Agrobacterium tumefaciens: a natural tool for gene transformation

1. AGROBACTERIUM MEDIATED
GENE TRANSFER
Prepared by:
Dr.E.Gayathiri
Department of Plant Biology and Plant Biotechnology
Subject: Plant Biotechnology
Subject Code : 16UPBTC09

2. What to Know:
Learning Objectives
 Overview:
Introduction
Agrobacterium species
Ti Plasmid
Organization of T.- DNA
Gene Required for transfer of T- DNA
Mode of action/ Transfer

3. Agrobacterium tumefaciencs
 Agrobacterium is a gram-negative, rod shaped , soil
pathogenic bacteria involved in causing crown gall
formation disease in dicot plant species.
 Ex: A.tumefaciens and A. rhizogenes
 It had been considered as natural genetic engineer,
so they can transform into the plant.
 It is serious pathogen of walnut, grape vines, stone
fruit, nut trees, sugar beets, horse radish etc,
 Optimum temperature:28⁰ C.
 Doubling time 2.5 to 4 hours
 Grow aerobically
 No spore formation
 Motile having on to six peritrichous flagella.

4. Agrobacterium tumefaciencs
 Bacterium contain tumour inducing plasmid
(Ti plasmid or pTi plasmid) contains T-DNA
and all gene necessary to transfer T-DNA into
plant cell.
 Many strain do not have pTi plasmid
 The bacteria parasitize the plant tissue by
intergrating its own DNA in plant genome and
changes the expression of the plant
metabolism.
Agrobacterium tumefaciencs is used as a
tools in genetic engineering to engineer
the desired gene.

5.  Agrobacterium species.
 1.A.tumefaciens has Ti
plasmid that is responsible
for tumor induction (crown
gall disease) in plants
 2. A.tumefaciens is also known
as A.radiobacter/Rhizobium
radiobater
 3.A.rhizogene had Ri plamid
which is analogue to Ti plasmid
 4. A.rubi cause cane gall in
sugar cane plant
 5. A.vitis gall in grape plant

6. Ti Plasmid
 Ti Plasmid (Tumor inducing)
 Large size plasmid of 200
kbp
 Ths Ti plasmid is lost when
grown above 28⁰C
 Plasmid have T-DNA, right
border, Left border, Vir
gene,Phytochrome region,
Origin of replication and
opine region

7. Organization of T- DNA
 T- DNA is the transferred DNA of the tumor inducing (Ti)
plasmid.
 The size of T- DNA is between 15-30 kb
 It had RB and LB, RB helps in transfer and integration of T-
DNA.
 T-DNA carry genes for Phytohormones (Auxin and Cytokinin)
and opine that are expressed in plant cell
 Over expression of the hormones at site of infection is
responsible for the proliferation of wounded cell in gall/
tumor.
 The tumor can harbor a plenty of bacteria.

8. Organization of T- DNA
 Opines are low molecular weight compounds found in
plants crown gall tumors or hairy root tumors
 Opines are used as important source of nitrogen,
carbon and energy
 Opines are condensation products of
 1. An amino acid and a keto acid or
 2. Amino acid and sugar
 Different types of opine may present: Nopaline,
Octopine and agropia

9. Organization of T- DNA
 Genes for auxin, cytokinin and opines are transcribed
and translated in plant cell
 Example of T-DNA genes in Ti plasmid

10. Genes required for transfer
of T- DNA

11. Mode of action/Transfer
 Steps in transformation
of plant cell
 1. Plant stress condition
 2. Phenolics production
 3. Signal of bacteria
 4. Virulence system
activation
 5. Generation of T-DNA
complex
 6. T-DNA transfer
 7.T-DNA integration in
plant genome.

12. Role as a genetic tool
 T- DNA region get replaced by any gene of
interest and then targeted to plant cell for
transformation
NOTE:
 Monocot are not good host for Agrobacterium
 There is a hypothetical believe that monocot
are resistant to Agrobacterium because they do
not produce phenolic that can induce virulence
genes

13. 22-06-2009 First Asian PGPR Congress, Hyderabad 21-24 June 2009 1
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