Populus canescens callus induction medium and a method for constructing a regeneration system

Abstract

This invention discloses a callus induction culture medium and a method for constructing a regeneration system for *Populus euphratica*, belonging to the field of plant tissue culture technology. The callus induction culture medium for *Populus euphratica* is prepared from the following raw materials: 1L of 1 / 2 MS medium, 0.5-1mg 6-BA, and 1-2mg NAA. The method for constructing a regeneration system using the callus induction culture medium includes the following steps: *Populus euphratica* seeds are disinfected and germinated in 1 / 2 MS medium to obtain sterile seedlings; cotyledons of the sterile seedlings are placed in the callus induction culture medium for callus induction culture, resulting in callus differentiation culture to obtain adventitious buds; rooting induction culture is then used to obtain regenerated sterile seedlings; stem segments with axillary buds are cut and rooted for propagation culture to obtain *Populus euphratica* seedlings; these seedlings are transplanted, covered with film, and cultured in the dark; the film is then removed, and the seedlings are allowed to grow naturally. This invention accelerates the propagation of superior germplasm resources of *Populus euphratica* and provides a preliminary foundation for the genetic transformation system and germplasm innovation of *Populus euphratica*.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and in particular relates to a method for constructing a callus induction culture medium and regeneration system of Populus euphratica. Background Technology

[0002] The grey poplar (Populus pruinosa Schrenk), also known as the grey poplar, belongs to the Populus euphratica section of the Salicaceae family. It is a precious forest resource unique to desert regions. It performs well in terms of high temperature resistance, wind resistance, and corrosion resistance, and is therefore a suitable afforestation tree species in Northwest China.

[0003] Plant tissue culture is a technique that involves inoculating isolated plant organs, tissues, or cells under sterile conditions into a culture medium containing appropriate nutrients and plant hormones, and then cultivating them under suitable conditions to obtain regenerated, complete plants. This technique can effectively preserve the original genetic characteristics of plants, making it an important tool for the propagation and preservation of superior plant germplasm resources. Research on plant tissue culture in my country began in the 1950s. To date, complete and stable plant tissue culture technology systems have been established for various crops and horticultural plants.

[0004] Among the Populus genus, species such as the silver poplar, European aspen, hairy-fruited poplar, American black poplar, and the white poplar hybrids 84K poplar (P. alba × P. glandulosa) and 717 hybrid poplar (P. tremula × P. alba) have already developed mature regeneration systems. Based on these systems, genetic transformation systems have been established and can be used for germplasm improvement and innovation.

[0005] However, within the Populus euphratica school, only reports on Populus euphratica regeneration systems have been found so far, while reports on Populus pulvinata regeneration systems are extremely rare. This hinders the preservation of superior germplasm resources of Populus pulvinata and also affects the establishment of Populus pulvinata genetic transformation systems. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for constructing a callus induction culture medium and regeneration system for Populus euphratica, which accelerates the propagation of superior germplasm resources of Populus euphratica and provides a preliminary foundation for the genetic transformation system and germplasm innovation of Populus euphratica.

[0007] To achieve the above objectives, the present invention provides a callus induction culture medium for Populus euphratica, which is prepared from the following raw materials: 1L of 1 / 2MS medium, 0.5-1mg of 6-BA, and 1-2mg of NAA.

[0008] Preferably, the callus induction culture medium is used for induction culture of cotyledons of Populus euphratica as explants.

[0009] Preferably, the 1 / 2MS medium comprises the following components at the following concentrations: MS 2.2 g / L, MES 0.5 g / L, sucrose 10 g / L and plant gel 4 g / L, with water as the solvent; the pH of the 1 / 2MS medium is 5.7.

