A tissue culture method for Zanthoxylum bungeanum

Abstract

The invention discloses a tissue culture method of pepper, belonging to the technical field of tissue culture. The method comprises the following steps: (1) callus induction: taking pepper tender stems and disinfecting them to carry out callus induction culture; (2) proliferation culture: inoculating callus into a proliferation liquid culture medium for oscillation and illumination culture, and spraying a dispersant during the culture process; (3) adventitious bud induction: carrying out adventitious bud induction culture on embryonic callus obtained by proliferation culture; (4) adventitious bud elongation: cutting the induced bud clusters into small clusters for adventitious bud elongation culture; (5) rooting culture: when the adventitious buds elongate to 3-4 cm, separating the adventitious bud clusters into single plants and carrying out rooting culture; (6) seedling hardening and transplanting: opening a bottle mouth to harden the seedlings for 3-5 days, then taking out the rooted seedlings and transplanting them, thereby solving the problem that it is difficult to simultaneously obtain high differentiation rate, high density, high quality and high survival rate in pepper tissue culture.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, in particular to a tissue culture method of Zanthoxylum bungeanum. Background Art

[0002] Zanthoxylum bungeanum Maxim. and Zanthoxylumarmatum DC. are widely planted in Shaanxi, Gansu, Shandong, Henan, Sichuan, Chongqing, Yunnan, Guizhou and other places as one of the important economic tree species in my country. Its peel can be used as seasoning, its seeds can be used to extract oil, and its roots, stems, leaves, peels and seeds can be used as medicine, which has high utilization value. The method of plant tissue culture can maintain the excellent traits of the variety, and the propagation speed is fast and efficient. Therefore, it is an effective way to obtain a large number of excellent Zanthoxylum seedlings with stable traits in a short period of time through in vitro culture, which is an effective way to cultivate Zanthoxylum seedlings in factories, and also provides technical support for genetic transformation, gene editing and germplasm resource preservation of Zanthoxylum bungeanum. At the same time, during the tissue culture of Zanthoxylum bungeanum, hormones are often added to the culture medium to induce bud differentiation and increase the bud differentiation rate. However, too high a bud differentiation rate will lead to too high a bud density. Too high a density of buds will squeeze each other and compete for nutrient resources in a limited space, resulting in weak and poor growth of the proliferated buds, which cannot be cultured for rooting. Even if they can take root, the survival rate of transplantation is not high. Currently, the main method is to adjust the inoculation density or separate the high-density buds to strengthen the seedlings, so as to reduce the competition between buds and ensure the healthy growth of buds, but this will reduce the efficiency of tissue culture and increase the cost.

[0003] Therefore, the present invention provides a method for tissue culture of Zanthoxylum bungeanum, which improves the survival rate of tissue culture while improving the differentiation rate of tissue culture buds. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a tissue culture method for Zanthoxylum bungeanum, which solves the problem that it is difficult to achieve high differentiation rate, high density, high quality and high survival rate during the tissue culture process of Zanthoxylum bungeanum.

[0005] The present invention solves the above technical problems by the following technical means:

[0006] A method for tissue culture of Zanthoxylum bungeanum, comprising the following steps:

[0007] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 minutes, then soak them in 75% alcohol for 10 seconds, disinfect them with 0.1% mercuric chloride for 3-5 minutes, and then cut them into small pieces of 0.5-0.6 cm in length to obtain explants, which are inoculated into callus induction solid medium at a temperature of 24-26°C. After culturing in the dark for 7-10 days, they are further cultured for 10-12 days under the conditions of 14 hours / day of light and a light intensity of 1500-2000 lx to obtain callus tissue, and sprayed with a dispersant during the light culture process;

[0008] (2) Proliferation culture: Cut the callus into 0.5-0.6 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid culture medium, and cultured under shaking illumination at a speed of 110-120 r / min, a temperature of 24-26°C, a light intensity of 1500-2000 lx for 7-10 days;

[0009] (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium and cultured for 14-16 days at a temperature of 24-26°C, a light intensity of 14 h / day and a light intensity of 1500-2000 lx;

[0010] (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to adventitious bud elongation solid medium, and cultured at a temperature of 24-26°C, a light intensity of 14 h / day, and a light intensity of 1500-2000 lx;

[0011] (5) Rooting culture: When the adventitious buds grow to 3-4 cm, separate the individual buds and inoculate them into a rooting solid culture medium at a temperature of 24-26°C, a light intensity of 14 h / day, and a light intensity of 1500-2000 lx;

[0012] (6) Hardening and transplanting: When the root length is 1.5-2 cm, open the bottle mouth and harden the seedlings for 3-5 days. Then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1.

