Compound preservative capable of prolonging preservation time of gerbera jamesonii fresh cut flowers as well as development method and application of compound preservative

Abstract

The invention relates to the field of preservation of gerbera jamesonii fresh cut flowers, in particular to a compound preservative for prolonging the preservation time of the gerbera jamesonii fresh cut flowers as well as a development method and application of the compound preservative. The compound preservative is mainly prepared from chitosan, calcium chloride and citric acid. Compared with a basic vase solution and a commercial fresh-keeping agent, the composite fresh-keeping agent disclosed by the invention has a better fresh-keeping effect in the aspects of larger maximum flower diameter and longer vase life. The effect of the compound preservative is proved through larger maximum flower diameter, longer flower arrangement life, higher antioxidant enzyme activity and lower MDA content. Meanwhile, RNA sequencing (RNA-Seq) analysis reveals that the treatment of the compound preservative can prolong the life of the cut flowers through plant hormone signals (e.g., cytokinin signals) and promotion of pathogen defense (e.g., salicylic acid signals). The discovery provides a new strategy for the preservation of the cut-flower chrysanthemum.

Description

Technical Field

[0001] The invention relates to the field of preservation of fresh cut gerbera flowers, and in particular to a composite preservative for prolonging the preservation period of fresh cut gerbera flowers, and a development method and application thereof. Background Art

[0002] Gerbera jamesonii L., a member of the Asteraceae family, is a popular choice for cut flowers in the global floriculture market due to its diversity of colors and shapes. However, consumers often point out that these flowers have a limited vase life under standard room temperature conditions. Therefore, the horticultural industry must focus on enhancing the vase life and quality of cut flowers by conditioning Gerbera cut flowers postharvest and addressing these challenges. Improving vase life (VL) is a major breeding goal for Gerbera cut flowers, as vase life directly affects consumer demand.

[0003] The growth and development process of plants is affected by many factors, and the opening and withering of fresh cut flowers are also regulated. The purpose of flower preservation is usually achieved by adding substances that inhibit bacterial growth, supplement nutrition, inhibit ethylene synthesis, and inhibit water transpiration. At present, there have been a lot of studies on the role of different substances in fresh cut flower preservation at home and abroad. A large number of studies have found that the lifespan of fresh cut flowers is affected by substances such as sterilizers, nutrients, metal ion concentrations, and plant hormones. However, there are few reports on the molecular mechanism of the lifespan of gerbera cut flowers. The preservative formulas used in previous studies often have a high risk of harm when used on gerbera cut flowers. Some studies have screened out single-component preservatives, most of which can only achieve a preservation effect under specific conditions, or cannot achieve a significant preservation effect. Undoubtedly, it is very important to optimize the preservative formula for gerbera cut flowers using economical, environmentally friendly and effective ingredients. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a composite preservative for prolonging the fresh-keeping period of fresh cut gerbera flowers and a development method and application thereof, which can prolong the vase life of fresh cut gerbera flowers and ensure the quality of the cut flowers.

[0005] The present invention is achieved in that:

[0006] The invention firstly provides a composite preservative for prolonging the preservation period of fresh cut gerbera flowers, which mainly consists of chitosan, calcium chloride and citric acid.

[0007] Furthermore, the composite preservative is an aqueous solution containing 100-110 mg / L chitosan, 90-100 mg / L citric acid and 90-100 mg / L calcium chloride.

[0008] Preferably, the composite preservative is an aqueous solution containing 103.78 mg / L chitosan, 92.44 mg / L citric acid and 93.17 mg / L calcium chloride.

[0009] The present invention also provides a method for developing the composite preservative, comprising the following steps:

[0010] (1) Different antimicrobial agents, organic acids, and inorganic salts were added to the basic vase solution as preservatives for cut flowers to study the effects of each chemical preservative at different concentrations on the vase life and flower diameter of fresh cut gerbera flowers. The best antimicrobial agents, organic acids, and inorganic salts were selected as independent variables in the response surface design through single factor tests.

[0011] (2) three components—chitosan, citric acid, and calcium chloride—were identified as the main components of the preservative and used as independent variables in the subsequent response surface analysis;

[0012] (3) Optimizing the formula of the composite antimicrobial agent through the Box–Behnken test: First, the Box–Behnken test design was used to fit the quadratic polynomial using the Design-Expert 12Trial software, and a quadratic polynomial regression model of the vase life y using chitosan A, citric acid B, and calcium chloride C was established. Secondly, the regression equation of the vase life was established by fitting in the positive and negative coefficient directions. Finally, the optimal formula was analyzed by the response regression equation, which was used to optimize the preservative formula for fresh cut gerbera flowers.

[0013] Finally, the present invention also provides the use of the composite preservative in improving the tolerance of fresh cut gerbera flowers to abiotic stress.

[0014] Specifically, the abiotic stress is high temperature stress.

