Tissue culture and rapid propagation seedling raising method for dendrobium officinale

Abstract

The invention discloses a dendrobium officinale tissue culture and rapid propagation seedling raising method, and belongs to the technical field of plant tissue culture. The method comprises the steps of explant selection and disinfection, primary culture, subculture multiplication culture, rooting and seedling strengthening culture, seedling hardening and transplanting. Wherein the explant disinfection adopts a two-step disinfection method of soaking the explant in a sodium hypochlorite solution with the effective chlorine concentration of 1%-5% for 8-12 minutes and soaking the explant in a mercury bichloride solution with the concentration of 0.1%-0.2% for 5-7 minutes; the subculture multiplication culture medium is based on an MS culture medium and is added with humic acid, and the rooting and seedling strengthening culture medium is based on a 1 / 2 MS culture medium and is added with activated carbon; seedling hardening adopts a gradient seedling hardening mode, and a culture medium is formed by mixing fermented pine barks, fermented peanut shells and broken stones according to the volume ratio of 2: 2: 1. The method is thorough in disinfection and efficient in proliferation, the sterile bud induction rate, the proliferation coefficient and the transplanting survival rate can be remarkably increased, the culture period is shortened, the production cost is reduced, and high-quality dendrobium officinale seedlings can be rapidly cultivated.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture seedling technology, and in particular to a method for rapid propagation of Dendrobium officinale through tissue culture. Background Technology

[0002] Dendrobium officinale ( Dendrobium offcinale ), also known as black knot grass, belongs to the Orchidaceae family ( Orchidaceae Dendrobium genus ( Dendrobium Dendrobium officinale is a valuable traditional Chinese medicine. Its stems can be used medicinally, possessing properties such as nourishing yin and clearing heat, promoting body fluid production and benefiting the stomach, and moistening the lungs and relieving cough. Modern pharmacological research shows that Dendrobium officinale also enhances human immunity, has anti-tumor effects, anti-aging properties, and improves diabetes symptoms. Because Dendrobium officinale has demanding requirements for its growing environment and a very low natural propagation rate, conventional propagation methods such as division and cuttings are inefficient and time-consuming, failing to meet the needs of large-scale production. Therefore, tissue culture for rapid propagation has become the main method for cultivating Dendrobium officinale seedlings.

[0003] Current tissue culture techniques for Dendrobium officinale have several shortcomings. First, the explant sterilization effect is poor, leading to contamination and a decreased success rate. Second, the culture medium formula is often unreasonable, resulting in low proliferation coefficients in the original corms, poor differentiation, and slow, uneven growth of the tissue-cultured seedlings. Third, the hardening-off and transplanting techniques are inadequate, leading to weak adaptability of the seedlings and low transplant survival rates. Fourth, the overall culture cycle is long, resulting in high production costs, making it difficult to meet the demands of large-scale industrial seedling production. Therefore, developing a rapid propagation method for Dendrobium officinale tissue culture that is thoroughly sterilized, highly efficient in proliferation, easy to harden, and suitable for practical production has become an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapid propagation of Dendrobium officinale through tissue culture, which solves the problems of high pollution rate, low proliferation coefficient, low transplant survival rate and long cultivation cycle in the existing technology, realizes rapid, efficient and high-quality cultivation of Dendrobium officinale seedlings, reduces production costs and meets the needs of large-scale production.

[0005] To achieve the above objectives, this invention provides a method for rapid propagation of Dendrobium officinale through tissue culture, comprising the steps of explant disinfection, primary culture, subculture proliferation culture, rooting and seedling strengthening culture, hardening and transplanting; the explants are disinfected using a two-step method of sodium hypochlorite and mercuric chloride, humic acid is added to the subculture proliferation culture medium, activated carbon is added to the rooting and seedling strengthening culture medium, hardening is carried out using a gradient hardening method, and the cultivation substrate is a mixed substrate of fermented pine bark, fermented peanut shells and gravel.

