CN107389411A - A kind of method of grape root tip chromosomes Conventional compression - Google Patents
A kind of method of grape root tip chromosomes Conventional compression Download PDFInfo
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- CN107389411A CN107389411A CN201710753892.6A CN201710753892A CN107389411A CN 107389411 A CN107389411 A CN 107389411A CN 201710753892 A CN201710753892 A CN 201710753892A CN 107389411 A CN107389411 A CN 107389411A
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 40
- 235000009754 Vitis X bourquina Nutrition 0.000 title claims abstract description 29
- 235000012333 Vitis X labruscana Nutrition 0.000 title claims abstract description 29
- 235000014787 Vitis vinifera Nutrition 0.000 title claims abstract description 29
- 230000006835 compression Effects 0.000 title claims abstract description 11
- 238000007906 compression Methods 0.000 title claims abstract description 11
- 240000006365 Vitis vinifera Species 0.000 title 1
- 241000219095 Vitis Species 0.000 claims abstract description 31
- 238000004043 dyeing Methods 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 13
- 238000000386 microscopy Methods 0.000 claims abstract description 11
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 9
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 6
- 230000005593 dissociations Effects 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 239000012153 distilled water Substances 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 20
- 238000005520 cutting process Methods 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 235000019441 ethanol Nutrition 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 229940088598 enzyme Drugs 0.000 claims description 12
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 claims description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 9
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical group OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 claims description 8
- 238000011010 flushing procedure Methods 0.000 claims description 7
- 239000012362 glacial acetic acid Substances 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000006059 cover glass Substances 0.000 claims description 4
- 108010059892 Cellulase Proteins 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 claims description 3
- 239000004576 sand Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 210000004700 fetal blood Anatomy 0.000 claims description 2
- 235000011054 acetic acid Nutrition 0.000 claims 1
- 150000001243 acetic acids Chemical class 0.000 claims 1
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 4
- 230000024321 chromosome segregation Effects 0.000 abstract description 3
- AEMOLEFTQBMNLQ-BKBMJHBISA-N alpha-D-galacturonic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-BKBMJHBISA-N 0.000 abstract description 2
- 230000032823 cell division Effects 0.000 abstract description 2
- 230000008618 cell wall macromolecule catabolic process Effects 0.000 abstract description 2
- 210000000805 cytoplasm Anatomy 0.000 abstract description 2
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000000442 meristematic effect Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 208000035199 Tetraploidy Diseases 0.000 description 3
- 208000026487 Triploidy Diseases 0.000 description 3
- 238000012214 genetic breeding Methods 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
- 230000002559 cytogenic effect Effects 0.000 description 2
- 125000005909 ethyl alcohol group Chemical group 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000745 plant chromosome Anatomy 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
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- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Sampling And Sample Adjustment (AREA)
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Abstract
The invention discloses a kind of method of grape root tip chromosomes Conventional compression, including material culture, pretreatment, fixation, preservation, dissociation, dyeing, tabletting and microscopy.The grape branch that the present invention cultivates is taken root, and method is easy, and tip of a root thickness is moderate, is easy to make chromosome tabletting, obtained squashed preparation cell is smooth, Chromosome spread；Tabletting success rate can be improved using the pretreatment of saturation paracide solution, shortens chromosome thicker, reduces cytoplasm viscosity, obtain more chromosome division phases；Digested using 3.5% mixed enzyme solution, make cell wall breakdown, the pectic substance between the histocyte of meristematic zone decomposes, and makes cell scattered, Chromosome spread space is bigger, and Chromosome spread is uniform, is easy to count.Preparation method of the present invention is easy, quick, and low manufacture cost is easily operated, and obtained cell division lover, the degree of accuracy is high, significant for understanding the selection of Parent and later stage identification in grape genetic background, breeding process.
Description
Technical field
The invention belongs to cytogenetics field, is one of vitis spp methods for ploidy determination, and in particular to Yi Zhongpu
The method of grape root tip chromosomes Conventional compression.
