CN110800612A - Efficient and low-consumption camptotheca acuminata tissue culture and transplantation method - Google Patents
Efficient and low-consumption camptotheca acuminata tissue culture and transplantation method Download PDFInfo
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- 241000759905 Camptotheca acuminata Species 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000002054 transplantation Methods 0.000 title claims abstract description 7
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- 238000004161 plant tissue culture Methods 0.000 abstract description 2
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Classifications
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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
-
- 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
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
-
- 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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
-
- 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
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Soil Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention relates to a high-efficiency and low-consumption camptotheca acuminate tissue culture and transplantation method, belonging to the technical field of plant tissue culture. The method comprises the steps of tissue culture of camptotheca acuminate seedlings, hardening and transplanting of the seedlings to vermiculite, transplanting of the seedlings to seedling cups, and transplanting to a field to establish a leaf garden; the step of hardening the seedlings and transplanting the seedlings to vermiculite comprises the following steps: before transplanting, 10-15mL of tap water is added into each seedling, the seedling is placed in indoor natural light to be hardened for 5-7 days, then the culture medium at the root of the seedling is cleaned by the tap water, the seedling is transplanted to vermiculite, the vermiculite absorbs the tap water from the bottom of the small pot to be saturated, the tap water is supplemented from the bottom of the small pot every 3-5 days, and the seedling is cultured for 21-28 days. The invention solves the problem of shortage of camptotheca acuminata resources.
Description
Technical Field
The invention relates to a high-efficiency and low-consumption camptotheca acuminate tissue culture and transplantation method, belonging to the technical field of plant tissue culture.
Background
Camptotheca acuminata (Camptotheca acuminata Decne) is a Camptotheca (Camptotheca Decne) plant of davidiaceae (nyssaae), is a perennial deciduous tree plant unique to China, is mainly distributed in Yangtze river basin and southwest provinces, and has the characteristics of high growth speed, less plant diseases and insect pests and the like. Different parts of camptotheca contain a secondary metabolite Camptothecin (CPT), which is the only natural plant active ingredient discovered so far to exert cytotoxicity by inhibiting topoisomerase I, and is a broad-spectrum anticancer and antitumor drug. At present, camptothecin drugs are praised as the first choice of anticancer drugs in the twenty-first century, and are deeply favored by the plant and medical communities. The demand of camptothecin in medicine is getting larger and larger, and the camptotheca acuminata also becomes a medicinal plant with a very promising application prospect.
Because of the low content of camptothecin in camptotheca acuminata, the existing method only depends on extracting camptothecin from harvested natural camptotheca acuminata, which can not only destroy the ecological environment but also can not meet the demand, but also has great difficulty in chemically synthesizing camptothecin. In addition, the wild camptotheca acuminata resource is unstable, and the camptothecin is expensive and in short supply along with the increasing demand of the camptothecin on the market. Camptotheca acuminata was put into the national level II key point by the forestry ministry of China to protect wild plants in 1999, but the consumption of Camptotheca acuminata in production is increasing, so that the wild Camptotheca acuminata resource in China is reduced sharply. Therefore, large-scale planting and cultivation of camptotheca acuminata is imperative.
Camptotheca acuminata is usually propagated by seeds, but the germination rate is not high due to the strong dormancy of the seeds. The cutting propagation of the camptotheca acuminate can be influenced by seasons. Tissue culture technology provides a cheap and rapid propagation mode for medicinal plants and endangered species, in order to utilize camptotheca acuminata resources in large-scale cultivation, tissue culture and rapid propagation of camptotheca acuminata are one of important ways and methods for propagating camptotheca acuminata seedlings in large quantities, and the tissue culture technology not only can realize preservation and development of camptotheca acuminata germplasm resources, but also is beneficial to basic research on molecular genetics and the like.
Camptotheca acuminata is a woody plant, and its tissue culture is relatively difficult. The tissue culture seedling of camptotheca acuminata is difficult to survive when being directly transplanted to soil, and the survival rate is only about 10 percent.
Disclosure of Invention
The invention establishes a high-efficiency and low-consumption rapid propagation method of the camptotheca acuminate tissue culture seedlings, improves the transplanting survival rate and adaptability, preserves, develops and cultivates and utilizes camptotheca acuminate resources in a large scale, realizes the medicinal and other values of the camptotheca acuminate resources, and overcomes the difficult problem of the camptotheca acuminate tissue culture seedlings.