[0013] Furthermore, the solid culture medium for callus induction in step (1) is: MS+6-BA 1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+antibiotics 40 mg / L+carrageenan 5.5 g / L.

[0014] Since intermittent spraying of dispersants is required during callus induction culture, which may cause contamination of the culture medium, antibiotics are added to the proliferation liquid culture medium to inhibit contamination of tissue culture seedlings.

[0015] Furthermore, the proliferation liquid culture medium in step (2) is: MS+6-BA 1.0 mg / L+IBA 0.2 mg / L+sucrose 30 g / L.

[0016] Furthermore, the solid culture medium for adventitious bud induction in step (3) is: MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+sucrose 30 g / L+carrageenan 5.5 g / L.

[0017] Furthermore, the solid culture medium for adventitious bud elongation in step (4) is: MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+sucrose 30 g / L+carrageenan 5.5 g / L.

[0018] Furthermore, the rooting solid culture medium in step (5) is: 1 / 2MS+IBA 1.0 mg / L+NAA 0.5 mg / L+carrageenan 5.5 g / L.

[0019] Furthermore, the antibiotic in step (1) is one of tetracycline, streptomycin and penicillin.

[0020] Furthermore, the method of using the dispersant in step (1) is: spray once every day in the first 5 days, and once every 2 days after 5 days, with 2-3g sprayed each time.

[0021] Furthermore, the raw materials of the dispersant are as follows: refined ammonium, sucrose fatty acid ester, sophorolipid, ferrous nitrate, and the mass ratio thereof is (1-1.5): (1.2-1.5): (0.8-1.2): (1-1.5).

[0022] Further, the preparation method of the dispersant is as follows:

[0023] Sucrose fatty acid ester is added to a 30 wt % ethanol solution, and stirred evenly to obtain a sucrose fatty acid ester solution. Subsequently, ammonium sulfate, sophorolipids, and ferrous nitrate are added to the sucrose fatty acid ester solution, and mixed evenly to obtain a dispersant.

[0024] During the callus induction light culture process, the dispersant is sprayed once a day for the first 5 days and once every 2 days after 5 days, so that the callus can proliferate to generate a loose and fragile structure. More callus can be formed after gently pressing, which is more conducive to obtaining more embryonic callus after proliferation culture and improving proliferation efficiency; the loose callus generated by callus induction is inoculated into a proliferation liquid culture medium for proliferation culture. The liquid culture medium can promote the proliferation and differentiation of callus and is conducive to the dispersion of callus cells, thereby further improving the looseness of callus; then the embryonic callus is induced to bud. Since the culture is spread more widely at this time, the competition for nutrients and space between the culture tissues is smaller, which is conducive to the growth of buds and roots.

[0025] The ammonium sulfate in the dispersant can increase the activity of pectinase in the callus cells of pepper, promote the decomposition of pectin between cell walls into water-soluble pectin, weaken the connection between cells, thereby increasing the gap between cells, and forming a loose callus structure between cells. A certain amount of sophorolipids is also added to the dispersant to further enhance the activity of pectinase, promote the increase of water-soluble pectin, and loosen the callus. Sucrose fatty acid esters can increase the permeability of cell membranes, allowing more pectinase to penetrate, hydrolyze pectin, reduce the bonding substances between cells, and increase the intercellular gap. Ferrous nitrate can increase the ionic strength of the dispersant. After spraying on the callus, it can reduce the connection between cells, promote the dispersion of cells, and form loose callus. The competition for nutrients and space between loose callus is reduced, which helps to fully utilize nutrients and is conducive to the growth of adventitious buds.

[0026] Beneficial effects:

[0027] The present invention can form loose callus during the tissue culture process, effectively improve the bud differentiation rate while evacuating the callus structure, reduce competition between buds, ensure the healthy growth of buds, and achieve the dual purposes of proliferation and strong seedlings. In addition, it can ensure that pepper tissue culture can be reproduced in large quantities in a short time, thereby improving the economic benefits of tissue culture. At the same time, the tissue culture seedlings treated by the present invention have a high subsequent rooting rate and strong adaptability, effectively improving the transplant survival rate of pepper tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Elongation diagram of adventitious buds after treatment in Example 1. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to specific embodiments:

[0030] Embodiment 1:

[0031] Preparation of dispersant:

[0032] 1.2 g of sucrose fatty acid ester was added to 120 ml of 30 wt% ethanol solution and stirred to obtain a sucrose fatty acid ester solution. Subsequently, 1 g of ammonium sulfate, 0.8 g of sophorolipids and 1 g of ferrous nitrate were added to the sucrose fatty acid ester solution and mixed to obtain a dispersant.

[0033] Tissue culture method of Zanthoxylum bungeanum:

[0034] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 min, then soak them in 75% alcohol for 10 s, disinfect them with 0.1% mercuric chloride for 3 min, and then cut them into small pieces of 0.5 cm in length to obtain explants, which are inoculated into a solid medium for callus induction. The solid medium for callus induction is: MS + 6-BA 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L, the culture temperature is 24°C, and after culturing in the dark for 7 days, continue to culture under the conditions of 14 h / day of light and 1500 lx of light intensity for 10 days to obtain callus, and spray the evacuation agent during the light culture process, spraying once every 1 day for the first 5 days, and spraying once every 2 days after 5 days, spraying 2 g each time;

[0035] (2) Proliferation culture: Cut the callus into 0.5 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid medium, the proliferation liquid medium was: MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L, and the culture was carried out under shaking light conditions of 110r / min, 24℃, 14h / day of light, and 1500lx of light intensity for 7 days;

[0036] (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium, which was: MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured for 14 days at 24°C, 14 h / day of light intensity, and 1500 lx;

[0037] (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to the adventitious bud elongation solid medium, which was: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured at 24°C, 14 h / day of light, and 1500 lx of light intensity;

[0038] (5) Rooting culture: When the adventitious buds grow to 3 cm, separate the individual buds and inoculate them into a rooting solid culture medium containing 1 / 2MS + 1.0 mg / L IBA + 0.5 mg / L NAA + 5.5 g / L carrageenan. Culture the culture medium at 24°C, 14 h / day of light, and 1500 lx of light intensity for 30 days.

[0039] (6) Hardening and transplanting: Open the bottle mouth to harden the seedlings for 3 days, then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1.

[0040] Embodiment 2:

[0041] Preparation of dispersant:

[0042] 1.35 g of sucrose fatty acid ester was added to 135 ml of 30 wt% ethanol solution and stirred to obtain a sucrose fatty acid ester solution. Subsequently, 1.25 g of ammonium sulfate, 1 g of sophorolipid and 1.25 g of ferrous nitrate were added to the sucrose fatty acid ester solution and mixed to obtain a dispersant.

[0043] Tissue culture method of Zanthoxylum bungeanum:

[0044] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 min, then soak them in 75% alcohol for 10 s, disinfect them with 0.1% mercuric chloride for 4 min, and then cut them into small pieces of 0.55 cm in length to obtain explants, which are inoculated into a solid medium for callus induction. The solid medium for callus induction is: MS + 6-BA 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L, the culture temperature is 25°C, and after culturing in the dark for 8 days, continue to culture under the conditions of 14 h / day of light and 1800 lx of light intensity for 11 days to obtain callus, and spray the evacuation agent during the light culture process, spraying once every 1 day for the first 5 days, and spraying once every 2 days after 5 days, spraying 2.5 g each time;

[0045] (2) Proliferation culture: Cut the callus into 0.55 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid medium, the proliferation liquid medium was: MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L, and the culture was carried out under shaking light conditions of 115r / min, 25℃, 14h / day of light, and 1800lx of light intensity for 8 days;

[0046] (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium, which was: MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured for 15 days at 24°C, 14 h / day of light intensity, and 1800 lx;

[0047] (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to the adventitious bud elongation solid medium, which was: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured at a temperature of 25°C, a light intensity of 14 h / day, and a light intensity of 1800 lx;

[0048] (5) Rooting culture: When the adventitious buds grew to 3.5 cm, the individual plants were separated and inoculated into a rooting solid culture medium containing 1 / 2MS + 1.0 mg / L IBA + 0.5 mg / L NAA + 5.5 g / L carrageenan. The culture was carried out at 25°C, 14 h / day of light, and 1800 lx of light intensity for 32 days.

