CN106896008A - A kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen - Google Patents
A kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen Download PDFInfo
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- CN106896008A CN106896008A CN201710263670.6A CN201710263670A CN106896008A CN 106896008 A CN106896008 A CN 106896008A CN 201710263670 A CN201710263670 A CN 201710263670A CN 106896008 A CN106896008 A CN 106896008A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The present invention proposes a kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen, and the method is comprised the following steps：The step such as root tip culture, tip of a root sampling and pre-treatment, tip of a root enzymolysis, chromosome sectioning and microscopy, is characterized in the whole strain culture root system of barrel plant；Tip of a root treatment is processed 3 ~ 4 hours for 25 ~ 28 DEG C using the 8 oxyquinoline aqueous solution of 0.002M, and wall Low Osmotic Method film-making is gone using enzymolysis.The present invention is workable, it is readily available the tip of a root of high activity meristematic zone, save the time of root tip culture, use manpower and material resources sparingly, film-making efficiency high, the division phases for obtaining meristematic zone cell are more, and the chromosome morphology of acquisition is clear, be uniformly dispersed, it is particularly suitable for the chromosome sectioning of spot thatch platymiscium, this method is equally applicable to the chromosome sectioning of sugarcane and its relative genus plant.
Description
Technical field
The present invention relates to the preparation method of the plant chromosome mid-term phase sample in plant cytogenetics, more particularly to spot
The preparation method of thatch plant root tip chromosome.
Technical background
Chromosome is the carrier of inhereditary material, and chromosome has relatively stable number and form in different species.
Cell chromosome genetic research has consequence in genetic breeding, and the preparation of chromosome specimen is cytogenetical study
Basic experiment method.Prepared by plant chromosome sample include following flow：Root tip culture, materials, pre-treatment, fixation, enzyme
Solution（Or dissociation）, film-making and microscopy.
Spot thatchErianthus arundinaceus (Retz.) Jeswiet. is perennial herb, is uprightly grown thickly, without ground
Lower stem, distributed pole is wide, and adaptability is extremely strong.It has drought resisting, waterlogging, resistance to lean, resistant to diseases and insects is strong, tiller is more, perennial root good, raw
The features such as power long is vigorous, with the excellent proterties that can be utilized by cane breeding that cane breeding person is pursued, so extremely generation
The favor of breeding man of various countries of boundary.Sugarcane has been breached both at home and abroad at present to be obtained more for material with the generic cross of spot thatch.Spot thatch
And its offspring hybridized with sugarcane has great importance to Sugarcane genetic improvement.
Chromosome number 2n=60 of most spot thatches, researcher also it has been found that there is 2n=40, but than paddy rice,
The crop chromosome bar number such as corn, sorghum is more, and chromosome sectioning difficulty is big.The culture of particularly spot lalang grass rhizome point is always a hardly possible
Point, method such as paddy rice, corn, the wheat of traditional plant root tip culture are obtained by tips of a root such as germinations.Spot thatch is
The general method with reference to sugarcane of clonal line, by obtaining the stem section of spot thatch plant, to root point moisturizing culture on the section of stem section
The tip of a root is taken to obtain the root of new life, length to 1cm or so.But spot thatch stem is generally the empty silk floss heart, the root point on section is less, energy after moisturizing
The root point for sending out roots is less, and conventional method culture root needs substantial amounts of spot thatch stem section, in addition the temperature during stem section is cultivated
Requirement with humid control is higher, and the tip of a root grows rear easily browning and stops growing, so as to obtain the stronger tip of a root of activity not allowing
Easily.Preferable chromosome sectioning difficulty is obtained greatly with traditional spot thatch flaking method, researcher is seeking a kind of efficient
Spot thatch method of chromosome preparation, can within a short period of time obtain the good spot lalang grass rhizome point of activity, with more split coil method cell, no
Need to cut stem culture repeatedly, take root, use manpower and material resources sparingly, improve spot thatch chromosome preparation efficiency.
The content of the invention
It is an object of the invention to provide a kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen, it has section
Save time and experiment material and labour, improve the effect of conventional efficient.
Technical scheme is as follows：
A kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen, comprises the following steps.
（1）Root tip culture：
The early spring, the tender plant of clip spot thatch children is planted in culture bucket, and side and the bear for cultivating bucket are permeable, and bucket inner bottom part is
Water conservation is breathed freely matrix, and by cultivation managements such as normal water, fertilizer and weedings, the root of spot thatch can be coiled in bucket after 3 ~ 4 months
Surrounding and bottom.
