Tissue culture rapid propagation method of aquatic plant ottelia alismoides
By relieving the dormancy of agave seeds, seed germination and tissue culture technology, the rapid reproduction of agave seeds was successfully achieved, solving the problems of difficult seeds and reproduction, and a large number of excellent seedlings suitable for transplantation were obtained.
Patent Information
- Application Number
- CN202311172207.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-12
- Publication Date
- 2025-03-14
AI Technical Summary
The seeds of Agavea have strong dormant properties and lack effective rapid breeding methods, which leads to a decline in its population and is difficult to meet the needs of preservation and rapid reproduction of germplasm resources.
By removing seed dormancy, the seed germination was obtained after disinfection, the seeds germinated, and then the buds were cultured in the induction medium, and then transferred to the proliferation medium for multiple transfers. Finally, the rooted plants were cultivated in the rooting medium, and the seedlings were refined and transplanted.
The rapid reproduction of agave grass has been achieved, and a large number of excellent seedlings suitable for transplantation have the advantages of simple operation, short cycle and high efficiency, which solves the problems of seed dormancy and reproduction difficulties.
Smart Images
Figure CN119605643A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of aquatic plant biotechnology, in particular to a tissue culture and rapid propagation method of an aquatic plant Agave tongueii. Background Art
[0002] Ottelia alismoides, Hydrocharitaceae, Hydrocharitaceae, is an annual submerged plant. The petals are often white or lavender, and the flowering period is from April to October. Because it is an aquatic plant and its leaves are shaped like tongues, it is named Ottelia alismoides. In addition to its ornamental value, Ottelia alismoides is also a delicious wild vegetable. It can also be used as medicine, mainly for carbuncle, burns from hot water or fire. Ottelia alismoides was once widely distributed in lakes, ditches, ponds or low-lying areas with accumulated water throughout China. However, in recent years, due to the increasingly serious problems such as eutrophication of water bodies and the discharge of pollutants, the habitat of Ottelia alismoides has been severely damaged, and its population has dropped sharply. Ottelia alismoides was listed as a national second-level key protected wild plant in 2021.
[0003] Agave has a simple vegetative body and does not have vegetative reproduction. Seed propagation is its only way of reproduction. In addition, the seeds are very dormant, which brings certain difficulties to reproduction. Cluster bud induction is the most common and most efficient form of tissue culture. By inducing cluster buds to obtain regenerated plants, a large number of regenerated plants can be multiplied in a short period of time. However, there are currently no reports on the rapid propagation method of Agave, a national protected aquatic plant, at home and abroad. Therefore, the development of a tissue culture rapid propagation method for Agave has become a problem that needs to be solved urgently. Summary of the invention
[0004] In view of the above, the object of the present invention is to provide a method for rapid propagation of the aquatic plant Agave tongueii through tissue culture, which can rapidly propagate a large number of excellent Agave tongueii seedlings suitable for transplanting.
[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical measures: a tissue culture rapid propagation method of an aquatic plant Agave tongueii, comprising relieving seed dormancy and disinfecting, germinating seeds to obtain sterile embryos, inducing culturing of clustered buds, multiplying culturing of clustered buds, rooting culturing of robust plants, hardening and transplanting, specifically comprising the following steps:
[0006] (1) Breaking dormancy and disinfecting seeds: Take mature Agave seeds, break dormancy, soak them in a 2% disinfectant hand sanitizer solution for 10-20 minutes, and rinse them under running water for 60 minutes. Rinse them with sterile water for 3-5 times on a clean bench, soak them in 70% ethanol for 30 seconds, and then soak them in a 0.1% HgCl2 solution for 1-3 minutes. Finally, rinse them with sterile water for 3-5 times, and use sterile filter paper to absorb the surface moisture to obtain explants.
[0007] (2) Germinating seeds to obtain sterile embryos: placing the explants obtained in step (1) in sterile water and culturing them in the dark at 25° C. for 4-5 days, and the seeds can germinate to obtain embryos;
[0008] (3) Induction culture of clustered buds: The embryos obtained in step (2) are placed in an induction culture medium and cultured under light at 25° C. for 25-30 days with a light intensity of 2000 lux and a light duration of 12 hours per day to obtain clustered buds;
[0009] The induction medium is a MS medium containing 6-BA at a concentration of 1 mg / L, NAA at a concentration of 0.5 mg / L, sucrose at a concentration of 30 g / L, and plant gel at a concentration of 3 g / L.
[0010] (4) Proliferation culture of clustered buds: The clustered buds obtained in step (3) are transferred into a proliferation culture medium and cultured for 20-30 days (a cycle of 7 days, 3-4 transfers), with a light intensity of 2000 lux and a light time of 12 hours / day to obtain a large number of clustered buds;
[0011] The proliferation medium is a MS medium supplemented with 2 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose and 3 g / L phytagel;
[0012] (5) Rooting culture of robust plants: placing the clustered buds obtained in step (4) in a rooting medium, culturing under light at 25° C. for 25-30 days, with a light intensity of 2000 lux and a light duration of 12 hours per day, to obtain complete plants with roots;
[0013] The rooting medium is prepared by adding 0.5 mg / L NAA, 30 g / L sucrose and 3 g / L phytogel to 1 / 2 MS medium, and adding sterile water to 1 / 3 of the bottle.
