A method for improving tissue culture efficiency

Abstract

The application belongs to the technical field of plant tissue culture, and discloses a method for improving tissue culture efficiency. The method comprises the following steps: preparing tomato aseptic seedlings; removing cotyledons and stem tip growth points of the aseptic seedlings, retaining hypocotyls and radicles as explants, horizontally inoculating the explants into full amount MS induction medium containing no plant growth regulator but adding activated carbon to culture, and differentiating to form adventitious buds; and then obtaining regenerated plants through subculture proliferation and rooting culture. The method selects the hypocotyls and radicles after removing the cotyledons as the explants, and combines with the induction medium containing no plant growth regulator, so that the occurrence of cotyledon explant browning and vitrification seedlings is reduced, the browning rate of the explants is reduced, the differentiation efficiency of the adventitious buds is improved, the quality of the tissue culture seedlings is improved, the induction period is shortened, and the overall efficiency of the tomato tissue culture is improved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a method for improving tissue culture efficiency. Background Technology

[0002] tomato( Solanum lycopersicum L. Tomato is an important vegetable crop, and tissue culture technology is often used for rapid propagation of seedlings and genetic improvement in production. Currently, most tomato tissue cultures use cotyledons as explants. The specific procedure is as follows: take cotyledons from sterile seedlings, cut them into small pieces, and inoculate them on MS medium supplemented with 6-BA (6-benzyladenine) and NAA (naphthaleneacetic acid) for induction.

[0003] Tomato cotyledons, as explants, possess advantages such as vigorous cell division, high regeneration frequency, and convenient material sourcing. However, they also present the following problems: Firstly, the large cut area of ​​the cotyledons, coupled with abundant thin-walled cells, leads to the leakage of large amounts of phenolic substances during cutting. These substances undergo enzymatic browning under the catalysis of polyphenol oxidase, and the resulting quinones further polymerize to form brown substances, causing the explants to lose vitality or even die. Secondly, the cotyledons, in direct contact with culture media containing high concentrations of cytokinins, have a strong water absorption capacity, easily inducing vitrified seedlings that are transparent and have high water content. Vitrified seedlings have translucent leaves and stems, lost stomatal function, and exhibit disordered water metabolism, resulting in extremely low survival rates after transplanting. Furthermore, under certain hormone ratios, the callus induced from cotyledons is loose, water-soaked, and has large intercellular spaces. This type of callus has a low proportion of embryogenic cells and poor ability to differentiate into adventitious shoots, leading to overall low tissue culture efficiency.

[0004] In existing techniques, cotyledons are typically cut into small pieces of 0.5-1 cm, and the conventional concentration range for 6-BA is 1.0-3.0 mg / L, while the conventional concentration range for NAA is 0.05-1.0 mg / L. Furthermore, vitamin C (50-200 mg / L) and activated charcoal (0.2-1 g / L) are commonly used as anti-browning agents in existing techniques, but their effectiveness is limited. Therefore, there is a need to develop a tomato tissue culture method that can reduce explant browning rate, shorten the induction period, and improve tissue culture efficiency. Summary of the Invention

[0005] This invention provides a method to improve tissue culture efficiency. By selecting hypocotyls and radicles after removing cotyledons as explants and combining them with an induction medium that does not contain plant growth regulators, the occurrence of browning and vitrification of cotyledon explants is reduced, the browning rate of explants is decreased, the efficiency of adventitious bud differentiation and the quality of tissue culture seedlings are improved, the induction period is shortened, and the overall efficiency of tomato tissue culture is enhanced.

[0006] This invention is implemented through the following technical solution: a method for improving tissue culture efficiency, comprising the following steps: S1. Preparation of sterile tomato seedlings: After disinfecting the tomato seeds, inoculate them into a germination carrier and culture them under culture conditions until the cotyledons are fully expanded or the first true leaf grows to obtain sterile seedlings. S2. Explant preparation: Take the sterile seedling, remove the cotyledons and shoot tip growth point, and retain the remaining hypocotyl and radicle as explants. Place the explants horizontally on the induction medium, so that the cut surfaces at both ends of the explants are in contact with the medium. S3. Induction Culture: The inoculated explants are placed in an induction medium without any plant growth regulators and cultured to differentiate into adventitious shoots; the induction medium includes MS basal medium, 0.4~0.6 g / L activated carbon, sucrose and agar, and no plant growth regulators are added; S4. Subculture: When the adventitious shoots grow to 1.5-2.5 cm, they are cut off from the explants and transferred to a subculture medium for proliferation culture. The subculture cycle is 18-22 days. S5. Rooting culture: Select tissue culture seedlings with a height of 3-5cm and 3-4 leaves that are intact, not curled, not wrinkled, and not vitrified. Transfer them to a rooting culture medium to induce rooting and obtain regenerated plants with roots and stems and leaves.

