CN110679489A - Rapid propagation method of traditional Chinese medicine plant ancient uncaria - Google Patents
Rapid propagation method of traditional Chinese medicine plant ancient uncaria Download PDFInfo
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- CN110679489A CN110679489A CN201911176415.3A CN201911176415A CN110679489A CN 110679489 A CN110679489 A CN 110679489A CN 201911176415 A CN201911176415 A CN 201911176415A CN 110679489 A CN110679489 A CN 110679489A
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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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- 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
Abstract
The invention discloses a tissue culture and rapid propagation method of traditional Chinese medicine ancient gambir plant, which comprises the following steps in sequence: obtaining an explant of uncaria rhynchophylla, inducing embryonic callus, inducing and culturing adventitious buds, carrying out proliferation culture on the adventitious buds, carrying out rooting culture, and transplanting; the culture medium for the induction culture of the embryogenic callus comprises: 1/2MS culture medium, 2-2.5 mg/L indolebutyric acid, 30mg/L adenine, 20-30 mg/L hydrolyzed casein, 30-40 g/L sucrose, 3.5-4.5 g/L agar, and the pH value is 5.5-5.8. The induction rate of the method reaches more than 80%, the transplanting survival rate reaches more than 85%, and the tissue culture and rapid propagation of the ancient uncaria are realized.
Description
Technical Field
The invention relates to a method for plant tissue culture in agricultural biotechnology, in particular to a tissue culture and rapid propagation method of a traditional Chinese medicine ancient gambir plant.
Background
Ancient ramulus Uncariae cum uncis (Cryptolepis buchananii Roem. et Schult), also known as Sedum maritime, caulis et folium Hedyotis Hedyotideae, caulis et folium Callicarpae Hortoriae, radix et rhizoma Rhei Limoniae, etc., is a plant of the genus Cynanchum of the family Asclepiadaceae. The fertilizer is mainly distributed in places such as Guangdong, Guangxi, Yunnan and the like in China, and is distributed in places such as India, Burma, Srilanka, Vietnam and the like abroad. Has effects in relaxing muscles and tendons, activating collateral flow, relieving swelling and pain, and removing toxic materials, and can be used for treating lumbago, abdominal pain, traumatic injury, fracture, carbuncle, sore, and tinea. The uncaria rhynchophylla has wide prospects in clinical and folk application in treatment of tumors, asthma, cough, rheumatoid arthritis and the like.
At present, researches on ancient uncaria are mainly in the aspects of drug effect, toxicology and the like, and related researches on artificial propagation and planting technologies are not reported. The problem to be solved for expanding artificial planting is the technical problems of breeding, breeding and cultivation of excellent germplasm. Therefore, the establishment of the tissue culture and rapid propagation method of the ancient uncaria is particularly important.
Disclosure of Invention
The invention aims to provide a tissue culture and rapid propagation method of traditional Chinese medicine uncaria rhynchophylla, which selects the top end or the young tip of the uncaria rhynchophylla in winter and spring as an explant, and performs the steps of embryogenic callus induction, adventitious bud proliferation, rooting culture, transplantation and the like, wherein the induction rate is more than 80%, the transplantation survival rate is more than 85%, and the tissue culture and rapid propagation of the uncaria rhynchophylla is realized.
The technical scheme provided by the invention is as follows: a tissue culture and rapid propagation method of traditional Chinese medicine ancient gambir plant comprises the following steps in sequence: obtaining an explant of the uncaria rhynchophylla, inducing embryonic callus, performing adventitious bud induction culture, performing adventitious bud proliferation culture, performing rooting culture and transplanting; the culture medium for the induction culture of the embryogenic callus comprises: 1/2MS culture medium, 2-2.5 mg/L indolebutyric acid (IBA), 30mg/L adenine, 20-30 mg/L hydrolyzed Casein (CH), 30-40 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.5-5.8.
In the tissue culture and rapid propagation method of the traditional Chinese medicine ancient uncaria, the method for the induction culture operation of the embryonic callus comprises the following steps: picking strong terminal buds or young shoots of the ancient uncaria which are collected and used as explants in winter and spring to be about 1-1.5 cm long, stripping young leaves, trimming to about 8mm, sterilizing, shearing to form stem tips with the size of 2-5 mm, inoculating to an induction culture medium, and culturing in a dark and full black environment at 25 ℃ for 40-50 days to induce and form embryonic callus.
In the tissue culture and rapid propagation method of the uncaria rhynchophylla, the induction culture medium used for the induction culture of the adventitious buds comprises: 1/2MS culture medium, 2-2.5 mg/L Zeatin (ZT), 2-2.5 mg/L Cytokinin (CTK), 30-40 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.4-5.7. Culturing in a culture medium for 40-50 days to induce the formation of adventitious buds.
