CN104458538B - A kind of quick method for determining peony pollen mother cell Meiosis - Google Patents
A kind of quick method for determining peony pollen mother cell Meiosis Download PDFInfo
- Publication number
- CN104458538B CN104458538B CN201410649179.3A CN201410649179A CN104458538B CN 104458538 B CN104458538 B CN 104458538B CN 201410649179 A CN201410649179 A CN 201410649179A CN 104458538 B CN104458538 B CN 104458538B
- Authority
- CN
- China
- Prior art keywords
- meiosis
- mother cell
- bud
- pollen mother
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of quick method for determining peony pollen mother cell Meiosis, key step includes sampling, fixed, dyeing, tabletting and microexamination, this method combination temperature, suitable bud is chosen according to the multiple sampling standard of anther color and bud diameter, dyeing tabletting is carried out again, is then observed, determined meiosis each period of peony pollen mother cell under the microscope.Using the method for the present invention, same day can after fixing of drawing materials observes the meiosis situation of detection pollen cell, and this can not only save time, cost-effective, raising conventional efficient, be also avoided that and miss meiophase.
Description
Technical field
The invention belongs to plant phenotype in biotechnology and cytology research method field, and in particular to a kind of quick determination
The method of peony pollen mother cell Meiosis.
Background technology
For the eucaryote of zoogamy, meiosis is a very important process, and it is thin from diploid
Haploid production gamete in born of the same parents, in this process DNA replication dna continuous nuclear fission twice, chromosome number once and then occurs
Mesh halves.The hereditary variation that half parental set of chromosome had not only been supplied in meiosis but also reorganization is present in parent.Therefore meiosis
Have to the evolution for maintaining the normal development of individual, the stability of holding species, the origin of announcement species and species
There is important leading role.
How a small amount of plant Meiosis, such as Xi Mengli determined by the methods of bud size studies have reported that crossing
Deng (Lilium longiflorum meiosis of microspore mother cell process and bud size correlation research, Nanjing Forestry University's journal,
2008) its Meiosis, Liu Tianlun (paddy pollen mother cell meiosis are determined by observing Lilium longiflorum bud size
Period, Chinese agriculture section science, 1981) paddy pollen mother cell meiophase, Gao Peng determined according to the length of rice grain husk flower
Deng (research of bark of eucommia Microspore and megaspore mother cell Tempo of Meiosis corresponding relation, Beijing Forestry University's journal, 2006) according to Du
Zhong Huaya length and the change of flower pesticide judge meiophase, yellow far new etc. (position observation of rye Meiosis, life
Thing is circulated a notice of, 2008) rye Meiosis is judged according to the length of flower pesticide, also Li Jing etc.s (aspen microspore generation
With Devflopment Ofmle Gametophyte process immediate observation, forestry science and technology, 2014) aspen meiophase determined according to the water planting time, wherein
It is related to the color change of flower pesticide to mention meiosis.But it is single that standard is investigated in these methods, and the subtrahend of plant cell point
Split that period typical time is very short, easily miss optimal observation period.
Although the report of existing a small amount of research Paeonia Pollen Mother Cell growth course, but without for determining it
The report of pollen mother cellses period method.Such as Han Li (research with Devflopment Ofmle Gametophyte occurs for tree peony microspore,
Journal of Shandong agri.Univ's (natural science edition), 2000,31 (1):27~31) in the generation of research tree peony microspore and male gametophyte
It is mid-March to mid-April once to be mentioned during development in the Taian Shandong sampling time, and its sampling time is up to one month；Due to year
Temperature Change between border is very big, even if being also to differ in the annual real Meiosis of peony pollen mother cell in areal
Sample, thus, the descriptive report according to this fuzzy time section is can not accurately to determine pollen mother cellses
Time.In addition, according to the sampling that one month is up on foregoing report, it is necessary to consume substantial amounts of experiment material, input is substantial amounts of
Personnel and time cost, it is clear that not only taken a lot of work but also time-consuming.
