Culture medium composition and method for inducing callus of woody plant to germinate

Abstract

The invention belongs to the field of woody plant callus culture and differentiation, and particularly relates to a culture medium composition and a method for inducing woody plant callus germination. The invention provides a culture medium combination and a method for inducing woody plant callus germination, NAA, 2, 4-D, 6-BA, gibberellin (GA3) GA3, glutamine, a scutellaria baicalensis extract, lactoalbumin hydrolysate, zeatin and cane sugar in the combined culture medium are beneficial for inducing callus formation and differentiation into buds, and callus browning is effectively prevented. The result of the embodiment shows that the culture medium composition can induce the callus of the woody plant to germinate in a short time, the browning rate is reduced, and the total flavone content of the cultured buds is high.

Description

Technical Field

[0001] The invention belongs to the field of woody plant callus culture and differentiation, and particularly relates to a culture medium combination and method for inducing woody plant callus budding. Background Art

[0002] The main functions of woody plants are to maintain ecological balance, regulate climate, prevent wind and sand, reduce noise pollution, purify air, conserve water resources, maintain water and soil, and regulate carbon-oxygen balance. Poplar (Populus L.) is a very important woody plant. Poplar is an important afforestation tree species in my country. It is a model woody plant with the characteristics of fast growth, high yield, and strong adaptability. Poplars are widely cultivated in my country. They are the main building materials, greening and afforestation tree species. They play an irreplaceable and huge role in timber production, urban greening and ecological construction, and have important economic and ecological values. However, during the planting process of poplars, they will be affected by the surrounding environment or pathogens, resulting in a decrease in yield and quality. Therefore, breeding improved varieties of poplars is the key to improving the adaptability of poplars to the surrounding environment. Compared with traditional asexual propagation methods such as cuttings and grafting, tissue culture methods have the characteristics of rapid reproduction, convenient management, short culture cycle, and easy industrialization. Plant callus is omnipotent and usually has the advantages of rapid growth, large reproduction, small space occupation, and factory production. It is also an ideal way to select and breed improved varieties. Studying callus regeneration plays an important role in studying plant stress resistance. However, when conducting tissue culture of woody plants, there are still problems such as high callus browning rate and long callus differentiation period into buds. Summary of the invention

[0003] The invention provides a culture medium combination and method for inducing callus budding of woody plants. The culture medium combination of the invention can shorten the time for callus of woody plants to regenerate buds, and the browning rate of callus is low.

[0004] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0005] The present invention provides a culture medium combination for inducing callus budding of woody plants, the culture medium combination comprising an induction culture medium and a budding 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 budding culture medium uses MS culture medium as a basic culture medium and further comprises: 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 budding medium is 5.5-5.8.

[0009] Preferably, the preparation method of the scutellaria baicalensis extract comprises: mixing scutellaria baicalensis with an organic solvent to obtain an extract; and filtering the extract after ultrasonic extraction to obtain the scutellaria baicalensis extract.

[0010] The present invention provides a method for inducing callus budding of woody plants, using the culture medium combination described in any one of the above technical solutions to induce callus budding of woody plants, and the method comprises the following steps:

[0011] Inoculating the explants of the woody plant on an induction medium for callus induction culture to obtain callus tissue;

[0012] The callus tissue is transferred to a sprouting medium for sprouting culture.

[0013] Preferably, the explant comprises a leaf.

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

[0015] Preferably, the induction culture and sprout culture are light-dark alternating cultures, the light culture of the induction culture comprises white light irradiation; the light culture of the sprout culture comprises red light irradiation.

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

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

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

[0019] Beneficial effects of the present invention: The present invention provides a culture medium combination for inducing callus budding of woody plants, the culture medium combination comprising an induction culture medium and a budding culture medium.

[0020] In the culture medium combination provided by the present invention, 2,4-dichlorophenoxyacetic acid (2,4-D) is used as a plant growth regulator to stimulate plant growth; naphthylacetic acid is used as a plant growth regulator to promote cell division, promote growth and enhance resistance; 6-benzylaminopurine is a synthetic cytokinin plant growth regulator, which plays the role of promoting cell division, inducing tissue differentiation and inducing bud production in the culture medium; glutamine provides the plant with a nitrogen source necessary for growth; hydrolyzed milk protein provides nutrients for plant growth; scutellaria baicalensis extract is rich in flavonoids, has an antioxidant effect, can regulate plant hormone transport, and increase the antibacterial activity and stress resistance of plants; gibberellin (GA3) can promote cell division and stimulate germination, and inhibit the browning of callus tissue to a certain extent; zeatin is a cytokinin of an adenine derivative, which has the effect of promoting cell division and promoting the production of young shoots in callus tissue; sucrose provides the plant with the energy required for growth and development, and also participates in various physiological activities and regulation of the plant, improving stress resistance; agar is a solid agent to support the plant, inhibit the growth of pollutants such as bacteria, and protect the culture medium.

