Efficient banana protoplast electrofusion method

Abstract

The invention belongs to the technical field of banana breeding, and particularly relates to an efficient banana protoplast electrofusion method. The banana fusion efficiency and protoplast activity are remarkably improved by providing a culture medium combination consisting of a starting culture medium M0-1, a callus induction culture medium M0-2, a callus subculture medium M0-3, an embryonic callus suspension culture medium M0-4, a protoplast culture medium M0-5 and a nursing culture medium M0-6; on the basis, the invention also provides an efficient banana protoplast electrofusion method, and a set of efficient and perfect system for culturing the protoplast until a regenerated plant is obtained is established by performing callus induction, ECS establishment, embryogenesis, plant regeneration and other operations on a banana explant. And a scientific technical reference is provided for introducing a target gene and creating an excellent plant variety.

Description

Technical Field

[0001] The invention belongs to the technical field of banana breeding, and in particular relates to a high-efficiency banana protoplast electrofusion method. Background Art

[0002] Banana (Musa spp.) is a perennial herbaceous plant of the genus Musa in the family Musaceae. It is an important economic and food crop in tropical and subtropical regions. It is widely distributed in tropical and subtropical regions around the world. It is listed as one of the world's four major fruits along with apples, grapes and citrus. Bananas are grown in more than 130 countries and regions around the world. In 2021, the global banana planting area was 80.0529 million mu, an increase of 1.266 million mu from 2020, a year-on-year increase of about 1.6%. In 2022, my country's banana planting area reached 5.05 million mu, a decrease of 351,200 mu from 2021, a year-on-year decrease of 6.5%; the output was 11.7768 million tons. Affected by comprehensive factors such as the decline in area, the year-on-year increase was only 52,600 tons, a year-on-year increase of 0.4%.

[0003] For a long time, bananas have mainly relied on asexual reproduction, and many varieties generally show sterility, which has led to a rapid reduction in its genetic diversity. Although protoplast fusion technology can overcome the sexual reproduction barriers of plants and is an important tool for exploring the beneficial genes of parents and cultivating excellent varieties, it still has the disadvantages of low fusion rate and weak protoplast vitality. Summary of the invention

[0004] The invention aims to provide an efficient banana protoplast electrofusion method, which has the characteristics of high fusion rate and strong protoplast activity.

[0005] The present invention provides a culture medium combination, which includes: one or more of a starting culture medium M0-1, a callus induction culture medium M0-2, a callus subculture culture medium M0-3, an embryonic callus suspension culture medium M0-4, a protoplast culture medium M0-5 and a nursing culture medium M0-6;

[0006] The M0-1 is based on MS medium and also includes the following components: 3.5-4.5 mg / L 2,4-D, 0.5-1.5 mg / L biotin, 0.5-1.5 mg / L glutamic acid, 0.5-1.5 mg / L 6-BA, 8-12 g / L agar and 25-30 g / L sucrose;

[0007] The M0-2 is based on MS medium and also includes the following components: 3.5-4.5 mg / L 2,4-D, 0.5-1.5 mg / L biotin, 0.5-1.5 mg / L glutamic acid, 0.5-1.5 mg / L auxin, 0.5-1.5 mg / L naphthaleneacetic acid, 8-12 g / L agar, 25-30 g / L sucrose and 0.1-0.2 g / L malt extract;

[0008] The M0-3 is based on MS medium and also includes the following components: 4.5-5.5 mg / L of Morel vitamins, 1.5-1.75 g / L of dipotassium hydrogen phosphate, 3.5-4.5 mg / L of 2,4-D, 8-12 g / L of agar and 25-30 g / L of sucrose;

[0009] The M0-4 is based on MS medium and also includes the following components: 0.5-1.5 mg / L biotin, 650-680 μmol / L glutamine, 80-120 mg / L MES, and 25-30 g / L sucrose;

[0010] The M0-5 uses MS medium as the basic medium and also includes the following components: 80-100 mg / L MES, 80-100 mg / L inositol, 0.5-1.5 mg / L biotin, 3.5-4.5 mg / L 2,4-D, 8-12 g / L agar, 35-40 g / L sucrose and 75-85 g / L glucose;

[0011] The M0-6 uses MS culture medium as the basic culture medium and also includes the following components: 4.5-5.5 mg / L of Morel and Wetmore vitamins, 1.5-2.5 mg / L of 2,4-D, 200-250 mg / L of glucose, 70-80 g / L of maltose, 20-25 g / L of sucrose and 1.5-2.5 mL / L of coconut milk;

[0012] The pH values ​​of M0-1, M0-2, M0-3, M0-4, M0-5 and M0-6 are 5.7-5.8 respectively.

