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Molecular Plant Breeding 2011, Vol.2, No.8, 48
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Table 1 Pedigree of transgenic rice cross lines used for transgene integration and expression analyses
Transgenic donors
Cross combinations * (Female parent (generation) /
Male parent (generation))
Plant generation for Southern and
Northern analysis
TR 5
T5
TR 5 (T2) / CJN3
F3
TR 5 (T2) / CJ601
F3
TR 5 (T2) / CJ683
F3
TR 5 (T2) / Bing 97-264
F3
Ming B
T6
Ming B (T2) / Jia 59
F3
Ming B (T2) / Jia 60
F3
Ming B (T2) / Xuzao
F3
TR 6
T6
TR 6(T2) / CJN2
F3
TR 6(T2) / Bing 95-13
F3
TR 5(T5) / TR 6 (T6)
F1
Jingyin 119
T12
C20 / Jingyin 119 (T3)
F8
Jingyin 119 (T3) /57
F8
Jingyin 119 (T3) / Bing 94-02
F8
Jingyin 119 (T3) / 59
F8
Jingyin 119 (T3) / 104
F8
Jingyin 119 (T3) /59 // L97-55
F7
Jingyin 119 (T3) /57 // 9522
F7
Jingyin 119 (T3) / 390 // S1
F7
Jingyin 119(T3) / 63 // T951
F7
Jingyin 119(T3) / Bing 94-02 // T951
F7
Jingyin 119(T3) / 02 // T951
F7
Jingyin 119(T3) / 63 // 390
F7
Jingyin 119 (T3) / 59 // DS4
F7
Jingyin 119(T3) / 503 // T951
F7
Jingyin 119(T3) / 59 // T951
F7
Jingyin 119(T3) / 57 // DS4 /// L97-55
F5
Jingyin 119 (T3) / 59 // DS4 /// Jingyin 119 (T3) / 31 // 9522 F4
*Note: ‘/’ stands for the first turn cross; ‘//’ stands for the second turn cross; ‘///’ stands for the third turn cross. The F
1
hybrid plants
were used as transgenic donors when multiple cross experiments were conducted
Figure 3 The probe structure and site on pCB
1
Note: Diagrams of the pCB
1
are not to scale; Probe 1:
bar
gene
probe used for Southern and Northern blotting; Probe 2:
cecropin B
gene probe used for Southern and Northern blotting;
Act: Rice actin-1 promoter;
CB
:
cecropin B
gene encoding
sequence; Pin: Potato proteinase inhibitor II terminator; 35S:
Cauliflower mosaic virus 35S promoter;
bar
:
bar
gene
encoding sequence; nos:
A. tumefaciens
Ti-plasmid nopaline
synthase terminator
blot hybridization and detection was carried out using
DIG Luminescent Detection Kit (Roche Company)
according to the manufacture’s instructions.
3.3 Southern blot hybridization
Genomic DNA was isolated from rice leaves using the
SDS DNA extraction method as described by Lu and
Zheng (1992) Aliquots (5 μg) were digested overnight
with appropriate restriction enzymes, fractionated by
0.8% agarose gel electrophoresis and blotted onto
Amersham N
+
Hybond membranes according to the
Southern blotting method (Sambrook et al., 1989).
The linear fragment (0.9 kb) comprising the open