Method for rapid propagation of saussurea involucrata by intermittent submersion tissue culture

Abstract

The application discloses a kind of lotus of Guanyin intermittent immersion tissue culture rapid propagation method, comprising the following steps: S10, preparation sterile explant;S20, in sterile environment, first sterile liquid medium is poured into the tank of the sterilized intermittent immersion type culture system, then according to the inoculation amount of 40-60 explants per liter of culture medium, sterile explant is inoculated into the tank of the intermittent immersion type culture system;S30, the tank of inoculation is connected to the intermittent immersion type culture system, set system culture parameters, then the whole system is placed in tissue room and is cultured, wherein, immersion time is 2-10min, immersion interval time is 4-12h, culture cycle is 75-100d, the light intensity in tissue room environment is 1800-2000lux, the light cycle is 14h / d, relative humidity is 60%, and temperature is 25±1℃.It has obvious proliferation effect, and the characteristics of short culture cycle.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of flower propagation, and in particular to a method for rapid propagation of Alocasia amazonica by intermittent submersion tissue culture. BACKGROUND

[0002] Alocasia amazonica is a perennial herbaceous plant of Araceae. Its leaves are shield-shaped, dark green, with obvious veins, silver-white, and purple-brown on the back. It is extremely shade-tolerant, and the leaf color is evergreen in all seasons. It is a leafy plant used for high-end indoor decoration and garden and waterscape design, and is popular in the flower market at home and abroad. Because the seeds of Alocasia amazonica are not easy to obtain, it is often propagated by division and tuber, with low propagation coefficient, and its large-scale production is limited. Rapid propagation of Alocasia amazonica by tissue culture is of great significance to meet the demand of the flower market.

[0003] Chinese patent CN1175725C discloses a method for propagating Alocasia amazonica, which is an ornamental flower. The method uses conventional solid and liquid tissue culture mode, small tubers as explants, first induces callus, then produces multiple shoots from callus, and finally forms complete plants. The method has the characteristics of high propagation rate and short culture period.

[0004] Although the prior art can solve the problem of slow propagation of Alocasia amazonica to some extent, it is still necessary to note that the conventional solid and liquid tissue culture technology has the problems of low quality of seedlings, low resistance, and low survival rate of transplanted seedlings. In addition, a large amount of labor and materials are consumed in large-scale production, and the production cost is high. Therefore, it is still a problem for those skilled in the art to research and solve how to achieve efficient and low-cost production of Alocasia amazonica seedlings, ensure good quality of seedlings, high survival rate of transplanted seedlings, and improve the convenience and repeatability of operation. SUMMARY

[0005] In view of the above defects or deficiencies in the prior art, it is desirable to provide a method for rapid propagation of Alocasia amazonica by intermittent submersion tissue culture, which is convenient to operate, has high repeatability, produces robust tissue culture seedlings, and has low production cost.

[0006] In a first aspect, the method for rapid propagation of Alocasia amazonica by intermittent submersion tissue culture comprises the following steps:

[0007] S10, preparing sterile explants;

[0008] S20, in a sterile environment, first pouring sterile liquid medium into the tank body of the intermittent submersion culture system, and then inoculating 40-60 explants per liter of medium into the tank body of the intermittent submersion culture system;

[0009] S30, connecting the inoculated tank to the batch submergence culture system, setting the system culture parameters, and then placing the whole system in the tissue room for culture, wherein the submergence time in the system culture parameters is 2-10 min, the submergence interval time in the system culture parameters is 4-12 h, the culture cycle in the system culture parameters is 75-100 d, the light intensity in the tissue room environment is 1800-2000 lux, the light cycle in the tissue room environment is 14 h / d, the relative humidity in the tissue room environment is 60%, and the temperature in the tissue room environment is 25±1℃.

[0010] According to the technical scheme provided by the embodiments of the present application, the obvious proliferation effect can be achieved by limiting the system culture parameters, and the culture cycle is short.

