A breeding method for excellent seedlings of Dendrobium nobile Lindl. in tropical rainforests

Abstract

The present invention belongs to the field of agricultural biotechnology and relates to a method for breeding excellent seedlings of Dendrobium nobile Lindl. in the tropical rainforest. Wild Dendrobium nobile Lindl. seeds in the rainforest area of Wuzhishan, Hainan are collected as explants, and after protocorm induction and proliferation, small seedlings are differentiated and then subjected to rooting culture to obtain complete regenerated plants. By using wild Dendrobium nobile Lindl. seeds in the rainforest area of Wuzhishan, Hainan as explants for tissue culture, the differentiation rate can be increased, the propagation speed can be improved, and the environmental factors in the rainforest area of Wuzhishan, Hainan are simulated as culture conditions, enabling the seedlings to be cultivated in a semi-wild environment. The obtained Dendrobium nobile Lindl. seedlings have stable genetic traits and small variations, shortening the field transplanting and domestication time of the seedlings, improving the survival rate of transplantation, accelerating the growth speed and growth amount after transplantation. Moreover, the content of the active ingredients in the products is higher than that of the similar products in other production areas, laying a material and technical foundation for promoting the sustainable development of the Dendrobium nobile Lindl. industry in Hainan region.

Description

Technical Field

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

[0002] Dendrobium nobile, as a precious variety among Orchidaceae plants and also a rare medicinal plant, has the effects of nourishing yin and clearing heat, promoting the production of body fluid and quenching thirst, nourishing the stomach and strengthening the kidney, etc., and is widely used in the field of traditional Chinese medicine. In addition, Dendrobium nobile also contains rich nutrients such as polysaccharides, amino acids, vitamins and trace elements, and has extremely high health care value. In recent years, due to its unique medicinal value and ornamental value, it has attracted much attention, and the market demand for Dendrobium nobile has been increasing continuously. However, the seeds of Dendrobium nobile are extremely small, the embryo development is incomplete, there is no endosperm tissue, and the germination rate in the natural state is extremely low (less than 5%). Coupled with the long-term predatory excavation, the wild resources have been nearly exhausted and are in an endangered state, far from meeting the domestic and foreign market demands, resulting in a shortage of market supply. Therefore, it is of great significance to carry out the research on the artificial cultivation of Dendrobium nobile.

[0003] Plant tissue culture has the characteristics of less demand for raw materials, being not restricted by time and location, high propagation coefficient, and being able to maintain the excellent traits of the mother plant, and is widely used in the breeding of plant seedlings. With the continuous increase in the area of artificial cultivation, the demand for high-quality seedlings of Dendrobium nobile is also increasing. Using the tissue rapid propagation technology to produce a large number of high-quality seedlings of Dendrobium nobile has, to a certain extent, solved the problem of the shortage of high-quality seedlings of Dendrobium nobile.

[0004] Currently, the main research directions of the tissue culture technology of Dendrobium nobile are the screening of explants, the selection and preparation of culture media, the selection of culture methods, the design of plant regeneration pathways, etc. Although it can effectively improve the efficiency of cell dedifferentiation and redifferentiation and provide a large number of excellent seedlings with consistent traits for the 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 poor quality when introducing excellent varieties, and even phenomena such as the shrinkage of plant leaves and the death of the whole plant.

[0005] Dendrobium nobile Lindl. is mainly distributed in the subtropical regions of southern China, growing in mountain forests at higher altitudes, including the Wuzhishan area in Hainan and the Chishui area in Guizhou. Due to the different ecological environments in different regions, there are significant interspecific differences among Dendrobium nobile Lindl. in different regions, and the compositions of the culture media for in vitro culture vary greatly. At present, the cultivation industry of Dendrobium nobile Lindl. in Hainan is developing rapidly, mainly concentrated in the Wuzhishan rainforest area (at a higher altitude), but the breeding of corresponding high-quality seedlings cannot keep up with the development needs. However, the excellent seedlings introduced from other provinces are prone to "acclimatization" due to the unique geographical environment and weather in Hainan Province when planted in Hainan, resulting in unsatisfactory production. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for breeding excellent seedlings of Dendrobium nobile Lindl. in tropical rainforests. Using the seeds of wild Dendrobium nobile Lindl. in the Wuzhishan rainforest area (high-altitude area in Hainan) as explants for tissue culture, the differentiation rate can be increased by screening the culture medium, and the propagation speed and efficiency of excellent seedlings of Dendrobium nobile Lindl. can be improved. By simulating the environmental factors in the Wuzhishan rainforest area as culture conditions, the seedlings can be cultivated in a semi-wild environment, greatly shortening the time for transplanting and domestication of the seedlings, facilitating the improvement of the survival rate of field transplantation, accelerating the growth speed after transplantation, and shortening the product harvesting time.

