CN104480184B - The germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity - Google Patents
The germination in vitro of turnip type rape pollen cultivates the method measuring Pollen Activity Download PDFInfo
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Abstract
The germination in vitro that the present invention be more particularly directed to turnip type rape pollen cultivates method and the turnip type rape in-vitro pollen germination culture fluid thereof measuring Pollen Activity.The germination in vitro of a kind of turnip type rape pollen cultivates the method measuring Pollen Activity, and it is mainly characterized by and comprises the following steps: 1) pollen collection: 2) in-vitro pollen germination and pollen tube growth；3) mensuration of Pollen Activity.It is an advantage of the invention that the turnip type rape in-vitro pollen germination fluid medium using the present invention to provide, with BK as solvent, comprising component has 50 g L‑1Polyethylene glycol 6000,150 g L‑1Sucrose, 2mg L‑1Gibberellins, 10mg L‑1Vitamin B1, pH is to cultivate under 6.5,35 DEG C of illumination conditions, and turnip type rape pollen germination rate can reach 75.3%, and pollen tube also can preferably be grown；Owing to the addition of PEG, turnip type rape pollen tube growth is more straight, it is simple to observes and measures.
Description
Technical field
The invention belongs to plant pollen vitality test technical field, particularly to sprouting in vitro of a kind of turnip type rape pollen
Send out fluid medium, and utilize this turnip type rape in-vitro pollen germination liquid culture based assays turnip type rape Pollen Activity
Method.
Background technology
Turnip type rape (Brassica rapa L.) is one of Brassica Crops diploid elementary species, precocious, cold-resistant and
And type enriches, it it is one of the three major types type of China's Cultivated rapeseed.Turnip type rape plant is the shortest and the smallest, and main root is more flourishing
Or undeveloped, side root is medium or flourishing.Leaf base embraces stem entirely.Spending yellowish to deep yellow, petal is circular, relatively big, petal side when blooming
Change or in imbricate；In the middle of inflorescence, alabastrum is less than open flower；Flower pesticide extroversion ftractures.The gorgeous U.S. flood dragon of rape flower is beautiful, can beautify ring
, there is higher ornamental value in border, and containing abundant nectar, is the good nectar source of development apiculture.
Brassica campestris L is the crop that economic worth is high, the Oleum Brassicae campestris squeezed out with Semen Brassicae campestris, containing abundant fatty acid and multiple
Vitamin, nutritious, it is easy to digestion, is comparatively ideal edible vegetable oil.Along with the raising of breeding technique in modern age, Oleum Brassicae campestris
Oil is improved therewith, the new varieties of low toxicity low erucic acid, the most expanded comes, and purposes is more and more extensive.Metallurgical, machine
The industry such as tool, rubber, chemical industry, paint, soapmaking, medicine, all using Oleum Brassicae campestris as raw material.The dregs of fat are commonly called as rapeseed cake, rich in albumen
Matter, crude fat, aminoacid and mineral element, be first-class organic fertilizer.Rapeseed cake is through detoxification treatment, or fabulous domestic animal
Concentrated feed.The dish bar of Brassica campestris L, branch and leaf, shell etc., containing the thick protein of more than 3%, the crude fat of about 0.5% and on a small quantity sugar
Class material, is embedded in soil, is converted into organic matter, can fertilize the soil and increase soil fertility, and is referred to as " tender stubble " by peasant, it is possible to be ground into feedstuff, be used for
Development animal raising.It addition, Pollen Brassicae campestris, big of fine quality, it is first-class nutriment, is referred to as " optimum food of longevity person " by people
In view of turnip type rape pollen is as the main carriers of turnip type rape genetic resources, therefore, Chinese cabbage type oil is carried out
The research of cauliflower powder vitality test, preserves Chinese cabbage type Rape Germplasm Resources and has the heaviest with innovation, the selection-breeding of improved seeds
The theory and practice meaning wanted.At present, Pollen Activity method for measuring mainly has microscope morphologic observation, mineral acid to detect
Method, staining, field pollination detection method, germination in vitro method etc..
