Construction method of purple perilla tissue culture regeneration system

Abstract

The invention provides a construction method of a purple perilla tissue culture regeneration system, and belongs to the technical field of tissue culture. Proliferation of cluster buds; preparing a regeneration unit; a circulating culture system; rooting and transplanting: the browning index pressure is reduced to 12.5% through an AC-PVP composite adsorption antioxidant, the survival rate is synchronously increased to 93.8%, and cluster buds are stimulated to proliferate to 5.2 buds / explants; in combination with a regeneration unit preparation standard of 45-degree beveling and 0.5 mm callus retention, the regeneration rate breaks through 96.3%, and the mitotic index reaches 12.4%; the purple perilla tissue culture medium has the advantages that the purple perilla tissue culture medium is combined with konjac glucomannan-methylcellulose temperature response gel, the viscosity of sol is suddenly reduced to 150 Pa.s, the root damage rate is compressed to 5.1%, the transplanting survival rate is increased to 95.3%, the three problems of out-of-control browning, low regeneration efficiency and transplanting damage in purple perilla tissue culture are cooperatively solved through the three modules, and a core support is provided for production of high-added-value purple perilla metabolites and gene editing breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, in particular to a method for constructing a perilla tissue culture regeneration system. Background Art

[0002] Perilla, an annual herbaceous plant of the genus Perilla in the Lamiaceae family, is a highly valuable economic crop for both food and medicine. However, its seeds are small nuts with a low germination rate, and seed propagation is prone to degradation, which limits its production. Using young, internoded stem segments as explants for tissue culture can yield large amounts of callus tissue, remove pathogens, and purify the complex.

[0003] Currently, cotyledons, hypocotyls or leaves are commonly used as explants in tissue culture of Perilla frutescens, and their regeneration efficiency is significantly restricted by genotype and hormone ratio; the proliferation stage mostly relies on high concentrations of cytokinins, which easily induce vitrification and mutation, and the proliferation coefficient shows a sharp downward trend after three subcultures; the browning problem of explants is prominent, and conventional anti-browning schemes such as adding VC can only reduce the browning rate to about 35%, while inhibiting bud differentiation; in the rooting stage, although the rooting rate of 1 / 2MS+NAA / IBA mixed hormones can reach 100%, the root system is weak, the transplant survival rate is less than 85%, and it lacks the function of synchronous enrichment of metabolites.

[0004] In view of the above problems, the present invention proposes a method for constructing a tissue culture regeneration system for Perilla frutescens. The method selects axillary bud stem segments combined with anatomical grade meristematic zone cuttings, adopts a special adsorptive antioxidant, combines the regeneration unit preparation standards, and uses konjac gum-methylcellulose temperature-responsive gel to solve the three problems of uncontrolled browning, low regeneration efficiency and transplant damage in Perilla frutescens tissue culture. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems in the existing technology and propose a method for constructing a perilla tissue culture regeneration system.

[0006] The purpose of the present invention can be achieved through the following technical solutions: A method for constructing a perilla tissue culture regeneration system, comprising: S1. Explant preparation: Stem segments with axillary buds from sterile Perilla seedlings were sterilized and inoculated into primary induction medium. The culture was conducted at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. S2. Cluster bud proliferation: transfer the germinated axillary buds to proliferation medium and culture them under a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form cluster buds; S3. Preparation of regeneration units: Select a meristem-rich section with a height of 2-3 mm from the clustered buds, cut it at a 45° angle under a dissecting microscope, and retain 0.3-0.7 mm of callus tissue at the base to obtain regeneration units; S4, Circulation Culture System: The regenerated units are inoculated into proliferation medium for secondary proliferation, or transferred into rooting medium for direct rooting induction; S5, rooting and transplanting: transplant the rooted seedlings to the substrate after hardening; The proliferation medium is MS modified medium + 1.0-2.0 mg / L 6-BA + 0.1-0.3 mg / L 1 BA + 150-250 mg / L adsorbable antioxidant; Wherein, the adsorptive antioxidant is an activated carbon-polyvinyl pyrrolidone complex, and the mass ratio of the activated carbon to polyvinyl pyrrolidone is 2:1; The rooting medium comprises 1 / 2MS, 0.5-1.0mg / IBA, 0.01-0.03mg / L methyl jasmonate, and a temperature-responsive gel matrix. The temperature-responsive gel matrix is a konjac gum-methylcellulose blend with a mass ratio of 3:1. The combination of 1 / 2MS, IBA, methyl jasmonate, and a temperature-responsive gel matrix (konjac gum-methylcellulose) provides essential rooting hormones while also promoting rooting with methyl jasmonate. This unique temperature-responsive gel matrix (with a phase transition point of 25°C) provides support in a solid state at room temperature. When heated and liquefied, it may facilitate material exchange or root growth and help protect active substances (such as methyl jasmonate).

