Method for fusion and callus regeneration of Yu valley and pennisetum alopecuroides protoplast

Abstract

The invention discloses a Yu-grain and pennisetum alopecuroides protoplast fusion and callus regeneration method, which comprises the following steps: step 1, callus suspension cell culture: using young ears of Yu-grain and pennisetum alopecuroides to induce granular calluses, and establishing a callus suspension cell line; step 2, protoplast separation and purification: taking the suspension cell line as a material, carrying out protoplast separation by an enzymolysis method, and purifying the protoplast by a filtration-centrifugation method; step 3, protoplast fusion and callus regeneration: fusing the Yu valley protoplast and the pennisetum alopecuroides protoplast by using an electric fusion method; and taking the fused protoplast, and carrying out moisturizing culture at 27-28 DEG C in the absence of light / scattered light to obtain the regenerated callus of the fused protoplast. All the steps involved in the method are a whole set of culture system, all links are tightly connected to form a whole, and a foundation is laid for obtaining a pennisetum interspecific protoplast fusion regeneration plant.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant biological breeding, and particularly relates to a method for protoplast fusion of Pennisetum glaucum and Pennisetum alopecuroides and its callus regeneration. Background Art

[0002] Plants of the genus Pennisetum Rich. belong to the Gramineae family, and are annual or perennial. They are widely planted in tropical and subtropical regions. There are about 140 species in the world, and 11 species and 2 varieties in China. The main Pennisetum plants cultivated and utilized in China are: Pennisetum glaucum (also known as pearl millet, scientific name: P. glaucum), Pennisetum purpureum, Pennisetum alopecuroides, Pennisetum polystachyon, Pennisetum clandestinum, and some interspecific hybrids. Among them, Pennisetum glaucum is an annual herb of the genus Pennisetum in the Gramineae family, native to north-central Africa, with strong stress resistance, high temperature resistance, drought resistance, or tolerance to infertile soil. It grows fast, has a high seed setting rate, is drought-resistant and water-saving in arid and high-temperature regions, has a high starch content in seeds, a high content of essential amino acids and unsaturated fatty acids, and is the sixth largest cereal crop in the world. It is the main food crop in arid and semi-arid tropical regions such as India and Africa, and is also a high-quality forage grass, with the characteristics of high forage yield, good palatability, and excellent forage quality. Pennisetum alopecuroides is a native plant in China, with a perennial habit, and its leaves and flowers have high ornamental value. The root system of the plant can reach 1.5 - 2.0 meters, with good soil and slope protection functions, tolerance to slight salinity, drought and infertile soil, and strong cold resistance. It can also survive safely at about -20°C, but has a low biological yield and poor palatability. Existing research and practice have shown that it is difficult to carry out sexual interspecific hybridization between Pennisetum glaucum and Pennisetum alopecuroides.

[0003] Protoplasts refer to naked cells from which the cell walls have been removed. Protoplast fusion refers to the technology in which protoplasts from different sources are induced to fuse under certain external conditions without sexual hybridization and are cultured in vitro to regenerate into hybrid plants, also known as somatic hybridization technology. Protoplast fusion can effectively overcome difficulties such as sexual hybridization incompatibility and distant hybridization barriers, and can also transfer cell nuclei, chromosome fragments, or cytoplasm in a targeted manner, effectively overcoming biological barriers such as distant hybridization incompatibility and male and female gamete sterility encountered in traditional breeding, and improving plant resistance and other traits. In recent years, protoplast fusion has played an important role in the improvement of plant varieties such as wheat and potato, and a series of progress and breakthroughs have been made.In the 1980s, the Vasil team of American scholars carried out research on the protoplast fusion of pearl millet with Guinea grass, sugarcane with pearl millet, and einkorn wheat with pearl millet (Peggy Ozias-Akins, Robert J. Ferl, and Indra K. Vasil, Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) + Panicum maximum Jacq. (Guinea grass), Molecular Genetics and Genomics, 1986: 203: 365-370; Zohreh Tabaeizadeh, Robert J. Ferl, and Indra K. Vasil*, Somatic hybridization in the Gramineae: Saccharum officinarum L. (sugarcane) and Pennisetum americanum (L.) Schum. (pearl millet), Proceedings of the National Academy of Sciences of the United States of America, 1986, Genetic, 83: 565616-5619; Vimla Vasil, Robert J. Ferl, and Indra K. Vasil*, Somatic hybridization in the Gramineae: Triticum monococcum L. (Einkorn) + Pennisetum americanum (L.) K. Schum. (Pearl Millet), Journal of Plant Physiology, 1988, 132: 160-163). Callus after protoplast fusion was obtained by the PEG method. Since then, there have been few research papers on the protoplast fusion of the genus Pennisetum.

[0004] In recent years, the Innovation Team for Forage Grass Genetic Breeding and Ecological Application of the Jiangsu Academy of Agricultural Sciences has continuously carried out interspecific hybridization breeding of Pennisetum to improve the cold resistance of pearl millet and the grass yield and forage quality of Pennisetum. Existing research and practice have found that using the methods published in the above-mentioned literature with the collected pearl millet resources cannot achieve high-frequency embryogenic callus suspension culture and a high protoplast fusion rate. It is speculated that this may be related to the interspecific differences between pearl millet / Pennisetum and the fusion method. In addition, using the PEG method for protoplast fusion will have a certain toxic effect on cells, and operations such as multiple washing and centrifugation will affect cell activity and subsequent regeneration ability, greatly reducing the protoplast fusion efficiency. At the same time, there are no reports on the methods for protoplast fusion and callus regeneration of annual pearl millet and highly cold-resistant perennial Pennisetum at home and abroad. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems of the existing technology and provide a method for protoplast fusion and callus regeneration of pearl millet and Pennisetum. In the present invention, granular callus is induced from the young ears of pearl millet and Pennisetum, and a high-frequency embryogenic callus suspension cell line of the donor and recipient is established. Using the suspension cell line as the material, protoplasts are isolated by an enzymatic method to establish a method for efficient isolation of pearl millet and Pennisetum protoplasts. Then, the electrofusion method is used to improve the fusion efficiency of the donor and recipient of Pennisetum and pearl millet protoplasts, and the regenerated callus after the fusion of pearl millet and Pennisetum protoplasts is obtained, laying a foundation for obtaining regenerated plants by protoplast fusion between species of Pennisetum.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention protects a method for protoplast fusion and callus regeneration of pearl millet and Pennisetum, and the method includes the following steps:

[0008] Step 1, callus suspension cell culture: Granular callus is induced from the young ears of pearl millet and Pennisetum, and a high-frequency embryogenic callus suspension cell line of the donor and recipient is established by suspension culture;

[0009] Step 2, protoplast isolation and purification: Using the suspension cell line as the material, protoplasts are isolated by an enzymatic method, and the protoplasts are purified by a filtration-centrifugation method;

[0010] Step 3, protoplast fusion and callus regeneration: Using the electrofusion method, pearl millet protoplasts and Pennisetum protoplasts are fused at a ratio of 1-5:1-5. The fused protoplasts are taken and cultured at 27-28°C in the dark / under scattered light with moisture retention to obtain the regenerated callus of the fused protoplasts.

