WO2023203253A1

WO2023203253A1 - Calcium phosphate nanoparticles and uses thereof

Info

Publication number: WO2023203253A1
Application number: PCT/EP2023/060675
Authority: WO
Inventors: José Manuel DELGADO LÓPEZ; Gloria Belén RAMÍREZ RODRÍGUEZ; Andrés CÁCERES BURBANO; Manuel José SÁNCHEZ DEL CASTILLO; Belén PARRA TORREJÓN; Francisco José BERMÚDEZ PÉREZ; Edgar GARCÍA FORTEA; Ana Isabel GUZMÁN CARRASCO
Original assignee: Nanointec, S.L.
Priority date: 2022-04-22
Filing date: 2023-04-24
Publication date: 2023-10-26

Abstract

The invention relates to calcium phosphate nanoparticles comprising amino acids and/or with metal ions or metal oxide ions and the uses thereof in agriculture for improving agronomic traits in crop plants.

Description

CALCIUM PHOSPHATE NANOPARTICLES AND USES THEREOF

FIELD OF THE INVENTION

The present invention belongs to the field of biotechnology and nanotechnology. It specifically relates to nanoparticles comprising metal ions, metal oxide ions and/or amino acids and its use in agriculture.

BACKGROUND OF INVENTION

Nutrition and plant defense is the key element in the growth and development of crops. Poor availability of fertilizers or nutrients to plants results in lack of proper growth and the plants become more susceptible to attack by pests. Other problems associated with agriculture are the soil condition such as drought, biotic and abiotic stress, which tend to create low and uncertain yields.

Fertilizers can be added to the soil or foliage of crops to supply the elements necessary for proper plant nutrition. Typically, elements such as nitrogen (N), phosphorus (P), and potassium (K) are used to make up the basic components of standard fertilizers. However, modern, complex fertilizers can contain a variety of organic or inorganic nutrients, micronutrients or growth enhancing mixtures, vitamins, amino acids, carbohydrates and polysaccharides in addition to the basic components.

Zinc (Zn) dietary deficiency is an important global public health problem in humans. Fortifying food crops with Zn fertilizers is a potential solution to alleviate Zn deficiency in human diet. However, Zn foliar and soil application shows low nutrient efficiency. Fertilizers containing Zn (i.e. , ZnSC>4, Zn(NOa)2, Zn-EDTA) have been applied in the soil or in the leaves to increase Zn content of grains and fruits. The Zn fortified crop products enter in the food chain after harvest being an effective strategy to treat Zn deficiency in world population. Zn is also a vital nutrient element for plants involved in various plant functions including increasing the rate of enzymes, chlorophyll, antioxidants, and is a necessary constituent of numerous proteins. Therefore, Zn fertilization not only improved Zn content of crops but also enhanced plant growth, development and yield. Nonetheless, the efficiency of Zn fertilizers depends on Zn absorption and translocation mechanism in plants which is governed by Zn transporters and metal chelators of plant system. Soil Zn fertilization can be inefficient and expensive since Zn²⁺ ions can be fixed in the soil through precipitation and sorption reactions and so be unavailable to plant roots. Foliar application of soluble Zn salts can overcome this issue but it can cause leaf burning due to high Zn dosage requiring repeated application of reduced Zn throughout the growing season.

Amino acids provide many different benefits to plant health. Amino acids will help in the production of chlorophyll, increase nutrient absorption, or fight stress. Amino acids also support the growth of phytohormones, which control the development of healthy plants by supporting tissues and cells. In addition, amino acids, added to the soil can help increase nitrogen content by limiting the need for fertilizer with a high nutrient concentration.

A large problem with several crop nutrients or fertilizers or plant growth promoting products is that, when applied it either remains in unavailable form and not adequately absorbed by the plant or it may rapidly leach through soil, due to either their rapid mobility in the soil or their physical form and characteristics. Thus, lesser amount of nutrition becomes available to plant and hence these products will have less nutrient use efficacy. In addition, leaching of nutrients may also contribute to groundwater contamination in regions with intensive agriculture. Thus, providing adequate nutrition in a manner such that there is a maximum uptake of nutrient by the plant along with protection to the crops pertaining to the environmental condition remains a great challenge. The use of fertilizer and micronutrient composition needs to be optimized and their uptake by crops on application needs to be improved in order to provide an economical result to the farmer and also reduce the burden on the environment.

SUMMARY OF THE INVENTION

The authors of the invention have successfully obtained nanoparticles comprising either the amino acid glycine or the metal ion Zn wherein the amino acid and the metal ion are encapsulated within the nanoparticle.

Regarding the amino acid glycine, the present application demonstrates the successful formation of calcium phosphate nanoparticles containing glycine. In particular, the new nanoparticles are calcium phosphate nanoparticles with low crystallinity, which is a desirable feature for their application as part of fertilizer since lower crystallinity favors greater solubility and therefore greater release of calcium, phosphate and glycine into the medium.

In addition, the authors of the invention have demonstrated that the use of nanoparticles of amorphous calcium phosphate doped with Zn (ZnACP) provide a series of benefits to a variety of crops. In particular, foliar application of ZnACP nanoparticles in tomato fruits triggered to a relevant enhancement of the yield and quality of the fruits respect to control samples, and provided tomatoes with the highest Zn content with regard to other fertilizers such as zinc sulphate (ZnSOt) or zinc lignosulfonate (Zn-LgSF). Also, the ZnACP application had an impact on pepper flowering, showing an increase of 50% or more flower buds compared to control plants and even higher than other treatments, and consequently affecting positively over pollen and seed production. Additionally also in pepper species, data supports the effect of ZnACP on the percentage of vacuolated microspores, the selection of BBCH3=5 as a reference and critical stage in the androgenesis, and also in the route between microsporogenesis and microgametogenesis. This relevant information could be of great interest in the development of double haploids.

Thus, in a first aspect the invention relates to a composition, hereinafter the composition of the invention, comprising calcium phosphate nanoparticles wherein the nanoparticles comprise an encapsulated compound selected from a metal ion or a metal oxide ion and an amino acid, or a combination thereof, wherein the calcium phosphate is amorphous calcium phosphate (ACP) and wherein the nanoparticles does not comprise CaCCh or CaBOs.

In another aspect, the present invention relates to a method for preparing the composition of the invention, wherein the method comprises: a) contacting a composition A comprising a calcium salt and an amino acid, or a combination of an amino acid and either a metal ion or a metal oxide ion with a composition B comprising a phosphate salt under conditions adequate for the formation of a precipitate formed by calcium phosphate nanoparticles, wherein the contacting is carried at basic or neutral pH; b) collecting the precipitate obtained in step a), and c) dispersing the precipitate obtained in step b) in an aqueous solvent.

In another aspect, the present invention relates to a method for promoting uptake of a metal ion and/or of an amino acid in plants which comprises applying the composition of the invention to the plant, to a propagule thereof or to the soil in which the plant is grown.

In another aspect, the present invention relates to a method for improving an agronomic trait in a plant which comprises applying the composition of the invention to the plant, to a propagule thereof or to the soil in which the plant is grown. In another aspect, the invention relates to the use of the composition of the invention for supplying nutrient elements to a plant and/or for improving an agronomic trait in a plant.

In another aspect, the invention relates to the use of the composition of the invention as a fertilizer.

DESCRIPTION OF THE FIGURES

Figure 1. (a) XRPD patterns and (b) FTIR spectra of AGP and ZnACP nanoparticles.

Figure 2. HAADF-STEM micrograph and corresponding EDS maps showing Ca (b), P(c) and Zn (d) distributions in ZnACP nanoparticles.

Figure 3. Average number of pepper flowers along evaluation period (days after transplanting-DAT), for ZnACP treatment and control plants.

Figure 4. Top) Development of flower bud until anthesis, described for BBCH3 levels (left to right, BBCH3=1 to BBCH3=8) (scale is 10 mm). Middle) Anthocyanin pigmentation and length increase of the anthers (scale is 10 mm). Bottom) Cell development in the pollen sac (left to right, young microspore, medium microspore, vacuolated microspore, young bicellular pollen, medium bicellular pollen and dehydrated pollen grain) (scale is 90 pm).

Figure 5. Average number of flowers/inflorescences counted at 30, 37 and 45 days (from top to bottom).

Figure 6. a) X-ray diffraction pattern and b) infrared spectrum of samples synthesized at 30 minutes at acidic pH, neutral pH and basic pH. DCPD: dicalcium phosphate dihydrate and AGP: amorphous calcium phosphate.

Figure 7. a) X-ray diffraction pattern and b) infrared spectrum of samples synthesized for 24 hours at acidic pH, neutral pH and basic pH. DCPD: dicalcium phosphate dihydrate and HA: hydroxyapatite.

Figure 8. Transmission electron microscopy images of calcium phosphate nanoparticles synthesized within 30 minutes (a) showing AGP nanoparticles of rounded morphology with a diameter of 20 nanometers and within 24 hours (b), showing elongated hydroxyapatite nanoparticles of approximately 20 nanometers in length and 2-3 nm in width approximately. Figure 9. (a and c) X-ray diffraction patterns and (b and d) infrared spectra of samples synthesized at 30 minutes (a-b) and 24 hours (c-d) in the presence of glycine at acidic pH, neutral pH and basic pH. DCPD: dicalcium phosphate dihydrate, ACP: amorphous calcium phosphate, HA: hydroxyapatite.

Figure 10. a) X-ray diffraction pattern and b) infrared spectrum of calcium phosphate nanoparticle nanocomposites obtained in the presence of glycine at different concentrations 0.2 M (CaP_Gly1), 0.4 M (CaP_Gly2) and 0.6 M (CaP_Gly3) after 30 minutes of maturation. ACP: amorphous calcium phosphate.

Figure 11. A) X-ray diffraction pattern and b) infrared spectrum of calcium phosphate nanoparticle nanocomposites obtained in the presence of glycine at different concentrations 0.2 M (CaP_Gly1), 0.4 M (CaP_Gly2) and 0.6 M (CaP_Gly3) after 24 hours of maturation. ACP: amorphous calcium phosphate.

Figure 12. Transmission electron microscopy images of CaP_Gly3 nanocomposites synthesized at 30 minutes (a) where ACP nanoparticles of rounded morphology and diameter of 20 nanometers and at 24 hours (b) showing elongated hydroxyapatite nanoparticles of approximately 20 nanometers in length and approximately 2-3 nm in width.

