CN111505157B

CN111505157B - Nicotine conversion tobacco plant color quality rapid screening method based on non-hatching treatment

Info

Publication number: CN111505157B
Application number: CN202010385199.XA
Authority: CN
Inventors: 李勇; 逄涛; 陈学军; 师君丽; 李永平; 孔光辉
Original assignee: Yunnan Academy of Tobacco Agricultural Sciences
Current assignee: Yunnan Academy of Tobacco Agricultural Sciences
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2022-11-15
Anticipated expiration: 2040-05-09
Also published as: CN111505157A

Abstract

The invention discloses a nicotine conversion tobacco plant chromaticness rapid screening method based on non-hatching treatment, which comprises seedling raising sampling, alkaloid extraction, instrument analysis and data analysis, and compared with the prior art, the method cancels the steps of sample grinding and sample weighing, and shortens the sample extraction time; the scheme also simplifies the determination method of nicotine and nornicotine, saves the sample incubation process, optimizes the gas chromatography-mass spectrometry analysis method, namely, adopts a 270 ℃ constant temperature mode instead of temperature programming, finally ensures that the whole identification process only needs 5-6 minutes, greatly improves the identification efficiency, and is suitable for screening large-scale tobacco plant samples; in addition, the invention introduces the PPNC as a simplified scheme of the PNC for the first time, the PPNC is easier to obtain than the PNC, and is not influenced by factors such as standard product purity, standard curve quality and the like, and the PPNC has a better screening effect than the PNC to a certain extent.

Description

Nicotine conversion tobacco plant color quality rapid screening method based on non-hatching treatment

Technical Field

The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a nicotine conversion tobacco plant chromaticness rapid screening method based on non-hatching treatment.

Background

Nicotine is the major alkaloid of cultivated tobacco (Nicotiana tabacum l.), and usually accounts for about 95% of the total alkaloid content of cultivated tobacco. Nornicotine is a demethylated product of nicotine and is present in lesser amounts in cultivated tobacco but in higher amounts in its ancestral species villiated tobacco and forest tobacco, the major alkaloids of these tobaccos. Siminszky et al demonstrated that nicotine conversion to nornicotine is regulated by cytochrome P450 monooxygenase, designated CYP82E47, whereas CYP82E47 is regulated by leaf-aging-associated signaling pathways. In tobacco cultivation, the conversion of nicotine to nornicotine occurs primarily during tobacco leaf senescence and conditioning. Mutant plants that can convert most of the nicotine to nornicotine in cultivated tobacco are referred to as "transformants". The nicotine content in the transformant is reduced, while the contents of nornicotine, N-nitrosonornicotine (NNN), a tobacco-specific nitrosamine, carcinogen), and macitein are multiplied. The change causes the smoking quality of the tobacco leaf of the transformant to be reduced and the carcinogenic risk to be increased. There are many reports on transformants in burley tobacco and flue-cured tobacco. In burley tobacco, 20% of the transformants can be found at most, the proportion of the transformants in the cured tobacco is about 0.6%, and the cured tobacco is called as cinnabar tobacco or cherry red tobacco, which is popular to a certain extent because of the special glutinous rice flavor. Because the NNN content of normal flue-cured tobacco is low relative to burley tobacco (approximately 1% or less of burley tobacco), the concentration of NNN in flue-cured tobacco transformants is much lower than in burley and burley tobacco transformants.

The identification and removal of transformants from a population of cultivated tobacco is very important for the production of tobacco leaves of stable quality. The common identification method of the transformant generally adopts ethylene, ethephon, sodium bicarbonate and the like as activators to promote the nicotine of the transformant to be converted into a large amount of nornicotine in a short time, and then the transformant is judged by measuring the content of the nicotine and the nornicotine.

The above methods are more heavily focused on field tobacco plants, which typically take several days to identify. Shi et al optimized the incubation conditions of the transformants (including temperature, relative humidity and activators) to achieve maximum conversion of nicotine, and then screened the transformants for maximum conversion. The method is very effective but time-consuming, takes 4-6 days for incubation reaction, and takes several hours for measuring the content of nicotine and nornicotine, so the method is not suitable for rapid screening of large-scale tobacco plant samples.

In summary, how to rapidly screen out transformants from large-scale samples is a problem that needs to be solved at present, and compared with screening and identifying field tobacco plants, the method for rapidly screening out transformants from seedling tray seedlings is more meaningful, but no report of related methods is found at present.

