DESC:FIELD OF INVENTION:
The present invention relates to agrochemical formulations and uses thereof for inducing abiotic stress tolerance in plants. More particularly, the present invention relates to a novel bio-formulation of carbon nanoparticles and carbon quantum dots in conjunction of specific naturally occurring bioactive substances and methods of employing these substances to induce abiotic stress tolerance in plants.

BACKGROUND OF THE INVENTION:
Agriculture is a multi-billion dollar industry. Fertile soils are required for improved plant growth. Fifteen essential nutrients are supplied by soil which is categorized into macronutrients and micronutrients. Nitrogen (N), phosphorus (P) and potassium (K) are primary nutrients or macronutrients. This is because (1) they are required by the plant in large amounts relative to other nutrients, and (2) deficiencies in these lead to growth and development impediment. Calcium (Ca), magnesium (Mg) and Sulphur (S) are termed secondary nutrients, because their absence is less likely to constitute growth-limiting factors in soil. Zinc (Zn), Chlorine (Cl), Boron (B), Molybdenum (Mo), Copper (Cu), Iron (Fe), Manganese (Mn), Cobalt (Co) and Nickel (Ni) are termed micronutrients, as they are required in very small amounts relative to other plant nutrients in the average plant, and (2) they are least likely to be limiting plant growth and development in many soil systems. Fertilizers are often used to improve the fertility of the soil for agricultural crops. But fertilizers, particularly synthetic fertilizers have a major potential to pollute soil, water and air; in recent years, many efforts were done to minimize these problems by agricultural practices and the design of the new improved fertilizers. Conventional fertilizers are generally applied on the crops by either spraying or broadcasting. But it has been estimated that around 40-70% of nitrogen, 80-90% of phosphorus, and 50-90% of potassium content of applied fertilizers are lost in the environment and could not reach the plant, which causes sustainable and economic losses.
In addition, abiotic stresses such as drought (water deficit), excessive watering (water-logging/flooding), extreme temperatures (cold, frost and heat), salinity and mineral (metal and metalloid) toxicity negatively impact growth, development, yield and seed quality of crop and other plants.Abiotic stress is defined as the negative impact of non-living factors on the living organisms in a specific environment. It includes numerous stresses caused by complex environmental conditions, e.g., strong light, UV, high and low temperatures, freezing, drought, salinity, heavy metals and hypoxia. These stresses will increase in the near future because of global climate change. According to reports from the Intergovernmental Panel on Climate Change on the World Wide Web at ipcc.ch, in the European heat wave of 2003, crop production was reduced by around 30%. Globally climate change affects more than 70% of all agriculture. Annually Indian agriculture suffers a loss of US$ 42.66 mil 17.5?% of this is due to insect and pests and climate change related abiotic stress.
Productivity is decelerating & the highest yields can only be obtained by using ever increasing amounts of inputs. Prolonged exposure to abiotic stresses results in greater susceptibility to biotic stresses such as pathogens and pests. It also has entailed increased building up of pest/diseases with higher resistance further reducing crop yield. These practices results in ever escalating input costs and produce with higher residues which are subjected to import restrictions internationally for failing to comply with safety norms. 30 % of total consumption of insecticides/pesticides fall under the extremely hazardous toxic Class I insecticides/pesticides. Many of those affected by this are smallholder farmers that make up the majority of the farming population. Farmers are also unaware of the environment friendly bio-pesticides. A sustainable commercially viable, effective alternative could prove to be a game changer for Indian Agriculture.
Recent trend to move away from heavy use of chemicals in agriculture has boosted use of organic products, however their quality and efficiency remains a bottle neck for organic agriculture. Modern technologies like Nano biotechnology, Bioactives and bio simulants etc applied to agricultural production could play a fundamental role to reduce applications of plant protection products, minimize nutrient losses in fertilization, increase yields through optimized nutrient management and the amount of chemicals released into the environment. Large scale production, economic viability and ease in usage have hindered implementation of these technologies by farmers, especially from developing nations like India. A cheaper, easier to use environmentally safe alternative is the requirement of the hour in the agriculture, horticulture, floriculture sectors in India.

Technical innovation in agriculture is of extreme importance, in particular to address global challenges such as population growth, climate change and the limited availability of important plant nutrients.
Traditionally, plant breeding is used to generate better adapted varieties to abiotic stresses. Success of breeding has vastly improved by incorporating molecular biology and genetics. Molecular biology has led to deeper understanding of genes responsible for adaptation and broadened our knowledge and understanding of the underlying mechanism. However breeding still remains a cumbersome and lengthy process. Regulatory hurdles in many countries do not permit or restrict the use of GMO’s in agriculture, limiting the scope of generating newer varieties tolerant to abiotic stresses.
Another approach used to overcome is applying various agrochemicals to crops to relieve stress. The largest share of agricultural biological today is of traditional microbial based bio stimulants or bio pesticides. These require optimum conditions and time to achieve the desired effect. Heavy residues of chemicals in the soils affect the microbial survival and performance; farmers therefore do not get the desired effect. Besides, to obtain the best results farmers are required to change their current agronomic practices. Shifting to sustainable alternatives thus becomes even more difficult for the farmers. 60% of all biostimulants sold today are simple formulations of either humic/fulvic acid derivatives or sea weed extracts.
Organic/residue free food market in India is slated to reach US $ 871 Million by 2021, these changing market trends means that famers need to adopt to organic or residue free food production. However farmers have to choose between fast acting precise chemical formulations and slow acting but safer more sustainable biological formulation.
Today farmers need a safer, rapid acting target specific formulation compatible with all forms or agriculture – be it chemical, organic, residue free or modern techniques like precision agriculture, hydroponics/aquaponics etc. A sustainable commercially viable, effective alternative could prove to be a game changer for Indian Agriculture.

Breeding is long and cumbersome process. In addition abiotic stress is mitigated through several pathways and is not a monogenic trait. Identifying and pyramiding all genes in a single cultivar is a problem. Varieties developed are usually region specific and do not work well in other agroclimatic zones. Not many products are available that are useful to elevate stress, increase resistance of plants; boosts root development and also increase plant growth, health and produce.
Chemical additives are environmentally not safe, leave traces, lead to bioaccumulation. They lead to scorching of leaves, and leave residual spots on plants that decreases plant value commercially. Currently available organic additives are mostly living organism based and cannot be combined with chemical fertigation/pesticide application as that causes death of the beneficial organisms.
PRIOR ART
WO2016100624A1 discloses compositions and methods for improving yield, yield stability, and/or drought stress tolerance in plants. Plants and/or plant parts identified, selected and/or produced using compositions and methods of the present invention are also provided.
US20090156404A1 is directed to methods and compositions for improving growth or yield of plants by reducing stress from abiotic factors without adversely affecting photosynthesis. In one embodiment, the compositions include particulate material and one or more plant growth regulating compounds such as non-gaseous plant hormones, amino acids and amino acid derivatives, and terpenes, and mixtures thereof. The composition is applied to at least part of the surface of the plant, forming a film on the plant. The effects of abiotic factors such as heat, cold, light, and water stress may be reduced or eliminated after application of the composition, and growth or yield may be increased while photosynthesis is not adversely affected.
Taking into consideration all the above factors, it is implied that there is a need of an hour to develop a sustainable commercially viable, effective, easier to use at the same time environmentally safe alternative that could prove to be a game changer for Agriculture. WO2010098337A1
OBJECT OF THE INVENTION:
The primary object of the present invention is to provide a bio-formulation to increase tolerance of the plants to abiotic stress that will result in improved yield.
Further object of the present invention is to provide a bio-formulation to promote plant growth, root development, cell wall strengthening and priming the plant to deal with biotic and abiotic stress.
Another object of the present invention is to provide a bio-formulation to minimize the contamination of the environment at large and help to support the regeneration of the ecosystem.