The invention provides a high-efficiency and low-consumption camptotheca tissue culture and transplantation method, which comprises the steps of tissue culture of camptotheca seedlings, hardening and transplanting the seedlings to vermiculite, transplanting the seedlings to a seedling cup, and transplanting the seedlings to a field to establish a leaf garden; the step of hardening the seedlings and transplanting the seedlings to vermiculite comprises the following steps: before transplanting, 10-15mL of tap water is added into each seedling, the seedling is placed in indoor natural light to be hardened for 5-7 days, then the culture medium at the root of the seedling is cleaned by the tap water, the seedling is transplanted to vermiculite, the vermiculite absorbs the tap water from the bottom of the small pot to be saturated, the tap water is supplemented from the bottom of the small pot every 3-5 days, and the seedling is cultured for 21-28 days.
The preferred steps of transplanting the seedlings to the seedling cup are as follows: and (3) filling 1/3-1/2 of soil in the seedling cup, sucking tap water from the bottom of the soil to saturation, transplanting the seedlings with vermiculite to the seedling cup, adding the soil until the seedling cup is full, supplementing tap water from the bottom of the seedling cup, supplementing 0.5% urea tap water solution from the bottom of the seedling cup every 3-5 days, and culturing for 28-35 days.
The invention preferably comprises the following steps of transplanting the seedlings into a field to establish a leaf garden: transplanting the seedlings in the seedling cup to the outdoor or in the field for hardening the seedlings for 5-7 days, removing the seedling cup during transplanting, directly transplanting the seedlings with soil to the field, wherein the transplanting specification is 0.45-0.50m multiplied by 0.45-0.50m, applying a fertilizer special for camptotheca acuminata, and establishing a camptotheca acuminata leaf garden.
The preferable tissue culture camptotheca acuminata seedling step comprises the steps of explant disinfection and pre-culture, single-plant aseptic seedling culture, cluster bud induction and culture, adventitious root induction and strong seedling culture; the induced adventitious root and strong seedling culture comprises the following steps: cutting rootless cluster buds growing to 2-3cm with 2-3 young leaves, inoculating to a rooting and seedling-strengthening culture medium: WPM culture medium, naphthylacetic acid 0.05mg/L, indolebutyric acid 0.5mg/L and cane sugar 25g/L, inducing rooting and strengthening seedling for 28-35 days.
The invention preferably induces and cultures the cluster buds as follows: cutting the single aseptic seedling into 1.5-2.0cm segments, each segment having 1-2 axillary buds, reserving 1-2 leaves, inoculating into a cluster bud induction culture medium: WPM culture medium + 6-benzylaminopurine 0.5mg/L + sucrose 30g/L, inducing for 28-35 days.
The invention preferably selects the single sterile seedling culture as follows: selecting a survival stem section, cutting a new branch growing at the axilla of the leaf of the stem section in a superclean workbench, and inoculating the new branch into a subculture medium: WPM culture medium, indoleacetic acid 0.1mg/L, indolebutyric acid 0.5mg/L and sucrose 30g/L, and culturing for 28-35 days.
The invention preferably provides that the explant sterilization and pre-culture is as follows: selecting new camptotheca acuminata branches, transversely cutting the branches into small sections of 2-2.5cm, wherein each section has 1-2 axillary buds, and disinfecting as explants, wherein the disinfection method comprises the following steps: washing with tap water, soaking with 70% alcohol in a clean bench, washing with sterile water, soaking with 0.1% mercuric chloride, washing with sterile water, air drying, cutting off the incision contacted with 0.1% mercuric chloride, and inoculating to initial culture medium: MS culture medium, 0.1mg/L naphthylacetic acid and 30g/L cane sugar are placed in a light culture chamber for pre-culture for 21-28 days.
In the present invention, preferably, the water for the culture medium is tap water.
Preferably, the artificial illumination is LED illumination, the illumination intensity is 1000-1500lx, and the illumination is 12h every day.
The invention has the beneficial effects that:
at present, camptotheca acuminate resources are very limited, and the cost is too high and the ecological environment is damaged by cutting wild camptotheca acuminate resources to extract medicinal components. The tissue culture method is adopted for rapid propagation of the camptotheca acuminata seedlings, which is not only beneficial to expanding the propagation coefficient of the camptotheca acuminata, but also can obtain a large amount of high-quality seedlings, realize large-scale, normalized and industrialized planting, ensure annual supply, provide a new way for accelerating the artificial propagation of the camptotheca acuminata and the cultivation of medicinal camptotheca acuminata forests in future, and solve the problem of shortage of camptotheca acuminata resources. Meanwhile, the asexual propagation can efficiently maintain various excellent properties of the stock plant and lay a material foundation for the research of genetic engineering of endangered species of the camptotheca acuminata.
