CYTOPLASMIC MALE STERILITY IN NICOTIANA, RESTORATION OF
FERTILITY, AND THE NUCLEOLUS. 11. N . DEBNEYZ CYTOPLASM1
J. A. BURNS
AND
D. U. GERSTEL
Department of Crop Science, North Carolina Staie University, Raleigh, North Carolina 27650
AND
S. A. SAND
Plant Genetics Section, Roswell Park Memorial Institutez, Orchard Park, N e w York 14127
Manuscript received March 13, 1978
ABSTRACT
Previously, it was shown that a fragment chromosome, apparently derived
from the Nicotiana repanda chromosomal complement, restores to normal the
morphology and fertility of the abortive and feminized anthers produced by
plants that possess the N . tabacum genome in cytoplasm from N . repanda.
Furthermore, that restorer chromosome organizes the nucleolus and inhibits
the nucleolus-forming activity of the nucleolar organizers of N . tabacum
chromosomes present in the same cells, particularly in pollen mother cells. To
determine whether these relations are basic or only coincidental, restorer
chromosomes for other cytoplasms are now being investigated. The present
paper describes a study of a chromosome, presumably derived from N . debneyi,
with partial restoring power. Acting in the cytoplasm of N . debneyi, it directs
formation of morphologically normal anthers, without, however, restoring
pollen fertility. W e find that this chromosome also has a functioning nucleolar
organizer, but only slightly inhibits the nucleolus-forming capacity of N .
tabacum chromosomes. The suggestion of a relationship between the nucleolar
apparatus and restoration of normal anthers is thus strengthened by the observation that restorers are found on nucleolus-forming chromosomes from two
very distinct Nicotiana species, as well as in several comparable cases cited
from the Triticinae. The manner in which the nucleolus, or its organizer, may
direct defeminization and restoration of anther morphology is not known;
BURNS
suggestions were offered in the preceding paper in this series (GERSTEL,
and BURK1978).
CYTOPLASMIC male sterility may result when the chromosomes of one
species are substituted by successive backcrosses in the cytoplasm of another. The history of hybridization in Nicotiana ( SMITH1968) and other higher
plants abounds with examples (GRUN 1976). Reintroduction of a specific
chromosome from the donor of the cytoplasm may restore male fertility. One
such case has been analyzed cytologically (GERSTEL,BURNS and BURK1978)
where the combination of Nicotidna repanda cytoplasm and N . tabacum chromPaper number 5549 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North
Carolina 27650.
a New York State Department of Health; Orchard Park Laboratories.
Genetlcs 90: 151-159 September, 1978
152
J. A. BURNS, D. U. GERSTEL A N D S. A. SAND
osomes was found to be male sterile, with the anthers replaced by pistillate
structures. Normal anther development and fertility could be restored by the
addition to the complement of a chromosome that was thought to have been
derived from N . repanda. This chromosome was highly modified; it was very
small, of fragment dimensions, and carried nucleolar organizers and satellites at
each end. Furthermore, this chromosome, when present, considerably reduced
the ability of the satellited chromosomes from N . ta8acum to form nucleoli; i.e.,
it exerted amphiplasty to use the term coined by NAWASCHIN
(1928).
The observed relation between fertility restoration and nucleolar organizer
could be coincidental, or it may be a functional one; i.e., development of uncommitted primordia into male structures may be controlled by nucleolar
organizers or by nucleoli. One way of attacking this question is to investigate
the frequency of such associations between restorers and nucleolar organizers in
other male-sterile systems.
A second cytological study will be reported here involving the male sterility
engendered when the chromosomes of N . debneyi Domin are replaced by those
of N . tabacum L. in N . debneyi cytoplasm. The male sterility that occurs in this
system, symbolized by [Deb] tbc, has been described by several investigators
(CLAYTON
1950; SAND1960; CHAPLIN1964; TSIKOV
and YANAKIEVA
1967;
NIKOVAand TSIKOV
1976), but only SAND(1968) and SANDand CHRISTOFF
(1973) have addressed the problem of fertility restoration and developed the
material necessary for the present work.
N . debneyi is an Australian species of the Section Suaveolentes (GOODSPEED
1954). In the present context it is important that N . debneyi is only distantly
related to South American N . repanda Willd. (Section Repandae) ,the subject of
the previous study. Significant differences exist between male sterilities in the
cytoplasms of N . repanda and N . debneyi. For the [Rep] tbc system, only two
forms were described by BURKand MANN(1970). Plants lacking restorers had
slightly modified corollas and abortive and feminized stamens, whereas the restored plants had normal stamens that produced functional pollen. In the [Deb]
tbc male sterility system, on the other hand, SAND(1968) noted extensive segregation. SANDand CHRISTOFF(1973) selected five distinct types, ranging stepwise from plants with highly abnormal split corollas and stigmas in place of
anthers (HICKS, BELLand SAND1977; HICKSand SAND1977) to those with
normal sympetalous corollas and nearly normal stamens. All were functional
females; however, even the most nearly restored type produced no functional
pollen.
