Method for protoplast fusion of pennisetum squarrosum and pennisetum villosum and callus regeneration thereof

Abstract

This invention discloses a method for the fusion of *Hymenochloa crus-galli* and *Phragmites australis* bioplasts and their callus regeneration, comprising the following steps: Step 1, callus suspension cell culture: Granular callus is induced using young spikelets of *Hymenochloa crus-galli* and *Phragmites australis* to establish a callus suspension cell line; Step 2, protoplast isolation and purification: Protoplasts are isolated using enzymatic digestion and purified using filtration-centrifugation; Step 3, protoplast fusion and callus regeneration: *Hymenochloa crus-galli* and *Phragmites australis* bioplasts are fused using an electrofusion method; the fused protoplasts are then cultured at 27-28°C in the dark / diffuse light environment to obtain regenerated callus from the fused protoplasts. The steps involved in this invention form a complete culture system, with each step closely connected to form a whole, laying the foundation for obtaining interspecific protoplast fusion and regeneration of plants within the *Phragmites australis* genus.

Description

Technical Field

[0001] This invention belongs to the field of plant biological breeding technology, specifically relating to a method for the fusion of *Erythrina variegata* and *Phragmites australis* bioplasts and their callus regeneration. Background Technology

[0002] Plants of the genus *Pennisetum* Rich. belong to the Poaceae family. They are annual or perennial and widely cultivated in tropical and subtropical regions. There are approximately 140 species worldwide, with 11 species and 2 varieties found in my country. The main species of *Pennisetum* cultivated in my country include: *P. glaucum* (also known as American pennisetum), *P. purpureum*, *P. alopecuroides*, *P. polystachyon*, *P. cladestinum*, and some interspecific hybrids. Among them, *P. glaucum* is an annual herb native to north-central Africa. It is highly resilient and tolerant of high temperatures, drought, and poor soil. It grows rapidly in arid and hot regions, has a high seed setting rate, is drought-resistant and water-saving, and its seeds are high in starch, essential amino acids, and unsaturated fatty acids. It is the world's sixth largest cereal crop and a major food crop in arid and semi-arid tropical regions such as India and Africa. It is also a high-quality forage grass, characterized by high yield, good palatability, and excellent feed quality. *Pennisetum alopecuroides* is a native plant of my country, distributed wild in areas ranging from the Liaodong Peninsula in the north to Hainan Island in the south, and west to Guanzhong Plain in Shaanxi Province. It is a perennial plant with high ornamental value for its leaves and flowers. Its root system can reach 1.5-2.0 meters, providing good soil stabilization and slope protection. It is tolerant of slightly saline-alkali soils and also drought-resistant and infertile soils. It is cold-resistant and can safely overwinter at around -20℃, but its biomass yield is low and its palatability is poor. Existing research and practice have shown that sexual hybridization between *Pennisetum alopecuroides* and *Pennisetum alopecuroides* is difficult.

[0003] Protoplasts are naked cells with their cell walls removed. Protoplast fusion refers to the technique of inducing the fusion of protoplasts from different sources without sexual hybridization, under certain external conditions, and then culturing them in vitro to regenerate a hybrid plant; it is also known as somatic cell hybridization. Protoplast fusion can effectively overcome difficulties such as sexual hybridization incompatibility and distant hybridization barriers. It can also directionally transfer cell nuclei, chromosomes, chromosome fragments, or cytoplasm, effectively overcoming biological obstacles encountered in traditional breeding, such as distant hybridization incompatibility and gamete sterility, thereby 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 potatoes, achieving a series of advances and breakthroughs.In the 1980s, the team of American scholar Vasil conducted research on protoplast fusion of millet and sorghum, sugarcane and millet, and wheat and millet (Peggy Ozias-Akins, Robert J. Ferl, and Indra K. Vasil, Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearlmillet) + 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), obtained callus tissue after protoplast fusion using the PEG method. Since then, there have been few research papers on protoplast fusion of the genus Pennisetum.

[0004] In recent years, the Grass Genetics, Breeding and Ecological Application Innovation Team of the Jiangsu Academy of Agricultural Sciences has been continuously conducting interspecific hybridization breeding of the genus *Pennisetum* to improve the cold resistance of *Eriocaulon buergerianum* and the forage yield and quality of *Pennisetum*. Existing research and practice have found that using the methods published in the aforementioned literature with collected *Eriocaulon buergerianum* resources, high-frequency embryogenic callus suspension culture and a high protoplast fusion rate cannot be achieved. This is speculated to be related to the differences between *Eriocaulon buergerianum* and *Pennisetum* species and the fusion method used. Furthermore, protoplast fusion using the PEG method can have a certain toxic effect on cells, and repeated washing and centrifugation can affect cell viability and subsequent regeneration capacity, greatly reducing protoplast fusion efficiency. Meanwhile, no methods for protoplast fusion and callus regeneration of annual *Eriocaulon buergerianum* and highly cold-resistant perennial *Pennisetum* have been reported domestically or internationally. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of existing technologies and provide a method for the fusion of protoplasts of *Centella asiatica* and *Pennisetum purpureus* and its callus regeneration. This invention induces granular callus tissue using young spikelets of *Centella asiatica* and *Pennisetum purpureus*, and establishes a high-frequency embryogenic callus suspension cell line for donor and recipient. Using this suspension cell line as material, protoplasts are separated by enzymatic digestion, establishing a method for efficient separation of *Centella asiatica* and *Pennisetum purpureus* protoplasts. Then, an electrofusion method is used to improve the fusion efficiency of protoplasts of *Pennisetum purpureus* and *Centella asiatica*, obtaining regenerated callus tissue after the fusion of *Centella asiatica* and *Pennisetum purpureus* protoplasts, laying the foundation for obtaining interspecific protoplast fusion regenerated plants of the *Pennisetum* genus.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention protects a method for the fusion of *Przewalski's grain* and *Przewalski's tail* bioplasts and the regeneration of their callus tissue, the method comprising the following steps:

[0008] Step 1, Callus suspension cell culture: Granular callus was induced with young spikelets of *Gnaphalium affine* and *Philodendron simsii*, and suspension culture was used to establish a donor-recipient high-frequency embryogenic callus suspension cell line.