Figure 13. Thermogravimetric analysis of CaP-Gly3 nanocomposite synthesized after 24 hours of maturation.

DETAILED DESCRIPTION OF THE INVENTION

Methods of the invention

In a first aspect, the invention relates to a method, hereinafter the first method of the invention, for promoting uptake of a metal ion and/or of an amino acid in plants which comprises applying to the plant, to a propagule thereof or to the soil in which the plant is grown a composition comprising calcium phosphate nanoparticles wherein the nanoparticles comprise an encapsulated compound selected from a metal ion or a metal oxide ion and an amino acid or a combination thereof, wherein the calcium phosphate is amorphous calcium phosphate (ACP) and wherein the nanoparticles does not comprise CaCOs or CaBOs.

In a second aspect, the invention relates to a method, hereinafter the second method of the invention, for improving an agronomic trait in a plant which comprises applying to the plant, to a propagule thereof or to the soil in which the plant is grown a composition comprising calcium phosphate nanoparticles wherein the nanoparticles comprise an encapsulated compound selected from a metal ion or a metal oxide ion and an amino acid, wherein the calcium phosphate is amorphous calcium phosphate (ACP) and wherein the nanoparticles does not comprise CaCCh or CaBCh.

The term “uptake”, as used herein refers to acquisition of nutrients or water by the roots and vegetative parts of a crop or plant following its application.

As used herein “ion” refers to a small particle having electrical charge. Ions are either single charged atoms (simple ions), or small charged “molecules” (polyatomic ions).

The invention refers to positively charged metal ions, metal referring to elements that form electropositive ions by donating electrons to form bonds. Metals are present in the periodic table under the groups: alkali metals, alkaline earth metals, and transition metals.

In a particular embodiment, the nanoparticles of the composition of the invention further comprise a compound adsorbed on the surface of the nanoparticles and wherein said compound is selected from a metal ion or a metal oxide ion, an amino acid or a combination thereof.

The expression “comprise a compound adsorbed on the surface of the nanoparticles and wherein said compound is selected from a metal ion or a metal oxide ion, an amino acid or a combination thereof" is used herein to define: nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising an amino acid adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising a metal ion adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising a metal oxide ion adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising an amino acid and a metal ion adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising an amino acid and a metal oxide ion adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising a metal oxide ion and metal ion adsorbed on the surface, nanoparticles comprising an encapsulated amino acid, a metal ion or a metal oxide ion, further comprising an amino acid, a metal oxide ion and metal ion adsorbed on the surface.

In a preferred embodiment, the nanoparticles contain a metal ion or a metal oxide ion encapsulated within the nanoparticles and an amino acid adsorbed on the surface of the nanoparticles.

“Metal oxide ion”, as used herein, refers to compound that contains at least one oxygen atom and metal element in its chemical formula. "Oxide" itself is the dianion of oxygen, an O^2- (molecular) ion. Metal oxides thus typically contain an anion of oxygen in the oxidation state of ~².

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers).

The term "natural amino acids" or “naturally occurring amino acid” comprises the 20 naturally occurring amino acids and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.

As used herein the term "non-natural amino acid" or “synthetic amino acid” refers to a carboxylic acid, or a derivative thereof, substituted at position “a” with an amine group and being structurally related to a natural amino acid. Illustrative non- limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2- aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3- diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxy lysine, alio hydroxy lysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N- methylglycine, N-methyliso leucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, etc. In an embodiment, the amino acid is selected from, proline, cysteine, glutamine, glycine, histidine, lysine, alanine, arginine, serine, tryptophan, valine, glutamic acid, phenylalanine and asparagine.

In another embodiment the metal ion is selected from Fe²⁺/Fe³⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺ and Ag⁺ and the metal oxide ion is selected from a boron oxide ion, a selenium oxide ion and a molybdenum oxide ion.

In another embodiment, the amino acid is selected from, proline, cysteine, glutamine, glycine, histidine, lysine, alanine, arginine, serine, tryptophan, valine, glutamic acid, phenylalanine and asparagine, the metal ion is selected from Fe²⁺/Fe³⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺ and Ag⁺ and the metal oxide ion is selected from a boron oxide ion, a selenium oxide ion and a molybdenum oxide ion.

In a preferred embodiment, the amino acid is glycine.

In another embodiment, the metal ion is Zn²⁺.

In another embodiment, the amino acid is glycine and the metal ion is Zn²⁺.

In an embodiment, the content of the amino acid in the nanoparticles is of between 1 % and 7 % (w/w) and/or the content of metal ion in the nanoparticles is of between 5 % and 12 % (w/w) and/or the content of the metal oxide ion is of between 1 % and 5 % (w/w).

In another embodiment, the content of the amino acid in the nanoparticles is of between 1 % and 7 % (w/w) and/or the content of metal ion in the nanoparticles is of between 5 % and 12 % (w/w).

In another embodiment, the content of the amino acid in the nanoparticles is of between 1 % and 10% (w/w), between 1 % and 9% (w/w), between 1 % and 8% (w/w), between 1 % and 7 % (w/w).

In a preferred embodiment, the content of the amino acid in the nanoparticles is of between 1% and 7% (w/w).

In another embodiment, the content of the amino acid in the nanoparticles of about 2% (w/w), about, 3% (w/w), about 5% (w/w) about 6% (w/w) and about 7% (w/w).

In another embodiment, the content of the metal ion in the nanoparticles is of between 5% and 15% (w/w), between 5% and 12% (w/w), between 6% and 12% (w/w), between 7% and 12% (w/w), between 8% and 12% (w/w), between 9% and 11% (w/w). In another embodiment, the content of the metal ion in the nanoparticles is of about 10% (w/w). In another embodiment, the content of the metal oxide ion in the nanoparticles is of between 1% and 5% (w/w).

As used herein, “calcium phosphate” refers to a family of minerals containing calcium ions (Ca2+), together with orthophosphates (PC>4³'), metaphosphates or pyrophosphates (P2O7⁴ ) and hydrogen or hydroxide ions. As used herein, “calcium phosphate” specifically includes amorphous calcium phosphate (ACP).

The term “amorphous calcium phosphate” or “ACP” is used to refer to a unique species among all forms of calcium phosphate in that it lacks long-range, periodic atomic scale order of crystalline calcium phosphates. This means that ACP can be recognized from its broad and diffuse X-ray diffraction pattern with a maximum at 25 degrees 2 theta, and no other different features compared, with well crystallized hydroxyapatite. Additionally or alternatively, amorphous calcium phosphates may be characterized as calcium phosphate materials in which analysis by XRD shows the typical broad band peaking at approximately 31 2-theta and extending from 22 to 36 2-theta. ACP is formed from spherical ion clusters called Posner clusters (characteristic diameter 9.5 A) and approximately 20 wt% of tightly bound water.

The ACP forming part of the nanoparticles of the invention include compounds with chemical formula Cas PO^ ntW and Ca/P molar ratio with a range of 1.34-1.50 in different pH and 1.50-1.67 when adding different amount of carbonates. In addition, the ACP of the invention also includes ACP with HPCU^2- ions instead of PO4³; leading to a lower Ca/P ratio, as low as 1.15.

Calcium phosphate nanoparticles comprise an amorphous calcium phosphate phase as small as 1 nm and as large as 250 nm, preferably between 1 nm and 250 nm, between 1 nm to 150 nm, between 1 nm and 75 nm, between 5 nm to 250 nm, between 5 to 150 nm, between 5 to 75 nm, between 10 to 250 nm, between 10 to 150 nm, between 10 to 100 nm, between 10 to 75 nm, between 10 to 50 nm, preferably between 10 to 25 nm, between 20 to 25 nm in diameter. In a preferred embodiment, the nanoparticles have a spherical morphology. In another embodiment, the amorphous calcium nanoparticles have a spherical morphology and a diameter of around 20 nm or 22 nm.

Methods for the production of ACP are known in the art and typically imply the mixing of a calcium salt and a phosphate. Alternatively, the present document provides, as a fourth aspect of the invention, a method of preparing calcium phosphate nanoparticles, together with an amino acid and/or metal ion or metal oxide ion according to the invention. The nanoparticles does not comprise calcium carbonate (CaCO₃) or calcium borate (CaBO₃).

In a particular embodiment, the nanoparticles does not comprise does not comprise urea, CaCO₃, CaBO₃, NO₃ or CO₃.

In another particular embodiment, when the nanoparticles comprises a metal ion, then, the nanoparticles further comprise a citric acid derivative

As used herein, “citric acid”, also known as 2-hydroxypropane-1 ,2,3-tricarboxylic acid or anhydrous citric acid is a tricarboxylic acid that is propane-1 ,2, 3-tricarboxylic acid bearing a hydroxy substituent at position 2. A particularly suitable citric acid derivative is a water- soluble alkali metal salt of citric acid, typically the lithium, potassium or sodium salt. It is preferred to use the sodium salt. Trisodium citrate dihydrate is particularly preferred.

The kind of the citric acid derivative is not particularly limited as long as it is a known citric acid derivative. For example, the citric acid derivative may be at least one selected from the group consisting of acetyl triethyl citrate, diethyl citrate, tributyl citrate, triethyl citrate, and acetyl tributyl citrate.

That is, the citric acid derivative may be contained in an amount between 1 % and 10 % w/w based on the total weight of the nanoparticle comprising the amino acid or the metal ion.

In a particular embodiment, the citric acid derivative is sodium citrate (NasCeHsO?). In another embodiment, the citric acid derivative is potassium citrate (KsCeHsO?).

“Adsorption”, as used herein refers to a surface process, the accumulation of a gas or liquid on a liquid or solid. Adsorption can be defined further based on the strength of the interaction between the adsorbent (the substrate onto which chemicals attach) and the adsorbed molecules. Adsorption can be physical or chemical. Physical adsorption or physisorption implies van der Waals interactions between substrate and adsorbate (the molecule that is adsorbed); chemical adsorption or chemisorption involves chemical bonds (covalent bonds usually) in sticking the adsorbate to the adsorbent.

Chemisorption involves more energy than physisorption. The difference between the two processes is loosely based on the binding energy of the interaction.

