SPONTANEOUS MORPHOLOGICAL ALTERATIONS OF
CHROMOSOMES IN NICOTIANA HYBRIDS
.r. MOAV, R. MOAV
AND
D. ZOHARY
Laboratory of Genetics
The Hebrew University of Jerusalem, Jerusalem, Israel
Received December 12, 1967
EVERAL workers have already reported that hybridization between Nicotiana
species can bring about chromosome instability. One conspicuous case is loss
of somatic plumbaginifolia chromosomes in derivatives of N . tabacum X N .
plumbaginifolia hybrids (MOAV1961). The present paper deals with another
peculiarity of alien chromosomes in tobacco hybrid derivatives. It reports on
heritable changes in size and stainability detected in N . plumbgginifolia chromosomes after their addition to N . tabacum genomes.
MATERIALS
The two species involved in this study are Nicotiana tabacum, variety Red Russian n = 24
(henceforth tbc), and N . plumbaginifolia, n = 10 (henceforth pbg). For details on the two species
and their cytogenetic afhities see GOODSPEED
(1954).
Backcrossed derivatives cif the sesquidiploid hybrid (2n tbc f n pbg) to tbc were examined.
The mode of their origin was described earlier (CAMERON
and Moav 1957; MOAV1961).
Two dominant markers were employed for marking individual pbg chromosomes: ( 1 ) WSthe normal, dominant allele of the White-seedling locus-necessary for chlorophyll production.
Inheritance of the white seedling character, i.e. inability to produce chlorophyll, is controlled by
a pair of alleles, the white seedling determining allele ws being the recessive. (2) KZ or Pollen
Killer, a pbg gene which interacts with tbc genomes to cause 85% pollen abortion (CAMERON
and MOAV1957).
CYTOLOGICAL TECHNIQUES
Aceto-orcein squashes of fresh pollen mother cells were studied during first meiotic metaphase.
The chromosomes were measured at a magnification of lux)^, with the aid of an ocular micrometer (one micrometer unit equals l .26 microns). Only width and length were measured,
hence whenever size of a chromosome is referred to, it means the estimated area of its microscopic
image. Each chromosome WEISmeasured five times, and the mean of the five measurements was
used for an estimate.
Even a slight change in the stage of the meiotic cycle affects the absolute size of the chromosomes. TOovercome this difficulty, the size of the univalents under study was assessed only relative to the mean of the biggest two bivalents of the same cell. “Size” of a chromosome is defined
here as width times length o r “area” of the microscopic image of the univalent under investigation divided by the average “area” of the t w o biggest bivalents of the same cell, multiplied by 100.
Mitotic plates were examiined in root-tips of cuttings. The root-tips were pretreated for one to
three hours with a saturated aqueous solution of para-dichlorobenzene, then transferred for 24
hours to a solution of 3 parts ethyl alcohol to one part glacial acetic acid. After fixation they were
kept in 70% alcohol. Before squashing the fixed root-tips were soaked for 24 hours in a 7: 1 solution of 2% aceto-orcein and 0.1 N HCl.
Genetics 5 9 : 57-63 May 1968.
58
J. MOAV, R. MOAV A N D D. ZOHARY
E X P E R I M E N T A L RESULTS
Changes in the “Pollen Killer” chromosome: A spontaneous increase in the
size of the pbg chromosome carrying the dominant K1 marker was discovered in
two plants. Both were offspring of a single hybrid derivative (plant No. 54630~7)
whose chromosomal constitution was 24 tbc bivalents plus three pbg univalents.
Two of the pbg univalents were marked by dominant genes. One designated
K l ( p ) was marked by Kl, and the second designated W s ( p ) , was marked by W s .
None of the three pbg univalents of the parental plant was outstanding in size or
appearance.
One offspring (plant No. 54R48p1) in which an enlarged K l ( p ) univalent was
found, resulted from selfing of the above (54630~7)parental plant, while the
second enlarged Kl(p) chromosome was found in an offspring (plant No.
54R14pl) of a back-cross of the same parent to a tbc plant homozygous for the
recessive ws (White seedling) allele.
The first off spring (54R48pl) possessed 24 tbc bivalents plus a single abnormally large univalent marked by the Kl marker. The second offspring (54214~1)
possessed 24 tbc bivalents plus two pbg univalents. One univalent marked by W s
maintained its original size, while the second, which was marked by K1, was
again abnormally large.
