CN118749425A - High-efficiency artificial propagation method of Dendrobium officinale - Google Patents

Abstract

The present invention belongs to the technical field of plant induced regeneration, and specifically relates to an efficient artificial propagation method of Dendrobium officinale, comprising: taking biennial stem segments with nodes as materials, cutting them into appropriate sizes and performing disinfection treatment; after the stem segments are connected to culture medium A and cultured for 90 days, the integration of proliferation and rooting is completed through a direct organogenesis pathway. The present invention realizes the cyclic production of Dendrobium officinale, and the entire culture process can be completed with only one culture medium, which greatly improves the efficiency of artificial in vitro propagation, and the proliferation coefficient is completely comparable to the non-symbiotic germination mode of seeds, and solves the problems of large separation of offspring traits and uneven quality caused by non-symbiotic germination of seeds, and greatly improves the quality and yield of seedlings. The present invention can provide a new technical support for the large-scale growth of Dendrobium officinale in factories, so as to achieve the purpose of cultivating genetically stable and high-quality seedlings to improve the efficiency of artificial planting, and has low cost, short cycle, high seedling quality and survival rate.

Description

High-efficiency artificial propagation method of Dendrobium officinale

Technical Field

The invention belongs to the technical field of plant induced regeneration, and particularly relates to a high-efficiency artificial propagation method of Dendrobium officinale.

Background Art

Dendrobium officinale Kimura et Migo, also known as black knot grass, is a perennial epiphytic herb of the genus Dendrobium in the family Orchidaceae. It grows on semi-shaded and moist rocks in the mountains at an altitude of 1600-2100m. It likes warm and humid climate and is not cold-resistant. The air humidity is 60%-80%, and the most suitable temperature in the growing season is 20-25℃. It is distributed in Anhui, Zhejiang, Fujian, Guangxi, Sichuan, Yunnan and other places in my country. The dried stem of Dendrobium candidum is commonly known as "Fengdou". It was first recorded in "Shen Nong's Herbal Classic" and listed as a top-grade product. It has the reputation of "the first of the nine immortal herbs". This species is a precious medicinal and edible plant in my country. It tastes sweet and is slightly cold in nature. It has the effects of nourishing yin and clearing heat, promoting body fluid and benefiting the stomach, moistening the lungs and relieving cough. It contains a variety of chemical components. In addition to polysaccharides, one of the main active ingredients, there are more than 190 compounds such as alkaloids, phenanthrenes, bibenzyls, fluorenones, sesquiterpenes, coumarins, steroids and volatile oils. Dendrobium candidum 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, hypertensive stroke, cataracts, weak constitution or other sub-health conditions. In recent years, due to the continuous development of Dendrobium officinale related products, it has gradually expanded into the fields of health care products and skin care products. The large market demand has led to over-exploitation of wild resources. In addition, its unique metabolic process - Crassulacean acid metabolism (CAM), low photosynthetic efficiency, long growth cycle; sexual reproduction of plants is difficult, and the fruiting rate and seed germination rate are low. Wild resources are now very scarce. The "Regulations on the Protection and Management of Wild Medicinal Resources" issued by the State Council in 1987 listed Dendrobium officinale as a second-level protected variety; in 1992, it was included in the "China Plant Red Book" as an endangered plant; internationally, it was listed in the "Convention on International Trade in Endangered Plants" (CITES), and wild varieties are prohibited from trading.

Dendrobium candidum has strict requirements on habitats and cannot grow in soil. It mostly grows on tree trunks or between stone walls. Its unique metabolic process - Crassulacean acid metabolism (CAM) makes it different from ordinary _C3 plants. It photosynthesizes at night and relies on endophytes to produce nutrients and aerial roots to absorb water and nutrients in the air. It has low photosynthetic intensity and a long growth cycle. Similar to most orchids, the seeds of Dendrobium plants including Dendrobium candidum are small and lack cotyledons and endosperm, resulting in incomplete embryo development. Under natural conditions, it needs to work with related symbiotic bacteria to germinate, which leads to a long germination time and a low germination rate (only 5%), and an even lower seedling rate. It generally takes 4-5 years or even longer from seed germination to flowering plants that can be identified for traits. What is confusing is that as early as more than 10 years ago, a large number of scholars or producers and operators have conducted research on artificial rapid propagation of Dendrobium candidum. From the perspective of literature level, most of them are non-mainstream journals with extremely low reproducibility; from the perspective of actual production, most of them use protocorms obtained by non-symbiotic germination of seeds for reproduction, but there are disadvantages such as complex process, long seedling cycle, weak regenerated plants with serious trait separation, and extremely low survival rate of direct cultivation. Therefore, it is urgent to seek a new artificial breeding method with low cost, short time, high quality and survival rate, and high reproducibility, which has become an inevitable choice for the sustainable utilization and large-scale reproduction of Dendrobium officinale.

