CN103451147A - Lactuca sativa L. pollen in vitro germination culture medium and method for measuring pollen activity - Google Patents
Lactuca sativa L. pollen in vitro germination culture medium and method for measuring pollen activity Download PDFInfo
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Abstract
The invention discloses a lactuca sativa L. pollen in vitro germination culture medium and a method for measuring pollen activity. The lactuca sativa L. pollen in vitro germination culture medium uses distilled water as a solvent and comprises components of ME3 (Monnior), PEG4000 (Polyethylene Glycol) and saccharose. The method comprises the following steps: (1), collecting pollen, namely, selecting an inflorescence flowering in that day, transferring into a freshness protection package filled with clear water, and bringing back in a room; (2), preparing a pollen in vitro liquid culture medium; (3), spreading and culturing pollen, namely, horizontally placing a glass slide loaded with the liquid culture medium on an experiment platform, removing petals of a capitulum by using tweezers, stripping off a synandrium stamen and then taking an anther out, enabling the anther to slide on the surface of the culture medium to ensure that pollen is uniformly spread and distributed on the surface of the culture medium in a single layer and a pollen tube grows; (4), measuring the pollen activity, namely, after in vitro culturing, observing, and counting a pollen germination rate, wherein the pollen germination rate reflects a proportion of vital pollens. The method is rapid, simple and easy to operate, good in culturing effect, and large in practical application value; abundant vital germinating pollens are obtained, and the pollen activity is effectively measured.
Description
Technical field
The invention belongs to vegetable plant Pollen Activity determination techniques field, particularly a kind of lettuce in-vitro pollen germination liquid nutrient medium, also relate to the method for utilizing this liquid nutrient medium isolated culture lettuce pollen germination to measure Pollen Activity.
Background technology
Lettuce (Lactuca sativa L.) belongs to composite family (Compositae), Lactuca (Lactuca), biennial herb plant, originates in Mediterranean, likes the weather that cools, non-refractory.In lettuce, carbohydrate content is lower, and inorganic salt and vitamin contents are abundant, and especially more nicotinic acid has diuresis, hypotensive etc. effect for hypertension, cardiac.Therefore, lettuce is extensively cultivated all over the world, has become the vegetables that people like.
In recent years, along with the human consumer, to the lettuce increase in demand, impel the demand to high-quality, excellent economic characters lettuce material also to increase sharply, yet, depend merely on existing lettuce resource and can not satisfy the demands, in the case, the fundamental research of lettuce germ plasm resource has generally been carried out in various places.Abundant germ plasm resource is the basic substance that carries out lettuce genetic research and germplasm improvement, and the research with special skill proterties lettuce germplasm identifies and preserve to be the prerequisite that science is carried out prevalent variety cultivation.In view of the main carriers of lettuce pollen as the lettuce genetic resources, therefore, carry out the research of lettuce Pollen Activity mensuration, to the very important theory and practice meaning of seed selection of the preservation of lettuce germ plasm resource and innovation, improved seeds.
Pollen germination is that after pollen absorbs nutrition, volume increases, and inner turgescence increases, and makes intine outwards outstanding along the germ pore place, forms the process of pollen tube.Starting pollen germination is a more complicated process, and the primary condition of its sprouting needs the participation of sugared source, boric acid and mineral element.It is comparatively stable that in-vitro pollen germination and pollen tube growth are measured the Pollen Activity measurement result, can more accurately reflect the practical situation of Pollen Activity.But in-vitro pollen germination needs suitable substratum, and substratum basal component commonly used is sugar and boric acid.Sucrose is sugared main source, and concentration is generally 10%-20%; Boric acid is 0.001%-0.005%, pH5.5-6.5.Recent study shows in minimum medium to add Ca
2+, Mg
2+, K
+, the element of the promotion pollen germinations such as PEG can effectively promote more difficult sprouting pollen germination.The people such as Zhao Hongbo have remarkable promoter action for PEG to the in vitro germination and growth of pollen powder of chrysanthemun flower.At present, successful isolated culture goes out the pollen of various plants, as quite ripe as Techniques of in Vitro Cultures such as lily, tobaccos, and less for the lettuce in-vitro pollen culture studies of composite family.
