A growth regulating system and method for a nutrient medium of a tissue culture seedling of miscanthus

Abstract

The application discloses a kind of fungus grass tissue culture seedling nutrient medium growth regulation system and regulation method, belong to plant tissue culture technical field.A kind of fungus grass tissue culture seedling nutrient medium growth regulation system and regulation method, comprising: nutrient medium deployment and supply unit, for deployment and provide at least two different components nutrient medium solution to tissue culture seedling according to control instruction;Environmental parameter regulation unit, for adjusting at least one physical environmental parameter in the growth environment of tissue culture seedling;Monitoring unit, for real-time or timing obtaining the growth state information of tissue culture seedling;Control unit is respectively connected with the nutrient medium deployment and supply unit, environmental parameter regulation unit and monitoring unit communication;The control unit is configured with the synergistic regulation strategy of fungus grass tissue culture seedling in different growth and development stages;The present application is to solve the significant problems in the prior art, such as static nutrition medium, mutual fragmentation of regulation factors, insufficient fungus grass variety adaptability.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a growth regulation system and method for a nutrient medium for Juncao tissue culture seedlings. Background Technology

[0002] Juncao refers to herbaceous plants that can be used as cultivation materials for edible and medicinal fungi, mainly including Poaceae varieties such as Giant Napier Grass, Oasis No. 1, and Oasis No. 3. Juncao is characterized by its large biomass, rapid growth, and strong adaptability, making it not only valuable as a substitute for wood in the cultivation of edible and medicinal fungi, but also showing broad application prospects in livestock feed, biomass energy, and ecological restoration. With the continuous expansion of the Juncao industrialization, the demand for high-quality, uniform, and pest-free Juncao seedlings is increasing. Tissue culture rapid propagation (referred to as "tissue culture rapid propagation") technology has become a key technical approach to ensure the supply of Juncao seedlings.

[0003] The following shortcomings still exist in existing technologies, which restrict the large-scale, industrialized, and high-quality production of Juncao tissue culture seedlings:

[0004] (1) Lack of dynamic nutrition regulation capability. Most of the existing Juncao tissue culture nutrient media are static systems with fixed formulas. They cannot dynamically adjust the culture medium components according to the real-time changes in nutritional requirements or growth status feedback of tissue culture seedlings at different developmental stages. The regulation process is lagging, has poor precision, and poses a risk of contamination.

[0005] (2) Nutrient substrate regulation and environmental factor regulation are separated. In the existing technology, nutrient substrate component regulation (culture medium formulation adjustment) and environmental factor regulation (control of physical parameters such as light, temperature and humidity) are usually analyzed and operated as independent modules. There is a lack of systematic synergistic regulation mechanism between the two. Under the condition of only regulating environmental parameters while ignoring the adaptive adjustment of nutrient substrate formulation (or vice versa), the growth status of tissue culture seedlings is often difficult to reach the optimal level.

[0006] (3) Lack of systematic adaptation to the differences in Juncao varieties. Different varieties and types of Juncao (such as giant Juncao, Oasis No. 1, Oasis No. 3, etc.) have significant genotypic differences in nutritional requirements and hormone responses. The existing general tissue culture environment regulation system is not targeted enough and lacks a specialized regulation parameter system and adaptive regulation strategy for different Juncao varieties, making it difficult to meet the technical needs of parallel development of multiple Juncao varieties. Summary of the Invention

[0007] The purpose of this invention is to address the significant technical defects in the existing technology, such as static nutrient substrates, fragmented regulatory factors, and insufficient compatibility with different varieties of Juncao grass, and to propose a growth regulation system and method for the nutrient substrate of Juncao grass tissue culture seedlings.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A growth regulation system for a nutrient medium for tissue culture seedlings of Juncao grass includes: a nutrient medium preparation and supply unit for preparing and supplying a nutrient medium solution of at least two different components to the tissue culture seedlings according to control instructions;

[0010] An environmental parameter adjustment unit is used to adjust at least one physical environmental parameter in the growth environment of tissue culture seedlings.

