Matrix-free rooting method for peach branches

Abstract

The invention provides a medium-free rooting method for peach branches, and belongs to the technical field of tissue culture. Inserting the lower part of the peach branch into the rooting composition hydrogel, taking out, attaching the hydrogel to the lower part of the peach branch, suspending, spraying water vapor to the lower part of the peach branch under a culture condition, culturing until the lower part of the peach branch roots, and continuing culturing to obtain a peach branch rooting seedling. Compared with conventional cuttage, the method has the advantages that water vapor is sprayed directly under the culture condition, a substrate is not used, labor, money and space are saved, control and management are convenient, the rooting effect is good, rapid rooting is easy to achieve, peach branch rooting seedlings are obtained rapidly, and the method has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and in particular to a method for rooting peach branches without substrate. Background Art

[0002] Peaches are known as "longevity peaches" and "immortal peaches." Due to their delicious flesh, they are also called "the best fruit in the world." Peach flesh contains protein, fat, carbohydrates, crude fiber, calcium, phosphorus, iron, carotene, vitamin B1, organic acids (primarily malic and citric), sugars (primarily glucose, fructose, sucrose, and xylose), and volatile oils. Every 100 grams of fresh peaches contains 88% water, approximately 0.7 grams of protein, 11 grams of carbohydrates, and only 180 kilojoules of calories. Peaches are suitable for those suffering from hypokalemia and iron deficiency anemia.

[0003] Peach has a long history of cultivation in my country and is widely distributed. Its advantages include early fruiting, high yields, and early returns. It is also easy to manage and can easily yield high yields. Traditionally, peach seedling cultivation has been done through grafting, but this process requires a long cultivation cycle, high investment, and low returns. To shorten seedling cultivation time and reduce costs, including the cost of establishing a garden, cuttings have been used to produce own-rooted seedlings. Compared to grafted seedlings, own-rooted seedlings produced through cuttings offer advantages such as stable traits, rapid seedling establishment, uniform growth, and early and high yields. Currently, two common methods for peach seedling cultivation are hardwood cuttings and softwood cuttings. Softwood cuttings are used during the growing season to take cuttings from leafy new shoots, while hardwood cuttings are used during the dormant season to take cuttings from lignified, leafless, one-year-old branches. Hardwood cuttings are easy to collect cuttings and can even be planted directly in the field for garden establishment, offering promising application prospects. However, seedling cultivation requires selecting a suitable substrate, such as river sand, vermiculite, or peat, and requires treatment, such as disinfection and formulation, to ensure proper air permeability, water retention, and nutrient supply. This process is complex and cumbersome. Furthermore, high-quality cutting substrates, such as imported peat, are expensive, and the large amounts used increase seedling cultivation costs.

[0004] Rooting agents are auxin compounds that belong to the class of plant growth regulators and promoters. Their application in fruit trees is to maintain apical dominance within the plant, induce transport of assimilated products to the fruit, and promote rooting. Currently, the most widely used method involves mixing the hormones with water or an organic solvent and applying or spraying them to the target area. A drawback of this method is that the concentration of hormones and other root-promoting substances in the target area gradually decreases over time. This is particularly evident in rainy fields, which reduces the effectiveness of hormones and other root-promoting substances during layering, resulting in a lower rooting rate and a poor rooting effect. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for rooting peach branches without a substrate. Compared with conventional cuttings, the method directly sprays water vapor under culture conditions without the need for a substrate, saves labor, effort, money, and space, is easy to control, and is easy to manage. The method also has good rooting effect, is easy to achieve rapid rooting, and can quickly obtain rooted peach branch seedlings. The method has broad application prospects.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The invention provides a method for rooting peach branches without a substrate. The method comprises the following steps: inserting the lower part of the peach branch into a hydrogel of a rooting composition, taking the branch out, adhering the gel to the lower part of the peach branch, and hanging the branch. Under culture conditions, water vapor is sprayed on the lower part of the peach branch, and the branch is cultured until the lower part of the peach branch takes root. The cultivation is continued to obtain a rooted peach branch seedling.

