Plt Genetic Eng

8/8/2019 Plt Genetic Eng

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Plant transformation

Objectives:

1. What is plant transformation?

2. What isAgrobacterium? How and why does it transform

plant cells?

3. How isAgrobacterium used as a tool in molecular genetics?

References:

Hooykaas and Schilperoort. 1992. Agrobacterium and plantgenetic engineering. Plant Molecular Biology 19: 15-38.

Westhoff et al. Molecular Plant Development:from gene to plant.Chapter 7, 236-243.


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Transient no incorporation of exogenous DNAinto the genome

Stable incorporation into genome

Introduction of exogenous DNA into a plant cell

Transformation of multicellular organisms:

Cannot directly transform every cell - Transformation

involves one cell which then regenerates an entire

organism


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Agrobacterium tumefaciens:

a natural tool for plant transformation

Soil gram positive bacterium

Agrobacterium tumefaciens attached to a plant cell

Martha Hawes


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a natural tool for plant transformation 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


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a natural tool for plant transformation Genes involved in crown gall disease are not present on

the chromosome ofA. tumefaciens but on a large plasmid,called the Ti (tumor-inducing) plasmid.

Ti

A. tumefaciens

Circular

chromosome

vir

genes

LB

RB

T-DNA

Ti plasmid

~ 120 kbp

ori


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A. tumefaciens T-DNA Structure

LB and RB cis acting DNA elementsNos - nopaline synthase opine biosynthetic gene

Shi - shoot inducing - 2 genes for auxin synthesis*

Roi - root inducing - gene for cytokinin synthesis*

Shi Nos

RB

Shi Roi

LB

*have eukaryotic promoters not expressed in

Agrobacterium!!!


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T-DNA transfer into plants

T-DNA transfer process is activated whenAgrobacterium gets in contact

with damaged plant tissue

T-DNA is nicked at the RB, single stranded DNA gets replicated to the

LB and moved into the plant cell - this catalyzed by products of vir genes

http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF


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T-DNA is inserted into plant nuclear genome at random sites

T-DNA transfer into plants

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)

http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF


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Agrobacterium tumefaciens as a tool

for genetic engineering

Problem: tumor

How can we engeneer 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


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Agrobacterium tumefaciens as a tool

for genetic engineering virgenes and T-DNA can be on separate plasmids

only left and right borders (LB & RB) are required forT-DNA to be transferred

vir

genesTi plasmid

ori ori(E.coli) ori

(Agrobacterium)

Selectable

marker(Bacteria)

RB

LB

T-DNA

Binary

vector

Cloning site for

plant genes

Selectable marker

(Plants)


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Steps in plant transformation

1. Propagate binary vector in E. coli

3. Re-introduce engineered binary vector into E. colito amplify

4. Isolate engineered binary vector and introduce into

Agrobacterium containing a modified (small) Ti plasmid

5. Infect plant tissue with engineeredAgrobacterium

(T-DNA containing the foreign gene gets inserted into a

plant cell genome)

2. Isolate binary vector from E.coliand engineer (introducea foreign gene)


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In each cell T-DNA gets integrated at a different site

in the genome


Consequences of the insertion:

- Foreign DNA inserted

- Insertional mutagenesis(does not kill the cell the organism is diploid!)

Each cell is hemizygous for the insertion only one

of the homologous chromosomes gets the insertion


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Problem:

We want to transform the whole organism, not one cell!!!


This is done by:

Transforming plant cells in culture, selecting transformed cells andregenerating the entire plant from the transformed cell (eg. tobacco)


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Problem:

We want to transform the whole organism, not one cell!!!


This is done by:

Transforming plant cells in culture, selecting transformed cells andregenerating the entire plant from the transformed cell (eg. tobacco)

In planta transformation ofArabidopsis

- Dip flowering plants intoAgrobacterium suspension- Harvest seed and select for transformants (they are

hemizygous!)

Systemic infection inArabidopsis was accomplished by

transformation of female gametes!

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