CN105647905A - Method using protoplast asymmetric fusion technology to obtain hybrid grape plants - Google Patents
Method using protoplast asymmetric fusion technology to obtain hybrid grape plants Download PDFInfo
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Abstract
The invention provides a method using protoplast asymmetric fusion technology to obtain hybrid grape plants. The method includes: taking fresh embryonic callus, and adding enzyme liquid for enzymolysis; respectively performing IOA passivation and UV passivation on protoplast to obtain receptor protoplast and donor protoplast, mixing the receptor protoplast and the donor protoplast according to the ratio of 1:1, sequentially adding PEG fusion induction liquid and a high-Ca2+ high-pH solution to obtain fused hybrid cells; using a KM8P solid culture medium to culture the fused hybrid cells in an embedded manner; sequentially transferring the particles of the embryonic callus to a differential culture medium and a rooting culture medium for culturing; when tissue cultured seedlings grow to 5-7cm, planting into a seedling exercising room for seedling exercising so as to obtain somatic cell hybrid plants. The method has the advantages that the grape embryonic callus is used as the material, the protoplast asymmetric fusion technology is used to successfully obtain the hybrid grape plants, and a foundation is laid on the culture of new grape varieties, grape resistance enhancing and grape quality increasing.
Description
Technical field
The invention belongs to Fructus Vitis viniferae somatocyte hybriding technology field, be specifically related to a kind of method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant.
Background technology
In plant sexual hybridization is only limitted between the nearer cultigen of part sibship and wild species or plants, the syngenesis ability of some plant is very low even not to be possessed, so being difficult to improved by the mode of sexual hybridization and cultivate new varieties, seriously limit the progress of breeding work. The method of south China Fructus Vitis viniferae conventional breeding includes cross-breeding, introduces a fine variety, grow directly from seeds seed selection, mutagenic breeding and Bud mutation etc. Somatic hybridization of plant technology then can pass through the fusion of allos protoplast, the hereditary character that recombinant plant circle is excellent widely, overcome remote edge sexual hybridization incompatibility, shortening the breeding cycle, expand gene recombinaton scope, the prospect of being widely applied is opened for creating novel plant, also a brand-new approach has been started for plant breeding and creation breeding material, thus have started to be applied to the breed improvement of crops, vegetable, fruit tree, Chinese crude drug and other plant, and obtain the hybrid somatic cell between kind, in kind, between genus or cybrid. Utilize cell-fusion techniques breeding to be by an effective way of Grape Germplasm resource innovation, it is possible to shorten the breeding time limit of Fructus Vitis viniferae, improve breeding efficiency, create new kind and type. Research about Grape Protoplast fusion aspect is also reported seldom, the genotype that screening is suitable for is that can Grape Protoplast cultivate successful key factor, Grape Protoplast culture studies has carried out substantial amounts of genotype screening, and finally only have a small amount of Genotype and obtain regeneration plant, also in the stage of fumbling. The optimal condition that Grape Protoplast is cultivated, the optimal dose of protoplast passivation, what after the best PEG concentration of protoplast fusion, fusion, the relevant suitable condition etc. of the somatic cultivation of hybrid and plant regeneration was all studied is few, but without forming ripe technical system.
The Chinese invention patent that Authorization Notice No. is CN101139582A discloses a kind of method utilizing protoplast asymmetric fusion technology to obtain banana somatic cell hybrid. The method is Fructus Musae embryonal suspension cell to carry out enzymolysis in enzymolysis solution obtain protoplast, receptor protoplast iodoacetamide processes, Donor primordial plastid uviol lamp is irradiated processing, and after Donor primordial plastid and receptor protoplast being mixed, merges with polyethylene glycol method. Fusion product is suspended in protoplast fluid medium and carries out accompanied culture, it is thus achieved that cell mass transfer to somatic embryo inducement culture medium cultivated, until somatic embryo is sprouted, then carry out cultivating and namely obtain complete somatic cell hybrid plant. The Chinese invention patent that Authorization Notice No. is CN102505004A discloses the extraction of a kind of strawberry protoplast and the method for fusion. The method is with the blade of Fructus Fragariae Ananssae for research material, and the mode combined by pre-treatment and enzymolysis obtains the protoplast of high vigor, utilizes 30-45%PEG6000 in conjunction with high Ca2+, both fusions of method induction of high pH, improve the probability that protoplast merges one to one, the protoplast vigor of separation is more than 85%, and fusion rate can reach 11.7% one to one. The Chinese invention patent that Authorization Notice No. is CN104429939A discloses a kind of method utilizing diploid cell to cultivate triploid Herba Dendrobii with pollen cell Cell-fusion breeding method.
