Method for increasing content of glucosinolate and total flavonoids in broccoli

Abstract

The invention provides a method for increasing the content of glucosinolate and total flavonoids in broccoli, which comprises the following steps: performing germination treatment on broccoli seeds for 47.5-48.5 hours to obtain broccoli bud seedlings; the broccoli seedlings are transplanted into a culture medium to be subjected to seedling culture for 71.5-72.5 h, and broccoli seedlings are obtained; carrying out exogenous methyl jasmonate treatment; the treatment time of the exogenous methyl jasmonate is 0.5-48 hours; the concentration of jasmonic acid methyl ester in the exogenous jasmonic acid methyl ester treatment is 25-100 [mu] mol / L. On the premise of not influencing the growth of plants, such as dry weight, fresh weight, chlorophyll fluorescence parameters and the like, the content of glucoraphanin, brassica glucosinolate, 4-methoxy brassica glucosinolate, neoglucose brassin, total aliphatic glucosinolate, total indole glucosinolate, total glucosinolate and total flavone can be remarkably increased. And economic, efficient, convenient and accurate measures can be taken in production.

Description

Technical Field

[0001] The present application relates to the technical field of plant growth and secondary metabolite accumulation regulation, and in particular to a method for increasing the content of glucosinolates and total flavonoids in broccoli. Background Art

[0002] Eating cruciferous vegetables such as broccoli can reduce the risk of cardiovascular disease and cancer to a certain extent, and can also reduce blood sugar to a certain extent. This is mainly attributed to the two very important secondary metabolites contained in broccoli, namely glucosinolates and total flavonoids. Among them, glucosinolates, also known as thioglucosinolates, can be hydrolyzed by myrosinase to produce a variety of active substances such as isothiocyanates, which have significant anti-inflammatory, anti-cancer, anti-obesity and neuroprotective functions. As a strong antioxidant, total flavonoids help prevent the occurrence of chronic diseases such as cardiovascular disease, cancer and neurodegenerative diseases, and also have antibacterial, antiviral, anti-allergic and other biological activities.

[0003] As high-value-added broccoli becomes more popular, finding efficient, economical and convenient methods to increase the content of glucosinolates and total flavonoids in broccoli is of great significance for increasing the nutritional value and health benefits of broccoli. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide a method for increasing the content of glucosinolates and total flavonoids in broccoli.

[0005] Based on the above purpose, the present application provides a method for increasing the content of glucosinolates and total flavonoids in broccoli, the method comprising:

[0006] Performing a germination treatment on broccoli seeds to obtain broccoli sprouts; the duration of the germination treatment is 47.5 hours to 48.5 hours;

[0007] Transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings; the seedling cultivation time is 71.5 hours to 72.5 hours;

[0008] The broccoli seedlings are treated with exogenous methyl jasmonate to increase the content of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucose brassinolide, total aliphatic glucosinolates, total indole glucosinolates and total glucosinolates as well as the content of total flavonoids without affecting the growth of the plants, such as dry weight, fresh weight and chlorophyll fluorescence parameters; the duration of the exogenous methyl jasmonate treatment is 0.5h to 48h; the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L to 100μmol / L.

[0009] In some of the embodiments, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25 μmol / L to 100 μmol / L.

[0010] In some embodiments, the duration of the exogenous methyl jasmonate treatment is 0.5 h to 48 h.

[0011] In some embodiments, the culture matrix comprises a solid matrix and a liquid matrix in a volume ratio of 4:5.5; the liquid matrix is ​​a 1 / 2MS liquid culture medium; and the exogenous methyl jasmonate treatment of the broccoli seedlings comprises:

[0012] Providing a 1 / 2MS liquid culture medium containing methyl jasmonate; wherein the final concentration of methyl jasmonate in the 1 / 2MS culture medium containing methyl jasmonate is 25 μmol / L to 100 μmol / L;

[0013] The 1 / 2 MS liquid medium in the culture matrix is ​​replaced with the 1 / 2 MS liquid medium containing methyl jasmonate.

