A method for rapid propagation of potato microtubers for artificial seeds

Abstract

The application discloses a potato microtuber rapid propagation method for artificial seeds, and steps are as follows: 1, detoxification; after heat treatment of potato seed tubers, apical buds are stripped, inoculated into sterile seedlings to obtain a culture medium for meristem culture, and sterile seedlings are obtained; 2, induction of callus of potato sterile seedling stems; the sterile seedling stems obtained in step 1 are transferred into a callus induction culture medium for induction culture, the sterile seedling stem incisions gradually swell, and callus tissues are formed; 3, microtuber induction from callus; the callus tissues obtained in step 2 are transferred into a callus tuberization culture medium, the callus tissues gradually granulate, and the granular parts develop into microtubers finally, wherein the callus tuberization culture medium is MS+2.0 mg / L TDZ+0.1 mg / L paclobutrazol. The method can rapidly and massively produce detoxified microtubers, and the microtubers can be further made into artificial seeds, so that the method has great application potential in production.

Description

Technical Field

[0001] This invention belongs to the field of potato microtubule propagation technology, specifically relating to a method for rapid propagation of potato microtubules using artificial seeds. Background Technology

[0002] potato( Solanum tuberosum L. is an annual herbaceous plant belonging to the Solanaceae family and the Solanum genus. It is best grown in loose, fertile sandy loam soil. It is highly adaptable, has a wide planting range, a short growth cycle, and high yield. It is the world's fourth largest food crop. Using virus-free seed potatoes is an important measure to prevent viral diseases and increase yield.

[0003] Virus-free seed potatoes are generally obtained through shoot tip meristem culture, which includes two methods. One method involves obtaining larger, virus-free mini-tubers through shoot tip meristem culture, which are then propagated in greenhouses for production. This method has disadvantages such as requiring large land areas, significant investment, and a long timeframe, and is limited by seasonality, preventing year-round production. The second method involves obtaining smaller, virus-free mini-tubers through shoot tip meristem culture, which are then further processed into virus-free mini-tuber artificial seeds for production. This method is carried out indoors, requiring less space, allowing for rapid propagation, and is not limited by season. However, the time required for in vitro culture of virus-free test-tube seedlings to form mini-tubers is relatively long, and the yield is low. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for rapid propagation of potato microtubers for artificial seeds. This method can rapidly and massively produce virus-free microtubers, which can be further processed into artificial seeds, and has enormous application potential in production.

[0005] This invention is achieved through the following technical solution:

[0006] This invention provides a method for rapid propagation of potato microtubers for artificial seeds, comprising the following steps:

[0007] Step 1: Detoxification;

[0008] After heat treatment, potato seed tubers are peeled off from the stem tips and inoculated into a culture medium for aseptic seedlings to obtain meristematic tissue culture, thus obtaining aseptic seedlings.

[0009] Step 2: Inducing callus formation in aseptic potato seedling stem segments;

[0010] The sterile seedling stem segments obtained in step one were transferred to callus induction medium for induction culture. The cut surfaces of the sterile seedling stem segments gradually swelled and formed callus tissue.

[0011] Step 3: Inducing microtubules from callus;

[0012] The callus tissue obtained in step two was transferred to a callus tuber culture medium. The callus tissue gradually granulated, and the granular part eventually developed into a micro-tuber.

[0013] Preferably, the culture medium for obtaining sterile seedlings in step one is MS + 0.2 mg / L TDZ + 50 ml / L coconut juice.

[0014] Preferably, the callus induction medium in step two is MS + 2.5 mg / L 6-BA + 1.0 mg / L NAA.

[0015] Preferably, the callus-forming culture medium in step three is MS + 2.0 mg / L TDZ + 0.1 mg / L paclobutrazol.

[0016] The preferred culture conditions are: culture room temperature 26±1℃, light intensity 3000Lux, photoperiod 14h light / 10h dark, and relative humidity 40-50%.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) In the tissue culture of sterile potato seedling stem segments, the present invention found that a large number of micro-potato-like particles were directly formed on the callus tissue. The particle size was small, only a few millimeters, which is very suitable for artificial seeds.

[0019] (2) In this invention, paclobutrazol was mainly added to the callus tuber culture medium. Paclobutrazol is a highly efficient and low-toxic plant growth retardant that inhibits callus growth, test-tube seedling growth and rooting. Its mechanism of action is that paclobutrazol inhibits the biosynthesis of gibberellin and the physiological effects of gibberellin, thereby delaying the differentiation rate of callus and causing the formation of micro-tubers on the callus instead of sprouting.

