Application and preparation method of alfalfa tissue culture hairy root tip chromosome slice

Abstract

The invention discloses application and a preparation method of an alfalfa tissue culture hairy root tip chromosome slice, and the preparation method comprises the following steps: (1) preparing an explant by using mature alfalfa leaves, and then mediating hairy roots to transform the explant by using agrobacterium rhizogenes to obtain hairy roots; and (2) carrying out chromosome slide preparation by using the hairy roots obtained in the step (1). According to the method disclosed by the invention, the hairy roots cultured in vitro are used as chromosome slide preparation materials, and the hairy roots cultured in vitro are generated by induction of agrobacterium rhizogenes, have the characteristics of hormone autotrophy and infinite growth, are rapid in growth, stable in heredity and easy to operate, can be subcultured and preserved for a long time under an aseptic condition, realize material taking as required, are not limited by seasons and growth cycles, and are suitable for large-scale production. And the flexibility and repeatability of the experiment are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of chromosome preparation, and in particular to the use and preparation method of chromosome slides from the root tips of alfalfa tissue culture hairy roots. Background Technology

[0002] Alfalfa, a perennial herbaceous plant belonging to the legume family, is known as the "King of Forage" and is one of the world's most important forage plants. Alfalfa is a cross-pollinated plant with a complex genetic background, containing diploid, tetraploid, and polyploid types in nature. To explore the genetic diversity of alfalfa germplasm resources, rapid identification of chromosome numbers is necessary. Chromosome counting is currently the most direct and accurate method for determining plant chromosome numbers. High-quality chromosome preparation is the foundation for cytogenetics studies such as karyotype analysis, fluorescence in situ hybridization (FISH), and genome in situ hybridization (GISH). Furthermore, chromosome analysis is crucial for alfalfa variety improvement, molecular breeding, and genomic research.

[0003] Currently, chromosome counting in alfalfa somatic cells typically employs root tip chromosome preparation. However, due to the small size of alfalfa chromosomes (average length <3μm per chromosome), critical steps such as sample collection, pretreatment, dissociation, and staining require higher sensitivity, necessitating extensive experimental testing and counting of root tips from numerous plants. Traditional preparation techniques usually rely on seed roots or shoot tip meristems, which are limited by factors such as long cultivation cycles, poor synchronicity of cell division, strong environmental dependence, and inconvenient sample collection. This results in a severe shortage of chromosome preparation material, significantly hindering further research.

[0004] Hairy roots are transgenic organs induced by *Agrobacterium rhizogenes* infection, characterized by hormone autotrophy, rapid growth, and vigorous branching. As a classic product of *Agrobacterium rhizogenes*-mediated genetic transformation, the application of hairy roots has long been concentrated in areas such as secondary metabolite production and gene function verification. Currently, there are no studies on using hairy root tip tissue as a core raw material for chromosome preparation.

[0005] This invention attempts to introduce hairy root tip tissue as the core raw material for chromosome preparation, to develop a more stable, efficient, and seasonally unrestricted chromosome preparation material, as well as a chromosome preparation method using this material. Summary of the Invention

[0006] The first objective of this invention is to provide the use of alfalfa tissue culture hairy root tips for chromosome preparation.

[0007] The second objective of this invention is to provide a method for preparing chromosome slides from the root tips of alfalfa tissue culture hairy roots.

[0008] The first objective of this invention is achieved through the following technical solution: using alfalfa tissue-cultured hairy root tips for chromosome preparation. Hairy roots, as a classic product of Agrobacterium rhizogenes-mediated genetic transformation, have long been primarily used in secondary metabolite production and gene function verification. This invention successfully breaks through this traditional cognitive boundary, developing and verifying for the first time a novel cytological application of hairy root tips as a high-quality material for chromosome preparation, achieving a leapfrog functional expansion of this experimental material from biochemical analysis to cytogenetic analysis.

[0009] The second objective of this invention is achieved by the following technical solution: a method for preparing chromosome slides from the root tips of alfalfa tissue culture hairy roots, characterized by comprising the following steps: (1) Explants were prepared using mature alfalfa leaves, and then hairy roots were transformed into explants using Agrobacterium rhizogenes-mediated transformation to obtain hairy roots; (2) Use the hairy roots obtained in step (1) to prepare chromosome slides.

