Method for increasing content of linarin in filial generation of wild chrysanthemum flower and application

Abstract

The invention relates to the technical field of chrysanthemum indicum breeding, in particular to a method for increasing the content of linarin in a wild chrysanthemum flower filial generation and application. According to the method for increasing the content of linarin in the filial generation of wild chrysanthemum, tetraploid chrysanthemum indicum is used as a male parent, diploid chrysanthemum indicum is used as a female parent, and the first filial generation is obtained through hybridization cultivation. According to the method, the diploid female parent and the tetraploid male parent are selected to be hybridized, the genetic characteristics of the filial generation are regulated through ploidy difference, dilution of the medicinal component linarin is avoided, the content of the linarin in the wild chrysanthemum flowers obtained by planting the first filial generation obtained through cultivation is high, and the yield of the linarin is high (the content of the linarin is larger than or equal to 0.8 wt%; and according with the Chinese Pharmacopoeia standard) more than or equal to 87.5%.

Description

Technical Field

[0001] The invention relates to the technical field of wild chrysanthemum breeding, and in particular to a method for increasing the content of montanin in wild chrysanthemum hybrid offspring and its application. Background Art

[0002] Chrysanthemum indicum is an important Chinese medicinal material. It contains a flavonoid glycoside compound - mongoside. In current research, mongoside has been found to have anti-inflammatory, antioxidant, antibacterial and antiviral effects. The current edition of the Chinese Pharmacopoeia clearly stipulates that mongoside in Chrysanthemum indicum medicinal materials should be no less than 0.80wt% according to the dry product calculation. However, there are the following problems in traditional breeding methods: 1) parents are not distinguished, multiple germplasms are mixed and planted, and the qualified rate of mongoside in offspring is low, which is a serious waste of resources; 2) parent selection is blind, and there is a lack of systematic research on the correlation between parental genetic characteristics (such as ploidy) and offspring efficacy mongoside content; 3) the purpose is to obtain triploid plants, and the difference in the influence of different ploidy hybrid combinations on offspring medicinal material components is not clear. At the same time, Chrysanthemum indicum was originally a wild species. In recent years, based on the improvement of medicinal material demand, a large number of wild species have been converted to domestic species. In this process, it is inevitable that wild diploid and tetraploid germplasms are introduced into production fields, resulting in uneven medicinal material quality.

[0003] The prior art discloses a processing method for improving the quality of chrysanthemums based on the introduction of chamomile glycosides through interspecific distant hybridization. Purple-flowered wild chrysanthemums are introduced through distant hybridization to create new germplasm offspring. However, through the detection of chamomile glycoside content in its hybrid offspring, it was found that the breeding efficiency of the first-generation individuals that meet the standards of the "Chinese Pharmacopoeia" (≥0.80%) was significantly low. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art that hybrid offspring with high chlorophyll content cannot be obtained through intraspecific hybridization and the breeding efficiency is low, thereby providing a method and application for increasing the chlorophyll content in hybrid offspring of wild chrysanthemum.

[0005] To this end, the present invention provides a method for increasing the content of montannin in hybrid offspring of wild chrysanthemum, wherein tetraploid wild chrysanthemum is used as the male parent and diploid wild chrysanthemum is used as the female parent, and the first generation is obtained through hybridization cultivation.

[0006] In some embodiments, the specific steps of the hybrid breeding include emasculation of the female parent, pollination, and obtaining the first-generation hybrid, and the pollination is artificial pollination.

[0007] In some embodiments, before castrating the female parent, the steps of asexually propagating and planting the male and female parents are also included.

[0008] In some embodiments, when the male and female parents are planted after asexual propagation, an isolation zone of at least 500 meters is set between the male and female parents, and it is ensured that there are no other wild chrysanthemums within 500 meters of the planting area.

[0009] In some embodiments, the hybridization cultivation step further includes raising seedlings and transplanting the second-generation hybrids to obtain the second-generation wild chrysanthemums.

