Nitraria tangutorum tissue culture rapid propagation method

Abstract

The invention discloses a nitraria tangutorum tissue culture rapid propagation method. The method comprises the following steps: (1) carrying out sterile treatment on seeds; (2) germination: putting the seeds on an inoculated germination culture medium; (3) induction: cutting a hypocotyl by 1-2cm and putting the hypocotyl into an induction culture medium; (4) proliferation and differentiation: putting into a proliferation and differentiation culture medium; and (5) rooting: finally putting into a rooting culture medium. According to the tissue culture rapid propagation technology, a large number of nitraria tangutorum plants can be obtained within a short time, the breeding requirement is met, large-scale production can be achieved, and the breeding speed is increased. Through a tissue culture rapid propagation technology, dependence on wild nitraria tangutorum bobr resources can be reduced, and protection of wild plant resources is facilitated. The nitraria tangutorum tissue culture and rapid propagation technology has a wide application prospect in the aspects of promoting psammophyte mass sustainable development and protecting plant resources.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a rapid propagation method of Nitraria tangutorum tissue culture. Background Art

[0002] Nitraria sibiricum is a shrub of the genus Nitraria in the family Tribulus. It has many dense branches and grows in clusters. It has a strong ability to fix and block sand. Nitraria sibiricum is a xerophytic positive plant. It is not tolerant to waterlogging and waterlogging, but is drought-tolerant and salt-tolerant. Its branches are generally spread on the ground, and sand is accumulated into mounds. It is an excellent sand-fixing plant. Nitraria sibiricum is distributed in northern Shaanxi, western Inner Mongolia, and Hexi, Gansu, China. It often grows in deserts and semi-desert lake basin sandy land, river terraces, piedmont plain sandy land, and clay land with wind-blown sand.

[0003] Generally, three methods are used for seeding, softwood cuttings and hardwood cuttings. However, from the perspective of seeding, seeding is the best method, with a higher survival rate than the other two methods. At present, the propagation method of Nitraria sibiricum is single, the breeding efficiency is low, and the total number of seedlings is small. Therefore, it is urgent to adopt a new seedling propagation method to increase the propagation speed of Nitraria sibiricum to meet production needs. Summary of the invention

[0004] In order to solve the problem of Nitraria tangutica breeding, the present invention provides a method for rapid propagation of Nitraria tangutica tissue culture. The Nitraria tangutica tissue culture rapid propagation technology of the present invention can break through the traditional breeding methods, solve the problems of long breeding years, single breeding methods, and slow propagation, and can achieve rapid propagation of multiple generations in one year through tissue culture methods, thereby solving the problem of breeding shortage. To this end, the present invention adopts the following technical solutions: A method for rapid propagation of Nitraria tangutorum tissue culture of the present invention comprises the following steps: (1) Sterile treatment of Nitraria tangutorum seeds; (2) Germination culture: placing on an inoculated germination medium; the germination medium is MS medium supplemented with naphthaleneacetic acid NAA, gibberellic acid GA3 and activated carbon AC; (3) Induction culture: 1-2 cm hypocotyl cuts are placed in an induction medium; the induction medium is MS medium supplemented with auxin 2AD, 6-benzylpurine 6-BA, naphthaleneacetic acid NAA and activated carbon AC; (4) Proliferation and differentiation culture: placing in a proliferation and differentiation medium; the proliferation and differentiation medium is a modified B5 medium supplemented with 6-benzylpurine 6-BA, kinetin KT and activated carbon AC; (5) Rooting culture: Finally, place the rooting medium; the rooting medium is 1 / 2 MS medium with indolebutyric acid IBA and activated carbon AC added. Indolebutyric acid can promote root growth and form a root system.

[0005] For the above method, it is preferred that in step (1) the seed sterilization treatment is to soak the Nitraria tangutorum seeds in 75% (v / v) alcohol and 0.1 wt% mercuric chloride successively.

