A method for quality cultivation of rich organo-selenium chives

Abstract

The application discloses a kind of rich organic selenium quality cultivation methods of chives, belong to plant cultivation technical field, comprising the following steps: step one, seed disinfection;Step two, germination;Step three, seedling;Step four, transplanting;Step five, routine management;Step six, selenium;Step seven, harvest.The total selenium content of chives treated by sodium selenite can reach 13.4~95.8mg / kg DW, compared with conventional cultivation of chives, increased by 111.32~1411.1 times.Selenium methionine is the main selenium form, the content can reach 1.7~6.9mg / kg DW, while significantly improving other nutritional quality such as soluble sugar, total phenolic acid and total flavonoids.The rich selenium chives cultivated by root application with exogenous sodium selenite as selenium source is not only simple, quick and safe in production operation, but also solves the problem of single nutritional value and low organic selenium content in existing cultivation technology, improves the added value of chives, and provides diversified sources for human selenium supplementation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant cultivation, and particularly relates to a selenium-rich garlic quality cultivation method and application thereof. BACKGROUND

[0002] Garlic (Allium ascalonicum) is a herbaceous plant of the Liliaceae family (Allium) and has important edible and medicinal values. Research shows that garlic has a certain selenium enrichment capacity. As one of the three major seasonings in vegetable varieties, garlic has a large demand and has become one of the characteristic vegetables of "south vegetables northward transportation" and "west vegetables eastward transportation".

[0003] Selenium is an essential trace mineral element for human and animals and plays an important role in maintaining the health of the body. It is reported that about 2 / 3 of the areas in China are selenium-deficient, among which the northeast region is the most concentrated, with an average selenium content of 0.318 ug / g in soil. Plants can convert inorganic selenium in nature into organic selenium that is easy for human body to absorb, and are excellent carriers for dietary selenium supplementation. Exogenous selenium fortification cultivation can effectively improve the selenium content in plants and can be used as an effective strategy to improve the selenium deficiency status of human body.

[0004] However, the garlic cultivated by the current cultivation technology has single nutritional value and low organic selenium content, and has great limitations in application. SUMMARY

[0005] The purpose of the present application is to provide a selenium-rich garlic quality cultivation method that can be applied in production, so as to improve the added value of garlic and promote the development of selenium-rich garlic industry, and provide diversified sources for human body selenium supplementation.

[0006] The present application is realized by the following technical solutions:

[0007] A selenium-rich garlic quality cultivation method, comprising the following steps:

[0008] Step 1: seed disinfection

[0009] The garlic seeds are soaked in warm water at 55 DEG C for 15 min;

[0010] Step 2: germination

[0011] The seeds soaked in step 1 are soaked at 30 DEG C for 24 h and then transferred to a culture dish for germination;

[0012] Step 3: seedling raising

[0013] After the seeds are white, they are sown in a plug tray and seedling raising is carried out in a light incubator;

[0014] Step 4: transplanting

[0015] When the chives seedlings grow 4-5 true leaves, transplant them into plastic flower pots in a greenhouse, cultivate them by mixing peat and soil, and irrigate them with Hoagland nutrient solution after transplanting to ensure their normal growth;

[0016] Step five, daily management:

[0017] Irrigate the chives with water 10 times per day, apply conventional fertilizer 2 times, weed 3 times, and apply insecticide 1 time.

[0018] Step six, selenium application:

[0019] After the chives tiller, irrigate the roots with sodium selenite;

[0020] Step seven, harvesting:

[0021] On the seventh day after the last time of selenium application, harvest the chives.

[0022] Further, the germination temperature in step two is 30°C.

[0023] Further, the white appearance in step three is 70% white appearance, and after 70% of the seeds have white appearance, use 72-hole plug seedlings, and the parameters of the light incubator are set as follows: day and night temperatures are 20 / 15°C, light period is 15 / 9h, relative humidity is 50%-70%, and light intensity is 2500Lx.

[0024] Further, the substrate used in step three is a mixture of peat, perlite and vermiculite with a mass ratio of 2:1:1.

[0025] Further, the flower pot size in step four is 14*12*12cm, and the peat and soil are fully mixed with a mass ratio of 1:1 and weighed at 2.5kg.

[0026] Further, the temperature of the greenhouse is controlled at 16-20°C, and the relative humidity is 65-75%.

[0027] Further, the Hoagland nutrient solution is irrigated every 7 days after transplanting, and 100-150mL is applied each time.

