A kind of culture method of begonia tissue culture seedling

Abstract

The present invention provides a method for cultivating begonia tissue culture seedlings, belonging to the technical field of plant tissue culture. The method comprises the following steps: (1) sterilizing begonia explants and inoculating them with a virus-free culture medium, performing virus-free culture, and obtaining clustered buds; (2) inoculating the clustered buds with an induction culture medium, performing induction culture, and obtaining new buds; (3) inoculating the new buds with a proliferation culture medium, performing proliferation culture, and obtaining clustered adventitious buds; and (4) inoculating the clustered adventitious buds with a rooting culture medium, performing rooting culture, and hardening the seedlings to obtain begonia tissue culture seedlings. The present invention uses a specific induction culture medium, a proliferation culture medium, and a rooting culture medium to cultivate begonia tissue culture seedlings, thereby significantly improving the proliferation coefficient, average number of roots, rooting rate, and number of adventitious buds of the begonia tissue culture seedlings. Furthermore, the present invention cleans the tissue culture seedlings after hardening, thereby improving the relative transplant survival rate of the tissue culture seedlings. The method can be used for factory-scale seedling cultivation, solving the problems of low reproduction rate and uneven growth uniformity in existing cultivation methods.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a culture method for begonia tissue culture seedlings. Background Art

[0002] Begonia is a large genus of plants found throughout the pantropics. Due to habitat degradation and human disturbance, many species are threatened to varying degrees, making their conservation urgent.

[0003] Begonias are typical shade-loving plants, boasting a rich variety of species, vibrant leaf shapes and colors, and vibrant, vibrant flowers. They possess exceptionally high ornamental and practical value, earning them the nickname "the king of foliage plants." Begonias can be propagated primarily through seeding, cuttings, division, and tissue culture. While the first three methods are effective, they are slow and unable to meet market demand. To meet the high demand for begonias in urban greening projects and among home gardening enthusiasts, a method for factory-scale begonia seedling cultivation is urgently needed.

[0004] Although tissue culture propagation is the best method for achieving large-scale production, the choice of culture medium and the concentration of various nutrients in the culture medium play a decisive role in the proliferation of seedlings during tissue culture propagation of begonias. For example, when the 6-BA concentration in the culture medium is too high, the growth of the seedlings is inhibited, resulting in glass seedlings. However, when the concentration is too low, the differentiation rate decreases, and the proliferation effect is not achieved. Therefore, the optimal culture medium formula for begonia seedling cultivation at different stages of industrialization is screened, thereby achieving the goal of rapid propagation while solving the problems of low propagation rate and uneven growth uniformity in existing cultivation methods. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a cultivation method for begonia tissue culture seedlings. The method of the present invention can improve the proliferation coefficient, average number of roots, rooting rate and number of adventitious buds of begonia tissue culture seedlings, and effectively solve the problems of low reproduction rate and uneven growth uniformity in existing cultivation methods.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for culturing begonia tissue culture seedlings, comprising the following steps:

[0008] (1) After the begonia explants are sterilized, they are inoculated into a virus-free culture medium and cultured to obtain clustered buds;

[0009] (2) inoculating the clustered buds into an induction culture medium, inducing culture, and obtaining new buds;

[0010] (3) inoculating the new shoots into a proliferation medium, and culturing them to obtain clustered adventitious shoots;

[0011] (4) Inoculate the clustered adventitious buds into the rooting medium, culture them for rooting, and harden them to obtain the begonia tissue culture seedlings.

[0012] Preferably, the detoxified culture medium comprises MS+0.5-1.5 mg / L6-BA+0.08-0.15 mg / LNAA+5-8 g / L agar+28-32 g / L sucrose.

[0013] Preferably, the virus-free culture time is 10 to 18 days, and the virus-free culture temperature is 22 to 26°C.

[0014] Preferably, the induction medium comprises MS+0.5-1.5 mg / L 6-BA+0.08-0.5 mg / L NAA+5-8 g / L agar+28-32 g / L sucrose.

[0015] Preferably, the induction culture time is 20 to 45 days, and the induction culture temperature is 22 to 26°C.

[0016] Preferably, the proliferation culture medium comprises MS+0.3-1.2 mg / L 6-BA+0.1-0.5 mg / L NAA+5-8 g / L agar+28-32 g / L sucrose.

[0017] Preferably, the proliferation culture time is 35 to 45 days, and the proliferation culture temperature is 22 to 26°C.

[0018] Preferably, the rooting medium comprises 1 / 2MS+0.5-1.5 mg / L NAA+5-8 g / L agar+28-32 g / L sucrose.

[0019] Preferably, the rooting culture time is 20 to 35 days, and the rooting culture temperature is 22 to 26°C;

[0020] The time for hardening the seedlings is 4 to 15 days.

[0021] Preferably, the begonia includes one or more of Begonia masoniana, Begonia picturata, Begonia bervirimosa ssp. exotica, Begonia 'High Way', Begonia Fireworks, and Begonia 'Zhou Hai Dan'.

