Tissue culture medium for hybrid dendrobium, tissue culture method and application

Abstract

The invention belongs to the technical field of tissue culture of plants, and particularly relates to a tissue culture medium and a tissue culture method of hybrid dendrobium and application of the tissue culture medium and the tissue culture method. The tissue culture medium of the hybrid dendrobium nobile comprises a seed germination medium, a proliferation induction medium and a rooting and seedling strengthening medium, under the combined action of 6-BA, NAA, potato juice, mashed banana and activated carbon, the tissue culture medium is beneficial to inducing germination of hybrid dendrobium seeds, proliferation of protocorm clusters and differentiation of the protocorm clusters into cluster buds, particularly, the potato juice is added to promote growth and development of the hybrid dendrobium protocorm clusters, a large number of flourishing cluster buds are obtained, and then a large number of tissue culture seedlings are obtained. The obtained hybrid dendrobium tissue culture seedlings are good in plant consistency and robust, the transplanting survival rate of the tissue culture seedlings is increased, and the culture time of the tissue culture seedlings is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a hybrid dendrobium tissue culture medium and a tissue culture method and application. Background Art

[0002] Dendrobium nobileLindl. is a plant of the Orchidaceae family and the genus Dendrobium. Spring Dendrobium is a deciduous species of Dendrobium in the genus Dendrobium. Its flowers are bright and colorful, and its flowering period is in spring. The flowering period is more than 20 days. It has the characteristics of compact plant shape, colorful flowers and fragrant flowers. It is an important member of the "Four Major Ornamental Orchids" and is often used as a gift potted flower. It is one of the main potted and cut flower varieties in the current international flower market.

[0003] Autumn Dendrobium mainly refers to the Phalaenopsis type, the Antelope Dendrobium type and the hybrids between these two groups. Their natural flowering period is mainly concentrated in autumn, hence the name. Autumn Dendrobium refers to the Phalaenopsis type of Dendrobium hybridized with the Phalaenopsis type as the main parent. It has excellent characteristics such as beautiful flower shape, bright color and long vase period.

[0004] The market potential of spring Dendrobium hybrids is huge and can lead the trend of potted plants. However, the traditional artificial propagation methods of spring Dendrobium hybrids, such as division and cutting, are slow and have low proliferation rates, and it is difficult to meet the requirements of large-scale production. Dendrobium seedlings obtained through tissue culture technology divide quickly and have good growth consistency. Therefore, tissue culture technology has been widely used in the production of hybrid spring Dendrobium. The rapid propagation technology of seed tissue culture of spring Dendrobium hybrids has become a research hotspot.

[0005] However, the tissue culture of hybrid spring Dendrobium can easily cause vitrification of the protocorm and yellowing to death. It also has the problems of low survival rate and slow growth of tissue culture seedlings, which leads to difficulties in later maintenance and high planting and maintenance costs, limiting the production scale of hybrid spring Dendrobium. Therefore, improving the survival rate of hybrid spring Dendrobium seedlings transplanting plays an important role in production. Summary of the invention

[0006] The purpose of the present invention is to provide a tissue culture medium and a tissue culture method and application of hybrid Dendrobium, which can improve the transplant survival rate of hybrid Dendrobium tissue culture seedlings and obtain plants with good consistency and robustness.

[0007] In order to solve the above problems, the present invention provides the following technical solutions:

[0008] The present invention provides a tissue culture medium for hybrid dendrobium, wherein the tissue culture medium comprises a seed germination medium, a proliferation induction medium and a rooting and seedling strengthening medium;

[0009] The seed germination medium uses 1 / 2MS medium as the basic medium, and also includes: 25-35 g / L sucrose and 6.5-7.5 g / L agar;

[0010] The proliferation induction medium uses 2 / 3MS medium as the basic medium, and also includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 75-85 g / L potato juice, 25-35 g / L sucrose and 6.5-7.5 g / L agar;

[0011] The rooting and seedling strengthening culture medium uses 1 / 2MS culture medium as the basic culture medium and also includes the following additional components: 0.2-0.5 mg / L NAA, 65-75 g / L banana mud, 0.15-0.25 g / L activated carbon, 25-35 g / L sucrose and 6.5-7.5 g / L agar.

[0012] The present invention provides a tissue culture method for hybrid dendrobium, wherein the tissue culture method adopts the tissue culture medium described in the above technical solution, and comprises the following steps:

[0013] The hybrid Dendrobium explants are inoculated on a seed germination medium for germination culture to obtain a protocorm mass;

[0014] Inoculating the protocorm mass into a proliferation induction medium for proliferation induction culture to obtain clustered buds;

[0015] Inoculating the clustered buds into a rooting and seedling strengthening medium for seedling strengthening and rooting culture to obtain a complete hybrid Dendrobium tissue culture seedling plant;

[0016] The hybrid Dendrobium tissue culture seedlings are hardened and transplanted to obtain hybrid Dendrobium tissue culture seedlings that can be used for planting.

[0017] Preferably, the explant comprises seeds in hybrid Dendrobium capsules with a maturity of 80% to 90%.

[0018] Preferably, before taking out the seeds from the hybrid Dendrobium capsule, the capsule is also cleaned and disinfected;

[0019] The cleaning includes running water washing and detergent washing, the running water washing time is 10 to 20 minutes, the detergent washing time is 8 to 15 minutes, and after the detergent washing, sterile water washing and alcohol wiping are performed;

[0020] The disinfection method comprises: firstly soaking in alcohol with a mass concentration of 70% to 75% for 10 to 30 seconds, and rinsing with sterile water for 1 to 2 times; then soaking in a 10% to 25% sodium hypochlorite solution for 15 to 30 minutes; and finally rinsing with sterile water for 5 to 6 times; and adding 1 to 2 drops of Tween when soaking in the 10% to 25% sodium hypochlorite solution.

