type of sectorial should be produced in a number
of different double-deficient plants.
A large number of sectorials have been found. These are classified as
simple mutant sectorials, involving a single recognizable change, and com-
pound mutant sectorials, which are combinations of the simple mutant
types. Only those sectorials which are readily recognizable because of their
good growth capacity are included in this classification. The simple mutant
sectorials are as follows: (I) translucent white ("onion skin") with colorless
cell walls, no plastids; (2) opaque z&z&, with colorless cell walls, colorless
plastids; (3) deficiency-brown-midrib, similar in detail to tissues homozy-
gous for the bml gene; (4) pink colored tissues with colorless cell walls,
colorless plastids; (5) blotched chlorophyll pattern. The following types of
compound mutant sectorials have been found: (I) pink, deficiency-bm,
viable in sunlight; (2) pink, deficiency-bm, dries in sunlight; (3) opaque
white, deficiency-bm; (4) blotch, dries in sunlight; (5) blotch, deficiency-
bm, dries in sunlight. The compound mutant sectorials are far more fre-
quent than the simple mutant sectorials. The summation of the characters
exhibited by these sectorials is equal to the characters present in tissues
homozygous for the full extent of the deficiency in Def 1, with the excep-
tion that the tissues with the total deficiency are incapable of growing at

s This is an estimate of the number of chromomeres. The chromomeres in maize appear to be
compound and may show more or less in a particular region depending upon the state of contrac-
tion or the degree of stretching in a particular preparation.


              RING CHROMOSOMES IN MAIZE          367
a rate which will result in a large sector, whereas the simple mutant
sectorials outlined above more nearly approximate the normal growth
rate.
The theory is proposed that the simple mutant effects are due to losses
of one particular region from the ring chromosome, each mutant effect
being related to one particular region. The compound mutant effects are
then produced by losses of several adjacent regions within the ring chromo-
some. On this theory, a linear arrangement of the mutant effects can be
referred to the chromosome in the following order: pink, deficiency-bm,
dries in sunlight and blotch. Translucent white must be removed from
deficiency-bm and opaque white close to it. The presence of a ring chromo-
some in the cells of a sectorial has been determined cytologically when the
sectorial included a part of the tassel. If an altered ring chromosome was
present in the mutant sectorials, streaks of tissue homozygous deficient
for the full extent of the deficiency in Defl should appear within these
sectors through loss of this altered ring chromosome. This is especially
easy to confirm in sectorials of deficiency-bm and blotch.
In double-deficient plants with two ring chromosomes, the same types
of sectorials are encountered. Here, however, all of the sectors are not
solid; many of them are variegated. The explanation is similar to vari-
egated patterns of Bml and bml in plants which have two normal chromo-
somes V carrying bml and two ring chromosomes carrying Bml and to the
patterns of homozygous deficient tissues in double-deficient plants with
two ring chromosomes. One ring chromosome has suffered a deletion. Pro-
duction of homozygous deficient tissues showing the character of the dele-
tion can occur only after loss of the second normal ring chromosome. Thus
the sectorial is a variegate of normal and mutant tissues. Should the altered
ring chromosome be lost in a somatic mitosis many generations later than
that which produced the alteration, the tissues resulting after this loss
will appear as normal one-ring double-deficient tissue inserted into
variegated tissue since only the one normal ring chromosome is left in
this tissue. These variegated sectors, mosaics of two types of patterns, are
frequently encountered in the two-ring double-deficient plants.
If the theory that the mutant sectorials are due to losses of specific
regions within the ring chromosome is correct, plants homozygous for
Def2 R2 should show mutant sectorials similar to those exhibited by
the double-deficient plants plus a number of types not exhibited by these
latter'plants since the extent of the deficiency in Def 2 includes the same
four chromomeres plus five more. This has been fully realized. Simple and
compound mutants of pink, deficiency-bm, blotch, and opaque white plus
an additional number of chlorophyll and growth types have been found.
Should a change occur in the ring chromosome, it should remain un-


