CN114946317A

CN114946317A - Method for promoting germination of switchgrass seeds

Info

Publication number: CN114946317A
Application number: CN202210662279.4A
Authority: CN
Inventors: 胡国富; 王琪; 李凤兰; 张攀; 秦立刚; 郭悦; 鲁亚林; 杨劼; 朱思齐; 华毅
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-08-30
Anticipated expiration: 2042-06-13
Also published as: CN114946317B

Abstract

The invention discloses a method for promoting germination of switchgrass seeds, which belongs to the technical field of seed germination and comprises the following steps of (1) seed selection: selecting seeds with uniform color and size; (2) seed disinfection: washing the seeds with distilled water for 3-5 times, soaking in 75% ethanol for 120s, and washing with distilled water for 5-8 times; (3) seed drying: drying the seeds in a drying box at 30 ℃ for 3 h; (4) infection of bacterial liquid: dip-dyeing the seeds with EM bacterial liquid with the concentration of 0.5-10 percent; (5) and (5) drying again: putting the dip-dyed seeds into a drying box at 30 ℃ again for drying for 4 hours; (6) culturing and germinating: the temperature in the illumination incubator is set to be 25-26 ℃, illumination is carried out for 12h, and germination is carried out in dark for 12h, so that the problems of irregular seedling emergence, low germination vigor and long germination time of switchgrass are solved.

Description

Method for promoting germination of switchgrass seeds

Technical Field

The invention relates to the technical field of seed germination, in particular to a method for promoting germination of switchgrass seeds.

Background

Switchgrass (Panicum virgatum L.) is a perennial C4 herb, belonging to the family Gramineae (Panicum) and Panicum (Panicum). Switchgrass and starfish are typical grasses of the grass family. Can be used as forage grass, water and soil conservation and wind barrier plants, and is also a raw material for producing biofuel and alternative energy.

Switchgrass seeds belong to deep dormant seeds, the germination rate of aged seeds in 1-2 years is only about 5 percent, the germination rate can be increased after five or six years, but the germination vigor is obviously reduced. At present, the dormancy method of switchgrass is commonly carried out by freezing stratification treatment, seed coat scratching, soaking and chemical agent treatment for breaking the dormancy of seeds, wherein the freezing stratification treatment has the best effect, but if the seeds are sowed in time after freezing stratification, the drying of the seeds can cause the phenomenon of seed dormancy recovery. The phenomenon of dormancy reversion can result in seeds being in a wet state, sticky together and unable to be sown mechanically, and secondly the wet seeds are neither storable nor difficult to package for seed makers. In addition, even if the seeds are wet by hand sowing, the seeds may undergo a re-drying process under the drought of soil or exposure to strong summer sun, thereby causing an undesirable phenomenon of dormancy restoration. The freezing stratification process is required to be performed for a plurality of times if the dormancy restoration phenomenon of the seeds is to be overcome. However, this method requires high environmental temperature and requires a long time, and is difficult to implement in actual production. Meanwhile, the seedling emergence of the switchgrass is irregular, the germination vigor is low, and the further difficulty in sowing the switchgrass is generated, so that the germination time required by the switchgrass seeds is shortened on the premise of ensuring the germination rate of the switchgrass seeds, the germination vigor is kept higher, and the uniformity of seedling emergence is a difficult problem faced by the switchgrass in the production at present.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for promoting germination of switchgrass seeds, which solves the problems of irregular seedling emergence, low germination vigor and long germination time of switchgrass.

The invention solves the technical problems by the following technical means:

a method of promoting germination of switchgrass seeds, the method comprising the steps of:

(1) seed selection: selecting seeds with uniform color and size;

(2) seed disinfection: washing the seeds with distilled water for 3-5 times, soaking in 75% ethanol for 120s, and washing with distilled water for 5-8 times;

(3) seed drying: drying the seeds in a drying box at 30 ℃ for 3 h;

(4) infection of bacterial liquid: dip-dyeing the seeds with EM bacterial liquid with the concentration of 0.5-10 percent;

(5) and (5) drying again: putting the dip-dyed seeds into a drying box at 30 ℃ again for drying for 4 hours;

(6) culturing and sprouting: setting the temperature in the illumination incubator to be 25-26 ℃, carrying out illumination for 12h, carrying out culture germination in darkness for 12h, and directly sowing seeds after culture and germination on the soil for carrying out switchgrass planting.