Morphologic observation is based on pollen sterile in growth course owing to being affected by some factor, ateliosis
Or bad pollen morphology is usually in deformity, and the normal well-regulated profile of pollen.The normal pollen of turnip type rape is oval
Shape, and pollen sterile is irregular shape, can differentiate pollen viability according to form.But, the Chinese cabbage type of storage a period of time
Brassica campestris L mature pollen, in storage, Pollen Activity can be gradually reduced, owing to the normal pollen of turnip type rape is in storage
Vigor may be lost, but form does not occur significant change；So that after pollen morphology observational method cannot differentiate storage
Normal Pollen Activity situation of change.
Inorganic acid system is that pollen there will be germinal aperature has the phenomenon expanding outer process to judge by after mineral acid treatment
Pollen is the most vibrant, but also there will be this phenomenon after pollen water suction, uses mineral acid can not judge that pollen the most really has
Vigor.
Staining is by using staining reagent to dye pollen grain, judges the height of Pollen Activity according to color change
Low, conventional staining reagent has I2-KI solution, aceto-camine and triphenyltetrazolium chloride (TTC) etc..And Determination Staining flower
Powder viability experiment repeatability is poor, can cause the biggest experimental error, can not Accurate Determining pollen actual vigor height.
Field pollination method is that the formational situation according to fruit and seed judges pollen viability, although with actual production
For close, reflect final setting percentage, but its time cycle is long, workload is big, and is vulnerable to weather condition, time place
Impact, is unfavorable for producing, and uses less in actual application.
In-vitro pollen germination is relatively stable with pollen tube growth measurement result, more can react the actual state of Pollen Activity.
But in-vitro pollen germination needs suitable culture medium, conventional culture medium basis is sugar and boron, and sucrose concentration is generally
10%～20%, boric acid is 0.001%～0.005%, pH 5.5～6.5.
The kinds of culture medium that different Plant Pollen Germinations need is different with concentration, and two caryogram pollen are easier to sprout, typically
Cultivate on minimal medium, and the three more difficult sproutings of caryogram pollen, to be added other on the basis of minimal medium and promote
The element of pollen germination, such as Ca2+, Mg2+, K+, the composition such as PEG.In recent years, people to crucifer in-vitro pollen germination and
Pollen tube growth has carried out more research, Song Hongxia etc. (PEG impact on 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 PEG6000 pair
The growth of Radix Dauci Sativae in-vitro pollen germination has the effect of remarkably promoting, and boric acid, Ca2+Radix Dauci Sativae pollen germination there is facilitation；Cao
Firm etc. report (cabbage type rape in-vitro pollen is cultivated, and 2010,30 (2): 0399-0404, northwest Botany Gazette) Wild cabbage type oil
The appropriate media of dish in-vitro pollen germination, Zhao Yanling etc. (Radix Raphani in vitro pollen is sprouted and is studied with pollen tube growth factor of influence,
The first phase in 2007, Jiangsu's agriculture science) have studied the sprouting of Radix Raphani in vitro pollen and the factor of influence of pollen tube growth.But, close
In the research of turnip type rape in-vitro pollen germination Yu pollen tube growth, there is not been reported both at home and abroad.
Summary of the invention
It is an object of the invention to, for avoiding the deficiencies in the prior art, it is provided that sprouting in vitro of a kind of turnip type rape pollen
Send out and cultivate the method measuring Pollen Activity.On the basis of determining turnip type rape in-vitro pollen germination cultivating system, to Chinese cabbage
Type Pollen Brassicae campestris isolated culture, mensuration turnip type rape Pollen Activity reliable, effective.
For achieving the above object, the technical scheme that the present invention takes is: the germination in vitro training of a kind of turnip type rape pollen
Supporting the method measuring Pollen Activity, it is mainly characterized by and comprises the following steps:
1) pollen collection:
Choose and grow healthy and strong turnip type rape plant normal, without disease pest harm, choose in turnip type rape full-bloom stage
The inflorescence bloomed, collects pollen；
2) in-vitro pollen germination and pollen tube growth:
Pollen is uniformly sprinkling upon on turnip type rape in-vitro pollen germination fluid medium, 30 DEG C～40 DEG C of 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 surpasses
Crossing pollen grain diameter, pollen tube starts fast-growth；
3) mensuration of Pollen Activity:
After isolated culture 45min～1h, with pollen tube length more than pollen grain a diameter of pollen germination standard, often process weight
Multiple 3 times, often repeating random observation 3～5 visuals field, every visual field observation pollen number 30～80, add up pollen germination rate, pollen is sprouted
The rate reaction of sending out has vitality pollen proportion.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, it is characterised in that step is also
Include:
4) the most just putting with LeicaDM6000B microscopic system carry micrometer measure pollen tube length, every visual field with
10～15 pollen measured by machine, often process 30～40 pollen tube lengths of survey altogether, calculate its meansigma methods, in-vitro pollen germination pollen
The quality of pipe growing state reaction pollen grain physiological situation.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, described turnip type rape
In-vitro pollen germination fluid medium, with BK as solvent, its component comprised has 50g L-1～100g L-1PEG 6000
(polyethylene glycol 6000), 130g L-1～150g L-1Sucrose, 1.5mg L-1～2.5mg L-1Gibberellins and 8.5m
g·L-1～10mg L-1Vitamin B1, pH is 6.0～7.0.
