Basic HTML Version
Molecular Plant Breeding 2013, Vol.5, No.5, 24
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http://mpb.biopublisher.ca
26
All
the five PCR positive plants were subjected to
feeding by neonate larvae of the bollworm
Helicoverpa armigera
. Bioassay on detached leaves
showed considerable variation among the transgenic
plants. The larval mortality percentage of five T
0
transgenic plants ranged from 33.0% to 67.0% (T
0
plant no: 3) (Table 2).
Table 2 Insect bioassay of transgenic cotton plants (T
0
) against
Helicoverpa armigera
Transgenic lines (T
0
)
Mortality rate after 3 days (%)
1
60.0
b
2
47.0
c
3
67.0
a
4
33.0
e
5
40.0
d
Control
0.0
Note: The values in a column with different superscript letters
represent significant differences by LSD (P=0.05)
The larvae fed on the transgenic plants were stunted
in growth when compared to larvae fed on
non-transformed control plant (Figure 3). Biotoxicity
assays conducted by Bakhsh et al. (2009) in transgenic
cotton leaves with 2
nd
instar Heliothis larvae exhibited
variable mortality rate of 60%~100%.
Figure 3 Insect Bioassay
Note: A: a) leaf of non-transgenic (control) plant; B: b) leaf of
transgenic plant
Variability in larval mortality and growth rate
reduction observed among transgenic cotton plants
might be due to the differences in the expression
levels of gene in these plants. In this study, however,
100% mortality has not been observed in the
transgenic plants analyzed. This could be because of
lower level of expression of the cry2Ab gene in
transgenic plants. However, with large number of
transformation experiments, it would be possible to
obtain large number of independent transgenic events.
2 Materials and methods
2.1 Induction of embryogenic calli
Delinted seeds of Coker 310 were surface sterilized with
70% ethanol for 1 min and washed thrice with sterile
distilled water. They were again surface sterilized with
0.1% (w/v) aqueous mercuric chloride solution for 10
min and washed thrice subsequently with sterile
distilled water and soaked overnight to soften the seed
coats. Seed coats were removed next day and the
sterile seeds were germinated on a half strength MS
(Murashige and Skoog, 1962) basal medium
supplemented with 15 g/L sucrose and 9 g/L agar. The
germination bottles were then incubated for one week
in the culture room at 25°C±2°C under 16/8 hr
photoperiod. Cotyledon (~15 mm
2
) sections were
excised from seven day old cotton seedlings and
inoculated in the MS medium supplemented with
0.1 mg/L 2, 4-D and 0.5 mg/L kinetin, 30 g/Lmaltose
solidified with 4 g/L phytagel for callus induction.
After one month of culture (total age of callus –30
days) light-yellow fresh callus were separated from
the explant and subcultured in the same medium for
another one month for callus proliferation. Highly
proliferating callus (total age of the callus -60 days)
were transferred to basal MS medium and maintained
for two months in the same medium to achieve callus
maturation. After callus maturation (total age of callus
-120 days) good proliferating callus were transferred
to MS medium supplemented with 1.9 g/L KNO
3
, 30
g/L maltose solidified with 4 g/L phytagel [referred to
as somatic embryo induction (SEI) media] for the
induction of embryogenic callus. After one month of
culture (total age of callus -150 days), vigorously
growing, friable, loose and white embryogenic calli
were used for transformation experiments.
2.2 Kanamycin sensitivity test
To determine the optimum concentration of
kanamycin to be used for
in vitro
selection of
transformed tissues, five month old embryogenic calli
of Coker 310 was cultured in the SEI medium
supplemented with 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). The
frequency of survived explants was calculated after
four rounds of selection (15 days per round of
selection). After assessing sensitivity of the explants,