[0021] Preferably, before the protocorm mass is inoculated into the proliferation induction medium, the method further comprises cutting the protocorm mass into pieces of size of (3-5) mm×(3-5) mm before inoculation.

[0022] Preferably, the temperatures of the germination culture, proliferation induction culture and seedling rooting culture are 25-27°C respectively;

[0023] The germination culture, proliferation induction culture and seedling rooting culture are all carried out under light-dark alternation conditions, the illumination time in the light-dark alternation is 12-14h / d, the illumination intensity of the germination culture and proliferation induction culture is 1500-2000lx, and the illumination intensity of the seedling rooting culture is 2000-3000lx.

[0024] Preferably, the germination culture time is 28 to 32 days, the proliferation induction culture time is 60 to 240 days, and the seedling rooting culture time is 30 to 40 days.

[0025] Preferably, the proliferation induction culture process further includes 1 to 3 subcultures, and the subculture begins after 58 to 60 days of proliferation induction culture; the time of each subculture is 58 to 60 days, and the subculture medium is a proliferation induction medium.

[0026] Preferably, the seedling hardening is completed in the seedling hardening room of the tissue culture room, and the time of the seedling hardening is 9 to 11 days;

[0027] The transplanting is completed in a greenhouse, and the composition of the transplanting matrix includes perlite and bark; the mass ratio of the perlite to the bark is (1-2): (1-2).

[0028] The present invention provides the application of the tissue culture medium described in the above technical solution or the tissue culture method described in the above technical solution in improving the propagation efficiency and / or transplant survival rate of hybrid Dendrobium tissue culture seedlings.

[0029] The beneficial effects of the invention are as follows: the invention provides a tissue culture medium for hybrid dendrobium, comprising a seed germination medium, a proliferation induction medium and a rooting and seedling strengthening medium; the seed germination medium uses a 1 / 2MS medium as a basic medium, and further comprises: 25-35 g / L sucrose and 6.5-7.5 g / L agar; the proliferation induction medium uses a 2 / 3MS medium as a basic medium, and further comprises: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 75-85 g / L potato juice, 25-35 g / L sucrose and 6.5-7.5 g / L agar; the rooting and seedling strengthening medium uses a 1 / 2MS medium as a basic medium, and further comprises: 0.2-0.5 mg / L NAA, 65-75 g / L banana puree, 0.15-0.25 g / L activated carbon, 25-35 g / L sucrose and 6.5-7.5 agar.

[0030] In the tissue culture medium provided by the present invention, 6-BA (6-benzylaminopurine) stimulates cell division and promotes the growth and development of hybrid Dendrobium protocorms; NAA (1-naphthaleneacetic acid) promotes the growth of hybrid Dendrobium adventitious buds and roots; potato juice promotes the growth and development of protocorms, banana puree has a promoting effect on cell differentiation into seedlings in the seedling rooting culture, and the addition of activated carbon is beneficial to rooting. Under the joint action of 6-BA, NAA, potato juice, banana puree and activated carbon, it is beneficial to induce hybrid Dendrobium seed germination, protocorm proliferation and differentiation into clustered buds, obtain a large number of tissue culture seedlings, and then obtain plants with good consistency and robustness, improve the transplant survival rate of tissue culture seedlings, and the number of differentiated clustered buds is more, the germination rate is high, and the growth is better. The results of the embodiment show that the use of the tissue culture medium of the present invention can improve the transplant survival rate of hybrid Dendrobium tissue culture seedlings, and the transplant survival rate is more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0032] Figure 1 The aseptic germination of the hybrid spring Dendrobium officinale seeds of Dendrobium nobile × Dendrobium japonici on the 10th day of germination culture in Example 1;

[0033] Figure 2 This is the situation of the 30th day of germination and cultivation of the hybrid spring Dendrobium of Dendrobium nobile × Dendrobium jasminoides in Example 1;

[0034] Figure 3 This is the situation of the 60th day of proliferation induction culture of the hybrid Dendrobium nobile × Dendrobium jasminoides in Example 1;

[0035] Figure 4 This is the situation of the 40th day of cultivation of the hybrid spring Dendrobium of Dendrobium nobile × Dendrobium jasminoides in Example 1;

[0036] Figure 5 This is the situation of the hybrid spring Dendrobium nobile tissue culture seedlings of Dendrobium nobile × Dendrobium jasminoides in Example 1 on the 60th day after transplantation. DETAILED DESCRIPTION

[0037] The present invention provides a tissue culture medium for hybrid dendrobium, wherein the tissue culture medium comprises a seed germination medium, a proliferation induction medium and a rooting and seedling strengthening medium;

[0038] The seed germination medium uses 1 / 2MS medium as the basic medium, and also includes: 25-35 g / L sucrose and 6.5-7.5 g / L agar;

[0039] The proliferation induction medium uses 2 / 3MS medium as the basic medium, and also includes: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 75-85 g / L potato juice, 25-35 g / L sucrose and 6.5-7.5 g / L agar;

[0040] The rooting and seedling strengthening culture medium uses 1 / 2MS culture medium as the basic culture medium and also includes the following additional components: 0.2-0.5 mg / L NAA, 65-75 g / L banana mud, 0.15-0.25 g / L activated carbon, 25-35 g / L sucrose and 6.5-7.5 g / L agar.

[0041] In the present invention, the seed germination medium uses 1 / 2MS medium as the basic medium, and also includes: 25-35g / L sucrose and 6.5-7.5g / L agar; more preferably, 1 / 2MS medium is used as the basic medium, and only 25-35g / L sucrose and 6.5-7.5g / L agar are added. The concentration of the additional sucrose in the seed germination medium of the present invention is 25-35g / L, more preferably 30g / L; the concentration of the additional agar in the seed germination medium is 6.5-7.5g / L, more preferably 7g / L. In the present invention, the pH value of the seed germination medium after sterilization is preferably 5.8-6.2, and more preferably 5.8.

