*3.5. Chlorophyll a Fluorescence*

The chlorophyll fluorescence rose from a low minimum level ("O" level or Fo) to a higher maximum level ("P" level or Fm) when exposed from dark to light. The maximum primary photochemical efficiency of PSII, estimated from Fv/Fm, was almost identical in the transgenics to that in the VC and WT (Figure 3f).

#### *3.6. Analysis of MDA, H2O2, Ion Leakage, and Antioxidant Response in Marker-Free PDH45 T1 Transgenic Plants*

The salt-induced changes in the ion leakage, H2O2, proline content, accumulation of MDA, RWC, and antioxidant machineries in T1 *PDH45* transgenic lines (L4, L7, L8, L11 and L13) were compared with WT and VC rice seedlings. We observed reduced levels of MDA, H2O2, and ion leakage, alongside an increase in proline content in *PDH45* transgenic lines in comparison to the WT and VC plants under salt stress at 200 mM NaCl (Figure 4a–d). The activities of CAT, APX, GPX, GR and RWC were increased in *PDH45* transgenic plants as compared to WT and VC plants (Figure 4e–i).

**Figure 4.** Biochemical analysis of *PDH45*-overexpressing T1 transgenic lines (L4, L7, L8, L11, L13, VC) and WT plants exposed to 24 h at 200 mM NaCl treatment. (**a**) Ion leakage. (**b**) Hydrogen peroxide (H2O2) content. (**c**) Lipid peroxidation expressed in terms of MDA content. (**d**) Level of proline accumulation. (**e**) Catalase (CAT) activity; one unit of enzyme activity defined as 1 μmol H2O2 oxidized min<sup>−</sup>1. (**f**) Ascorbate peroxidase (APX) activity; one unit of enzyme activity defined as 1 μmol of ascorbate oxidized min−1. (**g**) Guaiacol peroxidase (GPX) activity. (**h**) Glutathione reductase (GR) activity; one unit of enzyme activity is defined as 1 μmol of GS-TNB formed min−<sup>1</sup> due to reduction of DTNB. (**i**) Percent relative water content (RWC). Values are mean ± SE (*n* = 3). Different letters on the top of bars indicate significant differences at *p* ≤ 0.05 level as determined by Duncan's multiple range test (DMRT).

#### *3.7. The Sugar and Hormone Content of Marker-Free PDH45 T1 Transgenic Plants*

The *PDH45* L4, L7, L8, L11 and L13 transgenic lines showed higher endogenous sugar (glucose and fructose) content in roots as well as in shoots when compared with WT and VC plants (Figure 5a,b). The endogenous hormones such as GA, zeatin, and IAA content were also higher in roots and shoots of *PDH45* transgenics as compared to WT and VC plants (Figure 5c–e). The potassium content in transgenic plants was higher, whereas sodium

content was lower in marker-free *PDH45* transgenic plant tissues as compared to WT and VC plants (Figure 5f).

**Figure 5.** Soluble sugar, hormones, and K<sup>+</sup> and Na<sup>+</sup> content in the roots and shoots of *PDH45* overexpressing marker-free transgenic lines (L4, L7, L8, L11 L13) as compared to WT and VC plants exposed to 24 h at 200 mM NaCl treatment. (**a**) Glucose content. (**b**) Fructose content. (**c**) Endogenous GA content. (**d**) Endogenous zeatin content. (**e**) Endogenous IAA content. (**f**) Endogenous potassium and sodium content. Values are mean ± SE (*n* = 3). Different letters on the top of bars indicate significant differences at *p* ≤ 0.05 level as determined by Duncan's multiple range test (DMRT).

#### **4. Discussion**

In the era of frequently changing global climatic conditions, shortage of irrigation water, reduced agriculturally suitable cultivable land area, degradation and salinization of the agricultural soil, and unpredictable onset of abiotic stresses, agricultural productivity is severely affected, posing a serious threat to food security. Therefore, it is imperative to develop genetically engineered stress-tolerant crops with all the qualifications of global acceptance. It has been reported that overexpression of helicases (*PDH45*/*PDH47*) in different model and crop plants provides salt/cold tolerance through increased antioxidant capacity, photosynthetic efficiency, and ion homeostasis, as well as by regulating the expression of various stress responsive genes [16,29–32,37,45]. Genetically engineered transgenic crops with selectable markers (antibiotic or herbicide resistance) have public and regulatory concerns; therefore, development of marker-free transgenic plants is needed in order to avoid public and biosafety concerns and to facilitate the commercialization of genetically engineered crop plants [17].

