Culture medium for improving wheat anther culture ability and application of culture medium in wheat breeding

Abstract

The invention belongs to the technical field of wheat breeding, and particularly relates to a culture medium for improving wheat anther culture capacity and application of the culture medium to wheat breeding. The invention provides a culture medium for improving wheat anther culture ability. The culture medium is prepared by adding saccharides and Arabic gum into a W14 culture medium, the culture medium suitable for various wheat varieties is obtained through cooperation of the Arabic gum and the carbon source, the culture medium is used for wheat anther culture, the callus induction rate is increased by 4.8 times, and the callus induction rates of the four wheat varieties all reach 50% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat breeding, and in particular relates to a culture medium for improving the culture ability of wheat anthers and an application thereof in wheat breeding. Background Art

[0002] The traditional method of wheat breeding is to breed varieties based on several rounds of hybridization, selfing and hybrid selection, and this process takes 8 to 10 years. Therefore, accelerating the wheat breeding process is an important direction of contemporary wheat breeding research, and anther in vitro culture can meet this demand. In addition to shortening the breeding period, the most important advantage of wheat anther culture is that it will produce spontaneous chromosome doubling, thereby producing a homozygous double haploid system. Although many advances have been made in wheat anther in vitro culture technology, it still faces some challenges in practical application, including genotype dependence and low callus induction rate. The basic culture medium for wheat anther in vitro culture has gone through potatoes, N6, MS, W14, C17, K3, K (decene culture medium), etc. At present, N6, W14, C17 and other culture media are the most widely used basic culture media in wheat anther in vitro culture. However, due to the influence of genotype, these culture media cannot be adapted to all wheat materials.

[0003] Improving the callus induction rate of wheat anthers is the key to wheat anther culture technology, and optimization of the culture medium is one of the strategies to improve the callus induction rate. Adding additives to the culture medium can greatly improve the efficiency of wheat anther culture. The most common and widely used additives include plant hormones, amino acids, polysaccharides, and plant organs and tissues, and their effects on callus induction rate have been widely confirmed.

[0004] Gum arabic is extracted from Robinia pseudoacacia, and its main components are arabinogalactan (AG) and arabinogalactan protein (AGP). Arabinogalactan protein is a highly glycosylated hydroxyproline-rich glycoprotein that is widely distributed in the cell walls and plasma membranes of plants and plays an important role in multiple processes of plant growth and development. Arabinogalactan protein can be detected in the floral organs of plants, including anthers, pollen tubes, ovaries, and ovules. In addition, Arabinogalactan protein also exists in the secretion fluid of plant roots in the form of water-soluble macromolecules and appears in the culture medium of in vitro suspended cells. Arabinogalactan protein can participate in programmed cell death during Arabidopsis cell culture. In wheat microspore culture, the addition of arabinogalactose can significantly reduce the mortality of microspores; the addition of gum arabic can increase the survival rate of microspores and the yield of microspore embryos (Jocelyne Letarte etc. Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture. Cell Biology and Morphogenesis Volume 24, pages 691-698, (2006)). Summary of the invention

[0005] In view of the poor applicability of the culture medium for wheat anther culture at this stage, the present application provides a culture medium for improving the anther culture ability of wheat. By combining gum arabic with a carbon source, a culture medium suitable for various varieties of wheat is obtained. When used for wheat anther culture, the callus induction rate is increased by 4.8 times, and the callus induction rates of four wheat varieties all reach more than 50%.

[0006] The technical solution of this application is as follows:

[0007] In a first aspect, the present application provides a culture medium for improving the culturing ability of wheat anthers, wherein sugars and gum arabic are added to W14 culture medium.

