CN104232561A - In-vitro culture method of salix saposhnikovii pollen - Google Patents
In-vitro culture method of salix saposhnikovii pollen Download PDFInfo
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- CN104232561A CN104232561A CN201410491744.8A CN201410491744A CN104232561A CN 104232561 A CN104232561 A CN 104232561A CN 201410491744 A CN201410491744 A CN 201410491744A CN 104232561 A CN104232561 A CN 104232561A
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Abstract
The invention discloses an in-vitro culture method of salix saposhnikovii pollen. The in-vitro culture method of salix saposhnikovii pollen comprises the following steps: (1) collecting pollen; (2) preparing a culture medium; (3) determining a culture method; (4) setting the culture temperature and the culture time. The salix saposhnikovii pollen viability measured by the in-vitro culture medium is more accurate than the pollen viability measured by an ordinary staining method; the salix saposhnikovii pollen has a good germinating effect in the culture medium and can be easily observed; the pollen is cultured in a solid culture medium, so that an experimental error caused by flowing of a liquid culture medium is avoided; the in-vitro culture method of salix saposhnikovii pollen is low in cost and easy to operate; the in-vitro culture method of salix saposhnikovii pollen has an important practical and promotional significance in propagation of salix saposhnikovii.
Description
Technical field
The present invention relates to a kind of method of plant pollen isolated culture, specifically relate to a kind of shrub willow in-vitro pollen cultural method.
Background technology
Salix (
salixl.) be the maximum genus of Salicaceae, about 550 kinds, China sallow 255 kinds, accounts for 46% of whole world sum, be whole world sallow at most, the abundantest area, extensively distribute in national each province, district, and the Salix overwhelming majority is shrub willow.Shrub willow is also important energy seeds in American-European countries.
Pollen is as one of the form of shrub willow kind matter, and containing all gene types of shrub willow, have abundant genetic diversity, is the important materials of shrub willow preserving seed and cross-breeding.But most of shrub willow dioecy, flowering asynchronism, and the pollen natural condition lower life-span is short, this brings great inconvenience to the matter research of shrub willow kind and cross-breeding work thereof, is also unfavorable for the inherited character research of shrub willow simultaneously.Although willow pollen collection having application with being housed in the breeding of European willow, it being extracted and preserves method does not specifically describe.1996, Stanto did to study in great detail to the collection of Poplar Pollen and storage, and the method is not also suitable for willow.Poplar flower is typical anemophilous pollination, and willow is entomophilous pollination, and there is one deck adhesive substance on willow pollen surface.Wang Yuanxiu utilizes MS
0substratum detects the Pollen Activity of five kinds of willows and Eclectics offspring thereof, finds that between willow kind, the pollen viability between cross-fertilize seed and between parent hybrid differs greatly.
Summary of the invention
The object of the present invention is to provide a kind of convenient and swift, can the in-vitro culture method of Accurate Determining shrub willow pollen viability.
The present invention is achieved through the following technical solutions: a kind of shrub willow in-vitro pollen cultural method, it comprises the steps:
(1) collection of pollen: adopted back and be placed in water planting in greenhouse by half-open shrub willow staminiferous plant spray, after spending and blooming, collects pollen, for subsequent use;
(2) preparation of substratum: substratum main component is sucrose, boric acid, soluble calcium salt and agar, is poured into by substratum in culture dish for subsequent use;
(3) pollen cultures: on the substratum that fresh shrub willow pollen uniformly dispersing step (1) gathered is prepared in step (2), be placed in illumination box illumination cultivation;
(4) culture temperature and time is set: the incubator temperature in step (3) is set as 20 ~ 25 degrees Celsius, and the illumination cultivation time is can carry out germination rate observation after 10 ~ 18 hours, and Pollen Activity can reach 60%-80%.
In step (1), described shrub willow staminiferous plant spray be spring on the shrub willow staminiferous plant of robust growth, in coppice shoot, the more branch of top clip bud satiation, bud number.This branch development is good, and nutritional sufficiency, ripening degree is high, and later stage pollen germination rate is high.
In step (1), described hot-house culture condition is: temperature 20 ~ 25 DEG C, illumination 2000 Lux, light application time 16 h/d, relative humidity 65% ~ 75%.
In step (2), described substratum is: 200 mg/L boric acid+100 ~ 200 mg/L nitrocalcite+50 ~ 200 g/L sucrose+7 g/L agar, and medium component ratio there are differences because shrub willow kind is different.Sucrose provides carbon source for willow in-vitro pollen germination; H
3bO
3main Function in pollen germination to increase sugared absorption, running and metabolism, promotes the synthesis of the composition pectin thing forming pollen tube wall; External source Ca
2+the growth dynamically affecting pollen tube in pollen tube is regulated, the Ca of lower concentration by calcium channel
2+concentration is conducive to willow pollen germination.
Step (4) shrub willow culture temperature is 20-25 degree Celsius.Described pollen germination is the result of a series of biochemical reaction in pollen cell, and one of suitable temperature primary condition that to be biochemical reaction normally carry out.Between different plant there is significant difference in the optimum temperuture of pollen germination and pollen tube growth.
