CN104745523A - Separation, transformation and regeneration system for oilseed rape protoplast - Google Patents
Separation, transformation and regeneration system for oilseed rape protoplast Download PDFInfo
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Abstract
The invention relates to a high-efficient separation, transformation and regeneration system for oilseed rape protoplast. The regeneration system comprises a reagent mixture which is specially used for separating, transforming and regenerating the oilseed rape protoplast and a separation, transformation and regeneration method of the oilseed rape protoplast, wherein the reagent mixture comprises a 1/2MS B5 culture medium, an MS culture medium, enzymatic hydrolysate, a C culture medium, W5 washing liquid, Evans Blue dyeing liquid, transformation liquid, an ethylene glycol solution, a C:B culture medium, an A culture medium and an MS regenerative culture medium. The transformation method comprises culturing of aseptic seedlings, separation and purification of protoplast, transformation of protoplast, culturing and regeneration of protoplast and the like. The method provided by the invention has the advantages of simple steps, standard operation, low pollution rate, high transformation efficiency and the like.
Description
Technical field
The present invention relates to transgenic engineering field, be specifically related to the high efficiency separation of swede type rape protoplastis, conversion and regeneration system.
Background technology
Swede type rape (Brassica napus L) is important oil crops, and not only can be used for human consumption, its grouts also can be used for the grocery trades such as animal-feed and industrial processes.Now, traditional breeding method means more and more cannot meet the variety requirement day by day changed.The expansion breadth and depth of functional gene is depended in the further improvement of rape variety to a great extent.The protoplastis operational meanss such as external evoked, cytogamy and genetic transformation are that heritable variation provides new source, and somatic hybridization is simultaneously also for the genetic material between edge species far away exchanges and restructuring provides new possibility.Genome editing technique is that functional genomics research opens a new field, will become the powerful of crop improvement with the protoplast transformants system of its combination.
The key of protoplast transformants system is the regenerative power of induced tissue, so the hypocotyl of swede type rape and blade are Protoplast isolation and culture important sources.Report before mainly utilizes the hypocotyl of three cabbage type rape varieties to obtain protoplast regenerated plant, and genotype-independent is large, and transformation efficiency height differs, and test operation repeatability is poor, and mesophyll protoplast regeneration plant exists few successful example.
Summary of the invention
There is efficiency for the Rapeseed Protoplast existed in prior art to differ, the defect of test operation repeatability difference, the invention provides a kind of separation of Rapeseed Protoplast, conversion and regeneration system.First object of the present invention is to provide a kind of Rapeseed Protoplast and is separated, transforms and the substratum combination of regeneration, comprises 1/2MS B5 medium, A substratum, B substratum, C substratum, MS substratum, MS regeneration culture medium, W5 washing lotion, conversion fluid, solution, enzymolysis solution, W5 washing lotion and C:B substratum containing polyoxyethylene glycol; The formula of described 1/2MS B5 medium, MS substratum, A substratum, B substratum, C substratum, MS regeneration culture medium comprises following component:
Described MS trace 100 × formula as following table:
| Composition | Final concentration mg/L |
| CoCl 2·6H 2O | 2.5 |
| KI | 83 |
| MnSO 4·H 2O | 1690 |
| H 3BO 3 | 620 |
| CuSO 4·5H 2O | 2.5 |
| Na 2MoO 4·2H 2O | 25 |
| ZnSO 4·7H 2O | 860 |
Described molysite 1000 × formula as following table:
| Composition | Final concentration mg/L |
| FeNaEDTA | 36700 |
The composition of described W5 washing lotion comprises sodium-chlor 8.9g/L, calcium chloride 18.4g/L, Repone K 0.37g/L, glucose 0.9g/L;
The composition of described conversion fluid comprises N.F,USP MANNITOL 91g/L, magnesium chloride 14.25g/L, ethyl sulfonic acid 1g/L;
The composition of the described solution containing polyoxyethylene glycol comprises PEG 400g/L, N.F,USP MANNITOL 72.9g/L, nitrocalcite 16.4g/L.
Substratum combination of the present invention, the composition of described enzymolysis solution is included in described C substratum the macerozyme R10 adding the sucrose of 10g/L, cellulase R10 and 1g/L of 10g/L.
Substratum combination of the present invention, the composition of described Evans Blue dye liquor is included in the Evans Blue powder that with the addition of 2.5g/L in described W5 washing lotion.
