Method for promoting regeneration of somatic embryos of sweet sorghum

Abstract

The invention discloses a method for promoting regeneration of somatic embryos of sweet sorghum. The method comprises the following steps: (1) disinfecting mature sorghum seeds; (2) preparing a culture medium with a first 2, 4-D concentration gradient; (3) sowing sorghum seeds on a first 2, 4-D concentration gradient culture medium, carrying out dark treatment for 1-5 weeks, and inducing to obtain calluses; (4) preparing a culture medium with a second 2, 4-D concentration gradient; and (5) subculturing the callus to the second 2, 4-D concentration gradient culture medium prepared in the step (4), carrying out photoperiod of 16h / 8h and treatment time of 1-5 weeks, and obtaining the type 2 embryogenic callus at the hypocotyl after culture. The method can efficiently and stably promote the regeneration of the somatic embryos of the sweet sorghum, and overcomes the defects of long preparation period and high cost of the immature embryos of the sorghum; the problem of annual supply of sorghum gene editing receptors is solved.

Description

Technical Field

[0001] This invention relates to the field of plant developmental biology, and more specifically to a method for promoting the regeneration of somatic embryos in sweet sorghum. Background Technology

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

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

[0004] The current technical challenges in sorghum genetic transformation are as follows: Sorghum recipient systems typically originate from immature zygotic embryos (IZEs), but producing IZEs requires a long period, is limited by the growing season, or necessitates costly artificial climate chambers. Furthermore, IZEs exhibit genotype dependence, with most successful reports focusing on inbred lines such as Tx430, Tx623, and P898102. Using mature seeds results in an embryogenic callus induction rate of approximately 40%, with even lower regeneration rates and a higher incidence of deformed seedlings. Both factors make integration into existing hybridization breeding systems difficult. Summary of the Invention

[0005] Purpose of the invention: The present invention addresses the problems existing in the prior art by disclosing a method for promoting somatic embryo regeneration in sweet sorghum.

[0006] Mature seeds of parental lines are ideal materials for producing somatic embryos. This invention utilizes multi-stage, high-concentration 2,4-D treatment, combined with the antioxidant lipoic acid and the deacetylation inhibitor TSA, to induce somatic embryos from mature parental seeds on MS medium. The relatively high concentration of 2,4-D effectively induces embryogenic callus tissue from the hypocotyl, with a somatic embryogenesis rate of approximately 60% and a significantly improved regeneration rate. The main objective of this research is to apply this technology to the genetic transformation or gene editing of sweet sorghum. This invention can efficiently and stably promote the regeneration of somatic embryos in sweet sorghum, overcoming the disadvantages of long preparation cycles, high costs, and cumbersome operations associated with using immature sorghum embryos; it also solves the problem of year-round supply of sorghum gene editing recipients.

[0007] Technical solution: A method for promoting somatic embryo regeneration in sweet sorghum, comprising the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the culture medium with the first 2,4-D concentration gradient prepared in step (2). Treat them in the dark for 1 to 5 weeks to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. The treatment time is 1 to 5 weeks, and type 2 embryogenic callus can be seen at the hypocotyl after culture.

[0008] Beneficial Effects: This invention discloses a method for promoting somatic embryo regeneration in sweet sorghum, which has the following beneficial effects: 1. This invention provides a method for promoting somatic embryo regeneration in sweet sorghum, with an induction efficiency of up to 60% (e.g., Figure 8 (as shown) 2. This invention replaces the traditional immature zygotic embryo receptor system, overcoming the disadvantages of the immature zygotic embryo receptor system, such as long preparation cycle, high cost, and genotype dependence.

[0009] 3. Compared with the traditional single inducing agent dosage, the present invention uses a stepwise inducing agent treatment to significantly improve the callus regeneration rate without reducing the embryogenic callus induction rate, thus solving the problem of year-round supply of gene editing receptors for sweet sorghum backbone parent lines. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 1, which only involved the first 2,4-D concentration gradient (short cycle of 1 week).

[0011] Figure 2 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 2, where only the first 2,4-D concentration gradient (long cycle of 2 weeks) was performed.

[0012] Figure 3 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 3, involving two 2,4-D concentration gradients (short-cycle single-dose treatment).

[0013] Figure 4 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 4, where only the first 2,4-D concentration gradient (long cycle 8 weeks) was performed.