Disclosure of Invention

The invention aims to provide a rapid screening method for the chromaticness of a nicotine-converted tobacco strain based on non-hatching treatment.

The invention aims to realize the method for quickly screening the color quality of the nicotine conversion tobacco strain based on non-hatching treatment, which comprises the following steps of seedling culture sampling, alkaloid extraction, instrument analysis and data analysis:

s1, seedling raising and sampling: sowing and cultivating tobacco seeds of varieties to be screened in a seedling tray, sampling tobacco seedling leaves 45-55 days later, and collecting uniform-sized and uniform-quantity leaf blocks on the leaves;

s2, alkaloid extraction: carrying out alkaloid extraction on the leaf blocks obtained in the step 1 by using an extractant to obtain an alkaloid extract sample;

s3, analyzing by an instrument: analyzing the nicotine and the nornicotine content in the sample by adopting a gas chromatograph-mass spectrometer;

s4, data analysis: performing automatic peak identification and integral on all samples and standards by using a gas chromatograph-mass spectrometer chemical workstation, and calculating a PNC (portable network controller) or PPNC (point-to-point network controller) value, wherein the PNC is the quotient of the nicotine reduction concentration divided by the total nicotine and nicotine reduction concentration, and the PPNC is the quotient of the nicotine reduction peak area divided by the total nicotine and nicotine reduction peak area; when the PNC or PPNC of the tobacco plant is larger than or equal to the corresponding judgment point, the tobacco plant is judged to be a transformant and removed, and when the PNC or PPNC of the tobacco plant is smaller than the corresponding judgment point, the tobacco plant is judged to be a non-transformant and reserved.

The invention uses the gas chromatograph-mass spectrometer to measure the alkaloid in the tobacco seedling leaves, uses the PPNC value as a screening judgment point, establishes a method for quickly identifying the tobacco transformant based on the seedling of the seedling tray, and only needs 5-6 minutes in the whole identification process. The scheme has the advantages that: firstly, compared with the prior art, the scheme cancels the steps of sample grinding and sample weighing, and shortens the time of sample extraction; the scheme also simplifies the measuring method of nicotine and nornicotine, saves the sample incubation process, optimizes the gas chromatography-mass spectrometry analysis method, namely, adopts a 270 ℃ constant temperature mode rather than temperature programming, finally ensures that the whole identifying process only needs 5-6 minutes, greatly improves the identifying efficiency, and is suitable for screening large-scale tobacco plant samples; and secondly, the scheme introduces the PPNC as a simplification scheme of the PNC for the first time, the PPNC is easier to obtain than the PNC, and is not influenced by factors such as standard product purity, standard curve quality and the like, and the PPNC has a better screening effect than the PNC to a certain extent.

Drawings

FIG. 1 is a diagram of the correlation between PNC and PPNC;

FIG. 2 is a graph showing the distribution of values of PNC between transformants and non-transformants;

FIG. 3 is a graph showing the distribution of values of PPNC between transformants and non-transformants;

FIG. 4 is a schematic flow chart of the screening method of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.

The invention relates to a nicotine conversion tobacco plant color quality rapid screening method based on non-hatching treatment, which is characterized by comprising seedling raising sampling, alkaloid extraction, instrument analysis and data analysis, and as shown in figure 4, the method specifically comprises the following steps:

s1, seedling raising and sampling: sowing and cultivating tobacco seeds of varieties to be screened in a seedling tray, sampling leaves of each tobacco seedling 45-55 days later, and collecting leaf blocks with uniform size and quantity on the leaves;

s2, alkaloid extraction: extracting the leaf blocks obtained in the step 1 with an extractant to obtain an alkaloid extract sample;

s4, data analysis: performing automatic peak identification and integration on all samples and standards by using a gas chromatograph-mass spectrometer chemical workstation, and calculating a PNC (potential noise ratio) or PPNC (point of interest) value, wherein the PNC value is the quotient of the molar concentration of the nornicotine divided by the total molar concentration of the nicotine and the nornicotine, and the PNC value is greater than or equal to 4% and is used as a judgment point for screening and removing the transformants; PPNC is the quotient of the peak area of the nicotine and the total peak area of the nicotine and the nicotine, the PPNC value is more than or equal to 0.8 percent and is used as a judgment point for screening and removing the transformed plants, the screened transformed tobacco seedlings are removed, and the non-transformed tobacco seedlings are reserved.

