Plant Transformation Technology:Agrobacterium‐Mediated Transformation
Authors: Komari Toshihiko , Ishida Yuji , Hiei Yukoh 
Published: July 2004

The soil phytopathogenAgrobacterium tumefaciensinduces tumours, known as crown galls, mainly on dicotyledonous plants. Such tumours are generated by a complex, multi‐step transformation process. Another species,A. rhizogenes, causes hairy root disease in higher plantsviaan identical process.Agrobacteriumhas been utilised for the transfer of genes to dicotyledonous plants. Now, monocotyledonous plants are routinely transformed byAgrobacteriumdespite the fact that these plants, including important cereals, were thought until recently to be outside the range of this technology.

Most of the common transformation methods heavily depend on tissue culture technology and we refer to such methods asin vitrotransformation. The only case of a routine tissue culture free method is the ‘in planta’ transformation ofArabidopsis thaliana. Numerous critical factors are involved in both approaches. In transformationin vitro, key factors include choice of vectors and bacterial strains, types of plant tissues to be infected, procedures of preparation the tissues, protocols of infection and co‐cultivation, methods for subsequent culture and selection of transformed cells, antibiotics to remove infecting bacteria, techniques for regeneration of transgenic plants and genotypes of plants. It is our opinion that the type and quality of the starting material is the most important one among them. The capacity to serve as a host plant for crown gall disease is no longer a prerequisite for a host of vector systems based onAgrobacterium. The real prerequisite forin vitrotransformation is the availability of technology for dedifferentiation of tissues and regeneration of plants in a given species. On the other hand, the biological processes involved inin plantatransformation are yet to be elucidated. Recent evidence suggested that ovules are the primary target, and further basic understanding is likely to help extend the number of species transformed by the method.

The plant species that are routinely transformed byAgrobacteriumare expanding rapidly. Some gymnosperms, several forest trees and fruit trees, various legumes, and cereal and non‐cereal monocotyledons, which were once considered very recalcitrant, are now in a long list of transformable plants.Arabidopsis, tobacco and rice are the top three species that were transformed during the last two years. Although it is only half a dozen years since the current procedure of rice transformation mediated byAgrobacteriumwas published, the economic importance and the accumulation of genomic information has made rice the species of focus in plant biotechnology.

The advantages ofAgrobacterium‐mediated transformation include the transfer of pieces of DNA with defined ends and minimal rearrangement, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes and the high quality and fertility of transgenic plants. However, transformation does not always produce such ‘clean’ events. Formation of repeats of transgenes, certain rearrangements, integration of non‐target DNA segments and unstable expression of transgenes are among the complications. Although the majority of transgenic plants usually appear ‘good’ in a particular test, accumulation of ‘dropped’ plants is significant after multiple rounds of characterisation and screening. Therefore, further improvement in each of steps is highly desired.

Application of the gene transfer mediated byAgrobacteriumis further expanding. Transient expression of genes delivered byAgrobacteriumis now a useful tool in the study of promoters and gene function. Vectors specifically designed to carry very large segments of DNA have been developed and extensively tested. Insertional mutagenesis by DNA transferred byAgrobacteriumis a routine technique in genomics study inArabidopsisand rice. Methods for targeted integration of transgenes to genomes of higher plants have been drawing considerable attention. Various technologies for the production of ‘selection marker free’ transgenic plants are now in place for better public acceptance of biotechnology products.