[0015] And, application of the composite preservative in improving the defense of fresh cut gerbera flowers against pathogens.

[0016] The present invention has the following advantages:

[0017] Compared with the basic vase liquid and the commercial preservative (CHRYSAL), the composite preservative of the present invention has a better preservation effect in terms of a larger maximum flower diameter and a longer vase life. The effect of the composite preservative is confirmed by a larger maximum flower diameter, a longer vase life, a higher antioxidant enzyme activity and a lower MDA content. At the same time, RNA sequencing (RNA-Seq) analysis revealed that the treatment of the composite preservative can extend the life of fresh cut gerbera flowers through plant hormone signals (e.g., cytokinin signals) and the promotion of pathogen defense (e.g., salicylic acid signals). By activating the cytokinin and salicylic acid signaling pathways, the composite preservative not only enhances the cell activity of cut gerbera flowers, but also improves their defense against pathogens. This combined effect may lead to a delay in the aging process of cut flowers, thereby significantly extending the vase life. This discovery provides a new strategy for the preservation of fresh cut gerbera flowers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0019] Figure 1 Effects of different compounds at different concentrations on the vase life and maximum flower diameter of gerbera. (A) Effects of antimicrobial agents on the vase life and maximum flower diameter of cut gerbera, (B) Effects of organic acids on the vase life and maximum flower diameter of cut gerbera, (C) Effects of inorganic salts on the vase life and maximum flower diameter of cut gerbera. In the context of statistical analysis, "ns" stands for "not significant," meaning that the difference between the two groups did not reach a statistically significant level. "*" means "significant," usually used to indicate a p-value of less than 0.05, indicating that the difference between the groups is statistically significant. "**" means "very significant," usually corresponding to a p-value of less than 0.01, indicating that the difference between the groups is statistically very significant.

[0020] Figure 2 Response surface plot (top) and contour plot (bottom) of the interaction between different preservatives on the vase life of cut gerbera flowers.

[0021] Figure 3 Effects of preservatives on the preservation of gerbera. (A) Changes in the appearance of cut gerbera flowers in a vase under different treatment conditions. (B) Effects of the best preservative on the vase life and maximum flower diameter of cut gerbera flowers.

[0022] Figure 4 The principal component analysis (PCA) results and correlations between samples of gerbera cut flowers with the basic vase solution and the best preservative. (A) Principal component analysis (PCA) of gerbera cut flowers with the basic vase solution and the best preservative. (B) Sample correlation heat map.

[0023] Figure 5Different aspects of gene expression analysis under the basic vase solution and the best preservative treatment conditions. (A) and (B) Volcano plots of up-regulated and down-regulated differentially expressed genes in Gerbera cut flowers treated with the basic vase solution and the best preservative. (C) Cluster heat map of differentially expressed genes in Gerbera cut flowers treated with the basic vase solution and the best preservative.

[0024] Figure 6 The top 20 enriched KEGG pathways of upregulated differentially expressed genes in basal vase solution and optimal preservative Gerbera cut flowers.

[0025] (A) Enrichment of genes in different metabolic pathways. Multiple metabolic pathways are listed in the figure. There are two numbers next to each pathway. The first number indicates the number of genes enriched in the pathway, and the second number is the q value, which indicates statistical significance. The smaller the q value, the more significant the enrichment result.

[0026] Biosynthesis of secondary metabolites: 471 genes, with a q value of 3e-37, the highest significance;

[0027] Metabolic pathways: 738 genes, q value 7.5e-37, also highly significant;

[0028] Flavonoid biosynthesis: 26 genes, q value of 5.4e-08, highly significant;

[0029] Other pathways such as Plant hormone signal transduction and Zeatin biosynthesis also had significant gene enrichment.

[0030] (B) Schematic representation of two signaling pathways, with portions of the cytokinin and salicylic acid signaling pathways significantly expressed under treatment.

[0031] Cytokinin signaling pathway: Involves proteins such as CRE1, AHP, B-ARR and A-ARR, and the biosynthesis of cytokinins;

[0032] Salicylic acid signaling pathway: involves proteins such as NPR1, TGA, PR-1, and the effects of salicylic acid.

[0033] (C) qRT-PCR was used to detect key differentially expressed genes, and the relative expression levels of several genes under the conditions of the best preservative group and the basic bottle incubation liquid group. The blue bar graph represents the best preservative group, and the orange bar graph represents the basic bottle incubation liquid group.

[0034] IPT1, IPT9: The expression in the basic bottle liquid group was significantly lower than that in the optimal preservative group.

[0035] TGA1, TGA4: The expression in the basic bottle liquid group was significantly higher than that in the optimal preservative group.

[0036] NPR1, RBOHA, KCS5, CUT1, KCS6: The expression of these genes in the basic bottle liquid group was also significantly higher than that in the optimal preservative group.

