A method for culturing alfalfa sterile seedlings

Abstract

This invention relates to the field of plant tissue culture technology, specifically disclosing a method for cultivating aseptic alfalfa seedlings. The method uses young stem segments from the top of alfalfa cuttings as explants, sterilizes them with a 15-20% NaClO solution, and induces rooting in 1 / 2 MS medium supplemented with 0.1-0.2 mg / L IBA to obtain aseptic alfalfa seedlings. The method provided by this invention, by selecting appropriate disinfectants and induction media, not only significantly improves the rooting rate but also avoids root contamination, thereby increasing the success rate of cultivating aseptic alfalfa seedlings.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for culturing aseptic seedlings of alfalfa. Background Technology

[0002] alfalfa ( Medicagosativa Alfalfa (L.) is my country's most important forage resource, known as the "King of Forage." With the rapid development of animal husbandry, breeding new varieties of alfalfa to adapt to the individualized environmental needs of different regions in my country remains a pressing issue. Gene editing breeding has a short cycle and can achieve precise improvement of crop traits, and has been widely used in molecular breeding of various plants. Efficient genetic transformation is a prerequisite for an efficient gene editing system. However, in alfalfa transformation systems, it has been found that disinfecting explants can lead to contamination later on. Furthermore, disinfection of explants can also affect transformation efficiency. Therefore, it is necessary to develop a sterile seedling cultivation technology. Summary of the Invention

[0003] To provide a method for cultivating alfalfa sterile seedlings with high rooting rate and short cultivation time, this invention provides a method for cultivating alfalfa sterile seedlings. The method provided by this invention, by selecting appropriate disinfectants and induction culture media, not only significantly improves the rooting rate but also avoids root contamination, thereby increasing the success rate of cultivating alfalfa sterile seedlings.

[0004] This invention provides a method for cultivating aseptic seedlings of alfalfa. The method uses the tender stem segments at the top of alfalfa cuttings as explants, sterilizes them with a NaClO solution with a mass fraction of 15-20%, and induces rooting in 1 / 2 MS + 0.1-0.2 mg / L IBA induction medium to obtain aseptic seedlings.

[0005] Disinfecting explants with a NaClO solution of 15-20% by mass can prevent contamination of the roots of cultured sterile seedlings.

[0006] For the cultivation of aseptic seedlings, sterilization and other steps are crucial; changes in any step or component can lead to tissue culture failure or contamination. This invention promotes rooting synergistically through sterilization with a specific concentration of NaClO solution and a specific induction culture medium, while effectively preventing contamination.

[0007] Furthermore, the concentration of the NaClO solution is 15% by mass.

[0008] Furthermore, the concentration of the NaClO solution is 20% by mass.

[0009] Furthermore, the induction medium was 1 / 2 MS + 0.2 mg / L IBA.

[0010] Further, the induction culture medium preparation process is as follows: Weigh 2.23 g MS powder, dissolve it in 800 mL of ultrapure water, add 15 g sucrose, add 7.5 g Agar, add 100-200 μL of IBA at a concentration of 1 mg / mL, and bring the volume to 1000 mL. Adjust the pH to 5.8, and autoclave at 121℃ for 21 min.

[0011] Furthermore, before disinfecting with the NaClO solution, the explants are disinfected with 75% alcohol for 10-12 seconds.

[0012] Furthermore, the explants were soaked in 1 / 1000 of Tween 20 for 10-12 minutes before being disinfected with alcohol.

[0013] Furthermore, the disinfection process using a 15-20% NaClO solution is as follows: the stem explants disinfected with alcohol are transferred to a 15-20% NaClO solution and quickly transferred to a sterile tissue culture bottle. They are then shaken and disinfected in a laminar flow hood for 12-14 minutes and rinsed 4-6 times with sterile water.

[0014] Furthermore, the stem explants are cut into small segments using a "flat top, slanted bottom" cutting method before use.

