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Molecular Plant Breeding 2013, Vol.5, No.5, 24
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http://mpb.biopublisher.ca
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kanamycin concentration of 25 mg/L was used for
selection of transformed tissues in transformation
experiments.
2.3 Transformation with cry2Ab gene
A binary construct harboring the cry2Ab gene under
the control of the cauliflower mosaic virus 35S
(CaMV 35S) promoter and the plant selectable marker
neomycin phosphotranferase (nptII) gene was used for
transformation (Figure 4).
Figure 4 Physical map of pBI121 harbouring
cry2Ab
and
nptll
genes
Five months old embryogenic calli (derived from 7
days old cotyledon explants) of Coker 310 were
precultured in the fresh SEI media
for different
durations (1, 7, 14, 21 days) before
Agrobacterium
infection. Embryogenic calli with different preculture
periods were infected with the bacterial suspension
with intermittent shaking for 20 min. The bacterial
suspension was removed by pipette and the calli were
blotted dry and transferred to Whatman no filter paper
(cat no. 1004070) placed on co-cultivation medium
composed of SEI media and 100
M acetosyringone.
After co-cultivation at 24°C in the dark for 72h, the
infected embryogenic calli were transferred to
selection medium containing SEI media, 25 mg/L
kanamycin and 500 mg/L cefotaxime. After four
rounds of selection (each with 15 days duration) in the
selection medium, the frequency of kanamycin
resistant embryogenic calli was recorded for the callus
o
Bt
ained from different preculture period (total age of
the callus from callus initiation was 7 months). Of the
different preculture periods, the embryogenic calli
precultured for 7 days were used for further somatic
embryo induction studies. For the induction of
somatic embryos, the rapidly proliferating kanamycin
resistant embryogenic calli were subcultured on fresh
selection medium for further two months (each with
one month duration) (total age of the callus from
callus initiation was 9 months) and the frequency of
somatic embryos formed were recorded.
2.4 Plantlet regeneration
The matured cotyledonary stage embryos (3~5 mm
size) o
Bt
ained were germinated on the plantlet
development medium containing full strength MS
supplemented with 30 g/L sucrose, 1.0 mg/L ets
reached 4~6 leaves stage with sufficient roots (30~45
days of culture in plantlet development media) they
were gently removed from the medium and washed
in running tap water carefully to remove the media
adhering to roots. They were then transferred to
paper cups containing a 1:1:1 (vol/vol) mixture of
sterilized planting substrates like soil: sand: compost.
The plants were covered with polyethylene bags to
maintain enough humidity and kept in culture room
for 15 days. After 15 days, polypropylene bags were
removed and well established plantlets were
transferred to glass house.
2.5 PCR analysis
To verify the presence of nptII and cry2Ab genes,
PCR was performed with the
genomic
DNA samples
of putative transgenic and non-transgenic (control)
plants using gene specific primers. Reactions were
performed in a final volume of 25 μl and the mixture
contained 50 ng of genomic DNA, 2.5 μl of 10× PCR
buffer (10 mM Tris-HCI (pH 9.0), 50 mM KCl, 1.5
mM MgCl) 200mM of each of dNTPs, 70 ng of
upstream and downstream primers and 2 units of Taq
DNA polymerase. Amplification was performed in a
MYCycler, (Biorad,USA). The primer pair used to
amplify a 800 bp long internal fragment of the nptII
gene were 5'ATG ATT GAA CAA GAT GGA TTG
CACG 3' (Forward primer); 5' TCA GAA GAA CTC
GTC AAG AAG GC 3' (Reverse primer). Temperature
profile used for amplification of nptII gene was as
follows: Pre-incubation at 94°C for 5 min leading to
30 cycles of melting at 94°C for 1 min, annealing at
55°C for 1 min, and synthesis at 72°C for 1 min
followed by an extension at 72°C for 15 min. The
primer pair used to amplify a 611 bp–long internal
fragment of the cry2Ab gene were 5' CTG AGC TCA