CN112666316A - Method for distinguishing cinnabar smoke from common smoke - Google Patents
Method for distinguishing cinnabar smoke from common smoke Download PDFInfo
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- 229910052956 cinnabar Inorganic materials 0.000 title claims abstract description 67
- 239000000779 smoke Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 30
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 64
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 63
- 241000208125 Nicotiana Species 0.000 claims abstract description 56
- 229910052742 iron Inorganic materials 0.000 claims abstract description 32
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims abstract description 31
- 229930002875 chlorophyll Natural products 0.000 claims abstract description 30
- 235000019804 chlorophyll Nutrition 0.000 claims abstract description 30
- 235000015097 nutrients Nutrition 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 206010022971 Iron Deficiencies Diseases 0.000 claims abstract description 16
- 230000035784 germination Effects 0.000 claims abstract description 9
- 238000005259 measurement Methods 0.000 claims description 3
- QPWHIXUCVYSLML-UHFFFAOYSA-K C(C)(=O)[O-].C(C)(=O)O.C(C)(=O)[O-].C(C)(=O)[O-].[Fe+2].C(CN)N.[Na+] Chemical compound C(C)(=O)[O-].C(C)(=O)O.C(C)(=O)[O-].C(C)(=O)[O-].[Fe+2].C(CN)N.[Na+] QPWHIXUCVYSLML-UHFFFAOYSA-K 0.000 claims description 2
- 150000002505 iron Chemical class 0.000 claims description 2
- 244000061176 Nicotiana tabacum Species 0.000 abstract description 8
- 238000012216 screening Methods 0.000 abstract description 4
- 238000012271 agricultural production Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 25
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical class CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 6
- 229960002715 nicotine Drugs 0.000 description 6
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 4
- 239000003501 hydroponics Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001973 epigenetic effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 244000006449 Nicotiana forgetiana Species 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 229930002868 chlorophyll a Natural products 0.000 description 1
- 229930002869 chlorophyll b Natural products 0.000 description 1
- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- -1 demethyl nicotine Chemical compound 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
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- 239000003205 fragrance Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of tobacco identification, and particularly discloses a method for distinguishing cinnabar smoke from common smoke, which comprises the following steps: preparing 1/2 Hoagland nutrient solution with iron deficiency and 1/2 Hoagland nutrient solution with iron; selecting tobacco which is sown at the same time and grows into seedlings, selecting tobacco which grows for 10 days or two months after germination, carrying out water culture, adapting to 1/2 Hoagland nutrient solution with normal iron concentration for 3 days, and then carrying out iron-deficiency water culture for 21 days; after 21 days of water culture, cinnabar and common smoke are distinguished by leaf phenotype or measuring chlorophyll content and SPAD value, under the condition of iron-deficiency culture, cinnabar is obtained if the chlorophyll content is higher or the leaf phenotype is dark green, and the used Hoagland nutrient solution is convenient to prepare and has lower cost; the method can well distinguish the cinnabar from the common tobacco, and the cultivated tobacco is beneficial to research on molecular mechanism of cinnabar formation by technologists, screening of early-stage seeds and identification of purity, and promotes agricultural production.
Description
Technical Field
The invention belongs to the technical field of tobacco identification, and particularly relates to a method for distinguishing cinnabar smoke from common smoke.
Background
Nicotiana, family Solanaceae, is an annual or limited perennial herb. The method has wide planting in all parts of the world, and has very high economic value, edible value and medicinal value. The cinnabar smoke refers to the condition that the appearance of fully-ripe tobacco leaves after being baked presents cinnabar stripes, is commonly called cinnabar smoke, is called Cherry Red tobacco (Cherry Red) abroad, and has prominent fragrance and unique flavor. Early foreignNicotiana tabacumCherry red and red-free varieties were obtained from line L, cv. 401. Einosuke Wada reported a significant increase in the demethylated nicotine content of the cherry red variety of tobacco during the tobacco curing process, consistent with the appearance of red color species, and suggested a correlation between them.
