A method for breeding and transplanting of in vitro cultured seedlings of brazilian ginseng

Abstract

The application discloses a kind of propagation and efficient transplanting method of Brazilian ginseng tissue culture seedlings, belong to plant tissue culture and medicinal plant seedling technology field.The method aims to solve the technical problem of low and unstable survival rate of tissue culture seedlings in the process of conventional transplanting due to sudden change of environment, weak root adaptability and easy infection of diseases and pests.Its technical scheme points are: first, MS is added to the specific concentration 6-BA and NAA to start induction and subculture multiplication culture, and obtain robust cluster buds;Then, it is transferred to WPM medium and the hormone ratio is adjusted for rooting culture, and the developed adventitious roots are induced;Finally, when the root system reaches the appropriate length, through a series of operations such as cover adaptation exercise, carbendazim solution disinfection, pure vermiculite substrate transplanting and simplified post-planting management, the smooth transition of tissue culture seedlings to the external environment is realized.The method is mainly used for large-scale, efficient factory seedling production of Brazilian ginseng, and can significantly improve the survival rate of transplanting and seedling quality.

Description

Technical Field

[0001] This invention relates to the fields of plant tissue culture and medicinal plant seedling cultivation. More specifically, this invention relates to a method for the propagation and efficient transplanting of Brazilian ginseng tissue culture seedlings. Background Technology

[0002] Brazilian ginseng ( Pfaffia paniculata *Amaranthus fulvidraco*, also known as fafia, is a perennial herbaceous plant belonging to the Amaranthaceae family. Its roots are used medicinally and are rich in active ingredients such as saponins, oleanolic acid, various amino acids, and trace elements. It possesses multiple pharmacological effects, including anti-tumor, anti-inflammatory, fatigue-relieving, and immune-enhancing properties, and has broad market development prospects. This plant is native to South America, and China successfully introduced it at the beginning of this century. However, its wild resources are increasingly depleted due to overexploitation, and its natural seed setting rate is low, seed germination is difficult, and traditional propagation methods cannot meet the seedling needs of large-scale production. Therefore, using plant tissue culture technology for rapid propagation has become an important research direction for solving the problem of its seedling source.

[0003] In tissue culture research on Brazilian ginseng, techniques have been explored for in vitro propagation using different culture media and plant growth regulators. Related studies have shown that suitable basic culture media and hormone ratios are crucial for obtaining regenerated plants. For example, research such as the establishment of a rapid tissue culture propagation system for Brazilian ginseng (Jiang Hua, *Journal of West China Forestry Science*, Vol. 50, No. 1, Feb. 2021) indicates that WPM medium has a certain effect on bud induction in Brazilian ginseng, and MS medium is also commonly used as a basic medium. However, existing technologies still face some systemic technical challenges in transitioning to stable and efficient industrialized seedling production.

[0004] First, in the in vitro culture stage of tissue culture seedlings, there is a challenge in adapting the culture system due to the different requirements of the culture medium at different physiological stages. The main goal of the initiation and subculture proliferation stages is to obtain a large number of robust shoot clusters, while the rooting stage requires efficient induction of well-developed adventitious roots. Studies have found that a single culture medium formulation often fails to achieve optimal results for both goals simultaneously. For example, some culture media that perform well in promoting shoot proliferation may not be ideal for inducing rooting; while some formulations that promote rooting may have relatively low proliferation efficiency. This differentiated requirement for nutritional and hormonal environments at different culture stages complicates the establishment of a coherent and efficient culture process from initiation and proliferation to rooting, increasing the difficulty of technical optimization.

[0005] Secondly, in the crucial hardening-out and transplanting stage of tissue culture seedlings from the sterile environment of sterile bottles to outdoor cultivation, existing technologies lack systematic and quantifiable process parameters. The success of hardening-out and transplanting directly determines the final outcome of the preceding tissue culture work. In practice, the timing of hardening-out, the environmental acclimatization of seedlings before transplanting (such as opening the container for hardening), the selection of transplanting substrate and container, and the initial management after transplanting are all key factors affecting the survival rate. However, existing technical materials often vaguely mention the need for hardening-out or only provide individual parameters based on specific conditions, failing to form a complete set of parameterized and validated operational guidelines. This leads to large fluctuations in hardening-out survival rates and poor repeatability in production practice, becoming a bottleneck for large-scale production. Summary of the Invention

[0006] This invention aims to solve the technical challenges of low and unstable survival rates in the entire process of Brazilian ginseng tissue culture seedling production, from in vitro culture to transplanting and planting. These challenges stem from poor coordination between the culture stage and the hardening-off stage, unclear process parameters, and a lack of systematic adaptation solutions. Specifically, in existing technologies, highly efficient bud proliferation media often result in poor rooting, while media that promote good rooting may have low proliferation efficiency. Furthermore, the lack of clear and synergistically optimized parameters for hardening-off timing, acclimatization methods, and transplanting conditions makes it difficult for the cultured seedlings to adapt to the external environment, thus limiting the efficiency and reliability of large-scale seedling production.

[0007] To achieve the above objectives, this invention provides a method for the propagation and efficient transplanting of Brazilian ginseng tissue culture seedlings, comprising the following steps: Step 1, using MS as the basic culture medium and adding 6-BA and NAA, initiation induction culture and subculture proliferation culture are performed to obtain clustered shoots; Step 2, using WPM as the basic culture medium and adjusting the ratio of 6-BA and NAA, the clustered shoots are subjected to rooting culture to obtain regenerated plants; Step 3, when the average length of the adventitious roots of the regenerated plants reaches 2.5 cm to 3.5 cm, open-top adaptation culture is started; Step 4, the plants are removed, the culture medium attached to the roots is washed off, and disinfection treatment is performed before transplanting; Step 5, the disinfected plants are planted in pure vermiculite substrate; Step 6, after transplanting, the substrate is kept moist, and post-planting management is carried out without the application of other chemical pesticides.

[0008] Preferably, in step 1, the added 6-BA concentration is 1.0-2.0 mg / L and the NAA concentration is 0.1-0.3 mg / L; in step 2, the added 6-BA concentration is 0.05-0.2 mg / L and the NAA concentration is 0.3-0.5 mg / L.

[0009] Preferably, in step 1, the concentration of 6-BA is 1.5 mg / L and the concentration of NAA is 0.2 mg / L; in step 2, the concentration of 6-BA is 0.1 mg / L and the concentration of NAA is 0.4 mg / L; in step 3, the adventitious roots are opened for adaptation training when the average length reaches 3 cm; in step 5, pure vermiculite is filled into the 32-well plate.

[0010] Preferably, the opening adaptation training in step 3 includes: initially covering the mouth of the culture bottle with a sterile breathable membrane to reduce the relative humidity inside the bottle to 75%-85% within 12 hours; then removing the breathable membrane, controlling the relative humidity of the environment between 65%-75% through intermittent spraying, while increasing the light intensity to 3000-4000 lux, and continuing the training for 1.5-2.5 days.

[0011] Preferably, in step 6, post-planting management, carbendazim-resistant Trichoderma harzianum is introduced for biocontrol. Specifically, before transplanting, a carbendazim-resistant Trichoderma harzianum spore suspension is mixed with a pure vermiculite substrate to achieve a viable bacterial content of (2-5) × 10⁻⁶. 6 CFU / g; When transplanting, immerse the plant roots in a 0.1% carbendazim solution containing carbendazim-resistant Trichoderma harzianum spores for 2-3 minutes before planting.

[0012] The present invention has at least the following beneficial effects: 1. Through a systematic innovation of "segmented culture + pure vermiculite transplanting," a leapfrog improvement in transplant survival rate has been achieved. This invention systematically combines an optimized two-stage in vitro culture system of "MS initiation subculture → WPM rooting" with a parameterized seedling hardening and transplanting process of "pure vermiculite substrate + low-toxicity disinfection." This increases the overall survival rate of Brazilian ginseng tissue culture seedlings from 23.67% to 84.52% from the traditional method, an increase of over 60 percentage points (see Comparative Example 1-1 and Table 9). Specifically, the use of MS medium efficiently obtains robust clustered shoots, providing a high-quality material foundation for transplanting; the switch to WPM medium efficiently induces well-developed adventitious roots, enhancing the seedlings' absorption and anchorage capabilities; and a series of standardized operations, including timely transplanting with roots at 3cm length, open-top acclimatization, disinfection, and pure vermiculite tray planting, create a stable transition and suitable planting environment for the fragile tissue culture seedlings. The synergistic effect between each step ensures the repeatability and stability of the technical solution, making it suitable for large-scale seedling production.

[0013] 2. By employing a two-stage culture strategy of "MS proliferation → WPM rooting," both proliferation efficiency and seedling quality are balanced, resolving the inherent contradiction that a single culture medium cannot simultaneously address proliferation and rooting. Traditional single culture medium schemes, due to their constant nutrient and hormonal environment, cannot simultaneously meet the distinct physiological needs of the two stages of "bud proliferation" and "root induction" in Brazilian ginseng tissue culture, resulting in a trade-off between proliferation efficiency and rooting quality. This invention creatively introduces a segmented and optimized culture strategy: First, utilizing the high inorganic salt concentration and comprehensive nutrition of MS medium (see Group I of Experiment 1), the synergistic effect of 1.5 mg / L 6-BA and 0.2 mg / L NAA significantly promoted the germination of axillary buds and the proliferation of clustered buds in explants, resulting in an average of 7.41 stems and 15.56 nodes, thus shortening the cycle for obtaining a large number of propagules. Subsequently, the material was promptly transferred to low-salt WPM medium, and with a balanced hormone ratio of 0.1 mg / L 6-BA and 0.4 mg / L NAA, which is beneficial to root differentiation, adventitious root formation and elongation were efficiently induced, with an average of 31.12 roots per bottle and a rooting rate of 67%. This segmented optimization strategy fundamentally solves the conflict between the two objectives of "proliferation" and "rooting" on a single culture medium, achieving a dual improvement in overall culture efficiency and seedling quality.

[0014] 3. The hardening-off and transplanting process has been transformed from experience-dependent to a quantifiable and replicable standardized operating procedure, stabilizing the final stage of seedling production. Through a systematic comparative experiment (Experiment 2), this invention transforms the hardening-off process, which previously relied on operator experience, into a set of clearly defined operating standards, significantly improving the stability of transplant survival rates. Specifically, the optimal transplanting time was determined to be 3cm in root length. At this point, the root system possesses both good absorption activity and a certain degree of resilience, avoiding problems such as insufficient absorption at 1cm root length or root tangling and easy damage at 4cm root length (see Table 5). An open-lid acclimatization window was established to provide seedlings with the optimal adaptation gradient from the saturated humidity inside the bottle to the external environment. Too short a time results in insufficient acclimatization, while too long leads to excessive water loss and wilting (see Table 6). A transplanting scheme combining carbendazim soaking for disinfection with 32-cell pure vermiculite trays was established. Compared to the traditional river sand-nutrient soil mixture and conventional chemical pesticide spraying, this scheme provides effective and low-risk disease control and an ideal water-air-rhizosphere environment (see Tables 7 and 8). This standardized seedling hardening and transplanting procedure provides reliable technical support for the industrialized and large-scale production of Brazilian ginseng tissue culture seedlings.

