Method for breeding excellent seedlings of dendrobium nobile in tropical rainforest

Abstract

The invention belongs to the technical field of agricultural biology, and relates to a breeding method of excellent seedlings of dendrobium nobile in tropical rainforests, which comprises the following steps: collecting wild dendrobium nobile seeds in a rainforest area of Wuzhishan in Hainan as explants, carrying out protocorm induction, proliferation and differentiation to obtain plantlets, and carrying out rooting culture to obtain complete regenerated plants. According to the method, the wild dendrobium nobile seeds in the Wuzhishan rainforest region in Hainan are used as the explants for tissue culture, the differentiation rate can be increased, the propagation speed can be increased, environmental factors in the Wuzhishan rainforest region in Hainan are simulated as culture conditions, the seedlings can be cultivated in a field-like environment, and the obtained dendrobium nobile seedlings are stable in hereditary character, small in variation and high in yield. The seedling field transplanting domestication time is shortened, the transplanting survival rate is increased, the growth speed and the growth amount after transplanting are increased, the content of effective components of the product is higher than those of similar products in other production areas, and a material and technical foundation is laid for promoting sustainable development of the dendrobium nobile industry in Hainan.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and relates to a method for breeding excellent Dendrobium nobile seedlings, in particular to a method for breeding excellent tropical rainforest Dendrobium nobile seedlings. Background Art

[0002] Dendrobium nobile is a precious species of orchids and a rare medicinal plant. It has the effects of nourishing yin and clearing heat, promoting body fluid and quenching thirst, nourishing the stomach and benefiting the kidneys, etc. It is widely used in the field of traditional Chinese medicine. In addition, Dendrobium nobile is rich in nutrients such as polysaccharides, amino acids, vitamins and trace elements, and has extremely high health value. In recent years, it has attracted much attention due to its unique medicinal and ornamental value, and the market demand for Dendrobium nobile has continued to increase. However, the seeds of Dendrobium nobile are extremely small, the embryo is not fully developed, and there is no endosperm tissue. The germination rate is extremely low (less than 5%) in the natural state. In addition, the long-term predatory excavation has caused the wild resources to be almost exhausted and endangered, which is far from meeting the domestic and foreign market demand, resulting in a shortage of market supply. Therefore, it is of great significance to carry out research on the artificial cultivation of Dendrobium nobile.

[0003] Plant tissue culture has the characteristics of low raw material demand, no restrictions on time and location, high reproduction coefficient, and can maintain the excellent traits of the mother plant. It is widely used in plant seedling breeding. With the continuous increase in artificial planting area, the demand for high-quality seedlings of Dendrobium nobile is also increasing. The use of tissue rapid propagation technology to produce large quantities of high-quality seedlings of Dendrobium nobile has solved the shortage of high-quality seedlings of Dendrobium nobile to a certain extent.

[0004] Nowadays, the main research directions of tissue culture technology of Dendrobium nobile are explant screening, culture medium selection and preparation, culture method selection, plant regeneration pathway design, etc. Although it can effectively improve the efficiency of cell dedifferentiation and redifferentiation, and provide a large number of high-quality seedlings with consistent traits for subsequent artificial cultivation of Dendrobium nobile, the existing tissue culture system of Dendrobium nobile does not fully consider ecological factors. In actual production, the growth of plants is not only affected by internal factors, but also by some external factors, such as ecological environment and climatic conditions. The specificity of the environment may cause problems such as low yield and quality decline when introducing excellent varieties, and even plant leaf atrophy and whole plant death.

[0005] Dendrobium nobile is mainly distributed in the subtropical areas of southern my country, growing in mountain forests at higher altitudes, including Wuzhishan in Hainan and Chishui in Guizhou. Due to the different ecological environments in different regions, the species of Dendrobium nobile in different regions vary greatly, and the composition of the culture medium for in vitro culture is also very different. At present, the cultivation of Dendrobium nobile in Hainan is developing rapidly, mainly concentrated in the Wuzhishan rainforest area (at a higher altitude), but the corresponding high-quality seedlings cannot keep up with the development needs, and the excellent seedlings introduced from other provinces are prone to "acclimatization" when planted in Hainan due to the unique geographical environment and weather of Hainan Province, resulting in unsatisfactory production. Summary of the invention

