Regeneration method of sainfoin

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to a sainfoin regeneration method which comprises the following steps: inoculating a hypocotyl of a sainfoin sterile seedling as an explant to a regeneration and subculture medium for culture, firstly performing dark culture to obtain an adventitious bud, then performing subculture to obtain a rootless seedling, and performing seedling culture to obtain the sainfoin. And inoculating the rootless seedlings to a rooting culture medium for rooting culture to obtain a sainfoin regeneration plant. According to the method, the time used for constructing a sainfoin regeneration system is greatly shortened, the efficiency is improved, the survival rate of transplanting is high, and a foundation is laid for germplasm resource protection, propagation and subsequent genetic transformation research of sainfoin.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and particularly relates to a sainfoin regeneration method. Background Art

[0002] Sainfoin Onobrychis viciifolia Scop. ) is a plant of the genus Leguminosae in the Leguminosae family. It is highly favored for its high yield, good palatability and easy cultivation. Known as the queen of forage grass, this reputation further highlights its importance in animal husbandry. In recent years, with the rapid development of biotechnology, research on sainfoin in tissue culture and genetic transformation has also made some progress. However, despite the relevant research, the establishment of the regeneration system of sainfoin still faces some challenges, among which the long cycle of regeneration system construction is an obvious problem.

[0003] The regeneration system of sainfoin is crucial for subsequent genetic improvement. In order to accelerate this process, further in-depth research and development of efficient regeneration systems are needed. By improving existing technologies, the time required to build a sainfoin regeneration system can be shortened, thus providing a more reliable basis for its genetic improvement. Only by overcoming the challenges currently faced by the research on sainfoin regeneration systems can the genetic improvement of sainfoin be promoted, thereby improving its economic and ecological benefits in animal husbandry. By focusing on solving the key issues of the regeneration system, we can better utilize this important resource of sainfoin and make positive contributions to the sustainable development of animal husbandry. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for regenerating sainfoin.

[0005] The present invention provides a method for regenerating sainfoin, which comprises the following steps: using the hypocotyl of a sterile seedling of sainfoin as an explant, inoculating it on a regeneration and subculture medium for cultivation, first dark culturing to obtain adventitious buds, then subculture to obtain rootless seedlings, inoculating the rootless seedlings on a rooting medium for rooting culture, and obtaining a regenerated plant of sainfoin; The regeneration and subculture medium is a solid medium based on MS solid medium, to which 6-BA and NAA are added, to obtain a solid medium with a 6-BA concentration of 1.0 mg / L, an NAA concentration of 0.5 mg / L, and a pH value of 5.8.

[0006] In the above method, the regeneration and subculture medium is a solid medium obtained by adding sucrose, a coagulant and a plant growth regulator (6-BA and NAA) to a MS liquid medium.

[0007] In the above method, the solidifying agent in the regeneration and subculture medium is agar.

[0008] In the above method, the content of sucrose in the regeneration and subculture medium is 30 g / L, and the content of agar is 7.8 g / L.

[0009] In the above method, the sterile seedlings of Sainfoin are obtained by placing sterilized seeds of Sainfoin after removing seed pods on moistened filter paper for dark germination, and culturing the germinated seeds for 4-6 days.

[0010] In the above method, the disinfection is to soak in a 0.3% by mass HgCl2 solution for 30 minutes and then rinse with sterile water.

[0011] In the above method, the conditions for culturing the germinated seeds are: day / night temperature of 25°C / 18°C, light duration of 12 hours per day, and light intensity of 1800 Lx.

[0012] In the above method, the rooting medium is 1 / 2MS solid medium with a pH value of 5.8.

[0013] In the above method, the rooting medium is a solid medium obtained by adding sucrose and a coagulant to a 1 / 2MS liquid medium.

[0014] In the above method, the solidifying agent in the rooting medium is agar.

[0015] In the above method, the content of sucrose in the rooting medium is 30 g / L, and the content of agar is 7.8 g / L.

[0016] In the above method, the temperature during dark culture is 25° C., and the culture is carried out for 5-7 days.

[0017] In the above method, the subculture culture conditions are as follows: day / night temperature is 25°C / 18°C, light duration is 12h per day, and light intensity is 1800Lx.

[0018] The method for regenerating sainfoin also includes the steps of hardening and transplanting.

