Efficient artificial propagation method of dendrobium officinale

Abstract

The present application belongs to the technical field of plant induced regeneration, and particularly relates to a high-efficiency artificial propagation method of Dendrobium candidum, comprising the following steps: taking two-year-old stem segments with nodes as materials, cutting the stem segments into appropriate sizes, and disinfecting the stem segments; and culturing the stem segments in a culture medium A for 90 days, and then completing the proliferation and rooting integration through a direct organogenesis route. The present application realizes the cyclic production of Dendrobium candidum, and the whole culture process can be completed only by using one culture medium, greatly improving the artificial in-vitro propagation efficiency, and the proliferation coefficient is completely comparable to the seed asymbiotic germination method, and the problems of large offspring trait separation and different qualities caused by the seed asymbiotic germination are solved, and the quality and yield of seedlings are greatly improved. The present application can provide a brand-new technical support for the factory-scale growth of Dendrobium candidum, so as to achieve the purpose of cultivating seedlings with genetic stability and excellent quality to improve the artificial planting efficiency, and the cost is low, the cycle is short, and the quality and survival rate of seedlings are high.

Description

Technical Field

[0001] This invention belongs to the field of plant induced regeneration technology, specifically relating to a highly efficient artificial propagation method for Dendrobium officinale. Background Technology

[0002] Dendrobium officinale ( Dendrobium officinale Kimura et Migo, also known as black-jointed grass, belongs to the genus Dendrobium in the family Orchidaceae. Dendrobium A perennial epiphytic herb; growing on semi-shaded, damp rocks in mountainous areas at altitudes of 1600-2100 m, it prefers warm and humid climates, is not cold-hardy, and thrives in air humidity of 60%-80% with an optimal growing temperature of 20-25℃. It is distributed in Anhui, Zhejiang, Fujian, Guangxi, Sichuan, and Yunnan provinces of my country. The dried stem of *Dendrobium officinale*, commonly known as "Fengdou," was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior herb, earning the reputation of "the first of the nine immortal herbs." This species is a precious medicinal and edible plant in my country, with a sweet taste and slightly cold nature, possessing the effects of nourishing yin and clearing heat, promoting body fluid production and benefiting the stomach, moistening the lungs and relieving cough. It contains a diverse range of chemical components, including polysaccharides (one of the main active ingredients), as well as alkaloids, phenanthrenes, bibenzyl groups, fluorenes, sesquiterpenes, coumarins, steroids, and volatile oils, totaling more than 190 compounds. Dendrobium officinale is used to treat a variety of diseases, including Sjögren's syndrome, gastric ulcers, alcoholic liver damage, chronic obstructive pulmonary disease, diabetes, obesity, rheumatoid arthritis, hypertension, stroke, cataracts, physical weakness, and other sub-health conditions. In recent years, due to the continuous development of Dendrobium officinale-related products, it has gradually expanded into health supplements, skincare products, and other fields. High market demand has led to over-harvesting of wild resources. Coupled with its unique metabolic process—Crassulacean acid metabolism (CAM)—its low photosynthetic efficiency, long growth cycle, and difficulty in sexual reproduction, resulting in low fruit set and seed germination rates, wild resources are now extremely scarce. In 1992, it was listed as an endangered plant in the *China Plant Red Data Book*; internationally, it is listed in the *Convention on International Trade in Endangered Species of Wild Fauna and Flora* (CITES), and the trade of wild species is prohibited.

[0003] Dendrobium officinale has demanding habitat requirements; it cannot grow in soil and is mostly epiphytic on tree trunks or in rock crevices. Its unique metabolic process—Crassulacean acid metabolism (CAM)—distinguishes it from ordinary C3 plants. Photosynthesis occurs at night, relying on endophytic bacteria to produce nutrients and aerial roots to absorb moisture and nutrients from the air. This results in low photosynthetic intensity and a long growth cycle. Similar to most orchids, the seeds of Dendrobium officinale and other Dendrobium species are small and lack cotyledons and endosperm, leading to incomplete embryo development. Under natural conditions, germination requires the cooperation of related symbiotic bacteria, resulting in a long germination time and a low germination rate (only 5%), with an even lower seedling rate. From seed germination to flowering plants suitable for phenotypic identification, it generally takes 4-5 years, or even longer. Confusingly, more than a decade ago, numerous scholars and producers conducted research on the artificial rapid propagation of Dendrobium officinale. From the perspective of literature quality, most of the existing publications are in non-mainstream journals and have extremely low reproducibility. Furthermore, in practical production, propagation is mostly carried out using protocorms obtained from non-symbiotic seed germination, but this method suffers from drawbacks such as complex processes, long seedling cycles, weak regenerated plants with severe phenotypic segregation, and extremely low survival rates after direct cultivation. Therefore, the urgent need to find a new, low-cost, short-time, high-quality, high-survival-rate, and highly reproducible artificial propagation method has become an inevitable choice for the sustainable utilization and large-scale propagation of Dendrobium officinale. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing propagation technologies and provide a highly efficient artificial propagation method for Dendrobium officinale. This method enables the cyclical production of Dendrobium officinale, requiring only one culture medium for the entire cultivation process, greatly improving the efficiency of artificial in vitro propagation. The proliferation coefficient is comparable to that of non-symbiotic seed germination, and it solves the problems of large segregation and inconsistent quality in offspring caused by non-symbiotic seed germination, significantly improving seedling quality and yield. This direct organogenesis-type method, with its high proliferation coefficient and simple seedling formation, not only revolutionizes traditional Dendrobium officinale production methods but also allows for a cyclical production process due to the emergence of protocorms, eliminating the need for annual sowing or repeated explant culture, thus greatly simplifying the artificial propagation process. This invention provides a new technological support for the large-scale industrial growth of Dendrobium officinale, achieving the goal of cultivating genetically stable, high-quality seedlings to improve artificial cultivation efficiency, while also being low-cost, short-cycle, and producing high-quality seedlings with high survival rates.

