Effect of biostimulants on vegetative growth parameters of broccoli (Brassica Oleracea L. Var Italica) under protected structure

Introduction

Broccoli (Brassica oleracea L. var. italica, 2n = 2x = 18) is an important cole vegetable belonging to the family Brassicaceae and is valued for its high nutritional and healthpromoting properties. It is a rich source of vitamins, minerals, dietary fibre and bioactive compounds, particularly glucosinolates, flavonoids and carotenoids (Talalay and Fahey, 2001; Martins et al., 2022) [12,18] . The increasing demand for nutritious and functional vegetables has enhanced the commercial importance of broccoli. Growth and productivity of broccoli are greatly influenced by environmental conditions, particularly temperature, light, humidity and soil moisture. Protected cultivation provides a favorable and relatively controlled environment for crop growth by regulating these factors and improving resourceuse efficiency (Singh and Sirohi, 2006) [16] . However, optimum vegetative growth under protected conditions also depends on proper nutrient management. Excessive dependence on synthetic fertilizers may contribute to increasing production costs, soil degradation and environmental pollution (Calvo et al., 2014) [3] . Therefore, sustainable nutrient-management approaches are required to maintain crop productivity and soil health. Biostimulants have emerged as promising inputs for sustainable crop production owing to their ability to stimulate plant growth, improve nutrient uptake and utilization, enhance physiological activity and increase tolerance to environmental stresses (Du Jardin, 2015; Rouphael and Colla, 2020) [6,14] . Their application can promote root development, chlorophyll antibacterial [17,18] activities. synthesis, photosynthetic activity and overall vegetative growth, thereby improving plant performance (Nardi et al., 2002) [13] . Among various biostimulants, seaweed extracts contain naturally occurring plant growth regulators, macro-and micronutrients that can promote plant growth and improve tolerance to environmental stresses (Durand et al., 2003; Sahoo, 2000; Arioli et al., 2015) [1,7,15] . Humic acid can improve nutrient availability, nutrient uptake and root development, thereby supporting vigorous plant growth (Susic, 2016) [17] . Similarly, Jeevamrit, a fermented organic preparation rich in beneficial microorganisms, may enhance microbial activity and nutrient mineralization, improving nutrient availability to plants (Devakumar et al., 2014) [5] . Novel Organic Liquid Nutrient (NOLN), prepared from banana pseudo stem sap and organic inputs, also has potential as an organic plant-growth-promoting input (Desai et al., 2016) [4] . Although biostimulants have shown beneficial effects in several horticultural crops, limited information is available on the combined use of humic acid, seaweed extract, Jeevamrit and NOLN for improving the vegetative growth of broccoli under protected conditions. Therefore, the present investigation was undertaken to evaluate the effect of different biostimulants on vegetative growth parameters of broccoli (Brassica oleracea L. var. italica) under protected structure and to identify suitable biostimulant treatments for vigorous crop growth.

Materials and Methods

Healthy and uniform seedlings of broccoli (Brassica oleracea L. var. italica) variety Saki were used for conducting the investigation. The experiment was carried out during the Rabi seasons of 2024-25 and 2025-26 at the Centre of Excellence on Protected Cultivation and Precision Farming (COE on PCPF), Indira Gandhi Krishi Vishwavidyalaya (IGKV), Raipur, Chhattisgarh. The experiment was laid out in Randomized Block Design (RBD) with 20 treatments and three replications. The experimental plot measured 4.6 m × 1.6 m, and the crop was planted at a spacing of 120 cm × 45 cm × 45 cm in zig-zag manner.

For preparation of humic acid solution, 5 mL of humic acid was diluted with water and the volume was made up to 1 L. The prepared solution was applied as foliar spray two times. The first spray was given at 25 days after transplanting (DAT) and the second spray at 45 DAT. The solution was uniformly sprayed over the foliage until the leaves were adequately wetted using a hand/knapsack sprayer.

For preparation of seaweed extract solution, 3 mL of seaweed extract was diluted with water and the volume was made up to 1 L. The prepared solution was applied as foliar spray two times. The first spray was given at 25 DAT and the second spray at 45 DAT. The solution was uniformly sprayed over the foliage using a hand/knapsack sprayer.

