Method for rapid germination of seeds of ferula sinkiangensis
By combining high-temperature water bath and disinfection with sterile cotton culture, the problem of slow seed germination in Xinjiang Ferula assa-foetida was solved, achieving rapid germination and high survival rate, thus promoting increased yield and ecological restoration of Xinjiang Ferula assa-foetida plants.
Patent Information
- Application Number
- CN202310810752.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-27
- Publication Date
- 2025-12-26
- Estimated Expiration
- 2043-06-27
AI Technical Summary
The seeds of Ferula assa-foetida from Xinjiang germinate slowly with an extremely low germination rate, and existing technologies lack effective methods to promote rapid germination, leading to a sharp decline in the plant's population and its endangerment.
After sterilization using a high-temperature water bath combined with ethanol and hydrogen peroxide solution, Xinjiang Ferula seeds were cultured in moistened sterile cotton, and temperature and light conditions were controlled to promote seed germination.
This method enables rapid germination of Ferula seeds in Xinjiang, improves germination and survival rates, reduces the harvesting of wild plants, provides a stable supply of medicinal resources, and lowers costs and environmental impact.
Smart Images
Figure CN117063659B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of seed germination, and particularly relates to a method for rapid germination of Ferula sinkiangensis K.M.Shen seeds. BACKGROUND
[0002] Ferula sinkiangensis K.M.Shen is a perennial once-flowering herb of the Ferula genus in the Apiaceae family, with a height of 0.5-1.5 meters, and a strong garlic-like odor. The root is spindle-shaped or conical, thick, and the root neck is covered with withered leaf sheath fibers. The stem is usually single and thick; the leaf blade is triangular-ovate in outline, ternately and ternately pinnately and fully lobed. The compound corymbose inflorescence is born at the top of the stem branch, with a diameter of 8-12 mm, no total bract, and the fruit is oval, flat and pressed, and the fruit ridge is protruding. It is an early spring short-lived plant with a long life cycle and a short growth period, and it prefers cool and dry conditions, is drought-tolerant and afraid of waterlogging, and prefers sunlight. The growth site should be selected in a place with flat terrain, convenient drainage and irrigation. It is mainly distributed in the desert at an altitude of about 850 meters in Yili and on the gravelly clay soil slope.
[0003] The available resources on the desert are scarce, and the growth environment of Ferula sinkiangensis K.M.Shen is relatively poor, resulting in a small number of plants. Because Ferula sinkiangensis K.M.Shen has the effects of removing accumulation and eliminating phlegm, removing dampness and relieving pain, and killing insects, it has unique effects on rheumatoid arthritis and stomach disease, and is widely used as a medicinal plant by the Uighur, Mongolian and Hui ethnic groups, and is a plant resource with important medicinal value. Studies have found that Ferula sinkiangensis K.M.Shen has the phenomenon of male sterility, and at the same time, the low ratio of bisexual flowers on the plant, the low female influencing factor, the high rate of fruit insect feeding and the high seed mortality rate are all factors causing the endangerment of Ferula sinkiangensis K.M.Shen. Ferula sinkiangensis K.M.Shen not only has a very low germination rate, but also is affected by human activities, over-picking and over-grazing, resulting in a sharp decrease in the number of Ferula sinkiangensis K.M.Shen. Now Ferula sinkiangensis K.M.Shen has been listed as a Chinese second-class protected important wild medicinal material species, and belongs to the critically endangered species in the Red List of Endangered Species of the World Conservation Union. Ferula sinkiangensis K.M.Shen is a perennial once-flowering herb, with a long growth cycle and a slow seed germination rate under natural conditions. Ferula sinkiangensis K.M.Shen is a perennial short-lived plant, and can only flower and bear fruit in the eighth year and be harvested for medicinal materials. Moreover, the seed germination rate is very low. In order to improve the seed germination rate of Ferula sinkiangensis K.M.Shen and quickly break the low-temperature accumulation of Ferula sinkiangensis K.M.Shen seeds, a method for rapidly promoting the germination of Ferula sinkiangensis K.M.Shen seeds is urgently needed.