[0010] The present invention also provides a method for constructing a regeneration system using the callus induction culture medium, comprising the following steps:

[0011] (1) Disinfect the seeds of Populus euphratica, rinse them with sterile water 3 to 5 times, and use the 1 / 2MS medium to germinate the seeds for 6 to 8 days to obtain sterile seedlings;

[0012] (2) Take the cotyledons of the sterile seedlings obtained in step (1) as explants, inoculate them in the callus induction culture medium, and culture them in the dark for 6-8 days for the first time, and then culture them in the callus induction culture for 30 days to obtain callus.

[0013] (3) The callus obtained in step (2) was cultured in callus differentiation medium for 30-50 days to obtain adventitious shoots;

[0014] (4) The adventitious buds obtained in step (3) are cultured in the rooting medium for a second time in the dark for 4-6 days, and then induced to root for 20-30 days to obtain regenerated sterile seedlings.

[0015] (5) Take the regenerated sterile seedlings obtained in step (4), cut the stem segments with axillary buds and place them in the rooting medium. Culture them in the dark for the third time for 4-6 days, and then culture them for rooting and propagation for 10-20 days to obtain small seedlings of Populus euphratica.

[0016] (6) The gray poplar seedlings obtained in step (5) were transplanted, covered with film, and cultured in the dark for the fourth time for 7 days. The film was then removed, and the seedlings were allowed to grow naturally.

[0017] Preferably, the disinfection treatment in step (1) uses a 1% NaClO aqueous solution and is soaked for 80-100 seconds; the 1 / 2MS culture medium in step (1) includes the following components: MS 2.2 g / L, MES 0.5 g / L, sucrose 10 g / L and plant gel 4 g / L, with water as the solvent, and the pH value of the 1 / 2MS culture medium is 5.7; the germination culture temperature in step (1) is 25-30℃, the light intensity of the germination culture is 1000 lx, and the photoperiod of the germination culture is 16 hours of light and 8 hours of darkness per day.

[0018] Preferably, the temperature of the first dark culture in step (2) is 24-26℃; the temperature of the callus induction culture in step (2) is 25-30℃; the light intensity of the callus induction culture is 1000 lx; ​​and the light cycle of the callus induction culture is 12 hours of light and 12 hours of darkness per day.

[0019] Preferably, the callus tissue in step (3) is light green; the temperature of the differentiation culture in step (3) is 25-30℃, the light intensity of the differentiation culture is 1000lx, and the light cycle of the differentiation culture is 12h light and 12h dark per day; the formula of the callus differentiation culture medium in step (3) is: 1L of 1 / 2MS medium, 0.5mg 6-BA, and 0.1mg NAA.

[0020] Preferably, the temperature of the second dark culture in step (4) is 24-26℃; the temperature of the rooting induction culture in step (4) is 25℃, the light intensity of the rooting induction culture is 1000 lx, and the light cycle of the rooting induction culture is 16h light and 8h dark per day; the number of axillary buds on the stem segment in step (5) is 1, and the length of the stem segment is 1-2cm; the temperature of the third dark culture in step (5) is 24-26℃; the temperature of the rooting and propagation culture in step (5) is 25℃, the light intensity of the rooting and propagation culture is 1000 lx, and the light cycle of the rooting and propagation culture is 16h light and 8h dark per day.

[0021] Preferably, the rooting medium formulation in steps (4) and (5) is: 1L of 1 / 2MS medium and 0.5mg of IBA.

[0022] Preferably, the height of the gray poplar seedlings in step (6) is 1.5-2.5cm; the temperature of the fourth dark culture in step (6) is 25℃; the light intensity of natural growth in step (6) is 1000lx, and the light cycle of natural growth is 16h light and 8h darkness per day.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] This invention provides a method for constructing a cotyledon regeneration system of Populus euphratica and a suitable callus induction medium. The callus induction medium is based on 1 / 2 MS medium with the addition of 0.5 mg / L 6-BA and 2.0 mg / L NAA, which can achieve a callus induction success rate of 95% for cotyledons of aseptic seedlings. At the same time, a callus differentiation medium based on 1 / 2 MS medium with the addition of 0.5 mg / L 6-BA and 0.1 mg / L NAA is used in conjunction, which can achieve an adventitious shoot induction rate of 64.44%. Finally, a rooting medium with the formulation of 1 / 2 MS + 0.5 mg / L IBA can achieve a rooting rate of 100%. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 These are the cotyledons formed from the germination of seeds of Populus euphratica.

