CN112666316A - Method for distinguishing cinnabar smoke from common smoke - Google Patents

Abstract

Translated fromChinese

本发明属于烟草鉴别技术领域，具体公开一种分辨硃砂烟和普通烟的方法，包括以下步骤：配制缺铁的1/2霍格兰营养液和含铁1/2霍格兰营养液；挑取同时播种下长成幼苗的烟草，取发芽后生长10天或两个月的烟草，进行水培，在正常铁浓度的1/2霍格兰营养液中适应3天，然后进行缺铁水培21天；水培21天后，通过叶片表型或测定叶绿素含量和SPAD值来区别硃砂烟和普通烟，在缺铁培养下，叶绿素含量较高或叶片表型为深绿色的则为硃砂烟，所用霍格兰营养液配制方便，成本较低；该方法能很好的区分硃砂烟和普通烟草，所培育的烟草，有助于科技工作者对硃砂烟形成的分子机理的研究，以及前期种子的筛选和纯度的鉴定，促进农业生产。

The invention belongs to the technical field of tobacco identification, and specifically discloses a method for distinguishing cinnabar tobacco from ordinary tobacco, comprising the following steps: preparing iron-deficient 1/2 Hoagland nutrient solution and iron-containing 1/2 Hoagland nutrient solution; Take the tobacco that has grown into seedlings sown at the same time, take the tobacco grown for 10 days or two months after germination, carry out hydroponics, adapt to 1/2 Hoagland nutrient solution of normal iron concentration for 3 days, and then carry out iron-deficient hydroponics 21 days; after 21 days of hydroponics, cinnabar tobacco and ordinary tobacco were distinguished by leaf phenotype or determination of chlorophyll content and SPAD value. The used Hoagland nutrient solution is convenient to prepare and has a low cost; the method can well distinguish cinnabar tobacco from ordinary tobacco, and the cultivated tobacco is helpful for scientific and technological workers to study the molecular mechanism of cinnabar tobacco formation, as well as the early seeds Screening and identification of purity to facilitate agricultural production.

Description

Method for distinguishing cinnabar smoke from common smoke

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)

Translated fromChinese

1.一种分辨硃砂烟和普通烟的方法，其特征在于，使用缺铁的1/2霍格兰营养液培育烟草幼苗后，通过叶片表型或测定叶绿素含量和SPAD值来区别硃砂烟和普通烟。1. a method for distinguishing cinnabar tobacco and common tobacco, is characterized in that, after using iron-deficient 1/2 Hoagland nutrient solution to cultivate tobacco seedlings, distinguish cinnabar tobacco and cinnabar tobacco by leaf phenotype or measure chlorophyll content and SPAD value. Ordinary cigarettes.2.根据权利要求1所述的一种分辨硃砂烟和普通烟的方法，其特征在于，包括以下步骤：2. a kind of method of distinguishing cinnabar smoke and common smoke according to claim 1, is characterized in that, comprises the following steps:（1）配制缺铁的1/2霍格兰营养液和含铁的1/2霍格兰营养液；(1) Prepare iron-deficient 1/2 Hoagland nutrient solution and iron-containing 1/2 Hoagland nutrient solution;（2）挑取同时播种下长成幼苗的烟草，取发芽后生长10天或两个月的烟草，进行水培，在含铁的1/2霍格兰溶液中适应3天，然后用缺铁1/2霍格兰溶液水培21天；(2) Pick the tobacco that has grown into seedlings sown at the same time, take the tobacco that has grown for 10 days or two months after germination, carry out hydroponics, adapt to 1/2 Hoagland solution containing iron for 3 days, and then use the Iron 1/2 Hoagland solution hydroponically for 21 days;（3）水培21天后，通过叶片表型或测定叶绿素含量和SPAD值来区别硃砂烟和普通烟，在缺铁培养下，叶绿素含量高或叶片表型为深绿色的则为硃砂烟。(3) After 21 days of hydroponics, cinnabar tobacco and common tobacco were distinguished by leaf phenotype or determination of chlorophyll content and SPAD value. Under iron-deficiency culture, cinnabar tobacco with high chlorophyll content or dark green leaf phenotype was classified as cinnabar tobacco.3.根据权利要求2所述的一种分辨硃砂烟和普通烟的方法，其特征在于，发芽后生长10天，水培21天后的烟草，采集烟草到三叶，进行叶片表型或测定叶绿素含量和SPAD值来区别硃砂烟和普通烟。3. a kind of method for distinguishing cinnabar tobacco and common tobacco according to claim 2, it is characterized in that, grow 10 days after germination, the tobacco after 21 days of hydroponics, collect tobacco to three leaves, carry out leaf phenotype or measure chlorophyll Content and SPAD value to distinguish cinnabar tobacco and ordinary tobacco.4.根据权利要求2所述的一种分辨硃砂烟和普通烟的方法，其特征在于，发芽后生长两个月，水培21天后的烟草，采集顶端第一片真叶进行叶片表型或测定叶绿素含量和SPAD值来区别硃砂烟和普通烟。4. a kind of method for distinguishing cinnabar tobacco and common tobacco according to claim 2, it is characterized in that, grow two months after germination, the tobacco after 21 days of hydroponics, collect top first true leaf to carry out leaf phenotype or Chlorophyll content and SPAD value were determined to distinguish cinnabar tobacco from common tobacco.5.根据权利要求2或3或4所述的一种分辨硃砂烟和普通烟的方法，其特征在于，所述含铁的1/2霍格兰溶液，其铁浓度为40μmol/L，霍格兰溶液配制时所加铁盐为乙二胺四乙酸铁钠盐。5. a kind of method for distinguishing cinnabar smoke and common smoke according to claim 2 or 3 or 4, is characterized in that, described iron-containing 1/2 Hoagland solution, its iron concentration is 40 μmol/L, and the iron concentration is 40 μmol/L. The iron salt added in the preparation of Glan's solution is ethylenediaminetetraacetic acid iron sodium salt.6.根据权利要求5所述的一种分辨硃砂烟和普通烟的方法，其特征在于，所述硃砂烟品种来源于云烟87-硃砂烟的种子库，普通烟的品种为云烟87。6 . The method for distinguishing cinnabar tobacco from common tobacco according to claim 5 , wherein the variety of said cinnabar tobacco comes from the seed bank of Yunyan 87-cinnabar tobacco, and the variety of common tobacco is Yunyan 87. 7 .

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