Curr Genet (1993[)19:121-127

Current Genetics
9 Springer-Verlag 1991

A 2.5 kb NcoI fragment of Ogura radish mitochondrial DNA is correlated with cytoplasmic male-sterility in Brassica cybrids
Sandrine Bonhomme, Fran~oise Budar, Madina F~rault, and Georges Pelletier Laboratoire de Biologic Cellulaire, INRA Centre de Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France Received September 12/October 21, 1990

Summary. Spontaneous reversion to fertility was studied in the progeny of a cytoplasmic male-sterile (CMS) Brassica napus cybrid containing recombinant B. napus/ Ogura radish mitochondrial genomes. This reversion is concomitant with the disappearance of a 2.5 kb NcoI fragment present in the mitochondrial D N A of Ogura radish, and of CMS cybrids derived from plants carrying Ogura cytoplasm, and absent in the mitochondrial genome of normal Brassicas and fertile cybrids. This specific fragment hybridizes to a 1.4 kb transcript found only in male-sterile plants bearing an Ogura derived cytoplasm. Key words: B r a s s i c a - Ogura cytoplasmic male-sterility Cybrids - Mitochondrial D N A

Introduction Many Brassica species have important properties, as vegetables, condiments and as sources of industrial or edible oils. Fz hybrid Brassicas offer agronomic advantages because of a large heterosis effect: as an example, the yield of rapeseed (B. napus) hybrids can be 30% higher than the mean yield of the two parents (Lefort-Buson and Datte6 1982, 1983). Until now, no cytoplasmic male-sterility (CMS) systems has ever been economically exploited in Brassicas for the production of F ~ hybrids because of instability of the male-sterile phenotype (Thompson 1972; Rousselle et al, 1983). However, a cytoplasm conferring complete male sterility is known in a related species, radish (Ogura 1968), and restorer genes for this CMS have been localised on radish chromosomes (Heyn 1976). In order to create an improved system for the production of F 1 hybrid Brassica varieties, Ogura male sterility has been transferred to Brassica species (B. napus and B. oleraeea) by interspecific crosses coupled with embryo rescue and back-crossing (Bannerot et al. 1977).
Offprint requests to. S. Bonhomme

9However, the radish chloroplasts did not function properly in a Brassica nuclear background and chlorophyll deficiencies prevented any agronomical use of the malesterile lines obtained (except for a few spring types). Assuming the Ogura CMS trait was mitochondrially determined, as has been shown for virtually all species so far (for reviews see Lonsdale 1987; Newton 1988; Levings and Brown 1989), protoplast fusions were made between fertile and Ogura CMS Brassicas in order to replace the radish chloroplasts with Brassica chloroplasts (Pelletier et al. 1983; Menczel et al. 1987; Jourdan et al. 1989). Cybrids were selected with a normal chlorophyll content; these were also fully male sterile, strongly suggesting the mitochondrial (rot) location of the CMS determinant(s). A complete map of the Ogura radish mitochondrial genome for five restriction enzymes has been published and compared to the map of the fertile radish mitochondrial genome (Makaroff and Palmer 1988), but the number of rearrangements between both genomes is high (at least ten inversion events) and prevents the simple location of the difference(s) related to the CMS phenotype. The occurrence of interparental mitochondrial DNA recombination in the cybrids obtained previously (Ch6trit et al. 1985; Vedel et al. 1986, 1987) suggested that a comparison of these cybrids could be used to define the region(s) of the genome carrying the CMS determinant, as has been done successfully with petunia CMS cybrids (for reviews see Hanson et al. 1989, 1990). As an added bonus, one of our cybrid lines reverted to fertility at high frequency, providing the possibility to compare extremely closely related CMS and fertile lines. This type of material was invaluable in the implication of the urfl3-T gene in the T-type CMS of maize (Umbeck and Gengenbach 1983; Rottmann et al. 1987; Fauron et al. 1990). Materials and methods
Plant material. Cybrid 13 was obtained among 820 regenerated plants from protoplast fusions betweena male-sterile triazine-resistant B. napus cybrid carrying Ogura mitochondria (progeny of cybrid 77 described in Pelletier et al. t983; Ch6trit et al. 1985)and the