[0011] According to the technical scheme provided by the embodiments of the present application, the culture is carried out based on the batch submergence culture system, which is simple to operate and has high automation. The tissue culture seedlings do not need to be moved and the culture medium does not need to be replaced during the whole culture process. Compared with the traditional cultivation method, the pollution rate is greatly reduced, the frequent transfer of culture and the cleaning process of large quantities of tissue culture bottles are saved, the workload is significantly reduced, the production cost is reduced, and one-step seedling is realized.

[0012] According to the technical scheme provided by the embodiments of the present application, compared with the traditional solid tissue culture method, the culture of the Bauhinia Blake seedlings using the batch submergence culture system can reduce the subculture frequency, reduce the variation rate, and carry out sufficient gas exchange during the culture process, reduce or even avoid vitrification of the seedlings, and the seedlings obtained by propagation are more robust.

[0013] According to the technical scheme provided by the embodiments of the present application, the culture is carried out under indoor conditions, and the culture process is not affected by natural climate and other uncertain factors, and has strong repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0014] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments made with reference to the accompanying drawings:

[0015] Figure 1 Picture of the Bauhinia Blake cultured in Example 1;

[0016] Figure 2 Picture of the Bauhinia Blake cultured in Example 4;

[0017] Figure 3 Picture of the Bauhinia Blake cultured in Comparative Example 1. DETAILED DESCRIPTION

[0018] The application will be described in further detail below with reference to the drawings and embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the application and are not intended to limit the application. In addition, it should be noted that only parts related to the application are shown in the drawings for ease of description.

[0019] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in further detail below with reference to the drawings and embodiments.

[0020] The application discloses a kind of intermittent immersion tissue culture rapid propagation methods of lotus, comprising the following steps: S10, preparation sterile explant;S20, in sterile environment, first the sterile liquid medium is poured into the tank of sterilized intermittent immersion type culture system, then according to the inoculation amount of 40-60 explants per liter of culture medium Inoculation amount of sterile explant is inoculated into the tank of intermittent immersion type culture system;S30, the tank of inoculation is connected to intermittent immersion type culture system, set system culture parameters, then the whole system is placed in tissue room and is cultured, wherein, immersion time in system culture parameters is 2-10 min, immersion interval time in system culture parameters is 4-12 h, culture cycle in system culture parameters is 75-100 d, light intensity in tissue room environment is 1800-2000 lux, light cycle in tissue room environment is 14 h / d, relative humidity in tissue room environment is 60%, and temperature in tissue room environment is 25±1 ℃.

[0021] Further, in step S10, if the explant is a plant grown in natural environment, the explant is first sterilized, and then the sterile explant is prepared;If the explant is a sterile tissue culture seedling, the sterile explant is directly prepared.

[0022] Further, the sterilization of the explant comprises: selecting a healthy plant free of disease, removing the roots, first soaking in potassium permanganate solution for 8-12 min, rinsing with water for 15-30 min, then removing the leaves and retaining the stems in a super-clean workbench, soaking in 75% alcohol for 15-20 s, rinsing with sterile water twice, and then soaking in 0.1% mercury for 5-10 min, rinsing with sterile water 5-7 times.

[0023] Further, the preparation of the sterile explant comprises: cutting the stems of the sterilized sterile plant or sterile tissue culture seedling into stem segments containing 1-3 leaf buds, first inoculating the stem segments into sterile solid culture medium, and then performing the next inoculation after 3-5 days of culture and observation without microbial contamination.

[0024] Further, the solid culture medium is prepared by adding agar to the liquid culture medium to a mass concentration of 0.7%, and the solid culture medium is divided into tissue culture bottles and sterilized for standby use.

[0025] Further, in step S20, the liquid medium is a MS liquid medium based medium, adding plant hormone NAA to a final concentration of 0.3-1.0 mg / L, adding sucrose to a final concentration of 20-25 g / L, and adjusting the pH value to 6.0-6.2.

[0026] Further, after the liquid medium is prepared, the liquid medium is placed in a flask and sterilized.

[0027] Further, the immersion time in the system culture parameters is 4-6 min.

[0028] Further, the immersion interval time in the system culture parameters is 5-8 h.

[0029] Further, before step S20, the tank of the intermittent immersion culture system is sterilized.