In step (4), described pollen germination needs the regular hour, and the incubation time of shrub willow is 10 ~ 18h, and with the prolongation of incubation time, pollen germination takes the lead in raising trend gradually; After certain hour, germination rate no longer increases, but along with the prolongation of incubation time, pollen tube growth is long, is not easy to observation.Best incubation time is 12h.
Beneficial effect: compared with prior art, the invention has the advantages that:
1, adopt isolated culture base of the present invention to cultivate pollen, the developmental state of pollen tube can be observed directly when surveying shrub willow pollen viability, reflecting the vitality of pollen more intuitively.The principal element concentration that in the present invention, three, sucrose, boric acid and nitrocalcite affect shrub willow pollen germination is suitable for, and the sucrose avoided because of excessive concentrations can cause pollen to produce plasmolysis, causes protoplast dehydration, suppresses the phenomenon of pollen germination.
2, isolated culture of the present invention is adopted to survey shrub willow pollen viability compared with other Determination Staining pollen viabilities, cost is low, easy to operate, and avoid in staining and enter cell completely and the error that causes because pollen cell in statistics germination rate process suffers to destroy dye liquor.
3, adopt the isolated culture base of the present invention cultivation shrub willow pollen test period short, culture condition is simple.
In sum: the pollen viability that the shrub willow pollen viability that isolated culture base of the present invention records records than ordinary stain method is more accurate, shrub willow pollen is sprouted effective on this substratum, easy observation, and the testing error avoided causing because of liquid nutrient medium flowing cultivated by pollen in solid medium, cost is low, easy to operate, shrub willow propagation production has important practice and dissemination.
Accompanying drawing explanation
Fig. 1 is the impact that incubation time is cultivated shrub willow in-vitro pollen; In figure, P63 represents Salix suchowensis; P102 represents silver-colored willow; 2333 represent wormwood artemisia willow X hair branch willow; 2383 represent Salix suchowensis X wormwood artemisia willow.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
embodiment 1
(1) collection of pollen: by half-open Yin Liu (
s. argyracea) staminiferous plant spray is adopted back and to be placed in water planting in greenhouse in full bloom to spending, temperature controls at 20 ~ 25 degrees Celsius, wait spend in full bloom after, collect pollen in the centrifuge tube of 5mL,
(2) preparation of substratum: the optimal medium filtering out silver-colored willow is 150g/L sucrose+200mg/L boric acid+100mg/L nitrocalcite+7g/L agar, for subsequent use;
(3) pollen cultures: on the substratum prepared in step (2) by fresh silver-colored willow pollen uniformly dispersing, and be placed in LRH-300G illumination box illumination cultivation, illuminance is 2000Lux;
(3) culture temperature is set: incubator temperature is set as 25 degrees Celsius;
(4) set incubation time: along with the prolongation of incubation time, pollen germination takes the lead in raising trend gradually; After 12h, germination rate no longer increases.Result is as shown in table 1.
Table 1 incubation time and silver-colored willow in-vitro pollen cultivate the relation that germination rate changes
| Incubation time (h) | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | 12 | 14 | 16 |
| Germination rate (%) | 3.8 | 9.0 | 12.0 | 15.6 | 22.0 | 39.7 | 47.6 | 60.0 | 60.7 | 60.7 |
| Standard deviation | 4.7 | 2.3 | 3.8 | 9.1 | 10.0 | 7.1 | 8.5 | 10.6 | 10.8 | 10.8 |
embodiment 2
(1) collection of pollen: by half-open Salix suchowensis (
s. suchowensis) staminiferous plant spray is adopted back and to be placed in water planting in greenhouse in full bloom to spending, temperature controls at 20 ~ 25 degrees Celsius, wait spend in full bloom after, collect pollen in the centrifuge tube of 5mL,
(2) preparation of substratum: the optimal medium filtering out silver-colored willow is 100g/L sucrose+200mg/L boric acid+200mg/L nitrocalcite+7g/L agar, for subsequent use;
(3) pollen cultures: on the substratum prepared in step (2) by fresh Salix suchowensis pollen uniformly dispersing, and be placed in LRH-300G illumination box illumination cultivation, illuminance is 2000Lux;
(3) culture temperature is set: incubator temperature is set as 25 degrees Celsius;
(4) set incubation time: along with the prolongation of incubation time, pollen germination takes the lead in raising trend gradually; After 12h, germination rate no longer increases.Result is as shown in table 2.