[0005] To solve the above technical problems, the present invention adopts the following technical solution:

[0006] The efficient artificial propagation method of Dendrobium officinale is characterized by the following steps: taking two-year-old fresh stems, cutting them into appropriate sizes, sterilizing them, cutting them into appropriate sizes, and inoculating them into a culture medium for cultivation;

[0007] Furthermore, the efficient artificial propagation method of Dendrobium officinale includes the following steps:

[0008] (1) Take two-year-old fresh strips, cut them into appropriate sizes, disinfect and sterilize them, and then inoculate them into a culture medium for culture;

[0009] (2) Cut the stem segments from step (1) into appropriate sizes and disinfect them;

[0010] (3) The segmented stem that has been sterilized and disinfected in step (2) is inoculated into the following culture medium A, wherein culture medium A comprises the following raw materials:

[0011] 1 / 3 MS basic culture medium

[0012] Banana puree

[0013] Mashed potatoes

[0014] Naphthaleneacetic acid (NAA)

[0015] 6-Benzylaminopurine (6-BA)

[0016] Kinetin (KT)

[0017] Activated carbon (AC)

[0018] sucrose

[0019] Agar powder

[0020] By controlling the light intensity, temperature, and light duration during cultivation, sterile test-tube seedlings with a clump-like structure and a 100% rooting rate can be obtained. After three generations of continuous cultivation, approximately 6.0-8.0% of the stem segments in each generation produce protocorm clusters (PLBs) at the cut surfaces.

[0021] (4) Proliferation and development culture of protocorms (PLBs): The protocorm clusters that appeared in step (3) were collected and inoculated into fresh culture medium A. Under controlled light intensity, temperature and light duration, the protocorms (PLBs) were proliferated and the seedlings were developed.

[0022] (5) The bud clusters in step (4) are divided into clumps of 2-3 plants each and cultured in fresh culture medium A until they become seedlings, while the protocorms (PLBs) are cultured in fresh culture medium A to continue to proliferate and develop.

[0023] (6) Hardening and transplanting: Take the seedlings from step (3) or (5) and harden them at room temperature for 3 days, then open the bottle cap and harden them for another 2 days; take the seedlings out of the culture medium, clean the residual culture medium, soak them in carbendazim solution, and then transplant them into sterilized pine bark for warm and moist culture to obtain transplanted seedlings.

[0024] Further, the stem segment disinfection method described in step (2) is as follows: Cut the fresh stems into 1.5-2.0 cm long pieces with 2 nodes, remove the leaves, wash with running water, soak in 10% laundry detergent solution (w / v) for 10 min, rinse with running water for 30 min, and place on a clean bench. Treat with 75% ethanol solution (v / v) for 10-15 s, and 0.1% mercuric chloride aqueous solution (w / v) for 10-12 min, shaking the bottle continuously during the process to achieve the best sterilization effect. Finally, rinse with sterile water 3 times, each time for no less than 3 min; place on sterile absorbent paper, absorb the surface moisture, and then cut off about 0.2 cm at the base of the stem with a sterile scalpel. Inoculate vertically into culture medium A in the physiological direction.

[0025] Further, the culture medium A mentioned in step (3) includes the following raw materials:

[0026] 1 / 3 MS basic culture medium

[0027] Banana puree 60000 mg / L

[0028] Mashed potatoes 40000 mg / L

[0029] Naphthaleneacetic acid (NAA) 1.0-1.5 mg / L

[0030] 3-Indolebutyric acid (IBA) 1.0-1.5 mg / L

[0031] 6-Benzylaminopurine (6-BA) 0.1-0.5 mg / L

[0032] Kinetin (KT) 0.1-0.5 mg / L

[0033] Activated carbon (AC) 1000 mg / L

[0034] sucrose 15000 mg / L

[0035] Agar powder 4700 mg / L.

