CN101768568A - Fabrication method of seagrass seedling protoplast - Google Patents
Fabrication method of seagrass seedling protoplast Download PDFInfo
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Abstract
The invention discloses a fabrication method of seagrass seedling protoplast, comprising the following steps: temporarily raise the seagrass seedlings in double-anti sea water, select the seedlings that just sprout and sequentially put them into alcohol with the concentration of 75 percent, sodium hypochlorite solution with the concentration of 1 percent and potassium iodide solution with the concentration of 1.5 percent for disinfection, then wash, soak and restore the seedlings by sterile seawater. Then, the seagrass seedlings are shredded and placed into centrifugal tube with enzyme solution for enzymolysis. After the enzymolysis, glucose liquid is added before filtration. Wash the seedlings thrice with hypertonic sea water and obtain the precipitate after centrifugation. Add medium to the precipitate and obtain the protoplast through centrifugation. The invention can avoid pollution and obtain sterile seagrass seedling protoplast and has laid a solid foundation for the protoplast fusion, genetic transformation, plant regeneration as well as new breeds breeding. Moreover, the invention can also provide technical reserves for seagrass propagation of cell engineering, rapid propagation of fine lines seagrass and improving seagrass genetic engineering.
Description
Technical field
The present invention relates to the preparation method of protoplastis, relate in particular to the preparation method of aseptic sea tangle protoplastis.
Background technology
Protoplastis is that vegetable cell is removed the spherule cell that exposes behind the cell walls, and it has totipotency, can carry out a large amount of breedings fast under the manual control condition, also can induce its fusion, forms hybrid cell, for somatic hybridization provides experiment material.Isolate protoplastis from the marine alga body methods such as mechanical process, microbial method, enzyme process are arranged.General enzyme process is a method comparatively desirable and commonly used.The damage that the protoplastis that mechanical process obtains is subjected to is bigger, is unfavorable for the carrying out of subsequent experimental.Microbial method causes living contaminants easily, brings pollution for follow-up callus formation and one-tenth seedling.Sea tangle (Zostera marina L.) is commonly called as the sea-tangle grass, belong to Angiospermae (Angiospermae), Monocotyledonae (Monocotyledoneae, or title Liliopsida Liliopsida), Alismatales (Alismales), Potamogetonaceae (Potamogetonaceae), sea tangle belongs to (Zostera L.), sea tangle is a per nnial herb, and root stock is crawled, and gives birth to fibrous root on the joint.Stem is dredged branch estranged, and is flat, light green.The leaf alternate, green, long strip shape reaches more than 50 centimetres, and is wide about 3～5 millimeters, the top circle, full edge has 5～7 arteries and veins, and stipule is membranous.Spadix is wrapped in the spathe at the beginning, and long 4～6 centimetres, rachis is flat, and the edge does not have the bracteal appurtenant; Spend little, unisexuality, female, male flower is lined up 2 row, no perianth mutually along rachis one top-cross; Ovary ovum shape oblong; Column cap 2, setiform.Seed square circle is about 4 millimeters, and longitudinal grin is arranged.Sea tangle is distributed in maritime provinces such as the Shandong, Hebei, Liaoning of China; And be distributed in countries and regions such as Korea, Japan, Europe, North America.Aseptic sea tangle seedling protoplast can merge for protoplastis, genetic transformation, and plant regeneration, rearing new variety lays the first stone.Though the report that obtains aseptic marine alga protoplastis by routine disinfection method and enzymatic hydrolysis condition is arranged at present, introduced the multiple routine disinfection method of aseptic marine alga protoplastis as the paper high-level efficiency of callus " band induce " (publication is the 652nd～657 page of " Oceanologia et Limnologia Sinica " 1999 the 06th phase), wherein a kind of routine disinfection method is that the tissue block that will cut was soaked 10 minutes in the liquor kalii iodide of 1.5% concentration, with the cotton ball soaked in alcohol sassafras of 75% concentration 3 times, use the high pressure steam sterilization seawater flushing again 4 times, put into the MS solid and increase rich substratum and cultivate.Paper " tissue culture of kelp and application thereof " (publication is the 605th～611 page of " Plant Physiology Communications " 2007 the 03rd phase), paper " carrageen (Chondrus ocellatusHolm) protoplastis separates; cultivate and Study on Regeneration " (publication is the 52nd～61 page of " Chinese Marine University's journal (natural science edition) " the 01st phase in 1991), paper " preliminary study of the unicellular solid culture of yezoensis laver " (publication is the 52nd～61 page of " ocean circular " the 05th phase in 2004) has been introduced the conventional enzymatic hydrolysis condition of aseptic marine alga protoplastis: employing cellulase and sea snail enzymes combine enzymolysis time 2～3 hours.The enzymolysis time here is unsuitable long, otherwise protoplasma is known from experience a large amount of fragmentations, perhaps loses activity, so the investigator generally will abandon follow-up experiment at enzymolysis after 12 hours.But use above-mentioned routine disinfection method and enzymatic hydrolysis condition when handling sea tangle, all can not obtain the protoplastis of sea tangle.Think that this kind of plant of sea tangle does not belong to marine alga, belong to a kind of angiosperm that lives in the seawater,, yet there are no relevant report relevant for the sea tangle method for preparing protoplast just because of these physilogical characteristics of sea tangle.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of aseptic sea tangle seedling protoplast.