[0027] Figure 2 Different callus morphologies were induced in the cotyledons of Populus euphratica, where A was yellowish-green callus and B was light green callus.

[0028] Figure 3 The morphology of adventitious buds induced from callus tissue of Populus euphratica;

[0029] Figure 4 The images depict the morphology of roots induced from stem segments with axillary buds in Populus euphratica. A shows the root morphology of seedlings formed 10 days after the stem segment with axillary buds; B shows the root morphology of seedlings formed 20 days after the stem segment with axillary buds; C shows the root morphology of seedlings formed 40 days after the stem segment with axillary buds; D shows the morphology of seedlings formed 10 days after the stem segment with axillary buds; E shows the morphology of seedlings formed 20 days after the stem segment with axillary buds; and F shows the morphology of seedlings formed 40 days after the stem segment with axillary buds.

[0030] Figure 5 To promote the rooting and propagation of the gray poplar;

[0031] Figure 6 Transplanted from gray poplar. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] In this invention, 6-BA is 6-benzylaminopurine, purchased from Beijing Solarbio Science & Technology Co., Ltd.; NAA is naphthaleneacetic acid, purchased from Beijing Solarbio Science & Technology Co., Ltd.; IBA is indolebutyric acid, purchased from Beijing Solarbio Science & Technology Co., Ltd.; MS, MES and Phytagel were all purchased from Beijing Coolplay Technology Co., Ltd.

[0038] The seeds of Populus euphratica used in this invention were collected from Populus euphratica forests in Alar City, Aksu Prefecture, Xinjiang Uygur Autonomous Region in August and September 2021.

[0039] Example 1

[0040] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.

[0041] Callus induction medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 2mg NAA.

[0042] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.1mg NAA.

[0043] Rooting medium: 1 L of 1 / 2 MS medium and 0.5 mg IBA.

[0044] (1) Seeds of *Populus euphratica* were disinfected by soaking in a 1% NaClO aqueous solution for 90 seconds, rinsed four times with sterile water, and the surface moisture was absorbed. Seeds were then cultured on 1 / 2 MS medium under the following conditions: 16 hours of light at 30℃ and 1000 lx, followed by 8 hours of darkness at 25℃, for 7 days to obtain sterile seedlings (e.g., ...). Figure 1 (as shown);

[0045] (2) Under aseptic conditions, cotyledons of sterile seedlings were explanted and inoculated into callus induction medium. The culture was initially carried out in the dark for 7 days at 25°C. Then, callus induction culture was performed daily at 30°C and 1000 lx for 12 hours of light followed by 12 hours of darkness at 25°C for 30 days, yielding callus tissue (e.g., ...). Figure 2 (as shown);

[0046] (3) Take tender green callus tissue and place it in callus differentiation medium. Under conditions of 30℃ and 1000 lx light intensity, perform 12 hours of light exposure daily, followed by 12 hours of darkness at 25℃ daily for 40 days to obtain adventitious shoots (such as...). Figure 3 (as shown);

[0047] (4) Select healthy adventitious buds and culture them in the rooting medium at 25℃ for 5 days in the dark. Then, culture them in the dark at 25℃ for 16 hours a day and at 25℃ for 8 hours in the dark for 25 days to obtain regenerated sterile seedlings.

