Analysis method of culture medium combination based on Pseudostellaria heterophylla seedling cultivation

Abstract

The present invention discloses a culture medium combination analysis method based on the cultivation of Pseudostellaria heterophylla seedlings. The method starts with obtaining detailed initial data and health status of Pseudostellaria heterophylla stem tip explants, and sets cultivation indicators based on these data. Then, multiple generations of culture medium rounds and components are designed and optimized, and the best culture medium formula is evaluated and screened out through the calculation of functional relevance and suitability index. The scheme also includes ultra-low temperature treatment of Pseudostellaria heterophylla stem tip explants to remove viruses, and using statistical methods to analyze growth data. Finally, the culture medium with the best performance is selected for large-scale production of Pseudostellaria heterophylla seedlings to ensure the health, quality and production efficiency of the seedlings.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-agriculture and related industries, and particularly relates to a culture medium combination analysis technology based on Pseudostellaria heterophylla seedling cultivation. Background Art

[0002] With the continuous expansion of the Chinese medicinal material market, Pseudostellaria heterophylla, a precious herb, has attracted widespread attention for its seedling cultivation technology. Traditional methods for cultivating Pseudostellaria heterophylla seedlings primarily include sowing and cutting propagation. While sowing is simple, it suffers from issues such as low germination rates and a long growth cycle. Cutting propagation, as an asexual method, maintains genetic stability but is limited by reproduction speed and seasonal conditions.

[0003] In order to improve the cultivation efficiency of Pseudostellaria baicalensis seedlings, tissue culture technology has been developed. This technology can rapidly reproduce a large number of seedlings through cell proliferation and differentiation under sterile conditions. However, tissue culture technology has high requirements for equipment and technicians, and the cost is relatively high. In addition, stratification treatment, as a method to break the dormancy period of seeds, can improve the germination rate of seeds, but the treatment process is cumbersome and the effect is not always ideal. Therefore, in the prior art, although a variety of methods have been proposed to improve the cultivation of Pseudostellaria baicalensis seedlings, there are still some problems that need to be solved. For example, the low germination rate and slow growth rate of traditional methods, as well as the high cost and technical threshold of tissue culture technology, are all key factors restricting the efficiency and quality of Pseudostellaria baicalensis seedling cultivation.

[0004] Plant detoxification technology is a modern biotechnology, primarily based on plant tissue culture. Its application has promoted the dissemination of improved varieties of fruit trees and crops, ushering in a new technological revolution in agriculture. Using plant detoxification technology to cultivate healthy Chinese herbal medicine seedlings can purify and rejuvenate them, enhancing their resistance and promoting the development of the Chinese herbal medicine seed industry ("Research Progress on Detoxification Technology of Chinese Herbal Medicines," Cai Shike et al., Guangdong Agricultural Sciences, 2021). Therefore, detoxification seedling cultivation is a viable approach for cultivating Pseudostellaria heterophylla seedlings.

[0005] In the prior art, some patent documents have disclosed this. For example, Chinese patent document CN115968763A discloses a method for breeding virus-free Pseudostellaria heterophylla seedlings in a closed environment, and Chinese patent document CN109197594A discloses a method for tissue culture and propagation of virus-free Pseudostellaria heterophylla seedlings.

[0006] In the virus-free seedling raising method, the selection and optimization of the culture medium in tissue culture is a key technical difficulty, which needs to be precisely designed according to the growth characteristics and needs of Pseudostellaria spp. In the virus-free seedling raising method, the components of the culture medium include plant growth regulators, inorganic salts and organic matter, which have an important influence on the growth and differentiation of plant cells. Different plant species and different growth stages have different requirements for the culture medium. For Pseudostellaria spp., how to design a suitable culture medium formula based on its biological characteristics is an important challenge to achieve efficient seedling cultivation.

[0007] Although previous studies have explored the tissue culture of Pseudostellaria heterophylla, some deficiencies remain in the existing technology. For example, most studies focus on optimizing a single factor in the culture medium, lacking a comprehensive consideration of the interactions between culture medium components. Furthermore, the suitability assessment of the culture medium often relies on empirical judgment and lacks a systematic quantitative evaluation method. Therefore, a more scientific and systematic approach is needed to analyze and optimize the culture medium formulation for Pseudostellaria heterophylla. Summary of the Invention

[0008] In response to the deficiencies in the prior art, the present invention proposes a culture medium combination analysis method based on Pseudostellaria pseudoginseng seedling cultivation. This method designs the first generation of culture medium rounds and components by comprehensively analyzing the initial data and health status of Pseudostellaria pseudoginseng stem apex explants, and achieves systematic optimization of the culture medium formula through quantitative evaluation of the functional relevance and suitability index of the culture medium components. In addition, the present invention also proposes a suitability index threshold setting method based on historical data and statistical methods, which provides a scientific basis for the iterative optimization of the culture medium formula. Through the method of the present invention, the cultivation efficiency and quality of Pseudostellaria pseudoginseng seedlings can be effectively improved, and the development of Chinese medicinal material seedling cultivation technology can be promoted.

