CN108719056B - Tissue culture seedling method of sea buckthorn - Google Patents
Tissue culture seedling method of sea buckthorn Download PDFInfo
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- CN108719056B CN108719056B CN201810392935.7A CN201810392935A CN108719056B CN 108719056 B CN108719056 B CN 108719056B CN 201810392935 A CN201810392935 A CN 201810392935A CN 108719056 B CN108719056 B CN 108719056B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention provides a tissue culture seedling method of sea buckthorn, which comprises the following steps: (1) selecting materials: taking tender stem tips as tissue culture seedling raising materials; (2) sterilizing the tender stem tips; (3) determining culture conditions; (4) induction of tender stem tips; (5) proliferation of tender stem tips; (6) rooting of the tender stem tip; (7) transplanting: inducing rooting in rooting culture medium for 40-60 days, transplanting, culturing in matrix, controlling humidity to over 95% in the first week, gradually prolonging ventilation and illumination time, and naturally ventilating completely for 14-16 days; meanwhile, the temperature is controlled well, the temperature is controlled to be 25 +/-1 ℃ in the daytime and 15-18 ℃ at night. The tissue culture seedling method of the sea-buckthorn can effectively improve the induction rate, the proliferation rate and the rooting rate of the tender stem tips of the sea-buckthorn in the tissue culture process, and the survival rate after transplanting is over 96 percent.
Description
Technical Field
The invention belongs to the technical field of plant regeneration through a tissue culture technology, and particularly relates to a tissue culture seedling method of sea buckthorn.
Background
Hippophae rhamnoides (Latin's name: Hippophe rhamnoides Linn.) is a perennial deciduous fruit tree, shrub or small tree. The fruit, pericarp, leaf, bark and their processed products contain more than 280 kinds of physiologically active substances, and have strong special effects of diminishing inflammation, sterilizing, relieving pain and promoting tissue regeneration. Many of the components show magical therapeutic effects in killing and inhibiting tumor cells, resisting radiation, resisting blood coagulation, lowering blood pressure, preventing vascular embolism, resisting aging and fatigue, enhancing body vitality and immunity and the like.
The sea-buckthorn has luxuriant branches and leaves, developed lateral roots and extremely strong root tillering property, can quickly bunch roots and self-tillering to form a dense colony, can be symbiotic with actinomycetes, bacteria, mycobacteria and the like to form a large number of root nodules, and has stronger nitrogen fixation capacity (180 kg of nitrogen can be fixed per hectare) than soybeans. In addition, the ecological fertilizer has the obvious functions of rapid growth, drought resistance, saline-alkali resistance and saline-alkali resistance, protection against severe cold and summer heat, maintenance of water and soil, wind prevention and sand fixation, soil improvement, strong adaptability and the like, and promotes ecological balance, and is not only a pioneer tree species for afforestation and greening in arid and windy sand areas, but also an important salary forest and feed forest tree species. Therefore, the planting of sea buckthorn has important nutritional value, ecological value and economic value in the current adjustment of the industrial structure of returning back to agriculture and forestry.
The characteristics of good varieties of sea-buckthorn are difficult to maintain in production by seed propagation of male and female sea-buckthorn plants; the root shoot propagation coefficient is low. The survival rate of the cutting propagation adopted in the prior production is not high, and the diseases are easy to spread. The industrial breeding of seedlings by using the tissue culture method can not only greatly improve the propagation coefficient, but also establish an in vitro asexual germplasm resource bank to replace a resource garden occupying a large land area.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a tissue culture seedling method of sea buckthorn.
The invention provides a tissue culture seedling method of sea buckthorn, which comprises the following steps:
(1) selecting materials: taking tender stem tips as tissue culture seedling raising materials;
(2) sterilizing the tender stem tips;
(3) the culture conditions are as follows: in the process of induction, proliferation and rooting of the tender stem tip, the culture temperature is 25 +/-1 ℃, the humidity is 50-70%, the illumination intensity is 2000-3000Lx, and the illumination time is 13-16 h/d;
(4) induction of tender stem tips: peeling the disinfected tender stem tip and basal leaves, and inoculating the tender stem tip and basal leaves in an induction culture medium for culture;
(5) proliferation of tender stem tips: inducing the tender stem tips in an induction culture medium for 23-27 days, and transferring the tender stem tips into a proliferation culture medium for proliferation culture;
(6) rooting of tender stem tips: cutting off the single plant of the clustered seedling obtained by the multiplication culture, and inoculating the cut plant into a rooting culture medium for induced rooting;
(7) transplanting: inducing rooting in rooting culture medium for 40-60 days, transplanting, culturing in matrix, controlling humidity to over 95% in the first week, gradually prolonging ventilation and illumination time, and naturally ventilating completely for 14-16 days; meanwhile, the temperature is controlled well, the temperature is controlled to be 25 +/-1 ℃ in the daytime and 15-18 ℃ at night.
