Agriculture Reference
In-Depth Information
control and transgenic tobacco plants, respectively. (2) Cut fresh leaf disks into
quarters. (3) Transfer disks to 1 ml of histochemical reagent (X-Gluc) in 1.5-ml
microfuge tubes. (4) Incubate at 37
C for 1 h to overnight. (5) After incubation,
rinse in 70 % ethanol for 5 min (Ohta et al.
1990
; Vitha et al.
1995
; Stangeland and
Salehian
2002
; Sun et al.
2006
; Godoy-hern
ยด
ndez et al.
2008
).
10.4.2 PCR for Genomic DNA
PCR-based methods rely on the detection of the specific genes or DNA in the crop
plant. There are several DNA-based methodologies for the detection of transgene;
PCR-based method is the most common method used for commercial testing as
well as by regulatory authorities for the detection of transgene. This method is
based on amplification of a specific target DNA, allowing the million- or billion-
fold copies of a particular gene by using a pair of oligonucleotide primers. The
process involves extraction and purification of DNA, amplification of the specific
DNA fragments by using a pair of primers, and confirmation of the amplified
product. In principle, PCR is efficient in the detection of a single target gene in a
complex DNA mixture (Seki et al.
1999
).
10.4.3 Southern-Blot Analysis
Southern-blot analysis is the method routinely used for the detection of a specific
gene in a DNA sample. To test for the presence of the transgene, gDNA can be
isolated and purified by using standard protocol given by Sambrook and Russell
(
2001
) followed by the digestion with restriction endonuclease like BamHI and
XbaI. Then digested DNA can be purified using the phenol/chloroform method and
concentrated by precipitation with ethanol (Sambrook and Russell
2001
). The
digested and purified DNA fragments can be separated by electrophoresis, and
separated DNA fragments are transferred to a filter membrane. The detection of
specific DNA fragment can be done by probe hybridization (Southern
1975
).
10.4.4 Protein Extraction and Western-Blotting Analysis
Detection of the protein expressed by the transgene can be performed by Western-
blot analysis. Western-blot analysis involves the extraction of the protein; the
protein can be extracted from leaf tissue (100 mg) according to a protocol reported
by Stoger et al. (
1999
). The standard protocol given by Towbin et al. (
1979
)
included the extraction of proteins and their separation by the gel electrophoresis.
The separated proteins are then transferred to a membrane, where detection of a
