A method for rapid seedling raising of volcanic kudzu vine tissue culture

Abstract

The invention relates to a method for rapid seedling cultivation of volcanic pueraria tissue culture, comprising the steps of collecting volcanic pueraria vines free of pests and diseases, removing leaves and pruning, cleaning, and disinfecting for use; cutting off the lateral buds or terminal buds of the volcanic pueraria vines that have been disinfected, inserting and inoculating them into an induction medium as explants for bud induction culture; grafting the buds onto a proliferation medium for proliferation culture to induce multiple buds; dividing the proliferated multiple buds into multiple groups, each group containing 2 to 3 buds, transferring them to a bud elongation and seedling culture medium for bud elongation culture to obtain robust and elongated adventitious buds; cutting off the elongated adventitious buds and transferring them to a rooting medium to induce root production, thereby cultivating new volcanic pueraria plants. The invention uses the lateral buds or terminal buds on the volcanic pueraria vines as explants, establishes a complete and mature tissue culture rapid propagation technology system, and provides technical support for volcanic pueraria virus-free seedling cultivation, standardized seedling cultivation, factory seedling cultivation, and biotechnology breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more specifically to a method for rapid seedling raising of Pueraria lobata tissue culture. Background Art

[0002] Pueraria thomsonii Benth is the dried root of Pueraria thomsonii Benth, a plant of the Leguminosae family. It was first recorded in Shennong's Herbal Classic. It has the effects of relieving muscle and fever, promoting fluid production and quenching thirst, and detoxifying alcohol. It is often used for symptoms such as exogenous fever, headache, thirst, thirst, dizziness, headache, and alcohol poisoning. Modern pharmacological studies have shown that puerarin in Pueraria thomsonii has antipyretic and antiviral activities, as well as protective effects on cardiovascular, brain, and liver damage, anti-tumor and diabetes treatment. Therefore, the breeding and cultivation of Pueraria thomsonii has economic value.

[0003] Volcanic Pueraria is called "Volcanic Pueraria" because it originates from Datang Volcano, Qujiang District, Shaoguan City. Compared with general Pueraria from other places, Volcanic Pueraria has more starch content, less fiber and no residue, and tastes powdery and glutinous. It is rich in protein, amino acids and various trace elements, and has the effects of moistening the intestines and clearing away heat. However, the current lack of excellent varieties and long-term cutting propagation of Volcanic Pueraria has led to the gradual loss of excellent germplasm resources, variety degradation, reduced quality, increased diseases and pests, and the low level of standardized planting technology. These problems have gradually emerged. At present, Datang Town basically adopts the traditional cutting planting method, which has been facing the problem of species degradation, with a series of defects such as increased branching of Pueraria blocks, low yield (600-750kg per mu of Pueraria), and reduced market commodity rate. There is an urgent need for excellent quality, high-quality seedlings and standardized planting technology to expand the industrial development of Volcanic Pueraria. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for rapid seedling cultivation by tissue culture of Pueraria lobata, using the lateral buds or terminal buds on the Pueraria lobata vines as explants instead of small sections of vines, and is not limited to young and tender vines. The focus is on observing the fullness of the buds, the freedom of material selection is improved, the material selection is convenient, and the browning rate is greatly reduced.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A method for rapid seedling raising of Pueraria lobata tissue culture, comprising the following steps:

[0007] S1. Material processing and surface disinfection: Collect pest-free volcanic kudzu vines, remove the leaves, trim them, clean them, and disinfect them for later use;

[0008] S2. Bud induction culture: Cut off the lateral buds or terminal buds of the sterilized volcanic powder vines, use them as explants, insert them into the induction culture medium for bud induction culture, place them in a culture room, control the culture temperature at 25±1°C, and culture them in the dark for 30 to 35 days to induce adventitious buds;

[0009] The pH value of the induction medium is 5.8-6.0, and the induction medium comprises a modified MS medium and a first additive, wherein the modified MS medium is an MS medium in which the contents of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaCl2·2H2O are halved while the contents of trace elements, organic elements, and iron salts remain unchanged;

[0010] S3, bud proliferation culture: the buds obtained in step S2 are inoculated on a proliferation culture medium for proliferation culture, placed in a culture room, the culture temperature is 25±1°C, the light intensity is 1800-2500LX, the light intensity is 12h / d, and the culture is carried out for 30-35 days to induce multiple buds;