Summary of the invention

The object of the present invention is to solve the defects of the above-mentioned existing propagation technology, and to provide an efficient artificial propagation method of Dendrobium officinale, which realizes the cyclic production of Dendrobium officinale, and the whole culture process can be completed with only one culture medium, which greatly improves the efficiency of artificial in vitro propagation, and the proliferation coefficient is completely comparable to the seed non-symbiotic germination mode, and solves the problems of large separation of offspring traits and uneven quality caused by seed non-symbiotic germination, and greatly improves the quality and yield of seedlings. The high proliferation coefficient and simple seedling method of direct organogenesis not only overturns the traditional production method of Dendrobium officinale, but also can make the whole production process cyclic due to the appearance of protocorms, without the need for regular sowing or repeated explant culture every year, which greatly simplifies its artificial breeding process. The present invention can provide a new technical support for the large-scale growth of Dendrobium officinale in factories, so as to achieve the purpose of cultivating genetically stable and high-quality seedlings to improve the efficiency of artificial planting, and has low cost, short cycle, high seedling quality and survival rate.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

The high-efficiency artificial propagation method of Dendrobium officinale is characterized by comprising the steps of: taking two-year-old fresh strips, cutting them into appropriate sizes, cutting them into appropriate sizes after disinfection and sterilization, and inoculating them into a culture medium for cultivation;

Furthermore, the efficient artificial propagation method of Dendrobium officinale comprises the following steps:

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

(2) cutting the stem segments obtained in step (1) into appropriate sizes and sterilizing them;

(3) The noded stem segments sterilized in step (2) are inoculated into the following culture medium A, wherein the culture medium A comprises the following raw materials:

1/3MS basic culture medium

Mashed banana

Mashed potatoes

Naphthaleneacetic acid (NAA)

6-Benzylaminopurine (6-BA)

Kinetin (KT)

Activated carbon (AC)

sucrose

Agar powder

By controlling the light intensity, temperature and illumination time, we can obtain sterile test tube seedlings with clump shape and 100% rooting rate. After three generations of continuous cultivation, about 6.0-8.0% of the stem segments in each generation produced protobulb-like bodies (PLBs).

(4) Proliferation and development of protocorms (PLBs): Collect the protocorms (PLBs) clusters produced in step (3) and inoculate them into fresh culture medium A, and carry out the proliferation of protocorms (PLBs) and the development of seedlings under controlled conditions of light intensity, temperature, and illumination time;

(5) The bud clusters in step (4) are transferred to fresh medium A with 2-3 plants as one cluster and cultured until they become seedlings, while the protocorm-like bodies (PLBs) are transferred to fresh medium A to continue proliferation and development;

(6) Hardening and transplanting: Take the bottle seedlings in step (3) or (5) and place them at room temperature for 3 days, then open the bottle cap to harden them for 2 days; remove the seedlings from the culture medium, clean the remaining culture medium, soak them in a carbendazim solution, and then transplant them to disinfected crushed pine bark for heat preservation and moisture retention, thereby obtaining transplanted seedlings.

Furthermore, the stem segment disinfection treatment method in step (2) is as follows: cut the fresh stem into 1.5-2.0 cm long with 2 nodes, remove the leaves, wash with running water, soak in 10% washing powder solution (w/v) for 10 minutes, rinse with running water for 30 minutes, and place on a clean bench. Treat with 75% ethanol solution (v/v) for 10-15 seconds, treat with 0.1% mercuric chloride aqueous solution (w/v) for 10-12 minutes, shake the bottle constantly during this period to achieve the best sterilization effect, and finally rinse with sterile water for 3 times, each time for not less than 3 minutes; 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 insert into culture medium A in the physiological direction.

Furthermore, the culture medium A in step (3) comprises the following raw materials:

1/3MS basic culture medium

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

Furthermore, the protocorm-like bodies (PLBs) in step (3) are collected and transferred to fresh culture medium A to proliferate and develop into seedlings.