[0011] The monitoring unit is used to acquire information on the growth status of tissue culture seedlings in real time or at regular intervals.

[0012] The control unit is communicatively connected to the nutrient base preparation and supply unit, the environmental parameter adjustment unit, and the monitoring unit, respectively.

[0013] The control unit is equipped with a synergistic regulation strategy for the tissue culture seedlings at different growth and development stages; this synergistic regulation strategy defines the dynamic matching relationship between nutrient component parameters and environmental parameters at each stage;

[0014] The control unit is configured to: determine the current growth stage of the tissue culture seedling based on the growth status information obtained by the monitoring unit, generate a set of coordinated regulation instructions according to the coordinated regulation strategy, and send them simultaneously to the nutrient substrate preparation and supply unit and the environmental parameter adjustment unit, so as to coordinately adjust the supplied nutrient substrate components and the physical environmental parameters within the same regulation cycle.

[0015] Preferably, the nutrient base preparation and supply unit includes multiple plant growth regulator stock solution storage tanks and nutrient element mother liquor storage tanks. The control unit dynamically changes the concentration ratio of cytokinins and auxins in the output nutrient base by adjusting the ratio of liquids in different storage tanks.

[0016] Preferably, the synergistic regulation strategy includes: when the tissue culture seedling is determined to be in the subculture proliferation stage, the generated synergistic regulation instruction increases the mass concentration ratio of cytokinin to auxin in the nutrient medium, while increasing the ambient light intensity and / or prolonging the photoperiod; when the tissue culture seedling is determined to be in the rooting induction stage, the generated synergistic regulation instruction decreases the mass concentration ratio, while decreasing the ambient light intensity and / or shortening the photoperiod.

[0017] Preferably, the monitoring unit includes an image acquisition device and an image processing module. The growth status information includes plant height, number of adventitious buds, leaf area and / or root length information extracted from the image. The control unit determines the growth stage of the tissue culture seedling based on this information and its rate of change.

[0018] Preferably, the control unit stores a database of dedicated synergistic regulation strategies for different Juncao varieties, including at least one of Giant Napier Grass, Oasis No. 1, and Oasis No. 3. The databases for different varieties define the optimal combination of nutrient substrate components and physical environment parameters specific to each variety at each stage of initiation culture, subculture proliferation, and rooting induction.

[0019] Preferably, the method includes the following steps: S1, selecting the target Juncao variety and calling the corresponding collaborative adjustment strategy database in the control unit;

[0020] S2. During the cultivation process, the monitoring unit acquires real-time growth status information of the tissue culture seedlings;

[0021] S3. The control unit compares the acquired growth status information with the preset stage transition conditions, determines the current growth stage, and calculates the target values ​​of the nutrient matrix components and environmental parameters to be adjusted according to the coordinated adjustment strategy of the stage.

[0022] S4. The control unit sends an adjustment command to the nutrient substrate preparation and supply unit to adjust the nutrient substrate components supplied to the tissue culture seedlings to the target value, and simultaneously sends an adjustment command to the environmental parameter adjustment unit to adjust the physical environmental parameters to the corresponding target value.

[0023] S5. Execute steps S2 to S4 cyclically throughout the entire culture cycle of the tissue culture seedlings to achieve continuous, coordinated, and dynamic adjustment of nutrient substrate components and environmental parameters.

[0024] Preferably, in step S3, the synergistic regulation strategy is a mathematical model based on the response surface methodology to determine the influence of nutrient factors and environmental factors on the growth rate of tissue culture seedlings, or a decision model based on expert experience rules.

[0025] Preferably, the method further includes: when the monitoring unit detects that the tissue culture seedlings exhibit browning or vitrification, the control unit generates an emergency adjustment command to compensate for and coordinate the osmotic pressure of the nutrient medium, the hormone ratio, and the temperature and light intensity of the environment.

