Salinity stress, genotype screening, vigour index, dendrogram clustering, Principal Component Analysis.

Millets, often referred to as "miracle crops," are gaining importance due to their resilience to various abiotic and biotic stresses, coupled with their nutritional benefits and adaptability to marginal environments (Adhikari et al., 2021). Among them, Echinochloa frumentacea (Barnyard millet) excels in its nutritional profile, including high dietary fibre, iron content, low glycaemic index, and gluten-free properties. Notably, it is one of the oldest domesticated millets, predominantly grown in arid and semi-arid regions of Asia and Africa, where environmental stresses such as salinity are prevalent (Jha et al., 2022).

Soil salinity has emerged as a major constraint to agricultural productivity in these regions, adversely affecting seed germination, early seedling establishment, and ultimately crop yield. In Barnyard millet, salinity stress has been reported to reduce seedling length, disrupt water uptake, and impair physiological processes critical to early plant development (Nedunchezhiyan et al., 2020). High salinity levels lead to osmotic stress, ionic imbalance and the excessive production of reactive oxygen species (ROS), that collapse cellular structure and cell metabolisms (Upadhyay et al., 2022). Furthermore, elevated salt concentrations can suppress the synthesis of essential metabolites like flavonoids, amino acids, and proteins, contributing to compromised growth and grain quality (Negrão et al., 2017).

Plants respond to salt stress through including osmotic adjustment through accumulation of compatible solutes (such as proline and sugars), hormonal regulation (notably abscisic acid), and enhanced activity of antioxidant defense systems that scavenge ROS (Saha et al., 2022). Genetic variability among different genotypes plays a major role in determining the degree of tolerance or sensitivity to salinity, especially during early developmental stages (Mukhthambica et al., 2023). Thus, identifying and characterizing salt-tolerant genotypes is essential for improving millet productivity under stress-prone conditions (Vijayalakshmi et al., 2014).

The present study investigates the differential response of selected Barnyard millet genotypes to incremental levels of salt stress. Salinity was imposed by pre-soaking seeds in sodium chloride (NaCl) solutions of 50 mM, 100 mM, 150 mM and 200 mM concentrations, while untreated seeds served as the control. Key early growth parameters such as root and shoot length, germination percentage, fresh and dry biomass, and vigour index were evaluated to assess the physiological and biochemical impacts of salinity. This work aims to identify genotypic variation in salt tolerance, which may inform breeding strategies for stress-resilient millet cultivars. The salt-tolerance in genotypes is analysed by principal component analysis and dendrogram clustering.

A. Experimental details

The experiment was conducted at the Genetics and Plant Breeding Laboratory, in affiliation with Seed Science and Technology Laboratory at SRM College of Agricultural Sciences, Baburayanpettai, Tamil Nadu, India. One hundred genotypes of barnyard millet were obtained from the International Crop Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad. The salinity tolerance in each genotype were assessed by applying varied concentrations of NaCl across six treatments: T0 (control, 0 mM NaCl), T1 (hydro priming) and T2 to T5 (50 mM NaCl, 100 mM NaCl, 150 mM NaCl and 200 mM NaCl, respectively). The analysis was done using Factorial Completely Randomized Design (FCRD) with four replicates. The sterilized sand is used to grow the seedlings and it was placed in plastic trays. The initial stress was introduced to the genotypes by soaking seeds in distilled water and NaCl solutions for three hours. Hundred seeds per genotype are soaked for each treatment. The NaCl solutions were applied to the sanitized sand trays before sowing of the seed. The seeds are allowed to grow in the sand tray and observations are taken. 

B. Growth parameters recorded

The experiment was carried out with in accordance with International Seed Testing Association (ISTA) guidelines (ISTA, 2012). The germination data was recorded every day and on the 14th day of emergence, the last counts were made and the percentage of germination was determined. Ten healthy seedlings were selected from each replication for observing the root length, on the 14th day of emergence and it was measured in ‘cm’. The root length was measured starting from the surface to tip of the main root. Ten randomly selected healthy seedlings were selected from each replication, on the 14th day of emergence and it is denoted in ‘cm’. The shoot length was measured from the leaf tip to the end of the shoot of seedling. Ten healthy seedlings were selected from each replication, on the 14th day of emergence and weighed immediately after taking it from the sand tray and the fresh weight of roots and shoots can be calculated. Seedlings are shade dried and kept in a hot air oven at 70ºC for 20 hours and it was weighed. Fresh and dry weights are expressed in ‘mg 10 seedlings-1’. The values of the vigor index were calculated by multiplying germination percentage with total seedling length and the average values were shown as whole numbers (Abdul Baki and Anderson 1973). 

