A method and its application for increasing the content of arbutin and 6′-O-caffeoylarbutin in tissue culture seedlings of *Vaccinium bracteatum*.

Abstract

This invention relates to the field of Vaccinium bracteatum tissue culture technology, specifically to a method and application for increasing the content of arbutin and 6′-O-caffeoyl arbutin in Vaccinium bracteatum tissue culture seedlings. The method includes: using 1 / 4 MS medium as the rooting medium for the Vaccinium bracteatum tissue culture seedlings, wherein the specific concentrations of CaCl2·2H2O are 165-220 mg / L; KNO3 is 0-237.5 mg / L or 712.5-950 mg / L; MgSO4·7H2O is 0-46.25 mg / L; CuSO4·5H2O is 0.0375-0.05 mg / L; and MnSO4·4H2O is 0-11.15 mg / L. This invention achieves the controlled increase of the content of active compounds CA and Arb in Vaccinium bracteatum tissue culture seedlings by changing the mineral element concentrations in the 1 / 4 MS medium. This is an effective way to obtain large quantities of high-yielding CA and Arb Vaccinium bracteatum materials.

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 content of arbutin and 6′-O-caffeoylarbutin 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). 6′-O-caffeoylarbutin (CA) and arbutin (Arb) are two major secondary metabolites present in high amounts in Vaccinium dunalianum (Zhao et al., 2008; Cheng Guiguang et al., 2010; Luo et al., 2015; Li Na et al., 2016). Arb has pharmacological value in whitening, anti-oxidation, and antibacterial properties and is widely used in the cosmetics and pharmaceutical industries (Liu Xiaoting and Wang Xinxuan, 2022; Wei et al., 2007; Lutfun et al., 2022); CA also has multiple effects such as whitening (Xu et al., 2014), anti-oxidation (Zhao et al., 2021), and lowering serum uric acid (Ling Lin, 2021), and will play an important role in the 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 school (Panlong District, Kunming, Yunnan). They found a significant decrease in CA and Arb contents. Further investigation revealed differences in the essential mineral element content 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 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 of *Vaccinium bracteatum* and the accumulation of CA and Arb are unknown. Currently, there are no methods to increase CA and Arb in tissue-cultured *Vaccinium bracteatum* seedlings, making it necessary to address the current resource scarcity of *Vaccinium bracteatum*.

[0004] Chinese patent document CN202010176530.7 discloses a method for increasing the 6′O-caffeoyl arbutin content in *Vaccinium camphora* tissue culture seedlings, belonging to the field of plant tissue culture technology. The method involves subculturing *Vaccinium camphora* tissue culture seedlings using a WPM basal medium supplemented with two different plant growth regulators, ZT and NAA. The ZT concentration is 0.1–5.0 mg / L, and the NAA concentration is 0.01–2.0 mg / L, with the pH adjusted to 5.0–6.0. ​​By applying different concentrations of ZT and NAA, the CA content in the leaves and stems of the tissue culture seedlings increased by 4–7 times and 2–5 times, respectively, compared to the control group under WPM conditions. This is an effective way to obtain a large quantity of *Vaccinium camphora* material with high CA content.

[0005] Existing methods all involve using chemical pesticides to increase the 6′O-caffeoyl arbutin content in Vaccinium bracteatum tissue culture seedlings. This not only makes the planting process cumbersome but also easily leads to pollution problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for increasing the content of arbutin and 6′-O-caffeoylarbutin in Vaccinium camphora tissue culture seedlings. This is achieved by adjusting the concentrations (increasing or decreasing) of potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and manganese (Mn) mineral elements in the 1 / 4 MS medium to regulate and increase the content of active compounds CA and Arb in the Vaccinium camphora tissue culture seedlings. This is an effective way to obtain large quantities of high-yielding CA and Arb Vaccinium camphora materials.

[0007] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0008] A method for increasing the content of arbutin and 6′-O-caffeoylarbutin in tissue culture seedlings of *Vaccinium bracteatum* includes the following steps:

[0009] The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium, in which the concentration of Ca mineral elements was adjusted to 1.5-2 times the standard concentration, specifically the CaCl2·2H2O concentration of 165-220 mg / L;

[0010] The concentration of potassium mineral element was adjusted to 0-0.5 times or 1.5-2 times the standard concentration, corresponding to a KNO3 concentration of 0-237.5 mg / L or 712.5-950 mg / L.

[0011] The concentration of Mg mineral element was adjusted to 0-0.5 times the standard concentration, corresponding to a MgSO4·7H2O concentration of 0-46.25 mg / L.