[0007] Through the S3 step, a 2-3mm meristem-enriched segment is precisely selected, and the callus tissue is cut at a 45° angle and retained to a specific thickness to establish a highly efficient "regeneration unit", which is the basis for cyclic culture and germplasm preservation, and significantly improves the proliferation efficiency and regeneration capacity; the S4 step enables the regeneration unit to choose secondary proliferation (expanded reproduction) or direct rooting (obtaining a complete plant), which improves the flexibility and application range of the system; through a specific ratio of MS modified culture medium + 6-BA + IBA + adsorptive antioxidant (activated carbon-PVP complex), the proliferation of clustered buds is effectively promoted. At the same time, the adsorptive antioxidant can remove harmful substances produced during the culture process, reduce oxidative stress, and improve the quality of seedlings.

[0008] Preferably, in step S3, the method for identifying the meristem-enriched segment is: observing under a dissecting microscope that the cell division activity in the base 0.3-0.8 mm area is 3-5 times that of the middle of the bud, ensuring that the tissue area with the greatest regeneration potential can be accurately located under the dissecting microscope, and screening based on cell division activity ensures that the selected regeneration units have strong division ability, which is the key to subsequent efficient proliferation and regeneration, and significantly improves the efficiency of the regeneration system.

[0009] Preferably, in step S4, before the secondary proliferation, the regeneration unit is placed in a dark environment for pre-culture for 2 hours as a physical stimulus (trauma simulation / stress), which can activate key regeneration-related genes (such as WUSCHEL), thereby enhancing the organogenesis ability of the regeneration unit (especially the regeneration ability of buds). This pretreatment step is intended to optimize the response of the regeneration unit in the secondary proliferation stage, which helps to obtain more and more robust clustered buds.

[0010] Preferably, the rooting medium also includes 0.1-0.3 mg / L salicylic acid. Adding low concentrations of salicylic acid (SA) to the rooting medium can, on the one hand, synergize with IBA and methyl jasmonate to further promote root development and robustness. Furthermore, SA is a known signaling molecule that can induce specific secondary metabolic pathways (such as the phenylpropanoid pathway). This is expected to increase the content of valuable secondary metabolites such as rosmarinic acid in regenerated plants, thereby enhancing the medicinal and economic value of Perilla frutescens.

[0011] Preferably, the proliferation culture medium further contains 0.05-0.1 mg / L AgNO3, which is used to inhibit ethylene synthesis, effectively block the ethylene signal transduction pathway, and significantly reduce the physiological disorders caused by ethylene accumulation commonly seen in tissue culture. In addition, by inhibiting the adverse effects of ethylene, such as promoting abnormal morphogenesis, this measure can effectively control the occurrence of vitrification of test tube seedlings, significantly reduce the proportion of vitrified seedlings (target <5%), obtain more morphologically normal and robust seedlings, and improve the quality of proliferation.

[0012] Preferably, in step S5, the seedling hardening process includes: After 2-3 days of hardening the seedlings, soak the roots in 0.1% carbendazim solution containing 0.5mg / L brassinolide for 10 minutes; The transplanting medium was vermiculite: peat soil = 1:2 (v / v), and 100 mg / L chitosan was added.

[0013] Through a specific seedling hardening process (after opening the bottle for hardening, the roots are soaked in a solution containing brassinolide and carbendazim) combined with an optimized transplanting matrix (vermiculite: peat soil + chitosan), transplanting stress can be effectively reduced, thereby improving the transplanting survival rate; brassinolide (BR) can enhance the stress resistance of seedlings, and carbendazim can prevent fungal infections; chitosan has natural antibacterial and growth-promoting effects. The three work synergistically to significantly improve the adaptability of tissue culture seedlings from sterile environments to bacterial environments and the transplanting survival rate.