Background technology
Grape（VitisviniferaL.）It is the bejuco of Vitaceae Vitis, is the most ancient fruit tree of China
One of seeds.No matter eating raw, still brewageing processing etc., be loved by people, constantly cultivate new excellent grape
Kind, it is the pursuit unremitting always of grape breeding scientists.
Methods of Ploidy Identification improves breeding efficiency, cultivated improved seeds with important guidance to improveing grape character
Meaning.Research of the observation of chromosome number, form and structure to genetic breeding is significant, the structure and number of chromosome
Change will cause the variation of organism.Therefore, chromosome counting and morphologic observation, it is to explore biosystem occur, be hereditary
Rule and the basis for carrying out genetic breeding, it is the technology that identification and Selection parent material must be grasped in seed selection, breeding.In recent years
Come, the features such as research and development in terms of grape genetic breeding is rapid, and number small yet with grape chromosome is more, existing plant
The general pressed-disc technique of thing using limitation be present on grape, extremely limit to by the research of its chromosome aspect.Therefore, improve existing
Generic plant Chromosome Technique, a kind of Chromosome Pressing Technology for being specially adapted for grape is developed, for its cytogenetics
Theoretical research and breeding practice are significant.
The content of the invention
The purpose of the present invention is the deficiency for the general Chromosome Pressing Technology of existing plant, there is provided a kind of vitis spp
The method of root tip chromosomes tabletting.This method have easy to operate, microscopy is easy, Chromosome spread is good, cytoplasm is less, the back of the body
Scape is shallow, is easy to the advantages that observation enumerating chromosomes, can obtain clearly split coil method.
The present invention provides following technical scheme to achieve the above object
A kind of method of grape root tip chromosomes Conventional compression, it is characterised in that comprise the following steps：
（1）The annotinous branch of robust growth is chosen when the material culture winter cuts, winter sand storage takes out branch, by branch after three months
It is 10 ~ 15 cm cuttings that bar, which cuts into length, and often root cutting stays 2 ~ 3 buds, and clip is gone up when cutting cutting apart from upper bud 1.5 ~ 2.0
Cm, straight snips, lower clip close to cut a portion, tiltedly cut, cutting is completely immersed in clear water and soaks 24 h, reach clip section be in it is bud green
Pull out and drain after color, then 24 h in the downward naphthalene acid solution for immersing 100 mg/L of base portion, cutting is put into conical flask and trained
Support, the cm of the depth of water 2 ~ 4, be placed in illumination box, the Lx of light intensity 2000, the h of illumination 12,24 DEG C of cultures of constant temperature, change water one within one week
It is secondary.After cultivating 15 ~ 25 d, the cm of the tip of a root 2~3 is cut with scissors, is put into conical flask.
（2）Pretreatment will（1）The obtained tip of a root is immersed in saturation paracide solution, sealing, the h of dark treatment 2 ~ 4 under normal temperature.
（3）It is fixed by the pretreated tip of a root with distilled water flushing three times, be placed in the Kano fixer now matched somebody with somebody, seal,
24 ~ 48 h are fixed under the conditions of 4 DEG C.
（4）The tip of a root after fixing is preserved if do not carried out tabletting immediately, material is crossed into alcohol gradient, 95% alcohol at normal temperatures
10 ~ 30 min, the min of 80% alcohol 10 ~ 30, most after 4 DEG C of Cord bloods in 70% alcohol.
（5）The tip of a root after fixation is taken out in dissociation, and distilled water cleans three times, is placed in 2 mL centrifuge tubes, then toward centrifuge tube
The volume ratio of 3.5% mixed enzyme solution of middle addition, mixed enzyme solution and the tip of a root digests 20 ~ 30 min, Ran Houyong for 30: 1,37 DEG C of constant temperature
Distilled water flushing 2 ~ 3 times, it is placed in standby in distilled water.