[0009] The present invention mainly provides a culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation, the method comprising:

[0010] S1, obtaining initial data of a Pseudostellaria chinensis stem apex explant, the initial data of the explant including the stem apex length, the maximum diameter of the stem apex, the number of buds, and the interception time; collecting images of the Pseudostellaria chinensis stem apex explant, analyzing its visual characteristics, and evaluating its health status; and setting Pseudostellaria chinensis seedling cultivation indicators based on the initial data and health status of the Pseudostellaria chinensis stem apex explant;

[0011] S2, designing a first-generation culture medium cycle and culture medium components according to the Pseudostellaria heterophylla seedling cultivation index; the culture medium cycle at least includes a primary culture medium, a secondary proliferation culture medium, and a seedling rooting culture medium; the culture medium components at least include one or a combination of plant growth regulators, inorganic salts, and organic substances;

[0012] S3, according to the culture medium rounds and culture medium components, analyzing the functional correlation of the culture medium rounds and culture medium components with the Pseudostellaria heterophylla seedling cultivation indicators, and analyzing the dependence of the culture medium rounds and culture medium components on the Pseudostellaria heterophylla seedling cultivation indicators;

[0013] S4, performing ultra-low temperature treatment on the Pseudostellaria stem tip explant to remove viruses, and recording the virus-free time; performing tissue culture on the Pseudostellaria stem tip explant, including bud proliferation induction culture, cluster bud induction culture, and seedling rooting culture, according to the first generation culture medium cycle and culture medium components, and monitoring and recording the growth performance of the Pseudostellaria stem tip explant;

[0014] S5, collecting the growth performance data of the Pseudostellaria spp. stem tip explants recorded in the process, including bud proliferation, cluster bud formation and rooting; using statistical methods to analyze the growth data to determine the effect of each culture medium cycle on the Pseudostellaria spp. seedling cultivation index; calculating the culture medium suitability index I suit , evaluate the degree of satisfaction with the cultivation indicators of Pseudostellaria heterophylla seedlings;

[0015] S6, according to the suitability index I of the culture medium calculated for the first time suit , set the suitability index I suit Threshold, and designing culture medium cycles and culture medium components from the second generation to the Nth generation, repeating steps S3-S5, calculating the suitability index of the culture medium from the second generation to the Nth generation, and comparing the suitability index of each generation with the set threshold, until the suitability index of the Nth generation culture medium is higher than the set threshold, and taking the Nth generation culture medium cycle and culture medium components as the optimized culture medium cycle and culture medium components;

[0016] S7, select optimized culture medium cycles and culture medium components for large-scale production of Pseudostellaria heterophylla seedlings.

[0017] In some embodiments, as a further preferred embodiment, the image acquisition of the Pseudostellaria chinensis stem apex explant, analysis of its visual characteristics, and assessment of its health status include performing visual characteristic analysis on the Pseudostellaria chinensis stem apex explant using image acquisition technology, and quantitatively assessing health status indicators including color saturation and texture uniformity using image processing software;

[0018] The setting of the Pseudostellaria spp. seedling cultivation indicators includes combining initial data and health status assessment results to set key growth parameter indicators for Pseudostellaria spp. seedling cultivation, including but not limited to bud length, root length, number of leaves within a specific time, and a detoxification rate for evaluating the detoxification effect.

[0019] In some embodiments, as a further preferred solution, the health status indicator is measured by a health index, and the health index is calculated according to the following formula:

[0020] H h =aC+bT+cS

[0021] Where a, b, and c are weight coefficients determined by importance, a + b + c = 1, and C, T, and S are quantitative scores for color, texture, and shape, respectively, determined by the visual analysis system through a machine learning model.

[0022] The key growth parameter indicators for the cultivation of Pseudostellaria heterophylla seedlings include setting the expected bud length growth rate based on the stem tip length and diameter; setting the minimum percentage of rooting based on the number of bud points; setting the number and size range of leaves within a specific time; and setting the expected proportion of healthy plants after detoxification treatment based on the health index.

[0023] In some embodiments, as a further preferred embodiment, the plant growth regulators in step S2 include but are not limited to auxins and cytokinins, inorganic salts include nitrogen, phosphorus, and potassium sources, and organic matter includes agar and peptone.

[0024] In some embodiments, as a further preferred embodiment, analyzing the functional association between culture medium cycles and culture medium components in step S3 includes calculating attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute association.

[0025] In some embodiments, as a further preferred embodiment, the step of calculating the attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute correlation comprises the following steps:

[0026] First, the attribute weights of the culture medium components are calculated; the attribute weights are determined by analyzing the effects of different component concentrations on growth indicators and using regression analysis to determine the weight of each component;

[0027] The formula is:

[0028]

[0029] Where W i is the weight of the i-th component, ΔG j is the change in growth index in the jth experiment, X ij is the concentration of the i-th component in the j-th experiment;

[0030] After calculating the attribute weights of all components, a correlation matrix is ​​constructed and the Pearson correlation coefficient is used to measure the relationship between the weights of two components:

[0031]

[0032] Where C ij is the element in the i-th row and j-th column of the matrix, W ik is the weight of the i-th component in the k-th experiment, and are the average weights of components i and j respectively;

[0033] Finally, the average correlation between the different components is determined by calculating the mean of the correlation matrix:

[0034]

[0035] Where M is the mean of the correlation matrix, C ij is the element in the i-th row and j-th column in the matrix, and N is the total number of components; the mean of the correlation matrix is ​​used to evaluate the synergistic effect of different culture medium components in the cultivation of Pseudostellaria heterophylla seedlings, thereby guiding the optimization and formula adjustment of the culture medium.

