Method for establishing a sesbania hirta callus regeneration system

Abstract

The application discloses a method for establishing a Sesbania cannabina callus regeneration system. The method uses aseptic seedling stem segments as explants, and through callus induction, adventitious bud differentiation and rooting culture, the success rate of aseptic seedling callus induction can reach 98%, the adventitious bud induction rate is 89.7%, and the rooting rate reaches 90%. The method can realize efficient and rapid propagation throughout the year, and lays a technical foundation for Sesbania cannabina genetic transformation and gene editing based on callus, establishes a short-period and high-efficiency tissue culture and rapid propagation system, and forms complete tissue culture offspring plants.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for establishing a Sesbania cannabina callus regeneration system. Background Art

[0002] Sesbania cannabina ( Sesbania cannabina (Retz.) Pers.) is an annual subshrub-like herbaceous plant of the genus Sesbania in the legume family, with pinnate compound leaves and a height of up to 2.0 - 3.5 m. It has a large biomass and is a high-quality summer green manure. It is widely distributed in the tropical regions of the Eastern Hemisphere and is also widely cultivated in large areas in Guangdong, Fujian, Zhejiang, Jiangsu, Taiwan and other places in China. Sesbania cannabina has strong salt tolerance, waterlogging tolerance and barren tolerance, and is a pioneer green manure crop for improving saline-alkali land. It also has a high ability to resist diseases, insects and weeds. The ancients regarded it as the essence of the field, so it got the beautiful name of "Sesbania cannabina". Sesbania cannabina not only has great promotion potential in saline-alkali areas, but also shows unique utilization value in the industrial and medical fields.

[0003] CN202211452332.4 discloses a genetic transformation method of Sesbania cannabina. This method uses the cotyledons or hypocotyls of Sesbania cannabina as the starting explants to establish a rapid and effective regeneration system. This method first established an Agrobacterium-mediated glyphosate screening transformation system for Sesbania cannabina, filling the blank of the genetic transformation system of Sesbania cannabina. CN202411946439.3 discloses an efficient genetic transformation method for Sesbania rostrata. Using the cotyledon node as the starting explant without inducing callus, and combining with optimized medium components, it can quickly and effectively establish the genetic transformation system of Sesbania rostrata, and can improve the genetic transformation efficiency, reaching more than 25%, and shorten the transformation time.

[0004] Tissue culture technology is a technology that utilizes the totipotency of plant cells. Through aseptic operation, part of the tissue or cells of the plant body are placed on an artificially prepared culture medium, and appropriate culture conditions are given to induce the formation of callus and cluster buds, and finally form a complete plant. This technology has the advantages of fast propagation speed, being not restricted by seasons, and maintaining the excellent traits of the parent. So far, complete and stable plant tissue culture technology systems have been established for many crops and horticultural plants.

[0005] Although Sesbania cannabina has many excellent characteristics and wide application values, its traditional propagation method mainly relies on seed propagation, which has problems such as slow propagation speed and being restricted by seasons. In order to meet the large market demand for Sesbania cannabina and improve the propagation efficiency, it is particularly important to establish a tissue culture regeneration system for Sesbania cannabina. By establishing a tissue culture regeneration system for Sesbania cannabina, rapid propagation of Sesbania cannabina can be achieved, the usage of seeds as the main industrial raw material can be reduced, the production cost can be lowered, and the economic and ecological benefits can be improved.

[0006] Therefore, it is necessary to further explore more efficient and rapid guar gum callus regeneration systems. Summary of the Invention

[0007] The purpose of this invention is to provide a method for establishing a sesbania callus regeneration system to solve the problems existing in the prior art. This invention uses stem segments as explants for tissue culture, compares and analyzes seed disinfection time, and optimizes the effects of different plant growth regulator ratios in the culture medium on callus induction, differentiation, and plant regeneration. It finds a tissue culture regeneration method with high callus induction rate, high adventitious bud differentiation rate, and good rooting traits, thus enriching the sesbania callus culture program.

