阅读说明：本技术 一种小球藻提取物的制备方法及其应用 (Preparation method and application of chlorella extract ) 是由 孙诗清 赵永军 胡长伟 刘娟 徐微 李俊峰 曹卫星 孙辰 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种小球藻提取物的制备方法,包括：S1：小球藻的培养；S2：将上述培养的小球藻按照1:5比例接种到无磷BG11培养基进行活化培养,培养至OD650nm＝0.6,藻液进行不断扩培转接至15L；S3：将上述藻液放入到透析袋中,将透析袋里的藻液进行离心合并上层清液,然后使用等体积EA萃取,旋转蒸发仪蒸发浓缩EA提取液,直到变干,称得提取物的质量,最后用10mL甲醇溶出,-4℃保存待用,得到小球藻提取物溶液；S4：将上述步骤得到的小球藻提取物溶液配置成不同浓度待用。该小球藻提取物用于促进种子萌发的应用。本发明采用简单的提取步骤,提取方便,且提取物能够对种子萌发有很好的的促进作用。(The invention discloses a preparation method of a chlorella extract, which comprises the following steps: s1: culturing chlorella; s2: inoculating the cultured chlorella into a phosphorus-free BG11 culture medium at a ratio of 1:5, performing activation culture until OD650nm is 0.6, and continuously expanding and transferring the algae solution to 15L; s3: putting the algae liquid into a dialysis bag, centrifuging the algae liquid in the dialysis bag, combining supernatant, extracting with equal volume of EA, evaporating and concentrating the EA extract by a rotary evaporator until the extract becomes dry, weighing the extract, dissolving out with 10mL of methanol, and storing at-4 ℃ for later use to obtain chlorella extract solution; s4: preparing the chlorella extract solution obtained in the above steps into different concentrations for later use. The chlorella extract is used for promoting seed germination. The invention adopts simple extraction steps, is convenient to extract, and the extract can well promote the seed germination.)

1. A preparation method of chlorella extract is characterized in that: the method comprises the following steps in sequence:

s1: culturing chlorella: under the aseptic condition, inoculating chlorella into BG11 culture medium for activation culture for 48-72 h;

s2: inoculating the cultured chlorella into a phosphorus-free BG11 culture medium at a ratio of 1:5, performing activation culture until OD650nm is 0.6, and continuously expanding and transferring the algae solution to 15L;

s3: putting the algae liquid into a dialysis bag, centrifuging the algae liquid in the dialysis bag, combining supernatant, extracting with equal volume of EA, evaporating and concentrating the EA extract by a rotary evaporator until the extract becomes dry, weighing the extract, dissolving out with 10mL of methanol, and storing at-4 ℃ for later use to obtain chlorella extract solution;

s4: preparing the chlorella extract solution obtained in the above steps into different concentrations for later use.

2. The method for preparing a chlorella extract according to claim 1, wherein the method comprises the steps of: in step S1, the specific procedure of the culture is to inoculate 10mL of chlorella into BG11 medium at a ratio of 1:5 for activation culture until OD650nm becomes 0.6, and the algae liquid is transferred to 15L without any expansion culture.

3. The method for preparing a chlorella extract according to claim 1, wherein the method comprises the steps of: in steps S1 and S2, the culture conditions were both at a temperature of 25 ℃ and under illumination of 2000Lux for a period of 12Hr day/12 Hr night, and the flask was shaken 2 times a day.

4. The method for preparing a chlorella extract according to claim 1, wherein the method comprises the steps of: in step S2, the phosphorus-free BG11 medium is BG11 medium without adding a phosphorus source.

5. The method for preparing a chlorella extract according to claim 1, wherein the method comprises the steps of: in step S3, EA extraction is performed 3-5 times, each EA extract is collected, and all EA extracts are combined.

