Application of melatonin in promoting accumulation of phenols and anthocyanin substances in grape root system and root system development

Abstract

The invention discloses application of melatonin in promoting accumulation of phenols and anthocyanin substances in grape root systems and root system development. The melatonin is applied to a subculture medium for rooting culture of grape tissue culture seedling strains for the first time, and determination of root development related characters shows that treatment with melatonin of different concentrations affects growth and development of grape roots to different degrees, and treatment with low-concentration melatonin can significantly promote root development of grape tissue culture seedlings. The influence of high-concentration melatonin treatment on the root system of the grape tissue culture seedling is small or even inhibited. In addition, the content of the total phenols and the total anthocyanin substances in the root system is measured, and it is found that accumulation of the total phenols and the total anthocyanin substances in the grape root system can be remarkably promoted through treatment of 10 micromol / L melatonin. The invention lays a theoretical foundation for field application of melatonin.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology and biochemistry, and in particular to the application of melatonin in promoting the accumulation of phenolic and anthocyanin substances in grape roots and root development. Background Art

[0002] With the promotion of plant factory seedling cultivation technology, efficiently and conveniently breeding a large number of virus-free seedlings has become the primary task of current grape breeding. Therefore, it is particularly important to explore the mechanism by which different types and concentrations of plant growth regulators induce the occurrence of adventitious roots in plants.

[0003] Melatonin (MT) is a powerful natural antioxidant that can improve the plant's ability to resist biotic and abiotic stresses and has multiple physiological functions. In recent years, research reports on MT's effects on grape roots have mainly focused on abiotic stress, and there is almost no research on its effects on the growth and development of grape roots. The root system is an important organ involved in plant growth and development, material and energy metabolism, information transmission and transduction, and directly determines the resistance of grapes to adverse stress and restricts their growth and development.

[0004] Phenolic compounds are compounds generated when hydrogen atoms on aromatic rings are replaced by hydroxyl groups or functional derivatives. They are of various types and are important secondary metabolites of plants. According to the number of hydroxyl groups directly connected to the aromatic ring, they can be divided into monophenols, diphenols and polyphenols. For example, phenol, o-cresol, m-cresol, etc. are common monophenols; catechol, resorcinol, hydroquinone, eugenol, isoeugenol, etc. are common diphenols. Polyphenols can be divided into flavonoids, phenolic acids, coumarins, cyclohexanes and tannins, such as shikimic acid, coumarin, cinnamic acid, chlorogenic acid and rutin. In a broad sense, phenolic compounds can be divided into simple phenols, flavonoids and quinone compounds. Among them, flavonoid compounds are extremely abundant in plants, usually containing a C6-C3-C6 basic skeleton of two aromatic rings, and mostly exist in the form of glycosides. Anthocyanidins belong to the class of flavonoid compounds. Due to their very active chemical properties, free anthocyanidins are extremely unstable. In the natural state, they usually combine with monosaccharides or disaccharides in the form of glycosidic bonds with their free hydroxyl groups to form various types of anthocyanins. The main types of anthocyanidins in higher plants include geranium pigments, cornflower pigments, delphinium pigments, peony pigments, morning glory pigments, and mallow pigments. Phenolic substances not only play important physiological roles such as antioxidant, antibacterial, and stress resistance in the organs of higher plants, but also damage the cell membranes of seedling roots by affecting cell membrane permeability and enzyme activity, thereby inhibiting their growth and development. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to promote the accumulation of phenolic and anthocyanin substances in the root system of grapes and the root system development.

[0006] In order to solve the above technical problems, the present invention first provides a new use of melatonin or a culture medium containing melatonin.

[0007] The present invention provides the use of melatonin or a culture medium containing melatonin in any one of the following a1)-a6):

[0008] a1) Promote the accumulation of total phenolic substances in grape roots;

[0009] a2) preparing a product for promoting the accumulation of total phenolic substances in grape roots;

[0010] a3) Promote the accumulation of total anthocyanins in grape roots;

[0011] a4) preparing a product for promoting the accumulation of total anthocyanidins in grape roots;

[0012] a5) Promote the development of grape root system;

[0013] a6) preparing a product for promoting the development of grape root system.

[0014] Furthermore, the culture medium containing melatonin is a subculture culture medium containing melatonin.

[0015] Furthermore, the concentration of melatonin in the subculture medium may be 1-10 μmol / L, preferably 10 μmol / L.

