CN102168060A - Method for observing dynamic growth process of pollen tube germination and growth - Google Patents
Method for observing dynamic growth process of pollen tube germination and growth Download PDFInfo
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- CN102168060A CN102168060A CN 201110000356 CN201110000356A CN102168060A CN 102168060 A CN102168060 A CN 102168060A CN 201110000356 CN201110000356 CN 201110000356 CN 201110000356 A CN201110000356 A CN 201110000356A CN 102168060 A CN102168060 A CN 102168060A
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Abstract
The invention discloses a method for observing the dynamic growth process of pollen tube germination and growth. The invention selects neutral red as coloring agent to dye plant pollen and comprises steps of collecting pollen, preparing culture medium, preparing neutral red gradient dyeing liquor, preparing pollen culture chamber, spreading pollen, controlling pollen culture conditions, and observing the dynamic growth process of pollen tube germination and growth via a microscope. The germination rate and growth of the dyed pollen tube is basically consistent with that of the undyed control group and the pollen still keeps population effect. The invention has the advantages of rapidness, simplicity, stability, and great observation effect and allows direct observation of the dynamic growth process of pollen tube germination and growth.
Description
Technical field: the present invention relates to a kind of method of observing the dynamic growth course of germination of pollen tube and growth, belong to biological technical field.
Background technology: pollen is the microgametophyte of higher plant.It is transmitting male parent's genetic information in sexual propagation.Pollen is the important object of researchs such as plant genetic, breeding, evolution, reproduction, is again the important materials of spore-pollen analysis, bee colony cultivation, medicine manufacturing, medical treatment and physiological test etc.Pollen germination and pollen tube growth are that plant is finished amphigenetic significant process, just seem very important of the growth course of therefore observing pollen germination and pollen tube growth.
Up to now, the observation of plant pollen tube germination and pollen tube growth process mainly relies on the fixed preparation staining technique to obtain, for example, and aceto-camine method, IKI method, aceto-camine-fast green method etc.But above method all can not directly show the dynamic growth course of germination of pollen tube and pollen tube growth, can only dye respectively in different steps according to the experiment needs and fixedly observe.Therefore, directly carry out the dynamic growth course of germination of pollen tube and pollen tube growth and observe, can provide new data from another angle far and away, help this important stage in understanding and the control fertilization process.Yet, the direct viewing that do not dye, the pollen color is more shallow, and especially pollen tube is transparence, is not easy to very much observe.
Summary of the invention:, the invention provides the method for the dynamic growth course of a kind of quick, easy observation germination of pollen tube and growth at above-mentioned the deficiencies in the prior art
For achieving the above object, the technical solution used in the present invention is: a kind of method of observing the dynamic growth course of germination of pollen tube and growth, and concrete steps are as follows:
1), the collection of pollen
Gather open pollen on the same day.
2), the preparation of pollen substratum
Sucrose 5%-20%, agar 0.3%-0.6%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.4-6.4.
3), the preparation of toluylene red staining fluid
Alcohol compound concentration scope with 30% is the toluylene red staining fluid of 0.01%-0.15%.
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum.
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then.
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably.
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun, with pollen staining 30 seconds.Culture dish is holded up, at the substratum edge dye liquor is fully blotted with thieving paper.
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 22 ℃-25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
9), dyeing back identification of indicator
The germination rate of a, pollen
The population effect of b, pollen
The observing effect of c, pollen germination and pollen tube growth process
Described toluylene red concentration is preferably in 0.01%-0.10%.
The present invention has set up quick, easy, stable toluylene red dyeing process, can very clearly observe the dynamic growth course of germination of pollen tube and pollen tube growth, do not influence the observation of population effect of growth, the pollen of pollen germination rate and pollen tube, observing effect is splendid.
Embodiment
Embodiment 1
1), the collection of pollen
Gather open Clivia miniata Reg pollen on the same day.
2), the preparation of pollen substratum
Sucrose 5%, agar 0.5%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.8.
