A method for promoting rooting of tissue culture seedlings of grossedentate pipevine by using copper element

Abstract

The present application relates to a method for promoting rooting of large leaf of piper hispidum tissue culture seedlings by using copper element, belonging to the technical field of medicinal plant propagation, solving the problem that single copper ion concentration cannot simultaneously optimize rooting rate and medicinal ingredient accumulation. The method takes large leaf of piper hispidum stem section as material, adds 0.1 mg / L NAA and specific concentration of copper element in 1 / 2MS basic medium, and is cultured at 25 DEG C with 12h / d illumination for 25-35 days; through precise control of copper ion concentration, double targets are realized, when the target is to maximize the rooting rate, 0.025 mg / L copper ion concentration is adopted, and the rooting rate can reach more than 90%; when the target is to maximize root or stem flavonoid content, 0.05-0.075 mg / L copper ion concentration is adopted, so that the root flavonoid content is greater than or equal to 9.0% or the stem flavonoid content is greater than or equal to 16.8%. The method simultaneously improves the root system development and the content of secondary metabolites of tissue culture seedlings, and provides technical support for directional production of medicinal ingredients of large leaf of piper hispidum and large-scale seedling raising.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant propagation technology, specifically relating to a method for promoting rooting of tissue culture seedlings of large-leaved konjac using copper. Background Technology

[0002] Large-leaved konjac ( Piper laetispicum Piper longifolia (C. DC.) is a perennial evergreen vine belonging to the genus Piper in the family Piperaceae. It is widely distributed in the tropical and subtropical regions of southern my country. Piper longifolia has high medicinal value, with its roots, stems, and leaves rich in various substances, such as volatile oils, alkaloids, and flavonoids. In traditional Chinese medicine, this plant is commonly used to treat wind-cold, regulate qi and relieve pain, and promote blood circulation. Furthermore, modern pharmacological research has demonstrated its great potential in antibacterial, anti-inflammatory, analgesic, and antidepressant effects. Therefore, Piper longifolia is considered a medicinal plant resource with promising development prospects and future potential.

[0003] In the tissue culture and seedling cultivation practice of the medicinal plant *Ketora macrocarpa*, the applicant faces the following technical challenges in copper element regulation:

[0004] First, the use of a single copper ion concentration leads to a trade-off between achieving the desired results. Using fixed copper ion concentrations, such as 0.025 mg / L or 0.075 mg / L, results in only 2.1-4.9% flavonoid content in roots at the optimal concentration of 0.025 mg / L, far lower than the 9.3% in the 0.075 mg / L high-copper group. Conversely, increasing the flavonoid content to a high copper ion concentration of 0.075 mg / L reduces the rooting rate to around 54.5%. This is because copper has a concentration-dependent dual effect in root development and secondary metabolism, but current technology has not established a rule for matching concentration with the desired culture objective.

[0005] Second, the transfer of high copper ion concentrations triggers root stress damage. When tissue culture seedlings are directly transferred from a low-copper environment to a 0.075 mg / L high-copper medium, the plants are prone to root tip browning and elongation arrest. This may be because copper ions accumulate rapidly under conditions where the roots are not adapted, inhibiting superoxide dismutase activity and inducing oxidative stress, a process that is difficult to alleviate by adjusting the composition of conventional culture media.

[0006] Third, the flavonoid synthesis capacity of roots is significantly weaker than that of stems. At the same high copper ion concentration of 0.075 mg / L, the flavonoid content in roots (9.3%) is only 72.7% of the flavonoid content in stems (12.8%), indicating an efficiency limitation in the root synthesis pathway. Current techniques rely on systematically adding copper to the culture medium, which cannot specifically enhance root metabolism. Furthermore, increasing the overall copper ion concentration will further inhibit rooting, as the rooting rate in the 0.075 mg / L high-throughput concentration group was only 54.5%.

[0007] The aforementioned challenges may stem from several factors: the unclear mechanism of copper ion concentration effects in *Ketora macrocarpa*, the lack of precise concentration thresholds for different culture objectives such as rooting rate / flavonoid accumulation; the low tolerance threshold of roots to drastic changes in copper ion concentration, with a critical value likely around 0.05 mg / L, leading to physiological shock due to direct translocation across this threshold; and the delayed activation of the root phenylpropanoid metabolic pathway compared to the stem, with high copper stress further inhibiting the expression of related enzymes. Overcoming these difficulties requires breakthroughs in establishing quantitative models of the relationship between copper ion concentration and multiple objectives, developing methods for gradual root stress adaptation, and targeted enhancement of root flavonoid synthesis efficiency without exacerbating growth inhibition. Summary of the Invention

[0008] The problem is that a single copper ion concentration cannot simultaneously address the rooting rate and secondary metabolite accumulation of tissue culture seedlings. Although 0.025 mg / L copper increases the rooting rate, the flavonoid content in roots / stems is only 40-60% of that in the 0.075 mg / L high copper ion concentration group. On the other hand, although the 0.075 mg / L high copper concentration increases the flavonoid content, the rooting rate drops sharply by more than 40%. Therefore, there is an urgent need to establish a dynamic adaptation mechanism between concentration and culture objectives.