The genetic inheritance of the cinnabar belongs to epigenetic inheritance, and the main reason for the emergence of the cinnabar in the flue-cured tobacco is that the CYP82E4 gene is induced to generate strong expression, so that nicotine in the flue-cured tobacco is converted into demethylnicotine. Gene major, environmental interactions, central-upper canonical, gene functional expression that determines the epigenetic trait, and is stably heritable. According to the comprehensive judgment of relevant information, the nicotine conversion rate of the cinnabar smoke is gradually increased along with the increase of the algebra, namely most of nicotine is converted into demethyl nicotine to cause the decrease of the nicotine content.
Under the existing seed bank resources of the yunyan 97-cinnabar and the yunyan 87-cinnabar, in order to meet the popularization and production of various big smoke areas and the requirements of smokers and facilitate scientifically formulating the identification standard of the cinnabar, a quick and convenient identification method is needed, for example, a method for screening cinnabar seeds based on the quantitative conversion rate and gene expression of nicotine in CN201911140582.2 in the prior art has the defects of complex process, high cost and inapplicability to quick identification in the production process, and a method for quickly identifying flue-cured tobacco cinnabar tobacco leaves in the patent CN202010111937.1 is also available, tends to further identification after the tobacco leaves are picked to be baked, and is not applicable to quick judgment of the purity of the seeds in the early stage. The two methods cannot directly distinguish the cinnabar smoke from the common smoke from the phenotype in the actual cultivation and screening of the cinnabar smoke, so that a method for quickly distinguishing the cinnabar smoke in the early stage of tobacco planting is needed at present.
Disclosure of Invention
The invention mainly provides a method for distinguishing cinnabar and common smoke at present, so as to be convenient for production requirements, quickly distinguish cinnabar and common smoke, and determine the purity of tobacco seeds in the early stage so as to ensure the reliability of final products.
In order to achieve the above purpose, the invention mainly provides the following technical scheme:
a method for distinguishing cinnabar from common tobacco comprises culturing tobacco seedlings with 1/2 Hoagland nutrient solution with iron deficiency, and distinguishing cinnabar from common tobacco by leaf phenotype or measuring chlorophyll content and SPAD value.
Further, the method for distinguishing the cinnabar smoke from the common smoke comprises the following steps:
(1) preparing 1/2 Hoagland nutrient solution with iron deficiency and 1/2 Hoagland nutrient solution with iron;
(2) selecting tobacco which is sown at the same time and grows into seedlings, selecting tobacco which grows for 10 days or two months after germination, carrying out water culture, adapting to 1/2 Hoagland solution containing iron for 3 days, and then carrying out water culture for 21 days by using 1/2 Hoagland solution lacking iron;
(3) and (3) after 21 days of water culture, distinguishing the cinnabar from the common cinnabar by virtue of leaf phenotype or measuring the chlorophyll content and the SPAD value, and under the condition of iron-deficiency culture, judging that the cinnabar is high in chlorophyll content or the leaf phenotype is dark green.
Preferably, the tobacco is grown for 10 days after germination and cultured in water for 21 days, and three leaves are collected and subjected to leaf phenotype or chlorophyll content and SPAD value measurement to distinguish the cinnabar smoke from the common smoke.
Preferably, the first true leaf at the top is collected for leaf phenotype or chlorophyll content and SPAD value measurement to distinguish cinnabar smoke from common smoke after growing for two months after germination and water culture for 21 days.
Preferably, the iron-containing 1/2 Hoagland solution has an iron concentration of 40 μmol/L, and the iron salt added during the preparation of the Hoagland solution is ethylenediamine tetraacetic acid iron sodium salt.
Preferably, the cinnabar product is derived from a seed bank of Yunnan 87-cinnabar, and the common cigarette is Yunnan 87.