[0014] In some embodiments, the step of transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings comprises:

[0015] Supplementary culture medium is added to the culture matrix every 23.5 hours to 24.5 hours, and the supplementary culture medium includes 1 / 2MS liquid culture medium; the volume ratio of the supplementary culture medium to the liquid matrix is ​​11:1.

[0016] In some embodiments, providing a 1 / 2MS liquid culture medium containing methyl jasmonate comprises:

[0017] 100 mmol / L methyl jasmonate mother solution is added to the supplemented culture medium, and the medium is diluted to obtain the 1 / 2MS liquid culture medium containing methyl jasmonate.

[0018] In some embodiments, the culture matrix is ​​disposed in a seedling tray, and the seedling tray is disposed in a bottom tray; and replacing the 1 / 2MS liquid culture medium in the culture matrix with a 1 / 2MS liquid culture medium containing methyl jasmonate comprises:

[0019] Lift the seedling tray, and pour the 1 / 2MS liquid culture medium containing methyl jasmonate into the culture matrix;

[0020] The seedling tray is placed in a replaced chassis; the replaced chassis is a chassis without moisture.

[0021] In some embodiments, the solid matrix includes vermiculite and perlite; the volume ratio of the vermiculite to the perlite is 3:1.

[0022] In some embodiments, the broccoli seeds are subjected to germination treatment to obtain broccoli sprouts, comprising:

[0023] After soaking the broccoli seeds in a 30°C constant temperature incubator for 3.9 h to 4.1 h, spread them into a culture dish and culture them for germination for 47.5 h to 48.5 h.

[0024] In some embodiments, the photoperiod of the germination culture is set to light:dark=16h:8h.

[0025] As can be seen from the above, the method for increasing the content of glucosinolate and total flavonoids in broccoli provided in the present application comprises: performing a germination treatment on broccoli seeds to obtain broccoli sprouts; the duration of the germination treatment is 47.5h to 48.5h; transplanting the broccoli sprouts into a culture matrix for seedling cultivation to obtain broccoli seedlings; the duration of the seedling cultivation is 71.5h to 72.5h; exogenous methyl jasmonate treatment is performed on the broccoli seedlings; the exogenous jasmonate treatment is performed. The duration of the methyl jasmonate treatment is 0.5h to 48h; the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L to 100μmol / L; it can significantly increase the content of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucose brassinolide, total aliphatic glucosinolates, total indole glucosinolates, total glucosinolates and total flavonoids without affecting the growth of the plant, such as dry weight, fresh weight and chlorophyll fluorescence parameters. It is helpful to take economical, efficient, convenient and accurate measures in production to increase the content of active biological products glucosinolates and total flavonoids in the seedling stage of broccoli. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 This is a diagram showing the growth status of 5-day-old broccoli seedlings that were not treated with exogenous methyl jasmonate.

[0028] Figure 2 This is a diagram of the growth status of 5-day-old broccoli seedlings after treatment with exogenous methyl jasmonate.

[0029] Figure 3 This is the NBT staining result of 5-day-old broccoli seedlings that were not treated with exogenous methyl jasmonate.

[0030] Figure 4 This is the NBT staining result of 5-day-old broccoli seedlings after treatment with exogenous methyl jasmonate.

[0031] Figure 5 The graph shows the fresh weight of the whole broccoli seedlings grown to 5 days old before and after treatment with exogenous methyl jasmonate.

[0032] Figure 6 The graph shows the dry weight of the whole broccoli seedlings grown to 5 days old before and after treatment with exogenous methyl jasmonate.

[0033] Figure 7 Chlorophyll fluorescence parameters of 5-day-old broccoli seedlings after being treated with exogenous methyl jasmonate for 48 hours.

[0034] Figure 8a The changes in glucoraphanin (GRA) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0035] Figure 8b The changes in the content of glucosinolate (GAL) in 5-day-old broccoli seedlings before and after treatment with exogenous methyl jasmonate.

