Basic HTML Version
Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.2
http://tgg.sophiapublisher.com
Page 2 of 7
1.1
Agrobacterium
mediated transformation
Agrobacterium
mediated transformation is the most
widely used indirect method for wheat transformation.
Agrobacterium
tumefaciens
, a soil dwelling bacteria
that naturally infects dicots and causes tumorous
growth resulting in crown gall disease. Tumor
formation results from incorporation of T-DNA, a part
of small independent DNA molecule outside the
bacterial genome called Ti (tumor inducing) plasmid.
Small phenolic compounds exuded from plant wounds
stimulate expression of vir genes, located on Ti
plasmid and responsible for its excision, transfer and
integration into plant genome. The natural capability
of
Agrobacterium
was manipulated in plant
transformation by replacing the genes causing
tumorous growth by genes of interest. However,
Agrobacterium
has a natural tendency to infect dicot
plants and monocots were considered recalcitrant to
Agrobacterium transformation
. Therefore, most of the
Agrobacterium
mediated transformation procedures
were established for dicots and monocots including
economically important cereals lagged behind for a
considerable time. Low frequency of T-DNA transfer
into the target genome was the major limitation.
Nonetheless,
improvements
of
co-cultivation
conditions, selection and regeneration methods for
transformed tissues, in addition to incorporation of
super binary vectors helped in extending the host
range of
Agrobacterium
to several recalcitrant cereals.
Transformation of wheat had been attempted since
1988 but stable transformation through
Agrobacterium
became possible through a reliable and relatively
efficient transformation procedure and construction of
a new plasmid vector (Cheng et al., 1997) with stable
integration, expression and inheritance of transgene to
next generation. Further improvement in the plasmid
vector and wheat transformation procedure increased
the transformation efficiency. Several investigators
obtained subsequent successful transformation of
wheat through
Agrobacterium
with more than 4%
transformation efficiency. Multiple factors are
involved in
Agrobacterium
mediated transformation
that determine the success or failure of gene transfer,
stable integration and expression into plants.
Agrobacterium
strain, plasmid vector, selected tissue,
duration of pre-culture, extent of time and conditions
of inoculation and co-cultivation, presence of
acetosyringone in the medium, etc. are some of the
factors that can affect the success of transformation.
Following infection with
Agrobacterium
the calli
remain for a long time on the media containing
selective chemicals and antibiotics reducing the
recovery of transformed plantlets. Supplementation of
polyamine spermidine improves regeneration from the
transformed calli (Khanna and Daggard, 2003).
Increasing
Agrobacterium
cell density and duration of
inoculation/co-cultivation also increases expression of
foreign gene in wheat (Amoah et al., 2001).
Agrobacterium
mediated gene transfer has remarkable
advantage over direct methods. These include low
copy number of transgene leading to fewer problems
of co-suppression and instability, no requirement of
special equipment and technique, defined and
preferential integration of alien gene into
transcriptionally active regions of chromosome (Hiei
et al., 1997).
Agrobacterium
system is reproducible,
has higher transformation efficiency as compared with
particle bombardment for wheat (Hu et al., 2003).
Agrobacterium
also has a good promise for
in planta
transformation of wheat apical meristem. The newly
growing seedling may be cut to expose the apical
meristem followed by injury and infection with
Agrobacterium
solution to transfer the gene of interest
into germ line precursor cells. The selection for the
putative transformants can be done at the inflorescence
stage or progeny level.
1.2 Direct method
In the direct method there is no involvement of a
biological vector or bacterial mediation for transfer of
gene construct into explants. The gene construct is
directly incorporated into plant cells/tissues. Variable
efforts were made to introduce exogenous DNA into
wheat in the last two decades. Initially attempts were
made to deliver alien gene into wheat protoplast.
Polyethylene glycol (PEG) mediated gene transfer was
the first technique applied for genetic transformation
of wheat. Transgenic fertile wheat plants were
obtained using electroporation method with 0.40%
efficiency (Sorokin et al., 2000). Use of silicon
carbide fiber, microinjection and liposome fusion