Method for screening out negative sorghum somatic embryos

Abstract

The invention discloses a method for screening negative sorghum somatic embryos. The method comprises the following steps: (1) disinfecting mature sweet sorghum seeds; (2) preparing a first 2, 4-D concentration gradient callus induction culture medium; (3) sowing the sweet sorghum seeds into a first 2, 4-D concentration gradient callus induction culture medium; (4) sealing, carrying out dark culture, and screening out type 2 embryogenic calluses; (5) preparing a second 2, 4-D concentration gradient callus induction culture medium; (6) subculturing the 2-type embryogenic callus to a second 2, 4-D concentration gradient callus induction medium, sealing, carrying out dark culture, and screening out 2-type yellow granular callus; (7) preparing a differential medium; (8) differentiation culture; (9) sealing, controlling the photoperiod to be 16h / 8h, and continuously culturing; (10) preparing a rooting culture medium; (11) rooting culture; (12) sealing, rooting after continuous culture, and transplanting strong roots into soil to grow into complete fertile plants.

Description

Technical Field

[0001] This invention relates to the technical fields of crop biotechnology, plant developmental biology, molecular plant breeding, and plant gene editing, specifically to a method for screening negative sorghum somatic embryos. Background Technology

[0002] Sweet sorghum is a widely cultivated forage crop worldwide, but its genetic transformation is difficult, limiting the industrial application of genome editing. Easily prepared and screenable recipient systems are prerequisites for genetic transformation.

[0003] Plant somatic embryos are zygotic embryo-like structures formed by the development of plant somatic cells under in vitro culture conditions, and can regenerate into complete plants. In 1958, Steward et al. first discovered this phenomenon in the suspension culture of carrot phloem cells, confirming the totipotency of plant cells and laying the foundation for in vitro cell culture technology.

[0004] Currently, a prominent technical problem in the genetic transformation of mature sorghum seeds is the unclear pattern of antibiotic resistance in embryogenic callus tissue derived from mature seeds. Screening systems referencing immature zygotic embryos (IZEs) frequently result in positive callus tissue turning brown and dying due to inappropriate antibiotic concentrations, severely hindering the practical application of gene editing technology. Therefore, how to efficiently screen out negative sorghum somatic embryos has become an urgent technical problem to be solved. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of the prior art, the present invention discloses a method for screening negative sorghum somatic cell embryos.

[0006] This invention utilizes high concentrations of 2,4-D to induce somatic embryos while simultaneously using chemical methods to screen out negative sorghum somatic embryos.

[0007] This invention utilizes antibiotics such as hygromycin and kanamycin to efficiently screen out negative sorghum somatic embryos, and its understanding of the antibiotic resistance patterns in mature seed embryogenic callus is of great significance. The main objective of this research is to apply this technology to the genetic transformation or gene editing of sweet sorghum.

[0008] Technical solution: A method for screening negative sorghum somatic cell embryos, the steps of which are as follows: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 1 to 8 weeks as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for dark culture for 1 to 8 weeks as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 50-500 nm. After continuous culture for 4 to 8 weeks, the callus treated with high concentration of TDZ (thiabendazole) solution will show a complete main stem. After the main stem grows to no less than 2 cm, it will be ready to be subcultured into rooting medium. (10) Prepare rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. After continuous cultivation for 4 to 6 weeks, roots will develop. After the roots are strengthened by hydroponics, the plants will be transplanted into soil and develop into complete fertile plants.

[0009] Beneficial effects: Compared with the prior art, the method for screening negative sorghum somatic cell embryos disclosed in this invention has at least the following beneficial effects: 1. Using mature sorghum seeds as receptors results in high induction efficiency and can replace the traditional IZE (immature zygotic embryo) receptor system; 2. The tolerance pattern of type 2 somatic embryos to hygromycin and kanamycin was verified, which significantly improved the screening efficiency of negative callus tissue; 3. It was clarified that type 2 somatic cell embryos are not sensitive to carbenicillin and termethin, and these two antibiotics can be used to inhibit the excessive proliferation of Agrobacterium and ensure the acquisition of positive regenerated plants. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the induction and regeneration of sweet sorghum somatic embryos in Example 1. Hygromycin was used to screen out negative sorghum somatic embryos during the induction and regeneration process.

[0011] Figure 2 This is a schematic diagram of sweet sorghum somatic embryo induction and regeneration in Example 2. During the induction and regeneration process, kanamycin was used to screen out negative sorghum somatic embryos.

[0012] Figure 3 This is a schematic diagram of sweet sorghum somatic cell embryo induction in Example 3, during which negative sorghum somatic cell embryos were treated with a carbenicillin concentration gradient.

[0013] Figure 4 This is a schematic diagram of sweet sorghum somatic cell embryo induction in Example 4, during which negative sorghum somatic cell embryos were treated with a concentration gradient of termethin. Detailed Implementation

[0014] The specific embodiments of the present invention are described in detail below.

[0015] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–50 is listed for a specific parameter, it is also expected that ranges of 10–40 and 20–50 are also included. Furthermore, if the minimum range values ​​are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0–5" means that all real numbers between "0–5" have been listed herein; "0–5" is merely a shortened representation of these numerical combinations.

[0016] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0017] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0018] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0019] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0020] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0021] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0022] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0023] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0024] A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 1 to 8 weeks as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for dark culture for 1 to 8 weeks as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 50-500 nm. After continuous culture for 4 to 8 weeks, the callus treated with high concentration of TDZ (thiabendazole) solution will show a complete main stem. After the main stem grows to no less than 2 cm, it will be ready to be subcultured into rooting medium. (10) Prepare rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. After continuous cultivation for 4 to 6 weeks, roots will develop. After the roots are strengthened by hydroponics, the plants will be transplanted into soil and develop into complete fertile plants.

