Hunan millet tissue culture regeneration system, culture method and application

Abstract

The application discloses a tissue culture regeneration system and culture method of Hunan millet, and relates to the field of biological engineering. The regeneration system is to take the Hunan millet seeds with removed hulls as explants, and utilizes a culture medium composed of 1 / 2MS basic culture medium, 0.6-0.99 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar and 0.1% PPM, with pH being 5.8, to make the explants form complete Hunan millet plants. The regeneration system is used for tissue culture of the Hunan millet, effectively avoiding the problems of long culture period, high requirement for personnel and high culture cost caused by diversification of culture medium configuration in large-scale factory production. The application provides a simpler and more efficient method for large-scale factory production, research on gene function and molecular breeding and the like.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a tissue culture regeneration system and culture method for *Sorghum sibiricum* from Hunan, which relates to the field of bioengineering. Background Technology

[0002] Hunan Jizi ( Echinochloa frumentacea *Sorghum chinensis*, a plant belonging to the genus *Sorghum* of the Poaceae family, is native to tropical regions and has a long history of cultivation. Due to its advantages such as soft stems, abundant leaves, good palatability, high utilization rate, and high nutritional value, as well as its strong tillering ability, drought resistance, waterlogging resistance, salt and alkali tolerance, lodging resistance, and high forage and seed yields, it is widely cultivated as a food or forage crop in tropical Africa, India, Southeast Asia, Japan, Korea, Australia, and North America. Wild species have been found in Yunnan, and artificially cultivated species exist in Northeast, North, and Northwest China. Current research on *Sorghum chinensis* in Hunan mainly focuses on physiological index determination. Due to the complex genetic structure and large genome of *Sorghum chinensis*, domestic research on it is limited, resulting in lagging progress in crop genetics, breeding, and improvement. The lack of efficient tissue culture regeneration and genetic transformation systems means that research on gene function and molecular breeding of *Sorghum chinensis* remains a blank area.

[0003] Although current research has established tissue culture regeneration systems for *Sorghum humilis*, these systems typically involve different explants, different callus induction media to induce callus formation, different differentiation media to differentiate regenerated seedlings, and finally different rooting media to induce rooting in the regenerated seedlings. For example, Wang Wenjing et al.'s publication, "Construction of CRISPR / Cas9 Targeted Editing Vector for *Sorghum humilis* EfPDS Gene and Establishment of Genetic Transformation System," successfully achieved tissue culture regeneration of *Sorghum humilis*. However, this method used carcinogenic mercuric chloride (HgCl2) to disinfect the seeds, required a 2,4-D concentration of 4 mg / L, and employed different basic media, hormone types, and concentrations at different stages of callus induction, differentiation, and rooting. While many species' tissue culture regeneration systems also use different basic media and hormone concentrations and types at different stages of callus induction, differentiation, and rooting, these methods are problematic for large-scale industrial production. They place high demands on the skill level of personnel preparing the media, are prone to errors, and can easily lead to excess media at certain stages, resulting in waste and increased costs. To address the aforementioned problems, this invention has developed a simpler and more efficient tissue culture regeneration system for Hunan millet. Summary of the Invention

[0004] To address the complexity of current tissue culture processes for Hunan millet, this invention provides a highly versatile culture system and method. This not only simplifies the cultivation process of Hunan millet but also significantly reduces the use of the hormone 2,4-D. It effectively avoids the problems inherent in large-scale industrial production, such as the high skill requirements for personnel, the susceptibility to errors in culture medium preparation due to the diverse nature of culture medium configurations, and the waste and increased costs caused by excess culture medium at certain stages. This provides a simpler and more efficient method for large-scale industrial production and research in gene function and molecular breeding.

[0005] To achieve the technical objective of this invention, one aspect of this invention provides a tissue culture regeneration system for *Sorghum huskensis*, which uses *Sorghum huskensis* seeds with the husk removed as explants. The explants are then cultured in a medium consisting only of 1 / 2 MS basal medium, 0.6-0.99 mg / L of 2,4-D, 30 g / L of sucrose, 7 g / L of agar, and 0.1% PPM at a pH of 5.8 to form complete *Sorghum huskensis* plants.

[0006] In particular, the dosage of 2,4-D is 0.8 mg / L.

[0007] In particular, the explants are obtained after being sterilized with sodium hypochlorite.

