WO2023078710A1 - Composition for use in plants and uses thereof - Google Patents
Composition for use in plants and uses thereof Download PDFInfo
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- WO2023078710A1 WO2023078710A1 PCT/EP2022/079541 EP2022079541W WO2023078710A1 WO 2023078710 A1 WO2023078710 A1 WO 2023078710A1 EP 2022079541 W EP2022079541 W EP 2022079541W WO 2023078710 A1 WO2023078710 A1 WO 2023078710A1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/10—Apiaceae or Umbelliferae [Carrot family], e.g. parsley, caraway, dill, lovage, fennel or snakebed
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/12—Asteraceae or Compositae [Aster or Sunflower family], e.g. daisy, pyrethrum, artichoke, lettuce, sunflower, wormwood or tarragon
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
Definitions
- the present invention relates to the field of plant growth stimulation and plant-derived metabolite and biomolecule production, more precisely to compositions able to stimulate, increase or accelerate said features, even more precisely, to supplementary plant preparations.
- CRISPR-cas9 While crop improvement through genetically modified organisms or gene edited crops (CRISPR-cas9) is rather far from being worldwide accepted; regulatory frameworks and consumer preferences have provided a new paradigm based on chemical-free agriculture. Thus, farmers and industry are looking for innovative, natural, low- environmental impact and cost-effective solutions to increase crop yield (to substitute current solutions based on chemical compositions which are harmful for the environment and for human health). This is, the need to ensure food safety and quality with minimal environmental impact and increase production and efficiency has emerged in the recent years, requiring, therefore, sustainable agricultural practices and the development of new products in accordance with this new framework.
- MAPs Medicinal and Aromatic Plants
- WHO 2014-2023 strategy paper about integration between traditional and modern medicine
- MAPs secrete and store, mainly in their trichomes (plant hairs), a lot of metabolites with biological activity of industrial interest.
- MAPs are a very promising source of active ingredients for the industry, given consumers preferences regarding personal care and environmental sustainability.
- Plant trichomes are specialized defensive epidermal protrusions on the surfaces of leaves and other aerial organs of many plants. Trichomes are present in most of the vascular plant surfaces, and their main role is to defend plants against insect herbivores, virus, UV light and/or excessive water loss (Werker E. (2000) Trichome diversity and development. Advances in Botanical Research, 31 , 1-35).
- a very important characteristic of plant trichomes, especially in GSTs (Glandular-shape trichomes) is that they are able to synthesize and store large amounts of unique metabolites or biomolecules (Schil miller, A.L., Last, R.L. and Pichersky, E. (2008) Harnessing plant trichome biochemistry for the production of useful compounds.
- IDP isopentenyl diphosphate
- DMADP dimethylallyl diphosphate
- FDP is subsequently cyclized into amorpha-4,11 -diene by amorpha-4, 11 -diene synthase (ADS), which is the first unique intermediate of artemisinin biosynthesis (Bouwmeester, H.J., Wallaart, T.E., Janssen, M.H., van Loo, B., Jansen, B.J., Posthumus, M.A., Schmidt, C.O., de Kraker, J.W., Konig, W.A. and Franssen, M.C. (1999) Amorpha-4,11 -diene synthase catalyses the first probable step in artemisinin biosynthesis.
- ADS amorpha-4, 11 -diene synthase
- Amorpha-4, 11 -diene is then oxidized by amorpha-4, 11 -diene 12-hydroxylase (CYP71AV1), reduced by artemisinic aldehyde A11 (13) reductase (DBR2) to dihydroartemisinic aldehyde and again oxidized by aldehyde dehydrogenase 1 (ALDH1) into the direct precursor of artemisinin, dihydroartemisinic acid (DHAA) (Teoh, K.H., Polichuk, D.R., Reed, D.W., Nowak, G. and Covello, P.S.
- the final production step is considered to be a non-enzymatic reaction (Brown, G.D. (2010) The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua Molecules, 15, 7603-7698; Covello, P.S. (2008) Making artemisinin, Phytochemistry, 69).
- a peroxidase enzyme or an alternative series of oxidations occurring exclusively in plants, may in fact catalyse the crucial last reaction that converts the precursor into the valuable artemisinin molecule (Bryant, L., Flatley, B., Patole, C., Brown, G.D., and Cramer, R.(2015).
- compositions comprising natural ingredients (this is, without the need of chemicals - chemical-free) and, hence, respectful with the environment and safe, which allows to:
- trichomes preferably GSTs
- plant derived ingredients preferably, biomolecules, even more preferably, metabolites
- plant-derived ingredients preferably, biomolecules, even more preferably, metabolites
- plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia, Lavandula hybrid
- the present invention refers to a composition comprising: fermented rice starch; and extract obtained from Urtica.
- the present invention refers to a composition
- a composition comprising: fermented rice starch; fungi of the genus Trichoderma', and extract obtained from Urtica.
- the present invention refers to the use of a composition of the present invention as supplementary plant preparation.
- the present invention refers to the use of a composition of the present invention to increase growth of a plant, to accelerate growth or lifecycle of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant.
- the present invention refers to method to increase growth of a plant, to accelerate growth or lifecycle of a plant, to increase trichome density of a plant and/or to increase a plant-derived ingredient production in a plant comprising the application of a composition of the present invention to the plant.
- MAP Medical and Aromatic Plant
- botanical raw materials also known as herbal drugs that are primarily used for therapeutic, aromatic and/or culinary purposes as components of cosmetics, medicinal products, functional foods and other natural health products. They are also the starting materials for value-added processed natural ingredients such as essential oils, dry and liquid extracts and oleoresins.
- MAPs are: Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp.
- supplementary plant preparation and its plural refer to naturally produced substance/s synthetized in plants and/or micro-organisms whose function when applied to plants or the rhizosphere surrounding the plants is to improve physiological processes such as crop quality traits, nutrient use efficiency or tolerance to biotic and abiotic stress.
- the present invention refers to a composition comprising: fermented rice starch; and extract obtained from Urtica.
- the present invention refers to a composition
- a composition comprising: fermented rice starch; fungi of the genus Trichoderma', and extract obtained from Urtica.
- fermented rice starch is preferably a single fermented rice starch (this is, fermented rice starch preferably from a single rice species) or a combination of fermented rice starches (preferably a combination of fermented rice starches from two or more rice species); Fermented rice starch can be obtained from any rice species, most preferably is from Oryza sativa. Most preferably, fermented rice starch in the composition of the present invention is a fermented rice starch from Oryza sativa.
- Fermented rice starch can be prepared by any method or process known in the state of the art.
- fermented rice starch is prepared by means of a process comprising the following steps: a) providing rice starch; b) applying a temperature of between 20 to 40 °C for between 24 hours and 120 hours to the rice starch to obtain a naturally fermented rice starch product; c) keeping the naturally fermented rice starch product obtained in step b) in water, under stirring; d) keeping the product obtained in step c) at ambient temperature (preferably, 22°C to 25°C) for between 10 and 30 hours; e) filtering the product of step d) and obtaining said filtrate to be used as fermented rice starch in the composition of the present invention.
- rice starch is provided in powder.
- rice starch can be from any rice species, most preferably rice starch is from Oryza sativa.
- step b) the temperature is between 35°C and 37°C for 72 hours.
- step b) is carried out in an incubator.
- Step b) as explained above provides for a spontaneous and natural lactic fermentation carried out by lactobacilli without the need for inoculation.
- step c) lasts for between 2 and 10 hours, more preferably between 3 and 4 hours.
- the stirring is preferably homogenous, slow and constant. More preferably, in step c) stirring is at 400 rpm (revolutions per minute).
- step d) lasts for 20 hours.
- step d) is performed without stirring.
- filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve.
- filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
- the product obtained can be dried to obtain a dried fermented rice starch.
- step b) mentioned above allows not only to break proteins into free peptides and amino acids, but also to transform precursor biomolecules into phytohormones (such as, gibberellins).
- the fermentation step can also eliminate hypoallergenic or anti-nutritional factors that may be present (for example, trypsin inhibitors, phytate, oligosaccharides such as raffinose and stachyose, and saponins in legumes).
- fermented rice starch provides for, in the composition of the present invention, bioactive peptides, soluble proteins, free amino acids and phytohormones.
- the fermented rice starch provides for one or more phytohormones.
- the fermented rice starch provides for at least one gibberellin.
- Gibberellins are plant hormones (phytohormones) and include gibberellic acid and derivatives thereof. Increases in these natural phytohormones usually occur due to fermentation processes with lactic bacteria (lactobacilli) or the Gibberela Fujimori fungus. The Gibberellin family is involved in many physiological functions from germination, formation of sexual organs, flower inductions, abiotic stresses, cell division, etc...
- the at least one gibberellin mentioned above is, preferably, Gibberellin A1 (GA1), Gibberellin A3 (GA3), Gibberellin A4 (GA4) or combinations thereof, more preferably, the at least one gibberellin is GA1 , GA3 or GA4, even more preferably, the at least one gibberellin is GA3.
- the activity provided by the fermented rice starch in the composition of the present invention is provided by bioactive peptides, soluble proteins and amino acids, more preferably by phytohormones, even more preferably by at least one gibberellin.
- the fermented rice starch (as explained above) is at a concentration of between 0.0066 mg/mL and 0.24 mg/mL, more preferably, of between 0.0132 and 0.12 mg/mL, even more preferably, 0.04 mg/mL.
- the fungi of the genus Trichoderma are fungi from one or more species of the genus Trichoderma.
- the fungi of the genus Trichoderma are Trichoderma virens fungi (preferably, of the strain TV 29.8, GV41 or combinations thereof), Trichoderma atroviride fungi (preferably, of the strain IMI 206040), Trichoderma harzianum fungi (preferably, Trichoderma harzianum RIFAI strain T-22) or combination thereof, more preferably Trichoderma harzianum fungi, even more preferably, Trichoderma harzianum RIFAI strain T-22 fungi.
- the concentration of the fungi of the genus Trichoderma is preferably between 0.0026 mg/mL and 0.096 mg/mL, more preferably between 0.0053 and 0.048 mg/mL, even more preferably, 0.016 mg/mL.
- the fungi from the genus Trichoderma (preferably, Trichoderma harzianum RIFAI strain T-22) in the composition of the present invention provide, at least, for an increase in productivity of the crop, provide nutrients and resistance to abiotic (for example, drought and salinity) and biotic stresses (for example, pathogens).
- abiotic for example, drought and salinity
- biotic stresses for example, pathogens
- the extract obtained from Urtica is preferably a single extract obtained from Urtica (this is, an extract obtained, preferably, from a single Urtica species) or a combination of extracts obtained from Urtica (preferably, a combination of extracts obtained from two or more Urtica species).
- the extract obtained from Urtica is an extract obtained from Urtica dioica.
- the concentration of the extract obtained from Urtica is between 0.00175 mg/mL and 0.0636 mg/mL, more preferably between 0.0035 mg/mL and 0.0318 mg/mL, even more preferably, 0.0106 mg/mL.
- the extract obtained from Urtica is obtained by any means or process available in the state of the art, more preferably, it is obtained by means of a process comprising the following steps: a) providing dry plant material from Urtica-, b) keeping the dry plant material provided in step a) in water, under heating and stirring; c) filtering the material of step b) and obtaining said filtrate to be used as an extract obtained from Urtica in the composition of the present invention.
- the dry plant material comprises dry leaves with secondary stems, more preferably, in step a) the dry plant material consists of dry leaves.
- the temperature is between 40 °C and 50 °C, more preferably 45 °C.
- step b) lasts for between 12 and 36 hours, more preferably, 24 hours.
- the dry plant material is at a concentration of between 5 g/L and 20 g/L in the water, more preferably at a concentration of 12 g/L.
- step b) the stirring is preferably homogenous, slow and constant. More preferably, in step b) stirring is at 400 rpm (revolutions per minute).
- filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve.
- filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
- the extract obtained from Urtica mainly comprises organic acids (preferably, acetic acid, citric acid, butyric acid), chlorophyll, flavonoids, tannins, mineral salts (iron, sulphur, manganese, potassium), carotenes, histamine and acetylcholine, vitamins A, B2, K1 and folic acid.
- the extract from Urtica (preferably, an extract obtained from Urtica dioica) in the composition of the present invention, at least, fortifies and stimulates the microbial flora of the soil and leaves; strengthens plants; is used as organic fertilizer rich in nitrate; fights against chlorosis; and promotes photosynthesis.
- composition of the present invention as described above, wherein fermented rice starch; and an extract obtained from Urtica (preferably, fermented rice starch; fungi of the genus Trichoderma', and an extract obtained from Urtica) are used in combination provides for a synergistic and unexpected effect, which goes beyond the individual effect observed for each of its components and which solves the technical problems observed in the state of the art.
- composition which can (as exemplified below in Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua) :
- plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia,
- composition of the present invention enables the use of plants as biofactories to obtain an increased production of plant-derived ingredients of interest.
- said composition of the present invention further comprises one or more of: extract obtained from Centella’, extract obtained from Echinacea’, extract obtained from Taraxacum’, or combinations thereof.