[0006] The invention aims to provide a breeding method for high-quality seedlings of Dendrobium nobile in tropical rain forests. The method comprises the following steps: using wild Dendrobium nobile seeds in a Wuzhishan rain forest area (a high-altitude area in Hainan) as explants for tissue culture, and improving the differentiation rate by screening a culture medium, thereby improving the propagation speed and efficiency of the high-quality seedlings of Dendrobium nobile. The method also comprises the following steps: simulating environmental factors in the Wuzhishan rain forest area as culture conditions, so that the seedlings can be cultivated in a field-like environment, greatly shortening the time for transplanting and domesticating the seedlings, facilitating improving the survival rate of field transplanting, accelerating the growth rate after transplanting, and shortening the product harvesting time.

[0007] The technical solution adopted by the present invention is: A method for breeding high-quality tropical rainforest Dendrobium nobile seedlings, comprising the following steps: Step S1, collecting wild Dendrobium nobile seeds from the Wuzhishan rainforest area, selecting seeds that are moderately mature, plump and free of pests and diseases, and then performing surface disinfection treatment as explants.

[0008] Step S2, inoculating the seeds of Dendrobium nobile into an induction culture medium, germinating the seeds to form protocorms, and the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment. Subculture is performed once every 20-30 days, and the number of subcultures is 1-2 times; the induction culture medium is a modified MS culture medium + peptone 200-250 mg / L + FeSO4·7H2O10-20 mg / L + K2SO4 150-250 mg / L + Na2-EDTA 10-20 mg / L + aminoacetic acid 3-5 mg / L + calcium pantothenate 3-6 mg / L + 6-BA 4-6 mg / L + 2,4-D 2-4 mg / L + NAA 0.1~0.3mg / L+sucrose 300~350mg / L +5.0g / L agar, pH is 5.8.

[0009] Step S3, inoculating the protocorm into a proliferation culture medium for proliferation culture, the protocorm continuously proliferates and forms a protocorm-like cluster, and the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and the environment maintains high negative oxygen ion air. Subculture is performed once every 20-30 days, and the number of subcultures is 1-3 times; the proliferation culture medium is a modified MS culture medium + peptone 200-250 mg / L + FeSO4·7H2O 10-20 mg / L + K2SO4 150-250 mg / L + aminoacetic acid 3-5 mg / L + calcium pantothenate 3-6 mg / L + 6-BA 1-2 mg / L + 2,4-D 1-2 mg / L + NAA 0.1-0.3 mg / L + sucrose 300-350 mg / L + 5.0 g / L agar, pH 5.8.

[0010] Step S4, selecting well-growing, emerald green, and densely structured protocorms from the proliferating protocorm-like body clusters and transferring them into a differentiation medium for differentiation culture to obtain clustered buds; the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment, subculture is performed once every 20-30 days, and the number of subcultures is 1-3; the differentiation medium is a modified MS medium + peptone 200-250 mg / L + FeSO4·7H2O 10-20 mg / L + K2SO4 150-250 mg / L + Na2-EDTA 10-20 mg / L+aminoacetic acid 3-5 mg / L+calcium pantothenate 3-6 mg / L+glutamine 100-150 mg / L+2ip (N6-isopentenyl adenine) 0.2-0.5 mg / L+6-BA 3-5 mg / L+NAA 0.2-0.5 mg / L+sucrose 300-350 mg / L +5.0 g / L agar, pH is 5.8.

[0011] Step S5, cutting the differentiated clustered buds into single buds, inoculating them on a rooting and seedling-strengthening medium for culture, and obtaining complete regenerated plants, the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment, subculture is performed once every 20-30 days, and subculture is performed 1-3 times; the rooting and seedling-strengthening medium is a modified MS medium + peptone 200-250 mg / L + aminoacetic acid 3-5 mg / L + biotin 3-6 mg / L + 6-BA 3-5 mg / L + NAA 0.2-0.5 mg / L + IBA 0.5-0.8 mg / L + sucrose 300-350 mg / L + 5.0 g / L agar, and the pH is 5.8.