[0049] (6) Hardening and transplanting: Open the bottle mouth and harden the seedlings for 4 days, then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1.

[0050] Embodiment 3:

[0051] Preparation of dispersant:

[0052] 1.5 g of sucrose fatty acid ester was added to 150 ml of 30 wt% ethanol solution and stirred to obtain a sucrose fatty acid ester solution. Subsequently, 1.5 g of ammonium sulfate, 1.2 g of sophorolipids and 1.5 g of ferrous nitrate were added to the sucrose fatty acid ester solution and mixed to obtain a dispersant.

[0053] Tissue culture method of Zanthoxylum bungeanum:

[0054] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 min, then soak them in 75% alcohol for 10 s, disinfect them with 0.1% mercuric chloride for 5 min, and then cut them into small pieces of 0.6 cm in length to obtain explants, which are inoculated into a solid medium for callus induction. The solid medium for callus induction is: MS + 6-BA 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L, the culture temperature is 26°C, and after culturing in the dark for 7 days, continue to culture under the conditions of 14 h / day of light and 2000 lx of light intensity for 12 days to obtain callus, and spray the evacuation agent during the light culture process, spraying once every 1 day for the first 5 days, and spraying once every 2 days after 5 days, spraying 3 g each time;

[0055] (2) Proliferation culture: Cut the callus into 0.6 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid medium, the proliferation liquid medium was: MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L, and cultured under shaking light conditions of 120r / min, 26℃, 14h / day, and 2000lx for 10 days;

[0056] (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium, which was: MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured for 16 days at 24°C, 14 h / day of light intensity, and 2000 lx;

[0057] (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to the adventitious bud elongation solid medium. The adventitious bud elongation solid medium was: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L. The culture was carried out at a temperature of 26°C, a light intensity of 14 h / day, and a light intensity of 2000 lx.

[0058] (5) Rooting culture: When the adventitious buds grow to 4 cm, separate the individual buds and inoculate them into a rooting solid culture medium containing 1 / 2MS + 1.0 mg / L IBA + 0.5 mg / L NAA + 5.5 g / L carrageenan. Culture the buds at 26°C, 14 h / day of light, and 2000 lx of light intensity for 35 days.

[0059] (6) Hardening and transplanting: Open the bottle mouth to harden the seedlings for 5 days, then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1.

[0060] Comparative Example 1:

[0061] This comparative example is in contrast to Example 1, the only difference being the different raw materials of the dispersant, specifically, no refined ammonium is added, and the specific method is as follows:

[0062] 1.2 g of sucrose fatty acid ester was added to 120 ml of 30 wt% ethanol solution, and stirred evenly to obtain a sucrose fatty acid ester solution. Subsequently, 0.8 g of sophorolipid and 1 g of ferrous nitrate were added to the sucrose fatty acid ester solution, and mixed evenly to obtain a dispersant.

[0063] The tissue culture method of Zanthoxylum bungeanum in this comparative example is the same as that in Example 1.

[0064] Comparative Example 2:

[0065] This comparative example is in contrast to Example 1, the only difference being the different raw materials of the dispersant, specifically, no sophorolipid is added, and the specific method is as follows:

[0066] 1.2 g of sucrose fatty acid ester was added to 120 ml of 30 wt% ethanol solution, and stirred evenly to obtain a sucrose fatty acid ester solution. Subsequently, 1 g of ammonium sulfate and 1 g of ferrous nitrate were added to the sucrose fatty acid ester solution, and mixed evenly to obtain a dispersant.

[0067] The tissue culture method of Zanthoxylum bungeanum in this comparative example is the same as that in Example 1.

[0068] Comparative Example 3:

[0069] This comparative example is in contrast to Example 1, the only difference being the different raw materials of the dispersant, specifically, no ferrous nitrate is added, and the specific method is as follows:

[0070] S2: 1.2 g of sucrose fatty acid ester is added to 120 ml of 30 wt% ethanol solution, and stirred to obtain a sucrose fatty acid ester solution. Subsequently, 1 g of ammonium sulfate and 0.8 g of sophorolipid are added to the sucrose fatty acid ester solution, and mixed to obtain a dispersant.

[0071] The tissue culture method of Zanthoxylum bungeanum in this comparative example is the same as that in Example 1.

