A method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3

Abstract

This invention belongs to the field of tobacco agricultural technology and discloses a method for non-destructive and rapid screening of tobacco plants with high flavonoid content based on Multiplex 3. This method standardizes the measurement time, tobacco growth stage, leaf position, leaf surface, and measurement location. First, Multiplex 3 is used to detect tobacco leaves in the control group to establish a judgment interval. Then, the experimental population undergoes a first screening and a second screening to obtain individual tobacco plants with high flavonoid content. This method can rapidly screen high-flavonoid individual plants from a large population of tobacco materials. Verification by spectrophotometry and high-performance liquid chromatography-triple quadrupole mass spectrometry shows that the total flavonoid, chlorogenic acid, and rutin contents of the screened materials are all higher than those of the control group. Compared with existing technologies, this invention has advantages such as high detection sensitivity, strong specificity, non-destructive detection, simplicity, speed, and safety, making it particularly suitable for field application.

Description

Technical Field

[0001] This invention belongs to the field of tobacco agricultural technology, specifically relating to a method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 and its application. Background Technology

[0002] Flavonoids are widely distributed secondary metabolites in plants, with over 10,000 compounds reported, exhibiting important pharmacological activities such as anticancer and anti-inflammatory effects. The stress-resistance effects of flavonoids in plants have received considerable attention, with their roles in regulating polar auxin transport and free radical scavenging mechanisms emphasized. Under stress, plants accumulate flavonoids by regulating the expression of flavonoid synthase genes, and flavonoids regulate polar auxin transport (PAT) by acting on the auxin efflux carrier PIN-FORMED (PIN) in the form of ATP-binding box subfamily B / P-glycoprotein (ABCB / PGP) transporters. [1] Phenylacetin and flavonoids play crucial roles in plant defense against biotic and abiotic stresses. They can mediate defense responses, such as accumulation in leaves and glandular trichomes, and are associated with plant resistance to fungi and oomycetes. [2] Therefore, plant flavonoids play an important role in biosynthesis, biological functions, and in plant growth, development, and environmental stress responses.

[0003] Multiplex3 has also been applied in plant defense mechanism research, for example, by measuring polyphenol content to assess plant responses to stress. [3] Plants produce volatile compounds when they sense environmental stimuli. These compounds can be sensed by surrounding plants and trigger a defensive response. Multiplex3 can be used to monitor polyphenol changes in this airborne immune process. [4] .

[0004] Flavonoids are important products of tobacco secondary metabolism, playing a crucial role in tobacco growth and development and serving as a key factor in evaluating tobacco quality. Their accumulation, transformation, and degradation during tobacco ripening, curing, aging, and combustion generate diphenols and furfural derivatives. These products directly influence the aroma of tobacco smoke, imparting a sweet and roasted fragrance, and directly affecting the aroma style, quality, and intensity of flue-cured tobacco. [5-7] Flavonoids and polysaccharide extracts from tobacco leaves exhibited good antioxidant activity in in vitro antioxidant studies. Flavonoids showed better free radical scavenging activity than polysaccharides, demonstrating the potential to become potent antioxidants. [8]Furthermore, studies on the disease resistance of flavonoids in tobacco have shown enhanced resistance to various diseases, such as those caused by *Ralstonia solanacearum*, *Anthracnose*, and *Aureobasidium aizoon*. [9] .