[0007] Preferably, in step S1, the age of the sterile seedling is 7-10 days, calculated from the date of seed germination.

[0008] Preferably, the seed disinfection treatment in step S1 is carried out according to the following steps: first, rinse the seeds with sterile water, then soak them in an ethanol solution with a volume fraction of 70%-80% for 1-3 minutes, rinse them with sterile water 2-4 times, then soak them in a sodium hypochlorite solution with a mass fraction of 8%-12% for 10-20 minutes, and finally rinse them with sterile water 4-6 times.

[0009] Preferably, the germination carrier in step S1 is a sterile petri dish lined with filter paper and containing sterile water, or MS medium containing 0.1-0.3 mg / L naphthaleneacetic acid and 0.8-1.2 mg / L 6-benzyladenine.

[0010] Preferably, the explants in step S2 do not require secondary cutting; the whole hypocotyl after removing the cotyledons is used directly for inoculation.

[0011] Preferably, the induction culture time in step S3 is 20-30 days, and the differentiated adventitious buds have 2-3 functional leaves that are not damaged, curled, wrinkled, or vitrified.

[0012] Preferably, the subculture proliferation medium in step S4 includes MS basal medium, 1.0~2.0 mg / L 6-benzyladenine, 0.01~0.5 mg / L naphthaleneacetic acid, sucrose and agar.

[0013] Preferably, the rooting medium in step S5 includes MS basal medium, 0.1~0.3 mg / L naphthaleneacetic acid, sucrose and agar.

[0014] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses hypocotyls and radicles with cotyledons and shoot apical meristems removed as culture materials. These tissues have smaller cut areas and denser structures, reducing the outward leakage of phenolic substances, alleviating enzymatic browning caused by polyphenol oxidase, and lowering the probability of explants experiencing decreased viability and death due to browning. Simultaneously, the use of activated charcoal in the induction medium further improves the control of browning compared to traditional methods relying solely on conventional anti-browning agents such as vitamin C and activated charcoal.

[0015] 2. This invention uses an induction culture medium without any plant growth regulators for induction culture, avoiding direct contact between explants and culture medium containing high concentrations of cytokinins; and since the hypocotyl and radicle have weaker water absorption capacity than the cotyledons, it can reduce the occurrence of vitrified seedlings that are transparent and have high water content, avoiding the problems of semi-transparent leaves and stems, loss of stomatal function, and disordered water metabolism in vitrified seedlings, thus improving the survival rate of transplanted tissue culture seedlings.

[0016] 3. The explants used in this invention induce callus with a compact structure, small intercellular spaces, a high proportion of embryogenic cells, and a strong ability to differentiate into adventitious shoots. This solves the problems of loose, water-soaked callus, low proportion of embryogenic cells, and poor differentiation ability induced by traditional cotyledon explants, and improves the efficiency of adventitious shoot differentiation.

[0017] 4. The induction stage of this invention does not require the addition of plant growth regulators such as naphthaleneacetic acid and 6-benzyladenine, which simplifies the culture medium preparation process and reduces the use of hormone reagents. At the same time, the explants used can continue to differentiate into new adventitious buds after the differentiated adventitious buds are removed, and have the ability to differentiate continuously multiple times, which reduces the workload of repeated material collection and inoculation. Attached Figure Description

