A culture medium composition and method for inducing callus sprouting in woody plants

Abstract

The present application belongs to the field of woody plant callus culture and differentiation, and particularly relates to a culture medium combination and method for inducing the germination of woody plant callus. The present application provides a culture medium combination and method for inducing the germination of woody plant callus. In the combined culture medium, NAA, 2,4-D, 6-BA, gibberellin (GA3) GA3, glutamine, extract of Scutellaria baicalensis Georgi, hydrolyzed milk protein, zeatin, and sucrose are conducive to the induction of callus formation and differentiation into buds, and effectively prevent callus browning. The results of examples show that the culture medium combination of the present application can induce the germination of woody plant callus in a short time, reduce the browning rate, and the total flavonoid content of the obtained seedlings is high.

Description

Technical Field

[0001] This invention belongs to the field of callus culture and differentiation of woody plants, and specifically relates to a culture medium combination and method for inducing callus sprouting in woody plants. Background Technology

[0002] The main functions of woody plants include maintaining ecological balance, regulating climate, preventing wind erosion and sandstorms, reducing noise pollution, purifying air, conserving water resources, maintaining soil and water, and regulating carbon and oxygen balance. Poplar (Populus L.) is a very important woody plant. It is a crucial afforestation species in my country, a model woody plant characterized by rapid growth, high yield, and strong adaptability. Widely cultivated in my country, poplar is a major building material, landscaping, and afforestation species, playing an irreplaceable role in timber production, urban greening, and ecological construction, possessing significant economic and ecological value. However, poplar cultivation is susceptible to environmental and pathogenic factors, leading to decreased yield and quality. Therefore, selecting superior poplar varieties is key to improving their adaptability to the surrounding environment. Compared to traditional asexual propagation methods such as cuttings and grafting, tissue culture offers advantages such as rapid propagation, convenient management, short cultivation cycle, and ease of industrialization. Plant callus is totipotent and typically possesses advantages such as rapid growth, high propagation volume, small footprint, and suitability for industrialized production, making it an ideal method for breeding superior varieties. Studying plantlets regenerated from callus plays a crucial role in investigating plant stress resistance. However, woody plants still face challenges in tissue culture, including high callus browning rates and long differentiation cycles into buds. Summary of the Invention

[0003] This invention provides a culture medium combination and method for inducing callus sprouting in woody plants. The culture medium combination of this invention shortens the time for callus regeneration and results in a low browning rate of the callus.

[0004] To address the above technical problems, the present invention proposes the following technical solution:

[0005] This invention provides a culture medium combination for inducing callus sprouting in woody plants, the culture medium combination comprising an induction culture medium and a sprouting culture medium;

[0006] The induction medium uses MS medium as the basic medium and also includes: 4-6 mg / L 2,4-D, 450-500 mg / L glutamine, 250-350 mg / L hydrolyzed milk protein, 25-30 g / L sucrose and 6.5-7.5 g / L agar;

[0007] The germination medium uses MS medium as the basic medium and also includes: 0.5-0.8 mg / L 6-benzylaminopurine, 0.2-0.4 mg / L naphthaleneacetic acid, 40-60 mg / L Scutellaria baicalensis extract, 0.15-0.25 mg / L gibberellin, 0.4-0.6 mg / L zeatin, 25-30 g / L sucrose, and 5.5-6.0 g / L agar.

[0008] Preferably, the pH value of the induction medium and the germination medium is 5.5 to 5.8.

[0009] Preferably, the preparation method of the Scutellaria baicalensis extract includes: mixing and extracting Scutellaria baicalensis with an organic solvent to obtain an extract; and ultrasonically extracting the extract and filtering it to obtain the Scutellaria baicalensis extract.

[0010] This invention provides a method for inducing callus sprouting in woody plants, using the culture medium combination described in any of the above-mentioned technical solutions to induce callus sprouting in woody plants. The method includes the following steps:

[0011] Explants of woody plants were inoculated onto an induction medium for callus induction culture to obtain callus tissue.

[0012] The callus tissue was transferred to a budding culture medium for budding culture.

[0013] Preferably, the explant comprises a leaf.

[0014] Preferably, before inoculating the explants of the woody plant into the induction medium, the procedure further includes: cleaning and disinfecting the explants; the cleaning includes rinsing with running water and rinsing with deionized water in sequence; the disinfection includes: rinsing with 75% ethanol for 40-50 seconds, rinsing with sterile water 3-4 times, then soaking in a sodium hypochlorite solution with an effective chlorine content of 3.5%-4.5% for 8-12 minutes, and rinsing with sterile water 3-4 times.

[0015] Preferably, the induction culture and budding culture are light-dark alternating cultures, wherein the light culture for the induction culture includes white light irradiation, and the light culture for the budding culture includes red light irradiation.

[0016] Preferably, the white light irradiation time is 18 hours / day, and the light intensity of the white light irradiation is 10,000 to 20,000 lux; the red light irradiation time is 18 hours / day, and the light intensity of the red light irradiation is 10,000 to 20,000 lux.

[0017] Preferably, the induction culture time is 30-60 days; the budding culture time is 50-130 days.

[0018] Preferably, the temperature for the induction culture and budding culture is 23-25°C, and the humidity for the induction culture and budding culture is 16%-17%.

[0019] The beneficial effects of the present invention are as follows: The present invention provides a culture medium combination for inducing callus sprouting in woody plants, the culture medium combination including induction culture medium and sprouting culture medium.

