A method for genetic transformation of medicago trunculata
By genetically transforming the hypocotyl of alfalfa bean, resistant shoots were directly induced to differentiate and complete transgenic plants were obtained, solving the problem of missing genetic transformation system of alfalfa bean and realizing efficient molecular breeding improvement.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INNER MONGOLIA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-02-11
- Publication Date
- 2026-07-21
AI Technical Summary
Existing technologies cannot construct a genetic transformation system adapted to alfalfa beans, resulting in a lack of technical support for molecular breeding improvement, especially the inability to improve the splitting pod trait.
Hypocotyls of alfalfa were used as explants. Agrobacterium tumefaciens carrying the target gene expression vector was used for infection. The plants were then co-cultured and screened in a specific culture medium to directly induce the differentiation of resistant shoots. Finally, complete transgenic plants were obtained in a rooting medium.
The transformation cycle was successfully shortened, the transformation efficiency was improved, and genetically stable transgenic plants were obtained, providing a technical basis for molecular breeding of alfalfa.
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Figure CN121852457B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic transformation technology for alfalfa beans, specifically to a method for genetic transformation of alfalfa beans. Background Technology
[0002] Alfalfa sprouts are a perennial, axial-rooted, drought-tolerant legume of the genus *Alfalfa*, known for their high-quality forage. They possess excellent drought resistance and tolerance to poor soil conditions, are rich in crude protein, and have good palatability, making them valuable for livestock farming, artificial grassland establishment, and ecological restoration. However, alfalfa sprouts suffer from severe pod splitting; the pod splitting rate in experimental plants reached as high as 92.8% after maturity, significantly reducing field yield and severely hindering their industrialization and application. Plant genetic transformation is a core technology for conducting gene function research and achieving molecular improvement of species traits. Currently, two mainstream genetic transformation methods have been developed for legumes: the *Agrobacterium rhizogenes*-mediated hairy root induction system and the *Agrobacterium tumefaciens*-mediated plant regeneration transformation system. Among these, hairy roots, due to their rapid growth, high differentiation rate, and strong genetic stability, have become a high-quality explant for gene function verification and have been successfully applied in legume forages such as alfalfa, cloverleaf, and birdsfoot. However, to date, there are no reports on the establishment of genetic transformation systems for alfalfa beans, either domestically or internationally. There is a lack of technical foundation for improving its pod-splitting trait through molecular breeding. Constructing an efficient genetic transformation and hairy root gene editing system adapted to alfalfa beans has become an urgent problem to be solved.
[0003] In existing technologies, improving the success rate of genetic transformation in legumes revolves around four main dimensions: optimization of the Agrobacterium system, regulation of infection conditions, adjustment of culture medium formulation, and adaptation of gene editing systems. This has led to a mature technical approach: First, strain selection optimization, prioritizing Agrobacterium rhizogenes and Agrobacterium tumefaciens, which have broader host adaptability; second, regulation of infection conditions, with bacterial concentration and the addition of phenolic substances being key; third, optimization of culture medium and regeneration system, by adjusting the ratio of auxin to cytokinin to regulate callus formation and adventitious shoot differentiation, with the direct differentiation pathway shortening the regeneration cycle and improving efficiency; and fourth, synergy between transformation and gene editing.
