*Article* **Marker-Free Rice (***Oryza sativa* **L. cv. IR 64) Overexpressing** *PDH45* **Gene Confers Salinity Tolerance by Maintaining Photosynthesis and Antioxidant Machinery**

**Ranjan Kumar Sahoo 1,2, Renu Tuteja 2, Ritu Gill 3, Juan Francisco Jiménez Bremont 4,\*, Sarvajeet Singh Gill 3,\* and Narendra Tuteja 2,\***


**Abstract:** Helicases function as key enzymes in salinity stress tolerance, and the role and function of *PDH45* (pea DNA helicase 45) in stress tolerance have been reported in different crops with selectable markers, raising public and regulatory concerns. In the present study, we developed five lines of marker-free *PDH45*-overexpressing transgenic lines of rice (*Oryza sativa* L. cv. IR64). The overexpression of *PDH45* driven by CaMV35S promoter in transgenic rice conferred high salinity (200 mM NaCl) tolerance in the T1 generation. Molecular attributes such as PCR, RT-PCR, and Southern and Western blot analyses confirmed stable integration and expression of the *PDH45* gene in the *PDH45*-overexpressing lines. We observed higher endogenous levels of sugars (glucose and fructose) and hormones (GA, zeatin, and IAA) in the transgenic lines in comparison to control plants (empty vector (VC) and wild type (WT)) under salt treatments. Furthermore, photosynthetic characteristics such as net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 (Ci), and chlorophyll (Chl) content were significantly higher in transgenic lines under salinity stress as compared to control plants. However, the maximum primary photochemical efficiency of PSII, as an estimated from variable to maximum chlorophyll a fluorescence (Fv/Fm), was identical in the transgenics to that in the control plants. The activities of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (GPX), were significantly higher in transgenic lines in comparison to control plants, which helped in keeping the oxidative stress burden (MDA and H2O2) lesser on transgenic lines, thus protecting the growth and photosynthetic efficiency of the plants. Overall, the present research reports the development of marker-free *PDH45*-overexpressing transgenic lines for salt tolerance that can potentially avoid public and biosafety concerns and facilitate the commercialization of genetically engineered crop plants.

**Keywords:** antioxidants; reactive oxygen species; oxidative stress; marker-free transgenic rice; mature seed-derived calli; pea DNA helicase 45; photosynthesis; salinity stress tolerance
