Method for rapidly inducing adventitious shoots of wild membranous pod astragalus
By using sterile young stems of wild Astragalus membranaceus seedlings as explants and inducing adventitious buds and roots in a culture medium with specific hormones, the problem of long seedling cultivation cycle of wild Astragalus membranaceus has been solved, achieving rapid and low-cost induction of adventitious seedlings. This method is suitable for the protection of germplasm resources and seedling propagation of wild Astragalus membranaceus.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NINGXIA UNIVERSITY
- Filing Date
- 2024-07-22
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, wild Astragalus membranaceus suffers from problems such as long seedling cultivation cycle, limited seed production, and degradation of germplasm, making it difficult to meet market demand. Furthermore, traditional tissue culture methods are time-consuming.
Wild aseptic seedlings of Astragalus membranaceus were used as explants. Adventitious buds were induced on MS medium supplemented with 6-BA and IAA, and rooting culture was carried out on 1/2 MS medium supplemented with IAA and IBA. Finally, the seedlings were hardened off and transplanted, and the culture conditions and parameters were optimized.
This method enables rapid induction of adventitious seedlings from wild Astragalus membranaceus, shortens the seedling cycle, reduces costs, and increases germination and rooting rates. It is suitable for the protection of germplasm resources and rapid propagation of seedlings of wild Astragalus membranaceus.
Smart Images
Figure CN118661648B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to plant tissue culture technology, specifically a method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus. Background Technology
[0002] Astragalus membranaceus (Fisch.) Bunge belongs to the genus Astragalus in the family Fabaceae. It is mainly distributed in Heilongjiang, Jilin, Liaoning, Shanxi, Shaanxi, Hebei, Ningxia, and Xinjiang in my country. Its root is used as a traditional Chinese medicine, possessing analgesic, immune-boosting, diuretic, microcirculation-promoting, lipid-peroxidation-increasing, and plasma cyclic adenosine monophosphate-increasing pharmacological effects. In addition, Astragalus membranaceus has been used for landscaping, windbreak and sand fixation, and as animal feed, and has a long history of development and utilization in my country. In recent years, due to ecological changes and over-harvesting, wild Astragalus membranaceus resources have decreased sharply, and market demand is mainly driven by artificial cultivation. However, this cultivation faces challenges such as long seedling transplanting cycles, limited seed production, and degradation of germplasm.
[0003] Plant tissue culture, which involves isolating and inoculating plant tissues, organs, cells, and protoplasts under sterile conditions and culturing them in different types of culture media to obtain adventitious seedlings, is a crucial method for artificial plant propagation. It is significant for solving technical challenges in vegetative propagation and achieving asexual propagation of superior varieties. There are existing reports on callus culture of *Astragalus membranaceus*, but most studies used artificially cultivated plants as explants, including leaves, petioles, cotyledons, seeds, young stems, and hypocotyls. The process typically involves explant-callus-adventitious buds-rooting-seeded seedlings, which is time-consuming. Therefore, utilizing wild species as material to rapidly obtain purified clones through tissue culture for germplasm resource propagation, and subsequently selecting and industrializing high-quality seedlings, is particularly important. Summary of the Invention
[0004] In view of this, the present invention provides a method for inducing adventitious seedlings from wild Astragalus membranaceus seedlings. By means of tissue culture, adventitious buds are directly induced from the sterile seedlings of wild Astragalus membranaceus. The adventitious buds then root to obtain adventitious seedlings. This method is quick and simple, shortens the cycle and reduces costs, and is beneficial to the development and protection of wild Astragalus membranaceus planting resources.
[0005] A method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus, characterized by the following steps:
[0006] S1. Acquisition of sterile seedlings: Take mature seeds of wild Astragalus membranaceus, disinfect them, and inoculate them into MS medium to obtain sterile wild Astragalus membranaceus complete plants.
[0007] S2. Induction of adventitious buds from young stems: Young stems of wild Astragalus membranaceus were taken and used as explants. MS was used as the basic culture medium, and hormones 6-BA and IAA were added to induce the differentiation of adventitious buds.