[0014] (6) Hardening and transplanting: Add a small amount of tap water to the culture medium of step (5) and harden the seedlings for one week. After the seedlings grow well, remove the seedlings, wash the roots of the seedlings, and immediately transplant them to the aquatic plant resource nursery for further transplanting to the lake habitat.
[0015] Preferably, in step (1), the method for releasing the dormancy of Agave tongue seeds is to treat them in the dark at 4° C. for 1-2 months.
[0016] Preferably, in the step (1), the best treatment method for the explants of Agave tongue is: soaking in a 2% volume fraction bactericidal hand soap aqueous solution for 20 minutes, rinsing with tap water for 60 minutes, rinsing with sterile water for 5 times, soaking in 70% ethanol for 30 seconds, and then soaking in a 0.1% HgCl2 solution for 2 minutes, and finally rinsing with sterile water for 5 times.
[0017] Preferably, in step (3), the induction culture medium composition is: 4.74 g / L MS culture medium + 1 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
[0018] Preferably, in step (4), the proliferation culture medium consists of: 4.74 g / L MS culture medium + 2 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
[0019] Preferably, in step (5), the rooting culture medium composition is: 2.47 g / L MS culture medium + 0.5 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
[0020] Preferably, the pH of all culture media is adjusted to 5.8-6.0.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] (1) The present invention uses mature seeds of Agave tongueii as explant materials, obtains sterile embryos through seed germination, and obtains clustered buds after culturing in an induction medium for 25-30 days. The obtained clustered buds are transferred to a preferred proliferation medium and cultured for 20-30 days to obtain a large number of clustered buds.
[0023] (2) The present invention constructs a method for rapid propagation of Agave tongueii tissue culture. It has the advantages of simple operation, short cycle, high efficiency, etc. The method can successfully obtain Agave tongueii regeneration plants, providing a methodological basis for the research of its germplasm resource preservation, rapid propagation production and in vitro regeneration system. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to clearly present the purpose, technical solution and beneficial effects of the present invention, the present invention provides the following drawings for illustration:
[0025] Figure 1 Obtain sterile embryos for germination of Agave seeds;
[0026] Figure 2 Inoculate embryos onto induction medium (1 day);
[0027] Figure 3Inoculate embryos onto induction medium (7 days);
[0028] Figure 4 Inoculate embryos onto induction medium (14 days);
[0029] Figure 5 For the induction culture of clustered buds;
[0030] Figure 6 For the proliferation of clustered buds;
[0031] Figure 7 To cultivate strong plants for rooting;
[0032] Figure 8 To cultivate roots for strong plants;
[0033] Fig. 9 For complete rooted plants;
[0034] Fig.10 To cultivate the seedlings of Agave;
[0035] Fig.11 Cultivate the second seedling of Agave. DETAILED DESCRIPTION
[0036] In order to better illustrate the purpose, technical solutions and advantages of the present invention, an example of the present invention will be described in detail below with reference to the accompanying drawings.
[0037] The tissue culture rapid propagation method of the aquatic plant Agave tongueii provided in this embodiment comprises the following steps:
[0038] (1) Breaking dormancy and disinfecting seeds: Take mature seeds of Agave, break dormancy, and use them as explants. Soak them in a 2% aqueous solution of disinfectant hand sanitizer for 20 minutes, and rinse them under running water for 60 minutes. Rinse them with sterile water 5 times in a clean bench, soak them in 70% ethanol for 30 seconds, and then soak them in a solution containing 0.1% HgCl2 for 2 minutes. Finally, rinse them with sterile water 5 times, and use sterile filter paper to absorb the surface moisture to obtain explants.