[0009] Step 2, Protoplast Isolation and Purification: Protoplasts were isolated using enzymatic digestion with suspension cell lines as material, and purified using filtration-centrifugation.

[0010] Step 3, protoplast fusion and callus regeneration: Using the electrofusion method, the protoplasts of the Ottogi and the protoplasts of the Napier are fused at a ratio of 1~5:1~5. The fused protoplasts are then cultured at 27~28℃ in the dark / diffuse light and moisturized to obtain the regenerated callus tissue of the fused protoplasts.

[0011] In a specific implementation scheme, in step one, 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; the pH of the above medium is adjusted to 5.7~5.8 with dilute hydrochloric acid and sodium hydroxide, and then autoclaved at 121℃ for 20 min.

[0012] In a more specific implementation scheme, the specific steps for the callus suspension cell culture are as follows:

[0013] (1) Preparation of materials: Use young spikelets of *Gnaphalium affine* and *Pontylla serrata* as explants;

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

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

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

[0017] The above culture medium was adjusted to pH 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, and then autoclaved at 121℃ for 20 min.

[0018] (3) Take the unemerged young spikes of *Gnaphalium affine* and *Pontylla serrata*, wipe the leaf sheaths of the young spikes with 75% alcohol cotton balls under sterile conditions, peel off the young spikes, place them in a culture dish containing sterilized and dried filter paper, cut them into 4-5 mm long spike segments, inoculate them onto callus induction medium, and culture them in the dark at 27-28℃. After 7-10 days, loose and transparent callus tissue is induced at the base of the young spikes. After 15-20 days of subculture, granular callus tissue is obtained.

[0019] (4) The granular callus tissue of *Gnaphalium affine* and *Phragmites australis* was inoculated into a 250 mL Erlenmeyer flask containing suspension medium at a ratio of 1 g: 30 mL (callus tissue: suspension medium). The flask was cultured in the dark at a temperature of 27-28℃ and a rotation speed of 120 rpm. After every 2-3 days of culture, the suspension in the flask was discarded and an equal volume of fresh suspension medium was added. This step was repeated 3 times to obtain the suspension cell lines of *Gnaphalium affine* and *Phragmites australis*. Subculture was then performed at a volume ratio of 1:5 (suspension cell line: suspension medium).

[0020] In a specific implementation scheme, in step two, the components of the enzymatic hydrolysate are as follows: 20 g / L cellulase R-10 + 6 g / L analyte 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, hydrolysis time 6~8 h.

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

[0022] (1) Materials preparation: Suspension cell lines of *Gnaphalium affine* and *Phragmites australis*;

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

[0024] Enzymatic hydrolysate: 20 g / L cellulase R-10 + 6 g / L dissociative 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;

[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 pH to 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, autoclave at 121℃ for 20 min, and store at 4℃ after cooling;

[0026] The KAO & Michayluk medium consists of 0.453 g / L CaCl2, 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.

[0027] (3) Protoplast isolation: Take the suspension cell lines of *Eriocaulon buergerianum* and *Phragmites australis* that have been subcultured for 3-4 days, discard 20-25 mL of the clearer upper layer of culture medium, keep 5 mL of the suspension cell line with particles at the bottom, add 20 mL of enzyme digestion solution, and digest under the conditions of 27-28℃, 50 rpm and darkness for 6-8 h. Filter the digested cell line through a 70 μm cell sieve, dispense into 50 mL centrifuge tubes, centrifuge at 200 × g for 3 min, remove the enzyme solution at the top of the centrifuge tube, and keep the protoplasts at the bottom;

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

[0029] In the specific implementation plan, in step three, the electrical fusion parameters are: AC electric field strength (AC) 20 V / cm, AC application time 25 s, and post-AC duration 7 s; DC pulse strength (DC) 80 V / cm, DC pulse time 25 us, and pulse interval time 0.5 s.

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

[0031] (1) Preparation of materials: purified Miko and Wolftail grassland bioplasts;

[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 was sterilized by filtration through a 0.22 μm cell filter;

[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 gel;

[0035] (3) Protoplast fusion: Take 1 mL of purified *Eriocheir sinensis* and *Phragmites australis* protoplasts respectively, and adjust the concentration to 1×10⁻⁶. 5Protoplasts were mixed at a ratio of 1:5:1:5 per mL. The mixed protoplasts were placed on an electrode plate. Electrofusion parameters were as follows: AC electric field strength (AC) 20 V / cm, AC action time 25 s, post-AC duration 7 s; DC pulse strength (DC) 80 V / cm, DC pulse time 25 μs, pulse interval 0.5 s; one cycle was performed, selecting the fusion mode. The fused protoplasts were placed at room temperature for 20-30 min, followed by centrifugation at 100 × g for 1 min, and the supernatant was removed. The electrofusion instrument used was a BEX GEB 15+ / CFB16-HB fusion instrument, with electrodes: LF501 series gold nugget electrodes (1 mm). Electrofusion was performed under aseptic conditions. Electrodes were disinfected with 75% alcohol for 15 min, rinsed with sterile water, and disinfected repeatedly.