In other a small amount of correlative studys, researchers also use one " executeaaafunction " mostly, from its result of study
As can be seen that because the sampling time does not know, fail complete observation to the process of meiosis.
In addition, the researcher such as Wei Le mainly observes meiosis process by paraffin section technology, but needed for paraffin section
Time is longer (about 1 week), is unfavorable for the fragmentation condition of quick detection pollen mother cell, and materials have limitation.Accordingly, with respect to such as
What accurate quick method for determining tree peony meiosis exact time point is not reported so far.
The content of the invention
It is an object of the invention to provide a kind of quick method for determining peony pollen mother cell Meiosis, this is utilized
Same day can is quick after method materials are fixed, each period of precise Identification peony pollen mother cell meiosis, can not only
Experimental period is enough saved, experiment material is saved, reduces experimental cost, can also avoid missing meiophase, and then greatly carry
High conventional efficient and quality of experiments.
In order to achieve the above object, technical scheme provided by the invention is：
A kind of quick method for determining peony pollen mother cell Meiosis, including,
1) sample：When daily mean temperature is 6.5~15.5 DEG C, selection anther color is light yellow, a diameter of 12~16mm's
Bud；
2) it is fixed：The bud that step 1) is obtained removes sepal petal, is immersed in the fixer of Kano, marks；Gu
After fixed 1~24 hour, with alcohol washes, and it is transferred in alcohol and is saved backup in -40~-20 DEG C；
3) dye：The bud that step 2) saves backup is taken out, tears and takes stamen；Alcohol above is sucked with blotting paper；Carrying
Acetic acid is dripped on slide, stamen is placed in the acetic acid on slide and softened 1~10 second, one section of 1~6mm is cut with dissecting needle
Long flower pesticide, 15~35 μ L carbolfuchsins are added dropwise to the chromosome dyeing on flower pesticide, is caught broken sample with tweezers, removes residue, obtain
To sample；
4) observe：Sample is first found into the visual field under 10~30 times of mirrors of microscope, then seen under 40~100 times of mirrors of microscope
Examine the chromosome in division stage.
Preferably, the set time is 1~3 hour in step 2).
Preferably, stamen is placed in acetic acid in step 3) and softened 3~8 seconds.
Again preferably, stamen is placed in acetic acid in step 3) after softening, the flower pesticide of 2mm length is cut with dissecting needle.
Further, in described method, before being observed under the microscope, in addition to tableting step, specifically, in sample
Covered on product, firmly tabletting, chromosome is pressed off, so as to the image become apparent from observation.
It is found through experiments that, the meiosis between 12~16mm of bud diameter is more vigorous, can look within this range
The cell in each period during to meiosis；Temperature samples preferably at 6.5~15.5 DEG C, bud diameter it is relatively large but
Meiosis cell is also shown in the case that mean daily temperature is relatively low；When most of pollen mother cells are in bud
During meiophase, anther color is mostly light yellow, now samples optimum；And when anther color is light yellow partially milky white, greatly
Most pollen mother cells have not been entered into meiophase, now sample then partially early；And when anther color is changed into yellow, then
Most pollen mother cell has had been subjected to Meiosis, now samples then too late.
Therefore, when gathering sample, bud diameter is between 12~16mm, and mean temperature is 6.5~15.5 DEG C, flower pesticide
For color to be light yellow, the sample meiosis gathered in the case where three conditions are satisfied by is more vigorous, and has and more be in the later stage
Chromosome.
Beneficial effect：
In the method for the present invention, temperature is combined in sampling, the multi-standard selection of anther color and bud diameter is suitable
Bud, then carry out dyeing tabletting, and then go to detect the Meiosis of the pollen mother cell of tree peony, can by this method
Fast Acquisition peony pollen mother cell meiophase cell, avoid when sampling in the prior art, due to contingency with it is not true
The presence of qualitative factor, and miss to obtain the best period of the peony pollen mother cell in Meiosis.
