By Michael W. Saul, Raymond D. Shillito, Ioan Negrutiu (auth.), Stanton B. Gelvin, Robbert A. Schilperoort, Desh Pal S. Verma (eds.)
During the prior ten years, nice advances were made within the quarter of plant molecular biology. Such previously esoteric recommendations as gene move and plant regeneration at the moment are in many instances played, making the dissection of regulatory parts of genes a standard perform in lots of laboratories. in addition to this new know-how has come a virtually bewildering array of swiftly altering recommendations, frequently making it tricky for the amateur to choose and practice the process appropriate for answering a given organic query. In 1986, a few of us felt that lots of those suggestions had turn into regimen adequate to warrant the ebook of a laboratory guide. The handbook is designed either for complex university point laboratory classes and as a 'bench consultant' to be used within the medical laboratory. spotting the quickly altering nature of plant molecular biology know-how, the editors have designed a laboratory guide that's either effortless to take advantage of within the laboratory and in order to be up to date because the thoughts swap and new applied sciences are devised. extra chapters which may change or be further to this primary version might be released periodically. The editors realize that a number of the concepts defined during this guide depend on really good plant genetic fabric, microbial traces, or recombinant plasmids. these humans wanting such fabric should still touch the correct authors without delay. a listing of a number of the members to this guide, together with their addresses, is included.
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Additional resources for Plant Molecular Biology Manual
Example text
Introduction of plasmids into agrobacteria Plasmids can be introduced into Agrobacterium either by conjugation or by transformation. The latter technique, which was developed by Holsters et al. g. C58) and is not very efficient since 1 Ilg of DNA may give only up to 1000 transformants. Therefore, in general the conjugation process is used to introduce plasmids into Agrobacterium [3]. As far as is known, only plasmids of the E. coli inc groups P, Q and W can be stably maintained by Agrobacterium.
A reasonable approach when trying to transform a new plant species would be to begin with the supervirulent Ti plasmid, pB0542, in the strain A281 [19,29]. This strain efficiently transforms a very wide range of plants. Limited host range isolates, and other strains [30, 31], may transform plants that are not susceptible PMAN-A3/2 to infection by the broad host range strain, A281. The Ti plasmid pAL4404 [ 11] or similar disarmed Ti plasmids may be used if wild-type helper Ti plasmids inhibit the regeneration efficiency.
Spread 50-100 III of this mixture onto a sterile membrane filter that is placed on top of an agar layer with rich growth medium in a petri dish. 6. Incubate for at least 1 h (overnight incubation may give higher frequencies of transfer). 7. 9% NaCI solution by vigorous shaking. 8. Plate dilutions of the suspension onto selective medium plates. 9. Purify the transconjugants obtained by single colony isolation. This should be repeated at least twice. 10. Characterize the transconjugants by phage typing and determine the plasmid content.