Further, the invention also carries out a seed inhibition and excitation step after the step of drying again, and the steps are specifically operated as follows: culturing the dried seeds in an incubator at 25 ℃ for 2 days, spraying an inhibition excitant on the surfaces of the seeds, heating to 28 ℃ for culturing for 1-2 days, cleaning the surfaces with clear water, drying, and transferring into an illumination incubator for culturing and sprouting.

Further, the inhibition excitant comprises the following raw materials: alanine, sodium chloride, magnesium chloride, vitamin B2 and polyvinyl alcohol.

Further, the mass ratio of the alanine to the sodium chloride to the magnesium chloride to the vitamin B2 is (3-5): 0.2: 0.1: (1-2).

Further, the mass ratio of the alanine to the sodium chloride to the magnesium chloride to the vitamin B2 is 3: 0.2: 0.1: 1.

further, the viscosity of the polyvinyl alcohol is 4.8 to 5.8 mPas.

Further, the mass ratio of the polyvinyl alcohol to the alanine is (10-12): 1.

the method is characterized in that during the conventional culture process, the panicum virgatum seeds begin to emerge in 1-2 days, radicles begin to develop, but some seeds can delay development due to the existence of a deep dormancy stage, so that the panicum virgatum starts to emerge in 4-5 days, the germination vigor is low, the germination is irregular, the seeds can be subjected to inhibition excitation treatment for improving the germination vigor of the panicum virgatum, the roots of the panicum virgatum just exposed are inhibited, the seeds which are not yet exposed to the white are waited for slow development, all the seeds can be in the same growth stage through the inhibition excitation step, the emergence speed is consistent after sowing, and the planting culture of the panicum virgatum is more facilitated.

After the seeds are impregnated by EM bacterial liquid, the seed coats swell and soften, various enzymes in the seeds start to be activated, respiration and metabolism are rapidly enhanced, the embryonic roots of the seeds start to develop, at the moment, the exciting agent is inhibited from being sprayed on the broken part of the seed coats, and due to the synergistic effect of high-concentration sodium and magnesium ions, alanine and vitamin B2, the internal active enzymes required by the seeds such as protease, lipase and the like with high activity are jointly inhibited and passivated, the development speed of the embryonic roots is promoted to be reduced, and the activation of the seeds without exposing white in the seed coats is jointly waited. After waiting for several days, cleaning the inhibition excitant with clear water, entering a culture germination stage, and then immediately breaking the mouth of the post-developed seeds to germinate, so that the germination vigor of the seeds of the switchgrass is higher, and the uniformity of the seedling emergence is highly consistent.

Further, the preparation method of the inhibition excitant comprises the following steps: mixing polyvinyl alcohol with water, heating to 60 deg.C, stirring to dissolve completely, cooling to room temperature, adding sodium chloride, magnesium chloride and alanine, stirring to dissolve, adding vitamin B2, and ultrasonic stirring for 10-15min to obtain the final product.

Furthermore, the seed germination method is also suitable for the starwort.

Has the advantages that:

the method for promoting seed germination provided by the invention effectively breaks the dormant state of switchgrass seeds, obviously shortens the seed germination time, has high seed germination uniformity and is suitable for large-batch seedling culture; meanwhile, the method is simple, low in cost and easy to operate, treated seedlings grow normally and are vigorous, and the breeding period can be shortened, so that the breeding process is accelerated, and the method is suitable for popularization and application in breeding practice.

Drawings

FIG. 1: and (4) growing graphs after germination and sowing of switchgrass seeds.

