CYTOGENETICS OF FLOWER MODIFICATION OF A CYTOPLASMIC MALE-STERILE TOBACCO1

D. U. GERSTEL AND J. A. BURNS

Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27650

AND

S. A. SAND

Plant Genetics Section, Roswell Park Memorial Institute2 Orchard Park, New York 141 27

Manuscript received February 1,1980 Revised copy received May 19, 1980

ABSTRACT

Plants combining the cytoplasm of Nicotiana debneyi and the 48 chromo- somes from N . tabacum are male sterile. Early backcross generations of the amphidiploid hybrid to male N . tabacum produced a great variety of plants from which a series of phenotypes with characteristic flower forms and trans- mission rates have been isolated. Type 1A possesses completely feminized stamens and deeply split corollas, breeds true when backcrossed to normal males and carries 48 N . tabacum chromosomes. Other phenotypes, 2C, 3E and 4H, range toward normal morphology of corollas and stamens. Like lA, 2C forms no anther tissue and has 48 chromosomes. This type is transmitted to 36.3% of the backcross progeny, the remainder being of type 1A; presumably 2C carries a chromosome segment from N . debneyi that is responsible for the partial restoration of flower structure. In contrast, both 3E and 4H produce anthers and possess an extra chromosome. The extra chromosomes are trans- mitted to only 19.9% and 7.1% of the progeny, respectively. Significantly, the extra chromosomes found in the anther-forming types are nuclcolus or- ganizing and carry a satellite froin N . debneyi. On the basis of these ob- servations, we surmise that differentiation of anthers in plants with N . debneyi cytoplasm may depend on the presence of a nucleolus-organizing chromo- s3me from that species. This chromosome is unstable; unaltered, it conditions a highly restored phenotype (4H), but when structurally modified, it may control different phenotypic expressions. Other examples of satellited restorer chromosomes had been reported for different cytoplasmically male-sterile combinations; therefore, the phenomenon may have more general significance.

OMBINING artificially the cytoplasm of one species with the nucleus of another frequently results in male sterility (EDWARDSON 1970; GRUN 1976).

LACADENA (1968) has called the sterility thus produced alloplasmic male steril- ity. For the genus Nicotiana, SMITH (1968) and GERSTEL (1980) reviewed nu- merous cases in which substitution of the chromosomes of N . tabacum (tobacco)

1 Paper number 6274 of the JoumaI Series of the North Carolina Agricultural Research Service, Raleigh, North

3 New York State Department of Health, Orchard Park Laboratories. Carolina 27650. Aided by a grant from the North Carolina Tobacco Foundation.

Genetics 96: 223-235 September, 1980

224 D. U. GERSTEL, J. A. BURNS AND S. A. SAND

in the cytoplasm of another species led to staminal sterility with more-or-less pronounced abortion of the stamens. Frequently, malformation of the androe- cium entails feminization of the stamens, which become tipped with stigmatic structures (CHAPLIN 1964; HICKS, BELL and SAND 1977; NIKOVA and TSIKOV 1976; SAND 1968; SAND and CHRISTOFF 1973) and may even form external ovules ( GERSTEL, BURNS and BURK 1978).

The original hybrid usually was an amphidiploid having a nontabacum species as the female parent, and male steriles were then extracted and genetically puri- fied by means of successive backcrosses to N . tabacum as the recurrent pollinator. Generally, only the forms with the most abnormal anthers have been isolated in the search for male-sterile tobaccos. The transmission of male sterility, unaltered through many generations, and its disappearance in reciprocal crosses, when they could be made, provided the major evidence for cytoplasmic control of the male sterility syndrome.

In a number of cases, diverse intermediate forms, with less abnormal anthers, also occurred in the early backcross generations. Examples are the series with the cytoplasms of N . debneyi and N . megalosiphon (CLAYTON 1950), N . bigelovii and N . repanda (BURK 1960) and N . benthamiana (RAMAVARMA, APPARAO and NARAYANAN 1978). We have made similar observations among the derivatives of hybrids with the cytoplasms from N . gossei and N . undulata (unpublished). Such complexities are thus common and probably typical of systems of this kind. It may reasonably be supposed that anther abortion is incomplete as long as specific chromosomes or chromosome segments from the nontabacum parent are retained in conjunction with the cytoplasm of the same parent (SAND 1968, 1978). This need not exclude additional effects imposed by the environment, modifications dependent upon genes of the particular N . tabacum cultivar em- ployed as pollen parent, or alterations arising in the cytoplasm (BELLIARD, VEDEL and PELLETIER 1979; SAND 1978,1979).