The term “encapsulated” or “nanoencapsulation” is defined as the technology of packaging nanoparticles of solid, liquid, or gas, also known as the core or active, within a secondary material, named as the matrix or shell, to form nanocapsules. The core contains the active ingredient (e.g., the amino acid, the metal ion or the metal oxide ion) while the calcium phosphate shell isolates and protects the core from the surrounding environment. This protection can be permanent or temporal, in which case the core is generally released by diffusion or in response to a trigger, such as shear, pH, or enzyme action, thus enabling their controlled and timed delivery to a targeted site.

It is not necessary for every nanoparticle to comprise a compound of interest. Only a subset of the nanoparticles may comprise the compounds of interest. For example, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% (i.e. all of the nanoparticles) of the nanoparticles can comprise the metal ion or metal oxide ion and/or the amino acid or a combination thereof. In some embodiments, not all of the nanoparticles comprise both, the metal and the amino acid. In another embodiment, not all the nanoparticles comprise any of the compounds of interests. In some embodiments, the nanoparticles comprise only one of the compounds of interest.

The term “applying”, also referred to as “treatment” is used here to the application of the calcium phosphate nanoparticles comprising the compound of interest to a crop, plant, and propagule or to the soil where the plant, crop or propagule is grown.

According to a specific embodiment, applying comprises pre-harvest applying. According to a specific embodiment, said applying comprises post-harvest applying.

According to a specific embodiment, said plant is at a post-blossom stage. According to a specific embodiment, said plant is at a blossom stage. According to a specific embodiment, said plant is at a pre-blossom stage. When indicated a specific stage, the application can be confined only to this stage or to the recited stage and more. For instance, when indicated applying at blossom, applying can be effected at blossom or blossom+ post-blossom (i.e., fruit), or pre-blossom + blossom or pre-blossom+blossom + post blossom.

According to a specific embodiment, applying is post-emergence. According to a specific embodiment, the calcium phosphate nanoparticles are formulated in a composition selected from the group consisting of a dip, a spray or a concentrate.

According to a specific embodiment, said applying is in the vicinity of or onto the roots, stems, trunk, seed, fruits or leaves of the plant. According to a specific embodiment, said applying is by irrigation, drenching, dipping, soaking, injection, coating or spraying.

According to a specific embodiment, said applying is in an open field. According to a specific embodiment, said applying is in a greenhouse.

According to a specific embodiment, said applying is in a storage facility (e.g., dark room, refrigerator).

According to a specific embodiment, said applying is effected once. According to a specific embodiment, said applying comprises repeated application (2 or more applications e.g., every week). Repeated applications are especially envisaged for field/greenhouse treatments.

According to a specific embodiment, said repeated application comprises weekly administration during blossom pre-harvest.

For example, suggested regimen include but are not limited to, spraying plants in open fields and greenhouse, adding to irrigation of plants grown in the open field, greenhouse and in pots, dipping the whole foliage branch in the solution post harvest, adding to vase of cut flowers after harvest and before shipment.

The term "plant" includes whole plants, shoot vegetative organs/structures ( e.g., leaves, stems and tubers), roots, flowers and floral organs/structures ( e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue ( e.g., vascular tissue, ground tissue, and the like) and cells ( e.g., guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae.

Also, the term plant may include a part thereof, meaning any complete or partial plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like. “Propagule” includes all products of meiosis and mitosis, including but not limited to, seed and parts of the plant able to propagate a new plant. For example, propagule includes a shoot, root, or other plant part that is capable of growing into an entire plant. Propagule also includes grafts where one portion of a plant is grafted to another portion of a different plant (even one of a different species) to create a living organism. Propagule also includes all plants and seeds produced by cloning or by bringing together meiotic products, or allowing meiotic products to come together to form an embryo or fertilized egg (naturally or with human intervention).

Soil refers to the mixture of organic matter, minerals, gases, liquids, and organisms that together support life.

The term “agronomic trait” or “agronomic character” relates to plant characters related to crop production usually observed during plant growth. Examples of agronomic traits include, without limitation, plant eight, maturity, tiller number, panicle size, yield and other factors such as water use efficiency, cold tolerance, increased yield, nitrogen use efficiency, seed protein and seed oil content. Thus, the method of the invention comprises the improvement or enhancement of at least one of these traits, so that, the application of the method would render plants with enhance eight, tiller number, panicle size, enhance cold tolerance, enhance nitrogen use efficiency, enhanced amount of seed and oil content. In an important aspect of the invention the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. "Yield" can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), seed number, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. In a particular embodiment, the application of the nanoparticles as referred herein, led to an increase in the number of fruits. In particular, the method of the invention lead to an increase of the fruit number of between 100% and 400%, more preferably between 180% to 300%, more preferably between 190% to 250% if compared to a control treatment.

The quantification of the number of fruits according to the invention may be performed by manual count of the total number of fruits per cluster at the commercial stage, that is, at fully red/ripe stage. When a treatment is applied, said treatment is generally applied to the fruits at the fourth cluster and manual count of the total number of fruits (at the commercial stage) per cluster is registered for each treatment.

In a particular embodiment the increase in the number of fruits is induced when a crop, plant or propagule is supplied with nanoparticles comprising a metal ion, a metal oxide ion, an amino acid or a combination thereof.

More preferably, the increase in the number of fruits is induced when the crop, plant or propagule is supplied with nanoparticles comprising a metal ion, more particularly, Zn. In a more particular embodiment, the increase in the number of fruits takes place when the fertilized comprising the calcium phosphate nanoparticles comprising Zn, are applied to a tomato crop, plant or propagule.

“Control treatment” as used in the present application, refers to a crop, plant or propagule which have not received the calcium phosphate nanoparticles comprising the compound of interest or the fertilizer comprising the calcium phosphate nanoparticles with the compound of interest, wherein the compound of interest is a metal ion, metal oxide ion, amino acid of a combination thereof. Also, the control group may refer to a crop, plant, propagule which have received a fertilizer comprising the compound of interest not encapsulated or adsorbed into the calcium phosphate nanoparticles, wherein the compound of interest is a metal ion, metal oxide ion, amino acid of a combination thereof, wherein the compound of interest in the control group is the same as in the treatment group and is selected from a metal ion, metal oxide ion, amino acid of a combination thereof.

In another particular embodiment, the application of the calcium phosphate nanoparticles comprising the compound of interest, that is, an amino acid, a metal ion, a metal oxide ion or combinations thereof, leads to an increase in the nutrient content of the fruits compared to a control group that has not received the doped calcium phosphate nanoparticles. The evaluation of the nutrient content of the fruits, within the context of the present invention may be performed by any method known by a skilled in the art.

In a particular embodiment, the evaluation of the nutrient content of the fruits is performed as follows. Selected fruits are repeatedly washed with distilled water and dried until constant humidity and ground until sample homogenization. The samples are mineralized by wet method using H2SO4 1 H2O2 until a transparent mineralization was obtained. The Zn and Ca content is measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The ICP-MS measurement may be performed using a XSERIES 2 ICP-MS (Thermo Fisher) spectrometer.

More particularly, the nitrogen (N) content may be analyzed by the Kjeldahl method. In an embodiment, the Kjeldahl method is applied to a 1 mL of mineralized sample and placed in a Kjeldahl flask, mixed with 25-30 ml of NaOH and connected to Bouat device. Ammonia vapor is then collected in a flask and assessed with Shiro-Tashiro colorant.

Within the context of the present invention, the phosphorus content is measured by a spectrophotometer at 700 nm using the phosphomolybdic complex method.

In a particular embodiment, the application of the nanoparticles of the invention is performed with calcium phosphate nanoparticles comprising glycine, and as a consequence, there is an increase in the glycine uptake of the plant or propagule. In another embodiment, the application of the nanoparticles of the invention is performed with calcium phosphate nanoparticles comprising Zn, and therefore, there is an increase in the Zn uptake of the plant or propagule.

In a particular embodiment the increase in the uptake of glycine and/or Zn leads to an increase in the glycine or Zn content in the fruits.

In a particular embodiment, the calcium phosphate nanoparticles comprise Zn and the increase in the Zn content in the fruit is of between 200% to 500%, between 300% and 400%, more preferably of about 350% compared to a control group. In another particular embodiment, the increase in the Zn content in the fruits is induced when the calcium phosphate nanoparticles comprising Zn are administered to the plant or propagule by foliar application. In another embodiment the fruits showing an increase in the Zn fruit content are tomato fruits.

In another embodiment, the application of nanoparticles comprising the compound of interest leads to an increase in the N, P and Ca content in the fruits. More preferably, the increase in the N, P and Ca content in the fruits is induced by the application to the plant, propagule or soil with calcium phosphate nanoparticles comprising Zn. In an even more preferred embodiment the application is performed in tomato plants of propagules and the fruits are tomato fruits.

The quantification of the number of flowers may be performed by any method known in the art. More particularly, the quantification after treatment with nanoparticles is carried out according to the following methodology. Nanoparticles are applied foliarly in the apical zone of the plants prior to the inflorescence emergence (Principal growth stage BBCH1=5, meaning BBCH the scale of the Biologische Bundesanstalt, Bundessortenamt Chemische Industrie). The application is carried out during three consecutive weeks with the purpose that each intermediate stage/level of differentiation or meso stage (BBCH2) is treated once. The first bifurcation of stems corresponds to BBCH2=0, the next bifurcation from the secondary stems is BBCH2=1. Nanoparticles are applied to BBCH2 from level 0 to 7. The evaluation of flowering behavior starts 15 days after first application and at 30, 37 and 45 days after transplanting. The number of flowers/inflorescences, is visually counted and registered each time in all treated plants, including all secondary differentiation stages (BBCH3), since 0 to 9 (BBCH3, O=First flower bud/inflorescence visible; 1=Pedicel with flower primordium erected; 2=Pedicel at 90°; 3=Pedicel at 180°; 4=Protruding petals with less length than sepals; 5=Petals and sepals with the same lenght - calyx:corolla relation 1 :1 ; 6=Petals longer than sepals; 7=Prior to anthesis; 8=Anthesis; 9=Sepals degenerated). The number of flowers/inflorescences are determined in a total of thirty pepper plants treated with nanoparticles.

