Rapid propagation method of gold bean tissue culture and application

Abstract

The present application relates to the technical field of plant tissue culture, in particular to a kind of golden bean tissue culture rapid propagation method and application;Select non-lignified young stem section as explant, clean and then use 75% alcohol, saturated bleaching powder solution, 0.05% mercury successively disinfection;The explant after disinfection is primary culture for 25-35 days, the single bud of the cluster buds of primary culture is cut and subculture for 40-45 days, and the single bud of the cluster buds obtained by subculture is cut and rooted for 40-45 days.The disinfection contamination rate of the present application can be controlled within 40%, the disinfection mortality rate is controlled within 10%, the success rate of establishing sterile system in laboratory can be effectively improved, the primary induction rate is more than 70%, the proliferation coefficient is more than 4, and the rooting rate is more than 95%.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method and application for rapid propagation of soybean tissue culture. Background Technology

[0002] *Fortunella venosa*, commonly known as the Golden Bean, is a plant belonging to the genus *Fortunella* in the family Rutaceae, and is endemic to China. The plant typically does not exceed 1 meter in height, with short thorns on its branches. Its leaves are leathery and narrowly ovate. The fruit turns orange-red when ripe, about 6-8 mm in diameter, and has a long fruiting period, making it valuable for both its foliage and fruit. Due to its strong adaptability and tolerance to poor soil, it can be artificially cultivated in bonsai to develop gnarled stems, making it a popular material in ornamental horticulture. Wild populations often grow on the edges of evergreen broad-leaved forests or in rock crevices. Habitat reduction due to climate change and over-harvesting for the bonsai trade have led to a decline in its numbers, and it has been listed as a Class II protected plant in China.

[0003] Golden bean trees are primarily propagated through sowing and grafting. Golden bean seeds generally mature in autumn, from September to October. After harvesting, they are sown in spring or late autumn. Seeds sown in autumn will remain dormant and germinate only in spring, resulting in a longer growth cycle for seed propagation. Grafting is the preferred method of propagation. Grafted seedlings are generally more fragile than naturally grown trees. Because a connection is formed between the rootstock and scion, this connection point is susceptible to damage, such as breakage, splitting, or infection by pathogens. Furthermore, grafting between different tree species or varieties is not always successful due to potential genetic and physiological incompatibility. This can lead to grafting failure or the scion failing to fully integrate with the rootstock, thus affecting growth and development. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in current propagation methods by providing a rapid propagation method and application for soybean tissue culture.

[0005] To achieve the above objectives, the technical solution adopted by this invention is: a method for rapid propagation of golden soybeans through tissue culture, comprising the following steps:

[0006] (1) Selection of explants: Select young, non-lignified stem segments as explants;

[0007] (2) Disinfection: After cleaning, the explants are disinfected with 75% alcohol for 50-60 seconds, rinsed with sterile water 2-3 times, disinfected with saturated bleaching powder solution for 20-25 minutes, rinsed with sterile water 4-5 times, and disinfected with 0.05% mercuric chloride for 10-12 minutes, rinsed with sterile water 7-8 times.

[0008] (3) Primary culture: After sterilization, the explants were inoculated into the primary culture medium and cultured in alternating light and dark conditions for 25 to 35 days at a temperature of 25±2℃, with light intensity of 2000 to 3000 lx for 10 to 14 hours per day.

[0009] The formulation of the primary culture medium is: WPM + IBA 0.05~0.08mg / L + 6-BA 3.0~3.5mg / L + ZT 1.0~1.2mg / L + activated carbon 0.5g / L;

[0010] (4) Subculture: The clustered shoots of the primary culture were cut into single shoots and inoculated into the subculture medium. The temperature was 25±2℃, the light intensity was 2000-3000lx, and the light and dark were alternately cultured for 40-45 days.

[0011] The subculture medium was formulated as follows: WPM + IBA 0.01–0.02 mg / L + ZT 0.5–0.8 mg / L + BR 0.01–0.02 mg / L;

[0012] (5) Rooting culture: The clustered shoots obtained from subculture were cut into single shoots and inoculated into rooting culture medium. The shoots were cultured in alternating light and dark conditions for 40 to 45 days at a temperature of 25±2℃, with light intensity of 2000 to 3000 lx for 10 to 14 hours per day.

