A high-efficiency propagation method for ananas comosus by transplanting and cultivating the cluster buds and circulating cuttage

Abstract

The application provides a kind of anaphalis contorta cluster bud transplanting cultivation and circulatory cutting efficient breeding method, relates to the field of anaphalis contorta cluster bud transplanting, including the disinfection of selected anaphalis contorta plant, after the disinfection and washing of anaphalis contorta plant with sterile water, the stem segment of 2-3cm length with node is cut, is inoculated to cluster bud germination medium and is induced to obtain anaphalis contorta cluster bud;Anaphalis contorta cluster bud grows to 2-3cm and is cut into 0.5-1cm tissue block and is transferred to proliferation growth medium, and is cultured under full light;Anaphalis contorta cluster bud grows to 5-6cm and is transplanted outdoors, is planted in straw debris and peat mixed matrix, and anaphalis contorta cluster bud grows to 8-10cm and is cut with root hair node segment for cutting, and cutting matrix is straw debris and peat mixed matrix, and anaphalis contorta cutting plant grows to 8-10cm and is cut for expanding propagation seedling;Cutting anaphalis contorta seedling is used for large-scale production transplanting when growing to 3-4cm.The present application is conducive to the full contact and attraction of external plant to the components in the induction medium, promotes the maturation of plant, thereby improving the survival rate of transplanting and seedling raising.

Description

Technical Field

[0001] This invention relates to the field of transplanting clustered buds of Anoectochilus roxburghii, and more specifically, to a method for inducing and cultivating clustered buds of Anoectochilus roxburghii through transplanting and cyclical cutting propagation for high efficiency. Background Technology

[0002] Golden Thread Orchid (Anoectochilus roxburghii) is a precious perennial herb belonging to the genus Anoectochilus of the Orchidaceae family. This genus contains more than 30 species. It mainly grows in hilly areas at altitudes of 300-1200m, preferring shade and moisture. It flowers from September to October and bears fruit from October to November. Golden Thread Orchid is mainly produced in Taiwan, Fujian, Guangxi, Yunnan, Guizhou and other places in China. It has high medicinal value and is rich in vitamin C, minerals, lipids and cellulose. Its leaves are often used to treat diseases such as hypertension, rheumatoid arthritis, and diabetes, and have the effects of clearing heat and detoxifying, and lowering blood sugar.

[0003] Currently, the formation and differentiation of callus tissue and clustered buds in *Anoectochilus roxburghii* explants are slow, the growth of these clustered buds is slow, and the survival rate of transplanted and hardened seedlings is low. This, in turn, affects the development of the *Anoectochilus roxburghii* industry and reduces the income of growers. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes a method for the efficient propagation of *Anoectochilus roxburghii* through induced bud formation, transplanting, and cyclic cutting. This method facilitates full contact and absorption of the components in the induction medium by the explants, promotes callus formation and differentiation of bud clusters, facilitates rapid elongation of bud clusters and induced root hair growth, and accelerates plant maturation, thereby increasing the survival rate of transplanting and hardening-off seedlings. This promotes the development of the *Anoectochilus roxburghii* industry and increases the income of growers.

[0005] To achieve the above-mentioned technical objectives, this invention provides a method for the efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings, comprising the following steps:

[0006] (1) After removing the leaves from the selected Anoectochilus roxburghii plant, clean the soil from the surface of the rootstock of the plant. Gently brush the plant with a brush to fully moisten it. Place the plant in a container and add 75% alcohol to it. Place the container on a clean bench and disinfect the plant by penetrating it with 75% alcohol. Replace the 75% alcohol in the container with 0.1% mercuric chloride. Shake the container and disinfect the plant by the 0.1% mercuric chloride. Remove the plant from the container and rinse it with sterile water. Cut a 2-3 cm long segment of the stem with nodes from the plant. Place the segment on a clean bench and disinfect its surface with an ultraviolet lamp.

[0007] (2) The segmented stem was inoculated into a bud germination medium and placed in the dark to induce the buds of Anoectochilus roxburghii;

[0008] (3) After the clustered shoots of the golden thread lotus grow to 2-3cm, they are cut into 0.5-1cm pieces along with the callus tissue and transferred to the proliferation and growth medium. They are placed in the dark to induce the shoots to grow to 4-5cm, and then cultured under full light conditions.