[0022] Compared with the prior art, the present invention has the following beneficial effects: by using a specific induction medium, proliferation medium, and rooting medium to cultivate begonia tissue culture seedlings, the present invention significantly improves the proliferation coefficient, average number of roots, rooting rate, and number of adventitious buds of the begonia tissue culture seedlings. Furthermore, the present invention also cleans the tissue culture seedlings after hardening, thereby improving the relative transplant survival rate of the tissue culture seedlings. Furthermore, the cultivation method provided by the present invention is suitable for cultivating a variety of begonia tissue culture seedlings, including Begonia masoniana, Begonia picturata, Begonia bervirimosa ssp.exotica, Begonia 'High Way', Begonia Fireworks, and Begonia 'ZhouHaiDan'. The cultivation method provided by the present invention can be used for factory-scale seedling cultivation, solving the problems of low reproduction rate and uneven growth uniformity in existing cultivation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 To obtain clustered buds by inoculating begonia explants into virus-free culture medium;

[0024] Figure 2 The clustered buds were inoculated into the induction medium for induction culture to obtain new buds;

[0025] Figure 3 The new shoots were inoculated into the proliferation medium for proliferation culture to obtain clustered adventitious shoots;

[0026] Figure 4 The clustered adventitious buds were inoculated into the rooting medium for rooting culture to obtain the Begonia tissue culture seedlings;

[0027] Figure 5 This is the height measurement diagram of Begonia tissue culture seedlings;

[0028] Figure 6 This is a diagram for hardening begonia tissue culture seedlings;

[0029] Figure 7 This is a diagram showing the washing process for tissue culture seedlings after hardening;

[0030] Figure 8 In order to transfer the washed begonia tissue culture seedlings into the plug tray to obtain the plug tray seedlings;

[0031] Figure 9 This is the status of the plug tray seedlings one week after planting;

[0032] Figure 10 To maintain the plug seedlings;

[0033] Figure 11 This is a diagram of shading treatment for plug seedlings;

[0034] Figure 12 This is a diagram of the growth of seedlings in trays when the humidity is high;

[0035] Figure 13 The state diagram of begonia seedlings when they can be planted in plug trays;

[0036] Figure 14 This is a picture showing crabapples grown in a greenhouse;

[0037] Figure 15 This is a display picture of Zhou Haidan's flower exhibition in the greenhouse;

[0038] Figure 16 This is a picture showing crabapples grown outdoors;

[0039] Figure 17 Schematic diagram of an outdoor begonia flower display. DETAILED DESCRIPTION

[0040] The present invention provides a method for culturing begonia tissue culture seedlings, comprising the following steps:

[0041] (1) After the begonia explants are sterilized, they are inoculated into a virus-free culture medium and cultured to obtain clustered buds;

[0042] (2) inoculating the clustered buds into an induction culture medium, inducing culture, and obtaining new buds;

[0043] (3) inoculating the new shoots into a proliferation medium, and culturing them to obtain clustered adventitious shoots;

[0044] (4) Inoculate the clustered adventitious buds into the rooting medium, culture them for rooting, and harden them to obtain the begonia tissue culture seedlings.

[0045] In the present invention, after the begonia explants are disinfected, they are inoculated into a virus-free culture medium and cultured to obtain clustered buds. The liquid medicine used for disinfection includes: one or more of 150-200 g / L of detergent, 84 disinfectant with a mass concentration of 3%-8%, Tween-80 with a mass concentration of 0.3%-0.7%, 70% ethanol by volume, 0.1% mercuric chloride solution by volume and sterile water, preferably one or more of 160-180 g / L of detergent, 84 disinfectant with a mass concentration of 4%-7%, Tween-80 with a mass concentration of 0.4%-0.6% and sterile water, and further preferably one or more of 170 g / L of detergent, 84 disinfectant with a mass concentration of 5%-6%, Tween-80 with a mass concentration of 0.5% and One or more of sterile water; during the disinfection, detergent and 84 disinfectant are used in sequence, and the order of using the detergent and 84 disinfectant is preferably to use the detergent for soaking and disinfection first, and then use the 84 disinfectant for soaking and disinfection; the disinfection time when using detergent or 84 disinfectant is 20 to 30 minutes, preferably 22 to 28 minutes, and more preferably 24 to 26 minutes; after the disinfection, flush with running water for 30 to 35 minutes, preferably 31 to 34 minutes, and more preferably 32 to 33 minutes; the detoxification culture medium includes: MS + 0.5 to 1.5 mg / L 6-BA + 0.08 to 0.15 mg / L NAA+5-8g / L agar+28-32g / L sucrose, preferably: MS+0.8-1.2mg / L 6-BA+0.09-0.12mg / L NAA+5.5-7.5g / L agar+29-31g / L sucrose, more preferably: MS+1.0mg / L 6-BA+0.1mg / L NAA+6g / L agar+30g / L sucrose; the time of the detoxification culture is 10-18d, preferably 13-16d, more preferably 15d; the temperature of the detoxification culture is 22-26°C, preferably 23-25°C, more preferably 24°C; the light intensity of the detoxification culture is 2000-2500Lx, preferably 2200-2400Lx, more preferably 2300Lx.

[0046] In the present invention, the clustered buds are inoculated into an induction culture medium, induced and cultured to obtain new buds. Before inoculating the clustered buds with the induction culture medium, the bottom 2 to 3 mm is preferably cut off to allow the bud segments to better absorb the nutrients in the proliferation culture medium; the induction culture medium comprises: MS + 0.5 to 1.5 mg / L 6-BA + 0.08 to 0.5 mg / L NAA + 5 to 8 g / L agar + 28 to 32 g / L sucrose, preferably: MS + 0.8 to 1.4 mg / L 6-BA + 0.15 to 0.4 mg / L NAA + 5.5 to 7.5 g / L agar + 29 to 31 g / L sucrose, further preferably: MS + 1.2 mg / L 6-BA + 0.3 mg / L NAA + 6 to 7 g / L agar + 30 g / L sucrose; the induction culture time is 20 to 45 days, preferably 30 to 43 days, further preferably 40 days; the induction culture temperature is 22 to 26°C, preferably 23 to 25°C, further preferably 24°C.