（5）The tip of a root after dissociation is placed on slide by dyeing, is blotted the moisture around the tip of a root with blotting paper, is used tweezers
The tip of a root removed is caught broken, a drop carbolfuchsin dyeing liquor is instilled, dyes 2 ~ 3 min.
（6）Covered on the tip of a root after tabletting dyeing, blotting paper covered, with the pencil with erasing rubber
Cover glass is beaten in one end, and tabletting is made.
（7）The slice, thin piece made is placed in microscopy under microscope by microscopy, and cell mass is first found under 100 times, then to 1000 times
Lower observation is taken pictures, and finally chromosome is counted according to picture.
Described saturation paracide solution compound method is：The g of paracide 10 is weighed, is dissolved in 100 ml distilled water,
Preserved under normal temperature.
Liquid making method is fixed in described Kano：Absolute ethyl alcohol（Mass fraction >=99.7%）With glacial acetic acid by volume
3:1 is formulated.
3.5% described mixed enzyme solution compound method is：Each 0.7g of cellulase, pectase is weighed, 20ml is added and steams
Distilled water, 4 DEG C of refrigerators are interior to be preserved.
Described carbolfuchsin dyeing liquid making method is as follows：（1）Stoste A：3 g basic fuchsins are dissolved in the wine of 100 ml 70%
In essence；（2）Stoste B：The ml of stoste A 10 are taken to be added in the aqua carbolisata solution of 90 ml 5%, 5% described aqua carbolisata solution
It is by 5g phenol constant volume to 100 mL, half a year is preserved under the conditions of 4 DEG C；（3）Stoste C：The ml of stoste B 55 are taken, add 6 ml ice
In acetic acid and 6 ml 38% formaldehyde；（4）Dyeing liquor：The m1 of C liquid 10 ~ 20 is taken, adds the ml of 45% glacial acetic acid 80~90, then add sorb
The g of alcohol 1.8, the carbol fuchsin liquid of 10%~20% concentration is made into, is used after placing two weeks.
The time that step 3 fixes the pretreated tip of a root with Kano fixer is different because drawing materials, and the length of set time is not
Together, it is directly proportional to the set time for ease of later observations enumerating chromosomes, chromosome number.
Cover glass is beaten with pencil one end with erasing rubber in the tabletting of step 6, is the chromosome point to make cell open and flat
Dissipate, to obtain good split coil method.
In carbolfuchsin dyeing liquid making method, stoste A and stoste C can be preserved for a long time, and stoste B is limited in two weeks and used.Dye
Color liquid uses after the completion of preparing, and colorability is poor, and Color is bad, is used after placing two weeks, then can reach good dyeing
Effect.Sorbierite in dyeing liquor is penetration-assisting agent, has the effect of stable dyeing liquor concurrently, and were it not for sorbierite can also dye, but
Effect is poor.
Described grape variety includes the beautiful red treasured of diploid, triploid summer is black, tetraploid capital is sub-.
The present invention is had the following advantages and beneficial effect relative to prior art：
1st, grape branch is promoted to take root using water culture, method is easy, and the tip of a root thickness that water planting goes out is moderate, is easy to make dyeing
Body tabletting, obtained squashed preparation cell is smooth, Chromosome spread.If the tip of a root is too thick, cell membrane, epidermal tissue are blocked up, make
Tabletting it is not smooth enough, it is difficult to observation count, if the tip of a root is too thin, chromosome division phases are few, and tabletting effect is undesirable.
2nd, tabletting success rate can be improved using the pretreatment of saturation paracide solution, shortens chromosome thicker, reduced thin
Kytoplasm viscosity, to obtain more chromosome division phases.
3rd, digested using 3.5% mixed enzyme solution, make cell wall breakdown, the pectic substance between the histocyte of meristematic zone decomposes, and can make
Cell is scattered, and Chromosome spread space is bigger, and Chromosome spread is uniform, is easy to count.
4th, preparation method of the present invention is easy, quick, and low manufacture cost is easily operated, obtained cell division lover, accurately
Degree is high, and for the genetic background of understanding grape, in breeding process, the selection of Parent and later stage, which are identified, has great importance.
