Preparation method of pinto bean cutting slips for cross-region and / or off-peak cutting and pinto bean cutting seedling raising method

Abstract

The invention belongs to the technical field of plant cultivation, and particularly relates to a preparation method of a pachyrhizus erosus cutting slip for cross-region and / or peak shifting cuttage and a pachyrhizus erosus cuttage seedling raising method.The method comprises the following steps that in the growth environment with the average temperature being 20-25 DEG C and the average humidity being 80%-90%, a pachyrhizus erosus branch with two bud points is selected, the distance between the two bud points is 12-20 cm, and the pachyrhizus erosus branch with two bud points is selected; respectively truncating at positions 4-6cm away from the two bud points to obtain cutting slips containing the two bud points; the morphological lower ends of the cuttings are placed in a water retention carrier containing a bacteriostatic agent, then the cuttings are placed in a water retention appliance, and the pinto bean cuttings are obtained. According to the preparation method, the high vitality of the cuttings after long-distance and long-time transportation can be guaranteed, and the highest cuttage survival rate of pinto beans can reach 95.10%.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, specifically relating to a method for preparing *Dendrobium nobile* cuttings for cross-regional and / or staggered cutting propagation, and a method for *Dendrobium nobile* cutting propagation seedlings. Background Technology

[0002] Honeydew Beans ( Spatholobus suberectus *Dunn* is a perennial woody vine belonging to the genus *Dunn* in the legume family. It is the only original plant of *Spatholobus suberectus*, a traditional Chinese medicine, listed in the *Pharmacopoeia of the People's Republic of China* from 1977 to 2025. Its traditional effects include nourishing blood, promoting blood circulation, clearing the meridians, and relieving pain. It is mainly used to treat blood deficiency and chlorosis, rheumatic pain, and various gynecological conditions. Modern pharmacology has confirmed that its flavonoids (such as catechins), phenolic acids, and steroidal compounds have multiple activities, including promoting hematopoiesis, anti-tumor activity, anti-inflammatory activity, and immunomodulatory activity. It is an important raw material for more than two hundred kinds of traditional Chinese medicine preparations, such as *Qianjin Pian* and *Shujin Jianyao Wan*.

[0003] *Spatholobus suberectus* is mainly distributed in the mountainous forest edges of Guangdong, Guangxi, Yunnan, and Fujian provinces. The raw materials for *Spatholobus suberectus* medicinal materials are primarily sourced from wild harvesting. Due to its long growth cycle and slow regeneration, wild resources are nearing depletion. Currently, about 80% of domestic demand relies on imported wild resources from Southeast Asia. Artificial cultivation is a crucial measure to ensure the sustainable use of *Spatholobus suberectus* medicinal materials. *Spatholobus suberectus* has a wide distribution and high phenotypic diversity, but existing resources are mostly located in areas with inconvenient transportation and significant environmental differences. Therefore, it is necessary to establish germplasm resource nurseries for ex-situ preservation. Current reports primarily involve on-site cuttings, with a survival rate of only about 65%. Ex-situ propagation of wild resources requires transporting cuttings from different populations over long distances and for extended periods to a fixed propagation base. This process leads to dehydration and fungal growth, further reducing the survival rate. Therefore, ensuring high vigor of cuttings after long-distance, long-term transportation and high survival rates after cutting propagation are urgent problems to be solved for the preservation of wild resources across regions. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing bean cuttings for cross-regional and / or staggered cutting propagation and a method for bean cutting propagation. The preparation method can ensure the high vigor of the cuttings after long-distance and long-term transportation, and the survival rate of bean cuttings can reach up to 95.10%.

[0005] This invention provides a method for preparing *Pterocarya stenoptera* cuttings suitable for cross-regional and / or staggered cuttings, comprising the following steps: under a growing environment with an average temperature of 20-25℃ and an average humidity of 80-90%, select *Pterocarya stenoptera* branches with two buds, the distance between the two buds being 12-20cm, and cut them horizontally at a distance of 4-6cm from the two buds to obtain cuttings containing two buds; Place the morphological lower end of the cutting in a water-retaining carrier containing a bacteriostatic agent, and then place the cutting in a water-retaining device to obtain the Vigna lutea cutting.

[0006] As a preferred solution, the water-retaining carrier comprises one or more of water-retaining cotton, sterile absorbent cotton, and sterile gauze.

[0007] As a preferred solution, the water-retaining device comprises a fresh-keeping bag or a fresh-keeping box.

[0008] As a preferred solution, the bacteriostatic agent comprises carbendazim, thiophanate-methyl, or chlorothalonil.

[0009] The application also provides a Vigna lutea cutting seedling raising method for cross-regional and / or off-peak cutting, comprising the following steps: taking out the Vigna lutea cutting obtained by the preparation method from the water-retaining device, cutting off the Vigna lutea cutting at a position 1.5-2 cm away from the morphological upper end of the bud point, and sealing with a moisturizing agent; forming an inclined plane on the other side opposite to the position of the bud point at the morphological lower end, the inclined plane has an angle of 55-65° with the horizontal, and the position of the bud point retained at the morphological lower end corresponds to the center of the inclined plane, to obtain a pruned Vigna lutea cutting. After the pruned Vigna lutea cutting is immersed in a treatment agent, the surface water is dried, the cutting is inserted into the culture medium in a direction with an angle of 55-65° with the horizontal, and after watering once, the cutting is cultured in the shade in a closed manner; after 55-65 days of cutting, the device for closed culture is subjected to micropore treatment, and then the pore size is increased every 6-8 days, so that the cutting gradually adapts to the external environment and completes the hardening-off; the treatment agent comprises a rooting agent and a bacteriostatic agent.