[0006] For the method described above, it is preferred that the germination medium composition of step (2) is: MS medium + NAA (0.1-0.5 mg / L) + GA3 (0.08-0.2 mg / L) + AC (0.5-1.5 mg / L), and a more preferred composition is: MS medium + NAA (0.2-0.3 mg / L) + GA3 (0.1-0.2 mg / L) + AC (0.8-1.2 mg / L), and the most preferred composition is: MS + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L.

[0007] For the method described above, it is preferred that the composition of the induction medium in step (3) is: MS medium + 2AD (0.5-1.2 mg / L) + 6-BA (0.1-0.5 mg / L) + NAA (0.1-0.5 mg / L) + AC (0.5-1.5 mg / L), and a more preferred composition is: MS medium + 2AD (0.8-1.2 mg / L) + 6-BA (0.1-0.3 mg / L) + NAA (0.1-0.3 mg / L) + AC (0.8-1.2 mg / L), and the most preferred composition is: MS + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L.

[0008] For the method described above, it is preferred that the proliferation and differentiation medium composition of step (4) is: modified B5 medium + 6-BA (0.1-0.3 mg / L) + KT (0.01-0.04 mg / L) + AC (0.5-1.5 mg / L) mg / L, a more preferred composition is: modified B5 medium + 6-BA (0.1-0.2 mg / L) + KT (0.01-0.02 mg / L) + AC (0.8-1.2 mg / L) mg / L, and the most preferred composition is: modified B5 + 6-BA 0.15 mg / L + KT 0.01 mg / L + AC 1 mg / L, wherein the modified B5 is prepared by adding 0.24-0.26 mg / L 2,4-D and 0.83-0.85 mg / L KT to the B5 medium, preferably adding 0.24 mg / L 2,4-D and 0.833 mg / L KT.

[0009] In the above method, it is preferred that the rooting medium in step (5) is composed of: 1 / 2 MS medium + IBA (0.2-0.8 mg / L) + AC (0.1-0.5 mg / L), a more preferred composition is: 1 / 2 MS medium + IBA (0.4-0.6 mg / L) + AC (0.1-0.3 mg / L), the most preferred composition is: 1 / 2 MS + IBA 0.5 mg / + AC0.2 mg / L.

[0010] Preferably, in the above method, the culture conditions of step (2) germination culture, step (3) induction culture, step (4) proliferation and differentiation culture, and step (5) rooting culture can all be: 23-26°C, 6-8 hours / day, and 1000-4000 lux. Generally, for common plants, the illumination time is preferably more than 12 hours, but the inventors have found that the illumination time of the present invention is too long and is not conducive to the tissue culture growth of Nitraria tangutorum, and the illumination intensity can be preferably 3000 lux.

[0011] Preferably, in the above-mentioned method, the time for germination culture in step (2) and induction culture in step (3) is preferably 12-20 days, and optimally 15-16 days, while the time for proliferation and differentiation culture in step (4) is preferably 50-80 days, and optimally 60-65 days, and the time for rooting culture in step (5) is preferably 25-40 days, and optimally 30-35 days.

[0012] As a more preferred technical solution, the Nitraria tangutorum tissue culture rapid propagation method of the present invention comprises the following steps: (1) Seed sterilization treatment: Soak the seeds of Nitraria tangutorum in 75% (v / v) alcohol for 10-30 seconds, then rinse with sterile water, then soak in 0.1 wt % mercuric chloride for 0.5-2 minutes, and then rinse with sterile water; (2) Germination culture: Place on germination medium for 15-16 days, light exposure for 6-8 hours at 23-26℃, light intensity 1000-4000 lux; germination medium composition: MS medium + NAA 0.3mg / L + GA3 0.1mg / L + AC 1mg / L; (3) Induction culture: Cut the hypocotyl into 1-2 cm pieces and place them in the induction medium for 15-16 days, at 23-26℃ for 6-8 hours, with a light intensity of 1000-4000 lux; the composition of the induction medium is: MS+2AD 1mg / L+6BA 0.2mg / L+NAA0.2mg / L+AC 1mg / L; (4) Proliferation and differentiation culture: Culture in proliferation and differentiation medium for 60-65 days, at 23-26°C with light for 6-8 hours, and light intensity of 1000-4000 lux; the composition of proliferation and differentiation medium is: modified B5+6-BA 0.15mg / L+KT 0.01mg / L+AC1mg / L; (5) Rooting culture: Finally, place the seeds in rooting medium and culture for 30-35 days at 23-26℃ with light exposure for 6-8 hours and light intensity of 1000-4000 lux; the composition of rooting medium is: 1 / 2 MS + IBA 0.5mg / + AC 0.2mg / L.

