A DEAD box helicase Psp68 positively regulates salt stress responses in marker-free transgenic rice plants.

Helicases are the motor proteins not only involved in transcriptional and post-transcription process but also provide abiotic stress tolerance in many crops. The p68, belong to the SF2 (DEAD-box helicase) family proteins and overexpression of Psp68 providing enhanced tolerance to transgenic rice plants. In this study, salinity tolerant marker-free transgenic rice has been developed by overexpressing Psp68 gene and phenotypically characterized. The Psp68 overexpressing marker-free transgenic rice plants were initially screened in the rooting medium containing salt stress and 20% polyethylene glycol (PEG). Stable integration and overexpression of Psp68 in marker-free transgenic lines were confirmed by molecular analyses including PCR, southern, western blot, and qRT-PCR analyses. The marker-free transgenic lines showed enhanced tolerance to salinity stress as displayed by early seed germination, higher chlorophyll content, reduced necrosis, more survival rate, improved seedling growth and more grain yield per plant. Furthermore, Psp68 overexpressing marker-free transgenics also accumulated less Na⁺ and higher K⁺ ions in the presence of salinity stress. Phenotypic analyses also revealed that marker-free transgenic rice lines efficiently scavenge ROS-mediated damages as displayed by lower H₂O₂ and malondialdehyde content, delayed electrolyte leakage, higher photosynthetic efficiency, membrane stability, proline content and enhanced activities of antioxidants enzymes. Overall, our results confirmed that Psp68 overexpression confers salinity stress tolerance in marker-free transgenics, hence the technique could be utilized to develop genetically modified crops without any biosafety issues.