6. The method for preparing a chlorella extract according to claim 1, wherein the method comprises the steps of: in step S4, the chlorella extract is prepared at a concentration of 10^-5g/L、10^-7g/L、10^-11g/L and 10^-15g/L。

7. Use of a chlorella extract according to claim 1 for promoting seed germination.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a chlorella extract.

Background

Chlorella (Chlorella sp.) belongs to Chlorella, Chlorococcus, oocystidae, Chlorella, and is a common unicellular alga. The chlorella can adapt to different growth environments, is easy to artificially culture, and can not only utilize light energy for autotrophy, but also perform heterotrophy culture. Chlorella is rich in various useful components, grows in different environments, has a changed metabolic pathway, and therefore has different accumulated products. Chlorella is a good source of single-cell protein due to its wide ecological distribution, easy to culture and fast to grow. Is a good material for biotechnology research and has rich nutrition. The chlorella has important economic value and huge application and development potential.

The extraction process of the seaweed active substances mainly comprises three processes of physical extraction, chemical extraction and biological extraction. The most commonly used method at present is ultrasonic disruption extraction, which uses the operations of cavitation, crushing, stirring and the like of ultrasonic waves to destroy plant cells and permeate organic solvent into the plant cells so as to dissolve effective components in the plant into the organic solvent, on the other hand, degrades macromolecular active substances into effective small molecular substances which are soluble and easy to absorb, and then obtains the required components through separation and purification.

In the extraction process, the chlorella needs to be scientifically cultured, but the existing culture conditions of the chlorella are not suitable, so that the extraction purity is not high, the extraction means is complicated, and the extraction is troublesome.

Disclosure of Invention

The invention aims to provide a preparation method and application of chlorella extract to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of chlorella extract comprises the following steps in sequence:

s1: culturing chlorella: under the aseptic condition, inoculating chlorella into BG11 culture medium for activation culture for 48-72 h;

s2: inoculating the cultured chlorella into a phosphorus-free BG11 culture medium at a ratio of 1:5, performing activation culture until OD650nm is 0.6, and continuously expanding and transferring the algae solution to 15L;

s3: putting the algae liquid into a dialysis bag, centrifuging the algae liquid in the dialysis bag, combining supernatant, extracting with equal volume of EA, evaporating and concentrating the EA extract by a rotary evaporator until the extract is dried, weighing the extract, dissolving the extract by 10mL of methanol, and storing at-4 ℃ for later use to obtain a chlorella extract solution.

Preferably, in step S1, the cultivation is specifically performed by inoculating 10mL of chlorella into BG11 medium at a ratio of 1:5, performing activated culture until OD650nm is 0.6, and continuously expanding and transferring algae liquid to 15L.

In any of the above embodiments, it is preferable that the culture conditions are both at a temperature of 25 ℃ and under illumination of 2000Lux for a period of 12Hr day/12 Hr night, and the flask is shaken 2 times a day in steps S1 and S2.

In any of the above embodiments, in step S2, the phosphorus-free BG11 medium is BG11 medium without a phosphorus source.

In any of the above embodiments, it is preferable that the number of times of extraction with EA is 3 to 5 in step S3, each EA extract is collected, and all EA extracts are combined.

In any of the above embodiments, preferably, in step S4, the chlorella extract is prepared at a concentration of 10^{- 5}g/L、10^-7g/L、10^-11g/L and 10^-15g/L。

An application of Chlorella extract in promoting seed germination is provided.

The invention has the technical effects and advantages that: the preparation method of the chlorella extract is characterized in that before extraction, chlorella is cultured, simple extraction steps are adopted, extraction is convenient, the extract can well promote seed germination, the germination rate, the germination vigor and the germination index of seeds are improved, the germination period is ended in advance, the elongation of radicles in the seed germination process is promoted, and the seed germination condition is comprehensively improved.

