CN115968884B - Plant antifreeze agent containing curcumin and raffinose as well as preparation method and application thereof - Google Patents
Plant antifreeze agent containing curcumin and raffinose as well as preparation method and application thereof Download PDFInfo
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- CN115968884B CN115968884B CN202310008967.3A CN202310008967A CN115968884B CN 115968884 B CN115968884 B CN 115968884B CN 202310008967 A CN202310008967 A CN 202310008967A CN 115968884 B CN115968884 B CN 115968884B
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- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 title claims abstract description 155
- 235000012754 curcumin Nutrition 0.000 title claims abstract description 77
- 229940109262 curcumin Drugs 0.000 title claims abstract description 77
- 239000004148 curcumin Substances 0.000 title claims abstract description 77
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 title claims abstract description 77
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 title claims abstract description 71
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a plant antifreeze agent containing curcumin and raffinose, a preparation method and application thereof, and relates to the technical fields of plant growth regulators and agricultural planting management. The plant antifreeze agent containing curcumin and raffinose mainly comprises the effective components of curcumin and raffinose, wherein the volume percentage of the curcumin is 0.1-0.5%, the mass percentage of the raffinose is 0.6-6%, and the antifreeze agent also comprises other components of monopotassium phosphate, calcium chloride, glycerol and tween 20. The invention breaks through the research of curcumin and raffinose in the low temperature resistance of plants, can improve the antioxidant enzyme activity of the plants, increase in-vivo permeation regulating substances to cope with low temperature stress, and strengthen the frost resistance of the plants. The composite antifreeze agent has the advantages of simple components, no hormone or pesticide components, safety, environmental protection, self configuration, low cost, simple operation and suitability for popularization and application.
Description
Technical Field
The invention relates to the technical field of plant growth regulators and agricultural planting management, in particular to a plant antifreeze containing curcumin and raffinose, and a preparation method and application thereof.
Background
Low temperature stress is an important abiotic stress affecting plant growth and development. Low temperature stress includes cold damage (< 15 ℃) and freeze damage (< 0 ℃) where the effect of freeze damage on plants is greatest. In north of China, the cold in the spring is a common natural disaster, and seriously affects the growth and the yield of crops. The season of the growth of crops such as vegetables, tobacco and the like in spring, the cold in the spring seriously affects the growth of plants, even causes frostbite of seedlings, and cannot grow normally, so that serious economic loss is caused.
Many crops are sensitive to low temperatures, such as tobacco, tomato, cucumber, rice, corn, and the like.
Tobacco (Nicotiana tabacum l.) is an important cash crop and a temperature-preferred plant, which is relatively sensitive to low temperatures. Tobacco seedlings are in a cross stage, namely a two-leaf one-heart stage, and the temperature in the shed is easy to be low when the tobacco seedlings suffer from the cold of the spring, so that the tobacco seedlings are frosted, the leaves are easy to be deformed and yellow, and the normal development of the tobacco seedlings is influenced. The tobacco is transplanted before and after about 5 months and is often damaged at low temperature, and the tobacco is delayed in development and even early blossoming due to the fact that the tobacco is in a period from five leaves to six leaves. The low temperature research of tobacco has practical application value.
Tomatoes and cucumbers are the main cultivated vegetable varieties in China. The optimal growth temperature of tomatoes is 20-30 ℃ and is sensitive to low-temperature reaction. The main harm of low temperature to tomatoes is shown in a seedling stage, the growth and development of tomato seedlings can be retarded at the temperature below 10 ℃, and abnormal fruits can be generated when two true leaves are subjected to low-temperature treatment at the temperature of 6 ℃. The optimal growth temperature of cucumber seedlings is 18-30 ℃ and is sensitive to low temperature. When the temperature is lower than 12 ℃, the leaves of the cucumber seedlings can be water-stained, and the growth is slow, so that the normal growth and development of the seedlings are affected. Early spring cucumber seedlings were injured by low temperature in two true She Shiyi. Therefore, the spring is positive to the seedling stage of tomatoes and cucumbers, the low-temperature resistance of the tomato seedlings and the cucumber seedlings is improved, and the method has very important significance for guaranteeing the yield of the seedlings and improving the economic benefit.
The existing antifreezing measures mainly comprise the steps of cultivating new plant varieties, raising seedlings in a greenhouse, laminating films and the like, but the measures are time-consuming and labor-consuming, have long consumption period and are high in cost. Plant antifreeze agents treated by foliar spraying or root irrigation are also available in the market, but the plant antifreeze agents commonly available in the market at present have complex components, contain more hormone or pesticide components and the like.
Curcumin (Curcumin) is a polyphenol active substance existing in turmeric, is composed of two methoxy groups and benzene rings of two hydroxyl groups, can remove active oxygen, is an antioxidant, has the functions of resisting oxidation, resisting tumors, resisting cancers, resisting inflammation, resisting bacteria and the like, plays an important role in improving the antioxidant activity of animals and relieving oxidative damage, has not been researched and reported about the role of curcumin in resisting low temperature of plants, and can greatly promote the research and development of low-temperature management of the curcumin if the curcumin is researched and popularized and applied in resisting low temperature of plants.