[0042] The seed germination culture medium of the invention does not need to add any other plant growth regulators, and the germination rate of hybrid spring dendrobium seeds can reach more than 90%.

[0043] In the present invention, the proliferation induction medium uses 2 / 3MS medium as the basic medium, and also includes the following additional ingredients: 0.1-0.3mg / L 6-BA, 0.1-0.2mg / LNAA, 75-85g / L potato juice, 25-35g / L sucrose and 6.5-7.5g / L agar; more preferably, the 2 / 3MS medium is used as the basic medium, and only the following additional ingredients: 0.1-0.3mg / L 6-BA, 0.1-0.2mg / LNAA, 75-85g / L potato juice, 25-35g / L sucrose and 6.5-7.5g / L agar.

[0044] The concentration of 6-BA added to the proliferation induction medium of the present invention is 0.1-0.3 mg / L, more preferably 0.2 mg / L; the concentration of NAA added to the proliferation induction medium is 0.1-0.2 mg / L, more preferably 0.1 mg / L.

[0045] The concentration of the potato juice added to the proliferation induction medium of the present invention is preferably 75-85 g / L, more preferably 80 g / L; the method for preparing potato juice of the present invention preferably comprises: selecting pest-free potatoes, peeling and weighing 80 g of potatoes, cutting into strips with a length of 5-10 cm and a width of 0.5-1 cm, boiling in water for 20-30 min, and filtering with gauze to obtain a filtrate, which is potato juice. The potato juice prepared by adding 80 g of potatoes to 1 L of proliferation induction medium.

[0046] In the present invention, the concentration of the additional sucrose in the proliferation induction medium is 25-35 g / L sucrose, more preferably 30 g / L; the concentration of the additional agar in the proliferation induction medium is preferably 6.5-7.5 g / L, more preferably 7 g / L.

[0047] In the present invention, the pH value of the proliferation induction medium after sterilization is preferably 5.8 to 6.2, more preferably 5.8. 6-BA and NAA in the proliferation induction medium of the present invention can jointly promote the proliferation of the protocorm mass to form adventitious buds at an appropriate concentration ratio, and the potato juice in the proliferation induction medium promotes the growth and development of the protocorm mass to obtain lush clustered buds.

[0048] In the present invention, the rooting and seedling strengthening culture medium uses 1 / 2MS culture medium as the basic culture medium, and also includes the following additional ingredients: 0.2-0.5mg / L NAA, 65-75g / L banana puree, 0.15-0.25g / L activated carbon, 25-35g / L sucrose and 6.5-7.5g / L agar; more preferably, 1 / 2MS culture medium is used as the basic culture medium, and only the following additional ingredients: 0.2-0.5mg / L NAA, 65-75g / L banana puree, 0.15-0.25g / L activated carbon, 25-35g / L sucrose and 6.5-7.5g / L agar. The concentration of NAA added to the rooting and seedling strengthening medium of the present invention is 0.2-0.5 mg / L, more preferably 0.5 mg / L; the concentration of banana puree added to the rooting and seedling strengthening medium is 65-75 g / L, more preferably 70 g / L; the concentration of activated carbon added to the rooting and seedling strengthening medium is 0.15-0.25 g / L, more preferably 0.2 g / L; the concentration of sucrose added to the proliferation induction medium is 25-35 g / L sucrose, more preferably 30 g / L; the concentration of agar added to the rooting and seedling strengthening medium is 6.5-7.5 g / L, more preferably 7 g / L. The banana puree in the rooting and seedling strengthening medium of the present invention promotes cell differentiation into seedlings, and NAA and activated carbon are conducive to rooting. In the present invention, the pH value of the rooting and seedling strengthening medium after sterilization is preferably 5.8-6.2, more preferably 5.8. The present invention has no special requirements for the preparation of the banana puree, and a conventional method can be used.

[0049] The 1 / 2MS culture medium of the present invention refers to a MS culture medium in which the content of macroelements is halved, while the content of trace elements, iron salts and organic matter components remains unchanged.

[0050] The 2 / 3MS mentioned in the present invention means that the content of the macroelements in the MS culture medium is reduced by 1 / 3 to 2 / 3 of the original content; the content of the trace elements, iron salts and organic matter components remains unchanged.

[0051] The proliferation induction medium provided by the present invention creatively adds potato juice to promote the growth and development of the protocorm mass and obtain lush clustered buds. The seed germination medium, proliferation induction medium and rooting and seedling medium provided by the present invention have high reproduction efficiency, and can obtain a large number of clustered buds and hybrid Dendrobium tissue culture seedlings. The obtained plants have good consistency and strong growth. In addition, the tissue culture medium provided by the present invention has good application effect and improves the transplant survival rate of hybrid spring Dendrobium tissue culture seedlings. In an embodiment of the present invention, the transplant survival rate of hybrid Dendrobium tissue culture seedlings reaches more than 90%.

[0052] The optimal pH value of the seed germination medium, proliferation induction medium and rooting and seedling strengthening medium provided by the present invention after sterilization is 5.8. Since the pH value of the medium will decrease after sterilization, the pH value of the medium is adjusted to 6.0-6.2 before being placed in the sterilizer after the medium is prepared.

[0053] The present invention has no special limitation on the sources of 6-BA, NAA, banana puree, activated carbon, sucrose and agar, and conventional commercial products can be used.

