A culture medium and culture method for reducing vitrification of plant tissue culture seedlings

Abstract

The present invention relates to the technical field of plant tissue culture, and in particular to a culture medium and a culture method for reducing vitrification of plant tissue culture seedlings. The culture medium of the present invention is a basic culture medium in which graphene oxide, amino acid-containing water-soluble fertilizer A and amino acid-containing water-soluble fertilizer B are added; the amino acid-containing water-soluble fertilizer A is a medium-element amino acid-containing water-soluble fertilizer, wherein the amino acid is ≥100g / L and Ga is ≥30g / L; the amino acid-containing water-soluble fertilizer B is a trace element amino acid-containing water-soluble fertilizer, wherein the amino acid is ≥100g / L and Fe+Zn+B is ≥20g / L. The present invention reduces the vitrification rate of tissue culture seedlings and improves the proliferation index of tissue culture seedlings by adding appropriate concentrations of graphene oxide, amino acid-containing water-soluble fertilizer A and amino acid-containing water-soluble fertilizer B to the primary culture medium and the proliferation culture medium of plant tissue culture, and has the advantages of simple and convenient operation.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for alleviating vitrification of plant tissue culture seedlings. Background Art

[0002] In plant tissue culture, some translucent deformed test tube plants can often be observed. This type of plant is called "glass seedling", and this phenomenon is called vitrification. Due to the abnormal tissue structure and physiological function of glass seedlings, the differentiation ability is low, which is manifested as physiological disorder, longitudinal curling or swelling of leaves, fragile leaves, lack of wax in the cuticle of the leaf epidermis, high water content of the seedlings, low nutrient content and low differentiation ability. It is difficult to proliferate and sprout, and it is difficult to take root and grow seedlings. It is even more difficult to survive in transplanting to the field, which has become a major problem to be solved in tissue culture. The current solution is mainly to overcome the vitrification phenomenon by cooling, reducing relative humidity, reducing hormone concentration and other methods. However, the existing method is not very ideal. Summary of the invention

[0003] Based on the above content, the present invention provides a culture medium and a culture method for alleviating vitrification of plant tissue culture seedlings, which can effectively alleviate the vitrification problem occurring during plant tissue culture.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a culture medium, wherein graphene oxide, amino acid-containing water-soluble fertilizer A and amino acid-containing water-soluble fertilizer B are added to a basic culture medium;

[0006] The amino acid-containing water-soluble fertilizer A is a medium-element amino acid-containing water-soluble fertilizer, wherein the amino acid content is ≥100 g / L and the Ga content is ≥30 g / L;

[0007] The amino acid-containing water-soluble fertilizer B is a trace element type amino acid-containing water-soluble fertilizer, wherein the amino acid is ≥100 g / L and Fe+Zn+B is ≥20 g / L.

[0008] Furthermore, the amino acid-containing water-soluble fertilizer A complies with the agricultural industry standard NY1429-2010 of the People's Republic of China, and contains 18 kinds of highly active free amino acids required for crop growth (alanine, glycine, serine, tyrosine, aspartic acid, threonine, arginine, lysine, glutamic acid, methionine, tryptophan, proline, cystine, valine, isoleucine, leucine, phenylalanine, and histidine, a total of 18 kinds);

[0009] The amino acid-containing water-soluble fertilizer B complies with the agricultural industry standard NY1429-2010 of the People's Republic of China, and contains 18 highly active free amino acids required for crop growth (alanine, glycine, serine, tyrosine, aspartic acid, threonine, arginine, lysine, glutamic acid, methionine, tryptophan, proline, cystine, valine, isoleucine, leucine, phenylalanine, and histidine, a total of 18 types).

[0010] Furthermore, 3-4 mg / L graphene oxide, 8-10 mg / L amino acid-containing water-soluble fertilizer A and 13-15 mg / L amino acid-containing water-soluble fertilizer B are added to the basal culture medium; more preferably, this culture medium is used as the primary culture medium for plant tissue culture.

[0011] Furthermore, 2-3 mg / L graphene oxide, 5-6 mg / L amino acid-containing water-soluble fertilizer A and 10-12 mg / L amino acid-containing water-soluble fertilizer B are added to the basal culture medium; more preferably, this culture medium is used as a proliferation medium for plant tissue culture.

[0012] Furthermore, 0.5-1 mg / L 6-BA, 7-8 g / L agar and 30 g / L sugar are added to the basal medium; more preferably, when used as a primary culture medium, the 6-BA content is 1 mg / L; when used as a proliferation culture medium, the 6-BA content is 0.5 mg / L;

[0013] The basic culture medium is 1 / 2MS culture medium or MS culture medium;

[0014] The 1 / 2MS culture medium comprises the following components: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 85 mg / L potassium dihydrogen phosphate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride;

[0015] The MS culture medium comprises the following components: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 440 mg / L calcium chloride, 370 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride.