[0013] The rooting medium formula is: modified WPM + IBA 0.2-0.4 mg / L + IAA 1.0-1.5 mg / L + coconut juice 50-80 mL / L;

[0014] The modified WPM was as follows: the amount of ammonium nitrate was adjusted to 200 mg / L, the amount of potassium dihydrogen phosphate was adjusted to 85 mg / L, the amount of magnesium sulfate was adjusted to 185 mg / L, the amount of calcium nitrate tetrahydrate was adjusted to 278 mg / L, the types and concentrations of trace elements were the same as those in MS medium, and the other components remained unchanged.

[0015] Furthermore, the formulation of the primary culture medium is: WPM + IBA 0.06 mg / L + 6-BA 3.3 mg / L + ZT 1.1 mg / L + activated carbon 0.5 g / L.

[0016] Furthermore, the subculture medium is formulated as follows: WPM + IBA 0.01 mg / L + ZT 0.6 mg / L + BR 0.02 mg / L.

[0017] Furthermore, the rooting medium is formulated as follows: modified WPM + IBA 0.3 mg / L + IAA 1.3 mg / L + coconut juice 65 mL / L.

[0018] The beneficial technical effects of this invention are:

[0019] 1. When establishing a sterile system for golden beans, the commonly used combination of 75% alcohol and 0.1% mercuric chloride can effectively reduce the contamination rate by extending the mercuric chloride disinfection time. However, mercuric chloride is extremely harmful to explants, causing a large number of explants to die or cells to become inactive and unable to be induced. When the mercuric chloride disinfection time is shortened, fewer explants die, but the disinfection contamination rate is almost 100%. This invention uses a saturated bleaching powder solution and reduces the mercuric chloride concentration to 0.05%, which can control the disinfection contamination rate to within 40% and the disinfection mortality rate to within 10%, effectively improving the success rate of establishing a sterile system in the laboratory.

[0020] 2. In the initial induction process, the present invention uses an appropriate combination of IBA, 6-BA and ZT concentrations, which can achieve an initial induction rate of over 70%.

[0021] 3. In the subculture process, this invention creatively combines BR with the primary culture, which enables the golden bean to produce a large number of clustered buds during the subculture process, and the proliferation coefficient can reach more than 4 after 40 days.

[0022] 4. In the rooting culture of this invention, a modified WPM is used, which can promote the starvation stress of the golden bean and thus promote root development. On the other hand, the trace elements of MS medium can make the seedling leaves greener.

[0023] 5. In the rooting culture of this invention, the combination of IBA and IAA is used, and an appropriate concentration of coconut juice is added at the same time, which can shorten the rooting time to 40-45 days and the rooting rate can be as high as 95% or more. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a photograph of the proliferation culture results in Example 1 of the present invention.

[0026] Figure 2 This is a photograph of the proliferation culture results in Example 2 of the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Part of the experimental process in this invention

[0029] (a) Disinfection test

[0030] Disinfection was carried out using 75% alcohol for 30 seconds, followed by disinfection tests using saturated bleaching powder solution, 0.1% mercuric chloride solution, and 0.05% mercuric chloride solution. The specific design is shown in Tables 1 and 2.

[0031] Table 1 Disinfection Experiment Design

[0032]

[0033] Table 2 Disinfection test results

[0034]

[0035] As shown in Tables 1 and 2 above, the disinfection contamination rate is 32.4%-39.0% and the disinfection mortality rate is 8.4%-20.9% when using 75% alcohol for 30 seconds, combined with saturated bleaching powder solution for 20 minutes and 0.05% mercuric chloride solution for 11-13 minutes.

[0036] (ii) Primary induction test

[0037] Using WPM as the basic culture medium and adding 0.05 mg / L of IBA, a combination experiment of 6-BA and ZT was conducted. The specific experimental design is shown in Tables 3 and 4.

[0038] Table 3 Initial Induction Experiment Design

[0039]

[0040] Table 4 Results of the initial induction test

[0041]

[0042] As shown in Table 3-4, when 6-BA 3.0 mg / L and ZT 1.0 mg / L are used, the primary induction rate reaches 73.9%.

[0043] (III) Subgeneration Proliferation Experiment

[0044] Using WPM as the basic culture medium and adding 0.02 mg / L IBA, a combination experiment of 6-BA, ZT and BR was conducted. The specific experimental design is shown in Tables 5 and 6.