The raffinose oligosaccharide (Raffinose Family Oligosaccharides, RFO) is composed of raffinose, stachyose and acteoside. Wherein raffinose (Raffinose) is produced from 1 alpha-1, 6-galactose linked to 6-glucosyl group on sucrose. RFOs are widely available in plants and have a wide range of functions. Research shows that under the stress, a large amount of RFOs is accumulated in the plant body and participates in the osmotic regulation of cells, so that the stress resistance of the plant is improved. The accumulation of raffinose in plants caused by low-temperature stress is not reported about whether the low-temperature resistance of tobacco seedlings can be improved by externally applying the raffinose, so that the raffinose has wide application prospect.
The application of the curcumin and the raffinose is not yet reported, so that the application and popularization of the curcumin and the raffinose in the application of the plant antifreeze can greatly promote the development of low-temperature stress management of plants.
Disclosure of Invention
The invention aims to provide a plant antifreeze agent containing curcumin and raffinose, which promotes the application and development of the curcumin and the raffinose in the aspect of plant low-temperature stress management, and solves the problems that the plant antifreeze agent commonly used in the market at present has complex components, contains more hormone or pesticide components and the like.
A method for improving low-temperature resistance of tobacco seedlings uses curcumin solution with volume percentage of 0.1% -0.5% to carry out foliage spraying treatment, wherein the foliage spraying treatment method is that liquid medicine is sprayed to the front and back surfaces of leaves to be dripped.
Based on the above scheme, preferably, the volume percentage of the curcumin solution is 0.1%, and the curcumin solution contains 0.02% of surfactant Tween 20.
Meanwhile, another method for improving the low-temperature resistance of tobacco seedlings is to spray the solution of raffinose with the mass percent of 0.6-6% on the leaf surfaces, wherein the method for spraying the solution on the front and back surfaces of the leaf surfaces is to drop the solution.
Based on the scheme, preferably, the mass percentage of the raffinose solution is 0.6%, and the raffinose solution contains 0.02% of surfactant Tween 20.
In order to achieve the aim, the application provides a plant antifreeze agent containing curcumin and raffinose, which mainly comprises the effective components of curcumin and raffinose, wherein the curcumin accounts for 0.1-0.5% by volume and the raffinose accounts for 0.6-6% by mass.
Based on the scheme, the fertilizer also comprises other components including monopotassium phosphate, calcium chloride, glycerol and Tween 20.
Preferably, the curcumin comprises 0.1% by volume, 0.6% by mass of raffinose, 0.01% by mass of potassium dihydrogen phosphate, 0.11% by mass of calcium chloride, 0.02% by volume of glycerin, 0.02% by volume of tween 20 and the balance of water.
Optionally, the method of using any one of the above, the period and method of using the plant antifreeze agent containing curcumin and raffinose is as follows:
Two leaves and one period seedling, the use time is before 10 am or after 4 pm, the use time is before 1 day at low temperature, the temperature is not lower than 0 ℃, the leaf surface is sprayed for 1 time, the plant antifreeze agent is sprayed until the liquid medicine is to be dripped on the front side and the back side of the leaf, or the plant antifreeze agent is sprayed according to the dosage of 750kg per hectare;
Five leaves and one heart stage seedling are sprayed for 1 time every other day and 4 times before the use time is 10 am or after 4 pm before low temperature comes; spraying the plant antifreeze agent until the liquid medicine is to be dripped on the front side and the back side of the blade, or spraying the plant antifreeze agent according to the dosage of 750kg per hectare.
Based on the above scheme, the plant antifreeze agent (herein or simply referred to as a composite plant antifreeze agent) containing curcumin and raffinose is most suitable for plants including tobacco, tomato and cucumber.
A preparation method of a plant frozen agent containing curcumin and raffinose, based on total volume of 1L, comprises the following steps:
s1: dissolving 0.0368g of plant active substance curcumin powder in 10ml of absolute ethyl alcohol to prepare curcumin mother liquor for later use;
S2: 1ml of curcumin mother liquor, 6g of raffinose, 0.1g of monopotassium phosphate, 1.1g of calcium chloride, 0.2ml of glycerol and 0.2ml of tween 20 are measured according to the proportion requirement for standby;
s3: adding the prepared materials in the step S2 into water, and fully stirring and uniformly mixing;
S4: after the preparation is completed, the mixture is transferred into a storage bottle and stored in a dark place.
The beneficial effects of the invention are as follows:
The application utilizes curcumin, raffinose, potassium dihydrogen phosphate, calcium chloride, glycerin and tween to compound into the efficient and environment-friendly composite plant antifreeze agent, and can obviously improve the survival rate of plant seedlings, enhance the antioxidant enzyme activity of plants, enhance the accumulation of osmotic adjusting substances and the like by spraying the composite plant antifreeze agent on leaf surfaces, thereby enhancing the low temperature resistance of plants and relieving the damage of low temperature to the plant seedlings.
The research of the application breaks through the research and application of curcumin in the low temperature stress resistance of plants, the research and application of raffinose in the low temperature stress resistance of plants, and the research and application of curcumin and raffinose, potassium dihydrogen phosphate, calcium chloride, glycerin and tween compound in the low temperature stress resistance of plants.
