*2.1. Cloning and Transformation of PDH45 Gene in IR64 Rice*

*PDH45* gene (accession number: Y17186) was used to establish the tissue culture technique. The coding region of *PDH45* gene (1.2 kb) was cloned in reporter gene-free plant transformation vector pCAMBIA1300 in place of hygromycin to generate complete reporter and antibiotic marker-free plasmid pCAMBIA1300-*PDH45*. An empty vector (pCAMBIA1300) construct, called vector control (VC), was used to compare the function of the gene, and the VC construct comprises all components except the *PDH45* gene. The above two constructs (pCAMBIA1300-*PDH45* and pCAMBIA1300) were used for the *Agrobacterium tumefaciens* (LBA4404)-mediated transformation method [28]. The same conditions were used to generate all the plants.

#### *2.2. Development of Selection Technique for Marker-Free Transgenic Plants*

A new selection technique was developed by adding 200 mM NaCl in selection media, shoot induction, and root induction media for the selection of *PDH45* marker-free transgenic plants during the plant induction stage. We modified the media described by Sahoo and Tuteja [28]. Here, we used 200 mM NaCl in place of hygromycin as the gene *PDH45* has already been reported as being responsible for salinity tolerance in different plants [29–34]. The other compositions of media were the same as described earlier [28].
