Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

Chapter 7 Cloning Vectors for Eukaryotes 115
Virulence
region
Plasmid A
~170 kb
Figure 7.12
The cointegration strategy.
Host
specificity
region
Small
E. coli-type
plasmid
Gene to
be cloned
New gene
T-DNA
T-DNA
T-DNA
T-DNA
fragment
Unique
restriction site
Plasmid B
~20 kb
Recombination
Recombinant Ti
plasmid
Virulence
T-DNA
Figure 7.11
The binary vector strategy. Plasmids A and B
complement each other when present together
in the same A. tumefaciens cell. The T-DNA
carried by plasmid B is transferred to the plant
chromosomal DNA by proteins coded by genes
carried by plasmid A.
Normal Ti
plasmid
Virulence
Host
specificity
Host
specificity
Production of transformed plants with the Ti plasmid
If A. tumefaciens bacteria that contain an engineered Ti plasmid are introduced into a
plant in the natural way, by infection of a wound in the stem, then only the cells in
the resulting crown gall will possess the cloned gene (Figure 7.13a). This is obviously
of little value to the biotechnologist. Instead a way of introducing the new gene into
every cell in the plant is needed.
There are several solutions, the simplest being to infect not the mature plant but a
culture of plant cells or protoplasts (p. 85) in liquid medium (Figure 7.13b). Plant cells
and protoplasts whose cell walls have re-formed can be treated in the same way as
microorganisms: for example, they can be plated onto a selective medium in order to