[0008] Specifically, the explants are sterilized with sodium hypochlorite by soaking the seeds (after removing the husks) in a sodium hypochlorite solution for 7-10 minutes in a clean bench environment, rinsing with sterile water, then sterilizing with 75% alcohol for 3 minutes, rinsing three times with sterile water, and finally placing them on filter paper that has been sterilized by high temperature and high pressure to absorb excess water, thus obtaining the explants.

[0009] The concentration of sodium hypochlorite used is 3%-5%.

[0010] To achieve the technical objective of this invention, a second aspect of this invention provides a method for tissue culture and regeneration of *Sorghum sibiricum* from Hunan, comprising:

[0011] Prepare a culture medium consisting only of 1 / 2 MS basal medium, 0.6-0.99 mg / L of 2,4-D, 30 g / L of sucrose, 7 g / L of agar, and 0.1% PPM, with a pH of 5.8;

[0012] After removing the husks of *Sorghum husk* seeds from Hunan, the seeds were disinfected using sodium hypochlorite and then dried to obtain explants.

[0013] The explants were inoculated into the culture medium and subjected to induction culture, differentiation culture and rooting culture in sequence to obtain Hunan millet seedlings.

[0014] In particular, the dosage of 2,4-D is 0.8 mg / L.

[0015] In particular, the method also includes hardening off Hunan millet seedlings using a culture medium before transplanting to obtain complete Hunan millet plants.

[0016] The process includes disinfection with sodium hypochlorite and drying to obtain explants, which involves: disinfecting Hunan millet seeds (after removing the husks) with sodium hypochlorite for 7-10 minutes in a clean bench, rinsing with sterile water, then disinfecting with 75% alcohol for 3 minutes, rinsing three times with sterile water, and placing them on filter paper that has been sterilized by high temperature and high pressure to absorb excess moisture, thus obtaining explants.

[0017] The step of inoculating the explants into the culture medium for induction culture involves placing the culture container in a dark incubator for dark culture.

[0018] Preferably, the induction culture temperature is 23°C.

[0019] The differentiation culture and rooting culture in the culture medium are carried out under light and dark conditions, and the culture medium is changed every 10-15 days.

[0020] In particular, the light conditions are 16 hours of light exposure at 25°C; the darkness conditions are 8 hours of darkness at 23°C.

[0021] The seedling hardening and transplanting process involves culturing Hunan millet seedlings in a culture medium for 20 days, then opening the culture bottle cap and placing them in a natural environment for 3 days. Afterward, the culture medium is washed off the roots, and the seedlings are transplanted into a sterilized substrate containing nutrient soil and vermiculite in a 2:1 ratio to continue growing, thus obtaining complete Hunan millet plants.

[0022] To achieve the technical objective of this invention, a third aspect of this invention provides the use of the above-described tissue culture regeneration system and / or the above-described method to construct a Hunan millet genetic regeneration system.

[0023] To achieve the technical objective of this invention, a fourth aspect of this invention provides the use of the above-described tissue culture regeneration system and / or the above-described method for culturing genetically regenerated plants of *Sorghum sibirica* from Hunan.

[0024] The tissue culture regeneration system provided by this invention only requires one culture medium to complete the induction of callus, tissue differentiation and rooting culture of *Sorghum hunanense*, with a seedling survival rate of 100%. Attached Figure Description

[0025] Figure 1 This is a diagram of the callus induction stage of *Sorghum bicolor* from Hunan. The callus tissue in the diagram is relatively firm and appears milky white or light yellow.

[0026] Figure 2 This is a diagram illustrating the callus induction and differentiation stages of *Sorghum sibiricum* in Hunan.

[0027] Figure 3 This is a diagram showing the differentiation stages and the initial rooting stage of adventitious buds in Hunan millet.

[0028] Figure 4 This is a diagram of the adventitious bud rooting stage of millet in Hunan.

[0029] Figure 5 This is a picture showing the growth of *Sorghum sibiricum* seedlings after transplanting from Hunan. The substrate is a mixture of nutrient soil and vermiculite in a 2:1 ratio. After washing the roots of the culture medium, the seedlings are transplanted into the sterilized substrate to continue growing. Detailed Implementation

[0030] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc., used in the following embodiments are commercially available unless otherwise specified.

[0031] Example 1

[0032] 1. Seed pretreatment

[0033] After removing the husks, the seeds of *Sorghum husk* from Hunan were disinfected by soaking them in a 3%-5% sodium hypochlorite solution for 7-10 minutes in a clean bench, then rinsed with sterile water, disinfected with 75% alcohol for 3 minutes, rinsed three times with sterile water, and then placed on filter paper that had been sterilized by high temperature and high pressure to absorb excess water, thus obtaining explants.