Using method of the invention, it is possible to adopt the cell sample of tree peony Meiosis, and it is able to observe that each
In individual period, time and manpower are saved, cost has been saved, has improved operating efficiency for research work, and be more comprehensively deep
Disclose meiosis this biological phenomena with entering provide experiment material this premise.
Brief description of the drawings
Fig. 1 is one of bud taken in the embodiment of the present invention.
Fig. 2 is the bud for removing sepal and petal in the embodiment of the present invention.
Fig. 3 is the pollen mother cell of subtrahend first division early stage in the embodiment of the present invention.
Fig. 4 is the pollen mother cell of subtrahend first metaphase in the embodiment of the present invention.
Fig. 5 is the pollen mother cell of subtrahend aⅠ in the embodiment of the present invention.
Fig. 6 is the pollen mother cell in subtrahend first division latter stage in the embodiment of the present invention.
Fig. 7 is the pollen mother cell of subtrahend second division early stage in the embodiment of the present invention.
Fig. 8 is the pollen mother cell of subtrahend second division mid-term in the embodiment of the present invention.
Fig. 9 is the pollen mother cell of subtrahend second anaphase in the embodiment of the present invention.
Figure 10 is the pollen mother cell in subtrahend second division latter stage in the embodiment of the present invention.
Figure 11 is the mature flower powder in the embodiment of the present invention.
Embodiment
Below in conjunction with specific embodiments and the drawings technical scheme is described in further detail, it is necessary to particularly point out
That these descriptions are only exemplary descriptions, and are not meant to limit the scope of the invention, various made to the present invention aobvious and
The modifications and variations being clear to also are included in the scope of the present invention.
Embodiment determines phoenix pellet pollen mother cellses period
1. material and method：
Equipment：Tweezers, scalpel, slide measure, ice chest, plastic bottle with cover, dissecting needle, slide, cover glass, culture
Ware, filter paper, blotting paper, ordinary optical microscope.
Reagent：Kano fixer, carbolfuchsin, 45% acetic acid (volume ratio), 70% alcohol (volume ratio).
Sample：Feng Dan, its bud is taken as sample.
Gather sample site：Shanghai Chenshan Botanical Garden.
This 56 bud sample of collection, sampling bud diameter span are that 11.50mm~18.75mm (refers to table 1 and figure
1), 727 photos altogether.
1.1 gather and fix：
1) preparation：Load Kano fixer (ethanol in plastic bottle：Acetic acid=3:1), it is placed in ice chest；
2) sample：The diameter of phoenix pellet bud is measured with slide measure, diameter range is in 11.50mm~18.75mm；
3) it is fixed：Bud is taken with tweezers, removes sepal petal, is immersed in the fixer of Kano, marks；It is fixed
After 1~3 hour, with 70% alcohol washes, and it is transferred in 70% alcohol and is saved backup in -20 DEG C of refrigerators, fixed cleaning
I.e. detectable afterwards, frequency acquisition can be half a day, adopt once within one day or two days.
1.2 chromosome sectionings and detection
1) bud is taken out, tears and takes a stamen, blotting paper sucks alcohol above；
2) (about 40 μ L) 45% acetic acid is dripped on slide, stamen is placed in acetic acid and softens 5s, uses dissecting needle
The flower pesticide of one section of 2mm length is cut, remaining be put into is preserved in liquid (70% alcohol)；
3) 20 μ L carbolfuchsins are added dropwise, is caught broken sample with tweezers, removes residue, obtain sample；
4) by sample covered, exerted oneself tabletting, chromosome is pressed off, under 10~30 times of mirrors of microscope with thumb
The visual field is found, 40~100 times of sem observations is then changed and photographs to record.
