Synthesis of a chitosan biopolymer for precision agriculture

Abstract

Polymeric compounds comprising chitosan polymers bound to trehalose molecules are disclosed, along with methods for their synthesis involving Schiff base formation, reduction, and transacetalization. Compositions include the polymers in buffered acidic solutions for seed coating. Methods encompass coating seeds to promote development in crops like chickpea, wheat, barley, corn, soybean, sunflower, Egyptian riverhemp, yellow mustard, triticale, sainfoin, and common vetch, improving germination, biomass, and pigments. Further methods integrate growth-promoting bacteria to enhance viability, nodulation, and symbiosis. The compounds and compositions of the disclosure mitigate abiotic stresses, such as salinity.

Description

PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699SYNTHESIS OF A CHITOSAN BIOPOLYMER FOR PRECISION AGRICULTURECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 679,275, filed August 5, 2024, which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under the MIT Climate Grand Challenge. The government has certain rights in the invention.BACKGROUND

[0003] In the post-green revolution era, the agricultural sector has advanced the formulation of novel fertilizers and appropriate crop management. However, excessive use of agricultural inputs has followed, as it is challenging to have a uniform application over the soil surface, inducing environmental losses and constraints (eutrophication, alteration of soil microbial diversity, an increase of soil salinity, etc.).

[0004] Food is the single, most important determinant of human health. However, the ability to provide sufficient, safe, and nutritious food to the global population, which is projected to reach 9.7 billion by 2050, is becoming a major challenge for the AgroFood infrastructure. Currently, more than 800 million people are living in conditions of food insecurity, and climate change is exacerbating the biotic and abiotic stressors that negatively affect crop yield and quality. Moreover, with the emergence of various severe challenges, we have witnessed a decrease in crop production, mainly due to climatic fluctuations, resulting in the emergence of new diseases, water scarcity, and soil salinization.

[0005] To date, overappli cation of fertilizers and pesticides is still a widely adopted agricultural practice to increase crop yield, due to the low efficiency in agrochemical delivery and utilization. The potential benefits of biomaterials-based innovation in agriculture and food production, however, remain underexplored. Some efforts have focused on the engineering ofPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 seed microenvironment. By modifying the seed surroundings, scientists increased the resistance of plants against physical damages, pathogen infections, and promoting the uptake of essential nutrients in the first stages of plant germination. Others have relied on the direct intervention on adult plants to enhance their strength by using precision and payload delivery. This method allows direct operation on the single plant, without any possible loss of active compounds by sowing or watering the soil (Kundu, A., et al., “Precision Vascular Delivery of Agrochemicals with Micromilled Microneedles (MMMNs)” Sci. Rep. 2019, 9 (1), 14008).

[0006] Seed coatings were initially developed to increase the size of small seeds or to make uniform their irregular shape to be properly managed by agricultural machinery. Seed coating has evolved into an efficient way to create a microenvironment for seed development through use of active coating materials and inclusion of other micronutrients or molecules.

[0007] Seed coating usually comprises an active nutrient, such as fertilizers, hormones and antibiotics, and a carrier, a polymeric material, synthetic or natural, that can create a protective film on the surface of the seeds. Among the carriers, the choice depends on several factors: adherence to seed surface, germination increase, and plant development. Typically, coatings are based on silica, carboxymethylcellulose, polyvinyl alcohol, gum arabic, bentonite, silk fibroin, or alginates (Ma, Y. “Seed Coating with Beneficial Microorganisms for Precision Agriculture” Biotechnol. Adv. 2019, 37 (7), 107423; Zvinavashe, A., et al., “Engineering the Plant Microenvironment To Facilitate Plant-Growth-Promoting Microbe Association” J. Agric. Food Chem. 2021, 69 (45), 13270-13285).

[0008] Chitosan is a natural biopolymer derived from the hydrolysis of chitin (a linear polysaccharide which monomeric unit is 2-acetamido-2-deoxy-P-D-glucopyranose, linked together by a P-1,4 glycosidic bond), the second most abundant biomaterial after cellulose, composing the exoskeletons of insects and crustaceans and cell walls of fungi. It is considered an agroalimentary waste, with an annual production of more than 1011tons, with almost 70% produced by the crustacean market.

[0009] Chitosan possesses certain antiviral, antimicrobial, and antifungal activity, and has been used as an insect repellent, for plant disease control, as a physical barrier, to chelate nutrients,PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 and to modulate plant defense responses (Zeng, D., et al. “Application of Bioactive Coatings Based on Chitosan for Soybean Seed Protection” Int. J. Carbohydr. Chem. 2012, 2012, 1-5).

[0010] Neither chitin or nor chitosan occur in nature as purely N-acetylated and N-deacetylated polysaccharides, respectively, but rather a fraction of acetamido-monomer for chitosan, and deacetylated monomer in chitin. The ratio between the acetamido-monomer and the deacetylated monomer is defined as Degree of Deacetylation (DDA). Usually, polymers with DDA lower than 50% are insoluble and classified as chitins, whilst the ones with higher DDA are soluble and identified as chitosan.

[0011] Obtaining chitin from inexpensive biological sources is desirable, but it is environmentally responsible to reduce seafood waste which tends to accumulate in coastal regions, causing pollution and modifying the ecosystems. Crustacean shell chemical composition may vary for each species, but the average composition is about 30% by mass of pure chitin, about 15% in proteins, and more than 50% CaCC . Shells also possess a small fraction of lipid (<3%) and some pigments, such as carotenoids.

[0012] Plant growth promoting rhizobacteria (PGPRs) can be used as biofertilizers to fix nitrogen and solubilize bioavailable phosphorous for roots uptake. The synergistic use of biomaterials and PGPRs has been proposed to deliver beneficial microorganisms to the soil, to boost crop production, mitigate abiotic stressors and reduce the use of synthetic fertilizers (Saberi Riseh, R., et al. “Encapsulation of Plant Biocontrol Bacteria with Alginate as a Main Polymer Material” Int J Mol Sci 22, 11165 (2021); ) Vejan, P., et al. “Encapsulation of plant growth promoting Rhizobacteria — prospects and potential in agricultural sector: a review” J Plant Nutr 42, 2600-2623 (2019)). However, PGPRs are often non-spore forming bacteria and need to be transported in suspension with osmoprotectants and using the cold chain before seeds priming occurs right before sowing. These steps hinder the broad diffusion of PGPRs, particularly in rural areas, and limit their successful deployment due to off target delivery and reduced viability.

[0013] Trehalose is a disaccharide that is ubiquitously used in nature to mitigate water stress and is receiving an increasing attention in agriculture due to its role in plant cell signaling, and influence on plants’ development stages (Mhada, M., et al. “Bioformulation of Silk-BasedPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699Coating to Preserve and Deliver Rhizobium Tropici to Phaseolus Vulgaris Under Saline Environments” Front. Plant Sci. 2021, 12). In seed coating formulations, trehalose adhesion on the seed surface is achieved using binders such as silica, carboxymethylcellulose, polyvinyl alcohol, gum arabic, bentonite, silk fibroin, alginates, and chitosan with the latter providing also the function of biostimulant (Chandrasekaran, M., et al. “Chitosan derivatives act as a bio-stimulants in plants: A review” Int J Biol Macromol 271, 132720 (2024)). Though while commonly used in coatings, trehalose has proven to limit PGPRs viability.

[0014] So, despite some progress, there exists a need in the art for new and improved agrochemical agents including for use in seed coating that are economical, safe and easy to prepare on a large scale, versatile, and ideally have improved properties over currently utilized agrochemical agents.SUMMARY

[0015] The present disclosure provides polymeric compounds comprising chitosan polymers bound to trehalose molecules, methods for their synthesis, compositions thereof for seed coating, and methods of use for promoting plant development, bacterial growth, and tolerance to abiotic stresses such as salinity. These compounds, exemplified by Chito- Trehalose, can enhance crop yield and PGPR viability under normal and saline conditions.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. lA to FIG. 1C show the processing of chitosan, chemical derivatization, and characterization of the new material Chito-Trehalose. FIG. 1A (panel a) shows the processing of chitosan. Pure chitosan was obtained from the seafood waste comprising shrimp, lobster, and crab exoskeletons. Through a preliminary deproteinization and demineralization of the shells with alkaline and acidic solution, respectively, pure chitin was obtained as a whitish powder. Then, chitin was hydrolyzed with concentrated hot NaOH solutions to obtain chitosan. The polymer was finally dissolved into an aqueous slightly acidic solution of acetic acid (pH 6.2-6.6) and subjected to rhizobacteria toxicity tests or used as a starting point for the synthesis of the new material Chito-Trehalose. FIG 1A (panel b) shows that chitosan was chemically modified through a three-step chemical process involving the chemistry of thePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 free -NH2 group in the polymer structure. The final polymer was then tested for toxicity studies and coating techniques. FIG. IB: All the intermediates and the final Chito-Trehalose were characterized by ATR-FTIR spectroscopy, highlighting the typical bands for each product, and dividing the spectra in the two regions 4000-2000 cm'1and 1800-600 cm'1. The -OH, -NH, -CHX, Amide I, Amide II, Amide III, and other characteristic bands were studied for the correct structural investigation. FIG. 1C: The products were also characterized via NMR techniques, e.g., 'H-NMR spectra. All intermediates and final polymer spectra are stacked together, showing the structural differences according to the chemical shifts and pattern of protons. Moreover, are also reported the spectra of digested chitosan and Chito- Trehalose, after being in contact with Rhizobacteria after 48h, strongly suggesting their role as a source of elements in the growth of bacteria. Chitosan was completely digested into acetic acid, whilst Chito-Trehalose was converted in a new derivative, in which an ethyl hydroxy moiety is attached to the -NH2 group.

[0017] FIG. 2A and FIG. 2B show NMR spectra for Intermediate I. FIG. 2A shows bidimensional 'H-'3C-NMR HSQC spectrum H 500 MHz, D2O) for Intermediate 1 (chitosan-imine). FIG. 2B shows a zoomed view of the carbonyl region.

[0018] FIG. 3 shows13C-NMR spectrum (125 MHz, D2O) for Intermediate 2 (chitosan-amine).

[0019] FIG. 4A and FIG 4B show 'H-NMR spectrum (500 MHz, D2O) for Chito-Trehalose. FIG. 4A shows the full spectrum; FIG. 4B shows an expansion of the signal range.

[0020] FIG. 5A and FIG 5B show13C-NMR spectrum (125 MHz, D2O) for Chito-Trehalose. FIG. 5A shows the full spectrum; FIG. 5B shows an expansion of the signal range.

[0021] FIG. 6 shows DLS values of trehalose, chitosan, chitosan + trehalose, and Chito- Trehalose in aqueous medium at progressively higher concentrations. All solutions were analyzed at pH 6.4.

[0022] FIG. 7 shows UV-Vis spectra of 0.1 wt / v% solutions of chitosan, Intermediates 1 and 2, Chito-Trehalose, trehalose, and chitosan and trehalose mixed together.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0023] FIG. 8 shows SEM images of chickpea coated seeds coated with chitosan solutions at different concentrations.

[0024] FIG. 9 shows the effect of the pH of chitosan solutions (orange (lower) curve, right-side scale on both graphs) on the germination yields (blue (upper) curve, left-side scale on both graphs) of Cicer arietinum (chickpea) coated seeds. On left panel, the results obtained with chitosan solution without buffering the pH. On right panel, results after buffering the chitosan solutions at pH 6.2-6.5.

[0025] FIG. 10 shows the effects of chitosan seed coatings on plant parameters. Left panel: growth curve of chickpeas until adult stage at day 18 with chitosan seed coatings at different concentrations (chitosan - medium molecular weight (MMW)). Right panel: variation of vital plant parameters in coated seeds, ranging the concentration of chitosan in the coating solution

[0026] FIG. 11 shows the appearance of chickpeas plants grown with different chitosan coating concentrations after day 18.

[0027] FIG. 12 shows the effect of chitosan on plant (chickpea) growth. Left panel: growth curve of chickpeas until adult stage at day 18 with chitosan seed coatings at different molecular weights (chitosan concentration 1.0% wt / v). Right panel: variation of vital plant parameters in coated seeds, ranging the molecular weight of chitosan in the coating solution.

[0028] FIG. 13 shows the appearance of chickpeas plants grown with different chitosan coating molecular weights after day 18.

[0029] FIG. 14 shows a table of results showing the effect of Chito-trehalose on plant growth versus various standards.

[0030] FIG. 15A through FIG. 15H show an evaluation of upper district vital plant parameters in selected crops after coating the seeds, including shoot height (FIG. 15 A), fresh shoot weight (FIG. 15B ), dry shoot weight (FIG. 15C), germination yield (FIG. 15D), plant weight (FIG. 15E), chlorophyll A (FIG. 15F), chlorophyll B (FIG. 15G), and carotenoids content (FIG. 15H). Six different coatings were used: water, chitosan 0.5% wt / v, trehalose 0.5% wt / v,PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 chitosan 0.5% wt / v + trehalose 0.5% wt / v, silk + trehalose 3 : 1 ratio, and Chito-Trehalose 0.5% wt / v. Chito-Trehalose resulted in the best coating material, since it beneficially affected all the plant parameters for all the tested crops, overcoming the effects of chitosan and silk + trehalose.

[0031] FIG. 16A to FIG. 16D show an evaluation of root district vital plant parameters in selected crops after coating the seeds: fresh root weight (FIG. 16A) and dry root weight (FIG. 16B), root length (FIG. 16C), and the development of Rhizobacteria on the plant roots (FIG. 16D). Six different coatings were used: water, chitosan 0.5% wt / v, trehalose 0.5% wt / v, chitosan 0.5% wt / v + trehalose 0.5% wt / v, silk + trehalose 3 : 1 ratio, and Chito-Trehalose 0.5% wt / v. As observed for aerial parts of the plants, also roots were beneficially affected by coatings, especially for Chito-Trehalose polymer. Moreover, development of nodules was observed with the use of genetically engineered bacteria with fluorescent proteins, creating fluorescent nodules within the first 3-5 cm of roots.

[0032] FIG. 17 shows the effect of chitosan concentration towards A. tropici growth. Top: Rhizobium tropici toxicity test with increasing amount of chitosan in aqueous phase (pH=6.4). Bottom: Toxicity of chitosan at different concentrations towards R. tropici growth (chitosan MMW). Bacteria solutions were incubated at 30°C under constant stirring at 200 rpm, using a starting bacteria Optical Density (OD) equal to 1.0. For each experiment, 5 mL of material solution and 1 mL of pellet were used.

[0033] FIG. 18 shows the effect of chitosan molecular weight (MW) towards R. tropici growth. Top: Rhizobium tropici toxicity test with different chitosan molecular weights in aqueous phase (pH=6.3-6.5, fixed concentration of 0.5 wt / v %). Bottom: Toxicity of chitosan at different molecular weights towards R. tropici growth (chitosan 1.0 wt / v). Bacteria solutions were incubated at 30°C under constant stirring at 200 rpm, using a starting bacteria Optical Density (OD) equal to 1.0. For each experiment, 5 mL of material solution and 1 mL of pellet.

[0034] FIG. 19A to FIG. 19F show the toxicity of chitosan derivatives on Rhizobacteria both in liquid and solid phase. FIG. 19A shows a schematic of the assay. Each of Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, and Bradyrhizobium japonicum, andPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699Pseudomonas fluorescens were tested in liquid phase (FIG. 19B to FIG. 19F, respectively, top panel) and solid phase conditions (FIG. 19B to FIG. 19F, respectively, bottom panel). For liquid phase experiments, Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, Bradyrhizobium japonicum, and Pseudomonas fluorescens were incubated in the following aqueous solutions: water, chitosan 0.5% wt / v, trehalose 0.5% wt / v, chitosan 0.5% wt / v + trehalose 0.5% wt / v, and Chito-Trehalose 0.5% wt / v. Toxicity in liquid phase was evaluated on a period of 2 days, counting the colonies after Oh, Ih, 6h, 24, and 48h (with the exception of Pseudomonas fluorescens, which lacks a Oh control), repeating the experiments thrice for the standard deviation. Liquid-based experiments are shown. Solid phase viability of bacteria is shown in different films: water, chitosan, Chito-Trehalose, and silk + trehalose 3: 1 ratio.

