CN104745523B - System for separating, transforming and regenerating rape protoplast - Google Patents
System for separating, transforming and regenerating rape protoplast Download PDFInfo
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Abstract
The invention relates to a high-efficiency separation, transformation and regeneration system for rape protoplasts, which comprises a reagent combination and a method special for the separation, transformation and regeneration of the rape protoplasts, wherein the reagent combination comprises 1/2MS B5 culture medium, MS culture medium, enzymolysis liquid, C culture medium, W5 washing liquid, Evans Blue dye solution, transformation liquid, polyethylene glycol solution, C: B culture medium, A culture medium and MS regeneration culture medium, the transformation method comprises the steps of culture of aseptic seedlings, separation and purification of the protoplasts, transformation of the protoplasts, culture and regeneration of the protoplasts and the like.
Description
Technical Field
The invention relates to the field of transgenic engineering, in particular to a high-efficiency separation, transformation and regeneration system of brassica napus protoplasts.
Background
Cabbage type rape (Brassica napus L) is an important oil crop, and can be used for human consumption, and the cake can also be used for food industry and industrial processing such as animal feed. Nowadays, traditional breeding means are increasingly unable to meet the changing variety requirements. The further improvement of rape varieties depends greatly on the extent and depth of the expansion of functional genes. Protoplast manipulation means such as in vitro induction, cell fusion and genetic transformation provide a new source for genetic variation, and meanwhile, somatic cell hybridization also provides a new possibility for genetic material exchange and recombination among distant species. Genome editing technology opens a new field for functional genomics research, and a protoplast transformation system combined with the technology will become a powerful tool for crop improvement.
The key of the protoplast transformation system lies in the regeneration capacity of the induced tissue, so the hypocotyl and the leaf of the brassica napus are important sources for separating and culturing the protoplast. The previous reports mainly use hypocotyls of three cabbage type rape varieties to obtain protoplast regeneration plants, and the protoplast regeneration plants have large genotype dependence, different transformation efficiency, poor test operation repeatability and few successful paradigms.
Disclosure of Invention
Aiming at the defects of different efficiency and poor test operation repeatability of rape protoplasts in the prior art, the invention provides a separation, transformation and regeneration system of rape protoplasts. The first object of the invention is to provide a culture medium combination for separating, transforming and regenerating rape protoplast, which comprises 1/2MS B5 culture medium, A culture medium, B culture medium, C culture medium, MS regeneration culture medium, W5 washing liquid, transformation liquid, solution containing polyethylene glycol, enzymolysis liquid, W5 washing liquid and C: B culture medium; the formula of the 1/2MS B5 culture medium, the MS culture medium, the A culture medium, the B culture medium, the C culture medium and the MS regeneration culture medium comprises the following components:
the MS trace 100X formulation is as follows:
| component (A) | Final concentration mg/L |
| CoCl2·6H2O | 2.5 |
| KI | 83 |
| MnSO4·H2O | 1690 |
| H3BO3 | 620 |
| CuSO4·5H2O | 2.5 |
| Na2MoO4·2H2O | 25 |
| ZnSO4·7H2O | 860 |
The formulation of the iron salt 1000 x is as follows:
| component (A) | Final concentration mg/L |
| FeNaEDTA | 36700 |
The W5 lotion comprises 8.9g/L of sodium chloride, 18.4g/L of calcium chloride, 0.37g/L of potassium chloride and 0.9g/L of glucose;
the composition of the conversion solution comprises 91g/L of mannitol, 14.25g/L of magnesium chloride and 1g/L of ethanesulfonic acid;
the solution containing the polyethylene glycol comprises 400g/L of PEG, 72.9g/L of mannitol and 16.4g/L of calcium nitrate.
The culture medium combination comprises the components of the enzymolysis liquid, wherein 10g/L of sucrose, 10g/L of cellulase R10 and 1g/L of eductase R10 are added into the culture medium C.
The culture medium combination comprises the Evans Blue dye solution and 2.5g/L of Evans Blue powder added into the W5 washing liquid.
The composition of the culture medium combination of the invention comprises the following components in percentage by weight in the ratio of 1: 1, 6g/L of marine spot agarose was added to the mixture.