[0013] The beneficial effects of the present invention are: 1. The tissue culture rapid propagation technology of the present invention is not limited by time and space. From a single Nitraria tangutorum seed to germination and callus induction, the callus can continuously differentiate and then take root, so that a large number of Nitraria tangutorum sand-fixing plants can be obtained in a relatively short period of time (about 3-4 months), and multiple generations of reproduction can be completed in one year, thus meeting breeding needs.

[0014] 2. Technical prospects: It can achieve large-scale production and speed up breeding.

[0015] 3. Protect wild resources: Through tissue culture rapid propagation technology, we can reduce dependence on wild white thorn resources and help protect wild plant resources.

[0016] 4. In general, the tissue culture rapid propagation technology of Nitraria tangutorum has broad application prospects in promoting the large-scale sustainable development of desert plants and protecting plant resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are pictures of proliferation, differentiation and rooting conditions. A is a photo of a large number of proliferation and differentiation, and B is a photo of a large number of rooting. DETAILED DESCRIPTION

[0018] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0019] In the embodiment of the present invention, NAA is naphthylacetic acid, 6-BA is 6-benzylpurine, GA3 is gibberellic acid, AC is activated carbon, KT is kinetin, 2AD is auxin, IBA is indolebutyric acid, and 2,4-D is 2,4-dichlorophenoxyacetic acid, all of which are commercially available commodities. The improved B5 medium is improved on the basis of the B5 medium (without sucrose and agar), wherein the addition amount of 2,4-D is 0.24 mg / L and the addition amount of KT is 0.833 mg / L. Example 1

[0020] A method for rapid propagation of Nitraria tangutorum tissue culture, comprising the following steps: (1) Seed sterilization treatment: The seeds of Nitraria tangutorum collected from Alxa League, Inner Mongolia (Inner Mongolia Alxa Ecosystem Research Center) were first soaked in 75% (v / v) alcohol for 20 seconds, then rinsed once with sterile water, then soaked in 0.1wt% mercuric chloride solution for 1 minute, and then rinsed three times with sterile water; (2) Germination culture: Place the seeds treated in step (1) on a germination medium for inoculation and expose to light at 24°C for 6 hours / day with a light intensity of 3000 lux. The composition of the germination medium is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. Inoculation should be carried out in a fume hood to ensure sterility. After the seeds are placed in the germination medium for about 7 days, small white buds can be seen growing on the top of the seeds. The selection of the germination medium allows the white thorn seeds to germinate well. After 15-16 days of cultivation, the seed buds grow to about 2-3 cm. The germination of the white thorn seeds further proves that the seeds are active, so that subsequent induction can be carried out to form callus.

[0021] (3) Induction culture: When the buds are 2-3 cm long, cut the hypocotyls by 1-2 cm and place them in the induction culture medium. Expose them at 24°C for 6 hours a day with a light intensity of 3000 lux. The composition of the induction culture medium is: MS culture medium 10mg / L+2AD 1mg / L+6-BA0.2mg / L+NAA 0.2mg / L+AC 1mg / L. After the seeds germinate, cut the hypocotyls by 1-2 cm and place them in the induction culture medium. In the induction culture medium, the hypocotyls will grow callus tissue in small pieces. After growing for about half a month (15-16 days), they can be proliferated and differentiated.

