CN112666316B - A method for distinguishing between cinnabar smoke and common smoke - Google Patents

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 deficient in iron and 1/2 Hoagland nutrient solution containing iron; selecting tobacco which grows into seedlings under the condition of sowing simultaneously, selecting tobacco which grows for 10 days or two months after germination, carrying out water culture, adapting to 3 days in 1/2 Hoagland nutrient solution with normal iron concentration, and then carrying out iron-deficiency water culture for 21 days; after 21 days of water culture, differentiating cinnabar smoke and common smoke by virtue of leaf phenotype or measuring chlorophyll content and SPAD value, and under the condition of iron-deficiency culture, determining cinnabar smoke if the chlorophyll content is higher or the leaf phenotype is dark green, wherein the used Hoagland nutrient solution is convenient to prepare and has low cost; the method can well distinguish the cinnabar smoke from the common tobacco, and the cultivated tobacco is beneficial to research on molecular mechanism of cinnabar smoke formation by science and technology workers, screening of early-stage seeds and identification of purity, and promotes agricultural production.

Description

A method for distinguishing between cinnabar smoke and common smoke

technical field

The invention belongs to the technical field of tobacco identification, and in particular relates to a method for distinguishing cinnabar smoke and common smoke.

Background technique

Nicotiana is an annual or limited perennial herbaceous plant of the family Solanaceae. It is widely planted all over the world and has high economic value, food value and medicinal value. Cinnabar tobacco refers to the appearance of fully mature tobacco leaves with cinnabar markings after roasting. It is commonly known as cinnabar tobacco, and it is called Cherry Red tobacco in foreign countries. It has a prominent aroma and unique flavor. Cherry-red and non-red varieties were obtained from Nicotiana tabacum , L. cv. 401 strain in the early stage abroad. Einosuke Wada reported a significant increase in nornicotine content of cherry red varieties of tobacco during tobacco curing, consistent with the appearance of red substances, and proposed a correlation between them.

The inheritance of cinnabar tobacco belongs to epigenetics, and the main reason for the appearance of cinnabar tobacco in flue-cured tobacco is that CYP82E4 gene is induced and strongly expressed, so that nicotine in flue-cured tobacco is transformed into normethyl nicotine. Gene main effect, environment interaction, and upper-middle typicality determine the functional expression of genes of apparent traits, and they can be stably inherited. According to the comprehensive judgment of relevant information, with the increase of algebra, the nicotine conversion rate of cinnabar nicotine gradually increases, that is, most of the nicotine is converted into normethyl nicotine, resulting in a decrease in nicotine content.

Under the existing seed bank resources of Yunyan 97-cinnabar tobacco and Yunyan 87-cinnabar tobacco, in order to meet the promotion of production in major tobacco areas and the needs of smokers, and facilitate the scientific establishment of identification standards for cinnabar tobacco, a fast and convenient identification is needed The method, such as CN201911140582.2 in the prior art, is based on the quantitative conversion rate of nicotine and the method of gene expression screening of Nicotiana cinnabar seeds. The process is complicated, the cost is high, and it is not suitable for rapid identification in the production process. Another patent is CN202010111937.1. The method for rapid identification of flue-cured cinnabar leaves tends to be further identified after the leaves are picked and cured, and is not suitable for rapid determination of seed purity in the early stage. These two methods cannot directly distinguish cinnabar tobacco and ordinary tobacco from the phenotype in the actual cultivation and screening of cinnabar tobacco. Therefore, a method for quickly identifying cinnabar tobacco in the early stage of tobacco planting is currently needed.

Contents of the invention

The main purpose of the present invention is to provide a method for distinguishing between cinnabar smoke and ordinary tobacco, so as to meet the needs of production, quickly distinguish between cinnabar smoke and ordinary tobacco, and measure the purity of tobacco seeds in the early stage to ensure the reliability of the final product.

To achieve the above object, the present invention mainly provides the following technical solutions:

A method for distinguishing cinnabar tobacco from common tobacco. After cultivating tobacco seedlings with iron-deficient 1/2 Hoagland nutrient solution, distinguish cinnabar tobacco from common tobacco by leaf phenotype or measuring chlorophyll content and SPAD value.

Further, a method for distinguishing cinnabar smoke from common smoke comprises the following steps:

(1) Prepare iron-deficient 1/2 Hoagland nutrient solution and iron-containing 1/2 Hoagland nutrient solution;

(2) Pick the tobacco that grows into seedlings after sowing 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's solution containing iron for 3 days, and then use the lack of Iron 1/2 Hoagland solution hydroponics for 21 days;

(3) After 21 days of hydroponic cultivation, cinnabar tobacco and common tobacco were distinguished by leaf phenotype or determination of chlorophyll content and SPAD value. Under iron-deficiency culture, those with high chlorophyll content or dark green leaf phenotype were cinnabar tobacco.

Preferably, grow 10 days after germination, hydroponic the tobacco after 21 days, collect tobacco to three leaves, carry out leaf phenotype or measure chlorophyll content and SPAD value to distinguish cinnabar tobacco and common tobacco.