[0025] Furthermore, the specific steps of step (1) are as follows: (11) Take 8-12 ml of mature sweet sorghum seeds, put them in a centrifuge tube, and then add 40 ml of ethanol solution containing 40 μl of surfactant Tween-20. Tighten the cap and shake on a shaker at 60-100 rpm for 30-40 minutes. (12) After discarding the liquid, add 40ml of 1% to 3% pasteurization solution and shake at 60 to 100 rpm for at least 3 hours. After discarding the liquid, wash with ultrapure water 2 to 4 times and set aside.

[0026] Furthermore, the specific steps of step (2) are as follows: (21) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for at least 20 minutes, cool to 60-80℃. (22) Add an appropriate amount of 2,4-D stock solution to the reagent bottle so that the concentration of 2,4-D is 1-20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle so that the concentration of lipoic acid is 10-1000 μM; (24) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle so that the concentration of hygromycin is 1-1000 mg / L, the concentration of kanamycin is 1-1000 mg / L, the concentration of carbenicillin is 1-1000 mg / L, the concentration of termethin is 1-1000 mg / L, and the concentration of acetylsuccione is 1-1000 mg / L. (25) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the reagent bottle so that the concentration of deacetylation inhibitor TSA is 0.1-100 μM; (26) Add an appropriate amount of CuSO4 to the reagent bottle to make the final concentration of CuSO4 1 µM. Shake well, pour the plate, and condense to obtain the first 2,4-D concentration gradient callus induction medium.

[0027] Further, step (3): the seeding density is controlled at 15 seeds / petal, with each 2 petri dishes as a biological replicate.

[0028] Furthermore, the specific steps of step (5) are as follows: (51) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for at least 20 minutes, cool to 60-80℃. (52) Add an appropriate amount of 2,4-D stock solution to the reagent bottle and adjust the final concentration of 2,4-D to 1-20 mg / L; (53) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle and adjust the concentration of lipoic acid to 10-1000 μM; (54) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, and adjust the concentration of hygromycin to 1-1000 mg / L, the concentration of kanamycin to 1-1000 mg / L, the concentration of carbenicillin to 1-1000 mg / L, the concentration of termethin to 1-1000 mg / L, and the concentration of acetylsuccione to 1-1000 mg / L. (55) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (56) Add an appropriate amount of CuSO4 to the reagent bottle, adjust the final concentration of CuSO4 to 1 µM, shake well, pour the plate, and condense to obtain the second 2,4-D concentration gradient callus induction medium.

[0029] Further, in step (6): 2 to 8 type 2 embryogenic callus tissues are sown in each culture dish.

[0030] Furthermore, the specific steps of step (7) are as follows: (71) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Add 30g of sucrose and 8-12g of agar powder to the reagent bottle. Sterilize at 120℃ for at least 20 minutes and then cool to 60℃-80℃. (72) Add appropriate amounts of IAA (indoleacetic acid) stock solution and TDZ (thiabendazole) stock solution to the reagent bottle respectively, and adjust the final concentration of IAA to 1 mg / L and the final concentration of TDZ to 1-5 mg / L. (73) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the final concentration of CuSO4 to 1 µM; (74) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, adjust the concentration of hygromycin to 1-1000 mg / L, the concentration of kanamycin to 1-1000 mg / L, the concentration of carbenicillin to 1-1000 mg / L, the concentration of termethin to 1-1000 mg / L, and the concentration of acetylsuccione to 1-1000 mg / L, and obtain the differentiation medium after condensation; (75) Pour 25 ml of the differentiation medium obtained in step (4) into a breathable glass culture bottle for later use.

[0031] Further, in step (8): every 3 type 2 yellow granular callus tissues are inoculated into a breathable glass culture bottle.

[0032] Furthermore, step (10) is as follows: (101) Put 4.4g of MS medium into a reagent bottle, add water to make up to 1L, adjust the pH to 5.6-6.0, then add 30g of sucrose and 8-12g of agar powder, autoclave at 120℃ for at least 20 minutes, and then cool to 60℃-80℃. (102) Add appropriate amounts of NAA (naphthaleneacetic acid) stock solution, IAA (indoleacetic acid) stock solution and IBA (indolebutyric acid) stock solution to the reagent bottle respectively, and adjust the final concentration of NAA to 0.1-5 mg / L, the final concentration of IAA to 0.1-5 mg / L, and the final concentration of IBA to 0.1-5 mg / L. (103) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the concentration of CuSO4 to 1 µM to obtain the rooting medium. (104) Pour 25 ml of the rooting medium obtained in step (103) into a breathable glass culture bottle and condense for later use. The rooting medium is not screened for antibiotics.

[0033] Further, in step (11), every 1 to 3 main stems are inoculated into a breathable glass culture bottle.

[0034] In one embodiment: A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 1 week as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for dark culture for 1 week as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture bottle and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 50. After continuous culture for 4 to 8 weeks, the callus treated with high concentration of TDZ (thiabendazole) solution will show a complete main stem. After the main stem grows to no less than 2 cm, it will be ready to be subcultured into rooting medium. (10) Prepare rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture bottle and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 50. After four weeks of continuous cultivation, roots develop. After hydroponics to strengthen the roots, the plants are transplanted into soil and develop into complete fertile plants.

