阅读说明：本技术 一种高效低耗的喜树组织培养和移栽方法 (Efficient and low-consumption camptotheca acuminata tissue culture and transplantation method ) 是由 刘志文 李班 于放 于 2019-12-06 设计创作，主要内容包括：本发明涉及一种高效低耗的喜树组织培养和移栽方法,属于植物组织培养技术领域。本发明所述方法包括组培喜树苗步骤、炼苗移栽至蛭石步骤、移栽至育苗杯步骤、移栽至大田建立叶用园步骤；所述炼苗移栽至蛭石步骤为：移栽前,每苗加10-15mL自来水,置于室内自然光下炼苗5-7天后,用自来水清洗苗根部的培养基,再将其移栽至蛭石,蛭石从小钵底部吸取自来水至饱和,每3-5天从小钵底部补充自来水一次,培养21-28天。本发明解决喜树资源短缺的问题。(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.)

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.

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.