[0035] FIG. 20 shows13C-NMR spectrum (125 MHz, D2O) for Digested Chito-Trehalose.

[0036] FIG. 21 shows bidimensional 'H-13C-NMR HSQC spectrum (rH 500 MHz, D2O) for digested Chito-Trehalose.

[0037] FIG. 22 shows germination yield, root length, and shoot length of different crop seeds under increasing salinity stress, from 0 mM to 400 mM.

[0038] FIG. 23 shows seeds germination of barley, com, chickpea, and soybean after 4 days in progressively saline solutions, from 0 mM to 400 mM.

[0039] FIG. 24 shows vital plant parameters for chickpea grown until day 18 in the saline range O mM - 150 mM.

[0040] FIG. 25 shows chickpea plant phenotype under different saline conditions.

[0041] FIG. 26 shows plant vital parameters for barley under normal and saline stress conditions using different seed coatings.

[0042] FIG. 27 shows plant vital parameters for chickpea under normal and saline stress conditions using different seed coatings.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0043] FIG. 28 shows plant vital parameters for corn under normal and saline stress conditions using different seed coatings.

[0044] FIG. 29 shows plant vital parameters for soybean under normal and saline stress conditions using different seed coatings.

[0045] FIG. 30 shows barley plant phenotype grown from seeds coated with water, chitosan, trehalose, and chito-trehalose in normal and 150 mM saline conditions.

[0046] FIG. 31 shows chickpea plant phenotype grown from seeds coated with water, chitosan, trehalose, and chito-trehalose in normal and 150 mM saline conditions.

[0047] FIG. 32 shows com plant phenotype grown from seeds coated with water, chitosan, trehalose, and chito-trehalose in normal and 150 mM saline conditions.

[0048] FIG. 33 shows soybean plant phenotype grown from seeds coated with water, chitosan, trehalose, and chito-trehalose in normal and 150 mM saline conditions.

[0049] FIG. 34 shows rhizobia nodules on plant roots grown in 150 mM NaCl saline stress.

[0050] FIG. 35 shows a full view of all vital plant parameters affected by different seed coating materials. Overall, the Chito-Trehalose 0.5% wt / v coating resulted in the most effective enhancing coating for plant growth, since every crop shown highest parameters values when they grew from seed coated with the derived polymer.

[0051] FIG. 36 shows a chemical synthesis scheme for the final chitosan derivative, Chito- trehalose, obtained from the linking of trehalose to the biopolymer. The first step involved the formation of a Schiff Base through reaction of free amino group in D-glucosamine moieties with 2,2-dimethoxyacetaldehyde. The second step consisted in the reduction of the Schiff Base with NaBFU Finally, trehalose was directly linked to the biopolymer via a transacetalization reaction between the two dimethoxy residues in intermediate 2 with the diol system in position C4-C6 in trehalose.

[0052] FIG. 37 shows the effect of trehalose, L-proline and glycine betaine towards R. tropici growth (0.5 wt / v% for each solution, pH buffered at 6.3-6.5). Bacteria solutions werePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 incubated at 3O°C under constant stirring at 200 rpm, using a starting bacteria OpticalDensity (OD) equal to 1.0. For each experiment, 5 mL of material solution and 1 mL of pellet bacteria were used. The starting colonies counting is highlighted with a dash horizontal line. The top panel shows the viability / toxicity data in tabular form, and the bottom panel shows the data in graphical form.

[0053] FIG. 38 shows the effect of water, chitosan and Chito-Trehalose on plant (yellow mustard; Sinapis alba) growth. Top panel: variation of vital plant parameters in coated seeds until adult stage at day 18. Bottom panel: appearance of yellow mustard plants grown with a chitosan coating versus a Chito-Trehalose coating.

[0054] FIG. 39 shows the effect of water, chitosan and Chito-Trehalose on plant (Triticale; x Triticosecale) growth. Top panel: variation of vital plant parameters in coated seeds until adult stage at day 18. Bottom panel: appearance of triticale plants grown with a chitosan coating versus a Chito-Trehalose coating.

[0055] FIG. 40 shows the effect of water, chitosan and Chito-Trehalose on plant (sainfoin Onobrychis viciifolia) growth. Top panel: variation of vital plant parameters in coated seeds until adult stage at day 18. Bottom panel: appearance of sainfoin plants grown with a chitosan coating versus a Chito-Trehalose coating.

[0056] FIG. 41 shows the effect of water, chitosan and Chito-Trehalose on plant (common vetch Vicia saliva) growth. Top panel: variation of vital plant parameters in coated seeds until adult stage at day 18. Bottom panel: appearance of common vetch plants grown with a chitosan coating versus a Chito-Trehalose coating with close-up of roots inset.

[0057] FIG. 42 shows ATR-FTIR spectroscopy at various steps of chitosan purification from insect exuviae.DETAILED DESCRIPTION

[0058] The inventors have surprisingly found that a novel compound, Chito-trehalose is a versatile and effective seed coating material, with superior properties for seed protection and plant growth, and also promotes symbiotic bacterial growth of growth promoting bacteria.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699The chemical reactions involved in the production of Chito-trehalose are mild, high-yielding, and based on commercially available, accessible, and non-toxic reagents, and purification steps and intermediates handlings are based on simple scalable techniques such as filtration, evaporation, and centrifugation. Chito-trehalose, when used as a coating material for commercially important and valuable food source crops (such as Cicer arietinum (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifolia (sainfoin), and Vida sativa (common vetch)), promoted plant development. Chito-trehalose was also found to promote growth of symbiotic nitrogen-fixing growth promoting bacteria, such as Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, Bradyrhizobium japonicum, and Pseudomonas fluorescens.

[0059] Furthermore, Chito-trehalose demonstrates enhanced efficacy under abiotic stress conditions, particularly salinity, by mitigating osmotic stress and supporting plant and bacterial resilience, as evidenced by maintained germination rates, biomass, and nodulation in environments with high soil salinity.

[0060] Without being bound by theory, it is believed that the trehalose transacetalization of Chito-trehalose prevents the folding of the chitosan backbone in globular conformations, imparting a more open web-like structure that facilitates the exposure of polar groups, which may explain, e.g. why the Chito-trehalose is superior to the simple mixture of chitosan and trehalose in preserving growth promoting bacteria.Definitions

[0061] As used herein, the term “about” means ±5% of the recited value, but the term may also be used to refer to any value below ±5%, e.g. ±4%, ±3%, ±2%, ±1%, or ±0% (i.e. the recited value itself).

[0062] When a range is recited herein, it is to be understood that the stated range is meant to additionally encompass any sub-range within the stated range, or any specific value falling within the range, even if not explicitly enumerated herein. For example, recitation of a rangePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 of 0-10 is also intended to include the ranges of, e.g., 1-5, 2-8, 4-6, etc. as well as specific values within the range, e.g. 1, 2, 3.5, 6.75, 8, etc. even if not specifically stated.

[0063] As used herein, the term “chitosan polymer(s)” refers to a random copolymer comprising monomers of D-glucosamine and N-acetyl-D-glucosamine.

[0064] The term “trehalose molecule(s)” is used herein in accordance with its meaning in the art, and may also be used to refer to trehalose covalently bound to a polymer and / or within a cross-linked polymer as disclosed herein, including covalently bound in accordance with the chemistry and formulas disclosed herein.

[0065] As used herein, the phrase “promoting plant development” means improving upon or increasing one or more parameters associated with plant growth and / or, in the case of food source crops, crop quality and / or yield, relative to those parameters, etc. in the absence of the stated actions and / or conditions. Such parameters include, but are not limited to, germination yield, shoot length, root length, plant weight, including fresh and dry weight of shoots and roots, and chlorophyll and carotenoid content in fresh leaves.

[0066] As used herein, the phrase “promoting bacterial growth” means increasing bacterial count relative to the bacterial count in the absence of the stated actions and / or conditions, or maintaining bacterial count where the bacterial count would otherwise decline in the absence of the stated actions and / or conditions.

[0067] As used herein, “growth promoting bacteria” refer to bacteria that improve plant development and / or promote soil bioremediation.

[0068] As used herein, "salinity stress" or "saline conditions" refers to soil or growth environments with elevated salt levels, typically measured as electrical conductivity exceeding 4 dS mor NaCl concentrations of about, e.g., 25-50 mM to about 200 mM, or to 300 mM, which adversely affect plant growth, germination, or microbial viability.Polymeric Compounds and Synthesis Thereof

[0069] An aspect of the disclosure provides polymeric compounds that include one or more chitosan polymers and one or more trehalose molecules, wherein the trehalose molecules arePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 bound to deacetylated monomers of the chitosan polymers. In some embodiments, the trehalose molecules are bound to the deacetylated monomers of the chitosan polymers through a transacetal linkage, such as the specific structure where the linkage forms involving the C4 and C6 hydroxyl groups of one glucose unit in trehalose and the linker attached to the amino group of the deacetylated glucosamine monomer.

[0070] In an embodiment, the disclosure provides trehalose molecules that are bound to the deacetylated monomers of the chitosan polymers as follows:

[0071] In a further embodiment, the disclosure provides a polymeric compound of the general formula: C-T-C, in which T denotes a trehalose molecule and C denotes a chitosan polymer.

[0072] In an embodiment, the disclosure provides a polymeric compound the includes the compound of Formula I:PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO25699(Formula I) in which each n and m are independently an integer of 0 or greater; each R is independentlyX is a bond to the amine of a deacetylated monomer of the same or a different chitosan polymeric chain.

[0073] The chains of the polymeric compound of Formula I can be of any suitable length or weight. In certain embodiments, the disclosure provides compounds where the n+m within the same polymer chain is between about 200 to about 1600. In some embodiments, n+m within the same polymer chain is between about 200 to about 1200, about 500 to about 1600, about 400 to about 1400, about 500 to about 1200, or about 50 kDa to about 400 kDa. In certain embodiments, the polymeric compound may have a molecular weight of about 50 kDa to about 400 kDa, and in some embodiments, the polymeric compound may have a molecular weight of about 100 kDa to about 200 kDa. In other aspects, polymeric compound may have a molecular weight of about 100 kDa to about 150 kDa, of about 150 kDa to about 200 kDa, of about 120 kDa to about 180 kDa, or of about 140 kDa to about 160 kDa.

[0074] In various embodiments, the chitosan chains are random copolymers where less than about 50% of the total monomers are N-acetyl glucosamine, such as less than about 35%, less than about 25%, less than about 15%, less than about 10%, or less than about 5%, ensuring high solubility in aqueous acidic media suitable for coating applications. In some embodiments, less than about 35% of the total monomers are N-acetyl glucosamine, and in some embodiments, less than about 5% of the total monomers are N-acetyl glucosamine.

[0075] The polymeric compounds of the disclosure include any ratio of monomers of the chitosan polymers to the trehalose molecules. In certain instances, the ratio of monomers toPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 trehalose molecules may be about 5:4, in certain instances, it may be about 5:3, in certain instances, it may be about 5:2, in certain instances, it may be about 5: 1, and in other instances, it may be about 10: 1.

[0076] Further embodiments include crosslinked polymer chains, where trehalose or additional linkers, e.g., facilitate inter-chain connections, enhancing structural stability and controlled release properties. The ratio of chitosan monomers to trehalose molecules can vary from about 20:1 to about 3: 1, including ratios such as about 10:1 to about 5:4, about 5:3 to about 5: 1, or specifically about 5:3 to about 10:1, to optimize osmoprotective effects and bacterial compatibility.

[0077] The polymeric compounds of the disclosure can be synthesized using a 3-step method that uses inexpensive, non-toxic, commercially available reagents and mild reaction conditions, and provides a high yield after simple purification steps. In the first step, starting with chitosan, the free amino groups are reacted with 2,2-dimethoxyacetaldehyde to form a Schiff s base as a first intermediate. In certain instances, the first step may be performed using a concentration of 1% or less of chitosan. In certain instances, the first step may be performed in aqueous media at about 70°C for about 24 hours, or at any temperature below about 70°C and at or above room temperature for the same amount of time without any significant difference in the final yield. In certain aspects of the disclosure, residual 2,2- dimethoxyacetaldehyde may be removed from the first intermediate after performing the first step of the synthesis, and in other aspects, the first intermediate may be concentrated to 2% wt / v prior to performing the second step of the synthesis.

[0078] In the second step, the first intermediate is reduced to form an amine as a second intermediate. In certain instances, the reducing agent may be NaBFU and the reaction may be performed under vigorous mixing at room temperature, with the NaBFU being added dropwise. In certain aspects of the disclosure, the second intermediate may be collected, filtered, and washed with distilled water one or more times prior to performing the third step of the synthesis.

[0079] In the third step, a transacetalization reaction between trehalose and the second intermediate is preformed, resulting in the polymeric compound of the disclosure. In certainPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 instances, the reaction may be performed in a mild acidic aqueous medium (pH 5-6) at about 80°C overnight, using a slight excess of trehalose. The final product, Chito-trehalose, may be purified through the addition of base to precipitate the final product, followed by filtration and washing with distilled water to obtain a purified product.

[0080] The present disclosure also provides a polymeric compound prepared by the above method.

[0081] The polymeric compounds and compositions disclosed herein are particularly effective in promoting plant development and bacterial growth under abiotic stresses, such as salinity. In embodiments, the compounds confer tolerance to salinity levels of about 50 mM to about 200 mM NaCl, maintaining or enhancing germination yields, root and shoot lengths, biomass, and pigment contents relative to uncoated controls. Without being bound by theory, the pronounced stress mitigation is attributed to the osmoprotective properties of the trehalose moiety and the structural stability provided by the chitosan backbone, which facilitates nutrient release and microbial symbiosis even in high-salt environments.Compositions

[0082] In another aspect, the disclosure provides a composition for seed coating comprising a polymeric compound as disclosed herein dissolved in an acidic solution. The acidic solution can comprise any known acid, including, but not limited to, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, citric acid, or lactic acid. In embodiments, the acidic solution is buffered to a pH range of about 5.0 to about 7.5, including sub-ranges such as about 5.5 to 7.0 or about 6.0 to about 6.6. In some embodiments, the acidic solution is buffered to a pH of between about 6.0 to about 6.6. In some embodiments, the acidic solution comprises acetic acid.

[0083] In some embodiments, the polymeric compound may be present in the seed coating composition at a concentration of about about 0.1% to about 5% wt / v, with ranges up to about 3% wt / v or less, including sub-ranges such as about 0.1-1%, about 0.5-2%, about 1- 3%, or about 1.0% wt / v or about 0.5% wt / v. In some embodiments, the polymeric compoundPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 may be present at a concentration of about 1.0% wt / v or at a concentration of about 0.5% wt / v.Methods

[0084] In a further embodiment, the disclosure provides a method of seed coating comprising submerging seeds in a seed coating composition for a suitable time to enable an functionally adequate and stable coating. In embodiments, seeds are submerged for about 1 to about 30 seconds, such as about 2-25 seconds, or about 2-20 seconds, or about 5-20 seconds, or about 10-20 seconds, or about 15-20 seconds. In some embodiments, the seeds are submerged for about 2-20 seconds, or about 15-20 seconds, or about 10 seconds, or about 5 seconds. After soaking, the seeds are preferably dried using any suitable drying technique that is not harmful to the seed or coating, including air drying. In some embodiments, the soaked seeds are airdried for between about 10-48 hours, including about 20-30 hours or about 24 hours, to form stable coatings.

