Method for improving anther culture efficiency of rice

Abstract

The present application relates to a method for improving rice anther culture ability, and belongs to the field of plant tissue culture. Specifically, the present application uses nanocarbon materials to develop a new technology for greatly improving rice anther culture ability.

Description

Technical Field

[0001] This invention relates to a method for improving the culture capacity of rice anthers, belonging to the field of plant tissue culture. Background Technology

[0002] Rice anther culture uses tissue culture technology to inoculate rice anthers that have developed to a certain stage onto an artificial culture medium using aseptic techniques. By altering the developmental program of pollen grains within the anthers, dedifferentiation is induced, and continuous mitosis is performed to form cell clusters, which in turn form a mass of undifferentiated thin-walled tissue—callus. Subsequently, the callus is redifferentiated into embryoids or into complete rice plants.

[0003] Traditional hybridization breeding provides solid technical support for the cultivation of superior crop varieties. However, traditional breeding requires 8-10 generations of continuous self-pollination and selection to obtain relatively stable materials, which has limitations such as long breeding cycles, low efficiency, and high degree of randomness, failing to meet the needs of modern agriculture for high efficiency and precision. Anther culture technology overcomes these shortcomings, obtaining pure lines of hybrid offspring in just two generations. Combined with molecular marker selection, it significantly shortens the breeding process and improves the accuracy of selection. Furthermore, this technology is virtually irreplaceable in significantly reducing the time to homozygosity in hybrid offspring. In addition, plants obtained through anther culture undergo gene recombination, and gene mutations may be induced during the culture process, resulting in a richer variety of genetic variations in anther-cultured offspring, which is beneficial for creating germplasm materials that are difficult to obtain through other methods.

[0004] Although rice anthers are used as explants during the culture process, the developmental program of pollen grains within the anthers is actually altered, and the resulting rice plants originate from the pollen. Rice pollen can only survive outside the plant for 10-15 minutes, making rice anther culture significantly more difficult to produce seedlings compared to plant tissue culture using somatic cells as explants. While rice anther culture technology has made significant contributions to japonica rice variety breeding, it remains strictly genotype-dependent and complex to operate. In particular, the anther culture efficiency of indica rice is significantly weaker than that of japonica rice, making it difficult to meet breeding requirements. Therefore, improving the efficiency of rice anther culture is crucial for fully leveraging its technological advantages, accelerating breeding and research processes, improving research efficiency, rapidly cultivating breakthrough rice varieties with high yield, high quality, and multiple resistance traits, revitalizing the national seed industry, and ensuring national food security.

[0005] Anther culture comprises two crucial stages: callus induction and callus differentiation. Increasing both the induction and differentiation rates significantly improves anther culture efficiency. The product of the induction and differentiation rates is the anther culture power, the ultimate indicator of overall anther culture efficiency.

[0006] Many factors influence the efficiency of anther culture, with genotype being the most important. Adding a certain concentration of chemical substances (such as organic matter and hormones) to the culture medium can optimize the physiological and biochemical environment during the culture process, promoting conditions conducive to callus formation and differentiation, thereby improving culture efficiency.

[0007] Recent studies have shown that nanomaterials have an impact on callus induction, differentiation, somatic clonal variation, and cell metabolites during plant tissue culture. Therefore, many researchers have tried to apply nanomaterials to explant sterilization and plant genetic transformation.

[0008] However, there are many types of nanomaterials, and it is still unclear which nanomaterial and what method of addition can more effectively promote rice anther culture and improve the overall anther culture efficiency.

[0009] This invention aims to develop a more efficient method for rice anther culture by testing the effect of nanomaterials on the efficiency of rice anther culture. Summary of the Invention

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention provides an induction culture medium for rice anther culture, characterized by the following culture medium formula:

[0012] N6+KT 1.0mg / L+2,4-D 2.0mg / L+NAA 3.0mg / L+proline 600mg / L+maltose 50mg / L+plant gel 3.0mg / L (pH 5.8)+4mg / L~8mg / L nano carbon sol.