- composition of the present invention further comprises an extract obtained from Centella.
- composition of the present invention further comprises an extract obtained from Echinacea. In a further embodiment, the composition of the present invention further comprises an extract obtained from Taraxacum.
- the composition of the present invention further comprises an extract obtained from Centella, an extract obtained from Echinacea and an extract obtained from Taraxacum.
- the extract obtained from Centella is preferably a single extract obtained from Centella (this is, an extract obtained, preferably, from a single Centella species) or a combination of extracts obtained from Centella (preferably, a combination of extracts obtained from two or more Centella species).
- the extract obtained from Centella is an extract obtained from Centella asiatica.
- the extract obtained from Centella is present in a concentration of between 0.0010 mg/mL and 0.0372 mg/mL, more preferably between 0.0020 mg/mL and 0.0186 mg/mL, even more preferably 0.062 mg/mL.
- the extract obtained from Taraxacum is preferably a single extract obtained from Taraxacum (this is, an extract obtained, preferably, from a single Taraxacum species) or a combination of extracts obtained from Taraxacum (preferably, a combination of extracts obtained from two or more Taraxacum species).
- the extract obtained from Taraxacum is an extract obtained from Taraxacum officinale.
- the extract obtained from Taraxacum is present in a concentration of between 0.00095 mg/mL and 0.0346 mg/mL, more preferably between 0.0019 mg/mL and 0.0173 mg/mL, even more preferably 0.0057 mg/mL.
- the extract obtained from Echinacea is preferably a single extract obtained from Echinacea (this is, an extract obtained, preferably, from a single Echinacea species) or a combination of extracts obtained from Echinacea (preferably, a combination of extracts obtained from two or more Echinacea species).
- the extract obtained from Echinacea is an extract obtained from Echinacea purpurea.
- the extract obtained from Echinacea is present in a concentration of between 0.0022 mg/mL and 0.0798 mg/mL, more preferably 0.0044 mg/mL and 0.0399 mg/mL, even more preferably 0.0133 mg/mL.
- the extract obtained from Centella-, the extract obtained from Echinacea-, and/or the extract obtained from Taraxacum are produced or obtained in the same ways as explained above for the extract obtained from Urtica. This is, they can be obtained by any means or process available in the state of the art, more preferably, they are obtained by means of a process comprising the following steps: a) providing dry plant material from the corresponding plant (Centella, Taraxacum or Echinacea, in accordance with what has been explained above); b) keeping the dry plant material provided in step a) in water, under heating and stirring; c) filtering the material of step b) and obtaining said filtrate to be used as an extract obtained from the corresponding plant (Centella, Taraxacum or Echinacea, in accordance with what has been explained above) in the composition of the present invention.
- the dry plant material comprises dry leaves with secondary stems, more preferably, in step a) the dry plant material consists of dry leaves.
- step b) the temperature is between 40 °C and 50 °C, more preferably 45 °C. Also preferably, step b) lasts for between 12 and 36 hours, more preferably, 24 hours.
- the dry plant material is at a concentration of between 5 g/L and 20 g/L in the water, more preferably at a concentration of 12 g/L.
- step b) the stirring is preferably homogenous, slow, and constant. More preferably, in step b) stirring is at 400 rpm (revolutions per minute).
- filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve, even more preferably a stainless-steel sieve with an internal height of 25mm and diameter of 200mm.
- filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
- the main components of the extract obtained from Centella are: pentacyclic triterpenes and their genins: asian and madecasic acids and heterosides (asiatoside, centelloside, madecasoside and terminoloside); tannins (up to 25% in weight); flavonoids; fatty acids (preferably, linoleic and/or palmitic); alkaloids (such as hydrocotiline, mucilages, phytosterols (campesterol, sitosterol, stigmasterol)); and free amino acids (alanine, serine, aminobutyrate, aspartate, glutamate, lysine and threonine).
- pentacyclic triterpenes and their genins asian and madecasic acids and heterosides (asiatoside, centelloside, madecasoside and terminoloside); tannins (up to 25% in weight); flavonoids; fatty acids (preferably, linoleic and/or palmitic); alkaloids (such as hydrocoti
- the extract obtained from Centella provides, in the composition of the present invention, at least, antimicrobial and antiviral activities.
- the main components of the extract obtained from Echinacea are: vitamins of groups C and B (such as riboflavin); beta-carotenes; minerals (such as, iron, sodium, magnesium and calcium); antioxidants; alkamides; and polysaccharides.
- the extract obtained from Echinacea provides, in the composition of the present invention, at least, a strengthening of the immune system of the plant.
- the main components of the extract obtained from Taraxacum are: iron, potassium, calcium, zinc, magnesium, phosphorus and vitamins such as vitamins A, K, and C.
- the extract obtained from Taraxacum provides, in the composition of the present invention, at least, antibiotic activity and increase the defences from the plant.
- the addition to the composition of the present invention of the extract obtained from Centella’, the extract obtained from Echinacea’, the extract obtained from Taraxacum’, or, preferably, of the combination of the three surprisingly improves the performance of the composition of the present invention and, hence, also solves the problems present in the state of the art providing for: an even accelerated growth of plants, allowing for an increased number of crops per year.
- GSTs glandular-shape trichomes
- plant-derived ingredients of interest preferably, biomolecules, more preferably, metabolites
- o artemisinin and flavonoids preferably, casticin and eupatorina
- plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia,
- composition of the present invention comprises further ingredients or components.
- carriers or excipients which do not alter or do not alter in an unacceptable manner the activity of the composition of the present invention.
- composition of the present invention in any of the above-mentioned embodiments is preferably applied in a quantity of between 45 and 576 litres per hectare, more preferably, between 90 and 288 litres per hectare. Said total quantity is preferably applied by means of 2 to 10 applications, more preferably, between 4 and 8 applications. Also preferably said applications are performed at least once per week, more preferably at least twice per week, even more preferably twice per week.
- the above-mentioned applications are preferably performed in the plant (preferably in the leaves) and in the soil. More preferably, between 75% and 90% of the volume of each application is applied in the plant (preferably in the leaves) and between 10% and 25% of the volume of each application is applied in the soil. Even more preferably, 85% of the volume of each application is applied in the plant (preferably in the leaves) and 15% of the volume of each application is applied in the soil.
- composition of the present invention can be carried out by any means known in the state of the art, more preferably, by means of sulphating backpack or trolley.
- composition of the present invention is preferably used in plants in the juvenile state.
- the composition of the present invention is in a concentrated form, this is, in a form which must be diluted before being used.
- the composition of the present invention can be in a concentrated form of between 10x to 500x, more preferably, between 10x and 100x, more preferably, between 50x and 100x, even more preferably, the composition of the present invention is in a 50x or 100x concentrated form. In these cases, all the concentration ranges noted above should be multiplied by the corresponding concentration factor to obtain the concentration in the concentrated form.
- this concentrated forms, before being used will have to be diluted the same number of times as they are concentrated (for example, a 50x concentrated composition will have to be diluted 50 times before being used and a 100x concentrated composition will have to be diluted 100 times before being used).
- composition of the present invention acts stimulating and accelerating the growth of plants, preferably, MAPs, more preferably, Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia
- composition of the present invention also increases the production of molecules and/or biomolecules of interest in plants (preferably, MAPs, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Artemisia annua, even more preferably Artemisia annua).
- MAPs molecules and/or biomolecules of interest in plants
- the composition of the present invention is, preferably, a supplementary plant preparation able to provide the above effects.
- the plant can be any plant known in the state of the art or which is discovered or generated in the future.
- the plant is a MAP, more preferably, Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscul
- the use in this second aspect of the present invention is in a plant in the juvenile state. This provides for an even increased acceleration of the flowering.
- composition of the present invention is as already explained in the first aspect of the present invention.
- the present invention refers to the use of a composition of the present invention to increase growth of a plant, to accelerate growth of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant.
- composition of the present invention is in accordance with the first aspect of the present invention.
- the increase in the growth of the plant is preferably an increase in the biomass thereof, more preferably, an increase in the number of leaves, an increase in the area of the leaves, an increase in the height of the plant, an increase in internode number or combinations thereof.
- the internodes were branches originate from the amin stem (therefore, an increase in the number of internodes will provide an increase in the number of branches and, on its turn, an increase in the number of leaves).
- the increase in the number of leaves and/or in the area of the leaves preferably, provides for an increase in the number of trichomes, an increase in the density of trichomes or combinations thereof.
- trichomes are glandular-shape trichomes (GSTs).
- Trichomes (preferably, GSTs) mature just before flowering. Therefore, an acceleration of the flowering provides an acceleration of the production of plant-derived ingredients of interest (preferably, biomolecules of interest, more preferably, metabolites of interest).
- plant-derived ingredients of interest preferably, biomolecules of interest, more preferably, metabolites of interest.
- the acceleration of the flowering provides for a reduction of the lifecycle of the plant and, therefore, an increased production (more harvests can be produced each year),
- any plant-derived ingredient is contemplated, for example, biomolecules, essential oils, dry/liquid extracts, oleoresins and active pharmaceutical ingredients. More preferably the plant-derived ingredient is an essential oil and/or a biomolecule.
- the biomolecule can be any biomolecule naturally produced by the plant or any biomolecule the production of which has been artificially induced (by any method or means know in the state of the art) in the plant. More preferably, the plant-derived ingredient is a biomolecule, even more preferably a metabolite, still more preferably, a metabolite produced in the trichomes (preferably, in the GSTs) of the plant.
- the plant is Salvia officinalis and then, preferably the plant- derived ingredient is: essential oil, viridiflorol, alpha-thujene, cariophyllene oxide, sabinene, alpha-pinene, trans-caryophyllene, spathulenol, camphor or combinations thereof.
- the plant is Origanum vulgare and then, preferably, the plant-derived ingredient is: essential oil, trans-carveol, Germacrene D, carvacrol, alpha-pinene or combinations thereof.
- the plant is Lavandula latifolia and then, preferably, the plant-derived ingredient is: essential oil, linalyl acetate, valencene, z-beta-farnesene, trans-caryophyllene, Borneol, 1 ,8 cineole or combinations thereof.
- the plant is Lavandula hybrida and then, preferably, the plant-derived ingredient is: essential oil, t-cadinol, alpha-bisabolol, Borneol, trans-carveol, geranyl acetate or combinations thereof.
- the plant is Rosmarinus officinalis and then, preferably, the plant-derived ingredient is: essential oil, camphor, beta-eudesmol, caryophillene oxide, limonene, terpinolene, alpha-trans bergamotene or combinations thereof.
- the plant is Artemisia annua and then, preferably, the metabolite is a sesquiterpene and/or a flavonoid, more preferably, casticin, euparotin and/or artemisinin, even more preferably, artemisinin.
- the use in this third aspect of the present invention is in a plant in the juvenile state. This provides for an even increased acceleration of the flowering.
- the plant the increase in the growth, the accelerated growth, the increase in the plant- derived ingredient production and the plant-derived ingredient are as explained above in the third aspect of the present invention.
- Figure 1 shows scanning electron microscope (SEM) of the results obtained in the different treatment groups tested in example 1 included below, for the number of trichomes.
- figure 1A refers to the control (no treatment applied)
- figure 1 B refers to the group to which cytokinins were applied
- figure 1C refers to the group to which jasmonic acid was applied
- figure 1 D refers to the group to which Trichoderma harzianum RIFAI strain T-22 were applied
- figure 1 E refers to the group to which the extract obtained from Urtica dioica was applied
- figure 1 F refers to the group to which fermented rice starch was applied
- figure 1G refers to the group to which Composition 1 (for details see example 1)
- figure 1 H refers to the group to which Composition 2 (for details see example 1).
- Figure 2 is a picture of the results obtained in the different treatment groups tested in example 4 included below. More precisely, figure 2A refers to the control (no treatment applied), figure 2B refers to the group to which cytokinins were applied, figure 2C refers to the group to which jasmonic acid was applied, figure 2D refers to the group to which the extract obtained from Urtica dioica was applied, figure 2E refers to the group to which Trichoderma harzianum RIFAI strain T-22 were applied, figure 2F refers to the group to which fermented rice starch was applied, figure 2G refers to the group to which Composition 1 (for details see example 1), and figure 2H refers to the group to which Composition 2 (for details see example 1).
- Figure 3 is a picture of results obtained in example 4 included below. More precisely, compared are plants treated with Composition 1 (left; see example 1 for the details on the composition) and control plants (right; plant not treated).
- Example 1 Analysis of trichome density in Artemisia annua.
- Seeds of Artemisia annua varieties Anamed (www.anamed.net) and Chongqing (Professor K. Tang, Shanghai Jiao Tong University) were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare.
- between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil.
- Each application was performed by means of sulphating backpack or trolley SG2.
- Trichome initiation was monitored using an Olympus DP71 microscope by counting all trichomes on the adaxial surface of individual and fully developed upper leaves. Diverse upper leaves were counted independently given that these leaves showed different trichome production. Leaves were chosen at 56 days (8 weeks) after germination due to the fact that at this stage they were fully developed, and the full treatment had been completed.
