CN102703599A - Improved preparation method of kelp chromosome - Google Patents
Improved preparation method of kelp chromosome Download PDFInfo
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- CN102703599A CN102703599A CN2012102328238A CN201210232823A CN102703599A CN 102703599 A CN102703599 A CN 102703599A CN 2012102328238 A CN2012102328238 A CN 2012102328238A CN 201210232823 A CN201210232823 A CN 201210232823A CN 102703599 A CN102703599 A CN 102703599A
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Abstract
The invention discloses an improved preparation method of a kelp chromosome. The steps are absorbing surplus moisture after kelp gamobiums are centrifuged, keeping the kelp gamobiums to be deposited, and adding colchicines solution for evenly shaking and stewing; conducting hypotension on the kelp gamobiums by using potassium chloride solution, fixing the kelp gamobiums by sing kano stationary liquid after hypotension, conducting enzymolysis and wall removal through mixed enzyme liquid of cellulose R-10 solution, pectinase solution and liquation enzyme R-10 solution, absorbing supernate by using a liquor relief gun after the kelp gamobiums are centrifuged, adding glacial acetic acid for evenly mixing to form cell suspension, absorbing the cell suspension by using a dropper to an upper dropping sheet of a centrifuging and pre-cooling glass slide, burning the bottom face of the glass slide by using an alcohol burner for one time, and naturally drying or stoving, dying the kelp gamobium chromosome adhered to the glass slide by using giemsa staining fluid, and observing and photographing under a microscope. The chromosomes obtained by using the improved preparation method are good in segregation, differentiable in shape and denumerable in chromosome split pahse, and the improved preparation method is simple and rapid in method, strong in repeatability and capable of opening in a general laboratory.
Description
Technical field
The present invention relates to chromosomal preparation method, relate in particular to the chromosomal preparation method of improved sea-tangle.
Background technology
The sporophyte of sea-tangle is individual big, is tame object.Kelp gametophyte is small filament, is the object of artificial culture seed, also is the basis that the kelp germplasm resource is preserved.Kelp gametophyte has the branch of male and female, and megagametophyte is made up of one or several cell, and microgametophyte generally is made up of a plurality of cells, and the microgametophyte cell is little than the megagametophyte cell individual.In developmental process, megagametophyte increases the volume of cell, and microgametophyte then carries out the division of cell, increases the number of cell, forms cellulous branch body or group's spherule together.
Sea-tangle karyomit(e) is less, because the difference of method of chromosome preparation causes the sea-tangle chromosome number to have dispute.Mainly be with section method and iron haematoxylin dyeing in early days.Section method is a comparison primary method, is used to calculate the time-consuming and out of true of karyomit(e).Many subsequently with pressed disc method and aceto-camine, Chloral Hydrate iron acetate phenodin or aceto-orcein dyeing.Twentieth century six the seventies Chinese scholars are observed chromosome number to the kelp gametophyte cell through pressed disc method, and most scholars think to have only 22, and minority scholar thinks has 31.To nineteen nineties; Japan scholar Hiroshi Yabu etc. thinks that the sea-tangle gametid [cell has 32 karyomit(e)s (to see paper " chromosome numbers of four main laminarias (brown alga) " approximately; " Japanese phycology journal, 1991 the 39th the 2nd phases of volume, 185-187 page or leaf).Chinese scholar Zhou Liran in 2004 etc. utilize compressing tablet and brazilwood extract dyeing to observe kelp gametophyte to have 31 karyomit(e)s.The karyomit(e) that pressed disc method obtains is point-like more; Can't the differential staining volume morphing, can not do karyotyping and (see paper " a kind of improved sea-tangle microgametophyte (heterokontae) method of chromosome preparation ", " hydrobiology "; 2004 the 512nd phases, the 141-144 page or leaf).Scholars such as Liu Yu (see paper " chromosomal DAPI dyeing of sea-tangle and caryogram initial analysis "; Publication is in " aquatic product journal " 2012 the 01st phases; The 50-54 page or leaf) utilizes the method for dripping sheet method and DAPI fluorescent dyeing; Observe the kelp gametophyte cell and have 31 karyomit(e)s, karyomit(e) disperses better, and the number form can be distinguished.But make fluorochromine can only pass through fluorescent microscope ability observations, fluorescent microscope market sale price is higher, can not popularize use.