[0009] (4) When the clustered buds of Anoectochilus roxburghii grow to 5-6cm, they are directly transplanted outdoors and planted in a seedling tray filled with a mixture of straw shreds and peat moss. They are then placed on a tidal seedbed for cultivation, and the substrate humidity is controlled between 40-50%.

[0010] (5) When the clustered buds of Anoectochilus roxburghii grow to 8-10cm, cut the segments with root hairs for cutting. The cutting substrate is a mixture of straw shreds and peat moss. The cutting substrate is placed in a seedling tray with small holes at the bottom, which is 5cm high. The seedling tray is placed on a tidal seedbed for cultivation. The humidity of the cutting substrate is controlled between 50-60%.

[0011] (6) Once the golden thread lotus cuttings have grown to 8-10cm, they can be cut and propagated to expand the seedlings.

[0012] (7) Cuttings of Anoectochilus roxburghii can be transplanted for large-scale production when they grow to 3-4 cm.

[0013] Preferably, in step (1), the *Anoectochilus roxburghii* plant is disinfected by penetrating it with 75% alcohol for 10-15 seconds.

[0014] Preferably, in step (1), the segmented stem is placed on a clean bench and its surface is disinfected with an ultraviolet lamp for 20 minutes.

[0015] Preferably, in step (1), the extracted *Anoectochilus roxburghii* plant is rinsed with sterile water 3 to 4 times.

[0016] Preferably, in step (1), the *Anoectochilus roxburghii* plant is disinfected with 0.1% mercuric chloride for 2–3 minutes.

[0017] Preferably, the budding culture medium in step (2) includes: MS medium, activated carbon at a concentration of 3 g / L, sucrose at a concentration of 30 g / L, agar at a concentration of 6 g / L, and kinetin at a concentration of 0.2 mg / L.

[0018] Preferably, the proliferation and growth medium in step (3) includes: 1 / 4MS medium, agar with a concentration of 6 g / L, sucrose with a concentration of 30 g / L, NAA with a concentration of 2 mg / L, and IBA with a concentration of 1.5 mg / L.

[0019] Preferably, the ratio of straw fragments to peat moss mixed substrate in step (4) is 2:1.

[0020] Preferably, the ratio of straw fragments to peat moss mixed substrate in step (5) is 1:2.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention proposes a highly efficient propagation method for *Anoectochilus roxburghii* through transplanting and cyclic cuttings, which facilitates full contact and absorption of the components in the induction medium by the explants; promotes callus formation and differentiation of clustered buds from the explants; facilitates rapid elongation of the clustered buds and the induction of root hair growth; promotes plant maturation, thereby improving the survival rate of transplanting and hardening off; promotes the development of the *Anoectochilus roxburghii* industry; and increases the income of growers. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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] This invention provides a method for the efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings, comprising the following steps:

[0025] (1) After removing the leaves from the selected Anoectochilus roxburghii plant, clean the soil from the surface of the rootstock of the plant. Gently brush the plant with a brush to fully moisten it. Place the plant in a container and add 75% alcohol to it. Place the container on a clean bench and disinfect the plant by penetrating it with 75% alcohol. Replace the 75% alcohol in the container with 0.1% mercuric chloride. Shake the container and disinfect the plant by the 0.1% mercuric chloride. Remove the plant from the container and rinse it with sterile water. Cut a 2-3 cm long segment of the stem with nodes from the plant. Place the segment on a clean bench and disinfect its surface with an ultraviolet lamp.

[0026] (2) The segmented stem was inoculated into a bud germination medium and placed in the dark to induce the buds of Anoectochilus roxburghii;

[0027] (3) After the clustered shoots of the golden thread lotus grow to 2-3cm, they are cut into 0.5-1cm pieces along with the callus tissue and transferred to the proliferation and growth medium. They are placed in the dark to induce the shoots to grow to 4-5cm, and then cultured under full light conditions.

[0028] (4) When the clustered buds of Anoectochilus roxburghii grow to 5-6cm, they are directly transplanted outdoors and planted in a seedling tray filled with a mixture of straw shreds and peat moss. They are then placed on a tidal seedbed for cultivation, and the substrate humidity is controlled between 40-50%.

