CN117063659A

CN117063659A - Method for rapidly germinating ferula seeds in Xinjiang

Info

Publication number: CN117063659A
Application number: CN202310810752.3A
Authority: CN
Inventors: 郭新勇; 赵静怡; 张丽; 朱芸; 耿乐; 王美; 田志佳; 余香雪
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2023-06-27
Filing date: 2023-06-27
Publication date: 2023-11-17

Abstract

The application provides a method for rapidly germinating ferula sinkiangensis seeds, which belongs to the technical field of seed germination and comprises the following steps: sterilizing the Sinkiang Ferula asafetida seeds in water bath at 30-45 deg.c for 1.5-2.5 hr to obtain sterilized Sinkiang Ferula asafetida seeds, and culturing the sterilized Sinkiang Ferula asafetida seeds in soaked sterilized cotton. The method can quickly promote the germination of the Sinkiang asafetida seeds, does not use any hormone treatment in the germination process, does not need to add a culture medium, is ecological and environment-friendly, has low cost, and is simple and easy to operate. The application is not only beneficial to the production increase of the ferula sinkiangensis and the establishment of a sterile system of the ferula sinkiangensis, but also can reduce the picking of wild ferula asafetida, provides stable and rich medicine source guarantee of the ferula asafetida for the traditional Chinese medicine industry, and lays a solid foundation for relieving the imminent extinction of the ferula asafetida and restoring the ecological environment of the ferula asafetida in the sinkiangensis in the future.

Description

Method for rapidly germinating ferula seeds in Xinjiang

Technical Field

The application belongs to the technical field of seed germination, and particularly relates to a method for rapidly germinating ferula sinkiangensis seeds.

Background

Sinkiang Ferula (academic name: ferula sinkiangensis K.M.shen) is herb of Ferula genus of Umbelliferae, and has a height of 0.5-1.5 m, and a strong odor of herba Alii Fistulosi and Bulbus Allii. The root is spindle-shaped or conical, thick and strong, and withered leaf sheath fibers remain on the root neck. The stems are usually single and thick; the outline of the blade is triangular oval, and the blade is three-outlet type and three-back feathered full cracks. The multiple umbrella-shaped inflorescence grows on the top of the stem branch, has the diameter of 8-12 cm, has no total bract, and is oval in meristematic fruit, flat in dorsum and abdomen and protruding in fruit edge. The plant belongs to early spring short-lived plants with long life cycle and short growth period, is warm and cool, drought-enduring, waterlogging-fearing and sun-loving, and the growing place is selected in a place with flat land, convenient drainage and irrigation. Is mainly distributed on desert with the altitude of 850 meters such as Yili and the like and clay slopes with gravels.

The available resources on the desert are deficient, the growth environment of the Xinjiang asafetida is worse, the number of plants is smaller, and the Xinjiang asafetida has the effects of resolving food stagnation, dispersing masses, eliminating dampness, relieving pain, killing insects and the like, has unique curative effects on rheumatic arthritis and stomach diseases, is widely used as medicinal plants in folks by minority nationalities such as vitamins, mongolia, gyrus and the like, and is a plant resource with important medicinal value. The research shows that the Xinjiang asafetida has male sterility, and meanwhile, the lower amphoteric flower proportion, lower female influence factor, higher fruit worm feeding rate and higher seed death rate on the Xinjiang asafetida plant are all factors causing the endangered of the Xinjiang asafetida, so that the Xinjiang asafetida is extremely low in germination rate, and can be influenced by human activity, excessive picking and excessive grazing, and the quantity of the Xinjiang asafetida is greatly reduced. The Xinjiang asafetida is now listed as a Chinese secondary protection important wild medicinal material species, belongs to an extremely dangerous species in red directory of the world natural protection alliance endangered species, belongs to herbaceous plants with perennial once fruiting, has long growth cycle, is a perennial short-lived plant, can bloom and fruit and collect medicinal materials only in the 8 th year, has extremely low seed germination rate, and is urgent to develop a method for rapidly promoting the germination of the Xinjiang asafetida seeds in order to improve the germination speed of the Xinjiang asafetida seeds and rapidly break the low-temperature lamination of the asafetida seeds.

Xinjiang asafetida has high scientific research value, but because of the relation of the growing regional environment of the Xinjiang asafetida, the research of Xinjiang is almost ecological to date. There is a need for further intensive research into the germination of ferula seeds using molecular biology methods. The plant tissue culture technology is a very important technology in molecular biology research, wherein the germination of plant seeds is also a key link in a plant tissue culture system, but the research on accelerating the germination method of the ferula seeds in Xinjiang has not been reported at present.