Another object of the present invention is to provide a method for the isolation, transformation and regeneration of canola protoplasts. The method of the present invention preferably comprises the following steps:
1) cultivation of aseptic seedlings
Sowing the rape seeds with the disinfected surfaces in a 1/2MS B5 culture medium, taking the stem tips and transferring the stem tips to a 1/2MS B5 culture medium to culture for 2-6 weeks when the seeds grow a first true leaf, and obtaining rape aseptic seedlings with young and tender leaves;
2) isolation and purification of protoplasts
Taking the young leaves, cutting into small pieces, soaking in the enzymolysis liquid, sealing an enzymolysis system, keeping out of the sun, and standing for 16-18 h at the temperature of 24-26 ℃;
filtering the mixture after enzymolysis, collecting filtrate, dissolving the filter residue again by using the culture medium C, filtering, collecting filtrate, repeating the operation until no protoplast is separated out, and mixing the obtained filtrates to obtain mixed filtrate;
adding the W5 washing solution into the mixed filtrate, mixing uniformly, centrifuging, and sucking a protoplast layer; adding the W5 lotion again, and obtaining the precipitate after centrifugation as the protoplast;
3) transformation of protoplasts
Disinfecting plasmid DNA, dissolving the plasmid DNA in ultrapure sterile water to form a plasmid DNA system, dissolving the protoplast in the transformation solution to form a protoplast suspension 1, mixing the two, adding the solution containing polyethylene glycol into the mixed system, and standing at room temperature for 10-20 min;
adding the W5 solution into the mixed system, uniformly mixing, and centrifuging to obtain a precipitate, namely the converted protoplast;
4) cultivation of protoplasts, selection of transformed lines and regeneration
Dissolving the transformed protoplast in the culture medium C to prepare a protoplast suspension 2, mixing the protoplast suspension 2 with the culture medium C and the culture medium B, and then culturing the protoplast cells until cell division occurs for multiple times;
and transferring the protoplast after cell division into the culture medium A added with antibiotics, transferring the protoplast to the MS regeneration culture medium after 5-6 weeks of callus, transferring the protoplast to the MS regeneration culture medium when the callus grows to 8-10mm, transferring the protoplast to the MS regeneration culture medium again to grow cluster buds, cutting the cluster buds after 1-2 weeks of growth, placing the cluster buds on the MS regeneration culture medium again, transferring the cluster buds to the MS regeneration culture medium when the buds grow to 3-4cm, inducing to root, transplanting and performing strong seedling culture to obtain the rape plant.
In step 2) of the method of the present invention, a filter membrane having a pore size of 70 μm is used for the filtration operation.
The culture conditions of the protoplasmic somatic cells in the culture medium C: B in the step 4) of the method are 22-26 h under the dark condition at the temperature of 22-25 ℃, then the cells are cultured for 5 d-7 d under the dark condition, preferably 24h under the dark condition at the temperature of 24 ℃, and then the cells are cultured for 6d under the dark condition.
In the method, the culture condition in the A culture medium added with the antibiotic is shaking culture at 60-100 rpm under the dark light condition of 22-26 ℃, preferably shaking culture at 80rpm under the dark light condition of 24 ℃;
the culture condition of the callus or the bud in the MS regeneration culture medium is 22-26 ℃ dark light culture, preferably 24 ℃ dark light culture;
the culture condition of the sprouts on the MS culture medium is 22-26 ℃ dark light culture, and preferably 24 ℃ dark light culture.
The dosage of each reagent in the invention is determined according to the actual experimental requirements.
The culture system is suitable for separating, transforming and regenerating protoplasts of all rape varieties, and preferably selects No. 10 of Zhongshuang, No. 41 of Nanyang, No. 49 of cloud oil, No. 11 of Ningza, No. 9603 and No. 11 of Qin you in cabbage type rape (Brassica napus L).
Advantageous effects
The invention discloses a high-efficiency separation, transformation and regeneration system of Brassica napus (Brassica napus L) protoplast and a special culture medium. The invention takes rape sterile seedlings as starting materials, adopts cellulase R-10 and isolation enzyme R-10 to digest rape mesophyll tissues and separates protoplasts by a density gradient sedimentation method, thus obtaining the high-purity protoplasts. Target genes are introduced into a rape protoplast genome through polyethylene glycol mediation, and a large number of rape transgenic plants are cultivated through liquid shallow layer culture and subsequent differentiation regeneration.