[0009] To achieve the objective of this invention, a method for promoting rooting of *Ketora macrocarpa* tissue culture seedlings using copper is provided, comprising:

[0010] Step 1: Select leafy konjac stem segments as tissue culture material;

[0011] Step 2: Using 1 / 2 MS as the basal medium, add 0.1 mg / L NAA and copper ions at a specific concentration of 0.025 mg / L~0.075 mg / L to the tissue culture material for rooting culture; the copper concentration must meet the following conditions:

[0012] When the goal is to maximize rooting rate, the copper ion concentration is 0.025 mg / L; when the goal is to maximize root / stem flavonoid content, the copper ion concentration is 0.05–0.075 mg / L.

[0013] Cultivating at 25℃ and under 12 h / d light for 25–35 days induces simultaneous root development and accumulation of secondary metabolites.

[0014] Different copper source chemical forms may affect the bioavailability of ions through differences in solubility. Therefore, preferably, the copper element of the present invention is added in the form of copper sulfate or copper chloride.

[0015] The inconsistent physiological age of stem segments may lead to differences in rooting rate. Therefore, the preferred stem segment of the large-leaved konjac of the present invention is the 2nd to 4th segment below the terminal bud.

[0016] Leafless stem segments may lead to delayed rooting, while too many leaves can cause water loss through transpiration. Therefore, it is necessary to optimize the balance between photosynthesis and water. Preferably, the large-leaved konjac stem segments with leaves in this invention retain 1–2 complete leaves.

[0017] Preferably, in step 2 of the present invention, when the goal is to simultaneously achieve a high rooting rate of ≥90% and a high root flavonoid content of ≥9.0%, the following steps are performed:

[0018] Step 21, in the first stage from day 0 to 15, the leafy konjac stem segments were placed in a rooting medium with a copper ion concentration of 0.025 mg / L and the rooting medium contained 1 / 2 MS and 0.1 mg / L NAA. The culture conditions were 25℃ and 12h / d light.

[0019] Step 22, during the transition phase on day 15, the tissue culture seedlings are transferred to a three-layer gradient buffer medium, which is constructed layer by layer from top to bottom:

[0020] Upper culture medium: 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L copper ions + 0.7% (w / v) agar;

[0021] Intermediate medium: 1 / 2 MS + 0.1 mg / L NAA + 0.05 mg / L copper ions + 0.005–0.01 mg / L phenylalanine + 0.7% (w / v) agar;

[0022] Lower layer culture medium: 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L copper ions + 0.7% (w / v) agar;

[0023] The cultivation conditions are 25℃ and a dark environment;

[0024] Step 23, in the second stage from day 16 to 30, the tissue culture seedlings were transferred to a final medium containing 0.075 mg / L copper ions + 0.005 mg / L salicylic acid. The final medium contained 1 / 2 MS and 0.1 mg / L NAA. The culture conditions were 25°C and 12 h / d light.

[0025] This embodiment addresses the core technical problem 1: stress damage caused by a sudden increase in copper ion concentration. If the concentration is directly transferred from a low copper ion concentration of 0.025 mg / L to a high copper ion concentration of 0.075 mg / L, exceeding the root tolerance threshold, excessive concentration can lead to root malformation or apoptosis. This embodiment sets up a copper ion gradient buffer layer, using a physical stratification design with concentration gradients of 0.025, 0.05, and 0.075 mg / L, allowing the roots to gradually adapt to the high copper environment over 12 hours. Table 1 of this invention shows that 0.05 mg / L is an intermediate safe concentration. Furthermore, the addition of quercetin precursors, using phenylalanine as a flavonoid synthesis precursor, pre-activates metabolic pathways and shortens the high copper induction period. Addressing the core technical problem 2: insufficient flavonoid synthesis efficiency under high copper conditions, Table 4 of this invention shows that although a high copper ion concentration of 0.075 mg / L results in 9.3% flavonoids in the roots, it is lower than the 12.8% in the stems, indicating that the root synthesis capacity is weaker than that of the stems. This embodiment incorporates salicylic acid as a synergist, adding 0.005 mg / L of salicylic acid to stimulate root defense responses and directionally enhance the efficiency of root flavonoid synthesis.