The sampling method comprises the following specific steps: the leaf is folded along the main vein in half, then folded in half, a puncher with the diameter of 5-7mm is used for punching the folded tobacco leaves for 2-4 times, all holes are distributed on the folded tobacco leaves uniformly as much as possible, and 8-16 circular leaf blocks are collected.

In the step 1, the leaf is the largest leaf of the tobacco plant, and the product of the length and the width of the largest leaf is more than 35cm & lt 2 & gt.

In step 2, the extractant is methyl butyl ethyl ether (MTBE), each seedling sample is placed in a 1.5mL centrifuge tube, 0.3mL MTBE containing 1. Mu.g/mL quinoline and 0.5mL 2.5% aqueous sodium hydroxide are added, vortex for 1 minute, 150. Mu.L of supernatant is taken after the extracts are statically layered and transferred to a gas chromatography autosampler injection vial for instrumental analysis. The reason why MTBE was chosen as the extractant is: MTBE can dissolve nicotine and nornicotine well, and has a large difference between polarity and water, so that emulsification phenomenon does not occur during extraction, and the MTBE can be separated from water rapidly.

In step 3, the gas chromatograph-mass spectrometer is in the model of Agilent 7890A-5977B.

In step 3, the gas chromatography conditions of the gas chromatograph-mass spectrometer are as follows: a DB-5MS capillary chromatographic column is adopted, the split flow ratio is 20, the sample injection amount is 2 mu L, the separation temperature is constant at 270 ℃, and the gas flow is 0.5-1.5mL/min. The invention adopts a gas chromatography-mass spectrometry method to separate samples, and the chromatographic analysis does not need complete separation, so that the isothermal separation is adopted. Compared with temperature programming, the separation time is greatly shortened in constant-temperature separation, the time period is longer because the temperature programming needs to spend a certain time in both temperature raising and temperature lowering, the time for constant-temperature separation is only 1.8min, and the total operation time of each sample instrument is 3.4-3.6min.

The carrier gas is helium.

In step 3, the mass spectrometry conditions of the gas chromatograph-mass spectrometer are as follows: selecting an ion mode for data acquisition, wherein the analytical ions of nicotine and nornicotine are respectively set to be 84 and 70; ionization is carried out in an electron bombardment mode, the ionization voltage is 70eV, the detector voltage is 1.0kV, and the temperatures of an ion source and a transmission line are respectively kept at 200 ℃ and 300 ℃.

The tobacco variety is flue-cured tobacco or burley tobacco.

When the non-hatching treatment based rapid screening method for the color and the quality of the nicotine-converted tobacco plants is applied to the identification of the transplanted field tobacco plants or the converted plants of mature tobacco plants before flue-curing, the sampling amount of non-seedling samples is properly reduced or the extracting solution is diluted according to the content of nicotine and nicotine reduction in tobacco leaves of the non-seedling samples, so that the method is ensured to be in a reasonable linear response range.

To verify the feasibility and accuracy of the method of the present invention, all experimental tobacco seedlings in the following examples were screened by the method of the present invention and incubated by a program based on the Shi method (see background), i.e., the samples were incubated at 37 ℃ and 80% relative humidity for 7 days, and then taken out for analysis to determine whether they were transformants, which were used as a reference to determine the accuracy of the method.

Example 1

S1, seedling raising and sampling

Flue-cured tobacco yunyan 85 tobacco seeds were sown in respective seedling trays (9X 18 holes, pore diameter: 3cm X3 cm) in a greenhouse, and sampled 45 days after sowing. For each seedling, the largest leaf (length-width product is more than 35 cm) ² ) The collected leaves are folded along the main vein in half, then folded in half, a hole puncher with the diameter of 6mm is adopted to punch holes on the folded tobacco leaves for 3 times (three holes are uniformly distributed on the folded tobacco leaves as much as possible during punching), 12 circular leaf blocks with the diameter of 6mm are collected, and the circular leaf blocks are placed into a 1.5mL plastic centrifuge tube.