122 fertile, triazine-susceptible B. napus spring variety Brutor. A triazine resistance test (Ducruet and Gasquez 1978) was performed on a leaf sample from each regenerant to determine the chloroplast type: either triazine-resistant chloroplasts, originating from parent 77, or triazine-susceptible chloroplasts, originating from the Brutor line. The plants were later observed at the flowering stage. Plants showing non-parental combinations of cytoplasmically encoded characters (either susceptible/male-sterile or resistant/male-fertile) were selected as eybrids. Cybrid 13 was of the susceptible/malesterile type. Male-sterile descendants and fertile-revertants of this cybrid were studied. Other Brassica cybrids which are referred to below were described previously (Pelletier et al. 1983) or obtained in the same or further fusion experiments between protoplasts from male-sterile and male-fertile parents. As controls we used cauliflower and rapeseed plants with their own cytoplasm or Ogura cytoplasm. 13 plants, five plants were totally male sterile (including plants 13-2 and 13-7), one was male fertile (13-6) and seven were sterile but with some male-fertile flowers (Fig. 1). The fertile plant 13-6 was self-pollinated and crossed with Brutor. In b o t h cases only fertile plants were obtained (43 and 42 respectively). I n crosses between the male-sterile plant 13-7 and Brutor, 24 descendants were entirely sterile and six showed some fertile flowers, a result similar to that o b t a i n e d with cybrid 13 itself (see above). Plant 13-2 was crossed to the restorer line R F which is heterozygous for specific restorer genes for O g u r a male-sterility in Brassica (Ch&rit et a l . / 9 8 5 ) . The progeny o f this cross was c o m p o s e d o f 53 male-sterile plants, 37 male-fertile plants and nine plants almost entirely sterile t h o u g h showing some fertile flowers. A transmission defect o f this restorer gene by the pollen has been observed by P e l l a n - D e l o u r m e (1986) and explains segregations statistically different f r o m 1:1. These results suggest that male-sterile plants o f the cybrid 13 family contain the O g u r a C M S determinant, as did other cybrids previously studied (Ch6trit et al. 1985; and see discussion). A t this stage o f the study, two possibilities were considered: either cybrid 13 contained a mixture o f "malefertile" and "male-sterile" m i t o c h o n d r i a l genomes, so that one could further select for b o t h phenotypes, or cybrid 13 contained a structurally unstable m i t o c h o n drial g e n o m e which reverted to a m o r e stable "fertile" configuration, so that it w o u l d be impossible to m a i n t a i n an h o m o g e n e o u s male-sterile p h e n o t y p e a m o n g further generations. Male-sterile plants, obtained f r o m the p r o g e n y o f the male-sterile plant 13-7, were p r o p a g a t e d b o t h by cuttings and by sexual crosses with Brutor. After a variable n u m ber o f generations ( 1 - 5 ) by b o t h p r o p a g a t i o n methods, all families gave some fertile revertants; but sexually p r o p a g a t e d fertile plants were never observed to give back sterile plants (Fig. 1). Considering these results together, we f a v o u r the seco n d explanation p r o p o s e d above, i.e., cybrid 13 bore an unstable m i t o c h o n d r i a l g e n o m e which lost the C M S determinant during the process leading to a "fertile" configuration, with no possibility o f reverting back to a sterile phenotype.