[0012] Furthermore, in step S1, the disinfection treatment is to soak the seeds in 75% alcohol for 8 to 12 minutes, then rinse with sterile water 2 to 4 times, each time for 5 minutes, to remove impurities and microorganisms on the surface of the seeds, and dry them under sterile conditions.

[0013] Furthermore, in steps S2, S3, S4 and S5, the high negative oxygen ion air refers to air in which the concentration of negative oxygen ions is maintained at more than 3000 per cubic centimeter.

[0014] Furthermore, in step S5, the mixed concentration of NAA and IBA in the rooting and seedling strengthening medium is 1 mg / L.

[0015] The present invention uses wild Dendrobium nobile seeds from the Wuzhishan rainforest area (a high altitude area in Hainan) as explants for tissue culture, which can reflect the regional ecological environment differences and plant quality differences. At the same time, the differentiation rate can be improved by screening the culture medium. By simulating the environmental factors (high negative oxygen ions, large temperature difference between day and night, and high humidity) in the high altitude area of ​​Hainan as culture conditions, the propagation speed and efficiency of excellent Dendrobium nobile seedlings can be improved, laying a foundation for large-scale production of Dendrobium nobile seedlings with consistent traits and large-scale cultivation. In addition, due to the setting of the culture conditions, the seedlings can be cultivated in a quasi-field environment. The obtained Dendrobium nobile seedlings have stable genetic traits and small variation, and have the characteristics of simple operation, low cost, high reproduction coefficient, etc., which greatly shortens the seedling transplanting and acclimation time, is conducive to improving the survival rate of transplanting, accelerates the growth speed and growth amount after transplanting, shortens the growth cycle, and the content of the active ingredients of the products is higher than that of similar products in other production areas, and the quality is greatly improved, laying a material and technical foundation for promoting the sustainable development of the Dendrobium nobile industry in Hainan. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention are further described in detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0017] The quantitative tests in the following examples were repeated three times, and the results were averaged. The modified MS medium involved was based on the MS medium, with the content of macroelements adjusted to 3 / 4 of the original content, the content of trace elements adjusted to 2 times the original content, and the other components remained unchanged.

[0018] 1. Tissue culture and seedling cultivation of Dendrobium nobile Embodiment 1

[0019] (1) Wild Dendrobium nobile capsules with moderate maturity, full fruit, no diseases, insect pests or cracks were collected from the Wuzhishan tropical rain forest area in Hainan. After cleaning and disinfection, the capsules were peeled and the seeds were taken out. The seeds were soaked in 75% alcohol for 10 minutes, then rinsed with sterile water for 3 times, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dried under sterile conditions as explants.

[0020] (2) The seeds of Dendrobium nobile were inoculated into an induction medium (modified MS medium + peptone 200 mg / L + FeSO4·7H2O 20 mg / L + K2SO4 200 mg / L + Na2-EDTA 20 mg / L + aminoacetic acid 4 mg / L + calcium pantothenate 5 mg / L + 6-BA 4 mg / L + 2,4-D 4 mg / L + NAA 0.2 mg / L + sucrose 300 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 3000 lx, photoperiod of 12 h light / 12 h dark, temperature of 25°C during light and 20°C during darkness, humidity of 80%, and negative oxygen ion concentration in the air above 3000 per cubic centimeter. The seeds were subcultured every 25 days, and the seeds germinated to form protocorms after one subculture.

[0021] (3) Select protocorms with good growth, emerald green color and compact structure and inoculate them into proliferation medium (modified MS medium + peptone 200 mg / L + FeSO4·7H2O 20 mg / L + K2SO4 200 mg / L + aminoacetic acid 4 mg / L + calcium pantothenate 5 mg / L + 6-BA 2 mg / L + 2,4-D 1 mg / L + NAA 0.2 mg / L + sucrose 300 mg / L + 5.0 g / L agar, pH 5.8) for proliferation culture. The culture conditions are as follows: light intensity of 3000 lx, light cycle of 12 hours light / 12 hours dark per day, temperature of 25℃ during light and 20℃ during dark, humidity of 80%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. Subculture once every 25 days, and obtain protocorm-like clusters after two subcultures.

