Method for culturing begonia tissue culture seedlings

Abstract

The invention provides a culture method of begonia tissue culture seedlings, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) sterilizing begonia explants, inoculating the begonia explants into a detoxification culture medium, and performing detoxification culture to obtain cluster buds; (2) inoculating the cluster buds into an induction culture medium, and performing induction culture to obtain new buds; (3) inoculating the new buds into a proliferation culture medium, and carrying out proliferation culture to obtain clustered adventitious buds; and (4) inoculating the clustered adventitious buds into a rooting culture medium, and performing rooting culture and seedling hardening to obtain begonia tissue culture seedlings. According to the method, the begonia tissue culture seedlings are cultured by using the specific induction culture medium, the proliferation culture medium and the rooting culture medium, so that the proliferation coefficient, the rooting average number, the rooting rate and the adventitious bud number of the begonia tissue culture seedlings are obviously improved. Meanwhile, the tissue culture seedlings are cleaned after seedling hardening, and the relative transplanting survival rate of the tissue culture seedlings is increased. The method can be used for industrialized seedling production, and solves the problems of low reproduction rate and non-uniform growth uniformity in the existing cultivation method.

Description

Technical Field

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

[0002] Begonia is a large genus in the plant kingdom, distributed in the pantropical regions of the earth. Due to habitat degradation and interference from human activities, many species are threatened to varying degrees, so its protection is urgent.

[0003] As a typical shade-loving plant, Begonia has a rich variety of species, colorful leaf shapes and colors, and beautiful flowers. It has extremely high horticultural ornamental and application value and is also known as the "king of foliage plants." There are four main ways to propagate Begonia: sowing, cuttings, division, and tissue culture. The first three methods can be used effectively, but the speed of propagation is slow and cannot meet market demand. In order to meet the large demand for Begonia by urban greening and home gardening enthusiasts, a method for factory-based Begonia seedling cultivation is urgently needed.

[0004] Although tissue culture propagation is the best method to achieve large-scale production. However, in the process of propagating begonias by tissue culture propagation, the selection of culture medium and the concentration of each nutrient element in the culture medium play a decisive role in the proliferation of seedlings. For example, when the concentration of 6-BA in the culture medium is too high, the growth of seedlings is inhibited and glass seedlings will appear. If the concentration is too low, the differentiation rate will decrease and the proliferation effect cannot be achieved. Therefore, the best culture medium formula for different cultivation periods of begonia factory seedlings is screened out, so as to achieve the purpose of rapid propagation while solving the problems of low reproduction 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, rooting average number, rooting rate and number of adventitious buds of begonia tissue culture seedlings, and effectively solves 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) sterilizing begonia explants and inoculating them into a virus-free culture medium, performing virus-free culture, and obtaining clustered buds;

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

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

[0011] (4) Inoculating the clustered adventitious buds into a rooting medium, performing rooting culture, and hardening the seedlings to obtain begonia tissue culture seedlings.

[0012] Preferably, the detoxified 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.

[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+0.5-1.5mg / LNAA+5-8g / L agar+28-32g / 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 'ZhouHaiDan'.

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

[0023] Figure 1 To obtain clustered buds by inoculating begonia explants into virus-free 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 are inoculated into a proliferation medium for proliferation culture to obtain a clustered adventitious shoot diagram;

[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 of tissue culture seedlings after hardening;

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

[0031] Fig. 9 This is the status of the plug tray seedlings one week after being placed on the tray;

[0032] Fig.10 To provide maintenance diagram for plug tray seedlings;

[0033] Fig.11 This is a diagram for shading the plug tray seedlings;

[0034] Fig.12 This is a diagram of the growth of plug tray seedlings when the humidity is high;

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

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

[0037] Fig.15 This is a display of Zhou Haidan’s flower show in the greenhouse;

[0038] Fig.16 Showing pictures of crabapples grown outdoors;

[0039] Fig.17 Schematic diagram of begonia flower display outdoors. DETAILED DESCRIPTION

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

[0041] (1) sterilizing begonia explants and inoculating them into a virus-free culture medium, performing virus-free culture, and obtaining clustered buds;

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

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

[0044] (4) Inoculating the clustered adventitious buds into a rooting medium, performing rooting culture, and hardening the seedlings to obtain begonia tissue culture seedlings.