[0085] In another aspect, the disclosure provides a method of promoting plant development. Such methods include coating plant seeds in a seed coating composition as disclosed above, germinating the seeds, and allowing the plants to grow. Germinating the seeds and allowing the plants to grow, and the conditions associated therewith, are within the purview of one of ordinary skill in the art. For example, germinating can be carried out under standard or stressed conditions (e.g., controlled humidity 50-90%, temperature 20-30°C). In certain embodiments, the coating thickness on the seeds is between about 10 pm to about 100 pm. In other aspects, the coating thickness on the seeds is about 10 pm to about 20 pm, about 40 pm to about 50 pm, or about 80 pm to about 50 pm.

[0086] The methods of promoting plant development disclosed herein may be used with any plant of interest, including, but not limited to commercial crops such as Cicer arietinum (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifolia (sainfoin), or Vicia sativa (common vetch).PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0087] In yet another embodiment, the disclosure provides a method of promoting growth of plant growth-promoting bacteria on a plant including coating plant seeds with a bacterial composition comprising a seed coating composition as disclosed herein and a growth promoting bacteria, germinating the seeds, and allowing the plant to grow. In some embodiments, the growth promoting bacteria are nitrogen fixing bacteria, including, but not limited to, Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, Bradyrhizobium japonicum, Pseudomonas fluorescens, and mixtures thereof. In embodiments, the bacteria form nodules on adult plant roots, enhancing nitrogen fixation and plant nutrition. These methods are suitable for any plant of interest, including, but not limited to, Cicer arietinum, Triticum aestivum, Hordeum vulgare, Zea mays, Glycine max, Helianthus annuus, Sesbania sesban, Sinapis alba, x Triticosecale, Onobrychis viciifalia, Vicia saliva, or equivalents.

[0088] In further embodiments, the methods disclosed herein are performed under salinity stress conditions, such as irrigation with high salinity water, e g., of 25 mM to about 200 mM NaCl or higher, wherein the polymeric compounds enhance tolerance, e.g., by maintaining viability, growth parameters, and symbiotic interactions.EXAMPLESExample 1: Materials and Methods

[0089] All chemicals and solvents were used without any further purification step. ATR-FTIR spectra were acquired with a Perkin Elmer (Waltham, MA) Spectrum Two Spectrophotometer equipped with a 2 x 2 mm Diamond crystal. Spectra were recorded in the range 4000-650 cm'1with a 2 cm'1resolution, using 0.5 cm'1acquisition interval and acquiring 16 scans for each sample.XH-NMR and13C-NMR spectra were recorded in deuterated solvents solution with an Agilent 500 spectrometer, operating at a frequency of 500 MHz for 'H, 125 MHz for13C; chemical shifts (5) values are given in parts per million (ppm) and coupling constants (J) in Hertz. For Dynamic Light Scattering, a Zeta Potential Analyzer (Brookhaven Instruments Corp., Holtsville, NY) was used to measure the particle size in resuspended solution at progressive dilutions. Each measurement was 180 s and at least three measurements were carried out per sample’s type. Ultraviolet- Visible SpectraPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 were acquired with a VWR UV-1600PC spectrophotometer, working at room temperature and with 1 cm optical path cuvettes. Scanning electron microscopy (SEM) images were taken using a Zeiss Merlin high-resolution scanning electron microscope. Samples were coated with 10 nm gold and observed on a 45° sample holder at 0.8-1 kV and 80 pA. Acquired data were analyzed and plotted with the software OriginPro 2017 (OriginLab Corporation, Northampton, MA).

[0090] Plant material and seed disinfection

[0091] All the seeds were purchased from local vendors (USA). The seeds were visually selected for each experiment, discarding the ones that had shown apparent damage or were split in more pieces. The seeds were disinfected through immersion in 70 % ethanol aqueous solution for 5’, rinsed with water and air dried for few hours on filter paper before using them. The following crops have been investigated: Cicer arietimim (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifolia (sainfoin), and Vicia sativa (common vetch).

[0092] Initial germination and growth

[0093] Lidded round plastic containers 12 cm in diameter were used as germination chambers. In each plate were placed three layers of cellulose paper sterilized by spraying them with ethanol and 15 seeds equally distant from each other’s and leaving them enough space to avoid overlap of roots. For germination experiment, in each plate were added 10 mL of a certain solution, the plate closed and let to incubate at 27°C for 4 days in a complete dark chamber at constant relative humidity of 70%. Seeds status was observed every day and eventually dried plates were rehydrated with 1-2 mL of bidistilled water for all the experiments. Each experiment was repeated thrice. Germination was taken daily for 4 days, considering germinated the seeds that had shown at least a root higher in 5 mm in length.After 4 days, 5 seeds per lid were selected and sowed in square pots 8.5x8.5x8 cm, at a depth of approximately 4 cm (Miracle-Gro Moisture Control Potting Soil Mix). Hence, a statistical sample of 15 adult plants was collected for each crop and for each experimental condition.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699Plants were grown in a plant growth chamber at 22 °C with day / night cycle 16h / 8h, at a relative humidity of 70-100%, with a light intensity of 100 pmol nr2s'1and watered regularly. Each crop had shown a specific gathering time, accordingly to the growth curves collected every day until a plateau growth was observed: chickpea for 18 days, wheat for 19 days, barley for 15 days, corn for 28 days, soybean for 19 days, sunflower for 33 days, and Egyptian riverhemp for 90 days.

[0094] After the specific growth time for each crop, plants were removed from pots and the following parameters were recorded: germination rate, shoot length for every day, root length at adult stage, shoot length at adult stage, total plant fresh mass, shoot fresh mass, root fresh mass, shoot dry mass, root dry mass, chlorophylls, and carotenoids content on fresh leaves. After collecting the plant sections, they were dried overnight in oven at 70 °C. The residual cotyledons were counted as part of the root.

[0095] Chlorophyll Content on fresh leaves

[0096] The total amount of Chlorophyll A (ChlA), Chlorophyll B (ChlB) and Carotenoids (Cars) was determined through spectroscopic measurements, recording the absorbance peak maxima at 470 nm, 649 nm, and 665 nm. For the determination of pigments concentrations, the following equations were used:

[0097] ChlA (|ig / mL)= 13.95 A665- 6.88 A649

[0098] ChlB (pg / mL)= 24.96 A649- 7.32 A665

[0099] Cars (pg / mL)= (1000 A47o - 2.05 ChlA -114.8 ChlB) / 245

[0100] Briefly, an exact amount of fresh leaves of adult stage plant (ca. 100 mg) were carefully weighted and incubated in 5 mL of 96% ethanol, letting the extraction occurs overnight at room temperature in a completely dark environment and shaking at 200 rpm. After this period, the leaves appeared bleached, and the green solution was filtered and centrifuged at 4800g for 20 minutes. The supernatant was collected and analyzed in a 1 cm light path cuvette in the UV-Vis range 300-800 nm. Final pigments values were corrected for the volume of ethanol and for the exact mass of the fresh tissue leaves.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0101] Bacteria Growth

[0102] Rhizobium tropici cell culture media

[0103] In an autoclavable glass bottle were introduced 2.5 g of Bacto-peptone, 1.5 g of Yeast extract, 5 mL of 0.7 M CaCh solution, and 500 mL of bidistilled water. The solution was autoclaved at 121 °C, 15 psi, for 45 minutes. After cooling to 60 °C, the following antibiotics were added: 400 pL of 25 mg / mL Rifampicin DMSO solution, 500 pL of 20 mg / mL Nalidixic acid H2O solution, 250 pL of 10 mg / mL Tetracycline H2O solution. The culture media was stirred and kept in fridge until its utilization.

[0104] Azorhizobium caulinodans cell culture media

[0105] In an autoclavable glass bottle were introduced 2.5 g of Triptone, 1.5 g of Yeast extract, 5 mL of 0.7 M CaCh solution, and 500 mL of bidistilled water. The solution was autoclaved at 121 °C, 15 psi, for 45 minutes. After cooling to 60°C, was added 500 pL of 100 mg / mL Ampicillin H2O solution. The culture media was stirred and kept in fridge until its utilization.

[0106] Klebsiella variicola cell culture media

[0107] In an autoclavable glass bottle were introduced 12.5 g of LB medium and 500 mL of bidistilled water. The solution was autoclaved at 121°C, 15 psi, for 45 minutes. After cooling to 60°C, was added 500 pL of 100 mg / mL Ampicillin H2O solution. The culture media was stirred and kept in fridge until its utilization.

[0108] Bradyrhizobium japonicum cell culture media

[0109] In an autoclavable glass bottle were introduced 77.0 g of African Violet Soil (local vendors, USA), 0.200 g of Na2CC>3, 400 mL of bidistilled water. The mixture was autoclaved at 121 °C for Ih. Then, the solution was filtered through cotton twice, and 200 mL of soil extract were used to prepare the culture media as following: 1.0 g of Yeast extract, 10.0 g of Mannitol, 200 mL of soil extract, and bidistilled water to reach 1.0 L of volume. The pH of the final broth was adjusted to pH 7.2 by addition of few drops of NaHCCh IM. The solution was then autoclaved at 121 °C for 45 minutes. The culture media was stirred and kept in fridge until its utilization.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0110] Pseudomonas fluoresce ns cell culture media

[0111] In an autoclavable glass bottle were introduced 11.5 g of Nutrient Broth DB medium and 500 mL of bidistilled water. The solution was autoclaved at 121 °C, 15 psi, for 45 minutes. The culture media was stirred and kept in fridge until its utilization.

[0112] Cell culture growth

[0113] Bacteria solutions were obtained by dissolving a scraped amount of freeze-dried (-80°C) glycerol stock solution in 4 mL of cell culture media. The solution was incubated at a certain temperature (30 °C for R. tropici, 37 °C for A. caulinodans, 27 °C for K. variicola, B. japonicum, and Pseudomonas fluorescens 200 rpm, for 24 h. After this period, optical density (OD) was measured by spectroscopic methods at 600 nm and adjusted to 1.0 by diluting with PBS solution. At this point, 1 mL of the stock solution was added to 50 mL of cell culture media and let incubate at 30°C, 200 rpm, for 24 h. Then, 25 mL of bacterial solution were centrifuged at 23°C, 2000 rpm for 15 minutes. The supernatant was discarded and replaced with 25 mL PBS solution and the solution was vortexed to disperse bacterial pellet. For toxicity experiments, 0.5 mL of bacteria solution were transferred to 15 mL screwed plastic vials, centrifuged at 23°C, 2000 rpm for 15 minutes, removed the supernatant, and added 5 mL of the toxic compound solution. Solutions were incubated at determined temperatures (30 °C for A. tropici, 37 °C for A. caulinodans, 27 °C for A. variicola, B. japonicum, and Pseudomonas fluorescens), 200 rpm, for 48 h, checking the bacteria population at Oh, Ih, 6h, 24h, 48h through spreading 100 pL into an agar plate obtained by adding 7.5 g agar powder to 500 mL of cell culture media.

[0114] Bacteria viability in solid phase

[0115] Bacteria survival rate in dry state was evaluated over a period of 28 days, observing the colonies counting at the following days: 0, 1, 7, 14, 21, and 28. Bacteria solutions were prepared as previously reported, and letting bacteria grow in the culture media until they reached a specific OD above reported in the following sections. After 48 hours, 20 mL of solution were centrifuged, the supernatant discarded and the pellet redissolved in 2.0 mL of testing solution. For each experiment, 100 pL of solution were cast on a 1.5 mL plastic vialPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 and let air dry overnight, repeating each data thrice, for a total of 1.8 mL per viability curve.The dry films / powders were then dissolved in 1.0 mL of PBS solution at established times of 0, 1, 7, 14, 21, and 28 days, vortexing and leaving in constant agitation at 200 rpm for 1 hour to ensure complete material dissolution. After appropriate dilutions, 100 pL of solution were spread on agar plates and incubated at a certain temperature for three days. The following testing solutions were used: bidistilled water (water), chitosan MMW 0.5% wt / v at pH 6.5 (chitosan); Bombyx mori silk fibroin 6% wt / v + trehalose 6% wt / v in a 1:3 v / v ratio (silk + trehalose), and the synthesized polymer Chito-Trehalose 0.5% wt / v at pH 6.4.

[0116] Rizhobium tropici viability in solid phase

[0117] Starting OD: 1.50; Dilutions at days 0, 1, 7, 14, 21, and 28: 107; 105; 103; 103; 102; 101, respectively. Plates were incubated at 30 °C.

[0118] Azorizhobium canlinodans viability in solid phase

[0119] Starting OD: 2.00; Dilutions at days 0, 1, 7, 14, 21, and 28: 107; 105; 104; 104; 103; 103, respectively. Plates were incubated at 37 °C.

[0120] Klebsiella variicola viability in solid phase

[0121] Starting OD: 2.06; Dilutions at days 0, 1, 7, 14, 21, and 28: 107; 106; 105; 105; 104; 103, respectively. Plates were incubated at 27 °C.

[0122] Bradyrhizobium japonicum viability in solid phase

[0123] Starting OD: 2.10; Dilutions at days 0, 1, 7, 14, 21, and 28: 1018;1016; 1015; 1014; 1013; 1013, respectively. Plates were incubated at 27 °C.

[0124] Pseudomonas fhiorescens viability in solid phase

[0125] Starting OD: 1.87; Dilutions at days 0, 1, 7, 14, 21, and 28: 107; 106; 105; 105; 104; 103, respectively. Plates were incubated at 28°C.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0126] Nodules analysis with fluorescent symbionts

[0127] Together with the viability of bacteria in solid phase, their ability to colonize the roots of plants was evaluated by fluorescence methods, using R. tropici GFP, A. caulinodans GFP, K. variicola FP , and B. japonicum on chickpeas, sesbania, corn, and soybean, respectively. Growing methods were the same as for non-fluorescent strains. In particular, the OD of the fluorescent strains were 1.48 for R. tropici-GFP , 2.5 for A. caulinodans-G , 2.2 for K. variicola-RFP , and 2.8 for B. japonicum. After growing the strains (48 hours) 20 mL of solution were centrifuged, the supernatant discarded and the pellet redissolved in 2.0 mL of testing solution. Disinfected seeds were then submerged in the bacteria solution for inoculation and let air dry overnight before sowing them into the soil. Plants were grown for longer time in respect to standard growth time in order to let nodules form on the root surface. More specifically, chickpea roots were analyzed after 30 days, sesbania roots after 90 days, corn roots after 30 days, and soybean roots after 40 days. No macroscopic nodules could be observed for R. tropici, A. caulinodans, and A. variicola, but they were detected through fluorescent microscopy as greenish or reddish, bumps near the crown region of the roots. B. japonicum formed macroscopic nodules on soybean roots.

[0128] Chitosan solutions and seed coating technique

[0129] Several chitosans (Ultra-Low MW (20 KDa, VWR), Very-Low MW (30 KDa, VWR), Low MW (50KDa, Sigma Aldrich), Medium MW (230 KDa, Sigma Aldrich), High MW (850 KDa, Sigma Aldrich), chitosan from hydrolyzed shells) were dissolved in 1% v / v CH3COOH aqueous solution in different concentrations, ranging from 0.25 to 3.0% wt / v. Solution was buffered with concentrated NaOH solution until pH reached a value of 6.3-6.6 close to the chitosan isoelectric point. Seeds were coated via deep-coating technique, submerging them in chitosan solution for 5 seconds and then letting them air-dry for 24 hours before sowing them in soil. Trehalose solution was prepared dissolving 0.5 g of trehalose in 100 mL of distilled water, thus obtaining a 0.5% wt / v coating solution. Chitosan + trehalose solution was prepared mixing together a 1.0% wt / v trehalose solution with 1% wt / v chitosan solution in equal volumes, reaching a final concentration of 0.5% wt / v both in trehalose and chitosan. Chito-Trehalose solution was prepared similarly to other solutions, dissolving 0.5 gPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 of dry polymer into 100 mL of distilled water. The final coating solution was buffered to pH 6.5 before using it for seeds coating. Silk + trehalose solution was prepared mixing in a 1 / 3 volume ratio a 6.0% wt / v silk fibroin solution (10 mL) prepared according to a reported procedure (Rockwood DN, et al. “Materials fabrication from Bombyx mori silk fibroin” Nat Protoc. 2011 Sep 22; 6(10): 1612-31) with a 6% wt / v trehalose solution (30 mL).