[0030] Further, the intermittent immersion culture system is a plant tissue culture system designed and made according to the principle of intermittent immersion culture, including a culture chamber, a liquid storage chamber, a power system, and a control system. The culture chamber is a cavity for plant growth, the liquid storage chamber is a cavity for storing liquid medium, the power system is a power device for providing liquid medium flow, and the control system is a device for controlling the operation of the power system, thereby controlling the immersion frequency of the culture system. The tank of the intermittent immersion culture system is a cavity of the culture chamber and the liquid storage chamber through a pipeline, which is isolated from the outside through sterile air filtration. The sterilization method is high-temperature high-pressure moist heat sterilization.

[0031] The liquid medium used in the embodiment is a MS liquid medium to which NAA and sucrose are added respectively and then compounded, and the pH value is adjusted to a certain range.

[0032] The concentration of each nutrient component in the MS liquid medium is (solvent is water): ① large components (mg / L): potassium nitrate (KNO3) 1900, ammonium nitrate (NH4NO3) 1650, magnesium sulfate heptahydrate (MgSO4·7H2O) 370, potassium dihydrogen phosphate (KH2PO4) 170, calcium chloride (CaCl2·2H2O) 440, ② trace components (mg / L): potassium iodide (KI) 0.83, boric acid (H3BO3) 6.2, manganese sulfate tetrahydrate (MnSO4·4H2O) 22.3, zinc sulfate heptahydrate (ZnSO4·7H2O) 8.6, sodium molybdate dihydrate (Na2MoO4·2H2O) 0.25, copper sulfate pentahydrate (CuSO4·5H2O) 0.025, cobalt chloride hexahydrate (CoCl2·6H2O) 0.025, ③ iron salt (mg / L): ferrous sulfate (FeSO4·7H2O) 27.8, sodium ethylenediaminetetraacetate (Na2EDTA·2H2O) 37.3, ④ organic components (mg / L): myo-inositol 100, nicotinic acid 0.5, glycine 2, thiamine hydrochloride (VB1) 0.1, pyridoxal hydrochloride (VB6) 0.1.

[0033] Example 1:

[0034] (1) According to the formula MS+NAA 0.5 mg / L+ sucrose 22.5 g / L, adjust pH 6.0, prepare 1000 mL of liquid medium, pour into triangular bottles; according to the formula of the liquid medium, add agar to 0.7% of mass concentration to prepare 500 mL of solid medium, and evenly distribute to 10 bottles of 200 mL of tissue culture bottles. The liquid medium and solid medium are subjected to high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1-1.0 MPa, sterilization time is 20 min) and then cooled for standby.

[0035] The intermittent immersion culture system is a plant bioreactor disclosed in Chinese patent CN104770304B, wherein the culture tank body is 6000 mL, the upper part of the culture chamber is 4000 mL, and the lower part of the liquid storage chamber is 2000 mL, and the two chambers are separated by a seedling raising disc. After the reactor tank body is wrapped and subjected to high temperature and high pressure wet heat sterilization treatment (heating temperature is 121℃, pressure is 0.1-1.0 MPa, sterilization time is 20 min), it is cooled for standby.

[0036] (2) The natural environment growing Lotus seed was used as the material to prepare the aseptic explant. The specific method was as follows. The healthy and disease-free plant was selected, the root was removed, the plant leaves were removed first, and the stem was soaked in potassium permanganate solution for 10 min, then washed with running water for 20 min, then in the clean bench, the plant leaves were removed, the stem was soaked in 75% alcohol for 15 s, washed with sterile water for 2 times, then soaked in 0.1% mercury for 8 min, washed with sterile water for 5-7 times, finally the stem was cut into stem segments containing 1-3 leaf buds, 10 stem segments were inoculated into the solid culture medium in each bottle, and the culture did not appear pollution after 3-5 days in the tissue culture room.

[0037] (3) 1000 mL of sterile liquid medium was poured into the tank body of the sterilized intermittent immersion culture system, and then 60 sterile explants were inoculated into the tank body.

