Efficient seeding method of ampelopsis japonica and method for forming tea potato common by grafting ampelopsis japonica on ampelopsis glandulosa

Abstract

The application discloses a high-efficiency seeding method of white yam and a method for forming tea-pot co-production by grafting ampelopsis brevipedunculata var. hupehensis onto white yam, and belongs to the technical field of plant cultivation. The high-efficiency seeding method of white yam comprises the following steps: white yam seeds are planted after being treated with 3500-4000 mg / L of gibberellin, and are cultivated by fertilizing once a month. The method for forming tea-pot co-production by grafting ampelopsis brevipedunculata var. hupehensis onto white yam comprises the following steps: the white yam is used as a stock, the ampelopsis brevipedunculata var. hupehensis tender branch with small buds is used as a scion, and the grafting is performed by using the wedge grafting technology; after the grafting, the plant base is watered once every 2-3 days; and after the formation layer is healed, the plant is transplanted to a field to perform seedling raising. The cultivation method of white yam provided by the application can increase the germination rate of white yam to more than 80%. In addition, the application realizes the development of the underground part of white yam and the aboveground part of ampelopsis brevipedunculata var. hupehensis, and further realizes the common utilization of the aboveground part and the underground part, and significantly improves the land utilization rate and the income.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation technology, specifically to an efficient sowing method for *Ampelopsis japonica* and a method for grafting *Ampelopsis japonica* onto *Aeonium spp.* to form a tea-sweet potato co-production. Background Technology

[0002] Ampelopsis japonica is a woody vine belonging to the genus *Ampelopsis* in the family Vitaceae. Its tuberous roots are rich in organic acids, sterols, and triterpenoids, exhibiting significant antitumor and anti-inflammatory pharmacological activities, and are widely used in traditional medicine and compound preparations. However, the industrial production of Ampelopsis japonica faces two major bottlenecks: firstly, the overwintering germination rate of its seeds is extremely low (<5%), and losses are severe due to factors such as rotting and damage from birds and animals; secondly, the growth cycle of Ampelopsis japonica is long, lasting 3-4 years, with tuberous roots only harvested at maturity, while the vines and branches completely die each winter, resulting in almost zero utilization of above-ground biomass. In contrast, the above-ground parts of its relative, *Nekemias grossedentata*, are significantly superior. The vine tea made from its tender leaves is rich in dihydromyricetin and other components, and has excellent anti-thrombotic and lipid-lowering effects. It is known as the "miracle herb" and has a strong market demand. Moreover, it yields a bumper harvest in the second year of planting and a stable harvest in the third year, resulting in high economic benefits. However, the underground parts are not fully developed.

[0003] Currently, there is a problem with the low germination rate of *Bai Lian*. In addition, how to develop the underground parts of *Bai Lian* and the above-ground parts of *Ampelopsis thunbergii* are technical problems that need to be solved by existing technologies. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an efficient sowing method for *Ampelopsis japonica* and a method for grafting *Ampelopsis japonica* onto *Alopecurus aequalis* to form a tea-sweet potato co-production, thus solving the technical problem of low germination rate of *Ampelopsis japonica* in the prior art.

[0005] This invention aims to construct a grafting system of *Ampelopsis japonica* and *Vitis thunbergii*, and to break through the industrial bottleneck of the spatiotemporal utilization efficiency of medicinal plant resources by integrating seed germination regulation technology and grafting technology, so as to achieve efficient resource allocation.

[0006] To achieve the above-mentioned technical objectives, the present invention provides a highly efficient sowing method for *Ampelopsis japonica*, comprising the following steps: treating *Ampelopsis japonica* seeds with 3500-4000 mg / L gibberellin before sowing, watering once every 2-3 days, and fertilizing once a month to cultivate the seeds.

[0007] In any embodiment, the gibberellin treatment time is 48-72 hours.

[0008] Furthermore, this invention also proposes a method for grafting *Ampelopsis thunbergii* onto *Vitis thunbergii* to form a tea-sweet potato co-production, comprising the following steps:

[0009] S1. Using *Ampelopsis japonica* as rootstock and *Ampelopsis thunbergii* shoots with small buds as scions, grafting is performed using cleft grafting technique.

[0010] S2. After grafting, water the base of the plant once every 2-3 days; spray the above-ground parts of the plant with water once in the morning, noon and evening; after the cambium has healed, transplant it to the field for hardening-off.

[0011] In any embodiment, in step S1, using *Ampelopsis japonica* as rootstock includes: selecting 4-month-old *Ampelopsis japonica* seedlings as rootstock, retaining one functional leaf at the top when the stem diameter is 2±0.5mm, and cutting the stem 5-6cm from the ground.

[0012] In any embodiment, in step S2, the grafted tea sweet potato is cultivated at 22-24°C with a relative humidity of 55-60% and a photoperiod of 14h / 10h.

