CN117887777A - Pollen vitality improver containing coenzyme Q - Google Patents
Pollen vitality improver containing coenzyme Q Download PDFInfo
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- CN117887777A CN117887777A CN202410058711.8A CN202410058711A CN117887777A CN 117887777 A CN117887777 A CN 117887777A CN 202410058711 A CN202410058711 A CN 202410058711A CN 117887777 A CN117887777 A CN 117887777A
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- 235000017471 coenzyme Q10 Nutrition 0.000 title claims abstract description 29
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 title claims abstract description 29
- 230000035899 viability Effects 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims description 17
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 claims description 15
- 229940110767 coenzyme Q10 Drugs 0.000 claims description 15
- ICFIZJQGJAJRSU-SGHXUWJISA-N ubiquinone-8 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ICFIZJQGJAJRSU-SGHXUWJISA-N 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000035784 germination Effects 0.000 abstract description 25
- 230000010152 pollination Effects 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 235000013399 edible fruits Nutrition 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 abstract description 2
- 230000026267 regulation of growth Effects 0.000 abstract description 2
- 230000001850 reproductive effect Effects 0.000 abstract description 2
- 239000011782 vitamin Substances 0.000 abstract description 2
- 229940088594 vitamin Drugs 0.000 abstract description 2
- 229930003231 vitamin Natural products 0.000 abstract description 2
- 235000013343 vitamin Nutrition 0.000 abstract description 2
- 150000003722 vitamin derivatives Chemical class 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 9
- 239000012869 germination medium Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 241000235058 Komagataella pastoris Species 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 235000009025 Carya illinoensis Nutrition 0.000 description 3
- 241001453450 Carya illinoinensis Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
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- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000007198 pollen germination Effects 0.000 description 3
- 239000012804 pollen sample Substances 0.000 description 3
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- 241000196324 Embryophyta Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
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- 239000002245 particle Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 2
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- UUGXJSBPSRROMU-UHFFFAOYSA-N 2,3-dimethoxy-5-methyl-2-<(all-E)-3',7',11',15',19',23',27',31',35'-nonamethylhexatriaconta-2',6',10',14',18',22',26',30',34',nonaenyl>cyclohexa-2,5-dien-1,4-dion Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O UUGXJSBPSRROMU-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000004129 EU approved improving agent Substances 0.000 description 1
- 101000860835 Homo sapiens Ubiquinone biosynthesis protein COQ9, mitochondrial Proteins 0.000 description 1
- 241000758791 Juglandaceae Species 0.000 description 1
- 241000521553 Pichia fermentans Species 0.000 description 1
- 102100028230 Ubiquinone biosynthesis protein COQ9, mitochondrial Human genes 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical class [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 239000006185 dispersion Substances 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 230000005094 fruit set Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 229910052900 illite Inorganic materials 0.000 description 1
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- 239000008297 liquid dosage form Substances 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- VGIBGUSAECPPNB-UHFFFAOYSA-L nonaaluminum;magnesium;tripotassium;1,3-dioxido-2,4,5-trioxa-1,3-disilabicyclo[1.1.1]pentane;iron(2+);oxygen(2-);fluoride;hydroxide Chemical compound [OH-].[O-2].[O-2].[O-2].[O-2].[O-2].[F-].[Mg+2].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[Al+3].[K+].[K+].[K+].[Fe+2].O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2.O1[Si]2([O-])O[Si]1([O-])O2 VGIBGUSAECPPNB-UHFFFAOYSA-L 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
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- 108700011257 plant PGP Proteins 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
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- UUGXJSBPSRROMU-WJNLUYJISA-N ubiquinone-9 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O UUGXJSBPSRROMU-WJNLUYJISA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to plant reproductive growth regulation, in particular to a composition for improving pollen activity. The invention provides a coenzyme Q-containing pollen viability improver which effectively improves the germination capacity of fresh and stored apocarya pollen. The coenzyme Q provided by the invention is an important vitamin substance required by human body, is harmless to human body and environment-friendly, can be used as a novel pollution-free biological improver, and is used for artificial supplementary pollination of fruit trees.
Description
Technical Field
The invention relates to plant reproductive growth regulation, in particular to a composition for improving pollen activity.
Background
In the fruit tree planting process, the influence of pollination failure and flower and fruit dropping on the economic benefit of fruit tree planting is very remarkable, and especially in the planting of important economic value tree species, the problem is more remarkable. Taking the apocarya as an example, the tree species with the most economic value in the genus apocarya of the family Juglandaceae has obvious cultivation economic benefit. However, the yield of apocarya is affected by a number of factors, of which pollination and fruit set rate are key. At present, the yield is improved mainly through the use of chemical agents and pesticides, and although the method controls diseases to a certain extent, hybridization pollination is affected, so that pollination efficiency is reduced, meanwhile, the fruit quality is reduced, and the potential safety hazard of food is increased. Aiming at the problem, the development of a safe and effective biological control method is particularly important. The pollination efficiency is improved, the yield is improved, and the economic benefit of fruit tree planting can be effectively improved while the fruit diseases are controlled; on the other hand, the apocarya is required to be subjected to artificial pollination to make up for insufficient pollination caused by weather problems, or to targeted artificial auxiliary breeding. To solve these problems, pollen needs to be collected and preserved in advance in production for artificial pollination at an appropriate time. These pollens collected and preserved in advance have a germination rate which decreases with the preservation time, and thus improvement of the vigor of the pollens is very important for improving the effect of artificial pollination. At present, methods for detecting pollen viability exist in production, and few recovery measures or improving agents are needed for the preserved pollen after the pollen viability is reduced.