According to the method, the iron-deficiency and iron-content concentration (40 mu M) 1/2 Hoagland nutrient solution is used for hydroponics of the tobacco, and the cinnabar and the common cloud 87 tobacco are distinguished through the leaf phenotype after 21 days, so that the effect is remarkable, the treatment method is simple, the popularization is facilitated, the preparation of the Hoagland nutrient solution is convenient, and the cost is low; the method can well distinguish the cinnabar from the common tobacco, particularly the tobacco cultivated aiming at the existing cinnabar 87-cinnabar seed bank, is beneficial to research on the molecular mechanism of the formation of the cinnabar by technologists, screening of early-stage seeds and identification of purity, and promotes agricultural production.
The invention uses the iron-deficiency 1/2 Hoagland nutrient solution and further cooperates with the culture method to ensure that the phenotype of the cinnabar smoke and the common smoke has obvious change, thereby being capable of rapidly identifying the tobacco, and having low cost and convenient operation.
Drawings
FIG. 1 is a table graph of tobacco seedlings grown for 10 days after 21 days from tobacco to three leaves (third true leaf from top to bottom) by hydroponics with 1/2 Hoagland nutrient solution of different iron concentrations (0, 40, 160, 320 μ M) according to the present invention;
FIG. 2 is a graph comparing the chlorophyll content and SPAD value of tobacco seedlings grown in water culture for 10 days in 1/2 Hoagland nutrient solution at different iron concentrations (0, 40, 160, 320 μ M) according to the invention after 21 days; wherein, the A graph is the chlorophyll content from tobacco to three leaves (the third true leaf counted from top to bottom), and the B graph is the SPAD value from tobacco to three leaves (the third true leaf counted from top to bottom);
FIG. 3 is a table graph of leaves of individual tobacco at different positions after 21 days when tobacco plants grown for 2 months in accordance with the present invention were hydroponically cultured with 1/2 Hoagland nutrient solution at different iron concentrations (0, 40. mu.M); wherein, the A picture is the phenotype of the leaves at different positions after the iron deficiency treatment of the single tobacco, and the B picture is the phenotype of the leaves at different positions after the normal iron treatment of the single tobacco;
FIG. 4 is a graph showing the SPAD values of leaves in different positions after 21 days, for 2 months of tobacco grown in hydroponics with 1/2 Hoagland nutrient solutions of different iron concentrations (0, 40. mu.M) according to the present invention.
Detailed Description
The invention is explained in more detail below with reference to the figures and examples, without limiting the scope of the invention. In The examples, unless otherwise specified, The Method is carried out according to conventional procedures, if no specific specification, The reagents are all conventional commercial reagents or reagents prepared according to conventional methods, and for The convenience of distinction, The cinnabar screened by The Yunyan 87 in The examples are collectively named as CR60, The Hoagland nutrient solution prepared by The Method is a classical formula, and The specific preparation Method can be referred to documents D.R. Hoagland, D.I. Arnon, The Water-cut Method for Growing Plants Without Soil, California Agricultural experiment Station cloth 347 (1937), and The expression of The iron concentration in The examples of The invention is abbreviated and has The unit of mu mol/L.
Example 1
Tobacco seedlings grown for 10 days were hydroponically cultured with 1/2 Hoagland nutrient solutions of different iron concentrations (0, 40, 160, 320. mu.M), comprising the following steps:
(1) the experimental material is tobacco; selecting tobacco seedlings which grow for 10 days after germination, transferring the tobacco seedlings to a hydroponic box filled with Hoagland nutrient solution with iron concentration (40 mu M), carrying out treatment with different iron concentrations after three days, and changing the Hoagland nutrient solution every 3 days;
(2) observing leaf phenotype after 21 days, and taking pictures from tobacco to three leaves (the third true leaf from top to bottom);
as shown in figure 1, the color of the iron-deficiency cloud 87 leaf is more yellow than that of the cinnabar (CR 60), and the cloud 87 and the cinnabar (CR 60) are well distinguished. There was no significant difference in leaf color between the cloud 87 and the cinnabar (CR 60) treated with the high iron concentration (160, 320. mu.M).