[0047] In the present invention, the new shoots are inoculated into a proliferation culture medium and cultured to obtain clustered adventitious shoots. The length of the new shoots is 4-5 mm, preferably 4.2-4.8 mm, and more preferably 4.4-4.6 mm; the proliferation culture medium comprises: MS + 0.3-1.2 mg / L 6-BA + 0.1-0.5 mg / L NAA + 5-8 g / L agar + 28-32 g / L sucrose, preferably: MS + 0.4-0.8 mg / L 6-BA + 0.2-0.4 mg / L NAA + 5.5-7.5 g / L agar + 29-31 g / L sucrose, and more preferably: MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 6-7 g / L agar + 30 g / L sucrose; the time of the proliferation culture is 35-45 days, preferably 38-41 days, and more preferably 40 days; the temperature of the proliferation culture is 22-26°C, preferably 23-25°C, and more preferably 24°C.

[0048] In the present invention, the clustered adventitious buds are inoculated into a rooting medium, rooted and cultured, and hardened to obtain begonia tissue culture seedlings. The length of the clustered adventitious buds is 1 to 1.5 cm, preferably 1.2 to 1.4 cm, and more preferably 1.3 cm; the rooting medium comprises: 1 / 2 MS + 0.5 to 1.5 mg / L NAA + 5 to 8 g / L agar + 28 to 32 g / L sucrose, preferably: 1 / 2 MS + 0.6 to 1.2 mg / L NAA+5.5-7.5g / L agar+29-31g / L sucrose, more preferably: 1 / 2MS+1.0mg / LNAA+6g / L agar+30g / L sucrose; the rooting culture time is 20-35d, preferably 25-33 days, more preferably 27-32d; the rooting culture temperature is 22-26°C, preferably 23-25°C, more preferably 24°C; the seedling hardening time is 4-15d, preferably 5-12d, more preferably 6-10d; the seedling hardening temperature is 20-28°C, preferably 22-26°C, more preferably 24-25°C; the seedling hardening is to place a culture bottle containing tissue culture seedlings with a growth height of ≥2cm, ≥3 leaves, and a diameter of 3 leaves ≥0.6cm in a transplanting ring. The transplanting environment is characterized in that the bottle mouth is opened and placed for 3 to 4 days, and then placed for 1 to 2 days in the manner of loosening the bottle mouth, half opening the bottle mouth and fully opening the bottle mouth; the temperature of the transplanting environment is 20 to 28°C, preferably 22 to 26°C, and more preferably 24 to 25°C; the humidity of the transplanting environment is 60% to 80%; the light intensity of the transplanting environment is 2000 to 5000Lx, preferably 2500 to 4500Lx, more preferably 3000 to 4000Lx, and even more preferably 3500Lx; after the hardening, the tissue culture seedlings with strong growth and root length exceeding 2 cm are taken out to obtain the begonia tissue culture seedlings, and the culture medium of the roots is washed with clean water to avoid the plants being soaked in clean water, thereby improving the relative transplant survival rate of the tissue culture seedlings; the begonia preferably includes Begonia masoniana (also known as 'Iron Armor' Begonia), Begonia picturata (also known as 'A Mouth of Blood' Begonia), Begonia bervirimosa ssp.exotica (also known as 'Red Vein' Begonia), Begonia 'HighWay' (also known as 'Haiwei' Begonia), Begonia Fireworks (also known as 'Fireworks' Begonia), Begonia 'ZhouHaiDan' (also known as 'Zhou Haidan' Begonia).

[0049] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] A method for cultivating begonia tissue culture seedlings comprises the following steps:

[0052] (1) The terminal buds of young plants of begonia 'Yikouxue' without pests and diseases were collected as explants of begonia 'Yikouxue', the leaves were cut off, and each bud was retained. After washing the surface dust with running water, the plants were placed in 200g / L detergent and soaked for 30min, and brushed with a brush while soaking, and rinsed with running water for 30min, and soaked in 84 disinfectant with a mass concentration of 5% for 30min, and brushed with a brush while soaking, and the residual disinfectant was rinsed with running water. Then, the plants were placed in a clean bench and soaked in sterile water for 30min, the sterile water was poured out, and the plants were immersed in 70% ethanol by volume for 30s, and then 0.5% Tween-80 was added and soaked in 0.1% mercuric chloride solution by volume for 2min, and gently shaken. The plants were rinsed with sterile water for 5 times, and the excess water was absorbed with sterile filter paper. The plants were then inoculated with MS+1.0mg / L In a detoxified medium consisting of 6-BA, 0.1 mg / L NAA, 6 g / L agar, and 30 g / L sucrose, inoculate only one terminal bud per bottle of culture medium. Incubate in the light for 14 hours and in the dark for 10 hours. Incubate on a culture rack at a light intensity of 2000 Lx and 24 ± 2°C for 15 days. If no contamination is observed, detoxification is complete. Remove any dead or contaminated explants that appear during the disinfection process to obtain clustered buds.