[0013] Preferably, the M0-1 uses MS medium as the basic medium and further comprises the following components: 4 mg / L 2,4-D, 1 mg / L biotin, 1 mg / L glutamic acid, 1 mg / L 6-BA, 10 g / L agar and 30 g / L sucrose;

[0014] The M0-2 is based on MS medium and also includes the following components: 4 mg / L 2,4-D, 1 mg / L biotin, 1 mg / L glutamic acid, 1 mg / L auxin, 1 mg / L naphthaleneacetic acid, 10 g / L agar, 30 g / L sucrose and 0.1 g / L malt extract;

[0015] The M0-3 is based on MS medium and also includes the following components: 5 mg / L Morel and Wetmore vitamins, 1.75 g / L dipotassium hydrogen phosphate, 4 mg / L 2,4-D, 10 g / L agar and 30 g / L sucrose;

[0016] The M0-4 uses MS medium as the basic medium and also includes the following components: 1 mg / L biotin, 680 μmol / L glutamine, 100 mg / L MES, and 30 g / L sucrose;

[0017] The M0-5 uses MS medium as the basic medium and also includes the following components: 100 mg / L MES, 100 mg / L inositol, 1 mg / L biotin, 4 mg / L 2,4-D, 10 g / L agar, 40 g / L sucrose and 80 g / L glucose;

[0018] The M0-6 is based on MS culture medium and also includes the following components: 5 mg / L Morel and Wetmore vitamins, 2 mg / L 2,4-D, 250 mg / L glucose, 72 g / L maltose, 20 g / L sucrose and 2 ml / L coconut milk;

[0019] The pH value of the M0-1 is 5.8;

[0020] The pH values ​​of M0-2, M0-3, M0-4, M0-5 and M0-6 are all 5.7.

[0021] The present invention provides application of the culture medium combination described in the above technical solution in promoting banana protoplast fusion.

[0022] Preferably, the banana varieties include: one or more of Brazilian banana, pink banana, Chinese banana, large banana, red banana, small-fruited wild banana, and wild banana.

[0023] Preferably, the fusion method includes: electrofusion.

[0024] The present invention provides a method for efficient banana protoplast electrofusion, which uses the culture medium combination described in the above technical solution to perform electrofusion, comprising the following steps:

[0025] Using M0-1 to initiate culture of banana explants to obtain cultured explants;

[0026] performing callus culture on the cultured explants in sequence using M0-2 and M0-3 to obtain embryonic callus tissue;

[0027] Using M0-4 to perform suspension culture on the embryonic callus to obtain embryonic suspension cells;

[0028] Sequentially performing enzymatic separation and purification on the embryonic suspension cells to obtain banana recipient protoplasts and banana donor protoplasts;

[0029] Electrofusion of the banana recipient protoplasts and the banana donor protoplasts at a volume ratio of 1:1 to obtain fused cells;

[0030] After adjusting the density of fusion cells by using M0-5, the cells were cultured in M0-6 to obtain banana protoplasm fusion hybrid cells.

[0031] The complex enzyme solution used for the enzymatic separation comprises cellulase, pectinase and macerate.

[0032] Preferably, the mass ratio of the cellulase, pectinase and macerate is 3:0.1:1.

[0033] Preferably, the enzymatic hydrolysis time is 10 hours; when performing the enzymatic hydrolysis separation, the volume ratio of the embryonic suspension cells to the complex enzyme solution is 1:5-10.

[0034] Preferably, the concentrations of the banana recipient protoplasts and banana donor protoplasts are 3-5×10 5 Pieces / mL.

