Culture medium and culture method for increasing length of ovule fibers of sea island cotton

Abstract

The invention discloses a culture medium and a culture method for increasing the length of sea island cotton ovule fibers, and relates to the technical field of cotton culture, the key point of the technical scheme is that the culture medium is formed by adding a second component into a basic culture medium, and the second component comprises cinnamic acid; the method comprises the following steps: binding color lines for marking when cotton blooms, and recording the blooming day as 0 day; carrying out in-vitro dark culture on young bell ovules 2 days after flowering; the culture medium is prepared by adding a second component into the basic culture medium; the culture conditions are as follows: the temperature is 30-35 DEG C and the culture time is 5-35 days.

Description

Technical Field

[0001] The invention relates to the technical field of cotton culture, and more particularly to a culture medium and a culture method for increasing the fiber length of sea island cotton ovules. Background Art

[0002] Sea Island Cotton (Gossypium barbadense) and Upland Cotton (Gossypium hirsutum) are currently the most important tetraploid cultivated cotton species. The output of Sea Island Cotton accounts for about 2% of the world's total cotton production, far lower than the output of Upland Cotton, which accounts for 95% of the world's total cotton production. Upland Cotton has high yield and good adaptability, but the fiber quality of Sea Island Cotton is superior to that of Upland Cotton. It is known as the "gem of fiber". Sea Island Cotton is favored by the high-end market and has unique economic properties. It is an important raw material for high-yield and high-value-added textiles. Therefore, Sea Island Cotton has become the leader among many cotton species with its excellent fiber quality.

[0003] In vitro culture of cotton ovules belongs to plant tissue culture technology. As an important component of cotton tissue culture, it is an important way to overcome the incompatibility of distant hybridization. By culturing fibers from in vitro ovules, the differences between them and field cotton fibers can be observed indoors, providing a reference for exploring fiber-related genes and improving fiber quality.

[0004] The fiber quality of Sea Island cotton is excellent and it has evolved into a cotton variety with excellent fiber quality. It also has key differences with Upland cotton in fiber quality-related traits, making it an ideal material for studying cotton fiber development. Currently, only Upland cotton ovules are raised, and there are no reports on ovule raising of Sea Island cotton. The reason is that the existing system is not suitable for Sea Island cotton. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a culture medium for increasing the fiber length of sea island cotton ovules, wherein the culture medium is a basal culture medium with a second component added thereto, wherein the second component includes cinnamic acid.

[0006] Furthermore, the added amount of cinnamic acid is 1-10 μmol / L.

[0007] Furthermore, the second component also includes indoleacetic acid.

[0008] Furthermore, the added amount of the indoleacetic acid is: 10-50 μmol / L.

[0009] Furthermore, the basic culture medium formula is: KH2PO4 270-280mg / L, CaCl2﹒2H2O 440-445mg / L, MgSO4﹒7H2O 490-495mg / L, KNO3 5050-5060mg / L, H3BO3 5-7mg / L, Na2MoO4﹒2H2O 0.2-0.3mg / L, KI 0.75-0.95mg / L, COCl2﹒6H2O 0.02-0.03mg / L, MnSO4﹒ H2O 16-18mg / L, CuSO4﹒5H2O 0.02-0.03mg / L, FeSO4﹒7H2O 7-10mg / L, VB1 1.2-1.5mg / L, VB3 0.4-0.6mg / L, VB6 0.7-1.0mg / L, VB8 170-190mg / L, C6H 12 O6 23000-25000mg / L, GA3 4-6μmol / L, KOH 0.5-1.5μmol / L.

[0010] Furthermore, the basic culture medium formula is: KH2PO4 272mg / L, CaCl2﹒2H2O 441mg / L, MgSO4﹒7H2O 493mg / L, KNO3 5055mg / L, H3BO3 6.2mg / L, Na2MoO4﹒2H2O 0.24mg / L, KI 0.83mg / L, COCl2﹒6H2O 0.024mg / L, MnSO4﹒H2O 16.9mg / L, CuSO4﹒5H2O0.025mg / L, FeSO4﹒7H2O 8.6mg / L, VB1 1.35mg / L, VB3 0.5mg / L, VB6 0.82mg / L, VB8 180mg / L, C6H 12 O6 24000mg / L, GA3 5μmol / L, KOH 1μmol / L.