In the production practice, tap water is used for replacing distilled water to prepare the culture medium, so that the cost is reduced, and the tap water contains more nutrient components such as ions and the like than the distilled water, and is more favorable for the growth of the test-tube plantlet of the camptotheca acuminata. The LED lamp is used as a novel light source and mainly comprises blue light with the wavelength of 400-500 and yellow light emitted by fluorescent powder excited by the blue light. The photosynthesis of the plants mainly absorbs blue light with the wavelength of 400-500nm and red light with the wavelength of 600-700nm, the wavelength of the traditional fluorescent lamp is 610-520 nm, and the LED lamp not only saves electricity and cost, but also is more beneficial to the photosynthesis and growth of the camptotheca acuminata.
The WPM culture medium mainly applied in the invention is a high-efficiency solid powder culture medium created according to the characteristics of woody plants, and is easy to purchase and convenient to prepare in the market.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
The following MS culture media were purchased from Qingdao Haibo Biotech, Inc.;
the following naphthylacetic acids were purchased from Beijing Solaibao Tech technologies, Inc.;
the following WPM medium was purchased from Qingdao Haibo Biotech Co., Ltd;
the following indoleacetic acids were purchased from Beijing Solaibao Tech technologies, Inc.;
the following indolebutyric acids were purchased from Beijing Solaibao Tech technologies, Inc.;
the following 6-benzylaminopurine was purchased from Beijing Solaibao Tech Co., Ltd;
the following vermiculite was purchased from north Heibei Dewoo fertilizers, Inc., product specifications: 1-3 mm;
the following camptotheca acuminata special fertilizer is purchased from Olympic agriculture science and technology Limited in Henan republic;
the following media were solidified at 8g/L agar when formulated according to the instructions provided;
the following culture medium water is tap water;
the pH of the following culture medium is adjusted to 5.8 by using 1mol/L NaOH or HCl;
sterilizing the culture medium in 20-25mL per bottle at 121 deg.C under high pressure and moist heat for 20 min;
the artificial illumination is LED illumination except natural light, the illumination intensity is 1000-1500lx, and the illumination is 12h every day.
Example 1
A high-efficiency and low-consumption camptotheca acuminate tissue culture and transplantation method comprises the following steps:
firstly, high-efficiency high-quality tissue culture of tree-like seedlings:
explant sterilization and preculture
Selecting new branches of camptotheca acuminate, transversely cutting the branches into small sections of 2-2.5cm, wherein each section is provided with 1-2 axillary buds, and using the branches as explants to carry out disinfection treatment;
repeatedly washing with running water for 50min, soaking in 70% ethanol for 45s on a clean bench, washing with sterile water for 3 times, and immediately adding 0.1% mercuric chloride (HgCl)2) Soaking for 10min, removing disinfectant, shaking for sufficient disinfection, washing with sterile water for 3 times, air drying, and removing disinfectant HgCl2Cutting the contacted cut, wherein the upper cut is cut flatly, the lower cut is cut obliquely at an angle of 45 degrees to enlarge the contact area with the culture medium, a small stem section of 1.5-2cm is left, the lower cut is vertically inserted and inoculated on a solid culture medium of an initial culture medium MS culture medium, naphthylacetic acid (0.1mg/L) + sucrose (30 g/L), and the solid culture medium is placed in a light culture chamber for culture and is pre-cultured for 25 d;
culture of single sterile seedling
Selecting the survival stem section which is thoroughly and cleanly disinfected and has no pollution, cutting new branches growing at the axilla of the stem section leaves on a superclean bench, inoculating into a subculture medium WPM culture medium, indoleacetic acid (0.1mg/L), indolebutyric acid (0.5mg/L), cane sugar (30 g/L) for subculture, and culturing for 35d to obtain a single seedling with roots which grows vigorously and is in a good state, wherein the height of the seedling can reach 6-8cm, the number of the roots can reach 5-10, but the roots are thin, the stem is weak, the leaves can reach 8-12, and the seedling is shown to have more knots and more axillary buds, so that the shape of the seedling at the moment is more favorable for propagation culture;
cluster bud induction and culture
The induction of the bud is the key for obtaining the camptotheca acuminate regenerated seedling, and the selection of high-concentration cytokinin is beneficial to the induction of cluster buds, so that a large amount of sterile camptotheca acuminate bud seedlings can be quickly obtained;