[0011] Wherein, the pH value of the proliferation medium is 5.8-6.0, and the proliferation medium comprises MS medium and a second additive;

[0012] S4. Elongation of adventitious buds and cultivation of strong seedlings: Divide the proliferated multiple buds into multiple groups, each group containing 2 to 3 buds, and transfer them to bud elongation and strong seedling culture medium for bud elongation culture. Place them in a culture room with a culture temperature of 25±1℃, a light intensity of 1800 to 2500LX, and a light duration of 12h / d;

[0013] Wherein, the pH value of the bud elongation and seedling strengthening medium is 5.8-6.0, and the bud elongation and seedling strengthening medium comprises MS medium and a third additive;

[0014] S5. Rooting culture: After the elongated adventitious buds are cut off, they are transferred to the rooting medium and placed in a culture room at a culture temperature of 25±1°C and light culture for 30 to 35 days to induce root production;

[0015] Wherein, the pH value of the rooting medium is 5.8-6.0, and the rooting medium comprises a modified MS medium and a fourth additive;

[0016] S6. Plant the tissue culture seedlings with roots to cultivate new Pueraria lobata plants.

[0017] In one of the embodiments, in step S1, after the kudzu vines are trimmed into small segments of 1.5 to 2.5 cm, the small segments of kudzu vines are cleaned with detergent or washing powder for 10 to 15 minutes, and rinsed under running water for 1 hour to complete the cleaning operation.

[0018] In one of the embodiments, in step S1, the cleaned volcanic kudzu vine strips are soaked in 1:100 84 disinfectant and kept shaking for 10 to 15 minutes, taken out and soaked in 75% alcohol for 30 seconds, then washed with sterile water for 3 to 5 times, then soaked in HgCl2 for disinfection for 8 to 10 minutes, and finally washed with sterile water for 3 to 5 times to clean them directly.

[0019] In one embodiment, in step S2, the first additive includes 0.1-1 mg / L 6-BA, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or 0.2-0.5 mg / L KT, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or 0.1-0.3 mg / L TDZ, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder.

[0020] In one embodiment, in step S3, the second additive includes 0.2-1 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 8-9 g / L carrageenan, or 0.1-0.3 mg / L TDZ, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 8-9 g / L carrageenan.

[0021] In one embodiment, in step S4, the third additive includes 0.1-0.3 mg / L 6-BA, 0.1 mg / L I BA, 8-10% coconut juice, 30 mg / L sucrose and 8-9 g / L carrageenan.

[0022] In one embodiment, in step S5, the fourth additive includes 0.2-1 mg / L IBA, 30 g / L sucrose and 6-7 g / L agar powder.

[0023] In summary, the present invention has the following beneficial effects:

[0024] The present invention applies tissue culture technology to establish an effective artificial rapid propagation system of Pueraria lobata, takes the lateral buds or terminal buds of the stem segment of Pueraria lobata as explants, takes MS as a basic culture medium, selects suitable adventitious bud induction, bud proliferation, bud elongation and rooting culture medium formulas through different hormone types and concentration ratios, and provides a theoretical basis and practical operation technology for the factory production of Pueraria lobata seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1In the figure, A: select pest-free Pueraria lobata vines with lateral or terminal buds and trim them to 1.5-2.5cm; B: cut off the lateral or terminal buds of the vines and culture them separately to induce adventitious buds; C: after the adventitious buds are proliferated and cultured, "multiple buds" are proliferated; D: elongation of adventitious buds and cultivation of strong seedlings; E: rooting of adventitious buds; F: growth status of the plant after rooting. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0027] It is worth noting that the directional words such as "upper" and "lower" involved in this article are all relative to the viewing angle of the drawings and are only for the convenience of description and cannot be understood as limitations on the technical solution.

[0028] Currently reported tissue culture of Pueraria lobata usually uses young vines as explants, with strict requirements on the time and location of the material. In addition, the young vines are prone to browning, resulting in unstable or low bud germination rate.

[0029] However, in the prior art, the tissue culture seedlings of Pueraria lobata still have the problems of high explant browning rate, unstable bud induction rate, low bud proliferation coefficient, difficulty in bud elongation, and callus differentiation. A complete tissue culture seedling technology system has not yet been established for volcanic Pueraria lobata. In view of the problems existing in the prior art, the present invention provides a method for rapid seedling cultivation of volcanic Pueraria lobata tissue culture, establishes a complete volcanic Pueraria lobata seedling cultivation technology system, and provides a technical approach for its factory seedling cultivation and standardized seedling cultivation.