[0004] Xinjiang asafetida has high scientific research value, but due to the relationship between the growth environment of Xinjiang asafetida, so far for Xinjiang research is almost all in the field of ecology. Now it is urgent to use the method of molecular biology to further explore the germination of Xinjiang asafetida seed. Plant tissue culture technology is a very important technology in molecular biology research, and the seed germination is also a key link in the plant tissue culture system, but the method of accelerating the germination of Xinjiang asafetida seed has not been reported. SUMMARY
[0005] Therefore, the purpose of the present application is to provide a Xinjiang asafetida seed rapid germination method, which can efficiently and quickly promote the germination of Xinjiang asafetida seed, without using any plant hormone treatment, without adding culture medium, and is ecological, low cost and simple.
[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:
[0007] The present application provides a Xinjiang asafetida seed rapid germination method, comprising the following steps:
[0008] The Xinjiang asafetida seed is sterilized by water bath at 30-45 DEG C for 1.5-2.5 h to obtain sterile Xinjiang asafetida seed, and then the sterile Xinjiang asafetida seed is cultured in the soaked sterile cotton.
[0009] Preferably, the soaked sterile cotton is soaked with sterile water.
[0010] Preferably, the culture temperature is 20-25 DEG C, the absolute humidity of the culture air is 30%-40%, the light intensity of the culture is 4000-5000 Lux, and the light duration of the culture is 12-16 h / day.
[0011] Preferably, the sterilization reagent comprises 70%-80% ethanol solution and 5%-7% hydrogen peroxide solution.
[0012] Preferably, the sterilization method comprises:
[0013] (1) the Xinjiang asafetida seed after water bath is soaked with ethanol solution for 80-100 s, and washed with sterile water to obtain Xinjiang asafetida seed after ethanol sterilization;
[0014] (2) the Xinjiang asafetida seed after ethanol sterilization is soaked with hydrogen peroxide solution for 8-12 min, and then washed with sterile water to obtain sterile Xinjiang asafetida seed.
[0015] Preferably, in steps (1) and (2), the washing time is 3-5 min, and the washing time is 3-5 min.
[0016] Preferably, in step (1) and step (2), the temperature of the soaking is 20-25℃.
[0017] Compared with the prior art, the present application has the following beneficial effects:
[0018] The present application provides a method for rapid germination of Xinjiang asafetida seeds, which can quickly promote the germination of Xinjiang asafetida seeds, and does not use any hormone treatment and does not need to add culture medium during the germination process, which is ecological, environmentally friendly, low in cost and easy to operate. The present application not only helps to increase the yield of Xinjiang asafetida plants and establish a sterile system of Xinjiang asafetida plants, but also reduces the picking of wild asafetida, provides stable and abundant Xinjiang asafetida resources for the traditional Chinese medicine industry, and lays a solid foundation for alleviating the imminent extinction of Xinjiang asafetida plants and restoring the ecological environment of Xinjiang asafetida. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 For the Xinjiang asafetida seeds cultured for 8 days in Example 3;
[0020] Figure 2 For the Xinjiang asafetida seeds cultured for 9 days in Example 1;
[0021] Figure 3 For the Xinjiang asafetida seeds cultured for 11 days in Example 2
[0022] Figure 4 For the Xinjiang asafetida seeds cultured for 10 days in Example 4
[0023] Figure 5 For the Xinjiang asafetida seeds cultured for 21 days in Comparative Example 2;
[0024] Figure 6 For the Xinjiang asafetida seeds cultured for 26 days in Comparative Example 1;
[0025] Figure 7 For the Xinjiang asafetida seeds cultured for 24 days in Comparative Example 3;
[0026] Figure 8 For the Xinjiang asafetida seeds cultured for 17 days in Comparative Example 4 (right side of the figure) and Comparative Example 6 (left side of the figure). DETAILED DESCRIPTION
[0027] The present application provides a method for rapid germination of Xinjiang asafetida seeds, comprising the following steps:
[0028] The Xinjiang asafetida seeds are sterilized by water bath at 30-45℃ for 1.5-2.5h to obtain sterile Xinjiang asafetida seeds, and then the sterile Xinjiang asafetida seeds are cultured in wet sterile cotton.
