Tissue culture method of Zhongshu 10 medicago sativa

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method of medicago sativa var. Sativa 10. The tissue culture method comprises the following specific steps: firstly disinfecting seeds for culture, then performing callus induction and subculture, then performing differentiation culture, and finally performing rooting culture and transplanting. The tissue culture method suitable for the Zhongshu 10 medicago sativa and four culture media of callus induction, subculture, differentiation and rooting are constructed for the first time; the germination rate is high, the differentiation rate is high, the tissue culture method is simple and convenient, the feasibility is high, and a technical guarantee is provided for protecting the Zhongshu 10 medicago sativa material; and a technical basis is provided for improving the Zhongqiu No.10 medicago sativa.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method for Zhongmu No. 10 alfalfa. Background Art

[0002] Alfalfa variety Zhongmu No. 10 is an important high-quality forage grass in northern my country. Establishing a tissue culture system for it, strengthening the cultivation and propagation of high-quality native grass varieties, is of great significance to the protection of high-quality forage grasses. At the same time, alfalfa tissue culture is a way to achieve high-quality alfalfa production. The establishment of an alfalfa genetic transformation system and the functional verification of some genes rely on its complete tissue culture technology. During the transformation process, the transformation conditions and culture conditions are improved on the basis of tissue culture. Therefore, it is particularly important to establish a complete tissue culture program. Due to the characteristics of autotetraploidy and cross-pollination, alfalfa varieties have significant genotypic differences, which greatly hinders the breeding of new varieties. Currently, many varieties of alfalfa have established tissue culture technology. For example, Chinese patent CN202311553960.6 discloses a tissue culture propagation method for alfalfa. The hypocotyls of sterile wild alfalfa seedlings in Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region are used as explants and inoculated on a callus induction medium for culture to obtain callus tissue, and then the callus tissue is induced to sprout. The adventitious buds on the clustered buds that have been differentiated and cultured are separated and inoculated on a rooting medium for culture to obtain regenerated seedlings, which are then hardened and transplanted to obtain alfalfa seedlings. By optimizing the callus induction medium, the sprouting induction medium and the rooting medium, the callus culture rate, the sprouting induction rate, the rooting induction rate and the hardening and transplanting survival rate are improved, which provides a technical basis for the tissue culture propagation of alfalfa in Xinjiang Uygur Autonomous Region. Chinese patent CN201210440085.6 discloses a method for the aseptic tissue culture and rapid propagation of wild alfalfa (Medicagao sativa L.) in Deqin. The method involves using young leaves of wild alfalfa for protocorm induction, differentiation, seedling strengthening, and rooting culture. The cultivated wild alfalfa has a proliferation coefficient of 10 within one month, an average rooting rate of 98%, and an average transplant survival rate of 95%.

[0003] However, there is currently no report on the tissue culture technology system for Zhongmu No. 10 alfalfa. As an important forage plant in northern my country, establishing a complete tissue culture system for Zhongmu No. 10 alfalfa is of great significance for the protection and improvement of excellent forage. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a tissue culture method for alfalfa Zhongmu No. 10, which constructs a tissue culture system for Zhongmu No. 10 through seed disinfection, callus induction, callus differentiation and rooting of differentiated seedlings.

[0005] In order to achieve the above object, the present invention provides a tissue culture method of alfalfa cultivar Zhongmu No. 10, which comprises the following steps:

[0006] (1) Seed disinfection and cultivation

[0007] Select plump Zhongmu No. 10 seeds, wrap them with gauze, rinse them under tap water for 10-30 minutes, then remove damaged seeds and seed coats, disinfect them in a sterile environment, first with alcohol, then with HgCl2 solution, then rinse with sterile water, then add sterile water to cover the seeds, and culture them in a 24°C incubator in the dark for 10-20 hours, then pour out the sterile water in a clean bench, and use sterile filter paper to absorb the moisture on the surface of the clean seeds, then inoculate them on MS culture medium and culture them in a 24°C light incubator with 16 hours of light per day. Seedlings will grow after about 7 days of culture;