Brief description of the drawings
Fig. 1 is the beautiful red precious grape root tip chromosomes microexamination figure of diploid of the present invention（Eyepiece × object lens=10 × 100,2n
=2X=38）.
Fig. 2 is the black grape root tip chromosomes microexamination figure of triploid summer of the present invention（Eyepiece × object lens=10 × 100,2n=
3X=57）.
Fig. 3 is tetraploid Jingya" grape root tip chromosomes microexamination figure of the present invention（Eyepiece × object lens=10 × 100,2n=
4X=76）.
Example 1
The beautiful red precious grape tip of a root dyeing chromosome tabletting method of diploid, including the step of following order：
（1）The annual beautiful red precious branch of robust growth is chosen when the material culture winter cuts, winter sand storage takes out branch after three months
Bar, it is 10 ~ 12cm cuttings that branch is cut into length, stays 2 ~ 3 buds per root cutting, cut during cutting upper clip apart from upper bud 1.5 ~
2 cm, straight snips, lower clip are tiltedly cut close to the portion of cutting, cutting are completely immersed in clear water and soaks 24 h, reach clip section in fresh
Green, pull out and drain, base portion immerses downwards 24 h in 100 mg/L naphthalene acid solution, hestening rooting, and cutting is put into taper
Cultivate, the cm of the depth of water 2 ~ 4, be placed in illumination box, the Lx of light intensity 2000, the h of illumination 12 in bottle, 24 DEG C of cultures of constant temperature, one week
Change water once.After cultivating 15 ~ 25 d, the cm of the tip of a root 2~3 is cut with scissors, is put into conical flask.
（2）Pretreatment immerses the beautiful red precious tip of a root in saturation paracide solution, sealing, the h of dark treatment 4 under normal temperature.
（3）It is fixed by pretreated beautiful red precious tip of a root material with distilled water flushing three times, be placed in Kano fixer afterwards
In（V absolute ethyl alcohols：V glacial acetic acid=3:1, Kano fixer is now with the current）, 24 h are fixed under 4 DEG C of low temperature.
（4）The tip of a root after fixation is taken out in dissociation, and distilled water cleans three times, is placed in 2 mL centrifuge tubes, then toward centrifuge tube
3.5% mixed enzyme solution of middle addition（Each 0.7g of cellulase, pectase is taken, 20ml distilled water is added, is preserved in 4 DEG C of refrigerators）,
The volume ratio of mixed enzyme solution and the tip of a root is 30: 1, and 37 DEG C of constant temperature digest 20 min, then with distilled water flushing 2 ~ 3 times, is placed in steaming
It is standby in distilled water.
（5）Dyeing tweezers gripping tip of a root material is placed on slide, moisture is sucked with blotting paper, with tweezers by material clip
It is broken, carbolfuchsin dyeing liquor is dripped, dyes 2 min.
（6）Covered on material after tabletting dyeing, blotting paper covered, with the pencil with erasing rubber
Cover glass is beaten in one end, makes cell open and flat, Chromosome spread, to obtain good split coil method.
（7）The slice, thin piece made is placed in microscopy under microscope by microscopy, first under low power（100×）Cell mass is found, then is arrived
Under high power（1000×）Observation is taken pictures, and finally chromosome is counted according to picture, and Fig. 1 is shown in microscopy result and microphotograph.
Described carbolfuchsin dyeing liquid making method is as follows：（1）Stoste A：3 g basic fuchsins are dissolved in the wine of 100 ml 70%
In essence；（2）Stoste B：The ml of stoste A 10 are taken to be added in the aqua carbolisata solution of 90 ml 5%, 5% described aqua carbolisata solution
It is by 5g phenol constant volume to 100 mL, half a year is preserved under the conditions of 4 DEG C；（3）Stoste C：The ml of stoste B 55 are taken, add 6 ml ice
In acetic acid and 6 ml 38% formaldehyde；（4）Dyeing liquor：The m1 of C liquid 10 ~ 20 is taken, adds the ml of 45% glacial acetic acid 80~90, then add sorb
The g of alcohol 1.8, the carbol fuchsin liquid of 10%~20% concentration is made into, is used after placing two weeks.
