CN104480184A - Method for determining viability of pollen by carrying out in-vitro germination culture on brassica rapa pollen - Google Patents
Method for determining viability of pollen by carrying out in-vitro germination culture on brassica rapa pollen Download PDFInfo
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Abstract
The invention particularly relates to a method for determining the viability of pollen by carrying out in-vitro germination culture on brassica rapa pollen and an in-vitro germination culture liquid for brassica rapa pollen. The method for determining the viability of pollen by carrying out in-vitro germination culture on brassica rapa pollen is mainly characterized by comprising the following steps: (1) collecting pollen, (2) carrying out in-vitro germination on pollen and growing pollen tubes; and (3) determining the viability of pollen. The method has the advantages that the liquid culture medium for in-vitro germination of brassica rapa pollen provided by the invention takes BK as a solvent, comprises the following components: 50g.L<-1> of polyethylene glycol (PEG) 6000, 150g.L<-1> of sucrose, 2mg.L<-1> of gibberellin and 10mg.L<-1> of vitamin B1 and has pH of 6.5; the brassica rapa pollen is cultured under the illumination condition at 35 DEG C, the germination rate of the brassica rapa pollen can reach 75.3% and the pollen tubes can well grow; since PEG is added, the brassica rapa pollen tubes straightly grow, so that observation and measurement are convenient.
Description
Technical field
The invention belongs to plant pollen vitality test technical field, particularly a kind of germination in vitro liquid nutrient medium of turnip type rape pollen, and utilize the method for this turnip type rape in-vitro pollen germination liquid culture based assays turnip type rape Pollen Activity.
Background technology
Turnip type rape (Brassica rapa L.) is one of Brassica Crops diploid elementary species, and precocious, cold-resistant and type is enriched, and is one of three major types type of China's Cultivated rapeseed.Turnip type rape plant is general shorter and smaller, and main root is more flourishing or undeveloped, and side root is medium or flourishing.Leaf base embraces stem entirely.Spend yellowish to deep yellow, petal is circular, comparatively large, and when blooming, petal side is repeatedly or in imbricate; In the middle of inflorescence, bud is lower than open flower; Flower pesticide extroversion cracking.The gorgeous U.S. flood dragon of rape flower is beautiful, can beautify the environment, and has higher ornamental value, and containing abundant nectar, is the good nectar source of development Apiculture.
Rape is the high crop of economic worth, and the rapeseed oil of squeezing out by Semen Brassicae campestris is containing abundant lipid acid and multivitamin, nutritious, and being easy to digestion, is comparatively ideal edible vegetable oil.Along with the raising of breeding technique in modern age, the oil of rapeseed oil is improved thereupon, and the new variety of low toxicity low erucic acid, are come by popularization gradually, and purposes is also more and more extensive.The industry such as metallurgy, machinery, rubber, chemical industry, paint, soapmaking, medicine, all using rapeseed oil as raw material.The dregs of fat are commonly called as rapeseed cake, rich in proteins, crude fat, amino acid and mineral element, are first-class organic fertilizer.Rapeseed cake is through detoxification treatment, or fabulous livestock concentrated feed.The dish bar, branches and leaves, shell etc. of rape, containing the crude protein of more than 3%, the crude fat of about 0.5% and a small amount of glucide, be embedded in soil, be converted into organic matter, can fertilize the soil and increase soil fertility, be called " tender stubble " by peasant, also can be ground into feed, be used for developing animal raising.In addition, Pollen Brassicae campestris, large of fine quality, be first-class nutritious prod, be called " optimum food of long-lived person " by people
In view of turnip type rape pollen is as the main carriers of turnip type rape genetic resources, therefore, carry out the research of turnip type rape Pollen Activity mensuration, Chinese cabbage type Rape Germplasm Resources is preserved and with the seed selection of innovation, improved seeds, there is very important theory and practice meaning.At present, Pollen Activity method for measuring mainly contains microscope morphologic observation, mineral acid detection method, staining, field pollination detection method, germination in vitro method etc.
Morphologic observation be according to pollen sterile in process of growth owing to being subject to the impact of some factor, ateliosis or bad pollen morphology are usually in deformity, and the normal well-regulated profile of pollen.The normal pollen of turnip type rape is oval, and pollen sterile is irregular shape, can differentiate pollen viability according to form.But, the turnip type rape mature pollen of storage for some time, in storage, Pollen Activity can decline gradually, and because the normal pollen of turnip type rape vigor in storage may be lost, but form considerable change does not occur; Thus normal Pollen Activity changing conditions after making pollen morphology observation just can not differentiate storage.