[0021] Under the joint action of NAA, 2,4-D, 6-BA, gibberellin GA3, glutamine, hydrolyzed milk protein, scutellaria extract, zeatin, and sucrose, callus formation and differentiation into buds are induced, and callus browning is effectively prevented. The results of the embodiment show that the culture medium combination of the present invention can induce woody plant callus budding in a short time, and reduce the browning rate, and the total flavonoid content of the young sprouts obtained by culture is high. The young sprouts can produce a large amount of flavonoid substances, which not only improves the plant's own stress resistance, but the young sprout extract can also be used in the prevention research of human diseases.

[0022] The present invention also provides a method for inducing callus budding of woody plants. The induction culture method of the present invention has a short production cycle and can realize factory production through amplification culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Diagram of callus tissue pattern of woody plant poplar cultured on different culture media;

[0024] Figure 2The results are as follows: The application of the induction medium to the differentiation of callus in Comparative Example 2-1; A is the growth state of callus at different culture times during budding induction, B is the budding rate, and C is the browning rate;

[0025] Figure 3 The results are as follows: The culture medium B used in Comparative Example 2-2 promotes the differentiation of callus tissue; wherein A is the growth state of callus tissue at different culture times during budding induction, B is the budding rate, and C is the browning rate;

[0026] Figure 4 The figure shows the promotion of callus differentiation by the budding medium used in Example 2; wherein A is the growth state of callus at different culture times during budding induction, B is the budding rate, and C is the browning rate;

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

[0028] Figures 2 to 5 "**" indicates significant difference at P < 0.001 level. DETAILED DESCRIPTION

[0029] The invention provides a culture medium combination for inducing callus budding of woody plants, wherein the culture medium combination comprises an induction culture medium and a budding culture medium.

[0030] The induction medium of the present invention 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. The solvent of the induction medium is water. As an embodiment, the concentration of 2,4-D in the induction medium provided by the present invention can be 4-6 mg / L; in a specific embodiment of the present 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 a specific embodiment of the present invention, the concentration of glutamine in the induction medium can be 450, 460, 470, 480, 490 or 500 mg / L. The concentration of the hydrolyzed milk protein in the induction medium can be 250-350 mg / L; in a specific embodiment of the present invention, the concentration of the 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 a specific embodiment 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, and in a specific embodiment 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 can jointly promote the formation of callus tissue of woody plants at an appropriate concentration ratio, and can effectively reduce the browning rate of callus tissue. In the present invention, the pH value of the induction medium may be 5.5 to 5.8.

[0031] The germination medium of the present invention uses MS medium as the basic medium, and further comprises: 0.5-0.8 mg / L 6-benzylaminopurine, 0.2-0.4 mg / L naphthaleneacetic acid, 40-60 mg / L scutellariae 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 an embodiment, the concentration of 6-benzylaminopurine in the germination medium provided by the present invention can be 0.5-0.8 mg / L, or 0.6-0.7 mg / L; in a specific embodiment of the present 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 in the budding medium can be 0.2-0.4 mg / L, or 0.3 mg / L; in a specific embodiment of the present invention, the concentration of naphthaleneacetic acid in the budding medium can be 0.2, 0.25, 0.3, 0.35 or 0.4 mg / L. The concentration of the scutellaria baicalensis extract in the budding medium can be 40-60 mg / L, or 50 mg / L; in a specific embodiment of the present invention, the concentration of the scutellaria baicalensis extract in the budding medium can be 40, 45, 50, 55 or 60 mg / L. The concentration of gibberellin (GA3) in the budding medium can be 0.15-0.25 mg / L, or 0.20 mg / L; in a specific embodiment of the present invention, the concentration of gibberellin GA3 in the budding medium can be 0.15, 0.17, 0.19, 0.20, 0.23 or 0.25 mg / L. The concentration of zeatin in the budding medium can be 0.4-0.6 mg / L, or 0.5 mg / L. The concentration of sucrose in the budding medium can be 25-30 g / L, or 26-28 g / L; in a specific embodiment of the present invention, the concentration of sucrose in the budding medium can be 25, 26, 27, 28, 29 or 30 g / L. The concentration of agar in the budding medium can be 5.5-6.0 g / L, or 6.0 g / L; in a specific embodiment of the present invention, the concentration of agar in the budding 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 can make the callus produced by woody plants sprout quickly and inhibit the production of brown callus at an appropriate concentration ratio. In the present invention, the pH value of the budding medium can be 5.5-5.8. The solvent of the budding medium is water. The preparation method of the budding medium preferably comprises: sterilizing the MS medium, 6-benzylaminopurine, naphthylacetic acid, scutellaria baicalensis extract, gibberellin GA3, sucrose, agar and water, and then adding zeatin when the temperature reaches about 50°C.