Disclosure of Invention

Therefore, the application aims to provide the method for rapidly germinating the ferula seeds in Xinjiang, which can efficiently and rapidly promote the germination of the ferula seeds in Xinjiang, does not need any phytohormone treatment, does not need to add a culture medium, is ecological and environment-friendly, has low cost, and is simple and easy to implement.

In order to achieve the above object, the present application provides the following technical solutions:

the application provides a method for rapidly germinating ferula sinkiangensis seeds, which comprises the following steps:

sterilizing the Sinkiang Ferula asafetida seeds in water bath at 30-45 deg.c for 1.5-2.5 hr to obtain sterilized Sinkiang Ferula asafetida seeds, and culturing the sterilized Sinkiang Ferula asafetida seeds in soaked sterilized cotton.

Preferably, the soaked sterile cotton is soaked with sterile water.

Preferably, the temperature of the culture is 20-25 ℃, the absolute humidity of the air of the culture is 30-40%, the illumination intensity of the culture is 4000-5000 Lux, and the illumination duration of the culture is 12-16 h/day.

Preferably, the disinfecting agent comprises an ethanol solution with a volume concentration of 70-80% and a hydrogen peroxide solution with a volume concentration of 5-7%.

Preferably, the method of sterilization comprises:

(1) Soaking the Xinjiang asafetida seeds after water bath in ethanol solution for 80-100 s, and washing with sterile water to obtain the Xinjiang asafetida seeds after ethanol disinfection;

(2) Soaking the Sinkiang Ferula asafetida seeds sterilized by ethanol in hydrogen peroxide solution for 8-12 min, and washing with sterile water to obtain the sterile Sinkiang Ferula asafetida seeds.

Preferably, in the step (1) and the step (2), the washing is performed for 4 to 5 times, and the time for each washing is 3 to 5 minutes.

Preferably, in the step (1) and the step (2), the soaking temperature is 20-25 ℃.

Compared with the prior art, the application has the following beneficial effects:

the application provides a method for rapidly germinating ferula seeds in Xinjiang, which can rapidly promote the germination of the ferula seeds in Xinjiang, does not use any hormone treatment in the germination process, does not need to add a culture medium, is ecological and environment-friendly, has low cost, and is simple and easy to operate. The application is not only beneficial to the production increase of the ferula sinkiangensis and the establishment of a sterile system of the ferula sinkiangensis, but also can reduce the picking of wild ferula asafetida, provides stable and rich medicine source guarantee of the ferula asafetida for the traditional Chinese medicine industry, and lays a solid foundation for relieving the imminent extinction of the ferula asafetida and restoring the ecological environment of the ferula asafetida in the sinkiangensis in the future.

Drawings

FIG. 1 shows the seeds of Ferula sinkiana on day 8 of the culture in example 3;

FIG. 2 shows the seeds of Ferula sinkiana on day 9 of the culture in example 1;

FIG. 3 shows the seeds of Ferula sinkiana on day 11 of the culture in example 2

FIG. 4 shows the seeds of Ferula sinkiana on day 10 of the culture in example 4

FIG. 5 shows the seeds of Ferula sinkiana on day 21 of culture in comparative example 2;

FIG. 6 shows the seeds of Ferula sinkiana on day 26 of culture in comparative example 1;

FIG. 7 shows the seeds of Ferula sinkiana on day 24 of culture in comparative example 3;

FIG. 8 shows the seeds of Sinkiang Ferula asafetida cultivated on day 17 in comparative example 4 (right side of the figure) and comparative example 6 (left side of the figure).

Detailed Description

The method adopts the high-temperature water bath of the ferula sinkiangensis seeds to quickly break dormancy of the ferula sinkiangensis seeds and promote germination of the ferula sinkiangensis seeds, and as a preferred implementation mode, the ferula sinkiangensis seeds are subjected to the water bath at the temperature of 35-42 ℃ for 100-130 min. The method comprises the steps of washing the Sinkiang Ferula seeds with water for 20-40 min at normal temperature before the Sinkiang Ferula seeds are subjected to high-temperature water bath, so as to remove surface impurities of the Sinkiang Ferula seeds, and adding a detergent in the washing process for further removing the surface impurities of the Sinkiang Ferula seeds.