[0008] The purpose of this invention is to provide a method for establishing a guar gum callus regeneration system, comprising the following steps: (1) Obtaining sterile vaccines Sterile seeds were placed on germination medium and cultured to obtain sterile seedlings. The germination medium comprises basal medium and NAA; (2) Subculturing of sterile seedlings by cuttings Take the stem tip of the sterile seedling and insert it into the subculture medium for subculture. Use the tender stem segment as the explant. The subculture medium comprises basal medium and NAA; (3) Induction of callus tissue The explants were placed in a callus induction medium for induction culture to obtain induced callus tissue. The callus induction culture medium contains basal culture medium, NAA, and 6-BA; (4) Differentiation of adventitious buds The callus tissue was placed in an adventitious bud induction medium for differentiation culture to obtain adventitious buds; The adventitious bud differentiation medium contains basal medium, NAA and 6-BA or basal medium, TDZ and IAA; (5) Rooting The adventitious buds were placed in a rooting medium for rooting culture, and after hardening, sesame seedlings were obtained. The rooting medium comprises basal medium and NAA; The basal culture medium contains 1 / 2 MS, 30 g / L sucrose and 6.8 g / L agar.

[0009] Preferably, the concentration of NAA is 0.2-0.5 mg / L based on the total volume of the germination medium.

[0010] More preferably, the concentration of NAA is 0.5 mg / L based on the total volume of the germination medium.

[0011] Preferably, the concentration of NAA is 0.2-0.5 mg / L NAA, based on the total volume of the subculture medium.

[0012] More preferably, the concentration of NAA is 0.3 mg / L NAA, based on the total volume of the subculture medium.

[0013] Preferably, based on the total volume of the callus induction culture medium, the concentration of NAA is 0.1-1 mg / L and the concentration of 6-BA is 1-3 mg / L.

[0014] More preferably, the concentration of NAA is 0.3 mg / L and the concentration of 6-BA is 1 mg / L, based on the total volume of the callus induction culture medium.

[0015] Preferably, based on the total volume of the adventitious bud differentiation medium, the concentration of TDZ is 0.1-1 mg / L and the concentration of IAA is 1-3 mg / L.

[0016] More preferably, the concentration of TDZ is 0.1 mg / L and the concentration of IAA is 1-3 mg / L, based on the total volume of the adventitious bud differentiation medium.

[0017] Preferably, based on the total volume of the adventitious bud differentiation medium, the concentration of 6-BA is 1-3 mg / L and the concentration of NAA is 0.5-2 mg / L.

[0018] Preferably, the concentration of NAA is 0.1-0.5 mg / L based on the total volume of the rooting medium.

[0019] More preferably, the concentration of NAA is 0.5 mg / L based on the total volume of the rooting medium.

[0020] Preferably, in step (1), the method for preparing the disinfected seeds is as follows: soaking the sesame seeds in alcohol, rinsing them with sterile water, treating them with sodium hypochlorite solution, rinsing them with sterile water, and then removing the surface moisture.

[0021] More preferably, the concentration of the alcohol is 75%, and the soaking time is 5 minutes.

[0022] More preferably, the concentration of the sodium hypochlorite solution is 1.5-3%, and the treatment time is 8-12 min; preferably, the treatment is with a 1.5% NaClO sodium hypochlorite solution for 12 min.

[0023] More preferably, the seeds are plump seeds that have not been soaked or had their seed coats removed.

[0024] Preferably, in step (1), the cultivation conditions are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h.

[0025] Preferably, in step (1), the culture time is 11-21 days.

[0026] More preferably, the culture time is 16 days.

[0027] Preferably, in step (2), the length of the stem tip is 2-2.5 cm, and it is subcultured once every two weeks for a total of 2 subcultures.

[0028] Preferably, in step (2), the subculture conditions are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h.

[0029] Preferably, in step (2), the subculture time is 10-20 days.

[0030] More preferably, the subculture time is 15 days.

[0031] Preferably, in step (3), the length of the explant is 1-1.5 cm.

[0032] Preferably, in step (3), the conditions for induction culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h.

[0033] Preferably, in step (3), the induction culture time is 15-25 days.

[0034] More preferably, the induction culture time is 20 days.

[0035] Preferably, in step (4), the conditions for differentiation culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h.

[0036] Preferably, in step (4), the differentiation culture time is 8-19 days.

[0037] More preferably, the differentiation culture time is 14 days.

[0038] Preferably, in step (5), the conditions for rooting culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h.

[0039] Preferably, in step (5), the rooting culture time is 10-20 days.

[0040] More preferably, the rooting culture time is 15 days.

