Methods of enhancing crop resilience against environmental stresses

Abstract

Disclosed herein is a plurality of doped carbon nanodots, comprising: a plurality of carbon nanodots; and a dopant comprising from one or more of the group consisting of Si, Ce and Se, wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 1.5 to 4 wt%. Also disclosed herein are a method of preparing a plurality of carbon nanodots, a method of enhancing plant stress resilience to an abiotic stressor, use of a plurality of doped carbon nanodots, and a fertilizer formulation comprising a plurality of doped carbon nanodots.

Description

[0001] METHODS OF ENHANCING CROP RESILIENCE AGAINST ENVIRONMENTAL

[0002] STRESSES

[0003] Field of Invention

[0004] The present disclosure generally relates to methods of enhancing crop resilience, and more particularly relates to methods of enhancing crop resilience against environmental stresses.

[0005] Background

[0006] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0007] The projected world population will increase to 9.7 billion in the next 30 years, and the requirement of overall food production is over 60% relative to 2010. Although the current agricultural industry and production can provide enough food, it is still unsustainable and negatively impacts the environment. Furthermore, the other resources spent on agriculture, including fertilizers, pesticides, water, and land resources, are staggering. There is an agreement that sustainable development in agriculture practice helps to achieve the capacity to fulfil the increasing food requirement and minimize environmental issues, resulting in longterm high product yield in the future.

[0008] The biotic and abiotic stresses during the agriculture crop growth period are one of the main factors influencing crop production and yield significantly. The common stresses include drought, salinity, extreme temperature, UV radiation, etc. As the global population increases, the demand for food also increases significantly, and these environmental stresses are presented worldwide that limit crop and food production. Thus, it is essential to overcome the stresses and promote crop yield.

[0009] Recently, nanoparticles have been studied as one of the solutions to provide plants with enough stress tolerance against these environmental stresses. Spinach is a kind of winter vegetable that has been widely grown in urban vertical farming in countries with limited land resources, such as Singapore. However, spinach is sensitive to abiotic stress, for example, the high temperature. Thus, the growing of spinach in urban farming is energy-intensive as it is necessary to maintain a low temperature and ventilation. So far, there has been limited research on the stress tolerance enhancement of spinach and the approaches to help spinach achieve stress tolerance by using nanotechnology have attracted the interest of research. With the enhancement in spinach stress tolerance, more energy-saving growth in urban farming could be achieved and the production yield and nutrient level could also be improved.

[0010] Silicon (Si) fertilizer in the form of monosilicic acid, orthosilicic acid, and inorganic complex has been used to supply the crop to improve the yield and quality. However, the uptake and absorption of Si by plants from soil is limited and the amount absorbed may not be sufficient to promote the growth of crops. Meanwhile, the high solubility and absorption rate of monosilicic acid and orthosilicic acid may also induce potential phytotoxicity in crops. One of the effective approaches for eliminating these disadvantages of direct application of Si fertilizer is to use nano-type Si. Currently, silica nanoparticles (SiNP) have attracted the interest of research and are being used as the Si fertilizer in agriculture applications due to their small size and high surface area properties. It has been preliminarily reported in some plant growth studies that Si nanoparticles showed potential in either production increment or stress tolerance enhancement, in watermelon, cucumber, and groundnut. Nevertheless, SiNP still have difficulty in real applications due to its poor stability in temperature and pH environments. Furthermore, silicon is not the essential micro or macronutrient plants require during growth. Thus, the recent studies on nano form Si utilization in agriculture applications are still limited and not systematic.

[0011] Therefore, to overcome at least one of the aforementioned problems, there exists a need for new materials and new methods of enhancing crop resilience against environmental stresses.

[0012] Summary of Invention

[0013] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0014] 1 . A plurality of doped carbon nanodots, comprising: a plurality of carbon nanodots; and a dopant comprising from one or more of the group consisting of Si, Ce and Se, wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 1 .5 to 4 wt%.

[0015] 2. The plurality of doped carbon nanodots according to Clause 1 , wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 2 to 3 wt%, such as from 2.07 to 2.5 wt%. 3. The plurality of doped carbon nanodots according to Clause 1 or Clause 2, wherein the plurality of doped carbon nanodots have one or more of the following properties:

[0016] (a) a hydrodynamic size of from 40 to 120 nm, such as from 50 to 100 nm, such as from 70 to 80 nm, such as about 78.71 nm;

[0017] (b) a stability in an aqueous solution of at least six months; and

[0018] (c) the plurality of doped carbon nanodots are substantially free of toxic components; and

[0019] (d) the plurality of doped carbon nanodots are suitable for use in agriculture.

[0020] 4. The plurality of doped carbon nanodots according to any one of the preceding clauses, wherein the dopant comprises Si.

[0021] 5. A method of preparing a plurality of carbon nanodots, the method comprising:

[0022] (i) providing an aqueous mixture comprising a carbon source precursor and a dopant precursor; and

[0023] (ii) subjecting the aqueous mixture to a hydrothermal reaction at an elevated temperature for a period of time to provide the plurality of carbon nanodots, wherein: the carbon source precursor is selected from one or more of the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine (EDA), citric acid, urea, glycerol, and chitosan; the dopant precursor is selected from one or more of the group consisting of a silica, a silicic acid, a silicate salt, ceric oxide, a ceric acid, a ceric salt, selenium oxide, a selenic acid, and a selenic salt.

[0024] 6. The method according to Clause 5, wherein the weight to weight ratio of the carbon source precursor to the dopant precursor is from 100:1 to 5:1 , such as from 50:1 to 9:1 , such as about 10:1.

[0025] 7. The method according to Clause 5 to Clause 6, wherein the hydrothermal reaction is conducted in an autoclave heated to a temperature of from 100 to 300 °C, such as from 120 to 240 °C, such as about 200 °C.

[0026] 8. The method according to any one of Clauses 5 to 7, wherein the hydrothermal reaction is conducted for a period of from 1 hour to 24 hours, such as from 2 to 16 hours, such as from 5 to 15 hours, such as about 10 hours.

[0027] 9. The method according to any one of Clauses 5 to 8, wherein the carbon source precursor is EDTA. 10. The method according to any one of Clauses 5 to 9, wherein the dopant precursor is a silica, optionally wherein the carbon source precursor is EDTA and wherein the dopant precursor is a silica.

[0028] 11. A method of enhancing plant stress resilience to an abiotic stressor, the method comprising providing a formulation comprising plurality of doped carbon nanodots according to any one of Clauses 1 to 5 and applying the formulation to a plant subjected to an abiotic stress.

[0029] 12. Use of a plurality of doped carbon nanodots according to any one of Clauses 1 to 5 in enhancing plant stress resilience to an abiotic stressor.

[0030] 13. The method according to Clause 11 or the use according to Clause 12, wherein the abiotic stressor is one or both of a heat stress and a salinity stress, where the level of heat and / or salinity to provide a stress is relative to the optimal growing conditions of a particular plant species.

[0031] 14. The method of Clause 1 1 or Clause 13 or the use of Clause 12 or 13, wherein the formulation comprises a concentration of the plurality of doped carbon nanodots of from 10 to 500 mg / L, such as from 20 to 400 mg / L, such as from 25 to 300 mg / L, such as from 50 to 200 mg / L, such as from 75 to 150 mg / L, such as from 25 to 100 mg / L.

[0032] 15. The method of any one of Clauses 1 1 and 13 to 14 or the use of any one of Clause 12 to 14, wherein the formulation is provided in an amount to provide from 1 to 100 mg of the plurality of doped carbon nanodots per plant, such as from 2 to 20 mg / plant, such as from 3 to 4 mg / plant, such as about 3.5 mg / plant.

[0033] 16. The method of any one of Clauses 1 1 and 13 to 14 or the use of any one of Clause 12 to 14, wherein a plant species to be treated is selected from one or more of the group consisting of spinach, arugula, and kalian.

[0034] 17. A fertilizer formulation comprising a plurality of doped carbon nanodots according to any one of Clauses 1 to 5.

