Populus euphratica callus induction medium and a method for constructing a regeneration system
By constructing a callus induction culture medium and regeneration system using Populus euphratica cotyledons as explants, the problem of rooting difficulties in Populus euphratica was solved, the regeneration rate was improved, and a basis for the preservation and genetic transformation of stress-resistant gene resources was provided.
Patent Information
- Application Number
- CN202410632473.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-21
- Publication Date
- 2026-08-25
- Estimated Expiration
- 2044-05-21
AI Technical Summary
The lack of existing technologies for constructing regeneration systems using Populus euphratica cotyledons as explants leads to difficulties in rooting Populus euphratica, short seed lifespan, and low survival rate of cuttings, making it difficult to effectively preserve superior germplasm resources and discover stress-resistant genes.
Using cotyledons obtained from the germination of Populus euphratica seeds as explants, a callus induction medium was constructed by screening suitable culture medium ratios, including a combination of 1/2 MS medium, 6-BA and NAA. Adventitious shoots were formed through dark culture and differentiation culture, and finally rooted and propagated in rooting medium to establish a complete regeneration system.
This study achieved efficient regeneration using Populus euphratica cotyledons as explants, reduced the risk of microorganisms carried by the explants, improved callus induction rate and adventitious bud rooting rate, and provided a basis for the discovery and genetic transformation of stress-resistant genes.
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Figure CN118370210B_ABST
Abstract
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 for Populus euphratica. Background Technology
[0002] Populus euphratica Oliv. belongs to the Populus section of the Salicaceae family. It is a precious forest resource unique to desert regions, with characteristics such as drought resistance, cold resistance and salt tolerance, and is a resource pool of stress-resistant genes.
[0003] However, Populus euphratica faces challenges in rooting, has short seed lifespan, and low cutting survival rates, making asexual propagation a better option. Several forest tree species have already established asexual propagation systems. For example, Populus yunnanensis, a species in the Salicaceae family, has established a complete in vitro culture system using readily available young stems. Redwoods in North America are primarily propagated through cuttings, and tissue culture research is also extensive, with efforts underway to establish a more comprehensive asexual propagation system. Populus tomentosa utilizes plant tissue culture for rapid propagation, and its in vitro rapid propagation technology has been widely applied in afforestation and seedling production. Populus 84K has established a new system for tissue culture and rapid propagation through experimental research. Currently, one approach involves using Populus 84K and Populus tomentosa as materials, constructing regeneration systems by considering explant type, culture medium selection, and hormone selection, producing a large number of complete plants, enabling large-scale, year-round seedling production. Another approach primarily utilizes explants such as shoot tips and stem segments for establishing regeneration systems. Based on the totipotency of plant cells, theoretically, any organ or tissue can be used as an explant. However, in practical applications, explants of different varieties, organs, and physiological states have vastly different differentiation capabilities. In order to improve the success rate of in vitro culture, the selection of materials should take into account many factors, such as the quality of the variety, the location and size of the material, the physiological state and developmental stage, and whether there is any bacterial infection.
[0004] Generally, reports on Populus euphratica tissue culture primarily use explant materials such as shoot tips, leaves, axillary buds, dormant winter buds, stem segments, inflorescence axes, and anthers. However, there are currently few reports on regeneration techniques using Populus euphratica cotyledons as explants. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention proposes a method for constructing a callus induction culture medium and regeneration system for Populus euphratica. Using cotyledons obtained from Populus euphratica seed germination as explants, and by adjusting the ratio of plant hormones and screening suitable culture media, the goal of constructing a regeneration system using Populus euphratica cotyledons as explants is achieved. This provides a preliminary foundation for preserving superior germplasm resources of Populus euphratica, identifying stress-resistance genes, and establishing a Populus euphratica genetic transformation system.
[0006] To achieve the above objectives, the present invention provides a Populus euphratica callus induction culture medium, which is prepared from the following raw materials: 1L of 1 / 2MS medium, 0.1-1mg of 6-BA, and 0.5-2mg of NAA.
[0007] Preferably, the callus induction culture medium is used for induction culture of Populus euphratica cotyledons as explants.
[0008] 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.
[0009] The present invention also provides a method for constructing a regeneration system using the callus induction culture medium, comprising the following steps:
[0010] (1) Populus euphratica seeds were disinfected by rinsing with sterile water 3 to 5 times and then germinating in the 1 / 2MS medium for 6 to 8 days to obtain sterile seedlings.
[0011] (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.
[0012] (3) The callus obtained in step (2) was cultured in callus differentiation medium for 30-50 days to obtain adventitious shoots;
[0013] (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.
[0014] (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 poplar seedlings.
[0015] 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.
[0016] 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 16 hours of light and 8 hours of darkness per day.
[0017] 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 16h light and 8h 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 0.02mg NAA.