[0036] Figure 8c The changes in sergenin (GER) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0037] Figure 8d The changes in the content of brassica glucosinolate (GBS) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0038] Figure 8e The changes in 4-methoxybrassica glucosinolate (4MBS) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0039] Figure 8f The changes in the content of neoglucose brassinolide (NGBS) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0040] Figure 8g The changes in total aliphatic glucosinolates (AGLs) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0041] Figure 8h The changes in the content of total indole glucosinolates (IGLs) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0042] Figure 8iThe changes in total glucosinolates (GLSs) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0043] Fig. 9 Total flavonoids content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. DETAILED DESCRIPTION

[0044] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0045] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should have the common meanings understood by persons with ordinary skills in the field to which the present application belongs.

[0046] Plant hormones, such as jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and brassinolide (BR), have been confirmed by a large number of studies to play an important role in promoting the accumulation of plant secondary metabolites. However, the effects of different types of plant hormones on plant secondary metabolites show significant differences. At the same time, because different plants have their own unique physiological characteristics, even the same plant hormone has different effects on secondary metabolites when acting on different plants. In addition, the physiological state and metabolic needs of the same plant change at different growth stages, and the effects of the same plant hormone on it, including the degree of influence on secondary metabolites, also vary.

[0047] Based on this, the embodiment of the present application provides a method for increasing the content of glucosinolates and total flavonoids in broccoli, by selecting broccoli in the seedling stage, and applying exogenous methyl jasmonate treatment to broccoli in the seedling stage (broccoli seedlings grown to the fifth day), and subjecting the broccoli in the seedling stage to instantaneous exogenous methyl jasmonate treatment, it is possible to significantly increase the content of glucosinolate, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucose brassinolide, total aliphatic glucosinolates, total indole glucosinolates, total glucosinolates and total flavonoids without affecting the growth of the plant, such as dry weight, fresh weight and chlorophyll fluorescence parameters. It is helpful to take economical, efficient, convenient and accurate measures in production to increase the content of active biological products glucosinolates and total flavonoids in the seedling stage of broccoli. In addition, the selection of broccoli in the seedling stage in the early stage of growth has the advantages of softer cell walls than broccoli in the mature / commercial period, which helps the human body to digest and absorb more easily. At the same time, the broccoli seedlings have richer glucosinolate content. The obtained broccoli seedlings can be used as an ideal raw material for nutritional supplements.

[0048] The method for increasing the content of glucosinolates and total flavonoids in broccoli provided in the embodiments of the present application may include:

[0049] S100, performing a germination treatment on the broccoli seeds to obtain broccoli sprouts; the duration of the germination treatment is 47.5 hours to 48.5 hours;

[0050] S200, transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings; the seedling cultivation time is 71.5 hours to 72.5 hours;

[0051] S300, treating the broccoli seedlings with exogenous methyl jasmonate to increase the content of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucose brassinolide, total aliphatic glucosinolates, total indole glucosinolates, total glucosinolates and total flavonoids without affecting the growth of the plants, such as dry weight, fresh weight and chlorophyll fluorescence parameters; the duration of the exogenous methyl jasmonate treatment is 0.5h to 48h; the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L to 100μmol / L.

[0052] In some embodiments, in step S100, the broccoli seeds are subjected to germination treatment to obtain broccoli sprouts, which may include: soaking the broccoli seeds in a 30°C constant temperature incubator for 3.9 hours to 4.1 hours, spreading them in a culture dish, and germinating and culturing them for 47.5 hours to 48.5 hours. The soaking may be soaking in distilled water, for example, distilled water greater than twice the volume of the seeds may be used for soaking, so that the seeds can fully absorb water.

[0053] In some embodiments, a layer of filter paper can be laid on the bottom of the culture dish, soaked with distilled water, and then the broccoli seeds can be laid flat. During the germination culture, the temperature of the culture chamber can be set to 25±1°C, the humidity can be set to 70%, and the photoperiod can be set to light:dark=16h:8h.

[0054] In some of the embodiments, distilled water may be added once during the germination culture process.

