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Huang et al., 2011, Integrating the
hrap
Gene from Sweet Pepper into Potato Enhances Resistance to
Phytopthora infestans
, Molecular Plant Breeding Vol.2
No.5 (doi: 10.5376/mpb.2011.02.0005)
34
al (2004) transferred
Bt
gene into Atlantic, only with a
positive rate of only 2.96%. In this experiment, we
observed both positive and negative PCR results from
different plants propagated by one line. It is possible
that the inserted exogenous gene was lost over the
course of multiple asexual propagations, or the
exogenous gene expression was blocked in the
transformant, or the PCR amplification stripe was
simply too weak to be discerned using the methods
employed in this study. Regenerative plants may
differentiate from a number of cells, some of which
may be non-transformed, thus passing those
characteristics on to the regenerated plants.
Multiple analyses employed in the current
investigation, i.e., PCR, Southern Blot, RT-PCR and
sequencing, illustrate the successful integration and
expression of the target gene in transformed plants.
Li and Fan (1999b) identified
P. infestans
resistance in
55 transformed plants with Harpin protein, and found
that two were highly resistant. Zhang et al (2001)
reported that transformed
go
gene regenerative plants
exhibited a delayed occurrence of late blight
symptoms
.
Li et al
(2002) screened seven lines (out of
24 positive PCR lines) that demonstrated a significant
difference in disease resistance compared to the
control. Wang et al
(2007) reported that
hrap
gene,
integrated into a tomato plant, improved the plant’s
resistance to
R. solacearum
. In this study, the
hypersensitive-response promotion protein gene
hrap
was introduced into potato, resulting in transgenic
potato plants that were resistant to
P. infestans
and
R.
solacearum
. The experimental results indicate that
transformation of
hrap
gene into potato to improve the
ability of disease resistance is feasible.
The integration of the pathogenesis-related (PR)
protein gene is considered to be significant in
producing disease resistant plants and in improving
the quality and yield of potato.
3 Materials and Methods
3.1 Plant materials
The tested potato cultivar called Burbank, a variety
from the United States was used in this research
purchased from the Institute of Vegetables and
Flowers, Chinese Academy of Agricultural Sciences
(CAAS), which is known to be susceptible to late blight.
The potato was subcultured in MS medium with 6 g /L
agarose and 30 g /L glucose; pH value was adjusted to
5.8. The leaf of the tissue cultured plantwas used as the
explant after grown at 3~4 weeks old.
3.2
hrap
gene and its expression plasmid
The
hrap
gene was regulated by the CaMV35S
promoter and its expression plasmid, pBI-HRAP was
transformed into
Agrobacterium
, which was kindly
provided by Prof. Teng-yung Feng from Institute of
Botany, Academia Sinica in Taiwan. The plasmid
structure is shown in Figure 5.
Figutre 5 The structure of plasmid harbouring HRAP in pBI121
3.3 Genetic transformation
The explant from the tissue cultured potato leaflets
were placed on CIM medium (medium formula: BAP
2.0 mg/L, NAA 0.2 mg/L, 2,4
-
D 0.2 mg/L,
glucose
16 g/L, agarose 6 g/L and finally adjusted pH to 5.8),
and pre-cultured for 2 days at 25
℃
in the dark. Then
the explants were immersed for 20 min in the
Agrobacterium
inoculum kept an optical density of
about 0.5 at 600 nm (
OD
600
), and co-cultured for 2
days with
Agrobacterium
in the same medium and
condition. Explants were transferred to GIM medium
(CIM medium (30 g/L glucose) supplemented with
500 mg/L carbenicillin and 50 mg/L kanamycin (Kan,
Sigma-Aldrich, St. Louis, MO, USA)). After about
three weeks, the leaves with calli are transferred into
SIM medium (GIM with BAP 2.0 mg/L, NAA 0.2
mg/L, 2,4
-
D 0.2 mg/L replaced by BAP 2.5 mg/L and
GA
3
5.0 mg/L) to induce adventitious bud. Once the
adventitious buds reached 2 to 3 cm in height, they
were cut and transferred to CIM for subculture. The
adventitious buds were then again removed as they
reached a height of 2 to 3 cm and transferred to
rooting medium (RM: MS medium supplemented with
75 mg/L Kan, 6 g/L agarose and 30 g/L glucose, pH
5.8). Regenerated plantlets rooted normally were