CN103837534B - A kind of method measuring paenoiae alba Pollen Activity - Google Patents
A kind of method measuring paenoiae alba Pollen Activity Download PDFInfo
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Abstract
The invention discloses a kind of method measuring paenoiae alba Pollen Activity, comprising: the pollen collecting paenoiae alba; Cultured in vitro is carried out on the surface described pollen being placed in nutrient culture media; Cultured in vitro terminates rear statistics pollen germination rate; Consisting of of described nutrient culture media: sucrose 80 ~ 150g/L, boric acid 15 ~ 30mg/L, agar 8 ~ 10g/L; The time of described cultured in vitro is 2.5 ~ 3.5h.The isolated culture condition of the present invention to medicinal paenoiae alba pollen is explored, mensuration for its Pollen Activity provides a kind of simple, quick, feasible, reliable method, for medicinal paenoiae alba and northern Herbaceous peony are hybridized, cultivate possess Zhejiang characteristic, the Herbaceous peony new varieties that adapt to Zhejiang weather serve place mat and facilitation.
Description
Technical field
The present invention relates to plant pollen vitality test field, particularly relate to a kind of method measuring paenoiae alba Pollen Activity.
Background technology
Chinese herbaceous peony is Chinese tradition flowers, nearly 4000 of cultivation history, just on the books in " Book of Songs Zheng wind ": " Wei Shi and female, Yi Qixiang teases, give with spoon medicine ".Chinese herbaceous peony is described as " flower phase ", mutually equally celebrated for their achievements with " flower king " tree peony, has good ornamental value and medical value.Chinese herbaceous peony property happiness illumination and cooling, non-refractory and humidity, the ground such as Heze, Luoyang of North China of state is Chinese herbaceous peony, town of peony still, and fancy breed is numerous, and flower pattern, pattern, plant type are rich and varied, can be widely used in colored border, potted plant or cut-flower views and admires.
Zhejiang Province because of weather damp and hot, native country is viewed and admired Cultivars of Chinese Herbaceous Peony and is cultivated almost nil, even if introduce northern kind, also many because weather is uncomfortable and disease and pest seriously causes appreciation effect to be had a greatly reduced quality.But just there is the record of planting medicinal Chinese herbaceous peony in Zhejiang Province from ancient times, the paenoiae alba produced as Panan County, Jinhua now and other places belongs to one of famous Chinese crude drug " eight Zhe's ", its root-like stock quality better, possesses soft liver of enriching blood, flat liver pain relieving, astringe yin receive the effects such as sweat; The flower of paenoiae alba is purplish red, pink, whitewash or pure white, also possesses good ornamental value; Meanwhile, paenoiae alba is long at Zhejiang cultivation history, the adaptability certain to the warm tool in Zhejiang Province.
The flower pattern extremely polyphyll of a lot of kind of tradition Herbaceous peony, stamen many lobeizations, without flower pesticide, more without pollen; Also have part kind to possess flower pesticide, but pollen amount is little, these all bring obstruction for utilizing artificial hybridization pollination to cultivate new varieties.Comparatively speaking, paenoiae alba in every respect on morphological feature and ecological habit closer to the wild ancestor of Chinese herbaceous peony.Such as, because its root-like stock is made for medicinal for a long time, the screening of flower pattern gets the brush-off more, mostly is single-lobe type or lotus type, stamen and flower pesticide clear, pollen amount is very big, is good male parent material.Therefore, using medicinal paenoiae alba as male parent, north Herbaceous peony kind, as female parent, is expected to cultivate and not only possesses good ornamental value, but also can adapt to the Herbaceous peony new varieties of Zhejiang Province's warm, thus solves the Zhejiang area Herbaceous peony kind embarrassed shape that there is a serious shortage in the supply.
The higher pollen viability of tool is the important indicator selecting male parent.The Pollen Activity of paenoiae alba is not quite clear, and its surveying work relevant does not have report so far.Therefore, set up the method for measuring of a set of effective mensuration paenoiae alba Pollen Activity, the crossbreeding work that can be Zhejiang paenoiae alba and northern Herbaceous peony brings very large Promotive effect.