In another embodiment, the application to a plant, propagule or soil of the calcium phosphate nanoparticles comprising the compound of interest leads to an increase in the number of flowers. In a particular embodiment, the increase in the number of flowers is induced when the calcium phosphate nanoparticles comprise with Zn. In another particular embodiment, the increase in the number of flowers is induced when the calcium phosphate nanoparticles comprise Zn and the plant is a pepper plant. In a more preferred embodiment, the application of the calcium phosphate nanoparticles comprising Zn in a pepper plant lead to an increase in the number of flowers is between 5% and 50%, more particularly between 10% and 40%, preferably about 13%, about, 18%, about 20%, about 25%, about 30%, about 35% or about 40% compared to a control group without treatment. In another embodiment, the application to a plant, propagule or soil of the calcium phosphate nanoparticles comprising the compound of interest leads to an increase in the pigmentation value, total cells per ml and/or percentage of vacuolated microspores. In a particular embodiment the increase in the pigmentation value, total cells per ml and/or percentage of vacuolated microspores is induced when the calcium phosphate nanoparticles comprise Zn. In another particular embodiment, the increase in the pigmentation value, total cells per ml and/or percentage of vacuolated microspores is induced when the calcium phosphate nanoparticles comprise Zn and the plant is a pepper plant. In a particular embodiment the increase in the percentage of vacuolated microspores is of about 10% to 80%, between 20% and 70%, between 30% and 70%, more preferably about 50%, about 55%, about 60%, about 65% or about 70% compared to a control group without treatment.

According to the present invention, the variable of pigmentation is measured based on a scale proposed by Parra and collaborators (Parra et al., 2010, 2013). This scale includes 4 values: 1 : anther no pigmented; 2: pigmentation only in the apical part of the concave side; 3: pigmentation to the middle of the concave side, with partially pigmented borders; and 4: anther completely purple. Anthers are evaluated from a total of 45 flower buds.

Within the context of the present invention, the evaluation of the percentage of vacuolated microspore is evaluated as followed. The internal content of anthers is collected in order to evaluate the type of cells (percentage of vacuolated microspore) present in the pollen sac using an inverted microscope Motic®. A specific scale is proposed to determine the cellular content as follow: 0: Stem cell in the anther tissue; 1 : Tetrads; 2: Young microspore; 3: Medium microspore; 4: ~50%Medium microspore/~50% Vacuolated microspore; 5: (-80%) Vacuolated microspore; 6: -50% Vacuolated microspore/~50% Young bi-celullar pollen; 7: (-80%) Young bi-celullar pollen; 8: (-80%) Medium bi-cell ular pollen; 9: (-80%) Mature bi-celullar pollen.

Regarding the pre-evaluation to determine the cell content, the highest percentage of vacuolated microspores is identified in flower buds at secondary differentiation stages (BBCH3=5). Consequently, the number of flower buds is counted at BBCH=5, when the relation between calyx:corolla was 1 :1.

In another embodiment, the application to a plant, propagule or soil of the calcium phosphate nanoparticles comprising the compound of interest leads to an increase in the number of flower buds. In a particular embodiment, the increase in the number of flower buds is induced when the calcium phosphate nanoparticles comprise Zn. In another particular embodiment, the increase in the number of flower buds is induced when the calcium phosphate nanoparticles comprise Zn and the plant is a pepper plant. In a particular embodiment the increase in the percentage of vacuolated microspores is of about 10% to 80%, between 20% and 70%, between 30% and 60%, more preferably about 30%, about 35%, about 40%, about 45% or about 50%, about 55% or about 60% compared to a control group without treatment.

In order to calculate the number of cell per mL (quantification of microspores), a total of 6 anthers is collected from one flower bud, with a total of fifteen flower buds per treatment. After maceration, 10 pL are analyzed at “Neubauer improved chamber”. Cell counting is visual using a manual counter.

In a particular embodiment, within the context of the methods of the invention, the application is carried out by impregnating the leaves of the plant with an aqueous suspension of the nanoparticles.

In another embodiment, the composition that is applied further comprises at least one additional agrochemical compound.

The agrochemical compounds to be used in the instant invention include herbicides, fungicides and insecticides as well as plant growth regulators and inhibitors and plant activators. Preferred are agrochemical compounds which show systemic or mesostemic properties, which means such compounds are transported by the plant to different loci of the plant.

Examples of suitable herbicides are: Pretilachlor, Clomeprop, Bifenox, Pyrazoxyfen, Pyrazolynate, Cinosulfuron, Dimepiperate, Bensulfuron-methyl, Pyrazosulfuron-ethyl, Naproanilide, Bromobutide, Mefenacet, Imazosulfuron, Daimuron, Bentazon, Simetryn, Etobenzanid, Cyhalofop-butyl, Cafenstrole, Azimsulfuron, Pyriminobac-methyl, Benzofenap, Pyributicarb, Thenylchlor, MCPB, Benfuresate, Butamifos, Cyclosulfamuron, Dimethametryn, Esprocarb, Fentrazamide, Indanofan, Isoprothiolane, Molinate, Oxadiclomefon, Oxaziclomefone, Paclobutrazol, Pentoxazone, Prohexadione- ca, Pyrazogyl, Simetryne, Thiobencarb and Uniconazole.

Examples of suitable fungicides are: Acibenzolar-S-Methyl, Isoprothiolane, Ipconazole, Iprodione, Oxolinic acid, Kasugamycin, Capropamid, Captan, Thiabendazole, Thiram, Thiophanate-methyl, Organocopper, Tricyclazole, Triflumizole, Validamycin, Azoxystrobin, Pyroquilon, Fludioxonil, Prochloraz, Probenazole, Benomyl, Methasulfocarb, TPN, BJL-002, BJL-003, Chlorothalonil, Copper, Diclocymet, Diclomezine, Edifenphos, Fenoxanil, Ferimzone, Flutolanil, Furametpyr, Hymexazol, Mepronil, Metominostrobin, Pefurazoate, Pencycuron, Tecloftalam and Thifluzamide.

Examples of suitable insecticides are: Imidacloprid, Etofenprox, Cartap, Thiamethoxam, Thiocyclam, Bensultap, Bendiocarb, Monocrotophos, Alprocarb, Pymetrozine, Benfuracarb, Buprofezin, Carbosulfan, Cycloprothrin, Fenitrothion, Fipronil, Isoxathion, Phenthoate, Silafluofen, Triazophos, Trichlorfon, Methoxyfenozide and Clothianidin.

Among these agrochemical compounds, Acibenzolar-S-methyl, Fludioxonil, Pyroquilon, Thiamethoxam, Thiocyclam, Pymetrozine, Pretilachlor, Cinosulfuron are particularly preferred.

In a further embodiment the plant is selected from the group consisting of cereals, legumes, fruit trees and vegetables.

In another embodiment, the plant is selected from the plant families comprising Solanaceas, Cucurbitaceas, Brassicacea, Poacea and Fabaceae.

In an additional embodiment, the plant is selected from Solanum licopersicum, Solanum melongena, Capsicum frutescens, Capsicum annuum, Solanum tuberosum, Lycium barbarum, Citrullus lanatus, Cucumis melo, Cucumis sativus, Cucurbita pepo, Cucurbita maxima, Cucurbita moschata, Cucurbita argyrosperma, Lactuca sativa, Brassica oleracea, Spinacia oleracea, Oryza sativa, Zea mays, Triticum sp.pl., Sorghum bicolor, Secale cereal, Hordeum vulgare, Saccharum officinarum, Bambusa sp.pl., Glycine max, Lens culinaris, Vida faba, Lens culinaris, Cicer arietinum, Pisum sativum, Malus domestica, Pyrus communis, Juglans regia, Prunus dulcis, Pistacia vera, Citrus sinensis, Ananas comosus, Musa paradisiaca, Musa acuminata x M. balbisiana, Fragaria x ananassa, Rubus ulmifolius, Vaccinium oxycoccus, Rubus idaeus and Olea europaea.

In a preferred embodiment, the plant is a tomato or a pepper plant.

Composition of the invention

In another aspect, the invention relates to a composition comprising calcium phosphate nanoparticles, wherein the calcium phosphate nanoparticles comprise an encapsulated compound selected from a metal ion or a metal oxide ion and an amino acid, wherein the calcium phosphate is amorphous calcium phosphate (ACP) and wherein the nanoparticles does not comprise CaCCh or CaBCh. Most of the definitions related to the composition of the invention have already been described within the context of the above referred methods. Accordingly all the definitions and embodiments related to the methods already described apply to the composition of the invention. In particular, the embodiments related to the nanoparticles applied in the methods of the invention.

In a particular embodiment, the nanoparticles further comprise a compound adsorbed on the surface of the nanoparticles and wherein said compound is selected from a metal ion or a metal oxide ion, an amino acid or a combination thereof.

In a more particular embodiment, the nanoparticles contain a metal ion or a metal oxide ion encapsulated within the nanoparticles and an amino acid adsorbed on the surface of the nanoparticles.

In another particular embodiment, the amino acid is selected from proline, cysteine, glutamine, glycine, histidine, lysine, alanine, arginine, serine, tryptophan, valine, glutamic acid, phenylalanine and asparagine.

In another particular embodiment, the metal ion is selected from Fe²⁺/Fe³⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Ag⁺, and the metal oxide ion is selected from BO3³; SeOa²' and MOO4².

In a preferred embodiment, the amino acid is glycine and/or the metal ion is Zn²⁺.

In another embodiment, the content of the amino acid in the nanoparticles is of between 1 % and 7 % (w/w) and the content of metal ion in the nanoparticles is of between 5 % and 12 % (w/w).

In a preferred embodiment, the content of the amino acid in the nanoparticles is of between 1 % and 7% (w/w). In another embodiment, the content of the amino acid in the nanoparticles of about 2% (w/w), about, 3% (w/w), about 5% (w/w) and about 6% (w/w).

In another embodiment, the content of the metal ion in the nanoparticles is of between 5% and 15% (w/w), between 5% and 12% (w/w), between 6% and 12% (w/w), between 7% and 12% (w/w), between 8% and 12% (w/w), between 9% and 11 % (w/w). In another embodiment, the content of the metal ion in the nanoparticles is of about 10% (w/w).

In a more particular embodiment, the content of the metal ion is of 10% (w/w). More particularly, when the metal ion is Zn, the content of this ion in the nanoparticles is of 10%.

In another embodiment, when the metal ion Zn comprised in the nanoparticles is of about 10% (w/w), then, the content of Ca is of about 10-23% (w/w), the content of K is of about 1-2% (w/w) and the content of P is of about 15-17% (w/w).

