Plant BIO stimulant formulation for enhancing plant growth and managing abiotic stress
Patent Information
- Application Number
- AU2026204218
- Authority / Receiving Office
- AU · AU
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-13
- Filing Date
- 2026-01-16
- Publication Date
- 2026-08-27
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Abstract
Description
TITLE: PLANT BIO STIMULANT FORMULATION FOR ENHANCING PLANT GROWTH AND MANAGING ABIOTIC STRESS TECHNICAL FIELD OF INVENTION: The invention relates to the field of agriculture, plant bio stimulants, and crop stress management, thus overall plant health and sustainable farming. The invention in particular relates to plant bio stimulant formulation of combination of seaweeds, botanicals for enhancing plant growth, improving abiotic stress tolerance, and promoting crop yield using water-ethanol percolation process. BACKGROUND: Considering the significant economic, social, and environmental challenges associated with modern agriculture, including declining soil fertility, increasing abiotic stress, and excessive reliance on chemical fertilizers, there is an urgent need for sustainable and effective bio stimulant solutions. Abiotic stress factors such as drought, salinity, and temperature fluctuations can severely impact crop yield and quality, making it essential to develop formulations that enhance plant resilience and crop performance. Seaweed-based agricultural inputs have been widely studied for their benefits in growth enhancement, stress mitigation, and soil health improvement. However, most commercial formulations predominantly utilize brown seaweed like Ascophyllum nodosum or red algae Kappahycus alverzi and employ fermentation or hydrolysis-based extraction methods. The global demand for biostimulants is projected to grow exponentially, placing significant strain on the limited natural resources of key seaweeds like brown seaweed (Ascophyllum nodosum) and red seaweed (Kappaphycus alvarezii). Current estimates suggest that the biostimulant industry's seaweed requirement is around 10-12 million metric tons annually, expected to increase by 30-40% over the next 5 years, driven by rising adoption in sustainable agriculture. This surge in demand will likely result in raw material shortages, significantly increasing sourcing costs by 20-25% annually, further pressuring supply chains. Ensuring sustainability and exploring alternative species or synthetic solutions will be crucial for mitigating this impact Fermentation- and hydrolysis-based extraction methods are the most prevalent in the biostimulant industry. These extraction methods inherently produce variable compositions because the underlying processes are non-targeted and degradative. In fermentation, the microbial enzymes break down the raw material in an uncontrolled, pathway-dependent manner, generating unpredictable mixtures of metabolites that vary with microorganism type, inoculum load, temperature, pH, and time. Similarly, acid- or alkali-based hydrolysis causes indiscriminate cleavage of biomolecules—polysaccharides, proteins, lipids, phenolics— leading to over-degradation, loss of functional actives, and batch-to-batch inconsistency. In contrast, solvent-based cold percolation is a non-destructive extraction approach that preserves native biochemical structures. Because no enzymatic or chemical breakdown occurs, the extraction selectively pulls out the desired class of metabolites while maintaining their original configuration and ratios. This results in far more standardized, reproducible compositions with improved retention of bioactive secondary metabolites—critical for consistent product performance. This approach ensures enhanced efficacy in stress tolerance and plant growth promotion while reducing thermal and enzymatic degradation of critical metabolites. Global agricultural practices are increasingly shifting towards sustainable and environmentally friendly alternatives to chemical fertilizers. Excessive fertilizer use leads to soil degradation, nitrogen leaching, and greenhouse gas emissions, necessitating solutions that enhance nutrient use efficiency (NUE) and reduce dependency on synthetic inputs. A review of existing patents and literature reveals several bio stimulant formulations utilizing seaweed extracts, each with unique compositions and extraction methods. While these formulations contribute to plant growth and stress management, they primarily rely on specific seaweed species, fermentation-based extraction methods, or limited bioactive compound retention, creating gaps that necessitate the development of more sustainable, efficient, and broad-spectrum bio stimulants. US20150351408A1 describes a bio stimulant formulation derived from acid hydrolysis of seaweeds and combining with humic / fulvic acids, focusing on delivering amino acids to reduce plant stress and enhance nutrient absorption. However, it does not explore a multi-species seaweed combination or the role of botanicals like wheatgrass extract in improving antioxidant activity and nutrient uptake. EP3294066A4 discloses a formulation combining seaweed juice and hydrolysate obtained from seaweed pulp post-juice extraction, aiming to promote plant growth. This method, however, relies on hydrolysis-based extraction and especially focusing on red seaweed Kappaphycus, which may degrade certain bioactive compounds, unlike the cold percolation water-ethanol extraction process proposed in the present invention. US20210323885A1 discusses fertilizers coated with seaweed extracts like Kappaphycus alvarezii, Ascophyllum nodosum, Ecklonia maxima, Durvillea potatorum, Macrocystis pyrifera, Sargassum spp., and Laminaria digitata or any combination thereof to improve crop yield and soil health but does not focus on direct bio stimulant effects, nor does it optimize the combination of seaweed and botanical extracts for stress tolerance. US10954170B2 outlines a method for producing aqueous seaweed extract concentrates, particularly from Sargassum species using fermentation, for use as bio stimulants. While effective, it does not incorporate the non-fermentative solvent-based extraction method, which ensures better bioactive compound preservation and product consistency. Taking into consideration all the above factors, it is implied that there is a need to develop a sustainable, commercially viable, and effective alternative that enhances plant growth, improves abiotic stress tolerance, and reduces fertilizer dependency while ensuring environmental safety and agricultural sustainability. OBJECT OF THE INVENTION: The primary object of the present invention is to provide a plant bio stimulant formulation to enhance plant growth, improve abiotic stress tolerance, and promote crop yield. Another object of the present invention is to provide a plant bio stimulant formulation that comprises of a combination of brown seaweeds (like Durvillaea potatorum), green seaweeds (like Ulva lactuca), and botanicals like wheatgrass (Triticum aestivum) extracts. Another object of the present invention is to provide a plant bio stimulant formulation that is developed using a gentler cold percolation method using solvents like water ethanol, ensuring the preservation of key bioactive compounds for optimal plant health. Another object of the present invention is to provide a plant bio stimulant formulation that improves stress resilience in plants under drought, salinity, and temperature fluctuations, while also enhancing root development, overall plant vigor and increase crop productivity. SUMMARY OF THE INVENTION: Before the present invention is described, it is to be understood that the present invention is not limited to specific methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. The invention relates to a plant bio stimulant formulation comprising a combination of combination of brown seaweeds (like Durvillaea potatorum), green seaweeds (like Ulva lactuca), and botanicals like wheatgrass (Triticum