[0072] Comparative Example 4:

[0073] This comparative example is in contrast to Example 1, the only difference being the different raw materials of the dispersant, specifically, no sucrose fatty acid ester is added, and the specific method is as follows:

[0074] S2: Add 1 g of ammonium sulfate to 120 ml of deionized water, stir evenly to obtain an ammonium sulfate solution, then add 0.8 g of sophorolipid and 1 g of ferrous nitrate to the ammonium sulfate solution, mix evenly to obtain a dispersant.

[0075] The tissue culture method of Zanthoxylum bungeanum in this comparative example is the same as that in Example 1.

[0076] Experiment: Zanthoxylum bungeanum tissue culture experiment

[0077] The tissue culture method of Zanthoxylum bungeanum is as follows:

[0078] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 minutes, then soak them in 75% alcohol for 10 seconds, disinfect them with 0.1% mercuric chloride for 3 minutes, and then cut them into small pieces of 0.5 cm long to obtain explants, which are inoculated into solid medium for callus induction. For each treatment, 50 bottles are inoculated with 2 pieces in each bottle. The solid medium for callus induction is: MS + 6-BA 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L. The culture temperature is 24°C. After 7 days of dark culture, continue to culture for 10 days under the conditions of 14 h / day of light and 1500 lx of light intensity to obtain callus. Spray the dispersant during the light culture process. Spray once every 1 day for the first 5 days, and spray once every 2 days after 5 days. Spray 2 g each time.

[0079] (2) Proliferation culture: Cut the callus into 0.5 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid medium, with 2 clumps in each bottle. The proliferation liquid medium was: MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L. The culture was cultured under shaking light at a speed of 110r / min, a temperature of 24℃, a light intensity of 14h / day, and a light intensity of 1500lx for 7 days.

[0080] (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium, and 3 pieces were inoculated in each bottle. The adventitious bud induction solid medium was: MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, and cultured for 14 days at 24°C, 14 h / day of light intensity, and 1500 lx;

[0081] (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to the adventitious bud elongation solid medium. Four buds were inoculated in each bottle. The adventitious bud elongation solid medium was: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L. The culture was carried out at a temperature of 24°C, a light intensity of 14 h / day, and a light intensity of 1500 lx.

[0082] (5) Rooting culture: When the adventitious buds extend to 3 cm, separate the individual plants from the adventitious bud clusters and inoculate them into solid rooting culture medium. Inoculate one section per bottle. The solid rooting culture medium is: 1 / 2MS + IBA 1.0 mg / L + NAA 0.5 mg / L + carrageenan 5.5 g / L. Culture at 24°C, 14 h / day of light, and 1500 lx of light intensity for 30 days.

[0083] (6) Hardening and transplanting: Open the bottle mouth to harden the seedlings for 3 days, then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1.

[0084] Referring to the above tissue culture method, the following experiments were carried out:

[0085] 1. Experimental group 1 used the dispersant prepared in Example 1;

[0086] 2. Comparative groups 1-4 respectively used the dispersants prepared in comparative examples 1-4;

[0087] 3. Comparative group 5 used the dispersant prepared in Example 1, but the solid medium for callus induction used in the tissue culture step (1) was: MS + 6-BA 2.0 mg / L + 2,4-D 1.0 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L;

[0088] 4. Comparative group 6 used the dispersant prepared in Example 1, but a proliferation solid medium was specifically used during the proliferation culture process. The proliferation solid medium was MS + 6-BA 1.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L. The specific steps (2) of the proliferation culture process are as follows:

[0089] (2) Proliferation culture: Cut the callus into 0.5 cm 2 The clumps were inoculated into the proliferation solid culture medium, 2 clumps were inoculated into each bottle, the proliferation solid culture medium was: MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L + carrageenan 5.5g / L, and cultured for 7 days at 24℃, 14h / day of light intensity, and 1500 lx;

[0090] 5. In the callus induction process of control group 7, no dispersant was used, and the tissue culture method was as follows:

[0091] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 min, then soak them in 75% alcohol for 10 s, disinfect them with 0.1% mercuric chloride for 3 min, and then cut them into small pieces of 0.5 cm in length to obtain explants. The explants were inoculated into a solid medium for callus induction. The solid medium for callus induction was: MS + 6-BA 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30 g / L + tetracycline 40 mg / L + carrageenan 5.5 g / L. The culture temperature was 24°C. After culturing in the dark for 7 days, the culture was continued for 10 days under the conditions of 14 h / day of light intensity and 1500 lx to obtain callus.