The germination in vitro of described turnip type rape pollen cultivates the method measuring Pollen Activity, the composition of described BK
For: H3BO3(boric acid) 100mg L-1、Ca(NO3)2·4H2O (calcium nitrate) 300mg L-1、MgSO4·7H2O (crystalline sulfuric acid magnesium)
200mg·L-1And KNO3(potassium nitrate) 100mg L-1.With distilled water as solvent.
Compared with prior art, the present invention has a following useful technique effect:
1, turnip type rape pollen is on the turnip type rape in-vitro pollen germination fluid medium that the present invention provides, 35 DEG C
Cultivating under illumination condition, turnip type rape pollen germination rate can reach 75.3%, and pollen tube also can preferably be grown；Owing to adding
Having added PEG, turnip type rape pollen tube growth is more straight, it is simple to observes and measures.
2, turnip type rape in-vitro pollen germination is utilized to measure Pollen Activity measurement result with pollen tube growth determination techniques
Reliable and stable, pollen germination rate reaction has vitality pollen proportion, the observation of pollen tube growth to react pollen grain with measuring
The quality of physiological situation, turnip type rape in-vitro pollen germination and pollen tube growth determination techniques are turnip type rape Pollen Activity
Mensuration provide a kind of method reliable, effective.
Accompanying drawing illustrates:
Fig. 1. the growth of turnip type rape in-vitro pollen germination is with the situation of change of incubation time
Fig. 2. during for pH6.5, turnip type rape pollen is at BK+150g L-1Sucrose+50g L-1PEG6000+
2mg·L-1Gibberellins+10m g L-1Vitamin B1 (35 DEG C of constant temperature) culture medium on sprout the photo gathered after 40min and show
It is intended to.
Fig. 3. during for pH6.5, turnip type rape pollen is at BK+150g L-1Sucrose+50g L-1PEG6000+
2mg·L-1Gibberellins+10m g L-1Vitamin B1 (35 DEG C of constant temperature) culture medium on sprout the photo gathered after 50min and show
It is intended to.
Fig. 4. during for pH6.5, turnip type rape pollen is at BK+150g L-1Sucrose+50g L-1PEG6000+
2mg·L-1Gibberellins+10m g L-1Vitamin B1 (35 DEG C of constant temperature) culture medium on sprout the photo signal gathered after 1h
Figure.
Detailed description of the invention
It is described in further detail below in conjunction with the preferred example shown in accompanying drawing:
Embodiment 1: see Fig. 1, the method that the germination in vitro cultivation of a kind of turnip type rape pollen measures Pollen Activity, it is main
Feature is wanted to be to comprise the following steps:
1) pollen collection:
Choose and grow healthy and strong turnip type rape plant normal, without disease pest harm, choose in turnip type rape full-bloom stage
The inflorescence bloomed, collects pollen；
2) in-vitro pollen germination and pollen tube growth:
Pollen is uniformly sprinkling upon on turnip type rape in-vitro pollen germination fluid 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
Grain diameter, pollen tube starts fast-growth；
3) mensuration of Pollen Activity:
After isolated culture 45min, with pollen tube length more than pollen grain a diameter of pollen germination standard, often process repetition 3
Secondary, often repeat 3 visuals field of random observation, every visual field is observed pollen number and is no less than 30, adds up pollen germination rate, pollen germination rate
Reaction has vitality pollen proportion.