In only one reported case has a series of modified phenotyes been systemati- cally selected and their inheritance studied. SAND (1968) , who undertook the investigation in a search for genetical restorers, chose the backcrosses from the amphidiploid of N . debneyi and N . tabacum. The same inbred line of N . taba- cum served as the recurrent pollen parent in each generation.

From the offspring, SAND and CHRISTOFF (1973) selected a number of distinct heritable types. The most abnormal one has terminal stigmas subtended by stig- matic lateral outgrowths instead of anthers. Furthermore, corollas are deeply split nearly to the base. Inheritance of this phenotype, named 1 A, is cytoplasmically controlled and persists unaltered through many backcross generations, during which the N . tabacum nucleus is substituted in N . debneyi cytoplasm. At the other end of the spectrum, a type with anthers of normal appearance and fully restored tubular corollas was isolated; the pollen, however, is aborted after meiosis. This 4H type segregates; when used as the female parent, it gives a low frequency of 4H offspring, with most of the siblings being 1A. Types 2C and 3E, also segregating in backcrosses, are intermediate in both anther development and the degree of splitting of corollas. Types IA, 2C, 3E and 4H represent more-or-

MODIFICATION O F MALE-STERILE TOBACCO 225

less stepwise changes toward normalcy, expressed in both the stamen and corolla whorls (for genetic details and illustrations, see SAND and CHRISTOFF 1973). There exists a qualitative dichotomy with respect to stamen structure; neither 1A nor 2C show any trace of anthers, and their filaments terminate in stigmatic structures; whereas, 3E and 4H possess anthers, much malformed in 3E but ostensibly normal in 4H.

In a preceding paper (BURNS, GERSTEL and SAND 1978), we described the chromosomal differences existing between 1A and 4H. The present report ex- tends the earlier cytological observations and provides chromosomal analyses of the types 2C and 3E.

MATERIALS AND METHODS

Seed samples came from advanced backcross generations of lines developed by S. A. SAND. The ancestral hybrid plant was an amphidiploid synthesized from N . debneyi Domin (2n = 48) used as female and N . tabacum L. (2n = 48) cv. Kupchunos (see pedigree in Figure 1). The same inbred cv. Kupchunos, a Connecticut Broadleaf type, served as the recurrent male parent in the succeeding backcrosses. Specification of the cultivars employed in studies of nucleolar or- ganizers is necessary because we find that the dimensions of the nucleolar organizing regions (NORs) vary among tobacco varieties.

Two plants with the 4H phenotype (nos. 27 and 19) were among those selected at BC,. The pedigrees developed from these 2 plants are shown in part in Figure 1. At BC,, the line on the left was split again; 1 lineage was carried to BC,, when cytological determinations were made. (This family, S73300, was the one investigated earlier by BURNS, GERSTEL and SAND 1978.) In the BC, generation, a variant plant with the 2C phenotype was selected and carried to BC,, (family S75314), when chromosomes of that line were analyzed in the present study. A second 4H line was also studied cytologically in the BC,, generation (family S77274).

In a second branch of the pedigree, another 2C variant and 2 3E types were selected in BC, and carried to BC,, (families S75313, S75317 and S75318, respectively), when cytological studies were undertaken. The chromosomes from representative sib plants of the 1A type were studied in all 6 families.

Plants were raised in the field at Raleigh, N.C., from seed produced at Orchard Park, N.Y. Corollas were prepared for chromosome counts. Metaphases of pollen mother cells (PMCs) were used for the same purpose and for analyses of meiotic pairing. Associations between spe- cific chromosomes and nucleoli were observed at diakinesis. Root tips are the material of choice for the study of NORs and were generated from cuttings rooted in the greenhouse.

The cytological techniques were those routinely employed in our laboratories (BURNS 1964; GERSTEL, BURNS and BURK 1978). Counts of chromosome numbers were based on 5 or more well-spread corolla cells per plant. Chromosome morphologies, i.e., arm lengths and satellites, were generally studied in 20 root-tip cells per plant in which the chromosome numbers were checked again. Numbers of PMCs used for meiotic studies are given in the text.

