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Molecular Plant Breeding 2011, Vol.2, No.5, 30
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Figure 3 Southern Blot
analysis of some transgenic plants
Note: 1: Positive control, plasmid Pbihrap, the relatively dark
color was a result of poor banding; 15: Negative control, an
un-transformed plant; 2~14: Transgenic plants; Bands of 2, 3,
4
，
6, 9 and 10 are weak, meaning the
hrap
gene was not for
sure transferred into plants; Lines 5, 7, 8, 11, 12, 13 and 14
were considered to be positive transgenic plants
1.5 RT-PCR analysis
RT-PCR analysis was performed on 10 transgenic
generation plants randomly selected from different
lines in order to verify the expression of transgenic
plants. No PCR product was detected in the negative
control (non-transformed) plant (Figure 4, lane 11).
All the 10 tested plants were confirmed positive by
RT-PCR using nested gene-specific primers with an
Figure 4 RT-PCR analysis of individual
hrap
transgenic
potatoes
Note: 1~10: Transformed plants; 11: Negative control; M:
Marker D2000
expected 720 bp fragment detected (Figure 4, lanes
1~10), confirming that the
hrap
gene was stably
integrated into the genome and expressed in these
plants.
1.6 Sequencing validation
DNA sequencing analysis was perdormed by
comparing the
hrap
genes isolated from
transformation plants in this experiment with that
registered in the Genebank (GenBank: AF168415).
Results demonstrated that there of the ten transgenic
plants were five bases different from AF168415, and a
similarity of 99.3%. (Nos of 226, 296, 438, 534 and
583) was detected.
1.7 Assay of disease resistance
According to Center International Potato (CIP) late
blight 9-score standards, the disease resistance to
P.
infestans
detection in transgenic lines indicated a
resistant line rate of 50%. Five plants (1-2-23, 1-2-32,
1-4-8, 1-10-9, 1-14-26) among the 42 tested plants
showed resistance to
P. infestans
, and the rate of
resistant plants was calculated to be 11.90% (Table 4).
According to the earliest wilting leaves time and
whether the entire plant wilted as disease resistance
and sensitivity standards, both the rates of resistant
lines and resistant plants to
R. solacearum
were 50%.
Two transgenic plants (1-4-5, 1-10-7) were
highly-resistant to
R. solacearum
, and 1-1-27 was
moderately resistant to the disease, while the control
was susceptible (Table 4).
Table 4 Detection of disease (
P.
infestans and
R.
solanacearum) resistance in transgenic potatoes
No. of detections
No. of plants (lines) of resistance
Rate of resistance
Lines
8
4
50.00%
P. infestans
Plants
42
5
11.90%
Lines
6
3
50.00%
R. solanacearum
Plants
6
3
50.00%
2 Discussion
In the analysis of the rooting ability of transformed
adventitious buds on MS medium containing 75 mg/L
Kan, about 24% of the buds could not grow root after
the first rooting-selection, and 14% after the second
rooting-selection. These results indicated that the
twice rooting-selection of the transformed plants was
effective. The results are consistent with the data
reported by Zhang et al (2004).
In this study, PCR positive rate of transgenic plants
obtained was 61.15%, which was similar to the rate
obtained by transforming
HarpinEa
gene into Atlantic
(55.14%) reported by Li et al
(2002). However, Su et