[0035] Preferably, the parameters of the electrofusion include: an alternating electric field of 200 V / cm, an action time of 30 s, a DC pulse voltage of 1500 V / cm, a pulse time of 40 μs, and a pulse number of 2 times.

[0036] Beneficial effects:

[0037] The invention provides a culture medium combination, which comprises: a start-up culture medium, a callus induction culture medium, a protoplast culture medium, and one or more of M1 and M2 culture mediums. By specifically screening the components of each culture medium, the banana fusion efficiency and the activity of the protoplasts are significantly improved.

[0038] Based on the above technical advantages, the present invention provides the application of the culture medium combination described in the above technical solution in promoting banana protoplast fusion. By carrying out operations such as callus induction, ECS establishment, embryogenesis and plant regeneration on banana explants, an efficient and complete system for protoplast culture is established until regenerated plants are obtained, and the fusion efficiency of banana and the vitality of protoplasts are significantly improved. Therefore, the technical solution of the present invention provides a scientific technical reference for introducing target genes and creating excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0040] Figure 1 The effects of different enzymolysis times provided by the present invention on protoplast separation;

[0041] Figure 2 The effects of different complex enzyme solutions provided by the present invention on protoplast separation;

[0042] Figure 3 A diagram of protoplast clusters provided by the present invention;

[0043] Figure 4 The banana leaf protoplast separation provided by the present invention;

[0044] Figure 5 Banana protoplast vitality detection provided by the present invention;

[0045] Figure 6 The invention provides banana protoplast electrofusion. DETAILED DESCRIPTION

[0046] In the present invention, unless otherwise specified, the reagents, equipment and methods used are all conventionally selected.

[0047] In order to further illustrate the present invention, the scheme provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0048] In order to verify the effect of different enzymatic hydrolysis times on protoplast separation, Experimental Examples 1-5 were carried out.

[0049] Test Example 1

[0050] (1) Sample collection: The flower buds of healthy plants (specifically: Musa Cavendish subgropp cv. Baxi, AAA) and Musa acumnata, AA) were collected one month after budding on sunny days, and the fruit axis was cut 6 cm above the base of the flower bud to obtain the samples to be processed;

[0051] (2) Material preparation and disinfection: Use a sterile knife to peel off the flower bud bracts in step (1) layer by layer from the base to about 5 cm in length and 3 cm in width, and wipe with 75% alcohol by volume for disinfection;

[0052] (3) Culture medium preparation and inoculation: Continue to peel the flower buds in step (2) under sterile conditions until the diameter is about 1 cm, then cut them into 6-8 pieces longitudinally and transfer them to M0-1 culture medium, culture them in the dark at 25°C to obtain immature inflorescences for start-up culture; wherein the M0-1 culture medium is: MS + 4 mg / L 2,4-D + 1 mg / L biotin + 1 mg / L glutamic acid + 1 mg / L 6-BA + 10 g / L agar + 30 g / L sucrose, pH 5.8;

[0053] (4) Callus induction and subculture: When the immature inflorescence tissue initiated in step (3) is enlarged, it is transferred to M0-2 medium (M0-2 medium is: MS + 4 mg / L 2,4-D, 1 mg / L biotin + 1 mg / L glutamic acid + 1 mg / L auxin + 1 mg / L naphthaleneacetic acid + 10 g / L agar + 30 g / L sucrose + 0.1 g / L malt extract, pH is 5.8) for callus induction culture to obtain light yellow loose embryonic callus growing around it; the light yellow loose embryonic callus is transferred to M0-3 medium (M0-3 medium is: MS + 5 mg / L Morel and Wetmore vitamin + 1.75 g / L potassium dihydrogen phosphate + 4 mg / L 2,4-D + 10 g / L agar + 30 g / L sucrose, pH 5.7) to obtain embryonic callus with uniform growth, loose texture and light yellow color;

[0054] (5) ECS establishment: About 2 g of embryonic callus tissue with uniform growth, loose texture and light yellow color in step (4) was selected and placed on sterile filter paper. The tissue was gently broken with forceps and a scalpel, and then added to a 100 mL Erlenmeyer flask containing 40 mL of liquid culture medium M0-4 (M0-4 culture medium is: MS + 1 mg / L biotin + 680 umol / L glutamine + 100 mg / L MES + 30 g / L sucrose, pH 5.7) and cultured in suspension on a shaking table at 110 r / min in the dark at a culture temperature of 27°C.