[0029] (5) When the clustered buds of Anoectochilus roxburghii grow to 8-10cm, cut the segments with root hairs for cutting. The cutting substrate is a mixture of straw shreds and peat moss. The cutting substrate is placed in a seedling tray with small holes at the bottom, which is 5cm high. The seedling tray is placed on a tidal seedbed for cultivation. The humidity of the cutting substrate is controlled between 50-60%.

[0030] (6) Once the golden thread lotus cuttings have grown to 8-10cm, they can be cut and propagated to expand the seedlings.

[0031] (7) Cuttings of Anoectochilus roxburghii can be transplanted for large-scale production when they grow to 3-4 cm.

[0032] Further, in step (1), the *Anoectochilus roxburghii* plant is disinfected by penetrating it with 75% alcohol for 10-15 seconds.

[0033] Furthermore, the segmented stem described in step (1) is placed on a clean bench and its surface is disinfected with an ultraviolet lamp for 20 minutes.

[0034] Further, in step (1), the extracted *Anoectochilus roxburghii* plant is rinsed with sterile water 3 to 4 times.

[0035] Further, in step (1), the *Anoectochilus roxburghii* plant is disinfected with 0.1% mercuric chloride for 2–3 minutes.

[0036] Furthermore, the budding culture medium in step (2) includes: MS medium, activated carbon at a concentration of 3 g / L, sucrose at a concentration of 30 g / L, agar at a concentration of 6 g / L, and kinetin at a concentration of 0.2 mg / L.

[0037] Further, the proliferation and growth medium in step (3) includes: 1 / 4 MS medium, agar at a concentration of 6 g / L, sucrose at a concentration of 30 g / L, NAA at a concentration of 2 mg / L, and IBA at a concentration of 1.5 mg / L.

[0038] Furthermore, in step (4), the ratio of straw fragments to peat moss mixed substrate is 2:1.

[0039] Furthermore, in step (5), the ratio of straw fragments to peat moss mixed substrate is 1:2.

[0040] The experimental material used in this study was *Anoectochilus roxburghii* from Fujian. Tissue culture was primarily conducted on stem segments with nodes to investigate the effects of different hormone concentrations and ratios on bud germination. *Anoectochilus roxburghii* seedlings were used as tissue culture material to study the effects of different hormone concentrations on rooting.

[0041] Explant treatment:

[0042] Select healthy Anoectochilus roxburghii plants, wash the soil off the roots with tap water, disinfect with 75% alcohol for 10-15 seconds, then disinfect with 0.1% mercuric chloride for 2-3 minutes, shake the container to ensure thorough disinfection, rinse with sterile water 3-4 times, and cut the stem segments with nodes, about 2-3 cm in length, with a scalpel that has been flammed over an alcohol lamp. Place the segments on a clean bench for ultraviolet disinfection for about 20 minutes.

[0043] Cultivation conditions:

[0044] The inoculated *Anoectochilus roxburghii* explants were placed in a culture temperature of (25±2)℃, and dark conditions were mainly used for inducing clustered shoots. Before transplanting, full light was provided with a light intensity of 1500–2000 lx.

[0045] Effects of different hormone concentrations and ratios on bud germination:

[0046] Sterilized explant stem segments were inoculated into media numbered 1, 3, 4, 8, and 10, for a total of 5 treatments (detailed formulations are shown in Table 1), with 25 replicates per treatment. Adventitious shoots were induced by adding 30 g / L sucrose, 6 g / L agar, and 3 g / L activated charcoal to MS medium. After 40 days, the total number of shoots, shoot length, and number of roots were recorded to screen for the optimal medium formulation for inducing shoot germination.

[0047] Table 1. Culture medium combinations for inducing adventitious buds from *Anoectochilus roxburghii* stem segments.

[0048]

[0049] The effect of different hormone concentrations on rooting:

[0050] In a sterile environment under a laminar flow hood, rooted buds (2-3 cm in length) were harvested from aseptic tissue-cultured *Anoectochilus roxburghii* seedlings and inoculated onto culture media numbered S1-S9 to induce bud proliferation and growth. Nine treatments were administered (detailed treatment formulations are shown in Table 2), with 20 replicates per treatment. The basal culture media included MS, 1 / 2 MS, and 1 / 4 MS, supplemented with 30 g / L sucrose and 7.8 g / L agar. The growth and development of the *Anoectochilus roxburghii* buds were observed and recorded. After 45 days, the number of roots, root length, rooting rate, and seedling height were counted to determine the optimal hormone ratio for inducing proliferation and growth.