[0035] Drawings FIG. 1 depicts (a) Si-doped carbon dots (CD-Si) solution under daylight, (b) CD-Si solution under ultraviolet (UV) light, (c) transmission electron microscopy (TEM) image, (d) TEM-EDX image, (d-i)-(d-iv) energy-dispersive X-ray (EDX) elemental mapping, including C, O, N, and Si, (e) dynamic light scattering (DLS) spectrum, (f) Ultraviolet-visible (UV-Vis) spectrum, (g) Photoluminescence spectra, and (h) Fourier transform infrared (FTIR) spectrum of CD-Si.

[0036] FIG. 2 depicts emission fluorescence of CD-Si over 6 months.

[0037] FIG. 3 depicts phenotype of spinach under treatments of (a) HT control, (b) LT control, (c) CD- Si 25 mg / L, (d) CD-Si 50 mg / L, (e) CD 25 mg / L, (f) CD 50 mg / L, (g) SiO225 mg / L, and (H) SiO250 mg / L on day 63 before harvesting.

[0038] FIG. 4 depicts growth behavior of spinach in vermiculite under heat stress (a) fresh leaf weight, (b) fresh stem weight, (c) fresh root weight, and (d) total fresh biomass. Data with different letters are considered significantly different (P < 0.05).

[0039] FIG. 5 depicts (a) chlorophyll content and (b) carotenoid content of fresh spinach leaf. Data with different letters are considered significantly different (P < 0.05).

[0040] FIG. 6 depicts the phenotype of spinach growth under three different salinity stress levels and the spinach growth under same salinity stress level but with CD-Si treatments.

[0041] FIG. 7 depicts growth parameter of spinach in vermiculite: (a) total number of leaves; (b) total fresh weight of leaves; (c) total fresh biomass, and (d) chlorophyll content. Data with different letters are considered significantly different (P < 0.05).

[0042] FIG. 8 depicts DLS size results for CD-Si synthesized using silicic acid and sodium silicate at different ratios (SA-1 , SA-2, SA-3, SS-1 , SS-2 and SS-3).

[0043] FIG. 9 depicts (a) DLS spectrum and (b) TEM image of CD-Se.

[0044] FIG. 10 depicts EDX image of CD-Se, including C, N, O, and Se elements mapping.

[0045] FIG. 11 depicts (a) DLS spectrum and (b) TEM image of CD-Ce.

[0046] FIG. 12 depicts EDX image of CD-Ce, including C, N, 0, and Ce elements mapping. Description

[0047] It has been surprisingly found that the treatment of plants with a plurality of doped carbon nanodots as disclosed herein assist the plants in fighting against environmental stresses such as heat and salinity. For example, plants treated with the plurality of doped carbon nanodots have improved stress tolerance and plant growth and recovery.

[0048] Thus, in a first aspect of the invention, there is provided a plurality of doped carbon nanodots, comprising: a plurality of carbon nanodots; and a dopant comprising from one or more of the groups consisting of Si, Ce and Se, wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 1 .5 to 4 wt%.

[0049] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0050] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greaterthan 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0051] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like. Where used herein, the term “a plurality of doped carbon nanodots” refers to two or more of carbon doped nanodots.

[0052] Where used herein, the term “a plurality of carbon nanodots” refers to two or more of carbon nanodots.

[0053] In some embodiments that may be mentioned herein, the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots may be from 2 to 3 wt%, such as from 2.07 to 2.5 wt%.

[0054] In some embodiments that may be mentioned herein, the plurality of doped carbon nanodots may have one or more of the following properties:

[0055] (a) a hydrodynamic size of from 40 to 120 nm, such as from 50 to 100 nm, such as from 70 to 80 nm, such as about 78.71 nm;

[0056] (b) a stability in an aqueous solution of at least six months; and

[0057] (c) the plurality of doped carbon nanodots are substantially free of toxic components; and

[0058] (d) the plurality of doped carbon nanodots are suitable for use in agriculture.

[0059] Details of the hydrodynamic size measurement technique are provided in the examples section below.

[0060] Without wishing to be bound by theory, the plurality of doped carbon nanodots can generally maintain their particle size and optical properties in an aqueous solution thus demonstrating their long-term stability under proper storage conditions (e.g. room temperature and in the dark).

[0061] When used herein, the term “substantially free of toxic components” refers to the plurality of doped carbon nanodots having less than 1 wt% of toxic components relative to the total weight of the plurality of doped carbon nanodots. For example, the plurality of doped carbon nanodots may have less than 0.5 wt%, such as less than 0.1 wt%, such as less than 0.01 wt%, such as no toxic components.

[0062] When used herein, the term “toxic components” refers to toxic carbon sources such as Rhodamine B (RhB), phthalic acid, phenol compounds, and thiourea, toxic Si sources such as SiCk, TEOS (Tetraethyl orthosilicate), and APTES ((3-Aminopropyl) triethoxysilane), and organic solvents such as acetone, ethanol, and dimethylformamide. Without wishing to be bound by theory, the plurality of doped carbon nanodots may be substantially free of toxic components because the methods of synthesizing the plurality of doped carbon nanodots avoid the use of toxic components. As will be appreciated, as the plurality of doped carbon nanodots can be substantially non-toxic, environment-friendly and chlorine-free, they may be suitable for use in agriculture.

[0063] In some embodiments that may be mentioned herein, the dopant may comprise Si.

[0064] In a second aspect of the invention, there is provided a method of preparing a plurality of carbon nanodots, the method comprising:

[0065] (i) providing an aqueous mixture comprising a carbon source precursor and a dopant precursor; and

[0066] (ii) subjecting the aqueous mixture to a hydrothermal reaction at an elevated temperature for a period of time to provide the plurality of carbon nanodots, wherein: the carbon source precursor is selected from one or more of the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine (EDA), citric acid, urea, glycerol, and chitosan; the dopant precursor is selected from one or more of the group consisting of a silica, a silicic acid, a silicate salt, ceric oxide, a ceric acid, a ceric salt, selenium oxide, a selenic acid, and a selenic salt.

[0067] Examples of suitable Si dopant precursors include, but are not limited to silicon dioxide (SiOz), silicic acid (e.g. [H2xSiOx+2]n, orthosilicic acid, metasilicic acid, and pyrosilicic acid), silicate salts, and combinations thereof.

[0068] Examples of suitable Ce dopant precursors include, but are not limited to Ce(OH)4, cerium acetate, cerium oxides, and combinations thereof.

[0069] Examples of suitable Se dopant precursors include, but are not limited to Na2SeOs, selenourea, selenocystine, selenium dioxide, and combinations thereof.

[0070] Details of the hydrothermal reaction are provided in the examples section below.

[0071] Any suitable elevated temperature may be used for the hydrothermal reaction. For example, the hydrothermal reaction may be conducted in an autoclave heated to a temperature of from 100 to 300 °C, such as from 120 to 240 °C, such as about 200 °C. Any suitable period of time may be used for the hydrothermal reaction. For example, the hydrothermal reaction may be conducted for a period of from 1 hour to 24 hours, such as from 2 to 16 hours, such as from 5 to 15 hours, such as about 10 hours.

[0072] Any suitable weight to weight ratio of the carbon source precursor to the dopant precursor may be used in the formulations. For example, the weight to weight ratio of the carbon source precursor to the dopant precursor may be from 100:1 to 5:1 , such as from 100:1 to 9:1 , such as from 100:1 to 10:1 , such as from 100:1 to 50:1 , such as from 50:1 to 5:1 , such as from 50:1 to 9:1 , such as from 50:1 to 10:1 , such as from 10:1 to 5:1 , such as from 10:1 to 9:1 , such as from 9:1 to 5:1 , such as about 10:1. In some embodiments that may be mentioned herein, the weight to weight ratio of the carbon source precursor to the dopant precursor may be from 100:1 to 5:1 , such as from 50:1 to 9:1 , such as about 10:1 .