[0008] S3. Rooting culture: Single adventitious shoots are transferred to 1 / 2 MS rooting medium supplemented with IAA and IBA to induce adventitious roots;
[0009] S4. Hardening off and transplanting: Take tissue culture seedlings that have undergone adventitious bud induction and rooting culture, harden them off, and then transplant them.
[0010] Preferably, the wild Astragalus membranaceus seeds in S1 need to be pretreated, specifically by rubbing with fine sand for 5 minutes, rinsing with running water for 2 hours, rinsing with sterile water 4-5 times in a clean bench, soaking in 75% anhydrous ethanol for 30 seconds, disinfecting with 15% NaClO for 5-15 minutes, soaking in 75% alcohol for 30 seconds after disinfection, rinsing with sterile water 4-5 times, blotting dry with sterile filter paper, inoculating the wild Astragalus membranaceus seeds into MS medium, observing the growth of sterile seedlings, and obtaining complete wild Astragalus membranaceus plants.
[0011] Preferably, the NaClO sterilization time is 10 minutes.
[0012] Preferably, the concentration of 6-BA in S2 is 1.5 mg / L and the concentration of IAA is 0.2 mg / L.
[0013] Preferably, the concentration of IBA in S3 is 0.5 mg / L and the concentration of IAA is 1.0 mg / L.
[0014] Preferably, the hardening time for S4 is 3-5 days, and the tissue culture bottle is half-open during hardening.
[0015] Preferably, the culture conditions are as follows: MS basal medium is selected, and agar 3.5 g / L and sucrose 15 g / L are added to form MS medium. The above components of 1 / 2 MS medium are halved. The pH is 5.8. The medium is sterilized at 115℃ for 30 min. The culture temperature is (23±1)℃, the illuminance is 3000 lx, and the photoperiod is 12 h / d.
[0016] The beneficial effects are as follows:
[0017] The greatest benefit of this invention is that it provides a method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus, while also determining the specific process and optimal parameters of the method, thus filling the gap in methods for rapidly inducing adventitious seedlings from wild Astragalus membranaceus.
[0018] Secondly:
[0019] (1) By using the aseptic seedlings of wild Astragalus membranaceus as explants and inoculating them onto a culture medium with added hormones to induce adventitious seedlings, time-saving and labor-saving effects can be achieved. Compared with the traditional method, the cultivation cycle can be reduced by 40 days.
[0020] (2) The 6-BA and IAA used are common hormones and are inexpensive;
[0021] (3) The experiment is short and can process a large amount of materials and induce adventitious seedlings;
[0022] (4) It is easy to operate and has low equipment requirements;
[0023] (6) From the perspective of propagating wild Astragalus membranaceus, this method can quickly induce adventitious seedlings, and the high germination rate of this method can be used to induce a large number of adventitious seedlings, which greatly shortens the seedling cultivation cycle of wild Astragalus membranaceus and reduces costs, laying a good foundation for the protection of wild Astragalus membranaceus germplasm resources, rapid propagation of seedlings and genetic improvement. Attached Figure Description
[0024] Figure 1 Flowchart for the rapid induction of adventitious seedlings from wild Astragalus membranaceus;
[0025] Figure 2 Aseptic seedlings of wild Astragalus membranaceus;
[0026] Figure 3 Wild Astragalus membranaceus induces adventitious buds;
[0027] Figure 4 Adventitious seedlings were obtained by rooting adventitious buds from wild Astragalus membranaceus. Detailed Implementation
[0028] To make the objectives, solutions, and advantages of the technical solutions of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of specific embodiments of the present invention.
[0029] Example 1:
[0030] like Figure 1 As shown, a method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus is characterized by the following steps:
[0031] S1. Acquisition of sterile seedlings: Take mature seeds of wild Astragalus membranaceus, disinfect them, and inoculate them into MS medium to obtain sterile wild Astragalus membranaceus complete plants.
[0032] S2. Induction of adventitious buds from young stems: Young stems of wild Astragalus membranaceus were used as explants, and MS was used as the basic culture medium. Hormones 6-BA and IAA were added to induce adventitious bud differentiation. The optimal concentration of 6-BA in S2 was 1.5 mg / L, and the optimal concentration of IAA was 0.2 mg / L.