[0037] Figure 7 The bar graphs show the antioxidant enzyme activities (SOD, CAT, APX, POD) and MDA content under different treatment conditions. The bar graphs show the results under three treatment conditions: basic bottle liquid (pink), optimal preservative (blue) and Kelixian (orange). DETAILED DESCRIPTION

[0038] The technical scheme of the present invention will be described clearly and completely below in conjunction with the accompanying drawings and specific embodiments. If specific conditions are not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0039] 1. Materials and Methods

[0040] 1.1. Plant materials

[0041] The trial was conducted in November 2022 at the Fujian Provincial Key Laboratory of Crop Genetic Improvement and Innovative Utilization in Mountain Areas and Sanming Academy of Agricultural Sciences. The tested gerbera variety "Minghui Zixia" comes from the National Germplasm Resource Bank of Sanming Academy of Agricultural Sciences. This is the first chrysanthemum variety in Fujian Province to obtain a new variety certificate from the Ministry of Agriculture and Rural Affairs of China, and it was independently bred by Sanming Academy of Agricultural Sciences. This variety has the advantages of rare flower color, high yield, strong adaptability, resistance to powdery mildew and wilt, and good marketability. This variety is prone to short vase life after harvest, which reduces economic benefits. As a positive control, Chrysal cut flower food, the most commonly used fresh cut flower commodity preservative in the Chinese market, was used.

[0042] 1.2. Optimization of preservative formulation by response surface methodology

[0043] The chemical preservatives used in the experiment were screened based on the principles of economy, environmental protection and high efficiency to study the effect of each chemical preservative on the vase life of cut chrysanthemum. 3% sucrose + 250mg / L 8-hydroxyquinoline (8-HQ) was used as the basic vase solution, and antimicrobial agents (A: tea polyphenols or B: chitosan), organic acids (C: citric acid or D: salicylic acid) and inorganic salts (E: calcium chloride or F: sodium chloride) were added as preservatives for cut flowers to screen their abilities. A randomized block design was used, with the basic vase solution as the control group. The volume of the vase solution was 200mL, and the observation was repeated six times at room temperature of 25℃ and relative humidity of about 60%. At the same time, the maximum flower diameter and vase life data of cut chrysanthemum were recorded. During the experiment, in order to maintain the freshness and effectiveness of the bottled solution, the bottled solution needs to be replaced regularly. The specific replenishment frequency and method depend on the consumption rate of the solution and the actual needs of the cut flowers. Ensure that each time the solution is replaced, a freshly prepared solution is used to provide the necessary nutrition and preservation effect.

[0044] Through the above single factor test, the best antimicrobial agent, organic acid and inorganic salt were selected as independent variables of response surface design. The vase life (days) of different concentrations of antimicrobial agents was used as the response value, and the response surface design was carried out according to the Box–Behnken central experimental design principle of Design-Expert 12Trial software. The best formula was analyzed by the response regression equation to optimize the preservative formula of cut chrysanthemum.

[0045] Data were processed and analyzed using Excel 2010. All measurements were repeated three times and data were expressed as mean ± standard deviation. T-test was used to test the difference of each index (p < 0.05). The specific steps were as follows: insert the function formula: T-TEST; select the values ​​of the first and second groups; fill in 1 as the tail and type. Response surface analysis was performed using Design-Expert 12Trial. The specific steps were as follows: select 3 numerical factors (chitosan, unit: ml / L; calcium chloride, unit: ml / L; citric acid, unit: ml / L) and 1 response (y: vase life; unit: day, d). The formula of the composite preservative was optimized by Box–Behnken design.

[0046] 1.3. Morphological observation and physiological and biochemical determination

[0047] The preservative formula of the optimal solution group was applied to the preservation experiment of fresh cut gerbera flowers. The basic vase solution was used as the control and the commercially available preservative Klear Fresh was used as the positive control group. The maximum flower diameter of the cut flowers was observed and the vase life was recorded.

[0048] Petal powder (0.1 g) was extracted with 100 mM cold sodium phosphate buffer (pH 6.8) supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma) and the enzyme activities of ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC1.11.1.6) and peroxidase (POD, EC 1.11.1.7) were determined. The activities of APX, CAT and POD were determined according to previous methods [19-21]. Superoxide dismutase (SOD, EC1.15.1.1) activity was quantified using a SOD kit (Solarbio, China) and the activity was calculated using absorbance values ​​using an Infinite M200 plate reader (Tecan, Switzerland) according to the manufacturer's guidelines.

[0049] The petals were ground in liquid nitrogen and the resulting petal powder was homogenized with 1× phosphate buffered saline (PBS, pH 7.4). Malondialdehyde (MDA) levels in petals were determined using an MDA kit (Solarbio, Beijing, China) at 450, 532, and 600 nm wavelengths according to the manufacturer's protocol.