[0045] In the present invention, after the begonia explants are disinfected, they are inoculated into a virus-free culture medium for virus-free culture to obtain clustered buds. The disinfection liquid used includes: 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 one or more of sterile water, preferably 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 one or more of sterile water, and further preferably 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 disinfection first, and then to use the 84 disinfectant for soaking 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, further 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, and further preferably 15d; the temperature of the detoxification culture is 22-26°C, preferably 23-25°C, and further preferably 24°C; the light intensity of the detoxification culture is 2000-2500Lx, preferably 2200-2400Lx, and further 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 medium, the bottom 2 to 3 mm is preferably cut off to allow the bud segments to better absorb the nutrients in the proliferation medium; the induction 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, and 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, and further preferably 40 days; the induction culture temperature is 22 to 26°C, preferably 23 to 25°C, and further preferably 24°C.

[0047] In the present invention, the new shoots are inoculated into a proliferation culture medium and cultured for proliferation to obtain clustered adventitious shoots. The length of the new sprout is 4-5 mm, preferably 4.2-4.8 mm, and more preferably 4.4-4.6 mm; the proliferation culture medium includes: 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 d, preferably 38-41 d, and more preferably 40 d; 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 the seedlings are 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 includes: 1 / 2MS + 0.5 to 1.5 mg / L NAA + 5 to 8 g / L agar + 28 to 32 g / L sucrose, preferably: 1 / 2MS + 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 described in detail. 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 more preferably 3500Lx; after the hardening, the tissue culture seedlings with strong growth and root length exceeding 2cm 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 immersed in clean water, so as to improve 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

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

[0052] (1) The terminal buds of young plants of begonias without diseases and insect pests were collected as explants of begonias, 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. Then, the plants were 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 then 0.5% Tween-80 was added and soaked in 0.1% mercuric chloride solution by volume for 2 min, and gently shaken, rinsed with sterile water for 5 times, and excess water was absorbed with sterile filter paper, and then inoculated into MS+1.0 mg / L In the detoxified medium of 6-BA+0.1mg / LNAA+6g / L agar+30g / L sucrose, only one apical bud was inoculated into each bottle of medium, and the light culture time was set to 14h, dark culture time was set to 10h, and cultured statically on a culture rack at a light intensity of 2000Lx and 24±2℃ for 15d. If no contamination occurs, it means that the detoxification is completed, and the dead explants and contaminated explants that appear during the disinfection process are removed to obtain clustered buds;

[0053] (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+1.2 mg / L 6-BA+0.3 mg / L NAA+6 g / L agar+30 g / L sucrose. Inoculate only one bud segment into each bottle of culture medium, and incubate at 24 ± 2 °C for 20 days to obtain new buds.

[0054] (3) In a clean bench, cut off the new shoots with a length of 4 to 5 mm and inoculate them one by one in a proliferation medium containing MS + 0.50 mg / L 6-BA + 0.30 mg / L NAA + 6 g / L agar + 30 g / L sucrose. Inoculate only one new shoot in each bottle of culture medium and incubate at 24 ± 2 °C for 20 days. Begin to observe the differentiation of the new shoots. It was found that the proliferation rate reached the highest level at 40 days of culture, and the callus 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 reaches 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 inoculate them into a bottle of rooting medium containing 1 / 2MS + 1.0 mg / L NAA + 6 g / L agar + 30 g / L sucrose (one bottle of propagated seedlings can be cut into the amount of two bottles of rooting seedlings). Incubate at 24±2℃ for 7 days until roots grow, and then transfer to a culture rack with sufficient light and 24±2℃ for further culturing for 20 days. Set the light culture time to 14 h and the dark culture time to 10 h, 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 20°C, a humidity of 60%-80%, and a light intensity of 2000Lx, open the bottle mouth and place it for 3 days, and then place it for 1 day with the bottle mouth loosened, half-opened, and fully opened respectively. Take out the tissue culture seedlings with healthy growth and root length exceeding 2 cm, wash the culture medium at the roots 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, and 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 then soaked in 0.1% mercuric chloride solution by volume for 2 min, and gently shaken, rinsed with sterile water 5 times, and the excess water was absorbed with sterile filter paper, and then inoculated with MS+1.0 mg / L In the detoxification medium of 6-BA+0.1mg / LNAA+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 statically cultured on a culture rack at a light intensity of 2500Lx and 24±2℃ for 15d. If no contamination occurred, it indicated that the detoxification was completed. The dead explants and contaminated explants that appeared during the disinfection process were removed to obtain clustered buds;

[0059] (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 g / L agar+30 g / L sucrose. Inoculate only one bud segment into each bottle of culture medium, and incubate at 24 ± 2 °C for 40 days to obtain new buds.