[0014] Preferably, the temperature-responsive gel matrix has a phase transition temperature of 25°C. At conventional culture temperatures (e.g., 26±1°C), it is liquid, facilitating the diffusion and absorption of water, nutrients, and hormones. At low temperatures (e.g., for storage or transportation), it is solid, providing physical support and effectively "locking" active substances such as methyl jasmonate. This physical protection mechanism helps significantly reduce the volatilization or degradation loss of heat-sensitive active substances (e.g., methyl jasmonate) during culture medium sterilization and storage, ensuring their continued effective function during culture.

[0015] Preferably, the regeneration unit is inoculated in MS preservation medium containing 0.5 mg / L paclobutrazol and cryotreated at 4°C for preservation of perilla germplasm in vitro. This method provides a specific method for in vitro preservation of perilla germplasm using regeneration units (MS medium containing paclobutrazol followed by cryotreatment at 4°C), establishing an in vitro preservation system. Paclobutrazol is a growth retardant that effectively inhibits cell division and elongation; cryotreatment further slows metabolism. This combination allows for long-term preservation of regeneration units in a slow-growing or growth-stagnant state (expected to last for several months or even more than a year), providing an effective and cost-effective means for the safe and secure preservation of perilla germplasm.

[0016] Compared with the existing technology, the method for constructing the perilla tissue culture regeneration system has the following beneficial effects: 1. The present invention provides a method for constructing a perilla tissue culture regeneration system, which uses an AC-PVP composite antioxidant to solve the problem of excessive hormone adsorption by traditional single activated carbon. PVP enhances the antioxidant synergistic effect, reduces the browning index to 12.5%, and increases the survival rate to 93.8%.

[0017] 2. The present invention provides a method for constructing a perilla tissue culture regeneration system. Through 45° oblique cutting + 0.5mm callus retention, the regeneration rate exceeded 96.3%, which was 12.6% higher than the 0.5mm vertical cutting group. The mitotic index was 12.4%, and the cell activity increased sharply.

[0018] 3. The present invention provides a method for constructing a perilla tissue culture regeneration system, using a temperature-responsive gel with a sol viscosity of 150 Pa·s. The damage rate decreased by 85%, and the transplant survival rate reached 95.3%, which was 36% higher than that of the traditional group. New roots germinated 3 days earlier.

[0019] In summary, the present invention provides a method for constructing a perilla tissue culture regeneration system, wherein the AC-PVP composite adsorption antioxidant is used to reduce the browning index to 12.5%, which is 82% lower than that of the control group, while simultaneously increasing the survival rate to 93.8% and stimulating the proliferation of clustered buds to 5.2 buds / explant. Combined with the regeneration unit preparation standard of 45° bevel cutting + 0.5mm callus retention, the WUS gene expression is activated, the regeneration rate exceeds 96.3%, and the mitotic index reaches 12.4%. Combined with the konjac gum-methylcellulose temperature-responsive gel, the sol viscosity is suddenly reduced to 150 Pa·s, achieving "zero mechanical stripping" of the root system, reducing the root damage rate to 5.1%, and the transplant survival rate jumps to 95.3%. Through the three modules, the three problems of uncontrolled browning, inefficient regeneration, and transplant damage in perilla tissue culture are overcome, providing core support for the production of high-value-added perilla metabolites and gene editing breeding. DETAILED DESCRIPTION

[0020] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments. Specific embodiment one: Genetically homogeneous sterile seedlings of the "Perilla frutescens No. 1" variety (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5±0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS-modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 150 mg / L of an adsorbent antioxidant (AC)-polyvinylpyrrolidone (PVP) complex at a mass ratio of 2:1. Thirty explants were used per group, with three replicates. Specific embodiment two: Genetically homogeneous sterile seedlings of the cultivar "Perilla 1" (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5±0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS-modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 250 mg / L of an adsorbent antioxidant (an activated carbon-polyvinylpyrrolidone complex, with a mass ratio of 2:1). Thirty explants were used per group, with three replicates. Specific embodiment three: Genetically homogeneous sterile seedlings of the "Perilla frutescens No. 1" variety (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5±0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS-modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 200 mg / L of an adsorbent antioxidant (an activated carbon-polyvinylpyrrolidone complex at a mass ratio of 2:1). Thirty explants were used per group, with three replicates.

[0024] Comparative Example 1: Genetically homogeneous sterile seedlings of "Perilla 1" (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5 ± 0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25 ± 1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a light intensity of 14 h / d and a temperature of 26 ± 1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS modified medium supplemented with 1.5 mg / L 6-BA and 0.2 mg / L IBA. Thirty explants were grown per group, with three replicates.