Inorganic acid system is that pollen there will be germ pore has the phenomenon of expansion outer process to judge whether pollen has vigor by after mineral acid treatment, but also there will be this phenomenon after pollen water suction, adopts mineral acid can not judge whether pollen really has vigor.
Staining is that judge the height of Pollen Activity according to colour-change, conventional staining reagent has I by using staining reagent to dye to pollen granule
2-KI solution, aceto-camine and triphenyltetrazolium chloride (TTC) etc.And Determination Staining Pollen Activity experimental repeatability is poor, very large experimental error can be caused, can not the actual vigor height of Accurate Determining pollen.
Field pollination method judges pollen viability according to the formational situation of fruit and seed, although the most close with actual production, reflect final setting percentage, but its time cycle is long, workload is large, and be vulnerable to the impact in weather condition, time place, be unfavorable for producing, adopt less in practical application.
In-vitro pollen germination and pollen tube growth measurement result comparatively stable, more can react the actual state of Pollen Activity.But in-vitro pollen germination needs suitable substratum, conventional substratum basal component is sugar and boron, and sucrose concentration is generally 10% ~ 20%, and boric acid is 0.001% ~ 0.005%, pH 5.5 ~ 6.5.
The kinds of culture medium that different Plant Pollen Germination needs is different with concentration, two caryogram pollen are more easily sprouted, and general minimum medium is cultivated, and the more difficult sprouting of three caryogram pollen, the element that other promotes pollen germination to be added, as Ca on the basis of minimum medium
2+, Mg
2+, K
+, the compositions such as PEG.In recent years, people have carried out more research to cress in-vitro pollen germination and pollen tube growth, and Song Hongxia etc. (PEG on the impact of Radix Dauci Sativae in-vitro pollen germination, 2011 (06): 22-24. the Changjiang river vegetables; Radix Dauci Sativae in-vitro pollen germination is studied, and 2010,38 (4): 25-27, Shanxi Agricultural science) think that PEG6000 has remarkable promoter action to the growth of Radix Dauci Sativae in-vitro pollen germination, and boric acid, Ca
2+promoter action is had to Radix Dauci Sativae pollen germination; Cao Yi etc. report, and (swede type rape in-vitro pollen is cultivated, 2010,30 (2): 0399-0404, northwest Botany Gazette) appropriate media of swede type rape in-vitro pollen germination, Zhao Yanling etc. (sprout and study with pollen tube growth factor of influence by radish in vitro pollen, the first phase in 2007, Jiangsu's agriculture science) have studied radish in vitro pollen and sprout the factor of influence with pollen tube growth.But there is not been reported both at home and abroad about the research of turnip type rape in-vitro pollen germination and pollen tube growth.
Summary of the invention
The object of the invention is to, for avoiding the deficiencies in the prior art, providing a kind of germination in vitro of turnip type rape pollen to cultivate the method measuring Pollen Activity.On the basis determining turnip type rape in-vitro pollen germination culture system, to Chinese cabbage type Pollen Brassicae campestris isolated culture, reliable, effective mensuration turnip type rape Pollen Activity.