[0010] In some embodiments, the content of limonene in the male parent is less than or equal to 0.8 wt %. Preferably, the content of limonene in the male parent is 0.

[0011] In some embodiments, the present invention does not limit the content of chlorophyllin in the mother plant. Based on existing research, whether the chlorophyllin content of the mother plant meets the requirements of the pharmacopoeia (greater than or equal to 0.8wt%) or does not meet the requirements of the pharmacopoeia (less than 0.8wt%), the chlorophyllin content of the off-generation wild chrysanthemum obtained by hybridization with tetraploid wild chrysanthemum meets the requirements of the pharmacopoeia.

[0012] Preferably, the content of limonene in the mother parent is 2.12 wt%.

[0013] More preferably, the variety of the female parent is 999 Huaju 03, and the variety of the male parent is 999 Huaju 04.

[0014] In some embodiments, the female plant is castrated, pollination is carried out in November of the planting year, and the seeds are harvested 40-45 days after pollination to obtain the first-generation hybrid.

[0015] In some embodiments, the first-generation hybrids are raised in March, transplanted in May of the same year, and harvested in November of the same year.

[0016] The invention provides a method for increasing the content of montana glycoside in wild chrysanthemum hybrid offspring, and uses the hybrids cultivated therefrom in increasing the content of montana glycoside in wild chrysanthemum and improving the yield of wild chrysanthemum.

[0017] The technical solution of the present invention has the following advantages:

[0018] The present invention provides a method for increasing the content of montanol in hybrid offspring of wild chrysanthemum. The method comprises hybridizing a tetraploid wild chrysanthemum as a male parent and a diploid wild chrysanthemum as a female parent to obtain a first-generation offspring. The method selects a diploid female parent for hybridization with a tetraploid male parent, utilizes ploidy differences to regulate the genetic characteristics of the hybrid offspring, avoids dilution of the medicinal ingredient montanol, and cultivates wild chrysanthemums with a high montanol content in the hybrid offspring after planting. The qualified rate of montanol (montanol content is greater than or equal to 0.8wt%, meeting the standards of the Chinese Pharmacopoeia) is greater than or equal to 87.5%. DETAILED DESCRIPTION

[0019] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0020] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0021] In the embodiments and comparative examples of the present invention, variety A is specifically 999 Huaju 03, and variety B is specifically 999 Huaju 04.

[0022] Example 1

[0023] This embodiment provides a method for increasing the content of chrysanthemum indicum hybrid offspring, and the specific steps and parameters are as follows:

[0024] (1) In 2023, variety A (diploid, mongoside content 2.12 wt%) was selected as the female parent, and variety B (tetraploid, mongoside content 0 wt%) was selected as the male parent. The seedlings after asexual propagation were planted in the planting field, and a 500-meter isolation belt was set up to ensure that there were no other wild chrysanthemums within 500 meters of the planting area and no possible contamination by non-target pollen. The planting field was located in the Xiawei group of Baisha Town, Yangxin County, Hubei Province;

[0025] (2) In November 2023, artificial assisted pollination was used to emasculate the female parent before flowering. After emasculation, the male parent pollen was artificially pollinated and bagged. After pollination for about 40 days, the diploid female parent F1 hybrid was harvested;

[0026] (3) In March 2024, the diploid female parent F1 hybrid seeds were raised into seedlings, and in May, 50 healthy seedlings were transplanted to the experimental field and managed as usual;

[0027] (4) In November 2024, 16 F1 hybrid wild chrysanthemums were harvested in full bloom.

[0028] (5) According to the detection method recorded in the Chinese Pharmacopoeia, the content of F1 generation mongoside was detected by high performance liquid chromatography (HPLC), and individuals that met the pharmacopoeia standard (≥0.80%) were screened. The results are shown in Table 1.

[0029] Table 1 Content of mongoside in the first generation of Chrysanthemum indicum hybrid harvested in Example 1

[0030]

[0031]

[0032] In this embodiment, a total of 16 first-generation hybrid plants of Chrysanthemum indicum were harvested (randomly sampled), 14 of which had a leucoside content greater than 0.80 wt%, and a qualified rate of offspring of 87.5%.