[0025] Comparative Example 2: Genetically homogeneous sterile seedlings of the cultivar "Perilla 1" (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5±0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS-modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 250 mg / L of an adsorbent antioxidant. The adsorbent antioxidant was activated carbon alone, with a mass ratio of activated carbon to polyvinylpyrrolidone of 2:1. Thirty explants were used per group, with three replicates.

[0026] Comparative Example 3: Genetically homogeneous sterile seedlings of "Perilla frutescens No. 1" (5th generation tissue culture) were selected. Stem segments with axillary buds (1.5 ± 0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25 ± 1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26 ± 1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 250 mg / L of an adsorbent antioxidant (polyvinyl pyrrolidone). Thirty explants were used per group, with three replicates.

[0027] Comparative Example 4: Genetically homogeneous sterile seedlings of "Perilla 1" (fifth generation tissue culture) were selected. Stem segments with axillary buds (1.5 ± 0.2 cm in length) were uniformly removed and sterilized with 75% ethanol for 30 seconds followed by 0.1% HgCl₂ for 8 minutes. These stem segments were then inoculated into primary induction medium and cultured at a light intensity of 1500-2000 lx and a temperature of 25 ± 1°C for 15-20 days to induce axillary bud germination. The germinated axillary buds were then transferred to proliferation medium and cultured at a photoperiod of 14 h / d and a temperature of 26 ± 1°C for 25-30 days to form clusters of buds. The proliferation medium consisted of MS modified medium supplemented with 1.5 mg / L 6-BA, 0.2 mg / L IBA, and 350 mg / LVC. Thirty explants were grown per group, with three replicates.

[0028] To monitor the dynamics of browning, explants were scanned using multispectral imaging on days 3, 7, and 14 after inoculation; Browning index = browning pixel area / total pixel area × 100% Browning level: Level 0 (no browning) → Level 4 (complete browning) Detection of oxidative stress indicators Phenolic substance accumulation (Day 7): 0.5 g of explants were extracted with 80% methanol, and the gallic acid and catechin contents were determined by HPLC (chromatographic conditions: C18 column, 280 nm). PPO enzyme activity (polyphenol oxidase): After enzyme extraction, catechol was used as substrate to measure OD 420 Change (U / gFW) Regeneration performance verification was performed by counting the survival rate (no browning and growth) after 30 days of culture.

[0029] Table 1 Comparison of anti-browning effects of different antioxidants (Cultivation Day 7)

[0030] The AC-PVP composites (mass ratio 2:1) used in specific Examples 1 to 3 exhibited significant synergistic enhancement within a concentration range of 150-250 mg / L. The browning index of Example 1 was reduced by 59% compared with the single activated carbon in Comparative Example 2 and by 49% compared with Comparative Example 4. The PPO activity of Example 1 was reduced to 26% of that in Comparative Example 1, and the accumulation of phenolic substances was reduced by 75%. The survival rate of Example 2 reached 93.8%, an increase of 121% compared with Comparative Example 1. Specific embodiment four: Preparation of regenerative units: The 30-day-cultured clusters of buds (2-3 mm in height) from Specific Example 2 were obtained and manipulated under a 20x dissecting microscope. The buds were cut by tilting the blade at a 45° angle, leaving 0.5 ± 0.05 mm. Fifty regenerative units were prepared for each group and inoculated into the same proliferation medium (MS + 1.5 mg / L 6-BA + 0.2 mg / L IBA).

[0032] Comparative Example 5: Preparation of regenerative units: 30-day-cultured clusters of buds (2-3 mm in height) from Specific Example 2 were obtained and manipulated under a 20x dissecting microscope. Sectioning was performed by tilting the blade at a 45° angle, leaving 0.8 ± 0.05 mm. Fifty regenerative units were prepared for each group and inoculated into the same proliferation medium (MS + 1.5 mg / L 6-BA + 0.2 mg / L IBA).

[0033] Comparative Example 6: Preparation of regeneration units: The 30-day-cultured clusters of shoots (2-3 mm in height) from Specific Example 2 were obtained and manipulated under a 20x dissecting microscope. Cutting was performed by vertically cutting with a scalpel, leaving 0.2 ± 0.05 mm of callus at the base. Fifty regeneration units were prepared for each group and inoculated into the same proliferation medium (MS + 1.5 mg / L 6-BA + 0.2 mg / L IBA).

