Molecular and General Genetics MGG

, Volume 251, Issue 3, pp 373–380

Selectable marker recycling in the chloroplast

Authors

  • N. Fischer
    • Departments of Molecular Biology and of Plant BiologyUniversity of Geneva
  • O. Stampacchia
    • Departments of Molecular Biology and of Plant BiologyUniversity of Geneva
  • K. Redding
    • Departments of Molecular Biology and of Plant BiologyUniversity of Geneva
  • J. -D. Rochaix
    • Departments of Molecular Biology and of Plant BiologyUniversity of Geneva
Original Paper

DOI: 10.1007/BF02172529

Cite this article as:
Fischer, N., Stampacchia, O., Redding, K. et al. Molec. Gen. Genet. (1996) 251: 373. doi:10.1007/BF02172529

Abstract

The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.

Key words

Chlamydomonas reinhardtiiChloroplast transformationaadARecombination

Copyright information

© Springer-Verlag 1996