（2）The tip of a root is sampled and pre-treatment：
In fair weather morning 8 summer:00~10:00, whole plant is separated together with matrix with culture bucket, select activity root high
Point, the tip of a root of about 0.5cm is cut with scalpel, is positioned over rinsing in distilled water, and it is the 8- hydroxyls of 0.002M that concentration is put into after rinsing
25 ~ 28 DEG C of the base quinoline aqueous solution is processed 3 ~ 4 hours, is rinsed with distilled water after treatment, after gauze wipes root system table secretion, then is put
It is placed in Ka Nuoshi fixers, 4 DEG C of refrigerators are processed 24 ~ 48 hours.
（3）The tip of a root is digested
From step（2）Refrigerator in take out the tip of a root, with distilled water rinse 2 ~ 3 times, clean fixer after, be positioned over 0.075M KCl
Hypotonic 1 hour before solution, the tip of a root is taken out afterwards, and to be positioned in the enzymolysis mixed solution containing cellulose and pectase enzymolysis 5 ~ 8 small
When, enzymolysis liquid is washed away with distilled water after enzymolysis, latter hypotonic 1 hour, it is transferred to Ka Nuoshi fixers and fixes 0.5 hour.
（4）Chromosome sectioning
With scalpel and tweezers by step（3）Meristernatic zone cell dissociation go out, and uniform application dries on slide.
（5）Microscopy
By step（4）In the slide that dries be placed under the light microscope with difference function and carry out microscopy, the selective staining bodily form
Good, the scattered cell of state, makes a record, and -80 DEG C save backup.
Prioritization scheme is：Step（3）The mass concentration difference of the concentration of described enzymolysis mixed liquor, cellulose and pectase
It is 3.5% and 1.75%.
The present invention has substantive distinguishing features following prominent and significant progress compared with prior art.
1. the present invention uses the whole strain of barrel plant and takes the method for root and carries out root tip culture, and tip of a root activity is strong, effect stability,
There is time-consuming, experiment material and labour, conventional efficient is improved.
2. the sectioning cells that the present invention is obtained are clean, and cell division phases are more, and Chromosome spread, form are good, can be used for
Chromosome carries out karyotyping or downstream experiment（Such as chromosome fluorescence in-situ hybridization）It is spot thatch germplasm Deng chromosomal inheritance research
Chromosomal inheritance research provides effective technological reserve.
Specific embodiment
With reference to embodiment, the present invention is described further.
Embodiment
A kind of preparation method of spot thatch plant root tip meristematic zone chromosome, comprises the following steps.
（1）Root tip culture：In the early spring, clip spot thatch children is planted straight in mouth under specification back cut diameter 35cm for tender plant 2-3 plants
A diameter of 2cm holes water-permeable and air permeable is beaten in footpath 27cm height 32cm Oxfords bucket, bucket four sides and bottom, and bucket inner bottom part is put into 3cm or so thickness
The pine bark of degree, with field of vegetables soil as planting matrix, thickness is about 15cm, by cultivation managements such as normal water, fertilizer and weedings, makes
Plant grows up strong and sturdy in a good condition, after 3 months plant enter growth animated period, spot lalang grass rhizome system can wind barrel inner bottom part and
Edge.
（2）The tip of a root is sampled and pre-treatment：Morning 9 summer：00~10：00 separates whole plant together with matrix with bucket, now root
System's growth is vigorous, and sturdy root system is climbed between full matrix and bucket, chooses bottom or edge white colour is more bright, and growth is vigorous,
The tip of a root of health, the tip of a root of about 0.5cm is cut with scalpel, is positioned over rinsing in distilled water, concentration is put into after rinsing and is
25 ~ 28 DEG C of the 8-hydroxyquinoline aqueous solution of 0.002M is processed 4 hours, after distilled water rinsing, after wiping root table secretion with gauze
Input Ka Nuoshi fixers are processed 24 ~ 48 hours in 4 DEG C of refrigerators.
（3）The tip of a root is digested：The tip of a root is taken out from refrigerator, after rinsing 2 ~ 3 times with distilled water, before being positioned over 0.075M KCl solution
Hypotonic 60min, takes out during the tip of a root is positioned over the enzymolysis mixed liquor of containing cellulose 3.5% and pectase 1.75% and digests 5 ~ 8h afterwards（Depending on
The size of its tip of a root determines enzymolysis time）, after wash away enzyme liquid with distilled water, rear hypotonic 60min is transferred to solid after Ka Nuoshi fixers
Determine 30min.
（4）Chromosome sectioning：With tweezers and scalpel, by step（3）Meristernatic zone cell dissociation go out, uniform application
In slide, dry.
（5）Microscopy：The slide that will be dried is positioned over the sediments microscope inspection with difference function, selective staining volume morphing is good,
Scattered cell, makes a record, and -80 DEG C save backup.
