Method for rapid tissue culture and regeneration of medicago sativa based on organogenesis pathway

Abstract

The invention belongs to the field of plant biotechnology breeding, and particularly relates to an organogenesis approach-based alfalfa rapid tissue culture regeneration method, which comprises the following steps: disinfecting and sterilizing alfalfa seeds, placing the seeds in a seed culture medium for dark culture and vernalization, and alternately culturing the vernalized seeds in a light environment and a dark environment, enabling the seeds to germinate cotyledons; cutting off cotyledons, transferring the cotyledons into a bud induction culture medium, alternately culturing in a light environment and a dark environment, and inducing to obtain cluster buds; transferring the cluster buds into a bud elongation culture medium, and alternately culturing in a light environment and a dark environment to enable the cluster buds to grow, so as to obtain highly developed cluster buds; and transferring the highly developed cluster buds into a rooting culture medium, alternately culturing in a light environment and a dark environment, and culturing and rooting to obtain a complete plant. According to the regeneration material obtained by the method, the conventional tissue culture period of 6-8 months can be shortened to only 1-2 months, and the tissue culture regeneration time is greatly shortened.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding, and in particular relates to a method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway. Background Technology

[0002] Alfalfa, a perennial high-protein forage, enjoys the reputation of "King of Forage" due to its high yield, quality, palatability, and adaptability, and occupies an important position in the development of animal husbandry. However, the current breeding quantity, speed, and quality of alfalfa are insufficient to meet production demands, resulting in a long-term reliance on imported alfalfa seeds and posing a challenge to the industry's security.

[0003] The emergence of CRISPR / Cas9 gene editing technology has provided a revolutionary technological path for alfalfa breeding. However, alfalfa, as a cross-pollinated, autotetraploid plant, presents unique challenges to bio-breeding due to its complex genetic background. Tracing its technological development, since the establishment of the callus-induced regeneration transformation system based on true leaf explants in the 1980s, despite nearly 40 years of continuous optimization (including systematic improvements to culture medium components and hormone ratios), the genetic transformation system based on the traditional callus pathway still faces three major technical bottlenecks: 1) a narrow range of recipient genotypes, applicable only to a few varieties; 2) a long regeneration cycle of approximately 6-8 months, with unstable transformation efficiency; and 3) high operating costs and poor reproducibility. The root cause of these technical bottlenecks lies in the inherent biological limitations of the traditional true leaf explant-callus regeneration system. To solve the problems in alfalfa bio-breeding development, it is essential to fundamentally innovate the technological path. Currently, constructing a novel, efficient, and stable regeneration system has become key to driving breakthroughs in genetic transformation technology.

[0004] In summary, there is an urgent need to establish a new regeneration system based on direct organogenesis to completely avoid the traditional callus formation stage, so as to shorten the cycle, increase efficiency, and broaden the applicability of alfalfa cultivation. Summary of the Invention

[0005] To address the above problems, this invention provides a method for rapid tissue culture and regeneration of alfalfa based on organogenesis pathway.

[0006] This invention is achieved through the following technical solution: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway includes the following steps: (1) After sterilizing alfalfa seeds, place them in seed culture medium and vernalize them in the dark at 0℃~10℃ for 1 to 2 days. After vernalization, alternate between 8h~16h light environment and 8h~16h dark environment for 1 to 3 days to allow the seeds to germinate cotyledons. (2) Cut off the cotyledons and transfer them into the bud induction medium. Culture them alternately in an 8h~16h light environment and an 8h~16h dark environment for 14 days to induce clustered buds. The bud induction medium is MSTB medium. (3) The obtained clustered buds were transferred into the bud elongation medium and cultured alternately in an 8h~16h light environment and an 8h dark environment for 14 days to allow the clustered buds to grow and obtain highly developed clustered buds; the bud elongation medium was MSZN medium. (4) The highly developed clustered shoots are transferred to the rooting medium and cultured alternately in an 8h-16h light environment and an 8h-16h dark environment for 22 to 37 days to cultivate roots and obtain complete plants; the rooting medium is MSSG rooting medium.

[0007] Preferably, the temperature of the illuminated environment is 20℃~26℃; the temperature of the dark environment is 18℃~22℃.

[0008] Preferably, the light intensity is 4000 lx to 4200 lx.

[0009] Preferably, the cut cotyledon includes the junction of the cotyledon and the hypocotyl.

[0010] Preferably, the seed culture medium is 1 / 2 MS medium.

[0011] Preferably, the MSTB culture medium is formulated as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 30μL thiabendazole, 500μL 6-benzylaminopurine, 3g~3.2g plant gel, 1mL cephalosporin and 1mL termethin, with distilled water to a final volume of 1L; the concentration of thiabendazole is 10mg / mL; the concentration of 6-benzylaminopurine is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

[0012] Preferably, the MSZN medium is formulated as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 0.05mL~0.5mL zeatin, 0.05mL~0.2mL naphthaleneacetic acid, 2.9g~3.1g plant gel, 1mL cephalosporin and 1mL termethin, with distilled water to a final volume of 1L; the concentration of zeatin is 1mg / mL; the concentration of naphthaleneacetic acid is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

[0013] Preferably, the MSSG rooting medium is formulated as follows: 1.11g~4.43g MS basal medium, 10g~20g sucrose, 0.1mL~0.2mL indole-3-butyric acid, 3.2g~3.4g plant gel, 1mL cephalosporin, 1mL termethin, and distilled water to a final volume of 1L; the concentration of indole-3-butyric acid is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

[0014] Preferably, alfalfa seeds are disinfected and sterilized by soaking in alcohol and sodium hypochlorite.

[0015] Preferably, the alfalfa seeds are Zhongmu No. 3 and Zhongmu No. 1 seeds.