4) the most just putting with LeicaDM6000B microscopic system carry micrometer measure pollen tube length, every visual field with
10 pollen measured by machine, often process 30 pollen tube lengths of survey altogether, calculate its meansigma methods, in-vitro pollen germination pollen tube growth feelings
The quality of condition reaction pollen grain physiological situation.
Embodiment 2: the germination in vitro of a kind of turnip type rape pollen cultivates the method measuring Pollen Activity, its main feature
It is to comprise the following steps:
1) pollen collection:
Choose and grow healthy and strong turnip type rape plant normal, without disease pest harm, choose in turnip type rape full-bloom stage
The inflorescence bloomed, collects pollen；
2) in-vitro pollen germination and pollen tube growth:
Pollen is uniformly sprinkling upon on turnip type rape in-vitro pollen germination fluid 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
Grain diameter, pollen tube starts fast-growth；
3) mensuration of Pollen Activity:
After isolated culture 50min, with pollen tube length more than pollen grain a diameter of pollen germination standard, often process repetition 3
Secondary, often repeat 4 visuals field of random observation, every visual field is observed pollen number and is no less than 50, adds up pollen germination rate, pollen germination rate
Reaction has vitality pollen proportion.
4) the most just putting with LeicaDM6000B microscopic system carry micrometer measure pollen tube length, every visual field with
12 pollen measured by machine, often process 35 pollen tube lengths of survey altogether, calculate its meansigma methods, in-vitro pollen germination pollen tube growth feelings
The quality of condition reaction pollen grain physiological situation.
Embodiment 3: the germination in vitro of a kind of turnip type rape pollen cultivates the method measuring Pollen Activity, its main feature
It is to comprise the following steps:
1) same as in Example 1.
2) in-vitro pollen germination and pollen tube growth:
Pollen is uniformly sprinkling upon on turnip type rape in-vitro pollen germination fluid 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
Grain diameter, pollen tube starts fast-growth；
3) mensuration of Pollen Activity:
After isolated culture 1h, with pollen tube length more than pollen grain a diameter of pollen germination standard, often process and be repeated 3 times,
Often repeating 5 visuals field of random observation, pollen number 80 is observed in every visual field, adds up pollen germination rate, and pollen germination rate reaction has life
Vigor pollen proportion.
4) the most just putting with LeicaDM6000B microscopic system carry micrometer measure pollen tube length, every visual field with
15 pollen measured by machine, often process 40 pollen tube lengths of survey altogether, calculate its meansigma methods, in-vitro pollen germination pollen tube growth feelings
The quality of condition reaction pollen grain physiological situation.
Embodiment 4: described turnip type rape in-vitro pollen germination fluid medium, with BK as solvent, it also comprises group
Divide and have 50g L-1PEG 6000 (polyethylene glycol 6000), 130g L-1Sucrose, 1.5mg L-1Gibberellins and 8.5m
g·L-1Vitamin B1, pH is 6.0.
Wherein the consisting of of BK: H3BO3(boric acid) 100mg L-1、Ca(NO3)2·4H2O (calcium nitrate) 300mg L-1、
MgSO4·7H2O (crystalline sulfuric acid magnesium) 200mg L-1And KNO3(potassium nitrate) 100mg L-1.With distilled water as solvent.
Embodiment 5: described turnip type rape in-vitro pollen germination fluid medium, with BK as solvent, it comprises component
There is 80g L-1PEG 6000 (polyethylene glycol 6000), 100g L-1Sucrose, 2.0mg L-1Gibberellins and 9m g L-1
Vitamin B1, pH is 6.5.
The wherein composition of BK such as embodiment 4.
Embodiment 6: described turnip type rape in-vitro pollen germination fluid medium, with BK as solvent, it comprises component
There is 100g L-1PEG 6000 (polyethylene glycol 6000), 150g L-1Sucrose, 2.5mg L-1Gibberellins and 10mg
L-1The BK culture fluid of vitamin B1, pH is 7.0.
The wherein composition of BK such as embodiment 4.
Experimental verification example: the germination in vitro of a kind of turnip type rape pollen cultivates the method measuring Pollen Activity,
1, the collection of turnip type rape pollen
Turnip type rape pollen is in picking up from Gansu Agriculture University's arid habitat key lab, acquisition method at the beginning of certain 10 months year
For: choosing and grow healthy and strong plant normal, without disease pest harm, choose the inflorescence bloomed in full-bloom stage, alabastrum is naturally open, flower
Powder sheds from flower pesticide, collects pollen standby.