Furthermore, the clusters of buds in step (4) are transferred to fresh culture medium A in groups of 2-3 plants each and cultured until they become seedlings, while the protocorm-like bodies (PLBs) are transferred to fresh culture medium A for further proliferation and development.

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

Furthermore, the size of the shredded pine bark in step (6) is 1.5×1.5 cm, and the sterilization method is boiling in boiling water for 2-3 hours; the culture temperature is (25±2)° C., and the humidity is 60-80%.

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

(1) The present invention uses tissue culture technology to achieve year-round production in the culture room, which not only saves land resources but also improves economic benefits, overcoming the difficulty of traditional vegetative propagation methods that cannot achieve year-round production;

(2) The present 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 of Dendrobium plants. Through the present invention, a large number of seedlings can be obtained in a short time, with low cost, short cycle, easy standardization and factory operation, which effectively improves the quality of seedlings, solves the problem of protection of Dendrobium officinale germplasm resources, and provides high-quality seedlings of unified standards for its large-scale promotion and planting.

(3) The present invention only requires one culture medium to complete the proliferation and rooting process by direct organogenesis, which can not only meet the needs of large-scale production, but also maintain the excellent traits of the mother plant. It is the most effective proliferation method for artificial rapid propagation and improves the quality of seedlings.

(4) The present invention achieves the purpose of efficient and rapid propagation. A proliferation culture cycle of 90 days is achieved. The propagation coefficient of direct organogenesis of stem segments into seedlings can reach more than 10.0; and the propagation coefficient of direct organogenesis of stem segments into seedlings can reach more than 10.0.

The reproduction coefficient of seedlings can reach above 30.0; the sum of the two can reach above 40.0.

(5) The present invention confirms that the stem segments of Dendrobium officinale have a strong regeneration ability. As long as the conditions are suitable (appropriate culture medium) and calculated on an annual basis, their proliferation efficiency is completely comparable to that of the seed-protocorm-regenerated seedling approach. Moreover, the trait differences caused by separation are avoided, thereby obtaining regenerated seedlings with a consistent genetic background, which is unattainable by the seed-protocorm-regenerated seedling approach.

(6) The present invention can repeatedly cycle from stem segments to axillary bud clusters and then to the production of protocorm-like bodies (PLBs). In addition, the integrated culture of proliferation and rooting makes it possible to artificially control the ratio of proliferated seedlings to commercial seedlings in the production of Dendrobium officinale with high efficiency and short cycle. At the same time, since protocorm-like bodies (PLBs) are produced in each cycle, the problem of their gradual disappearance with the increase of the number of transfer generations can be avoided.

(7) The Dendrobium officinale test tube seedlings produced by the present invention have a high survival rate after transplantation and grow rapidly because each clump/plant has an unusually developed adventitious root system. Currently, they have been cultivated by the cultivation base of Xiangxin Agricultural Technology Development Co., Ltd. in Yuxi City, Yunnan Province (102°31′25″E, 24°26′22″N, Alt:

1688m) for demonstration cultivation, the effect was very good;

(8) The present invention has important significance and value for the artificial rapid propagation of Dendrobium officinale, and can also provide a technical reference for the rapid propagation of other Dendrobium plants.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram of each growth stage of the integrated culture of stem segment proliferation and rooting;

Among them, 1A is the growth condition diagram after 30 days of culture; 1B is the growth condition diagram after 45 days of culture; 1C is the growth condition diagram after 60 days of culture; 1D is the growth condition diagram after 75 days of culture; 1E and 1F are the growth condition diagrams after 90 days of culture;

FIG2 is a diagram showing the various growth stages of the occurrence, proliferation and seedling formation of protocorm-like bodies (PLBs);

Among them, 2A is the source of culture materials; 2B is the growth of protocorm-like bodies just after being inoculated into the culture medium; 2C is the growth after 30 days of culture; 2D and 2E are the growth after 45 days of culture; 2F and 2G are the growth of a single protocorm-like body after 45 days of culture; 2H, 2G, and 2I are the growth after 60 days of culture; 2J and 2K are the growth of clustered buds developed from protocorms-like bodies after 30 days of culture; 2L is the growth of clustered buds developed from protocorms-like bodies after 60 days of culture;

Fig. 3 is a diagram of various growth stages for domestication and transplanting;