[0055] In the first month of culture, the fresh M0-4 liquid medium was replaced every 7 days, and the large suspended particles that were difficult to disperse were filtered with a 900 μm (18 mesh) pore size screen until there were no brown particles in the suspended culture; after the first month, the medium was replaced every 15 days, and 1.5% (v / v) subculture was performed. After the culture was relatively dispersed, the larger cell clusters were removed with a 154 μm (100 mesh) filter; the growth of callus tissue and the growth changes of suspended cells or cell clusters were regularly observed until uniform and stable embryonic suspended cells were obtained;

[0056] (6) Protoplast separation and purification: Take the embryonic cell suspension system that has been subcultured for 15 days in step (5) and has a stable physiological state, filter it through a 200 μm cell sieve, place the uniform small cell clusters in the culture solution in M0-4 medium and culture it for another 7 days, let it stand and settle, and then remove part of the supernatant; centrifuge again to remove the supernatant and add M0-4 liquid culture medium to obtain embryonic suspension cells; add 15 mL of composite enzyme solution (composite enzyme solution: 3 wt% cellulase + 0.1 wt% pectinase + 1 wt% matricase + 15.7 g / L KCl + 7.8 g / L Cacl) according to the volume ratio of embryonic suspension cells to composite enzyme solution of 1:5-10. 2+ 100mg / L MES (2-(N-morpholino)ethanesulfonic acid), fixed to volume with sterile water) to obtain a mixture; mannitol was used as an osmotic pressure regulator to adjust the pH of the complex enzyme solution to 5.7, and then sterilized by filtering through a 0.22μm diameter microporous filter membrane, and then enzymolyzed on a shaker at 50r / min under 27°C dark conditions for 8h. After the enzymolysis was completed, the enzyme solution was filtered with a 100μm cell sieve, centrifuged at 1000r / min for 5 minutes, and the supernatant was carefully aspirated and washed twice with M0-5 medium (M0-5 medium is MS+100mg / L MES+100mg / L inositol+1mg / L biotin+4mg / L 2,4-D++40g / L sucrose+80g / L glucose, pH 5.7), the cell counting plate was used to calculate the density, and the density was adjusted to 3-5×10 5 / mL, and obtain parental protoplasts (see Figure 4 ) ; FDA fluorescence staining was used to detect protoplast viability (see Figure 5 ).

[0057] (7) Protoplast fusion and fusion cell culture: The electrofusion instrument is the "CRY-3" model of Ningbo Xinzhi Biotechnology Co., Ltd.;

[0058] The banana parent protoplasts treated in step (6) are mixed in a ratio of 1:1 to obtain a protoplast mixture; the protoplast mixture is added to the slide electrode groove by pipetting, and the fusion parameters are set (specific parameters are: alternating electric field (AC): 200V / cm, AC action time: 30s, direct current pulse voltage (DC): 1500V / cm, DC pulse time: 40μs, pulse number: 2 times), and the electrofusion experiment is performed under room temperature. The protoplasts are first clustered under the alternating electric field (see Figure 3 ), and then fused by DC pulses (see Figure 6 ).

[0059] After fusion is complete, let it stand for 20 minutes, aspirate the protoplast suspension from the slide with a pipette and place it in a 1.5 mL centrifuge tube. Remove the supernatant after centrifugation. Add M0-5 liquid medium (M0-5 liquid medium is: MS + 100 mg / L MES + 100 mg / L inositol + 1 mg / L biotin + 4 mg / L 2,4-D + 40 g / L sucrose + 80 g / L glucose, pH 5.7) and centrifuge to remove the supernatant. Repeat three times to adjust the cell density to 5×10 5 / mL to obtain fused cells; then the fused cells were added to M0-6 medium (M0-6 medium: MS + 5 mg / L Morel and Wetmore vitamins + 2 mg / L 2,4-D + 250 mg / L glucose + 72 g / L maltose + 20 g / L sucrose + 2 mL / L coconut milk; pH value is 5.7) and cultured at 28 ° C in the dark to obtain stable fused cells with regenerated cell walls.