368                BARBARA McCLINTOCK
altered for most of the cell generations and thus should be capable of being
passed from one generation to the next, provided the original alteration
occurred in tissues which give rise to the gametes. A female gamete con-
taining a deficient rod chromosome plus an altered ring chromosome united
with a male gamete with the deficient rod chromosome and a normal ring
chromosome would give rise to plants which are complete mosaics of
normal and changed tissues. The changed tissues should be of the same
type in an individual plant. In some cases there should be many female
gametes in the original plant with this same altered ring chromosome if the
alteration occurred early in the ontogeny of the ear. Thus there should be
many individuals in a culture resulting from the outlined cross each
exhibiting the same type of variegated pattern involving the identical
mutant characters. This has been realized in one culture. These plants are
useful for cytological determinations of the nature of the modification in
the ring chromosome but a detailed account of this will appear in a later
paper. It is not the purpose of this paper to consider all the evidence
concerning changed rings and their mutant effects. It is too extensive. It
is necessary to mention it, however, since a comprehension of the mitotic
behavior of ring chromosomes would lead one to anticipate the presence of
such changed rings with phenotypic effects.

DISCUSSION

The presence of ring-shaped chromosomes and a suggestion as to their
inconstant behavior in somatic tissues was first published by NAWASHIN
(1930; see also, 1936) for a single plant of Crepis tectorum. Since this time,
ring-shaped chromosomes have been found or produced in Drosophila
(L. V. MORGAN 1933; STURTEVANT and BEADLE 1936; SIDOROV, SOKOLOL
and TROFIMOV 1936; SCHULTZ and CATCHESIDE 1937), Trillium (HUSKINS
and HUNTER 1935), Locusta (WHITE 1935), Pisum (ATABEKOWA 1936).
Tradescantia (HUSTED 1936), Tulipa (UPCOTT 1937) and Nicotiana
(CLAUSEN, unpublished). In none of these cases has an intensive cyto-
logical study been made to determine the mitotic behavior of the ring
chromosomes. In Zea a number of ring-shaped chromosomes have been
found (MCCLINTOCK 1932, 1933, and unpublished; RHOADES and Mc-
CLINTOCK 1935; CREIGHTON, unpublished; CAMERON, unpublished). In all
cases studied, the mitotic behavior of these ring chromosomes has been
similar. Deletions of sectors from the ring, duplication of sectors, additions
in numbers of ring chromosomes of varying constitutions and total loss
of the ring chromosome from the nuclei have been observed. In Drosophila,
phenotypic effects which could be definitely ascribed to alterations in
constitution of the ring-shaped X chromosome have not been described.
The chances of detecting such an altered ring chromosome would depend


              RING CHROMOSOMES IN MAIZE          369
on the rate at which aberrant anaphase figures would be formed. As
pointed out in section II, the rate at which alterations occur in ring
chromosomes in maize depends upon the length of the chromonema com-
posing the ring. Since the primary cause of double-sized and interlocking
rings, the first step in the production of altered ring chromosomes, has not
been determined, it is difficult to argue that the behavior found in Crepis,
Nicotiana (R. E. CLAUSEN, unpublished) and Zea would likewise be found
in Drosophila.
HUSTED (1936) reported double-sized, continuous ring-shaped chromo-
somes, in some of which the two chromonemata made a half turn around
each other, and interlocked rings in the microspores of Tradescantia fol-
lowing irradiation. Such configurations should be expected if, before irra-
diation, the chromosomes were split and the two chromatids were rela-
tionally coiled about one another. Although the Tradescantia cases were
not followed beyond the microspore stage, HUSTED attempted to explain
the method by which such figures could be produced throughout the life
of a plant. The presence of a continuous ring with two spindle fiber attach-
ment regions or two interlocked sister ring chromatids at somatic ana-
phase, whatever the method by which it originally arose, would result in
a chromatin bridge the strands of which would eventually break. HUSTED
assumes that each chromosome is split at somatic anaphase. "A broken
end of an anaphase chromatid (caused by breakage of continuous or
interlocking rings) may unite as often with the broken end of its sister
as with its other broken end. Ring chromosomes which are continually
breaking might persist in this way. Whenever the two broken ends of one
chromatid unite, however, and the two sister strands are not t-wisted, a
`disjunctional' [two sister halves free to disjoin] ring-shaped chromosome
would result. There would be a trend toward displacement of the `con-
tinuous' and `interlocked' types by `disjunctional' rings which separate
without breaking unless relational coiling of chromatids is increased before
each union of broken ends" (page 551). The disjunctional rings, once
established, should remain free from further complications. Such a theory
of the continuous appearance of double-sized and interlocked ring chromo-
somes throughout the life of a plant cannot account for the origin of such
configurations in maize, although it may contribute to some of the cases.
This arises from the following consideration. In plants homozygous for
Def 2 and R 2, the two split halves of the ring chromosomes separate freely
from one another at anaphase I in most of the sporocytes, that is, are
"disjunctional," not continuous or interlocked. Thus, the ring chromosome
in the majority of the spores has been derived from a ring chromosome
whose two split halves have separated freely in the previous division. As
seen in section VII, only those gametes which possess a ring chromosome