[0073] In some embodiments that may be mentioned herein, the carbon source precursor may be EDTA.

[0074] In some embodiments that may be mentioned herein, the dopant precursor may be a silica. In further embodiments that may be mentioned herein, the carbon source precursor may be EDTA and the dopant precursor may be a silica.

[0075] In a third aspect of the invention, there is provided a method of enhancing plant stress resilience to an abiotic stressor, the method comprising providing a formulation comprising plurality of doped carbon nanodots according to the first aspect of the invention and applying the formulation to a plant subjected to an abiotic stress.

[0076] Details of the method of enhancing plant stress resilience to an abiotic stressor are provided in the examples section below.

[0077] In a fourth aspect of the invention, there is provided use of a plurality of doped carbon nanodots according to the first aspect of the invention in enhancing plant stress resilience to an abiotic stressor.

[0078] Examples of abiotic stressors include, but are not limited to heat stress, salinity stress, and moisture stress. In some embodiments that may be mentioned herein, the abiotic stressor may be one or both of a heat stress and a salinity stress, where the level of heat and / or salinity to provide a stress is relative to the optimal growing conditions of a particular plant species. Any suitable concentration of the plurality of doped carbon nanodots may be used in the formulations. In some embodiments that may be mentioned herein, the formulation may comprise a concentration of the plurality of doped carbon nanodots of from 10 to 500 mg / L, such as from 20 to 400 mg / L, such as from 25 to 300 mg / L, such as from 50 to 200 mg / L, such as from 75 to 150 mg / L, such as from 25 to 100 mg / L. For example, the concentration of the plurality of doped carbon nanodots may be 25 mg / L or 50 mg / L. As will be appreciated, the concentration of the plurality of doped carbon nanodots used in the formulations provides improved Si delivery efficiency and optimal plant stress resilience enhancement.

[0079] Any suitable amount of the plurality of doped carbon nanodots per plant may be used in the formulations. For example, the amount of the plurality of doped carbon nanodots required by plants to overcome an abiotic stressor is dependent on the plant species. For example, the formulation may be provided in an amount to provide from 1 to 100 mg of the plurality of doped carbon nanodots per plant, such as from 1 to 20 mg / plant, such as from 1 to 4 mg / plant, such as from 1 to 3 mg / plant, such as from 1 to 2 mg / plant, such as from 2 to 100 mg / plant, such as from 2 to 20 mg / plant, such as from 2 to 4 mg / plant, such as from 2 to 3 mg / plant, such as from 3 to 100 mg / plant, such as from 3 to 20 mg / plant, such as from 3 to 4 mg / plant, such as from 4 to 100 mg / plant, such as from 4 to 20 mg / plant, such as from 20 to 100 mg / plant. In some embodiments that may be mentioned herein, the formulation may be provided in an amount to provide from 1 to 100 mg of the plurality of doped carbon nanodots per plant, such as from 2 to 20 mg / plant, such as from 3 to 4 mg / plant, such as about 3.5 mg / plant.

[0080] Any suitable plant species to be treated may be used. In some embodiments that may be mentioned herein, a plant species to be treated may be selected from one or more of the group consisting of spinach, arugula, and kailan.

[0081] In a fifth aspect of the invention, there is provided a fertilizer formulation comprising a plurality of doped carbon nanodots according to the first aspect of the invention.

[0082] The fertilizer formulation may comprise the following:

[0083] (a) a plurality of doped carbon nanodots according to the first aspect of the invention; or

[0084] (b) a plurality of doped carbon nanodots according to the first aspect of the invention, mixed with a conventional fertilizer.

[0085] For example, the fertilizer formulation may comprise the plurality of doped carbon nanodots according to the first aspect of the invention, mixed with a Hoagland solution. As will be appreciated, the present disclosure provides advantages such as improved plant growth, restoration and recovery effect, and enhanced plant abioic stress tolerance.

[0086] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0087] Examples

[0088] Materials

[0089] Baby spinach (Spinacia oleracea Platypus RZ) seeds were obtained from Bioflora Pte Ltd. 14- day-old spinach seedlings (Spinacia oleracea Platypus RZ) were obtained from Sustenir Pte Ltd. The chemicals used for CD-Si preparation and Hoagland solution preparation, including ethylenediaminetetraacetic (EDTA), silicon dioxide (SiOa), sodium molybdate (Na2MoC>4), copper sulfate (CuSCU), zinc sulfate (ZnSC>4), ethylenediaminetetraacetic acid ferric sodium salt (FeEDTA), manganese(ll) chloride (MnCh), boric acid (H3BO3), calcium nitrate (Ca(NO3)2), magnesium sulfate (MgSCU), monopotassium phosphate (KH2PO4), and potassium nitrate (KNO3) were purchased from Sigma-Aldrich and were used directly without any further treatment and purification.

[0090] The structure and morphology of synthesized CD-Si were characterized using transmission electron microscopy (TEM, JEOL 2100). The elemental distribution and mapping were analyzed through energy-dispersive X-ray (EDX). Dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) was conducted to measure the hydrodynamic size of CD-Si in the aqueous solution. Ultraviolet-visible (UV-Vis, Shimazu UV-2700) and photoluminescence (PL, Agilent Technologies Cary Eclipse) spectrums were performed to study the optical properties of CD-Si. Fourier transform infrared (FTIR, PerkinElmer Frontier) spectrums were performed to identify the functional groups, bonding types, and molecular conformations of CD-Si by the infrared spectrum of transmission or absorption. The element content in the CD and leaf tissue was measured with the help of Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES, Perkin Elmer Avio 550 Max).

[0091] Example 1. Synthesis of Si-doped carbon dots (CD-Si) and Carbon Dots (CD)

[0092] CD-Si was synthesized via a one-step hydrothermal method, in which 0.5 g of ethylenediaminetetraacetic acid (EDTA) and 0.05 g of SiO2 were generally dispersed in 50 mL of deionized water. The solution was stirred to let EDTA and SiO2 be fully dispersed and then poured into a 100 mL stainless-steel equipped Teflon autoclave and heated typically, but not exclusively at 120-240 °C (i.e. 200 °C) for typically, but not exclusively 2-16 hours (i.e. 10 hours). After the reaction, the solution was left to cool down to room temperature. The solution was first taken out and centrifuged at 5000 rpm for 20 minutes. The solution was filtered with filter paper to remove the remaining large particles. Then, the solution was filtered again with a 0.22 pm syringe filter. Finally, the solution was dialyzed with a 2kD dialysis membrane bag for 24 hours. The final solution was filtered again with a 0.22 pm syringe filter and kept in the dark for further characterization and application.

[0093] CD were synthesized via the same method as CD-Si, in which 0.5 g of EDTA was generally dispersed in 50 mL of deionized water and poured into a 100 mL stainless-steel equipped Teflon autoclave and heated at 200 °C for 10 hours. The following post-treatment of CD is the same as CD-Si.

[0094] Example 2. CD-Si Characterization

[0095] FIGS. 1 a and b show the CD-Si solution under daylight and 365 nm UV torch light, respectively, which emitted a blue-green fluorescence in the dark environment. The TEM image in FIG. 1 c reveals that CD-Si formed aggregates easily and had an average particle size of 10-20 nm, which agree with the observation and DLS analysis results. The elemental content was analyzed through EDX, and the results are shown in FIG. 1d, which indicate the presence of Si within the CD-Si particles. The overlapping images of C, N, O, and Si elemental mapping confirm the corresponding TEM morphology. The Si content within the CD-Si was measured and quantified with the help of ICP-OES to be 2.3 wt%, while the theoretical Si content calculated based on reaction precursors was 4.2 wt% instead. The Si content within CD-Si was much lower than the theoretical value, and the possible reason could be the formation of insoluble silica gel during the hydrothermal reaction. This silica gel was filtered and did not participate in forming CD-Si. When CD-Si was dissolved in an aqueous solution, the DLS analysis indicates that the CD-Si had an average hydrodynamic size of 78.71 nm with a uniform size distribution, which is shown in FIG. 1 e. The DLS result indicates a relatively small particle size and shows the potential of CD-Si to be used in further applications. However, the zeta potential value was measured to be -4.91 mV only, which shows that the synthesized CD-Si had an incipient instability, which will rapidly cause coagulation, flocculation, or aggregation within the aqueous solution, thus there is the possibility to result in a relatively large hydrodynamic size upon a long term storage. However, based on the actual long-term storage in the dark under room temperature, apparent aggregation was not observed, and the particle size slightly increased, with no significant changes in the emission fluorescence properties. The shelf-life of CD-Si in aqueous form lasts at least 6 months with no apparent changes in properties. No significant precipitation is observed upon long term storage, and uniform particle dispersion is achieveable by applying sonication. The emission fluorescence also showed no significant change in the emission peak wavelength (FIG. 2). In addition, longterm storage stability when mixed with other fertilizers lasted at least 6 months without any apparent precipitation. Thus, long-term storage stability of CD-Si has been achieved.

