By Kevan M. A. Gartland, Michael R. Davey
Agrobacterium Protocols bargains starting and skilled researchers the main complete number of step by step protocols for the genetic manipulation of vegetation utilizing Agrobacterium. the themes diversity from the upkeep of bacterial tradition collections to facets of the metabolism and body structure of remodeled tissues and transgenic vegetation. Drawing at the paintings of major scientists from laboratories world wide, Agrobacterium Protocols presents a wealth of thoughts for introducing particular DNA sequences into aim plant species and discusses the environmental implications of genetically engineered crops. Its targeted strategies will facilitate swift move of complicated options to different laboratories and their exploitation in primary and utilized plant biology.
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A..? (vir gene induction assay ) Normal vir Fig. 3. Summary of procedure for analysis of the nature of the mutations in the avirulent clones. Clones are analyzed for presence of the mutation on the Ti plasmid or elsewhere in the genome. They are then tested for their ability to induce a virB::lucZ fusion m the presence of acetosyringone and to be complemented by a variety of clones containing wild-type genes involved in oncogenicity. Avirulent mutants recovered after treatment with acetosyringone are often altered in virA and vi&, which are the two loci responsible for detection and transduction of the acetosyringone &-gene inducing signal.
4. Cultures on AT nopaline medium are incubated for 7 d and abihty of each of the mdivrdual clones to grow on this medium IS then monitored. 5. 0, and are stored as frozen glycerol stocks. 6. The presence of a Ti plasmid m the clones IS mvestrgated by agarose gel electrophoresrs following in-well lysts. Numerous varrations on the original protocol of Eckhardt (8) exist, and this basic protocol may have to be adapted to the partrcular strain being used and the condmons prevailing m the laboratory.
Hooykaas, P J J , and Schilperoort, R. A (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Tr-plasmrd. Nature 303, 179,180. 8. Stachel, S. E and Nester, E W. (1986) The genetic and transcriptional organization of the vir region of the A6 TI plasmid of Agrobacterium tumefaciens. EMBO J. $1445-1454 9. Stachel, S E and Zambryski, P (1986) virA and virG control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens.
Agrobacterium Protocols by Kevan M. A. Gartland, Michael R. Davey