Summary of the invention
The objective of the invention is to be to provide a kind of in vitro germination medium of lettuce pollen, this substratum is liquid nutrient medium, preparation is simple, the pollen germination required time is short, just can observe the lettuce pollen germination imagination after 30 minutes, can clearly observe the sprouting of pollen after 2 hours, be a kind of liquid nutrient medium of high-efficient culture lettuce pollen germination.
Another object of the present invention is to be to provide a kind of liquid nutrient medium isolated culture lettuce pollen germination to measure the method for Pollen Activity, easy to implement the method, easy and simple to handle, on the basis of determining lettuce in-vitro pollen germination culture system, the lettuce in-vitro pollen is cultivated to accurate and effective mensuration lettuce Pollen Activity.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
A kind of lettuce in-vitro pollen germination liquid nutrient medium, take distilled water as solvent, and its component comprised is ME
3, 170-190gL
-1pEG4000 and 90-110gL
-1sucrose, pH is 5.8-6.0.
Monnior substratum (ME
3substratum), polyoxyethylene glycol (PEG), English name polyethylene glycol.
Described ME
3component be: saltpetre (KNO
3) 950-1050mgL
-1, ammonium chloride (NH
4cl) 261.25-288.75mgL
-1, four water-calcium nitrate (CaNO
34H
2o) 1520-1680mgL
-1, potassium primary phosphate (KH
2pO
4) 80.75-89.25mgL
-1, Repone K (KCl) 166.25-183.75mgL
-1, magnesium sulfate heptahydrate (MgSO
47H
2o) 475-525mgL
-1, potassiumiodide (KI) 0.83mgL
-1, boric acid (H
3bO
3) 100mgL
-1, cupric sulfate pentahydrate (CuSO
45H
2o) 0.025mgL
-1, Sodium Molybdate Dihydrate (Na
2moO
42H
2o) 0.25mgL
-1, CoCL2 6H2O (CoCl
26H
2o) 0.025mgL
-1, disodium ethylene diamine tetraacetate (Na
2eDTA) 7.45mgL
-1, iron vitriol (FeSO
47H
2o) 0.025, pyridoxine hydrochloride (V
b6) 1.0mgL
-1and thiamine hydrochloride (V
b1) 1.0mgL
-1.
A kind of preparation method of lettuce in-vitro pollen germination liquid nutrient medium, its step is as follows:
1. according to a series of mother liquors of described recipe configuration (table 1): macroelement (concentrated 20 times), trace element (concentrated 200 times), molysite (concentrated 200 times), organism (concentrated 200 times).The preparation these 4 kinds of mother liquors in, accurately take in proportion every kind of composition, and make every kind of composition be dissolved in respectively distilled water, and then they are mixed with each other, constant volume, to 1L, is stored in vial, puts Refrigerator store standby, while wherein preparing mother liquid of iron salt by FeSO
47H2O and Na
2eDTA is placed in respectively 450ml distilled water, and under 78-82 ℃ of condition, heating continuous the stirring make it abundant dissolving, then by iron vitriol (FeSO
47H2O) and disodium ethylene diamine tetraacetate (Na
2eDTA) these 2 kinds of solution mix, and adding distil water, to final volume 1L, is stored in Brown Glass Brown glass bottles and jars only, puts Refrigerator store standby.
2. take 180g PEG4000 and 100g sucrose, be placed in respectively 1000mL(PEG4000) and 500mL(sucrose) glass beaker, add respectively 500ml and 250ml distilled water that PEG and sucrose are fully dissolved, sucrose solution after fully dissolving is sneaked in PEG4000 solution, fully mix.The order that is respectively 50ml, 5ml, 5ml, 5ml according to macroelement, trace element, molysite, organic composition adds PEG and sucrose mixed solution, then with distilled water, is settled to final volume (1L), fully mixes.