[0016] A second technical solution of the present invention is a method for preparing the above-mentioned culture medium, the method for preparing the culture medium comprising the following steps:

[0017] First, graphene oxide, amino acid-containing water-soluble fertilizer A and amino acid-containing water-soluble fertilizer B are uniformly mixed and then uniformly mixed with other raw materials. Then, the pH value is adjusted to 5.8-6.0 with acid or alkali, sterilized, and shaken until solidified to obtain the culture medium.

[0018] Graphene oxide has a strong adsorption effect on macromolecular organic matter. Firstly, mixing graphene oxide, amino acid water-soluble fertilizer A (taking Guoguang Angai as an example) and amino acid water-soluble fertilizer B (taking Guoguang Amino Acid Iron as an example) evenly and then mixing them evenly with other raw materials is beneficial for graphene oxide to fully adsorb amino acid water-soluble fertilizer A and amino acid water-soluble fertilizer B. Then, dispersing them in the culture medium can better promote plants to absorb the nutrients of the two amino acid water-soluble fertilizers.

[0019] Furthermore, the acid is 1 mol / L HCL; and the base is 1 mol / L NaOH.

[0020] The third technical solution of the present invention is the application of the above culture medium in plant tissue culture.

[0021] A fourth technical solution of the present invention is a method for reducing vitrification of plant tissue culture seedlings, comprising: using the above-mentioned culture medium (more preferably the primary culture medium) for primary culture in the primary culture stage of plant tissue culture, and / or using the above-mentioned culture medium (more preferably the proliferation culture medium) for proliferation culture in the proliferation culture stage of plant tissue culture.

[0022] Furthermore, the conditions for the primary culture are: temperature of (25±2)°C, light intensity of 2000LUX, light duration of 8h / d, and humidity of 60-65% for continuous culture for 20-25d.

[0023] Furthermore, the conditions of the proliferation culture are: temperature of (25±2)°C, light intensity of 2000LUX, light duration of 12h / d, humidity of 60-65% for continuous culture of 25-30d.

[0024] Furthermore, in the primary culture stage of plant tissue culture, stem segments with buds are used as the source of explants; the method for selecting stem segments with buds is as follows: in early summer, stem segments with buds on branches of vigorous growing mother trees without diseases and insect pests are selected, the buds are required to be full, and the stem segment length is 1 to 2 cm, and the explants are cut; then the explants are placed in 2 mg / L graphene oxide and soaked for 2 minutes (graphene oxide has a bactericidal effect), and then rinsed with sterile water for 3 times; then 75% alcohol is added for disinfection for 30 seconds, and then quickly rinsed with sterile water for 3 to 4 times; after rinsing, it is disinfected with 0.1% mercuric chloride for 3 to 5 minutes, rinsed with sterile water 8 times, and then inoculated into the primary culture medium, and stable sterile tissue culture seedlings are formed after primary culture.

[0025] In the plant tissue culture proliferation stage, the sterile tissue culture seedlings formed after the primary culture are inoculated on the proliferation medium, and induced to differentiate under budding conditions to obtain a large number of adventitious buds.

[0026] Graphene oxide (graphie oxide GO) is one of the special graphene family nanomaterials (Graphene Family Nanomaterials, GFNs) modified by surface functionalization. The basal plane of its single-layer carbon atoms contains forces such as π-π stacking, and is connected to rich functional groups such as hydroxyl, carboxyl, and epoxy bonds. It is an oxide form of graphene. It has a high specific surface area and rich functional groups, with hydroxyl and epoxy groups on the bottom and carboxyl groups on the edge. These functional groups give graphene oxide good hydrophilicity, dispersibility and biocompatibility. The surface of graphene oxide contains a large number of hydrophilic functional groups, which can remain stable when dispersed in water. The graphene oxide with good surface modification can be stably present whether in a high salt solution or under physiological conditions, which lays the foundation for its application in biology. The present invention finds that low concentrations of graphene oxide play a positive role in the growth and development of plants, can promote the absorption of calcium and iron by plants, and improve the water status of plants at the same time, and reduce the vitrification phenomenon of plant tissue culture seedlings.

[0027] The present invention discloses the following technical effects:

[0028] The present invention reduces the vitrification rate of tissue culture seedlings and improves the proliferation index of tissue culture seedlings by adding graphene oxide, amino acid water-soluble fertilizer A (Guoguang Angai) and amino acid water-soluble fertilizer B (Guoguang Amino Acid Iron) of appropriate concentrations into a primary culture medium and a proliferation culture medium for plant tissue culture; graphene oxide has strong adsorption properties for organic pollutants, heavy metal ions and macromolecular substances (DNA, protein and humic acid); after being fully mixed with amino acid water-soluble fertilizer A (Guoguang Angai) and amino acid water-soluble fertilizer B (Guoguang Amino Acid Iron) and added into the culture medium, the absorption of metal ions such as calcium and iron and organic nutrients by explants and plants can be improved, and the contents of lignin and chlorophyll can be increased, thereby reducing the vitrification rate of plant tissue culture seedlings.

[0029] The present invention reduces the vitrification of plant tissue culture seedlings by adding graphene oxide and amino acid-containing water-soluble fertilizers into a culture medium, and has the advantage of simple and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 These are normal raspberry seedlings at different growth stages in Example 16 of the present invention; the left picture is the proliferation stage, and the right picture is the rooting stage.