[0026] Preferably, the physical environment parameters in step S4 include at least two of temperature, humidity, light intensity, light cycle, and CO2 concentration; the target values ​​of the nutrient substrate components include a combination of macro-element concentration, micro-element concentration, carbon source concentration, and types and concentrations of plant growth regulators.

[0027] Preferably, in step S4, when the control unit performs coordinated adjustment of environmental parameters and nutrient substrate components, it adopts a time-delay strategy: first adjusts the environmental parameters, and after a preset plant physiological response delay time, gradually adjusts the nutrient substrate components to reduce the physiological stress caused by the same step to the tissue culture seedlings.

[0028] Compared with the prior art, the present invention provides a growth regulation system and method for the nutrient medium of fungal tissue culture seedlings, which has the following beneficial effects:

[0029] 1. This invention achieves deep synergistic regulation of nutrient substrate components and environmental parameters, significantly improving the growth quality of tissue culture seedlings. Through a preset synergistic regulation strategy in the control unit, the optimal ratio of nutrient substrate hormones and environmental factors such as light and temperature are matched synchronously at different developmental stages of the tissue culture seedlings. This overcomes the defect of the separation between nutrient regulation and environmental regulation in the prior art. Since the absorption and response of plants to hormones are highly dependent on environmental conditions, this synergistic regulation can keep the tissue culture seedlings in the optimal physiological state at all times, effectively reducing the incidence of physiological abnormalities such as vitrification and browning, increasing the proliferation coefficient of adventitious buds and root quality, thereby obtaining tissue culture seedlings with robust growth and high uniformity.

[0030] 2. This invention has the ability to dynamically and in a closed loop to regulate the growth status, freeing it from the dependence on static formulas and human experience. The system can obtain the growth status information of tissue culture seedlings in real time through the monitoring unit and automatically determine its growth stage, thereby generating closed-loop regulation instructions. Compared with the traditional method of manually changing the fixed culture medium periodically, this invention not only greatly reduces the intensity of manual operation and the risk of contamination, but also makes the regulation timing more precise and the response speed faster, which can effectively avoid problems such as growth stagnation or poor differentiation of tissue culture seedlings caused by regulation lag.

[0031] 3. This invention meets the specific regulatory needs of different Juncao varieties, improving the system's versatility and adaptability. By embedding a dedicated collaborative regulation strategy database for different Juncao varieties such as Giant Juncao, Oasis No. 1, and Oasis No. 3 into the control unit, a single system can flexibly adapt to the rapid propagation of multiple Juncao varieties through tissue culture. This fundamentally solves the shortcomings of existing general-purpose environmental control systems that cannot perform fine-tuning for variety differences, providing technical support for the parallel development of multiple Juncao varieties. It can also provide emergency collaborative compensation for abnormal physiological states, further ensuring production safety. When the system identifies characteristic traits such as browning and vitrification in tissue culture seedlings, it can automatically perform compensatory collaborative adjustments to nutrient substrate osmotic pressure, hormone ratio, and environmental temperature and light, promptly curbing abnormal development and mitigating potential losses. This is of great significance for continuous industrial production. Attached Figure Description

[0032] Figure 1 This is a block diagram of the overall structure of a growth regulation system for a nutrient medium for tissue culture seedlings of the present invention;

[0033] Figure 2 This invention relates to a growth regulation system and method for a nutrient medium for tissue culture seedlings. Figure 1 A schematic diagram of the specific structure of the medium-nutrient base formulation and supply unit;

[0034] Figure 3 This is a flowchart of a method for regulating the growth of tissue culture seedlings using a nutrient medium according to the present invention.