aestivum) extracts for enhancing plant growth, improving abiotic stress tolerance, and promoting crop yield. According to an aspect of the present invention, the invention relates to the use of a cold percolation extraction method using solvents like water-ethanol to obtain a stable and highly bioactive formulation, ensuring the preservation of polyphenols, flavonoids, tannins, antioxidants, and other secondary metabolites essential for plant health. According to an aspect of the present invention, the formulation uses synergistic effects of Durvillaea potatorum, Ulva lactuca, and wheatgrass extracts, which collectively enhance root development, and stress resilience, improving plant tolerance to drought, salinity, and temperature fluctuations. The formulation works by enhancing plant metabolism, root architecture, and cellular defense mechanisms, leading to improved water retention, increased biomass accumulation, and higher photosynthetic efficiency. The inclusion of wheatgrass extract provides an additional source of antioxidants, tannins which further protects plants from oxidative stress and improves their overall physiological response under challenging environmental conditions. According to an aspect of the present invention, the primary mode of delivery of the bio stimulant formulation includes foliar spray, root drench, granule coating and seed treatment, ensuring efficient uptake and maximum efficacy across diverse crop species. For agricultural applications, the formulation is designed for use in varied environmental conditions, including regions experiencing water scarcity, salinity stress, and high-temperature fluctuations. The formulation has been validated through laboratory and field studies, demonstrating a 15- 20% increase in yield, making it a viable and cost-effective alternative to conventional bio stimulants. The comparative trials conducted across different crop species, including tomato, rice, chili, soybean and cotton have shown significant improvements in plant vigor, and overall yield, further establishing the formulation’s superior performance in both controlled and real-world agricultural conditions. BRIEF DESCRIPTION OF DRAWINGS A complete understanding of the present invention may be made by reference to the following detailed description, which is to be taken in conjunction with the accompanying drawings. The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate one or more embodiments of the present invention and, together with the detailed description, serve to explain the principles and implementations of the invention. Figure 1 depicts the graphs illustrating the tank-mix compatibility of the composition with a wide range of foliar fertilizers and agrochemicals, Figure 2 depicts the graph that show the consistency in the composition batches produced over 3 years, Figure 3 depicts the graph that exhibits high formulation stability; Figure 4a to 4f depicts how the bio stimulant formulation of the present invention regulates different pathways. DETAILED DESCRIPTION OF INVENTION Before the present invention is described, it is to be understood that this invention is not limited to methodologies described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. Various embodiments of the present invention are described below. It is, however, noted that the present invention is not limited to these embodiments, but rather the intention is that modifications that are apparent are also included. The present invention discloses a plant bio-stimulant formulation of brown seaweed (Durvillaea potatorum), green seaweed (Ulva lactuca), and wheatgrass (Triticum aestivum) extracts, utilizing a cold percolation method using solvents like water-ethanol, and methods of employing the formulation to enhance plant growth and yield and induce abiotic stress tolerance in crops. Unlike conventional fermentation-based seaweed extracts, this formulation utilizes a water:ethanol solvent extraction method, ensuring the preservation of key bioactive compounds such as polyphenols, flavonoids, terpenes, antioxidants, and polysaccharides. These bioactive molecules work synergistically to mitigate oxidative stress, improve membrane stability, enhance root architecture, and promote overall plant vigor. The inclusion of wheatgrass extract provides an additional antioxidant boost, further protecting plants against environmental stressors. The formulation is designed to improve stress tolerance under conditions of drought, salinity, and high-temperature fluctuations while promoting plant health and yield. Comparative trials demonstrate significant improvements in plant water retention, antioxidant activity, and biomass accumulation compared to standard seaweed-based formulations. The invention also offers a more efficient and scalable alternative to fermentation processes, ensuring consistent product quality and activity. The raw materials used in the composition comprises of Brown seaweed that is Durvillaea species that is selected from Brown seaweed - Durvillaea species like Durvillaea antarctica, Durvillaea potatorum Durvillaea poha, Durvillaea willana, Durvillaea fenestrate, Durvillaea amatheiae Durvillaea incurvate Durvillaea chathamensis. Green seaweed is selected from Ulva australis, Ulva clathrata, Ulva intestinalis, and Ulva linza, Ulva lactuca, Ulva compressa Ulva fasciata, Ulva reticulata, Ulva quilonensis, Ulva gujaratensis, Ulva rigid, Ulva flexuosa, Ulva paschima etc. Botanical extracts are selected from Triticum aestivum, Triticum durum, Triticum dicoccu, Triticum sphaerococcum Triticum sphaerococcum Triticum boeoticum Triticum dicoccoides riticum spelta, Triticum macha, Triticum vavilovii etc or other gramine members like Hordeum spontaneum, Hordeum distichum, Hordeum distichum Hordeum distichum Hordeum agriocrithon Hordeum lagunculiforme, Avena sterilis, Avena byzantine Avena fatua Avena sativa, Panicum miliaceum Sorghum bicolor Oryza sativa. According to the embodiment of the present invention, the drying and pulverization of the seaweed and botanical biomass comprises of the following steps. Freshly harvested seaweed biomass is first rinsed with clean water to remove surface salts, sand, and epiphytes. Freshly harvested botanical biomass is first rinsed with clean water to remove sand, and other impurities. The cleaned biomass of both is then subjected to solar shade drying under ambient conditions. The drying process is conducted in a well-ventilated covered structure that allowed diffused sunlight and airflow while preventing direct UV exposure, ensuring preservation of bioactive compounds. The temperature during drying is maintained below 45°C, with continuous monitoring to avoid degradation of thermolabile secondary metabolites. Drying is continued until the moisture content of the biomass is reduced to <10%, or until the biomass became crisp and brittle to touch. Once fully dried, the seaweed and botanical biomass is milled using a hammer mill and passed through a 40-mesh sieve to obtain a fine, uniform powder suitable for solvent extraction. The resulting powder is stored in airtight, light-resistant containers at room temperature until use. According to the embodiment of the present invention, the dried and milled Durvillaea, Ulva, and Triticum powders are subjected to a solvent-based extraction process designed to selectively isolate secondary metabolites such as polyphenols, phlorotannin, tannins, flavonoids, alkaloids, and terpenoids, while minimizing the recovery of bulk polysaccharides like alginate and mannitol. Each powder (10 kg) was first immersed in deionized water at a 1:10 w / v ratio for 24 hours at 25°C to allow cold aqueous pre-soaking, which hydrated the cell wall matrix and facilitated the release of water-soluble impurities, thereby improving extraction efficiency. After pre-soaking, the hydrated biomass was transferred to a percolation chamber and subjected to slow percolation with a hydroalcoholic solvent mixture containing 60% water