[0016] Furthermore, the secondary culture medium is MS medium.

[0017] In order to solve the above technical problems, the present invention further provides a grape subculture culture medium.

[0018] The grape subculture medium provided by the invention is a subculture medium containing melatonin.

[0019] Furthermore, the concentration of melatonin in the subculture medium may be 1-10 μmol / L, preferably 10 μmol / L.

[0020] Furthermore, the secondary culture medium is MS medium.

[0021] In some embodiments, the grape subculture medium consists of melatonin and the above-mentioned MS medium.

[0022] In order to solve the above technical problems, the present invention also provides a new use of the above grape subculture culture medium.

[0023] The present invention provides the use of a grape subculture medium in any one of the following a1)-a6):

[0024] a1) Promote the accumulation of total phenolic substances in grape roots;

[0025] a2) preparing a product for promoting the accumulation of total phenolic substances in grape roots;

[0026] a3) Promote the accumulation of total anthocyanins in grape roots;

[0027] a4) preparing a product for promoting the accumulation of total anthocyanidins in grape roots;

[0028] a5) Promote the development of grape root system;

[0029] a6) preparing a product for promoting the development of grape root system.

[0030] In order to solve the above technical problems, the present invention finally provides a method for promoting the accumulation of total phenolic substances and / or total anthocyanins in grape roots or promoting the development of grape roots.

[0031] The method for promoting the accumulation of total phenolic substances and / or total anthocyanins substances in grape roots or promoting the development of grape roots provided by the present invention comprises the following steps: culturing grape tissue culture seedlings in the above-mentioned grape subculture medium.

[0032] In the above method, the culture conditions are a temperature of 28° C., a photoperiod of 16 hours of light / 8 hours of darkness, and a light intensity of 2000-3000 lx.

[0033] In the above method, the culturing time may be at least 30 days, specifically 30 days.

[0034] Furthermore, the culture method comprises the following steps: cutting off the tissue containing the growth point and 2-3 cotyledons from the grape tissue culture seedlings, inserting the lower stem segment of the tissue into the above-mentioned grape subculture medium (3-4 mm) for culture, and subculturing once every 30 days.

[0035] In the above method, the grape tissue culture seedlings are 'Sweet Sapphire' grape tissue culture seedlings.

[0036] The solvent of any of the above-mentioned MS culture media is water, and the solutes and their concentrations are potassium nitrate 1900 mg / L; ammonium nitrate 1650 mg / L; potassium dihydrogen phosphate 170 mg / L; magnesium sulfate heptahydrate 370 mg / L; calcium chloride 330 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; manganese sulfate monohydrate 17 mg / L; zinc sulfate heptahydrate 8.6 mg / L; sodium molybdate dihydrate 0.25 mg / L; copper sulfate pentahydrate 0.025mg / L; cobalt chloride hexahydrate 0.025mg / L; disodium ethylenediaminetetraacetic acid 37.3mg / L; ferrous sulfate heptahydrate 27.8mg / L; inositol 100mg / L; glycine 2mg / L; thiamine hydrochloride 0.1mg / L; pyridoxine hydrochloride 0.5mg / L; niacin 0.5mg / L; IBA 0.3mg / L; sucrose 30g / L; agar 7g / L.

[0037] Any of the above-mentioned total phenolic substances include phenolic acid, flavonoids, tea polyphenols, lignans, resveratrol, catechins, chlorogenic acid, caffeic acid, ferulic acid, benzoic acid, kaempferol, naringin, morin, quercetin, etc. The total phenolic substances are determined by the Folin-phenol method.

[0038] Any of the above-mentioned total anthocyanidins includes cyanidin, delphinium pigment, peony pigment, morning glory pigment, malva pigment and geranium pigment, etc. The content of the total anthocyanidins is determined by the method of Neff and Chory (Neff MM, Chory J..Genetic interactions between phytochrome A, phytochrome B, and Cryptochrome 1 during Arabidopsis development. Plant Physiology, 1998, 118: 27-35.).

[0039] Any of the above-mentioned promoting the development of grape root system is embodied in any of the following b1)-b7):

[0040] b1) Improve the rooting rate of grapes;

[0041] b2) Increase the fresh weight of grape roots;

[0042] b3) Increase the number of grape adventitious roots;

[0043] b4) Increase the average diameter of grape adventitious roots;

[0044] b5) Increase the total root length of grapes;

[0045] b6) Increase the surface area of ​​grape roots;

[0046] b7) Increase the vitality of grape roots.

