A method for inducing young stem callus of red plume sorghum

Abstract

The application discloses a method for inducing callus of tender stems of red-awn sorghum, comprising the following steps: S1, taking red-awn sorghum seeds, inoculating the seeds in a seed culture medium after surface sterilization, and obtaining aseptic seedlings through illumination culture, and taking tender stems as explants; and S2, cutting the tender stems, inoculating the tender stems in a callus induction culture medium for dark culture until callus is formed; the callus induction culture medium takes MS as a basic culture medium and contains sucrose, agar, glycine, 2,4D, KT and citric acid. Through the addition of citric acid and hormone regulation, the method effectively inhibits the browning of the tender stem explants, improves the callus induction rate, and obtains high-quality callus with vigorous growth and dense texture, thereby providing technical support for genetic transformation and breeding of red-awn sorghum.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for inducing callus tissue from young stems of *Sorghum sibirica*. Background Technology

[0002] Red-tasseled sorghum is a distinctive sorghum variety developed through decades of systematic breeding. While significant progress has been made in its breeding and promotion, major technical bottlenecks remain in germplasm resource innovation and genetic improvement. The primary obstacle in sorghum tissue culture is the frequent oxidation and browning of phenolic substances. Sorghum tissues are rich in polyphenolic compounds. When explants are cut and mechanically damaged, polyphenol oxidase catalyzes the oxidation of phenolic substances to form quinones. These quinones covalently bind with proteins and amino acids in the culture medium, producing a dark brown precipitate that not only inhibits cell division and meristem activity but can even lead to explant necrosis and death. Secondly, the poor embryogenicity retention of sorghum callus is another deep-seated obstacle. Sorghum somatic cells easily lose their differentiation and regeneration potential during repeated subcultures under in vitro culture conditions, and different genotypes of sorghum exhibit vastly different responses to tissue culture.

[0003] In existing technologies, researchers have explored various sorghum explants for callus induction. For example, using young spikelets as explants yields high callus emergence rates and good embryogenicity, but is subject to strict seasonality, as young sorghum spikelets only appear briefly around flowering time, making year-round, all-weather tissue culture impossible. Using mature seeds or mature embryos as explants is another option, but the callus induction rate of mature embryos is highly dependent on genotype, with significant differences in callus emergence rates among different genotypes, and browning rates increasing significantly with each generation. Young embryos are widely recognized as one of the best explants for callus induction, exhibiting excellent callus emergence and differentiation rates. However, obtaining young embryos requires embryo removal within a strict post-pollination time window, demanding extremely high operator skill.

[0004] Current methods are not specifically optimized for the Hongyingzi sorghum variety. Different genotypes of sorghum exhibit significant differences in their response to tissue culture conditions. Directly applying methods suitable for other sorghum varieties to Hongyingzi sorghum often fails to achieve ideal results. As a variety specifically for Maotai-flavor liquor, Hongyingzi sorghum possesses unique physicochemical characteristics (such as high amylopectin and phenolic content), which constitute advantages for brewing but also increase the risk of browning induced by phenolic oxidation during tissue culture. This places higher demands on the formulation of plant growth regulators and the construction of antioxidant systems in the induction medium. Current technology lacks induction culture protocols specifically designed for the physiological and biochemical characteristics of Hongyingzi sorghum. Summary of the Invention

[0005] The present invention aims to provide a method for inducing callus tissue from young stems of red sorghum, using young explants as starting material, and an induction system that can stably obtain material at any time of year and achieve high induction rate and good embryogenicity maintenance.

[0006] To achieve the above objectives, the first aspect of this application provides the following technical solution: A method for inducing callus from young stems of *Sorghum sibirica* includes the following steps: S1, taking *Sorghum sibirica* seeds, surface-sterilizing them, inoculating them into a seed culture medium, and culturing under light to obtain sterile seedlings, then using young stems as explants; S2, cutting the young stems into segments, inoculating them into a callus induction medium, and culturing them in the dark until callus forms; the callus induction medium uses MS as the basal medium and contains sucrose, agar, glycine, and 2,4-dimethylformamide. D, KT and citric acid.

