Method for increasing content of chlorogenic acid in vitro seedlings of vaccinium uliginosum var. kanchingense and application thereof

Abstract

The present application relates to the technical field of tissue culture of Vaccinium dunalianum, and particularly relates to a method for increasing the content of chlorogenic acid in tissue culture seedlings of Vaccinium dunalianum and application, wherein a rooting culture medium for the tissue culture seedlings of Vaccinium dunalianum is 1 / 4MS culture medium, wherein the concentration of K mineral element is adjusted to 1.5-2 times of the standard concentration, and the concentration of KNO3 is specifically 712.5-950 mg / L. By changing the concentration of K mineral element (increasing or decreasing) in the 1 / 4MS culture medium, the content of active compound CGA in the tissue culture seedlings of Vaccinium dunalianum can be controlled and increased. It is an effective way to obtain high-yield CGA Vaccinium dunalianum materials in large quantities.

Description

Technical Field

[0001] This invention relates to the field of Vaccinium bracteatum tissue culture technology, specifically to a method and application for increasing the chlorogenic acid content in Vaccinium bracteatum tissue culture seedlings. Background Technology

[0002] Vaccinium dunalianum Wight, a distinctive ethnic resource plant belonging to the genus Vaccinium in the family Ericaceae, has important edible (Li Guoze et al., 2021; Kong Lingpeng et al., 2019; Cheng Guiguang et al., 2010), medicinal (Gao et al., 2022), and skin-whitening chemical applications (Zhao et al., 2008; Luo et al., 2015). Chlorogenic acid (CGA) is one of the most abundant secondary metabolites in Vaccinium dunalianum Wight, second only to 6'-O-caffeoylarbutin (CA) (Zhao et al., 2008; Cheng Guiguang et al., 2010; Luo et al., 2015; Li Na et al., 2016). CGA has strong anti-aging, free radical scavenging, bactericidal, antiviral, anti-inflammatory, lipid-lowering, and cardiovascular protective effects (Thammarat, 2023; Huang, et al., 2023), and is widely used in food, pharmaceutical, and chemical industries.

[0003] This research group introduced *Vaccinium bracteatum* from a wild plant cultivation base (Wuding County, Chuxiong, Yunnan) to the experimental field of the author's university (Panlong District, Kunming, Yunnan) and found a significant decrease in CGA content. Further investigation revealed differences in the content of essential mineral elements in the soils of the two growing locations. Many factors influence plant growth and compound accumulation, among which essential mineral elements are a crucial one. Essential mineral elements not only play important physiological functions in plants but also directly participate in metabolism, affecting compound accumulation. Ca 2+ Excessive potassium (K) concentration can lead to narrow leaves and reduced chlorophyll content (Peng et al., 2020); excessive potassium (K) application can inhibit the growth of hemp plants in terms of height, stem diameter, and leaf area, reduce sucrase activity, and decrease the accumulation of dry matter and sucrose (Xu et al., 2015); potassium deficiency can cause yellowing, browning, and scorching of seedling leaf tips or margins, brown spots or patches on leaves, curling of a few leaves, and inhibition of root growth (Yang et al., 2019; Liu et al., 2018). The effects of different mineral elements on the growth and CGA accumulation of *Vaccinium bracteatum* are unknown. Currently, there is no method to increase CGA in tissue-cultured *Vaccinium bracteatum* seedlings, making it necessary to address the current resource scarcity of *Vaccinium bracteatum*.

[0004] Existing Chinese patent document CN201710816137.8 discloses a planting method for increasing the chlorogenic acid content in honeysuckle, including: fertilization, shading, spraying, and harvesting. The method is simple and easy to operate, and significantly increases the chlorogenic acid content in honeysuckle to 7.14%. After spring pruning, organic fertilizer is applied, providing balanced nutrition, promoting absorption, accelerating new branch growth, promoting flowering, and increasing honeysuckle yield. Shading and sufficient watering are carried out 20-25 days before each flower harvest to reduce light exposure, delay flower opening, improve the stability of chlorogenic acid, and promote the accumulation of chlorogenic acid in the flower. 8-10 days before flower harvest, a nutrient solution is sprayed twice consecutively. The nutrient solution is rich in macro- and micro-mineral elements, providing balanced nutrition, promoting absorption, and the added multivitamins and chlorophyll can accelerate the plant's synthesis of chlorogenic acid, delay flower opening, and increase the chlorogenic acid content in honeysuckle.