[0036] In some embodiments, as a further preferred embodiment, after the Pseudostellaria zizyphus stem tip explants are subjected to ultra-low temperature treatment in step S4, virus-free seedlings are identified by observing the sections using a transmission electron microscope.

[0037] In some embodiments, as a further preferred embodiment, the suitability index I of the culture medium is calculated in step S5. suit Use the following formula:

[0038]

[0039] Where G i is the growth index of the i-th experiment, is the average growth index, G max and G min are the maximum and minimum growth indicators, respectively.

[0040] In some embodiments, as a further preferred solution, the suitability index I in step S6 is suit The threshold is set based on the following principles:

[0041] I s =I opt +kσ I

[0042] Where, I opt is the best suitability index in history, k is the confidence level coefficient, ranging from 1 to 3, σ I is the standard deviation of the suitability index.

[0043] Advantages and beneficial effects of the present invention:

[0044] This invention significantly improves the efficiency of Pseudostellaria pseudoginseng seedling cultivation through a systematic culture medium combination analysis method. By accurately obtaining initial data and health status of Pseudostellaria pseudoginseng stem apex explants, combined with quantitative health index assessment, the scientific and targeted culture medium design is ensured. This method reduces the uncertainty in traditional cultivation processes, accelerates the growth cycle of seedlings, and thus improves overall cultivation efficiency.

[0045] At the same time, the suitability index introduced in this invention provides a quantitative indicator for evaluating culture medium formulas, making the quality control of seedling cultivation more accurate and objective. By analyzing growth data using statistical methods, the specific impact of each culture medium cycle on the cultivation indicators of Pseudostellaria heterophylla seedlings can be clearly determined, thereby achieving strict control of seedling quality.

[0046] On the other hand, the present invention achieves dynamic optimization of culture medium formulations by iteratively calculating the suitability index and setting a threshold. Comparing the suitability index of each generation of culture medium with the threshold guides the adjustment of culture medium components until the optimal culture medium formulation is found. This method not only improves culture medium utilization efficiency but also reduces resource waste.

[0047] Since the present invention has verified the effectiveness of the culture medium formula through repeated iterations, large-scale production can ensure the consistency and stability of seedlings, meeting the market's large demand for high-quality Pseudostellaria heterophylla seedlings. Therefore, overall, the present invention not only improves the efficiency and quality of Pseudostellaria heterophylla seedling cultivation, but also provides a feasible path for the modernization and standardization of Chinese medicinal materials. This is of great significance for promoting the development of the Chinese medicinal materials industry and improving the international competitiveness of Chinese medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying any creative work, other drawings obtained based on these drawings still fall within the scope of the present invention.

[0049] Figure 1 A flowchart showing an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention will be described in detail below so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the following embodiments are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0051] In the following description, for the purpose of illustrating the various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with this application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0052] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0053] Example 1:

[0054] The present invention mainly provides a culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation, such as Figure 1 As shown, the method includes:

[0055] S1, obtaining initial data of a Pseudostellaria chinensis stem apex explant, the initial data of the explant including the stem apex length, the maximum diameter of the stem apex, the number of buds, and the interception time; collecting images of the Pseudostellaria chinensis stem apex explant, analyzing its visual characteristics, and evaluating its health status; and setting Pseudostellaria chinensis seedling cultivation indicators based on the initial data and health status of the Pseudostellaria chinensis stem apex explant;

[0056] In this step, the explant is first accurately measured to record the length, maximum diameter, number of buds and specific time of interception of the Pseudostellaria chinensis stem tip. These data provide basic growth characteristic information for the cultivation process. Subsequently, the visual characteristics of the Pseudostellaria chinensis stem tip explant are analyzed using image acquisition technology, and indicators such as color saturation and texture uniformity are quantitatively evaluated using image processing software to assess the health status of the explant. Combining the initial data and health assessment results, key growth parameter indicators for Pseudostellaria chinensis seedling cultivation are set, such as bud length, root length, number of leaves and detoxification rate.

[0057] S2, designing a first-generation culture medium cycle and culture medium components according to the Pseudostellaria heterophylla seedling cultivation index; the culture medium cycle at least includes a primary culture medium, a secondary proliferation culture medium, and a seedling rooting culture medium; the culture medium components at least include one or a combination of plant growth regulators, inorganic salts, and organic substances;

[0058] In some embodiments, as a further preferred embodiment, the plant growth regulators in step S2 include but are not limited to auxins and cytokinins, inorganic salts include nitrogen, phosphorus, and potassium sources, and organic matter includes agar and peptone.

[0059] The specific functions of each component are described as follows:

[0060] (1) Plant growth regulators:

[0061] Auxin: Indoleacetic acid (IAA) or naphthaleneacetic acid (NAA), used to promote cell elongation and differentiation, often used in rooting culture media.

[0062] Cytokinin: 6-benzyladenine (6-BA), used to promote cell division and bud proliferation, suitable for proliferation culture medium.

[0063] (2) Inorganic salts:

[0064] Nitrogen source: ammonium nitrate (NH4NO3) and potassium nitrate (KNO3), provide nitrogen elements and promote plant protein synthesis.

[0065] Phosphorus source: Potassium dihydrogen phosphate (KH2PO4) or diammonium hydrogen phosphate (NH4H2PO4), which provides phosphorus, which is essential for plant energy conversion and cell structure.

[0066] Potassium source: Potassium sulfate (K2SO4), provides potassium, which plays an important role in plant enzyme activity and photosynthesis.

