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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37-49
http://mpb.sophiapublisher.com
38
emerged on the optimization, improvement of
protocols and use of these protocols for the transfer of
genes of agronomic and economic importance. Many
attempts have been done to engineer fungal resistance
in wheat using single gene transfer technology which
has brought partial resistance against pathogens under
question. The strategy has been to target fungal cell
wall or fungal metabolism by using the genes
encoding proteins which either digest and degrade
fungal cell wall or interfere metabolic processes, the
examples has been glucanases, chitinases, thaumatin
like protein and ribosomal inactivating proteins
(Muehlbauer and Bushnell, 2003, Bliffeld et al., 1999;
Bieri et al., 2003, Oldach et al., 2001; Chen et al.,
1999; Makandar et al.,
2006; Balconi et al., 2007;
Mackintosh et al., 2007; Shin et al., 2008).
Structurally fungal cell wall is consisted of β-glucan
and amino glucans chitin (Ply-GluNAc), some portion
of which is always acetylated in the form of chitosan.
The attempts till present have targeted either β-glucan
with the help of glucanase gene or chitin with the help
of chitinase (Oldach et al.,
2001; Mackintosh et al.,
2007). In both the cases single genes are unable to
completely disrupt the fungal cell wall, additionally
the chitosan is never degraded by chitinase and this
portion helps the fungus to survive. A successful
strategy will be to target both the chitin and chitosan
by overexpressing chitinase and chitosanase genes. By
this strategy not only both amino glucans will be
targeted but the impact of the degradative enzymes
will be boosted synergistically resulting in completely
dismantled cell wall structure.
Constitutive expression of transgene is generally
desirable but in disease resistance related experiments
it has not proved as rule. Sometimes the transgenes
have shown disease like symptoms under normal
conditions when the pathogen attack was not there.
This is probably due to cellular reprogramming in the
presence of proteins which are desired only under
specific conditions. Similarly the examples have
proved that constitutive promoters are not necessarily
constitutive all the time and there is different
expression in different parts of the plant. Under such
conditions it was not possible to get the disease control
in those plant parts where constitutive promoter did not
express at desired level. Inducible promoter is the
solution of such pitfalls.
1 Results
With the emergence and development of molecular
biology, the phenomenon of disease resistance and
pathogen virulence is being studied at molecular level.
The factors responsible for pathogen virulence and
plant defense responses against pathogen attacks are
being studied extensively. Parasites of pathogens are
being used as tools for biological control of plant
diseases. All these attributes of plants and the
pathogens are controlled by genes and the manipulation
of these genes by genetic transformation can be used
for increasing resistance of plants against diseases. In
this research, two antifungal genes
HarChit
and
HarCho
from
Trichoderma harzianum
(myco-parasite)
were co-expressed separately under constitutive
Ubiquitin
promoter as well as under stress cum
disease inducible
Vst
-
1
promoter in wheat. These
genes hydrolyse the chitin present in the fungal cell
wall. Inducible and constitutive promoters were used
to get advantages of both the promoters and compare
the effect on the disease resistance. To evaluate any of
their effect on disease resistance wheat fungal
pathogen
Erysiphe graminis
f. sp.
tritici
, the causal
organism of powdery Mildew of wheat was used in
infection assays using transgenic lines and non
transgenic controls.
1.1 Genetic transformation of wheat
Winter wheat genotype Florida was selected for
transformation purpose. This genotype has already
been reported to be readily transformable by many
authors. Immature Zygotic Embryos were bombarded
in a batch of
pUbi-HarChit
,
pUb-HarCho and
P35SAcS
and then separately making a batch of
pVst-HarChit
,
pVst-HarCho
and
P35SAcS. P35SAcS
is a selection marker gene which
confers resistance
against
Basta
.
The bombarded IZEs were maintained
on a regime of callus induction and regeneration
media containing selection agent Basta. The results
are summarised in Table 1. The plants recovered upon
selection media were checked by PCR and Southern
Blot analysis for the presence or absence of genes of
interest. All the plants recovered through selection did