[0036] Furthermore, the pH value of the culture medium A is 5.4-5.6.

[0037] Furthermore, the protocorms (PLBs) from step (3) are collected and transferred to fresh culture medium A for proliferation and development into seedlings.

[0038] Further, the bud clusters from step (4) were divided into clusters of 2-3 plants each and cultured in fresh culture medium A until they became seedlings, while the protocorms (PLBs) were cultured in fresh culture medium A to continue to proliferate and develop.

[0039] Furthermore, the mass concentration of the carbendazim solution in step (6) is 0.1-0.5%.

[0040] Furthermore, in step (6), the size of the pine bark fragments is 1.5 × 1.5 cm, the disinfection method is boiling water for 2-3 hours, the cultivation temperature is (25 ± 2) ℃, and the humidity is 60-80%.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The present invention uses tissue culture technology to achieve year-round production in the culture room, which saves land resources and improves economic efficiency, and overcomes the difficulty of traditional vegetative propagation methods that cannot carry out year-round production.

[0043] (2) This invention solves the problems of separation of maternal traits, long cycle, weak regenerated seedlings and difficulty in survival caused by non-symbiotic germination of seeds in Dendrobium plants. Through this invention, a large number of seedlings can be obtained in a short time, with low cost, short cycle, easy standardization and industrial operation, effectively improving the quality of seedlings, solving the problem of Dendrobium officinale germplasm resource protection, and providing high-quality seedlings with unified standards for its large-scale promotion and planting.

[0044] (3) The present invention can complete the proliferation and rooting process by direct organogenesis in only one culture medium, which can meet the needs of large-scale production while maintaining the excellent traits of the parent plant. It is the most effective proliferation method for artificial rapid propagation and improves the quality of seedlings.

[0045] (4) This invention achieves the goal of efficient and rapid propagation. A propagation culture cycle is 90 days. The propagation coefficient of seedlings produced directly from stem segments can reach more than 10.0; while the propagation coefficient of seedlings produced through protocorms (PLBs) can reach more than 30.0; the sum of the two can reach more than 40.0.

[0046] (5) This invention confirms that Dendrobium officinale stem segments have a strong regeneration ability. As long as the conditions are suitable (appropriate culture medium) and the calculation is in years, its proliferation efficiency can be comparable to that of the seed-protocorm-regenerated seedling pathway. Moreover, it avoids the phenotypic differences caused by separation, thereby obtaining regenerated seedlings with consistent genetic background, which is something that the seed-protocorm-regenerated seedling pathway cannot achieve.

[0047] (6) The present invention can repeatedly cycle from stem segments to axillary bud clusters and then to the production of protocorms (PLBs). In addition, the integrated cultivation of proliferation and rooting allows the production of Dendrobium officinale to be completely controlled by human intervention, with high efficiency and short cycle. At the same time, since protocorms (PLBs) are produced in each cycle, the problem of them gradually disappearing with the increase of the number of transfer generations can be avoided.

[0048] (7) The Dendrobium officinale test-tube seedlings produced by this invention have a high transplant survival rate and grow rapidly because each clump / plant has an exceptionally well-developed adventitious root system. They have been demonstrated and cultivated at the cultivation base of Xiangxin Agricultural Technology Development Co., Ltd. in Yuxi City, Yunnan Province (102°31′25″ E, 24°26′22″ N, Alt: 1688 m), with very good results.

[0049] (8) This invention is of great significance and value for the rapid artificial propagation of Dendrobium officinale, and can also provide technical reference for the rapid propagation of other Dendrobium species. Attached Figure Description

[0050] Figure 1 Diagrams showing the various growth stages of integrated stem segment propagation and rooting culture;

[0051] Among them, 1A shows the growth after 30 days of culture; 1B shows the growth after 45 days of culture; 1C shows the growth after 60 days of culture; 1D shows the growth after 75 days of culture; and 1E and 1F show the growth after 90 days of culture.

[0052] Figure 2 Diagrams showing the various growth stages of protocormoids (PLBs) during their occurrence, proliferation, and seedling formation;

[0053] In this diagram, 2A shows the source of the culture material; 2B shows the growth of the protocormoids immediately after inoculation into the culture medium; 2C shows the growth after 30 days of culture; 2D and 2E show the growth after 45 days of culture; 2F and 2G show the growth of a single protocormoid after 45 days of culture; 2H, 2G, and 2I show the growth after 60 days of culture; 2J and 2K show the growth of the clustered buds developed from the protocormoids after 30 days of culture; and 2L shows the growth of the clustered buds developed from the protocormoids after 60 days of culture.