Preferably, in the step (2), the specific method for sterilizing is as follows: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, flowing tap water for 8-12min, washing with distilled water for 3 times, placing on a sterilized 30min ultra-clean bench, and adding 0.1% HgCl2Sterilizing for 2-2.5min while shaking, and washing with sterilized distilled water for 6-7 times.
Preferably, in step (3), the culture temperature is 25 ℃, the humidity is 60%, the illumination intensity is 2500Lx, and the illumination time is 14 h/d.
Preferably, in step (4), the formulation of the induction medium is: 1/2B5+6-BA 0.2-0.4mg/L + sucrose 30g/L + agar 6 g/L.
Preferably, in step (4), the formulation of the induction medium is: 1/2B5+6-BA 0.3mg/L + sucrose 30g/L + agar 6 g/L.
Preferably, in step (5), the formula of the proliferation medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
Preferably, in the step (6), the formula of the rooting medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
Preferably, in the step (7), the matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
The tissue culture seedling method of the sea-buckthorn can effectively improve the induction rate, the proliferation rate and the rooting rate of the tender stem tips of the sea-buckthorn in the tissue culture process, and the survival rate after transplanting is over 96 percent.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
In the present invention, 1/2MS and 1/2B5 are common general knowledge known to those skilled in the art, which are mass halved MS medium and B5 medium. MS medium and B5 medium are likewise known to the person skilled in the art.
The tissue culture seedling method of the sea buckthorn comprises the following steps:
(1) test materials: the tender stem tips harvested in the field can be harvested in the whole growing season as long as the stem tips are not degenerated into thorns.
(2) The stem tip disinfection method comprises the following steps: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, flowing tap water for 8-12min, washing with distilled water for 3 times, placing on a sterilized 30min ultra-clean bench, and adding 0.1% HgCl2Sterilizing for 2-2.5min while shaking, and washing with sterilized distilled water for 6-7 times. Browning is very severe with alcohol, but with the disinfection method described above, the browning rate is within 1%.
(3) The culture conditions are as follows: in the processes of induction, proliferation and rooting of the stem tip, the culture temperature is 25 +/-1 ℃, the humidity is 50-70%, the illumination intensity is 2000-3000Lx, and the illumination time is 13-16 h/d.
(4) Induction of stem tips: placing the sterilized tender stem tip on sterilized filter paper, sucking water, cutting into 0.5-1.5 cm length, peeling off leaf blade at stem tip base, and inoculating into induction culture medium for culturing.
The formula of the induction culture medium is as follows: 1/2B5+6-BA 0.2-0.4mg/L + sucrose 30g/L + agar 6 g/L.
The applicant tested various varieties of sea buckthorn, such as male plants, QX-0601, QX-0104, QX-0102, QX-0105 and the like, and found that the induction rate of the induction medium of the present application is higher than that of the induction medium using 1/2MS as a basal medium, and is between 91% and 99%.
(5) Proliferation of stem tips: the tender stem tip is induced in an induction culture medium for 23-27 days and then transferred into a proliferation culture medium for proliferation culture, and the subculture is generally carried out once in 25-30 days.
The formula of the proliferation culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
The applicant tests a plurality of sea buckthorn varieties, such as male plants, QX-0601, QX-0103, Liaofu, QX-0102, QX-0105 and the like, and finds that the proliferation rates of the varieties are greatly improved to 87-98% and the growth vigor is good when compared with the proliferation culture medium of the application which uses 1/2MS culture medium or MS culture medium as a basic culture medium.
(6) Rooting of the stem tip: cutting off the single plant of the cluster seedling obtained by the propagation culture, and inoculating the cut plant into a rooting culture medium for inducing rooting.
The rooting medium comprises the following components: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
The applicant tests a plurality of varieties of sea buckthorn, such as male plants, QX-0601, QX-0103, Liaofun and the like, and finds that the rooting rate of the male plants and the QX-0601 is 90-95%, and the rooting rate of the QX-0103 and the Liaofun is 70-80%.