For achieving the above object, the technical scheme that the present invention takes is: a kind of germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity, and its principal feature is to comprise the following steps:
1) pollen collection:
Choose the healthy and strong turnip type rape plant growing the harm of normal, anosis worm, choose the inflorescence of having bloomed in turnip type rape full-bloom stage, collect pollen;
2) in-vitro pollen germination and pollen tube growth:
Pollen is evenly sprinkling upon on turnip type rape in-vitro pollen germination liquid nutrient medium, 30 DEG C ~ 40 DEG C constant temperature culture; PH is 6.0 ~ 7.0;
In-vitro pollen germination is cultivated 15min ~ 25min rear section pollen and is started to sprout, and after pollen germination, pollen tube length exceedes pollen granule diameter, and pollen tube starts quick growth;
3) mensuration of Pollen Activity:
After isolated culture 45min ~ 1h, be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 3 ~ 5 visuals field, pollen number 30 ~ 80 is observed in every visual field, statistics pollen germination rate, and pollen germination rate reaction has vitality pollen proportion.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, it is characterized in that step also includes:
4) automatically just put microscopic system with LeicaDM6000B and carry micrometer measurement pollen tube length, every visual field random measurement 10 ~ 15 pollen, often process and survey 30 ~ 40 pollen tube lengths altogether, calculate its mean value, the quality of in-vitro pollen germination pollen tube growth situation reaction pollen granule physiological situation.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, and described turnip type rape in-vitro pollen germination liquid nutrient medium, take BK as solvent, its component comprised has 50gL
-1~ 100gL
-1pEG 6000 (polyethylene glycol 6000), 130gL
-1~ 150gL
-1sucrose, 1.5mgL
-1~ 2.5mgL
-1plant hormones regulators,gibberellins and 8.5m gL
-1~ 10mgL
-1vITMAIN B1, pH is 6.0 ~ 7.0.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, and described BK consists of: H
3bO
3(boric acid) 100mgL
-1, Ca (NO
3)
24H
2o (nitrocalcite) 300mgL
-1, MgSO
47H
2o (crystalline sulfuric acid magnesium) 200mgL
-1and KNO
3(saltpetre) 100mgL
-1.Take distilled water as solvent.
Compared with prior art, the present invention has following useful technique effect:
1, turnip type rape pollen is on turnip type rape in-vitro pollen germination liquid nutrient medium provided by the invention, and cultivate under 35 DEG C of illumination conditions, turnip type rape pollen germination rate can reach 75.3%, and pollen tube also can better be grown; Owing to the addition of PEG, turnip type rape pollen tube growth is comparatively straight, is convenient to observe and measures.
2, turnip type rape in-vitro pollen germination and pollen tube growth determination techniques is utilized to measure Pollen Activity measurement result reliable and stable, pollen germination rate reaction has vitality pollen proportion, the quality of pollen granule physiological situation is reacted in observation and the measurement of pollen tube growth, and turnip type rape in-vitro pollen germination and pollen tube growth determination techniques are that the mensuration of turnip type rape Pollen Activity provides a kind of reliable, effective means.
Accompanying drawing illustrates:
Fig. 1. the growth of turnip type rape in-vitro pollen germination is with the changing conditions of incubation time
Fig. 2. during for pH6.5, turnip type rape pollen is at BK+150gL
-1sucrose+50gL
-1pEG6000+2mgL
-1plant hormones regulators,gibberellins+10m gL
-1vITMAIN B1 (35 DEG C of constant temperature) substratum on sprout 40min after the photo schematic diagram that gathers.
Fig. 3. during for pH6.5, turnip type rape pollen is at BK+150gL
-1sucrose+50gL
-1pEG6000+2mgL
-1plant hormones regulators,gibberellins+10m gL
-1vITMAIN B1 (35 DEG C of constant temperature) substratum on sprout 50min after the photo schematic diagram that gathers.
Fig. 4. during for pH6.5, turnip type rape pollen is at BK+150gL
-1sucrose+50gL
-1pEG6000+2mgL
-1plant hormones regulators,gibberellins+10m gL
-1vITMAIN B1 (35 DEG C of constant temperature) substratum on sprout 1h after the photo schematic diagram that gathers.
Embodiment
Below in conjunction with accompanying drawing shownschematically preferred example be described in further detail:
Embodiment 1: see Fig. 1, a kind of germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity, and its principal feature is to comprise the following steps:
1) pollen collection:
Choose the healthy and strong turnip type rape plant growing the harm of normal, anosis worm, choose the inflorescence of having bloomed in turnip type rape full-bloom stage, collect pollen;
2) in-vitro pollen germination and pollen tube growth:
Pollen is evenly sprinkling upon on turnip type rape in-vitro pollen germination liquid nutrient medium, 30 DEG C of constant temperature culture; PH is 6.0;
In-vitro pollen germination is cultivated 15min rear section pollen and is started to sprout, and after pollen germination, pollen tube length exceedes pollen granule diameter, and pollen tube starts quick growth;
3) mensuration of Pollen Activity:
After isolated culture 45min, be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 3 visuals field, every visual field is observed pollen number and is no less than 30, statistics pollen germination rate, and pollen germination rate reaction has vitality pollen proportion.