Substratum of the present invention combination, the composition of described C:B substratum is included in the extra large spot agarose adding 6g/L in the mixed solution that described C substratum and described B substratum form by 1:1.
Another object of the present invention is to provide a kind of method being separated Rapeseed Protoplast, transforming and regenerating.Method of the present invention, preferably includes following steps:
1) cultivation of aseptic seedling
Semen Brassicae campestris after surface sterilization is seeded in 1/2MS B5 medium, when seed grows rough leaf, get stem apex be transferred in 1/2MS B5 medium cultivate 2 ~ 6 weeks, the rape aseptic seedling of young leaflet tablet must be grown;
2) abstraction and purification of protoplastis
Get described young leaflet tablet, be cut into small pieces and be dipped in described enzymolysis solution, by enzymatic hydrolysis system sealing, lucifuge, at 24 DEG C ~ 26 DEG C temperature, leave standstill 16 ~ 18h;
Mixture after enzymolysis is filtered, collects filtrate, filter residue is dissolved again with described C substratum, filters, collect filtrate, repeat this operation until no longer include protoplastis precipitation, gained filtrate is mixed, must filtrate be mixed;
In described mixing filtrate, add described W5 washing lotion, mix rear centrifugal, draw protoplastis layer; Again add W5 washing lotion, the throw out after centrifugal is protoplastis;
3) conversion of protoplastis
Be dissolved in ultrapure sterilized water after plasmid DNA is sterilized, form plasmid DNA system, described protoplastis is dissolved in described conversion fluid, form Protoplast suspension 1, the two is mixed, and add the described solution containing polyoxyethylene glycol in system upon mixing, left at room temperature 10 ~ 20min;
In described mixed system, add described W5 solution, centrifugal after mixing, throw out is the protoplastis after conversion;
4) cultivation of protoplastis, the screening of conversion system and regeneration
Protoplastis after described conversion is dissolved in described C substratum, makes Protoplast suspension 2, mix with described C:B substratum, then protoplasm somatocyte is cultured to and occurs repeatedly cell fission;
Protoplastis after described cell fission is proceeded to and with the addition of in antibiotic described A substratum, occur that callus is after 5 ~ 6 weeks, transfer on described MS regeneration culture medium, during described callus growth to 8 ~ 10mm, again transferred on described MS regeneration culture medium, grow Multiple Buds, to described Multiple Buds growth after 1 ~ 2 week, cut and be again placed on MS regeneration culture medium, when young shoot grows to 3 ~ 4cm, it transfers on MS regeneration culture medium, root induction, strong seedling culture is carried out in transplanting, obtains rapeseed plants.
The step 2 of the method for the invention) in use in filter operation aperture to be the filter membrane of 70 μm.
The step 4 of the method for the invention) in the culture condition of protoplasm somatocyte in described C:B substratum be under 22 ~ 25 DEG C of dark conditions cultivation 22 ~ 26h, 5d ~ 7d is cultivated under moving to half-light again, cultivate 24h under being preferably 24 DEG C of dark conditions, then cultivate 6d under moving to half-light.
The method of the invention is under the described culture condition that with the addition of in antibiotic A substratum is 22 ~ 26 DEG C of subdued light conditions, and 60 ~ 100rpm wave and culture, is preferably 80rpm wave and culture under 24 DEG C of subdued light conditions;
Described callus or the culture condition of bud in described MS regeneration culture medium are that 22 ~ 26 DEG C of half-lights are cultivated, and are preferably 24 DEG C of half-lights and cultivate;
The culture condition of described young shoot on described MS substratum is that 22 ~ 26 DEG C of half-lights are cultivated, and is preferably 24 DEG C of half-lights and cultivates.
The consumption of each reagent of the present invention is determined according to the experiment demand of reality.
Culture system of the present invention is applicable to the separation of the protoplastis of all kinds of rape, conversion and regeneration, in preferred swede type rape (Brassica napus L) two No. 10, Nanyang 41, No. 49, cloud oil, peaceful assorted No. 11,9603, No. 11, Qin You.
Beneficial effect
The invention discloses the high efficiency separation of a kind of swede type rape (Brassica napus L) protoplastis, conversion and regeneration system and special culture media.The present invention for parent material, adopts cellulase R-10 and macerozyme R-10 with rape aseptic seedlings, digests and utilizes the method for density gradient sedimentation to be separated protoplastis, obtain highly purified protoplastis to rape leave meat tissue.By poly-di-alcohol mediation, goal gene is imported in Rapeseed Protoplast genome, through swallow liquid culture and follow-up differentiation and regeneration, cultivated a large amount of rape transfer-gen plant.