Jeevamrit was prepared and diluted to obtain a 3 per cent solution. It was applied through both foliar spray and soil drenching in the treatments where it was included. For foliar application, the prepared solution was uniformly sprayed over the foliage, whereas for soil application, the solution was applied as a drench near the root zone. The application was carried out according to the treatment schedule. Fresh Jeevamrit solution was prepared before each application.

Novel organic liquid nutrient (NOLN)

NOLN was applied at 1.5 and 2.0 per cent concentrations. For preparation of 1 L of solution, 15 mL and 20 mL of NOLN were diluted with water to obtain 1.5 and 2.0 per cent concentrations, respectively. The prepared solutions were applied as foliar sprays according to the treatment schedule. Fresh solutions were prepared immediately before spraying. The treatment combinations were as follows: T₁ -Control (water spray); T₂ -Humic acid @ 5 mL L⁻¹; T₃ -Seaweed extract @ 3 mL L⁻¹; T₄ -Jeevamrit @ 3%; T₅ -NOLN @ 1.5%; T₆ -NOLN @ 2.0%; T₇ -Humic acid @ 5 mL L⁻¹ + NOLN @ 1.5%; T₈ -Humic acid @ 5 mL L⁻¹ + NOLN @ 2.0%; T₉ -Seaweed extract @ 3 mL L⁻¹ + NOLN @ 1.5%; T₁₀ -Seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%; T₁₁ -Jeevamrit @ 3% + NOLN @ 1.5%; T₁₂ -Jeevamrit @ 3% + NOLN @ 2.0%; T₁₃ -Humic acid @ 5 mL L⁻¹ + seaweed extract @ 3 mL L⁻¹ + NOLN @ 1.5%; T₁₄ -Humic acid @ 5 mL L⁻¹ + seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%; T₁₅ -Jeevamrit @ 3% + seaweed extract @ 3 mL L⁻¹ + NOLN @ 1.5%; T₁₆ -Jeevamrit @ 3% + seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%; T₁₇ -Jeevamrit @ 3% + humic acid @ 5 mL L⁻¹ + NOLN @ 1.5%; T₁₈ -Jeevamrit @ 3% + humic acid @ 5 mL L⁻¹ + NOLN @ 2.0%; T₁₉ -Humic acid @ 5 mL L⁻¹ + seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 1.5%; and T₂₀ -Humic acid @ 5 mL L⁻¹ + seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 2.0%. The seedlings were transplanted at the 3-4 true-leaf stage on raised beds. Farmyard manure and recommended basal fertilizers were incorporated into the soil, while watersoluble fertilizers were subsequently applied through fertigation. The crop was maintained under uniform cultural practices throughout the experimental period. Irrigation was provided through drip irrigation, and necessary intercultural and plant protection measures were followed uniformly in all treatments. Five healthy plants were randomly selected and tagged from each experimental plot for recording observations on vegetative growth. The observations recorded included plant height at 15, 30 and 60 DAT and at harvest; number of leaves per plant at 15, 30 and 60 DAT and at harvest; stem diameter at 30 DAT and at harvest; leaf width at 30 and 60 DAT; leaf length at 30 and 60 DAT; leaf area at 30 and 60 DAT; plant spread in north-south direction at 30 and 60 DAT; and plant spread in east-west direction at 30 and 60 DAT. The recorded observations were subjected to statistical analysis to assess the effect of different biostimulant treatments on vegetative growth of broccoli under protected conditions.

Observations recorded on vegetative growth parameters

Five healthy and representative plants were randomly selected and tagged from each experimental plot for recording observations on vegetative growth. The following eight vegetative growth parameters were recorded at the specified stages of crop growth: 1. Plant height (cm): Plant height was recorded at 15, 30 and 60 days after transplanting (DAT) and at harvest. The height was measured from the base of the plant to the growing tip using a measuring scale and expressed in centimetres. 2. Number of leaves per plant: The total number of fully developed leaves on each tagged plant was counted at 15, 30 and 60 DAT and at harvest, and the mean was calculated and expressed as number of leaves per plant. 3. Stem diameter (cm): Stem diameter was measured at 30 DAT and at harvest using a digital/vernier caliper and expressed in centimetres. 4. Leaf width (cm): The maximum width of a fully expanded representative leaf was measured at 30 and 60 DAT using a measuring scale and expressed in centimetres.