[0007] Furthermore, the seed culture medium is MS basal medium, with added sucrose, agar and glycine.

[0008] Furthermore, the seed surface disinfection includes: disinfection with 75% ethanol, rinsing with sterile water, disinfection with 0.1% mercuric chloride, and rinsing thoroughly with sterile water.

[0009] Furthermore, the conditions for light culture are: 25-28℃, 12 hours of light / 12 hours of darkness, and culture for 14-15 days.

[0010] Furthermore, the length of the young stem segment is 0.3–0.5 cm.

[0011] Furthermore, the concentration of citric acid in the callus induction culture medium is 10-30 mg / L.

[0012] Furthermore, the concentration of the citric acid is 20 mg / L.

[0013] Furthermore, the conditions for dark culture are: 22-25℃, dark culture for 5-8 days.

[0014] Furthermore, the pH of the callus induction culture medium is 5.8.

[0015] The second aspect of this application provides the application of the method based on the first aspect in the genetic transformation or breeding of red sorghum.

[0016] Working principle and beneficial effects of the present invention: Young stems obtained from the aseptic germination of red sorghum seeds have cells in an active state of division, with thin cell walls and dense protoplasm, making them highly responsive to exogenous plant hormones and easy to initiate dedifferentiation. Furthermore, seeds can be preserved for a long time and germinate at any time, overcoming the seasonal limitations of obtaining explants such as young spikelets and embryos.

[0017] 2,4 D, as an auxin, is a key signaling molecule that induces somatic cell dedifferentiation. It activates the expression of cell cycle-related genes, causing differentiated stem cells to revert to a meristematic state. KT (kinetin), as a cytokinin, is associated with 2,4-... The synergistic effect of D promotes cell division and callus proliferation on the one hand, and helps induce embryogenic callus with differentiation potential on the other. The combination of the two overcomes the problems of callus being prone to waterlogging and loose texture under single hormone conditions.

[0018] Citric acid is an organic acid that can inhibit the activity of polyphenol oxidase by lowering the local pH of the culture medium, thus reducing the rate of catalytic oxidation of phenolic substances. Simultaneously, citric acid has the ability to chelate copper ions at the active site of polyphenol oxidase, further reducing enzyme activity and thereby inhibiting browning at its source. Compared to traditional PVP and activated carbon, citric acid does not non-selectively adsorb nutrients and hormones in the culture medium, therefore it does not negatively affect the normal growth of callus tissue.

[0019] This application significantly inhibited the browning rate of young stem explants from *Sorghum sibirica*, reducing browning and death of explants. Simultaneously, it effectively improved the callus induction rate. The induced callus was milky white and dense, rather than water-soaked or loose, exhibiting good embryogenic potential, thus laying a material foundation for subsequent differentiation, regeneration, and genetic transformation. Furthermore, the culture system of this invention is stable and easy to operate; experiments can be conducted year-round using seeds as starting material, unaffected by seasonal limitations. The citric acid used is a common and inexpensive reagent, requiring no special equipment, making it low-cost and easy to promote. Attached Figure Description

[0020] Figure 1 Image showing the collection of explants from sterile sorghum seedlings and young stems; Figure 2 The growth of sterile seedlings obtained by culturing sorghum seeds under light at different time points; Figure 3 The growth status of callus tissue in young stems at different time points under the same citric acid concentration; Figure 4 Comparison of callus growth in young stems under different citric acid concentrations; Figure 5 The graph shows the changes in callus formation rate and browning rate of young stem callus tissue with citric acid concentration. Detailed Implementation

[0021] The following detailed description illustrates the specific implementation methods: Example 1: Weigh MS and sucrose, dissolve in water, adjust pH to 5.8, add agar and boil, then add glycine, dispense into containers and sterilize at 121℃ for 30 min to obtain seed inoculation medium; MS medium was used as the basal medium, with the addition of sucrose, agar, glycine, and 2,4-dimethylformamide. D, KT, and citric acid (with different concentration gradients). To prepare, first add citric acid to adjust the pH to 5.8, then add agar and boil, followed by glycine and hormones. After dispensing, sterilize to obtain MS solid medium.

