阅读说明：本技术 植物的组织培养方法及其装置以及代谢产物的生产方法 (Plant tissue culture method and device and production method of metabolite ) 是由 饶君凤 吕伟德 凌霄 林紫怡 胡玉林 罗煦钦 边天煜 何国强 于 2019-11-29 设计创作，主要内容包括：本发明公开了植物的组织培养方法及其装置以及代谢产物的生产方法,属于植物组织培养和代谢产物领域。植物的组织培养方法及其装置以及代谢产物的生产方法,通过明确植物的组织培养方法,优化愈伤组织培养流程,加快了愈伤组织的培养过程,并且通过优化生长素起到了对愈伤组织的诱导及生长作用,增长了其生长代谢物产量,由于目前的生态环境造成的破坏和人们对于野生资源的盲目采集,造成许多野生植物趋于濒危,导致众多的生物天然活性物质来源减少,并且化学合成方法由于工艺复杂和费用高,同时易造成新的环境污染,本发明可以有效的增长植物天然活性物质的生产量。(The invention discloses a plant tissue culture method and a device thereof and a production method of a metabolite, belonging to the field of plant tissue culture and metabolites. The invention relates to a plant tissue culture method, a plant tissue culture device and a production method of metabolites, wherein the callus culture process is optimized by defining the plant tissue culture method, the callus culture process is accelerated, the callus is induced and grown by optimizing auxin, the yield of the growing metabolites is increased, a plurality of wild plants tend to be endangered due to the damage caused by the current ecological environment and the blind collection of wild resources by people, so that the sources of a plurality of biological natural active substances are reduced, and the production of the plant natural active substances can be effectively increased due to the complex process and high cost of a chemical synthesis method and the easy new environmental pollution.)

1. A plant tissue culture method and a device thereof and a production method of metabolites are characterized by comprising the following steps:

s1, collecting young stems and leaves of the current year, cutting the stems into small segments with the length of 3-4cm, cutting the leaves into long strips with the width of 1cm, washing the long strips with 2% hydrogen peroxide by volume fraction, repeatedly washing the long strips with tap water, washing the long strips with running water for 1.5h, sterilizing the long strips with 65% ethanol by volume fraction for 40S, washing the long strips with sterile water for 3-4 times, and then adopting 1g/L HgCl₂Sterilizing for 3-7min, and washing with sterile water for 4-5 times;

s2, selecting the current year plump seeds, soaking for 22h, peeling off the seed coat, repeatedly washing with clear water, then adopting 70% alcohol by volume fraction, disinfecting for 1min, washing with sterile water for 3-4 times, 1g/L HgCl₂Sterilizing for 15min, washing with sterile water for 4-5 timesInoculating into 1/2MS culture medium, and culturing at 22-27 deg.C under natural illumination;

s3, cutting the young stem which is developed in the S2 into 0.7cm long sections, cutting young leaves into 0.5cm by 0.5cm squares, inoculating the squares on the standby material collected in the S1, and then placing the squares in a callus culture medium for callus culture;

s4, culturing the callus induced in the S3 on a first generation culture medium to obtain a first generation callus, and performing subculture on the callus on a subculture medium after the culture is finished;

s5, collecting the third generation callus of stem subculture in S4, adding 30ml B into 100ml three-leg bottle₅The culture medium is used for callus extraction culture;

s6, collecting the callus cultured in S5, placing the callus in an oven to be dried to constant weight at 80 ℃, placing the callus into a flask after being crushed, adding alcohol with the volume fraction of 60% into a water bath at 75 ℃, condensing and refluxing for extraction twice for 1 hour each time, and filtering and combining the filtrate to obtain the callus metabolite.

2. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: 35g/L of sucrose in the callus culture medium in the S3 is solidified by 8g/L of agar, the pH is adjusted to 6.3 before the callus culture medium is autoclaved, the temperature of a culture room is controlled to be about 22-27 ℃, a culture bottle is placed on a non-lighting culture frame and covered by black cloth during dark light culture, and the light is irradiated for 12h/d at the light intensity of 1, 000-1 and 500lx during light culture.

3. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: the first generation culture medium is B₅Medium and 0.5mg/LNAA and 0.5mg/LBA were added.

4. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: b is added in the subculture medium₅Culture medium, and adding 0.5mg/L2, 4-D and 0.5mg/LNAA。

5. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: the callus powder in the S6 is mixed with alcohol with the volume fraction of 60% according to the ratio of 1: 16 in proportion by mass.

6. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: and culturing and germinating for 30d in the S3 for later use.

7. The tissue culture method of a plant and the device thereof and the method for producing a metabolite according to claim 1, wherein: harvested after 20 days of culture in said S5.