[0005] References [1]Sabir IA, Shah IH, Riaz MW, et al. Flavonoids: a review onbiosynthesis and transportation mechanism in plants[J]. Functional &Integrative Genomics, 2023, 23(4): 315. doi: 10.1007 / s10142-023-01147-4. [2]Kumar PA ,Bhasker K ,Nikhil KSB , et al.Role ofPhenylpropanoids and Flavonoids in Plant Defense Mechanism[J].InternationalJournal of Environment and Climate Change,2023,13(9):2951-2960. [3]Liu Q ,Wang C ,Jiang J , et al.Multi-source data fusion improved the potential of proximal fluorescence sensors in predicting nitrogennutrition status across winter wheat growth stages[J].Computers and Electronics in Agriculture,2024,219108786-. [4]Qian G, Wang Y, Huang F, et al. Molecular basis of methylsalicylate-mediated plant airborne defense. Nature, 2023, 622(7981): 139-148.DOI: 10.1038 / s41586-023-06533-3. [5] Yang Tiezhao, Li Qinkui, Li Wei. Plant secondary metabolism and aroma substances in tobacco [J]. Chinese Tobacco Science, 2005, (04): 23-26. [6] Zhou Kun, Zhou Qingming, Hu Xiaolan. Research progress on aroma substances in flue-cured tobacco [J]. Chinese Tobacco Science, 2008, (02): 58-61. [7] Liu Caiyun, Liu Hongxiang, Chang Zhilong, et al. Research progress on aroma quality of tobacco [J]. Chinese Tobacco Science, 2010, 31(06):75-78. [8]Ru Q ,Wang L ,Li W , et al.In Vitro Antioxidant Properties ofFlavonoids and Polysaccharides Extract from Tobacco (Nicotiana tabacum L.)Leaves[J].Molecules,2012,17(9):11281-11291. [9] Li Ming. Heterologous expression of flavonoid regulatory gene AtMYB12 and its contribution to plant disease resistance [D]. Shandong Agricultural University, 2012. DOI:10.7666 / d.Y2345720. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a simple, rapid, accurate and non-destructive analytical method suitable for field analysis, so as to realize rapid and real-time monitoring of tobacco flavonoid content in the field, and provide theoretical basis and technical support for tobacco flavonoid content monitoring, tobacco field management and tobacco agricultural technology.

[0007] This invention is based on a preliminary study of Multiplex 3 in the detection of flavonoid content in tobacco leaves, and provides a method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3.

[0008] The objective of this invention is achieved through the following technical solution: A method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 includes the following steps: Step S1: Select the tobacco plant to be tested and test the tobacco leaves under weak light conditions; reduce the impact of excessive light intensity on the Multiplex 3 test results. Step S2: Standardize the growth period, leaf position, and measurement surface of the tobacco leaves to be tested, and select tobacco leaves with the same growth period and leaf position for flavonoid content determination; Step S3: Selection of the measurement location on tobacco leaves. Avoid leaf veins and select the same leaf surface location for flavonoid content measurement; Step S4: Use Multiplex 3 to determine the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group. Use the measured values ​​of relevant parameters of flavonoid content to plot the Multiplex 3 measured values-Density normal distribution curve, and observe the mean and dispersion of the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group measured by Multiplex 3. Step S5: By analyzing the values ​​of relevant parameters of flavonoid content in the control group tobacco leaves measured by Multiplex 3, the range of values ​​for the relevant parameters of high flavonoid content in tobacco leaves is obtained. Step S6: The first detection of flavonoid content-related parameters was performed on tobacco leaves in the Multiplex 3 experimental group to preliminarily screen tobacco plants with high flavonoid content and mark them; using the measured values ​​of flavonoid content-related parameters, a normal distribution curve of Multiplex 3 measured values-Density was plotted to observe the mean and dispersion of flavonoid content-related parameters in tobacco leaves of the Multiplex 3 experimental group. Step S7: Use Multiplex 3 to perform a second screening of tobacco plants with high flavonoid content in the initial screening of tobacco leaves in Step S6. This second screening should be conducted at a different time, leaf position, and leaf surface measurement location than in Step S6. After the second screening, tobacco plants with high flavonoid content in tobacco leaves are obtained and marked. Using the measured values ​​of flavonoid content-related parameters, a Multiplex 3 measurement value-Density normal distribution curve is plotted to observe the mean and dispersion of the values ​​of flavonoid content-related parameters in the tobacco leaves of the Multiplex 3 test group. Thus, tobacco plants with high flavonoid content in tobacco leaves are obtained.