SUMMARY:

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 the particular embodiments only and is not intended to limit the scope of the present invention.
The present invention describes a novel bio-formulation comprising of carbon nanoparticles and carbon quantum dots in conjunction of specific naturally occurring bioactive substances and methods of employing these substances to induce abiotic stress tolerance in plants. The bio-formulation promotes plant growth, root development, cell wall strengthening and priming the plant to deal with biotic and abiotic stress. This enables plants to get more nutrients and water, thus making them healthy and able to sustain water shock, intermittent periods of drought, transplantation shock and/or transpiration loss. The bio-formulation induces responses in plants, such as an increase in osmotic adjustment (proline content), cell wall strengthening, and stress protein synthesis resulting in increases in crop yield, relative to plants treated with fertilizer only. The bio-formulation employees carbon nanoparticles and carbon dots of the particle size 162-216nm which modify the plants’ physiological response, flavonoids that help in better uptake of nutrients resulting in signalling heat and temperature stress, sphingolipid responsible for improved tolerance of plants to biotic and abiotic stress. The bio-formulation also contains carboxylic acid and terpenes. For experimental purposes, a number of bio-formulations comprising the aforementioned components are formulated and their respective effect was tested on various stages of plants and various methods of plant propagation. They were tested for their effect on seed germination/establishment of cuttings/hardening of plants (tissue culture or from Green house/polyhouse to shade net). The plants tested include chilli, tomatoes, eggplant, cabbage, cauliflower. Cuttings such as marigold, ixora, pentas, poinsettia, chrysanthemum, hibiscus, rose were also taken into consideration. The effect of the best bio-formulation is also tested in field. Plants tested in field include tomato and rice, with tomato being an exemplary representative of dicots and rice being a representative of monocots. Hence, the bio-formulation of the present invention may also be further used for a wide range of other plants and is not limited tomato and rice. The novel bio-formulation of the present invention may further be used typically in any physical form such as a liquid formulation or in the form of granular composition or a foliar formulation.
BRIEF DESCRIPTION OF DRAWINGS:
FIG.1 illustrates the treatment schedule of the novel bio-formulation at different stages of plant development.
FIG. 2 illustrates water stress recovery study format.
FIG. 3 illustrates the effect of bio-formulation on tomato plants after transplantation.
FIG.4 illustrates the effect of bio-formulation on vegetative growth of tomato plants.
FIG.5 illustrates the water stress and recovery response for Formulation 1.
FIG. 6 illustrates the water stress and recovery response for Formulation 1 (granular).
FIG.7 illustrates the water stress and recovery response for Formulation 2.
FIG.8 illustrates the water stress and recovery response for Formulation 3.
FIG.9 illustrates the water stress and recovery response for Commercial seaweed1.
FIG.10 illustrates the water stress and recovery response for Commercial seaweed2.
FIG. 11 illustrates the leaf rolling index for Formulation 1.
FIG. 12 illustrates the leaf rolling index for Formulation 1 (granular).
FIG. 13 illustrates the leaf rolling index for Formulation 2.
FIG. 14 illustrates the leaf rolling index for Formulation 3
FIG. 15 illustrates the leaf rolling index for Commercial Biostimulant 1.
FIG. 16 illustrates the leaf rolling index for Commercial Biostimulant 2.
FIG. 17 illustrates the leaf rolling in tomato plants under high stress conditions on day 8 of water withdrawal.
FIG.18 illustrates the increased fruit settings in tomato plant after treatment with
Formulation 3.
FIG.19 illustrates the effect of granular form of the bio-formulation on establishment of plants after 10 and 20days of transplantation.
FIG.20 illustrates the effect of bio-formulation on spikelet formulation in rice.
FIG.21 illustrates the liquid chromatographic analysis of Formulation 3.
FIG.22 illustrates the mass spectra of Formulation 3.
TABLE. 1 illustrates the components of the bio-formulations used in the present invention.
TABLE. 2 illustrates the alternative compounds of the proposed bio-formulations.
TABLE. 3 illustrates the composition of the different formulations used for the study.
TABLE. 4 illustrates the treatment of the formulations.
TABLE. 5 illustrates the effect of the bioformulations at the seed germination stage.
TABLE. 6 illustrates the physiological parameters on Day 8 after withdrawal of water in tomato plant under high stress conditions.
TABLE. 7 illustrates the antioxidant levels on Day 8 after withdrawal of water in tomato plant under high stress conditions.
TABLE. 8 illustrates the effect of Formulation 3 on vegetative growth of tomato plants at field conditions 60 DAT.
TABLE. 9 illustrates the effect of Formulation 3 on vegetative growth of tomato plants at field conditions 60 DAT.
TABLE. 10 illustrates the effect of the bio-formulations on growth of rice plants 50 DAT.
TABLE. 11 illustrates the effect of the bio-formulations on panicle formation in rice.

DETAILED DESCRIPTION OF THE INVENTION:

Before the present invention is described, it is to be understood that this invention is not limited to particular 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 “comprises”, 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.

The present invention discloses novel bio-formulations of carbon nanoparticles and carbon quantum dots in conjunction with specific naturally occurring bioactive substances and methods of employing these substances to induce abiotic stress tolerance in plants. The said bio-formulations comprise of the ingredients described in TABLE. 1.