[0130] Statistical Analysis

[0131] All results collected are expressed as average value based on all the seeds and plants observed. Specifically, seeds experiments involved 15 seeds per plate, repeated thrice, for a total of 45 seeds. For adult plants, 5 germinated seeds were selected and sowed, for a total of 15 samples. ANOVA variance test was conducted for all data sets.Example 2: Synthesis and Characterization of Chitosan-Trehalose Polymer (Chito- Trehalose)

[0132] The final product Chito-Trehalose was prepared according to chemical design of the scheme shown in FIG. 36. Starting from chitosan, a first reaction with 2,2- dimethoxyacetaldehyde was carried out to prepare the intermediate 1 in which an imine bond was formed between the aldehyde and the free amino-group of the biopolymer. The imine bond was subsequently reduced to amine bond through reaction of intermediate 1 with NaBH4 to prepare intermediate 2. Finally, a transacetalization reaction between the two methoxy-groups of Intermediate 2 and trehalose was performed, obtaining the final product Chito-Trehalose.

[0133] Inventors considered an amino-group chemical modification as a simplest and reliable type of modification on chitosan-based materials due to quantitative yields, mild conditions that prevent or limit biopolymer hydrolysis, chemical stability, and ease of purification of intermediates. Thus to stabilize the polymer structure, the imine was reduced into an amine group and the sugar was chemically bound to the polymer chain via a transacetalization, through the reactivity of the hydroxyl groups in trehalose. Inventors describe trehalose experiments here, among the plethora of sugars that could be used.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0134] In order to chemically bind trehalose to chitosan polymer chain, the reactivity of both amino-group in chitosan and the special site of trehalose based on the two hydroxyl groups in C4 and C6 skeleton, since they react with aldehydes to form acetals, were exploited.Inventors found it nontrivial to find a linker between the polymer and the sugar that ensured a quantitative yield in both endings, affording stable intermediates. A double, short aldehyde was chosen, since aldehydes can both react with amines and diols, to form imines (Schiff Bases) and acetals, respectively. Finally, to avoid cross-reactions between the amines in chitosan and diols in trehalose, 2,2-dimethoxyacetaldehyde was used. This chemical, considered non-toxic, has one readily available aldehyde on Cl, and a masked one as a dimethoxy acetaldehyde at C2 that can be successively activated by hydrolysis. FIG. 1 A (Panel b) shows the general synthetic process of Chito-Trehalose, starting from a chitosan solution and involving the major steps for Intermediate 1 and Intermediate 2.

[0135] Processing of crustacean shells and obtaining of chitosan

[0136] Pure chitin was obtained from commercially available shells through a deproteinization and demineralization procedure. The first step involved a heating bath of shells with 0.1 M NaOH in order to hydrolyze the proteins bound to the shell structure, while the removal of minerals, mainly CaCCh, was achieved by submerging the deproteinized shells into a 0.3 M HC1 solution. After rinsing the material, pure chitin was obtained as a pale white powder. At this point, the chemical hydrolysis of chitin was performed using a highly concentrated NaOH solution (12 M) and heating the reaction media at 120 °C for 6 hours. The final powder was then rinsed with water until liquors were at neutral pH, affording pure chitosan as white flakes.

[0137] Chitosan was dissolved into a 1% v / v CH3COOH aqueous solution and pH buffered until pH 6.3-6.6, using different amounts of biopolymer according to the desired concentration (FIG. 1A, Panel a).

[0138] Chitin extraction, hydrolysis and chitosan purification from insect exuviae .

[0139] Commercially available frass, a mixture of black soldier fly (Hermetia illucens)' fecal matter and exuviae, was purchased from Boogie Brew (Rohnert Park, CA). The powder wasPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 dispersed in aqueous 1.25 M NaOH at 90 °C for 4h in a ratio of 1 :20 wt / v frass / solution. After the required time, the frass was washed several times with bidistilled water until the liquors became colorless. The deproteinization was repeated two more times. Complete removal of proteins and fecal matter was assessed with ATR-FTIR spectroscopy.Deproteinized frass was treated with a aqueous solution of HC1 0.5 M at 40°C for 2 h in a ratio of 1 :20 wt / v powder / solution. The powder was washed with bidistilled water until liquors reached neutral pH. The demineralization procedure was repeated two more times. Complete demineralization was confirmed by ATR-FTIR spectroscopy, thus obtaining a mixture of chitin and melanins (FIG. 42). A final step of bleaching was achieved to remove black pigments, soaking the powder in NaOCl 6% at 60 °C for 1 h. The pure chitin was then washed several times with distilled water. Pure chitosan was then obtained following the procedure described above herein for chitin obtained from crustacean shells. Composition in terms of weight of frass: 78% fecal matter, 22% insects exuviae. Composition in terms of weight of exuviae: 24% proteins, 30% minerals, 39% melanins, 7% chitin.

[0140] Synthesis of Intermediate 1

[0141] A few chitosan Schiff Bases are known, based on both aromatic and aliphatic aldehydes. Reaction conditions are usually mild, based on aqueous media and performed at room temperature or with slight temperature increase. Moreover, reactions often afford quantitative yields in few hours. A common result is based on the final consistency of chitosan Schiff Bases, since hydrogels are obtained in most instances depending on the temperature, aldehyde amounts and biopolymer concentration. 2,2-dimethoxyacetalehyde is considered a non-toxic chemical, useful for many reactions in which a masked aldehyde is required. The reaction was conducted in an aqueous environment at 70 °C for 24 hours, and a concentration of chitosan 1% was used. If significantly higher polymer concentrations were used, an increase of solution thickness and gelification was observed, preventing a complete chemical reaction and making the purification tedious. Due to the tendency of gelification, the Intermediate 1 was partially purified with removal of excess aldehyde with prolonged heating at 80°C for 24h since this compound is volatile and possesses a low boiling point (b.p.63.9°C). Complete absence of residual aldehyde was confirmed with Fehling test on a small aliquot of Intermediate 1, a very sensitive test to detect even small traces of aldehydes in aPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 sample. The chemical structure of the Intermediate 1 was assessed through infrared and nuclear magnetic resonance techniques.

[0142] Attenuated Total Reflectance Fourier Transformed Infrared Radiation (ATR-FTIR) is one of the most useful and fast analytical tools for determination of polymers structures. Chitosan is a polysaccharide chemically analogous to cellulose, composed by D-glucosamine and N- Acetyl-D-glucosamine monomers bound together via 0-(l -4) glycosidic bonds. The FTIR spectra in FIG. IB (black curve, i.e., top left panel, top trace; top right panel, top trace) show many characteristic peaks. More specifically, the bands observed for commercially available chitosan are 3355 and 3289 cm for O-H stretching, 2878 cm'1for C-H stretching, 1653 cm'1for Amide I band, 1588 cm'1for Amide II band, 1422 cm'1for CH2 bending and CH3 deformation, 1375 cm'1for symmetric CH3 deformation and CH bending, 1318 cm'1for Amide III band together with CH2 wagging, and finally 1252, 1150, 1068, 1031 cm'1are typical bands associated with saccharide skeletons, corresponding to C-O-C stretching bands. 891 and 661 cm'1were observed and associated with skeletal ring vibrations.

[0143] ATR-FTIR spectrum of Intermediate 1 in FIG. IB (green curve, i.e., top left and right panels, third trace from top; bottom left and right panels, bottom trace) shows some significant differences compared to chitosan. First, the composite band at 3500-300 cm'1appears simplified due to absence of N-H stretching band at 3290 cm'1, suggesting the complete reaction of free -NH2 groups. Because of the addition of the linker dimethoxy acetaldehyde, new bands are now visible at 2950 cm'1, corresponding to O-CH3 stretching. The presence of imine bond is highlighted by the new C=N stretching band at 1741 cm'1. Moreover, a lowering of the intensity of the N-H bending band (Amide II) at 1588 cm'1is also detected.

[0144] 'H-NMR. spectroscopy is a useful technique to confirm the chemical modification of chitosan (FIG. 1C). The biopolymer itself shows definite peaks: 5.26 ppm and 4.96 ppm can be assigned to the protons at the Cl position of D-glucosamine and N-acetyl-D-glucosamine moieties, respectively. Then, peaks in the range 4.51-3.84 ppm group the other protons in positions C2-C6 of both monomers. The peak at 3.56 ppm is associated to the proton at C2 of D-glucosamine. Finally, the singlet at 2.40 ppm refers to the CH3 group of the N-acetyl-D-PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 glucosamine. According to Equation (1), is it possible to determine the degree of deacetylation of chitosan. In particular, the peaks at 5.26 ppm and 2.40 ppm must be integrated and then calculate their ratio according to the following equation: f Hl(1) Degree of Deacetylation (DDA) = - - ■ 100 f Hl + if HAc

[0145] The DDA of commercially available chitosan was 83%, determined by 'H-NMR techniques.

[0146] The 'H-NMR spectrum of Intermediate 1 was acquired on a sample prepared by dialysis of the polymer with bidistilled water and then D O and it is reported in FIG. 1C. Due to tendency of gelification in neutral environments, the sample was purified by slow dialysis against bidistilled water until no residual 2,2-dimethoxyacetalehyde was present in the sample, based on1H-NMR analysis and Fehling test. Once the polymer was purified, it was diluted with D2O and spectra were acquired. The 'H-NMR spectrum (FIG. 1C) revealed the presence of two molecules of acetic acid together with the product, suggesting a stabilizing effect of acidic solvent molecules in preventing gelification of the material. The two acid molecules were responsible of the singlet at 1.83 ppm whose peak integrates for 6 protons. Another important difference from pristine chitosan spectrum is the absence of the proton peak at 5.26 ppm corresponding to the proton at Cl of D-glucosamine moiety, suggesting that, in the Intermediate 1, no residual free -NEE were present, due to complete reaction with dimethoxy acetaldehyde. Technically, the DDA of Intermediate 1 is 0%, since all amino groups of the polymer are present in the form of amides and imines. Further evidence of successful Schiff Base formation is the presence of new peaks in the range 3.87-3.58 ppm due to the methoxy groups in the Intermediate 1 structure. Moreover, according to the integration peaks, in the same region also falls the proton of the acetal group of the linker - CH(OCH3)2. The imine proton of the linker -N=CH-C falls proximal to solvent signal at 4.6 ppm and hence it was not possible to correctly assign it. Presence of those signals were confirmed also with HSQC 'H-13C-NMR spectra with a direct correlation of the13C peak at 74.81 ppm with some proton in the 'H range 3.87-3.58 ppm. See FIG. 2A and FIG. 2B.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0147] Regarding the13C-NMR, other interesting features can be found by comparison of spectra of chitosan and Intermediate 1. In particular, chitosan shows the typical peaks 98.01 ppm; 77.52 ppm; 75.31 ppm; 70.57 ppm; 60.84 ppm; 56.46 ppm, corresponding to Cl, C4, C3, C5, C6, and C2 atoms in the fully deacetylated polymer (only D-glucosamine moieties). Investigation of the13C spectrum of Intermediate 1 shows a new set of peaks: 181.26 ppm and 23.18 ppm from acetic acid molecules; the carbonyl of N-acetyl-D-glucosamine moiety at 174.67 ppm, together with the relative methyl group in N-acetyl pendant at 22.16 ppm; the imine carbon at 143.17 ppm whose proton correlation falls in the solvent peak at 4.6 ppm, as observed from HSQC experiments; a new peak at 100.25 ppm corresponding to methoxy groups of the imine, and finally the -CH(OCH3)2 imine peak at 74.81 ppm. Other peaks are relative to the sugar structure: 100.01 ppm, 77.20 ppm, 72.34 ppm, 60.09 ppm, and 56.34 ppm.

[0148] Synthesis of Intermediate 2

[0149] Intermediate 1, prepared at a concentration of 1% wt / v, was concentrated to 2% by letting evaporate half of the volume of the solution. At this point, the imine bond was reduced to amine bond using NaBFU as a mild reducing agent added in slight excess to the mixture under vigorous mixing at room temperature. The reducing agent, dissolved in a minimal amount of ethanol, was added dropwise to prevent exothermic reactions, and minimize the formation of bubbles. Addition of reducing agent progressively caused a white colloid precipitation from the orange viscous solution, until a complete discoloration was observed after 24 hours of reaction. Then, the white colloid was collected by filtration and was washed several times with distilled water, mixing and dispersing the Intermediate 2 pulp each time and repeating the filtration with Buchner funnel.

[0150] Confirmation of complete reduction was assessed by ATR-FTIR and NMR analyses. The ATR-FTIR spectrum of Intermediate 2, shown in FIG. IB (blue curve, i.e., top left and right panels, bottom trace), compared to the spectrum of Intermediate 1 (green curve; as noted above), had shown a new broad peak at 3290 cm'1due to N-H bond stretching derived from imine reduction. Moreover, a complete disappearance of C=N stretching band at 1741 cm'1PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 was observed. Together with this evidence, the Amide II band at 1586 cm-1increased its intensity, as a result of new amines in the polymer structure.

[0151] 'H-NMR. spectrum in FIG. 1C shows a set of 10H peaks in the range 4.00-3.60 ppm; a singlet at 3.42 ppm integrating for 6 protons corresponding to the two methoxy groups; another singlet at 3.26 ppm corresponds to -CH(OCH3)2, while a broadened peak at 1.97 ppm, integrating for 2H, was assigned to the -N-CH2-C moiety. Other peaks are at 3.08 ppm, integrating for glucosamine residues, and a singlet at 1.99 ppm relative to methyl group on N-acetyl-D-glucosamine moiety.

[0152] 13C-NMR spectrum (FIG. 3), despite the poor quality even after more than 15000 scans due to the very low solubility of Intermediate 2, still had shown some diagnostic peaks. It is present the carbonyl peak of N-Acetyl-D-Glucosamine at 184.82 ppm, together with the - CH3 group at 32.59 ppm; Methoxy groups of the -amino linker gave a peak at 55.83 ppm, while -NH-CH2-C and the -NH-CFF-CHIOCH h fall at 60.01 and 137.71 ppm, respectively.

[0153] Synthesis of Chito-Trehalose

[0154] The final product was synthesized by conjugation of trehalose with Intermediate 2 through transacetalization reaction, in which the two hydroxy groups at positions C4 and C6 of the sugar substituted the two methoxy groups of Intermediate 2.

[0155] A prior method where trehalose is chemically bound to chitosan does so through a series of tedious, long, and low yielding reactions involving harsh chemicals in order to synthesize a cross-linked drug delivery system of a different structure (Iglesias, N., et al., “Biodegradable Double Cross-Linked Chitosan Hydrogels for Drug Delivery: Impact of Chemistry on Rheological and Pharmacological Performance” Int. J. Biol. Macromol. 2020, 165, 2205-2218). According to this reported procedure, a diiodo-derivative of trehalose was prepared involving anhydrous DMF, THF, pyridine, acetic anhydride, methanol, DCM, triphenylphosphine, sodium methoxide, preliminary purification with acidic resins and a final column chromatography, yielding the desired diiodo-trehalose after several days in a 25% yield. The conjugation with chitosan was achieved through stirring the chemicals with citric acid. The final polymer derivative hence was based on a different type of chemical reactionPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 between a trehalose derivative and chitosan, resulting in a structure that is chemically distinct from the presently claimed compounds.