The method for measuring the chlorophyll content of the tobacco by using the tobacco seedlings treated in the example 1 comprises the following steps:
A. respectively sampling tobacco leaves treated by 1/2 Hoagland nutrient solution with different iron concentrations (0, 40, 160 and 320 mu M) for 21 days, collecting four tobacco plants to different parts of three leaves (the third true leaf counted from top to bottom), and accurately weighing 0.1g fresh weight by using a balance;
B. cutting the leaves into 1-2mm filaments, adding into 2 mL of extractive solution (containing 99.5% dimethyl sulfoxide), and extracting chlorophyll in water bath at 65 deg.C for 160 min;
C. after cooling, 8 mL of acetone (volume fraction of 80%) is added and mixed evenly;
D. measuring chlorophyll content by ultraviolet spectrophotometry: respectively measuring the light absorption values under OD663 and OD 646;
E. chlorophyll a (Ca (mg/L)) = 12.27 × a 663-2.52 × a646 (a represents absorbance);
chlorophyll b (Ca (mg/L)) = 20.10 × a 646-4.92 × a 663; total chlorophyll concentration C (a + b) = Ca + Cb;
chlorophyll content (mg/g fresh weight) = chlorophyll concentration × extract volume × dilution factor)/(sample fresh weight);
as shown in FIG. 2, graph A shows that the chlorophyll content of tobacco leaves treated by 1/2 Hoagland nutrient solution for 21 days at different iron concentrations (0, 40, 160 and 320 mu M), the chlorophyll content of the cloud 87 is obviously lower than that of cinnabar smoke (CR 60) in the iron deficiency state, and the chlorophyll content has no significant difference in the treatment at high iron concentration (160 and 320 mu M). Consistent with the color of the leaf shown in fig. 1.
The SPAD value of tobacco was determined using the tobacco seedlings treated in example 1, comprising the following steps:
A. SPAD values of tobacco from example 1 to three leaves (third true leaf from top to bottom) were measured with a SPAD-502 Plus (Konica Minolta, Japan) chlorophyll apparatus;
as shown in FIG. 2, panel B is the SPAD value of tobacco leaves treated with 1/2 Hoagland nutrient solution at different iron concentrations (0, 40, 160, 320 μ M) for 21 days, which value characterizes the chlorophyll content, and is consistent with the trend of chlorophyll content measured by ultraviolet spectrophotometry, the chlorophyll content of cloud 87 is obviously lower than that of cinnabar (CR 60) in iron deficiency, and the chlorophyll content has no significant difference under the treatment of high iron concentration (160, 320 μ M). Consistent with the color of the leaf shown in fig. 1.
Example 2
Tobacco grown for 2 months in hydroponics with 1/2 Hoagland nutrient solutions of different iron concentrations (0, 40. mu.M) was prepared by the following steps:
(1) the experimental material is tobacco; selecting tobacco growing for 2 months after germination, moving the tobacco to a water culture box filled with 1/2 Hoagland nutrient solution with iron concentration (40 mu M), processing different iron concentrations after three days, and changing the Hoagland nutrient solution every 3 days;
(2) observing the leaf phenotype after 21 days, and taking a picture of the single tobacco leaf;
as shown in FIG. 3, Panel A is a picture of single leaf of Cinnabaris (CR 60) and cloud 87 in iron-deficiency water culture for 21 days; the B picture is a picture of a single leaf cultivated in water for 21 days at normal iron concentration of cinnabar and cloud 87. The color of the top leaf of the cinnabar (CR 60) in the A picture is obviously greener than that of the cloud 87, so that the cinnabar and the cloud 87 can be well distinguished.