[0053] (2) Cut off the 2-3 mm bottom of the clustered buds to expose the fresh interface, and inoculate the remaining bud segment into an induction medium consisting of MS + 1.2 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose. Only one bud segment was inoculated into each bottle of culture medium, and the culture was statically cultured at 24 ± 2 °C for 20 days to obtain new buds.

[0054] (3) In a clean bench, new shoots with a length of 4 to 5 mm were cut off and inoculated one by one with the proliferation medium of MS+0.50 mg / L 6-BA+0.30 mg / L NAA+6 g / L agar+30 g / L sucrose. Only one new shoot was inoculated into each bottle of culture medium and cultured at 24 ± 2 °C for 20 days. The differentiation of the new shoots was observed. It was found that the proliferation rate reached the highest when the culture reached 40 days, and the callus tissue was a healthy yellow-green color. The yellow leaves of the clustered adventitious buds were few, which was the optimal turnover date. At this time, the sterile seedlings performed normally, and the clustered adventitious buds reached the rooting standard. After continuing to culture for 9 to 10 generations, each sterile seedling could produce 10 to 15 adventitious roots, and a complete tissue culture seedling was obtained.

[0055] (4) When the number of sterile seedlings in the bottle is expanded to 100, cut the clustered adventitious buds with a length of 1 to 1.5 cm in the clean bench, and use tweezers to take 10 clustered adventitious buds and insert them into a bottle of 1 / 2MS + 1.0 mg / L NAA + 6 g / L agar + 30 g / L sucrose rooting medium (one bottle of propagation bottle seedlings can be cut into the amount of two bottles of rooting seedlings). Incubate at 24 ± 2 ° C for 7 days until roots grow out, and then transfer to a culture rack with sufficient light and 24 ± 2 ° C for further culturing for 20 days. Set the light culture time to 14 hours and the dark culture time to 10 hours, place the culture bottle containing the tissue culture seedlings with a growth height of ≥2 cm and ≥3 leaves in a transplanting environment with a temperature of 20°C, a humidity of 60% to 80%, and a light intensity of 2000Lx, open the bottle mouth and place it for 3 days, and then place it for 1 day in different ways according to the bottle mouth being loosened, half-opened, and fully opened. Take out the tissue culture seedlings with strong growth and root length exceeding 2 cm, wash the culture medium at the root with clean water, and avoid soaking the plants in clean water to obtain tissue culture seedlings.

[0056] Example 2

[0057] A method for cultivating begonia tissue culture seedlings, comprising the following steps:

[0058] (1) The terminal buds of young plants of 'red-striped' begonia without pests and diseases were collected as 'red-striped' begonia explants, the leaves were cut off, and each bud was retained. After washing the surface dust with running water, the plants were placed in 200 g / L detergent and soaked for 30 min, and brushed with a brush while soaking, and rinsed with running water for 30 min, and soaked in 84 disinfectant with a mass concentration of 5% for 30 min, and brushed with a brush while soaking, and the residual disinfectant was rinsed with running water. The plants were then placed in a clean bench and soaked in sterile water for 30 min, the sterile water was poured out, and the plants were immersed in 70% ethanol by volume for 30 s, and soaked in 0.1% mercuric chloride solution by volume for 2 min, and gently shaken. The plants were rinsed with sterile water 5 times, and the excess water was absorbed with sterile filter paper, and the plants were inoculated with MS+1.0 mg / L. In a detoxified medium consisting of 6-BA + 0.1 mg / L NAA + 6 g / L agar + 30 g / L sucrose, only one terminal bud was inoculated into each bottle of culture medium. The light culture time was set to 14 h, and the dark culture time was set to 10 h. The culture was statically cultured on a culture rack at a light intensity of 2500 Lx and 24 ± 2°C for 15 days. If no contamination was found, the detoxification was complete. Dead explants and contaminated explants that appeared during the disinfection process were removed to obtain clustered buds.

[0059] (2) Cut off the 2-3 mm bottom of the clustered buds to expose the fresh interface, and inoculate the remaining bud segment into an induction medium consisting of MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose. Only one bud segment was inoculated into each bottle of culture medium, and the culture was statically cultured at 24 ± 2 °C for 40 days to obtain new buds.

[0060] (3) In a clean bench, new shoots with a length of 4 to 5 mm were cut off and inoculated one by one into a proliferation medium of MS + 0.50 mg / L 6-BA + 0.30 mg / L NAA + 6 g / L agar + 30 g / L sucrose. Only one new shoot was inoculated into each bottle of culture medium and cultured at 24 ± 2 °C. The differentiation of the new shoots was observed at 20 days. It was found that the proliferation rate reached the highest at 40 days of culture, and the callus tissue was a healthy yellow-green color. There were few yellow leaves in the clustered adventitious buds, which was the optimal turnover date. At this time, the sterile seedlings performed normally, and the clustered adventitious buds reached the rooting standard. After continuing to culture for 9 to 10 generations, each sterile seedling could produce 10 to 15 adventitious roots, and a complete tissue culture seedling was obtained.