[0022] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium, and expose them to light for 6 hours / day at 24°C with a light intensity of 3000 lux. The composition of the proliferation and differentiation medium is: modified B5 medium 10mg / L + 6-BA 0.15mg / L + KT0.01mg / L + AC 1mg / L. In the proliferation and differentiation medium, callus tissue will proliferate and differentiate in large quantities. The calli need to be separated and placed in different culture media. After about one month of culture, leaves can be observed growing on the callus. At this time, fresh culture media needs to be replaced to provide nutrients for its growth in time. After about 60 days of culture, a large amount of callus tissue will proliferate and differentiate. Figure 1 A.

[0023] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 6 hours / day at 24℃ with a light intensity of 3000 lux. The composition of the rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After being placed in the rooting medium, roots will grow in about 20 days. Figure 1B. After the roots become strong (about 30 days), the plant grows into a complete plant and can be preserved permanently or hardened. Example 2

[0024] A method for rapid propagation of Nitraria tangutorum tissue culture, comprising the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: Place the seeds treated in step (1) on a germination medium for inoculation and expose to light at 23°C for 8 hours / day with a light intensity of 3000 lux. The composition of the germination medium is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. Inoculation should be carried out in a fume hood to ensure sterility. The selection of the germination medium allows the seeds of Nitraria tangutorum to germinate well. The seeds germinate after 15 days of cultivation. The germination of the seeds further proves that the seeds are active, so that subsequent induction can be carried out to form callus.

[0025] (3) Induction culture: When the buds are 2-3 cm, cut the hypocotyl into 1-2 cm pieces and place them in the induction culture medium. Expose the cells to 8 hours of light per day at 23°C with a light intensity of 3000 lux. The composition of the induction culture medium is: MS medium 10 mg / L + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L. Callus tissue will grow after 15 days of induction culture.

[0026] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium and illuminate them at 23°C for 8 hours per day with a light intensity of 3000 lux. The proliferation and differentiation medium consists of: modified B5 medium 10 mg / L + 6-BA 0.15 mg / L + KT 0.01 mg / L + AC 1 mg / L. The proliferation and differentiation culture lasts for about 60 days, and a large amount of callus tissue undergoes proliferation and differentiation.

[0027] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 8 hours / day at 23℃ with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After about 30 days of rooting culture, the plants will take root in the medium and become complete plants. Example 3

[0028] A method for rapid propagation of Nitraria tangutorum tissue culture, comprising the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: The seeds treated in step (1) were placed on a germination medium for inoculation and illuminated at 25°C for 7 hours / day with a light intensity of 3000 lux. The germination medium composition is: MS medium 10 mg / L + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L. After 18 days of germination culture, the seeds germinated, and subsequent induction to form callus was performed.

[0029] (3) Induction culture: When the buds are 2-3 cm, cut the hypocotyl into 1-2 cm pieces and place them in the induction culture medium. Expose them at 25°C for 7 hours per day with a light intensity of 3000 lux. The composition of the induction culture medium is: MS medium 10 mg / L + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L. Callus tissue will grow after 18 days of induction culture.

[0030] (4) Proliferation and differentiation culture: Place the induced plants in a proliferation and differentiation medium and illuminate them at 25°C for 7 hours per day with a light intensity of 3000 lux. The proliferation and differentiation medium consists of: modified B5 medium 10 mg / L + 6-BA 0.15 mg / L + KT 0.01 mg / L + AC 1 mg / L. The proliferation and differentiation culture lasts for about 65 days, and a large amount of callus tissue undergoes proliferation and differentiation.

[0031] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 7 hours / day at 25℃ with a light intensity of 3000 lux. The composition of the rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After about 30 days of rooting culture, the plants will take root in the medium and become complete plants.

