A method for rooting peach branches without substrate

Abstract

This invention proposes a substrate-free rooting method for peach branches, belonging to the field of tissue culture technology. The lower part of a peach branch is inserted into a rooting hydrogel composition, then removed, with the gel adhering to the lower part of the branch. The branch is then suspended and, under culture conditions, water vapor is sprayed onto the lower part of the branch. Cultivation continues until roots develop at the lower part of the branch. Continued cultivation yields rooted peach branch seedlings. Compared to conventional cuttings, this invention directly utilizes water vapor under culture conditions, eliminating the need for a substrate. It saves labor, money, and space, is easy to control and manage, and exhibits excellent rooting results, facilitating rapid rooting and the quick acquisition of rooted peach branch seedlings, thus possessing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology, specifically to a method for rooting peach branches without a substrate. Background Technology

[0002] Peaches are known as "longevity peaches" and "immortal peaches," and are also called "the number one fruit in the world" due to their delicious flesh. Peach flesh contains protein, fat, carbohydrates, crude fiber, calcium, phosphorus, iron, carotene, vitamin B1, as well as organic acids (mainly malic acid and citric acid), sugars (mainly glucose, fructose, sucrose, and xylose), and volatile oils. Every 100 grams of fresh peach contains 88% water, approximately 0.7 grams of protein, 11 grams of carbohydrates, and only 180 kilojoules of energy. Peaches are suitable for people with hypokalemia and iron-deficiency anemia.

[0003] Peaches have a long history of cultivation in my country and are widely distributed. They offer advantages such as early fruiting, early high yields, and early profits, and are easy to manage and produce high yields. Traditionally, peach seedlings are propagated through grafting, but this method is time-consuming, requires significant investment, and yields low returns. To shorten the seedling time, reduce costs, and lower orchard establishment expenses, cutting propagation has become a popular method for producing self-rooted seedlings. Compared to grafted seedlings, self-rooted seedlings obtained through cutting propagation offer advantages such as stable traits, rapid seedling growth, uniform seedling size, and early high yields. Currently, two common methods for peach tree cutting propagation are hardwood cuttings and softwood cuttings: softwood cuttings are taken from leafy new shoots during the growing season, while hardwood cuttings are taken from lignified one-year-old leafless branches during dormancy. Hardwood cuttings are easier to collect and can even be directly planted in the open ground for orchard establishment, showing promising application prospects. However, propagation by cuttings requires the selection of suitable substrates, such as river sand, vermiculite, and peat moss, and the substrate must be treated, such as by disinfection and proper mixing, to ensure its aeration, water retention, and nutrient supply properties. This process is quite complex. Furthermore, high-quality propagation substrates, such as imported peat moss, are expensive, and the amount of substrate used is large, increasing the cost of propagation.

[0004] Rooting agents are auxin compounds belonging to the plant growth regulator and promoter class. In fruit trees, they maintain apical dominance, induce the transport of assimilates to the fruit, and promote root formation. Currently, a common method of applying hormones is to mix them with water or organic solvents and then apply or spray them onto the target area. However, this method has a drawback: over time, the concentration of hormones and other rooting promoters at the target site gradually decreases, especially noticeable in rainy conditions. This reduces the effectiveness of the hormones and other rooting promoters in layering, resulting in a lower rooting rate and affecting the overall rooting effect. Summary of the Invention

[0005] The purpose of this invention is to propose a substrate-free rooting method for peach branches. Compared with conventional cuttings, this method involves spraying water vapor directly under cultivation conditions, eliminating the need for a substrate. This saves labor, money, and space, and is easy to control and manage. It also results in good rooting performance, facilitates rapid rooting, and allows for the quick acquisition of rooted peach branch seedlings, thus having broad application prospects.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for rooting peach branches without a substrate. The lower part of the peach branch is inserted into a rooting hydrogel composition, then removed. The hydrogel adheres to the lower part of the peach branch, which is then suspended. Under culture conditions, water vapor is sprayed onto the lower part of the peach branch. The branch is cultured until roots develop at the lower part. Culture is continued to obtain rooted peach branch seedlings.

[0008] As a further improvement of the present invention, the cultivation conditions are: temperature of 15-20℃, humidity of 50-60%RH, light of 8-10h / day; the spraying rate of water vapor is 1-2L / h, spraying for 1-2h, resting for 1-2h, the cultivation time is 7-10d, and the continued cultivation time is 3-5d.

[0009] As a further improvement of the present invention, the preparation method of the rooting composition hydrogel is as follows:

[0010] S1. Preparation of the intermediate: Indomethacin anhydride and 3-dimethylaminopropylamine were reacted by stirring, water was added for lysis, and the solvent was removed by vacuum in the organic phase to obtain the intermediate, the structure of which is as follows: ;

[0011] S2. Preparation of rooting agent A: Indolebutyric acid was added to an organic solvent, NHS and EDC were added, the mixture was stirred to activate the agent, an intermediate was added, the reaction was stirred, water was added to liquidize the mixture, the solvent was removed from the organic phase under reduced pressure, and the mixture was recrystallized from acetone to obtain rooting agent A, with the following structure: ;

[0012] S3. Preparation of rooting agent B: Naphthaleneacetic acid was added to an organic solvent, NHS and EDC were added, and the mixture was stirred to activate the agent. An intermediate was added, and the mixture was stirred to react. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The acetone / ethyl acetate mixture was recrystallized to obtain rooting agent B, with the following structure: ;