[0028] Further, the sodium selenite solution concentration in step six is 20-240mg / L, 100mL is irrigated per pot each time, the interval between selenium applications is 5-7 days, and the number of times of selenium application is 2-3 times.

[0029] Further, the sodium selenite solution concentration is 160mg / L, 7 days once, and a total of 3 times.

[0030] Compared with the prior art, the present application has the following advantages:

[0031] 1. The chives selenium-enriched cultivation method of the present application adds a step of root application of sodium selenite to the conventional cultivation, i.e. after tillering of the chives, sodium selenite solution is applied to the roots every 7 days for a total of 3 times, the application concentration of the sodium selenite ranges from 20 to 240 mg / L, the total selenium content can reach 13.4 to 95.8 mg / kg DW, which is 166.5 to 1411.1 times higher than that of the chives cultivated conventionally. Selenium methionine is the main selenium form, and the content can reach 1.7 to 6.9 mg / kg DW, and the application of sodium selenite significantly improves the contents of other nutritional qualities such as soluble sugar, total phenolic acid and total flavonoids.

[0032] 2. The method provided by the present application improves the contents of soluble sugar, flavonoids and total phenolic acid while improving the selenium content of chives. The method can guide the field cultivation of selenium-enriched chives and improve the added value of chives.

[0033] 3. The present application guides the production of stable and high-quality selenium-enriched chives, solves the problems of single nutritional value and low organic selenium content in the existing cultivation technology, promotes the development of selenium-enriched chive industry, and provides diversified sources for human selenium supplementation.

[0034] 4. The selenium-enriched chives cultivated by the present application using exogenous sodium selenite as the selenium source through root application are not only simple and rapid to produce and operate, but also relatively safe. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a schematic diagram of the effects of sodium selenite of different concentrations on the plant weight (A), plant height (B), diameter (C) and root length (D) of chives provided by the present application;

[0036] Figure 2 is a schematic diagram of the effects of sodium selenite on the contents of chlorophyll a (A), chlorophyll b (B), chlorophyll a+b (C), soluble protein (D) and soluble sugar (E) of chives provided by the present application;

[0037] Figure 3 is a schematic diagram of the effects of sodium selenite treatment on the contents of total flavonoids (A) and total phenolic acid (B) in the aboveground parts of chives provided by the present application;

[0038] Figure 4 is a schematic diagram of the effects of sodium selenite on the antioxidant enzyme activity in the aboveground parts of chives provided by the present application;

[0039] Figure 5 is a schematic diagram of the effects of sodium selenite treatment on the total selenium contents in the aboveground and underground parts of chives provided by the present application;

[0040] Figure 6is a schematic view of the effect of different concentrations of sodium selenite on the total sulfur content of the above-ground parts of chives provided by the embodiment of the present application. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0042] EMBODIMENT

[0043] A chive organoselenium-rich quality cultivation method, comprising the following steps:

[0044] Step one, seed disinfection:

[0045] Chive seeds are soaked in warm water at 55°C for 15 minutes;

[0046] Step two, germination:

[0047] After soaking at 30°C for 24 hours, the seeds are transferred to a culture dish for germination at a temperature of 30°C;

[0048] Step three, seedling raising:

[0049] After the seeds have 70% of the white part exposed, 72-hole plug seedlings are raised, the substrate is uniformly mixed with peat, perlite and vermiculite at a mass ratio of 2:1:1, the seedling substrate is loaded into the tray one day before sowing, water is poured through, and the surface is smoothed, the germinated chive seeds are sown in the plug, and placed in a light incubator for culture, the light incubator parameters are set as follows: day and night temperatures are 20 / 15°C, light period is 15 / 9h, relative humidity is 50%-70%, and light intensity is 2500Lx;

[0050] Step four, transplanting:

[0051] When the chive seedlings grow 4-5 true leaves, they are transplanted into a greenhouse plastic pot containing a mixture of grass charcoal and soil (1:1 mixed thoroughly) weighing 2.5 kg, in order to ensure normal growth of the chives, Hoggan nutrient solution is manually irrigated every 7 days after transplanting, and 100-150 mL is applied each time;

[0052] The Hoagland nutrient solution is developed by Qingdao Haibo Bio (HB8870-1), and its usage is as follows: 1.26 g of the Hoagland nutrient solution is weighed in 1000 ml of distilled water, heated and dissolved, and then divided and sterilized at 115°C for 20 minutes for standby use. After dissolution, the solution composition includes: potassium sulfate 607 mg / L, ammonium dihydrogen phosphate 115 mg / L, magnesium sulfate 493 mg / L, EDTA iron sodium salt 20 mg / L, ferrous sulfate 15 mg / L, boric acid 2.86 mg / L, borax 4.5 mg / L, manganese sulfate 2.13 mg / L, copper sulfate 0.05 mg / L, zinc sulfate 0.22 mg / L, ammonium sulfate 0.02 mg / L;