- Cytokinins used at a concentration of 0.02 mg/mL
- Jasmonic acid used at a concentrarion of 0.02 mg/mL
- Trichoderma harzianum RIFAI strain T-22 used at a concentration of 0.016 mg/mL
- Extract obtained from Urtica dioica used at a concentration of 0.0106 mg/mL Fermented rice starch (Oryza sativa), used at a concentration of 0.04 mg/mL Composition 1 : Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Trichomes SEM images required for the analysis were obtained as previously described by Sanchez-Chardi et al, 2010 (Sanchez-Chardi A, Olivares F, Byrd TF, Julian E, Brambilla C, Luquin M (2011) Demonstration of cord formation by rough Mycobacterium ab-scessus variants: implications for the clinical microbiology laboratory. JCIin Microbiol 49:2293-2295.).
- samples were fixed in 2.5% (volume/volume) glutaraldehyde in 0.1 M Phosphate buffer saline (PBS) (pH 7.4) for 2 h at 4°C, washed 4 times for 10 min each time in 0.1 M PBS, postfixed in 1 % (volume/volume)osmium tetraoxide with 0.7% (volume/volume) ferrocyanide in PBS, washed in water, dehydrated in an ascending ethanol series (50, 70, 80, 90, and 95% (volume/volume) for 10 min each and twice with 100% ethanol), and dried by critical-point drying with CO2.
- PBS Phosphate buffer saline
- Results obtained in this example appear summarized in Table 1 and Figure 1 .
- Compositions 1 and 2 showed a synergistic and unexpected increase in trichome density in the Artemisia annua plants.
- Table 1 the individual components of the Compositions showed a little to moderate effect in increasing the thrichome density.
- thay acted synergistically and produced and unexpectedly superior increase in trichome density (even higher in combination 2).
- the amount of artemisinin in the plants of example 1 was determined by extraction and analysis on a Waters Alliance 2695 HPLC system coupled with a Waters 2420 ELSD detector as previously described (Jiang W, Fu X, Pan Q, Tang Y, Shen Q, Lv Z, Yan T, Shi P, Li L, Zhang L, Wang G, Sun X, and Tang K (2016). Overexpression of AaWRKYI Leads to an Enhanced Content of Artemisinin in Artemisia annua. Biomed Res Int: 7314971).
- transitions used for the identification and quantification of artemisinin were: from 283.17 ppb (parts per billion) to 151.09 ppb and from 283.17 ppb to 209.08 ppb, all in mass/charge number of ions (m/z), with a quantification limit of 2.5 ppb.
- Compositions 1 and 2 showed a superior and unexpected increase in artemisinin content in the Artemisia annua plants, which can be considered as a result of a synergistic interaction between the different constituents.
- Table 4 the individual components of the Compositions showed a little to moderate effect in increasing artemisinin content.
- thay acted synergistically and produced and unexpectedly superior increase in artemisinin content (even higher in Composition 2).
- the primers for each of the genes artemisinic aldehyde A11 (13) reductase (DBR2), amorpha-4, 11 -diene 12-hydroxylase (CYP71AV1), amorpha-4,11 -diene synthase (ADS), and p-ACTIN are listed below in Table 5. Obtained data were normalized using the p-ACTIN gene as reference.
- RT-qPCR reverse transcription quantitative polymerase chain reaction
- qPCR quantitative polymerase chain reaction
- SYBR Green I Master Mix and Light Cycler 480 Roche
- PCR polymerase chain reaction efficiency was calculated and determined as previously described in Talke et al., 2006 (Talke, LN., Hanikenne, M. and Kramer, II. (2006) Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant physiology, 142, 148- 167). The results obtained in this example appear summarized in Tables 6 to 8.
- Example 4 Analysis of the increase in biomass production in Artemisia annua.
- Leaf area was monitored using an Olympus DP71 microscope by measuring leaf area (pixels/cm 2 ) on individual and fully developed medium leaves. Medium leaves area was measured independently 63 days (9 weeks) after germination due to the fact that at this stage medium leaves were fully developed and the full treatment for the different groups had been previously completed. The results obtained appear summarized in Table 9 included below and Figures 2 and 3. Table 9. Results of leaf area (mean and standard deviation) obtained for the different experimental groups tested in example 4.
- Leaf number per plant was monitored by counting all leaves from 10 independent Artemisia annua plants for each treatment group. Leaves were counted 70 days (10 weeks) after germination when they were completely developed. Results obtained appear summarized in Table 10 included below and Figures 2 and 3.
- Trichoderma harzianum RIFAI strain T-22 and the extract obtained from Urtica dioica showed a little effect while fermented rice starch (Oryza sativa) showed a moderate effect.
- Compositions 1 and 2 showed a synergistic and unexpected increased effect in both parameters, which exceedes the individual performance of the individual components and which is even greater for Composition 2 (showing the improving effect of the extract obtained from Echinacea, the extract obtained from Centella asiatica and of the extract obtained from Taraxacum officinale).
- Cytokinins and Jasmonic acid as can be derived from Tables 9 and 10 had a minimal o even a negative effect in the two analysed biomass parameters.
- Example 5 Analysis of the acceleration of the flowering in Artemisia annua.
- Flowering time measurements plants were randomized with the respective controls and grown on soil in controlled environment growth chambers. Flowering time was determined as the number of days from sowing to the appearance of the floral bud of at least 20 individual plants for each treatment. The number of days to flowering was determined when the first floral buds were visible to the naked eye. Indeed, Artemisia annua plants were carefully checked for visible signs of flowering every two days. All flowering time assays were performed at least twice. Flowering time data were subjected to analyses of variance (ANOVA). Post-hoc tests were performed using Tukey’s multiple comparisons test after two-way ANOVA. Statistical analyses were performed with Prism 6 software (GraphPad Software, Inc).
- the treatment groups tested in this example were:
- Cytokinins used at a concentration of 0.02 mg/mL
- Jasmonic acid used at a concentrarion of 0.02 mg/mL
- Trichoderma harzianum RIFAI strain T-22 used at a concentration of 0.016 mg/mL
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Compositions 1 and 2 which produce a very significant decrease in flowering time with regard to the control (from 7 months to approximately 2.5 months).
- the effect observed for this two Compositions is superior to the addition or sum of the effects seen individually for Trichoderma harzianum RIFAI strain T-22, the extract obtained from Urtica dioica and fermented rice starch (Oryza sativa), demonstrating a synergistic effect, which is even greater in Composition 2 (showing the improving effect of the extract obtained from Echinacea, the extract obtained from Centella asiatica and of the extract obtained from Taraxacum officinale).
- Seeds of Salvia officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare.
- between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil.
- Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Plant Biomass (kg/plant) was measured as follows:
- Plant material (leaves and secondary branches) were collected at the beginning of the flowering and fresh biomass/plant was determined by weighing in a certified precision balance. Afterwards plant material was dried in a drying chamber at a temperature of between 32 °C and 40 °C until they reach a constant weight. Then, the dried plant material was weighed in a certified precision balance.
- Carrier gas Helium (1.0 mL/min) Volume injected: 0.5 pL
- the peaks for each of the secondary metabolites or biomolecules was particular for each plant species. Then the area of each of the peaks of interest was measured.
- results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 14. Table 14. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Salvia officinalis in example 6. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
- composition 2 was effective in increasing the biomass of Salvia officinalis, as well as the production of essential oils and of several biomolecules with therapeutic properties.
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Plant biomass (both, fresh and dried) was measured as explained in Example 6.
- the quantity of the biomolecules of interest was measured as explained in Example 6.
- results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 17.
- Table 17. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Origanum vulgare in example 7. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained. Therefore, as it can be derived from tables 15 to 17, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Origanum vulgare, as well as the production of essential oils and of several biomolecules with therapeutic properties.
- Example 8 Analysis of the effect of the present invention in Lavandula latifolia
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Plant biomass (both, fresh and dry) was measured as explained in Example 6.
- composition 2 was effective in increasing the biomass of Lavandula latifolia, as well as the production of essential oils and of several biomolecules with therapeutic properties.
- Seeds of Lavandula hybrida were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle. In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Plant biomass (both, fresh and dry) was measured as explained in Example 6.
- the quantity of the biomolecules of interest was measured as explained in Example 6.
- composition 2 was effective in increasing the biomass of Lavandula hybrida, as well as the production of essential oils and of several biomolecules with therapeutic properties.
- Seeds of Rosmarinus officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Plant biomass (both, fresh and dry) were measured as explained in Example 6.
- the quantity of the biomolecules of interest was measured as explained in Example 6.
- composition 2 was effective in increasing the biomass of Rosmarinus officinalis, as well as the production of several biomolecules with therapeutic properties.
- Seeds of Ocimum basilicum were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare.
- between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil.
- Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- Plant biomass (both, fresh and dry) was measured as explained in Example 6.
- composition 2 was effective in increasing the biomass of Ocimum basilicum.
- Example 12 Analysis of the effect of the present invention in Cannabis spp
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 2 Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
- the woody stem was obtained, and the number of internodes was determined by means of visual inspection. In addition, the height of the woody stems was also measured.
- Table 30 Results obtained for height and number of internodes in Cannabis spp in example 12. Therefore, as it can be derived from tables 29 and 30, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Cannabis spp.
- the present invention in all its aspects solves the technical problems present in the state of the art and provides for:
- MAPs accelerated the growth of plants
- Increase in plant growth and in biomass production of plants (more preferably, MAPs), including, an increased number of leaves and bigger leaves.
- plant-derived ingredients of interest preferably, essential oil or biomolecules
- plant-derived ingredients of interest preferably, essential oil or biomolecules
- Example 13 Analysis of the effect of a composition of the present invention in Salvia officinalis
- Seeds of Salvia officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare.
- between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil.
- Each application was performed by means of sulphating backpack or trolley SG2.
- the following treatment groups were:
- Composition 3 Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
- Fermented rice starch (Oryza sativa) (0.04 mg/mL).
- Plant Biomass (kg/plant) was measured as follows: Plant material (leaves and secondary branches) were collected at the beginning of the flowering and fresh biomass/plant was determined by weighing in a certified precision balance.
- Example 14 Analysis of the effect of a composition of the present invention in Ocimum basilicum
- Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
- the total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
- Composition 3 Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
- Fermented rice starch (Oryza sativa) (0.04 mg/mL).
- Plant biomass and height were measured as explained above.
- Table 33 Results obtained for the number of leaves in Ocimum basilicum in example 14. Therefore, as it can be derived from tables 32 and 33, the composition of the present invention (as exemplified by composition 3) was effective in increasing in a synergistic way the biomass of Ocimum basilicum (fresh biomass, height and number of leaves per plant).
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Abstract
Disclosed herein is a composition comprising: fermented rice starch and extract obtained from Urtica useful to increase and accelerate growth of plants. Also disclosed herein are uses and methods of use of the composition.
Description
COMPOSITION FOR USE IN PLANTS AND USES THEREOF
TECHNICAL FIELD
The present invention relates to the field of plant growth stimulation and plant-derived metabolite and biomolecule production, more precisely to compositions able to stimulate, increase or accelerate said features, even more precisely, to supplementary plant preparations.
BACKGROUND OF THE INVENTION
Today’s population growth, decreasing agricultural land, increasing input costs and limited availability thereof, need to reduce fertilizer usage, water usage constrains, sustainability, climate change impacts and increased abiotic stress are main global drivers and threats for agricultural sector worldwide.
While crop improvement through genetically modified organisms or gene edited crops (CRISPR-cas9) is rather far from being worldwide accepted; regulatory frameworks and consumer preferences have provided a new paradigm based on chemical-free agriculture. Thus, farmers and industry are looking for innovative, natural, low- environmental impact and cost-effective solutions to increase crop yield (to substitute current solutions based on chemical compositions which are harmful for the environment and for human health). This is, the need to ensure food safety and quality with minimal environmental impact and increase production and efficiency has emerged in the recent years, requiring, therefore, sustainable agricultural practices and the development of new products in accordance with this new framework.
Medicinal and Aromatic Plants (MAPs) are botanical raw materials, also known as herbal drugs, primarily used for therapeutic, aromatic and/or culinary purposes as components of cosmetics, medicinal products, functional foods and other natural health products. According to WHO (2014-2023 strategy paper about integration between traditional and modern medicine) there are 21 ,000 plants used for medicinal purposes since ancient times to treat diseases. They are also the starting materials for value-added processed natural ingredients such as essential oils, dry/liquid extracts, oleoresins and Active Pharmaceutical Ingredients (APIs). MAPs secrete and store, mainly in their trichomes (plant hairs), a lot of metabolites with biological activity of industrial interest. MAPs are a very promising source of active ingredients for the industry, given consumers preferences regarding personal care and environmental sustainability. Nowadays, much
of these compounds are synthesized through chemical processes using synthesis routes which involve a very complex ensemble of chemical reactions, because effective production through MAPs is not technically feasible due to the lack of consistency, lack of reliability and, mainly, lack of required quantities, production efficiency and yield.
