Method for artificially and efficiently breeding anoectochilus roxburghii by using protocorm-like bodies

Abstract

The invention relates to the technical field of plant seedling culture, and discloses a method for artificially and efficiently breeding anoectochilus roxburghii by using protocorm-like bodies, which specifically comprises the following steps: step (1), obtaining explants; step (2), disinfecting the explants; step (3): pre-culturing the explant: thickening the explant material in a culture medium A, and expanding joints; step (4), inducing the explants in a culture medium B to generate a large number of protocorm-like bodies, further proliferating the protocorm-like bodies to generate new protocorm-like bodies, developing into seedlings, and enabling the proliferation coefficient to reach 35.0 or above; (5) rooting culture: cutting clumpy buds developed from protocorm-like bodies in the step (4) into proper sizes, and inoculating the clumpy buds into a culture medium C to induce rooting; and (6) domesticating and transplanting. According to the induction method, the protocorm-like body can be directly induced from the explant, seed germination is not needed, the cost is low, the period is short, the seedling quality and the survival rate are high, and application and popularization value is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant seedling cultivation, and in particular to a method for artificially and efficiently propagating roxburghii using protocorm-like bodies. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Anoectochilus is a perennial terrestrial herbaceous plant of the Orchidaceae family. There are about 60 species in the genus, mainly distributed in tropical and subtropical regions of Asia. There are 20 species in China, among which A. roxburghii (Wall.) Lindl. has the widest distribution and is the most used. It is also known as golden thread lotus, golden silkworm, golden stone pine, tree grass lotus, bird ginseng, golden thread tiger head banana, golden thread bone elimination, etc. Its Chinese name is golden thread orchid orchid orchid with flowers and leaves. It is listed as the basal plant of traditional Chinese medicine Anoectochilus along with Taiwan A. formosanus and Yunnan A. chapaensis. Anoectochilus has creeping fleshy roots with nodes, black purple or dark purple leaves, and golden red shiny reticular veins, which has extremely high ornamental value. At the same time, the whole plant of golden thread orchid is used as medicine. It is sweet and neutral in nature. It has the effects of clearing heat and cooling blood, removing dampness and detoxifying. It can treat hemoptysis caused by tuberculosis, diabetes, nephritis, cystitis, myasthenia gravis, spermatorrhea, rheumatic and rheumatoid arthritis, infantile convulsions, leucorrhea in women, and snake bites. Pharmacological studies have shown that golden thread orchid contains alkaloids, saponins, polysaccharides and other ingredients, which have good therapeutic effects on cancer, hyperlipidemia, diabetes, inflammation, and liver damage. The unique leaf structure, beautiful vein color and huge biological activity give golden thread orchid a very high commodity value. The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) lists it as a protected species in Appendix II. China lists it as a second-level protected plant.

[0004] Due to huge market demand and continuous habitat destruction, wild populations of Golden Line Orchid are extremely rare. In order to achieve sustainable development of the high commercial value of Golden Line Orchid, it is urgent to carry out artificial breeding research on it. Seed propagation is a common way of regeneration for many plants, but it is difficult for Golden Line Orchid to regenerate through seeds. Similar to most orchids, the seeds of Golden Line Orchid are small in size and lack endosperm. Their germination requires the combination of mycorrhizae, which provide nutrients for the germinated seeds until the seedlings grow green leaves and can be self-sustaining. Therefore, seed propagation under natural or quasi-natural conditions has the disadvantages of long germination time, low germination rate and seedling rate. For example, the comparative document CN116195516B discloses a non-symbiotic germination culture medium and breeding method for seeds of plants of the same genus as Goldenrod, which uses an improved B5 culture medium to sow seeds of plants of the same genus as Goldenrod to obtain protocorms after seed germination; and continues to subculture the protocorms in the B5 culture medium; then carries out proliferation culture in a proliferation culture medium, which requires 30 days for germination, subculture once every 60 days, requires at least two subcultures, and then requires about 30 days for proliferation, and a single cycle takes three months; and the reproduction coefficient of various Goldenrods for this method is only 4.8-6.5.

