Basic HTML Version
Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37-49
http://mpb.sophiapublisher.com
39
not contain genes of interest rather only 8 out of 34
Basta selected plants contained at least one gene of
interest in case of Experiment
-
1 (
pUbi-HarChit+Ubi
-HarCho+P35SAcS
) and 6 out of 31 plants contained
at least one gene of interest in case of Experiment
-
2
(
pVst-HarChit+Vst-HarCho+P35SAcS
). This made a
transformation frequency of 0.26 and 0.17% for first
and second combination. If talked only based upon
Basta resistance which is conferred by
P35SAcS,
the
transformation frequency proved to be 1.08 and 0.87%
for both combinations as a total of 3128 and 3540
IZEs were bombarded respectively
.
Table 1 Primer pairs used to amplify
HarChit
and
HarCho
gene
1.2 Molecular verification of
pUbi-Harchit
and
pUbi-Harcho
transgenic plants and their progenies
The plants which were resistant to
Basta
were checked
for the presence of
pUbi-HarChit
and/or
pUbi-HarCho
by Southern Blot analysis. Details of the procedure
can be seen in the material and Methods. A 400 bp
long fragment from
HarChit
gene and another 400 bp
long fragment from
HarCho
gene were labelled with
DIG. Chemiluminescence’s detection was done with
CSPD® and the images were developed on X-rays
film. A total of seven plants (I.A
-
1, I.A
-
2, I.A
-
3,
I.A
-
4, I.A
-
4, I.A
-
5, I.A
-
6, and I.A
-
18) had
pUbi-Harchit
construct with intact promoter, gene of
interest and terminator. Out of these seven plants three
had single copy of
pUbi-Harchit
, three had double
copy integration and one had three to four copies
integrated in the genome. Seven plants (I.A
-
1, I.A
-
3,
I.A
-
4, I.A
-
4, I.A
-
5, I.A
-
6, I.A
-
17 and I.A
-
18) were
positive for
pUbi-Harcho
and out of these seven
plants six were already positive for
pUbi-Harchit.
I.A
-
2 was positive for
pUbi-Harchit
but proved
negative for
pUbi-Harcho.
Similarly I.A
-
17 was
negative for
pUbi-Harchit
but proved positive for
pUbi-Harcho.
Out of the seven plants positive for the
pUbi-Harcho
three showed single copy integration,
one showed two copies, others showed four, five and
seven copy integration. In summery six plants showed
the co-integration of both
pUbi-Harchit
and
pUbi-Harcho
constructs while single construct (once
pUbi-Harchit
and once
pUbi-Harcho
) was present in couple of
plants. The rest of the plants were either having only
bar
gene or were wrongly selected.
1.3 Molecular verification of
pVst-HarChit
and
pVst-HarCho
transgenic plants
The results of Southern Blot analysis showed that out
of 31 plants only five were positive for both
pVst-HarChit
and
pVst-HarCho
while one plant was
having only
pVst-HarCho.
The rest of the plants were
either having only
bar
gene or were false selection.
Only one plant had single copy integration for
pVst-HarChit
, the rest of the plants were having
multicopy integrations for both the gene constructs.
The copy number of integration varied from 1
-
5 for
pVst-HarChit
and 2
-
7 for
pVst-HarCho
. In the plant
“I.A
-
8” one band in the lane reserved for cassette out
showed heavier band than the expected while the
bands in the lane reserved for linearized plasmid were
normal. The rest of the plants showed at least one
band equal to the band in the positive control lanes.
The figure 1 shows the Southern Blot analysis for
pVst-HarCho
construct of all the positive plants. DNA
of all the probes along with positive and negative
probes were run on 0.8% agarose gel and transferred
to nylon membrane by capillary method and cross
linked later. DNA on this membrane was hybridized to
the DIG labelled 400 bp single stranded DNA probe
from
HarCho
and the detection was made with CSPD
substrate. Southern Blot analysis of the putative
transgenic plants showed that 6 plants are positive for
pVst-HarCho
. Five out of them were also positive for
pVst-HarChit
while I.A-8 was negative for
pVst-HarChit
. They showed high copy number in
general except for Plant2 who had 1
-
2 copies.
Primer name
Primer Sequence (5
'
→
3
')
Length (bp)
Product (bp)
Iqrar/Cho upper
GACTAGTCATGCGATCCAACGCTCTCCTTGGCCTCG
36
780
Iqrar/Cho lower
CGTCGACGCGTTAAGCCTTGAGGCCGGCAAC
31
Iqrar/Chit upper
GACTAGTCATGTTGAGCTTCCTCGGAAAATCCGTAG
36
1275
Iqrar/Chit lower
GACGTCGACGCGTTAGTTCAGACCATTCTTGATGTT
36