CN103868842A - Method for detecting quantity of stigma pollens after pollination of flowering plants - Google Patents
Method for detecting quantity of stigma pollens after pollination of flowering plants Download PDFInfo
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- CN103868842A CN103868842A CN201410047329.3A CN201410047329A CN103868842A CN 103868842 A CN103868842 A CN 103868842A CN 201410047329 A CN201410047329 A CN 201410047329A CN 103868842 A CN103868842 A CN 103868842A
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Abstract
The invention provides a method for detecting the quantity of stigma pollens after the pollination of flowering plants. The method comprises the following steps: stigma dyeing, separating and collecting pollens, fabricating glass slides, scanning the glass slides, counting the quantity of the pollens by using counting software, counting under a microscope and the like. The method has the high accuracy and applicability, the error caused by a human factor and the like can be avoided to the maximal extent, and the method also can be properly adjusted and popularized to the statistics of the quantity of the stigma pollens of a plurality of flowering crops.
Description
Technical field
The invention belongs to agricultural cience and farming techniques field, specifically, relate to the detection method of the crop pollination rear pillar head pollen quantity of blooming.
Background technology
Peach is one of main temperate zone fruit, extensively cultivation in the world, and Chinese peach cultivated area and output all rank first in the world.Peach belongs to seasonal fresh fruit, not storage endurance, and the envirment factor that establishment planting technology can affect peach growth by change is as illumination, temperature, moisture, CO
2, edaphic condition etc., reach the object that regulates the fructescence, meet consumer to demand fresh, pollution-free, anti-Ji Tao, meanwhile, can be grower and bring rich profit.
Florescence pollination is to affect the solid key factor of crop setting of blooming, and peach blossom belongs to typical entomophilous flower, and insect pollination can significantly improve peach output.But closely during the last ten years, along with the adjustment of agricultural planting structure, industrial crops single variety implant mass pattern (county's one Pin Huo mono-village's one product phenomenon) is progressively promoted, make wild insect pollinator lose the food source of perching in the anniversary, often cause the wild insect pollinator quantity in main producing region sharply to decline.The peach florescence is shorter, and the wild insect pollinator in main producing region is less, easily occurs the not foot phenomenon of pollinating.Cause the phenomenon of peach production declining in facility cultivation, to show more obvious because pollination is not enough.Facility peach is in growth course, and especially, before and after the florescence, in the environment in isolation relative to the external world, air flow property is poor and lack natural pollination insect, and the fruit drop phenomenon causing because Pollination Fertilization is insufficient is particularly serious.Fully pollinate for guarantee peach blossom, improve output, grower is that peach blossom is pollinated in florescence normal employing manual type or introducing insect pollinator.Although artificial pollination can significantly improve percentage of fertile fruit, need to expend a large amount of manpower and materials, by comparison, adopting insect is peach blossom pollination, not only can reduce production costs, and can improve peach output, improves fruit quality, has clear superiority.
In peach produces, widely used insect pollinator mainly comprises several large classes such as honeybee, bumblebee and wall honeybee, because the aspects such as different insect pollinator morphosis and biological habit exist larger difference, efficiency difference while causing it to be peach pollination, thereby peach ultimate capacity is impacted; Meanwhile, Pollinating Insect configuration quantity also can affect peach setting situation.Therefore,, in peach plant development process, according to peach blossom bloom Biological characteristics and planting environment, the insect of selecting the insect pollinator that Pollination Efficiency is higher and being equipped with right quantity pollinates, and becomes the indispensable link of peach high-yield cultivating.And select suitable index to evaluate the Pollination Efficiency of insect at the peach florescence, and according to index in time the quantity to insect pollinator etc. regulate and control, can farthest improve undoubtedly peach output.
At present the research of insect pollination efficiency is focused mostly on and visiting flower frequency, visiting and take time, contact column cap frequency, carries in the indexs such as pollen amount.Although such index can reflect the visit flower behavior difference of different insects to a certain extent, arbitrary single index all cannot directly impact the pollination fertilization process of peach, can not effectively evaluate insect pollination efficiency.Research shows, plant pistil stigma pollen sedimentation quantity and percentage of fertile fruit height correlation.Within the scope of the pollen quantity can bear at column cap, pollen sedimentation quantity is more, is more conducive to the carrying out of fertilization process, and successfully fertilization is the solid prerequisite of plant setting.Therefore add up peach blossom pollen deposition quantity after Insect Pollination, can directly reflect the height of insect pollination efficiency.
