Method for promoting fruit callus by using cinnamic acid or caffeic acid

Abstract

The invention discloses a method for promoting fruit callus by using cinnamic acid or caffeic acid, and belongs to the technical field of agricultural product storage. Cinnamic acid or caffeic acid is applied to fruit callus treatment, and the disease resistance to diseases is enhanced while callus is promoted. The method for promoting fruit callus through cinnamic acid or caffeic acid has the advantages of being obvious in effect, environmentally friendly, remarkable in economic benefit and the like, an efficient and sustainable fruit treatment method is provided for fruit farmers, and wide application prospects and market potential are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural product storage, and in particular to a method for promoting fruit healing by utilizing cinnamic acid or caffeic acid. Background Art

[0002] Citrus (Citrus reticulata Blanco) belongs to the genus Citrus of the Rutaceae family. It is one of the most important economic crops in the world. It has a large production volume and a wide planting area in my country. It is mainly eaten fresh in my country. Transportation is an important link in the fruit and vegetable supply chain. Citrus fruits are usually transported with leaves during transportation. In this process, it is inevitable that the citrus fruits will be squeezed and collided with each other, causing mechanical damage. Mechanical damage will directly cause the cell structure of the injured part of the fruit to be destroyed, resulting in the loss of water and juice, and rapid softening of the tissue; mechanical damage also accelerates the invasion of microorganisms, leading to the occurrence of post-harvest diseases, which greatly reduces the quality and economic benefits of citrus fruits.

[0003] In existing research reports, fruits and vegetables such as potatoes, apples, and pears have the ability to heal after harvest. Callus refers to the ability of fruits and vegetables to form callus tissue under suitable conditions after mechanical trauma caused by the outside world. The callus marker substance formed is suberin (Transcriptional regulation of wound suberin deposition in potato cultivars with differential wound healing capacity. Plant J, 2021, 107 (1): 77-99.). The callus process includes two stages: the formation of a "closed layer" and a "wound periderm". The former refers to the suberization of thin-walled cells at the wound site, and the latter is a layer of corked cells formed below the closed layer. Existing studies have shown that fruits and vegetables after callus have the characteristics of reducing the incidence of pathogen infection and the diameter of lesions, which is of great significance for ensuring the quality of fruits and vegetables after harvest and extending their shelf life. However, the natural healing process of fruits and vegetables is slow. Therefore, how to effectively improve the post-harvest healing process of fruits and vegetables while adopting scientific and effective measures to slow down the mechanical damage of fruits is a technology and problem that technicians in this field need to solve.

[0004] Existing research reports indicate that spraying chitosan on cassava roots can promote wound healing and induce disease resistance, thereby reducing the occurrence of postharvest diseases (Spraying chitosan on cassava roots reduces postharvest deterioration by promoting wound healing and inducing disease resistance. Carbohydrate Polymers, 2023, 318.); postharvest benzothiazide (BTH) treatment can promote the callus of pear fruit, and the results show that phenylpropanoid metabolism as well as peroxidase and polyphenol oxidase play an important role in callus formation (Postharvest benzothiazide treatment promotes the callus of pear fruit. Food Science, 2018, 39(9): 190-195); low-voltage electrostatic field treatment can promote the callus of postharvest potato tubers (Effect of low-voltage electrostatic field on the callus effect of postharvest potato tubers. Food Research and Development, 2022, 43(10): 62-69).

[0005] Currently, there are few studies on post-harvest callus of citrus fruits. The development of safe and environmentally friendly chemical agents suitable for citrus fruit callus treatment is expected to bring new development opportunities to the citrus industry. Summary of the invention

[0006] The purpose of the present invention is to provide a safe and effective natural compound preparation capable of promoting fruit healing, which is applied to the treatment of mechanical damage to post-harvest fruits to promote the healing of damaged tissues without affecting the edible quality of the fruits.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides the use of cinnamic acid or caffeic acid in the preparation of a preparation for promoting fruit wound healing. The present invention has found that compared with the natural wound healing of the fruit, the content of suberin in the wound of the fruit treated with exogenous cinnamic acid or caffeic acid is significantly increased, indicating that cinnamic acid or caffeic acid has the ability to promote fruit wound healing. Therefore, it is applied to the wound healing treatment of post-harvest fruit.

[0009] The present invention selects two naturally occurring compounds, cinnamic acid and caffeic acid, as treatment agents, which can not only promote the healing of citrus fruits but also have the advantages of environmental protection and safety.

[0010] Furthermore, the effective concentration of cinnamic acid or caffeic acid in the preparation is ≥0.1 g / L.

[0011] Furthermore, the effective concentration of cinnamic acid or caffeic acid in the preparation is 0.1-1.0 g / L.