[0041] The beneficial technical effects of this invention are: Compared to existing technologies, this invention provides a complete method for constructing a sesquiterpene callus culture regeneration system. Using sterile seedling stem segments as explants, a short-cycle, high-efficiency tissue culture rapid propagation system is established through callus induction, adventitious bud differentiation, and rooting culture, resulting in complete tissue culture progeny plants. This system achieves 100% regeneration efficiency using sterile seedling stem tip cuttings for subculturing. Using 1 cm sterile seedling stem segments as explants, adding 0.3 mg / L NAA and 1 mg / L 6-BA to 1 / 2 MS basal medium resulted in a 98% success rate for callus induction; adding 0.1 mg / L TDZ and 3 mg / L IAA resulted in an 89.7% adventitious bud induction rate; and adding 0.5 mg / L NAA resulted in a 90% rooting rate. This technology not only enables efficient and rapid propagation throughout the year but also lays the technological foundation for sesquiterpene genetic transformation and gene editing based on callus tissue. Attached Figure Description

[0042] Figure 1 This invention relates to seed inoculation, germination, and the acquisition of sterile seedlings.

[0043] Figure 2 This invention relates to the screening of a sterile seedling subculture rooting medium.

[0044] Figure 3 This is a diagram illustrating the succession process of the present invention.

[0045] Figure 4 This invention relates to the induction and proliferation of callus tissue.

[0046] Figure 5 This refers to the redifferentiation of callus tissue according to the present invention.

[0047] Figure 6 This is a schematic diagram of the guar gum regeneration system of the present invention. Detailed Implementation

[0048] To better understand the purpose, structure, and function of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0050] In the following examples, the sesame variety is Yanjingjiao No. 2, which is sourced from the Yancheng Academy of Agricultural Sciences in Jiangsu Province.

[0051] Example 1: Investigation on the effects of different concentrations of disinfectant solution on seed germination rate and contamination rate after treatment for different durations. Select plump seeds that have not been soaked or had their seed coats removed. Rinse them under running water for 4-6 hours, then soak them in 75% alcohol for 5 minutes in a clean bench. Rinse them 3-4 times with sterile water, then treat them with sodium hypochlorite of different concentrations for different times. Finally, rinse them 4-6 times with sterile water to obtain sterilized sesame seeds. Place them on sterile, dry filter paper to absorb the surface moisture and set aside for later use.

[0052] Then, sesbania seeds were inoculated onto 1 / 2 MS germination medium (Phytotech, catalog number: M519) containing 0.5 mg / L NAA and cultured at 23℃ for 10 days. Each treatment was repeated in triplicate, and the seed germination rate and contamination rate were calculated after 10 days to examine the effect of different treatments on seed germination.

[0053] Table 1. Screening of Seed Disinfection Time

[0054] As shown in Table 1, under the three treatment conditions, the seed contamination rate of treatment group ③ was the lowest, but the germination rate decreased significantly due to the excessively high NaClO concentration. Under treatment group ②, although the NaClO concentration was higher than that of treatment group ①, the seed contamination rate was higher than that of treatment group ①, possibly due to the slightly shorter disinfection time. Considering both NaClO concentration and disinfection time, treatment group ① had the best effect among the three disinfection conditions, with a seed germination rate of over 90% and a contamination rate controlled below 9%.

[0055] In conclusion, sterilization with 1.5% NaClO for 12 minutes is the optimal treatment, resulting in high seed germination rates and relatively low contamination rates, ensuring the acquisition of sterile seedlings. After approximately two weeks of growth following inoculation, the sterile seedlings showed good growth. Figure 1 .

[0056] Example 2: Investigation of the effect of subculture medium on stem segment growth and rooting 2.1 Germination Culture The plant material used was the shoot tip of sterile seedlings formed after being treated with treatment group ③ in Example 1 and inoculated into 1 / 2 MS germination medium containing 0.5 mg / L NAA and cultured at 23°C for 15 days; the sucrose content was 30 g / L and the agar content was 6.8 g / L.

[0057] 2.2 Subgeneration The culture medium used for subculture was 1 / 2 MS as the basal medium, with different concentrations of NAA added (see Table 2), and the pH adjusted to 5.6-5.8. Sterile seedlings were held with sterile forceps, and stem tips with leaflets were cut off. The leaflets were removed, and 2-2.5 cm of the stem tip was inserted into subculture medium containing different NAA concentrations. The culture was placed in a tissue culture room (temperature 23±2 ℃, light intensity 2000-2500 lx, photoperiod 16 / 8 h) for two weeks. Stem growth and rooting were observed, and the results are shown in Table 2. Figure 2 .