[0006] Preferably, the explant disinfection is specifically performed as follows: Select healthy, disease-free, one-year-old Dendrobium officinale plants, cut stem segments with axillary buds as explants, remove excess leaves, rinse thoroughly with clean water, place in a sterile environment, first soak in a sodium hypochlorite solution with an effective chlorine concentration of 1%-5% for 8-12 minutes, remove and rinse 3 times with sterile water, then soak in a mercuric chloride solution with a concentration of 0.1%-0.2% for 5-7 minutes, remove and rinse 5 times with sterile water, and finally use sterile filter paper to absorb the surface moisture to obtain the disinfected explants.

[0007] Preferably, the primary culture specifically involves: cutting the sterilized explants into small segments, retaining one axillary bud in each segment, inoculating them onto the primary culture medium, and placing them in a culture room for primary culture. The culture temperature is controlled at 25-27℃, the light intensity is controlled at 2000-2500 lux, the light duration is 16 hours per day, and the culture is carried out for 30 days to induce axillary bud germination and the formation of aseptic buds. The primary culture medium is based on MS medium, with the addition of naphthaleneacetic acid, gibberellin, sucrose, and agar. The concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of gibberellin is 0.1 mg / L, the concentration of sucrose is 30 g / L, the concentration of agar is 6 g / L, and the pH of the culture medium is adjusted to 5.8-6.0.

[0008] Preferably, the subculture proliferation culture specifically involves: cutting the aseptic shoots obtained from the primary culture from the explants, inoculating them onto the subculture proliferation medium, placing them in a culture room for subculture proliferation culture, controlling the culture temperature at 25-27℃, the light intensity at 2500-3000 lux, the light duration at 16 hours per day, subculturing once every 30 days, and repeating the subculture twice to obtain a large number of robust clustered shoots; The subculture medium was based on MS medium, with the addition of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose and agar. The concentrations of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose, and agar were 1.5 mg / L, 0.3 mg / L, 5 g / L, 30 g / L, and 6 g / L. The pH of the medium was adjusted to 5.8-6.0.

[0009] Preferably, the rooting and seedling cultivation specifically involves: selecting robust clustered shoots with a height of 2-3 cm obtained from subculture proliferation, dividing them into individual plants, inoculating them onto a rooting and seedling cultivation medium, and placing them in a cultivation room for rooting and seedling cultivation. The cultivation temperature is controlled at 24-26℃, the light intensity is controlled at 3000-3500 lux, the light duration is 16 hours per day, and the cultivation lasts for 40 days to obtain tissue culture seedlings with well-developed root systems and robust growth. The rooting and seedling strengthening medium is based on 1 / 2 MS medium, with the addition of naphthaleneacetic acid, indolebutyric acid, activated carbon, sucrose and agar. The concentration of naphthaleneacetic acid is 0.5 mg / L, the concentration of indolebutyric acid is 0.3 mg / L, the concentration of activated carbon is 1 g / L, the concentration of sucrose is 25 g / L, the concentration of agar is 6 g / L, and the pH of the medium is adjusted to 5.8-6.0.

[0010] Preferably, the hardening-off and transplanting process specifically involves: removing the tissue culture seedlings obtained from the rooting and strengthening culture along with the culture bottle from the culture room and placing them in a greenhouse for hardening-off. The greenhouse temperature is controlled at 26-28℃, the humidity at 70-80%, natural light, and ventilation for 2 hours per day. After 10 days of hardening-off, the caps of the culture bottles are opened, and hardening-off continues for another 3 days. After hardening-off, the tissue culture seedlings are removed from the culture bottles, the root culture medium is cleaned, and they are transplanted into the pretreated cultivation substrate. After transplanting, the temperature and humidity are controlled and shading is provided. After 30 days, surviving seedlings are obtained.

[0011] Preferably, the cultivation substrate is composed of fermented pine bark, fermented peanut shells and gravel mixed in a volume ratio of 2:2:1. The pretreatment method is to expose the cultivation substrate to sunlight for 3 days, then spray it with carbendazim solution, and dry it for later use.

[0012] Therefore, the method for rapid propagation of Dendrobium officinale through tissue culture of the present invention has the following beneficial effects: (1) The present invention optimizes the explant disinfection process by using a two-step disinfection method of sodium hypochlorite and mercuric chloride. Compared with the existing single disinfection method, the disinfection is more thorough, effectively reducing the explant contamination rate, while reducing the damage of disinfection reagents to axillary buds and improving the induction rate of aseptic buds.