3. finally use 1molL
-1naOH or 1molL
-1hCl adjust the pH value to 5.8-6.0, obtain the in-vitro pollen liquid nutrient medium.
Table 1ME
3the preparation of substratum mother liquor
A kind of liquid nutrient medium isolated culture lettuce pollen germination is measured the method for Pollen Activity, and its step is as follows:
(1) gather pollen:
Choose grow normal, without the plant of disease and pest, robust growth, choose inflorescence to be opened on the same day in the lettuce full-bloom stage, together with spray, take, move into the freshness protection package that fills clear water, take back in laboratory.
(2) preparation of in-vitro pollen liquid nutrient medium:
Take distilled water as solvent, and contained component is: saltpetre (KNO
3) 950-1050mgL
-1, ammonium chloride (NH
4cl) 261.25-288.75mgL
-1, four water-calcium nitrate (CaNO
34H
2o) 1520-1680mgL
-1, potassium primary phosphate (KH
2pO
4) 80.75-89.25mgL
-1, Repone K (KCl) 166.25-183.75mgL
-1, magnesium sulfate heptahydrate (MgSO
47H
2o) 475-525mgL
-1, potassiumiodide (KI) 0.83mgL
-1, boric acid (H
3bO
3) 100mgL
-1, cupric sulfate pentahydrate (CuSO
45H
2o) 0.025mgL
-1, Sodium Molybdate Dihydrate (Na
2moO
42H
2o) 0.25mgL
-1, CoCL2 6H2O (CoCl
26H
2o) 0.025mgL
-1, disodium ethylene diamine tetraacetate (Na
2eDTA) 7.45mgL
-1, iron vitriol (FeSO
47H
2o) 0.025mgL
-1, pyridoxine hydrochloride (V
b6) 1.0mgL
-1and thiamine hydrochloride (V
b1) 1.0mgL
-1, Macrogol 4000 (PEG4000) 170-190gL
-1, sucrose 90-110gL
-1, mix, finally regulate the pH value to 5.8-6.0, obtain the in-vitro pollen liquid nutrient medium.
(3) spreading of pollen and cultivation:
The slide glass that is loaded with liquid nutrient medium is placed horizontally on experiment table, extract the petal of head titbit with tweezers, strip off synandrium pistil takes out flower pesticide gently, clamp flower pesticide below filigree, flower pesticide slided in media surface gently and make the pollen individual layer, be scattered and be distributed on media surface equably, at 300-400 μ molm
-2s
-1constant temperature culture under the light intensity illumination condition, in relative air humidity 90%, 24-26 ℃ constant incubator, in-vitro pollen germination is cultivated 28-32min rear section pollen and is started to sprout, after pollen germination, pollen tube length surpasses the pollen granule diameter, and pollen tube starts Fast Growth.
(4) mensuration of Pollen Activity:
After isolated culture 1-3h, the pollen tube length of usining is greater than the standard of pollen granule diameter as pollen germination; Three visuals field of each random observation, each visual field is observed the pollen number and is no less than 30, the statistics pollen germination rate, triplicate is averaged as the pollen germination rate measurement result, and the pollen germination rate reaction has vitality pollen proportion.
The above; be only the present invention's embodiment preferably, but protection scope of the present invention is not limited to this, anyly is familiar with in technical scope that those skilled in the art disclose in the present invention; the variation that can expect easily or replacement, 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.
The present invention compared with prior art, has the following advantages and effect:
1, lettuce pollen germination substratum of the present invention provides essential envrionment conditions for pollen germination and growth, not only quick, simple, and culture effect is good, can obtain and have in a large number blodynamic sprouting pollen.