(7) Transplanting: inducing rooting in rooting culture medium for 40-60 days, transplanting the seedling until the root grows to 3.5-4.5cm, washing the seedling in bottle, transplanting to substrate, and culturing to obtain regenerated plant. The matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
The key point of the success of seedling transplanting is the control of the humidity in the first week, the humidity is controlled to be more than 95% in the first week after seedling transplanting, the ventilation and illumination time is gradually prolonged, and the curtain can be completely uncovered for about 15 days to carry out natural ventilation. Meanwhile, the temperature is controlled well, the temperature is controlled to be 25 +/-1 ℃ in the daytime and 15-18 ℃ at night.
After the test-tube plantlets are transplanted for 20 days, the survival rate can reach more than 96%.
Example 1
The tissue culture seedling method of the sea buckthorn comprises the following steps:
(1) test materials: the tender stem tips harvested in the field can be harvested in the whole growing season as long as the stem tips are not degenerated into thorns.
(2) The stem tip disinfection method comprises the following steps: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, flowing tap water for 10min, washing with distilled water for 3 times, placing on a sterilized 30min ultra-clean bench, and adding 0.1% HgCl2Sterilizing for 2min, shaking, and washing with sterilized distilled water for 6 times. The browning rate was 1%.
(3) The culture conditions are as follows: in the processes of induction, proliferation and rooting of the stem tip, the culture temperature is 25 ℃, the humidity is 60%, the illumination intensity is 2500Lx, and the illumination time is 14 h/d.
(4) Induction of stem tips: placing the sterilized tender stem tip on sterilized filter paper, sucking water, cutting into 1cm length, peeling the base leaf of the stem tip, and inoculating into induction culture medium for culture.
The formula of the induction culture medium is as follows: 1/2B5+6-BA 0.3mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the inductivity is 95 percent; when the sea-buckthorn variety is QX-0601, the inductivity is 97 percent; when the sea-buckthorn variety is QX-0104, the inductivity is 93%; when the sea-buckthorn variety is QX-0102, the inductivity is 96%; when the sea-buckthorn variety is QX-0105, the inductivity is 99%.
(5) Proliferation of stem tips: and (3) inducing the tender stem tips in an induction culture medium for 25 days, transferring the tender stem tips into a proliferation culture medium for proliferation culture, and performing subculture once in 28 days.
The formula of the proliferation culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the proliferation rate is 93 percent; when the sea-buckthorn variety is QX-0601, the proliferation rate is 97 percent; when the sea buckthorn variety is QX-0103, the proliferation rate is 96%; when the sea buckthorn variety is Liaofu, the proliferation rate is 92 percent; when the sea-buckthorn variety is QX-0102, the proliferation rate is 94 percent; when the sea-buckthorn variety is QX-0105, the proliferation rate is 98%.
(6) Rooting of the stem tip: cutting off the single plant of the cluster seedling obtained by the propagation culture, and inoculating the cut plant into a rooting culture medium for inducing rooting.
The rooting medium comprises the following components: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the rooting rate is 93%; when the sea-buckthorn variety is QX-0601, the rooting rate is 95 percent; when the sea-buckthorn variety is QX-0103, the rooting rate is 76%; when the sea buckthorn variety is Liaofu, the rooting rate is 80%.
(7) Transplanting: inducing in rooting culture medium for 50 days, transplanting the seedling with root of 3.5-4.5cm, washing the seedling, transplanting to substrate, and culturing to obtain regenerated plant. The matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
The key point of the success of seedling transplanting is the control of the humidity in the first week, the humidity is controlled to be more than 95% in the first week after seedling transplanting, the ventilation and illumination time is gradually prolonged, and the curtain can be completely uncovered after 15 days. Meanwhile, the temperature is controlled well, the temperature is controlled to be 25 ℃ in the daytime and 15-18 ℃ at night.
After the test-tube plantlets are transplanted for 20 days, the survival rate can reach 98 percent.
Example 2
The tissue culture seedling method of the sea buckthorn comprises the following steps:
(1) test materials: the tender stem tips harvested in the field can be harvested in the whole growing season as long as the stem tips are not degenerated into thorns.