[0020] In the culture medium combination provided by this invention, 2,4-dichlorophenoxyacetic acid (2,4-D) acts as a plant growth regulator, stimulating plant growth; naphthaleneacetic acid, as a plant growth regulator, promotes cell division, growth, and enhances resistance; 6-benzylaminopurine is a synthetic cytokinin-like plant growth regulator that promotes cell division, induces tissue differentiation, and induces bud formation in the culture medium; glutamine provides the nitrogen source necessary for plant growth; hydrolyzed milk protein provides nutrients for plant growth; Scutellaria baicalensis extract is rich in flavonoids, has antioxidant effects, can regulate plant hormone transport, and increase the antibacterial activity and stress resistance of plants; gibberellin (GA3) can promote cell division, stimulate bud formation, and inhibit callus browning to a certain extent; zeatin is a cytokinin derived from adenine that promotes cell division and the formation of young buds from callus tissue; sucrose provides the energy required for plant growth and development, participates in various physiological activities and regulation of plants, and enhances stress resistance; agar acts as a solid support for plants, inhibits the growth of bacteria and other contaminants, and protects the culture medium.

[0021] The combined effects of NAA, 2,4-D, 6-BA, gibberellin GA3, glutamine, hydrolyzed lactic acid, Scutellaria baicalensis extract, zeatin, and sucrose promote callus formation and shoot differentiation, effectively preventing callus browning. Results from the examples show that the culture medium combination of this invention can induce callus sprouting in woody plants in a short time, reduce the browning rate, and produce young shoots with high total flavonoid content. These young shoots can produce a large amount of flavonoids, which not only enhances the plant's own stress resistance, but the extract can also be applied to research on the prevention of human diseases.

[0022] This invention also provides a method for inducing callus sprouting in woody plants. The induction culture method of this invention has a short production cycle and can be industrialized through amplification culture. Attached Figure Description

[0023] Figure 1 Diagrams illustrating callus formation from poplar trees cultured on different culture media;

[0024] Figure 2The results show the effect of induction medium on callus differentiation in Comparative Example 2-1; where A represents the growth status of callus at different culture times during budding induction, B represents the budding rate, and C represents the browning rate.

[0025] Figure 3 The results show the effect of culture medium B used in Comparative Example 2-2 on the differentiation of callus; where A represents the growth status of callus at different culture times during budding induction, B represents the budding rate, and C represents the browning rate.

[0026] Figure 4 The budding culture medium used in Example 2 promotes the differentiation of callus tissue; where A represents the growth status of callus tissue at different culture times during budding induction, B represents the budding rate, and C represents the browning rate.

[0027] Figure 5 Figure showing the total flavonoids determination and survival rate statistics of callus or shoots cultured in different types of culture media;

[0028] Figures 2-5 In the figure, "**" indicates that the difference is significant at the P < 0.001 level. Detailed Implementation

[0029] The present invention provides a culture medium combination for inducing callus sprouting in woody plants, the culture medium combination comprising an induction medium and a sprouting medium.

[0030] The induction medium of this invention uses MS medium as the basal medium and further comprises: 4–6 mg / L 2,4-D, 450–500 mg / L glutamine, 250–350 mg / L hydrolyzed milk protein, 25–30 g / L sucrose, and 6.5–7.5 g / L agar. The solvent of the induction medium is water. As one embodiment, the concentration of 2,4-D in the induction medium provided by this invention can be 4–6 mg / L; in specific embodiments of this invention, the concentration of 2,4-D in the induction medium can be 4, 4.5, 5, 5.5, or 6 mg / L. The concentration of glutamine in the induction medium can be 450–500 mg / L; in specific embodiments of this invention, the concentration of glutamine in the induction medium can be 450, 460, 470, 480, 490, or 500 mg / L. The concentration of hydrolyzed milk protein in the induction medium can be 250–350 mg / L; in specific embodiments of the present invention, the concentration of hydrolyzed milk protein in the induction medium can be 250, 270, 290, 310, 330, or 350 mg / L. The concentration of sucrose in the induction medium can be 25–30 g / L, or 26–29 g / L; in specific embodiments of the present invention, the concentration of sucrose in the induction medium can be 25, 26, 27, 28, 29, or 30 g / L. The concentration of agar in the induction medium can be 6.5–7.5 g / L; in specific embodiments of the present invention, the concentration of agar in the induction medium can be 6.5, 6.7, 6.9, 7.0, 7.1, 7.3, or 7.5 g / L. In the present invention, 2,4-D, glutamine, and hydrolyzed milk protein, at appropriate concentration ratios, can jointly promote the formation of callus tissue in woody plants and effectively reduce the browning rate of callus tissue. In this invention, the pH value of the induction culture medium can be 5.5 to 5.8.

[0031] The germination medium of this invention uses MS medium as the basic medium and further includes: 0.5–0.8 mg / L 6-benzylaminopurine, 0.2–0.4 mg / L naphthaleneacetic acid, 40–60 mg / L Scutellaria baicalensis extract, 0.15–0.25 mg / L gibberellin, 0.4–0.6 mg / L zeatin, 25–30 g / L sucrose, and 5.5–6.0 g / L agar. As one embodiment, the concentration of 6-benzylaminopurine in the germination medium provided by this invention can be 0.5–0.8 mg / L or 0.6–0.7 mg / L; in specific embodiments of this invention, the concentration of 6-benzylaminopurine in the germination medium can be 0.5, 0.6, 0.7, or 0.8 mg / L. The concentration of naphthaleneacetic acid (NAA) in the germination medium can be 0.2–0.4 mg / L, or 0.3 mg / L; in specific embodiments of the present invention, the concentration of NAA in the germination medium can be 0.2, 0.25, 0.3, 0.35, or 0.4 mg / L. The concentration of Scutellaria baicalensis extract in the germination medium can be 40–60 mg / L, or 50 mg / L; in specific embodiments of the present invention, the concentration of Scutellaria baicalensis extract in the germination medium can be 40, 45, 50, 55, or 60 mg / L. The concentration of gibberellin (GA3) in the germination medium can be 0.15–0.25 mg / L, or 0.20 mg / L; in specific embodiments of the present invention, the concentration of gibberellin (GA3) in the germination medium can be 0.15, 0.17, 0.19, 0.20, 0.23, or 0.25 mg / L. The concentration of zeatin in the germination medium can be 0.4–0.6 mg / L, or 0.5 mg / L. The concentration of sucrose in the germination medium can be 25–30 g / L, or 26–28 g / L; in specific embodiments of the present invention, the concentration of sucrose in the germination medium can be 25, 26, 27, 28, 29, or 30 g / L. The concentration of agar in the germination medium can be 5.5–6.0 g / L, or 6.0 g / L; in specific embodiments of the present invention, the concentration of agar in the germination medium can be 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 g / L. In the present invention, naphthaleneacetic acid, 6-benzylaminopurine, Scutellaria baicalensis extract, gibberellin GA3, and zeatin, when mixed at appropriate concentrations, can promote rapid germination of callus tissue produced by woody plants and inhibit the formation of browned callus tissue. In the present invention, the pH value of the germination medium can be 5.5–5.8. The solvent of the germination medium is water, and the preferred method for preparing the germination medium includes: mixing and sterilizing the MS medium, 6-benzylaminopurine, naphthaleneacetic acid, scutellaria baicalensis extract, gibberellin GA3, sucrose, agar and water, and then adding zeatin when the temperature reaches about 50°C.

