CN102876768A - Method for measuring pollen vitality of lycoris plants using in-vitro germination method - Google Patents
Method for measuring pollen vitality of lycoris plants using in-vitro germination method Download PDFInfo
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- CN102876768A CN102876768A CN2012103523115A CN201210352311A CN102876768A CN 102876768 A CN102876768 A CN 102876768A CN 2012103523115 A CN2012103523115 A CN 2012103523115A CN 201210352311 A CN201210352311 A CN 201210352311A CN 102876768 A CN102876768 A CN 102876768A
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Abstract
The invention discloses a method for measuring pollen vitality of lycoris plants using an in-vitro germination method, comprising the following steps: collecting pollens and placing the pollens into culture solution to perform in-vitro culture, wherein formula of the culture solution consists of 30-80 g/L of sucrose and 5-25 mg/L of boric acid; the temperature for the in-vitro culture is 25-35 degrees centigrade; the time for the in-vitro culture is 60-90 min; and the relative humidity for the in-vitro culture is 70-90%. According to the invention, in-vitro culture conditions of the pollens of lycoris plants are explored; a simple, rapid and feasible method for measuring the pollen vitality of the lycoris plants is provided; and basis for cross breeding of the lycoris plants is provided.
Description
Technical field
The present invention relates to plant pollen vitality test field, relate in particular to the method that a kind of stripped sprouting method is measured the lycoris plants Pollen Activity.
Background technology
Lycoris plants is the plant that a class has ornamental value, and flower is very large, and is bright in luster, because its shade tolerance is strong, in afforestation, planted in sylvan life, border, edge, meadow etc. more.The lycoris plants resource of China is very abundant, but the overwhelming majority also is in wild state, and the artificial growth kind is still very single, so the new variety of seed selection short-tube lycoris, helps to promote the application of lycoris plants in afforestation.
In the conventional breeding of plant, in order to carry out artificial supplementary pollination or hybridization pollination, need the early stage pollen that gathers and store, especially in cross-breeding work, the basic work that is absolutely necessary of the vigor of Study of Pollens, usually before using the pollen that gathers and store, need to do the mensuration of Pollen Activity.Measuring Pollen Activity is basis and the key link of conventional hybridization breeding.
Mensuration for Pollen Activity mainly contains three class methods at present, and the first kind is the pollen staining method, and the Equations of The Second Kind method is field pollination method, and the 3rd class is the in-vitro pollen germination method.
The pollen staining method is by using chemical reagent that pollen is dyeed, and judges the vigor situation of pollen according to the depth of the pollen color after the dyeing, and staining mainly contains IKI staining, acetic red dyeing, TTC staining etc.These class methods are quick, easy, but owing to different plant pollen wall thickness, starch content, Some Related Enzymes content difference, the difference on effect that staining is used different plants is also larger.
Field pollination method is by artificial collection pollen and to filigree pollination, calculates the pollination setting percentage behind the fruit maturation, this be a kind of directly, reliable method, but time-consuming, effort is subjected to season, weather effect larger.
The stripped sprouting method of pollen is that the pollen that will gather carries out external sprouting experiment, the statistics germination rate, this kind method fast, simply, accuracy is also higher, but because the condition of the external sprouting of pollen of different sorts plant can be had any different, substratum for example, incubation time, culture temperature etc. need to have to the isolated culture condition of plant pollen reasonable assurance.
The POLLEN MORPHOLOGY of lycoris plants is zygomorphy, and equatorial view is boat-shaped or kidney shape, and polar view is ellipse or oblong.At present, the research for Lycoris pollen both at home and abroad concentrates on classification aspect between Lycoris phylogenetic relationship and kind mostly, finds effectively to identify the method for Lycoris Pollen Activity.
Summary of the invention
The invention provides a kind of stripped sprouting method and measure the method for lycoris plants Pollen Activity, the method can be measured the Pollen Activity of lycoris plants quickly and efficiently.
A kind of stripped sprouting method is measured the method for lycoris plants Pollen Activity, comprises collection pollen, places nutrient solution to carry out isolated culture described pollen,
The prescription of described nutrient solution is: sucrose 30～80g/L, boric acid 15～25mg/L;
The temperature of described isolated culture is 25～35 ℃, and the time is 60～90min, relative humidity 70～90%.
The stripped sprouting method of pollen is to measure the effective ways of Pollen Activity, and reproducibility of results is high, generally comprises following steps: collect pollen, place nutrient solution to carry out isolated culture pollen, microscopically is observed the pollen germination situation, the statistics pollen germination rate.