RESULTS

Transmission ratios: SAND and CHRISTOFF (1973) studied the segregation ratios of the various types over eight backcross generations and observed char- acteristically different transmissioc frequencies for each type. Type 1A never segregated. The ratios obtained for the partially restored types 2C, 3E and 4H in the combined 1978 and 1979 plaiitings are listed in Table 1. These ratios are similar to those recorded previously, though the maternal phenotypes occurred with somewhat higher frequencies than reported before. Differences were ob-

226 D. U. GERSTEL, J. A. BURNS AND S. A. SAND

4n (N. debneyi X N. tabacuml X N. tabacum

I

- , ( # lo )

12 S75314 S77274 S75313 S75317 S75318 2C line 4H-2 line 2C line 3E line 3 E line - - - - -

FIGURE 1 .-Pedigree of lines examined cytologically. Numbers in parentheses are plant numbers. Plant numbers in the same row represent sibs.

served between similar lines (last column), but were nonsignificant by chi- square tests. Combined, the 2C lines gave 36.3% 2C offspring; the 3E type segre- gated 19.9% 3E, and 4H gave 7.1 % of that type. The major class in every case was again 1A.

In addition to the respective maternal types, 3E and 4H have each produced in many backcross generations plants resembling the more feminized types, but at very low frequencies (SAND and CHRISTOPF 1973; SAND 1978). In generations BC, to BC,, raised at Orchard Park and among a total of 2,486 plants, 4H has given about 1.6% offspring resembling 2C or 3E, and 3E types yielded about 1.8% 2C-like progeny (SAND 1979). Similarly, rare 2C and 3E phenotypes occurred also in the 4H cultures grown at Raleigh (Table 1).

Chromosomal classes: The chromosome complements of 1A and 4H types segregating in the BC, generation (family S73300) have been previously de- scribed (BURNS, GERSTEL and SAND 1978). The chromosomes of 1A were indis-

MODIFICATIOh’ OF MALE-STERILE TOBACCO

TABLE I

Segregution frequencies.

22 7

P w n y phenotypes “I

Maternal plienntjpe Families 1A 2C 3E 411 Om-types Total ,o

2C S75313 55 31 - - - 86 36.05 (2C) S75314 24 14 - - - 38 36.84 (2C)

3E 875317 80 - 25 - - 105 23.81 (3E) S75318 65 - 11 - - 76 14.47 (3E)

4H S73300 264 1 - 15 1 281 5.34 (4H) 877274 229 - 1 2 3 1 254 9.06 (4H)

Data are from combined 1978 and 1979 field cultures. They represent BC,, generation sam- ples, with the exception of family S73300, which was taken a t BC,. Preceding backcross genera- tions gave similar, though somewhat lower, frequencies of the maternal phenotypes (SAND and CHRISTOIT 1973). The differences in transmission between families of similar phenotypes are nonsignificant.

Backcrosses of type 1A have given uniform IA progenies without segregation in any genera- tion (not tabulated).

tinguishable from those of the Kupchunos parent, as expected on the basis of the breeding results. Plants of both types have the normal tobacco complement of 48 chromosomes with two pairs of satellited, nucleolus-organizing chromosomes (Figure 2). (A third pair of satellites may exist in tobacco (GOODSPEED 1954), but we have not observed it in cv. Kupchunos.)

Type 4H plants from family S73300 possessed 49 instead of 48 chromosomes. In somatic metaphases, the extra chromosome resembled one of the satellited chromosomes of the N . debneyi complement, as can be seen by comparing Figure 2d and f. At meiotic metaphase, 4H plants usually formed 24” + 1’ and rarely 23” + 3’; failure to pair indicated that the extra chromosome was of alien origin.

t b c deb 3E AH-1 AH. 2

I

r X

a b c d

2

e f 9

FIGURE 2.-Photomimgraphs of the satellited chromosonies from mot tips: (a) and (b) from the N. tubacum cv. Kupchunos complement: (a) was identified as the F chromosome ( C u u s w and CAMERON 19W); the other (b), unidentified, will be called “X.” (c) and (d) illustrate the satellited chromosomes of N. debneyi. (e) represents the extra chromosome of the 3E phenotypes. (f) is from 4H-1 and ( g ) is the doubly satellited chromosome from 4H-2.

228 D. U. GERSTEL, J. A. BURNS AND S. A. SAND

In analyzable meiotic prophase cells, the extra chromosome was regularly as- sociated with the nucleolus. This relationship is consistent with the hypothesis that the NOR may play some role in anther development and that, for anther restoration, the NOR must match the cytoplasm and be derived from the same species ( GERSTEL, BURNS and BURK 1978).