One aspect of the present invention separable go out a large amount of highly purified protoplastis, plasmid DNA success can be proceeded in protoplastis efficiently on the other hand, protoplastis can successfully be trained and can obtain the transfer-gen plant of a large amount of high expression level simultaneously.For the batch production of follow-up rape prevalent variety cultivation is laid a good foundation.
The present invention can be the breed improvement such as the research of the functional genomics such as Subcellular Localization, promoter expression and plant soma fusion, genetic transformation, genome editor and provides possibility.The present invention's success has been applied in Chinese main planting rape kind, for the improvement of rape variety and genetics research have done desk study.
Accompanying drawing explanation
Fig. 1 is the protoplastis carrying out after dyeing counting;
Fig. 2 is the first time cell fission of protoplastis;
Fig. 3 is the cultivation situation of protoplastis in culture medium A after repeatedly dividing.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
One, protoplast electrofusion, the mother liquor of conversion and regeneration and substratum is prepared
Formula and the preparation method of the B5 medium of 1/2MS described in the present invention, MS substratum, A substratum, B substratum, C substratum, MS regeneration culture medium, W5 washing lotion, conversion fluid, enzymolysis solution, EvansBlue dye liquor, enzymolysis solution, C:B substratum and the solution containing polyethylene glycol are as follows:
The formula of 1/2MS B5 medium, MS substratum, A substratum, B substratum, C substratum, MS regeneration culture medium is as table 1:
Table 1
Described micro-100 × formula as table 2:
Table 2 MS trace 100 × formula
| Composition | Final concentration mg/L | 500mL mother liquor (g) |
| CoCl 2·6H 2O | 2.5 | 0.00125 |
| KI | 83 | 0.0415 |
| MnSO 4·H 2O | 1690 | 0.845 |
| H 3BO 3 | 620 | 0.31 |
| CuSO 4·5H 2O | 2.5 | 0.00125 |
| Na 2MoO 4·2H 2O | 25 | 0.0125 |
| ZnSO 4·7H 2O | 860 | 0.43 |
Described molysite 1000 × formula as table 3:
Table 3 molysite 1000 × formula
| Composition | Final concentration mg/L | 500mL mother liquor (g) |
| FeNaEDTA | 36700 | 18.35 |
W5 washing lotion: get sodium-chlor 8.9g, calcium chloride 18.4g, Repone K 0.37g, glucose 0.9g, adds water and is settled to 1L, adjusts pH to 5.8-6.0,121 DEG C of autoclaving 20min with 1M KOH.
Conversion fluid: get N.F,USP MANNITOL 9.1g, magnesium chloride 1.425g, ethyl sulfonic acid 0.1g, adds water and is settled to 100mL, adjusts pH to 5.6, filtration sterilization with 1M KOH.
Solution containing polyoxyethylene glycol (PEG): the PEG of 40g is dissolved into 100mL and with the addition of in the liquid of 7.29g N.F,USP MANNITOL, 1.64g nitrocalcite, adjust pH to 8.0 with 1M KOH, 121 DEG C of autoclaving 20min, under being stored in-20 DEG C of conditions after packing.
Enzymolysis solution: add the sucrose that concentration is 10g/L, cellulase R101g, macerozyme R100.1g in every 100mL C substratum, adjust pH to 5.6 with 1M KOH, filtration sterilization, under being stored in-20 DEG C of conditions after packing.
Evans Blue dye liquor: by the Evans Blue powder dissolution of 0.25g in 100mL W5 washing lotion, filtration sterilization.
C:B substratum: be added in the mixing solutions of 100mL C and B in 1:1 ratio by the extra large spot agarose of 0.6g, microwave oven high fire heating 5s, dissolves completely to agarose.
Two, the separation of Rapeseed Protoplast, conversion and regeneration
1, the cultivation of aseptic seedling
1.1 Semen Brassicae campestriss weighing about 1g (raising No. 7, oil) put into 15mL centrifuge tube, abandon waste liquid after adding the spirituous solution jog 5min of 5mL 75%, and with aseptic water washing 1 time; With the mixing solutions surface sterilization 15min of volume percent 0.05%Tween20 and 10% hydrogen peroxide, then use aseptic water washing 5 times.