and 40% ethanol (v / v), using a solvent-to-seaweed ratio of 10:1 (v / w). Percolation was carried out at ambient temperature under light-protected conditions to prevent oxidative degradation of light-sensitive metabolites, and continued for 12-14 days until the percolate was visually and analytically confirmed to reach exhaustion. The resulting extract was then filtered to remove particulate matter, after which the clear supernatant was stored at 25°C under light-protected conditions prior to concentration or further analysis, ensuring preservation of thermolabile bio actives. Different solvents and methods were tested for extraction process. The extraction process was carried out continuously for 12 hours to 14 days, allowing complete depletion of soluble metabolites from the biomass. Following extraction, the solvent was removed using rotary evaporation under reduced pressure at 40-45 °C and 150 mbar until a viscous dark-brown concentrate was obtained. The resulting extract was then standardized to a final concentration of 1000 ppm total polyphenols, quantified as gallic acid equivalents using the Folin-Ciocalteu assay, and 1000 ppm tannins, measured using the DMBA method. Below is the details of the test results: Solvent / Method Yiel d (% w / w dry) Total Polyph enols (% w / w, GAE) Phlorot annin (% w / w) Terpen oids (% w / w) Alginic Acid (% w / w) Mannitol (% w / w) Solvent Cost (USD / L) Scalabilit yIndex (1-5) Notes Water (cold macerati on) 7.5 4.8 0.9 0.3 4.5 2.8 0.00 5 Low phenolic extraction, high polysaccharide co-extraction Ethanol 95% 11.2 10.6 2.3 0.8 1.2 0.6 1.20 3 Good extraction, high cost, flammability concerns Methanol 80% 12.0 11.4 2.6 0.9 0.9 0.5 0.90 2 Strong extraction, but high toxicity & regulatory barrier Water-Ethanol 60:40 (selected ) 12.5 12.0 2.8 0.95 0.6 0.3 0.60 5 Balanced solvent system, best ratio of actives to cost, scalable Supercrit ical CO2 (EtOH 5-10%) 9.2 8.7 1.5 1.2 0.3 0.2 6-12 equival ent 2 High purity, expensive equipment, energy-intensive TABLE 1 Solvent selection was guided by both extraction performance and practical considerations related to safety, cost, and scalability. Water, while safe and inexpensive, was found to be ineffective for extracting most secondary metabolites, yielding primarily polysaccharides. Ethanol at 95% offered good extraction efficiency but posed challenges due to higher cost and flammability. Methanol at 80% demonstrated excellent solvent performance; however, its toxicity and regulatory restrictions made it unsuitable for agricultural input applications. Supercritical CO2 provided strong selectivity for certain metabolites but was dismissed due to its high operational cost and energy demand, which are impractical for large-scale production. Ultimately, a 60:40 water-ethanol mixture was selected as the optimal solvent system, providing high recovery of polyphenols and phlorotannin while minimizing extraction of alginates and mannitol, and offering a safe, cost-effective, and scalable solution. According to the embodiment of the present invention, the plant bio stimulant formulation comprising 70% w / v of brown seaweed (Durvillaea potatorum) extract, 10% w / v of green seaweed (Ulva lactuca) extract, and 20% w / v of wheatgrass (Triticum aestivum) extract. The extracts are obtained using the water-ethanol solvent extraction process. The bio-stimulant acts for enhancing plant growth, improving abiotic stress tolerance, and promoting crop yield. The formulation ensures the preservation of key bioactive compounds, including polyphenols, flavonoids, tannins, antioxidants, and other secondary metabolites, which synergistically contribute to mitigating oxidative stress, enhancing membrane stability, improving root architecture, and increasing plant vigor. Sr. No. Ingredient Scientific name Role Function % w / v in formulation 1. Brown Seaweed Extract Durvillaea potatorum Abiotic stress tolerance enhancer Improves drought, salinity, and heat resistance 70 2. Green Seaweed Extract Ulva lactuca Growth stimulator Enhances plant metabolic activity and growth 10 3. Wheatgrass Extract Triticum aestivum Antioxidant and metabolic booster Enhances photosynthesis, reduces oxidative stress 20 TABLE 2 According to the embodiment of the present invention the extract obtained using the described extraction process contains a range of secondary metabolite classes in concentrations typically spanning 10 ppm to 50,000 ppm (w / w). Total polyphenols, expressed as gallic acid equivalents, are recovered at high levels due to the efficiency of the 40% ethanol solvent system and are quantified using the Folin-Ciocalteu assay. Phlorotannin, which are characteristic of brown seaweeds, are similarly present within the 10-50,000 ppm range and are measured using the DMBA method. Terpenoids appear within a comparable concentration range and are primarily associated with the lipid-soluble fraction, with quantification performed via GC-MS. Flavonoids, including catechins and quercetin derivatives, are detected at concentrations between 10 ppm and 20,000 ppm. In contrast, alginic acid and mannitol occur only at trace to low levels (0.01% to 20,000 ppm), as their extraction is minimized by the non-acidic, cold, non-fermentative solvent conditions employed, thereby reducing the presence of bulk polysaccharides and sugar alcohol impurities in the final extract. Compatibility: According to the embodiment of the present invention, the formulation is designed for broad-spectrum compatibility with commonly used fertilizers, micronutrient blends, and crop protection products. Laboratory and field-scale evaluations have confirmed no adverse interactions, ensuring consistent efficacy and performance when applied in standard agronomic programs. The bio composition of the present invention is readily miscible in water and demonstrates excellent tank-mix compatibility with a wide range of foliar fertilizers and fertilizers, including agrochemicals. Concentrated, ready-to-use doses formulation were mixed with fertilizers, micronutrients, insecticides, and pesticides to assess tank-mix compatibility. The mixtures were evaluated for any changes in their spectral signatures at 0, 2, 4, and 6 hours after mixing as illustrated in FIG. 1. Active ingredient Brand names Present biostimulant formulation Conventional Fermented Ascophyllum formulation NPK, Micronutrients All grades of NPK Compatible even with Cu, Zn, sulphur Not Compatible with Cu, Zn Azoxystrobin Amistar Compatible Compatible Chlorpyrifos Saaf, Kavach, Bunos, Tafban, Dursban Compatible Compatible Captan Captan, Captaf, Taqat, Captol, Foltaf Compatible Compatible Mancozeb Mancozeb, Curzate M8, Ridomil Compatible Compatible Carbendazim Carbendazim, Dhanustin, Cammando Compatible Compatible Benomyl B enomyl / B enofit Compatible Compatible Wettable sulphur Sulphur Compatible Not Compatible Glyphosate Round -up Compatible Not Compatible O-S Dimethyl acetylphosphoramidothioate Acephate / Asatof / Starthene Compatible Not Compatible Thiamethoxam Actara Compatible Compatible Imidacloprid Admire, Confidor Compatible Compatible Cypermethrin Cymbush Compatible Compatible Fipronil Jump, Regent Compatible Compatible Lambda-cyhalothrin Karate Compatible Not Compatible Melathion Melathion Compatible Compatible Dichlorvos Nuvan Compatible Compatible Diafenthiuron Pegasus Compatible Not Compatible Acetamiprid Pride / Rekord Compatible Compatible TABLE 3 Stability and consistency: The scale-up and manufacturing process enables production of a highly reproducible bio formulation with a consistent and well-defined composition. For this spectral signature of separate batches from three separate years was done. The graph as illustrated in FIG. 2 reveals very high adherence to the spectral signature with very minute variation indicating consistency in batches produced over 3 years, this represents a significant advancement compared to fermented Ascophyllum-based biostimulants, which often exhibit considerable