Efficient transformation mediated by Agrobacterium tumefaciens with a ternary plasmid in Pharbitis nil

Abstract

Pharbitis nil Choisy has a large number of mutants and is well known in classical genetics. The mutants exhibit particularly rich and varied floral colors and patterns compared to other plants. P. nil is a typical short-day plant and has therefore been used as a model plant for the genetic analysis of floral colors and patterns and for the photoperiodic induction of flowering. In this paper, we describe an efficient transformation protocol mediated by Agrobacterium tumefaciens in P. nil. The binary vector pBI121, containing the neomycin phosphotransferase II (NPT II) gene, was used as a selectable marker, and the luciferase (Luc⁺) gene was used as a reporter instead of β-glucuronidase (GUS). Agrobacterium tumefaciens strain GV3101 is carried in the binary vector, while that of LBA4404 is contained the binary and ternary vector, which expresses a constitutive virG mutant gene (virGN54D). We infected 393 somatic embryos with the Agrobacterium strain LBA4404/virGN54D/pBI121-Luc⁺. Fifty-seven kanamycin-resistant shoots were obtained after 2–3 months of culturing in a selection medium, and over 20% of the regenerated shoots were transgenic. Transformation was confirmed with PCR analysis, Southern hybridization, and Luc assay of the transgenic plants. All transgenic plants were morphologically normal and were fertile. The transformation efficiency in this study reached 3.1% of treated explants, which is high enough to produce transgenic P. nil with genes of interest.