[0011] In a specific embodiment, in the first step, the induction medium used for induction is: MS + 2 mg / L 2,4-dichlorophenoxyacetic acid + 30 g / L sucrose + 3.75 g / L gel; the suspension medium used for suspension culture is: MS + 30 g / L sucrose + 50 mL / L coconut juice + 2 mg / L 2,4-dichlorophenoxyacetic acid; after adjusting the pH of the above medium to 5.7 - 5.8 with dilute hydrochloric acid and sodium hydroxide, it is autoclaved at 121 °C for 20 min.

[0012] In a more specific embodiment, the specific steps for culturing callus suspension cells are as follows:

[0013] (1) Prepare materials: Select the young spikes of Pennisetum glaucum and Pennisetum alopecuroides as explants;

[0014] (2) Preparation of induction medium and suspension medium:

[0015] Induction medium: MS + 2 mg / L 2,4-dichlorophenoxyacetic acid + 30 g / L sucrose + 3.75 g / L gel; the subculture medium is the same as the induction medium, and the subculture period is 15 - 20 d;

[0016] Suspension medium: MS + 30 g / L sucrose + 50 mL / L coconut juice + 2 mg / L 2,4-dichlorophenoxyacetic acid, adjust the pH to 5.8 with dilute hydrochloric acid and sodium hydroxide; the subculture suspension medium is the same as the suspension medium, and the subculture period is 7 - 14 d;

[0017] After adjusting the pH of the above medium to 5.7 - 5.8 with dilute hydrochloric acid and sodium hydroxide, it is autoclaved at 121 °C for 20 min;

[0018] (3) Take the unemerged young spikes of Pennisetum glaucum and Pennisetum alopecuroides, wipe the leaf sheaths outside the young spikes with 75% alcohol cotton balls under sterile conditions, peel out the young spikes, place them in a culture dish with sterilized and dried filter paper, cut them into spike segments 4 - 5 mm long, inoculate them onto the callus induction medium, and culture them under dark conditions at a temperature of 27 - 28 °C. After 7 - 10 d, loose and transparent callus is first induced at the base of the young spikes. After transferring to the subculture medium and proliferating for 15 - 20 d, granular callus is obtained;

[0019] (4) Inoculate the granular callus of Pennisetum glaucum and Pennisetum alopecuroides into a 250 mL Erlenmeyer flask filled with suspension medium, and transfer them in a ratio of (callus: suspension medium) 1 g: 30 mL. Culture them under dark conditions at a temperature of 27 - 28 °C and a rotation speed of 120 rpm. After culturing for 2 - 3 d each time, pour out the suspension in the flask and add an equal volume of fresh suspension medium. Repeat this step 3 times to obtain the suspension cell line of Pennisetum glaucum and Pennisetum alopecuroides; then subculture according to the volume ratio of (suspension cell line: suspension medium) 1:5.

[0020] In a specific embodiment, in the second step, the components of the enzyme digestion solution for enzymatic hydrolysis are as follows: enzyme digestion solution: 20 g / L cellulase R-10 + 6 g / L macerozyme R-10 + 0.69 g / L 2-(N-morpholino)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate + 0.4 M mannitol, pH 5.7 - 5.8, and the enzymatic hydrolysis time is 6 - 8 h.

[0021] In a more specific embodiment, the specific steps of the protoplast isolation and purification method are as follows:

[0022] (1) Prepare materials: pearl millet and Pennisetum americanum suspension cell lines;

[0023] (2) Prepare the solutions required for protoplast isolation and purification:

[0024] Enzyme digestion solution: 20 g / L cellulase R-10 + 6 g / L macerozyme R-10 + 0.69 g / L 2-(N-morpholino)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate + 0.4 M mannitol, pH 5.7 - 5.8;

[0025] W1 solution: KAO&Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-BA; adjust the pH to 5.7 - 5.8 with dilute hydrochloric acid and sodium hydroxide, autoclave at 121 °C for 20 min, and store at 4 °C after cooling;

[0026] The KAO&Michayluk medium consists of 0.453 g / L CaC12, 0.3 g / L KCl, 0.17 g / L KH2PO4, 1.9 g / L KNO3, 0.147 g / L MgSO4, 0.6 g / L NH4NO3, 0.000025 g / L CoCl2·6H2O, 0.000025 g / L CuSO4·5H2O, 0.0367 g / L FeNaEDTA, 0.003 g / L H3BO3, 0.00075 g / L KI, 0.01 g / L MnSO4·H2O, 0.00025 g / L Na2MoO4·2H2O, 0.002 g / L ZnSO4·7H2O;

[0027] (3) Protoplast isolation: Take the pearl millet and Pennisetum americanum suspension cell lines that have been subcultured for 3 - 4 d, pour out 20 - 25 mL of the relatively clear medium on the upper layer, retain 5 mL of the suspension cell line with particles at the bottom, add 20 mL of the enzyme digestion solution, and perform enzymatic hydrolysis at 27 - 28 °C, 50 rpm, and in the dark for 6 - 8 h. Filter the enzymatically hydrolyzed cell line through a 70 μm cell sieve, aliquot it into 50 mL centrifuge tubes, centrifuge at 200 × g for 3 min, remove the enzyme solution in the upper part of the centrifuge tube, and leave the protoplasts at the bottom;

[0028] (4) Protoplast purification: Add an equal volume of W1 solution, centrifuge at 200×g, discard the upper solution, and retain the protoplasts at the bottom. Repeat this step once to obtain purified protoplasts.

[0029] In a specific embodiment, in the third step, the electrofusion parameters are as follows: alternating current (AC) electric field strength 20 V / cm, AC application time 25 s, post - AC duration 7 s; direct current (DC) pulse strength 80 V / cm, DC pulse time 25 μs, pulse interval time 0.5 s.

[0030] In a more specific embodiment, the specific steps of protoplast fusion and callus regeneration are as follows:

[0031] (1) Prepare materials: Purified protoplasts of Pennisetum glaucum and Pennisetum alopecuroides.

[0032] (2) Prepare the solutions required for protoplast fusion and callus regeneration:

[0033] Electrofusion solution: 0.6 M mannitol + 5 mM CaCl2; The electrofusion solution is filtered and sterilized using a cell filter (0.22 μm).

