Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Distinguishing among male sterile and fertile lines of tomatoes (Lycopersicon esculentum Mill.) by means of electrophoretic protein patterns

Vertical block SDS-PAAG electrophoresis of individual tomato seeds was used to examine the potential of seed proteins as gene markers for distinguishing male sterile from fertile lines and cultivars. Seeds of three genic male sterile lines and four fertile cultivars and lines were investigated. The line 6944 is carrier of the gene ms 10 ³⁵for pollen male sterility and the lines P-1a and PPS-3 carry the gene ps-2 for positional sterility. A modified extract solution which includes 1M tris — buffer pH 7.0, 7 % SDS, dimethylformamide and distilled water was applied. Qualitative variation in the A protein locus which could be used as a marker for distinguishing male sterile from fertile tomato lines was established.     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Ultrastructural aspects of cytoplasmic male sterility inPetunia hybrida

Summary Anther development of isogenic male fertile and cytoplasmic male sterile types ofPetunia hybrida cv. Blue Bedder is studied by electron microscopy. First deviation in sporogenesis of the sterile type, is observed during leptotene stage of the meiocytes. Initial aberration is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells. These vacuoles reveal the first aspects of degeneration; no other ultrastructural differences are observed. Vacuolation is accompanied by the condensation of cytoplasmic organelles. The tapetal cells become distorted and ultrastructural aberrations in mitochondria do occur. The mitochondria elongate and contain several tubular cristae.Substantial evidence suggests, that cytoplasmic male sterility in petunia is encoded by the mitochondrial genome (Boeshore el al. 1983). However, before degeneration becomes manifest, no consistent ultrastructural differences in mitochondrial organization are observed.Abortion of the tapetum and the sporogenous tissue in cytoplasmic male sterile plants, generally follows a corresponding pattern. Ultimately, the cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Reciprocal transfer of male sterile and normal plasmons in Petunia

Summary The goal in this experiment was to achieve direct plasmon transfer via cell fusion. Two lines were used — a normal fertile line of P. hybrida, and a cytoplasmic male sterile (cms) line with the nuclear background of P. parodii. Two plants phenotypically similar to the original male sterile line were developed from protoplasts, but instead of being cms they were male fertile. On the other hand, two plants typical of the original normal line developed from protoplasts, but they were cms instead of fertile. Chromosome counts were done and in all cases the expected diploid number (=14) was found. Genetic analysis showed that sorting out of cms and fertile segregants was evident in the first and second backcross of the cms cybrids. The fertile type cybrids were stable fertile for several generations of selfing and proper backcrossing. These results are discussed in the light of an earlier fusion experiment in which these two parental lines were involved.Contribution from the Department of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 991-E, 1984 series     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Restriction endonuclease analysis of mitochondrial DNA from sorghum with fertile and male-sterile cytoplasms

Summary Mitochondrial DNA from four paired (fertile and male-sterile) lines and six isocytoplasmic strains of sorghum (Sorghum bicolor (L.) Moench) were fragmented by endonucleases and their electrophoretic patterns were examined. Cytoplasmic male sterile lines differed from their male-fertile counterparts consistently. Among the isocytoplasmic strains, KS 36A (S. verticilli-florum cytoplasm), KS 38A (S. conspicum cytoplasm), and KS 39A (S. niloticum cytoplasm) showed minor differences from the other strains. Results suggest that restriction endonuclease patterns are useful in detecting differences in mitochondrial genomes.This study was supported by a research grant from Kansas Grain Sorghum Commission, Kansas Board of Agriculture. Contribution 89-28-J from the Kansas Agricultural Experiment Station.     

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.     

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids

We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped. Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.     

Detection of an open reading frame related to the CMS-associated urf-s in fertile Petunia lines and species and in other fertile Solanaceae species

Summary In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 3511-E, 1991 series     

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.     

Cytoplasmic male sterility inBeta is associated with structural rearrangements of the mitochondrial DNA and is not due to interspecific organelle transfer

Summary Chloroplast (ct) and mitochondrial (mt) DNAs from four cytoplasmic male sterile (cms) and 22 normal fertile sugar beet lines and accessions of wild beets from the genusBeta have been compared with restriction analyses and Southern hybridizations. We have used restriction analyses of ctDNA as a phylogenetic marker to confirm the taxonomic relationships between the different cytoplasms. According to the ctDNA data, all four cms cytoplasms belong to the same taxonomic section,Beta. Restriction patterns of ct and mtDNA from fertile accessions produced analogous trees of similarity and showed a close correlation between the organellar DNA diversity and the accepted taxonomic classification of the species studied. However, the mtDNA restriction profiles of the four cms types differed dramatically from each other and from those of all fertile accessions from the genus. No indication of cytoplasmic introgression was found in any of the four investigated cms types. Southern hybridization to mtDNA revealed variant genomic arrangements in the different fertile and cms cytoplasms, indicating that rearrangement of the mitochondrial genome is a common denominator to the different cms systems inBeta. It may, indeed, be a common property to spontaneously occurring cms in all or most species.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Asymmetric protoplast fusion aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne L.

Summary Techniques have been developed for the production of cybrids in Lolium perenne (perennial ryegrass). Gamma-irradiated protoplasts of a cytoplasmically male-sterile breeding line of perennial ryegrass (B200) were fused with iodoacetamide-treated protoplasts of a fertile breeding line (Jon 401). After fusion 25 putative cybrid calli were characterized to determine mitochondrion type and composition of the nuclear genome. Analysis of phosphoglucoisomerase isozyme profiles and determination of the ploidy level by flow cytometry indicated that all of the calli tested essentially contained the nuclear DNA of the fertile line. However, the presence of parts of the nuclear DNA from the sterile line could not be excluded. Southern blotting of total DNA isolated from the parental lines and putative cybrids combined with hybridizations using the mitochondrial probes cox1 and atp6 revealed that the mitochondria of the calli originated from the fertile line (5 calli), the sterile line (5 calli) or from both parental lines (15 calli). The hybridization patterns of the mtDNA from the cybrid calli showed extensive quantitative and qualitative variation, suggesting that fusion-induced inter- or intramolecular mitochondrial recombination had taken place.