In the following, we shall expand the observations on plants of the 4H pheno- type and then describe types 2C and 3E. The results are summarized in Table 2.

4H segregants of family S77274 also had 49 chromosomes like 4H plants from S73300. Six plants were analyzed. They exhibited a very unusual extra chromo- some possessing satellites at both ends. The short satellited arm was similar to that of the extra chromosomes of the previously analyzed 4H type, but the long arm was visibly longer and carried a second satellite (Figures 2g and 3 ) . In dia- kinesis preparations, both ends of that chromosome tended to be attached to the nucleolus (Figure 4). In meiotic metaphases, 24 pairs and a univalent were usu- ally found, though rare cells exhibited 23” f 1’” (in 5 of 60 cells). To distinguish between the chromosomally different 4H plant types from families S73300 and S77274, the former are called 4H-1 and the latter 4H-2. Two 1A segregants chosen from family S77274 had only the genome of N . tabacum, like the 1A sibs from S73300 analyzed in the earlier investigation.

Chromosomes from 3E segregants of two families were first cowted in corolla preparations; five plants of family S75317 and four from S75318 each possessed 49 chromosomes, but one plant in S75317 had 48. Cuttings were then rooted from six plants with 49 chromosomes (three from each S75317 and S75318) and from the plant with 48 chromosomes. All seven plants were found to possess a satellited extra chromosome in addition to the satellited chromosomes char- acteristic of cv. Kupchunos. The exceptional plant with 48 chromosomes was therefore monosomic for some unidentified chromosome of the N . tabacum complement, but had the distinctive satellited extra chromosome. The extra chromosomes from both lines looked alike; one example is shown in Figure 2e. As controls, chromosomes were counted from 1A type sibs segregating from S75317 and S75318 (five plants each). All had 48 chromosomes,

TABLIS 2

Chromosome complements of the plant types with N. debneyi cytoplasm

Number of Phenotype Fa m 11 i e s Anthers’ chromosomes Extra chromosomes ___ _ _ _ _ _ _ _ _ _ ~ ~

4H- 1 S73330 normal, but sterile 49 satellited 4H-2 S77274 normal, but sterile 49 double satellites 3E S75317 malformed 491- satellited 3E S75318 malformed 49 satellited 2 c S 753 1 3-4 no anthers 48 - 1A All 6 lines no anthers 48 -

* For detailed illustrated descriptions of the stamen types see SAND and CHRISTOFF (1973). -f One exceptional plant had 48 chromosomes; but the missing one was not the satellited extra

chromosome (see text).

MODIFICATION OF MALE-STERI1.E TOBACCO 229

Lb a = d I:IGURE 3.-(a) Root-tip cell of a plant of type 4H-2 in which two satellited F chromosomes

of tobacco and the doubly satellited restorer chromosome (labeled 4H-2) can be identified. The plants also possess a second pair of sntellited tobacco chromosomes (“Xs”; see Figure 2b), but these are not often recognizable in cells with highly contracted chromosomes. ( X 2500). (b), (c) and (d) are more highly enlarged parts of three cells from type 4H-2, showing the restorer chromosome with two satellites. (d) is from the same cell as 2g; (b) and (c) are from different cells.

Meiotic analyses of 3E plants can be performed only with difficulty because the teratological anthers produce few PMC’s and then not a t all times. Many PMCs were highly abnormal and some cells contained more than 100 pairs of chromosomes in addition to a few univalents. In normal PMCs, typically, the extra chromosome was unpaired. This was true for all 30 metaphase cells counted from one plant of S75318. However, two plants from S75317 formed trivalents (in 3 of 16 and 4 of 30 cells). These data were obtained in the winter season, but could not be confirmed because no analyzable PMCs could be found in the following summer. The question of a structural difference between the extra chromosomes of the two 3E families remains in abeyance.

A total of five clear cells in diakinesis could be observed in PMCs from one 3E plant of each of the two families; in all cells, the extra univalent was attached to

230 D. U. GERSTEL, J. A. BURNS A N D S. A. S A N D

4

FIGURE 4.-Diakinesis with 24"+1' from a 4H-2 plant showing association of the univalent restorer chromosome with the nucleolus at lower left. ( x 2500).

the nucleolus. This observation, together with the finding of a satellited extra chromosome in root tips, suggests that the extra chromosomes of 3E possess a functional NOR.