[0096] The optical properties including UV-Vis and fluorescence are shown in FIGS. 1f and g. The UV-Vis results indicate a significant peak at 220 nm and a broad peak shoulder at 263 nm, which demonstrate the TT-TT* transition of C=C in the sp2core and n-o* transition of hydroxyl, amine, and possible siloxane compounds on the surface of CD-Si. The fluorescence of CD-Si was measured, and from excitation wavelength of 350 to 370 nm, the emission intensity gradually increased with an unshifted blue color emission wavelength of 422 nm. When the excitation wavelength further increased above 370 nm, the emission wavelength experienced a redshift with a gradually decreased intensity. The fluorescent emission results of CD-Si were found to overlap with the chloroplast absorption spectrum from 400 nm to 650 nm, which indicate an excellent light-conversion ability and can be used to absorb UV light and convert it into blue-violet or red light for plants to absorb to improve the photosynthetic activity and as a result, promote the plant growth.

[0097] The FTIR spectrum in FIG. 1 h demonstrates that the CD-Si contained abundant hydrophilic groups O-H / N-H stretching bonding around 3200-3400 cm1, which ensures the excellent water solubility property of CD-Si. Furthermore, the Si-related functional groups and bonds are also defined in the FTIR spectrum, including Si-O-C stretching (1 176 cm-1), Si-O-Si asymmetrical stretching (1078 cm1), Si-O-Si symmetrical stretching (797 cm1), and Si-0 functional groups (460 cm1). These functional groups indicate that Si was present and linked / doped with CD via covalent bonding. The characterization results indicate that CD-Si were successfully synthesized and the properties would be sufficient for further plant cultivation application.

[0098] Example 3. Spinach Cultivation under Heat Stress

[0099] Spinach Cultivation under Heat Stress

[0100] Baby spinach (Spinacia oleracea Platypus RZ) seeds were obtained from Bioflora Pte Ltd., which pre-treatment had been done, and no further treatment or sterilization was required. Spinach seeds of similar size were selected and washed with deionized water three times. Then, the seeds were soaked in 50 mL of deionized water and stored in the fridge at 4 °C for 24 hours. This process simulates the vernalization conditions to trigger faster and better germination. Then, the seeds were removed, rinsed with deionized water, and dried carefully with tissue paper.

[0101] Under the heat stress plant growth, the spinach growth was carried out in small pots of 10 x 10 x 10 cm containing vermiculite as the growth matrix. One spinach seed was sown in one pot for germination. The germination and growth of spinach were performed in a temperature- controlled growth chamber (19 °C / 17 °C day / night) with a relative humidity of 60%, and the fluorescent light supplied the light with a period of 12 h / 12 h. After 14 days of sowing, the halfstrength Hoagland solution with 3 mL per seedling was added to the seedlings after day 14 to promote true leaf growth every 2 days. Spinach seedlings with similar sizes were divided into different groups: one low-temperature control treatment was kept under the same condition (labeled as LT), and the other groups were moved to an environment with heat stress of 28 °C / 27 °C day / night with different treatments including high-temperature control (i.e. 28 °C / 27 °C day / night), and pure CD 25 / 50 mg / L, CD-Si 25 / 50 mg / L, and SiOa 25 / 50 mg / L (labeled as HT, CD 25 / 50, CD-Si 25 / 50, and SiO2 25 / 50 respectively). The half-strength Hoagland solution was used as the control treatment, and the pure CD and CD-Si were mixed with the same half-strength Hoagland solution and applied to the spinach. The fertilizer was added to the spinach with 5.0 mL per seedling at three-day intervals starting from day 21 after sowing. The total growth cycle for the spinach growth under heat stress was 63 days.

[0102] Plant Growth Measurement

[0103] The spinach plants were harvested on day 63 in both batches of growth, and the parameters, including leaf number, fresh biomass of root, stem, and leaves, were measured immediately after harvesting. Then, the fresh spinach was dried in the 80 °C oven for 72 hours for further elemental analysis study as described in the following examples.

[0104] Pigment Content Measurement

[0105] The total chlorophyll and carotenoid content was measured after the plant harvesting. The freshly harvested leaves were cut into small pieces and added to 10 mL of 95% ethanol solvent. The tubes were kept in the dark at 4 °C for seven days before UV-Vis measurement. The absorbance value was measured and recorded at wavelengths of 470 nm, 648.6 nm, and 664.2 nm, respectively. The concentration of chlorophyll content chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll content (ChlT), and carotenoid content (Cc) were then calculated and determined by the following equations:

[0106] Chla= 13.36A6642—5.194648 6

[0107] Chlb= 27.43A648 6— 8.1246642

[0108] Results and discussion

[0109] The phenotype of spinach growth under heat stress conducted in the vermiculite is shown in FIG. 3. The picture in FIG. 3b clearly shows that spinach growth without heat stress under low temperatures had the best growth condition with a larger leaf size and darker green color among all treatments. The spinach grown under heat stress without any further treatment shown in FIG. 3a had the worst growth with smaller leaf size and yellow color of leaf. Furthermore, one of the seedlings was observed to be dead during the growth on day 35 before harvesting in HT, SiOs 25 / 50 treatments. With the addition of CD-Si and CD to the spinach under heat stress, the difference could be observed and shown in FIGS. 3c-f. The CD-Si 50 mg / L treatment could improve the spinach growth most significantly compared to CD and HT, with larger leaf size and green color leaf, which indicates that CD-Si shows the potential to enhance the heat stress tolerance of spinach and could recover the growth. Meanwhile, CD treatment showed a limited recovery effect compared to CD-Si treatment, and the leaf size and color was not fully recovered.

[0110] The growth behavior of spinach growth under heat stress conducted in the vermiculite with a growth cycle of 63 days is shown in FIG. 4. By comparing the leaf size shown in FIG. 3, it was clear that heat stress inhibited the average leaf size development, especially in the HT control treatment. With the CD-Si treatments to the spinach, the leaf size could be remarkably recovered compared to the spinach leaf size grown under low temperatures. However, the CD-alone treatments at both 25 and 50 mg / L concentrations did not show significant recovery effects compared to the leaves under low temperatures and only displayed a limited increment compared to the high-temperature control treatment.