370                BARBARA McCLINTOCK
are functional. If HUSTED'S theory of the continuous appearance of double-
sized and interlocked rings were correct for maize, most of the individuals
resulting from the cross of normal bml by homozygous Def 2 R2 should
be totally Bm 1 through elimination of the cause of loss of the ring chromo-
somes in somatic mitoses. As shown on page 364, all plants resulting from
the cross are variegated for Bm 1 and bml. The cause of the interlocked
or double-sized rings arises anew and is dependent upon the length of the
chromonema in the ring for its frequency. The primary cause of these con-
figurations may be related to the occurrence of a crossover between the
two split halves of a ring chromosome. The high frequency of normally
disjoining ring chromosomes, even when the chromonema of the ring is
long, leads one to conclude that the plane of splitting or method of repro-
duction of a new. chromonema from an old, is definitely predetermined
along a given plane and that trouble might arise only during or after the
split had occurred at some point of tension or torsion in the chromosome,
that is, a tension relieved by an interchange of segments at this point,
resulting in a somatic crossing over between the two chromatids. Unless
two crossovers occurred in a chromosome, only continuous, double-sized
rings would result. As noted in section II, there was a high frequency of
such continuous, double-sized rings as compared to other possible compli-
cated configurations. The maize chromosomes are too small in somatic
cells to give a clear picture of the configurations in each cell other than
in those with simple continuous rings. On this theory, interlocked rings
could arise (I) after two somatic crossovers in which the second crossover
did not counteract the first, or (2) following a previous anaphase break
and reunion of broken ends in which a twist in the chromonema was
present before the union occurred. In this latter case, it is not necessary
to assume that the chromosome is split before union of broken ends occurs
as interlocked or continuous rings could arise depending upon the plane
of splitting or reproduction of the chromonema assumed. On this hy-
pothesis, the proportion of interlocked rings to continuous rings would be
expected to be greater with large ring chromosomes than with small ring
chromosomes. Likewise, sister nuclei in these plants should show aberrant
ring configurations more frequently than sister nuclei in plants with small
ring chromosomes.
Since no rod-shaped fragment chromosomes have been observed to arise
from ring-shaped chromosomes through breakages in somatic anaphase
and telophases, it has been assumed that union of broken ends must occur.
In an effort to determine if two broken ends which enter a nucleus would
unite, the following experiment was outlined. A plant was made heterozy-
gous for two inversions on two different chromosomes. The inversions did
not include the spindle fiber attachment regions and therefore, should give