Isolation of nucleic acids. Total DNA was isolated from 4-week-old plant leaves according to Dellaporta et al. (1983). Mitoehondrial DNA was extracted from 8-week-old plant leaves as described in Vedel and Mathieu (1982), except that the mitochondria were not purified on sucrose gradients before lysis and lysis was performed in 4% sarcosyl with 0,5 mg/ml proteinase K (Boehringer Mannheim GmbH, Mannheim, W-Germany), in 50 mM Tris-HC1 pH 8, 20 mM EDTA. After precipitation, mitochondrial DNA was purified by caesium chloride-ethidium bromide gradient centrifugation (method 1 of Vedel and Mathieu 1982) in polyallomer centrifuge tubes (quick-seal Beckman no 344625, Beckman Instruments Inc., Palo Alto, CA). Total RNAs were isolated from leaves or buds according to Logemann et al. (1987). Mitochondrial RNAs were extracted from 8 weekold cauliflower heads as described in Stern and Newton (1986). Mitochondrial or total DNA restriction analysis and agarose gel eleetrophoresis. Were all performed as described in Ch4trit et al. (1985); mt or total RNAs were separated on electrophoresis gels containing formaldehyde as described in Sambrook et al. (1989). Transfer of DNA or RNA. Using nylon filters (Hybond-N, Amersham, Bucks, CA) transfer was carried out by capillary blotting in 6 x SSC or 10 x SSPE respectively, following the manufacturer's instructions. Prehybridization and hybridization were conducted according to Amersham, using probes labelled with the multiprime DNA labelling system (Amersham) and purified on Sephadex G50 columns (Sambrook et al. 1989). Cloning of mtDNA. A mtDNA library of the male-sterile cybrid (13-7) was constructed in the phage lambda vector EMBL3, grown on the restrictive E. coli strain NM539 (Frishauf et al. 1983). Restriction fragments resulting from partial digestion of the mtDNA with MboI were cloned in the BamHI site of the vector. Approximately 2.5 x 10* clones were obtained per gg of mtDNA. The mtDNA library was titred and plated out in order to obtain isolated plaques which were transferred onto nylon filters (Hybond-N Amersham) as described in Sambrook et al. (1989). The hybridization probe used to screen the mtDNA library was prepared as follows: the CMS-specific mtDNA fragment was eluted using the Gene-clean TM (Bio 101 Inc., La Jolla, CA) procedure from a mtDNA digest loaded on a preparative agarose gel. Eluted DNA was then labelled as described above. Lambda DNA extraction, subcloning of the 2.5kb NcoI fragment into the NcoI site of pTrc99A (Amann et al. 1988), and plasmid DNA extractions followed the protocols in Sambrook et al. (1989). Recombinant plasmids were grown in E. coli strain NM522 (Gough and Murray 1983).

Comparison between mitochondrial D N A s o f male-sterile and fertile-revertant plants." isolation o f a fragment specific to male-sterile plants
M i t o c h o n d r i a l D N A was extracted f r o m leaves o f malesterile 13-7 descendants and fertile revertants (13-6 or /3-7 descendants) and digested with several restriction enzymes, in order to c o m p a r e their restriction patterns. M i t o c h o n d r i a l genomes of b o t h types were very similar, since no difference could be observed between male-sterile and fertile-revertant m i t o c h o n d r i a l restriction profiles using m o s t enzymes tested (data n o t shown). H o w e v e r ,

Results
Reversion to fertility in the progeny o f male-sterile cybrid 13
I n the first p r o g e n y generation obtained by pollination o f cybrid 13 with the rapeseed cultivar Brutor, c o m p o s e d o f

123 13 x MS nucleus: B. napus chloroplasts: B. napus (triazine susceptible) mitochondria: recombinant Ogura/Brutor
cybrid

B. napus cv Brutor

--I
13.2 MS

1
13.4 MS/F

I
13.6 F

I
13.7 MS x Brutor

MS

Brutor

MS

MS

MS

I MS

Brutor

MS

MS

MS

1
MS

MS

Brutor

MS

A /,, I I/,, I
MS F F MS F F F F MS F

MS

MS x Brutor

F MS

Fig. 1. Progeny of cybrid 13. Instability of the male-sterile phenotype was observed in the first sexual generation, with the occurrence of one fertile-revertant plant (13-6). After one to five generations, revertant plants were observed for all the male-sterile descendants

F MS MS F of the plant 13-7. The fertile-revertant phenotype was stable through several generations of self pollination. Vertical lines, sexual cross; arrows, cuttings; MS, male-sterile; E male fertile; MS/F, male sterile with some fertile flowers

