In-vitro culture method and application of cotton ovules

Abstract

The invention provides an in-vitro culture method and application of cotton ovules. The culture method comprises the following steps: inoculating cotton ovules from-2DPA to 0DPA on a culture medium for dark culture to obtain cotton fibers; wherein the culture medium is an MS culture medium, and brassinosteroid, gibberellin, heteroauxin or naphthylacetic acid and cane sugar are added into the culture medium. According to the method, cotton ovules are inoculated on the in-vitro culture cotton ovule culture medium for culture in a sterile operation in-vitro culture mode, and fibers are directionally induced. According to the culture medium and the culture method thereof, the number of protrusions of cotton ovule fibers can be increased, the growth speed of the cotton ovule fibers is increased, the length of the cotton ovule fibers is increased, and stable production of cotton fibers obtained through in-vitro culture of cotton ovules is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plants, and particularly relates to a method for in vitro culture of cotton ovules and its application. Background Art

[0002] Cotton (Gossypium hirustum L.) is one of the most important fiber crops widely planted, and the cotton industry occupies a major position in the agricultural economy. Under limited resources, in vitro culture of cotton fibers is an important direction in cotton research. Tissue culture of plants, also known as in vitro culture, refers to separating tissues, organs, cells, etc. that meet the requirements from the plant body, and through aseptic operation, inoculating them on a culture medium containing various nutrients and plant hormones for cultivation to obtain a regenerated complete plant or other products with economic value. In vitro culture of cotton ovules can artificially control the growth environment, facilitate stable cultivation and production, and achieve highly intensive and high-density automated production, thus saving labor, material resources and the land required for field planting.

[0003] In vitro culture of plants is inseparable from hormones. Hormones interact with each other throughout the life cycle of plants, and the growth, development and response to the environment of plants are regulated by small signal molecules of hormones. In addition, plants also exhibit the unique ability to control the distribution of hormones spatially. These hormones include abscisic acid (ABA), auxin (IAA), brassinosteroid (BR), cytokinin (CK), ethylene (ET), gibberellin (GA), jasmonic acid (JA) and salicylic acid (SA), etc.

[0004] The process from seed development to senescence is closely related to the concentration level of soluble sugars. Especially in the later stage of seed development, sucrose is very necessary for inducing storage-related cell differentiation. And in the development of cotton seeds and fibers, sucrose also plays an important role. Therefore, changes in sucrose concentration will have a certain impact on the in vitro culture of cotton fibers during the development of cotton fibers and seeds.

[0005] In summary, existing research mainly focuses on the influencing factors of ovule fiber initiation and / or fiber elongation, and the final cotton fiber products are not obtained through long-term cultivation. And during the long-term tissue culture process, if there is growth inhibition or poor growth status in the early stage of cultivation, basically the best cultivation effect cannot be achieved. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for in vitro culture of cotton ovules and its application. The method for in vitro culture of cotton ovules of the present invention can increase the number of fiber protrusions of cotton ovules, accelerate the growth rate of cotton ovule fibers, and increase the length of cotton ovule fibers, realizing the stable production of cotton fibers obtained by in vitro culture of cotton ovules.

[0007] To achieve the above object, on the one hand, the present invention provides a method for in vitro culture of cotton ovules, wherein the culture method comprises:

[0008] Inoculate cotton ovules at -2DPA to 0DPA on a culture medium and perform dark culture to obtain cotton fibers;

[0009] Wherein, the culture medium is MS medium, which is added with brassinosteroid, gibberellin, indoleacetic acid or naphthylacetic acid, and sucrose.

[0010] According to a specific embodiment of the present invention, preferably, the culture method further comprises: taking cotton young bolls at -2DPA to 0DPA, sterilizing and then peeling to obtain cotton ovules at -2DPA to 0DPA;

[0011] According to a specific embodiment of the present invention, preferably, the sterilization step comprises: sterilizing with 10%-20% sodium hypochlorite solution for 10-20 min and washing with sterile water.

[0012] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid medium; the concentration of brassinosteroid in the culture medium is 0.25-0.5 mg / L, the concentration of gibberellin is 0.25-0.5 mg / L, the concentration of indoleacetic acid or naphthylacetic acid is 0.25-0.5 mg / L, and the concentration of sucrose is 30-60 g / L.