Mensuration at present for Pollen Activity mainly contains 3 class methods, is respectively pollen staining method, field pollination method, in-vitro pollen germination method.Pollen staining method uses chemical reagent to dye to pollen, and judge vigor condition according to the depth of pollen color after dyeing, method is quick, easy, mainly contains K2-KI decoration method, acetic red dyeing, TTC decoration method etc.In the document such as " Chinese herbaceous peony Pollen viability and hybrid strain select Primary Study ", " research of Chinese herbaceous peony pollen viability measuring method ", " research of Chinese herbaceous peony Pollen Activity and Pistil recipient phase ", " long-term storage Chinese herbaceous peony Pollen viability technique study ", just record and utilized decoration method to measure the Pollen Activity of different flower pattern Herbaceous peony kind.But the pollen that can be colored may not be sprouted, not necessarily can stretch out pollen tube, but therefore, it is possible to be colored the pollen that cannot bear pollen tube, after hybridization pollination, also may not necessarily reach fertilization, therefore the Pollen Activity of Determination Staining is often higher.Field pollination method is the experiment that is directly cross-breeding in field, after fruit maturation, calculate setting percentage.The method is the most original, directly and reliable, but also time-consumingly to take a lot of work, by season and weather effect comparatively large, be difficult to meet the active demand of cultivating new varieties.
Manually give the condition of pollen germination, allow it grow pollen tube, measure Pollen Activity according to sprouting ratio, be reliably, method easily, the germination in vitro method of Here it is pollen.Have paper report to utilize germination in vitro method to measure the Pollen Activity of northern Herbaceous peony kind more, as " long-term storage Chinese herbaceous peony Pollen viability technique study ", " Pollen viability of different flower pattern Chinese herbaceous peony and comparative studies ", " medium component is on the impact of Chinese herbaceous peony pollen germination and pollen tube growth ", the storage characteristics of Chinese herbaceous peony pollen " under three kinds of reserve temperature conditions ", the shelf-life of Chinese herbaceous peony pollen " under the different condition ", " viability of Chinese herbaceous peony pollen Excised Embryos after 4 years detects ", " optimization of Chinese herbaceous peony in-vitro pollen germination nutrient media components " etc.These reports relate to the solid-state of nutrient culture media or liquid form, different composition proportion etc., but research material is substantially all the Herbaceous peony kind in the north, about the Pollen Activity report of southern area medicinal Chinese herbaceous peony never occurs.
The practice that applicant is observed by Pollen Activity finds, the medium component proportioning of paenoiae alba pollen germination and effective observation time etc., compared with traditional Herbaceous peony kind, there is difference, and pollen also exists comparatively serious sag in incubation, this does not see and relates in report in the past.Therefore, set up the scientific approach that a set of applicable paenoiae alba Pollen Activity measures, the significant theory and practice meaning of tool, better can promote the medicinal paenoiae alba in native country, Zhejiang and the crossbreeding work of northern Herbaceous peony kind, in the hope of the Herbaceous peony new varieties possessing independent intellectual property right and Zhejiang region feature can be obtained early.
Summary of the invention
The invention provides a kind of method measuring paenoiae alba Pollen Activity, the Pollen Activity of paenoiae alba can be measured quickly and accurately.
Measure a method for paenoiae alba Pollen Activity, comprising: the pollen collecting paenoiae alba; Cultured in vitro is carried out on the surface described pollen being placed in nutrient culture media; Cultured in vitro terminates rear statistics pollen germination rate;
Consisting of of described nutrient culture media: sucrose 80 ~ 150g/L, boric acid 15 ~ 30mg/L, agar 8 ~ 10g/L;
The time of described cultured in vitro is 2.5 ~ 3.5h.
The germination in vitro method of pollen is the effective ways measuring Pollen Activity, and reproducibility of results is high, generally comprises following steps: collect pollen, be placed in by pollen on nutrient culture media and carry out cultured in vitro, examine under a microscope pollen germination situation, statistics pollen germination rate.
