Transcript Methods of profucing transgenic plants

Lecture 3
Ti plasmid derived vector system
Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumorproducing
genes
Opine catabolism
Virulence region
ORI
T-DNA encoded
genes can be
substituted by
target genes
Ti-plasmid based vectors
Binary systems
Co-integrated vectors
Needs 3 vectors
Needs 2 vectors:
Disarmed Ti plasmid
with gene of interest
(no vir genes)
Helper vector
for infection
(with vir genes)
Form
co-integrated plasmid
after homologous
recombination on T-DNA
Disarmed Ti plasmid
capable for infection
Intermediate vector
with T-region
and gene of interest
(transferred by conjugation)
Helper vector
for transfer of
intermediate plasmid into A.tum
Co-integrated vectors
(hybrid ti-plasmids)
Right now
rarely used
DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E. coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use
2) Relatively inefficient gene transfer compared to the binary vecto
Ti plasmid vector systems
are often working as binary vectors
T DNA region removed
Gene of interest
Plant selectable marker
Bacterial selectable
marker
Disarmed
Ti
plasmid
ori for E.coli
Virulence
region
ori for A. tumefaciens
HELPER
plasmid
ori for A. tum
DISADVANTAGE: Depending on the orientation,
plasmids with two different origins of replication may be unstable in E. coli
ADVANTAGE: small vectors are used, which increases transfer efficiency
from E. coli to Agrobacterium.
No intermolecular recombination is needed