[0030] The present invention uses the lateral buds or terminal buds on the volcanic kudzu vines as explants for separate bud induction culture, and improves the culture effect through a specific culture medium, such as Figure 1 As shown, it provides a theoretical basis and practical operation technology for the factory production of volcanic kudzu seedlings. The specific cultivation steps are as follows:

[0031] S1. Material handling and surface disinfection:

[0032] Collect disease- and insect-free Kudzu vines, remove the leaves, trim them, clean them, and disinfect them for later use.

[0033] Specifically, after the volcanic kudzu vines are trimmed into small segments of 1.5 to 2.5 cm, the small segments of the volcanic kudzu vines are cleaned with detergent or washing powder for 10 to 15 minutes, and rinsed under running water for 1 hour to complete the cleaning operation. The volcanic kudzu vines are collected from the wild, and there are epidermal hairs on the vines, and there is more dust and more microorganisms on the surface, which is not easy to clean and requires a long time to rinse.

[0034] Soak the cleaned Pueraria lobata vines in 1:100 84 disinfectant and keep shaking for 10 to 15 minutes. After taking out, soak them in 75% alcohol for 30 seconds, then wash them with sterile water for 3 to 5 times, then soak them in HgCl2 for disinfection for 8 to 10 minutes, and finally wash them with sterile water for 3 to 5 times to clean them directly.

[0035] It should be noted that different microorganisms have different tolerances to different disinfectants. The present invention uses different disinfectants to kill different types of microorganisms and reduce the contamination rate. Before cutting the rattan, the surface is disinfected with 1:100 84 disinfectant to kill some microorganisms. Then, 75% alcohol is used for disinfection for 30 seconds, and HgCl 2 is used for disinfection to kill some stubborn microorganisms.

[0036] S2. Bud induction culture:

[0037] The lateral buds or terminal buds of the sterilized Kudzu vines are cut off and used as explants. They are inserted into the induction medium for bud induction culture. The culture temperature is controlled at 25±1°C in a culture room and cultured in the dark for 30 to 35 days to induce adventitious buds.

[0038] The pH value of the induction medium is 5.8-6.0, and the induction medium includes a modified MS medium and a first additive. The modified MS medium is an MS medium in which the content of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaCl 2·2H2O is halved while the content of trace elements, organic elements, and iron salts remains unchanged.

[0039] The first additive includes 0.1-1 mg / L 6-BA, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or 0.2-0.5 mg / L KT, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or 0.1-0.3 mg / L TDZ, 0.1 mg / L I BA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder.

[0040] The present invention found that a low concentration of cytokinin combined with auxin can promote the germination of kudzu buds, and the three combinations of 6-BA / IBA, KT / I BA, and TDZ / I BA can all achieve the germination of volcanic kudzu buds. In the prior art, most of the tissue cultures of other kudzu buds use a culture medium of 6-BA / NAA combination, which is very different from the culture medium components used in the present invention. In some of the schemes, the present invention also uses a higher amount of PVP, which helps to reduce the browning of the explants.

[0041] The invention performs separate bud induction culture on the lateral buds or terminal buds on the volcanic kudzu vines, adds a combination of cytokinin 6-BA, KT or TDZ and auxin I BA into an improved MS culture medium, and after 15 days, the lateral buds or terminal buds begin to germinate, and a small amount of sterile small buds are formed. After continuing to culture for 30 days, 2 to 3 buds will be generated around the small buds. The invention enables the bud induction rate of the volcanic kudzu vines to be higher, reaching more than 80%.

[0042] S3. Bud proliferation culture:

[0043] The buds obtained in step S2 are inoculated on a proliferation medium for proliferation culture, placed in a culture room, the culture temperature is 25±1°C, the light intensity is 1800-2500LX, the light intensity is 12h / d, and cultured for 30-35 days to induce multiple buds.

[0044] Among them, the pH value of the proliferation culture medium is 5.8-6.0, and the proliferation culture medium includes MS culture medium and a second additive. The MS culture medium is an existing conventional culture medium, and the second additive includes 0.2-1 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 8-9 g / L carrageenan, or 0.1-0.3 mg / L TDZ, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 8-9 g / L carrageenan.