We developed the method to select marker and reporter free transgenic lines using the previously published reports [46,47]. In this research, marker-free *PDH45* transgenic rice plants were raised using Agrobacterium-mediated transformation followed by screening with 200 mM NaCl in selection, shoot, and root regeneration media to select only the transformed calli for plant regeneration because *PDH45* is responsible for salinity tolerance [31–33,45]. The elevated stress tolerance in *PDH45*-expressing plants correlated with MH1 (*M*. *sativa* helicase 1) transgenic plants, showing that MH1 functions in abiotic stress tolerance by elevating reactive oxygen species (ROS) burden and through osmotic adjustment [48]. Five independent transgenic lines (L4, L7, L8, L11 and L13) along with empty VC and WT plants were used for functional validation under salt stress. These lines express almost similar levels of *PDH45* protein. Similar to previous reports, these *PDH45* transgenic rice plants also showed high salinity tolerance. This was indicated by the presence of higher chlorophyll content in the leaf disks of salinity-stressed T1 transgenic plants, whereas VC and WT plant leaves became yellow. Moreover, the transgenic plants were able to grow in the continuous presence of 200 mM NaCl stress. These results indicate that the introduced trait is functional in transgenic plants and that it is also stable. The transgenic lines also maintained higher endogenous nutrient contents as compared to the VC and WT plants under salinity stress, which revealed the salinity tolerance potential of the transgenic lines. Similar findings have been reported earlier [12,32,33,45,49]. Higher concentration of potassium and lower concentration of sodium were found in leaves of *PDH45*-overexpressing transgenic lines as compared with VC and WT plants.

*PDH45*-overexpressing marker-free transgenic lines maintained higher endogenous nutrient contents under salinity stress as compared with WT and VC plants, which proved the salt stress tolerance potential of the marker-free *PDH45* transgenic lines, which is in agreement with the previous reports [31–33]. The higher potassium and lower sodium concentration in T1 transgenic plants indicates that the lower Na+/K+ ratio in the transgenic lines might be responsible for imparting better stress tolerance to salinity stress in comparison to the VC and WT plants. The better photosynthetic activities such as net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, CO2 release and transpiration rate, and photosynthetic yield (Fv/Fm) were observed in *PDH45* transgenic lines as compared to the VC and WT plants. The retention of chlorophyll content in transgenic lines indicates the better control over the photosynthetic apparatus under salt stress. Our data are in agreement with the earlier reports on *PDH45-*, SUV3-, and BAT1-overexpressing rice plants under stress [12,31,32,49].

Sugars such as glucose and fructose may play a key role in salt defense mechanisms through ROS detoxification [49–53]. The sugar content in *PDH45*-overexpressing marker-free transgenic lines was higher as compared to VC and WT plants. The *PDH45* overexpressing transgenic rice plants showed significantly higher endogenous content of plant hormones in leaf, stem, and root, directing the molecular and biochemical mechanisms to confer increased stress tolerance [54]. A similar trend of endogenous plant hormone profile was also reflected in OsSUV3 and OsBAT1 transgenic rice under stress conditions [7,49]. This is a very simple, reproducible, and improved protocol for selection of marker-free transgenic rice plants using Agrobacterium-mediated transformation of mature seed-derived callus tissues of indica rice variety, IR64.

#### **5. Conclusions**

The present study provides reporter and marker-free transgenic rice plants that has a scope for future commercialization and approval from regulatory agencies as they are focusing on the removal of reporter and marker genes from transgenic plants. We developed a unique successful salt screening method for screening transgenic lines during tissue culture and also utilized the unique function of *PDH45* helicase in providing salt tolerance in marker-free transgenic rice cv. IR64. It also provides a good example for the exploitation of helicases for enhanced agricultural production, while withstanding extreme climatic conditions, maintaining biosafety regulations, and ensuring food security.

**Author Contributions:** R.T. and N.T. designed the research; R.K.S. performed the experiments; R.K.S., R.T. and N.T. analyzed the data; R.K.S., R.G., S.S.G., J.F.J.B. and N.T. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. The APC was funded by JFJB.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

**Acknowledgments:** Work on plant abiotic stress tolerance in N.T.'s laboratory was partially supported by the Department of Science and Technology (DST), Government of India, and Department of Biotechnology (DBT), Government of India. S.S.G. & R.G. acknowledges partial support from DBT-BUILDER grant, Department of Biotechnology, Govt. of India (BT/INF/22/SP43043/2021). JFJB acknowledges funding support by CONACYT (Ciencia Básica A1-S-25233). The authors gratefully acknowledge the help of Govindjee for his critical review and suggestions for the improvement of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

