Screening of natural biostimulants and optimization of seed priming concentrations for enhancing germination, seedling vigour and reserve mobilization in mungbean (Vigna radiata L. Wilczek)

Introduction

Mungbean (Vigna radiata L. Wilczek) is one of the most important pulse crops cultivated in tropical and subtropical regions because of its high protein content, short duration and ability to improve soil fertility through biological nitrogen fixation. It serves as an inexpensive source of dietary protein, vitamins and minerals and plays a vital role in sustainable cropping systems. However, poor seedling establishment caused by slow or uneven germination frequently limits productivity, particularly under adverse environmental conditions such as high temperature, moisture deficit and soil salinity. Improving early seedling vigour therefore remains a major objective in mungbean production. Seed priming is a simple and effective pre-sowing treatment in which seeds are partially hydrated to initiate metabolic activities associated with germination without allowing radicle emergence. Following drying, primed seeds exhibit faster and more synchronized germination, improved enzyme activity, greater stress tolerance and enhanced seedling establishment. Seed priming has become an important agronomic technique because it is inexpensive, environmentally safe and easily adopted by farmers. Recently, natural biostimulants have attracted considerable attention as alternatives to synthetic growth regulators due to increasing concerns regarding environmental sustainability and chemical inputs. Plant-derived extracts, seaweed products and animal-derived organic formulations contain diverse bioactive compounds, including phytohormones, amino acids, vitamins, phenolics, osmoprotectants and essential mineral nutrients, which stimulate physiological and biochemical processes in plants. These compounds improve nutrient uptake, activate antioxidant defence systems, enhance enzyme activities and promote plant growth under both normal and stressful conditions. Consequently, natural biostimulants are increasingly being incorporated into sustainable crop production systems. Seaweed extracts are among the most widely studied biostimulants because they contain naturally occurring auxins, cytokinins, gibberellins, betaines and polysaccharides that stimulate germination and seedling growth. Similarly, cow urine has long been recognized in traditional agriculture as a source of macro-and micronutrients, growth-promoting substances and beneficial microorganisms. Several medicinal plants such as Leucas aspera, Ocimum tenuiflorum, Acalypha indica, Euphorbia hirta and Heliotropium indicum possess diverse secondary metabolites with antioxidant and antimicrobial properties that may influence seed metabolism during germination. Nevertheless, comparative information regarding their suitability as seed priming agents and the optimization of their application concentrations remains limited. The beneficial effects of seed priming are largely mediated through enhanced reserve mobilization during germination. Activation of hydrolytic enzymes, particularly α-amylase, facilitates the degradation of starch into soluble sugars that supply energy for embryo growth and seedling development. Enhanced reserve mobilization is therefore closely associated with improved germination, seedling vigour and biomass accumulation. Measuring α-amylase activity together with mobilization efficiency provides valuable insights into the physiological mechanisms underlying the action of different biostimulants. Although numerous studies have demonstrated the beneficial effects of individual biostimulants, systematic screening of a broad range of natural biostimulants under identical experimental conditions is scarce. Furthermore, information on the optimum concentrations required to maximize seedling performance while avoiding inhibitory effects is limited. Determining the most effective priming concentration is essential because excessive concentrations may induce osmotic stress or phytotoxicity, reducing germination and seedling growth. Therefore, the present investigation was undertaken to (i) compare the efficacy of different natural biostimulants as seed priming agents for mungbean, (ii) determine the optimum concentration for enhancing germination and seedling vigour, and (iii) investigate their influence on αamylase activity and reserve mobilization during early seedling development. The findings are expected to identify promising eco-friendly seed priming agents that can improve crop establishment and provide a scientific basis for their subsequent evaluation under pot and field conditions.