At present the method for test column headdress flower powder sedimentation quantity mainly comprises directly dissecting Microscopic observation column cap and using the solution such as hydrochloric acid that pollen is separated with column cap, utilizes blood counting chamber to add up pollen quantity.Directly examine under a microscope column cap and add up pollen granule number needs and want assessor correctly to identify the pollen granule on column cap, but peach blossom pollen color is organized similar to column cap, and pollen granule is adsorbed on stigma surface diverse location after column cap is sprouted, while causing counting, be difficult to observe and distinguish pollen granule, in addition the pollen quantity on peach blossom column cap is sometimes up to more than thousands of grains, cause workload large, statistical difficulty, often there is larger error in different assessors' statistics, cannot ensure the reliability of result; Utilize particular solution that pollen granule is separated with column cap, re-using blood counting chamber counts under the microscope, although avoided the interference that column cap tissue is distinguished pollen granule also to improve to a certain extent reliability, but still need to expend the plenty of time with naked eyes statistics pollen granule quantity, and personnel's operability is had relatively high expectations.
Summary of the invention
The object of this invention is to provide a kind of detection method of the flowering phase of crop pollination rear pillar head pollen quantity of blooming, thus the Pollination Efficiency of effective and Fast Evaluation insect pollinator.
In order to realize the object of the invention, the detection method of a kind of flowering phase of crop pollination rear pillar head pollen quantity of blooming of the present invention, comprise column cap dyeing, separate and collect pollen, microslide making, scan microslide, utilize and the step such as under Counting software statistics pollen granule quantity and microscope, count.
Above-mentioned detection method comprises the following steps:
1) column cap dyeing: gather the column cap of blooming after flowering phase of crop pollination and be placed in centrifuge tube, add malachite green dyeing liquor to dye in centrifuge tube, collect dyeing liquor as solution I;
2) separate and collect pollen: column cap is transferred in another flat based tubes that fills malachite green dyeing liquor, utilize the broken column cap tissue of ultrasonic cell disruption instrument, realize separating of pollen granule and column cap, collect dyeing liquor as solution II, with 0.85%-1.15%(preferably 1%) NaCl solution rinses the pollen granule adhering on cell crushing instrument ultrasonic transformer, collects washing fluid as solution III; Merge solution I, II and III, the pollen granule in above-mentioned amalgamation liquid is collected on filter membrane with vacuum filtration bottle;
3) making of microslide and scanning: after pollen granule is collected on filter membrane, filter membrane is loaded with to pollen granule to face up, open and flat docile has 15%-40%(preferably 30% in dripping) on the microslide of glycerite, be made into Temporary slide, with scanner be electronic photo by the filter membrane scanning on microslide;
4) adopt pollen granule quantity on Image J software statistics filter membrane, and by step 2) in remaining column cap tissue be placed on microslide, count under the microscope residual pollen granule quantity, the residual pollen granule quantity sum of pollen granule quantity and column cap on filter membrane, is the flowering phase of crop pollination rear pillar head pollen quantity of blooming.
Separate in pollen process at suction filtration solution, due to the use of vacuum pump, continuous negative pressure in liquid-collecting bottle can form twitch force, good and filter core without stop up in the situation that at filtration unit leakproofness, this power acts on filter membrane surface equably, make flow through solution and the filter membrane uniform contact of filter membrane, thus while guaranteeing that pollen granule separates with solution stressed evenly, pollen granule can be adsorbed on filter membrane fully dispersedly; Meanwhile, in whole separation pollen process, due to through solution suction filtration repeatedly, pollen granule constantly suspended dispersed, be adsorbed on filter membrane again, improve the dispersiveness of pollen granule, reduced the overlapping probability of pollen granule, avoided because of the overlapped statistical error that may cause between pollen granule.
Aforesaid detection method, also comprise with 0.85%-1.15%(preferably 1%) NaCl solution rinsing step 1) described in centrifuge tube, to collect the pollen granule being attached on tube wall, as solution IV, and with 0.85%-1.15%(preferably 1%) NaCl solution rinsing step 2) described in flat based tubes, to collect the pollen granule being attached on tube wall, as solution V; After being merged, solution I, II, III, IV and V carry out vacuum filtration.