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

[0008] A method for breeding excellent seedlings of Dendrobium nobile Lindl. in tropical rainforests comprises the following steps:

[0009] Step S1: Collect the seeds of wild Dendrobium nobile Lindl. in the Wuzhishan rainforest area, and select the seeds with moderate maturity, plumpness and no diseases and pests for surface disinfection treatment and then use them as explants.

[0010] Step S2: Inoculate the seeds of Dendrobium nobile Lindl. into the induction medium, and the seeds germinate to form protocorms. The culture conditions are as follows: the light intensity is 2800 - 3300 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 - 27 °C, the temperature during darkness is 18 - 20 °C, the humidity is 75 - 85%, maintain high negative oxygen ion air in the environment, subculture once every 20 - 30 days, and subculture 1 - 2 times; the induction medium is 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 + glycine 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, and the pH is 5.8.

[0011] Step S3: Inoculate the protocorms into a proliferation medium for proliferation culture. The protocorms continuously proliferate and form protocorm-like body clusters. The culture conditions are as follows: the light intensity is 2800 - 3300 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 - 27 °C, the temperature during darkness is 18 - 20 °C, the humidity is 75 - 85%, maintain high negative oxygen ion air in the environment, subculture once every 20 - 30 days, and subculture 1 - 3 times; the proliferation medium is modified MS medium + 200 - 250 mg / L of peptone + 10 - 20 mg / L of FeSO4·7H2O + 150 - 250 mg / L of K2SO4 + 3 - 5 mg / L of glycine + 3 - 6 mg / L of calcium pantothenate + 1 - 2 mg / L of 6-BA + 1 - 2 mg / L of 2,4-D + 0.1 - 0.3 mg / L of NAA + 300 - 350 mg / L of sucrose + 5.0 g / L of agar, and the pH is 5.8.

[0012] Step S4: Select well-grown, bright green, and relatively dense protocorm-like bodies from the proliferated protocorm-like body clusters and transfer them to a differentiation medium for differentiation culture to obtain clustered shoots; the culture conditions are as follows: the light intensity is 2800 - 3300 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 - 27 °C, the temperature during darkness is 18 - 20 °C, the humidity is 75 - 85%, maintain high negative oxygen ion air in the environment, subculture once every 20 - 30 days, and subculture 1 - 3 times; the differentiation medium is modified MS medium + 200 - 250 mg / L of peptone + 10 - 20 mg / L of FeSO4·7H2O + 150 - 250 mg / L of K2SO4 + 10 - 20 mg / L of Na2-EDTA + 3 - 5 mg / L of glycine + 3 - 6 mg / L of calcium pantothenate + 100 - 150 mg / L of glutamine + 0.2 - 0.5 mg / L of 2ip (N6-isopentenyladenine) + 3 - 5 mg / L of 6-BA + 0.2 - 0.5 mg / L of NAA + 300 - 350 mg / L of sucrose + 5.0 g / L of agar, and the pH is 5.8.