[0034] This invention uses non-toxic, non-carcinogenic sodium hypochlorite to disinfect Hunan millet seeds after the husk has been removed, thus avoiding harm to operators. In particular, this application controls the concentration of sodium hypochlorite at 3%-5%, achieving seed disinfection without inhibiting seed embryogenicity.

[0035] 2. Preparation of culture medium

[0036] Add 0.8 mg / L of 2,4-D, 30 g / L of sucrose, 7 g / L of agar, and 0.1% PPM (plant tissue culture antibacterial agent) to 1 / 2 MS basal medium, and control the pH of the medium to 5.8, and set aside for later use.

[0037] 3. Tissue culture of explants

[0038] The sterilized seeds were inoculated into the culture medium prepared in step 2, and the culture container was placed in a dark incubator (23℃) for dark culture. Callus tissue was induced in 8-14 days. According to statistics, the callus tissue induction rate reached 86.43%. The induced callus tissue was as follows: Figure 1As shown in the figure, the callus tissue is relatively firm and milky white or light yellow, indicating that the callus tissue has good embryogenicity.

[0039] The obtained callus tissue was re-inoculated into the culture medium prepared in step 2 and cultured for differentiation at 25℃ (16h light) / 23℃ (8h darkness). The culture was transferred to fresh culture medium from step 2 every 10-15 days. Adventitious shoots differentiated within 3-4 weeks. Statistically, the budding rate reached 65.95%. The differentiated adventitious shoots... Figure 2 As shown in the figure, a small portion of the callus induction medium is in the callus stage, while most of the larger callus tissues have begun to differentiate into green adventitious buds, indicating that this callus culture medium can also be used for callus differentiation culture.

[0040] The obtained adventitious shoots were re-inoculated into the culture medium prepared in step 2 and cultured at 25℃ (16h light) / 23℃ (8h darkness), transferring to fresh culture medium every 10-15 days. The adventitious shoots will root in about 2-3 weeks. Figure 3 As shown, while most adventitious buds differentiated, a small portion of the larger adventitious buds had already begun to root, indicating that adventitious bud differentiation and rooting occurred simultaneously on the same culture medium. Statistically, the rooting rate reached 100%, and the rooting state was as follows: Figure 4 As shown in the figure, the regenerated seedlings after rooting are growing healthily.

[0041] After the plants began to root, they were cultured in the culture medium for another 20 days. Then, the culture bottle was opened and left in a natural environment for 3 days. Afterward, the culture medium was washed off the roots, and the plants were transplanted into a sterilized substrate containing nutrient soil and vermiculite in a 2:1 ratio to continue growth, thus obtaining complete *Sorghum hunanense* plants. Figure 5 As shown.

[0042] During their research on the tissue culture regeneration system of Hunan millet, the inventors unexpectedly discovered that using a culture medium with the same components allows Hunan millet to complete the entire process from callus induction, differentiation, and rooting. This offers advantages for industrial production, including simple operation, low cost, and material savings. The entire process can be completed within three months, promoting the upgrading and transformation of related industries in Hunan millet.

[0043] The following are some of the experiments conducted by the inventor during the research process.

[0044] Experiment Example 1: Partial Experiments in the Callus Induction Stage

[0045] In the early stages of their research on tissue culture of *Sorghum humilis*, the inventors attempted to prepare callus induction culture media with different hormone concentrations to observe the callus induction rate and callus phenotype of *Sorghum humilis*, as detailed below:

[0046] This experimental example only demonstrates MS-type basal culture medium and experimental treatments that only change the components of 2,4-D and 6-BA hormones. The components of 2,4-D and 6-BA are shown in Table 1. Other components include 30 g / L sucrose, 7 g / L agar, 0.1% PPM (plant tissue culture antibacterial agent), and a pH of 5.8.

[0047] Table 1. Composition of culture media with different hormone concentrations

[0048]

[0049] The sterilized seeds were inoculated onto the callus induction medium for each treatment (three replicates per treatment). The culture containers were placed in a dark incubator (23°C) for incubation. The inventors observed that callus tissue was induced on day 8, accounting for 28.78%. By day 14, the number of induced callus tissues had stabilized. The callus induction rate for each treatment was then calculated and the average value was determined. The statistical results of the callus induction rate are shown in Table 2.

