A tissue culture rapid propagation method for hybrid Primula

Abstract

The present invention relates to a tissue culture rapid propagation method for hybrid primula, and the method comprises the following steps: disinfection and sterilization of explants; induction culture; proliferation culture; rooting medium for strong seedlings and planting out of bottles; and transplanting of tissue culture seedlings. The growth cycle of the entire regeneration system in the present invention is about 100 days. The transplant survival rate of the hybrid primula tissue culture seedlings bred by the present invention reaches more than 95%, and the rooting rate and survival rate are very high, which can provide high-quality seedlings for large-scale planting.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a tissue culture rapid propagation method of hybrid Primula vera. Background Art

[0002] Gesneriaceae plants are rich in species, including many genera, the more common ones include Primula, Radix Gesneriae, Gesneriae Myrrha, etc., which have high ornamental value and are very popular ornamental flowers in the world in recent years. China has rich resources of Gesneriaceae plants, many types, and outstanding characteristics, but the development of their ornamental value is relatively small.

[0003] Primula is a highly ornamental group in the Gesneriaceae family, with a distribution center in the karst limestone areas of southern my country, including Guangxi, Guangdong, and Hainan. Outside of China, only Vietnam has more than a dozen species. Its flower colors are rich and varied, and some species have white stripes on the leaves. Most of them like shade and are easy to cultivate. They have high horticultural ornamental value and can be used as indoor potted flowers. They are a good new native flower resource in my country.

[0004] The propagation methods of Primulina plants are mainly divided into two categories, one is traditional sowing propagation; the other is asexual propagation, mainly including cuttings, division and tissue culture. Seed propagation of Primulina is limited by seed sources, small seeds that are difficult to separate, and an imperfect seed production system. It is not easy to achieve standardized production, and the management of seedling division and transplanting after germination is relatively complicated. Cutting propagation is easy to operate and manage, and is currently a commonly used propagation method for Primulina plants, but it still has problems such as long production cycle and low propagation coefficient. Efficient and stable tissue culture rapid propagation technology is an effective way to mass-produce seedlings. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide a tissue culture rapid propagation method for hybrid Primula, and the technical problem to be solved is to use tissue culture rapid propagation to make the transplant survival rate of Primula tissue culture seedlings reach more than 98%.

[0006] The purpose of the present invention and the technical problem to be solved are achieved by adopting the following technical solutions. The present invention proposes a tissue culture rapid propagation method of hybrid primrose, comprising the following steps:

[0007] 1) Disinfection and sterilization of explants

[0008] Soak the newly grown 2-3 cm long young leaves at the top of the stems of pest-free plants in a diluted detergent solution for 3-4 minutes, rinse under running water for 30 minutes, and then sterilize them.

[0009] 2) Induction culture

[0010] The leaves obtained in step 1) are cut into a size of 0.5-1 cm×0.5-1 cm, and are laid flat with the leaves facing upward and the back facing downward into the induction medium and cultured for 30-40 days;

[0011] 3) Proliferation culture: the adventitious buds with 2-3 leaves obtained in step 2) are cut off together with the explant leaves below, and then the leaves are placed flat and transferred to the proliferation medium for culture for 20-30 days;

[0012] 4) inoculating the leaves obtained in step 3) onto a seedling rooting medium and culturing for 20-30 days, and inoculating the leaves onto a seedling rooting medium again when roots grow and culturing for 20-30 days, and removing the leaves from the bottle and planting after roots of 2-3 cm grow;

[0013] 5) Transplantation of tissue culture seedlings: Take out the seedlings obtained in step 4) from the culture bottle, wash off the culture medium, and transfer them to a 10*10 cm nutrient pot.

[0014] The purpose of the present invention and the technical problems to be solved can be further achieved by adopting the following technical measures.

[0015] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 1), the sterilization operation includes: first soaking the leaves in a 75% (v / v) ethanol solution for 30-40s, and rinsing with sterile water 3-4 times; then soaking and sterilizing with a 2wt% NaClO solution for 2-3min, and rinsing with sterile water 3-5 times after sterilization.

