Culture medium as well as culture method and application thereof

Abstract

The invention provides a culture medium and a culture method and application thereof. The culture medium takes an MS culture medium as a basic culture medium, and cane sugar, brassinosteroid, gibberellin, naphthylacetic acid and cystine are also added. According to the culture medium and the culture method thereof, the number of cotton ovule fiber protrusions can be increased, the weight of cotton ovule fibers can be increased, and stable production of cotton fibers obtained through in-vitro culture of cotton ovules is achieved.

Description

Technical Field

[0001] The present invention relates to the field of cotton fibers, and in particular, to a culture medium, a culture method thereof, and an application thereof. Background Art

[0002] Cotton fibers are differentiated from single cells on the outer epidermis of ovules and are a special type of trichome. Based on this characteristic, cotton fibers can be used as an ideal model for studying plant cell wall formation and cell elongation. Since fiber protrusion is strictly regulated, ultimately only 25% - 30% of the cells can protrude and develop into long fibers. Therefore, improving cotton fiber protrusion is an important indicator for cotton yield.

[0003] The development process of cotton fibers can be divided into five stages, namely fiber cell protrusion, elongation, transition, secondary wall thickening, and dehydration and maturation. Approximately - 3 to 3 DPA, cotton fibers begin to protrude; after fiber protrusion, rapid elongation begins and continues until 16 DPA; from 16 to 20 DPA, fiber elongation slows down and the secondary wall begins to be synthesized; from 20 to 40 DPA, fiber elongation stops and the secondary wall gradually thickens; thereafter, the fiber cells enter programmed cell death and undergo dehydration and maturation.

[0004] Cystine is a non - essential amino acid of sulfur in plants and can promote cellular redox reactions. The main research focuses on the medical field, such as the cystine / glutamate antiporter (System Xc -), which takes up cystine and excretes glutamate, providing raw materials for intracellular glutathione synthesis and participating in the regulation of extracellular glutamate concentration. It can play roles such as antioxidant stress and regulation of nerve signal transmission, and is closely related to various diseases such as central nervous system diseases, tumor growth, and drug resistance formation. In the feed industry, cystine can be used as an additive in animal feed to provide essential amino acids and promote animal growth. In plants, the research on cystine is less.

[0005] The tissue culture of plants, also called in vitro culture, refers to the technology of separating the required tissues, organs, or cells from the plant body, through aseptic operation, inoculating them on a culture medium containing various nutrients and plant hormones for culture to obtain a regenerated complete plant or other products with economic value. Plant in vitro propagation has the following four characteristics: 1. High propagation efficiency; 2. Strong controllability of culture conditions; 3. Small occupied space; 4. Convenient management and conducive to automated control.

[0006] In summary, there is an urgent need for an in vitro culture medium for cotton ovules to increase cotton yield. Summary of the Invention

[0007] To solve the above - mentioned technical problems, the present invention provides a culture medium, a culture method thereof, and an application thereof. This culture medium can increase the number of cotton ovule fiber protrusions and the weight of cotton ovule fibers.

[0008] To achieve the above object, on the one hand, the present invention provides a culture medium, which is based on the MS culture medium and further contains sucrose, brassinosteroid, gibberellin, naphthylacetic acid and cystine.

[0009] According to a specific embodiment of the present invention, the culture medium is a liquid culture medium;

[0010] In the culture medium, the content of brassinosteroid is 0.25 - 0.5 mg / L, the content of gibberellin is 0.25 - 0.5 mg / L, the content of naphthylacetic acid is 0.25 - 0.5 mg / L, the content of sucrose is 30 - 45 g / L, and the content of cystine is 25 - 100 mg / L.

[0011] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid culture medium; the concentration of brassinosteroid in the culture medium is 0.5 mg / L, the concentration of gibberellin is 0.5 mg / L, the concentration of naphthylacetic acid is 0.25 - 0.5 mg / L, the concentration of sucrose is 30 - 45 g / L, and the content of cystine is 25 - 100 mg / L.

[0012] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid culture medium; the concentration of brassinosteroid in the culture medium is 0.5 mg / L, the concentration of gibberellin is 0.5 mg / L, the concentration of naphthylacetic acid is 0.5 mg / L, the concentration of sucrose is 30 g / L, and the content of cystine is 25 - 100 mg / L.