[0032] Comparative Example 1 A method for culturing Nitraria tangutorum tissue comprises the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: the same as in Example 1; (3) Induction culture: the same as in Example 1; (4) Proliferation and differentiation culture: Proliferation and differentiation culture is based on unmodified B5 medium, and the specific composition is: B5 medium 10mg / L + 6-BA 0.15mg / L + KT 0.01mg / L + AC 1mg / L. The callus tissue proliferates and differentiates a small amount. After one month of culture, no leaves are seen growing from the callus tissue. After about 50 days of culture, a small amount of callus leaves appear, and rooting culture is carried out.

[0033] (5) Rooting culture: Finally, place in rooting medium, and light for 6 hours / day at 24℃, with a light intensity of 3000 lux. The composition of rooting medium is: 1 / 2 MS medium 5mg / L + IBA 0.5mg / L + AC 0.2mg / L. After being placed in the rooting medium, a very small number of tiny roots will grow in about 35 days, and then the tiny roots will grow slowly after 10-15 days of cultivation.

[0034] Comparative Example 2 A method for culturing Nitraria tangutorum tissue comprises the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: the same as in Example 1; (3) Induction culture: the same as in Example 1; (4) Proliferation and differentiation culture: The proliferation and differentiation culture uses MS medium as the basic medium. The specific composition is: MS medium 10 mg / L + 6-BA 0.15 mg / L + KT 0.01 mg / L + AC 1 mg / L. The proliferation and differentiation of callus tissue was not obvious. No leaves were seen growing in the callus tissue after two months of culture.

[0035] (5) Rooting culture: Finally, place the plants in the rooting medium and light them for 6 hours / day at 24°C with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5 mg / L + IBA 0.5 mg / L + AC 0.2 mg / L. No rooting was observed after about 30 days of culture.

[0036] By comparing Example 1, Comparative Example 1 and Comparative Example 2, the improved B5 medium is more suitable for the proliferation and differentiation growth of the Nitraria tangutorum callus of the present application than the unmodified B5 medium and the commonly used MS medium, so that it can quickly grow callus, which is then conducive to its rooting and growth.

[0037] Comparative Example 3 A method for culturing Nitraria tangutorum tissue comprises the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: the same as in Example 1; (3) Induction culture: the same as in Example 1; (4) Proliferation and differentiation culture: Proliferation and differentiation culture is based on low-concentration modified B5 medium, with the specific composition of modified B5 medium 6mg / L + 6-BA 0.15mg / L + KT 0.01mg / L + AC 1mg / L. No callus growth leaves were observed after 50 days of culture, and a small amount of callus leaves appeared after about three months of culture, and subsequent rooting culture was carried out.

[0038] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 6 hours per day at 24°C with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5 mg / L + IBA 0.5 mg / L + AC 0.2 mg / L. A small amount of roots can be seen after about 60 days of culture.

[0039] Comparative Example 4 A method for culturing Nitraria tangutorum tissue comprises the following steps: (1) Seed sterilization treatment: the same as in Example 1; (2) Germination culture: the same as in Example 1; (3) Induction culture: the same as in Example 1; (4) Proliferation and differentiation culture: Proliferation and differentiation culture is based on high-concentration modified B5 medium, and its specific composition is: modified B5 medium 15mg / L + 6-BA 0.15mg / L + KT 0.01mg / L + AC 1mg / L. After 20 days of culture, incomplete bean sprout-like (deformed) leaves can be seen in the callus tissue. After about 50 days of culture, a large number of incomplete and deformed bean sprout-like leaves can be seen in the callus tissue. At the same time, the callus tissue becomes obviously thicker and is accompanied by a small number of nodular blocks, and rooting culture is carried out.

[0040] (5) Rooting culture: Finally, place the plants in the rooting medium and expose them to light for 6 hours per day at 24°C with a light intensity of 3000 lux. The rooting medium consists of 1 / 2 MS medium 5 mg / L + IBA 0.5 mg / L + AC 0.2 mg / L. After about 20 days of culture, a large number of tuberous roots can be seen.