The technical scheme that the present invention solves the problems of the technologies described above is:
A kind of preparation method of aseptic sea tangle seedling protoplast the steps include:
(1) the sea tangle seedling being put into two anti-seawater supported 2 days temporarily;
(2) choose the sea tangle seedling of firm sprouting;
(3) the sea tangle seedling after will supporting temporarily is immersed in the alcohol 10 seconds of 75% concentration;
(4) the sea tangle seedling is immersed in the chlorine bleach liquor 5 minutes of 1% concentration;
(5) the sea tangle seedling is immersed in the liquor kalii iodide 10 minutes of 1.5% concentration;
(6) with the sea tangle seedling with aseptic seawater flushing 3 times, use aseptic sea water immersion 10 minutes again, soaked 20 minutes with aseptic restoring sea water then;
(7) pulling the sea tangle seedling out back from aseptic seawater blots with aseptic cotton, then the sea tangle seedling is put into centrifuge tube and adds enzyme liquid, the enzyme liquid formula is seawater 1000 weight parts, cellulase 470 weight parts, polygalacturonase 5 weight parts, sorbyl alcohol 90 weight parts shred the sea tangle seedling in centrifuge tube with scissors again, and the sea tangle seedling that shreds carries out enzymolysis in enzyme liquid;
(8) after enzymolysis finishes, in enzyme liquid, add the equal-volume liquid of glucose, leave standstill, get supernatant liquor, filter then;
(9) the configuration height oozes seawater, add the equal-volume height in the supernatant liquor after filtration and ooze seawater, ooze seawater with height and wash 3 times: in supernatant liquor, add isopyknic high ooze seawater and shake up after, rotation is 5 minutes under whizzer 2000r/min rotating speed, abandon supernatant liquor after centrifugal, obtain throw out; In throw out, add once more isopyknic high ooze seawater and shake up after, rotation is 5 minutes under whizzer 2000r/min rotating speed, abandons supernatant liquor after centrifugal, obtains throw out; In throw out, add once more isopyknic high ooze seawater and shake up after, rotation minute under whizzer 2000r/min rotating speed is abandoned supernatant liquor after centrifugal, obtains throw out;
Add nutrient solution in the throw out of (10) the 3rd centrifugal acquisitions, it is centrifugal under the rotating speed of 2000r/min with whizzer to shake up the back, can collect the sea tangle seedling protoplast.
The enzymolysis optimum condition of above-mentioned steps (7) is that enzyme liquid pH value is 4, and temperature keeps 15 ℃, 0.6 gram cellulase concentration, enzymolysis time 48 hours.
The Glucose Liquid bulk concentration of above-mentioned steps (8) is 2 moles/cubic centimetre, and described filtering screen distance is 300 orders.
