阅读说明：本技术 一种烟草包衣种子活力的检测方法 (Method for detecting activity of tobacco coated seeds ) 是由 周向平 胡日生 杨全柳 彭曙光 肖钦之 王锡春 麻浩 李清 刘卉 张洪波 于 2021-01-04 设计创作，主要内容包括：本发明公开了一种烟草包衣种子活力的检测方法。在对不同烟草品种包衣种子以及同一烟草品种不同包衣种子批的活力进行检测时,对需要检测的包衣种子首先进行低温胁迫试验,之后再进行标准发芽试验；与此同时对包衣种子处理和标准发芽过程中的萌发指标、活力指标以及生理指标进行测定,并通过对这些指标进行综合分析判断包衣种子活力的高低。本方法简便易操作,对试验条件要求简单,且能准确的鉴定出不同烟草品种包衣种子以及同一烟草品种不同包衣种子批的活力,可为后续的烟草育苗以及优质高效生产提供保障。(The invention discloses a method for detecting the activity of tobacco coated seeds. When the vitality of different coated seeds of different tobacco varieties and different coated seed batches of the same tobacco variety is detected, firstly, a low-temperature stress test is carried out on the coated seeds to be detected, and then, a standard germination test is carried out; meanwhile, the germination index, the vitality index and the physiological index in the coating seed treatment and standard germination processes are measured, and the vitality of the coating seeds is judged by comprehensively analyzing the indexes. The method is simple and easy to operate, has simple requirements on test conditions, can accurately identify the coated seeds of different tobacco varieties and the vitality of different coated seed batches of the same tobacco variety, and can provide guarantee for subsequent tobacco seedling culture and high-quality and high-efficiency production.)

1. A method for detecting the activity of tobacco coated seeds is characterized by comprising the following steps:

carrying out low-temperature stress test on the tobacco coated seeds under the conditions of 14-18 ℃, humidity of 100%, illumination of 11-13 h and darkness of 11-13 h, and setting 3-5 times of repetition;

after the stress test is carried out for 6-8 days, the tobacco coated seeds are turned to 24-25 ℃, the humidity is 90-100%, the standard germination test is carried out for 6-8 days under the conditions of light illumination for 11-13 hours and dark illumination for 11-13 hours, the tobacco coated seeds are enabled to grow for 14-16 days, meanwhile, the germination index, the vitality index and the physiological index in the germination process of the tobacco coated seeds are measured, and the vitality of the coated seeds is judged through comprehensive analysis of the indexes.

2. The method according to claim 1, further comprising measuring and calculating the germination index, vitality index and physiological index of the seedlings grown for 14-16 days and 14-16 days as follows:

(1) the germination indexes are as follows: counting the percentage of the number of the germination coated seeds in the total number of the tobacco coated seeds from the beginning of the test to the end of 14-16 days, and recording as the germination rate;

counting the percentage of the number of the germinated coated seeds in the total number of the coated seeds to be tested from the 7 th or 8 th day after the tobacco coated seeds are put into the test, and recording the percentage as the germination potential;

taking 50 seedlings after 14-16 days of germination, washing the 50 seedlings with tap water, drying the seedlings at 80 ℃ after absorbing the surface water to constant weight, weighing the seedlings until the weight is mg, and taking the average value as the dry weight S; randomly taking 10 normal growing seedlings, respectively measuring the total length of the seedlings by using a ruler, and taking an average value to be recorded as the bud length;

(2) the vitality index is as follows: GI ═ sigma (Gt/DT), wherein GI is germination index, Gt is germination number on day t, and DT is germination number on day t;

VI is S multiplied by GI, wherein VI is vitality index, GI is germination index, and S is dry weight of seedlings;

MGT ═ Σ (Gt × Tt)/∑ Gt; wherein MGT is the average germination time and Gt is the number of newly germinated coated seeds over time Tt;