[0051] Table 2. Culture medium combinations for inducing the proliferation of clustered shoots of Anoectochilus roxburghii.

[0052]

[0053] The effect of substrate ratio on the transplanting effect of Anoectochilus roxburghii test-tube seedlings:

[0054] Designing different straw fragments: A comparative analysis of the effects of transplanting *Anoectochilus roxburghii* test-tube seedlings and cutting and propagating *Anoectochilus roxburghii* stem segments after survival and growth;

[0055] Effects of different hormone concentrations and ratios on stem bud germination and rooting:

[0056] Aseptic stem segments with nodes from *Anoectochilus roxburghii* were inoculated into prepared culture medium, with a blank control group prepared on MS medium. Approximately 7 days later, 1-2 white buds sprouted from the inoculated stem nodes, with aerial roots beginning to develop at the base of the buds; these aerial roots were white. After 10 days, terminal buds emerged, and around 20 days later, dark green new leaves appeared. The buds elongated to 2-3 stem nodes, and by day 40, the bud induction rate of the inoculated stem segments approached 100%. The buds were generally robust, with large, dark green leaves, and robust white roots emerging from the base of the buds. Detailed statistical results of the experiment are shown in Table 3.

[0057] Table 3 Effects of hormone types and ratios on bud induction

[0058]

[0059] Values ​​labeled with different letters showed significant differences at the P=0.05 level, and data were compared in columns. Table 3 shows that, compared to the MS blank control group, the culture media supplemented with cytokinin KT, 2,4-D, and auxin NAA and IBA all had a positive effect on bud elongation and the germination of clustered buds, with highly significant differences among treatments. Experimental data showed that the highest average bud length was achieved on medium 10 (1.968 cm), followed by medium 4 (1.605 cm), and the lowest was on medium 3 (1.033 cm). Regarding the average number of buds, the highest was on medium 1 (2.000), followed by medium 4 (1.920), and the lowest was on medium 8 (0.840). In terms of growth coefficient, mediums 1 and 4 had the highest growth coefficients, both at 2. Furthermore, the addition of 3% activated carbon to the culture medium appropriately inhibited the growth of *Anoectochilus roxburghii*, reduced fungal damage, significantly thickened buds, and resulted in mostly dark green leaves.

[0060] Effects of different hormone ratios on the proliferation and growth of clustered shoots:

[0061] Tissue blocks of 2-3 cm long rooted buds were taken from aseptic tissue-cultured *Anoectochilus roxburghii* seedlings and inoculated into a growth medium. After about 7 days, white protrusions appeared at the stem nodes, root hairs grew at the base, and the roots further elongated, with an increase in the number of root hairs. Results were observed and recorded after 45 days. Table 4 shows that in terms of vegetative growth, the average height increase was greatest in medium S8 (2.033 cm), followed by medium S7 (1.302 cm), and smallest in medium S3 (0.300 cm). The average number of newly added leaves was also greatest in medium S8 (1.350 leaves), followed by medium S7 (1.050 leaves), and lowest in medium S6 (0.600 leaves). Medium S1, S4, and S5 were at similar levels. The number of newly added buds was highest in treatment S8, followed by S4, and lowest in treatment S6.

[0062] The effect of auxin concentration on root induction in seedlings was determined using orthogonal design, as shown in Table 4. T1, T2, and T3 represent the total number of new roots under the same influencing factor (concentration levels of macroelements and NAA in MS medium). X1, X2, and X3 are the average values ​​under the same influencing factor level. Comparing the differences between the maximum and minimum values ​​of the influencing factor, it can be seen that the macroelement content in the basal medium has a greater impact on root induction than the NAA content. When the hormone type remains constant, the number of new roots per plant and the average root length increase with decreasing macroelements in MS, 1 / 2 MS, and 1 / 4 MS. Therefore, the most suitable basal medium for root growth is 1 / 4 MS medium. Taking the maximum value (T3X2) of T1, T2, T3, and X1, X2, X3, a suitable medium is 1 / 4 MS medium (Number 8) + 2 mg / L NAA + 1.5 mg / L IBA, which is suitable for root induction and growth.