batch-to-batch variability. Achieving consistent formulation quality is critical, as it forms the foundation for delivering reliable and reproducible field performance. Shelf life: The formulation exhibits high formulation stability with no significant phase separation, precipitation, or viscosity changes under storage temperatures between 5 °C and 45 °C. For this spectral signature of a single batches was done over a period four separate years was done. The graph as illustrated in FIG. 3, reveals very high adherence to the spectral signature with almost no variation. Its active components retain biological efficacy for a minimum of 4 years under recommended storage conditions. Heavy Metal reduction: The extraction method of the present invention also significantly reduces the heavy metal content in the final formulation compared to traditional fermentationbased Ascophyllum biostimulants, enhancing both product safety and environmental sustainability as shown in Table 4. Heavy metal Limit Present bio-composition Conventional Ascophyllum Biostimulant Chromium (IV) as Cr <0.1mg / L Bellow detection limit Bellow detection limit Lead <0.1mg / L Bellow detection limit Above threshold Arsenic as (As2O3) <0.1mg / L Bellow detection limit Above threshold Cadmium <0.1mg / L Bellow detection limit Above threshold Copper <0.1mg / L Bellow detection limit Bellow detection limit Zinc <0.1mg / L Bellow detection limit Bellow detection limit Mercury <0.1mg / L Bellow detection limit Above threshold TABLE 4 Toxicity: Toxicological assessments confirm that the present biostimulant is non-toxic to humans, animals, and beneficial non-target organisms when used according to label directions. The formulation is classified as low-risk, with no reported phytotoxicity on tested crops as illustrated in TABLE 5. It meets safety standards for biostimulant use under prevailing agricultural regulations. Toxicology Test Present formulation Conventional Ascophyllum composition Oral LD50 (rat) Non-toxic (max tested dose 5,000 mg / kg) Non-toxic (max tested dose 5,000 mg / kg) Dermal LD50 (rat) Non-toxic (max tested dose 2,000 mg / kg) Non-toxic (max tested dose 2,000 mg / kg) Inhalation LC50 (rat) Non-toxic (max tested dose 10.04 ml / L) Non-toxic (max tested dose 10.04 ml / L) Eye Irritation (rabbit) Minimally irritating (scoring index 2) Irritating, reported mild conjunctival redness (scoring index 4) Skin Irritation (rabbit) Non-irritant Mildly irritating FISH Toxicity (96h LC50, Labeo rohita) Non-toxic (max tested dose 100 mg / L) Mildly toxic, due to high pH AVIAN Toxicity (Oral LC50, Gallus domesticus) Non-toxic (max tested dose 5 ml / kg) Non-toxic (max tested dose 5 ml / kg) BEE Toxicity (Contact LD50, Apis mellifera) Non-toxic (max tested dose 100 pl / bee) Non-toxic (max tested dose 100 pl / bee) Earthworm Toxicity (Contact LC50, Eisenia foetida) Non-toxic (max tested dose 1000 ml / kg of soil) Non-toxic (max tested dose 1000 ml / kg of soil) TABLE 5 Application: Materials and Methods: According to the embodiment of the present invention, the primary mode of delivery of the bio stimulant formulation includes foliar spray, root drench, granule coating and seed treatment, ensuring efficient uptake and maximum efficacy across diverse crop species. The formulation can be mixed with other agriculture chemicals like fertilizers, micronutrients, hormones, amino acids, protein hydrolyzates, humic or fulvic acids, vitamins, insecticides, pesticides etc. for integrated applications as well. For agricultural applications, the formulation is designed for use in varied environmental conditions, including regions experiencing water scarcity, salinity stress, and high-temperature fluctuations. The formulation has been validated through laboratory and field studies demonstrating stress tolerance, growth and yield enhancement. According to the embodiment of the present invention, for field application, comparative trials conducted across different crop species, including tomato, rice, and chili, soybean cotton have shown significant improvements in plant vigor, and overall yield, further establishing the formulation’s superior performance in both controlled and real-world agricultural conditions. The bio stimulant formulation enhances plant metabolism, root architecture, and cellular defense mechanisms, leading to improved water retention, increased biomass accumulation, and higher photosynthetic efficiency. The inclusion of wheatgrass extract provides an additional source of antioxidants, tannins which further protects plants from oxidative stress and improves their overall physiological response under challenging environmental conditions Plant Material and Growth Conditions: Rice (Oryza sativa L., variety DRR Dhan 65) and tomato (Solanum lycopersicum) seedlings were cultivated in 12-inch pots containing a sterilized soil-sand-compost mixture in a 2:1:1 ratio. The plants were maintained in a controlled greenhouse environment at 28°C with 60-70% relative humidity and a 14-hour light / 10-hour dark photoperiod. Seedlings were grown for 21 days under these conditions prior to the initiation of stress treatments. Biostimulant Preparation and Application: Two biostimulant formulations were evaluated in the study. The first was a seaweed-botanical combination consisting of extracts from brown and green seaweeds together with botanical components; this formulation was applied as a foliar spray at 4 mL / L, corresponding to 800 mL per acre. The second treatment consisted of a commercially available fermented Ascophyllum extract, characterized by a high polysaccharide content, and applied at the manufacturer’s recommended dose of 2.5 mL / L. Both biostimulants were applied twice, once at 15 days after sowing (DAS) and again at 21 DAS, which was three days prior to the imposition of stress conditions. Stress Treatments: Following acclimation, plants were subjected to abiotic stress conditions for a duration of seven days. Drought stress was induced by withholding irrigation, maintaining soil moisture below 15% (v / v). Salinity stress was imposed by irrigating the plants daily with a 150 mM NaCl solution. Heat stress was applied by transferring the plants to a controlled-environment chamber maintained at 45°C ± 1°C during the light period for six hours each day. Control plants were maintained under normal irrigation without any stress imposition. Morphological and Physiological Measurements a. Root Architecture: Plants were harvested seven days after the onset of stress, and their root systems were analyzed either using a WinRHIZO root-scanning system or through manual imaging. Quantitative parameters recorded included total root length, root surface area, root volume, and the total number of root tips. b. Growth Metrics: Shoot length, fresh weight, and dry weight were measured for all experimental plants. Relative water content (RWC) was calculated using the formula RWC = [(FW - DW) / (TW - DW)] x 100, where FW denotes fresh weight, TW represents turgid weight obtained after four hours of hydration, and DW indicates dry weight measured after drying samples for 48 hours at 70°C. c. Membrane Stability Index (MSI): Leaf discs of 0.5 cm diameter were excised from treated and control plants and incubated in 10 mL of distilled water at 25°C. Electrical conductivity of the solution was measured after four hours to obtain the initial conductivity value (C1). The samples were then boiled at 100°C for ten minutes, cooled, and re-measured to obtain the total conductivity (C2). Membrane stability index was calculated as MSI (%) = [1 - (C1 / C2)] x 100. Reactive Oxygen Species (ROS) Detection in Leaves a. DAB Staining for Hydrogen Peroxide (H2O2): Leaves were infiltrated with a 1 mg / mL solution of 3,3‘-diaminobenzidine (DAB) adjusted to pH 3.8 and incubated for six hours under light. After staining, chlorophyll was removed by ethanol bleaching, and the leaves were imaged under brightfield conditions. Brown precipitate formation was used