Method for screening tobacco variety with high flavonoid content based on Multiplex 3
By using a Multiplex 3 sensor and a normal distribution model to screen tobacco varieties with high flavonoid content, the problem of rapid, accurate, and non-destructive monitoring of flavonoid content in field tobacco breeding was solved, achieving efficient tobacco breeding screening.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XUCHANG COMPANY OF HENAN TOBACCO
- Filing Date
- 2026-03-09
- Publication Date
- 2026-05-08
AI Technical Summary
Existing technologies make it difficult to monitor the flavonoid content in tobacco leaves quickly, accurately, and non-destructively in the field, resulting in complex operations, high costs, and poor timeliness in tobacco breeding.
The flavonoid content of tobacco leaf samples was measured using a Multiplex 3 sensor under the same environmental parameters. Screening thresholds were set, and tobacco varieties with high flavonoid content were screened out using a normal distribution model. A second screening was conducted by combining differential conditions, and a standardized process and statistical model were constructed.
It enables large-scale, non-destructive, rapid, and low-cost screening with high accuracy in tobacco breeding, improves the repeatability and comparability of field testing, and enhances breeding efficiency and precision.
Smart Images
Figure CN121995012A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tobacco agricultural technology, specifically relating to a method for screening tobacco varieties with high flavonoid content based on Multiplex 3. 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 export carrier PIN-FORMED (PIN) in the form of ATP-binding box subfamily B / P-glycoprotein (ABCB / PGP) transporters. Phenylpropanols 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. Therefore, plant flavonoids play important roles in biosynthesis, biological function, and plant growth, development, and environmental stress responses.
[0003] 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. Flavonoids and polysaccharide extracts from tobacco leaves have demonstrated good antioxidant activity in in vitro antioxidant studies. Flavonoids show better free radical scavenging activity than polysaccharides, showing potential as potent antioxidants. Furthermore, studies on the disease resistance of tobacco by flavonoids have shown enhanced resistance to various diseases, such as *Ralstonia solanacearum*, *Anthracnose*, and *Agrostis spp.*
[0004] Currently, the determination of flavonoid content in tobacco leaves is mostly achieved through field sampling and laboratory chemical analysis. However, if the sample size is small, it cannot accurately reflect the flavonoid content level of tobacco in the field, making it difficult to achieve large-scale monitoring. While increasing the sample size can improve accuracy, it is complicated to operate, has poor timeliness, requires destructive collection of large quantities of samples, and is costly. Therefore, it is urgent to develop a simple, rapid, accurate, low-destructive method suitable for field analysis. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides a method for screening tobacco varieties with high flavonoid content based on Multiplex 3. This method is simple, rapid, accurate, non-destructive, low-impact, and not limited by application scenarios. It enables rapid and real-time monitoring of flavonoid content in tobacco in the field, providing a theoretical basis and technical support for the monitoring of tobacco flavonoid content, tobacco field management, and tobacco agricultural technology.
[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a method for screening tobacco varieties with high flavonoid content based on Multiplex 3, the method comprising the following steps: Under the same environmental parameters and sampling conditions, the flavonoid content of control tobacco leaf samples and experimental tobacco leaf samples was measured using a Multiplex 3 sensor. Based on the flavonoid content results of the control tobacco leaf samples, the screening threshold was obtained. Based on the screening threshold and the flavonoid content of the experimental tobacco sample leaves, candidate tobacco plants with high flavonoid content were initially screened. Under differentiated conditions by changing at least one of the environmental parameters or sampling conditions, the flavonoid content of the candidate tobacco leaves is measured again using a Multiplex 3 sensor. Tobacco varieties with flavonoid content still higher than the screening threshold under differentiated conditions are considered to have high flavonoid content.
[0007] Furthermore, the environmental parameters and sampling conditions include the following three types: 1) Select a time period with light intensity of 300 lux to 500 lux for measurement; 2) Select tobacco leaves from the same growth stage and leaf position; 3) Take measurements on the same area on the leaf, avoiding the veins.
[0008] Furthermore, the leaf area of the tobacco sample leaves is ≥28 cm². 2 If the leaf area of the tobacco sample is <28 cm² 2 A black material was used to block the light source; the area of the black material was equal to the area of the tobacco sample leaf plus 28 cm². 2 The area of the difference between them is used to ensure that the total coverage area reaches 28cm. 2 .
[0009] Furthermore, the leaf is a fresh leaf.
[0010] Furthermore, setting the screening threshold includes the following steps: The measured flavonoid content values were subjected to a normal distribution test, and a measured value-density curve was plotted. Calculate the mean μ and standard deviation σ of the control group; Set the filtering threshold to a numerical range from μ+σ to μ+2σ.
[0011] Furthermore, the differentiation conditions include the following three types: 1) Select a testing time different from the initial screening time; 2) Select tobacco leaf positions different from those used in the initial screening; 3) Select measurement locations on tobacco leaves that are different from those used in the initial screening.
