Transcript Slide 1
Plant transformation • Agrobacterium • Plasmids • Methods Vacuum infiltration Tissue culture Agrobacteria are soil-borne, bacterial plant pathogens which cause tumorous growths or roots to develop on infected plants. Infected plant cells produce opine used for the proliferation of bacteria Opine synthesis is under the control of genes in T-DNA T-DNA is part of the Ti plasmid present in Agrobacterium Characteristics of the Ti-Plasmid Large circular plasmid containing genes for: Virulence Catabolism of specific opines Host-directed opine synthesis Host-directed, bacterial-type plant hormones Genes for synthesis of opines and plant hormones are contained on the TDNA; T-DNA is bordered by 25 bp direct repeats; only the TR border is required for transfer although the TL border increased the efficiency when it is present; no sequences other that the borders are required for transfer Two primary steps in transformation • Binding of Agrobacterium to a plant cell (up to 200/cell) • Transfer of DNA to the plant cell (multiple T-DNAs can be transferred) Each step requires a different set of genes. Binding to plant cell requires chromosomal Agrobacterium genes: chvA and chvB - mutants at these two loci result in a marked reduction in Agrobacterium binding to plant cells; chvA encodes a transport factor and chvB encodes a protein involved in 2-linked betaglucan synthesis pscA – Involved in the synthesis of the major neutral and acid extracellular polysaccharides Transfer of T-DNA requires the presence of several virulence genes Wounded cells secrete low-molecular weight molecules that induce the expression of vir genes. Acetosyringone and hydroxy-acetosyringone R1 R2 The two molecules stimulate the synthesis of several vir genes. Further, acetosyringone can act as a chemical attractant in vitro and thus may act as chemotactic agent in nature. Vir Gene Function VirA encodes a single protein which resembles a transmembrane chemoreceptor found in other bacteria; constitutively expressed; monocistronic VirG a positive regulatory protein which relays environmental information (when plant inducible factors are present) to other vir loci; requires virA; strongly affects virB,C,D, E: monocistronic VirE a single-stranded DNA binding protein that appears to coat the T-strand during transfer to the plant cell; polycistronic VirC and virD site-specific endonuclease that cleaves at the 25 bp direct repeats borders of the T-DNA; produces a T-strand that is the intermediate molecule that is transported to the plant cell; polycistronic VirB may play a role in directingT-DNA transfer events at the bacterial cell surface Plant transformation An analysis of 161 transformants of Arabidopsis was performed to determine the type of integration events. 55% single event 20% two unlinked events 6% three unlinked events 1% four unlinked events 12% non-Mendelian ratios In tomato, in ten different transformants, integration occurred at nine different chromosomes. The target DNA sites are not specific except they are AT rich. Plant transformation Plasmids Plant transformation Plasmids Plant transformation Plasmids Plant transformation Plasmids Plant transformation Plasmids Plant transformation Plasmids Plant transformation Plasmids Tissue localization Tissue localization using GUS reporter gene From: http://www.jic.bbsrc.ac.uk/science/cdb/exotic/index.htm Plant transformation Methods Vacuum infiltration Cut the primary Inflorescence Wait for the secondary Inflorescence to appear Dip the plant in Agrobacteria suspension Apply mild vacuum www.rz.uni-karlsruhe.de/~db26/Fotos-Knoch Plant transformation Methods Tissue culture All steps are performed Under sterile conditions Cut the cotyledons Dip the cut surface of the cotyledons In Agrobacteria suspension Introduce the cut size in the growth medium Wait…… Plant transformation Methods Tissue culture http://www.fp.ucalgary.ca/moloneylab/bnapus.htm Plant transformation Methods Tissue culture http://www.fp.ucalgary.ca/moloneylab/bnapus.htm Plant transformation Methods Tissue culture http://www.fp.ucalgary.ca/moloneylab/bnapus.htm Describe the steps required for the production of homozygous transgenic lines containing single insertions Make sure you identify the seed generation