[0014] Figure 5 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 5, involving two 2,4-D concentration gradients (short-cycle dose increase / decrease treatments).

[0015] Figure 6 This is a schematic diagram of sweet sorghum somatic embryo induction in Example 6, where only the first 2,4-D concentration gradient (medium-cycle high and low dose treatment) was performed.

[0016] Figure 7 This is a schematic diagram of sweet sorghum somatic embryo induction with two 2,4-D concentration gradients (long-cycle dose increase and decrease treatment) in Example 7.

[0017] Figure 8 This is a schematic diagram of somatic embryo induction of sweet sorghum cells with different 2,4-D concentration gradients in Example 8. Detailed Implementation

[0018] The specific embodiments of the present invention are described in detail below.

[0019] 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.

[0020] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0021] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0022] 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.

[0023] 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.

[0024] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0025] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0026] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0027] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0028] In the following examples, MS medium was purchased from Duchefa Reagents Ltd., Netherlands.

[0029] A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the culture medium with the first 2,4-D concentration gradient prepared in step (2). Treat them in the dark for 1 to 5 weeks to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. The treatment time is 1 to 5 weeks, and type 2 embryogenic callus can be seen at the hypocotyl after culture.

[0030] Furthermore, the procedure also includes step (6), preparing an MS solid medium containing cytokinins and auxins, then subculturing the type 2 embryogenic callus obtained in step (5) onto the MS solid medium containing cytokinins and auxins prepared in step (6) to induce aboveground regeneration for 4 to 8 weeks, followed by rooting culture for 4 to 8 weeks, followed by hydroponic rooting, and then transplanting into the soil to obtain fertile regenerated plants.

[0031] 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.

[0032] Furthermore, the specific steps of step (2) are as follows: (21) Add 2-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, add 20-50g of sucrose and 10g of agar powder, then autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (22) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1-20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (24) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (25) Add an appropriate amount of CuSO4 to the culture flask and adjust the final concentration of CuSO4 to 1 µM; (26) After shaking well, pour the culture medium into petri dishes, 20-40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the first 2,4-D concentration gradient culture medium.

[0033] Furthermore, in step (3): the seeding density is controlled at 5 to 50 seeds per petri dish.

[0034] Furthermore, step (4) is as follows: (41) Add 2-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, add 20-50g of sucrose and 10g of agar powder, then autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (42) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1-20 mg / L; (43) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (44) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (45) After shaking well, pour the culture medium into petri dishes, 20-40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the culture medium for the second 2,4-D concentration gradient.

[0035] Furthermore, in step (5): the seeding density is controlled at 5 to 50 seeds per petri dish.

[0036] Furthermore, the specific steps for preparing the MS solid culture medium containing cytokinins and auxins in step (6) are as follows: (61) Add 0.1-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, then add 20-50g of sucrose and 10g of agar powder, autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (62) Add an appropriate amount of TDZ (thiadiazuron) stock solution to the culture flask and adjust the concentration of TDZ to 0.1-20 mg / L; (63) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (64) Add an appropriate amount of IAA (indoleacetic acid) stock solution to the culture flask and adjust the concentration of IAA (indoleacetic acid) to 0.1-20 mg / L; (65) Add 6-10g of agar to the culture flask, shake well, and then dispense the culture medium into other culture flasks, 20-40ml per flask. After capping, cover with a sterile plastic bag and set aside. This will give you MS solid culture medium containing cytokinins and auxins.

[0037] In one embodiment: A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the culture medium with the first 2,4-D concentration gradient prepared in step (2). Treat them in the dark for 1 week to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 50 ppm. The treatment time was 5 weeks, and type 2 embryogenic callus tissue could be seen at the hypocotyl after culture.

[0038] Furthermore, the procedure also includes step (6), preparing an MS solid medium containing cytokinins and auxins, then subculturing the type 2 embryogenic callus obtained in step (5) onto the MS solid medium containing cytokinins and auxins prepared in step (6), inducing aboveground regeneration for 4 weeks, followed by rooting culture for 4 weeks, and after hydroponics to strengthen the roots, transplanting into the soil to obtain fertile regenerated plants.

[0039] 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.