[0055] In some embodiments, in step S200, the culture matrix may include a solid matrix and a liquid matrix in a volume ratio of 4:5.5. The solid matrix may include vermiculite and perlite. The volume ratio of the vermiculite to the perlite is 3:1. The liquid culture medium may be a 1 / 2MS liquid culture medium. The liquid culture medium may be prepared by adding 2.17g of 1 / 2MS solid powder to 1L of distilled water. The culture matrix with this ratio can provide suitable support and suitable nutrition.

[0056] In some of the embodiments, the culture matrix can be arranged in a seedling tray, and the seedling tray is arranged in a bottom tray.

[0057] In some embodiments, the step of transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings may include:

[0058] Supplementary culture medium is added to the culture matrix every 24 hours, and the supplementary culture medium includes 1 / 2MS liquid culture medium; the volume ratio of the supplementary culture medium to the liquid matrix can be 11:1.

[0059] In some embodiments, in step S300, treating the broccoli seedlings with exogenous methyl jasmonate may include:

[0060] Providing a 1 / 2MS liquid culture medium containing methyl jasmonate; wherein the final concentration of methyl jasmonate in the 1 / 2MS culture medium containing methyl jasmonate is 25 μmol / L to 100 μmol / L;

[0061] The 1 / 2MS liquid medium in the culture matrix is ​​replaced with the 1 / 2MS liquid medium containing methyl jasmonate. In this way, the broccoli seedlings can be treated with exogenous methyl jasmonate.

[0062] In some embodiments, the 1 / 2MS liquid culture medium containing methyl jasmonate is prepared by the following method:

[0063] In the supplemented culture medium, 100mmol / L of methyl jasmonate mother solution is added and diluted so that the final concentration of the methyl jasmonate is 25μmol / L to 100μmol / L, thereby obtaining the 1 / 2MS liquid culture medium containing methyl jasmonate. Methyl jasmonate can be dissolved in 10% ethanol solution to obtain a 100mmol / L methyl jasmonate mother solution, which is stored in a 4°C refrigerator for standby use. It can be diluted with water to a desired concentration before hormone treatment.

[0064] In some embodiments, replacing the 1 / 2MS liquid culture medium in the culture matrix with a 1 / 2MS liquid culture medium containing methyl jasmonate may include:

[0065] The seedling tray is lifted, and the 1 / 2MS liquid culture medium containing methyl jasmonate is poured into the culture matrix. Specifically, the 1 / 2MS liquid culture medium containing methyl jasmonate can be poured from top to bottom to replace the liquid in the culture matrix.

[0066] The seedling tray is placed in a replaced chassis; the replaced chassis is a chassis without moisture. The replaced chassis can be a new chassis.

[0067] In some embodiments, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25 μmol / L, 50 μmol / L or 100 μmol / L.

[0068] In some embodiments, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment can be 98 μmol / L to 100 μmol / L, and the treatment time can be 6 h to 10 h or 46 h to 48 h. Or the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment can be 25 μmol / L to 26 μmol / L, and the treatment time can be 46 h to 48 h. Methyl jasmonate with this concentration and treatment time can significantly increase the content of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucosinolate, total aliphatic glucosinolates, total indole glucosinolates, and total glucosinolates; at the same time, it can also significantly increase the content of total flavonoids.

[0069] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0070] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0071] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0072] Example 1

[0073] 1. Cultivation Methods

[0074] Step (1) Germination of broccoli seeds

[0075] Place broccoli seeds in a 10mL centrifuge tube, add distilled water twice the volume of the seeds, and soak in a 30℃ constant temperature incubator for 4 hours to allow the seeds to fully absorb water. Prepare 3 clean transparent glass culture dishes, spread a layer of filter paper on the bottom of the dish, soak it with distilled water, evenly spread the seeds soaked for 4 hours on the filter paper in the culture dish, add an appropriate amount of water, cover it with a top cover, and place it in an artificial culture room for germination for 48 hours. Replenish water once during this period. The temperature of the culture room is set to 25±1℃, the humidity is 70%, and the light cycle is set to light: dark = 16h:8h.

