Construction method of ormosia calluses induced regeneration system

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a construction method of a sainfoin callus induction regeneration system, which comprises the following steps: preparing sterile seedlings of sainfoin; cotyledons of the sterile seedlings are taken, wounds are formed at the tops of the cotyledons to prepare explants, and the explants are induced to form calluses; inoculating the callus to a differential medium for induced differentiation to form buds; performing regeneration culture on the bud points to obtain regenerated seedlings; and inoculating the regenerated seedlings on a rooting culture medium for culture and rooting induction. According to the method, the sainfoin cotyledons are adopted as the explants, and compared with hypocotyls and other tissues which are complex in structure and high in lignification degree, the cotyledons are easier to conduct wound induction and hormone response, and dedifferentiation and starting of the regeneration process are promoted. Due to the selection of the explant, the method disclosed by the invention has obvious advantages in the aspects of simplicity and convenience in operation, controllability and adaptability.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and particularly relates to a method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop. Background Art

[0002] Onobrychis viciaefolia Scop. ( Onobrychis viciifolia Scop. ) as a leguminous plant of the genus Onobrychis, is widely used in agricultural production and ecological restoration due to its drought tolerance, salt tolerance, and high nitrogen fixation ability. Moreover, the high yield and palatability of Onobrychis viciaefolia Scop. play an important role in feed production. At the same time, the excellent adaptability of Onobrychis viciaefolia Scop. to arid and barren areas makes it an excellent species for ecological restoration and sustainable agricultural development. Its deep root system can effectively fix the soil, reduce soil erosion, and form an efficient nitrogen fixation system in symbiosis with rhizobia, significantly improving soil fertility. However, traditional breeding methods are limited by problems such as self-incompatibility, difficult hybridization, and long growth cycle of Onobrychis viciaefolia Scop., making it difficult to quickly screen for excellent traits such as disease resistance and salt-alkali tolerance.

[0003] Regarding the establishment of the Onobrychis viciaefolia Scop. regeneration system, it is extremely difficult to induce callus formation from some explants of Onobrychis viciaefolia Scop. during in vitro culture. Even if a small amount of callus can be produced from individual explants, these calli often show growth stagnation, browning, and even death during subsequent subculture. In addition, even if the callus survives, its differentiation potential is low, and it is often difficult to induce adventitious buds or adventitious roots, thus unable to complete the regeneration process. Summary of the Invention

[0004] Due to the phenomenon that it is difficult to induce callus formation in Onobrychis viciaefolia Scop. and it is prone to death during subsequent subculture, it is impossible to successfully construct an Onobrychis viciaefolia Scop. regeneration system. Therefore, the present invention provides a method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop.

[0005] The technical solution adopted by the present invention is as follows: The present invention provides a method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop., comprising the following steps: Preparing sterile seedlings of Onobrychis viciaefolia Scop.; Taking the cotyledons of sterile seedlings, forming wounds at the top of the cotyledons to prepare explants, and inducing the explants to form callus; Inoculating the callus onto a differentiation medium to induce the formation of bud points. The formula of the differentiation medium is: each liter of the differentiation medium contains 4 g - 5 g of MS powder, 0.1 mg - 8 mg of plant growth regulator, 28 g - 30 g of sucrose, 7 g - 8 g of agar, and the rest is made up with water; Performing regeneration culture on the bud points to obtain regenerated seedlings; Inoculate the regenerated seedlings on the rooting medium for rooting induction. The formula of each liter of rooting medium is as follows: each liter of rooting medium contains 7 g - 8 g of SH dry powder, 7 g - 8 g of agar, 8 g - 12 g of sucrose, and the rest is made up with water.

[0006] Preferably, the plant growth regulator is at least one of 6 - BA, NAA, CH, and KT.

[0007] Preferably, the concentration of 6 - BA is 0.5 mg / L - 3.0 mg / L; the concentration of NAA is 0.1 mg / L - 1.0 mg / L; the concentration of CH is 1 mg / L - 2 mg / L; the concentration of KT is 0.5 mg / L - 2 mg / L.