[0004] Although genetic transformation technology for legumes has become increasingly mature, these technologies cannot be directly applied to alfalfa beans. Existing technologies have significant shortcomings and research gaps: First, species specificity leads to technology transfer failure. Existing optimization schemes are all based on other legume forages. Alfalfa beans have unique genotypes and tissue physiological characteristics, and there are no reference bases for key conditions such as Agrobacterium strain suitability, optimal infection parameters, and hormone ratios. Direct application easily leads to low transformation efficiency and induction failure. Second, a specific transformation system for alfalfa beans is lacking. Existing research does not involve a complete technical chain of explant screening, callus differentiation, and plant regeneration, failing to form a closed loop from transformation to trait verification. Third, the improvement of the splitting pod trait lacks technical support. Editing the splitting pod gene in alfalfa beans requires a specific genetic transformation system, and existing technologies cannot meet the needs of gene editing and functional verification. In summary, existing technologies cannot solve the core problems missing in the construction of a genetic transformation system for alfalfa beans. There is an urgent need to construct a genetic transformation system adapted to alfalfa beans to provide technical support for its molecular breeding. Summary of the Invention
[0005] The present invention aims to provide a genetic transformation method for alfalfa beans to solve the technical problem that the existing technology has not yet established a complete genetic transformation system for alfalfa beans, which makes it impossible to carry out precise molecular breeding improvement of alfalfa beans.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for genetic transformation of alfalfa beans, comprising the following steps: S1. Using the hypocotyl of sterile alfalfa seedlings as explants, Agrobacterium resuspension carrying the target gene expression vector was used to infect the explants. The infected explants were then co-cultured. The co-cultured explants were then destered and screened in a differentiation selection medium containing antibiotics and cytokinins to directly induce the differentiation of resistant shoots. The resistant shoots that had grown to a certain height were then transferred to a rooting medium to induce rooting. After hardening off, complete transgenic alfalfa plants were obtained. The target gene is MrAGL8 Gene or MrPdh1 Genes, the ones mentioned MrAGL8 The nucleotide sequence of the gene is shown in SEQ_ID_NO.1; MrPdh1 The nucleotide sequence of the gene is shown in SEQ_ID_NO.2; S2. Screening and identification of transgenic plants: PCR and sequencing identification of the target gene were performed on the above-mentioned transgenic alfalfa plants.
[0007] Preferably, as an improvement, the explant is a hypocotyl obtained from alfalfa seedlings cultured for 14 days, with 1-2 wounds made.
[0008] Preferably, as an improvement, the resuspension includes a resuspension and an Agrobacterium strain; the resuspension needs to be prepared on the same day and includes the following raw materials at the following mass concentrations: MS liquid medium, 20 g / L sucrose, 1 μg / mL zeatin, 100 μM acetylsylgenone, and pH 5.8; the Agrobacterium strain is GV3101, and the OD600 value of the Agrobacterium strain in the resuspension is 0.4.
[0009] Preferably, as an improvement, the infection includes the following steps: placing the cut hypocotyl in the infection solution for 10 minutes, placing it on sterile filter paper to blot dry the bacteria, and then placing the hypocotyl in a new infection solution for 10 minutes, gently shaking it during the infection process.
[0010] Preferably, as an improvement, the culture medium used for co-culture comprises the following raw materials at the following mass concentrations: MS solid medium, 20 g / L sucrose, 7.5 g / L agar, 1 μg / mL zeatin, 100 μM acetylsylgenone, and pH 5.8; the co-culture is performed by placing the dried hypocotyl with the wound side facing up on the culture medium and incubating it in the dark for 2-3 days until small colonies appear around the hypocotyl; rinsing the agrobacterium on the hypocotyl with sterile water until there are no impurities in the rinsing water; then rinsing twice more with sterile water containing 200 μg / mL termethin, and finally placing the hypocotyl on sterile filter paper to air dry.
[0011] Preferably, as an improvement, the differentiation screening medium comprises the following raw materials at the following mass concentrations: MS solid medium, 20 g / L sucrose, 7.5 g / L agar, 1 μg / mL zeatin, 200 μg / mL termethin, 50 μg / mL kanamycin, and pH 5.8.
[0012] Preferably, as an improvement, the rooting medium comprises the following raw materials at the following mass concentrations: MS liquid medium, 10 g / L sucrose, 6 g / L agar, and pH 5.8.
[0013] Preferably, as an improvement, the conditions for seedling hardening are as follows: temperature 21±1℃, light and dark culture for 16 / 8 h, light intensity 2000 lx, and the ratio of vermiculite to nutrient soil is 2:1.
[0014] Preferably, as an improvement, the target gene expression vector is... pCanG-HA-MrAGL8 or zmpl-bar- MrPdh1 .
[0015] Preferably, as an improvement, this scheme also provides a hyacinth bean genetic transformation system, which is a complete transgenic hyacinth bean plant constructed by the above method.
[0016] The principles and advantages of this scheme are: 1. This scheme has successfully obtained genetically transformed plants of alfalfa for the first time, breaking through the long-standing bottleneck of immature genetic transformation technology of alfalfa and the inability to obtain stable transgenic plants.
[0017] 2. Compared to the existing alfalfa tissue regeneration system with a cycle of 60-80 days, this scheme shortens the cycle to 30 days, greatly reducing the time to obtain complete plants.