Technical Field

The invention relates to the field of plant tissue culture and metabolites, in particular to a plant tissue culture method and a device thereof and a production method of metabolites.

Background

A great variety and vast amount of plants on the earth are valuable resources on which human beings live, and organic matters from the plants comprise primary metabolites and secondary metabolites. The primary metabolite is directly related to the growth, development and reproduction of plants, is the metabolism of the plants for obtaining energy, and provides energy and intermediate products for the survival, growth, development and reproduction of plants. The secondary metabolites are relative to the primary metabolites, and take some intermediate products of the primary metabolism as raw materials or precursors to carry out further metabolic processes. The secondary metabolite of the plant is a substance generated in the process of releasing energy, and has no direct relation with the growth, development and propagation of the plant. However, these secondary metabolites often have strong biological activity and economic utilization value, and are natural active substances (i.e. medicinal active ingredients) for preventing and treating diseases of human beings or natural products with unique economic utilization value. The secondary metabolism mechanism is still hazy and is well-known to be difficult to research so far, but is a research field with great application prospect.

The prior plant tissue culture method and device and the metabolite production method have low metabolite yield and can not meet the prior requirements.

Disclosure of Invention

The invention aims to solve the problems that the existing metabolite yield is low and the existing needs cannot be met, and provides a plant tissue culture method and a device thereof and a metabolite production method.

In order to achieve the purpose, the invention adopts the following technical scheme:

a plant tissue culture method and a device thereof and a production method of metabolites comprise the following steps:

s1, collecting young stem and leaf of the current year, cutting stem into 3-4cm long segments, cutting leaf into 1cm wide strips, washing with 2% hydrogen peroxide solution, and repeatedly washing with tap waterWashing with running water for 1.5 hr, sterilizing with 65 vol% ethanol for 40s, washing with sterile water for 3-4 times, and washing with 1g/L HgCl₂Sterilizing for 3-7min, and washing with sterile water for 4-5 times;

s2, selecting the current year plump seeds, soaking for 22h, peeling off the seed coat, repeatedly washing with clear water, then adopting 70% alcohol by volume fraction, disinfecting for 1min, washing with sterile water for 3-4 times, 1g/L HgCl₂Sterilizing for 15min, washing with sterile water for 4-5 times, inoculating into 1/2MS culture medium, and culturing at 22-27 deg.C under natural illumination;

s3, cutting the young stem which is developed in the S2 into 0.7cm long sections, cutting young leaves into 0.5cm by 0.5cm squares, inoculating the squares on the standby material collected in the S1, and then placing the squares in a callus culture medium for callus culture;

s4, culturing the callus induced in the S3 on a first generation culture medium to obtain a first generation callus, and performing subculture on the callus on a subculture medium after the culture is finished;

s5, collecting the third generation callus of stem subculture in S4, adding 30ml B into 100ml three-leg bottle₅The culture medium is used for callus extraction culture;

s6, collecting the callus cultured in S5, placing the callus in an oven to be dried to constant weight at 80 ℃, placing the callus into a flask after being crushed, adding alcohol with the volume fraction of 60% into a water bath at 75 ℃, condensing and refluxing for extraction twice for 1 hour each time, and filtering and combining the filtrate to obtain the callus metabolite.

Preferably, the callus culture medium in S3 contains 35g/L sucrose, is solidified with 8g/L agar, pH is adjusted to 6.3 before the callus culture medium is autoclaved, the temperature of the culture room is controlled to be about 22-27 ℃, the culture bottle is placed on a non-lighting culture frame and covered with black cloth during dark light culture, and the light culture is performed for 12h/d with the light intensity of 1, 000-1, 500 lx.

Preferably, the primary medium is B₅Medium and 0.5mg/LNAA and 0.5mg/LBA were added.

Preferably, B is added to the subculture medium₅Medium and 0.5mg/L2, 4-D and 0.5 mg/LNAA.

Preferably, the callus powder in S6 is mixed with 60% alcohol by volume according to a ratio of 1: 16 in proportion by mass.

Preferably, the culture in the S3 is cultured and germinated into seedlings for 30d for standby.

Preferably, the cells are harvested after 20 days of culture in S5.

Compared with the prior art, the invention provides a plant tissue culture method and a device thereof and a production method of metabolites, and has the following beneficial effects:

1. the invention optimizes the callus culture process by defining the plant tissue culture method, accelerates the callus culture process, plays a role in inducing and growing the callus by optimizing auxin, increases the yield of the growth metabolite, causes a plurality of wild plants to be endangered due to the damage caused by the current ecological environment and the blind collection of wild resources by people, leads to the reduction of a plurality of biological natural active substance sources, and easily causes new environmental pollution due to the complex process and high cost of the chemical synthesis method.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations and positional relationships for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.