Method for tissue culture and rapid propagation of radix puerariae

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for tissue culture and rapid propagation of radix puerariae. The method specifically comprises the following steps: (1) collecting radix puerariae tissues, sterilizing, inoculating to a starting culture medium, and culturing to obtain radix puerariae aseptic seedlings; (2) inoculating the aseptic seedlings of the radix puerariae into a callus induction culture medium for culture to obtain radix puerariae callus; (3) inoculating the radix puerariae callus into an adventitious bud differentiation culture medium for culturing to obtain radix puerariae adventitious buds; (4) inoculating the adventitious buds of the radix puerariae into a proliferation culture medium for culturing to obtain proliferation test-tube plantlets of the radix puerariae; (5) inoculating the proliferation seedlings of the kudzuvine roots into a rooting culture medium for culturing to obtain rooting test-tube seedlings of the kudzuvine roots; and (6) transplanting after seedling hardening. The propagation coefficient of radix puerariae tissue culture seedlings prepared by the method is 3.2-4.7, the quality is relatively good, radix puerariae rooting test-tube seedlings are subjected to acclimatization and transplantation, the root development is good, and the survival rate can reach 91%.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of kudzu root tissue culture. Background Art

[0002] Pueraria root is the dried root of Pueraria lobata (Willd.) Ohwi, a plant of the Leguminosae family. It is used in the food, beverage, and pharmaceutical industries. The edible part of Pueraria root is the tuber, which has the effects of clearing away heat and detoxifying, nourishing nutrition, preventing heatstroke and cooling down. It also contains a variety of trace elements and has special therapeutic effects on coronary heart disease, angina pectoris, and diabetes. The root tuber can also be processed into Pueraria powder, which can be made into a series of products such as Pueraria vermicelli, Pueraria cake, Pueraria beverage, Pueraria jelly, Pueraria wine, and Pueraria biscuits. Pueraria tea and Chinese herbal medicine slices are also gradually entering the market, which has broad market prospects.

[0003] The traditional method of propagation of kudzu root is cutting propagation, which has caused serious accumulation of viruses over the years. At present, virus disease has become an important reason for the degeneration of kudzu species, reduced yield and deterioration of quality. The virus-free kudzu can improve yield and quality. The use of stem tip culture in tissue culture technology can effectively remove viruses from plants, produce virus-free healthy plants, significantly improve the quality and yield of kudzu root, and play an important role in promoting the kudzu industry economy.

[0004] Therefore, how to provide a method for culturing kudzu root tissue to solve the above technical problems is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] The invention aims to provide a method for rapid propagation of kudzu root tissue culture.

[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 rapid propagation of kudzu root tissue culture, comprising the following steps:

[0008] (1) collecting kudzu root tissue, disinfecting it, and inoculating it into a starting culture medium to obtain kudzu root sterile seedlings;

[0009] (2) inoculating the sterile Pueraria seedlings obtained in step (1) into a callus induction medium for cultivation to obtain Pueraria callus tissue;

[0010] (3) inoculating the kudzu root callus obtained in step (2) into an adventitious bud differentiation medium for cultivation to obtain kudzu root adventitious buds;

[0011] (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium for cultivation to obtain kudzu root proliferation test tube plantlets;

[0012] (5) inoculating the kudzu root proliferating seedlings obtained in step (4) into a rooting medium for cultivation to obtain kudzu rooting test tube seedlings;

[0013] (6) hardening the kudzu root test tube seedlings obtained in step (5) and then transplanting them;

[0014] Preferably, the starting medium in step (1) is: MS+6-KT 2.0-2.2 mg / L+NAA 0.3-0.4 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, with a pH value of 5.8-6.0;

[0015] Preferably, the callus induction medium in step (2) is: 1 / 4MS + IBA 1.0-1.2 mg / L + 2,4-D 0.2-0.3 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, with a pH value of 5.8-6.0;

[0016] Preferably, the adventitious bud differentiation medium in step (3) is: MS + 6-KT 3.0-3.2 mg / L + TDZ 0.5-0.6 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, with a pH value of 5.8-6.0;

[0017] Preferably, the proliferation medium in step (4) is: 1 / 2MS + 6-BA 0.5-1.5 mg / L + IAA 0.2-0.6 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L

[0018] Preferably, the rooting medium in step (5) is: 1 / 2MS + NAA 0.5-0.7 mg / L + IBA 0.3-0.4 mg / L + activated carbon 0.8-0.9 g / L + sucrose 25-30 g / L + agar 7.0-7.5 g / L, with a pH value of 5.8-6.0.