[0050] Table 2. Statistical results of callus induction rate for each treatment

[0051]

[0052] Statistical results show that under pH 5.8 conditions, the inventors found that the callus induction rate was higher without 6-BA than with 6-BA. When the 2,4-D concentration was 0.8 mg / L, 3 mg / L, and 4 mg / L, the callus induction rate was higher than 80%, with the highest rate of 86.43% at 0.8 mg / L. This dosage is very rare in the field of tissue culture of *Smilax china* from Hunan and was an unexpected discovery by the inventors.

[0053] Furthermore, to investigate whether the basal culture medium affects the callus tissue of *Sorghum hunanense* when only 2,4-D hormone is used, the inventors set up an experimental treatment to compare and analyze the effects of different basal culture media (as shown in Table 3) on the callus induction rate. Except for the use of only 2,4-D hormone, the other culture media compositions were the same as those in the previous section, using 30 g / L sucrose, 7 g / L agar, 0.1% PPM (plant tissue culture antibacterial agent), and a pH of 5.8. The callus induction rate of each treatment was calculated, and the results are shown in Table 3.

[0054] Table 3. Effects of different basal culture media on callus induction rate under conditions where only 2,4-D hormone is used.

[0055]

[0056] Based on experimental treatment and statistical results, it can be found that 1 / 2MS basal medium has a higher callus induction rate than MS basal medium.

[0057] Experiment Example 2: Partial Experiments in the Differentiation Stage

[0058] During their research on the tissue culture of *Sorghum sibiricum* from Hunan, the inventors were surprised to discover that on the callus induction medium 1 / 2 MS basal medium and 0.8 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, and 0.1% PPM (plant tissue culture antibacterial agent), at a pH of 5.8, a small portion of the callus remained in the callus stage, while most of the larger callus developed green adventitious buds. This demonstrates that the callus induction medium provided in this application can also differentiate into adventitious buds. Therefore, the inventors continued to use the callus induction medium to attempt to differentiate callus tissue. Since this culture medium contains only one hormone, 2,4-D, the inventors hypothesized that 2,4-D also plays an important role in the differentiation of *Sorghum hunanense* callus. Therefore, to explore the effect of different concentrations of hormone 2,4-D on callus differentiation, we prepared different concentrations of 2,4-D culture media based on a medium (1 / 2 MS basal medium and 0.8 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, 0.1% PPM, pH 5.8) for callus differentiation culture to observe the callus differentiation of *Sorghum hunanense*, as detailed below:

[0059] After inducing callus from *Sorghum hunanense*, the callus was detached from the explants using a sterilized scalpel and transferred to the culture media of each treatment group as shown in Table 4. At the same time, the explants that had already induced callus were placed in the culture media of each treatment group as controls (CK) for observation. The culture was carried out at 25℃ (16h light) / 23℃ (8h darkness) and transferred to new culture media every 10-15 days. When the number of adventitious shoots was relatively stable, the callus differentiation rate data were counted (as shown in Table 4).

[0060] Table 4. Effects of different concentrations of 2,4-D on callus differentiation rate.

[0061]

[0062] Our experiments showed that, without peeling, a clear differentiation state could be observed 36 days after the explants were inoculated onto the culture medium, at which point the differentiation rate reached 14.20%. This demonstrates that, with direct culture medium replacement, explants can directly differentiate into adventitious shoots from callus tissue without the need for specially formulated different differentiation media. This simple and convenient approach is highly beneficial for industrial production and widespread adoption.

[0063] Furthermore, the experimental statistics shown in Table 4 also reveal that the culture medium with high callus induction efficiency also exhibits the best differentiation rate during the differentiation culture stage. Especially when cultured after callus removal, the differentiation rate reaches 65.95%. When differentiation is performed directly without removal, the differentiation rate can reach 75.60%. This unexpected discovery changes the traditional understanding that different culture media are needed for successful and efficient induction and differentiation at different tissue culture stages.

[0064] Experiment Example 3: Partial Experiments in the Rooting Stage

[0065] During their research on tissue culture of *Sorghum sibiricum* from Hunan, the inventors were surprised to discover that while callus tissue differentiated on the culture medium, roots formed from larger adventitious buds. This discovery led them to speculate whether the culture medium used for induction could also be used for rooting. The inventors also found that the culture medium originally used for induction contained only 2,4-D, suggesting that 2,4-D might also be important for the rooting of adventitious buds from *Sorghum sibiricum*. Therefore, to explore the effect of different concentrations of the hormone 2,4-D on the rooting of adventitious buds, the inventors prepared different concentrations of 2,4-D culture media based on the original induction medium and used them for adventitious bud rooting culture to observe the rooting behavior of *Sorghum sibiricum* adventitious buds, as detailed below:

[0066] Differentiated plants with adventitious buds reaching approximately 4 cm in length were transferred to the original induction medium (as shown in Table 5). At the same time, differentiated plants whose medium was directly changed without selecting the length of adventitious buds were also transferred to the same medium as a control (CK). The plants were cultured at 25℃ (16h light) / 23℃ (8h darkness) and transferred to a new medium every 10-15 days. When the root growth of the adventitious buds was relatively stable and no longer changed, the rooting data were collected, as shown in Table 5.

