Efficacy of seed priming with Gibberellic Acid (GA₃), Plant Growth Promoting Rhizobacteria (PGPR), and botanical extracts on germination and enzyme activity of sorghum (Sorghum bicolor L. Moench)

Introduction

Sorghum (Sorghum bicolor L. Moench) is one of the major cereal crops cultivated worldwide for food, fodder and industrial purposes. Due to its remarkable adaptability to drought, high temperature and poor soil conditions, it plays an important role in sustainable agriculture, particularly in semi-arid and marginal production environments. Sorghum grains are nutritionally important because they contain carbohydrates, proteins, dietary fibre, minerals and bioactive compounds beneficial for human and animal nutrition. However, productivity of sorghum is often limited by poor seed germination, weak crop establishment and environmental stresses during different growth stages (Singh et al., 2022; Kumar and Verma, 2023) [22,29] . Seed quality is a key determinant of crop performance as it influences germination, seedling vigour and subsequent plant growth. Poor seed vigour results in delayed emergence, uneven plant stand and reduced biomass accumulation, ultimately affecting yield potential. Seed priming has emerged as an effective seed enhancement technique that improves physiological readiness of seeds by controlled hydration before sowing. This process activates metabolic activities, enhances enzymatic reactions, improves membrane stability and promotes rapid and uniform germination under field conditions (Rao et al., 2021) [28] . Different approaches of seed priming, including hormonal priming, biopriming and botanical priming, have gained importance due to their ability to enhance crop performance in an ecofriendly manner.

Priming improves germination through activation of hydrolytic enzymes, mobilization of stored reserves, repair of cellular damage and regulation of endogenous hormones (Patel and Singh, 2023) [20] . Among plant growth regulators, gibberellic acid (GA₃) is widely recognized for its role in breaking seed dormancy, stimulating enzyme activity, promoting cell elongation and enhancing vegetative growth. GA₃ priming improves α-amylase activity, carbohydrate mobilization and early seedling establishment, resulting in improved growth and yield attributes (Mehta et al., 2024) [12] . Previous studies have reported that GA₃ application enhances germination percentage, seedling vigour, shoot and root growth, chlorophyll content and biomass production in different crops. In sorghum, GA₃ priming has been associated with improved metabolic activation, enhanced antioxidant capacity and better crop establishment under stress conditions (Rao et al., 2022) [25] . However, limited information is available regarding the influence of GA₃ priming on combined physiological, yield and nutritional quality traits of sorghum grains. Plant growth-promoting rhizobacteria (PGPR) have gained considerable attention as biological seed enhancement agents due to their beneficial effects on plant growth and stress tolerance. PGPR promotes plant development through nitrogen fixation, phosphorus solubilization, phytohormone production, siderophore synthesis and improved nutrient acquisition. These microorganisms also enhance root development, chlorophyll content and antioxidant defence mechanisms, thereby improving plant growth and productivity (Rao and Singh, 2023; Patel et al., 2024) [27,21] . In cereals, PGPR inoculation has been reported to improve germination, biomass accumulation and yield by enhancing physiological efficiency and stress adaptation. Botanical seed priming is another sustainable approach that utilizes plant extracts rich in natural growth-promoting substances, antioxidants, vitamins, minerals and bioactive compounds. Plant extracts such as moringa leaf extract contain naturally occurring cytokinins, amino acids and nutrients that stimulate enzymatic activity, improve seedling vigour and enhance plant growth (Mehta et al., 2023) [13] . Botanical priming has also been associated with improved antioxidant defence, delayed senescence and enhanced tolerance against environmental stresses. Although several studies have demonstrated the individual benefits of GA₃, PGPR and botanical priming on seed germination and crop growth, comparative evaluation of these priming agents under uniform field conditions is still limited. Moreover, their influence on physiological traits, yield components and nutritional quality parameters of sorghum grains requires further investigation. Therefore, the present study was undertaken to evaluate the effect of GA₃, PGPR and botanical seed priming treatments on germination behaviour, biochemical responses, growth, yield performance and grain quality of sorghum.