[0036] (4) Callus regeneration: The fused protoplasts were resuspended in W1 solution and the concentration was adjusted to 1×10⁻⁶. 5 For each protoplast, take 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass culture dish (3.5 cm in diameter). Incubate at 27-28°C in the dark / diffuse light and with humidity. Every 7 days, add 0.1 mL of fresh W1 solution to the culture dish until callus regenerates. 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 this invention is young spikelets. Because the outer surface is disinfected and does not come into contact with the young spikelets, the callus induction rate is as high as 100%, which is 7.15% higher than the existing induction rate.

[0039] (2) The granular callus tissues of *Gnaphalium affine* and *Phragmites australis* induced by the method of the present invention are easy to establish a suspension cell line with embryogenicity, and the suspension does not turn brown, with high proliferation efficiency. After 14 days of suspension culture, the dense area of ​​the callus tissue is 23.8 times that of the initial value.

[0040] (3) The present invention uses the filtration-centrifugation method to purify protoplasts, which is simple and easy to implement. The protoplasts obtained are morphologically complete, have high yield, and have a protoplast viability of up to 80%, laying the foundation for subsequent somatic cell fusion and the establishment of a gene instantaneous transformation system.

[0041] (4) The present invention uses electrofusion method, which does not have the problem of cytotoxicity. The electrofusion efficiency is 37.83% higher than the reported PEG method, and the post-fusion processing is simple.

[0042] (5) The method of callus suspension culture, protoplast separation, protoplast fusion and callus regeneration involved in this invention is a complete culture system. Each link is closely connected to form a whole, and the implementation of any step is crucial to the whole process. Attached Figure Description

[0043] Figure 1 Granular callus tissue of Otani (left) and Pennisetum (right).

[0044] Figure 2 Screening of *Napierus oleraceus* suspension culture medium. Among them, Figure 2 AB: Add 5-15% CM to M24 and MS suspension and incubate for 7 days; Figure 2 CD: Seven days after the first subculture, 5-15% CM was added to the M24 and MS suspension to morphologically represent cells. Figure 2 EF: Callus tissue with 5-15% CM M24 and MS suspension added 7 days after the first subculture.

[0045] Figure 3 Suspension cell lines of Miyako (left) and Napier grass (right).

[0046] Figure 4 Protoplasts of Miyako (left) and Napier grass (right).

[0047] Figure 5 The bioplasma of Yugu and Wolf Tail Grassland fused together.

[0048] Figure 6 Regenerated callus tissue resulting from the fusion of Miyako and Wolftail grassland bioplasts. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents or instruments used are considered to be 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; subculture medium was the same as the induction medium, and the subculture period was 15-20 days.

[0051] Suspension medium: MS + 30 g / L sucrose + 50 mL / L coconut juice + 2 mg / L 2,4-dichlorophenoxyacetic acid, pH adjusted to 5.8 with dilute hydrochloric acid and sodium hydroxide; Subculture medium is the same as the suspension medium described above, and the subculture cycle is 7-14 days.

[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 CaCl2, 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] The above culture medium was adjusted to pH 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, then autoclaved at 121℃ for 20 min, cooled, and stored at 4℃.

[0056] Enzymatic hydrolysate: 20 g / L cellulase R-10 + 6 g / L dissociative 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;

[0057] Electrofusion solution: 0.6 M mannitol + 5 mM CaCl2; both the above enzymatic hydrolysate and electrofusion solution were sterilized by filtration using a cell filter (0.22 μm).

[0058] This invention protects a method for the fusion of *Przewalski's grain* and *Przewalski's tail* bioplasts and the regeneration of their callus tissue, the method comprising the following steps:

[0059] 1. Callus suspension cell culture:

[0060] Take the unemerged young spikelets of *Gnaphalium affine* and *Phragmites australis*, wipe the leaf sheaths of the young spikelets with 75% alcohol cotton balls under sterile conditions, peel off the young spikelets, place them in a culture dish containing sterilized and dried filter paper, cut them into 4-5 mm long segments, inoculate them onto callus induction medium, and culture them in the dark at 27-28℃. After 7-10 days, loose and transparent callus tissue is induced at the base of the young spikelets. After subculturing on subculture medium for 15-20 days, granular callus tissue is obtained.

[0061] Granular callus tissues of *Gnaphalium affine* and *Polygonum hydropiper* were inoculated into 250 mL Erlenmeyer flasks containing suspension culture medium at a ratio of 1 g callus tissue to 30 mL suspension culture medium. The flasks were cultured in the dark at 27–28 °C and 120 rpm. Every 2–3 days, the suspension was discarded, and an equal volume of fresh suspension culture medium was added. This process was repeated three times to obtain *Gnaphalium affine* and *Polygonum hydropiper* suspension cell lines. Subculture was then performed at a cell line to suspension culture medium volume ratio of 1:5.

[0062] 2. Protoplast isolation and purification:

[0063] Protoplast isolation: Take the suspension cell lines of *Eriocaulon buergerianum* and *Phragmites australis* that have been subcultured for 3-4 days, discard 20-25 mL of the clearer upper layer of culture medium, and retain 5 mL of the suspension cell line with particles at the bottom. Add 20 mL of enzyme digestion solution and digest the cells at 27-28℃, 50 rpm, and in the dark for 6-8 h. Filter the digested cell lines through a 70 μm cell sieve, aliquot them into 50 mL centrifuge tubes, centrifuge at 200×g for 3 min, remove the enzyme solution at 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 supernatant, and 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 *Eriocheir sinensis* and *Phragmites australis* protoplasts respectively, and adjust the concentration to 1×10⁻⁶. 5Protoplasts were mixed at a 1:1 ratio (number of protoplasts per mL). The mixed protoplasts were placed on an electrode plate. Electrofusion parameters were: AC electric field strength (AC) 20 V / cm, AC action time 25 s, post-AC duration 7 s; DC pulse strength (DC) 80 V / cm, DC pulse time 25 μs, pulse interval 0.5 s; one cycle was performed, selecting the fusion mode. The fused protoplasts were incubated at room temperature for 20–30 min, followed by centrifugation at 100 × g for 1 min, and the supernatant was removed. The electrofusion instrument used was a BEX GEB15+ / CFB16-HB fusion instrument, with electrodes: LF501 series gold nugget electrodes (1 mm). Electrofusion was performed under aseptic conditions. Electrodes were disinfected with 75% alcohol for 15 min, then rinsed with sterile water, and this disinfection was repeated 3–5 times.