Useful technique effect of the present invention: because this kind of plant of sea tangle does not belong to marine alga, be that the angiosperm of planting and sprouting is bloomed, tied to a kind of finishing in seawater, just because of these physilogical characteristics of sea tangle, so sea tangle on composition and the physiological property between marine alga and angiosperm, more approach angiosperm.Sea tangle seedling of the present invention is disinfected the back through alcohol, chlorine bleach liquor, liquor kalii iodide and obtains aseptic seedlings; Process shreds, the enzymolysis aftertreatment obtains the sea tangle seedling protoplast.Because the fibre content of sea tangle cell walls is higher than other phycophyta, so the present invention's enzyme solution of having selected cellulase and polygalacturonase to combine, and improve the content of cellulase in enzyme liquid.Do not influencing under the active prerequisite of sea tangle seedling protoplast, prolonging enzymolysis time, enzymolysis 48 hours was a breakthrough, Chang enzymolysis time so, general marine alga is known from experience dead, and sea tangle is really different, enzymolysis 48 hours, sea tangle seedling protoplast reach the quantity peak and have activity.The inventive method can overcome pollution, obtains aseptic sea tangle seedling protoplast, for protoplastis merges, and genetic transformation, plant regeneration, work such as rearing new variety lay the first stone.In addition, the inventive method also can be for setting up sea tangle cell engineering breeding technology, and the good sea tangle strain of fast breeding improves the sea tangle genetic engineering tachnical storage is provided.
Description of drawings
1, Fig. 1 is the inventive method flow chart of steps.
2, the sea tangle seedling protoplast photo that obtains after 48 hours for enzymolysis processing of the present invention of Fig. 2.
3, Fig. 3 measures the photo of sea tangle seedling protoplast diameter after 48 hours for enzymolysis processing of the present invention.
4, Fig. 4 is the sea tangle seedling protoplast photo of enzymolysis processing of the present invention toluylene red and Methylene blue dyeing optics microscopically after 48 hours.
5, Fig. 5 is enzymolysis processing of the present invention sea tangle seedling protoplast photo under toluylene red and the Methylene blue dyeing optics phase microscope after 48 hours.
6, Fig. 6 is the broken line graph of cellulase concentration and sea tangle seedling protoplast burst size relation.
7, Fig. 7 is the broken line graph of enzymolysis time and sea tangle seedling protoplast burst size relation.
8, Fig. 8 is the broken line graph of pH value and sea tangle seedling protoplast burst size relation.
9, Fig. 9 is that temperature and sea tangle seedling protoplast burst size concern broken line graph.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
As shown in Figure 1, a kind of preparation method of aseptic sea tangle seedling protoplast, its concrete steps are:
(1) the sea tangle seedling is put into two anti-seawater and support temporarily, in the two anti-seawater of 4 degrees centigrade of temperature maintenances, supported 2 days temporarily.The sea tangle seedling is spontaneous growth both, also propagate artificially or cultivate, sea tangle seedling culture method see this claimant application for a patent for invention " a kind of quick germination method of grass wrack seeds ", application number is 200910223107.1.
The two anti-seawater prescriptions of present embodiment are: penicillin concn is every milliliter 100 unit (100units/ml), Streptomycin sulphate concentration is every milliliter 100 unit (100units/ml), promptly 1 liter of seawater needs the penicillin 0.06 of 0.48g (800,000 unit) to restrain, and Streptomycin sulphate 0.1 gram that needs 1,000,000 units, mix and get final product by method such as stirring or shake up.
(2) choose one month sea tangle seedling of seed germination, it is 0.025 gram sea tangle seedling that present embodiment is got fresh weight, and at this moment the sea tangle seedling grows two cotyledons, gets complete sea tangle seedling, and removes sea tangle seedling endosperm part.The unsuitable growth time of the sea tangle seedling of choosing is long, growth time is long, and sea tangle seedling cell fibrosis composition is long, needs the time of enzymolysis long more, injury to the sea tangle protoplastis can strengthen like this, is unfavorable for that later stage sea tangle protoplastis becomes the carrying out of seedling follow-up work.Growth time is too short, and the sea tangle seedling is less, does not grow cotyledon, and the protoplastis kind of acquisition is incomplete, and it is too light to lack leaf sea tangle seedling weight partly, can not get the sea tangle seedling protoplast of ideal quantity.
(3) put into the alcohol of 75% concentration in the sterilisable chamber Bechtop, the sea tangle seedling after will supporting temporarily then is immersed in the alcohol 10 seconds of 75% concentration.
Bechtop will carry out the UV-lamp irradiation before use, and opens aseptic wind, and irradiation time is 30 minutes, and the aseptic processing room also will shine 30 minutes with UV-lamp simultaneously, closed UV-lamp after 15 minutes, and personnel enter sterilisable chamber and carry out concrete operations.