              RING CHROMOSOMES IN MAIZE             371
bridges at anaphase I (or II) and free fragments after a crossover within
the inverted segment (MCCLINTOCK 1933; M~~NTZING 1934;Saar~~ 1935;
RICHARDSON 1936; DARLINGTON 1936; UPCOTTI~~~;SAXI~~~ andothers).
With normal crossing over, the size of the fragment is constant for any
particular inversion. In the two inversions chosen, the size of the fragment
produced by each was readily distinguishable. In many sporocytes of the
plants with the inversions, two chromatin bridges with their respective
fragments produced by a crossover in each of the two inversions, were
present at anaphase I. In most cases, the bridges of chromatin had broken
by late telophase and the broken ends had been drawn into the telophase
nuclei. Thus broken ends from two chromosomes entered the same
nucleus. If fusion of these broken ends occurred, the product of this fu-
sion should be obvious in some of the second division figures in the cells of
which two recognizable fragments were present. In maize, the two second
meiotic anaphase figures are oriented in one plane and thus can be observed
together. However, the evidence for fusions was negative. It was then
considered that the broken ends might be too far apart from one another,
in many cases, to join together before the second meiotic mitosis. There-
fore, cases of double-crossing over in plants heterozygous for a single
inversion were investigated. When a four-strand double crossover occurs
within the inverted region, a double bridge involving all four chromatids,
and two free fragments of similar size are found in anaphase I. The strands
composing these bridges break and the two broken ends, lying side by side,
enter the same nucleus. If fusions of these broken ends occurs in each
telophase I nucleus, each sister cell in anaphase II should show a chromo-
some involved in an anaphase bridge. The sister cells to be examined can
be distinguished by the two fragments of recognizable dimensions. The
evidence for fusions of broken ends entering the same nucleus was like-
wise mainly negative in this case. On the supposition that each chromatid
might already be split in anaphase I and that fusions occurred between
broken ends of the two split halves of a chromatid rather than between
the broken ends of each chromatid, anaphase configurations in the micro-
spores of these plants were examined. Such fusions should give rise to a
chromatin bridge at anapha.se of the first mitosis in the spore (see Sax
1937). Upon examination, a chromosome showing a bridge configuration
was found in a number of spores. By a method which will be described
in a separate publication, it was possible to show that the spores which
have a bridge configuration likewise possess a chromosome which was
broken during the meiotic mitoses. The examinations indicated, also, that
such fusions probably always occur. Such evidence illustrates, directly,
the tendency of broken ends to fuse. One would be tempted to use this
information and transfer the process to the somatic chromosomes. How-


372                 BARBARA McCLINTOCK
ever, the evidence at present indicates that one is not justified in doing so.
Until the contradictory features of this evidence are completely analysed,
the author is unwilling to interpret the ring chromosome behavior on this
basis.
Viable homozygous deficiencies in Drosophila giving effects similar to
"genes" known to be located in the region which has been made deficient,
have been described by MULLER (1935) for yellow and achaete, EPHRUSSI
(1934), STERN (193j) and DEMEREC and HOOVER (1936) for yellow,
STURTEVANT and BEADLE (1936) for scute, EMMENS (1937) for roughest-2,
and OLIVER (1937) for facet. Viable individuals homozygous deficient for
a region possessing no known genes have been described by DEMEREC and
HOOVER. Homozygous deficiencies producing immediate or delayed effects
in zygotes and embryos, which eventually result in death to the individual,
have been described by POULSON (1937). As far as the author is aware,
homozygous deficiencies in plants which simulate a gene known to be
located in the deleted segment, have been found only in the bml case
described in this paper. Evidence that the known genes in the Drosophila
cases might be due to deficiencies in the regions involved, has been given
only for the gene roughest-e. Simulation of the known gene by a region
deficient for its locus has been claimed for the other cases and applies
likewise to the bml case in Zea. It is unprofitable at present to estimate
to what extent homozygous deficiencies are responsible for known genie
effects. That some of these may be related to position effects seems pos-
sible from the accumulating evidence in Drosophila. In how many of
these cases the factor of a minute deficiency can be eliminated, remains
to be decided, granting that the presence of a deficiency introduces the
possibility of a position effect. In the case described in this paper, the two
independent segments of chromosome V, the deficient rod and the un-
altered ring fragment, produce effects, with regard to the Bml character,
which are indistinguishable from that produced by a normal chromosome
V carrying Bml. The only evidence so far obtained of a "position effect"
is derived from the appearance of plants homozygous for Def2 and R2
and from the lack of expected transmission through the pollen of gametes
with Def 1 R 1 or Def 1 R2. In neither case can the factor of a minute
deficiency be eliminated, the deficiency affecting plant growth in the former
case and pollen transmission in the latter case.
The method of producing phenotypic effects by alterations in ring
chromosomes in plants with two deficient chromosomes plus a covering
ring fragment, briefly described in section VIII, should prove useful in
analysing in considerable detail the genetic composition of small sections
of chromosomes. The effects produced have been ascribed to minute
homozygous deficiencies rather than to position effects since homozygous