[0016] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 2), the composition of the induction medium is: MS is used as the basic medium, and 0.1-0.5 mg·L -1 NAA and 0.5-1.0 mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization.

[0017] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 2), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light duration is 15-16 h·d. -1 .

[0018] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 3), the composition of the proliferation medium is: MS is used as the basic medium, and 0.1-1.5 mg·L -1NAA and 0.5-2mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization.

[0019] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 3), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light duration is 15-16 h·d. -1 .

[0020] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primulina, in step 4), the composition of the seedling rooting medium is: 1 / 2MS as the basic medium, supplemented with 28-30 g·L sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization.

[0021] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 4), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light duration is 15-16 h·d. -1 .

[0022] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 5), the cultivation medium in the nutrient pot is exposed to sunlight for 1 day in advance and is watered thoroughly in advance.

[0023] Preferably, in the aforementioned tissue culture rapid propagation method of hybrid Primula, in step 5), the cultivation medium in the nutrient pot is composed of peat, vermiculite and perlite in a mass ratio of (5-6): (2-3): 2.

[0024] Compared with the prior art, the tissue culture rapid propagation method of hybrid Primula serrata described in the present invention has the following beneficial effects:

[0025] The invention utilizes plant tissue culture technology to carry out tissue culture of hybrid primula, and establishes a tissue culture rapid propagation system and method of hybrid primula.

[0026] The method of the invention is simple, easy to implement and economical; it shortens the seedling raising period, saves costs and saves outdoor seedling raising space.

[0027] The growth cycle of the entire regeneration system in the present invention is about 100 days, the survival rate of explant disinfection and sterilization is more than 85%, the differentiation rate of adventitious bud induction culture is about 90%, and the rooting rate of tissue culture seedlings is more than 95%. The transplant survival rate of hybrid primula tissue culture seedlings bred by the present invention reaches more than 95%, which can provide high-quality seedlings for large-scale planting.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. DETAILED DESCRIPTION

[0029] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation method, structure, characteristics and effects of the tissue culture rapid propagation method of hybrid primrose according to the present invention are described in detail below in combination with the preferred embodiments. In the following description, different "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments may be combined in any suitable form.

[0030] Unless otherwise specified, the materials, reagents, etc. involved below are all commercially available products well known to those skilled in the art; unless otherwise specified, the methods described are all methods known in the art. Unless otherwise defined, the technical terms or scientific terms used should have the usual meanings understood by ordinary technicians in the field to which the present invention belongs. If no specific experimental steps or conditions are specified below, the operations or conditions of the conventional experimental steps described in the literature in the field can be followed.

[0031] Some embodiments of the present invention provide a method for rapid tissue culture propagation of hybrid Primula, comprising the following steps:

[0032] 1) Disinfection and sterilization of explants

[0033] Take the young leaves about 2-3 cm long that have just grown from the top of the stem of a plant without disease and insect pests, soak them in a detergent diluent (the detergent diluent is prepared by the following steps: 1-2 ml of Omo detergent is mixed evenly with about 200 ml of water; experimental tests show that if it is greater than 2 ml, the detergent is too much and the rinsing is not clean, affecting the subsequent sterilization; if it is less than 1 ml, the detergent is too little and the sterilization effect is poor) for 3-4 minutes (experimental tests show that the leaf contamination rate is as high as 80% when it is less than 3 minutes, and the leaf mortality rate increases when it is greater than 5 minutes), then rinse under running water for 30-40 minutes, if it is less than 30 minutes, the detergent cannot be rinsed clean, and if it is greater than 40 minutes, the subsequent browning rate of the leaves increases; then transfer to an ultra-clean workbench for sterilization. First, soak the leaves in 75% (v / v) ethanol solution for 30-40s, and rinse with sterile water 3-4 times; experimental tests show that if it is more than 4 times, the leaves are prone to browning due to too many rinses, and if it is less than 3 times, the alcohol attached to the surface of the leaves may not be rinsed clean enough; then soak and sterilize with 2-5wt% NaClO solution (experimental tests show that if it is greater than 5wt%, the concentration is too high and the leaves cannot survive, and if it is less than 2wt%, the sterilization effect is poor), and the sterilization time is 2-3min (the gradient experiment sets three gradients of 2min, 2min30s, and 3min. The time varies according to the explant leaves. The survival rate of young leaves is the highest when the sterilization time is 2min, and the survival rate of mature leaves is the highest when the sterilization time is 3min; after sterilization, rinse with sterile water for 3-5 Times. Experimental tests have shown that if it is more than 5 times, the leaves will turn brown too many times. If it is less than 3 times, the sodium hypochlorite attached to the surface of the leaves may not be rinsed cleanly. During the sterilization process, gently shake the leaves to allow them to fully contact the solution. After the sterilization is completed, use sterile filter paper (you can choose low-speed, medium-speed or high-speed filter paper to ensure that the surface of the leaves is fully dried and no liquid remains) to absorb the moisture of the sterilized explants. If there is no oscillation, the leaves will squeeze each other and some surfaces will not be able to fully contact the disinfectant. If the oscillation is too intense, it may damage the leaves and increase the mortality rate of the leaves. Specifically, the plant is 'Beilin Spring' Primulella ( Primula The hybrid Primula 'Spring of Beilin' has been applied for international registration, and its registration number is 181421. That is, the hybrid Primula 'Spring of Beilin' Primula '.

[0034] 2) Induction culture

[0035] Take the disinfected leaves from step 1) and cut them into 0.5-1cm×0.5-1cm (the tender leaf explants are small, so this size is more suitable for cutting), and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. Considering the growth of the leaves, the culture temperature is (23±2)℃, the light intensity is 2000-3000lx, and the light time is 15-16h·d.-1 The induction medium is composed of: MS as the basic medium, and 0.1-0.5 mg·L -1 NAA and 0.5-1.0 mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 (If the agar content is less than 6g·L -1 , which may cause the culture medium to not solidify). The pH of the culture medium is adjusted to 5.8-6.0 before sterilization. Preferably, the composition of the induction culture medium is: MS is used as the basic culture medium, 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0036] 3) Proliferation culture: After 30-40 days of culture in step 2 (different explants have different growth rates, and according to experimental statistics, the growth time can grow into 2-3 leaves within this range), cut off the adventitious buds with 2-3 leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. Considering the growth of the leaves, the culture temperature is (23±2)℃, the light intensity is 2000-3000lx, and the light time is 15-16 h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1-1.5 mg·L -1 NAA and 0.5-2mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 The pH of the culture medium is adjusted to 5.8-6.0 before sterilization. Preferably, the composition of the proliferation medium is: MS is used as the basic culture medium, 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0037] 4) Seedling rooting culture: After 20-30 days of culture in step 3), the seeds are inoculated onto the seedling rooting medium at a temperature of (23±2)°C, a light intensity of 2000-3000 lx, and a light duration of 15-16 h·d. -1After culturing for 20-30 days under the conditions of , root growth can be seen, and the seedlings are inoculated into the rooting medium again. After culturing for 20-30 days, the number of leaf rounds of the seedlings increases by naked eye observation, and it can be seen that the number of leaf rounds grows from 1 to 2-3 rounds, with a total of about 5-6 leaves. After growing 2-3 cm (if it is less than 2 cm, the root system is too short and the root system is relatively fragile, resulting in damage to the root system and a low survival rate; if it is greater than 3 cm, it may be time-consuming; 2-3 cm is the length of the root system, which is relatively strong and saves time) roots, the seedlings can be taken out of the bottle for planting; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 28-30 g·L -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization.

[0038] 5) Transplanting of tissue culture seedlings: Take out the seedlings from step 4 from the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The culture medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance. The mass ratio of the culture medium is: peat: vermiculite: perlite = (5-6): (2-3): 2.