[0008] Preferably, the alcohol disinfection time is 1 min; the HgCl2 disinfection time is 6-8 min;

[0009] (2) Callus induction and subculture: The cotyledons of Zhongmu No. 10 seedlings were used as explants. The cotyledons were cut off with a knife and cut into two halves horizontally. The leaves were spread flat on the callus induction medium with the leaves facing upwards. The culture medium was placed in a 24°C incubator in the dark for about 20-30 days for callus to grow well. The callus induction medium was based on MS medium, supplemented with sucrose, plant gel, 2,4-D and 6-BA. The concentration of 2,4-D was 0.3-1 mg / L, the concentration of 6-BA was 1 mg / L, the concentration of sucrose was 30 g / L, and the concentration of plant gel was 3 g / L. The optimal concentration of 2,4-D was 0.5 mg / L.

[0010] After callus tissue is induced, the excess leaves are cut off, and the induced embryonic callus of Zhongmu No. 10 is cut into soybean-sized pieces and placed in a subculture medium in a dark incubator at 24°C for subculture. The subculture medium is based on MS medium supplemented with 1 mg / L 6-BA and 0.1 mg / L NAA. Subculture is performed once every 15 days, and a total of 1-2 subcultures are performed until the callus becomes dense and crystal-clear.

[0011] (3) Callus differentiation

[0012] After the callus subculture is completed, dense and compact calli are selected and placed in differentiation medium. Five calli are placed in each culture bottle and placed in a 24°C incubator with 16 hours of light per day. Differentiation seedlings will grow after about 20-30 days of culture.

[0013] The differentiation medium is based on MS medium, supplemented with sucrose, phytagel, NAA, KT, 6-BA, and hydrolyzed casein. The concentrations of NAA, KT, 6-BA, 6-BA, and hydrolyzed casein are 0.1-1 mg / L, 0.1 mg / L, 0.2-1 mg / L, 3 mg / L, 30 g / L, and 3 g / L, respectively.

[0014] Preferably, the concentration of NAA is 1 mg / L, and the concentration of 6-BA is 0.2 mg / L;

[0015] (4) Differentiation seedlings take root

[0016] After the differentiated seedlings grow to 4-6 cm, they are inoculated into a rooting medium using 1 / 2 MS medium as the base medium, supplemented with 20 g / L sucrose, 3 g / L phytogel, and 0.2-0.6 mg / L IBA hormone. They are first cultured in a 24°C incubator in the dark for 2 days, then placed in a 24°C incubator with 16 hours of light per day for 10-15 days for roots to emerge.

[0017] Preferably, the concentration of IBA is 0.6 mg / L;

[0018] (5) Transplantation After the differentiated seedlings take root, wait until they grow to about 8-10cm, open the tissue culture bottle, add a small amount of pure water, and harden the seedlings at room temperature for one day. Then take out the tissue culture seedlings, rinse the culture medium at the roots under tap water, move them into nutrient soil, soak them with pure water, cover them with film and protect them from light, then open the lid and grow them at room temperature.

[0019] In view of the problems that the current tissue culture method of alfalfa has poor tissue culture effect on Zhongmu No. 10, low germination rate, low differentiation rate, etc., the present invention has developed a tissue culture method suitable for Zhongmu No. 10 alfalfa, innovatively studied disinfection reagents and disinfection methods suitable for Zhongmu No. 10, and at the same time clarified the nutritional components and ratios of the culture medium required for each stage of the tissue culture of Zhongmu No. 10 alfalfa. Using this tissue culture method, the germination rate was improved to 86.67%.

[0020] Compared with the prior art, the present invention selects the Inner Mongolia dominant forage grass Zhongmu No. 10 alfalfa material, and constructs for the first time a tissue culture method suitable for Zhongmu No. 10 alfalfa, including five parts: seed disinfection, callus induction, callus subculture, callus differentiation, rooting of differentiated seedlings, and transplanting, as well as four culture media for callus induction, subculture, differentiation, and rooting; the present invention has a high germination rate, a high differentiation rate, a simple tissue culture method, and high feasibility, providing technical guarantee for protecting the Zhongmu No. 10 alfalfa material and a technical basis for improving the Zhongmu No. 10 alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a germination diagram of the Zhongmu No. 10 seeds involved in the present invention after being disinfected by method 1.

