T Dna Transfer Process, Virulence, Agrobacterium, Chromosome, Gene Transfer, Plasmid, Glucons, Cell Surface Proteins, Cellulose Fibrils

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Gene Transfer Methods in Plants Gene Transfer Methods in Plants Introduction Target Cells for Transformation Theoretical Routes for Production of Transgenic Plants Vectors of Gene Transfer Vectors based on Ti and Ri Plasmids of Agrobacterium Structure and Function of Ti and Ri Plasmids Selectable Marker Genes in Vectors Used for Gene Transfer T-DNA Transfer Process Vectors based on Ti and Ri Plasmids Virus Vectors Transformation Techniques Using Agrobacterium Prerequisites for Production of Transgenic Plants Explants Used for Transformation Marker Genes for Selection and Scoring of Tranformed Cells/Calli or Shoots Reporter Genes Used as Scoreable Markers and their Enzyme Assays Neomycin Phosphotransferase Gene (nptII) Chloramphenicol Acetyl Transferase Gene (cat)

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T-DNA Transfer Process.
An early event in the T-DNA transfer process is the nicking of T-DNA of the Ti plasmid at two specific sites, each between the third and fourth base of the bottom strand of each 25bp repeat. This initiates DNA synthesis from the nick in the right hand 25bp repeat sequence in 5' - 3' direction thus displacing a single T-DNA strand. This T-DNA single strand forms a complex with protein VirE and gets transported to the point nucleus. The virD operon encodes an endonuclease that produces the nicks in the border sequences.

Molecular Events in the Transfer of the T-DNA to plant cells (Redrawn from D. Grierson 'Plant Genetic Engineering, 1991).

Molecular Events in the Transfer of the T-DNA to plant cells (Redrawn from D. Grierson 'Plant Genetic Engineering, 1991).

1.Vir B Protein Complex	2.Vir D2 protein binds to 25 bp repeats and introduces site specific nicks in bottom strand	3.A new bottom strand synthezied producing the T-strand
4. Polymerase	5.T-strand	6.T-strand is complexed with Vir E protein and exported to plant nucleus

Several gene products of the virB operon have been identified in the bacterial envelope, a location, which suggests that they may playa role in directing T-DNA transfer extracellularly. The functions of several other vir gene products are largely unknown.

Apart from the role of Ti plasmid, the genes located on Agrobacteriu mchromosome also help in virulence. These genes are involved in the synthesis and secretion of glucons, cellulose fibrils and cell surface proteins. These loci are constitutively expressed and are also found in other soil bacteria associated with higher plants. Thus, these loci playa more general role in the virulence of Agrobacteriumand thus also in the Agrobacteriummediated gene transfer.

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β-Glucuronidase (GUS)Gene (uidA) Luciferase Gene (lux) Green Fluorescent Protein Gene Phosphomannose Isomerase Gene (pmi) Agrobacterium-Mediated Transformation in Monocots Agroinfection and Gene Transfer Physical Delivery or DNA Mediated Gene Transfer (DMGT) Chemically Stimulated DNA Uptake by Protoplasts Microinjection and Macroinjection Microprojectiles for Gene Transfer Electroporation Method for Gene Transfer Liposome Mediated Gene Transfer Calcium Phosphate Precipitation Method Transformation Using Pollen or Pollen Tube Incubation of Dry Seeds,Embryos,Tissues or Cell in DNA Transformation by Ultrasonication Removal of Selectable Marker Gene from Transgenic Plants Need for Removal of Selectable Marker Gene Genetic Systems Used for Removal of Marker Gene Examples of North American Plant Transformation Facilities Plant Transformation Facilities (PTFs)