[0067] Callus regeneration: The fused protoplasts were resuspended in W1 solution, and the concentration was adjusted to 1×10⁻⁶. 5 For each protoplast, take 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass culture dish (3.5 cm in diameter). Incubate at 27-28°C in the dark / diffuse light and with humidity. Every 7 days, add 0.1 mL of fresh W1 solution to the culture dish until callus tissue regenerates. When the callus tissue grows to 1.5-2 mm, pick it out and transfer it to W2 medium for proliferation.

[0068] This invention has screened and illustrated methods for callus suspension culture, protoplast isolation, protoplast fusion, and callus regeneration after fusion of *Gnaphalium affine* and *Phragmites australis*, but the invention is not limited thereto.

[0069] Example 1: Callus induction and suspension culture screening of *Gnaphalium affine* and *Phragmites australis*

[0070] 1. Materials and Methods

[0071] 1.1 Materials

[0072] Miya Yuho

[0073] 1.2 Experimental Design

[0074] (1) Screening of callus induction media. The callus induction media used MS + 30 g / L sucrose + 3.75 g / L gel as the basic medium, and added 0, 0.5, 1.0, 2.0 and 3.0 mg / L of 2,4-D and 0, 0.05, 0.1, 0.5 and 1 mg / L of kinetin-6-furfurylaminopurine (KT) respectively, for a total of 25 media formulations. The media were sterilized at 121℃ for 20 min. Ten explants were inoculated into each medium, with 5 replicates. The purpose was to screen the hormone ratio with the highest induction rate. The callus induction rate (%) = number of induced callus / total number of inoculated explants × 100%, and the callus browning rate (%) = number of browned callus / total number of inoculated explants × 100%. The specific design is as follows:

[0075] Table 1. Design of combinations of 2,4-D and KT hormones at different concentrations

[0076]

[0077] (2) Screening of suspension culture media. Two types of culture media were used: MS basal medium (MS + 2 mg / L 2,4-D + 30 g / L sucrose) and M24 basal medium (MS + 30 g / L sucrose + 100 mg / L inositol + 2 mg / L 2,4-D + 500 mg / L hydrolyzed casein). Different concentrations of coconut water (CM) were added to each medium, as follows: MS + 5% CM, MS + 10% CM, MS + 15% CM, M24 + 5% CM, M24 + 10% CM, and M24 + 15% CM. The dense area of ​​callus tissue refers to the area occupied by the callus tissue spread out flat.

[0078] 1.3 Cultivation Methods

[0079] Callus induction method: Take the unemerged young spikelets of the panicle, wipe the leaf sheaths of the young spikelets with a cotton ball soaked in 75% alcohol under sterile conditions, and peel off the young spikelets. Place them in a culture dish containing sterilized and dried filter paper, cut them into 4-5 mm long segments, and inoculate them onto the callus induction medium. Culture them in the dark at 27-28℃. After 7-10 days, loose and transparent callus tissue is induced at the base of the young spikelets. Transfer them to the selected induction medium for subculture. After 15-20 days of proliferation, granular callus tissue is obtained.

[0080] Suspension cell line culture method: Miya granular callus was inoculated into a 250 mL Erlenmeyer flask containing suspension medium at a ratio of (callus: suspension medium) of 1.0 g: 30 mL. The flask was cultured in the dark at a temperature of 27-28℃ and a rotation speed of 120 rpm. After every 2-3 days of culture, the suspension in the flask was discarded and an equal volume of fresh suspension medium was added. This step was repeated 3 times to obtain the Miya suspension cell line. Subculture was then performed at a volume ratio of (suspension cell line: suspension medium) of 1:5.

[0081] 2 Results and Analysis

[0082] 2.1 Effects of different concentrations of 2,4-D combined with KT on callus induction rate and browning rate

[0083] Different concentrations of 2,4-D and KT significantly affected the induction rate of *Codonopsis pilosula* callus. As shown in Table 2, in treatment 4, the highest induction rate of *Codonopsis pilosula* spikelets (100%) was achieved with the addition of 2 mg / L 2,4-D alone to the induction medium, which was 7.15% higher than the previously reported induction rate (93.33%) using *Codonopsis pilosula* seeds as explants. It was also found that treatments 9 and 14, i.e., with the addition of 0.05 and 0.1 mg / L KT and 2 mg / L 2,4-D respectively, achieved callus induction rates of 90% and 94%, respectively. The absence of 2,4-D significantly reduced the callus induction rate 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, as seen in treatments 19, 20, and 24. Therefore, treatment 4, i.e., MS medium supplemented with 2 mg / L 2,4-D, and further supplemented with 30 g / L sucrose and 3.75 g / L gel, is the optimal induction medium. After subculturing in this medium for 15-20 days, granular callus tissue was obtained. Figure 1 ).

[0084] Table 2. Effects of different concentrations of 2,4-D combined with KT on callus induction rate and browning rate.

[0085]

[0086] Note: Different letters in the same column indicate significant differences in data (P<0.05), and the same applies below.