[0012] Furthermore, the fruit is a citrus fruit. Exogenous treatment with cinnamic acid or caffeic acid significantly increases the healing rate of the citrus fruit peel. The applicable treatment objects of the present invention are not limited to citrus.

[0013] Furthermore, the preparation enhances the disease resistance of post-harvest fruits by promoting fruit callus. The present invention shows that the probability of disease occurrence in fruits treated with cinnamic acid or caffeic acid callus is significantly reduced compared with natural callus.

[0014] Furthermore, the post-harvest diseases of citrus fruits include green mold disease. The diseases that can be prevented by the present invention are not limited to green mold disease.

[0015] Furthermore, the application of the preparation includes: applying the preparation to the mechanically damaged part of the fruit, storing it in a dark environment to heal the wound, or soaking it to make the preparation adhere to the damaged part of the fruit.

[0016] The present invention also provides a method for promoting wound healing of citrus fruits, the method comprising: inoculating a treatment agent containing cinnamic acid or caffeic acid into the mechanically damaged part of the citrus fruit peel, and performing wound healing in a dark environment.

[0017] Furthermore, the concentration of cinnamic acid or caffeic acid in the treatment agent is 0.1-1.0 g / L. Studies have shown that under this treatment concentration condition, the formation of suberin is significantly promoted, and the thickness of the suberin layer gradually increases with the increase of concentration.

[0018] Furthermore, after the treatment agent is inoculated, the treatment agent is allowed to stand at room temperature until the treatment agent is completely absorbed.

[0019] Further, the wound healing was carried out at 20-25°C.

[0020] The present invention has the following beneficial effects:

[0021] (1) Significant wound healing effect: The present invention provides the use of cinnamic acid or caffeic acid in promoting fruit wound healing. Compared with the fruit with natural wound healing, the growth rate of suberin in the fruit tissue treated with cinnamic acid and caffeic acid is significantly accelerated, indicating that cinnamic acid and caffeic acid treatment have a promoting effect on the wound healing process.

[0022] (2) Significantly reducing the incidence of disease and the diameter of lesions after fruit harvest: The present invention applies cinnamic acid or caffeic acid to fruit callus treatment, which not only promotes callus but also significantly reduces the incidence of post-harvest fruit diseases. The treatment method of the present invention prolongs the storage time of the fruit, thereby reducing the losses caused by the disease and increasing the market competitiveness of the fruit.

[0023] (3) Environmentally friendly: Compared with traditional chemical methods, cinnamic acid and caffeic acid as treatment agents to promote wound healing have the advantage of being environmentally friendly. Because these substances are naturally occurring compounds, they have no negative impact on the environment and will not leave harmful substances in the fruit. This is in line with the green concept of modern agricultural development, helps reduce pesticide residue problems, and protects the ecological environment.

[0024] (4) Economic benefits: The present invention uses cinnamic acid and caffeic acid to promote wound healing, which can not only improve the quality and market competitiveness of fruits, but also bring significant economic benefits. By reducing post-harvest losses and reducing the occurrence of diseases, fruit farmers can save expenses in the pre-storage stage, and reduce the risk of disease occurrence during the storage of fruits, thereby increasing the economic value of fruits and bringing considerable profits to fruit farmers.

[0025] In summary, the method of using cinnamic acid and caffeic acid to promote fruit healing has the advantages of obvious effect, environmental friendliness, and significant economic benefits. It provides fruit farmers with an efficient and sustainable fruit treatment method with broad application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Observation diagram of suberin in the exogenous cinnamic acid, caffeic acid treatment group and the control group.

[0027] Figure 2 Shown is the disease occurrence of fruits in the exogenous cinnamic acid, caffeic acid treatment group and the control group.

[0028] Figure 3 It is the statistical chart of the incidence rate of the exogenous cinnamic acid, caffeic acid treatment group and the control group.

[0029] Figure 4 Statistical graph of lesion diameters of the exogenous cinnamic acid, caffeic acid treatment groups and the control group.

[0030] Figure 5 The suberin observation diagram of the groups treated with different concentrations of cinnamic acid and caffeic acid and the control group.

[0031] Note: The columns and vertical lines in the figure represent the mean and standard deviation of biological replicates, respectively. Different letters at the same time point indicate significant differences. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] Example 1: Effects of exogenous treatment with cinnamic acid and caffeic acid on callus formation in citrus fruit I. Research materials and methods

[0035] 1. Preparation of cinnamic acid and caffeic acid solution

[0036] Weigh appropriate amounts of cinnamic acid (CAS No.: 621-82-9) and caffeic acid (CAS No.: 331-39-5) with a purity of 98%, prepare them into solutions with a concentration of 0.1 g / L, and place them in a 4°C refrigerator for later use.

[0037] 2. Selection of citrus fruit varieties

[0038] The citrus variety used in this experiment was Tarocco blood orange (Citrus sinensis cv. Tarocco).