[0013] In some implementations, the carbon quanta in the aforementioned nano-carbon sol are 1-10 nm.

[0014] In some implementations, the above-mentioned nano-carbon sol is 4 mg / L.

[0015] The present invention also provides a differentiation culture medium for rice anther culture, characterized in that the culture medium formula is as follows:

[0016] MS + KT 2.0 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 mg / L + plant gel 3.0 mg / L (pH 5.8) + 4 mg / L ~ 8 mg / L nano carbon sol.

[0017] In some implementations, the carbon quanta in the aforementioned nano-carbon sol are 1-10 nm.

[0018] In some implementations, the above-mentioned nano-carbon sol is 4 mg / L.

[0019] The present invention also provides the application of the above-mentioned culture medium in rice anther culture.

[0020] In some implementation schemes, the rice variety mentioned above is Ejing 403.

[0021] This invention also provides a method for rice anther culture, characterized by comprising the following steps:

[0022] 1) Take young rice panicles, pretreat them at 8℃ for 8 days, and then inoculate them onto the above-mentioned induction medium. Incubate in the dark at 28℃;

[0023] 2) When the callus tissue grows to a diameter of 2-3 mm, transfer it to the differentiation medium mentioned above and culture it at 28℃ for 14h / 10h light / dark to grow seedlings.

[0024] In some implementation schemes, the rice variety mentioned above is Ejing 403.

[0025] The advantages and beneficial effects of the present invention are as follows: Tests have shown that adding 4 mg / L to 8 mg / L nano carbon sol to rice anther induction or differentiation medium can significantly improve the rice anther culture capacity. Attached Figure Description

[0026] Figure 1 Effects of different concentrations of nano-carbon sol on callus induction. The left figure shows the callus induction of Ejing 403 via anthers after adding nano-carbon sol at concentrations of 0, 4 mg / L, 6 mg / L, 8 mg / L, and 16 mg / L, respectively, with induction medium I7 as the control. The right figure shows the callus induction rate and the results of Duncan's analysis of variance after adding different nano-carbon sols.

[0027] Figure 2 The effect of different concentrations of nano-carbon sol on green seedling differentiation. The left figure shows the differentiation of callus tissue into green seedlings of Ejing 403 after adding nano-carbon sol at concentrations of 0, 4 mg / L, 6 mg / L, 8 mg / L and 16 mg / L from top to bottom, with differentiation medium F1 as the control; the right figure shows the green seedling differentiation rate and the results of Duncan's analysis of variance after adding different nano-carbon sols. Detailed Implementation

[0028] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0029] As used herein, “rice” means any rice plant and includes all plant varieties that can be bred with rice, including the whole plant, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which the plant can regenerate, plant callus, and complete plant cells in a plant or plant part, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, root, root tip, anther, etc. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0031] Example

[0032] Example 1: Selection and Testing of Nanomaterials

[0033] Nanomaterials are diverse, and existing research data shows that nanomaterials used in tissue culture or genetic transformation include nanolayered composite hydroxides (ZL2018110364500), nano-gold, magnetic nanomaterials (Nature Plants, 2017, 3: 956-964), nano-selenium, nano-silicon (ZL2012100703671), nano-titanium dioxide (ZL201510061294), and nano-carbon (Biotechnology Bulletin, 2017, 33(4): 5). Since there is no research data showing which nanomaterial should be used for rice anther culture, the inventors first tested the effects of some nanomaterials on rice anther culture.

[0034] The method for rice anther culture was carried out in accordance with publicly available technical data (e.g., Hubei Agricultural Sciences, 2018, 57(24): 164-166), specifically as follows:

[0035] Using the rice variety Ejing 403 (bred by the Institute of Food Crops, Hubei Academy of Agricultural Sciences) as the experimental material, young panicles were collected, wrapped in damp gauze, and placed in a foam box (with ice packs) before being brought back to the laboratory. After 8 days of low-temperature pretreatment at 8℃, the anthers were inoculated onto the induction medium. Each bottle contained approximately 100 anthers, with 20 bottles per treatment, and the media were incubated in the dark at 28℃.