Fructus Vitis viniferae is slow compared with other fruit trees progress in Protoplast cuhnre and plant regeneration technique, and is putative be difficult to one of fruit variety carrying out plant regeneration and genetic transformation. There is few successful example in relevant Grape Protoplast separation purification, cultivation and fusion aspect.
Summary of the invention
The technical problem to be solved is the deficiency overcoming above prior art problem, it is provided that a kind of method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant. The method with Fructus Vitis viniferae embryo callus for material, to protoplast separate purification, the cultivation of protoplast, the fusion of protoplast, the selection of hybrid cell, hybrid cell cultivation etc. study. The present invention tentatively establishes cultivation and the integration technology system of Grape Protoplast, lays a good foundation for cultivation new grape variety, the resistance of enhancing Fructus Vitis viniferae, raising grape quality, has highly important theory and actual application value.
The technical solution adopted in the present invention is:
A kind of method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant, including following operating procedure:
(1) separation of protoplast and purification: take the enzyme-added liquid of fresh embryo callus, 26-28 DEG C, under dark condition, 60-70r/min vibrates enzymolysis 2-12h, is then peeled off purification and obtains Grape Protoplast;
(2) protoplast Passivation Treatment:
A () IOA is passivated: taking the protoplast use CPW solution suspension that step (1) obtains, adjusting protoplast density is 0.5-1 �� 105The suspension of individual/mL, 4.0-8.0mmol/L iodoacetamide (IOA) solution passivation being subsequently adding 15 proportionings (volume ratio) processes 9-11min, uses CPW solution washing 2-3 time, use KM after having processed8P fluid medium suspends, and protoplast density is adjusted to 0.5-1 �� 105Individual/mL, obtains receptor protoplast;
B () UV is passivated: taking the protoplast use CPW solution suspension that step (1) obtains, adjusting protoplast density is 0.5-1 �� 105The suspension of individual/mL, at 1250-1350 �� W/cm2Irradiate 0.5-16min under ultraviolet lighting intensity, obtain Donor primordial plastid;
(3) fusion of protoplast: the Donor primordial plastid that the receptor protoplast obtain step (3) (a) and step (3) (b) obtain mixes by 11 volume ratios, obtain parent's suspension, the PEG adding 1-4 times of parent's suspension vol merges induction liquid, at 25-30 DEG C, it is incubated 15-30min, adds the high Ca of 2-4 times of parent's suspension vol2+-high pH solution, is incubated 10-15min at 25-30 DEG C, then uses CPW solution washing 2-3 time after mixing, the centrifugal hybrid cell obtaining merging;
(4) somatic cultivation is merged: use KM8The blendling cell that P solid medium embedding culture step (3) obtains, at 25-27 DEG C, quiescent culture under dark condition, until growing regeneration embryo callus granule;
(5) regenerating the induction of embryo callus embryoid: be forwarded on division culture medium by regeneration embryo callus granule inducing embryoid body, until growing adventitious bud, then adventitious bud being transferred on root media cultivation;
(6) seedling exercising and transplanting: when tissue cultured seedling grows to 5-7cm and root growth is vigorous, is transplanted in seeding room by tissue cultured seedling and carries out seedling exercising, namely obtains somatic cell hybrid plant.
In described step (1), the formula of enzyme liquid is: 2.50% (g/100mL, lower with) cellulase, 0.25% pectase, 0.75% macerozyme, 0.40mol/L mannitol, 0.1%MES (2-(N-morpholino)-ethane sulfonic acid), pH5.8.
In described step (1), the amount of every enzyme-added liquid of 1g embryo callus is 10mL.
In described step (3), the formula of PEG fusion induction liquid is: with CPW solution for base fluid, containing PEG30-50g, CaCl in every 100mL2��2H2O150mg��KH2PO410mg, mannitol 3g.