[0092] 6. In the callus induction process of control group 8, no dispersant was used. In the proliferation culture process, the proliferation liquid culture medium was replaced with the proliferation solid culture medium. The specific culture process is as follows:

[0093] (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 minutes, then soak them in 75% alcohol for 10 seconds, disinfect them with 0.1% mercuric chloride for 3 minutes, and then cut them into small pieces of 0.5 cm in length to obtain explants, which are inoculated into solid medium for callus induction. For each treatment, 50 bottles are inoculated with 2 pieces in each bottle. The solid medium for callus induction is: MS+6-BA 1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+tetracycline 40 mg / L+carrageenan 5.5 g / L. After culturing at 24°C in the dark for 7 days, continue culturing for 10 days under the conditions of 14 h / day of light and 1500 lx of light intensity to obtain callus;

[0094] (2) Proliferation culture: Cut the callus into 0.5 cm 2 The clumps were inoculated into the proliferation solid culture medium, 2 pieces were inoculated in each bottle, the proliferation solid culture medium was MS + 6-BA 1.0mg / L + IBA 0.2mg / L + sucrose 30g / L + carrageenan 5.5g / L, and cultured for 7 days at 24℃, 14h / day of light intensity, and 1500lx.

[0095] During the callus induction process, the growth of callus tissue and the subsequent growth of adventitious buds were observed and recorded. The growth of callus tissue and adventitious buds is shown in Table 1; at the same time, the proliferation coefficient of adventitious buds was detected 20 days after the induction of adventitious buds, and then rooting culture was carried out. After 30 days of rooting culture, the rooting rate was detected, and the results are shown in Table 2.

[0096] Table 1

[0097]

[0098]

[0099] Table 2

[0100]

[0101] By analyzing Table 1 and Table 2, we can get:

[0102] 1. Compared with experimental group 1, comparison group 1 differs only in that no ammonium sulfate is added. Compared with experimental group 1, comparison group 2 differs only in that no sophorolipid is added. The callus obtained by its proliferation has too much pectin as the adhesive substance between cells, resulting in a smaller gap between cells. The callus is tighter than Example 1, and the competition for nutrients and space is larger, resulting in weaker growth of the adventitious buds of comparative examples 1 and 2, small leaf spread, and weaker rooting ability. Compared with experimental group 1, comparison group 3 differs only in that ferrous nitrate is not added, the ionic strength of the dispersant is lower, the looseness of the callus is lower, and the competition for nutrients and space is larger, resulting in weaker growth of adventitious buds and lower rooting rate. Compared with experimental group 1, comparison group 4 differs only in that sucrose fatty acid ester is not added, the adhesive substance pectin between cells is more, the intercellular gap is larger, and the callus is tighter, resulting in weak growth of adventitious buds and low rooting rate.

[0103] 2. Compared with experimental group 1, the only difference between control group 5 and experimental group 1 is that the solid culture medium for callus induction is: MS+6-BA2.0mg / L+2,4-D 1.0mg / L+sucrose 30g / L+tetracycline 40mg / L+carrageenan 5.5g / L. The hormone concentration is relatively high, which causes the callus structure obtained by proliferation to become gradually more compact, and the competition for nutrients and space is greater, resulting in a lower bud proliferation coefficient, weaker growth of the generated adventitious buds, and lower rooting ability.

