By Kan Wang
A superior choice of Agrobacterium-mediated transformation innovations for state of the art plant genetic engineering, useful genomic research, and crop development. quantity 1 information the main up to date suggestions to be had for twenty-six plant species drawn from cereal plants, business vegetation, legume crops, and vegetable crops, and offers a number of tools for introducing DNA into 3 significant version plant species, Arabidopsis thaliana, Medicago truncatula, and Nicotiana. The authors additionally define the elemental equipment in Agrobacterium manipulation and techniques for vector development. quantity 2 includes one other thirty-three confirmed thoughts for root crops, turf grasses, woody species, tropic vegetation, nuts and culmination, decorative crops, and medicinal crops. extra chapters supply equipment for introducing DNA into non-plant species, equivalent to micro organism, fungi, algae, and mammalian cells. The protocols keep on with the profitable equipment in Molecular Biology™ sequence layout, every one delivering step by step laboratory directions, an creation outlining the foundations in the back of the strategy, lists of the mandatory apparatus and reagents, and pointers on troubleshooting and keeping off recognized pitfalls.
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Additional resources for Agrobacterium Protocols, Second Edition: Volume I (Methods in Molecular Biology Vol 343)
Useful tips related to vector construction are given in Notes 12–18. 1. Obtain plasmids and other DNA fragments necessary for constructions of vectors from appropriate sources. 2. Combine the bacteria-selectable marker and the plasmid replication functions for E. coli. 3. Insert the plasmid replication functions for A. tumefaciens, if necessary. 4. Insert the plasmid mobilization functions, if necessary. 5. Insert the RB, the LB, and the MCS to give the empty vector. 6. Construct the expression unit of the selectable marker gene separately.
3. Reporter Gene Cassette 1. Promoters. It is convenient to have a reporter gene expressed in various tissues and organs, and so the promoter is often chosen from the same group of promoters that may be used for selectable marker genes, which include 35S, Ubi, Act, Nos, and other T-DNA promoters. In some of the vectors, the promoter for the selectable marker and the reporter is the same, but, generally speaking, avoidance of duplication of the same components is recommended. 24 Komari et al. 2.
It is important to handle the culture quickly to prevent thawing and refreezing. If handled properly, the same culture can used repeatedly for retrieving the bacteria strain. 4. Notes 1. Media ingredients are available from a number of biological supply companies including Fisher Scientific, BD-BBL/Difco, and Sigma-Aldrich. 2. Autoclave-generated steam at a temperature of 121°C and pressure near 15 lb/in2 is the most common method of sterilizing bacterial growth media. Recommended autoclave times range from 15 min to 1 h.