[0013] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid culture medium; the concentration of brassinosteroid in the culture medium is 0.5 mg / L, the concentration of gibberellin is 0.5 mg / L, the concentration of naphthylacetic acid is 0.5 mg / L, the concentration of sucrose is 30 g / L, and the content of cystine is 50 - 100 mg / L.

[0014] According to a specific embodiment of the present invention, the content of cystine in the culture medium is 50 - 100 mg / L, preferably 50 - 75 mg / L, and more preferably 50 mg / L.

[0015] According to a specific embodiment of the present invention, the MS culture medium includes the MS culture medium containing B5 vitamins;

[0016] According to a specific embodiment of the present invention, preferably, the gibberellin includes gibberellin A3.

[0017] According to a specific embodiment of the present invention, the pH of the culture medium is 5.6 - 5.8.

[0018] On the other hand, the present invention also provides a method for preparing the above-mentioned culture medium, and the method includes:

[0019] Adding MS powder containing B5 vitamin and sucrose into water, adjusting the pH of the culture medium to 5.6 - 5.8, making up the volume, cooling after autoclaving; then adding brassinolide, gibberellin, naphthylacetic acid and cystine and mixing to obtain the culture medium.

[0020] According to a specific embodiment of the present invention, the temperature of the autoclaving is 115 - 121 °C and the time is 10 - 15 min.

[0021] On the other hand, the present invention also provides a culture method, and the culture method is carried out using the above-mentioned culture medium.

[0022] According to a specific embodiment of the present invention, the culture method includes:

[0023] Taking cotton ovules and inoculating them on the above-mentioned culture medium for dark culture to obtain cotton fibers.

[0024] In the above-mentioned culture method, preferably, the culture method further includes: taking cotton young bolls, sterilizing them and then peeling to obtain cotton ovules;

[0025] In the above-mentioned culture method, preferably, the sterilization step includes: sterilizing with 10% - 20% sodium hypochlorite solution for 10 - 20 min and washing with sterilized water.

[0026] In the above-mentioned culture method, preferably, the embryo age of the cotton ovules is -2 DPA to 0 DPA, more preferably 0 DPA;

[0027] In the above-mentioned culture method, preferably, the number of days of the dark culture is 21 - 30 days, more preferably 30 days;

[0028] In the above-mentioned culture method, preferably, the temperature of the dark culture is 25 - 30 °C, more preferably 30 °C.

[0029] On the other hand, the present invention also provides an application of the above-mentioned culture medium and the above-mentioned culture method in increasing the number of fiber protrusions of cotton ovules and / or increasing the fiber weight of cotton ovules.

[0030] On the other hand, the present invention also provides an application of cystine in preparing a composition for increasing the number of fiber protrusions of cotton ovules and / or increasing the fiber weight of cotton ovules.

[0031] The beneficial effects of the present invention are as follows:

[0032] The culture medium provided by the present invention is based on the MS culture medium, and sucrose, brassinosteroid, gibberellin, naphthylacetic acid, and cystine are added thereto. The combination and special ratio of various substances in the culture medium provided by the present invention can significantly increase the fiber length, the number of fiber protrusions, and the fiber weight. Compared with the culture medium without cystine added, adding cystine can significantly increase the number of fiber protrusions and the fiber weight, but there is no obvious change in the fiber length.

[0033] The present invention further tested the culture media added with methionine, cysteine, reduced glutathione, or oxidized glutathione, and found that they all had inhibitory effects to varying degrees and would cause the fibers to become shorter. Culturing cotton fibers with an appropriate amount of cystine added not only results in no obvious change in the fiber length, but also significantly increases the number of fiber protrusions observed by electron microscopy, and the weighing results also show that the fiber amount is significantly increased. Therefore, on the basis of the culture medium ratio of the present invention, adding cystine is more conducive to the in vitro culture of ovule fibers, providing the possibility for the subsequent industrialized in vitro production of cotton fibers.

[0034] A culture medium for increasing cotton fiber protrusions provided by the present invention adopts the method of in vitro culture of cotton ovules, and artificially controls the culture conditions to study the early development mechanism of cotton fibers; the fiber amount of the cotton fibers obtained by culturing with this culture medium is greatly improved, which is of great significance for improving the quality of cotton. Description of the Drawings

[0035] Figure 1 It is an electron micrograph of fiber protrusions of ovules at -1 DPA in Example 1 after being cultured for one day under different treatments.

[0036] Figure 2 It is a statistical result graph of the number of fiber protrusions under different treatments observed by electron microscopy in Example 1.

