Chromosomal chimeras in Crepis

CHROMOSOMAL CHIMERAS IN CREPIS

LILLIAN HOLLINGSHEAD

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University of California Publications in Agricultural Sciences

Volume 2, No. 12, pp. 343-354, plates 54, 55, 2 figures in text

Issued March 26, 1928

University of California Press
Berkeley, California

Cambridge University Press
London, England

CHROMOSOMAL CHIMERAS IN CREPIS

BY

LILLIAN HOLLINGSHEAD

Gates (1924) stated that "it is unknown at the present time how
widespread polyploidy in somatic tissues may be." Several writers
have since that time reported cases of somatic polyploidy and it was
thought worth while to put on record instances of this condition
recently discovered in the Genetics Laboratory of the University of
Califoi'nia.

In the course of an examination of root tips of various Crepis
species and species hybrids, two plants which were partly tetraploid
have been found. ^ These are not the first cases of chromosomal chi-
meras reported in Crepis, Lesley (1925) and Nawaschin (1926) having
previously recorded the phenomenon. Lesley 's report is a note stating
that in an P^ between C. biennis (« = 20) and C. foetida (w^5)
a few neighboring cells were found having about twice 25 chromo-
somes, whereas most of the cells contained the expected 25. Nawaschin
reported the occurrence of a tetraploid area in the form of a narrow
sector in a diploid root of C. Bioscoridis (w^4).

The first of the two cases to be described was that of a chimeral
root of a derivative from a cross between Crepis biennis {n = 20) and
C. setosa (^ = 4) which had 24 chromosomes in most of the somatic
cells. In this root, however, a large number of cells was found which
obviously had many more than the noi*mal 24 chromosomes, several
approximated 48, and two cells gave clear accounts of 48 chromosomes.
The normal and tetraploid chromosome complexes are shown in
figure A. By an examination of successive sections it was determined
that the tetraploid cells were confined to a definite region which
extended from at least vei-y close to the root cap to a point where no
division figures could be found. Apparently the longitudinal outlines
of the tetraploid area were fairly regular, since only one case occurred
in which diploid and tetraploid cells were found in different sections
occupying the same position relative to the circumference, and this
was on the line of demarcation between the two areas.

1 Since writing the above a root of C. Hakelei (/i^8) containing several
neighboring cells with about twice the normal chromosome number and one of
C. mothtana (w^6) with one tetraploid cell have been found.

344 University of California Publications in Agricultural Sciences [Vol. 2

Figure B was made up from an examination of the whole root and
shows that the tetraploid area occupied the major portion of the root
and that its cross-section was very irregular in shape. The dotted
lines indicate the portions of the boundary between the 2n and 4«
areas which could not be accurately determined. It is uncertain
whether the tetraploid area extended into the central cylinder or not.
The tetraploid area at a is two cell layers deep, at h it is only one, but
opposite a at c it occupies most or all of the cortex. While there is
considerable variation in cell size \vithin both areas the average size
of the tetraploid cells is larger than that of the diploid.

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^•^

Fig. A. 1, Diploid chromosome complex of biennis-setosa liybrid derivative
(2n = 24). 2, Tetraploid complex (2w^48) from chimeral root of the same
plant.

The shape of the tetraploid area is of some interest from the point
of view of development as presumably the doubling of chromosomes
took place in one of the initial cells from which the root developed.
The area does not show the comparative regularity in shape exhibited
by Nawaschin's tetraploid sector. On the other hand, tetraploidy here
is confined to one definite area, which was not the case with Lesley's
(1925) tomato chimeras, where isolated areas of tetraploid cells were
observed. The condition is similar to that which Langlet (1927)
found in two roots of Thalictrum but differs from that reported by
Winge (1927) in Tragopogon hybrids where the tetraploid parts of
two roots by reason of their larger cells rendered the cross-sections
of the roots eccentric. The plant bearing this chimeral root was of
normal appearance in the rosette stage but unfortunately died before
flowering.

The second case showing a chimeral condition was a plant of
Crepis Burcniana (?^ = 4). Of the thirty roots of this plant which
were examined two were tetraploid, having 16 chromosomes in all the

1928]

Hollingsliead : Chromosomal Chimeras in Crcpis

345

plates observed. Plate 54, figures a and b, shows the chromosome com-
plexes with surrounding areas from the outer cortex in comparable
regions of the diploid and tetraploid roots. Each of the chromosomes
of the diploid complex could be recognized in the tetraploid and the
longest one of the set could be identified four times in the best tetra-
ploid plate. Undoubtedly there has been a doubling of the diploid
set. An examination of plate 54 shows that the average size of cells
and nuclei is larger in the tetraploid root.