[0029] The present application adopts high-temperature water bath Xinjiang asafetida seeds, quickly breaks the dormancy of Xinjiang asafetida seeds, and promotes the germination of Xinjiang asafetida seeds. As a preferred embodiment, the Xinjiang asafetida seeds are water bathed at 35-42°C for 100-130 min. Before the high-temperature water bath of the Xinjiang asafetida seeds, the surface impurities of the Xinjiang asafetida seeds are removed by washing with water at room temperature for 20-40 min. In order to further remove the surface impurities of the Xinjiang asafetida seeds, washing liquid can also be added during the washing process.
[0030] In the present application, the Xinjiang asafetida seeds are water bathed at 30-45°C for 1.5-2.5 h to obtain water-bathed Xinjiang asafetida seeds, which are then sterilized. After obtaining the water-bathed Xinjiang asafetida seeds, the present application further includes the step of washing the water-bathed Xinjiang asafetida seeds with sterile water for 4-5 times before sterilization, in order to remove dust or impurities on the seeds. The sterilizing agent preferably includes an ethanol solution with a volume concentration of 70%-80% and a hydrogen peroxide solution with a volume concentration of 5%-7%. The solvent used in the ethanol solution or the hydrogen peroxide solution is water. For example, the preparation of a 70%-80% ethanol solution includes diluting anhydrous ethanol with water to a 70%-80% ethanol solution, and mixing well. The preparation of a 5%-7% hydrogen peroxide solution includes diluting 30% hydrogen peroxide with sterile water to a 5%-7% hydrogen peroxide solution, and mixing well. The present application does not have special limitations on the source of anhydrous ethanol and 30% hydrogen peroxide, which can be prepared by commercially available or commonly known methods in the art.
[0031] As a preferred embodiment, the sterilization method includes: (1) soaking the water-bathed Xinjiang asafetida seeds with an ethanol solution for 80-100 s, and washing with sterile water to obtain ethanol-sterilized Xinjiang asafetida seeds; (2) soaking the ethanol-sterilized Xinjiang asafetida seeds with a hydrogen peroxide solution for 8-12 min, and washing with sterile water to obtain sterile Xinjiang asafetida seeds. In a preferred embodiment, in steps (1) and (2), the washing is performed 4-5 times, each time for 3-5 min, and the soaking temperature is 20-25°C. The present application uses Xinjiang asafetida seeds soaked in an ethanol solution and a hydrogen peroxide solution, thereby inhibiting the activity of bacteria and fungi, preventing bacterial contamination of the sterile environment, and promoting the germination of Xinjiang asafetida seeds.
[0032] In the present application, after sterilization, the sterile Xinjiang asafetida seeds are cultured in wet sterile cotton. The wet sterile cotton is preferably soaked with sterile water, and the wetness of the wet sterile cotton is preferably 3-5 g / cm 3The temperature of the culture is preferably 20-25℃, the absolute humidity of the culture is preferably 30%-40%, the light intensity of the culture is preferably 4000-5000 Lux, and the light duration of the culture is preferably 12-16 hours per day. During the culture, the Xinjiang asarum seed is observed, and the results show that the seed germinates on the 8th day by using the method of the application, compared with other methods such as low temperature (such as 4℃) method, temperature alternating treatment method, freezing treatment method and plant hormone treatment method, the method of the application achieves the purpose of rapid germination of Xinjiang asarum seed.
[0033] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the protection scope of the application.
[0034] In the following comparative examples, the 1 / 2MS medium is prepared as follows: 2.47g of 1 / 2MS medium (without agar and sucrose) provided by Haibo Biotechnology Co., Ltd. is weighed and dissolved in 1000mL of distilled water, and then autoclaved at 116℃ for 30 minutes.