Plants of type 2C were studied from two families (four from S75313 and six plants from S75314). All corolla cells examined had 48 chromosomes and none possessed an extra chromosome. Root tip cells from cuttings of three of the plants did not reveal any satellites other than those found in cv. Kupchunos. PMCs from type 2C could not be obtained, of course, since the plants had no anthers.

The earlier observations that the chromosomes of 1A plants are indistinguish- able from those of cv. Kupchunos were confirmed by analyzing corolla cell chromosomes from five 1A segregants in each of the families S75313 and S75314. The two minority types obtained in the 4H-2 family S77274, listed in Table 1,

were cytologically analyzed. One was classified as type 3E on the basis of corolla conformation and the shape of the aberrant anthers. This plant had only 48 chromosomes, but one chromosome was evidently derived from the extra chro- mosome of the 4H-2 parent. I t carried the extra satellite distinguishing the short arm of that chromosome. The satellite on the long arm was deleted.

MODIFICATION O F MALE-STERILE TOBACCO 23 1

The second plant was scored as “off-type” since it differed from all other plants observed in this study. The corollas were intermediate between classes 2 and 3, while the anthers were replaced by stigma-tipped petaloid structures. NO anther tissue was evident. This plant had 48 chromosomes plus a very small frag- ment without satellite, probably a derivative from the extra chromosome of the 4H-2 parent.

The satellited chromosomes: Two pairs of satellited chromosomes can be seen in Kupchunos tobacco. One very small satellite, usually set off by a long con- striction or stalk, characterizes the F chromosome, as located by CLAUSEN and CAMERON (1944). A much larger satellite with a very short constriction can be recognized in some cells on an as yet unidentified chromosome to be called “X.” N. debneyi has two pairs with satellites that are clearly distinguishable from those of N . tabacum by their intermediate sizes and stalk lengths, as well as by the lengths of the adjacent arms. As shown in Figure 2, the satellited chromo- somes of N . tabacum have small short arms; whereas, one satellited chromosome of N . debneyi is submetacentric and the other acrocentric.

As mentioned, the extra chromosome of 4H-1 (Figure 2f) is indistinguishable from one of the satellited N . debneyi chromosomes (Figure 2d), while the extra chromosome from 4H-2 (Figure 2g) has a distinctly longer long arm bearing a second satellite. This satellite is of the same small size as that of the F chromo- some. The extra chromosomes from the two 3E lines are indistinguishable (Figure 2e). They differ from the extra chromosome of 4H-I, and the similar N . debneyi chromosome, in having a somewhat smaller short arm and a slightly longer long arm.

The satellites of the short arms of the extra chromosomes of both 4H types and of the 3E plants are of the same size and resemble the N . debrzeyi satellite shown in Figure 2d. On the other hand, the satellited short arms from 3E and the 4H types differ perceptably from those of the two satellited N . tabacum chromo- somes and the satellited acrocentric of N . debrzeyi (Figure 2) .

DISCUSSION

The restorer chromosomes: The presence of satellites on the extra chromo- somes in plants of the types 3E and 4H that possess anthers and the absence of extra satellites from the antherless types (IA, 2C and the “off -type” plant) may be the most interesting findings of this study.

The chromosomes of the 1A and 2C types cannot be distinguished from a normal N . tabacum complement at the degree of resolution afforded by carmine- squash technique. This is not unexpected for IA, since the genetical evidence in- dicates that in 1A an intact N . tabacum genome is substituted in N . debneyi cy- toplasm. For type 2C, the genetical data and the selection procedure required for its maintenance make it probable that a chromosomal segment from N . debneyi has introgressed into the N . tabacum complement. The size and the location of that alien segment are unknown; nucleolar organizers do not seem to be involved. The phenotypic effect of that segment, observed in heterozygotes, is a relatively minor modification of the stamen and corolla whorls.