In another embodiment the molar ratio (Ca+Zn)/P is of between 1 and 2, more preferably of about 1.3.

In another embodiment, when the molar ratio (Ca+Zn)/P is of about 1-2, then the zeta potential is of between -15 to -20 mV or between -16 to -18 mV.

In a particular embodiment, when the nanoparticle comprises a metal ion, then, the nanoparticles further comprise a citric acid derivative.

A particularly suitable citric acid derivative is a water-soluble alkali metal salt of citric acid, typically the lithium, potassium or sodium salt. It is preferred to use the sodium salt. Trisodium citrate dihydrate is particularly preferred.

That is, the citric acid derivative may be contained in an amount between 1 % and 10 % w/w based on the total weight of the nanoparticle comprising the metal ion or the amino acid.

In a particular embodiment, the citric acid derivative is sodium citrate (NasCeHsO?). In another embodiment, the citric acid derivative is potassium citrate (K3C6H5O7).

In another aspect, the invention relates to a composition which has been obtained by a method for preparing the composition according to the invention as explained below.

Method for preparing the composition of the invention In another aspect, the present invention relates to a method, hereinafter the third method of the invention, for preparing the composition of the invention, wherein the method comprises: a) contacting a composition A comprising a calcium salt and a metal ion or a metal oxide or an amino acid, or a combination of an amino acid and either a metal ion or a metal oxide ion with a composition B comprising a phosphate salt under conditions adequate for the formation of a precipitate formed by calcium phosphate nanoparticles, wherein the contacting is carried at basic or neutral pH; b) collecting the precipitate obtained in step a), and c) dispersing the precipitate obtained in step b) in an aqueous solvent.

The term "contacting”, as used herein refers to the process by which the composition A comes into contact with the composition B. The contacting step includes any possible conventional method that allows both compositions to react with each other.

The "adequate conditions" are those known by the person skilled in the art that allows the composition A and B to react and which include the specific concentration of composition A and composition B, temperature, pH and time sufficient to permit the mixing of the components of compositions A and B.

In a particular embodiment, composition A and composition B are mixed at equal volume, that is, at 1 :1 (v/v).

In a particular embodiment, the contacting step a) is carried out for a period of between 1 minute and 48 hours, between 1 minute and 24 hours, between 1 minute and 12 hours, between 1 minute and 10 hours, between 1 minute and 5 hours, between 1 minute and 1 hour, preferably between 1 minute and 30 minutes.

In addition, the type of particles formed after mixing composition A and B depends on the time of mixing (i.e., maturation time). Typically, the particles formed immediately after mixing composition A and B, are ACP, which may last in the precipitate for a period of time between 1 minute and 30 minutes, and then transformed into nanocrystalline hydroxyapatite at longer maturation times. Thus, the longer the maturation time, the higher the crystallinity of the nanoparticles. Therefore, depending on the maturation time, the proportion of ACP within the precipitate may vary according to the embodiments already described within the context of the composition of the invention. Increasing the maturation time to 24 hours would increase the amount of amino acid or a combination of an amino acid and a either a metal ion or a metal oxide ion incorporated in the calcium phosphate nanoparticles. In an embodiment, when the maturation time is of about 30 minutes, the ACP represents at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the calcium phosphates of the composition of the invention.

The temperature is preferably between 10 °C and 45 °C, more preferably between 15 °C and 40 °C, even more preferably between 20 °C and 30 °C. In a preferred embodiment, the temperature is between 18 °C and 25 °C.

Step a) is performed in alkaline media, preferably in a pH range between 8 and 12, between 9 and 11 , between 8 and 10 or between 9 and 12.

In a particular embodiment, the contacting step a) is carried out at basic pH for at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 5 hours, at least 10 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least, 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours or at least 24 hours.

In a particular embodiment, the contacting between the calcium phosphate nanoparticles and the amino acid, the metal ion or the metal oxide, or with a combination of an amino acid and a either a metal ion or a metal oxide ion, is performed under agitation.

In a particular embodiment, the weight ratio of the amino acid or with a combination of an amino acid, a metal ion or a metal oxide ion, and a either a metal ion or a metal oxide ion in the nanoparticles obtained in step c) is between 1 and 15% (w/w).

Preferably, once finished the contacting period, the nanoparticles comprising the amino acid, the metal ion or the metal oxide ion, or with a combination of an amino acid and a either a metal ion or a metal oxide ion are isolated from unbound compounds by centrifugation (12000 rpm, 15 min, 18 °C) and stored at 4 °C.

In another embodiment, when the composition A comprises a metal ion or a metal oxide ion, then the composition A additionally comprises a citric acid derivative.

Suitable citrate acid derivatives have already been described within the context of the composition of the invention and equally apply to the method of the invention. In a particular embodiment the citric acid derivative is sodium citrate (NasCeHsO?). In another embodiment, the citric acid derivative is potassium citrate (K3C6H5O7). In another embodiment, the citric acid derivative is at a concentration in a range from 0.01 M to 0.5 from 0.1 to 0.3 M, preferably about 0.2 M. In a preferred embodiment the citric acid derivative is potassium citrate (K3C6H5O7) at a concentration of 0.2 M.

That is, the citric acid derivative may be contained in an amount between 1% and 10 % w/w based on the total weight of the nanoparticle comprising the amino acid or the metal ion.

In another embodiment, the calcium salt is calcium chloride (CaCh) or calcium nitrate (Ca(NOs)2) and/or the phosphate is provided as a phosphate salt. Examples of phosphate salts include without limitation K3PO4, K2HPO4, Na2HPC>4, NasPC In a preferred embodiment the phosphate salt is selected from K2HPO4, K3PO4, KH2PO4, Na2HPC>4 and NasPC In another embodiment the phosphate salt is K2HPO4.

In a preferred embodiment, the calcium salt is at a concentration in a range from 0.05 M to 0.8 M, from 0.1 M to 0.3 M, more preferably about 0.2 M. In a particular embodiment, the calcium salt is selected from CaCh and Ca(NOs)2. In a particular embodiment the calcium salt is Ca(NOs)2 or CaCh at a concentration of 0.2 M. In a preferred embodiment, the calcium salt is CaCh at a concentration of 0.2 M.

In an embodiment, the phosphate is at a concentration in the range from 0.05 M to 0.3 M, from 0.1 to 0.2 M, preferable about 0.12 M. In another embodiment, the phosphate is K2HPO4 at a concentration of 0.12 M.

In a more particular embodiment, the calcium salt is CaCh at a concentration of 0.2 M, the phosphate is K2HPO4 at a concentration of 0.12 M and the citric acid derivative is KsCeHSO? at a concentration of 0.2 M.

In a preferred embodiment, the amino acid is glycine and the metal ion is Zn²⁺.

In another embodiment, the molar ratio between the metal ion or metal oxide ion and the calcium salt in the composition A is of between 1% and 100%, between 2% and 80%, between 3% and 60 %, between 4% and 50 %, between 5% and 40%, between 5% and 30 %, more preferably between 5% and 20%.

In a particular embodiment, the concentration of the amino acid in the composition A is of between 0.1 M to 10 M, between 0.1 and 5 M, between 0.1 and 2 M, between 0.1 and 1 M, more particularly between 0.2 M and 0.6 M. In a particular embodiment, the weight ratio of the amino acid or with a combination of an amino acid and an either a metal ion or a metal oxide ion in the nanoparticles is of about 1-15% (w/w).

In another embodiment the composition B further comprises a carbonate salt.

As used herein, a carbonate is a salt of carbonic acid (H2CO3), characterized by the presence of the carbonate ion, a polyatomic ion, a polyatomic ion with the formula CO3². The carbonate ion may be contained in an amount between 0.1 and 7% w/w based on the total weight of the nanoparticle comprising the compounds of interest.

In a particular embodiment, the carbonate salt is sodium or potassium carbonate.

The contacting of compositions A and B leads to the formation of a precipitate of calcium phosphate, which is collected in step b). The collecting of the precipitate may be performed by any conventional method known in the art, such as filtration, centrifugation or evaporation.

Typically, the type of particles formed after the interaction of composition A and B, are ACP. ACP, may transform into hydroxyapatite microcrystalline in the presence of water. The lifetime of the ACP precursor in aqueous solution is a function of the presence of additive molecules and ions, pH, ionic strength, and temperature. The precipitate obtained in step b) is dispersed in an aqueous solution according to the step c) of the method of the invention. As used herein “dispersing” is used as the process by which distributed particles of one material are dispersed in a continuous phase of another material. The two phases may be in the same or different states of matter. Typically, the precipitate is dispersed in an aqueous solvent, more preferably, water.

Optionally, prior to the dispersion step, the precipitate may be washed with ultrapure water by centrifugation, for example at 5000 rpm for 15 min at 18 °C for the removal of non-reacted ions.

In a particular embodiment, the method further comprises contacting the nanoparticles obtained in step (c) with a composition comprising a metal ion or a metal oxide ion, an amino acid or a combination thereof under conditions adequate for the adsorption of said metal ion or metal oxide ion, amino acid or combination thereof onto the surface of the nanoparticles.

At the end of the step of removal of non-reacted ions, a suspension of nanoparticles is obtained that can be subjected to addition of bidistilled water and freeze dried to obtain the calcium phosphate nanoparticles. Alternatively, the product of step b) can be freeze- dried to obtain powders.

Preferably, once finished the contacting period, the nanoparticles comprising the amino acid or with a combination of an amino acid and a either a metal ion or a metal oxide ion are isolated from unbound compounds by centrifugation (12000 rpm, 15 min, 18 °C) and stored at 4 °C.

In another aspect, the invention relates to a composition obtainable by the third method of the invention.

Further uses of the compositions of the invention

In another aspect, the invention relates to the use of the composition of the invention for supplying nutrient elements to a plant and/or for improving an agronomic trait in a plant.

The invention will be described by way of the following examples, which are to be considered as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

Materials and Methods

Materials

Potassium citrate tribasic dihydrate (^(CeHsOy^F^O, >99.0% pure), potassium phosphate dibasic anhydrous (K2HPO4, >99.0% pure), potassium hydroxide (KOH 85% pellet for analysis), calcium chloride dihydrate (CaCh 2H2O >99.0% pure) and zinc chloride (ZnCh, >97% ACS reagent) were purchased from Sigma-Aldrich. Glycine was purchased from Agrointec. Ultrapure water (0.22pS, 25°C, Milli-Q, Millipore) was used to prepare all the solutions.