[0012] Furthermore, when using the Multiplex 3 sensor, each sample must be measured at least three times consecutively.
[0013] Furthermore, the Multiplex 3 sensor is the Force-A Multiplex 3 MX315411.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for screening tobacco varieties with high flavonoid content based on Multiplex 3 sensors. The method involves measuring the flavonoid content of tobacco leaf samples using a Multiplex 3 sensor to obtain a screening threshold. Based on this threshold, samples undergo primary and secondary screening to ultimately obtain individual tobacco plants with leaves exhibiting high flavonoid content. This invention transforms general-purpose detection equipment into a dedicated screening tool for tobacco breeding, innovatively constructing a system solution of "standardized operation, dynamic threshold, and high-throughput screening," meeting the urgent need in tobacco molecular breeding for large-scale, non-destructive, simple, rapid, highly accurate, and low-cost screening.
[0015] (1) A standardized field rapid detection procedure was established: By limiting the process and screening conditions of the screening method, environmental variables and biological variations were systematically controlled, which significantly improved the repeatability, comparability and reliability of field detection data, and provided a standard operating procedure for large-scale application.
[0016] (2) A dynamic screening model based on statistics was constructed: by measuring the control population and establishing the normal distribution curve of flavonoid content, the threshold of "high content" was objectively defined by the mean plus standard deviation (e.g., μ+1σ~μ+2σ). This model transforms the general FLAV index from a relative value that is difficult to quantify into a relative screening standard that can be used in breeding, thus solving the problem of the lack of objective screening basis in traditional methods.
[0017] (3) Achieved high-throughput, low-cost non-destructive field screening: The method provided by this invention simplifies the determination of complex physiological indicators into a linear process of "measurement-comparison-decision", which can quickly and non-destructively screen tens of thousands of tobacco plants in the field, greatly improving the breeding efficiency of high flavonoid tobacco and reducing screening costs.
[0018] (4) Improved screening accuracy and breeding efficiency: By combining standardized processes with statistical models, the limitations of relative instrument measurements were effectively avoided, and the focus was on identifying the relatively optimal individuals in a specific population, providing reliable technical support for the targeted breeding of tobacco with high flavonoid content. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 The flowchart shows the method for rapid screening of tobacco with high flavonoid content based on Multiplex 3 non-destructive screening provided by the present invention.
[0021] Figure 2 The numerical-density normal distribution curve was determined for Multiplex 3.
[0022] Figure 3 * indicates a comparison of total flavonoid content in tobacco leaves with high flavonoid content and control tobacco leaves. P <0.05, ** indicates P <0.01.
[0023] Figure 4 * indicates a comparison of chlorogenic acid content in tobacco leaves with high flavonoid content and control tobacco leaves. P <0.05, ** indicates P <0.01.
[0024] Figure 5 To compare the rutin content in tobacco leaves with high flavonoid content and control tobacco leaves, ** indicates P <0.01. Detailed Implementation
[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0026] Example 1: A method for screening tobacco varieties with high flavonoid content based on Multiplex 3. Biological material: Cultivated tobacco K326, 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 was K326 tobacco, and the experimental group was the M4 generation of the EMS mutant of K326 tobacco. The number of K326 tobacco plants in the control group was 200 individual plants, and there were 110 lines of EMS mutant of K326 in the experimental group, with 200 individual plants planted in each line.
[0027] The flowchart for this method can be found here. Figure 1 Specifically, it includes the following steps: Step S1: Select the measurement time. Choose a time with a light intensity of about 400 lux to reduce the impact of excessive light intensity on the Multiplex 3 detection results.
[0028] Step S2: Selection of tobacco growth period, tobacco leaf position, tobacco leaf surface and tobacco leaf back. Select tobacco leaves with the same growth period and leaf position for flavonoid content determination.
[0029] Step S3: Select the measurement location on the tobacco leaf surface, avoiding the leaf veins, and select the same leaf surface location for flavonoid content measurement.
[0030] Step S4: Use the Force-A Multiplex 3 MX315411 to determine the flavonoid content in the tobacco leaves of the control group: Select "Flav" on the instrument panel, press the button on the instrument handle to start the test, and acquire the measurement data after about 1 to 3 seconds; then, based on the measurement values obtained for each tobacco leaf of the control group, plot the normal distribution curve of the multiplex 3 measurement value-density of the control group, calculate the mean (μ) and standard deviation (σ) of the measurement values of the control group, and set the screening threshold to (μ+σ).
[0031] Step S5: By analyzing the measured values of the control group tobacco leaves obtained by Multiplex 3, the range of measured values of the tobacco leaves is determined.
[0032] Step S6: Referring to Step S4, obtain the measurement data of the tobacco leaves in the experimental group. Using the measured data of the experimental group, plot the normal distribution curve of the multiplex 3 measured values versus density of the experimental group. Calculate the mean and standard deviation of the measured values of the experimental group to ensure that the measured data of the experimental group conforms to a normal distribution. Preliminarily screen out tobacco plants with higher measured data and label them.