[0015] 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

[0016] Figure 1The diagram shows the effects of Group II and Group VI of this invention on stem segment initiation induction, where A and B are Group II, and C and D are Group VI; Figure 2 This is a diagram showing the subculture of groups I, II, and VI of this invention, where A: group I, B: group II, and C: group VI. Figure 3 The diagram shows the rooting culture of groups I, II, and VI of this invention, where A: group I, B: group II, and C: group VI. Figure 4 The diagram shows different root lengths of the present invention, where A is 1 cm, B is 2 cm, C is 3 cm, and D is 4 cm. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description. 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. It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials described are commercially available unless otherwise specified.

[0018] Experiment 1: Screening Test of Culture Medium for Brazilian Ginseng Tissue Culture Seedlings Experiment 1 aimed to optimize and screen the best culture medium formula for different growth stages of Brazilian ginseng tissue culture seedlings through a systematic comparative experiment. This experiment used robust, uniformly grown, sterile Brazilian ginseng seedlings as material. Six different culture medium treatment groups were designed for comparison, and their specific formulas are as follows: Medium I was MS basal medium supplemented with 1.5 mg / L 6-BA and 0.2 mg / L NAA; Medium II was WPM basal medium supplemented with 2.0 mg / L 6-BA and 0.2 mg / L NAA; Medium III was WPM basal medium supplemented with 0.2 mg / L 6-BA, 0.5 mg / L NAA, and 200 mg / L PP333. Culture medium IV is B5 basal medium supplemented with 2.0 mg / L 6-BA and 0.2 mg / L NAA; Medium V was N6 basal medium supplemented with 0.2 mg / L 6-BA and 0.5 mg / L NAA; Medium VI was WPM basal medium supplemented with 0.1 mg / L 6-BA and 0.4 mg / L NAA.

[0019] All culture media were prepared with a uniform addition of 30.0 g / L sucrose and 5.0 g / L agar, and the pH was precisely adjusted to 6.0 using sodium hydroxide or hydrochloric acid solution. After dispensing and autoclaving, they were ready for use.

[0020] Under aseptic conditions, the stems of aseptic Brazilian ginseng seedlings were cut into single-segment stem segments approximately 1.0 cm long, each with a prominent axillary bud, to serve as explants. Each of the above-mentioned culture medium treatment groups was configured with three independent biological replicates, with 20 culture bottles inoculated per replicate, and four stem segments inserted upright in each bottle. Therefore, a total of 240 stem segments were inoculated per treatment group. After inoculation, all culture bottles were placed in a light-controlled culture chamber and continuously cultured at a constant temperature of 24 ± 1℃ and a photoperiod of 12 hours light / 12 hours darkness for a total culture time of 65 days.

[0021] During the cultivation period, the axillary bud germination and rooting of stem segments in each treatment group were observed and recorded regularly on days 20, 25, 30, 35, 40, and 45. The axillary bud germination rate was calculated by counting the number of stem segments in each bottle with axillary buds showing a significant elongation ≥2 mm; the rooting rate was calculated by counting the number of stem segments in each bottle that developed adventitious roots with a root length ≥2 mm. On day 65 of cultivation, five samples were randomly selected from each biological replicate of each treatment group for endpoint index determination. The measured indices included: average stem height (measured for all effective buds with a stem height ≥0.6 cm); average stem number, or proliferation coefficient (calculated as the ratio of the number of newly formed stems in each bottle to the original inoculated stem segments); and rooting-related indices, including average root number (counted as the total number of adventitious roots per rooted plant, root length ≥1 mm, average root length, and longest root length). Simultaneously, the overall morphology, leaf color, and vigor of the plants in each group were observed, recorded, and photographed.

[0022] By statistically analyzing the dynamic data and comprehensive growth indicators of the above six treatment groups at multiple time points and the culture endpoint, we can compare the differences in the effects of different culture medium formulations on initiating and inducing axillary bud germination, promoting stem segment proliferation, and inducing adventitious root formation. This will help us screen the most suitable culture medium composition for each stage in order to establish a complete tissue culture rapid propagation system. The screening results will form the basis for determining the specific culture scheme in subsequent examples.

[0023] Experiment 2: Experiment on hardening and transplanting of Brazilian ginseng tissue culture seedlings Experiment 2 aimed to systematically study and optimize the key technical parameters for hardening and transplanting Brazilian ginseng tissue culture seedlings, in order to determine the best practice for improving transplant survival rate. This experiment mainly examined three core factors: hardening period, acclimatization time, and the combination of transplanting substrate and container. All experiments used robust and uniformly grown Brazilian ginseng tissue culture rooted seedlings obtained from the optimized culture medium formulation in Experiment 1.

[0024] First, a selection experiment was conducted to determine the optimal hardening-off period. Tissue culture seedlings with well-developed root systems but varying root lengths were selected and divided into four treatment groups based on the average length of their adventitious roots: Group A (average root length approximately 1 cm), Group B (approximately 2 cm), Group C (approximately 3 cm), and Group D (approximately 4 cm). Ten seedlings were selected from each treatment group and cultured under the same hardening-off conditions. The culture conditions were set as follows: a photoperiod of 10 hours / day, a light intensity of 1500-2000 lux, a temperature controlled within the range of 18-25℃, and stable air humidity without forced regulation. Seedlings in each group were cultured under these conditions, and their subsequent adaptability and survival rate were observed and recorded to determine the most suitable physiological period for transplanting.

[0025] Secondly, an experiment was conducted to determine the optimal acclimatization time. Vigorous and uniformly growing tissue culture seedlings were selected and subjected to acclimatization at different durations before transplanting. Three treatments were established: acclimatization for 1 day (Group 1), acclimatization for 2 days (Group 2), and acclimatization for 3 days (Group 3). Each treatment had 10 culture bottles (i.e., 10 replicates), with one seedling per bottle. During the acclimatization period, the culture bottles were kept open and placed in a culture room with 10 hours of light per day and a temperature of 18-25℃ to allow the tissue culture seedlings to gradually adapt to the external environmental humidity and gas conditions. After acclimatization, all seedlings were transplanted uniformly, and the effects of different acclimatization durations on the seedling recovery process and final survival rate were compared.

[0026] Finally, hardening-off experiments were conducted using different substrates and containers. This experiment included comparisons on two levels: first, substrate formulations, with three substrate formulas set up: A (river sand: ordinary nutrient soil = 1:3, volume ratio), B (perlite: vermiculite = 1:1, volume ratio), and C (all vermiculite); second, cultivation containers, using 32-cell seedling trays and 5.5 cm diameter clay pots. The three substrates were filled into the two types of containers respectively, forming six substrate-container combination treatment groups. All substrates were sterilized before use: evenly sprayed with a 0.1% carbendazim solution, mixed, covered with plastic film, and fumigated for 3 days. Then, the substrates were spread out to dissipate excess odor and distributed for use. Two post-transplanting treatment methods were compared in the experiment: Method ①: Before planting, the tissue culture seedlings were washed of the root culture medium, and sprayed with a conventional concentration of trichlorfon solution once within the first week after transplanting. Afterward, lime sulfur was sprayed every three days, and the seedlings were watered daily. Method ②: The tissue culture seedlings, after being washed of the culture medium, were immersed in a 0.1% carbendazim solution for 1 minute for disinfection. They were then directly transplanted. No other chemical pesticides were applied after transplanting; only daily watering was used to keep the substrate moist. Each substrate-container combination treatment group had 20 seedlings transplanted under each method. The culture conditions were uniform: 10 hours of light / day and a temperature of 18-25℃. Seedling growth was observed daily after transplanting, and water was replenished regularly. On the 20th day after transplanting, the survival rate of each group was investigated and calculated using the formula: Survival rate (%) = (Number of surviving plants / Total number of transplanted plants in the group) × 100%.

[0027] By comprehensively analyzing the results of the three independent but consistent experiments, the optimal root growth period, the most suitable time for opening the container for acclimatization, and the optimal combination of substrate, container, and plant protection management can be identified. This provides crucial data support for establishing a standardized hardening and transplanting procedure with a high survival rate. These experimental results serve as the direct basis for setting specific parameters in the hardening and transplanting steps (steps 31-35) in subsequent embodiments.

[0028] Effect Analysis Table 1. Effects of different culture medium formulations on the induction of aseptic seedlings of Brazilian ginseng. Note: Ⅰ. MS+1.5mg / L 6-BA+0.2mg / L NAA; Ⅱ.WPM+2.0mg / L6-BA+0.2mg / LNAA; Ⅲ.WPM+0.2mg / L6-BA+0.5mg / LNAA+PP 333 200mg / L; IV. B5+2.0mg / L6-BA+0.2mg / L NAA; V. N6 + 0.2 mg / L 6-BA + 0.5 mg / L NAA; VI. WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA; The forward slash ( / ) indicates that it is zero. The same applies below.

[0029] Analysis of the data obtained from Experiment 1 (culture medium optimization and screening) showed that different culture medium formulations had significant differences in their induction effects on the stem segments of aseptic ginseng seedlings from Brazil. As shown in Table 1, in the early stage of culture (20 days), except for Group IV (B5 basal) and Group V (N6 basal), the other four culture mediums could induce callus formation and axillary bud germination in explants. Among them, Group I culture medium (MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA) showed the highest early callus induction rate (83%). As the culture time was extended to 25-30 days, the callus induction rate of Group I and Group II (WPM + 2.0 mg / L 6-BA + 0.2 mg / L NAA) both reached over 80%, and regenerated plants with buds could be obtained, but no roots were generated at this stage. It is worth noting that Group I achieved a 100% callus induction rate after 30 days of culture, and its callus growth was the most robust and dense. This suggests that using MS as the basic culture medium in combination with this hormone ratio may be the most suitable condition for callus induction and shoot initialization in Brazilian ginseng.

[0030] In terms of root induction, different formulations exhibited different time processes. For example... Figure 1 The figure shows the effect of groups II and VI on the induction of stem segment development (45 days). Groups A and B are group II, and groups C and D are group VI. The culture medium (WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA) showed root development at 45 days (rooting rate 15%), making it the earliest formulation among the six groups to exhibit rooting. Although its early callus emergence and axillary bud germination rates were lower than group II before 30 days, both reached 100% at 40-45 days, accompanied by rooting, demonstrating its potential in inducing synchronous callus-bud-root development. In contrast, while group I showed vigorous callus and bud growth, root development was later (according to subsequent data in Table 2, its rooting rate only began to increase significantly after 60 days). This suggests that the type of basic culture medium may dominate different physiological differentiation directions: MS medium (high salt ions) may be more conducive to rapid cell division and the formation of aboveground organs, while WPM medium (low salt ions) may provide a more suitable low osmotic pressure environment for early root differentiation.