Fig. B. A cross-section of root of hiennis-setosa hybrid derivative showing
the extent of the tetraploid area. The outer ring is the extension of the root cap
and contains no dividing cells.

The occurrence of two tetraploid roots in the same plant may be
taken as evidence that part of the central cylinder from which
branches arise had become tetraploid. It was thought possible that
the plant above ground might have been affected similarly, and if so,
the pollen produced on a tetraploid branch would be larger than that
on a diploid. Examination of pollen fi*om various branches, however,
showed no noticeable size differences, so it was concluded that tetra-
ploidy was probably confined to the roots.

In this same plant a root was examined which contained a number
of large cells. Most of them Avere clearly multinucleate, from two to
four nuclei having been counted in single cells (pi. 55). Plate 55 h
shows one of the smallest of these with 2 nuclei, and 2 nuclei may be
observed in one of the cells in a. In the larger cells nuclei were to be
found in successive sections. These cells were scattered throughout

346 University of California Puhlicaticns in Agricultural Sciences [Vol. 2

the cortex, mostly near the i^eriphery, and in one region a group of
them seems to be responsible for the somewhat misshapen appearance
of the root in cross-section (pi. 55 a,c). The cells vary in size, some-
times extending through three or four sections 7/x thick. They are
more or less vacuolated, depending on their size, but even the smallest
ones could be distinguished almost at a glance by their less densely ,
staining cytoplasm.

The normal chromosome complex of 8 was to be seen in several
plates of normal cells. In one very large vacuolated cell a large
number of chromosomes was observed, apparently in metaphase
(pi. 55 c?). Note the V-shaped arrangement of the metaphase plate.
The chromosomes could be seen in 3 successive sections of 7/i, thickness.

Nawaschin (1926) has reported a giant cell with over 500 chi'omo-
somes in a root tip of Crcpis tectorum and takes up favorably the
theory that it has arisen by successive chromosome divisions without
accompanying cell divisions. The cell he shows is not greatly unlike
• some of tliose seen in this material. Here, however, there seems to be
some evidence that the large cells are the result of fusion of several
smaller ones. The V-shape of the one plate observed in a giant cell
indicates that it may be a combination of two plates and that a nuclear
fusion has taken place. Plate 55 c gives the impression that two large
cells are in the process of fusion and indeed the cell wall has practic-
ally disappeared. It seems likely that such cell fusion might be asso-
ciated with an abnormal or pathological condition of the root, further
evidence of which was to be seen in small black areas probably repre-
senting degenerated cells (pi. 55 b).

The origin of tetraploidy in diploid tissue has been discussed by
various investigators. In some cases it has been associated with specific
outside influences. The effect of narcotics in inducing tetraploidy has
been investigated by Nemec (1903) and Sakamura (1920). Blakeslee
and Belling (1924) found tetraploid shoots in Datura plants subjected
to cold. Lesley (1926) found tetraploid areas in tomato plants
affected by mosaic and thought it might be jiossible that local changes
due to this disease might affect mitotic processes. Cases of polyploid
cells have been attributed to abnormal processes related to degenera-
tion as in the investing cells of the ovaries of Anasa tristis (Wilson,
1906). The doubling of chromosome numbers in Winkler's (1916)
Avell-kno^Mi chimeras has been attributed to the effect of wounding.
Jorgensen and Crane (1927) have recently secured tetraploidy in
Solaitum by the use of Winkler's method. Winge (1927) finds that

3928] Hnllingshead : Chromosomal Chimeras in Crepis 347

most of the cells of the "crown galls" on sugar beets which can be
induced by inoculation with Bacterium tumefaciens have the tetra-
ploid chromosome number.

Nawaschin did not venture any suggestion as to causal agencies in
connection with his tetraploid sector in Crepis Dioscoridis. Lesley
believed that it was unlikely that cold played any part as a causal
factor in tomato chimeras, as only the roots seemed to be affected. It
has been suggested by Mr. C. W. Haney that watering greenhouse
plants with cold water would pro\'ide the necessary conditions if sud-
den lowering of temperature has anything to do with the production
of tetraploid root cells. The plants described here were entirely
normal as far as could be observed and tetraploidy could not be
ascribed to any special factor in the environment.

Winkler had suggested that certain tissues may regularly become
polyploid and Breslawetz (1926) has reported tetraploidy as the
universal condition in the dermatogen of the root tips of Cannabis
sativa.- De Litardiere (1923) found tetraploid and octoploid cells
in the deraiatogen of Spinacia oleraeea. In both these cases it would
seem that the transforming of diploid into tetraploid cells must have
oceui-red many times in the same root.