[0054] Figure 3 Diagrams showing the various growth stages of transplanted plants after domestication;

[0055] Among them, 3A shows the growth status 60 days after transplanting; 3B shows the growth status 180 days after transplanting; 3C shows the growth status 240 days after transplanting; and 3D shows the growth status 16-17 months after transplanting.

[0056] Figure 4 This diagram illustrates the regeneration of Dendrobium officinale stem segments and the cyclic regeneration of protocorms (PLBs). Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0058] Based on this invention, the efficient artificial propagation method of Dendrobium officinale includes the following steps:

[0059] S1 Select healthy plants with good growth and no pests or diseases. Take fresh stems that have formed apical buds from two-year-old plants and cut them into 1.5-2.0cm stem segments with nodes, each with 2 nodes. Disinfect and set aside for use.

[0060] S2. Inoculate the treated stem segments with nodes into culture medium A, and culture at 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day for 90 days to obtain clump-like, sterile plantlets with 100% rooting rate (see details for each growth stage). Figure 1 ), and protocorm-like clusters are produced at the stem segment cut;

[0061] like Figure 1 As shown (scale bar = 2.0 cm), from Figure 1 As can be seen in A, after 30 days of cultivation, axillary buds on both the upper and lower nodes of the stem segment sprouted into new branches, and new leaves began to unfold; from Figure 1 As can be seen in B, after 45 days of cultivation, the new shoots elongated further, the leaves grew larger, and adventitious roots began to appear; from Figure 1 As can be seen in section C, after 60 days of cultivation, with the rapid growth of adventitious roots, 2-4 tillers began to appear at the basal nodes of each material; from Figure 1 As can be seen in D, after 75 days of cultivation, the barberry seedlings grew vigorously, almost reaching the height of the axillary buds, at which point they were ready for exposure to the open for acclimatization; from Figure 1 As seen in E and 1F, continued culture for 90 days yields sterile test-tube seedlings with a clump-like growth pattern and a 100% rooting rate, at which point the proliferation coefficient reaches 11.25. After three generations of continuous culture, approximately 6.0-8.0% of the stem segments at the cut site produce protocorm clusters (PLBs) in each generation.

[0062] S3. Collect the protocorm clusters produced in each generation of S2 and inoculate them into fresh culture medium A. Maintain a light intensity of 1800-2500 lx and a daily light exposure of 9-11 h for 60 days to form a protocorm / clustered bud mixture with a proliferation coefficient >30 (see details for each growth stage). Figure 2 );

[0063] like Figure 2 As shown (Figure AL scale = 2cm; Figure FH scale = 0.2cm), where, from Figure 2 As can be seen in Figure C, after 30 days of cultivation, the small clusters significantly enlarged and small buds appeared; from... Figure 2 As seen in D and 2E, after 45 days of cultivation, the small clusters continued to swell into patches with villous rhizoids, upon which numerous small spheres appeared, accompanied by the emergence of conical pointed leaves; from Figure 2 As seen in F and 2G, when the small spheres are peeled off, obvious bipolar growth can be observed, thus identifying them as protocorms; from Figure 2 H, 2G, and 2I show that after 60 days of culture, these protocorms not only develop into seedlings but also proliferate to a certain extent. At this point, the entire surface of the culture medium is covered with a mixture of seedlings and protocorms, with a proliferation coefficient exceeding 30.0. Figure 2 As seen in J and 2K, when the bud clusters developed from protocorms were cut into 3-4 plants per cluster and transferred to fresh culture medium, the bud clusters began to turn green and exhibited a "bud-to-bud" phenomenon after 30 days; from Figure 2 As can be seen, the multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 seedlings per clump, and transferred to an integrated culture medium for propagation and rooting. After 60 days of cultivation, it can be acclimatized and transplanted.

[0064] S4. Transfer the robust buds in the mixture into the culture medium to form rooted seedlings. Repeat S3-S4 with the protocorms in the mixture. After 30 days, the robust buds in the culture medium begin to turn green and exhibit the phenomenon of "bud-to-bud" growth. After 60 days of culture, a multi-bud cluster culture is formed. The protocorms can be recycled throughout the entire production process. In addition, the integrated culture of propagation and rooting allows for the artificial control of the ratio of propagated seedlings to commercial seedlings, greatly simplifying the artificial propagation process.

[0065] S5. After hardening off the rooted seedlings from S4 at room temperature for 3-5 days, sterilize them and transfer them into a substrate for warm and moist cultivation to obtain transplanted seedlings (see details for each growth stage). Figure 3 );

[0066] like Figure 3 As shown (scale bar = 1.0 cm), from Figure 3 As can be seen in Figure A, the test-tube seedlings showed obvious signs of growth 60 days after transplanting, with elongated internodes, larger and more expanded leaves, and a survival rate of over 90%. Figure 3 As can be seen in B, after 180 days, lateral buds at the base of the plant continuously develop and grow to about 5.0 cm, the stem nodes thicken, and the leaves become larger; from Figure 3 As can be seen in Figure C, after 240 days, the leaf edges turned light red, the stem nodes turned light black, and the plant grew vigorously; from Figure 3 As can be seen in D, by April to May of the second year after transplanting, the branches are basically covered with flowers, and at this time, spring buds also continue to emerge from the base of the plant, showing promising growth.