C. Statistical Analysis 

The data from each observation were analyzed using the standard deviation from the replicates was used. The Factorial Completely Random Design (FCRD) was used for experimental set up. The principal component analysis (PCA) and Dendrogram Clustering of the growth parameters were done using SPSS version 16.0 (SPSS Inc., Chicago, USA).

A. Dendrogram

The clustering of genotypes was done using dendrogram to screen the genotypes for salinity tolerance. The genotypes within similar cluster represented the same extent of tolerance to the salinity stress. The clustering was done for the key traits that are used to assess the salinity tolerance in the millet seedlings and the traits include germination percentage and vigour index of the genotypes (Tanwar et al., 2023).

The cluster I (red color) included genotypes G34, G18, G25, G28, G31, G33, G30, G40, G43 and G44 (Fig. 1). These clusters represent highest germination percentage among other genotypes and they exhibited similar response to the exposure of different treatments of salinity. The halophytic mechanism in certain genotypes was exhibited by maintenance of cell turgor and the enzymatic production (Afzal et al., 2023). These genotypes responded significantly well, due to the regulated uptake of water, which potentially directs to increased germination percentage.

The cluster II (green) and cluster III (blue) encompasses moderately salt tolerant genotypes (Fig. 1). The cluster II included 28 genotypes and showed moderate variability in salt stress adaptation. The cluster III formed a smaller group of genotypes with comparatively lower level of tolerance to salinity exposure. The cluster IV (purple) represented the highly divergent genotypes with sensitivity to salinity (Fig. 1). It included 44 genotypes whose germination percentage is highly reduced due to the salt stress. This revealed that the barnyard millet exhibit sensitivity to various salinity levels (Mukhopadhyay et al., 2021). The reduction in germination percentage was observed due to the oxidative stress and ion toxicity. The reactive oxygen species (ROS) production within the seeds will be influenced by the ionic toxicity (Nedunchezhiyan et al., 2020). It disrupts the cellular components and compartmentalization.

The vigour index of the genotypes was computed and subjected to dendrogram analysis (Fig. 2). The cluster IV (purple) consisted of genotypes that show higher vigour index. The genotypes G30, G43, G34, G44, G18 and G25 showed highest degree of variability. The consistency in seedling establishment can be positively related with the growth parameters like germination percentage (Powell, 2022).  The cluster III (blue) was the smallest cluster with fewer genotypes, showing moderate variability. The cluster II (green) also showed moderate variability & vigour index, but this cluster includes majority of the genotypes. The cluster I (red) expressed lesser variability among all the clusters. This result proves that the vigor index of genotypes varied across treatments and showed moderate divergence. The moderate divergence showed distinct clusters, which indicates medium level of dissimilarity (Saddiq et al., 2021). 

According to results of both the dendrogram, vigour index was considered as the discriminative trait among other growth parameters. The vigour of the seedlings is not only important for filed emergence, but also correlates with stress mitigation mechanisms (Williams et al., 2019). The results of clustering pattern will help the breeders to identify elite genotypes, resilient to salinity stress. 

B. Principal Component Analysis (PCA)

The Principal Component Analysis was used to evaluate the variation in the barnyard millet genotypes, which were subjected to different salinity levels. This PCA was used to identify the principal components and distinguishing traits, that contribute to the salt tolerance mechanisms in the barnyard millet genotypes (Prabu et al., 2020). Germination percentage, root length, shoot length, total seedling length, fresh weight, dry weight and vigour index are the traits subjected to the principal component analysis.