[0005] Chinese patent document CN201710541724.0 discloses a method for increasing the yield of fresh medicinal chrysanthemum flowers and the content of total flavonoids and chlorogenic acid in the flowers by foliar spraying. The method includes (1) preparing a mixed aqueous solution with a salicylic acid concentration of 0.5-2 mmol / L and a magnesium sulfate mass fraction of 0.02%-0.1%; (2) spraying the prepared mixed solution onto the leaves of the growing medicinal chrysanthemum during the flower bud differentiation period or early budding stage on a rainless morning after the dew has dried, once every 10 days, for a total of 3 sprays. Medicinal chrysanthemums treated with this method were harvested in October when 50% of the flowers were in full bloom in the field. Compared with those under the same conditions without foliar spraying, the yield of fresh flowers was 20%-30% higher, and the content of total flavonoids and chlorogenic acid in the flowers was 20%-30% and 20%-35% higher, respectively. This method is simple to operate in the field, cost-effective, and easy to promote and apply on a large scale.

[0006] Currently, there is no method to increase the chlorogenic acid content of blueberries with camphor leaves. Existing methods all rely on chemical pesticides, which are not only cumbersome to cultivate but also prone to pollution problems. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for increasing the chlorogenic acid (CGA) content in *Vaccinium camphora* tissue culture seedlings. This method achieves increased CGA content in the seedlings by adjusting the potassium (K) concentration in the 1 / 4 MS medium (by increasing or decreasing it). This is an effective way to obtain large quantities of high-yield CGA *Vaccinium camphora* seedlings.

[0008] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0009] A method for increasing the chlorogenic acid content in *Vaccinium bracteatum* tissue culture seedlings includes the following steps:

[0010] The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium, in which the concentration of potassium mineral element was adjusted to 1.5-2 times the standard concentration, and the corresponding KNO3 concentration was 712.5-950 mg / L.

[0011] Furthermore, the concentration of potassium mineral element in the 1 / 4MS medium was adjusted to 1.5 times the standard concentration, corresponding to a KNO3 concentration of 712.5 mg / L.

[0012] Furthermore, the concentration of Ca mineral elements in the 1 / 4MS medium was adjusted to 1.5-2 times the standard concentration, corresponding to a CaCl2·2H2O concentration of 165-220 mg / L.

[0013] Furthermore, the 1 / 4MS medium also includes IBA 2 mg / L, sucrose 15 g / L, activated carbon 0.1 g / L, agar powder 4.5 g / L, and pH 5.8-6.0.

[0014] Furthermore, the tissue culture seedlings of *Vaccinium bracteatum* were subcultured using the following culture media: the starting medium was WPM + 6-BA 3.0 mg / L + NAA 0.2 mg / L, the subculture medium was WPM + ZT 2.0 mg / L, the culture temperature was 25 ± 2℃, the light intensity was 3000 lx, the photoperiod was 12 h / d, and the culture period was 70 days.

[0015] On the other hand, this invention proposes the application of the above method in the study of the growth of camphor blueberry and its chlorogenic acid accumulation.

[0016] The beneficial effects of this invention are:

[0017] Chlorogenic acid (CGA), a multifunctional phenolic acid, is produced in plants through specific biosynthetic pathways. This invention increases CGA synthesis by regulating potassium concentration, particularly by a 1.5-fold increase, resulting in a significant increase in CGA content. Existing research has found that increases or decreases in potassium (K) mineral concentration lead to a decrease in plant CGA content. However, this invention achieves a significant increase in CGA content by raising the potassium concentration, demonstrating the intrinsic mechanism of increasing CGA content through adjusting the potassium concentration in the culture medium from a molecular biology perspective.