[0076] Step (2) Cultivation of broccoli seedlings

[0077] Prepare 32-hole seedling trays, the solid matrix is ​​vermiculite: perlite = 3: 1, each seedling tray requires about 4L of solid culture medium for support, the liquid matrix is ​​1 / 2MS culture medium (2.17g 1 / 2MS solid powder is added to 1L distilled water), and each seedling tray requires about 5.5L of 1 / 2MS culture medium. Transplant the germinated broccoli sprouts from the culture dish to the culture medium of the seedling tray, and evenly transplant 9 broccoli sprouts in each hole in a 3×3 layout, and supplement 500mL of 1 / 2MS culture medium every 24h after transplanting.

[0078] Step (3) Exogenous methyl jasmonate treatment

[0079] Methyl jasmonate was dissolved in 10% ethanol solution to prepare 100mmol / L mother solution and stored in a 4℃ refrigerator for standby use, and diluted to the required concentration before each hormone treatment. In order to measure the influence of the ethanol-water solvent added during the methyl jasmonate hormone treatment, the present invention designed a corresponding Mock control for each concentration hormone treatment experiment. As above, the 10% ethanol solution was stored in a 4℃ refrigerator as a mother solution for standby use, and diluted to the required concentration before each treatment.

[0080] Broccoli seeds were transplanted 48 hours after germination, and 500 mL of 1 / 2MS liquid culture medium was added every day after transplanting. Broccoli seedlings were treated once, that is, on the 5th day after sowing. Samples were taken at 0 hours on the 5th day, and the liquid was replaced immediately after taking, that is, the original 1 / 2MS liquid culture medium was replaced with 1 / 2MS culture medium containing methyl jasmonate. During the operation, the seedling tray was lifted, and the 1 / 2MS liquid culture medium containing methyl jasmonate was watered from top to bottom to replace the liquid in the solid culture medium, and the seedlings were placed in a new bottom tray after the replacement was completed.

[0081] 2. Detection Method

[0082] Samples were collected at 0.5h, 2h, 8h, 24h and 48h after treatment. The seedlings were photographed after sampling and after NBT staining, and the dry weight and fresh weight of the plants, chlorophyll fluorescence parameters, glucosinolate and total flavonoids contents were measured.

[0083] Among them, plant superoxide anion staining (NBT method) is a common method for detecting the content of superoxide anion in plant tissues. This method is based on the principle that NBT (nitro blue tetrazolium) dye reacts with superoxide anions to form dark blue granular formazan.

[0084] The specific method of the glucosinolate detection method is as follows: weigh 40 mg of dry broccoli sample, add 1 mL of preheated methanol, add 25 μL of 5 mmol / L benzyl glucosinolate as internal standard, and boil in a boiling water bath for 10 min. Collect the supernatant, add 1 mL of preheated methanol and repeat the operation, and mix the supernatant extracted twice to obtain the crude extract of glucosinolate. Add the crude extract to an activated DEAE-Sephadex A-25 chromatography column, rinse once with methanol, rinse twice with ddH2O, and finally rinse once with acetate buffer. Add 0.5 mL of sulfatase, desulfurize overnight at 25°C, and elute three times with 300 μL of ddH2O. After desulfurization of glucosinolate, chromatographic analysis was performed on a Sciex Exion LC system (Sciex, CA, USA) using a Waters ACQUITYT3 column (50×2.1 mm, 1.8 μm). The mobile phase was ddH2O and methanol, and the flow rate was 0.5 mL / min. Desulfurized glucosinolates were separated by ultra-high performance liquid chromatography (UPLC), and the product peaks were integrated respectively. The content of each product was calculated by the peak area ratio of each product to the internal standard GTP, i.e., the peak area quantitative method.

[0085] Among them, the specific method of the total flavonoids detection method is as follows: after the broccoli sample is ground into powder with liquid nitrogen, about 0.2g of the sample is weighed and 0.5mL of 60% ethanol solution is added. Ultrasonic extraction is performed at 60℃ for 30min, and centrifugation is performed at 10000g at 25℃ for 10min, and the supernatant is taken for testing. 20μL of the supernatant is taken into a 96-well plate, 6μL of 5% sodium nitrite is added, mixed, and allowed to stand at room temperature for 6min; then 6μL of 10% Al(NO3)3 solution is added, mixed, allowed to stand at room temperature for 6min, and then 80μL of 4% NaOH solution and 88μL of distilled water are added, mixed, allowed to stand at room temperature for 15min, and then the absorbance value is measured at a wavelength of 510nm.