cutting the single seedling into small sections of 1.5-2.0cm, culturing 1-2 axillary buds per section with 1-2 leaves reserved, inoculating the small sections to a cluster bud induction culture medium WPM culture medium + 6-benzylaminopurine (0.5mg/L) + sucrose 30g/L, and inducing cluster buds, wherein 1 section is cultured in each bottle, and the large number of cluster buds are mainly induced and the growth of the cluster buds are promoted at the moment, the height of the seedling is small, the maximum is only 4.0cm, generally 3.0cm, the growth of adventitious roots is small or not, after 30 days, 20-30 cluster buds can grow per seedling, and the propagation coefficient can reach 30-40;
inducing adventitious root and strong seedling culture
The formation of adventitious roots is a very critical link in the process of tissue culture of camptotheca acuminate, which directly influences the transplanting survival rate of tissue culture seedlings and is related to the success or failure of tissue culture;
cutting the rootless cluster single buds growing to 2-3cm and having 2-3 young leaves, transferring the rootless cluster single buds into a rooting and strong seedling culture medium WPM culture medium, naphthylacetic acid (0.05mg/L) + indolebutyric acid (0.5mg/L) + sucrose (25 g/L) for culture, inducing the rootage and the strong seedling, 1 bud in each bottle, and obtaining a well-developed and strong root system after 1 month, wherein the newly grown adventitious roots of the cluster buds are white in color, the rooting percentage is 95.0%, the average rootage number is 5.4 pieces/plant, and the shortest root induction period is 20 d;
after 35d rooting and strong seedling culture, aseptic seedlings have leaves which are green, roots which are 2-5cm long, thick roots, developed roots, thick stems which can reach 1.5-2.0mm, plant height which can reach 4-6cm and leaves number which can reach 7-10, and hardening seedlings and transplanting are carried out;
secondly, hardening seedlings and transplanting to vermiculite
Transplanting tissue culture seedling of Camptotheca acuminata into external environment from sterile culture medium and illumination culture environment, adapting to temperature, humidity and natural illumination change, hardening seedling before transplanting, selecting small seedling with strong growth, vigorous growth and dark green leaf color, opening tissue culture bottle cap, adding 10-15mL tap water into each bottle, placing in room under natural light, hardening seedling for 5-7d, taking out bud seedling, washing culture medium at root of tissue culture seedling with tap water, transplanting into small pot (7 × 7cm) of vermiculite, filling vermiculite into small pot, placing in tray, allowing vermiculite to fully absorb tap water from tray bottom to saturation, not directly watering from small pot upper part, digging a 3-4cm deep hole in the vermiculite pot during transplanting, placing all root systems together gently, placing the root systems into a hole gently, covering the vermiculite on the hole lightly by using tools such as tweezers and the like to cover all the roots, taking care in the operation process to plant the roots in place once without damaging each root as much as possible, pouring tap water from a tray every 4d, keeping the small pots moist, culturing for 20d, and enabling the survival rate to reach 95%;
thirdly, transplanting the seedlings to a seedling cup
Firstly, a seedling cup (9 multiplied by 9cm) is filled into 1/3 field soil and placed on a tray, tap water is sucked from the bottom of the tray until the soil is fully saturated, the small pot can be slightly squeezed along four sides during transplanting, so that the vermiculite in the small pot can be conveniently moved together with seedlings, the vermiculite seedlings and the vermiculite are transplanted into the small pot filled with the field soil together, tap water is timely supplemented from the bottom of the tray after the field soil is added until the small pot is full, one seedling is transplanted in one cup, the vermiculite is transplanted as completely as possible without damaging the root system as much as possible, the tap water solution (0.5%) of urea is poured into the tray once every 4 days, the field soil in the small pot is kept moist, the seedlings are promoted to grow, the seedling cup is moved into a greenhouse for cultivation, the cultivation lasts for 1 month, and the survival rate can reach 98%;
fourthly, transplanting the leaves to a field to establish a leaf garden
Transferring the seedlings in the seedling cup to the field, placing for 7 days, carrying out outdoor and field hardening, removing the seedling cup during transplanting, directly transferring the camptotheca acuminata seedlings to the field with soil, applying camptotheca acuminata special fertilizer according to the instructions, establishing a camptotheca acuminata leaf garden, wherein the transplanting specification is 0.5m multiplied by 0.5m, 2660 plants per mu, the survival rate of the transplanted tissue culture seedlings exceeds 95%, and the leaf yield of camptotheca acuminata in unit area is the maximum at the moment, so that higher-yield camptothecine can be obtained, and the later cultivation management is the same as the normal camptotheca acuminata management without special management.
