A culture method for inducing Dendrobium nobile Lindl. tissue culture seedlings to bloom

Abstract

The application discloses a culture method for inducing Dendrobium nobile Lindl. tissue culture seedlings to blossom, and belongs to the technical field of medicinal plant tissue culture. The whole culture process is divided into two stages: a vegetative growth culture stage, in which sterile explants are inoculated into a tank of an intermittent immersion type bioreactor, modified liquid medium is added into the tank, culture parameters are set, and the tank is cultured in a tissue culture room for 75-90 days; and an induction of blossom culture stage, in which TDZ is supplemented into the tank, culture parameters are changed, and the tank is further cultured in the tissue culture room for 45-60 days. The method is completed with the aid of the bioreactor, has high automation degree, and does not need to replace the culture medium during the culture process. Only TDZ needs to be added in the induction of blossom culture stage, the culture method does not harm the tissue culture seedlings, can improve the culture efficiency, has obvious blossom promoting effect, has the characteristics of short culture period, high induction of blossom rate and strong tissue culture seedlings, can improve the medicinal and ornamental values of commodities, and promotes the development of the Dendrobium nobile Lindi. industry.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant tissue culture technology, specifically to a method for cultivating Dendrobium nobile seedlings, and more particularly to a method for inducing flowering in Dendrobium nobile tissue culture seedlings. Background Technology

[0002] Dendrobium nobile Lindl., belonging to the genus Dendrobium in the family Orchidaceae, is a perennial epiphytic herb that mainly grows in tropical and subtropical rainforests and similar warm and humid environments. It is susceptible to frost and high temperatures. The stems of Dendrobium nobile are thin at both ends and thick in the middle, with the entire stem flattened and golden in color. Its shape resembles an ancient "golden hairpin" ornament, hence the name "Golden Hairpin."

[0003] Dendrobium nobile is a traditional and precious Chinese medicinal herb. Its medicinal use was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and it has been included in all editions of the *Chinese Pharmacopoeia*. It is ranked first among the nine immortal herbs and historically there is a saying, "Southern Dendrobium, Northern Ginseng." Both the stems and flowers of Dendrobium nobile have strong medicinal value. The stems are used medicinally; they are cold in nature, bitter and slightly salty in taste, and have effects such as enhancing immunity, strengthening yin and replenishing essence, promoting body fluids and nourishing the stomach, moistening the lungs and relieving cough, and anti-tumor effects. The flowers of Dendrobium nobile are excellent for nourishing yin and moistening the lungs, nourishing the stomach and promoting body fluids, improving eyesight and clearing heat, relieving depression, enhancing immunity, and anti-aging effects. They are also beneficial for respiratory diseases, diabetes, chronic hepatitis, and chronic gastritis. In addition to its medicinal value, Dendrobium nobile also has high ornamental value due to its vibrant colors and unique flower shape. With the improvement of people's living standards and the gradual enhancement of their awareness of physical and mental health, Dendrobium nobile is highly favored by the market due to its excellent health benefits and ornamental value.

[0004] However, Dendrobium nobile seeds are tiny and the embryos are not fully developed, making it extremely difficult for them to germinate under natural conditions. Wild resources have been drastically reduced, and long-term harvesting has led to a shortage of germplasm resources, bringing it to the brink of extinction. In addition, since orchids have a relatively long juvenile period of up to 30 months and cannot flower and bear fruit during this period, Dendrobium nobile flowers also have the characteristics of short flowering period and low yield, making it difficult to meet market demand. Therefore, artificial propagation techniques are often needed for assisted cultivation. While the application of plant tissue culture methods for the propagation of Dendrobium nobile can alleviate the shortage of Dendrobium nobile seedlings to some extent and shorten the time required for the transition from vegetative to reproductive growth, propagation by division or axillary buds is time-consuming and has a low propagation coefficient. Chinese Patent CN 105532473 B discloses a tissue culture method for Dendrobium nobile seedlings. This method establishes an intermittent immersion liquid culture system, inoculates explants into an intermittent immersion bioreactor containing MS culture medium, and propagates Dendrobium nobile seedlings after setting parameters. This method has the characteristics of high proliferation rate, high degree of automation, low labor intensity, good quality of tissue culture seedlings, and strong adaptability.

