CN110558230A - Tissue culture method of detoxified radix pseudostellariae root tuber - Google Patents
Tissue culture method of detoxified radix pseudostellariae root tuber Download PDFInfo
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- CN110558230A CN110558230A CN201910947614.3A CN201910947614A CN110558230A CN 110558230 A CN110558230 A CN 110558230A CN 201910947614 A CN201910947614 A CN 201910947614A CN 110558230 A CN110558230 A CN 110558230A
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- radix pseudostellariae
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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 relates to the technical field of seed breeding, and discloses a tissue culture method of detoxified radix pseudostellariae root tuber, which comprises the steps of processing and disinfecting an explant, inducing a stem tip of radix pseudostellariae, carrying out proliferation culture on radix pseudostellariae seedlings, culturing the root tuber in a radix pseudostellariae bottle and the like. The invention can shorten the seed production time and cycle, has good detoxification effect, and has the advantages of high speed, high efficiency, simple operation and low cost; the proliferation culture medium adopts 1/2MS, the addition of potassium dihydrogen phosphate can make the leaf of radix Pseudostellariae seedling more extended and grow more vigorously without adding hormone, and the root tuber induction culture medium adopts 1/2MS with potassium dihydrogen phosphate and high concentration (60g/L) sucrose to make the root tuber more expanded.
Description
Technical Field
the invention relates to the technical field of seed breeding, in particular to a tissue culture method of detoxified radix pseudostellariae root tuber.
background
radix pseudostellariae is the root tuber of the pseudostellaria heterophylla of perennial herbaceous plants of the Caryophyllaceae family, has the effects of benefiting qi, strengthening spleen, promoting the production of body fluid and moistening lung, has obvious effects on night sweat, inappetence and insomnia caused by asthenia of the old, has more mild drug property compared with ginseng, is suitable for deficiency tonifying and qi invigorating of children, and is also called as pseudostellaria heterophylla or juvenile pseudostellaria heterophylla. Radix pseudostellariae is a tonifying traditional Chinese medicine which can be used for health-care food, can be used as a substitute of ginseng or even American ginseng, has huge development space in the traditional Chinese medicine and pharmaceutical industry and the health-care product industry, and has a steadily rising trend in recent years.
The planting of the radix pseudostellariae is concentrated in Guizhou, Fujian, Anhui, Shandong, Jiangsu and other places, and the radix pseudostellariae is mainly bred by root tubers in production. Due to long-term vegetative propagation of root tubers, the viral diseases of the radix pseudostellariae are common and serious, the incidence rate of the viral diseases in partial planting areas is over 90 percent, the disease is a main disease influencing the yield and the quality of the radix pseudostellariae, and great obstacles are caused to the income of farmers and the development of the radix pseudostellariae industry. The types of viruses infecting radix pseudostellariae are many, and Tobacco Mosaic Virus (TMV), turnip mosaic virus (TuMV), Cucumber Mosaic Virus (CMV) and the like are mainly reported, and the agricultural control measures have little effect.
In view of the asexual propagation characteristics of the radix pseudostellariae planting, the cultivation of the detoxified seedlings or the seed chickens is a key means for overcoming the current production situation of the radix pseudostellariae with the toxicity, and related attempts are made by technical personnel, so that the detoxified seedlings or the seed chickens of the radix pseudostellariae can be obtained. The conventional method for detoxifying and cultivating the radix pseudostellariae is complex in operation technology, high in technical requirement on operators and easy to cause bacterial pollution, so that the cultivation cost is high, on the other hand, the field planting density of the radix pseudostellariae is high, nearly 7 thousands of plants per mu are used, the seed consumption is large, and the seed production speed of the radix pseudostellariae is urgently needed to be improved.
Disclosure of Invention
Based on the problems, the tissue culture method of the detoxified radix pseudostellariae root tuber provided by the invention can shorten the seed production time and period, has a good detoxification effect, and has the advantages of high speed, high efficiency, simplicity in operation and low cost.