[0010] Furthermore, step (1) specifically includes the following steps: S1: Explants were prepared using mature alfalfa leaves; S2: Preparation of Agrobacterium rhizogenes bacterial suspension; S3: Agrobacterium rhizogenes infects explants to obtain transformed explants; S4: Place the transformed explants on co-culture medium and incubate in the dark for 24-48 h; S5: After co-culturing, the transformed explants are cleaned of bacterial solution from their surface and transferred to a selection medium. They are then cultured in the dark to induce hairy roots, which are obtained after 10 to 15 days.

[0011] Further, S1: preparing explants using mature alfalfa leaves specifically involves: selecting mature alfalfa leaves, adding Tween20 to a 7.5%~8% sodium hypochlorite solution to obtain a mixture, soaking the alfalfa leaves in the mixture for 10 minutes to disinfect the leaves, and rinsing with sterile water until odorless; wherein the volume ratio of Tween20 to sodium hypochlorite solution is 1:500; after absorbing the moisture from the alfalfa leaves, cutting them to 1~2 cm. 2The size of the explants was determined by using a sterile scalpel to make a 45° oblique incision along the leaf veins, preserving the midrib structure. Adding Tween 20 reduces surface tension and enhances wettability, ensuring the disinfectant fully contacts the entire leaf surface (including tiny crevices, pits, and pores) without leaving any blind spots—a prerequisite for effective disinfection. Simultaneously, it promotes emulsification and removes organic matter. Tween 20 emulsifies and disperses hydrophobic organic matter into the disinfectant solution, removing it from the surface or making it easier for the disinfectant to penetrate, thereby exposing the protected microorganisms and allowing the disinfectant to directly act on them.

[0012] Furthermore, S2: preparing Agrobacterium rhizogenes bacterial suspension specifically involves: adding the shaken Agrobacterium rhizogenes competent cells to TY liquid culture medium, shaking at 28°C until OD = 0.6 ~ 0.8, centrifuging, discarding the waste liquid, adding 1 / 2 MS infection solution to resuspend, shaking again until OD = 0.6 ~ 0.8, and diluting with 1 / 2 MS infection solution to OD = 0.25 ~ 0.3.

[0013] Further, S3: Obtaining transformed explants by Agrobacterium rhizogenes infection of explants specifically involves: placing the explants in Agrobacterium rhizogenes bacterial solution, soaking for 20 minutes with shaking during infection, and appropriately shaking and sonicating at 45kHz for 3-6 minutes. Then, the explants are removed, placed on sterile filter paper to air dry, and transformed explants are obtained. Ultrasonic treatment can assist gene delivery and improve transformation efficiency: 1. Creating micro-wounds: Ultrasound can create numerous micro-damages on the surface cells of plant tissues. These micro-wounds provide Agrobacterium with more invasion channels, making it easier for it to access the internal cells. 2. Removing gases and air bubbles: Air often exists on the leaf surface and in the interstitial spaces, hindering sufficient contact between the Agrobacterium suspension and the explant surface. Ultrasound can effectively remove these air bubbles, allowing the bacterial solution to better wet and adhere to the entire tissue surface. 3. Enhancing Agrobacterium invasion: Micro-wounds significantly increase the probability of Agrobacterium successfully attaching and entering plant cells. 4. Promote T-DNA delivery and integration: The entry of more Agrobacterium means that more T-DNA (carrying the target gene) is injected into plant cells, which ultimately increases the probability of T-DNA integration into the plant genome.

[0014] Furthermore, the TY liquid culture medium comprises the following components in the following amounts: tryptone 5.5 g / L; yeast extract 2.8 g / L; 1M CaCl2 solution 10 ml / L; and rifampin 0.05 mg / mL. Rifampin is used to control Agrobacterium and prevent over-contamination.

[0015] Furthermore, the co-culture medium comprises the following components in the following amounts: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; acetylsyringone 0.02 mg / mL.

[0016] Furthermore, the screening medium comprises the following components in the following amounts: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; termethin 0.4 mg / mL.

[0017] Furthermore, step (2) involves using the hairy roots obtained in step (1) to prepare chromosome slides, specifically including the following steps: S1: Pretreatment: Select hairy roots 1-2 cm long and cut off root tips 0.3-0.5 cm long; place the cut hairy root tips in centrifuge tubes containing distilled water and place them in crushed ice. Place the ice in a 4°C freezer for 24 hours to accumulate metaphase cells of mitosis. Pretreatment can enrich cells in metaphase, making the chromosomes moderately concentrated, thus making it easier to obtain high-quality chromosomes, which is beneficial for chromosome dispersion and slide preparation.