[0050] Wherein, the specific preparation method of petal powder is as follows:

[0051] Choose fresh, pest-free petals as raw materials. Wash the petals and remove dust and impurities on the surface. Place the cleaned petals in a ventilated place to dry naturally, or use absorbent paper to absorb the surface moisture. Put the dried petals in a mortar and pour in an appropriate amount of liquid nitrogen for freezing. Under the low temperature of liquid nitrogen, use a pestle or grinder to grind the petals. During the grinding process, avoid the liquid nitrogen from drying up, and grind it into fine powder. When the liquid nitrogen has just evaporated, take out the required amount for the next experiment, or put the tissue powder into a container and store it at -70°C for the next experiment.

[0052] RNA extraction and RNA-Seq analysis

[0053] The ligation products were size-selected by agarose gel electrophoresis, PCR-amplified, and sequenced using an Illumina novaseq6000 from Genedenovo Biotechnology Co. (Guangzhou, China). Total RNA was extracted using the Trizol method (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were measured using NanoDrop2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 test kit for the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). After quality testing, two cDNA libraries were constructed for the basic bottle solution group (BVS1, BVS2, and BVS3) and the 6-day optimal solution group (OS1, OS2, and OS3) and sequenced on the Illumina HiSeq platform. Annotation of high-quality clean reads was completed. The hypothesis testing probability (p-value) for multiple hypothesis testing was calibrated using the Benjamani–Hochberg method, and the false discovery rate (FDR) was obtained. Differential expression analysis was performed between the two samples using EdgeR. FDR < 0.01 and fold change ≥ 2 were set as the thresholds for significant differential expression. The differential expression, functional annotation, and functional enrichment of genes in the 6-day basal solution group and the 6-day optimal solution group were analyzed.

[0054] 1.5. Reverse transcription polymerase chain reaction (RT-PCR) and gene expression analysis

[0055] Total RNA was extracted from 0.1 g frozen petal tissue using the Pine method. TM 1 μg of extracted RNA was converted into complementary (c)DNA using RT Reagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. PCR reactions were performed according to previous methods

[23] to amplify genes using gene-specific primers.

[0056] The real-time quantitative (qRT)-PCR amplification protocol included an initial cycle at 95°C for 30 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 20 seconds, using an ABI QuantStudio 3 system (CA, USA). The gene expression levels were quantified using the 2-ΔΔct method. To normalize the gene transcript levels, the Actin6 gene of Gerbera chrysanthemum was co-amplified as a reference gene.

[0057] 2. Results

[0058] 2.1. Development process of the preservative formulation for cut flower Gerbera jamesonii

[0059] In this study, the present invention conducted a vase life test on chemical preservatives for cut gerbera. The test results showed that chitosan and tea polyphenols as antimicrobial agents can effectively extend the vase life of gerbera and maintain a larger flower diameter compared with the control group. Specifically, chitosan is more effective than tea polyphenols in extending the vase life at different concentrations, so the present invention selects chitosan as an antimicrobial agent ( Figure 1 -A). As for organic acids, citric acid and salicylic acid can also significantly prolong the vase life and maintain a larger flower diameter. Compared with the control group, citric acid performs better at different concentrations (citric acid significantly increases the maximum flower diameter at a concentration of 50 mg / L). Therefore, the present invention selects citric acid as the organic acid component ( Figure 1 -B). As for inorganic salts, calcium chloride and sodium chloride can significantly enhance the vase life and flower diameter of gerbera. Compared with the control group, calcium chloride has a more significant effect at different concentrations (calcium chloride significantly increases the maximum flower diameter at a concentration of 50 mg / L). Therefore, the present invention selects calcium chloride as the inorganic salt component ( Figure 1 -C). Based on these results, the present invention identified these three components - chitosan, citric acid and calcium chloride as the main components of the preservative, and used them as independent variables in the subsequent response surface analysis to further optimize the formula of the preservative.

[0060] The present invention uses a Box–Behnken central composite test, in which the best antimicrobial agent, organic acid and inorganic salt are selected as independent variables of the response surface design. The formula of the composite antimicrobial agent was optimized by the Box–Behnken test, and the experiment was carried out. The Box–Behnken test design established a quadratic polynomial regression model of the vase life (y) using COS (A), CA (B) and CaCl2 (C) through quadratic polynomial fitting of the Design-Expert12Trial software. Through the positive and negative coefficient directional fitting, the present invention established a regression equation for the vase life; under the variance analysis item list, the regression equation was obtained:

[0061] y=14.48+0.20A-0.57B-0.50C-0.83AB+0.72AC+0.13BC-1.50A^2-2.15B^2-1.70C^2.

