Plant transformation

Transcription

1 Plant transformation Objectives: 1. What is plant transformation? 2. What is Agrobacterium? How and why does it transform plant cells? 3. How is Agrobacterium used as a tool in molecular genetics? References: Hooykaas and Schilperoort Agrobacterium and plant genetic engineering. Plant Molecular Biology 19: Westhoff et al. Molecular Plant Development:from gene to plant. Chapter 7,

2 Plant transformation Introduction of exogenous DNA into a plant cell Transient no incorporation of exogenous DNA into the genome Stable incorporation of introduced exogenous DNA into the genome Transformation of multicellular organisms: C t di tl t f ll T f ti Cannot directly transform every cell - Transformation involves one cell which then regenerates an entire organism

3 Agrobacterium tumefaciens: a natural tool for plant transformation ti Soil gram positive bacterium Martha Hawes Agrobacterium tumefaciens attached to a plant cell

4 Agrobacterium tumefaciens: a natural tool for plant transformation ti Causes Crown Gall disease - tumors (galls) form at base of stem in many dicots Photographs supplied by Sharon von Broembsen, Oklahoma State University production of tumors is caused by the transfer of bacterial DNA to the plant, which integrates into the plant genome

5 Agrobacterium tumefaciens: a natural tool for plant transformation ti Genes involved in crown gall disease are not present on the chromosome of A. tumefaciens but on a large plasmid, called the Ti (tumor-inducing) plasmid. Ti A. tumefaciens Circular chromosome vir genes LB T-DNA Ti plasmid ~ 120 kbp ori RB

6 A. tumefaciens T-DNA Structure LB RB Shi Shi Roi Nos LB and RB 25 bp repeats Nos - nopaline synthase opine biosynthetic i gene* * Shi - shoot inducing - 2 genes for auxin synthesis* Roi - root inducing - gene for cytokinin synthesis* *have eukaryotic promoters these genes are not expressed in Agrobacterium!!!

7 T-DNA transfer into plants T-DNA transfer process is activated when Agrobacterium gets in contact with damaged plant tissue T-DNA is nicked at the RB, T-DNA gets replicated to the LB and moved into the plant cell this is catalyzed by products of vir genes

8 T-DNA transfer into plants T-DNA is inserted into plant nuclear genome at random sites Transformed cell starts proliferating upon DNA integration resulting in tumor formation. Why? Transformed cells make opines - specific nutrients (type of amino acids) for bacterium ( Genetic colonization )

9 Agrobacterium tumefaciens as a tool for genetic engineering i Problem: tumor How can we engineer the Ti plasmid to make it useful? Delete auxin and cytokinin genes Retain vir genes, LB&RB, ori Ti plasmid is huge (~120 kb) need to make it smaller

10 Agrobacterium tumefaciens as a tool for genetic engineering i vir genes and T-DNA can be on separate plasmids only left and right borders (LB & RB) are required for T-DNA to be transferred Cloning site for plant genes LB Selectable marker (Plants) vir genes Ti plasmid T-DNA Binary vector RB Selectable marker (Bacteria) ori ori (E.coli) ori (Agrobacterium)

11 Steps in plant transformation 1. Propagate binary vector in E. coli 2. Isolate binary vector from E.coli and engineer (introduce a foreign gene) 3. Re-introduce engineered binary vector into E. coli to amplify 4. Isolate engineered binary vector and introduce into Agrobacterium containing a modified (smaller) Ti plasmid 5. Infect plant tissue with engineered Agrobacterium (T-DNA containing the foreign gene gets inserted into a plant cell genome)

12 Plant transformation In each cell T-DNA gets integrated at a different site in the genome Each cell is hemizygous for the insertion only one of the homologous chromosomes gets the insertion Consequences of the insertion: - Foreign DNA is inserted - Insertional mutagenesis (does not kill the cell the organism is diploid!)

13 Plant transformation Problem: We want to transform the whole organism, not one cell!!! This is done by: Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco)

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15 Plant transformation Problem: We want to transform the whole organism, not one cell!!! This is done by: Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco) In planta transformation of Arabidopsis - Dip flowering plants into Agrobacterium suspension - Harvest seed and select for transformants (they are hemizygous!)

16 Plant transformation plbio.life.ku.dk In planta transformation of Arabidopsis (Floral dip method) Systemic infection in Arabidopsis is accomplished by transformation of female gametes!