[0087] 2.2 Effects of different coconut milk (CM) concentrations on suspended callus tissue

[0088] Coconut juice concentration has a significant impact on the establishment and morphology of suspension cell lines. After culturing callus tissue in M24 and MS suspensions with 5% CM for 7 days, both the suspension and the callus tissue were pale yellow. However, the suspensions with 10% CM and 15% CM were significantly darker in color than the 5% CM suspension, and the callus tissue was prone to browning. Figure 2 (AB); After three initial suspension cultures, and 7 days after the first subculture, the original large callus began to proliferate into small callus. Figure 2 (EF) The M24 and MS suspensions with 5% CM showed a large number of cell clusters composed of spherical cells and very few elongated cells; while the suspensions with 10% CM and 15% CM contained mostly elongated cells and yielded fewer cells. Figure 2 (CD). MS suspension with 5% CM showed more uniform cell morphology than M24 suspension. Therefore, MS suspension with 5% CM can produce high-yield suspension cell lines with intact morphology.

[0089] 2.3 Effects of suspension culture medium on the proliferation of suspension cell lines

[0090] Approximately 1g of granular callus tissue from *Pennisetum affine* was inoculated into 30 mL of 5% CM MS suspension medium and cultured in suspension for 7–10 days. During the first 1–3 days, the callus density area did not change significantly; proliferation was slow during this stage, indicating an adaptation phase. After 7–10 days of subculture in suspension, the suspension system entered a rapid proliferation phase, with the callus density area reaching 56.4 cm². 2 The surface area of ​​the callus after 7 days of suspension culture was 23.8 times that of the initial suspension culture, clarifying the effect of suspension culture on the proliferation of suspension cell lines. Figure 3 ).

[0091] Example 2 Screening of protoplast isolation conditions

[0092] 1. Materials and Methods

[0093] 1.1 Materials

[0094] Granular callus and suspension cell lines of *Gnaphalium affine* and *Phragmites australis*.

[0095] 1.2 Experimental Design

[0096] (1) Screening of enzymatic hydrolysis conditions. To investigate the effects of different enzymatic hydrolysis conditions on the yield and morphology of Yugu protoplasts, a four-factor, three-level orthogonal experiment was designed to screen the optimal enzymatic hydrolysis solution formulation for cellulase (15, 20, 25 g / L), analyte (6, 8, 10 g / L), mannitol concentration (0.3, 0.4, 0.5 M), and hydrolysis time (4, 6, 8 h).

[0097] (2) Analysis of the enzymatic hydrolysis effect of granular callus and suspension cell line. Take 10 g of granular callus and 10 mL of suspension cell line respectively. Remove the clear liquid on the upper layer of suspension cell line and add 40 mL of enzymatic hydrolysis solution to each. Set up 4 replicates for each treatment. Under the conditions of 28℃ and darkness, the enzymatic hydrolysis was carried out at 50 rpm for 8 h.

[0098] (3) Effects of different cellulase brands on the enzymatic hydrolysis of suspension cell lines. Four commonly used cellulase brands on the market, namely YAKULT, Yuanye, Aladdin, and Maclean, were selected and evaluated from the aspects of enzymatic hydrolysis effect and cost-effectiveness. The enzymatic hydrolysis solution consisted of 20 g / L cellulase, 6 g / L analyte R-10 (YAKULT, Japan), 0.69 g / L 2-(N-morpholine) 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.

[0099] 1.3 Test Methods

[0100] (1) Enzymatic hydrolysis method. After subculturing the *Calvatia japonica* and *Phragmites australis* suspension cell lines 1-2 times, on days 3-5 after subculturing, select 5 mL of healthy *Calvatia japonica* callus suspension cell lines, add 20 mL of enzyme solution, seal with sealing film, and hydrolyze at 50 rpm in the dark at 28℃. Filter the hydrolyzed cell lines through a 70 μm cell sieve, aliquot into 50 mL centrifuge tubes, centrifuge at 200×g for 3 min, remove the enzyme solution from the top of the centrifuge tube, and retain the protoplasts at the bottom. Add an equal volume of W1 solution, centrifuge at 200×g, remove the upper layer of solution, and retain the protoplasts at the bottom. Repeat this step to obtain purified protoplasts. The enzymatic hydrolysate consisted of cellulase R-10 (YAKULT, Japan), analyte R-10 (YAKULT, Japan), 0.69 g / L 2-(N-morpholine) 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 the solution was filtered to remove bacteria.

[0101] (2) Analysis of the enzymatic digestion effect of granular callus and suspension cell line. Using the granular callus and suspension cell line cultured by the experimental design (1) and (2) in Example 1 as materials, the protoplasts were enzymatically digested and purified using the conditions and methods screened by the experimental design (1) in Example 2.

[0102] (3) Effect of different brands of cellulase on the enzymatic hydrolysis effect of suspension cell lines. Take 5 mL of Napier grass suspension cell line and add 20 mL of enzymatic hydrolysis solution of different brands of cellulase respectively. Other enzyme solution components and enzymatic hydrolysis methods are the same as the formulation, screening conditions and methods of experimental design (1) in Example 1. Each treatment is set with 4 replicates, and 4 fields of view are counted for each replicate.

[0103] (4) Method for determining protoplast yield: 10 μL of protoplast resuspension is dropped onto a hemocytometer, covered with a coverslip, and counted under an inverted microscope. Count four times consecutively and take the average value. Method for determining protoplast viability: 5 mg FDA is dissolved in 1 mL of acetone as the working solution. Then, 20 μL of FDA working solution is added to 1 mL of suspended protoplasts. After reacting in the dark at room temperature for 3-4 min, a small amount of solution is placed on a slide and observed under a fluorescence microscope. Protoplasts that emit green fluorescence are viable. Protoplast viability = viable protoplasts / total number of protoplasts in the field of view × 100%. Four representative fields of view are selected for statistical analysis. All methods for calculating protoplast yield and viability described in this patent application are implemented according to this method.