Materials and Methods

The present investigation entitled "Efficacy of Seed Priming with Gibberellic Acid (GA₃), Plant Growth-Promoting Rhizobacteria (PGPR), and Botanical Extracts on Germination and Enzyme Activity of Sorghum (Sorghum bicolor L. Moench)" was conducted during two consecutive Kharif cropping seasons, 2021-22 and 2022-23. at Central crop research field and Laboratory investigations were carried out at Jacob Institute of Biotechnology & Bioengineering (JIBB), Sam Higginbottom University of Agriculture, Technology and Sciences (SHUATS), Prayagraj, Uttar Pradesh, India. The experiment was designed to evaluate the influence of different seed priming treatments on germination behaviour, biochemical responses, growth attributes, yield performance and nutritional quality parameters of sorghum. Healthy and uniform seeds of sorghum were subjected to different priming treatments including gibberellic acid (GA₃), plant growth-promoting rhizobacteria (PGPR), and botanical extracts. The treated seeds were evaluated under experimental conditions for various physio-chemical, biochemical and quality parameters. The experiment consisted of nine treatments including untreated control (T₀), GA₃ @ 100 µM (T₁), GA₃ @ 200 µM (T₂), PGPR @ 250 ppm (T₃), PGPR @ 500 ppm (T₄), moringa leaf extract @ 2% (T₅), moringa leaf extract @ 5% (T₆), custard apple leaf extract @ 2% (T₇), and custard apple leaf extract @ 5% (T₈). The experiment was conducted in a randomized block design (RBD) with appropriate replications for field related parameters while Complete Randomized Design was used for laboratory trails.

Preparation and Application of Seed Priming Treatments

Seed priming treatments were prepared using hormonal, biological and botanical agents under laboratory conditions to ensure uniformity and reproducibility. Gibberellic acid (GA₃) solutions were prepared at 100 and 200 µM concentrations using freshly prepared solutions for hormonal priming. Biological priming was performed using PGPR cultures containing Bacillus spp. and Pseudomonas spp., multiplied in LB medium and applied at 250 and 500 ppm concentrations. For botanical priming, aqueous leaf extracts of moringa (Moringa oleifera) and custard apple (Annona squamosa) were prepared at 2% and 5% concentrations from shade-dried leaf powder. Healthy sorghum seeds were obtained and surface sterilized before treatment application. The seeds were separately soaked in respective priming solutions for 12 hours at 25 °C, whereas distilled water-soaked seeds were maintained as control. After completion of priming, excess solution was removed, and seeds were air-dried under room temperature until the original moisture level was restored. The treated seeds were subsequently used for germination studies, biochemical analysis, growth evaluation and yield assessment under experimental conditions.

Observation Recorded

Germination Percentage: Germination percentage was calculated by counting the number of normal seedlings at the final count and expressing it as a percentage of the total number of seeds sown. This parameter reflects the overall viability and effectiveness of seed priming treatments. Speed of germination was determined using daily germination counts to evaluate the rate at which seeds germinated. It indicates the rapidity and uniformity of seed emergence under different priming treatments.

Germination Index: Germination index was calculated by assigning greater weight to seeds that germinated earlier, thereby integrating both germination speed and total germination. Higher GI values indicate faster and more synchronized germination.

Vigour Index I: Vigour index I was computed by multiplying germination percentage with mean seedling length (shoot + root). This index provides a combined measure of germination performance and early seedling growth.

