A method for propagating and renewing germplasm resources of *Axypsis cuspidatum*
By using a one-step aseptic root regeneration bud tissue culture and heterologous root replacement, the root system of radish was used to replace the root system of mustard greens, which solved the problem of poor root adaptability of mustard greens in planting in different places, and achieved the effect of completing the growth period and seed propagation in farmland.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INSTITUTE OF VEGETABLES & FLOWERS CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2024-06-04
- Publication Date
- 2026-05-26
AI Technical Summary
Axew has difficulty reproducing and regenerating in its native habitat and in other places, especially because its root system is not adapted to the soil in the interior, which leads to early death of the plant and failure to complete its growth period and reproduction.
A one-step aseptic root regeneration method for rapid propagation through tissue culture was adopted. The original root system was replaced by a heterologous root system. Radish roots were used to replace the roots of *Strombus haematomarginatus*. Combined with radish materials that are highly resistant to clubroot, grafting and off-site planting were carried out.
This technology enables *Sinomenium acutum* to complete its entire growth cycle in farmland, improving seed survival rate and regeneration capacity, solving root adaptability issues, avoiding clubroot infection, simplifying operational procedures, and reducing pollution risks.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of rapid plant propagation technology, specifically disclosing a method for the propagation and renewal of germplasm resources of *Aristolochia debilis*. Background Technology
[0002] *Pugionium dolabratum*, a species of sand mustard in the Brassicaceae family, is an endangered wild plant with significant economic and ecological value, including edible, medicinal, feed, and windbreak and sand-fixing uses. It also exhibits strong resistance to drought, cold, sand burial, and ultraviolet radiation, making it a valuable wild relative for the biobreeding of Brassicaceae crops. Over the past few decades, due to increasingly deteriorating habitats and excessive human exploitation, the distribution and population of *Pugionium dolabratum* have declined year by year. It was included in the *Flora of Rare and Endangered Plants of Inner Mongolia* in 1978, classifying it as a rare and endangered wild plant.
[0003] *Symplocos buergeriana* has a narrow distribution range. It grows on shifting or semi-shifting sand dunes in desert and semi-desert areas, exhibiting strict habitat requirements. These stringent requirements, along with its autotoxicity, low regrowth rate, and susceptibility to pests and diseases, make the propagation and regeneration of *Symplocos buergeriana* extremely difficult. Two long-standing problems hinder its propagation: 1. In its native habitat, most *Symplocos buergeriana* plants fail to overwinter and regrow, with an even smaller proportion producing seeds. 2. When cultivated in other locations, *Symplocos buergeriana* cannot complete its entire growth cycle, resulting in failure to propagate and regenerate. Research has revealed that the primary cause of plant death when *Symplocos buergeriana* is planted in other locations is its root system's inability to adapt to inland soil, leading to root rot and premature death, thus preventing propagation and regeneration. To protect this endangered resource and ensure the long-term preservation and regular propagation of *Symplocos buergeriana* seeds, it is necessary to establish a method for propagating *Symplocos buergeriana* in inland farmland. Summary of the Invention
[0004] This invention is a method for artificial propagation and renewal of *Sinocyclocheilus fasciatus* in farmland in different locations. This method uses a simple and efficient one-step aseptic root regeneration method to propagate a large number of *Sinocyclocheilus fasciatus* tissue culture seedlings. By replacing the original root system with a heterologous root system, the environmental adaptability of *Sinocyclocheilus fasciatus* is changed, enabling *Sinocyclocheilus fasciatus* to complete its entire growth cycle and produce seeds when densely planted in farmland.
[0005] The present invention provides a method for the propagation and regeneration of *Sinomenium acutum*, comprising the following steps:
[0006] S1 Axewing Sand Mustard Seed Disinfection and Sowing:
[0007] Take plump seeds of *Gnaphalium affine* and soak them in 50℃ warm water for 6-8 hours. Wrap them in gauze and rinse them under running water for 1 hour. Surface disinfect them with 70% anhydrous ethanol and a 1:1 diluted 84 disinfectant solution, with 70% ethanol disinfecting for 15-60 seconds and the 1:1 diluted 84 disinfectant solution disinfecting for 5-15 minutes. Then rinse them three times with sterilized distilled water. Remove the seeds, blot them dry with filter paper, and sow 5-20 seeds in each Erlenmeyer flask containing M1 culture medium. All the above operations are performed in a clean bench.
[0008] The M1 culture medium formula of this invention is: 2-2.3 g / L MS powder, 25-35 g / L sucrose, and 6-9 g / L agar powder, preferably 2.20-2.30 g / L MS powder, 28-32 g / L sucrose, and 7-8 g / L agar powder, specifically 2.215 g / L MS powder, 30 g / L sucrose, and 7.5 g / L agar powder. After autoclaving, it is dispensed for use.