[0156] While trehalose can be conjugated to other molecules and polymers through the reactivity of its hydroxyl groups, known trehalose conjugation has required harsh conditions, such as prolonged exposition to high temperatures (e.g., more than 100°C), toxic chemicals, such as DMF, and long reaction times, all of which conditions are detrimental for natural compounds and biopolymers that irreversibly hydrolyze and decompose.

[0157] The synthesis of Chito-Trehalose derived polymer disclosed herein was achieved with commercially available trehalose in a mild acidic aqueous medium (pH 5-6) at 80 °C overnight, using a slight excess of trehalose compared to reactive sites of Intermediate 2. Since trehalose is a symmetrical molecule with two active positions at carbon C4 / C6 and C47C6’, the final polymer resulted in a double transacetalization with the sugar. After completion, a viscous orange colloid was observed, and it was purified by addition of few drops of concentrated NaOH until pH was raised to 9-10, causing the precipitation of the desired material. After filtration with a Buchner funnel, Chito-Trehalose was resuspended in bidistilled water and thoroughly mixed until a homogeneous suspension was obtained. Material was then recovered via centrifugation at 3000 rpm, removing the upper liquors. The procedure was repeated several times, until the supernatant aqueous phase appeared colorless and both UV-Vis and ATR-FTIR confirmed the absence of residual trehalose.

[0158] The chemical structure of Chito-Trehalose was confirmed with ATR-FTIR and NMR techniques.

[0159] Comparing the ATR-FTIR spectra of Chito-Trehalose with the intermediates (FIG. IB), it is possible to observe a new band at 2920-2910 cm'1for C-H stretching, at 988 cm'1for asymmetric a-l-l-glycosidic bond stretching, and a set of trehalose characteristic bands between 1200 cm'1and 900 cm'1, due to C-O-C bendings.

[0160] An investigation of 'H-NMR. spectrum in FIG. 1C shows two different classes of peaks, according to the chemical structure of Chito-Trehalose. First, the trehalose peaks are clearly distinguishable from the chitosan backbone due to their resolution and coupling constants:PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 256995.24 (d, J= 3.8 Hz, 2H); 3.69 (dd, J = 10.0, 3.8 Hz, 3H); 3.49 (t, J= 9.3 Hz, 2H), and the others in the range 4.10-3.75 ppm. Other peaks are the one at 4.65 (br, 2H), corresponding to the Hl’ proton in the former D-Glucosamine moiety, 3.24 (br. t, 2H), resulting in the presence of both H2 and H2’ protons, 2.12 (s, 2H) due to the -CH3 group of acetyl residue in N-Acetyl-D-Glucosamine. The peak at 4.29 (s, 2H) is imputable to the acetal proton in the bridge (-NH-CH2-CH(OCH3)2, while the broadened peaks at 3.92 and 3.57 are the diasterotopic protons in the bridge (-NH-CH -CH(OCH02.

[0161] Also, considering the integration curves, a total of 40 protons were observed in the Chito- Trehalose spectrum (See FIG. 4A and FIG. 4B). If the ratio between the N-Acetyl- Glucosamine and D-Glucosamine moieties in the polymer backbone is not significantly different from the starting chitosan, as observed from Equation (1) (roughly 1:3), the total amount of protons can be assigned as follows:14H for the D-Glucosamine now bearing the trehalose through the N-linking, 6H for N-Acetyl -D-Glucosamine, 14H for trehalose, and 6H for the N-linker between chitosan and trehalose.

[0162] Regarding the13C-NMR, three different peak clusters were detected (See FIG. 5 A and FIG. 5B). The chitosan backbone (22.04; 55.72; 59.90; 69.92; 74.68; 76.19; 97.39; 168.78), the trehalose sugar (60.42; 69.60; 70.94; 72.04; 72.41; 93.12), and the linker with 47.23 for (- NH-CH2-CH(OCH3)2 and 101.19 for (-NH-CH2-CH(OCH3)2).

[0163] Additionally, the NMR characterization of Chito-Trehalose depicted peak sets that are not repeated and are sharper than the ones of pure chitosan, suggesting that the trehalose transacetalization prevents the folding of the chitosan backbone in globular conformations, imparting a more open web-like structure that facilitates the exposure of polar groups.

[0164] Dynamic Light Scattering (DLS) analyses were also performed on Chito-Trehalose, chitosan, trehalose and the mixture of chitosan and trehalose, as reported in FIG. 6.

[0165] The measurement of the Z-average diameter in solutions is a reliable analysis of the degree of solvation and structural arrangement of macromolecules according to their molecular weight and chemical features. Chitosan’s average hydrodynamic diameters strongly depend on pH, temperature, concentration, degree of deacetylation, and molecularPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 weight. As can be seen in FIG. 6, chitosan (cyan whiskers; i.e., second column for each condition) Z-average diameter progressively decreases with dilution, starting from ca. 1400 nm at the highest concentration, and reaching ca. 350 nm at the lowest concentration, suggesting a macromolecule rearrangement in the medium. In fact, concentrated chitosan solution promotes macromolecule-macromolecule aggregation, due to electrostatic interactions between hydroxy groups and free -NH2 groups that can be partially protonated in the ammonium form, thus causing repulsion between macromolecules (and hence increasing the average aggregate dimensions). Other weak interactions between the polymer chains take place and they are in competition with strong solvent-macromolecule ones. With the increase of solvent molecules (i.e. dilution of macromolecules), the macromolecule-macromolecule interactions are minimized in favor of solvent-macromolecule systems, thus decreasing the Z-average diameter, until the theoretical infinite dilution where only one macromolecule is completely surrounded by solvent molecules.

[0166] Analogously, chitosan and trehalose not chemically bound (FIG. 6, blue whiskers; i.e., third column for each condition) show a similar behavior when the concentration of the solution is progressively decreasing. Contrary to polymers and other molecules with big molecular weight, small molecules often exhibit a much greater mobility, and they strongly interact with solvent molecules instead of other solute molecules. For this reason, trehalose (FIG. 6, gray whiskers; i .e., first column for each condition) does not show any change of the Z-average diameter upon dilution. Interestingly, Chito-Trehalose (FIG. 6, magenta whiskers; i.e., fourth column for each condition) seems not to be dependent on the dilution effect, since it exhibits the same Z-average diameter at any concentration value, from 270 nm at its highest concentration, to 230 nm at highest dilution. This evidence, strongly supporting the different chemical and physical structure of derived polymer compared to chitosan, could be interpreted as the result of insertion of trehalose in the polymer structure, with a considerable increase in highly polar hydroxy groups and simultaneously canceling the aggregation behavior of free -NH2 group in chitosan. The highly polar Chito-Trehalose strongly interacts with polar and small solvent molecules instead of creating macromolecular aggregates with other polymer structures, independently from its concentration.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0167] UV-Vis spectra of the intermediates were acquired, using a 0.1 % wt / v concentration for all the compounds (FIG. 7). For comparison, also pure trehalose and the mixture of trehalose and chitosan were analyzed. As can be seen from FIG. 7, all the curves present a peak at 292 nm corresponding to electronic transition in C-0 bonds n— >7i. In fact, all systems are based on saccharide units and share many common spectroscopic features. The spectrum of Chito- Trehalose (FIG 7, blue (top) curve) also shown a new broad peak at 326 nm. Moreover, despite the same concentration of all samples, Chito-Trehalose exhibited higher absorption intensities compared to other curves. As further evidence of the different chemical nature of Chito-Trehalose, a solution obtained by simply mixing chitosan and trehalose was also analyzed (FIG. 7, purple (third from bottom) curve). As a result, any significant difference between the simple mix and pure chitosan and trehalose could be observed, suggesting that Chito-Trehalose is chemically different from all other intermediates and reagents.

[0168] Exemplary Reaction Protocols

[0169] Intermediate 1 : In a IL beaker, 5.0 g of chitosan was dissolved in 500 mL of AcOH 1% aqueous solution (1 eq, 0.031 mol), obtaining a biopolymer solution concentration of 1.0% wt / v. Working with diluted chitosan solutions is mandatory since higher concentrations will cause gelification of the final material, preventing a complete reaction with the aldehyde. Once the chitosan was completely dissolved, 2,2- dimethoxyacetaldehyde was added to the reaction media (1.5 eq, 0.046 mol, 60 % wt / v, 7.0 mL) and let the reaction occur at 70 °C for 24 h. The solution progressively increased its viscosity and became yellowish. After completion, the solution was let evaporate at 80°C for 24h, in order to remove the residual 2,2-dimethoxyacetaldehyde (b.p. 63.9 °C) and concentrate the derivative to 2% wt / v (final volume 250 mL). For structural investigations, a small portion of the viscous solution was dialyzed against bidistilled water for 2 days until no residual 2,2-dimethoxyacetaldehyde was present in the sample, based on1H-NMR analysis and Fehling test, and then dried for ATR- FTIR analysis or diluted with D2O for NMR analysis. For NMR spectra, two molecules of Acetic Acid were observed in a salt form with intermediate 1 after the dialysis purification. As a consequence, their presence prevented gelification of the product. According to the DDA, the intermediate 1 was collected with a yield of 91% (6.5g) as orange shiny flakes.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0170] Intermediate 2: In a 500 mL beaker, the concentrated 2% intermediate 2 (6.5g) was mixed with 1 eq of NaBH4 referring to the amount of 2,2-dimethoxyacetaldehyde previously added (0.046 mol, 1.74g). The reducing agent was slowly added to the biopolymer viscous solution and vigorously stirred to prevent excessive heating and gas bubbles formation. After complete addition of NaBTU, the mixture was let to proceed at room temperature for 24 hours. At this point, a viscous white colloid was formed, and its precipitation was forced by water evaporation at 80 °C until volume was reduced to 100 mL. The intermediate 2 was collected as a white pulp by filtration and was washed with several amounts of water, mixing thoroughly the polymer each time, and repeating the filtration under vacuum. The final product was dried in oven at 70 °C overnight. According to the DDA the intermediate 2 was collected with a yield of 78% (3.84g) as a white solid. In a large scale experiment, starting with 100g of chitosan, Intermediate 2 was obtained in a yield of 99-100% (quantitative) obtaining 110g of Intermediate 2.

[0171] Chito-Trehalose: In a 100 mL round bottom flask, 3.84 g of the intermediate 2 was added and dissolved under vigorous stirring in 150 mL of distilled water at pH 5.0 due to addition of concentrated HC1. Temperature was increased to 60 °C to promote polymer dissolution. Then, 0.75 eq of trehalose were added, referring to the amount of aldehyde used for the synthesis of intermediate 2 (0.023 mol, 7.68g). Reaction was let to proceed at 80 °C overnight. After completion, the solution appeared as a dark orange viscous liquid. After cooling to room temperature, few drops of concentrated NaOH were added until pH 9-10, thus promoting product precipitation as an orange colloid. The solution was centrifuged at 3000 rpm for 15’ and the supernatant was removed. The crude product was thoroughly washed several times with distilled water, repeating the mixing and the centrifuge steps, until liquors became colorless and at neutral pH. The last liquor was also checked through ATR- FTIR and UV-Vis spectroscopy, confirming the absence of residual trehalose in the final product. According to the DDA, chito-trehalose was collected with a yield of 70 % (3.8g) as a pale orange solid, with an overall yield of 51 % starting from commercially available chitosan. In a large scale experiment, starting with 100g of commercially available chitosan, Chito-Trehalose was obtained in a yield of 96%. In a separate large scale experiment, starting with 100g of chitosan prepared from crustacean shells as described herein, Chito-Trehalose was obtained in a yield of 92%, obtaining 145g of Chito-Trehalose.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699Example 3: Protocol Optimization: Effect of Chitosan on Crops

[0172] Chitosan Solutions and Seed Coating Technique

[0173] The effect of chitosan was investigated on plant vital parameters of several commercially important and food valuable crops: Cicer arietinum (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifalia (sainfoin), and Vicia sativa (common vetch). Among the parameters observed in plants, were studied the germination rates, the root length, plant length, fresh root and plant weight, dry root and plant weight, and chlorophyll content of adult plant leaves.

[0174] First, an optimization protocol was evaluated to find the best conditions for polymer concentration, pH, coating techniques, and the toxicity towards rhizobacteria. Using commercially available chitosan (MMW, Sigma Aldrich) and Rhizobium tropici as a model system, preliminary experiments were carried out.

[0175] Several chitosan solutions in l%v / v CH3COOH were prepared, ranging from 0.25% up to 2.5% wt / v. A slightly acidic medium is required to promote dissolution of chitosan, since protonation of the free -NH2 groups in C2 position causes chain-to-chain repulsion due to Coulomb effects and allows more solvent molecules to enter within the polymer chain, assisting dissolution. While many acidic environments are known (e.g., HC1, HCOOH, and the like), acetic acid was utilized to minimize chemical hydrolysis of glycosidic bonds at pH proximal to chitosan isoelectric point (pH 6.3-6.6).

[0176] Due to progressive increase in viscosity of chitosan solution, systems whose concentration was higher than about 3% wt / v were more difficult to prepare and therefore somewhat less desirable for seed coating processing.

[0177] After the complete dissolution of chitosan in acidic solutions, chickpeas (Cicer arietinum) seeds were processed through coating via simple immersion in chitosan solutions for 5 seconds and then rinsed and let air dry overnight on a cotton grid to allow draining of excess solution. A single coating step was performed for every seed.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0178] As can be seen in FIG 8, Cross-Sectional Scanning Electron Microscopy images were collected to observe the texture and thickness of the coatings.

[0179] As the concentration of chitosan was increased, the coating thickness increased. The thinnest coating was observed for 0.5% wt / v, whereas a non-homogeneous coating of 10-20 pm is visible. At chitosan 1% wt / v, a homogeneous coating of 40-50 pm fully covered the seeds. A 2% wt / v chitosan solution produced a thick and rough coating of 80-100 pm, while the 2.5% wt / v polymer solution gave a dense coating whose thickness could not be clearly determined as the instrument could not focus on the seed surface. Coating thickness was estimated to be more than 100 pm thick. Higher polymer concentrations also tended to cause globular aggregations of microparticles in the coating layer. Once dried, coated seeds of chickpea were sowed in pots and evaluated their germination upon chitosan coating treatment.

[0180] A first coating attempt resulted in very low germination yields due to the acidic coating layer that negatively affected seeds vitality. Indeed, as can be seen in FIG. 9, left panel, by comparison of germination yields (blue (upper) line) with pH of chitosan solutions (orange (lower) lines), overly acidic pH is detrimental to seeds. When pH was increased, germination yields increased also, suggesting that chitosan solutions are preferably buffered to near neutral pH values before applying the coatings, dependent on specific seed preferences.

[0181] A second coating was performed on fresh seeds, using a buffered solution of chitosan in acetic acid. Results are shown in FIG. 9, right panel. Chitosan solutions were buffered with few drops of concentrated NaOH until pH reached a value comprised between 6 and 6.6. It was not possible to reach higher pH values since chitosan precipitated at pH near its isoelectric point (pH ~ 6.5), whereas all -NH3+groups are neutralized and any charge that helps solubilization disappears. As can be seen, chitosan coating has a positive effect on germination yields, raising the value from a control 70% up to 100% for almost all concentrations.

[0182] With buffered chitosan solutions, chickpea seeds were sowed and allowed to grow until they reached a growth limit due to limited occupancy of the pots. Shoot height was measured each day, until day 18. Chitosan exhibits a consistent enhancement on plant growth, with aPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 boost in shoot emergence starting from day 2, whereas water treated seeds sprouted at day 6, as can be observed in FIG. 10. Moreover, despite the wider standard deviation, chitosan also increased the maximum height of the adult plant at day 18. It seems that concentration of chitosan has a minimal effect on germination and shoot growth.