[0008] As a further improvement of the present invention, the culture conditions are a temperature of 15-20°C, a humidity of 50-60RH%, and a light intensity of 8-10h / day; the spraying rate of the water vapor is 1-2L / h, the spraying is 1-2h, and the rest is 1-2h. The culture time is 7-10d, and the continued culture 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 intermediate: Isatoic anhydride and 3-dimethylaminopropylamine were stirred and reacted, water was added to separate the organic phase, and the solvent was removed under reduced pressure to obtain an intermediate having the following structure: ;

[0011] S2. Preparation of Rooting Agent A: Indolebutyric acid was added to an organic solvent, followed by NHS and EDC, and the mixture was stirred for activation. The intermediate was added, the mixture was stirred for reaction, and water was added to separate the mixture. The organic phase was decompressed to remove the solvent, and the mixture was recrystallized from acetone to obtain Rooting Agent A, which has the following structure: ;

[0012] S3. Preparation of Rooting Agent B: Naphthaleneacetic acid was added to an organic solvent, followed by NHS and EDC, and the mixture was stirred for activation. The intermediate was added, the mixture was stirred for reaction, and water was added to separate the mixture. The organic phase was decompressed to remove the solvent, and the mixture was recrystallized from an acetone / ethyl acetate mixture to obtain Rooting Agent B. The structure is as follows: ;

[0013] S4. Preparation of a slow-release rooting agent: Rooting agent A and rooting agent B are mixed uniformly and added to an organic solvent to obtain an organic phase; gelatin is dissolved in water, the organic phase is added, homogenized, the pH of the homogenate is adjusted to a first pH value, the reaction is stirred, the temperature is lowered, the pH of the solution is adjusted to a 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 an emulsifier are added, stirred, emulsified, a 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: adding a slow-release rooting agent to water, adding tannic acid and a catalyst, heating and stirring the reaction, filtering, washing, and drying to obtain a modified rooting agent;

[0015] S6. Preparation of a rooting composition hydrogel: dissolving a silk fibroin peptide in water to obtain a silk fibroin peptide solution, dissolving chitosan in an acid solution to obtain a chitosan solution, uniformly mixing the silk fibroin peptide solution and the chitosan solution, adding a modified rooting agent, adding NHS and EDC, and stirring to react to form a gel state to prepare a rooting composition hydrogel.

[0016] As a further improvement of the present invention, the molar ratio of isatoic anhydride to 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 minutes, and the stirring reaction time is 7-10 hours.

[0018] As a further improvement of the present invention, the molar ratio of naphthylacetic 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 minutes, and the stirring reaction time is 6-8 hours.

[0019] As a further improvement of the present invention, the mass ratio of the 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 a pH of 8-9, the temperature of the heating and stirring reaction is 45-55°C, and the time is 3-5h.

[0021] As a further improvement of the present invention, the mass ratio of the 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 5-7h, and the acid solution is 1-3wt% acetic acid or lactic acid solution.

[0022] The present invention further provides a rooting composition hydrogel, the preparation method of which is as follows:

[0023] S1. Preparation of intermediate: 1 molar equivalent of isatoic anhydride and 1.1-1.2 molar equivalents of 3-dimethylaminopropylamine were stirred for 1-2 hours, the mixture was separated by adding water, and the solvent was removed from the organic phase under reduced pressure to obtain an intermediate with the following structure: ;

[0024] S2. Preparation of Rooting Agent A: 1.1-1.2 molar equivalents of indolebutyric acid were added to an organic solvent, followed by 1.3-1.5 molar equivalents of NHS and 1.2-1.3 molar equivalents of EDC. The mixture was stirred and activated for 20-40 minutes. 1 molar equivalent of the intermediate was added and the mixture was stirred for 7-10 hours. Water was added to separate the mixture, and the organic phase was decompressed to remove the solvent. The mixture was recrystallized from acetone to obtain Rooting Agent A, which has the following structure: ;

[0025] S3. Preparation of rooting agent B: 1.1-1.2 molar equivalents of naphthaleneacetic acid were added to an organic solvent, followed by 1.3-1.5 molar equivalents of NHS and 1.2-1.3 molar equivalents of EDC. The mixture was stirred and activated for 20-40 minutes. 1 molar equivalent of the intermediate was added and the mixture was stirred for 6-8 hours. Water was added to separate the mixture, and the organic phase was stripped of solvent under reduced pressure. The mixture was recrystallized from an acetone / ethyl acetate mixture to obtain rooting agent B. The structure is as follows: ;

[0026] S4. Preparation of a slow-release rooting agent: 3-5 parts by weight of a rooting agent A and 2-4 parts by weight of a rooting agent B were mixed uniformly and added to an organic solvent to obtain an organic phase; 5-6 parts by weight of gelatin were dissolved in water, added to the organic phase, homogenized, the pH of the homogenate was adjusted to 4-4.5, stirred for 20-40 min, cooled to 3-8 ° C, the pH of the solution was adjusted to 6.3-6.7, 2-3 parts by weight of transglutaminase was added, stirred for 2-4 h, filtered, washed, and dried to obtain microcapsules, added to water, 10-12 parts by weight of sodium alginate and 0.5-1 parts by weight of an emulsifier were added, stirred, emulsified, 3-5 parts by weight of a 2-5wt% calcium chloride solution was added dropwise, cured at room temperature for 20-40 min, filtered, washed, and dried to obtain a slow-release rooting agent;

[0027] S5. Preparation of a modified rooting agent: 10 parts by weight of a slow-release rooting agent was added to water, 5-7 parts by weight of tannic acid and 1-2 parts by weight of a catalyst were added, heated to 45-55 ° C, stirred for 3-5h, filtered, washed, and dried to obtain a modified rooting agent;

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

[0029] S6. Preparation of a 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 1-3wt% acetic acid or lactic acid solution to obtain a chitosan solution, mix the silk fibroin peptide solution and the chitosan solution evenly, add 5-7 parts by weight of a 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 prepare a rooting composition hydrogel.

