An induction medium and its application in obtaining endangered plant gynostemma pentaphyllum plants

Abstract

The present application relates to a kind of induction medium and its application in obtaining endangered plant Gynostemma pentaphyllum plant, the induction medium, with MS medium as base medium, each component is according to the composition of final concentration and includes: 6-benzylaminoadenine, 1~1.5mg / L;Gibberellin, 1.25~10mg / L;1-naphthalene acetic acid, 0.005~0.01mg / L, the aforementioned component is dissolved in the MS medium.The induction medium not only realizes the induction of embryonic callus, but also subculture can induce a large number of differentiated seedlings while proliferating embryonic callus, compared with prior art first through callus induction medium induction callus, after several subcultures, then using differentiation medium induction bud differentiation culture mode, not only simplifies experimental procedure, but also greatly shortens the period of Gynostemma pentaphyllum tissue rapid propagation system establishment.

Description

Technical Field

[0001] This invention relates to the field of plant propagation technology, specifically to an induction culture medium and its application in obtaining plants of the endangered plant *Gymnocladus chinensis*. Background Technology

[0002] Gymnocarpos przewalskii Maxim. is a perennial semi-shrub belonging to the genus Gymnocarpos in the family Caryophyllaceae. It is a relatively rare relict plant in the desert regions of Central Asia and one of the important tree species constituting the vegetation of stony deserts. It has the characteristics of drought resistance, salt and alkali tolerance, barrenness tolerance, wind erosion and sand burial resistance, deep root system and long life. It plays a positive role in preventing desertification and protecting and maintaining the balance of desert ecosystems.

[0003] Due to the harsh natural environment in the distribution area of ​​*Gymnocladus chinensis*, natural reproduction is difficult, and in recent years, human activities have caused the population of *Gymnocladus chinensis* to decline day by day.

[0004] Natural regeneration and artificial propagation of *Gymnocladus chinensis* are difficult in the wild, with a survival rate of only 20%–30% for cuttings. Regarding tissue culture research, Wang Zhibo successfully obtained regenerated plants in 2004 using hypocotyls of *Gymnocladus chinensis* through callus induction. The key technique involved placing plump seeds in a petri dish lined with moist filter paper and culturing them at 26–28 °C for 5 days. The hypocotyls were then excised, sterilized, and used to establish a regeneration system through callus induction. However, because *Gymnocladus chinensis* is mainly distributed in the arid northwest region, the annual flowering season (late May to early June) is particularly dry, leading to decreased pollen viability, pollen abortion, and even flower drop. Therefore, obtaining plump seeds is very difficult, limiting the work of establishing a regeneration system using hypocotyls. In October 2021, Yang Haifeng et al. applied for an invention patent entitled "A Method for Establishing a Tissue Culture Regeneration System for the Organogenesis Pathway of Gymnocarpus macrocarpus" (application number CN202111208051X). The main technical point of this patent is to use Gymnocarpus macrocarpus stem segments collected from the wild as explants, and obtain regenerated plants through explant disinfection, primary callus induction culture, adventitious bud induction culture, adventitious bud subculture, and rooting culture. However, because the explants are collected from the wild, incomplete disinfection can easily cause contamination, and excessive disinfection time can easily kill the tissue. Therefore, the technical requirements are very high, and the success rate is not high. In addition, as a shrub, Gymnocarpus macrocarpus has a high degree of lignification of its branches and trunks. Coupled with the arid environment and the withering of branches in autumn and winter, it is very difficult to induce callus tissue in the stem segments. Therefore, the source of materials is severely limited by climate and season. Summary of the Invention

[0005] In view of this, and in response to the problems of high difficulty in inducing callus formation in *Gymnocladus chinensis* using existing artificial methods and the numerous difficulties in artificially propagating *Gymnocladus chinensis* seedlings, this invention provides an induction culture medium and its application in obtaining *Gymnocladus chinensis* plants, an endangered plant.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a special induction medium for callus tissue of *Gymnocladus chinensis*, with MS (Murashige and Skoog) medium as the base medium. The components, according to the final concentration, include: 6-benzyladenine (6-BA), 1-1.5 mg / L; gibberellin (GA3), 1.25-10 mg / L; and 1-naphthaleneacetic acid (NAA), 0.005-0.01 mg / L. All of the aforementioned components are dissolved in the MS medium.