[0019] Compared with the prior art, the beneficial effect of the present invention is that it greatly shortens the time used to construct the regeneration system of red clover, improves efficiency, and has a high survival rate of transplantation, which lays a foundation for the protection, propagation and subsequent genetic transformation research of red clover germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A flow chart is established for the regeneration system of Sainfoin in Example 1 of the present invention, wherein: Figure 1 A is a photo of seed disinfection. Figure 1 B is a photo of seed germination. Figure 1 C is a photo of hypocotyl inoculation. Figure 1 D is a photo of explant sprouts. Figure 1 E is a photo of the growth of the subcultured tissue culture bottle. Figure 1 F is the growth diagram of inoculation into rooting medium. Figure 1 G is a photo of the growth after transplanting into the seedling pot.

[0021] Figure 2 This is a diagram of the tissue culture results of Sainfoin in Example 2-5 of the present invention. Figure 2 A is the tissue culture result of Example 2, Figure 2 B is the tissue culture result of Example 3, Figure 2 C is the tissue culture result of Example 4, Figure 2 D is the tissue culture result of Example 5. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] Sainfoin Onobrychis viciifolia Scop. ) Variety Caohong No. 1 is a product of Gansu Daxing Agricultural Science and Technology Development Co., Ltd.

[0025] The following test reagents involved in the following examples are all commercially available: MS dry powder (Phytotech M153); 6-BA (6-benzyladenine, Phytotech B130); NAA (naphthaleneacetic acid, Coolaber PH101); HgCl2 (mercuric chloride); sucrose; agar; alcohol; sodium hypochlorite; sodium hydroxide; hydrochloric acid.

[0026] Example 1 In this embodiment, the grass red No. 1 red bean grass ( Onobrychis viciifolia Scop. ) The regeneration method is as follows: 1. Obtaining sterile seedlings: First, use sandpaper to remove the seed pods of Sainfoin No. 1.

[0027] Prepare a HgCl2 solution (solute is HgCl2, solvent is water) with a solute mass percentage of 0.3%.

[0028] The method for disinfecting seeds is to soak the seeds of Red Bean Grass No. 1 with the seed pods removed in a 0.3% HgCl2 solution by mass for 40 minutes, pour out the HgCl2 solution and then rinse with sterile water for 6 times.

[0029] Place sterilized filter paper in a petri dish, moisten it with sterile water, and then place the rinsed seeds on top (see Figure 1 A), wrapped in tin foil and germinated in the dark at 25℃ (room temperature), and new moistened filter paper was replaced every day. The contamination rate was calculated on the third day of seed germination, and the seeds were germinated on the fourth day (see Figure 1 B) After counting the number of germinations and the germination rate, the germinated seeds were placed in an artificial climate incubator for cultivation under the conditions of 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 5-7 days to obtain sterile seedlings.

[0030] 2. Preparation of required culture medium: Regeneration and subculture medium: formula is MS + 1.0 mg·L －１ 6-BA+0.5mg·L －１ NAA+30g / L sucrose+7.8g / L agar, pH=5.8. Taking 1 L volume as an example, the preparation method of regeneration and subculture medium is to dissolve 4.43 g MS dry powder, 1.0 mg 6-BA (6-benzyladenine), 0.5 mg NAA (naphthaleneacetic acid), 30 g sucrose and 7.8 g agar in water, dilute to 1 L, use standard concentration of HCl and NaOH to adjust pH=5.8, put it in a high temperature sterilizer at 120℃ for 60 min, cool to 65℃ and pour into a culture dish to cool and solidify.

[0031] Rooting medium: The formula is 1 / 2MS + 7.8 g / L agar + 30 g / L sucrose, pH = 5.8. Taking 1 L volume as an example, the preparation method of rooting medium is to dissolve 2.215g MS dry powder, 30 g sucrose and 7.8 g agar in water, dilute to 1L, use standard concentrations of HCl and NaOH to adjust pH = 5.8, put it in a high-temperature sterilizer at 120℃ for 60 min, cool it to 65℃, pour it into the culture bottle and cool it to solidify.

[0032] 3. Regeneration culture of hypocotyls: Use a sterile scalpel to cut the hypocotyls of the sterile seedlings obtained in step 1 and inoculate them as explants on the regeneration and subculture medium (see Figure 1 C), wrapped in tin foil and cultured in the dark at 25°C for 5-7 days, the explants produced adventitious buds (see Figure 1 D).

