This document provides an overview of Agrobacterium-mediated gene transfer. It discusses that Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants by transferring tumor-inducing (Ti) plasmid DNA (T-DNA) into the plant genome. The Ti plasmid contains the T-DNA region flanked by borders, and virulence genes required for T-DNA transfer. Upon sensing plant signals, the virulence genes activate and the T-DNA is transferred to plant cells, where it integrates into the genome and expresses genes that cause tumor formation by increasing phytohormone levels. This natural plant genetic transformation ability makes Agrobacterium an important tool in genetic engineering

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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