[0032] The induction culture medium and germination culture medium described in this invention are used after sterilization. This invention does not have any special limitations on the sterilization method; conventional methods can be used.

[0033] This invention does not impose any particular limitation on the preparation of the Scutellaria baicalensis extract; conventional methods can be used. As one embodiment, the preparation method of the Scutellaria baicalensis extract includes: mixing and extracting Scutellaria baicalensis with an organic solvent to obtain an extract; ultrasonically extracting the extract and then filtering to obtain the Scutellaria baicalensis extract. As one embodiment, this invention uses Scutellaria baicalensis powder mixed with an organic solvent; as one example, the water content of the Scutellaria baicalensis powder can be 8%; the mass-to-volume ratio of the Scutellaria baicalensis powder to the organic solvent can be 1g:100mL; the organic solvent can be 70% ethanol (volume concentration). As one embodiment, the extraction method can be soaking, and the soaking time can be 24 hours; the ultrasonic extraction time can be 30 minutes, the ultrasonic extraction power can be 80W, and the ultrasonic frequency can be 40kHz. This invention does not impose any particular limitation on the filtration method; conventional methods can be used. The filtrate obtained after filtration is the Scutellaria baicalensis extract. The Scutellaria baicalensis extract obtained by organic solvent extraction has high quercetin and total flavonoid content, which is beneficial for improving the stress resistance of buds.

[0034] This invention does not have any special limitations on the source of the components in the culture medium combination; conventional commercially available products can be used.

[0035] This invention also provides a method for inducing callus sprouting in woody plants, using the culture medium combination described in the above technical solution to induce callus sprouting in woody plants. The method includes the following steps:

[0036] Explants of woody plants were inoculated onto an induction medium for callus induction culture to obtain callus tissue.

[0037] The callus tissue was transferred to a budding culture medium for budding culture.

[0038] This invention involves inoculating explants of woody plants onto an induction medium for callus induction culture to obtain callus tissue. Preferably, before inoculating the explants into the induction medium, the process further includes cleaning and disinfecting the explants. The cleaning preferably includes rinsing with running water and then rinsing with deionized water. This invention does not have specific limitations on the cleaning method, as long as thorough cleaning is achieved. As one possible implementation, the running water rinsing time can be 4 hours, and the deionized water rinsing time can be 3-4 times. The disinfection preferably includes rinsing with 75% ethanol for 40-50 seconds, or 42-44 seconds; rinsing with sterile water 3-4 times; then soaking in a sodium hypochlorite solution with an effective chlorine content of 3.5%-4.5% for 8-12 minutes, or 9-11 minutes; followed by rinsing with sterile water 3-4 times. In a specific embodiment of the present invention, the leaf is first rinsed with 75% ethanol for 45 seconds and then rinsed with sterile water 3 to 4 times; then it is soaked in a sodium hypochlorite solution with an effective chlorine content of 4% for 10 minutes and rinsed with sterile water 3 to 4 times.

[0039] This invention preferably uses leaves of woody plants as explants for seedling cultivation. The leaves are separated into individual leaflets and then cleaned and disinfected. In this invention, the leaves of the woody plant are preferably young leaves from the top of the plant; these young leaves are those that have grown for 2-5 months. Young leaves are used because they lack obvious lignification, have a larger surface area for easier handling, and are more likely to develop callus tissue. The woody plants used in this invention preferably include poplar and / or pigeon pea, with the poplar including at least one of Populus tomentosa, Populus microphylla, and Populus tomentosa.

[0040] After cleaning and disinfecting the leaves as described in this invention, single leaves are preferably inoculated into an induction medium for callus induction culture. In this invention, the induction culture temperature can be 23–25°C or 24°C; the induction culture time can be 30–60 days or 25–35 days; bud differentiation begins within 20–40 days of callus culture, and callus tissue with a diameter of 0.5–2 cm is obtained after 30–60 days of culture. The humidity of the induction culture can be 16%–17% or 16.2%. The induction culture of this invention is preferably a light-dark alternating culture. Light culture is carried out under light conditions, more preferably under white light irradiation; the white light irradiation time can be 18 hours / day; the white light intensity can be 10,000–20,000 lux. White light irradiation in this invention is achieved by using an LED light source. The dark culture time can be 6 hours / day. The purpose of dark culture is to promote callus formation, avoid photoinhibition, reduce photo-oxidation, maintain plant hormone balance, and promote cell division.

[0041] Callus tissue with a diameter of 0.5–2 cm was obtained. In this invention, the callus tissue was transferred to a budding culture medium for budding culture. During the transfer process, the callus tissue may or may not be cut into pieces.

[0042] In one implementation, the temperature for germination culture can be 23–25°C or 24°C. The germination culture time can be 50–130 days. The humidity for germination culture can be 16%–17% or 16.2%.