(3) physiological indexes are as follows:

determination of superoxide dismutase enzyme activity:

weighing coated seed seedlings W which grow for 14-16 days_{Sample (I)}0.500g of each of 2mL of a phosphate buffer solution which had been preliminarily cooled and had a concentration of 50mmol/L and a pH of 7.8 was added to the sterilized grinding tube, and the mixture was quickly ground on ice to give a slurryAdding phosphate buffer solution to the final volume of 5mL, centrifuging at 4 deg.C at 10000rpm for 15min, sucking the supernatant from the tube to another new 1.5mL centrifuge tube, and measuring the volume V_TThen the obtained product is used as crude enzyme liquid of superoxide dismutase for standby;

taking 7 transparent 5mL test tubes with the same texture, wherein 3 test tubes are used as a determination tube, 4 test tubes are used as a reference tube, 3 test tubes in the reference tube are used as a maximum photoreduction tube, and 1 test tube is used as a reference tube, and sequentially adding all solutions or reagents according to the table 1;

thirdly, after all reaction systems are fully and uniformly mixed, 1 contrast tube is placed in a dark place, and the other 3 contrast tubes are used as maximum photoreduction tubes and placed under 4000lux illumination for reaction for 20 min;

the tube was shielded to stop the reaction. Taking a reference tube without illumination as a reference, and respectively measuring the absorbance value A of other tubes at the wavelength of 560 nm;

calculating a result:

it is known that the superoxide dismutase activity Unit is expressed as one enzyme activity Unit (Unit, U) of 50% inhibition of the photochemical reduction of nitroblue tetrazolium, and the superoxide dismutase activity is calculated by the following formula:

wherein: v_QThe dosage (mL) of the sample is measured; a. the_{Maximum light reduction tube}The absorbance value of the maximum reduction tube; a. the_{Sample tube}Is the absorbance value of the sample tube;

TABLE 1 superoxide dismutase Activity measurement reaction System

Determination of malonaldehyde content:

weighing 0.500g of each of a control group and fresh leaves of coated seed seedlings which grow for 14-16 days after each treatment in a precooled mortar, adding 5mL of trichloroacetic acid with the mass percentage of 5%, immediately grinding, transferring the homogenate into another new centrifugal tube after sufficient grinding, and centrifuging at room temperature and 5000rpm for 10 min;

carefully sucking 2mL of supernatant, adding thiobarbituric acid with the equal volume concentration of 0.67%, uniformly mixing, performing boiling water bath for 30min, cooling, and centrifuging;

thirdly, respectively measuring the absorbance of each tube at 450nm, 532nm and 600nm by using a spectrophotometer, and calculating the content of malondialdehyde (mu mol g)^-1Fresh weight:

malondialdehyde content (. mu. mol g)^-1Fresh weight ═ 6.45 × (a)₅₃₂–A₆₀₀)–0.56×A₄₅₀

(4) And (4) carrying out variance analysis on the measured related indexes, and comprehensively judging the vitality of the coated seeds of the tobacco variety through the difference level.

3. A method according to claim 1 or 2, characterized in that for the moisture control in the germination culture dish no clear water is visible in the culture dish, but the filter paper is not allowed to dry.

4. The method according to claim 1, wherein the production process of the naked seeds for coating, the quality of the naked seeds and the coating process all meet the requirements of national standard GB/T2138-2019 tobacco seeds on the germination rate of the coated seeds being more than or equal to 92%, the water content being less than or equal to 3%, the single seed rate being more than or equal to 98%, the seed rate being more than or equal to 99%, the cracking degree being more than or equal to 99%, the particle size being more than or equal to 95%, and the compressive strength being more than or equal to 90%.

5. The method of claim 1, wherein the method can be used for viability testing of coated seeds of different tobacco varieties and batches of coated seeds of the same tobacco variety.

Technical Field

The invention relates to a method for detecting the activity of tobacco coated seeds, belonging to the field of seed quality detection.