[0013] (2) The present invention optimizes the culture medium formula at each stage. Adding humic acid to the subculture proliferation medium can promote the proliferation and growth of clustered buds, increase the proliferation coefficient, and make the clustered buds grow stronger. Adding activated carbon to the rooting and seedling strengthening culture medium can adsorb harmful substances in the culture medium, reduce the browning phenomenon of tissue culture seedlings, and promote root growth, thereby improving the rooting rate and root quality.

[0014] (3) The present invention optimizes the seedling hardening and transplanting process, adopts a gradient hardening method to enable tissue culture seedlings to gradually adapt to the external environment and improve the stress resistance of tissue culture seedlings; the cultivation substrate is selected by mixing fermented pine bark, fermented peanut shells and gravel, which has good air permeability and water retention, and is suitable for the root growth needs of Dendrobium officinale. Combined with temperature and humidity and shading management after transplanting, the survival rate of transplanting is significantly improved.

[0015] (4) The entire cultivation cycle of this invention is short, and it only takes about 170 days from explant disinfection to transplant survival. Compared with the existing technology, the cultivation cycle is shortened by more than 30 days, and the proliferation coefficient and transplant survival rate are significantly improved. It can quickly cultivate a large number of high-quality Dendrobium officinale seedlings, reduce production costs, and meet the needs of large-scale and industrialized production.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Results for aseptic bud induction rate; Figure 2 The results are for transplant survival rate and rooting rate. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0022] Example 1 A method for rapid propagation of Dendrobium officinale through tissue culture includes the following steps: S1. Selection and disinfection of explants: Select healthy, disease-free, one-year-old Dendrobium officinale plants, cut stem segments with axillary buds as explants, remove excess leaves, rinse with clean water, and place in a sterile environment. First, soak in a 2% sodium hypochlorite solution for 8 minutes, then rinse 3 times with sterile water. Next, soak in a 0.1% mercuric chloride solution for 5 minutes, then rinse 5 times with sterile water. Finally, use sterile filter paper to absorb the surface moisture to obtain disinfected explants.

[0023] S2. Primary culture: The sterilized explants were cut into small segments, each retaining one axillary bud. These segments were inoculated onto primary culture medium and placed in a culture room for primary culture. The culture temperature was controlled at 25℃, the light intensity at 2000 lux, and the light duration at 16 hours per day. After 30 days of culture, axillary buds were induced to germinate and form aseptic buds.

[0024] The primary culture medium was based on MS medium, with the addition of naphthaleneacetic acid (NAA), gibberellin, sucrose, and agar. The concentrations of NAA, gibberellin, sucrose, and agar were 0.2 mg / L, 0.1 mg / L, 30 g / L, and 6 g / L. The pH of the culture medium was adjusted to 5.8, and it was then autoclaved before use.

[0025] S3. Subculture and Proliferation: The aseptic shoots obtained from the primary culture were cut from the explants, inoculated onto the subculture and proliferation medium, and placed in a culture room for subculture and proliferation. The culture temperature was controlled at 25℃, the light intensity at 2500 lux, and the light duration at 16 hours per day. Subculture was performed every 30 days, for a total of 2 subcultures, to obtain a large number of robust clustered shoots.

[0026] The subculture proliferation medium was based on MS medium, with the addition of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose and agar. The concentrations of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose, and agar were 1.5 mg / L, 0.3 mg / L, 5 g / L, 30 g / L, and 6 g / L. The pH of the medium was adjusted to 5.8, and the medium was autoclaved before use.

[0027] S4. Rooting and seedling cultivation: Healthy, clustered shoots with a height of 2-3 cm obtained from subculture were selected, divided into individual plants, and inoculated onto a rooting and seedling strengthening medium. These plants were then placed in a culture room for rooting and seedling strengthening culture. The culture temperature was controlled at 24℃, the light intensity at 3000 lux, and the light duration at 16 hours per day. After 40 days of culture, tissue culture seedlings with well-developed root systems and robust growth were obtained.