[0038] (4) The inoculated tank body was connected to the intermittent immersion culture system, the immersion time was set to 5 min, the interval time was set to 6 h, the culture cycle was set to 90 d, and the culture was placed in the tissue culture room, and the environment of the tissue culture room was set to light intensity 1800-2000 lux, light cycle 14 h / d, relative humidity 60%, and temperature 25±1℃.

[0039] The picture of the Lotus seed cultured in Example 1 is shown in Figure 1

[0040] Example 2:

[0041] (1) According to the formula MS+NAA 0.3 mg / L+sucrose 20 g / L, adjust pH 6.2, prepare 1000 mL of liquid medium, pour into a triangular bottle; according to the formula of the liquid medium, add agar to a mass concentration of 0.7% to prepare 200 mL of solid culture medium, and evenly divide into 10 200 mL tissue culture bottles. The liquid and solid culture media were treated by high temperature and high pressure wet heat sterilization (heating temperature 121℃, pressure 0.1-1.0 MPa, sterilization 20 min) and then cooled for standby.

[0042] The intermittent immersion culture system is a plant bioreactor disclosed in Chinese patent CN104770304B, wherein the culture tank body is 6000 mL, the upper part of the culture room is 4000 mL, and the lower part of the liquid storage room is 2000 mL, and the two rooms are separated by a seedling raising plate. The reactor tank body was wrapped and then treated by high temperature and high pressure wet heat sterilization (heating temperature 121℃, pressure 0.1-1.0 MPa, sterilization 20 min) and then cooled for standby.

[0043] (2) The tissue culture seedlings of Lotus seed were used as the explants, the leaves were removed, and the stem was cut into stem segments containing 1-3 leaf buds, 10 stem segments were inoculated into the solid culture medium in each bottle, and the culture did not appear pollution after 3-5 days in the tissue culture room. ​

[0044] (3) Pour 1000 mL of sterile liquid medium into the tank of the sterilized batch submersion culture system, and then inoculate 40 explants into the tank.

[0045] (4) Connect the inoculated tank to the batch submersion culture system, set the submersion time to 6 min, the interval time to 8 h, and the culture cycle to 100 d, and place it in the tissue culture room for culture, with the environment of the tissue culture room set to an illumination intensity of 1800-2000 lux, an illumination cycle of 14 h / d, a relative humidity of 60%, and a temperature of 25±1°C.

[0046] Example 3:

[0047] (1) Prepare 500 mL of liquid medium according to the formula MS+NAA 1.0 mg / L+ sucrose 25 g / L, adjust the pH to 6.0, pour into a flask; prepare 250 mL of solid medium according to the formula of the liquid medium by adding agar to a mass concentration of 0.7%, and evenly divide and pack into 5 bottles of 200 mL of tissue culture bottles. The liquid and solid media are subjected to high-temperature and high-pressure wet heat sterilization treatment (heating temperature of 121°C, pressure of 0.1-1.0 MPa, sterilization for 20 min), and then cooled for standby.

[0048] The batch submersion culture system is a batch submersion plant tissue organ culture reactor disclosed in Chinese patent CN201420083Y, wherein the culture tank is a double-bottle type, the culture chamber is a 1000 mL spherical glass bottle, and the liquid storage chamber is a 1000 mL triangular bottle, and the two chambers are connected in communication through a silica gel pipe. The reactor tank is wrapped and subjected to high-temperature and high-pressure wet heat sterilization treatment (heating temperature of 121°C, pressure of 0.1-1.0 MPa, sterilization for 20 min), and then cooled for standby.

[0049] (2) Prepare sterile explants from Hoya carnosa grown in a natural environment, and the specific method is as follows: select healthy and disease-free plants, remove the roots, first immerse in a potassium permanganate solution for 12 min, wash with running water for 30 min, then in a clean bench, remove the leaves and retain the stems, immerse in 75% alcohol for 20 s, wash with sterile water for 2 times, immerse in 0.1% mercury chloride for 10 min, and wash with sterile water for 5-7 times, finally cut the stems into stem segments containing 1-3 leaf buds, inoculate 10 stem segments per bottle into the solid medium, and place in the tissue culture room for culture for 3-5 days without contamination.