Claims (3)
1. the preparation method of a kind of spot thatch plant root tip meristematic zone chromosome specimen, it is characterised in that comprise the following steps：
（1）Root tip culture：The early spring, the tender plant of clip spot thatch children is planted in culture bucket, and side and the bear for cultivating bucket are saturating
Water, bucket inner bottom part is the ventilative matrix of water conservation, by cultivation managements such as normal water, fertilizer and weedings, the root meeting of spot thatch after 3 ~ 4 months
Coil in the surrounding in bucket and bottom；
（2）The tip of a root is sampled and pre-treatment：Whole plant is separated together with matrix with culture bucket, the activity tip of a root high is selected, with operation
Knife or operation clip length are about the tip of a root of 0.5cm, are positioned over rinsing in distilled water, and it is the 8- of 0.002M that concentration is put into after rinsing
The oxyquinoline aqueous solution, 25 ~ 28 DEG C process 3 ~ 4 hours, cleaned with distilled water after treatment, then in being positioned over Ka Nuoshi fixers, 4
DEG C refrigerator is processed 24 ~ 48 hours；
（3）The tip of a root is digested：From step（2）Refrigerator in take out the tip of a root, with distilled water rinse 2 ~ 3 times, clean fixer after, place
Hypotonic 1 hour in 0.075M KCl solution, the tip of a root is taken out afterwards and is positioned in the enzymolysis mixed liquor containing cellulose and pectase
Enzymolysis 5 ~ 8 hours, hypotonic 1 hour after being cleaned with distilled water after enzymolysis, is transferred to Ka Nuoshi fixers and fixes 0.5 hour；
（4）Chromosome sectioning：With tweezers and scalpel by step（3）Meristernatic zone cell dissociation go out, and uniform application in
On slide, dry.
（5）Microscopy：By step（4）The slide for drying is positioned over the sediments microscope inspection with difference function, selective staining volume morphing
Good, scattered cell, makes a record, and -80 DEG C save backup.
2. the method that as claimed in claim 1 prepared by a kind of acquisition of spot thatch plant root tip meristematic zone and chromosome, it is special
It is the step to levy（2）The tip of a root sample preferred period be the spot thatch jointing stage be 6-9 months, sample time is preferably fine day
Morning 8:00~10:00.
3. the method that as claimed in claim 1 prepared by a kind of acquisition of spot thatch plant root tip meristematic zone and chromosome, it is special
It is step to levy（3）The mass concentration of the concentration of described enzymolysis mixed liquor, cellulose and pectase is respectively 3.5% He
1.75%, hydrolysis temperature is 37 DEG C.
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| CN201710263670.6A CN106896008A (en) | 2017-04-21 | 2017-04-21 | A kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen |
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| CN201710263670.6A CN106896008A (en) | 2017-04-21 | 2017-04-21 | A kind of preparation method of spot thatch plant root tip meristematic zone chromosome specimen |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108731994A (en) * | 2018-05-21 | 2018-11-02 | 遵义医学院 | A kind of production method of climbing groundsel root tip chromosomes sample slice |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| CN113375989A (en) * | 2021-04-16 | 2021-09-10 | 中国热带农业科学院热带生物技术研究所 | Separation method and research method of rubber tree latex pipe network |
| CN114938807A (en) * | 2022-05-06 | 2022-08-26 | 中国热带农业科学院海口实验站 | Method for preparing chromosome specimen of root tip meristematic region of passionfruit subgenus plant |
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| CN103993095A (en) * | 2014-06-10 | 2014-08-20 | 中国科学院西北高原生物研究所 | Preparation method of cell chromosome metaphase specimen of psathyrostachys nevski plant |
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| CN102492767A (en) * | 2011-11-15 | 2012-06-13 | 四川农业大学 | Preparation method of chromosome suitable for karyotype analysis of rubus plants |
| CN103993095A (en) * | 2014-06-10 | 2014-08-20 | 中国科学院西北高原生物研究所 | Preparation method of cell chromosome metaphase specimen of psathyrostachys nevski plant |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108731994A (en) * | 2018-05-21 | 2018-11-02 | 遵义医学院 | A kind of production method of climbing groundsel root tip chromosomes sample slice |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| CN113375989A (en) * | 2021-04-16 | 2021-09-10 | 中国热带农业科学院热带生物技术研究所 | Separation method and research method of rubber tree latex pipe network |
| CN113375989B (en) * | 2021-04-16 | 2023-12-15 | 中国热带农业科学院热带生物技术研究所 | Separation method and research method of rubber tree milk tube network |
| CN114938807A (en) * | 2022-05-06 | 2022-08-26 | 中国热带农业科学院海口实验站 | Method for preparing chromosome specimen of root tip meristematic region of passionfruit subgenus plant |
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