[0019] Preferably, the light intensity during the Pueraria tissue culture rapid propagation is 2000-3000 lx, the temperature is 23-27° C., the humidity is 65%-75%, and the daily light duration is 11-13 h.

[0020] Preferably, the culturing time in step (1) is 5 to 7 days; the culturing time in step (2) is 10 to 15 days; the culturing time in step (3) is 13 to 20 days; the culturing time in step (4) is 25 to 40 days, and the culturing time in step (5) is 17 to 25 days.

[0021] Preferably, the transplanting standard of the Pueraria rooted test tube seedlings in step (6) is: root length 3-5 cm, seedling height 5-7 cm.

[0022] Preferably, the time for hardening the seedlings in step (6) is 4 to 7 days.

[0023] Preferably, the transplanting method in step (6) is: washing the rooted test tube seedlings of Pueraria lobata and then soaking them, transplanting them into the seedling culture medium, covering them with a film and culturing them for 7 to 9 days, and then removing the film.

[0024] Preferably, the soaking of seedlings is to soak the Pueraria rooting test tube seedlings in a 500-fold thiophanate-methyl solution for 20 to 30 minutes.

[0025] Preferably, the seedling medium is vermiculite and peat soil in a mass ratio of 1:1 to 3.

[0026] Preferably, the variety of Pueraria root is Gange No. 1 (Powder Pueraria), Kuju Pueraria or Wild Pueraria.

[0027] Preferably, the disinfection method in step (1) is: first disinfect with alcohol having a volume concentration of 70-80% for 30-60 seconds, rinse with water 2-3 times, then disinfect with mercuric chloride having a volume concentration of 0.08-0.12% for 4-6 minutes, and rinse with water 3-5 times.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The results of tissue culture research on kudzu root show that kudzu root callus can be induced by using stem segments with buds, wherein the best induction medium for callus differentiation adventitious buds is MS+3.0mg / L6-KT+0.5mg / LTDZ+7.0-7.5g / L agar+25-30g / L sucrose, and the induction rate can reach 91.3%.

[0030] The kudzu root tissue culture seedlings prepared by the invention have a proliferation coefficient between 3.2 and 4.7 and good quality. Meanwhile, the kudzu root test tube seedlings are hardened and transplanted, and the root system is well developed and the survival rate is high, which can reach more than 91%. In addition, tissue culture bags can be used to replace tissue culture bottles for kudzu root tissue culture, thereby greatly reducing material costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 The Pueraria lobata callus cultured for 10 days in Example 3;

[0033] Figure 2 The adventitious buds of the kudzu root test tube seedlings of Example 3 were cultured for 20 days;

[0034] Figure 3 The growth state of the kudzu root test tube seedlings after 30 days of proliferation culture in Example 3

[0035] Figure 4 This is the growth state of the kudzu root test tube seedlings after 40 days of proliferation culture in Example 3;

[0036] Figure 5 The rooting culture of the kudzu root test tube seedlings of Example 3;

[0037] Figure 6 Transplanting and hardening of the kudzu root test tube seedlings of Example 3;

[0038] Figure 7 This is the Pueraria greenhouse seedling raising in Example 3. DETAILED DESCRIPTION

[0039] The present invention provides a method for rapid propagation of kudzu root tissue culture, comprising the following steps:

[0040] (1) collecting kudzu root tissue, disinfecting it, and inoculating it into a starting culture medium to obtain kudzu root sterile seedlings;

[0041] (2) inoculating the sterile Pueraria seedlings obtained in step (1) into a callus induction medium for cultivation to obtain Pueraria callus tissue;

[0042] (3) inoculating the kudzu root callus obtained in step (2) into an adventitious bud differentiation medium for cultivation to obtain kudzu root adventitious buds;

[0043] (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium for cultivation to obtain kudzu root proliferation test tube plantlets;