[0053] Step five, daily management:

[0054] The normal growing chives are watered 10 times, fertilized 2 times, weeded 3 times, and de-insected once;

[0055] Step six, selenium application:

[0056] After the chives tiller, select the plants with consistent growth, 50 plants for a small area, set three biological repeats, use sodium selenite as the main selenium source, set 6 selenium concentration gradients, i.e. 0, 20, 40, 80, 160, and 240 mg / L, to perform root irrigation treatment, once every 7 days, 100 mL of sodium selenite solution is applied to each pot each time, and the treatment is performed 3 times;

[0057] Step seven, harvesting:

[0058] The chives are harvested 7 days after the last selenium application.

[0059] During harvesting, the aboveground and underground parts are taken, the underground part is washed with clean water, and then rinsed with deionized water and dried after the surface moisture is removed, 10 plants are taken as a group in a random sampling manner, the plant weight, plant height, diameter, and root length of the chives are measured, and the values are recorded.

[0060] The chive seeds involved in this example are purchased from the agricultural market in Jingzhou City, Hubei Province, and the variety name is Four Seasons Small Chives. Unless otherwise specified, the raw materials, reagents, and equipment involved in the following examples are obtained through commercial channels.

[0061] Please refer to Figure 1 After applying different concentrations of sodium selenite solution, the plant weight and plant height of the chives show a downward trend, but there is no significant difference between 160 mg / L and the control, and the plant weight and plant height are significantly lower than the control when the sodium selenite concentration is 240 mg / L. Figure 1 A, Figure 1 B). When measuring the diameter, the upper part of 1 cm of the white onion is measured using a vernier caliper, Figure 1C) Sodium selenite treatment had no significant effect on the diameter of chives. Roots are important organs for plant nutrition. After sodium selenite treatment, the root length of chives showed an upward trend and then decreased, and was significantly higher than the control group at 40 mg / L and 80 mg / L sodium selenite treatment (D). Figure 1 D). This shows that applying low concentrations of sodium selenite does not cause stress or toxicity to chive growth, but inhibits its growth at high concentrations of 240 mg / L.

[0062] After the growth measurement was completed, the physiological indicators were determined, including chlorophyll, soluble protein and soluble sugar content.

[0063] The determination of chlorophyll content in chives was carried out according to the book "Plant Physiology Experiment Guide", and the specific operation was as follows:

[0064] Accurately take 96 mL of anhydrous ethanol in a 100 mL volumetric flask, add water to the calibration line, accurately weigh 0.2 g of chive leaves in a clean mortar, add a small amount of calcium carbonate powder and a small amount of 96% ethanol, grind to homogenate, then add 5 mL of 96% ethanol, continue to grind until the tissue turns white. Take a filter paper and place it in the funnel, wet it with 96% ethanol, pour the extract along the glass rod into the funnel, and filter the filtrate into a 10 mL centrifuge tube wrapped with tin foil paper, wash the mortar and rod with a small amount of 96% ethanol, and finally pour the filtrate into the funnel, wash the filter paper and residue with 96% ethanol several times until no green color is left. Finally, make up with 96% ethanol, shake well. Measure the absorbance values at wavelengths of 470 nm, 649 nm and 665 nm, respectively. The results are calculated as follows: chlorophyll a = 13.95*OD 665 -6.88*OD 649 ; chlorophyll b = 24.96*OD 649 -7.32*OD 665 ;

[0065] The soluble protein content of chive aboveground part was determined by Coomassie brilliant blue G-250, and the specific steps were as follows:

[0066] (1) Solution preparation:

[0067] ① 100 μg / mL bovine serum protein solution: accurately weigh 100 mg of bovine serum protein in a clean beaker, dissolve in water and make up to 100 mL, which is 1000 μg / mL protein stock solution. Accurately take 1 mL of stock solution in a 10 mL volumetric flask and make up with deionized water, which is 100 μg / mL protein solution. ② Coomassie brilliant blue G-250 solution: accurately weigh 100 mg of Coomassie brilliant blue G-250 powder in a clean beaker, add 50 mL of 90% ethanol solution to dissolve, then add 100 mL of 85% phosphoric acid, and finally make up to 1000 mL with distilled water.