Among them, 3A is the growth chart after 60 days of transplanting; 3B is the growth chart after 180 days of transplanting; 3C is the growth chart after 240 days of transplanting; 3D is the growth chart after 16-17 months of transplanting;

FIG. 4 is a diagram showing the regeneration of stem segments and protocorm-like bodies (PLBs) of Dendrobium officinale.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Based on the present invention, the efficient artificial propagation method of Dendrobium officinale comprises the following steps:

S1 Select healthy plants with good growth potential and no pests and diseases, take the two-year-old fresh top branches, trim them into 1.5-2.0 cm node stem segments with 2 nodes, disinfect them and set aside for use;

S2. The treated stem segments with nodes were inoculated into medium A at 21-23°C, 1800-2500 lx, 9-11 hours of light per day, and cultured for 90 days to obtain clump-shaped sterile test tube seedlings with a rooting rate of 100% (see Figure 1 for each growth stage), and protocorm-like masses were produced at the cuts of the stem segments;

As shown in Figure 1 (scale = 2.0 cm), from Figure 1A, it can be seen that after 30 days of cultivation, the axillary buds on the upper and lower nodes of the stem segment all sprouted into new branches, and the new leaves began to stretch; from Figure 1B, it can be seen that after 45 days of cultivation, the new buds further elongated, the leaves grew up and adventitious roots began to appear; from Figure 1C, it can be seen that after 60 days of cultivation, with the rapid growth of adventitious roots, 2-4 tillering seedlings began to appear at the base of each material stem node; from Figure 1D, it can be seen that after 75 days of cultivation, the barberry seedlings grew vigorously and were almost as high as the axillary buds, and at this time they could be exposed for domestication; from Figures 1E and 1F, it can be seen that after continuing to cultivate for 90 days, a clump-shaped sterile test tube seedling with a rooting rate of 100% can be obtained, and the proliferation coefficient at this time can reach 11.25; after three generations of continuous cultivation, about 6.0-8.0% of the stem segment incisions produced protocorm-like bodies (PLBs) in each generation;

S3. Collect the protocorm-like bodies produced in each generation of S2 and inoculate them into fresh culture medium A, with a light intensity of 1800-2500 lx and a light intensity of 9-11 hours per day for 60 days to form a protocorm-like body/clustered strong bud mixture with a proliferation coefficient of >30 (see Figure 2 for each growth stage);

As shown in Figure 2 (Figure A-L scale = 2 cm; Figure F-H scale = 0.2 cm), it can be seen from Figure 2C that after 30 days of culture, the small group swelled significantly and small buds appeared; it can be seen from Figures 2D and 2E that after 45 days of culture, the small group continued to swell into a patchy shape with villi-like rhizoids attached, with a large number of small spheres on it, accompanied by the appearance of conical pointed leaves; it can be seen from Figures 2F and 2G that after the small spheres were peeled off, obvious bipolar growth could be observed, which can be judged as a protocorm-like body; it can be seen from Figures 2H, 2G, and 2I that after 60 days of culture, this While developing into seedlings, some protocorms-like bodies also proliferate to a certain extent. At this time, the entire surface of the culture medium is covered with a mixture of seedlings and protocorms-like bodies, and the proliferation coefficient can reach above 30.0; as can be seen from Figures 2J and 2K, the clusters of buds developed from protocorms-like bodies are cut into clusters of 3-4 plants and transferred to fresh culture media. After 30 days, the clusters of buds begin to turn green and the "bud multiplication" phenomenon appears; as can be seen from Figure 2L, the multi-bud cluster cultures formed after 60 days of cultivation can be divided into single seedlings or clusters of 2-3 plants, which can be transferred to the integrated culture medium for proliferation and rooting, and can be domesticated and transplanted after 60 days of cultivation.

