Application of melatonin in promoting the accumulation of phenolic and anthocyanin substances and root development in grape roots

Abstract

This invention discloses the application of melatonin in promoting the accumulation of phenolic and anthocyanin substances and root development in grape roots. This invention is the first to apply melatonin to the rooting culture of grape tissue culture seedlings in a subculture medium. Measurements of root development-related traits revealed that different concentrations of melatonin treatment affected grape root growth and development to varying degrees. Low concentrations of melatonin significantly promoted root development in grape tissue culture seedlings, while high concentrations had little or no effect on root development. Furthermore, measurements of total phenolic and anthocyanin content in roots showed that 10 μmol / L melatonin treatment significantly promoted the accumulation of total phenolic and anthocyanin substances in grape roots. This invention lays the theoretical foundation for the field application of melatonin.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and biochemistry, specifically to the application of melatonin in promoting the accumulation of phenolic and anthocyanin substances in grape roots and root development. Background Technology

[0002] With the promotion of plant factory seedling production technology, the efficient and convenient propagation of a large number of virus-free seedlings has become the primary task of grape breeding. Therefore, it is particularly important to explore the mechanism by which different types and concentrations of plant growth regulators induce adventitious root formation in plants.

[0003] Melatonin (MT), a powerful natural antioxidant, not only enhances a plant's resistance to biotic and abiotic stresses but also possesses various physiological functions. Recent research reports on MT in grape roots have primarily focused on abiotic stresses, with almost no research on its effects on grape root growth and development. Roots are crucial organs involved in plant growth and development, metabolism, and information transmission and transduction, directly determining the strength of grape's resistance to abiotic stresses and thus constraining its growth and development.

[0004] Phenolic compounds are compounds formed when hydrogen atoms on aromatic rings are replaced by hydroxyl groups or functional derivatives. They are diverse and important plant secondary metabolites. Based on the number of hydroxyl groups directly attached to the aromatic ring, they can be classified into monohydric phenols, dihydric phenols, and polyhydric phenols. Common monohydric phenols include phenol, o-cresol, and m-cresol; common dihydric phenols include catechol, resorcinol, hydroquinone, eugenol, and isoeugenol. Polyhydric phenols can be further divided into flavonoids, phenolic acids, coumarins, cyclohexanes, and tannins, such as shikimic acid, coumarins, cinnamic acid, chlorogenic acid, and rutin. In a broader sense, phenolic compounds can be divided into simple phenols, flavonoids, and quinones. Flavonoids are extremely abundant in plants, typically containing a C6-C3-C6 basic skeleton with two aromatic rings, and often exist in glycoside form. Anthocyanins belong to the flavonoid family of compounds. Due to their highly reactive chemical properties, free anthocyanins are extremely unstable. In their natural state, they typically combine with monosaccharides or disaccharides via glycosidic bonds through their free hydroxyl groups, forming various anthocyanins. The main types of anthocyanins found in higher plants include pelargonidin, cyanidin, delphinidin, peonydin, morning glorydin, and mallowdin. Phenolic compounds not only play important physiological roles in higher plant organs, such as antioxidation, antibacterial activity, and stress resistance, but they also inhibit the growth and development of seedling roots by affecting cell membrane permeability and enzyme activity, thereby damaging the cell membranes. Summary of the Invention

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

[0006] To address the aforementioned technical problems, the present invention first provides a new use for melatonin or a culture medium containing melatonin.

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

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

[0009] a2) Prepare products that promote the accumulation of total phenolic substances in grape roots;

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

[0011] a4) Prepare products that promote the accumulation of total anthocyanins in grape roots;

[0012] a5) Promotes grape root development;

[0013] a6) Prepare products that promote grape root development.

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

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

[0016] Furthermore, the subculture medium is MS medium.

[0017] To address the aforementioned technical problems, the present invention provides a grape subculture medium.

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

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

[0020] Furthermore, the subculture medium is MS medium.

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

[0022] To address the aforementioned technical problems, this invention also provides a new use for the above-mentioned grape subculture medium.

[0023] This invention provides the use of grape subculture medium in any of the following a1)-a6):

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

[0025] a2) Prepare products that promote the accumulation of total phenolic substances in grape roots;

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

[0027] a4) Prepare products that promote the accumulation of total anthocyanins in grape roots;

[0028] a5) Promotes grape root development;

[0029] a6) Prepare products that promote grape root development.