The sprouting ability of pollen is one of index of reflection Pollen Activity, sprouts and grows pollen tube, could pass style, enter ovary, complete amphigamy because pollen only has.The sprouting ability of pollen represents with pollen germination rate usually, can be measured the vigor of pollen by the germination rate adding up pollen.
The nutrient culture media of cultured in vitro, incubation time are the principal elements that impact measures accuracy rate, the plant of not of the same race, kind, its pollen the composition of nutrient culture media that is suitable for often widely different, nutrient culture media of the present invention is solid medium, be conducive to follow-up microscopical observation, and be made up of water, sucrose, boric acid and agar.Wherein, sucrose is the Major Nutrient material source of pollen germination, and boric acid then can promote the lasting elongation of the sprouting of pollen and pollen tube, and sucrose and boric acid are only in suitable concentration range, can make the sprouting that pollen is good.Preferably, the concentration of described sucrose is 100g/L, and the concentration of described boric acid is 20g/L.
Secondly, the sag that the pollen that the present invention is directed to paenoiae alba exists in incubation, controls the agar content in the nutrient culture media of cultured in vitro and incubation time, improves the accuracy that Pollen Activity measures.The time of described cultured in vitro is preferably 3h, and in nutrient culture media, the concentration of described agar is preferably 8g/L.In addition, the cultured in vitro time of the present invention has also taken into account the population effect of pollen germination, if incubation time is shorter, then pollen germination amount is few, can not reflect Pollen Activity strictly according to the facts.Within the cultured in vitro time of the present invention, the pollen germination rate of paenoiae alba tends towards stability.In addition paenoiae alba of the present invention originates from Panan County, Jinhua, Zhejiang Province city.
The method of described collection pollen is: the flower pesticide gathering paenoiae alba, be placed in dry vessel dry 24 ~ 48 hours, until pollen instant of complete cracking, then collect pollen, wherein, the flower of paenoiae alba when described flower pesticide preferably takes from that internal layer petal slightly bursts forth, flower pesticide mays be seen indistinctly.
During cultured in vitro, can not solidified nutrient culture media be joined in the spherical concave surface of spill microslide, clear for focusing, improve statistical accuracy, the surfacing of nutrient culture media should be kept as far as possible, after culture medium solidifying, then pollen is seeded to the surface of nutrient culture media.
Suitable cultivation temperature is conducive to paenoiae alba pollen and sprouts fast, and preferably, the temperature of described cultured in vitro is 23 ~ 28 DEG C, is more preferably 25 DEG C.
In the cultured in vitro process of pollen, in order to prevent the sprouting of the Influence of Evaporation pollen of moisture in described nutrient culture media, during cultured in vitro, the relative humidity keeping environment is 80% ~ 85%, is preferably 80%.
After in-vitro pollen cultivates a period of time, negative polliniferous microslide is placed in basis of microscopic observation, reaches 1 times of pollen granule diameter with pollen tube length and above pollen is considered as sprouting, statistics germination rate.Described microscope specifically can adopt OlympusBH-2 fluorescent microscope, is more conducive to the germination rate adding up paenoiae alba pollen.
Compared with prior art, beneficial effect of the present invention is:
Method measurement result of the present invention is reliable and stable, has verified the nutrient culture media, condition of culture, incubation time, observation condition etc. that are comparatively applicable to of paenoiae alba pollen germination, first for providing quick, the reliable method that measure Pollen Activity for the paenoiae alba of pharmacy.
Accompanying drawing explanation
Fig. 1 is applicable to petal stretching degree when gathering paenoiae alba pollen, observation Pollen Activity.
Fig. 2 utilizes the nutrient culture media of 3 kinds of agar powder content to cultivate paenoiae alba pollen, the germination rate variation diagram of process in time after the inoculation of its pollen.
Fig. 3 is the sprouting figure of process in time after the inoculation of paenoiae alba pollen;
A: the pollen germination situation after inoculation 1h; B: the pollen germination situation after inoculation 3h;
C: the pollen germination situation after inoculation 5h; D: the pollen germination situation after inoculation 6h.
