Linked Life Data

8680308

Source:http://linkedlifedata.com/resource/pubmed/id/8680308

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1996-8-16
pubmed:abstractText
A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
B
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
0032-0935
pubmed:author
pubmed:issnType
Print
pubmed:volume
199
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
193-201
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.
pubmed:affiliation
Botanisches Institut der Universität München, Germany. koop@botanik.biologie.uni-muenchen.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't