(4) in the sterilisable chamber Bechtop, put into clorox (NaCIO) solution of 1% concentration, will soak the chlorine bleach liquor 5 minutes that sea tangle seedling behind the alcohol is immersed in 1% concentration then.
(5) in the sterilisable chamber Bechtop, put into potassiumiodide (KI) solution of 1.5% concentration, will soak potassiumiodide (KI) solution 10 minutes that sea tangle seedling behind the chlorine bleach liquor is immersed in 1.5% concentration then.
(6) will soak sea tangle seedling behind the liquor kalii iodide with aseptic seawater flushing 3 times, and be about to the sea tangle seedling and put into the culture dish that aseptic seawater is housed, clamp seedling, and in water, rinse and wash, and rinse successively and wash 3 times with tweezers.Use aseptic sea water immersion 10 minutes again, purpose is to remove residual potassiumiodide, alcohol, clorox and give birth to the residual of seawater; Soaked 20 minutes with aseptic restoring sea water then, purpose is that the cell of sea tangle seedling can be recovered from the environment that stimulates, and makes cell be in a kind of good state, for next step enzymolysis is prepared.Aseptic sea water immersion will carry out in different culture dish with recovery sea tangle seedling.
The preparation method of aseptic seawater is: get living seawater (this life seawater is the seawater that boiled of cool to room temperature), in Bechtop, through 121 ℃, 1030 pascal (N/m
2) stainless steel filter behind the autoclaving carries out suction filtration, this stainless steel filter carries out suction filtration with the supporting use of no oily diaphragm vacuum pump, and suction filtration filter membrane aperture is 0.22 micron, obtains aseptic seawater by suction filtration.
(7) pulling the sea tangle seedling out back from aseptic seawater blots with aseptic cotton, then the sea tangle seedling is put into centrifuge tube (EP pipe) and add 1.25 milliliters of enzyme liquid, use scissors (present embodiment adopts long 10 centimetres medical operation scissors) again, centrifuge tube inclination miter angle, from 5～10 centimetres of spirit lamps, in centrifuge tube the sea tangle seedling is shredded, the time that generally shreds cut sea tangle seedling tissue block and is advisable for 1 cubic millimeter between 15～20 minute.The centrifuge tube that the sea tangle seedling that shreds and enzyme liquid will be housed then placed under 15 ℃ of temperature enzymolysis 48 hours.
Aseptic cotton is cut into the square of 5 * 5 centimetres of sizes.In the present embodiment, the aseptic utensil of aseptic cotton and needs all will be with high-pressure sterilizing pot at 121 ℃, 1030 pascal (N/m
2) down sterilization 25 minutes of high pressure.
The enzyme liquid formula is seawater 1000 weight parts, Mierocrystalline cellulose 470 weight parts, and polygalacturonase 5 weight parts, sorbyl alcohol 90 weight parts, present embodiment is an example to dispose 1.25 milliliters of enzyme liquid, (promptly 1.275 restrain seawater, the density of seawater is by 1.02 gram per centimeters to need 1.25 milliliters of seawater
3Calculate), cellulase 0.6 gram, polygalacturonase 0.0062 gram, sorbyl alcohol 0.115 gram is got 1.25 milliliters of seawater with liquid-transfering gun and is joined in the test tube, adds cellulase 0.6 gram then respectively, polygalacturonase 0.0062 gram, sorbyl alcohol 0.115 gram.After shaking up, with whizzer under the 500r/min rotating speed centrifugal 10 minutes, go precipitation, will go sedimentary supernatant liquor to pour in the new centrifuge tube, this supernatant liquor be enzyme liquid.Be that the hydrochloric acid of 1 mol (being 1N HCL) is 4 with enzyme liquid furnishing pH value with concentration then, the enzyme liquid for preparing at the clean worktable suction filtration of operation, is made aseptic enzyme liquid by the syringe needle filter, and syringe needle filter filter membrane aperture is 0.22 micron.Enzyme liquid storage temperature does not surpass 20 ℃, avoids enzyme liquid inactivation.Present embodiment adopts the enzyme liquid for preparing is put into 4 ℃ of refrigerator cold-storages preservations temporarily, takes out from refrigerator during use, returns to normal temperature and use in sterilisable chamber, and enzyme liquid was preferably joined the same day same day and used.