[0035] Furthermore, the specific steps of step (1) are as follows: (11) Take 8 ml of mature sweet sorghum seeds, put them in a centrifuge tube, and then add 40 ml of ethanol solution containing 40 μl of surfactant Tween-20. Tighten the cap and shake on a shaker at 60 rpm for 40 minutes. (12) After discarding the liquid, add 40ml of 1% pasteurization solution, shake at 60rpm for 3 hours, discard the liquid, wash twice with ultrapure water, and set aside.

[0036] Furthermore, the specific steps of step (2) are as follows: (21) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 20 minutes, cool to 60℃. (22) Add an appropriate amount of 2,4-D stock solution to the reagent bottle so that the concentration of 2,4-D is 1 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle so that the concentration of lipoic acid is 10 μM; (24) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle so that the concentrations of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione are 1 mg / L. (25) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the reagent bottle so that the concentration of deacetylation inhibitor TSA is 0.1 μM; (26) Add an appropriate amount of CuSO4 to the reagent bottle to make the final concentration of CuSO4 1 µM. Shake well, pour the plate, and condense to obtain the first 2,4-D concentration gradient callus induction medium.

[0037] Further, step (3): the seeding density is controlled at 15 seeds / petal, with each 2 petri dishes as a biological replicate.

[0038] Furthermore, the specific steps of step (5) are as follows: (51) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. Sterilize at 120℃ for 20 minutes and then cool to 60℃. (52) Add an appropriate amount of 2,4-D stock solution to the reagent bottle and adjust the final concentration of 2,4-D to 1 mg / L; (53) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle and adjust the concentration of lipoic acid to 10 μM; (54) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, and adjust the concentration of hygromycin to 1 mg / L, the concentration of kanamycin to 1 mg / L, the concentration of carbenicillin to 1 mg / L, the concentration of termethin to 1 mg / L, and the concentration of acetylsuccione to 1 mg / L. (55) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle and adjust the concentration of deacetylation inhibitor TSA to 0.1 μM; (56) Add an appropriate amount of CuSO4 to the reagent bottle, adjust the final concentration of CuSO4 to 1 µM, shake well, pour the plate, and condense to obtain the second 2,4-D concentration gradient callus induction medium.

[0039] Further, in step (6): two type 2 embryogenic callus tissues are seeded in each culture dish.

[0040] Furthermore, the specific steps of step (7) are as follows: (71) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Add 30g of sucrose and 8g of agar powder to the reagent bottle. Sterilize at 120℃ for at least 20 minutes and then cool to 60℃. (72) Add appropriate amounts of IAA (indoleacetic acid) stock solution and TDZ (thiabendazole) stock solution to the reagent bottle respectively, and adjust the final concentration of IAA to 1 mg / L and the final concentration of TDZ to 1 mg / L. (73) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the final concentration of CuSO4 to 1 µM; (74) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle respectively, adjust the concentration of hygromycin to 1 mg / L, the concentration of kanamycin to 1 mg / L, the concentration of carbenicillin to 1 mg / L, the concentration of termethin to 1 mg / L, and the concentration of acetylsuccione to 1 mg / L, and obtain the differentiation medium after condensation; (75) Pour 25 ml of the differentiation medium obtained in step (4) into a breathable glass culture bottle for later use.

[0041] Further, in step (8): every 3 type 2 yellow granular callus tissues are inoculated into a breathable glass culture bottle.

[0042] Furthermore, step (10) is as follows: (101) Put 4.4g of MS medium into a reagent bottle, add water to make up to 1L, adjust the pH to 5.6, then add 30g of sucrose and 8g of agar powder, autoclave at 120℃ for 20 minutes, and then cool to 60℃. (102) Add appropriate amounts of NAA (naphthaleneacetic acid) stock solution, IAA (indoleacetic acid) stock solution and IBA (indolebutyric acid) stock solution to the reagent bottle respectively, and adjust the final concentration of NAA to 0.1 mg / L, the final concentration of IAA to 0.1 mg / L, and the final concentration of IBA to 0.1 mg / L. (103) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the concentration of CuSO4 to 1 µM to obtain the rooting medium. (104) Pour 25 ml of the rooting medium obtained in step (103) into a breathable glass culture bottle and condense for later use. The rooting medium is not screened for antibiotics.

[0043] Further, in step (11), each main stem is inoculated into a breathable glass culture bottle.

[0044] In another embodiment: A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 8 weeks as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for dark culture for 8 weeks as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 500. After 8 weeks of continuous culture, the callus treated with high concentration of TDZ (thiabendazole) solution showed a complete main stem. After the main stem grew to no less than 2 cm, it was ready to be subcultured into rooting medium. (10) Prepare rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 500 ppm. After 6 weeks of continuous cultivation, roots will develop. After the roots are strengthened by hydroponics, the plants will be transplanted into soil and develop into complete fertile plants.

[0045] Furthermore, the specific steps of step (1) are as follows: (11) Take 12 ml of mature sweet sorghum seeds, put them in a centrifuge tube, and then add 40 ml of ethanol solution containing 40 μl of surfactant Tween-20. Tighten the cap and shake at 100 rpm for 30 minutes on a shaker. (12) After discarding the liquid, add 40ml of 3% pasteurization solution, shake at 100rpm for 5 hours, discard the liquid, wash with ultrapure water 4 times, and set aside.