The plant antifreeze agent containing curcumin and raffinose provided by the application has the advantages of simple components, no hormone or pesticide components, safety, environmental protection, no toxicity and no pollution; the plant antifreeze agent containing curcumin and raffinose can be prepared by self, is low in cost and simple to operate, and is worthy of popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is apparent that the drawings in the following description are only one embodiment of the present invention, and that other embodiments of the drawings may be derived from the drawings provided without inventive effort for a person skilled in the art.
Fig. 1: the embodiment of the invention firstly externally applied curcumin affects the phenotype of tobacco seedlings subjected to low-temperature stress;
Fig. 2: the embodiment of the invention has the effects of externally applied curcumin on the physiological index of tobacco seedlings in five-leaf-one-heart period under low-temperature stress;
fig. 3: the influence of the external Shi Mianzi sugar on the phenotype of the tobacco seedlings subjected to low-temperature stress is disclosed in the embodiment of the invention;
Fig. 4: the influence of the external Shi Mianzi sugar on the physiological index of the low-temperature stress five-leaf first-heart tobacco seedling is provided;
fig. 5: the fourth external application of the composite plant antifreeze agent influences the phenotype of the tobacco seedlings under low-temperature stress;
Fig. 6: the fourth external application compound plant antifreeze agent provided by the embodiment of the invention has influence on the physiological index of the tobacco seedlings in the five-leaf-one-heart period under low-temperature stress;
fig. 7: the fourth external application of the composite plant antifreeze agent affects the tobacco seedlings grown in the field;
Fig. 8: the fifth external application compound plant antifreeze agent of the embodiment of the invention has influence on the phenotype of low-temperature stress tomato seedlings;
Fig. 9: the fifth external application compound plant antifreeze agent of the embodiment of the invention has influence on the physiological index of the tomato seedlings in the two-leaf one-heart period under low temperature stress;
fig. 10: the fifth external application compound plant antifreeze agent of the embodiment of the invention has influence on the phenotype of low-temperature stress cucumber seedlings;
Fig. 11: the fifth external application compound plant antifreeze agent provided by the embodiment of the invention has influence on the physiological index of the cucumber seedlings in the period of two leaves and one heart under low temperature stress.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
Embodiment one: influence of external Shi Jianghuang on low-temperature stress tobacco seedlings screening and verifying experiment 1 and experimental materials:
Tobacco (Nicotiana tabacum l.) 'K326' variety, maintained for the plant stress physiology and molecular biology laboratory of the peninsula agricultural university;
curcumin (Curcumin) was purchased from Shanghai Rui Yongsheng biotechnology Co., ltd;
tween 20 (Tween 20) was purchased from Guozhu Chemicals Co.
2. The experimental method comprises the following steps:
phenotypic observation adopts tobacco seedlings in two periods as experimental materials, namely two-leaf one-heart period and five-leaf one-heart period.
Two-leaf one heart stage:
1) Material culture: sowing tobacco seeds in seedling raising trays containing turf wood nutrient soil, culturing in a greenhouse with the temperature of 25 ℃ and the humidity of 55% and the photoperiod of 16/8h, and selecting tobacco seedlings with consistent growth vigor after two weeks, and transplanting the tobacco seedlings into the seedling raising trays for subsequent treatment.
2) Two-leaf one-heart stage tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment 1: spraying curcumin solution with the volume percentage of 0.1% on the leaf surface;
③ Treatment 2: spraying curcumin solution with the volume percentage of 0.5% on the leaf surface;
the treatment method comprises the following steps: spraying curcumin solution (containing 0.02% of surfactant Tween 20) with volume percentage of 0, 0.1 and 0.5% on two leaves and one period of tobacco seedling, spraying the front and back surfaces of the leaves until the liquid medicine is to be dropped, standing for 12h, standing in a low-temperature illumination incubator for gradient cooling treatment, treating at 4 ℃ for 2h, treating at 0 ℃ for 1h, treating at-1 ℃ for 1h, treating at-2 ℃ for 1h, treating at-3 ℃ for 1h, standing in a greenhouse (25 ℃) for 3d, observing phenotype, counting survival rate of the seedling, and repeating each treatment for more than 3 times.
Five-leaf one heart stage:
1) Material culture: sowing tobacco seeds in a seedling raising tray containing turf wood nutrient soil, placing the tobacco seeds in a greenhouse with the temperature of 25 ℃ and the humidity of 55% and the photoperiod of 16/8h for cultivation, selecting single tobacco seedlings with consistent growth vigor after two weeks, transplanting the single tobacco seedlings into a flowerpot for continuous cultivation until the single tobacco seedlings with consistent growth vigor are in a four-leaf one-heart period, and selecting the seedlings with consistent growth vigor for subsequent treatment.