[0008] Preferably, the concentration of 6 - BA is 1 mg / L; the concentration of NAA is 0.5 mg / L; the concentration of CH is 1.5 mg / L; the concentration of KT is 1 mg / L.

[0009] Preferably, each liter of differentiation medium contains 4.43 g of MS dry powder, 3 mg of plant growth regulator, 30 g of sucrose, 7.8 g of agar, and the rest is made up with water; Each liter of rooting medium contains 7.105 g of SH dry powder, 7 g of agar, 10 g of sucrose, and the rest is made up with water.

[0010] Preferably, the medium formula for inducing the explant to form callus is as follows: Each liter of medium contains 4.43 g of MS dry powder, 1 mg of 6 - BA, 0.5 mg of NAA, 30 g of sucrose, 7.8 g of agar, and the rest is made up with water.

[0011] Preferably, the medium formula for regeneration culture is as follows: Each liter of medium contains 4.43 g of MS dry powder, 7 g of agar, 10 g of sucrose, and the rest is made up with water.

[0012] Preferably, the culture conditions for inducing differentiation are: day / night temperature is 25°C / 18°C, light duration is 12 h per day, and light intensity is 1800 Lx.

[0013] Preferably, the method for preparing aseptic seedlings of Onobrychis viciifolia is as follows: After disinfecting the seeds of Onobrychis viciifolia, germinate them in the dark environment. After germination, transfer them to a light culture at a day / night temperature of 25°C / 18°C, a light duration of 12 h per day, a light intensity of 1800 Lx for 7 days, and then aseptic seedlings can be obtained.

[0014] Preferably, the temperature for germination in the dark environment is 25°C.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method for constructing a callus induction and regeneration system of Onobrychis viciaefolia, comprising the following steps: Prepare aseptic seedlings of Onobrychis viciaefolia; take the cotyledons of the aseptic seedlings, form wounds at the top of the cotyledons to prepare explants, and induce the explants to form callus; inoculate the callus onto a differentiation medium to induce the formation of bud points, and the formula of the differentiation medium is: each liter of the differentiation medium contains 4 g - 5 g of MS powder, 0.1 mg - 8 mg of plant growth regulator, 28 g - 30 g of sucrose, 7 g - 8 g of agar, and the rest is made up with water; perform regeneration culture on the bud points to obtain regenerated seedlings; inoculate the regenerated seedlings onto a rooting medium for culturing to induce root growth, and the formula of each liter of the rooting medium is: each liter of the rooting medium contains 7 g - 8 g of SH powder, 7 g - 8 g of agar, 8 g - 12 g of sucrose, and the rest is made up with water. The present invention uses the cotyledons of Onobrychis viciaefolia as explants, and after culturing with the differentiation medium described in the present invention, callus is successfully induced and adventitious buds are efficiently differentiated; at the same time, the present invention also provides a supporting rooting medium, enabling all adventitious buds to take root and obtaining regenerated seedlings of Onobrychis viciaefolia, and successfully establishing a set of regeneration system for Onobrychis viciaefolia.

[0016] Genetic improvement of Onobrychis viciaefolia is realized through the callus induction and regeneration system of the present invention, providing technical support for the germplasm innovation of Onobrychis viciaefolia. In addition, combined with the callus induction and regeneration system, an efficient genetic transformation system is established, providing a key technical path for the function verification of stress-resistant genes, gene editing breeding and high-quality germplasm production of Onobrychis viciaefolia, and promoting its sustainable development in the livestock industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a flow chart for the construction of the callus induction and regeneration system. A - G are, in sequence: seed disinfection diagram, explant inoculation diagram, callus subculture diagram, callus differentiation diagram, regenerated seedling diagram of Onobrychis viciaefolia, complete plant diagram of Onobrychis viciaefolia, and growth condition diagram of Onobrychis viciaefolia after transplantation.

[0018] Figure 2 It is a diagram of the regenerated buds induced and differentiated in each example. A - I are, in sequence: Example 1 - Example 9.

[0019] Figure 3 It is a comparison diagram of root growth between Example 1 and Comparative Example 1. A: Comparative Example 1; B: Example 1.