[0016] This invention utilizes the cotyledons of germinating alfalfa seeds. Through a series of cultivation processes, the complete tissue culture cycle of alfalfa is shortened to less than two months. It features rapid callus formation, strong differentiation and reproductive capabilities, ultimately yielding several alfalfa materials with improved tissue culture rates. This provides important initial techniques and materials for alfalfa tissue culture and subsequent genetic transformation. Furthermore, the results are of high quality and obtained quickly.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway, comprising the following steps: (1) after sterilizing alfalfa seeds, place them in a seed culture medium and vernalize them in the dark at 0℃~10℃ for 1~2 days. After vernalization, the seeds are cultured alternately in an 8h~16h light environment and an 8h~16h dark environment for 1~3 days to induce the seeds to germinate cotyledons; (2) cut off the cotyledons and transfer them to a bud induction culture medium, and culture them alternately in an 8h~16h light environment and an 8h~16h dark environment for 14 days to induce clustered buds. (3) The obtained clustered buds are transferred to the bud elongation medium and cultured alternately in an 8h~16h light environment and an 8h dark environment for 14 days to allow the clustered buds to grow and obtain highly developed clustered buds; the bud elongation medium is MSZN medium; (4) The highly developed clustered buds are transferred to the rooting medium and cultured alternately in an 8h~16h light environment and an 8h~16h dark environment for 22~37 days to culture for rooting and obtain complete plants; the rooting medium is MSSG rooting medium. This invention addresses the technical bottlenecks of existing tissue culture systems, such as long regeneration cycles (6-8 months) and low regeneration efficiency. It develops a rapid tissue culture regeneration method for alfalfa based on the organogenesis pathway. The innovation lies in establishing a highly efficient direct organogenesis system, avoiding the long-cycle limitations of the traditional callus pathway. Through the selection of specific explants, hormone ratios, and screening of culture conditions, the regeneration cycle is shortened to 50-65 days. This method is highly practical and operable, with low cost, and has significant practical implications for the efficient and rapid screening of alfalfa germplasm resources. It can also provide a reference for research on tissue culture of other plants, possessing broad application prospects and potential for technology transfer.

[0018] Furthermore, the rapid tissue culture regeneration method for alfalfa based on organogenesis pathway provided by this invention offers an efficient and stable recipient system and regeneration platform to address the long-standing technical bottlenecks in its genetic transformation. This application mainly demonstrates three outstanding beneficial effects: 1. Innovation in Alfalfa Regeneration Theory: Since the 1980s, the genetic transformation system of alfalfa has relied on callus induction from true leaves or cotyledons for tissue regeneration, which suffers from problems such as long transformation cycles, strong genotype dependence, and system instability. This invention breaks away from this traditional approach, innovatively utilizing the cotyledon organogenesis pathway to achieve regeneration, thereby expanding the regeneration pathway of alfalfa and providing new theoretical basis and technical directions for research related to alfalfa regeneration and genetic transformation.

[0019] 2. Providing a novel genetic transformation approach for gene engineering research and bio-breeding: The cotyledonary explants used in this method exhibit high regeneration activity and uniform state, independent of genotype. This significantly enhances the acceptance and integration of exogenous vector plasmids, laying a material foundation for efficient transformation. Furthermore, their stable and high-frequency regeneration characteristics ensure efficient transformation of positive cells and efficient regeneration of complete plants, resulting in a significant and stable increase in the final yield of transgenic positive plants. The entire operational system is standardized, closely integrated with the genetic transformation screening process, and highly reproducible, providing reliable and efficient technical support for gene function research and genetic engineering breeding of alfalfa.

[0020] 3. Significantly shortens the genetic transformation cycle: By combining this rapid regeneration system with the genetic transformation process, the traditional genetic transformation system (based on callus tissue) takes 6-8 months (which may actually take more than 8 months) to the system (based on organogenesis) in 1-2 months, which greatly accelerates the breeding process. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a diagram showing the state of alfalfa seeds after they have been evenly spread over 1 / 2 MS. Figure 1 In the diagram, A shows the state of Alfalfa No. 3 seeds after being evenly spread over 1 / 2 MS; B shows the state of Alfalfa No. 1 seeds after being evenly spread over 1 / 2 MS.

[0023] Figure 2 This is a diagram showing the state of alfalfa seeds germinating with two cotyledons, as described in this invention. Figure 2 In the diagram, A shows the germination state of Alfalfa No. 3 with two cotyledons; B shows the germination state of Alfalfa No. 1 with two cotyledons.

[0024] Figure 3 This is a diagram showing the state of alfalfa cotyledons after they have been cut off and placed in a bud induction culture medium according to the present invention. Figure 3 In the image, A shows the state of alfalfa No. 3 after its cotyledons were cut off and placed in the bud induction medium; B shows the state of alfalfa No. 1 after its cotyledons were cut off and placed in the bud induction medium.

[0025] Figure 4 This is a diagram showing the state of alfalfa cotyledons after being induced to grow clustered buds according to the present invention. Figure 4In the diagram, A shows the state of *Amygdalus var. mongolica* after its cotyledons were induced to grow into clustered buds; B shows the state of *Amygdalus var. mongolica* after its cotyledons were induced to grow into clustered buds.

[0026] Figure 5 This is a diagram illustrating the rapid elongation of alfalfa shoots after they have been transplanted into a shoot elongation medium, according to the present invention. Figure 5 In the diagram, A shows the rapid elongation of the clustered buds of *Amyda sinensis* var. *zhongmu* 3 after being transferred to the bud elongation medium; B shows the rapid elongation of the clustered buds of *Amyda sinensis* var. *zhongmu* 1 after being transferred to the bud elongation medium.

[0027] Figure 6 This is a diagram showing the state of alfalfa buds after differentiation into complete plants according to the present invention. Figure 6 In the diagram, A shows the state of *Amyda sinensis* var. *mongolica* after its buds differentiated into complete plants; B shows the state of *Amyda sinensis* var. *mongolica* after its buds differentiated into complete plants.

[0028] Figure 7 This is a diagram showing the state of an intact alfalfa plant after it has been transplanted into nutrient soil and cultured according to the present invention. Figure 7 In the image, A shows the state of a complete *Amyda sinensis* var. *mongolica* after transplanting into nutrient soil; B shows the state of a complete *Amyda sinensis* var. *mongolica* after transplanting into nutrient soil.

[0029] Figure 8 This invention describes the entire process of callus culture and regeneration based on true leaves of Alfalfa Species No. 3. Figure 8 In the figures, A shows the state of alfalfa leaves of variety Zhongmu 3 after 7 days of dark culture on SH3a medium; B shows the state of leaves after culture on A medium followed by 30 days of dark culture on SH3a medium; C shows the state of leaves after culture on B medium followed by 30 days of subculture with 16 hours of light / 24℃, 8 hours of darkness / 20℃, and a light intensity of 4000 lx on SH3a medium; D shows the state of leaves after culture on C medium followed by one month of subculture on MSBK medium; E shows the state of leaves after culture on D medium followed by 70 days of culture on SH9a medium; and F shows the state of regenerated shoots selected from E medium and cultured on MSO medium for one month.