In the tissue culture and rapid propagation method of the uncaria rhynchophylla, the propagation culture medium used in the propagation culture operation of the adventitious buds comprises: 1/2MS culture medium, 3-3.5 mg/L Zeatin (ZT), 30-40 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.4-5.7. The adventitious bud multiplication culture operation specifically comprises the following steps: the adventitious bud obtained by the adventitious bud induction culture is cut into stem sections with the length of 1.5-2.0 cm, and the stem sections are inoculated into a proliferation culture medium for culturing for 30-40 days, so that the proliferation of the adventitious bud can be realized.
In the tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant, the rooting culture medium used in the rooting culture operation comprises the following components: WS medium, 30-50 mg/L hydrolyzed Casein (CH), 0.5-1.0 mg/L Ethylene (ETH), 20-30 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.4-5.8.
In the tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant, the rooting culture operation method comprises the following steps: and (3) cutting the adventitious bud of 2.0-3.0 cm from the base part of the adventitious bud obtained after proliferation culture of the adventitious bud, inoculating the adventitious bud into a rooting culture medium, and culturing for 40-50 days to realize rooting of the test-tube plantlet.
In the tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant, the specific method of the transplanting operation is as follows: hardening the test-tube plantlets with good root growth obtained by rooting culture operation under natural light of a greenhouse for 10 days, transplanting the culture medium with the cleaned roots into humus soil and vermiculite (the mass ratio is 1:4) to cultivate the test-tube plantlets into seedlings, and obtaining the ancient uncaria seedlings.
In the tissue culture and rapid propagation method of the ancient uncaria, the culture conditions in the adventitious bud multiplication culture operation and the rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
Has the advantages that:
the invention adopts plant tissue culture technology to establish the tissue culture and rapid propagation of the ancient uncaria, has the characteristics of short period, low cost, high propagation coefficient, consistent seedling growth and the like, can be directly used for the industrial production of the ancient uncaria seedlings, and has great significance for promoting the popularization and the planting of the ancient uncaria and protecting wild ancient uncaria plant resources.
Detailed Description
Example one
(1) Explant collection: selecting the top end or young tip of strong-growing uncaria rhynchophylla in winter and spring as an explant in the wild of Shoping county in Guangxi, collecting, preserving water and moisture, and bringing back to a laboratory in time.
(2) Induction of embryogenic callus: and (2) removing young leaves of the explants collected in the step (1) back to the laboratory, soaking the young leaves in saturated bleaching powder supernatant for 3-5 minutes, washing the explants with sterile water for 5 times, then sucking the water with sterile filter paper, disinfecting the explants in 0.5% sodium hypochlorite solution for 10 minutes, washing the explants with sterile water for 8 times, then sucking the water with sterile filter paper, carefully peeling the shoots into 2-5 mm-sized stem tips, inoculating the stem tips into an induction culture medium, and culturing the shoots in 25 ℃ all-black dark for 40 days to induce and form embryonic callus, wherein the induction rate is 88%. The induction culture medium is as follows: 1/2MS +2.5mg/L IBA +30mg/L adenine +30mg/L CH +40g/L sucrose +4.5g/L agar, and the pH value is 5.5-5.8.
(3) Adventitious bud induction culture: inoculating the embryogenic callus obtained by culturing in the step (2) into an adventitious bud induction culture medium for culturing for 40 days to induce adventitious buds. The adventitious bud induction culture medium comprises: 1/2MS +2.5mg/L ZT +2.5mg/LCTK +40g/L sucrose +4.5g/L agar, and the pH is 5.4-5.7.
(4) And (3) adventitious bud multiplication culture: and (3) cutting the adventitious bud obtained by induction in the step (2) into stem sections with the length of about 1.5-2.0 cm, inoculating the stem sections into an adventitious bud multiplication culture medium, and culturing for 30 days to realize the multiplication of the adventitious bud, wherein the multiplication coefficient reaches 6.2. The adventitious bud multiplication culture medium comprises: 1/2MS +3.5mg/L ZT +40g/L sucrose +4.5g/L agar, and the pH is 5.4-5.7.
(5) Rooting culture: and (3) cutting the adventitious bud with the length of about 2.0-3.0 cm obtained by proliferation in the step (4) from the base part, inoculating the adventitious bud into a rooting culture medium, and culturing for 40 days to realize rooting of the test-tube plantlet, wherein the rooting rate reaches 90%. The rooting culture medium comprises: WS +50mg/L CH +1.0mg/L ETH +30g/L sucrose +4.5g/L agar, and pH is 5.4-5.7.
(6) Transplanting: and (3) hardening the test-tube seedlings with good root growth in the step (5) under natural light of a greenhouse for 10 days, transplanting the culture medium with cleaned roots into humus soil and vermiculite (the mass ratio is 1:4) to be cultivated into seedlings, namely the ancient uncaria seedlings are obtained, and the survival rate reaches 92% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
Example two
(1) Explant collection: selecting the top end or young tip of strong-growing uncaria rhynchophylla in winter and spring as an explant in the wild of Guangxi Jinxiu county, collecting, preserving water and moisture, and bringing back to a laboratory in time.