(2) The stem tip disinfection method comprises the following steps: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, cleaning with running tap water for 12min, washing with distilled water for 3 times, placing on a sterilized 30min ultra-clean bench, and cleaning with 0.1% HgCl2Sterilizing for 2min, shaking, and washing with sterilized distilled water for 6 times. The browning rate was 1%.
(3) The culture conditions are as follows: in the processes of induction, proliferation and rooting of the stem tip, the culture temperature is 24 ℃, the humidity is 50%, the illumination intensity is 3000Lx, and the illumination time is 13 h/d.
(4) Induction of stem tips: placing the sterilized tender stem tip on sterilized filter paper, sucking water, cutting into 1.5cm length, peeling the base leaf of the stem tip, and inoculating into induction culture medium for culture.
The formula of the induction culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the inductivity is 93 percent; when the sea-buckthorn variety is QX-0601, the inductivity is 95 percent; when the sea-buckthorn variety is QX-0104, the inductivity is 92 percent; when the sea buckthorn variety is QX-0102, the inductivity is 91%; when the sea-buckthorn variety is QX-0105, the inductivity is 97%.
(5) Proliferation of stem tips: and (3) inducing the tender stem tips in an induction culture medium for 23 days, transferring the tender stem tips into a proliferation culture medium for proliferation culture, and performing subculture once in 25 days.
The formula of the proliferation culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the proliferation rate is 90 percent; when the sea-buckthorn variety is QX-0601, the proliferation rate is 94 percent; when the sea buckthorn variety is QX-0103, the proliferation rate is 93 percent; when the sea buckthorn variety is Liaofu, the proliferation rate is 87 percent; when the sea buckthorn variety is QX-0102, the proliferation rate is 89%; when the sea-buckthorn variety is QX-0105, the proliferation rate is 92%.
(6) Rooting of the stem tip: cutting off the single plant of the cluster seedling obtained by the propagation culture, and inoculating the cut plant into a rooting culture medium for inducing rooting.
The rooting medium comprises the following components: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the rooting rate is 90%; when the sea-buckthorn variety is QX-0601, the rooting rate is 93 percent; when the sea-buckthorn variety is QX-0103, the rooting rate is 72%; when the sea buckthorn variety is the Liaofu, the rooting rate is 77 percent.
(7) Transplanting: inducing rooting in rooting culture medium for 60 days, transplanting the seedling until the root grows to 3.5-4.5cm, washing the seedling in bottle, transplanting to substrate, and culturing to obtain regenerated plant. The matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
The key point of the success of seedling transplanting is the control of the humidity in the first week, the humidity is controlled to be more than 95% in the first week after seedling transplanting, the ventilation and illumination time is gradually prolonged, and the curtain can be completely uncovered after 16 days. Meanwhile, the temperature is controlled well, the temperature is controlled at 24 ℃ in the daytime and 15-18 ℃ at night.
After the test-tube plantlets are transplanted for 20 days, the survival rate can reach 96%.
Example 3
The tissue culture seedling method of the sea buckthorn comprises the following steps:
(1) test materials: the tender stem tips harvested in the field can be harvested in the whole growing season as long as the stem tips are not degenerated into thorns.
(2) The stem tip disinfection method comprises the following steps: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, flowing tap water for 8min, washing with distilled water for 3 times, placing on sterilized 30min ultra-clean bench, and connectingThen 0.1% of HgCl was used2Sterilizing for 2.5min, shaking, and washing with sterilized distilled water for 7 times. The browning rate was 1%.
(3) The culture conditions are as follows: in the processes of induction, proliferation and rooting of the stem tip, the culture temperature is 26 ℃, the humidity is 70%, the illumination intensity is 2000Lx, and the illumination time is 16 h/d.
(4) Induction of stem tips: placing the sterilized tender stem tip on sterilized filter paper, sucking water, cutting into 0.5cm length, peeling the base leaf of the stem tip, and inoculating into induction culture medium for culture.
The formula of the induction culture medium is as follows: 1/2B5+6-BA 0.4mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the inductivity is 92 percent; when the sea-buckthorn variety is QX-0601, the inductivity is 96 percent; when the sea buckthorn variety is QX-0104, the inductivity is 91%; when the sea-buckthorn variety is QX-0102, the inductivity is 92 percent; when the sea-buckthorn variety is QX-0105, the inductivity is 98%.
(5) Proliferation of stem tips: and (3) inducing the tender stem tips in an induction culture medium for 27 days, transferring the tender stem tips into a proliferation culture medium for proliferation culture, and performing subculture once in 30 days.