The genetic data obtained by SANDand CHRISTOFF
(1973) wereconsistent with
the assumption that the most extreme male sterile type (IA) had only the
chromosomal complement from N . tabacum in N . d e b n q i cytoplasm, whereas
the more or less restored forms (lB, 2C, 3E, and 4H) possessed additional single
chromosomes derived from N . debneyi, either unchanged or modified. To simplify matters initially for this cytological study, we have neglected the interm-diate types lB, 2C and 3E and concentrated on an analysis of the restorer
chromosome in the most normalized phenotype 4H. Since the pollen of 4H plants
M A L E STERILITY A N D N U C L E O L U S
153
is aborted, we use the term restoration here in a restricted sense to mean normalization or defeminization of anther morphology.
MATERIALS A N D METHODS
The male-sterile lines were originally derived from an amphidiploid hybrid synthesized from
N . debneyi as female and N. tubacum cu. Kupchunos as male parent. The various male-sterile
forms mentioned in the preceding paragraph occurred as segregants in subsequent backcross
generations with Kupchunos tobacco as recurrent male parent and have been previously illu1973. Two families were employed in the present investigation.
strated (SANDand CHRISTOFF
One was an advanced backcross line (BC,) that segregated into the extreme male sterile type 1A
and the most highly restored form 4H.The other family was inbred cu. Kupchunos. Seeds of
both families were sown on wet filter paper; all germinants were transplanted to seedling pots
and finally to nursery rows near Raleigh, North Carolina.
The cytological techniques employed were described by GERSTEL,
BURNSand BURK (1978).
Pollen mother cells (PMC’s) were collected in the field for observations of diplotenes and metaphases. Corollas were also taken in the field. For the study of root tips, plants were established
in the greenhouse. For each cell type, i.e., diplotenes, meiotic metaphases and somatic metaphases
from corollas and from root tips, fixations were made on the same day for direct comparisons.
RESULTS
From the cross 4H ( 0 ) x Kupchunos, 220 seeds produced 176 mature plants,
of which seven were of the 4H type and the rest IA. Transmission of the restored
condition was thus very low, i.e., 4.0%, but comparable to the average 2% reported by SANDand CHRISTOFF(1973) for several larger trials. These authors
also had recovered intermediate types among progenies of 4H, but no intermediates were obtained in this sample.
The course of meiosis proceeded normally in the restored 4H-type plants and
chromosomes were counted in six of the seven. They all possessed one extra
medium-sized chromosome, in addition to the normal 2n = 48 of N . tabacum.
At metaphase of meiosis the extra chromosome remained unassociated (Figure
1); of a total of 55 cells, 52 had 24, f 1, and three cells exhibited 2311 4-31. Mitotic chromosomes were counted in three of these plants; their corolla metaphases had uniformly 49 chromosomes (total of 30 cells). Type 1A sister plants
lacked the extra chromosome. Since this type does not produce anthers, only
somatic cells were counted. Five corolla metaphases scored from each of six 1A
plants uniformly exhibited 48 chromosomes.
The information gained from the cytological observation that all six of the
analyzed 4H restorer plants carried the extra chromosome, when added1 to the
previous indication from the genetic data of SANDand CHRISTOFF(1973) , leads
to the conclusion that the extra chromosome is necessary for restoration.
The relation of the extra chromosome to nucleoli was studied at diplotene in
P M C ’ s and in root tips. Scoring at diplotene had of necessity to be selective, insoBy the use of Bayes’ formula (ANDERSON
and BANCROFT
1952) the a priori degree of belief in the necessity of the
extra chromosome for restoration can be combined with the cytological evidence to arrive at a new Q posteriori probability or degree of belief. Even a moderate initial degrse of belief of 2/3 leads to an Q posteriori value of 0.93, while an
initial degree of belief of 0.9, based on the breeding data, leads to an a posteriori value of 0.98. (The authors are
for this analysis).
grateful to H. E. SCHAFFER
154
J. A. B U R N S , D. U . GERSTEL A N D S. A. S A N D
far as at this stage, in N . tabacum, the chromosomes are often too crowded for
analysis. However, many clear cells could be obtained, and Table 1 shows the
results. In all 53 cells analyzed from four plants of the 4H type with 24, +ll;
the univalent was joined to the nucleolus (Figure 2); the connection was often
drawn out by the pressure exerted during slide preparation, indicating a firm
attachment and not just an overlap. The univalent, which presumably is the
carrier of the restorer gene(s) and was derived from N . deb’neyi, is thus consistently involved in nucleolus formation.