[0033] Example 2

[0034] This embodiment provides a method for increasing the content of montanol in hybrid offspring of Chrysanthemum indicum. The specific steps and parameters are the same as those in Example 1, except that the planting field is located in Xinwuxia Group, Baisha Town, Yangxin County, Hubei Province.

[0035] The results of the determination of the content of monanthus serrataecarpa in the first generation of Chrysanthemum indicum hybrids collected in this example are shown in Table 2.

[0036] Table 2 Content of mongoside in the first generation of Chrysanthemum indicum hybrid harvested in Example 2

[0037]

[0038] In this embodiment, a total of 16 first-generation hybrid plants of Chrysanthemum indica were harvested (randomly sampled), 15 of which had a leucoside content greater than 0.80 wt%, and the qualified rate of their offspring was 93.75%.

[0039] Example 3

[0040] This embodiment provides a method for increasing the content of monardoside in the hybrid offspring of Chrysanthemum indicum. The specific steps and parameters are the same as those in Example 1, except that the method is carried out in Tuku Formation, Baisha Town, Yangxin County, Hubei Province.

[0041] The results of the determination of the content of monanthus glycosides in the first generation of Chrysanthemum indicum hybrids harvested in this example are shown in Table 3.

[0042] Table 3 Content of mongoside in the first generation of Chrysanthemum indicum hybrid harvested in Example 3

[0043]

[0044] In this embodiment, a total of 16 first-generation hybrid plants of Chrysanthemum indica were harvested (randomly sampled), 15 of which had a leucoside content greater than 0.80 wt%, and the qualified rate of their offspring was 93.75%.

[0045] Comparative Example 1

[0046] This comparative example provides a method for increasing the content of chlorogenic acid in the hybrid offspring of wild chrysanthemum. The specific steps and parameters are the same as those in Example 1, except that in step (1), variety B (tetraploid, chlorogenic acid content of 0wt%) is used as the female parent, and variety A (diploid, chlorogenic acid content of 2.12wt%) is used as the male parent.

[0047] Through testing, the results of determination of the content of monanthus glycoside in the first generation of Chrysanthemum indicum hybrid harvested in this comparative example are shown in Table 4.

[0048] Table 4 Comparative Example 1 harvested Chrysanthemum indicum hybrid first generation monarda content

[0049]

[0050]

[0051] In this comparative example, 16 first-generation hybrid plants of Chrysanthemum indica were harvested (randomly sampled), 8 of which had a montana glycoside content greater than 0.80 wt%, and the qualified rate of their offspring was 50%.

[0052] Comparative Example 2

[0053] This comparative example provides a method for increasing the content of chlorogenic acid in the hybrid offspring of wild chrysanthemum. The specific steps and parameters are the same as those in Example 2, except that in step (1), variety B (tetraploid, chlorogenic acid content of 0wt%) is used as the female parent, and variety A (diploid, chlorogenic acid content of 2.12wt%) is used as the male parent.

[0054] Through testing, the results of determination of the content of monanthus serrataecarpa in the first generation hybrid of Chrysanthemum indicum harvested in this comparative example are shown in Table 5.

[0055] Table 5 Comparative Example 2 harvested chrysanthemum hybrid first generation mongoose content

[0056]

[0057]

[0058] In this comparative example, 16 first-generation hybrid plants of Chrysanthemum indica were harvested (randomly sampled), 9 of which had a montana glycoside content greater than 0.80 wt%, and the qualified rate of their offspring was 56.25 wt%.

[0059] Comparative Example 3

[0060] This comparative example provides a method for increasing the content of chlorogenic acid in the hybrid offspring of wild chrysanthemum. The specific steps and parameters are the same as those in Example 3, except that in step (1), variety B (tetraploid, chlorogenic acid content of 0wt%) is used as the female parent, and variety A (diploid, chlorogenic acid content of 2.12wt%) is used as the male parent.