[0010] As a preferred solution, the whole-day temperature during the culture in the shade in a closed manner is maintained at 10-42°C, the whole-day humidity is maintained at 23%-100%, and the shading degree is 85%-95%.

[0011] As a preferred solution, the pore size during the micropore treatment is 0.5-1.5 cm, and the distance between the pores is ≥15 cm; when the pore size is increased, the pore size is increased by 2-3 cm each time.

[0012] As a preferred solution, the moisturizing agent comprises vaseline, horticultural wax, beeswax, paraffin, or plant wound healing paste.

[0013] As a preferred solution, the rooting agent comprises ABT rooting powder or Sima rooting powder, and the bacteriostatic agent comprises carbendazim, thiophanate-methyl, or chlorothalonil.

[0014] As a preferred solution, the time for immersion in the treatment agent is 3-5 h.

[0015] Beneficial effects: the application provides a preparation method of dense flower bean cutting for cross-regional and / or staggered cutting, comprising the following steps: selecting dense flower bean branches with two bud points in a growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90%, the distance between the two bud points is 12-20 cm, and the branches are cut flat at 4-6 cm away from the two bud points respectively to obtain cutting with two bud points; the morphological lower end of the cutting is placed in a water-retaining carrier containing a bacteriostatic agent, and then the cutting is placed in a water-retaining device to obtain dense flower bean cutting. The branches are collected in a growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90%, that is, the dense flower bean branches are selected in a specific time from November to March (dry season, dormant period), the photosynthesis and respiration of the dense flower bean plant are weakened in the dormant period, and the consumption rate of the nutrients stored by the cutting itself is significantly reduced; and the dry season environment has low humidity, which can effectively inhibit the reproduction of root rot pathogens, thereby reducing the root rot probability of the cutting after cutting; selecting branches with appropriate distance between two bud points and performing specific treatment can make the cutting store water and nutrients to the maximum extent, effectively maintain the activity of the cutting, ensure the high activity of the cutting after long-distance and long-time transportation, and the survival rate of dense flower bean cutting can reach 95.10%. Not only the problem of cross-regional resource preservation is solved, but also a technical foundation is laid for the construction of wild germplasm nursery and the selection of excellent single plants.

[0016] The application also provides a dense flower bean cutting seedling raising method for cross-regional and / or staggered cutting, comprising the following steps: taking out the dense flower bean cutting obtained by the above preparation method from the water-retaining device, cutting flat at a position 1.5-2 cm away from the bud point at the morphological upper end of the dense flower bean cutting, and sealing with a moisture-retaining agent; forming an inclined surface on the other side opposite to the position of the bud point at the morphological lower end, the inclined surface has an angle of 55 DEG -65 DEG with the horizontal, and the position of the bud point reserved at the morphological lower end of the cutting corresponds to the center of the inclined surface, to obtain the pruned dense flower bean cutting; after the pruned dense flower bean cutting is immersed in a treatment agent, the surface water is dried, the cutting is inserted into the culture medium in a direction with an angle of 55 DEG -65 DEG with the horizontal, water is poured once, and then the cutting is cultured in the shade in a closed manner; after 55-65 days of cutting, the closed culture device is treated with micropores, and then the pore size is enlarged every 6-8 days to gradually adapt to the external environment, and the seedling raising is completed; the treatment agent comprises a rooting agent and a bacteriostatic agent. The cutting method effectively solves the in vitro stress problem caused by long-distance and long-time transportation in the process of migration cutting of dense flower bean wild resources, and increases the survival rate of dense flower bean cutting to 95.10%, thereby providing an efficient solution for the ex situ protection of rare wild resources. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The pruning schematic diagram before cross-regional and / or staggered cutting; Figure 2 The pruning schematic diagram after transportation or placement; Figure 3 Pruning physical map before transportation for Example 1; Figure 4 Pruning physical map after transportation for Example 1; Figure 5 Growth state of cutting seedling after cutting for 60 days in Example 1; Figure 6 Growth state of cutting seedling after cutting for 90 days in Example 1; Figure 7 Growth state of cutting bed after cutting for 90 days in Example 4. DETAILED DESCRIPTION

[0018] The application provides a preparation method of dense flower bean cutting twigs for cross-regional and / or staggered cutting, comprising the following steps: selecting a dense flower bean branch with two bud points in a growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90%, the distance between the two bud points being 12-20 cm, and cutting the branch at a distance of 4-6 cm from the two bud points respectively to obtain the cutting twigs containing the two bud points. Placing the morphological lower end of the cutting twigs in a water-retaining carrier containing a bacteriostatic agent, and then placing the cutting twigs in a water-retaining device to obtain the dense flower bean cutting twigs.

[0019] Unless otherwise specified, the application does not have special requirements for the raw materials, and commercially available goods known to those skilled in the art can be used.

[0020] The application selects a dense flower bean branch with two bud points in a growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90%. The growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90% in the application refers to a specific period from November to the following March, which belongs to winter, and the dense flower bean is in a dormant period, and the period is a dry season, and the photosynthesis of the dense flower bean plant is weakened, so that the nutrients stored in the cutting twigs are consumed slowly, and the root rot probability is low. As a preferred embodiment, the dense flower bean branch selected in the application is a healthy, disease-free and lignified hard branch. The lignified branch has the characteristics of high storage, strong water retention and stable support, and the bud points and cambium of the hard branch are more full and mature, so that the hard branch can start the 'rooting program' faster after cutting.