Plant trichomes are specialized defensive epidermal protrusions on the surfaces of leaves and other aerial organs of many plants. Trichomes are present in most of the vascular plant surfaces, and their main role is to defend plants against insect herbivores, virus, UV light and/or excessive water loss (Werker E. (2000) Trichome diversity and development. Advances in Botanical Research, 31 , 1-35). A very important characteristic of plant trichomes, especially in GSTs (Glandular-shape trichomes), is that they are able to synthesize and store large amounts of unique metabolites or biomolecules (Schil miller, A.L., Last, R.L. and Pichersky, E. (2008) Harnessing plant trichome biochemistry for the production of useful compounds. The Plant journal: for cell and molecular biology, 54, 702-711), as artemisinin in Artemisia annua plant. Indeed, the biosynthetic pathway of artemisinin, an antimalarial and anticancer substance, only takes place in Artemisia annua GSTs’; however, as already introduced previously, artemisinin content in this plant is too low to cover the worldwide demand of the product. Also, trichomes are able to produce other metabolites or biomolecules that act in synergy with the main therapeutic molecules. Artemisia annua is still the main resource for artemisinin (AN). However, as stated above, the plant extraction-based Artemisinin industry (WHO (2014) World Malaria Report 2014. http://www.who.int/malaria/publications/world_malaria_report_2014/en/) is hindered by the fact that the content of Artemisinin in Artemisia annua is extremely low (0.1-1% /g dry weight), which makes the price still too high for a higher number of malarial patients (Abdin, M.Z., Israr, M., Rehman, R.U. and Jain, S.K. (2003) Artemisinin, a novel antimalarial drug: Biochemical and molecular approaches for enhanced production. Planta Med, 69, 1-11 ; Zhang, Y., Teoh, K.H., Reed, D.W., Maes, L., Goossens, A. and Olson, D.J. (2008) The molecular cloning of artemisinic aldehyde A11 (13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. The Journal of biological chemistry, 283, 21501-21508). Therefore, it is critical to improve the artemisinin yield in Artemisia annua (Pulice, G., Pelaz, S. and Matias- Hernandez L. (2016) Molecular Farming in Artemisia annua, a Promising Approach to Improve Anti-malarial Drug Production. Front Plant Sci 18;7:329.).
Many studies have been carried out to investigate the artemisinin biosynthetic pathway. Two molecules of isopentenyl diphosphate (IDP) and one molecule of dimethylallyl
diphosphate (DMADP) are condensed by farnesyl diphosphate synthase (FDS) to farnesyl diphosphate (FDP). FDP is subsequently cyclized into amorpha-4,11 -diene by amorpha-4, 11 -diene synthase (ADS), which is the first unique intermediate of artemisinin biosynthesis (Bouwmeester, H.J., Wallaart, T.E., Janssen, M.H., van Loo, B., Jansen, B.J., Posthumus, M.A., Schmidt, C.O., de Kraker, J.W., Konig, W.A. and Franssen, M.C. (1999) Amorpha-4,11 -diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry, 52, 843-854; Mercke, P., Bengtsson, M., Bouwmeester, H.J., Posthumus, M.A. and Brodelius, P.E. (2000) Molecular cloning, expression, and characterization of amorpha-4,11 -diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua Archives of biochemistry and biophysics, 381 , 173-180). Amorpha-4, 11 -diene is then oxidized by amorpha-4, 11 -diene 12-hydroxylase (CYP71AV1), reduced by artemisinic aldehyde A11 (13) reductase (DBR2) to dihydroartemisinic aldehyde and again oxidized by aldehyde dehydrogenase 1 (ALDH1) into the direct precursor of artemisinin, dihydroartemisinic acid (DHAA) (Teoh, K.H., Polichuk, D.R., Reed, D.W., Nowak, G. and Covello, P.S. (2006) Artemisia annua (Asteraceae) trichome-specific cDNAs reveal CYP71AV1 , a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. . FEBS letters, 580, 1411-1416; Zhang et al., 2008, already mentioned above; Teoh, K.H., Polichuk, D.R., Reed, D.W. and Covello, P.S. (2009) Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany 87, 635-642). The final production step is considered to be a non-enzymatic reaction (Brown, G.D. (2010) The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua Molecules, 15, 7603-7698; Covello, P.S. (2008) Making artemisinin, Phytochemistry, 69). However, recent results have proposed that a peroxidase enzyme or an alternative series of oxidations, occurring exclusively in plants, may in fact catalyse the crucial last reaction that converts the precursor into the valuable artemisinin molecule (Bryant, L., Flatley, B., Patole, C., Brown, G.D., and Cramer, R.(2015). Proteomic analysis of Artemisia annua - towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin. BMC Plant Biol; 15: 175).
The limitations explained above for artemisinin also apply to other MAPs and ingredients, metabolites and biomolecules derived thereof.
As stated above, the agriculture sector has today an increased focus on improving crop yield per hectare, availability of arable land, and adaptation to global climate change (drought, salinity, higher temperatures or floods). Due to that reasons, several solutions have already been proposed, as, for example: Bryosei (SEIPASA), Mixfol B or BialMicro
(BIAGRO), Equilibrium (BIOIBERICA), Ikasu or Algamix (LIDA), Plus (MANVERT), Biotryg (YARA), Citogrower or Frutaliv (FUTURECO), Agrialgae (ALGAENERGY) and SM6 (PLYMAG), which are directed at nutrient and water use efficiency, soil improvements, abiotic stress mitigation and plant nutrition mainly in fruit and vegetables productions, but not in MAPs (Dunham Trimmer, International Bio Intelligence, 2017; http://dunhamtrimmer.com/).
Therefore, there is the need in the state of art of non-contaminating, chemical-free compositions (reducing, hence, contamination of the environment and of the products produced within the crops and improving safety of the compositions) which allow to increase productivity and yield of crops (preferably, MAPs, more preferably, Artemisia annua).
DESCRIPTION OF THE INVENTION
The inventors of the present invention, after extensive and exhaustive research, have surprisingly found compositions comprising natural ingredients (this is, without the need of chemicals - chemical-free) and, hence, respectful with the environment and safe, which allows to:
Increase and/or accelerate growth of plants.
Increase biomass of plants.
- Accelerate flowering of plants.
Increase the number or density of trichomes, preferably GSTs, allowing the use of said trichomes as natural bio-factories for the increased production of plant derived ingredients (preferably, biomolecules, even more preferably, metabolites) of interest.
Increase the production of plant-derived ingredients (preferably, biomolecules, even more preferably, metabolites) of interest in plants.
In the context of the present invention, plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia,
Lavandula hybrida, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis, Origanum vulgare, Hypericum perforatum, Cannabis spp, Thymus spp, Mentha spp, Cnicus benedictus, Humulus lupulus, Urtica dioica, Melissa officinalis, Moringa olifeira, Hyssopus officinalis, Echinacea purpurea, Cassia angustifolia, Equisetum arvense, Coriandrum sativum, Pelargonium graveolens, Nicotiana tabacum, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua, even more preferably Artemisia annua.
In a first aspect, the present invention refers to a composition comprising: fermented rice starch; and extract obtained from Urtica.
More preferably, in this first aspect, the present invention refers to a composition comprising: fermented rice starch; fungi of the genus Trichoderma', and extract obtained from Urtica.
In a further aspect, the present invention refers to the use of a composition of the present invention as supplementary plant preparation.
In a third aspect, the present invention refers to the use of a composition of the present invention to increase growth of a plant, to accelerate growth or lifecycle of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant.
In a final aspect, the present invention refers to method to increase growth of a plant, to accelerate growth or lifecycle of a plant, to increase trichome density of a plant and/or to increase a plant-derived ingredient production in a plant comprising the application of a composition of the present invention to the plant.
As used herein, “Medicinal and Aromatic Plant”, “MAP” and their plurals are used interchangeably and acquire the common meaning they have in the state of the art, this is, they are botanical raw materials, also known as herbal drugs that are primarily used for therapeutic, aromatic and/or culinary purposes as components of cosmetics,
medicinal products, functional foods and other natural health products. They are also the starting materials for value-added processed natural ingredients such as essential oils, dry and liquid extracts and oleoresins. Examples of MAPs are: Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp.
Also, as used herein, “supplementary plant preparation” and its plural refer to naturally produced substance/s synthetized in plants and/or micro-organisms whose function when applied to plants or the rhizosphere surrounding the plants is to improve physiological processes such as crop quality traits, nutrient use efficiency or tolerance to biotic and abiotic stress.
Therefore, as noted above, in a first aspect, the present invention refers to a composition comprising: fermented rice starch; and extract obtained from Urtica.
More preferably, in this first aspect, the present invention refers to a composition comprising: fermented rice starch; fungi of the genus Trichoderma', and extract obtained from Urtica.
In the composition of the present invention, fermented rice starch is preferably a single fermented rice starch (this is, fermented rice starch preferably from a single rice species) or a combination of fermented rice starches (preferably a combination of fermented rice starches from two or more rice species); Fermented rice starch can be obtained from any rice species, most preferably is from Oryza sativa. Most preferably, fermented rice starch in the composition of the present invention is a fermented rice starch from Oryza sativa.
Fermented rice starch can be prepared by any method or process known in the state of the art. In a preferred embodiment, fermented rice starch is prepared by means of a process comprising the following steps:
a) providing rice starch; b) applying a temperature of between 20 to 40 °C for between 24 hours and 120 hours to the rice starch to obtain a naturally fermented rice starch product; c) keeping the naturally fermented rice starch product obtained in step b) in water, under stirring; d) keeping the product obtained in step c) at ambient temperature (preferably, 22°C to 25°C) for between 10 and 30 hours; e) filtering the product of step d) and obtaining said filtrate to be used as fermented rice starch in the composition of the present invention.
Preferably, in step a) rice starch is provided in powder. In addition, as noted above, rice starch can be from any rice species, most preferably rice starch is from Oryza sativa.
Also preferably, in step b) the temperature is between 35°C and 37°C for 72 hours. In addition, in a preferred embodiment, step b) is carried out in an incubator.
Step b) as explained above provides for a spontaneous and natural lactic fermentation carried out by lactobacilli without the need for inoculation.
Preferably, step c) lasts for between 2 and 10 hours, more preferably between 3 and 4 hours. In addition, in step c) the stirring is preferably homogenous, slow and constant. More preferably, in step c) stirring is at 400 rpm (revolutions per minute).
Preferably, step d) lasts for 20 hours. In addition, in a preferred embodiment, step d) is performed without stirring.
In addition, in step e) mentioned above, filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve. Preferably, in step e) filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
After step e), optionally, the product obtained can be dried to obtain a dried fermented rice starch.
The fermentation carried out in step b) mentioned above allows not only to break proteins into free peptides and amino acids, but also to transform precursor biomolecules into
phytohormones (such as, gibberellins). In addition, the fermentation step can also eliminate hypoallergenic or anti-nutritional factors that may be present (for example, trypsin inhibitors, phytate, oligosaccharides such as raffinose and stachyose, and saponins in legumes).
Hence, fermented rice starch provides for, in the composition of the present invention, bioactive peptides, soluble proteins, free amino acids and phytohormones. In a preferred embodiment of the composition of the present invention the fermented rice starch provides for one or more phytohormones. In the most preferred of the composition of the present invention the fermented rice starch provides for at least one gibberellin.
Gibberellins are plant hormones (phytohormones) and include gibberellic acid and derivatives thereof. Increases in these natural phytohormones usually occur due to fermentation processes with lactic bacteria (lactobacilli) or the Gibberela Fujimori fungus. The Gibberellin family is involved in many physiological functions from germination, formation of sexual organs, flower inductions, abiotic stresses, cell division, etc...
The at least one gibberellin mentioned above is, preferably, Gibberellin A1 (GA1), Gibberellin A3 (GA3), Gibberellin A4 (GA4) or combinations thereof, more preferably, the at least one gibberellin is GA1 , GA3 or GA4, even more preferably, the at least one gibberellin is GA3.
The activity provided by the fermented rice starch in the composition of the present invention is provided by bioactive peptides, soluble proteins and amino acids, more preferably by phytohormones, even more preferably by at least one gibberellin.
Also preferably, the fermented rice starch (as explained above) is at a concentration of between 0.0066 mg/mL and 0.24 mg/mL, more preferably, of between 0.0132 and 0.12 mg/mL, even more preferably, 0.04 mg/mL.
Moreover, in the composition of the present invention it is contemplated that the fungi of the genus Trichoderma are fungi from one or more species of the genus Trichoderma. Preferably, the fungi of the genus Trichoderma are Trichoderma virens fungi (preferably, of the strain TV 29.8, GV41 or combinations thereof), Trichoderma atroviride fungi (preferably, of the strain IMI 206040), Trichoderma harzianum fungi (preferably, Trichoderma harzianum RIFAI strain T-22) or combination thereof, more preferably Trichoderma harzianum fungi, even more preferably, Trichoderma harzianum RIFAI
strain T-22 fungi.
The concentration of the fungi of the genus Trichoderma (as explained above) is preferably between 0.0026 mg/mL and 0.096 mg/mL, more preferably between 0.0053 and 0.048 mg/mL, even more preferably, 0.016 mg/mL.