[0061] (4) When the number of sterile seedlings in the bottle is expanded to 100, cut the clustered adventitious buds with a length of 2 to 2.5 cm in the clean bench, and use tweezers to take 10 clustered adventitious buds and insert them into a bottle of 1 / 2MS + 1.0 mg / L NAA + 6 g / L agar + 30 g / L sucrose rooting medium (one bottle of propagation bottle seedlings can be cut into the amount of two bottles of rooting seedlings). Incubate at 24 ± 2 ° C for 7 days until roots grow out, and then transfer to a culture rack with sufficient light and 24 ± 2 ° C. Continue to culture for 20 days, place the culture bottles containing tissue culture seedlings with a growth height of ≥2 cm and ≥3 leaves in a transplanting environment with a temperature of 28°C, a humidity of 60% to 80%, and a light intensity of 5000Lx, open the bottle mouth and place it for 3 days, and then place it for 1 day in the respective ways of loosening the bottle mouth, half-opening the bottle mouth, and fully opening the bottle mouth. Take out the tissue culture seedlings with healthy growth and root length exceeding 2 cm, wash the culture medium at the root with clean water, and avoid soaking the plants in clean water to obtain tissue culture seedlings.

[0062] Example 3

[0063] A method for cultivating begonia tissue culture seedlings, comprising the following steps:

[0064] (1) The terminal buds of young plants of 'Fireworks' begonia without pests and diseases were collected as 'Fireworks' begonia explants, the leaves were cut off, and each bud was retained. After washing the surface dust with running water, the plants were placed in 200g / L detergent and soaked for 30min, and brushed with a brush while soaking, and rinsed with running water for 30min, and soaked in 84 disinfectant with a mass concentration of 5% for 30min, and brushed with a brush while soaking, and the residual disinfectant was rinsed with running water. The plants were then placed in a clean bench and soaked in sterile water for 30min, the sterile water was poured out, and the plants were immersed in 70% ethanol by volume for 30s, and soaked in 0.1% mercuric chloride solution by volume for 2min, and gently shaken. The plants were rinsed with sterile water for 5 times, and the excess water was absorbed with sterile filter paper. The plants were then inoculated with MS+1.2mg / L 6-BA+0.1mg / L In the virus-free medium of NAA+6g / L agar+30g / L sucrose, only one terminal bud was inoculated into each bottle of culture medium, and the light culture time was set to 14h and the dark culture time was set to 10h. The culture was placed on a culture rack with a light intensity of 2300Lx and 24±2℃ for 15d. If no contamination occurred, the virus-free process was completed. The dead explants and contaminated explants that appeared during the disinfection process were removed to obtain clustered buds ( Figure 1 );

[0065] (2) Cut off 2-3 mm of the bottom of the clustered buds to expose the fresh interface, and inoculate the remaining bud segments into the induction medium of MS+0.8 mg / L 6-BA+0.3 mg / L NAA+6.5 g / L agar+30 g / L sucrose. Only one bud segment was inoculated into each bottle of culture medium, and the culture was kept at 24 ± 2 ° C for 30 days to obtain new buds ( Figure 2 );

[0066] (3) In the clean bench, the new shoots with a length of 4-5 mm were cut off and inoculated one by one with a proliferation medium of MS+0.5 mg / L6-BA+0.3 mg / L NAA+6.5 g / L agar+30 g / L sucrose. Only one new shoot was inoculated into each bottle of culture medium. The culture was kept at 24±2℃ for 20 days and the differentiation of the new shoots was observed. It was found that when the culture was cultivated to 40 days, the proliferation rate reached the highest, and the callus tissue was healthy yellow-green. There were few yellow leaves of the clustered adventitious buds. This was the optimal turnover date. At this time, the sterile seedlings performed normally and the clustered adventitious buds reached the rooting standard. After continuing to culture for 9-10 generations, each sterile seedling could produce 10-15 adventitious roots, and a complete tissue culture seedling was obtained ( Figure 3 );

[0067] (4) When the number of sterile seedlings in the bottle is expanded to 100, cut buds with a length of 1 to 1.5 cm in the clean bench, and use tweezers to take 10 buds from each bottle and insert them into a bottle of 1 / 2 MS + 1.5 mg / L NAA + 6 g / L agar + 30 g / L sucrose rooting medium (one bottle of propagation bottle seedlings can be cut into the amount of two bottles of rooting seedlings). Incubate at 24 ± 2 ° C for 7 days until roots grow. Figure 4 ), and then transferred to a culture rack with sufficient light and 24±2℃ for further culturing for 25 days, and the tissue culture seedlings ( Figure 5 ) were placed in a transplanting environment with a temperature of 25°C, a humidity of 60% to 80%, and a light intensity of 4000 Lx. The bottles were opened for 4 days, and then placed for 2 days with the bottles opened loosely, half-opened, and fully opened. Figure 6 ), take out the healthy and strong tissue culture seedlings with roots longer than 2 cm, and wash the culture medium at the roots with clean water ( Figure 7 ) and avoid soaking the plants in clean water to obtain tissue culture seedlings.

[0068] Example 4

[0069] Begonia tissue culture seedlings were cultured using the method of Example 1, with the only difference being that the "proliferation medium comprising MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose" was replaced with a "proliferation medium comprising MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose".

[0070] Table 1 Comparison of proliferation rates of different species in different culture media

[0071] Variety processing A mouthful of blood armor Red veins Haiwei Fireworks Zhou Haidan Control (CK) 1.4 1.8 2.8 1.8 3.8 2 Medium 1 9.4 7.8 6.4 6.2 8.2 6.8 Medium 2 16.2 17.4 15.2 13.6 18.2 12.2 Medium 3 7.8 9.4 8.6 7.6 10.4 8

[0072] Note: CK is MS + 6 g / L agar + 30 g / L sucrose; Medium 1 is MS + 0.3 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose; Medium 2 is MS + 0.5 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose; Medium 3 is MS + 0.8 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose.