[0044] (5) inoculating the kudzu root proliferating seedlings obtained in step (4) into a rooting medium for cultivation to obtain kudzu rooting test tube seedlings;

[0045] (6) hardening the kudzu root test tube seedlings obtained in step (5) and then transplanting them;

[0046] In the present invention, the starting culture medium in step (1) is: MS + 6-KT 2.0-2.2 mg / L + NAA 0.3-0.4 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, with a pH value of 5.8-6.0; preferably, MS + 6-KT 2.1 mg / L + NAA 0.3 mg / L + agar 7.1-7.4 g / L + sucrose 26-29 g / L, with a pH value of 5.9; further preferably, MS + 6-KT 2.1 mg / L + NAA 0.3 mg / L + agar 7.2-7.3 g / L + sucrose 27-28 g / L, with a pH value of 5.9; more preferably, MS + 6-KT 2.1 mg / L + NAA 0.3 mg / L + agar 7.2 g / L + sucrose 27 g / L, with a pH value of 5.9.

[0047] In the present invention, the callus induction medium in step (2) is: 1 / 4MS + IBA 1.0-1.2mg / L + 2,4-D 0.2-0.3mg / L + agar 7.0-7.5g / L + sucrose 25-30g / L, with a pH value of 5.8-6.0; preferably 1 / 4MS + IBA 1.1mg / L + 2,4-D 0.2mg / L + agar 7.1-7.4g / L + sucrose 26-29g / L, with a pH value of 5.9; further preferably 1 / 4MS + IBA 1.1mg / L + 2,4-D 0.2mg / L + agar 7.2-7.3g / L + sucrose 27-28g / L, with a pH value of 5.9; more preferably 1 / 4MS + IBA 1.1mg / L + 2,4-D 0.2mg / L+agar 7.2g / L+sucrose 27g / L, pH value is 5.9.

[0048] In the present invention, the adventitious bud differentiation medium in step (3) is: MS + 6-KT 3.0-3.2 mg / L + TDZ 0.5-0.6 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L; preferably MS + 6-KT 3.1 mg / L + TDZ 0.6 mg / L + agar 7.1-7.4 g / L + sucrose 26-29 g / L; further preferably MS + 6-KT 3.1 mg / L + TDZ 0.6 mg / L + agar 7.2-7.3 g / L + sucrose 27-28 g / L; more preferably MS + 6-KT 3.1 mg / L + TDZ 0.6 mg / L + agar 7.3 g / L + sucrose 28 g / L.

[0049] In the present invention, the proliferation medium in step (4) is: 1 / 2MS + 6-BA 0.5-1.5mg / L + IAA 0.2-0.6mg / L + agar 7.0-7.5g / L + sucrose 25-30g / L, with a pH value of 5.8-6.0; preferably 1 / 2MS + 6-BA 0.7-1.3mg / L + IAA 0.3-0.6mg / L + agar 7.1-7.4g / L + sucrose 26-29g / L, with a pH value of 5.9; further preferably 1 / 2MS + 6-BA 0.9-1.1mg / L + IAA 0.4-0.6mg / L + agar 7.2-7.3g / L + sucrose 27-28g / L, with a pH value of 5.9; more preferably 1 / 2MS + 6-BA 1mg / L + IAA 0.6 mg / L+agar 7.2g / L+sucrose 27g / L, pH value is 5.9.

[0050] In the present invention, the rooting medium in step (5) is: 1 / 2MS + NAA 0.5-0.7mg / L + IBA 0.3-0.4mg / L + activated carbon 0.8-0.9g / L + sucrose 25-30g / L + agar 7.0-7.5g / L, pH value is 5.8-6.0; preferably 1 / 2MS + NAA 0.6mg / L + IBA 0.3mg / L + activated carbon 0.8g / L + sucrose 26-29g / L + agar 7.1-7.4g / L, pH value is 5.9; more preferably 1 / 2MS + NAA 0.6mg / L + IBA 0.3mg / L+activated carbon 0.8g / L+sucrose 27-28g / L+agar 7.2-7.3g / L, pH value is 5.9; more preferably 1 / 2MS+NAA 0.6mg / L+IBA 0.3mg / L+activated carbon 0.8g / L+sucrose 27g / L+agar 7.2g / L, pH value is 5.9.

