2. Molecular analysis of the putative
transgenic plants
• The genomic DNA (gDNA)was isolated using
GenElute Plant Genomic DNA Miniprep Kit
(Sigma-St Louis, USA).
• PCR was performed using NPTII/APS2 primers
to detect the transgene.
• Southern blotting was done (Sambrook et al.
1989).
3. • Sample digestion at 37 C using BamH1.
• Electrophoresis.
• A biotin-labeled probe of NPTII gene fragment was
prepared.
• The chemiluminescent signals were detected.
4. • The regenerated transgenic lines transferred to the
greenhouse after they attained sufficient growth.
• A total of 61 independent T0 transgenic lines.
• Selected three transgenic lines were used for
testing their potential to tolerate and accumulate
heavy metals.
5. • One-month-old WT plants were used as controls.
• A 10-cm long shoot segments of the clonally
propagated WT and T0 independent transgenic
events were used for the test.
• The tolerance of transgenic lines to a mixture of
heavy metals was analyzed using a hydroponic
system.
6. The hydroponic medium was amended with a
mixture of five heavy metals
• Pb(NO3)2
• CdSO4
• CuSO4
• NiSO4
• Na3VO4
7. • At the end of 2 months, the plant shoots and
roots were harvested and the dry weight of
each plant was measured.
• Translocation factor was calculated by using
the formula TF=Cshoot/Croot. Where Cshoot
and Croot are metals concentration in the
shoot (mg kg1) and root of plant.
8. • The heavy metals were measured by coupled
plasma atomic emission spectroscopy.
• One gram of dry powdered plant tissues was
digested in10 ml concentrated nitric acid, 5ml
concentrated hydrochloric acid, and 0.5 ml
30% hydrogen peroxide.
• Samples were diluted to 50 ml with deionized
water prior to analysis.
• Calibration standard and internal standards
were used for quality control.
9. • Percentage recovery was calculated using the
following formula, % recovery=observed
concentration 100/true value.
• Three biological replicates were used for wild-
type plants and each of the transgenic line .
• ANOVA was performed to compare the
significant differences in mean values.