[0007] Furthermore, the induction culture medium also contains: 28-32 g / L granulated sugar; 4.2-5.4 g / L agar; and a pH of 5.0-6.5. Preferably, the components in the induction medium, according to their final concentrations, are: 6-benzyladenine (6-BA), 1.5 mg / L; gibberellin (GA3), 7.5 mg / L; 1-naphthaleneacetic acid (NAA), 0.005 mg / L; granulated sugar, 30 g / L; agar, 4.8 g / L; and pH adjusted to 5.8–6.0. ​​All of the aforementioned components are dissolved in the MS medium.

[0008] Furthermore, the induction culture medium is prepared by mixing all components into a solution; preferably, the pH is further adjusted to 5.8-6.0; and then sterilized.

[0009] Secondly, the present invention provides a method for inducing callus tissue of naked-fruit wood embryotype, comprising the following steps: (1) Using healthy test-tube seedling leaves as explant material, the leaf tips were removed and the leaves were horizontally inoculated into the above induction medium; (2) After inoculation, the culture dish was cultured for 25 to 30 days at a temperature of 23±2℃, light intensity of 16h / d and light intensity of 2000~2500lx.

[0010] Furthermore, the method for preparing the explant material includes: (1-1) Collect branches of naked-fruited wood, cut them into sections of 2.5-5.0 cm, rinse them with running water for 1.5-2 hours, and then disinfect them; (1-2) Cut tender stem tips of 1-2 cm in length from sterilized branches of *Gnaphalium affine* and inoculate them into solid MS medium. Culture them for 25-30 days at a temperature of 23±2℃, a light intensity of 16h / d and a light intensity of 2000-2500lx to obtain sterile test-tube seedlings with 5-6 stem nodes. Use the leaves of the sterile test-tube seedlings as explant materials. Alternatively, the sterile test-tube seedlings obtained in step (1-2) can be further propagated, and the leaves of the propagated test-tube seedlings can be used as explant materials.

[0011] Preferably, in step (1-1), branches of the naked-fruited tree are collected in spring or summer.

[0012] Preferably, in step (1-1), the disinfection process involves sequentially soaking in 75% alcohol for 30 seconds, disinfecting with a 0.1% HgCl2 solution for 5-6 minutes, and then rinsing with sterile water 5-6 times.

[0013] Preferably, in step (1-2), the illumination uses LED pure white light as the light source.

[0014] Preferably, the method for propagating the sterile test-tube seedlings includes: 1) Using the sterile test-tube seedlings as the base seedlings, the seedlings were cut into single stem segments with one axillary bud under sterile conditions, inoculated into solid MS medium, and further cultured for 25 to 30 days at a temperature of 23±2℃, a light intensity of 16h / d, and a light intensity of 2000 to 2500 lx, to obtain 5 to 6 times more sterile test-tube seedlings than the number of sterile test-tube seedlings described above. 2) Using the sterile test-tube seedlings obtained in step 1) as the base seedlings, obtain 5 to 6 times more sterile test-tube seedlings than those obtained in step 1) under the same culture conditions. Repeat this process to continuously obtain propagated test-tube seedlings that can be used as explant materials.

[0015] Preferably, in step 1), the illumination uses LED pure white light as the light source.

[0016] Preferably, in step (2), the illumination uses LED pure white light as the light source.

[0017] Thirdly, the present invention provides a method for the rapid proliferation and differentiation of embryogenic callus into naked-fruited tree seedlings, comprising: Step 1: Obtain callus tissue using the above-mentioned method for inducing callus tissue in *Gymnocarpus nagi*. Step 2: Transfer the obtained callus tissue to the above-mentioned induction medium for subculture until seedlings differentiate.

[0018] Furthermore, the subculture conditions in step 2 are: temperature 23±2℃, light intensity 16h / d, light intensity 2000~2500lx, and culture time 35~40 days.

[0019] Preferably, the illumination for the subculture is provided by using LED pure white light as the light source.

[0020] Fourthly, the present invention provides a method for obtaining a complete regenerated plant of *Gymnocladus chinensis*, comprising: Naked-fruit tree seedlings were obtained by using the above-mentioned method of rapid proliferation and differentiation of embryogenic callus tissue; The naked-fruited tree seedlings were cut from the callus tissue and transferred to the rooting medium to induce rooting; After rooting, transplant into vermiculite, and a complete regenerated plant will form in 15–20 days.