[0013] In any embodiment, in step S1, the length of the scion is 3.5-4.5 cm.

[0014] In any embodiment, in step S1, the grafting using the cleft grafting technique includes: sharpening both sides of the bottom of the scion, then cutting a cleft in the upper part of the rootstock, inserting the sharpened scion into the cleft, then wrapping the cleft with a film to ensure that the cambium layers of the scion and the rootstock are in close contact, and inserting the grafted scion into the sand.

[0015] In any embodiment, before step S1, the method further includes: using a single vine of *Avicennia marina* as the parent plant, cultivating it in a substrate, irrigating it with a substrate saturated with water every 72±2 hours; and applying top dressing to the roots monthly at a concentration of 1-1.2 g / L.

[0016] In any embodiment, the matrix comprises peat, perlite and vermiculite, wherein the mass ratio of the peat, the perlite and the vermiculite is 3:(1-1.2):(1-1.2).

[0017] In any implementation, biological control of grape thrips is also included during the cultivation of Ampelopsis lanceolata, with insecticides sprayed at 10-11 day intervals.

[0018] Compared with the prior art, the beneficial effects of the present invention include: the cultivation method of white peony proposed in the present invention can increase the germination rate of white peony to more than 80%.

[0019] Furthermore, this invention proposes a highly efficient cultivation technique for grafting tea seedlings onto *Ampelopsis japonica* and *Aeonium adenophorum*. *Ampelopsis japonica* is used as the rootstock, and young shoots of *Aeonium adenophorum* with existing buds are used as scions, employing a cleft grafting technique. After grafting, the base of the plant is watered every 2-3 days; the above-ground parts of the plant are sprayed with water three times a day (morning, noon, and evening); after the cambium has healed, the plant is transplanted to the field for hardening off, thus constructing a composite plant—tea seedling—combining a "fast-growing above-ground economic module (for annual vine tea harvest) + a reserve-type underground medicinal module." This innovative plant has brought about a structural change in the annual output value per unit area: the traditional monoculture of *Ampelopsis japonica* yields a total output value of about 48,000 yuan / mu over three years; while the tea-potato system, with its above-ground vine tea yielding an annual income of about 30,000 yuan / mu, plus the late-stage income from the underground *Ampelopsis japonica* tubers, achieves an average annual output value of 46,000 yuan / mu, nearly tripling the output value, doubling land utilization, and shortening the production cycle. This technology provides a replicable solution for the cross-species resource integration of medicinal plants and economic crops, allowing for the simultaneous acquisition of the enlarged roots of *Ampelopsis japonica* and the tender leaves of *Ampelopsis lanceolata*, realizing the development of both the above-ground and underground parts of *Ampelopsis japonica*, thereby achieving the joint utilization of both above-ground and underground parts, significantly improving land utilization and income. Attached Figure Description

[0020] Figure 1 These are detailed diagrams of the scion, rootstock, and grafting method in Embodiment 3 of the present invention; Figure 1 A: Front view of the scion; Figure 1 B: Side view of the scion; Figure 1 C: Rootstock diagram; Figure 1 D: Joining diagram.

[0021] Figure 2 This is a diagram of the grafting and binding of the scion and rootstock in Embodiment 3 of the present invention.

[0022] Figure 3 This is a picture of a 32-day rooted plant grafted with *Arabidopsis thunbergii* in sand culture, as described in Example 3 of this invention.

[0023] Figure 4 This is a diagram of the healing process at the graft union of a 40-day-old grafted *Alopecurus dentata* seedling after untying, according to Example 3 of this invention.

[0024] Figure 5 This is an overall view of the grafted *Alopecurus dentata* seedling after being untied, according to Embodiment 3 of the present invention. Detailed Implementation

[0025] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0026] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0027] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0028] This specific embodiment provides an efficient sowing method for *Ampelopsis japonica*, including the following steps: treating *Ampelopsis japonica* seeds with 3500-4000 mg / L gibberellin for 48-72 hours before sowing, watering once every 2-3 days, and fertilizing once a month to cultivate the seeds.

[0029] This specific embodiment provides a method for grafting *Ampelopsis thunbergii* onto *Vitis thunbergii* to form a tea-sweet potato co-production, including the following steps:

[0030] S1. Using *Ampelopsis japonica* as rootstock and young shoots of *Vitis thunbergii* with existing buds as scions, grafting is performed using the cleft grafting technique. The *Ampelopsis japonica* rootstock is selected as follows: 4-month-old *Ampelopsis japonica* seedlings are selected as rootstock; when the stem diameter is 2±0.5mm, one functional leaf is retained at the top, and the stem is cut 5-6cm above the ground. The length of the scion is 3.5-4.5cm. The cleft grafting technique involves: sharpening both sides of the scion's base, then making a cleft in the upper part of the rootstock, inserting the sharpened scion into the cleft, and then wrapping the cleft with a film to ensure close contact between the cambium layers of the scion and the rootstock. The grafted scion is then inserted into sandy soil.