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

[0031] The present invention prepares rooting agents A and B based on intermediates by coupling carboxyl groups and amino groups. The preparation method is simple, the conditions are mild, and industrial application is easy to achieve. The rooting agents can be absorbed by the roots, stems, leaves, and fruits of plants and then transported to the active parts. The rooting agents have the effects of accelerating cell division, inducing callus growth, promoting rooting and germination, and fruit enlargement. In particular, the rooting promoting effect on peach branches is obvious, and the rooting of plants is promoted, and the yield and quality are improved. The rooting agents A and B have a synergistic effect.

[0032] However, when a rooting agent is attached to the target area using conventional smearing or spraying methods, the concentration of the rooting agent at the target area will significantly decrease over time or under the influence of rain, thereby greatly reducing the efficiency of promoting rooting. The present invention, by embedding rooting agent A and rooting agent B through a double-coagulation emulsion method and then further embedding the surface through sodium alginate cross-linking, obtains a slow-release rooting agent that can slowly release the rooting agent components, thereby prolonging the efficacy and achieving a long-term promoting effect. However, simply embedding the rooting agent still has the risk of a large amount of slow-release rooting agent falling off after rain, which also reduces the efficiency of promoting rooting.

[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, so that the modified rooting agent can adhere well to the surface of the peach branch, thereby exerting a long-term effect of promoting cell separation and promoting rooting.

[0034] The hydrogel can bind water molecules in a spatial network and retain moisture, and has a structure similar to that of an extracellular matrix. The thermal stability of pure silk fibroin peptide hydrogel is poor and the degradation rate is too fast, which limits its application. The present invention blends silk fibroin peptide and chitosan, integrating the advantages of the two materials, and can form hydrogen bonds or electrostatic interactions with the silk fibroin peptide, thereby improving the performance of the silk fibroin peptide. By cross-linking with EDC and NHS, the adverse effects of introducing aldehyde cross-linking agents on plant growth are avoided. The obtained hydrogel has better thermal stability and a suitable degradation rate. The present invention further disperses the modified rooting agent in the silk fibroin peptide-chitosan hydrogel. The hydrogel can better adhere to the peach branches, thereby further improving the adhesion of the rooting agent on the surface of the peach branches, giving play to the effect of long-term promotion of cell separation and promoting rooting. At the same time, it can also provide nutrients such as organic matter and nitrogen sources for rooting growth and provide water, thereby promoting rapid rooting.

[0035] Compared with conventional cuttings, the present invention sprays water vapor directly under culture conditions without using a substrate, saves labor, effort, money, and space, is easy to control, and is easy to manage. It also has a good rooting effect, is easy to achieve rapid rooting, and can quickly obtain rooted peach seedlings, thus having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1This is a photo of the rooted peach seedlings in Example 3 of the present invention. DETAILED DESCRIPTION

[0038] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts 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] Here’s how:

[0042] S1. Preparation of intermediates:

[0043]

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

[0045] NMR results: 1H NMR (300 MHz, CDCl3) δ8.0 (br, 1H), 7.75 (d, J=6.5 Hz, 1H), 7.26 (m, 1H), 6.82 (m, 1H), 6.61 (d, J=5.7 Hz, 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, followed by 13 mmol of NHS and 12 mmol of EDC, and the mixture was stirred and activated for 20 min. 10 mmol of the intermediate was added and the mixture was stirred and reacted for 7 h. Water was added to separate the mixture, and the organic phase was decompressed to remove the solvent. 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, found: 407.2, yield: 92%.

[0049] NMR results: 1H NMR (300 MHz, CDCl3) δ10.2 (br, 1H), 8.2 (br, 2H), 7.94 (d, J=6.2 Hz, 1H), 7.89 (d, J=6.7 Hz, 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, followed by 13 mmol of NHS and 12 mmol of EDC. The mixture was stirred and activated for 20 min. 10 mmol of the intermediate was added and the mixture was stirred and reacted for 6 h. Water was added to separate the mixture. The organic phase was decompressed to remove the solvent. The mixture was recrystallized from an acetone / ethyl acetate mixed solution (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B. ESI-MS calculated value: C 24 H 28 N3O2(M+H) + 390.21, found: 390.2, yield 90%.