[0086] 3. Test results

[0087] Test results such as Figures 1 to 9 As shown. Among them, Figure 1 This is a diagram showing the growth status of 5-day-old broccoli seedlings that were not treated with exogenous methyl jasmonate. Figure 2 This is a diagram of the growth status of 5-day-old broccoli seedlings after treatment with exogenous methyl jasmonate. Figure 3 This is the NBT staining result of 5-day-old broccoli seedlings that were not treated with exogenous methyl jasmonate. Figure 4 This is the NBT staining result of 5-day-old broccoli seedlings after treatment with exogenous methyl jasmonate. Figure 5 The graph shows the fresh weight of the whole broccoli seedlings grown to 5 days old before and after treatment with exogenous methyl jasmonate. Figure 6The graph shows the dry weight of the whole broccoli seedlings grown to 5 days old before and after treatment with exogenous methyl jasmonate. Figure 7 Chlorophyll fluorescence parameters of 5-day-old broccoli seedlings after being treated with exogenous methyl jasmonate for 48 hours. Figure 8a The changes in glucoraphanin (GRA) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8b The changes in the content of glucosinolate (GAL) in 5-day-old broccoli seedlings before and after treatment with exogenous methyl jasmonate. Figure 8c The changes in sergenin (GER) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8d The changes in the content of brassica glucosinolate (GBS) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8e The changes in 4-methoxybrassica glucosinolate (4MBS) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8f The changes in the content of neoglucose brassinolide (NGBS) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8g The changes in total aliphatic glucosinolates (AGLs) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8h The changes in the content of total indole glucosinolates (IGLs) in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Figure 8i The changes in total glucosinolates (GLSs) content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment. Fig. 9 Total flavonoids content in 5-day-old broccoli seedlings before and after exogenous methyl jasmonate treatment.

[0088] 4. Results Analysis

[0089] 4.1 Effects of exogenous methyl jasmonate on the growth of broccoli seedlings

[0090] Depend on Figure 1 and Figure 2 It can be seen that after collecting broccoli seedling samples before and 0.5h, 2h, 8h, 24h, and 48h after exogenous methyl jasmonate treatment and taking pictures and observing, there was no significant difference between the broccoli seedlings treated with different concentrations of exogenous methyl jasmonate and the broccoli seedlings treated with the corresponding concentrations of Mock. Therefore, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment was 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment was 0.5h~48h, which did not affect the growth of broccoli seedlings.

[0091] 4.2 Effects of exogenous methyl jasmonate on reactive oxygen species (ROS) levels in broccoli seedlings

[0092] Depend on Figure 3 and Figure 4 It can be seen that compared with the corresponding concentration Mock treatment, the ROS level was reduced after treatment with 25μmol / L, 50μmol / L and 100μmol / L methyl jasmonate, among which the treatment effects of 25μmol / L and 50μmol / L methyl jasmonate were more obvious. Therefore, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment was 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment was 0.5h~48h, which can reduce the level of reactive oxygen (ROS) in broccoli seedlings.

[0093] 4.3 Effect of exogenous methyl jasmonate on the fresh weight of broccoli seedlings

[0094] Depend on Figure 5 It can be seen that the fresh weight of broccoli seedlings was measured before and 0.5h, 2h, 8h, 24h, and 48h after the treatment with exogenous methyl jasmonate. There was no significant difference between the fresh weight of broccoli seedlings treated with different concentrations of exogenous methyl jasmonate and the fresh weight of broccoli seedlings treated with the corresponding concentration of Mock. Therefore, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment was 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment was 0.5h~48h, which had no effect on the fresh weight of broccoli seedlings.