[0046] Furthermore, the specific steps of step (2) are as follows: (21) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. Sterilize at 120℃ for 40 minutes and then cool to 80℃. (22) Add an appropriate amount of 2,4-D stock solution to the reagent bottle so that the concentration of 2,4-D is 20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle so that the concentration of lipoic acid is 1000 μM; (24) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle so that the concentrations of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione are 1000 mg / L, 1000 mg / L, 1000 mg / L, and 1000 mg / L, respectively. (25) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the reagent bottle so that the concentration of deacetylation inhibitor TSA is 100 μM; (26) Add an appropriate amount of CuSO4 to the reagent bottle to make the final concentration of CuSO4 1 µM. Shake well, pour the plate, and condense to obtain the first 2,4-D concentration gradient callus induction medium.

[0047] Further, step (3): the seeding density is controlled at 15 seeds / petal, with each 2 petri dishes as a biological replicate.

[0048] Furthermore, the specific steps of step (5) are as follows: (51) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 40 minutes, cool to 80℃. (52) Add an appropriate amount of 2,4-D stock solution to the reagent bottle and adjust the final concentration of 2,4-D to 20 mg / L; (53) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle and adjust the concentration of lipoic acid to 1000 μM; (54) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, and adjust the concentration of hygromycin to 1000 mg / L, the concentration of kanamycin to 1000 mg / L, the concentration of carbenicillin to 1000 mg / L, the concentration of termethin to 1000 mg / L, and the concentration of acetylsuccione to 1000 mg / L. (55) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle and adjust the concentration of deacetylation inhibitor TSA to 100 μM; (56) Add an appropriate amount of CuSO4 to the reagent bottle, adjust the final concentration of CuSO4 to 1 µM, shake well, pour the plate, and condense to obtain the second 2,4-D concentration gradient callus induction medium.

[0049] Further, in step (6): eight type 2 embryogenic callus tissues are seeded in each culture dish.

[0050] Furthermore, the specific steps of step (7) are as follows: (71) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Add 30g of sucrose and 12g of agar powder to the reagent bottle. Sterilize by autoclaving at 120℃ for 40 minutes and then cool to 80℃. (72) Add appropriate amounts of IAA (indoleacetic acid) stock solution and TDZ (thiabendazole) stock solution to the reagent bottle respectively, and adjust the final concentration of IAA to 1 mg / L and the final concentration of TDZ to 5 mg / L. (73) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the final concentration of CuSO4 to 1 µM; (74) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, adjust the concentration of hygromycin to 1000 mg / L, the concentration of kanamycin to 1000 mg / L, the concentration of carbenicillin to 1000 mg / L, the concentration of termethin to 1000 mg / L, and the concentration of acetylsuccione to 1000 mg / L, and obtain the differentiation medium after condensation; (75) Pour 25 ml of the differentiation medium obtained in step (4) into a breathable glass culture bottle for later use.

[0051] Further, in step (8): every 3 type 2 yellow granular callus tissues are inoculated into a breathable glass culture bottle.

[0052] Furthermore, step (10) is as follows: (101) Put 4.4g of MS medium into a reagent bottle, add water to make up to 1L, adjust the pH to 6.0, then add 30g of sucrose and 12g of agar powder, autoclave at 120℃ for 40 minutes, and then cool to 80℃. (102) Add appropriate amounts of NAA (naphthaleneacetic acid) stock solution, IAA (indoleacetic acid) stock solution and IBA (indolebutyric acid) stock solution to the reagent bottle respectively, adjust the final concentration of NAA to 5 mg / L, adjust the final concentration of IAA to 5 mg / L, and adjust the final concentration of IBA to 5 mg / L. (103) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the concentration of CuSO4 to 1 µM to obtain the rooting medium. (104) Pour 25 ml of the rooting medium obtained in step (103) into a breathable glass culture bottle and condense for later use. The rooting medium is not screened for antibiotics.

[0053] Further, in step (11), every 3 main stems are inoculated into a breathable glass culture bottle.

[0054] In yet another embodiment: A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 4 weeks as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for 6 weeks in the dark as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 200. After 6 weeks of continuous culture, the callus treated with high concentration of TDZ (thiabendazole) solution showed a complete main stem. After the main stem grew to no less than 2 cm, it was ready to be subcultured into rooting medium. (10) Prepare rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 300 ppm. After 5 weeks of continuous cultivation, roots will develop. After the roots are strengthened by hydroponics, the plants will be transplanted into soil and develop into complete fertile plants.

[0055] Furthermore, the specific steps of step (1) are as follows: (11) Take 10 ml of mature sweet sorghum seeds, put them in a centrifuge tube, and then add 40 ml of ethanol solution containing 40 μl of surfactant Tween-20. Tighten the cap and shake on a shaker at 80 rpm for 35 minutes. (12) After discarding the liquid, add 40ml of 1% to 3% pasteurization solution, shake at 80rpm for 4 hours, discard the liquid, wash 3 times with ultrapure water, and set aside.

[0056] Furthermore, the specific steps of step (2) are as follows: (21) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. Sterilize at 120℃ for 30 minutes and then cool to 70℃. (22) Add an appropriate amount of 2,4-D stock solution to the reagent bottle so that the concentration of 2,4-D is 10 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle so that the concentration of lipoic acid is 300 μM; (24) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle so that the concentration of hygromycin is 200 mg / L, the concentration of kanamycin is 300 mg / L, the concentration of carbenicillin is 400 mg / L, the concentration of termethin is 500 mg / L, and the concentration of acetylsuccione is 500 mg / L. (25) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the reagent bottle so that the concentration of deacetylation inhibitor TSA is 50 μM; (26) Add an appropriate amount of CuSO4 to the reagent bottle to make the final concentration of CuSO4 1 µM. Shake well, pour the plate, and condense to obtain the first 2,4-D concentration gradient callus induction medium.