[0040] Furthermore, the specific steps of step (2) are as follows: (21) Add 2g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5, add 20g of sucrose and 10g of agar powder, then autoclave at 110℃ for 10 minutes and cool to 50℃. (22) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10 μM; (24) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1 μM; (25) Add an appropriate amount of CuSO4 to the culture flask and adjust the final concentration of CuSO4 to 1 µM; (26) After shaking well, pour the culture medium into petri dishes, 20 ml per dish, cover with a sterile plastic bag and set aside. This gives you the first 2,4-D concentration gradient culture medium.

[0041] Furthermore, in step (3): the seeding density is controlled at 5 seeds / petal.

[0042] Furthermore, step (4) is as follows: (41) Add 2g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5, add 20g of sucrose and 10g of agar powder, then autoclave at 110℃ for 10 minutes and cool to 50℃. (42) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1 mg / L; (43) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10 μM; (44) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1 μM; (45) After shaking well, pour the culture medium into petri dishes, 20 ml per dish, cover with a sterile plastic bag and set aside. This gives you the culture medium for the second 2,4-D concentration gradient.

[0043] Furthermore, in step (5): the seeding density is controlled at 5 seeds / petal.

[0044] Furthermore, the specific steps for preparing the MS solid culture medium containing cytokinins and auxins in step (6) are as follows: (61) Add 0.1g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5, then add 20g of sucrose and 10g of agar powder, autoclave at 110℃ for 10 minutes, and cool to 50℃. (62) Add an appropriate amount of TDZ (thiadiazuron) stock solution to the culture flask and adjust the concentration of TDZ to 0.1 mg / L; (63) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10 μM; (64) Add an appropriate amount of IAA (indoleacetic acid) stock solution to the culture flask and adjust the concentration of IAA (indoleacetic acid) to 0.1 mg / L; (65) Add 6g of agar to the culture flask, shake well, and then dispense the culture medium into other culture flasks, 20ml per flask. After capping, cover with a sterile plastic bag and set aside. This gives you MS solid culture medium containing cytokinins and auxins.

[0045] In another embodiment: A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the culture medium with the first 2,4-D concentration gradient prepared in step (2). Treat them in the dark for 5 weeks to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 500 ppm. The treatment time was 1 week, and type 2 embryogenic callus tissue could be seen at the hypocotyl after culture.

[0046] Furthermore, the procedure also includes step (6), preparing an MS solid medium containing cytokinin and auxin, then subculturing the type 2 embryogenic callus obtained in step (5) onto the MS solid medium containing cytokinin and auxin prepared in step (6), inducing aboveground regeneration for 8 weeks, followed by rooting culture for 8 weeks, and after hydroponics to strengthen the roots, transplanting into the soil to obtain fertile regenerated plants.

[0047] 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 3 hours, discard the liquid, wash with ultrapure water 4 times, and set aside.

[0048] Furthermore, the specific steps of step (2) are as follows: (21) Add 8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 6.0, add 50g of sucrose and 10g of agar powder, then autoclave at 130℃ for 15 minutes and cool to 80℃. (22) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 1000 μM; (24) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 100 μM. (25) Add an appropriate amount of CuSO4 to the culture flask and adjust the final concentration of CuSO4 to 1 µM; (26) After shaking well, pour the culture medium into petri dishes, 40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the first 2,4-D concentration gradient culture medium.

[0049] Furthermore, in step (3): the seeding density is controlled at 50 seeds / petal.

[0050] Furthermore, step (4) is as follows: (41) Add 8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 6.0, add 50g of sucrose and 10g of agar powder, then autoclave at 130℃ for 20 minutes and cool to 80℃. (42) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 20 mg / L; (43) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 1000 μM; (44) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 100 μM; (45) After shaking well, pour the culture medium into petri dishes, 40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the culture medium for the second 2,4-D concentration gradient.

[0051] Furthermore, in step (5): the seeding density is controlled at 50 seeds / petal.

[0052] Furthermore, the specific steps for preparing the MS solid culture medium containing cytokinins and auxins in step (6) are as follows: (61) Add 8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 6.0, add 50g of sucrose and 10g of agar powder, autoclave at 130℃ for 20 minutes, and cool to 80℃. (62) Add an appropriate amount of TDZ (thiadiazuron) stock solution to the culture flask and adjust the concentration of TDZ to 20 mg / L; (63) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 1000 μM; (64) Add an appropriate amount of IAA (indoleacetic acid) stock solution to the culture flask and adjust the concentration of IAA (indoleacetic acid) to 20 mg / L; (65) Add 10g of agar to the culture flask, shake well, and then dispense the culture medium into other culture flasks, 40ml per flask. After capping, cover with a sterile plastic bag and set aside. This gives you MS solid culture medium containing cytokinins and auxins.