2, the present invention utilizes in vitro culture method to measure the in vitro culture method of lettuce Pollen Activity, can effectively measure Pollen Activity, has larger actual application value.
3, lettuce pollen is on lettuce in-vitro pollen germination liquid nutrient medium provided by the invention, under illumination condition, under relative air humidity 90%, 25 ℃ of constant temperature, cultivate, the interpolation that the lettuce pollen germination rate can reach 91%, PEG4000 can make the lettuce pollen tube growth more straight, is convenient to observe.
4, utilize lettuce in-vitro pollen germination technical measurement Pollen Activity measurement result reliable and stable, pollen germination rate reacts viable pollen proportion, and the mensuration that the lettuce in-vitro pollen germination is the lettuce Pollen Activity provides a kind of reliable, effective means.
Embodiment
The present invention, on the basis of determining lettuce in-vitro pollen germination substratum, cultivates the lettuce in-vitro pollen, measures pollen germination rate, reliable and effective mensuration lettuce Pollen Activity.Below the present invention is described further, the explanation of the invention is not limited.
Embodiment 1:
A kind of lettuce in-vitro pollen germination liquid nutrient medium, take distilled water as solvent, and its component comprised is ME
3, 170 or 180 or 190gL
-1pEG4000 and 90 or 100 or 110gL
-1sucrose, the Medium's PH Value of adjusting is 5.8 or 5.9 or 6.0.
Described ME
3component be: saltpetre (KNO
3) 950 or 1000 or 1050mgL
-1, ammonium chloride (NH
4cl) 261.25 or 275 or 288.75mgL
-1, four water-calcium nitrate (CaNO
34H
2o) 1520 or 1600 or 1680, potassium primary phosphate (KH
2pO
4) 80.75 or 85 or 89.25mgL
-1, Repone K (KCl) 166.25 or 175 or 183.75mgL
-1, magnesium sulfate heptahydrate (MgSO
47H
2o) 475 or 500 or 525mgL
-1, potassiumiodide (KI) 0.83mgL
-1, boric acid (H
3bO
3) 100mgL
-1, cupric sulfate pentahydrate (CuSO
45H
2o) 0.025mgL
-1, Sodium Molybdate Dihydrate (Na
2moO
42H
2o) 0.25mgL
-1, CoCL2 6H2O (CoCl
26H
2o) 0.025mgL
-1, disodium ethylene diamine tetraacetate (Na
2eDTA) 7.45m
gl
-1, iron vitriol (FeSO
47H
2o) 0.025mgL
-1, pyridoxine hydrochloride (V
b6) 1.0mgL
-1and thiamine hydrochloride (V
b1) 1.0mgL
-1.
A kind of preparation method of lettuce in-vitro pollen germination liquid nutrient medium, its step is as follows:
1. according to a series of mother liquors of described recipe configuration (table 1): configuration macroelement (concentrated 20 times), trace element (concentrated 200 times), molysite (concentrated 200 times), organism (concentrated 200 times), accurately take in proportion every kind of composition, and make every kind of composition be dissolved in respectively distilled water, and then they are mixed with each other to constant volume to 1L, be stored in respectively in vial, put Refrigerator store standby, while wherein preparing mother liquid of iron salt by FeSO
47H
2o and Na
2eDTA is placed in respectively 450ml distilled water, and under 78 or 79 or 80 or 81 or 82 ℃ of conditions, heating continuous the stirring make it abundant dissolving, then by FeSO
47H
2o and Na
2these 2 kinds of solution of EDTA mix, and adding distil water, to final volume 1L, is stored in Brown Glass Brown glass bottles and jars only, puts Refrigerator store standby.
2. take 170 or 180 or 190gL
-1with 90 or 100 or 110gL
-1sucrose, be placed in respectively 1000mL(PEG4000) and 500mL(sucrose) glass beaker, add 500ml and 250ml distilled water that PEG4000 and sucrose are fully dissolved, the sucrose solution after fully dissolving is sneaked in PEG4000 solution, fully mix.Be respectively and add PEG and sucrose mixed solution according to the order of macroelement (47.5ml or 50mL or 52.5mL), trace element (5mL), molysite (5mL), organic composition (5mL), then with distilled water, be settled to 1L, fully mix.