as an indicator of H2O2 accumulation. b. NBT Staining for Superoxide Radicals: To assess superoxide levels, leaves were incubated in a 0.1% nitro blue tetrazolium (NBT) solution prepared in 10 mM potassium phosphate buffer at pH 7.8 for four hours in the dark. After staining, leaves were decolorized in ethanol, and blue formazan deposition was imaged. Quantification of staining intensity was performed digitally using ImageJ software. Statistical Analysis: All experimental treatments were conducted with a minimum of five biological replicates. Data were subjected to analysis of variance (ANOVA), and significant differences among treatment means were determined using Tukey’s HSD post-hoc test at a significance threshold of p < 0.05. Where applicable, root architectural traits and ROS staining intensities were quantified using digital image-analysis tools. The Comparison of Biostimulant Effects (Combination Biostimulant vs. single source biostimulant) in drought conditions is illustrated in TABLE 6 below: Parameter Absolute control Control (No Biostimulant) Ascop Extract Durvillaea Extract (A) Ulva Extract (B) Triticum Extract (C) Combination of Biostimulant (A+ B+ C) Shoot Length (cm) 20 ± 0.6 10.4 ± 0.7 11.7 ± 0.4 15.9 ± 0.7 14.6 ± 0.4 12.7 ± 0.2 17.7 ± 0.6 Shoot Dry Weight (g) 1.1 ± 0.1 0.6 ± 0.2 0.63 ± 0.3 0.88 ± 0.4 0.75 ± 0.6 0.8 ± 0.5 0.97 ± 0.7 Canopy area (cm2) 55.6 ± 0.3 15.73 ± 0.9 34.13 ± 1.1 40.59 ±1.3 39.1± 0.8 35.03 ± 1.5 48.59 ±1.3 Relative Water Content (%) 85.7 ± 1.1 33.5 ± 2.1 41.3 ± 1.7 63.4 ± 1.9 58.73 ± 2.2 55.9 ± 1.8 75.3 ± 1.5 Membrane Stability Index (MSI %) 87.7 ± 4.2 42.5 ± 2.3 51.6 ± 2.7 64.1± 3.1 68.29 ± 2.7 62.3 ± 2.1 77.1± 1.9 Leaf rolling index (%) 0 25.24 ± 1.1 41..47 ± 1.3 15.46 ± 1.4 13.45 ± 1 12.24 ± 1.3 11.06 ± 0.9 Chlorophyll content 42.62 ± 1.4 49.41 ± 1.1 43.82 ± 1.6 52.52± 1.9 56 ± 2.0 58.70 ± 1.8 63.63 ± 2.2 DAB Staining Intensity (A.U.) - 210 ± 9 175 ± 11 121 ± 14 115 ± 10 95 ± 9 85 ± 5.4 NBT Staining Intensity (A.U.) - 198 ± 9 152 ± 12 108 ± 7 99 ± 2 90 ± 6 88 ± 3 Seedling Survival (%) 95 ± 2 21 ± 5 33 ± 5 75 ± 4 80 ± 6 86 ± 3 91 ± 1 TABLE 6 The Comparison of Biostimulant Effects (Combination Biostimulant vs. single source biostimulant) in salinity conditions is illustrated in TABLE 7 below: Parameter Absolut e control Control (No Biostimulant ) Ascop Extrac t Durvillae a Extract (A) Ulva Extract (B) Triticu m Extract (C) Combinatio n of Biostimulant (A+ B+ C) Shoot Length (cm) 21 ± 0.7 10.8 ± 0.5 12.7 ± 0.4 15.9 ± 0.9 16.6 ± 0.4 15.7 ± 0.2 18.5 ± 0.3 Shoot Dry Weight (g) 1.1 ± 0.2 0.6 ± 0.4 0.82 ± 0.3 0.98 ± 0.6 1.03 ± 0.5 0.98 ± 0.4 1.05 ± 0.9 Canopy area (cm2) 62.8 ± 0.2 39.73 ± 1.3 41.13 ± 0.8 48.59 ±1.1 49.1± 0.6 44.03 ± 1.2 59.4 ±1.1 Relative Water 90.7 ± 2.2 73.5 ± 2.4 76.3 ± 1.7 83.4 ± 2.1 85.63 ± 1.6 85.9 ± 1.7 89.3 ± 2.1 Content (%) Membrane Stability Index (MSI %) 80.7 ± 3.1 41.9 ± 2.8 48.2 ± 2.1 61.4± 2.4 58.29 ± 1.9 65.3 ± 1.5 71.1± 1.2 Chlorophyl l content 40.6 ± 1.9 25.1 ± 1.6 27.82 ± 1.3 33.5 ± 1.5 36 ± 1.9 35 ± 2.1 39 ± 2.3 DAB Staining Intensity (A.U.) - 201 ± 11 155 ± 8 131 ± 9 115 ± 12 101 ± 10 89 ± 6 NBT Staining Intensity (A.U.) - 189± 9 108 ± 8 87 ± 7 90 ± 5 80 ± 6 78 ± 4 Seedling Survival (%) 96 ± 1 15 ± 3 43 ± 6 81 ± 7 84 ± 5 87 ± 2 92 ± 3 TABLE 7 The Comparison of Biostimulant Effects (Combination Biostimulant vs. single source biostimulant) in heat conditions is illustrated in TABLE 8 below: Parameter Absolut e control Control (No Biostimulant ) Ascop Extrac t Durvillae a Extract (A) Ulva Extract (B) Triticu m Extract (C) Combinatio n of Biostimulant (A+ B+ C) Shoot Length (cm) 26.9 ± 0.6 17.4 ± 0.5 19.7 ± 0.4 22.1 ± 0.6 22.6 ± 0.8 21.7 ± 0.3 24.7 ± 0.2 Shoot Dry Weight (g) 1.1 ± 0.2 0.56 ± 0.1 0.61 ± 0.2 0.77 ± 0.3 0.85 ± 0.4 0.83 ± 0.6 0.91 ± 0.3 Canopy area (cm2) 52.7 ± 0.5 35.3 ± 1.1 38.2 ± 1.2 45.9 ± 0.9 48.1 ± 0.3 40.3 ± 1.2 50.9 ±1.1 Relative Water Content (%) 96.7 ± 1.6 69.5 ± 2.3 76.3 ± 1.5 85.4 ± 2.1 92.63 ± 1.7 90.9 ± 1.8 95.3 ± 1.5 Membrane Stability Index (MSI %) 82.4 ± 3.3 55.9 ± 3.0 59.2 ± 2.4 71.4± 2.1 67.29 ± 2.3 72.3 ± 2.8 77.1± 2.2 Chlorophyl l content 41.32 24.31 ± 2.3 23.82 ± 1.6 30.52 ± 2.1 31.5 ± 3.9 32 ± 5.2 35 ± 3.5 DAB Staining Intensity (A.U.) - 168 ± 9 145 ± 7 109 ± 11 111 ± 5 95 ± 3 85 ± 2 NBT Staining Intensity (A.U.) - 149 ± 6 116 ± 9 101 ± 11 98 ± 7 86 ± 3 75 ± 2 Seedling Survival (%) 95 ± 3 19 ± 2 68 ± 4 79 ± 2 82 ± 3 84 ± 2 91 ± 1 TABLE 8 Field study: A Comparative Study of Combination Biostimulant vs. Single-Source Biostimulant was carried out in Tomato, Cotton, Soybean, and Chilli Objective: The study was conducted to evaluate and compare the agronomic performance, physiological responses, and yield-quality attributes of multiple crop species—including tomato, cotton, soybean, and chili—when treated with two different categories of biostimulants. The first treatment consisted of a polyphenol-rich Durvillaea solvent extract, while the second comprised a polysaccharide-rich conventional fermented Ascophyllum extract. An untreated control group receiving no biostimulant was included to establish baseline physiological and yield performance. The principal objective of the study was to determine whether the combination biostimulant provided superior enhancement of plant vigor, stress tolerance, and crop productivity compared with a conventional single-source biostimulant. Experimental Design: Field experiments were conducted using cucumber as the model crop under a randomized complete block design with five replications per treatment. Each plot measured 3 m x 2 m and contained ten uniformly spaced plants. Three treatments were imposed: a control receiving no biostimulant; a fermented Ascophyllum extract applied as a foliar spray at 5 mL / L; and a combination biostimulant applied at the same concentration. Applications were delivered via foliar spray and root drench at 15, 30, 45, and 60 days after transplanting. Trials were carried out across three agroclimatic zones: Dapoli, Dharwad, and Rahuri, India, representing diverse environmental conditions to ensure robustness of performance outcomes. All plots received uniform irrigation and fertilization, including standardized NPK inputs, and were maintained under recommended cultivation practices appropriate to each location. Parameters Measured: Morphological, physiological, and yield-related parameters were recorded at multiple growth stages to quantify treatment effects. Plant height was measured at 30, 45, and 60 days after transplanting (DAT) by recording the distance from the soil surface to the apical meristem. Canopy spread was assessed at 45 and 60 DAT by measuring the maximum horizontal diameter of the plant. Chlorophyll concentration was measured at the same time points using a SPAD-502 chlorophyll meter. Reproductive development was monitored through daily recording of the number of days required for 50% flowering and by manually counting the number of flowers produced per plant between 45 and 60 DAT. Yield was assessed at harvest by determining total fruit weight per plant and calculating corresponding values in tonnes per hectare. Post-harvest fruit quality parameters were evaluated through measurement of total soluble solids (TSS) using a digital refractometer, firmness using a penetrometer, color using visual scoring or a chromameter, and titratable acidity through standard acid-base titration. These combined assessments enabled a comprehensive comparison of the performance of the combination biostimulant against the single-source biostimulant and the untreated control. The results of the field study is presented below as average of all studies in TABLE 9. The seaweed-botanical combination demonstrated statistically significant superiority over individual seaweed or botanical applications, with improvements observed in vegetative growth, earliness, yield, and fruit quality. On average, the combination delivered a 13.6% enhancement compared to the best-performing single component, indicating a synergistic interaction