Method for shortening fructus amomi tissue culture time
By regulating the tissue culture of Amomum villosum using leaf extract, the problems of long explant induction cycle and exogenous hormone risks have been solved, achieving efficient and low-cost tissue culture that is suitable for large-scale application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHWEST FORESTRY UNIVERSITY
- Filing Date
- 2026-03-25
- Publication Date
- 2026-05-12
AI Technical Summary
The current method of inducing explants in Amomum villosum tissue culture involves a long induction period, and the use of exogenous hormones carries residual risks and high costs, making it difficult to meet the needs of large-scale production.
Extracts were prepared from Amomum villosum leaves. Natural active ingredients were used to regulate explant differentiation and shorten the induction time. Leaves with a material-to-liquid ratio of 25-100 g/L were extracted at 25-30℃ and filtered through four layers of sterile gauze. The explants were then cultured in MS medium containing the extract.
It significantly shortens the induction period to 2-3 weeks, increases induction efficiency by 54.76%, reduces the risk of deformities, simplifies the operation process, reduces costs, and maintains excellent germplasm traits.
Smart Images
Figure CN122004133A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture technology, and specifically relates to a method for shortening the tissue culture time of Amomum villosum. Background Technology
[0002] Amomum villosum Lour. is a perennial herb belonging to the genus Amomum in the ginger family. Its dried fruit is a valuable traditional Chinese medicine with edible, medicinal, and economic value, and is in high demand in the market. Tissue culture technology is a key means for the large-scale seedling production and breeding of superior varieties of Amomum villosum. However, the existing tissue culture process for Amomum villosum has a prominent problem of long explant induction period: traditional methods rely on exogenous hormones (such as 6-BA and NAA) for regulation, and the induction time from inoculation to the formation of callus or adventitious buds usually takes 4-6 weeks, resulting in low induction efficiency, which seriously restricts the large-scale production of Amomum villosum tissue culture seedlings.
[0003] Meanwhile, studies have shown that Amomum villosum leaves contain abundant natural active ingredients (such as volatile oils, flavonoids, and phenolic compounds), which may participate in the regulation of Amomum villosum's own growth and development and have potential tissue differentiation-promoting activity. However, current technologies mostly focus on optimizing the ratio of exogenous hormones, and there are no reports on using Amomum villosum leaves as raw materials to obtain natural active liquids through simple extraction and applying them to autologous tissue culture to shorten the induction time. Traditional methods not only pose a risk of exogenous hormone residues, but also involve complex hormone ratio adjustments and high costs, making it difficult to meet the needs of efficient and environmentally friendly large-scale seedling cultivation. Summary of the Invention
[0004] This invention aims to provide a natural, efficient, and low-cost process that uses Amomum villosum leaves to prepare an extract, which is then applied to Amomum villosum tissue culture. By regulating explant differentiation through natural active ingredients, the induction time can be shortened.
[0005] A method for shortening the tissue culture time of Amomum villosum includes the following steps:
[0006] (1) Preparation of Amomum villosum leaf extract: Collect Amomum villosum leaves, rinse with sterile water, air dry, cut into pieces, and then soak in sterile water at a material-to-liquid ratio of 25-100 g / L. Extract at 25-30℃, and filter through 4 layers of sterile gauze and 0.22 μm microporous membrane to obtain the extract.
[0007] (2) Explant treatment: The creeping stems of Amomum villosum were selected as explants. After cleaning and disinfection, they were inoculated into MS medium containing extract. Culture conditions: 25±2℃, 2500lx light, 12h / d photoperiod, until the explants formed callus or adventitious buds.
[0008] Preferably, in the preparation of the extract, the leaves are cut into 0.5-1cm pieces, the extraction time is 24 hours, and the mixture is stirred once every 8 hours.
[0009] Preferably, the explant disinfection steps are as follows: soaking in 75% ethanol for 10 seconds, rinsing with sterile water 3 times, disinfecting with 0.1% mercuric chloride for 8 minutes, and rinsing with sterile water 5-6 times.
[0010] Preferably, in step (2), the amount of extract added to the MS medium is 0.5 mg / L.