Detailed Description

The invention will be described in detail below with reference to specific examples and figure 1:

example 1: germination of switchgrass seeds

(1) Seed selection: selecting mature switchgrass seeds with uniform color and size;

(2) seed disinfection: washing the seeds with distilled water for 5 times, soaking the seeds in 75% alcohol for 120s, and then washing the seeds with distilled water for 8 times;

(3) seed drying: drying the disinfected seeds in a drying box at 30 ℃ for 3h until the surfaces of the seeds are completely dried;

(4) bacterial liquid infection: in an aseptic environment, dip-dyeing seeds by using an EM bacterial liquid stock solution with the concentration of 5%, wherein the dip-dyeing dosage is based on the dosage of soaking but not completely submerging the seeds;

(5) and (5) drying again: putting the dyed seeds into a drying box at 30 ℃ again for drying for 4 hours until the surfaces of the seeds are completely dried;

(6) culturing and germinating: the temperature in the illumination incubator is set to 25 ℃, illumination is carried out for 12 hours, and germination is cultured in the dark for 12 hours.

Example 2: germination of switchgrass seeds

In the present example, germination tests were performed on switchgrass seeds by dip-dyeing supernatant, sterilized solution and stock solution of different concentrations of EM bacterial solution based on example 2.

The seeds were aliquoted into 14 aliquots of 200 seeds in a sterile environment and placed in a petri dish in a uniform and aligned arrangement. Taking out 1 part of seeds and taking the seeds as a blank control group without any treatment, infecting the rest seeds with supernatant, sterilized solution and stock solution of EM bacterial solution with different concentrations respectively, wherein the concentration of each bacterial solution is 100%, 10%, 5%, 0.5% and 0%, the dosage of the infecting solution is based on the dosage of soaking the seeds without completely submerging the seeds, and repeating the test for 3 times.

The germination date of the first seed is taken as the initial germination date, the 'white exposure' of the seed is taken as the standard for counting the germination, the germination number of the seed is counted day by day, the test is carried out for 7 days, and the data obtained after 7 days are shown in table 1.

Preparing a supernatant fluid: and (3) taking a certain amount of EM stock solution by using a pipette gun, adding the EM stock solution into a centrifugal tube, setting the rotating speed to 3000r/s, and operating the equipment for 15 min. Then the upper part 2/3 of the solution is taken, namely the stock solution of the supernatant;

preparing a sterilizing solution: adding a certain amount of EM stock solution into a centrifuge tube by using a liquid transfer gun, and placing the centrifuge tube into a sterilization pot for sterilization at 120 ℃ for 90min to obtain a sterilization solution stock solution;

and measuring each bacteria liquid by using a liquid transfer gun, diluting the bacteria liquid to a required concentration to obtain the final required staining liquid, paying attention to the fact that the preparation of the EM bacteria liquid needs to be carried out in an aseptic environment, wearing the disinfection experimental clothes in the whole operation process, and wearing a mask and gloves.

TABLE 1

Note: the different lower case letters in the same column in the table indicate the significance of the difference at the 5% level.

As can be seen from the data in Table 1, the germination potential and germination percentage of EM bacterial liquid with concentration of 0.5-10% are both greater than 60%, and the germination potential of the EM bacterial liquid with concentration of 100% is lower, so that the germination of the switchgrass seeds is inhibited by the EM bacterial liquid with excessively high concentration. The immersion liquid adopts the supernatant, the sterilization liquid and the stock solution of the EM bacterial liquid to have smaller influence difference on the germination vigor and the germination rate of the seeds, the immersion liquid adopts the supernatant or the sterilization liquid or the stock solution to have smaller influence on the switchgrass seeds in the EM bacterial liquid with the concentration of 0.5 percent, 5 percent and 10 percent, and the growth vigor of the seedlings after the planting is also obviously higher than that of the seeds in a control group or the concentration of 100 percent.