Seed Points after sterilization is sowed at (30, every ware) in the culture dish of the 90mm × 15mm filling 1/2MS B5 medium by 1.2, and be positioned over 24 DEG C, the photoperiod is 16 h light/8 h dark, intensity of illumination is 84 μm of ol/m
2in the growth room of/s condition.After 1.5-2 week, seed cuts the healthy and strong free of contamination stem apex of blade and transplants to the square cultivation box of 10cm (suitable for reading) × 7cm (the end opening) × 10cm filling 1/2MS B5 medium (strain of every box two) when growing rough leaf, 24 DEG C, the photoperiod is 16 h light/8 h dark, intensity of illumination is 84 μm of ol/m
2cultivate 4 weeks under/s condition.
2, the abstraction and purification of protoplastis
Cut the young leaflet tablet that 2-4 sheet launches completely under 2.1 aseptic conditions, be dipped in enzymolysis solution (in 9cm culture dish about 12mL), remove main lobe arteries and veins, be cut into 8-12mm
2square, continue to add 3mL enzymolysis solution, culture dish with sealed membrane sealing and with aluminium foil parcel, in 25 DEG C of hold over night (be the best with 16-18 hour).
2.2 softly rock culture dish makes protoplastis fully be discharged into out, with the mixture after the membrane filtration enzymolysis of 70 μm, collect filtrate, with pasteur pipet, the residue on filter membrane is carefully drawn in culture dish, add 5mL C substratum, again rock culture dish to clean zymolyte, again by membrane filtration until no longer include protoplastis and separate out, and gained filtrate to be mixed.
Described mixing filtrate to be dispensed in 14mL round bottom centrifuge tube and slowly to add the W5 washing lotion of 1mL by 2.3, in the centrifugal 5min of 400r/min, after solution layering, the protoplastis layer sucking-off of middle green is transferred in new 14ml centrifuge tube with pasteur pipet.In centrifuge tube, add 7mL W5 washing lotion, softly inclination centrifuge tube makes protoplastis mix back and forth, in the centrifugal 5min of 400r/min.Remove supernatant liquor, add 2-5mL W5 washing lotion, same way mixes and is placed on stand-by on ice.
After 2.4 dilutions 10 times, get 5 μ L and drop on blood cell counting plate, drip 5 μ L0.25%Evans Blue dye liquors simultaneously, after left at room temperature 5min, in the undyed living cell rate of counted under microscope more than 90%, the output of protoplastis is 7.1 × 10
6(as Fig. 1).
3, the conversion of protoplastis
3.1 plasmid DNA (pVec8-GFP:35S-hpt-Nos+Ubi-gfp-Nos) are separated out in dehydrated alcohol and after sterilizing, being dissolved to final concentration minimum with aseptic ultrapure water is 0.7 μ g/ μ L.After protoplastis has counted, in the centrifugal 5min of 400r/min, remove supernatant liquor, add conversion fluid in proportion, make resuspended protoplastis obtain concentration and reach 1.6 × 10
6individual/mL.
3.2 add 300 μ L in new 14mL centrifuge tube, and (protoplastis number is 5 × 10
5) Protoplast suspension, add 30 μ L plasmid DNA, limit rotating centrifugal tube edge slowly adds from tube wall surrounding the solution (as figure) that 300 μ L contain PEG, and direction finding is softly rocked centrifuge tube back and forth and made it abundant mixing, under room temperature condition, static 15min, rocks once every 3min.
3.3 according to the order of 1mL, 2mL, 3mL respectively to W5 solution in centrifuge tube, every minor tick 2min fully mixing, in the centrifugal 5min of 400r/min.Finally add 6mL W5 solution fully to mix, in the centrifugal 5min of 400r/min.
In 3.4 transient expression experiments, remove supernatant liquor, add 2.5mL C substratum resuspended after, observe cultivate 24h under 25 DEG C of dark conditions after, the protoplastis more than 80% presents blueness.
The detailed process of stably express experiment is as step 4.
4, the cultivation of protoplastis, the screening of conversion system and regeneration
Protoplastis to be resuspended in C substratum and to form suspension by 4.1, and (protoplastis number is 5 × 10 to get 0.5mL Protoplast suspension
5) add in the culture dish of 60mm × 8mm, then add 4.5mL and shift to an earlier date preheating, the C:B substratum of about 45 DEG C, fully mixing makes it just to be laid in culture dish.After substratum cooling curing, culture dish sealed and cultivates 24h under being placed in 24 DEG C of dark conditions, then cultivating 6d under transferring to subdued light conditions.First time and repeatedly cell fission subsequently (as Fig. 2) can be observed in culturing process.
