um Mediated Plant Tra

30th April2012

Presented by,
Prabhu Thirusangu,
Research Scholar,

Transgenic plants An introduction

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 crops

herbicide resistance
Insecticide resistance
Bt genes in field corn (maize)
virus-resistance (coat-protein) genes

Plant Transformation Methods

Physical
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Silica/carbon fibers
Lazer mediated
SAT

Chemical
PEG
DEAE-dextran
Calcium
phosphate
Artificial lipids
Proteins
Dendrimers

Biological
A. Tumefaciens
A. Rhizogenes
Virus-mediated

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
4
transferred.

Scientific classification

Kingdom
Phylum
Class
Order
Family
Genus
Species

:Bacteria
:Proteobacteria
:Alproteobacthaperia
:Rhizobiales
:Rhizobiaceae
:Agrobacterium
: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 Agrobacteriumhost 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

Male-sterility

Sethoxydimr herbicide

Modified fatty acid

Bromoxynilr herbicide

Flower colour

Glyphosater herbicide

Flower life

Sulfonylurear herbicide

Delayed fruit ripening

Bt gene

Virus resistance

Construction of vector
with disired genes

Binary vector
t-DNA
Ampicillin
resistance
Bacterial ORI

Plasmid DNA
VIR genes
Bacterial
Chromosome

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

Problems and potential

Benefits and Problems with

Agrabacteria.

Scientists can insert any gene they want into the plasmid in place of the tumor
causing genes and subsequently into the plant cell genome

By varying experimental materials, culture conditions, bacterial strains, etc.

scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn

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

The vast majority of approved genetically engineered agriculture has been

transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer

Original problems existed in that Agrobacterium tumefaciens only affects

dicotyledonous plants

Monocotyledon plants are not very susceptible to the bacterial infection

Conclusion..
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.

References
Introduction to plant biotechnology by H.S
Chawla
Molecular biology of the cell by Albert et
al
Online
References
http://www.nepadbiosafety.net/forregulators/resources/subjects/biotechnology/
plant-transformation-agro
http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/ds
meth/ds.htm
http://arabidopsis.info/students/agrobacterium/uses.html
http://microbewiki.kenyon.edu/index.php/Agrobacterium_tumefacien0
073
http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/Cro

Thank you all

By,
T.Prabhu,
30th April2012