[0009] Furthermore, the tobacco includes fresh tobacco leaves; the leaf position in step (1) is the fifth leaf from bottom to top at maturity.

[0010] Furthermore, the weak light conditions mentioned in step 1 are the detection under natural light conditions in the early morning or evening.

[0011] Furthermore, the leaf area of ​​the tobacco leaf should be greater than 28 cm²; when the leaf area is less than 28 cm², a light-blocking material should be used to block part of the external light source before testing.

[0012] Furthermore, step S4 must be performed before each step S6, in order to test the tobacco leaves of the control group and update the distribution range of the control group before screening and testing the experimental group.

[0013] Furthermore, when using Multiplex 3 for detection, each sample is tested three times consecutively, and the average value is used as the test result for that sample. The Multiplex 3 used is the Multiplex 3 MX315411 from Force-A, France.

[0014] Furthermore, the total flavonoid content of the selected tobacco plants with high flavonoid content was higher than that of the control group by visible spectrophotometry; and the chlorogenic acid and / or rutin content was higher than that of the control group by high performance liquid chromatography-triple quadrupole mass spectrometry.

[0015] The method of this invention can be applied to the screening of tobacco germplasm resources with high flavonoid content, the initial screening of breeding materials, or the rapid monitoring of flavonoid content in tobacco in the field.

[0016] Compared with traditional destructive sampling and laboratory analysis, this invention has the following advantages: It can perform non-destructive testing on live tobacco leaves in the field; it can be used directly for initial screening in the field without the need for large-scale destructive sampling of the target population. It has a fast testing speed and a short single testing time, making it suitable for initial screening of large groups. By establishing a control group reference distribution and conducting a second test, the stability and reliability of the screening results can be improved. It can identify high-flavonoid candidate plants in advance and then use chemical analysis methods for further verification, which can reduce the risk of false screening.

[0017] It is helpful for screening high-flavonoid tobacco materials and for early-stage evaluation in breeding. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method of the present invention; Figure 2 Measure the numerical-Density normal distribution curve for Multiplex 3; Figure 3 This study compares the total flavonoid content in tobacco leaves with high flavonoid content and control tobacco leaves.

[0019] Figure 4 This study compares the chlorogenic acid content in tobacco leaves with high flavonoid content and control tobacco leaves.

[0020] Figure 5 This study compares the rutin content in tobacco leaves with high flavonoid content and those of control tobacco leaves. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Before introducing the embodiments, the specific experimental background information of the following embodiments is briefly introduced as follows. biomaterials

[0022] Cultivated tobacco K326 is a widely planted cultivated tobacco. In the following examples, cultivated tobacco K326 was planted in Jinhua Town, Fenggang County, Zunyi City, Guizhou Province. The control group consisted of K326 tobacco and the experimental group consisted of the EMS mutant M4 generation tobacco of K326. 200 individual plants were planted in the control group K326 and 110 lines of the EMS mutant of K326 were planted in the experimental group. 200 individual plants were planted in each line.