[0058] Table 2 Subculture medium experimental design Formula Name Specific concentration N1 1 / 2MS + NAA 0.1 mg / L N2 1 / 2MS + NAA 0.2 mg / L N3 1 / 2MS + NAA 0.3 mg / L N4 1 / 2MS + NAA 0.4 mg / L N5 1 / 2MS + NAA 0.5 mg / L from Figure 2 It can be seen that the five culture media exhibited different growth states due to their varying NAA concentrations. Among them, N1 medium failed to promote rooting; N2, N3, and N4 media were able to induce rooting to some extent; however, comparatively, the rooting speed was fastest on N5 medium, and the root system was thicker and healthier.

[0059] Therefore, based on the growth status of the crown and roots of the subcultured sterile seedlings, 1 / 2MS + 0.5 mg / L NAA (N5 medium) was finally determined as the optimal subculture medium formula.

[0060] The results of subculturing sterile seedlings in N5 medium for 15 days are shown in the figure. Figure 3 .

[0061] Example 3: Effect of callus induction methods on callus induction rate Using sterile sesame seedlings subcultured in N5 medium for 20 days as material, sesame stem segments were cut into 1 cm long pieces using a scalpel in a clean bench as explants. The callus induction medium was based on 1 / 2 MS medium, with different concentrations of NAA and 6-BA added, resulting in 18 different ratio combinations (Table 3).

[0062] Under normal light conditions and at a culture temperature of 23℃ in the tissue culture room, the prepared explants were transferred into 18 different callus induction media. After 20 days, the callus growth was observed and statistically analyzed, and the optimal callus induction medium was determined based on the callus induction rate. Callus induction culture was then performed.

[0063] Callus induction rate (%) = number of stem segments that induced callus / total number of inoculated stem segments × 100%.

[0064] Table 3 Effects of different culture media on callus induction from sesbania stem segments deal with NAA (mg / L) 6-BA (mg / L) Number Healing number Induction rate (%) 1 0.1 1 30 3 10 2 0.1 2 32 4 10.5 3 0.1 3 31 6 9.7 4 0.15 1 30 5 16.7 5 0.15 2 35 7 20 6 0.15 3 40 23 57.5 7 0.2 1 18 13 72.2 8 0.2 2 30 20 66.6 9 0.2 3 70 40 57.1 10 0.25 1 58 43 74.1 11 0.25 2 38 31 81.6 12 0.25 3 30 18 60 13 0.3 1 48 47 98 14 0.3 2 41 40 97.6 15 0.3 3 36 35 97.2 16 1 1 52 43 82.7 17 1 2 55 45 81.8 18 1 3 58 44 75.9 Table 3 shows that when the NAA concentration was 0.1 mg / L, the induction rate was relatively low when combined with different concentrations of 6-BA, ranging from only 9.7% to 10.5%. However, as the NAA concentration increased to 0.3 mg / L, the induction rate significantly improved, reaching over 97% (treatments 13-15), and the callus tissue exhibited clumping, large volume, and good growth. However, when the NAA concentration continued to increase to 1 mg / L, the induction rate showed a decreasing trend (75.9%–82.7%), indicating that excessively high NAA concentrations may inhibit callus formation. When the NAA concentration was fixed, changing the 6-BA concentration within a certain range showed relatively little effect on callus formation.

[0065] The induction rate of treatment group 6 (a combination of 0.15 mg / L NAA and 3 mg / L 6-BA) (57.5%) was significantly higher than that of treatment group 4 (a combination of 0.15 mg / L NAA and 1 mg / L 6-BA) (16.7%) and treatment group 5 (a combination of 0.15 mg / L NAA and 2 mg / L 6-BA) (20%).

[0066] Therefore, the optimal culture medium for inducing callus using aseptic stem segments of *Senecio scandens* seedlings as explants was 1 / 2 MS + NAA 0.3 mg / L + 6-BA 1 mg / L, achieving an induction rate of 98% (treatment 13). Furthermore, the callus tissue exhibited clump-like formation, large volume, and good growth. Figure 3 ).

[0067] Callus tissue was transferred to a new callus induction medium (1 / 2MS + NAA 0.3 mg / L + 6-BA 1 mg / L) every two weeks for proliferation, and this process was repeated three times before bud differentiation induction was performed.

[0068] Example 4: Investigation of the effect of adventitious shoot differentiation medium on callus induction rate The adventitious shoot differentiation medium was based on 1 / 2 MS medium, with different concentrations of 6-BA, NAA and TDZ, IAA added respectively, resulting in 18 different ratio combinations (Table 4).