[0035] Example 1
[0036] A method for rapid germination of Xinjiang asarum seed, the steps are as follows:
[0037] (1) The Xinjiang asarum seed collected from the wild is washed with running water at room temperature for 30 minutes, and a small amount of detergent is added during the washing process to remove the surface impurities;
[0038] (2) The Xinjiang asarum seed is water-bathed in a water bath kettle at 30℃ for 2 hours to obtain the water-bathed Xinjiang asarum seed;
[0039] (3) The 150mL conical flask, distilled water and absorbent cotton are sterilized in a high-pressure steam sterilization kettle at 121℃ for 20 minutes;
[0040] (4) In the clean bench, the seed is placed in a 150mL sterile conical flask and washed with sterile water for 4-5 times;
[0041] (5) The seed is sterilized by immersing in a 75% ethanol solution at 25℃ for 90s, and the sterile conical flask is shaken multiple times during the process, then taken out and washed with sterile water for 4 times;
[0042] (6) The seed is immersed in a 6% hydrogen peroxide solution at 25℃ for 10 minutes, and the conical flask is shaken multiple times during the process, then taken out and washed with sterile water for 4 times to obtain the sterile Xinjiang asarum seed;
[0043] (7) In the clean bench, 200g of sterile water is used to soak 56cm 3Sterile cotton is wetted, and then 25 sterile Xinjiang asafetida seeds are placed on the wetted sterile cotton, and cultured at 23℃, with an absolute humidity of air being 35%, and an illumination intensity being 4000-5000 Lux, under the condition that the illumination time is 14h per day, and the germination time of the seeds is observed and recorded.
[0044] Example 2
[0045] The difference between this example and Example 1 is that the step (2) of this example is that the Xinjiang asafetida seeds are water-bathed in a water bath kettle at 35℃ for 2h to obtain water-bathed Xinjiang asafetida seeds, and the remaining steps are the same as those of Example 1.
[0046] Example 3
[0047] The difference between this example and Example 1 is that the step (2) of this example is that the Xinjiang asafetida seeds are water-bathed in a water bath kettle at 40℃ for 2h to obtain water-bathed Xinjiang asafetida seeds, and the remaining steps are the same as those of Example 1.
[0048] Example 4
[0049] The difference between this example and Example 1 is that the step (2) of this example is that the Xinjiang asafetida seeds are water-bathed in a water bath kettle at 45℃ for 2h to obtain water-bathed Xinjiang asafetida seeds, and the remaining steps are the same as those of Example 1.
[0050] Example 5
[0051] The difference between this example and Example 1 is that the step (2) of this example is that the Xinjiang asafetida seeds are water-bathed in a water bath kettle at 40℃ for 1.5h to obtain water-bathed Xinjiang asafetida seeds, and the remaining steps are the same as those of Example 1.
[0052] Example 6
[0053] The difference between this example and Example 1 is that the step (2) of this example is that the Xinjiang asafetida seeds are water-bathed in a water bath kettle at 40℃ for 2.5h to obtain water-bathed Xinjiang asafetida seeds, and the remaining steps are the same as those of Example 1.
[0054] Example 7
[0055] A method for rapid germination of Xinjiang asafetida seeds, the steps are as follows:
[0056] (1) The Xinjiang asafetida seeds collected from the wild are washed with running water at room temperature for 30min, and a small amount of detergent is added during the washing process to remove surface impurities;
[0057] (2) The Xinjiang asafetida seeds are water-bathed in a water bath kettle at 30℃ for 2h to obtain water-bathed Xinjiang asafetida seeds;
[0058] (3) using high-pressure steam sterilization pot, 150 mL conical flask, distilled water, sterilization of defatted cotton at 121 ℃ for 20 minutes;
[0059] (4) on the clean bench, the seeds were placed in 150 mL sterile conical flask, washed with sterile water 5 times;
[0060] (5) soaked with 70% ethanol solution in volume concentration at 25 ℃ for 100 s for seed disinfection, during which the sterile conical flask was shaken several times, taken out, washed with sterile water 4 times;
[0061] (6) soaked with 7% hydrogen peroxide solution in volume concentration at 25 ℃ for 8 min, during which the conical flask was shaken several times, taken out, washed with sterile water 5 times, to obtain sterile Xinjiang asafetida seeds;
[0062] (7) in the clean bench, 50 cm 3 Sterile cotton was soaked, and then 25 sterile Xinjiang asafetida seeds were placed on the soaked sterile cotton, cultured at 25 ℃, absolute humidity of air was 30%, light intensity was 4000-5000 Lux, and the light length was 12 h per day, and the germination time of the seeds was observed and recorded.