[0021] The distance between the two selected bud points is 12-20 cm Figure 1 L3), and the branch is cut at a distance of 4-6 cm Figure 1 L1 and L2) from the two bud points respectively to obtain the cutting twigs containing the two bud points, and the pruning schematic diagram of the cutting twigs is shown in Figure 1The distance between the two bud points can be any value in the range of 12-20 cm, for example 12, 15, 18 or 20 cm. If the distance between the two bud points is too long, the surface area of the branch is large, the transpiration is strong, the cutting cannot efficiently absorb water before rooting, and is prone to dehydration and wilting. At the same time, the nutrients stored in the branch itself need to be supplied to the longer vine, which will be quickly consumed, making it difficult to support the formation of callus and root system at the base. If the distance between the two bud points is too short, it is not conducive to cutting, and the upper bud point will be buried in the soil and difficult to sprout new branches. In the present application, the cutting is performed within a range of 4-6 cm from the bud point, for example 4, 4.5, 5, 5.5 or 6 cm. The plane formed during cutting can minimize the contact area between the cutting surface and the air, reducing the rate of water loss. If the plane is too long from the two bud points, it will increase the transportation cost, and if it is too short, the water and nutrient storage outside the bud point will be too low, accelerating the rate of loss of bud vitality. As a preferred embodiment, a small bevel is cut at the lower end of the cutting to distinguish the polarity and mark the lower end of the cutting seedling, which is convenient for identifying the correct cutting direction in the later stage. In the present application, polarity differentiation refers to the fixed growth direction of the plant branch, i.e. the morphological upper end always sprouts branches and leaves, and the morphological lower end always forms root system, and the direction is irreversible. Therefore, prior to field work, it is necessary to mark the cutting according to the proximity to the plant body (morphological lower end) and the distance from the plant body (morphological upper end) to facilitate correct cutting in the later stage. The present application does not have specific limitations on the small bevel, which is only a marker symbol and can be seen with the naked eye. The length of the small bevel can be any value in the range of 5-10 mm, for example 5, 6, 6.5, 7, 8, 8.5, 9 or 10 mm; the included angle (θ) between the small bevel and the horizontal direction can be any value in the range of 30°-45°, for example 30°, 35°, 40° or 45°. Figure 1

[0022] The morphological lower end of the cutting is placed in a water-retaining carrier containing a bacteriostatic agent, and then the cutting is placed in a water-retaining device to obtain the vigna unguiculata cutting. The water-retaining carrier includes one or more of water-retaining cotton, sterile absorbent cotton and sterile gauze. The bacteriostatic agent includes carbendazim, thiophanate-methyl or chlorothalonil. The present application does not have special limitations on the concentration of the bacteriostatic agent, which can be used according to the product instructions. In the specific embodiments of the present application, the bacteriostatic agent is diluted by 1000 times before use. The water-retaining device includes a fresh-keeping bag or a fresh-keeping box. As a preferred solution, the two ends of the cutting are placed in the water-retaining device. As a preferred solution, the cutting can be tied and fixed when placed in the water-retaining carrier and the water-retaining device. The tying tool includes a rope and / or a rubber band.

[0023] ​The dense-flowered bean cuttings obtained by the present invention can be used for cross-regional and / or staggered cutting propagation. When cross-regional ex-situ cutting propagation and / or staggered cutting propagation are carried out, the problem of in vitro stress can be effectively solved during long-distance and long-term transportation or placement.

[0024] This invention also provides a method for propagating *Phyllostachys edulis* cuttings for cross-regional and / or staggered-peak propagation, comprising the following steps: removing *Phyllostachys edulis* cuttings prepared using the above method from a water-retaining container, and cutting the cuttings horizontally at a distance of 1.5-2 cm from the bud at the morphological upper end of the cuttings. Figure 2 (L4), seal with a humectant; form a sloping surface on the opposite side of the morphological lower end at the location of the bud point, the sloping surface forming an angle with the horizontal ( Figure 2 The angle (β) is 55°~65°, so that the bud point retained at the lower morphological end of the cutting corresponds to the center of the slant, resulting in the pruned *Dendrobium nobile* cutting. See the pruning diagram below. Figure 2 ; Immerse the base of the pruned bean cuttings in the treatment agent, remove them and let them dry. Insert them into the culture medium at a 55°~65° angle to the horizontal. Water them thoroughly once and then keep them in a shaded and sealed environment for cultivation. After 55~65 days of cultivation, treat the sealed cultivation container with micropores. Then enlarge the pore size every 6~8 days to gradually adapt the seedlings to the external environment and complete the hardening-off process.

[0025] Before removing the bean cuttings prepared using the above method from the water-retaining container, they can be transported or placed. As a preferred embodiment, during transport, the bean cuttings (along with the water-retaining container) prepared using the above method are placed in a transport container, with holes made at random locations within the container. The number of holes needs to be determined based on the size of the transport container to ensure air circulation. This invention does not impose any special limitations on the transport container; any conventional transport container can be used. The diameter of the holes can be any value within the range of 0.5 to 2 cm, for example, 0.5, 1, 1.5, or 2 cm. During transport, it is preferred to use a courier service near the collection point. The transport or placement time can be any value within the range of 1 to 5 days, for example, 1, 2, 3, 4, or 5 days.