[0067] Table 5. Effects of different concentrations of 2,4-D on the rooting rate of adventitious shoots.

[0068]

[0069] In our experiments, we observed that when only the culture medium was changed without selecting the length of adventitious buds, obvious roots could be observed on the culture medium 60 days after the explants were inoculated, with 15.82% of the plants having roots. This demonstrates that by directly changing the culture medium, explants can not only differentiate adventitious buds directly from callus tissue, but also directly root from callus tissue into adventitious buds, without the need for specially formulated differentiation and rooting media. This is very simple and convenient, and more conducive to the large-scale production of *Sorghum sibiricum* in Hunan.

[0070] The experimental statistics in Table 5 also show that the culture medium with high induction and differentiation rates also exhibits outstanding effects in the rooting culture stage, with a rooting rate of 100%. This unexpected discovery not only greatly simplifies the culture medium used for the propagation and production of *Sorghum sibiricum* in Hunan, but also shortens the culture cycle. The same culture medium only needs to be updated at appropriate growth times, simplifying the method and reducing the operational requirements for technicians.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A tissue culture regeneration system for *Sorghum sibiricum* from Hunan, characterized in that, Using seeds of *Sorghum husk* with the glumes removed as explants, complete *Sorghum husk* plants were formed from the explants using a medium consisting only of 1 / 2 MS basal medium, 0.6-0.99 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, and 0.1% PPM at pH 5.

8.

2. The regeneration system as described in claim 1, characterized in that, The dosage of 2,4-D is 0.8 mg / L.

3. A method for tissue culture and regeneration of *Sorghum sibiricum* from Hunan, characterized in that... include: Prepare a culture medium consisting only of 1 / 2 MS basal medium, 0.6-0.99 mg / L of 2,4-D, 30 g / L of sucrose, 7 g / L of agar, and 0.1% PPM, with a pH of 5.8; After removing the husks of *Sorghum husk* seeds from Hunan, the seeds were disinfected using sodium hypochlorite and then dried to obtain explants. The explants were inoculated into the culture medium and subjected to induction culture, differentiation culture and rooting culture in sequence to obtain Hunan millet seedlings.

4. The method as described in claim 3, characterized in that, The dosage of 2,4-D is 0.8 mg / L.

5. The method as described in claim 3, characterized in that, The process includes disinfection with sodium hypochlorite and drying to obtain explants. The process involves: disinfecting Hunan millet seeds (after removing the husks) with sodium hypochlorite for 7-10 minutes in a clean bench, rinsing with sterile water, then disinfecting with 75% alcohol for 3 minutes, rinsing three times with sterile water, and finally placing them on filter paper that has been sterilized by high temperature and high pressure to absorb excess moisture, thus obtaining explants.

6. The method as described in claim 3, characterized in that, Inducing explant culture by inoculating them into a culture medium involves placing the culture container in a dark incubator for dark culture.

7. The method as described in claim 6, characterized in that, The induction culture temperature was 23℃.

8. The method as described in claim 3, characterized in that, Differentiation and rooting cultures were carried out in the culture medium under alternating light and dark conditions, with the culture medium being changed every 10-15 days.

9. The method as described in claim 8, characterized in that, The light conditions were 16 hours of light exposure at 25°C; the darkness conditions were 8 hours of darkness at 23°C.

10. The method as described in claim 3, characterized in that, It also includes a seedling hardening and transplanting step, which involves culturing Hunan millet seedlings in a culture medium for 20 days, opening the culture bottle cap and placing them in a natural environment for 3 days, then washing the culture medium from the roots and transplanting them into a sterilized substrate containing nutrient soil: vermiculite = 2:1 to continue growth, thereby obtaining complete Hunan millet plants.

11. The tissue culture regeneration system according to claims 1-2 or the method according to any one of claims 3-10 is used to construct the Hunan millet genetic regeneration system.

12. The tissue culture regeneration system according to claims 1-2 or the method according to any one of claims 3-10 is used to cultivate genetically regenerated plants of *Sorghum sibiricum* from Hunan.

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