In the application, the seeds of the ferula sinkiangensis are subjected to water bath for 1.5 to 2.5 hours at the temperature of 30 to 45 ℃ to obtain the seeds of the ferula sinkiangensis after water bath, and the seeds of the ferula sinkiangensis are sterilized. After the Sinkiang asafetida seeds are obtained after the water bath and before disinfection, the method also comprises the step of flushing the Sinkiang asafetida seeds for 4-5 times after the water bath with sterile water so as to remove dust or impurities on the seeds. The sterilizing agent preferably comprises an ethanol solution with the volume concentration of 70-80% and a hydrogen peroxide solution with the volume concentration of 5-7%. The solvent used for the ethanol solution or the hydrogen peroxide solution is water, for example, the preparation of the ethanol solution with the concentration of 70-80 percent comprises the steps of diluting the ethanol solution with absolute ethanol to the ethanol solution with the concentration of 70-80 percent by adding water, and uniformly mixing. The preparation of the 5% -7% hydrogen peroxide solution comprises the steps of diluting the hydrogen peroxide solution to the 5% -7% hydrogen peroxide solution by 30% hydrogen peroxide and sterile water, and uniformly mixing. The source of the absolute ethanol and 30% hydrogen peroxide is not particularly limited, and the absolute ethanol and 30% hydrogen peroxide can be prepared by a method commercially available in the art or known in the art.

As a preferred embodiment, the method of sterilization comprises: (1) Soaking the Xinjiang asafetida seeds after water bath in ethanol solution for 80-100 s, and washing with sterile water to obtain the Xinjiang asafetida seeds after ethanol disinfection; (2) Soaking the Sinkiang Ferula asafetida seeds sterilized by ethanol in hydrogen peroxide solution for 8-12 min, and washing with sterile water to obtain the sterile Sinkiang Ferula asafetida seeds. In a preferred embodiment, in the step (1) and the step (2), the number of times of flushing is 4 to 5, the time of each flushing is 3 to 5Min, and the soaking temperature is 20 to 25 ℃. The application adopts the Sinkiang asafetida seeds soaked in the ethanol solution and the hydrogen peroxide solution, thereby inhibiting the bacterial and fungal activity of the Sinkiang asafetida seeds, preventing bacteria from polluting the sterile environment, and promoting the germination of the Sinkiang asafetida seeds.

In the application, after disinfection, the sterilized Sinkiang asafetida seeds are placed in soaked sterilized cotton for culture. The soaked sterile cotton is preferably soaked with sterile water, and the humidity of the soaking is preferably 3-5 g/cm ³ . The temperature of the culture is preferably 20-25 ℃, the absolute humidity of the air of the culture is preferably 30-40%, the illumination intensity of the culture is preferably 4000-5000 Lux, and the illumination duration of the culture is preferably 12-16 h/day. As a result of observing the condition of the Sinkiang asafetida seeds during the culture period, the method disclosed by the application is used for sprouting on the 8 th day, and compared with other methods such as a low-temperature (e.g. 4 ℃) method, a temperature alternation treatment method, a freezing treatment method and a plant hormone treatment method, the method disclosed by the application achieves the aim of fast sprouting of the Sinkiang asafetida seeds.

The technical solutions provided by the present application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present application.

In the following comparative examples, 1/2MS medium was prepared: 2.47g of the 1/2MS culture medium (without agar and sucrose) provided by the Haibo biotechnology Co., ltd was weighed and dissolved in 1000mL of distilled water, and the mixture was autoclaved at 116℃for 30 minutes.

Example 1

A method for rapidly germinating ferula seeds in Xinjiang comprises the following steps:

(1) Washing the Sinkiang Ferula asafetida seeds collected from the field with running water at normal temperature for 30min, adding a small amount of detergent during washing, and removing surface impurities;

(2) Placing the Xinjiang asafetida seeds in a water bath kettle at 30 ℃ for 2 hours to obtain the Xinjiang asafetida seeds after water bath;

(3) Sterilizing 150mL conical flask, distilled water and absorbent cotton at 121deg.C for 20 min;

(4) On an ultra-clean workbench, placing seeds into a 150mL sterile conical flask, and flushing with sterile water for 4-5 times;

(5) Soaking the seeds in 75% ethanol solution at 25 ℃ for 90s, sterilizing the seeds, shaking the sterile conical flask for a plurality of times during the sterilization, taking out the sterilized conical flask, and flushing the sterilized conical flask with sterile water for 4 times;

(6) Soaking in 6% hydrogen peroxide solution at 25deg.C for 10min, shaking the conical flask for multiple times, taking out, and washing with sterile water for 4 times to obtain sterile Sinkiang Ferula seed;

(7) In an ultra clean bench, 200g of sterile water was used to remove 56cm ³ Soaking sterilized cotton, placing 25 sterilized Sinkiang Ferula asafetida seeds on the soaked sterilized cotton, culturing at 23deg.C under the conditions of air absolute humidity of 35% and illumination intensity of 4000-5000 Lux, illuminating for 14h each day, and observing and recording germination time of the seeds.