[0034] W2 medium: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4 - D + 1 mg / L 6 - BA + 0.5 mg / L zeatin + 3.75 g / L agarose.

[0035] (3) Protoplast fusion: Take 1 mL of purified protoplasts of Pennisetum glaucum and Pennisetum alopecuroides respectively, adjust the concentration to 1×10 5 cells / mL, and mix them at a ratio of 1 - 5:1 - 5; Aspirate the mixed protoplasts and place them in the electrode plate. The electrofusion parameters are as follows: alternating current (AC) electric field strength 20 V / cm, AC application time 25 s, post - AC duration 7 s; direct current (DC) pulse strength 80 V / cm, DC pulse time 25 μs, pulse interval time 0.5 s; Cycle once and select the fusion mode; Let the fused protoplasts stand at room temperature for 20 - 30 min, then centrifuge at 100×g for 1 min to remove the supernatant; The electrofusion instrument used is: GEB 15+ / CFB16 - HB fusion instrument from BEX company, and the electrodes are LF501 series gold block electrodes (1 mm); The electrofusion is carried out in a sterile environment. After disinfecting the electrodes with 75% alcohol for 15 min, rinse them with sterile water and disinfect repeatedly.

[0036] (4) Callus regeneration: Resuspend the fused protoplasts with W1 solution and adjust the concentration to 1×10 5per mL. Aspirate 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass petri dish (diameter 3.5 cm), and culture them in the dark / scattered light at 27 - 28 °C with moisturizing; every 7 days, add 0.1 mL of fresh W1 solution to the petri dish until callus is regenerated; when the callus grows to 1.5 - 2 mm, pick it out and transfer it to W2 medium for proliferation.

[0037] Compared with the prior art, the method of the present invention has the following beneficial effects:

[0038] (1) The explant material used in the present invention is young ears. Since the outer surface disinfection does not contact the young ears, the callus induction rate is as high as 100%, which is 7.15% higher than the existing induction rate.

[0039] (2) The granular callus of Pennisetum glaucum and Pennisetum alopecuroides induced by the method of the present invention is easy to establish an embryogenic suspension cell line, and the suspension does not turn brown, with high proliferation efficiency. After 14 days of callus suspension culture, the compact area of the callus is 23.8 times that of the initial value.

[0040] (3) The present invention purifies protoplasts by the filtration - centrifugation method, which is simple and easy to operate. The obtained protoplasts have a complete morphology and high yield, and the protoplast viability is as high as 80%, laying a foundation for subsequent somatic cell fusion, establishment of gene transient transformation system, etc.

[0041] (4) The present invention adopts the electrofusion method. The electrofusion method has no cytotoxicity problem, and the electrofusion efficiency is 37.83% higher than the reported PEG method, and the post - fusion treatment is simple.

[0042] (5) The methods of callus suspension culture, protoplast isolation, protoplast fusion and callus regeneration after fusion involved in the present invention are a complete set of culture systems. Each link is closely connected to form a whole, and the implementation of any step is crucial for the whole process. Description of the Drawings

[0043] Figure 1 Granular callus of Pennisetum glaucum (left) and Pennisetum alopecuroides (right).

[0044] Figure 2 Screening of Pennisetum americanum suspension medium. Among them, Figure 2 A - B: M24 and MS suspensions supplemented with 5 - 15% CM were cultured for 7 days; Figure 2 C - D: Cell morphology in M24 and MS suspensions supplemented with 5 - 15% CM after the first sub - culture for 7 days; Figure 2 E - F: Callus in M24 and MS suspensions supplemented with 5 - 15% CM after the first sub - culture for 7 days.

[0045] Figure 3Sorghum sudanense (left) and Pennisetum alopecuroides (right) suspension cell lines.

[0046] Figure 4 Protoplasts of Sorghum sudanense (left) and Pennisetum alopecuroides (right).

[0047] Figure 5 Protoplast fusion of Sorghum sudanense and Pennisetum alopecuroides.

[0048] Figure 6 Regenerated callus after protoplast fusion of Sorghum sudanense and Pennisetum alopecuroides. Detailed implementation mode

[0049] The present invention will be further described in detail below in conjunction with embodiments. Reagents or equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0050] Induction medium: MS + 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 g / L sucrose + 3.75 g / L gel; The subculture medium is the same as the induction medium, and the subculture period is 15 - 20 d.

[0051] Suspension medium: MS + 30 g / L sucrose + 50 mL / L coconut milk + 2 mg / L 2,4-Dichlorophenoxyacetic acid, adjusted to pH 5.8 with dilute hydrochloric acid and sodium hydroxide; The subculture suspension medium is the same as the suspension medium, and the subculture period is 7 - 14 d.

[0052] W1 solution: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-benzylaminopurine (6-BA).

[0053] W2 medium: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-BA + 0.5 mg / L zeatin + 3.75 g / L gel.

[0054] The KAO & Michayluk medium consists of 0.453 g / L CaC12, 0.3 g / L KCl, 0.17 g / L KH2PO4, 1.9 g / L KNO3, 0.147 g / L MgSO4, 0.6 g / L NH4NO3, 0.000025 g / L CoCl2·6H2O, 0.000025 g / L CuSO4·5H2O, 0.0367 g / L FeNaEDTA, 0.003 g / L H3BO3, 0.00075 g / L KI, 0.01 g / L MnSO4.H2O, 0.00025 g / L Na2MoO4·2H2O, and 0.002 g / L ZnSO4·7H2O.

[0055] After adjusting the pH of the above medium to 5.7 - 5.8 with dilute hydrochloric acid and sodium hydroxide, it is autoclaved at 121 °C for 20 min, and stored at 4 °C after cooling.

[0056] Enzyme solution: 20 g / L cellulase R - 10 + 6 g / L macerozyme R - 10 + 0.69 g / L 2-(N - morpholino)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate + 0.4 M mannitol, pH 5.7 - 5.8;

[0057] Electrofusion solution: 0.6 M mannitol + 5 mM CaCl2; both the above enzyme solution and electrofusion solution are filtered and sterilized with a cell filter (0.22 μm).

[0058] The present invention protects a method for protoplast fusion and callus regeneration of Pennisetum glaucum and Pennisetum americanum, and the method includes the following steps:

[0059] 1. Suspension cell culture of callus:

[0060] Take the unemerged young spikes of Pennisetum glaucum and Pennisetum americanum, wipe the leaf sheaths outside the young spikes with 75% alcohol cotton balls under sterile conditions, peel out the young spikes, place them in a culture dish with sterilized and dried filter paper, cut them into spike segments 4 - 5 mm long, inoculate them onto the callus induction medium, and culture them under dark conditions at a temperature of 27 - 28 °C. After 7 - 10 d, loose and transparent callus is first induced at the base of the young spikes. After transferring to the subculture medium and proliferating for 15 - 20 d, granular callus is obtained.