The length of a fully expanded representative leaf was measured from the base of the leaf to the tip at 30 and 60 DAT and expressed in centimetres. 6. Leaf area (cm²): Leaf area was recorded at 30 and 60 DAT using the adopted leaf-area measurement method/instrument and expressed in square centimetres. 7. Plant spread (cm) -North-South (N-S): Plant spread in the north-south direction was measured at 30 and 60 DAT from the maximum horizontal spread of the plant and expressed in centimetres. 8. Plant spread (cm) -East-West (E-W): Plant spread in the east-west direction was measured at 30 and 60 DAT from the maximum horizontal spread of the plant and expressed in centimetres.

The data on plant height (cm) were recorded at 15, 30 and 60 days after transplanting (DAT) and at harvest and statistically analyzed. At 15 DAT, plant height differed non-significantly among the treatments during 2024-25, 2025-26 and in the pooled analysis. However, numerically higher plant height was recorded in T5 (NOLN @ 1.5%), with values of 8.48, 9.45 and 8.97 cm during 2024-25, 2025-26 and pooled analysis, respectively. The lowest plant height was recorded in T1 (Control), with corresponding values of 7.38, 7.56 and 7.47 cm, respectively. At 30 DAT, plant height also differed non-significantly among the treatments during 2024-25, 2025-26 and in the pooled analysis. However, numerically higher plant height was recorded in T19 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 1.5%), with values of 33.02, 40.83 and 36.92 cm during 2024-25, 2025-26 and pooled analysis, respectively. The lowest plant height was observed in T1 (Control), with corresponding values of 32.93, 34.42 and 33.67 cm, respectively.

In the pooled analysis at 60 DAT, the maximum plant height (67.64 cm) was recorded in T19, which was statistically at par with T20 (66.40 cm), T16 (64.28 cm), T18 (60.75 cm) and T14 (60.21 cm). The minimum pooled plant height (50.32 cm) was recorded in T1 (Control).

In the pooled analysis at Harvest, the maximum plant height (76.84 cm) was recorded in T19, which was statistically at par with T10 (75.62 cm). The minimum pooled plant height (58.25 cm) was recorded in T1 (Control). The non-significant differences observed at 15 and 30 DAT may be attributed to the relatively early stage of crop growth, when the effects of biostimulant application were not sufficiently pronounced. The significant differences observed at 60 DAT and harvest indicate that the beneficial effects of biostimulants became more evident with advancement of crop growth. The superior performance of T19, comprising humic acid, seaweed extract, Jeevamrit and NOLN, may be attributed to their complementary effects on nutrient availability, root activity, cell division and elongation, and overall plant growth. Humic substances are known to stimulate root development and improve nutrient uptake, thereby promoting vegetative growth. Similar positive effects of biostimulants on vegetative growth and plant height in broccoli have been reported by Baldaniya et al. (2023) [2] . and Ismael et al. (2024) [10] .

Number of leaves plant -1

The data on the number of leaves plant⁻¹ were recorded at 15, 30 and 60 days after transplanting (DAT) and at harvest and statistically analyzed. In the pooled analysis at 15 DAT, the differences among treatments were non-significant. The maximum number of leaves plant⁻¹ (5.60) was recorded in T17 (Jeevamrit @ 3% + Humic acid @ 5 mL L⁻¹ + NOLN @ 1.5%), followed by T19 (5.57), whereas the minimum (4.97) was recorded in T1 (Control). In the pooled analysis at 30 DAT, the differences among treatments remained nonsignificant. The maximum number of leaves plant⁻¹ (8.90) was recorded in T14 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%), followed by T17 (8.84) and T15 (8.81). The minimum (7.32) was recorded in T1 (Control). In the pooled analysis at 60 DAT, the differences among treatments were non-significant. The maximum number of leaves plant⁻¹ (18.23) was recorded in T8 (Humic acid @ 5 mL L⁻¹ + NOLN @ 2.0%) and T20 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 2.0%), followed by T10 (18.15). The minimum (16.43) was recorded in T12 (Jeevamrit @ 3% + NOLN @ 2.0%). In the pooled analysis at harvest, the differences among treatments remained nonsignificant. The maximum number of leaves plant⁻¹ (22.28) was recorded in T17, followed by T20 (22.00) and T8 (21.30). The minimum (17.30) was recorded in T1 (Control).Overall, the number of leaves plant⁻¹ showed nonsignificant differences among the treatments at all growth stages and in both individual years as well as pooled analysis. Similar observations regarding the influence of biostimulants on vegetative growth attributes in broccoli have been reported by Mahmood et al. (2023).