The fungi from the genus Trichoderma (preferably, Trichoderma harzianum RIFAI strain T-22) in the composition of the present invention provide, at least, for an increase in productivity of the crop, provide nutrients and resistance to abiotic (for example, drought and salinity) and biotic stresses (for example, pathogens).
In the composition of the present invention, the extract obtained from Urtica is preferably a single extract obtained from Urtica (this is, an extract obtained, preferably, from a single Urtica species) or a combination of extracts obtained from Urtica (preferably, a combination of extracts obtained from two or more Urtica species).
In a preferred embodiment, in the composition of the present invention, the extract obtained from Urtica is an extract obtained from Urtica dioica. In addition, the concentration of the extract obtained from Urtica (preferably, Urtica dioica) is between 0.00175 mg/mL and 0.0636 mg/mL, more preferably between 0.0035 mg/mL and 0.0318 mg/mL, even more preferably, 0.0106 mg/mL.
Moreover, in the composition of the present invention it is contemplated that the extract obtained from Urtica is obtained by any means or process available in the state of the art, more preferably, it is obtained by means of a process comprising the following steps: a) providing dry plant material from Urtica-, b) keeping the dry plant material provided in step a) in water, under heating and stirring; c) filtering the material of step b) and obtaining said filtrate to be used as an extract obtained from Urtica in the composition of the present invention.
Preferably, in step a) the dry plant material comprises dry leaves with secondary stems, more preferably, in step a) the dry plant material consists of dry leaves.
Preferably, in step b) the temperature is between 40 °C and 50 °C, more preferably 45 °C. Also preferably, step b) lasts for between 12 and 36 hours, more preferably, 24 hours.
Also preferably, in step b) the dry plant material is at a concentration of between 5 g/L and 20 g/L in the water, more preferably at a concentration of 12 g/L.
In step b) the stirring is preferably homogenous, slow and constant. More preferably, in step b) stirring is at 400 rpm (revolutions per minute).
In addition, in step c) mentioned above, filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve. Preferably, in step c) filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
The extract obtained from Urtica (preferably, an extract obtained from Urtica dioica) mainly comprises organic acids (preferably, acetic acid, citric acid, butyric acid), chlorophyll, flavonoids, tannins, mineral salts (iron, sulphur, manganese, potassium), carotenes, histamine and acetylcholine, vitamins A, B2, K1 and folic acid.
The extract from Urtica (preferably, an extract obtained from Urtica dioica) in the composition of the present invention, at least, fortifies and stimulates the microbial flora of the soil and leaves; strengthens plants; is used as organic fertilizer rich in nitrate; fights against chlorosis; and promotes photosynthesis.
As can be directly derivable from the examples included below the composition of the present invention as described above, wherein fermented rice starch; and an extract obtained from Urtica (preferably, fermented rice starch; fungi of the genus Trichoderma', and an extract obtained from Urtica) are used in combination provides for a synergistic and unexpected effect, which goes beyond the individual effect observed for each of its components and which solves the technical problems observed in the state of the art. This is, it provides for a composition which can (as exemplified below in Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua) :
- Accelerate the growth of plants, allowing for an increased number of crops per year.
Increase biomass production of plants, including, an increased number of leaves and bigger leaves.
Increase the number and density of trichomes, more preferably glandular- shape trichomes.
Hence, increase the production of plant-derived ingredients of interest (preferably, biomolecules, more preferably, metabolites) (faster and in increased amounts), as exemplified by artemisinin and flavonoids (preferably, casticin and eupatorin) production in Artemisia annua.
As already stated above, in the context of the present invention, plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia, Lavandula hybrida, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis, Origanum vulgare, Hypericum perforatum, Cannabis spp, Thymus spp, Mentha spp, Cnicus benedictus, Humulus lupulus, Urtica dioica, Melissa officinalis, Moringa olifeira, Hyssopus officinalis, Echinacea purpurea, Cassia angustifolia, Equisetum arvense, Coriandrum sativum, Pelargonium graveolens, Nicotiana tabacum, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua, even more preferably Artemisia annua.
Therefore, the composition of the present invention enables the use of plants as biofactories to obtain an increased production of plant-derived ingredients of interest.
In a further preferred embodiment of the composition of the present invention, said composition of the present invention further comprises one or more of: extract obtained from Centella’, extract obtained from Echinacea’, extract obtained from Taraxacum’, or combinations thereof.
Therefore, in an embodiment, the composition of the present invention further comprises an extract obtained from Centella.
In another embodiment, the composition of the present invention further comprises an extract obtained from Echinacea.
In a further embodiment, the composition of the present invention further comprises an extract obtained from Taraxacum.
In a most preferred embodiment, the composition of the present invention further comprises an extract obtained from Centella, an extract obtained from Echinacea and an extract obtained from Taraxacum.
In the composition of the present invention, the extract obtained from Centella is preferably a single extract obtained from Centella (this is, an extract obtained, preferably, from a single Centella species) or a combination of extracts obtained from Centella (preferably, a combination of extracts obtained from two or more Centella species). Preferably, the extract obtained from Centella is an extract obtained from Centella asiatica. Also preferably, the extract obtained from Centella (preferably, Centella asiatica) is present in a concentration of between 0.0010 mg/mL and 0.0372 mg/mL, more preferably between 0.0020 mg/mL and 0.0186 mg/mL, even more preferably 0.062 mg/mL.
In the composition of the present invention, the extract obtained from Taraxacum is preferably a single extract obtained from Taraxacum (this is, an extract obtained, preferably, from a single Taraxacum species) or a combination of extracts obtained from Taraxacum (preferably, a combination of extracts obtained from two or more Taraxacum species). Preferably, the extract obtained from Taraxacum is an extract obtained from Taraxacum officinale. Also preferably, the extract obtained from Taraxacum (preferably, Taraxacum officinale) is present in a concentration of between 0.00095 mg/mL and 0.0346 mg/mL, more preferably between 0.0019 mg/mL and 0.0173 mg/mL, even more preferably 0.0057 mg/mL.
In the composition of the present invention, the extract obtained from Echinacea is preferably a single extract obtained from Echinacea (this is, an extract obtained, preferably, from a single Echinacea species) or a combination of extracts obtained from Echinacea (preferably, a combination of extracts obtained from two or more Echinacea species). Preferably, the extract obtained from Echinacea is an extract obtained from Echinacea purpurea. Also preferably, the extract obtained from Echinacea (preferably, Echinacea purpurea) is present in a concentration of between 0.0022 mg/mL and 0.0798 mg/mL, more preferably 0.0044 mg/mL and 0.0399 mg/mL, even more preferably 0.0133 mg/mL.
The extract obtained from Centella-, the extract obtained from Echinacea-, and/or the extract obtained from Taraxacum are produced or obtained in the same ways as explained above for the extract obtained from Urtica. This is, they can be obtained by any means or process available in the state of the art, more preferably, they are obtained by means of a process comprising the following steps: a) providing dry plant material from the corresponding plant (Centella, Taraxacum or Echinacea, in accordance with what has been explained above); b) keeping the dry plant material provided in step a) in water, under heating and stirring; c) filtering the material of step b) and obtaining said filtrate to be used as an extract obtained from the corresponding plant (Centella, Taraxacum or Echinacea, in accordance with what has been explained above) in the composition of the present invention.
Preferably, in step a) the dry plant material comprises dry leaves with secondary stems, more preferably, in step a) the dry plant material consists of dry leaves.
Preferably, in step b) the temperature is between 40 °C and 50 °C, more preferably 45 °C. Also preferably, step b) lasts for between 12 and 36 hours, more preferably, 24 hours.
Also preferably, in step b) the dry plant material is at a concentration of between 5 g/L and 20 g/L in the water, more preferably at a concentration of 12 g/L.
In step b) the stirring is preferably homogenous, slow, and constant. More preferably, in step b) stirring is at 400 rpm (revolutions per minute).
In addition, in step c) mentioned above, filtering can be carried out by any means known in the state of the art, more preferably, by using a strainer or a sieve, even more preferably a stainless-steel sieve with an internal height of 25mm and diameter of 200mm. Preferably, in step c) filtering is carried out using a pore size of between 63 pm and 150 pm, more preferably, by means of a strainer or a sieve with said pore size.
Preferably, the main components of the extract obtained from Centella (preferably, an extract obtained from Centella asiatica) are: pentacyclic triterpenes and their genins: asian and madecasic acids and heterosides (asiatoside, centelloside, madecasoside
and terminoloside); tannins (up to 25% in weight); flavonoids; fatty acids (preferably, linoleic and/or palmitic); alkaloids (such as hydrocotiline, mucilages, phytosterols (campesterol, sitosterol, stigmasterol)); and free amino acids (alanine, serine, aminobutyrate, aspartate, glutamate, lysine and threonine).
The extract obtained from Centella provides, in the composition of the present invention, at least, antimicrobial and antiviral activities.
Preferably, the main components of the extract obtained from Echinacea (preferably, an extract obtained from Echinacea purpurea) are: vitamins of groups C and B (such as riboflavin); beta-carotenes; minerals (such as, iron, sodium, magnesium and calcium); antioxidants; alkamides; and polysaccharides.
The extract obtained from Echinacea provides, in the composition of the present invention, at least, a strengthening of the immune system of the plant.
Preferably, the main components of the extract obtained from Taraxacum (preferably, an extract obtained from Taraxacum officinale) are: iron, potassium, calcium, zinc, magnesium, phosphorus and vitamins such as vitamins A, K, and C.
The extract obtained from Taraxacum provides, in the composition of the present invention, at least, antibiotic activity and increase the defences from the plant.
As it is evident from the examples included below the addition to the composition of the present invention of the extract obtained from Centella’, the extract obtained from Echinacea’, the extract obtained from Taraxacum’, or, preferably, of the combination of the three, surprisingly improves the performance of the composition of the present invention and, hence, also solves the problems present in the state of the art providing for: an even accelerated growth of plants, allowing for an increased number of crops per year.
Higher increase in biomass production of plants, including, an increased number of leaves and bigger leaves.
Increased number and density of trichomes, more preferably glandular-shape trichomes (GSTs).
Hence, increase the production of plant-derived ingredients of interest (preferably, biomolecules, more preferably, metabolites) (faster and in increased amounts), as exemplified by: o artemisinin and flavonoids (preferably, casticin and eupatorina) production in Artemisia annua. o essential oil, viridiflorol, alpha-thujene, cariophyllene oxide, sabinene, alpha-pinene, trans-caryophyllene, spathulenol and camphor production in Salvia officinalis. o essential oil, trans-carveol, Germacrene D, carvacrol and alphapinene production in Origanum vulgare. o essential oil, linalyl acetate, valencene, z-beta-farnesene, transcaryophyllene, Borneol and 1 ,8 cineole production in Lavandula latifolia. o essential oil, t-cadinol, alpha-bisabolol, Borneol, trans-carveol and geranyl acetate production in Lavandula hybrida. o essential oil, camphor, beta-eudesmol, caryophillene oxide, limonene, terpinolene and alpha-trans bergamotene production in Rosmarinus officinalis.
As already stated above, in the context of the present invention, plants are, preferably, MAPs, more preferably Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia, Lavandula hybrida, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis, Origanum vulgare, Hypericum perforatum, Cannabis spp, Thymus spp, Mentha spp, Cnicus benedictus, Humulus lupulus, Urtica dioica, Melissa officinalis, Moringa olifeira, Hyssopus officinalis, Echinacea purpurea, Cassia angustifolia, Equisetum arvense, Coriandrum sativum, Pelargonium graveolens, Nicotiana tabacum, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua, even more preferably Artemisia annua.
It is contemplated that the composition of the present invention comprises further ingredients or components. For example, carriers or excipients which do not alter or do
not alter in an unacceptable manner the activity of the composition of the present invention.
The composition of the present invention, in any of the above-mentioned embodiments is preferably applied in a quantity of between 45 and 576 litres per hectare, more preferably, between 90 and 288 litres per hectare. Said total quantity is preferably applied by means of 2 to 10 applications, more preferably, between 4 and 8 applications. Also preferably said applications are performed at least once per week, more preferably at least twice per week, even more preferably twice per week.
The above-mentioned applications are preferably performed in the plant (preferably in the leaves) and in the soil. More preferably, between 75% and 90% of the volume of each application is applied in the plant (preferably in the leaves) and between 10% and 25% of the volume of each application is applied in the soil. Even more preferably, 85% of the volume of each application is applied in the plant (preferably in the leaves) and 15% of the volume of each application is applied in the soil.
The above-mentioned application of the composition of the present invention can be carried out by any means known in the state of the art, more preferably, by means of sulphating backpack or trolley.
The composition of the present invention is preferably used in plants in the juvenile state.
A person skilled in the art will be able to make the required adaptations to the number of applications, the total quantity applied, and the moment and frequency of application based on the particulars of the situation and of the selected plant.