[0005] While existing technologies can address the shortage of Dendrobium nobile germplasm resources to some extent, it is important to note that conventional solid and liquid tissue culture techniques only play a significant role in seedling production. They are unlikely to induce flowering, and a low number of flowers will severely impact flower yield, thereby significantly reducing the commercial value and hindering the development of the Dendrobium nobile industry. In their article "Study on In vitro flowering induced by protocorms of Dendrobium nobile", Huang Jing et al. pointed out that using protocorms of Dendrobium nobile as raw material, cytokinin BA, phenylthiazolylurein (TDZ), and abscisic acid (ABA) were added to the culture medium to induce flower bud formation. The article explored the in vitro flowering process of Dendrobium nobile, optimized its early flowering system, and pointed out that certain concentrations of TDZ and ABA had a better effect on flower bud induction. However, the biggest drawback of this method is that the stems and leaves of the flowering plants in the test tubes are not well developed, and only a small number of flower buds can flower normally, resulting in a low flowering rate, which in turn affects the flower yield. In addition, the culture medium needs to be changed every month during the culture process, which is not only labor-intensive, but also damages the roots of the tissue culture seedlings during the culture process, affecting the normal growth of the plants and thus affecting the cultivation efficiency.

[0006] In conclusion, achieving good results in promoting flowering of Dendrobium nobile while ensuring that the cultivation method is not affected by unpredictable factors such as natural climate, maintaining a stable flowering rate, solving the problem of flowering period regulation in promoting flowering, and simultaneously improving the ease of operation and repeatability are still problems that urgently need to be studied and solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to solve the problems existing in the prior art and provide a cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings. The method is convenient to operate, highly reproducible, has a significant effect on promoting flowering, and produces robust tissue culture seedlings with a high flowering rate.

[0008] To achieve the above-mentioned technical objectives, the present invention is implemented through the following technical solution: a cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings, the specific process of which is as follows:

[0009] (1) Prepare the modified liquid culture medium and TDZ stock solution, sterilize and cool before use;

[0010] (2) The tank of the intermittent submerged bioreactor is sterilized and cooled for later use;

[0011] (3) Preparation of sterile explants of Dendrobium nobile;

[0012] (4) Vegetative growth culture stage: Under sterile conditions, sterile explants of Dendrobium nobile were inoculated into the tank of a sterilized reactor, sterile modified liquid culture medium was poured into the tank, the reactor was assembled, culture parameters were set, and the tank was placed in a tissue culture room for 75-90 days.

[0013] (5) Flowering induction culture stage: Under sterile conditions, TDZ stock solution is added to the modified liquid culture medium in the tank, the culture parameters are changed, and the tank is placed in the tissue culture room for further culture for 45-60 days.

[0014] Further, in step 1), the modified liquid culture medium is prepared by adding 0.1% peptone, 8% potato juice, and 2.5% sucrose to the modified MS liquid culture medium and adjusting the pH to 6.0.

[0015] Furthermore, the modified MS liquid culture medium uses water as a solvent, and the contents of each component are as follows: potassium nitrate 1425 mg / L, ammonium nitrate 1237.5 mg / L, magnesium sulfate heptahydrate 277.5 mg / L, potassium dihydrogen phosphate 127.5 mg / L, calcium chloride 330 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, ferrous sulfate 27.8 mg / L, sodium EDTA 37.3 mg / L, inositol 250 mg / L, nicotinic acid 1.25 mg / L, glycine 5 mg / L, thiamine hydrochloride 1.25 mg / L, and pyridoxine hydrochloride 1.25 mg / L.

[0016] Furthermore, in step 3), the sterile explant is a protocorm or a young seedling obtained by solid tissue culture.