Claims (9)
1. A high-efficiency and low-consumption camptotheca acuminate tissue culture and transplantation method is characterized in that: the method comprises the steps of tissue culture of camptotheca acuminate seedlings, hardening and transplanting of the seedlings to vermiculite, transplanting to seedling cups, and transplanting to fields to establish leaf garden;
the step of hardening the seedlings and transplanting the seedlings to vermiculite comprises the following steps: before transplanting, 10-15mL of tap water is added into each seedling, the seedling is placed in indoor natural light to be hardened for 5-7 days, then the culture medium at the root of the seedling is cleaned by the tap water, the seedling is transplanted to vermiculite, the vermiculite absorbs the tap water from the bottom of the small pot to be saturated, the tap water is supplemented from the bottom of the small pot every 3-5 days, and the seedling is cultured for 21-28 days.
2. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 1, wherein: the step of transplanting to the seedling cup is as follows: and (3) filling 1/3-1/2 of soil in the seedling cup, sucking tap water from the bottom of the soil to saturation, transplanting the seedlings with vermiculite to the seedling cup, adding the soil until the seedling cup is full, supplementing tap water from the bottom of the seedling cup, supplementing 0.5% urea tap water solution from the bottom of the seedling cup every 3-5 days, and culturing for 28-35 days.
3. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 2, wherein: the steps of transplanting the seedlings to a field and establishing the leaf garden are as follows: transplanting the seedlings in the seedling cup to the outdoor or in the field for hardening the seedlings for 5-7 days, removing the seedling cup during transplanting, directly transplanting the seedlings with soil to the field, wherein the transplanting specification is 0.45-0.50m multiplied by 0.45-0.50m, applying a fertilizer special for camptotheca acuminata, and establishing a camptotheca acuminata leaf garden.
4. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 3, wherein: the tissue culture camptotheca acuminate seedling step comprises explant disinfection and pre-culture, single-plant aseptic seedling culture, cluster bud induction and culture, adventitious root induction and strong seedling culture;
the induced adventitious root and strong seedling culture comprises the following steps: cutting rootless cluster buds growing to 2-3cm with 2-3 young leaves, inoculating to a rooting and seedling-strengthening culture medium: WPM culture medium, naphthylacetic acid 0.05mg/L, indolebutyric acid 0.5mg/L and cane sugar 25g/L, inducing rooting and strengthening seedling for 28-35 days.
5. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 4, wherein: the cluster bud induction and culture comprises the following steps: cutting the single aseptic seedling into 1.5-2.0cm segments, each segment having 1-2 axillary buds, reserving 1-2 leaves, inoculating into a cluster bud induction culture medium: WPM culture medium + 6-benzylaminopurine 0.5mg/L + sucrose 30g/L, inducing for 28-35 days.
6. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 5, wherein: the single sterile seedling culture comprises the following steps: selecting a survival stem section, cutting a new branch growing at the axilla of the leaf of the stem section in a superclean workbench, and inoculating the new branch into a subculture medium: WPM culture medium, indoleacetic acid 0.1mg/L, indolebutyric acid 0.5mg/L and sucrose 30g/L, and culturing for 28-35 days.
7. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 6, wherein: the explant disinfection and pre-culture comprises the following steps: selecting new camptotheca acuminata branches, transversely cutting the branches into small sections of 2-2.5cm, wherein each section has 1-2 axillary buds, and disinfecting as explants, wherein the disinfection method comprises the following steps: washing with tap water, soaking with 70% alcohol in a clean bench, washing with sterile water, soaking with 0.1% mercuric chloride, washing with sterile water, air drying, cutting off the incision contacted with 0.1% mercuric chloride, and inoculating to initial culture medium: MS culture medium, 0.1mg/L naphthylacetic acid and 30g/L cane sugar are placed in a light culture chamber for pre-culture for 21-28 days.
8. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 7, wherein: the water used for the culture medium is tap water.
9. The method for culturing and transplanting camptotheca acuminata tissue with high efficiency and low consumption according to claim 8, wherein: the artificial illumination is LED illumination with the illumination intensity of 1000-1500lx and the illumination time of 12h every day.
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