Development of Chemical Inducible Deletion Technologies for Citrus Marker-Free Transformation

摘要

Antibiotic or herbicide resistant selective genes are widely used as markers in transformation experimentation for the selection of transgenic regenerants from wild type tissues. However, the potential hazards on ecological environment and human health of such marker genes remaining in the transgenic plants have caused common social worries and concerns. In the present study, a chemical-inducible auto-excision marker free vector system with CrelloxP site-specific recombination system and an inducible expression system XVE was used in citrus. The result indicated that marker gene excision in this vector could occur in citrus transformants. When regenerated buds reached to 2-4 mm with 1-2 mm explants, they were cut and transformed to chemical-inducible medium (MS+ lmg/L 6-BA + 4 μM β-estradiol). After reaching 1 cm, shoots were cut and subcultured on chemical-inducible medium for another 30 days. Molecular detection and fluorescence observation indicated that different gene recombination and partially excisions existed in various transgenic citrus clones. The complete excision of marker genes was detected in 2 of the 80 transgenic clones. The suitable conditions for the induction of removing marker genes were discussed.