[0043] The budding culture described in this invention is preferably carried out under alternating light and dark conditions. The light culture is conducted under illumination, more preferably under red light illumination. The red light illumination time can be 18 hours per day; the light intensity of the red light illumination can be 10,000–20,000 lux. The dark culture time can be 6 hours per day. Red light illumination in this invention is achieved by adjusting the LED light source to produce only red light. Red light is one of the most effective light sources for plant photosynthesis in the full spectrum. Irradiating plants with red light can promote plant growth, improve stress resistance, promote the absorption of nutrients in the culture medium, and increase photosynthesis. Irradiation with red light promotes the formation of tender buds from callus tissue and reduces the browning rate of callus tissue.

[0044] After budding culture, young shoots are obtained, with a height of 0.5–2 cm. The total flavonoid content in these young shoots is higher than that of ordinary callus or its budding tissue, resulting in stronger stress resistance. This strong stress resistance is due to the high flavonoid content. Therefore, applying the technical solution of this invention to the study of the molecular mechanisms of woody plant growth and development and the cultivation of resistant seedlings has significant value.

[0045] The culture medium combination and culture conditions of the present invention can induce callus tissue to generate buds in woody plants in a short time and reduce the browning rate of callus tissue.

[0046] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] The reagents or instruments used in this invention are all conventional products that can be purchased from the market.

[0048] 1. The culture medium components used in the following examples and comparative examples are as follows:

[0049] The induction medium (denoted as medium A) consists of MS medium as the base medium, supplemented with 2,4-D, glutamine, hydrolyzed milk protein, sucrose, and agar.

[0050] The amounts of each additive added to culture medium A are as follows: 5 mg / L 2,4-D; 500 mg / L glutamine; 300 mg / L hydrolyzed milk protein; 30 g / L sucrose; 7 g / L agar; pH = 5.8.

[0051] The components of culture medium B are: MS medium as the basal medium, supplemented with 1 mg / L naphthaleneacetic acid, 2 mg / L 6-benzylaminopurine, 500 mg / L hydrolyzed milk protein, 700 mg / L proline, 30 g / L sucrose, and 7 g / L agar. The pH of the medium is 5.8. Proline, as an osmotic protectant, helps plants maintain intracellular water balance and cell structure stability, enhances stress resistance, and regulates plant development.

[0052] The germination medium (denoted as medium C) consisted of MS medium as the basal medium, supplemented with 0.2 mg / L naphthaleneacetic acid, 0.6 mg / L 6-benzylaminopurine, 0.2 mg / L gibberellin GA3, 30 g / L sucrose, 6 g / L agar, and 5 mL / L crude Scutellaria baicalensis extract, with a flavonoid content of 10 mg / mL in the Scutellaria baicalensis extract. The remaining components of the medium were sterilized and then supplemented with 0.5 mg / L zeatin. The pH of the medium was 5.8.

[0053] 2. Methods for determining total flavonoids in cultured callus tissue or young shoots of understory economic plants.

[0054] The total flavonoid content was determined by a spectrophotometric method using aluminum salt in an alkaline medium. Under alkaline conditions, flavonoids form complexes with aluminum salts, which have a maximum absorption peak at a wavelength of 500 nm.

[0055] Taking young poplar buds as an example, the specific steps of the measurement method are as follows:

[0056] (1) First, the standard curve is created.

[0057] Accurately weigh 5 mg of rutin standard, dissolve it in 70% ethanol (v / v), and dilute to 25 mL in a volumetric flask. Shake well to obtain a 0.2 mg / mL standard solution. Accurately pipette 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the standard solution into separate 10 mL volumetric flasks. Add 0.4 mL of reagent A, shake well, and let stand for 6 min. Add 0.4 mL of reagent B, shake well, and let stand for 6 min. Add 4.0 mL of reagent C, then add water to the mark, shake well, and let stand for 15 min. Use the reagent blank as the reference solution. Measure the absorbance at 500 nm using a 1 cm cuvette and plot the standard curve. The standard curve is shown below. Figure 5 The standard curve equation is: y = 0.3372x + 0.0413, where x is the content of the flavonoid standard rutin; and y is the absorbance value at 500 nm.

[0058] The assay reagent A is a 5% NaNO2 solution (mass concentration).

[0059] The assay reagent B is a 10% Al(NO3)3 solution;

[0060] The reagent C used for the determination is a 5% NaOH solution.

[0061] (2) Extraction of total flavonoids from Populus euphratica samples.

[0062] Fresh Populus tomentosa samples were dried at low temperature until the moisture content was less than 8%, and then made into a dry powder. 1.0 g of the dry powder was accurately weighed and placed in a 100 mL volumetric flask. 30 mL of 70% ethanol was added, and the mixture was soaked for 24 hours. The mixture was then ultrasonically extracted for 30 minutes, filtered, and the filtrate was diluted to a final volume with 70% ethanol in a 100 mL volumetric flask to obtain the flavonoid extract, which was then set aside for use.

[0063] (3) Determination of total flavonoid content in the sample.

[0064] Take 1.00 mL of the flavonoid extract from step (2) and place it in a 10 mL volumetric flask. Add 0.4 mL of reagent A, shake well, and let stand for 6 min. Then add 0.4 mL of reagent B, shake well, and let stand for 6 min. Then add 4.0 mL of reagent C, and add water to the 10 mL mark of the volumetric flask. Shake well and let stand for 15 min. Use the reagent blank as the reference solution. Measure the absorbance at 500 nm using a 1 cm cuvette, and calculate the total flavonoid content using the standard curve method. Reagent A is a 5% NaNO2 solution; reagent B is a 10% Al(NO3)3 solution; and reagent C is a 5% NaOH solution.

[0065] 3. Methods for preparing Scutellaria baicalensis extract and determining its flavonoid content

[0066] The preparation method of Scutellaria baicalensis extract is as follows: Fresh Scutellaria baicalensis sample is dried at low temperature to a moisture content of less than 8% to obtain a dry powder. 1.0 g of the dry powder is accurately weighed and placed in a 100 mL volumetric flask. 30 mL of 70% ethanol is added, and the mixture is soaked for 24 hours. Ultrasonic extraction is then performed for 30 minutes. After two filtrations (first using gauze, then using a 0.22 μm filter membrane), the resulting filtrate is the Scutellaria baicalensis extract.