[0008] Arabinogalactoprotein (AGP) is one of the main components of gum arabic. Many of the functions of AGP in growth were suggested using the Yariv reagent, the addition of which inhibited the growth of pollen tubes (Roy, Stéphane, et al. "Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube." Planta 204 (1998): 450-458.), somatic embryos and root tissues (Willats, William GT, and J. Paul Knox. "A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of β-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana." The Plant Journal 9.6 (1996): 919-925; Ding, Lei, and Jian-Kang Zhu. "A role for arabinogalactan-proteins in root epidermal cell expansion." Planta 203 (1997): 289-294.). This inhibitory effect should be related to the expansion and proliferation of cells. In plants, 4-hydroxy-3-methoxycinnamic acid combines with polysaccharides to produce a bridge that aggregates polysaccharides through oxidation, thereby limiting the expansion of the cell wall and giving the cell wall rigidity. The binding mode of 4-hydroxy-3-methoxycinnamic acid with polysaccharides is similar to the structure of Yariv reagent. AGPs may compete with polysaccharides to bind 4-hydroxy-3-methoxycinnamic acid, thereby preventing the formation of bridge structures, resulting in reduced rigidity of the cell wall, thereby promoting cell expansion and proliferation.

[0009] Maltose produces 2 molecules of glucose after hydrolysis, while sucrose produces one part of glucose and one part of fructose after hydrolysis. Glucose has a smaller molecular weight and may be more easily absorbed by plant cells to form polysaccharides, which combine with AGP to promote cell expansion and growth.

[0010] According to some embodiments of the present application, the sugar is sucrose and / or maltose; the added amount of the sugar is: 60-100 g / L.

[0011] According to some preferred embodiments of the present application, the amount of gum arabic added is: 300-00 mg / L.

[0012] According to some preferred embodiments of the present application, the mass ratio of the sugar to gum arabic is 180-220:1.

[0013] The second aspect of the present application provides a method for improving the culturing capacity of wheat anthers, using the above-mentioned culture medium for improving the culturing capacity of wheat anthers.

[0014] According to some embodiments of the present application, a cultivation method for improving the culturability of wheat anthers comprises the following steps:

[0015] (1) Preparation and sterilization of culture medium;

[0016] (2) Cultivation after inoculation: Cultivate at room temperature for 40-55 days.

[0017] According to some preferred embodiments of the present application, the sterilization conditions in step (1) are high pressure sterilization at 100-200° C. for 5-60 minutes;

[0018] And / or, step (2) pre-treating the seeds before inoculation; preferably, the pre-treatment process is to remove the young ears and sterilize with ethanol;

[0019] And / or, the inoculation amount in step (2) is: 1-4 ears / 15 mL.

[0020] The third aspect of the present application is to protect the use of maltose and gum arabic in a culture medium for improving the culturing ability of wheat anthers.

[0021] The fourth aspect of the present application is to protect the application of maltose and gum arabic in wheat breeding.

[0022] According to some embodiments of the present application, the mass ratio of maltose to gum arabic is 180-220:1.

[0023] Beneficial effects of the present invention

[0024] In the culture medium with maltose added, the callus induction rate of Jimai 38 in culture medium 3-1 was the highest, which was 54.5%, 5.5 times higher than that of the control, followed by 12% in culture medium 2-1, 1.2 times higher than that of the control; the callus induction rate of Jimai 4075 in culture medium 3-1 was the highest, which was 13.5%, 9 times higher than that of the control ( Figure 1 ); the callus induction rate of Jimai 22 in medium 3-1 was the highest, which was 4.5%, followed by medium 4-1, with a callus induction rate of 2.0%, and no callus was produced in the control medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The diagrams are callus images of Jimai 4075 grown in various culture media;

[0026] Figure 2 Callus induction rates of three wheat materials in maltose and sucrose medium. DETAILED DESCRIPTION

[0027] The wheat varieties used in this application include: Jimai 38, Jimai 4075, Jimai 22, Jimai 262, Jimai 4072, Kenong 199 and Yannong 24.

