<Li> Agrobacterium - mediated transformation is the easiest and most simple plant transformation . Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid containing suspended Agrobacterium . The bacteria will attach to many of the plant cells exposed by the cut . The plant cells secrete wound - related phenolic compounds which in turn act to upregulate the virulence operon of the Agrobacterium . The virulence operon includes many genes that encode for proteins that are part of a Type IV secretion system that exports from the bacterium proteins and DNA (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus . The transferred DNA (called T - DNA) is piloted to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein VirD2, which is covalently attached to the end of the T - DNA at the Right border (RB). Exactly how the T - DNA is integrated into the host plant genomic DNA is an active area of plant biology research . Assuming that a selection marker (such as an antibiotic resistance gene) was included in the T - DNA, the transformed plant tissue can be cultured on selective media to produce shoots . The shoots are then transferred to a different medium to promote root formation . Once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the T1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an herbicide resistance gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants . Some plants species, such as Arabidopsis thaliana can be transformed by dipping the flowers or whole plant, into a suspension of Agrobacterium tumefaciens, typically strain C58 (C = Cherry, 58 = 1958, the year in which this particular strain of A. tumefaciens was isolated from a cherry tree in an orchard at Cornell University in Ithaca, New York). Though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this manner . </Li> <Li> Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant . If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells . However, genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell . For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome . The progeny of the infected plants is virus - free and also free of the inserted gene . </Li> <P> Some vector-less methods include: </P> <Ul> <Li> Gene gun: Also referred to as particle bombardment, microprojectile bombardment, or biolistics . Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos . Some genetic material will stay in the cells and transform them . This method also allows transformation of plant plastids . The transformation efficiency is lower than in Agrobacterium - mediated transformation, but most plants can be transformed with this method . </Li> <Li> Electroporation: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria . </Li> </Ul>

Where did the modified bacterium approach to gene insertion come from