[0053] In yet another embodiment: A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the first 2,4-D concentration gradient culture medium prepared in step (2). Treat them in the dark for 3 weeks to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 300 ppm. The treatment time was 2 weeks, and type 2 embryogenic callus tissue could be seen at the hypocotyl after culture.

[0054] Furthermore, the procedure also includes step (6), preparing an MS solid medium containing cytokinins and auxins, then subculturing the type 2 embryogenic callus obtained in step (5) onto the MS solid medium containing cytokinins and auxins prepared in step (6), inducing aboveground regeneration for 6 weeks, followed by rooting culture for 6 weeks, and after hydroponics to strengthen the roots, transplanting into the soil to obtain fertile regenerated 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 2% pasteurization solution, shake at 80rpm for 4 hours, discard the liquid, wash with ultrapure water 3 times, and set aside.

[0056] Furthermore, the specific steps of step (2) are as follows: (21) Add 4.4g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.8, add 30g of sucrose and 10g of agar powder, then autoclave at 121℃ for 15 minutes and cool to 60℃. (22) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 15 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 200 μM; (24) Add an appropriate amount of deacetylation inhibitor TSA (Trichostatin A) to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 20 μM; (25) Add an appropriate amount of CuSO4 to the culture flask and adjust the final concentration of CuSO4 to 1 µM; (26) After shaking well, pour the culture medium into petri dishes, 30 ml per dish, cover with a sterile plastic bag and set aside. This gives you the first 2,4-D concentration gradient culture medium.

[0057] Furthermore, in step (3): the seeding density is controlled at 20 seeds / petal.

[0058] Furthermore, step (4) is as follows: (41) Add 4.4g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.8, add 30g of sucrose and 10g of agar powder, then autoclave at 121℃ for 15 minutes and cool to 60℃. (42) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 10 mg / L; (43) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 200 μM; (44) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 20 μM; (45) After shaking well, pour the culture medium into petri dishes, 30 ml per dish, cover with a sterile plastic bag and set aside. This gives you the culture medium for the second 2,4-D concentration gradient.

[0059] Furthermore, in step (5): the seeding density is controlled at 10 seeds / petal.

[0060] Furthermore, the specific steps for preparing the MS solid culture medium containing cytokinins and auxins in step (6) are as follows: (61) Add 4g of MS medium to the culture flask, 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 121℃ for 10 minutes, and cool to 60℃. (62) Add an appropriate amount of TDZ (thiadiazuron) stock solution to the culture flask and adjust the concentration of TDZ to 10 mg / L; (63) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 200 μM; (64) Add an appropriate amount of IAA (indoleacetic acid) stock solution to the culture flask and adjust the concentration of IAA (indoleacetic acid) to 10 mg / L; (65) Add 8g of agar to the culture flask, shake well, and then dispense the culture medium into other culture flasks, 30ml per flask. After capping, cover with a sterile plastic bag and set aside. This gives you MS solid culture medium containing cytokinins and auxins.

[0061] Example 1 A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Take 10ml of mature sweet sorghum seeds and place them in a 50ml centrifuge tube. Add 40ml of ethanol solution containing the surfactant Tween-20, tighten the cap and shake at 100rpm for 40 minutes on a shaker; discard the liquid and then add 40ml of 3% pasteurization solution, shake at 100rpm for 3 hours to sterilize, discard the liquid and wash 4 times with ultrapure water.

[0062] (2) Preparation of the first 2,4-D concentration gradient callus induction medium: Add 3g of MS medium to a culture flask, add water to bring the volume to 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder. Autoclave at 120℃ for 20 minutes, cool to 70℃, add 2,4-D stock solution, adjust the final concentration to 6 mg / L, the concentration of antioxidant lipoic acid (LA) to 100μM, the concentration of deacetylation inhibitor TSA to 10μM, and the final concentration of CuSO4 to 1 µM. Shake well and pour the medium into petri dishes, 30ml per dish, cover with a sterile plastic bag for later use. This gives the first 2,4-D concentration gradient culture medium. (3) The seeds treated in step (1) are evenly sown onto the culture medium with the first 2,4-D concentration gradient prepared in step (2), and the sowing density is controlled at 15 seeds / petition, with each 2 petitions as a biological replicate. (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 1 week as the first 2,4-D concentration gradient treatment.