[0018] 3. Initiation of the "direct organogenesis" pathway: This invention selects the hypocotyl of alfalfa with high differentiation potential as explants. By adding zeatin to the basal medium MS, the hypocotyl directly differentiates into shoots, shortening the transformation cycle and thus skipping the step of dedifferentiation to form callus tissue.
[0019] 4. Direct selection and propagation of transgenic shoots: After co-culture, explants were placed in a shoot induction medium containing kanamycin. In this medium, untransformed cells were inhibited or killed by antibiotics, while transformed cells successfully infused with the resistance gene could utilize their inherent direct shoot-forming ability to germinate resistant shoot clusters directly from the explant cut. This process eliminates the need for the lengthy stages of resistant callus induction, proliferation, and redifferentiation, significantly shortening the transformation cycle and minimizing somatic clonal variation.
[0020] 5. Regeneration and rooting: The resistant shoots that have grown to a certain height are transferred into a rooting medium to induce the formation of a complete root system, thereby obtaining genetically stable transgenic plants and providing reliable materials for subsequent genetic improvement. Attached Figure Description
[0021] Figure 1 This illustrates the effect of different concentrations of zeatin on the differentiation rate in Example 3 of the present invention.
[0022] Figure 2 This illustrates the effect of different concentrations of kanamycin on the differentiation rate in Example 3 of this invention.
[0023] Figure 3 This illustrates the effect of different concentrations of herbicide on the differentiation rate in Example 3 of the present invention.
[0024] Figure 4 This illustrates the effect of different bacterial concentrations on the differentiation rate in Example 3 of the present invention.
[0025] Figure 5 This is the hypocotyl explant of *Alfalfa* in Example 3 of the present invention.
[0026] Figure 6 The hypocotyl (7 days) on the differentiation medium (50 μg / mL KAN) in Example 3 of this invention.
[0027] Figure 7 The hypocotyl (14 d) on the differentiation medium (50 μg / mL KAN) in Example 3 of this invention.
[0028] Figure 8 The 21-day-old bud of the flat alfalfa bean with KAN resistance is shown in Example 3 of this invention.
[0029] Figure 9 This refers to the genetically modified alfalfa bean in Example 3 of the present invention. MrAGL8 Molecular detection electrophoresis image.
[0030] Figure 10 This refers to the genetically modified alfalfa bean in Example 3 of the present invention. MrAGL8 Molecular detection electrophoresis image.
[0031] Figure 11 The transgenic alfalfa bean G8M14 that was successfully sequenced in this embodiment of the invention.
[0032] Figure 12 The transgenic alfalfa bean G8M15 that was successfully sequenced in this embodiment of the invention.
[0033] Figure 13 The transgenic alfalfa bean G8M16 that was successfully sequenced in this embodiment of the invention.
[0034] Figure 14 The rooting transgenic organism in this embodiment of the invention MrAGL8 Alfalfa bean plant.
[0035] Figure 15 Transgenic seedlings used in the embodiments of the present invention MrAGL8 Alfalfa bean plant.
[0036] Figure 16 This embodiment of the invention is for alfalfa beans. MrPdh1 Electrophoresis diagram of gene-edited resistant differentiated shoots.
[0037] Figure 17 Rooting in the embodiments of the present invention MrPdh1 Gene-edited alfalfa bean plants.
[0038] Figure 18 Seedling hardening in the embodiments of the present invention MrPdh1 Gene-edited alfalfa bean plants. Detailed Implementation
[0039] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.
[0040] Overview of the Plan This protocol provides a genetic transformation method for alfalfa beans, including the following steps: S1. Using the hypocotyls of aseptic seedlings of *Alfalfa* as explants, a vector carrying the target gene (the target gene is...) was used. MrAGL8 Gene or MrPdh1 The gene and the target gene expression vector are p CanG-HA-MrAGL8 or zmpl-bar- MrPdh1 Agrobacterium resuspension (OD600 value of 0.4-0.8) was used to infect explants, and the infected explants were co-cultured. The co-cultured explants were then subjected to sterilization and screening in a differentiation selection medium containing antibiotics (including 50 μg / mL kanamycin and 1 μg / mL herbicide) and cytokinins (including 1-1.5 μg / mL zeatin) to directly induce the differentiation of resistant shoots. The resistant shoots that grew to a certain height were transferred to a rooting medium to induce rooting, and after hardening off, complete transgenic alfalfa plants were obtained. This solution is for reference only. MrAGL8 The nucleotide sequence of the gene is shown in SEQ_ID_NO.1; MrPdh1 The nucleotide sequence of the gene is shown in SEQ_ID_NO.2; S2. Screening and identification of transgenic plants: The obtained KAN-resistant transgenic seedlings (i.e., the above-mentioned transgenic alfalfa plants) were subjected to PCR and sequencing identification of the target gene.