4) automatically just put microscopic system with LeicaDM6000B and carry micrometer measurement pollen tube length, every visual field random measurement 10 pollen, often process and survey 30 pollen tube lengths altogether, calculate its mean value, the quality of in-vitro pollen germination pollen tube growth situation reaction pollen granule physiological situation.
Embodiment 2: a kind of germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity, and its principal feature is to comprise the following steps:
1) pollen collection:
Choose the healthy and strong turnip type rape plant growing the harm of normal, anosis worm, choose the inflorescence of having bloomed in turnip type rape full-bloom stage, collect pollen;
2) in-vitro pollen germination and pollen tube growth:
Pollen is evenly sprinkling upon on turnip type rape in-vitro pollen germination liquid nutrient medium, 35 DEG C of constant temperature culture; PH is 6.5;
In-vitro pollen germination is cultivated 20min rear section pollen and is started to sprout, and after pollen germination, pollen tube length exceedes pollen granule diameter, and pollen tube starts quick growth;
3) mensuration of Pollen Activity:
After isolated culture 50min, be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 4 visuals field, every visual field is observed pollen number and is no less than 50, statistics pollen germination rate, and pollen germination rate reaction has vitality pollen proportion.
4) automatically just put microscopic system with LeicaDM6000B and carry micrometer measurement pollen tube length, every visual field random measurement 12 pollen, often process and survey 35 pollen tube lengths altogether, calculate its mean value, the quality of in-vitro pollen germination pollen tube growth situation reaction pollen granule physiological situation.
Embodiment 3: a kind of germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity, and its principal feature is to comprise the following steps:
1) identical with embodiment 1.
2) in-vitro pollen germination and pollen tube growth:
Pollen is evenly sprinkling upon on turnip type rape in-vitro pollen germination liquid nutrient medium, 40 DEG C of constant temperature culture; PH is 7.0;
In-vitro pollen germination is cultivated 25min rear section pollen and is started to sprout, and after pollen germination, pollen tube length exceedes pollen granule diameter, and pollen tube starts quick growth;
3) mensuration of Pollen Activity:
After isolated culture 1h, be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 5 visuals field, pollen number 80 is observed in every visual field, statistics pollen germination rate, and pollen germination rate reaction has vitality pollen proportion.
4) automatically just put microscopic system with LeicaDM6000B and carry micrometer measurement pollen tube length, every visual field random measurement 15 pollen, often process and survey 40 pollen tube lengths altogether, calculate its mean value, the quality of in-vitro pollen germination pollen tube growth situation reaction pollen granule physiological situation.
Embodiment 4: described turnip type rape in-vitro pollen germination liquid nutrient medium, take BK as solvent, it also comprises component 50gL
-1pEG 6000 (polyethylene glycol 6000), 130gL
-1sucrose, 1.5mgL
-1plant hormones regulators,gibberellins and 8.5m gL
-1vITMAIN B1, pH is 6.0.
Wherein the consisting of of BK: H
3bO
3(boric acid) 100mgL
-1, Ca (NO
3)
24H
2o (nitrocalcite) 300mgL
-1, MgSO
47H
2o (crystalline sulfuric acid magnesium) 200mgL
-1and KNO
3(saltpetre) 100mgL
-1.Take distilled water as solvent.
Embodiment 5: described turnip type rape in-vitro pollen germination liquid nutrient medium, be solvent with BK, it comprises component 80gL
-1pEG 6000 (polyethylene glycol 6000), 100gL
-1sucrose, 2.0mgL
-1plant hormones regulators,gibberellins and 9m gL
-1vITMAIN B1, pH is 6.5.
Wherein the composition of BK is as embodiment 4.
Embodiment 6: described turnip type rape in-vitro pollen germination liquid nutrient medium, be solvent with BK, it comprises component 100gL
-1pEG 6000 (polyethylene glycol 6000), 150gL
-1sucrose, 2.5mgL
-1plant hormones regulators,gibberellins and 10mgL
-1the BK nutrient solution of VITMAIN B1, pH is 7.0.
Wherein the composition of BK is as embodiment 4.
Experimental verification example: a kind of germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity,
1, the collection of turnip type rape pollen
Turnip type rape pollen picks up from arid habitat key lab of Gansu Agriculture University at the beginning of certain 10 months year, acquisition method is: choose the healthy and strong plant growing the harm of normal, anosis worm, choose the inflorescence of having bloomed in full-bloom stage, and bud is naturally open, pollen sheds from flower pesticide, collects pollen for subsequent use.