[0021] Furthermore, the rooting medium uses MS (Murashige and Skoog) medium as the basic medium, and the components according to the final concentration include: 0.001 mg / L of 1-naphthaleneacetic acid (NAA), 30 g / L of white sugar, and 4.8 g / L of agar.

[0022] Furthermore, the method for preparing the rooting medium includes: mixing the ingredients according to the composition of the rooting medium, adjusting the pH to 5.8-6.0, dispensing it into culture bottles, and then autoclaving at 121°C for 20 minutes to obtain the final product.

[0023] Furthermore, the controlled conditions for inducing root growth are as follows: temperature 23±2℃, blue-white LED beads (preferably color temperature 30000K) and purple-red LED beads (preferably color temperature 1600K) in a 2:1 ratio as light source, light exposure 16h / d, light intensity 2000~2500lx, and culture for 20~25d.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses leaves from sterile test-tube seedlings of the endangered plant *Gymnocladus chinensis* as explants. During the vigorous growth period of *Gymnocladus chinensis* in spring or summer, young branches are collected from the wild, disinfected, and then used to establish sterile test-tube seedlings. These sterile test-tube seedlings are then used as the base seedlings to propagate the seedlings asexually indefinitely, thus continuously obtaining explants. The leaves from these test-tube seedlings are then used as explants to establish a regeneration system. Compared with existing technologies that ① use hypocotyls cultured from plump seeds or ② use stem segments collected from the wild as explants to establish a regeneration system, this invention not only facilitates explant propagation and provides convenient material sourcing with a low contamination rate, but also avoids the disadvantages of explant sourcing being limited by the natural environment and season.

[0025] 2. This invention utilizes plant tissue culture methods and employs a special induction medium for callus tissue of *Gymnocarpus nagi*. This not only achieves the induction of embryogenic callus tissue, but also induces a large number of differentiated seedlings during subculture while rapidly proliferating embryogenic callus tissue. Compared with the existing technology that first induces callus tissue using a callus induction medium, and then induces bud differentiation using a differentiation medium after several subcultures, this invention simplifies the experimental process and greatly shortens the cycle of establishing a rapid propagation system for *Gymnocarpus nagi* tissue. Attached Figure Description

[0026] Figure 1 The figure shows the induction of callus tissue on eight different culture media after 26 days of induction in Example 2 of this invention. The numbers on each culture dish in the figure correspond to the culture medium numbers.

[0027] Figure 2 The images show the embryogenic callus and initially differentiated seedlings after 15 days of subculture on fresh culture medium No. 5 in Example 3 of this invention.

[0028] Figure 3 The seedlings differentiated after 40 days of subculture on fresh culture medium No. 5 in Example 3 of this invention.

[0029] Figure 4 This shows the root induction of the differentiated plantlets in Example 4 of the present invention after 15 days of growth on the rooting medium.

[0030] Figure 5 The above are regenerated plants of *Gymnocladus chinensis* obtained in Example 4 of this invention. Figures A, B, and C show plants 3 days, 20 days, and 30 days after the rooted seedlings were transplanted into vermiculite, respectively. Detailed Implementation

[0031] The lateral branches of *Gymnocladus chinensis* used in this embodiment of the invention were collected from the Anxi Extremely Arid Desert National Nature Reserve in Gansu Province.

[0032] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1

[0034] This embodiment provides a special induction medium for callus tissue of *Gymnocladus orientalis*, which uses MS (Murashige and Skoog) medium as the basal medium and adds other components at the following final concentrations: 6-benzyladenine (6-BA), 1.5 mg / L; gibberellin (GA3), 7.5 mg / L; 1-naphthaleneacetic acid (NAA), 0.005 mg / L; granulated sugar, 30 g / L; and agar, 4.8 g / L. The induction medium is prepared by mixing all the aforementioned components thoroughly, adjusting the pH to 5.8–6.0 with sodium hydroxide solution, dispensing into Erlenmeyer flasks, and sterilizing at 121°C and 0.1 MPa for 20 min.