Example 2
The black root tip chromosomes tabletting method of triploid summer, including the step of following order：
Step 1 and 2 is the same as embodiment 1.
Step 3 is fixed pretreated summer black root point material distilled water flushing three times, is placed in Kano fixer afterwards
In（V absolute ethyl alcohols：V glacial acetic acid=3:1, Kano fixer is now with the current）, 36 h are fixed under 4 DEG C of low temperature.
Step 4, which dissociates, takes out the summer black root point after fixation, and distilled water cleans three times, is placed in 2 mL centrifuge tubes, then past
3.5% mixed enzyme solution is added in centrifuge tube（With embodiment 1）, the volume ratio of mixed enzyme solution and the tip of a root is 30: 1,37 DEG C of constant temperature enzymes
25 min are solved, then with distilled water flushing 2 ~ 3 times, are placed in standby in distilled water.
Step 5,6 and 7 are the same as embodiment 1.Fig. 2 is shown in microscopy result and microphotograph.
Example 3
Tetraploid capital Asia root tip chromosomes tabletting method, including the step of following order：
In addition to the min of enzymolysis 30 that step 3 set time is 48 h, step 4 dissociates, remaining step is the same as embodiment 2.Microscopy result
See Fig. 3 with microphotograph.
The result that practices on Three Represents grape variety shows above, and this method is a kind of effective grape dye
The method of colour solid Conventional compression, and the tabletting method is simple to operate, and Chromosome spread is uniform and background is shallower, chromosome
Number easily counts, and the chromosome number degree of accuracy drawn using this method film-making is high.
Claims (5)
- A kind of 1. method of grape root tip chromosomes Conventional compression, it is characterised in that comprise the following steps：（1）The annotinous branch of robust growth is chosen when the material culture winter cuts, winter sand storage takes out branch, by branch after three months It is 10 ~ 15 cm cuttings that bar, which cuts into length, stays 2 ~ 3 buds per root cutting, cut during cutting upper clip apart from the upper cm of bud 1.5 ~ 2, Straight snips, lower clip close to cut a portion, tiltedly cut, cutting is completely immersed in clear water and soaks 24 h, it is in emerald green to reach clip section, Pull out and drain, then 24 h in the downward naphthalene acid solution for immersing 100 mg/L of base portion, cutting is put into conical flask and cultivated, water Deep 2 ~ 4 cm, are placed in illumination box, the Lx of light intensity 2000, the h of illumination 12, and 24 DEG C of cultures of constant temperature a, Zhou Huanshui once, is cultivated After 15 ~ 25 d, the cm of the tip of a root 2~3 is cut with scissors, is put into conical flask；（2）Pretreatment will（1）The obtained tip of a root is immersed in saturation paracide solution, sealing, the h of dark treatment 2 ~ 4 under normal temperature；（3）It is fixed by the pretreated tip of a root with distilled water flushing three times, be placed in the Kano fixer now matched somebody with somebody, seal, 4 DEG C Under the conditions of fix 24 ~ 48 h；（4）Preserve it is fixed after the tip of a root if do not carried out tabletting immediately, material normal temperature can descend to alcohol gradient, 95% alcohol 10 ~ 30 min, the min of 80% alcohol 10 ~ 30, most after 4 DEG C of Cord bloods in 70% alcohol；（5）Dissociation will it is fixed after the tip of a root take out, distilled water cleans three times, is placed in 2 mL centrifuge tubes, then toward adding in centrifuge tube The volume ratio of 3.5% mixed enzyme solution, mixed enzyme solution and the tip of a root is that 30: 1,37 DEG C of constant temperature digest 20 ~ 30 min, then uses distilled water Rinse 2 ~ 3 times, be placed in standby in distilled water；（5）The tip of a root after dissociation is placed on slide by dyeing, is blotted the moisture around the tip of a root with blotting paper, will be taken with tweezers Under the tip of a root be caught broken, instill one drop carbolfuchsin dyeing liquor, dye 2 ~ 3 min；（6）Covered on the tip of a root after tabletting dyeing, blotting paper covered, with pencil one end with erasing rubber Cover glass is beaten, tabletting is made；（7）The slice, thin piece made is placed in microscopy under microscope by microscopy, cell mass is first found under 100 times, then seen under 1000 times Examine and take pictures, finally chromosome is counted according to picture.