The possibility of fragmentation giving rise to these increased
numbers is easily excluded in most cases. The two most favorably
received theories to account for doubling are: (1) the fusion of nuclei
from two cells; (2) the division of the chromosome complex without
cytoplasmic division. Breslawetz has brought forward evidence that
nuclear fusion gave rise to the tetraploid cells which made up the
dermatogen of the roots in Cannabis sativa. As no diploid complexes
were to be seen in that region of the root one would conclude that
fusion of diploid to form tetraploid nuclei had taken place before any
normal diploid divisions occurred, or at least at an early stage in the
development of the root. If so, one wonders why evidences of nuclear
fusion were still to be found in well developed roots. On the other
hand, the paired condition of the chromosomes in some of the tetra-
ploid cells in Spinacia oleraeea convinced de Litardiere that these
cells had just completed a chromosome division without separation of
the resulting daughter chromosomes.

The occurrence of multinucleate cells in a root of the Crepis
Bureniana plant which was partly tetraploid has been noted above.
The significance of this phenomenon in the origin of the tetraploid

2 De Litardiere (1924) found rare diploid cells in the peribleni and in one
ease a tetraploid cell in the plerome of roots of this species.

348 University of California Publications in A gricttltwal Sciences [Vol. 2

roots is questionable. The presence of several nuclei in one cell and
the abnormal appearance of these large cells would incline one to
belittle the possibility that tetraploidy here had originated by cell and
nuclear fusion. However, it has been pointed out that the one chromo-
some complex observed in a giant cell indicated that nuclear fusion
had taken place. We cannot, therefore, dismiss the possibility that a
fusion of nuclei from two cells gave rise to a cell which was tetraploid
and thence to tetraploid roots. It seems more likely, however, that the
occurrence of tetraploidy and of a root with giant multinucleate cells
in the same plant was merely a coincidence.

Whatever the method of origin, the frequent occurrence of tetra-
ploidy in somatic tissues throws some light on two much discussed
questions. First, there is that of the mode of origin of diploid gametes.
Rosenberg (1926-27), Karpechenko (1927), and others have described
processes in the reduction divisions of apogamous species and inter-
specific hybrids bj' which diploid gametes are formed. The increasing
frequency with which tetraploidy has been recorded in root tips makes
it seem likely that it would be found in other tissues were they
examined as consistently. Its occurrence in the cells of the germinal
line would lead to the formation of gametes with twice the normal
chromosome number. This lias been shown to occur in Datura where
tetraploid shoots were found. Presumably a smaller area might be
affected and only a portion of the gametes formed on one shoot might
be diploid.

In the second place, the frequent occurrence of somatic tetraploidy
has a bearing on the origin of tetraploids and tetraploid hybrids.
Prirmda kewensis arose as a bud sport probably from an Fi hybrid of
P. verticillata and P. florihunda. It has the sum of the diploid chro-
mosome numbers of the parent species and Clausen and Goodspeed

(1925) have suggested that it is a true tetraploid hybrid, the bud sport
having arisen by a doubling of somatic chromosomes. A similar
explanation with the doubling occurring immediately subsequent to
fertilization was suggested by these investigators to explain the origin
of a tetraploid hybrid between Nicotiana tabacum and N. glutinosa.
Rosenberg (1926) has recently proposed an explanation for the origin
of the tetraploid AcgUops — Tritic^im hybrid of Tschermak and Bleier

(1926) which depends on the chance meeting of diploid gametes
formed by a " semi-heterotypic " division. In the light of the fore-
going facts it seems much simpler to suppose that a doubling of the
chromosomes took place in the fertilized egg, or in some cell of the

1928] Hollingshead : Chromosomal Chimeras in Crepis 349

young embryo which gave rise to the growing point of tlie stem.
Nawaschin was led to favor this theory of the origin of tetraploids by
the frequency of 4j( plants in Crepis tecforum. He calculated the fre-
quency of diploid gametes from the number of triploid plants obtained
in over 4,000 individuals, and on this basis determined the number of
tetraploids which should occur by chance meeting of those gametes.
He found the expected number of tetraploids to be much less than that
actually occurring. He concluded, therefore, that tetraploids arose
through the doubling of chromosomes in the fertilized egg cells.

In view of the increasing number of cases in which tetraploidy has
arisen in normal diploid tissue, one is justified in concluding that it
may play a part in the origin of polyploid species and interspecific
hybrids.

I acknowledge with pleasure my indebtedness to Dr. J. L. Collins
and Professor E. Bi Babcock for the material used in this study.

SUMMARY

Tetraploidy was observed in the roots of two different plants. One,
a C. biennis x C. setosa hybrid derivative, had one root partly tetra-
ploid. The other, a plant of C. Bureniana, had two roots wholly
tetraploid.

No external factors could be associated with the tetraploidy.

Giant multinucleate vacuolated cells occurred in another root of
the same C. Bureniana plant. Evidence of cell and nuclear fusion was
observed. It is doubtful whether this phenomenon has any significance
in relation to the origin of the tetrajjloid roots.

