CN101613755B - Method for identifying carnation chromosome number by bud - Google Patents
Method for identifying carnation chromosome number by bud Download PDFInfo
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- CN101613755B CN101613755B CN 200910094743 CN200910094743A CN101613755B CN 101613755 B CN101613755 B CN 101613755B CN 200910094743 CN200910094743 CN 200910094743 CN 200910094743 A CN200910094743 A CN 200910094743A CN 101613755 B CN101613755 B CN 101613755B
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Abstract
The invention provides a method for identifying carnation chromosome number by bud, comprising: obtaining bud anther, ovary wall and corolla tissue, pre-treating ice water mixture, fixing by stationary liquid, dyeing, dissociating with hydrochloric acid, tabletting and microscopic examining. Compared with the traditional method that a root tip tissue is used for chromosome tabletting, the invention solves the disadvantages of few visual filed cells, difficult microscopic examination and poor chromosome dispersion during root tip chromosome tabletting, and overcomes the problems of long rootage period for carnation cutting seedling and time and labor wasting for cutting; compared with stem tip chromosome tabletting, the invention overcomes the interference problem of non-meristematic cell or reserve substance or secretion, chromosome tabletting can be realized only by using buds, which does not damage plant growing state and has convenient material resource; in addition, a plurality of metaphase cells exist, so that the difficulties of small carnation chromosome, huge quantity and hard counting of the chromosome can be solved. Thus, when plants bloom, the effective method is that the bud is used for chromosome tabletting.
Description
Technical field
The present invention relates to a kind of method of identifying carnation chromosome number, especially a kind of method with bud evaluation carnation chromosome number belongs to cytobiology and Cytogenetic techniques field.
Background technology
Oeillet (Dianthus caryophyllus Linn) has another name called carnation, for the perennial perennial root herbage of Caryophyllaceae Carnation, is one of world-renowned fresh cutting flower, also is one of topmost large outlet flowers in Yunnan Province.In the oeillet cross-breeding, exist and hybridize not affinity, the phenomenon that setting percentage is on the low side.One of them reason is unclear to the cytology background of a lot of germplasms.Therefore, success ratio, the raising cross-breeding efficient of hybridization be increased, carnation chromosome ploidy analysis problem should be solved earlier.
Karyomit(e) is the carrier of gene, and the preparation chromosome specimen is undoubtedly the most basic technology of cytogenetics, and good chromosome sectioning is the prerequisite of carrying out chromosome banding, group type analysis, in situ hybridization etc.The observation of plant cell chromosome and analysis, significant for genetics phenomenon Study such as cytometaplasia in the action of chromosome karyotype analysis, cytogamy after stain colour solid and the culturing process.
The chromosome length of oeillet is 2.5-4.0um, belongs to the microchromosome type, has normal kinetochore, because carnation chromosome number is many, and belongs to microchromosome, the compressing tablet difficulty is bigger, therefore needs to seek the high material of cell division index and carries out chromosome counting.The tip of a root is the most frequently used material of chromosome counting, and especially the seed root-tip cells is a research karyomit(e) material the most reliably.The oeillet setting percentage is low, most plants shaky and by nourishing and generating, but cuttage seeding is taken root the cycle long, and cuttage wastes time and energy, and therefore carries out chromosome sectioning if get the tip of a root with the slotting seedling rooting of fibre, then need spend longer for some time, the difficulty of drawing materials is big.When getting stem apex in addition and carrying out chromosome sectioning; Because stem apex is drawn materials and is subject to the restriction of the season of growth, and the interference of different non-meristematic cell or repertory or secretory product is arranged, make metacinesis phase cell few or do not have; Stem apex strips difficulty, thereby influences chromosome sectioning and counting.Therefore, must improve prior art.
Summary of the invention
The object of the present invention is to provide a kind of drawing materials easily, time saving and energy saving, and the many mutually methods with bud evaluation carnation chromosome number of metaphase.