Summary of the invention
The present invention provides a kind of karyomit(e) scattered, and form can be distinguished and the denumerable chromosomal preparation method of improved sea-tangle.
Technical solution of the present invention is:
The chromosomal preparation method of a kind of improved sea-tangle the steps include:
(1) kelp gametophyte pre-treatment: the centrifugal back of kelp gametophyte is absorbed excessive moisture and is kept the kelp gametophyte deposition, and adds the colchicine solution vibration and shake up and leave standstill;
(2) kelp gametophyte is hypotonic: kelp gametophyte is hypotonic with Klorvess Liquid;
(3) kelp gametophyte is fixed: the kelp gametophyte after hypotonic is fixed with the Kano stationary liquid;
(4) enzymolysis removes wall: carry out enzymolysis with the mixed enzyme solution of cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution and remove wall.
(5) cold sheet: enzymolysis goes to the centrifugal back of the kelp gametophyte behind the wall to absorb supernatant with liquid-transfering gun; Add glacial acetic acid; Vibration is mixed into cell suspension; Draw cell suspension at a sheet above the slide glass of precooling with dropper, the slide glass bottom surface is burnt once the back through spirit lamp flame and is dried naturally or dry;
(6) Ji's nurse Sa dyeing: dye to adopt Ji's nurse Sa staining fluid attached to the kelp gametophyte karyomit(e) on the slide glass.
(7) microscopically is observed and is taken pictures.
The colchicine solution of above-mentioned steps (1) is that weightmeasurement ratio is 0.2% colchicine solution, under 4 ℃, leaves standstill 7～8 hours behind the colchicine solution of kelp gametophyte adding 0.2%.
The concentration of the Klorvess Liquid of above-mentioned steps (2) is 0.075mol/L, and kelp gametophyte deposition adds to vibrate behind the Klorvess Liquid that concentration is 0.075mol/L and shakes up, and at room temperature leaves standstill 30～40 minutes.
The Kano stationary liquid of above-mentioned steps (3) is by the volume ratio configuration by 3: 1 of methyl alcohol and glacial acetic acid, 4 ℃ of Kano stationary liquids fixing three times down, each 20 minutes, 4 ℃ of Kano stationary liquids that renews again at last fixing 24 hours down.
In the above-mentioned steps (4), cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution mix by 2: 1: 1 volume ratio becomes mixed enzyme solution after shaking up, and joins existing usefulness, 4 ℃ of held at present.
In the above-mentioned steps (5), drip sheet from the height of 30～40 centimetres of the slide glasss of precooling.
Technique effect of the present invention is: the present invention adopts the method for cold sheet and Giemsa staining, has obtained karyomit(e) and has disperseed better, and form can be distinguished and denumerable karyomit(e) division phase.Ji's nurse Sa dyeing simultaneously is to whole chromosome dyeing, the olistherozone territory can not occur, and uses simple microscope to get final product observations.The inventive method adopts cold sheet and the painted method of Ji's nurse Sa, has obtained karyomit(e) and has disperseed better, and form can be distinguished and denumerable karyomit(e) division phase.In addition, the inventive method is easy fast, and repeatable strong, common laboratory all can be operated.
Description of drawings
1, Fig. 1 is No. 2 hybridization in the east kelp gametophyte karyomit(e) photo of the inventive method preparation.
2, Fig. 2 utilizes the kelp gametophyte karyomit(e) photo of pressed disc method and brazilwood extract dyeing for Chinese scholar Zhou Liran.