[0009] S2 Aseptic seedling culture of *Sinomenium axeoniflorum*:
[0010] After sowing, place the seeds in a tissue culture room and cultivate them in the dark. *Axe-winged Sand Mustard* seeds have a dormancy period, and germination time varies considerably, ranging from 5 to 30 days. Close observation is necessary to determine the germination period based on actual conditions.
[0011] S3 Heterogeneous Root System Replacement:
[0012] 1) Preparation of rootstock radish:
[0013] After the radish seedlings have been cultivated to the breaking-belly stage, when the hypocotyl diameter is 5mm-15mm, use a sharp scalpel to make a horizontal cut 2cm-5cm away from the substrate, removing the cotyledons and true leaves, leaving only the hypocotyl with roots, and make a 1cm longitudinal cut at the upper end of the hypocotyl.
[0014] In this invention, radish seeds for the rootstock are sown in a substrate composed of a 1:1 mixture of peat moss and vermiculite, with a pH of 5.5-6.5; the photoperiod is 16 hours of light followed by 8 hours of darkness; and the temperature is 25-28℃. Hoagland's nutrient solution is applied regularly. After root replanting, the surviving plants are transplanted into 10cm diameter pots for hardening off for 2-3 weeks; subsequently, they are transplanted to the field.
[0015] 2) Grafting:
[0016] Cultivate *Gnaphalium affine* seedlings until the stem diameter reaches 2mm-5mm. Using a sharp scalpel, make a slanted cut on the stem 2cm-5cm from the leaf tip, creating a slanted surface. Take a portion with leaves and the growing point at the stem tip, and insert the slanted cut stem end into the hypocotyl suture of the radish, clamping it securely with a small clip. Subsequently, place it in a high-temperature (25-30℃) and high-humidity (relative humidity >90%) environment for 1-2 weeks to allow the interface to heal.
[0017] The process after S2 includes the following step: one-step regeneration of shoots from *Axypsis cusia*:
[0018] Seven days after germination, the seedlings are cut off, leaving the roots in the culture medium. They are then placed in a tissue culture room for further cultivation under a 12-hour light-12-hour dark photoperiod. No further intervention is required; regenerated buds will gradually differentiate from the roots. Bud differentiation can continue from week 3 to week 20. Once the regenerated buds have grown on the original culture medium for more than 4-6 weeks, they can be transferred to fresh M1 medium and cultured into tissue culture seedlings according to the normal procedure.
[0019] Cultivation of S4 after root replacement:
[0020] After root replanting, *Axypsis cuspidatum* plants are planted in sandy soil, specifically in sandy soil in North China (such as Beijing and Hebei). To prevent waterlogging and ensure good aeration, the planting field is prepared as raised beds. In winter, they are transplanted to an unheated greenhouse for vernalization. During cultivation, pests and diseases are controlled, especially spider mites, aphids, and powdery mildew.
[0021] Each resource plot is planted and isolated with 40-mesh netting, and pollinating insects such as mason bees are released to assist in pollination. After the seeds mature, they are harvested promptly, tested, dried, and stored at low temperatures.
[0022] Beneficial effects
[0023] The native habitat of *Axypsis cuspidatum* is the Gobi Desert and sand dunes. Due to its narrow ecological adaptability, artificial propagation is extremely difficult. When artificially propagated in farmland in Beijing and Hebei, *Axypsis cuspidatum* can germinate, but subsequently die off and fail to flower or bear fruit. Compared with existing techniques, this technology effectively solves the problem of *Axypsis cuspidatum*'s root system being unsuitable for soil, leading to early plant death and hindering propagation and regeneration.
[0024] Axew is susceptible to clubroot disease, and most cruciferous plants are also susceptible to clubroot disease. Highly resistant radish materials can be used to solve the problem of clubroot disease and even plant death.
[0025] Radish roots can be harvested and stored over winter, maintaining a very high survival rate the following year. Compared to other cruciferous crops such as cabbage and mustard, radishes, when used as root donors, facilitate the overwintering and subsequent regrowth and reproductive growth of *Strombus haematomarginatus* the following year. Therefore, they can be used for the propagation and renewal of *Strombus haematomarginatus* germplasm resources.
[0026] Furthermore, the one-step tissue culture regeneration system of *Gnaphalium affine* of this invention is simple and easy to operate. It does not require explant cutting, complex culture media, the addition of exogenous hormones, or multiple culture media changes, saving manpower and financial resources, and preventing the risk of contamination during culture media changes. See Table 1 for comparison.
[0027] Table 1. Comparison of the beneficial effects of the present invention with existing tissue culture techniques.
[0028] . Attached Figure Description
[0029] Figure 1 Aseptic seedlings of *Axewing Sand Mustard*.