[0037] Figure 3 It is a graph of the fiber state of ovules at 0 DPA in Example 2 after being cultured for one month under different treatments.

[0038] Figure 4 It is a graph of the fiber length results of ovules at 0 DPA in Example 2 after being cultured for one month under different treatments.

[0039] Figure 5 It is a statistical graph of the weighing results of fiber-bearing ovules under different treatments in Example 2.

[0040] Figure 6 It is a statistical graph of the weighing results of fiber-free ovules under different treatments in Example 2.

[0041] Figure 7 It is a graph of the fiber state of ovules at 0 DPA in Comparative Example 1 after being cultured for one month under different treatments.

[0042] Figure 8 The figure shows the fiber length results of the ovules of 0DPA in Comparative Example 1 after being cultured for one month under different treatments.

[0043] Figure 9 The figure shows the fiber state of the ovules of 0DPA in Comparative Example 2 after being cultured for one month under different treatments.

[0044] Figure 10 The figure shows the fiber length results of the ovules of 0DPA in Comparative Example 2 after being cultured for one month under different treatments.

[0045] Figure 11 The figure shows the fiber state of the ovules of 0DPA in Comparative Example 3 after being cultured for one month under different treatments (reduced glutathione).

[0046] Figure 12 The figure shows the fiber length results of the ovules of 0DPA in Comparative Example 3 after being cultured for one month under different treatments (reduced glutathione).

[0047] Figure 13 The figure shows the fiber state of the ovules of 0DPA in Comparative Example 3 after being cultured for one month under different treatments (oxidized glutathione).

[0048] Figure 14 The figure shows the fiber length results of the ovules of 0DPA in Comparative Example 3 after being cultured for one month under different treatments (oxidized glutathione). Detailed implementation manners

[0049] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention will be described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0050] In actual production, those skilled in the art can understand that, on the premise of obtaining qualified products, some relevant process steps can be adjusted or increased or decreased.

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0052] The materials, reagents, instruments, and test methods used in the following examples are as follows:

[0053] 1. Test materials, reagents, and instruments

[0054] 1.1 Test materials

[0055] The test materials were Jinmian 15 (R15), and all materials were self-crossed and preserved by this laboratory every year.

[0056] 1.2 Test reagents

[0057] The sucrose used in the experiment was domestic analytical pure. The plant growth regulators included gibberellin (GA3, manufacturer Coolaber), brassinolide (brassinosteroid BR, product number B21633-20mg purchased from Yuanye Bio), naphthylacetic acid (NAA, product number N605-100ml purchased from Phytotech), MS (containing B5 vitamins) medium (purchased with product number: PM1031-50L, manufacturer Coolaber), methionine (product number M9625-25G, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.), cysteine (product number 168149-25G, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.), reduced glutathione (product number 60342ES60, purchased from Yeasen), and oxidized glutathione (product number MG1544-1G, purchased from MesGen Biotech).

[0058] 1.3 Experimental instruments

[0059] (1) Instruments: Laminar flow hood (Shanghai Hujing SW-CJ-1D / 2F), distilled water generator (Licon SmartPlus-EP), autoclave (Panasonic MLS-3781-PC), constant temperature incubator (Ningbo Jiangnan Instrument RXZ-500), electronic balance (Shanghai Hengping JY60001).

[0060] (2) Tools: Forceps, scalpel, gloves, alcohol sprayer, alcohol cotton balls, alcohol lamp.

[0061] 2. Preparation of medium

[0062] Preparation of the basic medium: Adjust the pH of the MS (containing B5 vitamins) medium to 5.8, and sterilize it under high pressure (121 °C, 20 min) for standby. Naphthylacetic acid (NAA), gibberellin (GA3), and brassinosteroid (BR) are added to the MS medium; the addition amount of naphthylacetic acid (NAA) is 0.5 mg / L, the addition amount of gibberellin (GA3) is 0.5 mg / L, and the addition amount of brassinosteroid (BR) is 0.5 mg / L.

