*2.4. Leaf Disk Senescence Assay and Chlorophyll Content*

The chlorophyll content after leaf disk senescence assay was measured using the method described earlier [37].

#### *2.5. Biochemical Analysis of Antioxidant Activities of Marker-Free PDH45 Transgenic Lines*

The seeds of *PDH45* transgenic, WT, and VC plants were kept in hydroponics for germination, and 21-d-old plants were dipped in 200 mM NaCl for 24 h. The experiments were conducted in green houses of the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, where 16 h light photoperiod at 25 ◦C temperature was maintained. Similar stress treatment and stress conditions as described were also used in the present study [12]. After 24 h salt stress, the plant tissues were used for biochemical analysis such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), proline, hydrogen peroxide (H2O2), lipid peroxidation, relative water content (RWC), and electrolytic leakage. All the parameters were measured using the methods described earlier [38].

#### *2.6. Measurement of Photosynthetic Activities and Agronomic Characteristics of PDH45 Transgenic Plants*

The different photosynthetic measurements such as photosynthetic yield, rate, intercellular CO2 concentration, CO2 release, stomatal conductance, and transpiration rate were recorded using an infrared gas analyzer (IRGA; LI-COR, http://www.licor.com, (accessed on 2 November 2021), on a sunny day between 11:00 and 12:00 noon. The plants were grown under 200 mM NaCl stress in a large tank, and all the parameters were measured using the expanded leaves of mature plants (60 d old). After 12 d of salt stress, different agronomic characteristics were measured using the method described earlier [12].

#### *2.7. Chlorophyll a Fluorescence Measurements*

Plants were grown in green houses of the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, where 16 h light [photosynthetically active radiation (750 μmol m−<sup>2</sup> s<sup>−</sup>1)] photoperiod at 25 ◦C temperature was maintained. Minimal fluorescence (Fo), maximal fluorescence (Fm), maximal variable fluorescence (Fv), and Fv/Fm ratio were included, where Fv = Fm − Fo.

Chlorophyll a (Chl a) fluorescence from the leaves of 25-day-old WT, VC, and transgenic rice seedlings was measured with a PAM-2100 fluorometer (Walz, Germany). Before each measurement, the leaf sample was kept in the dark for 20 min [39]. Optimum quantum efficiency (uPSII, also referred to as Y) of Photosystem II (PSII) was inferred from Fv/Fm = (Fm − Fo)/Fm [40].

#### *2.8. Estimation of Sugar, Hormones (GA, Zeatin and IAA), and Ion Contents*

Shoots and roots from mature (60 d old) *PDH45* T1 transgenic, VC, and WT plants after 12 d of salt stress were used in this study. The sugar content was estimated as described earlier [41]. The endogenous plant hormones (GA, zeatin and IAA) were estimated as described earlier [42]. The flame ionization photometer was used for the estimation of potassium, as described by Chapman and Pratt [43]. The sodium content was estimated as described by Munns et al. [44].

#### *2.9. Salinity Tolerance of Transgenic Plant under 200 mM NaCl Stress*

The *PDH45* transgenic lines (L4, L7, L8, L11 and L13) and VC and WT rice plants (60 d old) were grown in one large metal pot filled with soil and dipped in 200 mM NaCl. The plants were allowed to grow up to maturity (harvest), and the phenotypic conditions of these plants were recorded.