              RING CHROMOSOMES IN MAIZE            373
deficiencies must be produced by alterations in the ring chromosomes. If
individual regions within the deficient segment produce particular effects
independent of their neighbors, combinations of these effects should be
produced by loss from the ring chromosome of two or more of these regions.
Since the method by which the ring chromosomes become altered should
delete adjacent segments from `the ring chromosome, an orderly arrange-
ment of compound effects should result. The order of the particular regions
within the ring chromosome which have specific effects could be de-
veloped from analyses of the individual effects contributing to the com-
pound effects. Since the results obtained so far substantially correspond
to the requirements of this theory, the notion of a particulate nature
of the composition of this region of the chromosome has been retained.
The development of this method of analysing the composition of sections
of chromosomes has just been started. It would be premature to draw
rigid conclusions from the results so far obtained. Three deficiencies
of chromosome V, each of which can be covered by a ring fragment, are
available for this study. Two of these fall within the range of the third.
Correlations of results from all three deficiencies should conform to a
predicted pattern if the above theory is correct. Until the evidence from
these studies has accumulated, no attempt will be made to force a par-
ticulate theory of the organization of the chromosome in contradistinction
to a continuum theory. The former will be retained as a working hypoth-
esis until the evidence definitely requires a modified view.

SUMMARY

I. Two cases of a deficiency adjacent to the spindIe fiber attachment
region in the short arm of chromosome V involving approximately 1/20
(Def 1) and 1/7 (Def 2) the Iength of the chromosome, respectively, were
produced by X-ray treatment. The piece deleted in each case formed a
small ring-shaped chromosome, Rl and R2, respectively. In each case a
section of the spindle fiber attachment region was removed to the ring
chromosome and a section was retained by the deficient rod chromosome.
Since the deficient rods and compensating ring chromosomes possessed a
functional section of the spindle fiber attachment region, each was capable
of participating successfully in the mitotic process. The ring chromosome
in each case carried the locus of Bml (allele of bml, brown mid-rib, pro-
ducing a brown color in the lignified cell walls). The rod chromosomes
lacked the locus for Bml.         *
2. Plants with two normal chromosomes V carrying bm 1 (or one normal

chromosome V carrying bml and a deficient chromosome V) plus either
ring chromosome are variegated for Bm 1 and bm 1 through frequent losses
of the ring chromosome from the somatic nuclei.


374                 BARBARA McCLINTOCK
3. Somatic loss of ring-shaped chromosomes is described.
4. The abnormal mitotic behavior of large and small ring-shaped chro-
mosomes is contrasted. Large ring-shaped chromosomes frequently change
in size and chromatin constitution during somatic mitotic cycles. Small
ring-shaped chromosomes are more frequently lost from nuclei during
mitotic cycles although changes,in size and chromatin content sometimes