[0032] The induction medium and budding medium of the present invention are sterilized before use. The present invention has no particular limitation on the sterilization method, and conventional methods can be used.

[0033] The present invention has no special restrictions on the preparation of the scutellaria extract, and conventional methods can be used. As an embodiment, the preparation method of the scutellaria extract includes: mixing scutellaria with an organic solvent to obtain an extract, and filtering the extract after ultrasonic extraction to obtain the scutellaria extract. As an embodiment, the present invention mixes scutellaria dry powder with an organic solvent; as an example, the water content of the scutellaria dry powder can be 8%; the mass volume ratio of the scutellaria dry powder to the organic solvent can be 1g:100mL; the organic solvent can be ethanol with a volume concentration of 70%. As an embodiment, the extraction method can be soaking, and the soaking time can be 24h; the ultrasonic extraction time can be 30min, the ultrasonic extraction power can be 80W, and the ultrasonic frequency is 40KHz. The present invention has no special restrictions on the filtering method, and conventional methods can be used. The filtrate obtained after filtration is the scutellaria extract. The scutellaria extract obtained by extraction with an organic solvent has high contents of quercetin and total flavonoids, which is beneficial to improving the stress resistance of the buds.

[0034] The present invention has no special limitation on the sources of the components in the culture medium combination, and conventional commercially available products may be used.

[0035] The present invention also provides a method for inducing callus budding of woody plants, using the culture medium combination described in the above technical solution to induce callus budding of woody plants, the method comprising the following steps:

[0036] Inoculating the explants of the woody plant on an induction medium for callus induction culture to obtain callus tissue;

[0037] The callus tissue is transferred to a sprouting medium for sprouting culture.

[0038] The present invention inoculates the explants of woody plants on an induction medium for callus induction culture to obtain callus. Before the explants of woody plants described in the present invention are inoculated on the induction medium, it is preferred that the explants are cleaned and disinfected; the cleaning preferably includes running water rinsing and deionized water rinsing in sequence; the present invention does not specifically limit the cleaning method, as long as it is clean. As an implementable method, the running water rinsing time can be 4 hours, and the deionized water rinsing time can be 3 to 4 times. The disinfection described in the present invention preferably includes: rinsing with ethanol with a volume concentration of 75% can be 40 to 50 seconds, or 42 to 44 seconds; the sterile water rinsing can be 3 to 4 times, and then soaking in a sodium hypochlorite solution with an effective chlorine content of 3.5% to 4.5% can be 8 to 12 minutes, or 9 to 11 minutes, and then rinsing with sterile water can be 3 to 4 times. In a specific embodiment of the present invention, the blades are first rinsed with 75% ethanol for 45 seconds, and then rinsed with sterile water for 3 to 4 times; then soaked in a sodium hypochlorite solution with an effective chlorine content of 4% for 10 minutes, and then rinsed with sterile water for 3 to 4 times.

[0039] The present invention preferably uses the leaves of woody plants as explants for seedling cultivation, and the leaves are divided into individual leaves before being cleaned and disinfected. In the present invention, the leaves of woody plants are preferably young leaves at the top of woody plants; the young leaves are leaves with a growth time of 2 to 5 months. The reason for using young leaves is that young leaves do not have obvious lignification, and are large in area and easy to operate, and are more likely to form callus tissue. The woody plants of the present invention preferably include poplars and / or pigeon peas, and the poplars include at least one of Populus cathayana, Populus simonii and Populus tomentosa.

[0040] After the leaves of the present invention are cleaned and disinfected, it is preferred that a single leaf is inoculated into an induction medium for callus induction culture. In the present invention, the temperature of the induction culture can be 23-25°C, or 24°C; the time of the induction culture can be 30-60d, or 25-35d; the callus culture begins to differentiate and sprout within 20-40d, and the callus culture is 30-60d to obtain a callus with a diameter of 0.5-2cm. The humidity of the induction culture can be 16%-17%, or 16.2%. The induction culture of the present invention is preferably light-dark alternating culture, and the light culture of the present invention is carried out under the condition of light, more preferably under the condition of white light irradiation; the time of white light irradiation can be 18h / d; the light intensity of the white light irradiation can be 10000-20000lux. The white light irradiation of the present invention is achieved by using an LED light source to irradiate white light. The time of dark culture of the present invention can be 6h / d. The role of dark culture is to promote the formation of callus, avoid light inhibition, reduce photooxidation, maintain plant hormone balance and promote cell division.

[0041] The callus with a diameter of 0.5 to 2 cm is obtained, and the callus is transferred to a sprout culture medium for sprout culture. The callus can be cut into pieces or not during the transfer.

[0042] As an embodiment, the temperature of the sprout culture can be 23-25° C., or 24° C. The duration of the sprout culture can be 50-130 days. The humidity of the sprout culture can be 16%-17%, or 16.2%.