[0111] The fresh leaf weight shown in FIG. 4a reflects a more significant recovery effect resulting from CD-Si treatments. The low-temperature control treatment had an average fresh leaf weight of 8.16 g per plant, while the fresh leaf weight from the high-temperature control treatment significantly decreased by 60.4% and only reached 3.23 g per plant. With the addition of CD-Si, the fresh leaf weight was significantly recovered to 6.67 g and 6.83 g per plant, which was 81.7% and 83.7% of the low-temperature control at 25 and 50 mg / L concentrations, respectively. In contrast, the addition of CD to the spinach did not show a significant recovery effect compared to the CD-Si treatments. The leaf weight of spinach with CD treatment was only 3.94 g and 4.18 g, respectively. The leaf weight only increased by 22.0% and 29.4% compared to the high-temperature control treatment and only recovered the fresh leaf by 48.3% and 51.2%. The addition of SiO2nanoparticles to the spinach under heat stress could have some recovery effects compared to HT control and CD treatments. The fresh leaf weight of spinach under SiO2treatments was 4.95 g and 5.18 g, respectively. The fresh leaf weight increased by 53.3% and 60.4% compared to HT control treatment, and 25.6% and 23.9% compared to CD treatments. However, the average fresh leaf weight with SiO2treatment was still much lower compared to the LT control treatment and also lower than that in CD-Si treatments. The CD-Si treatments could increase the fresh leaf weight by 34.7% and 31 .9% compared to the SiO2treatments alone. The fresh leaf weight results indicated that the Si could participate in the stress tolerance enhancement to recover the plant leaf development but the proper format or carrier such as CD could help to further improve the effects. There was an interesting observation that under heat stress, the addition of CD-Si and SiO2could help to extend the stem growth, which can be seen in FIG. 3 that longer and thinner stems were grown in the CD-Si and SiO2treatments compared to the LT control treatment. Furthermore, the heat stress during spinach growth would depress the root development compared to the LT control treatment. The fresh root weight was only 0.62 g, 0.64 g, and 0.68 g in the HT control and CD treatments, which was only 27.1 %-29.7% of LT control treatment. With the addition of CD-Si and SiO2nanoparticles, root development could be significantly recovered compared to the others. The root fresh weight recovered to around 1 .6 g, which was about 70% of the LT control treatment. The better root development during plant cultivation indicates better nutrient absorption and uptake ability and thus, resulted in better stem and leaf development. These results demonstrate that Si played an important role in stress tolerance enhancement during the plant cultivation.

[0112] The fresh biomass results further indicate that the addition of Si to CD plays an important role in stress tolerance enhancement during plant growth. CD-Si could significantly recover the spinach growth under heat stress while pure CD only showed limited effects on the growth. The CD-Si 25 and 50 mg / L treatments could recover the spinach’s fresh weight to 10.50 g and 11.04 g, respectively, which was 87.2% and 91.7% recovered compared to the control LT treatment; 12.04 g per plant on average. The SiO2could also recover the total fresh biomass to 71 .4% and 73.3% of LT control treatment. Si in the nano form could be the solution for the stress tolerance enhancement, and the CD as the carrier for Si to translocate to the higher part of the plant might be more efficient compared to the application of nano Si directly, thus improve the plant growth more significantly.

[0113] Similar results were observed in the chlorophyll and carotenoid contents in the fresh leaves of spinach among different treatments. As shown in FIG. 5a, the chlorophyll content was at the highest when no heat stress was applied to the spinach with Hoagland solution only which was 2.289 mg / g fresh leaf as LT control. However, when heat stress was applied to the spinach, the HT control treatment resulted in the chlorophyll content decreased by 38.4% compared to LT. The lower chlorophyll content in the leaf indicates that the photosynthesis activity was reduced compared to the normal condition, as a result, the production of carbohydrates and biomass of the leaf was significantly reduced, which matched the previous biomass results. Furthermore, the addition of CD alone to the spinach under heat stress did not show a significant improvement in the chlorophyll content compared to the HT control treatment, and the chlorophyll content was only 1 .061 and 0.948 mg / g fresh leaf weight, respectively. However, the improvement and recovery effects became more significant when CD-Si was added to the spinach at different concentrations. The chlorophyll content recovery effect increased significantly when the CD-Si concentration increased from 25 mg / L to 50 mg / L, which was 1 .818 and 2.094 mg / g fresh leaf weight, respectively. The chlorophyll content under CD-Si treatments was recovered to 79.4% and 91 .5% of the LT treatment. In contrast, SiC>2 treatment did not show a significant improvement in the leaf chlorophyll content compared to HT control and CD treatments. The result demonstrates that under heat stress, the addition of nano-silica directly to the spinach might not have straightforward effects on the chlorophyll content enhancement compared to the CD-Si treatments and such CD carrier or pathway could be a key component for Si to achieve the recovery effect. The higher chlorophyll content indicates a higher photosynthesis activity which resulted in a higher production of carbohydrates and better plant growth, which matched the trend of fresh leaf weight shown previously. The possible reason for the increment of chlorophyll at 50 mg / L concentration could be that CD-Si exhibited a higher bioavailability due to improved solubility and wettability and, thus, could be more absorbed and translocated to the leaf parts. The above results indicate that Si had been absorbed and transported to the higher part of the spinach to reinforce plant development and restore the chlorophyll content to help enhance the heat stress tolerance with the help of CD rather than CD itself.

[0114] Another type of important photosynthesis pigment carotenoid content in the leaves was also measured together with the chlorophyll content (FIG. 5b). The results indicate that the heat stress and addition of CD-Si and CD at different concentrations did not show a significant difference among treatments. This result demonstrates that the carotenoid content would not be influenced under abiotic heat stress and the addition of CD-Si / CD fertilizer would also have limited or no effects on the carotenoid under the stress.

[0115] Example 4. Elemental Content Study for Spinach Grown under Heat Stress in Example 3 Elemental Content Analysis

[0116] The spinach root, stem, and leaf were cut and dried at 80 °C for 72 hours after 63 days’ growth. Then, the dried plant parts were ground and digested with a mixture of H2O2and HNO3(1 :1 volume ratio) at 105 °C for 2 hours. The total elemental contents were measured by ICP-OES analysis.

[0117] Statistical Analysis

[0118] Each treatment was repeated with six replicates, with the results presented as mean and standard deviation. The data analysis was performed using OriginLab 2021 for one-way analysis of variance (ANOVA) analysis on the basis of Turkey’s Test (p < 0.05).

[0119] Results and discussion

[0120] The elemental contents including Fe, Zn, Mg, and Si in the spinach leaf, stem, and root were measured and investigated separately. The elemental contents in the leaf are shown in Table 1.

[0121] Table 1. Elemental contents concentration (mg / kg) in the spinach leaf.a'b'c'dData are considered significantly different (P < 0.05). It was clear that with the heat stress to spinach up to 27-28 °C in the HT control treatment, the elemental content including Fe, Zn, and Mg all decreased significantly compared to the LT control treatment. However, with the addition of CD-Si to the spinach under heat stress, the element contents in the leaf could be significantly recovered compared to HT and LT control treatments. The Fe contents in the leaf with CD-Si 25 / 50 treatments were 306.0 and 342.7 mg / kg, respectively, which were recovered to 72.3% and 80.9% compared to the LT control treatment. The addition of CD alone to the spinach did not affect the Fe content in the leaf significantly compared to the HT control. Similar to the Fe content, the Zn and Mg contents in the leaf, the CD-Si treatments could improve and recover the weight percentage efficiently compared to HT control and CD treatments. The Zn content was recovered to 68.5%-74.1%, and the Mg content was recovered to 77.9%-79.5% of the LT control treatment. For the Si content, the treatments without Si addition during cultivation did not show a significant difference in Si contents and only CD-Si treatments resulted in a significant increase in the Si weight percentage in the leaf compared to the other treatments by 54.1%-64.4%. Meanwhile, the addition of SiC>2 nanoparticles showed limited recovery in the Fe content in the leaf. The Fe content increased by 28.3%-46.5% compared to the HT control treatment. These results indicate that the addition of CD-Si during spinach cultivation under heat stress could help to promote the nutrient element uptake to the higher part of the plant to promote growth and overcome the stress. The mineral content results were identical to the plant growth behavior and chlorophyll content results shown in Example 3.