[0017] Further, in step 4), the inoculation amount of sterile explants is 10-15g of protocorms per liter of modified liquid culture medium or 300-500 tissue culture seedlings per liter of modified liquid culture medium.

[0018] Furthermore, in step 4), the cultivation parameters for the vegetative growth stage are: immersion frequency 5 min / 4-6 h, aeration rate 2.5 L / min; temperature of the tissue culture room 25±1℃, relative humidity 50-70%, light intensity 1800-2200 lux, and photoperiod 14 h / d.

[0019] Furthermore, in step 5), the initial concentration of the TDZ stock solution added to the modified liquid culture medium is 1 mg / mL.

[0020] Furthermore, in step 5), the final concentration of TDZ in the modified liquid culture medium after supplementing with TDZ stock solution is 1.5–3 mg / L.

[0021] Furthermore, in step 5), the cultivation parameters for the induction of flowering culture stage are: immersion frequency 5 min / 8-10 h, aeration rate 2.5 L / min; temperature of the tissue culture room 20±1℃, relative humidity 40-60%, light intensity 2200-2500 lux, and photoperiod 16 h / d.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. The cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings disclosed in this application is based on an intermittent immersion liquid culture system. The entire cultivation process mainly includes two stages: vegetative growth culture and flowering induction culture. By limiting the culture parameters of each stage, a significant flowering promotion effect can be achieved. It has the characteristics of short cultivation cycle and high flowering induction rate.

[0024] 2. The cultivation method disclosed in this application can induce flowering in tissue culture seedlings of Dendrobium nobile, resulting in a high flowering rate and significantly increasing the yield of Dendrobium nobile flowers. This enhances the medicinal and ornamental value of the product, improves the economic value of Dendrobium nobile, and promotes the development of the Dendrobium nobile industry.

[0025] 3. The cultivation method disclosed in this application is based on an intermittent immersion liquid culture system, which is simple to operate, highly automated, and highly reproducible. The tissue culture seedlings do not need to be moved or the culture medium needs to be changed during the entire cultivation process. Compared with traditional cultivation methods, it eliminates the frequent transfer of culture materials and the large-scale bottling and cleaning process of tissue culture bottles, which significantly reduces the workload and production costs. Moreover, only a certain concentration of plant hormones needs to be added during the induction of flowering culture stage. This operation method can ensure that the tissue culture seedlings are not damaged and significantly improve the cultivation efficiency.

[0026] 4. Compared with traditional solid tissue culture methods, the use of intermittent immersion plant bioreactors for the culture of Dendrobium nobile explants can reduce the number of subcultures, lower the mutation rate, and ensure sufficient gas exchange during the culture process, reducing or even avoiding vitrification of seedlings, resulting in more robust seedlings.

[0027] 5. The cultivation method disclosed in this application is carried out under indoor conditions. The cultivation process is not affected by uncertain factors such as natural climate. Not only is the flowering rate high, but the flowering rate is also stable. This solves the difficulty of controlling the flowering period in promoting flowering. The flowering of Dendrobium nobile is no longer restricted by a fixed flowering period, and there will be no problem of "flowers without market or market without flowers". Attached Figure Description

[0028] Figure 1 These are photographs of Dendrobium nobile cultivated in Example 1 and Comparative Example 1. In the example, (A) is a photograph of Dendrobium nobile in bloom cultivated in Example 1, and (B) is a photograph of Dendrobium nobile in bloom cultivated in Comparative Example 1. Detailed Implementation

[0029] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the essence of the invention are within the scope of the present invention.

[0030] The modified liquid culture medium used in the examples was prepared by adding 0.1% peptone, 8% mashed potatoes, and 2.5% sucrose to modified MS liquid culture medium and adjusting the pH to 6.0. The potato juice was the juice obtained by simmering potato slices. The potato juice obtained by simmering can satisfy the purpose of destroying tissues and cells to obtain intracellular contents without destroying the macromolecular structure of cells.