[0061] Through testing, the results of determination of the content of monanthus serrataecarpa in the first generation hybrid of Chrysanthemum indicum harvested in this comparative example are shown in Table 6.

[0062] Table 6 Comparative Example 3 harvested chrysanthemum hybrid first generation mongoose content

[0063]

[0064]

[0065] In this comparative example, 16 first-generation hybrid plants of Chrysanthemum indica were harvested (randomly sampled), 9 of which had a montana glycoside content greater than 0.80 wt%, and the qualified rate of their offspring was 56.25 wt%.

[0066] The content of monanthoside in the first generation of Chrysanthemum indicum hybrids harvested from Examples 1-3 and Comparative Examples 1-3 was statistically tested, and the results are shown in Table 7.

[0067] Table 7 Statistical test of the content of mongoside in the first generation of Chrysanthemum indicum hybrids

[0068]

[0069] According to Table 7, the qualified rate of the first generation of wild chrysanthemum hybrids cultivated in the embodiment of the present invention is significantly higher than that of the control example, and the chi-square test result is extremely significant, indicating that the method for increasing the content of glycosides in the hybrid offspring of wild chrysanthemum provided by the present invention can effectively increase the qualified rate of glycosides in wild chrysanthemum.

[0070] The average yield per plant of the first-generation hybrid chrysanthemum harvested from Examples 1-3 and Comparative Examples 1-3 was statistically tested, and the results are shown in Table 8.

[0071] Table 8 Statistical test of the average yield of individual plants of the first generation of wild chrysanthemum hybrids

[0072]

[0073]

[0074] According to the data in Table 8, the P-value is 0.027312, which is less than the significance level of 0.05, and the null hypothesis is rejected. There is a significant difference between the average single-plant yield of the comparative example and the embodiment; the F statistic is 11.55067, which is greater than the critical value of 7.708647, and further shows that there is a significant difference between the embodiment and the comparative example groups, indicating that the method for increasing the content of chrysanthemum flos-fermentans in the hybrid offspring provided by the present invention can increase the single-plant yield of the hybrid offspring.

[0075] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for increasing the content of chrysanthemum flos-Hybrid offspring, characterized in that: The first generation was obtained through hybridization using tetraploid wild chrysanthemum as the male parent and diploid wild chrysanthemum as the female parent.

2. The method for improving the content of chrysanthemum flos-nucifera in hybrid offspring according to claim 1, wherein: The specific steps of the hybrid breeding include emasculation of the female plant, pollination, and obtaining the first-generation hybrid, wherein the pollination is artificial pollination.

3. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 2, wherein: Before castrating the female plant, the steps of asexual propagation and planting of the male and female plants are also included.

4. The method for improving the content of chrysanthemum flos-nucifera in hybrid offspring according to claim 3, wherein: When planting the male and female plants after asexual propagation, set up an isolation zone of at least 500 meters between the male and female plants, and ensure that there are no other wild chrysanthemums within 500 meters of the planting area.

5. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 2, wherein: The hybridization cultivation step also includes raising seedlings and transplanting the second-generation hybrids to obtain the second-generation wild chrysanthemums.

6. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 1, wherein: The content of limonene in the male parent is less than or equal to 0.8 wt % and greater than or equal to 0.

7. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 6, wherein: The content of limonene in the male parent is 0, and the content of limonene in the female parent is 2.12 wt %.

8. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 1, wherein: The female plant is emasculated and pollinated in November of the planting year. The seeds are harvested 40-45 days after pollination to obtain the first-generation hybrid.

9. The method for improving the content of chrysanthemum flos-indica in hybrid offspring according to claim 5, wherein: The seedling raising time of the first-generation hybrid is March, the transplanting time is May of the same year, and the harvesting time is November of the same year.

10. Use of a hybrid cultivated by the method for increasing the content of montana glycoside in wild chrysanthemum hybrid offspring according to any one of claims 1 to 9 in increasing the content of montana glycoside in wild chrysanthemum and improving the yield of wild chrysanthemum.

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