[0031] S2. After grafting, water the base of the plant once every 2-3 days; spray the above-ground parts of the plant with water once in the morning, noon and evening; after the cambium heals, transplant to the field for hardening off; after grafting, cultivate the tea sweet potato under the conditions of 22-24℃, 55-60% relative humidity and 14h / 10h photoperiod.

[0032] In some embodiments, before step S1, the method further includes: using a single vine of *Arabidopsis thaliana* as the parent plant, employing substrate cultivation, and irrigating the substrate with saturated water content every 72±2 hours; using grape-specific slow-release compound fertilizer, applying it to the roots monthly at a concentration of 1-1.2 g / L; the substrate includes peat, perlite, and vermiculite, with the mass ratio of peat, perlite, and vermiculite being 3:(1-1.2):(1-1.2); during the cultivation of *Arabidopsis thaliana*, the method also includes biological control of grape thrips, spraying insecticides at 10-11 day intervals.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0035] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0036] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0037] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0038] Example 1

[0039] Standardized propagation technology of Grapevine scions

[0040] This embodiment uses a high-quality single vine of *Aeonium adenophorum* from Huaihua, Hunan Province as the female parent to construct an asexual propagation system. A substrate cultivation model is adopted, with a substrate ratio of peat:perlite:vermiculite = 3:1:1. Precise water and fertilizer management is implemented: irrigation to saturated substrate moisture content is completed every 72±2 hours; nutrient supply uses grape-specific slow-release compound fertilizer (from existing technology), applied monthly as root fertilization at a concentration of 1 g / L. For biological control of grape thrips (*Drepanothrips reuteri*), a novel low-toxicity insecticide, Ailvshi (active ingredient: ethyl spinosad, (from existing technology)), is used for targeted and precise application at a concentration of 1000 times dilution, with an application interval of 10 days.

[0041] Example 2

[0042] White peony rootstock seed germination regulation technology

[0043] Gibberellin concentration gradient screening

[0044] This study used robust seedlings of *Ampelopsis japonica* as rootstock. However, the germination rate of *Ampelopsis japonica* seeds under natural conditions is less than 5%. To improve the germination rate of *Ampelopsis japonica* seeds, this study used seeds that had naturally matured for 6 months as experimental material and investigated the effect of gibberellin (GA3) concentration gradients (1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, 3500 mg / L, 4000 mg / L, and 5000 mg / L) for 24 hours. Each treatment group was replicated three times, with 50 seeds per replicate, and germination experiments were conducted in a constant temperature soil culture room at 25 ± 2℃. One-way ANOVA and Duncan's multiple comparisons were used to evaluate the germination rate. The results are shown in Table 1. Treatment with 4000 mg / L GA3 for 24 h resulted in a seed germination rate of 70.00 ± 0.00% a and a germination potential of 46.67 ± 3.05% a. The combined germination rate and germination potential were the highest among treatments with non-complete significance.

[0045] Table 1. Effects of different GA3 concentrations on seed germination of *Ampelopsis japonica*.

[0046]

[0047] Processing time optimization

[0048] Based on the determination that 4000 mg / L is the optimal GA3 concentration, the effects of treatment time (12 h, 24 h, 48 h, and 72 h) on germination characteristics were further investigated. The results showed that the germination rate of the 72 h treatment group was significantly increased to 84.0 ± 3.5%, significantly higher than other treatment times, and 151.9% higher than the CK group (33.33 ± 8.08%) (Table 2). The conclusion is that treatment with 4000 mg / L gibberellin for 72 h can significantly increase the germination rate of *Ampelopsis japonica* seeds to approximately 85%, which is about 2.5 times the germination rate of seeds naturally stored for six months and then subjected to 24 h of water immersion treatment (CK).

[0049] Table 2. Effects of different treatment times with 4000 mg / L GA3 on seed germination of *Ampelopsis japonica*.

[0050]

[0051] Note: Germination rate: The percentage of test seeds that germinated out of the total number of test seeds;

[0052] Germination potential: The percentage of seeds germinated during the peak of the daily germination rate out of the total number of seeds in the tested sample.

[0053] After treatment with 4000 mg / L gibberellin for 72 hours, the seeds were sown in seedling trays. *Ampelopsis japonica* and *Aeonium arboreum* have similar growth habits, requiring watering every three days until thoroughly soaked. Grape-appropriate fertilizer was applied once a month. *Ampelopsis japonica* is susceptible to powdery mildew; early treatment with pesticides is necessary. In case of outbreaks, diseased leaves should be pruned and the affected area isolated. During normal cultivation, attention should be paid to powdery mildew to prevent it from harming the rootstock. *Aeonium arboreum* is also susceptible to grape thrips; *Agrostis spp.* pesticide can be used, spraying areas with concentrated infestations.