The malachite green dyeing liquor using in the present invention is by 0.1% malachite green solution and 0.85%-1.15%(preferably 1%) NaCl solution forms by the volume ratio mixed preparing of 1:400.
Aforesaid detection method, in step 1), dyeing time is 15h, dyeing temperature is 37 ℃, carries out shaking table concussion dyeing with 200rpm rotating speed.
Aforesaid detection method, step 2) in utilize the broken column cap tissue of ultrasonic cell disruption instrument to set program as follows: omnidistance ultrasonic time 60s, ultrasonic time 60s; quiescent interval 0s; temperature protection is set as 50 ℃, ultrasonic power 100W, and ultrasonic transformer specification is Φ 6.
Aforesaid detection method, the filter cup volume of collecting pollen vacuum filtration bottle used in step 3) is 300mL, and liquid-collecting bottle volume is 2000mL, and main material is borax glass, and core filter base material is ground glass, filtering core diameter is 20mm; Filter membrane material is nylon, and filter membrane aperture is 10-30 μ m, and filter membrane is shaped as the circle of 20mm < diameter < 25mm, or the square of 20mm < length of side < 25mm; When use, amalgamation liquid is poured in filter cup, through vacuum filtration, pollen granule is adsorbed on filter membrane, residual liquid flow through filter membrane and filter core, be collected in liquid-collecting bottle.
Aforesaid detection method, in the time that the described crop that blooms is peach, in step 4), adopt pollen granule quantity on ImageJ software statistics filter membrane, target particles color parameter setting range is: color-values 120-150, saturation degree 70-255, brightness 0-255, target particles size setting value is 40-80, circular contour setting value is 0.5-0.8.And in step 3), filter membrane aperture is 20 μ m.
The flowering phase of crop Pollination Modes of blooming relating in the present invention is insect pollination.
The detection method of the flowering phase of crop pollination rear pillar head pollen quantity of blooming provided by the invention, there is higher accuracy and applicability, can avoid largely the error causing due to human factor etc., also can extend in the statistics of column cap pollen quantity of the multiple crop that blooms through suitable adjustment.
(1) this method can reduce largely or avoid systematic error in operating process.Collecting pollen link, collect the pollen in dyeing liquor and the washing fluid etc. in the dyeing liquor in flat based tubes, centrifuge tube, effectively improve statistics accuracy, reduce systematic error.
(2) the present invention, in pollen count link according to pollen self character, sets filter membrane pore size and software parameter, has improved accuracy, has avoided the error causing due to the difference between observer's individuality.Also can select the filter membrane of different pore size size and suitably adjust software parameter, the method being extended in the statistics of column cap pollen quantity of other crops, there is stronger applicability simultaneously.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment detects the method for peach blossom pollen deposition quantity
Comprise the following steps:
1, column cap dyeing: add 1.4mL malachite green dyeing liquor (0.1% malachite green and 1%NaCl solution are formulated by 1:400 volume ratio) in every 1.5mL Eppendolf centrifuge tube that sample column cap is housed, shaking table concussion dyeing 15h (37 ℃, 200rpm; Higher temperature makes pollen granule easily painted, and even dyeing is guaranteed in shaking table concussion), collect dyeing liquor as solution I.
2, separate pollen: use clean tweezers to take out column cap, be placed in the flat based tubes that 10mL malachite green dyeing liquor is housed, (program setting is as follows: omnidistance time 60s to utilize the ultrasonic processing column cap tissue of ultrasonic cell disruption instrument (JY92-II DN, China), ultrasonic time 60s, quiescent interval 0s, temperature protection is set as 50 ℃, ultrasonic power 100W, ultrasonic transformer specification is Φ 6), make pollen granule depart from column cap, after ultrasonic end, take out flat based tubes, collect dyeing liquor as solution II; Change another clean flat based tubes and be placed in ultrasonic transformer lower end, prevent that dyeing liquor residual on ultrasonic transformer from dripping and causing the loss of pollen granule quantity, in addition, rinse the pollen granule adhering on ultrasonic transformer with 1%NaCl solution, collect washing fluid as solution III.