[0047] Any of the above mentioned grape varieties is 'Sweet Sapphire'.

[0048] The structural formula of any of the above melatonins is shown in Formula I, the chemical name is N-acetyl-5-methoxytryptamine, and the CAS number is 73-31-4.

[0049]

[0050]

[0051] The present invention applies melatonin to the subculture medium for the rooting culture of grape tissue culture seedlings for the first time. By measuring the root development-related traits, it is found that different concentrations of melatonin treatment affect the growth and development of grape roots to varying degrees. Low concentrations of melatonin treatment can significantly promote the root development of grape tissue culture seedlings, while high concentrations of melatonin treatment have little effect on the root system of grape tissue culture seedlings or even inhibit it. Except for the rooting rate, the values ​​of all other indicators show a trend of first increasing and then decreasing with the increase of the concentration of added melatonin. Among them, the 10μmol / L melatonin treatment has the highest increase multiple: the root fresh weight, adventitious root number, adventitious root average diameter, total root length, root surface area and root activity of 'Sweet Sapphire' grapes are 1.84 times, 1.42 times, 1.86 times, 1.46 times, 1.55 times and 1.55 times of the treatment without melatonin (0μmol / L), respectively, all of which are significantly different. In addition, by measuring the content of total phenolic and anthocyanidin substances in the root system, it was found that 10 μmol / L melatonin treatment can also significantly promote the accumulation of total phenolic and anthocyanidin substances in the root system of grapes. The present invention lays a theoretical foundation for the field application of melatonin. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the effect of different concentrations of MT on the root development of grape.

[0053] Figure 2 This is the effect of 10 μmol / L MT treatment on the contents of total phenolic substances and total anthocyanins in grape roots. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0057] The 'Sweet Blue Sapphire' grape tissue culture seedlings in the following examples are recorded in the literature "de Freitas LaiberPascoal G., de Almeida Sousa Cruz MA, Pimentel de Abreu J., Santos MCB, Bernardes Fanaro G., Júnior MRM, Freitas Silva O., Moreira RFA, Cameron L.C., Larraz Ferreira M.,Teodoro AJ.Evaluation of the antioxidantcapacity,volatile composition and phenolic content of hybrid Vitis viniferaL.varieties sweet sapphire and sweet surprise.Food Chemistry,2022,366:130644."

[0058] The melatonin in the following examples is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., China, with a product number of M118674. Melatonin belongs to the indole heterocyclic compound, also known as pinealogen, melatonin, and melatonin. Its structural formula is shown in Formula I, and its chemical name is N-acetyl-5-methoxytryptamine, with a CAS number of 73-31-4.

[0059]

[0060] The solvent of the MS culture medium in the following examples is water, and the solutes and their concentrations are potassium nitrate 1900 mg / L; ammonium nitrate 1650 mg / L; potassium dihydrogen phosphate 170 mg / L; magnesium sulfate heptahydrate 370 mg / L; calcium chloride 330 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; manganese sulfate monohydrate 17 mg / L; zinc sulfate heptahydrate 8.6 mg / L; sodium molybdate dihydrate 0.25 mg / L; copper sulfate pentahydrate 0.025mg / L; cobalt chloride hexahydrate 0.025mg / L; disodium ethylenediaminetetraacetic acid 37.3mg / L; ferrous sulfate heptahydrate 27.8mg / L; inositol 100mg / L; glycine 2mg / L; thiamine hydrochloride 0.1mg / L; pyridoxine hydrochloride 0.5mg / L; niacin 0.5mg / L; IBA 0.3mg / L; sucrose 30g / L; agar 7g / L.

[0061] Example 1: Effect of melatonin on rooting of 'Sweet Sapphire' grape tissue culture seedlings

[0062] The subculture medium was used to subculture the 'Sweet Sapphire' grape tissue culture seedlings. The specific steps are as follows: in a sterile clean bench, the tissue containing the growth point and 2-3 cotyledons was cut from the 'Sweet Sapphire' grape tissue culture seedlings, and the lower stem segment of the tissue was inserted into the MS subculture medium 3-4 mm, and the temperature was 28°C, the photoperiod was 16 hours of light / 8 hours of darkness, and the light intensity was 2000-3000lx. The subculture was performed once every 30 days. The subculture medium was the MS medium containing melatonin, and according to the different melatonin concentrations, it was divided into the following groups:

[0063] 0 μmol / L group: the concentration of melatonin in MS medium was 0 μmol / L.

