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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37 -49
http://mpb.sophiapublisher.com
44
expressed under inducible promoter. Recently Anand
et al., 2003, reported that 20 out of 24 wheat
transgenic plants transformed with rice chitinase and
glucanse genes using bar as marker gene showed
complete gene silencing in T1 generation and onwards.
They observed heavy methylation of their transgene.
Silensing of marker gene in our results may also be
following the same phenomenon. We also noted an
additional thing that many of the putative transgenic
plants resistant to selectable marker were not
containing our gene(s) of interest. Similarly there were
instances when only one gene of interest was present
in a transgenic instead of both. Transformation
experiments were done over a year but the transgenic
plants were found between November and April, this
was what found by Brettschneider et al., 1997 during
Maize transformation under Hamburg conditions. This
indicates that there is something related with the
explants which comes from outer environment of
green house and affects its efficiency for transformation.
The primary transgenics having the genes of interest
were grown to maturity and the harvested seed was
grown to get T1 generation where seven plants out
eight for experiment-1 (genes of interest under
constitutive promoter) showed single locus integration
while only two out of six for could show the single
locus integration for experiment-2 (genes of interest
under constitutive promoter). This might be due to
silencing of selection marker gene or any unknown
reason.
2.2 Co-expression of HarChit and HarCho
The defence response genes function in a variety of
ways to inhibit fungal infection and expression of
these genes in transgenic plants has been shown to
enhance fungal resistance (Muehlbauer and Bushnell,
2003). Wheat like other plants has an innate defence
response against the fungal pathogens that involves
the induction of PR genes at the rear end. These PR
genes defend by attacking different organs of the
pathogen. For example chitinases and 1, 3-β- glucanases
hydrolyse the fungal cell wall by targetting fungal cell
wall chitin (poly-GlcNAc) and 1, 3- β -Glucan as
substrate. The presence of chitinase genes in plants
and lower organisms have been known and the
chitinolytic activity is also established invivo and
invitro for these genes (Yanai et al., 1992; Blaisean
and Lafay, 1992; Botha et al., 1998; Singh et al.,
2007). Chitosanase is an enzyme similar to
chitinase, capable of hydrolysing the β-1,4- linkages
between N-acetyl-D-glucosamine and D-glucosamine
residues in a partially acetylated fungal cell wall
polymer. Chitosanases have the potential of
slowing or preventing fungal infection by degrading
the structural chitosan (poly-GlcN) found in the cell
wall of many fungi (Hendrix and Stewart, 2002).
Glucose amine oligomers, released from fungal cell
walls after hydrolysis with chitinase or chitosanase,
are elicitors of plant defence response (Lee et al., 1999;
Vander et al., 1998). The response elicited by these
molecules depends on the length and degree of
acetylation of the oligomers released (Vander et al.,
1998). Oligomers that are relatively short (e.g.
products of chitosanase hydrolysis) are active elicitors of
plant defence systems.
T. harzianum, a soil-borne fungus known to be a
control agent of fungal plant pathogens (Papavizas,
1985), is one of the fungi that produce degrading
enzymes which destroy key cell wall structural polymers
of fungal pathogens (Hendrix and Stewart, 2002). The
purified enzymes from T. harzianum are substantially
more chitinolytic and glucanolytic than the enzymes
from other known sources (upto 100 times more
active than the corresponding plant enzymes and
effective on a much wider range of pathogens) (Lorito
et al., 1994; Lorito et al., 1996). They are also non
toxic to plants even at high concentration (Carsolio et
al., 1999). Furthermore, the anti-fungal activity is
synergistically enhanced when different Trichoderma
cell wall degrading enzymes are used together or in
combination with plant PR-proteins, commercial
fungicides, cell membrane-affecting toxins or biocontrol
bacteria (Lorito et al., 1996; Steyaert et al., 2004).
Co-expression of chitinase and chitosanase genes from
T. harzianum could therefore result in a synergistic
enhancement of anti-fungal activity of wheat.
Constitutive promoters have been used in maximum
transgenic experiments in all the plants. In some
experiments specially related to disease resistance the
expressed protein is not required all the time or it is