The sprouting ability of pollen is one of index of reflection Pollen Activity, because pollen only has sprouting and grows pollen tube, just can pass style, enters ovary, finishes fertilization.The sprouting ability of pollen represents with pollen germination rate usually, can measure the vigor of pollen by the germination rate of statistics pollen.
The nutrient solution of isolated culture, temperature, time are the principal elements that accuracy rate is measured in impact, and the present invention is optimized culture temperature, time, nutrient solution prescription, vigor that can the described pollen of Fast Measurement.
Lycoris plants of the present invention refers to Lycoris aurea, changes brocade flower or Lycoris.
The method of described collection pollen is: gather the flower pesticide that is about to cracking, placed dry vessel dry 3～6 days, until then the pollen instant of complete cracking collects pollen.For the ease of collecting pollen, can in vessel, put into one deck template.Certainly, also can directly gather at the flower pesticide that ftractures.
During isolated culture, can drip 2～3 nutrient solutions with the pollen uniformly dispersing on the spill slide glass, mixing leaves standstill cultivation gently, and too much nutrient solution overflows slide glass easily.
Different plant pollens exsomatize to sprout needs different nutrient solutions, the sprouting of each components influence pollen of nutrient solution, and the nutrient solution of isolated culture of the present invention is comprised of sucrose, boric acid and water.
Sucrose is not only pollen germination and pollen tube growth Major Nutrient material source, also keeping the osmotic pressure of external environment, sucrose concentration is excessively low, can cause extraneous sucrose concentration to be lower than the osmotic pressure of pollen inside, pollen wall breaks, and content is separated out, pollen loses vitality, and sucrose concentration is when too high, thereby can cause again the plasmolysis of pollen to suppress to sprout, and sucrose concentration of the present invention is preferably 40～60g/L.
Boric acid can increase pollen to absorption, running and the metabolism of sucrose, form sugared boric acid complex body, increase oxygen absorption, and the composition of promotion formation pollen tube-pectin thing is synthetic, thereby promote pollen germination and pollen tube growth, the concentration of described boric acid is preferably 20～25mg/L.
The sprouting of pollen is the result of a series of biochemical reactions in the pollen cell, and biochemical reactions to need to carry out in order the temperature that suits, behind the isolated culture certain hour, germination rate and the pollen tube growth of pollen are in steady state, preferably, the temperature of described isolated culture is 27～30 ℃.
In addition, in the isolated culture process of pollen, in order to prevent the evaporation of described nutrient solution, during isolated culture, keeping the relative humidity of environment is 70%～90%.
After in-vitro pollen is cultivated for some time, will bear polliniferous slide glass and place microscopically to observe, and reach 1 times of the length of pollen granule own and above pollen with pollen tube length and be considered as sprouting the statistics germination rate.
The present invention explores the isolated culture condition of lycoris plants pollen, for the mensuration of lycoris plants Pollen Activity provides a kind of simple, quick, feasible method, for the cross-breeding of lycoris plants provides the basis.
Description of drawings
Fig. 1 changes the image that brocade flower in-vitro pollen is cultivated 60min;
Fig. 2 changes the image that brocade flower in-vitro pollen is cultivated 80min;
Fig. 3 is the image that the Lycoris aurea in-vitro pollen is cultivated 40min;
Fig. 4 is the image that the Lycoris aurea in-vitro pollen is cultivated 60min;
Fig. 5 is the image that the Lycoris in-vitro pollen is cultivated 60min;
Fig. 6 is the image that the Lycoris in-vitro pollen is cultivated 90min.
Embodiment
Embodiment 1
(1) the uncracked brocade flower flower pesticide that changes is put into the desiccation culture ware that template is housed, culture dish is placed the room dryer 4 days that fills discolour silica gel, shed to pollen.
(2) get an amount of pollen with dissecting needle and enter in the spill slide glass, drip gently and contain sucrose 50g/L, 2～3 of the nutrient solutions of boric acid 20mg/L, gently mixing.
(3) slide glass is put into gently the moisture preservation box of relative humidity 80%, placed 27 ℃ environment to cultivate 60min.
(4) take out gently slide glass, prevent strenuous vibration, in case the pollen tube fracture, place and observe the pollen germination situation under 60 power microscopes, observe the great-hearted pollen germination of tool in the visual field and go out pollen tube, be about 1～4 times of the length of pollen granule own, be convenient to add up the pollen germination rate that changes the brocade flower this moment.
