CN103087975A - Apocarya pollen in-vitro germination liquid culture medium and application thereof in measuring pollen activity - Google Patents
Apocarya pollen in-vitro germination liquid culture medium and application thereof in measuring pollen activity Download PDFInfo
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- CN103087975A CN103087975A CN2013100108401A CN201310010840A CN103087975A CN 103087975 A CN103087975 A CN 103087975A CN 2013100108401 A CN2013100108401 A CN 2013100108401A CN 201310010840 A CN201310010840 A CN 201310010840A CN 103087975 A CN103087975 A CN 103087975A
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Abstract
The invention discloses apocarya pollen in-vitro germination liquid culture medium and application thereof in measuring pollen activity. The culture medium uses distilled water as solvent and contains sucrose, H3BO3 and Ca(NO3)2.4H2O. The cultivation method comprises hydration treatment, cultivation temperature and time adjustment. According to the apocarya pollen in-vitro germination liquid culture medium disclosed by the invention, the technical problems that the apocarya pollen germination rate is low and the germination tube growth is slow are solved, so that the pollen germination can be more than 70%. According to the culture method and the cultivation method disclosed by the invention, the pollen germination and growth of the apocarya are improved, the operation is simple, the cost is low, the period is short and the germination rate is high, so that the apocarya pollen in-vitro germination liquid culture medium has an extensive application prospect in the juglandaceae plant biology.
Description
Technical field
The invention belongs to plant pollen vitality test technical field, relate in particular to a kind of liquid nutrient medium of apocarya in-vitro pollen germination, and utilize this substratum to measure the method for apocarya Pollen Activity.
Background technology
Apocarya (Carya illinoinensis) is Juglandaceae (Julandaceae) hickory (Carya nutt) plant, has another name called pecan tree, pecan.Apocarya is one of world-renowned dry fruit tree variety, and its nut is large, shell is thin, and kernel percent is high, gets benevolence easy, and output is high.Apocarya is also important traditional oil tree, and its fat content is up to more than 70%, and unsaturated fatty acid content up to 97%, is first-class oil for cooking.Apocarya or good material are used and flower garden afforestation tree, and its woody texture is fine and smooth, and is strong but pliable in texture, is the ideal material of building, military project, upholstery and making top-grade furniture; Its tree-like tall and big, tree vigo(u)r is tall and straight, is well received viewing and admiring, shade and avenue tree planting.Apocarya originates in the U.S. and northern Mexico, now producing region centered by the U.S., be distributed in the ground such as the U.S., Canada, Mexico, Brazil, Argentina, Peru, Italy, France, Australia, Egypt, South Africa, Israel, Japan, China.
Apocarya was introduced a fine variety to China existing more than 100 year, failed so far to form industrialization, and carrying out fine-variety breeding is an important job.Carry out the research that the apocarya Pollen Activity is measured, the seed selection of germ plasm resource preservation and innovation, improved seeds is had the theory and practice meaning.
Apocarya is the different flowering plant of monoecism, mostly the kind dichogamy.Therefore in the cross-breeding process, need to preserve the pollen that gathers, after storage, the vigor of pollen is related to the success or failure of artificial pollination.The main method that Pollen Activity is measured has staining and stripped sprouting method.
Staining is by use scene of mountains reagent, pollen granule to be dyeed, and according to the height of colour-change judgement Pollen Activity, staining reagent commonly used has I2-KI solution and triphenyltetrazolium chloride (TTC) etc.Have at present the report that apocarya pollen is carried out its vigor of TTC dyeing mensuration, but can only reflect metabolism situation or the nutrition content of pollen due to staining, and the color error in judgement is larger, can not show directly and accurately the vigor of pollen.
The condition that the condition that the in-vitro pollen germination method provides and pollen are sprouted at column cap is more approaching, is to detect Pollen Activity method the most reliably.In-vitro pollen germination needs suitable substratum and culture condition, and nutrient media components commonly used is sucrose, boric acid, 20 ℃ ~ 25 ℃ of temperature.
Apocarya pollen germination difficulty, prior art fail to break through always.In juglandaceae plant, the pollen germination that is detected in walnut (Juglans regia L.) pollen is studied, but germination rate can not reflect Pollen Activity exactly only up to 2.22%.
Summary of the invention
The technical problem that solves: the application of a kind of apocarya in-vitro pollen germination liquid nutrient medium and mensuration Pollen Activity thereof is provided, on the basis of determining apocarya in-vitro pollen germination culture system, the apocarya in-vitro pollen is cultivated, and can reliable and effective mensuration apocarya Pollen Activity.