[0013] S4. Preparation of slow-release rooting agent: Rooting agent A and rooting agent B are mixed evenly and added to an organic solvent to obtain an organic phase; gelatin is dissolved in water, added to the organic phase, homogenized, the pH of the homogenate is adjusted to the first pH value, the reaction is stirred, the temperature is lowered, the pH of the solution is adjusted to the second pH value, transglutaminase is added, the reaction is stirred, filtered, washed, and dried to obtain microcapsules, which are added to water, sodium alginate and emulsifier are added, stirred, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, filtered, washed, and dried to obtain a slow-release rooting agent;

[0014] S5. Preparation of modified rooting agent: Add slow-release rooting agent to water, add tannic acid and catalyst, heat and stir to react, filter, wash, and dry to obtain modified rooting agent;

[0015] S6. Preparation of rooting composition hydrogel: Dissolve silk fibroin peptide in water to obtain silk fibroin peptide solution, dissolve chitosan in acid solution to obtain chitosan solution, mix the silk fibroin peptide solution and chitosan solution evenly, add modified rooting agent, add NHS and EDC, stir to react, form gel state, and obtain rooting composition hydrogel.

[0016] As a further improvement of the present invention, the molar ratio of indomethacin anhydride and 3-dimethylaminopropylamine in step S1 is 1:1.1-1.2, and the reaction time is 1-2 hours.

[0017] As a further improvement of the present invention, the molar ratio of indolebutyric acid, intermediate, NHS and EDC in step S2 is 1.1-1.2:1:1.3-1.5:1.2-1.3, the stirring activation time is 20-40 min, and the stirring reaction time is 7-10 h.

[0018] As a further improvement of the present invention, the molar ratio of naphthaleneacetic acid, intermediate, NHS and EDC in step S3 is 1.1-1.2:1:1.3-1.5:1.2-1.3, the stirring activation time is 20-40 min, and the stirring reaction time is 6-8 h.

[0019] As a further improvement of the present invention, the mass ratio of rooting agent A, rooting agent B, gelatin, transglutaminase, sodium alginate and emulsifier in step S4 is 3-5:2-4:5-6:2-3:10-12:0.5-1, the first pH value is 4-4.5, the second pH value is 6.3-6.7, the temperature is lowered to 3-8°C, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

[0020] As a further improvement of the present invention, the mass ratio of the slow-release rooting agent, tannic acid and catalyst in step S5 is 10:5-7:1-2, the catalyst is a Tris-HCl solution with pH=8-9, and the heating and stirring reaction is carried out at a temperature of 45-55°C for 3-5 hours.

[0021] As a further improvement of the present invention, the mass ratio of silk fibroin peptide, chitosan, modified rooting agent, NHS and EDC in step S6 is 6-8:10-12:5-7:3-5:3-5, the stirring reaction time is 5-7 hours, and the acid solution is a 1-3 wt% acetic acid or lactic acid solution.

[0022] This invention further protects a rooting composition hydrogel, the preparation method of which is as follows:

[0023] S1. Preparation of the intermediate: 1 molar equivalent of indomethacin anhydride and 1.1-1.2 molar equivalents of 3-dimethylaminopropylamine were stirred and reacted for 1-2 hours. Water was added for lysis, and the solvent was removed from the organic phase under reduced pressure to obtain the intermediate, the structure of which is as follows: ;

[0024] S2. Preparation of rooting agent A: 1.1-1.2 mol equivalents of indolebutyric acid were added to an organic solvent, along with 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the reaction was stirred for 7-10 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from acetone to obtain rooting agent A, with the following structure: ;

[0025] S3. Preparation of rooting agent B: 1.1-1.2 mol equivalents of naphthaleneacetic acid were added to an organic solvent, followed by 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the mixture was stirred and reacted for 6-8 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The acetone / ethyl acetate mixture was recrystallized to obtain rooting agent B, with the following structure: ;

[0026] S4. Preparation of slow-release rooting agent: Mix 3-5 parts by weight of rooting agent A and 2-4 parts by weight of rooting agent B evenly, add to an organic solvent to obtain an organic phase; dissolve 5-6 parts by weight of gelatin in water, add to the organic phase, homogenize, adjust the pH of the homogenized solution to 4-4.5, stir for 20-40 min, cool to 3-8℃, adjust the pH of the solution to 6.3-6.7, add 2-3 parts by weight of transglutaminase, stir for 2-4 h, filter, wash, dry to obtain microcapsules, add to water, add 10-12 parts by weight of sodium alginate and 0.5-1 parts by weight of emulsifier, stir, emulsify, add 3-5 parts by weight of 2-5 wt% calcium chloride solution, solidify at room temperature for 20-40 min, filter, wash, dry to obtain the slow-release rooting agent;

[0027] S5. Preparation of modified rooting agent: Add 10 parts by weight of slow-release rooting agent to water, add 5-7 parts by weight of tannic acid and 1-2 parts by weight of catalyst, heat to 45-55℃, stir and react for 3-5 hours, filter, wash, and dry to obtain modified rooting agent.