3, Fig. 3 is Ji's nurse Sa dyeing slide glass bridging vertical view.
4, Fig. 4 is Ji's nurse Sa dyeing slide glass bridging side-view.
Among the figure, 1, sheet glass, 2, have chromosome specimen slide glass, 3, slide glass, 4, have chromosome specimen slide glass and the hole between the sheet glass.
Specific embodiments
Specify below in conjunction with accompanying drawing and embodiment:
One, the present embodiment kelp gametophyte is taken from " No. 2, east " hybridization kelp gametophyte that preserve in country of Shandong Oriental Ocean Sci-tech Co., Ltd., marine alga Engineering Technical Research Centre quality saving storehouse.
Kelp gametophyte also can be according to following method collection: in the sea-tangle mature period; Select healthy and strong sporophyte; Taken off young sporangial main laminaria piece; Clean multipass, removing dirt settling and assorted algae with sterilization seawater (living seawater boils and promptly obtained the seawater of sterilizing in 2 minutes) and sterilization cotton (degreasing cotton is gone into pressure steam sterilizer 120 ℃ of temperature after wrapping up with kraft paper, and pressure 0.165MPa sterilization promptly obtained the cotton of sterilizing in 15 minutes); After the main laminaria piece dries in the shade; Place the sterilization seawater nutrient solution that has added SODIUMNITRATE and potassium primary phosphate, kelp spore is concentrated diffused 1～3 hour, then kelp spore water is poured in the staining jar that is placed with slide glass; Make kelp spore attached to forming the sea-tangle sporoblast on the slide glass, (cultivate about a week under 1000～1500Lx) conditions and promptly form gametophyte female, that hero can be distinguished in suitable temperature (10 ± 2 ℃) and illumination.At this moment; Adopt the microcapillary partition method that one female, microgametophyte is taken out respectively at microscopically; Female, microgametophyte places different sterilization seawater nutrient solutions to carry out single culture respectively; Female, microgametophyte continues under the 1500Lx illumination condition, to nourish and grow in 24 hours in sterilization seawater nutrient solution, just can form clone, i.e. gametophyte clone.Get into kelp gametophyte then and preserve the stage; Kelp gametophyte is continuous light (1500Lx) in sterilization seawater nutrient solution, changes the seawater nutrient solution of once sterilizing in per 7 days, is about to former sterilization seawater nutrient solution and pours out; Keep kelp gametophyte, add the sterilization seawater nutrient solution of equivalent again.
Two, present embodiment adopts following laboratory apparatus:
A, illumination box: SPX-300B-G type, the rich Medical Equipment Plant of industry ltd that proves to be true after interrogation in Shanghai
B, desk centrifuge: TGL-16C type, Anting Scientific Instrument Factory, Shanghai
C, refrigerator: BCD-216TX type, Qingdao HaiEr Co., Ltd
D, microscope: XSP-C204 type, the rich Medical Equipment Plant of industry ltd that proves to be true after interrogation in Shanghai
E, digital camera: the IXUS 310HS of Canon
F, full temperature are cultivated shaking table: QYC-200 type, Shanghai new talent medicine equipment Manufacturing Co., Ltd
Three, present embodiment adopts following experiment reagent
1) configuration sodium nitrate solution: take by weighing 121 gram SODIUMNITRATE (molecular formula NaNO3, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure), after the dissolving of sterilization seawater, be settled to 500ml with the sterilization seawater.The sterilization seawater of the present embodiment seawater of all making a living boils 2 minutes, and naturally cools to the sterilization seawater of room temperature.
2) configuration potassium dihydrogen phosphate: take by weighing 17.5 gram potassium primary phosphates (molecular formula KH2PO4, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure), after the dissolving of sterilization seawater, be settled to 1000ml with the sterilization seawater.