[0028] The rooting and seedling strengthening medium was based on 1 / 2 MS medium, with the addition of naphthaleneacetic acid (NAA), indolebutyric acid (IBA), activated carbon, sucrose, and agar. The concentrations of NAA, IBA, activated carbon, sucrose, and agar were 0.5 mg / L, 0.3 mg / L, 1 g / L, 25 g / L, and 6 g / L. The pH of the medium was adjusted to 5.8, and the medium was autoclaved before use.

[0029] S5. Hardening off and Transplanting: The tissue culture seedlings obtained from the rooting and strengthening process are moved out of the culture room along with the culture bottles and placed in a greenhouse for hardening off. The greenhouse temperature is controlled at 26℃, the humidity at 70%, natural light, and ventilation for 2 hours per day. After 10 days of hardening off, the caps of the culture bottles are opened, and hardening off continues for another 3 days to allow the tissue culture seedlings to gradually adapt to the external environment.

[0030] After hardening off, remove the tissue culture seedlings from the culture bottles, gently wash off the culture medium adhering to the roots, and transplant them into the pretreated cultivation substrate. Water and keep moist after transplanting, maintain the greenhouse temperature at 25℃ and humidity at 70%, and provide shade to avoid direct sunlight. Calculate the survival rate 30 days after transplanting.

[0031] The cultivation substrate is made of fermented pine bark, fermented peanut shells and gravel in a volume ratio of 2:2:1. The pretreatment method is to expose the cultivation substrate to sunlight for 3 days, then spray it with carbendazim solution, and then let it dry before use.

[0032] Example 2 A method for rapid propagation of Dendrobium officinale through tissue culture includes the following steps: S1. Explant selection and disinfection: One-year-old Dendrobium officinale plants with robust growth and free from pests and diseases were selected. Stem segments with axillary buds were cut as explants. Excess leaves were removed, and the plants were rinsed with clean water and placed in a sterile environment. First, the plants were soaked in a sodium hypochlorite solution with an effective chlorine concentration of 3% for 10 minutes. After rinsing with sterile water 3 times, the plants were soaked in a mercuric chloride solution with a concentration of 0.15% for 6 minutes. After rinsing with sterile water 5 times, the surface moisture was finally absorbed with sterile filter paper to obtain sterilized explants.

[0033] S2. Primary culture: The sterilized explants were cut into small segments, each retaining one axillary bud. These segments were inoculated onto primary culture medium and placed in a culture room for primary culture. The culture temperature was controlled at 26℃, the light intensity at 2200 lux, and the light duration at 16 hours per day. After 30 days of culture, axillary buds were induced to germinate and form aseptic buds.

[0034] The primary culture medium was based on MS medium, with the addition of naphthaleneacetic acid (NAA), gibberellin, sucrose, and agar. The concentrations of NAA, gibberellin, sucrose, and agar were 0.2 mg / L, 0.1 mg / L, 30 g / L, and 6 g / L. The pH of the culture medium was adjusted to 5.9, and it was then autoclaved before use.

[0035] S3. Subculture and Proliferation: The aseptic shoots obtained from the primary culture were cut from the explants, inoculated onto the subculture and proliferation medium, and placed in a culture room for subculture and proliferation. The culture temperature was controlled at 26℃, the light intensity at 2800 lux, and the light duration at 16 hours per day. Subculture was performed every 30 days, for a total of 2 subcultures, to obtain a large number of robust clustered shoots.

[0036] The subculture proliferation medium was based on MS medium, with the addition of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose and agar. The concentrations of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose, and agar were 1.5 mg / L, 0.3 mg / L, 5 g / L, 30 g / L, and 6 g / L. The pH of the medium was adjusted to 5.9, and the medium was autoclaved before use.

[0037] S4. Rooting and seedling cultivation: Healthy, clustered shoots with a height of 2-3 cm obtained from subculture were selected, divided into individual plants, and inoculated onto a rooting and seedling strengthening medium. These plants were then placed in a culture room for rooting and seedling strengthening culture. The culture temperature was controlled at 25℃, the light intensity at 3200 lux, and the light duration at 16 hours per day. After 40 days of culture, tissue culture seedlings with well-developed root systems and robust growth were obtained.