S2, extracting alkaloid

Methyl butyl ether (MTBE) is used as an extracting agent, and quinoline is used as an internal standard. For each seedling sample, 0.3mL MTBE (containing 1. Mu.g/mL quinoline) and 0.5mL 2.5% aqueous sodium hydroxide were added simultaneously, vortexed for 1 minute, and 150. Mu.L of the supernatant, after standing the extracts to separate layers, was transferred to a gas chromatography autosampler vial for instrumental analysis.

S3, instrumental analysis

Analysis was performed using an Agilent 7890A gas chromatograph with a 5977B mass spectrometer. The analysis conditions of the gas chromatography were: the chromatographic column is a DB-5MS capillary chromatographic column (30 m multiplied by 0.25mm multiplied by 0.25 mu m), the flow ratio is 20, the sample injection amount is 2 mu L, the separation temperature is constant at 270 ℃, the separation time is 1.8min, the total operation time of each sample instrument is about 3.5min, and the flow rate of carrier gas (helium gas) is 1.0mL/min. The analysis conditions of the mass spectrum are as follows: data acquisition was performed using a Selective Ion Mode (SIM), with analytical ions for nicotine, nornicotine and quinoline set at 84, 70, respectively; ionization in Electron Impact (EI) mode with ionization voltage of 70eV, detector voltage of 1.0kV, ion source and transmission line temperature maintained at 200 deg.C and 300 deg.C, respectively.

S4, data analysis

Automated peak identification and integration of all samples and standards were performed using an agilent MSD ChemStation f.01.03.2357 chemical workstation, manually checked for integration errors and re-integrated if necessary. The concentration ranges of nicotine and nornicotine used for the calibration curves were 0.78-50. Mu.g/mL and 0.15-10. Mu.g/mL, respectively, with an internal standard (quinoline) concentration of 1.0. Mu.g/mL. Quantitative calibration was done using MSD chemistry workstation and the resulting data was transferred to Microsoft Excel for further analysis: the transformant is distinguished from the non-transformant by the Percentage of Nicotine Conversion (PNC), PNC is defined as the quotient of the nornicotine concentration (mol/L) divided by the total nicotine and nornicotine concentration (mol/L), and the tobacco seedlings with the PNC value of more than or equal to 4% are identified as the transformant, so that the complete elimination of the Yunyan 85 transformant can be realized under the condition of 21% loss of the non-transformant.

Example 2

The method of example 2 was identical to that of example 1, except that in this example, the transformant and the non-transformant were distinguished by PPNC, and tobacco shoots having a PPNC value of 0.9% or more were identified as the transformant, and the method allowed complete elimination of the yunyan 85 transformant at a loss of 7% of the non-transformant.

Example 3

S1, seedling raising and sampling

Sowing burley tobacco TN90 seeds inRespective seedling trays (9X 18 wells, pore diameter: 3cm X3 cm) in the greenhouse were sampled on the 50 th day after sowing. For each seedling, the largest leaf (length-width product greater than 35 cm) is taken ² ) The collected leaves are folded along the main vein, then folded again, a perforator with the diameter of 5mm is adopted to punch 3 times on the folded tobacco leaves (three holes are uniformly distributed on the folded tobacco leaves as much as possible during punching), 12 circular leaf blocks with the diameter of 5mm are collected, and the circular leaf blocks are placed into a 1.5mL plastic centrifuge tube.

S2, alkaloid extraction

Methyl butyl ether (MTBE) is used as an extractant, and quinoline is used as an internal standard. For each seedling sample, 0.3mL MTBE (containing 1. Mu.g/mL quinoline) and 0.5mL 2.5% aqueous sodium hydroxide were added simultaneously, vortexed for 1 minute, and 150. Mu.L of the supernatant, after standing the extracts to separate layers, was transferred to a gas chromatography autosampler vial for instrumental analysis.

S3, instrumental analysis

Analysis was performed using an Agilent 7890A gas chromatograph with a 5977B mass spectrometer. The analysis conditions of the gas chromatography were: the chromatographic column is a DB-5MS capillary chromatographic column (30 m multiplied by 0.25mm multiplied by 0.25 mu m), the flow ratio is 20, the sample injection amount is 2 mu L, the separation temperature is constant at 270 ℃, the separation time is 1.8min, the total operation time of each sample instrument is about 3.5min, and the flow rate of carrier gas (helium gas) is 1.0mL/min. The analysis conditions of the mass spectrum are as follows: data acquisition was performed using a Selective Ion Mode (SIM), with analytical ions for nicotine and nornicotine set at 84, 70, respectively; ionization is carried out in an Electron Impact (EI) mode, the ionization voltage is 70eV, the detector voltage is 1.0kV, and the ion source temperature and the transmission line temperature are respectively maintained at 200 ℃ and 300 ℃.