Technical scheme:
A kind of apocarya in-vitro pollen germination liquid nutrient medium, this substratum includes sucrose, H take distilled water as solvent
3BO
3And Ca (NO
3)
24H
2O。
Described solute is by the sucrose of 200g/L, the H of 200 ~ 300mg/L
3BO
3Ca (NO with 500mg/L
3)
24H
2O forms.
Above-mentioned substratum is measured the application of Pollen Activity, comprises the following steps:
1) pollen collection
Select to grow normal, without the healthy and strong apocarya plant of disease and pest, pluck the male flower inflorescence when the flower pesticide flavescence, it is spread out on template, be placed in wind sheltering dry place, next day, flower pesticide got final product loose powder; Sift out pollen, in the sealing bag of packing into, refrigerate standby;
2) the pollen rehydration is processed
In airtight container, use K
2SO
4Or CuSO
45H
2The O saturated salt solution is regulated relative humidity RH=97%, and pollen is positioned in this container, carries out the rehydration of 4 ~ 8h and processes;
3) in-vitro pollen germination and pollen tube growth
While obtaining liq substratum, its component is: 200gL
-1Sucrose+200～300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O； Drip 50 μ L liquid nutrient mediums on the groove slide glass, dip with pin the pollen that rehydration processes it is sprinkling upon on substratum equably, slide glass is put into the culture dish that is covered with moistening absorbent cotton, moisturizing is cultivated under dark, 25 ℃ of conditions; After 4h, pollen begins to sprout, the pollen tube growth growth, and after 24h, the pollen growth tends towards stability;
4) mensuration of Pollen Activity
After isolated culture 24h, each slide is chosen 5 unduplicated visuals field at random, and each visual field pollen number is no less than 50, is considered as sprouting greater than the pollen granule diameter with pollen tube length, and chooses 50 and measure pollen tube length, and each test all repeats 3 times.
Beneficial effect:
1, apocarya pollen on liquid nutrient medium provided by the invention, is cultivated under 25 ℃ of dark conditions, and its pollen germination rate can reach 74.46%, and pollen tube also can better be grown.
2, utilize this apocarya in-vitro pollen germination liquid nutrient medium and pollen tube growth determination techniques to measure the Pollen Activity measurement result reliable and stable, the pollen germination rate reflection has vigor pollen proportion, the observation of pollen tube growth and the quality of measuring reflection pollen granule upgrowth situation are for the mensuration of apocarya Pollen Activity provides a kind of reliable and effective method.
Description of drawings
Fig. 1 is that the apocarya in-vitro pollen is cultivated the pollen germination rate of different time and the statistical graph of pollen tube; Wherein, A: the pollen of sprouting; B: the pollen tube of growth 6h; C: the pollen tube of growth 24h.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
According to technical scheme of the present invention, at first prepare apocarya in-vitro pollen germination liquid nutrient medium, this substratum is take distilled water as solvent, wherein contains the sucrose of 200g/L, the H of 200 ~ 300mg/L
3BO
3, 500mg/L Ca (NO
3)
24H
2O。
On the basis of determining apocarya in-vitro pollen germination culture system, the apocarya in-vitro pollen is cultivated, and can reliable and effective mensuration apocarya Pollen Activity.
Be below the embodiment that the contriver provides, this embodiment is the more excellent example of the present invention, is mainly used in further explaining and understanding the present invention, the invention is not restricted to this embodiment.
Embodiment 1:
1, the collection of apocarya pollen
Select to grow normal, without the healthy and strong apocarya plant of disease and pest, in time pluck the male flower inflorescence when the flower pesticide flavescence, put into the sulfuric acid paper bag and take back rapidly the laboratory, it is spread out on template, be placed in wind sheltering dry place, next day, flower pesticide got final product loose powder.Sift out pollen with 120 order pharmacopeia, in the vacuum of packing into waterproof sealing bag, be placed on 20 ℃ of refrigerator cold-storages of ﹣ standby.
2, the screening of pollen rehydration time
Use K
2SO
4Or CuSO
45H
2The O saturated salt solution is regulated RH(Relative Humidity, relative humidity), allow pollen be placed on rehydration in 25 ℃, the encloses container of RH=97%, place respectively 0,1,2,3,4,8,12, after 24h, (contain 10%wt sucrose, 0.01%wtH with the BK substratum
3BO
3, 0.03%wtCa (NO
3)
24H
2O, 0.02%wtMgSO
47H
2O, 0.01%wtKNO
3) measure the germination rate of different rehydration time pollen.