[0030] To address the aforementioned technical problems, the present invention ultimately provides a method for promoting the accumulation of total phenolic substances and / or total anthocyanins in grape roots or for promoting grape root development.

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

[0032] In the above method, the cultivation 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 culture time can be at least 30 days, specifically 30 days.

[0034] Furthermore, the cultivation method includes the following steps: cutting off tissue containing the growth point and 2-3 cotyledons from grape tissue culture seedlings, inserting the lower stem segment of the tissue into the above-mentioned grape subculture medium (3-4 mm) for cultivation, 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 for any of the MS culture media described above is water, and the solutes and their concentrations are as follows: 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. / L; Copper sulfate pentahydrate 0.025mg / L; Cobalt chloride hexahydrate 0.025mg / L; Disodium EDTA 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; Nicotinic acid 0.5mg / L; IBA 0.3mg / L; Sucrose 30g / L; Agar 7g / L.

[0037] The total phenolic substances mentioned above include phenolic acids, 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 using the Folin-Ciocalteu method.

[0038] The total anthocyanins mentioned above include cyanidin, delphinidin, peonyin, morning gloryin, mallowin, and geraniumin, etc. The content of the total anthocyanins was determined using the Neff and Chory method (Neff MM, Chory J. Genetic interactions between phytochrome A, phytochrome B, and Cryptochrome 1 during Arabidopsis development. Plant Physiology, 1998, 118:27-35.).

[0039] The aforementioned promotion of grape root development is manifested in any one 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 adventitious roots in grapevines;

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

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

[0045] b6) Increase the root surface area of ​​grapes;

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

[0047] The grape variety mentioned above is 'Sweet Sapphire'.

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

[0049]

[0050]

[0051] This invention is the first to apply melatonin to the rooting culture of grape tissue culture seedlings in a subculture medium. Measurements of root development-related traits revealed that different concentrations of melatonin treatment affected grape root growth and development to varying degrees. Low-concentration melatonin treatment significantly promoted root development in grape tissue culture seedlings, while high-concentration melatonin treatment had little or no effect on root development, and even inhibited it. Except for the rooting rate, all other indicators showed a trend of first increasing and then decreasing with increasing melatonin concentration. The 10 μmol / L melatonin treatment showed the highest upregulation: the fresh weight of 'Sweet Sapphire' grape roots, the number of adventitious roots, the average diameter of adventitious roots, the total root length, the root surface area, and the root activity were 1.84 times, 1.42 times, 1.86 times, 1.46 times, 1.55 times, and 1.55 times higher than those in the treatment without melatonin (0 μmol / L), respectively, all showing significant differences. Furthermore, measurements of total phenolic and anthocyanin content in the roots revealed that treatment with 10 μmol / L melatonin significantly promoted the accumulation of total phenolic and anthocyanin substances in grape roots. This invention lays the theoretical foundation for the field application of melatonin. Attached Figure Description

[0052] Figure 1 The effects of different concentrations of MT treatment on grape root development.

[0053] Figure 2 The effect of 10 μmol / L MT treatment on the content of total phenolic substances and total anthocyanins in grape roots. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0056] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0057] The 'Sweet Sapphire' grape tissue culture seedlings in the following examples are described 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 used in the following examples is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., China, with product number M118674. Melatonin belongs to the indole heterocyclic class of compounds, also known as pineal hormone, melatonin, or melatonin extract. Its structural formula is shown in Formula I, and its chemical name is N-acetyl-5-methoxytryptamine, with CAS number 73-31-4.