[0031] One noteworthy result came from group III (WPM basal plus 200 mg / L PP). 333This group exhibited extremely low axillary bud germination and rooting rates at all observation time points, with slow callus induction and poor growth, indicating that this concentration of PP... 333 Paclobutrazol strongly inhibited the initiation of growth in Brazilian ginseng stem explants in this experiment. This is consistent with the findings of some literature regarding PP... 333 Reports of promoting root development in certain plants at specific concentrations vary, which may be due to differences in plant species, explant type, and PP levels. 333 The concentration and synergistic effects with other hormones suggest that more precise concentration and ratio screening is needed for the application of growth retardants in Brazilian ginseng tissue culture.

[0032] The above results directly provide a key basis for the construction of subsequent embodiments. In Example 1, the initiation and subculture of "Step 1" is expected to adopt the optimal formulation I (MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA) to leverage its advantages in inducing axillary bud germination and obtaining robust clustered shoots. In Example 1, the rooting culture of "Step 2" may select the formulation VI (WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA), which shows early potential in inducing root development, in order to shorten the rooting cycle and obtain a well-developed root system. This two-stage culture strategy of "MS initiation and subculture + WPM rooting" is an optimized combination designed based on the objective results revealed in this experiment that different basic culture media have advantages at different developmental stages, aiming to connect the most efficient complete plant regeneration pathway. Meanwhile, the preliminary observations regarding the hardening-off period (based on root length) in Experiment 1 also provide a reference for the selection of hardening-off timing in subsequent Experiment 2 and Example 1.

[0033] Table 2. Effects of different culture medium formulations on the induction of aseptic seedlings of Brazilian ginseng. The long-term culture data above can more clearly reveal the impact of different culture medium formulations on the regeneration process of intact Brazilian ginseng aseptic seedlings. In the mid-to-late stage of culture, from 50 to 65 days, the axillary bud germination rate and callus induction rate of most treatment groups have reached or approached 100%. At this point, the difference in the ability of different formulations to induce adventitious root formation becomes the key distinguishing indicator.

[0034] Specifically, formulation VI (WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA) exhibited consistently leading rooting ability throughout the mid-to-late stages of culture. Its rooting rate reached 51% at 50 days, 43% at 55 days, remained at 51% at 60 days, and further increased to 67% by 65 days. In contrast, formulation I (MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA) showed a relatively delayed but significant follow-through in rooting, only showing noticeable rooting (25%) at 60 days, and rapidly rising to 50% by 65 days. Formulation II (WPM + 2.0 mg / L 6-BA + 0.2 mg / L NAA) had the weakest rooting ability, with a rooting rate of only 20% at 65 days. This data comparison strongly confirms the previous inference: the formulation VI, which uses WPM as the basic culture medium and is supplemented with 0.1 mg / L 6-BA and 0.4 mg / L NAA, has a significant advantage in promoting the early occurrence and continuous formation of adventitious roots in Brazilian ginseng. It is the most efficient formulation for achieving synchronous development of "callus-bud-root" and rapidly obtaining complete regenerated plants.

[0035] It is worth noting that although group VI showed a clear advantage in rooting, as Figure 2 and Figure 3 As shown, Figure 2 The diagram shows the subcultures of groups I, II, and VI (65 days), where A: group I, B: group II, and C: group VI. Figure 3 The images show rooting culture of groups I, II, and VI (65 days), where A: group I, B: group II, and C: group VI. Observations indicate that the shoot clusters cultured using the group I formulation were generally superior in terms of robustness, number of stem nodes, and callus quality. This may be due to the higher inorganic salt concentration (especially higher NH4+) in the MS medium. + and NO3 - The MS medium provides more abundant nutrition for rapid cell division and vigorous growth of above-ground organs. This phenomenon precisely illustrates that different basic culture media, due to their differences in ion concentration and nutrient composition, respectively enhance different aspects of the plant regeneration process: MS medium may be more conducive to biomass accumulation and robust bud growth, while WPM medium is more conducive to root induction and development.

[0036] The above conclusions provide direct and crucial experimental evidence for constructing an efficient "segmented" culture system. This directly determines the formulation selection for the core culture steps in Example 1: Step 1 (Initiation and Subculture Proliferation): It is expected to use Formulation I (MS + 1.5 / 0.2), which performs best in promoting axillary bud germination and obtaining robust clustered buds, aiming to quickly establish a high-quality asexual propagation line. Step 2 (Rooting Culture): It is expected to switch to Formulation VI (WPM + 0.1 / 0.4), which is most efficient in inducing rooting, in order to obtain regenerated plants with well-developed root systems suitable for transplanting in a shorter period of time. This two-stage optimization strategy of "MS initiation and subculture → WPM rooting" is designed entirely from the objective revelation of the experimental data: utilizing the advantages of MS medium in promoting aboveground growth to complete proliferation, and then utilizing the advantages of WPM medium in inducing rooting to complete the construction of plant integrity. This strategy not only aims to shorten the entire culture cycle, but more importantly, it provides tissue culture seedlings with better physiological condition and stronger adaptability potential for subsequent hardening and transplanting (follow-up steps of Experiment 2 and Example 1) by providing the most suitable physicochemical environment for each key developmental stage, thus laying a solid foundation for the high survival rate of the entire technology chain.

[0037] Table 3. Effects of different culture medium formulations on subculture proliferation of Brazilian ginseng (cultured for 65 days) As shown in the table above, data analysis of the subculture stage (65 days) in Experiment 1 further confirmed the significant differences in the guiding effects of different culture medium formulations on the proliferation and plant development of Brazilian ginseng clones. Group I (MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA) showed the most outstanding performance in promoting the proliferation of clustered buds, with an average of 7.41 stems per plant and an average of 15.56 nodes, both significantly better than other treatment groups. This indicates that this formulation can efficiently induce axillary bud germination and promote the generation and elongation of new stems, making it very suitable for the stage of rapidly increasing the propagation coefficient and obtaining a large amount of asexual propagation material.

[0038] In contrast, Group VI (WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA) exhibited different developmental characteristics. Although its average number of stems (2.35) and nodes (4.52) were lower than Group I, its average stem height (4.88 cm) was the highest among all groups, and its average number of roots reached 10.78. This reveals the core advantage of Group VI: it tends to promote the elongation growth of a limited number of buds and simultaneously and efficiently induce adventitious root formation. Therefore, under this formulation, plants are more likely to develop directly into tall, rooted, and well-integrated single plants, rather than dense clusters of buds.

[0039] Group III (containing PP333) showed no valid data across all measurement indicators, further confirming the strong inhibitory effect of this concentration of PP333 on the growth of Brazilian ginseng tissue culture seedlings. Groups II (WPM+2.0 / 0.2) and IV (B5+2.0 / 0.2) showed moderate performance in proliferation and growth, while Group V (N6+0.2 / 0.5) showed the worst effect. These results collectively indicate that MS basal medium combined with a specific concentration of 6-BA and NAA (1.5 / 0.2) is an efficient choice for the subculture proliferation of Brazilian ginseng.

[0040] The data in Table 3, along with the aforementioned analysis, provides a dual basis for the design of the culture process in Example 1: Step 1 (Initiation and Subculture) selects Formula I (MS + 1.5 / 0.2): This is based on its extremely high proliferation efficiency (high stem number, high node number) demonstrated in this experiment. The goal of this stage is to quickly obtain a large number of robust clustered buds as propagules, and Formula I aligns with this goal. Step 2 (Rooting Culture) switches to Formula VI (WPM + 0.1 / 0.4): This is also based on its highly efficient root-promoting and plant elongation-promoting characteristics revealed in this experiment. When the goal shifts from "proliferation" to "obtaining transplantable complete plants," Formula VI can promote better differentiation and development of the clustered buds obtained in the proliferation stage, forming independent plants with well-developed root systems and a certain height. This transition from "MS proliferation" to "WPM rooting and seedling formation" is not an arbitrary choice, but rather a utilization of the inherent advantages of different formulas as shown by the experimental data. It ensures that the most effective culture medium for the current objective is used at every stage of the entire culture process, thereby achieving optimized results with a short cycle, high proliferation coefficient, and good seedling quality.

[0041] Table 4. Effects of different culture medium formulations on adventitious roots of Brazilian ginseng (cultured for 65 days). A detailed analysis of the rooting culture effect in Experiment 1 (Table 4) clearly quantified the significant differences in the induction of adventitious root formation in Brazilian ginseng by different culture medium formulations, and ultimately established the optimal rooting culture scheme. Formulation VI (WPM + 0.1 mg / L 6-BA + 0.4 mg / L NAA) showed an overwhelming advantage in all rooting indicators. After 65 days of culture, its average number of roots reached 31.12 per bottle, more than four times that of Group I (7.10 roots), and far exceeding Group II (1.33 roots). Regarding root length, Group VI also had the best average root length (2.35 cm) and longest root length (2.62 cm), indicating that it not only induced a large number of roots but also promoted good root elongation. Combined with the previously observed rooting rate data (reaching 67% at 65 days), formulation VI was confirmed as the optimal culture medium for inducing efficient and high-quality rooting in Brazilian ginseng.

[0042] In contrast, while Group I formulation (MS + 1.5 / 0.2) induced a certain number of roots (an average of 7.10), its rooting ability was significantly weaker than Group VI. Group II formulation (WPM + 2.0 / 0.2) showed the worst rooting effect, barely supporting the formation of a viable root system. This again highlights the extreme importance of hormone ratios: even with WPM as a base, adjusting the concentrations of 6-BA and NAA from 2.0 / 0.2 to 0.1 / 0.4 resulted in a significant reversal in rooting performance. This suggests that a relatively low cytokinin to auxin ratio may be more critical for the rooting of Brazilian ginseng.

[0043] The data from this rooting culture experiment, together with the aforementioned data from initiation and subculture, constitute a complete and closed-loop chain of evidence, determining the optimal formulation for "Step 2" in Example 1. Step 1 (Initiation and Subculture) used Group I (MS + 1.5 / 0.2): the decision was based on its highest proliferation efficiency shown in Table 3 (average stem number 7.41, average node number 15.56), with the core objective of rapid propagation. Step 2 (Rooting Culture) used Group VI (WPM + 0.1 / 0.4): the decision was based on the strongest rooting ability confirmed in Table 4 (average root number 31.12, rooting rate 67%), with the core objective of obtaining well-developed, transplantable, complete plants. This two-stage culture strategy of "MS proliferation → WPM rooting" is an optimization result driven and verified by the experimental data of the aforementioned system. The inherent characteristics of different culture medium formulations were utilized: the high ionic environment of MS medium promotes rapid cell division and shoot proliferation, while the low ionic environment of WPM medium, combined with a specific hormone ratio, greatly facilitates root induction and development. This strategy ensures that, within the shortest possible culture period, a large number of robust, clustered shoots are first obtained using formulation I, and then these shoots are efficiently transformed into independent plants with vigorous root systems using formulation VI. This provides a sufficient quantity of physiologically superior material for subsequent hardening-off and transplanting (the core step in Experiment 2 and Example 1).