Result is as shown in table 1, and it is necessary that pollen is put into that the rehydration of carrying out certain hour before substratum processes.The pollen germination rate of not doing the rehydration processing is very low, is only 2.51%.After processing by rehydration, pollen germination rate significantly raises.After rehydration 4h, germination rate reaches maximum 51.78%, for the contrast 20.68 times.Rehydration 8h, germination rate and rehydration 4h are without significant difference.Rehydration 12h, germination rate reduces.After different rehydration times, under same medium, pollen tube growth is without significant difference.Therefore, the best rehydration time of apocarya pollen germination is 4 ~ 8h.
Table 1: the impact of different rehydration times on the apocarya in-vitro pollen germination
3, the screening of in-vitro pollen germination culture condition
3.1 the screening of sucrose concentration
Increase respectively 0gL in distilled water
-1, 50gL
-1, 100gL
-1, 150gL
-1, 200gL
-1, 250gL
-1, 300gL
-1Sucrose (described 0gL
-1Namely do not add sucrose), the relatively impact of the sucrose of different concns on pollen germination and pollen tube growth.
Sucrose is a kind of osmotic adjustment, is also the energy substance of pollen tube growth simultaneously.Result is as shown in table 2, and sucrose is the requisite component of apocarya pollen germination, and in the sucrose-free substratum, pollen is not all sprouted.Along with the raising of sucrose concentration, pollen germination rate significantly raises, and pollen tube length also significantly increases.When sucrose concentration reaches 200gL
-1The time, pollen germination rate is the highest, is 46.64%, and this moment, corresponding pollen tube length was 177.60 μ m.
The impact of table 2 different concns sucrose on pollen germination and pollen tube growth
3.2 the screening of boron concentration
At 200gL
-1Increase respectively 0mgL in sucrose solution
-1, 100mgL
-1, 200mgL
-1, 300mgL
-1, 400mgL
-1, 500mgL
-1H
3BO
3(described 0mgL
-1Namely do not add H
3BO
3), the relatively impact of the boron of different concns on pollen germination and pollen tube growth.
Boron can promote absorption and the metabolism of sugar, and promotes the elongation growth of pollen tube.Result is as shown in table 3, and in the finite concentration scope, the growth of pollen germination rate and pollen tube increases with the increase of boric acid concentration, plays restraining effect but surpass finite concentration.When boric acid concentration is 300mgL
-1The time, pollen germination rate is the highest, is 69.24%, is 1.43 times of contrast, and this moment, corresponding pollen tube length was 225.40 μ m.When boric acid concentration is 200mgL
-1The time, pollen tube length reaches maximum value 239.71 μ m.Variance analysis shows, boric acid concentration is 200mgL
-1And 300mgL
-1The time, pollen tube length is without significant difference.Therefore the suitableeest boric acid concentration of apocarya pollen germination is 300mgL
-1
Table 3 different concns boric acid is done the impact on pollen germination and pollen tube growth mutually
3.3Ca
2+The screening of concentration
Containing 200gL
-1Increase respectively 0gL in sucrose solution
-1, 100mgL
-1, 200mgL
-1, 300mgL
-1, 400mgL
-1, 500mgL
-1, 700mgL
-1, 900mgL
-1Ca (NO
3)
24H
2O (described 0mgL
-1Namely do not add Ca (NO
3)
24H
2O), the Ca that compares different concns
2+Impact on pollen germination and pollen tube growth.
External source Ca
2+The trend that affects on the apocarya pollen germination is similar to boron, as seen from Table 4, and Ca in the finite concentration scope
2+Pollen germination and pollen tube growth are played a driving role.Ca (NO
3)
24H
2O concentration is 100mgL
-1~ 500mgL
-1The time, pollen is sprouted rate with Ca
2+The increase of concentration and improving, pollen tube length also have more obviously elongation.As Ca (NO
3)
24H
2O concentration reaches 500mgL
-1The time, pollen germination rate is the highest, and namely 61.76%, the pollen tube length of this moment also reaches maximum value 220.87 μ m.