[0068] (2) Standard curve drawing:

[0069] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1 mL of bovine serum albumin solution (100 μg / mL) into clean volumetric flasks, respectively, add 1, 0.8, 0.6, 0.4, 0.2, 0 mL of distilled water, continue to add 5 mL of Coomassie brilliant blue G-250 solution to each tube, mix well, prepare protein solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively, measure the absorbance value at 595 nm after standing for 2 min, and draw the standard curve.

[0070] (3) Sample extraction:

[0071] Accurately weigh 0.2 g of fresh chives leaves in a mortar, add 2 mL of distilled water and grind until homogenized, then wash the mortar with 4 mL of distilled water and transfer it into a 10 mL centrifuge tube, centrifuge at 4000 rpm for 20 min, and measure.

[0072] (4) Determination of protein in sample:

[0073] Accurately pipette 0.2 mL of the sample to be tested into a 10 mL clean centrifuge tube, add 0.8 mL of distilled water and 5 mL of Coomassie brilliant blue solution, mix well, stand for 2 min, and measure the absorbance value at 595 nm.

[0074] The soluble sugar content in chives is determined by anthrone colorimetry, and the specific steps are as follows:

[0075] (1) Solution preparation:

[0076] ① Glucose standard solution: accurately weigh 100 mg of anhydrous glucose powder into a beaker, dissolve in water, and dilute to 1000 mL to prepare a 1000 μg / g glucose standard solution, which is diluted 10 times for use. ② Anthrone ethyl acetate: accurately weigh 1 g of analytical pure anthrone into a beaker, add 50 mL of ethyl acetate, dissolve, and store in a brown bottle in the dark.

[0077] (2) Standard curve drawing:

[0078] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1 mL of glucose solution (100 μg / mL) into 6 clean 10 mL centrifuge tubes, add 2 mL, 1.8 mL, 1.6 mL, 1.4 mL, 1.2 mL, and 1 mL of distilled water, respectively, then add 0.5 mL of anthrone ethyl acetate and 5 mL of concentrated sulfuric acid, mix well, and heat in boiling water for 1 min, then measure the absorbance value at 630 nm after cooling, and draw the standard curve.

[0079] (3) Sample extraction:

[0080] Accurately weigh 0.2g of fresh scallion aerial parts into a 10mL centrifuge tube, stir in a boiling water bath for 30min, remove and cool to room temperature before centrifugation for analysis.

[0081] (4) Determination of soluble sugar content in the sample:

[0082] Accurately pipette 0.2 mL of each sample into a clean centrifuge tube, add 1.8 mL of distilled water, then add 0.5 mL of ethyl anthrone and 5 mL of concentrated sulfuric acid, mix well, incubate in a boiling water bath for 1 min, cool to room temperature, and measure the absorbance at 630 nm using the blank of the standard curve as a reference.

[0083] (5) Calculation of results:

[0084] Soluble sugar content (mg / g) = C*Vt / V1*m*1000

[0085] Please see Figure 2 240 mg / L sodium selenite significantly reduced the content of chlorophyll a and chlorophyll a+b in the aboveground parts of scallions. Figure 2 A, 2C), but had no significant effect on chlorophyll b. Figure 2 B). This study found that low-concentration sodium selenite treatment promoted the accumulation of chlorophyll a, chlorophyll a+, and chlorophyll a+b, but high-concentration treatment had an inhibitory effect. The literature "Feng R, Wei C, Tu S. The roles of selenium in protecting plants against abiotic stresses. Environmental and Experimental Botany, 2013, 87:58-68." reports that selenium can affect chlorophyll synthesis by influencing electron transport and mineral element absorption during photosynthesis. Therefore, changes in chlorophyll content may be related to the effects of selenium on photosynthetic processes and mineral element absorption.

[0086] Selenium also has a certain effect on the accumulation of soluble proteins. With increasing sodium selenite concentration, the soluble protein content in the aboveground parts of scallions showed a trend of first increasing and then decreasing, and was significantly higher than the control group at 80 mg / L, while slightly decreasing at 160 mg / L and 240 mg / L. Figure 2 D).

[0087] Soluble sugars are important isotonic regulators and nutrients in plants. Figure 2 E-test results showed that treatment with 160 mg / L sodium selenite significantly increased the soluble sugar content in the aboveground parts of scallions, and increased it by 16.78% compared with the control group.

[0088] The nutritional quality of chives was determined, including the following two physiological indicators: total flavonoids and total phenolic acids.