[0030] Figure 2 New shoots are generated on the roots of the axe-winged sand mustard.
[0031] Figure 3 Radish grafted onto axe-winged sand mustard.
[0032] Figure 4 Transplanting of *Axewing Sand Mustard* seedlings.
[0033] Figure 5 Axew winged sand mustard is infected with clubroot disease.
[0034] Figure 6 Highly resistant to clubroot disease, replanted seedlings. Detailed Implementation
[0035] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.
[0036] Example 1:
[0037] Populations 1 and 2 of *Strombax ceiba* from Ordos, Inner Mongolia, and populations from Gansu were collected; and seeds were brought back to Beijing for propagation.
[0038] 1. Take 100 plump seeds of *Gnaphalium affine*, soak them in 50℃ warm water for 6-8 hours, wrap them in gauze, and rinse them under running water for 1 hour.
[0039] 2. Disinfect the seeds with 70% ethanol for 30 seconds, then disinfect them with 1:1 diluted 84 disinfectant for 10 minutes. Sow them on M1 solid culture medium.
[0040] The M1 medium formula is: 2.215 g / L MS powder, 30 g / L sucrose, and 7.5 g / L agar powder; autoclave.
[0041] 3. Place in a tissue culture room under shade; one week after germination, cut off the plantlets from the culture medium, leaving the roots in the original culture medium, and seal the bottle. Figure 1 (The following example uses the No. 1 population of *Sinomenium acutum* as an example; the same applies to subsequent cases). The tissue culture was then placed in a culture room for further cultivation.
[0042] 4. Approximately 2-4 months later, new buds will gradually regenerate on the root system. Figure 2 ).
[0043] 5. Take the regenerated buds and transplant them onto fresh M1 culture medium, providing sufficient nutrition for the buds to grow.
[0044] 6. 100 radish seeds (R41 was used as an example in this experiment) were sown in the same culture medium. They were cultured in an artificial climate chamber with a photoperiod of 16 hours light: 8 hours darkness; temperature 25-28℃. Hoagland's solution was applied regularly. Radishes grown to the point of bud break, with a hypocotyl diameter of 5mm-15mm, were suitable for use as rootstock.
[0045] 7. Using a sharp scalpel blade, cut the radish seedling horizontally 2-5 cm from the substrate, removing the cotyledons and true leaves, leaving only the hypocotyl with roots. Make a 1 cm longitudinal cut at the upper end of the hypocotyl. Using a sharp scalpel blade, obliquely cut the stem of the *Vitex negundo* winged radish 2-5 cm from the heart leaf, creating an oblique cut surface. Take the portion containing the heart leaf and the apical growing point. Figure 3 Insert the diagonally cut axe-winged mustard stem into the hypocotyl cut of the radish, and clamp it tightly with a small clip. Figure 4 ).
[0046] 8. Place the grafted plants in a high-temperature (25-30℃) and high-humidity (relative humidity >90%) environment for 1-2 weeks to allow the graft union to heal. Experimental statistics show that the survival rates of grafted plants in populations 1, 2, and Gansu reached 60%, 65%, and 70%, respectively.
[0047] 9. After the roots have been replaced, transplant the surviving plants into 10cm diameter flowerpots to harden them off for 2-3 weeks; then transplant them into the field.
[0048] 10. After hardening off, plant the regenerated *Axypsis cuspidatum* seedlings in sandy soil. Specific management requirements: prevent waterlogging, ensure good aeration, and cultivate in raised beds. Control pests and diseases during cultivation, especially spider mites, aphids, and powdery mildew.
[0049] For propagation, each resource plot can be planted, isolated with 40-mesh netting, and pollinating insects such as mason bees can be introduced to assist pollination. After the seeds mature, they should be harvested promptly, tested, dried, and stored at low temperatures.
[0050] Example 2: Experiment on resistance to clubroot disease
[0051] After planting the *Sinocyclocheilus fasciatus* populations 1, 2, and Gansu population obtained in Example 1, artificial inoculation with clubroot pathogens was carried out, with 10 plants per plant sample.
[0052] Disease incidence was assessed 40 days after inoculation. Results showed that clubroot disease was present in populations 1, 2, and Gansu of *Symplocos edulis* that had not undergone root replacement. Figure 5(As shown in the disease situation) The disease rate was 100% in all populations, while the disease rate was 0% in the *Vitex negundo* populations 1, 2, and Gansu population after root replacement, meaning there was no disease in any of them. Figure 6 Plants free from clubroot infection were displayed. The inoculation and disease survey methods followed those described in the published paper: Ma, Y.; Wang, H.; Song, J.; Yang, W.; Jia, H.; Agerbirk, N.; Chen, Y.; Li, C.; Piao, Y.; Li, S.; et al. Identification of Clubroot-Resistant Germplasm in a Radish (Raphanussativus L.) Core Collection. Agronomy 2024, 14, 157 (https: / / doi.org / 10.3390 / agronomy14010114).