[0033] Step S7: Use Multiplex 3 to conduct a second screening of tobacco leaves with higher initial screening data from Step S6. The second screening should be conducted at a different time, leaf position, and measurement location than in Step S6. Select tobacco plants with higher measurement data and label them. Based on the measurement values obtained from the second screening, plot a normal distribution curve of Multiplex 3 measurement values versus density, and calculate the mean and standard deviation of the experimental group's measurement values to ensure that the experimental group's measurement data conforms to a normal distribution. Finally, select samples from the control group whose measurement values are greater than the screening range in Step S4, thus obtaining tobacco plants with high flavonoid content.
[0034] Experimental Example 1 According to the method disclosed in Example 1, 200 control group K326 tobacco plants and 22,000 experimental group K326 EMS mutant tobacco plants were selected for the experiment.
[0035] 1. Selection of measurement time: Select a time period with light intensity of about 400 lux for measurement.
[0036] 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 measured.
[0037] 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.
[0038] 4. Determine the flavonoid content in tobacco leaves of the control and experimental groups using Multiplex 3: Select "Flav" on the instrument panel, press the button on the instrument handle to start the test, and complete data acquisition after approximately 1 to 3 seconds. The mean μ of the control group data was 0.582953425, the standard deviation σ was 0.162833879, and the screening threshold (μ+σ) was set to 0.745787304. Figure 2 ).
[0039] 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.
[0040] Furthermore, the present invention used visible light spectrophotometry and high performance liquid chromatography-triple quadrupole mass spectrometry to determine the flavonoid content in dried tobacco leaf samples, in order to determine the accuracy of the screening method provided by the present invention.
[0041] The results of the determination of total flavonoid content in dried tobacco leaves by visible spectrophotometry are as follows: Figure 3 As shown in the figure, the total flavonoid content of the 13 tobacco plants of the EMS mutant with high flavonoid content obtained by screening using Multiplex 3 was 1.54 to 3.13 times higher than that of the control group K326 tobacco.
[0042] The contents of chlorogenic acid and rutin in dried tobacco leaves were determined by high performance liquid chromatography-triple quadrupole mass spectrometry. The results are as follows: Figure 4 and Figure 5 As shown in the figure, the 13 tobacco plants with high flavonoid content obtained by multiplex 3 screening under EMS mutant leaf conditions had higher chlorogenic acid and rutin content than 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.
[0043] The results of the above experimental examples show that the method for screening tobacco varieties with high flavonoid content based on Multiplex 3 provided by this invention is comparable to existing standard methods in terms of both accuracy and reliability. Moreover, the method provided by this invention does not require cumbersome sample preparation and processing steps. It can conveniently, quickly, and instantly determine the flavonoid content of field-grown tobacco without destructive sample collection, which is significantly better than existing standard methods.
[0044] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0045] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
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: Under the same environmental parameters and sampling conditions, the flavonoid content of control tobacco leaf samples and experimental tobacco leaf samples was measured using a Multiplex 3 sensor. Based on the flavonoid content results of the control tobacco leaf samples, the screening threshold was obtained. Based on the screening threshold and the flavonoid content of the experimental tobacco sample leaves, candidate tobacco plants with high flavonoid content were initially screened. Under differentiated conditions by changing at least one of the environmental parameters or sampling conditions, the flavonoid content of the candidate tobacco leaves is measured again using a Multiplex 3 sensor. Tobacco varieties with flavonoid content still higher than the screening threshold under differentiated conditions are considered to have high flavonoid content.
2. The method according to claim 1, characterized in that, The environmental parameters and sampling conditions include the following three types: 1) Select a time period with light intensity of 300 lux to 500 lux for measurement; 2) Select tobacco leaves from the same growth stage and leaf position; 3) Take measurements on the same area on the leaf, avoiding the veins.
3. The method according to claim 1, characterized in that, The leaf area of the tobacco sample leaves is ≥28 cm². 2 If the leaf area of the tobacco sample is <28cm² 2 A black material was used to block the light source; the area of the black material was equal to the area of the tobacco sample leaf plus 28 cm². 2 The area of the difference between them.
4. The method according to claim 1, characterized in that, The leaves are fresh leaves.
5. The method according to claim 1, characterized in that, Setting the filtering threshold includes the following steps: The measured flavonoid content values were subjected to a normal distribution test, and a measured value-density curve was plotted. Calculate the mean μ and standard deviation σ of the control group; Set the filtering threshold to a numerical range from μ+σ to μ+2σ.
6. The method according to claim 1, characterized in that, The differentiation conditions include the following three: 1) Select a testing time different from the initial screening time; 2) Select tobacco leaf positions different from those used in the initial screening; 3) Select measurement locations on tobacco leaves that are different from those used in the initial screening.
7. The method according to claim 1, characterized in that, When using the Multiplex 3 sensor, each sample must be measured at least three times consecutively.
8. The method according to claim 1, characterized in that, The Multiplex 3 sensor is a Force-A Multiplex 3 MX315411.