[0039] (2) Seed germination to obtain sterile embryos: The explants obtained in step (1) are placed in sterile water and cultured in a dark environment at 25° C. for 5 days. After the seeds germinate, sterile embryos are obtained. Figure 1 As shown;
[0040] (3) Induction culture of clustered buds: The embryos obtained in step (2) are placed in an induction culture medium and cultured under light at 25° C. for 30 days. Figure 2 , Figure 3 , Figure 4As shown, the light intensity is 2000 lux, the light duration is 12 hours / day, and the sterile seedlings are obtained. Figure 5 As shown; the composition of the induction medium is: 4.74g / L MS medium + 1mg / L 6-BA + 0.5mg / L NAA + 30g / L sucrose + 3g / L plant gel;
[0041] (4) Proliferation culture of clustered buds: The clustered buds obtained in step (3) were transferred to a proliferation culture medium and cultured for 25 days at a light intensity of 2000 lux and a light duration of 12 hours per day to obtain a large number of clustered buds, such as Figure 6 As shown; the proliferation medium composition is: 4.74g / L MS medium + 2mg / L 6-BA + 0.2mg / L NAA + 30g / L sucrose + 3g / L plant gel;
[0042] (5) Rooting culture of robust plants: The sprouts obtained in step (4) were placed in a rooting medium and cultured under light at 25° C. for 25 days. Figure 7 As shown, the light intensity is 2000 lux, the light time is 12 hours / day, and complete plants with roots are obtained, such as Figure 8 , Fig. 9 As shown; the rooting medium composition is: 2.47g / L MS medium + 0.5mg / L NAA + 30g / L sucrose + 3g / L plant gel, and sterile water is added to the culture bottle to 1 / 3 of the bottle body;
[0043] (6) Hardening and transplanting: Add a small amount of tap water to the culture medium in step (5), such as Fig.10 , Fig.11 As shown, the seedlings are hardened for a week. When the seedlings grow well, they are taken out and the roots are cleaned. They are immediately transplanted into a water tank containing soil and cultured at room temperature.
[0044] In this example, the pH value of all culture media was adjusted to 6.0.
[0045] In this embodiment, the seed germination rate is more than 90%, the induction rate of clustered buds can reach more than 90%, the rooting rate can reach more than 95%, the survival rate of clustered buds in the transplanted seedbed is 100%, the growth rate is fast, and the plants are strong. According to statistics, the adventitious buds induced by one Agave seed can differentiate into 15-20 seedlings within 2 months.
[0046] A tissue culture rapid propagation method for the aquatic plant Agave tongueii established using the above technical method has broadened the reproduction range and efficiency of Agave tongueii and has the advantages of simple operation, low pollution rate, high survival rate, short culture cycle, etc. It is worthy of wide promotion.
[0047] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A tissue culture rapid propagation method for an aquatic plant Agave tongueii, characterized in that: The following steps are involved: (1) Breaking dormancy of seeds and disinfecting: taking mature Agave seeds, breaking dormancy, soaking them in a 2% aqueous solution of bactericidal hand sanitizer for 10-20 minutes, and rinsing them with running water for 60 minutes; rinsing them with sterile water for 3-5 times on a clean bench, soaking them in 70% ethanol for 30 seconds, and then soaking them in a solution containing 0.1% HgCl2 for 1-3 minutes, and finally rinsing them with sterile water for 3-5 times, and drying the surface moisture with sterile filter paper to obtain explants; (2) Germinating seeds to obtain sterile embryos: placing the explants obtained in step (1) in sterile water and culturing them in the dark at 25° C. for 4-5 days, and the seeds can germinate to obtain embryos; (3) Induction culture of clustered buds: The embryos obtained in step (2) are placed in an induction culture medium and cultured under light at 25° C. for 25-30 days with a light intensity of 2000 lux and a light duration of 12 hours per day to obtain clustered buds; (4) Proliferation culture of clustered buds: the clustered buds obtained in step (3) are transferred to a proliferation culture medium and cultured for 20-30 days at a light intensity of 2000 lux and a light duration of 12 hours per day to obtain a large number of clustered buds; (5) Rooting culture of robust plants: placing the clustered buds obtained in step (4) in a rooting medium, culturing under light at 25° C. for 25-30 days, with a light intensity of 2000 lux and a light duration of 12 hours per day, to obtain complete plants with roots; (6) Hardening and transplanting: Add a small amount of tap water to the culture medium of step (5) and harden the seedlings for one week. After the seedlings grow well, remove the seedlings, wash the roots of the seedlings, and immediately transplant them to the aquatic plant resource nursery for further transplanting to the lake habitat.
2. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 1, characterized in that: In the step (1), the method for releasing the dormancy of Agave tongue seeds is to treat them in the dark at 4° C. for 1-2 months.
3. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 1, characterized in that: In the step (1), the best treatment method for the explants of Agave tongueii is: soaking in a 2% volume fraction bactericidal hand sanitizer aqueous solution for 20 minutes, rinsing with running water for 60 minutes, rinsing with sterile water for 5 times, soaking in 70% ethanol for 30 seconds, soaking in a solution containing 0.1% HgCl2 for 2 minutes, and finally rinsing with sterile water for 5 times.
4. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 1, characterized in that: In the step (3), the induction culture medium composition is: 4.74 g / L MS culture medium + 1 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
5. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 1, characterized in that: In the step (4), the proliferation culture medium is composed of: 4.74 g / L MS culture medium + 2 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
6. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 1, characterized in that: In the step (5), the rooting culture medium composition is: 2.47 g / L MS culture medium + 0.5 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel.
7. The method for rapid propagation of the aquatic plant Agave tongueii by tissue culture according to claim 4, 5 or 6, characterized in that: The pH value of the culture medium was adjusted to 5.8-6.0.