[0005] In addition, tillering propagation is another common regeneration method for golden thread orchids. Although it can maintain the characteristics of the parent, one rhizome only produces 1-2 buds, and there are also low efficiency and long cycles, which are not suitable for factory production. In 1992, Knudson's sterile non-symbiotic germination made it possible to mass-produce many valuable orchids. In the process of obtaining strong seedlings, several different forms of regeneration pathways appeared in the micropropagation system. Among them, the emergence of protocorms and protocorm-like bodies (PLBs) greatly improved the reproduction efficiency of orchids. Generally, protocorms-like bodies (PLBs) refer to embryonic tissues that form morphology similar to protocorms in test tubes by explants other than seeds. This structure similar to protocorms (Protocorms) formed in vitro by explant tissues or callus tissues is considered to be somatic embryos with extremely high regeneration potential. The high reproduction rate, short cycle and easy operation of tissue culture make it still an important means of large-scale rapid asexual propagation of excellent hybrid varieties and commercial cut flower varieties of orchids. Therefore, the induction and occurrence of protocorms (Protocorms) or protocorms-like bodies (PLBs) are still one of the research hotspots of orchids. Tissue culture technology not only has the advantages of fast growth, short cycle and strong repeatability, but also has a single source of materials, which can retain the original excellent traits of the plant and obtain a large number of seedlings with highly consistent genetic properties in a short period of time. The development of plant in vitro culture technology provides an effective guarantee for the scarcity of medicinal plant resources. There are some reports on the artificial rapid propagation of golden thread orchid, but they all have low-level publication journals, lack of substantive data, extremely poor repeatability, and unsatisfactory culture cycle and proliferation coefficient, so the overall application of its tissue culture seedlings is still at a low level. Therefore, it is necessary to seek a new asexual reproduction method with strong repeatability, low cost, short time, high quality and survival rate, and the ability to fix excellent traits to expand the reproduction of golden thread orchid, especially high-quality seedlings, and carry out factory production of high-quality seedlings to meet planting needs. Summary of the invention

[0006] The purpose of the present invention is to provide a method for artificially and efficiently propagating golden thread orchid using protocorms in view of the defects of the existing propagation technology of golden thread orchid, solve the problems of poor propagation repeatability and low propagation coefficient of golden thread orchid in the existing technology, and lay a technical foundation for fixing the characteristics of high-quality varieties and developing artificial planting. Through the present invention, high-quality seedlings with consistent genotype background can be provided to meet the needs of artificial planting.

[0007] The technical solution of the present invention is as follows:

[0008] A method for artificially and efficiently breeding Anoectochilus roxburghii using protocorm-like bodies, comprising the following steps:

[0009] Step (1): Obtaining explants: Select healthy plants with good growth potential and no pests or diseases, take stem segments with 3-4 nodes, and remove the leaves;

[0010] Step (2): explant disinfection;

[0011] Step (3): explant pre-culture: the explants treated in step (1) and step (2) are inoculated into a stem segment pre-culture medium and pre-cultured under conditions of controlled light intensity, temperature, and illumination time;

[0012] Step (4): Induction, proliferation and development of protocorm-like bodies (PLBs) into seedlings: The explants pre-cultured in step (3) are transferred into culture medium B for induction, proliferation and development of protocorm-like bodies (PLBs) into seedlings;

[0013] A large number of protocorms (PLBs) are induced by the pretreatment in step (3) and the culture medium culture in step (4). The protocorms (PLBs) in the present invention occur in the deformed axillary buds on one side of the stem node. The axillary buds have extremely shortened internodes and yellow-white spherical granular protrusions on the surface. As the culture time increases, a mass of protocorms (PLBs) is formed on the axillary bud nodes. No callus tissue is observed during the occurrence of protocorms (PLBs). Protocorms (PLBs) are directly generated from the nodes. Unlike some other orchid plants, protocorms (PLBs) are induced directly or indirectly from the stem tip incision.

[0014] Step (5): Rooting culture: the bud clusters developed from the protocorm-like bodies (PLBs) in step (4) are cut into appropriate sizes and inoculated into medium C to induce rooting of the induced new buds;

[0015] Step (6): domestication and transplanting: after the rooted seedlings have grown strong and thick roots, they are transplanted into a substrate for cultivation to obtain transplanted seedlings.

[0016] According to a preferred embodiment, the stem segment preculture medium in step (3) is a culture medium A comprising the following components: MS basic culture solution: 2.0-2.5 mg / L 6-benzylaminopurine (6-BA), 0.5-1.0 mg / L α-naphthylacetic acid (NAA), 5.0-6.0 mg / L uniconazole (S-3307), 30000 mg / L sucrose and 4700 mg / L agar powder, with a pH value of 5.4-5.6.

[0017] The purpose of the pre-culture in step (3) is to make the material thicker and the nodes swell. The inventors of the present application found that S-3307 played a greater role in the culture medium. It stimulated the abnormal activity of various tissue cells in the stem nodes of the explants, producing a structure similar to a "meristematic nodule", from which protocorms (PLBs) were derived; and the effect of S-3307 can last for at least 5 generations, indicating that it has a high affinity with the stem segments of the golden thread orchid, and is easily absorbed by the stem segments as endogenous substances and released from generation to generation; it well explains the origin of protocorms (PLBs) in golden thread orchid. S-3307 has not been disclosed in the prior art to have such an effect.