[0050] (3) Pour 500 mL of sterile liquid medium into the triangular bottle liquid storage chamber of the sterilized batch submersion culture system, and then inoculate 30 sterile explants into the spherical glass bottle culture chamber.

[0051] (4) The inoculated tank is connected to the batch submersion culture system, the submersion time is set to 4 min, the interval time is set to 5 h, the culture cycle is set to 75 d, and the tank is placed in the tissue culture room for culture. The environment of the tissue culture room is set to light intensity of 1800-2000 lux, light cycle of 14 h / d, relative humidity of 60%, and temperature of 25±1℃.

[0052] Example 4:

[0053] (1) A liquid medium of 500 mL is prepared according to the formula MS+NAA 0.8 mg / L+ sucrose 24 g / L, with pH adjusted to 6.2. A solid medium of 100 mL is prepared by adding agar to the liquid medium to a mass concentration of 0.7%, and then evenly divided into 5 bottles of 200 mL tissue culture bottles. The liquid and solid media are subjected to high-temperature and high-pressure wet heat sterilization treatment (heating temperature of 121℃, pressure of 0.1-1.0 MPa, sterilization time of 20 min), and then cooled for standby.

[0054] The batch submersion culture system is a batch submersion plant tissue organ culture reactor disclosed in Chinese patent CN201420083Y. The culture tank is a double-bottle type, the culture chamber is a 1000 mL spherical glass bottle, and the liquid storage chamber is a 1000 mL triangular bottle. The two chambers are connected through a silica gel tube. After the reactor tank is wrapped, it is subjected to high-temperature and high-pressure wet heat sterilization treatment (heating temperature of 121℃, pressure of 0.1-1.0 MPa, sterilization time of 20 min), and then cooled for standby.

[0055] (2) The tissue culture seedlings of Hymenocallis are used as explants, the leaves are removed, and the stems are cut into stem segments each containing 1-3 leaf buds. Each bottle is inoculated with 10 stem segments into the solid culture medium, and then placed in the tissue culture room for culture for 3-5 days without contamination.

[0056] (3) 500 mL of sterile liquid medium is poured into the sterilized triangular bottle liquid storage chamber of the batch submersion culture system, and then 20 sterile explants are inoculated into the spherical glass bottle culture chamber.

[0057] (4) The inoculated tank is connected to the batch submersion culture system, the submersion time is set to 10 min, the interval time is set to 12 h, the culture cycle is set to 80 d, and the tank is placed in the tissue culture room for culture. The environment of the tissue culture room is set to light intensity of 1800-2000 lux, light cycle of 14 h / d, relative humidity of 60%, and temperature of 25±1℃.

[0058] The picture of the cultured Hymenocallis in Example 4 is shown in Figure 2 .

[0059] Comparative Example 1:

[0060] The comparative example uses a solid culture method to cultivate the tissue culture seedlings of the Lotus plant, uses 200 mL tissue culture bottles as the culture container, the solid culture medium formula used is the same as the liquid culture medium formula in Example 1, and 0.7% agar is added; the medium addition amount is 50 mL / bottle, 3 sterilized Lotus plant stem segments are inoculated in each bottle, and the tissue culture room is set to a light intensity of 1800-2000 lux, a light cycle of 14 h / d, a relative humidity of 60%, and a temperature of 25±1°C for 90 d of culture.

[0061] The picture of the Lotus plant cultured in Comparative Example 1 is shown in Figure 3

[0062] Comparative Example 2:

[0063] The comparative example uses a solid culture method to cultivate the tissue culture seedlings of the Lotus plant, uses 200 mL tissue culture bottles as the culture container, the solid culture medium formula used is the same as the liquid culture medium formula in Example 4, and 0.7% agar is added; the medium addition amount is 50 mL / bottle, 1 Lotus plant tissue culture seedling stem segment is inoculated in each bottle, and the tissue culture room is set to a light intensity of 1800-2000 lux, a light cycle of 14 h / d, a relative humidity of 60%, and a temperature of 25±1°C for 80 d of culture.