[0028] Configuration of W14 medium: potassium nitrate, 2000 mg; potassium dihydrogen phosphate 380 mg, magnesium sulfate heptahydrate,

[0029] 200mg; calcium chloride dihydrate 140mg, potassium sulfate 700mg; manganese sulfate monohydrate 8mg, zinc sulfate heptahydrate 3mg;

[0030] Boric acid 3mg; potassium iodide 0.5mg; copper sulfate pentahydrate 0.025mg; cobalt chloride hexahydrate 0.025mg; sodium molybdate 0.005mg; ferrous sulfate heptahydrate 27.8mg; Na2EDTA.2H2O 37.3mg; glycine 2mg; vitamin B12mg; vitamin B60.5mg; niacin 0.5mg; 2,4-D 2mg; KT 250ul; add water to make up to 1000mL, adjust the pH to 5.8, and then add Ficoll 100g.

[0031] Example 1

[0032] A method for improving the culture ability of wheat anthers, characterized in that it comprises the following steps:

[0033] (1) Preparation and sterilization of culture medium: Add 80 g maltose to W14 culture medium, add 0 mg (control), 200 mg, 400 mg, 600 mg, 800 mg of gum arabic, and name them as culture medium 1-1, culture medium 2-1, culture medium 3-1, culture medium 4-1 and culture medium 5-1 respectively. Add 80 g sucrose to W14 culture medium, add 0 mg (control), 200 mg, 400 mg, 600 mg, 800 mg of gum arabic, and name them as culture medium 1-2, culture medium 2-2, culture medium 3-2, culture medium 4-2 and culture medium 5-2 respectively. The culture medium needs to be sterilized by high pressure at 120°C for 20 minutes. Cool and set aside.

[0034] (2) Post-inoculation culture: Before inoculation, peel off the young ears, sterilize with 75% ethanol for 30 seconds, and rinse with sterile distilled water in a clean bench until there is no foam. Use tweezers to take out about 50 anthers (2 ears) and place them in a culture dish containing 15 mL of induction medium. Seal the culture dish with sealing film, place it in a 30°C incubator for dark culture for 3 days, and then transfer it to a 28°C incubator for continued dark culture for 40 to 50 days. Inoculate 4 dishes for each material, and repeat 3 times.

[0035] Example 2

[0036] A method for improving the culture ability of wheat anthers, characterized in that it comprises the following steps:

[0037] (1) Preparation and sterilization of culture medium: Add 80 g sucrose to W14 culture medium, add 0 mg (control), 200 mg, 400 mg, 600 mg, 800 mg of gum arabic, and name them as culture medium 1-1, culture medium 2-1, culture medium 3-1, culture medium 4-1 and culture medium 5-1 respectively. Add 80 g sucrose to W14 culture medium, add 0 mg (control), 200 mg, 400 mg, 600 mg, 800 mg of gum arabic, and name them as culture medium 1-2, culture medium 2-2, culture medium 3-2, culture medium 4-2 and culture medium 5-2 respectively. The culture medium needs to be sterilized by high pressure at 120°C for 20 minutes. Cool and set aside.

[0038] (2) Post-inoculation culture: Before inoculation, peel off the young ears, sterilize with 75% ethanol for 30 seconds, and rinse with sterile distilled water in a clean bench until there is no foam. Use tweezers to take out about 50 anthers (2 ears) and place them in a culture dish containing 15 mL of induction medium. Seal the culture dish with sealing film, place it in a 30°C incubator for dark culture for 3 days, and then transfer it to a 28°C incubator for continued dark culture for 40 to 50 days. Inoculate 4 dishes for each material, and repeat 3 times.

[0039] Implementation effect example

[0040] In the control medium of sucrose and maltose, there was no significant difference in the callus induction rate of the three wheat materials.

[0041] However, when gum arabic was added, the callus induction rate of the medium containing maltose was higher than that of the medium containing sucrose with the highest gum arabic concentration ( Figure 2 ).