Table 4 different concns calcium is done the impact on pollen germination and pollen tube growth mutually
3.3 the screening of optimal medium
Comprehensive this 3 factor is carried out orthogonal experimental design (table 5).His-and-hers watches 5 data are carried out variance analysis, and result shows that the factor of significant difference is sucrose, H
3BO
3(P＜0.05), Ca
2+And the interaction between each factor all not significantly (P〉0.05), the size that affects of 3 factor pair apocarya pollen germinations is: sucrose＞H
3BO
3＞Ca
2+Further do the sum of squares comparative analysis, under each concentration of sucrose, pollen germination rate and pollen tube length sum of squares are with 20gL
-1The highest, H
3BO
3With 300mgL
-1The highest, Ca
2+With 500mgL
-1The highest, therefore optimal medium is combined as 200gL
-1Sucrose+300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O。But this is combined in orthogonal test and does not occur, and in table 1, best of breed is No. 13, i.e. 200gL
-1Sucrose+200mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O。For finding out the optimal medium combination, use 20mgL
-1Sucrose+300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2The substratum of O increases battery of tests.Result shows, 200gL
-1Sucrose+300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O and 200gL
-1Sucrose+200mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2Under the substratum of O, without significant difference.Therefore the optimal medium of apocarya pollen germination and pollen tube growth is combined as: 200gL
-1Sucrose+200 ~ 300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O。
Table 5 apocarya pollen germination L
25(5
3) orthogonal experiments
The above; only for the better embodiment of the present invention, but protection scope of the present invention is not limited to this, anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement are within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (3)
1. apocarya in-vitro pollen germination liquid nutrient medium, it is characterized in that: this substratum includes sucrose, H take distilled water as solvent
3BO
3And Ca (NO
3)
24H
2O。
2. apocarya in-vitro pollen germination liquid nutrient medium according to claim 1, is characterized in that described solute is by the sucrose of 200g/L, the H of 200 ~ 300mg/L
3BO
3Ca (NO with 500mg/L
3)
24H
2O forms.
3. the described substratum of above-mentioned arbitrary claim is measured the application of Pollen Activity, it is characterized in that, comprises the following steps:
1) pollen collection
Select to grow normal, without the healthy and strong apocarya plant of disease and pest, pluck the male flower inflorescence when the flower pesticide flavescence, it is spread out on template, be placed in wind sheltering dry place, next day, flower pesticide got final product loose powder; Sift out pollen, in the sealing bag of packing into, refrigerate standby;
2) the pollen rehydration is processed
In airtight container, use K
2SO
4Or CuSO
45H
2The O saturated salt solution is regulated relative humidity RH=97%, and pollen is positioned in this container, carries out the rehydration of 4 ~ 8h and processes;
3) in-vitro pollen germination and pollen tube growth
While obtaining liq substratum, its component is: 200gL
-1Sucrose+200～300mgL
-1H
3BO
3+ 500mgL
-1Ca (NO
3)
24H
2O； Drip 50 μ L liquid nutrient mediums on the groove slide glass, dip with pin the pollen that rehydration processes it is sprinkling upon on substratum equably, slide glass is put into the culture dish that is covered with moistening absorbent cotton, moisturizing is cultivated under dark, 25 ℃ of conditions; After 4h, pollen begins to sprout, the pollen tube growth growth, and after 24h, the pollen growth tends towards stability;
4) mensuration of Pollen Activity
After isolated culture 24h, each slide is chosen 5 unduplicated visuals field at random, and each visual field pollen number is no less than 50, is considered as sprouting greater than the pollen granule diameter with pollen tube length, and chooses 50 and measure pollen tube length, and each test all repeats 3 times.
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Cited By (7)
| 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 |
| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN104004702A (en) * | 2014-05-20 | 2014-08-27 | 四川农业大学 | Culture medium for germination of tetraploid hemarthria compressa pollen |
| CN104232561A (en) * | 2014-09-23 | 2014-12-24 | 江苏省林业科学研究院 | In-vitro culture method of salix saposhnikovii pollen |
| 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 |
| CN107593689A (en) * | 2017-10-26 | 2018-01-19 | 中国林业科学研究院亚热带林业研究所 | A kind of thin shell mountain pecan peach flower powder processing and freezing and storing method |
| CN112430566A (en) * | 2020-11-27 | 2021-03-02 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Culture method for carambola pollen in-vitro germination |
-
2013
- 2013-01-11 CN CN2013100108401A patent/CN103087975A/en active Pending
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| 《HortScience》 20110430 Patrick J.Conner optimization of in vitro pecan pollen germination 572 3 第46卷, 第4期 * |
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Cited By (9)
| 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 |
| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN104004702A (en) * | 2014-05-20 | 2014-08-27 | 四川农业大学 | Culture medium for germination of tetraploid hemarthria compressa pollen |
| CN104232561A (en) * | 2014-09-23 | 2014-12-24 | 江苏省林业科学研究院 | In-vitro culture method of salix saposhnikovii pollen |
| 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 |
| CN107593689A (en) * | 2017-10-26 | 2018-01-19 | 中国林业科学研究院亚热带林业研究所 | A kind of thin shell mountain pecan peach flower powder processing and freezing and storing method |
| CN112430566A (en) * | 2020-11-27 | 2021-03-02 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Culture method for carambola pollen in-vitro germination |
| CN112430566B (en) * | 2020-11-27 | 2022-11-18 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Culture method for carambola pollen in-vitro germination |
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Application publication date: 20130508 |