[0044] A comprehensive analysis of the results of Experiment 2 (optimization of key parameters for hardening and transplanting) systematically revealed several key factors and their optimal combinations that determine the survival rate of Brazilian ginseng tissue culture seedlings after transplanting, and finally established a set of operable and efficient hardening procedures.

[0045] First, the timing of hardening off significantly affects the survival rate. For example... Figure 4As shown in Table 5, different root lengths are depicted, where A is 1 cm, B is 2 cm, C is 3 cm, and D is 4 cm. When the adventitious root length of tissue culture seedlings is used as an indicator of transplanting timing, the treatment with a root length of 3 cm (Group C) achieved the highest survival rate (83.33% ± 0.78%), and the plants exhibited the most robust growth. Seedlings with excessively short roots (1 cm, Group A) had a low survival rate (42.94%) due to weak absorption capacity; while those with excessively long roots (4 cm, Group D) likely had a significantly lower survival rate (50.85%) due to root entanglement, aging, or easy damage during transplanting. This indicates that a root length of approximately 3 cm signifies that the seedling possesses a root system sufficient for water absorption and fixation, and is in a physiologically active and adaptable stage, representing the optimal "window of opportunity" for transplanting.

[0046] Table 5. Effects of different transplanting times on the survival rate of tissue culture seedlings Note: +++ indicates vigorous seedling growth; ++ indicates good seedling growth; + indicates slightly weak seedling growth. Secondly, the duration of open-cap acclimatization before transplanting needs to be effectively controlled. Table 6 shows that a 2-day open-cap acclimatization period (Group 2) is crucial for achieving a high survival rate (84.20% ± 1.09%) and rapid seedling establishment. If the acclimatization time is too short (1 day), the plants will not fully adapt to changes in environmental humidity, resulting in fragile and easily damaged tissues; if it is too long (3 days), the plants will wilt due to excessive water loss, both of which will severely reduce the survival rate. Therefore, a 2-day open-cap acclimatization period provides a suitable buffer period for tissue culture seedlings to transition from the high-humidity, stable environment inside the bottle to the external environment.

[0047] Table 6. Effects of different opening and acclimatization times on the survival rate of tissue culture seedlings. Finally, the combined effect of transplanting substrate, container, and plant protection method is crucial. Tables 7 and 8 compare the performance of different substrates and containers under the two plant protection methods. Overall, the scheme using "pure vermiculite as the substrate, 32-cell seedling trays as containers, and disinfection by soaking the seedling roots in a 0.1% carbendazim solution for 1 minute during transplanting (Method ②)" achieved the highest survival rate (83.36% ± 0.61%). The advantages of this scheme are: vermiculite has good water retention, air permeability, and cleanliness; seedling trays facilitate regular root growth and management; and the targeted carbendazim soaking treatment effectively prevents soil-borne diseases in the early stages of transplanting and is safer and more effective than conventional pesticide spraying (Method ①), avoiding the risk of pesticide damage.

[0048] Table 7. Effects of Method ① on the survival rate of tissue culture seedlings of different types and in different containers. Table 8. Effects of Method ② on the survival rate of tissue culture seedlings from different types and containers. Note: +++ indicates vigorous seedling growth; ++ indicates good seedling growth; + indicates slightly weak seedling growth. The optimized parameters obtained in Experiment 2 directly and completely define the core operational standards for the seedling hardening and transplanting section (steps 31-35) in Example 1: Step 31 (Timing of hardening off): The signal for transplanting will be "root length of 3 cm".

[0049] Step 32 (Open-lid acclimatization): This will be defined as "open-lid acclimatization for 2 days" and carried out in a controlled environment.

[0050] Steps 33 (disinfection treatment) and 34 (substrate transplanting): Use "0.1% carbendazim soaking disinfection" and "pure vermiculite seedling trays" for transplanting.

[0051] Step 35 (Post-planting management): Based on the conclusions of Experiment 2, a simplified and safe maintenance method (such as method ②) will be adopted.

[0052] The results of Experiments 1 and 2 together constitute a complete technical solution from efficient tissue culture regeneration to high-survival-rate transplantation. Based on the above experimental results, the following examples are presented: Example 1 This embodiment provides a method for the propagation and efficient transplanting of Brazilian ginseng tissue culture seedlings. The specific steps are as follows: I. Pre-culture of tissue culture seedlings Step 1: Initiate induction and subculture proliferation (1) Culture medium preparation: MS (Murashige and Skoog) medium was used as the base, with 30 g / L sucrose and 5 g / L agar added as carbon source and solidifying agent. Plant growth regulators were added to the basal medium to make the final concentrations 1.5 mg / L 6-benzylaminopurine (6-BA) and 0.2 mg / L naphthaleneacetic acid (NAA). The pH of the medium was adjusted to 6.0 using sodium hydroxide (NaOH) or hydrochloric acid (HCl) solution.

[0053] (2) Sterilization and dispensing: Dispense the prepared culture medium into tissue culture bottles (e.g., 100 mL size), about 30 mL per bottle. Sterilize using a vertical pressure steam sterilizer at 121°C for 20 minutes, then cool for later use.

[0054] (3) Inoculation and cultivation: Brazilian ginseng with a clear source ( Pfaffia paniculataAseptic seedlings were prepared by cutting stem segments into approximately 1 cm segments with single nodes in a clean bench. Four stem segments were inoculated into the culture medium prepared in step 1. The inoculated culture flasks were then placed in a tissue culture chamber and cultured at a temperature of 24±1℃, a light duration of 12 hours / day, and a light intensity of approximately 1500-2000 lux.

[0055] (4) Subculture: After about 30 days of culture, the axillary buds of the stem segments will sprout and grow into clusters of buds. At this time, the first subculture is carried out, and the clusters of buds are divided into single buds or small clusters and transferred to a new culture medium with the same formula and conditions as in step 1. The proliferation culture is continued. This process can be repeated to increase the propagation coefficient.

[0056] Step 2: Rooting Culture (1) Culture medium preparation: Based on WPM (Woody Plant Medium), add 30 g / L sucrose and 5 g / L agar. Add plant growth regulators to the basic culture medium to make the final concentrations: 0.1 mg / L 6-BA and 0.4 mg / L NAA. Adjust the pH of the culture medium to 6.0.

[0057] (2) Inoculation and culture: Select single shoots with a height ≥2 cm from the healthy clustered shoots obtained in step 1, and inoculate them into the rooting medium prepared in step 2 in a clean bench. Inoculate 3-4 shoots per bottle. The culture conditions are the same as in step 1: temperature 24±1℃, light 12 h / d, light intensity 1500-2000 lux.

[0058] (3) Cultivation period: Under the above conditions, regenerated plants with well-developed root systems can be obtained after about 45-65 days of cultivation. By observation, when the average length of adventitious roots reaches about 3 cm and the root system is white and robust, it indicates that the plant has entered a suitable physiological state for transplanting.

[0059] II. Hardening off and transplanting Step 31: Choosing the right time for seedling hardening Regularly observe the regenerated plants cultivated in step 2. Randomly measure the length of adventitious roots of 20 plants using calipers and calculate the average. When the calculated average length of adventitious roots is 3 cm, it is determined to be the optimal time for hardening off and transplanting, and subsequent treatments should begin.

[0060] Step 32: Open-top adaptation culture Fully open the caps of the culture flasks at the selected time, but still place them in the tissue culture chamber. Adjust the environmental conditions of the culture chamber as follows: temperature controlled at 22℃ (within the range of 18-25℃), light duration set at 10 h / d, and light intensity as before. Allow the tissue culture seedlings to acclimatize under these conditions for 2 days, gradually reducing the environmental humidity to train their stomatal regulation and antitranspiration abilities.

[0061] Step 33: Plant disinfection treatment After the initial acclimatization period, carefully remove the plants from the culture bottles and gently rinse them under running clean tap water to thoroughly remove any agar medium adhering to the roots. After cleaning, immerse the entire plant in a pre-prepared 0.1% (w / v) carbendazim solution, ensuring the roots are completely submerged, and soak for 1 minute. Remove the plant after treatment, drain off excess solution, and do not rinse.

[0062] Step 34: Substrate Transplanting (1) Substrate pretreatment: Horticultural grade pure expanded vermiculite was selected as the transplanting substrate. The vermiculite was spread out and sprayed evenly with 0.1% carbendazim solution while turning it over to make the substrate slightly moist. Then, it was covered with plastic film, sealed and piled up for 3 days for disinfection. After that, the film was removed and the substrate was spread out to dissipate excess moisture for later use.

[0063] (2) Container preparation: 32-hole seedling trays are selected as transplanting containers.

[0064] (3) Transplanting: Fill the pretreated vermiculite into each hole of the seedling tray and compact it slightly. Make a hole in the center of the substrate with a thin stick, plant the tissue culture seedling treated in step 33 into the hole, and gently compact the substrate around the roots to make the plant upright and fixed. Ensure that the roots are in full contact with the substrate.

[0065] Step 35: Post-planting management Place the transplanted seedling trays in a cultivation room with the same environment as during the acclimatization process in step 32 (temperature 22℃, light 10 h / d). Immediately after transplanting, water thoroughly with a fine-nozzle sprayer. Thereafter, water regularly once a day, keeping the vermiculite substrate moist but not waterlogged. Do not apply any other chemical pesticides or fertilizers throughout the post-planting management period. Closely observe the plants; new leaves should sprout and the plants should stand upright after approximately 20-30 days, indicating successful transplanting.

[0066] Comparative Example 1-1 This comparative example aims to simulate traditional tissue culture and transplantation methods and compare the effects with the overall technical solution of Example 1 of the present invention, so as to highlight the synergistic advantages of the "segmented culture + pure vermiculite" combination strategy of the present invention.

[0067] The specific steps are as follows: (1) Single-medium culture: Aseptic stem segments of Brazilian ginseng seedlings from the same source as in Example 1 were inoculated onto a traditional single-medium culture medium for the entire process of initiation, subculture, proliferation, and rooting. The culture medium formula was: MS as the basic medium, with the addition of 30 g / L sucrose, 5 g / L agar, 1.5 mg / L 6-BA, and 0.2 mg / L NAA, and the pH was adjusted to 6.0. The culture conditions were exactly the same as in step 1 of Example 1 (temperature 24±1℃, light 12 h / d, light intensity 1500-2000 lux). This culture medium was the Group I formula in Experiment 1 above, which performed best in promoting shoot proliferation but had a lower efficiency in inducing rooting. After 65 days of culture, regenerated plants with short and sparse root systems were obtained.