[0028] The catalyst is a Tris-HCl solution with a pH of 8-9;

[0029] S6. Preparation of rooting composition hydrogel: Dissolve 6-8 parts by weight of silk fibroin peptide in water to obtain a silk fibroin peptide solution. Dissolve 10-12 parts by weight of chitosan in 1-3 wt% acetic acid or lactic acid solution to obtain a chitosan solution. Mix the silk fibroin peptide solution and chitosan solution evenly, add 5-7 parts by weight of modified rooting agent, add 3-5 parts by weight of NHS and 3-5 parts by weight of EDC, stir and react for 5-7 hours to form a gel state, and obtain the rooting composition hydrogel.

[0030] The present invention has the following beneficial effects:

[0031] Based on an intermediate, this invention prepares rooting agent A and rooting agent B through the coupling of carboxyl and amino groups. The preparation method is simple, the conditions are mild, and it is easy to realize industrial application. The rooting agent can be absorbed by the roots, stems, leaves, and fruits of plants and then transported to the site of action. It has the effects of accelerating cell division, inducing callus growth, promoting rooting and budding, and fruit enlargement. In particular, it has a significant effect on promoting rooting of peach branches, and has the effect of promoting plant rooting, increasing yield and improving quality. Rooting agent A and rooting agent B have a synergistic effect.

[0032] However, when rooting agents are applied to the target area using ordinary smearing or spraying methods, their concentration at the target area decreases significantly over time or due to rainwater runoff, greatly reducing the efficiency of promoting root growth. This invention addresses this by encapsulating rooting agents A and B using a coagulation method, followed by further surface cross-linking with sodium alginate. The resulting slow-release rooting agent slowly releases its components, prolonging its efficacy and providing a long-lasting promoting effect. However, simply encapsulating the rooting agents still results in significant amounts of the slow-release agent detaching after rainwater runoff, further reducing the efficiency of promoting root growth.

[0033] Therefore, the present invention further modifies the surface of the prepared slow-release rooting agent by coating it with tannic acid. The tannic acid on the surface has good adhesion, which allows the modified rooting agent to adhere well to the surface of peach branches, thereby exerting a long-lasting effect of promoting cell separation and rooting.

[0034] Hydrogels can retain water molecules by binding them in a spatial network, exhibiting an extracellular matrix-like structure. However, pure silk fibroin peptide hydrogels suffer from poor thermal stability and excessively rapid degradation, limiting their applications. This invention blends silk fibroin peptides and chitosan, combining the advantages of both materials. The blend allows for hydrogen bonding or electrostatic interactions with the silk fibroin peptides, improving their performance. Crosslinking with EDC and NHS avoids the adverse effects of introducing aldehyde crosslinking agents on plant growth, resulting in a hydrogel with better thermal stability and a suitable degradation rate. Furthermore, this invention disperses a modified rooting agent within the silk fibroin peptide-chitosan hydrogel. This allows the hydrogel to adhere better to peach branches, further enhancing the adhesion of the rooting agent to the branch surface. This provides a long-lasting effect in promoting cell separation and rooting, while also providing organic matter, nitrogen, and water for root growth, thus promoting rapid rooting.

[0035] Compared with conventional cuttings, this invention directly sprays water vapor under cultivation conditions, without the need for a substrate, saving labor, money, and space. It is also easy to control and manage, and has a good rooting effect, making it easy to achieve rapid rooting and quickly obtain rooted peach branch seedlings, thus having broad application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1This is a photograph of a peach branch rooted seedling in Embodiment 3 of the present invention. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

[0040] Preparation Example 1: Preparation of Rooting Composition Hydrogel

[0041] The method is as follows:

[0042] S1. Preparation of intermediates:

[0043]

[0044] 10 mmol of indomethacin and 11 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 1 h. The mixture was then diluted with water, and the solvent was removed under reduced pressure using the organic phase to obtain the intermediate. ESI-MS calculated value: C 12 H 20 N3O(M+H) + 222.15, measured value: 222.2, yield: 97%.

[0045] NMR results: 1H NMR (300MHz, CDCl3) δ 8.0 (br, 1H), 7.75 (d, J=6.5Hz, 1H), 7.26 (m, 1H), 6.82 (m, 1H), 6.61 (d, J=5.7Hz, 1H), 4.1 (br, 2H), 3.2 (t, 2H), 2.37 (t, 2H), 2.28 (s, 6H), 1.67 (m, 2H).

[0046] S2. Preparation of rooting agent A:

[0047]

[0048] 11 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, along with 13 mmol of NHS and 12 mmol of EDC. The mixture was stirred and activated for 20 min. Then, 10 mmol of the intermediate was added, and the reaction was stirred for 7 h. Water was added for liquefaction, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A. ESI-MS calculated value: C 24 H31 N4O2(M+H) + 407.24, measured value: 407.2, yield: 92%.

[0049] NMR results: 1H NMR (300MHz, CDCl3) δ 10.2 (br, 1H), 8.2 (br, 2H), 7.94 (d, J=6.2Hz, 1H), 7.89 (d, J=6.7Hz, 1H), 7.45 (m, 1H), 7.20 (m, 5H), 6.8 (s, 1H), 3.2 (t, 2H), 2.4–2.45 (m, 4H), 2.25–2.28 (m, 8H), 1.92 (m, 2H), 1.69 (m, 2H).