2) Four-leaf first-season tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment group: spraying curcumin solution with the volume percentage of 0.1% on the leaf surface;
The treatment method comprises the following steps: spraying curcumin solution (containing 0.02% of surfactant Tween 20) with volume percentage of 0.1% on four leaves and one period of tobacco seedling, spraying the solution on the front and back sides of the leaves until the solution is to be dripped, spraying for 1 time every 24 hours, after 4 times of total treatment, putting the four leaves and one period of tobacco seedling in a low-temperature illumination incubator, carrying out gradient cooling treatment, treating at 4 ℃ for 2 hours, treating at 0 ℃ for 1 hour, -1 ℃ for 1 hour, -2 ℃ for 1 hour, -3 ℃ for 1 hour, -4 ℃ for 1 hour, putting the four leaves and one period of tobacco seedling in a greenhouse (25 ℃) for recovering growth for 3 days, observing the phenotype, counting the survival rate of the seedlings, and repeating the treatment for more than 3 times each time.
3) And (3) measuring a physiological index: selecting tobacco seedlings with four leaves and one period and consistent growth, carrying out leaf surface spraying treatment by using curcumin solution (containing 0.02% of surfactant Tween 20) with the volume percentage of 0.1%, spraying for 1 time every 24 hours, carrying out total treatment for 4 times, respectively sampling and mixing the third and the fourth true leaves before and after low-temperature treatment (2 hours at 4 ℃ and 1 hour at 0 ℃ and 1 hour at 1 ℃), and detecting low-temperature related physiological indexes.
Measuring the index: cell membrane permeability, MDA (malondialdehyde), SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), pro (proline), SS (soluble sugar), active oxygen content (H 2O2 and O 2 -).
Cell membrane permeance assay reference (Zhang et al, 2021) method, MDA content reference (Tang et al, 2017) method, O 2.- and H 2O2 content assay reference (Wang et al, 2019) method, SOD, POD, CAT activity reference (Shen et al, 2021) method, pro content assay according to (Quan et al, 2022) method, SS content assay reference (Gurrieri et al, 2020) method.
3. Experimental data:
TABLE 1 Effect of topical curcumin on Low temperature stress tobacco seedling survival
TABLE 2 influence of topical curcumin on physiological index of low temperature stress five-leaf one-heart tobacco seedlings
4. Experimental results and analysis:
As can be seen from fig. 1a, b and table 1, after the curcumin solution with the volume percentage of 0.1% is externally applied at the low temperature stress, the survival rate of the tobacco seedlings in the two-leaf one-heart period is improved by 2.48 times compared with the control, and after the curcumin solution with the volume percentage of 0.5% is externally applied, the survival rate of the tobacco seedlings in the two-leaf one-heart period is improved by 0.82 times compared with the control, so that the effect of improving the survival rate of the tobacco seedlings in the two-leaf one-heart period by the curcumin with the volume percentage of 0.1% under the low temperature stress is more remarkable. Therefore, the optimum concentration was selected for subsequent experiments to continue the study.
As can be seen from FIGS. 1C and D and Table 1, the application of the 0.1% by volume curcumin solution to the young five-leaf-one-heart tobacco plants also increases the survival rate of young five-leaf-one-heart tobacco plants by 1.54 times as compared with the control.
Curcumin solution with volume percentage of 0.1% is selected for detection of low-temperature related physiological indexes, as shown in fig. 2 and table 2. Compared with the control, the tobacco seedlings treated by the curcumin solution with the volume percent of 0.1 percent are subjected to low-temperature stress, the MDA content and the H 2O2、O2 .- content in leaves of the tobacco seedlings are obviously reduced, the contents of Soluble Sugar (SS) and proline (Pro) of osmotic adjusting substances are obviously increased, and the relative permeability of cell membranes can be reduced and the in-vivo biological enzyme (SOD, POD, CAT enzyme) activity can be improved by the curcumin solution with the volume percent of 0.1 percent.
The experimental data show that the curcumin solution with the volume percentage of 0.1 percent can effectively relieve the influence of low-temperature stress on tobacco seedlings and improve the self-resistance of the tobacco seedlings.
Embodiment two: screening and verifying experiment of influence of externally applied raffinose on low-temperature stress tobacco seedlings
1. Experimental materials:
Tobacco (Nicotiana tabacum l.) 'K326' variety, maintained for the plant stress physiology and molecular biology laboratory of the peninsula agricultural university;
Raffinose (Raffinose) was purchased from Shanghai Rui Yongshengmbh;
tween 20 (Tween 20) was purchased from Guozhu Chemicals Co.
2. The experimental method comprises the following steps:
phenotypic observation adopts tobacco seedlings in two periods as experimental materials, namely two-leaf one-heart period and five-leaf one-heart period.
Two-leaf one heart stage:
1) Material culture: sowing tobacco seeds in seedling raising trays containing turf wood nutrient soil, culturing in a greenhouse with the temperature of 25 ℃ and the humidity of 55% and the photoperiod of 16/8h, and selecting tobacco seedlings with consistent growth vigor after two weeks, and transplanting the tobacco seedlings into the seedling raising trays for subsequent treatment.