S4. The clustered strong buds in the mixture are transferred to the culture medium for cultivation to form rooted seedlings, and the protocorms in the mixture are repeated with S3-S4; the clustered strong buds transferred to the culture medium begin to turn green and show the phenomenon of "bud multiplication" after 30 days, and multi-bud cluster cultures are formed after 60 days of cultivation; the protocorms can cycle the entire production process, and the integrated cultivation of proliferation and rooting can artificially control the ratio of its proliferation seedlings to commercial seedlings, which greatly simplifies its artificial breeding process;

S5. The rooted seedlings of S4 were placed at room temperature for 3-5 days, sterilized, and moved into the substrate for heat preservation and moisture cultivation to obtain transplanted seedlings (see Figure 3 for each growth stage);

As shown in Figure 3 (scale = 1.0 cm), it can be seen from Figure 3A that the test tube seedlings showed obvious signs of growth 60 days after transplantation, with elongated internodes, enlarged and stretched leaves, and a survival rate of more than 90%; it can be seen from Figure 3B that 180 days later, lateral buds at the base of the plant continued to occur and grew to about 5.0 cm, the internodes became thicker, and the leaves became enlarged; it can be seen from Figure 3C that 240 days later, the edges of the leaves appeared light red, the internodes were light black, and the plants grew vigorously; it can be seen from Figure 3D that the branches were basically full of flowers in April and May of the second year after transplantation, and at this time, spring buds also continued to appear from the base of the plant and grew well.

The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

Example 1

The efficient artificial propagation method of Dendrobium officinale comprises the following steps:

(1) Obtaining explants: Observe for two consecutive years and select healthy plants with good growth potential and no pests or diseases. Take the two-year-old fresh top-bearing shoots.

(2) Cut the fresh strips in step (1) into 1.5-2.0 cm long strips with 2 nodes, remove the leaves, wash with running water, soak in 10% washing powder 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 min, shaking the bottle constantly during this period to achieve the best sterilization effect, and finally rinse with sterile water for 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 for use.

(3) The noded stem segments sterilized in step (2) are inoculated into the following culture medium A, wherein the culture medium A comprises the following raw materials:

1/3MS basic culture medium

Culture conditions: Under the conditions of light intensity of 1800-2500lx, light time of 10h/d, temperature controlled at 22±1℃, after 30 days of culture, the axillary buds on the upper and lower nodes of the stem segment all germinate into new branches, and new leaves begin to stretch; after 45 days of culture, the new buds further elongate, the leaves grow up and adventitious roots begin to appear; after 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillering seedlings begin to appear at the base of each material stem node; after 75 days of culture, the barberry seedlings grow vigorously, almost as high as the axillary buds, and can be exposed for domestication; continue to culture for 90 days, the test tube seedlings are tall and strong, the leaves are fully expanded, the adventitious root system is well developed, and the proliferation coefficient can reach 11.25 at this time. After 3 generations of continuous culture, about 6.0-8.0% of the stem segment cuts produce protocorm-like bodies (PLBs) in each generation.

(4) Proliferation and development of protocorm-like bodies (PLBs): The protocorm-like bodies 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 time of 10 h/d, and temperature controlled at 22±1°C, these nodular bodies were collected and cut into 0.5×0.5 cm pieces and transferred into fresh culture medium A. After 30 days of culture, the small bodies swelled significantly and small buds appeared. After 45 days of culture, the small bodies continued to swell into plaques with villi-like rhizomes, on which there were many small spheres and round Conical pointed leaves appear; after 60 days of cultivation, these protocorms-like bodies not only develop into seedlings, but also proliferate to a certain extent. At this time, the entire surface of the culture medium is covered with a mixture of seedlings and protocorms-like bodies, and the proliferation coefficient can reach 32.50; at this time, the clusters of buds developed from protocorms-like bodies can be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 30 days, the clusters of buds begin to turn green and the "bud-multiple buds" phenomenon appears; the multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 plants per cluster, transferred to fresh culture medium A, and can be acclimated and transplanted after 60 days of cultivation.

(5) Hardening and transplanting: Take the bottle seedlings in step (3) or (4) and place them at room temperature for 3 days, then open the bottle cap and harden them for 2 days; remove the seedlings from the culture medium, clean the residual culture medium, place them in a 0.5% carbendazim solution for disinfection for 3 minutes, and then transplant them into a flower pot with a substrate of crushed pine bark (about 1.5×1.5 cm) boiled in boiling water for 2.5 hours. After keeping warm (25±2° C.) and moisturizing (60-80%) for 60 days, transplanted seedlings are obtained, and the survival rate is 100%.

Example 2

The efficient artificial propagation method of Dendrobium officinale comprises the following steps:

(1) Obtaining explants: Observe for two consecutive years and select healthy plants with good growth potential and no pests or diseases. Take the two-year-old fresh top-bearing shoots.