[0026] Preferably, the pouring sequence of the three-layer gradient buffer culture medium is as follows: the lower layer culture medium is cooled to 40°C and aseptically poured into the culture container, with a thickness of 8-10 mm, and allowed to stand at room temperature of 20-25°C until completely solidified; the middle layer culture medium is cooled to 45°C and poured onto the surface of the solidified lower layer culture medium, with a thickness of 4-5 mm, and allowed to stand at room temperature of 20-25°C until semi-solidified; the upper layer culture medium is cooled to 50°C and slowly poured along the container wall onto the semi-solidified middle layer culture medium, with a thickness of 5-6 mm, and allowed to stand at room temperature of 20-25°C until all three layers are completely solidified.

[0027] In this embodiment, tissue culture seedlings are placed in the uppermost low-copper-ion concentration zone. As the roots grow downwards, they are sequentially exposed to copper ion concentration gradients of 0.025, 0.05, and 0.075 mg / L, achieving gradual stress adaptation. The lower layer is first filled with a low-temperature medium at 40°C, followed by a higher-temperature medium at 50°C to prevent the bottom layer from being dispersed by the hot fluid before solidification, thus improving the stability of the medium. The middle layer is filled at 45°C to protect phenylalanine, and the semi-solid state ensures that the active molecules are located in the main root growth zone. "Semi-solid state" refers to a surface capable of supporting a weight of 0.5 g / cm³. 2 (It does not collapse when lightly touched with a glass rod with a diameter of 1mm).

[0028] Preferably, in the transition phase of step 22, the concentration of phenylalanine in the intermediate layer culture medium is 0.005 mg / L; after the tissue culture seedlings are transferred to the three-layer gradient buffer medium, they are first cultured in the dark at 25°C for 6 hours to promote phenylalanine pre-absorption; then, combined induction with ultraviolet and blue light is performed, first with 295 nm ultraviolet light at an intensity of 4.5 W / m 2 Irradiate for 10 minutes, then use 450 nm blue light at an intensity of 30 μmol•m-2 •s -1 Irradiate for 20 minutes; continue culturing in the dark at 25℃ for the remaining 4 hours. In this embodiment, as shown in Table 4 of the instruction manual, at the same copper ion concentration, the root flavonoid content (5.1%~9.3%) is significantly lower than that of the stem flavonoid content (5.3%~19.1%), indicating that the root system's ability to transform precursors is weaker than that of the stem. This may be because exogenous phenylalanine needs to be transported to the roots via the vascular system, but the roots of tissue culture seedlings are immature. As shown in Table 1, the average root length is only 18~28 mm, resulting in insufficient precursor utilization. To address this problem, this embodiment uses photoinduction to precisely solve the root metabolic defects. 295 nm ultraviolet light (UV) penetrates the surface of the culture medium to directly stimulate root epidermal cells, inducing increased PAL enzyme activity and accelerated endogenous phenylalanine synthesis, reducing exogenous transport losses. 450 nm blue light activates photoreceptors, promoting the expression of chalcone synthase (CHS) genes related to flavonoid synthesis, and copper ions accumulate in the enzyme active site (copper is a CHS cofactor).

[0029] Preferably, before the transition phase in step 22 ends and before step 23 begins, the following treatment is performed: a) Immerse the roots of the tissue culture seedlings in a 0.1% (w / v) activated carbon suspension for 5 minutes; b) Rinse the roots three times with sterile water; c) Immediately transfer them to the final culture medium. Because the roots of the tissue culture seedlings are attached with a high concentration of copper ions, these ions are continuously released after transfer to the final culture medium, easily causing local concentration exceedances. This embodiment uses activated carbon treatment after the transition phase and before transfer to the final culture medium, which does not interfere with the photoinduction phase. Activated carbon can adsorb free copper ions, avoiding copper ion toxicity stress, and the short 5-minute treatment does not affect the total 30-day culture cycle.

[0030] Preferably, when the goal is to maximize stem flavonoid content, the following steps are performed: On day 20 of cultivation, a 0.05 mg / L copper ion solution is sprayed onto the surface of the stems and leaves of *Ketrapandia macrophylla*, with a spraying volume of 0.1 mL / plant (calculated as CuSO4·5H2O); cultivation continues at 25°C under 12 h / d light conditions until day 30. In this embodiment, Table 4 of the specification shows that the stem flavonoid content reaches 16.8% at a copper ion concentration of 0.05 mg / L, and 12.8% in the 0.075 mg / L group. The specification indicates that 0.05 mg / L is the optimal concentration for stem flavonoid synthesis. This invention, through foliar spraying of a 0.05 mg / L copper ion solution, allows copper to be absorbed into the vascular bundles through stomata / epidermis, directionally enhancing stem metabolism. Furthermore, the spraying on day 20 falls in the mid-to-late stage of the 30-day cultivation period, perfectly aligning with the period of vigorous secondary metabolism, while the synthesis of flavonoid active ingredients is enhanced in the mid-to-late growth stage.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. This invention, through dual-target concentration regulation, maintains a high rooting rate while increasing the accumulation of flavonoids in rhizomes to more than twice that of the low copper ion concentration group, breaking through the trade-off between rooting and metabolism under a single concentration, improving the biomass uniformity of tissue culture seedlings and increasing the batch qualification rate.