[0036] When the callus tissue grows to a diameter of 2-3 mm, it is transferred to differentiation medium. Each treatment consists of 10 bottles, with 5 callus tissues per bottle. The culture is carried out at 28°C for 14 h / 10 h in light / dark.

[0037] During the induction phase, I7 medium was used as a control. After 40 days of culture, the number of induced callus tissues and the induction rate were counted per bottle. During the differentiation phase, F1 medium was used as a control. After 40 days of culture, the number of differentiated green seedlings per bottle and the green seedling differentiation rate were counted. The formulations of I7 and F1 mediums are as follows—

[0038] I7: N6+KT 1.0mg / L+2,4-D 2.0mg / L+NAA 3.0mg / L+proline 600mg / L+maltose 50mg / L+plant gel 3.0mg / L (pH 5.8);

[0039] F1: MS + KT 2.0 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 mg / L + plant gel 3.0 mg / L (pH 5.8).

[0040] The inventors tested three additives: loofah sap, nano-selenium, and nano-carbon. The loofah sap was added to the induction medium, while the nano-selenium and nano-carbon were added to the differentiation medium. Specific test results are shown in Tables 1-3.

[0041] Table 1. Results of the test with added loofah sap.

[0042]

[0043] Note: L1, L2, and L3 are loofah sap solutions with volume concentrations of 10%, 15%, and 20% added to I7, respectively.

[0044] Report 2: Results of the test with added nano-selenium

[0045]

[0046] Note: The differentiation medium used during the differentiation stage was F1 as a control, while the rest were cultured with different concentrations of nano-selenium solution added.

[0047] Table 3. Test results for the addition of nano-carbon

[0048]

[0049] Test results showed that adding loofah sap to the induction medium not only failed to improve anther induction and differentiation, but also reduced the final culture capacity. Adding nano-selenium and nano-carbon to the differentiation medium did improve the differentiation rate. Specifically: adding 0.1 mg / L of nano-selenium significantly increased the differentiation rate, but adding more had no effect; adding 4 mg / L and 8 mg / L of nano-carbon significantly increased the differentiation rate, but adding more had no effect.

[0050] Example 2: The promoting effect of nano-carbon on rice anther culture

[0051] According to existing research, the addition of nano-carbon generally promotes callus induction and differentiation at a concentration of 40–60 mg / L (Biotechnology Bulletin, 2017, 33(4): 5), which differs significantly from the test results obtained in this invention. To further confirm the effect of nano-carbon on rice anther culture, the inventors repeated the test experiment of Example 1 and added a 6 mg / L addition parameter between the previously tested 4 mg / L and 8 mg / L addition amounts. Simultaneously, the addition stage was extended from the differentiation stage to both the induction and differentiation stages. The specific experimental design is shown in Table 4.

[0052] Table 4. Culture medium and nano-carbon concentration used for anther culture

[0053]

[0054] C2, Cx, C3, and C4 are identical to I7 in terms of culture medium composition except for the concentration of nano-carbon sol; F12, F1x, F13, and F14 are identical to F1 in terms of culture medium composition except for the concentration of nano-carbon sol.

[0055] The callus induction rate and differentiation rate of anther tissues cultured in different culture media were statistically analyzed. The results are shown in Table 5:

[0056] 1. During the induction phase, adding a certain concentration of nano-carbon sol to I7 medium can effectively improve the callus induction rate. According to Duncan's analysis of variance, the callus induction rate was significantly different from the control after adding 4 mg / L nano-carbon sol, and the same was true after adding 6 mg / L nano-carbon. Further increasing the concentration of nano-carbon did not significantly increase the callus induction rate; in fact, excessively high concentrations inhibited callus formation and reduced the induction rate. Figure 1 ).

