*2.10. Statistical Analysis*

The experimental data were statistically analyzed, and standard error was calculated from three independent observations. Analysis of variance (ANOVA) was performed on the data using SPSS (10.0 Inc., Chicago, IL, USA) to determine the least significant difference (LSD) for the significant data to identify the differences between means and presented as mean ± SE. The means were separated by Duncan's multiple range tests. Different letters indicate significant difference at *p* < 0.05.

#### **3. Results**

#### *3.1. Molecular Analysis of Marker-Free PDH45 Transgenic Lines*

The marker-free *PDH45* transgenic IR64 rice plants were developed using the pCAMBIA1300- *PDH45* gene construct (Figure 1a). Phenotypically, the transgenic rice plants were not significantly different from WT and VC plants (Figure 1b). The desired *PDH45* gene (1.2 kb) fragment was detected by PCR (Figure 1c). The Southern blot results confirmed the integration of a single-copy *PDH45* gene in transgenic rice plants in all the five transgenic lines (L4, L7, L8, L11, and L13) (Figure 1d). The real-time PCR (qRT-PCR) provided ≈8-fold induction in the transcript level of *PDH45* in transgenic lines (L4, L7, L8, L11 and L13) (Figure 1e). The Western blot results showed that *PDH45* protein was expressed to almost similar levels in all the transgenic lines (L4, L7, L8, L11 and L13) as compared to WT and VC plants (Figure 1f).

#### *3.2. PDH45 Transgenic Lines Showed Salinity Tolerance*

The damage caused in the leaf pieces by salt stress was observed in all the plants after 72 h; however, the *PDH45*-overexpressing lines displayed darker green leaves, in contrast to the yellowish leaves of the WT and VC plants (Figure 2a). In this sense, the reduction of chlorophyll content in leaf tissues was lesser in transgenic lines as compared to WT and VC plants under salt stress (Figure 2b). The lesser chlorophyll content in the leaf tissues of WT and VC plants as compared to transgenic lines provided strong evidence towards tolerance against salinity stress (Figure 2a,b). The transgenic lines (L4, L7, L8, L11 and L13) along with WT and VC plants were allowed to grow up to maturity in a metal tank filled with 200 mM NaCl. After 3d, WT and VC plants showed dropping characteristics, whereas *PDH45*-overexpressing transgenic lines L4, L7, L8, L11 and L13 grew well and produced viable seeds (Figure 2c,d).

**Figure 1.** Screening and analysis of *PDH45* marker-free transgenic lines. (**a**) T-DNA construct of pCAMBIA 1300-*PDH45.* (**b**) Transgenic lines (L4, L7, L8, L11, L13, VC, and WT). (**c**) PCR conformation of the *PDH45*-overexpressing transgenic (T1) lines showed the amplification of 1.2 Kb fragment. (**d**) Southern blot analysis showing the integration and copy number of the *PDH45* gene. (**e**) Relative gene expression of *PDH45* transgenic lines. (**f**) Western blot analysis showing the *PDH45* protein (≈45 kDa).

**Figure 2.** Salinity tolerance of *PDH45*-overexpressing transgenic T1 IR64 rice lines. (**a**) Leaf disk senescence assay under 100 and 200 mM NaCl treatment. (**b**) Chlorophyll content (mg/g fw) in *PDH45* transgenic lines after salt treatment. (**c**) Third day in 200 mM NaCl treatment. (**d**) After 15 days of NaCl treatment.

#### *3.3. Agronomic Performance of Marker-Free PDH45 Transgenic Plants under Stress*

The agronomic performance of T1 transgenic lines under 200 mM NaCl treatment was compared with WT and VC without NaCl treatment. Better agronomic characteristics were observed in *PDH45* transgenic plants as compared to WT and VC plants. Several phenotypic characteristics of transgenic plants were recorded and found to be almost similar to the WT and VC plants grown in 0 mM NaCl. However, under 200 mM NaCl treatment, the WT and VC plants did not survive until flowering stage (Figure 2d).