[0039] 3. Citrus fruit cleaning and wound simulation

[0040] Soak the citrus fruit in 2% sodium hypochlorite for 2 minutes, rinse slowly with clean water, dry it, and then use a sterilized hole puncher to make two holes at equal distances at the equator of the fruit. The wound depth is 3 mm and the diameter is 3 mm.

[0041] 4. Effects of exogenous treatment with cinnamic acid and caffeic acid on callus formation in citrus fruit

[0042] Inoculate 20 μL of the following liquids at the simulated wound site of the citrus fruit:

[0043] (1) 0.1 g / L cinnamic acid (CIN);

[0044] (2) 0.1 g / L caffeic acid (CA);

[0045] After standing at room temperature and the reagent was completely absorbed by the citrus fruit, the fruit was packaged with PE film and stored in a dark environment at 25°C for 3 days. The control treatments were: (1) natural callus for 3 days without exogenous treatment; (2) citrus fruit without callus.

[0046] When sampling, a sterilized puncher was used to take peel tissue with a diameter of 10 mm at the wound. The sampled citrus peel tissue was stained with toluidine blue and neutral red, washed with 75% ethanol and ultrapure water, and the changes in suberin were observed under a fluorescent upright microscope. After staining with toluidine blue and neutral red, suberin will show blue fluorescence under the observation of a fluorescent upright microscope.

[0047] 2. Experimental Results

[0048] like Figure 1 As shown, under the observation of fluorescence upright microscope, no blue fluorescent suberin layer could be seen in the 0-day callus group, but with the increase of callus days, the blue fluorescent suberin layer could be seen in the control group after 3 days of natural callus, and the suberin content in the 3-day callus group of citrus fruit treated with caffeic acid and cinnamic acid was significantly higher than that in the 3-day natural callus group, indicating that exogenous treatment with caffeic acid and cinnamic acid has the ability to promote citrus fruit callus.

[0049] Example 2: Effects of exogenous treatment of cinnamic acid and caffeic acid on disease resistance of citrus fruit after wound healing

[0050] 1. Research Methods

[0051] 1. Preparation of Penicillium digitatum spore suspension

[0052] 10 μL of Penicillium digitatum was taken from the glycerol tube stored in the laboratory, and after culturing on PDA solid medium for 7 days, the spore stock solution was collected with sterile water. The collected spore stock solution was graded diluted to 3×10 4 spores / mL and placed in a 4°C refrigerator until use.

[0053] 2. Citrus fruit varieties

[0054] The citrus variety used in this experiment was Tarocco blood orange (Citrus sinensis cv. Tarocco).

[0055] 3. Effects of exogenous treatment of cinnamic acid and caffeic acid on disease resistance of citrus fruit after wound healing

[0056] The preparation methods of the simulated wound of citrus fruit and the cinnamic acid and caffeic acid treatment agents are the same as those in Example 1. 20 μL of the following liquids are inoculated at the locations of the simulated wounds of citrus fruit:

[0057] (1) 0.1 g / L cinnamic acid (CIN);

[0058] (2) 0.1 g / L caffeic acid (CA);

[0059] After standing at room temperature and the reagent was completely absorbed by the citrus fruit, the fruit was packaged with PE film and stored in a dark environment at 25°C for 3 days. The control treatments were: (1) natural callus for 3 days without exogenous treatment; (2) citrus fruit without callus.

[0060] After 3 days of wound healing, 10 μL of 3×10 4spores / mL of Penicillium digitatum spore suspension, after the spore suspension is fully absorbed by the fruit, it is packaged in single fruit with PE film, stored at 25°C (temperature) and relative humidity of 85% to 90%, and covered with PE film to maintain humidity.

[0061] The incidence rate and lesion diameter were recorded 3 days after inoculation, and the incidence rate (Diseaseincidence) and lesion diameter (Lesiondiameter) were calculated every 1 day according to the following method; the calculation formula is as follows:

[0062]

[0063] 2. Experimental Results

[0064] Figure 2 The picture shows the disease of citrus fruits inoculated with Penicillium digitatum on the 6th day after callus 0 days, callus 3 days, and callus 3 days after cinnamic acid treatment and caffeic acid treatment. The growth trend of the pathogen is: a soft water-soaked circle is produced at the inoculation position at the beginning, and then the water-soaked circle gradually becomes larger, accompanied by the production of white and green spores. Figure 2 It can be seen that compared with the control group, the cinnamic acid and caffeic acid treatment groups had a significant effect in controlling diseases on citrus fruits.