Fig. 2a, b. Detection of a mitochondrial restriction fragment specific to male-sterile plants, a agarose gel electrophoresis of m t D N A extracted from plant leaves and restricted with NruI. Bru, Brassica napus cv Brutor; O, Brassica oleracea bearing Ogura mitochondria; F, fertile revertant from a descendant of the plant 13-7; MS, malesterile descendant of the plant 13-7. b Autoradiograph of hybridization of the m t D N A shown in a, with the N6.8 fragment labelled and used as a probe (in this case eluted from a lambda clone)

one restriction fragment of 6.8 kb was detected in the restriction profile of male-sterile plants mtDNA digested with Nrul, and never observed in mt patterns of corresponding fertile revertants (Fig. 2 a). This 6.8 kb NruI fragment (called N6.8) was eluted from a mtDNA digest of a 13-7 male-sterile descendant, labelled, and used as a probe against NruI mtDNA restriction profiles. A strong signal at 6.8 kb was observed in all male-sterile descendants ofcybrid 13, while no fragment of this size hybridized to the probe in the fertile-revertant mitochondrial genome (data not shown). A lambda library containing inserts of mtDNA from malesterile (13-7) plants was screened with the labelled eluted fragment, and two recombinant phages were isolated containing the entire N6.8 fragment and adjacent sequences. The N6.8 fragment was eluted from a NruI digest of one of these clones, labelled and used as a probe against Nru I mtDNA restriction profiles. It hybridized to a NruI fragment of 6.8 kb in Ogura and male-sterile descendants of cybrid 13 mtDNAs (but not in Brutor or in fertile-revertant mt genomes), showing the Ogura origin of this fragment, and to a NruI fragment of 11.5 kb in B. napus cv Brutor (Fig. 2 b). It also hybridized to a 15 kb NruI fragment in the mtDNA of both sterile and fertilerevertant descendants of cybrid 13, indicating that at least part of the N6.8 fragment is repeated elsewhere in their mt genomes.

124
lkb

SalI NruI PstI

I ~\\\\\\\\\\\\\\\\~ I 1

I I

//

NruI PstI NcoI BglII XhoI

ile pattern, several NcoI fragments hybridized in both male-sterile and fertile-revertant profiles, namely at 2.2, 10 and 14 kb. A NcoI fragment of 2.7 kb hybridized strongly in the mt genome of fertile-revertants but very faintly in the sterile one. Analysis of this hybridization profile leads to the conclusion that the 2.5 kb NcoI fragment, though specific to male-sterile mtDNA, contains sequences which are repeated elsewhere in the mt genome (on 2.2, 10 and 14 kb NcoI fragments), and these repeated sequences are also present in fertile-revertant m t D N A (the same 2.2, 10 and 14 kb NcoI fragments), in addition to a 2.7 kb specific fragment probably created during the reversion process.

Fig. 3. Alignment of restriction maps of part of the Ogura radish mitochondrial genome (a) (see Makaroff and Palmer 1988) with the lambda clone containing the entire N6.8 fragment (b). The N6.8 fragment is indicated by hatched lines. The 2.5 kb NcoI fragment is shaded. Arrowsindicate the cloning site of the lambda vector (MboI site in mtDNA). Slight size differences oserved between both maps are likely due to different size estimations from the stained gels. The letter A indicates a region of Ogura mtDNA absent in fertile radish, or at least highly recombined (Makaroff et al. 1989)

The Nco2.5 fragment is specific for Brassica cybrids 9 expressing Ogura CMS Southern hybridization of mitochondrial or total D N A with the labelled Nco2.5 fragment used as a probe showed that it is present in more than 30 Brassica cybrids expressing Ogura CMS, obtained in independant protoplast fusion experiments. No signal at 2.5 kb was observed in fertile segregants from these cybrids when they exist (9 independant cases), or in cybrids which were fertile from the beginning (five cases). Hybridization signals at different molecular weights are observed for all fertile plants, showing that part of the sequence of the Nco2.5 fragment is present in their genomes (Fig. 5).