[0015] This invention also provides an application of a tissue culture disinfectant in the disinfection of explants, wherein the tissue culture disinfectant is a NaClO solution with a mass fraction of 15-20%, which not only promotes rooting but also avoids contamination.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. This invention selects young stem segments at the top as explants, sterilizes them with a NaClO solution with a mass fraction of 15-20%, and induces rooting in 1 / 2 MS + 0.1-0.2 mg / L IBA induction medium to obtain sterile seedlings, which not only improves the rooting efficiency but also reduces the contamination rate. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Rooting status of different stem segments;

[0020] In the diagram, A is a schematic diagram of stem segment selection, which are named a, b, and c respectively; a is the tender stem segment at the top, and b and c are the middle stem segments;

[0021] B represents the rooting status of different stem segments;

[0022] C represents the different above-ground growth conditions of the stem tip.

[0023] Figure 2 The effects of different NaClO concentrations and induction media on the rooting and growth of aseptic seedlings were investigated. The NaClO concentrations were 10%, 15%, 20%, and 30%, and the induction media included 1 / 2 MS and MS media.

[0024] In the figure, A represents the rooting of aseptic seedlings under the conditions of treatment with 10% NaClO and culture in 1 / 2 MS medium;

[0025] B represents the rooting status of aseptic seedlings under the conditions of treatment with 15% NaClO and culture in 1 / 2 MS medium;

[0026] C represents the rooting status of aseptic seedlings under the conditions of treatment with 20% NaClO and culture in 1 / 2 MS medium;

[0027] D represents the rooting status of aseptic seedlings under the conditions of treatment with 30% NaClO and culture in 1 / 2 MS medium;

[0028] E represents the rooting status of aseptic seedlings under conditions of 10% NaClO treatment and MS medium culture;

[0029] F indicates the rooting status of aseptic seedlings under conditions of 15% NaClO treatment and MS medium culture;

[0030] G indicates the rooting status of aseptic seedlings under conditions of 20% NaClO treatment and MS medium culture;

[0031] H indicates the rooting status of aseptic seedlings under conditions of 30% NaClO treatment and MS medium culture.

[0032] Figure 3 The effect of different IBA concentrations on the rooting of aseptic seedlings;

[0033] In the figure, A represents the rooting of aseptic seedlings under the conditions of 1 / 2 MS medium + 0 mg / L IBA culture;

[0034] B indicates the rooting status of aseptic seedlings under the conditions of 1 / 2 MS medium + 0.1 mg / L IBA culture;

[0035] C represents the rooting status of aseptic seedlings under the conditions of 1 / 2 MS medium + 0.2 mg / L IBA culture;

[0036] D indicates the rooting status of aseptic seedlings under the conditions of 1 / 2 MS medium + 0.5 mg / L IBA culture;

[0037] E indicates the rooting status of aseptic seedlings under the conditions of 1 / 2 MS medium + 1 mg / L IBA culture;

[0038] F indicates the rooting status of aseptic seedlings under the conditions of 1 / 2 MS medium + 2 mg / L IBA culture;

[0039] G indicates the rooting status of aseptic seedlings cultured in 1 / 2 MS medium + 5 mg / L IBA.

[0040] Figure 4 The effects of different coagulants on the rooting and growth of aseptic seedlings;

[0041] In the figure, a represents the growth status of the underground part of the sterile seedling when Agar is used as a coagulant;

[0042] b represents the growth status of the aboveground parts of the sterile seedlings when Agar is used as a coagulant;

[0043] c represents the underground growth status of sterile seedlings when plant gel is used as a coagulant.

[0044] d represents the growth status of the sterile seedbed when plant gel is used as a coagulant. Detailed Implementation

[0045] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0046] This invention provides a method for cultivating aseptic seedlings of alfalfa, specifically including the following steps:

[0047] Selection of alfalfa stem explants: Select young stem segments (top stem segments) from vigorous alfalfa cuttings as explants. Cut the stem segments into small sections (remove leaves) using a "flat top, slanted bottom" cutting method. Soak the young stem segments in 1 / 1000 Tween 20 (1 mL of Tween added to 1000 mL of water) and shake for 10-12 minutes. Rinse with water until no foam remains, and set aside.

