**1. Introduction**

Rice (*Oryza sativa* L., family Gramineae (Poaceae)) is an important staple food crop that is produced (518 million tonnes, milled), cultivated, and consumed globally in >122 countries (excluding Antarctica), being susceptible to salt amongst cereal crops [1–3]. Major abiotic stresses (salinity, drought, extreme temperatures, heavy metal, etc.) are a significant limitation in rice cultivation globally [1]. Soil salinity is a major problem that reduces

**Citation:** Sahoo, R.K.; Tuteja, R.; Gill, R.; Jiménez Bremont, J.F.; Gill, S.S.; Tuteja, N. Marker-Free Rice (*Oryza sativa* L. cv. IR 64) Overexpressing *PDH45* Gene Confers Salinity Tolerance by Maintaining Photosynthesis and Antioxidant Machinery. *Antioxidants* **2022**, *11*, 770. https://doi.org/10.3390/ antiox11040770

Academic Editor: Nafees A. Khan

Received: 23 March 2022 Accepted: 5 April 2022 Published: 12 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

productivity of crops in irrigated as well as in tropical fields, where the deterioration of agricultural lands occur due to salinity [4–6]. It brings series of changes at the physiological, biochemical, and molecular levels by affecting the photosynthetic machinery (partial stomatal closure and hampered photosystem II (PSII), reactive oxygen species (ROS)-led molecular injury, restricted water/nutrient availability, and disturbed sodium (Na+)/potassium ion (K+) homeostasis), which ultimately poses serious yield penalty [7–12]. Due to rapidly growing global population and urbanization, it is impossible to increase the cultivated land area, and therefore to fulfill the demand of rice consumers, it becomes imperative to discover new techniques for developing salinity-tolerant crop plants by protecting the photosynthetic machinery (net photosynthetic rate, stomatal conductance, chlorophyll content), efficient ROS scavenging, membrane integrity, Na<sup>+</sup> exclusion, etc. [13–15]. Robust antioxidant machinery consisting of enzymatic (SOD, CAT, APX, GPX, GR, etc.) and non-enzymatic antioxidants (glutathione (GSH) and ascorbic acid (AsA) is efficient enough to protect the photosynthetic machinery, cellular components, and membranes under various abiotic stresses [8]. Therefore, strong antioxidant machinery can be well correlated with salinity stress tolerance in crop plants [8]. Nidumukkala et al. [16] reviewed the fact that overexpression of helicases in different model and crop plants provides salt tolerance though increased antioxidant capacity, photosynthetic efficiency, and ion homeostasis, as well as by regulating the expression of various stress responsive genes. Therefore, introduction of a stress-tolerant gene in rice is one of the effective ways to develop stress-tolerant cultivars without yield penalty. The presence of selectable marker genes (SMGs, antibiotic or herbicide resistance genes) in genetically engineered crops may arouse public and regulatory concerns due to biosafety issues because the weeds or pathogenic microorganisms present in soil may become resistant to herbicides or antibiotics and can harm public health [17]. The problem of transgene expression arises due to the sexual crossing, which can lead to homology-dependent gene silencing in the genome [17]. Due to consumer, environmental, and biosafety concerns, the regulatory bodies also encourage the development of marker-free transgenic crops with an array of different transformation strategies such as homologous recombination, sitespecific recombination, co-transformation, transposon-mediated transgene reintegration system, and CRISPR/Cas9 system [17–20]. The tissue culture methods are generally used to understand the mechanisms underlying salt tolerance of transgenic lines [21,22]. Several techniques have been developed to improve Agrobacterium-mediated transformation of indica rice [23–25]. The development of an efficient large-scale transformation system requires a large number of transformants for successful gene transfer [24]. Previously, many researchers developed a transformation protocol for marker-free transgenic rice plants using anther culture [26,27], but the unavailability of explants (anther) throughout the year is a major limitation of this method and it is very laborious to screen the transgenic plants by a PCR-based method.

In the present study, we report that overexpression of *PDH45* gene in an elite indica rice variety IR64 (*Oryza sativa* L., cv. IR64) showed tolerance against salinity stress as well as improved growth, photosynthesis, and better antioxidant machinery in the transgenic rice. We exploited the potential of transgenic technologies for crop improvement through developing marker-free transgenic *PDH45* rice. Thus, we also successfully developed a screening technique using 200 mM NaCl salt to screen marker-free *PDH45* transgenic rice plants. Development of rice transgenic lines overexpressing the *PDH45* gene without the antibiotic marker gene for stable expression of the stress-tolerant trait in a predictable manner avoids the transfer of undesirable transgenic material to non-transgenic crops and related species.