[0022] Figure 2 This is a callus induction diagram of Zhongmu No. 10 involved in the present invention.

[0023] Figure 3 This is a diagram of the callus subculture of Zhongmu No. 10 involved in the present invention.

[0024] Figure 4 This is the differentiation diagram of Zhongmu No. 10 involved in the present invention.

[0025] Figure 5 This is the rooting diagram of Zhongmu No. 10 involved in the present invention, wherein A is the rooting process diagram, B is the SG3 group root diagram, and C is the SG2 group root diagram.

[0026] Figure 6 This is a diagram of transplanting the tissue culture seedlings of Zhongmu No. 10 involved in the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0028] Example 1:

[0029] This embodiment relates to a tissue culture method of alfalfa cultivar Zhongmu No. 10, and the specific steps are as follows:

[0030] (1) Seed disinfection and cultivation Five disinfection methods were set up. Method 1: Wrap an appropriate amount of Zhongmu No. 10 seeds with gauze and rinse under tap water for about 20 minutes. Then put them in a small beaker, add pure water to cover the seeds, pour out the damaged seeds and seed coats suspended on the water surface, repeat several times, then put the full seeds in a sterile flask in a sterile environment, disinfect with 75 v / v% alcohol for 1 minute, and then disinfect with 0.1wt% HgCl2 aqueous solution for 6-8 minutes, shaking continuously during the period, and rinse with sterile water 5 times after disinfection; add sterile water to cover the seeds, seal the flask and place it in a 24℃ incubator for dark incubation for about 15 hours; Method 2: Take an appropriate amount of Zhongmu No. 10 seeds and place them in a small beaker. After washing them several times with tap water, soak them in purified water for 3 hours, discard the damaged seeds and seed coats suspended on the water surface, and repeat several times. Then, place the full seeds in a sterile Erlenmeyer flask in a sterile environment, disinfect them with 75v / v% alcohol for 30 seconds, and soak them in 0.1wt% HgCl2 aqueous solution for 8 minutes, shaking them continuously during the process. After disinfection, rinse them with sterile water 5 times;

[0031] Method 3: Take an appropriate amount of Zhongmu No. 10 seeds and place them in a small beaker. After washing them several times with tap water, soak the seeds in purified water for 40 minutes, discard the damaged seeds and seed skins suspended on the water surface, and repeat several times. Then, place the full seeds in a sterile Erlenmeyer flask in a sterile environment, soak them in 70v / v% alcohol for 40-60 seconds, rinse them with sterile water 5 times, soak them in 20wt% sodium hypochlorite aqueous solution for 25 minutes, and rinse them with sterile water 5 times.

[0032] Method 4: Take an appropriate amount of Zhongmu No. 10 seeds and place them in a small beaker. After washing them several times with tap water, soak the seeds in purified water for 3 hours, disinfect them with 75 v / v% alcohol for 1 minute, and soak them in 0.15 wt% HgCl2 aqueous solution for 5 minutes, shaking them continuously. After disinfection, rinse them with sterile water 5 times. Method 5: Wrap an appropriate amount of Zhongmu No. 10 seeds with gauze and rinse under tap water for about 20 minutes. Then put them in a small beaker and add pure water to cover the seeds. Pour out the damaged seeds and seed coats suspended on the water surface. Repeat several times. Then put the full seeds in a sterile Erlenmeyer flask in a sterile environment, disinfect with 75 v / v% alcohol for 1 minute, and then disinfect with 0.1wt% HgCl2 aqueous solution for 6-8 minutes, shaking continuously during the process. After disinfection, rinse with sterile water 5 times; The seeds disinfected by the above five methods were poured out of sterile water in a clean bench, and the water on the surface of the clean seeds was absorbed with sterile filter paper. The seeds were inoculated on MS culture medium and placed in a 24℃ light incubator for cultivation. The light intensity was 4000 LX and the seedlings grew out after about 7 days of cultivation ( Figure 1 ), the germination rate and contamination situation were counted, and the results are shown in Table 1;

[0033] Table 1 Sprouting conditions under different disinfection methods

[0034] The germination rates of the five disinfection methods were 86.67%, 5.00%, 36.67%, 6.67%, and 14.17%, respectively. Method 3 had a high contamination rate, while the other three methods were uncontaminated. Method 1 had the highest germination rate and was the most suitable method for seed disinfection of Zhongmu No. 10.

