Method for improving expression of anthocyanin in maize young embryo and application

Abstract

This invention provides a method and application for enhancing anthocyanin expression in maize immature embryos, belonging to the field of crop genetics and breeding technology. By adding specific concentrations of naringenin and hesperidin to the basal culture medium for haploid embryo identification, this invention significantly enhances anthocyanin accumulation and color intensity in immature embryos obtained from hybridization of haploid inducible lines. This effectively overcomes existing limitations. R1-nj The marker system significantly improves the accuracy of haploid identification, overcoming the problems of weak color development and difficulty in identification caused by germplasm background and environmental factors. It also has the advantages of strong universality, simple operation, and non-GMO nature, making it suitable for large-scale maize haploid breeding practices.

Description

Technical Field

[0001] This invention relates to the field of crop genetics and breeding technology, and in particular to a method and application for improving anthocyanin expression in maize immature embryos. Background Technology

[0002] Haploid breeding (DH) technology in maize can significantly improve breeding efficiency and shorten the breeding cycle, and has become an important maize breeding technology both domestically and internationally. In maize haploid breeding, since only about 10% of haploids are induced by hybridization, the remaining diploids have little practical application value in breeding. Therefore, haploid identification is a crucial step in DH breeding. Currently, the most basic method for haploid identification is morphological identification, but this method requires large-scale field planting, is costly, and time-consuming. Root tip squash is the most accurate method for counting haploid chromosomes, but it is complex to operate and slow in identification. Other methods include fluorescence in situ hybridization, flow cytometry, and molecular marker methods. While these methods offer high accuracy, they suffer from drawbacks such as complex operation, high cost, and inability to achieve early identification, thus failing to meet the needs of commercial breeding.

[0003] Identifying maize haploids using pigment labeling systems is simpler and more efficient, for example, based on... R1-nj The systematic labeling method produces seeds with purple embryos and aleurone layers. When crossed with maize seeds lacking pigment labeling, the resulting haploid kernels have a purple label on the aleurone layer but a colorless embryo. Diploid kernels have both a purple label on the aleurone layer and a purple embryo. This allows for direct identification of haploids with high accuracy. Identifying and doubling maize haploid embryos through tissue culture offers advantages such as high doubling efficiency and the ability to further shorten the breeding cycle, making it a primary technique in modern commercial maize haploid breeding. Haploid embryo identification requires... R1-nj Marked, but R1-nj Color intensity depends on anthocyanin expression and is influenced not only by the light, temperature, and climate conditions of the growing environment but also by germplasm background. Some maize germplasms, especially about 30% of tropical resources, possess... C1-I and C2-idf Genes that inhibit anthocyanin synthesis suppress anthocyanin synthesis during the embryo and grain stages, making accurate identification of haploids impossible and limiting the utilization of this germplasm resource. Studies have revealed the impact of... R1-nj The main factors for color development in the embryonic stage are light intensity and culture medium composition. Increasing light intensity and sucrose content in the culture medium can enhance color development intensity in embryonic embryos and has been applied in actual production. However, it still cannot solve the problem of color development difficulties in some germplasms. Currently, there are several methods to improve the color development intensity of pigment markers or overcome the difficulties. C1-I Genes that hinder haploid identification. One approach is to cultivate haploid inducible lines with enhanced pigment synthesis, such as those possessing both... R1-njInducing lines for grain pigments, red roots, purple leaf sheaths, and purple stems, or the tissue culture-specific inducing line CS2 which can enhance the color intensity of tissue-cultured embryos, can improve the accuracy of haploid identification. However, the cultivation cycle of these inducing lines is long, and there are issues with the ecological adaptability of varieties, limiting their widespread application. Secondly, there are oil and near-infrared haploid identification systems developed based on differences in oil content or near-infrared transmission spectra between haploid and diploid embryos. This method does not rely on anthocyanin labeling, achieves an accuracy rate of over 90%, and the accompanying equipment can enable high-throughput haploid grain identification. However, this method has limitations, being limited to grain identification, and requiring significant differences in oil content between the hybrid parents. With the development of transgenic technology, haploid identification systems based on genetic engineering have been successively developed and utilized. One approach utilizes embryo-specific bidirectional expression promoters to drive transcription factors that regulate anthocyanin synthesis. ZmC1 and ZmR2 This significantly enhanced anthocyanin synthesis in maize embryos; the inducible line with this enhancement marker showed a haploid identification accuracy exceeding 97%. The betaine synthesis system... RUBY By introducing an inducible line, betalains accumulate significantly in hybrid embryos, enabling direct identification of haploids with an accuracy rate of up to 100%. Although genetic engineering methods can overcome the colorimetric challenge, they are still not practically applicable due to regulations on transgenic organisms.