In order to solve the technical problems, the invention provides the following technical scheme:
A tissue culture method of detoxified radix pseudostellariae root tuber comprises the following steps:
S1: treatment and disinfection of explants
Refrigerating radix Pseudostellariae with robust bud at 1-4 deg.C, and cutting to obtain germinated bud as explant after the bud of radix Pseudostellariae germinates; washing the germinated bud with tap water, air drying, sequentially soaking in ethanol for 30s, 0.1% mercuric chloride for 7-8min, and sterile water for 3-5 times on a clean bench, and transferring to filter paper in a sterile culture dish;
S2: induction of stem tip of pseudostellaria root
Placing the explant processed in the step S1 under a stereoscope, stripping a stem tip of the radix pseudostellariae with the size of 0.5mm from the explant, inoculating the stem tip of the radix pseudostellariae on an induction culture medium for culturing at the temperature of 15-20 ℃, the illumination intensity of 1500-2000lx and the illumination period of 10h/d, and germinating the stem tip of the radix pseudostellariae to give out 1-2 adventitious buds of the radix pseudostellariae after culturing for 20-25 days;
s3: proliferation culture of radix pseudostellariae seedlings
Transferring the adventitious buds of the radix pseudostellariae induced in the step S2 onto a multiplication culture medium for culture, wherein the culture temperature is 15-20 ℃, the illumination intensity is 1500-2000lx, the illumination period is 10h/d, cutting the radix pseudostellariae seedlings into 4-5 sections after the radix pseudostellariae seedlings grow high, and repeating the transfer multiplication culture process by using the radix pseudostellariae seedling sections;
S4: cultivation of root tuber in radix pseudostellariae bottle
Transferring the radix pseudostellariae seedlings obtained in the step S3 to a root tuber culture medium for culture at the temperature of 15-20 ℃, growing 2-3 root tubers at stem nodes of the radix pseudostellariae after culturing for 50-60 days without opening a tissue culture lamp in a culture room, taking out and cleaning when the root tubers grow to be more than 0.5cm, refrigerating at the temperature of 1-4 ℃ for 20-30 days, then directly inoculating to a field, and harvesting from the field to be used as the radix pseudostellariae.
Further, the induction medium in step S2 is an improved MS medium, specifically, the MS medium is supplemented with 0.5-1mg/L6-BA, 0.1mg/L LNAA, 30g/L sucrose and 5g/L agar powder, and the PH value is 5.8.
Further, the proliferation medium in step S3 is an improved 1/2MS medium, specifically, 85mg/L monopotassium phosphate, 30g/L sucrose and 5g/L agar powder are added to the 1/2MS medium, and the PH value is 5.8.
Further, the period of the transfer proliferation culture in step S3 is 18-24 days/generation.
Further, the period of the transfer proliferation culture in step S3 was 20 days/generation.
further, the root tuber culture medium in the step S4 is an improved 1/2MS culture medium, specifically, 85mg/L potassium dihydrogen phosphate, 60g/L sucrose and 5g/L agar powder are added to the 1/2MS culture medium, and the pH value is 5.8.
Further, after the radix pseudostellariae seedlings grow to 4-6cm high in the step S3, the radix pseudostellariae seedlings are cut into 4-5 sections of radix pseudostellariae seedlings.