Materials and Methods

The present investigation was conducted during 2024 at Dr. Rajendra Prasad Central Agricultural University, Pusa, Bihar, India, to evaluate the effect of ethnobotanical plant extracts, seaweed extract and cow urine on Mungbean seedling traits. The study comprised an in vitro screening and dose optimization experiment followed by a pot experiment under controlled conditions. Healthy seeds of mungbean local variety 'HUM-16' sourced from TCA Dholi, RPCAU were surface sterilized with 0.1% HgCl₂ solution for 2-3 min followed by washing with distilled water. Seeds were primed with 16 different treatments at four concentrations viz., 2%, 3%, 4% and 5%. The treatments were extracts made from the ethnobotanical plants i.e., Lantana Camara, Moringa, Catnip, Leucas aspera, E. hirta, Achyranthes, Calotropis, Oxalis, Datura, T. procumbens, A. indica, Ocimum and I. heliotrope. The ethnobotanicals materials for extract preparation are easily available locally. Fresh, fully expanded young and tender leaves of ethnobotanical plants were collected, washed and air-dried briefly to remove surface moisture. For the stock extract, 250 g of leaves were blended with 1000 mL Cow Urine (1:4 w/v), and the slurry was left for 12 hrs and filtered first through muslin cloth and then through Whatman No. 1 paper to produce a clear solution. Working solutions of 2%, 3%, 4% and 5% were prepared by diluting the 2 mL, 3 mL 4 mL, 5 mL stock solution with 100 mL of distilled water respectively. These mixtures were prepared fresh on the day of priming. Seeds were primed with each priming agent at respective concentrations for 12 hours at room temperature. The experiment was set up in petri plates with each replication having 20 seeds of Mungbean followed by incubation. Control seeds were soaked only in distilled water, and all other aspects of their handling were the same as for treated seeds. Data were collected from 7 days old seedling. The parameters recorded from the experiment along with their formulae are as follows: Germination percentage (G%) = (Bernfeld, 1955) [15] . Length was measured with the aid of scale ruler, while dry weight was measured on electronic weighing balance (with precision of 0.1 mg) after drying the seedlings in hot air oven at 65±2 °C for one day.

Statistical analysis

The experiment was conducted in a Completely Randomized Design (CRD) with three replications to evaluate the effects of different seed priming treatments. All statistical analyses were conducted in R software using the GRAPESAgri1 package developed for agricultural data analysis.

Results and Discussion

Effect of biostimulants and priming concentration on germination percentage

Seed priming with different natural biostimulants significantly (p ≤ 0.05) influenced germination percentage of mungbean (Table 1). Significant effects were observed for treatment (T), concentration (C) and their interaction (T × C), indicating that the response of mungbean seeds depended on both the type of biostimulant and the concentration applied.

Among the tested biostimulants, Indian heliotrope recorded the highest mean germination (89.25%), followed by cow urine (90.50%), Leucas aspera (86.67%), catnip (85.42%) and seaweed extract (84.17%). Hydroprimed control maintained a consistently high germination (91.67%) across all concentrations. The lowest mean germination was observed with Aloe (67.50%), followed by Achyranthes (71.67%) and Calotropis (72.92%). Considering concentration means, 3% priming produced the highest overall germination (85.25%), which was statistically comparable to 2% (84.76%) but significantly higher than 4% (80.61%) and 5% (73.51%). Germination progressively declined with increasing concentration beyond 3%, indicating that excessive concentrations inhibited seed germination. Among individual treatment × concentration combinations, the maximum germination (95.33%) was recorded with Indian heliotrope (3%), closely followed by seaweed extract (2%) and Leucas aspera (2%) (95.00%). Conversely, the lowest germination (50.00%) was observed with Aloe (5%), demonstrating a pronounced inhibitory effect of higher extract concentration. [6,2,5] .