[0067] (3) Organic matter:

[0068] Solidifying agent: Agar, used to make solid culture medium and provide fixed support for plant tissue.

[0069] Nutritional supplement: Peptone, which provides amino acids and other nutrients to promote cell growth and division.

[0070] Step S2 designs the round and component of the first generation culture medium based on the cultivation index set in step S1. This step relates to selecting suitable plant growth regulators, such as auxin and cytokinin, to promote cell division and proliferation. Simultaneously, according to the demand of inorganic salts such as nitrogen, phosphorus, potassium source, and the interpolation of organic matter such as agar, peptone, the basic formula of culture medium is constructed. Every kind of culture medium round is all for the specific stage in the Pseudostellaria heterophylla growth cycle, and as the primary generation culture medium focuses on cell adaptation and growth, the secondary generation proliferation culture medium promotes the proliferation of bud, and the strong seedling rooting culture medium strengthens root development.

[0071] Before starting to select plant growth regulators and culture medium components, first refer to the effective plant growth regulators and culture medium components in existing studies and conduct preliminary pilot experiments to determine the approximate formulation range. Determine the specific nutrients and hormones required according to the different growth stages of Pseudostellaria heterophylla (such as primary culture, secondary proliferation, and seedling rooting). For example, primary culture may require a higher concentration of auxin to promote cell division, while the seedling rooting stage may require cytokinin to promote root development.

[0072] Experiments were designed to test different combinations of plant growth regulators and culture medium components. Multifactorial experimental design methods, such as orthogonal experimental design, were used to simultaneously examine the effects of multiple factors on the growth of Pseudostellaria heterophylla. The experimental results were then quantitatively analyzed, and growth indicators of Pseudostellaria heterophylla, such as bud length, root length, and number of leaves, were recorded. Statistical methods, such as analysis of variance (ANOVA) and regression analysis, were used to evaluate the effects of different components on growth indicators and determine the optimal culture medium formula.

[0073] Finally, the interactions and functional correlations between the culture medium components were analyzed. By constructing a correlation matrix and calculating attribute weights, the synergistic effects of different components on the growth of P. heterophylla were evaluated. Based on the experimental results and statistical analysis, the culture medium formula was iteratively optimized. Multiple rounds of experiments are required to fine-tune the concentrations of the components to achieve the best culture effect. In addition, when determining the culture medium formula, the feasibility of large-scale production, including cost-effectiveness, availability of components, and ease of operation, was considered. The impact of laboratory environmental conditions on the culture medium effect was also considered to ensure the stability and consistency of the selected culture medium formula in actual application.

[0074] The following is an example of the first-generation culture medium design for different growth stages, which is divided into three rounds, namely primary culture medium, secondary proliferation culture medium and seedling rooting culture medium.

[0075] The purpose of the primary culture medium is to help explants adapt to the culture environment and activate cell division. In practice, this medium can be directly adapted from Murashige and Skoog (MS) medium. MS is a widely used plant tissue culture medium developed by J. Murashige and F. Skoog in 1962. It contains a range of macronutrients, trace elements, organic compounds, and plant growth regulators, making it suitable for tissue culture of a wide variety of plants.

[0076] For example, the following components are used:

[0077] 1MS culture medium base.

[0078] 1.0 mg / L 6-benzyladenine (6-BA) to promote cell division.

[0079] 0.1 mg / L indoleacetic acid (IAA) was added to promote the initial formation of the root system.

[0080] 30 g / L sucrose was used as the carbon source.

[0081] 8 g / L agar was used as a coagulant.

[0082] The purpose of the subculture proliferation medium is to accelerate the proliferation rate of Pseudostellaria heterophylla and increase the number of buds. The following components are used:

[0083] 1 / 2MS medium to reduce salt concentration and promote faster cell division.

[0084] 2.0 mg / L indolebutyric acid (IBA) replaced IAA to enhance the proliferation effect.

[0085] 0.5mg / L kinetin (KT) promoted the proliferation of buds.

[0086] 15g / L sucrose, adjust the carbon source to meet the needs of rapid proliferation.

[0087] 7g / L agar.

[0088] The purpose of this medium is to promote the rooting of seedlings in preparation for transplanting into solid medium. It uses the following components:

[0089] 1 / 4MS culture medium further reduces the nutrient concentration and prepares the seedlings to adapt to the external environment.

[0090] 0.2mg / L naphthaleneacetic acid (NAA) promotes root development.

[0091] 10g / L sucrose, gradually reducing the carbon source to adapt to the metabolic changes of seedlings.

[0092] No agar is used, so that the culture medium is in liquid state, which is convenient for the roots to absorb nutrients.

[0093] 1%-2% activated carbon can be added to improve water quality and provide microbial inhibition.

[0094] Depending on the specific needs of your P. heterophylla, you may need to add the following: a trace element and vitamin blend to ensure all the nutrients necessary for growth. Antioxidants, such as ascorbic acid, to protect cells from oxidative stress. Osmolytes, such as polyvinylpyrrolidone (PVP), to help plant cells absorb water.

[0095] When designing a culture medium, the concentrations of the above components should be continuously adjusted based on experimental results and statistical analysis to achieve the best culture effect. In addition, the pH value of the culture medium is usually adjusted to around 5.8 to meet the growth requirements of plant cells.