[0053] NMR results: 1H NMR (300 MHz, CDCl3) δ7.95-8.1 (m, 3H), 7.77-7.82 (m, 2H), 7.62 (d, J=6.0 Hz, 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 a slow-release rooting agent: 3 g of rooting agent A and 2 g of rooting agent B were mixed and added to 100 mL of ethyl acetate to obtain an organic phase. 5 g of gelatin was dissolved in 200 mL of water and added to the organic phase. The mixture was homogenized at 20 MPa for 20 min. The pH of the homogenized solution was adjusted to 4 and stirred for 20 min. The mixture was cooled to 3°C and the pH of the solution was adjusted to 6.3. 2 g of transglutaminase was added and stirred for 2 h. The mixture was filtered, washed, and dried to obtain microcapsules. The mixture was added to 200 mL of water, 10 g of sodium alginate, and 0.5 g of Tween-80 were added. The mixture was stirred and emulsified at 8000 rpm for 15 min. 3 g of a 2 wt% calcium chloride solution was added dropwise and cured at room temperature for 20 min. The mixture was filtered, washed, and dried to obtain a slow-release rooting agent.

[0055] S5. Preparation of modified rooting agent: 10 g of slow-release rooting agent was added to 200 mL of water, 5 g of tannic acid, and 1 g of catalyst. The mixture was heated to 45°C and stirred for 3 h. The mixture was filtered, washed, and dried to obtain the modified rooting agent.

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

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

[0058] Preparation Example 2 Preparation of Rooting Composition Hydrogel

[0059] Here’s how:

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

[0061] S2. Preparation of rooting agent A: 12 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 10 h. Water was added to separate the two phases, and the solvent was removed from the organic phase under reduced pressure. The phase was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A in a yield of 93%.

[0062] S3. Preparation of rooting agent B: 12 ​​mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 8 h. Water was added to separate the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from a mixture of acetone and ethyl acetate (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B in a yield of 91%.

[0063] S4. Preparation of a slow-release rooting agent: 5 g of rooting agent A and 4 g of rooting agent B were mixed and added to 100 mL of ethyl acetate to obtain an organic phase; 6 g of gelatin was dissolved in 200 mL of water, added to the organic phase, homogenized at 20 MPa for 20 min, the pH of the homogenate was adjusted to 4.5, stirred for 40 min, cooled to 8°C, the pH of the solution was adjusted to 6.7, 3 g of transglutaminase was added, stirred for 4 h, filtered, washed, and dried to obtain microcapsules, which were added to 200 mL of water, 12 g of sodium alginate and 1 g of Tween-40 were added, stirred, emulsified at 8000 rpm for 15 min, 5 g of a 5 wt% calcium chloride solution was added dropwise, cured at room temperature for 40 min, filtered, washed, and dried to obtain a slow-release rooting agent;

[0064] S5. Preparation of modified rooting agent: 10 g of slow-release rooting agent was added to 200 mL of water, 7 g of tannic acid, and 2 g of catalyst. The mixture was heated to 55°C and stirred for 5 h. The mixture was filtered, washed, and dried to obtain the modified rooting agent.

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

[0066] S6. Preparation of a rooting composition hydrogel: 8 g of silk fibroin peptide was dissolved in 200 mL of water to obtain a silk fibroin peptide solution, 12 g of chitosan was dissolved in 200 mL of a 3 wt % lactic acid solution to obtain a chitosan solution, the silk fibroin peptide solution and the chitosan solution were mixed for 15 minutes, 7 g of a modified rooting agent, 5 g of NHS, and 5 g of EDC were added, and the mixture was stirred for 7 hours to form a gel state, thereby obtaining a rooting composition hydrogel.

[0067] Preparation Example 3 Preparation of Rooting Composition Hydrogel

[0068] Here’s how:

[0069] Preparation of intermediate S1: 10 mmol of isatoic 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 separate the organic phase, and the solvent was removed under reduced pressure to obtain the intermediate in 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, followed by 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 reaction was stirred for 8 h. Water was added to separate the mixture, 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 in a 94% yield.

[0071] S3. Preparation of rooting agent B: 11.5 mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 7 h. Water was added to separate the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from a mixture of acetone and ethyl acetate (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B in a yield of 92%.

[0072] S4. Preparation of a slow-release rooting agent: 4 g of rooting agent A and 3 g of rooting agent B were mixed and added to 100 mL of ethyl acetate to obtain an organic phase; 5.5 g of gelatin was dissolved in 200 mL of water, added to the organic phase, homogenized at 20 MPa for 20 min, the pH of the homogenate was adjusted to 4.2, stirred for 30 min, cooled to 5°C, the pH of the solution was adjusted to 6.5, 2.5 g of transglutaminase was added, stirred for 3 h, filtered, washed, and dried to obtain microcapsules, which were added to 200 mL of water, 11 g of sodium alginate and 0.7 g of Tween-85 were added, stirred, emulsified at 8000 rpm for 15 min, 4 g of a 3 wt % calcium chloride solution was added dropwise, cured at room temperature for 30 min, filtered, washed, and dried to obtain a slow-release rooting agent;

[0073] S5. Preparation of modified rooting agent: 10 g of slow-release rooting agent was added to 200 mL of water, along with 6 g of tannic acid and 1.5 g of catalyst. The mixture was heated to 50°C and stirred for 4 h. The mixture was filtered, washed, and dried to obtain the modified rooting agent.

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

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

[0076] Comparative Preparation Example 1

[0077] Compared with Preparation Example 3, the difference is that no rooting agent A is added in step S4.

[0078] The details are as follows:

[0079] S4. Preparation of slow-release rooting agent: Add 7g of rooting agent B to 100mL of ethyl acetate to obtain an organic phase; dissolve 5.5g of gelatin in 200mL of water, add the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH value of the homogenate to 4.2, stir and react for 30min, cool to 5°C, adjust the pH value of the solution to 6.5, add 2.5g of glutamine transaminase, stir and react for 3h, filter, wash, and dry to obtain microcapsules, add 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, and dry to obtain a slow-release rooting agent.

[0080] Comparative Preparation Example 2

[0081] Compared with Preparation Example 3, the difference is that no rooting agent B is added in step S4.

[0082] The details are as follows:

[0083] S4. Preparation of slow-release rooting agent: Add 7g of rooting agent A to 100mL of ethyl acetate to obtain an organic phase; dissolve 5.5g of gelatin in 200mL of water, add the organic phase, homogenize at 20MPa pressure for 20min, adjust the pH value of the homogenate to 4.2, stir and react for 30min, cool to 5℃, adjust the pH value of the solution to 6.5, add 2.5g of glutamine transaminase, stir and react for 3h, filter, wash, and dry to obtain microcapsules, add 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, and dry to obtain a slow-release rooting agent.

[0084] Comparative Preparation Example 3

[0085] Compared with Preparation Example 3, the difference is that step S5 is not performed.

[0086] The details are as follows:

[0087] S1 Preparation of intermediate: 10mmol isatoic anhydride and 11.5mmol 3-dimethylaminopropylamine were added to 50mL of ethyl acetate, stirred for 1.5h, split with water, and the organic phase was separated by removing the solvent under reduced pressure to obtain an intermediate;

[0088] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 8 h. Water was added to separate the mixture, and the organic phase was solvent-freeed under reduced pressure. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A.

[0089] S3. Preparation of rooting agent B: 11.5 mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 7 h. Water was added to separate the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from a mixture of acetone and ethyl acetate (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B.

[0090] S4. Preparation of a slow-release rooting agent: 4 g of rooting agent A and 3 g of rooting agent B were mixed and added to 100 mL of ethyl acetate to obtain an organic phase; 5.5 g of gelatin was dissolved in 200 mL of water, added to the organic phase, homogenized at 20 MPa for 20 min, the pH of the homogenate was adjusted to 4.2, stirred for 30 min, cooled to 5°C, the pH of the solution was adjusted to 6.5, 2.5 g of transglutaminase was added, stirred for 3 h, filtered, washed, and dried to obtain microcapsules, which were added to 200 mL of water, 11 g of sodium alginate and 0.7 g of Tween-85 were added, stirred, emulsified at 8000 rpm for 15 min, 4 g of a 3 wt % calcium chloride solution was added dropwise, cured at room temperature for 30 min, filtered, washed, and dried to obtain a slow-release rooting agent;

[0091] S5. Preparation of a rooting composition hydrogel: 7 g of silk fibroin peptide was dissolved in 200 mL of water to obtain a silk fibroin peptide solution, and 11 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain a chitosan solution. The silk fibroin peptide solution and the chitosan solution were mixed for 15 minutes, and 6 g of a sustained-release rooting agent, 4 g of NHS, and 4 g of EDC were added. The mixture was stirred and reacted for 6 hours to form a gel state, thereby obtaining a rooting composition hydrogel.

[0092] Comparative Preparation Example 4

[0093] Compared with Preparation Example 3, the difference is that steps S4 and S5 are not performed.

[0094] The details are as follows:

[0095] S1 Preparation of intermediate: 10mmol isatoic anhydride and 11.5mmol 3-dimethylaminopropylamine were added to 50mL of ethyl acetate, stirred for 1.5h, split with water, and the organic phase was separated by removing the solvent under reduced pressure to obtain an intermediate;

[0096] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 8 h. Water was added to separate the mixture, and the organic phase was solvent-freeed under reduced pressure. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A.

[0097] S3. Preparation of rooting agent B: 11.5 mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 7 h. Water was added to separate the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from a mixture of acetone and ethyl acetate (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B.

[0098] S4 rooting agent composition preparation: 4g rooting agent A and 3g rooting agent B were mixed for 15min to obtain a rooting agent composition;

[0099] S5. Preparation of a rooting composition hydrogel: 7 g of silk fibroin peptide was dissolved in 200 mL of water to obtain a silk fibroin peptide solution, 11 g of chitosan was dissolved in 200 mL of a 2 wt% acetic acid solution to obtain a chitosan solution, the silk fibroin peptide solution and the chitosan solution were mixed for 15 minutes, 6 g of a rooting agent composition was added, 4 g of NHS and 4 g of EDC were added, and the mixture was stirred for 6 hours to form a gel state, thereby obtaining a rooting composition hydrogel.

[0100] Comparative Preparation Example 5

[0101] Compared with Preparation Example 3, the difference is that step S6 is not performed.

[0102] The details are as follows:

[0103] S1 Preparation of intermediate: 10mmol isatoic anhydride and 11.5mmol 3-dimethylaminopropylamine were added to 50mL of ethyl acetate, stirred for 1.5h, split with water, and the organic phase was separated by removing the solvent under reduced pressure to obtain an intermediate;

[0104] S2. Preparation of rooting agent A: 11.5 mmol of indolebutyric acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 8 h. Water was added to separate the mixture, and the organic phase was solvent-freeed under reduced pressure. The mixture was recrystallized from acetone, filtered, washed, and dried to obtain rooting agent A.

[0105] S3. Preparation of rooting agent B: 11.5 mmol of naphthaleneacetic acid was added to 50 mL of ethyl acetate, followed by 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 reaction was stirred for 7 h. Water was added to separate the mixture, and the solvent was removed from the organic phase under reduced pressure. The mixture was recrystallized from a mixture of acetone and ethyl acetate (volume ratio 1:1), filtered, washed, and dried to obtain rooting agent B.

[0106] S4. Preparation of a slow-release rooting agent: 4 g of rooting agent A and 3 g of rooting agent B were mixed and added to 100 mL of ethyl acetate to obtain an organic phase; 5.5 g of gelatin was dissolved in 200 mL of water, added to the organic phase, homogenized at 20 MPa for 20 min, the pH of the homogenate was adjusted to 4.2, stirred for 30 min, cooled to 5°C, the pH of the solution was adjusted to 6.5, 2.5 g of transglutaminase was added, stirred for 3 h, filtered, washed, and dried to obtain microcapsules, which were added to 200 mL of water, 11 g of sodium alginate and 0.7 g of Tween-85 were added, stirred, emulsified at 8000 rpm for 15 min, 4 g of a 3 wt % calcium chloride solution was added dropwise, cured at room temperature for 30 min, filtered, washed, and dried to obtain a slow-release rooting agent;

[0107] S5. Preparation of modified rooting agent: 10 g of slow-release rooting agent was added to 200 mL of water, along with 6 g of tannic acid and 1.5 g of catalyst. The mixture was heated to 50°C and stirred for 4 h. The mixture was filtered, washed, and dried to obtain the modified rooting agent.

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

[0109] S6. Preparation of rooting composition: 6 g of modified rooting agent was added to 400 mL of water and ultrasonically dispersed at 1000 W for 15 min to prepare a rooting composition.

[0110] Example 1

[0111] A method for rooting peach branches without a substrate comprises inserting the lower part of the peach branch into the rooting composition hydrogel prepared in Preparation Example 1, taking out the branch, allowing the gel to adhere to the lower part of the peach branch, and hanging the branch. Under culture conditions, water vapor is sprayed on the lower part of the peach branch at a spray rate of 1 L / h. The spraying is continued for 1 hour, followed by a rest period of 1 hour, and the branch is cultured for 7 days until the lower part of the peach branch takes root. The branch is cultured for another 3 days to obtain rooted peach branch seedlings. The culture conditions are a temperature of 15° C., a humidity of 50 RH%, and a light intensity of 10 hours per day.

