An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus

Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transferring genes across species, the presence of selectable marker genes, and bacteria-derived vector backbone sequences have raised considerable health and environmental concerns. Intragenic vector system-based intragenesis/cisgenesis is a new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the associated shortcomings. We report here the development of an intragenic vector by assembling a T-DNA-like fragment and a buffering sequence following the left border from Citrus clementina into the backbone of the binary vector pCB302. Recovery of citrus regenerants is performed under non-selective conditions and positive intra-/cisgenic regenerants were identified through PCR analysis. Transformation efficiencies obtained in Arabidopsis and “Duncan” grapefruit were ~3% and ~0.67%, respectively, demonstrating the potential of the system for development of “foreign DNA-free” intra-/cisgenic citrus cultivars.