[0030] Figure 9 This invention describes the entire process of callus culture and regeneration of alfalfa variety Zhongmu No. 1 based on true leaves. Figure 9In the figures, A shows the state of alfalfa leaves of variety Zhongmu 1 after 7 days of dark culture on SH3a medium; B shows the state of leaves after culture on A medium followed by 42 days of dark culture on SH3a medium; C shows the state of leaves after culture on B medium followed by 42 days of subculture on SH3a medium with 16 hours of light / 24℃, 8 hours of darkness / 20℃, and a light intensity of 4000 lx; ​​D shows the state of leaves after culture on C medium followed by one month of subculture on MSBK medium; E shows the state of leaves after culture on D medium followed by three months of culture on SH9a medium; and F shows the state of regenerated shoots selected from E medium and cultured on MSO medium for one month.

[0031] Figure 10 This is a complete schematic diagram of the method for rapid tissue culture and regeneration of alfalfa based on the organogenesis pathway of the present invention.

[0032] Figure 11 This is the pCAMBIA3301 vector map of the present invention, obtained from SnapGene software.

[0033] Figure 12 This invention presents the genetic transformation process of cotyledon organogenesis in alfalfa plantlet No. 3. A shows the growth status of cotyledons infected with Agrobacterium in a co-culture medium; B shows the status of buds selected and cultured on a bud induction selection medium; C shows the elongation and rooting of the selected induced buds; D is an agarose gel electrophoresis image showing the positive identification results of plants induced after organogenesis genetic transformation, where + represents a positive control (template is Agrobacterium EHA105 containing pCAMBIA3301 plasmid); - represents a negative control (template is water); and 1-10 represent the identification of ten regenerated seedlings, with each well containing DNA from different regenerated plants.

[0034] Figure 13 This is a schematic diagram illustrating the genetic transformation process of the method for rapid tissue culture regeneration of alfalfa based on the organogenesis pathway of this invention.

[0035] Figure 14 The following diagrams show the results of screening cotyledons at suitable developmental stages according to the present invention. Among them, A shows the germination of cotyledons of different sizes at different growth stages after 1-5 days of cultivation in a light incubator, as well as the subsequent bud induction. B shows the effect of different sizes of cotyledons on subsequent bud induction, where removing the cotyledons after 1-3 days of seed growth can significantly improve the bud induction rate.

[0036] Figure 15The diagram shows the seed culture process of this invention, where the cotyledons were removed on the 3rd day and the seeds were cultured in mediums with different concentrations of hormones (TDZ and 6-BA). In the diagram, A represents the state after 7 days of culture, and B represents the state after 14 days of culture. The numbers below the diagram represent the actual amounts of 6-BA and TDZ added to the medium, and the stock solution concentrations are 6-BA (1 mg / L) and TDZ (10 mg / L), respectively.

[0037] Figure 16 The diagram shows the screening results of different hormone concentration combinations of the present invention; where A is a diagram showing the bud induction culture medium of 9 different hormone concentrations; and B is a diagram showing the bud induction rate corresponding to different hormone concentration combinations. Detailed Implementation

[0038] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0040] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0041] The following is an explanation and source of the terminology used in this invention: Seed source of Zhongmu No. 1 and Zhongmu No. 3: Both Zhongmu No. 1 and Zhongmu No. 3 were developed by Yang Qingchuan of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0042] Unless otherwise specified, all materials in this application are commercially available products.

[0043] MS (Murashige & Skoog Basal Medium With Vitamins): A commonly used basal medium for tissue culture.

[0044] 1 / 2MS solid medium: 2.22g MS basal medium, 10g sucrose, 7g agar, and distilled water to a final volume of 1L; the preparation method of the medium is as follows: weigh each material, dissolve it in distilled water and mix well, adjust the pH to 5.8 with KOH, bring the volume to a final volume, autoclave and cool to about 50℃, dispense the temporarily unsolidified culture medium into disposable petri dishes in a laminar flow hood (aseptic environment), air dry and store at 4℃.

[0045] Example 1: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 3 were used for cultivation. The specific steps are as follows: (1) Seed preparation: Alfalfa seeds of variety 3 were washed with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were blotted dry with filter paper in a laminar flow hood and then air-dried. They were then evenly spread on 1 / 2 MS medium. Figure 1 As shown in A; after vernalization in the dark at 4℃ for 1 day, the seeds were then cultured in an incubator with a light intensity of 4000 lx, alternating between 24℃ light for 16 hours and 20℃ darkness for 8 hours for 1 day, until the seeds germinated into two cotyledons. Figure 2 As shown in A in the diagram.

[0046] (2) Cut off the two cotyledons and the hypocotyl connection point, transfer them to the bud induction medium, and culture them for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 h of light at 24℃ and 8 h of darkness at 20℃, to obtain clustered buds, such as Figure 3 and Figure 4 As shown in A; the bud induction medium is MSTB medium; the formula of MSTB medium is as follows: MSTB medium: 4.43g MS basal medium, 30g sucrose, 30μL thiabendazole, 500μL 6-benzylaminopurine, 3.1g plant gel, 1mL cephalosporin, and 1mL tim, with distilled water to a final volume of 1L. Thiabendazole (TDZ) has a concentration of 10mg / mL; 6-benzylaminopurine (6-BA) has a concentration of 1mg / mL; cephalosporin (cef) and tim are both purchased from Phytotech and obtained by dissolving in sterile water and sterilizing by filtration. The concentrations of cephalosporin and tim are both 200mg / mL.

[0047] The preparation method of MSTB culture medium is as follows: Weigh the above-mentioned materials, dissolve them in distilled water and mix well. Adjust the pH to 5.8 with KOH, make up the volume, autoclave and cool to about 50°C. Dispense the temporarily solidified culture medium into disposable petri dishes in a laminar flow hood (sterile environment), dry and seal and store at 4°C.

[0048] (3) The obtained clustered buds were transferred into a bud elongation medium to promote rapid elongation. They were then cultured for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C, to obtain highly developed clustered buds, such as... Figure 5As shown in A; the bud elongation medium is MSZN medium; the formula of MSZN medium is as follows: 4.43g MS basal medium, 30g sucrose, 0.1mL zeatin, 0.1mL naphthaleneacetic acid (NAA), 3g plant gel, 1mL cephalosporin, and 1mL termethin, with distilled water to a final volume of 1L; the concentrations of zeatin, NAA, cephalosporin, and termethin are 1mg / mL, 1mg / mL, 200mg / mL, and 200mg / mL, respectively. Zeatin is abbreviated as Zeatin; NAA is abbreviated as NAA.

