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Molecular Plant Breeding 2013, Vol.5, No.5, 24
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http://mpb.biopublisher.ca
25
that are extremely amenable for transformation by
Agrobacterium
.
In the transformation studies, the embryogenic calli
was first subjected to kanamycin sensitivity test to
standardize the concentration of kanamycin for
effecting the selection of transformed callus.
Kanamycin selection for transgenic tissue using the
npt II
gene as a selectable marker is well-known for
cotton and has been commonly used in cotton gene
transformation (Wilkins et al., 2004; Khan et al.,
2010). Plant cells transformed with the
npt II
gene
can detoxify the antibiotics in the selection medium.
In this study, the embryogenic calli when cultured
on the medium containing different concentrations
of kanamycin (0, 5 mg/L, 10 mg/L, 15 mg/L, 20
mg/L, 25 mg/L, 30 mg/L, 35 mg/L and 40 mg/L)
none of the calli survived beyond the concentration
of 20 mg/L (Table 1). Therefore, 25 mg/L
concentration of kanamycin was chosen for the
selection of transformed tissues in transformation
experiments.
Table 1 Effect of different concentrations of kanamycin on the
survival of Coker 310 embryogenic calli
Kanamycin concentration (mg/L) Embryogenic calli survival (%)
0
100.0
a
5
80.0
b
10
62.2
c
15
37.7
d
20
17.7
e
25
0.0
30
0.0
35
0.0
40
0.0
Note: The values in a column with different superscript letters
represent significant differences by LSD (P=0.05)
Seven days old pre-cultured embryogenic calli after
cocultivation with
Agrobacterium
for 72 h, were
transferred on to the selection medium containing 25
mg/L kanamycin. During the first round of selection,
the co-cultivated calli became dark brown or black.
The callus remained dark brown or black also during
second selection. Fresh growth was observed from the
kanamycin resistant calli in the third selection. After
four rounds of selection (each round with 15 days
duration), the kanamycin resistant embryogenic calli
were at an average frequency of 37.0%. Further two
subcultures (each with one month duration) in the
same media the kanamycin resistant embryogenic calli
resulted in the formation of somatic embryos at an
average frequency of 23.7%. The matured cotyledonary
stage embryos (3~5 mm size) were germinated on the
plantlet development medium without kanamycin
selection. Out of 500 embryogenic calli cultured, eight
plants were regenerated at a regeneration frequency of
1.6%. All the eight plants were transferred to glass
house after hardening.
The eight putative transgenic plants (T
0
) were
confirmed for the presence of
nptII and cry2Ab
gene using specific primers. PCR analysis resulted
in the expected sizes of 800bp for the nptII (Figure
1) and 611 bp (Figure 2) for cry2Ab gene in five
putative transgenic plants. Thus, out of 500
explants co-cultivated, 5 plants were found to be
positive at the end that resulted in the transformation
efficiency of 1.0%.
Figure 1: PCR amplification of npt ll gene in putaive transgenic
lines of Coker 310
Note: 1: 100 bp ladder (Figure 1) and 1 kb ladder (Figure 2);
2: positive control; 3: negative control; 4-6: non-transformed
lines; 7-11: putative transgenic lines
Figure 2: PCR amplification of
cry2Ab
gene in putaive
transgenic lines of Coker 310
Note: 1: 100 bp ladder (Figure 1) and 1 kb ladder (Figure 2); 2:
positive control; 3: negative control; 4-6: non-transformed lines;
7-11: putative transgenic lines