[0054] The present invention provides a tissue culture method for hybrid dendrobium, wherein the tissue culture method adopts the tissue culture medium described in the above technical solution, and comprises the following steps:

[0055] The hybrid Dendrobium explants are inoculated on a seed germination medium for germination culture to obtain a protocorm mass;

[0056] Inoculating the protocorm mass into a proliferation induction medium for proliferation induction culture to obtain clustered buds;

[0057] Inoculating the clustered buds into a rooting and seedling strengthening medium for seedling strengthening and rooting culture to obtain a complete hybrid Dendrobium tissue culture seedling plant;

[0058] The hybrid Dendrobium tissue culture seedlings are hardened and transplanted to obtain hybrid Dendrobium tissue culture seedlings that can be used for planting.

[0059] In the present invention, the hybrid dendrobium preferably includes hybrid spring dendrobium; the present invention has no special restrictions on the source of hybrid dendrobium, and the technical scheme of the present invention is suitable for hybrid dendrobiums well known to those skilled in the art. In the embodiment of the present invention, the hybrid spring dendrobium obtained by Dendrobium nobile × Dendrobium jasminoides is used as an example for verification. The explant preferably includes seeds in hybrid dendrobium capsules with a maturity of 80% to 90%, and more preferably includes seeds in hybrid spring dendrobium capsules with a maturity of 80% to 90%.

[0060] In the present invention, after collecting the hybrid Dendrobium capsule, it is preferred to clean and disinfect the hybrid Dendrobium capsule. The present invention preferably includes: running water washing and detergent washing, the running water washing time is 10 to 20 minutes, the detergent washing time is 8 to 15 minutes, and the detergent washing is followed by sterile water washing and alcohol wiping. The running water washing time of the present invention is preferably 10 to 20 minutes, more preferably 15 minutes; the detergent washing time is preferably 8 to 15 minutes, more preferably 10 minutes. In the present invention, the detergent is preferably one or more of washing powder, hand soap and soap, further preferably one or two of washing powder and soap, more preferably washing powder. The number of sterile water washings of the present invention is preferably until there is no foam in the capsule; the number of alcohol wipings is preferably once.

[0061] After cleaning, the present invention preferably disinfects the capsule, and the disinfection method of the present invention preferably includes: first soaking in 70% to 75% alcohol by mass concentration for 10 to 30 seconds, and rinsing with sterile water 1 to 2 times; then soaking in 10% to 25% sodium hypochlorite solution for 15 to 30 minutes; finally rinsing with sterile water 5 to 6 times; adding 1 to 2 drops of Tween during soaking in the 10% to 25% sodium hypochlorite solution. The volume concentration of the alcohol of the present invention is preferably 70% to 75%, more preferably 75%; the alcohol soaking time is preferably 10 to 30 seconds, more preferably 12 to 26 seconds, and more preferably 20 seconds; after alcohol soaking, preferably rinse with sterile water 1 to 2 times, more preferably 2 times.

[0062] The concentration of sodium hypochlorite in the sodium hypochlorite solution of the present invention is preferably 10% to 25%, more preferably 25%; the soaking time of the sodium hypochlorite solution is preferably 15 to 30 minutes, more preferably 18 to 25 minutes, more preferably 20 minutes; after soaking in the sodium hypochlorite solution, it is preferably rinsed with sterile water 5 to 6 times, more preferably 6 times. The present invention has no special restrictions on sterile water, preferably obtained by sterilizing distilled water; the amount of the alcohol and sodium hypochlorite solution is preferably enough to submerge the capsule. The present invention uses Tween as a surfactant to enhance the sterilization effect of sodium hypochlorite. Sodium hypochlorite is used as a disinfectant, which is easy to rinse and remove, has a small residual amount, has a good disinfection effect, and has little effect on the tissue culture and breeding of capsules after disinfection.

[0063] After the capsule is cleaned and disinfected, the present invention preferably takes out the seeds in the capsule and inoculates them on a seed germination medium for germination culture to obtain a protocorm mass.

[0064] The present invention uses seeds in hybrid dendrobium capsules as explants for tissue culture rapid propagation of seedlings. In the embodiment of the present invention, since the amount of hybrid spring dendrobium seeds is extremely large, one capsule contains 6000 to 8000 seeds, and there is no need to go through the dedifferentiation and redifferentiation stages, a large number of seedlings can be obtained in a short time, shortening the culture time of tissue culture seedlings.

[0065] In the present invention, the temperature of the germination culture is preferably 25-27°C, more preferably 26°C; the germination culture is preferably carried out under alternating light and dark conditions, and the illumination time in the alternating light and dark is preferably 12-14h / d, more preferably 12h / d; the intensity of the illumination is preferably 1500-2000lx, more preferably 1800lx; the time of the germination culture is preferably 28-32d, more preferably 30d; the humidity of the germination culture is preferably 60%-80%, more preferably 70%.

[0066] After obtaining the protocorm mass, the present invention inoculates the protocorm mass into a proliferation induction culture medium for proliferation induction culture to obtain clustered buds.

[0067] The present invention preferably includes cutting the protocorm mass into pieces of (3-5) mm×(3-5) mm in size before inoculating the protocorm mass into a proliferation induction medium.

[0068] In the present invention, the temperature of the proliferation induction culture is preferably 25-27°C, more preferably 26°C; the proliferation induction culture is preferably carried out under alternating light and dark conditions, and the illumination time in the alternating light and dark is preferably 12-14h / d, more preferably 12h / d; the intensity of the illumination is preferably 1500-2000lx, more preferably 1700lx; the humidity of the proliferation induction culture is preferably 60%-80%, more preferably 70%.