[0104] 5. Compared with experimental group 1, control group 6 differs only in that a proliferative solid culture medium is used. The solid culture medium is not conducive to the differentiation and dispersion of callus tissue, resulting in a low proliferation coefficient of the obtained embryonic callus tissue during bud induction, weak growth of adventitious buds, and weak rooting ability. Compared with control group 1, control group 7 does not use a dispersant, and its callus tissue is more compact, poor growth, and adventitious buds grow weakly, the marginal leaves turn yellow and fall off, and the later growth is slower, which shows that the dispersant can effectively promote the formation of callus tissue with a sparse structure and promote the growth of subsequent adventitious buds. Compared with control group 1, control group 8 uses a proliferative solid culture medium and does not use a dispersant. Its callus tissue is more compact than the control group, and the growth of adventitious buds is worse, indicating that the use of a proliferative liquid culture medium combined with a dispersant can generate loose callus tissue and promote the growth and proliferation of buds.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A method for tissue culture of Zanthoxylum bungeanum, characterized in that: The method comprises the following steps: (1) Callus induction: Take the young stems of Zanthoxylum bungeanum and rinse them under running tap water for 30 minutes, then soak them in 75% alcohol for 10 seconds, disinfect them with 0.1% mercuric chloride for 3-5 minutes, and then cut them into small pieces of 0.5-0.6 cm in length to obtain explants. Inoculate them into solid medium for callus induction at a temperature of 24-26°C. After culturing in the dark for 7-10 days, continue culturing for 10-12 days under the conditions of 14 hours / day of light and a light intensity of 1500-2000 lx to obtain callus tissue, and spray dispersants during the light culture process; (2) Proliferation culture: Cut the callus into 0.5-0.6 cm 2 The clumps were gently crushed and inoculated into the proliferation liquid culture medium, and cultured under shaking illumination for 7-10 days at a speed of 110-120 r / min, a temperature of 24-26°C, a light intensity of 1500-2000 lx, and a light intensity of 14h / day; (3) Adventitious bud induction: The embryonic callus obtained from the proliferation culture was transferred to the adventitious bud induction solid medium and cultured for 14-16 days at a temperature of 24-26°C, a light intensity of 14 h / day and a light intensity of 1500-2000 lx; (4) Adventitious bud elongation: The induced bud clusters were cut into small clusters and transferred to the adventitious bud elongation solid culture medium for cultivation at a temperature of 24-26°C, a light intensity of 14 h / day, and a light intensity of 1500-2000 lx; (5) Rooting culture: When the adventitious buds grow to 3-4 cm, separate the individual buds and inoculate them into rooting solid culture medium. Culture them for 30-35 days at 24-26°C, 14 h / day of light, and 1500-2000 lx of light intensity. (6) Hardening and transplanting: Open the bottle mouth to harden the seedlings for 3-5 days, then take out the rooted seedlings and transplant them into a substrate with a volume ratio of sand: perlite: humus of 1:1:1; The solid medium for callus induction in step (1) is: MS+6-BA 1.0 mg / L+2,4-D 0.5 mg / L+sucrose 30 g / L+antibiotics 40 mg / L+carrageenan 5.5 g / L; The proliferation liquid culture medium in step (2) is: MS+6-BA 1.0 mg / L+ IBA 0.2 mg / L+ sucrose 30 g / L; The raw materials of the dispersant are as follows: ammonium sulfate, sucrose fatty acid ester, sophorolipids, and ferrous nitrate, and the mass ratios thereof are (1-1.5): (1.2-1.5): (0.8-1.2): (1-1.5).

2. The tissue culture method of Zanthoxylum bungeanum according to claim 1, wherein: The solid culture medium for adventitious bud induction in step (3) is: MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+sucrose 30 g / L+carrageenan 5.5 g / L.

3. A tissue culture method for Zanthoxylum bungeanum according to claim 2, characterized in that, The solid culture medium for adventitious bud elongation in step (4) is: MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+sucrose 30 g / L+carrageenan 5.5 g / L.

4. A tissue culture method for Zanthoxylum bungeanum according to claim 3, characterized in that, The rooting solid culture medium in step (5) is: 1 / 2MS+IBA 1.0 mg / L+NAA 0.5 mg / L+carrageenan 5.5 g / L.

5. A tissue culture method for Zanthoxylum bungeanum according to claim 4, characterized in that, The antibiotic in step (1) is one of tetracycline, streptomycin and penicillin.

6. The tissue culture method of Zanthoxylum bungeanum according to claim 5, characterized in that: The method of using the dispersant in step (1) is: spray once every day for the first 5 days, and once every 2 days after 5 days, with 2-3g sprayed each time.

7. A tissue culture method for Zanthoxylum bungeanum according to claim 6, characterized in that, The preparation method of the dispersant is as follows: Sucrose fatty acid ester is added to a 30wt% ethanol solution, and stirred evenly to obtain a sucrose fatty acid ester solution. Subsequently, ammonium sulfate, sophorolipids, and ferrous nitrate are added to the sucrose fatty acid ester solution, and mixed evenly to obtain a dispersant.

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