(2) Cut the fresh strips in step (1) into 1.5-2.0 cm long strips with 2 nodes, remove the leaves, wash with running water, soak in 10% washing powder 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 11 min, shaking the bottle constantly to achieve the best sterilization effect, and finally rinse with sterile water for 3 times, each time for no less than 3 min; place on sterile absorbent paper, absorb the surface moisture, and cut off about 0.2 cm at the base of the stem with a sterile scalpel for use.

(3) The noded stem segments sterilized in step (2) are inoculated into the following culture medium A, wherein the culture medium A comprises the following raw materials:

1/3MS basic culture medium

Culture conditions: Under the conditions of light intensity of 1800-2500lx, light time of 10h/d, and temperature controlled at 22±1℃, after 30 days of culture, the axillary buds on the upper and lower nodes of the stem segment all germinate into new branches, and new leaves begin to stretch; after 45 days of culture, the new buds further elongate, the leaves grow up and adventitious roots begin to appear; after 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillering seedlings begin to appear at the base of each material stem node; after 75 days of culture, the barberry seedlings grow vigorously, almost as high as the axillary buds, and can be exposed for domestication; continue to culture for 90 days, the test tube seedlings are tall and strong, the leaves are fully expanded, the adventitious root system is well developed, and the proliferation coefficient can reach 11.45 at this time. After 3 generations of continuous culture, about 6.0-8.0% of the stem segment cuts produce protocorm-like bodies (PLBs) in each generation.

(4) Proliferation and development of protocorm-like bodies (PLBs): The protocorm-like bodies 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 time of 10 h/d, and temperature controlled at 22±1°C, these nodular bodies were collected and cut into 0.5×0.5 cm pieces and transferred into fresh culture medium A. After 30 days of culture, the small bodies swelled significantly and small buds appeared. After 45 days of culture, the small bodies continued to swell into plaques with villi-like rhizomes, on which there were many small spheres and round Conical pointed leaves appear; after 60 days of cultivation, these protocorms-like bodies not only develop into seedlings, but also proliferate to a certain extent. At this time, the entire surface of the culture medium is covered with a mixture of seedlings and protocorms-like bodies, and the proliferation coefficient can reach 31.50; at this time, the clusters of buds developed from protocorms-like bodies can be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 30 days, the clusters of buds begin to turn green and the "bud-multiple buds" phenomenon appears; the multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 plants per cluster, transferred to fresh culture medium A, and can be acclimated and transplanted after 60 days of cultivation.

(5) Hardening and transplanting: Take the bottle seedlings in step (3) or (4) and place them at room temperature for 3 days, then open the bottle cap and harden them for 2 days; remove the seedlings from the culture medium, clean the remaining culture medium, place them in a 0.3% carbendazim solution for disinfection for 3 minutes, and then transplant them into a flower pot with a substrate of crushed pine bark (about 1.5×1.5 cm) boiled in boiling water for 3 hours. After culturing for 60 days, the transplanted seedlings are obtained, and the survival rate is 100%.

Example 3

The efficient artificial propagation method of Dendrobium officinale comprises the following steps:

(1) Obtaining explants: Observe for two consecutive years and select healthy plants with good growth potential and no pests or diseases. Take the two-year-old fresh top-bearing shoots.

(2) Cut the fresh strips in step (1) into 1.5-2.0 cm long strips with 2 nodes, remove the leaves, wash with running water, soak in 10% washing powder 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 12 min, shaking the bottle constantly during this period to achieve the best sterilization effect, and finally rinse with sterile water for 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 for use.

(3) The noded stem segments sterilized in step (2) are inoculated into the following culture medium A, wherein the culture medium A comprises the following raw materials:

1/3MS basic culture medium

Culture conditions: Under the conditions of light intensity of 1800-2500lx, light time of 10h/d, temperature controlled at 22±1℃, after 30 days of culture, the axillary buds on the upper and lower nodes of the stem segment all germinate into new branches, and new leaves begin to stretch; after 45 days of culture, the new buds further elongate, the leaves grow up and adventitious roots begin to appear; after 60 days of culture, with the rapid growth of adventitious roots, 2-4 tillering seedlings begin to appear at the base of each material stem node; after 75 days of culture, the barberry seedlings grow vigorously, almost as high as the axillary buds, and can be exposed for domestication; continue to culture for 90 days, the test tube seedlings are tall and strong, the leaves are fully expanded, the adventitious root system is well developed, and the proliferation coefficient can reach 11.85 at this time. After 3 generations of continuous culture, about 6.0-8.0% of the stem segment cuts produce protocorm-like bodies (PLBs) in each generation.