Disclosure of Invention
In order to solve the technical problems existing in the prior production, the invention provides a pollen viability improver containing coenzyme Q;
the coenzyme Q can be synthesized by an artificial method, and can also be extracted from plants or microorganisms;
The improver consists of 15-30% of sucrose, 2-4mM boric acid, 1-3mM calcium chloride, 0.2-1mM potassium chloride, 5-20mM MES-Tris (pH 5.8) and 0.1ppm-100ppm coenzyme Q;
The coenzyme Q in the improver is one of coenzyme Q8, coenzyme Q9 or coenzyme Q10;
The improver is characterized in that the improver can be in a liquid or solid dosage form;
the improver is safe and environment-friendly, wherein coenzyme Q is an important vitamin substance required by a human body, and other required substances are conventional reagents required by plant pollen germination.
Description of the embodiments
The technical scheme of the patent is further described in detail below with reference to the specific embodiments. The following examples do not limit the invention.
Embodiment one: extraction of coenzyme Q8 from 15B1 bacterial liquid
Culturing 15B1 bacteria:
Taking frozen 15B1 bacteria (Pichia pastoris PICHIA FERMENTANS strain 15B1 is a patent strain with the preservation number of CGMCC No.19317 and the patent number of ZL 2021100326148) from a refrigerator at the temperature of minus 80 ℃, thawing at normal temperature, sucking and mixing uniformly in an ultra-clean bench by a pipetting gun, adding 1% of bacteria liquid into a PDB culture medium, culturing for 12 hours in a shaking table at the temperature of 150 revolutions at 32 ℃, taking 100 mu L of a coating plate from the well-shaken bacteria liquid, placing the coating plate in a incubator at the temperature of 32 ℃ for 12 hours, observing the growth form and the color of colonies on a flat plate, selecting bacteria particles meeting the conditions from the coating plate, scribing on a PDA culture flat plate, culturing for 12 hours in the incubator at the temperature of 32 ℃, taking one better bacteria particle in a PBD culture medium, culturing for 12 hours in a shaking table at the temperature of 150 revolutions at the temperature of 32 ℃, centrifuging for 1 minute at 4000g/s, and taking supernatant;
efficient separation and collection:
Heat-treating the supernatant of 15B1 at 85 ℃ for 20 minutes, extracting with n-hexane 1:1, concentrating by 10 times with absolute ethanol, centrifuging the concentrated solution to obtain supernatant, separating the supernatant by using angilent liquid phase 1260, and carrying out the active substance separation by using a Hypersil BDS column 4.6 x 150 of the chromatographic column illite, wherein the loading amount is as follows: 50 μl, wavelength: 265nm, mobile phase: the absolute ethanol is used for collecting peaks of 2.5-3min, and the separated effective matters are concentrated by 10 times after rotary evaporation and collection, and then a Hypersil BDS chromatographic column of Dalian Lite is used. The mass spectrum is a quadrupole-electrostatic field Orbitrap mass spectrometer (Thermo FISHER SCIENTIFIC, USA)
Mass spectrometry:
Embodiment two: improving agent for promoting frozen pollen viability recovery
Collecting fresh Carya illinoensis pollen in the same year, air drying, and storing at-80deg.C in a freezing tube. Freezing pollen at-80deg.C, thawing in ice water bath for 1 hr, and standing at room temperature for 2 days at 25deg.C;
Preparing a liquid germination medium and a solid germination medium plate for pollen germination before use for standby; preferably, the liquid germination medium is prepared from 20% sucrose, 4mM boric acid, 3mM calcium chloride, 0.67mM potassium chloride, 10mM MES-Tris, and pH5.8; the solid germination culture medium is formed by adding 1% agarose (Biowest, spain) on the basis of the liquid germination culture medium;
the pollen germination steps are as follows:
1) Thawed pollen was rehydrated at 16 ℃ for 6 hours. The specific rehydration process comprises the following steps: taking a sealed container, and accommodating 1/3 of saturated copper sulfate therein; spreading the pollen sample placed at room temperature of 25 ℃ for 2 days on a culture dish, and suspending the culture dish on the liquid surface of a sealed container at a proper temperature;
2) Weighing rehydrated pollen into a 1.5 ml centrifuge tube, and then fully mixing the rehydrated pollen with the liquid germination medium according to a proportion;
Preferably, a group of: the ratio of rehydrated pollen to liquid germination medium was 500 μl of germination fluid mixed with 100mg pollen; another group: the ratio of rehydrated pollen to liquid germination medium was 500. Mu.L of germination medium mixed with 100mg pollen, and 10. Mu.L of the concentrated solution containing coenzyme Q8 isolated in example I was added;
3) Uniformly dispersing pollen suspension on the solid germination medium plate by unidirectional dispersion using a cell spreader with a side length of 1 cm, and immediately covering the plate after spreading to prevent drying;
4) Suspending the plate on the water surface in a sealed box, and germinating for 12 hours in the dark at 25 ℃;