[0067] The flavonoid content of the obtained Scutellaria baicalensis extract was determined as follows: The Scutellaria baicalensis extract was diluted to a 100 mL volumetric flask with 70% ethanol to obtain a flavonoid extract. 1.00 mL of the flavonoid extract was transferred to a 10 mL volumetric flask, and 0.4 mL of reagent A was added. The mixture was shaken well and allowed to stand for 6 min. 0.4 mL of reagent B was added, and the mixture was shaken well and allowed to stand for 6 min. 4.0 mL of reagent C was added, and water was added to the mark. The mixture was shaken well and allowed to stand for 15 min. A reagent blank was used as the reference solution. The absorbance was measured at 500 nm using a 1 cm cuvette, and the total flavonoid content was calculated using the standard curve method.

[0068] Example 1

[0069] Table 1 summarizes the types of understory economic plants and their main functional metabolites reported in the prior art, and Table 2 shows the flavonoid content of understory economic plants.

[0070] Table 1 Summary of types of economic plants under forest and their main functional metabolites

[0071] Understory economic plants Major metabolite 1 Major metabolite 2 Main metabolite 3 Main metabolite 4 Major metabolite 5 Sanqi saponins Flavonoids polysaccharides volatile oil amino acids Polygonatum polysaccharides Flavonoids Steroidal saponins Lignans Nitrogen compounds Dendrobium officinale polysaccharides Flavonoids Bibenzyl compounds amino acids Trace elements Three-leaf green Flavonoids Phenylacetic compounds polysaccharides Terpenoids alkaloids Wintergreen Flavonoids Terpenoids polysaccharides organic acids amino acids Scutellaria baicalensis Flavonoids Polyphenolic compounds organic acids volatile oil amino acids Red peony Glycosides Flavonoids organic acids volatile oil polysaccharides

[0072] Table 2. Flavonoid content and types in three types of understory economic plants with flavonoids as the main compounds.

[0073]

[0074] As shown in Tables 1 and 2, the main flavonoid compound in Scutellaria baicalensis is quercetin, and the total flavonoid content is relatively high. Therefore, crude extract of Scutellaria baicalensis was selected as an additive to the germination medium.

[0075] Example 2 and Comparative Examples 2-1 to 2-5 used the same batch of collected poplar leaves.

[0076] The schematic diagrams for the rapid bud induction and flavonoid content determination using *Populus tomentosa* leaves as explants in Examples 2, 2-1, and 2-2 are shown below. Figure 1 .

[0077] Example 2: Establishment of a rapid bud-inducing system using poplar leaves as explants

[0078] The culture medium combination used in Example 2 was A+C.

[0079] (1) Preparation of explants. Young poplar leaves were used as explants. The preparation process was as follows: First, the young leaves at the top of the poplar were cut off along with the young branches. They were rinsed with running water for about 4 hours to remove surface dust and contaminants. Then, they were rinsed with deionized water 3 to 4 times. In a clean bench, all the young leaves on the obtained young branches were separated into individual leaves and placed on sterile filter paper for later use.

[0080] (2) Sterilize the poplar leaves. Rinse the dried poplar leaves from step (1) with 75% ethanol for 45 seconds, then rinse with sterile water 3-4 times; then soak in sodium hypochlorite solution with 4% available chlorine for 10 minutes, then rinse with sterile water 3-4 times, and finally use sterile filter paper to absorb the surface moisture of the sterilized poplar leaves.

[0081] (3) Callus induction. The sterilized leaves from step (2) were cut into 1cm x 1cm pieces and placed on culture medium A with the underside of the leaves facing upwards. After 30-60 days of culture, callus tissue with a diameter of approximately 1cm grew from the leaf edges. The culture temperature was 24℃, and the humidity was 16.2%. The leaves were exposed to 100% white light for 18 hours daily, followed by 6 hours of dark culture. (1) to (3) in Example 2 correspond to... Figure 1 The A process in the middle.

[0082] (4) Sprouting induction. Callus tissue with a diameter greater than 1 cm grown in step (3) was transferred to culture medium C and cultured for a further period. Obvious young buds emerged from the callus tissue. The culture temperature was 24℃, humidity 16.2%; 100% red light was applied for 18 hours daily, followed by 6 hours of darkness. The effect of culture medium C on callus differentiation is shown in [see figure]. Figure 4 .

[0083] Comparative Example 2-1

[0084] Similar to Example 2, the only difference is that the callus tissue is not transferred to culture medium C, but rather to a new culture medium A for continued culture. Only by continuing culture in culture medium A can the callus tissue produce tender shoots. The effect of culture medium A on callus differentiation is shown in [the following text is incomplete and requires further context]. Figure 2 The culture medium combination used in Comparative Example 2-1 was A+A.

[0085] Comparative Example 2-2

[0086] Same as Example 2, the only difference being that the callus tissue was transferred to culture medium B, and culture medium C was replaced with culture medium B. Culture continued in culture medium B, and only continued culture in culture medium B could produce tender shoots. The effect of culture medium A on callus differentiation is shown in [see...]. Figure 3 The culture medium combination used in Comparative Example 2-2 was A+B.

[0087] Comparative Examples 2-3

[0088] Same as Example 2, the only difference is that the leaves are only soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. Fungi or bacteria are generated before callus tissue is formed on the leaves, which affects leaf growth.

[0089] Comparative Examples 2-4

[0090] Same as Example 2, the only difference is that the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, and the leaves will turn white and cannot produce healing.

[0091] Comparative Examples 2-5

[0092] Same as Example 2, the only difference is that the leaves are not washed with running water, and the contamination rate will be as high as 100%.