[0064] 1 μmol / L group: the concentration of melatonin in MS medium was 1 μmol / L.

[0065] 10 μmol / L group: The concentration of melatonin in MS medium was 10 μmol / L.

[0066] 50 μmol / L group: The concentration of melatonin in MS medium was 50 μmol / L.

[0067] 100 μmol / L group: The concentration of melatonin in MS medium was 100 μmol / L.

[0068] After about 30 days of cultivation, the phenotype was recorded by taking photos, and the root-related traits were counted. The number of adventitious roots, the average diameter of adventitious roots, the total root length, and the root surface area were all obtained by plant root scanner (Epson Perfection V850 Pro). The rooting rate was calculated by the following formula: rooting rate = number of rooted plants / total number of plants. The fresh root weight was obtained by weighing the mass of the new root system using an electronic balance. The root activity was determined by the trichlorothiazolidine chloride (TTC) method. For specific steps, please refer to the method in the literature "Zhang X., Huang G., Bian X., Zhao Q.. Effects of root interaction and nitrogen fertilization on the chlorophyll content, root activity, photosynthetic characteristics of intercropped soybean and microbial quantity in the rhizo-sphere. Plant, Soil and Environment, 2013, 59: 80-88." The data were analyzed by variance analysis using Microsoft Excel software (Student's t-test, P < 0.05). Three biological replicates were set for each treatment, and the results were averaged.

[0069] The results are shown in Table 1 and Figure 1 The results showed that different concentrations of melatonin treatment affected the growth and development of grape roots to varying degrees. Low concentrations of MT treatments of 1μmol / L and 10μmol / L could significantly enhance the root development of grape seedlings, while high concentrations of MT treatments of 50μmol / L and 100μmol / L had little effect on the root development of grape seedlings or even inhibited it. Except for the rooting rate, the values ​​of all other indicators showed a trend of first increasing and then decreasing with the increase of the concentration of melatonin added. Among them, the 10μmol / L melatonin treatment had the highest increase multiple: the root fresh weight, number of adventitious roots, average diameter of adventitious roots, total root length, root surface area and root activity of 'Sweet Sapphire' grapes were 1.84 times, 1.42 times, 1.86 times, 1.46 times, 1.55 times and 1.55 times of those without melatonin addition (0μmol / L), respectively, all showing significant differences. After treatment with 50μmol / L melatonin, all indicators except root activity were significantly reduced.

[0070] Table 1. Changes of grape root indexes under different concentrations of melatonin

[0071] MT concentration (μmol / L) 0 1 10 50 100 Rooting rate (%) 89.00±1.00a 86.67±6.01ab 83.33±4.74ab 70.00±8.00b 50.00±8.20c Root fresh weight (g) 0.51±0.03d 0.61±0.02c 1.12±0.04a 0.81±0.04b 0.32±0.03e Number of adventitious roots 10.33±0.58c 19.67±2.08b 28.00±4.36a 11.00±1.73c 8.33±2.31cd Average diameter of adventitious roots (mm) 1.74±0.12c 2.43±0.10b 3.24±0.25a 2.25±0.34bc 1.89±0.28bc Total root length (cm) 151.93±25.71b 138.81±19.50bc 202.87±8.25a 87.85±7.42c 64.83±15.49cd <![CDATA[Root surface area (cm 2 )]]> 30.93±4.01ab 25.94±3.89bc 40.25±6.71a 20.59±2.90c 13.53±3.13d <![CDATA[Root activity TTC mg·(gh) -1 > 0.14±0.01b 0.11±0.01d 0.17±0.01a 0.14±0.00b 0.13±0.01c

[0072] Example 2: Effect of melatonin on the content of phenolic and anthocyanin substances in 'Sweet Blue Sapphire' grape tissue culture seedlings

[0073] The 'Sweet Sapphire' grape tissue culture seedlings were cultured in a subculture medium under the following conditions: temperature of 28°C, photoperiod of 16 hours light / 8 hours dark, and light intensity of 2000-3000 lx. The subculture medium was MS medium containing 10 μmol / L melatonin, and MS medium without melatonin was used as a control. After about 30 days of culture, the contents of total phenols and total anthocyanins were determined.