[0073] Example 5

[0074] Begonia tissue culture seedlings were cultured using the method of Example 1, with the only difference being that the rooting medium comprising 1 / 2MS + 1.0 mg / L NAA + 6 g / L agar + 30 g / L sucrose was replaced with the rooting medium comprising 1 / 2MS + 1.5 mg / L NAA + 6 g / L agar + 30 g / L sucrose.

[0075] Table 2 Comparison of rooting rates of different varieties in different culture media

[0076] Variety processing A mouthful of blood armor Red veins Haiwei Fireworks Zhou Haidan Control (CK) 61.1％ 69.80％ 55.2％ 51.3％ 47.9％ 45.7％ Medium 1 79.0％ 77.90％ 75.0％ 76.9％ 76.3％ 78.4％ Medium 2 97.2％ 98.80％ 97.8％ 95.6％ 97.9％ 92.8％ Medium 3 87.1％ 90.60％ 85.8％ 86.3％ 89.1％ 84.3％

[0077] Note: CK is 1 / 2MS + 6g / L agar + 20g / L sucrose; medium 1 is 1 / 2MS + 0.5mg / L NAA + 6g / L agar + 30g / L sucrose; medium 2 is 1 / 2MS + 1.0mg / L NAA + 6g / L agar + 30g / L sucrose; medium 3 is 1 / 2MS + 1.5mg / L NAA + 6g / L agar + 30g / L sucrose.

[0078] Example 6

[0079] Begonia tissue culture seedlings were cultured using the method in Example 1, with the only difference being that the "induction medium containing MS + 1.2 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose" was replaced with "induction medium containing MS + 1.5 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose".

[0080] Table 3 Comparison of induction rates of different varieties in different culture media

[0081] Variety processing A mouthful of blood armor Red veins Haiwei Fireworks Zhou Haidan Control (CK) 54.1％ 51.5％ 44.0％ 47.6％ 50.4％ 43.1％ Medium 1 75.4％ 71.5％ 62.5％ 65.3％ 64.0％ 57.8％ Medium 2 87.8％ 85.4％ 80.4％ 80.6％ 81.9％ 72.0％ Medium 3 78.8％ 76.0％ 67.9％ 68.4％ 68.9％ 63.0％

[0082] Note: CK is MS + 6 g / L agar + 30 g / L sucrose; Medium 1 is MS + 1.0 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose; Medium 2 is MS + 1.2 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose; Medium 3 is MS + 1.5 mg / L 6-BA + 0.3 mg / L NAA + 6 g / L agar + 30 g / L sucrose.

[0083] Comparative Example 1

[0084] Begonia tissue culture seedlings were cultured using the method of Example 1, with the only difference being that the proliferation medium was replaced with a medium consisting of MS+6 g / L agar+30 g / L sucrose.

[0085] Comparative Example 2

[0086] Begonia tissue culture seedlings were cultured using the method of Example 1, with the only difference being that the rooting medium was replaced with a medium consisting of MS+6 g / L agar+30 g / L sucrose.

[0087] Comparative Example 3

[0088] Begonia tissue culture seedlings were cultured using the method of Example 1, with the only difference being that the induction medium was replaced with a medium consisting of MS+6 g / L agar+30 g / L sucrose.

[0089] Test Example 1

[0090] According to the methods in Example 1 and Comparative Example 1, tissue culture seedlings of Begonia masoniana (also known as 'Iron Armor' Begonia), Begonia picturata (also known as 'A Mouth of Blood' Begonia), Begonia bervirimosa ssp. exotica (also known as 'Red Stripe' Begonia), Begonia 'HighWay' (also known as 'Haiwei' Begonia), Begonia Fireworks (also known as 'Fireworks' Begonia), and Begonia 'ZhouHaiDan' (also known as 'Zhou Haidan' Begonia) were cultured respectively. During the proliferation culture, new shoots of the next generation were selected every 30 days and inoculated into a new proliferation medium. This was repeated 9 times. The rooting of the sterile seedlings of each generation of begonia was observed, and the proliferation coefficient was calculated. The results are shown in Table 4. The proliferation coefficient calculation formula is as follows:

[0091] Proliferation coefficient = number of explants that can be cut into pieces for proliferation / total number of single buds at the time of inoculation.

[0092] Table 4 Proliferation coefficients of different tissue culture seedlings

[0093]

[0094] The results showed that each generation of begonia sterile seedlings could grow normally, with leaves unfolding and green, and produced 12 to 18 adventitious roots. At the same time, the adventitious roots produced met the rooting standards.

[0095] Test Example 2

[0096] Tissue culture seedlings of Begonia masoniana (also known as 'Iron Armor' Begonia), Begonia picturata (also known as 'A Mouth of Blood' Begonia), Begonia bervirimosassp. exotica (also known as 'Red Stripe' Begonia), Begonia 'HighWay' (also known as 'Haiwei' Begonia), Begonia Fireworks (also known as 'Fireworks' Begonia), and Begonia 'ZhouHaiDan' (also known as 'Zhou Haidan' Begonia) were cultured according to the methods of Examples 1, 6, and 3, respectively. The growth of their adventitious bud clusters was observed, the number of buds was measured, and the induction rate of adventitious bud clusters was calculated. The results are shown in Table 5.