[0004] Anthocyanins are secondary plant metabolites of flavonoids. In higher plants, they mainly include six forms: pelargonidin, cyanidin, delphinidin, paeoniflorin, iridoside, and malvidin. Its biosynthetic pathway is mainly divided into three stages. In the first stage, phenylalanine is gradually converted into 4-coumaroyl-CoA by phenylalanine ammonia-lyase, cinnamic acid hydroxylase, and coumaroyl-CoA ligase. In the second stage, 4-coumaroyl-CoA is gradually synthesized into chalcone, naringenin, and dihydrokaempferol under the action of chalcone synthase (CHS), chalcone isomerase (CHI), and flavanon-3-hydroxylase (F3H). Hesperidin is then catalyzed into dihydroquercetin and dihydromyricetin by flavanon 3'-hydroxylase (F3'H) and flavanon 3'5'-hydroxylase (F3'5'H), respectively. The synthesis of chalcone and naringenin are the key rate-limiting steps. In the third stage, hesperidin, dihydroquercetin, and dihydromyricetin are progressively synthesized into various anthocyanin glucosides and anthocyanins under the action of key catalytic enzymes such as dihydroflavonol reductase. Naringenin is the first flavanone backbone molecule formed in the main anthocyanin biosynthetic pathway, and its metabolic flow directly determines the initial flux of anthocyanin synthesis. Hesperidin occupies a secondary position and is also crucial in the anthocyanin biosynthetic network. Structural genes involved in anthocyanin synthesis directly regulate anthocyanin formation, while differences in anthocyanin accumulation mechanisms are regulated by transcription factors. Currently, precise regulation of anthocyanin synthesis in maize requires further research for precision breeding of maize. For example, based on... R1-nj The late accumulation and low content of anthocyanins in haploid-induced embryos affect the accuracy of haploid embryo identification. Precise regulation of anthocyanin synthesis and improved anthocyanin expression in the embryo can enhance the accuracy of haploid embryo identification and shorten the time required for this step. This will further optimize the anthocyanin-based color labeling haploid breeding technology system, improving its application effect and reliability. Therefore, this application uses naringenin and hesperidin, intermediate products in the anthocyanin biosynthesis pathway, as key components of the embryo culture medium, directly as substrates to regulate anthocyanin synthesis in embryos, thereby enhancing the color intensity of the embryos. Summary of the Invention

[0005] The purpose of this invention is to provide an application of naringenin and hesperidin in improving the color intensity of maize hybrid embryo identification. By optimizing the key components of the embryo culture medium and directly supplementing with naringenin and hesperidin, the color intensity of anthocyanins in the embryo is enhanced, thereby improving the accuracy and efficiency of haploid identification.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of naringenin and hesperidin in improving the color intensity of maize hybrid embryo identification.

[0007] Preferably, when the concentration of naringenin is 14-18 mg / L and the concentration of hesperidin is 1.4-1.8 mg / L, the color intensity of maize hybrid embryos with sweet corn, waxy corn and common corn as the female parent and haploid inducing line as the male parent can be improved. The sweet corn includes Ketian 8, Ganketian 10 or Guangliangtian 27; the waxy corn includes Wannuo 2000, Wannongtiannuo 158 or Zhenuoyu 14; and the common corn includes Zhengdan 958, Demeya 3 or Dongdan 119; the haploid induction lines include CS2, CAU5, CAU6, CAU7 or CHOI4.