[0020] Figure 4 It is a comparison diagram of the growth conditions of regenerated seedlings in two tissue culture bottles. A: Example 1; B: Comparative Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The present invention will be further described below through specific embodiments, but the scope of the present invention is not limited. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0022] The inventive concept of the present invention is as follows: For the establishment of the Onobrychis viciaefolia Scop. regeneration system, it is extremely difficult to induce callus formation from some explants of Onobrychis viciaefolia Scop. during in vitro culture. Even if a small amount of callus can be produced from individual explants, these calli often exhibit growth arrest, browning, and even death during subsequent subculture. In addition, even if the callus survives, its differentiation potential is low, and it is often difficult to induce adventitious buds or adventitious roots, thus unable to complete the regeneration process. These problems may be related to factors such as the complex regulation mechanism of endogenous hormone levels in Onobrychis viciaefolia Scop., the low sensitivity of callus to exogenous hormones, and the limited regulation of gene expression. Therefore, establishing a highly efficient and stable callus induction and regeneration system remains a major technical bottleneck in the current tissue culture research of Onobrychis viciaefolia Scop.

[0023] As a nutritional organ in the early stage of seedlings, cotyledons have the characteristics of tender tissue structure and strong cell division potential. Compared with tissues such as hypocotyls with complex structures and higher lignification degrees, cotyledons are more prone to wound induction and hormone response, which is conducive to promoting the initiation of dedifferentiation and regeneration processes. In addition, cotyledons have a uniform morphology and are easy to operate standardly, facilitating batch culture and system repeatability control, and are suitable for the establishment and promotion of tissue culture procedures. The selection of this explant gives the method of the present invention obvious advantages in terms of simplicity, controllability, and adaptability of operation.

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0025] The Onobrychis viciaefolia Scop. Onobrychis viciifolia Scop. used in the present invention is the wild-type Onobrychis viciaefolia Scop.

[0026] The following test reagents involved in the following embodiments are all commercially available: MS powder is purchased from Phytotech, with the product number M153; 6-BA, that is, 6-benzyladenine, is purchased from Phytotech, with the product number B130; SH powder is purchased from Phytotech, with the product number S813; NAA, that is, naphthaleneacetic acid, is purchased from Coolaber, with the product number PH101; KT, that is, 6-aminopurine, is purchased from Phytotech, with the product number K483.

[0027] HgCl2, sugar, agar, alcohol, sodium hypochlorite, sodium hydroxide, and hydrochloric acid are all commercially available.

[0028] The Chinese-English comparison of the present invention is shown in Table 1.

[0029] Table 1 Chinese-English comparison table Example 1 A method for constructing a callus induction and regeneration system for Onobrychis viciaefolia, specifically as follows: 1. Prepare aseptic seedlings of Onobrychis viciaefolia.

[0030] First, use sandpaper to remove the seed pods of common Onobrychis viciaefolia seeds. Prepare an HgCl2 solution with a solute of HgCl2, a solvent of water, and a solute mass percentage of 0.3%.

[0031] The disinfection method of Onobrychis viciaefolia seeds is as follows: In a laminar flow hood, soak the common Onobrychis viciaefolia seeds without seed pods in an HgCl2 solution with a mass percentage of 0.3% for 40 minutes. After pouring out the HgCl2 solution, add sterile water and rinse 6 times. Lay sterilized filter paper in a petri dish, moisten it with sterile water, and then lay the rinsed seeds on it. Wrap it with tin foil and germinate in the dark at 25°C. New moist filter paper needs to be replaced daily in the laminar flow hood. Count the contamination rate on the third day of seed germination. When the seeds germinate on the fourth day, place the germinated seeds in an artificial climate incubator for cultivation. The conditions are a day / night temperature of 25°C / 18°C, a light duration of 12 hours per day, a light intensity of 1800 Lx, and light cultivation for 7 days to obtain aseptic seedlings.

[0032] 2. Take the cotyledons of the aseptic seedlings, form wounds at the top of the cotyledons to prepare explants, and induce the explants to form callus.