Chemical Class Active ingredient Role Function Formulation range
Carbon Nanoparticles, quantum dots.
Particle size 162 nm- 216 nm -Modify physiological responses -Abiotic stress, growth 0.01-400 mg/L
Flavonoid Luteolin, Luteolin 6- glycoside -Act as a specific signal for Rhizobium bacteria to initiate symbiosis
-Protect plants against UV/temperature damage -Better uptake of nutrients
-Stress-heat stress tolerance 0.01-10 %
Sphingolipid Icosanamide, dihydrosphingosine, Sphinganine, ceramide
-Improved tolerance of plants to biotic and abiotic stresses.
-Pollen development, signal transduction and in the response to biotic and abiotic stress.
-ABA-dependent stomata closure and the response to drought. -Cell wall modification
-Heat and temperature stress signalling 0.01-10 %
Carboxylic acid Propionic acids, ß-hydroxypropionate -Converted into malate, citrate, aspartate, and glutamate. Ready energy 0.01-10 %
Tricarboxylic acids- citric acids -Ready energy
-Stress 0.01-15 %
Terpenes Monoterpenes- Aucubin iridoid glycoside. defensive compounds- anti feedeants Defence priming 0.01-10 %
sesquiterpenoids Juvenile hormone II, Punctaporin -Interfere with the insect endocrine system involved in the regulation of developmental processes such as metamorphosis and reproduction in most insect species. 0.01-10 %
oleanolic acid isomer, usolic acid- triterpene aglycones of saponins -Epicuticular waxes where they act as a barrier against pathogens and water.
-Affects Surface signaling in pathogenesis potent anti-oxidant. Cuticle modification and protection from pathogens 0.01-5%
12 hydroxy 10 dodecenoic acid/ Traumatin -Plant hormone produced in response to wound. Wound healing 0.01-2 %

TABLE. 1

TABLE. 2 provides a list of alternative compounds that may be used in the bio-formulations instead of or in addition to the ones provided in TABLE. 1.

Chemical Class Active ingredient Alternate compounds
Carbon Nanoparticles, quantum dots
Particle size 162 nm- 216 nm -
Flavonoid Luteolin, Luteolin 6- glycoside Apigenin, Naringenin, Eriodictyol.
Daidzein, Genistein, Hesperetin, Kaempferol Rutin
Sphingolipid Icosanamide, dihydrosphingosine, Sphinganine, ceramide
long-chainorsphingoidbase- phytosphingosine sphinganine, sphingosine-1-phosphate, dihydrosphingosine,
ceramides - phytoceramide inositol-phosphorylceramides, glucosylceramides
Phospholipids,includingphosphatidicacid(PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphoinositides
carboxylic acid Propionic acids, ß-hydroxypropionate Butanoic acid
Tricarboxylic acids- citric acids Isocitric acid, succinic acid (SA), fumaric acid (FmA), and malic acid (MA).
Terpenes Monoterpenes- Aucubin iridoid glycoside. Catalpol 8-oxogeranial, ocimene, myrcenes, geraniol, citral, citronellal, citronellol, linalool, menthol, limonene, carvone

sesquiterpenoids Juvenile hormone II, Punctaporin Iso Caryophyllene, Longifolene, a-Cuparenone, Zingiberene, Caryophyllene, Geosmin,

oleanolic acid isomer, usolic acid- triterpene aglycones of saponins Ursolic acid, Betulinic acid, Moronic acid
12 hydroxy 10 dodecenoic acid/ Traumatin Traumatic acid, dodecenoic acid, traumatates.

TABLE. 2

The present invention is based on using carbon nanoparticles and quantum dots, and specific bioactive molecules that modulate physiological pathways in plants to induce stress tolerance, individually or in combination. The mechanism of action is by biochemical modulation of plant internal processes. Carbon quantum dots or CQDs (less than 10 nm in size) possess the attractive properties of high stability, good conductivity, low toxicity, good biocompatibility, environmental friendliness and simple synthetic routes. In addition, unlike carbon and carbon nanoparticles, CQDs have high water solubility. CQDs can be prepared by any methods described in literature. In the present invention, the CQDs are synthesized from natural inexpensive material such as coal, lignite etc. The detailed synthesis procedure is known and described. Additionally CQDs can also be synthesized by using Citric acid as a carbon source using hydro-thermal method. CQDs with various concentrations (0 to 400mg/L) are tested for abiotic stress tolerance in the present invention. CQDs are used individually in the formulation as CQDs being the sole component or in combination with metabolites, amino acids, NPK, micro nutrients, hormones, bio stimulants etc. TABLE. 3 describes the different bio-fomulations namely Formulation-1, Formulation-2 and Formulation-3 of the present invention wherein, the bio-formulations promote plant growth, root development, cell wall strengthening and priming the plant to deal with biotic and abiotic stress. The formulations increase and accelerate plant seed germination. They also increase the root formation and number resulting in treated plants having bigger root ball with increased number of tertiary roots with root hairs. This enables plants to get more nutrients and water, thus making them healthy and able to sustain water shock, intermittent periods of drought, transplantation shock or transpiration loss.

Chemical Class Active ingredient Formulation 1 Formulation 2 Formulation 3
Carbon Nanoparticles, quantum dots
Particle size 162 nm- 216 nm 0-400 mg/L - 0.01-400 mg/L
flavonoid Luteolin, Luteolin 6- glycoside - 0-10 % 0.01-10 %
Sphingolipid Icosanamide, dihydrosphingosine, Sphinganine, ceramide
- 0-10 % 0.01-10 %
carboxylic acid Propionic acids, ß-hydroxypropionate - 0-10 % 0.01-10 %
Tricarboxylic acids- citric acids - 0-15 % 0.01-15 %
Terpenes Monoterpenes- Aucubin iridoid glycoside. - 0-10 % 0.01-10 %
sesquiterpenoids Juvenile hormone II, Punctaporin - 0-10 % 0.01-10 %
oleanolic acid isomer, usolic acid- triterpene aglycones of saponins - 0-5% 0.01-5%
12 hydroxy 10 dodecenoic acid/ Traumatin - 0-2 % 0.01-2 %

TABLE. 3

The bio-formulations of the present invention induce responses in plants, such as an increase in osmotic adjustment (proline content), cell wall strengthening, stress protein synthesis and consequential and accompanying increases in crop yield, relative to plants treated with fertilizer only. This confers on treated plants an increase in resistance to drought, frost, salinity, flood, heat, wind and ultraviolet light exposure. The bio-formulation treated plants have higher chlorophyll A & B, sugars and protein, an increase in the quality of crop and yield.

The bio-formulations’ effect was tested on various stages of plants and various methods of plant propagation. The bio-formulations were tested for their effect on seed germination/establishment of cuttings/hardening of plants (tissue culture or from Green house/polyhouse to shade net). Plants tested include chilli, tomatoes, egg plan, cabbage, cauliflower and cuttings such as marigold, ixora, pentas, poinsettia, chrysanthemum, hibiscus, rose.

The effect of the bio-formulations of the present invention on the abiotic stress tolerance of the tested plants was also checked by carrying out different kind of analyses such as phenotypic measurements, image analysis, biomass measurement, leaf rolling, biochemical parameters analysis and antioxidant enzyme assay. The methodology and results of these experiments is described elsewhere in the description.

