slide is about how a A.tumifacian is transfer ed on to host plant.

3.  Today’s seminar is about how a
genetically engineered Agrobacterium
tumefacien is transfer on a target host
plant
 But before that we must know what
exactly a agrobacterium is. . .?

4. Agrobacterium is a genus of Gram-
negative bacteria established by H. J. Conn that
uses horizontal gene transfer to cause tumors in
plants.
Its belongs to the bacterial family Rhizobiaceae.
Agrobacterium tumefaciens is the most commonly
studied species in this genus.
 Agrobacterium is well known for its ability to
transfer DNA between itself and plants, and for this
it has become an important tool for genetic
engineering.
It is often found near soil level at the junction of
plant stem and root

5.  General approaches for inoculation of
Agrobacterium.
1. Infection of wounded plant.
2. Co-cultivation.
3. Leaf disc method.

6. INFECTION OF WOUNDED
PLANT We take well established in vitro plant
seedling
 It is best traditional way to obtain
agrobacterium transformed cells in aseptic
condition.
 On seedling freshly cut surface wound is
inoculated with an overnight culture of
agrobacterium.
 Then after few day the tumor produced is
excised and grow as callus culture
 Then the transformed calli pick off and
regenerated.

7. CO-CULTIVATION
 The protoplast are isolated during cell wall
reformation stage.
 Then the protoplast are incubated for 24-40
hr in a suspension of agrobacterium.
 The ratio of bacteria to protoplast is 100:1.
 The transformation occur during the
subsequent few day of co-cultivation.

8. LEAF DISC
 The procedure is carried out on any explant that
provides a good source for initiation of whole
plant.
 For this newly emerged cotyledons are used.
 In this tissue segment is excised from a explant
and allow to incubate in agrobacterium
suspension for few hour to 3 days.
 Then it is cultured on a bacteriological media for
growth of bacteria inoculated.
 After that tissue explant moved into media
containing a bacteriostatic agent eg.carbenicillin
to eliminate the bacteria.

10.  Then explant is moved to a media which is
designed for the selection of transformed
plant cells i.e. contain the antibiotics
eg.kanamycin
 Leaf disc method is technically simple and
produce transgenic plant rapidly therefore
used more than other methods.

11. Flow chart of agrobacterium
transformation
Explant
(seedling,cell,protoplast,leaf disc etc.)
Addition of wounding
Asetosyringone agrobacterium
co-cultivation to 30 mint in liquid medium
Allow infection
Medium without bactrio-
Statical agent(3day)

12. Infection process
(transfer of gene from agrobacterium to plant cells)
Medium with
bacteriostatic agent
(carbencillin,cefotaxime)to kill
bacteria
Transformed and non transformed tissue
Selection cycles Selective media to kill
non-transformed tissue
(kanamycin,hygromycin)
Transformed tissue

13. Transformed shoot
Rooted shoot
Adult putative transgenic plants
Testing of transgenic plant
Using PCR,
southern hybridization etc.
Seeds

15. References
 Introduction to plant biotechnology-
H.S.Chawla
 Image source- google image