[0032] Figure 2 These are normal Populus tremula seedlings at different growth stages in Example 32 of the present invention; the left picture is the proliferation stage, and the right picture is the rooting stage. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The "%" described in the present invention, unless otherwise specified, refers to mass percentage.

[0039] Unless otherwise specified, the raw materials used in the embodiments of the present invention can be obtained through commercial sources.

[0040] The 1 / 2MS medium used in the embodiment of the present invention includes the following components: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 85 mg / L potassium dihydrogen phosphate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride.

[0041] The MS medium used in the embodiment of the present invention comprises the following components: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 440 mg / L calcium chloride, 370 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride, and 0.1 mg / L thiamine hydrochloride.

[0042] The culture medium commonly used in specific experiments can be prepared into 100-fold stock solutions, stored in a refrigerator, and diluted proportionally when used. Plant hormone (6-BA) must be prepared separately into a stock solution with a concentration of 1 mg / mL.

[0043] The graphene oxide (Grapherie oxide GO) used in the embodiment of the present invention is derived from the School of Chemical Engineering / Carbon Materials Research Institute of Datong University in Shanxi Province. The graphene oxide sheet is 4 to 5 layers, spherical in water, with an aggregate diameter of about 200nm and a purity of>95%. Graphene oxide obtained by other means, such as purchase, is also applicable to the present invention.

[0044] The amino acid-containing water-soluble fertilizer A used in the embodiment of the present invention is specifically Guoguang Angai (amino acid-containing water-soluble fertilizer (middle element type)), purchased from Sichuan Runer Technology Co., Ltd., amino acid ≥ 100g / L, Ga ≥ 30g / L, registration certificate number Agricultural Fertilizer (2012) Standard No. 2441, implementation standard number: NY 1429; it is a yellow-brown soluble solution. After dilution, it is separately prepared into a mother solution with a concentration of 1 mg / mL.

[0045] The amino acid-containing water-soluble fertilizer B used in the embodiment of the present invention is specifically Guoguang Amino Acid Iron (amino acid-containing water-soluble fertilizer (trace element type)), purchased from Sichuan Guoguang Agrochemical Co., Ltd., amino acid ≥ 100g / L, Fe + Zn + B ≥ 20g / L, fertilizer registration certificate number: Agricultural Fertilizer (2018) Standard No. B0661, implementation standard number: NY 1429; it is a soluble solution. After dilution, it is prepared into a mother solution separately, with a concentration of 1mg / mL.

[0046] The sugars described in the embodiments of the present invention, unless otherwise specified, refer to sucrose.

[0047] The present invention is described in detail by using plant tissue culture of Rubus corchorifolius and European poplar (Populus tremula linn) for primary culture and proliferation culture.

[0048] 1. Primary culture of raspberries

[0049] Example 1

[0050] Step 1, preparation of primary culture medium:

[0051] The culture medium was prepared with distilled water according to the following formula: 1 / 2MS culture medium + 2mg / L graphene oxide + 1mg / L 6-BA + 5mg / L Guoguang Angai (containing amino acid water-soluble fertilizer) + 20mg / L Guoguang Amino Acid Iron (containing amino acid water-soluble fertilizer) + 7g / L agar + 30g / L sugar;

[0052] The graphene oxide is firstly mixed thoroughly with Guoguang Angai (containing amino acid water-soluble fertilizer) and Guoguang Amino Acid Iron (containing amino acid water-soluble fertilizer) solutions, and then mixed evenly with other raw materials, and the pH is adjusted to 5.8-6.0 with 1 mol / L HCL or NaOH, and then sterilized and packaged at high temperature and high pressure. After packaging, it is slowly shaken on a laboratory shaker until the agar solidifies to obtain the primary culture medium.

[0053] Step 2, explant disinfection and primary culture: select explants from the stem segments with buds on the mother tree branches that are vigorous and free of pests and diseases in early summer, with full buds and a stem segment length of 1 to 2 cm, and cut the explants with pruning shears; then place the explants in a triangular bottle, add 2 mg / L graphene oxide and soak for 2 minutes, then rinse with sterile water 3 times; then add 75% alcohol for disinfection for 30 seconds, then quickly rinse with sterile water 3 to 4 times; after rinsing, disinfect with 0.1% mercuric chloride for 3 to 5 minutes, and rinse with sterile water 8 times. Then access the primary culture medium prepared in step 1 for primary culture, inoculate 50 bottles; the primary culture conditions are: culture temperature is (25 ± 2) ° C, light intensity is 2000LUX, light time is 8h / d, humidity is 60 to 65% and cultured for 25 days.

[0054] Step 3, index determination: After 25 days of primary culture, collect and record indicators such as seedling height, germination rate, proliferation index, and vitrification rate.

[0055] Germination rate = number of stem segments with buds that germinate after the ndth bud / number of stem segments with buds at the time of inoculation × 100%;

[0056] Vitrification rate = number of explants vitrified / total number of explants × 100%;

[0057] Step 4: Data collection and organization:

[0058] EXCEL2003 was used for data collection and preliminary processing, and SPSS17.0 Statistics software LSD method was used for significant difference test, with the significance level of P<0.05.