The Stem Diameter data was noted at growth stages i.e., 30 DAT and at Harvest and statistically evaluated. The pooled mean data at 30 DAT, showed non-significant difference for this trait. The data revealed that maximum stem diameter (5.60) was recorded under the treatment T20 (Humic Acid @ 5 ml/lit + Seaweed Extract @ 3 ml/lit + Jeevamrit @ 3 % + NOLN @ 2.0 %). Moreover, the treatment T1 (control) was recorded minimum stem diameter (0.97). On the basis of pooled mean, the stem diameter data differed nonsignificantly. The data showed that the treatment T19 (Humic Acid @ 5 ml/lit + Seaweed Extract @ 3 ml/lit + Jeevamrit @ 3 % + NOLN @ 1.5 %) was recorded for highest stem diameter (3.35). However, lowest stem diameter (2.98) was observed under the treatment T1 (control). The non-significant variation in stem diameter at 30 DAT across the treatments indicates that the applied biostimulants did not produce a sufficiently pronounced response in stem thickening during the early vegetative stage. This may be attributed to the variation in response according to the type and concentration of the biostimulant and the prevailing growing conditions. Similar findings were observed in broccoli, where lower concentrations of Isabion and seaweed extract did not significantly increase stem diameter, whereas higher concentrations produced greater stem diameter, indicating that stem thickening may depend on the concentration of the applied biostimulant (Iqbal et al., 2023) [9] . In pooled data, the maximum Leaf width (18.75 cm) was noted in T10 (Seaweed Extract @ 3 ml/lit + NOLN @ 2.0 %), which was statistically at par with T7 (18.60 cm), T8 (17.91 cm), T19 (17.83 cm), thereby indicating the consistent superiority of these treatments. The lowest pooled Leaf width (12.41 cm) was recorded in T1 (control). The comparatively greater leaf width under the superior treatments may be attributed to the combined influence of biostimulants on nutrient availability, nutrient uptake and physiological activity, which favoured better leaf expansion and vegetative development. Seaweed extracts contain bioactive compounds and nutrients that can support plant growth, while organic nutrient sources may further contribute to improved nutrient supply. The variation in the superior treatment between the two years may be associated with differences in environmental conditions and the interaction between the applied biostimulants and crop growth conditions. Similar enhancement of vegetative growth and leaf development through biostimulant application has been observed by Iqbal et al., 2023 [9] . in broccoli.

The Leaf length data was noted at different growth stages i.e., 30 and 60 DAT and statistically evaluated.

In pooled data, the maximum Leaf length (15.63 cm) was again noted in T14, which was statistically at par with T5 (15.23 cm), T15 (15.20 cm), T18 (14.91 cm), and T13 (14.53 cm), thereby indicating the consistent superiority of T14. The lowest pooled Leaf length (12.02 cm) was recorded in T1 (control).

In pooled data, the maximum Leaf length (26.75 cm) was noted in T20 (Humic Acid @ 5 ml/lit + Seaweed Extract @ 3 ml/lit + Jeevamrit @ 3% + NOLN @ 2.0%), which was statistically at par with T14 (26.66 cm), T19 (26.65 cm), T10 (25.96 cm), and T7 (25.84 cm), thereby indicating the consistent superiority of these treatments. The lowest pooled Leaf length (21.73 cm) was recorded in T1 (control).

The greater leaf length recorded under T14 during the first year and T20 during the second year may be attributed to the favorable influence of the combined biostimulants on nutrient availability, nutrient uptake and vegetative growth.