On one hand, the invention can separate out a large amount of high-purity protoplasts, on the other hand, the plasmid DNA can be successfully and efficiently transferred into the protoplasts, and simultaneously, the protoplasts can be successfully cultured into transgenic plants with a large amount of high expression. Lays a foundation for the mass production of the subsequent rape fine breed cultivation.
The invention can provide possibility for functional genomics research such as subcellular localization, promoter expression and the like and variety improvement such as plant somatic cell fusion, genetic transformation, genome editing and the like. The invention is successfully applied to the Chinese main-cultivated rape variety, and makes a preliminary exploration for the improvement and the genetics research of the rape variety.
Drawings
FIG. 1 shows protoplasts counted after staining;
FIG. 2 is the first cell division of a protoplast;
FIG. 3 shows the culture of protoplasts after multiple divisions in medium A.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
First, mother liquor and culture medium for preparing protoplast separation, transformation and regeneration
The formula and the preparation method of the 1/2MS B5 culture medium, the MS culture medium, the A culture medium, the B culture medium, the C culture medium, the MS regeneration culture medium, the W5 washing liquid, the transformation liquid, the enzymolysis liquid, the Evans Blue dye liquid, the enzymolysis liquid, the C, B culture medium and the solution containing the polyhexamethylene glycol are as follows:
1/2 formula of MS B5 medium, MS medium, A medium, B medium, C medium, MS regeneration medium is shown in Table 1:
TABLE 1
The trace 100 x formulation is shown in table 2:
TABLE 2MS trace 100X formulation
| Component (A) | Final concentration mg/L | 500mL of mother liquor (g) |
| CoCl2·6H2O | 2.5 | 0.00125 |
| KI | 83 | 0.0415 |
| MnSO4·H2O | 1690 | 0.845 |
| H3BO3 | 620 | 0.31 |
| CuSO4·5H2O | 2.5 | 0.00125 |
| Na2MoO4·2H2O | 25 | 0.0125 |
| ZnSO4·7H2O | 860 | 0.43 |
The formulation of the iron salt 1000 x is shown in table 3:
TABLE 3 iron salt 1000 × formulation
| Component (A) | Final concentration mg/L | 500mL of mother liquor (g) |
| FeNaEDTA | 36700 | 18.35 |
W5 lotion: taking 8.9g of sodium chloride, 18.4g of calcium chloride, 0.37g of potassium chloride and 0.9g of glucose, adding water to a constant volume of 1L, adjusting the pH value to 5.8-6.0 by using 1M KOH, and sterilizing at 121 ℃ for 20min under high pressure.
Conversion solution: adding 9.1g of mannitol, 1.425g of magnesium chloride and 0.1g of ethanesulfonic acid into water to a constant volume of 100mL, adjusting the pH value to 5.6 by using 1M KOH, filtering and sterilizing.
Solution containing polyethylene glycol (PEG): dissolving 40g PEG in 100mL solution containing 7.29g mannitol and 1.64g calcium nitrate, adjusting pH to 8.0 with 1M KOH, autoclaving at 121 deg.C for 20min, packaging, and storing at-20 deg.C.
Enzymolysis liquid: adding 10g/L sucrose, cellulase R101g, and Segrenase R100.1g into 100mL C culture medium, adjusting pH to 5.6 with 1M KOH, filtering, sterilizing, packaging, and storing at-20 deg.C.
Evans Blue dye solution: 0.25g of Evans Blue powder was dissolved in 100mL of W5 lotion, and filtered and sterilized.
C, culture medium B: 0.6g of sea spot agarose was added to 100mL of C and B as 1: heating the mixed solution at the ratio of 1 for 5s with a microwave oven at high temperature until the agarose is completely dissolved.
Second, the separation, transformation and regeneration of rape protoplast
1. Cultivation of aseptic seedlings
1.1 weighing about 1g of rape seeds (Yangyou No. 7) and putting the rape seeds into a 15mL centrifuge tube, adding 5mL of 75% alcohol solution, shaking for 5min, discarding the waste liquid, and washing with sterile water for 1 time; surface sterilization with a mixed solution of 0.05% Tween20 and 10% hydrogen peroxide by volume for 15min, followed by 5 washes with sterile water.