[0034] Comparative Example 7: Preparation of regeneration units: The 30-day-old clustered buds (2-3 mm in height) from Specific Example 2 were obtained and manipulated under a 20x dissecting microscope. Sectioning was performed by vertically cutting, leaving 0.5 ± 0.05 mm. Fifty regeneration units were prepared for each group and inoculated into the same proliferation medium (MS + 1.5 mg / L 6-BA + 0.2 mg / L IBA).

[0035] 20 days after inoculation, the proportion of regeneration units that have rooted or sprouted is counted: Regeneration rate (%) = (number of successful regenerations / total number of inoculations) × 100% Cell division activity assay: The meristem of the regeneration unit was obtained on the 5th day of culture, fixed with FAA for 24 hours, embedded in paraffin, and sectioned at a thickness of 8 μm. The cells were stained with toluidine blue (0.1% dye, pH 4.2) and observed under a microscope. Mitotic index (%) = (number of cells in mitotic phase / total number of cells) × 100% (average value of 5 visual fields).

[0036] Table 2 Optimization of regeneration unit cutting criteria

[0037] As shown in the table above, the 45° bevel cut + 0.5mm callus used in Specific Example 4 was significantly optimal, with a regeneration rate of 96.3%, a 33% increase over the perpendicular cut + 0.2mm cut used in Comparative Example 6 and a 12.6% increase over the perpendicular cut + 0.5mm cut used in Comparative Example 7. The mitotic index of the cut method used in Specific Example 4 was 12.4%, 1.8 times that of Comparative Example 6, indicating a surge in meristematic cell proliferation activity. This is because the 45° bevel cut increases the wound contact area by 30%, activating WUS gene expression and promoting endogenous hormone secretion in the callus. The 0.5mm callus maintains a precise balance of protection and supply. Too thin (0.2mm) causes oxidative damage, while too thick (0.8mm) triggers cell differentiation and increases lignin deposition. The 0.8mm bevel cut used in Comparative Example 5 causes excessive callus growth to squeeze the meristematic zone, resulting in a regeneration rate of 88.2%. Specific embodiment five: The stem segments with axillary buds of sterile perilla seedlings were sterilized and inoculated into primary induction medium after surface disinfection. The axillary buds were induced to germinate under a light intensity of 1500-2000 lx and a temperature of 25±1℃ for 15-20 days. The germinated axillary buds were transferred to a proliferation medium and cultured for 25-30 days under a light cycle of 14h / d and a temperature of 26±1℃ to form clustered buds. The meristem-rich sections with a height of 2-3mm in the clustered buds were selected and cut at a 45° angle under a dissecting microscope, retaining 0.3-0.7mm callus tissue at the base to obtain regeneration units. The regeneration units were inoculated into a proliferation medium for secondary proliferation, or transferred into a rooting medium for direct rooting induction. The rooted seedlings were hardened and then transplanted into a substrate.

[0039] The rooting culture medium includes 1 / 2MS+0.5-1.0 mg / L IBA+0.01-0.03 mg / L methyl jasmonate+temperature-responsive gel matrix, wherein the temperature-responsive gel matrix is a konjac gum-methyl cellulose blend, and the mass ratio of the konjac gum to the methyl cellulose is 3:1.

[0040] Performance testing of the temperature-responsive gel matrix: A gel matrix was prepared as described above, with a 3:1 (w / w) ratio of konjac gum to methylcellulose, dissolved in deionized water (1.5%) and degassed using magnetic stirring. Rooted seedlings (n = 30) were cultured in the aforementioned responsive gel matrix. The gel was allowed to dissolve spontaneously (no rinsing was required) after incubation at 25°C for 10 minutes.

[0041] Comparative Example 8: The difference from the specific embodiment 5 is that the rooted seedlings were cultured in a 1 / 2MS+0.8% agar solidified matrix (n=30). The transplanting treatment was to violently peel off the agar with tweezers and rinse the roots with running water for 1 minute.

[0042] A comparative transplanting test was conducted under the following transplanting conditions: the matrix was vermiculite: peat soil = 1:2 (v / v), the environment was 25℃ / 70%RH, and the light intensity was 1000 lx.

[0043] Determine the phase transition temperature by DSC: Use a differential scanning calorimeter (DSC) with a temperature ramp from 20°C to 30°C (1°C / min heating rate) in a nitrogen atmosphere at 50 mL / min. The phase transition point is the temperature at the apex of the endothermic peak.

[0044] Rheological analysis: A rotational rheometer (MCR 302, cone and plate diameter 25 mm, gap 0.1 mm) was used. The temperature was constant at 25°C and the shear rate was 0.1 → 100 s. -1 scanning.