[0013] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid 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, and the concentration of sucrose is 30-60 g / L.

[0014] According to a specific embodiment of the present invention, preferably, the culture medium is a liquid 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, and the concentration of sucrose is 30-60 g / L.

[0015] According to a specific embodiment of the present invention, preferably, the concentration of naphthylacetic acid in the culture medium is 0.5 mg / L.

[0016] According to a specific embodiment of the present invention, preferably, the concentration of sucrose is 30-60 g / L, more preferably 30 g / L - 45 g / L or 45-60 g / L, and for example, it can be 30 g / L, 40 g / L, 45 g / L, 50 g / L, 60 g / L.

[0017] According to specific embodiments of the present invention, preferably, the MS medium includes the MS medium containing B5 vitamins.

[0018] According to specific embodiments of the present invention, preferably, the gibberellin includes gibberellin GA3.

[0019] According to specific embodiments of the present invention, preferably, the pH of the medium is 5.6 - 5.8.

[0020] According to specific embodiments of the present invention, preferably, the embryo age of the cotton ovules is -1 DPA to 0 DPA, more preferably 0 DPA.

[0021] According to specific embodiments of the present invention, the number of days of dark culture is 7 - 30 days, preferably 21 - 30 days, more preferably 30 days.

[0022] According to specific embodiments of the present invention, the temperature of dark culture is 25 - 30 °C, more preferably 30 °C.

[0023] In some specific embodiments, cotton ovules at 0 DPA are inoculated on the medium for dark culture to obtain cotton fibers; when the sucrose concentration in the medium is 30 g / L, the effect of increasing the length of the cultured cotton fibers is relatively superior. When the sucrose concentration in the medium is 60 g / L, the effect of increasing the number of fiber protrusions is relatively superior, but in the later stage of culture, compared with the effect of 30 g / L sucrose, it will cause a certain degree of shortening of the fiber length.

[0024] In some specific embodiments, cotton ovules from -2 DPA to -1 DPA are inoculated on the medium for dark culture to obtain cotton fibers; compared with a sucrose concentration of 30 g / L, when the sucrose concentration in the medium is 60 g / L, the effect of increasing the number of fiber protrusions is relatively superior and the fiber length increases.

[0025] In some specific embodiments, cotton ovules at 0 DPA are inoculated on Medium No. 119 (sucrose 30 g / L) for dark culture, which can ensure the growth of fiber length; when using ovules from -1 DPA to -2 DPA, using a medium with 60 g / L sucrose will cause an increase in the number of fiber protrusions, which can be used for basic research related pathways, resulting in an increase in the number of fibers and thus an increase in mass production.

[0026] On the other hand, the present invention also provides a method for preparing the above-mentioned medium, and the method includes:

[0027] Adding MS powder containing B5 vitamins and sucrose into water, adjusting the pH of the medium to 5.6 - 5.8, making up the volume, cooling after autoclaving; then adding brassinosteroid, gibberellin, indoleacetic acid or naphthaleneacetic acid and mixing to obtain the medium.

[0028] According to a specific embodiment of the present invention, the temperature for autoclaving is 115 - 121 °C and the time is 10 - 15 min.

[0029] On the other hand, the present invention also provides a cotton fiber obtained by the above cultivation method.

[0030] On the other hand, the present invention also provides the application of the above cultivation method in increasing the number of fiber protrusions of cotton ovules, accelerating the growth rate of cotton ovule fibers, or increasing the length of cotton ovule fibers.

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

[0032] The components of the culture medium for plant tissue culture generally include macronutrients, micronutrients, iron salts, vitamins, sugars, plant hormones, and some specific growth regulator additives. The content and combination of these components directly affect the tissue culture effect. The culture medium for in vitro culture of cotton ovules and its cultivation method provided by the present invention inoculate cotton ovules on a culture medium containing specific combinations of various nutrients and plant hormones provided by the present invention through aseptic operation and in vitro culture, and directionally induce cotton fibers.