[0033] 2. The gradient buffer culture medium of this invention allows roots to gradually adapt to a high-copper environment within 12 hours, reducing the root tip browning rate to below 5%. Photoinduction increases phenylalanine uptake efficiency to 3.5 times that of conventional dark culture, significantly increases the accumulation of flavonoid synthesis precursors, and shortens the metabolic activation lag period. Activated carbon treatment removes adsorbed copper ions from the root surface, preventing local concentration exceedances after transfer to the final culture medium, and reducing root oxidative damage markers to below the detection limit.

[0034] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0035] Figure 1 The rooting status of *Ketora macrocarpa* after treatment in each of the embodiments;

[0036] Figure 2 The water content of the rhizome of *Ketora macrocarpa* after treatment in each embodiment;

[0037] Figure 3 The chlorophyll content of *Ketora macrocarpa* after treatment in each of the embodiments is shown. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0039] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0040] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0041] Example 1: Low copper ion concentration group (0.0125 mg / L Cu) 2+ )

[0042] A method for promoting rooting of *Ketora macrocarpa* tissue culture seedlings using copper includes:

[0043] Step 1, prepare rooting medium: 1 / 2 MS + 0.1 mg / L NAA + 0.0125 mg / L Cu 2++ 0.7% (w / v) agar, pH 5.8. Among them, Cu 2+ Derived from copper sulfate pentahydrate, the same applies below.

[0044] Step 2: Inoculate the leafy stem segments into the above-mentioned rooting medium and culture them for 30 days at 25°C and 12 hours of light per day.

[0045] Results: The rooting rate reached 80.9%, with an average of 15.91 roots per plant. The root system was well developed, but the accumulation of secondary metabolites was low, with root flavonoid content at 2.1% and stem flavonoid content at 6.2%.

[0046] Example 2: Medium copper ion concentration group (0.025 mg / L Cu) 2+ )

[0047] A method for promoting rooting of *Ketora macrocarpa* tissue culture seedlings using copper includes:

[0048] Step 1, prepare rooting medium: 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L Cu 2+ + 0.7% (w / v) agar, pH 5.8.

[0049] Step 2, the cultivation conditions and methods are the same as in Example 1.

[0050] Results: The rooting rate reached a maximum of 91.7%, with an average root area of ​​63.81 mm. 2 This indicates that this concentration is most favorable for initial root development and root formation. However, flavonoid accumulation is still not ideal, with root flavonoid content at 4.9% and stem flavonoid content at 5.3%.

[0051] Example 3: Medium-high copper ion concentration group (0.05 mg / L Cu) 2+ )

[0052] A method for promoting rooting of *Ketora macrocarpa* tissue culture seedlings using copper includes:

[0053] Step 1, prepare rooting medium: 1 / 2 MS + 0.1 mg / L NAA + 0.05 mg / L Cu 2+ + 0.7% (w / v) agar, pH 5.8.

[0054] Step 2, the cultivation conditions and methods are the same as in Example 1.

[0055] Results: The rooting rate decreased to 55.5%. Notably, the stem flavonoid content reached a peak of 16.8%, indicating that this concentration was particularly favorable for the accumulation of secondary metabolites in the stem. The root flavonoid content was 8.6%, and the average root length was 24.40 mm.

[0056] Example 4: High copper ion concentration group (0.075 mg / L Cu) 2+ )

[0057] A method for promoting rooting of *Ketora macrocarpa* tissue culture seedlings using copper includes:

[0058] Step 1, prepare rooting medium: 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L Cu 2+ + 0.7% (w / v) agar, pH 5.8.

[0059] Step 2, the cultivation conditions and methods are the same as in Example 1.

[0060] Results: The rooting rate was low at 54.5%, and the roots showed signs of growth inhibition or stress. The fresh root weight was also low at 4.84 g. However, the root flavonoid content reached a high of 9.3%, indicating that high copper ion concentration strongly induced root flavonoid synthesis. The stem flavonoid content was 12.8%.

[0061] Comparative Example 1: Copper-free control group

[0062] The steps include:

[0063] Step 1, prepare the rooting medium: containing only 1 / 2 MS + 0.1 mg / L NAA + 0.7% (w / v) agar, pH 5.8, without adding any copper.

[0064] The cultivation conditions and methods are the same as in Example 1.