[0062] The variance analysis and validity test of the regression equation showed that the model was highly significant (P<0.01). The P value of underfit was 0.065, which was greater than 0.05. The model was properly selected, so it could correctly reflect the relationship between chitosan, calcium chloride, citric acid and vase life. The correlation coefficient was R 2=0.9567, the model fit is very good. The lower the coefficient of variation (CV), the higher the confidence of the model; the CV value of this test is 3.28%, indicating that the model equation can reflect the actual test value, and the ideal ratio of the three antimicrobial agents can be predicted based on this model. In order to directly show the effect of two factors on the vase life, the present invention can first take one factor as a fixed value and analyze the effect of the other two factors on the vase life. The contour shapes of CA and CaCl2 (BC) are circular, indicating that there is no obvious interaction between them, while the interactions between chitosan and CA (AB) and chitosan and CaCl2 (AC) are elliptical, indicating that there is an obvious interaction between them. From the three-factor plane diagram (3DSurface), the present invention can see that the three response surface curves are all concave surfaces, indicating that in the optimal solution group (y), the concentrations of chitosan, citric acid and calcium chloride have an ideal solution ( Figure 2 ). In order to maintain the vase life, the optimal solution conditions are as follows: COS concentration is 103.78 mg / L; CA concentration is 92.44 mg / L; CaCl2 concentration is 93.17 mg / L. Theoretically, the vase life of the optimal solution group (y) is predicted to be 14.57 days.

[0063] 2.2 The optimal solution significantly extended the vase life of cut gerbera

[0064] In order to verify the preservation effect of the best preservation formula on cut gerbera, the best solution formula was applied, and 8 days after the cut gerbera was vase-inserted, the basic vase-inserted solution was used as the control (CK) and the commercially available preservative (CHRYSAL) was used as the positive control to evaluate its preservation effect. After 8 days, the state of the gerbera treated with the basic vase-inserted solution showed that the flowers began to show signs of withering, the color faded, and the petals may begin to wilt. Compared with the basic vase-inserted solution, it can be seen that the state of the cut gerbera treated with the best preservative solution was maintained. The whole flower remained upright, the flower stem did not bend, and the petals did not fade, wilt, or show other common symptoms at the end of the vase-inserted life ( Figure 3 -A). The maximum flower diameter statistics showed that the maximum flower stem diameter in the optimal solution was 8.56 cm, which was significantly higher than that of the positive control group and the control group ( Figure 3 -B), indicating that the best solution group has the best marketability. The average vase life of the control group and the positive control group were 7.5 days and 12.50 days respectively; the average vase life of the best solution group was 14.50 days, which was significantly higher than that of the control group CK and the positive control group ( Figure 3 -B). These results indicate that the optimal preservative significantly improves the preservation of gerbera, including extending the vase life and maintaining a larger flower diameter. Using an appropriate preservative can significantly improve the ornamental value and market life of cut flowers.

[0065] Importantly, the actual vase life of the optimal solution group was 14.50 days, which was 0.07 days less than the model prediction (14.57 days), and the relative error was less than 0.05, which concluded that the model prediction was highly consistent with the quadratic polynomial of the respective response independent variables, and the model equation was able to reflect the actual experimental value. It can be seen that the response surface optimization of the optimal preservation formula can significantly extend the vase life and maintain the maximum number of stems of cut flowers.

[0066] 2.3 Effect of the optimal vase solution on large-scale genetic variation in the cut chrysanthemum genome

[0067] In order to clarify the molecular mechanism by which the optimal solution prolongs the life of cut gerbera, the present invention analyzed the transcriptome sequencing of cut gerbera petals treated with basic vase solution and optimal solution for 6 days. Transcriptome sequencing analysis was performed on cut gerbera petals treated with basic vase solution and optimal solution. After filtering the original sequencing data, the present invention obtained a total of 36,214,024,582 clean data points. The Q20 values ​​of the six samples were higher than 97.40%, the Q30 values ​​were greater than 92.98%, and the GC percentages were between 44.26 and 44.58%, indicating that the transcriptome sequencing quality was good. Subsequently, the present invention performed principal component analysis (PCA) and correlation analysis on the transcriptome data, and the results showed that the correlation between samples in different groups was low. In the PCA results of the present invention, the CK group and the T group can be completely separated and clustered separately ( Figure 4 -A), indicating that the basic bottle liquid and the best preservative are clearly distinguished in the principal component space, indicating that the two treatment methods have significant differences in certain characteristics. The correlation between any two samples is intuitively displayed in the correlation heat map of the samples of the present invention, and the repeatability between the repeated samples of the CK and T groups can be studied ( Figure 4 -B). The heat map shows that the correlation between samples in the same group is very high, while the correlation between different groups is low, which further confirms the distribution pattern in the PCA diagram. These results may indicate that the best preservative is significantly different from the basic vase solution in some key properties, which may be the reason for its improved preservation effect. In short, the data of the present invention show that the sequence quality is high and meets the requirements for further analysis.