[0039] In the above technical scheme, the present invention conducts rapid tissue culture propagation of hybrid primrose by regulating the sterilization method, the type and concentration of plant growth regulators, and the rooting of seedlings. The growth cycle of the entire regeneration system of the hybrid primrose is about 100 days. After testing, the survival rate of explant sterilization is more than 85%, the differentiation rate of adventitious bud induction culture is about 90%, and the rooting rate of tissue culture seedlings is more than 95%. The transplant survival rate of the hybrid primrose tissue culture seedlings bred by the present invention reaches more than 95%.

[0040] The present invention will be further described below in conjunction with specific embodiments, but this should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by technicians in this field based on the above-mentioned contents of the present invention still fall within the scope of protection of the present invention.

[0041] Example 1

[0042] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0043] 1. Sterilization of Explants

[0044] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0045] 2. Induction Culture

[0046] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0047] 3. Proliferation culture: After 30 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0048] 4. Rooting culture for strong seedlings: After 25 days of culture from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1After culturing for 20 days under the conditions of 50 °C, root growth was observed, and the seedlings were inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increased, and after growing 3 cm roots, they could be taken out of the bottle for planting. The composition of the rooting medium for seedlings was: 1 / 2MS as the basic medium, 30 g·L sucrose was added -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0049] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0050] The growth cycle of the entire regeneration system of this embodiment is 100 days, wherein the survival rate of explant disinfection and sterilization is 97%, the differentiation rate of adventitious bud induction culture is 98%, the plant growth is excellent, the color is emerald green, the rooting rate of tissue culture seedlings is 100%, and finally the survival rate of tissue culture seedlings transplanting reaches 100%.

[0051] Example 2

[0052] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0053] 1. Sterilization of Explants

[0054] Take a 5 cm long leaf from the top of the stem of a plant without pests and diseases, soak it in a detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse it under running water for 30 minutes, and then transfer it to a clean bench for sterilization: first soak the leaf in a 75% (v / v) ethanol solution for 30 seconds, rinse it with sterile water 3 times; then soak it in a 2wt% NaClO solution for sterilization for 2 minutes, and rinse it with sterile water 3 times after sterilization; during the sterilization process, gently shake it so that the leaf is fully in contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explant.

[0055] 2. Induction Culture

[0056] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0057] 3. Proliferation culture: After 35 days of culture in step 2, cut off the adventitious buds with 3 leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0058] 4. Rooting culture for seedlings: After 30 days of cultivation from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0059] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0060] The growth cycle of the entire regeneration system of this embodiment is 115 days, wherein the survival rate of explant disinfection and sterilization is 85%, the differentiation rate of adventitious bud induction culture is 92%, the plants grow well and are green in color, the rooting rate of tissue culture seedlings is 95%, and the final survival rate of tissue culture seedlings transplanting reaches 95%.

[0061] Example 3

[0062] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0063] 1. Sterilization of Explants

[0064] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them with 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0065] 2. Induction Culture

[0066] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.5 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0067] 3. Proliferation culture: After 30 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0068] 4. Rooting culture for strong seedlings: After 25 days of culture from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0069] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0070] The growth cycle of the entire regeneration system of this embodiment is 105 days, wherein the survival rate of explant disinfection and sterilization is 97%, the differentiation rate of adventitious bud induction culture is 94%, the plants grow well, are emerald green in color, have partial vitrification, the rooting rate of tissue culture seedlings is 97%, and the final survival rate of tissue culture seedlings transplanting reaches 95%.

[0071] Example 4

[0072] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0073] 1. Sterilization of Explants

[0074] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them with 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0075] 2. Induction Culture

[0076] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1NAA and 2mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0077] 3. Proliferation culture: After 30 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0078] 4. Rooting culture for strong seedlings: After 25 days of culture from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0079] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0080] The growth cycle of the entire regeneration system of this embodiment is 105 days, wherein the sterilization survival rate of the explants is 97%, the differentiation rate of adventitious bud induction culture is 90%, the growth is average, some of the colors are a little yellow, the rooting rate of the tissue culture seedlings is 95%, and the final survival rate of the tissue culture seedlings transplanting reaches 98%.

