FlashBack DNA Project By Isabel Cutter
Series 9 Protocol Treaty Poisoning of the Earth
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Let me retrieve the email from Lan Ying Lee and Stanton B Gelvin
Dear Alexis And Atom
For more than two decades, scientists have used Agrobacterium-mediated genetic transformation to generate transgenic plants. Initial technologies to introduce genes of interest (goi) into Agrobacterium involved complex microbial genetic methodologies that inserted these goi into the transfer DNA (T-DNA) region of large tumor-inducing plasmids (Ti-plasmids). However, scientists eventually learned that T-DNA transfer could still be effected if the T-DNA region and the virulence (vir) genes required for T-DNA processing and transfer were split into two replicons. This binary system permitted facile manipulation of Agrobacterium and opened up the field of plant genetic engineering to numerous laboratories. In this review, we recount the history of development of T-DNA binary vector systems, and we describe important components of these systems. Some of these considerations were previously described in a review by Hellens et al. (2000b).
DEVELOPMENT OF BINARY VECTOR SYSTEMS
Initial efforts to introduce goi into T-DNA for subsequent transfer to plants involved cumbersome genetic manipulations to recombine these genes into the T-DNA region of Ti-plasmids (co-integrate or exchange systems; Garfinkel et al., 1981; Zambryski et al., 1983; Fraley et al., 1985; Fig. 1A ). This was because Ti/Ri-plasmids are very large, low copy number in Agrobacterium, difficult to isolate and manipulate in vitro, and do not replicate in Escherichia coli, the favored host for genetic manipulation. T-DNA regions from wild-type Ti-plasmids are generally large and do not contain unique restriction endonuclease sites suitable for cloning a goi. In addition, scientists wanted to eliminate oncogenes from T-DNA to regenerate normal plants. Opine synthase genes were also generally deemed superfluous in constructions designed to deliver goi to plants.
Figure 1.Schematic diagram of co-integration/exchange systems and T-DNA binary vector systems to introduce genes into plants using Agrobacterium-mediated genetic transformation. A, Co-integration/exchange systems. Genes of interest (goi) are exchanged into the T-DNA region of a Ti-plasmid (either oncogenic or disarmed) via homologous recombination. Following exchange, the exchange/co-integration vector can be cured (removed) from the Agrobacterium cell; B, T-DNA binary vector systems. Genes of interest are maintained within the T-DNA region of a binary vector. Vir proteins encoded by genes on a separate replicon (vir helper) mediate T-DNA processing from the binary vector and T-DNA transfer from the bacterium to the host cell. The selection marker is used to indicate successful plant transformation. ori, Origin of replication; Abr, antibiotic-resistance gene used to select for the presence of the T-DNA binary vector in E. coli (during the initial stages of gene cassette construction) or in Agrobacterium.
We hope this helps.
Lan Ying Lee and Stanton B Gelvin
Scholars of Biology
Federation of American Societies for Experimental Biology (FASEB) 9650 Rockville Pike, Bethesda, MD 20814 | Phone: 301.634.7000 | Fax: 301.634.7001
Isabel M Cutter