[0013] Step S5: Cut the differentiated cluster buds into single buds, inoculate them on the rooting and strong-seedling culture medium, and culture to obtain complete regenerated plants. The culture conditions are as follows: the light intensity is 2800 - 3300 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 - 27 °C, the temperature during darkness is 18 - 20 °C, the humidity is 75 - 85%, maintain high negative oxygen ion air in the environment, subculture once every 20 - 30 days, and subculture 1 - 3 times; the rooting and strong-seedling culture medium is improved MS medium + 200 - 250 mg / L of peptone + 3 - 5 mg / L of glycine + 3 - 6 mg / L of biotin + 3 - 5 mg / L of 6-BA + 0.2 - 0.5 mg / L of NAA + 0.5 - 0.8 mg / L of IBA + 300 - 350 mg / L of sucrose + 5.0 g / L of agar, and the pH is 5.8.

[0014] Further, in step S1, the disinfection treatment is to soak the seeds in 75% alcohol for 8 - 12 minutes, then rinse them with sterile water 2 - 4 times, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dry them under sterile conditions.

[0015] Further, in steps S2, S3, S4, and S5, the high negative oxygen ion air means that the concentration of negative oxygen ions in the air remains above 3000 per cubic centimeter.

[0016] Further, in step S5, in the rooting and strong-seedling culture medium, the combined concentration of NAA and IBA is 1 mg / L.

[0017] The present invention uses the seeds of wild Dendrobium officinale in the Wuzhishan rainforest area (high-altitude area in Hainan) as explants for tissue culture, which can reflect the differences in regional ecological environment and plant quality. At the same time, by screening the culture medium, the differentiation rate can be improved. By simulating the environmental factors in the high-altitude area of Hainan (high negative oxygen ions, large day-night temperature difference, high humidity) as culture conditions, it can not only improve the propagation speed and efficiency of excellent seedlings of Dendrobium officinale, lay a foundation for large-scale production of Dendrobium officinale seedlings with consistent traits and large-scale cultivation, but also due to the setting of culture conditions, the seedlings can be cultivated in a quasi-wild environment. The obtained Dendrobium officinale seedlings have stable genetic traits, small variations, and characteristics such as simple operation, low cost, and high propagation coefficient, greatly shortening the seedling transplanting and acclimatization time, being beneficial to improving the survival rate of transplanting, accelerating the growth speed and growth amount after transplanting, shortening the growth cycle, and moreover, the content of effective components in the product 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 officinale industry in Hainan. Specific embodiments

[0018] The following will further describe in detail the specific implementation manners of the present invention in conjunction with embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0019] For the quantitative tests in the following embodiments, three repeated tests are set, and the results are averaged. The improved MS medium involved is based on the MS medium, with the macroelements adjusted to 3 / 4 of the original content and the microelements adjusted to 2 times the original content, and other components remain unchanged.

[0020] I. Tissue culture and seedling raising of Dendrobium nobile

[0021] Example 1

[0022] (1) Collect the wild Dendrobium nobile capsules with moderate maturity, plump fruits, no pests and diseases, and no cracks in the tropical rainforest area of Wuzhishan, Hainan. After cleaning and disinfecting, peel the Dendrobium nobile capsules and take out the seeds. Soak the seeds in 75% alcohol for 10 minutes, then rinse them 3 times with sterile water, 5 minutes each time, to remove the impurities and microorganisms on the seed surface, and dry them under sterile conditions as explants.

[0023] (2) Inoculate the Dendrobium nobile seeds into the induction medium (improved MS medium + peptone 200 mg / L + FeSO4·7H2O 20mg / L + K2SO4 200mg / L + Na2-EDTA 20mg / L + glycine 4mg / L + calcium pantothenate 5mg / L + 6-BA 4mg / L + 2,4-D 4mg / L + NAA 0.2mg / L + sucrose 300mg / L + 5.0g / L agar, pH 5.8) for culture. The culture conditions are as follows: the light intensity is 3000 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25°C, the temperature during darkness is 20°C, the humidity is 80%, and the concentration of negative oxygen ions in the environment air remains above 3000 per cubic centimeter. Subculture once every 25 days, and after 1 subculture, the seeds germinate to form protocorms.