[0059] Embodiment 2-9

[0060] The method of Example 1 was used to carry out the primary culture of raspberry plant tissue culture. The only difference from Example 1 was that the concentration of graphene oxide, Guoguang Angai concentration, and Guoguang amino acid iron concentration in step 1 were changed. Examples 2-9 were carried out. The experiment adopted a three-factor three-level orthogonal experiment. The three factors were graphene oxide, Guoguang Angai, and Guoguang amino acid iron. The experiment was carried out according to L9(3 4 ) Orthogonal array design, with 9 treatments and 2 replications, see Table 1 for details.

[0061] Table 1 Raspberry primary culture test plan

[0062]

[0063] 2. Survey results of primary culture of raspberries

[0064] The survey results are shown in Table 2. It can be seen that in the primary culture of raspberry, the maximum range of germination rate of different concentrations of graphene oxide can reach 34%, the maximum range of vitrification rate is 33.33%, and the seedling height is 0.92 cm; the maximum range of germination rate of different concentrations of Guoguang·Angai can reach 23.33%, the maximum range of vitrification rate is 29%, and the seedling height is 0.82 cm; the maximum range of germination rate of different concentrations of Guoguang·amino acid iron can reach 12.67%, the maximum range of vitrification rate is 29.67%, and the seedling height is 1.13 cm; the variance analysis of Tables 4, 5, and 6 also shows that in the primary culture of raspberry, graphene oxide, Guoguang·Angai, and Guoguang·amino acid iron all have a significant effect on the germination rate, vitrification rate, and seedling height, reaching a significant level (P < 0.05). Among them, the germination rate: graphene oxide > Guoguang · Angai > Guoguang · amino acid iron; vitrification rate: graphene oxide > Guoguang · amino acid iron > Guoguang · Angai; seedling height: Guoguang · amino acid iron > graphene oxide > Guoguang · Angai. Q test Table 6 shows that the vitrification rate of graphene oxide, Guoguang · Angai, and Guoguang · amino acid iron in the primary culture of raspberry reached significant differences (P < 0.05); the seedling height of Guoguang · amino acid iron reached significant differences (P < 0.05).

[0065] The results showed that the most suitable ingredients for the primary culture of raspberries were 4 mg / L graphene oxide, 10 mg / L Guoguang Angai, and 15 mg / L Guoguang amino acid iron, which could effectively reduce the vitrification rate and increase the germination rate and seedling height.

[0066] Table 2 Survey results of primary culture of raspberry (25 days)

[0067]

[0068]

[0069] Table 3 Analysis of variance of germination rate of primary culture of raspberry (unit: %)

[0070]

[0071] aR square = 0.905 (adjusted R square = 0.854) Table 4 Analysis of variance of vitrification rate of primary culture of raspberry (unit / %)

[0072]

[0073] aR square = 0.978 (adjusted R square = 0.967)

[0074] Table 5 Analysis of variance of height of primary cultured raspberry seedlings (unit / cm)

[0075]

[0076] aR square = 0.961 (adjusted R square = 0.940)

[0077] Table 6. Comparison of differences in primary culture of raspberry

[0078]

[0079] Note: Different lowercase letters indicate significant differences among the data of different concentration levels of the three factors (P<0.05);

[0080] 3. Raspberry proliferation culture test plan

[0081] Example 10

[0082] Step 1, preparation of proliferation medium:

[0083] Prepare the culture medium with distilled water according to the following formula: MS culture medium + 6-BA 0.5 mg / L + graphene oxide 1 mg / L + Guoguang · Angai (containing amino acid water-soluble fertilizer) 2 mg / L + Guoguang · Amino acid iron (containing amino acid water-soluble fertilizer) 15 mg / L + agar 8 g / L + sugar 30 g / L. Graphene oxide is first fully mixed with Guoguang · Angai (containing amino acid water-soluble fertilizer) and Guoguang · Amino acid iron (containing amino acid water-soluble fertilizer) solution, and then mixed evenly with other raw materials, and the pH is adjusted to 5.8-6.0 with 1 mol / L HCL or NaOH, and then sterilized and packaged at high temperature and high pressure. After packaging, the laboratory shaker is used to slowly shake until the agar solidifies to obtain the proliferation culture medium.

[0084] Step 2, adventitious bud preparation:

[0085] The sterile raspberry tissue culture seedlings in the primary culture stage were selected, trimmed into 2 cm long stem segments, and inoculated into proliferation culture medium for proliferation culture under aseptic operation. 50 bottles were inoculated and repeated twice. The proliferation culture conditions were as follows: temperature of (25±2)°C, light intensity of 2000LUX, light duration of 12h / d, and humidity of 60-65% for continuous culture for 30 days.

[0086] Step 3, indicator determination:

[0087] After 30 days of proliferation culture, the seedling height, proliferation index, leaf color and other indicators were collected and recorded.