[0050] S3. Preparation of rooting agent B:

[0051]

[0052] 11 mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, along with 13 mmol of NHS and 12 mmol of EDC. The mixture was stirred and activated for 20 min. Then, 10 mmol of the intermediate was added, and the reaction was stirred for 6 h. The mixture was then liquefied with water, and the solvent was removed under reduced pressure using the organic phase. The resulting acetone / ethyl acetate mixture (1:1 v / v) was recrystallized, filtered, washed, and dried to obtain rooting agent B. ESI-MS calculated value: C 24 H 28 N3O2(M+H) + 390.21, measured value: 390.2, yield: 90%.

[0053] NMR results: 1H NMR (300MHz, CDCl3) δ 7.95-8.1 (m, 3H), 7.77-7.82 (m, 2H), 7.62 (d, J=6.0Hz, 1H), 7.49-7.52 (m, 2H), 7.29-7.32 (m, 2H), 7.18-7.2 (m, 3H), 3.92 (m, 2H), 3.22 (t, 2H), 2.37 (t, 2H), 2.29 (s, 6H), 1.67 (m, 2H).

[0054] S4. Preparation of slow-release rooting agent: Mix 3g of rooting agent A and 2g of rooting agent B evenly, add to 100mL of ethyl acetate to obtain the organic phase; dissolve 5g of gelatin in 200mL of water, add to the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH of the homogenate to 4, stir for 20min, cool to 3℃, adjust the pH of the solution to 6.3, add 2g of transglutaminase, stir for 2h, filter, wash, dry to obtain microcapsules, add to 200mL of water, add 10g of sodium alginate and 0.5g of Tween-80, stir, emulsify at 8000r / min for 15min, add 3g of 2wt% calcium chloride solution dropwise, solidify at room temperature for 20min, filter, wash, dry to obtain the slow-release rooting agent;

[0055] S5. Preparation of modified rooting agent: Add 10g of slow-release rooting agent to 200mL of water, add 5g of tannic acid and 1g of catalyst, heat to 45℃, stir and react for 3h, filter, wash, and dry to obtain modified rooting agent;

[0056] The catalyst is a Tris-HCl solution with pH=8;

[0057] S6. Preparation of rooting composition hydrogel: 6g of silk fibroin peptide was dissolved in 200mL of water to obtain a silk fibroin peptide solution. 10g of chitosan was dissolved in 200mL of 1wt% acetic acid solution to obtain a chitosan solution. The silk fibroin peptide solution and chitosan solution were mixed for 15min. 5g of modified rooting agent, 3g of NHS and 3g of EDC were added. The mixture was stirred and reacted for 5h to form a gel state, thus obtaining the rooting composition hydrogel.

[0058] Preparation Example 2: Preparation of Rooting Composition Hydrogel

[0059] The method is as follows:

[0060] S1. Preparation of intermediate: 10 mmol of indomethacin anhydride and 12 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 2 h. Water was added to dissolve the intermediate, and the solvent was removed under reduced pressure in the organic phase to obtain the intermediate with a yield of 98%.

[0061] S2. Preparation of rooting agent A: 12 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, along with 15 mmol of NHS and 13 mmol of EDC. The mixture was stirred and activated for 40 min. 10 mmol of the intermediate was added, and the mixture was stirred and reacted for 10 h. Water was added to the mixture for liquefaction. The solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A with a yield of 93%.

[0062] S3. Preparation of rooting agent B: 12 ​​mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, along with 15 mmol of NHS and 13 mmol of EDC. The mixture was stirred and activated for 40 min. 10 mmol of the intermediate was added, and the mixture was stirred and reacted for 8 h. Water was added to liquidize the mixture, and the solvent was removed under reduced pressure using the organic phase. The acetone / ethyl acetate mixture (volume ratio 1:1) was recrystallized, filtered, washed, and dried to obtain rooting agent B with a yield of 91%.

[0063] S4. Preparation of slow-release rooting agent: Mix 5g of rooting agent A and 4g of rooting agent B evenly, add to 100mL of ethyl acetate to obtain the organic phase; dissolve 6g of gelatin in 200mL of water, add to the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH of the homogenate to 4.5, stir for 40min, cool to 8℃, adjust the pH of the solution to 6.7, add 3g of transglutaminase, stir for 4h, filter, wash, dry to obtain microcapsules, add to 200mL of water, add 12g of sodium alginate and 1g of Tween-40, stir, emulsify at 8000r / min for 15min, add 5g of 5wt% calcium chloride solution, solidify at room temperature for 40min, filter, wash, dry to obtain the slow-release rooting agent;

[0064] S5. Preparation of modified rooting agent: 10g of slow-release rooting agent was added to 200mL of water, along with 7g of tannic acid and 2g of catalyst. The mixture was heated to 55℃ and stirred for 5h. After filtration, washing, and drying, the modified rooting agent was obtained.

[0065] The catalyst is a Tris-HCl solution with pH=9;

[0066] S6. Preparation of rooting composition hydrogel: Dissolve 8g of silk fibroin peptide in 200mL of water to obtain silk fibroin peptide solution, dissolve 12g of chitosan in 200mL of 3wt% lactic acid solution to obtain chitosan solution, mix the silk fibroin peptide solution and chitosan solution for 15min, add 7g of modified rooting agent, add 5g of NHS and 5g of EDC, stir and react for 7h to form a gel state, and obtain rooting composition hydrogel.

[0067] Preparation Example 3: Preparation of Rooting Composition Hydrogel

[0068] The method is as follows:

[0069] S1. Preparation of intermediate: 10 mmol of indomethacin anhydride and 11.5 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 1.5 h. Water was added to dissolve the intermediate, and the solvent was removed by vacuum in the organic phase to obtain the intermediate with a yield of 98%.

