Gene Transfer Through Agrobacterium Tumefaciens
Steady increase in world population is forcing the scientists to increase agriculture production. Earlier crop improvement techniques based on hybridization of genotypes have had different characteristics. However, crop improvement through conventional breeding methods requires a wide gene pool in genetically close plant species. By the discovery of recombinant DNA technology, genetic engineering has become the most widely used tool in crop improvement.
One of the most important developments in plant biotechnology is the ability to transfer foreign genes into the plant genome. The gene transfers between crops and other unrelated organisms, which have potential candidate genes, lead to the production of improved varieties in terms of yield and resistance to disease, pest, and herbicides. Many different genetic transformation techniques were developed to obtain transgenic plants over the last three decades.
Among various gene delivery techniques, Agrobacterium-mediated genetic transformation and particle bombardment is the most widely used for the genetic engineering of plants. Low copy number transgenesis and the production of high-quality transgenic plants are the most important advantages of Agrobacterium-mediated gene transfer when compared to the particle bombardment or biolistic (Dai et al., 2001). The first genetically modified plant was produced in 1982 with A. tumefaciens by using tobacco leaf tissue (Fraley et al., 1983). Up to now, gene transfer with disarmed (nontumorigenic) Agrobacterium strains has been achieved by using more than 120 plant species such as maize, wheat, soybean, cotton, tobacco, and rice . A. tumefaciens, a member of Rhizobiaceae family, has been used for genetic transformation studies in plants. This ubiquitous gram-negative soil bacterium has an ability to transfer its segment of plasmid (Ti-Ri plasmid) surrounded by repeated nucleotides into plant genome naturally. The typical Ti plasmid, with a crucial role in crown gall disease,is about 200kb. Naturally grown Agrobacterium cells carry two types of gene on T-DNA region. The first one known as oncogenic genes includes auxin and cytokinin genes. The others are responsible for opine and agropine synthesis in infected plant tissues . The proteins coded by vi(virulence) genes carried out the transfer of T-DNA region into the plant cells. Phenolic substances released from wounded plant tissues induce the activation of vir genes located in tumor-inducing (Ti) plasmid of A. tumefaciens. There are about 30 genes in A. tumefaciens vir regulon, and about 20 of them are required for tumor formation in plant tissues. This regulon consists of at least six operon (VirA, VirB, VirC, VirD, VirG, and VirE) required for single-stranded T-DNA generation and transfer into the host plant cell genome. The improvement of plants through the gene transfer mainly relies on the tissue-culture response of genotypes or species. In order to generate transgenic plants, suitable transformation methods and a robust regeneration protocol are required. By this context, some of the species and explants may not be suitable for Agrobacterium-mediated gene transfer. Especially, monocotyledon plant species are recalcitrant to Agrobacterium-mediated transformation. These groups of plants are not naturally infected by A. tumefaciens due to the lack of phenolic substances required for induction of vir genes. By using artificial phenolic substances and hypervirulent strain, monocot plants such as cereal can be transformed by A. tumefaciens. Another group of plants also cannot be genetically engineered because of their low regeneration potential. As previously mentioned, the production of transgenic plants requires tissue- culture steps. Recently, tissue-culture-independent methods have been demonstrated to work in a limited number of plant species.
Single-stranded T-DNA generation and transfer into the host plant cell genome