[0038] The rooting and seedling strengthening medium is based on 1 / 2 MS medium, with the addition of naphthaleneacetic acid, indolebutyric acid, activated carbon, sucrose and agar. The concentration of naphthaleneacetic acid is 0.5 mg / L, the concentration of indolebutyric acid is 0.3 mg / L, the concentration of activated carbon is 1 g / L, the concentration of sucrose is 25 g / L, and the concentration of agar is 6 g / L. The pH of the medium is adjusted to 5.9, and it is then autoclaved before use.

[0039] S5. Hardening off and Transplanting: The tissue culture seedlings obtained from the rooting and strengthening process are moved out of the culture room along with the culture bottles and placed in a greenhouse for hardening off. The greenhouse temperature is controlled at 27℃, the humidity at 75%, natural light, and ventilation for 2 hours per day. After 10 days of hardening off, the caps of the culture bottles are opened, and hardening off continues for another 3 days to allow the tissue culture seedlings to gradually adapt to the external environment.

[0040] After hardening off, remove the tissue culture seedlings from the culture bottles, gently wash off the culture medium adhering to the roots, and transplant them into the pretreated cultivation substrate. Water and keep moist after transplanting, maintain the greenhouse temperature at 26℃ and humidity at 75%, and provide shade to avoid direct sunlight. Calculate the survival rate 30 days after transplanting.

[0041] The cultivation substrate is made of fermented pine bark, fermented peanut shells and gravel in a volume ratio of 2:2:1. The pretreatment method is to expose the cultivation substrate to sunlight for 3 days, then spray it with carbendazim solution, and then let it dry before use.

[0042] Example 3 A method for rapid propagation of Dendrobium officinale through tissue culture includes the following steps: S1. Explant selection and disinfection: One-year-old Dendrobium officinale plants with robust growth and free from pests and diseases were selected. Stem segments with axillary buds were cut as explants. Excess leaves were removed, and the plants were rinsed with clean water and placed in a sterile environment. First, the plants were soaked in a sodium hypochlorite solution with an effective chlorine concentration of 4% for 12 minutes. After rinsing with sterile water three times, they were soaked in a mercuric chloride solution with a concentration of 0.2% for 7 minutes. After rinsing with sterile water five times, the surface moisture was finally absorbed with sterile filter paper to obtain sterilized explants.

[0043] S2. Primary culture: The sterilized explants were cut into small segments, each retaining one axillary bud. These segments were inoculated onto primary culture medium and placed in a culture room for primary culture. The culture temperature was controlled at 27℃, the light intensity at 2500 lux, and the light duration at 16 hours per day. After 30 days of culture, axillary buds were induced to germinate and form aseptic buds.

[0044] The primary culture medium was based on MS medium, with the addition of naphthaleneacetic acid (NAA), gibberellin, sucrose, and agar. The concentrations of NAA, gibberellin, sucrose, and agar were 0.2 mg / L, 0.1 mg / L, 30 g / L, and 6 g / L. The pH of the culture medium was adjusted to 6.0, and it was then autoclaved before use.

[0045] S3, Subculture Proliferation: The aseptic shoots obtained from the primary culture were cut from the explants and inoculated onto the subculture proliferation medium, then placed in a culture room for subculture proliferation. The culture temperature was controlled at 27℃, the light intensity at 3000 lux, and the light duration at 16 hours per day. Subculture was performed every 30 days, for a total of 2 subcultures, resulting in a large number of robust clustered shoots.

[0046] The subculture proliferation medium was based on MS medium, with the addition of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose and agar. The concentrations of 6-benzylaminopurine, naphthaleneacetic acid, humic acid, sucrose, and agar were 1.5 mg / L, 0.3 mg / L, 5 g / L, 30 g / L, and 6 g / L. The pH of the medium was adjusted to 6.0 and then autoclaved before use.

[0047] S4. Rooting and seedling cultivation: Healthy, clustered shoots with a height of 2-3 cm obtained from subculture were selected, divided into individual plants, and inoculated onto a rooting and seedling strengthening medium. These plants were then placed in a culture room for rooting and seedling strengthening culture. The culture temperature was controlled at 26℃, the light intensity at 3500 lux, and the light duration at 16 hours per day for 40 days, resulting in tissue culture seedlings with well-developed root systems and robust growth.

