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Huang et al., 2011, Integrating the
hrap
Gene from Sweet Pepper into Potato Enhances Resistance to
Phytopthora infestans
, Molecular Plant Breeding Vol.2
No.5 (doi: 10.5376/mpb.2011.02.0005)
31
and indirect pollution effects of pesticides on non-target
insects and animals, including humans. Regarding these
public and environmental concerns, the identification,
development and utilization of
P. infestans
-resistance
genes are important priorities that might greatly benefit
the potato breeding and production.
Recent studies of the mechanisms in the fields of plant
disease resistance and pathogenicity had identified
some protein factors related to plant defense responses.
The resistant ability to
P. infestans
can be improved by
transforming the pathogenesis-related (PR) gene in
potatoes. It is reported that transgenic potato plants
carrying the tobacco osmotin protein were able to
delay the appearance of
P. infestans
spots and reduce
the frequency of
P. infestans
infection, which
indicated an improved capacity in late blight
resistance in the plant leaves (Liu et al (1994), Zhu et
al
(1996) and Li et al (1999a)). Jin et al (2001)
introduced the thaumatin protein gene (
tlp
) with the
selective marker of the herbicide-resistant
bar
gene
into a potato plant mediated by
Agrobacterium
tumefaciens
to delay onset of late blight symptoms
following inoculation with late blight spores. Yang et
al
(2001) introduced the gene,
avrD
, to potato plants
and comfirmed that disease resistance was enhanced
while infected with the
Phytopthora
protein,
elicitin.
Transgenic potato plants haeboring the glucose
oxidase gene (
go
) can accumulate H
2
O
2
inside the
plant and acquire distinct resistance to
P. infestans
(Zhang et al., 2001). Nan et al (2006) also reported
that the transgenic potato plants with the bean
chitinase gene improved the anti-pathogen
characteristics more than 30% over the control group
without the chitinase gene.
The hypersensitive-response assisting protein (HRAP),
identified in sweet peppers (
Capsicum annuum
cv.
ECW), might intensify the harpinPss-mediated
hypersensitive response (HR), which a defense
mechanism often found in plants exhibiting disease
resistance. The
hrap
gene encoding the HRAP can
split the harpinPss polymer into monomer and dimmer
components, the later will cause much more elevated
allergic necrosis and retarding the propagation of
bacterial spot virus and the bacterial pathogen,
Xanthomonas
, in sweet pepper, resulting in alleviating
symptoms (Chen et al, 1998 and 2000; Ger et al,
2002). For example, the
hrap
gene was introduced
into tobacco, the resulting transgenic plants were
acquired resistant to tobacco wildfire and soft-rotting
bacteria (Ger et al, 2002). Similarly, Pandey et al
(2005) introduced
hrap
into
Arabidopsis thaliana
me-
diated by
Agrobacterium
to generate transgenic plants
with resistant to soft-rotting bacteria. In present study,
the
hrap
gene was introduced into potato cultivar by
Agrobacterium
mediation and the resistant phenotypes
of the transgenic plant were evaluated.
1 Results
1.1 The
hrap
gene introduced into potato mediated
by
Agrobacterium
The transformation was performed using an
Agrobacterium tumefaciens standard binary (T-DNA
and vir regions) vector system, tissue cultured potato
leaflets as explants. Callus was observed two weeks
after the leaflets was cultured in the callus induction
medium. The callus induction rate was over 80%. The
adventitious buds were observed after the calli were
transferred into SIM for 3~4 weeks. The bud induction
rate was 50%. About ten days after the adventitious
buds were transferred into rooting medium,
regenerated plantlets were obtained. Finally , we had
obtained over 20 transgenic lines, more than 400
transgenic plants, and over 100 transgenic potatoes.
1.2 Anti-Kan rooting-selection
The non-transformed adventitious buds (control) grew
roots only in the medium with Kan free, they
exhibited only a small number of aerial roots in the
MS-Kan medium (Table 1). However, the transformed
adventitious buds readily grew roots in the MS-Kan
media. These data showed that the MS medium
containing 75 mg/L Kan was suitable for the
rooting-selection of transgenic plants (Figure 1). The
rooting-selection on the midium containing 75 mg/L
for transformed adventitious buds was conducted
twice. The rooting rate of the transformed adventitious
in the Kan medium was 76.32% and 86.21%,
respectively (Table 2). The second rooting rate was
about 10% higher than the first, which indicates that
as for Burbank, twice rooting-selection is efficient and
imperative.