[0070] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, along with 14 mmol of NHS and 12.5 mmol of EDC. The mixture was stirred and activated for 30 min. 10 mmol of the intermediate was added, and the mixture was stirred and reacted for 8 h. Water was added to the mixture for liquefaction. The solvent was removed by vacuum treatment of the organic phase. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A with a yield of 94%.

[0071] S3. Preparation of rooting agent B: 11.5 mmol naphthaleneacetic acid was added to 50 mL ethyl acetate, along with 14 mmol NHS and 12.5 mmol EDC. The mixture was stirred and activated for 30 min. 10 mmol of the intermediate was added, and the mixture was stirred and reacted for 7 h. Water was added to liquidize the mixture, and the solvent was removed under reduced pressure using the organic phase. The mixture was recrystallized from an acetone / ethyl acetate mixture (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B with a yield of 92%.

[0072] S4. Preparation of slow-release rooting agent: Mix 4g of rooting agent A and 3g of rooting agent B evenly, add to 100mL of ethyl acetate to obtain the organic phase; dissolve 5.5g of gelatin in 200mL of water, add to the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH of the homogenate to 4.2, stir for 30min, cool to 5℃, adjust the pH of the solution to 6.5, add 2.5g of transglutaminase, stir for 3h, filter, wash, dry to obtain microcapsules, add to 200mL of water, add 11g of sodium alginate and 0.7g of Tween-85, stir, emulsify at 8000r / min for 15min, add 4g of 3wt% calcium chloride solution dropwise, solidify at room temperature for 30min, filter, wash, dry to obtain the slow-release rooting agent;

[0073] S5. Preparation of modified rooting agent: 10g of slow-release rooting agent was added to 200mL of water, along with 6g of tannic acid and 1.5g of catalyst. The mixture was heated to 50℃ and stirred for 4h. After filtration, washing, and drying, the modified rooting agent was obtained.

[0074] The catalyst is a Tris-HCl solution with pH=8.5;

[0075] S6. Preparation of rooting composition hydrogel: 7g of silk fibroin peptide was dissolved in 200mL of water to obtain a silk fibroin peptide solution. 11g of chitosan was dissolved in 200mL of 2wt% acetic acid solution to obtain a chitosan solution. The silk fibroin peptide solution and chitosan solution were mixed for 15min. 6g of modified rooting agent, 4g of NHS and 4g of EDC were added. The mixture was stirred and reacted for 6h to form a gel state, thus obtaining the rooting composition hydrogel.

[0076] Comparative Preparation Example 1

[0077] The difference from preparation example 3 is that rooting agent A was not added in step S4.

[0078] Specifically as follows:

[0079] S4. Preparation of slow-release rooting agent: 7g of rooting agent B was added to 100mL of ethyl acetate to obtain the organic phase; 5.5g of gelatin was dissolved in 200mL of water, added to the organic phase, homogenized at 20MPa pressure for 20min, the pH of the homogenate was adjusted to 4.2, stirred for 30min, cooled to 5℃, the pH of the solution was adjusted to 6.5, 2.5g of transglutaminase was added, stirred for 3h, filtered, washed, and dried to obtain microcapsules, added to 200mL of water, 11g of sodium alginate and 0.7g of Tween-85, stirred, emulsified at 8000r / min for 15min, 4g of 3wt% calcium chloride solution was added dropwise, solidified at room temperature for 30min, filtered, washed, and dried to obtain the slow-release rooting agent.

[0080] Comparative Preparation Example 2

[0081] The difference compared to Preparation Example 3 is that rooting agent B was not added in step S4.

[0082] Specifically as follows:

[0083] S4. Preparation of slow-release rooting agent: 7g of rooting agent A was added to 100mL of ethyl acetate to obtain an organic phase; 5.5g of gelatin was dissolved in 200mL of water, added to the organic phase, homogenized at 20MPa pressure for 20min, the pH of the homogenate was adjusted to 4.2, stirred for 30min, cooled to 5℃, the pH of the solution was adjusted to 6.5, 2.5g of transglutaminase was added, stirred for 3h, filtered, washed, and dried to obtain microcapsules, added to 200mL of water, 11g of sodium alginate and 0.7g of Tween-85, stirred, emulsified at 8000r / min for 15min, 4g of 3wt% calcium chloride solution was added dropwise, solidified at room temperature for 30min, filtered, washed, and dried to obtain the slow-release rooting agent.

[0084] Comparative preparation example 3

[0085] The difference from preparation example 3 is that step S5 was not performed.

[0086] Specifically as follows:

[0087] S1. Preparation of intermediate: 10 mmol of indomethacin anhydride and 11.5 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 1.5 h. Water was added to dissolve the solvent in the organic phase under reduced pressure to obtain the intermediate.

[0088] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, 14 mmol of NHS and 12.5 mmol of EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol of intermediate was added, and the mixture was stirred and reacted for 8 h. Water was added to liquidize the mixture, the solvent was removed by vacuum in the organic phase, and the mixture was recrystallized from acetone. The mixture was filtered, washed, and dried to obtain rooting agent A.