[0096] S3, according to the culture medium rounds and culture medium components, analyzing the functional correlation of the culture medium rounds and culture medium components with the Pseudostellaria heterophylla seedling cultivation indicators, and analyzing the dependence of the culture medium rounds and culture medium components on the Pseudostellaria heterophylla seedling cultivation indicators;

[0097] In step S3, the functional correlation and dependency between the culture medium rounds and components and the Pseudostellaria spp. seedling cultivation indicators are further analyzed. This includes calculating the attribute weights of the culture medium components, constructing a correlation matrix, and quantitatively evaluating the synergistic effects between different components by calculating the matrix mean. In this way, the key components that have a significant impact on the Pseudostellaria spp. seedling cultivation effect, as well as the interactions between them, can be identified. This analysis helps to optimize the culture medium formula and ensure that the culture medium can meet the specific nutritional and environmental needs of Pseudostellaria spp. at different growth stages. Through these steps, a set of scientific culture medium design and optimization processes can be formed to provide strong support for the efficient cultivation of Pseudostellaria spp. seedlings.

[0098] S4, performing ultra-low temperature treatment on the Pseudostellaria stem tip explant to remove viruses, and recording the virus-free time; performing tissue culture on the Pseudostellaria stem tip explant, including bud proliferation induction culture, cluster bud induction culture, and seedling rooting culture, according to the first generation culture medium cycle and culture medium components, and monitoring and recording the growth performance of the Pseudostellaria stem tip explant;

[0099] In step S4, the explant is first subjected to ultra-low temperature treatment to remove viruses. This step utilizes the inhibitory effect of low temperature on viruses, by exposing the Radix Pseudostellariae stem tip explant to an ultra-low temperature environment (usually liquid nitrogen temperature, about -196 ℃), and the duration is based on the experimental preset, generally ranging from a few minutes to tens of minutes. After ultra-low temperature treatment, the specific detoxification time is recorded. This data is crucial for evaluating the detoxification effect. Subsequently, the explant treated with ultra-low temperature is transferred to a pre-designed first-generation culture medium. These culture media are prepared according to the cultivation indicators and culture medium components determined in steps S1-S3, and at least include primary culture medium, subculture proliferation culture medium and seedling rooting culture medium. During tissue culture, bud value-added induction, cluster bud induction and seedling rooting culture are carefully monitored, and the growth performance of the explant, such as bud length, root length and leaf development, is recorded.

[0100] S5, collecting the growth performance data of the Pseudostellaria stem tip explants recorded in the process, including bud proliferation, cluster bud formation, and rooting; using statistical methods to analyze the growth data to determine the effects of each culture medium cycle on the Pseudostellaria seedling cultivation indicators; calculating the suitability index Isuit of the culture medium to evaluate its satisfaction with the Pseudostellaria seedling cultivation indicators;

[0101] In step S5, collect the Pseudostellariae stem apex explant growth performance data recorded in step S4. These data include but are not limited to the quantity of bud increment, the formation situation of clustered buds and the efficiency of rooting. In order to ensure the accuracy and comparability of the data, all measurements and records should be carried out according to standardized operating procedures. Utilize statistical methods, such as analysis of variance (ANOVA) and regression analysis, the growth data collected are deeply analyzed. Through these analyses, the specific impact of different culture medium rounds on the Pseudostellariae seedling cultivation index can be determined, and the most effective culture medium formula is identified. In addition, calculate the suitability index I of the culture medium. suit This index comprehensively considers the relationship between growth indicators and culture medium formula, providing a quantitative evaluation tool for evaluating culture medium formula.

[0102] S6, according to the suitability index I of the culture medium calculated for the first time suit , set the suitability index I suit Threshold, and designing culture medium cycles and culture medium components from the second generation to the Nth generation, repeating steps S3-S5, calculating the suitability index of the culture medium from the second generation to the Nth generation, and comparing the suitability index of each generation with the set threshold, until the suitability index of the Nth generation culture medium is higher than the set threshold, and taking the Nth generation culture medium cycle and culture medium components as the optimized culture medium cycle and culture medium components;

[0103] In step S6, we first calculate the suitability index I in step S5. suit To set a threshold for the suitability index. This threshold can be based on the historical optimal suitability index I op t, plus a confidence level coefficient k multiplied by the standard deviation σ of the suitability index I When choosing k, consider the robustness of the experiment and your risk tolerance, typically choosing a value between 1 and 3. Next, design the second through Nth generation culture media and their composition. These designs should be based on the functional associations analyzed in step S3 and the growth data from step S5. Repeat steps S3 through S5, calculating the suitability index for each generation and comparing it to a threshold. This iterative process continues until the suitability index for a given generation exceeds the threshold, at which point the culture media for that generation is considered the optimized formulation.

[0104] If, in step S6, the suitability index of the Nth generation culture medium is always lower than the set threshold, it indicates that the current culture medium formula fails to achieve the expected cultivation effect. In this case, one or a combination of the following methods should be adopted for adjustment:

[0105] (1) Review the calculation method of the suitability index and the growth data on which it is based to ensure the accuracy of the data and the scientific nature of the analysis method. Check for any experimental errors or data interpretation bias.

[0106] (2) Review the functional correlation analysis of culture medium cycles and components in step S3, and further explore the interactions between different components and their specific effects on the growth of P. heterophylla.

[0107] (3) Based on existing experimental data and functional correlation analysis, adjust the culture medium formula, including changing the type or concentration of plant growth regulators, adjusting the ratio of inorganic salts and organic matter, etc.