High Ca in described step (3)2+-high pH solution is by glycine-NaOH buffer and Ca (NO3)2Solution even mixes. The formula of wherein said glycine-NaOH buffer is: glycine 3.75g/L, mannitol 15g/L, and uses 0.2mol/LNaOH solution to adjust pH to 9-10.5; Described Ca (NO3)2The concentration of solution is 94.4g/L.
In described step (5), the formula of division culture medium is the MS culture medium containing 6-benzyl aminoadenine 0.1mg/L, naphthalene acetic acid (NAA) 0.1mg/L, pH5.8.
In described step (5), the formula of root media is containing naphthalene acetic acid (NAA) 1.0mg/L, indolebutyric acid (IBA) 0.5mg/L-11/2MS culture medium, pH5.8.
The formula of described CPW solution is: potassium dihydrogen phosphate (KH2PO4) 27.2mg/L, potassium nitrate (KNO3) 101.0mg/L, CALCIUM CHLORIDE DIHYDRATE (CaCl2��2H2O) 1480.0mg/L, bitter salt (MgSO4��7H2O) 246.0mg/L, potassium iodide (KI) 0.16mg/L, copper sulfate pentahydrate (CuSO4��5H2O)0.025mg/L��
Protoplast is from separating purification, cultivation, embryoid induction and the extremely very long complexity of process to regeneration plant, and cultivation and the plant regeneration of protoplast are suffered from conclusive effect by each step in this process. Plant regeneration is all had a certain impact by suitable genotype, separate material, the concentration of homeo-osmosis agent, enzymolysis time, centrifugal rotational speed and time, cultivation temperature and the pH value etc. of protoplast, and the above influence factor affects also bigger in the incubation of protoplast.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
(1) with the protoplast of Fructus Vitis viniferae embryo callus separation for material, more higher than the material differentiation capability that blade, stem section, common callus etc. separate, it is more easy to acquisition regeneration plant, and draw materials conveniently, not by the restriction of the weather conditions such as time, season, the inducing culture of Fructus Vitis viniferae embryo callus can be carried out as required at any time;
(2) protoplast culture medium adopted is KM8P culture medium, than MS, B5��D2And the Ca of the culture medium such as CPW-13M2+Concentration is high, NH4+Concentration is low, and carbon source is enriched, and the required various nutrient substance of protoplast growth enrich, and such as vitamin, caseinhydrolysate, trace element etc., therefore, achieves good culture effect;
(3) receptor and Donor primordial plastid are passivated respectively processing before fusion, can reach only fusion product can regeneration plant, therefore the regeneration plant overwhelming majority obtained is hybrid, can reduce the workload of incubation and later stage and to the screening of hybrid and identify work;
(4) what protoplast fusion adopted is asymmetric amalgamation mode, and the hybrid plant obtained is asymmetric hybrid;
(5) step of the present invention is simple, consuming time short, the requirement of equipment and condition of culture is relatively low, efficiently solving the problem that Grape Protoplast is cultivated and merged difficulty, the mature technology system for setting up Grape Protoplast separation purification, cultivation and fusion provides certain theoretical foundation and technical support.
Accompanying drawing explanation
Shown in Fig. 1 is the protoplast photo of embryo callus of the present invention separation;
Shown in Fig. 2 is the photo of Grape Protoplast of the present invention fusion;
Shown in Fig. 3 is the photo of Grape Protoplast of the present invention cultivation;
Shown in Fig. 4 is the photo of Fructus Vitis viniferae embryo callus somatic embryo of the present invention;
Shown in Fig. 5 is the hybrid plant photo of the fused acquisition of Grape Protoplast of the present invention;
Shown in Fig. 6 is that Fructus Vitis viniferae hybrid plant of the present invention is taken root the photo of seedling exercising.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described in detail. It it is noted that detailed description below is all exemplary, it is intended to provide further instruction to the present invention. Except as otherwise noted, all Science and Technology terms that the present invention uses have the identical meanings being generally understood that with the technical field of the invention personnel.
