Plant Transformation

Abstract

The genetic constitution of plants can be altered in the laboratory by a process called transformation, whereby a segment of DNA ( deoxyribonucleic acid ) is introduced that becomes inserted in one of the plant chromosomes. Several methods to accomplish plant transformation have been devised. In all these methods, single cells are transformed and thereafter regenerated into complete, fertile plants by tissue culture procedures. Some of the transformation methods can only be applied to protoplasts (cells from which the walls have been removed). Nowadays, particle bombardment and the natural vector Agrobacterium tumefaciens are preferred because they can also cope with whole plant tissues such as roots and leaves, which are easier to handle, more stable and require less of the lengthy steps that are required for plant regeneration. Transgenes are integrated at random positions in the plant genome, but procedures for targeted integration become more and more efficient. Key concepts: Transgenic plants are plants derived from cells in which genes (often of nonplant origin) have been stably introduced by transformation to give the plant a new and useful trait. Transgenic plants can be obtained after transformation of single cells and the subsequent regeneration into complete, fertile plants by tissue culture protocols. Transformed plant cells can be identified by their ability to grow on selective media containing an antibiotic or a herbicide as transformation vectors contain selection genes conferring such properties. Novel functions are expressed in transformed plant cells if the coding regions are surrounded by promoter and terminator regions that are recognized by the plant transcription machinery. The most preferred methods for plant transformation use either the particle gun or the natural transformation system of Agrobacterium tumefaciens , as they can cope with cells present in whole plants or tissues. Agrobacterium tumefaciens can be disarmed by deletion of the onc ‐genes that are naturally present between the 25‐bp repeats of the T‐DNA. Any gene introduced between these repeats is translocated into plant cells by Agrobacterium tumefaciens. Transformed cells with a single copy of the transgene usually show higher and more stable expression than multicopy lines, in which expression may suffer from posttranscriptional gene silencing. (T)‐DNA integration in plant cells occurs at random sites in the genome by nonhomologous end‐joining and related backup pathways. Targeted integration of transgenes can be accomplished in plant cells by using either a site‐specific recombination system (e.g. Cre‐ lox ) or by homology‐directed integration in combination with a site‐specific nuclease (e.g. a zinc‐finger nuclease). Agrobacterium tumefaciens ‐mediated transformation can be applied not only in dicotyledonous plants, but also in monocots such as cereals and in yeasts and fungi. The T‐DNA can be used as a mutagen causing insertion mutations. Libraries of Arabidopsis thaliana T‐DNA transformants are in use now in mutant screens to identify insertion mutations in genes of interest (reverse genetics).

Development of a Molecular Breeding Strategy for the Integration of Transgenic Traits in Outcrossing Perennial Grasses

“…Genetic transformation technology allows for the introduction of novel genetic variation through the introgression of genes from related or unrelated species through transformation [1] and have been extensively reviewed [29][30][31][32][33]. The uncontrolled nature of both Agrobacterium-mediated transformation and biolistic transformation could lead to multiple insertion sites and complex integration patterns that could potentially lead to insertional mutagenesis [34].…”