Compared with the prior art, the invention has the beneficial effects that: the invention can shorten the seed production time and cycle, has good detoxification effect, and has the advantages of high speed, high efficiency, simple operation and low cost; the proliferation culture medium adopts 1/2MS, the addition of potassium dihydrogen phosphate can make the leaf of radix Pseudostellariae seedling more extended and grow more vigorously without adding hormone, and the root tuber induction culture medium adopts 1/2MS with potassium dihydrogen phosphate and high concentration (60g/L) sucrose to make the root tuber more expanded.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example (b):
a tissue culture method of detoxified radix pseudostellariae root tuber comprises the following steps:
s1: treatment and disinfection of explants
Refrigerating radix Pseudostellariae with robust bud at 1-4 deg.C, and cutting to obtain germinated bud as explant after the bud of radix Pseudostellariae germinates; washing the germinated bud with tap water, air drying, sequentially soaking in ethanol for 30s, 0.1% mercuric chloride for 7-8min, and sterile water for 3-5 times on a clean bench, and transferring to filter paper in a sterile culture dish; the contamination rate of the explants treated by the step is not more than 10%;
S2: induction of stem tip of pseudostellaria root
Placing the explant processed in the step S1 under a stereoscope, stripping the stem tip of the radix pseudostellariae with the size of about 0.5mm from the explant, inoculating the stem tip of the radix pseudostellariae on an induction culture medium for culturing at the temperature of 15-20 ℃, the illumination intensity of 1500-2000lx and the illumination period of 10h/d, and after 20-25 days of culturing, sprouting the stem tip of the radix pseudostellariae to give out 1-2 adventitious buds of the radix pseudostellariae; the induction culture medium in the step is an improved MS culture medium, specifically, 0.5-1mg/L6-BA, 0.1mg/L LNAA, 30g/L sucrose and 5g/L agar powder are added to the MS culture medium, the pH value is 5.8, wherein 6-BA is 6-benzylaminopurine, NAA is naphthylacetic acid, and the induction rate of the adventitious buds of the radix pseudostellariae in the step is more than 90%;
S3: proliferation culture of radix pseudostellariae seedlings
Transferring the adventitious buds of the radix pseudostellariae induced in the step S2 onto a proliferation culture medium for culture, wherein the culture temperature is 15-20 ℃, the illumination intensity is 1500-2000lx, the illumination period is 10h/d, the radix pseudostellariae seedlings are cut into 4-5 sections of radix pseudostellariae seedling sections after growing to be high, the radix pseudostellariae seedlings in the embodiment are cut into 4-5 sections of radix pseudostellariae seedling sections after growing to be high to be 4-6cm, the transfer proliferation culture process is repeated by the radix pseudostellariae seedling sections, the transfer proliferation culture period is 18-24 days/generation, namely one generation of transfer proliferation culture is performed every 18-24 days, the transfer proliferation culture period in the embodiment is 20 days/generation, and the proliferation quantity of each generation is increased at a speed of 4-5 times; the multiplication culture medium in the step is an improved 1/2MS culture medium, specifically, 85mg/L potassium dihydrogen phosphate, 30g/L sucrose and 5g/L agar powder are added to the 1/2MS culture medium, the pH value is 5.8, and the improved 1/2MS culture medium in the step is not added with hormones and is added with potassium dihydrogen phosphate, so that the seedlings of the radix pseudostellariae are higher and stronger in length, the leaves of the seedlings of the radix pseudostellariae are more extended, and the seedlings of the radix pseudostellariae grow more vigorously;
S4: cultivation of root tuber in radix pseudostellariae bottle
Transferring the radix pseudostellariae seedlings obtained in the step S3 into a root tuber culture medium for culture, wherein the culture temperature is 15-20 ℃, after the culture room is not provided with a tissue culture lamp for culture for 50-60 days, the stem nodes of the radix pseudostellariae grow 2-3 root tubers, the root tubers grow for 60 days in the embodiment, the root tubers grow to be more than 0.5cm in diameter, the root tubers are taken out and cleaned, the root tubers are directly inoculated into the field after being refrigerated for 20-30 days at the temperature of 1-4 ℃, the root tubers can be used as the radix pseudostellariae seeds after being harvested from the field, and finally, most of the harvested seeds are virus-removed and have high detoxification rate; the root tuber culture medium in the step is an improved 1/2MS culture medium, specifically, 85mg/L potassium dihydrogen phosphate, 60g/L sucrose and 5g/L agar powder are added to the 1/2MS culture medium, the pH value is 5.8, the expansion of the root tuber is facilitated by adding 85mg/L potassium dihydrogen phosphate and 60g/L sucrose in the culture medium, and in addition, a tissue culture lamp is not required to be turned on in the step, so that the production cost is greatly saved.
the above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.