Example 3: switchgrass seed germination II

3g of alanine, 0.2g of sodium chloride, 0.1g of magnesium chloride, 1g of vitamin B2 and 30g of polyvinyl alcohol having a viscosity number of 4.8 to 5.8 mPas are weighed out.

Firstly, mixing polyvinyl alcohol with water, wherein the mass ratio of the polyvinyl alcohol to the water is 1: 10, stirring and heating to 60 ℃, cooling to room temperature after complete dissolution, adding sodium chloride, magnesium chloride and alanine, stirring to dissolve, then adding vitamin B2, and stirring for 15min by using an ultrasonic stirrer with the frequency of 28KHz to obtain the excitation inhibitor.

(2) seed disinfection: washing the seeds with distilled water for 5 times, soaking with 75% ethanol for 120s, and washing with distilled water for 8 times;

(4) infection of bacterial liquid: in an aseptic environment, dip-dyeing seeds by using EM (effective microorganisms) bacterial liquid with concentration, wherein the dip-dyeing dosage is based on the dosage of soaking but not completely submerging the seeds;

(5) and (5) drying again: putting the dip-dyed seeds into a drying box at 30 ℃ again for drying for 4 hours until the surfaces of the seeds are completely dried;

(6) seed inhibition stimulation: culturing the dried seeds in an incubator at 25 ℃ for 2 days, spraying an inhibition excitant on the surfaces of the seeds, soaking the surfaces of the seeds, heating to 28 ℃ for culturing for 2 days, cleaning the surfaces with clear water, drying, and transferring into an illumination incubator for culturing and sprouting;

(7) culturing and germinating: the temperature in the illumination incubator is set to 25 ℃, illumination is carried out for 12 hours, and germination is cultured in the dark for 12 hours.

Example 4:

on the basis of example 3, this example prepares a comparative example of the inhibition excitant, and the formulation of the test raw materials is shown in table 2:

TABLE 2

The reagents of the comparative example were prepared as in example 3.

The example 3 and the comparative examples 1 to 9 were compared with each other in terms of cultivation and germination using 100 seeds of the same conditions, and the germination vigor was counted after 3 days of cultivation in the germination cultivation stage after the seed inhibition excitation, the germination rate was counted after 7 days, and the average was taken after repeating 3 times.

The germination method of switchgrass of example 1 was used as a control, and the germination potential was calculated on the third day of the germination period and the germination rate was calculated on the 11 days of the germination period.

The data obtained are shown in table 3:

TABLE 3

	Germination vigor (%)	Percentage of germination (%)		Germination vigor (%)	Percentage of germination (%)
						Example 3	90	91.3	Comparative example 6	52	69
Comparative example 1	29	35	Comparative example 7	42	55.3
						Comparative example 2	37	42.3	Comparative example 8	35.7	43
Comparative example 3	40.3	47	Comparative example 9	27	34
						Comparative example 4	42	53	Example 1 (control)	72	86.3
Comparative example 5	46.6	63	-	-	-

From the data in table 3, it can be seen that the germination vigor of example 3 reaches 90%, the germination rate reaches 91.3%, and compared with example 1, the germination vigor is improved by 18%, and the overall germination rate is improved by 5%, which indicates that the germination vigor of switchgrass can be obviously improved through the excitation inhibition treatment step, and the emergence is more orderly.

Example 3 compares with comparative examples 1-5 to know that the germination potential of example 3 is significantly higher than that of comparative examples 1-5, sodium magnesium ions are essential elements in the germination process of seeds, but the required amount in the germination process is in a dynamic process, the radicle development stage can increase the osmotic potential, but when the radicle develops after exposure to white, the required amount of sodium magnesium ions is reduced, so that the growth of the radicle can be inhibited by increasing the content of sodium magnesium ions just at the development stage of the radicle, but because the germination process of the seeds can be adjusted automatically and is adaptive to the environment, the pure high content of sodium magnesium ions is not enough to completely inhibit the growth of the radicle, and the addition of alanine and vitamin B2 inhibits the development of the radicle together.