[0060] Test Example 2

[0061] The difference from Experimental Example 1 is that the enzymatic hydrolysis for 10 h in step (6) is replaced by enzymatic hydrolysis for 4 h.

[0062] Test Example 3

[0063] The difference from Experimental Example 1 is that the enzymatic hydrolysis for 10 h in step (6) is replaced by enzymatic hydrolysis for 6 h.

[0064] Test Example 4

[0065] The difference from Experimental Example 1 is that the enzymatic hydrolysis for 10 h in step (6) is replaced by enzymatic hydrolysis for 8 h.

[0066] Test Example 5

[0067] The difference from Experimental Example 1 is that the enzymatic hydrolysis for 10 h in step (6) is replaced by enzymatic hydrolysis for 12 h.

[0068] The yield and activity of different fusion products in Experimental Examples 1-5 were measured respectively. The results are shown in Figure 1 .

[0069] Depend on Figure 1 It can be seen that under the condition of different enzymatic hydrolysis time, the yield of fusion product was the highest at 8.2×10 5 / mL, but the activity of the fusion product was only 82%; when the enzymatic hydrolysis was 8h, although the yield of the fusion product was not high, the activity was as high as 91%.

[0070] In order to verify the effects of different complex enzyme solutions on protoplast separation, Experimental Examples 4 and Experimental Examples 6-10 were carried out.

[0071] Test Example 6

[0072] The difference from Experimental Example 4 is that the enzymes in the complex enzyme solution of step (6) consist of 2 wt% of cellulase, 0.1 wt% of pectinase and 1 wt% of macerate.

[0073] Test Example 7

[0074] The difference from Experimental Example 4 is that the enzymes in the complex enzyme solution of step (6) consist of 2 wt% of cellulase, 0.2 wt% of pectinase and 1 wt% of macerate.

[0075] Test Example 8

[0076] The difference from Experimental Example 4 is that the enzymes in the complex enzyme solution of step (6) consist of 2.5 wt % of cellulase, 0.1 wt % of pectinase and 1 wt % of macerate.

[0077] Test Example 9

[0078] The difference from Experimental Example 4 is that the enzymes in the complex enzyme solution of step (6) consist of 2.5 wt % of cellulase, 0.2 wt % of pectinase and 1 wt % of macerate.

[0079] Test Example 10

[0080] The difference from Experimental Example 4 is that the enzymes in the complex enzyme solution of step (6) consist of 3 wt % of cellulase, 0.2 wt % of pectinase and 1 wt % of macerate.

[0081] The yield and activity of different fusion products in Experimental Examples 6-10 were measured respectively, and the yield and activity of the fusion product in Experimental Example 4 were statistically analyzed. The results are shown in Figure 2 (exist Figure 2 In the figure, A represents the result of Test Example 6; B represents the result of Test Example 7; C represents the result of Test Example 8; D represents the result of Test Example 9; E represents the result of Test Example 4; and F represents the result of Test Example 10).

[0082] Depend on Figure 2It can be seen that under the same enzymatic hydrolysis time, the addition amount of different enzymes in the complex enzyme solution affects the enzymatic hydrolysis effect. Among them, the combination of cellulase, pectinase and macerate in the ratio of 3wt%:0.1wt%:1wt% has the best effect, and the yield of its fusion product is the highest at 7×10 5 / mL, and the best activity is up to 91%.

[0083] Example 1

[0084] An efficient banana protoplast electrofusion method, comprising the following steps:

[0085] (1) Sample collection: On sunny days, flower buds were collected from healthy plants (specifically Musa Cavendish subgroppcv. Baxi, AAA, Musa acumnata, AA) that had bloomed one month later, and the fruit axis was cut 6 cm above the base of the flower bud to obtain the sample to be processed;

[0086] (2) Material preparation and disinfection: Use a sterile knife to peel off the flower bud bracts in step (1) layer by layer from the base to about 5 cm in length and 3 cm in width, and wipe with 75% alcohol by volume for disinfection;

