High-protein shrub four-winged chenopodium integrated breeding and high-yield cultivation method

Abstract

The application relates to the technical field of seedling cutting propagation, and discloses a high-protein shrub Atriplex canescens cultivation method integrating breeding and propagation, which comprises the following steps: cutting preparation, hormone treatment, substrate preparation, cutting and post-cutting management. The cutting is a tender branch at the base of a one-year-old transplanted seedling, which is cut into 12-15 cm, the base is bevelled, the top is flat cut, and 1-2 leaves are reserved; the hormone treatment adopts analytical pure IAA, IBA or NAA with a concentration of 100-300 mg / L; a single-factor complete random test design is further arranged; water is used as a control; the rooting rate is counted 20 days after cutting; the root system morphology, biomass, root-shoot ratio and physiological indexes such as chlorophyll, soluble sugar and soluble protein are measured 80 days after cutting; and comprehensive evaluation is carried out by combining with a membership function. The application can significantly improve the cutting rooting rate of Atriplex canescens, promote the development of the root system and the growth of seedlings, is simple and convenient to operate, low in cost, and suitable for large-scale seedling propagation of Atriplex canescens in arid regions.

Description

Technical Field

[0001] This invention relates to the field of seedling cutting propagation technology, specifically to a high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii. Background Technology

[0002] Currently, the propagation of *Acer tetrawingense* seedlings still faces severe challenges. On the one hand, seed propagation relies on imported seeds, which are costly and have unstable germination rates, resulting in inconsistent seedling sizes and difficulty in meeting the needs of large-scale production. On the other hand, while asexual propagation techniques, mainly softwood cuttings, can maintain the superior traits of the parent plant and improve propagation efficiency, many technical bottlenecks still exist in the process of industrialization. These factors severely restrict the large-scale, standardized production of high-quality *Acer tetrawingense* seedlings. Cuttings are an important propagation method for *Acer tetrawingense*, as they are convenient to operate, root easily, and have a high survival rate. Plant growth regulators play a key regulatory role in the development of adventitious roots and root system in cuttings, effectively promoting rooting in most difficult-to-root tree species. In the propagation of *Trifolium repens* cuttings, immersing the base of the cuttings in 100 mg / L NAA for 2-3 cm for 2 hours resulted in a rooting rate of up to 93.6%. In the hardwood cuttings of *Gymnocladus orientalis*, soaking the cuttings in 200 mg / L ABT-1 for 20 minutes resulted in a rooting rate of up to 62.22%. In the root cuttings of *Russula ovata*, treatment with 300 mg / L ABT-1 rooting powder significantly promoted the formation of adventitious roots and effectively improved the rooting effect of cuttings.

[0003] Currently, research on the effects of plant growth regulators on rooting and seedling quality is still not systematic enough. There is a lack of in-depth comparison of their effects on root morphology, biomass distribution, and physiological and biochemical characteristics, which cannot provide theoretical basis and technical support for the rapid propagation, large-scale seedling cultivation, and production application of improved varieties of Trichoderma tetragonum. Summary of the Invention

[0004] Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a high-yield cultivation method integrating the propagation and breeding of high-protein shrub Trichoderma tetrapanax. This method solves the problem that research on the effects of plant growth regulators on rooting and seedling quality is still not systematic enough, and there is a lack of in-depth comparison of their effects on root morphology, biomass distribution, and physiological and biochemical characteristics. As a result, it cannot provide theoretical basis and technical support for the rapid propagation, large-scale seedling cultivation, and production application of Trichoderma tetrapanax.

[0006] Technical solution To achieve the above objectives, the present invention provides the following technical solution: a high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii, comprising the following steps: Step 1: Cutting preparation: Select four-winged pheasant cuttings, cut and trim them, retaining the leaves; Step 2, Hormone Treatment: Immerse the base of the cuttings obtained in Step 1 in an aqueous solution of plant growth regulator; Step 3, substrate preparation: Use humus soil as the cutting substrate, and fill it into the cutting tray after sterilization; Step 4, Cuttings: Insert the cuttings treated in Step 2 into the substrate from Step 3; Step 5: Post-cutting management: Control the ambient temperature and humidity, and carry out regular disinfection.

[0007] As a further description of the above technical solution, the preparation of cuttings in step one is as follows: Select tender branches sprouting from the base of one-year-old transplanted seedlings of *Trifolium repens* that are growing well and free from pests and diseases, cut off excess branches and leaves while retaining intact nodes, cut the cuttings into 12-15cm long cuttings, make a slanted cut 1cm away from the base leaf bud, and make a horizontal cut 1cm away from the top leaf bud, retaining 1-2 leaves, and place the cut cuttings in a cool, shady place to keep them moist for later use.

[0008] As a further description of the above technical solution, the plant growth regulator mentioned in step two is any one of analytical grade indoleacetic acid, indolebutyric acid, or naphthaleneacetic acid, with a concentration of 100-300 mg / L.

[0009] As a further description of the above technical solution, the substrate preparation in step three is specifically as follows: disinfect the humus soil with a 500-fold dilution of 40% carbendazim wettable powder, fill the disinfected humus soil into the cutting tray with a thickness of 4cm, the total length of the cutting tray is 54cm, the width is 28cm, the single hole diameter is 4.5cm, the depth is 4.5cm, and the bottom diameter is 2cm.

[0010] As a further description of the above technical solution, the post-cutting management in step five is as follows: adjust the spraying interval and time according to the temperature and humidity of the greenhouse, control the temperature inside the greenhouse to 28±2℃, the relative humidity of the air to above 95%, and disinfect once a week with a 500-fold dilution of 40% carbendazim wettable powder.

[0011] As a further description of the above technical solution, the four-winged quinoa seedlings are one-year-old transplanted seedlings with a height of 50-60cm.

[0012] As a further description of the above technical solution, the method adopts a single-factor completely randomized experimental design, setting three plant growth regulators, namely IAA, IBA and NAA, with three concentrations of 100, 200 and 300 mg / L for each regulator, and water as a control, for a total of 10 treatments, with each treatment replicated 3 times.

[0013] As a further description of the above technical solution, the following steps are also included: index measurement steps: S1. The rooting rate is calculated on the 20th day after cutting. Rooting rate = number of rooted cuttings / total number of cuttings × 100%; S2. On the 80th day after cutting, root morphology, biomass, root-to-shoot ratio, and physiological indicators were measured.

[0014] As a further description of the above technical solution, the root morphology indicators include total root length, root surface area, root volume, number of root tips and number of branches, which are measured using a root scanner and root analysis software. The biomass determination includes weighing the fresh weight of the aboveground and underground parts of the plant, blanching them at 105℃ for 30 min, drying them at 75℃ to constant weight, determining the dry matter weight, and calculating the root-to-shoot ratio. The physiological indicators include the determination of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll content using the ethanol extraction method, the determination of soluble sugar content using the anthrone colorimetric method, and the determination of soluble protein content using the Coomassie Brilliant Blue G-250 method.

[0015] As a further description of the above technical solution, it also includes data processing steps: organizing the data, performing analysis of variance and multiple comparisons, performing principal component analysis and plotting charts, and calculating the membership function value R using the following formula:

[0016] In the formula: Let be the measured or calculated value of the i-th index for a certain treatment, where i ranges from 1 to 16, representing rooting rate, total root length, root surface area, root volume, number of root tips, number of branches, aboveground fresh weight, underground fresh weight, dry matter weight, root-to-shoot ratio, chlorophyll a, chlorophyll b, carotenoids, total chlorophyll, soluble sugar, and soluble protein, respectively. and These are the minimum and maximum values ​​among all those that process this metric.

[0017] Beneficial effects Compared with existing technologies, this invention provides a high-yield cultivation method integrating the breeding and propagation of high-protein shrub Trichosanthes kirilowii, which has the following beneficial effects: 1. By standardizing the cutting process and soaking the base of the cuttings in an appropriate concentration of plant growth regulator, the formation of adventitious roots at the base of the *Pleurotus eryngii* cuttings can be effectively promoted, significantly improving the rooting rate. Under optimal treatment conditions, the rooting rate can reach a high level.

[0018] 2. This invention uses humus soil as a substrate and disinfects it with 40% carbendazim at a dilution of 500 times to create a sterile, loose, and breathable cutting environment. Combined with precise control of temperature and humidity, it is conducive to the vigorous growth of the root system after the cuttings take root. The root morphology indicators of the cuttings treated by the method of this invention are significantly better than those of the control, such as total root length, root surface area, root volume, number of root tips, and number of branches.

[0019] 3. This invention demonstrates that the cuttings cultivated by this method have higher biomass accumulation, coordinated root and above-ground growth, reasonable root-to-shoot ratio, and better survival rate and stress resistance after transplanting by measuring the fresh weight of the above-ground and underground parts, dry matter weight, and root-to-shoot ratio of the seedlings 80 days after cutting.

[0020] 4. The present invention, through the determination of the content of photosynthetic pigments, soluble sugars and soluble proteins in leaves, shows that the cuttings cultivated by this method have enhanced photosynthetic capacity, increased accumulation of osmotic regulatory substances, good nutritional status and improved overall physiological quality.

[0021] 5. This invention employs a single-factor, completely randomized experimental design, setting multiple types and concentration gradients of plant growth regulators, with water as a control, and each treatment is replicated three times to ensure the reliability and reproducibility of the experimental results. Through analysis of variance, principal component analysis, and membership function comprehensive evaluation, the optimal combination of hormone types and concentrations can be scientifically screened, providing reliable technical parameters for the large-scale cutting propagation of Trichoderma tetragonum. Attached Figure Description

[0022] Figure 1 This is a flowchart of a high-yield cultivation method for the integrated breeding and propagation of the high-protein shrub Trichosanthes kirilowii, as proposed in this invention. Figure 2 This invention illustrates the effects of different plant growth regulator treatments on the rooting rate and root morphology of Trifolium repens cuttings in experimental examples. Figure 3 This invention illustrates the effects of different plant growth regulator treatments on biomass accumulation and root-to-shoot ratio of Trifolium repens cuttings in experimental examples. Figure 4 This invention illustrates the effects of different plant growth regulator treatments on the photosynthetic pigment content of leaves from *Trifolium repens* cuttings in experimental examples. Figure 5 This invention illustrates the effects of different plant growth regulator treatments on the soluble sugar and soluble protein content of Trifolium repens cuttings in experimental examples. Figure 6 Mantel assay for the effects of different plant growth regulator treatments on Trifolium repens cuttings in the experimental examples of this invention; Figure 7 This is a heatmap showing the loadings of different plant growth regulator treatments on various indicators of Trifolium repens cuttings in the first and second principal components of the experimental examples of this invention. Figure 8 The distribution of principal component scores and comprehensive evaluation values ​​of different plant growth regulator treatments on the cuttings of Trifolium repens in the experimental examples of this invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0024] Example: See attached document Figure 1 According to the appendix Figure 1 The steps shown are to prepare as follows: I. Preparation of Experimental Materials The *Trichoderma tetragonum* seedlings used in this experiment were obtained from an industrial demonstration base in Northwest China. They were one-year-old transplanted seedlings, 50-60cm in height. Healthy, disease-free *Trichoderma tetragonum* shrubs were selected and pruned. Cuttings were made from tender shoots sprouting from the base, removing excess branches and leaves while retaining intact nodes. The collected cuttings were placed in a shady place and kept moist for later use.

[0025] The plant growth regulators tested were IAA (indoleacetic acid), IBA (indolebutyric acid), or NAA (naphthaleneacetic acid), all of analytical grade.

[0026] Table 1 Information on plant growth regulators tested

[0027] II. Test Methods 1. Experimental Design The experiment employed a single-factor completely randomized design with three plant growth regulator treatments: IAA, IBA, and NAA. The three concentration treatments were IAA (100, 200, 300) mg / L, IBA (100, 200, 300) mg / L, and NAA (100, 200, 300) mg / L. Water was used as the control (CK). There were a total of 10 treatments, with each treatment replicated three times.

[0028] 2. Cuttings and Management The cutting propagation experiment was conducted at an industrial demonstration base in Northwest China. The cutting trays were 54cm long and 28cm wide, with individual holes measuring 4.5cm in diameter, 4.5cm in depth, and 2cm in bottom diameter, filled with a 4cm thick layer of substrate. Humus was used as the propagation substrate, and the cuttings were disinfected with a 500-fold dilution of 40% carbendazim wettable powder before planting. The cuttings were 12-15cm long, cut obliquely 1cm from the base leaf bud and horizontally 1cm from the top leaf bud, leaving 1-2 leaves. After planting, the cuttings were disinfected weekly with carbendazim. The spraying interval and time were adjusted according to the temperature and humidity of the greenhouse, maintaining a temperature of (28±2)℃ and a relative humidity above 95%.

[0029] 3. Indicator Measurement On day 20 after cutting, the rooting status of cuttings in each treatment was recorded. The formation of adventitious roots at the base of the cutting was used as the standard for rooting, and the rooting rate was calculated. Rooting rate % = Number of rooted cuttings / Total number of cuttings × 100%. On day 80 after cutting, cuttings with uniform growth were randomly selected from each treatment for various growth and physiological indicators. After removing the cuttings from the substrate, the roots were washed with clean water and the surface moisture was absorbed. Root images were acquired using a root scanner, and root morphology indicators such as total root length, root surface area, root volume, number of root tips, and number of branches were measured using root analysis software. The plants were then divided into aboveground and underground parts, and the fresh weight of the aboveground and underground parts was measured separately. They were then placed in an oven at 105℃ for 30 minutes to blanch, and then dried at 75℃ to constant weight. The dry matter mass was measured, and the root-to-shoot ratio was calculated. Fresh functional leaves from each treatment were collected, and the contents of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll were determined by ethanol extraction. The contents of soluble sugars were determined by anthrone colorimetric method, and the contents of soluble proteins were determined by Coomassie Brilliant Blue G-250 method.

[0030] 4. Data Processing Data processing can be performed using existing software, such as Excel 2019 for data organization, SPSS 23.0 for analysis of variance and Duncan's multiple comparisons, and Origin 2021 for principal component analysis and chart creation.

[0031] The formula for calculating the membership function value R is as follows:

[0032] In the formula: Let be the measured or calculated value of the i-th index for a certain treatment, where i ranges from 1 to 16, representing rooting rate, total root length, root surface area, root volume, number of root tips, number of branches, aboveground fresh weight, underground fresh weight, dry matter weight, root-to-shoot ratio, chlorophyll a, chlorophyll b, carotenoids, total chlorophyll, soluble sugar, and soluble protein, respectively. and These are the minimum and maximum values ​​among all those that process this metric.

[0033] Experimental example: I. Effects of different plant growth regulators on rooting and root morphology of Trifolium repens cuttings; like Figure 2 As shown, different plant growth regulators significantly affected the rooting and root morphology of *Trifolium repens* cuttings. P <0.05).

[0034] Compared with the control (CK) treatment, the rooting rate, total root length, root surface area, root volume, number of root tips, and number of branches increased by 7.27%–18.18%, 11.16%–125.77%, 1.89%–75.39%, 4.35%–82.61%, 3.61%–75.00%, and 0–25.65%, respectively, under each plant growth regulator treatment. Among these, the NAA 200 mg / L treatment showed the highest rooting rate (86.67%), which was significantly higher than the CK treatment. P <0.05) Figure 2 A). Compared with the control (CK) treatment, the total root length of the IBA 200 mg / L and NAA 200 mg / L treatments was significantly increased by 125.77% and 107.26%, respectively. P <0.05) Figure 2 B). Except for the IBA 200 mg / L treatment, the root surface area of ​​the other treatments was not significantly different from that of the CK treatment. Figure 2 C), the root volume treated with IBA 200 mg / L was significantly increased by 82.61% compared with the CK treatment. Figure 2 D). Except for the IBA 200 mg / L and NAA 200 mg / L treatments, the number of root tips in the other treatments was not significantly different from that in the control group (CK). Figure 2 E). The number of branching points varied from 526.00 to 778.67 in each treatment, with the IBA 200 mg / L treatment reaching a maximum of 778.67, significantly higher than the CK (E). P <0.05) Figure 2 F).

[0035] II. Effects of different plant growth regulators on biomass accumulation and root-shoot ratio of Trifolium repens cuttings; like Figure 3 As shown, different plant growth regulators significantly affected the biomass accumulation and root-to-shoot ratio of Triplophysa brevis cuttings.

[0036] Compared with the control (CK) treatment, the aboveground fresh weight, underground fresh weight, dry matter weight, and root-to-shoot ratio of each plant growth regulator treatment increased by 3.70%–53.09%, 2.88%–40.80%, 2.31%–16.76%, and 12.50%–75.00%, respectively. Compared with the CK treatment, the aboveground fresh weight of IBA 200 mg / L, NAA 200 mg / L, and IBA 300 mg / L treatments significantly increased by 53.09%, 45.68%, and 24.69%, respectively. P <0.05) Figure 3 A); Compared with the control (CK) treatment, the fresh weight of underground parts treated with IBA 200 mg / L and NAA 200 mg / L was significantly increased by 40.80% and 31.49%, respectively. P <0.05) Figure 3 B). The dry matter mass of the IAA 200 mg / L, IBA 200 mg / L, IBA 300 mg / L, and NAA 200 mg / L treatments was significantly higher than that of the CK treatment. P <0.05) Figure 3 C), the root-to-shoot ratios of the IBA 200 mg / L and NAA 200 mg / L treatments were 0.14 and 0.13, respectively, significantly higher than the control (CK), and significantly increased by 75.00% and 62.50% compared to the CK. P <0.05) Figure 3 D).

[0037] III. Effects of different plant growth regulators on the physiological characteristics of Trifolium repens cuttings; like Figure 4 As shown, different plant growth regulators have a significant effect on the photosynthetic pigments in the leaves of Tripterygium wilfordii. The chlorophyll a contents of the IAA 300 mg / L, NAA 100 mg / L, and NAA 200 mg / L treatments were 0.803, 0.731, and 0.770 mg / g, respectively, which were significantly increased by 32.95%, 21.03%, and 27.48% compared with the CK treatment. P <0.05) Figure 4 A). Except for the NAA 300 mg / L treatment, the chlorophyll b content in other treatments was not significantly different from that in the CK treatment. Figure 4 B). Compared with the control (CK) treatment, the carotenoid contents of the IAA 300 mg / L, NAA 200 mg / L, and NAA 100 mg / L treatments reached 0.262, 0.257, and 0.227 mg / g, respectively, which were significantly higher than those of the CK treatment by 34.36%, 31.79%, and 16.41%. P <0.05), while the carotenoid content was lowest in the IBA 100 mg / L treatment ( Figure 4 C). The total chlorophyll content of the IAA 300 mg / L, NAA 200 mg / L, NAA 300 mg / L and NAA 100 mg / L treatments were 1.170, 1.152, 1.143 and 1.137 mg / g, respectively, all significantly higher than that of the CK (C). P <0.05) Figure 4 D).

[0038] IV. Content of soluble sugars and soluble proteins; like Figure 5 As shown, the addition of different plant growth regulators has a significant effect on the soluble sugars and soluble proteins in the leaves of Trichoderma tetragonum.