[0067] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0068] Example 1

[0069] The efficient artificial propagation method of Dendrobium officinale includes the following steps:

[0070] (1) Obtaining explants: After two years of continuous observation, select healthy plants with good growth and no pests or diseases, and take fresh shoots that have formed apical buds after two years.

[0071] (2) Cut the fresh stems from step (1) into pieces 1.5-2.0 cm long with two nodes. Remove the leaves and wash them with running water. Soak them in a 10% laundry detergent solution (w / v) for 10 min, then rinse them with running water for 30 min. Place them on a clean bench. Treat them with a 75% ethanol solution (v / v) for 10-15 s, and a 0.1% mercuric chloride solution (w / v) for 10 min, shaking the bottle continuously to achieve the best sterilization effect. Finally, rinse them three times with sterile water for at least 3 min each time. Place them on sterile absorbent paper to dry the surface moisture, and then cut off about 0.2 cm from the base of the stem with a sterile scalpel.

[0072] (3) The segmented stem that has been sterilized in step (2) is inoculated into the following culture medium A, wherein culture medium A includes the following raw materials:

[0073] 1 / 3 MS basic culture medium

[0074] Banana puree 60000 mg / L

[0075] Mashed potatoes 40000 mg / L

[0076] Naphthaleneacetic acid (NAA) 1.0 mg / L

[0077] 3-Indolebutyric acid (IBA) 1.0 mg / L

[0078] 6-Benzylaminopurine (6-BA) 0.1 mg / L

[0079] Kinetin (KT) 0.1 mg / L

[0080] Activated carbon (AC) 1000 mg / L

[0081] sucrose 15000 mg / L

[0082] Agar powder 4700 mg / L

[0083] pH 5.4

[0084] Culture conditions: Under light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature controlled at 22±1℃, after 30 days of culture, axillary buds on both the upper and lower nodes of the stem segment sprouted into new branches, and new leaves began to unfold. After 45 days of culture, the new shoots further elongated, the leaves grew larger, and adventitious roots began to appear. After 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillers began to appear at the basal stem node of each material. After 75 days of culture, the tillers grew vigorously, almost reaching the height of the axillary buds, and at this time they could be exposed for acclimatization. Continuing culture to 90 days, the test-tube seedlings were tall and robust, with fully unfolded leaves and well-developed adventitious root systems, at which point the proliferation coefficient reached 11.25. After three generations of continuous culture, approximately 6.0-8.0% of the stem segment cuts produced protocormoids (PLBs) in each generation.

[0085] (4) Proliferation and development culture of protocorms (PLBs): The protocorm clusters that appeared in step (3) were collected and inoculated into fresh culture medium A. Under the conditions of light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control at 22±1℃, these nodular clusters were collected and divided into 0.5×0.5 cm pieces and transferred to fresh culture medium A. After 30 days of culture, the small clusters swelled significantly and small buds appeared. After 45 days of culture, the small clusters continued to swell into patches with villous rhizoids, which had many small spheres and conical pointed leaves. After 60 days of culture, these protocorms developed into seedlings and also proliferated to a certain extent. At this time, the entire surface of the culture medium was covered with a mixture of seedlings and protocorms, and the proliferation coefficient could reach 32.50. At this time, the bud clusters developed from the protocorms could be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 60 days, the buds begin to turn green and exhibit the phenomenon of "buds multiplying". The multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 seedlings per cluster, transferred to fresh culture medium A, and cultured for 60 days before acclimatization and transplanting.

[0086] (5) Hardening and transplanting: Take the seedlings from step (3) or (4) and harden them at room temperature for 3 days, then open the bottle cap and harden them for another 2 days. Take the seedlings out of the culture medium, clean the residual culture medium, disinfect them in a 0.5% carbendazim solution for 3 minutes, and then transplant them into flower pots with pine bark (about 1.5×1.5 cm) that has been boiled in boiling water for 2.5 hours as the substrate. Keep them warm (25±2℃) and moist (60-80%) for 60 days. The transplanted seedlings have a survival rate of 100%.

[0087] Example 2

[0088] The efficient artificial propagation method of Dendrobium officinale includes the following steps:

[0089] (1) Obtaining explants: After two years of continuous observation, select healthy plants with good growth and no pests or diseases, and take fresh shoots that have formed apical buds after two years.