[0065] Results: All indicators were poor, with the lowest rooting rate at only 33.3%, an average of only 4.56 roots per plant, and large variation in root length (sample variance 20.65). The root flavonoid content was 5.1%, and the stem flavonoid content was 19.1%, these abnormally high values ​​may be due to basal stress response, but overall plant growth was poor. This control group clearly demonstrates the necessity of copper for normal rooting and growth of *Ketora macrocarpa* tissue culture seedlings.

[0066] The complete effect experiments and data of the above embodiments and comparative examples are as follows:

[0067] Determination of chlorophyll content and total flavonoid content in *Ketora macrocarpa*

[0068] Tissue culture seedlings of *Ketrapandia macrophylla* were cultivated using the methods described in Examples 1-4 and Comparative Example 1, respectively. Agronomic traits of *Ketrapandia macrophylla* were measured, including rooting rate, fresh root weight, dry root weight, average total root length, average total root surface area, and average number of roots. Rooting rate = (number of rooted seedlings / total number of inoculated individual seedlings) × 100%. Fresh (dry) root weight = total fresh (dry) weight of roots from randomly selected plants / number of randomly selected plants. Average root length = total root length of randomly selected plants / number of randomly selected plants. Fresh (dry) plant weight = total fresh (dry) weight of randomly selected plants / number of randomly selected plants. Dry plant weight and dry root weight were calculated by drying the harvested plants or roots at 40℃ to constant weight. Average total root length = total root length of randomly selected plants / number of randomly selected plants. The results are shown in Tables 1-2. Chlorophyll and total flavonoid content were also measured, and the results are shown in Tables 3-4. Figure 1 In the diagram, 1 corresponds to Example 1, 2 to Example 1, 3 to Example 2, 4 to Example 3, and 5 to Example 4; each indicator is expressed as mean ± standard deviation, n=3; different lowercase letters in each treatment group indicate significant differences in content between different treatments (P<0.05).

[0069] Table 1

[0070]

[0071] Table 2

[0072]

[0073] Table 3

[0074]

[0075] Table 4

[0076]

[0077] From Table 1 and Figure 1 As can be seen, the concentration of exogenous copper ions significantly affects the rooting of *Ketora chinensis* tissue culture seedlings. In the 0 mg / L group, the average number of roots was only 4.56 per plant, with a rooting rate of 33.3% and an average root length of 28.29 mm. However, the sample variance of root length was as high as 20.65, indicating uneven root development. In the 0.0125 mg / L group, the average number of roots increased to 15.91 per plant, and the rooting rate improved to 80.9%, but the average root length decreased to 18.39 mm, indicating that copper promotes root growth but inhibits initial elongation. In the 0.025 mg / L group, the average number of roots was 14.57 per plant, slightly lower than the 0.0125 mg / L group, with a peak rooting rate of 91.7%, and the average root area decreased to 63.81 mm². 2This reflects a more compact root system. In the 0.05 mg / L group, the average number of roots sharply decreased to 8.32 per plant, the rooting rate dropped to 55.5%, and the average root length was 24.40 mm, indicating that high copper levels began to inhibit rooting. In the 0.075 mg / L group, the average number of roots was 11.46 per plant, the rooting rate was 54.5%, and the average root area further decreased to 65.41 mm². 2 This indicates that high copper levels continuously inhibit root development. Therefore, 0.025 mg / L is the optimal rooting concentration, achieving a rooting rate of 91.7%.

[0078] Table 2 and Figure 2 The study showed a dual effect of copper ion concentration on biomass. In the 0 mg / L group, the stem fresh weight was only 3.86 g, indicating severely limited growth, while the root water content was as high as 93.12%, with low metabolic activity. In the 0.0125–0.025 mg / L groups, the stem / root fresh weight increased significantly, from 14.71 g to 15.83 g, and from 6.43 g to 6.66 g, with water content remaining stable above 90%, indicating healthy growth. In the 0.05 mg / L group, the stem fresh weight decreased slightly to 14.42 g, but the root fresh weight plummeted to 4.41 g, possibly indicating that the roots were more sensitive to high copper levels. The stem water content rose to 91.40%, possibly accompanied by mild stress. In the 0.075 mg / L group, the stem fresh weight reached a peak of 20.55 g, with high copper promoting stem biomass accumulation, but the root fresh weight was only 4.84 g, significantly lower than the medium-low copper groups, and the root water content dropped to 89.88%, indicating intensified stress. Therefore, when the copper ion concentration is >0.05 mg / L, root growth is significantly inhibited, indicating that high copper concentrations suppress rooting.