[0081] Example 5

[0082] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0083] 1. Sterilization of Explants

[0084] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0085] 2. Induction Culture

[0086] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0087] 3. Proliferation culture: After 30 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0088] 4. Rooting culture for strong seedlings: After 25 days of culture from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1After culturing for 20 days under the conditions of 50 °C, root growth was observed, and the seedlings were inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increased, and after growing 3 cm roots, they could be taken out of the bottle for planting. The composition of the rooting medium for seedlings was: 1 / 2MS as the basic medium, 30 g·L sucrose was added -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0089] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0090] The growth cycle of the entire regeneration system of this embodiment is 100 days, wherein the survival rate of explant disinfection and sterilization is 97%, the differentiation rate of adventitious bud induction culture is 90%, the growth is good, the color is green, the rooting rate of tissue culture seedlings is 95%, and the final survival rate of tissue culture seedlings transplanting reaches 98%.

[0091] Example 6

[0092] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0093] 1. Sterilization of Explants

[0094] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0095] 2. Induction Culture

[0096] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L-1 6-BA, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0097] 3. Proliferation culture: After 30 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16 h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0098] 4. Rooting culture for strong seedlings: After 25 days of cultivation from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 20 days under the conditions of 50 °C, root growth was observed, and the seedlings were inoculated into the rooting medium again. After culturing for 25 days, the number of leaf rings of the seedlings gradually increased, and after growing 3 cm roots, they could be taken out of the bottle for planting. The composition of the rooting medium for seedlings was as follows: 1 / 2MS was used as the basic medium, and 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0099] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0100] The growth cycle of the entire regeneration system of this embodiment is 100 days, wherein the survival rate of explant disinfection and sterilization is 97%, the differentiation rate of adventitious bud induction culture is 98%, the plant growth is excellent, the color is emerald green, the rooting rate of tissue culture seedlings is 100%, and the effect is the same as that of Example 1, but hormones are additionally added to the rooting medium, and finally the survival rate of tissue culture seedlings transplanting reaches 100%.

[0101] Comparative Example 1

[0102] Except for using IBA instead of NAA, the other steps or treatments in this comparative example are exactly the same as those in Example 1. This comparative example provides a tissue culture rapid propagation method of hybrid primrose, comprising the following steps:

[0103] 1. Sterilization of Explants

[0104] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0105] 2. Induction Culture

[0106] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 IBA and 0.5mg·L -1 6-BA, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0107] 3. Proliferation culture: After 35 days of culture in step 2, cut off the adventitious buds with 3 leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 of IBA and 1.0 mg·L -1 6-BA, 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0108] 4. Rooting culture for strong seedlings: After 30 days of cultivation from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 30 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0109] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0110] The growth cycle of the entire regeneration system of this comparative example is 120 days, during which the survival rate of explants after disinfection and sterilization is 97%, the differentiation rate of adventitious bud induction culture is 88%, the plants grow well, some are yellow in color, the rooting rate of tissue culture seedlings is 98%, and the final survival rate of tissue culture seedlings transplanting reaches 88%.

[0111] Comparative Example 2

[0112] Except for using TDZ instead of 6-BA, the other steps or treatments in this comparative example are exactly the same as those in Example 1. This comparative example provides a tissue culture rapid propagation method of hybrid primrose, comprising the following steps:

[0113] 1. Sterilization of Explants

[0114] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 2 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0115] 2. Induction Culture

[0116] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 TDZ, with 30 g·L sucrose -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0117] 3. Proliferation culture: After 35 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16 h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 TDZ, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0118] 4. Rooting culture for seedlings: After 30 days of cultivation from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 30 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0119] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0120] The growth cycle of the entire regeneration system of this comparative example is 120 days, during which the sterilization survival rate of the explants is 97%, the differentiation rate of adventitious bud induction culture is 85%, the plants grow well, some plants turn yellow, the rooting rate of tissue culture seedlings is 95%, and the final survival rate of tissue culture seedlings transplanting reaches 89%.