2, the screening of in-vitro pollen germination condition of culture:
The screening of 2.1 in-vitro pollen germination culture medium:
Kinds of culture medium has considerable influence to plant pollen germination in vitro, and the appropriate media of different Plant Pollen Germinations is not
With.With BK culture medium as minimal medium, screen the PEG6000 of suitable concn, sucrose, GA the most successively3And VB1,
Finally regulate suitable pH.
BK consists of (mg L-1): H3BO3100mg·L-1、Ca(NO3)2·4H2O 300mg·L-1、MgSO4·7H2O
200mg·L-1And KNO3100mg·L-1.With distilled water as solvent.
2.2PEG concentration screening
PEG6000 is a kind of high-molecular weight compounds, primarily serves Osmoregulation effect.Fresh pollen is the most uniformly dissipated
Cloth with the addition of 0g L-1、50g·L-1、100g·L-1、150g·L-1、200g·L-1PEG6000 BK culture medium (BK train
Foster base is liquid；Here 0g L-1Represent the BK culture medium without PEG.Distilled water is added the most again in BK) in,
Concrete operations are as follows: (1) regulates the pH to 7.0 of above-mentioned different PEG concentration culture medium with the NaOH of 1mol/L；(2)
Draw 70 μ L culture medium with liquid-transfering gun, drop in formation dome shape drop on microscope slide gently, the pollen on two stamens is sowed
In sphere culture medium, during sowing, do not stir culture medium, in order to avoid pollen grain sinks to liquid culture basilar part, in germination process
Affect experimental result due to anoxia, preferably pollen grain is layered in culture medium uniformly；(3) above-mentioned microscope slide is put into paving
There is two-layer to moisten in the culture dish of filter paper (keeping certain humidity, it is to avoid sprout time culture medium evaporation too for a long time), do not add a cover glass
Sheet (there to be the oxygen of abundance during guaranteeing pollen germination), covers tightly after culture dish lid cultivates 1h in 35 DEG C of incubators and is showing
Micro-Microscopic observation statistics sprouts number and pollen tube length.(4) with pollen tube length more than pollen grain a diameter of pollen germination standard,
Often processing and be repeated 3 times, often repeat 3 visuals field of random observation, every visual field is observed pollen number and is no less than 30, adds up pollen germination
Rate；The most just putting microscopic system simultaneously with LeicaDM6000B and carrying micrometer measurement pollen tube length, every visual field is random
Measure 10 pollen, often process 30 pollen tube lengths of survey altogether, calculate its meansigma methods.Test is in triplicate.
Result is as shown in table 1, adds the PEG6000 of variable concentrations in BK culture medium, can significantly improve turnip type rape flower
Powder germination rate, wherein adds 50g L-1PEG6000 effect is the most notable, and pollen germination rate reaches 41.8%；Low concentration PEG (50
～150g L-1) promote pollen tube growth, and increase with concentration, facilitation gradually weakens, and high concentration PEG is (more than 200g
L-1) suppression pollen tube growth；Also find when microexamination, in the culture medium that with the addition of low concentration PEG, turnip type rape flower
Tube cell growth is relatively straight, and does not adds PEG or added the culture medium pollen tube growth bending of high concentration PEG, normal weave in, inconvenience
Observe and measure.
The impact that pollen germination is grown by table 1 variable concentrations PEG
2.3 sucrose concentration tests
Sucrose is a kind of osmotic adjustment, is also the energy substance of pollen tube growth simultaneously.At 50g L-1PEG6000+
The culture medium of BK increases 100g L respectively-1、150g·L-1、200g·L-1、250g·L-1Sucrose, compare variable concentrations
Sucrose is on pollen germination and the impact of pollen tube growth.
Result is as shown in table 2, BK+50g L-1In PEG culture medium, variable concentrations sucrose is to Chinese cabbage type Pollen Brassicae campestris germination rate
Different with pollen tube growth impact, low concentration sucrose (150g L-1) pollen germination and pollen tube growth had be obviously promoted work
With, and the sucrose of high concentration (>=200g L-1) can substantially suppress pollen germination and pollen tube growth, and along with sucrose concentration
Increase, be suppressed degree the most obvious.Containing 150g L-1The BK+50g L of sucrose-1Turnip type rape in PEG culture medium
Pollen growth is best, cultivates after 1h, and its pollen germination rate and pollen tube length respectively reach 65.1% and 91.1 μm.