The present invention accomplishes through following technical proposal: a kind of method with bud evaluation carnation chromosome number is characterized in that through following process steps:
A gets the bud of the long 1.0-1.9cm of oeillet;
B puts into mixture of ice and water pre-treatment 22-26h with bud;
C after pretreated bud cleaned, immerses the fixing 2-24h of processing in the stationary liquid of following volume ratio: absolute ethyl alcohol: Glacial acetic acid min. 99.5=3: 1;
D after the bud after fixing the processing cleaned, under room temperature, immerses concentration and is to dissociate in the HCL solution of 1mol/L and handle 10-20min;
E after the bud cleaning of dissociating after handling, places on the slide glass, tears the bud tissue to shreds with scalper, drips a 1-3 and drips the carbol fuchsin dye liquor, dyeing 5-10min;
F, the bud tissue covered after dyeing is handled, thieving paper in the covering touches the bud tissue, and cell is disperseed rapidly;
G with sealing around the deckglass, promptly gets chromosome number with observation by light microscope with nail varnish.
Said bud is specifically got its flower pesticide or ovary wall or corolla tissue.
The cleaning of said C, D, E step adopts zero(ppm) water to clean 2-3 time, and implement is a syringe.
The present invention compared with prior art has advantage and effect: adopt such scheme, promptly carry out chromosome sectioning with immature bud tissue, thereby carnation chromosome number is counted; Carry out chromosome sectioning with organization of root tips and compare with traditional; Solved in the root tip chromosomes film-making, few, the difficult microscopic of visual field cell, karyomit(e) disperses bad shortcoming; It is long to have overcome oeillet cuttage seeding cycle of taking root simultaneously, and the problem that wastes time and energy of cuttage; Compare with the stem tip chromosome film-making, overcome the interference problem of non-meristematic cell or repertory or secretory product, only need get bud can carry out chromosome sectioning; Can not destroy the growth conditions of plant; Draw materials conveniently, and metacinesis phase cell is many, it is little to have solved carnation chromosome; Quantity is many, the difficult problem that karyomit(e) is difficult to count.Therefore, when plant blossom, utilizing bud to carry out chromosome sectioning is efficient ways.
Embodiment
Below in conjunction with embodiment the present invention is done and to further describe.
Embodiment 1
With oeillet kind " autumn harvest " is material, wins the long bud of 1.9cm, strips out ovary, and ovary is vertically cut, and gets its ovary wall and puts into mixture of ice and water pre-treatment 24h; After drawing waste liquid with the 5ml syringe, pretreated ovary wall is cleaned 3 times with zero(ppm) water, immerse volume ratio and be: absolute ethyl alcohol: fixing 2h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind identical syringe absorption waste liquid, the ovary wall after fixing the processing is cleaned 3 times with zero(ppm) water, under room temperature, immerse 20min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind identical syringe absorption waste liquid, clean ovary wall 3 times with zero(ppm) water again, place on the slide glass, tear the ovary wall tissue to shreds with scalper; Drip 2 carbol fuchsin dye liquors, dyeing 10min, covered, thieving paper in the covering; Knock the ovary wall tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Use observation by light microscope immediately, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, meristematic cell was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.Dyed body analysis of accounts, oeillet kind " autumn harvest " chromosome number is 30, diploid.
Embodiment 2
With oeillet kind " Huang Fei's letter " is material, wins the long bud of 1.3cm, strips out ovary, and ovary is vertically cut, and gets its ovary wall and puts into mixture of ice and water pre-treatment 22h; Behind the waste liquid after draw handling with the 5ml syringe, ovary wall is cleaned 2 times with zero(ppm) water, immerse volume ratio afterwards and be: absolute ethyl alcohol: fixing 4h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 3 times with zero(ppm) water, under room temperature, immerse 15min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 3 times with zero(ppm) water again, place on the slide glass, tear the ovary wall tissue to shreds with scalper; Drip 3 carbol fuchsin dye liquors, dyeing 5min, covered, thieving paper in the covering; Knock the ovary wall tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Use observation by light microscope after four days, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, the meristematic cell number was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.Dyed body analysis of accounts, oeillet kind " Huang Fei's letter " chromosome number is 60, tetraploid.