[0018] 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 1000lx, and the light cycle of the rooting induction culture is 16h light and 8h dark per day.
[0019] Preferably, in step (5), the number of axillary buds on the stem segment is 1, and the length of the stem segment is 1-2 cm; 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 16 hours of light and 8 hours of darkness per day.
[0020] Preferably, the rooting medium formulation in steps (4) and (5) is: 1L of 1 / 2MS medium and 0.1mg of IBA.
[0021] Compared with the prior art, the present invention has the following advantages and technical effects:
[0022] This invention uses cotyledons obtained from the germination of Populus euphratica seeds as explants. By adjusting the ratio of plant hormones and screening suitable culture media, the goal of constructing a regeneration system using Populus euphratica cotyledons as explants was achieved. This provides a preliminary foundation for the preservation of superior Populus euphratica germplasm resources, the discovery of stress-resistance genes, and the establishment of a Populus euphratica genetic transformation system. Compared with regeneration systems constructed using leaves or other materials as explants, the cotyledons of sterile seedlings used in this invention reduce the microorganisms carried by the explants from the source. Furthermore, cotyledon materials have a higher callus induction rate due to their lower physiological age and lower content of secondary metabolites. Attached Figure Description
[0023] 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.
[0024] Figure 1 The diagram shows the morphology of cotyledons formed by the germination of Populus euphratica seeds. In the diagram, A represents seeds that have been cultured for 0 days, and B represents cotyledons formed after 10 days of germination.
[0025] Figure 2 Different callus morphologies were induced from Populus euphratica cotyledons, where A was a white, loose callus, B was a light green, loose callus, and C was a light yellow, loose callus.
[0026] Figure 3 A diagram showing the morphology of adventitious buds formed by callus differentiation in Populus euphratica.
[0027] Figure 4 These are morphological images of Populus euphratica rooting induction culture. A shows the root morphology of a stem segment with axillary buds cultured on rooting medium for 10 days; B shows the root morphology of a stem segment with axillary buds cultured on rooting medium for 20 days; C shows the root morphology of a stem segment with axillary buds cultured on rooting medium for 40 days; D shows the seedling morphology of a stem segment with axillary buds cultured on rooting medium for 10 days; E shows the seedling morphology of a stem segment with axillary buds cultured on rooting medium for 20 days; and F shows the seedling morphology of a stem segment with axillary buds cultured on rooting medium for 40 days.
[0028] Figure 5 This is a diagram showing the morphology of a young poplar seedling. Detailed Implementation
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] The poplar seeds used in this invention were collected from the poplar forests of Shaya County, Aksu Prefecture, Xinjiang Uygur Autonomous Region, in August and September 2021. After being dried, they were stored in a seed storage cabinet at -4℃ for later use.
[0036] Example 1
[0037] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.
[0038] Callus induction medium: 1L of 1 / 2MS medium, 0.1mg 6-BA and 2mg NAA.
[0039] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.02mg NAA.
[0040] Rooting medium: 1 L of 1 / 2 MS medium and 0.1 mg IBA.
[0041] (1) Populus euphratica seeds 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);
[0042] (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 for 30 days under conditions of 30°C, 1000 lx light intensity, 16 h of light, and 25°C, 8 h of darkness, to obtain callus tissue (e.g., ...). Figure 2 (as shown);
[0043] (3) Take tender green callus tissue and place it in callus differentiation medium. Under conditions of 30℃ and 1000 lx light intensity, perform 16 hours of light exposure daily, followed by 8 hours of darkness at 25℃, for 40 days to obtain adventitious shoots (such as...). Figure 3 (as shown);
[0044] (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.
[0045] (5) Regenerated aseptic seedlings: Stem segments with one axillary bud, 1.5 cm in length, were cut and 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 for 16 hours of light followed by 8 hours of darkness at 25℃ for 15 days, yielding Populus euphratica seedlings (e.g., ...). Figure 4 and Figure 5 (As shown).
[0046] Example 2
[0047] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.
[0048] Callus induction medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 1mg NAA.
[0049] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.02mg NAA.
[0050] Rooting medium: 1 L of 1 / 2 MS medium and 0.1 mg IBA.
[0051] (1) Populus euphratica seeds 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 at 30℃ and 1000 lx light intensity for 16 hours a day, and then in darkness at 25℃ for 8 hours a day for 6 days to obtain sterile seedlings.
[0052] (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 24℃ for 8 days. Then, the callus induction culture was carried out for 30 days at 30℃ and 1000 lx light intensity for 16 hours a day, and at 25℃ and 8 hours in the dark to obtain callus tissue.
[0053] (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 16h and the darkness is 8h at 25℃ for 30 days to carry out differentiation culture and obtain adventitious shoots.
[0054] (4) Select healthy adventitious buds and culture them in the rooting medium at 24℃ 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 20 days to induce rooting and obtain regenerated sterile seedlings.