[0079] Table 3 and Figure 3 Data on chlorophyll content showed that chlorophyll (SPAD value) responded to the "low-promoting, high-inhibiting" effect of copper ion concentration. In the low copper groups (0 and 0.0125 mg / L), the average chlorophyll values ​​were 41.35 and 40.17, respectively, with no significant difference, indicating that copper concentration had little effect on chlorophyll at this range. In the medium copper groups (0.025 and 0.05 mg / L), chlorophyll significantly increased to 50.62 and 54.43, respectively, showing that copper concentration had a significant impact on chlorophyll synthesis, indicating that copper promotes photosynthetic pigment synthesis through enzyme cofactor action. In the high copper group (0.075 mg / L), the average chlorophyll value decreased to 38.25, indicating that excessive copper damaged chloroplast structure. Therefore, the optimal copper ion concentration for chlorophyll synthesis is 0.025–0.05 mg / L, also demonstrating the dual effect of copper ion concentration.

[0080] Table 4 shows that the total flavonoid content exhibits organ-specific accumulation. Root flavonoids continuously increase with increasing copper ion concentration, reaching a peak of 9.3% in the 0.075 mg / L group, indicating that copper directly induces secondary metabolism in roots. The optimal content of stem flavonoids is 16.8% in the 0.05 mg / L group; however, it decreases to 12.8% in the 0.075 mg / L group, indicating that excessive copper inhibits synthesis efficiency. The copper-free group, at 19.1%, is abnormally high, possibly due to a growth stress response, indicating an unhealthy state. Therefore, the optimal concentrations for maximizing root flavonoids are 0.075 mg / L and for stem flavonoids are both 0.05 mg / L, showing different optimal concentrations. The high flavonoid content in the copper-free group is unsustainable, further demonstrating that a single concentration cannot simultaneously address both root development and medicinal component issues.

[0081] Based on the above experimental results, the scheme was further optimized, and the following embodiments were carried out.

[0082] Example 5

[0083] A method for promoting rooting of *Ketora chinensis* tissue culture seedlings using copper employs a three-stage gradient culture method to simultaneously optimize rooting rate and root flavonoid accumulation, including:

[0084] The first stage, from day 0 to 15, is for rooting initiation. The culture medium is: 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L Cu. 2+ Rooting initiation culture was initiated using 0.7% (w / v) agar at 25℃ under 12 h / d light conditions, with an optimal rooting concentration of 0.025 mg / L to promote efficient root development. The rooting rate reached over 90% by the end of this stage.

[0085] The transition phase lasted approximately 12 hours on day 15 for stress adaptation. A three-layer gradient buffer culture medium was constructed and poured from bottom to top to ensure physical stratification stability. The lower layer of medium was cooled to 40°C before pouring and contained 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L Cu. 2+ + 0.7% (w / v) agar, 8-10 mm thick, let stand until completely solidified. Cool the middle layer of medium to 45°C and pour it onto the solidified lower layer of medium, containing 1 / 2 MS + 0.1 mg / L NAA + 0.05 mg / L Cu. 2+ Add 0.005 mg / L phenylalanine and 0.7% (w / v) agar to a thickness of 4-5 mm, and let it stand until semi-solid to facilitate root contact with the active molecules. Cool the upper culture medium to 50°C and slowly pour it along the wall onto the semi-solid middle culture medium, containing 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L Cu. 2+ + 0.7% (w / v) agar, 5-6 mm thick, let stand until the three layers are completely cured.

[0086] Carefully transfer the tissue culture seedlings that have completed the first stage to the upper layer of this three-layer gradient buffer medium.

[0087] Then, light-induced treatment was performed in a dark environment. First, the cells were cultured at 25°C in the dark for 6 hours to promote the pre-absorption of phenylalanine from the intermediate culture medium by the roots. Phenylalanine is a precursor for flavonoid synthesis. Next, combined light-induced treatment was performed, first using 295 nm ultraviolet light at an intensity of 4.5 W / m². 2 Irradiation for 10 minutes penetrates and stimulates the root epidermis, inducing PAL enzyme activity; then 450 nm blue light at an intensity of 30 μmol·m⁻¹ is applied. -2 ·s -1 Irradiation for 20 minutes activates photoreceptors and promotes the expression of chalcone synthase (CHS), with copper as a cofactor. Finally, the cells are cultured in the dark at 25°C for the remaining 4 hours.

[0088] After the transition phase and before the start of the second phase, activated carbon treatment is performed by carefully immersing the roots of the tissue culture seedlings in a 0.1% (w / v) activated carbon suspension for 5 minutes to adsorb excess free copper ions on the root surface. The roots are then thoroughly rinsed three times with sterile water.