The present embodiment utilizes the method that protoplast asymmetric fusion technology obtains Fructus Vitis viniferae hybrid plant, comprises the following steps:
1. the separation of Grape Protoplast and purification
The separation of protoplast is the successful important step of Protoplast cuhnre, the impact of the enzymolysis of protoplast not only segregated material, but also relevant with the kind of enzyme and concentration, enzymolysis time, temperature, pH, osmotic pressure and other factors. As table 1, table 2 list different cellulase, pectase, macerozyme, mannitol and the enzymolysis time impact on protoplast electrofusion quantity and vigor.
The fresh embryo callus of pre-made protoplast electrofusion is placed in dark cultivation 24h by the present invention, takes 1g callus and adds 10mL enzyme liquid, 27 �� 1 DEG C, 60-70r min under dark condition-1, vibrate enzymolysis 2-12h. In protoplast enzymolysis process, after enzymolysis, 2h starts sampling, is put in for 1 time under inverted microscope every 2h resampling afterwards and observes protoplast enzymolysis situation and count, and stops enzymolysis when not being further added by protoplast number than front once counted number.
After enzymolysis terminates, superclean bench passes sequentially through mixed liquor 200 orders and 400 order rustless steel cells sieve, removes the tissue of non-thorough enzymolysis, with the CPW solution washing net of 1-2mL, collect filtrate and be placed in 15mL sterile centrifugation tube, at 700r min-1Centrifugal 8min, makes protoplast settle down. Discard upper strata enzyme liquid and microcell relic, the precipitation of residue about 1-2mL after centrifugal end, draw 10mLCPW solution with liquid-transfering gun and be slowly added to the protoplast at the bottom of suspension pipe along centrifugal tube wall. Suspension 700r min-1Centrifugal 8min., abandoning supernatant, then with CPW solution washing 1-2 time. Precipitation is resuspended in after 1-2mL protoplast cleaning mixture laying in 15mL sterile centrifugation tube on 5mLCPW-23S sucrose liquid layer, 500r min-1Centrifugal 5min, gentle aspiration 0 and the protoplast of CPW23S sucrose interface, go in 15mL sterile centrifugation tube in 5mL cleaning mixture, at 700r min-1Centrifugal 8min down, removes supernatant, then with CPW solution washing 2-3 time.Finally, add CPW solution, make Protoplast suspension, regulate density (10 further according to needs5-106Left and right) standby afterwards.
The impact on Grape Protoplast separation quantity of the table 1 each factor
As can be seen from Table 1, different combinations is different on the impact of protoplast enzymolysis quantity, and best with the effect of No. 10 combinations, the yield of protoplast reaches 12.33 �� 105Individual mL-1. Can being drawn by range analysis, the enzymolysis quantity of Grape Protoplast is affected significant difference by different disposal combination. The size order that protoplast enzymolysis quantity is affected by each factor level is: R (pectase concentration) > R (cellulase concentration) > R (mannitol concentration) > R (enzymolysis time) > R (macerozyme concentration), illustrate that in 5 factors, protoplast enzymolysis quantity is had the greatest impact by pectase concentration, next to that cellulase concentration, mannitol concentration, enzymolysis time and macerozyme concentration. The optimal level of each factor is mannitol concentration the first level respectively, enzymolysis time the second level, pectase concentration and macerozyme density control three level, cellulase concentration the 4th level.
The table 2 each influence factor impact on Grape Protoplast separation vigor
As can be seen from Table 2, different test combinations is different on the impact of Grape Protoplast vigor. Best with the effect of No. 3 combinations, the vigor of protoplast is up to 76%. Can being drawn by range analysis, Grape Protoplast vigor is affected significant difference by different disposal combination. The size order of protoplast effect of vigor is by each factor level: R (enzymolysis time) > R (macerozyme concentration) > R (mannitol concentration) > R (pectase concentration) > R (cellulase concentration), illustrate that in 5 factors, enzymolysis time is maximum to protoplast effect of vigor, next to that macerozyme concentration, mannitol concentration, pectase concentration, it it is finally cellulase concentration. The optimal level of each factor is mannitol concentration the first level respectively, enzymolysis time the 3rd level, cellulase concentration the first level, pectase concentration the first level and macerozyme concentration the first level.