[0018] Potassium is not only an important nutrient for plants, but also participates in various aspects such as water regulation, enzyme activity control, and disease defense. In this technical solution, appropriately increasing the potassium concentration to 1.5 times not only promotes the accumulation of CGA, but also has a positive impact on the overall growth status of the plant, such as improving plant height, root length, rooting rate, and the number of roots per plant. This demonstrates that this invention, while increasing the content of active ingredients, also comprehensively considers the optimization of plant physiological functions, ensuring healthy and robust plant growth.

[0019] This invention avoids the use of chemical pesticides and synthetic additives, employing a method that adjusts the concentration of naturally occurring mineral elements in the culture medium, making it a green and environmentally friendly production method. This method reduces the environmental pollution risks associated with chemical reagents while enhancing the natural purity of the blueberry product, thus facilitating consumer acceptance and market promotion.

[0020] Because this invention is simple and easy to implement, requiring no special equipment or high-cost chemical reagents, it significantly reduces production costs and improves economic efficiency. Furthermore, since chlorogenic acid (CGA) has a wide range of applications, increasing its content will directly enhance the market value and competitiveness of blueberry products, bringing greater economic benefits to producers.

[0021] The successful implementation of this invention not only provides an effective way to produce CGA in *Vaccinium bracteatum*, but also offers research directions and methodological ideas for increasing the content of specific secondary metabolites in other specialty plant resources. In particular, this scheme highlights the important role of mineral elements in regulating plant secondary metabolism, providing new research ideas for the field of plant metabolic engineering.

[0022] In summary, this technical solution, by fine-tuning the potassium concentration in the culture medium, achieved the goal of significantly increasing the chlorogenic acid content in *Vaccinium bracteatum* tissue culture seedlings while ensuring healthy plant growth, demonstrating its innovation and application potential in both theory and practice.

[0023] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The effect of different concentrations of potassium mineral element on the CGA content in tissue culture seedlings of *Vaccinium camphora*.

[0026] Figure 2 A schematic diagram showing the effect of different concentrations of Ca mineral element on the CGA content in tissue culture seedlings of Vaccinium camphora.

[0027] Figure 3 A schematic diagram showing the effect of different concentrations of Mg mineral element on the CGA content in Citrus camphora leaf tissue culture seedlings.

[0028] Figure 4A schematic diagram showing the effect of different concentrations of Cu mineral element on the CGA content in tissue culture seedlings of Vaccinium camphora.

[0029] Figure 5 This is a schematic diagram showing the effect of different concentrations of Mn mineral element on the CGA content in Citrus camphora tissue culture seedlings. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] The method for increasing the chlorogenic acid content in *Vaccinium bracteatum* tissue culture seedlings described in this embodiment includes the following steps:

[0033] The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium, in which the concentration of potassium mineral element was adjusted to 1.5-2 times the standard concentration, and the corresponding KNO3 concentration was 712.5-950 mg / L.

[0034] In this embodiment, the concentration of potassium mineral element in 1 / 4MS medium was adjusted to 1.5 times the standard concentration, corresponding to a KNO3 concentration of 712.5 mg / L.

[0035] In this embodiment, the concentration of Ca mineral element in 1 / 4MS medium was adjusted to 1.5-2 times the standard concentration, corresponding to a CaCl2·2H2O concentration of 165-220 mg / L.

[0036] In this embodiment, the 1 / 4MS medium also includes IBA 2 mg / L, sucrose 15 g / L, activated carbon 0.1 g / L, agar powder 4.5 g / L, and pH 5.8-6.0.

[0037] In this embodiment, the tissue culture seedlings of *Vaccinium bracteatum* were subcultured using the following culture media: the starting medium was WPM + 6-BA 3.0 mg / L + NAA 0.2 mg / L, the subculture medium was WPM + ZT 2.0 mg / L, the culture temperature was 25 ± 2℃, the light intensity was 3000 lx, the light duration was 12 h / d, and the culture period was 70 days.