rather than an additive effect. Cont rol Brown Seaweed Green seaweed Botanical extract Combin ation % incre ase over % incre ase over Brow % incre ase over green % increa se over botani contr ol n seaw eed seaw eed cal extrac t Plant height 12.97 14.2 15.5 15.5 15.1 16.42 6.34 -2.58 -2.58 Leaves / p lant 4.7 5 5.1 5.1 5.3 12.77 6.00 3.92 3.92 Male flowers / plant 8.55 6.8 7.1 6.5 10.8 26.32 58.82 52.11 66.15 Female flowers / plant 4.4 4.65 4.7 5.2 5.85 32.95 25.81 24.47 12.50 Days to 50% flowerin g 38.25 37.5 38.5 39 35.5 -7.19 -5.33 -7.79 -8.97 Fruits / pl ant 7.15 7.3 7 7.32 8.9 24.48 21.92 27.14 21.58 fruit length 14.35 14.77 14.9 15.16 16.81 17.14 13.81 12.82 10.88 Fruit girth 30.18 30.58 30.1 31.23 32.72 8.42 7.00 8.70 4.77 Single fruit weight 72 81.5 73.5 102.5 115.5 60.42 41.72 57.14 12.68 Yield / ac re 33.32 37.31 36.1 36.91 41.79 25.42 12.01 15.76 13.22 TABLE 9 Transcriptomics study- mode of action Method for Sample Collection and Transcriptomic Study (RNA-Seq) in Tomato Treated with Biostimulants: To compare transcriptomic changes in tomato plants treated with solvent-extracted Combination formulation vs. fermented Ascophyllum biostimulants under field conditions and identify differentially expressed genes (DEGs) related to growth, stress response, hormone signaling, and secondary metabolism. Same treatments as outlined in the field design: • T1 - Untreated control • T2 - Ascophyllum extract- conventional formulation • T3 - Combination extract- present formulation Sample Collection for Transcriptomics: Parameter Description Tissue Type Fully expanded 5th leaf from the apex (vegetative stage) and young floral buds (reproductive stage) Sampling Time 48 hours after final biostimulant application (peak transcriptomic response), ideally at 10:00-11:00 AM Biological Replicates 3 plants per treatment (n = 3), sampled independently Handling Immediately cut leaf tissue (~100 mg), snap-freeze in liquid nitrogen in the field Storage Transported in dry ice and stored at -80°C until RNA extraction RNA Extraction: Extract total RNA using Qiagen RNeasy Plant Mini Kit or equivalent. Include on-column DNase treatment to remove genomic DNA. Check RNA quality using: Nanodrop (260 / 280 and 260 / 230 ratios), Agilent Bioanalyzer or TapeStation (RIN > 7.5 required) Quantify with Qubit RNA HS Assay. Library Preparation and Sequencing: Use poly(A) selection for mRNA enrichment Library prep: RNA Library Prep Kit or equivalent. Index all samples and pool. Perform paired-end sequencing (150 bp) on Illumina NovaSeq or HiSeq platform. Aim for \~30 million reads per sample. Bioinformatic Analysis Step Tools / Pipelines Quality check FastQC, Trimmomatic Read alignment HISAT2 or STAR against tomato reference genome (Solanum lycopersicum) Transcript assembly & quantification StringTie or featureCounts Differential Expression Analysis DESeq2 or edgeR Functional Annotation Blast2GO, KEGG Mapper Pathway Enrichment GO, MapMan, PlantReactome Analysis: Transcriptomic analysis of treated plants revealed different pathways regulated by the formulation of the present invention as illustrated in TABLE 10. Biological Function / Pathway Formulation with only Seaweed Formulation with only botanical extract Formulation of present invention (combination) Synergistic Advantage in present formulation Photosynthesis (PSII / PSI) Upregulates PSII (10x), light harvesting maintained (LHCBlf), PSI downregulated to limit ROS Upregulates PSII f3.3x, PSI J3.3x Increases energy generation through 3X ATP synthase upregulation Increases ATP Synthase (7X), Complex I / V (6.7x), PSII (3x), reduces PET5 for ROS control Combined effect leads to both enhanced light capture and optimized photophosphorylation, with balanced ROS Chlorophyll / Protoheme synthesis — — Protoheme $20x Maximized chlorophyll production & energy transfer Rubisco / Carbon Fixation — $2.5x $3x Stable CO2 assimilation across growth phases Sucrose Transport Sucrose transport f10x Redirects carbon from lipids to polyphenolics; limits sugar breakdown Malate, Glycerate f10x Enhances glyoxylate cycle, carbon fixation (7.4^), photorespiration-linked recycling Sucrose transport for active growth Dual control of carbon: Fortisea conserves, 7525 recycles—improved carbon economy and anaplerotic recovery Oxidative Phosphorylation Complex I / V f10x, IROS complexes ETC f (Cytochrome bc1 / C oxidase) f across key ETC enzymes Sustained ATP production under both growth and reproduction GABA Shunt / Redox Regulation GABA f20x, Putrescine f9x — GABA f3x, Dual redox and pH buffering under stress Amino Acid Rewiring (BCAAs, Thr) — BCAAs f, Thr f3x BCAAs f3x, Thr f3x Energy supply + reproductive tissue support More robust osmoprotection, detoxification, and cell integrity under stress Arginine / Polyamine Pathway Putrescine f9x (via SAM) Arginine, Ornithine, Citrulline f Active Fertility support + osmoprotective nitrogen flow Methionine Cycle / SAM Homocysteine f2x, SAM f>20x — Active Epigenetic control + cell stability EF-Tu (Protein Synthesis) — f f Supports high-protein demand during fruit set Hormonal Regulation (GA / IAA / Zeatin) GA f10x, Aux1 / GH3 f11x GA f20x, IAA f3.5x, Zeatin f6.6x GA f10x, Zeatin f7x, IAA transport f20x, Brassinosteroids f2X ABA modulated stress f10x Hormonal rebalancing: Fortisea supports growth; 7525 enhances rooting and stress priming— together offer adaptive plasticity MAPK MAPK18 Drought, salinity, heat stress mitigation pathways upregulated Ethylene Modulation — Repression f2.5x Active Delayed senescence, improved shelf-life Volatile Organic Compounds (VOCs) 3,6- Nonadien-1-ol f2.5x Not explicit but BCAAs active Aroma boost inherited from Chiron Fruit Softening / Ripening Enzymes PG f7x, Pectin f3x Enhanced texture and ripening from Chiron input Lipid Signals (PG / PA) PG & PA f PG & PA f Florigen transport, gametogenesis, embryo development Phenylpropanoid (PAL activity) PAL $10¾ ^ redirection to soluble phenolics Moderate (via PR1) Chalcone synthase f7x Fortisea's ROS shield + Chiron’s flavonoid signalling ^ antioxidant + regulatory dual function Lignin Biosynthesis | (bypass to soluble pool) f7.2x Balanced Maintains firmness in fruits without sacrificing flexibility Flavonoids (Quercetin, Kaempferol) Downregulated (X9x) f (multiple flavonoids) ff (multiple active classes) UV protection, auxin modulation, floral induction support Anthocyanins / Carotenoids Delphinidin, Pelargonidin f; Astaxanthin f4x Indirect through ripening Active via inheritance Pigmentation + ROS defense synergy Phytoalexins / PR Proteins PR1 f5.1x, phytoalexins f PR1 + Chalcone-derived phytoalexins f Systemic immunity + localized defense + antioxidant priming PAMP-Triggered Immunity (PTI) Flagellin & fungal PAMP f20x Pathogen defense from early recognition to late-stage reinforcement Combines passive (chemical) and active (immune) defense— broad-spectrum protection Cuticle Wax Cell wall Phospholipids, MECR J2x Thicker cuticles, cell wall re enforcement and wax coatings TABLE 10 The bio stimulant formulation of the present invention regulated different pathways as illustrated in FIG. 4a to 4f. Fig. 4a- The biostimulant significantly enhanced photosynthetic performance by increasing chlorophyll synthesis seven-fold and boosting Photosystem I and II activity by three- and seven-fold, respectively, leading to greater light capture and stronger electron transport. This resulted in elevated ATP and NADPH production, while Rubisco levels doubled and protoheme synthesis increased twenty-fold, collectively strengthening carbon fixation and chlorophyll biosynthesis. FIG. 4b shows a significant increase in oxidative phosphorylation (6-fold), malate (6-8 fold), and D-glycerate (7-8 fold), leading to improved ATP synthesis and reduced metabolic waste. Malate plays a key role as a redox and carbon carrier, bridging organelles and supporting C4 / CAM carbon concentrating strategies. This metabolic rebalancing replenishes TCA intermediates and promotes stress tolerance by balancing