[0048] The rooting and seedling strengthening medium is based on 1 / 2 MS medium, with the addition of naphthaleneacetic acid, indolebutyric acid, activated carbon, sucrose and agar. The concentrations of naphthaleneacetic acid are 0.5 mg / L, indolebutyric acid is 0.3 mg / L, activated carbon is 1 g / L, sucrose is 25 g / L, and agar is 6 g / L. The pH of the medium is adjusted to 6.0, and it is then autoclaved before use.

[0049] S5. Hardening off and Transplanting: The tissue culture seedlings obtained from the rooting and strengthening process are moved out of the culture room along with the culture bottles and placed in a greenhouse for hardening off. The greenhouse temperature is controlled at 28℃, the humidity at 80%, natural light, and ventilation for 2 hours per day. After 10 days of hardening off, the caps of the culture bottles are opened, and hardening off continues for another 3 days to allow the tissue culture seedlings to gradually adapt to the external environment.

[0050] After hardening off, remove the tissue culture seedlings from the culture bottles, gently wash off the culture medium adhering to the roots, and transplant them into the pretreated cultivation substrate. Water and keep moist after transplanting, maintain the greenhouse temperature at 28℃ and humidity at 80%, and provide shade to avoid direct sunlight. Calculate the survival rate 30 days after transplanting.

[0051] The cultivation substrate is made of fermented pine bark, fermented peanut shells and gravel in a volume ratio of 2:2:1. The pretreatment method is to expose the cultivation substrate to sunlight for 3 days, then spray it with carbendazim solution, and then let it dry before use.

[0052] Comparative Example 1 A method for rapid propagation of Dendrobium officinale through tissue culture, compared with Example 2, only involves disinfecting the explants by soaking them in a single concentration of 0.15% mercuric chloride solution for 10 minutes; the remaining steps are exactly the same as in Example 2.

[0053] Comparative Example 2 A method for rapid propagation of Dendrobium officinale through tissue culture is provided. Compared with Example 2, humic acid is not added to the subculture proliferation medium, but the remaining steps are exactly the same as in Example 2.

[0054] Comparative Example 3 A method for rapid propagation of Dendrobium officinale through tissue culture, compared with Example 2, does not add activated carbon to the rooting and seedling strengthening culture medium, while the remaining steps are exactly the same as in Example 2.

[0055] Comparative Example 4 A method for rapid propagation of Dendrobium officinale through tissue culture is provided. Compared with Example 2, the seedling hardening method involves direct transplanting without gradient hardening, while the remaining steps are exactly the same as in Example 2.

[0056] Comparative Example 5 The existing conventional method for rapid propagation of Dendrobium officinale through tissue culture was adopted. Explants were disinfected by soaking in 0.1% mercuric chloride solution for 8 minutes. The primary culture medium was MS medium supplemented with 0.3 mg / L naphthaleneacetic acid and 30 g / L sucrose. The subculture proliferation medium was MS medium supplemented with 2 mg / L 6-benzylaminopurine and 0.2 mg / L naphthaleneacetic acid. The rooting and seedling strengthening medium was 1 / 2 MS medium supplemented with 0.4 mg / L naphthaleneacetic acid. After 3 days of hardening off, the seedlings were directly transplanted. The cultivation substrate was single fermented pine bark.

[0057] Experimental testing: Test materials: Dendrobium officinale tissue culture seedlings cultured in Examples 1 to 3 and Comparative Examples 1 to 5. Each example and comparative example had 3 replicates. Each replicate was inoculated with 100 explants and transplanted with 100 tissue culture seedlings.

[0058] Test metrics and methods: ① Contamination rate: The number of contaminated explants was counted 30 days after the initial culture.

[0059] .

[0060] ② Aseptic bud induction rate: After 30 days of initial culture, the number of explants that induced aseptic buds was counted.

[0061] .

[0062] ③Proliferation coefficient: After two generations of subculture, the total number of clustered shoots is counted.

[0063] .

[0064] ④ Rooting rate: After 40 days of rooted and robust seedling culture, the number of rooted tissue culture seedlings was counted.

[0065] .

[0066] ⑤ Browning rate: After 40 days of rooting and seedling cultivation, the number of tissue culture seedlings that showed browning was counted.

[0067] .

[0068] ⑥ Transplant survival rate: The number of surviving tissue culture seedlings was counted 30 days after transplanting.