[0065] Figure 3 and Figure 4 The following are the statistical graphs of the incidence and lesion diameter of Penicillium digitatum inoculated on citrus fruits after 0 days of callus, 3 days of callus, and 3 days of callus after treatment with cinnamic acid and caffeic acid. The incidence and lesion diameter were recorded 3 days after the inoculation of Penicillium digitatum on citrus fruits. Figure 3 and Figure 4 It can be seen that when inoculated with P.digitatum for 3 days, the morbidity of the two groups treated with cinnamic acid and caffeic acid for 3 days was reduced by 93.33% compared with the 0 day callus group, and the diameter of the lesion was reduced by 97.70% and 94.09% respectively; the morbidity was reduced by 75.00% compared with the 3 day callus group, and the diameter of the lesion was reduced by 85.71% and 60.00% respectively. When inoculated with P.digitatum for 6 days, the morbidity of the two groups treated with cinnamic acid and caffeic acid for 3 days was reduced by 84.20% and 82.22% compared with the 0 day callus group, and the diameter of the lesion was reduced by 93.37% and 89.36% respectively; the morbidity was reduced by 68.00% and 64.00% compared with the 3 day callus group, and the diameter of the lesion was reduced by 65.39% and 44.41% respectively. The morbidity and lesion diameter of the 3 day callus group treated with cinnamic acid and caffeic acid were lower than those of the control group (P<0.05). Example 3: Effects of exogenous treatment with different concentrations of cinnamic acid and caffeic acid on callus formation in citrus fruit

[0066] 1. Research Materials and Methods

[0067] 1. Preparation of cinnamic acid and caffeic acid solution

[0068] Weigh appropriate amounts of cinnamic acid and caffeic acid with a purity of 98%, prepare them into solutions with concentrations of 0.1 g / L, 0.5 g / L, and 1.0 g / L, respectively, and place them in a 4°C refrigerator for later use.

[0069] 2. Citrus fruit varieties

[0070] The citrus fruit variety used in this experiment was mandarin orange (Citrus unshiu Marc.).

[0071] 3. Effects of exogenous treatment with cinnamic acid and caffeic acid on callus formation in citrus fruit

[0072] The method for simulating wounds on citrus fruits is the same as in Example 1. 20 μL of the following liquids are inoculated at the locations of simulated wounds on citrus fruits:

[0073] (1) 0.1 g / L, 0.5 g / L, 1.0 g / L cinnamic acid (CIN);

[0074] (2) 0.1 g / L, 0.5 g / L, 1.0 g / L caffeic acid (CA);

[0075] After standing at room temperature and the reagent was completely absorbed by the citrus fruit, the fruit was packaged with PE film and stored in a dark environment at 25°C for 3 days. The control treatments were: (1) natural callus for 3 days without exogenous treatment; (2) citrus fruit without callus.

[0076] When sampling, a sterilized puncher was used to take peel tissue with a diameter of 10 mm at the wound. The sampled citrus peel tissue was stained with toluidine blue and neutral red, washed with 75% ethanol and ultrapure water, and the changes in suberin were observed under a fluorescent upright microscope. After staining with toluidine blue and neutral red, suberin will show blue fluorescence under the observation of a fluorescent upright microscope.

[0077] 2. Experimental Results

[0078] like Figure 5 As shown, the blue fluorescent suberin layer could not be seen in the callus 0d group, but with the increase of callus days, the blue fluorescent suberin layer could be seen in the control group after natural callus 3d, and the suberin content of the citrus fruit callus 3d group treated with caffeic acid and cinnamic acid was significantly higher than that of the natural callus 3d group, and the thickness of suberin gradually increased with the increase of concentration of the same phenolic acid treatment. This shows that exogenous treatment with caffeic acid and cinnamic acid has the ability to promote the callus of citrus fruit.

[0079] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of cinnamic acid or caffeic acid in the preparation of preparations for promoting fruit wound healing.

2. The use according to claim 1, characterized in that The effective concentration of cinnamic acid or caffeic acid in the preparation is ≥0.1 g / L.

3. The use according to claim 2, characterized in that The effective concentration of cinnamic acid or caffeic acid in the preparation is 0.1-1.0 g / L.

4. The use according to claim 1, characterized in that The fruit is a citrus fruit.

5. The use according to claim 1 or 4, characterized in that: The preparation enhances the disease resistance of post-harvest fruits by promoting fruit wound healing.

6. The use according to claim 5, characterized in that Fruit diseases include green mold.

7. A method for promoting wound healing of citrus fruits, characterized in that: The method comprises: inoculating a treatment agent containing cinnamic acid or caffeic acid into a mechanically damaged part of the citrus fruit peel, and healing the wound in a dark environment.

8. The method according to claim 7, characterized in that The concentration of cinnamic acid or caffeic acid in the treating agent is 0.1-1.0 g / L.

9. The method according to claim 7, characterized in that After the treatment agent is inoculated, it is left to stand at room temperature until the treatment agent is completely absorbed.

10. The method according to claim 7, characterized in that The wound healing was carried out at 20-25℃.

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