[0069] The proliferating callus tissue obtained after three consecutive subcultures in Example 3 was inoculated into adventitious shoot differentiation medium and cultured normally in the tissue culture room for 2 weeks. The adventitious shoot callus induction rate was then calculated. Two weeks after induction, the callus redifferentiation rate and growth status were statistically analyzed and recorded. Figure 4 ).

[0070] Adventitious shoot callus induction rate (%) = (Number of callus tissues induced to form adventitious shoots / Total number of callus tissues) × 100%.

[0071] Table 4 Effects of different hormones on adventitious shoot induction deal with 6-BA (mg / L) NAA (mg / L) TDZ (mg / L) IAA / (mg / L) Adventitious bud induction rate (%) 1 1 0.5 / / 0 2 2 0.5 / / 4.5 3 3 0.5 / / 10.6 4 1 1 / / 31.3 5 2 1 / / 37.3 6 3 1 / / 34.1 7 1 2 / / 83.7 8 2 2 / / 57.9 9 3 2 / / 57.1 10 / / 0.1 1 18.9 11 / / 0.5 1 30.4 12 / / 1 1 22.7 13 / / 0.1 2 54.2 14 / / 0.5 2 51.2 15 / / 1 2 42.1 16 / / 0.1 3 89.7 17 / / 0.5 3 82.7 18 / / 1 3 73.8 Table 4 shows that when the NAA concentration was 0.5 mg / L, the redifferentiation rate increased with increasing 6-BA concentration (0-10.6%). When the NAA concentration was 1 mg / L, the redifferentiation rate significantly increased to 31.3-37.3%, and different 6-BA concentrations did not have a significant effect on the redifferentiation rate. The best effect was achieved when the NAA concentration was 2 mg / L and the 6-BA concentration was 1 mg / L, with a redifferentiation rate of 83.7%. In summary, in the combination of 6-BA and NAA, the NAA concentration plays a dominant role, and the highest redifferentiation rate (83.7%) is obtained when the NAA concentration is 2 mg / L and the 6-BA concentration is 1 mg / L.

[0072] Table 4 shows that when TDZ and IAA are combined, the redifferentiation rate is 18.9-30.4% when the IAA concentration is 1 mg / L; 42.1-54.2% when the IAA concentration is 2 mg / L; and 73.8-89.7% when the IAA concentration is 3 mg / L. This indicates that when the IAA concentration remains constant, the adventitious shoot induction rate is not dependent on the TDZ concentration gradient. Under this hormone combination, the IAA concentration is the key factor; the callus redifferentiation rate reaches 89.7% under the conditions of 3 mg / L IAA + 0.1 mg / L TDZ, which is the optimal ratio among all experimental combinations.

[0073] Therefore, medium 16 showed the best differentiation and redifferentiation effects (0.1 mg / L TDZ and 3 mg / L IAA), with a redifferentiation rate of 89.7%, and the plants exhibited robust growth and good vigor. (See results below.) Figure 5 .

[0074] Example 5: Screening of adventitious shoot rooting induction medium After the adventitious buds from medium 16 in Example 4 differentiated to 2-3 cm, the buds were cut off and transferred to 1 / 2 MS medium containing different NAA concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg / L) for rooting induction. The buds were then cultured normally in a tissue culture growth chamber for 3 weeks, and the rooting rate was recorded.

[0075] Rooting rate (%) = Number of rooted adventitious buds / Total number of adventitious buds × 100%.

[0076] Table 5 Effects of NAA on rooting induction NAA concentration (mg / L) Number Number of inducements Induction rate (%) 0.1 45 0 0 0.2 50 5 10 0.3 45 12 26.7 0.4 46 22 47.8 0.5 50 45 90 As shown in Table 5, the rooting induction rate exhibited a significant dose-dependent increase with increasing NAA concentration. When the NAA concentration reached 0.5 mg / L, the rooting induction rate reached as high as 90%, significantly higher than other treatment groups. Therefore, 1 / 2 MS + 0.5 mg / L NAA was selected as the most suitable culture medium formula for rooting induction of adventitious shoots from Sesbania serrata.

[0077] The embodiments described above can be further combined or replaced according to actual needs, and are merely descriptions of preferred embodiments of the present invention, not intended to limit the concept and scope of the present invention. Any adjustments, modifications, or improvements made to the technical solutions by those skilled in the art without departing from the design concept of the present invention should be included within the protection scope of the present invention. The protection scope of the present invention is determined by the appended claims and their equivalents.