[0063] (5) Perform embryogenic callus induction rate statistics and microscopic examination directly on the callus induced in step (4).

[0064] Figure 1 This is a schematic diagram illustrating the somatic embryo induction of sweet sorghum cells using only the first 2,4-D concentration gradient (short cycle of 2 weeks) in Example 1. It can be obtained... Figure 1 A1, Figure 1 The callus cluster and typical callus tissue on the right side of B1 ( Figure 1 A2 and Figure 1 (B2 in the text).

[0065] Example 2 Example 2 is basically the same as Example 1, the main difference being that: in the callus induction phase, the treatment time of the first 2,4-D concentration gradient was extended from 1 week to 2 weeks, and the treatment concentration was 2 mg / L (see Example 1). Figure 2 E in the middle) and 8 mg / L ( Figure 2 The culture was then subjected to a 2-week recovery culture without 2,4-D. The experimental setup is shown in the left-hand coordinate graph. Figure 2 E) and ( Figure 2 F in the image). Microscopic examination revealed the callus cluster on the right side (F). Figure 2 E1 in Figure 2 (F1 in the middle) and typical callus tissue ( Figure 2 E2 in Figure 2 (F2 in the middle).

[0066] Example 3 A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Take 8 ml of mature sweet sorghum seeds, put them in a 50 ml centrifuge tube, add 40 ml of ethanol solution containing the surfactant Tween-20, tighten the cap and shake at 100 rpm for 40 minutes; discard the liquid and add 40 ml of 3% pasteurization solution, shake at 100 rpm for 3 hours to sterilize, discard the liquid and wash with ultrapure water 4 times.

[0067] (2) Preparation of the first 2,4-D concentration gradient callus induction medium: Add 3g of MS medium to a culture flask, add water to bring the volume to 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder. Autoclave at 120℃ for 20 minutes, cool to 80℃, add 2,4-D stock solution, and adjust the final concentration to 8 mg / L (corresponding to...). Figure 3C) and 2 mg / L (corresponding to Figure 3 In step (2), the concentration of antioxidant lipoic acid (LA) is 100 μM, the concentration of deacetylation inhibitor TSA is 10 μM, and the final concentration of CuSO4 is 1 µM. After shaking well, the culture medium is poured into petri dishes, 30 ml per dish, and covered with sterile plastic bags for later use. Thus, the first 2,4-D concentration gradient culture medium is obtained. (3) The seeds treated in step (1) are evenly sown onto the first 2,4-D concentration gradient culture medium prepared in step (2), and the sowing density is controlled at 15 seeds / petri dish. Each 2 dishes is a biological replicate.

[0068] (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 4 weeks as the first 2,4-D concentration gradient treatment.

[0069] (5) Preparation of the second 2,4-D concentration gradient callus induction medium: MS medium with 3% sucrose (w / v), pH 5.8, and 10 g agar powder. After autoclaving at 120°C for 20 minutes, cool to 70°C, add 2,4-D stock solution, and adjust the final concentration to 2 mg / L (corresponding to...). Figure 3 (C) and 8 mg / L (corresponding to Figure 3 The concentrations of the antioxidant lipoic acid (LA) and the deacetylation inhibitor TSA were 100 μM, and the final concentration of CuSO4 was 1 µM. After shaking well, the mixture was poured onto a plate, condensed, and prepared for subculture.

[0070] (6) Subculture the type 2 embryogenic callus induced in step (4) onto the second 2,4-D concentration gradient callus induction medium prepared in step (5), with 5 type 2 embryogenic callus per dish. After sealing with sealing film, place in an incubator at 25°C and culture in the dark for 6 weeks as the second 2,4-D concentration gradient treatment.

[0071] (7) The callus tissue induced in step (6) was directly subjected to embryogenic callus induction rate statistics. After microscopic examination, the callus population on the right side can be obtained. Figure 3 C1 in Figure 3 D1 in the middle) and typical callus tissue ( Figure 3 C2 in Figure 3 (D2 in the middle).