The impact that pollen germination is grown by table 2 variable concentrations sucrose
2.4GA3Concentration tests
Gibberellins can suppress the activity of auxin decomposing system in plant, to ensure the phase of tumor growth cellulose content level
To increase, certain density Exogenous gibberellic acid processes can promote pollen germination and pollen tube growth.At BK+50g L-1PEG6000
+150g·L-1Sucrose medium increases 1mg L respectively-1、2mg·L-1、3mg·L-1GA3, the GA of detection variable concentrations3
Chinese cabbage type Pollen Brassicae campestris is sprouted and the impact of pollen tube growth.
Result is as shown in table 3, the GA of variable concentrations3On Chinese cabbage type Pollen Brassicae campestris sprout and pollen tube growth impact not
With, low concentration GA3(2mg·L-1) pollen germination and pollen tube growth had the effect of being obviously promoted, and GA3Concentration too low or
Too high all it is unfavorable for pollen germination and pollen tube growth.Containing 2mg L-1GA3BK+50g L-1PEG+150g·L-1Sugarcane
On sugar culture-medium, the growth of turnip type rape pollen is best, and after cultivating 1h, its pollen germination rate and pollen tube length respectively reach
69.2% and 157.2 μm.
Table 3 variable concentrations GA3Impact on pollen germination growth
2.5VB1 concentration tests
Forefathers' research shows, pollen germination speed is increased by physiologically active class Vitamins B1, has reached quick
Purpose.At BK+50g L-1PEG6000+150g·L-1Sucrose+2mg L-1The culture medium of GA3 increases 5mg L respectively-1、
10mg·L-1、15mg·L-1VB1, research variable concentrations VB1 to Chinese cabbage type Pollen Brassicae campestris sprout and the shadow of pollen tube growth
Ring.
Result is as shown in table 4, the VB1 of variable concentrations Chinese cabbage type Pollen Brassicae campestris is sprouted and pollen tube growth impact not
With, containing 10mg L-1VB1 at BK+50g L-1PEG6000+150g·L-1Sucrose+2mg L-1GA3In culture medium in vain
The growth of dish type Pollen Brassicae campestris is best, and after cultivating 30min, its pollen germination rate and pollen tube length respectively reach 58.4% and 76.4
μm。
The impact that pollen germination is grown by table 4 variable concentrations VB1
2.6pH gradient test
The BK+50g L of 5.5,6.0,6.5,7.0,7.5,8.0,8.5 it is respectively at pH-1PEG6000+150g·L-1Sugarcane
Sugar+2mg L-1GA3+10mg·L-1Cultivate turnip type rape fresh pollen in VB1 culture medium, detect culture medium difference pH dialogue
Dish type Pollen Brassicae campestris is sprouted and the impact of pollen tube growth.
Result is as shown in table 5, and Chinese cabbage type Pollen Brassicae campestris is sprouted in Medium's PH Value change and pollen tube growth all has significantly
Impact, pH value, in the range of 5.5～8.5, all becomes in " unimodal " curve with the increase of pH value, pollen germination rate and pollen tube length
Changing, i.e. when pH value is less, pollen germination rate and pollen tube length increase with the rising of pH, reach maximum, afterwards during pH 6.5
Begin to decline.Show culture medium H+Excessive concentration or too low be all unfavorable for that turnip type rape pollen germination grows.
The impact that pollen germination is grown by the different pH value of table 5
Turnip type rape in-vitro pollen germination and pollen tube growth under 2.7 different incubation times:
Sprinkle even for fresh turnip type rape pollen at the BK+50g L that pH is 6.5-1PEG6000+150g·L-1Sugarcane
Sugar+2mg L-1GA3+10mg·L-1In the culture medium of VB1, it is placed in bottom and is lined with in the culture dish of double-layer filter paper, cover tightly cultivation
Ware lid also puts into constant incubator 35 DEG C cultivation, and after cultivation, 10min, 15min, 30min, 45min, 1h, 2h, 3h, 4h are fixed
Time observation pollen tube growth situation.