The effect of the best formulation of the present invention was also tested on field. Plants tested include tomato and rice with tomato being a representative of dicots and rice being a representative of monocots. It is to be noted that tomato and rice are simply exemplary representatives of the two said plant groups and that the bio-formulations of the present invention may also be further used for a wide range of other plants and hence are not limited to tomato and rice. The effect of method of administration of the formulation is evaluated by either soaking/dipping, drenching or spraying.

FIG.1 illustrates the treatment schedule of the novel bio-formulations at different stages of plant development. Effect of the novel bio-formulations of the present invention can be better understood from the following experiments carried out in different plants.
A. Effect of different bio-formulations on growth and stress tolerance in plants:
The present invention relates to bio-formulations that provide natural active ingredients to plants for development of better health, growth and produce. These bio formulations when applied to plants or to the rhizosphere are to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, and tolerance to abiotic stress, crop quality and yield. More particularly natural active ingredients of the bio-formulation do not contaminate the environment at large and helps to support the regeneration of the ecosystem. Several formulations were prepared comprising plant extracts in different combinations and concentrations.

Example 1:
An experiment was conducted to study comparative efficacy of various bio-formulations in commercially important crops like Tomato, at seedling stage and preliminary study at field stage.

Material and Methods:
1. Seedling stage experiment:
Best selling Tomato hybrid variety in local market Syngenta var. 1057, was selected for the experiment. Seeds were sown in protrays filled with cocopit mixture and maintained in a nursery. The trays were placed in the greenhouse with controlled conditions: average temperature 26–28°C, relative air humidity 50–60%, photoperiod 12/12 hours. Various bio-formulations (Formulation 1 (liquid and granular form), Formulation 2, Formulation 3, Commercial Biostimulant 1 and Commercial Biostimulant 2) were applied (1 ml/L) after 5 days of seed sowing. To evaluate the hormone like effect hormones like IAA and BAP were also applied. Seedlings were allowed to germinate and observations like seed germination, stem length, root number root length, root dry biomass were recorded after 30 days of seed sowing. Control seeds didn’t receive any treatment.
To study effect of bio-formulations after transplantation, seedlings were drenched with all the bio-formulations at concentration 1 ml/L, maintained in nursery for next 2 days and transplanted in field.
Effect of the best performing formulation was tested on field during summer season from March to June, 2018 in field area of 1 acre. Readings were recorded after 90 days of transplantation. Best formulation was applied to plants through drip system with 1 ml/L concentration after 10 and 25 days after transplantation. All experiments were performed with 10 plants per treatment and average of ten values is presented here.
2. Abiotic stress studies:
The study was conducted in Rabi season of 2020 in a Green house at Narayangaon, Dist Pune, Maharashtra, India. Tomato plants (VIRANG, F1 Hybrid, Seminis, Monsanto) were grown in a greenhouse under natural ambient light conditions. 28 day old tomato seedlings with approximately 15-20cm height, 4-5 well developed leaves and profuse root system were selected for transplantation. They were transplanted in polybags (3 L capacity) filled with mixture of Red soil: organic matter: sand (4:2:1 proportion). Plants were given fertigation once a week throughout experiment except drought period. The fertigation was as follows: NPK (19:19:19) + MgSo4 + NPK (13:0:45) at concentration 1gm/L. Temperature at the time of experiment was recorded to be maximum 38°C and minimum 15°C, and humidity was around 40-70%.
Application of biostimulants were carried out at 5 days after transplantation (DAT) and 15 DAT. Application stages are illustrated in a pictorial form in FIG. 2. Drought stress was induced 20 DAT and continued till 30 DAT. The control plants were irrigated with every day both before and during the drought stress experiment. Drought stress was imposed by withholding irrigation. For those plants subjected to drought stress, irrigation was completely suspended until 30DAT (at day 10 of drought stress) when water was re- administered. It was determined from previous experiments that plants do not recover from a water stress beyond 10 days. Therefore for these experiments water stress was studied for 10 days. Watering was resumed at 30 DAT and recovery from drought was studied till 38 DAT. Formulations treatment was performed on Day 5 and 15, by drenching the desired concentration of the bio-formulations described in TABLE. 3 over the leaves, using an agricultural pressure sprayer. The experimental scheme made use of 13 replica plants for each test and was completely randomized.
Plants used for sampling leaves were excluded from subsequent imaging or additional leaf sampling. Leaf samples were taken on the same days as imaging. The pre-drought samples were used to establish a baseline of the plants. All samples were taken at the same time of day (9 am), to minimize variation.
a. Phenotypic measurements of the plants were carried out non-destructively by plant imaging. Images of plants were taken over the course of the experiment. RGB images were used to evaluate the state of health of the plant via colour classification (i.e. green healthy tissue, yellow chlorotic tissues, and brown necrotic tissue), as well as for morphometric measurements, such as digital biovolume and height.

b. Image analysis was done using Image J software. In order to extract phenotypic measurements from the images, the plant had to be separated from the background. This separation was carried out either by converting the image colour space to HSI (hue, saturation and intensity) or using the intensity channel. This grey-scale image was then subjected to thresholding to create a binary mask to extract the plant from the image. The measurement of digital biomass, correlated with fresh weight.

Sr. No Treatment Form Concentration
1 Control
2 Formulation 1 Liquid 1 ml/L
3 Formulation 1 (granular) Granular 100 mg/seed
4 Formulation 2 Liquid 2 ml/L
5 Formulation 3 Liquid 1 ml/L
6 Commercial biostimulat 1 Liquid 1 ml/L
7 Commercial biostimulat 2 Liquid 1 ml/L
(Commercial biostimulant formulation usually comprising of a complex of vitamins, aminoacids, proteins and betaines, trace elements, natural growth promoting compounds)
TABLE. 4

c. Biomass measurement: Growth was measured in terms of fresh weight and dry weight during drought period and after resumption of watering. 3 plants were harvested at regular interval. Soil were removed from root zone and fresh weight was recorded. Plants were dried in hot air oven at 60°C to get constant dry weight.

d. Leaf rolling: The relationship between leaf rolling and physiological traits under imposed water stress conditions was analysed. Leaf rolling analysis was studied at during drought period and after resumption of watering. For leaf roll analysis, 3 and 4th mature leaf above cotyledons were selected and used for taking measurement throughout experiment. Leaf rolling was calculated by using formula:

LR=[(LW-LN)/LW]x100
Where,
LW: maximum leaf blade
LN: natural distance of leaf distance margin

e. Biochemical parameters: All assays were carried out on 8th day of drought condition. Plants watered throughout and drought exposed but without Biostimulant treatment were considered as a control. Various physiological parameters were studied post recovery from drought stress. Chlorophyll pigment was determined by extracting 200 mg of fresh leaves in acetone (80%) and measured spectrophotometrically at 663 and 645 nm expressed as mg/g of fresh weight. Sucrose and Glucose were analyzed from leaf tissue according to method described by Zhang et. al., (2006). Proline content was determined by method described by Bates et. Al., (1973). All experiments were replicated in triplicates.

f. Antioxidant enzyme assay: For antioxidant enzyme activity, frozen leaf samples (50 gm) were ground to fine powder using liquid nitrogen and extracted in 1.8 ml of Ice cold 50mM sodium phosphate buffer (pH 7.0) containing 0.2 mM EDTA and 1%PVP. The homogenate was centrifuged at 12000 g for 20 min at 4°C. Supernatant was immediately used for antioxidant enzyme activity. Catalase (CAT) was determined by using method described by Chance and Maehly (1955). Ascorbate peroxidase (APX) was determined by using method described by Zhang et. al., (2015). All experiments were replicated in triplicates.