Drawings

FIG. 1 shows the concentration of 10^-7Influence of different chlorella extracts in g/L on germination potential of arabidopsis seeds;

FIG. 2 shows the concentration of 10^-11Influence of different chlorella extracts in g/L on germination potential of arabidopsis seeds;

FIG. 3 shows the concentration of 10^-15Influence of different chlorella extracts in g/L on germination potential of arabidopsis seeds;

FIG. 4 shows a concentration of 10^-7Influence of different chlorella extracts on germination vigor of the green bristlegrass seeds in g/L;

FIG. 5 shows a concentration of 10^-11Influence of different chlorella extracts on germination vigor of the green bristlegrass seeds in g/L;

FIG. 6 shows the concentration of 10^-15Influence of different chlorella extracts on germination vigor of green bristlegrass seeds in g/L.

Detailed Description

The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1:

a preparation method of chlorella extract comprises the following steps in sequence:

s1: culturing chlorella: under the aseptic condition, inoculating chlorella into BG11 culture medium for activation culture for 48-72 h;

s2: inoculating the cultured chlorella into a phosphorus-free BG11 culture medium at a ratio of 1:5, performing activation culture until OD650nm is 0.6, and continuously expanding and transferring the algae solution to 15L;

s3: putting the algae liquid into a dialysis bag, centrifuging the algae liquid in the dialysis bag, combining supernatant, extracting with equal volume of EA, evaporating and concentrating the EA extract by a rotary evaporator until the extract is dried, weighing the extract, dissolving the extract by 10mL of methanol, and storing at-4 ℃ for later use to obtain a chlorella extract solution.

Specifically, in step S1, the culture method includes the specific steps of inoculating 10mL of chlorella into BG11 medium at a ratio of 1:5, performing activated culture until OD650nm is 0.6, and continuously expanding and transferring algae liquid to 15L.

Specifically, in steps S1 and S2, the culture conditions were both at a temperature of 25 ℃, with illumination of 2000Lux for a period of 12Hr day/12 Hr night, and the flask was shaken 2 times a day.

Specifically, in step S2, the phosphorus-free BG11 medium is BG11 medium without adding a phosphorus source.

Specifically, in step S3, EA extraction is performed 3 to 5 times, each EA extract is collected, and all EA extracts are combined.

Specifically, in step S4, the chlorella extract is prepared at a concentration of 10^-5g/L、10^-7g/L、10^{- 11}g/L and 10^-15g/L。

Respectively taking out the chlorella cultured in the absence of phosphorus on the 7 th day and the 15 th day, centrifuging the chlorella liquid in a dialysis bag, combining supernatant, breaking cells by using ultrasound to facilitate dissolution of intracellular substances, extracting the supernatant and the chlorella cells for 3 times by using equal volume of EA respectively, combining respective EA extract, evaporating and concentrating the EA extract by using a rotary evaporator until the extract becomes dry, weighing the mass of the extract, dissolving the extract by using 10mL of methanol, and storing the extract at-4 ℃ for later use; thus obtaining different chlorella extract solutions under phosphorus deficiency for later use.

Example 2: seed germination experiment of Arabidopsis thaliana

1. Preparation of sample solution

Preparing an extract solution:

weighing 1.1mg (estimated according to molecular weight 330.37 of 5-Deoxystrigol, content 30%) of the supernatant extract after 7 days of phosphorus-deficient culture, and adding into a 100mL volumetric flask (equivalent to 10%^-5g/L), then sucking 1mL, and fixing the volume to 100mL, which is equivalent to 10^-7g/L, at which time 1mL of the solution was taken out and the arrangement was continued 10^-11g/L of solution, 10 of the rest^-7The g/L extract solution was filtered with a sterile filter and then placed in a container (No. 21-1).

1mL of 10^-7The g/L solution is continuously diluted to constant volume (the method is the same as 10)^-7g/L, 2 times of constant volume is needed), 10 is obtained^-11g/L solution, at which time 1mL of solution was taken out and 10 was continuously prepared^-15g/L of solution, 10 of the rest^-11The g/L extract solution was filtered with a sterile filter and then placed in a container (No. 21-2).