The formula of the proliferation culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the proliferation rate is 89%; when the sea-buckthorn variety is QX-0601, the proliferation rate is 95 percent; when the sea buckthorn variety is QX-0103, the proliferation rate is 91%; when the sea buckthorn variety is Liaofu, the proliferation rate is 90 percent; when the sea buckthorn variety is QX-0102, the proliferation rate is 91%; when the sea-buckthorn variety is QX-0105, the proliferation rate is 94%.
(6) Rooting of the stem tip: cutting off the single plant of the cluster seedling obtained by the propagation culture, and inoculating the cut plant into a rooting culture medium for inducing rooting.
The rooting medium comprises the following components: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L.
When the sea-buckthorn variety is male, the rooting rate is 91%; when the sea-buckthorn variety is QX-0601, the rooting rate is 94%; when the sea-buckthorn variety is QX-0103, the rooting rate is 70%; when the sea buckthorn variety is Liaofu, the rooting rate is 73%.
(7) Transplanting: inducing rooting in rooting culture medium for 40 days, transplanting the seedling until the root grows to 3.5-4.5cm, washing the seedling in bottle, transplanting to substrate, and culturing to obtain regenerated plant. The matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
The key point of the success of seedling transplanting is the control of the humidity in the first week, the humidity is controlled to be more than 95% in the first week after seedling transplanting, the ventilation and illumination time is gradually prolonged, and the curtain can be completely uncovered after 14 days. Meanwhile, the temperature is controlled to be 26 ℃ in the daytime and 15-18 ℃ at night.
After the test-tube plantlets are transplanted for 20 days, the survival rate can reach 96%.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The tissue culture seedling method of sea buckthorn is characterized by comprising the following steps: the method comprises the following steps:
(1) selecting materials: taking tender stem tips as tissue culture seedling raising materials;
(2) the method is characterized by comprising the following steps of (1) sterilizing tender stem tips: taking tender stem tip, stripping peripheral leaves, cleaning with tap water, flowing tap water for 8-12min, washing with distilled water for 3 times, placing on a sterilized 30min ultra-clean bench, and adding 0.1% HgCl2Sterilizing for 2-2.5min while shaking, and washing with sterilized distilled water for 6-7 times;
(3) the culture conditions are as follows: in the process of induction, proliferation and rooting of the tender stem tip, the culture temperature is 25 +/-1 ℃, the humidity is 50-70%, the illumination intensity is 2000-3000Lx, and the illumination time is 13-16 h/d;
(4) induction of tender stem tips: peeling the disinfected tender stem tip and basal leaves, and inoculating the tender stem tip and basal leaves into an induction culture medium for culture, wherein the induction culture medium comprises the following components in parts by weight: 1/2B5+6-BA 0.2-0.4mg/L + sucrose 30g/L + agar 6 g/L;
(5) proliferation of tender stem tips: the tender stem tip is induced in an induction culture medium for 23-27 days and then transferred into a proliferation culture medium for proliferation culture, wherein the formula of the proliferation culture medium is as follows: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L;
(6) rooting of tender stem tips: cutting off the single plant of the clustered seedling obtained by the multiplication culture, and inoculating the cut plant to a rooting culture medium for induced rooting, wherein the formula of the rooting culture medium comprises: 1/2B5+6-BA 0.2mg/L + sucrose 30g/L + agar 6 g/L;
(7) transplanting: inducing rooting in rooting culture medium for 40-60 days, transplanting, culturing in matrix, controlling humidity to over 95% in the first week, gradually prolonging ventilation and illumination time, and naturally ventilating completely for 14-16 days; meanwhile, the temperature is controlled well, the temperature is controlled to be 25 +/-1 ℃ in the daytime and 15-18 ℃ at night.
2. The method of claim 1, wherein: in the step (3), the culture temperature is 25 ℃, the humidity is 60%, the illumination intensity is 2500Lx, and the illumination time is 14 h/d.
3. The method of claim 1, wherein: in the step (4), the formula of the induction medium is as follows: 1/2B5+6-BA 0.3mg/L + sucrose 30g/L + agar 6 g/L.
4. The method of claim 1, wherein: in the step (7), the matrix is prepared by mixing turfy soil and perlite, and the volume ratio of the turfy soil to the perlite is 1: 1.
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