The pollen of paenoiae alba is seeded on the nutrient culture media that holds with double dish by Fig. 4, observes the image of pollen germination under anatomical lens;
A: do not pad observation during black cloth bottom double dish; B: be lined with observation during black cloth bottom double dish.
When Fig. 5 to be the nutrient culture media upper surface of concave surface microslide be plane and protruding sphere, the corresponding diagram of paenoiae alba pollen germination observation;
A: sectional view when nutrient culture media is plane on concave surface microslide; B: sectional view when nutrient culture media is protruding sphere on concave surface microslide; C is paenoiae alba pollen germination observation figure corresponding in A figure situation; D is paenoiae alba pollen germination observation figure corresponding in B figure situation.
Embodiment
The present invention is explained further below in conjunction with embodiment.
The collection of 1 paenoiae alba flower pesticide
Choose robust plant, be acquisition target (the paenoiae alba place of production selected by this test is Panan County, Jinhua, Zhejiang Province city) without the paenoiae alba of disease and pest, within it layer petal slightly burst forth, may be seen indistinctly inner golden yellow flower pesticide time be acquisition time (Fig. 1).Select mid or late April calm fine day the morning 9 ~ 10 time gather flower pesticide, clamp the filigree of some stamens with sharp mouth tweezers, take off flower pesticide, flower pesticide is put into sulfuric acid paper bag and store.
The drying of 2 pollen
Template is spread out the double dish being placed in dried and clean, by extruding by template and double dish inwall close contact.Flower pesticide is evenly sprinkling upon on template, avoids the buttress between flower pesticide to fold.Double dish is placed in the hermetically drying device filling discolour silica gel, dry 24 ~ 48 hours, pollen shed stand-by.
The foundation of 3 pollen germination nutrient culture media
Select sucrose 100g/L and boric acid 20mg/L, using distilled water as solvent.Whether are the use amount of agar powder and the use of anhydrous calcium chloride, impact can sprout effect and observation to a great extent, therefore need establish the proportioning both this.
The selection of 3.1 agar powder use amounts
Add agar powder 5g/L, 8g/L and 10g/L respectively in the medium, find in the pollen germination in later stage: the nutrient culture media quality adding 5g/L agar powder is softer, pollen granule starts to sink after cultivation 2.5h, pollen granule and pollen tube are all gradually by nutrient culture media Surface mulch, when causing pollen viability also not reach stationary value, just do not see partial pollen and sprouted the pollen tube, thus make the statistical value of pollen germination rate (Fig. 2) less than normal compared with actual value.
The nutrient culture media adding 8g/L and 10g/L agar powder is comparatively solid, the sagging time can be delayed to pollen inoculation 4h after, thus make pollen granule and pollen tube can keep clear and legible at about the 3h that pollen germination rate is comparatively stable, be easy to observation and counting (in Fig. 3 B).But after 5h, pollen granule and pollen tube still can sink, gradually by nutrient culture media Surface mulch, do not see the sprouting of pollen tube, more difficult observation; Simultaneously pollen tube is long and appearance dish knot tangles, and has been unwell to observation germination rate (in Fig. 3 C); After 6h, pollen granule and pollen tube are obviously covered by nutrient culture media, and the pollen tube growth being still positioned at media surface is longer, and partial pollen occurs that drying shrinkage is hung splits, and are not suitable for observing (in Fig. 3 D).
Therefore, the agar powder consumption of 5g/L is very few, and to add 8g/L and 10g/L agar powder be comparatively suitable, and the nutrient culture media of the two on pollen sink time and degree without obvious gap.Take saving as principle, select the agar powder of 8g/L most suitable.
Whether the selection of 3.2 anhydrous calcium chlorides
Part research shows, calcium ion can promote the sprouting of pollen tube to a certain extent, therefore can add calcium ion in the nutrient culture media measured at the Pollen Activity of some plants, mostly is calcium nitrate or lime chloride.Add the anhydrous calcium chloride of 25mg/L in the medium and do not add lime chloride, comparing result finds: not adding pollen in the nutrient culture media of anhydrous calcium chloride and possess higher and stable germination rate, is aforesaid 63.301%.Add in the nutrient culture media of anhydrous calcium chloride, pollen germination rate is very low on the contrary, even if when observed reading is the highest, i.e. postvaccinal 3h, germination rate is also only 21.057%, and all the other times all below 20% (table 1).