[0026] As a preferred solution, after transportation, the V. sinensis cutting is taken out of the water-retaining device, and the morphological upper end is cut off at a position 1.5-2 cm away from the bud point, and the cut end is sealed with a moisturizing agent. When cutting, the position can be within a range of 1.5-2 cm away from the retained bud point, for example, 1.5 cm, 1.8 cm, or 2 cm; after this cutting, two bud points are still retained on the cutting. During transportation, the cross section will lose water and the activity will decrease, so it is necessary to cut again. If the cutting position is too close to the bud point, the cutting edge will easily damage the growth tissue (i.e., the bud primordium) of the bud point, causing the bud point to necrose; if it is too far away, the excess stem section will consume the limited nutrients and water of the cutting, and will also cause faster water loss due to the increased water evaporation area, affecting the survival rate of the cutting. The smallest cutting area can minimize water loss, and the flat cross section is less likely to accumulate rainwater and dew, reducing the risk of rotting at the upper end. The moisturizing agent used in the present application includes: Vaseline, horticultural wax, beeswax, paraffin, or plant wound healing paste. Sealing the cut end with a moisturizing agent can seal the vessels and intercellular spaces of the cut surface, prevent the branch from wilting due to "upper end water loss", and physically block the contact between the pathogen and the wound, reducing the risk of rotting at the upper end. The morphological lower end is beveled to form a bevel on the side opposite the position of the bud point, the bevel has an angle of 55°-65° with the horizontal, and the position of the bud point retained at the morphological lower end corresponds to the center of the bevel, to obtain the V. sinensis cutting after pruning. When the position of the bud point corresponds to the center of the bevel, it is easier to absorb nutrients and promote root formation. The angle of the bevel with the horizontal in the present application can be any value within the range of 55°-65°, for example, 55°, 60°, or 65°; under the premise of ensuring the strength of the cutting structure, the area of the cambium is maximized to promote root formation; the water absorption area is expanded to prevent the cutting from wilting; it is convenient to operate when cutting, and reduces human error and insertion damage.

[0027] The base of the cutting after pruning is immersed in a treatment agent. In the present application, the base is a position below the morphological lower end bud point. As a preferred embodiment, the treatment agent includes: a rooting agent and an antibacterial agent; the rooting agent includes: ABT rooting powder or Sima rooting powder; the antibacterial agent includes carbendazim solution, thiophanate-methyl, or chlorothalonil. The mass ratio of the rooting agent and the antibacterial agent in the present application can be any value within the range of 1:2-10, for example, 1:2, 1:4, 1:6, 1:8, or 1:10. In a specific embodiment of the present application, the antibacterial agent is diluted by 1000 times before use. The immersion time in the present application can be any value within the range of 3-5 h, for example, 3 h, 3.5 h, 4 h, 4.5 h, or 5 h; the immersion time can ensure that the rooting solution is fully absorbed by the branch to stimulate the differentiation of the root primordium, and can also effectively kill the pathogens at the cutting, while avoiding excessive use of the agent due to prolonged immersion, which may cause damage to the branch.

[0028] After taking out and drying the surface moisture, the cutting is inserted into the culture medium at an angle of 55° to 65° with the horizontal. The base of the cutting of dense-flower beans is pruned to have a fresh wound, and the wound is in a moist state after soaking. If the cutting is directly inserted into the medium, fungi and bacteria in the medium will quickly grow through the unhealed wound, causing the cut to rot and leading to the failure of cutting. In the present application, the angle with the horizontal can be any value within the range of 55° to 65°, such as 55°, 58°, 60°, 62°, or 65°. As a preferred embodiment, the culture medium is the surface soil of the original weed-covered area, and the depth of the surface soil can be any value within the range of ≤20 cm, such as 1, 5, 10, 15, or 20 cm; the soil is used as needed. The original weed-covered area is an area that has been in a natural weed-covered state for a long time, and the soil after removing the surface weeds and stones can be used as the culture medium. The soil in this area has not been subjected to any artificial improvement treatment such as screening, disinfection, and fertilization, which can reduce the operation cost. The culture medium retains the natural physical and chemical properties of the original soil and contains humus and original microbial communities formed by the decomposition of weed roots, which can simulate the soil microenvironment of the original growth of wild plants and reduce the adaptive stress of the cutting on artificial medium. The container for holding the culture medium includes a seedling raising bag, a seedling raising box, a seedling raising pot, or a seedbed. In the specific embodiments of the present application, the container for holding the culture medium is a non-woven fabric seedling raising bag, which is made of degradable material, and the bag does not need to be removed during subsequent transplanting. The entire seedling raising bag can be placed in the soil, which avoids root damage and simplifies the transplanting process.

[0029] After being inserted into the culture medium, the cutting is watered once and then subjected to shade and closed culture. In the present application, the whole-day temperature during shade and closed culture can be maintained at any value within the range of 10 to 42℃, such as 10, 12, 16, 18, 20, 23, 26, 29, 33, 36, 38, 40, or 42℃; and the whole-day humidity during shade and closed culture can be maintained at any value within the range of 23% to 100%, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Under this temperature and humidity condition, the cell division rate at the base of the cutting is the fastest, which can accelerate the formation of callus, activate the enzyme activity required for rooting, and shorten the rooting period; and high air humidity can prevent the leaves from wilting due to water loss. In the embodiments of the present application, the container for shade and closed culture includes a greenhouse, and the greenhouse includes a plastic film greenhouse. The shade degree during shade and closed culture can be any value within the range of 85% to 95%, such as 85%, 87%, 80%, 82%, or 95%. In the embodiments of the present application, a sunshade net is installed above and around the greenhouse to prevent strong light from shining on the cutting and causing the cutting to lose water.