[0069] .

[0070] ⑦ Culture cycle: From the start of explant disinfection to transplant survival, the average number of culture days for each example and comparative example was recorded.

[0071] The results of aseptic bud induction rate are as follows: Figure 1 As shown, the transplant survival rate and rooting rate results are as follows: Figure 2 As shown in the figure, the remaining test results are shown in Table 1.

[0072] Table 1

[0073] The test results above show that the contamination rate of Examples 1 to 3 was less than 2.3%, and the sterile bud induction rate was higher than 96.7%. In contrast, Comparative Example 1, using only mercuric chloride for disinfection, had a contamination rate of 8.7% and a sterile bud induction rate of 88.3%. Comparative Example 5, using conventional disinfection methods, had a contamination rate as high as 10.3% and a sterile bud induction rate of only 85.7%. This demonstrates that the two-step disinfection method of the present invention can effectively reduce the contamination rate and improve the sterile bud induction rate.

[0074] The proliferation coefficients of Examples 1 to 3 were all higher than 6.8, while the proliferation coefficient of Comparative Example 2, which did not contain humic acid in its subculture proliferation medium, dropped to 5.1, and the proliferation coefficient of Comparative Example 5 was only 4.8. This indicates that the addition of humic acid to the subculture proliferation medium in this invention can significantly improve the proliferation coefficient and increase the number of clustered shoots.

[0075] The rooting rates of Examples 1 to 3 were all above 95.3%, and the browning rates were all below 1.7%. In contrast, in Comparative Example 3, where no activated carbon was added to the rooting and seedling strengthening medium, the rooting rate dropped to 88.7%, and the browning rate increased to 8.3%. In Comparative Example 5, the rooting rate was only 86.3%, and the browning rate was as high as 9.7%. This demonstrates that adding activated carbon to the rooting and seedling strengthening medium of this invention can effectively improve the rooting rate, reduce the browning rate, and improve the growth of tissue culture seedlings.

[0076] The transplant survival rates of Examples 1 to 3 were all above 93.0%, while the transplant survival rate of Comparative Example 4, which did not undergo gradient hardening, dropped to 78.3%; and the transplant survival rate of Comparative Example 5 was only 75.0%. This indicates that the gradient hardening method of the present invention can effectively improve the stress resistance of tissue culture seedlings and increase the transplant survival rate.

[0077] The cultivation periods for Examples 1 to 3 were all around 170 days, while the cultivation period for Comparative Example 5 was as long as 202 days. This demonstrates that the present invention can significantly shorten the cultivation period, improve seedling efficiency, and reduce production costs.

[0078] In summary, the rapid propagation method for Dendrobium officinale tissue culture of the present invention effectively solves the problems of high contamination rate, low proliferation coefficient, low transplant survival rate and long culture cycle in the prior art by optimizing the explant disinfection process, culture medium formula and seedling hardening and transplanting process. It can quickly and efficiently cultivate high-quality Dendrobium officinale seedlings, meet the needs of large-scale production, and has high practical value and promotion prospects.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for rapid propagation of Dendrobium officinale through tissue culture, characterized in that: The method comprises the steps of explant disinfection, primary culture, subculture propagation culture, rooting and seedling culture, seedling hardening and transplanting; the explant is disinfected by two-step disinfection method of sodium hypochlorite and mercury bichloride, humic acid is added in the subculture propagation medium, activated carbon is added in the rooting and seedling culture medium, the seedling hardening adopts gradient seedling hardening mode, and the cultivation substrate adopts mixed substrate of fermented pine bark, fermented peanut shell and crushed stone.

2. The method for tissue culture rapid propagation and seedling raising of Dendrobium candidum according to claim 1, characterized in that, The explant disinfection specifically comprises the following steps: The explant is obtained by the following steps: 1-year-old Dendrobium candidum plants without diseases and pests are selected, stem segments with axillary buds are cut as explants, the excess leaves are removed, the explants are washed with clean water, and then are placed in a sterile environment; the explants are soaked in sodium hypochlorite solution with an effective chlorine concentration of 1%-5% for 8-12 min, washed with sterile water for 3 times, then soaked in mercury bichloride solution with a concentration of 0.1%-0.2% for 5-7 min, washed with sterile water for 5 times, and finally the surface water is absorbed by sterile filter paper to obtain the disinfected explants.