2 results
It is found through experiments that, peony pollen mother cell is in the bud diameter of meiophase between 12~16mm
(referring to table 1), mean temperature are 6.5~15.5 DEG C, and anther color is light yellow (see Fig. 2), the sample subtrahend gathered under the conditions of being somebody's turn to do
Division is more vigorous, meiosis can be detected from 10~90% pollen mother cell, and have the more chromosome in the later stage.
Meiosis main process and pollen grain forming process are as shown in Fig. 3~11, and Fig. 3 is before subtrahend first division
The pollen mother cell of phase, Fig. 4 are the pollen mother cell of subtrahend first metaphase, and Fig. 5 is subtrahend aⅠ
Pollen mother cell, Fig. 6 are the pollen mother cell in subtrahend first division latter stage, and Fig. 7 is the flower of this subtrahend second division early stage
Powder mother cell, Fig. 8 are the pollen mother cell of subtrahend second division mid-term, and Fig. 9 is that the pollen of subtrahend second anaphase is female
Cell, Figure 10 are the pollen mother cell in subtrahend second division latter stage, and Figure 11 is mature flower powder.
Meanwhile find that asynchrony phenomenon occurs in flower bud development during chromosome sectioning, and in same bud, middle stamen
Development is very fast, and edge is slower, and develop in same stamen it is also asynchronous, developed in morphology upper end it is fast compared with lower end, so
Can be according to development asynchrony phenomenon selection sample during materials.
Division period cell number percentage corresponding to the bud diameter of table 1, mean temperature and anther color
Peony pollen mother cell Meiosis is determined by the detection method of the present invention, tree peony subtrahend point can be adopted
The cell sample in period is split, and is able to observe that each period, is easy to follow-up study to draw materials, the time is saved for research work
With manpower, saved cost, improve operating efficiency, provided more in depth to disclose meiosis this biological phenomena comprehensively
Premise.
Claims (4)
1. a kind of quick method for determining peony pollen mother cell Meiosis, comprises the following steps,
1) sample：When daily mean temperature is 6.5~15.5 DEG C, selection anther color is light yellow, a diameter of 12.10~16mm flower
Flower bud；Wherein, the bud is the bud of Varieties of Peony ' Feng Dan '；
2) it is fixed：The bud that step 1) is obtained removes sepal petal, is immersed in the fixer of Kano, marks；Fix 1~
After 3 hours, with alcohol washes, and it is transferred in alcohol and is saved backup in -40~-20 DEG C；
3) dye：The bud that step 2) saves backup is taken out, tears and takes stamen；Suck alcohol above；Dripped on slide
Acetic acid, stamen is placed in the acetic acid on slide and softened 1~10 second, the flower pesticide of one section of 1~6mm length, drop are cut with dissecting needle
Add 15~35 μ L carbolfuchsins, to the chromosome dyeing on flower pesticide, be caught broken sample with tweezers, remove residue, obtain sample；
4) observe：Sample is first found into the visual field under 10~30 times of mirrors of microscope, then at 40~100 times of Microscopic observations of microscope
In the chromosome of division stage.
2. the quick method for determining peony pollen mother cell Meiosis as claimed in claim 1, it is characterised in that step
It is rapid 3) in stamen be placed in acetic acid softened 3~8 seconds.
3. the quick method for determining peony pollen mother cell Meiosis as claimed in claim 1 or 2, its feature exist
In, in step 3) by stamen be placed in acetic acid soften after, with dissecting needle cut 2mm length flower pesticide.