[0024] (3) Select well-grown, emerald green, and relatively dense protocorms and inoculate them into the proliferation medium (modified MS medium + 200 mg / L peptone + 20 mg / L FeSO4·7H2O + 200 mg / L K2SO4 + 4 mg / L glycine + 5 mg / L calcium pantothenate + 2 mg / L 6-BA + 1 mg / L 2,4-D + 0.2 mg / L NAA + 300 mg / L sucrose + 5.0 g / L agar, pH 5.8) for proliferation culture. The culture conditions are as follows: light intensity is 3000 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 °C, the temperature during darkness is 20 °C, the humidity is 80%, and the concentration of negative oxygen ions in the environment air is maintained above 3000 per cubic centimeter. Subculture once every 25 days, and after subculturing 2 times, protocorm proliferation obtains protocorm-like clusters.

[0025] (4) Select well-grown, emerald green, and relatively dense protocorm-like bodies from the proliferated protocorm-like clusters and transfer them to the differentiation medium (modified MS medium + 220 mg / L peptone + 15 mg / L FeSO4·7H2O + 180 mg / L K2SO4 + 17 mg / L Na2-EDTA + 3 mg / L glycine + 6 mg / L calcium pantothenate + 100 mg / L glutamine + 0.5 mg / L 2ip + 4 mg / L 6-BA + 0.4 mg / L NAA + 330 mg / L sucrose + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain clustered shoots; the culture conditions are as follows: light intensity is 2800 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 27 °C, the temperature during darkness is 18 °C, the humidity is 75%, and the concentration of negative oxygen ions in the environment air is maintained above 3000 per cubic centimeter. Subculture once every 30 days, the protocorm germinates after subculturing 1 time, and continue to subculture 1 time to obtain small seedlings.

[0026] (5) Inoculate the small seedlings on the rooting and strong-seedling medium (modified MS medium + 250 mg / L peptone + 3 mg / L glycine + 6 mg / L biotin + 3 mg / L 6-BA + 0.3 mg / L NAA + 0.7 mg / L IBA + 320 mg / L sucrose + 5.0 g / L agar, pH 5.8) for culture. The culture conditions are as follows: light intensity is 3200 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 °C, the temperature during darkness is 20 °C, the humidity is 85%, and the concentration of negative oxygen ions in the environment air is maintained above 3000 per cubic centimeter. Subculture once every 25 days, and after subculturing 2 times, complete roots can be generated to obtain complete regenerated plants.

[0027] Example 2

[0028] (1)Collect wild Dendrobium nobile Lindl. capsules with moderate maturity, plump fruits, no pests, diseases or cracks in the Hainan Wuzhishan tropical rainforest area. After cleaning and disinfection, peel the Dendrobium nobile Lindl. capsules and take out the seeds. Soak the seeds in 75% alcohol for 10 minutes, then rinse them with sterile water 3 times, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dry them under sterile conditions to be used as explants.

[0029] (2)Inoculate the Dendrobium nobile Lindl. seeds into the induction medium (modified MS medium + peptone 240 mg / L + FeSO4·7H2O 13mg / L + K2SO4 160mg / L + Na2-EDTA 17mg / L + glycine 3mg / L + calcium pantothenate 4mg / L + 6-BA 6mg / L + 2,4-D 2mg / L + NAA 0.1mg / L + sucrose 350mg / L + 5.0g / L agar, pH 5.8) for culture. The culture conditions are as follows: light intensity is 2900 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 27℃, the temperature during darkness is 20℃, the humidity is 80%, and the concentration of negative oxygen ions in the air environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days. After 1 subculture, the seeds germinate to form protocorms.

[0030] (3)Select well-grown, bright green and relatively dense protocorms and inoculate them into the proliferation medium (modified MS medium + peptone 240 mg / L + FeSO4·7H2O 13mg / L + K2SO4 160mg / L + glycine 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 is 2900 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 27℃, the temperature during darkness is 20℃, the humidity is 80%, and the concentration of negative oxygen ions in the air environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days. After 2 subcultures, the protocorms proliferate to obtain protocorm-like clusters.