[0031] The concentrations of various nutrients in the modified MS liquid culture medium (solvent: water) are as follows: ① Macronutrients (mg / L): Potassium nitrate (KNO3) 1425, Ammonium nitrate (NH4NO3) 1237.5, Magnesium sulfate heptahydrate (MgSO4·7H2O) 277.5, Potassium dihydrogen phosphate (KH2PO4) 127.5, Calcium chloride (CaCl2·2H2O) 330; ② Micronutrients (mg / L): Potassium iodide (KI) 0.83, Boric acid (H3BO3) 6.2, Manganese sulfate tetrahydrate (MnSO4·4H2O) 22.3, Zinc sulfate heptahydrate (ZnSO4·4H2O) 22.3. ① Iron salts (mg / L): ferrous sulfate (FeSO4·7H2O) 27.8, sodium ethylenediaminetetraacetate (Na2EDTA·2H2O) 37.3; ② Organic components (mg / L): inositol 250, nicotinic acid 1.25, glycine 5, thiamine hydrochloride (VB1) 1.25, pyridoxine hydrochloride (VB6) 1.25.

[0032] Preparation of TDZ stock solution: Accurately weigh 50 mg of TDZ, add dropwise to 10 mol / L NaOH solution until completely dissolved, then add distilled water and dilute to a 50 mL volumetric flask. TDZ was purchased from Sinopharm Chemical Reagent Co., Ltd., Sinopharm product number XW517075521.

[0033] Example 1

[0034] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0035] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the young seedlings of Dendrobium nobile obtained by solid tissue culture.

[0036] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 400 Dendrobium nobile juvenile tissue culture seedlings on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 6h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 75 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 2000lux, and photoperiod to 14h / d.

[0037] The intermittent submerged plant bioreactor used in this step includes a tank, a power system, and a control system. The power system generates gas and supplies it to the tank through connected pipes. The control system is connected to the power system via an electrical circuit. The tank has an upper and lower structure, sealed apart by a seedling tray. The upper part is a growth chamber, and the lower part is a liquid storage chamber. The growth chamber has a volume of 4000 mL, and the liquid storage chamber has a volume of 2000 mL. The two chambers are connected by a conduit connected to the seedling tray. The power system provides gas pressure to force the liquid culture medium from the liquid storage chamber into the growth chamber through the conduit. After the gas pressure is released, the liquid culture medium flows back to the liquid storage chamber through the conduit under the action of gravity and atmospheric air pressure. The control system controls the operation of the power system. The power system is an air pump, and the control system is a timer. The gas inside and outside the tank is at the same pressure. During the aeration phase, excess gas and outside air after aeration stops are discharged and introduced through a flat, round air filter sterilization device on the top left side of the tank to maintain sterility inside the tube. The above-mentioned intermittent submerged plant bioreactor is existing technology and will not be described in detail here.

[0038] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 2 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 8 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 45 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2400 lux, and the photoperiod to 16 h / d.

[0039] Example 2

[0040] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0041] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the young tissue culture seedlings of Dendrobium nobile obtained by solid tissue culture method.

[0042] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 300 Dendrobium nobile juvenile tissue culture seedlings on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 4h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 80 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 1800lux, and photoperiod to 14h / d.

[0043] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 1.5 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 9 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 50 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2200 lux, and the photoperiod to 16 h / d.

[0044] Example 3

[0045] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0046] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the young tissue culture seedlings of Dendrobium nobile obtained by solid tissue culture method.

[0047] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 500 Dendrobium nobile juvenile tissue culture seedlings on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 5h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 90 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 2200lux, and photoperiod to 14h / d.

[0048] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 3 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 10 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 60 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2500 lux, and the photoperiod to 16 h / d.

[0049] Example 4

[0050] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0051] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the protocorms of Dendrobium nobile obtained by solid tissue culture.

[0052] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 10g of Dendrobium nobile protocorms on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 6h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 75 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 2000lux, and photoperiod to 14h / d.

[0053] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 2 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 8 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 45 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2400 lux, and the photoperiod to 16 h / d.

[0054] Example 5

[0055] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0056] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the protocorms of Dendrobium nobile obtained by solid tissue culture.