[0035] This induction medium is a special induction medium for the regeneration of naked-fruited wood. It not only achieves the induction of embryogenic callus, but is also suitable for the rapid proliferation of embryogenic callus and the induction of a large number of differentiated seedlings in subculture. Example 2

[0036] This embodiment provides a method for inducing callus embryotype in naked-fruit wood, using different induction culture media as shown in Table 1. The specific induction method is as follows: (1) Using healthy test-tube seedling leaves as explant materials, the leaf tips were removed and the leaves were horizontally inoculated into 8 kinds of induction culture media (the composition and formulation of the culture media are shown in Table 1). The method for preparing the explant material includes: a) The side branches of *Gymnocladus orientalis* collected from the Gansu Anxi Extremely Arid Desert National Nature Reserve were placed in self-sealing bags, placed in an insulated box with ice packs, and brought back to the laboratory. They were cut into 2.5-5cm sections, rinsed with running water for 1.5-2 hours, soaked in 75% alcohol for 30 seconds on a clean bench, then disinfected with 0.1% HgCl2 for 5-6 minutes, and finally rinsed with sterile water 5-6 times before use. b) Cut tender stem tips of 1-2 cm in length from sterilized naked-fruited wood branches, inoculate them into solid MS medium, and culture them for 30 days at a temperature of 23±2℃, using LED pure white light as the light source, with a light intensity of 2000-2500 lx for 16 h / d to obtain sterile test-tube seedlings with 5-6 stem nodes; c) Using the sterile test-tube seedlings obtained in b) as the base seedlings, they were cut into single-segment stem segments with one axillary bud under sterile conditions, and further cultured for 30 days at a temperature of 23±2℃, a light intensity of 16h / d, and a light intensity of 2000~2500lx to obtain 5~6 times more test-tube seedlings than in b). d) Using the sterile test-tube seedlings obtained in c) as the base seedlings, under the same culture conditions and cycle as in c), obtain 5 to 6 times more test-tube seedlings than in c). Repeat this process to propagate the seedlings continuously, thereby obtaining a continuous supply of sterile test-tube seedlings and explant materials.

[0037] (2) After inoculation, the culture dish was placed on the culture rack and LED pure white light was used as the light source. The culture was carried out at a temperature of 23±2℃, light intensity of 16h / d and light intensity of 2000~2500lx. The induction and growth of callus on 8 different culture media were observed and recorded every day. The callus induction rate and embryonic callus induction rate were statistically analyzed, and the best culture medium for callus induction was screened.

[0038] Callus induction rate (%) = Total number of callus tissues / Total number of explants inoculated × 100%.

[0039] Embryotype callus induction rate (%) = number of embryotype callus / total number of inoculated tissues × 100%.

[0040] Table 1. Composition and ratio of callus induction culture medium 1 MS 1.50 10.0 0.005 30 4.8 2 MS 1.50 2.50 0.01 30 4.8 3 MS 1.50 1.25 0.01 30 4.8 4 MS 1.50 0.00 0.01 30 4.8 5 MS 1.50 7.50 0.005 30 4.8 6 MS 1.00 7.50 0.01 30 4.8 7 MS 0.00 7.50 0.01 30 4.8 8 MS 1.50 7.50 0.00 30 4.8 The experimental results are as follows: Observations revealed that after 5 days of culture, leaves began to swell. At 15 days, callus tissue grew from the ends of some leaves on media 1, 2, 3, 4, 5, 6, 7, and 8, with induction rates of 46.15%, 47.62%, 43.75%, 71.56%, 87.50%, 25.00%, 66.67%, and 21.74%, respectively (Table 2). Media 5 showed the highest callus induction rate. On media 6, although some leaves showed callus formation at one end, the surface had white hair-like structures. On media 8, while some leaves showed callus formation at one end, most leaves turned brown and died. With prolonged callus induction time, the growth status of callus tissue in different media showed significant differences at 26 days. Figure 1The callus clumps on medium 1 were small and mostly firm. On media 2, 3, and 7, the callus clumps were slightly larger than those on medium 1, some were yellowish-green and firm, while others were loose and covered with white flocculent material. On media 4 and 5, the callus clumps were larger, mostly yellowish-green, firm, and growing well, with some clumps showing differentiation into small plantlets. On media 8, the callus clumps were soft and covered with white flocculent material. Statistical results showed that the embryogenic callus induction rates on media 1–8 varied greatly after 30 days of culture, being 23.08%, 19.05%, 25.00%, 59.44%, 70.83%, 0%, 46.67%, and 0 respectively (Table 2). Media 5 showed the highest embryogenic callus induction rate, significantly higher than the other media; therefore, media 5 was the optimal medium for callus induction.