[0063] Table 4. Effects of different hormone ratios on the proliferation and growth of clustered buds.

[0064]

[0065]

[0066] The effect of different substrate ratios on the transplanting effect of Anoectochilus roxburghii test-tube seedlings:

[0067] Table 5. Effects of different substrates on the transplanting of *Anoectochilus roxburghii* test-tube seedlings

[0068]

[0069]

[0070] Different substrates significantly affected the overall growth of *Anoectochilus roxburghii* test-tube seedlings after transplanting (see Table 5). Relatively speaking, the 2:1 ratio of straw fragments to peat moss showed the best overall performance, with the highest survival rate, increased bud length, increased number of buds, and number of leaves, resulting in robust plants with dense foliage. Pure peat moss, on the other hand, performed the worst, with all statistical indicators at their lowest values. This was mainly manifested in robust plants with glossy black leaves, but also with a significant amount of rotten leaves and branches.

[0071] The effect of different substrates on the propagation effect of Anoectochilus roxburghii stem cuttings:

[0072] Table 6. Effects of different substrates on stem cuttings of Anoectochilus roxburghii.

[0073]

[0074]

[0075] Table 6 shows the effects of *Anoectochilus roxburghii* stem cuttings under different substrate conditions. The 1:2 ratio of straw fragments to peat moss showed the best overall performance, with the highest survival rate, bud length, leaf number, and root growth. In contrast, peat moss promoted bud elongation and increased leaf number but hindered root growth, even causing some rot, possibly due to excessive moisture and nutrients. Adding straw fragments improved aeration and promoted root elongation.

[0076] In tissue culture, controlling the cleanliness of the operating environment and culture room, and minimizing contamination, is crucial for obtaining large-scale sterile tissue culture seedlings. Common types of contamination include bacterial and fungal contamination. Bacterial contamination is characterized by a yellow or white, gelatinous substance with an unpleasant odor. Fungal contamination is mainly white, yellow, or grayish-black, with a fuzzy appearance and a musty smell. Fungi reproduce by spores; fungal hyphae extend into the surrounding air, releasing a large number of spores, which exacerbates the contamination of the culture medium. Bacterial or fungal colonies can generally be observed with the naked eye 4-5 days after inoculation. Besides fungal infection of the explant surface and during experimental procedures, endophytic bacteria also exist within the plant.

[0077] Thoroughly scrubbing the explants removes most microorganisms and debris. Simultaneously, the penetrating power of 75% alcohol allows it to penetrate the surface of the explants, facilitating thorough contact and disinfection with subsequent disinfectants, thus significantly enhancing the disinfection effect. Applying ultraviolet light for 20-30 minutes helps to more thoroughly kill any remaining microorganisms, especially endophytes that surface disinfection cannot kill, achieving a good killing effect.

[0078] Browning of the culture medium: During the rooting culture of *Anoectochilus roxburghii*, the phenolic substances secreted from the cut buds are oxidized to quinones, causing browning of the culture medium. If browning is not treated promptly, it will reduce the viability of *Anoectochilus roxburghii* and inhibit its growth and development. The main ways to combat the harmful effects of browning on tissue culture seedlings are: accelerating the timely transfer of browned seedlings from the culture medium; generally, the browning phenomenon will weaken after multiple transfers to fresh culture medium; adding a certain concentration of activated charcoal (0.1%–0.5%) can reduce browning of the culture medium; and using antioxidants such as citric acid and ascorbic acid can also alleviate browning symptoms.

[0079] In summary, the culture medium formula for promoting the formation of shoot clusters from stem segments is MS medium + 6 g / L agar + 3 g / L activated carbon + 30 g / L sucrose + 0.2 mg / L KT. The suitable culture medium formula for promoting the proliferation and growth of shoot cluster tissue blocks is 1 / 4 MS medium + 7.8 g / L agar + 30 g / L sucrose + 2 mg / L NAA + 1.5 mg / L IBA. The optimal substrate ratio for transplanting test-tube seedlings is a mixture of straw fragments and peat moss (2:1). However, for stem segment cuttings of *Anoectochilus roxburghii* plants that have survived transplanting and matured, the optimal substrate ratio is a mixture of straw fragments and peat moss (1:2).