[0048] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10-12 seconds; then transfer them to a 15-20% NaClO solution (BGI, CAS NO.7681-52-9), and quickly transfer them to sterile tissue culture bottles. Shake and disinfect in a laminar flow hood for 12-14 minutes, and rinse with sterile water 4-6 times.

[0049] In this invention, the functions of 75% alcohol and NaClO are as follows: 75% alcohol has strong penetrating power, allowing it to easily penetrate into bacteria, absorb moisture from bacterial proteins, causing them to dehydrate, denature, and coagulate, thus losing their function and achieving disinfection. Simultaneously, it also has a strong wetting effect, expelling air from the material and facilitating the penetration of other disinfectants. Sodium hypochlorite (NaClO) hydrolyzes to form hypochlorous acid, which has strong oxidizing properties and can destroy the protein structure of bacteria and viruses, thereby achieving disinfection.

[0050] Induction culture: Sterilized explants were inserted into 1 / 2 MS medium + 0.1-0.2 mg / L IBA (Sigma, CAS NO. 133-32-4) to induce rooting and obtain sterile seedlings;

[0051] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0052] I. The effects of explant selection on rooting and aboveground growth were studied through Example 1 and Comparative Examples 1-2.

[0053] Example 1: A method for cultivating aseptic seedlings of alfalfa.

[0054] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0055] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 15% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0056] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.2 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0057] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0058] Example 2: A method for cultivating aseptic seedlings of alfalfa.

[0059] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0060] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 20% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0061] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.2 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0062] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0063] Example 3: A method for cultivating aseptic seedlings of alfalfa.

[0064] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0065] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 15% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0066] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.1 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0067] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0068] Example 4: A method for cultivating aseptic seedlings of alfalfa.

[0069] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0070] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 20% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0071] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.1 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0072] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0073] Comparative Example 1: A method for cultivating aseptic seedlings of alfalfa.

[0074] Selection of alfalfa stem explants: Select stem segments from the middle part of vigorous alfalfa cuttings (e.g., ... Figure 1Select section A (or section b), and cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the middle stem segment in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0075] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 15% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0076] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.2 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0077] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0078] Comparative Example 2: A method for cultivating aseptic seedlings of alfalfa.

[0079] Selection of alfalfa stem explants: Select stem segments from the middle part of vigorous alfalfa cuttings (e.g., ... Figure 1 Select section A (c section), and cut the stem section into small sections using the "flat top, slanted bottom" cutting method (remove leaves). Soak the middle stem section in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until there is no foam, and set aside.

[0080] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 15% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0081] Induction culture: Sterilized explants were inserted into 1 / 2 MS + 0.2 mg / L IBA induction medium to induce rooting and obtain sterile seedlings;

[0082] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0083] The rooting and aboveground growth of the seedlings in Example 1 (using the young top stem segment as the explant), Comparative Example 1 (using the middle stem segment b as the explant), and Comparative Example 2 (using the middle stem segment c as the explant) were recorded and statistically analyzed. By comparing the rooting and aboveground growth of the aseptic seedlings in Example 1 and Comparative Examples 1-2, it was found that explant selection has a significant impact on rooting and aboveground growth. The results are as follows: Figure 1 As shown, in the preparation of sterile seedlings, selecting the top stem segment as the explant is beneficial for rooting and ensures good above-ground growth.

[0084] II. Effects of NaClO concentration and induction medium on rooting.

[0085] 1. Experimental reagents and culture media

[0086] The NaClO concentrations were set to 10%, 15%, 20%, and 30%.

[0087] The induction media include 1 / 2 MS medium and MS medium.