[0072] Example 4 Example 4 is basically the same as Example 3, the main difference being: in the callus induction phase, the first 2,4-D concentration gradient treatment time is 8 weeks, and the 2,4-D concentration is 2 mg / L (corresponding to...). Figure 4 A) and 8 mg / L (corresponding to Figure 4(B) was used to maintain the 2,4-D concentration constant for 8 weeks, and the cells were subcultured into fresh callus induction medium every two weeks. After 8 weeks of culture, the induced callus tissue was directly subjected to induction rate statistics and microscopic examination. The callus population on the right side could be obtained after microscopic examination. Figure 4 A1 in Figure 4 B1 in the middle) and typical callus tissue ( Figure 4 A2 in Figure 4 (B2 in the text).

[0073] Example 5 Example 5 is basically the same as Example 3, the main difference being: in the callus induction phase, the first 2,4-D concentration gradient treatment time is 2 weeks, and the 2,4-D concentration is 2 mg / L (corresponding to...). Figure 5 E in the middle) and 8 mg / L (corresponding to Figure 5 In the F), the second 2,4-D concentration gradient treatment time was 2 weeks, and the 2,4-D concentration was 8 mg / L (corresponding to F). Figure 5 E in the middle) and 2 mg / L (corresponding to Figure 5 (F in the text), after 4 weeks of culture, the induced callus tissue was directly subjected to induction rate statistics and microscopic examination. After microscopic examination, the callus population on the right side can be obtained (F). Figure 5 E1 in Figure 5 F1 in the middle) and typical callus tissue ( Figure 5 E2 in Figure 5 (F2 in the middle).

[0074] Example 6 Example 6 is basically the same as Example 1, the main difference being: in the callus induction phase, the first 2,4-D concentration gradient treatment time is 4 weeks, and the 2,4-D concentration is 2 mg / L (corresponding to...). Figure 6 C) and 8 mg / L (corresponding to Figure 6 D) was subcultured every two weeks. After 4 weeks of culture, no second-gradient induction was performed. The induction rate was directly statistically analyzed and examined under a microscope. The callus population on the right side could be obtained after microscopic examination. Figure 6 C1 in Figure 6 D1 in the middle) and typical callus tissue ( Figure 6 C2 in Figure 6 (D2 in the middle).

[0075] Example 7 Example 7 is basically the same as Example 3, the main difference being: in the callus induction stage, the first 2,4-D concentration gradient treatment time is 2 weeks, and the 2,4-D concentration is 8 mg / L (corresponding to...). Figure 7 (G) and 2 mg / L (corresponding to Figure 7 (H in the text). The second 2,4-D concentration gradient treatment lasted for 2 weeks, with a 2,4-D concentration of 2 mg / L (corresponding to...). Figure 7 (G) and 8 mg / L (corresponding to Figure 7 (H in the text). Then, a 4-week recovery culture treatment without 2,4-D was performed. After 8 weeks of culture, the induced callus tissue was directly subjected to induction rate statistics and microscopic examination. Microscopic examination revealed the callus population on the right side (…). Figure 7 G1 in Figure 7 H1 in the middle) and typical callus tissue ( Figure 7 G2 in Figure 8 (G2 in the middle).

[0076] Example 8 A method for promoting somatic embryo regeneration in sweet sorghum includes the following steps: (1) Take 12ml of mature sweet sorghum seeds and place them in a 50ml centrifuge tube. Add 40ml of ethanol solution containing surfactant, tighten the cap and shake at 100rpm for 40 minutes on a shaker; discard the liquid and then add 40ml of 3% pasteurization solution, shake at 100rpm for 3 hours to sterilize, discard the liquid and wash 4 times with ultrapure water; (2) Preparation of a single 2,4-D concentration callus induction medium: Add 3g of MS medium to a culture flask, add water to bring the volume to 1L, adjust the pH to 5.8, then add 30g of sucrose and 10g of agar powder. Autoclave at 120℃ for 20 minutes, cool to 60℃, and add appropriate amounts of 2,4-D stock solution to adjust the final concentrations of 2,4-D to 0mg / L, 2mg / L, 8mg / L, and 16mg / L, respectively (see details). Figure 8 (Parameter settings): The concentration of the antioxidant lipoic acid (LA) is 100 μM, the concentration of the deacetylation inhibitor TSA is 10 μM, and the final concentration of CuSO4 is 1 µM. After shaking well, pour the culture medium into petri dishes, 30 ml per dish, and cover with a sterile plastic bag for later use. This yields the first 2,4-D concentration gradient culture medium. (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. (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 8 weeks.