(5) if slide glass is continued to put into moisture preservation box to be cultivated, observe in microscopically behind the 20min, it is longer to can be observed germination of pollen tube, mutually is intertwined.
The standard of pollen germination is: pollen tube length is more than 1 times of the own length of pollen granule reaches.
The pollen number of germination rate=sprouting/total pollen granule number
Select 10 visuals field, the pollen granule number is greater than 50 in each visual field, and the germination rate mean value that calculates 10 visuals field is the germination rate of pollen.
Table 1 changes brocade flower pollen germination rate
| Time | 60min | 80min |
| Germination rate | 65.747％ | 70.122％ |
Shown in Fig. 1, Fig. 2 and table 1, after changing brocade flower pollen sprouts 60min in control environment, the great-hearted pollen of tool has all grown pollen tube, and length is more suitable, is convenient to observe add up pollen germination rate, and germination rate is 65.747%; If continue to cultivate 20min, then germination of pollen tube is more long, mutually covers and twines, and the pollen of easily will not sprouting is mistaken for to be sprouted, and therefore the germination rate of statistics is slightly high, and germination rate is 70.122%.
Embodiment 2
The Lycoris aurea flower pesticide that (1) will soon ftracture is evenly put into the desiccation culture ware that template is housed, and culture dish was placed room dryer 4 days, sheds to pollen.
(2) get an amount of pollen with dissecting needle and enter the spill slide glass, drip gently and contain sucrose 40g/L, 2～3 of the nutrient solutions of boric acid 20mg/L, gently mixing.
(3) slide glass is put into gently the moisture preservation box of relative humidity 80%, placed 30 ℃ environment to cultivate 40min.
(4) take out gently slide glass, prevent strenuous vibration, in case the pollen tube fracture places and observes the pollen germination situation under 60 power microscopes, part has blodynamic pollen and has just sprouted pollen tube, and length is not as good as 1 times of the length of pollen granule own.
(5) slide glass is put into moisture preservation box again, observe after cultivating 20min in 30 ℃ the environment, under 60 power microscopes, observe the pollen tube that the great-hearted pollen germination of visible tool goes out 1～4 times of himself length.
Lycoris aurea pollen germination rate method of calculation are with embodiment 1.
Table 2 Lycoris aurea pollen germination rate
| Time | 40min | 60min |
| Germination rate | 1.135％ | 32.511％ |
The standard of Lycoris aurea pollen germination is consistent with the standard of changing brocade flower pollen germination, and pollen tube length is then to be sprouting more than 1 times of the own length of pollen granule reaches, and determines that the time of sprouting is the prerequisite of accurate statistics germination rate.Shown in Fig. 3, Fig. 4 and table 2, after Lycoris aurea pollen was cultivated 40min, the partial pollen pipe was just sprouted as shown in FIG., length is not as good as 1 times of the length of pollen granule own, the great-hearted pollen of part tool is not also sprouted, and therefore be not the best period of observing pollen germination this moment, and germination rate is 1.135%; After continuing to cultivate 20min, the great-hearted pollen germination of tool goes out long pollen tube, and be easy to observe the pollen germination situation this moment, and germination rate is 32.511%.
Embodiment 3
(1) uncracked Lycoris flower pesticide is evenly put into the desiccation culture ware that template is housed, culture dish was placed room dryer 6 days, shed to pollen.
(2) get an amount of pollen with dissecting needle and enter in the spill slide glass, drip gently and contain sucrose 60g/L, 2～3 of the nutrient solutions of boric acid 25mg/L, gently mixing.
(3) slide glass is put into gently the moisture preservation box of relative humidity 75%, placed 27 ℃ environment to cultivate 60min.
(4) take out gently slide glass, prevent strenuous vibration, in case pollen tube fracture places and observe the pollen germination situation under 60 power microscopes, see that partial pollen sprouts longer pollen tube, part is still sprouted continuing.
(5) the spill slide glass is replaced gently continue to be cultivated, again observe under 60 power microscopes behind the 30min, can see that the great-hearted pollen germination of tool goes out long pollen tube, not vigourous pollen does not change.
Lycoris pollen germination rate method of calculation are with embodiment 1.