[0122] The element contents in the stem including Fe, Zn, Mg, and Si are shown in Table 2. In the case of the stem, the element contents under all treatments showed limited differences among the measurements, except the LT control treatment. The Fe, Zn, and Mg content from HT, CD-Si, CD and SiOs treatments showed similar concentrations, while were all lower than that in the LT control treatment. The possible reason could be that the heat stress will affect the uptake, translocation, and storage of nutrient elements in the stem, and the addition of CD-Si and CD treatments could not help to recover the adverse effects. Only the Si contents in the CD-Si treatments in the stem were significantly increased by 100.5%-178.4% compared to the others, which is reasonable that the stem was used for the Si uptake and translocation from root to leaf during plant growth. However, the Si content in the stem from SiO2 treatments was lower than that in the CD-Si treatments, which was only about 61.3%-65% of the content in the CD-Si treated stem. The results indicate that the CD-Si could be a more efficient way for the Si uptake and translocation through the stem to leaf compared to SIO2 nanoparticles directly. Table 2. Elemental contents concentration (mg / kg) in the spinach stem.a b cData are considered significantly different (P < 0.05).

[0123] The element contents in the roots of spinach were measured and shown in Table 3. The results show obvious trends and differences among different treatments. The spinach without heat stress in LT control showed the highest Fe, Zn, and Mg content concentrations. In contrast, the heat stress in HT, CD-Si, and CD treatments significantly depressed the elemental contents, which indicates that heat stress has great effects on the root nutrient absorption, storage, and translocation ability and capacity. However, the treatments with CD-Si showed an efficient recovery compared to the LT control treatment. The Fe content was 14647.6 and 2590.0 mg / kg in the LT and HT control, and the CD-Si treatments recovered the content up to 11266.1 and 8281.2 mg / kg, which was recovered by 76.9% and 56.5%, respectively. Similar results were also observed in the case of Mg content, the CD-Si treatments could recover the Mg content to 87.3% and 79.0% of the LT control level. The SiO2nanoparticle treatments recovered the Fe and Mg contents in the roots less significantly compared to the CD-Si treatments. The Fe content was recovered to 5384.3 and 7506.9 mg / kg, which was 36.8%- 51 .3% of the LT control treatment. The Mg content was recovered to 65.6%-69.2% of LT control treatment. Conversely, the Zn content did not show significant differences among all treatments except the LT control. The result demonstrates that the Zn absorption and storage were greatly affected by the heat stress but the following treatments did not help to recover and restore the Zn contents in the root part. The reduced uptake absorption, uptake, and storage of nutrient elements Fe, Zn, and Mg by roots would result in the inhibition effects on the growth of spinach, which matched the biomass and chlorophyll contents results shown in Example 3.

[0124] Table 3. Elemental contents concentration (mg / kg) in the spinach root.a b'c’d eData are considered significantly different (P < 0.05). The element contents analysis results from the spinach leaf, stem and root, demonstrate that CD-Si have great potential in the restoration and recovery effect of nutrient elements in the spinach under heat stress and it is more efficient compared to the direct application of SiC>2 nanoparticles to the spinach. Example 5. Spinach Growth under Salinity Stress

[0125] Spinach Cultivation under Salinity Stress 14-day-old spinach seedlings (Spinacia oleracea Platypus RZ) were obtained from Sustenir Pte Ltd. in the salinity stress study for a more uniform seedling size control. Under the salinity stress plant growth, the spinach growth was carried out in small pots of 10 x 10 x 10 cm containing vermiculite as the growth matrix. One spinach seedling was transplanted in one pot for further growth. The growth of spinach was performed in the temperature-controlled growth chamber (20 °C / 17 °C day / night) with a relative humidity of 60%, and the fluorescent light supplied the light with a period of 12 h / 12 h. The half-strength Hoagland solution with 0.5 mL per seedling was added to the seedlings when seedlings were transplanted to the pots to promote true leaf growth with a 3-day interval from the day of transplanting (labeled as Day 14). The spinach seedlings of similar sizes were divided into control (SO and NS) and three other groups with different concentrations of 0.1 / 0.5 / 1 .0 M NaCI (S1 , S2, and S3). At the same time, the other conditions (i.e. temperature, relative humidity, and light period) were controlled to be the same. Four treatments were conducted in the groups treated with NaCI solution, including control and CD-Si 25 / 50 / 100 mg / L (labeled as CK, C1 , C2, and C3). The halfstrength Hoagland solution with CD-Si fertilizer addition was added to the spinach with 3.0 mL per seedling at three-day intervals starting from day 21 , and the NaCI solution with different concentrations was added to the vermiculite with 10 mL per pot at a 7-day interval. Spinach leaves larger than 4 cm were cut starting from day 35 at a 7-day interval. The spinach grown under salinity was harvested on day 63.

[0126] Plant growth measurement and pigment content measurement were carried out by following the protocols described in Example 3.

[0127] Results and discussion

[0128] The spinach growth under salinity stress was conducted with a growth cycle of 63 days, and the spinach leaves were cut from day 35 with a 7-day interval until spinach harvesting. The phenotype of spinach is shown in FIG. 6. The spinach growth under salinity stress was significantly depressed and with the CD-Si treatments, the growth could be significantly recovered.

[0129] The growth behavior of spinach under salinity stress is shown in FIG. 7. FIG. 7a shows the total number of leaves harvested from the spinach over 63 days’ growth cycle. Under nonsalinity stress, the control treatment resulted in 18 leaves. As the salinity stress was applied and NaCI concentration increased, the total number of leaves significantly decreased by 44.4%, 51.8%, and 57.4%, respectively, to 10, 8.67, and 7.67. However, when the CD-Si was added to the spinach under salinity stress, the total number of leaves harvested gradually increased as the CD-Si concentration increased to 50 mg / L. At the lowest salinity stress (S1 ), the 50 mg / L CD-Si treatment significantly increased the total number of leaves by 26.7% from 10 to 12.67, which was recovered to 70.4% of the control treatment without salinity stress. A similar trend was also observed from the other salinity stress conditions. These results demonstrated that the salinity stress severely inhibits spinach leaf development with increased NaCI concentrations. The recorded death rates for spinach were 17% under moderate salinity stress (S2) and 50% under severe salinity stress (S3). However, with the addition of CD-Si, the death rate under severe stress (S3) decreased back to 17%, and there was no death of spinach observed under moderate stress (S2). These findings suggest that salinity stress has a significant impact on spinach mortality throughout its growth cycle, and the CD-Si could help spinach to overcome such stresses.

[0130] The fresh leaf weight was measured and shown in FIG. 7b. The results indicate that spinach leaf growth was inhibited under salinity stress, and the weight was dramatically decreased. The control treatment without salinity stress had an average total leaf weight of 15.5 g per plant. However, the fresh leaf weight significantly reduced to 3.65 g per plant under S1 level salinity stress, only 23.55% of the control treatment. With a further increment in NaCI concentration added, the fresh leaf weight gradually decreased to 2.94 g per plant, which was only 18.97% of the control treatment. However, there was an increment in fresh leaf weight observed when the CD-Si was added to the spinach under salinity stress. At the lowest salinity stress S1 , adding CD-Si at 25 and 50 mg / L concentrations improved the fresh leaf by 54.25% and 110.9% compared to the S1 control treatment without CD-Si addition, respectively. However, the fresh leaf weight was decreased again to the control level when the CD-Si concentration increased to 100 mg / L. The possible reason could be that the CD-Si was overdosed under this salinity stress condition and inhibited the growth. This result indicates that CD-Si could help to alleviate the low salinity stress, and the spinach leaf development could be partially recovered to 50% of the control treatment under normal conditions at a certain concentration. Similar results were observed from the other salinity stress level treatments, S2 and S3. The fresh leaf weight gradually decreased as the salinity stress level increased. With the addition of CD-Si at 50 mg / L concentration, the spinach fresh leaf weight could be promoted and recovered more significantly to 40-50% of the control treatment under normal conditions.