Eleven offspring resulting from selfing of plant 54R48pl and showing the
expression of the K l marker (about 85% pollen abortion) were examined cytologically in first metaphase of pollen mother cells. All these offspring (group
No. 36) revealed the 24 bivalents of tbc plus the enlarged Kl(p) univalent (Table
1, row 3 and Figure 2,l).
Selfing of plant 54Rl4pl yielded only two offspring that showed the high
TABLE 1
Relative magnitude of spontaneously enlarged N. plumbaginifolia
chromosomes in N. tabacum background
Relative size of chromosome
Row
Group Number
823
38
36
37
37
11
38
35
Measured chromosome
KKP)
KUP)
enlarged K l ( p )
enlarged K l ( p )
W4P)
WS(P)
enlarged W s ( p )
unmarked
Number
of plants
Number
of PMC’s
Mean percent
4
4
11
2
7
2
10
5
18
34
35
8
33
8
63
13
58.6
60.2
90.8
89.6
62.4
69.6
114.0
105.4
Range percent
56.0-61.2
57.8-65.2
80.6-99.8
822-96.9
46.5-67.7
65.8-73.5
93.9-118.1
101.4-1 11.5
(plumbaginifolia
cytoplasm)
The parent of group 37 (plant 54R14pl) carried an enlarged K l ( p ) chromosome and a
normal W s ( p ) chromosome. The parent of group 3& (plant 5 4 6 3 1 ~ 7 )carried an enlarged W S ( P )
and a normal Kl(p). The plants within each group segregated with respect to the two marked
plumbaginifolia univalents, hence each group was used for studying one enlarged chromosome
and the normal control of the second enlarged chromosome.
59
CHROMOSOME ALTERATIONS I N N I C O T I A N A
pollen abortion characteristic of KZ (group No. 37). Both possessed the enlarged
KLCp) chromosome of their parent (table 1, row 4). Seven other sibs of the same
group did not show the Pollen Killer effect and none possessed an enlarged chromosome.
The unaltered pbg chromosome marked by KL was measured in two control
populations (Table 1, rows 1 and 2). Its mean size in the two groups was, respectively. 58.6% and 60.276 of the mean size of the two biggest bivalents of the cells
examined. A comparison between the areas of the enlarged K l ( p ) chromosome
and the areas of the unaltered control chromosome shows an increase of about
50% (compare Table I., rows 1 , 2 with rows 3,4). The consistency of this difference is illustrated in Figiure 1.
The altered appearance of the KL(p) chromosome did not have any apparent
*
Grwp 38
*
(control)
*
-?
Group 823 (control)
700
I
*
600..
a
U
m
500
--
k
I
*
#
*
*
*
*
* *
*
* *
**
* * **
* *
*
*
0
a
C
*
$
300
EOO
600
700
800
900
Mean area of the two biggest bivalents
FIGURE
1.-The bivariate adistributions of the areas of the microscopic images of three K l ( p )
univalents (two control and one enlarged) and the mean areas of the two biggest bivalents of the
same cells. (Measurements were taken during first meiotic metaphase of pollen mother cells. The
units of measurement are those of the ocular micrometer).
60
J. MOAV, R. M O A V A N D D. ZOHARY
effect on the transmission or morphological expression of its marker. Even the
degree of somatic instability of this chromosome appeared to be unmodified in
the wcmd generation.
Mitotic plates of root tips of cuttings of several plants of group 36 with enlarged
K l ( p ) univalent, revealed 4.9 chromosomes. In every cell one chromosome was
considerably larger than all the others. The large chromosome was sub-metacentric with a secondary constriction at its long arm (Figure 2.2). In contrast, the
mitotic plates of the control (group 823) which also possessed 49 chromosomes.
did not reveal any outstanding chromosomes.
One member of group No. 36 which had 82% pollen abortion and possessed
the enlarged K l ( p ) chromosome developed a single shoot with only 5.3% pollen
abortion. Cytological examination of this “bud sport’’ showed that the enlarged
univalent was replaced by a small fragment. Clearly, the “somatic mutation”
responsible for this event was a somatic breakage of the enlarged KL(p) which
left only a small centric fragment. not carrying K l in the resulting cell lineage.