It is contemplated that the composition of the present invention is in a concentrated form, this is, in a form which must be diluted before being used. For example, the composition of the present invention can be in a concentrated form of between 10x to 500x, more preferably, between 10x and 100x, more preferably, between 50x and 100x, even more preferably, the composition of the present invention is in a 50x or 100x concentrated form. In these cases, all the concentration ranges noted above should be multiplied by the corresponding concentration factor to obtain the concentration in the concentrated form. As it is self-evident, this concentrated forms, before being used will have to be diluted the same number of times as they are concentrated (for example, a 50x
concentrated composition will have to be diluted 50 times before being used and a 100x concentrated composition will have to be diluted 100 times before being used).
Preferably, the present composition is stored in a concentrated form. In addition, preferably, said storage is in cold, more preferably at between 4 °C and 12 °C, more preferably at between 8 °C and 9 °C.
The composition of the present invention acts stimulating and accelerating the growth of plants, preferably, MAPs, more preferably, Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia, Lavandula hybrida, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis, Origanum vulgare, Hypericum perforatum, Cannabis spp, Thymus spp, Mentha spp, Cnicus benedictus, Humulus lupulus, Urtica dioica, Melissa officinalis, Moringa olifeira, Hyssopus officinalis, Echinacea purpurea, Cassia angustifolia, Equisetum arvense, Coriandrum sativum, Pelargonium graveolens, Nicotiana tabacum, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua, even more preferably Artemisia annua. The composition of the present invention also increases the production of molecules and/or biomolecules of interest in plants (preferably, MAPs, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Artemisia annua, even more preferably Artemisia annua). Hence, the composition of the present invention is, preferably, a supplementary plant preparation able to provide the above effects.
In a second aspect, the present invention refers to the use of a composition of the present invention (as explained above in the first aspect of the present invention) as supplementary plant preparation.
In this second aspect, the supplementary plant preparation is for the treatment of a plant.
It is contemplated that the plant can be any plant known in the state of the art or which is discovered or generated in the future. Preferably, the plant is a MAP, more preferably, Artemisia spp, Lavandula spp, Cannabis spp, Ocimum spp, Salvia spp, Rosmarinus
spp, Origanum spp, Mentha spp, Humulus spp, Cnicus spp, Thymus spp, Urtica spp, Nicotiana spp, Hypericum spp, Melissa spp, Moringa spp, Hyssopus spp, Echinacea spp, Cassia spp, Equisetum spp, Coriandrum spp, Pelargonium spp, more preferably, Artemisia spp (preferably, Artemisia annua, Artemisia absinthium, Artemisia afra, Artemisia vulgaris, Artemisia thuscula, Artemisia capillaris), Lavandula latifolia, Lavandula hybrida, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis, Origanum vulgare, Hypericum perforatum, Cannabis spp, Thymus spp, Mentha spp, Cnicus benedictus, Humulus lupulus, Urtica dioica, Melissa officinalis, Moringa olifeira, Hyssopus officinalis, Echinacea purpurea, Cassia angustifolia, Equisetum arvense, Coriandrum sativum, Pelargonium graveolens, Nicotiana tabacum, more preferably, Salvia officinalis, Origanum vulgare, Lavandula latifolia, Lavandula hybrida, Rosmarinus officinalis, Ocimum basilicum, Cannabis spp or Artemisia annua, even more preferably Artemisia annua.
Preferably, the use in this second aspect of the present invention is in a plant in the juvenile state. This provides for an even increased acceleration of the flowering.
In addition, the composition of the present invention, the quantity applied thereof and how it is applied is as already explained in the first aspect of the present invention.
In a third aspect, the present invention refers to the use of a composition of the present invention to increase growth of a plant, to accelerate growth of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant.
The composition of the present invention, the quantity applied thereof and how it is applied is in accordance with the first aspect of the present invention.
The plant is as already explained in the second aspect of the present invention.
The increase in the growth of the plant is preferably an increase in the biomass thereof, more preferably, an increase in the number of leaves, an increase in the area of the leaves, an increase in the height of the plant, an increase in internode number or combinations thereof. As it is known in the state of the art, the internodes were branches originate from the amin stem (therefore, an increase in the number of internodes will provide an increase in the number of branches and, on its turn, an increase in the number of leaves).
The increase in the number of leaves and/or in the area of the leaves, preferably, provides for an increase in the number of trichomes, an increase in the density of trichomes or combinations thereof.
Preferably, trichomes are glandular-shape trichomes (GSTs).
The accelerated growth of a plant preferably comprises an accelerated flowering (this is, a reduction of the flowering time), an accelerated biomass production (preferably, number of leaves and/or area of the leaves) or combinations thereof.
Regarding the flowering, its acceleration provides several advantages:
Trichomes (preferably, GSTs) mature just before flowering. Therefore, an acceleration of the flowering provides an acceleration of the production of plant-derived ingredients of interest (preferably, biomolecules of interest, more preferably, metabolites of interest).
The acceleration of the flowering provides for a reduction of the lifecycle of the plant and, therefore, an increased production (more harvests can be produced each year),
With regard to the increase in a plant-derived ingredient, any plant-derived ingredient is contemplated, for example, biomolecules, essential oils, dry/liquid extracts, oleoresins and active pharmaceutical ingredients. More preferably the plant-derived ingredient is an essential oil and/or a biomolecule. The biomolecule can be any biomolecule naturally produced by the plant or any biomolecule the production of which has been artificially induced (by any method or means know in the state of the art) in the plant. More preferably, the plant-derived ingredient is a biomolecule, even more preferably a metabolite, still more preferably, a metabolite produced in the trichomes (preferably, in the GSTs) of the plant.
In a preferred embodiment, the plant is Salvia officinalis and then, preferably the plant- derived ingredient is: essential oil, viridiflorol, alpha-thujene, cariophyllene oxide, sabinene, alpha-pinene, trans-caryophyllene, spathulenol, camphor or combinations thereof.
In another preferred embodiment, the plant is Origanum vulgare and then, preferably, the plant-derived ingredient is: essential oil, trans-carveol, Germacrene D, carvacrol, alpha-pinene or combinations thereof.
In a further preferred embodiment, the plant is Lavandula latifolia and then, preferably, the plant-derived ingredient is: essential oil, linalyl acetate, valencene, z-beta-farnesene, trans-caryophyllene, Borneol, 1 ,8 cineole or combinations thereof.
In an additionally preferred embodiment, the plant is Lavandula hybrida and then, preferably, the plant-derived ingredient is: essential oil, t-cadinol, alpha-bisabolol, Borneol, trans-carveol, geranyl acetate or combinations thereof.
In a further preferred embodiment, the plant is Rosmarinus officinalis and then, preferably, the plant-derived ingredient is: essential oil, camphor, beta-eudesmol, caryophillene oxide, limonene, terpinolene, alpha-trans bergamotene or combinations thereof.
In a most preferred embodiment, the plant is Artemisia annua and then, preferably, the metabolite is a sesquiterpene and/or a flavonoid, more preferably, casticin, euparotin and/or artemisinin, even more preferably, artemisinin.
Preferably, the use in this third aspect of the present invention is in a plant in the juvenile state. This provides for an even increased acceleration of the flowering.
In a fourth and final aspect, the present invention refers to a method to increase growth of a plant, to accelerate growth of a plant, to increase trichome density of a plant and/or to increase a plant-derived ingredient production in a plant comprising the application of a composition of the present invention to the plant.
The composition of the present invention, the quantity applied thereof and how it is applied is in accordance with the first aspect of the present invention.
The plant, the increase in the growth, the accelerated growth, the increase in the plant- derived ingredient production and the plant-derived ingredient are as explained above in the third aspect of the present invention.
As used herein, the term “comprise” and its plural, do not exclude the terms “consisting” and “consisting essentially of”. Therefore, also within the scope of the present invention are all the above embodiments in which the term “comprising” is substituted by “consisting” or by “consisting essentially of”.
SEQUENCE LISTING FREE TEXT
SEQ ID NO: 1
<223> ADS forward primer
SEQ ID NO: 2
<223> ADS reverse primer
SEQ ID NO: 3
<223> CYP71AV1 forward primer
SEQ ID NO: 4
<223> CYP71AV1 reverse primer
SEQ ID NO: 5
<223> DBR2 forward primer
SEQ ID NO: 6
<223> DBR2 reverse primer
SEQ ID NO: 7
<223> p-ACTIN forward primer
SEQ ID NO: 8
<223> p-ACTIN reverse primer
BRIEF DESCRIPTION OF DRAWINGS
To allow a better understanding, the present invention is described in more detail below with reference to the enclosed figures, which are presented by way of example, and with reference to illustrative and non-limitative examples.
Figure 1 shows scanning electron microscope (SEM) of the results obtained in the different treatment groups tested in example 1 included below, for the number of trichomes. More precisely, figure 1A refers to the control (no treatment applied), figure 1 B refers to the group to which cytokinins were applied, figure 1C refers to the group to which jasmonic acid was applied, figure 1 D refers to the group to which Trichoderma harzianum RIFAI strain T-22 were applied, figure 1 E refers to the group to which the extract obtained from Urtica dioica was applied, figure 1 F refers to the group to which fermented rice starch was applied, figure 1G refers to the group to which Composition 1 (for details see example 1), and figure 1 H refers to the group to which Composition 2 (for details see example 1).
Figure 2 is a picture of the results obtained in the different treatment groups tested in example 4 included below. More precisely, figure 2A refers to the control (no treatment applied), figure 2B refers to the group to which cytokinins were applied, figure 2C refers to the group to which jasmonic acid was applied, figure 2D refers to the group to which the extract obtained from Urtica dioica was applied, figure 2E refers to the group to which Trichoderma harzianum RIFAI strain T-22 were applied, figure 2F refers to the group to which fermented rice starch was applied, figure 2G refers to the group to which Composition 1 (for details see example 1), and figure 2H refers to the group to which Composition 2 (for details see example 1).
Figure 3 is a picture of results obtained in example 4 included below. More precisely, compared are plants treated with Composition 1 (left; see example 1 for the details on the composition) and control plants (right; plant not treated).
EXAMPLES
Example 1. Analysis of trichome density in Artemisia annua.
Seeds of Artemisia annua varieties Anamed (www.anamed.net) and Chongqing (Professor K. Tang, Shanghai Jiao Tong University) were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
For all the phenotypic analysis, all the experiments were repeated at least twice. Unless
otherwise specified, a minimum of 20 plants was used for trichome analysis for each developmental stage and treatment combination. Treatments with the different compounds and combinations were done between week 4 and week 8 (from sowing), twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
Trichome initiation was monitored using an Olympus DP71 microscope by counting all trichomes on the adaxial surface of individual and fully developed upper leaves. Diverse upper leaves were counted independently given that these leaves showed different trichome production. Leaves were chosen at 56 days (8 weeks) after germination due to the fact that at this stage they were fully developed, and the full treatment had been completed.
In the present example, the following treatment groups were:
Control: no treatment applied.
Cytokinins, used at a concentration of 0.02 mg/mL
Jasmonic acid, used at a concentrarion of 0.02 mg/mL
Trichoderma harzianum RIFAI strain T-22, used at a concentration of 0.016 mg/mL
Extract obtained from Urtica dioica, used at a concentration of 0.0106 mg/mL Fermented rice starch (Oryza sativa), used at a concentration of 0.04 mg/mL Composition 1 : Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Data is reported as mean value and standard deviation of the trichome density for each treatment group. Trichomes SEM images required for the analysis were obtained as
previously described by Sanchez-Chardi et al, 2010 (Sanchez-Chardi A, Olivares F, Byrd TF, Julian E, Brambilla C, Luquin M (2011) Demonstration of cord formation by rough Mycobacterium ab-scessus variants: implications for the clinical microbiology laboratory. JCIin Microbiol 49:2293-2295.). In short, samples were fixed in 2.5% (volume/volume) glutaraldehyde in 0.1 M Phosphate buffer saline (PBS) (pH 7.4) for 2 h at 4°C, washed 4 times for 10 min each time in 0.1 M PBS, postfixed in 1 % (volume/volume)osmium tetraoxide with 0.7% (volume/volume) ferrocyanide in PBS, washed in water, dehydrated in an ascending ethanol series (50, 70, 80, 90, and 95% (volume/volume) for 10 min each and twice with 100% ethanol), and dried by critical-point drying with CO2.
Table 1. Results of Trichome density (mean and standard deviation) obtained for the different experimental groups tested in example 1.
Results obtained in this example appear summarized in Table 1 and Figure 1 . As can be directly derivable from the results obtained in this example, Compositions 1 and 2 showed a synergistic and unexpected increase in trichome density in the Artemisia annua plants. As shown in Table 1 , the individual components of the Compositions
showed a little to moderate effect in increasing the thrichome density. However, when they were used in combination (Compositions 1 and 2), thay acted synergistically and produced and unexpectedly superior increase in trichome density (even higher in combination 2).
In addition, in this experiment Cytokinins and Jasmonic acid were tested as their activity seemed to make them candidates susceptible to increase trichome density (both are very important phytohormones with activities related with the health and growth of plants), but, as it can be seen in Tables 1 and Figure 1 , their effect was minimal or even negative.
Example 2. Analysis of artemisinin production in Artemisia annua.