[0131] The entire spinach growth was measured and shown in FIG. 7c. The fresh plant biomass also matched the fresh leaf biomass weight trend. The control treatment under normal conditions had an average total biomass of 20.45 g. In contrast, the control treatment under S1 , S2, and S3 salinity stress levels decreased the plant fresh biomass to 5.56 g, 5.55 g, and 4.48 g, respectively, only 21.90-27.19% of the control treatment. It was observed that the salinity stress not only inhibited the leaf growth but also the stem and root development. The root biomass under salinity stress was only 18.12-35.57% of the control treatment, and the stem biomass was only 26.30-56.07% of the control treatment. Similar to the leaf weight, adding CD-Si could alleviate the adverse effects of salinity stress on both root and stem development at a certain concentration of 50 mg / L. These results further demonstrate that CD-Si show potential in spinach plant growth recovery under salinity stress.

[0132] The photosynthetic pigment contents are shown in FIG. 7d. The results indicate that salinity stress on spinach would affect the chlorophyll content more significantly than the carotenoid content. The chlorophyll content of the control treatment under normal conditions was the highest at 1 .739 mg / g fresh leaf weight. When the salinity stress was applied to the spinach with increased NaCI concentration, the chlorophyll content decreased significantly to 0.686, 0.374, and 0.232 mg / g fresh leaf weight. The chlorophyll content was observed to be recovered when CD-Si was added with an increased concentration of up to 50 mg / L. At the lowest salinity stress S1 , the chlorophyll content increased to 1 .447 and 1 .576 mg / g fresh leaf weight with the addition of 25 and 50 mg / L CD-Si. The increment was about 1 10.9% and 129.7% compared to the S1 control. The chlorophyll content was shown to recover to 83.21% and 90.63% of the control treatment under normal conditions. However, the chlorophyll content was not affected and was slightly reduced when the CD-Si concentration increased to 100 mg / L. When the salinity stress level increased to S2 and S3, the improvement and recovery effects were more limited regardless of CD-Si concentration. Under the S2 salinity stress level, the chlorophyll content was not much influenced and varied from 0.691 to 0.742 mg / g fresh leaf weight. The further increment in salinity stress level to S3 with CD-Si addition also resulted in a close final chlorophyll content varied from 0.433 to 0.678 mg / g fresh leaf weight. This result indicates that the extremely high salinity stress during the spinach growth would have irreversible damage to the chlorophyll content, and the addition of CD-Si had limited effects on the chlorophyll enhancement. On the other hand, the carotenoid content in the spinach leaf did not show a significant change among all the treatments, which varied from 0.2 to 0.3 mg / g fresh leaf weight.

[0133] These results indicate that the Si in CD-Si does participate in plant growth and acts in a defensive role to prevent the destruction of chlorophyll contents. Furthermore, the possible mechanism for enhancing chlorophyll content under salinity is that the Si helps to increase the leaf rigidity by making it tougher and maintaining the photosynthesis activity. The plant growth parameter results above demonstrate that CD-Si could meditate positive effects on spinach growth under salinity stress by reducing damage to the plant and strengthening the plant to maintain normal growth and restore the production yield within certain limits. Example 6. Salinity Stress Element Content

[0134] The element content analysis was carried out by following the protocol in Example 4.

[0135] Results and discussion

[0136] The elemental contents, including Fe, Zn, Mg, and Si, in different parts of spinach are shown in Tables 4-6. Table 4. Elemental contents concentration (mg / kg) in the spinach leaf.a b c d eData are considered significantly different (P < 0 05) e S3C3 3841 .1 ±474.5d60.5±10.9b c5562.9±929.5b d5797.4±532.8b c

[0137] The results clearly indicate that salinity stress significantly depressed the element uptake by the spinach through the root-stem-leaf routine. The Fe content of the control treatment without salinity stress in the leaf was 1835.0 mg / kg. However, this value significantly decreased as the salinity stress level increased. The Fe content in the leaf was only 1351.7 mg / kg, 487.4 mg / kg, and 214.2 mg / kg under S1 , S2, and S3, respectively. Adding CD-Si under the lowest salinity stress level S1 positively affected the Fe content enhancement. At 25 mg / L concentration of CD-Si, the Fe content was recovered to 79.5% of the control treatment to 1459.2 mg / kg. However, when the salinity stress level was higher than 0.5 M NaCI, it was found that the damage to the spinach was irreversible, and the nutrient uptake recovery was limited even with the help of CD-Si. Similar results were also observed in the case of Zn and Mg micronutrients. Furthermore, since Mg is the essential element in chlorophyll and participates in photosynthetic activity, the reduction in Mg content in the spinach leaf also indicated a lower content of chlorophyll, which matched our previous results that the salinity stress could significantly decrease the chlorophyll content in the leaf with the increment of NaCI concentration. In the case of Si, it was observed that salinity stress did not show significant effects on Si content but only slightly decreased. With the CD-Si treatment and concentration increment, the Si content gradually increased, achieving the highest at 50 mg / L concentration.

[0138] The stem's elemental contents also showed a similar trend with contents in the leaf under salinity stress and CD-Si treatments. With the CD-Si treatment under salinity stress, the Fe, Zn, and Mg content significantly recovered. The Fe, Zn, and Mg content in the control treatment was 1205.7 mg / kg, 63.1 mg / kg, and 1 1336.5 mg / kg, respectively. At low salinity stress level S1 , the Fe, Zn, and Mg content was significantly recovered with CD-Si treatments. However, the higher salinity stress levels S2 and S3 would cause more severe damage to the spinach and the Fe, Zn, and Mg contents would not be greatly changed and recovered with CD-Si treatments. The element contents concentration in the stem was generally similar to that in the leaf; the possible reason could be that the stem was mainly used for nutrient uptake and translocation rather than storage.

[0139] When the elemental contents in the roots were investigated, it was found that the Fe and Mg contents showed a significant accumulation in the root part. When no salinity stress is applied, the Fe contents in the root significantly increased to 10414.5 mg / kg and the Mg content in the root significantly increased to 12991.3 mg / kg. The salinity stress to the spinach significantly decreased the Fe and Mg content in the roots with increased NaCI concentration from 0.1 M to 1 .0 M. The addition of CD-Si to the spinach under salinity stress could help to recover the Fe and Mg uptake and storage in the roots at lower salinity stress. The Fe content was recovered to 79.5%-89.7% of the control treatment, and the Mg content was recovered to 66.8%-77.5% of the control treatment at 0.1 M salinity stress level with CD-Si treatments. However, similar to the leaf and stem element content, the salinity stress higher than 0.5 M NaCI resulted in the damage to the spinach was irreversible, and the addition of CD-Si had limited effects on the element content recovery. On the other hand, the Zn content in the root did not show significant accumulation under salinity. The results demonstrate that under salinity stress, essential micronutrients such as Fe, Zn, and Mg would accumulate more in the roots rather than be translocated to the higher parts of the spinach. Thus, the lack of these micronutrients in the higher parts of spinach resulted in a server decrement in spinach growth and photosynthetic activity. When the CD-Si was added to the spinach under salinity stress with certain concentration ranges, the CD-Si could act as the transporter of the micronutrients such as Fe, Zn, and Mg to deliver the nutrients to higher parts of plants, just like the previous results observed in the heat stress study discussed in Examples 3 and 4. The possible mechanism of this CD-aided transportation could be: (1 ) the negatively charged CD-Si can absorb and interact with these positive elemental ions and bring them to higher parts of the plants through the apoplastic pathway; and (2) the CD-Si could open the apoplastic pathway for the micronutrients including Fe, Zn, and Mg to transfer into cells directly more easily. The results also report that the nutrients, Si and CD, could transfer from root to stem and finally reach higher leaf parts by the vascular system through the apoplastic pathway. The Si transferred through the apoplastic pathway could also protect the plants from the apoplastic bypass toxicants induced during abiotic stresses, such as heavy metals and salts. In the present disclosure, it was observed that salinity stress and CD-Si treatments under different concentrations did not greatly affect the Zn content. The possible reason could be the limited amount of Zn in the nutrient solution and the existence of Fe-Zn elemental competition within the spinach plant.