[0061] The granular callus of the safflower and the Pennisetum was inoculated into a 250 mL triangular flask filled with a suspension medium, and the transfer ratio was (callus: suspension medium) 1 g: 30 mL, and cultured in the dark, at a temperature of 27-28°C, and a rotation speed of 120 rpm. After every 2-3 days of culture, the suspension in the bottle was poured out, and an equal volume of fresh suspension medium was added. After repeating this step 3 times, the safflower and the Pennisetum suspension cell lines were obtained; and then the subculture was performed at a volume ratio of (suspension cell line: suspension medium) of 1:5;

[0062] 2. Protoplast separation and purification:

[0063] Protoplast isolation: Take the suspended cell lines of Miguel and Pennisetum that have been subcultured for 3 to 4 days, pour out 20 to 25 mL of the clearer upper culture medium, retain 5 mL of the suspended cell line with particles at the bottom, add 20 mL of enzymatic hydrolysis solution, and perform enzymatic hydrolysis for 6 to 8 hours at 27 to 28°C, 50 rpm, and in the dark. Filter the enzymatically hydrolyzed cell line with a 70 μm cell sieve, divide it into 50 mL centrifuge tubes, centrifuge at 200×g for 3 min, remove the enzyme solution on the top of the centrifuge tube, and retain the protoplasts at the bottom.

[0064] Protoplast purification: add an equal volume of W1 solution, centrifuge at 200×g, remove the upper solution, keep the protoplasts at the bottom, repeat this step to obtain purified protoplasts.

[0065] 3. Protoplast fusion and callus regeneration:

[0066] Protoplast fusion: Take 1 mL of purified protoplasts from Miguel and Pennisetum, respectively, and adjust the concentration to 1×10 5 / mL, 1:1 mixture; aspirate the mixed protoplasts and place them on the electrode plate, electrofusion parameters: alternating current electric field strength (AC)

[0067] 20V / cm, AC action time 25s, duration after AC 7s; DC pulse intensity (DC) 80V / cm, DC pulse time 25us, pulse interval 0.5s; cycle 1 time, select fusion mode; place the fused protoplasts at room temperature for 20-30min, then centrifuge at 100×g for 1min, and remove the supernatant. The electrofusion instrument used is: BEX GEB 15+ / CFB16-HB fusion instrument, electrode: LF501 series gold block electrode (1mm); electrofusion is performed in a sterile environment, the electrode is disinfected with 75% alcohol for 15min, then rinsed with sterile water, and disinfected repeatedly 3-5 times.

[0068] Callus regeneration: The fused protoplasts were resuspended in W1 solution and the concentration was adjusted to 1×10 5cells / mL. Absorb 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass petri dish (diameter 3.5 cm), and culture them at 27 - 28 °C in the dark / under scattered light with moisture retention; every 7 days, add 0.1 mL of fresh W1 solution to the petri dish until callus regenerates; when the callus grows to 1.5 - 2 mm, pick it out and transfer it to the W2 medium for proliferation.

[0069] The present invention screened and exemplified the methods for callus suspension culture, protoplast isolation, protoplast fusion and callus regeneration after fusion of Pennisetum glaucum and Pennisetum alopecuroides, but the present invention is not limited thereto.

[0070] Example 1 Induction of Callus and Screening of Suspension Medium for Pennisetum glaucum and Pennisetum alopecuroides

[0071] 1. Materials and Methods

[0072] 1.1 Materials

[0073] Young spikes of Pennisetum glaucum

[0074] 1.2 Experimental Design

[0075] (1) Screening of callus induction medium. The callus induction medium was based on MS + 30 g / L sucrose + 3.75 g / L gel, and 5 concentrations of 2,4-D at 0, 0.5, 1.0, 2.0, and 3.0 mg / L and 5 concentrations of kinetin 6-furfurylaminopurine (KT) at 0, 0.05, 0.1, 0.5, and 1 mg / L were added respectively, for a total of 25 medium formulations. Sterilize at 121 °C for 20 min. Inoculate 10 explants in each medium, with 5 replicates. The purpose was to screen the hormone ratio with the highest induction rate. The callus induction rate (%) = the number of induced calli / the total number of inoculated explants × 100%, and the callus browning rate (%) = the number of browned calli / the total number of inoculated explants × 100%. The specific design is as follows:

[0076] Table 1 Design of Combinations of Different Concentrations of 2,4-D and KT Hormones

[0077]

[0078] (2) Suspension medium screening. The media are divided into two types. One is the MS basic medium: MS + 2 mg / L 2,4-D + 30 g / L sucrose; the other is the M24 basic medium: MS + 30 g / L sucrose + 100 mg / L inositol + 2 mg / L 2,4-D + 500 mg / L casein hydrolysate. The above two media are respectively supplemented with different concentrations of coconut milk (CM), and the specific design is as follows: MS + 5% CM, MS + 10% CM, MS + 15% CM, M24 + 5% CM, M24 + 10% CM, and M24 + 15% CM. The compact area of the callus refers to the area occupied by the flattened callus.

[0079] 1.3 Culture method

[0080] Callus induction method: Take the unemerged young panicles of Pennisetum glaucum (L.) R. Br., wipe the leaf sheaths outside the young panicles with 75% alcohol cotton balls under sterile conditions, peel out the young panicles, place them in a culture dish with sterilized and dried filter paper, cut them into spike segments 4 - 5 mm long, inoculate them onto the callus induction medium, and culture them under dark conditions at a temperature of 27 - 28 °C. After 7 - 10 days, loose and transparent callus is first induced at the base of the young panicles, transfer it to the selected induction medium for subculture. After 15 - 20 days of proliferation, granular callus is obtained.

[0081] Suspension cell line culture method: Inoculate the granular callus of Pennisetum glaucum (L.) R. Br. into a 250 mL Erlenmeyer flask containing suspension medium, and the transfer ratio is (callus: suspension medium) 1.0 g: 30 mL. Culture it under dark conditions at a temperature of 27 - 28 °C and a rotation speed of 120 rpm. After culturing for 2 - 3 days each time, pour out the suspension in the flask and add an equal volume of fresh suspension medium. Repeat this step 3 times to obtain the suspension cell line of Pennisetum glaucum (L.) R. Br.; then subculture it according to the volume ratio of (suspension cell line: suspension medium) 1:5.