[0033] Use a sterile scalpel to cut off the cotyledons of the aseptic seedlings, and make a cut at the top of the cotyledons to form a wound, which is beneficial for the subsequent formation of callus. Use it as an explant and inoculate it into the callus induction medium. Wrap it with tin foil and culture it in the dark at 25°C. Callus will form at the wound after about 30 days. Use forceps to pick off the callus and inoculate it onto the subculture medium for continuous subculture. During the subculture process, the browning callus needs to be removed.

[0034] The formula of the callus induction medium is: MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8. Taking 1 L as an example, the preparation method is as follows: Dissolve 4.43 g of MS powder, 1 mg of 6-BA, 0.5 mg of NAA, 30 g of sucrose, and 7.8 g of agar in water, make up the volume to 1 L, adjust the pH to 5.8 using standard-concentration HCl or NaOH, put it into a high-temperature sterilizer and sterilize at 120°C for 60 minutes, and pour it into a petri dish and cool to solidify after cooling to 65°C.

[0035] The formula of the subculture medium is: MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8. The preparation method is the same as that of the callus induction medium.

[0036] 3. Inoculate the callus onto the differentiation medium to induce the formation of bud points.

[0037] Select calli with good growth status and inoculate them onto the differentiation medium for light culture. After about 15 days, green bud points will appear on the surface of the Onobrychis viciaefolia Scop. callus, which are the regenerated buds. Culture conditions: day / night temperature is 25°C / 18°C, light time is 12 h per day, and light intensity is 1800 Lx. Then count the budding rate.

[0038] The formula of the differentiation medium is: MS + 3.0 mg / L 6-BA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8. Taking 1 L volume as an example, the preparation method of the differentiation medium is: dissolve 4.43 g of MS powder, 3.0 mg of 6-BA, 30 g of sucrose and 7.8 g of agar in water, make up the volume to 1 L, adjust the pH = 5.8 with standard concentration of HCl or NaOH, put it into a high-temperature sterilizer and sterilize at 120°C for 60 min, and pour it into a culture dish to cool and solidify after cooling to 65°C.

[0039] 4. Conduct regeneration culture on the bud points to obtain regenerated seedlings.

[0040] Inoculate the callus with green bud points on the surface onto the regeneration medium for light culture. After about 30 days, new Onobrychis viciaefolia Scop. regenerated seedlings will grow from the callus. During the regeneration culture process, the browned callus at the base of the Onobrychis viciaefolia Scop. regenerated seedlings needs to be peeled off to prevent it from affecting the growth of the regenerated seedlings. Culture conditions: day / night temperature is 25°C / 18°C, light time is 12 h per day, and light intensity is 1800 Lx.

[0041] The formula of the regeneration medium is: MS + 7 g / L agar + 10 g / L sucrose, pH = 5.8. Taking 1 L volume as an example, the preparation method of the regeneration medium is to dissolve 4.43 g of MS powder, 10 g of sucrose and 7 g of agar in water, make up the volume to 1 L, adjust the pH = 5.8 with standard concentration of HCl or NaOH, put it into a high-temperature sterilizer and sterilize at 120°C for 60 min, and pour it into a culture bottle to cool and solidify after cooling to 65°C.

[0042] 5. Inoculate the regenerated seedlings onto the rooting medium for culture to induce root growth.

[0043] The regenerated seedlings of Onobrychis viciaefolia were inoculated onto the rooting medium for light-induced rooting culture, and the complete plants of Onobrychis viciaefolia could be obtained after 60 days. Culture conditions: day / night temperature was 25°C / 18°C, light duration was 12 h per day, and light intensity was 1800 Lx.

[0044] The formula of the rooting medium was: 1 / 2SH + 7 g / L agar + 10 g / L sucrose, pH = 5.8. Taking 1 L volume as an example, the preparation method of the rooting medium was: dissolve 7.105 g of SH dry powder, 10 g of sucrose and 7 g of agar in water, make up the volume to 1 L, adjust the pH to 5.8 with standard concentration of HCl or NaOH, put it into a high-temperature sterilizer and sterilize at 120°C for 60 min, and pour it into a culture bottle after cooling to 65°C and let it cool and solidify.