occur.
5. The frequency of aberrant mitotic configurations of the ring chromo-
somes, leading to loss or change in size, is related to the length of the
chromonema composing the ring. The longer the chromonema the more
frequent the aberrant configurations. With small ring-shaped chromo-
somes, whose aberrant mitotic configurations usually lead to loss of the
ring chromosome from the nuclei, the extent of variegation (z above) is
proportional to the size of the ring chromosome.
6. Functional gametes with Defl, Defl plus Rl, and Def2 plus R2 were
obtained. The functional capacities of these two deficiencies with various
combinations of the ring chromosomes were tested. Some of these were
functional, others were not.
7. Plants with Def l/Def2/R2 (R2 covers the deficiencies in the rod
chromosomes) were a uniform mosaic of tissues heterozygous and homo-
zygous for the full extent of the deficiency in Def 1 (the smaller deficiency)
through losses of the ring chromosome during somatic mitosis. The pat-
terns of homozygous deficient tissues in plants with these two deficient
rod chromosomes plus various combinations of ring chromosomes (R 1,
R2, two R2, Rl plus R2, two Rl plus one R2, two R2 plus one Rl)
have been compared and agree with expectancy on the basis of the cyto-
logical analysis of ring chromosome behavior in mitosis and the analysis
of variegation (z above) produced in plants with these same combinations
of ring cliromosomes.
8. The homozygous deficient tissues, lacking a locus for Bml, have the
phenotypic expression-of bml in their cell walls.
9. Plants homozygous for Def 2 R2 have 22 chromosomes in their
zygotes. Although the chromosome number has been increased, there has
been no increase in the genome. Losses of the R2 chromosomes during
development produce cells homozygous deficient for the extent of the
deficiency in Def 2. These cells are abnormal in appearance and are short
lived, degenerating before maturity of the surrounding cells.
IO. In plants with two deficient chromosomes and one or more com-
pensating ring chromosomes, somatic alteration in constitution of a ring
chromosome is reflected in modified tissues having mutational character-
istics. A number of repeatedly encountered, distinct types are briefly
described. One type is indistinguishable from normal bml.


RING CHROMOSOMES IN MAIZE            37.5

ACKNOWLEDGbfENTS

Much of the investigation described in this paper was undertaken at
CORNELL UNIVERSITY with the aid of a grant from the ROCKEFELLER
FOUNDATION. The original material for this investigation was supplied by
Dr. L. J. STADLER to whom the author is very grateful. To Dr. R. A.
EMERSON of CORNELL UNIVERSITY the author is particularly indebted for
his generosity in placing the facilities of the Department of Plant Breeding
at her disposal and for his continued encouragement.

LITERATURE CITED

ATABEKOWA, A. J., 1936 Die Wirkung der Rontgenstrahlen auf die Samen und Keimlinge der
Erbsen. J. Biol. (U.S.S.R.) 5: 99-116.

BURNHAM, C. R., 1932 An interchange in maize giving low sterility and chain configurations.
  Proc. Nat. Acad. Sci. Wash. 18: 434-440.

DARLINGTON, C. D., 1936 Crossing over and its mechanical relationships in Chorthippus and
  Stauroderus. J. Genet. 33: 460-500.

DE~~EREC, M., 1936 Frequency of "cell-lethals? among lethals obtained at random in the X
chromosome of Drosophila melwtogader. Proc. Nat. Acad. Sci. Wash. 22: 350-354.
DEMEREC, M., and HOOVER, M. E., 1936 Three related X chromosome deficiencies in Drosophila.
  J. Hered. 27: zos--212.

EKERSON, R. A., BEADLE, G. W., and FRASER, A. C., I935 A summary of linkage studies in maize.
  Cornell Univ. Agr. Ezp. Sta. Mem. 180: 1-83.
EONS, C. W., 1937 Salivary gland cytology of roughestainversion and reinversion, and roughst*.
  J. Genet.34: 191-202.

EPHRUSSI, B., 1934 The absence of autonomy in the development of the effects of certain de-
ficiencies in Drosophila melanogaster. Proc. Nat. Acad. Sci. Wash. 20: 420-422.
HUSKINS, C. L., and HUNTER, A. W. S., I935 The effects of X-radiation on chromosomes in the
microspores of Trillium erectjim Linn. Proc. Roy. Sot. London, Series B 117: 22-43.
HUSTED, L., 1936 An analysis of chromosome structure and behavior with the aid of X-ray in-
  duced rearrangements. Genetics 21: 537-553.
MCCLINTOCK, B., 1931 Cytological observations of deficiencies involving known genes, tranSlOca-
  tions and an inversion in Zea mays. Missouri Agr. Exp. Sta. Res. Bull. 163: 1-30.
1932 A correlation of ring-shaped chromosomes with variegation in Zea mays. Proc. Nat.
  Acad. Sci. Wash. 18: 677-681.
  1933 The association of non-homologous parts of chromosomes in the mid-prophase of meio-
  sisin Zea mays. Z. Zellf. mik. Anat. Ig: I9I-237.
  1937 The production of maize plants mosaic for homozygous deficiencies: Simulation of the
  bmf phenotype through loss of the Bml locus. (Abstract) Genetics 22: 200.
  1938 A method for detecting potential mutations of a specific chromosomal region. Genetics
   23:x59.