[0121] Comparative Example 3

[0122] This embodiment provides a tissue culture rapid propagation method of hybrid Primula, comprising the following steps:

[0123] 1. Sterilization of Explants

[0124] Take the 2 cm long tender leaves growing from the top of the stem of the disease-free and insect-free plants, soak them in detergent dilution (1.5 ml of Omo detergent + about 200 ml of water) for 3 minutes, rinse them under running water for 30 minutes, and then transfer them to the clean bench for sterilization: first soak the leaves in 75% (v / v) ethanol solution for 30 seconds, rinse them with sterile water 3 times; then soak and sterilize them in 2wt% NaClO solution for 3 minutes, and rinse them with sterile water 3 times after sterilization; during the sterilization process, gently shake the leaves to make them fully contact with the solution. After sterilization, use sterile filter paper to absorb the moisture of the sterilized explants.

[0125] 2. Induction Culture

[0126] Take the leaves disinfected in step 1, cut them into 0.5 cm × 0.5 cm size, and place them flatly in the induction medium with the leaf surface facing up and the back of the leaf facing down. The culture temperature is 23 ° C, the light intensity is 2000 lx, and the light time is 16 h·d. -1 The induction medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 0.5mg·L -1 6-BA, medium supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0127] 3. Proliferation culture: After 35 days of culture in step 2, cut off the adventitious buds with three leaves on the leaves, lay the leaves flat and transfer them to the proliferation culture medium. The culture temperature is 25 ℃, the light intensity is 2000lx, and the light time is 16 h·d. -1 The proliferation medium is composed of: MS as the basic medium, adding 0.1 mg·L -1 NAA and 1.0mg·L -1 6-BA, medium supplemented with sucrose 30 g·L-1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0128] 4. Rooting culture for strong seedlings: After 35 days of cultivation from step 3, the adventitious buds with 6 leaves were inoculated on the rooting medium for strong seedlings. The culture temperature was 25 °C, the light intensity was 2000 lx, and the light time was 16 h·d. -1 After culturing for 25 days under the conditions of , roots can be seen growing, and the seedlings are inoculated into the rooting medium again. After culturing for 35 days, the number of leaf rings of the seedlings gradually increases, and after growing 3 cm roots, they can be taken out of the bottle and planted; the composition of the rooting medium for seedlings is: 1 / 2MS as the basic medium, supplemented with sucrose 30 g·L -1 , agar 6 g·L -1 , the pH of the culture medium was adjusted to 5.9 before sterilization.

[0129] 5. Transplantation of tissue culture seedlings: Take the seedlings from step 4 out of the culture bottle, wash off the culture medium with clean water, and transfer them to a 10*10 cm nutrient pot. The cultivation medium in the nutrient pot has been exposed to sunlight for 1 day and watered in advance; the mass ratio of the cultivation medium is: peat: vermiculite: perlite = 3:1:1.

[0130] The growth cycle of the entire regeneration system of this comparative example is 130 days, during which the survival rate of explants after disinfection and sterilization is 55%, the differentiation rate of adventitious bud induction culture is 89%, most plants grow in general, some plants grow poorly and turn yellow, the rooting rate of tissue culture seedlings is 95%, and the final survival rate of tissue culture seedlings transplanting reaches 88%.

[0131] From the data of Examples 1-6 and Comparative Examples 1-3, it can be seen that the performance data of Examples 1 and 6 are the best, and the survival rate of explant disinfection and sterilization, adventitious bud differentiation rate, rooting rate and tissue culture seedling transplant survival rate are the highest, which are 97%, 98%, 100% and 100% respectively, but Example 6 adds more hormones than Example 1, so Example 1 is the best regeneration scheme. The growth cycle of Examples 2-4 is 5-15 days longer than that of Example 1, and the survival rate of explant disinfection and sterilization, the differentiation rate of adventitious bud culture are lower than that of Example 1, and the growth is not as good as that of Example 1. The growth cycle of Example 5 is the same as that of Example 1, but its differentiation rate of adventitious bud culture is lower than that of Example 1, and the growth of the plant is worse than that of Example 1. Regarding the adjustment of hormone concentration, when comparing Examples 3, 4, and 5 with Example 1, it is obvious that the differentiation rate of adventitious bud induction is lower than that of Example 1, and the growth is not as good as that of Example 1, which is manifested as general growth and yellow color. In general, the growth cycles of Examples 2-6 were shorter, the differentiation rates of adventitious bud culture were greater than or equal to 90%, and the overall regeneration was good.