Claims

1. A method for establishing a guar gum callus regeneration system, characterized in that, Includes the following steps: (1) Obtaining sterile vaccines Sterile seeds were placed on germination medium and cultured to obtain sterile seedlings. The germination medium comprises basal medium and NAA; (2) Subculturing of sterile seedlings by cuttings Take the stem tip of the sterile seedling and insert it into the subculture medium for subculture. Use the tender stem segment as the explant. The subculture medium comprises basal medium and NAA; (3) Induction of callus tissue The explants were placed in a callus induction medium for induction culture to obtain induced callus tissue. The callus induction culture medium contains basal culture medium, NAA, and 6-BA; (4) Differentiation of adventitious buds The callus tissue was placed in an adventitious bud induction medium for differentiation culture to obtain adventitious buds; The adventitious bud differentiation medium contains basal medium, NAA and 6-BA or basal medium, TDZ and IAA; (5) Rooting The adventitious buds were placed in a rooting medium for rooting culture, and after hardening, sesame seedlings were obtained. The rooting medium comprises basal medium and NAA; The basal culture medium contains 1 / 2 MS, 30 g / L sucrose and 6.8 g / L agar.

2. The method as described in claim 1, characterized in that, Based on the total volume of the germination medium, the concentration of NAA is 0.2-0.5 mg / L; preferably, it is 0.5 mg / L. And / or, based on the total volume of the subculture medium, the concentration of NAA is 0.2-0.5 mg / L NAA; And / or, based on the total volume of the callus induction medium, the concentration of NAA is 0.1-1 mg / L and the concentration of 6-BA is 1-3 mg / L; And / or, based on the total volume of the adventitious shoot differentiation medium, the concentration of TDZ is 0.1-1 mg / L and the concentration of IAA is 1-3 mg / L; And / or, based on the total volume of the adventitious shoot differentiation medium, the concentration of 6-BA is 1-3 mg / L and the concentration of NAA is 0.5-2 mg / L; And / or, based on the total volume of the rooting medium, the concentration of the NAA is 0.1-0.5 mg / L.

3. The method as described in claim 2, characterized in that, The concentration of NAA is 0.5 mg / L, based on the total volume of the germination medium. And / or, based on the total volume of the subculture medium, the concentration of NAA is 0.3 mg / L NAA; And / or, based on the total volume of the callus induction medium, the concentration of NAA is 0.3 mg / L and the concentration of 6-BA is 1 mg / L; And / or, based on the total volume of the adventitious shoot differentiation medium, the concentration of TDZ is 0.1 mg / L and the concentration of IAA is 1-3 mg / L; And / or, based on the total volume of the rooting medium, the concentration of the NAA is 0.5 mg / L.

4. The method as described in claim 1, characterized in that, In step (1), the method for preparing the disinfected seeds is as follows: soak the sesame seeds in alcohol, rinse with sterile water, treat with sodium hypochlorite solution, rinse with sterile water, and then remove the surface moisture.

5. The method as described in claim 4, characterized in that, The alcohol concentration is 75%, and the soaking time is 5 minutes; And / or, the concentration of the sodium hypochlorite solution is 1.5-3%, and the treatment time is 8-12 min; preferably, the treatment is with a 1.5% NaClO sodium hypochlorite solution for 12 min.

6. The method as described in claim 4, characterized in that, The seeds are plump seeds that have not been soaked or had their seed coats removed.

7. The method as described in claim 1, characterized in that, In step (2), the length of the stem tip is 2-2.5 cm, and it is subcultured once every two weeks for a total of 2 subcultures.

8. The method as described in claim 1, characterized in that, In step (3), the length of the explant is 1-1.5 cm.

9. The method as described in claim 1, characterized in that, Includes one or more of the following technical features: In step (1), the cultivation conditions are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8h; In step (1), the culture time is 11-21 days; In step (2), the conditions for subculture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h; In step (2), the subculture time is 10-20 days. In step (3), the conditions for induction culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h; In step (3), the induction culture time is 15-25 days; In step (4), the conditions for differentiation culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h; In step (4), the differentiation culture time is 8-19 days; In step (5), the conditions for rooting culture are: temperature 21-25 ℃, light intensity 2000-2500 Lx, and photoperiod 16 / 8 h; And / or, in step (5), the rooting culture time is 10-20 days.

10. The method as described in claim 9, characterized in that, Includes one or more of the following technical features: In step (1), the culture time is 16 days; In step (2), the subculture time is 15 days. In step (3), the induction culture time is 20 days; In step (4), the differentiation culture time is 14 days; In step (5), the rooting culture time is 15 days.

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