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  • 1
    ISSN: 1432-2145
    Keywords: Nicotiana ; Cytoplasmic male sterility ; Flower morphogenesis ; mtDNA organization ; In organello protein synthesis ; Mitochondrial transcripts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A detailed description of in planta floral ontogeny based on scanning electron microscopy, developmental histology and morphology is presented for three different alloplasmic gene-cytoplasmic male-sterile (CMS)Nicotiana tabacum isonuclear lines with cytoplasms ofN. bigelovii, N. debneyi and N. suaveolens and compared to the corresponding nuclear donorN. tabacum genotype. This allowed the precise determination of the developmental stages affected in the mutant forms as well as a thorough phenotypic characterization of them. The organization of the mitochondrial genome and expression of mitochondrial DNA (mtDNA) was investigated in the three different alloplasmic CMS tobacco analogs and compared to the corresponding malefertile parentalNicotiana species. Southern hybridizations of total cellular DNA and mtDNA from the different sets of lines with probes specific for mitochondrial genes coding for cytochrome oxidase subunits I, II and III, apocytochrome b, ATPase subunits α and 9 as well as 18S-5S ribosomal RNA indicated that: (a) mtDNA organization is different between mitochondrial genomes of fertile and sterile lines but identical in two different fertile tobacco lines; however genetic similarity among different mitochondrial genome types can be revealed by restriction fragment patterns; (b) although several differences were detected between the male-sterile and male-fertile plants, most of these were related to the origin of the mitochondria (cytoplasm donorNicotiana species); (c) identical mtDNA rearrangements — distinct to the cytoplasm donor — occur in cytoplasmic malesterile tobacco analogs bearing cytoplasm fromN. bigelovii in two differentN. tabacum nuclear backgrounds, indicating that mitochondrial genome structure inNicotiana is altered by substitution of the nuclear back-ground, since (d) inter- and intraspecific mitochondrial genome diversity among differentNicotiana species and the corresponding alloplasmic CMS tobacco analogs can be determined by hybridization with mtDNA specific probes. Analysis of in organello translation products in the three CMS-systems described confirmed the presence of variant proteins synthesized by mitochondria from CMS and male-fertileNicotiana isonuclear lines. In addition, differences due to the origin of both the nucleus and the cytoplasm, which involve changes in the presence or size of particular polypeptides, are apparent. A common feature of all three systems — including two different nuclear backgrounds — is the enhanced synthesis of a 31-kDa polypeptide in the strictly isonuclear CMS lines compared to the male-fertile tobacco. In addition, organellar gene expression was studied by Northern blot analysis of transcripts homologous to mitochondrial gene probes, revealing variant mtRNA species associated with some CMS lines.
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  • 2
    ISSN: 1432-203X
    Keywords: forage grasses ; Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions. Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10-3 to 10-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.
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  • 3
    ISSN: 1432-203X
    Keywords: Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration ; somaclonal variation ; genetic fidelity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials. The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 81 (1991), S. 477-486 
    ISSN: 1432-2242
    Keywords: One-to-one electrofusion ; Cybridization ; Cytoplast (enucleated protoplast) ; Cytoplasmic male sterility ; Nicotiana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Defined cybridization was performed by one-to-one electrofusion (microfusion) of preselected protoplast-cytoplast pairs of male-fertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile, streptomycin-sensitive N. tabacum cms (N. bigelovii), followed by microculture of the fusion products until plant regeneration. Dominant selectable markers, namely, kanamycin resistance (nptII) and hygromycin B resistance (hpt) genes had been previously integrated in the nuclear genomes of the otherwise almost fully isogenic parental strains using direct gene transfer to protoplasts. In addition to chromosome counts indicating the expected allotetraploid tobacco count of 48, the absence of the nucleus from the cytoplast donor line was confirmed by Southern blot hybridization using nptII and hpt probes, as well as by an in vitro selection test with leaf expiants and the corresponding enzyme assays for 30 cybrids. The cytoplasmic composition of the cybrids obtained was analyzed for chloroplast type using the streptomycin resistance/sensitivity locus. The fate of mitochondria in cybrids was checked by species-specific patterns in Southern analysis of restriction endonuclease digests of total DNA with N. sylvestris mitochondrial DNA probes.
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  • 5
    ISSN: 1432-2242
    Keywords: Electric field-induced microfusion ; Microculture ; Nicotiana ; Somatic hybridization ; Cytoplasmic male sterility
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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