[0018] According to a preferred embodiment, the culture medium B in step (4) comprises the following components: MS basic culture medium: 1.5-2.0 mg / L 6-benzylaminopurine (6-BA), 0.1-0.5 mg / L kinetin (KT), 0.1-0.5 mg / L α-naphthylacetic acid (NAA), 30000 mg / L sucrose and 4700 mg / L agar powder, with a pH value of 5.4-5.6.

[0019] According to a preferred embodiment, the culture medium C in step (5) comprises the following components: 0.5-1.0 mg / L of α-naphthylacetic acid (NAA), 30000 mg / L of sucrose and 4700 mg / L of agar powder, and the pH value is 5.4-5.6.

[0020] According to a preferred embodiment, step (6) is specifically as follows: when the rooted seedlings are strong and have thick roots, about 5-7 cm in height, the culture bottle is placed under natural light for 5 days, the sealing film of the culture bottle is removed and placed for 2 days, the rooted seedlings are taken out, the residual culture medium is carefully washed, the seedlings are placed in a carbendazim solution for disinfection, and then the seedlings are transplanted into a disinfected matrix for heat preservation and moisture retention, thereby obtaining transplanted seedlings.

[0021] According to a preferred embodiment, the matrix in step (6) is peat soil:perlite=4:1.

[0022] According to a preferred embodiment, the culture conditions in the matrix in step (6) are a culture temperature of 20-25° C. and a humidity of about 70% (65%-75%).

[0023] According to a preferred embodiment, the method for disinfecting the explants in step (2) is as follows: rinse with running water for 30 minutes, place on a clean bench, treat with 75% ethanol solution (v / v) for 10 seconds, treat with 0.1% mercuric chloride aqueous solution (w / v) for 8-10 minutes, 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 use a sterile scalpel to cut the stem segments into 2-3 cm long with 1-2 nodes.

[0024] According to a preferred embodiment, the culture conditions of steps (3), (4) and (5) are: pre-cultivating the stem segments under the conditions of a light intensity of 1500-2000 lx, a light duration of 8 h / d, and a temperature controlled at 23±2°C.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. A method for artificially and efficiently propagating golden thread orchid using protocorms (PLBs). The method of the present application has high efficiency, good proliferation effect, and a propagation coefficient of up to about 35; the culture cycle is short, and in addition to the pre-culture of the explant stem segment, the entire bottle rapid propagation can be completed in 120 days, wherein the induction, proliferation and seedling formation of the protocorms (PLBs) only take 60 days;

[0027] 2. A method for artificially and efficiently breeding golden thread orchid using protocorm-like bodies (PLBs). Based on the optimization of culture medium and culture steps, the induced protocorm-like bodies (PLBs) are large in number, have good proliferation effect, and have strong seedling-forming ability. Each stem segment of the regenerated plant can be used as a transfer material, and the proliferation coefficient is high, which far exceeds the existing reports on the genus Asteraceae. It 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 way for artificial rapid propagation and improves the quality of seedlings. At the same time, only three culture media are needed to complete the cycle culture, which is easy to standardize and factory-operate, and can quickly obtain a large number of seedlings, solve the problem of germplasm resource protection, and provide excellent seedlings of unified standards for large-scale planting.

[0028] 3. A method for artificially and efficiently breeding Rosa rugosa using protocorm-like bodies (PLBs), which has the advantages of low cost, short cycle, high seedling quality and high survival rate compared to the traditional method of non-symbiotic germination of Rosa rugosa seeds; it solves the problems of separation of maternal traits, long cycle, uneven quality of regenerated seedlings, weakness and difficulty in survival caused by non-symbiotic germination of Rosa rugosa seeds, and can keep all seedlings in the same genotype background, which is easy to standardize and operate in a factory, and effectively improves the quality of seedlings.

[0029] 4. 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, and overcomes the difficulty that traditional breeding methods cannot produce year-round; the golden thread orchid test tube seedlings produced by the present invention have a high transplant survival rate and grow rapidly because each clump / plant has an abnormally developed adventitious root system. At present, it has been demonstrated and 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), and the effect is very good;

[0030] 5. The present invention has important significance and value for the artificial rapid propagation of Goldenrod, and can also provide a technical reference for the rapid propagation of other Acrocanthus plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the results of pre-culture of stem segment explants. Scale bar = 2.0 cm.

[0032] in Figure 1 -A is a picture showing axillary buds at each node 15 days after the noded stem segment was grafted; Figure 1 -B After 45 days, the stem segment explants became obviously thicker, the axillary buds grew rapidly, and each stem segment node began to swell;

[0033] Figure 1 -C: After 60 days, the growth of axillary buds was inhibited, while the explant stem segment and the upper-level axillary bud stem node were obviously enlarged.