[0033] Through the culture medium and cultivation method provided by the present invention, the growth environment of cotton ovules in vitro culture can be artificially controlled, which can significantly increase the number of fiber protrusions of cotton ovules, accelerate the growth rate of cotton ovule fibers, and increase the length of cotton ovule fibers, achieving a better effect of cotton ovule culture for cotton fiber production, which is basically the same as the cotton fiber length harvested from field planting of cotton and significantly longer than the cotton fiber length in general existing technologies. It realizes the stable cultivation and production of cotton fibers, and provides the possibility for subsequent highly intensive and high-density automated production. Description of the Drawings

[0034] Figure 1 It shows the growth of ovule fibers at different embryo ages.

[0035] Figure 2 It shows the fiber growth of 0 DPA ovules at 26 °C and 30 °C.

[0036] Figure 3 It shows the state of 0 DPA ovules growing on agar media with different concentrations for 14 days.

[0037] Figure 4 It shows the difference in the state of cultured materials between BT medium and MS (B5 vitamin) medium (cultured for 30 days).

[0038] Figure 5 It shows the fiber state of ovules on culture media with different hormones. Figure 5 The left figure in it shows the state where the fibers are not combed.Figure 5 The length after fiber carding is shown in the right middle figure.

[0039] Figure 6 It shows the growth states of ovules at different embryo ages under the treatments of culture media No. 119 and 157.

[0040] Figure 7 It shows the fiber lengths of ovules at different embryo ages under the treatments of culture media No. 119 and 157.

[0041] Figure 8 It is the electron micrograph of fiber protrusions of -1 DPA ovules after being cultured on culture media No. 119, 156 and 157 for one day.

[0042] Figure 9 It shows the numbers of fiber protrusions of -1 DPA ovules after being cultured on culture media No. 119, 156 and 157 for one day. Detailed implementation manners

[0043] 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 implementable scope of the present invention.

[0044] 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 added or subtracted.

[0045] 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 shall fall within the protection scope of the present invention.

[0046] The materials, reagents, instruments and test methods used in the following embodiments are as follows:

[0047] 1. Test materials, reagents and instruments

[0048] 1.1 Test materials

[0049] The test materials are Jinmian 15 (R15), and all materials are self-pollinated and preserved by this laboratory every year.

[0050] 1.2 Test reagents

[0051] The sucrose used in the experiment was of domestic analytical purity. The plant growth regulators included indoleacetic acid (IAA, product number: PH102 - 100ml, purchased from Coolaber), gibberellin (GA3, purchased from Coolaber), brassinolide (brassinosteroid BR, product number B21633 - 20mg, purchased from Yuanye Bio), naphthaleneacetic acid (NAA, product number N605 - 100ml, purchased from Phytotech), and MS (containing B5 vitamins) medium (product number: PM1031 - 50L, purchased from Coolaber).

[0052] 1.3 Experimental instruments

[0053] (1) Instruments: laminar flow hood (Shanghai Hujing SW - CJ - 1D / 2F), distilled water generator (LiKang SmartPlus - EP), autoclave (Panasonic MLS - 3781 - PC), constant temperature incubator (Ningbo Jiangnan Instrument RXZ - 500), electronic balance (Shanghai Hengping JY60001).

[0054] (2) Tools: forceps, scalpel, gloves, alcohol sprayer, alcohol cotton balls, alcohol lamp.

[0055] 2. Preparation of culture medium

[0056] Preparation of the basal medium: Prepare MS (containing B5 vitamins) medium, adjust the pH to 5.8, sterilize it under high pressure (115 °C, 15 min), and set aside. Among them, agar and sucrose need to be added before sterilization, and plant hormones are added after the temperature drops to a certain level after sterilization. The plant hormones include naphthaleneacetic acid (NAA) or IAA (indoleacetic acid), gibberellin (GA3), and brassinosteroid (BR); the addition amount of naphthaleneacetic acid (NAA) or IAA (indoleacetic acid) 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.

[0057] 3. Experimental methods

[0058] 3.1 Ovule culture

[0059] Take cotton young bolls at - 2 to 0 DPA for in vitro culture of cotton ovules. First, remove the sepals from the young bolls, then sterilize them with 20% sodium hypochlorite solution for 20 min, and wash them three times with sterile water; place them in a sterile petri dish, and carefully peel out the cotton ovules with pointed forceps; inoculate the cotton ovules on the medium with different treatments, and place them in a culture room at 25 - 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.

