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  • 1
    Electronic Resource
    Electronic Resource
    Structure, organization and expression of transferred DNA in Nicotiana plumbaginifolia crown-gall tissues (1987)
    Springer
    Planta 171 (1987), S. 393-405 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Crown-gall ; DNA, transferred ; Nicotiana (T-DNA) ; T-DNA structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Data are provided which show that transferred DNA (T-DNA) present in Nicotiana plumbaginifolia crown-gall lines in most cases was scrambled and not intact. Both wild-type, and ‘rooter’- and ‘shooter’-type mutants of octopine-type Agrobacterium tumefaciens were used to infect N. plumbaginifolia plantlets, cultured in vitro. Resulting tumors were excised from the plantlets and cultured for more than three years. During subculturing the tumor lines were scored for the following phenotypic traits: phytohormone autonomous growth in vitro (Aut+), spontaneous shoot regeneration (Reg+), root deficiency of shoots (Rod+), octopine production (Ocs+) and mannopine and agropine production (Mas+Ags+). An unexpectedly large variety of phenotypes was observed. For instance, two out of three tumor lines induced on haploid plantlets by the rooter mutant LBA4210 regenerated shoots, a phenomenon which is not observed for octopine tobacco tumor lines. Fifty percent of the crown-gall lines studied did not contain octopine. Only one line out of six independent lines analyzed was found to have a ‘regular’ T-DNA structure. Occurrence of aberrant T-DNA structures was not correlated with the ploidy level of infected plantlets, nor with the T-region structure of the inciting bacterial strain. The pattern of TL-DNA transcripts was studied for one line and correlated well with the aberrant T-DNA structure detected. Segments of TR-DNA, having irregular structures as well, were detected in two out of the six lines studied. The scrambled nature of the TR-DNA explained the absence of mannopine and agropine in these two lines. In addition, it was observed that N. plumbaginifolia tissue lines which did not carry T-DNA, became readily phytohormone autotrophic (habituated) at an early stage in tissue culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00398685
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization, T-DNA methylation and conditional expression of opine genes (1986)
    Springer
    Plant molecular biology 7 (1986), S. 285-299 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; crown gall ; Nicotiana tabacum ; T-DNA structure ; DNA methylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut+), shoot regeneration (Reg+) and octopine synthesis (Ocs+)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut+Reg+ but shows little or no octopine synthesis activity (Ocs-). Subclones of TSO38, however, are either Ocs- or Ocs+. Ocs- shoots become Ocs+ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs+ and an Ocs- subclone of TSO38, which were negative for the presence of mannopine (Mas-) and agropine (Ags-), became Mas+Ags+ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs- line and in the Ocs+ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs- and an Ocs+ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs+ and an Ocs- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs- and the Ocs+ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00752901
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations (1986)
    Springer
    Plant molecular biology 7 (1986), S. 265-284 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; crown gall ; Nicotiana tabacum ; T-DNA structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having a Tn1831 insertion in the auxin locus, were investigated for their T-DNA structure and expression. In addition to clones with the expected phenotype, i.e. phytohormone autonomy, regeneration of non-rooting shoots and octopine synthesis (Aut+Reg+Ocs+ 'type I' clones), clones were obtained with an aberrant phenotype. Among these were the Aut-Reg-Ocs+ 'type II' clones. Two shooty type I clones and three type II callus clones (all randomly chosen) as well as a rooting shoot regenerated from a type II clone via a high kinetin treatment, all had a T-DNA structure which differed significantly from ‘regular’ T-DNA structures. No Tn1831 DNA sequences were detected in these clones. The two type I clones were identical: they both contained the same highly truncated T-DNA segments. One TL-DNA segment of approximately 0.7 kb, originating form the left part of the TL-region, was present at one copy per diploid tobacco genome. Another segment with a maximum size of about 7 kb was derived from the right hand part of the TL-region and was present at minimally two copies. Three copies of a truncated TR-DNA segment were detected, probably starting at the right TR-DNA border repeat and ending halfway the regular TR-region. Indications have been obtained that at least some of the T-DNA segments are closely linked, sometimes via intervening plant DNA sequences. The type I clones harbored TL-DNA transcripts 4, 6a/b and 3 as well as TR-DNA transcript 0′. The type II clones harbored three to six highly truncated T-DNA segments, originating from the right part of the TL-region. In addition they had TR-DNA segments, similar to those of the type I clones. On Northern blots TR-DNA transcripts 0′ and 1′ were detected as well as the TL-DNA transcripts 3 and 6a/b and an 1800 bp hybrid transcript (tr.Y) containing gene 6b sequences. Possible origins of the observed irregularities in T-DNA structures are discussed in relation to fidelity of transformation of plant cells viaAgrobacterium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00752900
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    Paper (German National Licenses)
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