In conjunction with data above and analysis, finally determine that enzyme formula of liquid of the present invention is as follows: 2.50% cellulase, 0.25% pectase, 0.25% macerozyme, 0.40mol/L mannitol, 0.1%MES, pH5.8. Enzymolysis time the best is 6h.
The protoplast photo that embryo callus of the present invention separates is as shown in Figure 1.
2. IOA and the UV Passivation Treatment of Grape Protoplast
(1) iodoacetamide (IOA) can destroy the organelle in Cytoplasm, causes that Cytoplasm inactivates. IOA is dissolved in KM8In P fluid medium, test arranges 5 Concentraton gradient (1,2,4,8,16mmol L-1), pH value is all adjusted to 5.8, and it is standby that solution is put in 4 DEG C of refrigerators after 0.45um filtering with microporous membrane sterilizing. Liquor-saturated gold preserved fragrant grape protoplast is stood Passivation Treatment 10min with under the dark 4 DEG C of conditions of the IOA of variable concentrations respectively so that it is Cytoplasm inactivates, and summer Black Grape protoplast does not process. Or summer black protoplast Passivation Treatment and the liquor-saturated fragrant protoplast of gold does not process. Protoplast 700r min after process-1Centrifugal 5min, abandons supernatant after centrifugal, after washing 2-3 time with CPW solution, adds KM8P culture medium is washed once, finally by the protoplast KM after passivation8P fluid medium suspends, and protoplast density is adjusted to 1 �� 105Individual mL-1Standby. Taking 1 Protoplast suspension and measure its vigor on microscope slide, all the other proceed in little culture dish (diameter 6cm).With KM8P is minimal medium, carries out low melting-point agarose solid entrapping cultivation under 27 �� 1 DEG C of dark conditions, with do not do Passivation Treatment for comparison. Its form and division situation is observed under 2d is positioned over inverted microscope.
The impact on the fragrant protoplast of liquor-saturated gold of the table 3 different IOA concentration
As known from Table 3, the IOA of variable concentrations processes the passivation significant difference to the fragrant protoplast vigor of liquor-saturated gold. Through 1.0,2.0,4.0,8.0mmol L-1After IOA processes, the fragrant protoplast vigor of liquor-saturated gold have dropped 33.8%, 52.2%, 74.96% and 76% than comparison respectively, it was shown that the fragrant protoplast of liquor-saturated gold is to ten points of sensitivities of IOA. Along with the increase of IOA concentration, the degree of crushing of protoplasm somatocyte is in adding main trend. Through 4.0mmol L-1IOA process after about 99% the liquor-saturated fragrant protoplast of gold be all inactivated and cell breakage degree is not as high. 4.0mmol L is described-1IOA can effectively be passivated the protoplast of liquor-saturated gold preserved fragrant grape.
(2) after the separated purification of the embryo callus of summer Black Grape, by its protoplast density furnishing 1 �� 105Individual mL-1, draw 1mL summer black protoplast respectively in the little culture dish of aseptic plastic (diameter 3cm) so that it is be just paved into one layer and do easy to remember, little culture dish is put into ultraviolet lamp box and (has 2 15W uviol lamps, bottom is from ultraviolet lamp tube 25cm) in Continuous irradiation 0.5min, 1min, 2min, 4min, 6min, 8min, 16min, each process 5 ware, repeats 3 times. Taking 1 Protoplast suspension after irradiation respectively and measure its vigor on microscope slide, all the other proceed in little culture dish (diameter 6cm). With KM8P is minimal medium, carries out low melting-point agarose solid entrapping cultivation under 27 �� 1 DEG C of dark, not do Passivation Treatment for comparison. Its form and division situation is observed under 2d is positioned over inverted microscope.
The different UV of table 4 processes the time impact on summer black protoplast
From table 4, it can be seen that the UV of variable concentrations processes the passivation significant difference to summer Black Grape protoplast vigor. After 0.5min, 1min, 2min, 4min, 6min, 8min, 16minUV process, the summer, black protoplast vigor have dropped 18.5% than comparison respectively, and 33.9%, 51.9%, 59.5%, 65.5%, 70.8% and 76%. Along with the prolongation of UV irradiation time, the passivation of summer black protoplast vigor is strengthened by UV, and cell breakage degree is deepened. Wherein the cell breakage degree of 8minUV treatment with irradiation protoplast starts to increase, and can strengthen the passivation of protoplast as extended the process time again, occurs that more protoplasm somatocyte is dead, broken. Therefore, UV irradiation 8min can effectively be passivated the protoplast of summer Black Grape.