Embodiment 3
" being crazy about " with the oeillet kind is material, gets the long bud of 1.5cm, and pollen was in monokaryon and kept to the side the phase this moment, took out flower pesticide, put into mixture of ice and water pre-treatment 24h; Behind the waste liquid after draw handling with the 5ml syringe, ovary wall is cleaned 3 times with zero(ppm) water, immerse volume ratio and be: absolute ethyl alcohol: fixing 2h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 3 times with zero(ppm) water, under room temperature, immerse 20min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 3 times with zero(ppm) water, place on the slide glass, tear anther tissue to shreds with scalper; Drip 2 carbol fuchsin dye liquors, dyeing 5min, covered, thieving paper in the covering; Knock anther tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Use observation by light microscope immediately, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, meristematic cell was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.It is 30 that dyed body analysis of accounts, oeillet kind " are crazy about " chromosome number, diploid.
Embodiment 4
" Luo Jiate " is material with the oeillet kind, gets the long bud of 1.2cm, and this moment, pollen was in the subtrahend first division diakinesis stage to the subtrahend a, took out flower pesticide, put into mixture of ice and water pre-treatment 22h; Behind the waste liquid after draw handling with the 5ml syringe, ovary wall is cleaned 3 times with zero(ppm) water, immerse volume ratio and be: absolute ethyl alcohol: fixing 4h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 2 times with zero(ppm) water, under room temperature, immerse 10min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind the waste liquid after the identical syringe absorption processing, ovary wall is cleaned 2 times with zero(ppm) water, place on the slide glass, tear anther tissue to shreds with scalper; Drip 3 carbol fuchsin dye liquors, dyeing 5min, covered, thieving paper in the covering; Knock anther tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Use observation by light microscope immediately, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, meristematic cell was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.Dyed body analysis of accounts, oeillet kind " Luo Jiate " chromosome number is 30, diploid.
Embodiment 5
With oeillet kind " high mallow titanium alloy " is material, gets the long bud of 1.2cm, takes out corolla, puts into mixture of ice and water pre-treatment 26h; Behind the waste liquid after draw handling with the 5ml syringe, corolla is cleaned 2 times with zero(ppm) water, immerse volume ratio and be: absolute ethyl alcohol: fixing 16h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind the waste liquid after the identical syringe absorption processing, corolla is cleaned 2 times with zero(ppm) water, under room temperature, immerse 15min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind the waste liquid after the identical syringe absorption processing, corolla is cleaned 3 times with zero(ppm) water, place on the slide glass, tear the corolla tissue to shreds with scalper; Drip 1 carbol fuchsin dye liquor, dyeing 5min, covered, thieving paper in the covering; Knock anther tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Used observation by light microscope on the 4th day, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, meristematic cell was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.Dyed body analysis of accounts, oeillet kind " high mallow titanium alloy " chromosome number is 30, diploid.
Embodiment 6
" powder beauty " is material with the oeillet kind, wins the long bud of 1.1cm, takes out corolla, puts into mixture of ice and water pre-treatment 26h; Behind the waste liquid after draw handling with the 5ml syringe, corolla is cleaned 2 times with zero(ppm) water, immerse volume ratio and be: absolute ethyl alcohol: fixing 16h in the Ka Nuoshi I stationary liquid of Glacial acetic acid min. 99.5=3: 1; Behind the waste liquid after the identical syringe absorption processing, corolla is cleaned 2 times with zero(ppm) water, under room temperature, immerse 20min in the HCL solution that concentration is 1mol/L, carry out the salt acid dissociation; Behind the waste liquid after the identical syringe absorption processing, corolla is cleaned 3 times with zero(ppm) water again, place on the slide glass, tear the corolla tissue to shreds with scalper; Drip 1 carbol fuchsin dye liquor, dyeing 8min, covered, thieving paper in the covering; Knock the corolla tissue with chopsticks on the thieving paper deckglass gently covering, cell is disperseed rapidly, around deckglass, carry out sealing with nail varnish; Use observation by light microscope after two days, its microscopy effect: under the situation of amplifying 1000 times (10 times of eyepiece * 100 times object lens), mid-term, meristematic cell was many; Karyomit(e) shrinks, and the karyomit(e) good dispersion is easy to counting.Dyed body analysis of accounts, oeillet kind " powder beauty " chromosome number is 60, tetraploid.