[0011] The second object of the present invention is to provide a method for increasing the fiber length of the ovule of sea island cotton, comprising the following steps:

[0012] When the cotton flowers bloom, they are marked with colored threads, and the day of flowering is recorded as day 0. The young boll ovules 2 days after flowering are taken and cultured in vitro in the dark. The culture medium is the above basic culture medium with the second component added. The culture conditions are: 30-35°C, and the culture time is 5-35 days.

[0013] In summary, the present invention has the following beneficial effects: the present invention solves the problem that the existing conventional basal culture medium cannot culture sea island cotton ovules, and the added CA can be used as a plant growth hormone. When the added concentration is 5 μmol / L, it is most suitable for the development, growth and maturation of sea island cotton ovule fibers. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The ovule phenotype of the sea island cotton of Example 1 and Comparative Examples 2-3 of the present invention;

[0015] Figure 2 The ovule phenotypes of the sea island cotton of Example 1 and Comparative Example 1 of the present invention; A: Example 1, B: Comparative Example 1;

[0016] Figure 3 The ovule phenotype of the sea island cotton of Examples 1-3 of the present invention;

[0017] Figure 4 It is the ovule phenotype of the sea island cotton of Example 1 and Example 5 of the present invention; wherein, CK: the second component of the culture medium is CA; IAA: the second component of the culture medium is CA and IAA;

[0018] Figure 5 It is a bar graph of fiber length of sea island cotton ovules in Example 1 and Example 5 of the present invention; wherein, CK: the second component of the culture medium is CA; IAA: the second component of the culture medium is CA and 30um IAA;

[0019] Figure 6 is a bar graph of fresh weight of sea island cotton germs of Examples 1 and 5 of the present invention;

[0020] Figure 7 is a bar graph of total fresh weight of sea island cotton ovules of Examples 1 and 5 of the present invention;

[0021] Figure 8 is a bar graph of the total dry weight of the sea island cotton ovules of Examples 1 and 5 of the present invention;

[0022] Fig. 9 It is a bar graph of the dry weight of the sea island cotton germs of Examples 1 and 5 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the embodiments.

[0024] The reagents used in the following examples are all commercially available.

[0025] Example 1

[0026] 1.1 The experimental material is the “Xinnongda H1” sea island cotton bred by Xinjiang Agricultural University and provided by the Department of Plant Genetics and Breeding, College of Agriculture, Xinjiang Agricultural University.

[0027] 1.2 The culture medium is a medium in which a second component is added to the basic medium. The second component is cinnamic acid (CA). The basic culture medium formula is as follows:

[0028] Table 1 is the basic culture medium formula of Example 1

[0029]

[0030] In the above culture medium, the amount of cinnamic acid (CA) added is 5 μmol / L, and the medium is sterilized after preparation for later use.

[0031] 1.3 Ovule culture:

[0032] When cotton flowers bloom, they are marked with colored threads, and the day of flowering is recorded as day 0; 2-day young bolls (young bolls 2 days after flowering) are taken for in vitro ovule culture; when collecting, at least 3-5 mm of pedicels are retained for easy dissection; the cotton bolls are quickly placed in an ice box for storage; the ovules are peeled and the in vitro ovule test is performed.

[0033] The specific steps include: first, remove the bracts and sepals of the young boll; second, disinfect the ovary with 75% alcohol for 10 minutes, then disinfect it with 0.1% HgCl2 for 10 minutes, and then rinse it with sterilized sterile water for 3 times, each time for at least 3 minutes, and dry it with sterilized filter paper each time; finally, take out the sterilized ovary under the clean bench, put it on a plate, cut the ovule ovary with a sterilized scalpel, select ovaries of the same size, and use sterilized pointed tweezers to peel the ovules to ensure that the ovules are not damaged, and inoculate them on the culture medium; each treatment is divided into 3 replicates, each replicate has 10 bottles, and each bottle has 20 ovules; finally, place it in a dark incubator at 32°C for constant temperature culture for 35 days to make it suspended culture and avoid shaking.

[0034] 1.4 Ovule phenotype determination: Determination of ovule morphology, weight and fiber length.

[0035] Example 2

[0036] The difference from Example 1 is that the added amount of cinnamic acid (CA) is 1 μmol / L.

[0037] Example 3

[0038] The difference from Example 1 is that the added amount of cinnamic acid (CA) is 10 μmol / L.

[0039] Example 4

[0040] The difference from Example 1 is that the culture medium is a medium in which a second component is added to the basal medium. The second component is cinnamic acid (CA) and indoleacetic acid. The amount of cinnamic acid (CA) added is 5 μmol / L, and the amount of indoleacetic acid added is 10 μmol / L.