As it is shown in figure 1, wherein, abscissa is incubation time to turnip type rape pollen germination growing state, left ordinate scale
Representing pollen germination rate (%), right ordinate scale represents pollen tube length (μm)；Turnip type rape pollen 15min starts to sprout,
20min pollen tube is prominent from pollen hole, and 30min pollen tube length has exceeded pollen grain diameter, and pollen tube starts fast-growth,
Improving rapidly with the prolongation Radix Platycodonis pollen germination rate of incubation time at the 1h starting to cultivate, after 1h, most vibrant pollen are sprouted the most
Sending out, 2h reaches maximum, and germination rate is not further added by afterwards；Pollen tube length is fast-growth before the 2h of initial incubation, spends afterwards
Tube cell growth gradually pauses, and 3h pollen tube growth weave in, pollen tube top starts to expand, 3.5h many pollen tubes top
Rupturing, content is released.
Therefore, at turnip type rape pollen at the BK+50g L that pH is 6.5-1PEG6000+150g·L-1Sucrose+2mg
L-1GA3+10mg·L-1In the culture medium of VB1, under 35 DEG C of constant temperatures after isolated culture 2h, measure germination rate and the pollen of pollen
The length of pipe, it is possible to the stable effective vigor reacting turnip type rape pollen.
This test instrument: RQX growth cabinet, JY/YP electronic balance, CAP type electronic balance, PHS-3C type pH
Meter, LeicaDM6000B is the most just putting microscope.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (2)
1. the germination in vitro of a turnip type rape pollen cultivates the method measuring Pollen Activity, it is characterised in that include following step
Rapid:
1) pollen collection:
Choose and grow healthy and strong turnip type rape plant normal, without disease pest harm, choose in turnip type rape full-bloom stage and bloom
Inflorescence, collect pollen；
2) in-vitro pollen germination and pollen tube growth:
Pollen is uniformly sowed on turnip type rape in-vitro pollen germination fluid medium, 30 DEG C～40 DEG C of constant temperature culture； pH
It 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 flower
Powder diameter, pollen tube starts fast-growth；
3) mensuration of Pollen Activity:
After isolated culture 45 min～1h, with pollen tube length more than pollen grain a diameter of pollen germination standard, often process repetition 3
Secondary, often repeat random observation 3～5 visuals field, every visual field observation pollen number 30～80, add up pollen germination rate, pollen germination
Rate reaction has vitality pollen proportion；
Described turnip type rape in-vitro pollen germination fluid medium, with BK as solvent, its component comprised has 50g L-1
～100 g L-1PEG 6000(polyethylene glycol 6000), 130 g L-1～150 g L-1Sucrose, 1.5mg L-1~
2.5mg·L-1Gibberellins and 8.5m g L-1～10 mg L-1Vitamin B1, pH is 6.0～7.0；The group of described BK
Become: H3BO3100 mg·L-1、Ca(NO3)2·4H2O 300 mg·L-1、MgSO4·7H2O 200 mg·L-1And KNO3 100
mg·L-1, with distilled water as solvent.
2. the germination in vitro of turnip type rape pollen as claimed in claim 1 cultivates the method measuring Pollen Activity, its feature
Being, step also includes:
4) the most just putting microscopic system with LeicaDM6000B and carrying micrometer measurement pollen tube length, every visual field is surveyed at random
Measuring 10～15 pollen, often process 30～40 pollen tube lengths of survey altogether, calculate its meansigma methods, in-vitro pollen germination pollen tube is raw
The quality of long situation reaction pollen grain physiological situation.
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| 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 |
| CN117402809B (en) * | 2023-11-21 | 2024-06-25 | 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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| CN101633902A (en) * | 2009-08-25 | 2010-01-27 | 商洛学院 | In-vitro pollen germination liquid medium of balloon flower and method using same to determine pollen viability thereof |
| CN101717747A (en) * | 2009-12-04 | 2010-06-02 | 西北农林科技大学 | Liquid medium for vitro scutellaria root pollen germination and method for testing activity of scutellaria root pollen |
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| CN101633902A (en) * | 2009-08-25 | 2010-01-27 | 商洛学院 | In-vitro pollen germination liquid medium of balloon flower and method using same to determine pollen viability thereof |
| CN101717747A (en) * | 2009-12-04 | 2010-06-02 | 西北农林科技大学 | Liquid medium for vitro scutellaria root pollen germination and method for testing activity of scutellaria root pollen |
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