The behavior of the paired N . tabacum chromosomes was variable in 4H
plants (Table 1 ) . I n many cells, two bivalents were attached to the nucleolus,
corresponding to the number of nucleolus-forming chromosomes normally found
1954; GERSTEL,BURNS and BURK (1978). Frein N . tabacum (GOODSPEED
quently only one N . tabacum pair was associated with the nucleolus, sometimes
none. In the controls, i.e., cu. Kupchunos, the proportion of attached pairs appeared to be larger ( P < 0.05). These observations show that the univalent,
when present, is not the sole organizer of nucleoli and does not completely suppress nucleolus formation by other chromosomes. However, the restorer chromosome may exert a weak degree of amphiplasty, since the number of nucleolusassociated bivalents was reduced in plants carrying it.
Usually only a single nucleolus was present in diplotene cells. There were a
few exceptions to this in the Kupchunos material. Five cells had one large and
one very small nucleolus, each attached to a separate pair. One unusual cell
had three nucleoli, one large and two small ones, joined to three chromosome
pairs.
Root-tip metaphases were studied for the purpose of observing the morphology
of the satellited chromosomes and to obtain further evidence on amphiplasty.
Kupchunos and 1A exhibited two pairs of satellited chromosomes, which resemBURNSand BURK (1978).
bled those of cu. Red Russian described by GERSTEL,
These were the F chromosomes and an undetermined chromosome pair “X”.
However, the F satellites of the present material were much smaller, and the
X satellites had much shorter stalks. The two 4H plants studied possessed a fifth
satellited chromosome ( d ) which was distinctly different, being shorter and
more nearly metacentric than either the F o r X ; it had a large satellite on a
short stalk (Figure 3 ) .
A rough method was thought satisfactory to determine whether amphiplasty
TABLE 3
Associations between chromosomes and nucleoli at diplotene*
TYPO
4H
Kupchunos
Number Number
plants
cells
4
3
53
29
Univalent
Attached
Free
2 attached
Bivalents
1 attached None attached
____
-~
- -~
53 (100.0)
0 (0.0)
22 (41.5) 21 (39.6)
___-
-
~
-
U)
(69.0)
* Figures in parentheses are percentages. x2 = 6.849; d.f. = 2; P
8 (27.6)
< 0.05
10 (18.9)
1 (3.4)
(for bivalents).
MA1.E STERII.ITY A N D NUCI.EOLUS
FIGURE1.-Meiotic metaphase of restored 4H plant with 2$,
valent. (Photomicrograph, x 1800).
FIGURE2.-Diplotene
155
+ ll. Arrow designates uni-
of 4H. The restorer univalent and one pair are attached to nucleolus
(n). (Photomicrograph, x 1800).
occurred in root tips (Table 2). The satellite stalks were divided by the lengths
of the adjacent short arms by visual estimation. Only small differences could be
observed for the average lengths of the satellite stalks of the I: chromosomes of
Kupchunos, 1A and 4H (2 plants). The stalks of the X chromosomes of all three
156
J. A. B U R N S , D. U. GERSTEL A N D S. A . S A N D
TABLE 2
Means of the ratios of satellite stalk lengths to short arm lengthst
“X”
P
Kupchunos
1A
4H-plant 1
4H-plant 2
1.8 +. 0.17
1.8 +- 0.14
2.1 k 0.12
2.0 & 0.15
0
0
0
0
Restorer
0.7 0.16
0.6 & 0.11
* Twenty-five cells were scored for each of the four plants listed, each containing two F and
two X chromosomes. Only one restorer chromosome was present per cell in the two 4H plants.
The data were obtained by dividing visually the length of each stalk by the length of the adjacent
short e m . Mere constrictions, o r their absence, were scored as zero.
types were very short and sometimes even no constriction was visible, so that
the X chromosomes could not always be identified. They were all scored as zero.
The short satellite arm of the restorer chromosome ( d ) was longer than that of
either the F or the X , which contributed to the small ratio of stalk/arm.
This method, though only approximate, established that the satellited chromosomes of N . tabacum were involved in nucleolus formation in root-tip cells, even
in the presence of the extra chromosome from N . debneyi. The observation of
satellite stalks at metaphase may be taken as evidence that the chromosome had
been associated with a nucleolus at the preceding stages (see MCCLINTOCK
s
a
__0
*
d
3
FIGURE
3.-Root-tip metaphase of 4H showing the five satellited chromosomes. Lettered are
F and “X” pairs and d restorer chromosome. Dotted lines represent faintly visible satellite stalks.
(Camera lucida,
x 2100).