[0063] Example 8
[0064] A method for rapid germination of Xinjiang asafetida seeds, the steps are as follows:
[0065] (1) the collected Xinjiang asafetida seeds from the wild were washed with running water at room temperature for 30 min, and a small amount of detergent was added during the washing process to remove surface impurities;
[0066] (2) the Xinjiang asafetida seeds were water-bathed at 30 ℃ for 2 h to obtain water-bathed Xinjiang asafetida seeds;
[0067] (3) using high-pressure steam sterilization pot, 150 mL conical flask, distilled water, sterilization of defatted cotton at 121 ℃ for 20 minutes;
[0068] (4) on the clean bench, the seeds were placed in 150 mL sterile conical flask, washed with sterile water 5 times;
[0069] (5) soaked with 80% ethanol solution in volume concentration at 25 ℃ for 80 s for seed disinfection, during which the sterile conical flask was shaken several times, taken out, washed with sterile water 5 times;
[0070] (6) soaked with 5% hydrogen peroxide solution in volume concentration at 25 ℃ for 12 min, during which the conical flask was shaken several times, taken out, washed with sterile water 4 times, to obtain sterile Xinjiang asafetida seeds;
[0071] (7) In the clean bench, 50 cm 3 The sterile cotton was wetted, and then 25 sterile Xinjiang Asafetida seeds were placed on the wetted sterile cotton. The seeds were cultured at 20℃, an absolute humidity of air of 40%, and a light intensity of 4000-5000 Lux, with a light duration of 16 hours per day, and the germination time of the seeds was observed and recorded.
[0072] Comparative Example 1
[0073] In this comparative example, the Xinjiang Asafetida seeds were treated by low-temperature treatment. The difference between this comparative example and Example 3 is that in step (7), in the clean bench, 25 sterile Xinjiang Asafetida seeds were placed in 25 mL of 1 / 2MS medium, and cultured at 4℃, a humidity of 40%, and a light intensity of 4000 Lux, with a light duration of 10 hours per day, and the germination time of the seeds was observed and recorded. The other steps were the same as in Example 3.
[0074] Comparative Example 2
[0075] In this comparative example, the Xinjiang Asafetida seeds were treated by temperature alternation treatment. The difference between this comparative example and Example 3 is that in step (7), in the clean bench, 25 sterile Xinjiang Asafetida seeds were placed in 25 mL of 1 / 2MS medium, and cultured at 4℃, a humidity of 40%, and a light intensity of 4000 Lux on the first day, with a light duration of 10 hours per day, and at 25℃, a humidity of 40%, and a light intensity of 4000 Lux on the second day, with a light duration of 10 hours per day. The culture mode of the first day and the second day was repeated subsequently (i.e., the seeds were cultured in an environment alternating between 4℃ and 25℃ every 24 hours), and the germination time of the seeds was observed and recorded. The other steps were the same as in Example 3.
[0076] Comparative Example 3
[0077] In this comparative example, the Xinjiang Asafetida seeds were treated by freezing treatment, i.e., the seeds were placed in a refrigerator at -20℃ for 24 hours, and then directly sowed in grass charcoal soil after thawing, and the germination of the seeds was observed at room temperature.
[0078] Comparative Example 4
[0079] The difference between this comparative example and Example 3 is that in step (7), in the clean bench, 25 sterile Xinjiang Asafetida seeds were placed in 25 mL of 1 / 2MS medium, and cultured at 23℃, a humidity of 30-40%, and a light intensity of 0-5000 Lux, with a light duration of 14 hours per day, and the germination time of the seeds was observed and recorded. The other steps were the same as in Example 3.