[0051] In the present invention, the light intensity during the Pueraria tissue culture rapid propagation is 2000-3000lx, the temperature is 23-27°C, the humidity is 65%-75%, and the daily illumination time is 11-13h; preferably, the light intensity is 2200-2800lx, the temperature is 24-26°C, the humidity is 67%-73%, and the daily illumination time is 12h; further preferably, the light intensity is 2400-2600lx, the temperature is 25°C, the humidity is 69%-71%, and the daily illumination time is 12h; more preferably, the light intensity is 2500lx, the temperature is 25°C, the humidity is 70%, and the daily illumination time is 12h.

[0052] In the present invention, the culturing time in step (1) is 5 to 7 days, preferably 6 days.

[0053] In the present invention, the culturing time in step (2) is 10 to 15 days; preferably 11 to 14 days; more preferably 12 to 13 days; and more preferably 12 days.

[0054] In the present invention, the culturing time in step (3) is 13 to 20 days; preferably 14 to 19 days; more preferably 15 to 18 days; and more preferably 17 days.

[0055] In the present invention, the culturing time in step (4) is 25 to 40 days; preferably 27 to 38 days; more preferably 29 to 36 days; and more preferably 32 days.

[0056] In the present invention, the culturing time in step (5) is 17 to 25 days; preferably 18 to 24 days; more preferably 19 to 23 days; and more preferably 20 days.

[0057] In the present invention, the standard for transplanting the kudzu root test tube seedlings in step (6) is: root length 3-5 cm, seedling height 5-7 cm; preferably, root length 4 cm, seedling height 6 cm.

[0058] In the present invention, the time for hardening the seedlings in step (6) is 4 to 7 days, preferably 5 to 6 days, and more preferably 6 days.

[0059] In the present invention, the transplanting method in step (6) is: washing the rooted test tube seedlings of Pueraria lobata and then soaking them, transplanting them into the seedling culture medium, covering them with a film and culturing them for 7 to 9 days, and then removing the film; preferably 8 days.

[0060] In the present invention, the soaking of seedlings is to soak the Pueraria test tube seedlings in 500 times thiophanate-methyl solution for 20 to 30 minutes; preferably 22 to 28 minutes; more preferably 24 to 26 minutes; more preferably 25 minutes.

[0061] In the present invention, the seedling culture medium is vermiculite and peat soil, and the mass ratio is 1:1 to 3; preferably 1:2.

[0062] In the present invention, the variety of Pueraria lobata is Gange No. 1 (Powder Pueraria), Ku Pueraria or Wild Pueraria; preferably Gange No. 1.

[0063] In the present invention, the disinfection method of step (1) is: first disinfect with alcohol with a volume concentration of 70-80% for 30-60s, rinse with water 2-3 times, and then disinfect with mercuric chloride with a volume concentration of 0.08-0.12% for 4-6min, and rinse with water 3-5 times; preferably, first disinfect with alcohol with a volume concentration of 72-78% for 40-50s, rinse with water 3 times, and then disinfect with mercuric chloride with a volume concentration of 0.09-0.11% for 6min, and rinse with water 4 times; further preferably, first disinfect with alcohol with a volume concentration of 75% for 45s, rinse with water 3 times, and then disinfect with mercuric chloride with a volume concentration of 0.1% for 6min, and rinse with water 4 times.

[0064] 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.

[0065] Example 1

[0066] A method for rapid propagation of kudzu root tissue culture, comprising the following steps:

[0067] (1) Obtaining sterile seedlings of Pueraria root: young stem segments and leaves with buds were collected at noon on a sunny day as tissue culture explants (Pueraria lobata), and after preliminary cleaning and disinfection, they were inoculated into the start-up culture medium and cultured for 5 days;

[0068] The disinfection method is: first disinfect with 70% volume concentration of alcohol for 30s, rinse with water twice, then disinfect with 0.08% volume concentration of mercuric chloride (HgCl2) for 4min, rinse with water three times;

[0069] The start-up culture medium is: MS+6-KT 2.0 mg / L+NAA 0.3 mg / L+agar 7.0 g / L+sucrose 25 g / L, pH value is 5.8;

