Seed nano-priming with zinc oxide nanoparticles enhances drought tolerance and reveals SSR-based genetic diversity in basmati rice (Oryza sativa L.)

Introduction

Rice (Oryza sativa L.) is the world's most important food crop, serving as the primary staple for approximately 40% of the global population and providing 23% of total caloric intake [1] . Its historical significance spans millennia, with evidence of rice cultivation in India dating back to 1500-1000 B.C. [2] . Rice has played a vital role in sustaining populations in the humid regions of Asia and parts of Western Africa, and its consumption has steadily increased in Europe and North America due to its spread. Currently, more than 120 countries produce rice, with China (148 million metric tons) and India (122 million metric tons) as the leading producers [3] . While rice can be cultivated in diverse agro-climatic environments, from high-altitude regions like Jammu to low-lying delta regions such as Sundarbans, the majority of global production and consumption occurs in Asia. In Southeast Asia, rice accounts for 60% of total food consumption, while it contributes around 35% in East and South Asia. Bangladesh, Cambodia, Indonesia, Laos, Myanmar, Thailand, and Vietnam have the highest per capita rice consumption, ranging from 130 to 180 kg per year [4] . Over the past five decades, global rice production has tripled, rising from 216 to 685 million tonnes, while productivity has more than doubled, from 1.87 to 4.30 tonnes per hectare. China currently leads in global rice production, while India, with the largest rice acreage of 45 million hectares, provides about one-fourth of Asia's total rice production [5] . However, rice production is increasingly threatened by both biotic and abiotic stresses, notably drought. Drought is a major limiting factor in rice cultivation, affecting up to 50% of the global rice crop and resulting in significant economic losses [6] . As global climate change exacerbates water scarcity, it is critical to develop strategies to enhance rice's resilience to drought.

Drought stress affects rice growth, development, metabolism, and physiological states by limiting water content, turgor, and overall plant water balance. It also leads to reduced leaf area, altered nutrient distribution, and diminished gas exchange, impacting photosynthesis and overall plant health [7,8] . Additionally, drought triggers the production of reactive oxygen species (ROS), which damage cell structures and metabolic processes [9] . In response, plants enhance their antioxidant enzyme activities, store osmolytes such as proline, and activate mechanisms to regulate stomatal closure and water use efficiency [10] . Nanotechnology has emerged as a promising solution to enhance agricultural sustainability and productivity. Seed priming with zinc oxide nanoparticles (ZnO-NPs) has shown significant potential to improve seed germination, seedling vigor, and overall plant resistance to drought stress [11] . Zinc plays a vital role in improving plant homeostasis, enhancing chlorophyll content, and regulating protein metabolism, thus supporting crops under water-deficit conditions [12,13] . In rice, ZnO-NPs have shown promising results in alleviating drought-induced stress by improving seed germination and plant vigor. Understanding genotype-specific responses to drought mitigation strategies is equally critical for effective crop improvement. Variability in drought tolerance among rice genotypes is governed not only by physiological adaptability but also by underlying genetic differences. Molecular characterization using Simple Sequence Repeat (SSR) markers has been widely employed to assess genetic diversity and establish genetic relationships among rice genotypes due to their high polymorphism, co-dominant inheritance, and reproducibility [14] . SSR-based analysis provides reliable insights into genetic distinctness and diversity, which are essential for identifying droughttolerant genotypes and supporting breeding programs [15] . Integrating morpho-physiological, biochemical, and molecular analyses offers a comprehensive approach to understanding drought tolerance mechanisms in rice. Therefore, the present study was undertaken to evaluate the effect of zinc oxide nanoparticle (ZnO-NP) seed priming on morpho-physiological and biochemical traits of selected Basmati rice genotypes under drought stress conditions. In addition, the study aimed to assess the genetic diversity and molecular differentiation among these Basmati rice genotypes using Simple Sequence Repeat (SSR) markers.