Example 2

The difference between this example and example 1 is that step (2) of this example is to put the seeds of Ferula sinkiangensis in a water bath at 35 ℃ for 2 hours to obtain the seeds of Ferula sinkiangensis after water bath, and the other steps are the same as example 1.

Example 3

The difference between this example and example 1 is that step (2) of this example is to put the seeds of Ferula sinkiangensis in a water bath at 40 ℃ for 2 hours to obtain the seeds of Ferula sinkiangensis after water bath, and the other steps are the same as example 1.

Example 4

The difference between this example and example 1 is that step (2) of this example is to put the seeds of Ferula sinkiangensis in a water bath at 45 ℃ for 2 hours to obtain the seeds of Ferula sinkiangensis after water bath, and the other steps are the same as example 1.

Example 5

The difference between this example and example 1 is that step (2) of this example is to put the seeds of Ferula sinkiangensis in a water bath at 40 ℃ for 1.5 hours to obtain the seeds of Ferula sinkiangensis after water bath, and the other steps are the same as example 1.

Example 6

The difference between this example and example 1 is that step (2) of this example is to put the seeds of Ferula sinkiangensis in a water bath at 40 ℃ for 2.5 hours to obtain the seeds of Ferula sinkiangensis after water bath, and the other steps are the same as example 1.

Example 7

(4) On an ultra-clean workbench, putting seeds into a 150mL sterile conical flask, and flushing with sterile water for 5 times;

(5) Soaking the seeds in 70% ethanol solution at 25 ℃ for 100s, sterilizing the seeds, shaking the sterile conical flask for a plurality of times during the sterilization, taking out the sterile conical flask, and flushing the sterile conical flask with sterile water for 4 times;

(6) Soaking in 7% hydrogen peroxide solution at 25deg.C for 8min, shaking the conical flask for multiple times, taking out, and washing with sterile water for 5 times to obtain sterile Sinkiang Ferula seed;

(7) In an ultra clean bench, 150g of sterile water was used to remove 50cm ³ Soaking sterilized cotton, placing 25 sterilized Sinkiang Ferula asafetida seeds on the soaked sterilized cotton, culturing under the conditions of 25 ℃ and air absolute humidity of 30% and illumination intensity of 4000-5000 Lux, wherein the illumination time of each day is 12 hours, and observing and recording the germination time of the seeds.

Example 8

(5) Soaking the seeds in 80% ethanol solution at 25 ℃ for 80s, sterilizing the seeds, shaking the sterile conical flask for a plurality of times during the sterilization, taking out the sterile conical flask, and flushing the sterile conical flask with sterile water for 5 times;

(6) Soaking in 5% hydrogen peroxide solution at 25deg.C for 12min, shaking the conical flask for multiple times, taking out, and washing with sterile water for 4 times to obtain sterile Sinkiang Ferula seed;

(7) In an ultra clean bench, 50cm of sterile water was used with 250g of water ³ Soaking sterilized cotton, placing 25 sterilized Sinkiang Ferula asafetida seeds on the soaked sterilized cotton, culturing under the conditions of 20 ℃ and air absolute humidity of 40% and illumination intensity of 4000-5000 Lux, wherein the illumination time of each day is 16h, and observing and recording the germination time of the seeds.

Comparative example 1

The comparative example uses a low temperature treatment method to treat the Sinkiang asafetida seeds, the comparative example is different from the example 3 in that in the step (7), 25 sterilized Sinkiang asafetida seeds are placed in 25mL of 1/2MS culture medium in an ultra clean workbench, cultured at 4 ℃ under the conditions that the humidity is 40% and the illumination intensity is 4000Lux, the illumination time of each day is 10 hours, the germination time of the seeds is observed and recorded, and the rest steps are the same as the example 3.