[0089] The total flavonoid content in chives was determined using a spectrophotometer, with the following specific steps:

[0090] (1) Solution preparation:

[0091] ① 80% ethanol: accurately measure 80 mL of anhydrous ethanol, dissolve in water, and then dilute to 100 mL.

[0092] ② 5% NaNO2: accurately weigh 5 g of sodium nitrite in a beaker, dissolve in deionized water, and then dilute to 100 mL.

[0093] ③ 10% AI(NO3)3: accurately weigh 10 g of AlCl3·6H2O in a beaker, dissolve in deionized water, and then dilute to 100 mL.

[0094] ④ 4% NaOH: accurately weigh 4 g of NaOH in a beaker, dissolve in deionized water, and then dilute to 100 mL.

[0095] (2) Rutin preparation:

[0096] Accurately weigh 100 mg of rutin and place it in a beaker, dissolve in 80% ethanol, and then dilute to 1000 mL, which is a 1000 μg / g rutin standard solution.

[0097] (3) Chive sample extraction:

[0098] Accurately weigh 0.2 g of dry sample powder into a 10 mL centrifuge tube, add 10 mL of 80% ethanol, heat in a 80°C water bath for 120 min, then centrifuge at 12000 rpm for 5 min at room temperature, and use the supernatant for detection.

[0099] (3) Standard curve preparation:

[0100] Respectively, take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of rutin into 10 mL volumetric flasks, add 5 mL of 80% ethanol, 0.3 mL of 5% NaNO2 to each test tube, mix well and stand for 5 min, continue to add 0.3 mL of 10% AI(NO3)3, mix well and stand for 5 min, finally dilute to 10 mL with 80% ethanol, mix well, stand for 10-20 min, and then measure the absorbance value at 510 nm. Linear regression is performed according to the absorbance value and the corresponding concentration to draw the standard curve.

[0101] (4) Sample determination:

[0102] Respectively take 1 mL of the sample to be tested in a 10 mL clean centrifuge tube, add 5 mL of 80% ethanol and 0.3 mL of 5% NaNO2, mix well and stand for 5 min. Continue to add 0.3 mL of 10% AI(NO3)3, mix well and stand for 5 min. Then use 80% ethanol to make up to 10 mL, mix well and stand for 10-20 min, and then measure the absorbance value at 510 nm.

[0103] The determination of the total phenolic acid content in chives refers to the method in the literature "Margraf T, Karnopp A R, Rosso N D, et al. Comparison between folin-ciocalteu and prussian blue assays to estimate the total phenolic content of juices and teas using 96-well microplates. Journal of Food Science, 2015, 80(11): C2397-C2403." with minor modifications, and the specific operation steps are as follows:

[0104] (1) Preparation of solution:

[0105] 0.3% sodium dodecyl sulfate: accurately weigh 0.3 g of sodium dodecyl sulfate in a beaker, dissolve and make up to 100 mL; 0.9% potassium ferricyanide: accurately weigh 0.9 g of potassium ferricyanide in a beaker, dissolve in deionized water and make up to 100 mL; 0.6% ferric chloride: weigh 0.6 g of ferric chloride, dissolve in deionized water and make up to 100 mL; preparation of gallic acid standard solution: accurately weigh 0.1 g of gallic acid powder, dissolve in 50 mL of deionized water and make up to 100 mL, store in a brown bottle for later use, which is 1000 μg / mL gallic acid standard solution; 0.1 mol / L hydrochloric acid preparation: accurately measure 0.9 mL of hydrochloric acid and make up to 100 mL with deionized water; 50% ethanol preparation: accurately measure 50 mL of anhydrous ethanol in a volumetric flask and make up to 100 mL with water.

[0106] (2) Extraction of chives above-ground sample:

[0107] Accurately weigh 0.2 g of chives above-ground dry sample in a 10 mL centrifuge tube, and add 10 mL of 50% ethanol. Soak for 30 min, ultrasonic extraction for 30 min, centrifuge at 5000 rpm for 10 min, and collect the supernatant for testing.

[0108] (3) Dilution of gallic acid standard solution:

[0109] Pipette 0 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of gallic acid standard solution into 100 mL volumetric flasks, respectively, and dilute to volume with deionized water to obtain standard solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively.

