CN105112492A - Detection method for kapok pollen viability - Google Patents
Detection method for kapok pollen viability Download PDFInfo
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- CN105112492A CN105112492A CN201510586503.6A CN201510586503A CN105112492A CN 105112492 A CN105112492 A CN 105112492A CN 201510586503 A CN201510586503 A CN 201510586503A CN 105112492 A CN105112492 A CN 105112492A
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Abstract
The invention relates to a detection method for kapok pollen viability. The detection method comprises the following steps that step 1, pollen is collected; step 2, culture media are prepared, wherein the agar culture media are prepared with distilled water and are prepared from 25-50 mg/L of boric acid, 90-110 g/L of sucrose and 7-9 g/L of agar, and the culture media are placed into culture dishes for standby application; step 3, pollen germination and culture are performed, wherein the pollen is dissolved in a culture solution, so that a pollen solution is formed, next, the pollen solution is sprayed onto the surfaces of the culture media, evenly spread and cultured at constant temperature under the condition of constant lighting; step 4, pollen viability detection is performed, wherein the culture dishes placed below an inverted microscope for observation after culture is completed, the amount of the pollen in all the culture dishes and the amount of the germinated pollen are counted, and then the pollen viability is calculated. According to the detection method, results are visual, data are reliable, the viability of fresh kapok pollen can be detected within 15 h, and therefore the detection method has important practical significance for hybrid seed production of kapok and cultivation of excellent new species of kapok.
Description
Technical field
The present invention relates to cross-breeding technology field, particularly relate to the detection method of kapok Pollen Activity.
Background technology
Kapok (BombaxceibaL.), has another name called silk cotton, Ying Xiongshu, Flos Bombacis Malabarici, Bombacaceae Gossampinus fallen leaves megaphanerophyte, Gao Keda 25m, bark canescence, and trunk has cone shape thick thorn usually; Branch is open and flat.Palmately compound leaf, petiole grows 10 ~ 20cm, 5 ~ 7, leaflet, and Long Circle is to oval shape lanceolar, and grow 10 ~ 16cm, wide 3.5 ~ 5.5cm, top is gradually sharp, and base portion is wealthy or gradually narrow, Quan Yuan, and two sides is all without hair, and petiolule grows 1.5 ~ 4cm; Stipule is little.Flower Dan Shengzhi top armpit, usually red, sometimes orange red, orange-yellow and yellow, diameter is 10 ~ 13cm about; Calyx cup-shaped, long 2 ~ 3cm, wide 2 ~ 3cm; Petal meat, the shape of falling ovum Long Circle, long 8 ~ 10cm, wide 3 ~ 4cm; Staminal tube is short, and base portion is thick, and upwards gradually thin, inside take turns part filigree upper part 2 and pitch, foreign steamer stamen is most, integrated 5 bundles, and every Shu Huasi more than 10 pieces is longer; Ovary 5 Room, style is longer than stamen, bar-shaped, and outstanding, column cap 5 splits.Capsule Long Circle, long 10 ~ 15cm, wide 4.5 ~ 8cm, close by the long pubescence of canescence; Seed is most, and obovate is smooth.In 3 ~ April of florescence, fruit is ripe for summer.
Kapok originate in Guangdong, Guangxi, Hainan, Hong Kong, Sichuan, Yunnan, Guizhou,
taiWan, China, the provinces and regions such as Fujian, South Asia, South East Asia and Australia
northern.Happiness light, deep-rootedness, drought-resistant, the warm hot climate of happiness; Be born in dry-hot valley and the savanna of height above sea level 1400 ~ below 1700m, also can grow in cheuch monsoonal forest.One of the Key species of south China area road, unit greening and scenic forest, often does shade tree, shade tree and landscape tree cultivation.
