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Molecular Plant Breeding 2013, Vol.4, No. 30, 247
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250
introduction of resistant genes into rape to improve its
resistance is therefore of considerable importance and
has obtained some achievement (Ganapathi et al.,
2001; May et al., 1995). Of the successful events, it
can be noted to be the application of wheat Oxalate
oxidase gene
OXO
in oilseed rape and its constitutive
expression in transgenic rape considerably increased
OXO activity and enhanced resistance to
S. sclera-
tiorum
with disease reductions from 88.4% to 90.2%
(Dong et al., 2008). In addition, it was also reported
that over-expression of a mitogen-activated protein
kinase (BnMPK4) led to increased tolerance to
S.
sclerotiorum
infection in transgenic rape canola
(Wang et al., 2009). The genes used in the studies
above belong to specific genes in functions which
generally lack stability and easily lose their resistance
when new pathogen races emerge due to mutation.
Hence genes with multi-resistance are preferred in
potato genetic breeding.
MSI-99
gene is a synthesized magainin II analogue
with high inhibitory effects to a wide spectrum of
microbial organisms, including bacteria, fungi and
protozoa (Zasloff, 1987). To this day,
MSI-99
has been
transferred into tomato (Alan et al., 2004), tobacco
(Chakrabarti et al., 2003; DeGray et al., 2001), banana
(Atkinson et al., 2003), potato (Ganapathi et al., 2007;
Hong et al., 2012) and grapevine (Vidal et al., 2006).
However it has not been reported in rape yet. In this
study, the
MSI-99m
gene was successfully transferred
into the rape cultivar Zhongyou 821 by
Agroba-
cterium
-mediated transformation and its expression
made transgenic rape plants more resistant to
S.
sclerotiorum
.
In this study, detached leaves
test and adult plant test
were respectively carried out for the evaluation of the
resistance of transgenic rape lines. Transgenic lines
except line M10 was more resistant to
S. sclerotiorum
than wild-type control, with the level of resistance
varying among the transgenic lines, and the results
were consistent in two assays. Notable variation
among the transgenic lines was observed and could be
attributed to the random inserting of the gene in
Agrobacterium
-mediated
genetic
transformation
(Alvarez et al., 2000; Butaye et al., 2004; Demeke et
al., 1999; Srivastava et al., 1996).
Rape suffers serious threats from pathogens, such as
Peronospora parasitica
and
Albugo candida
.
There is
an urgent need for multi-resistant varieties in rape
production. Previous studies have demonstrated that
MSI-99
possesses a wide spectrum of resistance
including
Sclerotinia sclerotiorum
,
Alternaria alternate
,
Botrytis cinerea
,
Pseudomonas syringae pv tabaci
,
Aspergillus flavus
,
Fusarium moniliforme
,
Verticillium
dahliae
,
Colletotrichum destructivum
etc. Further
analysis on the resistance of the transgenic rape lines
against other pathogens would provide more valuable
evidence for the application of
MSI-99m
gene in the
improvement of rape diseases resistance.
3 Materials and methods
3.1 Plant and fungal pathogen
The rape (
Brassica napus
L.) cultivar Zhongyou 821
was used as recipient in this transformation
experiment. Seeds of Zhongyou 821 were soaked in
75% ethanol for 30 s, immersed in 0.1% HgCl
2
for 1
min, and surface-sterilized for 5 min in a 2%
Na-hypochlorite solution. The seeds were subse-
quently rinsed three times with sterile distilled water
and germinated on hormone-free MS medium
(Murashige and Skoog, 1962) at 25
℃
under a 16/8 h
photoperiod at a light intensity of 44 μ mol·m
-2
·s
-1
and 60%~90% relative humidity. The sclerotia of the
fungus
S. sclerotiorum
were cultured to produce
mycelial inoculum on potato dextrose agar at 28
℃
.
3.2 Bacterial strains
Agrobacterium tumefaciens
strain GV3101 containing
vector pGS-
MSI-99m
constructed by our laboratory
was cultured in LB medium (pH 7.0) supplemented
with 100 mg/L kanamycin (Kan) and 50 mg/L
rifampicin (Rif) at 28
℃
. The structure of the vector
pGS-
MSI-99m
was showed in Figure 6.
3.3 Genetic transformation and plant regeneration
Rape plants were transformed with
MSI-99m
gene
using
Agrobacterium
-mediated method. A single
colony of
A. tumefaciens
strain GV3101 harboring the
vector pGS-
MSI-99m
was cultured in liquid LB
medium with 100 mg/L kanamycin (Kan) and 50
mg/L rifampicin (Rif) at 28°C overnight. The bacteria
was then harvested by centrifugation at 5000×g for 10
min and resuspended
to an optical density of OD
600
=