2, the screening of in-vitro pollen germination culture condition:
The screening of 2.1 in-vitro pollen germination substratum:
Kinds of culture medium has considerable influence to plant pollen germination in vitro, and the appropriate media of different Plant Pollen Germination is different.With BK substratum for minimum medium, screen the PEG6000 of suitable concn on its basis successively, sucrose, GA
3and VB1, finally regulate suitable pH.
BK consists of (mgL
-1): H
3bO
3100mgL
-1, Ca (NO
3)
24H
2o 300mgL
-1, MgSO
47H
2o 200mgL
-1and KNO
3100mgL
-1.Take distilled water as solvent.
2.2PEG concentration screening
PEG6000 is a kind of high-molecular weight compounds, mainly plays osmotic adjust action.Fresh pollen respectively uniformly dispersing with the addition of 0gL
-1, 50gL
-1, 100gL
-1, 150gL
-1, 200gL
-1the BK substratum of PEG6000 (BK substratum is liquid; Here 0gL
-1the BK substratum of PEG is not added in representative.In BK, do not add distilled water again) in,
Concrete operations are as follows: (1) regulates the pH to 7.0 of above-mentioned different PEG concentration cultures with the NaOH of 1mol/L; (2) 70 μ L substratum are drawn with liquid-transfering gun, drop in gently on slide glass and form dome shape drop, by the pollen sowing on two stamens on sphere substratum, substratum is not stirred during sowing, in order to avoid pollen granule sinks to liquid culture basis pontis, in germination process, affect experimental result due to anoxic, preferably pollen granule is layered on substratum uniformly; (3) above-mentioned slide glass is put into be covered with two-layer moistening filter paper and (keep certain humidity; avoid sprout time substratum evaporation too for a long time) culture dish in; do not add cover glass (to guarantee having sufficient oxygen in pollen germination process), cover tightly after culture dish lid cultivates 1h in 35 DEG C of incubators and examine under a microscope statistics sprouting number and pollen tube length.(4) be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 3 visuals field, every visual field is observed pollen number and is no less than 30, statistics pollen germination rate; Automatically just putting microscopic system with LeicaDM6000B simultaneously and carrying micrometer measurement pollen tube length, every visual field random measurement 10 pollen, often process and survey 30 pollen tube lengths altogether, calculate its mean value.Test in triplicate.
Result is as shown in table 1, adds the PEG6000 of different concns in BK substratum, can significantly improve turnip type rape pollen germination rate, wherein add 50gL
-1pEG6000 effect is the most remarkable, and pollen germination rate reaches 41.8%; Lower concentration PEG (50 ~ 150gL
-1) promote pollen tube growth, and increase with concentration, promoter action weakens gradually, and high density PEG (is greater than 200gL
-1) suppress pollen tube growth; Also find when microscopic examination, in the substratum that with the addition of lower concentration PEG, turnip type rape pollen tube growth is comparatively straight, and the substratum pollen tube growth not adding PEG or added high density PEG bends, and normal weave in, inconvenience is observed and measured.
The impact that table 1 different concns PEG grows pollen germination
2.3 sucrose concentration tests
Sucrose is a kind of osmotic adjustment, is also the energy substance of pollen tube growth simultaneously.At 50gL
-1100gL is increased respectively in the substratum of PEG6000+BK
-1, 150gL
-1, 200gL
-1, 250gL
-1sucrose, the sucrose comparing different concns is on the impact of pollen germination and pollen tube growth.
Result is as shown in table 2, BK+50gL
-1in PEG substratum, different concns sucrose is different with pollen tube growth impact on Chinese cabbage type Pollen Brassicae campestris germination rate, lower concentration sucrose (150gL
-1) to pollen germination and pollen tube growth, there is obvious promoter action, and the sucrose (>=200gL of high density
-1) can obviously suppress pollen germination and pollen tube growth, and along with the increase of sucrose concentration, be suppressed degree more obvious.Containing 150gL
-1the BK+50gL of sucrose
-1on PEG substratum, the growth of turnip type rape pollen is best, and after cultivating 1h, its pollen germination rate and pollen tube length reach 65.1% and 91.1 μm respectively.