Northern analysis Total RNAs were extracted from leaves or buds of descendants from cybrid 13, other male-sterile or fertile cybrids (from other fusion experiments), one male-sterile cybrid carrying Ogura mitochondria, and Brutor. Northern blots were hybridized with part of the Nco2.5 fragment (Fig. 6 a). One transcript of 1.4 kb was detected in all male-sterile cybrids, including two descendants of cybrid 13-7 and a restored cybrid, whereas no transcript was observed at that size in Brutor, nor in the fertile cybrids (including two revertants from cybrid 13 descendants). However, the B. napus cv Brutor, the fertile cybrids and all descendants of cybrid 13 (male-sterile and revertants) showed a 1.1 kb transcript hybridizing to the probe, which was absent in Ogura mitochondria and all other male-sterile cybrids tested. We checked that mitochondrial transcripts could be detected in total R N A samples by hybridizing the same Northern blot with a D N A fragment containing a mt atpA gene sequence (from Nicotiana plumbaginifolia, a gift from M. Boutry). We did not observe any differences in the mt atpA transcript patterns between male-sterile and fertile-revertant plants, all of them showing an m R N A at 1.8 kb (data not shown). A 1.4 kb transcript was also found in mitochondrial RNAs extracted from cauliflower heads of a line carrying Ogura cytoplasm, using the Neo2.5 fragment as a probe (Fig. 6 b).

Fig. 4. Autoradiograph of hybridization ofmtDNA from cybrid 13 descendants (MS, male sterile; E fertile revertant) restricted with NcoI, with the Nco2.5 fragment labelled and used as a probe A detailed restriction map of the region including the N6.8 fragment was obtained (Fig. 3) and aligned to part of the Ogura radish mt genome map (Makaroff and Palmer 1988); m t D N A from fertile and sterile descendants of cybrid l 3 was digested with various restriction enzymes and hybridized with subclones of the N6.8 fragment as probes. This allowed us to limit the region specific to the male-sterile genotype to a 2.5 kb NcoI fragment. This 2.5 kb NcoI fragment (called Nco2.5) was labelled and used as a probe against m t D N A from 13-7 and 13-6 descendants digested with Nco I (Fig. 4). In addition to the signal at 2.5 kb, which was specific to the male-ster-

125

Fig. 5 a, b. Autoradiographs of hybridization of total DNA from different Brassica napus cybrids (either male-sterile, MS, or fertile, F) restricted with NcoI, with the Nco2.5 fragment used as a probe. Bru, B. napus cv Brutor; O, B. oleracea bearing Ogura mitochondria; 13S and 13F, respectively, male-sterile and fertilerevertant descendants of cybrid 13

Fig. 6 a, b. Transcript analysis, a Northern analysis of total RNAs hybridized with part of the Nco2.5 fragment as a probe (indicated on the scheme below the autoradiogram). MS, male-sterile B. napus cybrids; R, restored B. napus cybrid; F, fertile B. napus cybrids; 13S and I3F,, respectively, male-sterile and fertile-revertant descendants Discussion
Alloplasmic Brassica lines bearing normal radish cytoplasm show incomplete male sterility ( M a c C o l l u m 1981). In contrast, Brassica plants with Ogura radish cytoplasm are completely male sterile. F r o m existing data, there is no direct evidence that the Ogura CMS determinant responsible for male sterility in radish is also active in Brassica male-sterile plants carrying Ogura cytoplasm. It is possible to imagine that the Ogura m t genome (which is

of cybrid 13; Ogu, B. napus cybrid carrying Ogura mitochondria; Bru, B. napus cv Brutor. RNAs were extracted from leaves (1) or buds (b). b Northern analysis of mtRNAs extracted from cauliflower heads, hybridized with the Nco2.5 fragment used as a probe. O, cauliflower having Ogura mitochondria; N, normal cauliflower

very different from the normal radish m t genome) could also lead to alloplasmic male sterility but with a more pronounced phenotypic effect, i.e., a completely malesterile phenotype. It is also possible to imagine that both types of CMS determinants (an alloplasmic interaction plus the Ogura CMS) are active in Brassica. The observation of two phenotypes (complete or incomplete male-sterility) a m o n g our cybrids strongly supports the latter possibility and suggests that at least two CMS determinants can be separated following m t D N A rearrangements (Pel-