[0088] 2. Cultivation methods for aseptic alfalfa seedlings

[0089] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0090] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to different concentrations of NaClO solution (10%, 15%, 20%, 30%), and quickly transfer them to sterile tissue culture bottles. Shake and disinfect in a laminar flow hood for 12 minutes, and rinse 5 times with sterile water.

[0091] Induction culture: Sterilized explants were inserted into 1 / 2 MS induction medium to induce rooting and obtain sterile seedlings;

[0092] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0093] 3. Experimental Results

[0094] Table 1. Screening of NaClO concentration and induction medium

[0095]

[0096] The results are as follows Figure 2 As shown in Table 1, the rooting performance of 1 / 2 MS medium was better than that of MS medium;

[0097] In the screening of NaClO concentrations, it was found that 10%, 15%, and 20% NaClO concentrations resulted in better rooting of stem explants than 30% NaClO. However, 10% NaClO concentration caused contamination during explant disinfection and is therefore not recommended. Explants disinfected with 15% NaClO all rooted by day 11 of rooting induction, while those disinfected with 20% NaClO all rooted by day 20. Therefore, 15% NaClO concentration is superior to 20% NaClO. Thus, using 15% or 20% NaClO for explant disinfection not only ensures good rooting but also avoids contamination.

[0098] Therefore, disinfecting explants with 15% NaClO and inducing rooting on 1 / 2 MS induction medium can improve rooting efficiency and reduce contamination rate.

[0099] III. Effects of different IBA (indole-3-butyric acid) concentrations on rooting of aseptic seedlings

[0100] 1. Experimental reagents and culture media

[0101] The IBA (Sigma, CAS NO.133-32-4) concentrations are mainly set at 0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, and 5 mg / L.

[0102] The induction medium was 1 / 2 MS medium.

[0103] 2. Cultivation methods for aseptic alfalfa seedlings

[0104] Selection of alfalfa stem explants: Select young stem segments (top stem segments, such as...) from vigorous alfalfa cuttings. Figure 1 (As shown in section A), cut the stem section into small segments using the "flat top, slanted bottom" cutting method (remove leaves). Soak the tender stem segments in 1 / 1000 of Tween 20 and shake for 10 minutes, then rinse with water until no foam remains, and set aside.

[0105] Disinfection of alfalfa stem explants: Disinfect the stem explants with 75% alcohol for 10 seconds; then transfer them to a 15% NaClO solution and quickly transfer them to a sterile tissue culture bottle. Disinfect them by shaking in a laminar flow hood for 12 minutes and rinse them 5 times with sterile water.

[0106] Induction culture: Sterilized explants were inserted into 1 / 2 MS + IBA (0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L) induction medium to induce rooting and obtain sterile seedlings;

[0107] The 1 / 2 MS induction medium formula is as follows: Weigh 2.22 g MS powder (Phytotech, M519), dissolve it in 800 mL of ultrapure water, add 15 g sucrose and 200 μL IBA (1 mg / mL), and bring the volume to 1000 mL. Adjust the pH to 5.8. Add 7.5 g Agar and autoclave at 121℃ for 21 min.

[0108] 3. Experimental Results

[0109] Table 2. Culture medium formulations with different IBA concentrations

[0110]

[0111] like Figure 3 As shown in Table 2, the rooting of explants was better when the IBA concentration was 0.1, 0.2, and 0.5 mg / L than at other concentrations. As the IBA concentration increased, the root growth weakened. Therefore, in the preparation of alfalfa sterile seedlings, it is recommended to add IBA concentrations of 0.1, 0.2, and 0.5 mg / L to 1 / 2 MS medium. However, from the perspective of rooting effect, 0.2 mg / L is more effective.

[0112] IV. Effects of Different Coagulants on Rooting and Growth of Aseptic Seedlings

[0113] After conducting the above screening tests, the coagulants in the 1 / 2 MS induction medium were further screened. The coagulants included Agar and plant gel. The specific components of the medium are shown in Table 3.