[0088] Proliferation coefficient = number of buds on the stem segment with buds after the ndth day / number of buds on the stem segment with buds at the time of inoculation × 100%;

[0089] Vitrification rate = number of explants vitrified / total number of explants × 100%

[0090] Step 4: Data collection and organization:

[0091] EXCEL2003 was used for data collection and preliminary processing, and SPSS17.0 Statistics software LSD method was used for significant difference test, with the significance level of P<0.05.

[0092] Examples 11-18

[0093] The method of Example 10 was used to carry out tissue culture and proliferation of raspberry plants. The only difference from Example 10 was that the concentrations of graphene oxide, Guoguang Angai, and Guoguang amino acid iron in step 1 were changed. Examples 11-18 were carried out. The experiment adopted a three-factor three-level orthogonal experiment. The three factors were graphene oxide, Guoguang Angai, and Guoguang amino acid iron. The experiment was carried out according to L9(3 4 ) Orthogonal array design, 9 treatments in total, 2 repetitions, see Table 7 for details.

[0094] Table 7 Raspberry proliferation culture test plan

[0095]

[0096] Note: Different lowercase letters indicate significant differences among the factors (P < 0.05)

[0097] 4. Results of the Raspberry Proliferation and Culture Experiment

[0098] The survey results are shown in Table 8. During the proliferation and cultivation stage of raspberries, the overall vitrification rate decreased. In the proliferation and cultivation of raspberries, the maximum range of the proliferation coefficient of different concentrations of graphene oxide can reach 1.16, the maximum range of the vitrification rate is 14.67%, and the seedling height is 2.54 cm; the maximum range of the proliferation coefficient of different concentrations of Guoguang·Angai can reach 0.67, the maximum range of the vitrification rate is 26%, and the seedling height is 1.95 cm; the maximum range of the proliferation coefficient of different concentrations of Guoguang·Amino Acid Iron can reach 0.75, the maximum range of the vitrification rate is 9.67%, and the seedling height is 0.58 cm; the variance analysis of Tables 9, 10, and 11 also shows that graphene oxide, Guoguang·Angai, and Guoguang·Amino Acid Iron have a significant effect on the proliferation coefficient, vitrification rate, and seedling height in the primary culture of raspberries, reaching a significant level (P < 0.05). Among them, the proliferation coefficient: graphene oxide > Guoguang · Angai > Guoguang · amino acid iron; vitrification rate: graphene oxide > Guoguang · amino acid iron > Guoguang · Angai; seedling height: graphene oxide > Guoguang · Angai > Guoguang · amino acid iron. Q test table 12 shows that the proliferation coefficient, vitrification rate, and seedling height of graphene oxide and Guoguang · Angai in raspberry proliferation culture are significantly different (P < 0.05); the proliferation coefficient and seedling height between Guoguang · amino acid iron 5 and 15 mg / L concentrations did not reach a significant difference, but 10 mg / L reached a significant difference with the two concentrations (P < 0.05).

[0099] The results showed that the most suitable ingredients for raspberry proliferation culture were 2 mg / L graphene oxide, 5 mg / L Guoguang Angai, and 10 mg / L Guoguang amino acid iron, which could effectively reduce the vitrification rate and increase the proliferation coefficient and growth amount.

[0100] Table 8 Raspberry proliferation culture survey results (30d)

[0101]

[0102]

[0103] Table 9 Analysis of variance of proliferation coefficient of raspberry proliferation culture

[0104]

[0105]

[0106] aR square = 0.966 (adjusted R square = 0.948)

[0107] Table 10 Analysis of variance of vitrification rate of raspberry proliferation culture (unit / %)

[0108]

[0109] aR square = 0.959 (adjusted R square = 0.937)

[0110] Table 11 Analysis of variance of raspberry proliferation culture seedling height (unit / cm)

[0111]

[0112] aR square = 0.983 (adjusted R square = 0.974)

[0113] Table 12 Comparison of differences in raspberry proliferation culture

[0114]

[0115] Note: Different lowercase letters indicate significant differences among the factors (P < 0.05)

[0116] 5. Primary Culture Experimental Program of Populus tremula

[0117] Embodiment 19

[0118] Step 1, preparation of primary culture medium:

[0119] The culture medium was prepared with distilled water according to the following formula: 1 / 2MS culture medium + 2mg / L graphene oxide + 1mg / L 6-BA + 3mg / L Guoguang Angai (containing amino acid water-soluble fertilizer) + 18mg / L Guoguang Amino Acid Iron (containing amino acid water-soluble fertilizer) + 7g / L agar + 30g / L sugar;

[0120] The graphene oxide is firstly mixed thoroughly with Guoguang Angai (containing amino acid water-soluble fertilizer) and Guoguang Amino Acid Iron (containing amino acid water-soluble fertilizer) solutions, and then mixed evenly with other raw materials, and the pH is adjusted to 5.8-6.0 with 1 mol / L HCL or NaOH, and then sterilized and packaged at high temperature and high pressure. After packaging, it is slowly shaken on a laboratory shaker until the agar solidifies to obtain the primary culture medium.