1.2 dibbling the sterilized seeds in 90mm × 15mm petri dishes (30 granules per dish) containing 1/2MS B5 medium, placing at 24 deg.C with 16 hr light/8 hr dark light cycle and 84 μmol/m light intensity2In a growth chamber under conditions of/s. After 1.5-2 weeks, cutting the strong and pollution-free stem tip when the first true leaf grows out of the seed, transplanting the stem tip into a square culture box (two plants per box) containing 1/2MS B5 culture medium and having the length of 10cm (upper opening) x 7cm (lower opening) x 10cm, and the light intensity is 84 mu mol/m2Cultured for 4 weeks under the condition of/s.
2. Isolation and purification of protoplasts
2.1 cutting 2-4 completely unfolded young leaves under aseptic condition, soaking in enzymolysis solution (about 12mL in 9cm culture dish), removing main vein, and cutting into 8-12mm2Squareness, 3mL of the enzymatic solution was added, the dish was sealed with a sealing film and wrapped with aluminum foil, and left to stand overnight at 25 deg.C (most preferably 16-18 hours).
2.2 gently shaking the petri dish to release the protoplasts sufficiently, filtering the mixture after the enzymolysis with a 70 μm filter membrane, collecting the filtrate, carefully sucking the residues on the filter membrane into the petri dish with a pasteur pipette, adding 5mL of C medium, shaking the petri dish again to clean the zymolyte, filtering again through the filter membrane until no more protoplasts are precipitated, and mixing the obtained filtrates.
2.3 the mixed filtrate was dispensed into 14mL round bottom centrifuge tubes and 1mL of W5 wash was added slowly and centrifuged at 400r/min for 5min, after the solution had separated, the middle green protoplast layer was aspirated with a Pasteur pipette and transferred to a new 14mL centrifuge tube. Add 7mL of W5 wash solution to the tube, gently tilt the tube back and forth to mix the protoplasts, and centrifuge at 400r/min for 5 min. The supernatant was removed, 2-5mL W5 wash was added, mixed in the same manner and placed on ice until use.
Diluting the mixture by 10 times at 2.4, adding 5 μ L dropwise onto a blood cell counting plate, simultaneously adding 5 μ L of 0.25% Evans Blue dye solution dropwise, standing at room temperature for 5min, and counting the number of unstained samples under a microscopeThe viable cell rate is over 90%, and the yield of protoplast is 7.1 × 106(see fig. 1).
3. Transformation of protoplasts
3.1 plasmid DNA (pVec 8-GFP: 35S-hpt-Nos + Ubi-GFP-Nos) was precipitated in absolute ethanol and sterilized, and then dissolved in sterile ultrapure water to a final concentration of 0.7. mu.g/. mu.L. After the counting of the protoplast is finished, centrifuging for 5min at 400r/min, removing supernatant, adding transformation liquid according to a proportion to ensure that the concentration of the resuspended protoplast reaches 1.6 multiplied by 106one/mL.
3.2 Add 300. mu.L (protoplast count 5X 10) to a fresh 14mL centrifuge tube5) Adding 30 μ L plasmid DNA into the protoplast suspension, slowly adding 300 μ L PEG-containing solution (shown in figure) from the periphery of the tube wall while rotating the centrifugal tube, gently shaking the centrifugal tube back and forth in the direction of the tube wall to mix well, standing at room temperature for 15min, and shaking once every 3 min.
3.3 the W5 solution was put into a centrifuge tube in the order of 1mL, 2mL and 3mL, with 2min intervals, and mixed well, and centrifuged at 400r/min for 5 min. Finally, 6mL of W5 solution is added and mixed well, and the mixture is centrifuged at 400r/min for 5 min.
3.4 transient expression experiments, remove the supernatant, add 2.5mL C medium heavy suspension, in 25 ℃ dark conditions after cultured for 24h observation, more than 80% of the protoplasts appear blue.
The specific procedure of the stable expression assay is as in step 4.