[0018] S2: Fixation: After removing the distilled water, use freshly prepared Carno fixative and fix at 4°C for 6-18 hours. After fixation, rinse quickly with distilled water until all residual fixative is removed, keep in 70% ethanol, and store at -20°C for later use. Using hairy roots to prepare chromosomes can shorten the fixation time compared with the ordinary chromosome preparation method.

[0019] S3: Dissociation: Take the root tip after fixation treatment, wash it several times with distilled water, dry it, and place it in 1 mol / L hydrochloric acid solution at 60℃ for 3~5 min to dissociate. After taking it out, wash it with distilled water until there is no hydrochloric acid residue in the root tip. S4: Staining. Use a scalpel to cut off about 0.3~0.5mm of the dissociated root tip meristem and place it on a glass slide. Quickly drop a drop of modified phenol-fuchsin staining solution on the root tip, which has been equilibrated in a 25℃ constant temperature incubator for 30 minutes in advance to avoid temperature fluctuations affecting the staining effect. After staining for 5 minutes, cover with a coverslip. S5: Slide preparation. Cover the coverslip with a filter paper and press vertically with your finger to absorb excess stain. Hold the edge of the coverslip with one hand to prevent it from moving and affecting the observation. Hold the biological dissecting needle above with the other hand and tap the root tip of the coverslip vertically for 1-2 minutes at a rhythm of once per second. After 1 minute, pause for 5 seconds to observe the cell dispersion. Then tap for another 30 seconds to ensure even force and spread the root tip cells into a thin layer to prepare a slide specimen for microscopic examination. S6: Microscopic examination.

[0020] Advantages of this invention: (1) The present invention uses in vitro cultured hairy roots as chromosome preparation material. Since the in vitro cultured hairy roots are induced by Agrobacterium rhizogenes, they have the characteristics of hormone autotrophy and unlimited growth. They also grow rapidly, are genetically stable, and are easy to operate. They can be subcultured and preserved for a long time under sterile conditions, so as to obtain materials as needed without being restricted by season or growth cycle, which greatly improves the flexibility and reproducibility of the experiment.

[0021] (2) Alfalfa has a long culture cycle, and an alfalfa plant usually has only one taproot and a few lateral roots. There are very few root tip meristems that can be used for slides, and each root tip harvest requires damage to an entire plant. However, the alfalfa in vitro culture system established in this invention can obtain a large amount (several grams to tens of grams) of fresh root tip material by culturing the hairy roots in shake flasks for 2 to 3 weeks. This material can be used at any time without damaging the mother plant, which significantly improves the efficiency of root tip harvesting and breaks through the seasonal harvesting limitation.

[0022] (3) The self-propagation characteristics of hairy root tips allow for repeated sampling of a single plant material ≥ 5 times, reducing the risk of damage to the mother plant by 78% compared to traditional methods.

[0023] (4) The successful application of alfalfa tissue culture hairy root tips in chromosome preparation technology proves that it has both experimental economic and ecological sustainability value, and has established a new paradigm for cytogenetics research of leguminous plants. Attached Figure Description

[0024] 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.

[0025] Figure 1 The diagram shows the process of preparing explants from mature alfalfa leaves in Example 1, followed by hairy root transformation of the explants using Agrobacterium rhizogenes-mediated transformation to obtain hairy roots.

[0026] Figure 2 This is a chromosome morphology diagram of a hairy root tip slide obtained in Example 1.

[0027] Figure 3 Chromosomal morphology for traditional root tip slides. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0029] Example 1: A method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots, comprising the following steps: (1) Explants were prepared using mature alfalfa leaves, and then hairy roots were transformed into hairy roots using Agrobacterium rhizogenes-mediated transformation, as shown in the example. Figure 1 As shown; specifically, it includes the following steps: S1: Explants were prepared using mature alfalfa leaves. Mature alfalfa leaves were selected, and Tween 20 was added to a 7.5% sodium hypochlorite solution to obtain a mixture. The alfalfa leaves were soaked in the mixture for 10 minutes to disinfect them, and then rinsed with sterile water until odorless. The volume ratio of Tween 20 to sodium hypochlorite solution was 1:500. After blotting the alfalfa leaves with filter paper, they were cut to 1-2 cm lengths. 2 The size was determined by using a sterile scalpel to make a 45° oblique cut along the leaf vein, preserving the main vein structure, to obtain explants for later use.

