The increasing availability of genomic information allows an unprecedented ability to link phenotypes with genotypes and subsequently to identify key genes involved in important agronomic traits. Crops with improved characteristics can then be produced by the targeted introduction of subtle mutations into such genes. KeyBase® is a targeted mutagenesis technology to introduce single point mutations into plant genes of interest, which is achieved by the introduction of chemically modified oligonucleotides into plant protoplasts. The sequence of the oligonucleotide is identical to the target gene except at the position of the desired mutation and this mismatch drives the alteration of the genomic sequence. The chemical structure and the design of the oligonucleotides have been optimized for plant cells. KeyBase oligonucleotides contain locked nucleic acid residues (LNAs) for increased targeting efficiency. Efficient protoplast techniques have allowed the regeneration of fully fertile plants carrying specific mutations after KeyBase treatment in tobacco, tomato, Petunia and oilseed rape. All KeyBase mutations have been confirmed by sequencing, and correct Mendelian transmission of the mutations to the offspring after selfing of the mutant plants demonstrate the stability of the mutations. KeyBase is now a routine method to produce herbicide-tolerant plants. Currently, the technology is being developed for rice and maize. As for rice, thousands of microcalli derived from protoplasts were generated in several independent experiments, reaching estimated plating efficiencies of up to 20%. The average plant regeneration efficiency from protoplast derived calli was 14%.
The KeyBase® technology is covered by patents and patent applications owned by Keygene N.V. KeyBase is a registered trademark of Keygene N.V. in one or more territories in the world.