The use of table 1 calcium ion is on the impact of paenoiae alba pollen germination rate
| Incubation time (h) | 0 | 0.5 | 1 | 1.5 | 2 | 2.5 | 3 |
| Germination rate (%, without calcium) | 0 | 0 | 0 | 14.303 | 32.731 | 60.285 | 63.300 |
| Germination rate (% has calcium) | 0 | 0 | 0 | 3.694 | 11.524 | 15.288 | 21.057 |
| Incubation time (h) | 3.5 | 4 | 4.5 | 5 | 5.5 | 6 | |
| Germination rate (%, without calcium) | 61.689 | 56.674 | 52.743 | 50.374 | 48.195 | 44.931 | |
| Germination rate (% has calcium) | 16.352 | 10.951 | 11.965 | 13.834 | 10.365 | 8.069 |
As can be seen here, the nutrient culture media of paenoiae alba pollen germination and traditional Herbaceous peony kind possess otherness.Generally, the use of calcium ion can promote the sprouting of traditional Herbaceous peony pollen tube to a certain extent, but seriously can hinder the sprouting of paenoiae alba pollen.Even cause the reason of this kind of phenomenon to be failed to understand, need to be studied further.
The selection of 4 observation instruments
Select OlympusBH-2 optical microscope as observation instrument, concave surface microslide, as the carrying tool of nutrient culture media, is equipped with the pipettor of 1mL rifle head, medical cotton stick is aid.
The instrument being generally used for observing Pollen Activity is anatomical lens and optical microscope.This team possesses OPLENICSZM745T anatomical lens and OlympusBH-2 optical microscope, when measuring the pollen viability of the flowers such as lilium flowers, fringed iris, all select anatomical lens, the nutrient culture media being about to just prepare is poured in double dish, after it solidifies or when liquid state, inoculate pollen, observe.But found by actual observation, the pollen granule of paenoiae alba compare lilium flowers and fringed iris little, even if use the most high magnification of anatomical lens, the visual field is still excessive, too small pollen is possessed too much in the visual field, and arachnoid can be formed in the whole visual field after germination of pollen tube, be difficult to count (in Fig. 4 A); Simultaneously because paenoiae alba pollen is golden yellow, very unintelligible under the light of anatomical lens, need bottom double dish, pad black cloth and just can see pollen granule and pollen tube clearly, also can bring very large difficulty (in Fig. 4 B) to observation.
Compare OPLENICSZM745T anatomical lens, OlympusBH-2 optical microscope possesses higher enlargement ratio, is applicable to the observation of pollen germination, but cannot puts into the larger double dish of volume because of hypotelorism between camera lens and objective table.Therefore, the Pollen Activity of observation paenoiae alba can select more frivolous concave surface microslide as the carrying tool of nutrient culture media, can put objective table smoothly.Select the Splan10PL of OlympusBH-2 optical microscope (0.30,160/0.17) camera lens to carry out observation most suitable, namely select the camera lens of 10 times of enlargement ratios.The enlargement ratio of its 4 times and 20 times adjacent camera lenses is too small and excessive respectively, is difficult to ensure suitable pollen observation quantity in the visual field.
The inoculation of 5 pollen
The injection of 5.1 nutrient culture media
When the nutrient culture media ot-yet-hardened configured, draw a little with the pipettor being furnished with 1mL rifle head, inject the spherical concave surface of concave surface microslide gently.Rifle head can draw circle gently, ensures being uniformly distributed and taking in right amount of liquid culture medium.
In spherical concave surface, the upper surface of nutrient culture media will keep smooth as far as possible, avoids presenting spherical surface hill, otherwise there will be the situation that partial pollen is clear, part void is burnt when observing, and very affects observed result.The depth of field of optical microscope is very shallow, and region clearly of focusing is very narrow, needs to ensure media surface smooth, and most of pollen granule just can be made all to drop on clearly in focusing area (in Fig. 5 in A and 5 C).On protruding sphere, the pollen that there will be in a part of region drops in field depth, focus clear, and another part drops on outside field depth, thus smudgy (in Fig. 5 in B and 5 D), make observed reading greatly less than normal.
