*2.3. Molecular Analysis (PCR, Southern Blot, qRT-PCR, and Western Blot) of T1 Transgenic PDH45 Plants*

The genomic DNA was extracted from the healthy leaves of marker-free *PDH45* transgenic plants and used to check the integration of the gene by PCR and Southern blot analysis. About 25 μg of genomic DNA was used for Southern blot analysis. First, the genomic DNA was digested with XbaI and resolved on 0.8% agarose gel followed by transfer to nylon membrane (Hybond-N, Amersham, Inc., Amersham, UK) as previously described [35]. The probe was radiolabelled by the gene amplification method using α–[32P] dCTP. Hybridization with the probe was conducted using the method described [35]. The qRT-PCR experiment was performed to check the transcript levels of the gene using gene-specific primers such as forward 5 -ATGGCGACAACTTCTGTG-3 and reverse 5 -TATATAAGATCACCAATATTCATTGG-3 . For Western blot analysis, the crude plant extract was denatured and separated by SDS PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane using the method described [36]. Polyclonal antibodies (1:1000 dilutions) from rabbit were used as a probe against the *PDH45* gene.