232 D. U. GERSTEL, J. A. BURNS A N D S. A. SAND

Restoration of anthers, whether malformed as in 3E or of normal appearance as in 4H plants, may require the presence of a NOR derived from a chromosome of the cytoplasm donor. The evidence presented here strengthens that provided by the early example studied by GERSTEL, BURNS and BURK (1978). There, res- toration was achieved by introducing a satellited chromosome from N . repanda into the cytoplasmically male-sterile form possessing the cytoplasm from N . repanda and the chromosomes of N . tabacum. Significantly, N . debneyi and N . repanda belong to distinct sections of the genus (GOODSPEED 1954). A prelim- inary study shows that in the male-sterile combination of N . undulata cytoplasm with N . tabacum chromosomes, an anther-restoring chromosome from N . un- dulata is also satellited (unpublished). N . undulata is a member of a third taxo- nomic section. Furthermore, associations between restorer chromosomes and NORs have also been reported by TSUNEWAKI (1974) for alloplasmic male- sterile wheats.

In that previous investigation of plants havicg the cytoplasm of N . repanda, the restorer chromosome exhibited what is known as either amphiplasty or nucleolar dominance. It was then suggested that for restoration the nucleolus might have to be formed exclusively by the alien chromosome (GERSTEL, BURNS and BURK 1978). However, in both the 4H and 3E types, pronounced secondary constrictions are observed in somatic N . tabacum chromosomes and nucleolar associations occur at diakinesis. Thus, it appears that for anther development it is crucial only that the restorer chromosome participate in nucleolus formation; whether NORs from N . tabacum are also active, or suppressed by amphiplasty, is not significant.

It is not known why differentiation of anthers may require the cytoplasm and NOR-bearing chromosomes to come from the same species. That the NOR itself, or its products, may be involved is suggested by an observation of TSUNEWAKI (1974) on wheat, where loss of that region rendered the chromosome ineffective in restoration. The alternative of a close linkage between restorer genes and NORs must also be considered, but then one may wonder why such a linkage has persisted during evolution. In either case, a functional relationship may be implied.

Segregation ratios: The transmission rates for the various types, on the whole, are in accordance with the cytological observations, but there are two discrepan- cies with respect to details. For the 2C type a 1: 1 backcross ratio would be ex- pected if the introgressed segment from N . debneyi is small. Instead, only 36.3% of the progeny were 2C ( P < 0.01). Occasional failure of pairing betweelz the segmentally substituted chromosome and its homologue from N . tabacum, fol- lowed by a loss of that chromosome, could explain the reduction of the 2C class. Alternatively, some adverse selection may occur. A transmission rate of about 20% observed for the 3E type is characteristic of univalents, whether they are from N . tabacum (CLAUSEN and CAMERON 1944) or of alien origin (GERSTEL 1943), but the much lower transmission of 5.3% and 9.1% fo r the two forms of the extra chromosomes of the 4H types remains unexplained.

MODIFICATION O F MALE-STERILE TOBACCO 233

Derivation of the restorer chromosomes: The evidence strongly supports the view that the extra chromosome of 4H-1 plants came from N . debneyi. This chromosome closely resembles the submetacentric satellited chromosome of N . debneyi (Figure 2) and fails to pair with N . tabacum chromosomes. I t also con- tains a biochemical marker from N . debneyi, namely the chromosomal gene coding for a specific polypeptide of the small subunit of Fraction 1 protein that is present in N . debneyi, but not in normal N . tabacum or 1A (CHEN and SAND 1979). Plants that must have had this alien chromosome were first isolated and selected in ~JI early backcross generation following the initial cross with N . debneyi (SAND 1968). This chromosome, which appears to be a primary alien addition type, was then carried along by selection from generation to generation.

SAND (1978) predicted on genetic grounds that structural alterations of the primary extra chromosome may have generated the other genetic modifiers of the basic male-sterile 1A phenotype. The genetical and cytological data now available bear out this prognosis. As discussed in MATERIALS AND METHODS and diagrammed in Figure 1, the various lines of 2C, 3E and probably 4H-2 origi- nated as rare events among the progenies of 4H type plants. The data do not re- veal which 4H type is ancestral, but it is unlikely that the unique extra chromo- some of 4H-2 preceded the N . debneyi-like 4H-1 type. The reappearance of 2C and 3E types in many backcross generations of 4H plants, and of 2C types in 3E lines, are supportive genetic evidence (SAND and CHRISTOFF 1973; SAND 1978). The independent and rare occurrences of these derivatives, with a frequency of less than 2%, indicate that they are “mutants” and not segregants. Once estab- lished, the 2C and 3E lines demonstrated true segregation and yielded 36.3% and 19.9% of 2C and 3E types, respectively.