The amount of artemisinin in the plants of example 1 was determined by extraction and analysis on a Waters Alliance 2695 HPLC system coupled with a Waters 2420 ELSD detector as previously described (Jiang W, Fu X, Pan Q, Tang Y, Shen Q, Lv Z, Yan T, Shi P, Li L, Zhang L, Wang G, Sun X, and Tang K (2016). Overexpression of AaWRKYI Leads to an Enhanced Content of Artemisinin in Artemisia annua. Biomed Res Int: 7314971).
Extraction of artemisinin from Artemisia annua.
First, 0.010 g of dried and milled leaves of Artemisia annua, then 1 mL of HPLC (high- performance liquid chromatography) quality acetone: milli-Q water (volume:volume, v:v; at 1 :1) was added and stirred under vortex for 1 minute. Afterwards, it was subject to 5 minutes of ultrasounds and centrifuged at 4,000 rpm (revolutions per minute) at 20 °C for 5 minutes and the supernatant was obtained. The obtained pellet was also obtained and reprocessed following this same process to obtain a further supernatant. The two supernatants obtained were mixed.
Measurement of artemisinin:
It was performed by means of UHPLC (ultra-high-performance liquid chromatography) adapting the HPLC (high-performance liquid chromatography) protocol disclosed in Bilia et al. 2006 (Bilia AR, Melillo de Malgalhaes P, Bergonzi M C, Vincieri F F(2006). Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia annua obtained from a commercial sample and a selected cultivar. Phytomedicine 2006 Jul;13(7):487-93).
Detection was performed by means of mass spectrometry (MS) MS/MS (triple quadrupole). The experimental conditions used were: Table 2. UHPLC conditions used in example 2.
The transitions used for the identification and quantification of artemisinin were: from 283.17 ppb (parts per billion) to 151.09 ppb and from 283.17 ppb to 209.08 ppb, all in mass/charge number of ions (m/z), with a quantification limit of 2.5 ppb.
The results obtained in this example appear summarized in Table 4.
Table 4. Results of artemisinin content (mean and standard deviation) obtained for the different experimental groups tested in example 2.
As can be directly derivable from the results obtained in this example, Compositions 1 and 2 showed a superior and unexpected increase in artemisinin content in the Artemisia annua plants, which can be considered as a result of a synergistic interaction between the different constituents. As shown in Table 4, the individual components of the Compositions showed a little to moderate effect in increasing artemisinin content. However, when they were used in in combination (Compositions 1 and 2), thay acted synergistically and produced and unexpectedly superior increase in artemisinin content (even higher in Composition 2).
In addition, in this experiment the effect of Cytokinins and Jasmonic acid was minimal or even negative.
The resuts obtained in this example are even more important, surprising and unexpected if we take into account that in the state of the art the maximum levels reached for artemisinin are around 1.5% (weight/weight - w/w, with regard to the dry weight of the plant) (and normally around 1 % w/w, with regard to the dry weight of the plant, in the control non-treated plants), while, with Compositions 1 and 2 almost a 2% w/w (with regard to the dry weight of the plant) of artemisinin was reached. This increase is very important to make feasible the production of biomolecules (more preferably, metabolites) in plants (preferably, MAPs), as exemplified by artemisinin in Artemisia annua.
Example 3. Analysis of the expression of several genes key in artemisinin production in Artemisia annua.
Upper young leaves of the plants used in example 1 (8 weeks after germination) were collected in order to analyze the correlation between the expression levels of genes encoding artemisinin biosynthetic enzymes. cDNA synthesis was carried out as described in Rieu and Powers, 2009 (Rieu, I. and Powers, S.J. (2009) Real-time quantitative RT-PCR: design, calculations, and statistics. The Plant cell, 21 , 1031-1033).
Briefly, for RT-qPCR (reverse transcription quantitative polymerase chain reaction) reactions, RNA was extracted from a pool of 20 plants with PureLink RNA Mini Kit (Ambion), treated with RNase-free DNasel (Ambion) and 1 pg was retrotranscribed with oligo(dT) and SuperScript III (Invitrogen). The expression levels of genes of interest were monitored by qPCR using SYBR Green I Master Mix and Light Cycler 480 (Roche) with the primers listed in table 5.
The primers for each of the genes artemisinic aldehyde A11 (13) reductase (DBR2), amorpha-4, 11 -diene 12-hydroxylase (CYP71AV1), amorpha-4,11 -diene synthase (ADS), and p-ACTIN are listed below in Table 5. Obtained data were normalized using the p-ACTIN gene as reference.
For reverse transcription quantitative polymerase chain reaction (RT-qPCR) reactions, the expression levels of genes of interest were monitored by quantitative polymerase chain reaction (qPCR) using SYBR Green I Master Mix and Light Cycler 480 (Roche) using the primers listed in Table 5. PCR (polymerase chain reaction) efficiency was calculated and determined as previously described in Talke et al., 2006 (Talke, LN., Hanikenne, M. and Kramer, II. (2006) Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant physiology, 142, 148- 167). The results obtained in this example appear summarized in Tables 6 to 8.
Table 6. Results for the expression of ADS gene (normalized by means of the 0- ACTIN gene) obtained for the different experimental groups tested in example 3.
Table 7. Results for the expression of DBR2 gene (normalized by means of the 0- ACTIN gene) obtained for the different experimental groups tested in example 3.
Table 8. Results for the expression of CYP gene (normalized by means of the 0- ACTIN gene) obtained for the different experimental groups tested in example 3.
As can be directly derived from Tables 6 to 8, the expression of the genes ADS, DBR2 and CYP follows a similar trend as that seen in example 2 for artemisinin content, as the highest increase in all cases was the one seen for Compositions 1 and 2.
Example 4. Analysis of the increase in biomass production in Artemisia annua.
The Artemisia annua plants of example 1 were analyzed.
In this phenotypic analysis, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves from at least 10 plants were used for leaf area measurement for each treatment combination.
Leaf area was monitored using an Olympus DP71 microscope by measuring leaf area (pixels/cm2) on individual and fully developed medium leaves. Medium leaves area was measured independently 63 days (9 weeks) after germination due to the fact that at this stage medium leaves were fully developed and the full treatment for the different groups had been previously completed. The results obtained appear summarized in Table 9 included below and Figures 2 and 3.
Table 9. Results of leaf area (mean and standard deviation) obtained for the different experimental groups tested in example 4.
Leaf number per plant was monitored by counting all leaves from 10 independent Artemisia annua plants for each treatment group. Leaves were counted 70 days (10 weeks) after germination when they were completely developed. Results obtained appear summarized in Table 10 included below and Figures 2 and 3.
Table 10. Results of the number of leaves per plant (mean and standard deviation) obtained for the different experimental groups tested in example 4.
Both parameters tested in this example (leaf area and number of leaves per plant) are indicative of the biomass of a plant. In both parameters a similar trend was seen.
More precisely, for both features, leaf area and number of leaves per plant (see Tables 9 and 10, respectively), Trichoderma harzianum RIFAI strain T-22 and the extract obtained from Urtica dioica showed a little effect while fermented rice starch (Oryza sativa) showed a moderate effect. However, Compositions 1 and 2 showed a synergistic and unexpected increased effect in both parameters, which exceedes the individual performance of the individual components and which is even greater for Composition 2 (showing the improving effect of the extract obtained from Echinacea, the extract obtained from Centella asiatica and of the extract obtained from Taraxacum officinale).
In addition, in this example Cytokinins and Jasmonic acid as can be derived from Tables 9 and 10 had a minimal o even a negative effect in the two analysed biomass parameters.
Example 5. Analysis of the acceleration of the flowering in Artemisia annua.
For flowering time measurements, plants were randomized with the respective controls and grown on soil in controlled environment growth chambers. Flowering time was determined as the number of days from sowing to the appearance of the floral bud of at least 20 individual plants for each treatment. The number of days to flowering was
determined when the first floral buds were visible to the naked eye. Indeed, Artemisia annua plants were carefully checked for visible signs of flowering every two days. All flowering time assays were performed at least twice. Flowering time data were subjected to analyses of variance (ANOVA). Post-hoc tests were performed using Tukey’s multiple comparisons test after two-way ANOVA. Statistical analyses were performed with Prism 6 software (GraphPad Software, Inc).
Treatments were applied in accordance with what has been explained in example 1 included above.
The treatment groups tested in this example were:
Control: no treatment applied.
Cytokinins, used at a concentration of 0.02 mg/mL
Jasmonic acid, used at a concentrarion of 0.02 mg/mL
Trichoderma harzianum RIFAI strain T-22, used at a concentration of 0.016 mg/mL
Extract obtained from Urtica dioica, used at a concentration of 0.0106 mg/mL Fermented rice starch (Oryza sativa), used at a concentration of 0.04 mg/mL Composition 1 : Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
The results obtained in this example appear summarized in Table 11 included below:
Table 11. Results obtained for the flowering time (mean and standard deviation) obtained for the different experimental groups tested in example 5. Flowering time,
herein, is understood as the number of days from sowing to the appearance of the floral bud of at least 20 individual plants for each treatment.
During their life cycle, plants undergo several developmental transitions. The timing of these transitions is essential for proper development and adjustment of growth to environmental conditions. After germination, plants undergo a juvenile phase of vegetative growth, in which they are unable to flower but still the florigen are being activated genetically. Following the juvenile period, there is a juvenile-to-adult transition, also termed vegetative phase change, leading to an adult phase in which plants become competent to flower under optimal environmental conditions. Although, the juvenile-to- adult transition is associated with diverse visible morphological changes, it is during the previous phase (juvenile phase) when all the floral activator and repressor are well- balanced and active as there is extensive crosstalk among the different genes acting in the flowering time network.
Generally, in response to environmental and endogenous signals, adult plants experience a vegetative-to-reproductive or floral transition. Plants have developed mechanisms to perceive and respond to environmental fluctuations by adjusting their growth as well as to predict upcoming daily and seasonal cues, which result in massive
developmental plasticity (Franklin, KA. Light and temperature signal crosstalk in plant development. Curr Opin Plant Biol. 2009 Feb;12(1):63-8. Review). The timing of developmental transitions is essential for proper growth and adaptation to environmental conditions. Therefore, floral induction has to be precisely controlled and tuned to the environment inputs to guarantee the reproductive success.
In normal conditions, Artemisia annua flowering occurs in month 7th; while at the same time GSTs only get mature (and produce artemisinin) just before the blooming phase. Therefore, it is important, to accelerate Artemisia annua flowering, but in the proper time and not too early in order to afford the proper development of the plant, in order to get artemisinin molecule and other therapeutic molecules produced within the trichomes much earlier.
Phenotypical analyses for the different treatment groups (see Table 11) reveal that Artemisia annua plants:
Flowering almost at the same time when treated with Trichoderma harzianum RIFAI strain T-22 or with Extract obtained from Urtica dioica (almost no difference with regard to the control).
Significant effect of fermented rice starch (Oryza sativa), which produces a much earlier flowering (approximately from 7 to 4 months) when compared with the control.
Synergistic and unexpected effect of Compositions 1 and 2 which produce a very significant decrease in flowering time with regard to the control (from 7 months to approximately 2.5 months). The effect observed for this two Compositions is superior to the addition or sum of the effects seen individually for Trichoderma harzianum RIFAI strain T-22, the extract obtained from Urtica dioica and fermented rice starch (Oryza sativa), demonstrating a synergistic effect, which is even greater in Composition 2 (showing the improving effect of the extract obtained from Echinacea, the extract obtained from Centella asiatica and of the extract obtained from Taraxacum officinale). These results are very interesting in terms of agricultural application, because they allow to have at least 3 crops/year (and up to maximum 5 crops/year, depending on the geographical location) instead of 1 crop per year, increasing hugely the production.
Example 6. Analysis of the effect of the present invention in Salvia officinalis
Seeds of Salvia officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant Biomass (kg/plant) was measured as follows:
Plant material (leaves and secondary branches) were collected at the beginning of the flowering and fresh biomass/plant was determined by weighing in a certified precision balance. Afterwards plant material was dried in a drying chamber at a temperature of between 32 °C and 40 °C until they reach a constant weight. Then, the dried plant material was weighed in a certified precision balance.
The results obtained for plant biomass appear summarized in table 12.
Table 12. Results obtained for fresh and dry biomass in Salvia officinalis in example 6.
In this example, also the percentage of essential oils and of secondary metabolites or biomolecules therein was analyzed. Briefly, once the plant material was dried as mentioned above, it was homogenized and subject to hydro-distillation in a Clevenger type apparatus for 3 hours to obtain the essential oils. The yield in each case was determined as ml of essential oils/mg of plant material. Once the essential oil samples were obtained, their chemical composition was analyzed by means of gas chromatography. The details of the gas chromatography are as follows:
Chromatograph: VARIAN 430 GC
Column: capillary: length 60 mm; internal diameter 0.25 mm
Thickness of film: 0.25 pm
Stationary phase: polydimethylsiloxane (VF-5ms)
Oven temperature:
From 90 °C to 116 °C at a rate of 2 °C/min, from 116 °C to 230 °C at a rate of 4 °C/min, end temperature of 230 °C during 5 minutes
Injector temperature: 250 °C
Detector temperature: 300 °C
Detector: flame ionization type
Carrier gas: Helium (1.0 mL/min)
Volume injected: 0.5 pL
Split ratio: 100:10
The peaks for each of the secondary metabolites or biomolecules was particular for each plant species. Then the area of each of the peaks of interest was measured.