[0045] 6. Hardening-off and transplanting of the regenerated seedlings.

[0046] Uncover the tissue culture bottle, seal the bottle mouth with a large sealing film and a rubber band, then make holes in the film for hardening-off, which takes 5 days. Then rinse the agar at the roots of the rooted seedlings and transplant them into a seedling-raising pot filled with a high-temperature sterilized seedling-raising substrate. The seedling-raising substrate was a mixture of nutrient soil and perlite in a volume ratio of 3:1, which was not agglomerated, did not drip water, and was preferably loose; the seedling-raising pot needed to be covered with a plastic wrap and have holes made in it, and watered regularly to obtain high-quality Onobrychis viciaefolia seedlings.

[0047] The above results are shown in Figure 1 。

[0048] Example 2 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia is as follows: Different from Example 1, the formula of the differentiation medium was MS + 0.5 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0049] Other steps were exactly the same as those in Example 1.

[0050] Example 3 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia is as follows: Different from Example 1, the formula of the differentiation medium was MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0051] Other steps were exactly the same as those in Example 1.

[0052] Example 4 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia is as follows: Different from Example 1, the formula of the differentiation medium is MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA + 1.0 mg / L CH + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0053] Other steps are exactly the same as those in Example 1.

[0054] Example 5 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop., is as follows: Different from Example 1, the formula of the differentiation medium is MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA + 2.0 mg / L CH + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0055] Other steps are exactly the same as those in Example 1.

[0056] Example 6 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop., is as follows: Different from Example 1, the formula of the differentiation medium is MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.5 mg / L KT + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0057] Other steps are exactly the same as those in Example 1.

[0058] Example 7 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop., is as follows: Different from Example 1, the formula of the differentiation medium is MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 2.0 mg / L CH + 2.0 mg / L KT + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0059] Other steps are exactly the same as those in Example 1.

[0060] Example 8 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia Scop., is as follows: Different from Example 1, the formula of the differentiation medium is MS + 0.5 mg / L 6-BA + 1.0 mg / L KT + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0061] Other steps are exactly the same as those in Example 1.

[0062] Example 9 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia, specifically as follows: Different from Example 1, the formula of the differentiation medium is MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar, pH = 5.8.

[0063] Other steps are exactly the same as those in Example 1.

[0064] Comparative Example 1 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia, specifically as follows: The formula of the differentiation medium is: MS + plant growth regulator + 30 g / L sucrose + 7.8 g / L agar. Taking 1 L volume as an example, the preparation method is: dissolve 4.43 g of MS dry powder, 30 g of sucrose and 7.8 g of agar in water, make up the volume to 1 L, adjust the pH = 5.8 with standard concentration of HCl or NaOH, put it into a high-temperature sterilization pot and sterilize at 120 °C for 60 min, and pour it into a petri dish and cool and solidify after cooling to 65 °C.

[0065] The formula of the rooting medium is: SH + 10 g / L sucrose + 7 g / L agar, pH = 5.8. Taking 1 L volume as an example, the preparation method of the rooting medium is to dissolve 14.21 g of SH dry powder, 10 g of sucrose and 7 g of agar in water, make up the volume to 1 L, adjust the pH value to 5.8 with standard concentration of HCl or NaOH, put it into a high-temperature sterilization pot and sterilize at 120 °C for 60 min, and pour it into a petri dish and cool and solidify after cooling to 65 °C.

[0066] Other steps are exactly the same as those in Example 1.

[0067] Comparative Example 2 A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia, specifically as follows: The formula of the rooting medium is: 1 / 2MS + 10 g / L sucrose + 7 g agar, pH is 5.

[0068] Different from Example 1 is the different tissue culture bottles used for the regenerated seedlings in the rooting stage, and other steps are exactly the same as those in Example 1.

[0069] From Figure 4 It can be seen that due to the poor light transmittance of the wide-mouth tissue culture bottle, the growth of the regenerated seedlings in the wide-mouth tissue culture bottle is relatively poor, the regenerated seedlings are relatively short and weak as a whole.