[0110] (4) Plotting the standard curve:

[0111] Pipe 0.2 mL of gallic acid at each dilution into a 10 mL centrifuge tube, add 2 mL of anhydrous ethanol, 0.4 mL of 0.3% sodium dodecyl sulfate solution, and 0.4 mL of 0.9% potassium ferricyanide / 0.6% ferric chloride (mixed in a 1:1 ratio before adding). Place in the dark for 5 min, then dilute to the mark with 0.1 mol / L hydrochloric acid. Mix thoroughly and react in the dark for 20 min. Measure the absorbance at 720 nm.

[0112] (5) Sample determination:

[0113] The total phenolic acid content in scallion extract was determined according to the determination steps in (4).

[0114] Flavonoids and phenolic acids are important secondary metabolites in plants, possessing antioxidant, anticancer, and antimutagenic effects. For example... Figure 3 As shown, the total flavonoids in the aerial parts of scallions after treatment with sodium selenite ( Figure 3 A) and total phenolic acids ( Figure 3 B) The content showed a continuous upward trend, reaching the maximum value at 160 mg / L, and increased by 1.94 and 2.04 times respectively compared with the control group, but decreased slightly at 240 mg / L.

[0115] The antioxidant enzyme activity of scallions was measured to analyze their antioxidant capacity.

[0116] Antioxidant enzyme activities such as peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were measured according to the kit instructions. The kits were purchased from Nanjing Jiancheng, and the product models were A084-3-1, A007-1-1, A001-3-2, and S0058, respectively.

[0117] Please see Figure 4 As the concentration of sodium selenite increased, the GPX activity in the aerial parts of scallions showed a trend of first increasing and then decreasing, reaching its maximum value at 160 mg / L sodium selenite treatment, which was 3.12 times higher than the control group, but slightly decreased at 240 mg / L treatment. Figure 4 A).

[0118] Figure 4Medium: (A) for glutathione peroxidase, (B) for catalase, (C) for peroxidase, (D) for superoxide dismutase;

[0119] Compared with the control, 20, 40, 160, 240 mg / L Na2SeO3 treatment significantly increased the CAT activity in the aboveground part of chives, but 80 mg / L Na2SeO3 treatment had no significant effect on the CAT activity in chives Figure 4 B).

[0120] Compared with the control, low concentration of Na2SeO3 treatment had no significant effect on the activities of POD and SOD in the aboveground part of chives, and significantly reduced the activities of POD and SOD in the aboveground part of chives at 240 mg / L Figure 4 C, Figure 4 D).

[0121] To clarify the effects of different concentrations of Na2SeO3 treatment on the accumulation of selenium in the aboveground and underground parts of chives, the total selenium, selenium form and sulfur content of chives were determined.

[0122] The total selenium content in the aboveground and underground parts of chives was determined by hydride generation-atomic fluorescence spectrometer (HG-AFS), and the specific operation was as follows:

[0123] (1) Preparation of solution:

[0124] 6 mol / L hydrochloric acid: accurately measure 50 mL of hydrochloric acid in a beaker, slowly add 40 mL of distilled water and stir uniformly with a glass rod, cool and constant volume to 100 mL; 100 g / L potassium ferricyanide preparation: accurately weigh 0.1 g of potassium ferricyanide in a beaker, dissolve in water and constant volume to 100 mL; 5% hydrochloric acid: accurately measure 50 mL of hydrochloric acid in a beaker, dissolve in water and cool to constant volume to 1000 mL. 2% potassium borohydride: weigh 20 g of potassium borohydride and 5 g of potassium hydroxide in a beaker, dissolve in water and constant volume to 1000 mL. This solution should be prepared and used immediately.

[0125] (2) Standard curve drawing:

[0126] 1000 μg / mL selenium standard purchased from China Institute of Metrology was diluted to different concentrations for drawing the standard curve. Three biological replicates were set.

[0127] (3) Sample pretreatment:

[0128] Accurately weigh 0.2 g of dried above-ground Allium fistulosum powder into a digestion tube, add 10 mL of HNO3 and 2 mL of H2O2 respectively, mix well, and then place in a microwave digestion instrument. The instrument parameters are set as follows: initial temperature 50°C, programmed temperature rise: 50-120°C, 12 min constant temperature 2 min. 120-150°C, 6 min constant temperature 5 min, 150-180°C, 10 min constant temperature 20 min. After cooling, transfer to a graphite digestion instrument and continue to heat to near dryness, then add 5 mL of hydrochloric acid (6 mol / L) and continue to heat until the solution becomes clear. After cooling, transfer to a 10 mL volumetric flask, add 2.5 mL of potassium ferricyanide solution (100 g / L), dilute to 10 mL with water, mix well, filter with a 0.45 μm filter membrane, and then measure.