[0089] S3. Preparation of rooting agent B: 11.5 mmol naphthaleneacetic acid was added to 50 mL ethyl acetate, 14 mmol NHS and 12.5 mmol EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol intermediate was added, and the mixture was stirred and reacted for 7 h. Water was added to lyse the mixture, and the solvent was removed by vacuum treatment of the organic phase. The acetone / ethyl acetate mixture (volume ratio 1:1) was recrystallized, filtered, washed, and dried to obtain rooting agent B.

[0090] S4. Preparation of slow-release rooting agent: Mix 4g of rooting agent A and 3g of rooting agent B evenly, add to 100mL of ethyl acetate to obtain the organic phase; dissolve 5.5g of gelatin in 200mL of water, add to the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH of the homogenate to 4.2, stir for 30min, cool to 5℃, adjust the pH of the solution to 6.5, add 2.5g of transglutaminase, stir for 3h, filter, wash, dry to obtain microcapsules, add to 200mL of water, add 11g of sodium alginate and 0.7g of Tween-85, stir, emulsify at 8000r / min for 15min, add 4g of 3wt% calcium chloride solution dropwise, solidify at room temperature for 30min, filter, wash, dry to obtain the slow-release rooting agent;

[0091] S5. Preparation of rooting composition hydrogel: Dissolve 7g of silk fibroin peptide in 200mL of water to obtain a silk fibroin peptide solution. Dissolve 11g of chitosan in 200mL of 2wt% acetic acid solution to obtain a chitosan solution. Mix the silk fibroin peptide solution and the chitosan solution for 15min. Add 6g of slow-release rooting agent, 4g of NHS and 4g of EDC. Stir and react for 6h to form a gel state and obtain the rooting composition hydrogel.

[0092] Comparative preparation example 4

[0093] The difference compared to preparation example 3 is that steps S4 and S5 were not performed.

[0094] Specifically as follows:

[0095] S1. Preparation of intermediate: 10 mmol of indomethacin anhydride and 11.5 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 1.5 h. Water was added to dissolve the solvent in the organic phase under reduced pressure to obtain the intermediate.

[0096] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, 14 mmol of NHS and 12.5 mmol of EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol of intermediate was added, and the mixture was stirred and reacted for 8 h. Water was added to liquidize the mixture, the solvent was removed by vacuum in the organic phase, and the mixture was recrystallized from acetone. The mixture was filtered, washed, and dried to obtain rooting agent A.

[0097] S3. Preparation of rooting agent B: 11.5 mmol naphthaleneacetic acid was added to 50 mL ethyl acetate, 14 mmol NHS and 12.5 mmol EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol intermediate was added, and the mixture was stirred and reacted for 7 h. Water was added to lyse the mixture, and the solvent was removed by vacuum treatment of the organic phase. The acetone / ethyl acetate mixture (volume ratio 1:1) was recrystallized, filtered, washed, and dried to obtain rooting agent B.

[0098] S4. Preparation of rooting agent composition: Mix 4g of rooting agent A and 3g of rooting agent B for 15min to obtain the rooting agent composition;

[0099] S5. Preparation of rooting composition hydrogel: Dissolve 7g of silk fibroin peptide in 200mL of water to obtain a silk fibroin peptide solution. Dissolve 11g of chitosan in 200mL of 2wt% acetic acid solution to obtain a chitosan solution. Mix the silk fibroin peptide solution and the chitosan solution for 15min. Add 6g of rooting agent composition, 4g of NHS and 4g of EDC. Stir and react for 6h to form a gel state, and obtain the rooting composition hydrogel.

[0100] Comparative preparation example 5

[0101] The difference from preparation example 3 is that step S6 was not performed.

[0102] Specifically as follows:

[0103] S1. Preparation of intermediate: 10 mmol of indomethacin anhydride and 11.5 mmol of 3-dimethylaminopropylamine were added to 50 mL of ethyl acetate and stirred for 1.5 h. Water was added to dissolve the solvent in the organic phase under reduced pressure to obtain the intermediate.

[0104] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, 14 mmol of NHS and 12.5 mmol of EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol of intermediate was added, and the mixture was stirred and reacted for 8 h. Water was added to liquidize the mixture, the solvent was removed by vacuum in the organic phase, and the mixture was recrystallized from acetone. The mixture was filtered, washed, and dried to obtain rooting agent A.

[0105] S3. Preparation of rooting agent B: 11.5 mmol naphthaleneacetic acid was added to 50 mL ethyl acetate, 14 mmol NHS and 12.5 mmol EDC were added, and the mixture was stirred and activated for 30 min. 10 mmol intermediate was added, and the mixture was stirred and reacted for 7 h. Water was added to lyse the mixture, and the solvent was removed by vacuum treatment of the organic phase. The acetone / ethyl acetate mixture (volume ratio 1:1) was recrystallized, filtered, washed, and dried to obtain rooting agent B.

[0106] S4. Preparation of slow-release rooting agent: Mix 4g of rooting agent A and 3g of rooting agent B evenly, add to 100mL of ethyl acetate to obtain the organic phase; dissolve 5.5g of gelatin in 200mL of water, add to the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH of the homogenate to 4.2, stir for 30min, cool to 5℃, adjust the pH of the solution to 6.5, add 2.5g of transglutaminase, stir for 3h, filter, wash, dry to obtain microcapsules, add to 200mL of water, add 11g of sodium alginate and 0.7g of Tween-85, stir, emulsify at 8000r / min for 15min, add 4g of 3wt% calcium chloride solution dropwise, solidify at room temperature for 30min, filter, wash, dry to obtain the slow-release rooting agent;

[0107] S5. Preparation of modified rooting agent: 10g of slow-release rooting agent was added to 200mL of water, along with 6g of tannic acid and 1.5g of catalyst. The mixture was heated to 50℃ and stirred for 4h. After filtration, washing, and drying, the modified rooting agent was obtained.