[0034] Figure 2 The diagram shows the induction, proliferation and seedling formation of protocorm-like bodies (PLBs). Scale bar = 2.0 cm.

[0035] in Figure 2 -A is a picture of the obviously enlarged stem nodes in contact with the culture medium after the formation of axillary buds on day 10 of culture; Figure 2 -B is a picture of axillary buds growing rapidly after 20 days of culture, with nodes continuing to expand and internode distance shortening, and protocorms (PLBs) appearing on the nodes at the base of the material; Figure 2 -C is the buds continue to grow after 30 days of cultivation, the leaves stretch, the stems elongate, the number of protocorms (PLBs) continues to increase, and some protocorms (PLBs) develop into new buds;

[0036] Figure 2 -D shows that after 40 days of culture, the axillary buds grew vigorously, the number of protocorms (PLBs) continued to increase and they continued to develop into new buds; Figure 2 -E is the picture where the growth of the earliest buds is inhibited after 50 days of culture, while new buds continue to grow;

[0037] Figure 2 -F is a picture of the entire culture bottle filled with buds and axillary buds of various levels developed from protocorm-like bodies (PLBs) after 60 days of culture; Figure 2 -G and H are images of protocorm-like bodies (PLBs) clumps cultured for 60-70 days.

[0038] Figure 3 It is a diagram showing the occurrence pattern of protocorm-like bodies (PLBs) on axillary buds of different levels. AE scale bar = 20 mm; FH scale bar = 2.0 cm.

[0039] in Figure 3 -A is a picture of axillary buds starting to appear on the nodes after 5 days of culture; Figure 3-B is the growth diagram of axillary bud primordium formed after 10 days of culture; Figure 3 -C is the picture of the axillary bud swelling and the appearance of nodes after 15 days of culture; Figure 3 -D is a picture showing several protrusions and white villi appearing on the surface of the axillary buds after 20 days of culture; Figure 3 -E is the picture of the axillary buds swelling and producing multiple bud primordia after 30 days of culture, forming a cluster of protobulbs (PLBs); Figure 3 -F is the development of protocorm-like bodies (PLBs) into young shoots after 40 days of culture; Figure 3 -G and H show that the basal nodes of axillary buds at all levels continued to expand after 60 days of culture, while protocorm-like bodies (PLBs) developed into a large number of regenerated buds with extremely shortened internodes, and bud spots continued to form on the basal nodes.

[0040] Figure 4 This is a picture of rooting culture and acclimatization transplanting, scale = 2.0 cm.

[0041] in, Figure 4 -A is a picture of white villi appearing on the material nodes after 15 days of culture, from which short white root tips emerge; Figure 4 -B shows the normal growth of the material after 30 days of culture, and the thick white adventitious roots can be clearly seen; Figure 4 -C is the material continued to grow after 45 days of culture, a large number of roots were formed, the roots turned from white to yellow-green and grew geotropically; Figure 4 -D and E show that the regenerated seedlings grew vigorously after 60 days of culture, with a large number of roots all over the culture bottle. The roots are numerous and strong, which is very conducive to the next step of transplanting and domestication; Figure 4 -F is the picture of normal growth of regenerated seedlings 30 days after transplanting; Figure 4 -G and H show that the stems of the regenerated seedlings are elongated, the leaves are unfolded, the root system is well developed, and the growth condition is good. DETAILED DESCRIPTION

[0042] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1 A method for efficient artificial propagation of golden thread orchid using protocorm-like bodies

[0046] The following steps are involved:

[0047] Step (1): Obtaining explants: Select healthy plants with good growth potential and no diseases or insect pests, take stem segments with 3-4 nodes, and remove the leaves.

[0048] Step (2): rinse the noded stem segments of step (1) with running water for 30 minutes, place them on a clean bench, treat them with 75% ethanol solution (v / v) for 10 seconds, treat them with 0.1% mercuric chloride aqueous solution (w / v) for 8 minutes, and finally rinse them with sterile water for 3 times, each time for no less than 3 minutes; place them on sterile absorbent paper, absorb the surface moisture, and then use a sterile scalpel to cut the stem segments into 2-3 cm long with 1-2 nodes.

[0049] Step (3): Pre-culture of stem segments: The noded stem segments sterilized in step (2) are vertically inoculated into the following culture medium A according to the physiological orientation:

[0050] MS basic culture medium:

[0051]

[0052] Culture conditions: Stem segments were pre-cultured under the conditions of 1500-2000 lx, 8 h / d of illumination, and 23 ± 2 °C of temperature. Figure 1 As shown, after 15 days, axillary buds occurred in each stem node; after 45 days, the stem segment explants became obviously thicker, the axillary buds on them grew rapidly, and each stem segment node began to swell; after 60 days, the growth of axillary buds was inhibited, while the explant stem segment and the upper axillary bud stem nodes were obviously swollen.