[0057] Further, step (3): the seeding density is controlled at 15 seeds / petal, with each 2 petri dishes as a biological replicate.

[0058] Furthermore, the specific steps of step (5) are as follows: (51) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 30 minutes, cool to 65℃. (52) Add an appropriate amount of 2,4-D stock solution to the reagent bottle and adjust the final concentration of 2,4-D to 8 mg / L; (53) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle and adjust the concentration of lipoic acid to 200 μM; (54) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, and adjust the concentration of hygromycin to 300 mg / L, the concentration of kanamycin to 400 mg / L, the concentration of carbenicillin to 500 mg / L, the concentration of termethin to 400 mg / L, and the concentration of acetylsuccione to 300 mg / L. (55) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle and adjust the concentration of deacetylation inhibitor TSA to 40 μM; (56) Add an appropriate amount of CuSO4 to the reagent bottle, adjust the final concentration of CuSO4 to 1 µM, shake well, pour the plate, and condense to obtain the second 2,4-D concentration gradient callus induction medium.

[0059] Further, in step (6): five type 2 embryogenic callus tissues are seeded in each culture dish.

[0060] Furthermore, the specific steps of step (7) are as follows: (71) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.8. Add 30g of sucrose and 10g of agar powder to the reagent bottle. Sterilize at 120℃ for at least 20 minutes and then cool to 60℃~80℃. (72) Add appropriate amounts of IAA (indoleacetic acid) stock solution and TDZ (thiabendazole) stock solution to the reagent bottle respectively, and adjust the final concentration of IAA to 1 mg / L and the final concentration of TDZ to 2 mg / L. (73) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the final concentration of CuSO4 to 1 µM; (74) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, adjust the concentration of hygromycin to 100 mg / L, the concentration of kanamycin to 200 mg / L, the concentration of carbenicillin to 300 mg / L, the concentration of termethin to 400 mg / L, and the concentration of acetylsuccione to 500 mg / L, and obtain the differentiation medium after condensation; (75) Pour 25 ml of the differentiation medium obtained in step (4) into a breathable glass culture bottle for later use.

[0061] Further, in step (8): every 3 type 2 yellow granular callus tissues are inoculated into a breathable glass culture bottle.

[0062] Furthermore, step (10) is as follows: (101) Put 4.4g of MS medium into a reagent bottle, add water to make up to 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder, autoclave at 120℃ for 30 minutes, and then cool to 72℃. (102) Add appropriate amounts of NAA (naphthaleneacetic acid) stock solution, IAA (indoleacetic acid) stock solution and IBA (indolebutyric acid) stock solution to the reagent bottle respectively, adjust the final concentration of NAA to 3 mg / L, adjust the final concentration of IAA to 2 mg / L, and adjust the final concentration of IBA to 4 mg / L. (103) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the concentration of CuSO4 to 1 µM to obtain the rooting medium. (104) Pour 25 ml of the rooting medium obtained in step (103) into a breathable glass culture bottle and condense for later use. The rooting medium is not screened for antibiotics.

[0063] Further, in step (11), every two main stems are inoculated into a breathable glass culture bottle.

[0064] Example 1 A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Take 10 ml of mature sweet sorghum seeds and place them in a 50 ml centrifuge tube. Add 45 ml of ethanol solution containing surfactant, tighten the cap and shake at 100 rpm for 30 minutes. Discard the liquid and add 40 ml of 2% pasteurization solution. Sterilize by shaking at 100 rpm for 3 hours. After discarding the liquid, wash with ultrapure water 4 times.

[0065] (2) Preparation of the first 2,4-D concentration gradient callus induction medium: Weigh 4.4g of MS basal medium and add it to a reagent bottle. Add water to bring the volume to 1L and adjust the pH to 5.8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 20 minutes, cool to 70℃ and add 2,4-D stock solution to adjust the final concentration to 5mg / L. In other embodiments, add 2,4-D stock solution to adjust the final concentration to 1mg / L. In other embodiments, add 2,4-D stock solution to adjust the final concentration to 20mg / L. The antioxidant lipoic acid (LA) concentration is 100μM, and the hygromycin concentrations are 0, 6.25, 2.5, and 25 mg / L (the concentration gradient is set as follows). Figure 1 (As shown in A and B); 0, 2, 4, 8, 16 mg / L (see details) Figure 1(Setting the concentration gradient of C); and setting the concentration of the deacetylation inhibitor TSA to 10 μM, with a final CuSO4 concentration of 1 µM. After shaking well, pour the mixture onto a plate, condense it, and then sow it.

[0066] (3) Sow the seeds treated in step (1) evenly onto the induction medium prepared in step (2), with a sowing density of 15 seeds / petal, and each 2 petals as a biological replicate.

[0067] (4) After sealing the petri dish from step (3) with sealing film, place it in an incubator at 25°C and incubate in the dark for 6 weeks as the first 2,4-D concentration gradient treatment.

[0068] (5) Preparation of the second 2,4-D concentration gradient callus induction medium: Weigh 4.4g of MS basal medium into a reagent bottle, add water to a final volume of 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 20 minutes, cool to 70℃, add 2,4-D stock solution, and adjust the final concentration to 10 mg / L. In other embodiments, add 2,4-D stock solution and adjust the final concentration to 1 mg / L. In other embodiments, add 2,4-D stock solution and adjust the final concentration to 20 mg / L. The antioxidant lipoic acid (LA) concentration is 100 μM, and the hygromycin concentrations are 0, 6.25, 2.5, and 25 mg / L (concentration gradient settings are as follows). Figure 1 (As shown in A and B); 0, 2, 4, 8, 16 mg / L (see details) Figure 1 The concentration gradient of C was set (and the concentration of the deacetylation inhibitor TSA was 10 μM, with a final concentration of CuSO4 of 1 µM). After shaking well, the plates were poured out, condensed, and prepared for subculture.