[0080] To protect the precious Chinese medicinal herb *Anoectochilus roxburghii* and prevent its wild species from becoming extinct, people have been using tissue culture and asexual reproduction to obtain large quantities of tissue-cultured seedlings, thus achieving the protection of the *Anoectochilus roxburghii* species. Future research should focus on developing more suitable tissue culture methods for *Anoectochilus roxburghii* growth with the lowest possible cost, thereby promoting the development of the *Anoectochilus roxburghii* industry and increasing the income of growers.

[0081] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. All should be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A kind of anaphalis contorta cluster bud transplanting cultivation and circulates cuttage high-efficiency breeding method, it is characterized in that, The method comprises the following steps: (1) After the leaves of the Anoectochilus roxburghii plant are removed, the soil on the rhizome surface of the Anoectochilus roxburghii plant is washed, the Anoectochilus roxburghii plant is brushed with a brush to fully wet the Anoectochilus roxburghii plant, the Anoectochilus roxburghii plant is placed in a container, 75% alcohol is added to the container, the container is placed on an ultraclean bench, the Anoectochilus roxburghii plant is sterilized by permeating 75% alcohol, the 75% alcohol in the container is replaced with 0.1% mercury, the container is shaken, the Anoectochilus roxburghii plant is sterilized by 0.1% mercury, the Anoectochilus roxburghii plant in the container is taken out, the taken-out Anoectochilus roxburghii plant is washed with sterile water, a stem segment with a length of 2-3 cm and a node is cut from the Anoectochilus roxburghii plant, and the stem segment with the node is placed on the ultraclean bench and sterilized by a UV lamp. (2) The stem segment with the node is inoculated into a cluster bud germination culture medium and placed in a dark condition to induce Anoectochilus roxburghii cluster buds. (3) When the Anoectochilus roxburghii cluster buds grow to 2-3 cm, the cluster buds are cut into 0.5-1 cm pieces with callus tissue, the pieces are transferred into a proliferation growth culture medium, and the pieces are placed in a dark condition to induce bud growth to 4-5 cm, and then full light is provided to culture the pieces. (4) When the Anoectochilus roxburghii cluster buds grow to 5-6 cm, the cluster buds are directly transplanted into a hole tray containing a mixture of straw debris and peat substrate, and the hole tray is placed on a tidal seedbed to culture the cluster buds, and the substrate humidity is controlled to be 40-50%. (5) When the Anoectochilus roxburghii cluster buds grow to 8-10 cm, the cluster buds are cut to obtain segments with roots and hairs, the segments are cut and planted, a mixture of straw debris and peat substrate is used as a cutting substrate, the cutting substrate is placed in a 5 cm high seedling square tray with small holes at the bottom, and the seedling square tray is placed on a tidal seedbed to culture the cutting substrate, and the cutting substrate humidity is controlled to be 50-60%. (6) When the Anoectochilus roxburghii cutting plants grow to 8-10 cm, the cutting plants can be cut again to propagate seedlings. (7) The cutting Anoectochilus roxburghii seedlings can be used for large-scale production when the seedlings grow to 3-4 cm.

2. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (1), the Anoectochilus roxburghii plant is sterilized by permeating 75% alcohol for 10-15 s.

3. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (1), the stem segment with the node is placed on the ultraclean bench and sterilized by the UV lamp for 20 min.

4. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (1), the taken-out Anoectochilus roxburghii plant is washed with sterile water for 3-4 times.

5. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (1), the Anoectochilus roxburghii plant is sterilized by 0.1% mercury for 2-3 min.

6. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (2), the cluster bud germination culture medium comprises: MS culture medium, 3 g / L active carbon, 30 g / L sucrose, 6 g / L agar, and 0.2 mg / L kinetin.

7. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (3), the proliferation growth culture medium comprises: 1 / 4 MS culture medium, 6 g / L agar, 30 g / L sucrose, 2 mg / L NAA, and 1.5 mg / L IBA.

8. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (4), the mixture of straw debris and peat substrate is mixed at a ratio of 2:

1.

9. The method for efficient propagation of *Anoectochilus roxburghii* through transplanting and cyclic cuttings according to claim 1, characterized in that... In step (5), the mixture of straw debris and peat substrate is mixed at a ratio of 1:2.

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