3) configuration sterilization seawater nutrient solution: measure above-mentioned 1ml sodium nitrate solution and 1ml potassium dihydrogen phosphate respectively, be added to 4000ml, shake up subsequent use through boiling 2 minutes and naturally cooling in the sterilization seawater of room temperature.
4) configuration 0.2% colchicine solution: take by weighing 0.2 gram NST-757 powder and (go up sea blue season development in science and technology ltd; The import packing); With behind the 1ml anhydrous alcohol solution, be settled to 100ml with the sterilization seawater more earlier, preserve with brown bottle splendid attire with cover and in 4 ℃ of refrigerators.
5) configuration 0.075mol/L Klorvess Liquid: take by weighing Repone K (chemical formula KCl, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure) 0.5588 gram, after the dissolving of sterilization seawater, be settled to 100ml with the sterilization seawater.
6) configuration Kano stationary liquid: with anhydrous methanol and 3: 1 by volume proportional arrangement of glacial acetic acid, join existing usefulness, 4 ℃ of refrigerators are placed at present.
7) cellulase R-10 (Cellulase " Onozuka " R-10) solution of configuration 1% concentration: take by weighing 0.1 gram cellulase R-10 powder (Japanese Yakult company produce), the pH that is dissolved in 10ml in 7.0 the 0.067mol/L phosphate buffered saline buffer, shakes up subsequent use.
8) macerozyme R-10 (Macerozyme R-10) solution of configuration 1% concentration: take by weighing 0.05 gram macerozyme R-10 powder (production of Beijing Suo Laibao Science and Technology Ltd.), the pH that is dissolved in 5ml in 7.0 the 0.067mol/L phosphate buffered saline buffer, shakes up subsequent use.
9) configuration 1% polygalacturonase solution: take by weighing 0.05 gram polygalacturonase powder (go up sea blue season development in science and technology ltd produce), the pH that is dissolved in 5ml in 7.0 the 0.067mol/L phosphate buffered saline buffer, shakes up subsequent use.
10) configuration pH is 7.0 0.067mol/L phosphate buffered saline buffer: configuration A liquid: 0.067mol/L potassium dihydrogen phosphate; Take by weighing potassium primary phosphate (molecular formula KH2PO4; Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces; Analytical pure) 9.112 restrains, after dissolving down with 60 ℃ of 50～100ml zero(ppm) water, be settled to 1000ml with zero(ppm) water.Configuration B liquid: 0.067mol/L disodium hydrogen phosphate solution; Take by weighing disodium hydrogen phosphate (molecular formula Na2HPO412H2O; Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces; Analytical pure) 23.986 restrains, after dissolving down with 60 ℃ of 50～100ml zero(ppm) water, be settled to 1000ml with zero(ppm) water.The B liquid of measuring A liquid and the 61.2ml of 38.8ml mixes, and both can obtain pH and be the phosphate buffered saline buffer of 7.0 0.067mol/L.
11) configuration Ji's nurse Sa (Giemsa) staining fluid: Ji's nurse Sa liquid concentrator and 1: 9 by volume mixed of Ji's nurse Sa diluent is existing with join at present, preserve with brown bottle with cover.Ji's nurse Sa liquid concentrator and Ji's nurse Sa diluent are Beijing Suo Laibao Science and Technology Ltd. and produce.
12) methyl alcohol, absolute ethyl alcohol, glacial acetic acid: be Shanghai Chemical Reagent Co., Ltd., Sinopharm Group and produce analytical pure.
Four, present embodiment kelp gametophyte method of chromosome preparation
(1) NST-757 pre-treatment
It is evenly big or small that the good kelp gametophyte of cultivation conditions means that microscopically is observed the kelp gametophyte cell; The kelp gametophyte that pigment is evenly distributed (female, microgametophyte all can); It is the 1.5ml plastic centrifuge tube that the good kelp gametophyte of cultivation conditions of getting volume and have big grain of rice size is put into volume; Rotating speed 1000rpm (rev/min), centrifugal 5min (minute) back absorbs excessive moisture with liquid-transfering gun and keeps the kelp gametophyte deposition, adds above-mentioned 0.2% colchicine solution 1ml; Vibration shakes up, and in 4 ℃ of refrigerators, leaves standstill 7～8 hours.