[0018] Figure 1 This is a diagram showing the induction state of cherry tomato explants cultured for 20 days according to an embodiment of the present invention. Figure 2 This is a diagram showing the induction state of epicotyl explants cultured for 46 days according to an embodiment of the present invention. Figure 3This is a diagram showing the induction state of epicotyl explants cultured for 44 days according to an embodiment of the present invention; Figure 4 This is a diagram showing the browning state of epicotyl explants after 20 days of culture, according to an embodiment of the present invention. Figure 5 This is a diagram showing the differentiation failure state of cotyledon explants cultured for 30 days according to an embodiment of the present invention. Figure 6 This is a diagram showing the differentiation failure state of cotyledon explants cultured for 20 days according to an embodiment of the present invention. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1-6 The present invention will be further described in detail with reference to specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0020] Culture medium preparation method The MS basic culture medium used in this invention is a complete MS medium containing 100 mg / L inositol, 0.1 mg / L vitamin B1, and 0.5 mg / L vitamin B6. Its macro- and micro-element composition is as follows: Macroelements: Ammonium nitrate 1650.0 mg / L, Potassium nitrate 1900.0 mg / L, Calcium chloride dihydrate 440.0 mg / L, Magnesium sulfate heptahydrate 370.0 mg / L, Potassium dihydrogen phosphate 170.0 mg / L Trace elements: Potassium iodide 0.83 mg / L, boric acid 6.20 mg / L, manganese sulfate tetrahydrate 22.30 mg / L, zinc sulfate heptahydrate 8.60 mg / L, sodium molybdate dihydrate 0.25 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L The specific preparation methods for each culture medium are as follows: (1) Induction medium: Mix MS basic medium, 0.5 g / L (0.05%) activated carbon, 30 g / L sucrose and 7 g / L agar, add distilled water to make up the volume, adjust the pH value to 5.7-5.8, autoclave at 121℃ for 15 min, and cool for later use.

[0021] (2) Subculture proliferation medium: Mix MS basic medium, 2.0 mg / L 6-benzyladenine, 0.03 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, add distilled water to make up the volume, adjust the pH to 5.7-5.8, autoclave at 121℃ for 15 min, and cool for later use.

[0022] (3) Rooting medium: Mix MS basic medium, naphthaleneacetic acid 0.2 mg / L, sucrose 30 g / L and agar 7 g / L, add distilled water to make up the volume, adjust the pH value to 5.7-5.8, autoclave at 121℃ for 15 min, and cool for later use.

[0023] General culture conditions The general culture conditions for all culture steps in this invention are as follows: temperature 25℃; light source is an LED lamp with a spectral composition of 13.5% red, 81.0% green, and 5.4% blue; light intensity 2000 lx, measured on the surface of the culture rack; illumination time 12 h / day. The culture container is a 240 mL tissue culture bottle, sealed with a matching cap containing a breathable membrane. The relative humidity of the culture room is controlled at 70%-80%, and the culture rack uses an internal circulation ventilation system.

[0024] Example 1: Tissue Culture Method for Yellow Cherry Tomatoes (1) Seed disinfection and germination: Select plump, disease-free yellow cherry tomato seeds. First, rinse the surface dust with sterile water, then soak in a 75% ethanol solution for 2 minutes for disinfection, and rinse 3 times with sterile water. Then, soak in a 10% sodium hypochlorite solution for 15 minutes for disinfection, gently shaking continuously during the process. Finally, rinse 5-6 times with sterile water. Place the disinfected seeds in a 90mm sterile petri dish lined with double-layered filter paper and 10mL of sterile water. Cultivate under the above general culture conditions until 7-10 days after germination, when the cotyledons are fully expanded, to obtain sterile seedlings.

[0025] (2) Explant preparation: Take the above-mentioned sterile seedlings in a clean bench, remove the cotyledons and shoot tip growth point by flat cutting, and retain the whole hypocotyl and radicle as explants. There are no special requirements for the flatness of the cut. The explants do not need to be cut again and can be used directly as whole sections. Place the explants horizontally on the induction medium, ensuring that both ends of the cut surface are in full contact with the medium.

[0026] (3) Induction culture: The inoculated explants were placed in an induction culture medium for 20 days. Under the general culture conditions described above, the explants differentiated into adventitious buds. The adventitious buds had 2-3 functional leaves, were plump, bright in color, and vigorous in growth. No browning, necrosis, or deformed seedlings appeared throughout the culture process.

[0027] (4) Subculture: When the adventitious shoots grow to about 2 cm, they are cut off from the explants and transferred to a subculture culture medium for proliferation. Under the general culture conditions described above, the adventitious shoots can proliferate to form cluster shoots after 20 days.

[0028] (5) Rooting culture: Select robust tissue culture seedlings with a height of 3-5cm and 3-4 leaves that are intact, not curled, not wrinkled, and not vitrified, and transfer them to a rooting medium to induce rooting. Under the general culture conditions described above, regenerated plants with well-developed root systems and complete stems and leaves can be obtained after 7-10 days of culture.

[0029] Example 2: Tissue Culture Method for Red Cherry Tomatoes The same seed disinfection, explant preparation, induction culture, subculture and rooting culture methods as in Example 1 were used, except that the tomato variety was changed to red cherry tomato.