[0108] (4) Evaluate the effects of the culture environment on the growth of P. heterophylla, such as temperature, humidity, and light, and consider the interactions between these factors and the culture medium formulation.

[0109] (5) In subsequent culture medium design, consider a wider range of variables, such as different culture medium brands, other types of organic matter, and inorganic salt sources.

[0110] (6) Increase the number of repetitions of each generation of culture medium experiments to obtain more reliable data and reduce the impact of accidental errors.

[0111] S7, select optimized culture medium cycles and culture medium components for large-scale production of Pseudostellaria heterophylla seedlings.

[0112] In step S7, select the optimized culture medium round and culture medium components determined in step S6, for the large-scale production of Radix Pseudostellariae seedling.This selection is based on the highest value of suitability index, ensures that selected culture medium can meet the growth demand of Radix Pseudostellariae to the greatest extent.Before large-scale production, carry out small-scale pilot test, to verify the performance of optimized culture medium in actual production environment.This includes monitoring the key indicators such as growth rate, rooting rate, leaf development of seedling, ensures stability and consistency of culture medium.Once the pilot test result is satisfactory, just can start large-scale production, utilize optimized culture medium round and component to cultivate Radix Pseudostellariae seedling in a large number.

[0113] On the whole, in the complete steps of the present invention, the explant initial data and the health status obtained in step S1 are the basis of culture medium design in step S2, and the culture medium formula of step S2 design has direct influence on the tissue culture effect in step S4. Functional correlation analysis and suitability index calculation in step S3 provide theoretical support for step S4, have guided the optimization direction of culture medium. The statistical analysis of growth data and the calculation of suitability index in step S5 provide scientific basis for the further optimization of culture medium in step S6, have guaranteed the scientificity and systematicness of whole culture medium optimization process. By this coherent process, can continuously iterate and optimize culture medium formula, finally realize the large-scale production of Pseudostellaria heterophylla seedling cultivation.

[0114] In some embodiments, as a further preferred embodiment, the image acquisition of the Pseudostellaria chinensis stem apex explant, analysis of its visual characteristics, and assessment of its health status include performing visual characteristic analysis on the Pseudostellaria chinensis stem apex explant using image acquisition technology, and quantitatively assessing health status indicators including color saturation and texture uniformity using image processing software;

[0115] The setting of the Pseudostellaria spp. seedling cultivation indicators includes combining initial data and health status assessment results to set key growth parameter indicators for Pseudostellaria spp. seedling cultivation, including but not limited to bud length, root length, number of leaves within a specific time, and a detoxification rate for evaluating the detoxification effect.

[0116] In some embodiments, as a further preferred solution, the health status indicator is measured by a health index, and the health index is calculated according to the following formula:

[0117] H h =aC+bT+cS

[0118] Where a, b, and c are weight coefficients determined by importance, a + b + c = 1, and C, T, and S are quantitative scores for color, texture, and shape, respectively, determined by the visual analysis system through a machine learning model.

[0119] The key growth parameter indicators for the cultivation of Pseudostellaria heterophylla seedlings include setting the expected bud length growth rate based on the stem tip length and diameter; setting the minimum percentage of rooting based on the number of bud points; setting the number and size range of leaves within a specific time; and setting the expected proportion of healthy plants after detoxification treatment based on the health index.

[0120] In some embodiments, as a further preferred embodiment, analyzing the functional association between culture medium cycles and culture medium components in step S3 includes calculating attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute association.

[0121] In some embodiments, as a further preferred embodiment, the step of calculating the attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute correlation comprises the following steps:

[0122] First, the attribute weights of the culture medium components are calculated; the attribute weights are determined by analyzing the effects of different component concentrations on growth indicators and using regression analysis to determine the weight of each component;

[0123] The formula is:

[0124]

[0125] Where W iis the weight of the i-th component, ΔG j is the change in growth index in the jth experiment, X ij is the concentration of the i-th component in the j-th experiment;

[0126] After calculating the attribute weights of all components, a correlation matrix is ​​constructed and the Pearson correlation coefficient is used to measure the relationship between the weights of two components:

[0127]

[0128] Where C ij is the element in the i-th row and j-th column of the matrix, W ik is the weight of the i-th component in the k-th experiment, and are the average weights of components i and j respectively;

[0129] Finally, the average correlation between the different components is determined by calculating the mean of the correlation matrix:

[0130]

[0131] Where M is the mean of the correlation matrix, C ij is the element in the i-th row and j-th column in the matrix, and N is the total number of components; the mean of the correlation matrix is ​​used to evaluate the synergistic effect of different culture medium components in the cultivation of Pseudostellaria heterophylla seedlings, thereby guiding the optimization and formula adjustment of the culture medium.

[0132] As the core indicator of the correlation matrix, the mean value M provides a quantitative perspective to evaluate the interactions and synergistic effects between different culture medium components. In the cultivation of Pseudomonas heterophylla seedlings, by calculating the mean of the correlation coefficients of each component in the matrix, M reflects the combined impact of each component in the culture medium on seedling growth indicators. A higher M value indicates that there is a positive synergistic effect between the culture medium components, which promotes the healthy growth of the seedlings, while a lower M value indicates mutual restraint between the components or no obvious synergistic effect. Therefore, the M value serves as the basis for optimizing the culture medium formula. By adjusting the concentration or type of each component, its synergistic effect can be enhanced, thereby improving the overall cultivation effect of the culture medium.