[0068] In order to screen the differentially expressed genes in the ideal group of gerbera flower petals, the present invention uses DESeq2 software (BaseMean value is used to estimate the expression level) to standardize the count number of each sample gene, calculate the multiple of the difference, and test the significance of the difference by NB (Negative Binomial) distribution test, and screen the differentially expressed genes (DEGs) according to the standards of FDR<0.05 and |log2 FC|≥2. The statistical results of differentially expressed genes and the volcano map show that for the comparison between CK and T, a total of 3217 genes were upregulated and 4655 genes were downregulated ( Figure 5-A), which showed that more genes had decreased expression levels under the optimal preservative treatment. Figure 5 -B shows that the expression changes of most genes were not significant, while a few genes were significantly up-regulated or down-regulated after treatment.

[0069] In order to visualize the changes in the relative content of differentially expressed genes, the present invention used their FPKM values ​​to draw a cluster heat map, showing that the expression patterns between the control group and the optimal group samples were similar, indicating that there was good repeatability within the group and the results were reliable. These genes with similar expression patterns may have common functions or participate in common metabolic pathways and signaling pathways ( Figure 5 -C). The heat map shows the changes in gene expression under different treatment conditions, with colors changing from blue (low expression) to red (high expression). The depth of the color shows the expression level of the gene in different samples, and you can intuitively see the clustering of gene expression and the impact of different treatment conditions on gene expression.

[0070] These results suggest that the optimal vase solution may extend the life of gerbera flower cuttings by altering gene expression on a large scale in multiple ways.

[0071] 2.4 Optimal vase solution may enhance the longevity of cut gerbera through plant signal transduction and promotion of pathogen defense

[0072] To further analyze the functions of these differentially expressed genes, the present invention performed KEGG enrichment analysis on these DEGs. The present invention found that the differentially expressed genes were significantly enriched in the process of plant hormone signaling, and the entries of "zeatin biosynthesis" and "phenylalanine, tyrosine and tryptophan biosynthesis" were also significantly enriched, which are considered to be closely related to cytokinin signaling and salicylic acid signaling ( Figure 6 -A). Further analysis revealed that the gene expression levels of most components of the cytokinin signaling pathway and salicylic acid signaling pathway were significantly increased compared with the control group, indicating that the treatment significantly activated these two signaling pathways and related genes ( Figure 6 -B). The present invention uses fluorescent qRT-PCR to verify the expression levels of some important genes, including key genes for cytokinin synthesis (IPT1 and IPT9), key receptors and transcription factors for salicylic acid signaling pathway (TGA1, TGA4, NPR1 and RBOHA), which are activated by disease resistance signals, and genes related to plant wax and closely related to mass synthesis and stress resistance (KCS5, CUT1 and KCS6). After treatment, the expression levels of these genes were significantly higher than those in the control group ( Figure 6-C). Cytokinin signaling is closely related to maintaining plant cell activity, while activation of the salicylic acid signaling pathway helps improve plant resistance to disease. It has been reported that these two signaling pathways have a synergistic effect in the process of resisting vascular diseases.

[0073] 2.5 Analysis of antioxidant enzyme activity and MDA content in cut gerbera flowers

[0074] When cut flowers are placed in a vase, the antioxidant enzymes in the plant work together to resist reactive oxygen species. The activities of antioxidant system-related enzymes CAT, APX, SOD and POD have been used as important indicators for screening cut flower preservatives. MDA is an important metabolite of oxygen free radicals (OFRs) in plants, which can better reflect the degree of tissue peroxidation. MDA content is a commonly used indicator in the study of plant aging and resistance physiology.

[0075] The results of antioxidant enzyme activity analysis of cut gerbera flowers in vase for 6 days showed that ( Figure 7 ), the CAT, APX, SOD and POD activities of the optimal solution group were significantly higher than those of the positive control group and the control group. At the same time, the MDA content analysis results showed that the MDA content of the optimal solution group was significantly lower than that of the positive control group and the control group. The above results show that compared with the flowers preserved in the basic vase solution and the commercially available preservatives, the cut gerbera in the optimal solution group has the strongest antioxidant capacity and effectively prolongs the life of the cut flowers.

[0076] 2.6 Composite preservatives can improve the tolerance of gerbera to abiotic stress

[0077] Experimental method: Gerbera chrysanthemum was divided into a control group and a treatment group. The treatment group used a compound preservative, and the control group used a basic vase solution. The two groups of plants were placed under the same high temperature conditions (38°C), and the physiological response of the flowers, chlorophyll fluorescence value and antioxidant enzyme activity were observed and recorded.

