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Molecular Plant Breeding 2013, Vol.4, No. 30, 247
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248
transferred into different plants and its expression can
enhance resistance against
Sclerotinia sclerotiorum
,
Alternaria alternata
and
Botrytis cinerea
,
Pseudomonas
syringae pv tabaci
,
Aspergillus flavus
,
Fusarium
moniliforme
,
Verticillium dahliae
, and
Colletotrichum
destructivum
in transgenic tobacco
(Chakrabarti et al.,
2003; DeGray et al., 2001),
Fusarium oxysporum f. sp.
cubense
and
Mycosphaerella musicola
in transgenic
banana (Atkinson et al., 2003), and
Pseudomonas
syringae pv. tomato
in transgenic tomato (Conlon and
Kim, 2000).
MSI-99m
, a synthesized
MSI-99
gene adapted for
expression in potato (Huada), was introduced in
potato and transgenic plants exhibited increased
resistance to
Phytophthora infestans
and
Ralstonia
solanacearum
(Hong et al., 2012). In this paper, we
reported the results in genetic transformation of rape
with the
MSI-99m
gene and the assessment of
transgenic plants for their resistance to
S. sclerotiorum
.
1 Results
1.1 Regeneration of transgenic rape plants
The hypocotyl explants infected were co-cultured with
A. tumefaciens
GV3101 on RM
1
medium for 2 days
(Figure 1A). Then they were transferred to the RM
2
medium for the induction of callus. Callus was visible
on the cut surface after 2 weeks of culture (Figure 1B).
Shoots appeared from the explants after one month on
shoot induction medium (RM
3
). Some of them could
grow to normal green plantlets, whereas the majority
bleached in the presence of Kan (Figure 1C). When
the green plantlets grew up to 3 cm length they were
inoculated on RM
4
medium with 15 mg/L Kan for
antibiotic resistance selection (Figure 1D~Figure 1E.
The rooting plantlets were finally transplanted in pots
and placed in the greenhouse (Figure 1F). In total, 25
independent Kan-resistant putative transformants were
regenerated, 9 of which were confirmed to be
PCR-positive (Figure 2).
1.2 Expression of
MSI-99m
Gene in Transgenic
Plants
Expression of
MSI-99m
gene in the 7 transgenic lines
was analyzed using quantitative RT-PCR.
MSI-99m
was expressed in all the transgenic lines but the levels
of transcripts among the transgenic lines were very
Figure 1 Regeneration of
MSI-99m
transformed
rape plants
Note: A: co-cultivation; B: callus formation; C: shoots
regenerated from hypocotyls and selected by 10 mg/L Kan ; D:
shoots grow and selected by 15mg/L Kan; E: rooting and
selection on medium with 15 mg/mL Kan; F: regenerated
plantlet transplanted in a pot
Figure 2 PCR detection of 9
MSI-99m
transgenic rape lines
Note: M: DNA marker; P: positive control (pGS); C: wild-type
plants as negative control; M3, M4, M10, M14, M16, M19,
M21: transgenic lines M3, M4, M10, M14, M16, M19, M21,
respectively
different (line M14 is 9 times of line M10). No
expression of
MSI-99m
was observed in the wild-type
control (Figure 3).
1.3 Evaluation of transgenic plant resistance to
Sclerotinia sclerotiorum
To evaluate resistance of the transgenic lines to
S.
sclerotiorum,
detached leaves
test, young plant
test
and adult plant test were applied in this experiment.
1.3.1 Detached leaf assay
Detached leaf test was carried out with 7 transgenic
lines and the wild-type control. After 4 days of
co-culture with
S. sclerotiorum
, wild-type leaves presented