Table 3 Lycoris pollen germination rate
| Time | 60min | 90min |
| Germination rate | 14.684％ | 29.637％ |
Shown in Fig. 5, Fig. 6 and table 3, the time of Lycoris pollen germination is comparatively long, may be relevant with self pollen structure, characteristic.If pollen is cultivated 60min and observed, the great-hearted pollen germination of part tool this moment goes out pollen tube, and the great-hearted pollen of part tool is still being sprouted, and add up its germination rate this moment can make germination rate on the low side, and germination rate is 14.684%; After continuing to cultivate 30min, the pollen tube that goes out of the great-hearted pollen germination of tool all long (be 1 times of the length of pollen granule own and more than) then, any variation does not occur in the great-hearted pollen of tool, and it is comparatively accurate that calculate germination rate this moment, and germination rate is 29.637%.
Can be drawn by three above embodiment results, measuring the Lycoris Pollen Activity can be slightly different observing time because of different kinds, different culture condition, but operation steps, culture condition are roughly the same, so this kind measuring method can be summed up as the method for measuring the Lycoris Pollen Activity.
Claims (8)
1. the method for a stripped sprouting method mensuration lycoris plants Pollen Activity comprises collection pollen, places nutrient solution to carry out isolated culture described pollen, it is characterized in that,
The prescription of described nutrient solution is: sucrose 30～80g/L, boric acid 15～25mg/L;
The temperature of described isolated culture is 25～35 ℃, and the time is 60～90min, relative humidity 70%～90%.
2. the method for claim 1 is characterized in that, described lycoris plants is Lycoris aurea, change brocade flower or Lycoris.
3. the method for claim 1 is characterized in that, the concentration of described sucrose is 40～60g/L, and the concentration of boric acid is 20～25mg/L.
4. the method for claim 1 is characterized in that, the temperature of described isolated culture is 27～30 ℃.
5. the method for claim 1 is characterized in that, the method for described collection pollen is: gather the flower pesticide that is about to cracking, placed dry vessel dry 3～6 days, until the pollen instant of complete cracking is collected pollen.
6. the method for claim 1 is characterized in that, described lycoris plants is Lycoris aurea; Described nutrient solution prescription is: sucrose 40g/L, boric acid 20mg/L; The temperature of described isolated culture is 30 ℃, and the time is 60min, relative humidity 80%.
7. the method for claim 1 is characterized in that, described lycoris plants is for changing the brocade flower; Described nutrient solution prescription is: sucrose 50g/L, boric acid 20mg/L; The temperature of described isolated culture is 27 ℃, and the time is 60min, relative humidity 80%.
8. the method for claim 1 is characterized in that, described lycoris plants is Lycoris; Described nutrient solution prescription is: sucrose 60g/L, boric acid 25mg/L; The temperature of described isolated culture is 27 ℃, and the time is 90min, relative humidity 75%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103430659A (en) * | 2013-09-05 | 2013-12-11 | 镇江瑞繁农艺有限公司 | Method for determining lotus pollen viability |
| CN103837534A (en) * | 2014-02-25 | 2014-06-04 | 浙江大学 | Method for testing pollen viability of white peony root |
| CN106337034A (en) * | 2016-08-24 | 2017-01-18 | 文山苗乡三七科技有限公司 | Method for determining vitality of pseudo-ginseng pollen |
| CN106520660A (en) * | 2016-10-11 | 2017-03-22 | 广西壮族自治区林业科学研究院 | Method for measuring pollen viability of bougainvillea spectabilis willd |
| CN113699095A (en) * | 2021-08-10 | 2021-11-26 | 江苏省中国科学院植物研究所 | Culture medium for in-vitro pollen germination of larch and method for determining pollen viability by using culture medium |
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2012
- 2012-09-20 CN CN2012103523115A patent/CN102876768A/en active Pending
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103430659A (en) * | 2013-09-05 | 2013-12-11 | 镇江瑞繁农艺有限公司 | Method for determining lotus pollen viability |
| CN103430659B (en) * | 2013-09-05 | 2015-03-04 | 镇江瑞繁农艺有限公司 | Method for determining lotus pollen viability |
| CN103837534A (en) * | 2014-02-25 | 2014-06-04 | 浙江大学 | Method for testing pollen viability of white peony root |
| CN106337034A (en) * | 2016-08-24 | 2017-01-18 | 文山苗乡三七科技有限公司 | Method for determining vitality of pseudo-ginseng pollen |
| CN106520660A (en) * | 2016-10-11 | 2017-03-22 | 广西壮族自治区林业科学研究院 | Method for measuring pollen viability of bougainvillea spectabilis willd |
| CN113699095A (en) * | 2021-08-10 | 2021-11-26 | 江苏省中国科学院植物研究所 | Culture medium for in-vitro pollen germination of larch and method for determining pollen viability by using culture medium |
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