3. The method for tissue culture rapid propagation and seedling raising of Dendrobium candidum according to claim 1, characterized in that, The primary culture specifically comprises the following steps: the disinfected explants are cut into small segments, each segment retains one axillary bud, and then the segments are inoculated on a primary culture medium and placed in a culture room for primary culture; the culture temperature is controlled at 25-27 DEG C, the light intensity is controlled at 2000-2500 lux, the light time is 16 h per day, and the culture is performed for 30 days to induce the axillary buds to germinate and form sterile buds; the primary culture medium is based on MS medium and added with naphthalene acetic acid, gibberellin, sucrose and agar, wherein the naphthalene acetic acid concentration is 0.2 mg / L, the gibberellin concentration is 0.1 mg / L, the sucrose concentration is 30 g / L, and the agar concentration is 6 g / L; the pH value of the culture medium is adjusted to 5.8-6.

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4. The method according to claim 1, wherein the method is characterized by, The subculture propagation culture specifically comprises the following steps: the sterile buds obtained by the primary culture are cut from the explants, inoculated on a subculture propagation medium, and placed in a culture room for subculture propagation culture; the culture temperature is controlled at 25-27 DEG C, the light intensity is controlled at 2500-3000 lux, the light time is 16 h per day, the subculture is performed once every 30 days, and the subculture is performed twice continuously to obtain a large number of robust clustered buds; The subculture propagation medium is based on MS medium and added with 6-benzylaminopurine, naphthalene acetic acid, humic acid, sucrose and agar, wherein the 6-benzylaminopurine concentration is 1.5 mg / L, the naphthalene acetic acid concentration is 0.3 mg / L, the humic acid concentration is 5 g / L, the sucrose concentration is 30 g / L, and the agar concentration is 6 g / L; the pH value of the culture medium is adjusted to 5.8-6.

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5. The method according to claim 1, wherein the method is characterized by the steps of, The rooting and seedling culture specifically comprises the following steps: the robust clustered buds with a height of 2-3 cm obtained by the subculture propagation culture are selected, inoculated on a rooting and seedling culture medium after being separated into single plants, and placed in a culture room for rooting and seedling culture; the culture temperature is controlled at 24-26 DEG C, the light intensity is controlled at 3000-3500 lux, the light time is 16 h per day, and the culture is performed for 40 days to obtain tissue culture seedlings with developed root systems and robust growth; The rooting and seedling strengthening culture medium is based on 1 / 2MS culture medium, and naphthalene acetic acid, indole butyric acid, activated carbon, sucrose and agar are added, wherein the concentration of naphthalene acetic acid is 0.5 mg / L, the concentration of indole butyric acid is 0.3 mg / L, the concentration of activated carbon is 1 g / L, the concentration of sucrose is 25 g / L, the concentration of agar is 6 g / L, and the pH value of the culture medium is adjusted to 5.8-6.

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6. The method according to claim 1, wherein the method is characterized by the steps of, The seedling training and transplanting are specifically as follows: the tissue culture seedlings obtained through the rooting and seedling strengthening culture are removed from the culture room together with the culture bottles, and are placed in a greenhouse for seedling training, the temperature of the greenhouse is controlled to be 26-28 DEG C, the humidity is controlled to be 70-80%, natural light is used, and ventilation is performed for 2 hours per day, after 10 days of seedling training, the culture bottle caps are opened, and seedling training is continuously performed for 3 days; after the seedling training is completed, the tissue culture seedlings are taken out of the culture bottles, the root culture medium is cleaned, and then the tissue culture seedlings are transplanted into pretreated culture medium, after the transplanting, the temperature and humidity are controlled, and shading treatment is performed, and after 30 days of transplanting, the survived seedlings are obtained.

7. The method for rapid propagation of Dendrobium officinale through tissue culture according to claim 6, characterized in that: The culture medium is mixed by fermenting pine bark, fermenting peanut shell and crushed stone at a volume ratio of 2:2:1, and the pretreatment method is that the culture medium is exposed to sunlight for 3 days, then a carbendazim solution is sprayed, and after being dried, the culture medium is ready for use.

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