The SPAD value of the leaves at different positions of the single plant was measured by using the tobacco treated in example 2, and the SPAD value of the 6 leaves of the single plant in example 2 was measured by using a SPAD-502 Plus (Konica Minolta, Japan) chlorophyll meter in the same manner as in example 1;
as shown in FIG. 4, the SPAD values of the leaves of the individual tobacco plants at different positions after 21 days of treatment with different iron concentrations were obtained. The SPAD value of the 1 st blade on the top end of the cinnabar (CR 60) is obviously higher than that of the cloud 87, which shows that the chlorophyll content of the cinnabar (CR 60) is higher than that of the cloud 87, and the color of the blade is consistent with that of the blade in figure 3.
Claims (6)
1. A method for distinguishing cinnabar smoke from common smoke is characterized in that after a tobacco seedling is cultured by using 1/2 Hoagland nutrient solution with iron deficiency, the cinnabar smoke and the common smoke are distinguished by virtue of leaf phenotype or by measuring chlorophyll content and SPAD value.
2. The method for differentiating cinnabar smoke and common smoke according to claim 1, comprising the steps of:
(1) preparing 1/2 Hoagland nutrient solution with iron deficiency and 1/2 Hoagland nutrient solution with iron;
(2) selecting tobacco which is sown at the same time and grows into seedlings, selecting tobacco which grows for 10 days or two months after germination, carrying out water culture, adapting to 1/2 Hoagland solution containing iron for 3 days, and then carrying out water culture for 21 days by using 1/2 Hoagland solution lacking iron;
(3) and (3) after 21 days of water culture, distinguishing the cinnabar from the common cinnabar by virtue of leaf phenotype or measuring the chlorophyll content and the SPAD value, and under the condition of iron-deficiency culture, judging that the cinnabar is high in chlorophyll content or the leaf phenotype is dark green.
3. A method of differentiating cinnabar smoke from common smoke according to claim 2, wherein the tobacco is grown for 10 days after germination, water cultured for 21 days, collected from three leaves, and subjected to leaf phenotype or chlorophyll content and SPAD value measurement to distinguish the cinnabar smoke from the common smoke.
4. A method of differentiating cinnabar smoke from common smoke according to claim 2, wherein the tobacco is grown in water culture for 21 days for two months after germination, and the first true leaf on the top is collected for leaf phenotype or the chlorophyll content and SPAD value are measured to differentiate cinnabar smoke from common smoke.
5. A method for distinguishing cinnabar from common smoke according to claim 2, 3 or 4, wherein said iron-containing 1/2 Hoagland solution has an iron concentration of 40 μmol/L, and the iron salt added in the Hoagland solution is ethylenediamine tetraacetic acid iron sodium salt.
6. The method for differentiating the cinnabar and the common smoke according to claim 5, wherein the cinnabar smoke is derived from a seed bank of cinnabar 87-cinnabar, and the common smoke is cinnabar 87.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584211A (en) * | 2021-08-09 | 2021-11-02 | 云南省烟草农业科学研究院 | Method for screening cinnabar plants based on CYP82E4 gene expression level |
| CN115478076A (en) * | 2021-09-28 | 2022-12-16 | 昆明理工大学 | Method for improving instantaneous expression of exogenous protein of cultured tobacco cloud 87 and application |
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| 潘义宏等: "不同品种中部烟叶SPAD值及其与叶绿素含量的相关性分析", 《河南农业大学学报》 * |
| 马林等: "烟草品种的SCAR标记鉴别", 《中国烟草学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584211A (en) * | 2021-08-09 | 2021-11-02 | 云南省烟草农业科学研究院 | Method for screening cinnabar plants based on CYP82E4 gene expression level |
| CN115478076A (en) * | 2021-09-28 | 2022-12-16 | 昆明理工大学 | Method for improving instantaneous expression of exogenous protein of cultured tobacco cloud 87 and application |
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