Agar block with division protoplastis is transferred in 250mL culture dish by 4.2, adds the A substratum that 20mL with the addition of 25mg/L Totomycin.80rpm wave and culture (as Fig. 3) under 24 DEG C of subdued light conditions.
After 4.3 3-4 weeks, first time the visible callus by protoplastis induced synthesis.Again after 5-6 week, transferred to by callus on MS regeneration culture medium, cultivate 1-2 week under 24 DEG C of subdued light conditions, callus is in continuous increase during this period.When callus reaches 8-10mm, transferred on new MS regeneration culture medium and continue to cultivate, obtained resistant calli.After 1-2 week, evoked callus forms the bud of growing thickly.After 1-2 week, normal for growth bud cut from callus and is placed on MS regeneration culture medium, cultivating under 24 DEG C of subdued light conditions.
4.4 when shoot growth is to 3-4cm, is transferred on MS regeneration culture medium, cultivates under 24 DEG C of subdued light conditions.After 1-3 week, induced bud is taken root.When the resistance Rice-rape fields upper part of taking root is grown to about 10cm and induces more than 2 strong root systems, transplant in the flowerpot of 12 × 12cm, in intensity of illumination 140 μm of ol/m
2/ s, photoperiod 14/10h, seedling stage diurnal temperature be 20/15 DEG C, booting and filling stage diurnal temperature be strong seedling and propagating in the growth room of 30/20 DEG C, grows up to healthy and strong plant.
Embodiment 2
With in swede type rape (Brassica napus L) two No. 10, Nanyang 41, No. 49, cloud oil, peaceful assorted No. 11,9603, the kind such as No. 11, Qin You, cultivate by the method in embodiment 1, all obtain rape transfer-gen plant.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. the agent combination for Rapeseed Protoplast high efficiency separation, conversion and regeneration, it is characterized in that, comprise 1/2MS B5 medium, MS substratum, A substratum, B substratum, C substratum, MS regeneration culture medium, W5 washing lotion, conversion fluid, solution, enzymolysis solution, W5 washing lotion and C:B substratum containing polyethylene glycol;
The formula of described 1/2MS B5 medium, MS substratum, A substratum, B substratum, C substratum, MS regeneration culture medium is as follows:
Described MS trace 100 × formula as following table:
Described molysite 1000 × formula as following table:
The composition of described W5 washing lotion comprises sodium-chlor 8.9g/L, calcium chloride 18.4g/L, Repone K 0.37g/L, glucose 0.9g/L;
The composition of described conversion fluid comprises N.F,USP MANNITOL 91g/L, magnesium chloride 14.25g/L, ethyl sulfonic acid 1g/L;
The composition of the described solution containing polyoxyethylene glycol comprises poly(oxyethylene glycol) 400 g/L, N.F,USP MANNITOL 72.9g/L, nitrocalcite 16.4g/L.
2. agent combination according to claim 1, is characterized in that, the composition of described enzymolysis solution is included in described C substratum the macerozyme R10 adding the sucrose of 10g/L, cellulase R10 and 1g/L of 10g/L.
3. agent combination according to claim 1 and 2, is characterized in that, the composition of described EvansBlue dye liquor is included in the Evans Blue powder adding 2.5g/L in described W5 washing lotion.
4. the agent combination according to claim 1 or 3, is characterized in that, the composition of described C:B substratum is included in the extra large spot agarose adding 6g/L in the mixed solution that described C substratum and described B substratum form by 1:1.
5. the agent combination according to any one of Claims 1 to 4, is characterized in that, the pH of described W5 washing lotion is 5.8 ~ 6.0; The pH of described conversion fluid is 5.6; The pH of the described solution containing polyoxyethylene glycol is 8.0, and the PH of described enzymolysis solution is 5.6.
6. the method utilizing the agent combination described in any one of claim 1 ~ 5 to be separated Rapeseed Protoplast, to transform and to regenerate.