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.05% toluylene red staining fluid.
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum.
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then.
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably.
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun, with pollen staining 30 seconds.Culture dish is holded up, at the substratum edge dye liquor is fully blotted with thieving paper.
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 23 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
9), dyeing back identification of indicator
The germination rate of a, dyeing group pollen and the control group basically identical that do not dye.
B, dyeing group pollen still have population effect.
The observing effect of c, dyeing group pollen germination and pollen tube growth process obviously is better than the control group that do not dye.
Embodiment 2
1), the collection of pollen
Gather the pollen of open lily on the same day.
2), the preparation of pollen substratum
Sucrose 10%, agar 0.6%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=6.0.
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.10% toluylene red staining fluid.
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum.
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then.
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably.
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun, with pollen staining 30 seconds.Culture dish is holded up, at the substratum edge dye liquor is fully blotted with thieving paper.
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 25 minutes.
9), dyeing back identification of indicator
The germination rate of a, dyeing group pollen and the control group basically identical that do not dye.
B, dyeing group pollen still have population effect.
The observing effect of c, dyeing group pollen germination and pollen tube growth process obviously is better than the control group that do not dye.
Embodiment 3
1), the collection of pollen
Gather the pollen of open tomato on the same day.
2), the preparation of pollen substratum
Sucrose 12%, agar 0.3%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.4.
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.08% toluylene red staining fluid.
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum.
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then.
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably.
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun, with pollen staining 30 seconds.Culture dish is holded up, at the substratum edge dye liquor is fully blotted with thieving paper.
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 22 ℃-25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
9), dyeing back identification of indicator
The germination rate of a, dyeing group pollen and the control group basically identical that do not dye.
B, dyeing group pollen still have population effect.
The observing effect of c, dyeing group pollen germination and pollen tube growth process obviously is better than the control group that do not dye.
Claims (5)
1. method of observing the dynamic growth course of germination of pollen tube and growth, concrete steps are as follows:
1), the collection of pollen
Gather open pollen on the same day;
2), the preparation of pollen substratum
Get sucrose 5%-20%, agar 0.3%-0.6%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.4-6.4;
3), the preparation of toluylene red staining fluid
Alcohol compound concentration scope with 30% is the toluylene red staining fluid of 0.01%-0.15%;
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum;
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then;
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably;
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun,, culture dish holded up, at the substratum edge dye liquor is fully blotted with thieving paper with pollen staining 30 seconds;
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 22 ℃-25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
2. a kind of method of observing the dynamic growth course of germination of pollen tube and growth as claimed in claim 1 is characterized in that: described toluylene red staining fluid concentration is between 0.01%-0.10%.
3. a kind of method of observing the dynamic growth course of germination of pollen tube and growth as claimed in claim 1 is characterized in that, the steps include:
1), the collection of pollen
Gather open Clivia miniata Reg pollen on the same day;
2), the preparation of pollen substratum
Sucrose 5%, agar 0.5%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.8;
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.05% toluylene red staining fluid;
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum;
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then;
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably;
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun,, culture dish holded up, at the substratum edge dye liquor is fully blotted with thieving paper with pollen staining 30 seconds;
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 23 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
4. a kind of method of observing the dynamic growth course of germination of pollen tube and growth as claimed in claim 1 is characterized in that, the steps include:
1), the collection of pollen
Gather the pollen of open tomato on the same day;
2), the preparation of pollen substratum
Sucrose 12%, agar 0.3%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=5.4;
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.08% toluylene red staining fluid;
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum;
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then;
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably;
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun,, culture dish holded up, at the substratum edge dye liquor is fully blotted with thieving paper with pollen staining 30 seconds;
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 22 ℃-25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 20 minutes.
