Co-Transfer of TALENs Construct Targeted for Chloroplast Genome and Chloroplast Transformation Vector Into Rice Using Particle Bombardment

Chloroplast transformation offers a number of advantages over nuclear transformation, such as high-level expression, multi-gene engineering in a single transformation event, maternal inheritance of transgene, and lack of gene silencing and position effects. To date, chloroplast transformation technology has become a valuable tool for genetic modification in higher plants, especially for dicotyledons and chloroplasts it offers a viable alternative to conventional bioreactor systems. However, this emerging technology has still not been efficiently applied in monocotyledonous cereal crops because of the current inability to obtain homoplasmic transplastomic plants. One of potentially major obstacles could be the inherently low transformation efficiency of exogenous DNA into chloroplast genomes by homologous recombination. Transcription activator-like effector nucleases (TALENs) have emerged as a newly powerful tool for precise genome editing in plants in recent years, and they show great stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) for gene insertion. Herein in this study, we constructed the TALENs targeted for rice chloroplast genome which was co-transferred with chloroplast homologous recombination plasmid containing hpt (encoding hygromycin phosphotransferase) and EGFP (encoding enhanced green fluorescence protein) genes by particle bombardment. Results showed that the transformation efficiency for the exogenous fragments with assistance from the TALENs was approximately two times more than that of solely transforming the chloroplast homologous recombination plasmid, preliminarily providing the foundation for future experiments to explore the establishment of chloroplast transformation system in monocotyledonous crops.