[0030] The micro-hole treatment is performed on the closed culture container after 55-65 days, for example, 55, 58, 60, 62 or 65 days, of cutting. At this time, the base of the Gliricidia sepium cutting has germinated more than 3-5 healthy new roots, and the root system is white, milky white or light brown in color; the apical bud point has germinated new leaves, and the new leaves are unfolded and bright in color, without wilting or yellowing. The pore size of the micro-hole treatment according to the present application can be any value in the range of 0.5-1.5 cm, for example, 0.5, 1 or 1.5 cm, and the pore spacing can be ≥15 cm. The micro-hole treatment can avoid large-area damage to the film structure, thereby affecting the moisturizing effect. The pore size is then expanded every 6-8 days, gradually adapting to the external environment. According to the present application, the pore size can be expanded every 6-8 days, for example, 6, 7 or 8 days, and the pore size can be increased by 2-3 cm, for example, 2, 2.5 or 3 cm each time. According to the method of the present application, the seedling raising can be completed after about 90 days of cutting. At this time, the cutting seedling has germinated more than 5 main roots at the base, and a large number of lateral roots have germinated, and the root system network can efficiently absorb water and nutrients, and is white or light brown (xylem root system) in color, without blackening, rotting or slimy feeling; the leaves are emerald green, and the new leaves grow.

[0031] In order to further illustrate the present application, a method for preparing Gliricidia sepium cuttings for cross-regional and / or staggered cutting and a Gliricidia sepium cutting seedling raising method according to the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0032] Example 1 1. In November 2024, two bud points were selected on a healthy, disease- and pest-free xylem hard branch of a wild Gliricidia sepium individual in the field, and the branch was cut at 5 cm from each bud point to ensure that the length of the branch between the two bud points was not more than 20 cm, thereby obtaining a cutting containing two bud points (total number of 160 cuttings). The pruning diagram of a randomly selected cutting is shown in Figure 3 A 5-10 mm small bevel was cut at the morphological lower end of the cutting, the small bevel and the horizontal direction formed an angle of 30°-45°, and the polarity was distinguished to determine the cutting direction.

[0033] 2. After the cutting in step 1 was treated, it was bundled with rubber bands, and the morphological lower end was wrapped with a water-retaining cotton soaked in a carbendazim solution. The cutting and the water-retaining cotton were further bundled, and finally a layer of fresh-keeping bag was wrapped around the upper and lower ends of the branch for further water retention. Carbendazim solution: carbendazim powder (effective ingredient carbendazim 50%; Dafusheng brand, purchased from Jiangsu Lanfeng Biological Chemical Co., Ltd., China; pesticide registration certificate number PD85150-8) was diluted with water according to a mass-volume ratio of 1 g:1000 mL to obtain a carbendazim solution.

[0034] 3. Mail from the express point near the collection site, after packing, open 3 holes with a diameter of about 1 cm around the box.

[0035] 4. Further pruning of the cuttings received after 3 days, i.e. cutting horizontally at a distance of 2 cm from the bud point on the morphological upper end, then sealing with vaseline, and cutting obliquely at a distance of 60° from the horizontal at the other side of the bud point on the lower end, so that the bud point position corresponds to the center of the cross section; obtain the pruned cuttings; see the pruning diagram of a randomly selected cutting Figure 4 .

[0036] 5. Immerse the base of the pruned cuttings in step 4 for 4 hours, then take them out; Treatment agent (mass ratio of rooting powder to carbendazim 1:10): ABT rooting powder 1g (total effective components of indole acetic acid and naphthalene acetic acid 50%; Beijing Abitt Bio-technology Co., Ltd., China; pesticide registration certificate number PD20081125), according to the instructions, mix indole acetic acid and naphthalene acetic acid with two bags of powder, then dissolve thoroughly with 100mL 75% ethanol to prepare ABT rooting mother liquor, then dilute with 10L water; add 10g carbendazim powder to the diluted solution to obtain the treatment agent.

[0037] 6. Dry the surface moisture of the cuttings taken out in step 5, and insert them into the non-woven fabric seedling raising bag (16cm x 18cm) in the direction of 60° from the horizontal, which is filled with culture medium (surface soil of original weed covered area with a depth ≤20cm).

[0038] 7. After cutting, pour water once in the seedling raising bag, use a plastic film greenhouse as a cutting bed, put the seedling raising bag into the cutting bed, keep the average temperature in the cutting bed at 20.87℃ (14.30~32.40℃), and the humidity at 87.63% (37.33%~99.16%), do not pour water in the film again before opening the film, and cover shading net with a shading degree of 90% on the top and around the plastic film greenhouse to prevent strong light from causing water loss in the cuttings.

[0039] 8. After 60 days of cutting, 3-5 or more healthy new roots have germinated at the base, the root system is white, cream or light brown in color; the top bud point germinates new leaves, the new leaves are unfolded and bright in color, without wilting and yellowing; see Figure 5The surface of the plastic film is first subjected to irregular micropore treatment, the initial pore diameter is 1 cm, and the pore spacing is greater than or equal to 15 cm, so as to avoid damage to the film structure and affect moisture retention; then the pore diameter is gradually increased every 7 days, and the pore diameter is increased by 2-3 cm each time, and the environmental gradient adaptation period is prolonged (about 4 weeks), so as to realize the gradual integration of indoor and outdoor temperature and humidity. After 90 days of cutting, the seedlings are finally completed. At this time, the base of the cutting seedling sprouts more than 5 main roots, and a large number of lateral roots sprout, the root system network can efficiently absorb water and nutrients, the color is white, light brown (lignified root system), no blackening, rotting, slimy feeling; the leaf is emerald green, and the new leaf grows; randomly select a cutting seedling to observe the growth of the cutting seedling after 90 days, as shown in Figure 6 .