[0108] The catalyst is a Tris-HCl solution with pH=8.5;

[0109] S6. Preparation of rooting composition: Add 6g of modified rooting agent to 400mL of water and disperse by ultrasonication at 1000W for 15min to obtain the rooting composition.

[0110] Example 1

[0111] A method for rooting peach branches without substrate involves inserting the lower part of a peach branch into a rooting composition hydrogel prepared in Preparation Example 1, removing the branch, and allowing the hydrogel to adhere to the lower part of the branch. The branch is then suspended and, under cultivation conditions, water vapor is sprayed onto the lower part of the branch at a rate of 1 L / h for 1 hour, followed by a 1-hour rest period. This process is repeated for 7 days until roots develop at the lower part of the branch. The cultivation continues for another 3 days to obtain rooted peach branch seedlings. The cultivation conditions are a temperature of 15℃, a humidity of 50% RH, and a light intensity of 10 h / day.

[0112] Example 2

[0113] A method for rooting peach branches without substrate involves inserting the lower part of a peach branch into a rooting composition hydrogel prepared in Preparation Example 2, removing the branch, and allowing the hydrogel to adhere to the lower part of the branch. The branch is then suspended and, under cultivation conditions, water vapor is sprayed onto the lower part of the branch at a rate of 2 L / h for 2 hours, followed by a 2-hour rest period. The branch is then cultivated for 10 days until roots develop at the lower part. Cultivation continues for another 5 days to obtain rooted peach branch seedlings. The cultivation conditions are a temperature of 20°C, a humidity of 60% RH, and a light intensity of 8 hours per day.

[0114] Example 3

[0115] A method for rooting peach branches without a substrate involves inserting the lower part of a peach branch into a rooting composition hydrogel prepared in Example 3, removing the branch, and allowing the gel to adhere to the lower part of the branch. The branch is then suspended and, under culture conditions, water vapor is sprayed onto the lower part of the branch at a rate of 1.5 L / h for 1.5 hours, followed by a 1.5-hour rest period. This process is repeated for 8 days until roots develop at the lower part of the branch. The branch is then cultured for another 4 days to obtain rooted peach branch seedlings. The results are shown in [the original text]. Figure 1 As shown in the figure, this method can clearly achieve rooting culture of peach branches without substrate; the culture conditions are a temperature of 17℃, a humidity of 55%RH, and a light intensity of 9h / day.

[0116] Comparative Example 1

[0117] The difference from Example 3 is that the rooting composition hydrogel was prepared by Comparative Preparation Example 1.

[0118] Comparative Example 2

[0119] The difference from Example 3 is that the rooting composition hydrogel was prepared from Comparative Preparation Example 2.

[0120] Comparative Example 3

[0121] The difference from Example 3 is that the rooting composition hydrogel was prepared by Comparative Preparation Example 3.

[0122] Comparative Example 4

[0123] The difference from Example 3 is that the rooting composition hydrogel was prepared from Comparative Preparation Example 4.

[0124] Comparative Example 5

[0125] The difference from Example 3 is that the rooting composition hydrogel was prepared from Comparative Preparation Example 5.

[0126] Test Example 1

[0127] Ten peach branches of the same variety, of uniform length and taken from the same section of the branch, were used in each group. Rooting culture was performed using the methods described in Examples 1-3 or Comparative Examples 1-5. The control group was prepared using the method described in Example 3, but without being inserted into the hydrogel containing the rooting composition prepared in Example 3. After culture, root parameters were analyzed using a root scanner, and total root length, number of root branches, and average root diameter were quantitatively analyzed using the WinRHIZO root analysis software. Root activity was measured using TTC. The results are shown in Table 1.

[0128] Table 1

[0129]

[0130] Note: * indicates P < 0.05 compared to the control group.

