Clustering of interactions of rapeseed-mustard genotypes and seed priming options as shown in Fig. 7 expressed the similarities between various combinations, especially, V6T1 and V5T1; between V1T1 and V2T1 and others. Clustering different physiological, biochemical and enzymatic quality parameters also showed the similarities between α- amylase activities at 24 and 48 h, peroxidase activities at 24 and 48 h, germination % and seedlings dry weight, root length and vigour index-I etc.
Inherent genetic variety and distinct interactions to the dominant agro-climatic situations during seed development are the two main causes of the difference in germination among genotypes. During seed filling, high ambient temperatures (> 30-35 °C) might hasten seed maturation, shortening the time needed for reserve accumulation and resulting in partial protein, lipid, and starch deposition. As a result, when seeds germinated, their metabolic potential was reduced. Furthermore, it was known that high temperatures inhibited the activity of α-amylase, which was essential for hydrolysing starch into soluble sugars required for the early growth of seedlings. High relative humidity (> 80%) during the late reproductive stages also raised the moisture content of the seeds, which weakened their vigour by encouraging either microbial infection or early sprouting. Cloudy weather or a shorter photoperiod reduced photosynthetic efficiency, which limited the amount of assimilates available to the developing seeds. This was especially true for oilseeds like rapeseed-mustard, where lipid synthesis was impacted. Besides, indeterminate flowering habit and exposure of developing seeds to existing weather conditions, probably exerted significant influence on biochemical activities and mobilization of food reserve on produced seeds and thereby, impacted on their germination. Seed priming with chemicals, specially, KHPO influenced germination probably due to altered enzyme activity, resulting in accelerated metabolism. Besides, consistent supply of energy through phosphorus, when seeds were primed with KHPO, for various physiological, biochemical and enzymatic activities during germination phase probably resulted in improvement of seed germination percentage. Further, Bewley and Black (1982) reported the activity of potassium in increasing ambient oxygen level by restricting oxygen availability for citric acid cycle. Sathish et al. (2011) observed similar improvement in germination of hybrid maize seeds and stated that the result might be due to influence of seed priming on seed membrane integrity, increase in protein and nucleic acid, repair of seed quality deterioration caused by lipid peroxidation, electrolyte leakage etc. PEG 6000 also exerted positive influence on seed germination perhaps due to its beneficial influence on activity enzymes such as protease, amylase which accelerated the growth of seed embryo.
Apart from complex interaction of the inherent genetic trait and the agro-climatic condition for production of seeds, variable food reserves in seeds of the genotypes also perhaps exerted significant variation on root and shoot growth of seedlings. Seed priming with KHPO probably resulted in increased food reserves which provided adequate supply of energy for various physiological, biochemical and enzymatic activities and resulted in ideal development of radicle and plumule lengths inside embryo. Presence of phosphorus in KHPO might also play important role in development of roots. Seed priming with PEG 6000 also recorded high root length and it was perhaps due to improved metabolic activities as well as radicle development inside seed. Seedling fresh and dry weights were the reflection of seedling growth (root and shoot). As emergence and initial seedling growth was totally dependent on food reserve of the seeds, variable food reserve possibly created variation in seedlings' fresh and dry weights of the genotypes. Seedlings' fresh and dry weights were improved under seed priming option probably due to greater accumulation of food reserve and thereby, developed high shoot and root growths (seedling growth) through translocation of dry matter to germinated seedlings. Presence of phosphorus in KHPO perhaps played important role in energy storage, energy transfer and oxidation reactions as phosphorus was the constituent of phospholipids, ADP and ATP. Thus, phosphorus utilized resources more efficiently in seeds and produced high seedlings' fresh weight. Further, seed priming with PEG 6000 also influenced seedling fresh and dry weights. Eesha et al. (2024) earlier reported positive effect of PEG 6000 on seedling fresh and dry weights of lentil. By establishing a low water-potential environment that permitted seeds to progressively absorb water, osmopriming with PEG-6000 greatly improved seed germination and seedling vigour. Without causing early radicle emergence, this treatment had started vital metabolic processes such DNA repair, enzyme activation, and reserve mobilization. Research on a variety of crops, including, pea, carrot, caraway, and lentil, showed that PEG-6000 treatments generated more homogeneous and robust seedlings with longer roots and shoots, as well as a higher germination percentage and a shorter mean germination time. Furthermore, PEG-primed seeds accumulated compatible solutes (proline, sugars) and activated antioxidant enzymes (APX, CAT, POD, SOD) under osmotic or salinity stress, which decreased lipid peroxidation and stabilized membranes, improving stress tolerance during early growth. The preconditioned seeds from PEG-6000 osmopriming resulted in robust seedlings and stronger, more synchronized germination, which were essential for improved crop performance and field emergence.
Vigour index was the product of germination and seedling length or dry weight. In the present study, genetic trait and agro-climate's influence on produced seeds significantly impacted on variability of seed germination and seedling length or dry weight, which in turn, imposed variation in seedling vigour index-I & II. Further, seed priming, specially, with KHPO improved germination, root and shoot lengths (seedling length) and dry weight and thereby, influenced vigour index-I &II. Specifically, it might repair the protein damage that occurred due to oxidative stress. Further, it probably maintained the normal function of metabolism pathways which, in turn, circulated energy required for biomass accumulation in essential structures and thus, produced vigorous seedling. Improvement of seed germination and seedling growth under chemical seed priming possibly due to improvement of ATPase activity as well as anti-oxidant activities, specially, peroxidase and their beneficial impacts on seed parts repair and embryo development reflected directly on seedling vigour index. Pandey et al. (2017) in cucumber and Dugesar et al. (2025) in black gram similarly reported improvement of seed germination and vigour index under seed priming with KHPO and PEG 6000, respectively.
