Agriculture Reference
In-Depth Information
Fig. 10.2 Way of nucleic acid attached to nanoparticles
sequences, i.e., promoter, terminator, and marker gene, which allow efficient
screening of the transformed and non-transformed. Binary vectors are the most
commonly used plant transformation vectors due to their ability to replicate in both
E. coli
and
Agrobacterium tumefaciens
(Fig.
10.2
).
For efficient plant transformation, three important steps are:
• Selection of appropriate plasmid
• Plasmid replication ability determining its copy number
• T-DNA region
The plant transformation vectors should have minimal heterologous sequences.
These sequences are required for plant transformation and selection of
transformants; they require all promoter and terminator sequences for plant expres-
sion of newly cloned genes. Generally the minimal selection vectors have either of
the two plant selection genes: they have either
hptII
, encoding resistance to
hygromycin, or
nptII
which encodes resistance to kanamycin. The selection gene
is under the control of double-enhancer version of the CaMV35S promoter in both
the cases. By subjecting to site-directed mutagenesis, interfering restriction sites are
removed within the coding sequence by converting them to the silent changes.
Kanamycin or chloramphenicol resistance marker allow a broad range of
Agrobacterium
or
E. coli
strains to be used in the transformation.
Various derivatives differing in the selectable marker, reporter, MCS, and other
factors are available in the pCAMBIA series (
www.cambia.org
), pGreen series, and
pPZP series. One of the derivatives is shown for each of the groups: empty vector
pGreen, selection vector pPZP111, and reporter vector pCAMBIA1302. A similar
variation is found in the derivatives of pBin19, which is considered to be a selection
vector, and reporter vector pBI121. An empty version of pBin19 may be obtained
by digesting pBin19 with
Cla
I and partially with
Sac
II, followed by
recircularization. These vector groups have been successfully employed in many
studies, which at a glance, account for two thirds of the recent publications in the
area of plant transformation.