[0033] S3. Rooting culture: Single adventitious shoots were transferred to 1 / 2 MS rooting medium supplemented with IAA and IBA to induce adventitious roots; the optimal concentration of IBA was 0.5 mg / L and the optimal concentration of IAA was 1.0 mg / L.
[0034] S4. Hardening off and Transplanting: Take tissue culture seedlings that have undergone adventitious bud induction and rooting culture, and harden them off before transplanting. The hardening-off time should be selected according to needs, preferably 3-5 days, and the tissue culture bottles should ideally be half-open during hardening off.
[0035] The above culture conditions are as follows: MS basal medium is selected, and agar 3.5 g / L and sucrose 15 g / L are added to form MS medium. The above components of 1 / 2 MS medium are halved, including MS basal medium, agar and sucrose. The pH is 5.8, sterilized at 115℃ for 30 min, culture temperature is (23±1)℃, light intensity is 3000 lx, and photoperiod is 12 h / d.
[0036] Example 2:
[0037] Based on Example 1, in order to achieve better cultivation, the wild Astragalus membranaceus seeds in S1 need to be pretreated. Specifically, they are rubbed with fine sand for 5 minutes, rinsed with running water for 2 hours, rinsed with sterile water 4-5 times in a clean bench, soaked in 75% anhydrous ethanol for 30 seconds, and disinfected with 15% NaClO for 5-15 minutes. The NaClO disinfection time is preferably 10 minutes. After disinfection, they are soaked in 75% alcohol for 30 seconds, rinsed with sterile water 4-5 times, and the water is absorbed with sterile filter paper. The wild Astragalus membranaceus seeds are then inoculated into MS medium, and the growth of sterile seedlings is observed to obtain complete wild Astragalus membranaceus plants.
[0038] Experimental Example 1:
[0039] I. Materials and Methods
[0040] (1) Materials
[0041] Mature seeds of wild Astragalus membranaceus (fisch.) Bunge were collected from the Liupan Mountains in Ningxia. A certain number of wild Astragalus membranaceus seeds were rubbed with fine sand for 5 minutes, then rinsed with running water for 2 hours. In a clean bench, the seeds were rinsed 4-5 times with sterile water, soaked in 75% anhydrous ethanol for 30 seconds, and then sterilized with different concentration gradients of NaClO (5%, 10%, 15%) for 5, 10, 15, and 20 minutes respectively. After sterilization, the seeds were soaked in 75% alcohol for 30 seconds, rinsed 4-5 times with sterile water, and dried with sterile filter paper. The wild Astragalus membranaceus seeds were inoculated into MS medium (4 seeds per bottle). The growth of sterile seedlings was observed to obtain complete wild Astragalus membranaceus plants.
[0042] (2) Induction of adventitious buds in young stems
[0043] Using young stems of wild Astragalus membranaceus as explants, and MS as the basic medium, adventitious shoot differentiation was induced by adding 6-BA (1.0, 1.5, 2.0 mg / L) and IAA (0.2 mg / L), respectively. The occurrence of adventitious shoots was counted after 30 days.
[0044] (3) Rooting culture
[0045] Single adventitious buds approximately 5 cm in height were cut from callus tissue and transferred to 1 / 2 MS rooting medium supplemented with different combinations of IAA (0.5, 1.0 mg / L) and IBA (0.5, 1.0, 2.0 mg / L) to induce adventitious roots. Results were analyzed after 35 days of culture.
[0046] (4) Hardening off seedlings and transplanting
[0047] Tissue culture seedlings induced from adventitious buds were subjected to hardening treatment for 0, 3, 5, and 7 days. The tissue culture bottles were opened either halfway or fully open. Sterilized distilled water was poured into the bottles to a height of 2-3 cm. After hardening, the seedlings were removed from the bottles, the culture medium was gently washed off the roots, and the seedlings were transplanted into a 1:1:1 substrate of humus soil, vermiculite, and perlite that had been sterilized by autoclaving. The seedlings were watered every 3 days, with 10 mL of water per bottle. The results were collected after 35 days.
