Coordinated Vacuolar Sequestration and Sodium Extrusion Improve Salinity Tolerance in Transgenic Rice: A Soil-Based Evaluation

Abstract Rice (Oryza sativa L.) is a primary global staple, yet its productivity is increasingly threatened by salinity stress, which impairs growth through ionic toxicity and osmotic imbalance. To combat this, plants must employ robust sodium (Na+) homeostasis mechanisms, specifically through the sequestration of Na+ into vacuoles or extrusion into the apoplast. This study investigated a multigene engineering strategy to enhance salt tolerance in the rice variety ‘Vikas’. Using a tissue culture-independent Agrobacterium-mediated in-planta transformation approach, we developed transgenic lines expressing various combinations of three key genes: AVP1 (Arabidopsis vacuolar H+-pyrophosphatase), NHX1 (vacuolar Na+/H+ antiporter), and SOS1 (plasma membrane Na+/H+ antiporter). The relative efficacy of multigene (AVP1-NHX1-SOS1) transgenics was compared against double-gene (AVP1-NHX1) and single-gene (AVP1, NHX1, and SOS1 individually) counterparts. Whole-plant performance was evaluated using a standardized soil system (CSSRI, Karnal) under two salinity levels: 4 and 8 dS/m. Results showed that multigene and double-gene transgenics significantly outperformed single-gene transgenic lines and wild-type controls. The AVP1-NHX1-SOS1 co-overexpressing lines exhibited superior photosynthetic rates, higher total dry matter, increased panicle weight, and improved spikelet fertility under stress. These findings demonstrate that stacking genes involved in both vacuolar sequestration and cellular extrusion provides a synergistic effect, effectively neutralizing cytosolic Na+ toxicity. This multigene approach offers a highly promising strategy for developing resilient rice cultivars capable of maintaining high yields in salt-affected environments.