1mL of 10^-11The g/L solution is continuously diluted to constant volume (the method is the same as 10)^-7g/L, 2 times of constant volume is needed), 10 is obtained^-15g/L solution, 10^-15g/L of the extract solution was filtered with a sterile filter and then placed in a container for use (No. 21-3).

The preparation method of the rest extract solution is the same as that of the supernatant extract after 7 days of phosphorus-deficient culture.

Preparation of control solution:

preparing GR24 solution: weighing extract 0.298mg (molecular weight 298.25) and adding into 100mL volumetric flask (equivalent to 10)^-5g/L), 3 concentrations (10) were prepared in the same manner^-7g/L、10^-11g/L、10^-15g/L) in the same manner as above.

Preparing a DMSO solution: weighing extract 0.078g (molecular weight 78.13) and adding into 100mL volumetric flask (equivalent to 10)^-3g/L), 3 concentrations (10) were prepared in the same manner^-7g/L、10^-11g/L、10^-15g/L) in the same manner as above.

2. Sterilization of instruments

And (3) sterilizing the used liquid and appliances:

preparation of sterile water: taking a proper amount of purified water, putting the purified water into a triangular flask, packing a cotton plug and newspaper, then putting the triangular flask into a sterilizing pot for sterilization, drying, cooling and using the triangular flask in an operation table.

Preparation of sterile petri dishes and filter paper: putting filter paper into culture dishes (2 pieces of filter paper in each dish), packaging the culture dishes with newspaper, sterilizing in a sterilizing pot, drying, cooling, and using in an operation table.

A needle head type filter: and (4) placing the needle head type filter into a sterilization pot for sterilization, drying, cooling and using in an operation table.

3. Preparation for use of super clean bench

Wiping a working table top with 75% alcohol 20min before use, placing articles required by an experiment on two sides of a working area in the middle of the table top, sterilizing a pipette suction head, a test tube, a culture dish and the like in advance, and starting an ultraviolet lamp to irradiate and sterilize the working area. When the operation is carried out, the illuminating lamp is turned on, the ultraviolet sterilizing lamp is turned off, hands are scrubbed by 75% alcohol, the alcohol lamp is ignited, the opened bottle mouth is exposed to flame, and the tweezers and the inoculating loop are burnt by the flame before being used.

4. Seed sterilization

1. Taking a certain amount of arabidopsis thaliana seeds, pouring the arabidopsis thaliana seeds into a 1.5mL centrifuge tube, adding sterile water, fully whirling and shaking for 1min, carrying out high-speed homeopathic centrifugation for 1min, and removing supernate and floating seeds. (operation in sterile Environment)

2. Adding 70% ethanol into a 1.5mL centrifuge tube (when the centrifuge tube is sterilized by 70% ethanol, the time is not suitable to be too long, the seed is easy to permeate and kill because the seed coat of the Arabidopsis thaliana seed is thin, 300uL of sterile water is added firstly, and 700uL of sterile anhydrous ethanol is added later) (operation in a sterile environment), carrying out vortex oscillation for 1min, carrying out high-speed homeopathic centrifugation for 15s, and carefully removing the supernatant by using a pipette (operation in a sterile environment).

3. Sterile water was added (operating in sterile environment), vortexed for 15s, high speed centrifugation for 15s, and the supernatant carefully removed with a pipette in sterile environment. This process was repeated three times.

4. Add 1% (v/v) sodium hypochlorite solution (working in sterile environment, ready for use) to the centrifuge tube, vortex on a vortex apparatus for 15min, and remove the supernatant in sterile environment.

5. Sterile water was added (operating in sterile environment), vortexed for 15s, high speed homeotropic centrifuged for 15s, and the supernatant carefully removed with a pipette in sterile environment. This process was repeated three times. After three washes, 500uL of sterile water was added and the seeds were poured onto sterile filter paper.