[0087] (3) Culture medium preparation and inoculation: Continue to peel the flower buds in step (2) under sterile conditions until the diameter is about 1 cm, then cut them into 6-8 pieces longitudinally and transfer them to M0-1 culture medium (M0-1 culture medium: MS + 4 mg / L 2,4-D + 1 mg / L biotin + 1 mg / L glutamic acid + 1 mg / L 6-BA + 10 g / L agar + 30 g / L sucrose, pH 5.8), and culture them in the dark at 25°C;

[0088] (4) Callus induction culture: When the immature inflorescence tissue initiated in step (3) is expanded, it is transferred to M0-2 medium (M0-2 medium: MS + 4 mg / L 2,4-D + 1 mg / L biotin + 1 mg / L glutamic acid + 1 mg / L auxin + 1 mg / L naphthaleneacetic acid + 10 g / L agar + 30 g / L sucrose + 0.1 g / L malt extract, pH 5.8) for callus induction culture to obtain light yellow loose embryonic callus growing around it; the light yellow loose embryonic callus is transferred to M0-3 medium (M0-3 medium: MS + 5 mg / L Morel andWetmore vitamin + 1.75g / L potassium dihydrogen phosphate + 4mg / L 2,4-D + 10g / L agar + 30g / L sucrose, pH 5.7) to obtain embryonic callus with uniform growth, loose texture and light yellow color;

[0089] (5) ECS establishment: About 2 g of embryonic callus tissue with uniform growth, loose texture and light yellow color in step (4) was selected and placed on sterile filter paper. The tissue was gently broken with forceps and a scalpel, and then added to a 100 mL Erlenmeyer flask containing 40 mL of liquid culture medium M0-4 (M0-4 culture medium: MS + 1 mg / L biotin + 680 umol / L glutamine + 100 mg / L MES + 30 g / L sucrose, pH 5.7) and cultured in suspension on a shaking table at 110 r / min in the dark at a culture temperature of 27°C.

[0090] In the first month of culture, fresh M0-4 liquid culture medium was replaced every 7 days, and large suspended particles that were difficult to disperse were filtered out with a 900 μm (18 mesh) pore size screen until there were no brown particles in the suspended culture; after one month, the culture medium was replaced every 15 days, and 1.5% (v / v) subculture was performed. After the culture was relatively dispersed, larger cell clusters were removed with a 154 μm (100 mesh) filter; the growth of callus tissue and the growth changes of suspended cells or cell clusters were regularly observed until uniform and stable embryonic suspended cells were obtained;

[0091] (6) Protoplast separation and purification: Take the embryonic cell suspension system that has been subcultured for 15 days in step (5) and has a stable physiological state, filter it through a 200 μm cell sieve, place the uniform small cell clusters in the culture solution in M0-4 medium and culture it for another 7 days, let it stand and settle, and then suck out part of the supernatant; centrifuge again to suck out the supernatant and add M0-4 liquid culture medium to obtain embryonic suspension cells; according to the volume ratio of embryonic suspension cells to complex enzyme solution of 1:5-10, add 10 mL of complex enzyme solution (complex enzyme solution: 3 wt% cellulase + 0.1 wt% pectinase + 1 wt% matricase + 15.7 g / L KCl + 7.8 g / L Cacl 2+ 100 mg / L MES, fixed to volume with sterile water) to obtain a mixture; adjust the pH of the complex enzyme solution to 5.7 with mannitol as an osmotic pressure regulator, and then filter and sterilize it through a 0.22 μm diameter microporous filter membrane, and then hydrolyze it on a shaker at 50 r / min at 27°C in the dark for 8 h, and wash the obtained complex enzyme solution with M0-5 culture medium (M0-5 culture medium: MS+100 mg / L MES+100 mg / L inositol+1 mg / L biotin+4 mg / L 2,4-D+40 g / L sucrose+80 g / L glucose, pH is 5.7) to obtain parental protoplasts.