[0041] Table 2. Callus induction on different culture media 1 232 46.15±1.03d 23.08±0.73d 2 239 47.62±0.98d 19.05±0.62e 3 234 43.75±1.52d 25.00±0.78d 4 240 71.56±0.89b 59.44±0.23b 5 219 87.50±1.03a 70.83±0.89a 6 228 25.00±0.67e 0 7 241 66.67±1.01c 46.67±0.59c 8 226 21.74±0.96e 0 Note: Lowercase letters in the same column of the table indicate significant differences between treatments (P<0.5). Example 3

[0042] This embodiment provides a method for the rapid proliferation and differentiation of embryogenic callus into naked-fruited tree seedlings, including: Step 1: Induction culture medium of Example 1 and induction method of Example 2 were used to induce callus tissue of naked fruit wood for 25-30 days to obtain callus tissue. Step 2: The embryogenic callus obtained in Step 1 was transferred to fresh induction medium from Example 1 for subculture. The culture conditions were: temperature 23±2℃, LED pure white light as the light source, light intensity 16h / d, and light intensity 2000~2500lx. After 7 days of culture, the embryogenic callus began to proliferate rapidly, and after 15~20 days of culture, it began to differentiate into plantlets. Figure 2 (This refers to callus tissue cultured for 15 days), which then proliferated and differentiated simultaneously. After 40 days of culture, each explant differentiated into an average of 30-40 seedlings. Figure 3 (For the differentiated seedlings cultured for 40 days). Example 4

[0043] This embodiment provides a method for obtaining a complete regenerated plant of *Gymnocladus chinensis*, including: On a clean bench, differentiated seedlings that had been subcultured for 40 days and reached a length of 3–3.5 cm in Example 3 were cut from the callus tissue and transferred to rooting medium to induce rooting. The rooting medium was MS (Murashige and Skoog) medium as the basal medium, and the components, according to the final concentration, included: 0.001 mg / L 1-naphthaleneacetic acid (NAA), 30 g / L granulated sugar, and 4.8 g / L agar. The rooting induction conditions were: temperature 23±2℃, LED light source: blue-white LED beads (color temperature 30000K) and purple-red LED beads (color temperature 1600K) in a 2:1 ratio, light exposure 16 h / d, light intensity 2000–2500 lx, and root formation occurred after 10–15 days of culture. Figure 4 The roots induced in the differentiated seedlings after 15 days of culture on rooting medium were transplanted to vermiculite after 20 days of culture, when the roots reached 0.5–1.0 cm in length. The seedlings were then thoroughly watered and covered with white plastic. The plastic was removed 7 days after transplanting, and new roots and shoots emerged after 15 days, forming a complete regenerated plant. Figure 5 A, B, and C are plants transplanted 3, 20, and 30 days after transplanting, respectively, with a survival rate of over 80%.

[0044] In summary, this invention only requires collecting tender branches from the wild during the vigorous growth season of the endangered plant *Gymnocladus chinensis* in spring and summer. After disinfection, sterile test-tube seedlings are established. These sterile test-tube seedlings are then used as the base seedlings to continuously propagate asexually. Finally, the leaves of these seedlings are used as explants to establish a regeneration system. Compared with existing technologies that use ① hypocotyls cultured from plump seeds or ② stem segments collected from the wild as explants to establish a regeneration system, this invention not only facilitates explant propagation and provides convenient material sourcing with a low contamination rate, but also avoids the disadvantages of explant sourcing being limited by the natural environment and season. Furthermore, this invention utilizes plant tissue culture methods and employs a special induction medium for callus tissue of *Gymnocarpus nagi*. This not only achieves the induction of embryogenic callus tissue, but also induces a large number of differentiated seedlings during subculture while rapidly proliferating embryogenic callus tissue. Compared with existing technologies that first induce callus tissue using a callus induction medium, and then induce bud differentiation using a differentiation medium after several subcultures, this invention simplifies the experimental process and significantly shortens the cycle for establishing a rapid propagation system of *Gymnocarpus nagi* tissue.