[0112] Example 2

[0113] A method for rooting peach branches without a substrate comprises inserting the lower part of the peach branch into the rooting composition hydrogel prepared in Preparation Example 2, taking out the branch, allowing the gel to adhere to the lower part of the peach branch, and hanging the branch. Under culture conditions, water vapor is sprayed on the lower part of the peach branch at a spray rate of 2 L / h. The spraying is continued for 2 hours, followed by a rest period of 2 hours, and the branch is cultured for 10 days until the lower part of the peach branch takes root. The branch is further cultured for 5 days to obtain rooted peach branch seedlings. The culture 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, wherein the lower part of the peach branch is inserted into the rooting composition hydrogel prepared in Preparation Example 3, taken out, the gel is attached to the lower part of the peach branch, and the peach branch is hung. Under culture conditions, water vapor is sprayed on the lower part of the peach branch at a spray rate of 1.5 L / h. The spraying is continued for 1.5 hours, and the peach branch is rested for 1.5 hours. The peach branch is cultured for 8 days until the lower part of the peach branch takes root. The peach branch rooted seedlings are obtained by continuing to culture for 4 days. Figure 1 As can be seen from the figure, this method can obviously achieve rooting culture of peach branches without substrate; the culture conditions are temperature of 17℃, humidity of 55RH%, and light of 9h / day.

[0116] Comparative Example 1

[0117] Compared with Example 3, the difference is that the rooting composition hydrogel is prepared by Comparative Preparation Example 1.

[0118] Comparative Example 2

[0119] Compared with Example 3, the difference is that the rooting composition hydrogel is prepared by Comparative Preparation Example 2.

[0120] Comparative Example 3

[0121] Compared with Example 3, the difference is that the rooting composition hydrogel is prepared by Comparative Preparation Example 3.

[0122] Comparative Example 4

[0123] Compared with Example 3, the difference is that the rooting composition hydrogel is prepared by Comparative Preparation Example 4.

[0124] Comparative Example 5

[0125] Compared with Example 3, the difference is that the rooting composition hydrogel is prepared by Comparative Preparation Example 5.

[0126] Test Example 1

[0127] Peach branches of the same variety, uniform in length and taken from the same section of the branches, were cultured for rooting using the methods described in Examples 1-3 or Comparative Examples 1-5. A blank control group was cultured using the method described in Example 3, but without inserting the rooting composition hydrogel prepared in Preparation Example 3. After the culture period, 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 root analysis software WinRHIZO. Root activity was measured using TTC. The results are shown in Table 1.

[0128] Table 1

[0129]

[0130] Note: *P<0.05 compared with the blank group.

[0131] As can be seen from the above table, the methods in Examples 1-3 of the present invention can effectively promote the growth of the root system of peach branches, and the root system activity is higher.

[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 in the scope of protection of the present invention.

Claims

1. A method for rooting peach branches without substrate, characterized in that: Insert the lower part of the peach branch into the rooting composition hydrogel, take it out, attach the gel to the lower part of the peach branch, hang it, spray water vapor on the lower part of the peach branch under culture conditions, culture until the lower part of the peach branch takes root, continue to culture, and obtain peach branch rooted seedlings.

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

3. The method for rooting peach branches without substrate according to claim 1, characterized in that: The preparation method of the rooting composition hydrogel is as follows: S1. Preparation of intermediate: isocyanic anhydride and 3-dimethylaminopropylamine were stirred for reaction, water was added to separate them, and the organic phase was decompressed to remove the solvent to obtain an intermediate with the following structure: ; S2. Preparation of rooting agent A: Indolebutyric acid is added to an organic solvent, NHS and EDC are added, stirred for activation, an intermediate is added, stirred for reaction, water is added to separate, the organic phase is decompressed to remove the solvent, and recrystallized from acetone to obtain a rooting agent A, the structure of which is as follows: ; S3. Preparation of rooting agent B: Add naphthaleneacetic acid to an organic solvent, add NHS and EDC, stir to activate, add the intermediate, stir to react, add water to separate, remove the solvent from the organic phase under reduced pressure, and recrystallize from an acetone / ethyl acetate mixed solution to obtain rooting agent B, the structure of which is as follows: ; S4. Preparation of a sustained-release rooting agent: mixing rooting agent A and rooting agent B evenly, adding to an organic solvent to obtain an organic phase; dissolving gelatin in water, adding the organic phase, homogenizing, adjusting the pH value of the homogenate to a first pH value, stirring for reaction, cooling, adjusting the pH value of the solution to a second pH value, adding glutamine aminotransferase, stirring for reaction, filtering, washing, drying, obtaining microcapsules, adding to water, adding sodium alginate and an emulsifier, stirring, emulsifying, dropping a calcium chloride solution, curing at room temperature, filtering, washing, drying, obtaining a sustained-release rooting agent; S5. Preparation of modified rooting agent: adding the slow-release rooting agent to water, adding tannic acid and a catalyst, heating and stirring the reaction, filtering, washing, and drying to obtain a modified rooting agent; S6. Preparation of rooting composition hydrogel: dissolve silk fibroin peptide in water to obtain a silk fibroin peptide solution, dissolve chitosan in acid solution to obtain a chitosan solution, mix the silk fibroin peptide solution and the chitosan solution evenly, add a modified rooting agent, add NHS and EDC, stir to react, form a gel state, and prepare a rooting composition hydrogel.