Here adopt NST-757 pre-treatment purpose to be: 1. to stop or destroy the formation of Spindle microtubule in the plant mitotic division process; Owing to there is not the effect of Spindle; The cell mitogen process was prevented in the metaphase in cell division stage, thus can cumulative comparison many chromosome images that is in metaphase in cell division; 2. promote karyomit(e) to concentrate and shorten that winding is overlapping each other between the minimizing karyomit(e), favourable chromosomal high dispersing; 3. change kytoplasm viscosity, promote chromosome clear, promote the tenuigenin cleaning.
(2) kelp gametophyte is hypotonic
At rotating speed 1000rpm, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, keep the gametophyte deposition, adding concentration is that Repone K (KCl) the solution 1ml of 0.075mol/L is hypotonic, vibration shakes up, and leaves standstill under the room temperature 30 minutes.
Hypotonic main effect has two: 1. promote cell plasmolysis, help enzymolysis process and remove cell walls; 2. promote the tenuigenin viscosity-modifying, it is clear to help chromosome image.
(3) kelp gametophyte is fixed
At rotating speed 1000rpm, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, keep the kelp gametophyte deposition.The Kano stationary liquid 1ml that adds 4 ℃ of placements of new configuration inhales dozen (suck also and release) solution 10 times gently with liquid-transfering gun, in 4 ℃ of refrigerators, leaves standstill 20min, at rotating speed 1000rpm, absorbs supernatant with liquid-transfering gun behind the centrifugal 5min, keeps the gametophyte deposition.Add the Kano stationary liquid 1ml of 4 ℃ of placements of new configuration again, inhale dozen solution gently 10 times, in 4 ℃ of refrigerators, leave standstill 20min,, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, keep the gametophyte deposition at rotating speed 1000rpm with liquid-transfering gun.Add the Kano stationary liquid 1ml of 4 ℃ of placements of new configuration once more, inhale dozen solution gently 10 times, in 4 ℃ of refrigerators, leave standstill 20min,, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, keep the gametophyte deposition at rotating speed 1000rpm with liquid-transfering gun.Add 1ml Kano stationary liquid at last, in 4 ℃ of refrigerators, leave standstill more than 24 hours.
Fixed mainly acts on: 1. rapid cell killing keeps split image.2. make the sex change of karyomit(e) nucleoprotein, deposition, demonstrate karyomit(e) true form and structure.3. make tenuigenin protoplastis protein denaturation, deposition, it is clear to help the karyomit(e) background.
(4) enzymolysis removes wall
At rotating speed 1000rpm, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, keep the kelp gametophyte deposition.Adding distil water 1ml goes into centrifuge tube, inhales gently with liquid-transfering gun and beats 10 flushings of solution kelp gametophyte deposition, at rotating speed 1000rpm, absorbs the zero(ppm) water in the centrifuge tube with liquid-transfering gun again behind the centrifugal 5min, notes not siphoning away kelp gametophyte, keeps kelp gametophyte.Add new zero(ppm) water 1ml and go into centrifuge tube, inhale gently with liquid-transfering gun and beat 10 flushings of solution kelp gametophyte deposition,, absorb the zero(ppm) water in the centrifuge tube with liquid-transfering gun again behind the centrifugal 5min, keep kelp gametophyte at rotating speed 1000rpm.Above-mentioned circulation step repeats to wash the kelp gametophyte deposition and amounts to 3～5 times.At rotating speed 1000rpm; Absorb supernatant with liquid-transfering gun behind the centrifugal 5min; Keep the kelp gametophyte deposition, add the mixed enzyme solution of newly joining and (cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution are shaken up after by 2: 1: 1 volume ratio mixing, join existing usefulness at present; 4 ℃ of refrigerators are placed) 1ml, cultivate on the shaking tables in 200rpm enzymolysis 18 hours 37 ℃ of full temperature.At rotating speed 1000rpm; 5min is centrifugal, and clear enzyme solution is absorbed gently with liquid-transfering gun in the back; Get 1ml zero(ppm) water with liquid-transfering gun again and inhale gently and beat 10 flushings of solution gametophyte and precipitate,, absorb zero(ppm) water with liquid-transfering gun behind the centrifugal 5min again at rotating speed 1000rpm; Add new 1ml zero(ppm) water and inhale dozen solution gently 10 times, the gametophyte deposition is washed.