Materials and Methods

The present study was conducted at the Rainout Shelter and Genomics Laboratory, Department of Agricultural Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India. Three Basmati rice (Oryza sativa L.) genotypes, namely Pusa Basmati 1718 (PB 1718), Pusa Basmati 1728 (PB 1728), and Vallabh Basmati 24 (VB 24), were used as experimental material. Seeds of all genotypes were surface sterilized and soaked in a 40 ppm zinc oxide nanoparticle (ZnO-NP) solution for 24 h prior to sowing, while untreated seeds served as the control. The experiment comprised seven treatments. Treatment T 0 represented the well-watered control. Drought stress treatments T 1 , T 2 , and T 3 were imposed by withholding irrigation for 5, 10, and 15 days, respectively. To evaluate the effect of ZnO-NP seed priming under drought conditions, primed seeds were subjected to the same durations of water withholding, constituting treatments T 4 , T 5 , and T 6 , corresponding to ZnO-NP priming combined with 5, 10, and 15 days of drought stress, respectively. The field experiment was conducted using a Randomized Block Design (RBD) with three replications. Thirty day old seedlings were transplanted into the field, and drought stress was imposed at the active tillering stage by withholding irrigation for the specified durations. Data were recorded on various morphological and yieldrelated traits, including flag leaf length, flag leaf width, primary leaf length, primary leaf width, plant height, panicle length, number of productive tillers per plant, days to 50% flowering, days to maturity, grain yield per plant, 1000grain weight, biological yield per plant, and harvest index. Physiological parameters such as chlorophyll content were measured using a Minolta™ SPAD chlorophyll meter [16] , relative water content (RWC) was determined following the method of Turner and Beg [17] , and membrane stability index (MSI) was estimated according to Sairam and Srivastava [18] . Biochemical analyses included estimation of total protein content using Lowry method [19] , proline content estimated using method of Bates [20] , and peroxidase (POD) enzyme activity as described by Curne and Galston [21] . For molecular characterization, genomic DNA was isolated from young leaf tissue using the cetyltrimethylammonium bromide (CTAB) method by Murray and Thompson [22] . Polymerase chain reaction (PCR) amplification was carried out using ten Simple Sequence Repeat (SSR) markers, namely RM-1, RM-16, RM-20, RM-201, RM-206, RM-212, RM-228, RM-252, RM-273, and RM-289 [23] . PCR amplification was performed with an initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 56-64 °C for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 8 min. Amplified products were resolved on agarose gels, and bands were scored as present [1] or absent [0] . Statistical analysis of the data was carried out using OPSTAT software. Standard error (SE) and critical difference (CD) at the 5% level of significance were calculated. Percentage reduction due to drought stress was calculated relative to the corresponding control, while percentage recovery under ZnO-NP treatment was calculated relative to the respective drought-stressed treatment using the formula: Percentage change (%) = [(Treatment -Reference)/Reference] × 100.

Results

Morphological Responses

Flag leaf length decreased across all rice genotypes under drought stress, with Vallabh Basmati 24 exhibiting the greatest reduction (37.9%), followed by Pusa Basmati 1728 (36.0%), while Pusa Basmati 1718 showed the least reduction (32.0%). ZnO-NP treatment significantly mitigated drought-induced damage, resulting in an improvement of 16