[0043] The bud culture of the present invention is preferably light-dark alternating culture, and the light culture of the present invention is carried out under the condition of light, more preferably under the condition of red light irradiation. The time of the red light irradiation can be 18h / d; the light intensity of the red light irradiation can be 10000-20000lux. The time of dark culture of the present invention can be 6h / d. The red light irradiation in the present invention is achieved by adjusting the LED light source to only produce 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 plant absorption of nutrients in the culture medium, and increase photosynthesis. Irradiation with red light promotes the formation of tender buds in callus tissue and reduces the browning rate of callus tissue.

[0044] After budding culture, young shoots are obtained, the height of the young shoots is 0.5 to 2 cm, the total flavonoid content in the obtained young shoots is higher than that of ordinary callus or budding tissue of ordinary callus, and the stress resistance is strong. The strong stress resistance is due to the high content of flavonoid substances. Therefore, the application of the technical solution of the present invention to the research on the molecular mechanism of growth and development of woody plants and the field of resistant seedling cultivation has important value.

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

[0046] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The reagents and instruments used in the present invention are all conventional products that can be purchased on the market.

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

[0049] The composition of the induction medium (referred to as medium A) is: MS medium as the basic medium, supplemented with 2,4-D, glutamine, hydrolyzed milk protein, sucrose, and agar.

[0050] The amounts of additives added to the 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 medium B are: MS medium as the base medium, 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, and the pH of the medium is 5.8. Proline, as an osmotic protective substance, helps plants maintain intracellular water balance and cell structure stability, improves stress resistance, and regulates plant development.

[0052] The components of the budding medium (referred to as medium C) are as follows: MS medium as the base 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, 5 ml / L crude extract of Scutellaria baicalensis, and the flavonoid content in the Scutellaria baicalensis extract is 10 mg / mL; after the remaining components in the medium are sterilized, 0.5 mg / L zeatin is added, and the pH of the medium is 5.8.

[0053] 2. Determination of total flavonoids in cultured callus or young shoots of economic plants under forests

[0054] The total flavonoid content was determined by spectrophotometry using an alkaline medium with aluminum salts for color development. Under alkaline conditions, flavonoids and aluminum salts form complexes with a maximum absorption peak at a wavelength of 500 nm.

[0055] Taking Populus cathayana buds as an example, the specific steps of the determination method are as follows:

[0056] (1) First, prepare a standard curve.

[0057] Accurately weigh 5 mg of rutin standard, dissolve it in 70% ethanol by volume, make up to volume in a 25 mL volumetric flask, shake well, and 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, place them in 10 mL volumetric flasks, add 0.4 mL of assay reagent A, shake well, and let stand for 6 min; add 0.4 mL of assay reagent B, shake well, and let stand for 6 min; add 4.0 mL of assay reagent C, add water to the scale, shake well, and let stand for 15 min. Use the reagent blank as the reference solution. Use a 1 cm cuvette to measure the absorbance at a wavelength of 500 nm and draw a standard curve. See the standard curve. Figure 5 A. The standard curve equation is: y = 0.3372x + 0.0413, where x is the content of rutin, a flavonoid standard; y is the absorbance value at 500 nm.

[0058] The assay reagent A is a 5% mass concentration NaNO2 solution;

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

[0060] The assay reagent C is a NaOH solution with a mass concentration of 5%.

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

[0062] Dry the fresh Populus cathayana sample at low temperature to make the water content less than 8% and make dry powder. Accurately weigh 1.0g of dry powder, place it in a 100mL volumetric flask, add 30mL of 70% ethanol by volume, and soak for 24h. Ultrasonic extraction for 30min, filter, and dilute the filtrate to a 100mL volumetric flask with 70% ethanol by volume to obtain flavonoid extract for standby use.

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

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

[0065] 3. Preparation of Scutellaria baicalensis Extract and Method for Determination of Flavonoid Content

[0066] The preparation method of the scutellaria baicalensis extract is as follows: a fresh scutellaria baicalensis sample is dried at low temperature to reduce the water content to less than 8% to prepare 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 continued for 30 minutes, and after two filtrations (first using gauze and then using a 0.22 μm filter membrane), the filtrate obtained is the scutellaria baicalensis extract.

[0067] The obtained Scutellaria baicalensis extract was subjected to flavonoid content determination: the Scutellaria baicalensis extract was fixed to volume with 70% ethanol in a 100mL volumetric flask to obtain a flavonoid extract, 1.00mL of the flavonoid extract was taken and placed in a 10mL volumetric flask, 0.4mL of assay reagent A was added, shaken, and allowed to stand for 6min; 0.4mL of assay reagent B was added, shaken, and allowed to stand for 6min; 4.0mL of assay reagent C was added, water was added to the scale, shaken, and allowed to stand for 15min. The reagent blank was used as the reference solution. The absorbance was measured at a wavelength of 500nm using a 1cm cuvette, and the total flavonoid content was calculated by the standard curve method.