[0022] (4) Select the well-growing, emerald green and compactly structured protocorms from the proliferating protocorm-like body clusters and transfer them into differentiation medium (modified MS medium + peptone 220 mg / L + FeSO4·7H2O 15 mg / L + K2SO4 180 mg / L + Na2-EDTA 17 mg / L + aminoacetic acid 3 mg / L + calcium pantothenate 6 mg / L + glutamine 100 mg / L + 2ip 0.5 mg / L + 6-BA 4 mg / L + NAA 0.4 mg / L + sucrose 330 mg / L + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain clustered buds; the culture conditions are as follows: light intensity of 2800 lx, light cycle of 12 hours light / 12 hours dark per day, temperature of 27°C during light and 18°C ​​in dark, humidity of 75%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. Subculture once every 30 days. Once the original corm germinates, continue subculture once to obtain seedlings.

[0023] (5) The seedlings were inoculated on a rooting and seedling medium (modified MS medium + peptone 250 mg / L + aminoacetic acid 3 mg / L + biotin 6 mg / L + 6-BA 3 mg / L + NAA 0.3 mg / L + IBA 0.7 mg / L + sucrose 320 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 3200 lx, daily photoperiod of 12 hours light / 12 hours dark, temperature of 25°C during light, 20°C during darkness, humidity of 85%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. Subculture was performed every 25 days, and a complete root system was generated after two subcultures to obtain a complete regenerated plant.

[0024] Embodiment 2 (1) Wild Dendrobium nobile capsules with moderate maturity, full fruit, no diseases, insect pests or cracks were collected from the Wuzhishan tropical rain forest area in Hainan. After cleaning and disinfection, the capsules were peeled and the seeds were taken out. The seeds were soaked in 75% alcohol for 10 minutes, then rinsed with sterile water for 3 times, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dried under sterile conditions as explants.

[0025] (2) The seeds of Dendrobium nobile were inoculated into an induction medium (modified MS medium + peptone 240 mg / L + FeSO4·7H2O 13 mg / L + K2SO4 160 mg / L + Na2-EDTA 17 mg / L + aminoacetic acid 3 mg / L + calcium pantothenate 4 mg / L + 6-BA 6 mg / L + 2,4-D 2 mg / L + NAA 0.1 mg / L + sucrose 350 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 2900 lx, photoperiod of 12 h light / 12 h dark, temperature of 27°C during light and 20°C during darkness, humidity of 80%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. The seeds were subcultured every 25 days, and the seeds germinated to form protocorms after one subculture.

[0026] (3) Select protocorms with good growth, emerald green color and relatively dense structure and inoculate them into proliferation medium (modified MS medium + peptone 240 mg / L + FeSO4·7H2O 13mg / L + K2SO4 160mg / L + aminoacetic acid 3mg / L + calcium pantothenate 4mg / L + 6-BA 1mg / L + 2,4-D 2mg / L + NAA 0.3mg / L + sucrose 350mg / L + 5.0g / L agar, pH 5.8) for proliferation culture. The culture conditions are as follows: light intensity 2900 lx, daily light cycle 12 hours light / 12 hours dark, temperature 27℃ during light, temperature 20℃ during dark, humidity 80%, and the concentration of negative oxygen ions in the air in the environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days, and protocorm-like clusters are obtained after two subcultures.

[0027] (4) Select the well-growing, emerald green and compactly structured protocorm-like bodies from the proliferating protocorm-like body clusters and transfer them into differentiation medium (modified MS medium + peptone 200 mg / L + FeSO4·7H2O 12 mg / L + K2SO4 220 mg / L + Na2-EDTA 11 mg / L + aminoacetic acid 4 mg / L + calcium pantothenate 4 mg / L + glutamine 150 mg / L + 2ip 0.3 mg / L + 6-BA 3 mg / L + NAA 0.2 mg / L + sucrose 300 mg / L + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain clustered buds; the culture conditions are as follows: light intensity of 3200 lx, light cycle of 12 hours light / 12 hours dark per day, temperature of 25°C during light and 20°C during dark, humidity of 75%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. Subculture once every 30 days. Once the original corm germinates, continue subculture once to obtain seedlings.