[0048] (5) Regenerated aseptic seedlings: Stem segments with one axillary bud, 1.5 cm in length, were inoculated upright into rooting medium. After a third dark culture at 25℃ for 5 days, rooting and propagation culture was carried out daily at 25℃ and 1000 lx light intensity for 16 hours, followed by 8 hours of darkness at 25℃ for 15 days, yielding small seedlings of *Populus euphratica* (e.g., ...). Figure 4 and Figure 5 (as shown);

[0049] (6) When the seedlings of *Populus euphratica* reach a height of 2cm, rinse the roots of the seedlings clean, transplant them into nutrient pots, water them thoroughly, cover them with plastic wrap, and incubate them in the dark for the fourth time for 7 days at a temperature of 25℃. Afterward, remove the film and allow them to grow naturally under a light intensity of 1000lx, with 16 hours of light and 8 hours of darkness per day (e.g., ...). Figure 6 (As shown).

[0050] Example 2

[0051] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.

[0052] Callus induction medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 1mg NAA.

[0053] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.1mg NAA.

[0054] Rooting medium: 1 L of 1 / 2 MS medium and 0.5 mg IBA.

[0055] (1) The seeds of Populus euphratica were soaked in a 1% NaClO aqueous solution for 80 seconds for disinfection, rinsed with sterile water 3 times, and the surface moisture of the seeds was dried. The seeds were cultured in 1 / 2 MS medium for 8 days under the conditions of 30℃ and 1000 lx light intensity, and 25℃ and 8 hours of darkness to obtain sterile seedlings.

[0056] (2) Under sterile conditions, the cotyledons of sterile seedlings were cut off as explants and inoculated into callus induction medium. The first dark culture was carried out at 25℃ for 8 days. Then, the callus induction culture was carried out for 30 days at 30℃ and 1000 lx light intensity for 12 hours a day, and at 25℃ and 12 hours in the dark to obtain callus tissue.

[0057] (3) Take tender green callus tissue and place it in callus differentiation medium. Under the conditions of 30℃ and 1000lx light intensity, the light is 12h for 12h and the darkness is 25℃ for 12h for 50 days to differentiate and culture, and adventitious shoots are obtained.

[0058] (4) Select healthy adventitious buds and culture them in the rooting medium at 25℃ for a second dark culture for 6 days. Then, culture them in the dark at 25℃ for 16 hours a day and at 25℃ for 8 hours a day for 30 days to induce rooting and obtain regenerated sterile seedlings.

[0059] (5) Regenerated sterile seedlings: Cut a stem segment with one axillary bud and a length of 1 cm and inoculate it in the rooting medium. Under the condition of 25℃, the third dark culture was carried out for 6 days. Then, under the condition of 25℃ and light intensity of 1000 lx, the light was 16 h per day and the darkness was 8 h per day at 25℃ for 15 days to carry out rooting and propagation culture to obtain gray poplar seedlings.

[0060] (6) When the seedlings of Populus euphratica are 2cm tall, rinse the roots of the seedlings clean, transplant them into nutrient pots, water them thoroughly, cover them with plastic wrap, and incubate them in the dark for the fourth time for 7 days at a temperature of 25℃. Then, remove the film and allow them to grow naturally under a light intensity of 1000lx, with 16 hours of light and 8 hours of darkness per day.

[0061] Example 3

[0062] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.

[0063] Callus induction medium: 1L of 1 / 2MS medium, 1mg 6-BA and 2mg NAA.

[0064] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.1mg NAA.

[0065] Rooting medium: 1 L of 1 / 2 MS medium and 0.5 mg IBA.

[0066] (1) The seeds of Populus euphratica were disinfected by soaking in a 1% NaClO aqueous solution for 100s, rinsed with sterile water 5 times, and the surface moisture of the seeds was dried. The seeds were cultured in 1 / 2 MS medium at 30℃ and 1000 lx light intensity for 16h per day, and then in darkness at 25℃ for 8h per day for 6 days to obtain sterile seedlings.

[0067] (2) Under sterile conditions, the cotyledons of sterile seedlings were cut off as explants and inoculated into callus induction medium. The first dark culture was carried out at 25℃ for 6 days. Then, the callus induction culture was carried out for 30 days at 30℃ and 1000 lx light intensity for 12 hours a day, and at 25℃ and 12 hours in the dark to obtain callus tissue.