Claims
1. A method for the propagation and regeneration of *Gnaphalium affine*, characterized in that, Includes the following steps: S1 Axewing Sand Mustard Seed Disinfection and Sowing: S2 Aseptic seedling culture of *Sinomenium axeoniflorum*: S3 heterogeneous root system replacement: 1) Preparation of rootstock radish: After the radish seedlings have been cultivated to the breaking-belly stage, when the hypocotyl diameter is 5mm-15mm, use a sharp scalpel to make a horizontal cut 2cm-5cm away from the substrate, cut off the cotyledons and true leaves, leaving only the hypocotyl with roots, and make a 1cm longitudinal cut at the upper end of the hypocotyl. 2) Grafting: Use a blade to obliquely cut the stem of the *Viburnum sibiricum* seedling from the centrifugal leaf, forming an oblique cut surface. Take a portion with leaves and the growing point at the stem tip, and insert the obliquely cut stem tip into the hypocotyl slit of the radish. Let it heal. Cultivate the *Viburnum sibiricum* seedling to a stem diameter of 2mm-5mm. Use a blade to obliquely cut the stem 2cm-5cm from the centrifugal leaf, forming an oblique cut surface. Take a portion with leaves and the growing point at the stem tip, and insert the obliquely cut stem tip into the hypocotyl slit of the radish, clamping it tightly with a small clip. Then, place it in an environment of 25-30℃ and relative humidity >90% for 1-2 weeks to allow the graft union to heal, obtaining a surviving plant after root grafting. S4 Cultivation after root replacement: Harden off and plant the regenerated *Sinocyclocheilus serrata* plants. The specific operation is as follows: Harden off the surviving plants after root replacement for 2-3 weeks; then transplant them to the field for planting. The planting involves planting the hardened seedlings in sandy soil.
2. The method for propagating and regenerating *Gnaphalium affine* as described in claim 1, characterized in that, S1 specific procedure: Take plump seeds of *Sargassum fusiforme*, soak them in 50℃ warm water for 6-8 hours, wrap them in gauze, rinse them with running water for 1 hour, and then surface disinfect them with 70% anhydrous ethanol and 1:1 diluted 84 disinfectant solution, with 70% ethanol disinfection for 15-60 seconds and 1:1 diluted 84 disinfectant solution surface disinfection for 5-15 minutes; then rinse them 3 times with sterilized distilled water; sow them in M1 medium. The M1 culture medium formula is: 2-2.3 g / L MS powder, 25-35 g / L sucrose, and 6-9 g / L agar powder.
3. The method for propagating and regenerating *Gnaphalium affine* as described in claim 2, characterized in that, The M1 medium formula is: 2.20-2.30 g / L MS powder, 28-32 g / L sucrose, and 7-8 g / L agar powder.
4. The method for propagating and regenerating *Gnaphalium affine* as described in claim 3, characterized in that, The M1 medium formula is: 2.215 g / L MS powder, 30 g / L sucrose, and 7.5 g / L agar powder.
5. The method for propagating and regenerating *Gnaphalium affine* as described in claim 1, characterized in that, In step S2, after sowing the seeds of *Sinocyclocheilus roxburghii* on M1 medium, they are placed in a tissue culture room for culture in the dark.
6. The method for propagating and regenerating *Gnaphalium affine* as described in claim 1, characterized in that, Step S2 is followed by a one-step method for cultivating regenerated shoots. The procedure is as follows: 5-9 days after germination, the seedlings are cut off, leaving the roots in the culture medium. The roots are then cultured under a 12-hour light: 12-hour dark light cycle to allow regenerated shoots to differentiate from the roots and serve as seedlings for the *Sinomenium acutum* in S3. Optionally, when the regenerated shoots have grown on the original culture medium for more than 4-6 weeks, they are transferred to fresh M1 culture medium.
7. The method for propagating and regenerating *Gnaphalium affine* as described in claim 1, characterized in that, The radish seeds were sown in a substrate consisting of a 1:1 mixture of peat moss and vermiculite with a pH of 5.5-6.5; the photoperiod was 16 hours of light followed by 8 hours of darkness; the temperature was 25-28°C; and the seeds were regularly irrigated with Hogland nutrient solution.
8. The method for propagating and regenerating *Gnaphalium affine* as described in claim 1, characterized in that, It also includes subsequent breeding steps, namely, isolating the seeds with mesh covers and releasing pollinating insects such as mason bees to assist in pollination; and promptly harvesting, testing, drying and storing the seeds at low temperatures after they mature as breeding material.