S4, data analysis

Automated peak identification and integration of all samples and standards were performed using an agilent MSD ChemStation f.01.03.2357 chemical workstation, manually checked for integration errors and re-integrated if necessary. The concentration ranges of nicotine and nornicotine used for the calibration curves were 0.78-50. Mu.g/mL and 0.15-10. Mu.g/mL, respectively, with an internal standard (quinoline) concentration of 1.0. Mu.g/mL. Quantitative calibration was done using MSD chemistry workstation and the resulting data was transferred to Microsoft Excel for further analysis: the transformant and the non-transformant are distinguished by nicotine conversion Percentage (PNC), wherein PNC is defined as the quotient of nornicotine concentration (mol/L) divided by total nicotine and nornicotine concentration (mol/L), and the tobacco seedlings with the measured PNC value of more than or equal to 4.5 percent are identified as the transformant, and the method can realize complete elimination of the burley tobacco N90 transformant under the condition of losing 23 percent of the non-transformant.

Example 4

The method of example 4 is identical to that of example 3, with the only difference that in this example the transformant and the non-transformant are distinguished by means of PPNC, a tobacco shoot with a PPNC value of greater than or equal to 0.8% is identified as the transformant, and the complete elimination of the N90 transformant of burley tobacco can be achieved with a loss of 8% of the non-transformant.

Test example 1

The following verification tests were carried out taking flue-cured tobacco variety Yunyan 85 as an example:

1. experiment of sampling stability

In order to realize the rapid screening of the transformants, the sample weighing step is omitted in the present experiment during the sampling. The stability of the weight of the sampled samples was experimentally evaluated (table 1), indicating that the relative standard deviation of the weight between 10 different tobacco leaf samples was between 4.1 and 4.6%, indicating that the sampling method used can collect samples of more stable weight. The reason for this may be that the growing environment in which the seedlings were grown in the seedling trays was almost identical, the harvested leaves had similar thickness and density, and the stability of the sample weight ensured the comparability of the calculated PNC or PPNC. The field tobacco leaves have larger difference in growing environment, and the weight difference between different samples can be larger if the method used in the experiment is adopted for sampling. However, since the PNC or PPNC is calculated independently of the sample weight, the difference in sample weight does not affect the calculated PNC and PPNC as long as the nicotine and nornicotine concentrations are within the linear response range of the instrument.

TABLE 1 weight distribution of samples obtained by sampling with a punch

	Leaf area (cm) ² )	Sample weight (mg)	RSD(％)
				Transformant (flue-cured tobacco)	68.1±12.3	46.1±2.1	4.6
Non-transformant (flue-cured tobacco)	69.3±13.2	46.0±1.9	4.1

Note that the leaf area is the average of the products of leaf length and leaf width for 10 samples, and the RSD is the relative standard deviation of the sample weight.

2. Experiment for influence of sampling position on analysis result on blade

In order to achieve rapid screening of transformants, 12 leaf pieces were collected from the leaf of seedling as one sample in this experiment, instead of collecting whole leaves or whole plants as specimens. The experiment compared the differences between the samples obtained from the edge and the middle of the seedling leaf (see table 2) and found that the content of nicotine and nornicotine in the edge leaf was higher than in the middle, while the PNC and PPNC values were not significantly different between the edge and middle sampling, which indicates that the sampling position on the leaf had little effect on the PNC and PPNC values, but it is still recommended to perform fixed position sampling of each leaf, for example, when the tobacco leaf is perforated after folding in this experiment, the perforation is performed 3 times evenly distributed on the tobacco leaf surface.

TABLE 2 influence of leaf sampling position on PNC and PPNC

Note that each set included 10 samples.

3. Experiment of influence of blade size on analysis result

Table 3 comparatively analyzes the effect of large leaf sampling and small leaf sampling on PNC and PPNC at 45 days of cured tobacco seedlings, and the results show that the contents of nicotine and nornicotine do not differ significantly between large leaf and small leaf, nor do PNC and PPNC differ significantly. Considering the difficulty of sampling too small leaves, it is recommended to choose a length-width product exceeding 35cm ² The blade of (a) is sampled.