[0140] Example 7. Synthesis of CD-Si

[0141] The synthesis of CD-Si covers a series of chemicals and biomass, including EDA, EDTA, citric acid, urea, glycerol, chitosan, and their mixtures. The synthesis of CD-Si using EDA, citric acid, urea, glycerol, or chitosan, is the same as described in Example 1 ; hydrothermal reaction at 200 °C for 10 hours. The dopant Si could be added to CD to form CD-Si during synthesis. Examples of the sources of Si include, but are not limited to the chemicals such as silicon dioxide (SiO2), silicic acid (SA), and silicate salts. Silicic acid (SA) and sodium silicate (SS) were used to produce CD-Si with different ratios with the carbon source EDA, using the same conditions of hydrothermal at 200 °C for 10 hours, as described in Example 1 . SA-1 refers to SA:EDA mole ratio of 2:1 , SA-2 refers to 5:1 , and SA-3 refers to 10:1. SS-1 refers to SS:EDA mole ratio of 2:1 , SS-2 refers to 5:1 , and SS-3 refers to 10:1 . The DLS size for these CD-Si was well controlled within the range of 60-130 nm (FIG. 8). Based on the different combinations and sources of Si, the dopant concentrations could be controlled within certain ranges upon the application requirements.

[0142] Example 8. Synthesis and Characterization of CD-Se / Ce

[0143] The CD for stress control purposes may include different types of dopants including silicon (Si), selenium (Se), and cerium (Ce). The general synthesis method and post-treatment for the CD-Se / Ce are the same as described in Example 1 with only changes in the precursors. For Cd-Se, the precursors used were EDTA and Na2SeO3. For Cd-Ce, the precursors used were EDTA + Ce(OH)4.

[0144] Results and discussion

[0145] FIGS. 9 and 10 depict the characterization of Cd-Se. The theoretical Se content and the actual Se content in CD-Se are 17.1 wt% and 2.07 wt%, respectively.

[0146] FIGS. 1 1 and 12 depict the characterization of Cd-Ce. The theoretical Ce content and the actual Ce content in Cd-Ce are 17.4 wt% and 1 .66 wt%, respectively.

[0147] Conclusion

[0148] Abiotic stresses such as drought, heat, and salinity stress during plant growth are common problems that will greatly affect plant growth and finally reduce the production yield of plants. The present disclosure involves CD-Si preparation, testing and characterization, and applications in plant growth to help the crop fight against environmental stresses such as heat and salinity. CD-Si was successfully synthesized and characterized in the present disclosure, and the relative physical properties of the CD-Si show potential for further utilization in the agriculture application for stress tolerance. The results obtained from the heat stress study demonstrate that CD-Si played an important role in the stress tolerance enhancement of the spinach. The spinach leaf development, photosynthetic pigments, and elemental contents in different parts were improved and recovered compared to the spinach without CD-Si treatments under stress. As such, with a treatment of CD-Si at a certain concentration range with appropriate particle size, the spinach could fight against heat stress and restore the plant growth parameters and nutrient levels such as fresh biomass, chlorophyll content, and mineral contents within certain limits. Si was found to have an important defensive role in the agriculture application to support better plant growth and help fight against various stresses.

[0149] CD-Si which have Si doped inside CD, could help to deliver Si and increase the utility efficiency, which could also help to decrease their amount used in agriculture. The present disclosure demonstrates that CD-Si could help to deliver the Si to strengthen the plant, and CD-Si also helped with the absorption and translocation of other nutrient elements such as Fe, Zn, and Mg from root to leaf to promote plant growth, and shows a potential and positive impact of CD- Si to be used in agriculture applications to enhance production even under various stresses. Thus, the CD and relative nanoparticles disclosed herein have been proven to have potential effects to be used in agriculture applications to promote plant growth, which makes using CD as a delivery tool or platform for micronutrient delivery in plant growth. The present disclosure provides a new perspective for improving plant stress tolerance by Si-doped carbon dots to achieve a more efficient and sustainable agriculture development.

Claims

1. Claims1 . A plurality of doped carbon nanodots, comprising: a plurality of carbon nanodots; and a dopant comprising from one or more of the group consisting of Si, Ce and Se, wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 1 .5 to 4 wt%.

2. The plurality of doped carbon nanodots according to Claim 1 , wherein the weight percentage of the dopant based on the weight of atomic Si, Ce and Se, relative to the total weight of the plurality of carbon nanodots is from 2 to 3 wt%, such as from 2.07 to 2.5 wt%.

3. The plurality of doped carbon nanodots according to Claim 1 or Claim 2, wherein the plurality of doped carbon nanodots have one or more of the following properties:(a) a hydrodynamic size of from 40 to 120 nm, such as from 50 to 100 nm, such as from 70 to 80 nm, such as about 78.71 nm;(b) a stability in an aqueous solution of at least six months; and(c) the plurality of doped carbon nanodots are substantially free of toxic components; and(d) the plurality of doped carbon nanodots are suitable for use in agriculture.

4. The plurality of doped carbon nanodots according to any one of the preceding claims, wherein the dopant comprises Si.

5. A method of preparing a plurality of carbon nanodots, the method comprising:(i) providing an aqueous mixture comprising a carbon source precursor and a dopant precursor; and(ii) subjecting the aqueous mixture to a hydrothermal reaction at an elevated temperature for a period of time to provide the plurality of carbon nanodots, wherein: the carbon source precursor is selected from one or more of the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine (EDA), citric acid, urea, glycerol, and chitosan; the dopant precursor is selected from one or more of the group consisting of a silica, a silicic acid, a silicate salt, ceric oxide, a ceric acid, a ceric salt, selenium oxide, a selenic acid, and a selenic salt.

6. The method according to Claim 5, wherein the weight to weight ratio of the carbon source precursor to the dopant precursor is from 100:1 to 5:1 , such as from 50:1 to 9:1 , such as about 10:1 .

7. The method according to Claim 5 to Claim 6, wherein the hydrothermal reaction is conducted in an autoclave heated to a temperature of from 100 to 300 °C, such as from 120 to 240 °C, such as about 200 °C.

8. The method according to any one of Claims 5 to 7, wherein the hydrothermal reaction is conducted for a period of from 1 hour to 24 hours, such as from 2 to 16 hours, such as from 5 to 15 hours, such as about 10 hours.

9. The method according to any one of Claims 5 to 8, wherein the carbon source precursor is EDTA.

10. The method according to any one of Claims 5 to 9, wherein the dopant precursor is a silica, optionally wherein the carbon source precursor is EDTA and wherein the dopant precursor is a silica.

11. A method of enhancing plant stress resilience to an abiotic stressor, the method comprising providing a formulation comprising plurality of doped carbon nanodots according to any one of Claims 1 to 5 and applying the formulation to a plant subjected to an abiotic stress.

12. Use of a plurality of doped carbon nanodots according to any one of Claims 1 to 5 in enhancing plant stress resilience to an abiotic stressor.

13. The method according to Claim 1 1 or the use according to Claim 12, wherein the abiotic stressor is one or both of a heat stress and a salinity stress, where the level of heat and / or salinity to provide a stress is relative to the optimal growing conditions of a particular plant species.

14. The method of Claim 1 1 or Claim 13 or the use of Claim 12 or 13, wherein the formulation comprises a concentration of the plurality of doped carbon nanodots of from 10 to 500 mg / L, such as from 20 to 400 mg / L, such as from 25 to 300 mg / L, such as from 50 to 200 mg / L, such as from 75 to 150 mg / L, such as from 25 to 100 mg / L.

15. The method of any one of Claims 11 and 13 to 14 or the use of any one of Claim 12 to 14, wherein the formulation is provided in an amount to provide from 1 to 100 mg of the plurality of doped carbon nanodots per plant, such as from 2 to 20 mg / plant, such as from 3 to 4 mg / plant, such as about 3.5 mg / plant.

16. The method of any one of Claims 11 and 13 to 14 or the use of any one of Claim 12 to 14, wherein a plant species to be treated is selected from one or more of the group consisting of spinach, arugula, and kailan.

17. A fertilizer formulation comprising a plurality of doped carbon nanodots according to any one of Claims 1 to 5.

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