Enzymolysis goes the effect of wall: 1. biology dissociates and handle to substitute physics and chemistry processings of dissociating, and has reduced karyomit(e) distortion, distortion, demonstrates really karyomit(e) firmly.2. remove cell walls fiber, pectin and kytoplasm to the influence of karyomit(e) quantity, make the karyomit(e) dispersion space bigger.Images such as 3. favourable kinetochore satellite are complete clear.
(5) cold sheet
At rotating speed 1000rpm, absorb supernatant with liquid-transfering gun behind the centrifugal 5min, add 100～200 μ L (microlitre) glacial acetic acids, vibration is mixed into cell suspension.Pick and place the clean slide that in 4 ℃ of refrigerator zero(ppm) water, soaks, control control water on filter paper is drawn cell suspension with dropper and is dripped sheet at the height from 30～40 centimetres of the slide glasss of precooling; Every slide glass drips 2～3, immediately slide glass one end is lifted then, and blows gently; Cell is disperseed rapidly, and the slide glass one side of not dripping cell suspension is then crossed once on spirit lamp flame, karyomit(e) is disperseed and expansion; Do not have an end of chromosomal one side to mark at slide glass, dry under the room temperature.
The effect of cold sheet: 1. utilize the certain altitude difference to let cell on slide glass, break, karyomit(e) is discharged in cell.2. there is not the disruptive cell to drop on the slide glass of precooling through behind the spirit lamp flame chromosomal losing when utilizing temperature head that cell rupture, this cell rupture mode can be avoided dripping sheet.
(6) Ji's nurse Sa dyeing
Like Fig. 3 and shown in Figure 4, get a sheet glass and lie on the experiment table, other gets two clean new slide glasss according to long parallel the sequencing of one side, and distance is less than the length of a slide glass.Room temperature is dried the slide glass that chromosome specimen is arranged put on two new slide glasss, and have chromosomal one to face down, as putting up a bridge; There is the slide glass of chromosome specimen to set successively other, between the slide glass that two have a chromosome specimen space do not arranged, just form the hole that two ends communicate between the slide glass that chromosome specimen arranged and the sheet glass; Draw Ji's nurse Sa staining fluid with dropper; By an end of hole, slowly inject Ji's nurse Sa staining fluid, note firmly evenly not producing bubble; After Ji's nurse Sa staining fluid all is full of hole, leave standstill 30～40min under the room temperature.The tap of fetching boiling water washes down staining fluid with very slow current along hole one end, notices that flow velocity is too not fast, prevents that the slide glass of chromosome specimen is flushed away, and will have the slide glass of chromosome specimen to take out then, and room temperature is dried.
(7) microscopically is observed and is taken pictures
Eyepiece (WF16 */10 of amplifying 16 times are selected in this experiment for use; Promptly amplify 16 times, field number is 10 millimeters) constant, rotate objective changement; Under 10 times of object lens, find earlier the scattered and denumerable karyomit(e) division phase of karyomit(e); Then cedar oil is dropped on the karyomit(e), forward to again under 100 times of object lens and take pictures, take pictures under the promptly oily mirror.