[0060] (3) In a clean bench, cut off the new shoots with a length of 4 to 5 mm and inoculate them one by one in a proliferation medium of MS + 0.50 mg / L 6-BA + 0.30 mg / L NAA + 6 g / L agar + 30 g / L sucrose. Inoculate only one new shoot in each bottle of culture medium and incubate at 24 ± 2 °C. Observe the differentiation of the new shoots at 20 days. It was found that the proliferation rate reached the highest at 40 days, and the callus 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;

[0061] (4) When the number of sterile seedlings in the bottle reaches 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 inoculate 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, and then transfer to a culture rack with sufficient light and 24 ± 2 ° C. Continue to cultivate for 20 days, place the culture bottles containing tissue culture seedlings with a growth height of ≥2cm and ≥3 leaves in a transplanting environment with a temperature of 28℃, a humidity of 60%-80%, and a light intensity of 5000Lx, open the bottle mouth and place it for 3 days, and then place it for 1 day with the bottle mouth loosened, half-opened, and fully opened respectively. Take out the tissue culture seedlings with healthy growth and root length exceeding 2cm, wash the culture medium at the roots 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 the surface dust was washed off with running water, the plants were placed in 200 g / L detergent and soaked for 30 min. The plants were brushed with a brush while soaking and rinsed with running water for 30 min. The plants were then soaked in 5% 84 disinfectant for 30 min. The plants were 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. The plants were then soaked in 0.1% mercuric chloride solution by volume for 2 min and gently shaken. The plants were rinsed with sterile water for 5 times. The excess water was absorbed with sterile filter paper and the plants were inoculated with MS+1.2 mg / L 6-BA+0.1 mg / L In the detoxification medium of NAA+6g / L agar+30g / L sucrose, only one apical bud was inoculated into each bottle of medium, and the light culture time was set to 14h, dark culture time was set to 10h, and static culture was placed on the culture rack at a light intensity of 2300Lx and 24±2℃ for 15d. If no contamination occurred, it means that the detoxification is 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. Inoculate only one bud segment into each bottle of culture medium, and incubate 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 into the 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°C for 20 days and the differentiation of the new shoots was observed. It was found that the proliferation rate reached the highest point when the callus was healthy yellow-green at 40 days. The yellow leaves of the clustered adventitious buds were few. This was the optimal turnover date. At this time, the sterile seedlings performed normally and the clustered adventitious buds reached the rooting standard. After 9-10 generations of continuous culture, 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 reaches 100, cut buds with a length of 1 to 1.5 cm in the clean bench, use tweezers to take 10 buds and inoculate them into a bottle of 1 / 2MS + 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 two bottles of rooting seedlings) and 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 cultivation for 25 days. 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 4000Lx. The bottles were opened for 4 days, and then placed for 2 days with the bottles loosened, 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 in 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 "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+6g / L agar+30g / L sucrose; Medium 1 is MS+0.3mg / L 6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose; Medium 2 is MS+0.5mg / L 6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose; Medium 3 is MS+0.8mg / L6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose.

[0073] Example 5

[0074] Begonia tissue culture seedlings were cultured using the method in Example 1, with the only difference being that "rooting medium comprising 1 / 2MS + 1.0 mg / L NAA + 6 g / L agar + 30 g / L sucrose" was replaced with "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+6g / L agar+30g / L sucrose; Medium 1 is MS+1.0mg / L 6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose; Medium 2 is MS+1.2mg / L 6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose; Medium 3 is MS+1.5mg / L6-BA+0.3mg / L NAA+6g / L agar+30g / L sucrose.