[0121] Step 2, explant disinfection and primary culture: select explants from stem segments with buds on mother tree branches that are vigorous and free of pests and diseases in early summer, with full buds and stem segments of 1 to 2 cm in length, and cut the explants with pruning shears; then place the explants in a triangular bottle, add 2 mg / L graphene oxide and soak for 2 minutes, then rinse with sterile water 3 times; then add 75% alcohol for disinfection for 30 seconds, then quickly rinse with sterile water 3 to 4 times; after rinsing, disinfect with 0.1% mercuric chloride for 3 to 5 minutes, and rinse with sterile water 8 times. Then access the primary culture medium prepared in step 1 for primary culture, and inoculate 50 bottles in total; the primary culture conditions are: culture temperature is (25 ± 2) ° C, light intensity is 2000 LUX, light time is 8 h / d, humidity is 60 to 65% and cultured for 25 days.

[0122] Step 3, index determination: After 25 days of primary culture, collect and record indicators such as seedling height, germination rate, proliferation index, and vitrification rate.

[0123] Germination rate = number of stem segments with buds that germinate after the ndth bud / number of stem segments with buds at the time of inoculation × 100%;

[0124] Vitrification rate = number of explants vitrified / total number of explants × 100%;

[0125] Step 4: Data collection and organization:

[0126] EXCEL2003 was used for data collection and preliminary processing, and SPSS17.0 Statistics software LSD method was used for significant difference test, with the significance level of P<0.05. The data were expressed as mean±standard deviation.

[0127] Examples 20-27

[0128] The method of Example 19 was used to carry out the primary culture of European Populus tremula plant tissue culture. The only difference from Example 19 was that the concentrations of graphene oxide, Guoguang Angai, and Guoguang amino acid iron in step 1 were changed. Examples 20-27 were carried out. The experiment adopted a three-factor three-level orthogonal experiment. The three factors were graphene oxide, Guoguang Angai, and Guoguang amino acid iron. The experiment was carried out according to L9(3 4 )Orthogonal array design, 9 treatments in total, 2 repetitions, see Table 13 for details.

[0129] Table 13 Primary cultivation technology scheme of European Populus

[0130]

[0131] 6. Survey results of primary cultivation of Populus tremula

[0132] The survey results are shown in Table 14, which shows that in the primary culture of European Populus, the maximum range of germination rate of different concentrations of graphene oxide can reach 27.67%, the maximum range of vitrification rate is 25.5%, and the seedling height is 1 cm; the maximum range of germination rate of different concentrations of Guoguang·Angai can reach 9%, the maximum range of vitrification rate is 16.17%, and the seedling height is 0.39 cm; the maximum range of germination rate of different concentrations of Guoguang·amino acid iron can reach 6.67%, the maximum range of vitrification rate is 10.83%, and the seedling height is 0.55 cm; The variance analysis of Tables 15, 16, and 17 also shows that in the primary culture of European Populus, graphene oxide, Guoguang·Angai, and Guoguang·amino acid iron all have significant effects on the germination rate and vitrification rate (P < 0.05). Among them, the germination rate: graphene oxide > Guoguang · Angai > Guoguang · amino acid iron; vitrification rate: graphene oxide > Guoguang · Angai > Guoguang · amino acid iron; Table 18 shows that graphene oxide and Guoguang · amino acid iron have a significant effect on seedling height in the primary culture of European poplar (P < 0.05), but Guoguang · Angai has no significant effect on seedling height (P < 0.05). Q test Table 18 shows the differences in germination rate, vitrification rate, and seedling height between the concentrations of graphene oxide, Guoguang · Angai, and Guoguang · amino acid iron in the primary culture of European poplar.

[0133] The results showed that the most suitable ingredients for the primary culture of European poplar are 3mg / L graphene oxide, 8mg / L Guoguang Angai, and 13mg / L Guoguang amino acid iron, which can effectively reduce the vitrification rate and increase the germination rate and seedling height.

[0134] Table 14 Survey results of primary cultivation of European Populus tremula (25 days)

[0135]

[0136]

[0137] Table 15 Analysis of variance of germination rate of primary culture of Populus tremula (unit: %)

[0138]

[0139] aR square = 0.957 (adjusted R square = 0.934)

[0140] Table 16 Analysis of variance of vitrification rate of primary culture of Populus tremula (unit: %)

[0141]

[0142]

[0143] aR square = 0.945 (adjusted R square = 0.916)

[0144] Table 17 Analysis of variance of height of primary cultured seedlings of Populus tremula (unit / cm)

[0145]

[0146] aR square = 0.841 (adjusted R square = 0.755)

[0147] Table 18 Comparison of primary culture differences of European Populus tremula

[0148]

[0149] Note: Different lowercase letters indicate significant differences between different concentrations of each factor (P < 0.05)

[0150] 7. European Populus multiplication and cultivation test plan

[0151] Embodiment 28

[0152] Step 1, preparation of proliferation medium:

[0153] Prepare the culture medium with distilled water according to the following formula: MS culture medium + 6-BA 0.5 mg / L + graphene oxide 1 mg / L + Guoguang · Angai (containing amino acid water-soluble fertilizer) 2 mg / L + Guoguang · Amino acid iron (containing amino acid water-soluble fertilizer) 16 mg / L + agar 7 g / L + sugar 30 g / L. Graphene oxide is first fully mixed with Guoguang · Angai (containing amino acid water-soluble fertilizer) and Guoguang · Amino acid iron (containing amino acid water-soluble fertilizer) solution, and then mixed evenly with other raw materials, and the pH is adjusted to 5.8-6.0 with 1 mol / L HCL or NaOH, and then sterilized and packaged at high temperature and high pressure. After packaging, the laboratory shaker is used to slowly shake until the agar solidifies to obtain the proliferation culture medium.