[0030] The results showed that adventitious shoots were successfully differentiated from explants of red cherry tomatoes on hormone-free induction medium, with a budding rate of 100% and a browning mortality rate of 0%. The induction culture time was 20 days, and the differentiated adventitious shoots had 2-3 functional leaves. There were no significant differences in callus status, shoot growth, and seedling quality at each stage compared to yellow cherry tomatoes.

[0031] Example 3: Tissue Culture Method for Black Cherry Tomatoes The same seed disinfection, explant preparation, induction culture, subculture and rooting culture methods as in Example 1 were used, except that the tomato variety was changed to black cherry tomato.

[0032] The results showed that black cherry tomato explants successfully differentiated adventitious shoots on hormone-free induction medium, with a budding rate of 100% and a browning mortality rate of 0%. Compared with yellow and red cherry tomatoes, black cherry tomatoes differentiated slightly slower, with an induction culture time of 20-30 days. Figure 1 As shown, after 20 days of explant culture, the differentiated adventitious buds also have 2-3 functional leaves, and the bud plumpness and growth vigor are consistent with the yellow and red varieties.

[0033] Example 4: Verification of the continuous differentiation ability of explants Using the same tissue culture method as in Example 1, after removing adventitious buds in step (4) of subculture proliferation, the remaining explant hypocotyl portion was kept on the original induction medium for further culture to verify its continuous differentiation ability.

[0034] The results showed that the explant hypocotyls could differentiate continuously for 5 rounds, with an average of 3 buds per round. By the 5th round, the explants began to show slight yellowing, but no browning or necrosis occurred. A single explant could yield approximately 15 complete plants with 3-4 functional leaves throughout the entire process, demonstrating excellent continuous propagation ability.

[0035] Comparative scale settings and test results The comparative examples were divided into an epicotyl group (comparative examples 1-3) and a traditional cotyledon group (comparative examples 4-5). The same type of explants were used with hormone-free medium and two commonly used hormone gradient mediums. All treatment materials were yellow cherry tomatoes, and the culture conditions were the same as in Example 1.

[0036] Comparative Example 1 (epigloaxial culture medium without hormones) Using the same yellow cherry tomato seeds and induction medium (containing 0.05% activated carbon but without plant growth regulators) as in Example 1, the only difference was that the explants were replaced with epicotyl explants, which were inoculated onto the medium in a horizontal manner. The culture conditions were the same as in Example 1.

[0037] The results show that, Figure 2 As shown, after 46 days of continuous culture, the callus tissue induced by the epicotyl was loose in texture and dark in color, with brown patches appearing in some areas; the germination time of adventitious buds was uneven and the differentiation cycle was disordered, some buds were thin and short, with weak growth, and only a single deformed leaf grew. The overall seedling quality was poor, and the browning phenomenon was obvious.

[0038] Comparative Example 2 (epigloaxial + hormone gradient 1) The same yellow cherry tomato hypocotyl explants as in Example 1 were used, but the induction medium was replaced with MS medium supplemented with 2.0 mg / L 6-BA and 0.01 mg / L NAA. The culture conditions were the same as in Example 1.

[0039] The results show that, Figure 3 As shown, after 44 days of continuous culture, the explants exhibited significant browning, and the callus tissue was dark and loose. Influenced by high concentrations of cytokinin, some adventitious buds showed vitrification characteristics, with thin, weak stems and curled, deformed leaves. Overall, the adventitious buds showed weak growth, lacked a uniform differentiation cycle, and resulted in a low number of effective seedlings.

[0040] Comparative Example 3 (epigloaxial + hormone gradient 2) The same yellow cherry tomato hypocotyl explants as in Example 1 were used, but the induction medium was replaced with MS medium supplemented with 1.5 mg / L 6-BA and 0.1 mg / L NAA. The culture conditions were the same as in Example 1.

[0041] The results show that: Figure 4 As shown, after 20 days of culture, large areas of browning and necrosis of explants can be observed, callus tissue is water-soaked and differentiation activity is greatly reduced; only some explants slowly germinate adventitious buds, and the overall differentiation effect is poor.

[0042] Comparative Example 4 (Cotyledon + Hormone Gradient 1, Traditional Formula) The same yellow cherry tomato cotyledon explants as in Example 1 were used, but the induction medium was replaced with MS medium supplemented with 2.0 mg / L 6-BA and 0.01 mg / L NAA. The culture conditions were the same as in Example 1.