Preferably, two months after germination, after 21 days of hydroponic tobacco, the first true leaf at the top is collected for leaf phenotype or the determination of chlorophyll content and SPAD value to distinguish cinnabar tobacco from ordinary tobacco.

Preferably, the iron-containing 1/2 Hoagland's solution has an iron concentration of 40 μmol/L, and the iron salt added when preparing the Hoagland's solution is iron sodium salt of ethylenediaminetetraacetate.

Preferably, the cinnabar tobacco variety comes from the seed bank of Yunyan 87-Cinnabar tobacco, and the common tobacco variety is Yunyan 87.

The present invention uses iron deficiency and iron concentration (40μM) 1/2 Hoagland nutrient solution to hydroponic tobacco, and distinguishes cinnabar tobacco and common cloud 87 tobacco through leaf phenotype after 21 days. The effect is remarkable, the treatment method is simple, and it is conducive to popularization. The Hoagland nutrient solution used is easy to prepare and the cost is low; this method can well distinguish cinnabar tobacco and common tobacco, especially for the existing Yunyan 87-cinnabar tobacco seed bank, and the cultivated tobacco is helpful for scientific and technological work The researchers' research on the molecular mechanism of cinnabar smoke formation, as well as the screening and purity identification of early seeds, promote agricultural production.

The present invention uses iron-deficiency 1/2 Hoagland nutrient solution, and further cooperates with the cultivation method, so that the phenotypes of cinnabar tobacco and ordinary tobacco are significantly changed, so that tobacco can be quickly identified, and the cost is low and the operation is convenient .

Description of drawings

Figure 1 shows the tobacco seedlings grown in hydroponics with 1/2 Hoagland nutrient solution with different iron concentrations (0, 40, 160, 320 μM) for 10 days, and after 21 days, the tobacco reached three leaves (the third from top to bottom) one true leaf) phenotype map;

Fig. 2 is a comparison chart of tobacco chlorophyll content and SPAD value after 21 days of 1/2 Hoagland nutrient solution hydroponic growth of tobacco seedlings with different iron concentrations (0, 40, 160, 320 μM) of the present invention; The chlorophyll content from tobacco to three leaves (the third true leaf from top to bottom), picture B is the SPAD value from tobacco to three leaves (the third true leaf from top to bottom);

Figure 3 is the phenotype diagram of the leaves of a single tobacco plant at different positions after 21 days of hydroponic growth of tobacco grown in 1/2 Hoagland nutrient solution with different iron concentrations (0, 40 μM) in the present invention; where Figure A is a single plant The phenotypes of leaves at different positions after the iron-deficiency treatment of tobacco plants, and the figure B is the phenotype of leaves at different positions after normal iron treatment of single tobacco plants;

Fig. 4 shows the SPAD values of leaves at different positions after 21 days of hydroponic growth of tobacco grown in 1/2 Hoagland nutrient solution with different iron concentrations (0, 40 μM) in accordance with the present invention.

detailed description

The present invention will be described in further detail below through the accompanying drawings and examples, but the protection scope of the present invention is not limited to the content described. Unless otherwise specified, the methods in the examples are carried out according to conventional operations. Unless otherwise specified, the reagents used are commercially available reagents or reagents prepared according to conventional methods. Named as CR60 for expression, the Hoagland nutrient solution prepared by the present invention is a classic configuration formula, the specific preparation method can refer to the literature D.R. Hoagland, D.I. Arnon, The Water-Culture Method for Growing Plants Without Soil, California Agricultural Experimental Station Bulletin 347 (1937), the expressions of iron concentration in the embodiments of the present invention are all abbreviations, and its unit is μ mol/L.

Example 1

Tobacco seedlings grown for 10 days were hydroponically grown in 1/2 Hoagland nutrient solution with different iron concentrations (0, 40, 160, 320 μM), including the following steps:

(1) The experimental material is tobacco; select the tobacco seedlings that have grown for 10 days after germination, and move them to the hydroponic box filled with Hoagland nutrient solution with iron concentration (40 μM). Change the Hoagland nutrient solution once a day;

(2) After 21 days, observe the leaf phenotype, and take photos of tobacco to three leaves (the third true leaf from top to bottom);

As shown in Figure 1, the leaves of Yun 87 are yellower than those of cinnabar tobacco (CR60) when iron is deficient, and it is easy to distinguish between Yun 87 and cinnabar tobacco (CR60). However, under the treatment of high iron concentration (160, 320 μM), there was no obvious difference in leaf color between Yun 87 and cinnabar smoke (CR60).