[0035] Figure 4 This is a table showing the synergistic regulation parameter settings for Oasis No. 1 tissue culture seedlings in Example 1 of the present invention, which describes a growth regulation system and method for a nutrient medium for Juncao tissue culture seedlings. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0037] Example 1

[0038] The system composition of this embodiment is as follows:

[0039] The nutrient medium preparation and supply unit includes six peristaltic pumps and storage bottles, respectively containing MS basal medium stock solution (10-fold concentration), sucrose solution, 6-BA stock solution (1 mg / mL), IBA stock solution (1 mg / mL), NAA stock solution (1 mg / mL), and sterile water. Each peristaltic pump has its flow rate independently controlled by a control unit. After online preparation via a mixing chamber, the solution is delivered to the tissue culture bottles through piping. The tissue culture bottles are equipped with vented, sealed caps, and the bottom of each bottle has a nutrient medium inlet and a waste liquid outlet.

[0040] Environmental parameter adjustment unit: including LED plant growth lights (adjustable light intensity and spectrum, mainly red and blue light), ultrasonic humidifier, circulating fan and air conditioner, which can realize automatic control of light intensity (0~200μmol·m⁻²·s⁻¹), temperature (22~28℃) and humidity (60%~85%RH).

[0041] Monitoring Unit: A high-resolution industrial camera is installed on the top of each culture rack to take photos of the plants inside the tissue culture bottles at regular intervals. The images are processed by an embedded computing module, and a trained convolutional neural network model is used to extract morphological indicators such as the number of adventitious buds, average bud length, and leaf color.

[0042] Control Unit: An industrial controller is used, with a built-in database of synergistic regulation strategies for Oasis No. 1 Juncao. This database contains the optimal parameter combinations for the three stages of initiation culture, subculture proliferation, and rooting induction (as shown in the table below), as well as the stage transition discrimination thresholds.

[0043] Table 1. Synergistic Regulation Parameters for Oasis No. 1 Juncao Tissue Culture Seedlings

[0044] Start cultivation 3.0 1.0 60 16 25±1 Adventitious buds that sprout and are ≥0.5cm in length are transferred to subculture for propagation. Subgeneration 5.0 1.0 120 18 26±1 The total number of adventitious buds is ≥15 and the average bud length is ≥1.5cm, or the buds are cultured for ≥18 days before being transferred to rooting induction. Rooting Induction 0 5.0* 80 12 24±1 Roots ≥2cm in length and ≥3 in number complete the culture.

[0045] Note: During the rooting induction stage, IBA can be replaced with NAA 5.0 mg / L. In this example, IBA is used.

[0046] The adjustment method flow in this embodiment is as follows:

[0047] The operator selects the variety "Oasis No. 1" through the human-machine interface, and the control unit retrieves the corresponding collaborative regulation strategy database to initialize each unit to the start-up culture parameters.

[0048] The nutrient preparation unit prepares MS medium online according to the start-up culture formula and injects it quantitatively into the tissue culture flask for inoculation of explants.

[0049] During the cultivation period, the monitoring unit collects images every 12 hours and extracts data on the number and length of adventitious buds. When the processing module identifies that more than 50% of the explant buds are ≥0.5cm in length, the control unit determines that the culture has entered the subculture propagation stage.

[0050] The control unit first sends a command to the environmental parameter adjustment unit to gradually increase the light intensity from 60 μmol·m⁻²·s⁻¹ to 120 μmol·m⁻²·s⁻¹, extend the photoperiod from 16 h to 18 h, and adjust the temperature to 26 °C. After 12 hours of physiological adaptation, the control unit sends a command to the nutrient medium preparation unit to switch the culture medium components: increase the 6-BA concentration to 5.0 mg / L, and maintain the IBA concentration at 1.0 mg / L.

[0051] During the subculture process, image analysis continuously tracks the process. When the total number of adventitious buds is ≥15 and the average length is ≥1.5cm, the system identifies that the conditions for rooting conversion have been met. At this point, the control unit initiates a coordinated switching process again: First, the environmental parameter adjustment unit reduces the light intensity to 80μmol·m⁻²·s⁻¹, shortens the photoperiod to 12h, and adjusts the temperature to 24℃; Second, after a delay of 8 hours, the nutrient preparation unit switches the hormone combination to a rooting medium containing 5.0mg / L IBA.