[0093] The germination rate and callus browning rate (hereinafter referred to as callus browning rate) of Example 2, Comparative Examples 2-1 and 2-2 were calculated using the following formulas, and the results are shown in Table 3. Figure 1 , Figure 5 :

[0094] Germination rate (%) = Number of buds / Number of calluses × 100%, the same below

[0095] Callus browning rate (%) = Number of browned calluses / Number of normal calluses × 100%, the same below

[0096] Table 3. Data on the effects of different culture media on bud induction and callus browning.

[0097]

[0098] Note: The callus germination rate and callus browning rate of the comparative examples are the average of three biological replicates at 300 days; the callus germination rate and callus browning rate of the examples are the average of three biological replicates at 150 days.

[0099] It is known that the culture medium combination of the present invention can improve the callus germination rate and reduce the callus browning rate, and shorten the germination time.

[0100] The flavonoid content and survival rate of normal callus and browned callus obtained in Comparative Example 2-2, the sprouting tissue obtained in Example 2, and the sprouting tissue obtained in Comparative Example 2-1 were determined. Survival rate (%) = (Number of sprouts that can continue to grow after transplantation / Total number of sprouts that appear) × 100%. Results are shown in […]. Figure 1 , Figure 5 And Table 4.

[0101] Table 4. Results of total flavonoid determination and survival rate of callus or buds cultured in different culture media.

[0102]

[0103] The stress resistance of the seedlings obtained in Example 2, Comparative Example 2-1, and Comparative Example 2-2 was verified. Taking drought stress as an example, the specific steps are as follows:

[0104] Poplar buds obtained in Example 2, Comparative Example 2-1 and Comparative Example 2-2 were cultured in water. PEG6000 at a mass concentration of 5% was added to the water to simulate drought stress. After culturing under drought stress for 4 hours, the electrical conductivity of the poplar buds was measured.

[0105] Conductivity Measurement Method: The young shoots were soaked in a conductive solution (i.e., deionized water) for 2 hours. The conductivity of the solution was then measured to indirectly reflect the relative conductivity of the leaves. The results are shown in Table 5. Under normal circumstances, plant cell membranes have selective permeability. However, when plants are subjected to adverse environmental conditions, the cell membranes may be damaged, leading to the leakage of electrolytes from the cells and thus increasing conductivity. The increase in relative conductivity indicates a greater degree of damage to the plant tissue, demonstrating the strong stress resistance of the young shoots obtained in Example 2.

[0106] Table 5. Conductivity measurements after stress under different culture media.

[0107] Culture medium classification Comparative Example 2-1 Comparative Example 2-2 Example 2 electrical conductivity 57.4±5.8μS 50.9±6.9μS 38.6±2.9μS

[0108] Example 3 and Comparative Examples 3-1 to 3-8 used leaves from the same batch of collected Populus tomentosa leaves.

[0109] Example 3: Establishment of a rapid bud-inducing system using Populus simonii leaves as explants

[0110] (1) Preparation of explants. Using poplar leaves as explants, the process of obtaining explants is as follows: Select young poplars planted in the culture room, cut off the young leaves at the top of the poplar along with the young branches, rinse with running water for about 4 hours to wash away surface dust and contaminants, and then rinse with deionized water 3 to 4 times; in a clean bench, separate all the young leaves on the cleaned young branches into individual leaves, place them on sterile filter paper, and set aside for later use.

[0111] (2) Sterilize the small-leaved poplar leaves. Rinse the dried small-leaved poplar leaves from step (1) with 75% ethanol for 45 seconds and rinse with sterile water 3-4 times; then soak them in sodium hypochlorite solution with 4% available chlorine for 10 minutes and rinse with sterile water 3-4 times. Finally, use sterile filter paper to absorb the surface moisture of the sterilized small-leaved poplar leaves.

[0112] (3) Callus induction. The sterilized leaves from step (2) were cut into 1cm*1cm pieces and placed on culture medium A with the underside of the leaves facing up. After 30-60 days of culture, callus tissue with a diameter of about 1cm grew from the leaf edges. The culture temperature was 24℃ and the humidity was 16.2%. The leaves were exposed to 100% white light for 18 hours and then cultured in the dark for 6 hours each day.

[0113] (4) Sprouting induction. The callus tissue with a diameter greater than 1 cm grown in step (3) was transferred into culture medium C and cultured for 80-100 days. Obvious tender buds could be seen growing on the callus tissue. The culture temperature was 24℃ and the humidity was 16.2%. The tissue was irradiated with 100% red light for 18 hours and cultured in the dark for 6 hours every day.

[0114] Comparative Example 3-1

[0115] Similar to Example 3, the only difference is that the callus tissue is not transferred into culture medium C, but into a new culture medium A for further culture. The callus tissue needs to be cultured in culture medium A for 230–250 days to produce tender shoots. Comparative Example 3-1 used a culture medium combination of A+A.

[0116] Comparative Example 3-2

[0117] Similar to Example 3, the only difference is that the callus tissue is transferred to culture medium B and cultured there. The callus remains intact for 180–200 days in culture medium B, and only after 250–270 days of culture in culture medium B can the callus tissue produce tender shoots. Comparative Example 3-2 uses a culture medium combination of A+B.

[0118] Comparative Example 3-3

[0119] Same as Example 3, the only difference is that the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. Before callus tissue is formed on the leaves, fungi or bacteria will grow on the leaves, affecting leaf growth.

[0120] Comparative Examples 3-4

[0121] Same as Example 3, except that when the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, the leaves will turn white and appear scorched, and will not heal.

[0122] Comparative Examples 3-5

[0123] Same as Example 3, except that the blades are not washed with running water.

[0124] Comparative Examples 3-6

[0125] Same as Example 3, except that Scutellaria baicalensis extract was not added to the germination medium. The results showed that the germination rate was reduced to a certain extent and the contamination was increased.

[0126] Comparative Examples 3-7

[0127] Same as Example 3, except that gibberellin GA3 was not added to the germination medium. The results showed that the browning rate of callus tissue increased and the germination rate decreased.

[0128] Comparative Examples 3-8

[0129] Same as Example 3, except that zeatin was not added to the germination medium, and the germination rate of callus tissue was found to be reduced.