[0053] Step (4): Protocorm-like body (PLBs) induction, proliferation and development into seedlings: Cut the old stem segments in step (3) into appropriate sizes and transfer them to the following culture medium B:

[0054] MS basic culture medium:

[0055]

[0056]

[0057] Culture conditions: Culture was carried out under the conditions of illuminance 1500-2000lx, light duration 8h / d, and temperature controlled at 23±2℃. Figure 2 As shown in the figure, after 10 days of culture, the nodes in contact with the culture medium after the occurrence of axillary buds were significantly enlarged; after 20 days of culture, the axillary buds grew rapidly, the nodes continued to swell and the internode distance shortened, and protocorm-like bodies (PLBs) appeared on the nodes at the base of the material; after 30 days of culture, the buds continued to grow, the leaves stretched, the stems elongated, the protocorm-like bodies (PLBs) continued to increase, and some protocorm-like bodies (PLBs) developed into new buds; after 40 days of culture, the axillary buds grew vigorously, the protocorm-like bodies (PLBs) continued to increase and continuously developed into new buds; after 50 days of culture, the growth of the earliest buds was inhibited, and the new buds continued to grow; after 60 days of culture, the entire culture bottle was filled with buds and axillary buds of various levels developed from protocorm-like bodies (PLBs); at this time, the proliferation coefficient was about 35.32.

[0058] It is worth noting that if Figure 2 and Figure 3 As shown, the old stem segments will gradually disappear in the subsequent culture due to shortening of the stems, expansion of the stem nodes, and the production of protocorms (PLBs); however, the above-mentioned culture process can be repeated for the induction of protocorms (PLBs) of the buds at all levels, until the expansion ability of the stem nodes weakens after the fifth generation of transfer. At this time, the pre-culture in step (3) needs to be repeated to obtain stem segment materials with the potential to produce protocorms (PLBs), and the cycle is repeated.

[0059] Step (5): Rooting culture: The bud clusters developed from the protocorm-like bodies (PLBs) in step (4) are cut into appropriate sizes and inoculated into the following culture medium C:

[0060] MS basic culture medium

[0061]

[0062] Culture conditions: Culture under the conditions of illuminance 1500-2000lx, light duration 8h / d, and temperature controlled at 23±2℃. Figure 4 As shown, white villi appeared on the material nodes after 15 days of culture, among which short white root tips were produced; after 30 days of culture, the material grew normally, and thick white adventitious roots could be clearly seen; after 45 days of culture, the material continued to grow, a large number of roots were formed, the roots turned from white to yellow-green and grew geotropically; at this time, the rooting rate was 100%.

[0063] Step (6): domestication and transplanting: Figure 4-F, when the rooted seedlings are strong and thick with a height of about 5-7 cm, the culture bottle is placed under natural light for 5 days, and then the sealing film of the culture bottle is removed and placed for 2 days, the rooted seedlings are taken out, the residual culture medium is carefully washed, and the seedlings are placed in a 0.1% carbendazim solution for treatment for 3 minutes, and then transplanted to a substrate (peat soil: perlite = 4:1) disinfected with a 0.05% potassium permanganate solution for heat preservation and moisture retention (temperature 20-25°C, humidity about 70%) for cultivation, and the transplanted seedlings are obtained, and the survival rate is 100%.

[0064] Example 2 A method for efficient artificial propagation of golden thread orchid using protocorm-like bodies (PLBs)

[0065] The following steps are involved:

[0066] Step (1): Obtaining explants: Select healthy plants with good growth potential and no diseases or insect pests, take stem segments with 3-4 nodes, and remove the leaves.

[0067] Step (2): rinse the noded stem segments of step (1) with running water for 30 minutes, place them on a clean bench, treat them with 75% ethanol solution (v / v) for 10 seconds, treat them with 0.1% mercuric chloride aqueous solution (w / v) for 9 minutes, and finally rinse them with sterile water for 3 times, each time for no less than 3 minutes; place them on sterile absorbent paper, absorb the surface moisture, and then use a sterile scalpel to cut the stem segments into 2-3 cm long with 1-2 nodes.

[0068] Step (3): Pre-culture of stem segments: The noded stem segments sterilized in step (2) are vertically inoculated into the following culture medium A according to the physiological orientation:

[0069] MS basic culture medium:

[0070]

[0071]

[0072] Culture conditions: Stem segments were pre-cultured under the conditions of 1500-2000lx illumination, 8h / d illumination time and temperature controlled at 23±2℃. Axillary buds occurred in each node after 15d. After 45d, the stem segment explants became obviously thicker, the axillary buds grew rapidly, and each node of the stem segment began to swell. After 60d, the growth of axillary buds was inhibited, while the nodes of the explant stem segment and the axillary buds above it swelled obviously.