[0052] During the rooting induction stage, continuous monitoring is conducted. When the root length and number of roots reach the seedling emergence standard, the system issues a prompt signal, completing a full cycle of cultivation.

[0053] Example 2: Emergency Coordinated Regulation – Addressing Browning

[0054] During the mid-stage of subculture propagation of Oasis No. 1 tissue culture seedlings, the monitoring unit detected brown patches on the base of some buds and leaves, with decreased color saturation, which the identification model determined to be early browning. The control unit then triggered an emergency collaborative adjustment program, performing the following compensatory adjustments under the same system architecture as the previous embodiment:

[0055] Regarding the nutrient base components: the concentration of 6-BA was reduced from 5.0 mg / L to 3.0 mg / L, and 1.0 g / L of polyvinylpyrrolidone (PVP) was added as an anti-browning agent (this addition was mixed in at a set ratio through an additional peristaltic pump channel).

[0056] Regarding environmental parameters: the culture temperature was lowered from 26℃ to 24℃, and the light intensity was temporarily reduced from 120μmol·m⁻²·s⁻¹ to 80μmol·m⁻²·s⁻¹.

[0057] Implementation strategy: First, adjust environmental parameters by lowering the temperature and reducing light intensity, then refresh the nutrient medium after 2 hours. After 48 hours of continuous monitoring, browning stopped, new growth points returned to normal green color, and then gradually returned to normal proliferation parameters.

[0058] This embodiment demonstrates the system's ability to dynamically and collaboratively intervene in abnormal situations, effectively preventing the entire bottle from being discarded due to the spread of browning.

[0059] Example 3: Multi-varietal adaptation—synergistic regulation of giant reed and Oasis No. 3

[0060] To verify the effectiveness of the variety-specific database, the system simultaneously stores the synergistic regulation parameter schemes for Giant Napier Grass and Oasis No. 3:

[0061] Giant Napier grass initiation culture stage: MS + 2,4-D 3.0 mg / L + IAA 0.2 mg / L, light intensity 100 μmol·m⁻²·s⁻¹, temperature 27℃, photoperiod 14 h; subculture proliferation stage: MS + 6-BA 2.0 mg / L + IBA 0.5 mg / L, light intensity 130 μmol·m⁻²·s⁻¹, temperature 27℃; rooting induction stage: 1 / 2 MS + IBA 0.5 mg / L, light intensity 70 μmol·m⁻²·s⁻¹.

[0062] The rooting induction stage of Oasis No. 3 was performed using MS + NAA 1.0 mg / L + IBA 0.5 mg / L, and the light intensity was increased by 10% compared to the rooting stage of Oasis No. 1.

[0063] When the operator switches the target variety on the control panel, the control unit automatically calls the corresponding variety's parameter package. All subsequent stage judgments and coordinated controls follow the variety-specific data, and the entire process requires no hardware modification. Actual operation results show that the tissue culture seedlings using the variety-specific database exhibit significantly better growth uniformity than those using general parameters, confirming the technical value of the variety adaptation strategy.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0065] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0066] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A growth regulation system for a nutrient medium for tissue culture seedlings, characterized in that, include: A nutrient substrate preparation and supply unit is used to prepare and supply nutrient substrate solutions of at least two different components to tissue culture seedlings according to control instructions; An environmental parameter adjustment unit is used to adjust at least one physical environmental parameter in the growth environment of tissue culture seedlings. The monitoring unit is used to acquire information on the growth status of tissue culture seedlings in real time or at regular intervals. The control unit is communicatively connected to the nutrient base preparation and supply unit, the environmental parameter adjustment unit, and the monitoring unit, respectively. The control unit is equipped with a synergistic regulation strategy for the tissue culture seedlings at different growth and development stages; this synergistic regulation strategy defines the dynamic matching relationship between nutrient component parameters and environmental parameters at each stage; The control unit is configured to: determine the current growth stage of the tissue culture seedling based on the growth status information obtained by the monitoring unit, generate a set of coordinated regulation instructions according to the coordinated regulation strategy, and send them simultaneously to the nutrient substrate preparation and supply unit and the environmental parameter adjustment unit, so as to coordinately adjust the supplied nutrient substrate components and the physical environmental parameters within the same regulation cycle.

2. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 1, characterized in that, The nutrient base preparation and supply unit includes multiple plant growth regulator stock solution storage tanks and nutrient element mother liquor storage tanks. The control unit dynamically changes the concentration ratio of cytokinins and auxins in the output nutrient base by adjusting the ratio of liquids in different storage tanks.

3. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 2, characterized in that, The synergistic regulation strategy includes: when the tissue culture seedling is determined to be in the subculture proliferation stage, the generated synergistic regulation instruction increases the mass concentration ratio of cytokinin to auxin in the nutrient medium, while increasing the ambient light intensity and / or prolonging the photoperiod; when the tissue culture seedling is determined to be in the rooting induction stage, the generated synergistic regulation instruction decreases the mass concentration ratio, while decreasing the ambient light intensity and / or shortening the photoperiod.

4. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 1, characterized in that, The monitoring unit includes an image acquisition device and an image processing module. The growth status information includes plant height, number of adventitious buds, leaf area and / or root length information extracted from the image. The control unit determines the growth stage of the tissue culture seedling based on this information and its rate of change.

5. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 1, characterized in that, The control unit stores a database of dedicated synergistic regulation strategies for different Juncao varieties, including at least one of Giant Juncao, Oasis No. 1, and Oasis No.

3. The databases for different varieties define the optimal combination of nutrient substrate components and physical environment parameters specific to each variety at each stage of initiation culture, subculture proliferation, and rooting induction.

6. A method for regulating the growth of tissue culture seedlings in a nutrient medium, applied to the system as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Select the target Juncao variety and call the corresponding collaborative adjustment strategy database in the control unit; S2. During the cultivation process, the monitoring unit acquires real-time growth status information of the tissue culture seedlings; S3. The control unit compares the acquired growth status information with the preset stage transition conditions, determines the current growth stage, and calculates the target values ​​of the nutrient matrix components and environmental parameters to be adjusted according to the coordinated adjustment strategy of the stage. S4. The control unit sends an adjustment command to the nutrient substrate preparation and supply unit to adjust the nutrient substrate components supplied to the tissue culture seedlings to the target value, and simultaneously sends an adjustment command to the environmental parameter adjustment unit to adjust the physical environmental parameters to the corresponding target value. S5. Execute steps S2 to S4 cyclically throughout the entire culture cycle of the tissue culture seedlings to achieve continuous, coordinated, and dynamic adjustment of nutrient substrate components and environmental parameters.

7. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 6, characterized in that, In step S3, the coordinated regulation strategy is a mathematical model based on the response surface methodology to determine the influence of nutrient factors and environmental factors on the growth rate of tissue culture seedlings, or a decision model based on expert experience rules.

8. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 6, characterized in that, The method further includes: when the monitoring unit detects that the tissue culture seedlings exhibit browning or vitrification, the control unit generates an emergency adjustment command to compensate for and coordinate the osmotic pressure of the nutrient medium, the hormone ratio, and the temperature and light intensity of the environment.

9. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 6, characterized in that, The physical environment parameters mentioned in step S4 include at least two of temperature, humidity, light intensity, photoperiod, and CO2 concentration; the target values ​​of the nutrient substrate components include a combination of macro-element concentration, micro-element concentration, carbon source concentration, and types and concentrations of plant growth regulators.

10. The growth regulation system and method for a nutrient medium for tissue culture seedlings according to claim 6, characterized in that, In step S4, when the control unit performs coordinated adjustment of environmental parameters and nutrient substrate components, it adopts a time-delay strategy: first adjusts the environmental parameters, and after a preset plant physiological response delay time, gradually adjusts the nutrient substrate components to reduce the physiological stress caused by the same step to the tissue culture seedlings.

Images