Use of Oncogene as Selectable Marker for the Production of Transgenic Marker-free Plant Kalânchoe spathulate

Abstract

Environmental safety of genetically modified plants has led to a demand for technologies allowing the production of transgenic plants without selectable markers. An effective transformation system using Multi-Auto-Transformation [MAT) vectors helps to generate such marker-free transgenic plants that also enable repeated transformation. The MAT vector system consists of positive selection, using the oncogene ipt with a site-specific recombination and DNA removal system that generates marker-free plant Kalanchoe spathulate which is an important ornamental and model plant. A agrobacterium tumefaciens strain harboring an ipt-type MAT vector containing model gene lacZ and the removable cassette with Gus’s gene in the T-DNA region is used to produce morphologically normal transgenic plants employing iii gene as the selectable marker gene and Gus gene as a reporter gene. Explants inoculated with iii type MAT vector were cultured on hormone- and selective agent-free Murashige and Skoog medium, and fifty nine percent of the regenerated shoots developed ipt shoot phenotype, morphologically abnormal shoots, within approximately three months after co-cultivation. Twenty-one morphologically normal shoots were produced from twelve transgenic ipt shoots nine months after co-cultivation. The normal shoots rooted well on hormone-free Murashige and Skoog medium. Molecular analysis of DNA from morphological normal shoots carne from ipt-shooty lines revealed that the normal shoots had only lacZ gene and the removable cassette consisting of ipt gene was excised. This study indicates that the ipf-type MAT vector could be used for the production of morphologically normal marker-free transgenic K. spathulate plants without using selective chemical agents and it will be efficiently used for the molecular breeding of plants with repeated transformation with various useful genes.