By the protoplast mixing for standby use in centrifuge tube of 1:1 by volume after IOA (liquor-saturated gold is fragrant) and UV (summer is black) Passivation Treatment.
3. the fusion of Grape Protoplast
The present invention adopts PEG-height pH-height Ca2+The fusion of method induction Grape Protoplast, factor affect PEG fusion mainly has the kind of PEG, molecular weight, process time, concentration, the density of protoplast and physiological status etc. PEG fusion method equipment needed thereby is simple, and simple to operate, treating capacity is big, it does not have significantly merge specificity. PEG fusion method is disadvantageous in that the process time is longer, and operating process is more complicated, and density required during to protoplast fusion is relatively big, and the injury of protoplast is also bigger. Therefore, process of the test strictly to control the impact on protoplast fusion of each factor.
The concentration of PEG is set to 20% by the present invention, and 30%, 40%, 50%.When carrying out fusion treatment with small test tube, taking amphiphilic this Protoplast suspension 0.5-1mL mixed, the PEG fusion induction liquid drawing 1-2mL is slowly added to along lab scale tube wall, rotates small test tube simultaneously, make PEG solution and protoplast be fully contacted, promote protoplast inter-adhesive. This process about needs 15-30min at 25-30 DEG C, and microscopy is it can be seen that this protoplast of amphiphilic assembles adhesion. After insulation, add the high Ca of about 2mL by same method2+-high pH solution, is incubated about 10-15min after mixing.
The impact that table 5PEG concentration versus cell merges
As known from Table 5, along with the increase of PEG concentration, heterolgous fusion rate is in the change declined afterwards that first rises, and when wherein PEG concentration is 40%, fusion rate is maximum, is 7.8%, but is higher than after 40%, and fusion rate declines. But when PEG concentration is 50%, fusion product can not be survived, substantial amounts of protoplast crushes, dead gradually. Owing to protoplast is had certain toxicity by PEG itself, for reducing its side effect to fusion product growth promoter, the PEG concentration of selection 40% is as the fragrant suitable concentration with summer black protoplast fusion of liquor-saturated gold, and now most protoplasts are merge one to one, and heterolgous fusion rate is 7.8%.
According to above test, finally determine that PEG of the present invention merges the formula of induction liquid and is: with CPW solution for base fluid, containing PEG30-50g, CaCl in every 100mL2��2H2O150mg��KH2PO410mg, mannitol 3g.
High Ca2+-high pH solution is by glycine-NaOH buffer and Ca (NO3)2Solution even mixes, and wherein the formula of glycine-NaOH buffer is: glycine 3.75g/L, mannitol 15g/L, and uses 0.2mol/LNaOH solution to adjust pH to 9-10.5; Ca (NO3)2The concentration of solution is 94.4g/L.
The photo that Grape Protoplast of the present invention merges is as shown in Figure 2.
4. merge somatic cultivation
By the hybrid cell after fusion, add KM8P fluid medium regulates density to 1 �� 105Individual mL-1, with KM8P is minimal medium, adopt low melting-point agarose solid entrapping method cultivate protoplast, sealed membrane sealing after in incubator 25 DEG C of constant temperature light culture. The protoplast separated by embryo callus is at KM8The multiple wall of about 6-7d protoplast in P culture medium, about 14-15d occurs that first division, 20-25d occur that the visible small callus of naked eyes occur in second division, about 65-70d. The photo that Grape Protoplast is cultivated is as shown in Figure 3.
5. regenerate the induction of embryo callus embryoid
Progressively, immediately regeneration embryo callus granule is forwarded to division culture medium MS+6-BA0.1mg L-1+NAA0.1mg��L-1Upper inducing embryoid body, at 25 DEG C light culture after 2 weeks dislocation intensity of illumination be under 1000-1500lx cultivate, irradiation bears adventitious bud after 1 week, then adventitious bud is transferred to root media 1/2MS+NAA1.0mg L-1+IBA0.5mg��L-1Upper cultivation. The photo of Fructus Vitis viniferae embryo callus somatic embryo is as shown in Figure 4.