[0049] The preparation method of MSZN medium is as follows: Weigh the above-mentioned materials, dissolve them in distilled water and mix well. Adjust the pH to 5.8 with KOH, make up the volume, autoclave and cool to about 50°C. Dispense the temporarily solidified culture medium into disposable petri dishes in a laminar flow hood (sterile environment), dry and seal and store at 4°C.

[0050] (4) The highly developed clustered shoots were transferred to rooting medium and cultured in an incubator with a light intensity of 4000 lx for 34 days under alternating conditions of 16 hours of light at 24°C and 8 hours of darkness at 20°C to obtain complete plants, such as Figure 6 As shown in A; the rooting medium is MSSG rooting medium, and the formula of the MSSG rooting medium is as follows: 4.43 g MS basal medium, 20 g sucrose, 0.2 mL indole-3-butyric acid, 3.4 g plant gel, 1 mL cephalosporin, 1 mL termethin, and distilled water to a final volume of 1 L; the concentrations of indole-3-butyric acid and termethin are 1 mg / mL, 200 mg / mL, and 200 mg / mL, respectively. The abbreviation for indole-3-butyric acid is IBA.

[0051] The preparation method of MSSG rooting medium is as follows: Weigh the above materials, dissolve them in distilled water and mix well. Adjust the pH to 5.8 with KOH, make up the volume, autoclave and cool to about 50°C. Dispense the temporarily solidified culture medium into disposable petri dishes in a laminar flow hood (sterile environment), dry and seal and store at 4°C.

[0052] (5) Transplant the intact plants from (4) into nutrient soil, cover them with plastic wrap, and harden them off in a greenhouse by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C for 2 days. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. Figure 7 As shown in A, the induction of cotyledons (A. 3) in alfalfa is summarized in Table 1. A complete schematic diagram of the rapid tissue culture regeneration pathway of alfalfa organogenesis is shown below. Figure 10 As shown.

[0053] Example 2: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 3 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 3 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried with filter paper in a clean bench and then air-dried. They were evenly spread on 1 / 2 MS medium. After vernalization in the dark at 0℃ for 1 day, the seeds were cultured in an incubator with a light intensity of 4100 lx for 2 days under alternating conditions of 20℃ light for 16 hours and 18℃ darkness for 8 hours to allow the seeds to germinate cotyledons.

[0054] (2) Cut off two cotyledons and the connection part of their hypocotyls, transfer them into the bud induction medium, and culture them for 14 days in an incubator with a light intensity of 4100 lx, alternating between 20°C light for 16 h and 18°C ​​darkness for 8 h, to obtain clustered buds.

[0055] (3) The obtained clustered buds were transferred into the bud elongation medium to make the clustered buds elongate rapidly. They were cultured for 14 days in an incubator with a light intensity of 4100 lx, alternating between 20°C light for 16 h and 18°C ​​darkness for 8 h, to obtain highly developed clustered buds. The bud elongation medium was MSZN medium.

[0056] (4) The highly developed clustered buds were transferred into the rooting medium and cultured in an incubator with a light intensity of 4100 lx for 22 days under alternating conditions of 16 h of light at 20°C and 8 h of darkness at 18°C ​​to obtain complete plants; the rooting medium was MSSG rooting medium.

[0057] (5) Transplant the intact plants from (4) into nutrient soil, cover them with plastic wrap and place them in a greenhouse. Harden them off for 2 days by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. The remaining steps in this embodiment are exactly the same as in Example 1.

[0058] Example 3: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 3 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 3 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried in a clean bench with filter paper and then air-dried. They were evenly spread on 1 / 2 MS medium. After vernalization in the dark at 10℃ for 2 days, the seeds were cultured in an incubator with a light intensity of 4200 lx for 3 days in an alternating environment of 26℃ light for 16 hours and 22℃ darkness for 8 hours to allow the seeds to germinate cotyledons.

[0059] (2) Cut off two cotyledons and the hypocotyl connection part, transfer them into the bud induction medium, and culture them alternately for 14 days in an incubator with a light intensity of 4200 lx at 26℃ for 16 h and at 22℃ for 8 h in the dark to obtain clustered buds.

[0060] (3) The obtained clustered buds were transferred into the bud elongation medium to make the clustered buds elongate rapidly. They were cultured for 14 days in an incubator with a light intensity of 4100 lx, alternating between 26°C light for 16 h and 22°C darkness for 8 h, to obtain highly developed clustered buds. The bud elongation medium was MSZN medium.

[0061] (4) The highly developed clustered buds were transferred into the rooting medium and cultured in an incubator with a light intensity of 4200 lx for 27 days under alternating conditions of 16 h light at 26°C and 8 h darkness at 22°C to obtain complete plants; the rooting medium was MSSG rooting medium.

[0062] (5) Transplant the intact plants from (4) into nutrient soil, cover them with plastic wrap and place them in a greenhouse. Harden them off for 2 days by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. The remaining steps in this embodiment are exactly the same as in Example 1.

[0063] Table 1 summarizes the induction of cotyledons of alfalfa plantlet No. 3.

[0064] Table 1 Summary of the induction of cotyledons of alfalfa No. 3 in this invention It should be noted that the state diagrams of the cultivation process in Examples 2 and 3 are similar to those in Example 1, therefore, the results will not be shown again.

[0065] Example 4: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 1 were used for cultivation. The specific steps are as follows: (1) Seed preparation: Clean, disinfect, and sterilize alfalfa seeds (Seed No. 1). After blotting with filter paper in a clean bench and air-drying, spread evenly on 1 / 2 MS medium. Figure 1 As shown in B; after vernalization in the dark at 4℃ for 1 day, the seeds were then cultured in an incubator with a light intensity of 4000 lx, alternating between 24℃ light for 16 hours and 20℃ darkness for 8 hours for 1 day, until the seeds germinated into two cotyledons. Figure 2 As shown in B in the diagram.

[0066] (2) Cut off the two cotyledons and the hypocotyl connection point, transfer them to the bud induction medium, and culture them for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 h of light at 24℃ and 8 h of darkness at 20℃, to obtain clustered buds, such as Figure 3 and Figure 4 As shown in B in the diagram.

[0067] (3) The obtained clustered buds were transferred into a bud elongation medium to promote rapid elongation. They were then cultured for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C, to obtain highly developed clustered buds, such as... Figure 5 As shown in B in the diagram.