[0045] Table 5: Orthogonal Design Table for Subgeneration Proliferation Experiments

[0046]

[0047] Table 6 Results of Propagation Experiment

[0048]

[0049] As shown in Tables 5-6, the proliferation coefficient was the highest and vitrification was minimal when ZT was 0.5 mg / L and BR was 0.02 mg / L.

[0050] Example 1

[0051] A rapid propagation method for golden bean tissue culture includes the following steps:

[0052] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0053] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 55 seconds, rinsed with sterile water 3 times, disinfected with saturated bleach solution for 23 minutes, rinsed with sterile water 5 times, and disinfected with 0.05% mercuric chloride for 11 minutes, and rinsed with sterile water 8 times.

[0054] (3) Primary culture: After sterilization, the explants were inoculated into the primary culture medium (formulation: WPM + IBA 0.06mg / L + 6-BA 3.3mg / L + ZT 1.1mg / L + activated carbon 0.5g / L), and cultured in alternating light and dark for 30 days at a temperature of 25±2℃, with 12h of light per day and a light intensity of 2500lx.

[0055] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into subculture medium (formulation: WPM + IBA 0.01 mg / L + ZT 0.6 mg / L + BR 0.02 mg / L). The medium was cultured at 25 ± 2℃ with alternating light and dark conditions for 43 days, with 12 hours of light per day and a light intensity of 2500 lx. The subculture proliferation results are as follows: Figure 1 As shown.

[0056] (5) Rooting culture: The clustered shoots obtained from subculture were cut into single shoots and inoculated into rooting medium (formulation: modified WPM + IBA 0.3mg / L + IAA 1.3mg / L + coconut juice 65mL / L). The temperature was 25±2℃, and the light intensity was 2500lx for 43 days with alternating light and dark conditions.

[0057] The modified WPM was as follows: the amount of ammonium nitrate was adjusted to 200 mg / L, the amount of potassium dihydrogen phosphate was adjusted to 85 mg / L, the amount of magnesium sulfate was adjusted to 185 mg / L, the amount of calcium nitrate tetrahydrate was adjusted to 278 mg / L, the types and concentrations of trace elements were the same as those in MS medium, and the other components remained unchanged.

[0058] Example 2

[0059] A rapid propagation method for golden bean tissue culture includes the following steps:

[0060] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0061] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 50 seconds, rinsed twice with sterile water, disinfected with saturated bleach solution for 20 minutes, rinsed four times with sterile water, and disinfected with 0.05% mercuric chloride for 10 minutes, and rinsed seven times with sterile water.

[0062] (3) Primary culture: After sterilization, the explants were inoculated into the primary culture medium (formulation: WPM + IBA 0.05mg / L + 6-BA 3.0mg / L + ZT 1.0mg / L + activated carbon 0.5g / L), and cultured in alternating light and dark for 35 days at a temperature of 25±2℃, with 10h of light per day and a light intensity of 3000lx.

[0063] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into a subculture medium (formulation: WPM + IBA 0.01 mg / L + ZT 0.5 mg / L + BR 0.01 mg / L). The medium was cultured at 25 ± 2℃ with 10 h of light per day and a light intensity of 3000 lx for 45 days using alternating light and dark conditions. The results of the subculture proliferation are as follows: Figure 2 As shown.

[0064] (5) Rooting culture: The clustered shoots obtained from subculture were cut into single shoots and inoculated into rooting medium (formulation: modified WPM + IBA 0.2mg / L + IAA 1.0mg / L + coconut juice 50ml / L). The temperature was 25±2℃, and the light intensity was 3000lx for 45 days with alternating light and dark conditions.

[0065] The modified WPM was as follows: the amount of ammonium nitrate was adjusted to 200 mg / L, the amount of potassium dihydrogen phosphate was adjusted to 85 mg / L, the amount of magnesium sulfate was adjusted to 185 mg / L, the amount of calcium nitrate tetrahydrate was adjusted to 278 mg / L, the types and concentrations of trace elements were the same as those in MS medium, and the other components remained unchanged.

[0066] Example 3

[0067] A rapid propagation method for golden bean tissue culture includes the following steps:

[0068] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0069] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 60 seconds, rinsed with sterile water 3 times, disinfected with saturated bleach solution for 25 minutes, rinsed with sterile water 5 times, and disinfected with 0.05% mercuric chloride for 12 minutes, and rinsed with sterile water 8 times.