Table 2 incubation time and Salix suchowensis in-vitro pollen cultivate the relation that germination rate changes
| Incubation time (h) | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | 12 | 14 | 16 |
| Germination rate (%) | 0.0 | 7.0 | 8.8 | 10.6 | 20.9 | 39.8 | 44.7 | 49.6 | 51.2 | 51.2 |
| Standard deviation | 0.0 | 3.3 | 1.3 | 1.8 | 5.2 | 2.8 | 6.5 | 8.3 | 4.6 | 4.6 |
embodiment 3
(1) collection of pollen: by half-open Salix suchowensis × wormwood artemisia willow hybrid (
s. suchowensis×
s. viminalis) staminiferous plant spray is adopted back and to be placed in water planting in greenhouse in full bloom to spending, temperature controls at 20 ~ 25 degrees Celsius, wait spend in full bloom after, collect pollen in the centrifuge tube of 5mL,
(2) preparation of substratum: the optimal medium filtering out Salix suchowensis × wormwood artemisia willow is 50g/L sucrose+200mg/L boric acid+100mg/L nitrocalcite+7g/L agar, for subsequent use;
(3) pollen cultures: on the substratum prepared in step (2) by fresh Salix suchowensis pollen uniformly dispersing, and be placed in LRH-300G illumination box illumination cultivation, illuminance is 2000Lux;
(3) culture temperature is set: incubator temperature is set as 25 degrees Celsius;
(4) set incubation time: along with the prolongation of incubation time, pollen germination takes the lead in raising trend gradually; After 12h, germination rate no longer increases.Result is as shown in table 3.
The relation that table 3 incubation time and Salix suchowensis × wormwood artemisia willow hybrid pollen isolated culture germination rate change
| Incubation time (h) | 0.5 | 1 | 2 | 4 | 6 | 8 | 10 | 12 | 14 | 16 |
| Germination rate (%) | 8.1 | 12.7 | 26.4 | 31.1 | 36.7 | 40.5 | 44.8 | 48.8 | 48.8 | 48.8 |
| Standard deviation | 2.0 | 3.5 | 5.1 | 11.6 | 7.8 | 8.1 | 7.3 | 7.7 | 7.7 | 7.7 |
The above discloses the present invention with preferred embodiment.So it is not intended to limiting the invention, and all employings are equal to replacement or the technical scheme that obtains of equivalent transformation mode, all drop within protection scope of the present invention.
Claims (7)
1. a shrub willow in-vitro pollen cultural method, it is characterized in that, it comprises the steps:
(1) collection of pollen: adopted back and be placed in water planting in greenhouse by half-open shrub willow staminiferous plant spray, after spending and blooming, collects pollen, for subsequent use;
(2) preparation of substratum: substratum main component is sucrose, boric acid, soluble calcium salt and agar, is poured into by substratum in culture dish for subsequent use;
(3) pollen cultures: on the substratum that fresh shrub willow pollen uniformly dispersing step (1) gathered is prepared in step (2), be placed in illumination box illumination cultivation;
(4) culture temperature and time is set: the incubator temperature in step (3) is set as 20 ~ 25 degrees Celsius, and the illumination cultivation time is can carry out germination rate observation after 10 ~ 18 hours.
2. according to the shrub willow in-vitro pollen cultural method described in claim 1, it is characterized in that: in step (1), described shrub willow staminiferous plant spray be spring on the shrub willow staminiferous plant of robust growth, in coppice shoot, the more branch of top clip bud satiation, bud number.
3., according to the shrub willow in-vitro pollen cultural method described in claim 1, it is characterized in that: in step (1), described hot-house culture condition is: temperature 20 ~ 25 DEG C, illumination 2000 Lx, light application time 16 h/d, relative humidity 65% ~ 75%.
4. according to the shrub willow in-vitro pollen cultural method described in claim 1, it is characterized in that: in step (2), described substratum is: 200 mg/L boric acid+100 ~ 200 mg/L nitrocalcite+50 ~ 200 g/L sucrose+7 g/L agar.
5., according to shrub willow in-vitro pollen cultural method described in claim 1, it is characterized in that: in step (3), illuminance is 2000Lux.
6. according to shrub willow in-vitro pollen cultural method described in claim 1, it is characterized in that: in step (4), the culture temperature of shrub willow is 25 DEG C.
7. shrub willow in-vitro pollen cultural method according to claim 1, it is characterized in that: in step (4), the incubation time of shrub willow pollen germination is 12h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105176910A (en) * | 2015-10-28 | 2015-12-23 | 山东省林业科学研究院 | Superior dry-land willow tree pollen in-vitro germination culture medium and method for determining viability of dry-land willow pollen |
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| CN103087975A (en) * | 2013-01-11 | 2013-05-08 | 南京林业大学 | Apocarya pollen in-vitro germination liquid culture medium and application thereof in measuring pollen activity |
| CN103627668A (en) * | 2013-11-20 | 2014-03-12 | 西北农林科技大学 | In-vitro wheat pollen germination method |
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Patent Citations (3)
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| CN102972296A (en) * | 2012-12-05 | 2013-03-20 | 广西壮族自治区林业科学研究院 | Acacia rachii pollen in-vitro culture activity detection technology |
| CN103087975A (en) * | 2013-01-11 | 2013-05-08 | 南京林业大学 | Apocarya pollen in-vitro germination liquid culture medium and application thereof in measuring pollen activity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105176910A (en) * | 2015-10-28 | 2015-12-23 | 山东省林业科学研究院 | Superior dry-land willow tree pollen in-vitro germination culture medium and method for determining viability of dry-land willow pollen |
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