[0068] (4) The highly developed clustered buds were transferred to a rooting medium and cultured in an incubator with a light intensity of 4000 lx for 37 days under alternating conditions of 16 hours of light at 24°C and 8 hours of darkness at 20°C to obtain complete plants, such as Figure 6 As shown in B in the diagram.

[0069] (5) Transplant the intact plants from (4) into nutrient soil, cover them with plastic wrap, and harden them off in a greenhouse by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C for 2 days. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. Figure 7 As shown in B in Example 1. The remaining steps are exactly the same as in Example 1.

[0070] Example 5: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 1 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 1 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried with filter paper in a clean bench and then air-dried. They were evenly spread on 1 / 2 MS medium. After vernalization in the dark at 0℃ for 1 day, the seeds were cultured in an incubator with a light intensity of 4100 lx for 2 days under alternating conditions of 20℃ light for 16 hours and 18℃ darkness for 8 hours to allow the seeds to germinate cotyledons.

[0071] (2) Cut off two cotyledons and the connection part of their hypocotyls, transfer them into the bud induction medium, and culture them for 14 days in an incubator with a light intensity of 4100 lx, alternating between 20°C light for 16 h and 18°C ​​darkness for 8 h, to obtain clustered buds.

[0072] (3) The obtained clustered buds were transferred into the bud elongation medium to make the clustered buds elongate rapidly. They were cultured for 14 days in an incubator with a light intensity of 4100 lx, alternating between 20°C light for 16 h and 18°C ​​darkness for 8 h, to obtain highly developed clustered buds. The bud elongation medium was MSZN medium.

[0073] (4) The highly developed clustered buds were transferred into the rooting medium and cultured in an incubator with a light intensity of 4100 lx for 34 days under alternating conditions of 16 h of light at 20°C and 8 h of darkness at 18°C ​​to obtain complete plants; the rooting medium was MSSG rooting medium.

[0074] (5) Transplant the intact plants from (4) into nutrient soil, cover them with plastic wrap and place them in a greenhouse. Harden them off for 2 days by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. The remaining steps in this embodiment are exactly the same as in Example 1.

[0075] Example 6: A method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway In this embodiment, alfalfa seeds of variety 1 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 1 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried in a clean bench with filter paper and then air-dried. They were evenly spread on 1 / 2 MS medium. After vernalization in the dark at 10℃ for 2 days, the seeds were cultured in an incubator with a light intensity of 4200 lx for 3 days in an alternating environment of 26℃ light for 16 hours and 22℃ darkness for 8 hours to allow the seeds to germinate cotyledons.

[0076] (2) Cut off two cotyledons and the hypocotyl connection part, transfer them into the bud induction medium, and culture them alternately for 14 days in an incubator with a light intensity of 4200 lx at 26℃ for 16 h and at 22℃ for 8 h in the dark to obtain clustered buds.

[0077] (3) The obtained clustered buds were transferred into the bud elongation medium to make the clustered buds elongate rapidly. They were cultured for 14 days in an incubator with a light intensity of 4100 lx, alternating between 26°C light for 16 h and 22°C darkness for 8 h, to obtain highly developed clustered buds. The bud elongation medium was MSZN medium.

[0078] (4) The highly developed clustered buds were transferred into the rooting medium and cultured in an incubator with a light intensity of 4200 lx for 35 days under alternating conditions of 16 h light at 26°C and 8 h darkness at 22°C to obtain complete plants; the rooting medium was MSSG rooting medium.

[0079] (5) Transplant the intact plants into nutrient soil, cover them with plastic wrap and place them in a greenhouse. Harden them off for 2 days by alternating between 16 hours of light at 24°C and 8 hours of darkness at 20°C. The light intensity in the greenhouse is 7500 lx. After that, remove the plastic wrap and continue to cultivate them in the same environment. The remaining steps in this embodiment are exactly the same as in embodiment 1.

[0080] Table 2 summarizes the induction of cotyledons in alfalfa plantlets No. 1.

[0081] Table 2 Summary of the induction of cotyledons of alfalfa in this invention It should be noted that the state diagrams of the cultivation process in Examples 5 and 6 are similar to those in Example 1, therefore, the results will not be shown again.

[0082] Furthermore, the culture medium formulations described in Examples 1 to 6 of this invention can all be used for rapid tissue culture regeneration of alfalfa within the following numerical ranges, and achieve similar effects.

[0083] The MSTB medium formula is as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 30μL thiamethoxam, 500μL 6-benzylaminopurine, 3g~3.2g plant gel, 1mL cephalosporin and 1mL termethin, and distilled water to make up to 1L.

[0084] The MSZN medium formula is as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 0.05mL~0.5mL zeatin, 0.05mL~0.2mL naphthaleneacetic acid, 2.9g~3.1g plant gel, 1mL cephalosporin and 1mL termethin, and distilled water to a final volume of 1L.

[0085] The formula for MSSG rooting medium is as follows: 1.11g~4.43g MS basal medium, 10g~20g sucrose, 0.1mL~0.2mL indole-3-butyric acid, 3.2g~3.4g plant gel, 1mL cephalosporin, 1mL termethin, and distilled water to a final volume of 1L.

[0086] Comparative Example 1: Tissue culture of leaves from alfalfa variety Zhongmu 3 (1) Explant preparation: Cut off the leaves of clean, insect-free and disease-free alfalfa plant No. 3 and place them in a clean bottle. Wash the leaves twice with clean water to remove surface dirt and dust. Then wash the leaves with alcohol for 30 seconds. Then wash the leaves with clean water again to remove the residual alcohol. Add 1 drop of Sodium Hypochlorite (to protect the leaves) to the bottle with a 1 mL pipette. Then add 20% sodium hypochlorite to the bottle for 5 min for disinfection. Wash with clean water 5 times. All washing processes are completed in a clean bench.

[0087] (2) Ultrasonic treatment: Place the cleaned leaves in a new clean bottle, add an appropriate amount of SH3a liquid culture medium (enough to cover the leaves in the bottle), and sonicate them for 10 seconds to create wounds on the back of the leaves.

[0088] (3) Callus induction: After sonication, the leaves were placed on clean filter paper, the moisture was absorbed, and then the wound was placed face down on SH3a solid medium. The leaves were cultured in the dark for 25 days to induce dedifferentiation. Then, they were cultured in an incubator for 30 days with alternating light (16 h at 24°C) and dark (8 h at 24°C), with a light intensity of 4000 lx. Subculture was performed every 14 days to induce callus formation. The entire callus induction process took approximately 2 months. Figure 8 As shown in A~C.