[0069] If the demand for hybrid Dendrobium cluster buds is large, subculture can be carried out. The proliferation induction culture process of the present invention preferably also includes multiple subcultures. When subculture is included, the time of the proliferation induction culture of the present invention is preferably 90 to 240 days, and more preferably 120 to 180 days. The subculture of the present invention preferably starts after the 58th to 60th day of the proliferation induction culture, and more preferably starts after the 60th day; the time of each subculture of the present invention is preferably 58 to 60 days, and more preferably 60 days. The number of subcultures in the proliferation induction culture process of the present invention preferably also includes 1 to 3 subcultures, and more preferably 2 times. When the proliferation induction culture process preferably also includes 1 subculture, the time of the proliferation induction culture is preferably 120 days. When the proliferation induction culture process preferably also includes 2 subcultures, the time of the proliferation induction culture is preferably 180 days. When the proliferation induction culture process preferably also includes 3 subcultures, the time of the proliferation induction culture is preferably 240 days. The subculture culture medium of the present invention is preferably a proliferation induction culture medium. The subculture of the present invention is to carry out continuous multi-generation culture by replacing fresh culture medium and continuously cutting or separating. The function of the subculture of the present invention is to obtain more luxuriant clustered buds of hybrid Dendrobium. If the demand for clustered buds of hybrid Dendrobium is small, subculture may not be carried out. When subculture is preferably not included in the proliferation induction culture process, the time of the proliferation induction culture is preferably 60d.

[0070] After obtaining the clustered buds, the present invention inoculates the clustered buds into a rooting and seedling strengthening medium for seedling strengthening and rooting culture to obtain a complete hybrid Dendrobium tissue culture seedling plant.

[0071] In the present invention, the temperature of the seedling rooting culture is preferably 25-27°C, more preferably 26°C; the seedling rooting culture is preferably carried out under alternating light and dark conditions, and the illumination time in the alternating light and dark is preferably 12-14h / d, more preferably 12-13h / d; the illumination intensity is preferably 2000-3000lx, more preferably 2500lx; the humidity of the seedling rooting is preferably 60%-80%, more preferably 70%; the seedling rooting culture time is preferably 30-40d.

[0072] After the strong seedling rooting culture of the invention, the hybrid dendrobium tissue culture seedlings are obtained. After the height of the tissue culture seedlings reaches 6 cm and the number of leaves is greater than 3 or the number of roots is 3, the seedling hardening transplanting and transplanting culture are carried out.

[0073] In the present invention, the seedling hardening is preferably carried out in a seedling hardening room in a tissue culture room, and the time for the seedling hardening is preferably 9 to 11 days, more preferably 10 days.

[0074] In the present invention, the transplanting is preferably completed in a greenhouse, and during the transplanting culture, the roots of the tissue culture seedlings after hardening are preferably washed, diseased plants are removed, the roots are disinfected and dried, and then transplanted into the substrate for culture. The root disinfection of the present invention is preferably carried out using one of carbendazim solution, ricinus, kresoxim-methyl, polymyxin and frostbite. In an embodiment of the present invention, the root disinfection is preferably carried out using a carbendazim solution. The carbendazim solution of the present invention is preferably a solution obtained by diluting carbendazim and water at a volume ratio of 1:1000.

[0075] The composition of the matrix used in the transplanting of the present invention is preferably perlite and bark; the mass ratio of the perlite and bark is preferably (1-2): (1-2), more preferably 1:1; the pH value of the matrix used in the transplanting of the present invention is preferably 5.8-6.2, more preferably 5.8. The transplanting is preferably carried out at a temperature of 20-25°C and between 10 and 12 o'clock in the morning. Before transplanting and culturing, the present invention preferably disinfects the matrix, and the present invention does not specifically limit the method of disinfection. In the embodiment of the present invention, 1.0g / L carbendazim solution is used to disinfect the matrix.

[0076] After the transplanting, the present invention preferably performs conventional management on the tissue culture seedlings to obtain hybrid Dendrobium plants. In the present invention, the conventional management preferably includes spraying potassium dihydrogen phosphate foliar fertilizer after the tissue culture seedlings are rooted in the matrix 30 days after transplanting; the potassium dihydrogen phosphate foliar fertilizer is preferably sprayed once every half a month, for a total of two times. The preparation method of the potassium dihydrogen phosphate foliar fertilizer of the present invention is preferably obtained by mixing 100g of potassium dihydrogen phosphate with 30kg of water.

[0077] The conventional management of the present invention preferably includes spraying carbendazim solution regularly 10 days after transplanting. The number of times the carbendazim solution of the present invention is sprayed is preferably 2 times, once every 10 days. The carbendazim solution of the present invention is preferably obtained by diluting carbendazim and water at a volume ratio of 1:1000.

[0078] The present invention provides application of the tissue culture medium described in the above technical solution or the tissue culture method described in the above technical solution in improving the survival rate of hybrid Dendrobium tissue culture seedlings after transplantation.

[0079] The hybrid dendrobium tissue culture seedlings obtained by the invention have a high transplant survival rate of more than 90%. The tissue culture medium and tissue culture conditions of the invention improve the transplant survival rate of the hybrid dendrobium tissue culture seedlings.

[0080] The present invention provides application of the tissue culture medium described in the above technical solution or the tissue culture method described in the above technical solution in improving the propagation efficiency of hybrid Dendrobium tissue culture seedlings.

[0081] The hybrid dendrobium tissue culture seedlings obtained by the present invention have a short culture time of only 6 to 8 months. In the embodiment of the present invention, the capsule seeds of the hybrid spring dendrobium obtained by Dendrobium nobile × Dendrobium jasminoides only take six months and 10 days from seed sowing to the end of rooting culture and the tissue culture seedlings are taken out of the bottle, and the culture time is short and the reproduction efficiency is high. The present invention improves the reproduction efficiency of the hybrid dendrobium tissue culture seedlings under the joint action of the tissue culture medium and the tissue culture conditions.

[0082] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0083] The hybrid spring Dendrobium capsules of Example 1 and Comparative Examples 1 to 3 were collected in the same batch.