[0038] On the other hand, this invention proposes the application of the above method in the study of the growth of camphor blueberry and its chlorogenic acid accumulation.

[0039] Example 2

[0040] Experimental materials

[0041] The experimental material was subcultured seedlings of wild blueberry leaves from Wuding County, Yunnan Province, after in vitro culture. Following the tissue culture formula of Luo et al. (2014), the culture was propagated using WPM + 6-BA 3.0 mg / L + NAA 0.2 mg / L as the starting medium, and WPM + ZT 2.0 mg / L as the subculture medium. The culture temperature was 25 ± 2℃, the light intensity was 3000 lx, and the photoperiod was 12 h / d.

[0042] Experimental methods

[0043] Selection and determination of mineral elements

[0044] Following the sampling principles of equal quantity, randomness, and multi-point mixing, soil samples were taken from three different locations in the native habitat (Wuding) and the transplanted site (school research base) of the blueberry plantation. Based on the soil texture, the soil was divided into three layers from top to bottom. After air drying, the soil samples were ground into powder and passed through an 80-mesh sieve. The soil samples were then tested by the Agricultural Product Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs (Kunming) according to the national standard NY / T 890-2004 to determine the content of available P, Zn, Fe, Mg, Mn, Ca, Cu, Na, and K mineral elements. Mineral elements with significant differences in content were screened as the basis for subsequent experimental treatment.

[0045] Mineral element concentration treatment and experimental material cultivation

[0046] The rooting formula for *Vaccinium bracteatum* tissue culture seedlings from Zhao Zhanping et al. (2019) (1 / 4 MS + IBA 2 mg / L + sucrose 15 g / L + activated carbon 0.1 g / L + agar powder 4.5 g / L, pH 5.8-6.0) was used as the basal medium. Mineral element concentrations were set using five compounds in the basal medium: CaCl2, KNO3, MgSO4, CuSO4, and MnSO4. The concentration of each of the five mineral elements in the basal medium was recorded as 1.0 times. Five treatment groups were set up with concentrations of 0, 0.5, 1.0, 1.5, and 2.0 times, labeled as Ca(0), Ca(0.5), Ca(1.0), Ca(1.5), and Ca(2.0). The other four elements were labeled the same as Ca. Each treatment group contained 30 bottles, with 10 tissue culture seedlings per bottle. Three biological replicates were set up for each treatment group. Tissue culture seedlings of the same batch and with similar phenotypes were inoculated into the culture media of the above-mentioned different treatment groups. After culturing under the same tissue culture environment for 70 days, the tissue culture seedlings were taken out of the bottle, and the culture medium was removed from the roots (base) for subsequent growth index and compound content determination.

[0047] Concentration values ​​of each element Ca, K, Mg, Cu, and Mn at different multiples in this experiment.

[0048]

[0049] Measurement and Statistical Analysis of Growth Indicators of Vaccinium bracteatum Tissue Culture Seedlings

[0050] Fifty tissue culture seedlings were randomly selected from each biological replicate group to determine plant height, root length, fresh weight, and dry weight. Root count, rooting rate, and water content were recorded, and growth phenotypic characteristics were described. Both fresh and dry weights were calculated for whole seedlings and weighed using an analytical balance. The tissue culture seedlings were first weighed for fresh weight, then growth phenotypic characteristics were observed and recorded. Plant height and root length were measured using vernier calipers, and the root count was observed and counted. Finally, following the method of Li Guoze et al. (2021), the samples were dried in a 45℃ oven to constant weight before weighing. Rooting rate = (number of rooted tissue culture seedlings / 50) × 100%, water content = (1 - dry weight / fresh weight) × 100%.