[0090] (2) Cut the fresh stems from step (1) into pieces 1.5-2.0 cm long with 2 nodes. Remove the leaves and wash with running water. Soak in 10% laundry detergent solution (w / v) for 10 min, then rinse with running water for 30 min. Place on a clean bench. Treat with 75% ethanol solution (v / v) for 10-15 s, and 0.1% mercuric chloride solution (w / v) for 11 min, shaking the bottle continuously to achieve the best sterilization effect. Finally, rinse with sterile water 3 times, each time for no less than 3 min. Place on sterile absorbent paper to absorb the surface moisture, and then cut off about 0.2 cm from the base of the stem with a sterile scalpel.

[0091] (3) The segmented stem that has been sterilized in step (2) is inoculated into the following culture medium A, wherein culture medium A includes the following raw materials:

[0092] 1 / 3 MS basic culture medium

[0093] Banana puree 60000 mg / L

[0094] Mashed potatoes 40000 mg / L

[0095] Naphthaleneacetic acid (NAA) 1.2 mg / L

[0096] 3-Indolebutyric acid (IBA) 1.2 mg / L

[0097] 6-Benzylaminopurine (6-BA) 0.3 mg / L

[0098] Kinetin (KT) 0.3 mg / L

[0099] Activated carbon (AC) 1000 mg / L

[0100] sucrose 15000 mg / L

[0101] Agar powder 4700 mg / L

[0102] pH 5.6

[0103] Culture conditions: Under light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature controlled at 22±1℃, after 30 days of culture, axillary buds on both the upper and lower nodes of the stem segment sprouted into new branches, and new leaves began to unfold. After 45 days of culture, the new shoots further elongated, the leaves grew larger, and adventitious roots began to appear. After 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillers began to appear at the basal stem node of each material. After 75 days of culture, the tillers grew vigorously, almost reaching the height of the axillary buds, and at this time they could be exposed for acclimatization. Continuing culture to 90 days, the test-tube seedlings were tall and robust, with fully unfolded leaves and well-developed adventitious root systems, at which point the proliferation coefficient reached 11.45. After three generations of continuous culture, approximately 6.0-8.0% of the stem segment cuts produced protocormoids (PLBs) in each generation.

[0104] (4) Proliferation and development culture of protocorms (PLBs): The protocorm clusters that appeared in step (3) were collected and inoculated into fresh culture medium A. Under the conditions of light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control at 22±1℃, these nodular clusters were collected and divided into 0.5×0.5 cm pieces and transferred to fresh culture medium A. After 30 days of culture, the small clusters swelled significantly and small buds appeared. After 45 days of culture, the small clusters continued to swell into patches with villous rhizoids, which had many small spheres and conical pointed leaves. After 60 days of culture, these protocorms developed into seedlings and also proliferated to a certain extent. At this time, the entire surface of the culture medium was covered with a mixture of seedlings and protocorms, and the proliferation coefficient could reach 31.50. At this time, the bud clusters developed from the protocorms could be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 60 days, the buds begin to turn green and exhibit the phenomenon of "buds multiplying". The multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 seedlings per cluster, transferred to fresh culture medium A, and cultured for 60 days before acclimatization and transplanting.

[0105] (5) Hardening and transplanting: Take the seedlings from step (3) or (4) and harden them at room temperature for 3 days, then open the bottle cap and harden them for another 2 days. Take the seedlings out of the culture medium, clean the residual culture medium, put them in a 0.3% carbendazim solution for 3 min for disinfection, and then transplant them into flower pots with pine bark (about 1.5×1.5 cm) that has been boiled in boiling water for 3 h as the substrate. Keep them warm (25±2℃) and moist (60-80%) for 60 days. The transplanted seedlings have a survival rate of 100%.

[0106] Example 3

[0107] The efficient artificial propagation method of Dendrobium officinale includes the following steps:

[0108] (1) Obtaining explants: After two years of continuous observation, select healthy plants with good growth and no pests or diseases, and take fresh shoots that have formed apical buds after two years.

[0109] (2) Cut the fresh stems from step (1) into pieces 1.5-2.0 cm long with two nodes. Remove the leaves and wash them with running water. Soak them in a 10% laundry detergent solution (w / v) for 10 min, then rinse them with running water for 30 min. Place them on a clean bench. Treat them with a 75% ethanol solution (v / v) for 10-15 s, and a 0.1% mercuric chloride solution (w / v) for 12 min, shaking the bottle continuously to achieve the best sterilization effect. Finally, rinse them three times with sterile water for at least 3 min each time. Place them on sterile absorbent paper to dry the surface moisture, and then cut off about 0.2 cm from the base of the stem with a sterile scalpel.