[0089] (4) Induction of bud differentiation: Well-developed callus tissue was placed in MSBK solid medium and cultured in an incubator with alternating light (16 h at 24°C) and darkness (8 h at 24°C) for 30 days, with a light intensity of 4000 lx. Subculture was performed every 14 days to induce bud regeneration. Figure 8 As shown in D; (5) Induction of bud growth and rooting: The callus tissue with buds from (4) was transferred into SH9a solid medium and cultured in an incubator with alternating light (16 h) and darkness (8 h) at 24°C until complete plants grew. The light intensity was 4000 lx. Subculture was performed every 14 days. This process took about 2 months. Figure 8 As shown in E in the figure.

[0090] Obtaining plants: The complete plants grown in (5) are transferred to small bottles containing MSO rooting medium for cultivation until they reach a height of 5cm. Figure 8 As shown in F in the figure. After subsequent hardening-off, the seedlings were transplanted into soil and cultured in a greenhouse with alternating periods of 16 hours of light at 22°C and 8 hours of darkness at 20°C, with a light intensity of 6700 lx. The tissue culture results of 120 leaves of Alfalfa Species No. 3 are summarized in Table 7.

[0091] Comparative Example 2: Tissue culture of leaves from Alfalfa Species No. 3 (1) Explant preparation: Cut off the leaves of clean, insect-free and disease-free alfalfa plant No. 3 and place them in a clean bottle. Wash the leaves with clean water 3 times to remove surface dirt and dust. Then wash the leaves with alcohol for 30 seconds. Then wash the leaves with clean water again to remove the residual alcohol. Add 2 drops of Sodium Hypochlorite (to protect the leaves) to the bottle with a 1mL pipette. Then add 20% sodium hypochlorite (by volume) to the bottle for disinfection for 5 minutes. Wash with clean water 6 times. All washing processes are completed in a clean bench.

[0092] (2) Ultrasonic treatment: Place the cleaned leaves in a new clean bottle, add an appropriate amount of SH3a liquid culture medium (enough to cover the leaves in the bottle), and sonicate them for 30 seconds to create wounds on the back of the leaves.

[0093] (3) Callus induction: After sonication, the leaves were placed on clean filter paper and the moisture was absorbed. Then, the wound was placed face down on SH3a solid medium and cultured in the dark for 35 days to induce leaf dedifferentiation. In the incubator, the leaves were cultured alternately at 24℃ for 16 hours of light and 24℃ for 8 hours of darkness for 40 days with a light intensity of 4200 lx. The cells were subcultured every 14 days to induce callus production. The entire callus induction time was about 2 months.

[0094] (4) Induction of bud differentiation: The callus tissue in good condition was placed in MSBK solid medium and cultured in an incubator with alternating light at 24℃ for 16h and darkness at 24℃ for 8h for 30 days, with a light intensity of 4000lx. The tissue was subcultured every 14 days to induce bud regeneration.

[0095] (5) Inducing bud growth and rooting: The callus tissue with buds in (4) was transferred into SH9a solid medium and cultured in an incubator with alternating light at 24°C for 16 hours and darkness at 24°C for 8 hours until a complete plant grew. Subculture was performed every 14 days. This process took about 3 months.

[0096] Obtaining plants: The complete plants grown in (5) were transferred into small bottles containing MSO rooting medium and cultured until they grew to 10cm in height. After hardening off, they were transferred into soil and cultured in a greenhouse with alternating light exposure of 22℃ for 16h and darkness exposure of 20℃ for 8h, with a light intensity of 7500lx.

[0097] Table 7 Summary of tissue culture results from 120 leaves of alfalfa variety Zhongmu 3 Comparative Example 3: Tissue culture of leaves from alfalfa variety Zhongmu 1 Comparative Example 3 was cultured using the same method as Comparative Example 1 to obtain regenerated plants. The total cycle was 6 months, and the culture process is shown in the figure below. Figure 9 As shown in Table 8, the tissue culture results of 120 leaves from alfalfa variety Zhongmu No. 1 are summarized in Table 8.

[0098] Table 8 Summary of tissue culture results from 120 leaves of Alfalfa No. 1 Comparative Example 4: Tissue culture of leaves from alfalfa variety Zhongmu 1 Comparative Example 4 was cultured using the same method as Comparative Example 2 to obtain regenerated plants, with a total cycle of 8 months.

[0099] Compared with the examples, the comparative examples have obvious disadvantages in terms of speed, ease of operation and other aspects. Both of the comparative examples mentioned above require complicated cleaning, sterilization and disinfection of the leaves during the experiment. The process involves dedifferentiation and redifferentiation of the tissue cells of alfalfa leaves, which is a long process, difficult to operate and easy to be infected by fungi.

[0100] The culture medium formulations used in the comparative examples are shown in Tables 3-6.

[0101] Table 3 SH3a solid culture medium Table 4 MSBK solid culture medium Table 5 SH9a solid culture medium Table 6 MSO solid culture medium Example 7 To verify the application value of the organogenesis-based rapid tissue culture regeneration method for alfalfa provided by this invention in genetic transformation, this embodiment uses the method of this invention for plant tissue culture during Agrobacterium-mediated genetic transformation. This method significantly shortens the cycle from infection to obtaining positive plants.

[0102] Genetic transformation: I. Vector selection: pCAMBIA3301 overexpression vector (empty vector).

[0103] Some important components in the carrier: 1. Promoter: CaMV 35S promoter (Tobacco Mosaic Virus 35S promoter), used to enhance gene expression.

[0104] 2. KanR: Kanamycin resistance tag, which acts as a prokaryotic resistance in this vector to induce kanamycin resistance in the strain.

[0105] 3. BIpR: Glufosinate resistance tag (PPT, phosphinothricin). In this vector, it is both true and resistant. When it is subsequently transferred into plants, positive plants will develop glufosinate resistance.

[0106] 4. GUS gene tag: The gene tag that comes with the vector.

[0107] Note: The plasmids used in this vector are commonly used by our research group and have been extensively preserved, so there is no need for repeated extraction.

[0108] The vector spectrum of pCAMBIA3301 is as follows: Figure 11 As shown.

[0109] II. Transformation of Competent Cells 1. Competent strain: Agrobacterium EHA105.