[0084] Example 1

[0085] The present invention provides a tissue culture method for hybrid spring dendrobium, which comprises the following steps: explant selection, cleaning and disinfection, seed germination, protocorm induction, proliferation and cluster bud induction, seedling and rooting culture, and acclimatization and transplanting. The specific steps are as follows:

[0086] (1) Explant selection: On July 25, 2021, seeds from full hybrid spring Dendrobium capsules with a maturity of 80% to 90% were collected as explants. The hybrid spring Dendrobium was obtained from Dendrobium nobile × Dendrobium candidum, and the seeds from its capsules were used as explants.

[0087] (2) Cleaning and disinfection of explants:

[0088] Cut the hybrid spring Dendrobium capsules from the hybrid spring Dendrobium plants, shave off the dirt on the surface of the capsules in the cleaning room outside the tissue culture room, rinse with running tap water for 20 minutes, soak and scrub with 5% laundry detergent solution for 10 minutes, rinse with sterile water several times until there is no foam, and then wipe it with alcohol. After the treatment is completed, take it to the inoculation room.

[0089] On a clean bench or in an inoculation box, immerse the capsule in 75% alcohol for 30 seconds, rinse once with sterile water, and then immerse in a 10% sodium hypochlorite solution for surface disinfection for 30 minutes. After disinfection, rinse five times with sterile water until there is no foam. When disinfecting with sodium hypochlorite solution, add 1 to 2 drops of Tween to the solution.

[0090] After disinfection, place the capsule on sterilized filter paper to absorb excess water for later use. Disinfection with sodium hypochlorite, the residual sodium hypochlorite has little effect on seed germination and the disinfectant is easy to rinse off.

[0091] (3) Seed germination culture: On July 25, 2021, the capsules of step (2) after disinfection were cut open on a sterilized steel plate, and a small amount of seeds were taken out and inoculated into the seed germination medium for culture to obtain the protocorm mass. The composition of the seed germination medium is: 1 / 2MS as the basic culture medium, and only contains 30g / L sucrose and 7g / L agar, and the pH value of the culture medium is 5.8. The temperature of seed culture is 27°C, the humidity is 70%, the light intensity is 1600lx, the light cycle is 12h / d, and the culture is 30d to obtain the protocorm mass. The composition of the 1 / 2MS culture medium is: KNO3950mg·L -1 ; NH4NO3825 mg·L -1 ;CaCl2·2H2O 220mg·L -1 ;MgSO4·7H2O 185mg·L -1 ; KH2PO485 mg·L -1 ;FeSO4·7H2O 5.56g·L -1 ;Na2-EDTA7.46g·L -1 ; MnSO4·4H2O 22.3mg·L -1 ; ZnSO4·7H2O 8.6mg·L -1 ;CuSO4·5H2O 0.025mg·L -1 ; H3BO36.2 mg·L -1 ;Na2MoO4·2H2O 0.25mg·L -1 ; KI 0.83mg·L -1 ; CoCl2·6H2O 0.025mg·L -1 ; Vitamin B1 0.1 mg·L -1; Vitamin B6 0.5 mg·L -1 ; Niacin 0.5mg·L -1 ; Glycine 2.0mg·L -1 and inositol 100mg·L -1 Seed germination culture on the 10th day Figure 1 Seed germination culture 30 days Figure 2 .

[0092] (4) Protocorm induction, proliferation and cluster bud induction culture (i.e. proliferation induction culture): On August 25, 2021, the protocorm group obtained in step (3) was inoculated into the proliferation induction medium (proliferation induction medium i.e. protocorm proliferation cluster bud induction medium). The composition of the proliferation induction medium is: 2 / 3MS as the basic culture medium, and only contains 0.2mg / L6-BA, 0.1mg / LNAA, 80g / L potato juice, 30g / L sucrose and 7g / L agar. The pH of the culture medium is 5.8. The temperature for the protocorm proliferation cluster bud induction culture is 25-27°C, the humidity is 60%-80%, the light intensity is 1500-2000lx, and the light cycle is 12h / d. After 60 days of culture, transfer once according to the situation to obtain lush cluster buds. On the 60th day of culture, see Figure 3 ; Proliferation induction culture for 120 days. The composition of 2 / 3MS medium is: KNO31266.7 mg·L -1 ; NH4NO3 1100 mg·L -1 ;CaCl2·2H2O 293.3mg·L -1 ;MgSO4·7H2O 246.7mg·L -1 ;KH2PO4113.3 mg·L -1 ;FeSO4·7H2O 5.56g·L -1 ;Na2-EDTA7.46g·L -1 ; MnSO4·4H2O 22.3mg·L -1 ; ZnSO4·7H2O 8.6mg·L -1 ;CuSO4·5H2O 0.025mg·L -1 ; H3BO36.2mg·L -1 ;Na2MoO4·2H2O 0.25mg·L -1 ; KI 0.83mg·L -1 ; CoCl2·6H2O 0.025mg·L -1 ; Vitamin B10.1 mg·L -1 ; Vitamin B6 0.5mg·L -1 ; Niacin 0.5mg·L -1 ; Glycine 2.0mg·L-1 and inositol 100mg·L -1 .

[0093] (5) Rooting and seedling cultivation: On December 27, 2021, the clustered buds obtained in step (4) were transferred to the rooting and seedling cultivation medium. The composition of the rooting and seedling cultivation medium is: 1 / 2MS as the basic culture medium, and only contains 0.5mg / LNAA, 70g / L banana puree, 30g / L sucrose, 7g / L agar and 0.2g / L activated carbon. The pH of the culture medium is 5.8. After 39 days of rooting and seedling cultivation, the plant height is 6cm, the number of leaves is greater than 3 or the number of roots is 3, and the clustered buds take root to form complete hybrid spring Dendrobium test tube seedling tissue culture seedlings. The temperature for rooting and seedling cultivation is 27°C, the humidity is 70%, the light intensity is 3000lx, and the light cycle is 12h / d. See on the 40th day of cultivation. Figure 4 .