[0040] Example 2 1. In January 2025, two bud points were selected on a healthy, disease-free lignified branch of a wild individual of dense-flowered beans in the wild, and the branch was cut flat at a distance of 5 cm from each bud point, ensuring that the length of the branch between the two bud points was not more than 20 cm, obtaining a cutting with two bud points (total of 210 branches). A small bevel of 5-10 mm was cut at the morphological lower end, the small bevel formed an angle of 30°-45° with the horizontal direction to determine the cutting direction.

[0041] 2. The cutting treated in step 1 was tied with a rubber band, and the morphological lower end was wrapped with a water-retaining cotton soaked in a carbendazim solution. The cutting and the water-retaining cotton were further tied, and finally a layer of fresh-keeping bag was wrapped around the upper and lower ends of the branch for further water retention. Carbendazim solution: configured in the manner of Example 1.

[0042] 3. The cutting was mailed from the nearby express point, and 3 holes with a diameter of about 1 cm were opened at random positions around the box after packing.

[0043] 4. The cutting received after 1 day was further pruned, i.e. cut flat at a distance of 2 cm from the bud point at the morphological upper end, then sealed with garden wax, and the lower end was cut obliquely at a position with an angle of 60° with the horizontal on the other side of the bud point to correspond to the center of the cross section.

[0044] 5. The cutting pruned in step 4 was immersed in the treatment agent for 4 hours and then taken out. Treatment agent: configured in the manner of Example 1.

[0045] 6. The cutting taken out in step 5 was dried to remove surface moisture, and then inserted into a non-woven fabric seedling raising bag (16 cm x 18 cm) filled with culture medium (the surface soil of the original grass covered area with a depth of less than or equal to 20 cm).

[0046] 7. After cutting, pour water once in the seedling bag, use a plastic film arched shed as a cutting bed, put the seedling bag into the cutting bed, keep the average temperature in the cutting bed at 18.72°C (12.60-31.00°C) and the average humidity at 88.60% (33.62%-98.13%), do not pour water in the film before the film is opened, put a shading net with a shading degree of 90% on the top and around the plastic film arched shed to prevent strong light from irradiating and causing the cutting to lose water.

[0047] 8. After 60 days of cutting, first perform irregular micropore treatment on the surface of the plastic film, the initial pore diameter is 1 cm and the pore spacing is ≥15 cm to avoid affecting moisture retention due to damage to the film structure; then gradually increase the pore diameter every 7 days, with an increase of 2-3 cm each time, gradually integrate the temperature and humidity inside and outside the shed by extending the environmental gradient adaptation period (about 4 weeks), and finally complete the hardening-off of the seedlings.

[0048] Example 3 1. In February 2025, two bud points were selected on a healthy, pest-free woody branch of a wild individual of dense-flowered bean in the wild, and the branch was cut flat at a distance of 5 cm from each bud point, ensuring that the length of the branch between the two bud points was not more than 20 cm, obtaining a cutting with two bud points (total number of 413 branches). A small bevel of 5-10 mm was cut at the morphological lower end, the small bevel formed an angle of 30°-45° with the horizontal direction to determine the cutting direction.

[0049] 2. After the treatment in step 1, the cutting was tied with a rubber band, and the morphological lower end was wrapped with a water-retaining cotton soaked in a carbendazim solution. The cutting and the water-retaining cotton were further tied, and finally a layer of fresh-keeping bag was wrapped around the upper and lower ends of the branch for further water retention. Carbendazim solution: configured in the manner of Example 1.

[0050] 3. The cutting was mailed from a nearby express point, and after packing, three holes with a diameter of about 1 cm were made at random positions around the box.

[0051] 4. The wild branch received after 2 days was further pruned, and the morphological upper end was cut flat at a distance of 2 cm from the bud point, then the wound was sealed with a plant wound healing paste, and the lower end was cut obliquely at an angle of 60° with the horizontal on the other side of the bud point to correspond to the center of the cross section.

[0052] 5. The cutting pruned in step 4 was immersed in the treatment agent for 4 hours and then taken out. Treatment agent: configured in the manner of Example 1.

[0053] 6. The cutting taken out in step 5 was dried to remove surface moisture, and then inserted into a non-woven fabric seedling bag (16 cm x 18 cm) filled with culture medium (the surface soil of the original grass-covered area with a depth of ≤20 cm).

[0054] 7. After cutting, pour water once in the seedling bag, use a plastic film arched shed as a cutting bed, put the seedling bag into the cutting bed, keep the average temperature in the cutting bed at 21.92°C (10.00~36.10°C), the average humidity at 82.90% (23.57%~98.45%), and do not pour water in the film before the film is opened, put a shading net with 90% shading degree on the top and around the plastic film arched shed to prevent strong light from irradiating and causing the cutting to lose water.

[0055] 8. After 60 days of cutting, first perform irregular micropore treatment on the surface of the plastic film, the initial pore diameter is 1 cm, and the pore spacing is ≥15 cm to avoid affecting moisture retention due to damage to the film structure; then gradually increase the pore diameter every 7 days, with an increase of 2~3 cm each time, gradually integrate the temperature and humidity inside and outside the shed by extending the environmental gradient adaptation period (about 4 weeks), and finally complete the hardening-off of the seedlings.

[0056] Example 4 1. In March 2025, select two bud points on a healthy, pest-free woody hard branch of a wild individual of dense-flowered beans in the wild, and cut the branch at 5 cm from each bud point to ensure that the length of the branch between the two bud points is not more than 20 cm, obtaining a cutting with two bud points (total number of 266 branches). Cut a 5~10 mm small bevel at the morphological lower end of the cutting, with the small bevel forming an angle of 30°~45° with the horizontal direction to determine the cutting direction.