[0068] (3) Take tender green callus tissue and place it in callus differentiation medium. Under the conditions of 30℃ and 1000 lx light intensity, the light is 12h for 30 days and the darkness is 12h for 25℃ to obtain adventitious shoots.

[0069] (4) Select healthy adventitious buds and culture them in the rooting medium at 25°C for a second dark culture for 4 days. Then, culture them in the dark at 25°C for 16 hours a day and at 25°C for 8 hours a day for 20 days to induce rooting and obtain regenerated sterile seedlings.

[0070] (5) Regenerated sterile seedlings: Cut a 2cm stem segment with one axillary bud and inoculate it in the rooting medium. Under the condition of 25℃, perform the third dark culture for 4 days. Then, under the condition of 25℃ and light intensity of 1000lx, perform the rooting and propagation culture for 10 days, and then perform the rooting and propagation culture for 8 hours at 25℃.

[0071] (6) When the seedlings of Populus euphratica are 2cm tall, rinse the roots of the seedlings clean, transplant them into nutrient pots, water them thoroughly, cover them with plastic wrap, and incubate them in the dark for the fourth time for 7 days at a temperature of 25℃. Then, remove the film and allow them to grow naturally under a light intensity of 1000lx, with 16 hours of light and 8 hours of darkness per day.

[0072] Experimental Example 1

[0073] In Example 1, the concentrations of 6-BA and NAA in the callus induction medium in step (2) were adjusted, and the effects of different concentrations on callus induction from Populus euphratica leaves were determined.

[0074] Table 1. Effects of different concentrations of 6-BA and NAA on callus induction in Populus tomentosa leaves.

[0075]

[0076]

[0077] As shown in Table 1, during the cotyledon callus induction process, by conducting experiments with different concentrations of 6-BA and NAA, it was found that when the callus induction medium described in Example 1 contained 0.5 mg / L 6-BA + 2.0 mg / L NAA, the cotyledon callus induction effect was the best at 95%, and the callus morphology was also better.

[0078] Experiment Example 2

[0079] In Example 1, the concentrations of 6-BA and NAA in the callus differentiation culture medium in step (3) were adjusted, and the effects of different concentrations on callus differentiation of Populus euphratica were determined.

[0080] Table 2. Effects of different concentrations of 6-BA and NAA on callus differentiation in Populus euphratica.

[0081]

[0082] As shown in Table 2, during the process of callus differentiation into adventitious shoots, the concentration of 6-BA and NAA was tested. It was found that when the callus differentiation medium described in Example 1 contained 0.5 mg / L 6-BA + 0.1 mg / L NAA, the callus differentiation rate was the highest at 64.44%, and the average number of shoots was 4.48.

[0083] Experimental Example 3

[0084] In Example 1, the concentration of IBA in the rooting medium in steps (4) and (5) was adjusted, and the effect of different concentrations on the rooting of regenerated aseptic seedlings of Populus euphratica was determined.

[0085] Table 3. Effects of different IBA concentrations on rooting of regenerated aseptic seedlings of Populus euphratica.

[0086] 1 — 0％ — 2 0.1 60 thin roots 3 0.5 100 The taproot is thick and strong, while the fibrous roots are fine and numerous. 4 2 75 Root formation from basal callus

[0087] As shown in Table 3, when the IBA concentration in the rooting medium is 0.5 mg / L, the adventitious shoot rooting rate is the highest at 100%.