(8) after enzymolysis finishes, add in enzyme liquid and the same volume of enzyme liquid phase that contains the sea tangle seedling tissue that shreds, concentration is 2 moles of/cubic centimetre (2mol/dm
3) liquid of glucose, make the sea tangle seedling protoplast swim in fluid surface, build the lid of centrifuge tube behind the adding liquid of glucose, slowly shake up then, the time of shaking up is 1 minute, establish after shaking up and leave standstill 20 minutes, wait one free sea tangle seedling protoplast cell to swim in the liquid upper strata, big tissue block sinks to the bottom.Get 1.5 milliliters of supernatant liquors by liquid-transfering gun, some muddiness of supernatant liquor at this moment, with 300 purpose nylon material filtering net filtering supernatant to remove sediment miscellaneous object, tissue block, cell debris that this sediment miscellaneous object main component is a not enzymolysis.Supernatant liquor main component after the filtration is the sea tangle seedling protoplast.
(9) the configuration height oozes seawater, add with supernatant liquor equal-volume height in the supernatant liquor after filtration and ooze seawater, oozing seawater with height washes 3 times: in supernatant liquor, add isopyknic high ooze seawater and shake up after, rotated 5 minutes down at whizzer 2000r/min rotating speed (being that per minute 2000 changes), abandon supernatant liquor after centrifugal, obtain throw out; After the height that adds same volume in throw out once more oozed seawater and shakes up, rotation was 5 minutes under whizzer 2000r/min rotating speed, abandoned supernatant liquor after centrifugal, obtained throw out; After the height that adds same volume in throw out once more oozed seawater and shakes up, rotation was 5 minutes under whizzer 2000r/min rotating speed, abandoned supernatant liquor after centrifugal, obtained throw out.At this moment sedimentary main component is exactly the sea tangle seedling protoplast.Increasing the purpose of oozing seawater is impurity such as the unnecessary enzyme liquid of flush away, glucose, and it is a kind of protection to protoplastis that the while height oozes seawater, makes protoplastis be in a kind of dewatering state, can reduce centrifugal physical abuse to protoplastis like this.The MS liquid medium suspension protoplastis that adds 1 milliliter in the last throw out that obtains utilizes blood counting chamber to carry out blood counting, and count results is 19.5 * 10
5Individual/milliliter, each visual field sea tangle protoplastis number is about 40 under 400 * mirror, and single sea tangle seedling protoplast diameter is 12～15 microns, as shown in Figure 3.By Methylene blue and toluylene red detection of active, sea tangle seedling protoplast survival rate is 80～90%, as shown in Figure 4 and Figure 5.
The present embodiment height oozes the seawater configuration proportion: 100 milliliters in sterilization seawater, sodium-chlor (Nacl) 1.5 grams, calcium chloride (CaCl
2) 0.11 gram, sodium-chlor, calcium chloride carry out suction filtration by the stainless steel filter at Bechtop after all being dissolved in the sterilization seawater, and stainless steel filter filter membrane aperture is 0.22 micron.The sterilization seawater here is the seawater that boiled, and sterilization seawater boiling time is 10 minutes.
MS liquid medium configuration proportion is: 100 milliliters of seawater need 0.48g (800,000 unit) penicillin 0.006 gram, Streptomycin sulphate 0.01 gram of 1,000,000 units, MS substratum 3.474 grams, concentration be 2 mg/litre 2,4 dichlorophenoxyacetic acid (2,4-D) 0.25 milliliter.Measure 100 milliliters of seawater with graduated cylinder, add MS substratum, penicillin, Streptomycin sulphate, 2,4 dichlorophenoxyacetic acid successively, shake all or stir and to obtain the MS liquid medium.
(10) the MS liquid medium that will dissolve sea tangle seedling protoplast thing uses whizzer under the rotating speed of 2000r/min, can collect the sea tangle seedling protoplast in centrifugal 5 minutes, the throw out of the sea tangle seedling protoplast of this moment for turning white slightly.
Present embodiment weight claims by electronic balance.