[0183] Regarding all other parameters, in FIG. 10, right panel, are summarized the variations of the plant vital parameters. Chitosan increased almost all the plant parameters (For 1% wt / v, root length +150%, plant weight +150%, shoot length + 120%, shoot fresh weight +200%, shoot dry weight +200%). The roots appeared to be negatively affected by the chitosan coating, since a reduction in both fresh and dry weight was observed upon coating the seed. Among all concentrations tested, 1% wt / v gave strong results in all plant parameters and offered ease of use in the coating process, resulting in a homogeneous coating along the seed surface (see FIG. 8 for SEM images).

[0184] Plants grown with different concentrations of chitosan in seed coating are visually depicted in FIG. 11.

[0185] Effect of chitosan on plant growth: Molecular Weight

[0186] Using a concentration of 1% wt / v of chitosan in buffered CH3COOH solutions, several molecular weights were tested, ranging from the lowest commercially available, namely Ultra-Low-Molecular-Weight (ULMW, 20 KDa) to the highest High-Molecular-Weight (HMW, 850 KDa). As stated for chitosan concentrations, different molecular weights did not significantly affect the growth of plant (FIG. 12).

[0187] Despite the slight differences in plant shoot lengths according to chitosan molecular weights, their major influence is observed for all other plant vital parameters. Indeed, as can be seen in FIG. 12, right panel, Medium Molecular Weight chitosan 1% wt / v coating increased almost all parameters, with a strong rise in aerial districts of shoots both in fresh and dry state.

[0188] Adult plants grown using different molecular weights for chitosan solutions are shown in FIG. 13.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0189] Example 4: Effect of Chito-Trehalose on Plant Growth

[0190] Once the positive effect of chitosan in the seed coating technology of chickpea as a model crop was established, the study was applied to the new synthesized material Chito- Trehalose. As a comparison, a coating based on trehalose, chitosan and trehalose mixed, and a known silk + trehalose coating system (Zvinavashe, A. T., et al., “A Bioinspired Approach to Engineer Seed Microenvironment to Boost Germination and Mitigate Soil Salinity” Proc. Natl. Acad. Sci. 2019, 116 (51), 25555-25561), were tested. Moreover, experiments were also extended to other selected valuable crops. Results are shown in Table 1 (FIG. 14).

[0191] Germination Yield

[0192] Germination represents the most critical parameter for a crop development, since low germination rates will reflect inevitably also poor crop gathering. FIG 15A to FIG. 15H show a general trend on the coating used and the relative effect on the germinability of seeds. All seeds show good to high germination yields, exception given for Sesbania seeds, whose starting value was ca. 35%. Chitosan (light blue whiskers; second column for each varietal) greatly increased the germination yields for all the crops: almost quantitative yields for chickpeas, soybean, and sunflower, with a strong increase for the Egyptian riverhemp, raising the yield up to -80%. Trehalose (dark blue whiskers; third column for each varietal), at a concentration of 0.5%wt / v, did not significantly affected the germination yields in the selected crops, showing only a slight increase of 2-3% of germinability compared to water as a reference. The coating composed by chitosan and trehalose mixed in a concentration of both 0.5% wt / v (pink whiskers; fourth column for each varietal) exhibited different effects according to the specific crop. For chickpea, sunflower, and Sesbania, gave better results than water and trehalose, but for com and soybean lowered the yields. Wheat and barley seemed not to be affected by the coating. For all the crops, the combination of chitosan and trehalose solutions resulted in a consistent lowering of the beneficial role of chitosan alone in the seed coating. Regarding the use of silk fibroin and trehalose mixed (orange whiskers; fifth column for each varietal), seeds increased their germination rates in chickpeas, wheat, barley, corn, soybean, and sunflower, while for Sesbania it was observed a lowering of the yields down to -20%. In all cases, silk + trehalose coating did not perform better thanPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 chitosan coating. Finally, Chito-Trehalose coating (brown whiskers; sixth column for each varietal) greatly improved the yields, with similar effects of chitosan for wheat, com, and sunflowers. For barley and Sesbania, the new derived polymer coating outperformed chitosan. Chickpea and soybean were positively affected by Chito-Trehalose, but not as much as for chitosan.

[0193] Plant Weight

[0194] Total plant weight was evaluated, as shown in FIG 15E. As a general observation, trehalose (dark blue whiskers; third column for each varietal) negatively affected all the crops, exception given for soybean, whereas a small weight increase was recorded. Chitosan coating (light blue whiskers; second column for each varietal) shown instead an increase of the total plant biomass, especially for wheat and corn. Regarding sunflower and Sesbania, chitosan coating limited the plant growth, lowering the final biomass production. Chitosan+ trehalose (pink whiskers; fourth column for each varietal) did not generally improve the plant development, as observed for chickpea, sunflower, and Sesbania, where the plant weight was inferior to the water treatment. Soybean, wheat, and barley increased their weight in presence of chitosan+ trehalose. Once again, for plant weight, the combination of chitosan and trehalose strongly limited the positive effect of chitosan. For silk + trehalose (orange whiskers; fifth column for each varietal) negative effects were observed for chickpea, soybean, barley, sunflower, while only small increases were present for wheat, corn, and Sesbania. However, the Chito-Trehalose coating (brown whiskers; sixth column for each varietal) strongly affected the plant development, increasing the biomass of chickpea (from -2800 mg to 4200 mg), soybean (from 1500 mg to 3400 mg), wheat (from 320 mg to 640 mg), barley (from 400 mg to 550 mg), com (from 4500 mg to 11000 mg), and Sesbania (from 22500 mg to 25500 mg). The only negative effect was observed for sunflower, with a lowering of the biomass from 17000 mg to 16000 mg. Apparently, sunflower did not well tolerate the coating materials for the plant development, giving best results when the seeds were treated only with water.

[0195] Shoot HeightPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0196] Shoot length was recorded on plants developed from coated seeds, and results are displayed in FIG 15 A. Trehalose (dark blue whiskers; third column for each varietal) had only shown non-significant or negative effects; results were detrimental for wheat, barley, soybean, and sunflower. A positive effect was observed only for com. Chitosan (light blue whiskers; second column for each varietal) generally outperformed all the coatings, but for sunflower it was not the best option for shoot development. Chito+trehalose (pink whiskers; fourth column for each varietal) had different effects depending on the crop investigated, showing good results for chickpea, barley, Sesbania, com, and sunflower, but being detrimental for wheat (shoot reduction from 27 cm to 20 cm) and soybean. Silk + trehalose (orange whiskers; fifth column for each varietal) moderately affected the shoot height, but for barley, soybean and sunflower, a decrease in shoot length was recorded. Chito-Trehalose (brown whiskers; sixth column for each varietal) had shown a positive effect on all the crops, overcoming the results observed for chitosan in chickpea, wheat, barley, Sesbania, and sunflower.

[0197] Fresh and Dry Shoot Weight

[0198] The weight of fresh and dry biomass is an important parameter to evaluate the tissue development in crops. Coatings on seeds also affected the weight of the aerial parts of the plant, whose results are reported in FIG. 15B (fresh) and FIG. 15C (dry). Regarding the fresh weight, trehalose (blue whiskers; third column for each varietal) had shown a negative effect for all the crops, especially in sunflower where the fresh weight decreased for 12000 mg to 8200 mg. Chitosan (light blue whiskers; second column for each varietal) had beneficial effects on almost all crops, even if it did not exhibit the highest performances. For sunflower, chitosan had a negative effect comparable to the one observed for trehalose, while in Sesbania chitosan had shown the worst results, lowering the fresh biomass from 15000 mg to 9500 mg. chitosan+ trehalose (pink whiskers; fourth column for each varietal) only has a moderate increase for barley and com, but considerably lowered the fresh biomass in sunflowers. Silk + trehalose (orange whiskers; fifth column for each varietal) had shown similar results for chitosan+ trehalose, being ineffective in most of the crops or lowering the fresh biomass, as observed for sunflower. Finally, Chito-Trehalose (brown whiskers; sixth column for each varietal) outperformed all the coatings, increasing the fresh weight in all thePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 crops, with strongest effects on corn (from 3700 mg to 8200 mg) and Sesbania (from 15000mg to 17400 mg). Once again, sunflower only tolerated water as a seed treatment, since all the coatings tested lowered the fresh biomass, exception given for Chito-Trehalose, which mitigated the adverse effect and hence exhibited similar results to the water coating.

[0199] Regarding the dry shoot weight, similar results were observed for trehalose (dark blue whiskers; third column for each varietal), that in all the cases gave negative effects. Chitosan (light blue whiskers; second column for each varietal) had shown the best performance for soybean and wheat, but results were detrimental for Sesbania, sunflower, and ineffective for barley. Chitosan+ trehalose (pink whiskers; fourth column for each varietal) was beneficial only for barley, and sunflower especially, giving the best results for dry shoot weight in this crop. In all the other crops, it had a negative effect. Silk + trehalose (orange whiskers; fifth column for each varietal) did not significantly appear different from trehalose and chitosan+ trehalose, generally lowering the dry biomass of aerial parts for all the crops. The only dry mass increase was observed for corn, whose results were comparable to chitosan. Chito- Trehalose (brown whiskers; sixth column for each varietal) increased the dry biomass of chickpea, Sesbania, barley, and corn, showing the highest values for these crops, but was detrimental for soybean, and almost ineffective for wheat and sunflower.

[0200] Chlorophylls and Carotenoids Content

[0201] The content of chlorophylls and carotenoids is directly related to the plant development, reaching high values for healthy tissues. The effects of the coating on the leaves pigments are not linear and predictable, but overall Chito-Trehalose had shown an increase of pigment production in almost all crops. As can be seen in FIG. 15F, Chlorophyll A (Chi A) content changed sensibly. Chitosan (light blue whiskers; second column for each varietal) lowered the ChlA levels in chickpea (recording the lowest value decrease, from 0.18 ug / mL to 0.11 ug / mL) and in wheat, but greatly increased the ChlA content in corn (reaching the highest value, from 0.18 ug / mL to 0.25 ug / mL), sunflower (from 0.20 ug / mL to 0.22 ug / mL) and Sesbania (from 0.12 ug / mL to 0.18 ug / mL). Barley and soybean were poorly affected by chitosan coating in terms of ChlA content. Trehalose (dark blue whiskers; sixth column for each varietal) only exhibited a negative effect on ChlA on all the crops, being particularlyPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 detrimental for corn (from 0.18 ug / mL to 0.10 ug / mL, the lowest value for this crop) and Sesbania (from 0.12 ug / mL to 0.09 ug / mL, the lowest value also for this crop). Chitosan+ trehalose (pink whiskers; third column for each varietal), similarly to trehalose, only resulted in diminishing the ChlA levels in all the crops, reaching the lowest value for sunflowers (from 0.20 ug / mL to 0.11 ug / mL). Also, Silk + trehalose (orange whiskers; fifth column for each varietal) only exhibited negative effects on ChlA contents in every plant, and for wheat the lowest value was recorded, from 0.20 ug / mL to 0.12 ug / mL. One exception is given for barley and Sesbania, where a small increase of leaf pigment was observed. Chito-Trehalose (brown whiskers; sixth column for each varietal) shown general positive effects, but it lowered the ChlA content in chickpea and soybean. The highest value of ChlA in wheat (from 0.20 ug / mL to 0.23 ug / mL), barley (from 0.13 ug / mL to 0.15 ug / mL), and Sesbania (from 0.12 ug / mL to 0.19 ug / mL) were observed in seed coated with Chito-Trehalose.

[0202] Chlorophyll B (ChlB; FIG. 15G) values shown a higher variability, compared to Chlorophyll A. Hence, small or null variations could be detected in some species. As for ChlA, also for ChlB unpredictable results were obtained. Chitosan (light blue whiskers; second column for each varietal) increased ChlB values in chickpea, barley, corn (with the highest value from 0.08 ug / mL to 0.085 ug / mL), sunflower (highest value, from 0.07 ug / mL to 0.083 ug / mL), and Sesbania (highest value, from 0,065 ug / mL to 0.083 ug / mL). Trehalose (dark blue whiskers; third column for each varietal) resulted in the lowest values for ChlB for all the crops but for barley, whereas it was observed the only increase of ChlB values. It shown the lowest levels for chickpea (from 0.073 ug / mL to 0.057 ug / mL), corn (from 0.08 ug / mL to 0.055 ug / mL), and Sesbania (from 0.065 ug / mL to 0.057 ug / mL). Chitosan+ trehalose (pink whiskers; fourth column for each varietal) had a similar effect of trehalose, being detrimental or just ineffective to most crops. In soybean and Sesbania has been observed the only significant ChlB increase, while ChlB was decreased in chickpea, wheat, and sunflower. Chito-Trehalose (brown whiskers; sixth column for each varietal), as for ChlA and ChlB, greatly increased Cars (carotenoid) content in all the crops, as for chickpea (highest value, 0.023 ug / mL), wheat (highest value, 0.023 ug / mL), barley (highest value, 0.022 ug / mL), corn (highest value, together with Silk + trehalose, 0.023 ug / mL), soybean (highest value, 0.021 ug / mL), sunflower (from 0.03 ug / mL to 0.021 ug / mL), and Sesbania (highest value, from 0.024 ug / mL to 0.09 ug / mL; see FIG 15H). Carotenoids are fundamentalPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 organic molecules involved in the photosynthetic apparatus for light conversion and for their antioxidative properties. The increase of carotenoids in a living vegetable tissue can be associated with an increase in photosynthetic activity and hence in a higher plant development.

[0203] Root Length

[0204] The coating effect on seeds was also evaluated at the root level, whose length was measured and reported in FIG. 16C. Generally, all the coatings slightly negatively affected the length of the roots, but some of them greatly increased their length. Chitosan (light blue whiskers; second column for each varietal) increased the root length in all the crops, giving the best result for chickpea (from 14 cm to 22 cm), but decreasing it for Sesbania. Trehalose (dark blue whiskers; third column for each varietal) exhibited small beneficial effects for barley, corn, and soybean. Chitosan + trehalose (pink whiskers; fourth column for each varietal) increased the root length in barley, com, and soybean, but was detrimental for chickpea, sunflower, and Sesbania. Silk + trehalose (orange whiskers; fifth column for each varietal) had a similar effect to chitosan+ trehalose, with a consistent positive effect only for Sesbania (from 14 cm to 18 cm). Chito-Trehalose (brown whiskers; sixth column for each varietal) increased the root length for all the selected crops, giving the best observed value for wheat (from 12 to 22 cm), barley (from 12 cm to 23 cm), com (from 12 cm to 22 cm), soybean (from 11 cm to 20 cm), and for Sesbania (from 14 cm to 21 cm).

[0205] Fresh and Dry Root Weight

[0206] In FIG. 16A and Fig. 16B are depicted the results of coatings in fresh and dry roots weight. Chitosan (light blue whiskers; second column for each varietal) did not increase the fresh root weight in all the crops, giving positive results only for wheat. For sunflower and Sesbania, a negative effect was observed. For all other crops, no significant variation was observed compared to water. Trehalose (dark blue whiskers; third column for each varietal) only shown negative results, especially for chickpea, barley, sunflower and Sesbania, while in the other crops no significant increase was observed. Chitosan+ trehalose (pink whiskers; fourth column for each varietal) and Silk + trehalose (orange whiskers; fifth column for each varietal) exhibited the same behavior, resulting almost ineffective in the development of thePCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 root fresh biomass, as for corn, wheat, barley, soybean. For Sesbania, a positive effect was recorded, increasing the biomass from 7500 mg to 8500 mg. Chito-Trehalose (brown whiskers; sixth column for each varietal) greatly increased the roots mass in corn (from 750 mg to 2250 mg), wheat (from 80 mg to 250 mg), soybean, and Sesbania. Regarding sunflower, this crop does not tolerate any coating since any material caused a decrease in root fresh weight.