7. method according to claim 6, is characterized in that, comprises the steps:
1) cultivation of aseptic seedling
Semen Brassicae campestris after surface sterilization is seeded in 1/2MS B5 medium, when seed grows rough leaf, get stem apex be transferred in 1/2MS B5 medium cultivate 2 ~ 6 weeks, the rape aseptic seedling of young leaflet tablet must be grown;
2) abstraction and purification of protoplastis
Get described young leaflet tablet, be cut into small pieces and be dipped in described enzymolysis solution, by enzymatic hydrolysis system sealing, lucifuge, at 24 DEG C ~ 26 DEG C temperature, leave standstill 16 ~ 18h;
Mixture after enzymolysis is filtered, collects filtrate, filter residue is dissolved again with described C substratum, filters, collect filtrate, repeat this operation until no longer include protoplastis precipitation, gained filtrate is mixed, must filtrate be mixed;
In described mixing filtrate, add described W5 washing lotion, mix rear centrifugal, draw protoplastis layer; Again add W5 washing lotion, the throw out after centrifugal is protoplastis;
3) conversion of protoplastis
Be dissolved in ultrapure sterilized water after plasmid DNA is sterilized, form plasmid DNA system, described protoplastis is dissolved in described conversion fluid, form Protoplast suspension 1, the two is mixed, and add the described solution containing polyoxyethylene glycol in system upon mixing, left at room temperature 10 ~ 20min;
In described mixed system, add described W5 solution, centrifugal after mixing, throw out is the protoplastis after conversion;
4) cultivation of protoplastis, the screening of conversion system and regeneration
Protoplastis after described conversion is dissolved in described C substratum, makes Protoplast suspension 2, mix with described C:B substratum, then protoplasm somatocyte is cultured to and occurs repeatedly cell fission;
Protoplastis after described cell fission is proceeded to and with the addition of wave and culture in antibiotic described A substratum, occur that callus is after 5 ~ 6 weeks, transfer on described MS regeneration culture medium, during described callus growth to 8 ~ 10mm, again transferred on described MS regeneration culture medium, grow Multiple Buds, to described Multiple Buds growth after 1 ~ 2 week, cut and be again placed on MS regeneration culture medium, when young shoot grows to 3 ~ 4cm, it transfers on MS regeneration culture medium, root induction, strong seedling culture is carried out in transplanting, obtains rapeseed plants.
8. method according to claim 7, it is characterized in that, described step 4) in the culture condition of protoplasm somatocyte in described C:B substratum be under 22 ~ 25 DEG C of dark conditions cultivation 22 ~ 26h, 5d ~ 7d is cultivated under moving to half-light again, cultivate 24h under being preferably 24 DEG C of dark conditions, then cultivate 6d under moving to half-light.
9. the method according to claim 7 or 8, is characterized in that, under the described culture condition that with the addition of in antibiotic A substratum is 22 ~ 26 DEG C of subdued light conditions, and 60 ~ 100rpm wave and culture, preferably 80rpm wave and culture under 24 DEG C of subdued light conditions;
Described callus or the culture condition of bud in described MS regeneration culture medium are that 22 ~ 26 DEG C of half-lights are cultivated, and are preferably 24 DEG C of half-lights and cultivate;
The culture condition of described young shoot on described MS substratum is that 22 ~ 26 DEG C of half-lights are cultivated, and is preferably 24 DEG C of half-lights and cultivates.
10. the method described in any one of claim 6 ~ 9 in swede type rape two No. 10, Nanyang 41, No. 49, cloud oil, peaceful assorted No. 11,9603, application in Qin You No. 11 protoplast electrofusion, conversion and regeneration.
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| CN114591882A (en) * | 2022-03-03 | 2022-06-07 | 东北林业大学 | Method for transient transformation of acanthopanax cells |
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| CN107267549B (en) * | 2017-07-06 | 2021-01-05 | 江苏省中国科学院植物研究所 | Method for separating, purifying and efficiently converting mesophyll protoplast of taxus chinensis variety 406 |
| CN108085335A (en) * | 2017-12-29 | 2018-05-29 | 河南科技学院 | A kind of method for cultivating the transgene cotton that fruit branch angle changes |
| CN108085335B (en) * | 2017-12-29 | 2021-04-27 | 河南科技学院 | Method for cultivating transgenic cotton with fruit branch included angle changed |
| CN109880788A (en) * | 2019-04-08 | 2019-06-14 | 天津吉诺沃生物科技有限公司 | The cabbage type rape protoplast electrofusion and genetic transforming method and regenerating system used not limited by genotype |
| CN111676183A (en) * | 2020-07-21 | 2020-09-18 | 华中农业大学 | Preparation method of rape mesophyll protoplast |
| CN114591882A (en) * | 2022-03-03 | 2022-06-07 | 东北林业大学 | Method for transient transformation of acanthopanax cells |
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