[0045] In the bud proliferation culture, under the appropriate 6-BA / I BA or TDZ / I BA concentration, the adventitious bud proliferation rate reached more than 90%, and the buds grew well, with many buds, forming "multiple bud bodies".

[0046] S4. Elongation of adventitious buds and cultivation of strong seedlings:

[0047] The proliferated multiple buds are cut into multiple groups, each containing 2 to 3 buds, and transferred to a bud elongation and seedling strengthening medium for bud elongation culture, and placed in a culture room with a culture temperature of 25±1°C, a light intensity of 1800 to 2500 LX, and a light duration of 12 h / d;

[0048] The pH value of the bud elongation and seedling strengthening medium is 5.8-6.0, and the bud elongation and seedling strengthening medium includes MS medium and a third additive, and the third additive includes 0.1-0.3 mg / L 6-BA, 0.1 mg / L I BA, 8-10% coconut juice, 30 mg / L sucrose and 8-9 g / L carrageenan. The present invention uses coconut juice instead of conventional hydrolyzed casein because coconut juice contains rich nutrients, and coconut juice can promote the elongation of volcanic pueraria lobata buds, bud growth, and make the buds grow strong.

[0049] In the culture of adventitious bud elongation, adding appropriate concentrations of 6-BA and coconut juice can promote the elongation of adventitious buds and make the adventitious buds grow healthily.

[0050] S5. Rooting culture:

[0051] After the elongated adventitious buds are cut off, they are transferred to rooting medium and placed in a culture room at a culture temperature of 25±1°C and light culture for 30 to 35 days to induce root production.

[0052] The pH value of the rooting medium is 5.8-6.0, and the rooting medium includes improved MS culture and a fourth additive, wherein the fourth additive includes 0.2-1 mg / L I BA, 30 g / L sucrose and 6-7 g / L agar powder.

[0053] S6. Plant the tissue culture seedlings with roots to cultivate new Pueraria lobata plants.

[0054] It should be noted that the present invention improves the culture medium and selects a scheme in which the content of some inorganic salts in the modified MS culture medium is halved while the content of iron salts remains unchanged. This is based on the growth and development characteristics of the volcanic kudzu vine and the properties of the material itself, and selects a suitable culture medium to facilitate plant growth. In the bud induction stage, the kudzu vine buds are prone to browning, and the halving of the inorganic salt content is beneficial to reducing the browning rate of the explants and improving the survival rate. In the rooting stage, it is beneficial to rooting.

[0055] The present invention adopts different culture media at different stages. For example, in the bud induction stage, the buds are small and do not need to absorb too many inorganic salt ions. Too many inorganic salt ions will accelerate the browning of the explants and reduce the survival rate. Therefore, in the bud induction stage, a culture medium with half the inorganic salt ions is used; in the bud proliferation and elongation stage, the buds grow rapidly and more inorganic salt ions are needed for rapid proliferation, and the inorganic salt ions in the culture medium do not need to be halved; in the rooting stage, a culture medium with half the inorganic salt ions is often used, which is conducive to the rooting of volcanic kudzu tissue culture seedlings.

[0056] The present invention selects suitable regeneration explants, takes plump lateral buds or terminal buds as explants, and configures an induction medium added with cytokinins 6-BA, KT and TDZ combined with auxin I BA, which is conducive to inducing bud germination, greatly reduces the browning rate, stabilizes bud germination, and the induction rate reaches more than 80%.

[0057] In the bud induction culture of the present invention, the tenderness of the rattan is no longer limited, and the fullness of the buds on the rattan is focused on. The buds are cut and cultured separately, the freedom of materials used for experiments is improved, the materials are convenient to obtain, the bud germination rate is greatly improved, the adventitious bud induction rate is above 80%, and in the culture of adventitious bud proliferation, the adventitious buds proliferate "multiple buds" to form "multiple bud bodies", the proliferation coefficient is greatly improved, the proliferation rate of the adventitious buds reaches above 90%, and to some extent determines the number of regenerated plants, and the buds are cut and transferred to a culture medium added with 8-10% coconut juice to utilize the elongation of the buds, proliferation and strong seedling culture. The test tube seedlings are easy to root, and they start to root 6 days after inoculation. The rooting rate of the test tube seedlings can reach 100%, the plants grow robustly, and the root system is well-developed, which provides a technical reference for other kudzu seedlings.