6. seedling exercising and transplanting
When tissue cultured seedling grows to 5��7cm, and when root growth is vigorous, being placed in seeding room by tissue cultured seedling and carry out seedling exercising 6��9d, corkage seedling exercising carries out stage by stage, and namely first pine covers 2��3d, and then partly uncap 2��3d, finally throws off lid completely. Test tube Seedling is progressively made to adapt to greenhouse, in case uppermost leaf sheet is wilted. Gently test tube Seedling is taken out from bottle with tweezers, is placed in water the culture medium thoroughly removing root, then with after the carbendazim immersion root 10��30min of 0.1%, move into sterilized after substrate in.Water permeable after transplanting, spray the organic 1/2MS nutritional solution of removing, and enclose plastic foil maintenance relative humidity. Remove the plastic film enclosed carry out opening wide seedling exercising moving into 5d, 10d, 15d, 20d after substrate, and take the blade of seedling under the same conditions, observe the pore opening of blade. Transplant early stage removing organic 1/2MS seedling is sprayed, when seedling cane color burn, water with tap water when blade is glossy or after growing young leaves. The hybrid plant photo of the fused acquisition of Grape Protoplast is as shown in Figure 5. Seedling exercising process is shown in Fig. 6.
The CPW solution formula that the present embodiments relate to: potassium dihydrogen phosphate 27.2mg/L, potassium nitrate 101.0mg/L, CALCIUM CHLORIDE DIHYDRATE 1480.0mg/L, bitter salt 246.0mg/L, potassium iodide 0.16mg/L, copper sulfate pentahydrate 0.025mg/L.
The main medium that the present embodiments relate to is as shown in table 6; The main agents used has a-naphthalene acetic acid (NAA), 6-benzyl aminoadenine (6-BA), indolebutyric acid (IBA), hydrochloric acid (HCL), sodium hydroxide (NaOH), agar, dehydrated alcohol, mannitol, mannitol, potassium dihydrogen phosphate (KH2PO4), potassium nitrate, calcium chloride dihydrate, Magnesium sulfate heptahydrate, potassium iodide, copper sulphate pentahydrate, sucrose, boric acid, inositol, folic acid, citric acid, malic acid, biotin, caseinhydrolysate, cellulase (Cellulase), macerozyme (Macerozyme), pectase (Pectolyase), 2-(N-morpholine) ethyl sulfonic acid, low melting-point agarose, polyethylene glycol 6000 (PEG), glycine, Ca (NO3)2��4H2O, iodoacetamide (IOA) etc., the key instrument used has high-pressure steam sterilizing pan, thunder magnetic Phs-3c precision pH meter, electronic analytical balance, superclean bench, micropipettor (Eppendorf), growth cabinet, water-bath freezing thermostat agitator (LSHE-300D), ordinary optical microscope (OLYMPUSDp70.), inverted microscope (NikonMODELC-LEDS), fluorescence inverted microscope (NikonECLIPSETi-S), blood counting chamber, cell sieves, low speed centrifuge (TDL-40B) etc., other materials used, reagent and experimental facilities, if no special instructions, be the commercially available prod meeting protoplast asymmetric fusion technical field.
The above, be only the preferred embodiments of the present invention, it should be pointed out that; for those skilled in the art; under the premise without departing from the core technology of the present invention, it is also possible to make improvements and modifications, these improvements and modifications also should belong to the scope of patent protection of the present invention. Any change in the implication suitable with claims of the present invention and scope, is all considered as being included in the scope of claims.