[0070] (2) Pueraria callus induction: Pueraria sterile seedlings were inoculated into callus induction medium in a tissue culture bag and cultured for 10 days. The callus induction medium was: 1 / 4MS + IBA 1.0 mg / L + 2,4-D 0.2 mg / L + agar 7.0 g / L + sucrose 25 g / L, pH 5.8;

[0071] (3) Culture of adventitious bud differentiation of kudzu root callus: When the kudzu root callus in step (2) grows densely, it is inoculated into an adventitious bud differentiation medium in a tissue culture bag and cultured for 13 days. The optimal culture medium is: MS + 6-KT 3.0 mg / L + TDZ 0.5 mg / L + agar 7.0 g / L + sucrose 25 g / L, with a pH value of 5.8.

[0072] (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium and culturing for 25 days to obtain kudzu root proliferation test tube plantlets; the proliferation medium is: 1 / 2MS + 6-BA 0.5 mg / L + IAA 0.2 mg / L + agar 7.0 g / L + sucrose 25 g / L, pH 5.8;

[0073] (5) Rooting culture of kudzu root test tube seedlings: The obtained kudzu root test tube seedlings were cultured to a plant height of 3.0 cm, and then transferred to the rooting medium in the tissue culture bag and cultured for 15 days;

[0074] The kudzu rooting medium is: 1 / 2MS+NAA0.5mg / L+IBA0.3 mg / L+activated carbon 0.8g / L+sucrose 25g / L+agar 7.0g / L, pH value is 5.8, the pH value of the rooting medium is 5.8;

[0075] (6) Hardening and transplanting of rooted test tube seedlings of Pueraria root: When the roots of the rooted test tube seedlings grow to 3 cm and the seedlings are 5 cm tall, remove the cap of the tissue culture bottle and harden the seedlings in a light incubator for 4 days. Take out the Pueraria root test tube seedlings, wash the agar at the base of the test tube seedlings, then soak them in 500-fold thiophanate-methyl solution for 20 minutes, and finally transplant them into seedling pots containing seedling medium for cultivation. Water them thoroughly, cover them with a film to keep them moist, pay attention to ventilation and maintain a suitable temperature. Remove the film after 7 days. The seedling medium is vermiculite: peat soil = 1:3.

[0076] (7) During the tissue culture of Pueraria root, the light intensity of the culture was 2000 lx, the temperature was between 23°C, the humidity was between 65%, and the light-dark cycle was 11 h of light and 11 h of darkness.

[0077] Example 2

[0078] A method for rapid propagation of kudzu root tissue culture, comprising the following steps:

[0079] (1) Obtaining sterile seedlings of Pueraria root: young stem segments and leaves with buds were collected at noon on a sunny day as tissue culture explants (Pueraria lobata), and after preliminary cleaning and disinfection, they were inoculated into the start-up culture medium and cultured for 7 days;

[0080] The disinfection method is: first disinfect with 80% volume concentration of alcohol for 60s, rinse with water 3 times, then disinfect with 0.12% volume concentration of mercuric chloride (HgCl2) for 6min, rinse with water 5 times;

[0081] The start-up culture medium is: MS+6-KT 2.2 mg / L+NAA 0.4 mg / L+agar 7.5 g / L+sucrose 30 g / L, pH value is 6.0;

[0082] (2) Pueraria callus induction: Pueraria sterile seedlings were inoculated into callus induction medium in a tissue culture bag and cultured for 15 days. The callus induction medium was: 1 / 4MS + IBA 1.2 mg / L + 2,4-D 0.3 mg / L + agar 7.5 g / L + sucrose 30 g / L, pH 6.0;

[0083] (3) Culture of adventitious bud differentiation of kudzu root callus: When the kudzu root callus in step (2) grows densely, it is inoculated into an adventitious bud differentiation medium in a tissue culture bag and cultured for 20 days. The optimal culture medium is: MS + 6-KT 3.2 mg / L + TDZ 0.6 mg / L + agar 7.5 g / L + sucrose 30 g / L, with a pH value of 6.0.