Fig. 2 is that the present invention is at PH4,15 ℃, 0.6 photos that restrain processing 400 * mirror downward view after 48 hours under the cellulase concentration condition, oval ball among Fig. 2 is the sea tangle seedling protoplast, approximate transparent irregular body is the remains that the protoplastis water inlet after of dying stays, i.e. cell debris.The individual number of sea tangle seedling protoplast is about 40 under each visual field, sea tangle seedling protoplast individual amount of the present invention is to count by blood counting chamber, done the gradient experiment on cellulase concentration, enzymolysis time, pH value, temperature respectively, the experimental result broken line graph of cellulase concentration, enzymolysis time, pH value, temperature and sea tangle seedling protoplast burst size relation is seen Fig. 6, Fig. 7, Fig. 8, Fig. 9 respectively.
Fig. 3 is enzymolysis processing of the present invention is measured sea tangle seedling protoplast diameter after 48 hours a photo.Sea tangle seedling protoplast diameter of the present invention is to utilize software to measure by the computer of being inverted allocating and measuring cell size on the shooting microscope.Blue line among Fig. 3 is the line segment that computer is measured the protoplastis diameter, and the numeral that shows the protoplastis diameter is arranged above the blue line, and there is red scale in Fig. 3 lower right corner, and red scale length is 100 microns.By computer measurement, single sea tangle seedling protoplast diameter is 12～15 microns.
Fig. 4 is the sea tangle seedling protoplast under PH4,15 ℃, 0.6 gram cellulase concentration condition, enzymolysis after 48 hours by toluylene red and Methylene blue dyed blended after, the photo of taking pictures under the simple microscope visual field, peach material is activated sea tangle seedling protoplast in Fig. 4 photo, navy blue material is not for there being active sea tangle seedling protoplast, and other thing is an impurity.
Fig. 5 is the sea tangle seedling protoplast under PH4,15 ℃, 0.6 gram cellulase concentration condition, enzymolysis after 48 hours by toluylene red and Methylene blue dyed blended after, the photo of under phase microscope, taking pictures.Atrament is not for there being active sea tangle seedling protoplast in Fig. 4 photo, pink material is activated sea tangle seedling protoplast, the rectangular thing of white shell and little grey stain material are the remains after impurity decomposes with the protoplastis of dying, i.e. cell debris.
The present invention by the dyeing of toluylene red and Methylene blue after, take at random and get 100 pictures, according to formula: survival rate=viable count/total cell count is calculated sea tangle seedling protoplast survival rate mean value.By toluylene red and Methylene blue dyed blended after, activated protoplastis understain is a pink at microscopically; The primary sound plastid engrain of inactivation is a mazarine at microscopically.Detect by activity, sea tangle seedling protoplast survival rate is 80～90%.
PH4 of the present invention represents the pH-value of enzyme liquid, and promptly pH value is 4; Described 15 ℃ of temperature that expression enzyme liquid keeps; 0.6 the gram cellulase concentration represents that 0.6 gram cellulase is dissolved in the concentration of cellulase in 1.25 milliliters of seawater, 1.0 the gram cellulase concentration represents that 1.0 gram cellulases are dissolved in the concentration of cellulase in 1.25 milliliters of seawater, the implication of other cellulase concentration and the like.
Fig. 6 is the experimental result broken line graph of cellulase concentration and sea tangle seedling protoplast burst size relation.Can see that by Fig. 6 in 0.075 gram～1.0 gram cellulase concentration scopes, the quantity that obtains the sea tangle seedling protoplast along with the rising of cellulase concentration improves gradually.Sea tangle seedling protoplast quantity and cellulase concentration are proportionate.To 1.0 gram cellulase concentrations, obtain the sea tangle seedling protoplast of comparatively high amts at 0.6 gram.Because the seawater amount is a fixed, increase along with cellulase concentration, it is thick that enzyme liquid is tending towards gradually, and most of cellulase all not have dissolving under 1.0 gram cellulase concentrations, and the sea tangle seedling protoplast quantity that obtains and 0.6 restrains cellulase concentration and is more or less the same.So it is best enzymolysis concentration that the present invention selects the cellulase concentration of 0.6 gram.In addition, along with the rising of cellulase concentration, contained in the enzyme liquid have toxic material to protoplastis and increase, and causes protoplastis to break, and also is a factor that influences sea tangle seedling protoplast quantity.
Fig. 7 is the experimental result broken line graph of enzymolysis time and sea tangle seedling protoplast burst size relation.As shown in Figure 7, in 0～80 hour time range, sea tangle seedling protoplast output begins to increase with the prolongation of enzymolysis time, and when reaching 48 hours, output reaches maximum value, is 3.9 * 10
6Individual/milliliter, subsequently, sea tangle seedling protoplast output begins to descend with the prolongation of enzymolysis time, and after enzymolysis surpassed 60 hours, protoplastis quantity tended to be steady.Therefore, enzymolysis 48 hours was best enzymolysis time.