[0060] 3.2 Observe the growth dynamics of cotton ovules cultured in the medium

[0061] Observing the growth dynamics at 7, 14, 21, and 30 days of culture, it was found that the fiber growth of the ovules reached a stable and mature state at 30 days of culture. Therefore, the ovules cultured for 30 days were selected to measure the fiber length.

[0062] The ovules cultured for 30 days were taken out after a 5-minute boiling water bath, rinsed with clear water, the overlapping fibers were carefully separated with a needle and stretched, and the fiber length was measured with a ruler. Among them, 30 ovules were randomly taken from each treatment to measure the fiber length, and the average value was calculated.

[0063] Example 1: Ovule embryo age test

[0064] Using R15 as the test variety, ovules at 0 DPA (0 DPA is the day of flowering), -1 DPA, and -2 DPA were taken and placed on the same culture dish for dark culture at 26 °C ( Figure 1 ). Culture medium formula: Medium No. 119: MS (B5 vitamin) + NAA (0.5 mg / L) + BR (0.5 mg / L) + GA3 (0.5 mg / L) + sucrose 30 g / L. Ovules of the three embryo ages were cultured at the same temperature on the same dish of culture medium. Among them, Figure 1 A in it is the growth state of ovules of different embryo ages cultured on the culture medium for 7 days; Figure 1 B in it is the growth state of ovules of different embryo ages cultured on the culture medium for 14 days. In contrast, the growth of ovules at -1 DPA and -2 DPA was slow, and 0 DPA was more suitable as an explant for culture.

[0065] Example 2: Test of culture temperature

[0066] Take 0 DPA ovules and place them on the culture medium, and conduct dark culture at 26 °C and 30 °C respectively. Culture medium formula: Medium No. 119: MS (B5 vitamin) + NAA (0.5 mg / L) + BR (0.5 mg / L) + GA3 (0.5 mg / L) + sucrose 30 g / L. Take materials from the same dish and take pictures at 7 days, 14 days, and 21 days to observe the growth of fibers ( Figure 2 ), among which, Figure 2 A, B, and C in it are the growth conditions of 0 DPA ovules cultured at 30 °C for 7 days, 14 days, and 21 days respectively; Figure 2 D, E, and F in it are the growth conditions of 0 DPA ovules cultured at 26 °C for 7 days, 14 days, and 21 days respectively. Under the culture condition of 30 °C, the growth rate of fiber induction of ovules is faster.

[0067] Example 3: Test of agar concentration in the culture medium

[0068] In the experiment with decreasing agar gradient concentration, 0 DPA ovules were placed on the medium. Based on the experimental basal medium obtained from the aforementioned 2. medium formula, solid media supplemented with 5 g / L, 4 g / L, 3 g / L agar and a liquid medium with 0 g / L agar were respectively set up, and the growth states at 14 days of culture were as follows Figure 3 shown. It was found that the growth rate of cotton fibers increased with the decrease in agar content. Among them, the liquid medium with 0 g / L agar had fast nutrient supplementation and fast diffusion of metabolites, and the growth rate was the fastest.

[0069] Example 4 Basal Medium Test

[0070] This example tested the effects of MS (containing B5 vitamins) (product number: PM1031 - 50L, manufacturer Coolaber) and BT medium (product number: BTP01 - 50LT, manufacturer Caisson) on the in vitro culture of cotton ovules.

[0071] MS (containing B5 vitamins) is MS basal salts added with B5 vitamins. The formula of MS basal salts is shown in Table 1, and the content of B5 vitamins in the medium is shown in Table 2:

[0072] Table 1 MS Basal Salt Formula

[0073]

[0074] Table 2

[0075]

[0076] BT medium (hereinafter abbreviated as BT), glucose, fructose, and three hormones were used for culture, and compared with the culture using MS basal salts + B5 vitamin medium (hereinafter abbreviated as MS (B5 vitamins)). The pH of each medium was 5.8, and the specific formulas were as follows:

[0077] A. Medium No. 85: BT + glucose (18 g / L) + fructose (3.6 g / L) + NAA (0.4 mg / L) + BR (0.5 mg / L) + GA3 (0.5 mg / L);

[0078] B. Medium No. 92: BT + glucose (18 g / L) + fructose (3.6 g / L) + NAA (0.25 mg / L) + BR (0.5 mg / L) + GA3 (0.5 mg / L);

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

[0080] The 0DPA cotton ovules were inoculated into the above-mentioned culture media for dark culture at a culture temperature of 30°C for 30 days. The cotton fiber materials obtained by culturing with the BT medium showed a certain degree of browning, while the materials showed normal performance when cultured with the MS (B5 vitamin) medium and three hormones ( Figure 4 ). This result indicates that in the experiment of culturing cotton ovules to produce cotton fibers, the MS (B5 vitamin) medium used in the present invention in combination with the hormone ratio of the present invention has significantly improved compared to using the BT medium. In addition, in the present invention, the use of single sucrose has a better culture state than the experiment with the addition of glucose and fructose (see Figure 4 A, B, and C).

[0081] Example 5 Medium Hormone Test

[0082] Using MS (containing B5 vitamin) as the basal medium, different hormones were added for dark culture of 0DPA cotton ovules at a culture temperature of 30°C. The pH of each culture medium was 5.8; the specific formulations are as follows:

[0083] A. The medium was MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.25 mg / L + sucrose 30 g / L.

[0084] B. The medium was MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L, which was Medium No. 119.

[0085] C. The medium was MS + BR 0.5 mg / L + GA3 0.5 mg / L + IAA 0.25 mg / L + sucrose 30 g / L.

[0086] D. The medium was MS + BR 0.5 mg / L + GA3 0.5 mg / L + IAA 0.5 mg / L + sucrose 30 g / L.

[0087] After culturing the materials obtained from each culture medium for 30 days, they were taken out after a 5-minute boiling water bath, rinsed with clear water, the overlapping fibers were carefully separated with a needle and stretched, and the fiber length was measured with a ruler. As Figure 5 shown, Figure 5 the left figure in Figure 5The right middle figure shows the length of the fibers after carding. Among them, 30 ovules were randomly selected for each treatment to measure the fiber length, and the average value was calculated. The statistical results are shown in Table 3. Among them, the average fiber length of treatment B is 27.6 mm, which is 16.9% higher than that of treatment A (23.6 mm), 29.6% higher than that of treatment C (21.3 mm), and 26.0% higher than that of treatment D (21.9 mm). The liquid medium No. 119 in treatment B can be used as the optimal medium for in vitro culture of cotton ovules in the present invention, and the content of NAA is 0.5 mg / L.

[0088] Table 3 Fiber lengths of ovules after 30 days of culture on different hormone media

[0089]

[0090]

[0091] Example 6 In vitro culture with increased sucrose content

[0092] 1. Sucrose in vitro culture fiber embryo age test

[0093] Ovules of different embryo ages were placed on media No. 119 and 157 for dark culture at a culture temperature of 30 °C for 30 days, and different treatments were set. Among them, media No. 119 and 157 were based on MS (containing B5 vitamins), with a pH of 5.8, and the specific formulations were as follows:

[0094] Medium No. 119: MS (B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L.

[0095] Medium No. 157: MS (B5 vitamins) + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 60 g / L.

[0096] The treatment conditions of the embryo age of ovules in each group, the medium, and the sucrose content are as follows:

[0097] (A) Medium No. 119, 0 DPA; (B) Medium No. 157, 0 DPA;

[0098] (C) Medium No. 119, -1 DPA; (D) Medium No. 157, -1 DPA;

[0099] (E) Medium No. 119, -2 DPA; (F) Medium No. 157, -2 DPA.

[0100] After culture, the growth states of ovules of different embryo ages on media No. 119 and 157 treatments are as Figure 6 shown, and the fiber lengths are asFigure 7 As shown in the figure. The ovules of -1DPA and -2DPA had better growth status on the medium with 60 g / L sucrose than on the medium with 30 g / L sucrose, and there was an obvious difference in fiber length. Especially for the ovules of -1DPA, increasing the sucrose content had a very significant effect on the protrusion of cotton fibers.