[0048] (5) Cultivation conditions
[0049] MS medium was used as the basal medium, with 3.5 g / L agar and 15 g / L sucrose added to form MS medium. The components of 1 / 2 MS medium were halved, including the basal MS medium, agar, and sucrose. The pH was 5.8, and the medium was sterilized at 115℃ for 30 min. The incubation temperature was (23±1)℃, the light intensity was 3000 lx, and the photoperiod was 12 h / d.
[0050] (6) Data Statistics
[0051] Callus induction rate = Number of callus explants produced from sterile explants / Total number of explants × 100%;
[0052] Adventitious bud induction rate = number of buds emerging from the inoculated callus / total number of inoculated callus × 100%;
[0053] Rooting rate = Number of rooted explants / Total number of inoculated explants × 100%;
[0054] Transplant survival rate = (Number of surviving tissue culture seedlings after transplanting / Total number of transplanted tissue culture seedlings) × 100%.
[0055] All data were analyzed using data processing systems such as Excel and SPSS 26.0.
[0056] II. Results and Analysis
[0057] (1) Acquisition of sterile vaccines
[0058] Seeds treated with different concentrations of NaClO and different disinfection times showed varying germination rates and contamination rates. With increasing NaClO concentration and disinfection time, the germination rate increased while the contamination rate decreased. Among the 12 treatments, seeds disinfected with 15% NaClO exhibited the highest germination rate and the lowest contamination rate, particularly at 10 min and 15 min, with a germination rate of (51.67±0.08)% and a contamination rate of 0%. Compared to seeds treated with 15% NaClO, the germination rates of seeds treated with 5% and 10% NaClO decreased. Although there was no significant difference in contamination rate between 15% NaClO disinfection for 10 min and 15 min, and the seeds were uncontaminated, the cotyledon formation time was shorter for seeds disinfected with 15% NaClO for 10 min. Therefore, the optimal disinfection method was 15% NaClO disinfection for 10 min (Table 1).
[0059] Sterilized wild Astragalus membranaceus seeds were inoculated onto MS basal medium. After 10 days, the radicle, hypocotyl, and cotyledons gradually developed, and after 20 days, they developed into complete plants, yielding sterile seedlings. Figure 2 ).
[0060] Table 1. Effects of NaClO concentration and disinfection time on seed germination
[0061]
[0062] Note: Germination rate and contamination rate are mean ± standard deviation; different lowercase letters after the data in the same column indicate significant differences (P < 0.05).
[0063] (2) Induction of adventitious buds in young stems
[0064] See Figure 3 Using sterile seedling stems as explants, the plants were inoculated into MS medium supplemented with 6-BA and IAA. Green buds emerged in about 5 days, and robust adventitious seedlings developed in about 30 days. Furthermore, different hormone concentrations resulted in different effects on the induction of adventitious buds from the seedling stems. Under the same IAA concentration, the induction rate of adventitious buds initially increased and then decreased with increasing 6-BA concentration, reaching a maximum of 90.3% at a 6-BA concentration of 1.5 mg / L. At lower 6-BA concentrations, the budding time was longer, some leaves quickly turned yellow, the buds were weaker, and the budding rate was also lower. Conversely, at higher 6-BA concentrations, the budding time was shorter, and the resulting adventitious bud plants were robust with thick stems, making them easier to induce rooting (Table 2).
[0065] Table 2 Effects of different hormone concentration ratios on adventitious bud induction in young stems
[0066]
[0067] Note: ++++: vigorous growth, rapid budding, and many buds; +++: relatively vigorous growth, relatively rapid budding, and many buds; ++: relatively slow growth, slow budding, and few buds; +: slow growth, slow budding, and few buds.