[0028] (5) The seedlings were inoculated on a rooting and seedling medium (modified MS medium + peptone 200 mg / L + aminoacetic acid 5 mg / L + biotin 4 mg / L + 6-BA 4 mg / L + NAA 0.5 mg / L + IBA 0.5 mg / L + sucrose 350 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 3000 lx, daily photoperiod of 12 hours light / 12 hours dark, temperature of 25°C during light, 18°C ​​during darkness, humidity of 85%, and the concentration of negative oxygen ions in the air maintained at more than 3000 per cubic centimeter. Subculture was performed every 25 days, and a complete root system was generated after two subcultures to obtain a complete regenerated plant.

[0029] Embodiment 3 (1) Wild Dendrobium nobile capsules with moderate maturity, full fruit, no diseases, insect pests or cracks were collected from the Wuzhishan tropical rain forest area in Hainan. After cleaning and disinfection, the capsules were peeled and the seeds were taken out. The seeds were soaked in 75% alcohol for 10 minutes, then rinsed with sterile water for 3 times, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dried under sterile conditions as explants.

[0030] (2) The seeds of Dendrobium nobile were inoculated into an induction medium (modified MS medium + peptone 210 mg / L + FeSO4·7H2O 16 mg / L + K2SO4 220 mg / L + Na2-EDTA 16 mg / L + aminoacetic acid 5 mg / L + calcium pantothenate 3 mg / L + 6-BA 5 mg / L + 2,4-D 3 mg / L + NAA 0.3 mg / L + sucrose 320 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 3200 lx, photoperiod of 12 h light / 12 h dark, temperature of 25°C during light and 18°C ​​during darkness, humidity of 75%, and negative oxygen ion concentration in the air above 3000 per cubic centimeter. The seeds were subcultured every 25 days, and after two subcultures, the seeds germinated to form protocorms.

[0031] (3) Select protocorms with good growth, emerald green color and relatively dense structure and inoculate them into proliferation medium (modified MS medium + peptone 210 mg / L + FeSO4·7H2O 16 mg / L + K2SO4 220 mg / L + aminoacetic acid 5 mg / L + calcium pantothenate 3 mg / L + 6-BA 2 mg / L + 2,4-D 1 mg / L + NAA 0.3 mg / L + sucrose 320 mg / L + 5.0 g / L agar, pH 5.8) for proliferation culture. The culture conditions are as follows: light intensity of 3200 lx, daily light cycle of 12 hours light / 12 hours dark, temperature of 25℃ during light, 18℃ during dark, humidity of 75%, and the concentration of negative oxygen ions in the air in the environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days, and obtain protocorm-like clusters after two subcultures.

[0032] (4) Select the well-growing, emerald green and compactly structured protocorm-like bodies from the proliferating protocorm-like body clusters and transfer them into differentiation medium (modified MS medium + peptone 250 mg / L + FeSO4·7H2O 20 mg / L + K2SO4 150 mg / L + Na2-EDTA 15 mg / L + aminoacetic acid 5 mg / L + calcium pantothenate 3 mg / L + glutamine 120 mg / L + 2ip 0.3 mg / L + 6-BA 5 mg / L + NAA 0.2 mg / L + sucrose 350 mg / L + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain clustered buds; the culture conditions are as follows: light intensity of 2800 lx, light cycle of 12 hours light / 12 hours dark per day, temperature of 27°C during light and 20°C during dark, humidity of 75%, and negative oxygen ion concentration in the air maintained at more than 3000 per cubic centimeter. Subculture once every 30 days. Once the original corm germinates, continue subculture once to obtain seedlings.

[0033] (5) The seedlings were inoculated on a rooting and seedling medium (modified MS medium + peptone 230 mg / L + aminoacetic acid 4 mg / L + biotin 3 mg / L + 6-BA 5 mg / L + NAA 0.4 mg / L + IBA 0.6 mg / L + sucrose 300 mg / L + 5.0 g / L agar, pH 5.8) and cultured under the following conditions: light intensity of 2800 lx, daily photoperiod of 12 hours light / 12 hours dark, temperature of 27°C during light, 20°C during darkness, humidity of 85%, and the concentration of negative oxygen ions in the air maintained at more than 3000 per cubic centimeter. Subculture was performed every 25 days, and a complete root system was generated after two subcultures to obtain a complete regenerated plant.