[0011] The technical effects achieved by this invention are as follows:
[0012] 1. Significantly shortens the induction period and improves propagation efficiency: This invention utilizes the natural growth-active substances (such as flavonoids, phytosterols, amino acids, etc.) contained in the leaves of Amomum villosum, which are directly applied to the explants through the form of extracts. This shortens the induction time of Amomum villosum explants from the conventional 4-6 weeks to 2-3 weeks, and increases the induction efficiency to about 54.76%, thus greatly accelerating the propagation process.
[0013] 2. Improve the survival rate of induced buds and reduce the risk of deformity: The leaf extract is a natural source of ingredients and has excellent physiological compatibility with the explants of Amomum villosum. This avoids problems such as browning and deformity caused by improper ratio of chemical hormones, and the adventitious buds grow vigorously.
[0014] 3. Simplified operation process and reduced production cost: The preparation process of the extract is simple, and the raw material is the leaves of Amomum villosum itself, which is readily available and inexpensive. It does not require precise proportions of complex chemical reagents, which significantly reduces the cost and difficulty of culture medium preparation and is suitable for large-scale promotion and application.
[0015] 4. Maintaining superior germplasm traits: The active ingredients in the natural extract have a high degree of compatibility with the metabolic system of Amomum villosum, which can effectively maintain the superior genetic characteristics of the original germplasm, avoid the potential impact of exogenous chemical substances on germplasm traits, and ensure the consistency of tissue culture seedling quality. Attached Figure Description
[0016] Figure 1 Statistical chart showing the effects of different hormone culture media on Amomum villosum tissue culture;
[0017] Figure 2 Statistical chart showing the effects of different concentrations of extract on Amomum villosum tissue culture. Detailed Implementation
[0018] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the embodiments described. Unless otherwise specified, the experiments and methods described in the embodiments are generally carried out according to conventional methods well known in the art and described in various references. The reagents and raw materials used in the present invention are all commercially available. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed.
[0019] Example 1
[0020] Step 1: Preparation of Amomum villosum leaf extract and explants
[0021] 1. Raw material collection and pretreatment: Collect fresh and old leaves from the pruning of the cardamom plantation, remove moldy and diseased leaves, rinse 5 times with sterile water to remove surface dust and impurities, place in a clean bench to air dry naturally for 4 hours until there is no moisture on the surface, and cut into small pieces of 0.5-1cm with sterile scissors.
[0022] 2. Preparation of extract: Weigh the above-mentioned chopped leaves and place them in a clean 1000mL sterile Erlenmeyer flask.
[0023] 3. Constant temperature extraction: After stirring evenly with a sterile glass rod, seal the triangular flask with sterile sealing film and extract at room temperature (25±2℃) for 24 hours. Stir with a sterile glass rod once every 8 hours to promote the full dissolution of natural active ingredients in the leaves.
[0024] 4. Filtration and sterilization: After extraction, the leaf residue is first removed by filtration through 4 layers of sterile gauze, and the filtrate is collected. Then, the filtrate is transferred to a sterile vacuum filtration flask and sterilized by vacuum filtration through a 0.22μm sterile microporous membrane to obtain a light yellow Amomum villosum leaf extract. The extracts are labeled and stored in a refrigerator at 4℃ for later use.
[0025] 5. Explant selection: Select the stolons of healthy, disease-free Amomum villosum plants as explants.
[0026] 6. Aseptic treatment: Place the stolons in a clean bench, first soak them in 75% ethanol solution for 10 seconds, then quickly rinse them 3 times with sterile water; then disinfect them in 0.1% mercuric chloride solution for 8 minutes, shaking constantly, and then rinse them 5-6 times with sterile water to remove residual disinfectant; finally, use sterile filter paper to absorb the surface moisture of the stolons and set them aside.
[0027] Step 2: Determination of the induction effect of the extract on Amomum villosum explants
[0028] This embodiment uses a comparative experiment to determine the effects of different concentrations of extract on the induction time and induction effect of Amomum villosum stem segment explants.