[0073] Step (4): Protocorm-like body (PLBs) induction, proliferation and development into seedlings: Cut the old stem segments in step (3) into appropriate sizes and transfer them to the following culture medium B:

[0074] MS basic culture medium:

[0075]

[0076] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 8h / d, and temperature controlled at 23±2℃. After 10d of culture, the nodes in contact with the culture medium swelled significantly after the occurrence of axillary buds. After 20d of culture, the axillary buds grew rapidly, the nodes continued to swell and the internode distance shortened, and protocorms (PLBs) appeared on the nodes at the base of the material. After 30d of culture, the buds continued to grow, the leaves stretched, the stems elongated, the protocorms (PLBs) continued to increase, and some protocorms (PLBs) developed into new buds. After 40d of culture, the axillary buds grew vigorously, the protocorms (PLBs) continued to increase and continuously developed into new buds. After 50d of culture, the growth of the earliest buds was inhibited, and the new buds continued to grow. After 60d of culture, the entire culture bottle was filled with buds and axillary buds of various levels developed from protocorms (PLBs); at this time, the proliferation coefficient was about 35.25.

[0077] It is worth noting that the old stem segments will gradually disappear in the subsequent culture due to the shortening of the stems, the expansion of the stem nodes, and the production of protocorms (PLBs); however, the above-mentioned culture process can be repeated for the buds of all levels to induce protocorms (PLBs) until the expansion ability of the stem nodes weakens after the fifth generation of transfer. At this time, the pre-culture in step 3 needs to be repeated to obtain the stem segment material with the ability to produce protocorms (PLBs) again, and the cycle is repeated.

[0078] Step (5): Rooting culture: The bud clusters developed from the protocorm-like bodies (PLBs) in step (4) are cut into appropriate sizes and inoculated into the following culture medium C:

[0079] MS basic culture medium

[0080]

[0081] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 8h / d, and temperature controlled at 23±2℃. After 15 days of culture, white villi appeared on the nodes of the material, among which short white root tips were produced; after 30 days of culture, the material grew normally, and thick white adventitious roots could be clearly seen; after 45 days of culture, the material continued to grow, a large number of roots were formed, the roots turned from white to yellow-green and grew geotropically; at this time, the rooting rate was 100%.

[0082] Step (6): domestication and transplanting: when the rooted seedlings are strong and thick, and about 5-7 cm in height, the culture bottle is placed under natural light for 5 days, and then the sealing film of the culture bottle is removed and placed for 2 days, the rooted seedlings are taken out, the residual culture medium is carefully washed, and the seedlings are placed in a 0.3% carbendazim solution for 4 minutes, and then transplanted to a substrate (peat soil: perlite = 4:1) disinfected with a 0.08% potassium permanganate solution for heat preservation and moisture retention (temperature 20-25°C, humidity about 70%) for cultivation, and the transplanted seedlings are obtained, and the survival rate is 100%.

[0083] Example 3 A method for efficient artificial propagation of golden thread orchid using protocorm-like bodies (PLBs)

[0084] The following steps are involved:

[0085] Step (1): Obtaining explants: Select healthy plants with good growth potential and no diseases or insect pests, take stem segments with 3-4 nodes, and remove the leaves.

[0086] Step (2): rinse the noded stem segments of step (1) with running water for 30 minutes, place them on a clean bench, treat them with 75% ethanol solution (v / v) for 10 seconds, treat them with 0.1% mercuric chloride aqueous solution (w / v) for 10 minutes, and finally rinse them with sterile water for 3 times, each time for no less than 3 minutes; place them on sterile absorbent paper, absorb the surface moisture, and then use a sterile scalpel to cut the stem segments into 2-3 cm long with 1-2 nodes.

[0087] Step (3): Pre-culture of stem segments: The noded stem segments sterilized in step (2) are vertically inoculated into the following culture medium A according to the physiological orientation:

[0088] MS basic culture medium:

[0089]

[0090] Culture conditions: Stem segments were pre-cultured under the conditions of 1500-2000lx illumination, 8h / d illumination time and temperature controlled at 23±2℃. Axillary buds occurred in each node after 15d. After 45d, the stem segment explants became obviously thicker, the axillary buds grew rapidly, and each node of the stem segment began to swell. After 60d, the growth of axillary buds was inhibited, while the nodes of the explant stem segment and the axillary buds above it swelled obviously.