[0034] 2. Effect Verification (1) Identification of the effect of tissue culture of Dendrobium nobile

[0035] 1. Identification of seed germination and proliferation of Dendrobium nobile

[0036] In order to investigate the germination and proliferation of Dendrobium nobile seeds, the number of protocorms formed by germination of Dendrobium nobile seeds and the proliferation of protocorms in the above embodiment were observed and counted, the germination rate and proliferation coefficient were calculated, and the average values ​​were taken. The results are shown in Table 1.

[0037] Table 1 Germination and proliferation of Dendrobium nobile seeds

[0038] *The number of seeds is based on the unit area (1cm 2 ) × total area. For ease of comparison, the initial number of seeds is set at 500.

[0039] The above-mentioned results of seed germination of Dendrobium nobile show that the improved MS-based culture medium of the present invention can effectively promote seed germination of Dendrobium nobile, improve the seed germination rate, and has a good effect of protocorm proliferation in the later stage and a high proliferation coefficient.

[0040] 2. Identification of protocorm differentiation effect In order to identify the differentiation effect of the protocorms, the differentiation conditions and the number of seedlings of the protocorms of the above examples were observed and counted, the differentiation rate was calculated, and the average value of each data was taken. The results are shown in Table 2.

[0041] Table 2 Differentiation of protocorms

[0042] The above-mentioned protocorm differentiation results show that the improved MS culture medium used in the present invention to induce the differentiation of protocorms into seedlings has a high differentiation rate, which can reach up to 96%, and has good differentiation efficiency.

[0043] 3. Identification of rooting effect of seedlings In order to identify the rooting effect of the seedlings, the rooting of the seedlings obtained in the above embodiment was observed and counted, the number of roots was counted, the rooting rate was calculated, and the average value of each data was taken. The results are shown in Table 3.

[0044] Table 3 Rooting of seedlings

[0045] The above seedling rooting results show that the present invention induces rooting of the obtained seedlings, and the rooting is fast, the root system is well developed, and the rooting rate reaches 100%, which is beneficial to improving the transplant survival rate of the rooted seedlings and the growth of the plants.

[0046] (2) Evaluation of field transplanting effects The Dendrobium nobile seedlings obtained in Example 1, Example 2, and Example 3 were planted in the wild in Wuzhishan, Hainan. Control 1: Wild Dendrobium nobile seeds in the Wuzhishan rainforest area were used as explants, and the culture medium at each stage was the same as that used in Example 1, but conventional culture conditions were used, specifically: the light intensity was 2800-3300 lx, the daily photoperiod was 12 hours of light / 12 hours of darkness, and the temperature was 25±2°C. Control 2: Commercially available Dendrobium nobile tissue culture seedlings.

[0047] Growth performance was evaluated 60 days after seedling transplantation, including indicators such as survival rate, growth rate, plant height, stem diameter, leaf area and root system status, as well as quantitative analysis of the content of medicinal ingredients such as dendrobium alkaloids, polysaccharides, amino acids, vitamin C and vitamin E in the materials, among which vitamin C and vitamin E were calculated by fresh weight. The average value of each data is shown in Tables 4 and 5.

[0048] Table 4 Growth of seedlings after transplanting

[0049] The above field transplantation results show that this application can effectively improve the quality of seedlings by screening culture media to improve the culture effect and simulating the Wuzhishan tropical rainforest environment (including light, temperature, humidity, air, etc.) as culture conditions. With the extension of planting time, the plant height, stem thickness and leaf area of ​​the seedlings gradually increase, and the root system is well developed, and the growth performance is significantly better than that of seedlings in ordinary culture environments (Control 1). Although Control 1 uses wild Dendrobium nobile seeds from the Wuzhishan tropical rainforest area as explants to breed seedlings, the transplanting situation is slightly better than the commercially available tissue culture seedlings (Control 2), but the advantage is not obvious.