The viscosity value of the polyvinyl alcohol selected in the comparative example 9 reaches 21-33 mPa.s, the growth potential and the germination rate are both low, the whole inhibition excitant is in a viscous state by adding the polyvinyl alcohol, the polyvinyl alcohol has good slow release capacity of active ingredients and a water retention effect, and the death caused by insufficient water content of seeds after the inhibition excitant is sprayed can be prevented. The viscosity of the polyvinyl alcohol is only selected within 4.8-5.8 mPa.s, and too high viscosity not only can lead to difficult release of effective components, but also can lead to isolation of oxygen or carbon dioxide after film forming, and the respiration is completely stopped, thus easily leading to seed death.

Comparative example 6 although the raw materials are the same as those of example 3, the mixture ratio is different, and the development of different ions or substances during the seed development process is directly or indirectly influenced, so the amounts of sodium and magnesium ions, alanine and vitamin B2 need to be strictly controlled, and the radicle is not continued to grow after being inhibited, and the seed germination is failed.

Comparative example 7 is that alanine is replaced by lysine, and comparative example 8 is that polyvinyl alcohol is replaced by Arabic gum, so that the germination vigor and the germination rate are reduced, and the alanine, sodium chloride, magnesium chloride and vitamin B2 of the invention are used for cooperatively inhibiting and exciting switchgrass seeds, so that the growth vigor is more consistent, and the germination rate is improved.

FIG. 1 shows switchgrass obtained after further cultivation for 30 days after sowing seeds of the present example, comparative example 1, comparative example 2, example 3, example 1, and comparative example 9, from left to right.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.

Claims

1. A method for promoting germination of switchgrass seeds, comprising the steps of:

(1) seed selection: selecting seeds with uniform color and size;

(2) seed disinfection: washing seeds with distilled water, soaking in 75% ethanol for 120s, and washing with distilled water;

(3) seed drying: drying the seeds in a drying box at 30 ℃ for 3 h;

(6) culturing and germinating: setting the temperature in the illumination incubator to be 25-26 ℃, illuminating for 12h, and culturing and germinating in the dark for 12 h.

2. The method of promoting the germination of switchgrass seeds of claim 1, wherein said redrying step is followed by a seed suppression stimulation step, comprising the steps of: culturing the dried seeds in an incubator at 25 ℃ for 2 days, spraying an inhibition excitant on the surfaces of the seeds, heating to 28 ℃ for culturing for 1-2 days, cleaning the surfaces with clear water, drying, and transferring into an illumination incubator for culturing and sprouting.

3. The method of promoting the germination of switchgrass seeds of claim 2, wherein said inhibition elicitor comprises the following raw materials: alanine, sodium chloride, magnesium chloride, vitamin B2 and polyvinyl alcohol.

4. The method for promoting germination of switchgrass seeds of claim 3, wherein said alanine, sodium chloride, magnesium chloride, vitamin B2 are present in a mass ratio of (3-5): 0.2: 0.1: (1-2).

5. The method of promoting germination of switchgrass seeds of claim 4, wherein said alanine, sodium chloride, magnesium chloride, vitamin B2 are present in a mass ratio of 3: 0.2: 0.1: 1.

6. the method of promoting germination of switchgrass seeds of claim 5, wherein said polyvinyl alcohol has a viscosity of 4.8 to 5.8 mPa-s.

7. The method of promoting germination of switchgrass seeds of claim 6, wherein said polyvinyl alcohol to alanine mass ratio is (10-12): 1.

8. the method of promoting the germination of switchgrass seeds of claim 7, wherein said inhibition stimulant is prepared by: mixing polyvinyl alcohol with water, heating to 60 deg.C, stirring to dissolve completely, cooling to room temperature, adding sodium chloride, magnesium chloride and alanine, stirring to dissolve, adding vitamin B2, and ultrasonic stirring for 10-15min to obtain the final product.

9. The method of promoting germination of switchgrass seeds of claim 8, wherein said method of seed germination is also applicable to starwort.