[0114] Table 3. Effects of different coagulants on rooting and growth of sterile seedlings

[0115]

[0116] The results are as follows Figure 4 As shown, comparison Figure 4 a, b, c, d show that adding agar as a solidifying agent to the culture medium during the preparation of sterile seedlings is more effective than using plant gel, as it not only promotes better rooting but also results in better growth of sterile seedlings.

[0117] In summary, this invention uses Xinjiang large-leaf alfalfa as the research material, employing stem segments from different parts of alfalfa cuttings as explants. These segments are sterilized with 10-30% NaClO solutions and cultured on different induction media to induce rooting. Aseptic alfalfa seedlings are then obtained through screening. The method provided by this invention mainly includes stem segment selection, NaClO concentration screening, induction medium screening, IBA concentration screening, and coagulant screening, aiming to improve the rooting rate of alfalfa and the success rate of cultivating aseptic alfalfa seedlings. Four screening experiments revealed that the top stem segments, sterilized in a 15% NaClO solution and induced to root on 1 / 2 MS + 0.2 mg / L IBA induction medium, exhibited the best rooting effect and the best growth of aseptic seedlings. Therefore, in the rapid propagation of alfalfa aseptic seedlings, it is recommended to use the top stem segment as the explant, disinfect the explant with 15% NaClO solution, and induce rooting on 1 / 2 MS (Agar) + 0.2 mg / L IBA induction medium.

[0118] This invention provides a method for rapidly obtaining sterile alfalfa seedlings under different screening conditions, providing excellent material for alfalfa tissue culture. Compared with using cut external stems and leaves as explants for tissue culture transformation, this method effectively reduces the contamination rate. By obtaining robust sterile seedlings, the sterilization steps before transformation can be reduced, thus effectively improving the conversion rate of alfalfa. This invention provides a basis for the propagation and preservation of rare alfalfa materials.

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for culturing alfalfa aseptic seedlings, characterized by, Young stem segments from the top of alfalfa cuttings were used as explants. The cuttings were sterilized with a 15-20% NaClO solution and induced to root using 1 / 2 MS medium with 0.2 mg / L IBA to obtain sterile seedlings.

2. The alfalfa sterile seedling culture method according to claim 1, characterized by, The concentration of the NaClO solution is 15% by mass.

3. The alfalfa sterile seedling culture method according to claim 1, characterized by, The concentration of the NaClO solution is 20% by mass.

4. The alfalfa aseptic seedling culture method according to claim 1, characterized by, The induction medium was prepared as follows: 2.22 g MS powder was weighed and dissolved in 800 mL of ultrapure water. 15 g sucrose and 7.5 g Agar were added. 200 μL of IBA with a concentration of 1 mg / mL was added and the volume was adjusted to 1000 mL. The pH was adjusted to 5.8 and the medium was autoclaved at 121 °C for 21 min.

5. The alfalfa seedling culture method according to claim 1, characterized by, Before disinfection with NaClO solution, the explants were disinfected with 75% alcohol for 10-12 seconds.

6. The method for cultivating aseptic alfalfa seedlings according to claim 5, characterized in that, Before alcohol disinfection, the explants were soaked in 1 / 1000 of Tween 20 for 10-12 minutes.

7. The method for cultivating aseptic alfalfa seedlings according to claim 6, characterized in that, The disinfection process using a 15-20% NaClO solution is as follows: After alcohol disinfection, the stem explants are transferred to a 15-20% NaClO solution and then quickly transferred to a sterile tissue culture bottle. The explants are then shaken in a laminar flow hood for 12-14 minutes and rinsed with sterile water 4-6 times.

8. The method for cultivating aseptic alfalfa seedlings according to claim 6, characterized in that, Before use, the stem explants should be cut into small segments using the "flat top, slanted bottom" cutting method.

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