(4) Proliferation and development of protocorm-like bodies (PLBs): The protocorm-like bodies 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 time of 10 h/d, and temperature controlled at 22±1°C, these nodular bodies were collected and cut into 0.5×0.5 cm pieces and transferred into fresh culture medium A. After 30 days of culture, the small bodies swelled significantly and small buds appeared. After 45 days of culture, the small bodies continued to swell into plaques with villi-like rhizomes, on which there were many small spheres and round Conical pointed leaves appear; after 60 days of cultivation, these protocorms-like bodies not only develop into seedlings, but also proliferate to a certain extent. At this time, the entire surface of the culture medium is covered with a mixture of seedlings and protocorms-like bodies, and the proliferation coefficient can reach 31.65; at this time, the clusters of buds developed from protocorms-like bodies can be cut into 3-4 plants as a cluster and transferred to fresh culture medium. After 30 days, the clusters of buds begin to turn green and the "bud-multiple buds" phenomenon appears; the multi-bud cluster culture formed after 60 days of cultivation can be divided into single seedlings or 2-3 plants per cluster, transferred to fresh culture medium A, and can be acclimated and transplanted after 60 days of cultivation.

(5) Hardening and transplanting: Take the bottle seedlings in step (3) or (4) and place them at room temperature for 3 days, then open the bottle cap and harden them for 2 days; remove the seedlings from the culture medium, clean the residual culture medium, place them in a 0.1% carbendazim solution for disinfection for 3 minutes, and then transplant them into a flower pot with a substrate of crushed pine bark (about 1.5×1.5 cm) boiled in boiling water for 2 hours. After culturing for 60 days, the transplanted seedlings are obtained, and the survival rate is 100%.

Technical principle of the present invention:

1. The stem segments of Dendrobium officinale are selected as explants, which ensures their stable genetic characteristics and solves the problem of germplasm decline caused by genetic drift and inbreeding.

2. According to the investigation, in the actual production of Dendrobium officinale in Yunnan, the seed-protocorm-regenerated seedling approach is adopted. The seeds are sown in September and October every year. The protocorms produced by non-symbiotic germination of seeds can produce test tube seedlings that can be transplanted after 12 months or even longer. It seems to be inefficient, but as long as the seeds are sown every year, the yield is considerable. The biggest disadvantage of this method is the uneven emergence of seedlings and the serious separation of seedling traits. However, the principle of yield first leads to the seed-protocorm-regenerated seedling approach having considerable commercial advantages, which is also one of the main reasons why most factory production adopts this approach. In the present invention, stem segments with two nodes are used as materials. At the same time as the two fixed buds occur, as the adventitious roots become more and more developed, their bases gradually become thicker, and root suckers unique to orchids gradually appear, which is the so-called "bud-sprouting" phenomenon. This is because the developed root system makes the whole plant have a high concentration of endogenous mitogens, resulting in 3-5 adventitious buds appearing at the part where the root system is connected to the stem, so that the test tube seedlings present a cluster structure; its proliferation coefficient far exceeds the same type of research. At the same time, this study also confirmed that the stem segments of Dendrobium officinale have a strong regeneration ability. As long as the conditions are suitable (appropriate culture medium and inoculation method) and calculated in years, its proliferation efficiency can be completely comparable to the seed-protocorm-regeneration seedling approach; and it avoids the difference in traits caused by separation, thereby obtaining regeneration seedlings with consistent genetic backgrounds, which is unattainable by the seed-protocorm-regeneration seedling approach.

3. The protocorms and protocorm-like bodies (PLBs) of orchids are usually defined as follows: those that originate from seeds are called protocorms, and those that originate from explant somatic embryos are called protocorms-like bodies. The biggest difference between the two is that protocorms-like bodies often have white rhizoid hairs, while protocorms do not have this structure. The protocorms-like bodies (PLBs) that appeared in this study have further significantly improved the proliferation efficiency of Dendrobium officinale in in vitro rapid propagation.

4. The core of the present invention is that the process from stem segments to axillary bud clusters and then to the production of protocorm-like bodies (PLBs) can be repeated, and the integrated cultivation of proliferation and rooting makes it possible to artificially control the ratio of proliferated seedlings to commercial seedlings in the production of Dendrobium officinale, with high efficiency and short cycle. At the same time, since PLBs are produced in each cycle, the problem of their gradual disappearance with the increase of the number of transfer generations can be avoided.