Claims (2)
1. identify with bud and it is characterized in that the method for carnation chromosome number for one kind through following process steps:
A gets the bud of the long 1.0-1.9cm of oeillet;
B puts into mixture of ice and water pre-treatment 22-26h with flower pesticide or ovary wall or corolla tissue;
C after pretreated flower pesticide or ovary wall or corolla tissue cleaned, immerses the fixing 2-24h of processing in the stationary liquid of following volume ratio: absolute ethyl alcohol: Glacial acetic acid min. 99.5=3: 1;
D after the flower pesticide after fixing processing the or ovary wall or corolla tissue cleaned, under room temperature, immerses concentration and is the processing 10-20min that dissociates in the HCL solution of 1mol/L;
E behind the flower pesticide or ovary wall or the cleaning of corolla tissue that dissociate after handling, tears flower pesticide or ovary wall or corolla tissue to shreds, places on the slide glass dyeing 5-10min;
F, flower pesticide or ovary wall or corolla tissue covered after dyeing is handled, thieving paper in the covering touches flower pesticide or ovary wall or corolla tissue, and cell is disperseed rapidly;
G with sealing around the deckglass, promptly gets chromosome number with observation by light microscope with nail varnish.
2. the method with bud evaluation carnation chromosome number as claimed in claim 1 is characterized in that the cleaning of said C, D, E step adopts zero(ppm) water to clean 2-3 time, and implement is a syringe.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200910094743 CN101613755B (en) | 2009-07-17 | 2009-07-17 | Method for identifying carnation chromosome number by bud |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200910094743 CN101613755B (en) | 2009-07-17 | 2009-07-17 | Method for identifying carnation chromosome number by bud |
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| CN101613755A CN101613755A (en) | 2009-12-30 |
| CN101613755B true CN101613755B (en) | 2012-09-05 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102277423B (en) * | 2011-07-13 | 2013-04-17 | 浙江万里学院 | Quick determination method of number of chromosomes of bivalve by utilizing ovum fluorescent microscope observation |
| CN105838786A (en) * | 2016-03-24 | 2016-08-10 | 西南大学 | Method of chromosome smearing of adult plants of loquat with young tender flower buds |
| CN110012748B (en) * | 2019-04-23 | 2021-10-01 | 南京林业大学 | Method for stripping single pistil in polymeric pistil |
| CN111238888A (en) * | 2020-01-16 | 2020-06-05 | 云南省农业科学院甘蔗研究所 | Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method |
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2009
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Non-Patent Citations (5)
| Title |
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| J.A.MORAKINYO等.CHROMOSOME BEHAVIOUR IN CAPSICUM ANNUUM,C.FRUTESCENS AND THEIR INTRA-AND INTERSPECIFIC HYBRIDS.《Nigerian Journal of Botany》.1992,第5卷135-143. * |
| 潘俊松等.海石竹的离体快繁及核型分析.《广西植物》.2004,第24卷(第1期),33-36. * |
| 瞿素萍等.香石竹的多倍体诱导及其变异研究.《西南农业大学学报(自然科学版)》.2004,第26卷(第5期),609-612. * |
| 莫锡君等.大花香石竹多倍体育种研究.《中国农学通报》.2005,第21卷(第11期),262-264. * |
| 闫素丽等.染色体核型分析及染色体显微分离技术研究进展.《生物技术通报》.2008,(第4期),70-74. * |
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