[0094] (6) Hardening and transplanting: On February 4, 2022, the rooted hybrid spring Dendrobium tissue culture seedlings obtained in step (5) were placed in a hardening room with bottles for 10 days and then transplanted to a greenhouse. Transplanting was carried out at 10-12 am at a temperature of 20-25°C. The temperature of the greenhouse was 24°C and the humidity was 70%. The transplanting culture was carried out on the 60th day. Figure 5 .

[0095] During the transplanting process, it is easy to be infected with bacterial soft rot. First, remove the diseased plants of hybrid spring Dendrobium tissue culture seedlings, and then soak the normal hybrid spring Dendrobium tissue culture seedlings in carbendazim solution for 30 minutes, then dry and transplant them. The carbendazim solution is obtained by diluting carbendazim and water in a volume ratio of 1:1000.

[0096] The transplanting matrix used in the greenhouse is a mixed matrix. The mass ratio of perlite to bark in the mixed matrix is ​​1:1, and the pH value of the mixed matrix is ​​5.8-6.2. The mixed matrix should be disinfected with 1.0g / L carbendazim solution before transplanting.

[0097] Spraying the plant regularly 10 days after transplanting, once every 10 days, twice in total, can make the hybrid spring Dendrobium tissue culture seedlings grow well. The sprayed drug is carbendazim solution.

[0098] After 30 days of transplanting, potassium dihydrogen phosphate foliar fertilizer solution was sprayed once every half a month. The potassium dihydrogen phosphate foliar fertilizer solution was obtained by adding 100 grams of potassium dihydrogen phosphate to 30 kg of water. The potassium dihydrogen phosphate foliar fertilizer solution was sprayed twice in total. The survival rate of hybrid spring Dendrobium tissue culture seedlings was investigated after 60 days of transplanting and cultivation.

[0099] The survival rate of the hybrid spring Dendrobium tissue culture seedlings of Example 1 was 94%.

[0100] Comparative Example 1

[0101] (1) Explant selection: Same as Example 1.

[0102] (2) Cleaning and disinfection of explants: Same as in Example 1.

[0103] (3) Seed germination culture: Same as Example 1.

[0104] Comparative Example 1 was only carried out to seed germination culture. The only difference between seed germination culture and Example 1 is that the basic culture medium in the culture medium formula is not 1 / 2MS, but a standard MS culture medium is used, and the rest of the steps are exactly the same as Example 1. Specifically, the seed germination culture medium is composed of: MS as the basic culture medium, and only contains 30g / L sucrose and 7g / L agar, and the pH value of the culture medium is 5.8.

[0105] The seed germination conditions of Example 1 and Comparative Example 1 after inoculation and culture for 10 days and 20 days were recorded respectively. After the hybrid spring Dendrobium seeds were inoculated and cultured in the culture medium, the seeds were in powder form and a large number of them could not be counted accurately. The approximate number of seed germination rates was obtained by visual observation. The approximate number of seed germination rates is shown in Table 1. According to Table 1, Example 1 uses 1 / 2MS as the basic culture medium, and the number of days used for seed germination is shorter, the germination rate is higher, and the growth is more robust.

[0106] Table 1 Germination of hybrid spring dendrobium seeds of Example 1 and Comparative Example 1

[0107]

[0108] Comparative Example 2

[0109] (1) Explant selection: Same as Example 1.

[0110] (2) Cleaning and disinfection of explants: Same as in Example 1.

[0111] (3) Seed germination culture: Same as Example 1.

[0112] (4) Protocorm induction, proliferation and cluster bud induction culture (i.e., proliferation induction culture): The only difference between the proliferation induction culture and Example 1 is that potato juice is not added to the culture medium formula, and the rest is exactly the same as Example 1. Specifically, the cluster bud proliferation induction culture uses 2 / 3MS as the basic culture medium and only contains 0.2 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose and 7 g / L agar. The culture medium pH is 5.8. Comparative Example 2 only performs proliferation induction culture.

[0113] Comparative Example 3

[0114] (1) Explant selection: Same as Example 1.

[0115] (2) Cleaning and disinfection of explants: Same as in Example 1.

[0116] (3) Seed germination culture: Same as Example 1.

[0117] (4) Protocorm induction, proliferation and cluster bud induction culture (i.e., proliferation induction culture): The only difference between the proliferation induction culture and Example 1 is that the plant growth regulator in the culture medium formula is IAA, and the rest is exactly the same as Example 1. Specifically, the proliferation induction culture is based on 2 / 3MS as the basic culture medium, and only contains 0.2 mg / L 6-BA, 0.1 mg / L IAA, 80 g / L potato juice, 30 g / L sucrose and 7 g / L agar. The culture medium pH is 5.8. Comparative Example 3 only carried out proliferation induction culture.

[0118] The induction of clustered buds 120 days after inoculation of Example 1 and Comparative Examples 2-3 was recorded respectively, as shown in Table 2. According to Table 2, compared with Comparative Examples 2 and 3, Example 1 has more clustered buds differentiated, a higher germination rate, and better growth potential. The calculation formula of the germination rate is: germination rate = number of buds / number of inoculated protocorms × 100%.

[0119] Table 2: Induction of clustered buds 120 days after inoculation in Example 1 and Comparative Examples 2-3

[0120]

[0121] Comparative Example 4

[0122] (1) Explant selection: Same as Example 1.

[0123] (2) Cleaning and disinfection of explants: Same as in Example 1.

[0124] (3) Seed germination culture: Same as Example 1.

[0125] (4) Protocorm induction, proliferation and cluster bud induction culture (i.e., proliferation induction culture): Same as Example 1.