5. The present 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 only 120 days at most. The invention has low cost and is easy to standardize and operate in a factory. It solves the traditional series of cumbersome cultivation processes from non-symbiotic germination of seeds, differentiation of seedlings, proliferation and rejuvenation, and rooting, and can achieve extremely high economic and social benefits.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (8)

1. The efficient artificial propagation method of the dendrobium candidum is characterized by comprising the following steps of:

(1) Acquisition of explants: continuously observing for two years, selecting a healthy plant with good growth vigor and no plant diseases and insect pests, and taking fresh strips of two-year old plants;

(2) Cutting the fresh strips obtained in the step (1) into proper sizes, sterilizing, cutting into proper sizes, and inoculating into a culture medium for culture;

(3) The sterilized stem segments with nodes in the step (2) are inoculated into a culture medium A which takes an MS culture medium as a basic culture medium and is added with 60000mg/L banana puree, 40000mg/L potato puree, 1.0-1.5mg/L naphthylacetic acid, 1.0-1.5 mg/L3-indolebutyric acid, 0.1-0.5 mg/L6-benzylaminopurine, 0.1-0.5mg/L kinetin, 1000mg/L active carbon, 15000mg/L sucrose and 4700mg/L agar powder, so that a cluster-shaped sterile test tube seedling with the rooting rate of 100% and a stem segment incision can be obtained;

(4) Collecting the protocorm-like clusters appearing in the step (3), and inoculating the protocorm-like clusters into a fresh culture medium A to perform proliferation of protocorm-like (PLBs) and development of seedlings;

(5) 1 cluster bud in the step (4) is transferred into a fresh culture medium A for culture until seedlings are formed according to 1 cluster of every 2-3 plants, and protocorm-like bodies are transferred into the fresh culture medium A for continuous proliferation and development;

(6) Hardening seedlings and transplanting: and (3) placing the seedlings in the step (3) or the step (5) at room temperature for hardening the seedlings for 3d, opening a bottle cap for hardening the seedlings for 2d, taking out the seedlings from the culture medium, cleaning the residual culture medium, soaking the seedlings in a carbendazim solution, and transplanting the seedlings into the sterilized crushed pine bark for heat preservation and moisture preservation culture to obtain the transplanted seedlings.

2. The efficient artificial propagation method of dendrobium candidum according to claim 1, wherein the method for sterilizing the fresh strips in the step (2) is as follows: cutting fresh strips to 1.5-2.0cm long with 2 sections, removing blade, washing with flowing water, soaking in 10% detergent solution (w/v) for 10min, washing with flowing water for 30min, and placing on a super clean bench. Treating with 75% ethanol solution (v/v) for 10-15s and 0.1% mercuric chloride aqueous solution (w/v) for 10-12min, shaking the bottle body continuously to achieve optimal sterilization effect, and washing with sterile water for 3 times each time not less than 3min; placing on sterile absorbent paper, absorbing water on the surface, cutting about 0.2cm at the basal stem with sterile scalpel, and vertically inoculating into culture medium A in physiological direction.

3. The efficient artificial propagation method of dendrobium candidum according to claims 1-2, wherein the pH value of the culture medium a is 5.4-5.6.

4. The efficient artificial propagation method of dendrobium candidum according to claims 1-2, wherein the environmental conditions in the step (3) are that the illumination intensity is 1800-2500lx at 21-23 ℃ and 9-11h are illuminated daily.

5. The efficient artificial propagation method of dendrobium candidum according to claim 4, wherein the environmental conditions in the step (4) are that the illumination intensity is 1800-2500lx at 21-23 ℃ and 9-11h per day.

6. A method of efficient artificial propagation of dendrobium candidum according to any one of claims 1, 2 or 5, wherein the protocorm-like in step (4) comprises continuous culture for 3 generations, and the protocorm-like clusters are generated at the stem segment cut in each generation of culture.

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

8. The efficient artificial propagation method of dendrobium candidum according to any one of claims 1,2 or 5, wherein the crushed pine bark in the step (6) is 1.5 x 1.5cm in size, and the disinfection method is boiling water steaming for 2-3 hours; the culture temperature is 25+ -2deg.C, and the humidity is 60-80%.