[0063] MS (containing B5 vitamins) is obtained by adding B5 vitamins to MS basic salts. The formula of MS basic salts is shown in Table 1, and the content of B5 vitamins in the medium is shown in Table 2:

[0064] Table 1 Formula of MS basic salts

[0065]

[0066]

[0067] Table 2

[0068]

[0069] 3. Test methods

[0070] 3.1 Ovule culture

[0071] Take cotton young bolls at 0 DPA for in vitro culture of cotton ovules. First, remove the sepals from the young bolls, then sterilize them with 10% sodium hypochlorite solution for 10 min, and wash them three times with sterile water. Put them into a sterile petri dish, and carefully dissect out the cotton ovules with sharp forceps. Inoculate the cotton ovules onto the media with different treatments and place them in a culture room at 30 °C for dark culture. Each treatment is cultured in 3 groups, with 3 petri dishes in each group, and 12 - 18 ovules are inoculated in each petri dish.

[0072] 3.2 Observe the growth dynamics of cotton ovules cultured in the medium

[0073] Observe the growth dynamics on the 7th, 14th, 21st, and 30th days of culture. It is found that when cultured for 30 days, the fiber growth of the ovules reaches a stable and mature state. Therefore, select the ovules cultured for 30 days to measure the length and weight of the fibers.

[0074] Length measurement: Take out the ovules cultured for 30 days, rinse them with water after a 5 - min boiling water bath, carefully separate and stretch the overlapping fibers with a needle, and measure the fiber length with a ruler. Among them, randomly take 30 ovules from each treatment to measure the fiber length and calculate the average value.

[0075] Weight measurement: Dry and weigh the ovules cultured for 30 days. Randomly take 30 materials from each treatment group, dry and weigh the ovules with fibers to obtain the weight of the ovules plus fibers for each material, and divide the sum by the number of materials to get the average weight of the ovules with fibers in this treatment group. Then remove the ovules and only weigh the weight of the fibers to obtain the average fiber weight.

[0076] Example 1 Electron microscopy observation of fiber protrusions of ovules cultured in different media

[0077] Inoculate the ovules at - 1 DPA onto the media with different treatments, and culture them in the dark at 30 °C for 1 day, and observe the fiber protrusions by electron microscopy, as Figure 1 shown.

[0078] The pH of each group of media is 5.8; the specific formula is as follows:

[0079] Control group: Medium No. 119: MS (containing B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0080] Experimental group:

[0081] (1) 25 mg / L cystine (Cys25): MS (containing B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cystine 25 mg / L.

[0082] (2) 50 mg / L cystine (Cys50): MS (containing B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cystine 50 mg / L.

[0083] As Figure 1 shown, where A is the overall electron microscope photograph of the ovule after being cultured on the control medium for one day, D is the corresponding local photograph; B is the overall electron microscope photograph of the ovule after being cultured on the medium supplemented with 25 mg / L cystine for one day, E is the corresponding local photograph; C is the overall electron microscope photograph of the ovule after being cultured on the medium supplemented with 50 mg / L cystine for one day, F is the corresponding local photograph.

[0084] The Prism software was used to analyze and statistically obtain the difference in the number of fiber protrusions (Table 3), and the statistical results are as Figure 2 shown.

[0085] Table 3 Statistical number of fiber protrusions observed by electron microscope under cystine treatment

[0086]

[0087]

[0088] As Figure 2 shown, the average number of fiber protrusions of the ovules cultured in Medium No. 119 of the control group is 382, the number of fiber protrusions of the experimental group (1) cultured with 25 mg / L cystine (Cys25) is 438, and the number of fiber protrusions of the experimental group (2) cultured with 50 mg / L cystine (Cys50) is 476. Compared with the control group, the fiber protrusions increased by 24.6%. In addition, according to the statistical analysis using the Prism software, there is no significant difference between the control group and the treatment of the experimental group (1), but there is a significant difference between the control group and the treatment of the experimental group (2).

[0089] Example 2 Fiber growth status of ovules cultured in different media

[0090] Based on the electron microscopy experimental results of Example 1, experiments were carried out around 50 mg / L cystine. Ovules at 0 DPA were inoculated on media with different treatments and cultured in the dark at 30 °C for 30 days to observe the fiber growth status.

[0091] The pH of each group of media was 5.8; the specific formulations were as follows:

[0092] Control group: MS (containing B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0093] Experimental group: MS (containing B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cystine 50 mg / L.

[0094] The fiber growth status was as Figure 3 shown, where A was the control group and B was the experimental group. After one month of culture, the fiber growth status of the B treatment was good, and it was slightly larger in volume compared to the control group, which might have a certain impact on the fiber weight.

[0095] The fiber length statistics were as Figure 4 shown, where A was the control group and B was the experimental group. The fiber lengths of the A and B treatments were both around 27 mm, indicating that the addition of cystine did not cause negative effects on the fiber length.