[0041] By comparing Example 1, Comparative Example 3 and Comparative Example 4, low-concentration modified B5 culture medium may lead to slower cell division, reduced biomass accumulation, affecting cell growth, delaying or inhibiting cell differentiation, and resulting in incomplete tissue or organ formation; while high-concentration modified B5 culture medium may accelerate cell division, increase biomass accumulation, induce excessive growth, lead to the formation of cell clumps (nodules), inhibit normal differentiation, lead to abnormal structure or incomplete development, interfere with normal morphogenesis, lead to deformed or overgrown tissues, and affect the healthy growth of plant cells.

[0042] Comparative Example 5 According to the method of Example 1, during the proliferation and differentiation culture in step (4), the B5 medium was subjected to an improvement test of adding 2,4-D and KT, and the rest was the same as in Example 1. The results are shown in Table 1: Table 1 Experiment on optimization and improvement of B5 medium

[0043] As shown in Table 1, when the B5 medium was improved and 0.24 mg / L 2,4-D and 0.833 mg / L KT were used as the basic medium for proliferation and differentiation, the callus was able to proliferate in large quantities and the white thorn was able to grow normal leaves after about one month of culture.

[0044] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A method for rapid propagation of Nitraria tangutorum tissue culture, characterized in that: The steps include: (1) Sterile treatment of Nitraria tangutorum seeds; (2) Germination culture: placing on an inoculated germination medium; the germination medium is MS medium supplemented with 0.1-0.5 mg / L NAA, 0.08-0.2 mg / L GA3 and 0.5-1.5 mg / L AC; (3) Induction culture: 1-2 cm hypocotyl cuts were placed in induction medium; the induction medium is MS medium supplemented with 0.5-1.2 mg / L 2AD, 0.1-0.5 mg / L 6-BA, 0.1-0.5 mg / L NAA and 0.5-1.5 mg / L AC; (4) Proliferation and differentiation culture: placing in a proliferation and differentiation medium; the proliferation and differentiation medium is a modified B5 medium supplemented with 0.1-0.3 mg / L 6-BA, 0.01-0.04 mg / L KT and 0.5-1.5 mg / L AC; the modified B5 medium is a B5 medium supplemented with 0.24-0.26 mg / L 2,4-D and 0.83-0.85 mg / L KT; (5) Rooting culture: Finally, place in rooting medium; the rooting medium is 1 / 2MS medium supplemented with 0.2-0.8 mg / L IBA and 0.1-0.5 mg / L AC; In the above steps, NAA is naphthylacetic acid, GA3 is gibberellic acid, AC is activated carbon, 2AD is auxin, 6-BA is 6-benzylpurine, KT is kinetin, and IBA is indolebutyric acid.

2. The method according to claim 1, characterized in that The seed sterilization treatment in step (1) is to soak the Nitraria tangutorum seeds in 75% alcohol and 0.1 wt % mercuric chloride successively.

3. The method according to claim 1, characterized in that The germination medium composition of step (2) is: MS medium + NAA 0.3 mg / L + GA3 0.1 mg / L + AC 1 mg / L.

4. The method according to claim 1, characterized in that The induction medium composition of step (3) is: MS medium + 2AD 1 mg / L + 6-BA 0.2 mg / L + NAA 0.2 mg / L + AC 1 mg / L.

5. The method according to claim 1, characterized in that The proliferation and differentiation culture medium in step (4) is composed of: modified B5 culture medium + 6-BA 0.15 mg / L + KT 0.01 mg / L + AC 1 mg / L.

6. The method according to claim 1, characterized in that The rooting medium composition of step (5) is: 1 / 2 MS medium + IBA 0.5 mg / L + AC 0.2 mg / L.

7. The method according to claim 1, characterized in that The culture conditions of step (2), step (3), step (4) and step (5) are: 23-26° C., illumination for 6-8 hours / day, and illumination intensity of 1000-4000 lux.

8. The method according to claim 1, characterized in that The culture time of step (2) and step (3) is 12-20 days.

9. The method according to claim 1, characterized in that The culture time of step (4) is 50-80 days.

10. The method according to claim 1, characterized in that The culture time of step (5) is 25-40 days.

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