[0082] 2 Results and analysis

[0083] 2.1 Effects of different combinations of 2,4-D and KT at different concentrations on callus induction rate and browning rate

[0084] There were significant differences in the induction rates of Pennisetum glaucum callus with different concentrations of 2,4-D and KT. As shown in Table 2, in treatment 4, the highest induction rate of Pennisetum glaucum young panicles reached 100% when 2 mg / L 2,4-D was added alone to the induction medium, which was 7.15% higher than the induction rate (93.33%) reported with Pennisetum glaucum seeds as explants. At the same time, it was found that in treatments 9 and 14, that is, when 0.05 and 0.1 mg / L of KT and 2 mg / L of 2,4-D were added, callus induction rates of 90% and 94% were obtained respectively, while the absence of 2,4-D significantly reduced the callus induction rate of Pennisetum glaucum, such as in treatments 1, 6, 11, 16 and 21; however, as the concentrations of both 2,4-D and KT increased, the callus induction rate decreased, such as in treatments 19, 20 and 24, etc. Therefore, treatment 4, that is, the MS medium supplemented with 2 mg / L 2,4-D and further supplemented with 30 g / L sucrose and 3.75 g / L gel, was the optimal induction medium. After subculture in this medium for 15 - 20 d, granular callus was obtained ( Figure 1 ).

[0085] Table 2 Effects of combinations of different concentrations of 2,4-D and KT on callus induction rate and browning rate

[0086]

[0087]

[0088] Note: Different letters in the same column indicate significant differences in data (P < 0.05), the same below.

[0089] 2.2 Effects of different concentrations of coconut milk (CM) on suspension callus

[0090] The concentration of coconut milk had a great influence on the establishment and morphology of the suspension cell line. After culturing callus in M24 and MS suspensions supplemented with 5% CM for 7 d, the colors of both the suspension and the callus were light yellow, while the colors of the suspensions supplemented with 10% CM and 15% CM were significantly darker than that of the 5% CM suspension, and the callus was prone to browning ( Figure 2 : A - B); when after 3 primary suspension cultures and 7 d of the first subculture, the original large - granular callus began to proliferate into small - granular callus ( Figure 2 : E - F), there were a large number of cell clusters composed of spherical cells and few elongated cells in the M24 and MS suspensions supplemented with 5% CM; while the suspensions supplemented with 10% CM and 15% CM mostly had elongated cells and fewer cells obtained ( Figure 2 : C - D). The round cells in the MS suspension supplemented with 5% CM had a more uniform morphology than those in the M24 suspension. Therefore, a suspension cell line with high yield and complete morphology could be obtained using the MS suspension supplemented with 5% CM.

[0091] 2.3 Effect of suspension medium on the proliferation of suspension cell line

[0092] About 1 g of granular Pennisetum americanum callus was respectively inoculated into 30 mL of MS suspension medium with 5% CM, and cultured in suspension for 7 - 10 d. In the first 1 - 3 d, the compact area of the callus did not change much, and the proliferation was very slow at this stage, being in the adaptation stage. After 7 - 10 d of subculture suspension culture, the suspension system entered the high-speed proliferation stage, and the compact area of its callus reached 56.4 cm 2 , which was 23.8 times the compact area of the callus after 7 d of primary suspension culture, clarifying the effect of suspension culture on the proliferation of suspension cell line( Figure 3 ).

[0093] Example 2 Screening of protoplast isolation conditions

[0094] 1. Materials and methods

[0095] 1.1 Materials

[0096] Pennisetum glaucum and Pennisetum americanum granular callus and suspension cell line.

[0097] 1.2 Experimental design

[0098] (1) Screening of enzymatic hydrolysis conditions. A 4-factor 3-level orthogonal experiment was designed for the effects of different enzymatic hydrolysis conditions on the yield and morphology of Pennisetum glaucum protoplasts, with cellulase (15, 20, 25 g / L), macerozyme (6, 8, 10 g / L), mannitol concentration (0.3, 0.4, 0.5 M), and enzymatic hydrolysis time (4, 6, 8 h) to screen the optimal enzymatic hydrolysis solution formula.

[0099] (2) Analysis of the enzymatic hydrolysis effects of granular callus and suspension cell line. 10 g of granular callus and 10 mL of suspension cell line were respectively taken. The upper clear liquid of the suspension cell line was removed, and 40 mL of enzymatic hydrolysis solution was added to each. Four replicates were set for each treatment. Under the conditions of 28 °C, dark, rotation speed of 50 rpm, and enzymatic hydrolysis for 8 h.

[0100] (3) Effects of cellulases of different brands on the enzymatic hydrolysis effect of suspension cell line. Four commonly used cellulase brands on the market, YAKULT, Yuanye, Aladdin, and Macklin, were selected and evaluated from two aspects of enzymatic hydrolysis effect and economy. The enzymatic hydrolysis solution consisted of 20 g / L cellulase, 6 g / L macerozyme R-10 (YAKULT, Japan), 0.69 g / L 2-(N-morpholino)ethanesulfonic acid monohydrate, 1.03 g / L calcium chloride dihydrate, and 0.4 M mannitol, and the pH was adjusted to 5.8 with dilute hydrochloric acid and sodium hydroxide.

[0101] 1.3 Experimental methods

[0102] (1) Enzymatic hydrolysis method. After the suspension cell lines of Pennisetum glaucum and Pennisetum alopecuroides were subcultured 1 - 2 times, on the 3rd - 5th day after subculture, 5 mL of the well - conditioned Pennisetum glaucum callus suspension cell line was selected, added to 20 mL of the enzyme solution mixture, sealed with a sealing film, and enzymatically hydrolyzed at 28 °C in the dark at a rotation speed of 50 rpm. The enzymatically hydrolyzed cell line was filtered through a 70 - μm cell sieve, aliquoted into 50 - mL centrifuge tubes, centrifuged at 200×g for 3 min, the enzyme solution in the upper part of the centrifuge tube was removed, and the protoplasts at the bottom were retained. An equal volume of W1 solution was added, centrifuged at 200×g, the upper solution was removed, and the protoplasts at the bottom were retained. This step was repeated to obtain purified protoplasts. The enzyme solution consisted of cellulase R - 10 (YAKULT, Japan), macerozyme R - 10 (YAKULT, Japan), 0.69 g / L 2 - (N - morpholino) ethanesulfonic acid monohydrate, 1.03 g / L calcium chloride dihydrate, and mannitol. The pH was adjusted to 5.8 with dilute hydrochloric acid and sodium hydroxide and filtered through a bacteria - proof filter.

[0103] (2) Analysis of the enzymatic hydrolysis effects of granular callus and suspension cell lines. Using the granular callus and suspension cell lines cultured by the methods of test designs (1) and (2) in Example 1 as materials, the conditions and methods screened in test design (1) of Example 2 were used for enzymatic hydrolysis and purification of protoplasts.

