CN105230482A - Method for establishing in-vitro regeneration system of Acrostichum aureurm - Google Patents
Method for establishing in-vitro regeneration system of Acrostichum aureurm Download PDFInfo
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Abstract
The invention discloses a method for establishing an in-vitro regeneration system of Acrostichum aureurm. According to the method, sporangiorus is used as explant; after surface disinfection, the explant is inoculated into a variety of mediums containing a 1/4 MS basal medium; prothallus is obtained through in-vitro germination of spores in sporangium; a part of the prothallus can further undergo enrichment culture, while the other part of the prothallus can be used for induced production of juvenile sporophyte; and a sapling is transplanted into a matrix after rooting culture, and the seedling of Acrostichum aureurm is formed eventually. The method can obtain a great number of high-quality seedlings in a short period of time, fills a technical blank in the prior art and provides powerful technical guarantee for garden cultivation, research and development of traditional Chinese medicine and ecological restoration of mangrove forest.
Description
Technical field
The invention belongs to agricultural biological technical field, the method that the halogen fern vitro Regeneration System of to be a kind of with sporangiorus be explant is set up.
Background technology
Halogen fern (
acrostichumaureaml.) be Acrostichaceae halogen Cyclosorus plant, be distributed in Asia, Africa, America, the torrid zone, Oceania and subtropical zone.Often being grown on Tidal Mangrove Species In China inner edge, seashore or tide energy embankment limit extremely, is the important symbol kind of Coastal beach.
The ornamental value that halogen fern tool is higher.Plant is tall and big tall and straight, can reach 2m.Root-like stock is upright, and top is close by brown brown wealthy lanceolar scale.Leaf heavy leather matter, clusters; Radix winglike compound leaf, it is 30 right that accessory pinna reaches, and the long 30 ~ 60cm of petiole, slightly can reach 2cm; Blade long 60 ~ 140cm, wide 30 ~ 60cm; Vein is netted, and two sides is visible.Sporangium is abound with below the fertile pinna of energy top.Halogen fern has very large development potentiality as horticultural species cultivation.
The medical value of halogen fern is also very high.In the traditional herbal medicines of halogen fern among the people as treatment wound, hemostasis, rheumatism, invermination, constipation, elephantiasis etc.Halogen fern not only has good antibacterial ability, and has very high antitumor activity.Wear the rich ethyl acetate extract activity waiting (Chinese Journal of Marine Drugs, 2005,24 (6): 44-46) to find halogen fern comparatively strong, IC50 reaches 6.3 μ g/ml, inhibits the growth activity of HeLa cell.Zhong Xiao etc. (Guangxi Teachers College's journal: natural science 2012,29 (3): 41-45) find that the main chemical compositions of halogen fern is the compound of steroid, triterpenes, flavonoids, glycoside, monose, amino acid and sulfuric acid ester.These compounds all have certain antibacterial, anti-inflammatory, the biologically active such as antitumor, anti-oxidant.These researchs show that halogen fern is the good material of developing anti-tumor medicaments and novel antibacterials.
Halogen fern can be able to play a significant role in mangrove forest ecological recovers.In 50 ~ nineties of 20th century, Chinese red woods area falls sharply 68.7%, and in existing standing forest, more than 80% is degeneration secondary forest, on the spot environmental deterioration.Plant at present for Mangrove restoration is all obtained by seminal propagation and the breeding of viviparous seedling, as pteridophyte monoid halogen Cyclosorus unique in true mangrove plant (
acrostichum) utilization rarely found, this is relevant with the seeling industry technical bottleneck of halogen fern, at present, there is not yet the report of halogen fern vitro Regeneration System method for building up.
Summary of the invention
The object of the invention is to provide a kind of method setting up halogen fern vitro Regeneration System, namely with the ripe sporangiorus of halogen fern for explant, through explant surface sterilization, spore axenic germination, prothallium propagation, sporophyte induction, hardening and transplanting and other steps, set up halogen fern vitro Regeneration System, realize the Fast-propagation object of halogen fern.
The present invention is realized by following technical proposals.Halogen fern (
acrostichumaureaml.) method of vitro Regeneration System foundation, comprises following sequential steps:
(1) explant collection and surface sterilization: clip halogen fern sporophyll, carries out surface sterilization.
(2) spore axenic germination: be separated the sporangiorus on sporophyll, be inoculated on spore germination aseptic culture medium and cultivate.Spore germination aseptic culture based formulas is: 1/4MS minimal medium+0 ~ 20g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+ agar 7g/L.
(3) prothallium Multiplying culture: prothallium montage step (2) obtained carries out prothallium Multiplying culture in prothallium proliferated culture medium.Every 50d changes a prothallium proliferated culture medium.Prothallium proliferation culture medium formula is: 1/4MS minimal medium+15g/L sucrose+6-BA0 ~ 0.6mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L.
(4) induction of sporophyte: prothallium step (3) formed pulverizes switching, induction produces juvenile sporophyte.Sporophyte Fiber differentiation based formulas is: 1/4MS minimal medium+gibberellin (GA
3) 0 ~ 1.2mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
(5) culture of rootage and hardening: separated by the juvenile sporophyte that step (4) is induced, switching induction juvenile sporophyte (seedling) is taken root.Culture medium prescription is: 1/4MS minimal medium+15g/L sucrose+2,4-D0.05mg/L or naa (NAA) 0.05mg/L+ agar 7g/L.After finding that the adventive root tip of a root is outstanding, unclamp in culturing room immediately, be left unlocked or unlatched bottle cap, hardening 10d.