5. Culture and preparation thereof

WeighingThe culture medium is added into a 500mL conical flask with a constant volume of 250mL, the mixture is sterilized in a sterilization pot (121 ℃, 20min) after being prepared, the mixture is cooled to about 40-50 ℃, and the mixture is poured into a flat plate with the thickness of less than 5mm, poured and laid in a sterile table, and the whole flat plate is required to be laid. Waiting for it to cool and solidify for use.

After the medium solidified, the extracts were applied separately, 2 dishes of each extract were applied evenly, and the control group was applied with 2ml of sterile water, 2ml of DMSO solution, and 2ml of GR24 solution, 2 each.

6. Seed inoculation and culture

Placing the seeds in order after the seeds are slightly dry, and dropping a sterile culture medium by using an inoculating loop (Medium), 30 per dish, the dishes were sealed with a preservative film to prevent bacteria from entering. And (3) putting the sealed culture dish inoculated with the arabidopsis seeds into an incubator at 4 ℃ for vernalization for 2 days, putting the vernalized seed plate into a 22-23 ℃ culture room for illumination culture, and performing a seed germination experiment.

7. Seed germination counting

The counting time of the seeds is 12h, 24h, 36h, 48h and 60h (timing from the beginning of culture), and the germination is observed by the way that the radicle grows and the leucocyte grows. Counting was completed until the number of sprouts did not change significantly.

8. Calculating germination rate and germination potential

Are respectively used 10^-7g/L、10^-11g/L、10^-15The germination rates of the arabidopsis thaliana seeds treated by different chlorella extract solutions in g/L are shown in tables 1, 2 and 3 respectively,

table 1: 10^-7g/L solution treated Arabidopsis thaliana seed germination rate

Table 2: 10^-11g/L solution treated Arabidopsis thaliana seed germination rate

Table 3: 10^-7g/L solution treated Arabidopsis thaliana seed germination rate

The above tables 1, 2 and 3 show that the chlorella extract solution has a greatly improved germination rate for seeds.

With a concentration of 10^-7g/L、10^-11g/L、10^-15The germination potential of Arabidopsis seeds treated by the chlorella extract solution in g/L is shown in figure 1, figure 2 and figure 3 respectively, and the chlorella extract can improve the germination potential of Arabidopsis seeds.

The germination rate and germination vigor are improved, so that the chlorella extract can improve the germination of seeds.

Example 3: sedum viridis seed germination experiment

1. Preparation in early stage of experiment

Taking a proper amount of purified water, putting into a 1000mL triangular flask, packaging with a cotton plug and kraft paper, putting into a sterilizing pot, sterilizing at 121 ℃ for 20 minutes, taking out after the sterilization is finished, and cooling.

Putting the three pieces of filter paper into culture dishes with uniform specifications respectively, packaging the culture dishes by kraft paper, putting the culture dishes into a sterilization pot, sterilizing at 121 ℃ for 20 minutes, taking out the culture dishes after the sterilization is finished, putting the culture dishes into a blast drying oven, and drying the culture dishes.

Packaging small beaker and volumetric flask with kraft paper, packaging conical flask with cotton plug and kraft paper, placing into a sterilizing pot, sterilizing at 121 deg.C for 20min, taking out, placing into a forced air drying oven, and drying glassware.

Before the super clean bench is used, the bench surface is wiped with alcohol cotton balls, the equipment related to the experiment is placed in the super clean bench, the ultraviolet lamp is turned on for sterilization, and the irradiation is generally carried out for 30 min. Before the ultraviolet sterilization is finished, the fan is turned on, the ultraviolet lamp is turned off, the illuminating lamp is turned on, the latex gloves are worn, the glass baffle plate is opened to the smallest possible gap, the hand enters the super-clean workbench, the alcohol lamp is ignited,

the hands were wiped with an alcohol cotton swab and the experiment was started after evaporation of the alcohol was complete.