The tall and big grandness of silk cotton tree, towering tall and straight, tree crown is neatly glorious.In 3 ~ April of spring, first spend posterior lobe, the florescence reaches 2 months, contains and spends period, completely sets redness, the sometimes flowering such as orange red, orange-yellow and yellow, affords a magnificent spectacle, add the gathering honey such as birds or honeybee, more add temperament and interest; Dan Huacong begins flower to withering 4 ~ 5 days, 5, petal, the shape of falling ovum Long Circle.Summer, green shade was as lid, and autumn and winter fallen leaves frontal lobe look turns yellow.Kapok is always considered as the symbol of hero by people, be the city flower in the city such as city of Guangzhou Guangdong and city of Sichuan, the excellent shade tree in south China area, shade tree and landscape tree.
Kapok be the south China area masses like road, unit greening and one of Landscape Forest seeds, wide accommodation is the ornamental flower with extensive exploitation application prospect.But kapok is naturally solid both by the ecological characteristics such as weather and soil, and again by the impact of the biological characteristics such as the age of tree and growing way, Natural seed setting rate is usually not high.The kapok florescence reaches 60 days, and excellent kapok individual plant ubiquity flowering asynchronism, is comparatively difficult to cultivate improved Varieties by spontaneous pollination.Carry out artificial hybridization controlled pollination, genetic resources is recombinated, it is the main path cultivating kapok improved Varieties, therefore measured by Pollen Activity, understand each batch of Flower of Common Bombax opaque amount, to carrying out hybridization controlled pollination combinations of pairs, the workload of prediction hybridization controlled pollination and success ratio, save the cost that pollen is preserved and hybridized controlled pollination, cultivate kapok improved Varieties and have important practical significance, good social benefit, ecological benefits and economic benefit can be obtained.
Summary of the invention
Based on this, the object of this invention is to provide a kind of detection method of kapok Pollen Activity.
Realize the concrete technical scheme of foregoing invention object as follows:
A detection method for kapok Pollen Activity, comprises the following steps:
(1) pollen collecting: gather bud, by the threshing in centrifuge tube of the pollen in pollen sac, dry;
(2) preparation of substratum: prepare nutrient agar with distilled water, wherein boronic acid containing 25 ~ 50mg/L, sucrose 90 ~ 110g/L, agar 7 ~ 9g/L, load culture dish by substratum stand-by;
(3) pollen germination is cultivated: be dissolved in nutrient solution by step (1) gained pollen, forms pollen liquid, then pollen liquid is sprayed onto the surface of substratum prepared by step (2), evenly spread out, constant temperature, permanent illumination cultivation; Described nutrient solution is the boric acid of 25 ~ 50mg/L and the sucrose solution of 90 ~ 110g/mL;
(4) Pollen Activity detects: observe under culture dish being placed in inverted microscope, the pollen that pollen tube length is greater than pollen granule diameter is considered as the pollen sprouted, each culture dish random selecting 3-6 nonoverlapping visual field, add up the quantity of the quantity of the pollen of each culture dish, the pollen of sprouting, calculate the vigor of pollen.
Wherein in an embodiment, described pollen collecting comprises the following steps:
(1) bud collection: 3 ~ April, clip flower bud body is full, N/D, the kapok bud of large flower bud phase, installs with sealed bag, for subsequent use;
(2) pollen threshing: petal tip is torn, below pollen sac, about 0.18 ~ 0.22cm cuts off, pollen sac is placed in culture dish, put into silica gel drier dry, pollen sac is split, the pollen sac split is loaded centrifuge tube, vibrate in eddy mixer, make pollen granule depart from pollen sac and be bonded at centrifuge tube inwall, stop operating, outwell sky pollen sac;
(3) pollen is dry: put back to by sticky polliniferous centrifuge tube in silica gel drier dry.
Wherein in some embodiments, described nutrient agar boronic acid containing 50mg/L, sucrose 100g/L, agar 8g/L.
Wherein in some embodiments, described nutrient agar boronic acid containing 25mg/L, sucrose 100g/L, agar 8g/L.
Wherein in an embodiment, the time of cultivating described in pollen germination culturing step is 2 ~ 3h.
Wherein in an embodiment, the temperature of cultivating described in pollen germination culturing step is 23 ~ 27 DEG C.
Wherein in an embodiment, the time dry described in pollen threshing step is 9 ~ 10h.
Wherein in an embodiment, described in pollen threshing step, the rotating speed of eddy mixer is 2700 ~ 2900 revs/min.