[0008] Preferably, when the haploid inducible line CS2 is the parent: When the concentration of naringenin is 6-10 mg / L or the concentration of hesperidin is 0.6-1.0 mg / L, it can improve the color intensity of the hybrid embryos with Wannong Tiannuo 158 as the female parent. When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8, Wannong Tiannuo 158, Zhe Nuoyu 14 or Demeya 3 as the female parent can be improved. When the concentration of naringenin is 14-26 mg / L or the concentration of hesperidin is 1.4-2.6 mg / L, it can improve the color intensity of the hybrid embryos with Dongdan 119 as the female parent. When the concentration of naringenin is 22-26 mg / L and the concentration of hesperidin is 2.2-2.6 mg / L, the color intensity of the hybrid embryos with Ketian 8 and Dongdan 119 as the female parent can be improved.

[0009] Preferably, when the haploid induction line CAU5 is used as the male parent, the color intensity of the hybrid embryos with naringenin concentration of 6-10 mg / L and / or hesperidin concentration of 0.6-1.0 mg / L can be improved.

[0010] Preferably, when the haploid inducible line CAU6 is the male parent: When the concentration of naringenin is 6-10 mg / L, it can improve the color intensity of hybrid embryos with Ketian 8 as the female parent; When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8, Wannuo 2000 or Demeya 3 as the female parent can be improved.

[0011] Preferably, when the haploid inducible line CAU7 is the male parent: When the concentration of naringenin is 6-10 mg / L, it can improve the color intensity of hybrid embryos with Demeya 3 as the female parent; When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8 or Demeya 3 as the female parent can be improved.

[0012] Preferably, when the haploid induction line CHOI4 is the male parent: When the concentration of naringenin is 6-10 mg / L and / or the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Wannuo 2000 or Demeya 3 as the female parent can be improved. When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of the hybrid embryos with Ketian 8 as the female parent can be improved.

[0013] This invention also provides a method for improving the color intensity in haploid identification of maize immature embryos using naringenin and hesperidin. The maize variety to be tested is used as the female parent and the inducing line is used as the male parent for hybridization. The immature embryos are harvested 15-17 days after hybridization and pollination. Depending on the male and female parent varieties, they are cultured in MS medium with naringenin and hesperidin concentrations specified in the above application. The MS medium was supplemented with a maize haploid doubling agent consisting of 0.05-0.4 g colchicine, 20 mL dimethyl sulfoxide, and 100-2000 mg calcium ions per L. The culture time is 22-26 h, and the light intensity during culture is 8000-12000 Lux.

[0014] The present invention also provides a culture medium for the above-described method, wherein the concentration of naringenin in the culture medium is 6-26 mg / L and the concentration of hesperidin in the culture medium is 0.6-2.6 mg / L.

[0015] The present invention also provides the application of the method or the culture medium described herein in increasing the anthocyanin content of maize hybrid embryos.

[0016] Beneficial effects

[0017] This invention significantly enhances anthocyanin accumulation and color intensity in hybrid embryos of maize materials with different genetic backgrounds by directly supplementing key anthocyanin synthesis precursors naringenin and hesperidin. Experiments show that the culture medium of this invention can significantly improve the accuracy of haploid identification. This invention exhibits good color enhancement effects on different types of maize (sweet, waxy, and common maize) and hybrid combinations with different inducible lines, breaking through the limitations of germplasm background on color development. R1-nj Limitations of colorimetric markers. This invention not only significantly enhances the colorimetric intensity of immature embryos and optimizes the identification technology of maize haploid immature embryos based on anthocyanin color markers, but also abandons traditional approaches that rely on long-cycle induction line cultivation or regulatory-restricted genetic engineering. It innovatively focuses on the targeted optimization of immature embryo culture medium components to improve identification efficiency, providing a safe, efficient, and easily integrated new technical approach to address technical challenges such as strong dependence on germplasm background and high environmental sensitivity, thus offering more possibilities for maize breeding. Attached Figure Description