[0207] For dry roots weight (FIG. 16C), chitosan (light blue whiskers; second column for each varietal) has a beneficial effect only for wheat, barley and slightly for soybean, while in all other cases decreased the dry biomass of roots. Trehalose (dark blue whiskers; third column for each varietal) had a detrimental effect on all the crops, with the lowest value recorded for corn (from 110 mg to 60 mg), sunflower (from 320 mg to 150 mg), wheat (from 26 mg to 21 mg), barley (from 14 mg to 10 mg), and Sesbania (from 60 mg to 55 mg). Chitosan+ trehalose (pink whiskers; fourth column for each varietal) was only moderately effective for barley but resulted ineffective of detrimental for other crops. Analogously for Silk + trehalose (orange whiskers; fifth column for each varietal), only a negative effect was recorded, comparable to trehalose coating. Only for wheat and barley was a small positive effect was observed. Chito-Trehalose (brown whiskers; sixth column for each varietal) outperformed all reported values for chickpea, barley, soybean, and Sesbania. The highest increase was observed for corn, whereas roots weight raised from 110 mg to 275 mg.

[0208] Example 5; Effect of Chitosan and Chito-Trehalose on Growth Promoting Bacteria

[0209] Toxicity of Chitosan towards Growth promoting Bacteria

[0210] After evaluating the growth increase of chitosan in chickpea plants, a study was conducted to assess the toxicity of this biopolymer in growth promoting bacteria.

[0211] Preliminary studies demonstrated the effect of chitosan both in concentration and MW towards R. tropici growth (FIG. 17 and FIG. 18). A concentration up to 0.5% wt / v has a beneficial effect on R. tropici growth, especially in the first incubation period between 0 h and 6 h. It seems that chitosan acts as a sugar source for bacteria, promoting their growth and maintaining their vitality during the time.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0212] Ultra-Low Molecular Weight did not influence the growth of bacteria, since colonies counts were similar to the ones obtained with Water experiment. Very Low and Low Molecular Weight have a beneficial effect on bacteria growth especially in the first contact hours (1 h-6h), but for prolonged times they did not affect the growth more than the Water experiment. More specifically, LMW increase bacteria colonies number at 6 h almost thrice compared to Water. MMW and HMW promoted instead R. tropici growth a prolonged time (24h and 48h). Molecular weight appears to have a crucial role in bacteria growth, showing a fast-promoting effect for lighter polymers, while a long-term positive effect was observed for longer and heavier polymer chains. Bacteria may use chitosan as a source of sugar to grow, and hydrolysis of chitosan is a length-driven polymer hydrolysis process. Hence, shorter chains are converted faster than longer chains, and bacteria grow faster in the first stages only for VLMW and LMW. Higher chains require more time to be digested, and thus bacteria show a maximum growth at late incubation times.

[0213] Toxicity of Chito-Trehalose Toward Growth Promoting Bacteria

[0214] The beneficial effect of chitosan and its derivative Chito-Trehalose was evaluated both in liquid and solid state for growth and preservation of selected nitrogen fixing bacteria, as generally depicted in FIG. 19A. Since coating of the seed relies on a simple and effective technology in promoting and protecting the first growing stage of plants, it is important that coating materials do not negatively affect the rhizosphere environment. Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, Bradyrhizobium japonicum, and Pseudomonas fluorescens were subjected to solutions and dry films of preserving materials. Those bacteria are fundamental in the proper plant development since a symbiosis is maintained with the roots of the plant. R. tropici has been naturally observed as a nitrogen fixing strain for Cicer arietinum (chickpea); A. caulinodans is a versatile strain originally observed in Sesbania sesban (Egyptian riverhemp); K. variicola is observed in the growth of Zea mays (com) and B. japonicum is the natural host of Glycine max roots (soybean).

[0215] A first trial was performed in liquid state, to evaluate the direct toxicity of coating materials towards selected bacteria strains, observing the number of colonies over time, without any other source of nutrients. As a reference, bacteria were also grown in distilledPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 water, chitosan, trehalose, chitosan and trehalose mixed together, and the final coating material Chito-Trehalose. All testing solutions were used at a concentration of 0.5% wt / v and buffered at pH 6.2-6.4. Colonies countings were collected at time 0, Ih, 6h, 24h, and 48h. Results are reported in FIG. 19B to FIG. 19E, top panels.

[0216] As can be observed from in FIG. 19B, top panel, R. tropici population decreased over time when using distilled water, as a natural consequence of lack in nutrients in the growth medium. Chitosan shown a beneficial effect on R. tropici growth, especially in the first stages after Ih and 6h. Chitosan does not preserve population over longer times. Trehalose, at the concentration of 0.5% wt / v, did not significantly affect bacteria, as observed comparing trehalose growth with the one in water. The mixture of chitosan with trehalose negatively affected R. tropici, especially after 6h, perhaps due to some interaction between polymer chains and sugar. Chito-Trehalose demonstrated the best results, able to promote the growth of R. tropici, in the short to medium term, but also preserving it even after 48h of incubation, strongly suggesting a positive effect of Chito-Trehalose in coating techniques for the Rhizosphere environments.

[0217] The chitosan coating had demonstrated its best results with Azorhizobium caulinodans. As can be seen in in FIG. 19C, top panel, chitosan increased the growth within the first period, but was ineffective for prolonged time periods. Trehalose did not significantly affect the growth, and chitosan together with trehalose seemed to limit the positive effects of chitosan. Chito-Trehalose strongly affected the growth and preservation of A caulinodans, especially for prolonged times, up to 48h, showing the best results after 24 hours of incubation.

[0218] Regarding B. japonicum, the bacteria did not well tolerate chitosan, as the growth was reduced after Ih of contact (FIG. 19E, top panel). Trehalose did not significantly affect the bacteria growth, being the same of water conditions. Analogously to chitosan, also chitosan+ trehalose had a small detrimental effect, while Chito-Trehalose prolonged the bacteria viability on a long period of time, compared to just water treatment.

[0219] Contrary to A. tropici, A. caulinodans and B. japonicum, chitosan-based coating materials were not suitable for promoting the growth of Klebsiella variicola. As can be seen in FIG.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 2569919D, top panel, chitosan itself completely inhibited the growth of K. variicola within the first hour, showing an extremely high toxicity towards the bacteria. Trehalose did not significantly affect the growth since no variation was observed compared to water. Similar to chitosan, chitosan and trehalose together were not able to preserve and promote K. variicola population. Chito-Trehalose, although it did not exhibit positive effects as observed for R. Tropici, A. caulinodans and B. japonicum, still preserved the bacteria population, especially comparing the effect of pristine chitosan, whose toxicity was observed almost instantaneously.

[0220] Preservation of Rhizobacteria in Solid Coatings

[0221] Once the beneficial role of Chito-Trehalose on liquid phase was established for the four strains of rhizobacteria, the role of the coating material was extended even to the solid phase, embedding cells into dry films, and evaluating their viability over time. Results are reported in FIG. 19B to FIG. 19E, bottom panels. As references, bare bacteria (water), chitosan coating, and Bombyx mori silk fibroin and trehalose were tested to study the positive effect of Chito-Trehalose among all other systems.

[0222] The common feature is the preserving effect of Chito-Trehalose for all varietals, as even after 28 days this coating demonstrated the best results among all other materials, with the best performance observed for R. tropici (FIG. 19B, bottom panel), compared to all other coatings. Interestingly, K. variicola well tolerated Chito-Trehalose, although its effect in solution was not as strong as, e.g., trehalose (FIG. 19D, bottom panel). As for liquid state, chitosan proved to be the worst coating system for K. variicola in liquid and dry states, demonstrating the lowest preservation among all coatings.

[0223] B. japonicum shown a high tolerance in solid phase viability even in bare conditions without any coating material (FIG. 19E, bottom panel). Nevertheless, with prolonged times, Chito-Trehalose better preserved the bacteria, significantly increasing their shelf-life over time.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0224] Chito-Trehalose resulted in a better viability for all the strains as compared to silk fibroin in liquid or solid phase, and the best among all coatings and all varietals in the solid phase by a very significant and meaningful margin.

[0225] The interaction between coatings and bacteria

[0226] It is not clear the effect of chitosan on bacteria growth and how the biopolymer could affect the population, especially because literature data often claim the toxicity of the biopolymer towards many microorganisms, as observed in this study for K. variicola.

[0227] Not to be bound by theory, but some chitosan toxicity may be caused by the acidic equilibria of the free amino group of the D-glucosamine moiety and, secondly, to its molecular weight, with the highest antimicrobial activity for shorter chains. This experimental evidence could explain a positive effect of Chito-Trehalose, compared to chitosan, on the preservation of all the bacteria strains, since the free-amino moieties are masked with the trehalose. However, such a hypothesis does not explain the increase of growth rate of bacteria - the microbial cells may use the material as a nutrient source that hydrolyzes over time.

[0228] To better understand the effects of chitosan and Chito-Trehalose, the culture solutions of R. tropici were examined through 'H-NMR spectroscopy after 48h of bacteria growth, to investigate any possible change in the polymer structure, whose stacked spectra are reported in FIG. 1C. As can be seen from comparison of 'H-NMR spectra, after 48 hours of incubation with R. tropici, the bare biopolymer chitosan was completely digested, since any residual peak could be detected in the final solutions. The only peak observed was the singlet of the methyl group of acetic acid, suggesting a digestion procedure involving chemical hydrolysis of chitosan into final acetic acid molecules. Hence, the biopolymer could be interpreted as a useful source of carbon and other elements for the correct growth of bacteria. The complete digestion of chitosan could be also responsible for the loss of population for all the strains after 24h or 48h, since no more nutrients source is available into the solution.

[0229] More interesting appears the 'H-NMR spectrum of Chito-Trehalose after 48 hours of digestion by R. tropici cultures. First, it can be observed that peaks of trehalose disappearedPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699(marked as * in the Chito-Trehalose spectrum), suggesting that trehalose is the first part of the derived biopolymer to be digested and used as a source of nutrients. Second, the partially hydrolyzed material has a chemical structure different from all other intermediates or pristine chitosan. Then, Integration of peaks in the region 4.00-3.30 ppm reveals a total of 10H, whereas other intermediates shown at least more than 6H signals due to the -OCH3 groups previously present in the N-linker. Since structural changes are minimal, it is not possible to determine the new structure only from1H-NMR. A deeper investigation of the digested product using13C-NMR was conducted, and interesting results follow (see FIG. 20): a peak at 173.50 ppm and 22.17 ppm confirmed the presence of the N-Acetyl residue in N-Acetyl- D-Glucosamine moiety in the final polymer structure. Then, peaks for skeletal sugar backbone were observed at 100.48, 77.25, 74.81, 72.50, 60.16, 56.42 ppm. More interestingly, further investigation on peaks at 60.16 ppm and 74.81 ppm shows a broadening compared to other structural peaks, suggesting that those two peaks are the result of more than one chemically similar carbon. Looking at the structure of the chitosan derivatives, the peak at 60.16 ppm corresponds to the C2 carbon, close to the N atom, slightly changing in its chemical shift according to the polymer structure (56.46 ppm for chitosan; 56.34 ppm for Intermediate 1; 55.83 ppm for Intermediate 2, together with the -CH2- proximal to N on the linker at 60.01 ppm; 55.72 ppm for Chito-Trehalose). This C should be then chemically analogous to C2 in D-Glucosamine moiety, considering also the chemical environment constituted by another C atom linked to -OH (C3 in D-Glucosamine). Applying a similar observation for the peak at 74.81 ppm, it could be pointed out that this signal corresponds to C6 in D-Glucosamine, with a CH2-OH pendant. Moreover, analysis of the HSQC spectra of Digested Chito-Trehalose (FIG. 21), reveals that13C peaks at 60.16 ppm and 74.81 ppm have a broad correspondence with 'H peaks, as a result of two close carbon atoms for each signal that couple with two sets of H atoms, thus confirming the presence of two additional C atoms chemically similar to the peaks at 60.16 ppm and 74.81 ppm. All together this information, also comparing the integration peaks in 'H-NMR, suggest that the final structure of the digested Chito-Trehalose could be a chitosan bearing an ethyl alcohol linked to the amine of D-glucosamine (-NH-CH2-CH2-OH) instead of the trehalose acetal.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0230] It is not clear the biochemical process involved in the polymer digestion, but the final structure of primary alcohol could be originated by the hydrolysis of the trehalose acetal, giving the aldehyde as a transient species, reduced finally into the primary -OH.

[0231] Bacteria Preservation and Root Nodules Formation in Plants

[0232] As a final application of Chito-Trehalose efficient coating, the ability of R. tropici, A. caulinodans, K. variicola, and B. japonicum to form nodules on the roots of chickpea, Egyptian riverhemp, and corn, and soybean, respectively, was evaluated using fluorescent strains to make them visible under optical microscope. F or B. japonicum, visual inspection was sufficient to observe nodules in soybean roots. After preparing the Chito-Trehalose solutions with dispersion of bacteria pellets, seeds of selected plants were dip-coated with the polymer-bacteria solution, let air dry overnight, and sowed in soil pots. Polarized optical microscopy techniques confirmed the inoculation of bacteria and small nodules on roots: R. tropici nodules on chickpea, A. caulinodans nodules on Egyptian riverhemp, K. variicola nodules on com (FIG. 16D). The apparent absence of macroscopic nodules was imputed to the N-rich soil where seeds were let grow and develop, that inhibited the growth of big nodules. Nevertheless, small fluorescent nodules were observed within the first 3-5 cm of root apparatus, commonly known as “root crown.” The exclusive allocation of nodules in the first section of roots is a strong suggestion that rhizobacteria nodules were formed by the bacteria contained into the seed coating material, instead of other strains naturally present in the soil.

[0233] Example 6: Salinity Stress Range and Plant Tolerance

[0234] The first salinity experiment was conducted in terms of seeds emergence and plant tolerance, using barley, chickpea, com, and soybean as model crops. Seeds were germinated in petri dishes under growing NaCl concentrations, from 0 mM (distilled water) to 400 mM, thus working in a wide range of salt stress. After day 4, seeds were examined and germination, root length and shoot length (if any) were measured, as reported in FIG. 22. As can be seen, for barley an initial germination yield increase was observed for 25 mM NaCl solutions, due to an osmotic effect compared to distilled water (0 mM). A similar positive effect was detected also for root and shoot lengths. Barley shown a salt resistance up to 100PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 mM, where higher salinities negatively affected seed health, with more intense detrimental effects at 300 mM and 400 mM.

[0235] For chickpea, germination yield remained constant up to 75 mM, with an increase in root length at 25 mM solution. In any case it was possible to observe sprout emergence and above 300 mM, no seeds survived the high salt stress.

[0236] Corn, among all crops investigated, showed the highest saline tolerance, with a constant germination yield of 90-100% even at 100 mM. Roots grew bigger in the range 50-100 mM, with a maximum at 75 mM, while shoots were negatively affected starting from 25 mM solutions.

[0237] Similar results were also observed for soybean, with a salt stress tolerance up to 75 mM in germination yield, and a maximum root growth at 50 mM. As for chickpea, no shoots were observed in the experiment timeline.

[0238] The increase in shoot and root lengths in seed emergence at low NaCl concentration can be explained by the osmotic effect of ions dissolved in water. Tap water and irrigation water typically have ionic strengths ranging from 1 to 20 mM, values comparable to NaCl concentration in these experiments. A visual representation of seeds grown in growing saline stress conditions is reported in FIG. 23.