[0050] Table 5 Content of each component in the material

[0051] The above-mentioned medicinal ingredient analysis results show that after the seedlings provided in the present application are planted, the contents of medicinal ingredients such as dendrobium alkaloids, polysaccharides, amino acids, vitamin C and vitamin E are significantly higher than those of seedlings grown under ordinary culture conditions (control one) and commercially available tissue culture seedlings (control two). The content of dendrobium alkaloids is about twice that of commercially available tissue culture seedlings (control two), showing its higher medicinal value.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for breeding high-quality tropical rainforest Dendrobium nobile seedlings, characterized in that: The steps are as follows: Step S1, collecting wild Dendrobium nobile seeds from the Wuzhishan rainforest area of ​​Hainan, selecting seeds that are moderately mature, plump and free of pests and diseases, and then performing surface disinfection treatment as explants; Step S2, inoculating the seeds of Dendrobium nobile into an induction culture medium, germinating the seeds to form protocorms, and the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment; the induction culture medium is a modified MS culture medium + peptone 200-250 mg / L + FeSO4·7H2O 10-20 mg / L + K2SO4 150-250 mg / L + Na2-EDTA 10-20 mg / L + aminoacetic acid 3-5 mg / L + calcium pantothenate 3-6 mg / L + 6-BA 4-6 mg / L + 2,4-D 2-4 mg / L + NAA 0.1-0.3 mg / L + sucrose 300-350 mg / L +5.0 g / L agar, pH 5.8; Step S3, inoculating the protocorm into a proliferation culture medium for proliferation culture, the protocorm continuously proliferates and forms a protocorm-like cluster, and the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and the environment is maintained with high negative oxygen ion air; the proliferation culture medium is a modified MS culture medium + peptone 200-250 mg / L + FeSO4·7H2O 10-20 mg / L + K2SO4 150-250 mg / L + aminoacetic acid 3-5 mg / L + calcium pantothenate 3-6 mg / L + 6-BA 1-2 mg / L + 2,4-D 1-2 mg / L + NAA 0.1-0.3 mg / L + sucrose 300-350 mg / L +5.0 g / L agar, pH 5.8; Step S4, selecting well-growing, emerald green, and densely structured protocorms from the proliferating protocorm-like body clusters and transferring them into a differentiation medium for differentiation culture to obtain clustered buds; the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment; the differentiation medium is a modified MS medium + peptone 200-250 mg / L + FeSO4·7H2O 10-20 mg / L + K2SO4 150-250 mg / L + Na2-EDTA 10-20 mg / L + aminoacetic acid 3-5 mg / L + calcium pantothenate 3-6 mg / L + glutamine 100-150 mg / L + 2ip 0.2-0.5 mg / L + 6-BA 3~5mg / L+NAA 0.2~0.5mg / L+sucrose 300~350mg / L +5.0g / L agar, pH 5.8; Step S5, cutting the differentiated clustered buds into single buds, inoculating them on a rooting and seedling-strengthening medium for culture, and obtaining complete regenerated plants, the culture conditions are as follows: the light intensity is 2800-3300 lx, the light cycle is 12 hours of light / 12 hours of darkness per day, the temperature is 25-27°C during light, the temperature is 18-20°C during darkness, the humidity is 75-85%, and high negative oxygen ion air is maintained in the environment; the rooting and seedling-strengthening medium is a modified MS medium + peptone 200-250 mg / L + aminoacetic acid 3-5 mg / L + biotin 3-6 mg / L + 6-BA 3-5 mg / L + NAA 0.2-0.5 mg / L + IBA 0.5-0.8 mg / L + sucrose 300-350 mg / L + 5.0 g / L agar, and the pH is 5.

8.

2. The method for breeding high-quality seedlings of a tropical rainforest Dendrobium nobile according to claim 1, characterized in that: In step S1, the disinfection treatment is to soak the seeds in 75% alcohol for 8 to 12 minutes, then rinse with sterile water 2 to 4 times, each time for 5 minutes to remove impurities and microorganisms on the surface of the seeds, and dry them under sterile conditions.

3. The method for breeding high-quality seedlings of a tropical rainforest Dendrobium nobile according to claim 1, characterized in that: In the steps S2, S3, S4 and S5, the high negative oxygen ion air refers to air in which the concentration of negative oxygen ions is maintained at more than 3000 per cubic centimeter.

4. The method for breeding high-quality tropical rainforest Dendrobium nobile seedlings according to claim 1, characterized in that: In step S5, the mixed concentration of NAA and IBA in the rooting and seedling strengthening medium is 1 mg / L.

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