[0083] Comparative Example 1

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

[0085] Comparative Example 2

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

[0087] Comparative Example 3

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

[0089] Test Example 1

[0090] According to the method in Example 1 and Comparative Example 1, 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), Begonia 'ZhouHaiDan' (also known as 'Zhou Haidan' Begonia) tissue culture seedlings were cultured respectively, and during proliferation culture, the next generation of new shoots were selected every 30 days and inoculated into a new proliferation medium, and repeated 9 times, and the rooting of each generation of Begonia sterile seedlings 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 have grown to the point where they can be cut and used 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 sterile begonia seedlings grew normally, with unfolded and emerald green leaves, and produced 12 to 18 adventitious roots. At the same time, the adventitious roots produced met the rooting standards.

[0095] Test Example 2

[0096] According to the methods of Example 1, Example 6 and Comparative Example 3, tissue culture seedlings of Begonia masoniana (also known as 'Iron Armor' Begonia), Begonia picturata (also known as 'One 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), Begonia 'ZhouHaiDan' (also known as 'Zhou Haidan' Begonia) were cultured respectively, and the growth of clustered adventitious buds was observed, and the number of adventitious buds was determined, and the induction rate of clustered adventitious buds 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 germinating explants / number of inoculated explants × 100%

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

[0100]

[0101] The results showed that at the 45th day of proliferation culture, the plants cultured in Comparative Example 3 were short, had thin and weak adventitious buds, and small leaves, which did not meet the standard of rooted seedlings (leaves ≥3, and the diameter of 3 leaves ≥0.6 cm); while the adventitious buds of the plants cultured in Examples 1 and 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), Begonia 'Zhou Hai Dan' (also known as 'Zhou Hai Dan' Begonia) were cultured respectively, and the rooting conditions were observed during rooting culture to the 15th and 20th days; 15 bottles were randomly selected from 60 bottles of culture bottles randomly selected, and the rooting conditions were counted; at the same time, the growth of tissue culture seedlings in different rooting culture media was observed, and the average number of roots and rooting rate were calculated. The results are shown in Table 6. The statistical formula for 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 most of the rooting seedlings had roots on the 15th day of rooting culture, and each rooting seedling had 4 to 10 fibrous roots on the 20th day of culture, with secondary roots and tertiary roots. The tissue culture seedlings cultured in Comparative Example 2 had light green leaves and few and thin roots, while the tissue culture seedlings cultured in Examples 1 and 5 grew luxuriantly, had dark green leaves, and had many and 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 leaves reach 4 leaves or more, take the culture bottle out of the culture room and put 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 state for one day each. When the light intensity exceeds 15000Lx, it is necessary to shade it. 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 promptly soaked in 5g / L potassium permanganate solution for 2 minutes for disinfection, and then washed with clean water for transplanting.

[0111] 2. Transplantation

[0112] 2.1 Transplanting medium

[0113] The transplanting medium is peat soil with 10% perlite, 3 Add 150g of 75% carbendazim or 50% carbendazim wettable powder to the substrate, and use 0.5g / L potassium permanganate solution (or 1000 times diluted phoxim granules) for sterilization and insecticide treatment every other day. The specific steps are to spray the prepared medicine on the prepared cultivation substrate with a sprayer, and stir the substrate continuously to mix it evenly until the water content reaches more than 20%, and the soil is held in a ball by hand and can be scattered on the ground (the 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×28cm, an upper diameter of 3.8cm, a lower diameter of 2cm, and a height of 4.5cm. Disinfect with 0.5% potassium permanganate solution 1 to 3 days before transplanting.

[0116] 2.3 Transplanting method

[0117] Take out the tissue culture seedlings from the culture bottle and clean the culture medium at the root of the seedlings with clean water in time (Note: Begonia is 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 on the survival rate of final transplantation

[0119]

[0120] Note: The relative transplant survival rate refers to the proportion 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 is significantly reduced, sometimes by 50% to 60%. This situation is 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 (those with a growth height of ≥3 cm and ≥5 leaves are large seedlings, and those with a growth height of <2 cm and ≥3 leaves are small seedlings) and transplant them in trays ( Figure 8 ), use a bamboo stick to make holes in the substrate, put the roots of the seedlings directly into the hole in the tray, ensure that the roots are stretched, pinch the soil tightly, and immediately water the roots with clean water after the tray is put on. Spray it lightly with a sprayer to fully fit the substrate and the tray together. (Note: The substrate has been irrigated after the tray is put on) After transplanting, cover the film to keep it moist, and punch holes in the film for ventilation. The operation is carried out in a shade shed, and the conditions of the shade shed are 60% to 80% shading, 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 plug tray is placed, it needs to be moisturized and heat-insulated, and shaded. The shade degree is 60% to 80% of the light intensity without shade, 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 carried out to cool down. Ventilation should be carried out for half an hour in the afternoon when the light is weak. When it is rainy, light and ventilation are needed. If there is no ventilation and the humidity is too high, mold and fungi will grow (such as Fig.12 ). Water once every 3-4 days, and spray once every 7-10 days with 500 times dilution of carbendazim wettable powder. The film can be removed after new leaves grow out, but shade is still required.