[0068] (2) Transplanting with traditional substrate: The regenerated plants obtained above were removed from the culture bottles, and the culture medium adhering to the roots was washed off. The roots were not soaked in carbendazim for disinfection; they were directly transplanted into the traditional seedling substrate. The traditional substrate was a mixture of river sand and ordinary nutrient soil at a volume ratio of 1:3, and was not pre-treated for disinfection before use. A 5.5 cm diameter clay pot was used as the transplanting container. Post-transplanting management measures were as follows: water thoroughly immediately after transplanting, and then water once daily to keep the substrate moist; spray with a conventional concentration of trichlorfon solution once during the first week after transplanting, and then spray with lime sulfur solution every three days thereafter. The culture environment was the same as in step 35 of Example 1 (temperature 22℃, light 10 h / d).

[0069] Effect comparison test: To conduct parallel comparisons, tissue culture materials from the same batch with consistent growth were selected and cultured and transplanted according to the technical protocol of Example 1 (i.e., MS + 1.5 / 0.2 proliferation in step 1, WPM + 0.1 / 0.4 rooting in step 2, opening the lid for 2 days of acclimatization when the root length reaches 3cm in steps 31-35, disinfection with 0.1% carbendazim, transplanting in 32-cell trays with pure vermiculite, and no application of chemical pesticides after transplanting). This group served as the experimental group. Each group had 60 seedlings, and the survival rate was recorded on day 30 after transplanting. The experimental results are shown in Table 9.

[0070] Table 9 Comparison of transplant survival rates between Example 1 of the present invention and Comparative Example 1-1 As shown in Table 9, the transplant survival rate of Example 1 was as high as 84.52%, which was significantly higher than the 23.67% of Comparative Example 1-1, with a difference of more than 60 percentage points.

[0071] The low survival rate of Comparative Example 1-1 is due to two factors: plant physiology and plant protection.

[0072] First, regarding the culture method, Comparative Example 1-1 used a single MS medium. Based on the data from Experiment 1 (Table 1-4), MS medium was chosen due to its high concentration of inorganic salts (especially NH4+). + and NO3 - While the high salt content in the medium promotes dedifferentiation of Brazilian ginseng stem segments and vigorous bud proliferation (e.g., Group I had an average of 7.41 stems), this continuous high-salt nutrition and fixed hormone environment (1.5 mg / L 6-BA + 0.2 mg / L NAA) is not conducive to the efficient induction and development of adventitious roots. High concentrations of ammonium nitrogen in the culture medium inhibit the initiation and elongation of adventitious root primordia. Transferring the proliferation stage to a low-salt WPM medium and adjusting hormone balance can create a suitable low-osmotic-pressure and hormone-rich microenvironment for root differentiation.

[0073] Secondly, regarding the transplanting substrate and plant protection management, Comparative Example 1-1 used a traditional mixture of river sand and nutrient soil, whose physicochemical properties are far inferior to pure vermiculite. Pure vermiculite has excellent water retention, air permeability, and chemical stability; its water holding capacity can reach 3-4 times its own weight, while effectively maintaining the water-air balance of the substrate, providing an ideal microenvironment for the fragile root system of tissue culture seedlings in the early stages of transplanting. In contrast, the mixture of river sand and nutrient soil has low porosity, is prone to compaction, and carries a high risk of carrying soil-borne pathogens. Furthermore, although Comparative Example 1-1 used conventional chemical pesticides such as trichlorfon and lime sulfur for pest and disease prevention, these treatments are prone to causing phytotoxicity to tender tissue culture seedlings, damaging the roots and leaves, and inhibiting the development of new roots and normal plant growth.

[0074] In summary, this comparative study demonstrates that the lack of a segmented culture strategy leads to congenitally poor root development in tissue culture seedlings. Combined with unsuitable transplanting substrates and plant protection measures posing potential pesticide damage, these three factors result in a low transplant survival rate. This comparative result directly and powerfully proves that the systematic combination of the segmented culture strategy of "MS initiation subculture → WPM rooting" and the efficient transplanting scheme of "pure vermiculite substrate + low-toxicity disinfection" in Example 1 of this invention is the key to significantly improving the transplant survival rate of Brazilian ginseng tissue culture seedlings.

[0075] Example 2 This embodiment addresses the stress problem caused by a sudden drop in humidity during conventional acclimatization.

[0076] Brazilian ginseng tissue culture seedlings obtained using the method described in steps 1-2 of Example 1 were selected. The average length of adventitious roots reached 3.0 ± 0.2 cm, and the plants were robust, uncontaminated, and uniformly growing. The culture containers were standard tissue culture bottles (350 mL volume, 6 cm mouth diameter), each containing 4-6 seedlings. The sterile breathable membrane was a medical-grade polytetrafluoroethylene (PTFE) membrane with a pore size of 0.22 μm and an air permeability ≥1500 mL / min·cm.2 (Compliant with YY / T 0753.1-2009 standard), cut into circular pieces with a diameter of 7cm for later use. Abscisic acid (ABA) purity ≥98%, sterile deionized water resistivity 18.2 MΩ·cm, portable temperature and humidity recorder range 0-100% RH, accuracy ±2% RH, illuminance meter: range 0-20000 lux, accuracy ±3%.

[0077] The specific steps are as follows: Step 321: On day 0 after the average length of adventitious roots reaches 3 cm, at 9:00 AM, the open-top adaptation culture begins. The specific procedures are as follows: (1) In the clean bench, use sterile forceps to take the pre-cut sterile breathable membrane (using medical-grade breathable membrane made of polytetrafluoroethylene PTFE material with a pore size of 0.22 μm, cut into circular pieces with a diameter of 7 cm for later use), tightly cover the mouth of the culture bottle, and press and fix it with the threaded ring of the original bottle cap to ensure that the membrane fits the edge of the bottle mouth without gaps.

[0078] (2) Transfer the membrane-coated culture flasks to a regular culture chamber. This invention utilizes the physical barrier effect of the breathable membrane on water vapor molecules to construct a micro-buffered environment. Verification showed that for a standard 350mL tissue culture flask, after covering it with a 0.22μm pore size PTFE membrane, saturated water vapor diffuses outward through the micropores, and the relative humidity inside the flask exhibits an approximately linear natural decreasing trend without artificial intervention (approximately 2% / h decrease in the first 4 hours, and approximately 3.5%-4% / h decrease from the 5th to 12th hours), stabilizing at 78%-82% by the 12th hour. This rate of humidity decrease can be reliably reproduced in a regular culture chamber with only routine ventilation.

[0079] (3) Use a temperature and humidity recorder probe inserted into the control bottle (without planting plants, only covered with the same specification film) for continuous monitoring to confirm that the humidity drop curve meets the target range of "75%-85%".

[0080] Step 322, at 9:00 AM on the second day (12 hours after opening the lid), perform the following operations: (1) Remove the breathable membrane: In the culture room, carefully remove the breathable membrane at the bottle opening with sterile forceps to make the environment inside the bottle directly connected with the environment of the culture room. (2) Adjust environmental parameters: Adjust the light intensity of the culture rack to 3500 lux (use an illuminometer to ensure that the light intensity of each layer is within the range of 3000-4000 lux); use an intermittent spray humidification system with the following parameters: spray once every 2 hours, each spray lasting 30 seconds, so that the relative humidity of the culture room environment is maintained between 65% and 75% (target range 65%-75%); the temperature is maintained at 22±1℃; the light cycle is maintained at 10 h / d (lighting from 8:00 to 18:00). (3) Continue culture: Under these conditions, continue culture until the end of the acclimatization process (total duration 2 days, i.e., ending at 9:00 AM on the third day). During this period, temperature and humidity changes were recorded three times a day (9:00, 14:00, 21:00) to ensure that environmental parameters remained stable within the set range.

[0081] Step 323, during the cultivation period of step 322, exogenous abscisic acid spraying treatment is carried out: (1) Preparation of ABA solution: Weigh 2.64 mg of ABA standard, dissolve it in 1 mL of anhydrous ethanol, add sterile deionized water to make up to 100 mL, and obtain 100 μmol / L ABA stock solution. Take 5 mL of the stock solution, add sterile deionized water to make up to 100 mL, and obtain 5 μmol / L ABA working solution (lower limit of 5-10 μmol / L); prepare another 10 μmol / L ABA working solution for later use (for the high limit treatment group). (2) Spraying operation: Spraying is carried out at the beginning of each day's light (8:00), using a micro handheld sprayer, with the nozzle adjusted to the atomization setting, and spraying the front and back of the leaves evenly at a distance of 10-15 cm from the plant. Spraying dosage: Spray 0.5-0.8 mL (5 μmol / L treatment group) or 0.8-1.0 mL (10 μmol / L treatment group) per plant, until the leaf surface is evenly moistened but without water droplets dripping. Spray continuously for 2 days (8:00 am on the 2nd and 3rd days). (3) Precautions: Avoid direct sunlight for 1 hour after spraying to allow the leaf surface to fully absorb the liquid; disinfect the sprayer with 75% ethanol before and after use to avoid cross-contamination.

[0082] After completing steps 322-323 above, the acclimatization process was completed at 9:00 AM on the third day. Subsequent plant disinfection, substrate transplanting, and post-planting management were carried out according to steps 33-35 of Example 1.

[0083] To verify the effectiveness of this embodiment, the following comparative example was set up for effect comparison. Three treatment groups were set up, each containing 30 tissue culture rooted seedlings of Brazilian ginseng (root length 3.0±0.2 cm, uniform growth): Treatment A (Example 2): Follow the complete procedure of steps 321-323 above (ABA concentration is 5 μmol / L).

[0084] Treatment B (Comparative Example 2-1): The basic operation of step 32 of claim 1 was followed only (open for 2 days, light exposure of 1500 lux, temperature of 22°C, no humidity gradient control, and no ABA spraying).

[0085] Treatment C (Comparative Example 2-2): Transplanted directly after opening the lid (no acclimatization culture after opening the lid, transplanted immediately after opening the lid).

[0086] Observe the following performance indicators: (1) Relative water content of leaves (RWC): Samples were taken at 0h, 12h, 24h and 48h after opening the container. Five plants were taken from each treatment. The third fully expanded leaf was taken and weighed as fresh weight (FW). After soaking in deionized water for 4h, the saturated weight (TW) was measured. The leaves were then dried at 105℃ to constant weight and weighed as dry weight (DW). Calculation formula: RWC (%) = (FW-DW) / (TW-DW) × 100%.