[0051] Compound content determination

[0052] Fifty tissue culture seedlings were randomly selected from each biological replicate group. Roots (bases) were removed, and stem and leaf tissues were mixed. The dry weight content of compounds was determined by HPLC, with three technical replicates. Powder samples were prepared according to the method of Li Guoze et al. (2021). Standard curves were plotted, compounds were extracted from the samples, and CGA content was determined according to the method of Li Na et al. (2016). Chromatographic conditions were as follows: F90104 CAPCELL PAK C18 MG(S-5) column (250 mm × 4.6 mm, 5 μm). Mobile phase: 1% glacial acetic acid [(filtered through a 0.45 μm pore size membrane - methanol (chromatographic grade)). Gradient elution: 0-5 min, 5% methanol; 5-10 min, 5%-15% methanol; 10-50 min, 15%-65% methanol; 50-55 min, 65%-95% methanol; 55-70 min, 95%-5% methanol. Column temperature: 25℃.

[0053] Data processing

[0054] Data are expressed as mean ± standard deviation (Mean ± SE). Excel 2013 was used to collect data, calculate the mean and standard deviation, and plot the data. SPSS 23.0 was used to analyze significance and correlation, employing ANOVA and LSD multiple comparison tests to determine if a significant difference existed between treatments at P = 0.05. Origin 2021 was used to plot the data.

[0055] Example 3

[0056] Results and analysis, such as Figure 1-5 As shown

[0057] Mineral element content determination and analysis of soil samples and selection of mineral elements for subsequent tests

[0058] The results of soil sample determination for nine mineral elements (P, Zn, Fe, Mg, Mn, Ca, Cu, Na, and K) in soil samples from the transplanting site and the original production site of *Vaccinium bracteatum* are shown in Table 1. Differences in mineral element content were observed between the two sites. The contents of Fe, Mg, Mn, Ca, Cu, Na, and K in the first soil layer showed significant differences between the two sites (P<0.05). The contents of all nine mineral elements in the second soil layer also showed significant differences between the two sites (P<0.05). The contents of P, Mg, Mn, Ca, Cu, and K in the third soil layer showed significant differences between the two sites (P<0.05). Furthermore, the differences in the contents of Ca, K, Mg, Cu, and Mn in all three soil layers were highly significant (P<0.05), with the differences in Ca, K, and Mg reaching a highly significant level (P<0.01). Therefore, five mineral elements—Ca, K, Mg, Cu, and Mn—were selected for subsequent experiments.

[0059] Table 1. Analysis of mineral element content in soil samples from the original and transplanted areas of *Vaccinium bracteatum*.

[0060]

[0061] Note: In the table, "A" represents "transplanting site" and "B" represents "place of origin". * indicates a significant difference (P < 0.05), ** indicates an extremely significant difference (P < 0.01), and no * indicates no significant difference (P > 0.05).

[0062] Effects of five mineral elements on the growth of Cinnamomum camphora tissue culture seedlings

[0063] The effect of calcium on the growth of *Vaccinium camphoratum* tissue culture seedlings

[0064] Among the different Ca concentration treatment groups, the rooting rate of *Vaccinium bracteatum* tissue culture seedlings was highest in Ca(0.5), Ca(1.5), and Ca(2.0), significantly higher than Ca(0), but not significantly different from Ca(1.0); water content showed no significant difference among the groups (Table 2). With increasing concentration, plant height first increased and then decreased, while root length and number of roots per plant showed a gradual decreasing trend. Plant height was highest in Ca(1.5), significantly higher than Ca(0) and Ca(2.0), but not significantly different from the Ca(0.5) and Ca(1.0) treatment groups. Except for the Ca(1.5) and Ca(1.0) groups, root length showed significant differences among the other groups (P<0.05). The number of roots per plant did not differ significantly among the Ca(0.5), Ca(1.0), and Ca(1.5) treatment groups, but was significantly higher in Ca(0) than in Ca(1.0), Ca(1.5), and Ca(2.0), and also significantly higher in Ca(0.5) than in Ca(2.0). This indicates that Ca deficiency in the culture medium significantly induces an increase in the number of roots and root elongation in *Vaccinium bracteatum* tissue culture seedlings, but significantly reduces the rooting rate and plant height. Regarding plant growth phenotypes, leaves were spread out and roots were well-developed in Ca(1.0), leaves were curled and roots were slender in Ca(0), roots were well-developed but leaves were wrinkled in Ca(0.5), and leaves were spread out but roots were short in both Ca(1.5) and Ca(2.0). In summary, Ca(1.0) is the optimal treatment concentration for the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0065] Table 2. Effects of different concentrations of Ca mineral element on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0066]

[0067]

[0068] Note: Letters in the table indicate differences in means within experimental groups at the P < 0.05 level. The same applies below.