3. Field analysis:
Example 1: Tomato
Tomato plants after field transplantation were analysed for vegetative and reproductive characteristics. Morphological observations were recorded on plant height, number of branches, shoot fresh weight, shoot dry weight, Chlorophyll content, days taken for onset of flowering, flower drop (%), total yield per plant and estimated yield per acre. Incidence of pest attack was also recorded by observing infection of leaf minor on leaves of tomato.
a) Sampling and analysis method: Composite leaves (leaf plate without the petiole) were sampled from the second, third and fourth fruit-bearing branch in order to get homogenous sample. Chlorophyll content was estimated as per Yoshida et al (1971).

b) Determination of total yield: For total fruit yield determination, all fruits were sampled from fruit-bearing branches and their mass was measured using technical balance and results are presented as gram/plant. All experiments were performed with 10 plants per treatment and average of ten values is presented here.

Example 2: Rice
Local Rice variety ‘Indrayani’ was selected for the experiment. Study was conducted in Kharif season 2018. Seeds were sown on bed and transplanted after 35 days of seed sowing. Climatic conditions at the time of experiment were, temperature between max 30°C during day time and minimum 19°C during night, relative air humidity 70–80%, photoperiod approximately 12/12 hours. Granular formulations were used at 6 kg per acre concentration after 20 and 60 days of transplantation. Approx 1 acre of area was used for all treatments.
Plants were harvested at 50 days of plantation to assess the effect of formulations on root formation, tillering and plant growth.
a) Growth parameters: The height of ten randomly labeled plants was recorded at 50 DAT. The height was recorded by measuring from the base to its growing tip and the mean value was worked out. Root system of harvested plants was washed thoroughly with water. Length of roots was recorded in similar fashion. Number of tillers was calculated separately from each treatment. All experiments were performed with 10 plants per treatment and average of ten values is presented here.

b) Determination of total yield: After second application of granular formulations plants were harvested at maturity. Number of panicles, length of panicles, and weight of 5 panicles were calculated in control and treated plants.
For total yield determination, all panicles were sampled from treated and control plants and their mass was measured using technical balance and presented as a total yield per acre in tones. All experiments were performed with 10 plants per treatment and average of ten values is presented here.
Results and discussion:
1. Seed germination:
The present study revealed that among different bio-formulations, formulation number 3 improved seed germination, early emergence, faster development of roots thereby favoured vigorous growth of shoots and roots compared to control. Leaves of treated plants were observed to be green thick and broader. Stem height and girth was also observed to be higher in most of the formulations compared to control as illustrated in FIG. 3. Such well grown plants treated with various formulations showed highest survival % post transplantation as illustrated in FIG. 4 The results can be better understood from the TABLE. 5.

Formulation Seed Germination (%) Stem length
(cm) Root number Root length
(cm) Root dry mass (gm/plant) Transplantation
Shock Survival
(%)
Control 62 ± 4 15.2 ± 1 20.3 ± 2 9 ± 0.7 0.014±0.003 78 ± 3
1 82 ± 3 14.3 ± 2 18.6 ± 2 8 ± 1.5 0.016±0.003 70 ±7
2 75 ± 2 14.5 ± 2 27.8 ± 1 9.4 ± 0.9 0.019±0.002 78 ± 5
3 96 ± 2 17.8 ± 1 29.1 ± 3 10 ± 1.2 0.02±0.005 92 ± 2
Commercial biostimulant 1 70 ± 5 16.2 ± 1 24.3 ± 2 8.5 ± 0.5 0.019±0.002 80 ± 6
Commercial biostimulant 2 73± 7 17.2± 2 25.4± 5 9.3± 1.3 0.018±0.004 85± 7
TABLE. 5
2. Abiotic stress studies:

a. Biomass changes in response to drought stress:
Changes in biomass for different treatment is represented in FIG.5 to FIG. 10. Plants with no stress served as a baseline for all compressions. Untreated plants under water deficit conditions severed as a baseline for drought stress. Plants treated with formulations served as an estimation of effect of formulation in optimum conditions. By comparing treated plants in stress against untreated plants without water deficit and with water deficit an estimation of stress mitigation ability of the formulations were made.

It was seen that untreated plants start experiencing drought effects from day 2 as shown in FIG. 5 after withdrawal of watering. Plants treated with Formulation 1 as shown in FIG. 5 and FIG. 6 were able to delay the onset by 4 days either in foliar or granular form. Formulation 2 was not effective in delaying the stress impact on plants as can be seen in FIG. 7. Formulation 3 was able to delay the onset of stress in plants for upto 6 days after water withdrawal as can be seen in FIG. 8, thereby reducing the stress window considerably. Commercial seaweed treated plants were not able to delay the onset atall as can be seen in FIG. 9 and FIG. 10. Stress related reduction in growth was observed as early as day 1 in all these.
In mid stressed conditions the untreated plants showed 40 % reduction in biomass, showing significant impact of growth due to stress. Formulation 1 was able to mitigate stress and effect on biomass was 24.4 % as can be seen in FIG. 5 and FIG. 6 where as Formulation 3 showed only moderate decrease of 14 % and maximum stress mitigation as can be seen in FIG. 8. Formulation 2 or commercially available seaweed based biostimulants under normal conditions were seen to boost growth and biomass of plants as can be seen in FIG. 7, FIG. 9 and FIG. 10 respectively. However, though they are used for stress mitigation, their effect on reducing stress impact and in recovery of plants was clearly not effective as formulation 3. Commercial seaweed formulations treated plants showed reduction on biomass by 41 % and 20 % as can be seen in FIG. 9 and FIG. 10, which was comparable to Formulation 2 with reduction of 40 % as can be seen in FIG. 7.
Once watering was resumed on day 10 of water withdrawal the recovery of plants in untreated and treated were compared (day 12). Though plants recovered after watering in all treatments, the degree of recovery was different. Untreated plants still had 16 % less biomass as compared to untreated plants which had received water throughout. Plants treated with Formulation 1 as shown in FIG. 5 and FIG. 6, recovered only 40% however plants treated with Formulation 3 as shown in FIG. 8 recovered almost totally with a difference of only 3.4 %. Plants treated with Formulation2 as shown in FIG. 7 showed 14 % less biomass however commercially available formulations showed 24 % and 20 % less biomass levels, indicating that only seaweed is not sufficient for plants to recover from water stress fully and restore normal growth.
This also clearly showed that it was a combination of QD and the actives (formulation 3) that showed all desired characters- delay on set of stress, reduce the impact of stress at highest stress points and enable plants to recover fully from water stress and attain normal growth.
b. Leaf rolling:
The relationship between leaf rolling and physiological traits under imposed water stress conditions was analysed and the results obtained were as illustrated in FIG. 11-17. Leaf rolling reflected the physiological changes occurring during water stress and is associated to an increased sub-stomatal CO2 concentration (Ci) and a decreased carboxylation efficiency. Moreover might be involved in protecting the PSII complex under water stress during the progressive inhibition of photosynthetic metabolism.