[0104] 2 Results and Analysis

[0105] 2.1 Screening of Enzymatic Digestion Conditions

[0106] Different enzymatic hydrolysis conditions significantly affected the yield and viability of protoplasts from the rice grain. Table 3 shows that treatment 4 yielded the highest protoplast production, reaching 2.1 × 10⁻⁶. 6 indivual / mL, significantly higher than treatments 2, 3, 8, and 9; treatment 4 also had the highest protoplast viability, reaching 80%, significantly higher than treatments 1, 3, 6, 8, and 9. Treatment 7 also achieved the same protoplast yield as treatment 4, but its protoplast viability was 2.4% lower than treatment 4. Combined with microscopic protoplast morphology, it was found that protoplasts in treatment 4 had clear edges and intact morphology. Figure 4 Finally, treatment 4 was determined to yield high-yield and highly active protoplasts under the conditions of 20 g / L cellulase R-10, 6 g / L sorbase R-10, and 0.4 M mannitol, followed by enzymatic hydrolysis for 8 h. The composition of the hydrolysate was determined to be: 20 g / L cellulase R-10 + 6 g / L sorbase R-10 + 0.4 M mannitol + 0.69 g / L 2-(N-morpholine)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate.

[0107] Table 3 Effects of different enzymatic hydrolysis conditions on protoplast yield and viability

[0108]

[0109] 2.2 Analysis of the Enzymatic Digestion Effect on Granular Callus and Suspension Cell Lines

[0110] The same enzymatic hydrolysis solution and conditions were used to enzymatically hydrolyze both the granular callus tissue and the suspension cell line from Yugu. The results are shown in Table 4. The suspension cell line yielded 1.345 × 10⁻⁶ cells. 8 The protoplast yield was 12.5 × 10⁶ cells / mL, with a protoplast viability of 78.61%. The protoplast yield and viability from enzymatic hydrolysis of granular callus were 12.5 × 10⁶ cells / mL. 6 The yield and activity of the enzyme were 22.6% and 90.71% and 71.25% lower than those of the suspension cell line, respectively.

[0111] Table 4. Analysis of the enzymatic hydrolysis effect on granular callus and suspension cell lines.

[0112]

[0113] 2.3 Effects of different brands of cellulase on the enzymatic hydrolysis effect of suspension cell lines

[0114] Cellulase and dissociation enzyme are important components of the enzymatic hydrolysate. Studies on the enzymatic hydrolysis effects of different brands of cellulase showed significant differences in their efficacy on the Yugu suspension cell line (Table 5). YAKULT cellulase exhibited the best hydrolysis effect, with the highest protoplast yield and activity, at 3.15 × 10⁻⁶. 6 indivual / mL and 80.37%; the Yuanye brand had the highest cellulase protoplast yield, reaching 3.43×10 mL. 6 indivual / The protoplast activity reached 75.06%, but its protoplast yield and activity were not significantly different from YAKULT cellulase. Aladdin and Maclean cellulase yields were not significantly different from the two brands mentioned above, but protoplast activity was significantly reduced. YAKULT cellulase showed the best enzymatic hydrolysis effect, but it was also the most expensive at 135 yuan / gram, which is 131.6 yuan per gram higher than Yuanye brand cellulase. Therefore, considering both economic efficiency and enzymatic hydrolysis effect, Yuanye brand cellulase is the best.

[0115] Table 5 Comparative analysis of the enzymatic hydrolysis effects of different brands of cellulase on suspension cell lines

[0116]

[0117] Example 3: Protoplast fusion and callus regeneration

[0118] 1. Materials and Methods

[0119] 1.1 Materials

[0120] Protoplasts purified from Miyako and Napier grass.

[0121] 1.2 Experimental Design

[0122] (1) Screening of electric fusion conditions. A four-factor, three-level experiment was designed to optimize the fusion parameters, which are (15, 20, 25 V / cm) AC electric field strength, (20, 30, 40 s) AC action time, (50, 80, 100 V / cm) DC pulse electric field strength and (10, 20, 30) DC pulse time, etc.

[0123] (2) Effect of PEG fusion method and electrofusion method on fusion efficiency. The PEG method was performed using the method of Vasil et al. (1986), and the electrofusion method was performed using the fusion conditions selected in the experimental design (1) of Example 3.

[0124] (3) Callus regeneration after protoplast fusion. The culture medium used was:

[0125] 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.

[0126] 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.

[0127] The above solutions and culture 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.

[0128] 1.3 Test Methods

[0129] (1) Screening of electrofusion conditions. The purified *Erythrina variegata* and *Pterocarya stenoptera* bioplasts were washed once with an electrofusion solution: 0.6 M mannitol + 5 mM CaCl2, and the concentration was adjusted to 1×10⁻⁶. 5 Protoplasts were mixed at a 1:1 ratio (number of protoplasts per mL). 10 μL of the mixed protoplasts were placed in an electrode plate, and the protoplast aggregation was observed under an inverted microscope. A DC pulse voltage was then applied to initiate fusion. The fusion rate was statistically analyzed under the inverted microscope; each data point represents the average of five fields of view. Autologous and allogeneic fusions were not differentiated, and the fusion rate was expressed as the total fusion rate. The electrofusion solution was sterilized by filtration through a 0.22 μm cell filter.

[0130] (2) Effect of PEG fusion method and electrofusion method on fusion efficiency. The fusion solution was prepared according to the method of Vasil et al. (1986), filtered for sterilization, and the PEG fusion time was 15 min. The protoplasts used were the purified protoplasts in the experimental design (1) of Example 3.

[0131] (3) Callus regeneration after protoplast fusion. After fusion, the protoplasts were rinsed with W1 solution and resuspended, and then cultured at 27~28℃ in the dark / diffuse light and humidified environment.