Background

"one seed can change one world". The tobacco seeds are special and irreplaceable production data in tobacco leaf production and are carriers of good varieties. The excellent tobacco seeds can improve the seeding quality and the seedling quality, thereby improving the yield and the quality of the tobacco leaves and achieving the purpose of increasing the economic benefit. The seed vigor plays a vital role in the actual production of tobacco, the high-vigor seeds are suitable for early sowing, the seed germination is shortened, the low-temperature germination period is avoided for the tobacco seeds, and the seedling growth time of the seeds is further shortened; meanwhile, the high-activity seeds have the advantage of resisting adverse environmental conditions, the high-activity seeds are deeply sown under the drought condition, water absorption is facilitated, sufficient soil jacking capacity is achieved, and the low-activity seeds cannot push out soil surface under the deep sowing condition; the high-activity seeds have high seedling rate and can reduce the seeding amount, and the low-activity seeds not only have low field emergence rate, but also are often lack of seedlings and broken ridges in the seedling stage, so the high-activity seeds not only save seeds, but also save labor cost, time and labor. The method for screening and identifying the seed vigor of the naked tobacco seeds by an artificial accelerated aging method in actual production is not popularized and used in a large area, but the method for measuring the seed vigor of the coated tobacco seeds is few, so that a scientific and practical method for detecting the seed vigor of the coated tobacco seeds is necessary to be established, the germination and growth conditions of the coated tobacco seeds in a low-temperature environment are accurately estimated, and the method has very important significance for the actual production of tobacco.

Disclosure of Invention

The invention aims to provide a method for detecting the activity of tobacco coated seeds, aiming at the defects, so that the activity of coated seeds of different tobacco varieties and different coated seed batches of the same tobacco variety can be accurately identified.

The purpose of the invention can be realized by the following technical scheme:

a method for detecting the activity of tobacco coated seeds is characterized in that low-temperature stress tests are carried out on tobacco coated seeds under the conditions of temperature of 14-18 ℃, humidity of 100%, illumination of 11-13 h and darkness of 11-13 h, and 3-5 times of repetition are set; after the seeds are stressed for 6-8 days, the seeds are subjected to standard germination tests for 6-8 days under the conditions of 24-25 ℃, humidity of 90-100%, illumination of 11-13 h and darkness of 11-13 h, so that the seeds grow for 14-16 days, and meanwhile, germination indexes, activity indexes and physiological indexes in the germination process of the coated seeds are measured, and the activity of the coated seeds is judged by comprehensively analyzing the indexes.

Further, the method for measuring and calculating the germination index, the vitality index and the physiological index of the seedlings in 14-16 days and 14-16 days after germination comprises the following steps:

(1) the germination indexes are as follows: counting the percentage of the number of the germinated coated seeds in the total number of the test coated seeds from the time when the coated seeds are put in to the 14 th to 16 th days, and recording the percentage as the germination rate; counting the percentage of the number of the germinated coated seeds in the total number of the coated seeds to be tested from the 7 th or 8 th day (belonging to the standard germination period) after the coated seeds are put into the test, and recording the percentage as the germination potential; taking 50 seedlings after 14-16 days of germination, washing the 50 seedlings with tap water, drying the seedlings at 80 ℃ after absorbing the surface water to constant weight, weighing the seedlings until the weight is mg, and taking the average value as the dry weight (S); randomly taking 10 normal growing seedlings, respectively measuring the total length (cm) of the seedlings by using a ruler, and taking an average value to record as the bud length;

(2) the vitality index is as follows: GI ∑ (Gt/DT), where GI is germination index, Gt is germination number on day t, and DT is germination day number. VI ═ S × GI, where VI is the vigor index, GI is the germination index, and S is the dry seedling weight (mg/plant). MGT ═ Σ (Gt × Tt)/∑ Gt. Wherein MGT is the average germination time and Gt is the number of newly germinated coated seeds over time Tt;

(3) physiological indexes are as follows:

determination of superoxide dismutase (SOD) enzyme activity:

weighing 0.500g (W) of coated seed seedlings which grow for 14-16 days_{Sample (I)}) 2mL of pre-chilled phosphate buffer (50mmol/L, pH 7.8) was added to each sterilized grinding tube and quickly ground to a slurry (on ice), phosphate buffer was added to a final volume of 5mL, centrifugation was carried out at 10000rpm at 4 ℃ for 15min, and the supernatant in the tube was carefully pipetted into another new 1.5mL centrifuge tube to measure its volume (V)_T) Then the crude SOD enzyme solution is used for standby;