2) Two-leaf one-heart stage tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment 1: spraying raffinose solution with the mass percentage of 0.6% on the leaf surface;
③ Treatment 2: spraying raffinose solution with the mass percentage of 6% on the leaf surface;
The treatment method comprises the following steps: spraying raffinose solution (containing 0.02% of surfactant Tween 20) with mass percentage of 0, 0.6 and 6% on both sides of the leaf until the liquid medicine is to be dropped, standing for 12h, standing in a low-temperature illumination incubator for gradient cooling treatment, treating at 4deg.C for 2h, treating at 0deg.C for 1h, -1 deg.C for 1h, -2 deg.C for 1h, -3 deg.C for 1h, standing in a greenhouse (25deg.C) for 3d, observing phenotype, counting survival rate of the seedling, and repeating each treatment for more than 3 times.
Five-leaf one heart stage:
1) Material culture: sowing tobacco seeds in a seedling raising tray containing turf wood nutrient soil, placing the tobacco seeds in a greenhouse with the temperature of 25 ℃ and the humidity of 55% and the photoperiod of 16/8h for cultivation, selecting single tobacco seedlings with consistent growth vigor after two weeks, transplanting the single tobacco seedlings into a flowerpot for continuous cultivation until the single tobacco seedlings with consistent growth vigor are in a four-leaf one-heart period, and selecting the seedlings with consistent growth vigor for subsequent treatment.
2) Four-leaf first-season tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment group: spraying raffinose solution with the mass percentage of 0.6% on the leaf surface;
The treatment method comprises the following steps: carrying out leaf surface spraying treatment on four-leaf first-period tobacco seedlings by using a raffinose solution (containing 0.02% of surfactant Tween 20) with the mass percentage of 0 and 0.6%, spraying the front and back surfaces of the leaves until the liquid medicine is to be dripped, spraying for 1 time every 24 hours, carrying out total treatment for 4 times until the five-leaf first-period tobacco seedlings are in a low-temperature illumination incubator, carrying out gradient cooling treatment, carrying out treatment at 4 ℃ for 2 hours, carrying out treatment at 0 ℃ for 1 hour, -carrying out treatment at 2 ℃ for 1 hour, -carrying out treatment at 3 ℃ for 1 hour, -carrying out treatment at 4 ℃ for 1 hour, placing the tobacco seedlings in a greenhouse (25 ℃) for recovering growth for 3d, observing the phenotype, counting the survival rate of the seedlings, and repeating the treatment for more than 3 times each time.
3) And (3) measuring a physiological index: selecting four-leaf first-period tobacco seedlings with consistent growth, carrying out leaf surface spraying treatment by using a raffinose solution (containing 0.02% of surfactant Tween 20) with the mass percentage of 0.6%, spraying for 1 time every 24 hours, carrying out total treatment for 4 times, respectively sampling and mixing the third and the fourth true leaves before and after low-temperature treatment (2 hours at 4 ℃ and 1 hour at 0 ℃ and 1 hour at 1 ℃), and detecting low-temperature related physiological indexes.
Measuring the index: cell membrane permeability, MDA (malondialdehyde), SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), pro (proline), SS (soluble sugar), active oxygen content (H 2O2 and O 2 .-).
Cell membrane permeance assay reference (Zhang et al, 2021) method, MDA content reference (Tang et al, 2017) method, O 2 . -and H 2O2 content assay reference (Wang et al, 2019) method, SOD, POD, CAT activity reference (Shen et al, 2021) method, proline content assay according to (Quan et al, 2022) soluble sugar content assay reference (Gurrieri et al, 2020) method.
3. Experimental data
TABLE 3 Effect of external application of raffinose on survival of Low temperature stress tobacco seedlings
TABLE 4 Effect of external application of raffinose on physiological index of Low temperature stress five-leaf first-period tobacco seedlings
4. Experimental results and analysis:
As shown in fig. 3a, b and table 3, after the 0.6% raffinose solution is externally applied at low temperature stress, the survival rate of the two-leaf one-heart tobacco seedlings is improved by 3.54 times compared with the control, and after the 6% raffinose solution is externally applied, the survival rate of the two-leaf one-heart tobacco seedlings is improved by 1.43 times compared with the control, so that the effect of improving the survival rate of the two-leaf one-heart tobacco seedlings by the 0.6% raffinose is more remarkable. Therefore, the optimum concentration was selected for subsequent experiments to continue the study.
As can be seen from FIGS. 3C and D and Table 3, the application of the 0.6% raffinose solution to the young plants in the five-leaf-one-heart stage also increases the survival rate of the young plants in the five-leaf-one-heart stage by 2.62 times as compared with the control.
The raffinose solution with the mass percentage of 0.6% is selected for detection of low-temperature related physiological indexes, as shown in fig. 4 and table 4. Compared with the control, the tobacco seedlings treated by the raffinose solution with the mass percent of 0.6% are subjected to low-temperature stress, the MDA content and the H 2O2、O2 .- content in leaves of the tobacco seedlings are obviously reduced, the contents of Soluble Sugar (SS) and proline (Pro) of osmotic adjusting substances are obviously increased, and the relative permeability of cell membranes can be reduced and the in-vivo biological enzyme (SOD, POD, CAT enzyme) activity can be improved by the raffinose solution with the mass percent of 0.6%.
The experiment shows that the raffinose solution with the mass percentage of 0.6% can effectively relieve the influence of low-temperature stress on tobacco seedlings and improve the self-resistance of the tobacco seedlings.