[0132] 2 Results and Analysis

[0133] 2.1 Screening of Electrofusion Conditions

[0134] The protoplast chaining effect influences the protoplast fusion rate. The protoplast chaining effect is determined by both the alternating current (AC) field and its duration; a larger electric field results in faster protoplast movement and faster chain formation, while a longer AC field duration leads to longer chain formation. Table 6 shows that when AC is 20 V / cm and the AC duration is 25 s, protoplasts move regularly and form relatively long chains, suitable for protoplast fusion. However, when DC is 80 V / cm and the DC duration is 25 μs, the highest fusion rate of 9.8% is achieved, significantly higher than other combinations except for treatment 6. Figure 5 The protoplasts are fusing. DC pulse intensity that is too low or too high, or duration that is too long or too short, will affect the protoplast fusion rate. In the experiment, AC 20 V / cm with an AC duration of 10 s and DC 80 V / cm with a DC duration of 25 μs were selected as the optimal conditions for protoplast electrofusion.

[0135] Table 6 Screening of Electrofusion Conditions

[0136]

[0137] 2.2 Effects of PEG fusion method and electrofusion method on fusion efficiency

[0138] To investigate the effects of PEG and electrofusion methods on fusion efficiency, we fused *Phyllostachys edulis* and *Phyllostachys edulis* bioplasts using PEG and electrofusion methods, respectively. The results are shown in Table 7. The electrofusion method achieved a significantly higher fusion rate than the PEG method, with an electrofusion rate of 9.4%, which is 37.83% higher than that of the PEG method.

[0139] Table 7. Effects of PEG fusion method and electrofusion method on fusion rate

[0140]

[0141] 2.3 Callus regeneration after protoplast fusion

[0142] The fused protoplasts were washed with W1 solution and resuspended, with the concentration adjusted to 1×10⁻⁶. 5 For each protoplast, take 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass culture dish (3.5 cm in diameter). Incubate at 27-28°C in the dark / diffuse light environment. Every 7 days, add 0.1 mL of fresh W1 solution to the culture dish. Callus tissue will generally regenerate in about 15-20 days. Figure 6 When the callus tissue grows to 1.5-2 mm, it is picked out and transferred to W2 medium for proliferation.

[0143] In summary, this invention investigated callus induction, suspension culture, protoplast isolation, protoplast fusion, and callus regeneration after fusion of *Polygonum hydropiper* and *Phragmites australis*. Screening of callus induction media revealed that MS medium containing 2 mg / L 2,4-D, 30 g / L sucrose, and 3.75 g / L gel was the optimal induction medium. Suspension medium containing MS medium containing 5% coconut milk, 30 g / L sucrose, and 2 mg / L 2,4-D yielded a high-yield suspension cell line with intact morphology. Approximately 1 g of *Phragmites australis* granular callus, after 14–20 days of culture, showed a callus density area 23.8 times that of the initial suspension culture after 7 days.

[0144] To optimize protoplast isolation, the optimal enzymatic hydrolysis solution was determined to be: 20 g / L cellulase R-10 + 6 g / L dissociative enzyme R-10 + 0.4 M mannitol + 0.69 g / L 2-(N-morpholine)ethanesulfonic acid monohydrate + 1.03 g / L calcium chloride dihydrate. Enzymatic hydrolysis at 28℃ in the dark, at 50 rpm for 8 h yielded protoplasts with high yield and viability. Using this hydrolysis solution and conditions, granular callus and suspension cell lines were hydrolyzed. The results showed that the yield and viability of suspension cell lines were significantly higher than those of granular callus. Different brands of cellulase significantly affected the hydrolysis effect on suspension cell lines. YAKULT cellulase showed the best hydrolysis effect, with relatively optimal protoplast yield and viability. However, considering both economic efficiency and hydrolysis effectiveness, the Yuanye brand cellulase was the best.

[0145] Regarding the protoplast fusion conditions, it was found that the highest protoplast fusion rate was achieved when AC was 20 V / cm and AC treatment time was 10 s, DC was 80 V / cm and DC treatment time was 25 μs. The fusion rate of the electrofusion method was significantly higher than that of the PEG method, and was 37.83% higher.

[0146] The fused protoplasts were washed with W1 solution and resuspended, with the concentration adjusted to 1×10⁻⁶. 5For each protoplast, take 0.6 mL of the fused protoplasts and spread them evenly in a sterile glass culture dish (3.5 cm in diameter). Incubate at 27-28°C in the dark / diffuse light and with humidity. Every 7 days, add 0.1 mL of fresh W1 solution to the culture dish. Callus tissue can usually regenerate in about 15-20 days.

[0147] The embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention are included within the protection scope of the present invention.

Claims

1. A method for protoplast fusion of Oryza sativa and Puccinellia distans and regeneration of callus therefrom, characterized by, The method comprises the following steps: Step one, callus suspension cell culture: granular callus is induced by young spikes of Oryzopsis and Wolf tail grass, and a high-frequency embryogenic callus suspension cell line is established for the donor and the recipient; Step two, protoplast isolation and purification: the suspension cell line is used as the material to isolate the protoplast by enzymatic method, and the protoplast is purified by filtration-centrifugation method; Step three, protoplast fusion and callus regeneration: the protoplasts of Oryzopsis and Wolf tail grass are fused according to 1-5:1-5 by using the electric fusion method, the fused protoplasts are taken, and the regenerated callus of the fused protoplasts is obtained by culture at 27-28 DEG C in the dark / diffused light and moisture preservation; In step one, the induction culture 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; after the above-mentioned medium is adjusted to pH 5.7-5.8 by dilute hydrochloric acid and sodium hydroxide, it is autoclaved at 121 DEG C for 20 min; In step two, the enzymatic hydrolysis liquid used for enzymatic hydrolysis comprises the following components: 20 g / L cellulase R-10+6 g / L isolated 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 the enzymatic hydrolysis time is 6-8 h; In step three, the electric fusion parameters are as follows: alternating current field strength 20 V / cm, alternating current field action time 25 s, and alternating current continuous time 7 s; direct current pulse strength 80 V / cm, direct current pulse time 25 s, and direct current pulse interval time 0.5 s.