carbon and nitrogen flow. FIG. 4c- illustrates how amino acid metabolism is rewired to enhance plant stress resilience and reproductive success. Key amino acids: serine, glycine, cysteine; valine, leucine, isoleucine; and glutamate show a threefold increase, supporting energy production, protein synthesis, and flowering. Threonine conversion boosts branched-chain amino acids (BCAAs), while the phosphoserine pathway enhances glutathione synthesis and redox balance, both crucial for floral development and stress tolerance. FIG. 4d- shows how hormonal reprogramming enables plant growth under stress conditions like drought and salinity. Key changes include a 7-fold increase in zeatin for cell division and root development, and a 20-fold rise in auxin transporter ABCB1 to strengthen growth orientation and vascular flow. Stress-responsive MAPK pathways are also activated, while ABA signaling increases 10-12 fold to enhance tolerance. Together, these shifts promote resilience and sustained growth under adverse environments. Fig. 4e highlights how phenylpropanoid and flavonoid pathways are rewired to boost plant defense and stress adaptation. Key changes include a twofold increase in chalcone synthase activity and mobile antioxidants, along with elevated pigment production like xanthoxin. Metabolites such as luteolin, quercetin, and naringenin enhance UV protection and antioxidant capacity, while shifts in alcohol derivatives favor mobile defense over structural reinforcement. These biochemical adjustments support growth modulation and resilience. Fig. 4f shows how upregulation of MECR enhances lipid metabolism in plants, boosting stress resilience. MECR activation (2.9-fold) stimulates mitochondrial fatty acid synthesis, increasing palmitic acids and phospholipids. This leads to improved membrane fluidity, cuticular wax biosynthesis, and structural lipid production, which strengthen drought tolerance, pathogen defense, and post-stress recovery. Lipid signaling and membrane stability are key outcomes of this metabolic reprogramming. The biostimulant formulation of the present invention exhibits a series of functional attributes that extend beyond the capabilities of conventional brown seaweed extracts, green seaweed extracts, or standalone botanical preparations. These enhanced properties arise from the synergistic interaction between its constituent classes of metabolites, creating coordinated physiological effects that are not generated by any single-source extract. One of the defining features of the composition is its ability to support a continuous transition between the vegetative and reproductive phases of plant development. This is achieved through an integrated delivery of metabolic energy substrates, biosynthetic precursors, and developmental signalling molecules that collectively maintain uninterrupted physiological activity as plants shift from biomass accumulation to flowering and fruit initiation. Another distinguishing characteristic of the biostimulant is its capacity to merge reactive oxygen species (ROS) defence mechanisms with immune-priming activity. The coordinated presence of phenolic antioxidants, pathogenesis-related (PR) proteins, and pattern-associated molecular pattern (PAMP) cues strengthens basal immunity while simultaneously moderating oxidative stress. This dual effect enhances stress tolerance without triggering deleterious hypersensitive responses, thereby contributing to sustained growth under abiotic and biotic stress conditions. The composition further supports organogenesis through finely tuned flavonoid-auxin interactions mediated by compounds such as luteolin, vitexin, and quercetin. These bio actives regulate auxin transport and sensitivity, promoting precise vascular differentiation and floral tissue development. As a result, the formulation enables more uniform reproductive initiation and improved structural integrity of developing organs. Importantly, the formulation avoids the hormonal overstimulation often encountered with single-source GA- or auxin-based products, instead producing a controlled amplification of endogenous hormonal pathways. This moderated hormonal response prevents growth abnormalities while maintaining optimal metabolic activation. Additionally, the formulation enhances sensory and post-harvest quality traits through a unique interplay of polysaccharide-modulating enzymes, lignin precursors, volatile organic compounds (VOCs), and antioxidant pigments. This coordinated action contributes to improved fruit firmness, aroma profile, and shelf stability, the attributes that are generally not observed when using single-source extracts lacking multipathway activity. Collectively, these features demonstrate that the bio stimulant formulation of the present invention represents a novel, non-obvious, and synergistic biostimulant composition that integrates seaweed-derived agents conferring vegetative vigor with botanical components enhancing reproductive development. The formulation uniquely converges redox stability with hormonal homeostasis, photosynthetic efficiency with developmental signaling, and immune priming with fruit-quality enhancement. These multi-layered effects correlate with distinct fold-change dynamics across primary and secondary metabolic pathways—responses that cannot be reproduced by either the seaweed fraction or the botanical fraction when applied independently. As a result, the formulation is a first-in-class, broad-spectrum biostimulant with clear functional differentiation and strong commercial potential. In conclusion, the bio stimulant formulation disclosed in this invention provides a scientifically validated, scalable, and sustainable solution for modern agriculture. By combining the unique bioactive properties of Durvillaea potatorum, Ulva lactuca, and wheatgrass extracts, and utilizing a non-fermentation, cold percolation process, the invention sets a new benchmark for plant bio stimulant efficacy, offering enhanced stress resistance, improved climate resilience, increased yields and long-term agricultural sustainability. Novelty and Differentiation of the present invention: • Seaweed Species Combination: Most seaweed-based formulations in the market use fermented Ascophyllum nodosum (brown seaweed) or red algae. However, the invention uses Durvillea potatorum (a less commonly commercialized brown seaweed species) and Ulva (green seaweed) giving a unique seaweed combination. The synergistic effect of Durvillea for abiotic stress tolerance and Ulva for growth enhancement is observed. • Addition of Botanical (Wheatgrass) Extract: The inclusion of wheatgrass extract is novel. Wheatgrass is known for its high antioxidant content (vitamins, minerals, amino acids, and chlorophyll), which may synergistically enhance stress mitigation in plants. Few, if any, commercially available agricultural formulations use wheatgrass extract. Seaweed combination with Botanical extract is very novel to derive a more synergistically acting formulation that combines bio actives from seaweeds and land plants Wheat grass addition significantly improved antioxidant effect, reducing oxidative damage, and improving photosynthetic efficiency under stress. • Extraction Method (Water: Ethanol Solvent-Based Extraction): The prevalent extraction process in the market relies on acid / alkali treatment of biomass followed by fermentationbased processes for Ascophyllum nodosum resulting in high pH 8-9 formulation. In contrast, the cold percolation extraction using water:ethanol-based solvent for Durvillea potatorum, Ulva, and wheatgrass stands out with its transparent, free flowing nature and low pH 3-4. This method could offer advantages like higher bioactive compound recovery, faster processing, and consistency of extract composition. The solvent-based extraction method preserves specific bioactive compounds especially the secondary metabolites that are degraded or modified during fermentation. • Synergistic Phase Bridging: The