[0095] 4.4 Effects of exogenous methyl jasmonate on the dry weight of broccoli seedlings

[0096] Depend on Figure 6 It can be seen that the dry weight of broccoli seedlings was measured before and 0.5h, 2h, 8h, 24h, and 48h after the treatment with exogenous methyl jasmonate. There was no significant difference between the dry weight of broccoli seedlings treated with different concentrations of exogenous methyl jasmonate and the dry weight of broccoli seedlings treated with the corresponding concentration of Mock. Therefore, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment was 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment was 0.5h~48h, which had no effect on the dry weight of broccoli seedlings.

[0097] 4.5 Effects of exogenous methyl jasmonate on chlorophyll fluorescence parameters in cotyledons of broccoli seedlings

[0098] Depend on Figure 7It can be seen that the broccoli seedlings were collected before and 48 hours after the exogenous methyl jasmonate treatment, and the chlorophyll fluorescence parameters in the cotyledons were measured. There was no significant difference between the chlorophyll fluorescence parameters in the cotyledons of broccoli seedlings treated with different concentrations of exogenous methyl jasmonate and the broccoli seedlings treated with the corresponding concentrations of Mock. Therefore, the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment was 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment was 48h, which had no effect on the chlorophyll fluorescence parameters in the cotyledons of broccoli.

[0099] 4.6 Effect of exogenous methyl jasmonate on glucosinolate content in broccoli seedlings

[0100] Depend on Figure 8a-8i It can be seen that the broccoli seedlings were collected before and 0.5h, 2h, 8h, 24h, and 48h after the exogenous methyl jasmonate treatment to determine the glucosinolate content. Although the total glucosinolate content between the different concentrations of Mock control groups decreased to different degrees, it showed a downward trend over time. The contents of total glucosinolates, total aliphatic glucosinolates, and total indole glucosinolates in the methyl jasmonate treatment group increased significantly, and the contents of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, and neoglucose brassinolide increased significantly.

[0101] It can be seen that the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment is 0.5h~48h, which can significantly increase the content of total glucosinolates, total aliphatic glucosinolates, and total indole glucosinolates, and the content of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, and neoglucose brassinolide. However, the effects of different concentrations and different treatment durations of methyl jasmonate treatment on the content of different types of glucosinolates in glucosinolates are not the same. And for the same type of glucosinolates, the effects of different concentrations and different treatment durations of methyl jasmonate treatment are not the same. Among them, the concentration of 25 μmol / L, the treatment time is 24h or 48h; or the concentration of 50 μmol / L, the treatment time is 24h; or the concentration of 50 μmol / L, the treatment time is 8h, 24h and 48h, can significantly increase the content of total glucosinolates, total aliphatic glucosinolates, total indole glucosinolates, glucoraphanin, brassica glucosinolate, 4-methoxy brassica glucosinolate, and neoglucosinolate. Among them, the concentration of 100 μmol / L, the treatment time is 48h, can better increase the content of total glucosinolates, total aliphatic glucosinolates, total indole glucosinolates, glucoraphanin, brassica glucosinolate, 4-methoxy brassica glucosinolate, and neoglucosinolate.

[0102] 4.7 Effect of exogenous methyl jasmonate on the total flavonoid content in broccoli seedlings

[0103] Depend on Fig. 9 It can be seen that the total flavonoids content of broccoli seedlings was measured before and 0.5h, 2h, 8h, 24h, and 48h after treatment with exogenous methyl jasmonate. The total flavonoids content increased significantly at 8h and 48h after treatment with different concentrations of methyl jasmonate; while the total flavonoids content after treatment with different concentrations of Mock showed an overall downward trend over time.

[0104] It can be seen that the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L~100μmol / L, and the duration of exogenous methyl jasmonate treatment is 0.5h~48h, which can significantly increase the total flavonoids. However, the effects of methyl jasmonate treatments with different concentrations and different treatment durations on the content of total flavonoids are not the same. And for the same concentration of methyl jasmonate treatment, the effects of different treatment durations are not the same. Among them, the concentration of 25μmol / L, or the concentration of 50μmol / L, or the concentration of 100μmol / L, and the treatment duration of 8h or 48h can all significantly increase the content of total brass. Among them, the concentration of 100μmol / L and the treatment duration of 8h or 48h can better increase the content of total flavonoids.

