Success Of Genetic Transformation Of A Cell Biology Essay


There are many ways to achieve the introduction of DNA into a cell or tissue but the most widely used method being based on the natural gene transfer system of the soil bacterium Agrobacterium tumefaciens. Although dicotyledonous plants were considered to be include the Agrobacterium tumefaciens host range (Raineri et al., 1990 and Gould et al., 1991), but successful transformation of rice by Agrobacterium has been reported in the last decade (Chan et al., 1992 and 1993 and Toki, 1997). However, some monocotyledonous species (japonica) are easily sensitive to transformation by Agrobacterium tumefaciens, except some species (indica) which remain recalcitrant to this method; but this problem can be overcome by inducing embryogenic callus as experiment material for Agrobacterium transformation or introducing DNA by direct gene transfer.

Generally, calli derived from immature embryos and mature seeds are used in rice for Agrobacterium-mediated transformation. Now-a-days, Agrobacterium-mediated transformation using scutellum derived calli from mature seeds have been established successfully in various cultivars of japonica rice as routine protocols (Nishimura et al., 2006; Toki et al., 2006; Hiei and Komari, 2008 and Toki, 2010). But many indica rice varieties are still recalcitrant using calli derived from mature seeds to Agrobacterium-mediated transformation (Kumar et al., 2005) and thus there is no more reports for efficient transformation.

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Conversely, immature embryos are more capable than mature seeds for Agrobacterium-mediated transformation in several rice genotypes of both indica and japonica (Hiei and Komari, 2008). But there is problem to prepare and manipulate of immature embryos because it is not available throughout the year at the proper developmental stage. Therefore, scutellum derived callus from mature seeds has become amenable for both japonica and indica rice.

Choosing a suitable vector where DNA sequences can be inserted is the important part for genetic transformation (Sambrook et al., 1989). Vector is an agent that facilitates to transfer foreign genetic material into plant genome.

In the early and mid-1980s, fundamental skeleton of the current vectors were developed for transformation of higher plants, afterward it had been revealed that crown gall tumorigenesis symbolized the genetic transformation of plant Cells (Fraley et al., 1986). The earliest success was the removal of wild-type T-DNA from Ti plasmids to generate “disarmed strainsâ€Â that causes tumors and inhibits plant regeneration (Hoekema et al., 1983). For transformation, interested genes in E. coli vectors could be introduced into a disarmed Ti plasmid (Fraley et al., 1986). Although, this system was efficient but there was limit that plasmid size was large in A. tumefaciens finally and structure confirmation was not simple. After that binary system was discovered, where T-DNA and virulence genes are located on different replicons in an A. tumefaciens and the transformation process of T-DNA was dynamic (Hoekema et al., 1983).

Later, transformation technologies for monocotyledons have much improved since the development of super binary vectors (Komari et al., 1996). Integration of an intermediate vector with an acceptor vector in A. tumefaciens, is the last step construction of a super-binary vector.

Interested genes are inserted into proper place of a binary vector by standard subcloning techniques. There are multiple cloning sites in standard vectors that are very useful in this regard. However, recently constructed vectors are more friendly for users because DNA fragments that are to be inserted into vector where is recognition sites, are very useful in this respect. A series of these rare sites are sited in the T-DNA in some of the recently created vectors (Chung et al., 2005).

There is another valuable cloning technology, GATEWAY system (Invitrogen) which based on phage lambda site-specific recombination mechanism. A DNA fragment may easily be substituted with another DNA fragment by this system. Thus GATEWAY system is a useful cloning system that is especially helpful in the production of T-DNA with several genes (Chen et al., 2006).

For overexpression and knock-down of effector genes, several binary vectors which based on GATEWAY system have been developed (karimi et al., 2002; Chung et al., 2005 and Earley et al., 2006). Most recently, IPKb vector set was developed combined with GATEWAY-cassettes for either over-expression or RNAi-constructs that contain a number of useful plasmid elements such as pVS1 origin of replication, promoters for monocot-compatible and selectable marker genes (Himmelbach et al., 2007).

Agrobacterium also facilitate to insert a particular segment of foreign DNA into the plant genome under the control of genetic elements such as the promoter. The promoter is a regulatory element and it is located at the immediate position of transcription start site (Beilmann et al., 1992). The promoter has the ability to binds RNA polymerase and start the RNA synthesis through enzymes (Oliver and Ward, 1985).

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Different promoters have different efficiencies and thus transcriptional levels vary. Mainly, promoters from Ti plasmids, such as nopaline synthase (Nos), octopine synthase (Ocs), mannopine synthase (Mas) and the 35S promoter derived from cauliflower mosaic virus are used for dicotyledonous plants and also the 35S promoter and the promoters derived from the ubiquitin (Ubi) gene of maize and the actin (Act) gene of rice are used for monocotyledonous plants (Vickers et al., 2006).

McElroy et al., (1990) reported that rice actin 1 gene (Actl) is an efficient promoter for controlling the constitutive expression of a foreign gene in transgenic rice.

Plant actin promoters can be active in all tissues since actin is a essential component of the plant cell cytoskeleton (Seagull, 1989). It was examined that the rice actin 1 gene, Actl, encoded a transcript that was reasonably abundant in all rice tissues and even all developmental stages (McElroy et al., 1990). Conversely, transformation frequency was much higher under the control of Ubi promoter in cereals (Ishida et al., 2004)