[0110] (3) The segmented stem that has been sterilized in step (2) is inoculated into the following culture medium A, wherein culture medium A includes the following raw materials:

[0111] 1 / 3 MS basic culture medium

[0112] Banana puree 60000 mg / L

[0113] Mashed potatoes 40000 mg / L

[0114] Naphthaleneacetic acid (NAA) 1.5 mg / L

[0115] 3-Indolebutyric acid (IBA) 1.5 mg / L

[0116] 6-Benzylaminopurine (6-BA) 0.5 mg / L

[0117] Kinetin (KT) 0.5 mg / L

[0118] Activated carbon (AC) 1000 mg / L

[0119] sucrose 15000 mg / L

[0120] Agar powder 4700 mg / L

[0121] pH 5.4

[0122] Culture conditions: Under light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature controlled at 22±1℃, after 30 days of culture, axillary buds on both the upper and lower nodes of the stem segment sprouted into new branches, and new leaves began to unfold. After 45 days of culture, the new shoots further elongated, the leaves grew larger, and adventitious roots began to appear. After 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillers began to appear at the basal stem node of each material. After 75 days of culture, the tillers grew vigorously, almost reaching the height of the axillary buds, and at this time they could be exposed for acclimatization. Continuing culture to 90 days, the test-tube seedlings were tall and robust, with fully unfolded leaves and well-developed adventitious root systems, at which point the proliferation coefficient reached 11.85. After three generations of continuous culture, approximately 6.0-8.0% of the stem segment cuts produced protocormoids (PLBs) in each generation.

[0123] (4) Proliferation and development culture of protocorms (PLBs): The protocorm clusters that appeared in step (3) were collected and inoculated into fresh culture medium A. Under the conditions of light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control at 22±1℃, these nodular clusters were collected and divided into 0.5×0.5 cm pieces and transferred to fresh culture medium A. After 30 days of culture, the small clusters swelled significantly and small buds appeared. After 45 days of culture, the small clusters continued to swell into patches with villous rhizoids, with many small spheres on them, accompanied by conical pointed leaves. After 60 days of culture, these protocorms developed into seedlings and also proliferated to a certain extent. At this time, the entire surface of the culture medium was covered with a mixture of seedlings and protocorms, and the proliferation coefficient could reach 31.65. At this time, the bud clusters developed from the protocorms could be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 60 days, the buds begin to turn green and exhibit the phenomenon of "buds multiplying". The multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 seedlings per cluster, transferred to fresh culture medium A, and cultured for 60 days before acclimatization and transplanting.

[0124] (5) Hardening and transplanting: Take the seedlings from step (3) or (4) and harden them at room temperature for 3 days, then open the bottle cap and harden them for another 2 days. Take the seedlings out of the culture medium, clean the residual culture medium, put them in a 0.1% carbendazim solution for 3 minutes to disinfect, and then transplant them into flower pots with pine bark (about 1.5×1.5 cm) that has been boiled in boiling water for 2 hours as the substrate. Keep them warm (25±2℃) and moist (60-80%) for 60 days. The transplanted seedlings have a survival rate of 100%.

[0125] Technical principle of the invention:

[0126] 1. The use of Dendrobium officinale stem segments as explants ensures stable genetic characteristics and solves the problem of germplasm degeneration caused by genetic drift and inbreeding.

[0127] 2. According to the survey, in the actual production of Dendrobium officinale in Yunnan, the seed-protocorm-regenerated seedling route is adopted. Sowing is done annually in September and October. Protocorms produced through non-symbiotic germination of seeds take 12 months or even longer to produce transplantable test-tube seedlings. While this method seems inefficient, the yield is considerable as long as sowing is done annually. The biggest drawback of this method is uneven germination and severe separation of seed and seedling traits. However, the principle of prioritizing yield makes the seed-protocorm-regenerated seedling route commercially advantageous, which is one of the main reasons why it is mostly used in factory production. This invention uses stem segments with two nodes as material. Simultaneously with the development of two terminal buds, as adventitious roots become more developed, their bases gradually thicken, eventually leading to the emergence of root suckers characteristic of orchids—a phenomenon known as "bud-to-bud proliferation." This is due to the high concentration of endogenous mitogens in the entire plant resulting from the developed root system, causing 3-5 adventitious buds to appear at the junction of the root system and stem, thus giving the test-tube seedlings a clustered structure. Its proliferation coefficient far exceeds that of similar studies. Furthermore, this study also confirms the strong regenerative capacity of *Dendrobium officinale* stem segments. Given suitable conditions (appropriate culture medium and inoculation method) and measured in years, its proliferation efficiency is comparable to the seed-protocorm-regenerated seedling pathway. Moreover, it avoids phenotypic differences caused by segregation, thus obtaining regenerated seedlings with a consistent genetic background, which is unattainable through the seed-protocorm-regenerated seedling pathway.

[0128] 3. In orchidaceae, protocorms and protocorm-like bodies (PLBs) are generally defined as follows: those arising from seeds are called protocorms, while those arising from somatic embryos of explants are called protocorms. The biggest difference between the two is that protocorms often have white rhizoids, while protocorms lack this structure. The protocorms (PLBs) observed in this study significantly improved the proliferation efficiency of Dendrobium officinale in in vitro rapid propagation.