[0133] For example, if the correlation coefficients of certain components are found to be lower than the mean M, the proportions of these components in the culture medium or their replacement components can be reconsidered to achieve better cultivation results. In this way, M not only quantifies the interactions between culture medium components but also provides scientific guidance for the precise design and adjustment of culture medium during the cultivation of Pseudomonas heterophylla seedlings.

[0134] In some embodiments, as a further preferred embodiment, after the Pseudostellaria zizyphus stem tip explants are subjected to ultra-low temperature treatment in step S4, virus-free seedlings are identified by observing the sections using a transmission electron microscope.

[0135] In step S4, the Pseudostellaria spp. stem tip explant is first subjected to ultra-low temperature treatment, the purpose of which is to use the destructive effect of low temperature on virus particles to achieve detoxification of seedlings. In specific implementation, the pre-treated Pseudostellaria spp. stem tip explant is placed in liquid nitrogen (-196°C) for short-term exposure, and the time is usually controlled within a few minutes to ensure that the virus can be effectively removed without causing irreversible damage to plant cells. After the treatment is completed, the explant is quickly transferred to a room temperature environment to restore its normal physiological state.

[0136] After cryogenic treatment, transmission electron microscopy (TEM) observation of the explants is necessary to verify the detoxification effect. First, the treated P. pseudoginseng stem tip explants are selected and aseptically cut into thin slices using a sharp blade. These slices must be thin enough to allow the electron microscope to penetrate and clearly observe the internal cellular structures. Next, the slices are mounted on slides designed for electron microscopy and stained as necessary to enhance the contrast of the intracellular structures.

[0137] Place the prepared slide in a transmission electron microscope for observation. By adjusting the microscope's magnification and focus, carefully observe the cells for the presence of virus particles. Virus particles typically appear as tiny structures with a specific morphology, distinct from the natural structure of plant cells. Through careful observation and comparison, determine whether the explant contains virus particles.

[0138] The effectiveness of the detoxification treatment is evaluated based on the results of transmission electron microscopy observations. If no virus particles are found in the observed cell samples, the explant can be considered to have met the detoxification standard. For detoxified seedlings confirmed to be free of virus particles, the treatment conditions, observation results, and related microscope parameters should be recorded in detail for subsequent research and production use. These records are of great significance for improving the detoxification process, increasing the detoxification rate, and ensuring the quality of seedlings. At the same time, these detoxified seedlings are used in subsequent tissue culture steps to cultivate healthy Pseudostellaria heterophylla seedlings.

[0139] In some embodiments, as a further preferred embodiment, the suitability index I of the culture medium is calculated in step S5. suit Use the following formula:

[0140]

[0141] Where G i is the growth index of the i-th experiment, is the average growth index, G max and G minare the maximum and minimum growth indicators, respectively.

[0142] In some embodiments, as a further preferred solution, the suitability index I in step S6 is suit The threshold is set based on the following principles:

[0143] I s =I opt +kσ I

[0144] Where, I opt is the best suitability index in history, k is the confidence level coefficient, ranging from 1 to 3, σ I is the standard deviation of the suitability index.

[0145] The confidence level coefficient k is determined based on the desired confidence level and risk tolerance. In statistics, the confidence level indicates how confident we are that a sample statistic is close to a population parameter. The confidence level coefficient k is used to determine the width of the confidence interval, which reflects our confidence in the result.

[0146] Choosing a confidence level requires first determining the desired confidence level. Common confidence levels are 90%, 95%, and 99%. The higher the confidence level, the wider the confidence interval and the larger the k value required. Use a standard normal distribution table to find the z-value corresponding to the selected confidence level. For example, for a 95% confidence level, the z-value is approximately 1.96.

[0147] At the same time, consider the risk tolerance of the experiment. If you can accept a greater risk (i.e. a wider confidence interval), you can choose a higher k value.

[0148] Value range: The value of k is usually between 1 and 3, depending on the required confidence level. For example: for a 90% confidence level, k is close to 1.645. For a 95% confidence level, k is about 1.96. For a 99% confidence level, k is close to 2.576. Applied in the threshold setting of the suitability index suitIsuit:

[0149] Suitability index I for seedling cultivation of Pseudostellaria heterophylla suit When selecting the k value for the threshold, consider the stability and reliability requirements of the training process. If the training process requires high precision, a higher confidence level may be necessary, resulting in a larger k value. Conversely, if the process can tolerate a certain degree of uncertainty, a lower k value can be selected.

[0150] Anything not described in detail in the present invention is well known to those skilled in the art.