Table 3 effect of leaf surface size on PNC and PPNC.

Note that the leaf area is the product of leaf length and leaf width

4. Distribution of PNC in unhatched seedlings

The contents of nicotine and nornicotine in the transformed and non-transformed plants were directly measured and PNC was calculated without incubation, as shown in table 4, showing that the nicotine content of the cured tobacco transformed plant was higher than that of the non-transformed plant, while the nornicotine content was lower than that of the non-transformed plant. This means that nicotine in the transformants already started to convert to nornicotine at the seedling stage.

The 221 flue-cured tobacco transformant-non-transformant population is analyzed, the average value of the PNC of the transformant is 4 times of that of the non-transformant, and the difference shows that the seedling transformants can be distinguished through the PNC. By examining the distribution of PNC in the transformants and non-transformants (FIG. 2), it was found that when the PNC value was 5% as a determination point for selecting and removing transformants, 99% of the transformants could be removed with a loss of 7% of the non-transformants, and when the PNC value was 4% as a determination point, the transformants could be completely removed with a loss of 21% of the non-transformants. Although part of non-transformants are lost in the method, the transformants can be rapidly screened out.

TABLE 4 PNC and PPNC value distribution of transformants and non-transformants

	Nicotine (ug/g)	Nicotine reduction (ug/g)	PNC(％)	PPNC(％)
					Flue-cured tobacco transformant (101 plants)	113.3±45.0	13.4±5.2	12.1±5.2	2.7±1.4
Non-transformed plant of flue-cured tobacco (120 plants)	144.4±45.0	4.1±1.3	3.2±1.4	0.6±0.3

5. Feasibility experiment Using PPNC

Since PNC is calculated from the absolute quantification of nicotine and nornicotine, the value of PNC is affected by the purity of the standard and the quality of the calibration curve. To simplify discrimination and eliminate possible errors, the experiment defines PPNC as the quotient of the nornicotine peak area divided by the total nicotine and nornicotine peak areas. A linear fit to PNC and PPNC can find that there is a strong linear correlation between the two (R2 =0.9968, see fig. 1): comparing the PNC and PPNC differentiation of the flue-cured tobacco transformant-non-transformant population, the PNC and PPNC of the transformant were found to be 3.8 times and 4.5 times of the non-transformant, respectively (see Table 4). Further examining the effect of both on the discrimination of the transformant (FIGS. 2 and 3), it was found that the discrimination of the transformant with the PPNC value of 0.9% could achieve the complete elimination of the transformant with only 8% loss of the non-transformant. When the PNC value is used for discrimination, 21% of non-transformants are lost to complete elimination of transformants. Therefore, the calculation of PPNC in this experiment is simpler than PNC and the effect is better when used for transformant screening.

As a result: from the above experiments, it was found that the average PNC and PPNC of the seedling transformants cultured without hatching were 3.8 and 4.5 times that of the non-transformants, respectively, and that complete rejection of the transformants could be achieved with only 8% loss of the non-transformants when screening was performed using PPNC. Therefore, in the case of rapid screening, screening can be performed in a manner of incubation-free culture.

The method simplifies the determination method of nicotine and nornicotine, eliminates the steps of sample grinding and sample weighing, shortens the extraction time of the sample, optimizes the GC-MS analysis method (adopts a 270 ℃ constant temperature mode instead of temperature programming), and finally ensures that the whole identification process only needs 5-6 minutes (as shown in figure 4).

The method established by the invention is also suitable for distinguishing the transplanted field tobacco plants or mature tobacco plants before curing. However, since nicotine and nornicotine are accumulated during the growth of tobacco, their contents are also much higher than those of young plants. Therefore, for non-seedling samples, the sampling amount should be properly reduced or the extracting solution should be diluted according to the content of nicotine and nornicotine in tobacco leaves, so as to ensure that the method is in a reasonable linear response range.