Embodiment III: preparation of composite plant antifreezing agent
1. Experimental materials:
Curcumin (Curcumin) and raffinose (Raffinose) were both purchased from Shanghai Rui Yongsheng biotechnology Co., ltd;
Tween 20 (Tween 20) and glycerol (glycerol) were purchased from national pharmaceutical group chemical reagent limited;
Calcium chloride (CaCl 2) and potassium dihydrogen phosphate (KH 2PO4) were purchased from Kande chemical Co., ltd.
2. The experimental method comprises the following steps: take the preparation of 1L composite plant antifreeze agent as an example
(1) Dissolving 0.0368g of curcumin powder as plant active substance in 10ml of absolute ethanol, heating and dissolving at 50 ℃ to prepare curcumine mother solution for later use;
(2) 1ml of curcumin mother liquor, 6g of raffinose, 0.1g of monopotassium phosphate, 1.1g of calcium chloride, 0.2ml of glycerol and 0.2ml of tween 20 are measured according to the proportion requirement for standby;
(3) Adding the materials prepared in the step (2) into water, fully and uniformly stirring, and fixing the volume;
(4) After the preparation is completed, the mixture is transferred into a storage bottle and stored in a dark place.
3. Plant antifreeze agent containing curcumin and raffinose
The components are as follows: curcumin 0.1%, raffinose 0.6%, potassium dihydrogen phosphate 0.01%, calcium chloride 0.11%, glycerin 0.02%, tween 20 0.02%, and water in balance.
Example four, influence of externally applied composite plant antifreeze on tobacco seedlings under low temperature stress 1, experimental materials:
potted experimental tobacco variety: tobacco (Nicotiana tabacum l.) 'K326' variety, maintained for the plant stress physiology and molecular biology laboratory of the peninsula agricultural university;
Tobacco variety grown in field: tobacco (Nicotiana tabacum l.) 'Zhongyan 301' variety, experimental field was located in the Qingdao yellow island Baoshan town.
2. The experimental method comprises the following steps:
phenotypic observation adopts tobacco seedlings in two periods as experimental materials, namely two-leaf one-heart period and five-leaf one-heart period.
Two-leaf one heart stage:
1) Material culture: sowing tobacco seeds in seedling raising trays containing turf wood nutrient soil, culturing in a greenhouse with the temperature of 25 ℃ and the humidity of 55 ℃ and the photoperiod of 16/8h, and selecting tobacco seedlings with consistent growth vigor after two weeks, and transplanting the tobacco seedlings into the seedling raising trays for subsequent treatment.
2) Two-leaf one-heart stage tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment group: spraying a composite plant antifreeze agent on leaf surfaces;
The treatment method comprises the following steps: treating tobacco seedlings in a period of two leaves and one period of heart by leaf surface distilled water and spraying a composite plant antifreeze agent, spraying the liquid medicine on the front and back surfaces of the leaves until the liquid medicine is to be dripped, standing for 12 hours, standing in a low-temperature illumination incubator for gradient cooling treatment, treating at 4 ℃ for 2 hours, treating at 0 ℃ for 1 hour, treating at-1 ℃ for 1 hour, treating at-2 ℃ for 1 hour, treating at-3 ℃ for 1 hour, standing in a greenhouse (25 ℃) for 3 days, observing the phenotype, counting the survival rate of the seedlings, treating 30 seedlings each time, and repeating the treatment for more than 3 times.
Five-leaf one heart stage:
1) Material culture: sowing tobacco seeds in seedling raising trays containing turf wood nutrient soil, placing the tobacco seeds in a greenhouse with the temperature of 25 ℃ and the humidity of 55 ℃ and the photoperiod of 16/8h for cultivation, selecting tobacco seedlings with consistent growth vigor after two weeks, transplanting the tobacco seedlings into a flowerpot for continuous cultivation until the seedlings with consistent growth vigor are in a period of four leaves and one heart, and selecting the seedlings with consistent growth vigor for subsequent treatment.
2) Four-leaf first-season tobacco seedlings were subjected to the following treatments:
① Control group: spraying distilled water on leaf surfaces;
② Treatment group: spraying a composite plant antifreeze agent on leaf surfaces;
The treatment method comprises the following steps: spraying distilled water and a composite plant antifreeze agent on leaf surfaces of four-leaf first-period tobacco seedlings, spraying the front and back surfaces of the leaves until the liquid medicine is to be dripped, spraying for 1 time every 24 hours, carrying out gradient cooling treatment for 2 hours at 4 ℃ in a low-temperature illumination incubator, carrying out treatment for 1 hour at 0 ℃ and 1 hour at-1 ℃, carrying out treatment for 1 hour at-2 ℃ and 1 hour at-3 ℃, carrying out treatment for 1 hour at-4 ℃, placing the tobacco seedlings in a greenhouse (25 ℃) for 3d of growth recovery, observing phenotypes, counting survival rate of the seedlings, and repeating the treatment for more than 3 times for 12 seedlings each time.