[0057] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 12.5g of Dendrobium nobile protocorms on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 4h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 80 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 1800lux, and photoperiod to 14h / d.

[0058] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 1.5 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 9 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 50 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2200 lux, and the photoperiod to 16 h / d.

[0059] Example 6

[0060] (1) Prepare 1000 mL of modified liquid culture medium and 50 mL of TDZ stock solution with a concentration of 1 mg / mL. After high temperature and high pressure wet heat sterilization (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization time is 20 min), cool and set aside.

[0061] (2) Preparation of sterile explants of Dendrobium nobile. The sterile explants used in this embodiment refer to the protocorms of Dendrobium nobile obtained by solid tissue culture.

[0062] (3) Assemble and wrap the tank of the intermittent immersion plant bioreactor, and perform high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1~1.0MPa, sterilization for 20min). After sterilization, cool it for later use. Under sterile conditions, pour 1000mL of sterilized modified liquid culture medium into the tank and inoculate 15g of Dendrobium nobile protocorms on the seedling tray. Then connect the tank to the power system and control system, set the intermittent immersion frequency to 5min / 5h and the aeration rate to 2.5L / min. Cultivate in the tissue culture room for 90 days to complete the vegetative growth culture. Adjust the temperature of the tissue culture room to 25±1℃, relative humidity to 60%, light intensity to 2200lux, and photoperiod to 14h / d.

[0063] (4) After the vegetative growth culture is completed, the tank is removed and moved to the ultra-clean workbench. After external sterilization, the prepared TDZ stock solution is added to the modified liquid culture medium in the intermittent immersion plant bioreactor tank under aseptic conditions, so that the final concentration of TDZ is 3 mg / L. During this process, the tissue culture seedlings do not need to be moved. Afterwards, the tank is removed and reconnected to the power and control system. The intermittent immersion frequency is set to 5 min / 10 h and the aeration rate is 5 L / min. The seedlings are cultured in the tissue culture room for 60 days to complete the induction of flowering culture. The temperature of the tissue culture room is adjusted to 20±1℃, the relative humidity to 50%, the light intensity to 2500 lux, and the photoperiod to 16 h / d.

[0064] Comparative Example 1

[0065] In the comparative case, solid-state culture was used to cultivate young tissue culture seedlings of Dendrobium nobile. A 500mL orchid bottle was used as the culture container. The culture medium formula was the same as the modified liquid culture medium formula in Example 1, with the addition of 0.72% agar. The culture medium addition amount was 100mL / bottle, and the inoculation amount was 40 young tissue culture seedlings / bottle. After 75 days of vegetative growth culture, 10mL of 2mg / L TDZ was added to the tissue culture bottle, and then flowering was induced for 45 days. The culture environment for the vegetative growth culture stage and the flowering induction culture stage was the same as that in Example 1.

[0066] Comparative Example 2

[0067] In the comparative case, the solid culture method was used to cultivate the young tissue culture seedlings of Dendrobium nobile. A 500mL orchid bottle was used as the culture container. The culture medium formula was the same as the modified liquid culture medium formula in Example 1, and 0.72% agar was added. The culture medium was added at a rate of 100mL / bottle, and the inoculation amount was 30 young tissue culture seedlings / bottle. After 80 days of vegetative growth culture, 10mL of 1.5mg / L TDZ was added to the tissue culture bottle, and then flowering was induced for 50 days. The culture environment for the vegetative growth culture stage and the flowering induction culture stage was the same as that in Example 2.

[0068] Comparative Example 3

[0069] In the comparative case, the solid culture method was used to cultivate the young tissue culture seedlings of Dendrobium nobile. A 500mL orchid bottle was used as the culture container. The culture medium formula was the same as the modified liquid culture medium formula in Example 1, and 0.72% agar was added. The culture medium was added at a rate of 100mL / bottle, and the inoculation amount was 12.5g protocorms / bottle. After 80 days of vegetative growth culture, 10mL of 1.5mg / L TDZ was added to the tissue culture bottle, and then flowering was induced for 50 days. The culture environment for the vegetative growth culture stage and the flowering induction culture stage was the same as that in Example 5.