[0070] (3) Primary culture: After sterilization, the explants were inoculated into the primary culture medium (formulation: WPM + IBA 0.08mg / L + 6-BA 3.5mg / L + ZT 1.2mg / L + activated carbon 0.5g / L), and cultured in alternating light and dark conditions for 25 days at a temperature of 25±2℃, with 14h of light per day and a light intensity of 2000lx.

[0071] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into the subculture medium (formulation: WPM + IBA 0.02mg / L + ZT 0.8mg / L + BR 0.02mg / L), and cultured in alternating light and dark for 40 days at a temperature of 25±2℃, with 14h of light per day and a light intensity of 2000lx.

[0072] (5) Rooting culture: The clustered shoots obtained from subculture were cut into single shoots and inoculated into rooting medium (formulation: modified WPM + IBA 0.4mg / L + IAA 1.5mg / L + coconut juice 80ml / L). The temperature was 25±2℃, and the light intensity was 2000lx for 40 days with alternating light and dark conditions.

[0073] The modified WPM was as follows: the amount of ammonium nitrate was adjusted to 200 mg / L, the amount of potassium dihydrogen phosphate was adjusted to 85 mg / L, the amount of magnesium sulfate was adjusted to 185 mg / L, the amount of calcium nitrate tetrahydrate was adjusted to 278 mg / L, the types and concentrations of trace elements were the same as those in MS medium, and the other components remained unchanged.

[0074] Examples 1-3 were statistically compared in terms of disinfection contamination rate, disinfection mortality rate, primary induction rate, proliferation coefficient, and rooting rate. The results are shown in Table 7 below.

[0075] Table 7 Statistical Results

[0076]

[0077] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A rapid propagation method for soybean tissue culture, characterized by the following steps: include: (1) Selection of explants: Select young, non-lignified stem segments as explants; (2) Disinfection: After cleaning, the explants are disinfected with 75% alcohol for 50-60 seconds, rinsed with sterile water 2-3 times, disinfected with saturated bleaching powder solution for 20-25 minutes, rinsed with sterile water 4-5 times, and disinfected with 0.05% mercuric chloride for 10-12 minutes, rinsed with sterile water 7-8 times. (3) Primary culture: After sterilization, the explants were inoculated into the primary culture medium and cultured in alternating light and dark conditions for 25 to 35 days at a temperature of 25±2℃, with light intensity of 2000 to 3000 lx for 10 to 14 hours per day. The formulation of the primary culture medium is: WPM + IBA 0.05~0.08mg / L + 6-BA 3.0~3.5mg / L + ZT 1.0~1.2mg / L + activated carbon 0.5g / L; (4) Subculture: The clustered shoots of the primary culture were cut into single shoots and inoculated into the subculture medium. The temperature was 25±2℃, the light intensity was 2000-3000lx, and the light and dark were alternately cultured for 40-45 days. The subculture medium was formulated as follows: WPM + IBA 0.01–0.02 mg / L + ZT 0.5–0.8 mg / L + BR 0.01–0.02 mg / L; (5) Rooting culture: The clustered shoots obtained from subculture were cut into single shoots and inoculated into rooting culture medium. The shoots were cultured in alternating light and dark conditions for 40 to 45 days at a temperature of 25±2℃, with light intensity of 2000 to 3000 lx for 10 to 14 hours per day. The rooting medium formula is: modified WPM + IBA 0.2-0.4 mg / L + IAA 1.0-1.5 mg / L + coconut juice 50-80 mL / L; The modified WPM was as follows: the amount of ammonium nitrate was adjusted to 200 mg / L, the amount of potassium dihydrogen phosphate was adjusted to 85 mg / L, the amount of magnesium sulfate was adjusted to 185 mg / L, the amount of calcium nitrate tetrahydrate was adjusted to 278 mg / L, the types and concentrations of trace elements were the same as those in MS medium, and the other components remained unchanged.

2. The method according to claim 1, characterized in that, The formulation of the primary culture medium is: WPM + IBA 0.06 mg / L + 6-BA 3.3 mg / L + ZT 1.1 mg / L + activated carbon 0.5 g / L.

3. The method according to claim 1, characterized in that, The subculture medium was formulated as follows: WPM + IBA 0.01 mg / L + ZT 0.6 mg / L + BR 0.02 mg / L.

4. The method according to claim 1, characterized in that, The rooting medium formula is: modified WPM + IBA 0.3 mg / L + IAA 1.3 mg / L + coconut juice 65 mL / L.

5. The application of the method according to any one of claims 1 to 4 in the rapid propagation of soybean tissue culture.

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