[0104] (3) Effects of different brands of cellulase on the enzymatic hydrolysis of suspension cell lines. 5 mL of the Pennisetum alopecuroides suspension cell line was taken and added to 20 mL of the enzyme solutions of different brands of cellulase respectively. The other components of the enzyme solution and the enzymatic hydrolysis method were the same as the formula, conditions, and methods screened in test design (1) of Example 1. Each treatment had 4 replicates, and 4 fields of view were counted for each replicate.

[0105] (4) Method for measuring protoplast yield: 10 μL of the protoplast resuspension was pipetted and dropped on a hemocytometer, covered with a cover glass, and counted under an inverted microscope. The counting was repeated four times continuously, and the average value was taken. Method for measuring protoplast viability: 5 mg of FDA was dissolved in 1 mL of acetone as the working solution. Then, 20 μL of the FDA working solution was added to 1 mL of the suspended protoplasts. After reacting at room temperature in the dark for 3 - 4 min, a small amount of the solution was taken and placed on a glass slide for observation under a fluorescence microscope. Protoplasts emitting green fluorescence were viable protoplasts. Protoplast viability = viable protoplasts / total protoplasts in this field of view × 100%. Four representative fields of view were selected for statistics. All the calculation methods for protoplast yield and viability described in this patent application were implemented according to this method.

[0106] 2 Results and Analysis

[0107] 2.1 Screening of enzymatic hydrolysis conditions

[0108] Different enzymatic hydrolysis conditions have a great impact on the yield and viability of pearl millet protoplasts. As can be seen from Table 3, under treatment 4, the highest protoplast yield was obtained, reaching 2.1×10 6 cells / mL, which was significantly higher than that of treatments 2, 3, 8, and 9; the protoplast viability of treatment 4 was also the highest, reaching 80%, which was significantly higher than that of treatments 1, 3, 6, 8, and 9. Treatment 7 also obtained the same protoplast yield as treatment 4, but its protoplast viability was 2.4% lower than that of treatment 4. Combining the protoplast morphology under the microscope, it was found that the protoplasts in treatment 4 had clear edges and complete morphology ( Figure 4 ). Finally, it was determined that under the conditions of 20 g / L cellulase R-10, 6 g / L macerozyme R-10, and 0.4 M mannitol, enzymatic hydrolysis for 8 h could obtain protoplasts with high yield and strong viability. The composition of the enzymatic hydrolysis solution was determined as: 20 g / L cellulase R-10 + 6 g / L macerozyme R-10 + 0.4 M mannitol + 0.69 g / L 2-(N-morpholino)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate.

[0109] Table 3 Effects of different enzymatic hydrolysis conditions on protoplast yield and viability

[0110]

[0111] 2.2 Analysis of enzymatic hydrolysis effects on granular callus and suspension cell line

[0112] Using the same enzymatic hydrolysis solution and conditions to enzymatically hydrolyze the granular callus and suspension cell line of pearl millet, the results are shown in Table 4. Using the suspension cell line, 1.345×10 8 cells / mL protoplasts can be obtained, and the protoplast viability reaches 78.61%; the protoplast yield and viability of the granular callus after enzymatic hydrolysis are 12.5×10 6 cells / mL and 22.6%, respectively, which are 90.71% and 71.25% lower than the yield and viability of the suspension cell line after enzymatic hydrolysis.

[0113] Table 4 Analysis of enzymatic hydrolysis effects on granular callus and suspension cell line

[0114]

[0115]

[0116] 2.3 Effects of different brands of cellulase on enzymatic hydrolysis of suspension cell line

[0117] Cellulase and maceration enzyme are important components of the enzymatic solution. The study on the enzymatic hydrolysis effect of different brands of cellulose showed that there were significant differences in the enzymatic hydrolysis effect of different brands of cellulose on the suspended cell line of Miguel (Table 5). YAKULT cellulase had the best enzymatic hydrolysis effect, and its protoplast yield and activity were relatively optimal, which were 3.15×10 6 / mL and 80.37%; the cellulase protoplast yield of Yuanye brand was the highest, reaching 3.43×10 6 / mL, the protoplast activity reached 75.06%, but there was no significant difference between its protoplast yield and activity and YAKULT cellulase. There was no significant difference between the yield of Aladdin and McLean and the above two brands of cellulase, but the protoplast activity was significantly reduced. YAKULT cellulase had the best enzymatic hydrolysis effect, but from the cost point of view, its price was the most expensive, reaching 135 grams / yuan, which was 131.6 yuan higher per gram than the source leaf brand cellulase. Therefore, from the perspective of economy and enzymatic hydrolysis effect, the source leaf brand cellulase is the best.

[0118] Table 5 Comparative analysis of the enzymatic hydrolysis effects of different brands of cellulases on suspension cell lines

[0119]

[0120] Example 3 Protoplast fusion and callus regeneration

[0121] 1. Materials and Methods

[0122] 1.1 Materials

[0123] Purified protoplasts of Mirifica ovata and Pennisetum truncatum.

[0124] 1.2 Experimental design

[0125] (1) Screening of electrofusion conditions. A four-factor three-level experiment was designed to optimize the fusion parameters: alternating current electric field strength (AC) (15, 20, 25 V / cm), AC action time (20, 30, 40 s), direct current pulse electric field strength (DC) (50, 80, 100 V / cm), and DC pulse time (10, 20, 30 s).

[0126] (2) Effects of PEG fusion and electrofusion on fusion efficiency: The PEG method was carried out using the method of Vasil et al. (1986), and the electrofusion method was carried out using the fusion conditions selected in the experimental design (1) in Example 3.

[0127] (3) Callus regeneration after protoplast fusion. The culture medium used was:

[0128] W1 solution: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-BA.

[0129] W2 medium: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-BA + 0.5 mg / L zeatin + 3.75 g / L gel.

[0130] The above solutions and media were adjusted to pH 5.7 - 5.8 with dilute hydrochloric acid and sodium hydroxide, autoclaved at 121 °C for 20 min, and stored at 4 °C after cooling.

[0131] 1.3 Test methods

[0132] (1) Screening of electrofusion conditions. The purified Pennisetum glaucum and Pennisetum alopecuroides protoplasts were rinsed once with electrofusion solution: 0.6 M mannitol + 5 mM CaCl2, and the concentrations were adjusted to 1×10 5 cells / mL and mixed at a ratio of 1:1; 10 μL of the mixed protoplasts was pipetted into the electrode plate, and the formation of protoplast clusters was observed under an inverted microscope, and then a DC pulse voltage was applied for fusion. The fusion rate was counted under an inverted microscope, and each data was the average of 5 fields of view, without distinguishing between self and allogeneic fusion, and the fusion rate was expressed as the total fusion rate. The above electrofusion solution was filtered and sterilized with a cell filter (0.22 μm).