5. a kind of method of observing the dynamic growth course of germination of pollen tube and growth as claimed in claim 1 is characterized in that, the steps include:
1), the collection of pollen
Gather the pollen of open lily on the same day;
2), the preparation of pollen substratum
Sucrose 10%, agar 0.6%, boric acid 0.04%, all the other add entry, wiring solution-forming pH value=6.0;
3), the preparation of toluylene red staining fluid
Alcohol compound concentration with 30% is 0.10% toluylene red staining fluid;
4), the preparation of pollen culturing room
Select for use culture dish as pollen culturing room, contain 2ml water at the bottom of the culture dish, the culture dish lid is coated with the thin layer substratum;
5), the preparation of thin layer substratum
Substratum takes out while hot, draws nutrient solution with liquid-transfering gun, at the inboard culture medium solution that drips 1ml of culture dish lid, with the glass transfering loop culture medium solution is launched lamellar substratum at once then;
6), the spreading of pollen
The culture dish that will have substratum is placed horizontally on the experiment table, extract the petal of titbit with tweezers, clamp flower pesticide below filigree, take out flower pesticide, flower pesticide is slided on media surface gently, and the tweezers that maybe will accompany flower pesticide striking at the culture dish edge gently makes the pollen individual layer under several, is scattered and is distributed on the media surface equably;
7), toluylene red staining fluid dyeing
On substratum, there is the pollen place to drip 0.5ml toluylene red staining fluid with liquid-transfering gun,, culture dish holded up, at the substratum edge dye liquor is fully blotted with thieving paper with pollen staining 30 seconds;
8), pollen is cultivated
Culture is placed in the thermostat container, and temperature is controlled at 25 ℃, begins to observe the dynamic growth course of germination of pollen tube and pollen tube growth after 25 minutes.
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| CN 201110000356 CN102168060A (en) | 2011-01-04 | 2011-01-04 | Method for observing dynamic growth process of pollen tube germination and growth |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110000356 CN102168060A (en) | 2011-01-04 | 2011-01-04 | Method for observing dynamic growth process of pollen tube germination and growth |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421737A (en) * | 2013-06-04 | 2013-12-04 | 塔里木大学 | Culture medium for promoting germination of Amygdalus L. pollen, culture method and application thereof |
| CN108827741A (en) * | 2018-07-19 | 2018-11-16 | 青岛农业大学 | A method of fluorescent dye is loaded in pollen tube using negative-pressure penetration |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624146A (en) * | 2004-12-17 | 2005-06-08 | 大连理工大学 | Process for obtaining transgenosis plant by ovary drop |
| CN101503468A (en) * | 2009-03-13 | 2009-08-12 | 北京林业大学 | Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof |
| CN101717747A (en) * | 2009-12-04 | 2010-06-02 | 西北农林科技大学 | Liquid medium for vitro scutellaria root pollen germination and method for testing activity of scutellaria root pollen |
-
2011
- 2011-01-04 CN CN 201110000356 patent/CN102168060A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624146A (en) * | 2004-12-17 | 2005-06-08 | 大连理工大学 | Process for obtaining transgenosis plant by ovary drop |
| CN101503468A (en) * | 2009-03-13 | 2009-08-12 | 北京林业大学 | Protein related to pollen germination and/or pollen tube growth, coding gene and use thereof |
| CN101717747A (en) * | 2009-12-04 | 2010-06-02 | 西北农林科技大学 | Liquid medium for vitro scutellaria root pollen germination and method for testing activity of scutellaria root pollen |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421737A (en) * | 2013-06-04 | 2013-12-04 | 塔里木大学 | Culture medium for promoting germination of Amygdalus L. pollen, culture method and application thereof |
| CN103421737B (en) * | 2013-06-04 | 2015-04-15 | 塔里木大学 | Culture medium for promoting germination of Amygdalus L. pollen, culture method and application thereof |
| CN108827741A (en) * | 2018-07-19 | 2018-11-16 | 青岛农业大学 | A method of fluorescent dye is loaded in pollen tube using negative-pressure penetration |
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Application publication date: 20110831 |