[0012] The concentration of Cu mineral elements was adjusted to 1.5-2 times the standard concentration, corresponding to a CuSO4·5H2O concentration of 0.0375-0.05 mg / L.

[0013] The concentration of Mn mineral element was adjusted to 0-0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 0-11.15 mg / L.

[0014] Furthermore, the concentration of Ca mineral elements in the 1 / 4MS medium was adjusted to twice the standard concentration, specifically corresponding to a CaCl2·2H2O concentration of 220 mg / L;

[0015] The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 or 712.5 mg / L.

[0016] The concentration of Mg mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MgSO4·7H2O concentration of 46.25 mg / L.

[0017] The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L.

[0018] The concentration of Mn mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 11.15 mg / L.

[0019] Furthermore, the concentration of Ca mineral elements in the 1 / 4MS medium was adjusted to 1.5 times the standard concentration, corresponding to a CaCl2·2H2O concentration of 165 mg / L.

[0020] The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 mg / L or 712.5 mg / L.

[0021] The concentration of Mg mineral element is adjusted to 0 or 0.5 times the standard concentration, which corresponds to a specific concentration of MgSO4·7H2O of 0 mg / L or 46.25 mg / L.

[0022] The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L.

[0023] The concentration of Mn mineral element is adjusted to 0 or 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 0 mg / L or 11.15 mg / L.

[0024] 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.

[0025] 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.

[0026] On the other hand, this invention proposes the application of the above method in the study of the growth of camphor blueberry and the accumulation of arbutin and 6′-O-caffeoylarbutin.

[0027] The beneficial effects of this invention are:

[0028] This technical solution significantly increases the content of arbutin (Arb), 6′-O-caffeoylarbutin (CA), by precisely controlling the concentration of mineral elements, particularly potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), and manganese (Mn), in the growth environment of *Vaccinium bracteatum* tissue culture seedlings. This achievement not only provides new ideas for the efficient cultivation of *Vaccinium bracteatum* but also provides technical support for the further development and utilization of its active ingredients.

[0029] First, increasing the concentration of calcium (Ca) in 1 / 4 MS medium to 1.5-2 times the standard concentration significantly increased the content of Arb and CA in tissue-cultured seedlings of *Vaccinium bracteatum*. Calcium (Ca) is an indispensable element for plant growth and development; it not only participates in cell wall construction but also involves signal transduction processes, playing a crucial role in the synthesis of secondary metabolites within plants. In this technique, appropriately increasing the Ca concentration promoted the synthesis of Arb and CA in *Vaccinium bracteatum*, possibly because the increased Ca improved the plant's physiological state, providing more favorable conditions for the synthesis of secondary metabolites.

[0030] Secondly, adjusting the concentration of potassium (K) to 0-0.5 times or 1.5-2 times the standard concentration, as well as adjusting the concentrations of Mg, Cu, and Mn, all positively impacted the content of Arb and CA. Potassium (K) is an essential nutrient for plant growth, participating in multiple physiological processes such as water regulation, enzyme activity control, and photosynthesis. In this technical scheme, appropriately adjusting the K concentration, especially increasing it to 1.5 times the standard concentration, significantly increased the content of Arb and CA. Adequate K promotes the efficient conversion and utilization of energy within plants, thus providing sufficient energy and precursor substances for the synthesis of Arb and CA.

[0031] Furthermore, precise control of the concentrations of Mg, Cu, and Mn can effectively promote the increase of Arb and CA content. Although these mineral elements are required in small amounts by plants, they participate in the regulation of many enzyme activities and biosynthetic pathways, playing an important role in normal plant growth and the synthesis of secondary metabolites. In this technical solution, appropriately increasing the concentrations of Cu and Mn or adjusting the Mg concentration can promote the synthesis of Arb and CA by affecting the activity of related enzymes or improving the plant's stress resistance. It 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.

[0032] 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.

[0033] 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. The successful implementation of this invention not only provides an effective way to produce CA and Arb in *Vaccinium bracteatum*, but also offers research directions and methodological approaches 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.

[0034] In summary, this technical solution effectively increased the content of Arb and CA by precisely controlling the concentration of specific mineral elements in the growth environment of *Vaccinium bracteatum* tissue culture seedlings. This achievement not only reveals the mechanism by which mineral elements affect the synthesis of secondary metabolites in *Vaccinium bracteatum*, but also provides a new technical approach for the cultivation and extraction of active ingredients in *Vaccinium bracteatum*.

[0035] 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

[0036] 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.

[0037] Figure 1 The effect of different concentrations of potassium mineral element on the Arb and CA content in tissue culture seedlings of *Vaccinium camphora*.