Tetraploidy arising in somatic tissue probably plays a part in the
origin of polyploid species and intei-specific hybrids.

350 University of California Puhlications in Agricultural Sciences [Vol.2

LITERATURE CITED

Blakeslee, a. F., and Belling, J.

1924. Chromosomal chimeras in the Jimson weed. Science, vol. 60, pp. 19-20.

Breslawetz, L.

1926. Polyploide Mitosen bei Cannabis sativa L. Berichte der deut. Bot.

Gesell., vol. 44, pp. 498-502.

Clausen, E. E., and Goodspeed, T. H.

1925. Interspecific hybridization in Nicotiana. II. A tetraploid glutinosa-

tahacum hybrid, an experimental verification of Wiiige's hypothesis.
Genetics, vol. 10, pp. 278-84.
Gates, E. E.

1924. Polyploidy. Brit. Jour. Exper. Biol., vol. 1, pp. 153-82.

JORGENSEN, C. A., AND CrANE, M. B.

1927. Formation and morphology of Solnnum chimeras. Jour. Gen., vol. 18,

pp. 247-73.

Kaepechenko, G. D.

1927. The production of polyploid gametes in hybrids. Hereditas, vol. 9,
pp. 349-68.

Langlet, O. F.

1927. Beitriige zur Zytologie der Eanunculazeen. Svensk. Bot. Tidskr.,
vol. 21, pp. 1-17.

Lesley, M. M.

1925. Chromosome chimeras in the tomato. Am. Nat., vol. 59, pp. 570-74.

LiTAKDIEEE, M. E. DE

1923. Les anomalies de la caryocinese somatique chez le Spinacia olcracea L.

Eevue Gen. Bot., vol. 35, pp. 369-81.

1924. Sur 1 'existence de figures didiploides dans le meristeme radiculaire

du Cannabis sativa L. La Cellule, vol. 35, pp. 21-25.

Nawaschin, M.

1926. Variabilitat des Zellkems bei Crepis Arten in Bezug auf die Artbildung.

Zeitschr. f. Zellf. u. Mikr. Anat., vol. 4, pp. 171-215.

Nemec, B.

1903. Tiber die Einwirkung des Chloral-hydrates auf die Kern und Zell-
teilung. Jahrb. Wiss. Bot., vol. 39, pp. 645-730.

EOSENBERG, O.

1926. tJber die Verdoppelung der Chromosomenzahl nach Bastierdung.

Berichte der deut. Bot. Gesell., vol. 44, pp. 455-60.
1926-27. Die semiheterotypische Teilung und ihre Bedeutung fiir die Entste-

hung verdoppelter Chromosomenzahlen. Hereditas, vol. 8, pp. 305-38.

1928] nollingsliead: Cliromosomal Chimeras in Crrpis 351

Sakamura, T.

1920. Experimcntelle Studien iibei" die Zell und Kernteihing mit besonderer
Riieksiclit auf Form, Grosse, mid Zalil der Chromosomoi. Jour.
Coll. Sci. Imp. Univ. Tokyo, vol. 39, i)p. 1-221.

TSCHEBMAK, E., UND BLEIEH, H.

1926. Tiber fruehtbare Aegilops Weizenbastarde. (Beispiele fiir die Eutste-

hung neuer Arten durch Bastardierung.) Berichte der deut. Bot.
Gesell., vol. 44, pp. 110-32.

Wilson, E. B.

1906. Studies on chromosomes III. Jour. Exper. Zool., vol. 3, pp. 1-40.

WlNGE, O.

1927. Zytologisfhe Untersuehungen iiber die Natur maligner Tumoren.

I. "Crown gall" der Zuckerriibe. Zeitsclir. f. Zellf. u. Mikr.
Anat., vol. 6, pp. 397-423.

Winkler, H.

1916. tJber die experimentelle Erzeugung von Pflanzen mit abweichenden
Chromosomenzahlen. Zeitsclir. f. Bot., vol. 8, pp. 417-.531.

PLATE 54
Comparable areas of a, diploid, b, tetrnploid roots of a Crepis Burenkina plant.

[352]

UNIV CALIF PUBL AGR. SCI . VOL 2

HOLLINGSHEAD I PLATE 54

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-r

PLATE 55

Photomicrographs of root of the Crepis Burcniana plant showing giant multi-
nucleate cells.

a. A cross-section showing a group of giant cells.
h. One of the smaller giant cells with two nuclei.

c. Two giant cells apparently fusing.

d. A large cell containing a V-shaped metaphase plate with many chromosomes.

[354]

UNIV. CALIF. PUBL. AGR. SCI . VOL 2

HOLLINGSHEAD I PLATE 55

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