The decline in germination at 5% concentration suggests that higher concentrations may create osmotic stress or introduce phytotoxic levels of secondary metabolites, thereby restricting water uptake and delaying metabolic activation. Similar concentration-dependent inhibitory responses have been documented for several botanical extracts, where excessive phenolics, alkaloids and other allelochemicals interfered with germination and early seedling growth (du Jardin, 2015; Rouphael and Colla, 2020) [1,2] . The superior performance of Indian heliotrope, cow urine and Leucas aspera indicates that these biostimulants possess favorable bioactive compounds capable of stimulating germination without causing toxicity at optimum concentrations. The overall findings demonstrate that 3% represents the optimum concentration for enhancing germination in mungbean under laboratory conditions, whereas concentrations above this threshold become progressively inhibitory.

Effect of biostimulants on Vigour Index I

Vigour Index I differed significantly among treatments, concentrations and their interaction (Table 2), indicating that seedling vigour was markedly influenced by both the nature of the biostimulant and priming concentration. Among the treatments, Leucas aspera recorded the highest mean Vigour Index I (2017.91), followed closely by Indian heliotrope (1999. Vigour Index I integrates germination percentage with seedling length and therefore reflects the combined effect of rapid germination and subsequent seedling growth. The enhanced vigour observed in Indian heliotrope, Leucas aspera and seaweed extract treatments indicates improved mobilization of seed reserves and more efficient utilization of stored carbohydrates for seedling elongation. Previous studies have shown that natural biostimulants enhance cell division, cell elongation and nutrient mobilization through increased synthesis of endogenous phytohormones and activation of metabolic enzymes (Calvo et al., 2014; Rouphael and Colla, 2020; Bulgari et al., 2019) [3,2,4] . The marked decline in Vigour Index I at 5% concentration suggests that excessive extract concentrations negatively affected seedling growth, possibly because of osmotic imbalance or inhibitory secondary metabolites that restricted root and shoot elongation.

Effect on Vigour Index II Vigour Index II also exhibited significant variation among treatments and concentrations (Table 3). Mean values ranged from 1.40 in Achyranthes to 2.44 in Indian heliotrope. Among all treatments, Indian heliotrope recorded the highest mean Vigour Index II (2.44), followed by Leucas aspera (2.21), seaweed extract (2.12), cow urine (2.09) and the control (2.08). The maximum individual value (3.02) was obtained with Indian heliotrope at 3% concentration.

The concentration effect followed a trend similar to that observed for germination and Vigour Index I. Mean Vigour Index II was highest at 3% (2.00), followed by 2% (1.97), while 4% (1.78) and 5% (1.57) showed a gradual decline. Figure 3. [6,2] .

Effect on α-amylase activity α-Amylase activity was significantly affected by treatment, concentration and their interaction (Table 4). The enzyme activity ranged from 0.18 to 0.81 mg maltose g⁻¹ FW min⁻¹, demonstrating considerable variation among the evaluated biostimulants. Among treatments, Indian heliotrope and cow urine recorded the highest mean α-amylase activity (0.72), followed closely by Leucas aspera (0.69) and seaweed extract (0.64). The lowest activity was observed in catnip (0.18). Across concentrations, the highest mean enzyme activity was observed at 3% (0.44), followed by 2% (0.42), whereas activity declined to 0.40 and 0.35 at 4% and 5%, respectively. The maximum α-amylase activity (0.81 mg maltose g⁻¹ FW min⁻¹) was recorded in Indian heliotrope at 3% concentration. [6,7,2] . The decline in enzyme activity at higher concentrations may be attributed to inhibitory effects of concentrated phytochemicals on enzyme synthesis or activity, resulting in reduced reserve mobilization and consequently poorer seedling performance.

Conclusion

Across all four parameters evaluated, 3% concentration consistently produced the best overall response, indicating that this concentration provided an optimal balance between stimulation of metabolic activity and avoidance of phytotoxic effects. Among the biostimulants, Indian heliotrope, Leucas aspera, seaweed extract and cow urine were consistently superior, exhibiting higher germination, greater seedling vigour and enhanced α-amylase activity. These findings identify these treatments as promising candidates for further evaluation under pot and field conditions.

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