Changes in the “White Seedling” chromosome: Another enlarged chromosome
was discovered in plant No. 5 4 6 3 1 ~ 7which was a backcross offspring of the
FIGURE
2.-Meiotic and mitotic metaphase plates showing spontaneously enlarged N. p l u m baginifolin chromosomes 1. First metaphase in r pollen mother cell containing 24 tobacco bivalents
and an enlarged K l f p ) univalent (marked by a n arrow). 2. Metaphase plate of a root-tip showing
the 48 tobacco chromosomes plus the enlarged K l ( p ) chromosome (marked by a n arrow). 3. Side
view of first metaphase in a pollen mother cell showing, in focus, the enlarged W s ( p ) univalent
(marked by an arrow) and another univalent of a n ordinary size (marked by Y). 4.5. Metaphase
in root-tips showing the sphere-like W s ( p ) (marked by arrows). Figures magnified 2 2 0 0 ~ .
61
CHROMOSOME ALTERATIONS I N NICOTIANA
sesquidiploid hybrid (2!n tbc -tn pbg) to a normal tbc. Metaphase plates of pollen
mother cells of this p1,ant revealed 24 bivalents plus three univalents. One univalent had the appearance of a darkly stained sphere and was larger than the
biggest tbc bivalent (Figure 2.3). Its bizarre appearance was due to its being not
only longer than expected but also thicker.
The plant possessed two doses of the recessive ws allele in its two tbc complements. The dominant 1Vs (necessary for chlorophyll production) was carried by
a pbg univalent which turned out to be the enlarged chromosome. This plant was
also carrying the K l marker on another one of its univalents.
The enlarged Ws(p) chromosome was traced and measured in metaphase
plates of ten offspring (group 38) resulting from self pollination of plant 54631~7
(Table 1, row 7). Its mean relative size was 114%, as compared with 65% of its
unmodified control (Talble 1, rows 5 and 6). Again there was a remarkable degree
of uniformity in the size and appearance of the enlarged chromosome in all the
plants in which it was present.
Corroborative examinations were made in root-tips of this offspring group,
(group 38). The sphere-shaped darkly stained odd chromosome was conspicuous
also in mitotic metaphase plates (Figure 2.4 and 5).
Two albino (chlorophylless) flower buds that appeared on the parent plant
54631~7were found to be lacking the enlarged sphere-like univalent. For details
on the nature of such (albino buds see MOAV(1961). Similarly albino (chlorophylless) shoots also grew occasionally from its mottled offspring (group 38).
The enlarged univalent. which was present in the green (mottled) parts of these
plants was missing in all the albino shoots (Table 2). This supplied direct evidence that the enlarged! univalent indeed carried the Ws locus.
The presence of the e.nlarged W s ( p ) chromosome appeared not to be correlated
with any other morphological, cytological or hereditary responses.
Spontaneously enlarged plumbaginifolia chromosome in plumbaginifolia cytoplasm: Diploid pbg plants were also used as female parents in a cross with autotetraploid tbc. The resulting sesquidiploid hybrid 2n tbc n pbg was repeatedly
backcrossed to normal tbc. I n all backcrosses tbc served as male parent. As a
+
TABLE 2
Chromonme numbers in green (mottled), and albino (chlorophylless)
shoots of the same plants
Albino shoots
Green shoots
Plant Number
38- 3
38-10
38-1 1
38- 4
38- 5
Karyotype.
+
+
+ +
24 I1 I $- G
2411 I t - G
24 I1 f I t-G
24 I1
2I
G
2411,+21+G
Number
of flowers
Number
of PMC
2
3
12
1
2
* G designates an abnormally large univalent
9
4
6
2
5
Karyotype
+
24 I1 I
2411 + I
2411 + I
2A. I1 2 I
2411+2I
,+
Number
of flowers
Number
of PMC
2
6
1
2
1
3
1
5
2
5
62
J. MOAV, R. MOAV A N D D. ZOHARY
result of this procedure most of the pbg chromosomes were eliminated and what
was essentially a tbc nucleus was transferred into pbg cytoplasm. In one hybrid
derivative obtained by this procedure, a single spontaneously enlarged pbg univalent was found together with the 24 normal tbc bivalents. The plant was selfed
and the relative size of the large univalent was determined in 5 of its offspring
(Table 1,row 8).
The finding of an enlarged pbg chromosome in a pbg cytoplasm seems to
eliminate cytoplasmic-chromosomal interaction as a causal mechanism for the
spontaneous chromosomal alteration.