[0129] (4) HG-AFS instrument parameter settings:

[0130] Negative high voltage 280V, lamp current 60mA, carrier gas flow rate 300mL / min, shielding gas flow 800mL / min, sample injection volume 1mL, delay time 1min. Each group of samples is set with three biological and technical repeats.

[0131] Use liquid chromatography-atomic fluorescence spectrometer (LC-AFS) to determine the selenium form of Allium fistulosum. The specific operation is as follows:

[0132] (1) Mobile phase preparation:

[0133] Accurately weigh 5.44 g of potassium dihydrogen phosphate powder and 1.49 g of potassium chloride into a beaker, add about 900 mL of water, stir to dissolve, adjust the pH to 6.0 with 1 mol / L potassium hydroxide, and dilute to 1000 mL. 35 g / mL potassium borohydride: accurately weigh 35 g of potassium borohydride and 5 g of potassium hydroxide into a beaker, dissolve in water, and dilute to 1 L. This solution needs to be prepared fresh. 10% hydrochloric acid: accurately measure 100 mL of hydrochloric acid solution into a beaker, stir evenly with a glass rod, and then dilute to 1000 mL. 0.15% potassium iodide digestion solution: accurately weigh 0.15 g of potassium iodide powder into a beaker, dissolve in water, and dilute to 1000 mL. 10% methanol solution: accurately measure 100 mL of methanol solution into a beaker, stir evenly with a glass rod, and then dilute to 1000 mL. All reagents are filtered with a 0.45 μm filter membrane, and then used after ultrasonic degassing.

[0134] (2) Standard curve preparation:

[0135] Five selenium standard compounds: SeCys2 (selenocysteine), MeSeCys (methyl selenocysteine), Se(IV) (tetravalent selenium), SeMet (selenomethionine), and Se(VI) (hexavalent selenium) were purchased from the National Standard Reference Material Research Center (Beijing, China).

[0136] (3) Sample processing:

[0137] Accurately weigh 0.2 g of chive sample powder into a 10 mL centrifuge tube, add 10 mL of protease XIV (8 mg / mL SigmaAldrich, USA) and mix well, place in a 120 rpm, 37°C shaking bed for 16 h, then place in an ultrasonic cleaner for 30 min, then centrifuge at 12000 rpm, 4°C for 15 min, then filter with a 0.22 μm filter membrane, and then measure.

[0138] (4) Instrument parameter settings:

[0139] Injection volume 100 μL, anion exchange column type: COLUMN PRP-X100 10 μm 250x2.1 mm, negative high voltage 320 V, lamp current 80 mA, carrier gas flow rate: 600 mL / min. Each group of samples was set with 3 biological replicates.

[0140] To clarify the metabolic relationship between selenium and sulfur in chives, the sulfur content in the aboveground part of chives was measured using a microcomputer coulometric sulfur analyzer (Jiangsu Guojing). Accurately weigh 0.5 g of chive sample into a magnetic boat, then place it in the coulometric sulfur analyzer for coulometric titration. The instrument parameter settings are as follows: furnace temperature: 900°C, sample injection stages: 3, power coefficient: 0.5.

[0141] As shown in Figure 5 With the increase of sodium selenite treatment concentration, the total selenium content in the aboveground and underground parts of chives showed a rising trend, and reached the maximum at 240 mg / L, which indicated that chives had a certain selenium absorption capacity. Among them, compared with the control group, the total selenium content in the aboveground part increased by 166.5 times, 381.1 times, 753.1 times, 111.32 times and 1411.1 times, respectively. The total selenium content in the underground part increased by 97.66 times, 238.04 times, 436.18 times, 523.17 times and 695.88 times, respectively, compared with the control group. In addition, the selenium accumulation in the underground part of chives after sodium selenite treatment was significantly higher than that in the aboveground part. This is consistent with the results of Wang et al. in the literature "Wang M, Peng Q, Zhou F, et al. Uptake kinetics and interaction of selenium species in tomato (Solanum lycopersicum L.) seedlings. Environmental Science and Pollution Research International, 2019, 26(10): 9730-9738."

[0142] As shown in Table 1, three selenium forms were detected, namely selenocysteine (SeCys), selenomethionine (SeMet) and selenate (SeVI), among which selenomethionine was the main selenium form in chives, and the content of organic selenium increased with the increase of selenium concentration, but in this study, two selenium forms, tetravalent selenium (Se) V and methyl-selenocysteine were not detected, and only sodium selenate was detected under the treatment of 240 mg / L sodium selenite.