126 letier et al. 1986). Among completely male-sterile cybrids, two different restoration patterns were observed after crossing to a rapeseed line (RF) carrying radish restorer genes (Chbtrit et al. 1985). Cybrid 13, like some other completely male-sterile cybrids, is restored by a single nuclear gene. These cybrids have always kept the Oguraspecific 2.5 kb NcoI fragment in their genome. Other cybrids restored by at least two genes have probably conserved two types of CMS (C. Primard, personal communication). Their mitochondrial genomes also carry the 2 . 5 k b NcoI fragment. Interspecific crosses between Ogura radish and Brassiea napus lines carrying restorer genes would help show whether the CMS determinant on the Neo2.5 fragment is also involved in the CMS phenotype of Ogura radish. The Ogura origin of the Nco2.5 fragment has been demonstrated, allowing us to reject the hypothesis that the CMS trait in our cybrids could be due to protoplast fusion, and was not inherited from the CMS parent. Moreover, this fragment has been localized on the Ogura radish m t D N A restriction map (Makaroff and Palmer 1988) overlapping the region A which is either absent in fertile radish m t D N A , or at least highly reorganised ( M a k a r o f f e t al. 1989). Successively, a 11.3 kb mitochondrial linear plasmid (Palmer et al. 1983), rearrangements near the Ogura mt atpA gene (Makaroff and Palmer 1988) and the mt atp6 gene ( M a k a r o f f e t al. 1989) have all been suggested as the cause of Ogura CMS. The Nco2.5 fragment is not linked to the atp6 or atpA genes on the Ogura map. In our cybrids, neither o f these genes appear to be associated with male sterility (D. Lancelin, personal communication). We presume that the Nco2.5 fragment gives rise to the 1.4 kb transcript seen only in male-sterile plants, whereas the 1.1 kb transcript seen in fertile cybrids, Brutor and all cybrid 13 descendants, comes from one of the other fragments of the mt genome which hybridize to the Nco2.5 probe. The different transcription patterns of the CMSassociated region between CMS and fertile-revertant plants suggest that the presence of the 1.4 kb transcript, rather than the absence of the 1.1 kb transcript, is involved in Ogura CMS, as male-sterile descendants of the plant 13-7 show both the 1.4 and the 1.1 kb transcripts. The CMS-specific 1.4 kb transcript is still present in a restored cybrid, implying a post-transcriptional or indirect effect of the nuclear restorer gene. Similar observations have been made on petunia cybrids obtained by interspecific protoplast fusions (Izhar et al. 1983). Comparison of m t D N A from CMS and fertile petunia cybrids showed a specific rearrangement involving structural genes, creating a new O R F (called pcf) whose expression is limited to male-sterile cybrids and restored plants (Young and Hanson 1987; for review see Hanson et al. 1990). We are now sequencing the Nco2.5 fragment in order to find the coding sequence corresponding to the 1.4 kb transcript detected on Northern blots. The occurrence of revertant plants among our CMS cybrids should allow us to follow the approach which was successfully used to study T-type CMS in maize. Fertilerevertants derived from maize CMS-T callus culture were isolated (Gengenbach et al. 1977), and shown to contain a deletion o f the gene urfl3-Tencoding a 13 kDa-specific polypeptide (Forde etal. 1978; Dewey etal. 1986), strongly implicating it as the CMS determinant (Rottmann et al. 1987; Fauron et al. 1990). Hybridization of NcoI m t D N A restriction profiles from CMS and fertilerevertant cybrid 13 descendants, using the Nco2.5 fragment as a probe, reveals fragments common to both mt genomes, and a 2.7 kb NcoI fragment specific to the m t D N A of fertile-revertants, which probably originates from the Nco2.5 fragments. Traces of this fragment are observed in the m t D N A of male-sterile descendants of cybrid 13, suggesting that an amplification of the 2.7 kb NcoI fragment is associated with the reversion. Cloning and sequencing of all these fragments should allow us to identify those involved in the reversion to fertility, where the recombination events take place, and whether any sequences are deleted from the revertant genome.

Acknowledgements. We are grateful to Ian Small for helpful discussions and support in writing the manuscript. We thank Jean Pierre Bourgin for critical reading of the manuscript, Catherine Primard and Dominique Lancelin for helpful discussions, and Alfred Martin-Canadell for taking care of the plants. This work was supported by the grant no 86C0949 of the Programme mobilisateur biotechnologies from the Minist6re de la Recherche et de la Technologic.

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