4. Cultivation of protoplasts, selection of transformed lines and regeneration
4.1 resuspending the protoplasts in C medium to form a suspension, 0.5mL of the protoplast suspension (number of protoplasts 5X 10)5) Adding into a 60mm × 8mm culture dish, adding 4.5mL of pre-preheated C: B culture medium at about 45 deg.C, and mixing well to make it just spread in the culture dish. And after the culture medium is cooled and solidified, sealing the culture dish, culturing for 24 hours in the dark at the temperature of 24 ℃, and then transferring to the dark for culturing for 6 days. First and subsequent multiple cell divisions can be observed during the culture (see FIG. 2).
4.2 transfer the agar blocks with dividing protoplasts to 250mL petri dishes and add 20mL of A medium supplemented with 25mg/L hygromycin. The culture was performed with shaking at 80rpm under a dark light condition at 24 ℃ (see FIG. 3).
After 4.33-4 weeks, callus formation induced by protoplasts was seen for the first time. After another 5-6 weeks, the calli were transferred to MS regeneration medium and incubated at 24 ℃ in the dark for 1-2 weeks during which time the calli continued to grow. When the callus reaches 8-10mm, transferring the callus to a new MS regeneration culture medium for continuous culture to obtain the resistant callus. After 1-2 weeks, callus was induced to form clumpy shoots. After 1-2 weeks, the normal shoots were excised from the callus and placed on MS regeneration medium and cultured in the dark at 24 ℃.
4.4 when the sprouts grow to 3-4cm, they are transferred to MS regeneration medium and cultured under the condition of 24 ℃ dark light. After 1-3 weeks, the shoots are induced to root. Transplanting the resistance rape to be rooted into 12 × 12cm flowerpot when the overground part of the resistance rape to be rooted grows to about 10cm and more than 2 strong roots are induced2And/s, the photoperiod is 14/10h, the day and night temperature in the seedling stage is 20/15 ℃, the temperature in the booting stage and the day and night temperature in the grouting stage are 30/20 ℃, strong seedlings are grown and propagated, and strong plants are grown.
Example 2
The Brassica napus (Brassica napus L) varieties of Zhongshuang No. 10, Nanyang No. 41, Yunyou No. 49, Ningza No. 11, 9603, Qinyou No. 11 and the like are cultured according to the method in the example 1, and rape transgenic plants are obtained.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (11)
1. A reagent combination for high-efficiency separation, transformation and regeneration of rape protoplast is characterized by comprising 1/2MSB5 culture medium, MS culture medium, A culture medium, B culture medium, C culture medium, MS regeneration culture medium, W5 washing liquid, transformation liquid, solution containing polyhexamethylene glycol, enzymolysis liquid, W5 washing liquid and C culture medium B;
media used in the isolation procedure: 1/2MS B5 culture medium, enzymolysis liquid, C culture medium and W5 washing liquid;
reagents used in the conversion process: a conversion solution, a solution containing the polyhexamethylene glycol, a W5 washing solution;
reagents used in the regeneration process: c culture medium, C is B culture medium, A culture medium, MS regeneration culture medium;
the 1/2MS B5 culture medium, the MS culture medium, the A culture medium, the B culture medium, the C culture medium and the MS regeneration culture medium have the following formulas:
the MS trace 100X formulation is as follows:
The formulation of the iron salt 1000 x is as follows:
The W5 lotion comprises 8.9g/L of sodium chloride, 18.4g/L of calcium chloride, 0.37g/L of potassium chloride and 0.9g/L of glucose;
the composition of the conversion solution comprises 91g/L of mannitol, 14.25g/L of magnesium chloride and 1g/L of ethanesulfonic acid;
the solution containing the polyethylene glycol comprises 400g/L of polyethylene glycol, 72.9g/L of mannitol and 16.4g/L of calcium nitrate;
the composition of the C-B culture medium comprises the following steps of 1: 1, adding 6g/L of marine spot agarose into the mixed solution; the composition of the enzymolysis liquid comprises that 10g/L of sucrose, 10g/L of cellulase R10 and 1g/L of eductase R10 are added into the culture medium C.
2. The reagent combination of claim 1, further comprising Evans Blue dye solution, wherein the Evans Blue dye solution comprises 2.5g/L Evans Blue powder added to the W5 washing solution.
3. The reagent combination according to claim 1 or 2, wherein the pH of the W5 lotion is 5.8 to 6.0; the pH of the conversion solution is 5.6; the pH value of the solution containing the polyethylene glycol is 8.0, and the pH value of the enzymolysis solution is 5.6.