Under mid stress conditions it was seen that lesser degree of early leaf rolling confers drought resistance. Untreated plants under stress showed significant leaf rolling on day 6 after withdrawal of water. Though earlier data shows that untested plant start experiencing stress as early as day 2. Lesser degree of early leaf rolling allows plants to reduce the impact of stress and maintain water levels in the plants while still continuing photosynthesis. Leaf rolling at 7-8% in formulation 1 treated plants was not significantly better than untreated stressed plants as can be inferred from FIG. 11 and FIG. 12. Formulation 2 treated plants too were just marginally better with 11% rolling index as illustrated in FIG. 13. Plants treated with formulation 3 as illustrated in FIG. 14 showed early leaf rolling (from day 3) of lower degree (lesser than 20-30 %). Commercial Biostimulant formulation 1 as illustrated in FIG. 15 was near identical to untreated control and formulation 2 as illustrated in FIG. 16 showed early leaf rolling of 35%.

It seemed Formulation 1 was able to mitigate stress through spontaneous response of leaf rolling to environmental conditions for maximizing carbon gain by increasing incident photosynthetic photon flux density (PPFD) or minimizes incident radiation, resulting in more favourable leaf temperatures and water status during drought as can be seen in FIG. 17.

Capony area was studied by computer based image analysis indicated that treated and untreated plants show similar trend under stress conditions, except in all treated plants canopy area recover was faster than that of control (Data not shown here). It is therefore interesting to note that at high stress condition drought tolerance was linked to the % that the leaf could remain open as opposed to 100 % rolled in untreated stressed plant. Plants treated with formulation 3 as illustrated in FIG. 14were able to retain the leaf open by 36 % and formulation 1 by 53% as can be seen in FIG. 11 and FIG. 12. Formulation 2 as illustrated in FIG. 13 and commercial seaweeds were able to keep leaves open by percentage between 15- 30 % as can be seen in FIG. 15 and FIG. 16 which is just marginally better. It was observed that not how open or how closed the leaves are, that determines tolerance but the optimum level of openness as per the stress conditions, thereby protecting and allowing photosynthesis to continue as can be seen in FIG. 17.

c. Biochemical parameters:
Chlorophyll content is directly related with the photosynthetic efficiency of the plants in stress conditions. Chlorophyll concentration has been known as an index for evaluation of source, therefore decrease of this can be consideration as a nonstomatal limiting factor in the drought stress conditions. Plants treated with formulation 3 showed 46% higher chlorophyll content as compared to the untreated plants in water stress. Plants treated with formulation 1 were not that was effective and showed 15 % increase while those treated with formulation 2 showed increase by 35 % as described in TABLE. 6.

Salinity and drought are the major osmotic stress limitations that affect plant growth and crop yield Overproduction of various compatible organic solutes is one of the most common stress responses of plants to environmental stresses. Proline accumulation is a well-known metabolic response of plants to drought and other stresses. Proline permits osmotic adjustment, stabilizes the structure of proteins and cell membranes, acts as a protective agent for enzymes, and is a free radical scavenger and antioxidant. At day 8 when plants were under high stress amount of proline was analysed and the results are provided in TABLE. 6. It was seen that plants treated with plants treated with formulation 3 had 57 % higher proline accumulation than untreated stressed plants. Formulation 1 treated plants showed an increase by 14 % only. Plants treated with formulation 2 showed an increase in proline accumulation by 35 %. Higher proline content shows ability of plants for osmoregulation in stress conditions.

Soluble sugars play an important role in maintaining the overall structure and growth of plants suggested that soluble sugar regulation in plants was a very complex manner. Soluble sugar maintains the leaf water content and osmotic adjustment of plants facing the conditions of drought stress. The released sugars and other derived metabolites support plant growth under stress, and function as osmoprotectants and compatible solutes to mitigate the negative effect of the stress. Other than osmoregulation, soluble sugars also act as signalling molecules to modulate the sensitivity of plants and thus help in cell responses. Plants treated with formulation 3 showed marked increase in osmolytes accumulations as indicated by 80 % higher levels of glucose and 77 % higher levels of sucrose as compared to untreated plants under water stress as can be seen in TABLE. 6. Plants treated with formulation 1 also showed increase in accumulation of soluble sugars although (12.5 % of glucose and 32 % of sucrose) not as effective as formulation 1. Formulation 2 and commercial seaweed formulations also led to increase in soluble sugar content with 30 % higher glucose and 56 % higher sucrose accumulation as compared to untreated plants in stress as shown in TABLE. 6.

It seems that seaweed induced stress tolerance may be mitigated though osmoregulation (accumulation of proline, glucose and sucrose) rather than through stomatal conductance, leaf rolling or canopy architecture changes as discussed above. Whereas formulation 3 and Formulation 1 were able to induce and mitigate stress using both these approaches.