[0089] The second phase, from days 16 to 30, involved fortifying flavonoid synthesis using a culture medium of 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L Cu. 2+ + 0.005 mg / L salicylic acid + 0.7% (w / v) agar, cultured at 25℃ with 12 h / d light. By transferring the roots to a high copper environment of 0.075 mg / L, salicylic acid was used to stimulate a defense response, synergistically with photoinduced pre-activated metabolic pathways, to directionally and efficiently synthesize root flavonoids.

[0090] Table 5

[0091]

[0092] Note: Root flavonoid yield = fresh root weight × root flavonoid content.

[0093] As shown in Table 5, traditional single copper ion concentration culture faces fundamental limitations. While a high rooting rate of 91.7% can be achieved using 0.025 mg / L copper, the root flavonoid content only stagnates at 4.9%. When the copper ion concentration is increased to 0.075 mg / L, although the root flavonoid content reaches 9.3%, the rooting rate drops sharply to 54.5%. Example 5, employing a gradient culture method with three-stage regulation, achieves a simultaneous leap in both objectives for the first time. The rooting rate breaks through to 94.2%, an increase of 2.5% compared to the single optimal group, Example 2, and the root flavonoid content jumps to 10.5%, surpassing the single high copper group, Example 4, by 12.9%. This means that within the same culture period, both healthy and well-developed root systems and plants rich in medicinal components are obtained simultaneously, solving the core bottleneck restricting the industrial-scale seedling cultivation of *Ketora macrocarpa*.

[0094] The toxic damage to roots caused by high copper ion concentrations has been a long-standing problem. In a single culture at 0.075 mg / L, 63.2% of the roots exhibited severe stress symptoms such as root tip browning and elongation arrest, resulting in less than 40% of the roots having absorption function. Example 5, using a gradient culture method with three layers of copper ion concentration buffer media (0.025, 0.05, and 0.075 mg / L), combined with activated carbon ion scavenging technology in the transition phase, completely eliminated the high copper stress effect. The root tip browning rate dropped to 0%, and the average root length recovered from 18.6 mm in the damaged state to a healthy 28.3 mm, with a functional root ratio as high as 89%. More importantly, while eliminating stress, the fresh root weight reached 7.8 g, a 61.2% increase compared to 4.84 g in Example 4, resulting in a single-plant root flavonoid yield of 819 mg, an 82% increase compared to the single high-copper group.

[0095] Example 5's breakthrough stems from precise intervention in secondary metabolic pathways. During the transition phase, combined irradiation with 295 nm ultraviolet light and 450 nm blue light increased the conversion efficiency of phenylalanine (a precursor to flavonoid synthesis) in the roots by 3.2 times. The addition of 0.005 mg / L salicylic acid to the final culture medium further activated PAL (phenylalanine ammonia-lyase), a key enzyme in the defense response. Simultaneously, the gradient stress adaptation mechanism maintained the activity of superoxide dismutase (SOD), ensuring a synergistic effect of metabolic activity and oxidative balance at the molecular level. This method, combining stress adaptation, metabolic activation, and damage protection, not only enabled *Piper kadsura* tissue culture seedlings to simultaneously achieve the dual industrial standards of a 94.2% rooting rate and 10.5% root flavonoid content within 30 days, but also boosted the yield of medicinal substances per plant to 819 mg, providing an irreplaceable solution for establishing an industrialized seedling production paradigm for medicinal Piperaceae plants.

[0096] Example 6

[0097] A method for promoting rooting of *Ketora chinensis* tissue culture seedlings using copper, employing foliar spraying to maximize stem flavonoids, includes:

[0098] During basal culture from day 0 to day 20, the culture medium and conditions of Example 3 were used, which was 1 / 2 MS + 0.1 mg / L NAA + 0.05 mg / L Cu. 2+ + 0.7% (w / v) agar, cultured at 25°C under 12 h / d light. Utilizing 0.05 mg / L Cu 2+ At the specified concentration, stem flavonoid accumulation was optimal, and after basal culture up to day 20, it entered a period of vigorous secondary metabolism.

[0099] Foliar treatment was performed on day 20, using a 0.05 mg / L Cu solution. 2+ Solution, Cu foliar spray 2+ Source: CuSO4·5H2O. Apply using a sterile sprayer, spraying 0.1 mL per plant onto the stem and leaf surface of *Ketrapandia macrophylla* tissue culture seedlings, avoiding direct spraying of the roots and culture medium, and ensuring uniform droplet adhesion. This procedure is performed in a sterile operating room. Through stomata and epidermal absorption, copper ions directly act on the stem and leaf tissues, further stimulating secondary metabolic pathways in the stem.

[0100] Continue culturing from day 20 to 30. Return the sprayed tissue culture seedlings to their original culture conditions of 25℃ and 12 h / d light, and continue culturing until day 30.

[0101] Table 6

[0102]

[0103] Note: Stem flavonoid yield = stem dry weight × stem flavonoid content; copper absorption efficiency is the cumulative amount of copper ions per unit stem dry weight.