[0038] Figure 2 A schematic diagram showing the effect of different concentrations of Ca mineral element on the Arb and CA content in tissue culture seedlings of Vaccinium camphora.

[0039] Figure 3 A schematic diagram showing the effect of different concentrations of Mg mineral element on the Arb and CA content in tissue culture seedlings of Vaccinium camphora.

[0040] Figure 4 A schematic diagram showing the effect of different concentrations of Cu mineral element on the Arb and CA content in tissue culture seedlings of Vaccinium camphora.

[0041] Figure 5 This is a schematic diagram showing the effect of different concentrations of Mn mineral element on the Arb and CA content in tissue culture seedlings of *Vaccinium bracteatum*. Detailed Implementation

[0042] 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.

[0043] Example 1

[0044] The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings described in this embodiment includes the following steps:

[0045] The rooting medium selected for the Citrus camphora tissue culture seedlings was 1 / 4 MS medium, in which the concentration of Ca mineral elements was adjusted to twice the standard concentration, specifically the CaCl2·2H2O concentration of 220 mg / L;

[0046] The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 or 712.5 mg / L.

[0047] The concentration of Mg mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MgSO4·7H2O concentration of 46.25 mg / L.

[0048] The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L.

[0049] The concentration of Mn mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 11.15 mg / L.

[0050] 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.

[0051] 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.

[0052] On the other hand, this invention proposes the application of the above method in the study of the growth of camphor blueberry and the accumulation of arbutin and 6′-O-caffeoylarbutin.

[0053] Example 2

[0054] The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings described in this embodiment includes the following steps:

[0055] The concentration of Ca mineral element in 1 / 4MS medium was adjusted to 1.5 times the standard concentration, which corresponds to a CaCl2·2H2O concentration of 165 mg / L.

[0056] The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 mg / L or 712.5 mg / L.

[0057] The concentration of Mg mineral element is adjusted to 0 or 0.5 times the standard concentration, which corresponds to a specific concentration of MgSO4·7H2O of 0 mg / L or 46.25 mg / L.

[0058] The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L.

[0059] The concentration of Mn mineral element is adjusted to 0 or 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 0 mg / L or 11.15 mg / L.

[0060] Example 3

[0061] Experimental materials

[0062] 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.

[0063] Experimental methods

[0064] Mineral element concentration treatment and experimental material cultivation

[0065] 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.

[0066] Concentration values ​​of each element Ca, K, Mg, Cu, and Mn at different multiples in this experiment.

[0067]

[0068] Measurement and Statistical Analysis of Growth Indicators of Vaccinium bracteatum Tissue Culture Seedlings

[0069] 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%.

[0070] Compound content determination

[0071] 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 the contents of CA and Arb were 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℃.

[0072] Data processing

[0073] 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.

[0074] Results and analysis, such as Figure 1-5 As shown

[0075] Effects of five mineral elements on the growth of Cinnamomum camphora tissue culture seedlings

[0076] The effect of calcium on the growth of tissue culture seedlings of *Vaccinium camphora*

[0077] Among the different Ca concentration treatment groups, the rooting rate of *Vaccinium bracteatum* tissue culture seedlings was the 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); the water content did not differ significantly among the groups (Table 1). 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 differed significantly 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 was 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.

[0078] Table 1. Effects of different concentrations of Ca mineral element on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0079]

[0080]

[0081] Note: Letters in the table indicate differences in means within experimental groups at the P < 0.05 level. The same applies below.

[0082] The effect of potassium on the growth of *Vaccinium camphoratum* tissue culture seedlings

[0083] In the different K concentration treatment groups, as the concentration gradually increased, the rooting rate, root length, and plant height of *Vaccinium bracteatum* tissue culture seedlings generally showed a trend of first increasing and then decreasing (Table 2). 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.

[0084] Table 2. Effects of different concentrations of mineral element K on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0085]

[0086]

[0087] 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 3). Regarding plant growth phenotype, at Mg(1.0), the leaves were spread out and the root system was robust and well-developed. At higher Mg concentrations, the plant had fewer roots and the leaves were curled, with the curling gradually worsening. At lower Mg concentrations or when Mg was absent, although the root system was well-developed, 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 plants. In summary, similar to Ca, Mg (1.0) is the optimal treatment concentration for the growth of Citrus camphora tissue culture seedlings.

[0088] Table 3. Effects of different Mg mineral element concentrations on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0089]

[0090] 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 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 4). 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.

[0091] Table 4. Effects of different Cu mineral element concentrations on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0092]

[0093] With increasing Mn concentration, rooting rate, root length, and number of roots per plant gradually increased, reaching their highest values ​​at Mn(2.0) (Table 5). 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 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.

