|Srivastava, Vibha - USDA/UCB PGEC|
Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 2, 2001
Publication Date: N/A
Interpretive Summary: This article describes the use of the Cre-lox site-specific recombination system to integrate DNA into a genomic lox site in the rice genome. Three plants were regenerated from the targeted cells and all were found to harbor the introduced DNA as a single copy at the intended target site. However, two of them also have additional copies of the introduced DNA elsewhere in the genome. This study demonstrates site-specific integration of DNA in an important crop plant.
Technical Abstract: Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.