Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

A unique cytoplasmic male sterility (CMS) determinant is present in three Phaseolus species characterized by different mitochondrial genomes

Previous results have shown that cytoplasmic male sterility (CMS) in lines from Phaseolus coccineus and Phaseolus vulgaris contain the same CMS-specific sequence, raising the question of whether this sequence rearrangement arose before divergence of the two species or afterward with subsequent transfer by introgression. Hybridization patterns of total DNA from eight P. vulgaris lines with cytoplasm from P. coccineus and three P. vulgaris lines were examined in order to analyze the mitochondrial DNA (mtDNA) diversity within each species and to determine differences between CMS lines derived from the two species. Three restriction enzymes and 17 heterologous mtDNA sequences were used. The analysis of the different hybridization patterns revealed a considerable diversity in mtDNA organization particularly within P. coccineus. We obtained distinctive hybridization patterns for the five CMS lines tested. The resulting classification showed that mitochondrial genomes from P. coccineus CMS lines group with those of fertile P. coccineus but not with CMS lines from P. vulgaris. The groupings concur with the taxonomic classification of these lines. The results support the hypothesis of a single ancient origin of the CMS determinant and exclude the transfer of cytoplasm by introgression from P. vulgaris to P. coccineus and P. coccineus ssp polyanthus.     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

Chondriome analysis in sexual progenies of Nicotiana cybrids

Summary We studied the chondriomes (the mitochondrial genomes) of sexual-progeny plants derived from eleven Nicotiana cybrids which resulted from donor-recipient protoplast fusions. The recipients were either N. tabacum or N. sylvestris and the donor (of the cytoplasm) was N. bigelovii. The chondriomes were characterized by the mitochondrial DNA (mtDNA) restriction-patterns. The differences in mtDNA restriction patterns were revealed after Sal I digestions and probing the respective Southern-blots with three mtDNA fragments. The hybridization patterns of mtDNAs from 35 second-generation plants (i.e. the sexual progeny derived from the cybrid plants) indicated only minor variations between plants derived from the same cybrid but pronounced variations among sibs derived from different cybrids. The mtDNA of 32 second-generation plants varied from both original fusion partners but the mtDNA of one (male-sterile) plant was apparently identical with the mtDNA of one of the original donor (N. bigelovii) and the mtDNA of two other (male-fertile) plants was apparently identical to the mtDNA of an original recipient (N. sylvestris). Generally, the mtDNAs of male-fertile, second-generation plants were similar to the mtDNAs of the original recipients while the mtDNAs of the male-sterile second-generation plants were similar to the mtDNA of the donor (N. begelovii). The analyses of mtDNAs from the thirdgeneration plants indicated stabilization of the chondriomes; no variations were detected between the mtDNAs of plants derived from a given second-generation plant.     

Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum

Summary Variation in mitochondrial genome organization and expression between male fertile and sterile nuclear-cytoplasmic combinations of sorghum has been examined. Cytoplasmic genotypes were classified into eleven groups on the basis of restriction endonuclease digestion of mitochondrial DNA (mtDNA) and five groups on the basis of mitochondrial translation products. These cytoplasms were further characterized by hybridization of specific gene probes to Southern blots of EcoRI digested mtDNA, and identification of the fragment location of four mitochondrial genes. Variation was observed in the genomic location and copy number of the F₁ ATPase -subunit gene, as well as the genomic location and gene product of the cytochrome c oxidase subunit I gene. The effect of nuclear genotype on mitochondrial genome organization, expression and the presence of two linear plasmid-like mtDNA molecules was examined. Our results indicate that nuclear-mitochondrial interactions are required for regulation of mitochondrial gene expression. When a cytoplasm is transferred from its natural to a foreign nuclear background some changes in the products of in organello mitochondrial protein synthesis occur. In a number of cytoplasmic genotypes these changes correlate with the expression of cytoplasmic male sterile phenotype, suggesting a possible molecular basis for this mutation.     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Non-radioactive organization and transcript analysis of the ATPase subunit 6 gene region in the mitochondrial genome from fertile and sterile (CMS) forms of wheat and triticale

In sterile triticale forms [with cytoplasmic male-sterile (CMS)-inducing timopheevi cytoplasm], fertile orms (with normal cytoplasm) and the corresponding wheat cross parents mitochondrial DNA (mtDNA) and RNA (messenger mtRNA) were characterized using total DNA and RNA material for Southern and Northern blots. A novel non-radioactive technique was applied by marking the probes with digoxenin. The fertile and sterile Triticum and triticale forms were analysed in three genes, atp 6, coxIII and rps13. These forms can be distinguished in the apt6 gene at the mtDNA and mtRNA levels.     

Organelle DNA variation in parental<Emphasis Type="Italic"> Solanum</Emphasis> spp. genotypes and nuclear-cytoplasmic interactions in<Emphasis Type="Italic"> Solanum tuberosum</Emphasis> (+)<Emphasis Type="Italic"> S. commersonii</Emphasis> somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC₁ clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.Contribution no. 24 from the Institute of Plant Genetics, Research Division of Portici     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Mitochondrial DMA variation in somatic embryogenic cultures ofLarix

outhern hybridization analysis using wheat mitochondrial gene-specific probes indicates that changes in mitochondrial genomic organization and the relative representation of certain genomic regions occur during in vitro somatic embryogenic cell culture ofLarix species. We observed differences in the mitochondrial (mt)DNA hybridization patterns between somatic embryogenic cell cultures and trees grown from seed forLarix leptolepis,L. decidua, and the reciprocal hybrids of these twoLarix species. This is the first study to describe the correlation of molecular changes in a gymnosperm mitochondrial genome with in vitro somatic embryogenic cell culture. Quantitative differences in mtDNA hybridization signals were also observed among a 4-year-old somatic embryogenic cell culture ofLarix ×eurolepis trees regenerated from this culture, and the seed source tree from which the somatic embryogenic cell cultures were initiated.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

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.     

Cybrid production based on mutagenic innactivation of protoplasts and rescuing of mutant plastids in fusion products: Potato with a plastome fromS. bulbocastanum andS. pinnatisectum

A procedure for cybrid production, based on double treatment of donor protoplasts by physical and afterwards chemical mutagens at superlethal doses (-irradiation at a dose of 1000 Gy was applied for the inactivation of nuclei; 3–5 mMN-nitroso-N-methylurea was used for the efficient induction of plastome mutation) and the rescuing of mutant plastids after fusion with untreated recipient protoplasts, was developed. For identification of mutant donor-type plastids in fusion products a selection for streptomycin was performed. In two sets of experiments, in whichS. tuberosum served as the recipient of foreign cytoplasm with the wild tuber-bearing speciesS. bulbocastanum andS. pinnatisectum as donors, a total of about 40 streptomycin-resistant colonies was isolated. Eight regenerants from theS. tuberosum+S. bulbacastanum fusion combination and four fromS. tuberosum+S. pinnatisectum were further investigated using chromosome counting, analysis of esterase isoenzymes, restriction analysis of organelle DNA, and blot hybridization. All but one plant from both combinations were characterised as potato cybrids possessing exclusively foreign plastids and retaining a morphology typical of the recipient. Only in one line was rearranged mtDNA detected. The availability of potato cybrids facilitates the analysis of plastome-encoded breeding traits and the identification of the most valuable source of cytoplasm among the wild potato species. The described system for producing cybrids without genetic selectable markers in the parental material offers the possibility for the rescue of cytoplasmic mutations which are impossible to isolate by conventional approaches.