[0077] (5) Perform embryogenic callus induction rate statistics and microscopic examination directly on the callus induced in step (4). This will yield the results. Figure 8 The relationship between callus population and concentration: When 2,4-D = 2 mg / L, a large amount of type 1 non-embryonic callus tissue will be induced; When 2,4-D = 8 mg / L, a large amount of type 2 embryogenic callus tissue will be induced.

[0078] 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 promoting somatic embryo regeneration in sweet sorghum, characterized in that, Includes the following steps: (1) Disinfect mature sorghum seeds; (2) Prepare culture media with the first 2,4-D concentration gradient; (3) Take the sorghum seeds disinfected in step (1) and sow them on the culture medium with the first 2,4-D concentration gradient prepared in step (2). Treat them in the dark for 1 to 5 weeks to induce callus tissue. (4) Prepare the culture medium with the second 2,4-D concentration gradient; (5) Subculture the callus obtained in step (3) onto the culture medium with the second 2,4-D concentration gradient prepared in step (4), with a photoperiod of 16h / 8h and a light intensity of 50-500 ppm. The treatment time is 1 to 5 weeks, and type 2 embryogenic callus can be seen at the hypocotyl after culture.

2. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, The process also includes step (6), preparing MS solid medium containing cytokinin and auxin, then subculturing the type 2 embryogenic callus obtained in step (5) onto the MS solid medium containing cytokinin and auxin prepared in step (6), inducing aboveground regeneration for 4 to 8 weeks, followed by rooting culture for 4 to 8 weeks, and after hydroponics to strengthen the roots, transplanting into the soil to obtain fertile regenerated plants.

3. The method for promoting somatic embryo regeneration in sweet sorghum 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.

4. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, The specific steps of step (2) are as follows: (21) Add 2-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, add 20-50g of sucrose and 10g of agar powder, then autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (22) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1-20 mg / L; (23) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (24) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (25) Add an appropriate amount of CuSO4 to the culture flask and adjust the final concentration of CuSO4 to 1 µM; (26) After shaking well, pour the culture medium into petri dishes, 20-40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the first 2,4-D concentration gradient culture medium.

5. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, In step (3): the seeding density is controlled at 5 to 50 seeds per petri dish.

6. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, The steps in step (4) are as follows: (41) Add 2-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, add 20-50g of sucrose and 10g of agar powder, then autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (42) Add an appropriate amount of 2,4-D stock solution to the culture flask and adjust the concentration of 2,4-D to 0.1-20 mg / L; (43) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (44) Add an appropriate amount of deacetylation inhibitor TSA to the culture flask and adjust the concentration of deacetylation inhibitor TSA to 0.1-100 μM; (45) After shaking well, pour the culture medium into petri dishes, 20-40 ml per dish, cover with a sterile plastic bag and set aside. This gives you the culture medium for the second 2,4-D concentration gradient.

7. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, In step (5): the seeding density is controlled at 5 to 50 seeds per petri dish.

8. The method for promoting somatic embryo regeneration in sweet sorghum as described in claim 1, characterized in that, The specific steps for preparing the MS solid culture medium containing cytokinins and auxins as described in step (6) are as follows: (61) Add 0.1-8.8g of MS medium to the culture flask, add water to make up to 1L, adjust the pH to 5.5-6.0, then add 20-50g of sucrose and 10g of agar powder, autoclave at 110-130℃ for at least 10 minutes, and cool to 50-80℃. (62) Add an appropriate amount of TDZ (thiadiazuron) stock solution to the culture flask and adjust the concentration of TDZ to 0.1-20 mg / L; (63) Add an appropriate amount of antioxidant lipoic acid to the culture flask and adjust the concentration of antioxidant lipoic acid to 10-1000 μM; (64) Add an appropriate amount of IAA (indoleacetic acid) stock solution to the culture flask and adjust the concentration of IAA (indoleacetic acid) to 0.1-20 mg / L; (65) Add 6-10g of agar to the culture flask, shake well, and then dispense the culture medium into other culture flasks, 20-40ml per flask. After capping, cover with a sterile plastic bag and set aside. This will give you MS solid culture medium containing cytokinins and auxins.

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