Observations related to Enzymes activity during germination α-Amylase activity in primed sorghum seeds was estimated following the method described by AOAC (2002), which measures the hydrolysis of starch into reducing sugars during germination. Crude enzyme extract obtained from germinating seeds was incubated with starch substrate under controlled conditions, and the released sugars were quantified spectrophotometrically. Increased α-amylase activity reflects enhanced starch degradation and improved reserve mobilization due to seed priming. β-Amylase activity was determined according to the procedure outlined by Nandi et al., (1994) by estimating the amount of maltose released from starch in germinating sorghum seeds. Enzyme extract was incubated with substrate and maltose content was measured colorimetrically. Higher β-amylase activity indicates efficient carbohydrate metabolism and greater energy availability during early seedling growth. Protease activity in primed sorghum seeds was analyzed using the method described by Dahot (1992), which quantifies amino acids released from storage proteins during germination. The enzyme extract was incubated with protein substrate and absorbance was recorded at a specific wavelength. Enhanced protease activity signifies improved protein mobilization and nitrogen availability as a result of seed priming treatments. Lipase activity was assessed following the protocol of Gadge et al., (2011) by estimating the free fatty acids liberated from lipid substrates in germinating sorghum seeds. The enzyme extract was incubated with lipid substrate and the activity was measured spectrophotometrically. Increased lipase activity indicates effective lipid breakdown, contributing to improved seedling establishment after priming. Phosphatase activity was estimated using the method suggested by Prazers et al., (2004), which measures the release of inorganic phosphate from organic phosphorus compounds in germinating seeds. Crude enzyme extract was incubated with suitable substrate and phosphate content was quantified. Higher phosphatase activity reflects enhanced phosphorus mobilization and metabolic efficiency in primed seeds. Dehydrogenase activity was determined according to Mrid et al., (2018) using a tetrazolium reduction assay to assess respiratory activity in germinating sorghum seeds. The intensity of color development indicated the level of enzymatic reduction and metabolic activity. Increased dehydrogenase activity denotes improved seed viability and vigor as influenced by seed priming treatments.

The data presented in the table 1, showed that the germination percentage was significantly influenced by different seed priming treatments during both years of experimentation. During the first year, germination ranged from 80.33% (T₀-Control) to 95.86% (T₃-PGPR 250 ppm), whereas in the second year it varied from 81.55% to 97.27%, respectively. The pooled analysis revealed maximum germination under T₃ (PGPR 250 ppm) (96.57%), followed by T₂ (GA₃ 200 µM) (92.33%) and T₇ (Custard apple leaf extract 2%) (91.34%), while the minimum was recorded in control (80.94%). The improvement in germination under PGPR and GA₃ priming may be due to enhanced metabolic activation, increased enzyme synthesis and improved mobilization of stored food reserves. Similar improvement in germination due to seed priming has been reported by Jisha et al., (2013) [10] , who observed enhanced germination performance in primed cereal seeds. The present findings suggest that priming treatments improved physiological readiness of seeds, resulting in faster and more uniform crop establishment.

Speed of Germination

Seed priming significantly improved the speed of germination during both years. The first-year values ranged from 10.04 (T₀) to 11.98 (T₃), while second-year values ranged from 11.04 to 13.58. The pooled analysis recorded maximum speed of germination under T₃ (PGPR 250 ppm) (12.78) followed by T₂ (GA₃ 200 µM) (12.21), whereas the lowest value was observed in control (10.54). Increased germination speed under priming treatments may be attributed to hydration-induced activation of enzymes and repair mechanisms within the seed. Similar observations were reported by Ashraf and Foolad (2005), who stated that priming enhances early metabolic activity and reduces germination time. The present results indicate that PGPR and GA₃ treatments accelerated the germination process by improving seed physiological efficiency.

Germination Index (GI)

The germination index showed significant variation among treatments. During the first year, GI ranged from 21.59 (control) to 34.35 (T₃), while during the second year it varied from 25.89 to 41.23. The pooled analysis recorded the highest GI under T₃-PGPR 250 ppm (37.79) followed by T₂-GA₃ 200 µM (36.09) and T₆-Moringa leaf extract 5% (35.22). Higher GI reflects rapid and uniform seedling emergence due to improved metabolic activity and faster reserve mobilization. Similar findings were reported by Kumar et al., (2021), who observed increased germination indices under microbial and hormonal priming treatments. The enhanced GI in treated seeds indicates improved seed vigour and better establishment potential under field conditions.