Comparative example 2

The comparative example uses the temperature alternation treatment method to treat the Sinkiang asafetida seeds, the comparative example is different from the example 3 in that in the step (7), 25 sterile Sinkiang asafetida seeds are placed in 25mL of 1/2MS culture medium in an ultra clean workbench, the first day is cultivated under the conditions of 4 ℃ and humidity of 40% and illumination intensity of 4000Lux, the illumination time period of each day is 10 hours, the second day is cultivated under the conditions of 25 ℃ and humidity of 40% and illumination intensity of 4000Lux, the illumination time period of each day is 10 hours, the cultivation modes of the first day and the second day (namely, cultivation is carried out in an alternating environment at 4 ℃ and 25 ℃ for 24 hours) are repeated, and the germination time of the seeds is recorded by observation, and the rest steps are the same as in the example 3.

Comparative example 3

The comparative example adopts a freezing treatment method to treat the ferula sinkiana seeds, namely the ferula sinkiana seeds are selected and placed in a refrigerator at the temperature of minus 20 ℃ to be frozen for 24 hours, then the seeds are directly inoculated in turfy soil after being thawed, and the germination condition of the seeds is observed at normal temperature.

Comparative example 4

The difference between this comparative example and example 3 is that in step (7), the sterilized seeds of Sinkiang Ferula asafetida were placed in 25mL of 1/2MS medium at 23℃under 30-40% humidity and 0-5000 Lux illumination intensity in an ultra clean bench, the illumination time per day was 14 hours, and the germination time of the seeds was observed and recorded, and the other steps were the same as in example 3.

Comparative example 5

The difference between this comparative example and comparative example 4 is that the sterile Sinkiang Ferula seeds were placed in 25mL 1/2MS medium of gibberellin at 0.2mg/L, and the rest of the procedure was the same as comparative example 4.

Comparative example 6

The difference between this comparative example and comparative example 5 is that gibberellin concentration of this comparative example is 0.5mg/L, and the remaining steps are the same as those of comparative example 5.

Comparative example 7

The difference between this comparative example and comparative example 5 is that gibberellin concentration of this comparative example is 1mg/L, and the remaining steps are the same as those of comparative example 5.

Comparative example 8

The difference between this comparative example and comparative example 5 is that gibberellin concentration of this comparative example is 2mg/L, and the remaining steps are the same as those of comparative example 5.

(1) The results of the statistically recorded time at which the seeds of examples 1 to 4 and comparative examples 1 to 8 began to germinate, and the seed germination rate on the 30 th day of cultivation are shown in table 1, wherein the seed germination rate= (number of germination/(number of seeds) ×100%.

TABLE 1 influence of different Ferula seed germination methods on seed germination time and germination rate

As shown in the results of Table 1, compared with the comparative example, the method of the application achieves the purpose of fast germination of the Sinkiang Ferula asafetida seeds, and the method does not need to add plant hormone and culture medium, has low cost and is ecological and environment-friendly, and the germination rate of the Sinkiang Ferula asafetida seeds treated by the water bath is higher than that of other treatment groups.

(3) All successfully germinated seeds of Sinkiang Ferula asafetida in examples 1 to 4 and comparative examples 1 to 8 were taken out of the culture medium or sterile cotton, transplanted into nutrient soil, respectively, 10mL of sterile water was sprayed every 2 days, recording was performed on day 60 and survival rate was calculated, survival rate= (number of Ferula asafetida seedlings surviving on day 60 +.total number of Ferula asafetida seedlings growing in nutrient soil) ×100%.

The results showed that the survival rates of the seeds of examples 1 to 4 were 95%, 92%, 97%, 91%, and the survival rates of comparative examples 1 to 8 were 80%, 89%, 83%, 85%, 70%, 88%, 80%, respectively, and it was found that the survival rates of the Sinkiang Ferula asafetida seeds after the water bath treatment of the present application were high compared to the other treatment groups.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims

1. The method for rapidly germinating the ferula seeds in Xinjiang is characterized by comprising the following steps of:

2. The method of claim 1, wherein the wetted sterile cotton is wetted with sterile water.

3. The method according to claim 1, wherein the temperature of the cultivation is 20-25 ℃, the absolute humidity of the air of the cultivation is 30-40%, the light intensity of the cultivation is 4000-5000 Lux, and the light duration of the cultivation is 12-16 h/day.

4. The method of claim 1, wherein the sanitizing agent comprises an ethanol solution having a concentration of 70% to 80% by volume and a hydrogen peroxide solution having a concentration of 5% to 7% by volume.

5. The method of claim 4, wherein the method of disinfecting comprises:

6. The method according to claim 5, wherein in the step (1) and the step (2), the number of times of flushing is 4 to 5, and the time of each flushing is 3 to 5 minutes.

7. The method according to claim 5, wherein the soaking temperature in step (1) and step (2) is 20 to 25 ℃.