[0018] Figure 1 This is a graph showing the results of the color development evaluation of maize hybrid embryos in Example 1; Figure 2 This is a diagram showing the enhancement effect of naringin and hesperidin on the color development of multi-background germplasm embryos in Example 2. Figure 3 The image shows the color intensity results of the hybrid embryos of Zheketian 8, Wannongtiannuo 158 and Dongdan 119 with CS2 in different culture media in Example 2. Figure 4 This is a graph showing the color enhancement effect of different inducing systems in Example 3; Figure 5 This is a graph showing the enhancement effect of different doses of naringenin and hesperidin on the color development of immature embryos in Example 4; Figure 6 The images show the results of the color enhancement effect of the naringin and hesperidin dual-factor culture medium on the inbred line G605 in Example 5, as well as the results of the detection of anthocyanin and flavonoid content. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] Forty-five sweet corn, 38 waxy corn, and 15 common corn hybrids were selected as test female parents. These varieties included representative regional varieties, nationally approved varieties, and some imported varieties, demonstrating broad regional representativeness. The tissue culture-specific induction line CS2 (derived from the team of Chen Shaojiang at China Agricultural University, exhibiting strong embryo color development) was selected as the male parent. Sixteen days after hybridization and pollination, embryos were harvested and placed on haploid tissue culture MS medium (supplemented with a maize haploid doubling agent consisting of 0.1 g colchicine, 20 mL dimethyl sulfoxide, and 500 mg calcium ions) and cultured in vitro at 10000 Lux for 24 h. The color development was determined according to the color development grade standard (…). Figure 1 a) Evaluation of embryo color development. The results showed that the color development intensity ranges of the embryos in sweet corn, waxy corn, and common corn populations were 1.18–3.73, 1.45–3.08, and 1.92–3.77, respectively, with average values ​​of 1.97, 2.15, and 2.68, respectively. Figure 1 b). There was no significant difference in embryo color intensity between sweet corn and waxy corn populations, but both were significantly lower than those of common corn. Analysis of embryo color distribution showed that the proportions of embryo color intensities below 2 in sweet corn, waxy corn, and common corn populations were 57.8%, 42.1%, and 6.7%, respectively. The existence of varieties with poor and significantly inhibited embryo color development in fresh corn (sweet corn and waxy corn) leads to low haploid identification efficiency. Figure 1 (b, c, d). To solve the problem of color development in maize germplasm embryos, it is necessary to optimize and upgrade the existing haploid embryo identification technology system.

[0022] Example 2

[0023] The synthesis pathways of anthocyanins in maize were analyzed, and key intermediate naringenin and rate-limiting step product hesperidin were screened as candidate enhancing factors. Both are flavonol compounds. The colorimetric effects of single-factor and multi-factor combinations were studied using naringenin at 8 mg / L (1N) and hesperidin at 0.8 mg / L (1D) as the basic concentration.

[0024] Sweet corn varieties Ketian 8, Ganketian 10, and Guangliangtian 27; waxy corn varieties Wannuo 2000, Wannongtiannuo 158, and Zhenuoyu 14; and common corn varieties Zhengdan 958, Demeiya 3, and Dongdan 119 were selected as the female parent for hybridization testing, with the haploid induction line CS2 as the male parent. Sixteen days after pollination, the embryos were cultured in vitro for 24 hours in different component media to evaluate the color development effect. The results showed that, except for Wannongtiannuo 158, adding a certain concentration of naringin or hesperidin alone could not significantly improve the color intensity of the hybrid embryos. Simultaneous addition of both factors at one-fold basic concentration significantly improved the color intensity of the embryos in Zheketian 8, Wannongtiannuo 158, Zhenuoyu 14, and Demeiya 3. Increasing the working concentration of the two factors to twice the basic concentration improved the color intensity of the embryos in all hybrids, and the improvement was significant or highly significant in all varieties except Guangliangtian 27 and Wannuo 2000. Figure 2 At twice the baseline concentration, the N and D dual factors increased the color intensity of immature embryos by 18.3%–71.8%. Among the three groups (sweet corn, waxy corn, and common corn), the varieties with the most significant increases were Zheke Sweet 8 (30.3%), Wannong Sweet Waxy 158 (71.8%), and Dongdan 119 (62.7%). Figure 3 The accuracy of haploid identification was improved by the addition of naringenin and hesperidin through molecular marker identification. The results of haploid identification accuracy are shown in Table 1. The experimental results show that at a certain concentration, the individual effects of the enhancing factors naringenin and hesperidin are limited, but they can significantly improve the color intensity of young embryos under the synergistic effect of specific concentrations.