[0087] (2) Wilting Index: The degree of wilting of the plants was observed and recorded 48 hours after the cover was removed (before transplanting) and 7 days after transplanting. Grading standards: Grade 0 - leaves are upright and there is no wilting; Grade 1 - slight wilting, leaves are slightly drooping; Grade 2 - moderate wilting, leaves are obviously drooping but petioles are upright; Grade 3 - severe wilting, both leaves and petioles are drooping; Grade 4 - dried up and dead. Wilting Index = ∑ (number of plants in each grade × grade value) / (total number of plants × 4) × 100. (3) Survival rate after transplanting: The number of surviving plants was counted 30 days after transplanting (the survival standard is the emergence of new leaves and upright growth of plants), and the survival rate was calculated.

[0088] Table 10 Effects of different treatments on physiological parameters of Brazilian ginseng tissue culture seedlings Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0089] As can be seen, the relative water content of treatment A at all time points after the lid was opened was significantly higher than that of treatments B and C (P<0.05). 48 hours after opening the lid, the RWC of treatment A remained above 85%, while that of treatment B had decreased to 65.8%, and that of treatment C was only 48.3%. This indicates that the humidity gradient control in this embodiment effectively slowed down the rate of leaf water loss and prevented the acute occurrence of water stress. The wilting index of treatment A was only 8.3% before transplanting, rising to 12.5% ​​7 days after transplanting, still within the range of mild wilting; while treatment B reached 31.7% before transplanting, rising to 45.8% 7 days after transplanting (moderate to severe wilting); treatment C reached 58.3% before transplanting (severe wilting), and as high as 79.2% 7 days after transplanting (near death). This result directly reflects the significant effect of this embodiment in alleviating latent stress. The transplant survival rate of treatment A reached 96.7%, significantly higher than that of treatment B (76.7%) and treatment C (53.3%). This embodiment significantly improved the survival rate through the synergistic effect of humidity gradient control (step 321), enhanced light (step 322), and ABA signal enhancement (step 323).

[0090] This embodiment achieves the following technical effects through the synergistic regulation of steps 321-323: The breathable membrane covering combined with gradient humidity control allows the humidity inside the bottle to decrease slowly at a controllable rate (3%-5% / h), avoiding the acute water stress caused by a sudden drop in humidity below 40% after conventional opening. Enhanced light intensity of 3000-4000 lux stimulates stomatal development and photosynthetic capacity, and combined with signal enhancement from a low concentration of ABA (5 μmol / L), it helps maintain a suitable water balance in the plants. A low concentration of ABA (0.5-1 mg / L, approximately 1.9-3.8 μmol / L) promotes the growth of tissue culture seedlings. The concentration of 5-10 μmol / L used in this embodiment is slightly higher than this range but far below the half-lethal concentration (approximately 19 μmol / L), thus leveraging the antitranspirant effect of ABA while avoiding excessive inhibition of photosynthesis. The synergistic effect of these three steps increases the transplant survival rate from 76.7% in the basic scheme to 96.7%.

[0091] Example 3 This study investigates the ecological control of latent diseases in Brazilian ginseng tissue culture seedlings after transplanting, addressing the risks of herbicide damage and disease outbreaks associated with conventional chemical control methods. In the later stages of transplanting Brazilian ginseng tissue culture seedlings, especially during hot and humid seasons, vermiculite substrate, while highly permeable, has strong water retention, making it susceptible to root rot pathogens (such as Fusarium oxysporum). Fusarium oxysporum This provides a breeding ground for disease. Example 1, while reducing the risk of pesticide damage by not using other chemical pesticides, may lead to disease outbreaks if chemical control is abandoned. This example introduces *Trichoderma harzianum* (…). Trichoderma harzianum A prevention, establishment, induction, and emergency response control system has been established.

[0092] I. Preparation of Experimental Materials The test material was Brazilian ginseng tissue culture seedlings obtained according to the methods described in steps 1-3 of Example 1. The average length of adventitious roots reached 3.0±0.2 cm. After acclimatization treatment in steps 321-323 of Example 2, the seedlings were disinfected in step 33 and transplanted into seed trays in step 34. At the time of transplanting, the plants were vigorous, uncontaminated, and grew uniformly.

[0093] Regarding the tested strains, Trichoderma harzianum ( Trichoderma harzianum Strains with good and stable tolerance to carbendazim, such as T22, were selected. Preliminary experiments verified that the selected strains exhibited a growth inhibition rate of <10% on PDA medium containing 0.1% carbendazim. The pathogen is *Fusarium oxysporum*, the main pathogen causing root rot in Brazilian ginseng. Fusarium oxysporum ).

[0094] The main reagents and consumables include: carbendazim (50% wettable powder, commercially available); Trichoderma harzianum spore suspension (Trichoderma harzianum is inoculated into PDA medium, cultured at 25°C for 7 days, spores are washed away with sterile water, counted using a hemocytometer, and adjusted to the required concentration); Trichoderma harzianum metabolic broth (Trichoderma harzianum is inoculated into PDB liquid medium, cultured at 28°C and 150 rpm for 7 days, and sterilized by filtration through a 0.22 μm filter membrane); pure vermiculite (particle size 2-4 mm, commercially available); 32-well trays (size 54 cm × 28 cm, volume of each well approximately 50 mL).

[0095] II. Specific Operating Steps Step 351 is the pretreatment with biocontrol agents, carried out 3 days before transplanting. First, a *Trichoderma harzianum* spore suspension is prepared: activated *Trichoderma harzianum* strains are inoculated onto PDA plates and cultured at 25°C for 7 days; after colonies have sporulated, 10 mL of sterile water is added, and the spores are gently scraped and washed with a sterile spreader; the spore suspension is collected by filtration through 4 layers of sterile gauze, counted using a hemocytometer, and the spore concentration is adjusted to 1×10⁻⁶. 7 CFU / mL was used as the stock solution. Then, matrix pretreatment was performed: following the method described in step 34 of Example 2, pure vermiculite was sprayed with a 0.1% carbendazim solution, mixed thoroughly, and covered with a film for 3 days for basic disinfection; on the third day, the film was removed, and the vermiculite was spread out to air dry for 6 hours to remove excess moisture; the pretreated vermiculite was then taken, and the spore suspension was evenly sprayed onto the vermiculite at a mass ratio of 1:80 (spore suspension: vermiculite). Specifically, 125 mL of spore suspension (1×10⁻⁶) was added to every 10 kg of vermiculite. 7 (CFU / mL) was added, and sterile water was simultaneously added to bring the final moisture content of the substrate to 60%-65%. The substrate was sprayed and agitated continuously to ensure even spore distribution. After mixing, it was covered again with a film and left at room temperature for 2 hours to allow spores to fully adsorb onto the surface of the substrate particles. Plate dilution counting showed that the viable bacteria content in the treated substrate reached 3.5 × 10⁻⁶. 6The CFU / g concentration meets the requirements. Finally, the treated bacterial vermiculite is filled into 32-well trays, each well filled to eight-tenths full, and then gently shaken to compact it for later use.

[0096] Step 352 is the rhizosphere colonization induction, which is carried out at transplanting. First, prepare the compound disinfection-colouring solution: Weigh 0.1 g of carbendazim (50% wettable powder), add a small amount of sterile water to make a paste, and dilute to 50 mL with sterile water to obtain a 0.1% carbendazim solution (based on active ingredient); take 50 mL of the above solution and add 1×10⁻⁶ Trichoderma harzianum spore suspension stock solution. 7 Add 15 mL of carbendazim (CFU / mL) and sterile water to a total volume of 100 mL to obtain a compound solution containing 0.1% carbendazim and 1.5 × 10⁻⁶ Trichoderma harzianum spores. 6 CFU / mL. Then, gently arrange the roots of the tissue culture seedlings washed in step 33, avoiding damage, and completely immerse the roots in the above compound solution, ensuring all root segments are submerged; soak for 2.5 minutes, gently shaking 2-3 times during this time to ensure the solution fully contacts the root surface; remove and drain excess solution, no rinsing with water is required. Finally, use a thin stick to poke holes in the bacterial vermiculite prepared in step 351, plant the soaked seedlings into the holes, gently compact the substrate around the roots, and immediately water thoroughly with a fine-nozzle sprayer (using clean water) after transplanting.

[0097] Step 353 involves inducing resistance during the latent period, performed on days 7 and 14 post-transplantation. First, a *Trichoderma harzianum* metabolic broth was prepared: *Trichoderma harzianum* was inoculated onto PDB liquid medium and cultured at 28°C and 150 rpm for 7 days with shaking. The culture broth was filtered through four layers of gauze to remove mycelia, and the filtrate was filtered through a 0.22 μm microporous membrane for sterilization, yielding a sterile stock solution of the metabolic broth. HPLC analysis showed that the Harzianolide content in the metabolic broth was approximately 0.5 mg / L. Then, a root irrigation solution was prepared: 10 mL of the stock metabolic broth was taken and diluted to 1000 mL with sterile water to obtain a 100-fold dilution. The Harzianolide content in the dilution was approximately 5 μg / L. The specific steps for root irrigation are as follows: The first root irrigation is carried out at 9:00 AM on the 7th day after transplanting. Using a 50mL measuring cup or a special root irrigation device, apply 12mL of diluted metabolic solution to each plant around the root zone. The second root irrigation is carried out at the same time on the 14th day after transplanting, using the same method to apply 12mL to each plant again. Do not water within 24 hours after root irrigation; just keep the substrate moist.

[0098] Step 354 is the emergency warning trigger, to be carried out within 20 days after transplanting. Plant observation should be conducted daily from 8:00 AM to 9:00 AM, observing the color and texture of the stem base, the presence of white mycelium on the root zone, and the presence of wilting or yellowing leaves. The location of any abnormal plants (planting tray number) should be recorded. Abnormality criteria are: leaf wilting (irreversible drooping of leaves at midday), stem base constriction (brown sunken lesions at the stem base, epidermal constriction), and white mycelium on the root zone (white, fluffy mycelium visible on the substrate surface or at the drainage holes of the planting tray). If a single plant meets any of the above abnormal criteria, it should be treated immediately (without waiting until the next day). The treatment area should be centered on the diseased plant, including two adjacent plants (those adjacent vertically and horizontally within the same planting tray), for a total of three plants. The emergency inoculum solution is prepared by taking a Trichoderma harzianum spore suspension stock solution (1×10⁻⁶). 7 50 mL of (CFU / mL) solution was added to sterile water and brought to a final volume of 100 mL to obtain 5 × 10⁻⁶ CFU / mL. 6 A high-concentration bacterial solution of CFU / mL was applied to each plant, with 20 mL of the solution around the root zone. This treatment was repeated for two consecutive days (treatment was initiated on day 1 upon discovery of the disease, and repeated at the same time on day 2). Recovery was observed on days 3, 5, and 7 post-treatment. Special cases included: if more than 3 scattered diseased plants appeared in the same planting tray, each plant was treated separately as described above; if ≥5 diseased plants appeared in a single day, a comprehensive prevention method was initiated, using 5 × 10⁻⁶ CFU / mL solution. 6 Apply CFU / mL bacterial solution to the roots of all plants once (20 mL / plant); if no new diseased plants appear within 7 days after the emergency treatment, resume normal management.