[0023] like Figure 1 As shown, the method of the present invention includes the following steps: Step S1: Select the tobacco plant to be tested and test the tobacco leaves under weak light conditions; reduce the impact of excessive light intensity on the Multiplex 3 test results. Step S2: Standardize the growth period, leaf position, and measurement surface of the tobacco leaves to be tested, and select tobacco leaves with the same growth period and leaf position for flavonoid content determination; Step S3: Selection of the measurement location on tobacco leaves. Avoid leaf veins and select the same leaf surface location for flavonoid content measurement; Step S4: Use Multiplex 3 to determine the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group. Use the measured values ​​of relevant parameters of flavonoid content to plot the Multiplex 3 measured values-Density normal distribution curve, and observe the mean and dispersion of the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group measured by Multiplex 3. Step S5: By analyzing the values ​​of relevant parameters of flavonoid content in the control group tobacco leaves measured by Multiplex 3, the range of values ​​for the relevant parameters of high flavonoid content in tobacco leaves is obtained. Step S6: The first detection of flavonoid content-related parameters was performed on tobacco leaves in the Multiplex 3 experimental group to preliminarily screen tobacco plants with high flavonoid content and mark them; using the measured values ​​of flavonoid content-related parameters, a normal distribution curve of Multiplex 3 measured values-Density was plotted to observe the mean and dispersion of flavonoid content-related parameters in tobacco leaves of the Multiplex 3 experimental group. Step S7: Use Multiplex 3 to perform a second screening of tobacco plants with high flavonoid content in the initial screening of tobacco leaves in Step S6. This second screening should be conducted at a different time, leaf position, and leaf surface measurement location than in Step S6. After the second screening, tobacco plants with high flavonoid content in tobacco leaves are obtained and marked. Using the measured values ​​of flavonoid content-related parameters, a Multiplex 3 measurement value-Density normal distribution curve is plotted to observe the mean and dispersion of the values ​​of flavonoid content-related parameters in the tobacco leaves of the Multiplex 3 test group. Thus, tobacco plants with high flavonoid content in tobacco leaves are obtained. Example 1

[0024] This Example 1 discloses a method for non-destructive and rapid screening of tobacco plants with high flavonoid content using Multiplex 3.

[0025] The samples tested were: 200 control group K326 tobacco plants and 22,000 experimental group K326 EMS mutant tobacco plants.

[0026] Combination Figure 1 The detection steps in this embodiment are as follows: 1. Selection of measurement time: Select early morning and evening for measurement; 2. Selection of tobacco samples for testing: The tobacco samples were selected from the mature control group K326 tobacco and the experimental group K326 EMS mutant tobacco. The leaf surface of the tobacco leaf from the 5th leaf from the bottom to the top was used for testing. 3. Selection of tobacco sample measurement location: Avoid leaf veins and select the left side of the main vein, in the middle of the leaf surface for measurement; 4. Use Multiplex 3 to determine the flavonoid content in tobacco leaves of the control and experimental groups: Select "Flav" on the instrument panel, press the button on the instrument handle to start the test, and complete the data acquisition after about 1 to 3 seconds.

[0027] 5. Thirteen tobacco plants with high flavonoid content were selected. The fifth leaf from the bottom was taken, blanched at 100℃ for 1 hour, and dried at 60℃ for 72 hours for testing. Example 2

[0028] Based on Experiment 1, the inventors further determined the flavonoid content in dried tobacco leaf samples using visible light spectrophotometry and high performance liquid chromatography-triple quadrupole mass spectrometry.

[0029] The total flavonoid content in dried tobacco leaves was determined by visible spectrophotometry, such as... Figure 3As shown in the figure, it can be seen that the 13 EMS mutant tobacco plants with high flavonoid content obtained by screening 22,000 experimental group K326 EMS mutant tobacco plants using Multiplex 3 had a total flavonoid content that was 1.54 to 3.13 times higher than that of the control group K326 tobacco.

[0030] The contents of chlorogenic acid and rutin in dried tobacco leaves were determined by high performance liquid chromatography-triple quadrupole mass spectrometry, as shown below. Figure 4 and Figure 5 As shown in the figure, it can be seen that among the 13 EMS mutant tobacco plants with high flavonoid content obtained by screening 22,000 experimental group K326 EMS mutant tobacco plants using Multiplex 3, the contents of chlorogenic acid and rutin were higher than those of the control group K326 tobacco. The chlorogenic acid content was 1.10 to 3.87 times higher than that of the control group K326 tobacco, and the rutin content was 1.56 to 3.14 times higher than that of the control group K326 tobacco. Based on the results of Examples 1-2 above, the method of the present invention for non-destructive rapid screening of tobacco with high flavonoid content using Multiplex 3 is comparable to the existing standard methods in terms of accuracy, reliability, and repeatability of the test results. However, the present invention does not require cumbersome sample preparation and processing steps, nor does it require destructive sample collection. It is convenient, rapid, and instantaneous for determining the flavonoid content of field-grown tobacco, which is significantly superior to the existing standard methods.