Table 6MS and KM8P culture medium prescription (mg/L)
Claims (10)
1. one kind utilizes the method that protoplast asymmetric fusion technology obtains Fructus Vitis viniferae hybrid plant, it is characterised in that include following operating procedure:
(1) separation of protoplast and purification: take the enzyme-added liquid of fresh embryo callus, 26-28 DEG C, under dark condition, 60-70r/min vibrates enzymolysis 2-12h, is then peeled off purification and obtains Grape Protoplast;
(2) protoplast Passivation Treatment:
A () IOA is passivated: taking the protoplast use CPW solution suspension that step (1) obtains, adjusting protoplast density is 0.5-1 �� 105The suspension of individual/mL, is subsequently adding the 4.0-8.0mmol/L iodoacetamide solution Passivation Treatment 9-11min of 15 proportionings, uses CPW solution washing 2-3 time, use KM after having processed8P fluid medium suspends, and protoplast density is adjusted to 0.5-1 �� 105Individual/mL, obtains receptor protoplast;
B () UV is passivated: taking the protoplast use CPW solution suspension that step (1) obtains, adjusting protoplast density is 0.5-1 �� 105The suspension of individual/mL, at 1250-1350 �� W/cm2Irradiate 0.5-16min under ultraviolet lighting intensity, obtain Donor primordial plastid;
(3) fusion of protoplast: the Donor primordial plastid that the receptor protoplast obtain step (3) (a) and step (3) (b) obtain mixes by 11 volume ratios, obtain parent's suspension, the PEG adding 1-4 times of parent's suspension vol merges induction liquid, at 25-30 DEG C, it is incubated 15-30min, adds the high Ca of 2-4 times of parent's suspension vol2+-high pH solution, is incubated 10-15min at 25-30 DEG C, then uses CPW solution washing 2-3 time after mixing, the centrifugal hybrid cell obtaining merging;
(4) somatic cultivation is merged: use KM8The blendling cell that P solid medium embedding culture step (3) obtains, at 25-27 DEG C, quiescent culture under dark condition, until growing regeneration embryo callus granule;
(5) regenerating the induction of embryo callus embryoid: be forwarded on division culture medium by regeneration embryo callus granule inducing embryoid body, until growing adventitious bud, then adventitious bud being transferred on root media cultivation;
(6) seedling exercising and transplanting: when tissue cultured seedling grows to 5-7cm and root growth is vigorous, is transplanted in seeding room by tissue cultured seedling and carries out seedling exercising, namely obtains somatic cell hybrid plant.
2. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterized in that: in described step (1), the formula of enzyme liquid is: 2.50% cellulase, 0.25% pectase, 0.25% macerozyme, 0.40mol/L mannitol, 0.1%MES, pH5.8.
3. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterised in that: in described step (1), the amount of every enzyme-added liquid of 1g embryo callus is 10mL.
4. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterized in that: in described step (3), the formula of PEG fusion induction liquid is: with CPW solution for base fluid, containing PEG30-50g, CaCl in every 100mL2��2H2O150mg��KH2PO410mg, mannitol 3g.
5. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterised in that: high Ca in described step (3)2+-high pH solution is by glycine-NaOH buffer and Ca (NO3)2Solution even mixes.
6. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 5, it is characterized in that: the formula of described glycine-NaOH buffer is: glycine 3.75g/L, mannitol 15g/L, and use 0.2mol/LNaOH solution to adjust pH to 9-10.5.
7. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 5, it is characterised in that: described Ca (NO3)2The concentration of solution is 94.4g/L.
8. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterized in that: in described step (5), the formula of division culture medium is the MS culture medium containing 6-benzyl aminoadenine 0.1mg/L, naphthalene acetic acid 0.1mg/L, pH5.8.
9. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterised in that: in described step (5), the formula of root media is containing naphthalene acetic acid 1.0mg/L, indolebutyric acid 0.5mg/L-11/2MS culture medium, pH5.8.
10. the method utilizing protoplast asymmetric fusion technology to obtain Fructus Vitis viniferae hybrid plant according to claim 1, it is characterised in that: the formula of described CPW solution is: potassium dihydrogen phosphate 27.2mg/L, potassium nitrate 101.0mg/L, CALCIUM CHLORIDE DIHYDRATE 1480.0mg/L, bitter salt 246.0mg/L, potassium iodide 0.16mg/L, copper sulfate pentahydrate 0.025mg/L.
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| CN112284791B (en) * | 2020-10-16 | 2023-02-28 | 扬州大学 | Method for separating xylem duct of grape fruit |
| CN115521897A (en) * | 2022-09-22 | 2022-12-27 | 鲁东大学 | Method for extracting protoplast from forest strawberry callus |
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