CN110800611A - Plant tissue culture method and device and production method of metabolite - Google Patents
Plant tissue culture method and device and production method of metabolite Download PDFInfo
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- CN110800611A CN110800611A CN201911197669.3A CN201911197669A CN110800611A CN 110800611 A CN110800611 A CN 110800611A CN 201911197669 A CN201911197669 A CN 201911197669A CN 110800611 A CN110800611 A CN 110800611A
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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
- 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
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Abstract
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.
Description
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.
Example 1:
a plant tissue culture method and a device thereof and a production method of metabolites comprise 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 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.
Further, it is preferable that 35g/L sucrose in the callus medium of S3 is solidified with 8g/L agar, pH is adjusted to 6.3 before autoclaving the callus medium, temperature of the culture chamber is controlled to be about 22-27 ℃, the culture bottle is placed on a non-lighting culture shelf and covered with black cloth during dark light culture, and the light culture is performed for 12h/d with light intensity of 1, 000-1, 500 lx.
Further, preferably, the primary medium is B5Culture medium, and adding0.5mg/LNAA and 0.5mg/LBA were added.
Further, preferably, B is present in the subculture medium5Medium and 0.5mg/L2, 4-D and 0.5 mg/LNAA.
Further, preferably, the callus powder in S6 is mixed with alcohol with a volume fraction of 60% in a ratio of 1: 16 in proportion by mass.
Further, preferably, the culture in S3 is performed for 30d after germination and seedling formation for standby.
Further, preferably, it is harvested after 20 days of culture in S5.
Example 2: based on example 1, but with the difference that:
TABLE 1 Effect of different concentrations of 2,4-D on callus induction
Table 1 shows that callus was inoculated on MS medium with 2,4-D of 0.0, 0.1, 03, 0.5, 1.0, 2.0 and 4.0 respectively to perform callus induction experiments with plant growth regulators. After the young stem is cultured for 3 days, the two ends of the young stem treated by each treatment begin to expand and become white, and most explants begin to grow callus from the two ends at 6 days. The control had no significant change and no callus was produced. Among the 2,4-D with different concentrations, the healing rate of 0.5mg/L is the highest, 100%, and the callus growth induced is the best. Too high or too low is not favorable for the induction and growth of the callus. At a concentration of 4.0 mg/L2, 4-D, the production of callus from explants was completely inhibited.
Example 3: based on examples 1 and 2, but with the difference that:
TABLE 2 Effect of different concentrations of NAA on callus induction
When the concentration of NAA is below 2.0mg/L, the healing rate is above 90%, and no obvious difference exists among treatments, but when the NAA with larger difference among healing time, callus color, size and growth condition after subculture is 0.1-0.5mg/L, the callus has better growth, but the color is dull, the color is not glossy, and the growth is slow after subculture. NAA1.0mg/L, the callus grows well, is cream-colored, bright and glossy, and grows well after transferring. The time for inducing callus by NAA at the concentration below 1.0mg/L is basically consistent and is 7 days; when NAA is 2.0mg/L, the induction rate and the growth vigor are good, the callus grows normally after subculture, but the time required for inducing the callus is 2-3d later than the concentration below 1.0 mg/L; when NAA is 4.0mg/L, the healing time is 2 days later than 2.0mg/L, and after only a small amount of callus grows out, the callus gradually turns brown and dies. The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
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.
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Citations (4)
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|---|---|---|---|---|
| CN1424395A (en) * | 2002-12-19 | 2003-06-18 | 中国人民解放军军事医学科学院生物工程研究所 | Snow lotus cell series with stable high productive flavone for mass plant cell culture and its establishing method |
| CN1635112A (en) * | 2003-12-26 | 2005-07-06 | 中国科学院过程工程研究所 | Method for constructing suspension cell culture system for breeding broomrape callus |
| CN1849882A (en) * | 2005-04-22 | 2006-10-25 | 甘肃省科学院生物研究所 | Method for producing podophyllotoxin induced from sino podophyllum hexandrum callus |
| CN109097319A (en) * | 2018-07-20 | 2018-12-28 | 中南大学 | A kind of application of the cultural method and Guava Leaf suspension cell of Guava Leaf suspension cell |
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- 2019-11-29 CN CN201911197669.3A patent/CN110800611A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424395A (en) * | 2002-12-19 | 2003-06-18 | 中国人民解放军军事医学科学院生物工程研究所 | Snow lotus cell series with stable high productive flavone for mass plant cell culture and its establishing method |
| CN1635112A (en) * | 2003-12-26 | 2005-07-06 | 中国科学院过程工程研究所 | Method for constructing suspension cell culture system for breeding broomrape callus |
| CN1849882A (en) * | 2005-04-22 | 2006-10-25 | 甘肃省科学院生物研究所 | Method for producing podophyllotoxin induced from sino podophyllum hexandrum callus |
| CN109097319A (en) * | 2018-07-20 | 2018-12-28 | 中南大学 | A kind of application of the cultural method and Guava Leaf suspension cell of Guava Leaf suspension cell |
Non-Patent Citations (1)
| Title |
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| 董峰丽等: "西红花组织培养研究进展 ", 《药物生物技术》 * |
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Application publication date: 20200218 |