[0022] It uses local red sorghum from Renhuai, with plump, intact seeds that are free from mold and pests.

[0023] A method for inducing callus tissue from young stems of *Sorghum sibirica* includes the following steps: Pretreatment: Select qualified red sorghum seeds and soak them in sterile water for 8-10 hours.

[0024] Surface disinfection: Disinfect with 75% ethanol, then rinse with sterile water; then disinfect with 0.1% mercuric chloride, and finally rinse thoroughly with sterile water.

[0025] Inoculation: Insert 10 sterilized seeds halfway into the seed inoculation medium per bottle. Sterilize the bottle mouth with a flame. Place in a 25℃ incubator and incubate for 14-15 days with 12 hours of light / 12 hours of darkness to obtain robust, sterile seedlings. Use the tender stems for later use.

[0026] Reference Figure 1 and Figure 2 Under aseptic conditions, young stems of the sterile seedlings were taken, their outer skin peeled off, and stem segments approximately 0.5 cm in length were cut as explants for callus induction. The cut young stem segments were inoculated into callus induction medium. Culture conditions: 25℃, dark culture. Observe daily and promptly clean any contaminated culture bottles.

[0027] Five days after culture, the callus induction was assessed. The callus emergence rate and browning rate were calculated using the following formulas: Callus emergence rate = (Number of explants that grew callus / Total number of inoculated explants) × 100%; Browning rate = (Number of explants that turned brown / Total number of inoculated explants) × 100%.

[0028] Example 2 (Optimal Condition Induction) Explants: Young stem segments from aseptic seedlings of *Sorghum sibirica*. Induction medium: MS medium + sucrose + agar + glycine + 2,4-dimethylformamide. D + KT + Optimal concentration of citric acid, pH 5.8. Culture conditions: 25℃, dark culture for 5 days. (Reference) Figure 3 Experimental results: High healing rate, lowest browning rate, and the callus tissue was milky white, dense, and grew vigorously. Example 3: A citric acid concentration gradient was set: 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / L, with all other conditions remaining the same. (Refer to...) Figure 4 and Figure 5 The results showed that low concentrations could increase the healing rate and reduce browning; while excessively high concentrations decreased the healing rate and increased browning.

[0029] Regarding browning rate: low concentrations (10~30 mg / L) do reduce browning, while excessively high concentrations (40 mg / L) cause browning rebound.

[0030] Regarding the cure rate: low concentrations did not increase the cure rate (10 mg / L even slightly decreased it), but 20 mg / L maintained the control level.

[0031] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for inducing callus tissue from young stems of *Sorghum sibirica*, characterized in that, Includes the following steps: S1. Take red sorghum seeds, disinfect them, and inoculate them into a seed culture medium. Culture under light to obtain sterile seedlings, and use young stems as explants. S2. Cut the young stems into segments and inoculate them into a callus induction medium for dark culture until callus formation. The callus induction medium uses MS as the basal medium and contains sucrose, agar, glycine, and 2,4-dimethylformamide. D, KT and citric acid.

2. The method according to claim 1, characterized in that, The seed culture medium is MS basal medium, supplemented with sucrose, agar and glycine.

3. The method according to claim 2, characterized in that, The seed surface disinfection includes: disinfection with 75% ethanol, rinsing with sterile water, disinfection with 0.1% mercuric chloride, and rinsing thoroughly with sterile water.

4. The method according to claim 3, characterized in that, The conditions for light culture are: 25-28℃, 12 hours of light / 12 hours of darkness, and culture for 14-15 days.

5. The method according to claim 4, characterized in that, The length of the young stem segments is 0.3–0.5 cm.

6. The method according to claim 5, characterized in that, The concentration of citric acid in the callus induction culture medium is 10-30 mg / L.

7. The method according to claim 6, characterized in that, The concentration of citric acid is 20 mg / L.

8. The method according to claim 7, characterized in that, The conditions for dark culture are: 22-25℃, dark culture for 5-8 days.

9. The method according to any one of claims 1 to 8, characterized in that, The pH of the callus induction medium is 5.

8.

10. The application of the method according to any one of claims 1 to 8 in the genetic transformation or breeding of red sorghum.

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