[0069] The effect of potassium on the growth of *Vaccinium camphoratum* tissue culture seedlings

[0070] In the different K concentration treatment groups, as the concentration gradually increased, the rooting rate, root length, and plant height of *Vaccinium camphora* tissue culture seedlings generally showed a trend of first increasing and then decreasing (Table 3). The rooting rate, root length, number of roots per plant, and plant height were all highest at K(1.5), and all showed significant differences from the K(0) and K(0.5) groups. The root length and number of roots per plant at K(1.5) also showed significant differences from K(1.0) and K(2.0), respectively (P<0.05). There were also significant differences in rooting rate between the K(1.5) and K(2.0) groups, and in plant height between the K(1.5) and K(1.0) groups (P<0.05), while there were no significant differences in rooting rate between the K(1.5) and K(1.0) groups, and in plant height between the K(1.5) and K(2.0) groups. The water content was lowest at K(1.5), significantly lower than at K(0), K(0.5), and K(2.0), but the plants exhibited the best growth at this concentration, with well-developed leaves and dense root systems. This indicates that K(1.5) is the optimal treatment concentration for the growth of *Vaccinium camphora* tissue culture seedlings.

[0071] Table 3. Effects of different concentrations of mineral element K on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0072]

[0073]

[0074] In different Mg concentration treatment groups, the rooting rate, root length, number of roots per plant, and plant height all showed a decreasing trend with increasing Mg concentration. Root length, number of roots per plant, and plant height at Mg(0) were significantly different from other treatment concentrations (P<0.05), while the rooting rate at Mg(0) was not significantly different from Mg(0.5) and Mg(1.0). The water content in each treatment group did not change significantly (Table 4). Regarding plant growth phenotype, leaves were spread out and roots were robust and well-developed at Mg(1.0). At higher Mg concentrations, the plant had fewer roots and the leaves were curled, with the curling gradually worsening. At lower or absent Mg concentrations, although the plant had well-developed roots, the leaves were wrinkled, or the roots were thin and long, and the leaves were curled and yellowed. This indicates that high Mg concentrations significantly inhibited the development and elongation of roots in *Vaccinium bracteatum* tissue culture seedlings. Although Mg deficiency could significantly induce an increase in the number of roots, root length, and plant height in *Vaccinium bracteatum* tissue culture seedlings, it was detrimental to the normal growth and development of the plant. In summary, similar to Ca, Mg (1.0) is the optimal treatment concentration for the growth of Citrus camphora tissue culture seedlings.

[0075] Table 4. Effects of different Mg mineral element concentrations on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0076]

[0077]

[0078] In the different Cu concentration treatment groups, the rooting rate, root length, and number of roots per plant all showed a decreasing trend with increasing Cu concentration. Furthermore, there were significant differences between Cu(0) and Cu(1.5) and Cu(2.0) groups (P<0.05), indicating that the induction and elongation of tissue-cultured seedling roots were gradually inhibited (Table 5). Plant height showed a wave-like trend of first increasing, then decreasing, and then increasing again, with the highest value in the Cu(1.0) group, which was significantly different from Cu(0), Cu(0.5), and Cu(1.5) (P<0.05). Water content showed a trend of first increasing and then decreasing, reaching its highest value in Cu(1.0), which was significantly different from Cu(0) and Cu(2.0) (P<0.05). Although the root system was well-developed in the Cu(0) and Cu(0.5) treatments, the leaves were curled. Although the leaves were spread out in the Cu(1.5) and Cu(2.0) groups, the root system was less developed. The tissue culture seedlings were in the best growth state at Cu(1.0), with both leaves spread out and roots well-developed.