[0097] The calculation formula of the induction rate of clustered adventitious buds is as follows:

[0098] Induction rate of clustered adventitious buds (%) = number of budding explants / number of inoculated explants × 100%

[0099] Table 5 Effects of different proliferation culture media on the number and induction rate of adventitious buds

[0100]

[0101] The results showed that when the proliferation culture reached the 45th day, the plants cultured in Comparative Example 3 were short, the adventitious buds were thin and weak, and the leaves were small, which did not meet the standard of rooted seedlings (leaves ≥3 pieces, and the diameter of 3 leaves ≥0.6 cm); while the adventitious buds of the plants cultured in Example 1 and Example 6 were tall and neat, and the adventitious buds were larger.

[0102] Test Example 3

[0103] According to the methods in Example 1, Example 5 and Comparative Example 2, tissue culture seedlings of Begonia masoniana (also known as 'Iron Armor' Begonia), Begonia picturata (also known as 'A Mouth of Blood' Begonia), Begonia bervirimosassp.exotica (also known as 'Red Stripe' Begonia), Begonia 'High Way' (also known as 'Haiwei' Begonia), Begonia Fireworks (also known as 'Fireworks' Begonia), and Begonia 'Zhou Hai Dan' (also known as 'Zhou Hai Dan' Begonia) were cultured, and the rooting conditions were observed on the 15th and 20th days of rooting culture; 15 bottles were randomly selected from 60 randomly selected culture bottles, and the rooting conditions were counted; at the same time, the growth of tissue culture seedlings in different rooting media was observed, and the average number of roots and the rooting rate were calculated. The results are shown in Table 6. The statistical formula for the rooting rate is as follows:

[0104] Rooting rate (%) = (number of rooting inoculations / total number of rooting inoculations) × 100%

[0105] Table 6 Effects of different rooting media on root number and rooting rate

[0106]

[0107] The results showed that by the 15th day of rooting culture, most of the rooting seedlings had grown roots. By the 20th day, each rooting seedling had 4 to 10 fibrous roots, including secondary and tertiary roots. The tissue culture seedlings cultured in Comparative Example 2 had light green leaves and few, thin roots, while the tissue culture seedlings cultured in Examples 1 and 5 grew luxuriantly, had dark green leaves, and had numerous, thick roots.

[0108] Test Example 4

[0109] 1. Training seedlings

[0110] When the height of the tissue culture seedlings reaches 4 to 9 cm or the number of leaves reaches 4 leaves or more, take the culture bottle out of the culture room and place it in the transplanting environment. Keep it in the open bottle mouth state for 3 to 4 days, and then keep it in the loose, half-open and fully open bottle mouth states for one day each. When the light intensity exceeds 15000Lx, it needs to be shading. After opening the bottle mouth, spray water frequently to keep it moist. If the culture medium is found to be contaminated with bacteria during the seedling training process, it should be soaked in 5g / L potassium permanganate solution for 2 minutes for disinfection, and then washed with clean water for transplanting.

[0111] 2. Transplanting

[0112] 2.1 Transplanting medium

[0113] The transplanting medium is peat soil with 10% perlite, and the amount of soil per 1m 3 Add 150g of 75% carbendazim or 50% carbendazim wettable powder to the substrate, and use 0.5g / L potassium permanganate solution (or 1000-fold dilution of phoxim granules) for sterilization and insecticide treatment every other day. Specifically, spray the prepared medicine on the prepared cultivation substrate with a sprayer, and stir the substrate continuously to mix it evenly until the moisture content reaches more than 20%. Hold the soil in your hand to form a ball that can fall apart when it falls to the ground (height is 3-5cm), and leave it for one day for use.

[0114] 2.2 Transplanting container

[0115] The seedling container should be a 72-hole tray with a size of 54cm x 28cm, with an upper diameter of 3.8cm, a lower diameter of 2cm, and a height of 4.5cm. Disinfect it with a 0.5% potassium permanganate solution 1 to 3 days before transplanting.

[0116] 2.3 Transplanting method

[0117] Remove the tissue culture seedlings from the culture bottle and clean the culture medium at the roots of the seedlings with clean water in time (Note: Begonias are relatively fragile, so be careful when cleaning). Try to avoid soaking the plants in clean water. Determine the effect of soaking time on the survival rate of transplantation. The results are shown in Table 7.

[0118] Table 7 Effect of soaking tissue culture seedlings and transplanting survival rate

[0119]

[0120] Note: The relative transplant survival rate refers to the ratio of transplant survival before and after soaking and washing.

[0121] As shown in Table 7, the survival rate of the transplanted tissue culture seedlings after immersion was significantly reduced, sometimes by 50% to 60%. This situation was particularly prominent in winter.

[0122] Transplant the cleaned tissue culture seedlings, transplant 10 trays (720 plants) for each species / variety, and select 3 trays for each species / variety. Classify the tissue culture seedlings (growing height ≥3cm, leaves ≥5 pieces are large seedlings, growing height <2cm, leaves ≥3 pieces are small seedlings) and transplant them in trays ( Figure 8 ), use bamboo sticks to insert holes in the substrate, and place the roots of the seedlings directly into the hole in the tray to ensure that the roots are stretched, pinch the soil tightly, and immediately water the roots with clean water after the tray is put on. Use a sprayer to lightly spray it again to fully fit the substrate and the tray together. (Note: The substrate has been thoroughly irrigated after the tray is put on) After transplanting, cover with film to keep it moist, and punch holes in the film for air permeability. The operation is carried out in a shade shed, and the conditions of the shade shed are 60% to 80% shade, 60% to 80% humidity, and 18 to 26°C. Just after planting the seedlings in the tray, the staff watered and pulled the shade net (the light was controlled below 5000Lx) ( Figures 9 to 11 ).