[0031] (4) Select well-grown, emerald green, and relatively dense protocorm-like bodies from the proliferated protocorm-like body clusters and transfer them to a differentiation medium (modified MS medium + 200 mg / L peptone + 12 mg / L FeSO4·7H2O + 220 mg / L K2SO4 + 11 mg / L Na2-EDTA + 4 mg / L glycine + 4 mg / L calcium pantothenate + 150 mg / L glutamine + 0.3 mg / L 2ip + 3 mg / L 6-BA + 0.2 mg / L NAA + 300 mg / L sucrose + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain clustered shoots. The culture conditions are as follows: the light intensity is 3200 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25°C, the temperature during darkness is 20°C, the humidity is 75%, and the concentration of negative oxygen ions in the air environment is maintained above 3000 per cubic centimeter. Subculture once every 30 days. The protocorms germinate after the first subculture, and small seedlings can be obtained after continuing to subculture once more.

[0032] (5) Inoculate the small seedlings on a rooting and strong-seedling medium (modified MS medium + 200 mg / L peptone + 5 mg / L glycine + 4 mg / L biotin + 4 mg / L 6-BA + 0.5 mg / L NAA + 0.5 mg / L IBA + 350 mg / L sucrose + 5.0 g / L agar, pH 5.8) for culture. The culture conditions are as follows: the light intensity is 3000 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25°C, the temperature during darkness is 18°C, the humidity is 85%, and the concentration of negative oxygen ions in the air environment is maintained above 3000 per cubic centimeter. Subculture once every 25 days. After subculturing twice, complete roots can be formed to obtain complete regenerated plants.

[0033] Example 3

[0034] (1) Collect wild Dendrobium nobile Lindl. capsules with moderate maturity, plump fruits, no pests and diseases, and no cracks in the Hainan Wuzhishan tropical rainforest area. After cleaning and disinfecting, peel the Dendrobium nobile Lindl. capsules and take out the seeds. Immerse the seeds in 75% alcohol for 10 minutes, then rinse them 3 times with sterile water, 5 minutes each time, to remove impurities and microorganisms on the seed surface, and dry them under sterile conditions to serve as explants.

[0035] (2) Inoculate the seeds of Dendrobium nobile Lindl. into an induction medium (modified MS medium + 210 mg / L peptone + 16 mg / L FeSO4·7H2O + 220 mg / L K2SO4 + 16 mg / L Na2-EDTA + 5 mg / L glycine + 3 mg / L calcium pantothenate + 5 mg / L 6-BA + 3 mg / L 2,4-D + 0.3 mg / L NAA + 320 mg / L sucrose + 5.0 g / L agar, pH 5.8) for cultivation. The cultivation conditions are as follows: light intensity is 3200 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 °C, the temperature during darkness is 18 °C, the humidity is 75%, and the concentration of negative oxygen ions in the air environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days, and after 2 subcultures, the seeds germinate to form protocorms.

[0036] (3) Select protocorms with good growth, bright green color, and relatively dense structure and inoculate them into a proliferation medium (modified MS medium + 210 mg / L peptone + 16 mg / L FeSO4·7H2O + 220 mg / L K2SO4 + 5 mg / L glycine + 3 mg / L calcium pantothenate + 2 mg / L 6-BA + 1 mg / L 2,4-D + 0.3 mg / L NAA + 320 mg / L sucrose + 5.0 g / L agar, pH 5.8.) for proliferation culture. The cultivation conditions are as follows: light intensity is 3200 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 25 °C, the temperature during darkness is 18 °C, the humidity is 75%, and the concentration of negative oxygen ions in the air environment is maintained at more than 3000 per cubic centimeter. Subculture once every 25 days, and after 2 subcultures, the protocorms proliferate to obtain protocorm-like clusters.

[0037] (4) Select protocorm-like bodies with good growth, bright green color, and relatively dense structure from the proliferated protocorm-like clusters and transfer them into a differentiation medium (modified MS medium + 250 mg / L peptone + 20 mg / L FeSO4·7H2O + 150 mg / L K2SO4 + 15 mg / L Na2-EDTA + 5 mg / L glycine + 3 mg / L calcium pantothenate + 120 mg / L glutamine + 0.3 mg / L 2ip + 5 mg / L 6-BA + 0.2 mg / L NAA + 350 mg / L sucrose + 5.0 g / L agar, pH 5.8) for differentiation culture to obtain cluster buds; the cultivation conditions are as follows: light intensity is 2800 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 27 °C, the temperature during darkness is 20 °C, the humidity is 75%, and the concentration of negative oxygen ions in the air environment is maintained at more than 3000 per cubic centimeter. Subculture once every 30 days, and after 1 subculture, the protocorms germinate, and continue to subculture once to obtain seedlings.