[0125] 3. Seedlings

[0126] When the root system of the plug seedlings is fully grown (such as Fig.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 management

[0128] Entering the maintenance and management stage, the greenhouse summer management: shading, moisturizing, ventilation. In summer, the temperature is kept below 25℃, watering once every 2-3 days, and the relative humidity of the air is controlled at about 60%. In winter, the temperature is kept above 5℃, and the temperature difference of watering in winter cannot exceed 5℃. Water once a week, and the key is to keep warm. Begonia needs N during its growth period. When using fertilizers with high nitrogen content such as urea, it is necessary to prevent seedlings from burning. You can often spray "Hua Duoduo 20-20-20" 1000 times liquid, spray once every 20 days in summer, and spray an appropriate amount (it is advisable to water the pot soil thoroughly). Snails and slugs are prone to occur in the seedling stage. Stem rot and root rot are prone to occur in the high temperature and high humidity environment of the greenhouse. The diseased plants should be removed immediately at the early stage of the disease, and 50% carbendazim 500-600 times liquid should be sprayed or irrigated with roots. Spray once every 10 days, and it is advisable to water the pot soil thoroughly. Spray 3-5 times in a row. You can also take methods such as spraying imidacloprid and more ventilation to prevent and control.

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

[0130] When planting greenhouse flower walls, avoid high temperatures and high humidity, which are conducive to the reproduction of pathogens and the formation of rot diseases. Therefore, close planting should be avoided to facilitate ventilation (such as Fig.14 and Fig.15 ). When planting in the field (such as Fig.16 and Fig.17 ), maintenance and management are relatively simple. Drainage should be done in time during the rainy season. In winter, seedlings need to be moved to greenhouses. In Shijiazhuang, they cannot overwinter in the open field. Snails and slugs are prone to occur in the seedling stage. Stem rot and root rot are prone to occur in the high temperature and high humidity environment of the greenhouse. In the early stage of the disease, the diseased plants should be removed immediately, and 50% carbendazim 500-600 times liquid should be sprayed or rooted, sprayed once every 10 days, and sprayed 3-5 times in a row. You can also take measures such as spraying imidacloprid and more ventilation to prevent and control.

[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 principle of the present invention. These improvements and modifications should also be regarded as 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) sterilizing begonia explants and inoculating them into a virus-free culture medium, performing virus-free culture, and obtaining clustered buds; (2) inoculating the clustered buds into an induction medium, inducing the buds, and obtaining new buds; (3) inoculating the new shoots into a proliferation medium, and culturing the proliferation 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.

2. The culture method according to claim 1, characterized in that The detoxified 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.

3. The culture method according to claim 2, characterized in that The time of the virus-free culture is 10 to 18 days, and the temperature of the virus-free culture is 22 to 26°C.

4. The culture method according to claim 1, characterized in that The induction culture 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.

5. The culture method according to claim 4, characterized in that The induction culture time is 20 to 45 days, and the induction culture temperature is 22 to 26°C.

6. The culture method according to claim 1, characterized in that 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.

7. The culture method according to claim 6, characterized in that: The proliferation culture time is 35-45 days, and the proliferation culture temperature is 22-26°C.

8. The culture method according to claim 1, characterized in that: The rooting culture medium comprises 1 / 2MS+0.5-1.5mg / LNAA+5-8g / L agar+28-32g / L sucrose.

9. The culture method according to claim 8, characterized in that: 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.

10. The culture method according to claim 1, characterized in that: The begonia includes one or more of Begoniamasoniana, Begonia picturata, Begonia bervirimosa ssp.exotica, Begonia'HighWay', Begonia Fireworks, and Begonia'ZhouHaiDan'.

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