[0088] In summary, the regeneration system construction method described in Example 1 improved the success rate of callus induction from Populus euphratica cotyledons, increased the callus differentiation rate, and improved the adventitious bud rooting rate.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a regeneration system using a callus induction culture medium, characterized in that, Includes the following steps: (1) Disinfect the seeds of Populus euphratica, rinse them with sterile water 3 to 5 times, and use 1 / 2MS medium for seed germination culture for 6 to 8 days to obtain sterile seedlings; (2) Take the cotyledons of the sterile seedlings obtained in step (1) as explants, inoculate them in callus induction culture medium, and culture them in the dark for 6-8 days for the first time, and then culture them in callus induction culture for 30 days to obtain callus tissue. (3) The callus obtained in step (2) is cultured in callus differentiation medium for 30-50 days to obtain adventitious shoots; (4) The adventitious buds obtained in step (3) are cultured in the rooting medium in the dark for a second time for 4-6 days, and then induced to root for 20-30 days to obtain regenerated sterile seedlings; (5) Take the regenerated sterile seedlings obtained in step (4), cut the stem segments with axillary buds and place them in the rooting medium. Culture them in the dark for the third time for 4-6 days, and then culture them for rooting and propagation for 10-20 days to obtain small seedlings of Populus euphratica. (6) The gray poplar seedlings obtained in step (5) were transplanted, covered with film, and cultured in the dark for the fourth time for 7 days. The film was then removed, and the seedlings were allowed to grow naturally. The callus induction medium mentioned in step (2) is prepared from the following raw materials: 1L of 1 / 2MS medium, 0.5~1mg of 6-BA, and 1~2mg of NAA; the callus induction medium is used for induction culture of Populus euphratica cotyledons as explants; the 1 / 2MS medium is prepared from the following concentration components: MS 2.2g / L, MES 0.5g / L, sucrose 10g / L and plant gel 4g / L, with water as the solvent; the pH value of the 1 / 2MS medium is 5.7; The 1 / 2MS medium in step (1) is prepared from the following components at the following concentrations: MS 2.2 g / L, MES 0.5 g / L, sucrose 10 g / L and plant gel 4 g / L, with water as the solvent, and the pH of the 1 / 2MS medium is 5.

7. The formula for the callus differentiation medium in step (3) is: 1L of 1 / 2MS medium, 0.5mg of 6-BA, and 0.1mg of NAA; The rooting medium formula described in steps (4) and (5) is: 1L of 1 / 2MS medium and 0.5mg of IBA.

2. The method for constructing a regeneration system according to claim 1, characterized in that, The disinfection treatment in step (1) uses a 1% NaClO aqueous solution and soaks for 80-100 seconds; the germination culture temperature in step (1) is 25-30℃, the light intensity of the germination culture is 1000 lx, and the light cycle of the germination culture is 16 hours of light and 8 hours of darkness per day.

3. The method for constructing a regeneration system according to claim 1, characterized in that, The temperature of the first dark culture in step (2) is 24~26℃; the temperature of the callus induction culture in step (2) is 25~30℃, the light intensity of the callus induction culture is 1000lx, and the light cycle of the callus induction culture is 12h light and 12h dark per day.

4. The method for constructing a regeneration system according to claim 1, characterized in that, The callus tissue described in step (3) is light green; the temperature of the differentiation culture in step (3) is 25~30℃, the light intensity of the differentiation culture is 1000lx, and the light cycle of the differentiation culture is 12h light and 12h darkness per day.

5. The method for constructing a regeneration system according to claim 1, characterized in that, The temperature of the second dark culture in step (4) is 24~26℃; the temperature of the rooting induction culture in step (4) is 25℃, the light intensity of the rooting induction culture is 1000lx, and the light cycle of the rooting induction culture is 16h light and 8h dark per day; the number of axillary buds on the stem segment in step (5) is 1, and the length of the stem segment is 1~2cm; the temperature of the third dark culture in step (5) is 24~26℃; the temperature of the rooting and propagation culture in step (5) is 25℃, the light intensity of the rooting and propagation culture is 1000lx, and the light cycle of the rooting and propagation culture is 16h light and 8h dark per day.

6. The method for constructing a regeneration system according to claim 1, characterized in that, The height of the gray poplar seedlings in step (6) is 1.5~2.5cm; the temperature of the fourth dark culture in step (6) is 25℃; the light intensity of natural growth in step (6) is 1000lx, and the light cycle of natural growth is 16h light and 8h darkness per day.

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