[0041] Example 5

[0042] The difference from Example 1 is that the culture medium is a medium in which a second component is added to the basal medium. The second component is cinnamic acid (CA) and indoleacetic acid. The amount of cinnamic acid (CA) added is 5 μmol / L, and the amount of indoleacetic acid added is 30 μmol / L.

[0043] Example 6

[0044] The difference from Example 1 is that the culture medium is a medium in which a second component is added to the basal medium. The second component is cinnamic acid (CA) and indoleacetic acid. The amount of cinnamic acid (CA) added is 5 μmol / L, and the amount of indoleacetic acid added is 50 μmol / L.

[0045] Comparative Example 1

[0046] The difference from Example 1 is that the culture medium is a basal culture medium.

[0047] Comparative Example 2

[0048] The difference from Example 1 is that the second component is jasmonic acid (JA).

[0049] Comparative Example 3

[0050] The difference from Example 1 is that the second component is strigolactone (SL).

[0051] Tests and results

[0052] Test method:

[0053] 1) Morphological determination: With the help of a stereo microscope, 9 ovules were randomly selected each time from the 0th day to the 35th day of ovule culture. The ovules were then carefully placed on black frosted cardboard with pointed tweezers (to avoid fiber damage and incompleteness) for photographing, and the height of the stereo microscope was adjusted.

[0054] 2) Ovule weight determination: Since the fiber growth of the ovules on day 0 was not obvious on day 5, 9 ovules were randomly selected from the ovule culture for weighing every 5 days, with 3 ovules in each group, and repeated 3 times. The total dry weight, total fresh weight, embryo fresh weight and embryo dry weight of the ovules were measured respectively.

[0055] Determination of fiber fresh weight: Use sharp forceps to peel off the fiber and ovules, and weigh the fiber fresh weight of the ovules using an electronic balance with an accuracy of 1 mg. Three ovules were peeled off for each group, and the process was repeated three times.

[0056] Determination of fiber dry weight: After weighing the fresh weight of the fiber and ovules, wrap them with tin foil, mark them and put them in a drying oven for drying. Adjust the temperature to 80℃ and dry for 40 minutes. After 40 minutes, take out the fresh fiber and ovules and weigh the total fresh weight of the ovules and the fresh weight of the fiber using an electronic balance with an accuracy of 1mg.

[0057] 3) Fiber length determination: Starting from the 5th day, 9 ovules were randomly selected every 5 days and placed on a glass slide. The cotton fibers were straightened with high-pressure water and the fiber length was measured with a vernier caliper.

[0058] Table 2 shows the fiber length and weight of the sea island cotton ovules of Examples 4-6

[0059] Number of days Fiber length (mm) Total weight (mg) 0day - 0.0047 5day - 0.007733 10day 11.33 0.018 15day 14.36 0.031667 20day 21.81 0.039667 25day 22.24 0.045 30day 27.54 0.050233 35day 28.33 0.0529

[0060] Depend on Figure 1 It can be seen that the second component is added to the culture medium of Example 1 of the present invention and Comparative Examples 2-3, and the ovule fibers of the sea island cotton cultured in the culture medium with cinnamic acid grow normally and the fiber length is elongated; while the ovule fibers cultured in the culture medium with jasmonic acid and strigolactone are short and the ovule fibers hardly grow.

[0061] Depend on Figure 2 It can be seen that the basic medium, that is, the medium of Comparative Example 1, cultured the sea island cotton ovules ( Figure 2 B), when the ovules of the sea island cotton were developed for 10 days, their ovule size was much smaller than that of the ovules developed in the culture medium of Example 1, indicating that the ovules of the sea island cotton could not develop normally in the basic culture medium for conventional upland cotton; from the perspective of the fiber coverage on the ovule surface, the surface coverage rate of the sea island cotton ovules cultured in the culture medium of Example 1 was almost half of that of the ovules, and the sea island cotton ovules cultured in the conventional basic culture medium had very little surface fiber.

[0062] Depend on Figure 3 It can be seen that in the culture medium with different concentrations of CA added, the ovules of Sea Island Cotton can grow normally and the fiber length increases, but the concentration of 5 μmol / L CA is most suitable for the growth of Sea Island Cotton ovules. The present invention finds that low concentrations of CA have a promoting effect on ovule fiber growth, and the SOD of the ovules with corresponding low concentrations of CA is significantly increased compared with the control group comparative example 1, and its POD expression is also higher than the control group comparative example 1, indicating that CA has the performance of low promotion and high inhibition in the development of Sea Island Cotton ovules.