3. finally use 1molL
-1naOH or 1molL
-1hCl adjust the pH value to 5.8-6.0, obtain the in-vitro pollen liquid nutrient medium.
Embodiment 2:
A kind of liquid nutrient medium isolated culture lettuce pollen germination is measured the method for Pollen Activity, and its step is as follows:
1, the collection of lettuce pollen:
Lettuce pollen picks up from vegetables Science Institute breeding base, Wuhan City lettuce colony in early June, 2013, acquisition method is: choose grow normal, bud is full, without the lettuce plant of disease and pest, robust growth, in full-bloom stage, 7:00～7:30 in the morning chooses the inflorescence of just blooming, together with spray, inflorescence is adopted down, move into and be equipped with in the freshness protection package of clear water rapidly, take back laboratory standby.2. the screening of lettuce in-vitro pollen germination condition: the screening of 2.1 lettuce in-vitro pollen germination PEG concentration:
PEG4000 is a kind of high-molecular weight compounds, mainly plays osmotic adjust action.Fresh pollen is evenly taken a walk and added respectively 0gL
-1, 90gL
-1, 180gL
-1, 240gL
-1the ME3+100gL of PEG4000
-1in the liquid sucrose substratum, regulate pH value to 5.8 or 5.9 or 6.0, described 0gL
-1for not adding PEG4000, covered is in relative air humidity 90%, 24 ℃ or 25 ℃ or 26 ℃ of constant temperature, light intensity 300-400 μ molm
-2s
-1in illumination box, cultivate.
Result is as shown in table 2, ME
3+ 100gL
-1sucrose, add the 90gL-of different concns in the liquid nutrient medium that pH is 5.6 or 5.7 or 5.8 or 5.9 or 6
1, 180gL-
1, 240gL-
1pEG4000, can obviously improve the lettuce pollen germination rate, wherein adds 180L
-1the PEG4000 effect is the most remarkable, and pollen germination rate reaches 91%; PEG4000 concentration is less than 180L
-1promote pollen tube growth, and weaken gradually 240gL along with concentration increases its promoter action
-1pEG4000 has significantly suppressed pollen tube growth; Observe under microscope that to have added in the PEG substratum lettuce pollen tube growth more straight, and the substratum pollen tube growth elongate curved of not adding PEG, weave in, inconvenience is observed and is measured.
The impact of table 2 different concns PEG on pollen germination
2.2 the screening of lettuce in-vitro pollen germination sucrose concentration:
Sucrose is the energy substance of pollen tube growth, is also a kind of osmotic adjustment.Fresh pollen is evenly taken a walk and added respectively 0gL
-1, 50gL
-1, 100gL
-1, 130gL
-1, 160gL
-1the ME of sucrose
3+ 180gL
-1in the liquid nutrient medium of PEG4000, regulate respectively pH value to 5.6 or 5.7 or 5.8 or 5.9 or 6, described 0gL
-1for not adding PEG4000, the relatively impact of the sucrose of different concns on pollen germination.Covered is in relative air humidity 90%, 25 ℃ of constant temperature, light intensity 300-400 μ molm
-2s
-1in illumination box, cultivate.
Result is as shown in table 3, ME
3+ 180gL
-150gL in the PEG4000 substratum
-1, 100gL
-1, 130gL
-1, 160gL
-1the affect difference of different concns sucrose on the lettuce pollen germination rate, 50gL
-1the sucrose of concentration has obvious promoter action to pollen germination, and higher concentration (>100gL
-1) sucrose obviously suppressed pollen germination, and, along with sucrose concentration increases, suppressed degree is more obvious.ME
3+ 180gL
-1add 100gL in the PEG4000 substratum
-1during sucrose, lettuce pollen growth is best, cultivate 12 or 3h after, its pollen germination rate reaches 92%.