[0029] 1. Comparison of different growth regulators:
[0030] Prepare MS basal medium (containing 30 g / L sucrose, 7 g / L agar, and pH adjusted to 5.8–6.0). For hormones that can be autoclaved, add 0.5 mg / L 6-BA and 0.5 mg / L NAA before sterilization, then autoclave at 121°C for 20 minutes. Add 0.5 mg / L CPPU and 0.5 mg / L extract (100 g / L concentration) to the medium after it has cooled to 50–55°C in a clean bench and mix thoroughly. The control group (CK) is MS medium without any added hormones. The final experimental treatments included: MS (hormone-free), MS + NAA, MS + 6-BA, MS + CPPU, and MS + extract.
[0031] 2. Comparison of extracts at different concentrations
[0032] Preparation of extract: Accurately weigh 25g, 50g, 75g and 100g of the above-mentioned chopped leaves and place them in four clean 1000mL sterile Erlenmeyer flasks respectively, and extract the extract according to step one.
[0033] Treatment group A (25g): Add 1000mL of sterile water;
[0034] Treatment group B (50g): Add 1000mL of sterile water;
[0035] Treatment group C (75g): Add 1000mL of sterile water;
[0036] Treatment group D (100g): Add 1000mL of sterile water.
[0037] Prepare MS basal medium (30 g / L sucrose, 7 g / L agar, pH adjusted to 5.8-6.0), autoclave at 121°C for 20 minutes, and cool to 50-55°C. In a clean bench, add the four prepared concentrations at 0.5 mg / L to the melted MS medium and mix thoroughly. Control medium (CK): Use MS medium without the extraction solution.
[0038] Under aseptic conditions, the explants of Amomum villosum stolons were inoculated into the culture media of the experimental and control groups, and the group information was marked. The culture flasks were placed in a culture room, and the culture conditions were: temperature 25±2℃, light intensity 2500lx, and light duration 12h / d.
[0039] 3. Observation and Recording:
[0040] Observation frequency: The growth status of explants was observed every 3 days, and the time of callus formation and adventitious bud differentiation were recorded.
[0041] Data statistics: When callus tissue that is visible to the naked eye forms on the surface of the explant, the induction time is recorded; when the length of the adventitious shoot reaches 1 cm, the differentiation time is recorded, and the callus formation rate and the robustness rate of the adventitious shoots in each group are statistically analyzed.
[0042] Table 1. Effects of different concentrations of Amomum villosum leaf extract on the induction effect of Amomum villosum explants.
[0043]
[0044] See Figure 1 , Figure 2 Experimental results showed that the addition of Amomum villosum leaf extract significantly shortened the induction time of Amomum villosum explants in all experimental groups. The 75 g / L extract had the best effect, shortening the adventitious bud differentiation time to 19 days, which was about 66.7% shorter than the control group. This proves that the extract of the present invention can effectively shorten the induction time of Amomum villosum tissue culture explants and the induction effect is stable, which has extremely high application value.
Claims
1. A method for shortening the tissue culture time of Amomum villosum, characterized in that, Includes the following steps: (1) Preparation of Amomum villosum leaf extract: Collect Amomum villosum leaves, rinse with sterile water, air dry, cut into pieces, and then soak in sterile water at a material-to-liquid ratio of 25-100 g / L. Extract at 25-30℃, and filter through 4 layers of sterile gauze and 0.22 μm microporous membrane to obtain the extract. (2) Explant treatment: The creeping stems of Amomum villosum were selected as explants. After cleaning and disinfection, they were inoculated into MS medium containing the extract. Culture conditions: 25±2℃, 2500lx light, 12h / d photoperiod, cultured until the explants form callus or adventitious buds.
2. The method according to claim 1, characterized in that, In the preparation of the extract, the leaves are cut into 0.5-1cm pieces, the extraction time is 24 hours, and the mixture is stirred once every 8 hours.
3. The method according to claim 1, wherein the explant disinfection steps are as follows: soaking in 75% ethanol for 10 seconds, rinsing with sterile water 3 times, disinfecting with 0.1% mercuric chloride for 8 minutes, and rinsing with sterile water 5-6 times.
4. The method according to claim 1, characterized in that, In step (2), the amount of extract added to MS medium is 0.5 mg / L.