[0068] (3) Adventitious seedlings take root
[0069] See Figure 4 Differentiated adventitious seedlings were cut at the base and inoculated into 1 / 2 MS medium supplemented with different concentrations of IAA and IBA for rooting culture for 30 days. The highest rooting rate (39.28%) was observed when the IBA concentration was 0.5 mg / L and the IAA concentration was 1.0 mg / L. Although rooting was slow, the seedlings were robust with thick stems. When the IBA concentration was 1.0 mg / L and the IAA concentration was 0.5 mg / L, the rooting rate was only 17.85%, and rooting was slow, with some leaves turning yellow and the plants showing weak growth.
[0070] (4) Hardening off seedlings and transplanting
[0071] Well-grown tissue culture seedlings were selected and hardened off for 3, 5, and 7 days before transplanting. The results showed that both hardening time and the method of opening the bottle cap significantly affected the transplant survival rate. With prolonged hardening time, the survival rate of transplanted tissue culture seedlings initially increased and then decreased, with the highest survival rate observed after 5 days of hardening. The survival rate was higher when the bottle cap was partially opened than when it was fully opened. Transplanting tissue culture seedlings after 3 days of hardening with the cap partially open resulted in a survival rate of 70%, with vigorous plant growth. Therefore, a hardening time of 3–5 days and a partially opened bottle cap are optimal.
[0072] This invention uses young stems to directly induce adventitious buds, resulting in a shorter budding time, a higher budding rate, and robust plants with the resulting adventitious buds. Compared to the traditional method of first obtaining callus tissue and then inducing adventitious buds, this method is more time-saving and lower in cost.
[0073] Furthermore, the study found that callus tissue obtained from young leaves and hypocotyls as explants could induce adventitious buds, while callus tissue obtained from young stems as explants could not. Therefore, this invention provides an optimal method for cultivating wild Astragalus membranaceus seedlings. Besides being time-saving and cost-effective, it also expands our understanding of wild Astragalus membranaceus seedling cultivation and offers a completely new approach.
Claims
1. A method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus, characterized in that... Includes the following steps: S1. Acquisition of sterile seedlings: Mature seeds of wild Astragalus membranaceus were collected, disinfected, and inoculated into MS medium for culture to obtain sterile, complete wild Astragalus membranaceus plants. The wild Astragalus membranaceus seeds in S1 need to be pretreated, specifically by rubbing with fine sand for 5 min, rinsing with running water for 2 h, rinsing with sterile water 4-5 times in a clean bench, soaking in 75% anhydrous ethanol for 30 s, disinfecting with 15% NaClO for 5-15 min, soaking in 75% alcohol for 30 s after disinfection, rinsing with sterile water 4-5 times, blotting dry with sterile filter paper, and inoculating the wild Astragalus membranaceus seeds into MS medium. S2. Induction of adventitious buds from young stems: Young stems of wild Astragalus membranaceus were taken as explants and MS medium was used as the basic medium. Adventitious bud differentiation was induced by adding hormones 6-BA and IAA respectively. The concentration of 6-BA was 1.5 mg / L and the concentration of IAA was 0.2 mg / L. S3. Rooting culture: Single adventitious shoots are transferred to 1 / 2 MS rooting medium supplemented with IAA and IBA to induce adventitious roots; S4. Hardening off and transplanting: Take tissue culture seedlings that have undergone adventitious bud induction and rooting culture, harden them off, and then transplant them.
2. The method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus according to claim 1, characterized in that: The NaClO disinfection time is 10 min.
3. The method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus according to claim 1, characterized in that: The concentration of IBA in S3 was 0.5 mg / L, and the concentration of IAA was 1.0 mg / L.
4. The method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus according to claim 3, characterized in that: In S4, the hardening time is 3-5 days, and the tissue culture bottle should be half-open during hardening.
5. A method for rapidly inducing adventitious seedlings from wild Astragalus membranaceus according to any one of claims 1-4, characterized in that: The culture conditions were as follows: MS basal medium was selected, and agar 3.5 g / L and sucrose 15 g / L were added to form MS medium. The above components of 1 / 2 MS medium were halved. The pH was 5.
8. The medium was sterilized at 115℃ for 30 min. The culture temperature was (23±1)℃, the light intensity was 3000 lx, and the photoperiod was 12 h / d.