[0033] 4. Subculture of regenerated shoots: After the explants sprout, place them in a tissue culture bottle containing regeneration and subculture medium for subculture, and culture them in an artificial climate incubator until rootless shoots are obtained (see Figure 1 Culture conditions: day / night temperature 25℃ / 18℃, light duration 12h per day, light intensity 1800Lx.

[0034] 5. Rooting culture of regenerated seedlings: Transfer the rootless seedlings to a tissue culture bottle containing rooting medium to promote rooting and root growth to obtain rooted seedlings (see Figure 1 F), which is the regenerated plant of Sainfoin.

[0035] 6. Plant hardening and transplanting: Remove the cover of the tissue culture bottle and seal the bottle mouth with a large sealing film and a rubber band, then pierce holes in the film for hardening, which takes 3-5 days, then rinse the agar at the root of the rooted seedlings and transplant them into a seedling pot filled with high-temperature sterilized seedling medium (the seedling medium is a mixture of nutrient soil and perlite in a volume ratio of 3:1, which is not lumpy, does not drip, and is relatively loose) (see Figure 1 G), the flower pots need to be covered with plastic wrap and pierced, and watered regularly to obtain high-quality red bean grass seedlings.

[0036] Example 2 The difference from Example 1 is that the formula of the regeneration and subculture medium is MS + 0.2 mg·L －１ 6-BA+0.5mg·L －１ NAA++30g / L sucrose+7.8g / L agar, pH=5.8. Taking 1 L volume as an example, the preparation method of regeneration and subculture medium is to dissolve 4.43 g MS dry powder, 0.2 mg 6-BA, 0.5 mg NAA, 30 g sucrose and 7.8 g agar in water, and adjust the volume to 1L. The pH value is adjusted to pH=5.8 using standard concentrations of HCl and NaOH, and then placed in a high-temperature sterilizer at 120℃ for 60 min. After cooling to 65℃, pour it into a culture dish and cool it to solidify.

[0037] The other steps are completely consistent with those in Example 1.

[0038] Results Figure 2 A: The explant began to swell and sprout, callus tissue was induced at the bottom, but no rooting occurred.

[0039] Example 3 The difference from Example 1 is that the formula of the regeneration and subculture medium is MS + 0.4 mg·L －１ 6-BA+0.5mg·L －１NAA+30g / L sucrose+7.8g / L agar, pH=5.8. Taking 1 L volume as an example, the preparation method of regeneration and subculture medium is to dissolve 4.43 g MS dry powder, 0.4 mg 6-BA, 0.5 mg NAA, 30 g sucrose and 7.8 g agar in water, dilute to 1 L, use standard concentration of HCl and NaOH to adjust pH=5.8, put it in a high temperature sterilizer at 120℃ for 60 min, cool it to 65℃ and pour it into a culture dish to cool and solidify.

[0040] The other steps are completely consistent with those in Example 1.

[0041] Results Figure 2 B: The explants began to swell, and some of them could produce buds, but callus tissue was induced at the base and no roots were formed. Some of the explants were directly induced into callus tissue.

[0042] Example 4 The difference from Example 1 is that the formula of the regeneration and subculture medium is MS + 0.6 mg·L －１ 6-BA+0.5mg·L －１ NAA++30g / L sucrose+7.8g / L agar, pH=5.8. Taking 1 L volume as an example, the preparation method of regeneration and subculture medium is to dissolve 4.43 g MS dry powder, 0.6 mg 6-BA, 0.5 mg NAA, 30 g sucrose and 7.8 g agar in water, dilute to 1 L, use standard concentration of HCl and NaOH to adjust pH=5.8, put it in a high temperature sterilizer at 120℃ for 60 minutes, cool it to 65℃ and pour it into a culture dish to cool and solidify.

[0043] The other steps are completely consistent with those in Example 1.

[0044] Results Figure 2 C: The explants began to swell, and some of them could produce buds, but callus tissue was induced at the base and no roots were formed. The callus tissue at the base of some explants turned slightly brown.

[0045] Example 5 The difference from Example 1 is that the formula of the regeneration and subculture medium is MS + 0.8 mg·L －１ 6-BA+0.5mg·L －１NAA++30g / L sucrose+7.8g / L agar, pH=5.8. Taking 1 L volume as an example, the preparation method of regeneration and subculture medium is to dissolve 2.22 g MS dry powder, 0.8 mg 6-BA, 0.5 mg NAA, 30 g sucrose and 7.8 g agar in water, dilute to 1 L, use standard concentration of HCl and NaOH to adjust pH=5.8, put it in a high temperature sterilizer at 120℃ for 60 minutes, cool it to 65℃ and pour it into a culture dish to cool and solidify.