(2) Induction of embryogenic callus: and (2) removing young leaves of the explants collected in the step (1) back to the laboratory, soaking the young leaves in a saturated bleaching powder supernatant for 3-5 minutes, washing the explants with sterile water for 5 times, then sucking the water with sterile filter paper, disinfecting the explants in a 0.5% sodium hypochlorite solution for 10 minutes, washing the explants with sterile water for 8 times, then sucking the water with sterile filter paper, carefully peeling the shoots into 2-5 mm-sized stem tips, inoculating the stem tips into an induction culture medium, and culturing the shoots in a 25 ℃ full-black dark environment for 45 days to induce the adventitious buds, wherein the induction rate reaches 85%. The induction culture medium is as follows: 1/2MS +2.3mg/L IBA +30mg/L adenine +25mg/L CH +35g/L sucrose +4.0g/L agar, and the pH value is 5.5-5.8.
(3) Adventitious bud induction culture: inoculating the embryogenic callus obtained by culturing in the step (2) into an adventitious bud induction culture medium for culturing for 45 days to induce adventitious buds. The adventitious bud induction culture medium comprises: 1/2MS +2.3mg/L ZT +2.3mg/LCTK +35g/L sucrose +4.0g/L agar, and the pH is 5.4-5.7.
(4) And (3) adventitious bud multiplication culture: and (4) cutting the adventitious bud obtained by induction in the step (3) into stem sections with the length of about 1.5-2.0 cm, inoculating the stem sections into an adventitious bud multiplication culture medium, and culturing for 35 days to realize the multiplication of the adventitious bud, wherein the multiplication coefficient reaches 5.5. The adventitious bud multiplication culture medium comprises: 1/2MS +3.3mg/L ZT +35g/L sucrose +4.0g/L agar, and the pH is 5.4-5.7.
(5) Rooting culture: and (3) cutting the adventitious bud which is obtained by proliferation in the step (4) and has the length of about 2.0-3.0 cm from the base part, inoculating the adventitious bud into a rooting culture medium, and culturing for 45 days to realize rooting of the test-tube plantlet, wherein the rooting rate is more than 88%. The rooting culture medium comprises: WS +40mg/L CH +0.8mg/L ETH +25g/L sucrose +4.0g/L agar, and the pH value is 5.4-5.7.
(6) Transplanting: and (3) hardening the test-tube seedlings with good root growth in the step (5) under natural light of a greenhouse for 10 days, transplanting the culture medium with cleaned roots into humus soil and vermiculite (the mass ratio is 1:4) to be cultivated into seedlings, namely the ancient uncaria seedlings are obtained, and the survival rate reaches 90% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
EXAMPLE III
(1) Explant collection: selecting the top end or young tip of strong-growing uncaria rhynchophylla in winter and spring as an explant in the wild of Guangxi Luchuan county, collecting, preserving water and moisture, and bringing the explant back to a laboratory in time.
(2) Induction of embryogenic callus: and (2) removing young leaves of the explants collected in the step (1) back to the laboratory, soaking the young leaves in a saturated bleaching powder supernatant for 3-5 minutes, washing the explants with sterile water for 5 times, then sucking the water with sterile filter paper, disinfecting the explants in a 0.5% sodium hypochlorite solution for 10 minutes, washing the explants with sterile water for 8 times, then sucking the water with sterile filter paper, carefully peeling the shoots into 2-5 mm-sized stem tips, inoculating the stem tips into an induction culture medium, and placing the shoots in a 25 ℃ full-black dark culture for 50 days to induce the adventitious buds, wherein the induction rate reaches 88%. The induction culture medium comprises: 1/2MS +2mg/L IBA +30mg/L adenine +20mg/L CH +30g/L sucrose +3.5g/L agar, pH 5.5-5.8
(3) Adventitious bud induction culture: inoculating the embryonic callus obtained by culturing in the step (2) into an adventitious bud induction culture medium for culturing for 50 days to induce adventitious buds. The adventitious bud induction culture medium comprises: 1/2MS +2.0mg/L ZT +2.0mg/L CTK +30g/L sucrose +3.5g/L agar, and the pH is 5.4-5.7.
(4) And (3) adventitious bud multiplication culture: and (4) cutting the adventitious bud obtained by induction in the step (3) into stem sections with the length of about 1.5-2.0 cm, inoculating the stem sections into an adventitious bud multiplication culture medium, and culturing for 40 days to realize the multiplication of the adventitious bud, wherein the multiplication coefficient reaches 4.8. The adventitious bud multiplication culture medium comprises: 1/2MS +3.0mg/L ZT +30g/L sucrose +3.5g/L agar, and the pH is 5.4-5.7.
