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Molecular Plant Breeding 2010, Vol.1 No.2
http://mpb.sophiapublisher.com
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This study is aim to evaluate and apply the three
Bt
genes which were constructed by the National Key
Laboratory of Crop Genetic Improvement. At the same
time, it could increase the efficiency of breeding by
marker-assist selection. In 2009, the group of Qifa
Zhang brought up the strategy of super green rice,
which requires that on the premise of continued yield
increase and quality improvement, Green Super Rice
should possess resistances to multiple insects and
diseases, high nutrient efficiency, and drought
resistance, promising to greatly reduce the consumption
of pesticides, chemical fertilizers, and water (Zhang,
2009). This study is the first step to realize the strategy
of super green rice, which exploits the resistant genes to
improve resistances of rice to the target insects by
transgenic techniques and marker-assist selection. In
addition, Huazhong Agricultural University got the first
GMO safety certificates of
cry1Ab/c
transgenic plants,
Huahui No.1 and
Bt
Shanyou63.
cry1Ab
and
cry1Ac
are
known as the first generation
Bt
genes, and
cry1C*
and
c
ry2A*
are considered as the second generation
Bt
genes which have better resistance and specificity to the
target insects. Therefore, we believe that the improved
lines in the study would supply many useful and
promising materials in the future for
Bt
hybrid rice
breeding program.
3 Materials and methods
3.1 Materials of donor and receptor parents
In this study, the donor parents are Minghui63 (
cry1Ac
),
Minghui63 (
cry1C*
) and Minghui63 (
cry2A*
) which
has already been constructed by National Key
Laboratory of Crop Genetic Improvement (Tang et al.,
2006; Chen et al., 2005). The receptor parents are
excellent hybrid lines in China, 9311 and Fuhui 838,
which are introduced separately from Lixiahe Research
Institute of Agriculture Sciences and Sichuan sc-nuclear
technique institute.
3.2 Technical route by maker-assist selection
Bt
genes were introgressed into 9311 and Fuhui 838 by
crossing them with the donor Minghui63 (
cry1Ac
),
Minghui63 (
cry1C*
) and Minghui63 (
cry2A*
), followed
by three generation of backcrossing and one generation
of selfing. In this scheme, the progeny of each backcross
was detected for the presence of the
Bt
genes both by
PCR detection and field assays for insect-resistant. Then,
the selected individuals were self-pollinated to produce
homozygote for each of the three
Bt
genes, thus
completing the whole breeding procedure.
3.3 Cultivation in the field
The selected
Bt
lines which followed by three
generation of backcrossing and one generation of
selfing randomly planted in the Experimental Farm of
Huazhong Agricultural University, Wuhan, China, in
the summer of 2009, all are 80 lines. Each line planted
24 individual, and the distance between plants within
a row was 16.5 cm and the rows were 39.6 cm apart.
During the whole growth period, there were no any
insecticides spray in the experiment field.
3.4 Marker-assisted selection
The primers for three different
Bt
genes are as
follows:
cry1C*
-F (5'
-
ttctactggggaggacatcg
-
3');
cry1C*
-R
(5'
-
cggtatctttgggtgattgg
-
5').
cry2A*
-F (5'
-
cgtgtcaatgctgacctgat
-
3');
cry2A*
-R
(5'
-
gatgccggacaggatgtagt
-
5’).
cry1Ac
-F
(5'-tcgagacgttagcgtgtttg
-
3');
cry1Ac
-R
(5'
-
gaggaaaggtaaactcgggc
-
5').
All the three primers are dominant makers which
could only detect the positive plants (Tang et al., 2006;
Chen et al., 2005).
PCR selection and agarose gel electrophoresis:
Total
cellular DNA of the plants was isolated from fresh leaf
tissue by the methods of CTAB (Murray and Thompson,
1980).The PCR reaction of all the three
Bt
gene was the
same and carried out as follows: one cycle at 94
℃
for
50s; one cycle at 57
℃
for 50s, one cycle at 72
℃
for
55s, the above steps run for 32 cycles, followed by
extension at 72
℃
for 7 min. The products were then
checked by agarose gel electrophoresis (3%, 250V,
30 min).
3.5 Assays of insect-resistant and the agronomic
performance
Assays of insect-resistant:
To assay the insect resistance
of the improved lines and controls to the leaffolders in
the natural field in Wuhan, 2009, and investigate all the
24 plants of each line.
Assays of agronomic performance:
The agronomic
traits of the improved lines which were homozygous