Sr. No Treatments Chlorophyll content (mg/g FW) Proline (µg/g of FW) Glucose (mg/g DW) Sucrose (mg/g DW)


1 Control (W) 1.55 ± 0.10 98.5 ± 4.1 11.6 ± 2.2 4.1 ± 2
2 Control (D) 1.26 ± 0.08 140.3 ± 7.45 15.2 ± 3.1 8.7 ± 1.5
3 Formulation 1 1.45 ± 0.011 160.3 ± 5.1 17.1 ± 1.8 11.5 ± 2.5
4 Formulation 1 (granular) 1.5 ± 0.05 175 ± 4 13.5 ± 2 10.3 ±1.2
5 Formulation 2 1.71 ± 0.04 195.2 + 8 19.8 ± 1.4 13.6 ±1.9
6 Formulation 3 1.85 ± 0.06 220.7 ± 10 22.1 ± 2.5 15.4 ± 2
7 Commercial seaweed Formulation 1 1.61 ± 0.03 189.2 + 6 17.8 ± 1.1 12.1 ±1.5
8 Commercial seaweedFormulation 2 1.76 ± 0.04 186.2 + 4 18.1 ± 1.1 11.8 ±1.7
*Legend: Control is untreated plants, CW are plants that are watered normally throughout the experiment. CD are plants whose water is withheld for 10 days of the experiment.
All formulations represent plants treated with designated formulations whose water is withheld for 10 days of the experiment.
TABLE. 6
d. Antioxidant activity:
Oxidative stress is crucial in relation to drought-induced injuries in plants. Drought stress exacerbates ROS production in plant cells. Excess production and the accumulation of ROS causes oxidative damage at cellular level, disrupts cellular membranes, and leads to enzyme inactivation, protein degradation, and ionic imbalance in plants. Mechanism of ROS production and its scavenging by high antioxidative capacity has been associated with tolerance of plants to abiotic stresses. At low concentrations, H2O2 acts as a signal molecule involved in the regulation of specific biological/physiological processes (photosynthetic functions, cell cycle, growth and development, plant responses to biotic and abiotic stresses). Oxidative stress and eventual cell death in plants can be caused by excess H2O2 accumulation. Since stress factors provoke enhanced production of H2O2 in plants, severe damage to biomolecules can be possible due to elevated and non-metabolized cellular H2O2. Plants are endowed with H2O2-metabolizing enzymes such as catalases (CAT), ascorbate peroxidases (APX). In particular, APX has a higher affinity for H2O2 and reduces it to H2O in chloroplasts, cytosol, mitochondria and peroxisomes, as well as in the apoplastic space, utilizing ascorbate as specific electron donor. APX and CAT are able to scavenge H2O2 with different mechanisms. Specifically, APX, contrary to CAT, requires an ascorbate and glutathione (GSH) regeneration system, the ascorbate-glutathione cycle. Instead, CAT directly converts H2O2 into H2O and 1/2 O2 and, on the contrary of APX, it is more involved in detoxification of H2O2 than the regulation as a signaling molecule in plants. APX levels were significantly increased in plants treated with formulation 3 (40%) and formulation 2 (38%) and seaweed treated plants as compared to untreated plants under water stress as shown in TABLE. 7. Formulation 1 treated plants did not show elevation in APX levels and was infact slightly lower (4%). This indicates that the actives in formulation 2 and 3 act as signalling molecules that in turn activate APX mediated signalling.

Elevated levels of CAT was found only in Formulation 3 treated plants (17% higher), Formulation 1 or seaweed actives were almost similar to untreated plants under stress of lower as shown in TABLE. 7. This clearly shows that upregulation of APX and CAT was mediated via the actives and not only QD.

Sr No Treatments CAT (nmol/g FW min-1) APX
(nmol/g FW min-1)


1 Control (W) 250.3 ± 4.2 10.2 ± 1.4
2 Control (D) 278.2 ± 7.1 12.1 ± 0.7
3 Formulation 1 260.6 ± 11.3 11.6 ± 1.1
4 Formulation 1 (granular) 265.2 ± 9.4 13.2 ± 1.2
5 Formulation 2 285.3 ± 4.5 16.8 ± 0.45
6 Formulation 3 325.5 ± 6.8 17 ± 0.9
7 Commercial seaweed Formulation 1 273.6 ± 12.3 16.2 ± 0.6
8 Commercial seaweed Formulation 2 278.2 ± 8 15. ± 1.2
*legend Control is untreated plants, CW are plants that are watered normally throughout the experiment. CD are plants whose water is withheld for 10 days of the experiment.
All formulations represent plants treated with designated formulations whose water is withheld for 10 days of the experiment.
TABLE. 7
3. Field analysis:
Example 1: Tomato
Among all the formulations studied, formulation 3 showed promising results at seedling stage, water stress tolerance and recovery. Hence formulation 3 was selected further for field analysis. Effect of formulation 3 on growth, flowering and fruiting is as presented in TABLE. 8 and TABLE. 9. Formulation 3 was observed to influence positively on plant height, branching number, shoot growth and chlorophyll content compared to control plants.
Formulation Plant Height
(cm) No of branches per plant Shoot FW
(gm) Shoot DW
(gm) Chlorophyll content
(mg/g FW)
Control 62 ± 8 7.2 ± 1.5 203 ± 41 141.5 ± 5 1.45

Formulation: 3 75 ± 6 9.5 ± 0.9 1340 ± 50 225.1 ± 10 1.88

TABLE. 8
Formulation 3 showed stimulatory effect on flowering and fruiting. Early flowering, more flowering, reduction in flower drop, more flower to fruit conversion, increase in fruit size and other qualitative characters was observed to be improved in formulation 3 treated plants compared to control.

Formulation Days for Flowering Flower drop (%) Average fruit wt (gm) Total Yield per plant Total estimated yield per acre (Ton)
Control 39 ± 2 40 ± 5 57.9 ± 8 2.56 ± 0.4 17.92

Formulation: 3 30 ± 1 15 ± 3 75.5 ± 5 3.9 ± 0.5 27.30
TABLE. 9
In addition to this, incidence of leaf minor attack was also observed to be less in treated plants and they were able to tolerate heat shock very well compared to control as illustrated in FIG. 18. Bio-formulation3 treated plants were able to grow well even at temperature higher than 40°C. In Control, leaves were rolled down with significant flower fruit drop. That ultimately affected growth and yield of crop. On the contrary the plants treated with formulation3 were able to grow well even at higher temperature. Leaves were with normal morphology and very less flower and fruit drop as illustrated in FIG. 18.

Overall appearances of bio-formulation treated plants indicated superior morphology in terms of stem and root quality. Leaves were broader; green in colour, with thick lamina. In addition formation of more number of white (active) roots ensures maximum uptake of water and nutrients enabling plants to get acclimatize to field conditions thereby significantly reducing mortality after transplantation.

This indicated that formulation 3 works as a seed germination enhancer and vegetative growth booster at seedling stage enabling establishment of plants at field stage.