[0046] The other steps are completely consistent with those in Example 1.

[0047] Results Figure 2 D: The explants began to swell, and some of them could produce buds, but callus was induced at the base, and no roots were formed, and some callus turned green. Some explants only produced buds, and callus was not induced at the base and no roots were formed.

[0048] Comparative Example 1 The difference from Example 1 is that the seeds are sterilized by soaking in 75% alcohol for 10 minutes, rinsing with sterile water three times, soaking in 1% sodium hypochlorite by mass for 3 minutes, and rinsing with sterile water 3-6 times. The other steps are completely consistent with Example 1.

[0049] Comparative Example 2 The difference from Example 1 is that the seeds are sterilized by sun drying for 8 hours, soaking in 0.3% HgCl2 for 20 minutes, and rinsing with sterile water for 3-6 times. The other steps are completely consistent with Example 1.

[0050] Comparative Example 3 The difference from Example 1 is that the seeds are sterilized by soaking them in alcohol, shaking them on a shaker for 30 minutes, rinsing them with sterile water for 3 times, soaking them in 1% sodium hypochlorite for 3 minutes, and rinsing them with sterile water for 3-6 times. The other steps are completely consistent with Example 1.

[0051] Comparative Example 4 The difference from Example 1 is that the seed disinfection method is to soak the seeds in alcohol for 10 minutes, rinse with sterile water 3 times, soak in 3% sodium hypochlorite for 20 minutes, and rinse with sterile water 3-6 times. The other steps are completely consistent with Example 1.

[0052] Comparative Example 5 The difference from Example 1 is that the seeds are sterilized by soaking in alcohol for 10 minutes, rinsing with sterile water for 3 times, soaking in 3% sodium hypochlorite for 15 minutes, and rinsing with sterile water for 3-6 times. The other steps are completely consistent with Example 1.

[0053] Comparative Example 6 The difference from Example 1 is that the seeds are sterilized by soaking in 0.3% HgCl2 for 30 minutes and then rinsed with sterile water for 3-6 times. The other steps are completely consistent with Example 1.

[0054] Each of the above embodiments and comparative examples was repeated three times. The total number of seeds inoculated in the three repetitions and the statistical results are shown in Table 1.

[0055] Table 1 Culture results

[0056] The above results show that the method of Example 1 is the most effective method. According to the culture cycle statistics, the construction period of the conventional regeneration system must reach 6-7 months, while the construction period of the regeneration system of the present invention is shortened to 3-4 months.

[0057] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for regenerating sainfoin, characterized in that: The method comprises the following steps: using the hypocotyl of the sterile seedlings of sainfoin as explants, inoculating them on a regeneration and subculture medium for cultivation, first dark culturing to obtain adventitious buds, then subculture to obtain rootless seedlings, inoculating the rootless seedlings on a rooting medium for rooting culture, and obtaining regenerated plants of sainfoin; The regeneration and subculture medium is a solid medium based on MS solid medium, to which 6-BA and NAA are added, to obtain a solid medium with a 6-BA concentration of 1.0 mg / L, an NAA concentration of 0.5 mg / L, and a pH value of 5.

8.

2. The method according to claim 1, characterized in that: The aseptic seedlings of Sainfoin are obtained by placing sterilized seeds of Sainfoin after removing seed pods on moistened filter paper for dark germination, and culturing the germinated seeds for 5-7 days.

3. The method according to claim 2, characterized in that: The disinfection is performed by soaking in a 0.3% by mass HgCl2 solution for 30 minutes and then rinsing with sterile water.

4. The method according to claim 2, characterized in that: The conditions for culturing the germinated seeds are as follows: a day / night temperature of 25°C / 18°C, a light duration of 12 hours per day, and a light intensity of 1800 Lx.

5. The method according to claim 1, characterized in that: The rooting medium is a 1 / 2MS solid medium with a pH value of 5.

8.

6. The method according to claim 1, characterized in that: The temperature during dark culture is 25° C., and the culture is carried out for 5-7 days.

7. The method according to any one of claim 6, characterized in that: The culture conditions of the subculture are as follows: the day / night temperature is 25°C / 18°C, the light duration is 12 hours per day, and the light intensity is 1800 Lx.

8. The method according to any one of claims 1 to 7, characterized in that: The method also includes the steps of hardening off the seedlings and transplanting.

Images