Enrichments of gene replacement events by Agrobacterium-mediated recombinase-mediated cassette exchange.

Hiroyasu Ebinuma,Katsuhiko Nakahama,Kazuya Nanto

doi:10.1007/s11032-015-0215-7

Hiroyasu Ebinuma, Katsuhiko Nakahama + Show 1 more

Open Access

https://doi.org/10.1007/s11032-015-0215-7

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Abstract

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium-mediated transformation.

Highlights

In current transformation methods, variable numbers of transgenes together with co-expressed marker genes are randomly inserted into the plant genome
We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacteriummediated transformation
Transgenic tobacco plants which have a single copy of negative marker genes at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method

Summary

Introduction

Variable numbers of transgenes together with co-expressed marker genes are randomly inserted into the plant genome. We have developed Agrobacterium-mediated transformation methods which enable us to introduce a markerfree transgene into pre-defined chromosomal loci by RMCE. The concept of removing a marker gene from targeted transgenic plants is demonstrated by repeated transformation (Srivastava and Ow 2004; Nanto and Ebinuma 2008). Gene replacement events are reproduced via HR, and co-delivery of the gene of interest as a template for HR with nucleases to specific genome loci remains a problem in plants. We reported that Agrobacterium could efficiently co-deliver the gene of interest to target genome loci with recombinases and reproduce gene replacement events (Nanto et al 2005). We demonstrate the enrichment protocols of gene replacement events by Agrobacterium-mediated RMCE and discuss the common problems of HR- and RMCEmediated gene replacements

Results and discussion

E EVHEVH

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Materials and methods

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