[0084] (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium and culturing for 40 days to obtain kudzu root proliferation test tube plantlets; the proliferation medium is: 1 / 2MS + 6-BA 1.5 mg / L + IAA 0.6 mg / L + agar 7.5 g / L + sucrose 30 g / L, pH 6.0;

[0085] (5) Rooting culture of kudzu root test tube seedlings: The obtained kudzu root test tube seedlings were cultured to a plant height of 5.0 cm, and then transferred to the rooting medium in the tissue culture bag and cultured for 25 days;

[0086] The Pueraria rooting medium is: 1 / 2MS+NAA0.7mg / L+IBA0.4 mg / L+activated carbon 0.9g / L+sucrose 30g / L+agar 7.5g / L, pH value is 6.0, the pH value of the rooting medium is 6.0;

[0087] (6) Hardening and transplanting of rooted test tube seedlings of Pueraria root: When the roots of the rooted test tube seedlings grow to 5 cm and the seedlings are 7 cm tall, remove the cap of the tissue culture bottle and harden the seedlings in a light incubator for 7 days. Take out the test tube seedlings of Pueraria root, wash the agar at the base of the test tube seedlings, and then soak them in 500-fold thiophanate-methyl solution for 30 minutes. Finally, transplant them into seedling pots containing seedling medium for cultivation, water them thoroughly, cover them with a film to keep them moist, pay attention to ventilation and maintain a suitable temperature. Remove the film after 9 days. The seedling medium is vermiculite: peat soil = 1:1.

[0088] (7) During the Pueraria tissue culture, the light intensity was 3000 lx, the temperature was between 27 °C, the humidity was between 75%, and the light-dark cycle was 13 h of light and 13 h of darkness.

[0089] Example 3

[0090] A method for rapid propagation of kudzu root tissue culture, comprising the following steps:

[0091] (1) Obtaining sterile seedlings of Pueraria root: young stem segments and leaves with buds were collected at noon on a sunny day as tissue culture explants (Gan Ge No. 1), and after preliminary cleaning and disinfection, they were inoculated into the start-up culture medium and cultured for 6 days;

[0092] The disinfection method is: first disinfect with 75% volume concentration of alcohol for 45s, rinse with water 3 times, then disinfect with 0.1% volume concentration of mercuric chloride (HgCl2) for 5min, rinse with water 4 times;

[0093] The start-up culture medium is: MS+6-KT 2.0 mg / L+NAA 0.3 mg / L+agar 7.3 g / L+sucrose 28 g / L, pH value is 5.9;

[0094] (2) Pueraria callus induction: Pueraria sterile seedlings were inoculated into callus induction medium in a tissue culture bag and cultured for 10 days. The callus induction medium was: 1 / 4MS + IBA 1.0 mg / L + 2,4-D 0.2 mg / L + agar 7.3 g / L + sucrose 28 g / L, pH 5.9;

[0095] (3) Culture of adventitious bud differentiation of kudzu root callus: When the kudzu root callus in step (2) grows densely, it is inoculated into an adventitious bud differentiation medium in a tissue culture bag and cultured for 15 days. The optimal culture medium is: MS + 6-KT 3.1 mg / L + TDZ 0.6 mg / L + agar 7.3 g / L + sucrose 28 g / L, with a pH value of 5.9.

[0096] (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium and culturing for 31 days to obtain kudzu root proliferation test tube plantlets; the proliferation medium comprises: 1 / 2MS + 6-BA 1 mg / L + IAA 0.6 mg / L + agar 7.3 g / L + sucrose 28 g / L, with a pH value of 5.9;

[0097] (5) Rooting culture of kudzu root test tube seedlings: The obtained kudzu root test tube seedlings were cultured to a plant height of 4 cm, and then transferred to the rooting medium in the tissue culture bag and cultured for 20 days;

[0098] The kudzu rooting medium is: 1 / 2MS+NAA 0.5mg / L+IBA0.3 mg / L+activated carbon 0.8g / L+sucrose 28g / L+7.3g / L agar, pH value is 5.9, the pH value of the rooting medium is 5.9;

[0099] (6) Hardening and transplanting of rooted test tube seedlings of Pueraria root: When the roots of the rooted test tube seedlings grow to 4 cm and the seedlings are 6 cm tall, remove the cap of the tissue culture bottle and harden the seedlings in a light incubator for 5 days. Take out the Pueraria root test tube seedlings, wash the agar at the base of the test tube seedlings, then soak them in 500-fold thiophanate-methyl solution for 25 minutes, and finally transplant them into seedling pots containing seedling medium for cultivation. Water them thoroughly, cover them with a film to keep them moist, pay attention to ventilation and maintain a suitable temperature. Remove the film after 8 days. The seedling medium is vermiculite: peat soil = 1:2.