combination uniquely supports continuity between vegetative and reproductive phases by integrating energy metabolism, biosynthesis, and signaling pathways. Neither seaweed nor botanicals alone confer this seamless transition thus helping in reducing flower drop, improving fruit set. • Integrated Stress Immunity System: The formulation elicits a dual defense mechanism: ROS scavenging from phenolics and immunity activation through PR proteins + PAMP-like signals. The measurable outcomes are reduced oxidative stress markers, increased disease tolerance. • Flavonoid-Auxin Crosstalk for Organogenesis: The specific flavonoids (luteolin, vitexin, quercetin) modulate auxin transport and signaling thus promoting vascular tissue integrity, floral differentiation, and fruit initiation. The precise flavonoid profile derived from botanical inclusion and seaweed base is novel. • Controlled Hormonal Amplification: Combination avoids harmful hormonal surges (GA, auxin) common in single-source products. Therefore, this combination is a balanced hormonal modulation system ensuring stable yield and reduced physiological disorders. • Post-Harvest Quality Enhancement: It provides unique synergy in extending shelf-life and sensory traits such as Pectin softening via PG enzymes, structural firmness from lignin deposition, volatile organic compounds (VOCs) contributing to aroma, pigments and antioxidants contributing to color stability and nutritional quality. • Superiority Over Single Inputs: The combination consistently delivers more than 13-15% better performance in terms of growth, yield, or quality when compared to seaweed or botanical extracts alone. ADVANTAGES: The advantages of the present invention are as follows: • Enhanced plant stress tolerance and improved growth efficiency through the synergistic combination of brown seaweed (Durvillaea potatorum), green seaweed (Ulva lactuca), and wheatgrass (Triticum aestivum) extracts. • Preservation of key bioactive compounds, including polyphenols, flavonoids, tannins, antioxidants, and other secondary metabolites, ensures that plants receive optimal support for mitigating oxidative stress, improving membrane stability, and enhancing root development. • Increased biomass accumulation, better water retention, and higher photosynthetic efficiency, particularly under drought, salinity, and temperature stress conditions. • The use of a cold percolation process using solvents like water-ethanol provides a highly stable and effective alternative to fermentation-based seaweed extracts, ensuring consistent bioactive compound retention and greater efficacy in plant growth promotion. • Unlike conventional fermentation methods, which may degrade essential bioactive, this process preserves and maximizes the potency of secondary metabolites critical for plant resilience. • The inclusion of wheatgrass extract further enhances the antioxidant activity of the formulation, offering improved stress protection, leading to better overall crop performance. • Additionally, the formulation can be applied as a foliar spray, root drench, granule coating or seed treatment, making it a versatile and adaptable solution for diverse agricultural applications. While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
Claims
1. A plant bio stimulant formulation for enhancing plant growth and managing abiotic stress characterized in thatthe bio composition comprises the extract of brown seaweed Durvillea at 70% w / v, green seaweed Ulva at 10% w / v and the wheatgrass Triticum extract at 20% w / v; andthe extraction is conducted using cold percolation method with water-ethanol solvent at the ratio of 60:40 v / v to maximize recovery of active metabolites; and the method of preparation of the formulation comprises the steps of• cleaning brown and green seaweed biomass by rinsing freshly harvested seaweed with clean water to remove surface salts, sand, and epiphytes;• cleaning botanical biomass by rinsing freshly harvested botanical material with clean water to remove sand and other impurities;• drying the cleaned biomass under shade in a well-ventilated covered structure that allows diffused sunlight while preventing direct UV exposure, maintaining the temperature below 45°c until the moisture content is reduced to less than 10% or the biomass becomes crisp and brittle;• pulverizing the dried biomass using a hammer mill and passing the resulting material through a 40-mesh sieve to obtain a fine, uniform powder;• storing the powdered biomass in airtight, light-resistant containers at room temperature;• extracting bioactive compounds from the powdered biomass by cold percolation using a water-ethanol solvent system at a volumetric ratio of approximately 60:40 (v / v), with a solvent-to-biomass ratio of about 10:1, at ambient temperature for a period ranging from 12 hours to 14 days;• removing the solvent from the extract by rotary evaporation under reduced pressure at a temperature between 40°c and 45°c;• standardizing the extract to contain not less than 1000 ppm polyphenols and not less than 1000 ppm tannins.
2. The bio composition as claimed in claim 1, wherein brown seaweed extract improves drought, salinity, and heat resistance, green seaweed extract enhances plant metabolic activity and growth and wheatgrass extract enhances photosynthesis, reduces oxidative stress.
3. The bio composition as claimed in claim 1, wherein the cold percolation extraction process using water:ethanol-based solvent for Durvillea, Ulva, and wheatgrass leads to higher bioactive compound recovery, faster processing, and consistency of extract composition and it preserves specific bioactive compounds especially the secondary metabolites that are degraded or modified during fermentation.
4. The bio composition as claimed in claim 1, wherein the formulation has low pH 3-4 and is readily miscible in water and demonstrates tank-mix compatibility with a wide range of foliar fertilizers and fertilizers, including agrochemicals.
5. The bio composition as claimed in claim 1, wherein the composition has a shelf life of at least four years when maintained within a temperature range of approximately 5-45 °C.
6. The bio composition as claimed in claim 1, wherein the formulation supports continuity between vegetative and reproductive phases by integrating energy metabolism, biosynthesis, and signaling pathways.
7. The bio composition as claimed in claim 1, wherein the formulation elicits a dual defense mechanism of ROS scavenging from phenolics and immunity activation through PR proteins and PAMP-like signals, thus showing reduced oxidative stress markers and increased disease tolerance.
8. The bio composition as claimed in claim 1, wherein the formulation contains specific flavonoids profile including luteolin, vitexin, quercetin, that modulate auxin transport and signaling thus promoting vascular tissue integrity, floral differentiation, and fruit initiation.
19. The bio composition as claimed in claim 1, wherein the formulation avoids harmful hormonal surges such as GA, auxin and the formulation is a balanced hormonal modulation system ensuring stable yield and reduced physiological disorders.
10. The bio composition as claimed in claim 1, wherein the formulation provides a unique synergy in extending shelf-life and sensory traits such as Pectin softening via PG enzymes, structural firmness from lignin deposition, volatile organic compounds (VOCs) contributing to aroma, pigments and antioxidants contributing to color stability and nutritional quality.
11. The bio composition as claimed in claim 1, wherein the primary mode of delivery of the formulation includes foliar spray, root drench, granule coating and seed treatment, ensuring efficient uptake and maximum efficacy across diverse crop species.
12. The bio composition as claimed in claim 1, wherein the formulation consistently delivers more than 13-15% better performance in terms of growth, yield, or quality when compared to seaweed or botanical extracts alone.