Fig. 8 is the experimental result broken line graph of pH value and sea tangle seedling protoplast burst size relation.As can be seen from Figure 8, increase with the pH value, sea tangle seedling protoplast burst size also progressively increases, and is 3～4 o'clock in the pH value, is fit to the generation of sea tangle seedling protoplast.When the pH value was 4.0, sea tangle seedling protoplast output was the highest, was 1.3 * 10
6Individual/milliliter, when the pH value greater than 4, sea tangle seedling protoplast burst size reduces significantly.
Fig. 9 is the experimental result broken line graph of temperature and sea tangle seedling protoplast burst size relation.As can be seen from Figure 9, during 15～20 ℃ of hydrolysis temperatures, be fit to the generation of sea tangle seedling protoplast.In the time of 15 ℃, sea tangle seedling protoplast output is the highest, reaches 4.4 * 10
6Individual/milliliter, hydrolysis temperature is lower than or when being higher than 15 ℃, sea tangle seedling protoplast output all descends.As seen, 15 ℃ is the optimum temperuture of enzymolysis.Be below or above this temperature, sea tangle seedling protoplast output all effected.The present invention selects 15 ℃ for use, and this temperature is lower than the life temperature of sea tangle, thereby does not influence the regeneration of follow-up sea tangle seedling protoplast.
The used instrument of present embodiment all can be bought on market, and its source and specifications and models see the following form:
| The instrument title | Manufacturer | Model | Specification | |
| Be inverted the shooting microscope | Japanese Olympus Co., Ltd. (OLYMPUS) | ??IX71/ | Eyepiece | 10 * |
| Whizzer | Shanghai Surgical Operation Equipment Factory | 800 types | ??500r/min～??4000r/min |
| The instrument title | Manufacturer | Model | Specification |
| Bechtop | Purifying Equipment Co., Ltd., Suzhou | ??SW-CJ-ID | Single/single face |
| High-pressure sterilizing pot | The rich Medical Equipment Plant of industry company limited that proves to be true after interrogation in Shanghai | ??YXQ-LS-50S?11 | |
| Electronic balance | Sai Duolisi scientific instruments equipment company limited | ??BSA224S-CW | ??0.0001g～120g |
| Blood counting chamber | Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai | ??XB-K-25 | ??0.10mm?1/400mm 2 |
| The stainless steel filter | New Asia, Shanghai company limited | ??1L | |
| The syringe needle filter | Shanghai AsiaSat company limited | Diameter 25mm | |
| Liquid-transfering gun | Qingdao hundred lucky commerce and trade difficult to understand company limited | ??100-1000ml | |
| Culture dish | Yantai China news chemical article company limited | Diameter 9cm | |
| Graduated cylinder | Yantai China news chemical article company limited | Capacity 5-100ml |
The present embodiment agents useful for same all can be bought on market, and raw materials used source and specification see the following form:
| Reagent name | Produce (sale) producer | Specification |
| Sorbyl alcohol | Tianjin recovery fine chemistry industry institute | Analytical pure |
| Cellulase | Last ingression Ke bio tech ltd | ??5g |
| Polygalacturonase | Go up sea blue season development in science and technology company limited | ??5g |
| Sorbyl alcohol | Tianjin recovery fine chemistry industry institute | Analytical pure |
| The MS substratum | Qingdao GaoKeYuan hypo Bioisystech Co., Ltd | ??250g |
| Reagent name | Produce (sale) | Specification | |
| 2,4 dichlorophenoxyacetic acid | Go up sea blue season development in science and technology company limited | ??100g | |
| Alcohol | Rui Jin, Tianjin special chemical company limited | ??500ml | |
| Clorox | Tianjin extensively becomes chemical reagent company limited | ??500g | |
| Potassiumiodide | Jinan China middle areas in Shandong Province is herded chemical industry company limited | ??500g | |
| Glucose (solid) | Tianjin section close europeanized reagent development centre | ??500g | |
| Penicillin | Last ingression Ke bio tech ltd | ??10g | |
| Streptomycin sulphate | Last ingression Ke bio tech ltd | ??50g | |
| Sodium-chlor | Tianjin Da Mao chemical reagent factory | ??500g | |
| Calcium chloride | Tianjin Da Mao chemical reagent factory | ??500g |