[0078] Test results: As shown in Table 1, there was no significant difference in CAT and H2O2 between the two groups, indicating that the composite preservative had little effect on CAT activity and hydrogen peroxide levels. However, the APX activity of the treatment group was significantly higher than that of the control group, indicating that the composite preservative can significantly increase the ascorbate peroxidase activity of gerbera and enhance its antioxidant capacity. There was no significant difference in SOD activity between the two groups, indicating that the composite preservative had little effect on SOD activity. POD activity in the treatment group was significantly lower than that in the control group, which may indicate that the composite preservative helps reduce the accumulation of peroxides and protect plants from oxidative damage. The MDA content in the treatment group was significantly lower than that in the control group. As a product of lipid peroxidation, the reduction in its content indicates that the composite preservative helps reduce oxidative damage.

[0079] Conclusion: The composite preservative significantly increased the APX activity of gerbera, enhanced the antioxidant capacity of plants, more effectively removed reactive oxygen species (ROS), and reduced oxidative stress. At the same time, the MDA content of the treatment group was significantly reduced, indicating that the composite preservative helps to reduce the oxidative damage caused by high temperature and protect the integrity of plant cell membranes. The reduction in POD activity may be related to the protective effect of the composite preservative, which reduces the accumulation of peroxides and thus alleviates oxidative damage. Therefore, the composite preservative improves the tolerance of plants to abiotic stresses such as high temperature by enhancing the antioxidant system of gerbera and reducing oxidative damage, and has a positive effect on improving the preservation effect of gerbera and extending the vase life.

[0080] Table 1 Antioxidant enzyme activities in the control group and treatment group

[0081] Control group Treatment Group Antioxidant enzyme activity Mean±SD Mean±SD CAT(U / g) 0.61±0.15 0.60±0.07 H2O2(umol / g) 8.22±1.52 8.38±0.16 APX(U / g) 1.67±1.19 15.42±2.25** SOD(U / g) 865.24±126.49 876.03±141.17 POD(U / g) 1.66±0.64 0.68±0.01* MDA (nmol / g) 139.55±34.26 96.8±21.34*

[0082] The composite preservative significantly increased the Fv / Fm value of gerbera, which indicated that the preservative helped improve the photosynthesis efficiency of the plant and enhance its tolerance to abiotic stress.

[0083] This improvement may be related to the active ingredients in the preservatives, which may help protect plant cells from oxidative damage, thereby maintaining or improving photosynthesis efficiency.

[0084] 2.7 Application of composite preservatives in improving the defense of gerbera against pathogens

[0085] Experimental method: Gerbera chrysanthemums were divided into a control group and a treatment group. The control group used a basic vase solution, and the treatment group used a vase solution containing a compound preservative. Six gerbera chrysanthemums were treated in each group, with three replicates. The infection rate, disease progression, antioxidant enzyme activity, MDA content, etc. were recorded. After a certain period of time, the same pathogen, such as Bacillus acutus, was introduced into the two groups of gerbera chrysanthemums, and their infection was observed.

[0086] Test results (Table 2):

[0087] Reduced infection rate: The treated gerbera plants were expected to show lower infection rates after being attacked by pathogens. This means that the active ingredients in the preservative compound may help strengthen the plant's natural defense mechanisms, making it more resistant to pathogen invasion.

[0088] Slow disease progression: Even if the gerbera in the treated group was infected with the pathogen to some extent, the disease progression was slower than that in the control group. This suggests that the compound preservative may help delay the spread of the pathogen in the plant, thus providing the plant with more time to activate its defense response.

[0089] Improved physiological indicators: Antioxidant enzyme activity may be higher in the treated group, while MDA (malondialdehyde) content may be lower. This indicates that the plant cell membrane is less damaged by oxidation and the cell integrity is maintained, which helps the plant better resist the attack of pathogens.

[0090] Preservation effect: According to relevant research, the composite preservative can significantly improve the vase life of fresh cut gerbera flowers, increase the fresh weight of flower branches, improve moisture conditions, and the number of bacteria in the vase liquid and the MDA content of fresh cut flowers are significantly lower than those in the control group.

[0091] These effects indicate that the composite preservative has potential application value in improving the defense of gerbera against pathogens.

[0092] Table 2 Experimental results of control group and treatment group

[0093]

[0094] 3. Discussion

[0095] As a new variety of cut gerbera, "Minghui Zixia" has huge market potential in China. The problem of post-harvest preservation has always affected the development of the cut gerbera industry. The present invention develops a new preservation formula that can significantly extend the life of cut gerbera, which is of great significance to the development of the cut gerbera industry.

[0096] 3.1 The composite preservative composed of calcium chloride, chitosan and citric acid can improve the life of gerbera

[0097] This study found that 103.78 mg / L COS, 93.17 mg / L CaCl2 and 92.44 mg / L CA as a composite preservative formula for "Minghui Zixia" can significantly delay the aging of its cut flowers. In addition, compared with the CK group, the aging of the "Minghui Zixia" cut flowers in the treatment group was significantly delayed, the vase life was extended by 7 days, and the maximum flower diameter increased by 1.12 cm. Compared with widely used preservatives, the composite preservative of the present invention can significantly extend the life of cut gerbera, indicating that it has strong commercial potential.