[0039] Compared with the control (CK) treatment, the soluble sugar content increased by 16.50% and 14.14% in the NAA (200 mg / L) and IBA (200 mg / L) treatments, respectively. P <0.05) Figure 5 (A) The soluble sugar content of all IAA concentrations was lower than that of the control (CK), with the lowest being 13.85 mg / g in the 200 mg / L IAA treatment, a decrease of 3.55% compared to the CK. P <0.05). The soluble protein content of IAA 200 mg / L, IBA 100 mg / L and NAA 200 mg / L treatments increased by 26.67%, 31.11% and 26.67% respectively compared with the CK treatment, all of which were significantly higher than ( P <0.05). The soluble protein content of the IBA 300 mg / L and NAA 300 mg / L treatments were 0.42 and 0.39 mg / g, respectively, which were 6.67% and 13.33% lower than that of the CK, respectively. Among them, the NAA 300 mg / L treatment was significantly lower than that of the CK. P <0.05) Figure 5 B).

[0040] V. Mantel assay for the effects of different plant growth regulator treatments on the cuttings of Trifolium repens; like Figure 6 Mantel correlation analysis showed that there was a significant correlation between rooting rate and root surface area, root volume, number of root tips, number of branches, root-to-shoot ratio, and chlorophyll a. P <0.05), indicating that root morphology and photosynthetic pigment accumulation have a significant impact on rooting of cuttings. Dry matter mass was significantly positively correlated with total root length, number of root tips, number of branches, aboveground fresh weight, underground fresh weight, root-to-shoot ratio, and chlorophyll a. P <0.05), among which the correlation between dry matter mass and total root length, aboveground fresh weight, underground fresh weight, and root-shoot ratio reached a highly significant level ( P ≤0.001).

[0041] VI. Principal component analysis of various indicators of Trifolium repens cuttings treated with different plant growth regulators; Based on correlation analysis, principal component analysis was performed on 16 growth and physiological indicators of *Trifolium repens* cuttings. Figure 7The variance contribution rates of the first principal component (PC1) and the second principal component (PC2) were 62.938% and 18.160%, respectively, with a cumulative variance contribution rate of 81.098%. In PC1, indicators such as total root length, root tip number, aboveground fresh weight, underground fresh weight, and dry matter weight had high loadings, mainly reflecting root development and biomass accumulation. In PC2, indicators such as chlorophyll a and carotenoid total chlorophyll had high loadings, mainly reflecting the level of photosynthetic pigment accumulation.

[0042] Principal component scores and overall evaluation results show that ( Figure 8 The IBA 200 mg / L treatment had the highest overall evaluation value of 1.259, ranking first; the NAA 200 mg / L treatment was second with 1.215; and the IAA 200 mg / L treatment ranked third.

[0043] Table 2. Membership function analysis of various indicators of *Trifolium repens* cuttings under different plant growth regulators.

[0044] Different plant growth regulator treatments showed varying effects on the rooting rate, root morphology, biomass, root-to-shoot ratio, and physiological parameters of Trifolium repens cuttings (Table 2). The IBA 200 mg / L treatment had the highest membership function values ​​for total root length, root surface area, root volume, number of root tips, number of branches, aboveground fresh weight, underground fresh weight, dry matter weight, and root-to-shoot ratio, with the highest average membership function value (0.8579), ranking first overall. The NAA 200 mg / L treatment ranked second overall (average membership function value 0.8136), with the highest membership function values ​​for rooting rate and soluble sugar among its indicators, and relatively high membership function values ​​for total root length, number of branches, underground fresh weight, root-to-shoot ratio, carotenoids, and total chlorophyll. The IAA 200 mg / L treatment ranked third overall (average membership function value 0.5690). The ranking of the other treatments is as follows: IBA 300 mg / L > IAA 300 mg / L > NAA 100 mg / L > NAA 300 mg / L > IBA 100 mg / L > IAA 100 mg / L > CK.

[0045] The results show that IBA 200 mg / L has the best rooting promoting effect on softwood cuttings of Tripterygium wilfordii, which can significantly improve the rooting rate of cuttings and effectively improve key root morphology indicators such as total root length, root surface area, root volume, number of root tips and number of root branches. By comprehensively analyzing multiple root morphology indicators, the quality of robust cuttings can be evaluated more comprehensively and objectively.