4. The method for rooting peach branches without substrate according to claim 3, characterized in that: In step S1, the molar ratio of isatoic anhydride to 3-dimethylaminopropylamine is 1:1.1-1.2, and the reaction time is 1-2h.

5. The method for rooting peach branches without substrate according to claim 3, characterized in that: In step S2, the molar ratio of indolebutyric acid, intermediate, NHS and EDC is 1.1-1.2:1:1.3-1.5:1.2-1.3, the stirring activation time is 20-40 minutes, and the stirring reaction time is 7-10 hours.

6. The method for rooting peach branches without substrate according to claim 3, characterized in that: In step S3, the molar ratio of naphthylacetic acid, intermediate, NHS and EDC is 1.1-1.2:1:1.3-1.5:1.2-1.3, the stirring activation time is 20-40 minutes, and the stirring reaction time is 6-8 hours.

7. The method for rooting peach branches without substrate according to claim 3, characterized in that: The mass ratio of the rooting agent A, the rooting agent B, the gelatin, the transglutaminase, the sodium alginate and the 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.

8. The method for rooting peach branches without substrate according to claim 3, characterized in that: 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 a pH of 8-9, the temperature of the heating and stirring reaction is 45-55° C., and the time is 3-5 hours.

9. The method for rooting peach branches without substrate according to claim 3, characterized in that: In step S6, the mass ratio of the silk fibroin peptide, chitosan, modified rooting agent, NHS and EDC is 6-8:10-12:5-7:3-5:3-5, the stirring reaction time is 5-7h, and the acid solution is 1-3wt% acetic acid or lactic acid solution.

10. A rooting composition hydrogel, characterized in that: The preparation method is as follows: S1. Preparation of intermediate: 1 molar equivalent of isatoic anhydride and 1.1-1.2 molar equivalent of 3-dimethylaminopropylamine were stirred for 1-2 hours, water was added to separate, and the organic phase was decompressed to remove the solvent to obtain an intermediate with the following structure: ; S2. Preparation of rooting agent A: 1.1-1.2 molar equivalents of indolebutyric acid are added to an organic solvent, 1.3-1.5 molar equivalents of NHS and 1.2-1.3 molar equivalents of EDC are added, and the mixture is stirred and activated for 20-40 minutes. 1 molar equivalent of the intermediate is added, and the mixture is stirred and reacted for 7-10 hours. Water is added to separate the mixture, and the organic phase is decompressed to remove the solvent, and the mixture is recrystallized from acetone to obtain a rooting agent A, the structure of which is as follows: ; S3. Preparation of rooting agent B: 1.1-1.2 molar equivalents of naphthylacetic acid are added to an organic solvent, 1.3-1.5 molar equivalents of NHS and 1.2-1.3 molar equivalents of EDC are added, and the mixture is stirred and activated for 20-40 minutes. 1 molar equivalent of the intermediate is added, and the mixture is stirred and reacted for 6-8 hours. Water is added to separate the mixture, and the organic phase is decompressed to remove the solvent, and the mixture is recrystallized from an acetone / ethyl acetate mixed solution to obtain a rooting agent B, the structure of which is as follows: ; S4. Preparation of a sustained-release rooting agent: 3-5 parts by weight of a rooting agent A and 2-4 parts by weight of a rooting agent B are mixed uniformly, and added to an organic solvent to obtain an organic phase; 5-6 parts by weight of gelatin are dissolved in water, added to the organic phase, homogenized, the pH value of the homogenate is adjusted to 4-4.5, stirred for reaction for 20-40 minutes, cooled to 3-8°C, the pH value of the solution is adjusted to 6.3-6.7, 2-3 parts by weight of glutamine transaminase are added, stirred for reaction for 2-4 hours, filtered, washed, dried, and microcapsules are obtained, added to water, 10-12 parts by weight of sodium alginate and 0.5-1 parts by weight of an emulsifier are added, stirred, emulsified, 3-5 parts by weight of a 2-5wt% calcium chloride solution are added dropwise, cured at room temperature for 20-40 minutes, filtered, washed, and dried to obtain a sustained-release rooting agent; S5. Preparation of modified rooting agent: 10 parts by weight of slow-release rooting agent was added to water, 5-7 parts by weight of tannic acid and 1-2 parts by weight of catalyst were added, heated to 45-55 ° C, stirred for 3-5h, filtered, washed, and dried to obtain a 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 1-3wt% acetic acid or lactic acid solution to obtain a chitosan solution, mix the silk fibroin peptide solution and the chitosan solution evenly, add 5-7 parts by weight of a 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 prepare a rooting composition hydrogel.

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