Wherein in an embodiment, the time of the vibration of eddy mixer described in pollen threshing step is 2 ~ 3 minutes.
Wherein in an embodiment, described in pollen drying step dry for the relative water content being dried to pollen be 20 ~ 25%.
When doing Pollen Activity and detecting, also can use staining, great-hearted kapok pollen all or locally can be dyed to redness or garnet in the TTC reagent of 0.5% and 1%, does not have vital pollen to be brown.But when adding up Pollen Activity, the pollen dying garnet or local dyeing is affected by human factors comparatively large, and do not have the visual result of film solid media method, estimation control pollination workload and success ratio exist certain error.Therefore, detection method visual result compared with TTC staining of kapok Pollen Activity of the present invention, data are reliable, method of the present invention can detect the vigor of fresh kapok pollen in 15h, to carrying out hybridization controlled pollination combination, the workload of prediction hybridization controlled pollination and success ratio, save the cost that pollen is preserved and hybridized controlled pollination, cultivates kapok improved Varieties and have important practical significance.
Accompanying drawing explanation
fig. 1for kapok pollen viability examination microscope photographing
figure.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
The vigor of kapok pollen is measured by the following method in the present embodiment:
1 pollen collecting
1.1 bud collections
In 3 ~ April, when fine, on excellent kapok individual plant, full, the N/D of clip flower bud body, the bud of large flower bud phase, install with sealed bag, stick to gather number and acquisition time to take back laboratory for subsequent use.
1.2 pollen threshings
With hand, petal tip is torn, littlely cut about 0.2cm below pollen sac with clean and cut off, pollen sac is placed in the culture dish being lined with pan paper, put into the dry 9 ~ 10h of silica gel drier, when pollen sac major part is split, pollen sac is put into the centrifuge tube of 2ml, build the lid of centrifuge tube, numbering, be placed on vibration 2 ~ 3min in the eddy mixer (its Bel VORTEX-5 eddy mixer of Haimen) that rotating speed is 2800 turns/min, when most pollen is bonded at centrifuge tube inwall, stop operating, open the lid of centrifuge tube, outwell sky pollen sac.
1.3 pollen are dry
Sticky polliniferous centrifuge tube is put back in silica gel drier dry, and in moisture eliminator, put 1 Hygrothermograph, when relative water content about 20% of Hygrothermograph in moisture eliminator, open the lid of moisture eliminator, again cover the lid of centrifuge tube, stay 3 centrifuge tubes to carry out vitality test, remaining installs with envelope and puts into sealing bag, envelope is indicated to gather number, acquisition time, collector, the content such as relative humidity, be placed in-20 degree refrigerators and preserve.
2 Pollen Activities detect
The preparation of 2.1 substratum
To prepare the substratum of 50mg/L boric acid+100g/L sucrose+8g/L agar, the preparation of substratum comprises the following steps:
(1) boric acid solution of 50mg/L is prepared with distilled water, stand-by;
(2) 10g sucrose is put into 100ml beaker, add 20 ~ 30ml boric acid solution that step (1) is prepared, after sucrose fully dissolves, be settled to 100ml, be mixed with 50mg/L boric acid+100g/L sucrose solution, stand-by as nutrient solution;
(3) 0.8g agar powder is put into the Erlenmeyer flask of 250ml, then the nutrient solution 100ml that step (2) is prepared is poured in Erlenmeyer flask, after agar powder fully dissolves, encase bottleneck with newspaper, and tie up with bungee.Erlenmeyer flask is placed in microwave oven and boils 1 ~ 2min, interval 30 ~ 40s takes out and rocks once, until boil, well-done substratum is poured in 9 90mm culture dish before curing, cover the lid of culture dish, naturally cooling, is mixed with the substratum of 50mg/L boric acid+100g/L sucrose+8g/L agar, stand-by.