[0074] The total phenolic content was determined by the Folin-phenol method. The specific steps refer to the method in the literature "Xu L., Yue Q., Bian F., Sun H., Zhai H., Yao Y..Melatonin enhances phenolics accumulation partially via ethylene signaling and resulted in high antioxidant capacity in grape berries. Frontiers in plant science, 2017, 8: 1426.", and the results were calculated using a calibration curve made with gallic acid. The relative content of total phenolic content in roots was expressed as mg gallic acid / g fresh weight (FW).

[0075] The content of total anthocyanidins was determined by the method of Neff and Chory. For specific steps, please refer to the method in the literature "Neff M.M., Chory J..Genetic interactions between phytochrome A, phytochrome B, and Cryptochrome 1 during Arabidopsis development. Plant Physiology, 1998, 118: 27-35." The specific steps include the following steps: grinding 1.5g of fresh grape roots with a 1% (v / v) hydrochloric acid-methanol solution extract. Ultrasonication for 3 minutes, incubation in the dark for 24 hours, centrifugation at 10000rpm for 10min, and using a spectrophotometer to measure the absorbance of the sample at 530nm and 657nm wavelengths. The relative content of total anthocyanins in the root system is expressed in mg / g fresh weight (FW).

[0076] The results are as follows Figure 2The results showed that 10 μmol / L melatonin treatment can significantly promote the accumulation of total phenolic and anthocyanin substances in the root system of grapes. Among them, the total phenolic and anthocyanin contents in the root system of grapes in the control group were 0.39 mg gallic acid / g and 0.27 mg / g, respectively, and the total phenolic and anthocyanin contents in the root system of grapes in the experimental group with 10 μmol / L melatonin added were 0.61 mg gallic acid / g and 2.77 mg / g, respectively.

[0077] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of melatonin or a culture medium containing melatonin in any of the following a1)-a6): a1) Promote the accumulation of total phenolic substances in grape roots; a2) preparing a product for promoting the accumulation of total phenolic substances in grape roots; a3) Promote the accumulation of total anthocyanins in grape roots; a4) preparing a product for promoting the accumulation of total anthocyanidins in grape roots; a5) Promote the development of grape root system; a6) preparing a product for promoting the development of grape root system.

2. The use according to claim 1, characterized in that: The culture medium containing melatonin is a subculture culture medium containing melatonin.

3. The use according to claim 2, characterized in that: The concentration of melatonin in the subculture medium is 1-10 μmol / L.

4. The use according to claim 2 or 3, characterized in that: The subculture medium is MS medium.

5. A grape subculture medium, wherein the grape subculture medium is a subculture medium containing melatonin.

6. The grape subculture medium according to claim 5, characterized in that: The concentration of melatonin in the subculture medium is 1-10 μmol / L.

7. The grape subculture medium according to claim 5 or 6, characterized in that: The subculture medium is MS medium.

8. Use of the grape subculture medium according to any one of claims 5 to 7 in any one of the following a1) to a6): a1) Promote the accumulation of total phenolic substances in grape roots; a2) preparing a product for promoting the accumulation of total phenolic substances in grape roots; a3) Promote the accumulation of total anthocyanins in grape roots; a4) preparing a product for promoting the accumulation of total anthocyanidins in grape roots; a5) Promote the development of grape root system; a6) preparing a product for promoting the development of grape root system.

9. A method for promoting the accumulation of total phenolic substances and / or total anthocyanins in grape roots, comprising the following steps: culturing grape tissue culture seedlings in the grape subculture medium according to any one of claims 5 to 7; Or, a method for promoting the development of grape root system, comprising the following steps: culturing grape tissue culture seedlings in the grape subculture medium according to any one of claims 5-7.

10. The use according to claim 1 or the use according to claim 8 or the method according to claim 9, characterized in that: The promotion of grape root development is embodied in any one of the following b1)-b7): b1) Improve the rooting rate of grapes; b2) Increase the fresh weight of grape roots; b3) Increase the number of grape adventitious roots; b4) Increase the average diameter of grape adventitious roots; b5) Increase the total root length of grapes; b6) Increase the surface area of ​​grape roots; b7) Increase the vitality of grape roots.

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