[0110] 2. Operating Instructions: a) Take Agrobacterium competent cells stored at -80℃ and place them at room temperature until they partially melt into an ice-water mixture, then insert them into ice.

[0111] b) Add 1 μg of plasmid DNA to every 100 μL of competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 min, liquid nitrogen for 5 min, water bath at 37°C for 5 min, and ice bath for 5 min in sequence.

[0112] c) Add 700 μL of antibiotic-free LB or YEB liquid medium and incubate at 28°C with shaking for 3 h.

[0113] d) Centrifuge at 6000 rpm for 1 min to collect bacteria, collect about 100 μL of supernatant, gently pipette to resuspend the bacterial block, spread it on LB agar plates containing the corresponding antibiotic (kanamycin in this invention) and rifampin, and incubate at 28°C for 3 days. The concentration of kanamycin is 50 µg / mL and the concentration of rifampin is 20 µg / mL.

[0114] e) Verify and confirm that the grown monoclonal strain is a positive monoclonal Agrobacterium carrying the correct vector, and use it as an engineered strain for the preparation of subsequent infection solutions.

[0115] f) Preparation of infection solution: Positive monoclonal Agrobacterium was inoculated into YEB liquid medium containing 50 µg / mL kanamycin and 20 µg / mL rifampin, and cultured at 28°C with shaking at 200 rpm until mid-logarithmic growth (OD).600 Under aseptic conditions, take 2 mL of bacterial suspension into a centrifuge tube, centrifuge at 4000 rpm for 5 minutes to collect the bacterial cells, and discard the supernatant. Gently resuspend the bacterial cells in an equal volume of infection buffer (1 / 2 MS liquid medium, with 100 µM acetylsalicylic acid added, pH adjusted to 5.2), and quantify the OD value of the bacterial suspension as 0.6. 600 The value is adjusted to 0.3 to obtain the inoculum.

[0116] III. Agrobacterium infection of cotyledons (1) The seeds of alfalfa No. 3 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried with filter paper in a clean bench and then spread evenly on 1 / 2 MS medium. After vernalization in the dark at 4℃ for 1 day, the seeds were cultured in an incubator with a light intensity of 4000 lx for 1 day under alternating conditions of 24℃ light for 16 hours and 20℃ darkness for 8 hours, so that the seeds germinated two cotyledons.

[0117] (2) Cut off the obtained cotyledons and place them in a clean vial containing the infection solution. After sealing, vacuum treatment is performed for 10 minutes. Then, in a clean bench, after rinsing with sterile water, the moisture is absorbed with sterile filter paper and spread flat in a co-culture medium for 24 hours.

[0118] (3) The cotyledons that have been co-cultured are placed in the bud induction selection medium and cultured alternately for 14 days under 24°C light for 16 hours and 20°C darkness for 8 hours to select and induce clustered buds; the bud induction selection medium is MSTB selection medium; the formula and preparation method of MSTB medium are exactly the same as in Example 1.

[0119] (4) The obtained clustered buds were transferred into the bud elongation medium to make the clustered buds elongate rapidly. They were cultured for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 h of light at 24°C and 8 h of darkness at 20°C, to obtain highly developed clustered buds. The bud elongation medium was MSZN medium. The formula and preparation method of MSZN medium were exactly the same as in Example 1.

[0120] (5) The highly developed clustered shoots were transferred to rooting medium and cultured for 34 days in an incubator with a light intensity of 4000 lx, alternating between 16 h of light at 24°C and 8 h of darkness at 20°C, to obtain complete plants. The rooting medium was MSSG rooting medium, and the formulation and preparation method of MSSG rooting medium were exactly the same as in Example 1. The obtained plant DNA was extracted and positively identified by PCR and agarose gel electrophoresis. A schematic diagram of the genetic transformation method of rapid tissue culture regeneration of alfalfa based on organogenesis pathway is shown below. Figure 13 As shown.

[0121] DNA was extracted from the plants using the CTAB method.

[0122] The procedure for extracting DNA from plants using the CTAB method is as follows: a) Mix 20 uI RNase with 40 mL CTAB (1:2000 ratio).

[0123] b) Take 0.2g of the leaf and put it into a 2mL centrifuge tube to homogenize it. Centrifuge briefly and shake all the homogenate from the mouth of the bottle to the bottom of the bottle. Add 700μL of CTAB.

[0124] c) Store at 65℃ for 30 minutes, then shake gently for 10 minutes.

[0125] d) Add 700uL of chloroform and shake thoroughly to mix completely.

[0126] e) Centrifuge at 13,000 rpm for 15 min at room temperature.

[0127] f) Take 600 μL of the supernatant, transfer it to a new centrifuge tube, add two-thirds volume of isopropanol, mix at -20°C and let stand for 15 min, centrifuge at 4°C for 15 min, remove the supernatant at 13000 rpm, and wash twice with 70% ethanol.

[0128] g) Centrifuge for five minutes to remove the upper layer of isopropanol, air dry under a clean bench, and then add 200uL of ddH2O.

[0129] DNA was obtained by placing the sample at -4℃ overnight and storing it at -20℃ for later use.

[0130] The obtained DNA was used for PCR. The primers for the PCR reaction are shown below: The base sequence of the upstream primer GUS-f is shown in SEQ ID NO.1: GCCGTATGTTATTGCCGGGAA.

[0131] The base sequence of the downstream primer GUS-r is shown in SEQ ID NO.2: ATCGGCTGATGCAGTTTCTCC.

[0132] PCR reaction procedure: 95℃ for 3 minutes (pre-denaturation), 95℃ for 15 seconds, 58℃ for 15 seconds, 72℃ for 60 seconds, 72℃ for 5 minutes; all programs are repeated 30 times.

[0133] The primers were obtained from Sangon Biotech (Shanghai) Co., Ltd.

[0134] After the PCR reaction was completed, the PCR products were recovered and positively identified by agarose gel electrophoresis.

[0135] The results of the identification are as follows Figure 12 As shown, positive plants carrying the pCAMBIA3301 vector were successfully obtained from Agrobacterium infection, and the PCR detection rate of positive plants remained at a high level; moreover, the entire cycle could be shortened to less than 2 months. This example fully demonstrates that the rapid tissue culture system established in this invention provides an efficient and stable technical platform for the genetic transformation of alfalfa, greatly shortening the transgenic breeding cycle.

[0136] It should be noted that the rapid tissue culture regeneration method of alfalfa based on organogenesis pathway described in Examples 2-6 of this invention can be used for plant genetic transformation to successfully obtain positive plants, and the PCR detection rate of positive plants remains at a high level; and the entire cycle can be shortened to less than 2 months.