2.2 pollen germinations are cultivated
Draw nutrient solution prepared by 300 μ l2.1 respectively with liquid-transfering gun and fill polliniferous centrifuge tube to 3, jiggle, pollen is made to be dissolved in solution, form pollen liquid, then with liquid-transfering gun, each pollen liquid is sprayed onto respectively the surface of 3 substratum, and with the clean glass rod that pushes away, pollen liquid is evenly spread out, cover culture dish lid, with sealed membrane sealing, numbering, is placed in 25 DEG C of artificial climate incubator constant temperature, permanent illumination cultivation.
2.3 Pollen Activities detect
After pollen germination, observe under culture dish being placed in 100 times of inverted microscopes, each culture dish random selecting 3 ~ 6 nonoverlapping visuals field, with microscope carry software take pictures (
as Fig. 1shown in), add up the quantity of the quantity of the pollen of each culture dish, the pollen of sprouting, statistics Pollen Activity, and the pollen frequency more than 100 requiring each culture dish to add up, and calculate the vigor of this batch of pollen.Calculation formula: quantity × 100% of the quantity/pollen of the pollen of Pollen Activity=sprouting, when calculating the quantity of pollen sprouted, the pollen that pollen tube length exceedes pollen granule diameter is then considered as the pollen sprouted.
3, detected result
The selection result of 3.1 substratum
With identical agar concentration, different boric acid and sucrose concentration make substratum, the pollen being numbered No. 1, mixing collected with aforesaid method carries out kapok pollen vitality test by the method described in above-mentioned " 2 Pollen Activities detect ", result shows: the substratum vigor of 50mg/L boric acid+100g/L sucrose+8g/L agar is the highest, average out to 73.9%, next is the substratum of 25mg/L boric acid+100g/L sucrose+8g/L agar, average out to 69.86% (
table 1).
table 1kapok pollen substratum is tested
The time the selection result that 3.2 pollen germinations are cultivated
With the 50mg/L boric acid+100g/L sucrose solution of 2.1 preparations for nutrient solution, with the 50mg/L boric acid+100g/L sucrose+8g/L agar of 2.1 preparations for substratum, carry out Pollen Activity by the method described in above-mentioned " 2 Pollen Activities detect " and measure.Kapok pollen tube length exceedes the pollen that pollen granule diameter is then considered as sprouting; Because pollen germination exists regular hour difference, the display of sprouting result (
table 2), when most of pollen tube length is pollen granule diameter 6 ~ 8 times, namely cultivate the Best Times that 2 ~ 3h is observation pollen germination, the Best Times that namely pollen germination is cultivated is 2 ~ 3h.
table 2pollen germination optimal viewing time test-results
The result that 3.3 Pollen Activities detect
The pollen being numbered FS16-13 and No. 1, mixing collected with aforesaid method cultivates 2 ~ 3h at the substratum of 50mg/L boric acid+100g/L sucrose+8g/L agar by the method described in 2.2 respectively, namely when most of pollen tube length is 6 ~ 8 times of pollen granule diameter, Pollen Activity is detected, detected result with method described in 2.3
as table 3shown in.
table 3method Pollen Activity measurement result sprouted by solid
4 pollen are preserved
The envelope that this batch of kapok pollen is housed is indicated the contents such as Pollen Activity, detection time, testing staff; Preserve in-20 DEG C of refrigerators, if fresh pollen vigor reaches more than 76.3%, when water content drops to 20%, preserve half a year Pollen Activity and reach more than 58.1%, preserve 1 year Pollen Activity still have more than 51.2% (
table 4), the demand of cross-breeding can be met.
table 4pollen Activity (%) under-20 DEG C of preservation conditions
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this specification sheets is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a detection method for kapok Pollen Activity, is characterized in that, comprises the following steps:
(1) pollen collecting: gather bud, by the threshing in centrifuge tube of the pollen in pollen sac, dry;
(2) preparation of substratum: prepare nutrient agar with distilled water, wherein boronic acid containing 25 ~ 50mg/L, sucrose 90 ~ 110g/L, agar 7 ~ 9g/L, load culture dish by substratum stand-by;
(3) pollen germination is cultivated: be dissolved in nutrient solution by step (1) gained pollen, forms pollen liquid, then pollen liquid is sprayed onto the surface of substratum prepared by step (2), evenly spread out, constant temperature, permanent illumination cultivation; Described nutrient solution is the boric acid of 25 ~ 50mg/L and the sucrose solution of 90 ~ 110g/L;
(4) Pollen Activity detects: observe under culture dish being placed in inverted microscope, the pollen that pollen tube length is greater than pollen granule diameter is considered as the pollen sprouted, each culture dish random selecting 3 ~ 6 nonoverlapping visuals field, add up the quantity of the quantity of the pollen of each culture dish, the pollen of sprouting, calculate the vigor of pollen.