[0068] Example 1

[0069] The types of forest economic plants and their main functional metabolites reported in the prior art are summarized in Table 1, and the amounts of flavonoids contained in forest economic plants are summarized in Table 2.

[0070] Table 1 Summary of understory economic plant types and main functional metabolites

[0071] Economic plants under forest Major metabolite 1 Major metabolite 2 Major metabolite 3 Major metabolite 4 Major metabolite 5 Panax notoginseng Saponin Flavonoids Polysaccharides Volatile Oil Amino Acids Polygonatum Polysaccharides Flavonoids Steroidal saponins Lignans Nitrogen compounds Dendrobium officinale Polysaccharides Flavonoids Bibenzyl substances Amino Acids Trace Elements Three Leaf Green Flavonoids Phenylpropanoids Polysaccharides Terpenoids Alkaloids Rabdosia rubescens Flavonoids Terpenoids Polysaccharides Organic Acid Amino Acids Scutellaria baicalensis Flavonoids Polyphenols Organic Acid Volatile Oil Amino Acids Red Peony Root Glycoside compounds Flavonoids Organic Acid Volatile Oil Polysaccharides

[0072] Table 2 The amount and types of flavonoids contained in three economic plants under the forest with flavonoids as the main compounds

[0073]

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

[0075] Example 2 and Comparative Examples 2-1 to 2-5 used Populus cathayana leaves collected from the same batch.

[0076] Example 2, Comparative Example 2-1 and Comparative Example 2-2 use Populus cathayana leaves as explants to quickly induce budding and determine the flavonoid content. Figure 1 .

[0077] Example 2 Establishment of a rapid budding induction system using Populus cathayana leaves as explants

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

[0079] (1) Explant preparation. Young leaves of Populus cathayana were used as explants. The preparation process was as follows: First, the young leaves at the top of Populus cathayana were cut off along with the young branches, rinsed with running water for about 4 hours to remove the dust and pollutants on the surface, and then rinsed with deionized water for 3 to 4 times; in a clean bench, all the young leaves on the young branches were separated into individual leaves, placed on sterile filter paper, and set aside.

[0080] (2) Sterilize the poplar leaves. The dried poplar leaves in step (1) were first rinsed with 75% ethanol by volume for 45 seconds, then rinsed with sterile water for 3 to 4 times; then soaked in a sodium hypochlorite solution with an effective chlorine content of 4% for 10 minutes, then rinsed with sterile water for 3 to 4 times, and finally the surface moisture of the sterilized poplar leaves was absorbed with sterile filter paper.

[0081] (3) Callus induction. Cut the sterilized leaves in step (2) into 1 cm*1 cm pieces, place the leaves with the back side facing up in culture medium A for cultivation. After 30 to 60 days of cultivation, callus tissue with a diameter of about 1 cm grows on the edge of the leaves. The culture temperature is 24°C and the humidity is 16.2%. Irradiate with 100% white light for 18 hours every day and culture in the dark for 6 hours. (1) to (3) in Example 2 correspond to Figure 1 A treatment in .

[0082] (4) Bud induction. The callus tissue with a diameter greater than 1 cm grown in step (3) was transferred to medium C and cultured continuously. The callus tissue grew obvious young buds. The culture temperature was 24°C and the humidity was 16.2%. The cells were exposed to 100% red light for 18 h and cultured in the dark for 6 h. The effect of medium C on the differentiation of callus tissue is shown in Table 2. Figure 4 .

[0083] Comparative Example 2-1

[0084] Same as Example 2, the only difference is that the callus tissue is not transferred to medium C, but transferred to a new medium A for continued cultivation. Only by continuing to cultivate the callus tissue in medium A can young shoots be produced. The effect of medium A on the differentiation of callus tissue is shown in 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 is that the callus tissue is transferred to medium B, medium C is replaced by medium B, and culture is continued in medium B. Only by continuing to culture in medium B can young shoots be produced. The effect of medium A on the differentiation of callus tissue is shown in Figure 3 The culture medium combination used in Comparative Example 2-2 is A+B.

[0087] Comparative Examples 2-3

[0088] The 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, and fungi or bacteria are produced when callus tissue is not produced on the leaves, affecting the growth of the leaves.

[0089] Comparative Examples 2-4

[0090] The same as Example 2, the only difference is that the leaves are immersed in the sodium hypochlorite solution with an effective chlorine content of 8% for only 10 minutes, and the leaves will appear bleached and cannot produce callus.

[0091] Comparative Examples 2-5

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

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

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

[0095] Browning rate of callus (%) = number of browning callus / number of normal callus × 100%, the same below

[0096] Table 3 Effects of different culture media on bud induction and callus browning

[0097]

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

[0099] It can be seen that the culture medium combination of the present invention can increase the callus germination rate and reduce the callus browning rate, thereby shortening the germination time.