[0069] (6) Subculture the type 2 embryogenic callus selected in step (4) onto the second 2,4-D concentration gradient callus induction medium prepared in step (5). Five type 2 embryogenic callus were placed in each dish. After sealing with sealing film, the dishes were placed in an incubator at 25°C and cultured in the dark for 6 weeks as the second 2,4-D concentration gradient treatment.

[0070] (7) Preparation of differentiation medium: Weigh 4.4g of MS basal medium into a reagent bottle, add water to a final volume of 1L, adjust the pH to 5.8, add 30g of sucrose and 8g of agar powder to the reagent bottle, autoclave at 120℃ for 20 minutes, cool to 70℃, add IAA stock solution and TDZ stock solution respectively, adjust the final IAA concentration to 1 mg / L and the final TDZ concentration to 2 mg / L; 1 µM CuSO4, hygromycin concentrations of 0, 6.25, 2.5, and 25 mg / L (concentration gradient settings are as follows). Figure 1(As shown in A and B); 0, 2, 4, 8, 16 mg / L (see details) Figure 1 (Set the concentration gradient of C); pour 25 ml into a breathable glass culture flask, condense and set aside.

[0071] (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto the differentiation medium prepared in step (7). Three yellow granular callus tissues were inoculated into one culture flask.

[0072] (9) After inoculation in step (8), seal the culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h (250). After 6 weeks of continuous culture, the callus treated with high concentration of TDZ showed a complete main stem. When the main stem grew to 2cm, it was ready to be subcultured to the rooting medium.

[0073] like Figure 1 As shown in A and B, hygromycin strongly inhibits somatic embryogenesis in sweet sorghum seeds, causing the seeds to turn brown and eventually die within a week of treatment.

[0074] like Figure 1 As shown in C, hygromycin exhibits strong inhibitory effects even at low concentrations.

[0075] like Figure 1 As shown in Figure D, negative embryogenic callus was completely killed by hygromycin. This indicates that hygromycin also strongly inhibits the shoot elongation stage. Somatic embryos regenerated from the negative control (callus induction stage containing only 2,4-D) typically turn brown and eventually die in shoot elongation cultures containing hygromycin, even at concentrations as low as 12.5 mg / L. Therefore, hygromycin is an effective selection marker for callus induction, shoot elongation, and root induction cultures.

[0076] Example 2 A method for screening negative sorghum somatic cell embryos, comprising the following steps: (1) Take 10 ml of mature sweet sorghum seeds and place them in a 50 ml centrifuge tube. Add 40 ml of ethanol solution containing surfactant, tighten the cap and shake at 80 rpm for 30 minutes. After discarding the liquid, add 40 ml of 2% pasteurization solution and shake at 80 rpm for 3 hours. After discarding the liquid, wash 4 times with ultrapure water.

[0077] (2) Preparation of the first 2,4-D concentration gradient callus induction medium: Weigh 4.4g of MS basal medium into a reagent bottle, add water to a final volume of 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder to the reagent bottle. Autoclave at 120℃ for 20 minutes, then cool to 60℃. Add 2,4-D stock solution and adjust the final concentration to 5 mg / L. The concentrations of the antioxidant lipoic acid (LA) are 100μM, and the concentrations of kanamycin are 0, 12.5, 25, and 50 mg / L (see details). Figure 2 (Setting the concentration gradients for A and B); and setting the concentration of the deacetylation inhibitor TSA to 10 μM, with a final CuSO4 concentration of 1 µM. After shaking well, pour the mixture onto a plate, condense, and then sow.

[0078] (3) Sow the seeds treated in step (1) evenly onto the induction medium prepared in step (2), with a sowing density of 15 seeds / petal, and each 2 petals as a biological replicate.

[0079] (4) After sealing the petri dish from step (3) with sealing film, place it in an incubator at 25°C and incubate in the dark for 6 weeks as the first 2,4-D concentration gradient treatment.

[0080] (5) Preparation of the second 2,4-D concentration gradient callus induction medium: Weigh 4.4g of MS basal medium into a reagent bottle, add water to a final volume of 1L, adjust the pH to 5.8, and then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for 20 minutes, cool to 70℃, add 2,4-D stock solution, and adjust the final concentration to 10 mg / L. The concentrations of the antioxidant lipoic acid (LA) are 100μM, and the concentrations of kanamycin are 0, 12.5, 25, and 50 mg / L (see details). Figure 2 (Set the concentration gradients for A and B); and set the concentration of the deacetylation inhibitor TSA to 10 μM, with a final CuSO4 concentration of 1 µM. After shaking well, pour the plate, condense, and prepare for subculture.

[0081] (6) Subculture the type 2 embryogenic callus selected in step (4) onto the second 2,4-D concentration gradient callus induction medium prepared in step (5). Four type 2 embryogenic callus tissues were placed in each dish. After sealing with sealing film, the dishes were placed in an incubator at 25°C and cultured in the dark for 4 weeks as the second 2,4-D concentration gradient treatment.

[0082] (7) Preparation of differentiation medium: MS medium with 3% sucrose (w / v), pH 5.8, and 10g agar powder. After autoclaving at 120℃ for 20 minutes, cool to 70℃, add IAA stock solution and TDZ stock solution respectively, and adjust the final IAA concentration to 1 mg / L and the final TDZ concentration to 2 mg / L; 1 µM CuSO4, kanamycin concentrations of 0, 12.5, 25, and 50 mg / L (see details). Figure 2 (Set the concentration gradient between A and B); pour 25 ml into a breathable glass culture flask, condense and set aside.

