Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred. Received: 26 April 2000 / Accepted: 28 August 2000     

Analyses of mitochondrial DNA structure and expression in three cytoplasmic male-sterile chicories originating from somatic hybridisation between fertile chicory and CMS sunflower protoplasts

Cytoplasmic male-sterile (CMS) chicories have been previously obtained by somatic hybridisation between fertile industrial chicory protoplasts and CMS sunflower protoplasts. In this study, we compared three different CMS chicory cybrids that originated from three different fusion events. The cybrids were backcrossed with different witloof chicories in order to transfer the three male-sterile cytoplasms from an industrial chicory nuclear environment to a witloof chicory nuclear context. Southern hybridisation, using different mitochondrial genes as probes, revealed that the three cybrid mitochondrial genomes were different and that they were stable throughout backcrossing generations regardless of the pollinator. However, pollinators were found to influence floral morphologies – with one being able to restore fertility – showing that nuclear context can affect the sterility of the cybrids. PCR and RFLP analyses revealed that the orf522 sequence, responsiblefor CMS in PET1 sunflower, was present in two out of the three cytoplasms studied, namely 411 and 523, but was absent from the other cytoplasm, 524. We thus concluded that orf522 is not responsible for CMS in the 524 cybrid. Although the orf522 gene is present in the 411 and 523 cytoplasms, it is probably not responsible for the sterile phenotype of these cybrids. Received: 3 June 1998 / Accepted: 30 April 1999     

Modifications of Mitochondrial DNA Cause Changes in Floral Development in Homeotic-like Mutants of Tobacco

To investigate the influence of mitochondrial genes on stamen development of higher plants, protoplasts from three different, male-sterile tobacco cultivars were fused. The fused cells were cultured individually into calli, from which plants were regenerated. Cybrid plants were obtained that exhibited flowers with recombined biparental male-sterile morphology and with novel male-sterile stamens that differed from any types from sexual or somatic hybridizations described previously. The male-sterile morphologies of these cybrids and their parents support the hypothesis that nuclear-mitochondrial interaction occurs at several stages in tobacco floral development and that several mitochondrial genes are necessary for normal stamen and corolla development. Analysis by restriction endonuclease digestion of mitochondrial DNA of male-sterile cybrids and their parents revealed that the mitochondrial DNA of male-sterile cybrids with parental floral morphology was unchanged when compared with parental mitochondrial DNA. Cybrids that were morphologically similar to one parent's male-sterile phenotype had mitochondrial DNA almost identical to that parent, whereas cybrids with recombined biparental or novel male-sterile phenotypes contained mitochondrial DNA different from both male-sterile parents and from each other. A set of mitochondrial DNA fragments could be correlated with split corollas, a feature found in several tobacco male-sterile cultivars. DNA gel blot analysis using a number of mitochondrial genes confirmed the conclusions based on ethidium bromide staining of mitochondrial DNA restriction digests.     

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures. Received: 4 June 1996 / Accepted: 2 August 1996     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

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.     

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.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana

Summary Variation in mitochondrial protein synthesis and genome organization was investigated. Three different alloplasmic cytoplasmic male-sterile Nicotiana tabacum cultivars, carrying N. repanda, N. suaveolens or N. debneyi cytoplasm, were analysed together with corresponding male-fertile parental and restored material. Although several differences were detected in the proteins synthesized by isolated mitochondria from the male-sterile and male-fertile plants, most of these were related to the origin of the mitochondria. However, a 23 kD protein was synthesized in the male-sterile cultivar carrying N. debneyi mitochondria, but not in other lines containing this cytoplasm. This protein was also present in the male-fertile parent containing N. tabacum mitochondria. Only the enhanced production of a 30 kD protein in the lines carrying mitochondria from N. repanda or N. debneyi was exclusively correlated with CMS. This protein was not present in any of the corresponding male-fertile parental and restored lines. Restriction enzyme analysis of mitochondrial DNA revealed a difference in abundance of a 5.6 kb XhoI fragment between lines containing N. debneyi mitochondria. No rearrangements of mitochondrial DNA was found between male-fertile and male-sterile lines carrying N. repanda or N. suaveolens cytoplasm. These results might indicate that CMS in alloplasmic Nicotiana cultivars is caused by alterations in the expression of mitochondrial genes, rather than by induced changes in the genome.     