[0132] In Comparative Example 1-2, the hormone types were adjusted from NAA and 6-BA to IBA and TDZ, respectively. Compared with Example 1, the growth period was extended by 20 days, the adventitious bud induction and differentiation rate was significantly lower, lower than 90%, and the growth was average. It is speculated that the reason for this result may be that NAA and 6-BA are more suitable for the adventitious bud induction and proliferation of Primula 'Beilin Spring'. In Comparative Example 3, the disinfection and sterilization time was adjusted, and the survival rate of explant disinfection and sterilization was significantly reduced. The disinfection and sterilization survival rates in Examples 1-6 were all higher than 85%, while those in Comparative Example 3 were only 55%. By comparison, it can be seen that Example 1 is the best regeneration system for Primula 'Beilin Spring'.

[0133] In the specification of the present invention, a lot of specific details are described. However, it is understood that embodiments of the present invention can be practiced without these specific details. In some embodiments, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this specification.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0135] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0136] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A tissue culture rapid propagation method for hybrid Primula, characterized in that: The following steps are involved: 1) Disinfection and sterilization of explants Soak the newly grown 2-3 cm long young leaves at the top of the stems of pest-free plants in a diluted detergent solution for 3-4 minutes, rinse under running water for 30 minutes, and then sterilize them. 2) Induction culture The leaves obtained in step 1) were cut into 0.5-1 cm × 0.5-1 cm sizes, and were laid flat with the leaves facing up and the backs facing down in an induction medium for 30-40 days. The induction medium was composed of: MS as the basic medium, 0.1-0.5 mg·L -1 NAA and 0.5-1.0 mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization; 3) Proliferation culture: the adventitious buds with 2-3 leaves obtained in step 2) are cut off together with the explant leaves below, and then the leaves are placed flat and transferred to the proliferation medium for 20-30 days; the composition of the proliferation medium is: MS as the basic medium, 0.1 mg·L -1 NAA and 0.5-2mg·L -1 6-BA, with 28-32 g·L of sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization; 4) The leaves obtained in step 3) are inoculated into a seedling rooting medium and cultured for 20-30 days. When roots grow, the leaves are inoculated into a seedling rooting medium again and cultured for 20-30 days. After the roots grow to 2-3 cm, the leaves are taken out of the bottle and planted. The composition of the seedling rooting medium is: 1 / 2MS as the basic medium, supplemented with 28-30 g·L sucrose -1 , agar 6-7 g·L -1 , the pH of the culture medium was adjusted to 5.8-6.0 before sterilization; 5) Transplanting of tissue culture seedlings: Take out the seedlings obtained in step 4) from the culture bottle, wash off the culture medium, and transfer them to a 10*10 cm nutrient pot; The hybrid Primulina pruriens is Primulina 'Beilinzhichun'.

2. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 1), the sterilization operation includes: first soaking the leaves in a 75% (v / v) ethanol solution for 30-40 seconds, and rinsing with sterile water 3-4 times; then soaking and sterilizing with a 2wt% NaClO solution for 2-3 minutes, and rinsing with sterile water 3-5 times after sterilization.

3. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 2), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light time is 15-16h·d -1 .

4. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 3), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light time is 15-16h·d -1 .

5. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 4), the culture conditions are: culture temperature is 21-25°C, light intensity is 2000-3000lx, and light time is 15-16h·d -1 .

6. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 5), the cultivation medium in the nutrient pot is exposed to sunlight for 1 day and watered thoroughly in advance.

7. The tissue culture rapid propagation method of hybrid Primula as claimed in claim 1, characterized in that: In step 5), the cultivation medium in the nutrient pot is composed of peat, vermiculite and perlite in a mass ratio of (5-6): (2-3): 2.