Claims

1. A nicotine conversion tobacco plant rapid screening method based on non-hatching treatment is characterized by comprising the steps of seedling raising sampling, alkaloid extraction, instrument analysis and data analysis, and specifically comprises the following steps:

s1, seedling raising and sampling: after tobacco seeds of varieties to be screened are sowed and cultured in a seedling raising tray for 45-55 days, sampling each tobacco seedling leaf, and collecting leaf blocks with uniform size and quantity on the leaf blocks;

s2, alkaloid extraction: extracting the leaf blocks obtained in the step S1 with an extractant to obtain an alkaloid extract sample;

s4, data analysis: performing automatic peak identification and integration on all samples and the standard substance by using a gas chromatograph-mass spectrometer chemical workstation, and calculating PNC (nicotine content index), wherein PNC is the quotient of the nicotine reduction concentration divided by the total concentration of nicotine and nicotine reduction; when the tobacco is Yunyan 85, identifying the tobacco seedlings with the PNC value being more than or equal to 4% of the judgment point as the transformants, and when the tobacco is burley TN90, identifying the tobacco seedlings with the PNC value being more than or equal to 4.5% of the judgment point as the transformants, and removing the transformants; when the PNC of the tobacco plant is smaller than the corresponding judgment point, judging the tobacco plant as a non-transformed plant, and keeping the non-transformed plant.

2. A nicotine conversion tobacco plant rapid screening method based on non-hatching treatment is characterized by comprising the steps of seedling raising sampling, alkaloid extraction, instrument analysis and data analysis, and specifically comprises the following steps:

s1, seedling raising and sampling: after sowing and cultivating tobacco seeds of varieties to be screened in a seedling tray for 45-55 days, sampling tobacco seedling leaves of each plant, and collecting leaf blocks with uniform size and quantity on the leaves;

s4, data analysis: performing automatic peak identification and integral on all samples and standard substances by using a gas chromatograph-mass spectrometer chemical workstation, and calculating a PPNC (point to point numerical control) value, wherein the PPNC is a quotient of a nicotine reduction peak area divided by a total nicotine and nicotine reduction peak area; when the tobacco is Yunyan 85, identifying the tobacco seedlings with the PPNC value being more than or equal to 0.9% of the judgment point as the transformants, and when the tobacco is burley tobacco TN90, identifying the tobacco seedlings with the PPNC value being more than or equal to 0.8% of the judgment point as the transformants, and removing the transformants; and when the PPNC of the tobacco plant is smaller than the corresponding judgment point, identifying the tobacco plant as a non-transformant and reserving the non-transformant.

3. The method according to claim 1 or 2, wherein the sampling is carried out by folding the lamina in half along the main vein, then folding it in half, perforating the folded tobacco leaves 2-4 times with a perforator having a diameter of 5-7mm, distributing the perforations as uniformly as possible on the folded tobacco leaves, and collecting 8-16 circular leaf blocks.

4. The method according to claim 1 or 2, wherein the leaf in step S1 is the largest leaf of the selected tobacco plant, and the product of length and width of the largest leaf is greater than 35cm ² 。

5. The method of claim 1 or 2, wherein the extractant in step S2 is MTBE, each seedling sample is placed in a 1.5mL centrifuge tube, 0.3mL MTBE containing 1 μ g/mL quinoline and 0.5mL 2.5% aqueous sodium hydroxide are added, vortexed for 1 minute, and 150 μ L of the supernatant is transferred to a gas chromatography autosampler vial for instrumental analysis after the extracts are allowed to stratify at rest.

6. The method according to claim 1 or 2, wherein the GC conditions of the GC-MS in step S3 are: and a DB-5MS capillary chromatographic column is adopted, the split flow ratio is 20, the sample injection amount is 2 mu L, the separation temperature is 270 ℃, the temperature is constant, the separation time is 1.8min, the total operation time of each sample instrument is 3.4-3.6min, and the carrier gas flow is 0.5-1.5mL/min.

7. The method of claim 6, wherein the carrier gas is helium.

8. The method according to claim 1 or 2, wherein the mass spectrometry conditions of the GC in step S3 are: selecting an ion mode for data acquisition, wherein the analytical ions of nicotine and nicotine reducing are respectively set to be 84 and 70; ionization is carried out in an electron bombardment mode, the ionization voltage is 70eV, the detector voltage is 1.0kV, and the temperatures of an ion source and a transmission line are respectively kept at 200 ℃ and 300 ℃.

9. Use of the method of claim 1 or 2 for the identification of post-transplant field tobacco plants or mature tobacco plant transformants prior to harvest.