[0126] (5) Rooting and seedling cultivation: The only difference between the rooting and seedling cultivation and Example 1 is that the plant growth regulator in the culture medium is IBA, and the other steps are exactly the same as Example 1. Specifically, 1 / 2MS is used as the basic culture medium, and only 0.5 mg / L IBA, 70 g / L banana puree, 30 g / L sucrose, 7 g / L agar, and 0.2 g / L activated carbon are contained.

[0127] (6) Hardening and transplanting: Same as in Example 1.

[0128] The rooting and seedling growth conditions of Example 1 and Comparative Example 4 at 39 days after inoculation into the rooting and seedling growth medium are shown in Table 3. The growth conditions of Example 1 and Comparative Example 4 at 15 days and 60 days after transplanting are shown in Table 4. According to Tables 3 and 4, it can be seen that Example 1 uses 0.5 mg / L NAA in the rooting medium formula, and the rooting quality is higher, the plants are stronger, and the transplanting survival rate is higher.

[0129] Table 3 Rooting and strengthening of seedlings in Example 1 and Comparative Example 4 on the 39th day of cultivation

[0130] Group Number of inoculations (strains) Average number of roots Average root length (cm) Average plant height (cm) Growth Example 1 100 4 8 6 Thick root Comparative Example 4 100 2 3 4 Root thin

[0131] Table 4 Growth conditions of Example 1 and Comparative Example 4 at 15d and 60d after transplanting

[0132]

[0133] In summary, the hybrid Dendrobium officinale in the culture medium of the present invention has a high germination rate, a larger number of differentiated clustered buds, a high germination rate, better growth, higher rooting quality, and stronger plants.

[0134] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture medium for hybrid Dendrobium, characterized in that: The tissue culture medium includes a seed germination medium, a proliferation induction medium and a rooting and seedling strengthening medium; The seed germination medium uses 1 / 2MS medium as the basic medium, and also includes: 25-35 g / L sucrose and 6.5-7.5 g / L agar; The proliferation induction medium uses 2 / 3MS medium as the basic medium, and also includes the following additional ingredients: 0.1-0.3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 75-85 g / L potato juice, 25-35 g / L sucrose and 6.5-7.5 g / L agar; The rooting and seedling strengthening culture medium uses 1 / 2MS culture medium as the basic culture medium and also comprises the following additional components: 0.2-0.5 mg / L NAA, 65-75 g / L banana mud, 0.15-0.25 g / L activated carbon, 25-35 g / L sucrose and 6.5-7.5 g / L agar.

2. A tissue culture method for hybrid Dendrobium, characterized in that: The tissue culture method adopts the tissue culture medium according to claim 1, comprising the following steps: The hybrid Dendrobium explants are inoculated on a seed germination medium for germination culture to obtain a protocorm mass; Inoculating the protocorm mass into a proliferation induction medium for proliferation induction culture to obtain clustered buds; Inoculating the clustered buds into a rooting and seedling strengthening medium for seedling strengthening and rooting culture to obtain a complete hybrid Dendrobium tissue culture seedling plant; The hybrid Dendrobium tissue culture seedlings are hardened and transplanted to obtain hybrid Dendrobium tissue culture seedlings that can be used for planting.

3. The tissue culture method according to claim 2, characterized in that The explants include seeds in hybrid dendrobium capsules with a maturity of 80% to 90%.

4. The tissue culture method according to claim 3, characterized in that Before taking out the seeds from the hybrid Dendrobium capsule, the capsule is also cleaned and disinfected; The cleaning includes running water washing and detergent washing, the running water washing time is 10 to 20 minutes, the detergent washing time is 8 to 15 minutes, and after the detergent washing, sterile water washing and alcohol wiping are performed; The disinfection method comprises: firstly soaking in alcohol with a mass concentration of 70% to 75% for 10 to 30 seconds, and rinsing with sterile water for 1 to 2 times; then soaking in a 10% to 25% sodium hypochlorite solution for 15 to 30 minutes; and finally rinsing with sterile water for 5 to 6 times; and adding 1 to 2 drops of Tween when soaking in the 10% to 25% sodium hypochlorite solution.

5. The tissue culture method according to claim 2, characterized in that Before the protocorm mass is inoculated into the proliferation induction medium, the method further includes cutting the protocorm mass into pieces with a size of (3-5) mm×(3-5) mm and then inoculating the pieces.

6. The tissue culture method according to claim 2, characterized in that The temperatures of the germination culture, proliferation induction culture and seedling rooting culture are 25-27°C respectively; The germination culture, proliferation induction culture and seedling rooting culture are all carried out under light-dark alternation conditions, the illumination time in the light-dark alternation is 12-14h / d, the illumination intensity of the germination culture and proliferation induction culture is 1500-2000lx, and the illumination intensity of the seedling rooting culture is 2000-3000lx.

7. The tissue culture method according to claim 2, characterized in that: The germination culture time is 28 to 32 days, the proliferation induction culture time is 60 to 240 days and the seedling rooting culture time is 30 to 40 days.

8. The tissue culture method according to claim 2 or 7, characterized in that: The proliferation induction culture process also includes 1 to 3 subcultures, and the subculture begins after the 58th to 60th day of the proliferation induction culture; the time of each subculture is 58 to 60 days, and the subculture medium is the proliferation induction medium.

9. The tissue culture method according to claim 2, characterized in that: The seedling hardening is completed in the seedling hardening room of the tissue culture room, and the time of the seedling hardening is 9 to 11 days; The transplanting is completed in a greenhouse, and the composition of the transplanting matrix includes perlite and bark; the mass ratio of the perlite to the bark is (1-2): (1-2).

10. Use of the tissue culture medium according to claim 1 or the tissue culture method according to any one of claims 2 to 9 in improving the propagation efficiency and / or transplant survival rate of hybrid Dendrobium tissue culture seedlings.

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