[0092] (7) Protoplast fusion and fusion cell culture: The electrofusion instrument is the "CRY-3" model of Ningbo Xinzhi Biotechnology Co., Ltd.;

[0093] The parent protoplasts treated in step (6) are mixed in a ratio of 1:1 to obtain a protoplast mixture; the protoplast mixture is added to the slide electrode groove by pipetting, and fusion parameters are set (specific parameters are: alternating electric field (AC): 200V / cm, AC action time: 30s, direct current pulse voltage (DC): 1500V / cm, DC pulse time: 40μs, pulse number: 2 times), and an electrofusion experiment is performed at room temperature to obtain electrofused protoplasts.

[0094] After fusion is complete, let it stand for 20 minutes, aspirate the protoplast suspension from the slide with a pipette and place it in a 1.5 mL centrifuge tube. Remove the supernatant after centrifugation. Add M0-5 culture medium and centrifuge to remove the supernatant. Repeat three times to adjust the cell density to 5×10 5 / mL to obtain fused cells; then the fused cells were added to M0-6 medium (M0-6 medium: MS + 5 mg / L Moreland Wetmore vitamins + 2 mg / L 2,4-D + 250 mg / L glucose + 72 g / L maltose + 20 g / L sucrose + 2 mL / L coconut milk; pH 5.7) and cultured at 28 ° C in the dark to obtain stable fused cells with regenerated cell walls.

[0095] Example 2

[0096] The difference from Example 1 is that in step (1), the sample of banana (genotype AAB) is used to replace the banana.

[0097] Identification of fused cells: The shape, size and staining of the cells in Examples 1 and 2 were observed to determine whether the cells were successfully fused, and the fused cells were fluorescently stained and ploidy analyzed.

[0098] By testing the fusion rate and vitality of the fusion products obtained in Examples 1 and 2, it was found that after adopting the technical solution of the present invention, the fusion rate of the banana with the AAB genotype in Example 2 could reach more than 53.5% and the vitality was 82.4%; the fusion rate of the banana with the AA genotype in Example 1 could reach more than 75.4% and the vitality was 96.1%.

[0099] In summary, the present invention establishes an efficient and complete system for protoplast culture until regenerated plants are obtained by performing callus induction, ECS establishment, embryogenesis and plant regeneration on banana explants, which provides a scientific technical reference for introducing target genes and creating excellent plant varieties.

[0100] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium combination, characterized in that: The culture medium combination includes: one or more of a starting culture medium M0-1, a callus induction culture medium M0-2, a callus subculture culture medium M0-3, an embryonic callus suspension culture medium M0-4, a protoplast culture medium M0-5 and a nursing culture medium M0-6; The M0-1 is based on MS medium and also includes the following components: 3.5-4.5 mg / L 2,4-D, 0.5-1.5 mg / L biotin, 0.5-1.5 mg / L glutamic acid, 0.5-1.5 mg / L 6-BA, 8-12 g / L agar and 25-30 g / L sucrose; The M0-2 is based on MS medium and also includes the following components: 3.5-4.5 mg / L 2,4-D, 0.5-1.5 mg / L biotin, 0.5-1.5 mg / L glutamic acid, 0.5-1.5 mg / L auxin, 0.5-1.5 mg / L naphthaleneacetic acid, 8-12 g / L agar, 25-30 g / L sucrose and 0.1-0.2 g / L malt extract; The M0-3 is based on MS medium and also includes the following components: 4.5-5.5 mg / L of Morel and Wetmore vitamins, 1.5-1.75 g / L of dipotassium hydrogen phosphate, 3.5-4.5 mg / L of 2,4-D, 8-12 g / L of agar and 25-30 g / L of sucrose; The M0-4 is based on MS medium and further includes the following components: 0.5-1.5 mg / L biotin, 650-680 μmol / L glutamine, 80-120 mg / L MES and 25-30 g / L sucrose; The M0-5 uses MS medium as the basic medium and also includes the following components: 80-100 mg / L MES, 80-100 mg / L inositol, 0.5-1.5 mg / L biotin, 3.5-4.5 mg / L 2,4-D, 8-12 g / L agar, 35-40 g / L sucrose and 75-85 g / L glucose; The M0-6 uses MS culture medium as the basic culture medium and also includes the following components: 4.5-5.5 mg / L of Morel and Wetmore vitamins, 1.5-2.5 mg / L of 2,4-D, 200-250 mg / L of glucose, 70-80 g / L of maltose, 20-25 g / L of sucrose and 1.5-2.5 mL / L of coconut milk; The pH values ​​of M0-1, M0-2, M0-3, M0-4, M0-5 and M0-6 are 5.7-5.8 respectively.

