Agrobacterium Mediated Plant Transformation

30th April’2012

Presented by,Prabhu Thirusangu,Research Scholar,

Transformation – the process of obtaining transgenic plants

Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome

Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants.

Most common genes (and traits) in transgenic or biotech cropsherbicide resistanceInsecticide resistanceBt genes in field corn (maize)virus-resistance (coat-protein) genes

Transgenic plants – An introduction

Plant Transformation Methods

Physical Chemical Biological

MicroinjectionPressureBiolistics - gene gun/particle bombardmentElectroporationSilica/carbon fibersLazer mediated SAT

PEGDEAE-dextranCalcium phosphateArtificial lipidsProteinsDendrimers

A. TumefaciensA. RhizogenesVirus-mediated

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Introduction to Agrobacteria…

A rod shaped, Gram negative soil bacterium

A natural genetic engineer

2 species A.tumefaciens (produces a gall) A. rhizogenes (produces roots)

Oncogenes (for auxin and cytokinin synthesis) + Opines

• Disease producing Agrobacterium are higly pathogenic and do not benefit the plant

• In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (VIR) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.

Scientific classification

• Kingdom :Bacteria• Phylum :Proteobacteria• Class :Alproteobacthaperia• Order :Rhizobiales• Family :Rhizobiaceae• Genus :Agrobacterium• Species :A. tumefaciens

Ti plasmid and virulence genes

• A Ti plasmid is a circular piece of DNA found in almost all Agrobacteria

• Three main regions T-DNA region(Between right and left T-DNA

border) Oncogene Opine

Virulence region Opine catabolism region

Cellular process of Agrobacterium–host interaction

Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129

Molecular involvement in t-DNA Transfer…

Images of Crown Gall Tumor

Approved Traits.....

• Glufosinater herbicide

• Sethoxydimr herbicide

• Bromoxynilr herbicide

• Glyphosater herbicide

• Sulfonylurear herbicide

• Bt gene

• Male-sterility

• Modified fatty acid

• Flower colour

• Flower life

• Delayed fruit ripening

• Virus resistance

Binary vector

t-DNA

VIR genes

Plasmid DNA

BacterialChromosome Bacterial ORI

Ampicillin resistance

Construction of vector with disired genes

Production of transgenic plants

Isolate and clone gene of interest

Add DNA segments to initiate or enhance gene expression

Add selectable markers

Introduce gene construct into plant cells (transformation)

Select transformed cells or tissues

Regenerate whole plants

Procedure for plant transformation

IMAGE: Mol bio of the cell by Albert (pg no:599)

Regeneration, Selection And Detection...

Regeneration: for shoot organogenesis, cytokinin (lower amounts of auxin) are required

Selection: two antibiotics are required• an antibiotic to kill the Agrobacterium, while not affecting the plant's

cell growth and division• a second antibiotic allows growth of transformed shoots (w/selectable

marker) but inhibits growth of untranformed plant cells.

Detection of the "trait" gene PCR methods can detect the presence of the "trait" DNA

protein detection methods are used where a gene product is produced that defines the trait

verification of the incorporation of the trait gene into the plant's chromosome

• by Southern hybridization• by demonstrating transfer of the trait to the original transformant's

progeny

Approved Transgenic plants.....

• Soybean• Corn • Cotton• Oil Seed rape• Sugarbeet• Squash• Tomato• Tobacco

• Carnations• Potato• Flax• Papaya• Chicory• Rice• Melon

Types of genetic modification

>99% of all transgenic crops are either herbicide or insect resistant

<1% have other traits

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Problems and potential

 Ø       Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome  Ø       By varying experimental materials, culture conditions, bacterial strains, etc. scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn Ø       This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this

Ø       The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer

Ø       Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants Ø       Monocotyledon plants are not very susceptible to the bacterial infection

Benefits and Problems with Agrabacteria….

Transformed plants will help the world to meet the human demands for food, energy, medicine, shelter, clothing, and a cleaner environment.

Gene transfer technology is being used to produce plants capable of hyper accumulating toxic metals from soil and water, thus contributing to a cleaner environment

Increases in plant production are needed to meet growing demands for food. Transformed Plants offers significant advantages over traditional plant breeding in developing improved crops.

Conclusion…..

References

Introduction to plant biotechnology by H.S Chawla

Molecular biology of the cell by Albert et al

Online Referenceshttp://www.nepadbiosafety.net/for-regulators/resources/subjects/biotechnology/ plant-transformation-agro

http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.htm

http://arabidopsis.info/students/agrobacterium/uses.html

http://microbewiki.kenyon.edu/index.php/Agrobacterium_tumefacien0073

http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/CrownGall.aspx

http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219w.htm

Thank you all

By, T.Prabhu,30th April’2012