3, collect pollen: through ultrasonic processing, most of pollen granule departs from column cap and is suspended in dyeing liquor, but still have minority pollen granule may be adsorbed in column cap tissue and splendid attire vessel, therefore use 1%NaCl solution to rinse column cap, tweezers, centrifuge tube and flat based tubes, collect washing fluid as solution IV, merge above-mentioned solution I, II, III and IV, be collected in and in filter cup, carry out vacuum filtration, finally use 1%NaCl solution washing and filtering cup, again carry out vacuum filtration, stigma surface pollen granule is collected in that on filter membrane, (vacuum flask model is FB-20T the most at last, Tianjin Ao Tesaiensi Instrument Ltd. of China produces, wherein filter cup specification 300mL, liquid-collecting bottle specification 2000mL, main material is borax glass, core filter base material is ground glass, core diameter is 20mm, filtering membrane model is 03-20/3, and its material is nylon, aperture 20 μ m, and Sai Fa company of Switzerland produces, and filter membrane is cut into 20mm × 20mm square, covers and filters core top, to carry pollen granule, vacuum filtration pump type is 2522Z-02, and WELCH company of the U.S. produces).
The column cap tissue rinsing through NaCl solution is placed on microslide, check and whether still have remaining pollen granule under the microscope, if pollen residual volume is few and can carry out under the microscope accurate counting, directly remaining pollen granule is counted and record, if pollen residual volume is excessive, cannot carry out accurate counting, ultrasonic processing column cap again according to the method described above.
4, scanning pollen: use pipettor to drip 30 μ L30% glycerites in clean microslide central authorities, use clean tweezers to take out the filter membrane that is loaded with pollen granule, be flattened on microslide central authorities, make glycerite soak filter membrane, require filter membrane smooth, without bubble.The scanner that use can scan slide is electronic photo (scanner models Nikon Coolscan9000ED, Japan) by the filter membrane scanning on microslide.
5, software counting: utilize pollen granule quantity on Image J software statistics filter membrane, design parameter is set as follows: tone value is 120-150, intensity value is 70-255, brightness: 0-255, target particles thing sizes values is 40-80, and circular contour value is 0.5-0.8, draws pollen granule quantity on filter membrane, be added with column cap residue pollen granule quantity, draw pollen deposition quantity.
A kind of detection method for peach blossom pollen deposition quantity of the present invention, design according to shape, size and the pollen granule of peach blossom powder and the coloured differently characteristic of column cap tissue, avoid largely manual operation error, improve detection accuracy, be also applicable to the detection of other crop pollen deposition quantity of blooming.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. the detection method of the flowering phase of crop of blooming pollination rear pillar head pollen quantity, is characterized in that, comprises that column cap dyes, separates and collect the making of pollen, microslide, scans microslide, utilizes the step of counting under Counting software statistics pollen granule quantity and microscope.
2. detection method according to claim 1, is characterized in that, comprises the following steps:
1) column cap dyeing: gather the column cap of blooming after flowering phase of crop pollination and be placed in centrifuge tube, add malachite green dyeing liquor to dye in centrifuge tube, collect dyeing liquor as solution I;
2) separate and collect pollen: column cap is transferred in another flat based tubes that fills malachite green dyeing liquor, utilize the broken column cap tissue of ultrasonic cell disruption instrument, realize separating of pollen granule and column cap, collect dyeing liquor as solution II, rinse the pollen granule adhering on cell crushing instrument ultrasonic transformer with 0.85%-1.15%NaCl solution, collect washing fluid as solution III; Merge solution I, II and III, the pollen granule in above-mentioned amalgamation liquid is collected on filter membrane with vacuum filtration bottle;
3) making of microslide and scanning: after pollen granule is collected on filter membrane, filter membrane is loaded with to pollen granule to face up, open and flat docile has on the microslide of 15%-40% glycerite in dripping, and is made into Temporary slide, and with scanner, the filter membrane on microslide being scanned is electronic photo;
4) adopt pollen granule quantity on Image J software statistics filter membrane, and by step 2) in remaining column cap tissue be placed on microslide, count under the microscope residual pollen granule quantity, the residual pollen granule quantity sum of pollen granule quantity and column cap on filter membrane, is the flowering phase of crop pollination rear pillar head pollen quantity of blooming.