Fig. 1 is the chromosomal photo of kelp gametophyte that utilizes the inventive method preparation, and visible by figure, it is scattered to dye, and length scale can distinguish that number is 31.
Fig. 2 is the kelp gametophyte karyomit(e) that Chinese scholar Zhou Liran in 2004 utilizes pressed disc method and brazilwood extract dyeing, and karyomit(e) is point-like, and the shape size can not be recognized.
Claims (6)
1. the chromosomal preparation method of improved sea-tangle the steps include:
(1) kelp gametophyte pre-treatment: the centrifugal back of kelp gametophyte is absorbed excessive moisture and is kept the kelp gametophyte deposition, and adds the colchicine solution vibration and shake up and leave standstill;
(2) kelp gametophyte is hypotonic: kelp gametophyte is hypotonic with Klorvess Liquid;
(3) kelp gametophyte is fixed: the kelp gametophyte after hypotonic is fixed with the Kano stationary liquid;
(4) enzymolysis removes wall: carry out enzymolysis with the mixed enzyme solution of cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution and remove wall;
(5) cold sheet: enzymolysis goes to the centrifugal back of the kelp gametophyte behind the wall to absorb supernatant with liquid-transfering gun; Add glacial acetic acid; Vibration is mixed into cell suspension; Draw cell suspension at a sheet above the slide glass of precooling with dropper, the slide glass bottom surface is burnt once the back through spirit lamp flame and is dried naturally or dry;
(6) Ji's nurse Sa dyeing: dye to adopt Ji's nurse Sa staining fluid attached to the kelp gametophyte karyomit(e) on the slide glass;
(7) microscopically is observed and is taken pictures.
2. according to the chromosomal preparation method of the said a kind of improved sea-tangle of claim 1; It is characterized in that: the colchicine solution of step (1) is that weightmeasurement ratio is 0.2% colchicine solution, under 4 ℃, leaves standstill 7～8 hours behind the colchicine solution of kelp gametophyte adding 0.2%.
3. according to the chromosomal preparation method of the said a kind of improved sea-tangle of claim 1; It is characterized in that: the concentration of the Klorvess Liquid of step (2) is 0.075mol/L; Kelp gametophyte deposition adds to vibrate behind the Klorvess Liquid that concentration is 0.075mol/L and shakes up, and at room temperature leaves standstill 30～40 minutes.
4. according to the chromosomal preparation method of the said a kind of improved sea-tangle of claim 1; It is characterized in that: the Kano stationary liquid of step (3) is by the volume ratio configuration by 3: 1 of methyl alcohol and glacial acetic acid; The Kano stationary liquid is fixed three times down for 4 ℃; Each 20 minutes, the Kano stationary liquid that renews was again at last fixed 24 hours down for 4 ℃.
5. according to the chromosomal preparation method of the said a kind of improved sea-tangle of claim 1; It is characterized in that: in step (4); Cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution mix by 2: 1: 1 volume ratio becomes mixed enzyme solution after shaking up, and joins existing usefulness, 4 ℃ of held at present.
6. according to the chromosomal preparation method of the said a kind of improved sea-tangle of claim 1, it is characterized in that: in step (5), drip sheet from the height of 30～40 centimetres of the slide glasss of precooling.
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| CN110749489A (en) * | 2019-11-21 | 2020-02-04 | 中南林业科技大学 | Method for rapidly detecting activity of nostoc sphaeroides seeds |
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| CN108007753A (en) * | 2018-01-22 | 2018-05-08 | 上海海洋大学 | A kind of method of chromosome preparation of Enteromorpha sporinite |
| CN110055209A (en) * | 2019-03-13 | 2019-07-26 | 海南省海洋与渔业科学院（海南省海洋开发规划设计研究院） | A kind of preparation method of Old Taylor algae protoplast |
| CN110055209B (en) * | 2019-03-13 | 2020-04-21 | 海南省海洋与渔业科学院 | Preparation method of Talaromyces protoplast |
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