[0025] Table 1. Accuracy (%) of identifying haploid embryos of the CS2 induction line and different hybrids in different culture media.

[0026] Example 3

[0027] To further investigate the effects of enhancing factors on different haploid inducing lines, the color intensity of embryos from four inducing lines (CAU5, CAU6, CAU7, and CHOI4) hybridizing Zheketian 8, Wannuo 2000, and Demeya 3 was studied under different culture media. The results showed that the addition of naringin and hesperidin significantly improved the color intensity of most combinations, but the enhancement effect varied among different hybrids and inducing line combinations. Except for Wannuo 2000 × CAU7, 2N2D significantly improved the color intensity compared to MS. Figure 4 (ac). The addition of naringenin and hesperidin, as well as the increase in dosage, can significantly improve the accuracy of haploid identification (Table 2). The above results demonstrate that the two factors of naringenin and hesperidin have universality in enhancing color development in different induction lines.

[0028] Table 2. Accuracy (%) of haploid identification under different haploid induction lines

[0029] Example 4

[0030] To further investigate the enhancing effects of different doses of naringin and hesperidin, and their combinations, on the color development of immature embryos, different folds of single-factor and two-factor culture media were set as independent variables. For Zheke Sweet 8 × CS2, adding 1, 2, and 3 times the concentration of single factor did not significantly improve the color development intensity; adding 1 times the concentration of two factor significantly improved the color development intensity, and the effect was comparable to that of 2 and 3 times the concentration of two factor. Other combinations did not significantly improve the intensity, and when the concentration of two factor reached 4 times, the color development intensity actually decreased. Figure 5 a). For Dongdan 119×CS2, single-factor combinations of 2x and 3x significantly improved color intensity; while for two-factor combinations, except for 1x and 4x combinations, all other combinations significantly improved color intensity, and were comparable to the effect of single-factor combinations of 2x or more. Figure 5 b). For Zhengdan 958×CS2, a single factor concentration of 1x can significantly improve the color intensity, and increasing the concentration of the single factor cannot further improve the color intensity; the optimal concentration for synergistic effect of the two factors is 2N2D, which can achieve the highest color intensity, while high concentrations of the two factors will actually reduce the intensity. Figure 5 c). The results of molecular marker identification showed that the addition of 2N2D could achieve a haploid identification accuracy of 100% (Table 3). In summary, under the experimental conditions, it was demonstrated that there may be a synergistic effect between naringenin and hesperidin. Although there were some differences among different parent materials, 2N2D could generally and significantly improve the color intensity of young embryos, thereby improving the haploid identification accuracy.

[0031] Table 3. Accuracy (%) of haploid embryo identification for different dosage combinations

[0032] Example 5

[0033] The color enhancement effect of naringenin and hesperidin on the sweet maize inbred line G605 was further investigated. The results showed that 2N2D medium could increase the color intensity of immature embryos on MS medium from 2.13 to 3.10, an increase of 44.61%. Figure 6 a, b). Consistent with the previous results, the combination of single-factor and low-concentration two-factor combinations could not significantly improve the color intensity of immature embryos ( Figure 6c) Naringenin and hesperidin have a synergistic effect on enhancing color development in young embryos, but a certain concentration is required to achieve this enhancement. Flavonoid and anthocyanin contents were detected in young embryos cultured in 2N2D and ordinary MS media. In the hybrid young embryos of sweet corn inbred lines G605 and CS2 cultured in 2N2D, cyanidin, pelargonidin, and peony anthocyanins were significantly increased, while delphinidin, morning gloryin, and malvidin were present at extremely low levels in both types of young embryos, with no significant difference. Figure 6 d). Among the key flavonoids in the anthocyanin synthesis pathway, the contents of naringenin chalcone, naringenin, hesperidin, and dihydroquercetin were significantly upregulated in immature embryos cultured in 2N2D. Figure 6 e).