[0057] 2. After the treatment in step 1, bundle the cutting with rubber bands, and then wrap the cutting with a water-retaining cotton soaked in a carbendazim solution at the morphological lower end position, further bundle the cutting with the water-retaining cotton, and finally wrap the cutting with a fresh-keeping bag at the upper and lower ends of the branch for further water retention. Carbendazim solution: configured in the manner of Example 1.

[0058] 3. Mail the cutting from the nearby express point after packing, and open 3 holes with a diameter of about 1 cm at random positions around the box.

[0059] 4. Further trim the cutting received after 4 days, i.e. cut the branch at 2 cm from the bud point at the morphological upper end, then seal the cut with beeswax, and cut the branch at an angle of 60° with the horizontal at the other side of the bud point at the lower end to make the bud point position correspond to the center of the cross section.

[0060] 5. Soak the base of the cutting trimmed in step 4 in the treatment agent for 4 hours and then take it out. Treatment agent: configured in the manner of Example 1.

[0061] 6. After removing the cuttings in step 5, let them dry on the surface and insert them into a non-woven fabric seedling bag (16cm×18cm) filled with culture medium (top soil of native weed-covered area with a depth ≤20cm) at a 60° angle to the horizontal.

[0062] 7. After cutting, water the seedling bags thoroughly. Use a plastic film arched greenhouse as the cutting bed, place the seedling bags in the cutting bed, and maintain the average temperature in the cutting bed at 24.12℃ (10.70~37.60℃) and the average humidity at 82.88% (27.33%~98.56%). Do not water the cuttings before lifting the film. Cover the top and sides of the plastic film arched greenhouse with a 90% shade net to prevent strong sunlight from causing the cuttings to lose moisture.

[0063] 8. Sixty days after cutting, first create irregular micropores on the surface of the plastic film, with an initial pore diameter of 1cm and a spacing of ≥15cm, to avoid damaging the film structure and affecting moisture retention. Then, gradually increase the pore diameter by 2-3cm every 7 days, extending the environmental gradient adaptation period (approximately 4 weeks) to achieve gradual integration of temperature and humidity inside and outside the greenhouse, ultimately completing the hardening-off process. For the growth status of the cuttings in the propagation bed after 90 days, please refer to [the relevant documentation / reference]. Figure 7 .

[0064] Example 5 1. In April 2025, healthy, disease-free, and lignified hard branches of wild *Dendrobium nobile* were selected from the wild. Two buds were chosen from each end of the lignified hard branch, and a horizontal cut was made 5cm from each end of the bud, ensuring that the length of the branch segment between the two buds did not exceed 20cm. This yielded 180 cuttings with two buds each. A small 5-10mm slant cut was made at the lower morphological end of each cutting, forming a 30°-45° angle with the horizontal direction, to distinguish the polarity and determine the direction of propagation.

[0065] 2. After binding the cuttings treated in step 1 with rubber bands, wrap the lower morphological end with water-retaining cotton soaked in carbendazim solution, further bind the cuttings and water-retaining cotton, and finally put a layer of plastic bag on both the upper and lower ends of the branch for further water retention. Carbendazim solution: Prepared according to the method in Example 1.

[0066] 3. The package will be mailed from a courier point near the collection site. After packing, make three holes with a diameter of about 1 cm at random locations around the box.

[0067] 4. Further prune the cuttings received 2 days later. Make a horizontal cut 2cm away from the bud at the upper morphological end, then seal the cut with paraffin wax. Make a slanted cut at the lower end at a 60° angle to the horizontal on the other side of the bud, so that the bud position corresponds to the center of the cross section.

[0068] 5. Immerse the base of the cuttings trimmed in step 4 in the treatment agent for 4 hours, then remove them; Treatment agent: Prepared according to the method of Example 1.

[0069] 6. After removing the cuttings in step 5, let them dry on the surface and insert them into a non-woven fabric seedling bag (16cm×18cm) filled with culture medium (top soil of native weed-covered area with a depth ≤20cm) at a 60° angle to the horizontal.

[0070] 7. After cutting, water the seedling bags thoroughly. Use a plastic film arched greenhouse as the cutting bed, place the seedling bags in the cutting bed, and maintain the average temperature in the cutting bed at 26.88℃ (12.95~39.21℃) and the average humidity at 83.28% (26.88%~99.95%). Do not water the cuttings before lifting the film. Cover the top and sides of the plastic film arched greenhouse with a 90% shade net to prevent strong sunlight from causing the cuttings to lose moisture.

[0071] 8. After 60 days of cutting, first treat the surface of the plastic film with irregular micropores. The initial pore diameter is 1cm and the pore spacing is ≥15cm to avoid damage to the film structure and affect moisture retention. Then, gradually increase the pore diameter every 7 days, increasing it by 2~3cm each time. By extending the environmental gradient adaptation period (lasting about 4 weeks), the temperature and humidity inside and outside the greenhouse will gradually merge, and finally the seedling hardening will be completed.

[0072] Example 6 1. In May 2025, healthy, disease-free, and lignified hard branches of wild *Dendrobium nobile* were selected from the wild. Two buds were chosen from each end of the lignified hard branch and cut horizontally 5cm from each bud, ensuring that the length of the branch segment between the two buds did not exceed 20cm. This yielded 93 cuttings with two buds each. A small 5-10mm slant cut was made at the lower morphological end of each cutting, forming a 30°-45° angle with the horizontal direction, to distinguish polarity and determine the direction of propagation.

[0073] 2. After binding the cuttings treated in step 1 with rubber bands, wrap the lower morphological end with water-retaining cotton soaked in carbendazim solution, further bind the cuttings and water-retaining cotton, and finally put a layer of plastic bag on both the upper and lower ends of the branch for further water retention. Carbendazim solution: Prepared according to the method in Example 1.