The principal components PC1 and PC2 of the control (T0) contributed to 70.87% and 10.75% accounted to 81.62% of cumulative variance (Table 1). The genotype differentiation was observed in shoot length, root length, total seedling length and vigour index in PC1. The PC2 was influenced by fresh weight (Fig. 3). It was observed that the biomass accumulation across genotypes was more in this treatment. It is because of the non-stress growth conditions, that the metabolism of seed germination and establishment is well maintained (Mishra et al., 2024). The hydro primed seeds (T1) showed 68.15% variance in PC1 with root length, shoot length, total seedling length and vigour index as distinguishing traits (Fig. 3). These traits had higher loading values. In PC2, the fresh weight was clearly dominant. Hydropriming possibly enhanced water uptake and vigour of the seedlings, due to improved rate of metabolism (Nedunchezhiyan et al., 2020).  

Under 50 mM NaCl concentrations, the fresh weight dominated PC2 and germination percentage contributed moderately to PC2. The PC1 & PC2 contributes to 75.47% and 8.15% respectively to the cumulative variance (Table 1). The mild salt stress interrupts the osmotic balance, reduces the water uptake and influences other growth parameters. The genotypes showing positive associations with growth parameters showed considerable tolerance. The principal component PC1 and PC2 accounted for 72.45% and 9.93% of total variation in 100 mM NaCl treatment. The major contributes to PC1 were vigour index, root length and shoot length. The dry weight showed very lower loading value in PC2. Fresh weight continued to influence PC2 in this treatment also (Fig. 3). The total seedling length was increased but the biomass was reduced due to the increased salt concentration (Narasimhulu et al., 2022). 

At 150 mM NaCl, PC1 had something loading values from root length, shoot length, fresh weight, total seedling length and germination percentage. The PC1 contributed to 73.29% of variance, whereas PC2 contributed to 10.07% of variance (Table 1). The strong loading value in PC2 was observed in dry weight. The above results indicate that some genotypes were able to germinate well and maintain the biomass, under salinity (Gupta and Khandelwal 2022). But few genotypes germinate well and fail to accumulate biomass. This phenomenon of reduction in accumulation of biomass is commonly observed in saline environments (Mukhthambica et al., 2023).

At the maximum concentration of 200 mM NaCl, the PC1 and PC2 explained 71.08% and 9.45% of total variance (80.52%) (Table 1). The PC1 influenced vigour index, shoot length, root length and total seedling length. PC2 was governed by germination percentage and dry weight (Fig. 3). The biplot revealed wider variability that is expressed due to salinity imposition (Durge et al., 2022). The reduction in growth of seedlings across the salinity treatments may be due to interruptions in membrane compartmentation, osmotic imbalance and ionic toxicity (Ladumor et al., 2021).

Fig. 1. Cluster dendrogram of germination percentage of Barnyard millet genotypes under various levels of salinity: Control (T0), Hydropriming (T1), and 50 mM NaCl (T2), 100 mM NaCl (T3), 150mM NaCl (T4) and 200mM NaCl (T5).

Fig. 3. Principal component biplot graphs of Barnyard millet genotypes under various levels of salinity treatments - Control (T0), Hydropriming (water) (T1), and 50mM (T2), 100mM (T3), 150mM (T4) and 200 mM (T5). The observed growth parameters include germination percentage (G%), shoot length (SL), root length (RL), total seedling length (TSL), fresh weight (FW), and dry weight (DW).

Table 1: Principal factors of principal component analysis and their eigenvalues, variability and cumulative variability for control, hydropriming and four different salinity treatments.

Table 2: Contribution of morphological and physiological traits in the principal factors under different salinity treatments (principal component loading values).

The findings from this study provide a strong foundation for future research aimed at improving salinity tolerance in Barnyard millet. The tolerant genotypes identified can be further evaluated under field conditions to validate their performance in real-world saline environments. Advanced molecular approaches such as gene expression profiling, genome-wide association studies and marker-assisted selection can be employed to identify and utilize key genes responsible for salt tolerance. By integrating the data with omics technologies will facilitate the development of elite varieties. The efforts will contribute to sustainable millet production in salt-affected regions, enhancing food security and supporting climate-resilient agriculture.