[0091] Step (4): Protocorm-like body (PLBs) induction, proliferation and development into seedlings: Cut the old stem segments in step (3) into appropriate sizes and transfer them to the following culture medium B:

[0092] MS basic culture medium:

[0093]

[0094] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 8h / d, and temperature controlled at 23±2℃. After 10d of culture, the nodes in contact with the culture medium swelled significantly after the occurrence of axillary buds. After 20d of culture, the axillary buds grew rapidly, the nodes continued to swell, and the internode distance shortened, and protocorms (PLBs) appeared on the nodes at the base of the material. After 30d of culture, the buds continued to grow, the leaves stretched, the stems elongated, the protocorms (PLBs) continued to increase, and some protocorms (PLBs) developed into new buds. After 40d of culture, the axillary buds grew vigorously, and the protocorms (PLBs) continued to increase and continuously developed into new buds. After 50d of culture, the growth of the earliest buds was inhibited, and the new buds continued to grow. After 60d of culture, the entire culture bottle was filled with buds and axillary buds of various levels developed from protocorms (PLBs); at this time, the proliferation coefficient was about 35.20.

[0095] It is worth noting that the old stem segments will gradually disappear in the subsequent culture due to the shortening of the stems, the expansion of the stem nodes, and the production of protocorms (PLBs); however, the above-mentioned culture process can be repeated for the buds of all levels to induce protocorms (PLBs) until the expansion ability of the stem nodes weakens after the fifth generation of transfer. At this time, the pre-culture in step 3 needs to be repeated to obtain the stem segment material with the ability to produce protocorms (PLBs) again, and the cycle is repeated.

[0096] Step (5): Rooting culture: The bud clusters developed from the protocorm-like bodies (PLBs) in step (4) are cut into appropriate sizes and inoculated into the following culture medium C:

[0097] MS basic culture medium

[0098]

[0099] Culture conditions: Culture was carried out under the conditions of light intensity of 1500-2000lx, light time of 8h / d, and temperature controlled at 23±2℃. After 15 days of culture, white villi appeared on the nodes of the material, among which short white root tips were produced; after 30 days of culture, the material grew normally, and thick white adventitious roots could be clearly seen; after 45 days of culture, the material continued to grow, a large number of roots were formed, the roots turned from white to yellow-green and grew geotropically; at this time, the rooting rate was 100%.

[0100] Step (6): domestication and transplanting: when the rooted seedlings are strong and thick, and about 5-7 cm in height, the culture bottle is placed under natural light for 5 days, and then the sealing film of the culture bottle is removed and placed for 2 days, the rooted seedlings are taken out, the residual culture medium is carefully washed, and the seedlings are placed in a 0.5% carbendazim solution for treatment for 5 minutes, and then transplanted to a substrate (peat soil: perlite = 4:1) disinfected with a 0.1% potassium permanganate solution for heat preservation and moisture retention (temperature 20-25°C, humidity about 70%) for cultivation, and the transplanted seedlings are obtained, and the survival rate is 100%.

[0101] Technical principle of the present invention:

[0102] 1. As Figure 3 As shown, the protocorm-like bodies (PLBs) of the present invention occur in the deformed axillary buds on one side of the stem node. The axillary buds have extremely shortened internodes and yellow-white spherical granular protrusions on the surface. As the culture time increases, a protocorm-like body (PLBs) mass is formed on the axillary bud node. No callus tissue is observed during the occurrence of protocorms (PLBs), and protocorms (PLBs) are directly produced from the nodes. Unlike some other orchid plants, protocorms (PLBs) are directly or indirectly induced from the stem tip incision. At the same time, under a stereoscope, it was found that this protocorm-like body (PLBs) mass is formed at the position of the base node of the axillary bud, and the young shoots developed from the protocorm-like body (PLBs) are connected by vascular bundles at the base. It can be judged that the protocorm-like body (PLBs) mass may come from the vascular bundle on the base node of the axillary bud. The inventors of the present application have discovered that under the action of exogenous hormones, especially succinylcholine (S-3307), the regenerated axillary buds have extremely shortened internodes in structure; under the influence of endogenous and exogenous hormones, the vascular parenchyma cells in the deformed bud swelling nodes are abnormally excited, and then differentiate into intrafascicular cambium, forming a nodule swelling similar to the secondary growth of dicotyledonous stems, producing a mass of meristem, and thus forming protobulbs (PLBs). This special swollen axillary bud node in the present invention should essentially be the result of continuous differentiation of axillary meristems, similar to the nodules of woody plants, morphologically a compact and independent cell mass, and in terms of internal structure, it contains at least meristematic cells, plastid-rich parenchyma cells and vascular cells, as well as epidermis, cortex and vascular tissue; under appropriate conditions, this nodule can directly differentiate into plant organs such as buds and roots, and the regenerated buds can develop into complete plants. From Figure 3 It can be seen that no bipolar structure was found in the development of these extremely large number of PLBs directly connected to the explants, suggesting that the regeneration of PLBs in A. roxburghii is an independent pathway. At the same time, the plantlets developed from PLBs have shortened internodes, and the bud primordia on the nodes differentiate vigorously to form bud clusters, further expanding their proliferation coefficient.