[0041] Specifically, let's take the construction of a genetic transformation system for the hairy roots of alfalfa as an example: Flat alfalfa beans are Tumed flat alfalfa beans ( Medicago ruthenica The seeds were a generous donation from Researcher Wang Zhaolan of the Grassland Research Institute, Chinese Academy of Agricultural Sciences. The alfalfa beans were cultivated in the Plant Function Research Laboratory of the College of Life Sciences, Inner Mongolia Agricultural University. The experiment used… pCanG-HA-MrAGL8 The overexpression vector was preserved in our laboratory; the vector used in the experiment was... zmpl-bar- MrPdh1 The vector was preserved in our laboratory; the plant genomic DNA extraction kit was from Yali Biotechnology (Jiangsu) Co., Ltd.; the DNA molecular weight Trans 2K DNA Maker was from TransGen Biotech (Beijing) Co., Ltd.; the primers were synthesized by BGI Genomics Co., Ltd. (the primers used in this protocol are shown in Table 1).
[0042] Table 1 List of primers used in the experiment
[0043] Example 1: The target gene is MrAGL8 Genetic transformation methods for alfalfa beans This embodiment provides a genetic transformation method for alfalfa beans, with the target gene being... MrAGL8 Gene, MrAGL8 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and includes the following steps: S1. Using the hypocotyl of sterile alfalfa seedlings as explants, the target gene expression vector p was used. CanG-HA- MrAGL8 Agrobacterium resuspension was used to infect explants, and the infected explants were co-cultured. The co-cultured explants were then debacted and screened in a differentiation selection medium containing antibiotics and cytokinins to directly induce the differentiation of resistant shoots. The resistant shoots that had grown to a certain height were transferred to a rooting medium to induce rooting, and after hardening off, complete transgenic alfalfa plants were obtained. S2. Screening and identification of transgenic plants: The obtained KAN-resistant transgenic seedlings (i.e., the above-mentioned transgenic alfalfa plants) were subjected to PCR and sequencing identification of the target gene.
[0044] The inventors screened different parameters, and the process was as follows: 1.1 Effect of different bacterial suspension concentrations on bud induction rate GV3101 Agrobacterium (carrying the target gene expression vector p) were used with different bacterial concentrations. CanG-HA-MrAGL8 The hypocotyl was infected in three biological replicates, and the results are shown in Table 2. After 14 days, the number of adventitious buds induced and the induction rate were observed and counted, and the final bacterial concentration (OD) was determined. 600 When the coefficient of variation (C=0.4) was 0.4, the adventitious shoot induction rate was the highest, with an average differentiation rate of 15.56%. Figure 1 (and Table 2).
[0045] Table 2. Effects of different bacterial suspension concentrations on differentiation shoot induction rate
[0046] 1.2 Effect of different zeatin concentrations on shoot induction rate Adventitious shoots were induced using different concentrations of zeatin in three biological replicates, and the results are shown in Table 3. The induction rate of adventitious shoots by different concentrations of zeatin showed a normal distribution, with 1 μg / mL of zeatin showing the highest induction rate, and an average differentiation rate of 47.78%. Figure 2 (and Table 3).
[0047] Table 3 Effects of different concentrations of zeatin on shoot differentiation induction
[0048] 1.3 Effects of different concentrations of kanamycin and herbicides on the selection of resistant shoots Hypocotyls were inoculated into differentiation media supplemented with different concentrations of kanamycin (0, 25, 50, 75, and 100 μg / mL) for screening, with three biological replicates. Adventitious shoot growth was observed and recorded after 14 days to determine the kanamycin resistance screening pressure. Hypocotyls were also inoculated into differentiation media supplemented with different concentrations of herbicides (0, 0.5, 1, 1.5, and 2 μg / mL) for screening, with three biological replicates. Adventitious shoot growth was observed and recorded after 14 days to determine the herbicide resistance screening pressure (Table 4). Finally, 50 μg / mL of kanamycin was determined as the optimal resistance level. Figure 3 ) and 1 μg / mL of herbicide ( Figure 4 It can be used for screening bud differentiation of alfalfa beans.