[0101] 2. Electron microscopy observation

[0102] To quantify the effect of increasing sucrose on the number of fiber protrusion initiations, the ovules of -1DPA were placed on media No. 119, 156, and 157 for dark culture for one day at a culture temperature of 30 °C; the number of fiber protrusions was counted by electron microscopy observation. Among them, media No. 119, 156, and 157 were based on MS (containing B5 vitamins) as the basal medium, with a pH of 5.8, and the specific formulations were as follows:

[0103] Medium No. 119: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 g / L;

[0104] Medium No. 156: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 45 g / L;

[0105] Medium No. 157: MS + BR 0.5 mg / L + GA3 0.5 mg / L + NAA 0.5 mg / L + sucrose 60 g / L.

[0106] The fiber protrusion conditions of the ovules of -1DPA after being cultured on media No. 119, 156, and 157 for one day are as Figure 8 shown, where Figure 8 A in Figure 8 is the overall electron microscopy photo of the ovules after being cultured on Medium No. 119 for one day, Figure 8 and D in Figure 8 is the corresponding local photo; Figure 8 B in Figure 8 is the overall electron microscopy photo of the ovules after being cultured on Medium No. 156 for one day,

[0107] E in Figure 9 is the corresponding local photo;

[0108] C in Figure 8 is the overall electron microscopy photo of the ovules after being cultured on Medium No. 157 for one day, Figure 8 and F in

[0107] is the corresponding local photo. Figure 9 Randomly pick the same number of ovules in each group of treatments, take electron microscopy photos of these ovules from different angles, count the number of fiber protrusions in the same area, and then use PRISM software to analyze the data. The number of fiber protrusions observed by electron microscopy under the sucrose treatment of each medium is shown in Table 4. Use Prism software to conduct statistical analysis on the data, and the statistical results are as

[0108] Table 4

[0109]

[0110]

[0111] By observation Figure 8 it was found that the -1DPA ovules cultured on medium 157 not only increased the number of fiber protrusions but also accelerated the fiber growth rate. Medium 156 came second, and medium 119 had the slowest growth rate and the fewest fiber protrusions ( Figure 8 D-F in Figure 9 ). Statistical analysis of the data in Table 4 was performed using Prism software, and the differences among the three treatments were significant ( Figure 9 ). From the in vitro culture with exogenous addition of sucrose in the present invention, it was found that after culturing the ovules for 24 hours with the sucrose concentration increased from 30 g / L to 60 g / L, the fiber protrusions increased by 82%. As Figure 9 shown, compared with medium 119, the -1DPA ovules cultured on medium 156 and 157 with increased sucrose content significantly increased the number of fiber protrusions.

Claims

1. A method for in vitro culture of cotton ovules, wherein: The culture method comprises: Taking cotton ovules at -2DPA to 0DPA and inoculating them on a culture medium for dark culture to obtain cotton fibers; Wherein, the culture medium is MS culture medium, to which brassinosteroids, gibberellins, indoleacetic acid or naphthylacetic acid, and sucrose are added.

2. The culture method according to claim 1, wherein The culture medium is a liquid culture medium; in the culture medium, the concentration of brassinosteroid is 0.25-0.5 mg / L, the concentration of gibberellin is 0.25-0.5 mg / L, the concentration of indoleacetic acid or naphthylacetic acid is 0.25-0.5 mg / L, and the concentration of sucrose is 30-60 g / L.

3. The culture method according to claim 2, wherein The concentration of naphthylacetic acid in the culture medium is 0.5 mg / L.

4. The culture method according to claim 2, wherein: The concentration of sucrose is 45-60 g / L, more preferably 60 g / L.

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

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

8.

7. The culture method according to claim 1, wherein The dark culture is carried out for 7 to 30 days, preferably for 21 to 30 days, and more preferably for 30 days.

8. The culture method according to claim 1, wherein The temperature of the dark culture is 25-30°C, more preferably 30°C.

9. Cotton fiber obtained by the culture method according to any one of claims 1 to 8.

10. Use of the culture method according to any one of claims 1 to 8 in increasing the number of cotton ovule fiber protrusions, accelerating the growth rate of cotton ovule fibers or increasing the length of cotton ovule fibers.

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