[0105] It can be seen that the method provided in the embodiment of the present application can significantly increase the content of total glucosinolates, total aliphatic glucosinolates, total indole glucosinolates, glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucosinolate and total flavonoids by adding exogenous methyl jasmonate to the culture medium of broccoli seedlings without affecting the growth of the plants, such as dry weight, fresh weight and chlorophyll fluorescence parameters. And it has the advantages of simple method and the like.

[0106] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0107] While the disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0108] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for increasing the content of glucosinolates and total flavonoids in broccoli, characterized in that: The method comprises: Performing a germination treatment on broccoli seeds to obtain broccoli sprouts; the duration of the germination treatment is 47.5 hours to 48.5 hours; Transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings; the seedling cultivation time is 71.5 hours to 72.5 hours; The broccoli seedlings are treated with exogenous methyl jasmonate to increase the contents of glucoraphanin, brassica glucosinolate, 4-methoxybrassica glucosinolate, neoglucose brassinolide, total aliphatic glucosinolates, total indole glucosinolates and total glucosinolates as well as the content of total flavonoids without affecting the growth of the plants; the duration of the exogenous methyl jasmonate treatment is 0.5h to 48h; and the concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 25μmol / L to 100μmol / L.

2. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 1, characterized in that: The concentration of methyl jasmonate in the exogenous methyl jasmonate treatment is 98 μmol / L to 100 μmol / L.

3. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 1, characterized in that: The duration of the exogenous methyl jasmonate treatment is 46 h to 48 h.

4. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 1, characterized in that: The culture matrix comprises a solid matrix and a liquid matrix in a volume ratio of 4:5.5; the liquid matrix is ​​a 1 / 2MS liquid culture medium; and the exogenous methyl jasmonate treatment of the broccoli seedlings comprises: Providing a 1 / 2MS liquid culture medium containing methyl jasmonate; wherein the final concentration of methyl jasmonate in the 1 / 2MS culture medium containing methyl jasmonate is 25 μmol / L to 100 μmol / L; The 1 / 2 MS liquid medium in the culture matrix is ​​replaced with the 1 / 2 MS liquid medium containing methyl jasmonate.

5. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 4, characterized in that: The step of transplanting the broccoli sprouts into a culture medium for seedling cultivation to obtain broccoli seedlings comprises: Supplementary culture medium is added to the culture matrix every 23.5 hours to 24.5 hours, and the supplementary culture medium includes 1 / 2MS liquid culture medium; the volume ratio of the supplementary culture medium to the liquid matrix is ​​11:

1.

6. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 5, characterized in that: The 1 / 2MS liquid culture medium containing methyl jasmonate is provided, comprising: 100 mmol / L methyl jasmonate mother solution is added to the supplemented culture medium, and the medium is diluted to obtain the 1 / 2MS liquid culture medium containing methyl jasmonate.

7. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 4, characterized in that: The culture matrix is ​​arranged in a seedling tray, and the seedling tray is arranged in a bottom tray; and the step of replacing the 1 / 2MS liquid culture medium in the culture matrix with the 1 / 2MS liquid culture medium containing methyl jasmonate comprises: Lift the seedling tray, and pour the 1 / 2MS liquid culture medium containing methyl jasmonate into the culture matrix; The seedling tray is placed in a replaced chassis; the replaced chassis is a chassis without moisture.

8. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 4, characterized in that: The solid matrix includes vermiculite and perlite; the volume ratio of the vermiculite to the perlite is 3:

1.

9. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 1, characterized in that: The broccoli seeds are subjected to germination treatment to obtain broccoli sprouts, comprising: Soak the broccoli seeds in a 30°C constant temperature incubator for 3.9h-4.1h, spread them into a culture dish, and culture them for germination for 47.5h-48.5h.

10. The method for increasing the content of glucosinolates and total flavonoids in broccoli according to claim 9, characterized in that: The photoperiod of the germination culture was set to light:dark=16h:8h.

Images