- 2. the method for a kind of grape root tip chromosomes Conventional compression according to claim 1, it is characterised in that described is full It is with paracide solution compound method：The g of paracide 10 is weighed, is dissolved in 100 ml distilled water, is preserved under normal temperature.
- A kind of 3. method of grape root tip chromosomes Conventional compression according to claim 1, it is characterised in that described card Liquid making method is fixed in promise：Absolute ethyl alcohol and glacial acetic acid by volume 3:1 is formulated, now with the current.
- 4. the method for a kind of grape root tip chromosomes Conventional compression according to claim 1, it is characterised in that described 3.5% mixed enzyme solution compound method is：Weigh each 0.7g of cellulase, pectase, add 20ml distilled water, in 4 DEG C of refrigerators Preserve.
- A kind of 5. method of grape root tip chromosomes Conventional compression according to claim 1, it is characterised in that described card Precious moral training liquid making method is as follows：（1）Stoste A：3 g basic fuchsins are dissolved in the alcohol of 100 ml 70%；（2）Stoste B：Take The ml of stoste A 10 are added in 90 ml5% aqua carbolisata solution, and 5% described aqua carbolisata solution is to arrive 5g phenol constant volumes 100 mL, half a year is preserved under the conditions of 4 DEG C；（3）Stoste C：Take the ml of stoste B 55, add 6 ml glacial acetic acids and 6 ml 38% In formaldehyde；（4）Dyeing liquor：The m1 of C liquid 10 ~ 20 is taken, adds the ml of 45% glacial acetic acid 80~90, then adds the g of sorbierite 1.8, it is made into 10%~ The carbol fuchsin liquid of 20% concentration, used after placing two weeks.
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| CN201710753892.6A CN107389411A (en) | 2017-08-29 | 2017-08-29 | A kind of method of grape root tip chromosomes Conventional compression |
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| CN201710753892.6A CN107389411A (en) | 2017-08-29 | 2017-08-29 | A kind of method of grape root tip chromosomes Conventional compression |
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| CN201710753892.6A Pending CN107389411A (en) | 2017-08-29 | 2017-08-29 | A kind of method of grape root tip chromosomes Conventional compression |
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| CN108168990A (en) * | 2017-12-27 | 2018-06-15 | 华中科技大学同济医学院附属协和医院 | A kind of frozen section fixer set agent and fixing means |
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| CN109490036A (en) * | 2018-11-22 | 2019-03-19 | 黑龙江大学 | A kind of distant hybridization Chromosomes in Sugarbeet flaking method |
| CN112255069A (en) * | 2020-10-22 | 2021-01-22 | 南京农业大学 | Dendrobium huoshanense root tip specimen tablet and preparation method and application thereof |
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Cited By (4)
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| CN108168990A (en) * | 2017-12-27 | 2018-06-15 | 华中科技大学同济医学院附属协和医院 | A kind of frozen section fixer set agent and fixing means |
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| CN109490036A (en) * | 2018-11-22 | 2019-03-19 | 黑龙江大学 | A kind of distant hybridization Chromosomes in Sugarbeet flaking method |
| CN112255069A (en) * | 2020-10-22 | 2021-01-22 | 南京农业大学 | Dendrobium huoshanense root tip specimen tablet and preparation method and application thereof |
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