Characterization of doped and non-doped Nanoparticles

Fourier transform infrared (FTIR) spectra of the samples were recorded on a Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). Powdered samples (2 mg) were mixed with 200 mg of anhydrous potassium bromide (KBr), set into a 12 mm diameter disc and pressed at 5 tons in a hydraulic press (Specac). Three pellets were produced for each sample and a KBr pellet without sample was used as blank. The infrared spectra were recorded from 400 cm^-1 to 4000 cm^-1 at a resolution of 4 cm^-1. X-Ray powder diffractograms (XRPD) were recorded on a Bruker AXS D8 Advance diffractometer using Cu Ka radiation (A = 1 .5418 A), from 8° to 60° (20) with a scan rate of 0.5 s step^-1, step size of 0.02° with an HV generator set at 40 kV and 40 mA. High-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) images and energy- dispersive X-ray (EDS) spectra of nanoparticles were acquired with STEM FEI TALOS F200X microscope equipped with a 4 Super-X SDDs (Thermo Fisher Scientific Waltham, MA, USA) of CIC-UGR. To this purpose, nanoparticles were ultrasonically dispersed in ultrapure water, and then, some drops of the slurry were deposited on 200 mesh copper grids covered with thin amorphous carbon films. Nanoparticle chemical composition was evaluated by inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 8300, PerkinElmer, from CIC-UGR). To this aim, 10 mg of the powdered samples were dissolved in 1 ml of ultrapure nitric acid and then, the mix was made up to 50 mL with ultrapure water. The samples were measured in triplicate at their correspondent emission wavelengths: 317.93 nm (Ca), 213.62 nm (P), 766.49 nm (K) and 206.20 nm (Zn). The surface charge of the nanoparticles (zeta potential, mV) was measured with Litesizer 500 (Anton Paar, Austria), through electrophoretic mobility.

The nitrogen and carbon content were measured by elemental analysis with a Thermo Scientific Flash 2000 organic elemental analyzer equipped with a microbalance (XP6, Mettler Toledo) from CIC-UGR.

Biofortification experiments in Tomato (Lycopersicon escullentum Mill.)

Commercial tomato (Lycopersicon escullentum Mill. var. cerasiforme, cv. HTL1708480 ®) was growth in a greenhouse at Universidad de Almeria (36°49'45"N 2°24'16"W) during the summer of 2018 under hydroponic conditions.

Automatic system (Himarcan R01A) was used to supply the basic nutrient solution to the crop with the following composition: 11 mM N (10 mM NOa' and 1 mM NH4⁺); 1 mM P; 8 mM K; 2 mM Ca; 1.5 mM Mg; 2 mM S; 4 pMol Fe; 2 pM Mn; 1 pM Zn; 1 pM Cu; 1 pM B and 0.5 pM Mo. Calcium restrictions were applied over 100% (Ca=2mM).

We evaluated the effect of the foliar application of different Zn source for a fixed Zn concentration (100 ppm): zinc doped amorphous calcium phosphate nanoparticles (ZnACP); zinc lignosulfonate (Zn-LgSf) and zinc sulfate (ZnSO4). For this study, control plants were sprayed with deionised water in the same volume and frequencies than Zn treatments. The Zn-containing compounds were dispersed in water with a magnetic stirrer (ANZESER SH-2) during 30 minutes and then foliarly applied into tomato plants, directly to the fruits, with an atomizer.

Fruit harvesting and sample analysis

At the commercial mature stage, only physiologically mature fruits were harvested and analysed. Yield (number of fruits, total weight, weight average), phenols content,⁴⁰ ° Brix (Digital refractometer Atago KERM), firmness (PSE-PTR 200 N), and colour (colorimeter Konica Minolta CR-400) were registered.

To evaluate the nutrient content of treated tomatoes, firstly each tomato was cut in 24 pieces and one to two pieces for each tomato were selected to obtain 31 g of “representative sample” per treatment. The samples were dried at 70°C for 72 hours on the oven. Following the Kjeldahl procedure, a dried sample of 0.25 g was passed through a Kjeldahl flask for mineralization to determine N, P, K, Ca, Mg, Fe, Mn, Zn and Cu. Nitrogen: 1 ml of mineralized sample and 25-30 ml of NaOH were mixed in the Kjeldahl flask and then connected to the Bouat device until the production of ammonia vapour. The latter was directed to the collecting flask, to be evaluated with Shiro-Tashiro dye.

The phosphorus content was determined by a spectrophotometer at 700 nm following the protocol of phosphomolybdic complex. The rest of elements (K, Ca, Mg, Fe, Mn, Zn and Cu) were analysed using an inductively coupled plasma mass spectrometry (ICP- MS, XSERIES 2, Thermo Fisher, Research facilities of Universidad de Almeria, UAL).

Results

Synthesis of ZnACP

Amorphous calcium phosphate (ACP) nanoparticles were synthesized in a clean, green, and scalable synthetic route inspired in bone mineralization. Two solutions of equal volume (100 mL) were mixed: (A) aqueous solution containing CaCh (0.2 M) and K3C6H5O7 (0.2 M) and (B) aqueous solution containing K2HPO4 (0.12M) with pH around 12 (adjusted with KOH). After stirring during 5 minutes at room temperature, the samples were centrifuged (5000 rpm, 10 minutes) to collect the nanoparticles and washed twice with ultra-pure water. Then, the nanoparticles were frozen at -20 °C and freeze-dried (Telstar) for further characterization. The same protocol was followed for the preparation of zinc-doped amorphous calcium phosphate (ZnACP) nanoparticles by adding ZnCh (40 mM) to the solution A at the initial molar Zn/Ca ratio of 20 %.

The XRD pattern of doped nanoparticles show a broad band centred at 30° (20) characteristic of ACP (Figure 1a). The diffraction data exclude the coprecipitation of highly crystalline zinc salts jointly with the nanoparticles. FTIR spectrum of Zn doped ACP nanoparticles displays the poorly defined phosphate vibrational bands characteristic of ACP along with the peaks ascribed to citrate and water (Figure 1 b). These vibrational bands were neither affected by the addition of Zn²⁺ ions.

The elemental composition of doped and non-doped ACP nanoparticles by ICP-OES was evaluated (Table 1). The mean Zn content in the as-prepared ZnACP sample was 10 wt.%. The incorporation of Zn²⁺ ions prompted to a slight decrease of Ca content as a result of the partial substitution of Ca²⁺ by Zn²⁺ ions. The (Ca+Zn)/P molar ratio of doped samples was lower than the control samples but it felt within the range typically found for ACP. ZnACP nanoparticles also show a less negative surface charge than control ACP nanoparticles, indicating that part of Zn²⁺ ions are adsorbed on the surface.

Table 1. Chemical composition and surface charge of as-prepared samples. Data are expressed as mean ± standard deviation.

~Ca K P Zn (Ca+Zn)/P

(wt.%) (wt.%) (wt.%) (wt.%) (Molarratio)

ACP 28.2 ± 4.2 1.8± 0.8 13.5± 2.0 “ 1.62 ± 0.01

ZnACP 21 .6 ± 0.7 1.5 ± 0.1 15.8 ± 0.3 10.6 ± 0.3 1.37 ± 0.02

HAADF-STEM image of the ZnACP sample (Figure 2) shows typical round shaped morphology of ACP nanoparticles, with average diameter of 22.7 ± 2.6 nm. The energy dispersive X-ray spectroscopy (EDS) mapping of the ZnACP reveals a uniform distribution of calcium, phosphorus and zinc within the nanoparticle. These findings confirmed that Zn is incorporated into amorphous calcium phosphate nanoparticles, as previously indicated the absence of Zn salt in XRD patterns. The Zn content of the nanoparticles remained constant for up to 7 months, as revealed by ICP-OES analysis of ZnACP samples storage at 4°C. After this period, the samples remained as ACP, confirming the long-term chemical stability of ZnACP. In a previous work, ACP nanoparticles synthesized through similar approach and functionalized with methyl jasmonate, remained stable in aqueous environment up to 49 days. The astonishing stability of ZnACP nanoparticles could be due to the synergetic effect of Zn and citrate on the stabilization of this amorphous phase. This aspect is of paramount importance for its future commercialization.

Use of nanoparticles for the biofortification in Tomato (Lycopersicon escullentum Mill.)

Biofortification assay was carried out in tomato plants, which are one of the most commonly grown, valuable and consumed vegetables worldwide. The nutritional composition of the tomato depends on the efficiency of nutrient uptake from the growing medium and thus an adequate amount of macro- and micro- nutrients are crucial to improve crop quality and yield. In this study, the effect of three different Zn sources on the yield and quality of tomato (Lycopersicon escullentum Mill) were evaluated: i) the most commonly used soluble Zn salt in the field (ZnSC>4); ii) Zn natural organic complexes (Zn-Lignosulfonate, Zn-LgSf) and iii) as-prepared Zn-doped amorphous calcium phosphate nanoparticles (ZnACP).

The effects of foliar application of three Zn sources on the yield and quality of tomato fruits are summarized in table 2. For all the tested variables, significant differences were obtained (p<0.05). Plants exposed to the foliar application of ZnACP nanoparticles produced the highest number of fruits, exceeding by 260% the control treatment. ZnSC>4 and Zn-LgSf also produced a significant increase of the fruit number, 197% and 247%, respectively. In the case of the fruit total weight, Zn-LgSf treatment achieved the highest value, followed by ZnACP and ZnSC>4 treatment. Zn application also increased the average fruit weight by 4.6%, 13% and 14% for ZnACP, ZnSC>4 and Zn-LgSf, respectively. All Zn treatments also enhanced the quality of tomato plants such as phenols content, total soluble solids (i.e., °Brix), firmness and colour.

The increase of number of fruit per cluster, fruit weight, number of fruits per plant, fruit yield per plant and higher total soluble solids (TSS, °Brix) as a result of Zn application in tomato plants had been widely proved. Whereas the foliar application of 0.35 mM of ZnSC>4 in two commercial cvs. (Blizzar and Liverto) of tomato (Lycopersicon esculentum L.) increased the fruit yield, average fruit weight and numbers of fruit respect to the control (0 mM Zn foliar application), the highest Zn foliar dosage (3.5 mM) triggered to a reduction of these values. It has been also demonstrated that the foliar application of ZnSC>4 (12.5 ppm or 100 ppm Zn) in combination with others micro- (B or Fe) or macronutrients (N, K and P) enhanced tomato yield. Zn can activate many enzymes involved in various biochemical pathways such as carbohydrate, protein and growth regulator metabolism, and thus, promote growth, yield and quality of crops.

In this study, the greatest improvement in tomato yield and quality was achieved with foliar application of Zn complexed with lignosulfonate (Zn-LgSf) and ZnACP nanoparticles. Other zinc nanofertilizer (i.e. , ZnO-NPs) also showed better enhancement of plant growth and fruit production than soluble ZnSO4 in coffee plants, habanero peppers and tomato, among others. Some authors pointed out that the low efficiency of the foliar application of soluble Zn salts is due to the fact that water soluble ions may have difficulty penetrating the lipophilic cuticle, thus limiting its availability. Zn enters the plant (leaf apoplast) directly through stomatai pore and increases Zn concentration in phloem tissue of leaf from where it can be directly translocated to growing sinks (J.e., foliage, grain, fruit). Therefore, Zn content of mature tomato was also evaluated in order to compare the effect of the foliar application of three different Zn sources (Table 3). ZnACP nanoparticles resulted in the highest Zn content in tomato fruit exceeding by 352% the control tomatoes. Zn-LgSf and ZnSC>4 treatment also increased Zn content in tomato by exceeding 279% and 174% to the control sample, respectively. Zn levels registered are close to the minimum required for the labelling of the fortified food products aimed at children (EC, 2008, 2012). This finding confirmed higher Zn efficiency for ZnACP nanoparticles than soluble Zn salts. The mechanism underlying the higher Zn efficiency for ZnACP nanoparticles needs further elucidation. It could be associated to the improvement of plant absorption and translocation and/or the gradual and controlled Zn supply provided to the plants along the slow dissolution of the nanoparticles. Another advantages of the foliar application Zn nanofertilizers compared to soluble Zn forms (ZnSOt) is the reduction of the likelihood of leaf burning due to high Zn dosage as well as reducing the need for repeated application of reduced dose of Zn throughout the growing season.

ZnACP treatment significantly improved the N, P and Ca content tomato fruits. Ca and P increase can be associated to the nanoparticle dissolution and gradual release of Ca and P (Table 3). Nonetheless, Ca and P content of tomato treated with ZnSC>4 and Zn- LgSF also increased compared to the control. Respect to N content, it has been pointed out that Zn helps plants absorb important nutrients, especially nitrogen responsible for protein synthesis. Table 2. Average values of yield and quality parameters in biofortified tomato fruits under different treatments.

Different letters in the same row show significant differences at 95% confidence level.

Table 3. Average values of element composition in biofortified tomato fruit.

Conclusions

Biocompatible and biodegradable amorphous calcium phosphate (ACP) nanoparticles were successfully doped with zinc ions (10 %w/w Zn content) without altering morphology and structure. EDX maps of these nanoparticles revealed a homogeneous Zn distribution along with calcium and phosphorus. These nanoparticles showed a longterm chemical stability, quite important issue for its potential commercialization. The Zn nutrient efficiency of ZnACP nanoparticles was assayed in vivo by means of greenhouse experiments on tomato plants. Foliar application of ZnACP nanoparticles triggered to a relevant enhancement of the yield and quality of the fruits respect to the control sample. More importantly, the treatment with ZnACP nanoparticles provided tomatoes with the highest Zn content (0.86 mg/100 g FW), followed by zinc sulphate (ZnSC>4, 0.52 mg/100 g FW) and zinc lignosulfonate (Zn-LgSF, (0.72 mg/100 g FW), two commercial Zn fertilizers widely used for biofortification. Compared to conventional fertilizers, ZnACP nanoparticles are promising strategy toward enhancing Zn efficiency in crops and producing Zn fortified products through sustainable agriculture practices.

Effect of ZnACP in flowering physiology in pepper crops

Five genotypes (breeding lines) of pepper were transplanted in a greenhouse at Universidad de Almeria (36°49'45"N 2°24'16"O) during spring 2021. Hydroponic System with 29 L bags of coconut fiber (Golden Grow - 85% dry matter) was used. The nutrient solution was prepared depending on daily consumption with the following composition: [(mMol)NO3: 13; NH4: 1.5; PO4: 1.5; K: 8; Ca: 3; Mg: 1.5; SO4: 2]; [(pMol) Fe: 15; Mn: 10; Zn: 0.5; Cu: 0.2; B: 2.5; Mo: 0.5], Biomimetic calcium phosphate nanoparticles (AGP) doped with Zn were synthesized though a biomimetic approach.

Total of 20 plants per each genotype were treated with ZnACP and 20 plants without treatment (Control). Foliar applications with electric spray were targeted to the apex, 12, 26, 36, and 49 days after transplanting (DAT).

The number of flowers increased with the time (days after transplanting-DAT) (Figure 3). ZnACP application showed 13.8% more total flowers with respect to the control. There were significant differences between cultivars. The Cv4 and Cv5 showed a higher number of flowers (18.5%) than the rest of cultivars, as well as the ZnACP showed almost one more flower than control (Table 4).

In table 4 is detailed the mean values of number of flowers per cultivar and treatment. Group 1 (CV1 , CV2, CV3) had a positive response, increasing around 30%, while Cv3 showed the highest increase in the number of flowers (40.8%).

Table 4. Average number of flowers and effect of ZnACP in five cultivars of pepper.

Different letters in the same column represent significant differences for ANOVA and LSD (<0.05).

There were significant differences between cultivars for phenological stages and morphological parameters. Regarding the treatment, there is an increase in pigmentation value, number of flower buds, total cells per mL and percentage of vacuolated microspores related to ZnACP (Table 5). The highest percentage (66.4%) of vacuolated microspore was reported in the BBCH3=5 stage compared to BBCH3=4 and BBCH3=6 (17.3 and 17.8, respectively) (Table 5) (Figure 4).

Table 5. Mean data for factors and the variables related to BBCH3=4. BBCH3=5. BBCH3=6

Different letters in the same column represent significant differences LSD (p>0.05).

Conclusions

The use of nanoparticles of amorphous calcium phosphate doped with Zn (ZnACP) have enhanced the pepper bloom at different levels depending on the cultivar. An increase of the number of flowers have been observed in most of the genotypes including in this assay. Zn has an important role in wall cell development and metabolism and enzyme synthesis in the plant. The ZnACP application seems to have an impact on pepper flowering, enhancing this process, and consequently affecting positively over pollen and seed production. Additionally data supports the effect of ZnACP on the percentage of vacuolated microspores, the selection of BBCH3=5 as a reference and critical stage in the androgenesis, and also in the route between microsporogenesis and microgametogenesis. This relevant information could be taken advantage of for a breeding approach in the development of double haploids. Impact of different Zn sources in the flowering and production of a commercial hybrid pepper

Plantlets of a commercial pepper (Capsicum annum L.) genotype (Hybrid F1 KLIMAN) were transplanted in an Almeria-type greenhouse located at El Ejido town (36°47'39"N 2°42'49"O) during summer 2021.

Five treatments were applied at the early stage of flower buds development in order to compare the effect of different sources of Zn including an extra nanoparticle without Zn and one treatment with boron because this element is the gold standard in flowering and fruit production. Evaluation in flowering development started 15 days after first application. Data obtained at 30, 37 and 45 days after transplanting are represented in Figure 5.

The sources of Zn and treatments were the following: T1 : ZnACP, T2: ACP (control), T3: Zn-LgSf (ZnMicro), T4: ZnO (Agroxilato), T5 (Codibor) and Control (No Zinc).

All fertilizers were calculated to supply 100 ppm of Zn.

Regarding flowering behavior, the effect of all Zn sources was remarkable. Zn applied in different forms enhanced and increased the total number of flower buds/inflorescences, more evident around thirty days after transplant (Figure 5).

At an early stage of development, the application of ZnACP showed an important increase of approximately 50% more flower buds compared to control plants and even higher than the rest of treatments. However, this effect tends to be less prominent over time.

Table 6. Percentage of flower bud increase (+) or decrease (-) in five treatments compared to control plants.

Conclusions

The use of ZnACP showed an effect more evident at early stages of development with an increase in the number of flower buds compared to control plants, and even slightly superior than other Zn sources.

Data showed that nanoparticles doped with Zn (100 ppm) can be applied in a commercial pepper hybrid (KLIMAN F1) with positive effects in flowering behavior. Complementary information has to be analyzed to understand the impact of Zn sources in yield, biofortification, seed and fruit quality (evaluation in progress).

Synthesis of calcium phosphate nanoparticles at different pHs

First, the synthesis of calcium phosphate nanoparticles is carried out at different pHs, to study the calcium phosphate phase that forms at acidic pH, neutral pH and basic pH. Calcium phosphate nanoparticles are wet-synthesized by mixing a calcium chloride solution (0.2 M CaCh) with a potassium phosphate solution (0.12 M K2HPO4). The synthesis is carried out at acidic pH, neutral pH by addition of 5 mL of sodium hydroxide (1M NaOH) and at basic pH by addition of 1.95 mL of NaOH (3M). The precipitation reaction is carried out at room temperature. Two maturation times are studied: 30 minutes and 24 hours. After this period, the reaction is stopped by centrifugation (5000 rpm, 15 minutes) and the samples obtained are washed with ultrapure water to remove excess reagents. The samples are then frozen at -20°C and lyophilized (pressure <1 mbar). The powder obtained is characterized by several techniques.

DCPD is obtained after 30 minutes at acidic pH, as indicated by the X-ray diffraction pattern (Figure 6a) as well as the infrared spectrum (Figure 6b). On the other hand, ACP is obtained at basic and neutral pH. Both the X-ray diffraction patterns as well as the infrared spectra of the calcium phosphate samples synthesized at different pHs show only the peaks associated with the calcium phosphate phases and do not show the presence of any impurities in any case, indicating that washing was effective.

When the reaction is kept under stirring for up to 24 hours at room temperature, the ACP is transformed into HA under neutral pH and basic pH conditions (Figure 7), whereas under acidic conditions, DCPD remains as the main phase and has not been transformed to any other phase. Both diffraction patterns and infrared spectra show no peaks associated with impurities or other phases in the final product, indicating that the reaction was carried out under the desired conditions and the washing was effective.