The superior performance of T20, comprising humic acid, seaweed extract, Jeevamrit and NOLN, may be associated with the complementary effects of these inputs in supporting plant nutrition and physiological activity, thereby favoring greater leaf expansion. Humic substances can influence nutrient uptake and plant growth, while seaweed-based biostimulants may support physiological processes associated with vegetative development. The variation in the best-performing treatment between the two years may be related to differences in environmental conditions and the interaction between the biostimulant treatments and crop growth. Similar enhancement of vegetative growth and leaf development through biostimulant application has been observed Iqbal et al., 2023 [9] . in broccoli. The enhanced leaf area under the superior treatments may be attributed to improved nutrient availability and uptake along with sustained physiological activity, which favored leaf expansion and development of a larger photosynthetic surface. The presence of bioactive compounds and nutrients in seaweed extracts may further contribute to improved vegetative growth and leaf development. Similar enhancement of leaf area and vegetative growth through biostimulant application has been observed in broccoli at harvest (Baldaniya et al., 2023) [2] .

The pooled mean data at 30 DAT, showed non-significant difference for this trait. The data revealed that maximum plant spread (42.14) was recorded under the treatment T13 (Humic Acid @ 5 ml/lit + Seaweed Extract @ 3 ml/lit + NOLN @ 1.5 %). Moreover, the treatment T1 (control) was recorded minimum plant spread (37.05 cm).

In pooled data, the maximum Plant Spread (71.49 cm) was again noted in T10, which was statistically at par with T17 (67.96 cm) and T20 (67.39 cm), thereby indicating the consistent superiority of these treatments. The lowest pooled Plant Spread (45.33 cm) was recorded in T1 (control).

The non-significant response at 30 DAT may be attributed to the comparatively limited influence of the applied biostimulants on lateral vegetative growth during the early stage of plant development. The greater plant spread under the superior treatments may be attributed to improved nutrient availability which promoted lateral growth and overall canopy development. Similar enhancement of vegetative growth through biostimulant application has been observed in broccoli, where seaweed extract and Jeevamrit treatments improved growth attributes (Baldaniya et al., 2023) [2] .

Conclusion

The present investigation demonstrated that the application of biostimulants, particularly their combined application, influenced several vegetative growth attributes of broccoli under protected conditions. Based on the pooled analysis of the two years, T19 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 1.5%) recorded the maximum plant height at 60 DAT (67.64 cm) and at harvest (76.84 cm), indicating its superior performance for plant growth. At 60 DAT, T19 was statistically at par with T20, T16, T18 and T14, whereas at harvest it was statistically at par with T10.

The differences in number of leaves per plant and stem diameter were non-significant at the respective stages of observation. Nevertheless, numerically, T17 (Jeevamrit @ 3% + Humic acid @ 5 mL L⁻¹ + NOLN @ 1.5%) recorded the maximum number of leaves at 15 DAT (5.60) and at harvest (22.28), while T20 recorded the maximum stem diameter at 30 DAT (1.07 cm) and T19 recorded the maximum stem diameter at harvest (3.35 cm). For leaf growth attributes, T15 (Jeevamrit @ 3% + Seaweed extract @ 3 mL L⁻¹ + NOLN @ 1.5%) recorded the maximum pooled leaf width (11.01 cm) and leaf area (129.28 cm²) at 30 DAT. At 60 DAT, T10 (Seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%) recorded the maximum leaf width (18.75 cm) and leaf area (365.55 cm²). The maximum leaf length at 30 DAT (15.63 cm) was recorded in T14 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + NOLN @ 2.0%), while at 60 DAT the maximum leaf length (26.75 cm) was recorded in T20 (Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 2.0%).

For plant canopy development, the maximum pooled plant spread in the N-S direction at 60 DAT (71.49 cm) was recorded in T10, which was statistically at par with T17 and T20. Similarly, the maximum pooled plant spread in the E-W direction at 60 DAT (74.56 cm) was recorded in T6 (NOLN @ 2.0%), which was statistically at par with T7, T8 and T17. At 30 DAT, plant spread in both N-S and E-W directions remained non-significant. Overall, the findings indicated that combined application of biostimulants was more effective in promoting vegetative growth and canopy development of broccoli under protected conditions than individual applications. Among the treatments, combinations involving seaweed extract, humic acid, Jeevamrit and NOLN performed consistently well across different growth parameters. Therefore, T19, comprising Humic acid @ 5 mL L⁻¹ + Seaweed extract @ 3 mL L⁻¹ + Jeevamrit @ 3% + NOLN @ 1.5%, emerged as the most promising treatment for overall plant growth, particularly with respect to plant height, while T10, T15, T14, T20 and T6 were superior for specific leaf and canopy attributes.

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