taking 7 test tubes with the same texture and better transparency and 5mL, wherein 3 test tubes are used as measuring tubes, 4 test tubes are used as reference tubes (3 test tubes are used as maximum photoreduction tubes, and the other test tube is used as a reference tube), and sequentially adding all solutions or reagents according to the table 1;

thirdly, after all reaction systems are fully and uniformly mixed, 1 control tube is placed in a dark place, and other tubes (the other 3 control tubes are used as maximum photoreduction tubes) are placed under 4000lux illumination for reaction for 20 min;

the tube was shielded to stop the reaction. Taking a reference tube without illumination as a reference, and respectively measuring the absorbance values (A) of other tubes at the wavelength of 560 nm;

calculating a result:

the SOD activity Unit is known to be expressed as 50% of inhibitory activity of Nitrobluetetrazolium (NBT) photochemical reduction as one enzyme activity Unit (Unit, U), and is calculated according to the following formula:

note: v_QThe dosage (mL) of the sample is measured;

TABLE 1 SOD Activity measurement reaction System

Determination of Malondialdehyde (MDA) content:

weighing 0.500g of each of a control group and fresh leaves of coated seed seedlings which grow for 14-16 days after each treatment in a precooled mortar, adding 5mL of trichloroacetic acid (TCA, 5 percent) and immediately grinding, transferring the homogenate into another new centrifugal tube after full grinding, and centrifuging at room temperature for 10min (5000 rpm);

carefully sucking 2mL of supernatant, adding thiobarbituric acid (TBA) with the equal volume concentration of 0.67% (prepared by 10% trichloroacetic acid), mixing completely, performing boiling water bath for 30min, cooling, and centrifuging;

thirdly, respectively measuring the absorbance of each tube at 450nm, 532nm and 600nm by using a spectrophotometer, and calculating the content of MDA (mu mol g)^-1Fresh weight):

MDA content (. mu.mol g)^-1Fresh weight ═ 6.45 × (a)₅₃₂–A₆₀₀)–0.56×A₄₅₀

(4) And (4) carrying out variance analysis on the measured related indexes, and comprehensively judging the vitality of the coated seeds of the tobacco variety through the difference level.

Further control of the moisture in the germination petri dish did not allow visible water to be seen in the petri dish, but did not allow the filter paper to dry.

The method is further used for the production process of naked seeds for coating, the quality of the naked seeds and the coating process all meet the requirements of national standards GB/T2138-2019 tobacco seeds that the germination rate of the coated seeds is more than or equal to 92 percent, the water content is less than or equal to 3 percent, the single seed rate is more than or equal to 98 percent, the seed rate is more than or equal to 99 percent, the cracking degree is more than or equal to 99 percent, the particle size conformity is more than or equal to 95 percent, and the compressive strength conformity is more than or equal to 90 percent.

The method can be applied to the activity detection of the coated seeds of different tobacco varieties and different coated seed batches of the same tobacco variety.

The detection method of the invention at least has the following beneficial effects:

the method for detecting the activity of the tobacco coated seeds is less, and the activity of different coated seeds cannot be well distinguished, so that part of coated seeds are slow in seedling emergence and poor in seedling emergence, manpower and material resources are wasted, and the economic benefit is finally influenced. Aiming at the problem, the invention provides (1) a method for detecting the vitality of the coated seeds of the tobacco under the condition of low temperature, which can accurately identify the vitality (the vitality is high or low) of the coated seeds of different tobacco varieties and different coated seed batches of the same tobacco variety, thereby ensuring the germination, seedling alignment and seedling strengthening of the coated seeds of the tobacco, and improving the yield and the quality; (2) the method can be carried out only by a refrigerator and a germination box under the condition of not measuring physiological indexes, so the method is particularly suitable for identifying the activity of the tobacco coating seeds on the base layer; (3) the method is simple, strong in operability, low in cost and free of environmental pollution.

Detailed Description

Example 1:

1. test tobacco variety and coated seed thereof: hunan No. 5 coated seeds coated by two coating materials are provided by the tobacco technology center of Yongzhou city, Hunan province, A is the coated seeds of Hunan No. 5, which adopt talcum powder as a coating agent, and B is the coated seeds of Hunan No. 5, which adopt attapulgite powder as main powder; xiangyan No. 5 is a local variety of Yongzhou in Hunan province, can be well adapted to local production environment, but grows slowly when facing low temperature conditions.