DISCUSSION
The observations on the spontaneously altered plumbaginifolia chromosomes
after their addition to tabacum nuclei may be summed up in the following points:
1) The phenomenon is not specific to a single pbg chromosome. Although it was
detected in only two marked chromosomes, it is rather likely that other pbg
chromosomes occasionally undergo similar changes. 2) There were no indications
that the altered condition increased the chromosome's level of instability. 3) Not
a single reversion to the original state was found. The only change of an altered
chromosome was discovered in a bud sport where somatic breakage of an enlarged
chromosome resulted in a small centric fragment. 4) Neither the phenotypic
expression nor the inheritance of the markers carried by the altered chromosome
appeared to undergo any changes. 5 ) Cytoplasmic-chromosome interactions probably can be rejected as a causal mechanism since the alterations took place in the
cytoplasms of both of the two species. 6) Translocations with members of the tbc
chromosome complement can also be rejected as an explanation for the phenomenon observed because there was no noticeable meiotic pairing of the altered
pbg univalents with the tbc chromosomes. The cytological observations were
supported by progeny tests #whichshowed that the transmission rate of the altered
chromosomes was not different from their unaltered original counterparts. 7)
Translocations between two pbg chromosomes can also be ruled out since they
do not fit with the quite unusual meiotic or mitotic appearance of the altered
W s ( p ) chromosome. Such simple explanation fits perhaps the mitotic appearance
of the altered K l ( p ) (Figure 2.2). However, it does not explain the conspicuous
change in condensation and staining of this chromosome in m ' o s i s . 8) The
morphology of the enlarged chromosomes rules out the possibility that they are
iso-chromosomes. 9) The high degree of uniformity in both size and appearance
of a given altered chromosome within a single plant or even within a group of
sibs seems to rule out the possibility that we are confronted here with a somatically unstable chromosome subjected to breakage-fusion-bridge cycles. 10) The
inability of orthodox mutational events to provide a satisfactory explanation to
chromosomal changes was also concluded for the spontaneous alterations in the
degree of somatic instability of the same pbg chromosomes in the same hybrid
derivatives (MOAV1961). Thus, it is tempting to speculate that the two phenomena, i.e., changes in morphology and changes in the stability level of the pbg
CHiROMOSOME ALTERATIONS I N NICOTIANA
63
chromosomes, are two manifestations of the same, still unexplained, mutational
event. 1 1 ) GERSTEL
and BURNS(1966) also observed occasional cells with individual chromosomes of very unusual length in hybrids between N . tabacum and
N . otophora. Unlike the present altered chromosomes which were transferred
regularly and uniformly from cell to cell and even from one generation to the
and BURNSapparently were not
next, the chromosomes described by GERSTEL
able to go through successivemitotic divisions.
The writers wish to express their sincere gratitude to Professor DONALD
R. CAMERON
for his
generosity in supplying the #seedstock needed for the present investigation.
SUMMARY
In hybrid derivatives of Nicotiana tabacum and N . plumbaginifolia, individual
plumbaginifolia chromosomes occasionally undergo spontaneous enlargement,
that may be accompanieed by changes of density and staining.-The phenomenon
was not restricted to a[ single chromosome. Once an added alien chromosome
undergoes spontaneous enlargement, it maintains its new state without further
changes during cell divisions, or even when transferred from one generation to
the next. Thus, the spontaneous enlargement may be considered a relatively
stable mutation of the morphology of the chromosome.-The phenomenon probably can not be explained by any of the orthodox chromosomal aberrations, or
by cytoplasmic-chromosomal interactions. The similarity between the present
morphological changes in the alien addition chromosomes and some features of
their somatic instability were pointed out and it was suggested that both may be
different manifestations of the same basic phenomenon.
LITERATURE CITED
D. R. and R. MOAV,
1957 Inheritance in Nicotiana tubucum XXVII: Pollen Killer, a n
CAMERON,
alien genetic locus inducing abortion of microspores not carrying it. Genetics 42 : 326-335.
GERSTFL,D. U. and J. A. BURNS,1966 Chromosomes of unusual length in hybrids between two
species of Nicotiana. In: Chromosomes Today. 1: 41-56. Edited by C. D. Darlington and
K. R. Lewis. Oliver & Boyd, Edinburgh.
GOODSPEED,
T. H. The Genus Nicotiana, 1954 Chronica Botanica. Waltham, Mass., USA.
MOAV,R., 1961 Instability in Nicotiana hybrids 11: A study of the Ws(pbg) locus of N . plumbaginifolia in N . tabucum nucleus. Genetics 46:1069-1088.
MOAV,R. and D. R. CAMERON,
1960 Iden I: The expression of instability in N . tabacum
plumbaginifolia. Am J. Botany 47: 87-93.
x N.