[0020] In conclusion, based on the establishment of sterile potato seedlings, this method can rapidly and massively produce virus-free microtubers, which can be further processed into artificial seeds, showing great potential for application in production. Attached Figure Description

[0021] Figure 1 This is a photograph of the callus induced by sterile seedling stem segments according to the present invention;

[0022] Figure 2 These are images of shoot clusters induced by callus tissue according to the present invention;

[0023] Figure 3 These are images of micro-potatoes induced by callus tissue according to the present invention;

[0024] Figure 4 This is a longitudinal section image of the potato microbud eye structure of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0026] Example:

[0027] A method for rapid propagation of potato microtubules for artificial seeds includes the following steps:

[0028] Step 1: Detoxification;

[0029] After heat treatment, potato seed tubers were peeled from the stem tips and inoculated into a sterile seedling acquisition medium for meristematic tissue culture to obtain sterile seedlings. The sterile seedling acquisition medium consisted of MS + 0.2 mg / L TDZ + 50 ml / L coconut juice.

[0030] Step 2: Inducing callus formation in aseptic potato seedling stem segments;

[0031] The sterile seedling stem segments obtained in step one were transferred to callus induction medium for induction culture. The cut surfaces of the sterile seedling stem segments gradually swelled and formed callus tissue, such as... Figure 1 As shown, its induction rate can reach 90%. The induction medium in step two is MS + 2.5 mg / L 6-BA + 1.0 mg / L NAA.

[0032] Step 3: Inducing microtubules from callus;

[0033] The callus tissue obtained in step two was transferred to a callus tuber culture medium. The callus tissue gradually granulated, and the granular portion eventually developed into microtubers, such as... Figure 3 As shown, the tuber-forming medium was MS + 2.0 mg / L TDZ + 0.1 mg / L paclobutrazol.

[0034] The cultivation conditions in the above steps are as follows: cultivation room temperature 26±1℃, light intensity 3000Lux, photoperiod 14h light / 10h dark, and relative humidity 40-50%.

[0035] Comparative Example

[0036] The difference from the example is that the culture medium in step three was replaced with MS + 2.0 mg / L TDZ + 2.5 mg / L GA3 + 0.2 mg / L 2,4-D, which resulted in shoot clusters growing on the callus tissue. Figure 2 As shown, the germination rate can reach 88%.

[0037] By comparing the culture media for callus sprouting and callus tuber formation, the main difference lies in hormones. Hormones, as important factors regulating explant cell division and differentiation, significantly influence their morphogenesis. The culture medium for callus sprouting primarily used 2,4-D and GA3. 2,4-D plays a crucial role in callus induction, while GA3 promotes the development of adventitious embryos into small plantlets and the elongation of young stems. However, high concentrations of GA3 inhibit tuber formation because GA3 promotes the expression of potato acid invertase, further degrading sucrose and thus inhibiting tuber formation.

[0038] Compared to callus sprouting, paclobutrazol was primarily added to the culture medium for callus tuber formation. Paclobutrazol, a highly effective and low-toxicity plant growth retardant, inhibits callus growth, in vitro seedling growth, and rooting. Its mechanism of action involves paclobutrazol inhibiting gibberellin biosynthesis and physiological effects, thereby slowing callus differentiation and leading to the formation of tubers on the callus instead of sprouting.

[0039] The microstructure of the prepared potato samples was observed using conventional paraffin sectioning method, such as... Figure 4 As shown, it was found to be a mini-tubers with eye structure characteristics. The specific steps are as follows:

[0040] (1) Collection of materials

[0041] Micro-potato particles formed on callus produced from sterile seedling stem segments were selected;

[0042] (2) Fixed

[0043] Treatment 1: Use formaldehyde fixative, immerse the selected material in the fixative, and fix for 30 minutes;

[0044] Treatment 2: Using FAA fixative (anhydrous ethanol: xylene: glacial acetic acid = 18:1:1 volume ratio), the selected material was placed in the fixative and fixed for 24 hours;

[0045] (3) Dehydration: The fixed material is soaked in a series of solutions.

[0046] Treatment 1: Soak in 50% alcohol for 15 minutes, 75% alcohol for 15 minutes, 85% alcohol for 15 minutes, 95% alcohol for 15 minutes, and 100% alcohol for 15 minutes.

[0047] Treatment 2: Soak in 50% alcohol for 2 hours, then vacuum for 30 minutes, then soak in 75% alcohol for 2 hours, then soak in 85% alcohol for 2 hours, then soak in 95% alcohol for 2 hours, then soak in 100% alcohol for 2 hours, then soak in 100% alcohol for 2 hours.

[0048] (4) Transparent, the dehydrated materials are soaked in the following order.

[0049] Treatment 1: Soak in a 1:1 mixture of 50% alcohol and xylene for 1 hour, then soak in pure xylene for 1 hour, and then soak in pure xylene for 40 minutes.