The impact of table 3 different concns PEG on pollen germination
2.3 the screening of lettuce in-vitro pollen germination temperature:
Temperature has considerable influence, the optimal temperature difference of different Plant Pollen Germinations in vitro sprouting of plant pollen.Fresh pollen evenly take a walk ME
3+ 180gL
-1pEG4000+160gL
-1on the liquid nutrient medium of sucrose, regulate pH value to 5.6 or 5.7 or 5.8 or 5.9 or 6, covered is respectively 20 ℃, 25 ℃, 30 ℃, 35 ℃ constant temperature, light intensity 300-400 μ molm in relative air humidity 90%, temperature
-2s
-1in illumination box, cultivate.Observe the pollen germination situation after cultivating 2h, the screening optimum culturing temperature.Temperature has remarkably influenced to the lettuce pollen germination as can be seen from Table 4, under 20 ℃ of conditions of low temperature, pollen germination rate is lower, rising along with temperature, germination rate significantly raises, under 25 ℃ of conditions, pollen germination rate reaches peak value, its germination rate is 91%, and the optimum culturing temperature that shows the lettuce pollen germination is 25 ℃.When temperature further raises, pollen germination is subject to obvious inhibition, and germination rate significantly descends, and under 35 ℃ of conditions, germination rate drops to 6%.
The impact of the different culture temperature of table 4 on pollen germination
Claims (2)
1. a lettuce in-vitro pollen germination liquid nutrient medium, take distilled water as solvent, it is characterized in that: its component comprised is ME
3, 170-190gL
-1pEG4000 and 90-110gL
-1sucrose, pH is 5.8-6.0;
Described ME
3component be: saltpetre 950-1050mgL
-1, ammonium chloride 261.25-288.75mgL
-1, four water-calcium nitrate 1520-1680mgL
-1, potassium primary phosphate 80.75-89.25mgL
-1, Repone K 166.25-183.75mgL
-1, magnesium sulfate heptahydrate 475-525mgL
-1, potassiumiodide 0.83mgL
-1, boric acid 100mgL
-1, cupric sulfate pentahydrate 0.025mgL
-1, Sodium Molybdate Dihydrate 0.25mgL
-1, CoCL2 6H2O 0.025mgL
-1, disodium ethylene diamine tetraacetate 7.45mgL
-1, iron vitriol 0.025, pyridoxine hydrochloride 1.0mgL
-1with thiamine hydrochloride 1.0mgL
-1;
A kind of preparation method of lettuce in-vitro pollen germination liquid nutrient medium, its step is as follows:
A, configuration mother liquor: macroelement, trace element, molysite, organism, when preparing mother liquor, take in proportion every kind of composition, make every kind of composition be dissolved in respectively distilled water, then mix, constant volume is to 1L, be stored in vial, put Refrigerator store standby, while wherein preparing mother liquid of iron salt by FeSO
47H
2o and Na
2eDTA is placed in respectively 450ml distilled water, and heating stirring and dissolving under 78-82 ℃ of condition, by FeSO
47H
2o and Na
2eDTA solution mixes, and adding distil water, to final volume 1L, is stored in Brown Glass Brown glass bottles and jars only, puts Refrigerator store standby;
B, take 180g PEG4000 and 100g sucrose, be placed in respectively 1000mL:PEG4000 and 500 mL: the sucrose glass beaker, add respectively 500ml and 250ml distilled water to make PEG and sucrose dissolved, sucrose solution after dissolving is sneaked in PEG4000 solution, mix, the order that is respectively 50ml, 5ml, 5ml, 5ml according to macroelement, trace element, molysite, organic composition adds PEG and sucrose mixed solution, then with distilled water, is settled to final volume 1L, mixes;
C, finally use 1molL
-1naOH or 1molL
-1hCl adjust the pH value to 5.8-6.0, obtain the in-vitro pollen liquid nutrient medium.