4. the quick method for determining peony pollen mother cell Meiosis as claimed in claim 1, it is characterised in that step
It is rapid 4) observed under the microscope before, in addition to tableting step, specifically, the covered on sample, firmly tabletting,
Chromosome is fully pressed off.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410649179.3A CN104458538B (en) | 2014-11-14 | 2014-11-14 | A kind of quick method for determining peony pollen mother cell Meiosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410649179.3A CN104458538B (en) | 2014-11-14 | 2014-11-14 | A kind of quick method for determining peony pollen mother cell Meiosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104458538A CN104458538A (en) | 2015-03-25 |
| CN104458538B true CN104458538B (en) | 2018-01-12 |
Family
ID=52904949
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410649179.3A Active CN104458538B (en) | 2014-11-14 | 2014-11-14 | A kind of quick method for determining peony pollen mother cell Meiosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104458538B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770226A (en) * | 2016-11-29 | 2017-05-31 | 河南中医药大学 | A kind of colouring method for observation of plant anther development structure change |
| CN108680418B (en) * | 2018-06-01 | 2021-03-23 | 广东金作农业科技有限公司 | Dyeing liquid and method for quickly dyeing cruciferous crop pollen |
| CN108827964A (en) * | 2018-07-05 | 2018-11-16 | 南京林业大学 | A kind of method of quick determining green bamboo Pistil And Stamen developmental process |
| CN110044827A (en) * | 2019-04-15 | 2019-07-23 | 南京农业大学 | A method of evaluation lily flower morphological feature and pollen development stage correlation |
| CN113575340B (en) * | 2021-07-08 | 2022-08-30 | 中国农业科学院蔬菜花卉研究所 | Method for determining developmental period of microspore mother cells of phalaenopsis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6383005A (en) * | 1986-09-29 | 1988-04-13 | Hokko Chem Ind Co Ltd | Male-sterilizing agent for paddy rice plant |
| CN101477763A (en) * | 2009-01-21 | 2009-07-08 | 路思谦 | Reduction division full period microscopic slide exemplar |
| CN102352352A (en) * | 2011-10-09 | 2012-02-15 | 山东农业大学 | Single chromosome producing method through micro cutting plant chromosomes |
-
2014
- 2014-11-14 CN CN201410649179.3A patent/CN104458538B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104458538A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104458538B (en) | A kind of quick method for determining peony pollen mother cell Meiosis | |
| CN102183394B (en) | Method for making hyacinth root tip chromosome slice | |
| CN103776657B (en) | A kind of method for making of coconut blade freezing microtome section | |
| CN102517384A (en) | Method for rapid identification of Gerbera jamesonii ploidy by using flow cytometry | |
| CN101266200A (en) | Paddy rice root tip chromosome slide-making method | |
| Liu et al. | Pistillate flower development and pollen tube growth mode during the delayed fertilization stage in Corylus heterophylla Fisch | |
| CN111238888A (en) | Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method | |
| CN101482515A (en) | Tabletting method for locust stem tip chromosome | |
| CN101595841B (en) | Induction method of poplar tetraploid plants | |
| CN103993095B (en) | A kind of Psathyrostachys plant cell chromosome division phases sample preparation method | |
| CN102492767A (en) | Preparation method of chromosome suitable for karyotype analysis of rubus plants | |
| CN103695557A (en) | Gingko karyotype analysis method based on stem tips | |
| CN104297034A (en) | Method applied to chromosome production of single tobacco plant by tender ovary | |
| CN102830002B (en) | Method for preparing pistacia chinensis bunge bud chromosome slide | |
| CN102564821A (en) | Tabletting method of plum blossom shoot tip chromosome | |
| CN107246987A (en) | A kind of method of Helminthosporium sativum chromosome sectioning | |
| CN101613755B (en) | Method for identifying carnation chromosome number by bud | |
| CN104006997B (en) | A kind of leymus plant root tip chromosome flaking method | |
| CN102352352A (en) | Single chromosome producing method through micro cutting plant chromosomes | |
| CN102277435A (en) | Plant ploidy identifying method and use thereof | |
| CN108007755A (en) | A kind of effectively observation tomato root knot megabacterium paraffin section method | |
| CN103710439A (en) | Method for quickly identifying chromosome number of rosa plant | |
| CN110044827A (en) | A method of evaluation lily flower morphological feature and pollen development stage correlation | |
| CN101477763A (en) | Reduction division full period microscopic slide exemplar | |
| CN106644636A (en) | Microdissection technology of tobacco chromosomes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |