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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37
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42
sequenced to confirm the
HarChit and HarCho
induction in transgenics. So we had co-expression of
HarChit
and and
HarCho
genes in cDNA was
synthesized from total RNA isolated from T
0
and T
1
plants and the amplification was taken using gene
specific primers. Positive control showed the band of
the expected size as did all the positive plants while
the negative controls never showed the presence of
any transcript from
HarChit.
PCR reaction showed the
presence of
HarChit
transcript after
Vst1
promoter
induction with wounding stress. 4A= T
0
plants and
4B= plants from T
1
generation. M= Marker, C
1
=
reagents control with H
2
O as template, C2 = negative
control with template from non transgenic plant. C
3
=
positive control with template from
pVst-Chit
plasmid
(Figure 4).
Figure 4 Induction of
HarChit
under stress inducible promoter
in T
0
and T
1
Generations
1.7 Phyto-pathological experiments
Erysiphe graminis
f.sp
. tritici
was used to check the
effect of
HarChit
and
HarCho
genes expressed
constitutively and induced under pathogen stress for
the enhancement of plant response against diseases.
1.8 Transgenic lines under constitutive promoter
Leaf segments of 4 centimetres length were cut from
plantlets at two leaves stage and cultured on the
anti-seniscence media. Non transgenic control was
compared with all the transgenic lines under study as
well as with the transgenic control. It was made sure
that 300-400 conidia should spread per cm
2
of the
inoculation plate. The results showed that there was
almost no difference in the number of colonies
developing on transgenic plants, transgenic control or
negative control in the beginning. The difference
started to be prominent 10 days post inoculation. The
colonies remained healthy on the transgenic control
and negative control while started withering and
burning on the transgenic plants. The difference in the
size was clearly visible Three weeks post inoculation.
This size was noted and compared with transgenic and
non transgenic control. The relative size of colonies
compared to size of colonies developed of non
transgenic control is shown in Figure 5 and Figure 6.
There was 34 to 60 percent less fungal growth on
tested transgenic lines compared to control lines.
I.A-18 showed 0.4 and I.A-6 showed 0.66 colony size
compared to 1.0 of control, the other transgenic lines
performed in between. Even the line I.A-1 that
expressed only
HarChit
showed reduction. It also
seems from these experiments that there is no role of
bar
or
gus
genes for fungal resistance.
The transgenics having
Harchit and HarCho
under the
control of constitutive promoter showed reduction in
fungal growth in general while the transgenics where
HarChit and HarCho were under inducible promoter, 3
out of six tested plants showed no affect on fungal
growth while in other three, the colony size of 0.74
and 0.64 was noted for I.A-7 and I.A-12 in
comparison to 1.0 of the controls. I.A-11 proved to
be the best in the lot and showed a colony size of 0.25
in comparison to 1 of the control. I.A-11 was the most
resistant against
Erysiphe
among all the transgenic
lines whether expressing antifungal genes under
constitutive or inducible promoter. Even the number
of developing colonies those were not less in number
in transgenic lines and the control plants were on
average 30% less than in number on I.A-11. Each
experiment for pathological analysis was repeated at
least thrice.
2 Discussions
2.1 Wheat transformation, transformation frequency
and segregation in the progenies
The objective of present study was the stable
transformation of wheat with antifungal genes from
Trichoderma harzianum
. This was to be done
separately under constitutive and inducible promoters
so that the difference in the performance of both type
of expression could be noted. Wheat transformation
via biolistic bombardment is being reported in many