Claims (3)
1. the preparation method of an aseptic sea tangle seedling protoplast is characterized in that, the steps include:
(1) the sea tangle seedling being put into two anti-seawater supported 2 days temporarily;
(2) choose the sea tangle seedling of firm sprouting;
(3) the sea tangle seedling after will supporting temporarily is immersed in the alcohol 10 seconds of 75% concentration;
(4) the sea tangle seedling is immersed in the chlorine bleach liquor 5 minutes of 1% concentration;
(5) the sea tangle seedling is immersed in the liquor kalii iodide 10 minutes of 1.5% concentration;
(6) with the sea tangle seedling with aseptic seawater flushing 3 times, use aseptic sea water immersion 10 minutes again, soaked 20 minutes with aseptic restoring sea water then;
(7) pulling the sea tangle seedling out back from aseptic seawater blots with aseptic cotton, then the sea tangle seedling is put into centrifuge tube and adds enzyme liquid, the enzyme liquid formula is seawater 1000 weight parts, cellulase 470 weight parts, polygalacturonase 5 weight parts, sorbyl alcohol 90 weight parts shred the sea tangle seedling in centrifuge tube with scissors again, and the sea tangle seedling that shreds carries out enzymolysis in enzyme liquid;
(8) after enzymolysis finishes, in enzyme liquid, add the equal-volume liquid of glucose, leave standstill, get supernatant liquor, filter then;
(9) the configuration height oozes seawater, add the equal-volume height in the supernatant liquor after filtration and ooze seawater, ooze seawater with height and wash 3 times: in supernatant liquor, add isopyknic high ooze seawater and shake up after, rotation is 5 minutes under whizzer 2000r/min rotating speed, abandon supernatant liquor after centrifugal, obtain throw out; In throw out, add once more isopyknic high ooze seawater and shake up after, rotation is 5 minutes under whizzer 2000r/min rotating speed, abandons supernatant liquor after centrifugal, obtains throw out; In throw out, add once more isopyknic high ooze seawater and shake up after, rotation is 5 minutes under whizzer 2000r/min rotating speed, abandons supernatant liquor after centrifugal, obtains throw out;
Add nutrient solution in the throw out of (10) the 3rd centrifugal acquisitions, it is centrifugal under the rotating speed of 2000r/min with whizzer to shake up the back, can collect the sea tangle seedling protoplast.
2. according to the preparation method of the described a kind of aseptic sea tangle seedling protoplast of claim 1, it is characterized in that: the enzymatic hydrolysis condition of step (7) is that enzyme liquid pH value is 4, and temperature keeps 15 ℃, 0.6 gram cellulase concentration, enzymolysis time 48 hours.
3. according to the preparation method of claim 1 or 2 described a kind of aseptic sea tangle seedling protoplasts, it is characterized in that: the described Glucose Liquid bulk concentration of step (8) is 2 moles/cubic centimetre, and described filtering screen distance is 300 orders.
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|---|---|---|---|---|
| CN101904304A (en) * | 2010-07-16 | 2010-12-08 | 中国海洋大学 | Cultivating method of zostera aseptic seedlings |
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- 2010-02-28 CN CN2010101325943A patent/CN101768568B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《Oecologia》 19821231 Dennison WC，et al. Photosynthetic responses of Zostera marina L.(eelgrass) to in situ manipulations of light intensity 137-144 1-3 , 2 * |
| 《植物学通报》 20021231 叶春江等 高等植物大叶藻研究进展及其对海洋沉水生活的适应 184-193 1-3 第19卷, 第2期 2 * |
| 《海洋湖沼通报》 20071231 于函，等 大叶藻的生态学特征及其与环境的关系 113-119 1-3 , 2 * |
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| CN103609422A (en) * | 2013-11-14 | 2014-03-05 | 青岛农业大学 | Zostera marina seed multiplication method |
| CN103609422B (en) * | 2013-11-14 | 2015-05-27 | 青岛农业大学 | Zostera marina seed multiplication method |
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| CN109880787A (en) * | 2019-03-13 | 2019-06-14 | 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 roundleaf silk powder algae protoplast |
| 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 |
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