[0130] Populus simonii leaf callus induction and budding induction were co-cultured for 150 days. The budding rate, callus browning rate and contamination rate of Example 3 and Comparative Examples 3-1 to 3-8 were calculated. The results are shown in Table 6. It can be seen that the technical solution of the present invention has a high budding rate and reduces the callus browning rate and contamination rate.

[0131] Contamination rate = (Number of contaminated callus / Total number of callus) * 100%, the same applies below.

[0132] Table 6. Sprouting rate, callus browning rate, and contamination rate (%) of Populus simonii leaves after 150 days of co-culture with callus and bud induction.

[0133]

[0134] Example 4 and Comparative Examples 4-1 to 4-8 used leaves from the same batch of collected Populus tomentosa leaves.

[0135] Example 4: Establishment of a rapid bud-inducing system using Populus tomentosa leaves as explants

[0136] (1) Preparation of explants. Using poplar leaves as explants, the process of obtaining explants is as follows: Select young poplars planted in the culture room, cut off the young leaves at the top of the poplar along with the young branches, rinse them with running water for about 4 hours to wash away surface dust and contaminants, and then rinse them with deionized water 3 to 4 times; in a clean bench, separate all the young leaves on the cleaned young branches into individual leaves, place them on sterile filter paper, and set them aside.

[0137] (2) Sterilize the leaves of Populus tomentosa. The Populus tomentosa leaves that have been dried on the sterile filter paper prepared in step (1) are first rinsed with 75% ethanol for 45 seconds and then rinsed with sterile water 3 to 4 times; then soaked in sodium hypochlorite solution with an effective chlorine content of 4% for 10 minutes and rinsed with sterile water 3 to 4 times. Finally, the surface moisture of the sterilized Populus tomentosa leaves is absorbed with sterile filter paper.

[0138] (3) Callus induction. The sterilized leaves from step (2) were cut into 1cm*1cm pieces and placed face down in culture medium A for culture. After 30-60 days of culture, callus tissue with a diameter of about 1cm grew from the leaf edges. The culture temperature was 24℃ and the humidity was 16.2%. The leaves were irradiated with 100% white light for 18 hours and then cultured in the dark for 1 hour each day.

[0139] (4) Sprouting induction. The callus tissue with a diameter greater than 1 cm grown in step (3) was transferred into culture medium C and cultured for 110-130 days. Obvious tender buds could be seen growing on the callus tissue. The culture temperature was 24℃ and the humidity was 16.2%. The tissue was irradiated with 100% red light for 18 hours and cultured in the dark for 6 hours every day.

[0140] Comparative Example 4-1

[0141] Similar to Example 4, the only difference is that the callus tissue is not transferred to culture medium C, but to a new culture medium A for further culture. After culturing in culture medium A for 260–280 days, most of the callus tissue is able to produce tender shoots. The culture medium combination used in Comparative Example 4-1 is A+A.

[0142] Comparative Example 4-2

[0143] Similar to Example 4, the only difference is that the callus tissue is transferred to culture medium B, and culture medium C is replaced with culture medium B. The tissue continues to be cultured in culture medium B, where it can maintain the callus state for 180–200 days. Young shoots only begin to develop after 250–270 days of culture in culture medium B. Comparative Example 4-2 uses a culture medium combination of A+B.

[0144] Comparative Example 4-3

[0145] Same as Example 4, the only difference being that the leaves were soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. The leaves did not produce callus tissue, but instead developed fungi or bacteria, affecting leaf growth.

[0146] Comparative Example 4-4

[0147] Same as Example 4, except that when the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, the leaves will turn white and cannot heal.

[0148] Comparative Example 4-5

[0149] Same as Example 4, except that the blades are not washed with running water.

[0150] Comparative Examples 4-6

[0151] Same as Example 4, except that Scutellaria baicalensis extract was not added to the germination medium. The results showed that the germination rate was reduced to a certain extent and the contamination was increased.

[0152] Comparative Examples 4-7

[0153] Same as Example 4, except that gibberellin GA3 was not added to the budding medium, and the result showed that the browning rate of callus tissue increased.

[0154] Comparative Examples 4-8

[0155] Same as Example 4, except that zeatin was not added to the germination medium, and the germination rate of callus tissue was found to be reduced.

[0156] Populus tomentosa leaves were co-cultured for 150 days to induce callus and budding. The budding rate, browning rate, and contamination rate of Examples 4 and Comparative Examples 4-1 to 4-8 were calculated, and the results are shown in Table 7. It can be seen that the culture medium of the present invention resulted in a high budding rate of Populus tomentosa leaves and reduced callus browning rate and contamination rate.

[0157] Table 7. Germination rate, browning rate, and contamination rate (%) of Populus tomentosa leaves after 150 days of co-culture with callus induction and bud induction.

[0158]

[0159] Example 5 and Comparative Examples 5-1 to 5-8 used leaves collected from the same batch of pigeon pea leaves.

[0160] Example 5: Establishment of a rapid budding system using pigeon pea leaves as explants

[0161] (1) Preparation of explants. Using pigeon pea leaves as explants, the process of obtaining explants is as follows: Select pigeon peas planted in the culture room, cut off the whole pigeon pea leaves, rinse them with running water for about 4 hours to wash away surface dust and contaminants, and then rinse them with deionized water 3 to 4 times; place the washed pigeon pea leaves on sterile filter paper in a clean bench for later use.

[0162] (2) Sterilize the pigeon pea leaves. Rinse the dried pigeon pea leaves on the sterile filter paper prepared in step (1) with 75% ethanol for 45 seconds, and rinse with sterile water 3 to 4 times; then soak them in a sodium hypochlorite solution with an effective chlorine content of 5% for 10 minutes, rinse with sterile water 3 to 4 times, and then use sterile filter paper to absorb the surface moisture of the sterilized pigeon pea leaves.