Vigour Index-I Vigour index-I differed significantly among treatments during both years. The values ranged from 540.87-999.40 during the first year and 655.67-1216.73 during the second year. The pooled data showed maximum vigour index under T₃ (PGPR 250 ppm) (1108.06) followed by T₂ (GA₃ 200 µM) (1073.98), while the lowest was recorded in control (598.27). The increased vigour index was mainly due to improvement in both germination percentage and seedling growth parameters. According to Moeinzadeh et al., (2010), seed priming improves seedling vigour by enhancing enzymatic activity and physiological processes during early growth. The present findings confirm that priming treatments improved seedling establishment and early growth performance in sorghum.

Enzyme Activities

The data shown in table 2, states that the activities of hydrolytic and metabolic enzymes were significantly influenced by different seed priming treatments, indicating enhanced biochemical activation during early seed germination. The pooled data revealed that the maximum enzyme activities were recorded under T₂ (GA₃ 200 µM), which showed α-amylase activity of 13.80 U mg⁻¹, βamylase activity of 8.70 U mg⁻¹, protease activity of 40.03 U/mL, lipase activity of 6.70 U/mL, phosphatase activity of 1.29 µmol pNP min⁻¹ g⁻¹ FW and dehydrogenase activity of 0.63 OD value. In comparison, the untreated control (T₀) recorded the lowest activities with α-amylase (8.40 U mg⁻¹), β-amylase (2.60 U mg⁻¹), protease (33.93 U/mL), lipase (3.86 U/mL), phosphatase (0.92 µmol pNP min⁻¹ g⁻¹ FW) and dehydrogenase (0.48 OD value). The enhancement of α-amylase and β-amylase activity under GA₃ priming may be attributed to the stimulatory effect of gibberellic acid on the synthesis and activation of hydrolytic enzymes during seed germination. Increased amylase activity facilitates rapid degradation of stored starch into soluble sugars, providing energy and carbon skeletons required for embryo growth and seedling establishment. Similar findings were reported by Bewley et al., (2013) [5] , who stated that hormonal priming accelerates reserve mobilization by activating hydrolytic enzymes during early germination stages. Table 2, revealed that the activity of protease was also increased significantly under priming treatments, with the highest value recorded in T₂ (40.03 U/mL) followed by T₅ (Moringa leaf extract 2%) (37.03 U/mL) and T₁ (GA₃ 100 µM) (36.74 U/mL). Enhanced protease activity indicates improved degradation of storage proteins into amino acids, which supports rapid cell division and seedling growth. The increased protein mobilization under primed seeds may have contributed to higher germination percentage and vigour index observed in the present study. Similar enhancement of proteolytic activity due to seed priming has been reported by Farooq et al., (2008) [6] .

Conclusion

The findings of the present study demonstrated that seed priming with gibberellic acid (GA₃), plant growthpromoting rhizobacteria (PGPR), and botanical extracts significantly enhanced germination and enzyme activityrelated traits of sorghum compared with the untreated control. Among the different treatments, PGPR @ 250 ppm proved most effective in improving germination, germination speed, germination index, and seedling vigour, while GA₃ @ 200 µM was superior in promoting hypocotyl and radicle growth as well as enhancing the activities of hydrolytic and metabolic enzymes. The improved enzymatic activities facilitated efficient mobilization of stored food reserves and accelerated early seedling development.

Overall, the study suggests that seed priming, particularly with PGPR @ 250 ppm and GA₃ @ 200 µM, is a promising and sustainable strategy for improving seed performance and ensuring vigorous crop establishment in sorghum.

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