[0042] The callus induction rates of three wheat materials and 10 culture media were different. Among the culture media with maltose added, the callus induction rate of Jimai 38 in culture medium 3-1 was the highest, 54.5%, 5.5 times higher than that of the control, followed by 12% in culture medium 2-1, 1.2 times higher than that of the control; the callus induction rate of Jimai 4075 in culture medium 3-1 was the highest, 13.5%, 9 times higher than that of the control ( Figure 1 ); the callus induction rate of Jimai 22 in medium 3-1 was the highest, which was 4.5%, followed by medium 4-1, with a callus induction rate of 2.0%, and no callus was produced in the control medium.

[0043] In the culture medium with sucrose added, the callus induction rate of Jimai 38 in culture medium 3-2 was the highest, which was 38.5%, 4.7 times higher than that of the control, followed by 10% in culture medium 4-2, 1.2 times higher than that of the control; the callus induction rate of Jimai 4075 in culture medium 3-2 was the highest, which was 10.5%, 6.1 times higher than that of the control; the callus induction rate of Jimai 22 in culture medium 3-2 was the highest, which was 2.0%, and no callus was produced in other culture media.

[0044] Table 1 Statistics of callus induction rate of wheat anthers treated with different concentrations of gum arabic (containing maltose)

[0045]

[0046] Table 2 Statistics of callus induction rate of wheat anthers treated with different concentrations of gum arabic (containing sucrose)

[0047]

[0048]

[0049] In summary, for the three wheat materials tested, the callus induction rates in medium 3-1 and 3-2 were higher than those in the control, but the overall callus rate in the medium with maltose added was higher than that in the medium with sucrose ( Figure 2 ). Therefore, adding 400 mg / L gum arabic to W14 containing maltose can increase the induction rate of wheat anther callus.

[0050] In view of this, we selected four materials, Jimai 262, Jimai 4072, Kenong 199 and Yannong 24, and added 400.0 mg / L of gum arabic to W14 containing maltose. The results showed that the callus induction rates were increased to 62.1%, 68.5%, 59.8% and 71.2%, respectively. In particular, the callus induction rate of Yannong 24 increased by 4.8 times. The callus induction rates of the four wheat varieties all reached more than 50%.

[0051] Table 3 Induction rate of wheat anther callus of four wheat varieties treated with gum arabic (containing maltose)

[0052]

Claims

1. A culture medium for improving the culturing ability of wheat anthers, characterized in that: The culture medium is prepared by adding sugars and gum arabic to W14 culture medium.

2. The culture medium for improving wheat anther culturing ability according to claim 1, characterized in that: The sugar is sucrose and / or maltose; the added amount of the sugar is 60-100 g / L.

3. The culture medium for improving wheat anther culturing ability according to claim 1 or 2, characterized in that: The added amount of the gum arabic is: 300-00 mg / L.

4. The culture medium for improving wheat anther culturing ability according to any one of claims 1 to 3, characterized in that: The mass ratio of the sugar to gum arabic is 180-220:

1.

5. A method for improving the culture ability of wheat anthers, characterized in that: A culture medium for improving the anther culturing ability of wheat as described in any one of claims 1 to 4 is used.

6. The method for improving the culture ability of wheat anthers according to claim 5, characterized in that: The following steps are involved: (1) Preparation and sterilization of culture medium; (2) Cultivation after inoculation: Cultivate at room temperature for 40-55 days.

7. The culture method according to claim 6, characterized in that: The sterilization conditions in step (1) are high pressure sterilization at 100-200° C. for 5-60 minutes; And / or, step (2) pre-treating the seeds before inoculation; preferably, the pre-treatment process is to remove the young ears and sterilize with ethanol; And / or, the inoculation amount in step (2) is: 1-4 ears / 15 mL.

8. Application of maltose and gum arabic in culture medium for improving wheat anther culturing capacity.

9. Application of maltose and gum arabic in wheat breeding.

10. The use according to claim 8 or 9, characterized in that: The mass ratio of maltose to gum arabic is 180-220:1.

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