MORGAN, L. v., 1933 A closed .X chromosome in Drosophila melanogaster. Genetics 18: 25-283.
MULLEn, H. J., 1935 A viable two-gene deficiency. J. Hered. 26: 469-478.
MULLER, H. J. and PROKOFJEVA, A. A., I935 The individual gene in relation to the chromomere
  and the chromosome. Proc. Nat. Acad. Sci. Wash. 21: 16-26.
M~TZING, A., 1934 Chromosome fragmentation in Crepis hybrids. Hereditas 19: 284-303.
NAWASEIN, M., 1930 Unbalanced somatic chromosomal variations in Crepis. Univ. Cal. Pub.
  Agr. Sci. 6: 95-106.
1936 Chromosomenanordnung und Chromosomenanomalien in somatischen Metaphasen
tmd ihre Bedeutung fiir die Theorie der Chromosomenindividualitst. Ber. deut. Bot. Ges. 54:
  279-290.

OLIVER, C. P., 1937 Evidence indicating that facet in Drosophila is due to a deficiency. Amer.
  Nat. 71:56~-566.


376                 BARBARA McCLINTOCK
POULSON, D. F., `937 Chromosomal deficiencies and the embryonic development of Drosophila
melanogaster. Proc. Nat. Acad. Sci. Wash. 23: 133-137.
RKOADES, M. M., 1936 A cytogenetic study of a chromosome fragment in maize. Genetics 21:
  4g1-502.
RHOADES, M. M., and MCCLINTOCK, B., rg35 The cytogenetics of maize. Bot. Rev. I: 292-325.
RICHARDSON, M. M., 1936 Structural hybridity in Lilium ntartagon album x L. Hansoni. J. Genet.
  32 : 411-450.
SAX, K., xg37a Chromosome behavior and nuclear development in Tradescantia. Genetics 22:
  523-533.
  r937b Chromosome inversions in Paeonie snffrulicosa. Cytologia, Fujii Jubilee Vol. 108-114,
SCHULTZ, J., and CATCHESIDE, D. G., 1937 The nature of closed X chromosomes in Drosophilrz
  melanogaster. J. Genet. 35: 315-320.
SIDOROW, B. N., SOKOLOW, N. N., and TROFIMOW, I. Eu., 1936 Crossing over in heterozygoten
  Inversionen. Genetica 18: 2g1-312.
SXITH, S. G., 1935 Chromosome fragmentation produced by crossing over in Trillium erectam L.
  J. Genet. 30: 227-233.
STADLER, L. J., 193s On the genetic nature of induced mutations in plants. II. A haplo-viable
  deficiency in maize. Missouri Agr. Exp. Sta. Res. Bull. 204: 1-29.
STERN, C.: 193s The effect of yellow-scute gene deficiency on somatic cells of Drosophila. Proc.
  Nat. Acad. Sci. Wash. 21: 374-379.
STURTEVANT, A. H., and BEADLE, G. W., 1936 The relations of inversions in the X chromosome
  of Drosophila melanogaster to crossing over and disjunction. Genetics 21: 554-604.
UPCOTT, M., 1937 The genetic structure of Tulipa. II Structural hydridity. J. Genet. 34: 339-398,
WHITE, M. J. D., 1935 The effects of X-rays on mitosis in the spermatogonial divisions of Locusta
  migratoria L. Proc. Roy. Sot. London. Series B 119: 61-84.