[0079] Table 5. Effects of different Cu mineral element concentrations on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0080]

[0081]

[0082] As the concentration of Mn applied increased, the rooting rate, root length, and number of roots per plant gradually increased, all reaching their highest values ​​at Mn(2.0) (Table 6). The rooting rate at Mn(2.0) was 100%, but the difference from Mn(1.5) was not significant. Significant differences in rooting rate were observed among the Mn(0), Mn(1.0), and Mn(1.5) groups (P<0.05). Root length showed significant differences among the Mn(2.0), Mn(1.5), and Mn(1.0) groups (P<0.05), but no significant differences were observed among the Mn(1.0), Mn(0.5), and Mn(0) groups. The number of roots per plant reached its maximum value at Mn(2.0), significantly higher than that at Mn(0), Mn(0.5), and Mn(1.0), but there was no significant difference compared to the Mn(1.5) group. The overall water content showed an increasing trend, with Mn(0) being significantly lower than the other four groups, but there were no significant differences among the Mn(0.5), Mn(1.0), Mn(1.5), and Mn(2.0) groups. Plant height was highest at Mn(1.0), showing a significant difference compared to the Mn(1.5), Mn(0.5), and Mn(0) groups (P<0.05). Plant growth was optimal at Mn(1.0); with increasing Mn concentration, although the root system was well-developed, the leaves curled; with decreasing or absent Mn concentration, the root system was sparse and the leaves wrinkled or curled. This indicates that high concentrations of Mn can induce increased root development, growth, and elongation in *Vaccinium bracteatum* tissue culture seedlings, but inhibit stem growth; low concentrations of Mn inhibited both root and stem growth and development, and both high and low concentrations of Mn were detrimental to leaf development. Comprehensive analysis shows that Mn(1.0) is the optimal Mn concentration for the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0083] Table 6. Effects of different concentrations of Mn mineral element on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0084]

[0085]

[0086] In tissue culture, when a culture medium is selected, the concentration of mineral elements is usually kept constant. Previous experiments in this invention yielded an ideal rooting medium for *Vaccinium bracteatum* (Zhao Zhanping et al., 2017). This experiment modifies the concentration of mineral elements in the culture medium by changing the amount of a single mineral element added, thereby regulating the growth of *Vaccinium bracteatum* tissue culture seedlings and the accumulation of CGA content. The operation is simple and easy to implement, facilitating widespread application.

[0087] In this invention, *Vaccinium bracteatum* tissue culture seedlings were used as experimental material. The concentrations of five mineral elements (Ca, K, Mg, Cu, and Mn) in 1 / 4 MS medium were used as baselines. Experiments were conducted at element concentration levels of 0, 0.5, 1.0, 1.5, and 2.0 times the standard concentration. After 70 days of cultivation, growth indicators such as plant water content, plant height, root length, rooting rate, and number of roots per plant, as well as the content of the compound CGA, were measured. The CGA content was determined by HPLC. The standard curve for this experiment was Y = 622753X - 60359, R0. 2 =0.9999.

[0088] The results showed that increasing the Ca concentration (>1-2 times) could increase the CGA content, but it was detrimental to the growth and development of *Vaccinium camphora* tissue culture seedlings. Decreasing the Mg concentration to Mg0.5 also increased the CGA content, but this was also detrimental to the growth and development of *Vaccinium camphora* tissue culture seedlings. Decreasing the Ca concentration, or increasing or decreasing the concentrations of Mg (except Mg0.5), Cu, and Mn, both reduced the CGA content and were detrimental to the growth and development of *Vaccinium camphora* tissue culture seedlings.

[0089] In this embodiment, the effect of potassium (K) concentration on CGA content was investigated. The chlorogenic acid (CGA) content was studied after K treatment was applied at 1.25 times and 1.75 times. The results showed that the CGA accumulation under K (1.25) treatment was 0.91 mg / g, and the CGA accumulation under K (1.75) treatment was 1.38 mg / g. The corresponding fold changes were 1.625 times that of the control K (1.0) and 2.464 times that of the control K (1.0).