The inoculation of 5.2 pollen
Dip a small amount of pollen with medical cotton stick, shake gently above the nutrient culture media of spherical concave surface, allow pollen fall to media surface, or smear to media surface gently.Guarantee is smeared evenly, prevents pollen buttress and folds or agglomerate, impact observation.If any agglomerate, available dissecting needle is sent out, but will prevent scratch from solidifying the surface of nutrient culture media, impact observation sharpness.Microslide is put into the growth cabinet that temperature is 25 DEG C, relative humidity is 80% to cultivate.
The mensuration of 6 Pollen Activities
The determination of 6.1 effective observation times
On the nutrient culture media of aforesaid " sucrose 100g/L+ boric acid 20mg/L+8g/L agar powder ", paenoiae alba pollen has pollen tube to stretch out at inoculation about 1h as seen, but can not count because pollen tube is shorter; Just in 2h have pollen germination more, can extend to the several times of pollen granule diameter; 2.5 ~ 3.5h is pollen germination rate comparatively stable period, substantially no longer rises; Start when 4h to occur pollen sag, affect counting because pollen tube is embedded by nutrient culture media, the observed reading of germination rate starts to reduce; Sink during 5 ~ 6h more obvious, a lot of pollen tube almost all sinks under nutrient culture media top layer, does not observe, and the pollen tube being still positioned at media surface then extends long, causes occurring that pollen tube tangles in the whole visual field, is more difficult to observation.Therefore select the postvaccinal 2.5 ~ 3.5h of pollen to be Optimal observational duration (Fig. 2).
6.2 observation procedures and result
When pollen cultures 2.5 ~ 3.5h, by concave surface microslide as sprouting and the elongation of observing pollen tube under OlympusBH-2 fluorescent microscope.5 visuals field observed by each microslide, are no less than 30 pollen in each visual field.Be greater than pollen granule diameter as sprouting standard using pollen tube length, each test all repeats 3 times.Germination rate is the ratio of pollen number and the total pollen granule number sprouted.
6.3 observed result
Under this cultivating system, the most stable in paenoiae alba pollen germination rate 2.5 ~ 3.5h after inoculation, wherein maximum in 3h value, be 63.301%(table 2).Visible, paenoiae alba germination rate is higher, can be cross-breeding as male parent material well.This team has acquired the pollen of paenoiae alba, and do with northern Herbaceous peony kind and hybridize, all cross combination all produces seeds and takes root, and the upgrowth situation of cross hybrid seedling needs to be observed further.
The pollen germination rate of nutrient culture media after cultivating 3h of table 2 different ratio
| The nutrient culture media of different ratio | Germination rate (%) |
| Agar powder is that 5g/L(is without calcium) | 57.569 |
| Agar powder is that 8g/L(is without calcium) | 63.301 |
| Agar powder is that 8g/L(has calcium) | 21.057 |
| Agar powder is that 10g/L(is without calcium) | 62.568 |
Claims (3)
1. measure a method for paenoiae alba Pollen Activity, comprising: the pollen collecting paenoiae alba; Cultured in vitro is carried out on the surface described pollen being placed in nutrient culture media; Cultured in vitro terminates rear statistics pollen germination rate; It is characterized in that,
Consisting of of described nutrient culture media: sucrose 100g/L, boric acid 20mg/L, agar 8g/L;
The temperature of described cultured in vitro is 23 ~ 28 DEG C, and the time is 3h, and relative humidity is 80 ~ 85%; Described paenoiae alba originates from Panan County, Jinhua, Zhejiang Province city.
2. the method for claim 1, is characterized in that, described nutrient culture media is in the spherical concave surface of concave surface microslide, and the upper surface of described nutrient culture media keeps smooth.
3. the method for claim 1, is characterized in that, during statistics pollen germination rate, adopts OlympusBH-2 observation by light microscope pollen germination situation.
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