By Kevan M. A. Gartland, Michael R. Davey
Agrobacterium Protocols bargains starting and skilled researchers the main complete choice of step by step protocols for the genetic manipulation of crops utilizing Agrobacterium. the themes variety from the upkeep of bacterial tradition collections to features of the metabolism and body structure of reworked tissues and transgenic crops. Drawing at the paintings of best scientists from laboratories worldwide, Agrobacterium Protocols offers a wealth of suggestions for introducing particular DNA sequences into goal plant species and discusses the environmental implications of genetically engineered crops. Its unique tactics will facilitate fast move of complex concepts to different laboratories and their exploitation in basic and utilized plant biology.
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Extra resources for Agrobacterium Protocols
Observation of Motility As a matter of routine, A. tumefaciens cultures are monitored for motility. Ten microliters of bacteria are mixed with 50 l,tL of chemotaxis medium in the well of an indented glass slide, and covered by a coverslip. Observations are made using 40X or 100X phase-contrast, in a Nikon Optiphot microscope (Nikon, Tokyo, Japan). This can reveal the state of the culture, from the proportion of motile cells. Moreover, it is possible to tell whether the culture is contaminated, as A.
5 g NaCl/L water. Autoclave. 3. 1. Measurement of Bacterial Growth Several experimental parameters are crucial to observation of growth inhibition in the presence of acetosyringone. These include concentration of acetosyringone, pH, and sugar composition of the growth medium, density of bacterial inoculum, and the particular bacterial strain being studied. With respect to the last factor, a limited survey suggested that A. tumefuciens strains carrying a nopaline-type Ti plasmid, such as strains C58 and T37, exhibit retarded growth in the presence of acetosyringone, but that strains carrying an octopine-type plasmid, such as strains A348, B6S3, and AchS, do not show this effect (I).
The ability of the avirulent mutants to respond to acetosyringone by inducing the vir genes is assessed using standard methodology, involving the use of a promoterless reporter gene fused to a vir promoter (II). The mutants are finally subjected to virulence complementation analysis by transfer of clones carrying specific wild-type genes involved in oncogenicity. Clones listed in Table 1 allow to cover the entire range of cases encountered to date in our laboratory. It is conceivable that other types of mutants may be recovered in the future.