[0094] Table 5. Effects of different concentrations of Mn mineral element on the growth of *Vaccinium bracteatum* tissue culture seedlings.

[0095]

[0096] 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 Arb and CA in vivo. The operation is simple and easy to implement, facilitating widespread application.

[0097] 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. Element concentration levels of 0, 0.5, 1.0, 1.5, and 2.0 times were set. 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 contents of compounds Arb and CA, were measured. Arb and CA contents were determined by HPLC. The standard curve for this experiment was Y = 622753X - 60359, R0. 2 =0.9999, and the results are shown in Tables 6 and 7.

[0098] Table 6. Variation of Arb and CA content in *Vaccinium bracteatum* tissue culture seedlings under the influence of different concentrations of elements Ca, K, Mg, Cu, and Mn.

[0099]

[0100] Table 7. Statistical values ​​of compound content under different treatments

[0101]

[0102]

[0103] This invention 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 found. Under Ca excess stress [Ca (2.0) and Ca (1.5)], the contents of CA and Arb in the plant were significantly increased. Mg (0.5) and Mg (0), Mn (0.5) and Mn (0) caused element deficiency stress in the growth of *Vaccinium bracteatum*, while Mg (2.0) and Mg (1.5), and Mn (2.0) and Mn (1.5) all caused element excess stress. Both Mg and Mn deficiency or excess stress induced a significant increase in the contents of CA and Arb in the plant.

[0104] Therefore, this invention can regulate and increase the content of active compounds CA and Arb in Vaccinium camphora tissue culture seedlings by changing the concentration of mineral elements Ca, K, Mg, Cu, and Mn in the 1 / 4MS medium (increasing or decreasing). This is an effective way to obtain large quantities of Vaccinium camphora materials with high CA and Arb yields.

[0105] 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 content of arbutin and 6′-O-caffeoylarbutin in tissue culture seedlings of *Vaccinium bracteatum*, characterized in that, The following steps are included: The rooting medium selected for the Citrus camphora tissue culture seedlings is 1 / 4 MS medium, in which the concentration of Ca mineral element is adjusted to 1.5-2 times the standard concentration, and the corresponding CaCl2·2H2O concentration is 165-220 mg / L. The concentration of potassium mineral element was adjusted to 0-0.5 times or 1.5-2 times the standard concentration, corresponding to a KNO3 concentration of 0-237.5 mg / L or 712.5-950 mg / L. The concentration of Mg mineral element was adjusted to 0-0.5 times the standard concentration, corresponding to a MgSO4·7H2O concentration of 0-46.25 mg / L. The concentration of Cu mineral elements was adjusted to 1.5-2 times the standard concentration, corresponding to a CuSO4·5H2O concentration of 0.0375-0.05 mg / L. The concentration of Mn mineral element was adjusted to 0-0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 0-11.15 mg / L.

2. The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings as described in claim 1, characterized in that: The concentration of Ca mineral elements in 1 / 4MS medium was adjusted to twice the standard concentration, specifically the CaCl2·2H2O concentration of 220 mg / L. The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 or 712.5 mg / L. The concentration of Mg mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MgSO4·7H2O concentration of 46.25 mg / L. The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L. The concentration of Mn mineral element was adjusted to 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 11.15 mg / L.

3. The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings as described in claim 1, characterized in that: The concentration of Ca mineral element in 1 / 4MS medium was adjusted to 1.5 times the standard concentration, which corresponds to a CaCl2·2H2O concentration of 165 mg / L. The concentration of potassium mineral element was adjusted to 0 or 1.5 times the standard concentration, corresponding to a KNO3 concentration of 0 mg / L or 712.5 mg / L. The concentration of Mg mineral element is adjusted to 0 or 0.5 times the standard concentration, which corresponds to a specific concentration of MgSO4·7H2O of 0 mg / L or 46.25 mg / L. The concentration of Cu mineral element was adjusted to twice the standard concentration, specifically the concentration of CuSO4·5H2O was 0.05 mg / L. The concentration of Mn mineral element is adjusted to 0 or 0.5 times the standard concentration, corresponding to a MnSO4·4H2O concentration of 0 mg / L or 11.15 mg / L.

4. The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings as described in claim 1, characterized in that: 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.

5. The method for increasing the content of arbutin and 6′-O-caffeoylarbutin in *Vaccinium bracteatum* tissue culture seedlings as described in claim 1, characterized in that: 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.

6. The application of the method according to any one of claims 1-5 in the study of the growth of camphor blueberry and the accumulation of arbutin and 6′-O-caffeoylarbutin.

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