[0054] Example 3

[0055] This embodiment proposes a method for grafting *Ampelopsis thunbergii* onto *Vitis thunbergii* to form a tea-sweet potato co-production, including the following steps:

[0056] S1. Select 4-month-old *Ampelopsis japonica* seedlings from Example 3 as rootstock. When the stem diameter reaches 2±0.5mm, retain one functional leaf at the top and cut the stem 5cm above the ground. Select tender shoots of *Vitis davidii* with small buds from Example 1 as scions, retaining about 1cm above the bud. The length of the scions should be uniformly about 4cm, and cleft grafting should be used. First, sharpen both sides of the bottom of the scion to ensure it can be easily inserted into the rootstock. Then, take the rootstock and remove the dehydrated upper part. Make a cleft cut in the upper part of the rootstock with a knife, insert the sharpened scion into the cleft, and leave a certain gap. Figure 1 Finally, wrap the grafting film from bottom to top to ensure close contact between the cambium layers of the scion and the rootstock, in order to promote healing and growth. Figure 2 After grafting, insert the cuttings into the sand and cover them to retain moisture.

[0057] S2. After grafting, place the grafted plant in a growth chamber at a temperature of 23℃, relative humidity (RH) of 60%, and a photoperiod of 14h / 10h (135μmol / m²). 2 s -1 Cultivate under the following conditions. Water the base of the plant every 2 days, watering thoroughly. Simultaneously, water the above-ground parts of the plant three times a day (morning, noon, and evening), with 6-hour intervals between each watering. Spray the entire plant with water during each watering. After about one week of cultivation, the axillary buds of *Ampelopsis grossedentata* will unfold their leaves. Figure 3 After about a month of cultivation, the cambium layer heals. Figure 4 Remove the binding straps and perform regular management. Once the plants are growing stably, transplant them to the field for hardening off, allowing them to adapt to outdoor conditions and then manage them in the field. Figure 5 We successfully cultivated Vibrio vitis-like grape grafted onto Leymus chinensis.

[0058] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for grafting *Ampelopsis japonica* onto *Ampelopsis thunbergii* to form a tea-sweet potato co-production, characterized in that, Includes the following steps: S1. Using *Ampelopsis japonica* obtained through an efficient sowing method as rootstock, and *Vitis thunbergii* shoots with existing buds as scions, grafting is performed using cleft grafting techniques. The *Ampelopsis japonica* rootstock is selected as follows: 4-month-old *Ampelopsis japonica* seedlings are selected as rootstock, and when the stem diameter is 2±0.5mm, the top functional leaf is retained, and the stem is cut 5-6cm above the ground. The *Ampelopsis japonica* is obtained as follows: *Ampelopsis japonica* seeds are treated with 3500-4000 mg / L gibberellin before sowing, watered every 2-3 days, and fertilized once a month. The gibberellin treatment time is 48-72 hours. S2. After grafting, water the base of the plant once every 2-3 days; spray the above-ground parts of the plant with water once in the morning, noon and evening; after the cambium heals, transplant to the field for hardening off; after grafting, the tea sweet potato is cultivated at 22-24℃ with a relative humidity of 55-60% and a photoperiod of 14 h / 10 h. Before step S1, the method further includes: using a single vine of *Aeonium arboreum* as the parent plant, cultivating it in a substrate, and irrigating it with the substrate saturated with water every 72±2 h; and applying top dressing to the roots monthly at a concentration of 1-1.2 g / L; the substrate includes peat, perlite and vermiculite, and the mass ratio of the peat, perlite and vermiculite is 3:(1-1.2):(1-1.2).

2. The method for grafting Ampelopsis japonica onto Viburnum odoratum to form a tea-sweet potato co-production according to claim 1, characterized in that, In step S1, the length of the scion is 3.5-4.5 cm.

3. The method for grafting Ampelopsis japonica onto Vitex negundo to form a tea-sweet potato co-production according to claim 1, characterized in that, In step S1, the grafting technique using cleft grafting includes: sharpening both sides of the bottom of the scion, then making a cleft on the upper part of the rootstock, inserting the sharpened scion into the cleft, then wrapping the cleft with a film to ensure that the cambium layers of the scion and the rootstock are in close contact, and then inserting the grafted scion into the sand.

4. The method for grafting Ampelopsis japonica onto Viburnum 'White Lotus' to form a tea-sweet potato co-production according to claim 1, characterized in that, Biological control of grape thrips is also included during the cultivation of Ampelopsis lanceolata, with insecticides sprayed every 10-11 days.

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