2. And (3) carrying out low-temperature stress treatment on Xiangyan No. 5 coated seeds, carrying out stress treatment for 7 days at the temperature of 16 ℃, and then carrying out standard germination test for 7 days at the temperature of 24 ℃.

3. Experimental treatment

And starting to distribute seeds in 2019, 10 and 20 days, putting wet filter paper in each culture dish, uniformly putting 100 coated seeds on the filter paper, putting the coated seeds into a light culture stress treatment with the temperature of 16 ℃, the humidity of 100%, the light of 12 hours and the darkness of 12 hours, recording the putting time, and starting to record the sprouting number day by day on the next day. And on the day of 2019, 10 and 27, adjusting the temperature of the illumination incubator to 24 ℃, keeping the rest conditions unchanged, continuing to culture and recording related data. In 2019, 11/3, the culture dish was taken out, and germination index (table 2), vitality index (table 2), physiological index (table 3), and the like were measured, and variance analysis was performed.

4. And (3) analyzing an experimental result:

indexes such as germination index, bud length, dry weight, SOD activity, vitality index and the like of the tobacco variety Xiangyan No. 5 coated seed B are obviously higher (P <0.05) than those of the coated seed A, and MDA content is obviously lower (P <0.05) than those of the coated seed A, which indicates that the vitality of the Xiangyan No. 5 coated seed B is obviously higher than that of the coated seed A. The method of the invention can better judge the vitality of the different types of coated seeds of Xiangyan No. 5.

TABLE 2 Germination index and vitality index of Xiangyan No. 5 different types of coated seeds

Note: dry weight is the average dry weight of 50 seedlings, LSD 0.05

TABLE 3 physiological indices of Xiangyan No. 5 different types of coated seeds

Example 2:

1. test tobacco variety and coated seed thereof: the coated seeds of two seed batches of the tobacco variety GX-8 are provided by the tobacco technology center of Yongzhou city, Hunan province. A is GX-8 coated seeds of one seed lot, and B is GX-8 coated seeds of another seed lot. GX-8 is a local variety of Yongzhou in Hunan province, can be well adapted to local production environment, but grows slowly in the face of low temperature conditions

2. And (3) carrying out low-temperature germination treatment on GX-8 coated seeds of two seed batches, carrying out stress treatment for 7 days at the temperature of 16 ℃, and then transferring to a standard germination test for 7 days at the temperature of 24 ℃.

3. The experimental treatment process comprises the following steps:

the seeds are distributed on 20 days in 10 months in 2019, wet filter paper is placed in each culture dish, then 100 coated seeds are uniformly placed on the filter paper, then the culture dish is placed in light culture at 16 ℃, the humidity is 100%, the light culture lasts for 12 hours in the light and the darkness lasts for 12 hours, the placing time is recorded, and the sprouting number of each day is recorded on the next day. And on the day of 2019, 10 and 27, adjusting the temperature of the illumination incubator to 24 ℃, keeping the rest conditions unchanged, continuing to culture and recording related data. In 2019, 11/3, the culture dish was taken out, and germination index (table 4), vitality index (table 4), physiological index (table 5), and the like were measured, and variance analysis was performed.

4. And (3) analyzing an experimental result:

indexes such as germination rate, germination vigor, germination index, bud length, dry weight, SOD activity, vitality index and the like of the tobacco GX-8 coated seed B are obviously (P <0.05) higher than those of the coated seed A, and MDA content is obviously (P <0.05) lower than that of the coated seed A, so that the vitality of the GX-8 coated seed B is obviously higher than that of the coated seed A. The method of the invention can be used for better judging the vitality of the coated seeds of GX-8 different seed batches.