Molecular Characterization

A total of ten SSR markers were employed to assess genetic diversity among three Basmati rice genotypes (PB-1718, PB-1728, and VB-24). All markers produced clear and reproducible amplification patterns. The amplified fragments ranged approximately from 100 to 200 bp, as estimated using a 100 bp DNA ladder. Out of the ten SSR markers analyzed, six markers (60%) were polymorphic, while four markers (RM-1, RM-212, RM-228, and RM-252) were monomorphic across all genotypes. Polymorphic markers, including RM-16, RM-20, RM-201, RM-206, RM-273, and RM-289, effectively differentiated the genotypes based on the presence or absence of alleles. Marker RM-273 showed variation in allele size, indicating multiple allelic forms among the genotypes. The Polymorphism Information Content (PIC) values ranged from 0.00 for monomorphic markers to moderate values (~0.40-0.45) for polymorphic markers, indicating a moderate level of marker informativeness and their effectiveness in detecting genetic variation among the studied genotypes. Cluster analysis based on SSR data using the UPGMA method grouped the genotypes into distinct clusters, revealing clear genetic relationships. PB-1718 and PB-1728 clustered closely, indicating higher genetic similarity, whereas VB-24 formed a separate cluster, reflecting greater genetic divergence. Overall, the SSR-based molecular analysis revealed moderate yet distinct genetic diversity among the three Basmati rice genotypes. This genetic differentiation corresponds well with the observed variation in drought tolerance at the morphological and physio-biochemical levels, supporting the genotype-specific responses to drought stress and ZnO-NP seed priming.

Discussion

Seed nano-priming with ZnO nanoparticles significantly enhanced drought tolerance in Basmati rice by partially mitigating drought-induced reductions in morphological, physiological, and yield-related traits. Under drought stress, all genotypes exhibited pronounced declines in leaf dimensions, plant height, panicle length, and grain yield; however, ZnO-NP application resulted in a consistent and measurable recovery of these traits. Similar improvements in growth and yield under ZnO-NP application have been reported earlier, highlighting the role of zinc in stress mitigation and plant development [24,25,26] . This improvement indicates enhanced water retention, better maintenance of photosynthetic capacity, and increased stabilization of cellular membranes under stress conditions. The higher chlorophyll content, relative water content, and membrane stability index observed in ZnO-NP-treated plants suggest that zinc-mediated regulation of cellular homeostasis and antioxidant defense mechanisms played a critical role in reducing drought-induced damage, thereby supporting improved growth and yield performance [27,28] . The moderated accumulation of proline in ZnO-NP treated plants further indicates a more efficient and regulated stressresponse mechanism compared to untreated droughtstressed plants. While drought stress alone induced a sharp increase in proline content particularly in Vallabh Basmati 24 ZnO-NP priming limited excessive proline accumulation, suggesting improved osmotic adjustment and reduced stress severity. Similar observations have been reported where lower proline accumulation under stress-mitigating treatments was associated with improved stress tolerance rather than stress injury [29,30] . Similarly, peroxidase activity increased under drought stress as part of the antioxidant defense response; however, comparatively lower enzyme activity in ZnO-NP-treated plants reflects a reduced oxidative burden, indicating effective mitigation of stressinduced reactive oxygen species [31] . Although Vallabh Basmati 24 has been reported as drought tolerant in earlier studies, its comparatively higher sensitivity observed in the present investigation can be attributed to the timing of stress imposition. In this study, drought stress was imposed at the early post-transplanting (active tillering) stage, a phase critically associated with root establishment and tiller initiation. Drought tolerance in rice is well known to be growth-stage specific, and tolerance expressed at reproductive or terminal stages does not necessarily confer resistance during early vegetative stress [32, 33] . The greater susceptibility of VB-24 under early-stage drought conditions suggests that its tolerance mechanisms may be more effective at later developmental stages rather than during initial establishment [34] . Molecular characterization using SSR markers revealed moderate but distinct genetic diversity among the three Basmati rice genotypes. Cluster analysis grouped PB-1718 and PB-1728 together, while VB-24 formed a genetically distinct cluster. This molecular differentiation corresponded well with the contrasting drought responses observed at the morphological and physio-biochemical levels. Similar associations between SSR based genetic diversity and drought response patterns have been reported in rice and other cereals [35,36] The genetic separation of VB-24 supports the conclusion that the observed differences in drought response and ZnO-NP efficacy are, at least in part, genetically governed rather than solely environmentally induced. The integration of molecular characterization with physiological screening therefore provides a robust framework for identifying stage-specific drought tolerance in rice genotypes [37] .

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