Basic HTML Version
Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37
-
49
http://mpb.sophiapublisher.com
45
needed only in some specific parts of the plant. When
it is expressed constitutively, there comes extensive
cellular reprogramming of defence components in the
parts of the plants which are not infected. This may
give some times rise to plants which are resistant to
diseases but weak in health and resulting productivity
(Gurr and Rushton, 2005). For example in Arabidopsis
NPR1 over expression using CaMV 35S promoter
brought broad spectrum resistance with normal
phenotype while in maize same was done using
Ubiquitin
promoter and it gave rise to diseased
genotype without infection (Cao et al., 1998; Pieterse
and Loon, 2004). Keeping in view these reasons and
the fact that chitinase and chitosanase enzymes of the
T. harzianum
are highly antifungal it was decided to
co-express
HarChit
and
HarCho
genes under constitutive
as well as under inducible promoter. This way we
could compare the impact of both the proteins on
fungal diseases when they were present constitutively
and induced under under pathogen attack.
Vst-1
promoter from grape was wine as a stress inducible
promoter that is also induced under disease in wheat
(Leckband and Loerz, 1998). Ubiquitin promoter from
Maize was used as constitutive promoter for the
co-expression of
HarChit
and
HarCho.
2.3 Performance of transgenics under constitutive
promoter
A total of four lines having
HarChit
and
HarCho,
one
line with
HarCho
and one transgenic control with
gus
gene under constitutive
Ubi-1
p omoter were evaluated
against non transgenic control line Florida. Powdery
mildew symptoms started to appear on the wheat
segments 5-6 days after inoculation with
E. graminis.
Observations were taken on the number of developing
colonies 9 dpi and colony size 21 dpi. The results
showed a little or no decrease in the number of
developing colonies per leaf segment on five lines and
transgenic control line having
Ubi-gus
gene compared
to the number of colonies per segment of leaf on non
transgenic control (Figure 5). 9 dpi the difference in
the size of the colonies for transgenic lines and
controls was not visibly noticeable. 21 dpi the size of
the fungal colonies was visibly different. On an
average the fungal colonies on transgenic plants were
40-60% smaller than the colonies on transgenic and
non transgenic controls. The colonies at the negative
control lines were looking healthier with a lot of
powdery mycelium while fungal colonies on transgenic
leaves were mostly dead with very less powdery
mycelium and the general appearance was brown and
smaller colonies. Moreover a little more yellowishness
was also observed in transgenic plants (Figure 6)
compared to transgenics which may be an indication
of accelerated Programmed cell death in the fight
against fungus.
2.4 Performance of transgenics under inducible
promoter
The behaviour of the T
1
progenies containing
HarChit
and
HarCho
genes under stress inducible gene was
relatively different from transgenic progenies containing
HarChit
and
HarCho
under constitutive promoter. Out
of the six T
1
progenies (
I.A
-
7-I.A
-
12
) tested with
E.
graminis
three showed almost the same number of
colonies as control lines while the rest showed
8%
-
29% less colonies than the controls. Progeny
I.A-8
had only
HarCho
but the resistance response of
it against the development of
E. graminis
colonies was
at par with other progenies having both the genes of
interest. The size and health of the
E. graminis
colonies developing on the leaf disks of transgenics
with the gene/genes of interest and control lines
showed visibly smaller colonies with less powdery
material while the leaf disks of the control lines
showed visibly larger colonies with a lot of white
powdery material. Out of the six lines tested, three
lines (
I.A
-
8, I.A
-
9
and
I.A
-
10
) showed the same size
of colonies the size of colonies was observed on
control lines. Three lines (
I.A
-
7, I.A
-
11
and
I.A
-
12
)
showed a reduction of 26, 75 and 36% in the colony
size respectively compared to the control lines. The
colonies on the controls were having more powdery
material and were healthier than those found on the
lines with over expressed
HaChit
and
HarCho
.
Various defence response genes have been used in
wheat transformation for powdery mildew resistance.
Oldach et al
(2001) observed a reduction of 32%
-
40%
in the number of developing
E. graminis
colonies on
wheat transgenic line overexpressing Barley class-II