[0031] The above embodiments 1-2 are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3, characterized in that, The method includes the following steps: Step S1: Select the tobacco plant to be tested and test the tobacco leaves under weak light conditions; reduce the impact of excessive light intensity on the Multiplex 3 test results. Step S2: Standardize the growth period, leaf position, and measurement surface of the tobacco leaves to be tested, and select tobacco leaves with the same growth period and leaf position for flavonoid content determination; Step S3: Selection of the measurement location on tobacco leaves. Avoid leaf veins and select the same leaf surface location for flavonoid content measurement; Step S4: Use Multiplex 3 to determine the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group. Use the measured values ​​of relevant parameters of flavonoid content to plot the Multiplex 3 measured values-Density normal distribution curve, and observe the mean and dispersion of the values ​​of relevant parameters of flavonoid content in tobacco leaves of the control group measured by Multiplex 3. Step S5: By analyzing the values ​​of relevant parameters of flavonoid content in the control group tobacco leaves measured by Multiplex 3, the range of values ​​for the relevant parameters of high flavonoid content in tobacco leaves is obtained. Step S6: The first detection of flavonoid content-related parameters was performed on tobacco leaves in the Multiplex 3 experimental group to preliminarily screen tobacco plants with high flavonoid content and mark them; using the measured values ​​of flavonoid content-related parameters, a normal distribution curve of Multiplex 3 measured values-Density was plotted to observe the mean and dispersion of flavonoid content-related parameters in tobacco leaves of the Multiplex 3 experimental group. Step S7: Use Multiplex 3 to perform a second screening of tobacco plants with high flavonoid content in the initial screening of tobacco leaves in Step S6. This second screening should be conducted at a different time, leaf position, and leaf surface measurement location than in Step S6. After the second screening, tobacco plants with high flavonoid content in tobacco leaves are obtained and marked. Using the measured values ​​of flavonoid content-related parameters, a Multiplex 3 measurement value-Density normal distribution curve is plotted to observe the mean and dispersion of the values ​​of flavonoid content-related parameters in the tobacco leaves of the Multiplex 3 test group. Thus, tobacco plants with high flavonoid content in tobacco leaves are obtained.

2. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, The tobacco includes fresh tobacco leaves.

3. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, The weak light conditions mentioned in step 1 refer to the detection being carried out under natural light conditions in the early morning or evening.

4. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, The leaf area of ​​the tobacco leaf should be greater than 28 cm²; when the leaf area is less than 28 cm², a light-blocking material should be used to block part of the external light source before testing.

5. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, Step S4 must be performed before each step S6. This is to test the tobacco leaves of the control group and update the distribution range of the control group before screening and testing the experimental group.

6. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, When using Multiplex 3 for detection, each sample is tested three times consecutively, and the average value is used as the test result for that sample.

7. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, The Multiplex 3 mentioned is the Multiplex 3 MX315411 from Force-A, a French company.

8. The method for non-destructive and rapid screening of tobacco with high flavonoid content based on Multiplex 3 according to claim 1, characterized in that, The leaf position mentioned in step (1) is the 5th leaf from bottom to top during the maturity stage.

9. The method according to any one of claims 1 to 8, characterized in that, The total flavonoid content of the selected tobacco plants with high flavonoid content was higher than that of the control group by visible spectrophotometry; and the chlorogenic acid and / or rutin content was higher than that of the control group by high performance liquid chromatography-triple quadrupole mass spectrometry.

10. The application of the method according to any one of claims 1 to 9 in the screening of tobacco germplasm resources with high flavonoid content, the initial screening of breeding materials, or the rapid monitoring of flavonoid content in tobacco in the field.

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