[0163] (3) Callus induction. The sterilized leaves from step (2) were cut into 1cm*1cm pieces and placed face down in culture medium A for culture. After 30-40 days of culture, callus tissue with a diameter of about 1cm grew from the leaf edges. The culture temperature was 24℃ and the humidity was 16.2%. The leaves were irradiated with 100% white light for 18 hours and cultured in the dark for 6 hours every day.

[0164] (4) Sprouting induction. The callus tissue with a diameter greater than 1 cm grown in step (3) is transferred into culture medium C and cultured for 50-70 days. Obvious tender buds can be seen growing on the callus tissue. The culture temperature is 24℃ and the humidity is 16.2%. The tissue is irradiated with 100% red light for 18 hours and cultured in the dark for 6 hours every day.

[0165] Comparative Example 5-1

[0166] Similar to Example 5, the only difference is that the callus tissue is not transferred into culture medium C, but rather into a new culture medium A for continued culture. It takes 130–150 days of culture for the tender shoots to develop. Comparative Example 5-1 used a culture medium combination of A+A.

[0167] Comparative Example 5-2

[0168] Similar to Example 5, the only difference is that the callus tissue was transferred into culture medium B and then cultured in culture medium B for 130–150 days to produce tender shoots. Comparative Example 5-2 used a culture medium combination of A+B.

[0169] Comparative Example 5-3

[0170] Same as Example 5, the only difference being that the leaves were soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. No callus tissue was formed on the leaves; instead, fungi or bacteria appeared, affecting leaf growth.

[0171] Comparative Example 5-4

[0172] Same as Example 5, except that when the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, the leaves will turn white and cannot heal.

[0173] Comparative Example 5-5

[0174] Same as Example 5, except that the blades are not washed with running water.

[0175] Comparative Examples 5-6

[0176] Similar to Example 5, the only difference was that Scutellaria baicalensis extract was not added to the germination medium. As a result, the germination rate was reduced to some extent and the contamination was increased.

[0177] Comparative Examples 5-7

[0178] Same as Example 5, except that gibberellin GA3 was not added to the budding medium, and the result showed that the browning rate of callus tissue increased.

[0179] Comparative Examples 5-8

[0180] Same as Example 5, except that zeatin was not added to the germination medium, and the result was that the callus germination rate was reduced.

[0181] Pigeon pea leaves were co-cultured with callus and bud induction for 100 days. The budding rate, callus browning rate, and contamination rate of Example 5 and Comparative Examples 5-1 to 5-8 were calculated, and the results are shown in Table 8. It can be seen that pigeon pea leaves cultured using the culture medium of the present invention have a high budding rate and a low browning rate.

[0182] Table 8. Sprouting rate, callus browning rate, and contamination rate (%) of pigeon pea leaves after 100 days of callus and bud induction.

[0183]

[0184] In summary, the technical solution provided by this invention induces callus sprouting in woody plants, reduces the browning rate of callus tissue, and shortens the sprouting time of callus tissue, laying the foundation for future cultivation of stress-resistant superior varieties of woody plants.

[0185] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A medium combination for inducing the germination of woody plant callus, characterized by comprising, The culture medium combination comprises an induction culture medium and a sprouting culture medium; The induction culture medium takes MS culture medium as a basic culture medium, and is added with 4-6 mg / L 2,4-D, 450-500 mg / L glutamine, 250-350 mg / L hydrolyzed milk protein, 25-30 g / L sucrose and 6.5-7.5 g / L agar; The sprouting culture medium takes MS culture medium as a basic culture medium, and is added with 0.5-0.8 mg / L 6-benzylaminopurine, 0.2-0.4 mg / L naphthalene acetic acid, 40-60 mg / L Scutellaria baicalensis extract, 0.15-0.25 mg / L gibberellin, 0.4-0.6 mg / L zeatin, 25-30 g / L sucrose and 5.5-6.0 g / L agar; The woody plant is Populus cathayana, Populus simonii, Populus tomentosa or Caesalpinia japonica; The explant is a leaf of the woody plant; The preparation method of the Scutellaria baicalensis extract comprises: mixing Scutellaria baicalensis with an organic solvent for leaching to obtain a leaching solution; The leaching solution is filtered after ultrasonic extraction to obtain the Scutellaria baicalensis extract.

2. The medium combination according to claim 1, characterized in that, The pH value of the induction culture medium and the sprouting culture medium is 5.5-5.

8.

3. A method of inducing shoot formation from woody plant callus, characterized by, The method for inducing the woody plant callus to sprout by using the culture medium combination in claim 1 or 2 comprises the following steps: The explant of the woody plant is inoculated on the induction culture medium for callus induction culture to obtain callus; The callus is transferred to the sprouting culture medium for sprouting culture; Before the explant of the woody plant is inoculated on the induction culture medium, the explant is also subjected to cleaning and disinfection; the cleaning comprises, in sequence, water flushing and deionized water flushing; the disinfection comprises: rinsing with ethanol with a volume concentration of 75% for 40-50 s, flushing with sterile water for 3-4 times, then soaking in sodium hypochlorite solution with an effective chlorine content of 3.5%-4.5% for 8-12 min, and flushing with sterile water for 3-4 times; The woody plant is Populus cathayana, Populus simonii, Populus tomentosa or Caesalpinia japonica; The explant is a leaf of the woody plant.

4. The method of claim 3, wherein, The induction culture and the sprouting culture are light-dark alternating culture, the light culture of the induction culture comprises white light irradiation, and the light culture of the sprouting culture comprises red light irradiation.

5. The method of claim 4, wherein, The time of the white light irradiation is 18 h / d, the light intensity of the white light irradiation is 10000-20000 lux, the time of the red light irradiation is 18 h / d, and the light intensity of the red light irradiation is 10000-20000 lux.

6. The method of claim 3, wherein, The time of the induction culture is 30-60 d, and the time of the sprouting culture is 50-130 d.

7. The method of claim 3 wherein, The temperature of the induction culture and the sprouting culture is 23-25 ℃, and the humidity of the induction culture and the sprouting culture is 16%-17%.

Images