[0038] (5) Inoculate the young seedlings on the rooting and strengthening seedling medium (modified MS medium + 230 mg / L peptone + 4 mg / L glycine + 3 mg / L biotin + 5 mg / L 6-BA + 0.4 mg / L NAA + 0.6 mg / L IBA + 300 mg / L sucrose + 5.0 g / L agar, pH 5.8) for cultivation. The cultivation conditions are as follows: the light intensity is 2800 lx, the daily light cycle is 12 hours of light / 12 hours of darkness, the temperature during light is 27 °C, the temperature during darkness is 20 °C, the humidity is 85%, and the concentration of negative oxygen ions in the air in the environment is maintained above 3000 per cubic centimeter. Subculture once every 25 days, and after 2 subcultures, complete roots can be formed to obtain complete regenerated plants.

[0039] II. Effect verification

[0040] (1) Identification of the tissue culture effect of Dendrobium nobile

[0041] 1. Identification of the germination and proliferation effects of Dendrobium nobile seeds

[0042] To investigate the germination and proliferation of Dendrobium nobile seeds, observe and count the number of protocorms formed from the germination of Dendrobium nobile seeds and the proliferation of protocorms in the above-mentioned examples, calculate the germination rate and proliferation coefficient, and take the average value of each data. The results are shown in Table 1.

[0043] Table 1 Germination and proliferation of Dendrobium nobile seeds

[0044]

[0045] * The number of seeds is calculated according to the density per unit area (1 cm 2 ) × total area after sowing. For the convenience of comparison, the initial number of seeds is set at 500 grains.

[0046] The above results of the germination of Dendrobium nobile seeds show that the present invention can effectively promote the germination of Dendrobium nobile seeds with modified MS as the basic medium, improve the seed germination rate, and has good later-stage protocorm proliferation effect and a high proliferation coefficient.

[0047] 2. Identification of the differentiation effect of protocorms

[0048] To identify the differentiation effect of protocorms, observe and count the differentiation of protocorms and the number of young seedlings in the above-mentioned examples, calculate the differentiation rate, and take the average value of each data. The results are shown in Table 2.

[0049] Table 2 Differentiation of protocorms

[0050]

[0051] The above results of protocorm differentiation indicate that the present invention uses an improved MS medium to induce and differentiate seedlings from protocorms, with a relatively high differentiation rate, up to 96% at most, and good differentiation efficiency.

[0052] 3. Identification of the rooting effect of seedlings

[0053] To identify the rooting effect of the seedlings, the rooting conditions of the seedlings obtained in the above examples were observed and counted. The number of roots was counted, and the rooting rate was calculated. The average value was taken for each data, and the results are shown in Table 3.

[0054] Table 3 Rooting conditions of seedlings

[0055]

[0056] The above results of seedling rooting indicate that the present invention induces rooting of the obtained seedlings, with fast rooting, well-developed roots, and a rooting rate of 100%, which is beneficial to improving the transplanting survival rate of rooted seedlings and the growth of plants.

[0057] (2) Identification of the field transplanting effect

[0058] The Dendrobium nobile Lindl. seedlings obtained in Example 1, Example 2, and Example 3 were planted in the wild in Wuzhishan area, Hainan. Control 1: Using wild Dendrobium nobile Lindl. seeds in the Wuzhishan rainforest area as explants, the culture media at each stage were the same as those used in Example 1, but conventional culture conditions were adopted, specifically: the light intensity was 2800 - 3300 lx, the daily light cycle was 12 hours of light / 12 hours of darkness, and the temperature was 25 ± 2°C. Control 2: Commercially available tissue-cultured seedlings of Dendrobium nobile Lindl.