[0100] (7) During the Pueraria tissue culture, the light intensity was 2500 lx, the temperature was between 25 °C, the humidity was between 70%, and the light-dark cycle was 12 h of light and 12 h of darkness.

[0101] Experimental Example 1

[0102] Based on the method of Example 3, the effects of different hormone ratios and types of culture medium in callus induction medium in step (2) on callus induction of kudzu root stem segments with buds were investigated. Specific grouping and results are shown in Table 1.

[0103] Table 1 Effects of different hormone ratios on callus induction of kudzu root stem segments with buds

[0104] Culture medium type IBA 2,4-D Callus growth status MS 0.1 0.1 Bright yellow, brownish color MS 0.5 0.2 Bright yellow color MS 1.0 0.3 No browning, tight texture 1 / 2MS 0.1 0.2 Browning 1 / 2MS 0.5 0.3 Loose texture 1 / 2MS 1.0 0.1 Light green color, loose texture 1 / 4MS 0.1 0.3 Light green color 1 / 4MS 0.5 0.1 Light green color, tight texture 1 / 4MS 1.0 0.2 Dark green color, tight texture

[0105] As shown in Table 1, 1 / 4MS is a suitable medium for the growth of Pueraria callus. The callus growth is best when IBA and 2,4-D are mixed in a ratio of 1:0.2.

[0106] Experimental Example 2

[0107] Based on the method of Example 3, the effects of different hormone ratios and types of culture medium in the Pueraria lobata proliferation culture medium on the differentiation of Pueraria lobata adventitious buds in step (4) were investigated. Specific grouping and results are shown in Table 2.

[0108] Table 2 Effects of different hormone ratios on callus proliferation culture of Pueraria lobata

[0109] Culture medium type 6-BA IAA Proliferation coefficient MS 0.5 0.2 3.5 MS 1.0 0.4 4.4 MS 1.5 0.6 3.8 1 / 2MS 0.5 0.4 3.6 1 / 2MS 1.0 0.6 4.7 1 / 2MS 1.5 0.2 4.3 1 / 4MS 0.5 0.6 3.9 1 / 4MS 1.0 0.2 3.2 1 / 4MS 1.5 0.4 3.4

[0110] As shown in Table 2, 1 / 2MS is a suitable medium for the growth of Pueraria callus. When 6-BA and IAA are mixed at a ratio of 1:0.6, the proliferation coefficient of adventitious buds differentiated from Pueraria callus is the highest.

[0111] Experimental Example 3

[0112] Based on the method of Example 3, the effects of different hormone ratios and types of culture medium in the Pueraria rooting culture medium on the differentiation of Pueraria adventitious buds in step (5) were investigated. Specific grouping and results are shown in Table 3.

[0113] Table 3 Effects of different hormone ratios on rooting culture of Pueraria lobata

[0114] Culture medium type NAA IBA Rooting rate (%) MS 0.1 0.1 82.5 MS 0.5 0.2 79.8 MS 1.0 0.3 85.4 1 / 2MS 0.1 0.2 82.6 1 / 2MS 0.5 0.3 91.5 1 / 2MS 1.0 0.1 87.9 1 / 4MS 0.1 0.3 72.4 1 / 4MS 0.5 0.1 85.7 1 / 4MS 1.0 0.2 79.2

[0115] As shown in Table 3, 1 / 2MS is a suitable medium for the growth of Pueraria callus. When NAA and IBA are in a ratio of 5:3, the differentiation rate of adventitious buds of Pueraria callus is the highest.

[0116] It can be seen that in the process of Pueraria tissue culture, the selection of culture medium type and the ratio of hormones are very important, which have a significant impact on the normal growth of Pueraria callus and the rooting process of Pueraria.

[0117] Experimental Example 3

[0118] Based on the method of Example 3, the effects of different seedling medium ratios in step (6) on the survival rate of kudzu seedlings after transplanting and hardening were investigated. The specific grouping and results are shown in Table 4.