[0049] Table 4. Screening pressure of differentiated shoots on kanamycin and herbicides
[0050] 1.4 Obtaining differentiated shoots of alfalfa 1.4.1 Preparation of Agrobacterium-infected bacterial suspension Two days before infection, single colonies on the bacterial plate were picked and placed in 1 mL of YEB liquid medium containing kanamycin (50 μg / mL) and rifampin (50 μg / mL), and cultured overnight in a sterile 2 mL centrifuge tube (28℃, 180 rpm).
[0051] One day before infection, 1 mL of overnight bacterial culture was added to 50 mL of YEB liquid medium containing kanamycin (50 μg / mL) and rifampin (50 μg / mL) at a ratio of 1:50 and cultured with shaking for 16 h (28℃, 180 rpm). The OD of the Agrobacterium culture was then measured. 600 Take 1 mL of the liquid culture medium without bacterial solution as the sample for OD measurement. 600 The blank control.
[0052] On the day of infection, wait for the bacterial culture to shake and incubate to reach OD. 600 The OD value was 0.6–0.8 (visually appearing as a wine-red turbid liquid); centrifuged at 4500 rpm for 10 min at room temperature, discarding the supernatant. The bacterial cells were resuspended in freshly prepared resuspension to OD value. 600 The concentration is 0.4~0.5, and the solution is prepared as an infiltration solution.
[0053] 1.4.2 Preparation and Infection of Alfalfa Explants Use sterilized tweezers and a scalpel to remove the roots, cotyledons, true leaves and buds from 2-week-old alfalfa seedlings to obtain the hypocotyl, and make 1-2 wounds on it (do not cut it off).
[0054] Cut the hypocotyl ( Figure 5Soak the explants in the inoculum solution for 10 minutes, then dry them on sterile filter paper. Next, soak the hypocotyls in a fresh inoculum solution for 10 minutes, gently shaking them during the inoculum process. No washing is required after inoculum inoculum. Place the inoculumed hypocotyl explants on sterile filter paper to dry completely.
[0055] Place the infected hypocotyl with the wound facing up on a co-culture medium and incubate in the dark for 2 days. Once small colonies appear around the hypocotyl, rinse the agrobacterium on the hypocotyl with sterile water until the water is free of impurities (about 5-6 times). Then, rinse twice with sterile water containing 200 μg / mL termethin and place the hypocotyl on sterile filter paper to dry completely.
[0056] The hypocotyls were transferred to a differentiation selection medium containing kanamycin and cultured under light for 16 hours for about one week. Some hypocotyls showed differentiated buds. Figure 6 After about two weeks, differentiated and undifferentiated hypocotyls can be distinguished. Figure 7 At this point, the differentiated hypocotyls can be subcultured. After three weeks of culture, some of the non-browning hypocotyls will no longer differentiate. Figure 8 ).against MrAGL8 Genetic transformation involved infecting 312 explants. After screening the infected explants with 50 μg / mL kanamycin, 81 resistant buds were obtained, representing a differentiation rate of 26%. Fourteen of the best-growing buds were selected for molecular identification, and nine of them tested positive. Figures 9-10 As shown, sequencing of sequences numbered G8M14, G8M15, and G8M16 confirmed that... MrAGL8 The sequencing results of the genetically modified alfalfa beans are as follows: Figure 11 , Figure 12 and Figure 13 As shown.
[0057] 1.5 Rooting The differentiated shoots of alfalfa were transferred to a rooting medium, and some roots appeared after about 30 days. Figure 14 For overexpression MrAGL8 Transgenic alfalfa bean rooted seedlings.
[0058] 1.6 Seedling hardening Seedlings numbered G8M14 and G8M15 were hardened off, and the results were as follows: Figure 15 As shown.