[0083] (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto the differentiation medium prepared in step (7). Three yellow granular callus tissues were inoculated into one culture flask.

[0084] (9) After sealing the culture flasks inoculated in step (8), place them in an incubator at 25°C with a photoperiod of 16h / 8h (300). After continuous culture for 4 to 8 weeks, the callus treated with high concentration of TDZ will show a complete main stem. When the main stem grows to 2 cm, it will be ready to be subcultured into rooting medium.

[0085] Figure 2 A in the diagram is a schematic diagram of sweet sorghum somatic cell embryo induction in Example 2. During the induction process, kanamycin was used to screen out negative sorghum somatic cell embryos. Figure 2 B in the figure represents the effect of kanamycin on somatic embryogenesis during the induction process in Example 2. The graph shows that kanamycin has no screening effect on somatic embryogenesis, and at certain concentrations (e.g., 25, 50 mg / L), it even exhibits a promoting effect.

[0086] Figure 2 C in the diagram is a negative control; the regeneration medium does not contain kanamycin and serves as a control setting. Figure 2 The "D" in the text refers to the process of gradual whitening and screening out negative calluses when kanamycin is at a concentration of 12.5 mg / L. Figure 2 E in the figure represents the process by which kanamycin at 25 mg / L causes severe whitening, directly eliminating negative callus. This is a very good (effective) result of concentration gradient treatment. Figure 2 C, D, and E in the diagram represent sorghum regenerated plants screened for negative results using kanamycin during the regeneration process; obvious whitening is observed, demonstrating a significant screening effect. Kanamycin slowly inhibits the shoot elongation process of somatic embryos (SE). In the negative control (where only 2.0 mg / L 2,4-D was added during callus induction), regenerated somatic embryos normally turned green on shoot elongation medium (SEM). Figure 2 (C in the text), and after 3 weeks of passage at 12.5 mg / L kanamycin, a slight greenish-whitening phenomenon gradually appeared (C in the text). Figure 2 D in the text). Typical whitening was observed on SEM at 25 mg / L kanamycin. Figure 2 (E in the text). Our results indicate that kanamycin is an effective selection marker during the shoot elongation stage, rather than the callus induction stage.

[0087] Example 3 Example 3 is basically the same as Example 2, the main difference being that during the callus induction stage, the concentration of carbenicillin was 0, 25, 50, and 100 mg / L, while the concentrations of other substances were exactly the same. The callus was subcultured into fresh callus induction medium every two weeks, and after 8 weeks of culture, the induced callus was directly subjected to induction rate statistics and microscopic examination. Figure 3 In Figure A, the curve representing the effect of carbenicillin on somatic embryogenesis during the induction process in Example 3 is shown. Figure 3 Example B illustrates the effect of carbenicillin on somatic embryogenesis. The graphs and morphological evidence demonstrate that carbenicillin has no inhibitory effect on somatic embryogenesis; therefore, it can be used to inhibit the overgrowth of bacteria such as Agrobacterium. Example 4 Example 4 The procedure was essentially the same as in Example 3, with the main difference being that during the callus induction phase, the concentrations of termethin were 0, 25, 50, and 100 mg / L, while the concentrations of other substances remained identical. The callus was subcultured into fresh callus induction medium every two weeks. After 8 weeks of culture, the induced callus was directly analyzed for induction rate and examined under a microscope. Figure 4 In Figure A, the curve representing the effect of termethin on somatic embryogenesis during the induction process of Example 4 is shown. Figure 4 B shows an example of the effect of termethin on somatic embryogenesis. The graphs and morphological evidence indicate that termethin has no inhibitory effect on somatic embryogenesis; therefore, it can be used to inhibit the excessive proliferation of bacteria such as Agrobacterium.

[0088] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for screening negative sorghum somatic cell embryos, characterized in that, The steps are as follows: (1) Disinfect mature sweet sorghum seeds; (2) Prepare the first 2,4-D concentration gradient callus induction medium; (3) The sweet sorghum seeds treated in step (1) are evenly sown onto a petri dish containing the first 2,4-D concentration gradient callus induction medium prepared in step (2). (4) After sealing the culture dish described in step (3) with sealing film, place it in an incubator at 25°C and culture it in the dark for 1 to 8 weeks as the first 2,4-D concentration gradient treatment to induce and screen out type 2 embryogenic callus. (5) Prepare a second 2,4-D concentration gradient callus induction medium; (6) Subculture the type 2 embryogenic callus selected in step (4) onto a culture dish containing the second 2,4-D concentration gradient callus induction medium prepared in step (5), seal it with sealing film and place it in an incubator at 25°C for dark culture for 1 to 8 weeks as the second 2,4-D concentration gradient treatment to induce and screen out type 2 yellow granular callus. (7) Preparation of differentiation culture medium; (8) Differentiation culture: The type 2 yellow granular callus induced in step (6) was inoculated onto a breathable glass culture bottle containing 25 mL of the differentiation medium prepared in step (7); (9) After inoculation in step (8), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and an illumination intensity of 50-500 nm. After continuous culture for 4 to 8 weeks, the callus treated with high concentration TDZ solution will show a complete main stem. After the main stem grows to no less than 2 cm, it will be ready to be subcultured into rooting medium. (10) Preparation of rooting culture medium; (11) Rooting culture: Cut off the main stem that was induced to be more than 2cm long in step (9), discard the remaining callus tissue, and inoculate it into a breathable glass culture bottle containing 25ml of rooting culture medium prepared in step (10); (12) After inoculation in step (11), seal the breathable glass culture flask and place it in an incubator at 25°C with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. After continuous cultivation for 4 to 6 weeks, roots will develop. After the roots are strengthened by hydroponics, the plants will be transplanted into soil and develop into complete fertile plants.

2. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, The specific steps of step (1) are as follows: (11) Take 8-12 ml of mature sweet sorghum seeds, put them in a centrifuge tube, and then add 40 ml of ethanol solution containing 40 μl of surfactant Tween-20. Tighten the cap and shake on a shaker at 60-100 rpm for 30-40 minutes. (12) After discarding the liquid, add 40ml of 1% to 3% pasteurization solution and shake at 60 to 100 rpm for at least 3 hours. After discarding the liquid, wash with ultrapure water 2 to 4 times and set aside.

3. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, The specific steps of step (2) are as follows: (21) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.

8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for at least 20 minutes, cool to 60-80℃. (22) Add an appropriate amount of 2,4-D stock solution to the reagent bottle so that the concentration of 2,4-D is 1-20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle so that the concentration of lipoic acid is 10-1000 μM; (24) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottle so that the concentration of hygromycin is 1-1000 mg / L, the concentration of kanamycin is 1-1000 mg / L, the concentration of carbenicillin is 1-1000 mg / L, the concentration of termethin is 1-1000 mg / L, and the concentration of acetylsuccione is 1-1000 mg / L. (25) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle so that the concentration of deacetylation inhibitor TSA is 0.1-100 μM; (26) Add an appropriate amount of CuSO4 to the reagent bottle to make the final concentration of CuSO4 1 µM. Shake well, pour the plate, and condense to obtain the first 2,4-D concentration gradient callus induction medium.

4. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, In step (3), the seeding density is controlled at 15 seeds / petal, with each 2 petals forming a biological replicate.

5. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, The specific steps of step (5) are as follows: (51) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.

8. Then add 30g of sucrose and 10g of agar powder to the reagent bottle. After autoclaving at 120℃ for at least 20 minutes, cool to 60-80℃. (52) Add an appropriate amount of 2,4-D stock solution to the reagent bottle and adjust the final concentration of 2,4-D to 1-20 mg / L; (53) Add an appropriate amount of antioxidant lipoic acid to the reagent bottle and adjust the concentration of lipoic acid to 10-1000 μM; (54) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, and adjust the concentration of hygromycin to 1-1000 mg / L, the concentration of kanamycin to 1-1000 mg / L, the concentration of carbenicillin to 1-1000 mg / L, the concentration of termethin to 1-1000 mg / L, and the concentration of acetylsuccione to 1-1000 mg / L. (55) Add an appropriate amount of deacetylation inhibitor TSA to the reagent bottle and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (56) Add an appropriate amount of CuSO4 to the reagent bottle, adjust the final concentration of CuSO4 to 1 µM, shake well, pour the plate, and condense to obtain the second 2,4-D concentration gradient callus induction medium.

6. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, In step (6): 2 to 8 type 2 embryogenic callus tissues are sown in each culture dish.

7. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, The specific steps of step (7) are as follows: (71) Weigh 4.4g of MS basal culture medium and add it to the reagent bottle. Add water to make up to 1L and adjust the pH to 5.

8. Add 30g of sucrose and 8-12g of agar powder to the reagent bottle. Sterilize at 120℃ for at least 20 minutes and then cool to 60℃-80℃. (72) Add appropriate amounts of IAA stock solution and TDZ stock solution to the reagent bottle respectively, and adjust the final concentration of IAA to 1 mg / L and the final concentration of TDZ to 1-5 mg / L. (73) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the final concentration of CuSO4 to 1 µM; (74) Add appropriate amounts of hygromycin, kanamycin, carbenicillin, termethin, and acetylsuccione to the reagent bottles respectively, adjust the concentration of hygromycin to 1-1000 mg / L, the concentration of kanamycin to 1-1000 mg / L, the concentration of carbenicillin to 1-1000 mg / L, the concentration of termethin to 1-1000 mg / L, and the concentration of acetylsuccione to 1-1000 mg / L, and obtain the differentiation medium after condensation; (75) Pour 25 ml of the differentiation medium obtained in step (4) into a breathable glass culture bottle for later use.

8. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, In step (8): every 3 type 2 yellow granular callus tissues are inoculated into a breathable glass culture bottle.

9. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, The steps of step (10) are as follows: (101) Put 4.4g of MS medium into a reagent bottle, add water to make up to 1L, adjust the pH to 5.6-6.0, then add 30g of sucrose and 8-12g of agar powder, autoclave at 120℃ for at least 20 minutes, and then cool to 60℃-80℃. (102) Add appropriate amounts of NAA stock solution, IAA stock solution and IBA stock solution to the reagent bottle respectively, adjust the final concentration of NAA to 0.1-5 mg / L, adjust the final concentration of IAA to 0.1-5 mg / L, and adjust the final concentration of IBA to 0.1-5 mg / L; (103) Add an appropriate amount of CuSO4 to the reagent bottle and adjust the concentration of CuSO4 to 1 µM to obtain the rooting medium. (104) Pour 25 ml of the rooting medium obtained in step (103) into a breathable glass culture bottle and condense for later use.

10. The method for screening negative sorghum somatic cell embryos as described in claim 1, characterized in that, In step (11), 1 to 3 main stems are inoculated into a breathable glass culture bottle.

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