2. the detection method of kapok Pollen Activity according to claim 1, it is characterized in that, pollen collecting comprises the following steps:
(1) bud collection: 3 ~ April, clip flower bud body is full, N/D, the kapok bud of large flower bud phase, installs with sealed bag, for subsequent use;
(2) pollen threshing: petal tip is torn, below pollen sac, 0.18 ~ 0.22cm cuts off, pollen sac is placed in culture dish, put into silica gel drier dry, pollen sac is split, the pollen sac split is loaded centrifuge tube, vibrate in eddy mixer, make pollen granule depart from pollen sac and be bonded at centrifuge tube inwall, stop operating, outwell sky pollen sac;
(3) pollen is dry: put back to by sticky polliniferous centrifuge tube in silica gel drier dry.
3. the detection method of kapok Pollen Activity according to claim 1 and 2, is characterized in that, described nutrient agar boronic acid containing 50mg/L, sucrose 100g/L, agar 8g/L.
4. the detection method of kapok Pollen Activity according to claim 1 and 2, is characterized in that, described nutrient agar boronic acid containing 25mg/L, sucrose 100g/L, agar 8g/L.
5. the detection method of kapok Pollen Activity according to claim 1 and 2, is characterized in that, the time of cultivating described in pollen germination culturing step is 2 ~ 3h.
6. the detection method of kapok Pollen Activity according to claim 1 and 2, is characterized in that, the temperature of cultivating described in pollen germination culturing step is 23 ~ 27 DEG C.
7. the detection method of kapok Pollen Activity according to claim 2, is characterized in that, the time dry described in pollen threshing step is 9 ~ 10h.
8. the detection method of kapok Pollen Activity according to claim 2, is characterized in that, described in pollen threshing step, the rotating speed of eddy mixer is 2700 ~ 2900 revs/min.
9. the detection method of kapok Pollen Activity according to claim 2, is characterized in that, the time of the vibration of eddy mixer described in pollen threshing step is 2 ~ 3 minutes.
10. the detection method of kapok Pollen Activity according to claim 2, is characterized in that, described in pollen drying step dry for the relative water content being dried to pollen be 20 ~ 25%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531441A (en) * | 2018-07-18 | 2018-09-14 | 中国热带农业科学院椰子研究所 | A kind of oil palm pollen sprouting culture medium and its germination method |
| CN108680418A (en) * | 2018-06-01 | 2018-10-19 | 广东金作农业科技有限公司 | A kind of rapid fluorescence colouring method of crop in cruciferae pollen |
Citations (1)
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|---|---|---|---|---|
| CN103837534A (en) * | 2014-02-25 | 2014-06-04 | 浙江大学 | Method for testing pollen viability of white peony root |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837534A (en) * | 2014-02-25 | 2014-06-04 | 浙江大学 | Method for testing pollen viability of white peony root |
Non-Patent Citations (2)
| Title |
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| ASHOKE BHATTACHARYA，SUDHENDU MANDAL: ""Pollination biology in Bombax ceiba Linn."，第1706-1712页", 《CURRENT SCIENCE》 * |
| 王利民: ""大花蕙兰杂交育种研究"，E048-3", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680418A (en) * | 2018-06-01 | 2018-10-19 | 广东金作农业科技有限公司 | A kind of rapid fluorescence colouring method of crop in cruciferae pollen |
| CN108680418B (en) * | 2018-06-01 | 2021-03-23 | 广东金作农业科技有限公司 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
| CN108531441A (en) * | 2018-07-18 | 2018-09-14 | 中国热带农业科学院椰子研究所 | A kind of oil palm pollen sprouting culture medium and its germination method |
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