[0154] Step 2, adventitious bud preparation:

[0155] Select sterile raspberry tissue culture seedlings in the primary culture stage, trim them into 2 cm long stem segments, and inoculate them into the proliferation culture medium prepared in step 1 for proliferation culture aseptically, inoculate 50 bottles, and repeat 2 times; the proliferation culture conditions are: temperature of (25±2)°C, light intensity of 2000LUX, light time of 12h / d, humidity of 60-65% for continuous culture for 30d.

[0156] Step 3, indicator determination:

[0157] After 30 days of proliferation culture, the seedling height, proliferation index, leaf color and other indicators were collected and recorded.

[0158] Proliferation index = number of buds on the stem segment with buds after the ndth day / number of buds on the stem segment with buds at the time of inoculation × 100%;

[0159] Vitrification rate = number of explants vitrified / total number of explants × 100%

[0160] Step 4: Data collection and organization:

[0161] EXCEL2003 was used for data collection and preliminary processing, and SPSS17.0 Statistics software LSD method was used for significant difference test, with the significance level of P<0.05.

[0162] Examples 29-36

[0163] The method of Example 28 was adopted to carry out tissue culture proliferation culture of raspberry plants. The only difference from Example 28 was that the concentrations of graphene oxide, Guoguang Angai, and Guoguang amino acid iron in step 1 were changed. Examples 29-36 were carried out. The experiment adopted a three-factor three-level orthogonal experiment. The three factors were graphene oxide, Guoguang Angai, and Guoguang amino acid iron. The experiment was designed according to the L9 (34) orthogonal table, with a total of 9 treatments and 2 repetitions. See Table 19 for details.

[0164] Table 19 European Populus multiplication culture test plan

[0165]

[0166] 8. Survey results of European Populus multiplication and cultivation

[0167] The survey results are shown in Table 20. During the proliferation and cultivation stage of European poplar, the overall vitrification rate decreased. In the proliferation and cultivation of European poplar, the maximum range of the proliferation coefficient of different concentrations of graphene oxide can reach 1.18, the maximum range of the vitrification rate is 20.33%, and the seedling height is 1.36cm; the maximum range of the proliferation coefficient of different concentrations of Guoguang·Angai can reach 0.31, the maximum range of the vitrification rate is 12%, and the seedling height is 1.04cm; the maximum range of the proliferation coefficient of different concentrations of Guoguang·Amino acid iron can reach 0.55, the maximum range of the vitrification rate is 12%, and the seedling height is 1.41cm; the variance analysis of Tables 21, 22, and 23 also shows that graphene oxide, Guoguang·Angai, and Guoguang·Amino acid iron in the primary culture of European poplar have a significant effect on the proliferation coefficient, vitrification rate, and seedling height, reaching a significant level (P < 0.05). Among them, the proliferation coefficient of graphene oxide>Guoguang·amino acid iron>Guoguang·Angai; the vitrification rate of graphene oxide>Guoguang·amino acid iron>Guoguang·Angai; the seedling height of graphene oxide>Guoguang·amino acid iron>Guoguang·Angai. Q test table 24 shows that the proliferation coefficient, vitrification rate, and seedling height of graphene oxide in the proliferation culture of European poplar all reached significant differences (P < 0.05); while there was no significant difference between some concentrations of Guoguang·amino acid iron and Guoguang·Angai (P < 0.05).

[0168] The results showed that the most suitable ingredients for the proliferation culture of European poplar are 3mg / L graphene oxide, 6mg / L Guoguang Angai, and 12mg / L Guoguang amino acid iron, which can effectively reduce the vitrification rate and increase the proliferation coefficient and growth amount.

[0169] Table 20 Results of the European Populus multiplication and cultivation survey (30 days)

[0170]

[0171]

[0172] Table 21 Analysis of variance of proliferation coefficients in European Populus multiplication culture

[0173]

[0174] aR square = 0.989 (adjusted R square = 0.983)

[0175] Table 22 Analysis of variance of vitrification rate of European Populus tremula proliferation culture (unit / %)

[0176]

[0177] aR square = 0.978 (adjusted R square = 0.966)

[0178] Table 23 Analysis of variance of height of primary cultured seedlings of Populus tremula (unit / cm)

[0179]

[0180] aR square = 0.953 (adjusted R square = 0.927)

[0181] Table 24 Comparison of primary culture differences of European Populus tremula

[0182]

[0183] Note: Different lowercase letters indicate significant differences among the factors (P < 0.05)

[0184] Figure 1 The normal raspberry seedlings in different growth stages of Example 16 of the present invention; in the figure, the left picture is the proliferation stage, and the right picture is the rooting stage. Figure 1 It can be seen that in the proliferation stage, the leaves of raspberry seedlings are tender green and the number of adventitious buds is large; in the rooting stage, normal raspberry seedlings have a well-developed root system and a large leaf area.

