THE PLANT CELL, Vol 4, Issue 1 39-45, Copyright © 1992 by American Society of Plant Biologists
Long Regions of Homologous DNA Are Incorporated into the Tobacco Plastid Genome by Transformation
J. M. Staub and P. Maliga
Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0759
We investigated the size of flanking DNA incorporated into the tobacco
plastid genome alongside a selectable antibiotic resistance mutation. The
results showed that integration of a long uninterrupted region of
homologous DNA, rather than of small fragments as previously thought, is
the more likely event in plastid transformation of land plants.
Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted
repeat region of the tobacco plastid genome. A spectinomycin resistance
mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a
streptomycin resistance mutation is encoded in exon II of the ribosomal
protein gene rps12. Transplastomic lines were obtained after introduction
of pJS75 DNA into leaf cells by the biolistic process and selection for the
spectinomycin resistance marker. Homologous replacement of resident
wild-type sequences resulted in integration of all, or almost all, of the
6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains
engineered cloning sites between two selectable markers, can be used as a
plastid insertion vector.