[0080] Comparative Example 5
[0081] The difference between the present comparative example and Comparative Example 4 is that the present comparative example is to place the sterile Ferula sinkiangensis seed in 25 mL of 1 / 2MS medium with 0.2 mg / L gibberellin, and the rest of the steps are the same as those of Comparative Example 4.
[0082] Comparative Example 6
[0083] The difference between the present comparative example and Comparative Example 5 is that the gibberellin concentration of the present comparative example is 0.5 mg / L, and the rest of the steps are the same as those of Comparative Example 5.
[0084] Comparative Example 7
[0085] The difference between the present comparative example and Comparative Example 5 is that the gibberellin concentration of the present comparative example is 1 mg / L, and the rest of the steps are the same as those of Comparative Example 5.
[0086] Comparative Example 8
[0087] The difference between the present comparative example and Comparative Example 5 is that the gibberellin concentration of the present comparative example is 2 mg / L, and the rest of the steps are the same as those of Comparative Example 5.
[0088] (1) The results of the time of starting germination of the seeds recorded in Examples 1-4 and Comparative Examples 1-8, and the seed germination rate on the 30th day of culture are shown in Table 1, wherein the seed germination rate = (the number of germinated seeds ÷ the number of seeds) x 100%.
[0089] Table 1 Effect of different Ferula seed germination methods on seed germination time and germination rate
[0090]
[0091]
[0092] From the results in Table 1, compared with the comparative examples, the method of the present application achieves the purpose of rapid germination of Ferula sinkiangensis seeds, and the method does not need to add plant hormones and culture medium, has low cost, is eco-friendly, and the germination rate of Ferula sinkiangensis seeds treated with water bath is higher than that of other treatment groups.
[0093] (3) All successfully germinated Ferula sinkiangensis seeds in Examples 1-4 and Comparative Examples 1-8 were taken out from the culture medium or sterile cotton, respectively transplanted into nutrient soil, sprayed with 10 mL of sterile water every 2 days, and recorded and calculated the survival rate on the 60th day, the survival rate = (the number of Ferula seedlings surviving on the 60th day ÷ the total number of Ferula seedlings grown in the nutrient soil) x 100%.
[0094] The results show that the survival rates of the seeds of Examples 1-4 are 95%, 92%, 97%, and 91% respectively, and the survival rates of Comparative Examples 1-8 are 80%, 80%, 89%, 83%, 85%, 70%, 88%, and 80% respectively, which shows that the survival rate of the Xinjiang asarum seed after the water bath treatment of the application is higher than that of other treatment groups.
[0095] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for rapid germination of seeds of Ferula sinkiangensis K. M. Shen, characterized in that, The method comprises the following steps: The seed of Ferula sinkiangensis K. M. Shen is sterilized in water bath at 30-45℃ for 1.5-2.5h to obtain the sterile seed of Ferula sinkiangensis K. M. Shen, and then the sterile seed of Ferula sinkiangensis K. M. Shen is cultured in the soaked sterile cotton; The culture temperature is 20-25℃, the absolute humidity of the culture air is 30%-40%, the light intensity of the culture is 4000-5000Lux, and the light time of the culture is 12-16h per day.
2. The method of claim 1, wherein, The soaked sterile cotton is soaked with sterile water.
3. The method of claim 1, wherein, The sterilization reagent comprises an ethanol solution with a volume concentration of 70%-80% and a hydrogen peroxide solution with a volume concentration of 5%-7%.
4. The method of claim 3, wherein, The sterilization method comprises: (1) soaking the seed of Ferula sinkiangensis K. M. Shen in the ethanol solution for 80-100s after water bath, and washing with sterile water to obtain the seed of Ferula sinkiangensis K. M. Shen sterilized by ethanol; (2) soaking the seed of Ferula sinkiangensis K. M. Shen sterilized by ethanol in the hydrogen peroxide solution for 8-12min, and washing with sterile water to obtain the sterile seed of Ferula sinkiangensis K. M. Shen.
5. The method of claim 4, wherein, In steps (1) and (2), the washing is performed 4-5 times, and each washing is performed for 3-5min.
6. The method of claim 4, wherein, In steps (1) and (2), the soaking temperature is 20-25℃.