[0035] (2) Callus induction and subculture

[0036] The cotyledons of Zhongmu No. 10 seedlings, which were cultured for about 7 days, were selected as explants. The cotyledons were cut off with a knife and cut into two halves horizontally. The leaves were spread flat on the callus induction medium with 40 explants per dish. The dishes were placed in a dark incubator at 24°C for about 20-30 days for callus to grow well.

[0037] The callus induction medium was based on MS medium supplemented with 30 g / L sucrose, 3 g / L phytagel, 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg / L 6-BA (6-benzylaminopurine). Three 2,4-D concentrations (Table 2) were set for callus induction experiments. The experimental results showed that the callus induction rate of Zhongmu No. 10 increased with increasing 2,4-D concentration, but high concentration (1 mg / L) of 2,4-D inhibited its callus induction. Among them, YD2 medium induced the best callus, with an induction rate of 93.08%. The optimal callus induction medium formula was: based on MS medium supplemented with 30 g / L sucrose, 3 g / L phytagel, 0.5 mg / L 2,4-D, and 1 mg / L 6-BA.

[0038] Table 2 Effects of different hormone concentration combinations on callus induction of Zhongmu No. 10

[0039] After the callus tissue was induced, the excess leaves were cut off and the induced embryonic callus of Zhongmu No. 10 ( Figure 2 ) cut into soybean-sized pieces and placed in subculture medium in a dark incubator at 24°C for subculture. The subculture medium is based on MS medium, supplemented with 1mg / L 6-BA and 0.1mg / L NAA (1-naphthaleneacetic acid). Subculture is performed once every 15 days, and a total of 1-2 subcultures are performed until the callus becomes dense and crystal-clear. Figure 3 ).

[0040] (3) Callus differentiation

[0041] After the callus subculture is completed, dense and compact calli are selected and placed in differentiation medium. Five calli are placed in each culture bottle and placed in a 24°C incubator with 16 hours of light and 8 hours of dark culture per day. The light intensity is 4000LX. Differentiation seedlings will grow after about 20-30 days of culture ( Figure 4 );

[0042] The differentiation medium used MS medium as the basal medium, supplemented with 30 g / L sucrose, 3 g / L phytogel, and NAA, KT (6-furfurylaminopurine), 6-BA, and CH hormone (hydrolyzed casein). Ten concentrations were used (Table 3). Experimental statistical results showed that FH2 medium had the best differentiation effect, with a differentiation rate of 33.33%.

[0043] Table 3 Effects of different hormone concentration combinations on differentiation of Zhongmu 10

[0044] (4) Differentiation seedlings take root

[0045] After the differentiated seedlings grew to about 5 cm, they were inoculated into rooting medium, which was a 1 / 2 MS medium with 20 g / L sucrose, 3 g / L plant gel, and different concentrations of IBA hormone (indolebutyric acid) added for rooting (Table 4). They were first cultured in a 24°C incubator in the dark for 2 days, and then cultured in a 24°C incubator with 16 / 8 h light and dark for 10-15 days to produce roots ( Figure 5 A), on the 15th day, the statistical results showed that the rooting rate was the highest when 0.6mg / L IBA hormone was added, reaching 26.67%. At the same time, SG3 group ( Figure 5 B) Compared with SG2 group ( Figure 5 C), the root system is thicker and more conducive to growth, so the SG3 medium is more suitable for rooting. The experimental results also show that too low a hormone concentration cannot promote the rooting of alfalfa seedlings, while too high a hormone concentration will inhibit rooting.

