Gene targeting and genetic transformation of plants

Abstract

A broad definition of gene targeting includes any method that can lead to permanent sitespecific modification of the genome [1], preferably with predetermined outcomes. More specifically, gene targeting is the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome, and often refers to the conversion of the endogenous gene into a designed sequence [2]. Rapid developments in the field of gene targeting, and the potential of the technology to revolutionalise genomics and plant biotechnology in particular has led to the adoption of this broad definition, over earlier definitions such as that by [3] and [4] that restricted gene targeting to homologous recombination mechanisms. While gene targeting does not necessarily lead to marker-free, vector backbone-free transforma‐ tion, gene targeting certainly brings these desired outcomes of plant transformation research closer. Such marker-free, vector backbone-free plants will be truly and precisely engineered plants, and might actually be non-transgenic, depending on the source of the sequences used. Gene targeting in Drosophila, mice and yeast is now more or less routine [5]. Transgenic organisms for use in research are ‘made-to-order’ via gene targeting and are sold by commercial compa‐ nies. Gene targeting in animals is accomplished via homologous recombination (HR). Howev‐ er, the same cannot be said of plants. Approaches adapted from gene targeting in yeast, insect and animal models have failed to give comparable results in plants mainly because the predom‐ inant mechanism of recombination in somatic cells of plants is not HR, but is non-homologous end joining, NHEJ, also known as illegitimate recombination