[0063] Depend on Figure 4It can be seen that by adding cinnamic acid CA, cinnamic acid CA and indoleacetic acid IAA to the culture medium respectively, the ovules of sea island cotton can grow normally and the fiber length increases; indoleacetic acid IAA is a known essential hormone for plant growth and plays an important role in fiber development; studies have shown that cinnamic acid is a new type of natural auxin efflux inhibitor that can promote lateral root growth, but there is no precedent for its application in fiber growth; by Figure 4 It can be seen that adding cinnamic acid CA to the basal culture medium has the same effect as adding indoleacetic acid IAA to the basal culture medium, that is, both can make the sea island cotton ovule fiber grow and extend, and the combined use of cinnamic acid CA and indoleacetic acid IAA has a better effect than adding only cinnamic acid CA, and the use of the two has the advantage of synergistic enhancement.

[0064] Depend on Figure 5-9 It can be seen that by adding cinnamic acid CA, cinnamic acid CA and indoleacetic acid IAA to the culture medium respectively, the ovules of sea island cotton can grow normally in them, the fiber length increases with the culture time, the embryo fresh weight, the total fresh weight of the ovule, the total dry weight of the ovule, and the embryo dry weight all increase with the culture time and tend to be stable after 25 days. Adding cinnamic acid CA and indoleacetic acid IAA to the basic culture medium can promote fiber growth and weight gain.

[0065] The invention solves the problem that the existing conventional basic culture medium cannot culture the sea island cotton ovules by adding the second component in the culture medium. The added cinnamic acid CA can be used as a plant growth hormone. When the added concentration is 5 μmol / L, it is most suitable for the development, growth and maturation of the sea island cotton ovule fibers.

[0066] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A culture medium for increasing the fiber length of sea island cotton ovules, characterized in that: The culture medium is prepared by adding a second component to a basic culture medium, wherein the second component includes cinnamic acid.

2. A culture medium for increasing the fiber length of sea island cotton ovules according to claim 1, characterized in that: The added amount of the cinnamic acid is 1-10 μmol / L.

3. A culture medium for increasing the fiber length of sea island cotton ovules according to claim 1, characterized in that: The second component also includes indoleacetic acid.

4. A culture medium for increasing the fiber length of sea island cotton ovules according to claim 1, characterized in that: The added amount of the indoleacetic acid is: 10-50 μmol / L.

5. A culture medium for increasing the fiber length of sea island cotton ovules according to claim 1, characterized in that: The basic culture medium formula is: KH2PO4 270-280mg / L, CaCl2﹒2H2O 440-445mg / L, MgSO4﹒7H2O 490-495mg / L, KNO3 5050-5060mg / L, H3BO3 5-7mg / L, Na2MoO4﹒2H2O0.2-0.3mg / L, KI 0.75-0.95mg / L, COCl2﹒6H2O 0.02-0.03mg / L, MnSO4﹒ H2O 16-18mg / L, CuSO4﹒5H2O 0.02-0.03mg / L, FeSO4﹒7H2O7-10mg / L, VB1 1.2-1.5mg / L, VB3 0.4-0.6mg / L, VB6 0.7-1.0mg / L, VB8 170-190mg / L, C6H 12 O623000-25000mg / L, GA3 4-6μmol / L, KOH 0.5-1.5μmol / L.

6. A culture medium for increasing the fiber length of sea island cotton ovules according to claim 1, characterized in that: The basic culture medium formula is: KH2PO4 272mg / L, CaCl2﹒2H2O 441mg / L, MgSO4﹒7H2O 493mg / L, KNO35055mg / L, H3BO3 6.2mg / L, Na2MoO4﹒2H2O 0.24mg / L, KI 0.83mg / L, COCl2﹒6H2O 0.024mg / L, MnSO4﹒H2O 16.9mg / L, CuSO4﹒5H2O0.025mg / L, FeSO4﹒7H2O 8.6mg / L, VB1 1.35mg / L, VB3 0.5mg / L, VB6 0.82mg / L, VB8 180mg / L, C6H 12 O6 24000mg / L, GA3 5μmol / L, KOH 1μmol / L.

7. A method for increasing the fiber length of sea island cotton ovules, characterized in that: The following steps are involved: When cotton flowers bloom, they are marked with colored threads, and the day of flowering is recorded as day 0. The young boll ovules are taken 2 days after flowering and separated from the in vitro dark Cultivation; the culture medium is a culture medium as described in any one of claims 1-6; the culture conditions are: 30-35°C, Cultivation time: 5-35 days.

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