[0090] Uniquely, both increasing and decreasing the potassium (K) concentration could increase the CGA content, with increases ranging from 1.1 to 4.34. For the growth and development of tissue culture seedlings, the optimal growth status of *Vaccinium bracteatum* tissue culture seedlings was observed when the K concentration was increased by 1.5 times; at this point, the accumulation of CGA under the K (1.5) treatment also reached its highest level, at 2.44 mg / g, which was 4.34 times that of the control K (1.0).

[0091] Table 7. Changes in CGA content in *Lonicera camphora* tissue culture seedlings under different concentrations of elements Ca, K, Mg, Cu, and Mn.

[0092]

[0093] Table 8. CGA content in *Lonicera camphora* tissue culture seedlings at different concentrations of elements Ca, K, Mg, Cu, and Mn.

[0094]

[0095] This study found that mineral element deficiency or excess stress can induce a significant increase in the synthesis of the main active ingredients in *Vaccinium bracteatum*. The optimal concentrations of Ca (1.0), Mg (1.0), Cu (1.0), and Mn (1.0) for the growth and development of seedling stems and leaves were determined. Under Ca excess stress [Ca (2.0) and Ca (1.5)], the CGA content in the plant significantly increased. Mg (0.5) and Mg (0), Mn (0.5) and Mn (0) caused element deficiency stress in *Vaccinium bracteatum*, while Mg (2.0) and Mg (1.5), and Mn (2.0) and Mn (1.5) all caused element excess stress. However, the CGA content was not affected by the increase induced by Mn and Cu deficiency or excess stress; instead, the content decreased significantly.

[0096] K + Plants play a vital role in various life activities, including nutrient transport, response to biotic and abiotic stresses, and growth and metabolic control. Therefore, plants require high levels of potassium during their growth and development. + Accumulation. The concentration of K (1.5) above the basal medium concentration is the optimal K concentration for the growth and development of *Vaccinium bracteatum*, and also the optimal concentration for the accumulation of the active compound CGA in vivo.

[0097] Therefore, this invention can regulate and increase the content of the active compound CGA in *Vaccinium bracteatum* tissue culture seedlings by changing the concentration of potassium mineral element in the 1 / 4MS medium (increasing or decreasing it). This is an effective way to obtain high-yield *Vaccinium bracteatum* materials with high CGA production.

[0098] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for increasing the chlorogenic acid content in *Vaccinium bracteatum* tissue culture seedlings, characterized in that, Includes the following steps: The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium + IBA 2 mg / L + sucrose 15 g / L + activated carbon 0.1 g / L + agar powder 4.5 g / L, with a pH of 5.8 to 6.

0. The concentration of KNO3 in the 1 / 4MS medium was adjusted from 712.5 mg / L to 950 mg / L.

2. The method for increasing the chlorogenic acid content in *Vaccinium camphora* tissue culture seedlings as described in claim 1, characterized in that: The concentration of KNO3 in the 1 / 4MS medium was adjusted to 712.5 mg / L.

3. A method for increasing the chlorogenic acid content in *Vaccinium bracteatum* tissue culture seedlings, characterized in that, Includes the following steps: The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium + IBA 2 mg / L + sucrose 15 g / L + activated carbon 0.1 g / L + agar powder 4.5 g / L, with a pH of 5.8 to 6.

0. The concentration of CaCl2·2H2O in the 1 / 4MS medium was adjusted to 165 mg / L to 220 mg / L.

4. The method for increasing the chlorogenic acid content in *Vaccinium camphora* tissue culture seedlings as described in claim 1 or 3, characterized in that: Before being transferred to the rooting medium for rooting culture, the *Vaccinium bracteatum* tissue culture seedlings were first cultured using a starting medium and a subculture medium. The starting medium consisted of WPM medium + 6-BA 3.0 mg / L + NAA 0.2 mg / L, and the subculture medium consisted of WPM medium + ZT 2.0 mg / L. The culture temperature was 25±2℃, the light intensity was 3000 lx, the light duration was 12 h / d, and the culture period was 70 days.

5. The application of the method described in claim 1 or 3 in the study of the growth of camphor blueberry and the accumulation of its main active components.

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