Split Selectable Genetic Markers Enable Multi-Gene Plant Transformation

A single marker for multiple genes

Diagram showing co-selection of two transgenes

System for gene stacking in woody and herbaceous plants. A split selectable genetic marker enables co-selection of two separate transgenic vectors simultaneously. (In this image, Kan and Hyg are genes for kanamycin or hygromycin antibiotic resistance used to select transformed seedlings. RUBY and eYGFPuv are reporter genes which enable phenotypic identification of transformed seedlings.)
[Reprinted under a Creative Commons license (CC BY) from Yuan et al. 2023. DOI:10.1038/s42003-023-04950-8]

The Science

Complex plant traits, such as yield, are controlled by multiple genes. The ability to introduce, or stack, multiple genes into plants can accelerate development of crops with desirable traits. However, gene stacking is challenging due to limited options for the selectable markers used during transformation.

To expand the toolkit of selectable markers, researchers demonstrated the functionality of a split selectable marker system in tobacco. First, a marker gene (RUBY) was divided into two fragments and tagged with intervening protein domains called inteins. The two partial fragments were then cloned into plasmid vectors and co-transformed into plants. The partial selectable marker fragments were then reassembled into full-length functional proteins via trans-splicing of the split inteins. This process demonstrated the functionality of the split selectable marker system in plants.

Next, the researchers demonstrated that this system could simultaneously introduce multiple genes using a single, split marker into both herbaceous (Arabidopsis thaliana) and woody (poplar) plants. This time they used split genes for kanamycin and hygromycin antibiotic resistance (split-KanR and split-HygR), which would enable selection of successfully transformed plants. Co-transformation with two reporter genes, RUBY and eYGFPuv, carried on the transforming plasmids along with the split marker gene was confirmed phenotypically. This demonstrated the functionality of using a single marker gene to introduce multiple genes into plants.

The Impact

Researchers demonstrate for the first time in plants that the split selectable marker systems split-KanR and split-HygR are effective for co-transformation of multiple genes in herbaceous and woody plants both in planta and plant tissue culture. Split selectable marker systems provide a valuable tool for gene-stacking, enabling markers to be used multiple times. This technology has great potential for accelerating improvement of plant traits.


This study aimed to develop split selectable markers using split inteins to enable single-selectable-marker-gene dependent co-transformation in plants.

Principal Investigator

Gerald Tuskan
The Center for Bioenergy Innovation, Oak Ridge National Laboratory
[email protected]

BER Program Manager

Shing Kwok

U.S. Department of Energy, Biological and Environmental Research (SC-33)
Biological Systems Science Division
[email protected]


This work was supported by the Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Biological and Environmental Research program.


Yuan, G., et al. 2023. “Split Selectable Marker Systems Utilizing Inteins Facilitate Gene Stacking in Plants,”
Communications Biology 6, 567. DOI:10.1038/s42003-023-04950-8.