2. The method of claim 1, wherein the method is characterized by, The specific steps of the callus suspension cell culture are as follows: (1) Prepare the material: the young spikes of Oryzopsis and Wolf tail grass are selected as the explants; (2) Prepare the induction medium and the suspension culture medium: The induction medium is: 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 15-20 d; The suspension culture medium is: MS+30 g / L sucrose+50 mL / L coconut juice+2 mg / L 2,4-dichlorophenoxyacetic acid; the subculture suspension culture medium is the same as the suspension culture medium, and the subculture cycle is 7-14 d; after the above-mentioned medium is adjusted to pH 5.7-5.8 by dilute hydrochloric acid and sodium hydroxide, it is autoclaved at 121 DEG C for 20 min; (3) Take the unelongated young spikes of Oryzopsis and Wolf tail grass, disinfect the leaf sheath outside the young spikes under sterile conditions, and peel off the young spikes, which are placed in a sterile and dried filter paper culture dish, cut into 4-5 mm long spike segments, inoculated on the callus induction medium, and cultured at a temperature of 27-28 DEG C in the dark to induce loose and transparent callus, which is transferred to the subculture medium to obtain granular callus; (4) The granular callus of Oryzopsis and Pennisetum is inoculated into a culture bottle containing a suspension medium, and the inoculation ratio of callus to suspension medium is 1 g:30 mL. The culture is carried out under dark conditions at a temperature of 27-28°C and a rotation speed of 120 rpm. The suspension in the bottle is discarded every 2-3 days, and an equal volume of fresh suspension medium is added. This step is repeated 3 times to obtain a suspension cell line of Oryzopsis and Pennisetum. Then, the suspension cell line is subcultured at a ratio of 1:5 (suspension cell line to suspension medium).

3. The method of claim 1, wherein the wheat and pennisetum protoplast fusion and the callus regeneration thereof are characterized by, The specific steps of the protoplast separation and purification method are as follows: (1) Material: Oryzopsis and Pennisetum suspension cell lines; (2) Solutions and media required for protoplast separation and purification: Enzymatic 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, the above enzymatic solution is filtered with a cell filter to remove bacteria; 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, the pH is adjusted to 5.7-5.8 with dilute hydrochloric acid and sodium hydroxide, and the solution is autoclaved at 121°C for 20 min and stored at 4°C after cooling; the KAO & Michayluk medium is composed of 0.453 g / L CaCl2, 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; (3) Protoplast separation: the Oryzopsis and Pennisetum suspension cell lines subcultured for 3-4 days are taken, the upper clear culture medium is discarded, and the granular suspension cell line at the bottom is retained. The enzymatic solution is added, and the enzymolysis is carried out at 27-28°C, 20-70 rpm, and in dark conditions for 6-8 h. The enzymolysed cell line is filtered, centrifuged, and the upper enzymatic solution is removed, and the bottom protoplast is retained; (4) Protoplast purification: an equal volume of W1 solution is added, centrifuged, the upper solution is removed, and the bottom protoplast is retained. This step is repeated to obtain purified protoplasts.

4. The method of claim 3, wherein the wheat and pennisetum protoplast fusion and the callus regeneration thereof are characterized by, The step (3) protoplast isolation specific steps as follows: take the 3~4 d after subculture millet and Chinese penisetum suspension cell lines, pour the clear culture medium, retain the bottom of the granular suspension cell lines, add enzyme solution, 27~28℃, 50 rpm, dark conditions for 6~8 h, filter the enzyme solution, centrifugal, remove the top enzyme solution, leave the bottom protoplast.

5. The method of claim 1, wherein the wheat and pennisetum protoplast fusion and the callus regeneration thereof are characterized by, The protoplast fusion and callus regeneration specific steps as follows: (1) preparation of materials: purified millet and Chinese penisetum protoplasts; (2) preparation of protoplast fusion and callus regeneration required solution: Electrofusion solution: 0.6 M mannitol + 5 mM CaCl2, filter sterilization; 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; (3) Protoplast fusion: 1 mL of purified Oryzopsis and Wolfson's protoplasts were taken respectively, and the concentration was adjusted to 1 x 10 5 9 / mL, 1:5 mixed; the mixed protoplasts were taken and placed in the electrode plate, and the electric fusion parameters were as follows: alternating current field strength 20 V / cm, alternating current field action time 25 s, alternating current continuous time 7 s; direct current pulse strength 80 V / cm, direct current pulse time 25 s, direct current pulse interval time 0.5 s; cycle 1 time, select fusion mode; the fused protoplasts were placed at room temperature for 20-30 min, then centrifuged at 100 x g for 1 min, and the supernatant was removed; The electrofusion instrument used is: BEX GEB 15+ / CFB16-HB fusion instrument, electrode: LF501 series 1 mm gold block electrode; Electrofusion is operated in a sterile environment, the electrode is sterilized with 75% alcohol and then washed with sterile water, repeated sterilization for several times; (4) Callus regeneration: the fused protoplasts are resuspended with W1 solution, the concentration is adjusted to 1 x 10 5 6 mL / mL, 0.6 mL of the fused protoplasts are taken and plated in a sterile glass culture dish, and cultured at 27~28℃ in the dark / light scattering light and moisture; every 7 days, 0.1 mL of fresh W1 solution is added to the culture dish until callus is regenerated; when the callus grows to 1.5~2 mm, it is picked out and transferred to W2 medium for proliferation.

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