[0100] The flavonoid content and survival rate of the normal callus and brown callus obtained in Comparative Example 2-2, the budding tissue obtained in Example 2 and the budding tissue obtained in Comparative Example 2-1 were measured. Survival rate (%) = young shoots that can continue to grow after transplantation / all young shoots that appear × 100%. The results are shown in Figure 1 , Figure 5 and Table 4.

[0101] Table 4 Total flavonoids determination and survival rate results of callus or sprouts cultured on different media

[0102]

[0103] The stress resistance of the young shoots 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] The Populus cathayana sprouts obtained in Example 2, Comparative Example 2-1 and Comparative Example 2-2 were cultured in water, PEG6000 with a mass concentration of 5% was added to the water to simulate drought stress conditions, and after culturing under drought stress conditions for 4 hours, the conductivity of the Populus cathayana sprouts was measured.

[0105] Conductivity determination method: soak the young shoots in a conductive liquid (conductive liquid is deionized water), soak for 2 hours, and then measure the conductivity of the conductive liquid. The relative conductivity of the leaves is indirectly reflected by measuring the conductivity of the conductive liquid. The results are shown in Table 5. Under normal circumstances, the plant cell membrane has selective permeability, but when the plant is affected by adverse environment, the cell membrane may be damaged, resulting in electrolyte extravasation in the cell, thereby increasing the conductivity. The increase in relative conductivity indicates that the greater the degree of damage to the plant tissue, it can be seen that the young shoots obtained in Example 2 have strong stress resistance.

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

[0107] Culture medium classification Comparative Example 2-1 Comparative Example 2-2 Example 2 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 Populus simonii leaves collected from the same batch.

[0109] Example 3 Establishment of a rapid budding induction system using Populus simonii leaves as explants

[0110] (1) Preparation of explants. The leaves of Populus simonii were used as explants. The process of obtaining explants was as follows: Select young Populus simonii planted in the culture room, cut off the young leaves at the top of the Populus simonii together with the young branches, rinse them with running water for about 4 hours to wash away the dust and pollutants on the surface, and then rinse them with deionized water for 3 to 4 times; in the clean bench, separate all the young leaves on the young branches after washing into individual leaves, place them on sterile filter paper, and set aside.

[0111] (2) Sterilize the leaves of Populus simonii. The dried leaves of Populus simonii in step (1) are first rinsed with 75% ethanol by volume for 45 seconds, and then rinsed with sterile water for 3 to 4 times; then, they are soaked in a sodium hypochlorite solution with an effective chlorine content of 4% for 10 minutes, and then rinsed with sterile water for 3 to 4 times. The surface moisture of the sterilized leaves of Populus simonii is then absorbed with sterile filter paper.

[0112] (3) Callus induction. Cut the sterilized leaves in step (2) into 1 cm*1 cm pieces, place the leaves with the back side facing up in culture medium A, and culture for 30 to 60 days. Callus with a diameter of about 1 cm grows on the edge of the leaves. The culture temperature is 24°C and the humidity is 16.2%. Irradiate with 100% white light for 18 hours and culture in the dark for 6 hours every day.

[0113] (4) Bud induction. The callus with a diameter greater than 1 cm grown in step (3) was transferred to medium C and cultured for 80 to 100 days. Young and tender buds could be seen growing on the callus. The culture temperature was 24°C and the humidity was 16.2%. The cells were exposed to 100% red light for 18 hours and cultured in the dark for 6 hours every day.

[0114] Comparative Example 3-1

[0115] The same as Example 3, the only difference is that the callus tissue is not transferred to medium C, but transferred to a new medium A for continued cultivation. The callus tissue can produce young shoots only after being cultured for 230 to 250 days in medium A. The medium combination used in Comparative Example 3-1 is A+A.

[0116] Comparative Example 3-2

[0117] Same as Example 3, the only difference is that the callus tissue is transferred to medium B, and culture is continued in medium B. The callus state can be maintained for 180 to 200 days in medium B, and the callus tissue can produce young shoots after 250 to 270 days of culture in medium B. The medium combination used in Comparative Example 3-2 is A+B.

[0118] Comparative Example 3-3

[0119] The 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. When callus tissue is not produced on the leaves, fungi or bacteria will be produced on the leaves, affecting the growth of the leaves.

[0120] Comparative Examples 3-4

[0121] The same as Example 3, the only difference is that the leaves are immersed in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, and the leaves will appear bleached and burnt, and no callus will be produced.

[0122] Comparative Examples 3-5

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

[0124] Comparative Examples 3-6

[0125] The same as Example 3, the only difference being that the 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] The same as Example 3, the only difference is that gibberellin GA3 is not added to the budding medium. The results show that the browning rate of callus tissue increases and the budding rate decreases.

[0128] Comparative Examples 3-8

[0129] The same as Example 3, the only difference being that zeatin was not added to the budding medium, and the result showed that the budding rate of callus tissue was reduced.