(5) Rooting culture: and (4) cutting the adventitious bud which is obtained by proliferation in the step (3) and has the length of about 2.0-3.0 cm from the base part, inoculating the adventitious bud into a rooting culture medium, and culturing for 50 days to realize rooting of the test-tube plantlet, wherein the rooting reaches more than 85%. The rooting culture medium comprises: WS +30mg/L CH +0.5mg/L ETH +20g/L sucrose +3.5g/L agar, and the pH value is 5.4-5.7.
(6) Transplanting: and (3) hardening the test-tube seedlings with good root growth in the step (5) under natural light of a greenhouse for 10 days, transplanting the culture medium with cleaned roots into humus soil and vermiculite (the mass ratio is 1:4) to be cultivated into seedlings, namely the ancient uncaria seedlings are obtained, and the survival rate reaches 85% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A tissue culture and rapid propagation method of traditional Chinese medicine ancient gambir plant is characterized by comprising the following steps in sequence: obtaining an explant of uncaria rhynchophylla, inducing embryonic callus, inducing and culturing adventitious buds, carrying out proliferation culture on the adventitious buds, carrying out rooting culture, and transplanting; the culture medium for the induction culture of the embryogenic callus comprises: 1/2MS culture medium, 2-2.5 mg/L indolebutyric acid, 30mg/L adenine, 20-30 mg/L hydrolyzed casein, 30-40 g/L sucrose, 3.5-4.5 g/L agar, and the pH value is 5.5-5.8.
2. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 1, which is characterized in that the method for the induction culture operation of the embryonic callus comprises the following steps: picking up 1-1.5 cm long terminal buds or young shoots of the collected ancient uncaria used as an explant in winter and spring, stripping young leaves, trimming to 8mm, sterilizing, shearing to 2-5 mm stem tips, inoculating to an induction culture medium, and culturing in a dark environment at 25 ℃ for 40-50 days to induce and form embryogenic callus.
3. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 1, wherein the induction culture medium used for the induction culture of the adventitious bud comprises: 1/2MS culture medium, 2-2.5 mg/L zeatin, 2-2.5 mg/L cytokinin, 30-40 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.4-5.7.
4. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 1, wherein the propagation culture medium used for the propagation culture operation of the adventitious bud comprises: 1/2MS culture medium, 3-3.5 mg/L zeatin, 30-40 g/L sucrose, 3.5-4.5 g/L agar, and pH is 5.4-5.7.
5. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 4, which is characterized in that the adventitious bud multiplication culture operation specifically comprises the following steps: cutting the adventitious bud obtained by adventitious bud induction culture into stem sections with the length of 1.5-2.0 cm, inoculating the stem sections into a proliferation culture medium, and culturing for 30-40 days to realize the proliferation of the adventitious bud; the culture conditions were all: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
6. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 1, wherein the rooting culture medium used in the rooting culture operation comprises: WS culture medium, 30-50 mg/L hydrolyzed casein, 0.5-1.0 mg/L ethylene, 20-30 g/L sucrose and 3.5-4.5 g/L agar, wherein the pH value is 5.4-5.8.
7. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 6, which is characterized in that the rooting culture operation method comprises the following steps: cutting 2.0-3.0 cm of adventitious buds from the base parts of the adventitious buds obtained after proliferation culture and proliferation, inoculating the adventitious buds into a rooting culture medium, and culturing for 40-50 days to realize rooting of the test-tube plantlet; the culture conditions were all: the culture temperature is 25-28 ℃, the illumination intensity is 2500-2800 lx, and the illumination time is 13-15 hours/day.
8. The tissue culture and rapid propagation method of the traditional Chinese medicine ancient gambir plant according to claim 1, which is characterized in that the specific method of the transplanting operation is as follows: and (3) hardening the test-tube plantlets with good root growth obtained by rooting culture operation under natural light of a greenhouse for 10 days, and transplanting the culture medium with cleaned roots into humus and vermiculite to cultivate into seedlings, thus obtaining the ancient uncaria seedlings.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111165358A (en) * | 2020-03-13 | 2020-05-19 | 临沧惠康中药材资源开发有限公司 | Method for inducing tetraploid plant by using adventitious bud of uncaria |
| CN115005096A (en) * | 2022-05-26 | 2022-09-06 | 桂林医学院 | Ethnic medicine alangium platanifolium seedling breeding method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111165358A (en) * | 2020-03-13 | 2020-05-19 | 临沧惠康中药材资源开发有限公司 | Method for inducing tetraploid plant by using adventitious bud of uncaria |
| CN115005096A (en) * | 2022-05-26 | 2022-09-06 | 桂林医学院 | Ethnic medicine alangium platanifolium seedling breeding method |
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