3. detection method according to claim 2, is characterized in that, described malachite green dyeing liquor is to be formed by the volume ratio mixed preparing of 1:400 by 0.1% malachite green solution and 0.85%-1.15%NaCl solution.
4. detection method according to claim 3, is characterized in that, in step 1), dyeing time is 15h, and dyeing temperature is 37 ℃, carries out shaking table concussion dyeing with 200rpm rotating speed.
5. detection method according to claim 2; it is characterized in that; step 2) in utilize the broken column cap tissue of ultrasonic cell disruption instrument to set program as follows: omnidistance ultrasonic time 60s; ultrasonic time 60s; quiescent interval 0s; temperature protection is set as 50 ℃, ultrasonic power 100W, and ultrasonic transformer specification is Φ 6.
6. detection method according to claim 2, is characterized in that, the filter cup volume of collecting pollen vacuum filtration bottle used in step 3) is 300mL, liquid-collecting bottle volume is 2000mL, main material is borax glass, and core filter base material is ground glass, and filtering core diameter is 20mm; Filter membrane material is nylon, and filter membrane aperture is 10-30 μ m, and filter membrane is shaped as the circle of 20mm < diameter < 25mm, or the square of 20mm < length of side < 25mm; When use, amalgamation liquid is poured in filter cup, through vacuum filtration, pollen granule is adsorbed on filter membrane, residual liquid flow through filter membrane and filter core, be collected in liquid-collecting bottle.
7. according to the detection method described in claim 1-6 any one, it is characterized in that, described in the crop that blooms be peach.
8. detection method according to claim 7, it is characterized in that, while adopting on Image J software statistics filter membrane pollen granule quantity in step 4), target particles color parameter setting range is: color-values 120-150, saturation degree 70-255, brightness 0-255, target particles size setting value is 40-80, circular contour setting value is 0.5-0.8.
9. detection method according to claim 7, is characterized in that, in step 3), filter membrane aperture is 20 μ m.
10. according to the detection method described in claim 1-6 any one, it is characterized in that, the flowering phase of crop Pollination Modes of blooming is insect pollination.
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| CN110132822A (en) * | 2019-05-05 | 2019-08-16 | 广西南亚热带农业科学研究所 | A kind of cassava pollen quantity measuring method |
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| CN110583634A (en) * | 2019-10-13 | 2019-12-20 | 安徽省农业科学院水稻研究所 | Preservation method for length of rice stigma |
| CN113917082A (en) * | 2021-10-11 | 2022-01-11 | 中国林业科学研究院资源昆虫研究所 | Method for identifying influence of local commercial crop pollination on exotic commercial crops |
| CN115220132A (en) * | 2022-07-04 | 2022-10-21 | 山东浪潮智慧医疗科技有限公司 | Method for forecasting pollen concentration in atmosphere |
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| CN104568869A (en) * | 2014-12-12 | 2015-04-29 | 中国热带农业科学院橡胶研究所 | Prediction method for fertility rate of cross pollination of rubber tree |
| WO2019236843A1 (en) * | 2018-06-06 | 2019-12-12 | Monsanto Technology Llc | Systems and methods for distinguishing fertile plant specimens from sterile plant specimens |
| US12019022B2 (en) | 2018-06-06 | 2024-06-25 | Monsanto Technology Llc | Systems and methods for distinguishing fertile plant specimens from sterile plant specimens |
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| CN110583634A (en) * | 2019-10-13 | 2019-12-20 | 安徽省农业科学院水稻研究所 | Preservation method for length of rice stigma |
| CN110583634B (en) * | 2019-10-13 | 2022-01-14 | 安徽省农业科学院水稻研究所 | Preservation method of rice stigma |
| CN113917082A (en) * | 2021-10-11 | 2022-01-11 | 中国林业科学研究院资源昆虫研究所 | Method for identifying influence of local commercial crop pollination on exotic commercial crops |
| CN115220132A (en) * | 2022-07-04 | 2022-10-21 | 山东浪潮智慧医疗科技有限公司 | Method for forecasting pollen concentration in atmosphere |
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Application publication date: 20140618 |