2. The culture medium combination according to claim 1, characterized in that: The M0-1 is based on MS medium and also includes the following components: 4 mg / L 2,4-D, 1 mg / L biotin, 1 mg / L glutamic acid, 1 mg / L 6-BA, 10 g / L agar and 30 g / L sucrose. The M0-2 medium is based on MS medium and also includes the following components: 4 mg / L 2,4-D, 1 mg / L biotin, 1 mg / L glutamic acid, 1 mg / L auxin, 1 mg / L naphthaleneacetic acid, 10 g / L agar, 30 g / L sucrose and 0.1 g / L malt extract. The M0-3 is based on MS medium and also includes the following components: 5 mg / L Morel and Wetmore vitamins, 1.75 g / L potassium dihydrogen phosphate, 4 mg / L 2,4-D, 10 g / L agar and 30 g / L sucrose. The M0-4 is based on MS medium and also includes the following components: 1 mg / L biotin, 680 μmol / L glutamine, 100 mg / L MES and 30 g / L sucrose. The M0-5 is based on MS medium and also includes the following components: 100 mg / L MES, 100 mg / L inositol, 1 mg / L biotin, 4 mg / L 2,4-D, 10 g / L agar, 40 g / L sucrose and 80 g / L glucose. The M0-6 is based on MS medium and also includes the following components: Morel and Wetmore vitamins, 1.75 g / L potassium dihydrogen phosphate, 4 mg / L 2,4-D, 10 g / L agar, 40 g / L sucrose and 80 g / L glucose. Wetmore Vitamin 2mg / L2,4-D 250mg / L Glucose 72g / L Maltose 20g / L Sucrose 2ml / L Coconut milk; The pH value of the M0-1 is 5.8; The pH values ​​of M0-2, M0-3, M0-4, M0-5 and M0-6 are all 5.

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3. Use of the culture medium combination according to claim 1 or 2 in promoting banana protoplast fusion.

4. The use according to claim 3, characterized in that: The banana varieties include: one or more of Brazilian banana, pink banana, Chinese banana, large banana, red banana, small-fruited wild banana, and wild banana.

5. The use according to claim 3, characterized in that: The fusion method includes: electrofusion.

6. A method for efficient banana protoplast electrofusion, characterized in that: The electrofusion is carried out using the culture medium combination as claimed in claim 1 or 2, comprising the following steps: Using M0-1 to initiate culture of banana explants to obtain cultured explants; performing callus culture on the cultured explants in sequence using M0-2 and M0-3 to obtain embryonic callus tissue; Using M0-4 to perform suspension culture on the embryonic callus to obtain embryonic suspension cells; Sequentially performing enzymatic separation and purification on the embryonic suspension cells to obtain banana recipient protoplasts and banana donor protoplasts; Electrofusion of the banana recipient protoplasts and the banana donor protoplasts at a volume ratio of 1:1 to obtain fused cells; After adjusting the density of fusion cells by using M0-5, the cells were cultured in M0-6 to obtain banana protoplasm fusion hybrid cells. The complex enzyme solution used for the enzymatic separation comprises cellulase, pectinase and macerate.

7. The method according to claim 6, characterized in that The mass ratio of the cellulase, pectinase and macerate is 3:0.1:

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8. The method according to claim 6, characterized in that The enzymatic hydrolysis time is 10 hours; when performing the enzymatic hydrolysis separation, the volume ratio of the embryonic suspension cells to the complex enzyme solution is 1:5-10.

9. The method according to claim 6, characterized in that The concentrations of banana recipient protoplasts and banana donor protoplasts were 3-5×10 5 Pieces / mL.

10. The method according to claim 6, characterized in that The parameters of the electrofusion include: an alternating electric field of 200 V / cm, an action time of 30 s, a direct current pulse voltage of 1500 V / cm, a pulse time of 40 μs, and a pulse number of 2 times.

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