[0030] S2: Prepare Agrobacterium rhizogenes culture. Take 100 μL of well-shaken Agrobacterium rhizogenes competent cells and add them to 50 mL of TY liquid medium. Shake at 28°C until the OD = 0.6-0.8. Centrifuge, discard the waste liquid, add 50 mL of 1 / 2 MS infection buffer to resuspend, shake again until the OD = 0.6-0.8, and dilute with 1 / 2 MS infection buffer to OD = 0.2-0.3. The TY liquid medium contains the following components: tryptone 5.5 g / L; yeast extract 2.8 g / L; 1 M CaCl2 solution 10 mL / L; rifampin 0.05 mg / mL.

[0031] S3: Agrobacterium rhizogenes was used to infect explants to obtain transformed explants. The explants were placed in Agrobacterium rhizogenes bacterial solution and soaked for 20 minutes with shaking. During this period, the explants were shaken appropriately and subjected to 45kHz sonication for 5 minutes. Then, the explants were removed, placed on sterile filter paper and dried to obtain transformed explants. S4: Place the transformed explants on a co-culture medium and incubate in the dark for 36 h. The co-culture medium consists of the following components: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; acetylsuccinone 0.02 mg / mL.

[0032] S5: After co-culturing, the transformed explants were cleaned of bacterial suspension on their surface with sterile filter paper and transferred to selection medium. They were then cultured in the dark to induce hairy roots, which were obtained after 10 to 15 days. The selection medium consisted of the following components: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; and termethin 0.4 mg / mL.

[0033] (2) Chromosome preparation using the hairy roots obtained in step (1); specifically including the following steps: S1: Pretreatment: Select hairy roots 1-2 cm long and cut off root tips 0.5 cm long; place the cut hairy root tips in centrifuge tubes containing distilled water and place them in crushed ice. Place the ice in a 4°C freezer and continue treatment for 24 hours to accumulate metaphase cells. S2: Fixation. After removing the distilled water, use freshly prepared Carno fixative, which consists of the following components in the following volume ratio: ethanol: glacial acetic acid 3:1. Fix at 4°C for 10 hours. After fixation, rinse quickly with distilled water until all residual fixative is removed. Keep in 70% ethanol and store at -20°C for later use. S3: Dissociation: Take the root tip after fixation treatment, wash it several times with distilled water, dry it, and place it in 1 mol / L hydrochloric acid solution at 60℃ for 5 min to dissociate. After taking it out, wash it with distilled water until there is no hydrochloric acid residue in the root tip. S4: Staining. Use a scalpel to cut off about 0.4 mm of the dissociated root tip meristem and place it on a glass slide. Quickly drop a drop of modified phenol-fuchsin staining solution on the root tip. After equilibration in a 25°C constant temperature incubator for 30 minutes, stain for 5 minutes and then cover with a coverslip. Pre-equilibration in a constant temperature incubator avoids temperature fluctuations affecting the staining effect.

[0034] S5: Slide preparation. Cover the coverslip with a filter paper and press vertically with your finger to absorb excess stain. Hold the edge of the coverslip with one hand to prevent it from moving and affecting the observation. Hold the biological dissecting needle above with the other hand and tap the root tip of the coverslip vertically for 1-2 minutes at a rhythm of once per second. After 1 minute, pause for 5 seconds to observe the cell dispersion. Then tap for another 30 seconds to ensure even force and spread the root tip cells into a thin layer to prepare a slide specimen for microscopic examination. S6: Microscopic examination. Place the prepared slide specimen under the eyepiece for observation. First, find a clear field of view under low magnification, then use high magnification for detailed observation, counting, and photographic recording. If overlapping mitotic phases are found, remove the slide and tap it repeatedly until clearly dispersed metaphase chromosomes are observed. Figure 2 As shown, the chromosomes are well dispersed, with few background impurities and clear main constriction marks.

[0035] The chromosome preparation rate using the method in Example 1 was 92.23%.

[0036] Slide yield = (Number of analyzable metaphase cells / Total number of observed cells) × 100% Chromosome preparation was performed according to the method in Example 1. The culture conditions and the amount of material were controllable, and the chromosome preparation rate was high and relatively stable.

[0037] In this example, the 1 / 2 MS infection solution was: sucrose 15 g / L, MS medium 2.215 g / L.

[0038] In this embodiment, the MS medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. (MS Medium (including vitamins), Lot: F1140114).