[0055] (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 24℃, it was cultured in the dark for the third time for 4 days. Then, under the condition of 25℃ and light intensity of 1000 lx, it was illuminated for 16 hours a day and then in the dark for 8 hours at 25℃ for 10 days to obtain Populus euphratica seedlings.
[0056] Example 3
[0057] 1 / 2MS medium: MS 2.2g, MES 0.5g, sucrose 10g and Phytagel 4g, with 1L water as solvent, pH 5.7.
[0058] Callus induction medium: 1L of 1 / 2MS medium, 1mg 6-BA and 0.5mg NAA.
[0059] Callus differentiation medium: 1L of 1 / 2MS medium, 0.5mg 6-BA and 0.02mg NAA.
[0060] Rooting medium: 1 L of 1 / 2 MS medium and 0.1 mg IBA.
[0061] (1) Populus euphratica seeds were soaked in a 1% NaClO aqueous solution for 100 seconds for disinfection, rinsed with sterile water 5 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.
[0062] (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 26℃ for 6 days. Then, the callus induction culture was carried out for 30 days at 30℃ and 1000 lx light intensity for 16 hours a day, and at 25℃ and 8 hours in the dark to obtain callus tissue.
[0063] (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 16h and the darkness is 8h at 25℃ for 50 days to obtain adventitious shoots.
[0064] (4) Select healthy adventitious buds and culture them in the rooting medium at 26℃ for a second dark culture for 4 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.
[0065] (5) Regenerated sterile seedlings: Cut a 2cm stem segment with one axillary bud and inoculate it in the rooting medium. Under the condition of 26℃, perform a third dark culture for 4 days. Then, under the condition of 25℃ and light intensity of 1000lx, perform light for 16h and dark for 8h at 25℃ for 20 days to obtain Populus euphratica seedlings.
[0066] Experimental Example 1
[0067] 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.
[0068] Table 1. Effects of different concentrations of 6-BA and NAA on callus induction in Populus euphratica leaves.
[0069]
[0070] The callus growth state of Populus euphratica can be divided into three categories: I. White, loose; II. Light green, loose; III. Light yellow, loose (e.g.) Figure 2 (As shown in A, B, and C). Table 1 shows that the induction rates of callus induction media were 100% in all media containing 0.1 mg / L 6-BA + 0.5 mg / L NAA, 0.1 mg / L 6-BA + 2.0 mg / L NAA, 0.5 mg / L 6-BA + 0.5 mg / L NAA, 0.5 mg / L 6-BA + 1.0 mg / L NAA, and 1.0 mg / L 6-BA + 1.0 mg / L NAA. Most of the callus was type II. However, considering the size of the callus fragments, the callus growth was best on the 1 / 2 MS medium containing 0.1 mg / L 6-BA + 2.0 mg / L NAA used in Example 1.
[0071] Experiment Example 2
[0072] In Example 1, the concentrations of 6-BA and NAA in the callus differentiation medium in step (3) were adjusted, and the effects of different concentrations on the differentiation of Populus euphratica callus were determined.
[0073] Table 2. Effects of different concentrations of 6-BA and NAA on callus differentiation in Populus euphratica.
[0074]
[0075] As shown in Table 2, based on the results of callus adventitious shoot differentiation rate, browning ratio and vitrification rate, the callus differentiation culture medium used in Example 1 containing 0.5 mg / L 6-BA + 0.02 mg / L NAA is the optimal one.
[0076] Experimental Example 3
[0077] 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 Populus euphratica regenerated sterile seedlings was determined.
[0078] Table 3. Effects of different IBA concentrations on rooting of Populus euphratica regenerated aseptic seedlings.
[0079]
[0080] As shown in Table 3, when the IBA concentration in the rooting medium is 0.1 mg / L, the adventitious shoot rooting rate is the highest at 100%, and the growth status is good.
[0081] 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.
[0082] 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) Populus euphratica seeds were disinfected by rinsing with sterile water 3 to 5 times and then germinating in 1 / 2 MS medium 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 poplar seedlings. The callus induction medium mentioned in step (2) is prepared from the following raw materials: 1L of 1 / 2MS medium, 0.1mg of 6-BA, and 2mg of NAA; the callus induction medium is used for induction culture of Populus euphratica cotyledons as explants; the 1 / 2MS medium consists of 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 of the 1 / 2MS medium is 5.7; The 1 / 2MS medium in step (1) consists of 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 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 6-BA, and 0.02mg NAA; The rooting medium formula described in steps (4) and (5) is: 1L of 1 / 2MS medium and 0.1mg 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 16h light and 8h 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 16 hours of light and 8 hours of 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.
6. The method for constructing a regeneration system according to claim 1, characterized in that, 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 darkness per day.