[0137] Experimental Example 1 Select cotyledons at the appropriate developmental stage as the main material for subsequent organogenesis.

[0138] In this experimental example, seeds of alfalfa variety Zhongmu 3 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 3 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried with filter paper in a clean bench and then spread evenly on 1 / 2 MS medium. After vernalization in the dark at 0℃ for 1 day, the seeds were cultured in an incubator with a light intensity of 4000 lx for 1 to 5 days in an alternating environment of 24℃ light for 16 hours and 20℃ darkness for 8 hours to allow the seeds to germinate cotyledons of different sizes.

[0139] (2) Cut off two cotyledons and the hypocotyl junction, transfer them to a bud induction medium, and culture them for 14 days in an incubator with a light intensity of 4000 lx, alternating between 16 h of light at 24°C and 8 h of darkness at 20°C, to obtain clustered buds. Count the number of clustered buds to determine the appropriate development time for the cotyledons in the light incubator. Finally, determine the cotyledons that germinate from seeds cultured in the light incubator for 1-3 days, such as... Figure 14 As shown, this is the most suitable. (The bud induction medium is the MSTB medium, with the following formula: 2.22g MS basal medium, 20g sucrose, 30μL thiamethoxam, 500mL benzylaminopurine, 3g plant gel, 1mL cephalosporin and 1mL termethin, with distilled water to a final volume of 1L; the drugs in this formula represent the minimum effective dose).

[0140] Experiment Example 2 The optimal hormone ratio was selected for use in the primary bud induction medium to induce organogenesis.

[0141] In this experimental example, seeds of alfalfa variety Zhongmu 3 were used for cultivation. The specific steps are as follows: (1) Seed preparation: The seeds of alfalfa No. 3 were cleaned with sterile water to remove dust, soaked in alcohol for 5 minutes for disinfection, soaked in sodium hypochlorite for 10 minutes for further disinfection and sterilization, and finally washed with sterile water to remove residue. After cleaning, disinfection and sterilization, the seeds were dried in a clean bench with filter paper and then air-dried. They were evenly spread on 1 / 2 MS medium. After vernalization in the dark at 0℃ for 1 day, the seeds were cultured in an incubator with a light intensity of 4000 lx, alternating between 24℃ light for 16 h and 20℃ darkness for 8 h for 1 day, so that the seeds could germinate cotyledons of different sizes.

[0142] (2) Cut off the two cotyledons and the hypocotyl junction, and transfer them into bud induction culture media with different hormone concentrations, such as... Figure 15 and 16 As shown, in an incubator with a light intensity of 4000 lx, the plants were cultured for 14 days under alternating conditions of 24℃ light for 16 h and 20℃ darkness for 8 h. The clustered shoots were observed, the number of clustered shoots was counted, and the optimal hormone concentration ratio for cotyledon induction was determined. Finally, the hormone ratio of 0.3 mg / L TDZ (with 30 μL of 10 mg / mL TDZ stock solution) and 0.5 mg / L 6-BA (with 500 μL of 1 mg / mL 6-BA stock solution) was found to have the highest shoot induction rate.

[0143] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention, and used in the establishment and application of genetic transformation systems, should fall within the protection scope defined by the claims of the present invention.

[0144] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for rapid tissue culture regeneration of Medicago sativa based on an organogenic pathway, characterized in that, Includes the following steps: (1) After sterilizing alfalfa seeds, place them in seed culture medium and vernalize them in the dark at 0℃~10℃ for 1 to 2 days. After vernalization, alternate between 8h~16h light environment and 8h~16h dark environment for 1 to 3 days to allow the seeds to germinate cotyledons. (2) Cut off the cotyledons and transfer them into the bud induction medium. Culture them alternately in an 8h~16h light environment and an 8h~16h dark environment for 14 days to induce clustered buds. The bud induction medium is MSTB medium. (3) The obtained clustered buds were transferred into the bud elongation medium and cultured alternately in an 8h~16h light environment and an 8h dark environment for 14 days to allow the clustered buds to grow and obtain highly developed clustered buds; the bud elongation medium was MSZN medium. (4) The highly developed clustered shoots are transferred to the rooting medium and cultured alternately in an 8h-16h light environment and an 8h-16h dark environment for 22 to 37 days to cultivate roots and obtain complete plants; the rooting medium is MSSG rooting medium.

2. The method of claim 1, wherein, The temperature of the illuminated environment is 20℃~26℃; the temperature of the dark environment is 18℃~22℃.

3. The method of claim 1, wherein, The light intensity is 4000 lx to 4200 lx.

4. The method of claim 1, wherein, The cut cotyledon includes the junction of the cotyledon and the hypocotyl.

5. The method of claim 1, wherein, The seed culture medium is 1 / 2 MS medium.

6. The method of claim 1, wherein, The MSTB medium formulation is as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 30μL thiabendazole, 500μL 6-benzylaminopurine, 3g~3.2g plant gel, 1mL cephalosporin and 1mL termethin, with distilled water added to a final volume of 1L; the concentration of thiabendazole is 10mg / mL; the concentration of 6-benzylaminopurine is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

7. The method of claim 1, wherein, The MSZN medium formulation is as follows: 2.22g~4.43g MS basal medium, 20g~30g sucrose, 0.05mL~0.5mL zeatin, 0.05mL~0.2mL naphthaleneacetic acid, 2.9g~3.1g plant gel, 1mL cephalosporin and 1mL termethin, with distilled water to a final volume of 1L; the concentration of zeatin is 1mg / mL; the concentration of naphthaleneacetic acid is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

8. The method of claim 1, wherein, The MSSG rooting medium formula is as follows: 1.11g~4.43g MS basal medium, 10g~20g sucrose, 0.1mL~0.2mL indole-3-butyric acid, 3.2g~3.4g plant gel, 1mL cephalosporin, 1mL termethin, and distilled water to a final volume of 1L; the concentration of indole-3-butyric acid is 1mg / mL; the concentration of cephalosporin is 200mg / mL; and the concentration of termethin is 200mg / mL.

9. The method of claim 1, wherein, Alfalfa seeds are disinfected and sterilized by soaking in alcohol and sodium hypochlorite.

10. The method according to any one of claims 1 to 9, characterized in that, The alfalfa seeds mentioned are Zhongmu No. 3 and Zhongmu No. 1 seeds.

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