[0039] Comparative Example 1: Chromosome preparation method using traditional root tips as raw material Step 1: Seed germination and root culture (obtaining experimental materials) (1) Seed disinfection: Place plump, disease-free, and newly produced alfalfa seeds into centrifuge tubes, add 75% ethanol and soak for 30 seconds (surface disinfection), discard the ethanol and rinse 3 times with sterile water, then soak in 0.1% mercuric chloride solution for 5 minutes (deep disinfection), and finally rinse 5 times with sterile water to remove residual disinfectant.

[0040] (2) Germination culture: Place two layers of sterile filter paper at the bottom of the petri dish, add a small amount of distilled water (enough to moisten the filter paper but not to accumulate water), spread the sterilized seeds evenly on the filter paper, cover the petri dish, and place it in a 25℃ constant temperature incubator to germinate in the dark.

[0041] (3) Selection of young roots: After culturing for 5 days, when the young roots grow to 0.5~1cm (at this time, the cells in the meristematic zone of the root tip of the young root are dividing vigorously, which is the best material for chromosome preparation), gently remove the young roots with tweezers and wash them once in distilled water.

[0042] Step 2: Preprocessing After cleaning, cut 0.5 cm long root tips from the young roots, place the cut root tips in centrifuge tubes containing distilled water, and place them in crushed ice. Keep the ice in a 4°C freezer for 24 hours to accumulate metaphase cells. Step 3: Carnot fixation After removing the distilled water, freshly prepared Carno fixative was used. The Carno fixative consisted of the following components in the following volume ratio: ethanol: glacial acetic acid 3:1. The fixative was placed in a 4°C refrigerator for 10 hours. After fixation, the fixative was quickly rinsed with distilled water until all residual fixative was removed. The fixative was then kept in 70% ethanol and stored in a -20°C refrigerator for later use. Step 4: Dissociation of hydrochloric acid After fixation, the root tips were washed several times with distilled water and dried. They were then placed in a 1 mol / L hydrochloric acid solution at 60°C for 5 min to dissociate. After removal, they were washed with distilled water until no hydrochloric acid residue remained in the root tips. Step 5: Staining Using a scalpel, cut off about 0.4 mm of the dissected root tip meristem and place it on a glass slide. Quickly drop a drop onto the root tip and stain with modified phenol-fuchsin solution that has been equilibrated in a 25°C incubator for 30 minutes beforehand. Stain for 5 minutes and then cover with a coverslip. Pre-equilibration in a 25°C incubator avoids temperature fluctuations from affecting the staining effect.

[0043] Step 6: Tableting Cover the coverslip with a filter paper, press vertically with your finger to absorb excess staining solution, hold the edge of the coverslip with one hand to prevent it from moving and affecting the observation effect; hold the biological dissecting needle above with the other hand and use its tip to gently tap the root tip area of ​​the coverslip vertically for 1-2 minutes, tapping once per second for 1 minute, then pause for 5 seconds to observe the cell dispersion, and then tap for another 30 seconds to ensure even force, so that the root tip cells are dispersed and spread out in a thin layer, and prepare a slide specimen for microscopic examination. Step 7: Microscopic examination Observe the prepared slide specimen under the eyepiece. First, find a clear field of view under low magnification, then use high magnification to observe, count, and photograph the specimen in detail. If overlapping mitotic phases are found, remove the slide and tap it repeatedly until clearly dispersed metaphase chromosomes are observed. Figure 3 As shown, due to interference from pigments / cell walls, chromosome dispersion is not obvious, there are many background impurities, and the main constriction is relatively clear.

[0044] The chromosome preparation rate using the method in Comparative Example 1 was 52.36%.

[0045] Slide yield = (Number of analyzable metaphase cells / Total number of observed cells) × 100%.

[0046] The slide preparation rate of Example 1 (92.23%) was significantly higher than that of Comparative Example 1 (52.36%). This indicates that the slide preparation rate is significantly affected by the root tip source; the slide preparation rate using hairy root tips is significantly higher than that using conventional root tips.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Applications of alfalfa tissue culture hairy root tip chromosome preparation.

2. A method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots, characterized in that, Includes the following steps: (1) Explants were prepared using mature alfalfa leaves, and then hairy roots were transformed into explants using Agrobacterium rhizogenes-mediated transformation to obtain hairy roots; (2) Use the hairy roots obtained in step (1) to prepare chromosome slides.

3. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 2, characterized in that, Step (1) specifically includes the following steps: S1: Explants were prepared using mature alfalfa leaves; S2: Preparation of Agrobacterium rhizogenes bacterial suspension; S3: Agrobacterium rhizogenes infects explants to obtain transformed explants; S4: Place the transformed explants on co-culture medium and incubate in the dark for 24-48 h; S5: After co-culturing, the transformed explants are cleaned of bacterial solution and transferred to selection medium. They are then cultured in the dark to induce hairy roots, which are obtained after 10 to 15 days.

4. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 3, characterized in that, S1: Explant preparation using mature alfalfa leaves, specifically: Select mature alfalfa leaves, add Tween20 to a 7.5%~8% sodium hypochlorite solution to obtain a mixture, soak the alfalfa leaves in the mixture for 10 minutes to disinfect the alfalfa leaves, and rinse with sterile water until odorless; wherein, the volume ratio of Tween20 to sodium hypochlorite solution is 1:500; after absorbing the water from the alfalfa leaves, cut them to 1~2cm. 2 The size was determined by using a sterile scalpel to make a 45° oblique cut along the leaf vein, preserving the main vein structure, to obtain explants for later use.

5. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 3, characterized in that, S2: Preparation of Agrobacterium rhizogenes bacterial suspension, specifically: add the shaken Agrobacterium rhizogenes competent cells to TY liquid culture medium, shake on a shaker at 28°C until OD = 0.6 ~ 0.8, centrifuge, discard waste liquid, add 1 / 2 MS infection solution to resuspend, shake again until OD = 0.6 ~ 0.8, and dilute with 1 / 2 MS infection solution to OD = 0.25 ~ 0.

3.

6. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 3, characterized in that, S3: Obtaining transformed explants by infecting explants with Agrobacterium rhizogenes, specifically: placing the explants in Agrobacterium rhizogenes bacterial solution, soaking for 20 minutes with shaking for infection, during which time the explants are shaken appropriately and subjected to 45kHz sonication for 3-6 minutes, then removing the explants, placing them on sterile filter paper to air dry, and obtaining transformed explants.

7. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 5, characterized in that, The TY liquid culture medium comprises the following components in the following amounts: tryptone 5.5 g / L; yeast extract 2.8 g / L; 1M CaCl2 solution 10 ml / L; rifampin 0.05 mg / mL.

8. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 3, characterized in that, The co-culture medium comprises the following components in the following amounts: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; acetylsyringone 0.02 mg / mL.

9. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 3, characterized in that, The screening medium comprises the following components in the following amounts: MS medium 2.215 g / L; sucrose 15 g / L; agar 7.8 g / L; termethin 0.4 mg / mL.

10. The method for preparing chromosome slides from the root tips of alfalfa tissue-cultured hairy roots according to claim 2, characterized in that, Step (2) involves preparing chromosome slides using the hairy roots obtained in step (1), specifically including the following steps: S1: Pretreatment: Select hairy roots 1-2 cm long and cut off root tips 0.3-0.5 cm long; place the cut hairy root tips in centrifuge tubes containing distilled water and place them in crushed ice. Place the ice in a 4°C freezer for 24 hours to accumulate metaphase cells. S2: Fixation: After removing the distilled water, use freshly prepared Carno fixative and fix at 4°C for 6-18 hours. After fixation, rinse quickly with distilled water until all residual fixative is removed, keep in 70% ethanol, and store at -20°C for later use. S3: Dissociation: Take the root tip after fixation treatment, wash it several times with distilled water, dry it, and place it in 1 mol / L hydrochloric acid solution at 60℃ for 3~5 min to dissociate. After taking it out, wash it with distilled water until there is no hydrochloric acid residue in the root tip. S4: Staining. Use a scalpel to cut off about 0.3-0.5 mm of the dissociated root tip meristem and place it on a glass slide. Quickly drop a drop of modified phenol-fuchsin staining solution on the root tip, which has been equilibrated in a 25°C incubator for 30 minutes in advance. Stain for 5 minutes and then cover with a coverslip. S5: Slide preparation. Cover the coverslip with a filter paper and press vertically with your finger to absorb excess stain. Hold the edge of the coverslip with one hand to prevent it from moving and affecting the observation. Hold the biological dissecting needle above with the other hand and tap the root tip of the coverslip vertically for 1-2 minutes at a rhythm of once per second. After 1 minute, pause for 5 seconds to observe the cell dispersion. Then tap for another 30 seconds to ensure even force and spread the root tip cells into a thin layer to prepare a slide specimen for microscopic examination. S6: Microscopic examination.

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