[0064] The average plant height, average root number, average root length, and proliferation coefficient of the Lotus plant tissue culture seedlings cultured under the conditions shown in Examples 1-4 and Comparative Examples 1 and 2 are counted, and the results are shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] The above description is merely preferred embodiments of the present application and a description of the principles of the technology used. Those skilled in the art should understand that the scope of the application disclosed in the present application is not limited to the technical solutions formed by the specific combinations of the technical features described above, and should also encompass other technical solutions formed by any combinations of the above technical features or equivalent features without departing from the inventive concept. For example, the above features can be replaced with technical features disclosed in the present application (but not limited to) having similar functions to form technical solutions.​

Claims

1. A method for rapid propagation of intermittent submersion tissue culture of a plant of the species Saussurea involucrata Maxim. having the taxonomic designation Saussurea involucrata Maxim. var. involucrata Maxim. Alocasia amazonica, characterized in that The method comprises the following steps: S10, preparing sterile explants; S20, in a sterile environment, first pouring sterile liquid medium into the tank of the sterilized intermittent immersion culture system, and then inoculating the sterile explants into the tank of the intermittent immersion culture system at an inoculation amount of 40-60 explants per liter of medium; S30, connecting the inoculated tank to the intermittent immersion culture system, setting the system culture parameters, and then placing the entire system in a tissue chamber for culture, wherein the immersion time in the system culture parameters is 2-10 min, the immersion interval time in the system culture parameters is 4-12 h, the culture cycle in the system culture parameters is 75-100 d, the light intensity in the tissue chamber environment is 1800-2000 lux, the light cycle in the tissue chamber environment is 14 h / d, the relative humidity in the tissue chamber environment is 60%, and the temperature in the tissue chamber environment is 25±1℃; In step S10, if the explants are plants grown in a natural environment, the explants are first sterilized, and then the sterile explants are prepared; if the explants are sterile tissue culture seedlings, the sterile explants are directly prepared; The method for preparing sterile explants is to cut the stems of the sterilized sterile plants or sterile tissue culture seedlings into stem segments containing 1-3 leaf buds, first inoculate the Alocasia explant stem segments into sterile solid medium, and after 3-5 days of culture and observation without microbial contamination, proceed to the next step of inoculation; The liquid medium is prepared by using MS liquid medium as the basic medium, adding plant hormone NAA to a final concentration of 0.3-1.0 mg / L, adding sucrose to a final concentration of 20-25 g / L, and adjusting the pH value to 6.0-6.2; The solid medium is prepared by adding agar to the liquid medium to a mass concentration of 0.7%.

2. The method for rapid propagation of intermittent submerged tissue culture of S. zeylanica according to claim 1, characterized in that, The sterilization of the explants includes: selecting healthy and disease-free plants, removing the roots, first soaking in potassium permanganate solution for 8-12 min, rinsing with water for 15-30 min, then removing the leaves and retaining the stems in the super-clean workbench, soaking in 75% alcohol for 15-20 s, rinsing with sterile water twice, and then soaking in 0.1% mercury for 5-10 min, rinsing with sterile water 5-7 times.

3. The method for rapid propagation of intermittent submerged tissue culture of Tirella angustata according to claim 1, characterized in that, After the solid medium is prepared, the solid medium is divided into tissue culture bottles and sterilized for standby use.

4. The method for rapid propagation of intermittent submerged tissue culture of Tirella angustifolia according to claim 1, characterized in that, After the liquid medium is prepared, the liquid medium is placed in a triangular flask and sterilized.

5. The method for rapid propagation of intermittent submerged tissue culture of Tirella angustata according to claim 1, characterized in that, The immersion time in the system culture parameters is 4-6 min.

6. The method for rapid propagation of intermittent submerged tissue culture of Tirella angustata according to claim 1, characterized in that, The immersion interval time in the system culture parameters is 5-8 h.

7. The method for rapid propagation of intermittent submerged tissue culture of Tirella angustata according to claim 1, characterized in that, Before step S20, the tank of the intermittent immersion culture system is sterilized.

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