[0133] (2) Effects of PEG fusion method and electrofusion method on fusion efficiency. The fusion solution was prepared by the method of Vasil et al. (1986), filtered and sterilized, the PEG fusion time was 15 min, and the protoplasts used were the purified protoplasts in the experimental design (1) of Example 3.

[0134] (3) Callus regeneration after protoplast fusion. The fused protoplasts were rinsed with W1 solution and resuspended, and then cultured at 27 - 28 °C in the dark / under scattered light with humidity maintained.

[0135] 2 Results and analysis

[0136] 2.1 Screening of electrofusion conditions

[0137] The clustering effect of protoplasts affects the protoplast fusion rate. The clustering effect of protoplasts is jointly determined by the alternating current (AC) electric field and its action time; the greater the electric field, the faster the protoplasts move, the faster the bead formation, and the longer the action time of the AC electric field, the longer the bead formation. As shown in Table 6, when the AC is 20 V / cm and the AC action time is 25 s, the protoplasts move regularly and can form long clusters, which is suitable for protoplast fusion; while when the direct current (DC) is 80 V / cm and the DC action time is 25 s, the highest fusion rate can be obtained, reaching 9.8%, which is significantly higher than other combinations except treatment 6. Figure 5 The protoplasts are in the process of fusion. Too low or too high DC pulse intensity, too long or too short action time will all affect the protoplast fusion rate. In the experiment, AC of 20 V / cm, AC action time of 10 s, DC of 80 V / cm, and DC action time of 25 s were selected as the optimal conditions for protoplast electrofusion.

[0138] Table 6 Screening of electrofusion conditions

[0139]

[0140] 2.2 Effects of PEG fusion method and electrofusion method on fusion efficiency

[0141] To explore the effects of PEG method and electrofusion method on fusion efficiency, we used PEG fusion method and electrofusion method to fuse the protoplasts of Pennisetum glaucum and Pennisetum alopecuroides respectively. The results are shown in Table 7. The fusion rate of electrofusion method is significantly higher than that of PEG method. The electrofusion method can obtain a fusion rate of 9.4%, which is 37.83% higher than that of PEG method.

[0142] Table 7 Effects of PEG fusion method and electrofusion method on fusion rate

[0143]

[0144] 2.3 Callus regeneration after protoplast fusion

[0145] The fused protoplasts were rinsed with W1 solution and then resuspended, and the concentration was adjusted to 1×10 5 cells / mL. 0.6 mL of the fused protoplasts was pipetted and spread evenly in a sterile glass petri dish (diameter 3.5 cm), and cultured at 27 - 28 °C in the dark / under scattered light with humidity maintained. Every 7 days, 0.1 mL of fresh W1 solution was added to the petri dish. Generally, callus can be regenerated in about 15 - 20 days ( Figure 6 ); when the callus grew to 1.5 - 2 mm, it was picked out and transferred to W2 medium for proliferation.

[0146] In summary, the present invention studied the callus induction, suspension culture, protoplast isolation, protoplast fusion, and callus regeneration after fusion of Pennisetum glaucum and Pennisetum americanum. By screening the callus induction medium, it was found that the medium of MS + 2 mg / L 2,4-D + 30 g / L sucrose + 3.75 g / L gel was the optimal induction medium; using the suspension medium of MS + 5% coconut milk + 30 g / L sucrose + 2 mg / L 2,4-D could obtain a suspension cell line with high yield and complete morphology; about 1 g of granular callus of Pennisetum americanum, after 14 - 20 days of culture, the dense area of its callus was 23.8 times that of the initial suspension culture for 7 days.

[0147] Regarding the conditions for protoplast isolation, the best enzyme digestion solution formula was screened out as: 20 g / L cellulase R-10 + 6 g / L macerozyme R-10 + 0.4 M mannitol + 0.69 g / L 2-(N-morpholino)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate. At 28 °C, under dark conditions, at a rotation speed of 50 rpm, and enzyme digestion for 8 h, protoplasts with high yield and strong vitality could be obtained. Using this enzyme digestion solution and enzyme digestion conditions to digest granular callus and suspension cell lines, it was found that the enzyme digestion yield and vitality of the suspension cell line were significantly higher than those of the granular callus. Different brands of cellulase had a significant impact on the enzyme digestion effect of the suspension cell line. The cellulase of YAKULT had the best enzyme digestion effect, and its protoplast yield and vitality were relatively optimal. However, in terms of both economy and enzyme digestion effect, the cellulase of Yuanye brand was the best.

[0148] Regarding the conditions for protoplast fusion, it was found that when the AC was 20 V / cm, the AC action time was 10 s, the DC was 80 V / cm, and the DC action time was 25 s, the protoplast fusion rate was the highest. The electrofusion method had a significantly higher fusion rate than the PEG method, and the fusion rate was 37.83% higher than that of the PEG method.

[0149] The fused protoplasts were rinsed with W1 solution and then resuspended, and the concentration was adjusted to 1×10 5 cells / mL. 0.6 mL of the fused protoplasts was pipetted and spread evenly in a sterile glass culture dish (diameter 3.5 cm), and cultured at 27 - 28 °C in the dark / scattered light with moisturization; every 7 days, 0.1 mL of fresh W1 solution was added to the culture dish, and callus could generally be regenerated in about 15 - 20 days.

[0150] The implementation modes of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A method for fusion of bioplasts of Milo and Pennisetum and callus regeneration, characterized in that: The method comprises the following steps: Step 1, callus suspension cell culture: using young spikelets of millet and pennisetum to induce granular callus, and establishing high-frequency embryonic callus suspension cell lines of donor and recipient through suspension culture; Step 2, protoplast separation and purification: using suspension cell lines as materials to separate protoplasts by enzymatic hydrolysis, and purifying protoplasts by filtration-centrifugation; Step 3, protoplast fusion and callus regeneration: using the electrofusion method, the protoplasts of Miscanthus annuus and Pennisetum truncatum were fused at a ratio of 1-5:1-5, and the fused protoplasts were cultured at 27-28°C in a dark / scattered light environment to obtain the regenerated callus tissue of the fused protoplasts.

2. The method for fusion of bioplasts of Milo and Pennisetum and callus regeneration according to claim 1, characterized in that: In the step 1, the induction medium used for induction is: MS + 2 mg / L 2,4-dichlorophenoxyacetic acid + 30 g / L sucrose + 3.75 g / L gel; the suspension culture medium used for suspension culture is: MS + 30 g / L sucrose + 50 mL / L coconut juice + 2 mg / L 2,4-dichlorophenoxyacetic acid; the above culture medium is adjusted to pH 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, and then sterilized at 121 ° C. for 20 minutes.