[0130] The callus induction and bud induction of Populus simonii leaves were co-cultured for 150 days, and 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 the callus browning rate and contamination rate are reduced.

[0131] Contamination rate = number of contaminated callus / total number of callus*100%, the same below.

[0132] Table 6 The sprouting rate, callus browning rate and contamination rate of Populus simonii leaves after 150 days of co-cultivation of callus induction and sprouting induction (%)

[0133]

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

[0135] Example 4 Establishment of a rapid budding induction system using leaves of Populus tomentosa as explants

[0136] (1) Preparation of explants. Populus tomentosa leaves were used as explants. The process of obtaining explants was as follows: young Populus tomentosa planted in the culture room was selected, and the young leaves at the top of the Populus tomentosa were cut off along with the young branches. They were rinsed with running water for about 4 hours to remove dust and pollutants on the surface, and then rinsed with deionized water for 3 to 4 times. All the young leaves on the washed young branches were separated into individual leaves in a clean bench and placed on sterile filter paper for later use.

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

[0138] (3) Callus induction. Cut the sterilized leaves in step (2) into 1 cm*1 cm pieces and place them in culture medium A with the back side facing up for culture. After 30 to 60 days of culture, callus with a diameter of about 1 cm grows on the edge of the leaves. The culture temperature is 24°C and the humidity is 16.2%. The leaves are exposed to 100% white light for 18 hours and cultured in the dark for 1 hour every day.

[0139] (4) Bud induction. The callus with a diameter greater than 1 cm grown in step (3) was transferred to medium C and cultured for 110 to 130 days. Young and tender buds could be seen growing on the callus. The culture temperature was 24°C and the humidity was 16.2%. The culture 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] The same as Example 4, the only difference is that the callus tissue is not transferred to medium C, but transferred to a new medium A for continued cultivation. Most of the callus tissue can produce young shoots after 260-280 days of continuous cultivation in medium A. The medium combination used in Comparative Example 4-1 is A+A.

[0142] Comparative Example 4-2

[0143] Same as Example 4, the only difference is that the callus tissue is transferred to medium B, medium C is replaced by medium B, and culture is continued in medium B. The callus state can be maintained for 180 to 200 days in medium B, and young shoots can be produced after 250 to 270 days of culture in medium B. The medium combination used in Comparative Example 4-2 is A+B.

[0144] Comparative Example 4-3

[0145] Same as Example 4, the only difference is that the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. The leaves do not produce callus tissue, which will produce fungi or bacteria and affect the growth of the leaves.

[0146] Comparative Example 4-4

[0147] The same as Example 4, the only difference is that the leaves are immersed in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, and the leaves will appear bleached and cannot produce callus.

[0148] Comparative Examples 4-5

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

[0150] Comparative Examples 4-6

[0151] The same as Example 4, the only difference being that the scutellaria baicalensis extract was not added to the germination medium, and the results showed that the germination rate was reduced to a certain extent and the contamination was increased.

[0152] Comparative Examples 4-7

[0153] The same as Example 4, the only difference is that gibberellin GA3 is not added to the budding medium. The results show that the browning rate of callus tissue increases.

[0154] Comparative Examples 4-8

[0155] The same as Example 4, the only difference being that zeatin was not added to the budding medium, and the results showed that the callus budding rate was reduced.

[0156] The callus induction and bud induction of Populus tomentosa leaves were co-cultured for 150 days, and the budding rate, browning rate and contamination rate of Example 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 budding rate of Populus tomentosa leaves using the culture medium of the present invention is high, and the callus browning rate and contamination rate are reduced.

[0157] Table 7 The budding rate, browning rate and contamination rate of Populus tomentosa leaves after 150 days of co-cultivation of callus induction and budding induction (%)

[0158]

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

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

[0161] (1) Preparation of explants. Pigeon pea leaves were used as explants. The process of obtaining the explants was as follows: Pigeon peas grown in the culture room were selected, and the whole pigeon pea leaf was cut off and rinsed with running water for about 4 hours to wash away the dust and pollutants on the surface, and then rinsed with deionized water for 3 to 4 times; the washed pigeon pea leaves were placed on sterile filter paper in a clean bench for later use.

[0162] (2) Sterilize the pigeon pea leaves. Rinse the pigeon pea leaves that have been dried 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. Cut the sterilized leaves in step (2) into 1 cm*1 cm pieces and place them in culture medium A with the back side facing up for culture. After 30 to 40 days of culture, callus with a diameter of about 1 cm grows on the edge of the leaves. The culture temperature is 24°C and the humidity is 16.2%. The leaves are exposed to 100% white light for 18 hours and cultured in the dark for 6 hours every day.