[0103] 2. In the actual production of golden thread orchid, most companies adopt the seed-protocorm-regenerated seedling approach. After pre-storing a sufficient amount of capsules, they can be sown at any time of the year. The protocorms produced by non-symbiotic germination of seeds generally take 18 months or even longer to produce test tube seedlings that can be transplanted; the biggest disadvantage of this method is the uneven emergence of seedlings and serious separation of seedling traits. The present invention uses stem segments with 1-2 nodes as materials. Due to the emergence of protocorms (PLBs), its proliferation coefficient far exceeds that of similar studies, and the seedling time is shortened to 120d, which is unattainable by the seed-protocorm-regenerated seedling approach. At the same time, the present invention avoids the trait differences caused by separation, thereby obtaining regenerated seedlings with consistent genetic backgrounds, which is also unattainable by the seed-protocorm-regenerated seedling approach.

[0104] 3. The present invention provides a method for artificially and efficiently breeding golden thread orchid using protocorm-like bodies (PLBs), which has low cost, short time, high quality and survival rate, and can fix excellent traits; this method can be used to expand the breeding volume of high-quality golden thread orchid seedlings, thereby carrying out factory production of high-quality seedlings to meet planting needs.

[0105] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for artificially and efficiently propagating Anoectochilus roxburghii using protocorm-like bodies, characterized in that: The steps include: Step (1): explant acquisition; Step (2): explant disinfection; Step (3): explant pre-culture: inoculating the explants treated in steps (1) and (2) into a stem segment pre-culture medium for pre-culture; Step (4): Protocorm-like body induction, proliferation and development into seedlings: the explants pre-cultured in step (3) are transferred to culture medium B for protocorm-like body induction, proliferation and development into seedlings; Step (5): Rooting culture: cutting the bud cluster developed from the protocorm-like body in step (4) into appropriate sizes and inoculating the buds induced in culture medium C to root; Step (6): domestication and transplanting: after the rooted seedlings have grown strong and thick roots, they are transplanted into a substrate for cultivation to obtain transplanted seedlings.

2. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 1, characterized in that: The stem segment preculture medium in step (3) is a culture medium A comprising the following components: MS basic culture solution: 6-benzylaminopurine, α-naphthylacetic acid, uniconazole, sucrose and agar powder.

3. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 2, characterized in that: The stem segment preculture medium in step (3) is a culture medium A comprising the following components: MS basic culture solution: 2.0-2.5 mg / L 6-benzylaminopurine, 0.5-1.0 mg / L α-naphthylacetic acid, 5.0-6.0 mg / L uniconazole, 30000 mg / L sucrose and 4700 mg / L agar powder, with a pH value of 5.4-5.

6.

4. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 1, characterized in that: The culture medium B in step (4) comprises the following components: MS basic culture solution: 6-benzylaminopurine, kinetin, α-naphthylacetic acid, sucrose and agar powder.

5. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 4, characterized in that: The culture medium B in step (4) comprises the following components: MS basic culture solution: 1.5-2.0 mg / L 6-benzylaminopurine, 0.1-0.5 mg / L kinetin, 0.1-0.5 mg / L α-naphthylacetic acid, 30000 mg / L sucrose and 4700 mg / L agar powder, with a pH value of 5.4-5.

6.

6. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 1, characterized in that: The culture medium C in step (5) comprises the following components: α-naphthylacetic acid, sucrose and agar powder.

7. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 6, characterized in that: The culture medium C in step (5) comprises the following components: 0.5-1.0 mg / L of α-naphthylacetic acid, 30,000 mg / L of sucrose and 4,700 mg / L of agar powder, and the pH value is 5.4-5.

6.

8. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 1, characterized in that: The culture conditions of steps (3), (4) and (5) are: pre-culture of stem segments under the conditions of light intensity of 1500-2000 lx, light duration of 8 h / d, and temperature controlled at 23±2°C.

9. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 1, characterized in that: The matrix in step (6) is peat soil:perlite=4:

1.

10. The method for artificial efficient propagation of Anoectochilus roxburghii using protocorm-like bodies according to claim 7, characterized in that: The culture conditions in the matrix in step (6) are a culture temperature of 20-25° C. and a humidity of 65%-75%.

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