[0043] The results showed that the cotyledon cut area was large and the content of thin-walled cells was high, resulting in a large amount of phenolic substances being released after cutting; for example... Figure 5 As shown, after 30 days of culture, the explants turned brown and died completely, the callus tissue necrotized and disintegrated, and no healthy adventitious buds differentiated, indicating complete differentiation failure.

[0044] Comparative Example 5 (Cotyledon + Hormone Gradient 2, Traditional Formula) The same yellow cherry tomato cotyledon explants as in Example 1 were used, but the induction medium was replaced with MS medium supplemented with 1.5 mg / L 6-BA and 0.1 mg / L NAA. The culture conditions were the same as in Example 1.

[0045] The results show that: Figure 6 As shown, after 20 days of culture, the cotyledon explants rapidly underwent enzymatic browning and necrosis, accompanied by obvious water staining and vitrification; the callus tissue was loose and inactive, and the differentiation of adventitious buds was completely inhibited, exhibiting typical defects of traditional tomato tissue culture.

[0046] Comparative analysis of core indicators In this experiment, 10 explants were used in each group, with 10 replicates for each treatment and 3 parallel trials conducted independently. Based on observation records and statistical data, the results were comprehensively compared across six dimensions: budding rate, browning mortality rate, induction period, callus state, adventitious shoot quality, and continuous differentiation and propagation ability. The results are as follows: (1) Sprouting rate (success rate of adventitious bud differentiation) Example 1: The budding rate was 100%, and all explants were able to differentiate into adventitious buds normally; Comparative Examples 1 and 2: No effective adventitious buds sprouted throughout the process, with a budding rate of 0%; Comparative Example 3: The budding rate was 44%; Comparative Examples 4 and 5: All explants turned brown and died, with no adventitious bud differentiation, and a budding rate of 0%. The differences in budding rates among the groups were highly significant (P<0.0001).

[0047] (2) Browning mortality rate Browning is a major cause of explant inactivation and culture failure in tomato tissue culture. Example 1: Browning mortality rate was 0%, with no browning or necrosis observed throughout the culture process. Comparative Examples 1 and 2: Explants showed severe browning, with mortality rates exceeding 50%. Comparative Example 3: Browning mortality rate was 16%, with some explants losing activity due to browning. Comparative Examples 4 and 5: Cotyledonary explants underwent severe enzymatic browning, with a browning mortality rate reaching 100%. The differences in browning mortality rates among the groups were statistically significant (P=0.0121).

[0048] (3) Induced growth cycle Example 1: In just 20 days, robust new shoots with a height of about 2cm can be differentiated, with a uniform differentiation rhythm and controllable cycle; Although 44% of the explants in Comparative Example 3 sprouted adventitious shoots, the growth rate was slow, and it took 82 days to reach the same growth state, with a significantly prolonged differentiation cycle; Comparative Examples 1, 2, 4, and 5 did not differentiate any effective adventitious shoots and had no normal growth cycle.

[0049] (4) Morphological state of callus tissue Example 1: The induced callus tissue was dense and firm, with a bright yellow color and a uniform and full structure. It was free of water staining, browning patches, and necrotic tissue, and had strong cell activity, which could continuously provide nutrition for bud differentiation. The callus tissues obtained by Comparative Examples 1 to 5 were generally loose in texture and dark in color. Among them, Comparative Examples 3, 4, and 5 also showed obvious water staining and water-soaked structures, some of which were accompanied by large areas of browning and necrotic patches. The proportion of embryonic cells was low and the differentiation potential was extremely poor. This is also the core reason for the low differentiation efficiency of traditional tissue culture.

[0050] (5) Morphology and growth quality of adventitious buds Example 1: The differentiated adventitious buds grew vigorously, with an average of 2 strong new buds sprouting from each explant. The buds were plump and bright in color, and each bud grew 2-3 complete, undamaged, and non-curled normal functional leaves. No deformed or vitrified seedlings were produced throughout the process. In contrast, the adventitious buds sprouted in Comparative Example 3 were weak, thin, and short. Most of them only grew a single deformed leaf and were accompanied by varying degrees of vitrification. In Comparative Examples 1, 2, 4, and 5, no healthy adventitious buds with seedling value were formed. Some of the sprouted buds had thin and weak stems and severely deformed leaves.