[0057] 2. During the differentiation stage, adding a certain concentration of nano-carbon sol to the F1 medium can effectively improve the green shoot differentiation rate of callus tissue. According to Duncan's analysis of variance, the green shoot differentiation rate was significantly different from the control after adding 4 mg / L nano-carbon sol. Adding other concentrations of nano-carbon did not produce significant differences compared to the control; in fact, excessively high concentrations inhibited callus differentiation and reduced the green shoot differentiation rate. Figure 2 ).

[0058] 3. Adding 4 mg / L of nano-carbon sol in the two aforementioned stages effectively improved callus induction rate and green seedling differentiation rate, thereby significantly enhancing the anther culture capacity of rice, with the final culture capacity being 3.57 times that of the control (highly significant). Adding 6 mg / L and 8 mg / L of nano-carbon sol also improved anther culture capacity to some extent, but the effect was not as good as that of adding 4 mg / L.

[0059] 4. Through observation, adding 4 mg / L of nano-carbon sol during the induction stage can not only improve the callus induction rate, but also accelerate the induction speed of callus, improve the state of callus, and help it to differentiate into green seedlings in the differentiation stage, increase the green seedling differentiation rate, and reduce the proportion of white seedlings.

[0060] Table 5. Culture medium and nano-carbon concentration used for anther culture

[0061]

[0062] Therefore, adding 4 mg / L to 8 mg / L of nano-carbon sol to rice anther induction or differentiation medium can significantly improve the anther culture capacity of rice.

[0063] The nano-carbon in this invention was prepared by Zhongke Daijun (Beijing) Technology Co., Ltd. The specific process is as follows: plant filter residue is dried to a moisture content of 2%-10%, then carbonized at high temperature to form carbon. The carbon is then pulverized into micron-sized carbon powder. An aqueous solution of the carbon powder is microwaved to obtain a carbon quantum (80%) solution with a size of 1-10 nm. Finally, the carbon solution is purified using a 3000 Da dialysis bag to obtain carbon quantum particles of 1-10 nm. The concentration of the nano-carbon sol stock solution is 0.4%, and 4 mg / L nano-carbon sol is obtained by diluting the stock solution 1000 times.

[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An induction culture medium for rice anther culture, characterized in that, The culture medium formula is as follows: N6 + KT 1.0 mg / L + 2,4-D 2.0 mg / L + NAA 3.0 mg / L + Proline 600 mg / L + Maltose 50 mg / L + Plant gel 3.0 mg / L pH 5.8 + 4 mg / L~8 mg / L Nano carbon sol.

2. The induction culture medium according to claim 1, characterized in that, The carbon quanta in the nano carbon sol are 1-10 nm.

3. The induction culture medium according to claim 1, characterized in that, The nano-carbon sol has a concentration of 4 mg / L.

4. A differentiation culture medium for rice anther culture, characterized in that, The culture medium formula is as follows: MS + KT 2.0 mg / L + 6-BA 0.5 mg / L + NAA 0.5 mg / L + sucrose 30 mg / L + plant gel 3.0 mg / L pH 5.8 + 4 mg / L~8 mg / L nano carbon sol.

5. The differentiation culture medium according to claim 4, characterized in that, The carbon quanta in the nano carbon sol are 1-10 nm.

6. The differentiation culture medium according to claim 4, characterized in that, The nano-carbon sol has a concentration of 4 mg / L.

7. The use of the culture medium according to any one of claims 1 to 6 in rice anther culture.

8. The application according to claim 7, characterized in that, The rice variety mentioned is Ejing 403.

9. A method for culturing rice anthers, characterized in that, Includes the following steps: 1) Take young rice panicles, pre-treat them at 8°C for 8 days, and then inoculate them onto the induction medium described in any one of claims 1 to 3, and incubate them in the dark at 28°C; 2) When the callus tissue grows to a diameter of 2-3 mm, it is transferred to the differentiation medium described in any one of claims 4 to 6 and cultured at 28°C for 14 h / 10 h in light / dark to form seedlings.

10. The method according to claim 9, characterized in that, The rice variety mentioned is Ejing 403.

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