2. Dilution of Chlorella extract

Dilution of the experimental group extracts: weighing Chlorella extract 1.1mg (estimated according to molecular weight of 5-Deoxystrigol 330.37, content of 30%), dissolving with DMSO, and adding sterile water to 100mL volumetric flask (equivalent to 10 mL)^-5g/L), then 1mL of 10 was aspirated^-5g/L solution, constant volume of 100mL, corresponding to 10^-7g/L of solution, at which time 1mL10 was removed^-7g/L solution is continuously prepared 10^-11g/L of solution, 10 will remain^-7g/L of the extract solution is placed in a container and marked for later use. 1mL of 10^-7The g/L solution is continuously diluted to constant volume (the method is the same as 10)^-7g/L dilution method, requireConstant volume 2 times) to obtain 10^-11g/L of solution, at which time 1mL of solution was taken out and continuously disposed 10^-15g/L of solution, 10 will remain^-11g/L of the extract solution is placed in a container and marked for later use. 1mL of 10^-11The g/L solution is continuously diluted to a constant volume (the constant volume is required to be 2 times in the same way as the 10-7g/L dilution method), and 10 is obtained^-15g/L solution of 10^-15g/L of the extract solution is placed in a container and marked for later use.

The chlorella extract can be diluted by the above method to obtain supernatant and algae cell extract of 7 days phosphorus-deficient culture, and 10 days of supernatant and algae cell extract of 15 days phosphorus-deficient culture^-7g/L、10^-11g/L、10^-15g/L of 3-concentration solution.

Dilution of control solution: taking GR24 solution as an example, weighing GR240.298mg (molecular weight 298.25) and using

DMSO was dissolved in an organic solvent, and the dissolved GR24 solution was made to a volume of 100mL (equivalent to 10) in a volumetric flask with sterile water^-5g/L), then 1mL of 10 was aspirated^-5g/L solution, constant volume to 100mL volumetric flask, corresponding to 10^-7The solution of the compound is added into the solution in g/L,

at this point, 1mL of the continuous arrangement 10 was taken out^-11g/L of solution, 10 of the rest^-7g/L GR24 solution was placed in a container and labeled for use.

The following dilution step of the solution and the dilution of the extracts of the experimental group 10^-11g/L、10^-15The 2 concentration methods of g/L are consistent to obtain GR24 solution 10^-7g/L、10^-11g/L、10^-15g/L of 3-concentration solution.

Preparing a DMSO solution: weighing extract 0.078g (molecular weight 78.13), and adding sterile water to a volume of 100mL and 8 measuring bottles (equivalent to 10)^-3g/L), then 1mL of 10 was aspirated^-3g/L solution, constant volume to 100mL volumetric flask, corresponding to 10^-5g/L of solution, another 1mL of 10^-5g/L solution, constant volume to 100mL volumetric flask, corresponding to 10^-7g/L DMSO solution is placed in a container and is used after marking.

The following dilution step of the solution and the dilution of the extracts of the experimental group 10^-11g/L、10^-15g/L ofThe concentration methods are consistent, and a DMSO solution 10 is obtained^-7g/L、10^-11g/L、10^-15g/L of 3-concentration solution.

15 days Normal culture supernatant and algae cell extract solution dilution test procedure and test group extract 10^{- 7}g/L、10^-11g/L、10^-15The 3 concentration methods of g/L are consistent, and 10 days of supernatant extract and algae cell extract of 15 days normal culture are obtained^-7g/L、10^-11g/L、10^-15g/L of 3-concentration solution.

3. Sterilization of solutions

After the dilution is finished, one bottle of the solution is opened, 10mL of the solution to be filtered is sucked, a 5mL syringe is used in the experiment, a sterile filter is replaced, the solution is poured into a sterilized small beaker for filtration, the steps are repeated for three times, and the solution is finally injected into a sterilized 15mL centrifuge tube and marked in order to ensure that the solution is sterile.

Other solutions to be sterilized were filter sterilized as described above.