[0051] (6) The ability of explants to continuously differentiate and proliferate The hypocotyl + radicle explants of Example 1 exhibited good continuous differentiation ability, capable of differentiating into 5 whorls of adventitious buds, with an average of 3 buds per whorl. By the 5th whorl, only slight yellowing was observed, without browning or necrosis. Each explant could cumulatively produce approximately 15 complete and robust plants, supporting multiple whorls of continuous propagation. In contrast, the explants of Comparative Examples 1 to 5 lost their differentiation activity after a single culture, failing to germinate new buds and lacking continuous propagation ability, resulting in low material utilization.

[0052] Adaptability verification of different tomato varieties This invention was simultaneously validated on three cherry tomato varieties: yellow, red, and black. The induction period for yellow and red cherry tomatoes was 20 days, with a 100% budding rate and 0% browning mortality, exhibiting identical callus status and bud growth. For black cherry tomatoes, the budding rate and browning mortality were also 100% and 0%, respectively, with only the differentiation period extended to 20-30 days. Adventitious bud morphology and seedling quality showed no significant differences compared to the other two varieties. The results indicate that this technology is highly applicable to different cherry tomato varieties.

[0053] In summary, this invention achieves 100% germination rate, 0% browning mortality, and a 20-day induction period by using hypocotyls and radicles (after removing cotyledons and shoot apical meristems) as explants, combined with an induction medium containing 0.05% activated carbon but without plant growth regulators. The synergistic effect of the explant type and the composition of the induction medium in this technical solution effectively addresses the technical problems of severe browning, low differentiation rate, long cycle, and inability to continuously propagate in traditional tomato tissue culture, significantly improving the efficiency of tomato tissue culture.

[0054] The scope of protection of this invention is defined by the appended claims. Any equivalent substitutions or improvements made based on the technical solution and inventive concept of this invention shall fall within the scope of protection of this invention.

Claims

1. A method for improving tissue culture efficiency, characterized in that, Includes the following steps: S1. Preparation of sterile tomato seedlings: After disinfecting the tomato seeds, inoculate them into a germination carrier and culture them under culture conditions until the cotyledons are fully expanded or the first true leaf grows to obtain sterile seedlings. S2. Explant preparation: Take the sterile seedling, remove the cotyledons and shoot tip growth point, and retain the remaining hypocotyl and radicle as explants. Place the explants horizontally on the induction medium, so that the cut surfaces at both ends of the explants are in contact with the medium. S3. Induction Culture: The inoculated explants are placed in an induction medium without any plant growth regulators and cultured to differentiate into adventitious shoots; the induction medium includes MS basal medium, 0.4~0.6 g / L activated carbon, sucrose and agar, and no plant growth regulators are added; S4. Subculture: When the adventitious shoots grow to 1.5-2.5cm, they are cut off from the explants and transferred to a subculture medium for subculture. The subculture cycle is 18-22 days. S5. Rooting culture: Select tissue culture seedlings with a height of 3-5cm and 3-4 leaves that are intact, not curled, not wrinkled, and not vitrified. Transfer them to a rooting culture medium to induce rooting and obtain regenerated plants with roots and stems and leaves.

2. The method according to claim 1, characterized in that, In step S1, the age of the sterile seedlings is 7-10 days, calculated from the date of seed germination.

3. The method according to claim 1, characterized in that, The seed disinfection treatment described in step S1 is carried out in the following steps: first, rinse the seeds with sterile water, then soak them in a 70%-80% ethanol solution for 1-3 minutes, rinse them with sterile water 2-4 times, then soak them in a sodium hypochlorite solution with an 8%-12% mass fraction for 10-20 minutes, and finally rinse them with sterile water 4-6 times.

4. The method according to claim 1, characterized in that, The germination carrier mentioned in step S1 is a sterile petri dish lined with filter paper and containing sterile water, or MS medium containing 0.1-0.3 mg / L naphthaleneacetic acid and 0.8-1.2 mg / L 6-benzyladenine.

5. The method according to claim 1, characterized in that, The explants described in step S2 do not require secondary cutting; the entire hypocotyl after removing the cotyledons can be used for inoculation.

6. The method according to claim 1, characterized in that, In step S3, the induction culture time is 20-30 days, and the differentiated adventitious buds have 2-3 functional leaves that are not damaged, curled, wrinkled, or vitrified.

7. The method according to claim 1, characterized in that, The subculture medium described in step S4 includes MS basal medium, 1.0–2.0 mg / L 6-benzyladenine, 0.01–0.5 mg / L naphthaleneacetic acid, sucrose, and agar.

8. The method according to claim 1, characterized in that, The rooting medium described in step S5 includes MS basal medium, 0.1-0.3 mg / L naphthaleneacetic acid, sucrose, and agar.

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