3) And (3) measuring a physiological index: selecting tobacco seedlings with four leaves and one period and consistent growth, respectively spraying distilled water and a composite plant antifreeze agent on the leaf surfaces, spraying for 1 time every 24 hours, after 4 times of total treatment, respectively sampling and mixing samples on the third and the fourth true leaves before and after low-temperature treatment (2 hours at 4 ℃ and 1 hour at 0 ℃ and 1 hour at-1 ℃), and detecting low-temperature related physiological indexes.
Measuring the index: cell membrane permeability, MDA (malondialdehyde), SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), active oxygen content (H 2O2 and O 2.-), pro (proline) content, SS (soluble sugar content).
Cell membrane permeance assay reference (Zhang et al, 2021) method, MDA content reference (Tang et al, 2017) method, O 2. - and H 2O2 content assay reference (Wang et al, 2019) method, SOD, POD, CAT activity reference (Shen et al, 2021) method, proline content assay according to (Quan et al, 2022) soluble sugar content assay reference (Gurrieri et al, 2020) method.
The tobacco seedlings growing in the field are respectively sprayed with a composite plant antifreeze agent once before transplanting (five leaves and one core period), sprayed on the leaves for 1 time before the cold tide comes, the phenotype is observed after 2 months, and indexes such as plant height, leaf area, lateral bud number and the like are counted.
3. Experimental data
TABLE 5 Effect of external application of composite plant anti-freeze to survival of Low temperature stress tobacco seedlings
TABLE 6 influence of external application of composite plant antifreeze on physiological index of low temperature stress five-leaf first-heart tobacco seedlings
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TABLE 7 influence of external application of composite plant antifreeze on growth index of field grown tobacco seedlings
4. Experimental results and analysis
As shown in fig. 5a, b and table 5, the survival rate of two-leaf one-heart tobacco seedlings externally applied with the composite plant antifreeze agent under low-temperature stress is improved by 5.67 times as compared with the control, and the survival rate is as high as 88.89%. As shown in fig. 5c, d and table 5, the survival rate of five-leaf first-period tobacco seedlings subjected to external application of the composite plant antifreeze agent under low-temperature stress is improved by 4.71 times as compared with the control, and the survival rate is up to 79.36%. The external application of the composite plant antifreeze agent can obviously improve the survival rate of tobacco seedlings in the two-leaf-one-heart period and the five-leaf-one-heart period.
As shown in fig. 6 and table 6, the physiological index change of tobacco seedlings after external application of the composite plant antifreeze agent is detected under low temperature stress, and compared with a control, the external application of the composite plant antifreeze agent can obviously reduce cell membrane damage, MDA content and H 2O2、O2.- content, increase the content of permeation regulating substances proline and soluble sugar, and improve the activity of biological enzymes.
As shown in fig. 7 and table 7, the leaf area and plant height of the field-grown tobacco seedlings are obviously improved compared with the control after the composite plant antifreeze agent is externally applied, and the lateral bud number of the plant is obviously reduced, so that the composite plant antifreeze agent plays a very important role in relieving the low-temperature damage of the tobacco.
Example five, influence of externally applied composite plant antifreeze on tomato and cucumber seedlings under low temperature stress 1, experimental materials:
Tomato (Solanum lycopersicum) 'golden shed number one' variety purchased from a nursery farm seed pesticide online store;
cucumber (culumis sativus l.) 'jin yan No. four' variety, purchased from farm No. one seed industry online store.
2. The experimental method comprises the following steps:
1) Material culture: sowing tomato and cucumber seeds in a seedling raising tray containing turf wood nutrient soil, placing the seedling raising tray in a greenhouse with the temperature of 25 ℃ and the humidity of 55 ℃ and the photoperiod of 16/8h for cultivation, transplanting the seedling raising tray into a flowerpot for continuous cultivation after one week until two leaves are in a period of one heart, and respectively selecting materials with consistent growth vigor for subsequent treatment.
2) The following treatments were set for tomato and cucumber seedlings, respectively:
① Control group: spraying distilled water on leaf surfaces;
② Treatment group: foliage spraying composite plant antifreeze agent
Phenotypic observation experiments: spraying distilled water and composite plant antifreeze agent on leaf surfaces of two-leaf one-heart-period tomato and cucumber seedlings, spraying the solution to the front and back surfaces of the leaf until the solution is to be dripped, standing for 12h, standing in a low-temperature illumination incubator for gradient cooling treatment, treating tomato seedlings (2 h at 4 ℃, 1h at 0 ℃, 1h at-1 ℃), 1h at-2 ℃), treating cucumber seedlings (2 h at 4 ℃, 1h at 0 ℃), 1h at-1 ℃ and 0.5h at-2 ℃), standing in a greenhouse (25 ℃) for recovering growth for 3d, observing phenotype, counting survival rate of seedlings, each treating 30 seedlings, repeating each treatment for more than 3 times,
3) And (3) measuring a physiological index: selecting two-leaf one-heart tomato seedlings and cucumber seedlings which are consistent in growth, respectively spraying distilled water and a composite plant antifreeze agent on leaf surfaces, standing for 12 hours, respectively sampling and mixing samples on a third real leaf and a fourth real leaf before low-temperature treatment and after low-temperature treatment (12 hours at 4 ℃), and detecting low-temperature related physiological indexes.