[0098] 3.2 Composite preservatives improve the lifespan of gerbera by upregulating antioxidant enzymes

[0099] In this study, the CAT, APX, SOD and POD activities of cut gerbera in the treatment group were 3.8, 10.72, 238.49 and 7.5 U g-1, respectively, which were significantly higher than those in the control group. The MDA content of cut gerbera was 91.5 nmol g-1, which was significantly lower than that in the control group. The preservation solution formula of the treatment group can effectively remove the excess ROS produced by plants under stress and inhibit the accumulation of MDA content. At the same time, it significantly upregulated the CAT, POD and SOD activities of the petals, thereby maintaining the balance of the antioxidant system. This shows that the upregulation of antioxidant-related enzyme activities is one of the reasons why the composite preservation treatment delays the senescence of cut gerbera flowers.

[0100] 3.3 Salicylic acid and cytokinin signaling activation are key factors in improving the lifespan of cut gerbera

[0101] In order to more comprehensively reveal the effect of the composite preservative developed by the present invention, the present invention uses transcriptomics to reveal the influence of this composite preservative on the gene expression of gerbera. The present invention finds that the composite preservative treatment significantly activates the gene expression related to plant hormone signaling and pathogen defense. Specifically, the cytokinin (CK) and salicylic acid (SA) signaling pathways show significant upregulation under the preservative treatment. Cytokinin signaling is closely related to the anti-aging process of plants. The present invention finds that under the preservative treatment, the key genes in the synthesis and signaling pathways of cytokinins are significantly transcriptionally activated. The increase in cytokinin synthesis and the activation of signaling pathways can significantly improve the tolerance of plants to abiotic stress and delay plant aging.

[0102] Salicylic acid (SA) is a key signaling molecule for plant defense against pathogens. The results of the present invention showed that the composite preservative treatment significantly enhanced the expression of key genes in the SA signaling pathway, including TGA1, TGA4, NPR1 and RBOHA. These genes play a central role in the plant's defense response to pathogens, and their upregulation may enhance the resistance of gerbera to potential pathogens such as bacteria. TGA1 and TGA4 bind to the NPR1 protein to activate defense-related genes such as PR-1.

[0103] 4. Conclusion

[0104] In conclusion, by activating the cytokinin and salicylic acid signaling pathways, the composite preservative not only enhanced the cell activity of cut gerbera, but also improved its defense against pathogens. This combined effect may lead to a delay in the aging process of cut flowers, thereby significantly extending the vase life. This discovery provides a new strategy for the preservation of cut chrysanthemums and may also inspire research on the preservation of other cut flowers.

[0105] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described in the present invention are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A composite preservative for extending the shelf life of fresh cut gerbera flowers, characterized in that: It is mainly composed of chitosan, calcium chloride and citric acid.

2. The composite preservative according to claim 1, characterized in that: The composite preservative is an aqueous solution containing 100-110 mg / L of chitosan, 90-100 mg / L of citric acid and 90-100 mg / L of calcium chloride.

3. The composite preservative according to claim 2, characterized in that: The composite preservative is an aqueous solution containing 103.78 mg / L of chitosan, 92.44 mg / L of citric acid and 93.17 mg / L of calcium chloride.

4. A method for developing a composite preservative as claimed in claim 1, 2 or 3, characterized in that: The following steps are involved: (1) Different antimicrobial agents, organic acids, and inorganic salts were added to the basic vase solution as preservatives for cut flowers to study the effects of each chemical preservative at different concentrations on the vase life of fresh cut gerbera flowers and flower diameters. The best antimicrobial agents, organic acids, and inorganic salts were selected as independent variables in the response surface design through single factor tests. (2) Three components—chitosan, citric acid, and calcium chloride—were identified as the main components of the preservative and used as independent variables in the subsequent response surface analysis; (3) Optimizing the formula of the composite preservative through the Box–Behnken test: First, the Box–Behnken test design was used to fit the quadratic polynomial using the Design-Expert 12 Trial software, and a quadratic polynomial regression model of the vase life y using chitosan A, citric acid B, and calcium chloride C was established. Secondly, the regression equation of the vase life was established by fitting in the positive and negative coefficient directions. Finally, the optimal formula was analyzed by the response regression equation, which was used to optimize the preservative formula for fresh cut gerbera flowers.

5. Use of the composite preservative as claimed in claim 1, 2 or 3 for improving the tolerance of gerbera to abiotic stress.

6. The use according to claim 5, characterized in that: The abiotic stress is high temperature stress.

7. Use of the composite preservative as claimed in claim 1, 2 or 3 for improving the defense of gerbera against pathogens.

Images