MALE STERILITY AND NUCLEOLUS
157
1934). Thus, while there was evidence for amphiplasty in anthers, root tip cells
did not express the phenomenon. It may be noted that in the previous study of
the restorer chromosome from N . repanda ( GERSTEL,
BURNSand BURK1978) a
pronounced degree of amphiplasty was observed in PMC’s, but the expression in
root tips, based on stalk length comparisons, was only weak.
DISCUSSION
Evidence that the extra chromosome comes from N . deb’neyi, the original female parent, appears quite convincing. No chromosome from N . tabacum has
been found to restore anthers during tests that have included a series of 25 backcrosses of type 1A to N . tabacum (SANDand CHRISTOFF1973). Second, the
extra chromosome is morphologically unlike any satellited chromosome from
N . tabacum (Figure 3), and lastly, the extra chromosome does not associate at
meiosis with the N . tabacum chromosomes, as shown.
The suggestion that there may be a functional relation between nucleolar
organizer and restorer is strengthened by this second example from Nicotiana,
particularly since N . debneyi and N . repanda are only distant relatives. Furthermore, in Triticum and related taxa, TSUNEWAKI
(1974) has shown that restorer genes are frequently, though not always located on satellited, nucleolusforming chromosomes. Close proximity of restorer to satellite was demonstrated
in the following two instances. In one wheat example, loss of a satellite was assciated with the loss of a restorer gene (TSUNEWAKI
1974). In the case with N .
repanda cytoplasm, the restorer chromosome with two satellites was a small
fragment, such that the restorer could not be far from either satellite.
We must point here to some differences between the Nicotianas and the wheat
group. In the wheats, male sterility generally affects only the pollen and is expressed after meiosis, whereas in Nicotiana the stamens are feminized and
aborted. The observation that the nucleolus may be involved in the restoration of
both types suggests that a related pathway may be affected in both forms of
male sterility. The difference could be one of timing. In the [Deb] tbc system,
anther development is nearly normal when the satellited extra chromosome is
present, but this does not suffice to restore the pollen in this case. Something
may be missing, possibly another element from N . debneyi, which could also be a
satellited chromosome, of which N . debneyi has two or possibly three (GOODSPEED 1954). In the sesquidiploid, i.e.,the first backcross of the amphidiploid N .
debneyi x N . tabacum to N . tabacum, which still possesses an intact complement of N . debneyi chromosomes, fertile pollen grains are formed, and an additional element for pollen restoration may be found there ( SANDand CHRISTOFF
1973). That post-meiotic pollen abortion in the 4H type may be due to an unfavorable interaction of N . tabacum chromosomal factors with the cytoplasm of
N . debneyi is less likely in view of the male fertility of the amphidiploid and
sesquidiploid generations.
It was perhaps fortunate that we observed pronounced amphiplasty in the
first Nicotiana case examined and that there, in analyzable PMC’s, only the re-
158
J. A. BURNS, D. U. GERSTEL A N D S. A. SAND
storer appeared to be associated with the nucleolus; thus the importance of the
nucleolus, o r the organizer, for male development was dramatically demonstrated. As the present case shows, exclusion of N . tabacum chromosomes from
nucleolar organization may not be the significant phenomenon, but rather the
contribution of the restorer chromosome. N . tabacum chromosomes may participate in formation o i the nucleolus, but their contribution must be neutral as
far as the restoration process is concerned.
The manner in which the nucleolar organizer, or the nucleolus itself, may
interact with cytoplasmic organelles to direct defeminization nad male development is not known. Suggestions were offered in the preceding paper (GERSTEL,
BURNSand BURK1978) and will not be repeated here.
One more point deserves to be mentioned. Male sterility occurred in the examples given when the cytoplasm of one species was combined with the chromosomes of another and restoration required the addition of a satellited chromosome from the cytoplasmic donor. In corn, the common restorers Rf,, Rfa and Rfs
are located on chromosomes other than the satellited sixth chromosome (DUVICK
1965; LAUGHNAN
and GABAY1975). This distinction may be based on differences in the origin of cytoplasmic male sterility, which in maize may be caused
by disruptive mutations in the cytoplasm rather than by introduction of an established alien cytoplasm (though the origin of the various types of cytoplasm
causing male sterility in maize may be a moot question, as DUVICK1965, has
mentioned). The resulting damage may be reparable by compensatory mutations somewhere in the maize genome. Of interest in this context is the sugand GABAY(1975) that newly arisen nuclear restorers in
gestion of LAUGHNAN
maize may be of the nature of episomes, a point which recently found support
in the molecular studies of PRING
et al. (1977). LAUGHNAN
and GABAY(1975),
furthermore, proposed that such restorer elements may become integrated at
various chromosomal sites. On the other hand, in Nicotiana and wheat, the
effect of an alien cytoplasm can be repaired by a specifically located chromosomal
element that is a normal component of the same alien species.
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Corresponding editor: N. W. GILLHAM