[0045] Root damage rate: transplanted roots were fixed with 2.5% glutaraldehyde, critical point dried, and root hairs were observed under a scanning electron microscope (SEM); damage rate (%) = (number of broken root hairs / total number of root hairs) × 100% (10 fields of view were counted).

[0046] Transplant survival rate (%) = (number of surviving seedlings after 14 days / total number of transplants) × 100% (the unfolding of new leaves is the survival standard).

[0047] Table 3 Phase change behavior and rheological properties

[0048] Table 4 Transplanting performance comparison

[0049] As can be seen from the above table, the temperature-responsive gel matrix achieves a sharp drop in viscosity to 150±12 Pa·s at 25°C through precise phase change properties, which is only 1.2% of that of Comparative Example 8 (traditional agar). This eliminates the need for mechanical peeling during the root dissolution process, compresses the root damage rate to 5.1±1.2%, an 85% decrease from 35.2±3.8% in Comparative Example 8 (traditional agar group), and retains the intact root hair structure and auxin transport channel. At the same time, the konjac gum remaining in the sol forms a water-retaining film on the roots, increasing the soil's water holding capacity by 40%. Combined with the no-rinse operation to avoid osmotic pressure fluctuations and reduce root electrolyte leakage, the transplanting survival rate ultimately reached 95.3±2.1%, a 36% increase from Comparative Example 8 (traditional agar group), and the time of new root germination was advanced by 3 days.

[0050] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A method for constructing a tissue culture regeneration system of Perilla frutescens, characterized in that: include: S1. Explant preparation: Stem segments with axillary buds from sterile Perilla seedlings were sterilized and inoculated into primary induction medium. The culture was conducted at a light intensity of 1500-2000 lx and a temperature of 25±1°C for 15-20 days to induce axillary bud germination. S2. Cluster bud proliferation: transfer the germinated axillary buds to proliferation medium and culture them under a photoperiod of 14 h / d and a temperature of 26±1°C for 25-30 days to form cluster buds; S3. Preparation of regeneration units: Select a meristem-rich section with a height of 2-3 mm from the clustered buds, cut it at a 45° angle under a dissecting microscope, and retain 0.3-0.7 mm of callus tissue at the base to obtain regeneration units; S4, Circulation Culture System: The regenerated units are inoculated into proliferation medium for secondary proliferation, or transferred into rooting medium for direct rooting induction; S5, rooting and transplanting: transplant the rooted seedlings to the substrate after hardening; The proliferation medium is MS modified medium + 1.0-2.0 mg / L 6-BA + 0.1-0.3 mg / L 1 BA + 150-250 mg / L adsorbable antioxidant; Wherein, the adsorptive antioxidant is an activated carbon-polyvinyl pyrrolidone complex, and the mass ratio of the activated carbon to polyvinyl pyrrolidone is 2:1; The rooting culture medium includes 1 / 2MS+0.5-1.0mg / LIBA+0.01-0.03mg / L methyl jasmonate+temperature-responsive gel matrix, wherein the temperature-responsive gel matrix is a konjac gum-methyl cellulose blend, and the mass ratio of the konjac gum to the methyl cellulose is 3:

1.

2. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: In step S3, the method for identifying the meristem-rich segment is: observing under a dissecting microscope that the cell division activity in the 0.3-0.8 mm area at the base is 3-5 times that of the middle part of the bud.

3. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: In step S4, before the secondary proliferation, the regeneration unit is placed in a dark environment for pre-culture for 2 hours.

4. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: The rooting medium also includes 0.1-0.3 mg / L salicylic acid.

5. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: The proliferation medium also contains 0.05-0.1 mg / L AgNO 3 .

6. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: In step S5, the seedling hardening process includes: After 2-3 days of hardening the seedlings, soak the roots in 0.1% carbendazim solution containing 0.5mg / L brassinolide for 10 minutes; The transplanting medium was vermiculite: peat soil = 1:2 (v / v), and 100 mg / L chitosan was added.

7. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: The phase transition temperature of the temperature-responsive gel matrix is 25°C.

8. The method for constructing a tissue culture regeneration system of Perilla frutescens according to claim 1, wherein: The regeneration unit is inoculated into an MS preservation medium containing 0.5 mg / L paclobutrazol, and is preserved after being treated at a low temperature of 4° C., so as to be used for preserving the in vitro germplasm of Perilla frutescens.

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