[0059] The growth performance was evaluated 60 days after the seedlings were transplanted, including indicators such as survival rate, growth rate, plant height, stem diameter, leaf area, and root condition, and the contents of medicinal components such as dendrobine, polysaccharide, amino acid, vitamin C, and vitamin E in the materials were quantitatively analyzed, where vitamin C and vitamin E were calculated based on fresh weight. The average value was taken for each data, and the results are shown in Tables 4 and 5.

[0060] Table 4 Growth conditions after seedling transplantation

[0061]

[0062] The above results of field transplantation show that while improving the cultivation effect by screening the culture medium, the present application simulates the Wuzhishan tropical rainforest environment (including light, temperature, humidity, air, etc.) as the cultivation condition, which can effectively improve the quality of seedlings. With the extension of the planting time, the plant height, stem diameter and leaf area of the seedlings gradually increase, and the root system is developed, and the growth performance is significantly better than that of the seedlings under the ordinary cultivation environment (Control 1). Although Control 1 uses the seeds of wild Dendrobium officinale in the Wuzhishan tropical rainforest area as explants to breed seedlings, the transplantation situation is slightly better than that of the commercially available tissue-cultured seedlings (Control 2), but the advantage is not obvious.

[0063] Content of each component in the material in Table 5

[0064]

[0065] The above results of the analysis of medicinal components show that after the seedlings provided by the present application are planted, the contents of medicinal components such as dendrobine, polysaccharide, amino acid, vitamin C and vitamin E in them are significantly higher than those of the seedlings under the ordinary cultivation environment (Control 1) and the commercially available tissue-cultured seedlings (Control 2). Among them, the content of dendrobine is about twice that of the commercially available tissue-cultured seedlings (Control 2), showing its higher medicinal value.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 Dendrobium nobile seeds into an induction culture medium, germinating the seeds to form protocorms, and the culture conditions are as follows: the light intensity is 2800-3300lx, 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 concentration of negative oxygen ions in the air is maintained at more than 3000 per cubic centimeter in the environment; the induction culture medium is a modified MS culture medium + peptone 200-250mg / L + FeSO4·7H2O 10-20mg / L + K2SO4 150-250mg / L + Na2-EDTA 10-20mg / L + aminoacetic acid 3-5mg / L + calcium pantothenate 3-6mg / L + 6-BA 4-6mg / L + 2,4-D 2-4mg / L + NAA 0.1~0.3mg / L+sucrose 300~350mg / L+5.0g / 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 2800-3300lx, 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 concentration of negative oxygen ions in the air is maintained at more than 3000 per cubic centimeter in the environment; the proliferation culture medium is a modified MS culture medium + peptone 200-250mg / L + FeSO4·7H2O 10-20mg / L + K2SO4 150-250mg / L + aminoacetic acid 3-5mg / L + calcium pantothenate 3-6mg / L + 6-BA 1-2mg / L + 2,4-D 1-2mg / L + NAA 0.1~0.3mg / L+sucrose 300~350mg / L+5.0g / 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 2800-3300lx, 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 concentration of negative oxygen ions in the air is maintained at more than 3000 per cubic centimeter in the environment; the differentiation medium is a modified MS medium + peptone 200-250mg / L + FeSO4·7H2O 10-20mg / L + K2SO4 150-250mg / L + Na2-EDTA 10-20mg / L + aminoacetic acid 3-5mg / L + calcium pantothenate 3-6mg / L + glutamine 100-150mg / L + 2ip 0.2~0.5mg / 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: light intensity is 2800-3300lx, 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 concentration of negative oxygen ions in the air is maintained at more than 3000 per cubic centimeter in the environment; the rooting and seedling-strengthening medium is a modified MS medium + peptone 200-250mg / L + aminoacetic acid 3-5mg / L + biotin 3-6mg / L + 6-BA 3-5mg / L + NAA 0.2-0.5mg / L + IBA 0.5-0.8mg / L + sucrose 300-350mg / L + 5.0g / L agar, pH is 5.8; The improved MS medium is 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 of the original content, and other components remaining unchanged.

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

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