4. A method for the isolation, transformation and regeneration of oilseed rape protoplasts using the reagent combination of any one of claims 1 to 3.
5. The method of claim 4, comprising the steps of:
1) cultivation of aseptic seedlings
Sowing the rape seeds with the disinfected surfaces in a 1/2MS B5 culture medium, taking the stem tips and transferring the stem tips to a 1/2MS B5 culture medium to culture for 2-6 weeks when the seeds grow a first true leaf, and obtaining rape aseptic seedlings with young and tender leaves;
2) isolation and purification of protoplasts
Taking the young leaves, cutting into small pieces, soaking in the enzymolysis liquid, sealing an enzymolysis system, keeping out of the sun, and standing for 16-18 h at the temperature of 24-26 ℃;
filtering the mixture after enzymolysis, collecting filtrate, dissolving the filter residue again by using the culture medium C, filtering, collecting filtrate, repeating the operation until no protoplast is separated out, and mixing the obtained filtrates to obtain mixed filtrate;
adding the W5 washing solution into the mixed filtrate, mixing uniformly, centrifuging, and sucking a protoplast layer; adding the W5 lotion again, and obtaining the precipitate after centrifugation as the protoplast;
3) transformation of protoplasts
Disinfecting plasmid DNA, dissolving the plasmid DNA in ultrapure sterile water to form a plasmid DNA system, dissolving the protoplast in the transformation solution to form a protoplast suspension 1, mixing the two, adding the solution containing polyethylene glycol into the mixed system, and standing at room temperature for 10-20 min;
adding the W5 solution into the mixed system, uniformly mixing, and centrifuging to obtain a precipitate, namely the converted protoplast;
4) cultivation of protoplasts, selection of transformed lines and regeneration
Dissolving the transformed protoplast in the culture medium C to prepare a protoplast suspension 2, mixing the protoplast suspension 2 with the culture medium C and the culture medium B, and then culturing the protoplast cells until cell division occurs for multiple times;
and transferring the protoplast after cell division into the culture medium A added with antibiotics for shake culture, transferring the protoplast to the MS regeneration culture medium after 5-6 weeks of callus, transferring the protoplast to the MS regeneration culture medium when the callus grows to 8-10mm, transferring the protoplast to the MS regeneration culture medium again to grow cluster buds, cutting the cluster buds after 1-2 weeks of growth, placing the cluster buds on the MS regeneration culture medium again, transferring the cluster buds to the MS regeneration culture medium when the young buds grow to 3-4cm, inducing rooting, and transplanting for strong seedling culture to obtain the rape plant.
6. The method according to claim 5, wherein the culturing conditions of the protoplast cells in the C: B culture medium in the step 4) are 22-26 h at 22-25 ℃ in the dark, and then culturing for 5-7 d in the dark.
7. The method according to claim 6, wherein the culture in step 4) is carried out at 24 ℃ for 24 hours in the dark and then transferred to the dark for 6 days.
8. The method according to any one of claims 5 to 7, wherein the culture conditions in the antibiotic-supplemented A medium are shaking culture at 60 to 100rpm under dark light conditions at 22 to 26 ℃,
the culture condition of the callus or the bud in the MS regeneration culture medium is 22-26 ℃ dark light culture;
and the culture condition of the sprouts on the MS culture medium is 22-26 ℃ dark light culture.
9. The method according to claim 8, wherein the culture conditions are 24 ℃ dark light culture.
10. The method according to claim 5 or 6, wherein the culture conditions in the antibiotic-supplemented A medium are: the culture was carried out with shaking at 80rpm under a dark light condition at 24 ℃.
11. Use of the method of any one of claims 5 to 10 for the isolation, transformation and regeneration of protoplasts of brassica napus No. 10, nanyang 41, yun oil No. 49, ninza No. 11, 9603, qin you No. 11.
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| CN105941148B (en) * | 2016-05-12 | 2018-08-03 | 上海交通大学 | Petunia mesophyll protoplast isolation and culture method |
| 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 |
| 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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| CN101139582A (en) * | 2007-08-10 | 2008-03-12 | 中山大学 | Method for acquiring banana somatic cell hybrid by using protoplast asymmetric fusion technology |
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