The evidence suggests that alterations of chromosome structure are the cause of these modifications. Structural instability of chromosomes in interspecific hybrids has often been reported in the Nicotiana literature (AR-RUSHDI 1975; BURNS and GERSTEL 1969; MOAV 1961: and others) and has also been observed in other genera (EHRENDORFER 1959; RUTISHAUSER and LACOUR 1956; WALTERS 1957). The kinds of alterations of the primary alien chromosome that may have occurred can be surmised from their morphology. If the extra chromosome of 4H-2 is derived from that of type 4H-1, as is probable, a reciprocal translocation between the long arm of 4H-1 and another chromosome may have produced 4H-2. The donor of the very small satellite together with an attached segment may have been the F chromosome of the N . tabacum complement.

The extra chromosomes found in both 3E lines are of the same total length as the 4H-I supernumerary, but differ from the latter in the position of the centromere. Such a change is achieved most economically by means of a peri- centric inversion. If so, the dissimilarity of the phenotypes 3E and 4H poses a problem. One might invoke a position effect in which the heterochromatin present in the NOR (MERRITT 1974) could be involved; however, this is a con- jecture. Additional small structural alterations cannot be excluded by the cyto- logical observations; a small deletion could be responsible for the phenotypic change from 4H to 3E. The trivalents formed occasionally in at least one 3E

234 D. U. GERSTEL, J. A. BURNS A N D S. A. SAND

line may indicate the presence of a segment translocated from a chromosome of N . tabacum. It is probable that the structure of extra chromosomes from 3E lines of independent origins would differ somewhat.

The N . debneyi segment postulated on genetic grounds to be present in 2C plants could also have been derived from the extra chromosome of 4H-1, though it might have a different origin. Like 3E phenotypes, 2C types are rarely, but repeatedly, obtained as new events in advanced backcrosses of 4H lines and also from 3E (SAND 1979). Here again, we do not know whether independently originating but phenotypically similar 2C plants carry translocations with iden- tical breakpoints.

One should not gain the impression that the variety of phenotypes existing in our material is limited to the forms that were studied cytologically. In early generations, a larger number of variations of anther and corolla whorls were observed than could be isolated and stabilized genetically (SAND 1968; SAND and CHRISTOFF 1973). Types IA, 2C, 3E, 4H and the “off type” represent the range of variations observed. But the effects of different and superposed N . debneyi chromosomes and their rearrangements are only partly analyzed. Par- ticularly, an additional N . debneyi element ( s ) presumably capable of restoring pollen fertility to 4H has not been retained in these backcross cultures. The al- ternative that homozygosity for the 4H extra chromosome may be required to restore pollen fertility has not been excluded.

In conclusion, one might suggest that the various modifications of the primary 4H addition type are caused, at least in part, by dissociations of regulatory ele- ments through spontaneous structural alterations of the restorer chromosome from N . debneyi.

LITERATURE CITED

AR-RUSHDI, A. H., 1957 The cytogenetics of variegation in a species hybrid in Nicotiana. Genetics 42: 312-325.

BELLIARD, G., F. VEDEL and G. PELLETIER, 1979 Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281 : 401403.

BURK, L. G., 1960 Male-sterile flower anomalies in interspecific tobacco hybrids. J. Heredity 51: 27-31.

BURNS, J. A., 1964 A technique for making preparations of mitotic chromosomes from Nico-

BURNS, J. A. and D. U. GERSTEL, 1969 Consequences of spontaneous breakage of heterochro- matic chromosome segments in Nicotiana hybrids. Genetics 63 : 427-439.

BURNS, J, A., D. U. GERSTEL and S. A. SAND, 1978 Cytoplasmic male sterility in Nicotiana, restoration of fertility, and the nucleolus. 11. N , debneyi cytoplasm. Genetics 90: 151-159.

CHAPLIN, J. F., 1964 Use of male-sterile tobaccos in the production of hybrid seed. Tob. Sci.

CHEN, K. and S. A. SAND, 1979 Nicotiana chromosome coding for a specific polypeptide of the

CLAUSEN, R. E. and D. R. CAMERON, 1944 Inheritance in Nicotiana tabamm. XVIII. Mono-

tiana flowers. Tobacco Sci. 8 : 1-2.

8 : 105-109.

small subunit of Fraction 1 Protein. Science 204: 179-180.

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MODIFICATION O F MALE-STERILE TOBACCO 235

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Corresponding editor: R. L. PHILLIPS

The Genus Nicotiana. Chronica Botanica, Waltham, Mass.