2. a kind of liquid nutrient medium isolated culture lettuce pollen germination claimed in claim 1 is measured the method for Pollen Activity, and its step is as follows:
(1) gather pollen:
Choose grow normal, without the plant of disease and pest, robust growth, choose open inflorescence on the same day in the lettuce full-bloom stage, together with spray, take, move into the freshness protection package that fills clear water, take back in laboratory;
(2) preparation of in-vitro pollen liquid nutrient medium:
Take distilled water as solvent, and contained component is: saltpetre 950-1050mgL
-1, ammonium chloride 261.25-288.75mgL
-1, four water-calcium nitrate 1520-1680mgL
-1, potassium primary phosphate 80.75-89.25mgL
-1, Repone K 166.25-183.75mgL
-1, magnesium sulfate heptahydrate 475-525mgL
-1, potassiumiodide 0.83mgL
-1, boric acid 100mgL
-1, cupric sulfate pentahydrate 0.025mgL
-1, Sodium Molybdate Dihydrate 0.25mgL
-1, CoCL2 6H2O 0.025mgL
-1, disodium ethylene diamine tetraacetate 7.45mgL
-1, iron vitriol 0.025mgL
-1, pyridoxine hydrochloride 1.0mgL
-1with thiamine hydrochloride 1.0mgL
-1, Macrogol 4000 170-190gL
-1, sucrose 90-110gL
-1, mix, regulate the pH value to 5.8-6.0;
(3) spreading of pollen and cultivation:
The slide glass that is loaded with liquid nutrient medium is placed horizontally on experiment table, extract the petal of head titbit with tweezers, strip off synandrium pistil takes out flower pesticide, clamp flower pesticide below filigree, flower pesticide slided in media surface and make the pollen individual layer, be scattered and be distributed on media surface equably, at 300-400 μ molm
-2s
-1constant temperature culture under the light intensity illumination condition, in relative air humidity 90%, 24-26 ℃ constant incubator, in-vitro pollen germination is cultivated 28-32 min rear section pollen and is started to sprout, and after pollen germination, pollen tube length surpasses the pollen granule diameter, and pollen tube starts growth;
(4) mensuration of Pollen Activity:
After isolated culture 1-3h, the pollen tube length of usining is greater than the standard of pollen granule diameter as pollen germination, three visuals field of each observation, each visual field is observed the pollen number and is no less than 30, the statistics pollen germination rate, triplicate is averaged as the pollen germination rate measurement result, and the pollen germination rate reaction has vitality pollen proportion.
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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 |
| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN104726391A (en) * | 2015-04-21 | 2015-06-24 | 河北双星种业有限公司 | Culture medium of sunflower pollen in vitro germination and method for determining sunflower pollen viability |
| 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 |
| CN107460229A (en) * | 2017-08-29 | 2017-12-12 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Spontaneum pollen vigor rapid identification method |
| CN107723270A (en) * | 2017-11-24 | 2018-02-23 | 玉溪中烟种子有限责任公司 | Half in-vitro culture method of tobacco pollen pipe |
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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 |
| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN104726391A (en) * | 2015-04-21 | 2015-06-24 | 河北双星种业有限公司 | Culture medium of sunflower pollen in vitro germination and method for determining sunflower pollen viability |
| CN104726391B (en) * | 2015-04-21 | 2018-04-13 | 河北双星种业股份有限公司 | A kind of culture medium of sunflower powder germination in vitro and the method for measuring sunflower powder vigor |
| 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 |
| CN107460229A (en) * | 2017-08-29 | 2017-12-12 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Spontaneum pollen vigor rapid identification method |
| CN107723270A (en) * | 2017-11-24 | 2018-02-23 | 玉溪中烟种子有限责任公司 | Half in-vitro culture method of tobacco pollen pipe |
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