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for rapid proliferation and differentiation of embryogenic callus into naked-fruited tree seedlings, characterized in that: include: Step 1: Callus tissue was obtained using the method of inducing callus tissue of naked-fruit wood embryotype; The method for inducing callus from naked-fruit wood embryotype includes the following steps: (1) Using healthy test-tube seedling leaves as explant material, the leaf tips are removed and the leaves are horizontally inoculated into the induction medium, wherein the induction medium is MS medium as the base medium, and the components according to the final concentration include: 6-benzylaminopurine, 1-1.5 mg / L; gibberellin, 1.25-10 mg / L; 1-naphthaleneacetic acid, 0.005-0.01 mg / L, and the aforementioned components are all dissolved in the MS medium; (2) After inoculation, the culture dishes were cultured for 25–30 days at a temperature of 23±2℃, a light intensity of 16h / d, and a light intensity of 2000–2500lx. Step 2: Transfer the obtained callus tissue to the induction medium for subculture until seedlings differentiate.

2. The method according to claim 1, characterized in that: The induction medium also contains: 28-32 g / L granulated sugar; 4.2-5.4 g / L agar; and pH 5.0-6.

5.

3. The method according to claim 1, characterized in that: The components of the induction medium, according to their final concentrations, are as follows: 6-benzylaminopurine, 1.5 mg / L; gibberellin, 7.5 mg / L; 1-naphthaleneacetic acid, 0.005 mg / L; granulated sugar, 30 g / L; agar, 4.8 g / L; pH adjusted to 5.8–6.0; all the aforementioned components are dissolved in the MS medium.

4. The method according to claim 1 or 2, characterized in that: The induction medium is prepared by mixing all the components into a solution.

5. The method according to claim 4, characterized in that: The method for preparing the explant material includes: (1-1) Collect branches of naked-fruited wood; cut them into sections of 2.5-5.0 cm, rinse them with running water for 1.5-2 hours, and then disinfect them; (1-2) Cut tender stem tips of 1-2 cm in length from sterilized branches of *Gnaphalium affine* and inoculate them into solid MS medium. Culture them for 25-30 days at a temperature of 23±2℃, a light intensity of 16h / d and a light intensity of 2000-2500lx to obtain sterile test-tube seedlings with 5-6 stem nodes. Use the leaves of the sterile test-tube seedlings as explant materials. Alternatively, the sterile test-tube seedlings obtained in step (1-2) can be further propagated, and the leaves of the propagated test-tube seedlings can be used as explant materials.

6. The method according to claim 5, characterized in that: The method for propagating the sterile test-tube seedlings includes: 1) Using the sterile test-tube seedlings as the base seedlings, the seedlings were cut into single stem segments with one axillary bud under sterile conditions, inoculated into solid MS medium, and further cultured for 25 to 30 days at a temperature of 23±2℃, a light intensity of 16h / d, and a light intensity of 2000 to 2500 lx, to obtain 5 to 6 times more sterile test-tube seedlings than the number of sterile test-tube seedlings described above. 2) Using the sterile test-tube seedlings obtained in step 1) as the base seedlings, obtain 5 to 6 times more sterile test-tube seedlings than those obtained in step 1) under the same culture conditions. Repeat this process to continuously obtain propagated test-tube seedlings as explant materials.

7. The method according to claim 1, characterized in that: The subculture conditions in step 2 are: temperature 23±2℃, light intensity 16h / d, light intensity 2000~2500lx, and culture for 35~40 days.

8. A method for obtaining a complete regenerated plant of *Gymnocladus chinensis*, characterized in that: include: The method for rapid proliferation and differentiation of naked-fruited tree seedlings using embryogenic callus tissue as described in claim 1 or 7 is used to obtain naked-fruited tree seedlings. The naked-fruited tree seedlings were cut from the callus tissue and transferred to the rooting medium to induce rooting; After rooting, transplant into vermiculite, and a complete regenerated plant will form in 15–20 days.

9. A method for obtaining a complete regenerated plant of *Gymnocladus chinensis* according to claim 8, characterized in that: The rooting medium uses MS medium as the basic medium, and the components, according to the final concentration, include: 0.001 mg / L 1-naphthaleneacetic acid, 30 g / L granulated sugar, and 4.8 g / L agar.

10. A method for obtaining a complete regenerated plant of *Gymnocladus chinensis* according to claim 8, characterized in that: The controlled conditions for induced rooting were: temperature 23±2℃, LED light source (blue and white LED beads), light intensity 2000~2500lx, and culture time 20~25d.

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