[0070] The budding situation table of the callus in different examples and comparative examples of the present invention is shown in Table 2; the situation of the regenerated buds induced and differentiated in each example is shown in Figure 2 ; the rooting comparison diagram of Example 1 and Comparative Example 1 is shown in Figure 3; The regenerated seedlings cultured in different tissue culture bottles are shown in Figure 4 .

[0071] Table 2 Bud emergence of callus under different differentiation medium formulations From Figure 3 it can be seen that although the base of the callus in Comparative Example 1 can take root, the growth state of the roots is not good, showing a semi-transparent state; and the growth rate is low, and there is no spread of the root system, and the regenerated seedlings are not easy to survive during the acclimatization process.

[0072] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0073] The above-described embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for constructing a callus induction and regeneration system of Onobrychis viciaefolia, characterized in that, It includes the following steps: Prepare aseptic seedlings of Onobrychis viciaefolia Scop.; Take the cotyledons of the aseptic seedlings, form wounds at the top of the cotyledons to prepare explants, and induce the explants to form callus; Inoculate the callus onto a differentiation medium to induce differentiation to form bud points. The formula of the differentiation medium is: each liter of the differentiation medium contains 4 g - 5 g of MS powder, 0.1 mg - 8 mg of plant growth regulator, 28 g - 30 g of sucrose, 7 g - 8 g of agar, and the rest is made up with water; Carry out regeneration culture on the bud points to obtain regenerated seedlings; Inoculate the regenerated seedlings onto a rooting medium for culture to induce root formation. The formula of each liter of the rooting medium is: each liter of the rooting medium contains 7 g - 8 g of SH powder, 7 g - 8 g of agar, 8 g - 12 g of sucrose, and the rest is made up with water.

2. The construction method according to claim 1, characterized in that, The plant growth regulator is at least one of 6-BA, NAA, CH, and KT.

3. The construction method according to claim 2, wherein The concentration of the 6-BA is 0.5 mg / L - 3.0 mg / L; The concentration of the NAA is 0.1 mg / L - 1.0 mg / L; The concentration of the CH is 1 mg / L - 2 mg / L; The concentration of the KT is 0.5 mg / L - 2 mg / L.

4. The construction method according to claim 3, wherein The concentration of the 6-BA is 1 mg / L; The concentration of the NAA is 0.5 mg / L; The concentration of the CH is 1.5 mg / L; The concentration of the KT is 1 mg / L.

5. The construction method according to claim 1, characterized in that Each liter of the differentiation medium contains 4.43 g of MS powder, 3 mg of plant growth regulator, 30 g of sucrose, 7.8 g of agar, and the rest is made up with water; Each liter of the rooting medium contains 7.105 g of SH powder, 7 g of agar, 10 g of sucrose, and the rest is made up with water.

6. The construction method according to claim 1, characterized in that The formula of the medium for inducing the explants to form callus is: Each liter of the medium contains 4.43 g of MS powder, 1 mg of 6-BA, 0.5 mg of NAA, 30 g of sucrose, 7.8 g of agar, and the rest is made up with water.

7. The construction method according to claim 1, characterized in that The formula of the medium for regeneration culture is: Each liter of the medium contains 4.43 g of MS powder, 7 g of agar, 10 g of sucrose, and the rest is made up with water.

8. The construction method according to claim 1, characterized in that The culture conditions for inducing differentiation are: day / night temperature is 25°C / 18°C, light time is 12 h per day, and light intensity is 1800 Lx.

9. The construction method according to claim 1, characterized in that, The method for preparing aseptic seedlings of Onobrychis viciaefolia Scop. is as follows: After disinfecting the seeds of Onobrychis viciaefolia Scop., germinate them in a dark environment. After germination, transfer them to a light culture at a day / night temperature of 25°C / 18°C, a light time of 12 h per day, and a light intensity of 1800 Lx for 7 d to obtain aseptic seedlings.

10. The construction method according to claim 9, characterized in that, The temperature for germination in the dark environment is 25°C.

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