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.     

Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological,genetic, and molecular characterization

Summary Somatic hybrid/cybrid plants were obtained by microfusion of defined protoplast pairs from malefertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile (cms), streptomycin-sensitive N. tabacum cms (N. bigelovii) after microculture of recovered fusants. Genetic and molecular characterization of the organelle composition of 30 somatic hybrid/cybrid plants was performed. The fate of chloroplasts was assessed by an in vivo assay for streptomycin resistance/ sensitivity using leaf explants (R₀ generation and R₁ seedlings). For the analysis of the mitochondrial (mt) DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA, with three DNA probes of N. sylvestris mitochondrial origin. In addition, detailed histological and scanning electron microscopy studies on flower ontogeny were performed for representative somatic hybrids/cybrids showing interesting flower morphology. The present study demonstrates that electrofusion of individually selected pairs of protoplasts (microfusion) can be used for the controlled somatic hybridization of higher plants.Abbreviations ac alternate current - BAP benzyl aminopurine - cms cytoplasmic male sterile - dc direct current - NAA naphthalenacetic acid - SEM scanning electron microscopy     

Fertility Restoration Is Associated with Loss of a Portion of the Mitochondrial Genome in Cytoplasmic Male-Sterile Common Bean

Restoration of pollen fertility to cytoplasmic male-sterile common bean by nuclear gene Fr is accompanied by mitochondrial (mt) DNA rearrangements within restored plants. These rearrangements are also observed upon spontaneous cytoplasmic reversion to fertility. An mtDNA fragment of at least 25 kilobases was lost from the genome upon restoration or reversion. This fragment contained DNA segments that were not repeated elsewhere in the genome and, therefore, were not detected within the genome upon fertility restoration. This result suggested that the particular mtDNA configuration absent from restored plants could not be maintained by a constant process of recombination but rather by autonomous replication. No evidence of excision of this region from the mt genome, in the form of a junction fragment associating flanking DNA regions, was detected in fertile restored plants. DNA gel blot hybridization of this mtDNA region, compared with hybridization to related regions of the mitochondrial genome that shared sequence homology, indicated that the mtDNA region associated with sterility was present in lower copy number. These observations, as well as the occurrence of similar or identical rearrangements upon spontaneous cytoplasmic reversion, indicate that the restoration of pollen fertility may be accompanied by loss of an independently replicating subgenomic DNA molecule from the mitochondrial genome.     

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.     

Cytoplasmic DNA variation in a potato protoclonal population

Summary Mitochondrial DNA variation was detected in potato plants (protoclones) regenerated from leaf mesophyll protoplasts. Two forms of variation were evident; (1) DNA sequence alterations within the high molecular weight mitochondrial chromosome and (2) the appearance of an additional low molecular weight mitochondrial DNA species. Variation in chloroplast DNA was not detected. The data suggests that protocloning can introduce molecular diversity into mitochondrial genomes and thereby assist in overcoming the cytoplasmic genetic uniformity prevalent in most major crops.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

Identification of Cytoplasmic Male Sterility in Chinese Radish Following PCR Analysis of Mitochondrial DNA

In order to understand the molecular characteristics of the Chinese radish, the mitochondrial DNA structure and sequence were analyzed in Chinese wild radish and cultivated varieties. A total of four male-sterile lines, four maintainer lines, 17 cultivars, and a single Chinese wild radish were used, along with 25 male-sterile individuals and 159 fertile plants. We found that the cytoplasm of Chinese radishes could be classified into two types: the normal type and the Ogura type. The Ogura-type cytoplasm was detected in 25 male-sterile plants. The 159 fertile plants had normal cytoplasm. Both the Ogura cytoplasm and the normal cytoplasm were detected in the male-sterile ??RA??. The orf138 gene in mitochondrial DNA was detected in cultivated Chinese radish cultivars but not in the wild radish. The Chinese radish orf138 nucleotide sequence was determined in four male-sterile lines and displayed complete homology to the known orf138 type A nucleotide sequence. Three types of mitochondrial orfB (type 1, type 2 and type 3) were found in Chinese radishes. Type 1 was only present in the male-sterile lines. Chinese cultivated radishes were divided into type 2 and type 3, while the Chinese wild radish only had type 3 cytoplasm.