[0099] III. Effect Verification Experiment To demonstrate the effectiveness of this embodiment, the following comparative examples were set up for effect comparison. The experimental design set up 4 treatment groups, each with 60 tissue-cultured rooted seedlings of Brazilian ginseng (underwent the same acclimatization treatment and had consistent growth): Treatment A (Example 3) was operated completely according to steps 351-354 above; Treatment B (Comparative Example 3-1) was operated only according to step 35 of Example 1 (without the application of any chemical pesticides or Trichoderma treatment); Treatment C (Comparative Example 3-2) was operated only with chemical control (spraying 50% carbendazim at 800 times dilution on the 7th and 14th days after transplanting, and spraying the entire plant when diseased); Treatment D (Comparative Example 3-3) was operated only with Trichoderma control (pretreatment in step 351 + soaking in step 352, but without root irrigation with metabolic solution in step 353 and emergency treatment in step 354).

[0100] To verify the control effect, the pathogen inoculum was first prepared: Fusarium oxysporum was inoculated into PDA medium and cultured at 25°C for 7 days. The spores were then washed away with sterile water, and the spore concentration was adjusted to 1×10⁻⁶. 6CFU / mL. Inoculation method: On the 3rd day after transplanting, each plant was drenched with 5 mL of pathogen spore suspension (in addition to the natural disease observation group, 30 plants were set up in each group for artificial inoculation challenge). Observation indicators included: natural disease incidence (the number of naturally diseased plants was recorded within 30 days after transplanting, no artificial inoculation was required), challenge disease incidence (the number of diseased plants was recorded within 30 days after artificial inoculation), disease index (calculated according to the 0-4 grade standard: 0-no disease; 1-slight yellowing of leaves; 2-wilted leaves, slight lesions at the base of the stem; 3-severe wilting of plants, obvious constriction at the base of the stem; 4-plant death), rhizosphere Trichoderma colonization (5 plants were taken from each treatment at 15 days and 30 days after transplanting, the rhizosphere substrate was shaken off, and the CFU / g of Trichoderma was determined by the dilution plate method), and transplant survival rate (the number of surviving plants was counted 30 days after transplanting).

[0101] Table 11. Effects of different treatments on disease control in Brazilian ginseng tissue culture seedlings Note: Different lowercase letters in the same column indicate significant differences (P<0.05). The experimental results showed that, in terms of natural disease incidence, treatment A had a rate of only 3.3%, significantly lower than treatment B's 26.7% (P<0.05), and also lower than treatment C's 10.0% and treatment D's 6.7%, indicating that the four-level control system in this embodiment can effectively prevent the occurrence of latent diseases under natural conditions. Under the strong stress conditions of artificial inoculation with pathogens, treatment A had a disease incidence of 10.0% and a disease index of 6.7, significantly lower than other treatments; treatment B (no control) had a disease incidence as high as 83.3% and a disease index of 71.3, approaching total crop failure; treatment C (chemical control) had a disease incidence of 46.7% and a disease index of 35.8, which had some effect, but the continuous use of carbendazim caused phytotoxicity to tissue culture seedlings, manifested as leaf edge scorching and stunted growth in some plants; treatment D (basal Trichoderma control only) had a disease incidence of 26.7% and a disease index of 18.3, which was better than chemical control but still not as good as treatment A. Regarding the colonization of *Trichoderma* in the rhizosphere, treatment A had a colonization rate of 28.6 × 10⁻⁶ at 15 and 30 days after transplanting. 5 CFU / g and 19.4×10 5 CFU / g was significantly higher than that of treatment D (15.2 × 10⁻⁶). 5 -CFU / g and 8.6×10 5(CFU / g), which demonstrates the synergistic effect of steps 351 and 352—utilizing the specific tolerance of Trichoderma to carbendazim to achieve "disinfection and inoculation simultaneously," enabling Trichoderma to establish a stable population advantage in the rhizosphere; the active substances in the metabolic solution in step 353 not only induce plant resistance but also provide nutrients for Trichoderma in the rhizosphere to promote its colonization; treatments B and C, because no Trichoderma was used, had no detectable Trichoderma in the rhizosphere. Regarding transplant survival rate, treatment A reached 96.7%, significantly higher than treatment B's 70.0% and treatment C's 83.3%, and also showing an upward trend compared to treatment D's 90.0%; compared to Example 1 step 35 where only no pesticide was applied, this example increased the survival rate by 26.7 percentage points, demonstrating significant economic value in large-scale production.

[0102] The core innovation of this embodiment lies in utilizing the specific tolerance of *Trichoderma* to carbendazim, enabling simultaneous chemical disinfection and biological inoculation. Specifically, in this embodiment, the substrate pretreatment in step 351 establishes the population base of *Trichoderma*, and the composite solution soaking in step 352 achieves simultaneous root surface disinfection and colonization—carbendazim kills the contaminants carried by the roots, creating colonization space for *Trichoderma*, which survives due to its tolerance to carbendazim and occupies the rhizosphere niche first. Step 353, diluting the metabolite 100 times and drenching the roots, not only induces resistance but also provides additional nutritional support for *Trichoderma* in the rhizosphere, explaining why the rhizosphere colonization rate in treatment A is significantly higher than in treatment D. Step 354 employs a point-to-line combined emergency strategy, avoiding interference from whole-plant application to disease-free plants, while using a high concentration (5×10⁻⁶) of the solution. 6 Trichoderma solution (CFU / mL) rapidly inhibits localized outbreaks.

[0103] Example 4 This embodiment addresses the resource waste caused by the direct rejection of abnormal ginseng tissue culture seedlings after transplanting through grading criteria and differentiated rejuvenation treatment, establishing a complete rescue system from mild symptom repair to severe symptom regeneration. In the large-scale production of Brazilian ginseng tissue culture seedlings, even after domestication optimization and disease control, approximately 5%-15% of plants still exhibit abnormal phenotypes such as wilting, yellowing, and growth retardation due to individual differences, latent stress, or mechanical damage. In practice, these abnormal seedlings are often directly rejected, resulting in wasted seedling resources and increased costs.

[0104] I. Preparation of Experimental Materials The test materials were Brazilian ginseng tissue culture seedlings cultured and transplanted in Examples 1-3, and plants exhibiting abnormal phenotypes within 7-21 days after transplanting. The main reagents and consumables included: 6-BA (6-benzylaminopurine, purity ≥98%); NAA (naphthaleneacetic acid, purity ≥98%); MS medium powder (hormone-free); Trichoderma harzianum metabolite broth (prepared according to step 353 of Example 3); paclobutrazol (PP) 33315% wettable powder, commercially available; portable lux meter (range 0-20000 lux, accuracy ±3%); temperature and humidity recorder (range 0-100%RH, accuracy ±2%RH).

[0105] Within 30 days after transplanting, the plants were observed daily from 8:00 to 9:00 AM. Plants exhibiting abnormal phenotypes were marked and graded, and a total of 360 abnormal seedlings were collected for the experiment in this embodiment.

[0106] II. Specific Operating Steps Step 361: Determining the degree of abnormality Phenotypic observations were conducted twice a week, on Tuesdays and Fridays from 8:00 AM to 9:00 AM, from the first 1 to 30 days after transplanting. At this time, sufficient light and suitable temperature facilitated accurate assessment of the degree of wilting. The grading criteria are as follows: Mild abnormality is characterized by slight wilting of leaves (slightly drooping at midday, recovering in the morning and evening), yellowing area <30%, surviving stem tips with visible new leaf sprouting, and sampling examination revealing white root tips with newly formed root hairs; Moderate abnormality is characterized by moderate wilting of leaves (drooping throughout but not completely dry), yellowing area 30%-60%, visible stem tips but stagnant growth with no new leaves, and some root tips showing browning and reduced root hairs; Severe abnormality is characterized by severely wilted or dried-out leaves losing turgor pressure, yellowing area >60%, browning or necrosis of stem tips, and most of the root system showing browning and rotting. For each abnormal seedling, observe and record the degree of leaf wilting and the percentage of yellowing area. Use a magnifying glass to observe the color and vitality of the stem tip. For plants suspected of being severely abnormal, gently separate the root zone substrate to observe the root color and texture (no more than 3 plants should be sampled per treatment group to avoid damage). After comprehensively judging the level, insert the corresponding color mark on the seedling tray: mild abnormality (green mark), moderate abnormality (yellow mark), and severe abnormality (red mark).

[0107] Step 362: Treatment for mild abnormalities A dedicated “rejuvenation and recovery zone” was marked out in the culture room. The light intensity was adjusted to 1000 lux using a shade net (required to be 800-1200 lux). An ultrasonic humidifier was used in conjunction with a timer to control the relative humidity of the environment at 82% (required to be 80%-85%). The temperature was maintained at 22±1℃, and the light cycle was maintained at 10h / d (lighting from 8:00 to 18:00). The method for preparing the rejuvenation nutrient solution is as follows: Prepare 1 mg / mL 6-BA stock solution and 1 mg / mL NAA stock solution separately (dissolve in a small amount of 1M NaOH and then bring to volume); weigh 1.1 g MS powder (without hormones) (normal MS is 4.4 g / L) and 7.5 g sucrose, add deionized water to bring to volume 1 L and adjust the pH to 5.8 to obtain 1 / 4 concentration MS nutrient solution; take 1 L of the above 1 / 4 concentration MS nutrient solution, add 0.5 mL of 6-BA stock solution (final concentration 0.5 mg / L) and 0.1 mL of NAA stock solution (final concentration 0.1 mg / L), mix well and set aside. Plants identified as having mild abnormalities were transferred to the rejuvenation and recovery area along with their seedling trays. A mini handheld sprayer was used to evenly spray the rejuvenation nutrient solution onto both sides of the leaves, ensuring the leaves were moist but not dripping (approximately 1.5-2.0 mL per plant). Spraying was done daily at 9:00 AM (one hour after the start of sunlight), once every three days for three consecutive treatments (days 0, 3, and 6). During the treatment period, the substrate was kept moist (watered once daily). On day 9 (three days after the third treatment), the recovery status was assessed. Recovery criteria included new leaf sprouting, upright leaves, and a reduction of more than 50% in the yellowed area. Plants meeting these criteria could be moved back to the normal management area.