TABLE 4 Germination index and viability index of different GX-8 seed lot coating seeds

Note: dry weight is the average dry weight of 50 seedlings, LSD 0.05

TABLE 5 physiological indices of different seed lots coated with GX-8

In both of the above-described embodiments,

(1) the germination indexes are as follows: counting the percentage of the number of the germinated coated seeds in the total number of the coated seeds to be tested from the beginning of the test to the end of the 14 th day, and recording the percentage as the germination rate; counting the percentage of the number of the germinated coated seeds in the total number of the coated seeds to be tested from the 8 th day after the coated seeds are put into the test, and recording the percentage as the germination potential; on the 14 th day of germination, 50 seedlings are taken and washed clean with tap water, dried at 80 ℃ after surface moisture is absorbed, weighed until the weight is constant, and the weight is accurate to mg, and the average value is taken and recorded as the dry weight (S); randomly taking 10 normal growing seedlings, respectively measuring the total length (cm) of the seedlings by using a ruler, and taking an average value to record as the bud length;

(2) the vitality index is as follows: GI ∑ (Gt/DT), where GI is germination index, Gt is germination number on day t, and DT is germination day number. VI ═ S × GI, where VI is the vigor index, GI is the germination index, and S is the dry seedling weight (mg/plant). MGT ═ Σ (Gt × Tt)/∑ Gt. Wherein MGT is the average germination time and Gt is the number of newly germinated coated seeds over time Tt;

(3) physiological indexes are as follows:

determination of superoxide dismutase (SOD) enzyme activity:

weighing 0.500g (W) of coated seed seedlings after 14 days of growth_{Sample (I)}) 2mL of pre-chilled phosphate buffer (50mmol/L, pH 7.8) was added to each sterilized grinding tube and quickly ground to a slurry (on ice), phosphate buffer was added to a final volume of 5mL, centrifugation was carried out at 10000rpm at 4 ℃ for 15min, and the supernatant in the tube was carefully pipetted into another new 1.5mL centrifuge tube to measure its volume (V)_T) Then the crude SOD enzyme solution is used for standby;

taking 7 test tubes with the same texture and better transparency and 5mL, wherein 3 test tubes are used as measuring tubes, 4 test tubes are used as reference tubes (3 test tubes are used as maximum photoreduction tubes, and the other test tube is used as a reference tube), and sequentially adding all solutions or reagents according to the table 1;

thirdly, after all reaction systems are fully and uniformly mixed, 1 control tube is placed in a dark place, and other tubes (the other 3 control tubes are used as maximum photoreduction tubes) are placed under 4000lux illumination for reaction for 20 min;

the tube was shielded to stop the reaction. Taking a reference tube without illumination as a reference, and respectively measuring the absorbance values (A) of other tubes at the wavelength of 560 nm;

calculating a result:

the SOD activity Unit is known to be expressed as 50% of inhibitory activity of Nitrobluetetrazolium (NBT) photochemical reduction as one enzyme activity Unit (Unit, U), and is calculated according to the following formula:

note: v_QThe dosage (mL) of the sample is measured;

TABLE 1 SOD Activity measurement reaction System

Determination of Malondialdehyde (MDA) content:

weighing 0.500g of each of the control group and the treated fresh leaves of the seedlings of the coated seeds which grow for 14 days into a precooled mortar, adding 5mL of trichloroacetic acid (TCA, 5 percent) into the mortar, immediately grinding the mixture until the mixture is sufficiently ground, transferring the homogenate into another new centrifugal tube, and centrifuging the mixture for 10min (5000rpm) at room temperature;

carefully sucking 2mL of supernatant, adding thiobarbituric acid (TBA) with the equal volume concentration of 0.67% (prepared by 10% trichloroacetic acid), mixing completely, performing boiling water bath for 30min, cooling, and centrifuging;

thirdly, respectively measuring the absorbance of each tube at 450nm, 532nm and 600nm by using a spectrophotometer, and calculating the content of MDA (mu mol g)^-1Fresh weight):

MDA content (. mu.mol g)^-1Fresh weight ═ 6.45 × (a)₅₃₂–A₆₀₀)–0.56×A₄₅₀

(4) And (4) carrying out variance analysis on the measured related indexes, and comprehensively judging the vitality of the coated seeds of the tobacco variety through the difference level.

The above description is only a preferred embodiment of the present patent, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the inventive concept, and these modifications and decorations should also be regarded as the protection scope of the present patent.