[0070] The flower bud induction rate, flowering rate, number of flowers, and maximum number of flowers per plant of *Dendrobium nobile* cultured under the conditions shown in Examples 1-6 and Comparative Examples 1-3 are as follows:

[0071]

[0072] Among them, the flower bud induction rate = (number of flower bud tissue culture seedlings / total number of tissue culture seedlings) × 100%, and the flowering rate = (number of flowering tissue culture seedlings / total number of tissue culture seedlings) × 100%.

[0073] As can be seen from the above data, the culture methods disclosed in Examples 1-6 for cultivating Dendrobium nobile tissue culture seedlings resulted in significantly better flower bud induction rate, flowering rate, and number of flowers per plant than the control example, which can greatly improve the flowering level and commercial value of Dendrobium nobile.

[0074] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A method for inducing flowering in tissue culture seedlings of Dendrobium nobile, characterized in that, The specific process is as follows: (1) Prepare the modified liquid culture medium and TDZ stock solution, sterilize and cool before use; (2) The tank of the intermittent submerged bioreactor is sterilized and cooled for later use; (3) Preparation of sterile explants of Dendrobium nobile; (4) Vegetative growth culture stage: Under sterile conditions, sterile explants of Dendrobium nobile were inoculated into the tank of a sterilized reactor, sterile modified liquid culture medium was poured into the tank, the reactor was assembled, culture parameters were set, and the tank was placed in the tissue culture room for 75-90 days. (5) Flowering induction culture stage: Under sterile conditions, TDZ stock solution was added to the modified liquid culture medium in the tank. After adding TDZ stock solution, the final concentration of TDZ in the modified liquid culture medium was 1.5~3 mg / L. After changing the culture parameters, the culture was placed in the tissue culture room for 45~60 days. In step (3), the sterile explants are protocorms or seedlings obtained by solid tissue culture. In step (1), the modified liquid culture medium is prepared by adding 0.1% peptone, 8% potato juice and 2.5% sucrose to the modified MS liquid culture medium and adjusting the pH to 6.

0. The modified MS liquid culture medium uses water as a solvent, and the contents of each component are as follows: potassium nitrate 1425 mg / L, ammonium nitrate 1237.5 mg / L, magnesium sulfate heptahydrate 277.5 mg / L, potassium dihydrogen phosphate 127.5 mg / L, calcium chloride 330 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate tetrahydrate 22.3 mg / L, zinc sulfate heptahydrate 8.6 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, ferrous sulfate 27.8 mg / L, sodium EDTA 37.3 mg / L, inositol 250 mg / L, nicotinic acid 1.25 mg / L, glycine 5 mg / L, thiamine hydrochloride 1.25 mg / L, and pyridoxine hydrochloride 1.25 mg / L.

2. The cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings as described in claim 1, characterized in that, In step (4), the amount of sterile explants inoculated is 10-15 g of protocorms per liter of modified liquid culture medium or 300-500 tissue culture seedlings per liter of modified liquid culture medium.

3. The cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings as described in claim 1, characterized in that, In step (4), the cultivation parameters for the vegetative growth stage are: immersion frequency 5 min / 4~6 h, aeration rate 2.5 L / min; temperature of the tissue culture room 25±1℃, relative humidity 50~70%, light intensity 1800~2200 lux, and photoperiod 14 h / d.

4. The cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings as described in claim 1, characterized in that, In step (5), the initial concentration of the TDZ stock solution added to the modified liquid culture medium is 1 mg / mL.

5. The cultivation method for inducing flowering of Dendrobium nobile tissue culture seedlings as described in claim 1, characterized in that, In step (5), the cultivation parameters for the induction of flowering culture stage are: immersion frequency 5 min / 8~10 h, aeration rate 2.5 L / min; temperature of tissue culture room 20±1℃, relative humidity 40~60%, light intensity 2200~2500 lux, and photoperiod 16 h / d.

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