[0034] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of naringin and hesperidin in improving the color intensity of maize hybrid embryo identification.

2. The application as described in claim 1, characterized in that, When the concentration of naringerin is 14-18 mg / L and the concentration of hesperidin is 1.4-1.8 mg / L, the color intensity of maize hybrid embryos with sweet corn, waxy corn and common corn hybrids as the female parent and haploid inducing lines as the male parent can be improved. The sweet corn includes Ketian 8, Ganketian 10 or Guangliangtian 27; the waxy corn includes Wannuo 2000, Wannongtiannuo 158 or Zhenuoyu 14; the common corn includes Zhengdan 958, Demeya 3 or Dongdan 119; the haploid induction lines include CS2, CAU5, CAU6, CAU7 or CHOI4.

3. The application as described in claim 1, characterized in that, When the haploid induction line CS2 is the male parent: When the concentration of naringenin is 6-10 mg / L or the concentration of hesperidin is 0.6-1.0 mg / L, it can improve the color intensity of the hybrid embryos with Wannong Tiannuo 158 as the female parent. When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8, Wannong Tiannuo 158, Zhe Nuoyu 14 or Demeya 3 as the female parent can be improved. When the concentration of naringenin is 14-26 mg / L or the concentration of hesperidin is 1.4-2.6 mg / L, it can improve the color intensity of the hybrid embryos with Dongdan 119 as the female parent. When the concentration of naringenin is 22-26 mg / L and the concentration of hesperidin is 2.2-2.6 mg / L, the color intensity of the hybrid embryos with Ketian 8 and Dongdan 119 as the female parent can be improved.

4. The application as described in claim 1, characterized in that, When the haploid induction line CAU5 is used as the male parent, the color intensity of hybrid embryos with naringenin concentration of 6-10 mg / L and / or hesperidin concentration of 0.6-1.0 mg / L can be improved.

5. The application as described in claim 1, characterized in that, When the haploid induction line CAU6 is the male parent: When the concentration of naringenin is 6-10 mg / L, it can improve the color intensity of hybrid embryos with Ketian 8 as the female parent; When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8, Wannuo 2000 or Demeya 3 as the female parent can be improved.

6. The application as described in claim 1, characterized in that, When the haploid induction line CAU7 is the male parent: When the concentration of naringenin is 6-10 mg / L, it can improve the color intensity of hybrid embryos with Demeya 3 as the female parent; When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Ketian 8 or Demeya 3 as the female parent can be improved.

7. The application as described in claim 1, characterized in that, When the haploid induction line CHOI4 is the male parent: When the concentration of naringenin is 6-10 mg / L and / or the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of hybrid embryos with Wannuo 2000 or Demeya 3 as the female parent can be improved. When the concentration of naringenin is 6-10 mg / L and the concentration of hesperidin is 0.6-1.0 mg / L, the color intensity of the hybrid embryos with Ketian 8 as the female parent can be improved.

8. A method for enhancing colorimetric intensity in haploid identification of maize immature embryos using naringin and hesperidin, characterized in that, Using the maize variety to be tested as the female parent and the inducing line as the male parent, hybridization was carried out. 15 to 17 days after hybridization and pollination, the immature embryos were removed and cultured in MS medium with the concentrations of naringenin and hesperidin specified in any one of the applications described in claims 1 to 8, according to the male and female parent varieties. The MS medium was supplemented with a maize haploid doubling agent consisting of 0.05-0.4 g colchicine, 20 mL dimethyl sulfoxide, and 100-2000 mg calcium ions per L. The culture time is 22-26 h, and the light intensity during culture is 8000-12000 Lux.

9. A culture medium according to claim 8, characterized in that, The concentration of naringenin in the culture medium is 6~26 mg / L, and the concentration of hesperidin in the culture medium is 0.6~2.6 mg / L.

10. The application of the method of claim 8 or the culture medium of claim 9 in increasing the anthocyanin content of maize hybrid embryos.

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