3. The method for fusion of sorghum and Pennisetum bioplastids and callus regeneration according to claim 2, characterized in that: The specific steps of callus suspension cell culture are as follows: (1) Preparation of materials: Select young spikelets of millet and Pennisetum as explants; (2) Prepare induction medium and suspension medium: Induction medium: MS + 2 mg / L 2,4-dichlorophenoxyacetic acid + 30 g / L sucrose + 3.75 g / L gel; the subculture medium is the same as the induction medium, and the subculture cycle is 15 to 20 days; Suspension medium: MS + 30 g / L sucrose + 50 mL / L coconut juice + 2 mg / L 2,4-dichlorophenoxyacetic acid; the subculture suspension medium is the same as the suspension medium, and the subculture period is 7 to 14 days. The above medium is adjusted to pH 5.7 to 5.8 with dilute hydrochloric acid and sodium hydroxide, and then sterilized at 121° C. for 20 minutes; (3) Take the unextracted young spikelets of Miguel and Pennisetum, sterilize the leaf sheath outside the young spikelets under sterile conditions, peel off the young spikelets, place them in a culture dish filled with sterilized and dried filter paper, cut them into 4-5 mm long spike segments, inoculate them on callus induction medium, culture them at a temperature of 27-28°C in the dark, induce loose and transparent callus tissue, and transfer them to a subculture medium to obtain granular callus tissue; (4) The granular callus of Mirella and Pennisetum was inoculated into a culture bottle containing a suspension culture medium, and then the callus: suspension culture medium = 1 g: 30 mL was inoculated. The culture was carried out in the dark at a temperature of 27-28°C and a rotation speed of 120 rpm. After every 2-3 days of culture, the suspension in the bottle was poured out and an equal volume of fresh suspension culture medium was added. After repeating this step 3 times, the Mirella and Pennisetum suspension cell lines were obtained; and then the cells were subcultured at a ratio of suspension cell line: suspension culture medium of 1:

5.

4. The method for fusion of bioplasts of Milo and Pennisetum and callus regeneration according to claim 1, characterized in that: In the step 2, the components of the enzymatic hydrolysis solution used for enzymatic hydrolysis are as follows: 20 g / L cellulase R-10 + 6 g / L cleavage enzyme R-10 + 0.69 g / L 2-(N-morpholine)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate + 0.4 M mannitol, pH 5.7-5.8, and enzymatic hydrolysis time 6-8 h.

5. The method for fusion of sorghum and Pennisetum bioplastids and callus regeneration according to claim 4, characterized in that: The specific steps of the protoplast separation and purification method are as follows: (1) Materials: Suspension cell lines of Mirifica serrata and Pennisetum truncatum; (2) Solutions and culture media required for protoplast separation and purification: Enzyme hydrolysate: 20 g / L cellulase R-10 + 6 g / L cleavage enzyme R-10 + 0.69 g / L 2-(N-morpholine)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate + 0.4 M mannitol, pH 5.7-5.8, the above enzyme hydrolysate is filtered and sterilized with a cell filter; W1 solution: KAO&Michayluk medium + 72.08g / L glucose + 1.25g / L sucrose + 2mg / L 2,4-D + 1mg / L6-BA, adjusted to pH 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, sterilized at 121°C for 20min, and stored at 4°C after cooling; the KAO&Michayluk medium consists of 0.453g / L CaC12, 0.3g / L KCl, 0.17g / L KH2PO4, 1.9g / L KNO3, 0.147g / L MgSO4, 0.6g / L NH4NO3, 0.000025g / L CoCl2·6H2O, 0.000025g / L CuSO4·5H2O, 0.0367g / L FeNaEDTA, 0.003g / L H3BO3, 0.00075g / L KI, 0.01g / L MnSO4·H2O, 0.00025g / LNa2MoO4·2H2O, 0.002g / L ZnSO4·7H2O; (3) Protoplast separation: Take the suspension cell lines of Miguel and Pennisetum that have been subcultured for 3 to 4 days, discard the upper clear culture medium, retain the suspension cell lines in the form of particles at the bottom, add enzymatic hydrolysis solution, and perform enzymatic hydrolysis for 6 to 8 hours at 27 to 28° C., 20 to 70 rpm, preferably 50 rpm, in the dark. Filter the enzymatically hydrolyzed cell lines, centrifuge, remove the enzyme solution at the top, and retain the protoplasts at the bottom; (4) Protoplast purification: Add an equal volume of W1 solution, centrifuge, remove the upper solution, and keep the protoplasts at the bottom. Repeat this step to obtain purified protoplasts.

6. The method for fusion of bioplasts of Milo and Pennisetum and callus regeneration according to claim 1, characterized in that: In the step three, the electrofusion parameters are: AC electric field strength 20 V / cm, AC electric field action time 25 s, AC duration 7 s; DC pulse strength 80 V / cm, DC pulse time 25 us, DC pulse interval 0.5 s.

7. The method for fusion of sorghum and Pennisetum bioplastids and callus regeneration according to claim 6, characterized in that: The specific steps of protoplast fusion and callus regeneration are as follows: (1) Prepare materials: purified bioplasts of Mirifica serrata and Pennisetum truncatum; (2) Prepare the solution required for protoplast fusion and callus regeneration: Electrofusion solution: 0.6 M mannitol + 5 mM CaCl2, sterilized by filtration; W2 medium: KAO & Michayluk medium + 72.08 g / L glucose + 1.25 g / L sucrose + 2 mg / L 2,4-D + 1 mg / L 6-BA + 0.5 mg / L zeatin + 3.75 g / L gelatin; (3) Protoplast fusion: Take 1 mL of purified protoplasts from Miguel and Pennisetum, respectively, and adjust the concentration to 1 × 10 5 / mL, 1-5:1-5 mixing; the mixed protoplasts were drawn and placed in the electrode plate, the electrofusion parameters were: AC electric field strength 20V / cm, AC electric field action time 25s, AC duration 7s; DC pulse intensity 80V / cm, DC pulse time 25us, DC pulse interval 0.5s; cycle once, select fusion mode; the fused protoplasts were placed at room temperature for 20-30min, then centrifuged at 100×g for 1min, and the supernatant was removed; the electrofusion instrument used was: BEX GEB 15+ / CFB16-HB fusion instrument, electrode: LF501 series 1mm gold block electrode; electrofusion was performed in a sterile environment, the electrodes were disinfected with 75% alcohol, rinsed with sterile water, and disinfected repeatedly for many times; (4) Callus regeneration: The fused protoplasts were resuspended in W1 solution and the concentration was adjusted to 1×10 5 / mL, aspirate 0.6mL of fused protoplasts and spread them on a sterile glass culture dish, and culture them at 27-28℃ in a moist environment away from light / scattered light; every 7 days, add 0.1mL of fresh W1 solution to the culture dish until callus tissue is regenerated; when the callus tissue grows to 1.5-2mm, pick it out and transfer it to W2 medium for proliferation.

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