[0096] The ovules with fibers cultured in different media treatments were dried and weighed. 30 materials were randomly selected from each treatment group, and the ovules with fibers were dried and weighed to obtain the average weight of the ovules plus fibers (Table 4). The average weight of the control group was 0.0278 g, and that of the experimental group was 0.0339 g. Then the ovules were removed, and only the fibers were weighed to obtain the average fiber weight. The average weight of the control group was 0.0120 g, and that of the experimental group was 0.0176 g. The Prism software was used to statistically analyze the differences between these two groups of data, and the results were as Figure 5 、 Figure 6 shown.

[0097] Based on the measured data of the weight of ovules with fibers ( Figure 5 ) and the statistical results of the data of only fiber weighing ( Figure 6 ), it can be concluded that the difference between the control group and the experimental group is extremely significant.

[0098] Table 4 Statistical data of the weight of ovules plus fibers and fiber weight cultured with 50 mg / L cystine

[0099]

[0100]

[0101] Combining the electron microscopy and fiber weighing results in Examples 1 and 2, it can be clearly seen that 50 mg / L of cystine has a positive effect on the number of protrusions of ovule epidermal cells and can significantly increase the fiber weight. The culture medium formula provided by the present invention is a system for artificially controlling high-yield and stable in vitro culture of cotton ovules, providing the possibility of implementation.

[0102] Comparative Example 1

[0103] Methionine is an essential sulfur-containing amino acid. The other two sulfur-containing amino acids are cysteine and cystine. The relationship among the three is that methionine can be converted into cysteine and cystine, and cysteine and cystine can be converted into each other, but the latter two cannot be converted into methionine. Gradients of methionine were added to the culture medium to culture the ovules, and its effect on fiber growth was tested.

[0104] Using MS (containing B5 vitamins) as the basal medium, different culture substances were added for dark culture of 0 DPA cotton ovules at a culture temperature of 30°C. The pH of each group of culture medium was 5.8; the specific formula was as follows:

[0105] A.119: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0106] B. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + methionine 25 mg / L.

[0107] C. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + methionine 50 mg / L.

[0108] D. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + methionine 100 mg / L.

[0109] After one month of culture, the fiber growth status of each group was as Figure 7 shown, and the fiber length statistics were as Figure 8 shown. The average fiber length of the control group A was 27 mm, and the lengths of groups B, C, and D were all around 17 mm, with significantly shorter fiber lengths.

[0110] Comparative Example 2

[0111] Cysteine can participate in glutathione metabolism, enhance the antioxidant capacity of cells, reduce the damage of free radicals to cells, and is also a metabolic precursor of many important molecular substances such as vitamins, cofactors, and many defense substances. Using MS (containing vitamin B5) as the basal medium, different culture substances were added for dark culture of 0DPA cotton ovules at a culture temperature of 30°C. The pH of each group of culture media was 5.8; the specific formula is as follows:

[0112] A. 119: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0113] B. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cysteine 31.25 mg / L.

[0114] C. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cysteine 62.5 mg / L.

[0115] D. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + cysteine 125 mg / L.

[0116] After one month of culture, the fiber growth status of each group was as shown in Figure 9 and the fiber length statistics were as shown in Figure 10 . The fiber length of the control group A was about 27 mm. With the increase in the amount of cysteine, the fiber length was about 22 mm; the fiber lengths of groups C and D were about 20 - 21 mm, and there was a significant decrease in the fiber amount.

[0117] Comparative Example 3

[0118] Glutathione is a tripeptide containing a sulfhydryl group formed by the combination of glutamate, cysteine, and glycine. It is mainly divided into two forms: reduced (GSH) and oxidized (GSSG).

[0119] (1) Reduced glutathione (GSH):

[0120] GSH is one of the most important antioxidant molecules in the body, which can neutralize free radicals and oxidizing substances in the body and protect cells from oxidative damage. As an important regulatory metabolite in cells, GSH participates in the tricarboxylic acid cycle and sugar metabolism in the body and can activate a variety of enzymes, such as sulfhydryl (SH) enzymes - coenzymes, etc., thus promoting the metabolism of carbohydrates, fats, and proteins.