[0050] Treatment 2: Soak in a 3:1 mixture of anhydrous ethanol and pure xylene for 2 hours, a 1:1 mixture of anhydrous ethanol and pure xylene for 2 hours, a 1:3 mixture of anhydrous ethanol and pure xylene for 2 hours, a pure xylene solution for 1 hour, and a pure xylene solution for 1 hour.

[0051] (5) Wax impregnation: Impregnate the transparent material with wax.

[0052] Treatment 1: Immerse the transparent material directly in molten pure paraffin wax (65℃) for 90 minutes;

[0053] Treatment 2: 2 hours in a wax impregnation solution of xylene and molten paraffin at a ratio of 3:1 (58-59℃), 2 hours in a wax impregnation solution of xylene and molten paraffin at a ratio of 1:1 (58-59℃), 2 hours in a wax impregnation solution of xylene and molten paraffin at a ratio of 1:3 (58-59℃), 2 hours in pure paraffin wax, and 2 hours in pure paraffin wax.

[0054] (6) Embedding

[0055] Process 1: Place the material soaked in paraffin wax into a mold, then wrap it with new paraffin wax, and then cool it at room temperature;

[0056] Process 2: Place the paraffin block in a -20°C refrigerator and cool for 3 minutes.

[0057] (7) Slicing, spreading and mounting

[0058] Using a paraffin microtome, the wrapped paraffin block is sliced. The sliced ​​wax strips are then spread in distilled water at 42°C. The material is then placed on a glass slide and dried in a slide dryer for 20 minutes to ensure that the material adheres firmly to the glass slide.

[0059] (8) Dewaxing: Immerse the glass slides with wax tape in the solution one by one.

[0060] Treatment 1: Soak in xylene for 10 min, pure xylene for 3 min, anhydrous ethanol for 2 min, 95% alcohol for 2 min, 90% alcohol for 2 min, and 75% alcohol for 2 min.

[0061] Treatment 2: Immerse in a 1:3 solution of xylene and anhydrous ethanol for 30 minutes, then in a 1:1 solution of xylene and anhydrous ethanol for 30 minutes, then in pure xylene for 30 minutes, then in pure xylene for 2 minutes, then in anhydrous ethanol for 2 minutes, then in 95% ethanol for 2 minutes, then in 90% ethanol for 2 minutes, then in 75% ethanol for 2 minutes, and finally in 50% ethanol for 2 minutes. After dewaxing, allow the paraffin sections to air dry naturally in a ventilated area. Avoid wiping them with paper towels or other items to prevent damage to the material on the slide.

[0062] (9) Staining: The dewaxed slides are then stained.

[0063] Treatment 1: Soak in safranin stain for 2 hours, stain with fast green stain for 10 seconds, and then wash away the excess color with anhydrous ethanol.

[0064] Treatment 2: Soak in safranin stain for 6 hours, stain with fast green stain for 15 seconds, and then wash away the excess color with distilled water.

[0065] (10) Observation.

[0066] The stained sections were placed under a microscope for observation and photographic recording.

[0067] It should be noted that although the present invention has been described through the above embodiments, the present invention may have many other embodiments. Without departing from the spirit and scope of the present invention, those skilled in the art can obviously make various corresponding changes and modifications to the present invention, but all such changes and modifications should fall within the scope of protection of the appended claims and their equivalents.

Claims

1. A method for rapid propagation of potato microtubers for artificial seeds, characterized in that: Includes the following steps: Step 1: Detoxification After heat treatment, potato seed tubers are peeled from the stem tips and inoculated into a sterile seedling acquisition medium for meristematic tissue culture to obtain sterile seedlings. The sterile seedling acquisition medium is MS + 0.2 mg / L TDZ + 50 ml / L coconut juice. Step 2: Inducing callus formation in aseptic potato seedling stem segments: The sterile seedling stem segments obtained in step one were transferred to callus induction medium for induction culture. The cut surfaces of the sterile seedling stem segments gradually swelled and formed callus tissue. The callus induction medium was MS + 2.5 mg / L 6-BA + 1.0 mg / L NAA. Step 3: Inducing microtubules from callus: The callus tissue obtained in step two was transferred to a callus tuber culture medium. The callus tissue gradually granulated, and the granular part eventually developed into microtubes. The callus tuber culture medium was MS + 2.0 mg / L TDZ + 0.1 mg / L paclobutrazol.

2. The method for rapid propagation of potato microtubules for artificial seeds according to claim 1, characterized in that: The cultivation conditions were as follows: cultivation room temperature 26±1℃, light intensity 3000Lux, photoperiod 14h light / 10h dark, and relative humidity 40-50%.

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