[0131] As can be seen from the table above, the methods in Examples 1-3 of this invention can effectively promote the growth of peach branch roots and increase root vitality.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rooting peach branches without substrate, characterized in that, The lower part of a peach branch is inserted into a rooting hydrogel composition. The branch is then removed, and the gel adheres to the lower part. The branch is then suspended and, under culture conditions, water vapor is sprayed onto the lower part of the branch. Culture continues until roots develop at the lower part of the branch. This process is repeated to obtain rooted peach branch seedlings. The preparation method of the rooting hydrogel composition is as follows: S1. Preparation of the intermediate: 1 molar equivalent of indomethacin anhydride and 1.1-1.2 molar equivalents of 3-dimethylaminopropylamine were stirred and reacted for 1-2 hours. Water was added for lysis, and the solvent was removed by vacuum treatment of the organic phase to obtain the intermediate, the structure of which is as follows: ; S2. Preparation of rooting agent A: 1.1-1.2 mol equivalents of indolebutyric acid were added to an organic solvent, along with 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the reaction was stirred for 7-10 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from acetone to obtain rooting agent A, with the following structure: ; S3. Preparation of rooting agent B: 1.1-1.2 mol equivalents of naphthaleneacetic acid were added to an organic solvent, followed by 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the mixture was stirred and reacted for 6-8 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The acetone / ethyl acetate mixture was recrystallized to obtain rooting agent B, with the following structure: ; S4. Preparation of slow-release rooting agent: Mix 3-5 parts by weight of rooting agent A and 2-4 parts by weight of rooting agent B evenly, add to an organic solvent to obtain an organic phase; dissolve 5-6 parts by weight of gelatin in water, add to the organic phase, homogenize, adjust the pH of the homogenized solution to 4-4.5, stir for 20-40 min, cool to 3-8℃, adjust the pH of the solution to 6.3-6.7, add 2-3 parts by weight of transglutaminase, stir for 2-4 h, filter, wash, dry to obtain microcapsules, add to water, add 10-12 parts by weight of sodium alginate and 0.5-1 parts by weight of emulsifier, stir, emulsify, add 3-5 parts by weight of 2-5 wt% calcium chloride solution, solidify at room temperature for 20-40 min, filter, wash, dry to obtain the slow-release rooting agent; S5. Preparation of modified rooting agent: Add 10 parts by weight of slow-release rooting agent to water, add 5-7 parts by weight of tannic acid and 1-2 parts by weight of catalyst, heat to 45-55℃, stir and react for 3-5 hours, filter, wash, and dry to obtain modified rooting agent. The catalyst is a Tris-HCl solution with a pH of 8-9; S6. Preparation of rooting composition hydrogel: Dissolve 6-8 parts by weight of silk fibroin peptide in water to obtain a silk fibroin peptide solution. Dissolve 10-12 parts by weight of chitosan in 1-3 wt% acetic acid or lactic acid solution to obtain a chitosan solution. Mix the silk fibroin peptide solution and chitosan solution evenly, add 5-7 parts by weight of modified rooting agent, add 3-5 parts by weight of NHS and 3-5 parts by weight of EDC, stir and react for 5-7 hours to form a gel state, and obtain the rooting composition hydrogel.

2. The method for rooting peach branches without substrate according to claim 1, characterized in that, The cultivation conditions are: temperature 15-20℃, humidity 50-60%RH, and light exposure 8-10h / day; the spraying rate of water vapor is 1-2L / h, spraying for 1-2h, resting for 1-2h, the cultivation time is 7-10d, and the continued cultivation time is 3-5d.

3. The method for rooting peach branches without substrate according to claim 1, characterized in that, The emulsifier mentioned in step S4 is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85.

4. A rooting composition hydrogel, characterized in that, The preparation method is as follows: S1. Preparation of the intermediate: 1 molar equivalent of indomethacin anhydride and 1.1-1.2 molar equivalents of 3-dimethylaminopropylamine were stirred and reacted for 1-2 hours. Water was added for lysis, and the solvent was removed by vacuum treatment of the organic phase to obtain the intermediate, the structure of which is as follows: ; S2. Preparation of rooting agent A: 1.1-1.2 mol equivalents of indolebutyric acid were added to an organic solvent, along with 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the reaction was stirred for 7-10 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from acetone to obtain rooting agent A, with the following structure: ; S3. Preparation of rooting agent B: 1.1-1.2 mol equivalents of naphthaleneacetic acid were added to an organic solvent, followed by 1.3-1.5 mol equivalents of NHS and 1.2-1.3 mol equivalents of EDC. The mixture was stirred and activated for 20-40 min. Then, 1 mol equivalent of an intermediate was added, and the mixture was stirred and reacted for 6-8 h. Water was added to liquidize the mixture, and the solvent was removed from the organic phase under reduced pressure. The acetone / ethyl acetate mixture was recrystallized to obtain rooting agent B, with the following structure: ; S4. Preparation of slow-release rooting agent: Mix 3-5 parts by weight of rooting agent A and 2-4 parts by weight of rooting agent B evenly, add to an organic solvent to obtain an organic phase; dissolve 5-6 parts by weight of gelatin in water, add to the organic phase, homogenize, adjust the pH of the homogenized solution to 4-4.5, stir for 20-40 min, cool to 3-8℃, adjust the pH of the solution to 6.3-6.7, add 2-3 parts by weight of transglutaminase, stir for 2-4 h, filter, wash, dry to obtain microcapsules, add to water, add 10-12 parts by weight of sodium alginate and 0.5-1 parts by weight of emulsifier, stir, emulsify, add 3-5 parts by weight of 2-5 wt% calcium chloride solution, solidify at room temperature for 20-40 min, filter, wash, dry to obtain the slow-release rooting agent; S5. Preparation of modified rooting agent: Add 10 parts by weight of slow-release rooting agent to water, add 5-7 parts by weight of tannic acid and 1-2 parts by weight of catalyst, heat to 45-55℃, stir and react for 3-5 hours, filter, wash, and dry to obtain modified rooting agent. The catalyst is a Tris-HCl solution with a pH of 8-9; S6. Preparation of rooting composition hydrogel: Dissolve 6-8 parts by weight of silk fibroin peptide in water to obtain a silk fibroin peptide solution. Dissolve 10-12 parts by weight of chitosan in 1-3 wt% acetic acid or lactic acid solution to obtain a chitosan solution. Mix the silk fibroin peptide solution and chitosan solution evenly, add 5-7 parts by weight of modified rooting agent, add 3-5 parts by weight of NHS and 3-5 parts by weight of EDC, stir and react for 5-7 hours to form a gel state, and obtain the rooting composition hydrogel.

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