5) During germination, germination was observed and recorded in all experiments at 10 x magnification using olympus inverted fluorescence IX71 microscope and DP71 camera;
preferably, the pollen tube sprouting length longer than the diameter of the pollen grains is regarded as the pollen grains sprouting;
6) The germination images were counted using software Image pro Plus. Set 3 experimental replicates. At each measurement, four fields were examined, each field containing approximately 200 pollen grains, and the arithmetic mean of the data from the four fields was repeated as one experiment. Exporting the data to Excel software for further analysis, including arithmetic mean and error analysis;
The analysis result shows (see Table 1) that the germination rate of pollen taken out from-80 ℃ is higher than that of pollen without coenzyme Q8 after the pollen is placed for 2 days at normal temperature, and the coenzyme Q8 has positive influence on the germination rate of pollen, so that the coenzyme Q8 promotes the recovery of pollen vitality;
Table 1: effects of coenzyme Q8 on germination of Carya illinoensis pollen samples
Germination rate of added substances%
Germination liquid 53.03+/-2.83
Germination liquid and coenzyme Q8.86+ -2.27
Embodiment III: coenzyme Q10 to promote recovery of frozen pollen viability
Preparing 10 mug/mL, 100 mug/mL and 1mg/mL coenzyme Q10 solution; the liquid pollen culture medium and the solid pollen culture medium are used for standby, and the test is carried out based on the pollen germination step;
preferably, 10. Mu.L of coenzyme Q10 is added into 500. Mu.L of germination liquid for use after dissolving coenzyme Q10 in n-hexane absolute ethanol=1:9, and the control group is liquid pollen culture medium without any bacteria or metabolites thereof;
The analysis result shows (see Table 2) that the coenzyme Q10 has a certain effect of promoting the pollen viability recovery, and the degrees of pollen viability recovery of the coenzyme Q10 with different concentrations are different, and 2 mug/mL of the coenzyme Q10 is more than 20 mug/mL of the coenzyme Q10 and more than 0.2 mug/mL of the coenzyme Q10, so that the effect of promoting the pollen viability recovery of the coenzyme Q10 with 2 mug/mL is the best;
TABLE 2 germination Effect of varying concentrations of coenzyme Q10 on Carya illinoensis pollen samples
Germination rate of added substances%
Control 34.71.+ -. 1.9
0.2. Mu.g/mL coenzyme Q10.58.+ -. 4.24
2 Μg/mL coenzyme Q10 38.33+ -1.41
20 Μg/mL coenzyme Q10.75.+ -. 3.2
Embodiment four: coenzyme Q10 to promote fresh pollen viability recovery
At early five months, several inflorescences are cut from the apocarya branches, and the apocarya branches are dried in the air at about 20 ℃ for 2-3 days, and extracting solution containing coenzyme Q8 from the pichia pastoris 15B1 is respectively concentrated by 10 times and not concentrated according to the first embodiment. Experimental results show that the germination rate of pollen of the control group added with the liquid germination culture solution is 55.74%, the germination rate of the germination culture solution added with the unconcentrated 15B1 bacterial solution is 72.79%, the germination rate of the germination culture solution added with the concentrated 10 times 15B1 bacterial solution is 72.66%, and from the analysis results of experimental data, the germination rates of pollen are improved after the unconcentrated fermented pichia pastoris 15B1 and the concentrated 10 times fermented pichia pastoris 15B1 are added, and the effects of both the germination rates on fresh pollen are obvious. This result also shows that fresh pollen is better than frozen pollen.
Claims (3)
1. A pollen viability improver containing coenzyme Q is characterized in that the improver consists of 15-30% of sucrose, 2-4mM boric acid, 1-3mM calcium chloride, 0.2-1mM potassium chloride, 5-20mM MES-Tris (pH 5-7) and 0.1ppm-100ppm of coenzyme Q.
2. The agent for improving pollen viability comprising coenzyme Q as claimed in claim 1, wherein the coenzyme Q is one or both of coenzyme Q8 and coenzyme Q10.
3. A coenzyme Q-containing pollen viability improver as claimed in claim 1 which may be in liquid or solid form.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410058711.8A CN117887777A (en) | 2024-01-16 | 2024-01-16 | Pollen vitality improver containing coenzyme Q |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410058711.8A CN117887777A (en) | 2024-01-16 | 2024-01-16 | Pollen vitality improver containing coenzyme Q |
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| Publication Number | Publication Date |
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| CN117887777A true CN117887777A (en) | 2024-04-16 |
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| CN202410058711.8A Pending CN117887777A (en) | 2024-01-16 | 2024-01-16 | Pollen vitality improver containing coenzyme Q |
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| Country | Link |
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| CN (1) | CN117887777A (en) |
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- 2024-01-16 CN CN202410058711.8A patent/CN117887777A/en active Pending
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