[0164] (4) Bud induction. The callus with a diameter greater than 1 cm grown in step (3) was transferred to medium C and cultured for 50 to 70 days. You can see obvious young buds growing on the callus. The culture temperature is 24°C and the humidity is 16.2%. The callus 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] The same as Example 5, the only difference is that the callus tissue is not transferred to the culture medium C, but transferred to the new culture medium A for continued cultivation, and young shoots can be produced after 130 to 150 days of cultivation. The culture medium combination used in Comparative Example 5-1 is A+A.

[0167] Comparative Example 5-2

[0168] Same as Example 5, the only difference is that the callus tissue is transferred to medium B, but continues to be cultured in medium B. Young shoots can only be produced after 130-150 days of culture in medium B. The medium combination used in Comparative Example 5-2 is A+B.

[0169] Comparative Example 5-3

[0170] Same as Example 5, the only difference is that the leaves are soaked in a sodium hypochlorite solution with an effective chlorine content of 2% for 10 minutes. No callus tissue is produced on the leaves, and fungi or bacteria are produced on the leaves, which affects the growth of the leaves.

[0171] Comparative Example 5-4

[0172] The same as Example 5, the only difference is that the leaves are immersed in a sodium hypochlorite solution with an effective chlorine content of 8% for 10 minutes, and the leaves will appear bleached and cannot produce callus.

[0173] Comparative Example 5-5

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

[0175] Comparative Examples 5-6

[0176] The same as Example 5, the only difference being that the scutellaria baicalensis extract was not added to the germination medium, and the results showed that the germination rate was reduced to a certain extent and the contamination was increased.

[0177] Comparative Examples 5-7

[0178] The same as Example 5, the only difference is that gibberellin GA3 is not added to the budding medium. The results show that the browning rate of callus tissue increases.

[0179] Comparative Examples 5-8

[0180] The same as Example 5, the only difference being that zeatin was not added to the budding medium, and the result showed that the budding rate of callus tissue was reduced.

[0181] The pigeon pea leaf callus induction and bud induction were co-cultured for 100 days, and 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 the pigeon pea leaf budding rate is high and the browning rate is low when the culture medium of the present invention is used to culture the pigeon pea leaf.

[0182] Table 8 The germination rate, callus browning rate and contamination rate of pigeon pea leaves after callus induction and germination induction for 100 days (%)

[0183]

[0184] In summary, the technical solution provided by the present invention induces callus budding in woody plants, reduces the browning rate of callus tissue, shortens the budding time of callus tissue, and lays a foundation for the future breeding of stress-resistant improved varieties of woody plants.

[0185] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium combination for inducing callus budding in woody plants, characterized in that: The culture medium combination comprises an induction culture medium and a sprouting culture medium; 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; The budding culture medium uses MS culture medium as a basic culture medium and further comprises: 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.

2. The culture medium combination according to claim 1, characterized in that: The pH values ​​of the induction medium and the sprouting medium are 5.5-5.

8.

3. The culture medium combination according to claim 1 or 2, characterized in that: The preparation method of the scutellaria baicalensis extract comprises: mixing scutellaria baicalensis with an organic solvent to obtain an extract; and ultrasonically extracting the extract and filtering it to obtain the scutellaria baicalensis extract.

4. A method for inducing callus budding in woody plants, characterized in that: The method of inducing callus budding of woody plants by using the culture medium combination according to any one of claims 1 to 3 comprises the following steps: Inoculating the explants of the woody plant on an induction medium for callus induction culture to obtain callus tissue; The callus tissue is transferred to a sprouting medium for sprouting culture.

5. The method according to claim 4, characterized in that: The explant includes a leaf.

6. The method according to claim 4, characterized in that: Before the explants of the woody plants are inoculated into the induction culture medium, the method further comprises: cleaning and disinfecting the explants; the cleaning comprises flushing with running water and flushing with deionized water in sequence; the disinfection comprises: rinsing with ethanol having a volume concentration of 75% for 40 to 50 seconds, flushing with sterile water for 3 to 4 times, then soaking with a sodium hypochlorite solution having an effective chlorine content of 3.5% to 4.5% for 8 to 12 minutes, and flushing with sterile water for 3 to 4 times.

7. The method according to claim 4, characterized in that: The induction culture and the sprout culture are light-dark alternating cultures, the light culture of the induction culture includes white light irradiation; the light culture of the sprout culture includes red light irradiation.

8. The method according to claim 7, characterized in that: The time of the white light irradiation is 18 h / d, and the illumination intensity of the white light irradiation is 10000-20000 lux; the time of the red light irradiation is 18 h / d, and the illumination intensity of the red light irradiation is 10000-20000 lux.

9. The method according to claim 4, characterized in that: The time of the induction culture is 30 to 60 days; the time of the sprout culture is 50 to 130 days.

10. The method according to claim 4, characterized in that: The temperature of the induction culture and the sprout culture is 23-25° C., and the humidity of the induction culture and the sprout culture is 16%-17%.

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