The results obtained for the percentage of essential oils appear summarized in table 13.
The time of appearance in the gas chromatography peaks of the biomolecules analyzed in this example were:
Viridiflorol: 34.16 minutes alpha-thujene: 11.08 minutes caryophyllene oxide: 33.81 minutes sabinene: 12.64 minutes alpha-pinene: 11.39 minutes trans-caryophyllene: 28.68 minutes spathulenol: 33.57 minutes camphor: 5.627 minutes
The results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 14.
Table 14. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Salvia officinalis in example 6. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
Therefore, as it can be derived from tables 12 to 14, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Salvia officinalis, as well as the production of essential oils and of several biomolecules with therapeutic properties.
Example 7. Analysis of the effect of the present invention in Origanum vulgare
Seeds of Origanum vulgare were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life- cycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant biomass (both, fresh and dried) was measured as explained in Example 6.
The results obtained for plant biomass appear summarized in table 15.
The quantity of essential oils was measured as explained in Example 6.
The results obtained for the percentage of essential oils appear summarized in table 16.
The quantity of the biomolecules of interest was measured as explained in Example 6.
The time of appearance in the gas chromatography peaks of the biomolecules analyzed in this example were: trans-carveol: 21.78 minutes - Germacrene D: 33.55 minutes
Carvacrol: 24.28 minutes alpha-pinene: 11.36 minutes
The results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 17. Table 17. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Origanum vulgare in example 7. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
Therefore, as it can be derived from tables 15 to 17, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Origanum vulgare, as well as the production of essential oils and of several biomolecules with therapeutic properties.
Example 8. Analysis of the effect of the present invention in Lavandula latifolia
Seeds of Lavandula latifolia were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant biomass (both, fresh and dry) was measured as explained in Example 6.
The results obtained for plant biomass appear summarized in table 18.
Table 18. Results obtained for fresh and dry biomass in Lavandula latifolia in example 8.
The quantity of essential oils was measured as explained in Example 6 The results obtained for the percentage of essential oils appear summarized in table 19.
Table 19. Results obtained for the percentage of essential oils in Lavandula latifolia in example 8.
The quantity of the biomolecules of interest was measured as explained in Example 6.
The time of appearance in the gas chromatography peaks of the biomolecules analyzed in this example were: linalyl acetate: 22.36 minutes valencene: 30.58 minutes - z-beta-farnesene: 29.16 minutes trans-caryophyllene: 28.69 minutes
Borneol: 20.12 minutes
1 ,8 cineole: 14.84 minutes
The results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 20.
Table 20. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Lavandula latifolia in example 8. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
Therefore, as it can be derived from tables 18 to 20, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Lavandula latifolia, as well as the production of essential oils and of several biomolecules with therapeutic properties.
Example 9. Analysis of the effect of the present invention in Lavandula hybrida
Seeds of Lavandula hybrida were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant biomass (both, fresh and dry) was measured as explained in Example 6.
The results obtained for plant biomass appear summarized in table 21.
Table 21. Results obtained for fresh and dry biomass in Lavandula hybrida in example 9.
The quantity of essential oils was measured as explained in Example 6.
The results obtained for the percentage of essential oils appear summarized in table 22.
The quantity of the biomolecules of interest was measured as explained in Example 6.
The time of appearance in the gas chromatography peaks of the biomolecules analyzed in this example were: t-cadinol: 35.32 minutes alpha-bisabolol: 36.35 minutes
Borneol: 20.12 minutes trans-carveol: 21.63 minutes geranyl acetate: 26.60 minutes
The results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 23.
Table 23. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Lavandula hybrida in example 9. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
Therefore, as it can be derived from tables 21 to 23, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Lavandula hybrida, as well as the production of essential oils and of several biomolecules with therapeutic properties.
Example 10. Analysis of the effect of the present invention in Rosmarinus officinalis
Seeds of Rosmarinus officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was
applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant biomass (both, fresh and dry) were measured as explained in Example 6.
The results obtained for plant biomass appear summarized in table 24.
The quantity of essential oils was measured as explained in Example 6.
The results obtained for the percentage of essential oils appear summarized in table 25.
Table 25. Results obtained for the percentage of essential oils in Rosmarinus officinalis in example 10.
The quantity of the biomolecules of interest was measured as explained in Example 6.
The time of appearance in the gas chromatography peaks of the biomolecules analyzed in this example were:
Camphor: 19.32 minutes beta-eudesmol: 35.77 minutes caryophyllene oxide: 33.78 minutes limonene: 14.64 minutes terpinolene: 16.67 minutes alpha-trans bergamotene: 28.67 minutes
The results obtained for the different biomolecules with therapeutic properties analyzed in this example appear summarized in table 26.
Table 26. Results obtained for the percentage of the different biomolecules with therapeutic properties analyzed in Rosmarinus officinalis in example 10. The percentage noted is the percentage of each of the biomolecules with regard to the total volume of essential oils obtained.
Therefore, as it can be derived from tables 24 to 26, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Rosmarinus officinalis, as well as the production of several biomolecules with therapeutic properties.
Example 11. Analysis of the effect of the present invention in Ocimum basilicum
Seeds of Ocimum basilicum were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa) (0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Plant biomass (both, fresh and dry) was measured as explained in Example 6.
The results obtained for biomass appear summarized in table 27.
In this example, also the number of leaves per plant was measured when the first signs of flowering appeared in the plants.
The results obtained for the leave number appear summarized in table 28.
Table 28. Results obtained for the number of leaves in Ocimum basilicum in example 11.
Therefore, as it can be derived from tables 27 and 28, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Ocimum basilicum.
Example 12. Analysis of the effect of the present invention in Cannabis spp
Seeds of Cannabis spp were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 2: Trichoderma harzianum RIFAI strain T-22 (0.016 mg/mL), Extract obtained from Urtica dioica (0.0106 mg/mL), fermented rice starch (Oryza sativa)
(0.04 mg/mL), extract obtained from Centella asiatica (0.0062 mg/mL), extract obtained from Echinacea purpurea (0.0133 mg/mL) and extract obtained from Taraxacum officinale (0.0057 mg/mL).
Fresh plant biomass was measured as explained in example 6. The results obtained for biomass appear summarized in table 29.
In this case the woody stem was obtained, and the number of internodes was determined by means of visual inspection. In addition, the height of the woody stems was also measured.
The results obtained for the height and number of internodes appear summarized in table 30.
Table 30. Results obtained for height and number of internodes in Cannabis spp in example 12.
Therefore, as it can be derived from tables 29 and 30, the composition of the present invention (as exemplified by composition 2) was effective in increasing the biomass of Cannabis spp.
Therefore, as it is directly derivable from the above examples, the present invention in all its aspects solves the technical problems present in the state of the art and provides for:
- An accelerated the growth of plants (more preferably, MAPs), even more allowing for an increased number of crops per year and yield thereof. Increase in plant growth and in biomass production of plants (more preferably, MAPs), including, an increased number of leaves and bigger leaves.
Increase in the number and density of trichomes, more preferably glandular- shape trichomes.
Increased production of plant-derived ingredients of interest (preferably, essential oil or biomolecules) (faster and in increased amounts).
All this, by means of an environmentally friendly composition and without chemicals harmful for the environment.
Example 13. Analysis of the effect of a composition of the present invention in Salvia officinalis
Seeds of Salvia officinalis were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant life-cycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of the volume of each application was applied to the leaves of the plants and between 10% and 25% of the volume of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 3: Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
Extract obtained from Urtica dioica (0.0106 mg/mL).
Fermented rice starch (Oryza sativa) (0.04 mg/mL).
Plant Biomass (kg/plant) was measured as follows: Plant material (leaves and secondary branches) were collected at the beginning of the flowering and fresh biomass/plant was determined by weighing in a certified precision balance.
Also the height of the plants was measured.
The results obtained for plant biomass and height appear summarized in table 31. Table 31. Results obtained for fresh biomass and height in Salvia officinalis in example 13.
Therefore, as it can be derived from table 31 , the composition of the present invention (as exemplified by composition 3) was effective in increasing in a clearly synergistic way the biomass and height of Salvia officinalis.
Example 14. Analysis of the effect of a composition of the present invention in Ocimum basilicum
Seeds of Ocimum basilicum were used in the experiments. Seeds were grown in soil under controlled long-day conditions at 22°C (16 h light/8 h dark) for 4 weeks; and later shifted to short-day conditions (8 h light/16 h dark) at 22°C until the end of the plant lifecycle.
In this example, all the experiments were repeated at least twice. Unless otherwise specified, a minimum of 20 leaves per group were used.
Treatments with the different compounds and combinations were done between week 4 and week 8, twice a week for 4 weeks (therefore, eight applications).
The total amount of treatment applied in each of the treatment groups was between 90 and 288 litres per hectare. In addition, between 75% and 90% of each application was applied to the leaves of the plants and between 10% and 25% of each application was applied to the soil. Each application was performed by means of sulphating backpack or trolley SG2.
In the present example, the following treatment groups were:
Control: no treatment applied.
Composition 3: Extract obtained from Urtica dioica (0.0106 mg/mL) and fermented rice starch (Oryza sativa) (0.04 mg/mL).
Extract obtained from Urtica dioica (0.0106 mg/mL).
Fermented rice starch (Oryza sativa) (0.04 mg/mL).
Plant biomass and height were measured as explained above.
The results obtained for biomass and height appear summarized in table 32.
Table 32. Results obtained for fresh biomass and height in Ocimum basilicum in example 14.
In this example, also the number of leaves per plant was measured when the first signs of flowering appeared in the plants.
The results obtained for the leave number appear summarized in table 33.
Table 33. Results obtained for the number of leaves in Ocimum basilicum in example 14.
Therefore, as it can be derived from tables 32 and 33, the composition of the present invention (as exemplified by composition 3) was effective in increasing in a synergistic way the biomass of Ocimum basilicum (fresh biomass, height and number of leaves per plant).
Claims
1. Composition comprising: fermented rice starch; and extract obtained from Urtica.
2. Composition in accordance with claim 1 , characterized in that it additionally comprises: fungi of the genus Trichoderma.
3. Composition in accordance with claim 1 or claim 2, characterized in that the fermented rice starch is fermented rice starch from Oryza sativa.
4. Composition in accordance with claim 2 or claim 3, characterized in that the fungi of the genus Trichoderma are Trichoderma harzianum fungi.
5. Composition in accordance with any one of claims 1 to 4, characterized in that the extract obtained from Urtica is an extract obtained from Urtica dioica.
6. Composition in accordance with any one of claims 1 to 5, characterized in that the concentration of the fermented rice starch is between 0.0066 mg/mL and 0.24 mg/mL.
7. Composition in accordance with any one of claims 2 to 6, characterized in that the concentration of the fungi of the genus Trichoderma is between 0.0026 mg/mL and 0.096 mg/mL.
8. Composition in accordance with any one of claims 1 to 7, characterized in that the concentration of the extract obtained from Urtica is between 0.00175 mg/mL and 0.0636 mg/mL.
9. Composition in accordance with any one of claims 1 to 8, characterized in that it additionally comprises: extract obtained from Centella’, extract obtained from Echinacea’, extract obtained from Taraxacum’, or combinations thereof.
10. Composition in accordance with claim 9, characterized in that the extract obtained from Centella is an extract obtained from Centella asiatica.
11 . Composition in accordance with claim 9 or 10, characterized in that the extract of Echinacea is an extract of Echinacea purpurea.
12. Composition in accordance with any one of claims 9 to 11 , characterized in that the extract obtained from Taraxacum is an extract obtained from Taraxacum officinale.
Composition in accordance with any one of claims 9 to 12, characterized in that the concentration of the extract obtained from Centella is between 0.0010 mg/mL and 0.0372 mg/mL, the concentration of the extract obtained from Echinacea is between 0.0022 mg/mL and 0.0798 mg/mL, and/or the concentration of the extract obtained from Taraxacum is between 0.00095 mg/mL and 0.0346 mg/mL. Use of a composition in accordance with any one of claims 1 to 13 as supplementary plant preparation. Use of a composition in accordance with any one of claims 1 to 13 to increase growth of a plant, to increase biomass of a plant, to accelerate growth of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant. Method to increase growth of a plant, to increase biomass of a plant, to accelerate growth of a plant, to increase trichome density in a plant and/or to increase a plant-derived ingredient production in a plant comprising the application of a composition in accordance with any one of claims 1 to 13 to the plant.
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JP2003250521A (en) * | 2002-07-04 | 2003-09-09 | Akita Konno Shoten:Kk | Method for producing microbial material for promoting plant growth using rice bean as culture substrate |
JP2006062984A (en) * | 2004-08-25 | 2006-03-09 | Yukihiro Hirose | Antiaging composition, cosmetic containing the same, and food and drink containing the same |
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