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Molecular Plant Breeding 2013, Vol.4, No. 30, 247
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253
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252
C
T.
EF-1α
)×T. X stands for different lines and T stands
for one time expression of
MSI-99m
after calibration
by
EF-1α.
Every experiment was repeated three times.
3.6 Evaluation of the disease resistance of transgenic
plants
S. Sclerotiorum
resistance of the transgenic lines was
assessed using detached leaf, young plant and adult
plant. Untransformed rape Zhongyou 821 was used as
a control.
3.6.1 Detached leaf inoculation test
For the detached leaf inoculation tests, second true
leaves from plants of four-true-leaf stage grown in the
glasshouse were collected as plant materials(Verma et
al., 2012). A pipette tip was used to gently wound the
leaf at the inoculation site. 30 μL spore suspension
(1×10
7
spores/mL) was dropped on sterile filter paper
discs (5 mm) and pieces of sterilized flower petals
were put onto each side of the filter paper discs. Then
the filter paper discs were placed onto the main vein
position of detached leaves. All leaves were arranged
on wet gauze in containers that were collectively
covered with transparent polyethylene bags
maintaining suitable moisture. The leaves in the
containers were incubated at 22
℃
in a dark room.
The leaf inoculation experiment was performed with
three biological replicates and eight leaves per
replicate. Four days post-inoculation, disease severity
was assessed on a 0~4 scale [0=no lesions;
1=percentage of lesion area on leaf≤10%; 2=11% ≤
percentage of lesion area on leaf≤30%; 3=31% ≤
percentage of lesion area on leaf≤50%; 4=percentage
of lesion area on leaf ≥51%]. The disease index was
calculated as following described (here
k
=4).
3.6.2 Adult plant inoculation test
Stems of rape in blooming adult plants were
inoculated with
S. sclerotiorum
. Mycelia of
S. sclera-
tiorum
were cultured on potato dextrose agar, and
some agar discs were excised from the edges of
growing colonies. A hole at the location of 35 cm
above ground on each plant stem made by a punch and
was filled with an agar disk. The tresis vulnuses were
wrapped with plastic laboratory film to prevent agar
plugs from desiccating or falling off. Stem inoculation
was performed with three biological replicates with at
least six plants per replicate. After 7 days of infection,
disease severity was assessed on a 0~4 scale and
disease index was calculated as following described
(here
k
=4). The standard of disease index was: 0=no
symptoms; 1=the lesion length on the stem was below
3.33 cm; 2=the lesion length on the stem was between
3.33 and 6.66 cm; 3=the lesion length on the stem was
between 6.66 and 9.99 cm; 4=the lesion length on the
stem was over 9.99 cm.
3.6.3 Statistical analysis
We used disease index to evaluate the ability of
resistance to
S. Sclerotiorum
. Disease index was
calculated by 100∑(
I n
1
)/(
N k
), where
I
is a disease
severity score on the 0–4 scale,
n
1
is the number of
plants with each score,
N
is total number of plants
assessed and
k
is the highest score. Statistical analysis
for these experiments was done using the SAS
programmer (SAS Institute, Raleigh, N.C.).
Acknowledgments
This study was supported financially by grants from the Jiangsu Agricultural
Science and Technology Innovation Fund (NO. cx (11)1020), National
Natural Science Foundation of China (NO.
11171155
) and
A Project Funded
by the Priority Academic Program Development of Jiangsu Higher
Education Institutions
:
Modern horticultural science (PAPD)
. We specially
thank Prof. Xiu-Qing Li and Miss Chidie Yang for his assistance
in writing
the manuscript.
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