[0239] Example 7: Plant Salt Tolerance in Chickpea

[0240] As observed for preliminary germination experiments, a useful 0-150 mM working range of salinity stress was set and its effect on adult plants was evaluated. Chickpea was used as a model crop and was grown until day 18 in different NaCl treated soils, namely at 0 mM (distilled water), 25 mM, 50 mM, 75 mM, 100 mM, 150 mM. Bottomless pots were used in order to avoid salinity decrease by leaching during watering of soil and a ratio of 1 seed per 200 g of soil was used to minimize NaCl variation over time due to plant tissues absorption. Results are reported in FIG. 24. Values are reported as relative data to normal growing conditions, highlighted as a pointed line with an average value of 100. As can be seen, Germination yield increased up to 110% in 25 mM solutions, due to the osmotic effect of low concentrated ionic solutions. Higher salinities negatively affected the germination ofPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699 chickpea, with a decrease down to 30% at 150 mM. Root length was negatively affected by salinity stress at any concentration, showing a maximum of 95% of the control value at 50 mM. Shoot length increased to 115% at 25 mM and progressively lowered down to 60% of the control value at 150 mM. Plant weights, both shoots and roots, were strongly affected by salinity in the soil. The fresh shoot weight decreased down to 80% of the control value at 25mM, and down to 40% at 150 mM. Fresh root weight showed an interesting trend, decreasing its value to 75 mM, and increasing up to 75% of the control at 150 mM. This effect is due to the NaCl absorption from plant tissues when salinity is high, even if the root apparatus is less developed at higher concentrations. A similar effect was observed also for dry tissues of both shoots and roots, with a decrease until 75 mM, and then an increase at higher NaCl concentration. For dry root tissues, the weight increased to 120% of the control when NaCl 150 mM was used as irrigation water. Chickpea plants phenotype grown in increasing salinity stress is depicted in FIG. 25.

[0241] Example 8: Crops Salinity Tolerance with Different Seed Coatings

[0242] Once the effects on plant development of increasing salinity up to 150 mM were evaluated, a threshold value of 150 mM was set to determine if seed coatings could impart a partial salt stress resistance to crops. Barley, chickpea, corn, and soybean were used as model crops, and the following seed coatings were used: water (no treatment), trehalose 0.5%wt / v, chitosan MMW 0.5%wt / v, chito-trehalose 0.5%wt / v. All coating solutions were buffered at pH 6.3-6.6 and seed coating was performed as described elsewhere herein.

[0243] For barley (FIG. 26), 150 mM salinity condition negatively affected plant development at all parameters, increasing the weight for both fresh and dry root weight due to NaCl absorption from plant tissues. Chitosan greatly increased plant tolerance to salinity, almost pairing the results obtained without salt stress. In contrast, trehalose did not mitigate the salt stress, only lowering the NaCl absorption from root tissues. Chito-Trehalose greatly increased plant defense to salinity stress, as the plant grew as in normal conditions without salt stress, limiting NaCl absorption at the root tissues. Barley plant phenotype is shown in FIG. 30.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0244] For chickpea (FIG. 27), chitosan offered some protection from high salinity stress, recovering some detrimental effects of NaCl, but not enough to fully recover plant parameters to those in absence of salt. Trehalose only showed negative effect, ever further increasing the plant damage from high salinity. Chito-Trehalose partially mitigated salt stress, and almost recovered the control condition without salt stress, although a decrease in dry root weight was observed. Chickpea plants phenotype is depicted in FIG. 31.

[0245] For corn (FIG. 28), chitosan greatly increased plant parameters in saline conditions, almost recovering the control experiments and mitigating the detrimental effects of NaCl . Trehalose beneficially affected the corn growth, with highest values in root length and fresh root weight. Also, trehalose coating completely recovered the parameters of corn grown in saline stress. Chito-Trehalose outperformed previous coatings, increasing all plant parameters, compared to control and saline conditions. Com plants phenotype is depicted in FIG. 32.

[0246] Soybeans (FIG. 29) are extremely sensitive to salinity stress, as can be seen by the decrease in all plant parameters. When using chitosan as a seed coating in salt conditions, all parameters increased, with a major effect on root apparatus, both in length and fresh and dry weights. In the case of trehalose, a full plant recovery was observed, although slightly less than chitosan coatings. For Chito-Trehalose, the highest protecting effect was observed, with plant parameters recovering to their control conditions and higher root length and fresh root weight. Soybean plant phenotype is shown in FIG. 33.

[0247] Example 9: Root Colonization Under Salinity Stress

[0248] In order to evaluate the protecting effects of Chito-Trehalose under salinity stress for the Rhizobia colonizing the crops root apparatus, a similar experiment to normal root colonization was carried out in salinity stress (150 mM NaCl). Seeds were coated and bacteria were grown as previously reported, as well as crop growing times. More specifically, Rhizobium tropici GFP was used with chickpea, Klebsiella variicola-RFP with barley, and Bradyrhizobium japonicum with soybean. For B. japonicum, the natural blue fluorescence of the bacteria was used to determine their presence at the root surfaces. As can be seen in FIG.PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 2569934, bacteria were preserved in the Chito-Trehalose coating and were able to colonize the crops roots even in highly saline stress conditions.

[0249] Example 10: Experimental Section for Salinity Studies

[0250] Germination yield and salinity level determination

[0251] The working NaCl concentration for soil experiments was established through preliminary germination yields of several seeds in a wide range of salt concentrations, from 0 mM to 400 mM. Together with germination yields, shoots and roots emergences were observed. Experiments were performed on seeds of chickpea (Cicer arietinum), barley (Hordeum vulgare), com (Zea mays), soybean (Glycine max). Seeds were placed in lidded petri dishes with a diameter of 15 cm and three cellulose sheets previously sterilized and imbued with 10 mL of aqueous NaCl, from 0 to 400 mM. For each dish, 15 seeds were germinated and put in a dark incubator for 4 days at 26 °C, checking their germination every day and adding distilled water to maintain constant water content and salt concentration. Each NaCl concentration was tested thrice.

[0252] The highest NaCl concentration at which seeds still germinated was 150 mM for all the crops tested, and hence this value was chosen to further grow adult plants and prepare the soil.

[0253] Soil conditioning

[0254] Once established the optimum NaCl concentration of 150 mM, soil was prepared in order to grow plants in salinity stress. More specifically, soil (Miracle-Gro Moisture Control Potting Soil Mix) was completely dried in oven at 60 °C for three days to remove all water content. The dried soil (5.0 kg) was watered with 2 L of NaCl 150 mM solution and thoroughly mixed to ensure homogenous conditions. Plastic 500 mL glasses without holes were used as pots to prevent NaCl leaching with watering. 400 g of conditioned soil were placed in each pot, and 2 seeds were sown, thus avoiding NaCl variations in the soil due to plant absorption over time. Experiments were repeated four times.

[0255] Example 11: SummaryPCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699

[0256] Overall, Chito-Trehalose demonstrated an enhancing effect of growth of plants in most all the parameters observed, such as increased germination yields, higher shoot heights, longer roots, higher biomass in both fresh and dry matter, and an increase in chlorophylls and carotenoids contents in leaves. A visual depiction of all the parameters investigated is shown in FIG. 35.

[0257] Chito-Trehalose was synthesized using inexpensive, non-toxic, commercially available reagents and mild reaction conditions, resulting in a high yield of product, following simple purification steps. Starting from chitosan, the Schiff Base with 2,2-dimethoxyacetaldehyde was prepared in aqueous media under slight heating. The excess of aldehyde was removed through evaporation exploiting the chemical low boiling point (b.p. 63.9°C), giving the pure Intermediate 1 in solution. The same solution was directly used for the next step, involving a chemical reduction of the imine bond to amine using NaBF at room temperature, affording Intermediate 2. The compound was then precipitated by raising the pH up to 8-9 and then recovered by filtration and washing it with water. Chito-Trehalose was prepared redissolving Intermediate 2 in water, together with trehalose in a slightly acidic media to promote the trans-acetalization reaction. As for the Intermediate 2, Chito-Trehalose was collected and purified by precipitation in alkaline media and then filtration steps after washing it with distilled water.

[0258] Chitosan, the intermediates, and Chito-Trehalose structures were fully characterized by ATR-FTIR, UV-Vis spectroscopies and mono- and bi-dimensional NMR techniques. Chito- Trehalose had shown a beneficial effect at all the plant parameters and its development stages, such as increased germination yields, higher shoots, higher fresh and dry weight of aerial parts and roots, length of the roots, and increased chlorophyll and carotenoid contents in fresh leaves. Several commercially and alimentary valuable crops were investigated: Cicer arietimim (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifalia (sainfoin), and Vida sativa (common vetch). Together with the selected crops, Chito- Trehalose had shown a growth promoting ability in solution and preservation for at least 28 days in solid phase for the symbiotic nitrogen-fixing bacteria Rhizobium tropici,PCT / US25 / 40476 04 August 2025 (04.08.2025)24-0414-WO 25699Azorhizobium caulinodans, Klebsiella variicola, B. japonicum, and Pseudomonas fluorescens.

[0259] Moreover, it was observed that bacteria can actively interact with the polymers, digesting them over time. Chitosan was completely converted into acetic acid, while Chito-Trehalose was transformed into a new derived polymer based on a hydroxy-ethyl pendant on the -NH2 group of chitosan skeleton, thus suggesting a nutritional effect for rhizosphere bacteria.

[0260] Finally, fluorescent strain of the above mentioned rhizobacteria were used to evaluate and demonstrate the ability of the microorganisms to form nodules on the roots of the plants in presence of the Chito-Trehalose coating material in seeds.

[0261] The Chito-Trehalose coating was also tested for high salinity stress on barley, chickpea, corn, and soybean as model crops, investigating both the tolerance in a wide NaCl range 0- 400 mM, and observing the effect of high salinity on plant growth. Among the seed coatings tested, Chito-Trehalose demonstrated the most robust protecting effect on seed emergence and plant growth, with a soil salinity value of 150 mM, mimicking the concentration observed in brackish irrigation water. Chito-Trehalose was also able to protect rhizobia (R. tropici, K. variicola, B. japonicum) embedded in the seed coating in highly saline soil, promoting their development at the root level under salinity stress.

[0262] All citations described herein are incorporated by reference in their entirety.

Claims

25699We Claim:

1. A polymeric compound comprising chitosan polymers and trehalose molecules, wherein the trehalose molecules are bound to deacetylated monomers of the chitosan polymers.

2. The polymeric compound of claim 1, wherein the trehalose molecules are bound to the deacetylated monomers of the chitosan polymers as follows:

3. The polymeric compound of claim 1 of the general formula:C - T - C wherein T is a trehalose molecule and wherein C is a chitosan polymer.

4. The polymeric compound of claim 3 comprising the compound of Formula I:wherein each n and m are independently an integer of 0 or greater; each R is independentlyX is a bond to the amine of a deacetylated monomer of the same or a different chitosan polymeric chain.

5. The polymeric compound of claim 4 wherein n+m within the same polymer chain is 200 to 1600.

6. The polymeric compound of claim 5 wherein n+m is 500 to 1200.

7. The polymeric compound of any one of claims 1-6 having a molecular weight of about 50 kDa to about 400 kDa.

8. The polymeric compound of claim 7 having a molecular weight of about 100 kDa to about 200 kDa.

9. The polymeric compound of any one of claims 1-8 wherein of the total polymer chain monomers, less than 35% are N-acetyl glucosamine monomers.

10. The polymeric compound of claim 9 wherein of the total polymer chain monomers, less than 5% are N-acetyl glucosamine monomers.

11. The polymeric compound of any one of claims 1-10 wherein the polymer chains are crosslinked.

12. The polymeric compound of any one of claims 1-11 wherein the ratio of monomers of the chitosan polymers to the trehalose molecules is about 5:3 to about 10: 1,13. A method of synthesizing a polymeric compound of Formula I(Formula I) wherein each n and m are independently an integer of 0 or greater;25699 each R is independentlyX is a bond to the amine of a deacetylated monomer of the same or a different chitosan polymeric chain, comprising: a) starting with chitosan, reacting the free amino groups with dimethoxy acetaldehyde to form a Schiff’s base as a first intermediate; b) reducing the first intermediate to form an amine as a second intermediate; and c) performing a transacetylation reaction between trehalose and the second intermediate to form the compound of Formula I.

14. The polymeric compound prepared by the method of claim 13.

15. A composition for seed coating comprising the polymeric compound of any one of claims 1-12 and 14 dissolved in an acidic solution.

16. The composition of claim 15 buffered to a pH of about 6.0 to about 6.6.

17. The composition of claim 15 wherein the acidic solution comprises acetic acid.

18. The composition of any one of claims 15-17 wherein the polymeric compound is present at a concentration of about 3% wt / v or less.

19. The composition of claim 18 wherein the polymeric compound is present at a concentration of about 1.0% wt / v.

20. The composition of claim 18 wherein the polymeric compound is present at a concentration of about 0.5% wt / v.

21. A method of seed coating comprising a) submerging seeds in a composition according to any one of claims 15-20 for 2-20 seconds; b) allowing the seeds to air dry for 20-30 hours.

22. The method according to claim 21 wherein the seeds are submerged in the composition for about 5 seconds.

23. The method according to any one of claims 21-22 wherein the seeds are allowed to air dry for around 24 hours.

24. A method of promoting plant development comprising coating plant seeds in a composition according to any one of claims 15-20, germinating the seeds, and allowing the plants to grow.

25. The method according to claim 24 wherein the coating thickness on the seeds is about 10 pm to about 100 pm.

26. The method according to claim 25 wherein the coating thickness is about 10 pm to about 20 pm.

27. The method according to claim 25 wherein the coating thickness is about 40 pm to about 50 pm.

28. The method according to claim 25 wherein the coating thickness is about 80 pm to about 50 pm.

29. The method according to any one of claims 24-28 wherein the plant is Cicer arietinum (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifolia (sainfoin), or Vicia sativa (common vetch).

30. A method of promoting bacterial growth of growth promoting bacteria on a plant comprising coating plant seeds with a bacterial composition comprising the composition according to any one of claims 15-20 and a growth promoting bacteria, germinating the seeds, and allowing the plant to grow.

31. The method of claim 30 wherein the growth promoting bacteria is a nitrogen fixing bacteria.

32. The method according to claim 30 wherein the growth promoting bacteria is Rhizobium tropici, Azorhizobium caulinodans, Klebsiella variicola, Bradyrhizobium japonicum, Pseudomonas fluorescens, or a mixture thereof.

33. The method of any one of claims 30-32 wherein the growth promoting bacteria form nodules on the roots of the adult plants.

34. The method of any one of claims 30-33 wherein the plant is Cicer arietinum (chickpea), Triticum aestivum (wheat), Hordeum vulgare (barley), Zea mays (corn), Glycine max (soybean), Helianthus annuus (sunflower), Sesbania sesban (Egyptian riverhemp), Sinapis alba (yellow mustard), x Triticosecale (triticale), Onobrychis viciifolia (sainfoin), or Vicia sativa (common vetch).

35. The method of any one of claims 24-29, wherein the promoting plant development occurs under salinity stress conditions comprising NaCl concentrations between about 25 mM to about 300 mM, between about 25 mM and about 250 mM, or between about 25 mM and about 200 mM, or between about 50 mM and about 250 mM.

36. The method of any one of claims 30-34, wherein the promoting bacterial growth occurs under salinity stress conditions comprising NaCl concentrations of between about 25 mM to about 300 mM, between about 25 mM and about 250 mM, or between about 25 mM and about 200 mM, or between about 50 mM and about 250 mM, and wherein bacterial viability and nodulation are maintained.

37. A method of mitigating salinity stress in plants, comprising coating seeds with the composition of any one of claims 15-20, germinating the seeds in soil conditioned with about 150 mM NaCl, and growing the plants, wherein plant parameters are recovered to at least about 60%, at least about 70%, at least about 80%, or at least about 90% of non-saline controls.

38. The method of claim 37, wherein the plants are barley, chickpea, com, or soybean.

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