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The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements

Published online by Cambridge University Press:  01 June 2000

NEIL W. ASHTON
Affiliation:
Department of Biology, University of Regina, Regina, S4S 0A2, Canada
CONNIE E. M. CHAMPAGNE
Affiliation:
Department of Biology, University of Regina, Regina, S4S 0A2, Canada
TRACEY WEILER
Affiliation:
Department of Biology, University of Regina, Regina, S4S 0A2, Canada
LAURENT K. VERKOCZY
Affiliation:
Department of Biology, University of Regina, Regina, S4S 0A2, Canada
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Abstract

Physcomitrella patens, recently renamed Aphanoregma patens, has been transformed with the plasmid, pBI426. On selective medium approx. 30% of regenerants expressed the transformed phenotype transiently (transients). The remaining 70% (transformants) retained their transformed phenotype (GUS-positive and resistant to G418) indefinitely when subcultured repeatedly on selective medium. However, most lost this phenotype after one or two passages through nonselective medium (unstable transformants). Approximately 0.2% of transformants retained their transformed phenotype after numerous passages through nonselective medium (stable transformants). Using PCR methodology, it has been shown that loss of the transformed phenotype by unstable transformants is invariably accompanied by disappearance of the transgenic DNA. Southern blot analysis data argue strongly that unstable transformants cultured under selective conditions contain unintegrated pBI426 as circular concatenates consisting of 3–40 copies of the plasmid. Under selective conditions, it appears that replication and/or partitioning of these extrachromosomal concatemers might be growth rate-limiting. This is the first report of a transgenic, autonomously replicating extrachromosomal element in a photosynthetic plant. A single copy of pBI426 has been inserted into the moss genome in each of three stable transformants analysed.

Type
Research Article
Copyright
© Trustees of the New Phytologist 2000

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