[0185] Figure 2 The normal seedlings of Populus tremula at different growth stages in Example 32 of the present invention are shown in the figure. The left picture is the proliferation stage, and the right picture is the rooting stage. Figure 2It can be seen that in the proliferation stage, the leaves of European Populus tremula seedlings are tender green, with a large number of adventitious buds and branches; in the rooting stage, the normal European Populus tremula seedlings have a well-developed root system and a large leaf area.

[0186] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for reducing vitrification of plant tissue culture seedlings, characterized in that: In the primary culture stage of plant tissue culture, a culture medium is used for primary culture, wherein the culture medium is prepared by adding graphene oxide, amino acid-containing water-soluble fertilizer A, amino acid-containing water-soluble fertilizer B, 0.5-1 mg / L 6-BA, 7-8 g / L agar, and 30 g / L sugar to a basic culture medium; The amino acid-containing water-soluble fertilizer A is Guoguang Angai; The amino acid-containing water-soluble fertilizer B is Guoguang amino acid iron; The basic culture medium is 1 / 2MS culture medium; The 1 / 2MS culture medium comprises the following components: 825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 85 mg / L potassium dihydrogen phosphate, 220 mg / L calcium chloride, 185 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride; When the stem segments with buds of raspberries are used as explants in the primary culture stage, the contents of graphene oxide, amino acid-containing water-soluble fertilizer A, and amino acid-containing water-soluble fertilizer B in the basal culture medium are 2 mg / L, 10 mg / L, and 15 mg / L, or 4 mg / L, 10 mg / L, and 20 mg / L, respectively; When the bud-bearing stem segments of Populus tremula were used as explants in the initial culture stage, the contents of graphene oxide, amino acid water-soluble fertilizer A and amino acid water-soluble fertilizer B in the basal culture medium were 1 mg / L, 8 mg / L and 13 mg / L, or 3 mg / L, 8 mg / L and 18 mg / L, respectively.

2. The method for reducing vitrification of plant tissue culture seedlings according to claim 1, characterized in that: In the plant tissue culture proliferation culture stage, the stem segments of the sterile plant tissue culture seedlings in the primary culture stage are used as explants, and proliferation culture is carried out using a proliferation medium, wherein the proliferation medium is prepared by adding graphene oxide, amino acid-containing water-soluble fertilizer A, amino acid-containing water-soluble fertilizer B, 0.5-1 mg / L 6-BA, 7-8 g / L agar, and 30 g / L sugar to a basic culture medium; The basic culture medium is MS culture medium; The MS culture medium comprises the following components: 1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 440 mg / L calcium chloride, 370 mg / L magnesium sulfate, 0.83 mg / L potassium iodide, 22.3 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L cobalt chloride, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L copper sulfate, 37.3 mg / L sodium ethylenediaminetetraacetic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 2 mg / L glycine, 0.5 mg / L pyridoxine hydrochloride and 0.1 mg / L thiamine hydrochloride; When the plant type in the proliferation culture stage is raspberry, the contents of graphene oxide, amino acid-containing water-soluble fertilizer A, and amino acid-containing water-soluble fertilizer B in the basal culture medium are 1 mg / L, 5 mg / L, and 10 mg / L, or 2 mg / L, 5 mg / L, and 15 mg / L, respectively; When the plant type in the proliferation culture stage is European poplar, the contents of graphene oxide, amino acid water-soluble fertilizer A and amino acid water-soluble fertilizer B in the basal culture medium are 1 mg / L, 6 mg / L and 12 mg / L, or 3 mg / L, 6 mg / L and 16 mg / L, respectively.

3. The method for reducing vitrification of plant tissue culture seedlings according to claim 1, characterized in that: The preparation method of the culture medium comprises the following steps: First, graphene oxide, amino acid-containing water-soluble fertilizer A and amino acid-containing water-soluble fertilizer B are uniformly mixed and then uniformly mixed with other raw materials. Then, the pH value is adjusted to 5.8-6.0 with acid or alkali, sterilized, and shaken until solidified to obtain the culture medium.

4. The method for reducing vitrification of plant tissue culture seedlings according to claim 1, characterized in that: The conditions for the primary culture are: temperature of (25±2)°C, light intensity of 2000LUX, light duration of 8h / d, humidity of 60-65% for continuous culture for 20-25d.

5. The method for reducing vitrification of plant tissue culture seedlings according to claim 2, characterized in that: The conditions for the proliferation culture are: temperature of (25±2)°C, light intensity of 2000LUX, light duration of 12h / d, humidity of 60-65% and continuous culture of 25-30d.

Images