Basic HTML Version
Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.4, 37-49
http://mpb.sophiapublisher.com
40
Figure 1 Southern Blot analysis of Primary Transforments (T
0
)
Note: U= Lane undigested g-DNA, 1= Lane with g-DNA
digested with endo-nuclease that linearizes the gene construct.
In this case it is
Kpn-I,
2 = g-DNA digested with endonuclease
that cuts the cassette out of the gene construct. In this case it is
sfi-I
. Lanes1
-
3 = I.A
-
7, Lanes 4
-
6 = I.A
-
8, Lanes 7
-
9 = I.A
-
9,
Lanes 10
-
12 = I.A
-
10, Lanes 13
-
15 = I.A
-
11, Lanes 16
-
18 =
I.A
-
12, Lanes 19
-
23 = controls and empty lanes in between.
1.4 Molecular verification of transgenes in segregating
generation
Self pollinated T
0
plants were harvested after they
reached the maturity. Segregation analysis based on
the
BASTA
resistance was done before going for
molecular verification of the segregating generations.
In all the lines except one from the first batch 3:1 ratio
in the surviving to dead was seen while from the
second batch of transgenics only two lines showed
normal 3:1 ratios and the others did not follow the
trend. It means single locus integration for
bar
gene
was there in some lines and absent in others. The
matter was not studied further in detail because it was
beyond the scope of this project. The plants were
advanced further for molecular analysis of genes of
interest and phyto-pathological studies.
Selected Basta resistant plants from the transgene
progenies were analyzed for the presence and
cosegregation of HarChit and HarCho genes with
bar
gene. For both the batches with constitutive and
inducible promoter respectively,
HarChit
and
HarCho
was always present in Basta resistant plants with the
same trend and integration pattern seen in the primary
transgenic. We have a case where all the plants died
upon basta spray in the progeny but southern showed
that
HarCho
and
HarChit
was still present in these
plants. Progenies were not only checked in T
1
but also in
T
2
example of one southern was shown in the figure 2.
Figure 2 Southern Blot analysis I.A-11in T
1
for Inducible
promoter construct with
HarChit
gene specific probe
DNA of all the probes along wit 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 T
1
selected plants showed that the integration of
pVst-HarChit
in the next generation is the same as it
was in the primary transforments. T
0
plant and the T
1
selected plants looked the same with a copy number of
three proving the successful transfer of
pVst-HarChit.
1.5 Expression analysis of
pUbi-HarChit
and
pUbi-HarCho
transgenes by Northern Blot
In the first batch of transgenics
HarChit
and
HarCho
were under the control of constitutive promoter.
Therefore all the plants which were proved to have
pUbi-Harchit
and/or
pUbi-Harcho
by Southern Blot
analysis were checked for the expression of
HarChit
and
HarCho
genes in primary transforments as well as
in T
1
generation. RNA was isolated from the leaves of
the transgenic and non transgenic control plants and
run on the degrading agarose gel at the rate of 15 µg
per lane per plant. Five lanes were reserved for each
transgenic plant, one lane for T
0
RNA and four for T
1
only one lane was reserved for non transgenic control
.
RNA was transferred to the positively charged Nylon
membrane.
400 bp long gene specific radio labelled
DNA probes generated using primer pairs given in
table 2 were used to detect the mRNA on memberane.
Hybridization detection by taken by capturing the
illumination on X-Rays film. Experiment example is
shown in figure 3. All the plants that contained
HarChit and/or HarCho
also showed expression of
these genes though in varying Freshly isolated RNA