[0059]

[0060] The MS medium used in the following examples was water as the solvent, and the solutes and their concentrations were as follows: 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. / L; Copper sulfate pentahydrate 0.025mg / L; Cobalt chloride hexahydrate 0.025mg / L; Disodium EDTA 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; Nicotinic acid 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] Subculture of 'Sweet Sapphire' grape tissue culture seedlings was performed using subculture medium. The specific steps are as follows: In a sterile laminar flow hood, tissue containing the growing point and 2-3 cotyledons was excised from the 'Sweet Sapphire' grape tissue culture seedlings. The lower stem segment of the tissue was inserted 3-4 mm into MS subculture medium and cultured at 28℃ with a photoperiod of 16 hours light / 8 hours dark and a light intensity of 2000-3000 lx. Subculture was performed every 30 days. The subculture medium was MS medium containing melatonin, and the subcultures were divided into the following groups according to different melatonin concentrations:

[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] Phenotypic data were recorded by photographing after approximately 30 days of cultivation, and root-related traits were statistically analyzed. The number of adventitious roots, average diameter of adventitious roots, total root length, and root surface area were all measured using a plant root scanner (Epson Perfection V850 Pro). Rooting rate was calculated using the following formula: Rooting rate = Number of rooted plants / Total number of plants. Root fresh weight was obtained by weighing the newly formed roots using an electronic balance. Root activity was determined using the chloropyridine (TTC) method; specific procedures are described 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 rhizosphere. Plant, Soil and Environment, 2013, 59:80-88.” Data were analyzed using Microsoft Excel (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 As shown, the results indicated that different concentrations of melatonin treatment affected the growth and development of grape roots to varying degrees. Low concentrations of MT (1 μmol / L and 10 μmol / L) significantly enhanced root development in grape tissue culture seedlings, while high concentrations of MT (50 μmol / L and 100 μmol / L) had little or no effect on root development, and some even inhibited it. Except for the rooting rate, all other indicators showed a trend of first increasing and then decreasing with increasing melatonin concentration. The 10 μmol / L melatonin treatment showed the highest upregulation: the fresh weight of 'Sweet Sapphire' grape roots, the number of adventitious roots, the average diameter of adventitious roots, the total root length, the root surface area, and the root activity were 1.84 times, 1.42 times, 1.86 times, 1.46 times, 1.55 times, and 1.55 times higher than those in the treatment without melatonin (0 μmol / L), respectively, all showing significant differences. After treatment with 50 μmol / L melatonin, all indicators except root activity decreased significantly.

[0070] Table 1. Changes in grape root indicators 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 Fresh root 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 (number of 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 tissue culture seedlings of 'Sweet Sapphire' grapes

[0073] 'Sweet Sapphire' grape tissue culture seedlings were cultured in subculture medium under the following conditions: temperature 28℃, 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, with MS medium without added melatonin serving as a control. After approximately 30 days of culture, the contents of total phenols and total anthocyanins were measured.

[0074] The total phenolic content was determined using the Folin-Ciocalteu method, with specific procedures described 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.” The results were calculated using a calibration curve prepared with gallic acid. The relative content of total phenols in roots is expressed as mg gallic acid / g fresh weight (FW).

[0075] The total anthocyanin content was determined using the method of Neff and Chory, with specific steps described 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 included: grinding 1.5 g of fresh grape roots with a 1% (v / v) hydrochloric acid-methanol solution extract; sonicating for 3 minutes; incubating in the dark for 24 hours; centrifuging at 10000 rpm for 10 minutes; and measuring the absorbance at 530 nm and 657 nm using a spectrophotometer. The relative content of total anthocyanins in the roots is expressed as mg / g fresh weight (FW).

[0076] The results are as follows Figure 2As shown, the results indicated that treatment with 10 μmol / L melatonin significantly promoted the accumulation of total phenols and anthocyanins in grape roots. Specifically, the total phenolic and anthocyanin contents in the control group grape roots were 0.39 mg gallic acid / g and 0.27 mg / g, respectively, while the contents in the experimental group treated with 10 μmol / L melatonin were 0.61 mg gallic acid / g and 2.77 mg / g, respectively.

[0077] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for promoting the accumulation of total phenolic substances in grape roots, comprising the following steps: culturing grape tissue culture seedlings in grape subculture medium; The grape subculture medium is a subculture medium containing melatonin; The concentration of melatonin in the subculture medium was 10 μmol / L; The subculture medium was MS medium.

2. A method for promoting the accumulation of total anthocyanins in grape roots, comprising the following steps: culturing grape tissue culture seedlings in grape subculture medium; The grape subculture medium is a subculture medium containing melatonin; The concentration of melatonin in the subculture medium was 10 μmol / L; The subculture medium was MS medium.

Images