[0046] Table 4 Effects of different hormone concentration combinations on alfalfa rooting

[0047] (5) Transplantation

[0048] After the differentiated seedlings take root, wait until they grow to about 8-10cm, open the tissue culture bottle, add a small amount of pure water, and harden the seedlings at room temperature for one day. Then take out the tissue culture seedlings, rinse the culture medium at the roots under tap water, move them into nutrient soil, soak them with pure water, cover them with film and protect them from light, then open the lid and grow them at room temperature.

Claims

1. A tissue culture method for alfalfa Zhongmu No. 10, characterized in that: The specific steps are as follows: (1) Seed disinfection and cultivation Wrap Zhongmu No. 10 alfalfa seeds in gauze and rinse under tap water for 10-30 minutes, then remove damaged seeds and seed coats; disinfect in a sterile environment, first with alcohol, then with HgCl2 solution, then rinse with sterile water, then add sterile water to cover the seeds, and culture in a 24°C incubator in the dark for 10-20 hours, then pour out the sterile water in a clean bench, and use sterile filter paper to absorb the moisture on the surface of the clean seeds, then inoculate on MS culture medium and culture in a 24°C light incubator with 16 hours of light per day, and grow seedlings after about 7 days of culture; (2) Callus induction and subculture: The cotyledons of Zhongmu No. 10 seedlings were used as explants. The cotyledons were cut off with a knife and cut into two halves horizontally. The leaves were spread flat on the callus induction medium with the leaves facing upwards. The culture was placed in a 24°C incubator in the dark for 20-30 days until the callus grew well. The callus induction medium was based on MS medium, supplemented with sucrose, plant gel, 2,4-D and 6-BA. The concentration of 2,4-D was 0.3-1 mg / L, the concentration of 6-BA was 1 mg / L, the concentration of sucrose was 30 g / L, and the concentration of plant gel was 3 g / L. After callus tissue is induced, the excess leaves are cut off, and the induced embryonic callus of Zhongmu No. 10 is cut into soybean-sized pieces and placed in a subculture medium in a dark incubator at 24°C for subculture. The subculture medium is based on MS medium supplemented with 1mg / L 6-BA and 0.1mg / L NAA hormone solution. Subculture is performed once every 15 days, and a total of 1-2 subcultures are performed until the callus becomes dense and crystal-clear. (3) Callus differentiation After the callus subculture is completed, dense and compact calli are selected and placed in differentiation medium, placed in an incubator at 24°C, and cultured for 16 hours per day. Differentiation seedlings will grow after 20-30 days of culture. The differentiation medium is based on MS medium, supplemented with sucrose, phytagel, NAA, KT, 6-BA, and hydrolyzed casein. In the differentiation medium, the concentration of NAA is 0.1-1 mg / L, the concentration of KT is 0.1 mg / L, the concentration of 6-BA is 0.2-1 mg / L, the concentration of hydrolyzed casein is 3 mg / L, the concentration of sucrose is 30 g / L, and the concentration of phytagel is 3 g / L. (4) Differentiation seedlings take root When the differentiated seedlings grow to 4-6 cm, they are inoculated into rooting medium and cultured in a 24°C incubator in the dark for 2 days, then placed in a 24°C incubator with 16 hours of light per day for 10-15 days for roots to emerge. (5) Transplantation After the differentiated seedlings take root, wait until they grow to 8-10cm, take out the tissue culture seedlings, move them into nutrient soil, and grow them at room temperature.

2. The tissue culture method of alfalfa No. 10 according to claim 1, wherein The concentration of 2,4-D in step (2) was 0.5 mg / L.

3. The tissue culture method of alfalfa No. 10 according to claim 1, characterized in that In step (3), the concentration of NAA is 1 mg / L, and the concentration of 6-BA is 0.2 mg / L.

4. The tissue culture method of alfalfa No. 10 according to claim 1, characterized in that The rooting medium is based on 1 / 2MS medium, supplemented with 20 g / L sucrose, 3 g / L plant gel and 0.2-0.6 mg / L IBA hormone.

5. The tissue culture method of alfalfa No. 10 according to claim 4, characterized in that, The concentration of IBA was 0.6 mg / L.

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