Adopt the tobacco seedling after embodiment 1 to measure the chlorophyll content of tobacco, comprise the steps:

A. Sampling tobacco leaves treated with different iron concentrations (0, 40, 160, 320 μM) 1/2 Hoagland nutrient solution for 21 days, collecting four tobacco plants to three leaves (the third true leaf from top to bottom) ) different parts, use a balance to accurately weigh 0.1g of fresh weight;

B. Cut the leaves into 1-2mm filaments, add them to 2 mL of extract solution (99.5% dimethyl sulfoxide), and extract chlorophyll in a water bath at 65°C for 160 minutes;

C. After cooling, add 8 mL of acetone (80% volume fraction) and mix well;

D, measure chlorophyll content with ultraviolet spectrophotometry: measure the absorbance value under OD663 and OD646 respectively;

E. Chlorophyll a (Ca (mg/L)) = 12.27×A663- 2.52×A646 (A means absorbance);

Chlorophyll b (Ca (mg/L)) = 20.10×A646- 4.92×A663; 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 Figure 2, Figure A shows the chlorophyll content of tobacco leaves treated with different iron concentrations (0, 40, 160, 320 μM) 1/2 Hoagland nutrient solution for 21 days, and the chlorophyll content of Yun 87 was significantly lower than that of cinnabar when iron was deficient Tobacco (CR60), under the treatment of high iron concentration (160, 320μM), there was no significant difference in chlorophyll content. Consistent with the leaf color shown in Figure 1.

The tobacco seedling after adopting embodiment 1 processing measures the SPAD value of tobacco, comprises the steps:

A, with SPAD-502 Plus (Konica Minolta, Japan) chlorophyll meter measuring the SPAD value of embodiment 1 tobacco to three leaves (the third true leaf counting from top to bottom);

As shown in Figure 2, Figure B shows the SPAD value of tobacco leaves treated with different iron concentrations (0, 40, 160, 320 μM) 1/2 Hoagland nutrient solution for 21 days, which represents the chlorophyll content and is determined by UV spectrophotometry The trend of chlorophyll content was consistent. When iron was deficient, the chlorophyll content of Yun 87 was significantly lower than that of cinnabar tobacco (CR60). Under the treatment of high iron concentration (160, 320 μM), there was no significant difference in chlorophyll content. Consistent with the leaf color shown in Figure 1.

Example 2

Tobacco grown for 2 months was hydroponically grown in 1/2 Hoagland nutrient solution with different iron concentrations (0, 40 μM), including the following steps:

(1) The experimental material is tobacco; select the tobacco that has grown for 2 months after germination, move it to a hydroponic box filled with 1/2 Hoagland nutrient solution with an iron concentration (40 μM), and treat it with different iron concentrations after three days of adaptation. Change the Hoagland nutrient solution every 3 days;

(2) After 21 days, observe the leaf phenotype and take photos of individual tobacco leaves;

As shown in Figure 3, picture A is a picture of a single plant leaf of cinnabar tobacco (CR60) and Yun 87 hydrocultured for 21 days with iron deficiency; picture B is a picture of a single plant leaf of cinnabar tobacco (CR60) and Yun 87 hydrocultured for 21 days with normal iron concentration. In picture A, the top leaf color of cinnabar smoke (CR60) is obviously greener than that of Yun 87, which can distinguish cinnabar smoke from Yun 87 very well.

Adopt the tobacco after the treatment of embodiment 2 to measure the SPAD value of the leaves of different positions of the single plant, measure the SPAD value of the 6 leaves of the single plant tobacco of the embodiment 2 with the SPAD-502 Plus (Konica Minolta, Japan) chlorophyll meter with embodiment 1;

As shown in Figure 4, it is the SPAD value of leaves at different positions of a single tobacco plant after 21 days of treatment with different iron concentrations. The SPAD value of the first leaf at the top of cinnabar tobacco (CR60) was significantly higher than that of Yun 87, indicating that the chlorophyll content of cinnabar tobacco (CR60) was higher than that of Yun 87, which was consistent with the leaf color in Figure 3.

Claims (2)

1. a method for distinguishing cinnabar smoke and common smoke, is characterized in that, comprises the following steps: (1) Prepare iron-deficient 1/2 Hoagland nutrient solution and iron-containing 1/2 Hoagland nutrient solution; (2) Pick the tobacco that grows into seedlings after sowing 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's solution containing iron for 3 days, and then use the lack of Iron 1/2 Hoagland solution hydroponics for 21 days; (3) After 21 days of hydroponic cultivation, the cinnabar tobacco and common tobacco were distinguished by leaf phenotype or SPAD value of chlorophyll content. Under iron-deficiency culture, the chlorophyll content was significantly different from common tobacco or the leaf phenotype was dark green The other is cinnabar smoke; The iron-containing 1/2 Hoagland's solution has an iron concentration of 40 μmol/L, and the iron salt added during the preparation of the Hoagland's solution is iron sodium ethylenediaminetetraacetate; When using tobacco grown 10 days after germination and 21 days after hydroponics, collect the tobacco to three leaves, perform leaf phenotype or measure the SPAD value that characterizes the chlorophyll content to distinguish cinnabar tobacco from ordinary tobacco; When using tobacco that has been grown for two months after germination and hydrocultured for 21 days, the first true leaf at the top is collected for leaf phenotype or the SPAD value that characterizes the chlorophyll content is used to distinguish cinnabar tobacco from ordinary tobacco. 2 . A method for distinguishing cinnabar tobacco from common tobacco according to claim 1 , wherein the cinnabar tobacco variety comes from the seed bank of Yunyan 87-cinnabar tobacco, and the common tobacco variety is Yunyan 87. 3 .

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