[0059] Example 2: The target gene is MrPdh1 Genetic transformation methods for alfalfa beans This embodiment is basically the same as Embodiment 1, except that: in this embodiment, the target gene is... MrPdh1 Gene, MrPdh1The nucleotide sequence of the gene is shown in SEQ_ID_NO.2. The target gene expression vector used in this embodiment is... zmpl-bar- MrPdh1 .
[0060] Inventors target gene editing MrPdh1 Genetic transformation involved infecting 836 explants. After screening with 1 μg / mL Basta, 209 resistant buds were obtained, representing a differentiation rate of 26.8%. Seventeen of the best-growing buds were selected for molecular identification, and all buds tested positive (e.g., ...). Figure 16 As shown in the figure, MPD7 is growing better.
[0061] Then, the differentiated shoots of alfalfa were transferred to a rooting medium. Some roots appeared after approximately 30-50 days. Figure 17 For hyacinth bean MrPdh1 Gene-edited rooted seedlings.
[0062] Finally, the seedlings with better growth, numbered MPD7, were hardened off, and the results were as follows: Figure 18 As shown.
[0063] In summary, this scheme achieved Agrobacterium tumefaciens-mediated genetic transformation by using the hypocotyl of *Alfalfa* as an explant, inducing direct differentiation of adventitious buds through organogenesis, and successfully obtaining complete transgenic plants, thus realizing a preliminary exploration of genetic transformation of *Alfalfa*.
[0064] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. A genetic transformation method for alfalfa beans, characterized by: Includes the following steps: S1. Using the hypocotyl of sterile alfalfa seedlings as explants, Agrobacterium resuspension carrying the target gene expression vector was used to infect the explants. The infected explants were then co-cultured. The co-cultured explants were then destered and screened in a differentiation selection medium containing antibiotics and cytokinins to directly induce the differentiation of resistant shoots. The resistant shoots that had grown to a certain height were then transferred to a rooting medium to induce rooting. After hardening off, complete transgenic alfalfa plants were obtained. The culture medium used for the co-culture includes the following ingredients: MS medium, 20 g / L sucrose, 7.5 g / L agar, 1 μg / mL zeatin, 100 μM acetylsylgenone, and pH 5.
8. The differentiation and screening medium comprises the following ingredients: MS medium, 20 g / L sucrose, 7.5 g / L agar, 1 μg / mL zeatin, 200 μg / mL termethin, 50 μg / mL kanamycin, and pH 5.
8. The target gene is MrAGL8 Gene or MrPdh1 Genes, the ones mentioned MrAGL8 The nucleotide sequence of the gene is shown in SEQ_ID_NO.1; MrPdh1 The nucleotide sequence of the gene is shown in SEQ_ID_NO.2; S2. Screening and identification of transgenic plants: PCR and sequencing identification of the target gene were performed on the above-mentioned transgenic alfalfa plants.
2. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The explants are hypocotyls obtained from 14-day-old cultured alfalfa seedlings, with 1-2 wounds created.
3. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The resuspension comprises a resuspension and an Agrobacterium strain; the resuspension must be prepared on the same day and includes the following ingredients: MS medium, 20 g / L sucrose, 1 μg / mL zeatin, 100 μM acetylsylgenone, pH 5.8; the Agrobacterium strain is GV3101, and the OD of the Agrobacterium strain in the resuspension is... 600 The value is 0.
4.
4. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The infection process includes the following steps: placing the cut hypocotyl in the infection solution for 10 minutes, absorbing the bacteria on sterile filter paper, and then placing the hypocotyl in a new infection solution for 10 minutes, gently shaking it during the infection process.
5. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The co-culture process involves placing the dried hypocotyl with the wound facing up on the culture medium and incubating it in the dark for 2-3 days until small colonies appear around the hypocotyl. The agrobacteria on the hypocotyl are then rinsed with sterile water until no impurities are found in the rinse water. After that, the hypocotyl is rinsed twice more with sterile water containing 200 μg / mL termethin, and then placed on sterile filter paper to air dry.
6. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The rooting medium comprises the following ingredients: MS medium, 10 g / L sucrose, 6 g / L agar, and pH 5.
8.
7. The method for genetic transformation of alfalfa beans according to claim 1, characterized in that: The conditions for seedling hardening were as follows: temperature 21±1℃, light and dark culture for 16 / 8 h, light intensity 2000 lx, and the ratio of vermiculite to nutrient soil was 2:1.