[0143] Table 1 Effect of sodium selenite treatment on selenium forms in the above-ground part of chives

[0144]

[0145] Note: The data are the average of three replicates ± standard error, and different letters represent significant differences between samples. nd: indicates not detected; SeMet: selenomethionine; SeCys2: selenocystine; SeMeCys: selenium-methyl selenocysteine; SeIV: tetravalent selenium; SeVI: hexavalent selenium

[0146] The results further prove that high concentration (160 μmol / L) sodium selenite treatment not only improves the nutritional quality of chives, but also significantly increases the total selenium content and organic selenium content of chives.

[0147] See Figure 6 Compared with the control group, sodium selenite treatment significantly promoted the accumulation of total sulfur in the above-ground part of chives, and increased by 1.15 times, 1.18 times, 1.22 times, 1.37 times and 1.48 times respectively compared with the control group, which indicates that there is no obvious competition between selenium and sulfur absorption in chives.

[0148] Conclusion:

[0149] Based on the results of the above examples, on the basis of conventional cultivation, sodium selenite root application for chives is not only simple and rapid in production operation, but also relatively safe. After treatment, the total selenium content of chives can reach 13.4-95.8 mg / kg DW, which is 111.32-1411.1 times higher than that of conventional cultivation of chives. Selenomethionine is the main selenium form, and the content can reach 1.7-6.9 mg / kg DW, which significantly improves other nutritional qualities such as soluble sugar, total phenolic acid and total flavonoid content.

[0150] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for quality cultivation of rich organo-selenium garlic, characterized in that, It comprises the following steps: Step one, seed disinfection: Chopped onion seeds are soaked in warm water at 55℃ for 15 min; Step two, germination: After soaking the seeds in step one for 24h at 30℃, they are transferred to a culture dish for germination; Step three, seedling raising: After the seeds have sprouted, they are sown in a plug tray and grown in a light incubator; Step four, transplanting: When the chopped onion seedlings have grown 4-5 true leaves, they are transplanted into a plastic flowerpot in a greenhouse, and are cultivated using a mixture of grass charcoal and soil, and are watered with Hoagland's nutrient solution to ensure normal growth after transplanting; Step five, daily management: The normally growing chopped onions are watered 10 days per time, are fertilized 2 times, are weeded 3 times, and are treated for pests 1 time; Step six, selenium application: After the chopped onions have tillered, they are treated with sodium selenite for root irrigation; The concentration of the sodium selenite solution is 80-160 mg / L, 100 mL is poured into each pot each time, the interval between the applications of selenium is 5-7 days, and the number of applications of selenium is 2-3 times; Step seven, harvesting: The chopped onions are harvested 7 days after the last application of selenium; The method increases the content of selenium in chopped onions, and also increases the contents of soluble sugar, flavonoids and total phenolic acid.

2. The method for quality cultivation of Allium fistulosum L. rich in organic selenium according to claim 1, characterized in that, The germination temperature in step two is 30℃.

3. The method for quality cultivation of Allium fistulosum L. rich in organic selenium according to claim 1, characterized in that, The sprouting in step three is 70%, and after 70% of the seeds have sprouted, the seedlings are grown in a 72-hole plug tray, and the parameters of the light incubator are set as follows: the day and night temperatures are 20 / 15℃, the light cycle is 15 / 9 h, the relative humidity is 50%-70%, and the light intensity is 2500 Lx.

4. The method for quality cultivation of Allium fistulosum L. rich in organic selenium according to claim 1, characterized in that, The substrate used for seedling raising in step three is a mixture of peat, perlite and vermiculite in a mass ratio of 2:1:

1.

5. The method for quality cultivation of Allium fistulosum L. rich in organic selenium according to claim 1, characterized in that, The flowerpot size in step four is 14*12*12 cm, and the grass charcoal and soil are mixed in a mass ratio of 1:1, and are weighed at 2.5 kg.

6. The method for quality cultivation of Allium fistulosum L. rich in organic selenium according to claim 1, characterized in that, The temperature of the greenhouse in step four is controlled at 16-20℃, and the relative humidity is 65-75%.

7. The method for quality cultivation of Allium fistulosum rich in organo-selenium according to claim 1, characterized in that, The Hoagland's nutrient solution is poured into the flowerpot every 7 days after transplanting, and 100-150 mL is applied each time.

8. The method for quality cultivation of Allium fistulosum rich in organo-selenium according to claim 1, characterized in that, The concentration of the sodium selenite solution is 160 mg / L, and the solution is applied once every 7 days for 3 times.

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