The impact that table 2 different concns sucrose grows pollen germination
2.4GA
3concentration tests
Plant hormones regulators,gibberellins can suppress the activity of growth hormone decomposing system in plant materials, and to ensure the relative increase of tumor growth cellulose content level, certain density Exogenous gibberellic acid process can promote pollen germination and pollen tube growth.At BK+50gL
-1pEG6000+150gL
-11mgL is increased respectively in sucrose medium
-1, 2mgL
-1, 3mgL
-1gA
3, detect the GA of different concns
3chinese cabbage type Pollen Brassicae campestris is sprouted and the impact of pollen tube growth.
Result is as shown in table 3, the GA of different concns
3sprout different with the impact of pollen tube growth on Chinese cabbage type Pollen Brassicae campestris, lower concentration GA
3(2mgL
-1) to pollen germination and pollen tube growth, there is obvious promoter action, and GA
3concentration too low or be too highly all unfavorable for pollen germination and pollen tube growth.Containing 2mgL
-1gA
3bK+50gL
-1pEG+150gL
-1in sucrose medium, the growth of turnip type rape pollen is best, and after cultivating 1h, its pollen germination rate and pollen tube length reach 69.2% and 157.2 μm respectively.
Table 3 different concns GA
3on the impact of pollen germination growth
2.5VB1 concentration tests
Forefathers' research shows, physiologically active class Vitamins B1 increases to pollen germination speed, reaches object efficiently.At BK+50gL
-1pEG6000+150gL
-1sucrose+2mgL
-15mgL is increased respectively in the substratum of GA3
-1, 10mgL
-1, 15mgL
-1vB1, the VB1 of research different concns sprouts and the impact of pollen tube growth Chinese cabbage type Pollen Brassicae campestris.
Result is as shown in table 4, and the VB1 of different concns sprouts different with the impact of pollen tube growth on Chinese cabbage type Pollen Brassicae campestris, containing 10mgL
-1vB1 at BK+50gL
-1pEG6000+150gL
-1sucrose+2mgL
-1gA
3on substratum, the growth of turnip type rape pollen is best, and after cultivating 30min, its pollen germination rate and pollen tube length reach 58.4% and 76.4 μm respectively.
The impact that table 4 different concns VB1 grows pollen germination
2.6pH gradient test
The BK+50gL of 5.5,6.0,6.5,7.0,7.5,8.0,8.5 is respectively at pH
-1pEG6000+150gL
-1sucrose+2mgL
-1gA
3+ 10mgL
-1vB1 substratum is cultivated turnip type rape fresh pollen, detect substratum different pH and Chinese cabbage type Pollen Brassicae campestris is sprouted and the impact of pollen tube growth.
Result is as shown in table 5, Medium's PH Value change is sprouted Chinese cabbage type Pollen Brassicae campestris and pollen tube growth all has remarkably influenced, pH value is in 5.5 ~ 8.5 scopes, with the increase of pH value, pollen germination rate and pollen tube length are all in " unimodal " curvilinear motion, namely when pH value is less, pollen germination rate and pollen tube length increase with the rising of pH, reach maximum during pH 6.5, start afterwards to decline.Show substratum H
+excessive concentration or be too lowly all unfavorable for that turnip type rape pollen germination grows.
The impact that the different pH value of table 5 grows pollen germination
Turnip type rape in-vitro pollen germination and pollen tube growth under 2.7 different incubation times:
To sprinkle at pH be the BK+50gL of 6.5 by even for fresh turnip type rape pollen
-1pEG6000+150gL
-1sucrose+2mgL
-1gA3+10mgL
-1on the substratum of VB1, be placed in the culture dish that bottom is lined with double-layer filter paper, cover tightly culture dish lid and put into constant incubator 35 DEG C cultivation, 10min, 15min, 30min, 45min, 1h, 2h, 3h, 4h observation at interval pollen tube growth situation after cultivating.