Measuring the index: cell membrane permeability, MDA (malondialdehyde), SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), active oxygen content (H 2O2 and O 2.-), pro (proline) content, SS (soluble sugar content).
Cell membrane permeance assay reference (Zhang et al, 2021) method, MDA content reference (Tang et al, 2017) method, O 2. - and H 2O2 content assay reference (Wang et al, 2019 a) method, SOD, POD, CAT activity reference (Shen et al, 2021) method, proline content assay according to (Quan et al, 2022) soluble sugar content assay reference (Gurrieri et al, 2020) method.
3. Experimental data
TABLE 8 Effect of external application of composite plant anti-freeze to Low temperature stress tomato and cucumber seedling survival
TABLE 9 Effect of external application of composite plant anti-freezing Agents on Low temperature stress two-leaf one-heart tomato seedling physiological index
TABLE 10 influence of external application of composite plant antifreeze on physiological index of low temperature stress two-leaf one-heart cucumber seedlings
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4. Experimental results and analysis
As shown in fig. 8, 10 and table 8, the control group showed lower survival rate of tomato and cucumber seedlings under low temperature stress, and the survival rate of seedlings treated by external application of the composite plant antifreeze agent was significantly improved. The survival rate of tomato seedlings subjected to external application of the composite plant antifreeze agent under low-temperature stress is improved by 3.57 times compared with that of control, and the survival rate of cucumber seedlings is improved by 4.00 times compared with that of control.
By detecting the change of physiological indexes of tomato and cucumber seedlings after low-temperature treatment, as shown in fig. 9, 11, table 9 and table 10, the external application of the composite plant antifreeze agent treatment is found to reduce cell membrane damage, MDA content and H 2O2、O2 .- content of tomatoes and cucumbers, increase the content of osmotic adjusting substances and improve the activity of biological enzymes. Therefore, the composite plant antifreeze agent can also relieve the low-temperature damage of tomatoes and cucumbers. The composite plant antifreeze agent has broad spectrum and can be popularized and used.
The present invention has been described above by way of example, but the present invention is not limited to the above-described embodiments, and any modifications or variations based on the present invention fall within the scope of the present invention.
Claims (5)
1. A plant antifreeze agent containing curcumin and raffinose, characterized in that: the main active ingredients of the anti-freezing agent are curcumin and raffinose, the anti-freezing agent is prepared from curcumin mother liquor and raffinose, the volume percentage of the curcumin mother liquor in the anti-freezing agent is 0.1-0.5%, and the mass percentage of the raffinose is 0.6-6%; the preparation method of the curcumin mother liquor comprises the following steps: 0.0368g of plant active substance curcumin powder is dissolved in 10ml absolute ethyl alcohol to prepare curcumin mother liquor; the plant antifreeze agent also comprises potassium dihydrogen phosphate, calcium chloride, glycerol and tween 20.
2. A plant jelly containing curcumin and raffinose as claimed in claim 1, wherein: the components are as follows: curcumin mother liquor volume percentage is 0.1%, raffinose mass percentage is 0.6%, monopotassium phosphate mass percentage is 0.01%, calcium chloride mass percentage is 0.11%, glycerin volume percentage is 0.02%, tween 20 volume percentage is 0.02%, and the balance is water.
3. A plant jelly containing curcumin and raffinose as claimed in claim 2, wherein: the using period and the method of the plant antifreeze agent containing curcumin and raffinose are as follows:
Two leaves and one period seedling, the use time is before 10 am or after 4 pm, the use time is before 1 day at low temperature, the temperature is not lower than 0 ℃, the leaf surface is sprayed for 1 time, the plant antifreeze agent is sprayed until the liquid medicine is to be dripped on the front side and the back side of the leaf, or the plant antifreeze agent is sprayed according to the dosage of 750kg per hectare;
Five leaves and one heart stage seedling are sprayed for 1 time every other day and 4 times before the use time is 10 am or after 4 pm before low temperature comes; spraying the plant antifreeze agent until the liquid medicine is to be dripped on the front side and the back side of the blade, or spraying the plant antifreeze agent according to the dosage of 750kg per hectare.
4. A plant antifreeze agent comprising curcumin and raffinose as claimed in claim 2, wherein: plants that are most suitable include tobacco, tomato, and cucumber.
5. A preparation method of a plant antifreeze agent containing curcumin and raffinose is characterized by comprising the following steps: based on the total volume of 1L, the method comprises the following steps:
S1: 0.0368g of plant active substance curcumin powder is dissolved in 10 ml absolute ethyl alcohol to prepare curcumin mother liquor for standby;
S2: 1ml curcumin mother liquor, 6g raffinose, 0.1g monopotassium phosphate, 1.1 g calcium chloride, 0.2 ml glycerin and 0.2 ml tween 20 are measured according to the proportion required for standby;
s3: adding the prepared materials in the step S2 into water, and fully stirring and uniformly mixing;
S4: after the preparation is completed, the mixture is transferred into a storage bottle and stored in a dark place.
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| WO2016118600A1 (en) * | 2015-01-20 | 2016-07-28 | Lipotec Laboratories, Llc | Methods and formulation for treatment of nuts, seeds, grains, cereals, plant growth media, and plants |
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