[0119] Table 4 Effects of different seedling medium ratios on the survival rate of kudzu seedlings after transplanting

[0120]

[0121]

[0122] It can be seen that in the process of kudzu root tissue culture, the selection of seedling medium and the ratio setting between the components are very important, which have a significant impact on the survival rate of kudzu root. The nutrient soil was purchased from Jiangsu Xingnong Matrix Technology Co., Ltd.

[0123] 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 rapid propagation of Pueraria root tissue culture, characterized in that: The steps include: (1) collecting kudzu root tissue, disinfecting it, and inoculating it into a starting culture medium to obtain kudzu root sterile seedlings; (2) inoculating the sterile Pueraria seedlings obtained in step (1) into a callus induction medium for cultivation to obtain Pueraria callus tissue; (3) inoculating the kudzu root callus obtained in step (2) into an adventitious bud differentiation medium for cultivation to obtain kudzu root adventitious buds; (4) inoculating the kudzu root adventitious buds obtained in step (3) into a proliferation medium for cultivation to obtain kudzu root proliferation test tube plantlets; (5) inoculating the kudzu root proliferating seedlings obtained in step (4) into a rooting medium for cultivation to obtain kudzu rooting test tube seedlings; (6) hardening the kudzu root test tube seedlings obtained in step (5) and then transplanting them; The starting culture medium in step (1) is: MS+6-KT 2.0-2.2 mg / L+NAA 0.3-0.4 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, with a pH value of 5.8-6.0; The callus induction medium in step (2) is: 1 / 4MS + IBA 1.0-1.2 mg / L + 2,4-D 0.2-0.3 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, with a pH value of 5.8-6.0; The adventitious bud differentiation medium in step (3) is: MS+6-KT 3.0-3.2 mg / L+TDZ 0.5-0.6 mg / L+agar 7.0-7.5 g / L+sucrose 25-30 g / L, with a pH value of 5.8-6.0; The proliferation medium in step (4) is: 1 / 2MS + 6-BA 0.5-1.5 mg / L + IAA 0.2-0.6 mg / L + agar 7.0-7.5 g / L + sucrose 25-30 g / L, with a pH value of 5.8-6.0; The rooting medium in step (5) is: 1 / 2MS + NAA 0.5-0.7 mg / L + IBA 0.3-0.4 mg / L + activated carbon 0.8-0.9 g / L + sucrose 25-30 g / L + agar 7.0-7.5 g / L, with a pH value of 5.8-6.

0.

2. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: During the period of rapid propagation of kudzu root tissue culture, the light intensity is 2000-3000 lx, the temperature is 23-27° C., the humidity is 65%-75%, and the light duration is 11-13 hours per day.

3. A method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The culturing time in step (1) is 5 to 7 days; the culturing time in step (2) is 10 to 15 days; the culturing time in step (3) is 13 to 20 days; the culturing time in step (4) is 25 to 40 days; and the culturing time in step 5 is 17 to 25 days.

4. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The standard for transplanting the rooted test tube seedlings of Pueraria lobata in step (6) is: root length 3 to 5 cm, seedling height 5 to 7 cm.

5. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The time for hardening the seedlings in step (6) is 4 to 7 days.

6. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The transplanting method in step (6) is as follows: washing the kudzu root test tube seedlings, soaking the seedlings, transplanting them into the seedling culture medium, covering them with a film and cultivating them for 7 to 9 days, and then removing the film.

7. The method for rapid propagation of Pueraria root tissue culture according to claim 6, characterized in that: The soaking of seedlings is to soak the kudzu root test tube seedlings in 500 times thiophanate-methyl solution for 20 to 30 minutes.

8. The method for rapid propagation of Pueraria root tissue culture according to claim 6, characterized in that: The seedling culture medium is vermiculite and peat soil, and the mass ratio is 1:1-3.

9. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The varieties of kudzu root are Gange No. 1, Kukudzu or Wild kudzu.

10. The method for rapid propagation of Pueraria root tissue culture according to claim 1, characterized in that: The disinfection method of step (1) is: first disinfect with alcohol with a volume concentration of 70-80% for 30-60s, rinse with water 2-3 times, then disinfect with mercuric chloride with a volume concentration of 0.08-0.12% for 4-6min, and rinse with water 3-5 times.

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