[0058] The present invention applies tissue culture technology to establish an effective artificial rapid propagation system of Pueraria lobata, takes the lateral buds or terminal buds of the stem segment of Pueraria lobata as explants, takes MS as a basic culture medium, selects suitable adventitious bud induction, bud proliferation, bud elongation and rooting culture medium formulas through different hormone types and concentration ratios, and provides a theoretical basis and practical operation technology for the factory production of Pueraria lobata seedlings.

[0059] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for rapid seedling cultivation of Pueraria lobata tissue culture, characterized in that: The following steps are involved: S1. Material processing and surface disinfection: Collect pest-free volcanic kudzu vines, remove the leaves, trim them, clean them, and disinfect them for later use; S2. Bud induction culture: Cut off the lateral buds or terminal buds of the sterilized Pueraria lobata vines, use them as explants, and inoculate them on the induction culture medium for bud induction culture to induce adventitious buds; Wherein, the induction medium is composed of a modified MS medium and a first additive, wherein the modified MS medium is an MS medium in which the contents of NH4NO3, KNO3, MgSO4▪7H2O, KH2PO4, and CaCl2▪2H2O are halved while the contents of trace elements, organic elements, and iron salts remain unchanged; S3, bud proliferation culture: the buds obtained in step S2 are inoculated on a proliferation medium for proliferation culture to induce multiple buds; Wherein, the proliferation medium consists of MS medium and a second additive; S4, elongation of adventitious buds and cultivation of strong seedlings: the proliferated multiple buds are cut into multiple groups, each group contains 2 to 3 buds, and transferred to the bud elongation and strong seedling culture medium for bud elongation culture, and placed in the culture room; Wherein, the bud elongation and seedling strengthening medium consists of MS medium and a third additive; S5, rooting culture: cut off the elongated adventitious buds and transfer them to rooting medium to induce root production; Wherein, the pH value of the rooting medium is 5.8-6.0, and the rooting medium is composed of improved MS culture and a fourth additive; S6, planting the tissue culture seedlings with root systems to cultivate new volcanic kudzu plants; Wherein, in step S2, the first additive consists of 0.1-1 mg / L 6-BA, 0.1 mg / L IBA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or consists of 0.2-0.5 mg / L KT, 0.1 mg / L IBA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder, or consists of 0.1-0.3 mg / L TDZ, 0.1 mg / L IBA, 0.5-4 g / L PVP, 30 g / L sucrose and 6-7 g / L agar powder; Wherein, in step S3, the second additive consists of 0.2-1 mg / L 6-BA, 0.05-0.1 mg / L IBA, 30 g / L sucrose and 8-9 g / L carrageenan, or consists of 0.1-0.3 mg / L TDZ, 0.05-0.1 mg / L IBA, 30 g / L sucrose and 8-9 g / L carrageenan; Wherein, in step S4, the third additive is composed of 0.1-0.3 mg / L 6-BA, 0.1 mg / L IBA, 8-10% coconut juice, 30 mg / L sucrose and 8-9 g / L carrageenan; Wherein, in step S5, the fourth additive is composed of 0.2-1 mg / L IBA, 30 g / L sucrose and 6-7 g / L agar powder; Wherein, the pH values ​​of the bud induction medium, the proliferation medium, the bud elongation medium and the rooting medium are all 5.8-6.

0.

2. The method for rapid seedling raising of Pueraria lobata tissue culture according to claim 1, characterized in that: In step S1, after the volcanic kudzu vines are trimmed into small sections of 1.5 to 2.5 cm, the small sections of the volcanic kudzu vines are washed with detergent or washing powder for 10 to 15 minutes, and rinsed under running water for 1 hour to complete the washing operation.

3. The method for rapid seedling raising of Pueraria lobata tissue culture according to claim 2, characterized in that: In step S1, the cleaned Pueraria lobata vine strips are soaked in 1:100 84 disinfectant and shaken for 10 to 15 minutes. After being taken out, they are soaked in 75% alcohol for 30 seconds, then washed with sterile water for 3 to 5 times, and then soaked in HgCl2 for disinfection for 8 to 10 minutes. Finally, they are washed with sterile water for 3 to 5 times to clean them directly.

4. The method for rapid seedling raising of Pueraria lobata tissue culture according to claim 1, characterized in that: In steps S3-S5, the light intensity is 1800-2500LX.

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