[0121] Using MS (containing vitamin B5) as the basal medium, different culture substances were added for dark culture of 0DPA cotton ovules at a culture temperature of 30°C. The pH of each group of media was 5.8; the specific formula is as follows:

[0122] A.119: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0123] B. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + reduced glutathione 12.5 mg / L.

[0124] C. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + reduced glutathione 25 mg / L.

[0125] D. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + reduced glutathione 50 mg / L.

[0126] After one month of culture, the fiber growth status of each group was as Figure 11 shown, and the fiber length statistics were as Figure 12 shown. Culturing 0DPA ovules with gradient GHS had a certain negative impact on the fiber amount. The fiber length of the control group A was about 27 mm, that of treatment B was 23 - 24 mm, that of treatment C was about 20 - 21 mm, and that of treatment D was about 20 - 21 mm, and there was no obvious change in the fiber amount.

[0127] (2) Glutathione (GSSG)

[0128] GSSG confers redox stability to cells and also acts as an interface between signal transduction pathways and metabolic reactions that promote growth and development, and also contributes to the assembly of cellular components, the biosynthesis of sulfur-containing metabolites, the inactivation of potentially harmful compounds, and the control of hormone signal strength.

[0129] Using MS (containing vitamin B5) as the basal medium, different culture substances were added for dark culture of 0DPA cotton ovules, and gradient concentrations of oxidized glutathione were added to test the effect on fiber growth. The culture temperature was 30°C. The pH of each group of media was 5.8; the specific formula is as follows:

[0130] A.119: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0131] B. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + Sucrose 30 g / L + GSSG 12.5 mg / L.

[0132] C. MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + Sucrose 30 g / L + GSSG 25 mg / L.

[0133] After one month of culture, the growth status of fibers in each group was as Figure 13 shown, and the fiber length statistics were as Figure 14 shown. Culturing 0DPA ovules with gradient concentrations of GSSG had a certain negative impact on the fiber yield. The fiber length of the control group was about 28 mm, that of treatment B was about 25 mm, and that of treatment C was about 21 mm.

[0134] From the above test results, compared with the control group, adding methionine, cysteine, reduced glutathione or oxidized glutathione all had inhibitory effects to varying degrees and would cause fiber shortening. However, culturing cotton fibers with an appropriate amount of cystine not only showed no obvious change in fiber length, but also significantly increased the number of fiber protrusions observed by electron microscopy, and the weighing results also showed that the fiber yield was significantly heavier. Therefore, compared with the above four substances, cystine is more beneficial to the in vitro culture of ovule fibers, providing the possibility for the subsequent industrialized in vitro production of cotton fibers.

Claims

1. A culture medium, wherein, The culture medium is based on the MS medium and further contains sucrose, brassinosteroid, gibberellin, naphthylacetic acid and cystine.

2. The culture medium according to claim 1, wherein, The culture medium is a liquid medium; In the culture medium, the content of brassinosteroid is 0.25 - 0.5 mg / L, the content of gibberellin is 0.25 - 0.5 mg / L, the content of naphthylacetic acid is 0.25 - 0.5 mg / L, the content of sucrose is 30 - 45 g / L, and the content of cystine is 25 - 100 mg / L.

3. The culture medium according to claim 2, wherein, In the culture medium, the content of cystine is 50 - 100 mg / L.

4. The culture medium according to claim 1, wherein, The MS medium includes the MS medium containing B5 vitamins; Preferably, the gibberellin includes GA3.

5. The culture medium according to claim 1, wherein, The pH of the culture medium is 5.6 - 5.

8.

6. A cultivation method, wherein, The culture method is carried out using the culture medium according to any one of claims 1 - 5.

7. The culturing method according to claim 6, wherein, The culture method includes: Taking cotton ovules and inoculating them on the culture medium according to any one of claims 1 - 5 for dark culture to obtain cotton fibers.

8. The cultivation method according to claim 7, wherein, The ovule age of the cotton ovules is -2 DPA to 0 DPA, preferably 0 DPA; Preferably, the number of days of dark culture is 21 - 30 days, more preferably 30 days; Preferably, the temperature of dark culture is 25 - 30 °C, more preferably 30 °C.

9. Use of the culture medium according to any one of claims 1 - 5 or the culture method according to any one of claims 6 - 8 in increasing the number of fiber protrusions of cotton ovules and / or increasing the weight of cotton ovule fibers.

10. Use of cystine in the preparation of a composition for increasing the number of fiber protrusions of cotton ovules and / or increasing the weight of cotton ovule fibers.

Images