Claims (7)
1. A tissue culture method of detoxified radix pseudostellariae root tuber is characterized by comprising the following steps:
S1: treatment and disinfection of explants
refrigerating radix Pseudostellariae with robust bud at 1-4 deg.C, and cutting to obtain germinated bud as explant after the bud of radix Pseudostellariae germinates; washing the germinated bud with tap water, air drying, sequentially soaking in ethanol for 30s, 0.1% mercuric chloride for 7-8min, and sterile water for 3-5 times on a clean bench, and transferring to filter paper in a sterile culture dish;
S2: induction of stem tip of pseudostellaria root
Placing the explant processed in the step S1 under a stereoscope, stripping a stem tip of the radix pseudostellariae with the size of 0.5mm from the explant, inoculating the stem tip of the radix pseudostellariae on an induction culture medium for culturing at the temperature of 15-20 ℃, the illumination intensity of 1500-2000lx and the illumination period of 10h/d, and germinating the stem tip of the radix pseudostellariae to give out 1-2 adventitious buds of the radix pseudostellariae after culturing for 20-25 days;
S3: proliferation culture of radix pseudostellariae seedlings
transferring the adventitious buds of the radix pseudostellariae induced in the step S2 onto a multiplication culture medium for culture, wherein the culture temperature is 15-20 ℃, the illumination intensity is 1500-2000lx, the illumination period is 10h/d, cutting the radix pseudostellariae seedlings into 4-5 sections after the radix pseudostellariae seedlings grow high, and repeating the transfer multiplication culture process by using the radix pseudostellariae seedling sections;
s4: cultivation of root tuber in radix pseudostellariae bottle
Transferring the radix pseudostellariae seedlings obtained in the step S3 to a root tuber culture medium for culture at the temperature of 15-20 ℃, growing 2-3 root tubers at stem nodes of the radix pseudostellariae after culturing for 50-60 days without opening a tissue culture lamp in a culture room, taking out and cleaning when the root tubers grow to be more than 0.5cm, refrigerating at the temperature of 1-4 ℃ for 20-30 days, then directly inoculating to a field, and harvesting from the field to be used as the radix pseudostellariae.
2. The tissue culture method of the apostichopus japonicus tuberous roots according to claim 1, wherein the induction medium in the step S2 is a modified MS medium, specifically, 0.5-1mg/L6-BA, 0.1mg/L LNAA, 30g/L sucrose and 5g/L agar powder are added to the MS medium, and the pH value is 5.8.
3. The tissue culture method of the detoxified radix pseudostellariae root tuber according to claim 1, wherein the multiplication medium in step S3 is an improved 1/2MS medium, specifically, the 1/2MS medium is supplemented with 85mg/L monopotassium phosphate, 30g/L sucrose and 5g/L agar powder, and the PH value is 5.8.
4. the tissue culture method of the root tuber of pseudostellaria yunnanensis of claim 1, wherein the period of the transfer proliferation culture in step S3 is 18-24 days/generation.
5. The tissue culture method of the root tuber of pseudostellaria yunnanensis of claim 4, wherein the period of the transfer proliferation culture in step S3 is 20 days/generation.
6. The tissue culture method of the apostichopus japonicus tuberous root according to claim 1, wherein the tuberous root culture medium in the step S4 is an improved 1/2MS culture medium, specifically, the 1/2MS culture medium is added with 85mg/L potassium dihydrogen phosphate, 60g/L sucrose and 5g/L agar powder, and the pH value is 5.8.
7. The tissue culture method of the detoxified radix pseudostellariae root tuber according to claim 1, wherein the radix pseudostellariae seedlings in step S3 are cut into 4-5 sections of radix pseudostellariae seedlings after growing to 4-6cm high.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115885854A (en) * | 2022-12-22 | 2023-04-04 | 宁德师范学院 | Method for removing pseudostellaria heterophylla virus by combining low-temperature induced germination and micro-stem tip culture |
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| CN108713487A (en) * | 2018-06-08 | 2018-10-30 | 宁德师范学院 | A kind of method that numerous radix pseudostellariae virus-free seed ginseng is expanded in soilless culture |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115885854A (en) * | 2022-12-22 | 2023-04-04 | 宁德师范学院 | Method for removing pseudostellaria heterophylla virus by combining low-temperature induced germination and micro-stem tip culture |
| CN115885854B (en) * | 2022-12-22 | 2024-01-26 | 宁德师范学院 | Method for removing pseudostellaria viruses by low-temperature induction germination combined with micro-stem tip culture |
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