[0108] Step 363: Treatment for moderate abnormalities First, environmental transfer and foliar spraying were performed as basic treatments according to step 362. The compound root irrigation solution was prepared as follows: 200 mL of Trichoderma harzianum metabolic solution (filtered and sterilized through a 0.22 μm filter membrane) prepared according to step 353 of Example 3 was mixed with 800 mL of 1 / 2 concentration MS nutrient solution (2.2 g MS powder, 15 g sucrose, and deionized water to a final volume of 1 L, adjusted to pH 5.8) to obtain a mixed solution (volume ratio 1:5). Before treatment, completely yellowed or withered old leaves were removed with sterilized scissors (leaving about 0.5 cm of the petiole base). Each plant retained the stem tip and 2-3 functional leaves (leaf parts with yellowing area <30%). Immediately after pruning, the scissors were wiped with 75% alcohol cotton balls for disinfection to prevent cross-infection. The root drenching operation is carried out simultaneously with the first foliar spraying (day 0), applying 18 mL of the mixed solution per plant (15-20 mL is required). On day 3, apply another 18 mL using the same method. Do not water for 24 hours after root drenching; simply keep the substrate moist. On day 7, assess the recovery status. The recovery criteria are new leaf sprouting, a reduction in the yellowing area of ​​the previously retained leaves, and significant elongation of the stem tip. If the recovery criteria are not met on day 7, repeat the foliar spraying + root drenching treatment (with the same metabolic solution concentration).

[0109] Step 364: Severe Abnormality Rescue Treatment Before attempting to rescue the plant, prepare fresh, pure vermiculite substrate and pretreat it according to step 34 of Example 1 (spray with 0.1% carbendazim and seal for 3 days). Prepare the hormone soaking solution by adding 0.2 mL of 6-BA stock solution (final concentration 0.2 mg / L) and 0.5 mL of NAA stock solution (final concentration 0.5 mg / L) to 1 L of sterile deionized water and mixing well. Separately prepare a paclobutrazol solution (add 0.067 g of 15% paclobutrazol wettable powder to sterile water to a final volume of 1 L to obtain a 10 mg / L paclobutrazol solution) for severe root rot. Carefully remove the severely affected plant from the seedling tray and gently shake off the substrate from the roots. Use sterilized scissors to cut off the above-ground portion (1-2 cm) 1.5 cm above the stem base, ensuring a clean cut to avoid crushing damage. The remaining basal stem segment should have 1-2 visible nodes or dormant bud primordia. Immerse the cut stem segments completely in the hormone solution for 30 minutes, gently shaking 2-3 times to ensure the solution fully contacts the cut and base. Remove and drain excess solution; no rinsing is necessary. Check the root system: if some roots are browned but the main root is viable, cut off the browned root segments, retaining the white ones; if all roots are browned and rotten, cut them all off, leaving only the stem base. Plant the treated stem segments in freshly sterilized pure vermiculite substrate, burying the base 1-1.5 cm deep. Water thoroughly immediately after planting (with sterile water). Place the planted trays in a shaded area within the "rejuvenation and recovery zone," adjusting the light intensity to 650 lux (ideally 500-800 lux), maintaining humidity at 80%-85%, and a temperature of 22±1℃. Keep the substrate moist but not waterlogged (use a misting system to water, avoiding heavy watering). Observe continuously for 21 days, noting whether new buds sprout from the base, whether callus tissue forms, and whether roots develop. Take photos to record the growth status once a week. After 21 days, calculate the survival rate (those with new buds sprouting or new roots developing are considered to have survived). Special cases: If no sprouting is seen after 21 days but the stem segment remains green, the observation period can be extended to 30 days; if basal rot (browning and softening) is found, remove and discard the stem immediately to avoid contaminating adjacent substrates.

[0110] To verify the effectiveness of this embodiment, the following comparative examples were set up for effect comparison. The experimental design set up 5 treatment groups, with 60 abnormal Brazilian ginseng tissue culture seedlings in each group (abnormal phenotypes appeared at similar times and were evenly distributed in each grade after being graded according to step 361): Treatment A (Example 4) was operated completely according to steps 361-364 above; Treatment B (Comparative Example 4-1) was managed normally according to Example 1 without any rejuvenation treatment (simulating conventional direct elimination); Treatment C (Comparative Example 4-2) only adopted mild abnormality treatment (step 362), and did not differentiate between moderate and severe abnormalities; Treatment D (Comparative Example 4-3) only adopted uniform rejuvenation treatment (no grading, all were treated according to the method in step 363); Treatment E (Comparative Example 4-4) only adopted severe abnormality treatment (no grading, all were treated by cutting off the aboveground parts according to step 364).

[0111] The observation indicators included: recovery rate at each level (the proportion of plants at each abnormal level that recovered to normal growth 21 days after treatment; recovery criteria were new leaf sprouting, upright plant growth, and upright leaves without wilting); overall rescue success rate (the proportion of all abnormal seedlings that successfully recovered to normal 21 days after treatment); recovery period (the number of days from the start of treatment to the appearance of obvious new leaf sprouting); stem diameter growth (the percentage increase in stem base diameter measured with calipers before and 21 days after treatment for successfully recovered plants); secondary abnormality rate (the proportion of successfully recovered plants that re-emerged with abnormal phenotypes within 14 days after being transplanted back to the normal management area); and transplant survival rate (the final number of surviving plants counted 45 days after treatment). The results are as follows: Table 12 Experimental Results of Abnormal Tissue Culture Seedlings of Brazilian Ginseng As can be seen, in terms of graded recovery rates, the recovery rate of mild abnormality treatment A reached 98.3%, which was not significantly different from treatment C (95.0%) but significantly higher than other treatments, indicating that foliar spraying combined with environmental control can effectively salvage mild abnormalities; the recovery rate of moderate abnormality treatment A reached 85.0%, which was significantly higher than treatment C (31.7%) and treatment D (76.7%), indicating that the synergistic treatment of root irrigation with metabolic solution and pruning of old leaves is crucial for moderate abnormalities; the recovery rate of severe abnormality treatment A reached 41.7%, which was not significantly different from treatment E (35.0%) but significantly higher than other treatments, indicating that stem segment regeneration is the only effective approach for plants whose above-ground parts have been severely damaged.

[0112] In terms of overall rescue success rate, treatment A reached 75.0%, which was significantly higher than treatment C (45.0%), treatment D (58.3%) and treatment E (18.9%), and much higher than treatment B (7.8%), indicating that the synergistic effect of the tiered treatment strategy is far superior to any single treatment method.

[0113] Regarding the recovery period, treatment A averaged 9.2 days, significantly shorter than treatments C (15.3 days) and D (12.5 days). Rapid recovery means that plants can return to normal production processes as soon as possible, reducing the occupation of resources in the rejuvenation zone. In terms of stem diameter growth, plants that successfully recovered under treatment A showed a 28.6% increase in stem diameter, significantly higher than other treatments.

[0114] Regarding the secondary abnormality rate, treatment A had a rate of only 5.6%, significantly lower than treatments C (22.2%), D (14.3%), and E (28.6%), indicating that the tiered treatment not only saved the plants but also enhanced their stress resistance and reduced the risk of recurrence through targeted measures. As for the 45-day survival rate, treatment A achieved a final survival rate of 71.7%, close to the overall rescue success rate (75.0%), indicating that most successfully recovered plants could survive stably. Treatment B (direct elimination) had only 7.8% natural recovery, verifying the reality that the vast majority of abnormal seedlings would die without intervention.

[0115] In this embodiment, regarding the synergy between hormone ratio and the degree of abnormality, mild abnormalities were treated with 0.5 mg / L 6-BA + 0.1 mg / L NAA (67% lower 6-BA concentration and 50% lower NAA concentration compared to the hormones in the subculture of Example 1), which promoted cell division without causing overstimulation; severe abnormalities were treated with 0.2 mg / L 6-BA + 0.5 mg / L NAA (similar to the 0.1 / 0.4 hormone ratio in the rooting medium of Example 1, but with slight adjustments), which was more conducive to the induction of adventitious roots. Regarding the synergy between the metabolic solution and the nutrient solution, Harzianolide in the Trichoderma harzianum metabolic solution significantly increased plant biomass and induced systemic resistance. When mixed with 1 / 2 concentration MS nutrient solution at a 1:5 ratio and applied as a root irrigation, it exerted both the induced resistance and growth-promoting effects of the metabolic solution and provided basic nutritional support. Regarding the synergistic effect of stem segment regeneration and hormone induction, after pruning the above-ground parts of severely abnormal plants, 0.2 mg / L 6-BA promotes the germination of dormant buds, and 0.5 mg / L NAA induces the formation of adventitious roots. The two work synergistically to stimulate the regeneration potential of stem segments.

[0116] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for the propagation and efficient transplanting of Brazilian ginseng tissue culture seedlings, characterized in that, Includes the following steps: Step 1: Using MS as the basic medium and adding 6-BA and NAA, start-up induction culture and subculture proliferation culture were carried out to obtain clustered shoots; Step 2: Using WPM as the basic culture medium and adjusting the ratio of 6-BA and NAA, rooting culture was carried out on the clustered shoots to obtain regenerated plants. Step 3: When the average length of the adventitious roots of the regenerated plants reaches 2.5 cm to 3.5 cm, open-top adaptation culture begins. Step 4: Remove the plant, wash off the culture medium adhering to the roots, and disinfect it before transplanting. Step 5: Plant the disinfected plants in a pure vermiculite substrate; Step 6: Keep the substrate moist after transplanting and carry out post-planting management without applying other chemical pesticides.

2. The method as described in claim 1, characterized in that, In step 1, the added 6-BA concentration is 1.0-2.0 mg / L and the NAA concentration is 0.1-0.3 mg / L; in step 2, the added 6-BA concentration is 0.05-0.2 mg / L and the NAA concentration is 0.3-0.5 mg / L.

3. The method according to claim 2, characterized in that, In step 1, the concentration of 6-BA was 1.5 mg / L and the concentration of NAA was 0.2 mg / L; in step 2, the concentration of 6-BA was 0.1 mg / L and the concentration of NAA was 0.4 mg / L; in step 3, the adventitious roots were opened to adapt when the average length reached 3 cm; in step 5, pure vermiculite was filled into the 32-well plate.

4. The method according to claim 1, characterized in that, Step 3, the acclimatization training, includes: initially covering the mouth of the culture bottle with a sterile breathable membrane to reduce the relative humidity inside the bottle to 75%-85% within 12 hours; then removing the breathable membrane and controlling the relative humidity of the environment between 65%-75% through intermittent spraying, while increasing the light intensity to 3000-4000 lux, continuing the training for 1.5-2.5 days.

5. The method according to claim 1, characterized in that, In step 6, post-planting management involves introducing carbendazim-resistant Trichoderma harzianum for biocontrol. Specifically, before transplanting, a carbendazim-resistant Trichoderma harzianum spore suspension is mixed with a pure vermiculite substrate to achieve a viable bacterial content of (2-5) × 10⁻⁶. 6 CFU / g; When transplanting, immerse the plant roots in a 0.1% carbendazim solution containing carbendazim-resistant Trichoderma harzianum spores for 2-3 minutes before planting.

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