Positive selection based on trehalose

Expressive Rsearch B.V. obtained an European patent claiming a method for the selection of transformed plant cells using trehalose. Related patent applications were also filed in the United States, Canada and some other jurisdictions such as Japan and China.

Technology overview

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. As a fact, trehalose does not accumulate to detectable levels in most plants. Cells, in particular plant cells, can not normally develop in a medium in which an increased concentration of trehalose is present without another metabolizable carbon source being present.

Positive selection methods were developed by transforming target plant cells with genes coding for trehalase, which is able to hydrolyze intracellular trehalose and/or its derivative to glucose for plant use, and selecting transformed cells on a medium containing trehalose as the sole carbon source. The genes coding for intracellular trehalases can be of bacterial origin (e.g. the TreF gene from E. coli) or of plant origin (e.g. the AtTRE1 gene from Arabidopsis).

The gene coding for an endogenous trehalase exists in the genomes of higher plants such as soybeen and Arabidopsis thaliana. However, these endogenous trehalase genes generally code for an extracellular trehalase, which is not active in the cell. Such endogenous genes can be modified by a number of ways such as deactivating the protein-secretion signal via deletion or mutagenesis, changing the protein targeting sequences, or the pH sensitivity of the enzymatically active site to make the modified trehalases intracellularly active.

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