4. Treatment of Setaria viridis seeds

Taking proper green bristlegrass seeds, pouring the proper green bristlegrass seeds into a 5mL centrifuge tube, adding sterile water (operating in a sterile environment), fully whirling and shaking for 1min, centrifuging at a high speed for 1min, removing supernatant and floating seeds (operating in a sterile environment), removing unsaturated seeds, and leaving the unsaturated seeds for germination experiments.

Adding 70% ethanol into the centrifuge tube (operating in sterile environment, adding 300uL sterile water first, then adding 700uL sterile anhydrous ethanol), vortexing and shaking for 1min, high-speed homokinetic centrifuging for 15s, and carefully removing the supernatant with a pipette (operating in sterile environment). This process is repeated once.

Washing with sterile water (operating in sterile environment), vortexing for 15s, high-speed centrifuging for 15s, and spraying with liquid-transfering gun

The supernatant was carefully removed (working in a sterile environment). This process was repeated twice.

Adding 1% (v/v) sodium hypochlorite solution into a centrifuge tube (operating in a sterile environment, when the solution is used at present, adding 990uL sterile water, then adding 10uL sterile sodium hypochlorite solution), performing vortex on a vortex instrument for 10min, and removing supernatant (operating in a sterile environment).

Sterile water was added (working in sterile environment), vortexed for 15s, high speed homeotropic centrifuged for 15s, and the supernatant carefully removed with a pipette (working in sterile environment). This process was repeated three times.

After being washed by sterile water, 500uL of sterile water is added into a centrifuge tube, the green bristlegrass seeds are poured on sterile filter paper, the green bristlegrass seeds are placed neatly after the water is slightly dried, and the three layers of sterile filter paper are placed into a sterile culture dish.

5. Inoculation of Setaria viridis seeds

Firstly, filter paper is soaked by 3ml of solutions with different concentrations (experimental components are respectively supernatant and algae cell extract of 7-day phosphorus-deficient culture, supernatant and algae cell extract of 15-day phosphorus-deficient culture, and 4 types of solutions, wherein each group is 2, and control components are respectively sterile water, DMSO solution, GR24 solution, supernatant of 15-day normal culture and 5 types of solutions of algae cell extract), then 30 seeds subjected to surface disinfection are uniformly paved on the filter paper, the filter paper is covered with a culture dish cover, and the culture dish cover is packaged by a preservative film to prevent the seeds from being affected by too fast water evaporation.

After inoculation is finished, putting culture dishes with uniform specifications into a constant-temperature incubator at 22 ℃ for germination acceleration, and supplementing sterile water in time during germination so as to ensure that water required by seed germination cannot exceed 3 mL.

6. Observation and recording of Setaria viridis seeds

After inoculation is finished, the germination condition of the green bristlegrass seeds is regularly observed every day, the number of the seeds normally germinated in each culture dish is counted, the germination number of the seeds is recorded, and the standard of judging whether the seeds germinate or not is adopted with the exposed white of 2mm as a standard.

7. Calculating germination rate and germination potential

Are respectively used 10^-7g/L、10^-11g/L、10^-15The germination rates of the arabidopsis thaliana seeds treated by different chlorella extract solutions in g/L are shown in tables 4, 5 and 6 respectively,

table 4: at a concentration of 10^-7Influence of chlorella extract in g/L on germination rate of Setaria viridis seeds

Table 5: at a concentration of 10^-11Influence of chlorella extract in g/L on germination rate of Setaria viridis seeds

Table 6: at a concentration of 10^-15Influence of chlorella extract in g/L on germination rate of Setaria viridis seeds

The above tables 4, 5 and 6 show that the chlorella extract solution has a greatly improved germination rate for seeds.

With a concentration of 10^-7g/L、10^-11g/L、10^-15The germination potential of Setaria viridis seeds treated with the chlorella extract solution in g/L is shown in fig. 4, fig. 5 and fig. 6, respectively, and the chlorella extract can improve the germination potential of Setaria viridis seeds.

The germination rate and germination vigor are improved, so that the chlorella extract can improve the germination of seeds.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.