[0074] 3. The package will be mailed from a courier point near the collection site. After packing, make three holes with a diameter of about 1 cm at random locations around the box.

[0075] 4. Further prune the cuttings received 3 days later. Make a horizontal cut 2cm away from the bud at the upper morphological end, then seal the cut with Vaseline. Make a slanted cut at the lower end at a 60° angle to the horizontal on the other side of the bud, so that the bud is positioned at the center of the cut.

[0076] 5. The base of the cutting pruned in step 4 is immersed in the treatment agent for 4 hours and then taken out; Treatment agent: configured in the manner of example 1.

[0077] 6. The cutting pruned in step 5 is dried to remove surface moisture, and then inserted into a non-woven fabric seedling raising bag (16 cm x 18 cm) in a direction with a horizontal angle of 60° after being filled with culture medium (topsoil of a native weed covered area with a depth of ≤20 cm).

[0078] 7. After cutting, the seedling raising bag is poured with water once to make it permeable, a plastic film shed is used as a cutting bed, the seedling raising bag is placed in the cutting bed, the average temperature in the cutting bed is maintained at 27.23°C (18.92~41.44°C), the average humidity is maintained at 84.74% (31.35%~99.96%), no water is poured into the film before the film is opened, a shading net with a shading degree of 90% is hung above and around the plastic film shed to prevent strong light from irradiating and causing the cutting to lose water.

[0079] 8. After 60 days of cutting, the surface of the plastic film is first treated with irregular micropores, the initial pore diameter is 1 cm, and the pore spacing is ≥15 cm to avoid damage to the film structure and affect moisture retention; then the pore diameter is gradually increased by 2~3 cm every 7 days, the environmental gradient adaptation period (about 4 weeks) is prolonged, the temperature and humidity inside and outside the shed are gradually integrated, and the seedling raising is finally completed.

[0080] Test example 1 The number of surviving cuttings of the dense-flowered bean cuttings in steps 1~6 after 60 days of cutting is counted, and the survival rate is calculated according to the following formula, and the results are shown in Table 1.

[0081] Survival rate = number of surviving cuttings / total number of cuttings x 100%.

[0082] Table 1 Survival rate after cutting

[0083] As can be seen, by using the preparation method and cutting method provided by the present application, the survival rate of the dense-flowered bean cuttings is increased to 95.10%, which provides an efficient solution for the ex situ conservation of rare wild resources.

[0084] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for the preparation of G. arborea cutting for cross-regional and / or off-peak cutting, characterized by, The method comprises the following steps: In a growth environment with an average temperature of 20-25 DEG C and an average humidity of 80-90%, select a branch of dense-flower beans with two bud points, the distance between the two bud points being 12-20 cm, and cut the branch at a distance of 4-6 cm from the two bud points respectively to obtain an insert with two bud points; Place the morphological lower end of the insert in a water-retaining carrier containing a bacteriostatic agent, and then place the insert in a water-retaining device to obtain a dense-flower bean insert.

2. The production method according to claim 1, characterized by, The water-retaining carrier comprises one or more of water-retaining cotton, sterile absorbent cotton and sterile gauze.

3. The preparation method according to claim 1, characterized in that, The water-retaining device comprises a fresh-keeping bag or a fresh-keeping box.

4. The preparation method according to claim 1, characterized in that, The bacteriostatic agent comprises carbendazim, thiophanate-methyl or chlorothalonil.

5. A method for cuttage of G. arborea for raising seedlings across the region and / or off-peak cuttage, characterized by, The method comprises the following steps: Take out the dense-flower bean insert prepared by the method of any one of claims 1-4 from the water-retaining device, cut the morphological upper end of the insert at a position 1.5-2 cm from the bud point, and seal the cut end with a moisturizing agent; form an inclined surface on the side opposite to the position of the bud point at the morphological lower end, the inclined surface having an angle of 55 DEG -65 DEG with the horizontal, and the position of the bud point remaining at the morphological lower end corresponding to the center of the inclined surface, to obtain a pruned dense-flower bean insert; Submerge the base of the pruned dense-flower bean insert in a treatment agent, dry the surface moisture, and insert the insert into a culture medium in a direction having an angle of 55 DEG -65 DEG with the horizontal, pour water once, and then perform shaded and closed culture; after 55-65 days of cutting, perform micropore treatment on the closed culture device, and then expand the pore size every 6-8 days to complete seedling raising; the treatment agent comprises a rooting agent and a bacteriostatic agent.

6. The microphityma bean cutting method according to claim 5, wherein The whole-day temperature of the shaded and closed culture is maintained at 10-42 DEG C; the whole-day humidity of the shaded and closed culture is maintained at 23%-100%; and the shading degree of the shaded and closed culture is 85%-95%.

7. The microphityma bean cutting method according to claim 5, characterized by, The pore size of the micropore treatment is 0.5-1.5 cm, and the distance between the pores is greater than or equal to 15 cm; and the pore size is expanded by 2-3 cm each time.

8. The microphityma bean cutting method according to claim 5, characterized by, The moisturizing agent comprises vaseline, horticultural wax, beeswax, paraffin wax or plant wound healing paste.

9. The microphit cutting propagation method according to claim 5, wherein The rooting agent comprises ABT rooting powder or Sima rooting powder; and the bacteriostatic agent comprises carbendazim, thiophanate-methyl or chlorothalonil.

10. The microphityma bean cutting method according to claim 9, characterized by, The time for submerging in the treatment agent is 3-5 h.

Images