[0129] 4. The core of this invention lies in the production of protocorms (PLBs) from stem segments to axillary bud clusters, which can be repeatedly cycled. Combined with the integrated cultivation of propagation and rooting, the production of Dendrobium officinale can be completely controlled by artificial means, with high efficiency and short cycle. At the same time, since PLBs are produced in each cycle, the problem of them gradually disappearing with the increase of the number of transfer generations can be avoided.

[0130] 5. This invention greatly simplifies the artificial rapid propagation process of Dendrobium officinale. Only one culture medium is needed to complete all stages of cultivation. The regeneration culture of test-tube seedlings can be achieved in a maximum of only 120 days. It is low in cost and easy to standardize and industrialize. It solves the traditional series of cumbersome cultivation processes from non-symbiotic germination of seeds, seedling differentiation, proliferation and rejuvenation and rooting, and can achieve extremely high economic and social benefits.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications 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.

Claims

1. A highly efficient artificial propagation method for Dendrobium officinale, characterized in that, Includes the following steps: (1) Obtaining explants: After two years of continuous observation, select healthy plants with good growth and no pests or diseases, and take fresh shoots that have formed apical buds after two years. (2) Cut the fresh strips from step (1) into appropriate sizes, and after sterilization, cut them into appropriate sizes and inoculate them into culture medium for culture; (3) The segmented stem segments that have been sterilized in step (2) are inoculated into the following culture medium A to obtain clump-shaped and rooted sterile test-tube seedlings and protocorm clusters produced at the cut ends of the stem segments. The culture medium A is based on MS medium and contains 60,000 mg / L banana puree, 40,000 mg / L potato puree, 1.0-1.5 mg / L naphthaleneacetic acid, 1.0-1.5 mg / L 3-indolebutyric acid, 0.1-0.5 mg / L 6-benzylaminopurine, 0.1-0.5 mg / L kinetin, 1,000 mg / L activated carbon, 15,000 mg / L sucrose, and 4,700 mg / L agar powder. (4) Collect the protocorm clusters that appeared in step (3) and inoculate them into fresh culture medium A to carry out the proliferation of protocorm clusters (PLBs) and the development of seedlings; (5) The buds in step (4) are divided into 2-3 clumps and cultured in fresh culture medium A until they become seedlings, while the protocorms are transferred to fresh culture medium A to continue to proliferate and develop. (6) Hardening and transplanting: Take the seedlings from step (3) or step (5) and harden them at room temperature for 3 days. After hardening them for another 2 days, remove the seedlings from the culture medium, clean off the residual culture medium, soak them in carbendazim solution, and then transplant them into sterilized pine bark for warm and moist culture to obtain transplanted seedlings.

2. The efficient artificial propagation method for Dendrobium officinale according to claim 1, characterized in that, The method for disinfecting the fresh stems described in step (2) is as follows: Cut the fresh stems into pieces 1.5-2.0 cm long with 2 nodes, remove the leaves, wash with running water, soak in 10% laundry detergent solution (w / v) for 10 min, rinse with running water for 30 min, and place on a clean bench; treat with 75% ethanol solution (v / v) for 10-15 s, and 0.1% mercuric chloride aqueous solution (w / v) for 10-12 min, shaking the bottle continuously during the process to achieve the best sterilization effect, and finally rinse with sterile water 3 times, each time for no less than 3 min; place on sterile absorbent paper, absorb the surface moisture, and then cut off about 0.2 cm at the base of the stem with a sterile scalpel, and vertically inoculate into culture medium A in the physiological direction.

3. The efficient artificial propagation method for Dendrobium officinale according to claim 1 or 2, characterized in that, The pH value of the culture medium A is 5.4-5.

6.

4. The efficient artificial propagation method for Dendrobium officinale according to claim 1 or 2, characterized in that, The environmental conditions in step (3) are 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day.

5. The efficient artificial propagation method for Dendrobium officinale according to claim 4, characterized in that, The environmental conditions in step (4) are 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day.

6. The efficient artificial propagation method for Dendrobium officinale according to any one of claims 1, 2, or 5, characterized in that, The protocormoids mentioned in step (4) include three generations of continuous culture, during which protocormoid clusters are generated at the cut ends of the stem segments in each generation of culture.

7. The efficient artificial propagation method for Dendrobium officinale according to any one of claims 1, 2, or 5, characterized in that, The mass concentration of the carbendazim solution in step (6) is 0.1-0.5%.

8. The efficient artificial propagation method for Dendrobium officinale according to any one of claims 1, 2, or 5, characterized in that, The size of the pine bark fragments mentioned in step (6) is 1.5×1.5 cm, and the disinfection method is boiling water for 2-3 hours; the cultivation temperature is 25±2℃, and the humidity is 60-80%.

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