[0104] As can be seen from Table 6, Example 6, through targeted delivery of copper ions on the leaves, increased the stem flavonoid content to 18.7% while maintaining root health. Because the spraying simultaneously promoted the accumulation of stem biomass, the dry weight increased significantly, and the stem flavonoid yield per plant reached 351.8 mg, which is 68.9% higher than that of Example 3, equivalent to extracting 1.43 kg more high-purity flavonoids per 10,000 seedlings.

[0105] Foliar spraying allows copper ions to be directly absorbed into the vascular bundles of the stem through stomata / epidermis, achieving a stem copper absorption efficiency of 8.3 μg / g DW, thus avoiding the utilization loss caused by copper fixation in the soil / culture medium. Targeted spraying ensures that copper ions do not come into contact with the culture medium, and the root flavonoid content of 8.5% is not significantly different from 8.6% in Example 3, completely avoiding the inhibitory effect of high copper on the root system.

[0106] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for promoting rooting of tissue-cultured seedlings of *Ketora macrocarpa* using copper, characterized in that, include: Step 1: Select leafy konjac stem segments as tissue culture material; Step 2: Using 1 / 2 MS as the basal medium, add 0.1 mg / L NAA and copper ions at a specific concentration of 0.025 mg / L-0.075 mg / L to the tissue culture material for rooting culture. The copper concentration should meet the following conditions: when the goal is to maximize the rooting rate, the copper ion concentration is 0.025 mg / L; when the goal is to maximize the root / stem flavonoid content, the copper ion concentration is 0.05-0.075 mg / L. Cultivating at 25℃ and under 12 h / d light for 25–35 days induces simultaneous root development and accumulation of secondary metabolites; In step 2, when the goal is to simultaneously achieve a high rooting rate and a high root flavonoid content, follow these steps: Step 21: In the first stage from day 0 to 15, the leafy konjac stem segments were placed in rooting medium containing 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L copper ions and cultured under the following conditions: 25°C and 12 h / d light. Step 22, during the transition phase on day 15, the tissue culture seedlings are transferred to a three-layer gradient buffer medium, which is constructed layer by layer from top to bottom: Upper culture medium: 1 / 2 MS + 0.1 mg / L NAA + 0.025 mg / L copper ions + 0.7% (w / v) agar; Intermediate medium: 1 / 2 MS + 0.1 mg / L NAA + 0.05 mg / L copper ions + 0.005-0.01 mg / L phenylalanine + 0.7% (w / v) agar; Lower layer culture medium: 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L copper ions + 0.7% (w / v) agar; The cultivation conditions are 25℃ and a dark environment; Step 23, in the second stage from day 16 to 30, the tissue culture seedlings were transferred to the final medium consisting of 1 / 2 MS + 0.1 mg / L NAA + 0.075 mg / L copper ions + 0.005 mg / L salicylic acid, and cultured at 25°C with a light intensity of 12 h / d.

2. The method according to claim 1, characterized in that, Copper ions are added in the form of copper sulfate or copper chloride.

3. The method according to claim 1, characterized in that, The stem segment of large-leaved konjac is the 2nd to 4th segment below the terminal bud.

4. The method according to claim 1, characterized in that, Large-leaved konjac stem segments with leaves should retain 1-2 intact leaves.

5. The method according to claim 1, characterized in that, The pouring sequence of the three-layer gradient buffer culture medium is as follows: the lower layer culture medium is cooled to 40°C and aseptically poured into the culture container, with a thickness of 8-10 mm, and allowed to stand at room temperature of 20-25°C until completely solidified; the middle layer culture medium is cooled to 45°C and poured onto the surface of the solidified lower layer culture medium, with a thickness of 4-5 mm, and allowed to stand at room temperature of 20-25°C until semi-solidified; the upper layer culture medium is cooled to 50°C and slowly poured along the container wall onto the semi-solidified middle layer culture medium, with a thickness of 5-6 mm, and allowed to stand at room temperature of 20-25°C until all three layers are completely solidified.

6. The method according to claim 1, characterized in that, Before starting step 23, perform the following treatments: a) Immerse the roots of the tissue culture seedlings in 0.1% (w / v) activated carbon suspension for 5 minutes; b) Rinse the roots 3 times with sterile water; c) Immediately transfer them to the final culture medium.

7. The method according to claim 6, characterized in that, When the goal is to maximize the content of stem flavonoids, the following steps are performed: On the 20th day of cultivation, a 0.05 mg / L copper ion solution is sprayed onto the surface of the stems and leaves of the large-leaved konjac at a rate of 0.1 mL / plant; cultivation continues at 25°C and 12 h / d light until the 30th day.

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