[0151] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation, characterized in that: The method comprises: S1, obtaining initial data of the Pseudostellaria chinensis stem apex explant, wherein the initial data of the explant includes the stem apex length, the maximum stem apex diameter, the number of buds and the interception time; collecting images of the Pseudostellaria chinensis stem apex explant, analyzing its visual characteristics and evaluating its health status; and setting the Pseudostellaria chinensis seedling cultivation index according to the initial data and health status of the Pseudostellaria chinensis stem apex explant; S2, designing the first generation culture medium cycle and culture medium components according to the Pseudostellaria heterophylla seedling cultivation index; the culture medium cycle at least includes primary culture medium, secondary proliferation culture medium, and seedling rooting culture medium; the culture medium components at least include one or a combination of plant growth regulators, inorganic salts, and organic substances; S3, according to the culture medium rounds and culture medium components, analyzing the functional correlation of the culture medium rounds and culture medium components with the Pseudostellaria heterophylla seedling cultivation indicators, and analyzing the dependence of the culture medium rounds and culture medium components on the Pseudostellaria heterophylla seedling cultivation indicators; S4, subjecting the Pseudostellariae Radix stem tip explant to ultra-low temperature treatment to remove viruses, and recording the virus-free time; subjecting the Pseudostellariae Radix stem tip explant to tissue culture including bud proliferation induction culture, cluster bud induction culture and seedling rooting culture according to the first generation culture medium round and culture medium components, and monitoring and recording the growth performance of the Pseudostellariae Radix stem tip explant; S5, collecting the growth performance data of the Pseudostellaria heterophylla stem tip explants recorded in the process, including bud proliferation, cluster bud formation and rooting; using statistical methods to analyze the growth data to determine the effect of each culture medium cycle on the Pseudostellaria heterophylla seedling cultivation index; calculating the culture medium suitability index I suit , evaluate the degree of satisfaction with the cultivation indicators of Pseudostellaria heterophylla seedlings; S6, according to the suitability index I of the culture medium calculated for the first time suit , set the suitability index I suit Threshold, and designing the culture medium cycles and culture medium components from the second generation to the Nth generation, repeating steps S3-S5, calculating the suitability index of the culture medium from the second generation to the Nth generation, and comparing the suitability index of each generation with the set threshold, until the suitability index of the Nth generation culture medium is higher than the set threshold, and taking the Nth generation culture medium cycles and culture medium components as the optimized culture medium cycles and culture medium components; S7, selecting optimized culture medium cycles and culture medium components for large-scale production of Pseudostellaria heterophylla seedlings; Step S5 calculates the suitability index I of the culture medium suit Use the following formula: In the formula, G i is the growth index of the ith experiment, is the average growth index, G max and G min are the maximum and minimum growth indices, respectively; The suitability index I in step S6 suit The threshold is set based on the following principles: IN s =I opt +kσ I In the formula, I opt is the best suitability index in history, k is the confidence level coefficient, ranging from 1 to 3, σ I is the standard deviation of the suitability index.

2. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 1, characterized in that, The image acquisition of the Pseudostellaria heterophylla stem tip explant is performed to analyze its visual characteristics and evaluate Its health status includes using image acquisition technology to analyze visual characteristics of the Pseudostellaria heterophylla stem tip explant, and quantitatively evaluating health status indicators including color saturation and texture uniformity through image processing software; The setting of the Pseudostellaria heterophylla seedling cultivation indicators includes combining initial data and health status assessment results to set key growth parameter indicators for Pseudostellaria heterophylla seedling cultivation, including but not limited to bud length, root length, number of leaves within a specific time, and a detoxification rate for evaluating the detoxification effect.

3. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 2, characterized in that, The health status indicator is measured by a health index, which is calculated according to the following formula: H h =aC+bT+cS In the formula, a, b, c are weight coefficients determined according to importance, a+b+c=1, C, T, S are quantitative scores of color, texture, and shape, respectively, which are determined by the visual analysis system through the machine learning model; The key growth parameter indicators for setting the cultivation of Pseudostellaria heterophylla seedlings include setting the expected bud length growth rate based on the stem tip length and diameter; setting the minimum percentage of rooting based on the number of bud points; setting the number and size range of leaves within a specific time; and setting the expected proportion of healthy plants after detoxification treatment based on the health index.

4. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 1, characterized in that, The plant growth regulators in step S2 include but are not limited to auxins and cytokinins, inorganic salts include nitrogen, phosphorus and potassium sources, and organic substances include agar and peptone.

5. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 1, characterized in that, Analyzing the functional associations between culture medium cycles and culture medium components in step S3 includes calculating attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute association.

6. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 5, characterized in that, The step of calculating the attribute weights corresponding to the culture medium components, constructing a correlation matrix, and calculating the matrix mean to determine the attribute correlation comprises the following steps: First, the attribute weights of the culture medium components are calculated; the attribute weights are determined by using regression analysis to determine the weight of each component by analyzing the effects of different component concentrations on growth indicators; The formula is: Where W i is the weight of the ith component, ΔG j is the change in growth index in the jth experiment, X ij is the concentration of the i-th component in the j-th experiment; After calculating the attribute weights of all components, a correlation matrix is ​​constructed, and the Pearson correlation coefficient is used to measure the relationship between the weights of two components: In the formula, C ij is the element in the i-th row and j-th column of the matrix, W ik is the weight of the i-th component in the k-th experiment, and are the average weights of components i and j respectively; Finally, the average correlation between the different components is determined by calculating the mean of the correlation matrix: Where M is the mean of the correlation matrix, C ij is the element in the i-th row and j-th column in the matrix, and N is the total number of components; the mean of the correlation matrix is ​​used to evaluate the synergistic effect of different culture medium components in the cultivation of Pseudospermum heterophyllum seedlings, thereby guiding the optimization and formula adjustment of the culture medium.

7. The culture medium combination analysis method based on Pseudostellaria heterophylla seedling cultivation according to claim 1, characterized in that, After the Pseudostellaria heterophylla stem tip explants are subjected to ultra-low temperature treatment in step S4, virus-free seedlings without virus particles are identified by transmission electron microscopy section observation.

8. A computer program product, characterized in that The product comprises a storage medium and computer-readable instructions stored on the medium, wherein the instructions are configured to perform the method of any one of claims 1 to 7 when executed.

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