[0123] 2.4 Management after transplanting

[0124] After the hole tray is placed, it is necessary to keep it moisturized and heat-insulated, and to provide shade. The shade degree is 60% to 80% of the light intensity of the unshaded area, the humidity is 60% to 80%, and the temperature is 22 to 26°C. If the temperature is too high on the day, proper ventilation should be performed to cool it down. Ventilation should be performed for half an hour in the afternoon when the light is weak. When encountering rainy weather, light and ventilation are required. If there is no ventilation and the humidity is too high, mold and fungi will grow (such as Figure 12 Water every 3-4 days and spray with 500 times diluted carbendazim wettable powder every 7-10 days. The film can be removed after new leaves grow, but shade is still required.

[0125] 3. Seedlings

[0126] When the root system of the seedlings is fully grown (such as Figure 13 The entire hole tray can be planted in a No. 10 two-color pot. The planting depth should not cover the center of the seedling. Press the seedling firmly when planting to prevent it from being washed out by watering.

[0127] 3.1 Maintenance and management

[0128] Entering the maintenance phase, greenhouse management in summer includes: shading, humidity control, and ventilation. Keep the temperature below 25°C in summer, water every two to three days, and keep relative humidity around 60%. Keep the temperature above 5°C in winter, and the temperature difference between watering and watering should not exceed 5°C. Water once a week, and insulation is key. Begonias require nitrogen during their growth period. Use high-nitrogen fertilizers like urea to prevent seedling burn. Frequently spray with a 1000x dilution of "Hua Duo Duo 20-20-20," spraying every 20 days in summer. Spray an appropriate amount (water the potting soil thoroughly). Seedlings are susceptible to snails and slugs. Stem and root rot are more likely to occur in the high temperature and humidity of the greenhouse. Remove diseased plants immediately at the early stages of the disease. Spray or irrigate the roots with a 500-600x dilution of 50% carbendazim, spraying every 10 days, watering the potting soil thoroughly. Spray three to five times continuously. Spraying with imidacloprid and frequent ventilation are also effective control measures.

[0129] 3.2 Maintenance of greenhouses, greenhouse flower walls and outdoor flower displays

[0130] When planting greenhouse flower walls, avoid high temperature and high humidity. High temperature and high humidity are conducive to the reproduction of pathogens and the formation of rot diseases. Therefore, dense planting should be avoided to facilitate ventilation (such as Figure 14 and Figure 15 When planting in the field (such as Figure 16 and Figure 17 Maintenance and management are relatively simple. Drainage should be done promptly during the rainy season. Seedlings need to be transplanted to a greenhouse in winter and should not be overwintered outdoors in the Shijiazhuang area. Snails and slugs are common during the seedling stage, and stem and root rots are more likely to occur in the high temperature and humidity of the greenhouse. Infected plants should be removed immediately at the early stages of the disease. A 50% carbendazim dilution (500-600 times diluted) should be sprayed or irrigated through the roots every 10 days for 3-5 consecutive sprays. Spraying with imidacloprid and frequent ventilation are also options.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for cultivating begonia tissue culture seedlings, characterized in that: The steps include: (1) After the begonia explants are sterilized, they are inoculated into a virus-free culture medium and cultured to obtain clustered buds; (2) inoculating the clustered buds into an induction culture medium, inducing culture, and obtaining new buds; (3) inoculating the new shoots into a proliferation medium, and culturing them to obtain clustered adventitious shoots; (4) inoculating the clustered adventitious buds into a rooting medium, performing rooting culture, and hardening the seedlings to obtain begonia tissue culture seedlings; The virus-free culture medium comprises MS+0.5-1.5 mg / L 6-BA+0.08-0.15 mg / L NAA+5-8 g / L agar+28-32 g / L sucrose; The induction medium includes MS+1.2 mg / L 6-BA+0.3 mg / L NAA+6-7 g / L agar+30 g / L sucrose; The proliferation culture medium includes MS+0.5mg / L 6-BA+0.3mg / L NAA+6-7g / L agar+30g / L sucrose; The rooting medium includes 1 / 2MS+1.0mg / L NAA+6g / L agar+30g / L sucrose; The begonia includes one or more of Begonia masoniana, Begonia picturata, Begonia bervirimosassp.exotica, Begonia'High Way', Begonia Fireworks, and Begonia'ZhouHaiDan'; The light intensity of the virus-free culture is 2000-2500Lx.

2. The culture method according to claim 1, wherein The virus-free culture time is 10 to 18 days, and the virus-free culture temperature is 22 to 26°C.

3. The culture method according to claim 1, wherein The induction culture time is 20 to 45 days, and the induction culture temperature is 22 to 26°C.

4. The culture method according to claim 1, wherein The proliferation culture time is 35 to 45 days, and the proliferation culture temperature is 22 to 26°C.

5. The culture method according to claim 1, wherein The rooting culture time is 20 to 35 days, and the rooting culture temperature is 22 to 26° C. The time for hardening the seedlings is 4 to 15 days.

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