Regarding oil content, rapeseed genotypes were better performer than mustard genotypes. In the present study, besides the unique genetic trait in each of the genotype, nutrient and water availability, prevailing weather (temperature, relative humidity, rainfall, sunshine hours etc.), drought or heat stress during seed development also played important role in variable oil content among the genotypes. Increase of oil content through seed priming with KHPO was observed due to the fact that phosphorus improved the oil content of seeds. Phosphorus was known to be the supplier of energy. In the present study, seed priming with KHPO probably provided adequate energy for biosynthesis of oil. Furthermore, Pusa Bold performed best in registering protein content in produced seeds. It might be due to its greater adaptation to the ago-climatic condition, proper vegetative growth, specially, roots for proper uptake and utilization of nitrogen from soil. Seed priming with KHPO recorded maximum total soluble protein content probably due to improved root system through which uptake and translocation of nutrients, specially, nitrogen from soil occurred to produced seeds. Accumulated nitrogen was the constituent of protein as observed in the present study. Besides KHPO, the positive impact of PEG 6000 on protein content of canola was earlier reported by Elahi et al. (2023).
Complex interaction of genetically unique genotypes with agro-climatic conditions created different impacts on seeds during development, specially, in their membrane integrity, moisture and lignin contents etc. which probably influenced the electrical conductivity of the seeds of genotypes differently. Higher electrical conductivity as observed in case of control indicated loss of membrane integrity which negatively impacted on seed quality. Reduction in electrical conductivity with seed priming, specially, using KHPO indicated that there might be restoration of seed membrane integrity, resulting in restriction of leaching of electrolytes. Further, low electrical conductivity under seed priming with PEG 6000 as observed in the present study was reported by Eesha et al. (2024) in lentil.
In the present study, rapeseed genotypes showed low α- amylase activity as compared to mustard genotypes probably due to the influence of rainfall, relative humidity and temperature on the genotypes during seed development and maturity. Since mustard genotypes were of longer duration than rapeseed genotypes, increasing atmospheric temperature and rainfall towards seed ripening and maturity stages perhaps created marked influence in elevating the α- amylase activity of the produced mustard seeds, specially, in Pusa Bold. Seed priming enhanced α- amylase activity of seeds over control perhaps due to its beneficial activity during seed hydration, starch hydrolysis and rapid conversion of starch in to reducing sugar. Further, there might be activation of α- amylase activity under seed priming and for such activity, energy was supplied at sufficient level from the food reserve which was high in produced seeds due to beneficial impact of KHPO or PEG 6000. α- amylase activity (48 h) was lower than α- amylase activity (24 h) which indicated depletion of food reserve due to energy generation for enzymatic activity.
In the present study, rapeseed genotypes exhibited lowest peroxidase activity than most of the mustard genotypes and it might be due to their short duration and less exposure to increasing temperature in spring or pre-summer times. Improved terminal heat or drought stress as observed in case of mustard genotypes probably exerted direct influence on higher peroxidase activity. Peroxidase activity was known to vary for drought or heat stress and acted as a defensive mechanism against oxidative damage. Higher activity of peroxidase indicated greater protection against stress.
Peroxidase activity increased under seed priming over control probably due to improvement of anti-oxidant properties as defense against oxidative stress. Adequate energy for such defense activity was supplied by food reserve of seeds which was accumulated due to beneficial impact of seed priming, specially, KHPO. Seed priming with PEG 6000, on the other hand, might improve cell membrane stability and thereby, checked lipid peroxidation, which was possibly accompanied by increased levels of various anti-oxidant enzymatic activity, specially, peroxidase. Increase of peroxidase activity (48 h) over that at 24 h under seed priming, thus, indicated seed quality maintenance. Valgimigli (2023) reported that a decrease in peroxidase activity was perhaps positively correlated with lipid peroxidation. PEG 6000 induced the early production of α-amylase by carefully controlling water uptake into seeds through the creation of a mild osmotic stress environment. These enzymes converted stored starch into soluble sugars, such as fructose and glucose, which drove increased energy production and metabolic activity, resulting in more rapid and consistent germination. PEG priming concurrently increased peroxidase both in terms of gene expression and enzyme activity. Peroxidase expression and activity significantly increased following PEG treatment, according to several investigations with Coronilla varia seeds. Reactive oxygen species were detoxified, lipid peroxidation was decreased, membranes were stabilized, and even lignin biosynthesis was supported by converting coumaryl alcohol to hydroxy phenyl lignin, which increased the vigour of seedlings.
The diversity among genotype × seed priming combinations based on important physiological, biochemical, and enzymatic seed quality parameters was depicted in hierarchical cluster analysis. The heat map showed patterns of similarity and divergence by visually grouping combinations into discrete clusters. With high values in germination percentage, seedling dry weight, vigour indices, and α-amylase and peroxidase activities, the combinations VT (Pusa Bold × KHPO and VT (Kranti × KH₂PO₄) notably established a separate cluster, indicating excellent seed quality and vigour. On the other hand, unprimed pairings with persistently low trait values, as VT (Anushka × Control) and VT (Sanchita × Control), showed poor seed performance without priming. A common physiological underpinning for early seedling vigour was suggested by the clustering of characteristics, which also showed strong interrelationships, particularly among root length, vigour index I, and germination percentage. Likewise, there was a tight clustering of α-amylase and peroxidase activities at 24 and 48 h, suggesting coordinated enzyme activation under priming conditions. There was little linkage between the features linked to vigour and characteristics like electrical conductivity and oil content, which seemed more independent. Considering all these, the research demonstrated the genotype-specific character of seed priming responses and validated the efficacy of KH₂PO₄ priming, especially in Pusa Bold. These results are useful for refining seed improvement procedures to increase oilseed yields in a range of environmental conditions.