[0046] It should be noted that the term "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii, characterized in that, Includes the following steps: Step 1: Cutting preparation: Select four-winged pheasant cuttings, cut and trim them, retaining the leaves; Step 2, Hormone Treatment: Immerse the base of the cuttings obtained in Step 1 in an aqueous solution of plant growth regulator; Step 3, substrate preparation: Use humus soil as the cutting substrate, and fill it into the cutting tray after sterilization; Step 4, Cuttings: Insert the cuttings treated in Step 2 into the substrate from Step 3; Step 5: Post-cutting management: Control the ambient temperature and humidity, and carry out regular disinfection.

2. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The specific preparation of cuttings in step one is as follows: Select tender shoots sprouting from the base of one-year-old transplanted seedlings of *Trifolium repens* that are growing well and free from pests and diseases. Cut off excess branches and leaves while keeping intact nodes. Cut the cuttings into 12-15cm long cuttings. Make a slanted cut 1cm from the base leaf bud and a horizontal cut 1cm from the top leaf bud, keeping 1-2 leaves. Place the cut cuttings in a cool, shady place to keep them moist for later use.

3. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The plant growth regulator mentioned in step two is any one of analytical grade indoleacetic acid, indolebutyric acid, or naphthaleneacetic acid, with a concentration of 100-300 mg / L.

4. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The substrate preparation in step three is as follows: disinfect the humus soil with a 500-fold dilution of 40% carbendazim wettable powder, fill the cutting tray with the disinfected humus soil to a thickness of 4cm, the total length of the cutting tray is 54cm, the width is 28cm, the diameter of a single hole is 4.5cm, the depth is 4.5cm, and the bottom diameter is 2cm.

5. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The post-cutting management described in step five is as follows: adjust the spraying interval and time according to the temperature and humidity of the greenhouse, control the temperature inside the greenhouse to 28±2℃, and the relative humidity of the air to above 95%, and disinfect once a week with a 500-fold dilution of 40% carbendazim wettable powder.

6. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The four-winged quinoa seedlings are one-year-old transplanted seedlings, with a height of 50-60cm.

7. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, The method employs a single-factor completely randomized experimental design, setting up three plant growth regulators, namely IAA, IBA, and NAA, with three concentrations of 100, 200, and 300 mg / L for each regulator, and using water as a control, for a total of 10 treatments, with each treatment replicated 3 times.

8. The high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, It also includes the steps for measuring the indicators: S1. The rooting rate is calculated on the 20th day after cutting. Rooting rate = number of rooted cuttings / total number of cuttings × 100%; S2. On the 80th day after cutting, root morphology, biomass, root-to-shoot ratio, and physiological indicators were measured.

9. A high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 8, characterized in that, The root morphology indicators include total root length, root surface area, root volume, number of root tips, and number of branches, which are measured using a root scanner and root analysis software. The biomass determination includes weighing the fresh weight of the aboveground and underground parts of the plant, blanching them at 105℃ for 30 minutes, drying them at 75℃ to constant weight, determining the dry matter mass, and calculating the root-to-shoot ratio. The physiological indicators include the determination of chlorophyll a, chlorophyll b, carotenoids and total chlorophyll content using the ethanol extraction method, the determination of soluble sugar content using the anthrone colorimetric method, and the determination of soluble protein content using the Coomassie Brilliant Blue G-250 method.

10. A high-yield cultivation method integrating the breeding and propagation of the high-protein shrub Trichosanthes kirilowii according to claim 1, characterized in that, It also includes data processing steps: organizing the data, performing analysis of variance and multiple comparisons, performing principal component analysis and plotting graphs, and calculating the membership function value R. In the formula: Let be the measured or calculated value of the i-th index for a certain treatment, where i ranges from 1 to 16, representing rooting rate, total root length, root surface area, root volume, number of root tips, number of branches, aboveground fresh weight, underground fresh weight, dry matter weight, root-to-shoot ratio, chlorophyll a, chlorophyll b, carotenoids, total chlorophyll, soluble sugar, and soluble protein, respectively. and These are the minimum and maximum values ​​among all those that process this metric.

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