As shown in Figure 1, wherein, X-coordinate is incubation time to turnip type rape pollen germination growing state, and left ordinate scale represents pollen germination rate (%), and right ordinate scale represents pollen tube length (μm); Turnip type rape pollen 15min starts to sprout, 20min pollen tube is outstanding from pollen hole, 30min pollen tube length has exceeded pollen granule diameter, pollen tube starts quick growth, improve rapidly with the prolongation balloonflower root pollen germination rate of incubation time at the 1h starting to cultivate, after 1h, majority has vigor pollen all to sprout, and 2h reaches maximum, and germination rate no longer increases afterwards; Pollen tube length grew fast before the 2h of initial incubation, and pollen tube growth pauses gradually afterwards, 3h pollen tube growth weave in, and pollen tube top starts to expand, and 3.5h breaks on many pollen tube tops, and content is released.
Therefore, be the BK+50gL of 6.5 at turnip type rape pollen at pH
-1pEG6000+150gL
-1sucrose+2mgL
-1gA
3+ 10mgL
-1on the substratum of VB1, under 35 DEG C of constant temperatures after isolated culture 2h, measure the germination rate of pollen and the length of pollen tube, the vigor of effective reaction turnip type rape pollen can be stablized.
This test instrument: RQX growth cabinet, JY/YP electronic balance, CAP type electronic balance, PHS-3C type pH meter, LeicaDM6000B automatically just puts microscope.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity, it is characterized in that comprising the following steps:
1) pollen collection:
Choose the healthy and strong turnip type rape plant growing the harm of normal, anosis worm, choose the inflorescence of having bloomed in turnip type rape full-bloom stage, collect pollen;
2) in-vitro pollen germination and pollen tube growth:
Pollen is evenly sowed on turnip type rape in-vitro pollen germination liquid nutrient medium, 30 DEG C ~ 40 DEG C constant temperature culture; PH is 6.0 ~ 7.0;
In-vitro pollen germination is cultivated 15min ~ 25min rear section pollen and is started to sprout, and after pollen germination, pollen tube length exceedes pollen granule diameter, and pollen tube starts quick growth;
3) mensuration of Pollen Activity:
After isolated culture 45min ~ 1h, be greater than pollen granule diameter for pollen germination standard with pollen tube length, often process repetition 3 times, often repeat random observation 3 ~ 5 visuals field, pollen number 30 ~ 80 is observed in every visual field, statistics pollen germination rate, and pollen germination rate reaction has vitality pollen proportion.
2. the germination in vitro of turnip type rape pollen as claimed in claim 1 cultivates the method measuring Pollen Activity, it is characterized in that step also includes:
4) automatically just put microscopic system with LeicaDM6000B and carry micrometer measurement pollen tube length, every visual field random measurement 10 ~ 15 pollen, often process and survey 30 ~ 40 pollen tube lengths altogether, calculate its mean value, the quality of in-vitro pollen germination pollen tube growth situation reaction pollen granule physiological situation.
3. the germination in vitro of turnip type rape pollen as claimed in claim 1 cultivates the method measuring Pollen Activity, and it is characterized in that described turnip type rape in-vitro pollen germination liquid nutrient medium, take BK as solvent, its component comprised has 50gL
-1~ 100gL
-1pEG 6000 (polyethylene glycol 6000), 130gL
-1~ 150gL
-1sucrose, 1.5mgL
-1~ 2.5mgL
-1plant hormones regulators,gibberellins and 8.5m gL
-1~ 10mgL
-1vITMAIN B1, pH is 6.0 ~ 7.0.
4. the germination in vitro of turnip type rape pollen as claimed in claim 3 cultivates the method measuring Pollen Activity, it is characterized in that consisting of of described BK: H
3bO
3100mgL
-1, Ca (NO
3)
24H
2o 300mgL
-1, MgSO
47H
2o 200mgL
-1and KNO
3100mgL
-1.Take distilled water as solvent.
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Cited By (3)
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| CN105746335A (en) * | 2016-03-08 | 2016-07-13 | 全椒井府富硒生态牧业有限公司 | Liquid pollination agent for early-maturing red pears |
| 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 |
| CN117402809A (en) * | 2023-11-21 | 2024-01-16 | 广州市林业和园林科学研究院 | Liquid culture medium of bougainvillea spectabilis and application thereof |
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|---|---|---|---|---|
| CN105746335A (en) * | 2016-03-08 | 2016-07-13 | 全椒井府富硒生态牧业有限公司 | Liquid pollination agent for early-maturing red pears |
| 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 |
| CN117402809A (en) * | 2023-11-21 | 2024-01-16 | 广州市林业和园林科学研究院 | Liquid culture medium of bougainvillea spectabilis and application thereof |
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