Effect of formulation 3 on seedling stage is well evident. Similar boosting effect is observed to be continued in on field studies. Profuse root system contributed to development of shoot system with more, broader, thicker leaves and healthier plants. This played probable role in more flowering, flower to fruit conversion, more fruiting and uniform quality of fruits. Well-developed stronger shoot system also found to be less affected by Leaf miner infection. So it is concluded that Formulation3 improves seed germination, seedling development, alleviate transplantation shock, relieves abiotic stress, impart tolerance to biotic stress, improve flowering, fruiting and overall qualitative and quantitative characteristics of harvest.
Example 2: Rice
Study the effect of different formulations on yield parameters of rice:-
Results of the present study revealed that granular formulation helped in establishment of plants after transplantation as shown in TABLE. 10 and FIG. 19. Treated plants developed profuse and showed faster root development which mainly contained active secondary and tertiary roots. Formulation 3 treated plants developed more tillering, greener leaves and height compared to control. Tillers in formulation 3 treated rice plants were 42 % higher than untreated plants.
Formulation Primary root length (cm) Secondary root no. Plant height (cm) Number of tillers
Control 9.5 ± 1.2 20 ± 3 58 ± 3 23.5 ± 1

Formulation 1 13.5 ± 1.7 18 ± 1.5 61 ± 2.5 21.7 ± 1.5

Formulation 2 11.7 ± 0.9 25 ± 1 65 ± 1.7 28.9 ± 1.9

Formulation 3 15.2 ± 1.1 32 ± 2 69 ± 2 33.5 ± 1
TABLE. 10
Similarly formulations stimulated reproductive growth in rice. Stimulatory effect was more prevalent in formulation 3 compared to control in terms of number of panicles, length of panicles and weight of panicles. This resulted in significant increase in estimated yield per acre as shown in TABLE. 11 and FIG. 20.

Formulation Number of Panicles Length of panicles (cm) Weight of 5 panicles (gm) Estimated yield/acre (Tons)
Control 12 ± 0.7 15 ± 1 16 ± 2.2 1.72 ± 0.1

1 10 ± 1.1 17 ± 1.5 18 ± 1.8 1.79 ± 0.07

2 15 ± 0.6 19 ± 1.9 19 ± 2.5 2.38 ± 0.11

3 17 ± 1.2 20 ± 2 28 ± 2 2.96 ± 0.05
TABLE. 11
Formulation 3 treated plants showed superior vegetative growth in terms of early rooting, more profuse rooting comprised more number of secondary and tertiary roots. Profuse rooting plays important role in early establishment of transplanted plants thereby helping fast recovery of plants from transplantation shock whereas secondary and tertiary rooting facilitate faster uptake of nutrient thereby promote more tillering. More number of greener and broader leaves increases photosynthetic efficiency of plants ensuring early completion of vegetative phase.
Vigorous vegetative growth induced by Formulation 3 followed by development of more number of panicle (41 % higher than untreated), higher length (33%), weight of panicles (75 %) and uniform flowering as shown in TABLE. 11 and FIG. 20. Total yield increased (72 %). This concludes that stimulatory effect of Formulation3 continues in reproductive phase of plants as well which resulted in significantly higher yield from treated plot compared to control.
Chemical analysis of selected formulations:
Spectral Analysis of Products:
All products were analyzed using LC MS at CAMS facility of NCL Venture Centre, Pune MH. Individual peak was analyzed using Mass Spectra and matched with Metlab Library for identification. Liquid chromatographic profile of Formulation 3 is presented in FIG.21. Mass spectral analysis of the formulation was conducted and presented in FIG.22.
The formulation of the present invention can be used as a liquid formulation for drip, drench. It can be coated on carrier material like but not limited to granules or sand and used for broadcast. It can be used as a foliar formulation by addition of known surfactants, adjuvants. It can also be made into gels by adding gelling agents, or packed into capsules or adhered to dissolvable membranes. The formulation can be used along with known osmo protectants like proline, sugars, and amino acids to relieve stress and boost growth. It can be used along with nutrients like NPK formulations or micronutrients or nano particles thereof. The formulation can also be used with microorganisms like rhizobium, azotobacter, micorhize etc. to relive stress.

Advantages of present invention:
The formulation can be used in variety of plant and plant product based industries such as Seedling industries, Nurseries, Plant hardening units (tissue culture or polyhouse grown plants), Polyhouse, Green houses, Orchards, Lawns, On field application, Agriculture, Floriculture and Horticulture.
The formulation can be used to increase germination rate, increase rooting, increase tertiary root formation, improve plant health, boost plant immunity, increase yield, Increase shelf life of product, reduce transplantation and transportation shock and protect from abiotic and biotic stress.

,CLAIMS:I claim,
1. A novel bio-formulation to manage abiotic stress in plants and to improve the yield comprising of at least one of the components selected from the group consisting of carbon in the form of carbon nanoparticles and carbon quantum dots, flavanoids, sphingolipids, carboxylic acids and terpenes wherein;
the formulation range of said carbon nanoparticles and carbon quantum dots is 0.01-400 mg/L;
the formulation range of said flavonoids is 0.01-10%;
the formulation range of said sphingolipids is 0.01-10%;
the formulation range of said carboxylic acids is 0.01-15%;
the formulation range of said terpenes is 0.01-10%;

2. A novel bio-formulation as claimed in claim 1, wherein the particle size of said carbon nanoparticles is 162 nm – 216 nm and the size of said carbon quantum dots is less than 10 nm preferably in the range of 1–2 nm.

3. A novel bio-formulation as claimed in claim 1, wherein said flavanoids are selected from a group consisting of Luteolin, Luteolin 5-glycoside, Apigenin, Naringenin, Eriodictyol, Diadzein, Genistein, Hesperetin and Kaemferol rutin.

4. A novel bio-formulation as claimed in claim 1, wherein said sphingolipids are selected from a group consisting of Icosamide, Dihyrosphingosine, Sphinganine, Ceramide, Phytosphingosine sphinganine, Sphingosine-1-phosphate, Phytoceramide inositol-phosphorylceramides, Glucosynceramides, and Phospholipids such as Phosphotidyl choline, Phosphotidyl ethanolamine, Phosphotidyl glycerol, Phosphotidyl serice, Phosphoinositides.

5. A novel bio-formulation as claimed in claim 1, wherein said carboxylic acids are selected from a group consisting of Propionic acid, ß-hydroxypropionate, Tricarboxylic acids, Citric acid, Butanoic acid, Isocitric acid, Succinic acid, Fumaric acid and Malic acid.

6. A novel bio-formulation as claimed in claim 1, wherein said terpenes are selected from a group consisting of Monoterpenes, Aucubin iridoid glycoside, Sesquiterpenoids juvenile hormone II, Punctaporin, Oleanolic acid isomer, Usolic acid, Triterpene aglycones of saponins, 12-hydroxy-10-dodecanoic acid/traumatin, Catalpol 8-oxogeranial, Ocimene, Mycerenes, Geraniol, Citral, Citronellal, Citronellol, Linalool, Menthol, Limonene, Carvone, Iso caryophyllene, Logifolene, a-Cuparenone, Zingiberene, Caryohyllene, Geosmin, Ursolic acid, Btulinic acid, Moronic acid, Traumatic acid, Dodecanoic acid and Traumatates.

7. A novel bio-formulation as claimed in claim 1, wherein said bio-formulation is prepared and used in at least one of the physical forms selected from a group consisting of liquid formulation, a coating on carries material such as but not limited to granules and sand, foliar formulation, gel, capsules and adherence to dissolvable membranes.