The morphology and dimensions of calcium phosphate nanoparticles (control) synthesized at basic pH after 30 minutes (ACP) and 24 hours (HA) have been analyzed by transmission electron microscopy (Figure 8). The nanoparticles obtained after 30 minutes have a spherical morphology typical of amorphous calcium phosphate. The diameter of these nanoparticles is around 20 nanometers. Within 24 hours, elongated nanoparticles with a morphology similar to the calcium phosphate nanoparticles that are part of our bones are obtained. Nanoparticles have a length of around 20 nm and thickness of 2-3 nm, but tend to aggregate both laterally and longitudinally.

2.2. Evaluation of the influence of pH and maturation time on glycine incorporation Glycine-functionalized calcium phosphate nanoparticles are wet-synthesized by mixing a solution of calcium chloride (0.2 M CaCh) and glycine (0.2 M) with a potassium phosphate solution (0.12 M K2HPO4). The synthesis is carried out at acidic pH, neutral pH by addition of 5 mL of sodium hydroxide (1 M NaOH) and at basic pH by addition of 15 mL of NaOH (1 M).

Figure 9 shows the X-ray diffraction patterns (a and c) as well as the infrared spectra (b and d) of the glycine-functionalized calcium phosphate nanoparticles at different pHs and after 30 minutes (a and b) and 24 hours (c and d) of maturation. The incorporation of glycine does not affect the calcium phosphate phase that precipitates, obtaining the same results as the control (Figure 6 and 7): at acidic pH, DCPD is obtained at 30 minutes and 24 hours, while at neutral and basic pH, ACP and HA are obtained at 30 minutes and 24 hours, respectively. The infrared spectra of the samples confirm the incorporation of glycine in nanoparticles synthesized at neutral pH and basic pH both at short maturation times (30 minutes, Figure 9b) and at long maturation times (Figure 9d, 24 hours).

The effective amount of amino acid incorporated in the amorphous calcium phosphate nanoparticles has been determined by elemental analysis (Table 8). The results indicate that there is no incorporation of glycine at acidic pH values whereas 1.98% and 1.72% of amino acid is incorporated when the synthesis is carried out at neutral pH and at basic pH for 30 minutes (Table 8). Increasing the maturation time to 24 hours doubles the amount of glycine incorporated at basic pH, reaching a value of 3.48 g of glycine incorporated in 100 g of hydroxyapatite nanoparticles.

Table 8. Composition (N, C and O in % by weight) of the nanocomposites of calcium phosphate glycine nanoparticles.

Optimization of glycine incorporation by increasing the initial amount in the reaction medium

The amount of glycine added in the calcium chloride solution is doubled (0.4 M glycine, CaP_Gly2) and tripled (0.6 M glycine, CaP_Gly3) to optimize the incorporation of glycine into calcium phosphate nanoparticles. The reaction is carried out at basic pH and will be evaluated both at short times (30 minutes) and at long times (24 hours).

At short times, amorphous calcium phosphate is obtained for all three conditions, as indicated by the diffraction pattern in Figure 10a. No other salts or impurities are present. The infrared spectra show the peaks associated with glycine for the three samples, being more intense for the sample prepared with the highest glycine concentration (CaP_Gly3), indicating greater incorporation of glycine as the amount of glycine in the reaction mixture increases. Elemental analysis values confirm that doubling and tripling the amount of glycine in the reaction doubles and triples the amount of glycine in the final product (T able 9), reaching 5.36% glycine in the nanocomposite synthesized at an initial glycine concentration of 0.6 M. Table 9. Composition (N, C and H in weight percent) of calcium phosphate nanoparticle nanocomposites obtained in the presence of glycine at different concentrations 0.2 M (CaP_Gly1), 0.4 M (CaP_Gly2) and 0.6 M (CaP_Gly3) after 30 minutes of maturation.

At 24 hours of maturation, low-crystalline hydroxyapatite is obtained for all three conditions, as indicated by the diffraction pattern in Figure 11a. No other salts or impurities are present. The infrared spectrum (Figure 11 b) shows the peaks associated with glycine, which are more intense for the sample called CaP_Gly3, indicating a greater incorporation of glycine as the amount of glycine in the reagent solutions increases. Elemental analysis values indicate that doubling and tripling the amount of glycine in the reaction yields 1.5 and 1.88 times more glycine in the final product (Table 10). The highest amount of glycine incorporated in nano-apatites is 6.54% for the reaction carried out with 0.6 M glycine. These results indicate that maturation time is a determining factor in the incorporation of glycine since at 30 minutes the maximum amount is 5.36% while at 24 hours it amounts to 6.54%.

Table 10. Composition (N, C and H in weight percent) of calcium phosphate nanoparticle nanocomposites obtained in the presence of glycine at different concentrations 0.2 M (CaP_Gly1), 0.4 M (CaP_Gly2) and 0.6 M (CaP_Gly3) after 24 hours of maturation.

CaP-Gly3 nanocomposites obtained after 30 minutes (Figure 12a) and 24 hours (Figure 12b) of maturation have a morphology similar to calcium phosphate nanoparticles synthesized under the same conditions in the absence of glycine (Figure 8).

Thermogravimetric analysis (Figure 13) of the nanocomposite obtained with the highest amount of glycine, CaP-Gly3 shows three main weight losses: from 30°C to 220°C associated with water loss that reaches a value of 10%, from 227°C to 645°C associated with glycine content that reaches a value of 6.5% and which is consistent with elemental analysis data and from 645°C to 871 °C associated with carbonate loss reaching a value of 2.1% by weight.

Claims

CLAIMS A composition comprising calcium phosphate nanoparticles wherein the nanoparticles comprise an encapsulated compound selected from a metal ion or a metal oxide ion, an amino acid or a combination thereof, wherein the calcium phosphate is amorphous calcium phosphate (ACP) and wherein the nanoparticles does not comprise CaCCh or CaBCh. The composition according to claim 1 , wherein the nanoparticles further comprise a compound adsorbed on the surface of the nanoparticles and wherein said compound is selected from a metal ion or a metal oxide ion, an amino acid or a combination thereof. The composition according to claim 2, wherein the nanoparticles contain a metal ion or a metal oxide ion encapsulated within the nanoparticles and an amino acid adsorbed on the surface of the nanoparticles. The composition according to any of claims 1 to 3, wherein the amino acid is selected from proline, cysteine, glutamine, glycine, histidine, lysine, alanine, arginine, serine, tryptophan, valine, glutamic acid, phenylalanine and asparagine. The composition according to any of claims 1 to 4, wherein the metal ion is selected from Fe²⁺/Fe³⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Ag⁺, and/or wherein the metal oxide ion is selected from BO3³; SeOa²' and MoCU^2-. The composition according to any of claims 4 or 5, wherein the amino acid is glycine and/or wherein the metal ion is Zn²⁺ The composition according to any of claims 1 to 6, wherein the content of the amino acid in the nanoparticles is of between 1 % and 7 % (w/w) and/or wherein the content of metal ion in the nanoparticles is of between 5 % and 12 % (w/w) and/or the content of the metal oxide ion is of between 1 % and 5 % (w/w).

8. The composition according to any of claims 1 to 7, wherein when the nanoparticles comprises a metal ion, then, the nanoparticles further comprise a citric acid derivative.

9. A method for preparing a composition according to any of claims 1 to 8, wherein the method comprises: a) contacting a composition A comprising a calcium salt and an amino acid, or a combination of an amino acid and either a metal ion or a metal oxide ion with a composition B comprising a phosphate salt under conditions adequate for the formation of a precipitate formed by calcium phosphate nanoparticles, wherein the contacting is carried at basic or neutral pH; b) collecting the precipitate obtained in step a), and c) dispersing the precipitate obtained in step b) in an aqueous solvent.

10. The method according to claim 9, wherein, the contacting step a) is carried out at basic pH for at least 24 hours.

11. The method according to any of claims 9 or 10, wherein, when the composition A comprises a metal ion or a metal oxide ion, then the composition A additionally comprises a citric acid derivative.

12. The method according to claim 11 , wherein the citric acid derivative is potassium citrate.

13. The method according to any of claims 9 to 12, wherein the calcium salt is calcium chloride (CaCI₂) or calcium nitrate (Ca(NO₃)₂) and/or wherein the phosphate is provided as a phosphate salt.

14. The method according to any of claims 9 to 13, wherein the phosphate salt is selected from K₂HPO₄, K₃PO₄, KH₂PO₄, Na₂HPO₄, NaH₂PO₄ and Na₃PO₄.

15. The method according to any of claims 9 to 14, wherein the calcium salt is at a concentration of 0.2 M and the phosphate is at a concentration of 0.12 M.

16. The method according to any of claims 11 to 15, wherein the citric acid derivative is at a concentration of 0.2 M.

17. The method according to any of claims 9 to 16, wherein the amino acid is glycine and/or wherein the metal ion is Zn²⁺.

18. The method according to any of claims 9 to 17, wherein the molar ratio between the metal ion and the calcium salt in the composition A is of between 5% and 20% (w/w).

19. The method according to any of claims 9 to 18, wherein the concentration of the amino acid in the composition A is of about 0.1 M to about 10 M, more particularly between 0.2 M and 0.6 M.

20. The method according to any of claims 9 to 19, wherein the method further comprises -

21. A composition obtainable by the method as defined in claims 9 to 20.

22. A method for promoting uptake of a metal ion and/or of an amino acid in plants which comprises applying the composition according to any of claims 1 to 8 or 21 to the plant, to a propagule thereof or to the soil in which the plant is grown.

23. A method for improving an agronomic trait in a plant which comprises applying the composition according to any of claims 1 to 8 or 21 to the plant, to a propagule thereof or to the soil in which the plant is grown.

24. The method according to any of claims 22 or 23 wherein the application is carried out by impregnating the leaves of the plant with an aqueous suspension of the nanoparticles.

25. The method according to any of claims 22 to 24 wherein the composition that is applied further comprises at least one additional agrochemical compound. The method according to any of claims 22 to 25 wherein the plant is a tomato or a pepper plant. Use of a composition as defined in any of claims 1 to 8 or 21 for supplying nutrient elements to a plant and/or for improving an agronomic trait in a plant. Use of a composition as defined in any of claims 1 to 8 or 21 as a fertilizer.