(6) transplant: the seedling of taking out step (5), wash away the gel entrapment culture base of root attachment, seedling replanting is entered in the matrix of high-temperature sterilization.Regularly water, cover film, keeps relative moisture more than 90%.
Above-mentioned 1/4MS minimal medium formula is: NH
4nO
3412.5mg/L, KNO
3475mg/L, KH
2pO
4100mg/L, MgSO
47H
2o92.5mg/L, CaCl
22H
2o110mg/L, KI0.83mg/L, H
3bO
36.2mg/L, MnSO44H
2o22.3mg/L, ZnSO
47H
2o8.6mg/L, Na
2moO
42H
2o0.25mg/L, CuSO
45H
2o0.025mg/L, CoCl
26H
2o0.025mg/L, FeSO
47H
2o6.95mg/L, Na
2-EDTA2H
2o9.325mg/L.Agar medium pH value is all adjusted to 5.8.
Above-mentioned condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
In above-mentioned steps (1), surface sterilization condition is preferably as follows: first use the alcohol-pickled 20s of 70%, abandon alcohol, add 0.1% mercuric chloride (HgCl
2) sterilization 2.5min.
Above-mentioned steps (2) spore axenic germination culture medium prescription is preferably: 1/4MS minimal medium+10g/L sucrose+6-BA0.2mg/L+ agar 7g/L
Above-mentioned steps (3) prothallium proliferation culture medium formula is preferably: 1/4MS minimal medium+15g/L sucrose+6-BA0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.
The Fiber differentiation based formulas of above-mentioned steps (4) sporophyte is preferably: 1/4MS minimal medium+GA
30.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.Vaccination ways is preferably the inoculation of fragment shape.
Above-mentioned steps (5) seedling prescription of rooting medium is preferably: 1/4MS minimal medium+15g/L sucrose+2,4-D0.05mg/L+ agar 7g/L.
The present invention establishes the method for halogen fern vitro Regeneration System first, has filled up prior art blank; Use the method for Fast-propagation of the present invention, highly intensive and high density factorial praluction can be realized, can obtain the halogen fern seedling of a large amount of high-quality within short-term, be the reliable technical guarantee that horticultural production, Chinese medicine cultivation and tropical Coast Mangrove restoration of the ecosystem provide.
Accompanying drawing explanation
Fig. 1 halogen fern prothallium cultivation effect figure.
Fig. 2 halogen fern sporophyte inducing effect figure.
Fig. 3 halogen fern transplants design sketch.
Embodiment
Prepare 1/4MS minimal medium, its formula is: NH
4nO
3412.5mg/L, KNO
3475mg/L, KH
2pO
4100mg/L, MgSO
47H
2o92.5mg/L, CaCl
22H
2o110mg/L, KI0.83mg/L, H
3bO
36.2mg/L, MnSO44H
2o22.3mg/L, ZnSO
47H
2o8.6mg/L, Na
2moO
42H
2o0.25mg/L, CuSO
45H
2o0.025mg/L, CoCl
26H
2o0.025mg/L, FeSO
47H
2o6.95mg/L, Na
2-EDTA2H
2o9.325mg/L.Agar medium pH value is all adjusted to 5.8.
The method that halogen fern culture in vitro system is set up, comprises the steps:
(1) explant collection and surface sterilization process: the ripe sporophyll of clip halogen fern is taken back in valve bag, and leaf back sporangiorus is dark brown, processes fresh material in time.Sporophyll surface sterilization: sporophyll is laterally cut to the ribbon small fragment of 1 × 3 ~ 4cm, puts in 100ml wide-mouth bottle, and saturated washing powder solution soaks, and add 1 Tween-80, lid bottle cap, embathes 20min, period every 3 ~ 5min is shaking by swirling bottle several gently; Running water slowly rinses sporophyll 10min; On superclean bench, abandon running water in bottle, add the alcohol-pickled 20s of 70%, abandon alcohol, add 0.1%HgCl
2sterilization 2.5min, then with aseptic water washing 5 times, for subsequent use.
(2) spore axenic germination: with the sporangiorus on scalpel scraping sporophyll, disperseed to be inoculated on spore axenic germination medium and cultivate.Spore axenic germination culture medium prescription is: 1/4MS minimal medium+0 ~ 20g/L sucrose+6-BA0.2mg/L+ agar 7g/L.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
(3) prothallium Multiplying culture: the prothallium that step (2) spore germination is formed is trimmed in prothallium proliferated culture medium and carries out prothallium Multiplying culture.Every 50d changes a subculture.Prothallium proliferation culture medium formula is: 1/4MS minimal medium+15g/L sucrose+6-BA0 ~ 0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
(4) Fiber differentiation of sporophyte: prothallium step (3) formed is pulverized and is forwarded to sporophyte inducing culture induction generation juvenile sporophyte.Cultivating basigamy formula is: 1/4MS minimal medium+GA
30 ~ 1.2mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
(5) culture of rootage and hardening: separated by the juvenile sporophyte that step (4) is induced, is forwarded to root media inducing spore body and forms adventive root.Cultivating basigamy formula is: 1/4MS minimal medium+15g/L sucrose+2,4-D0.05mg/L+ agar 7g/L.After the 10-15d naked eyes visible juvenile sporophyte adventive root tip of a root is just outstanding, unclamps in culturing room immediately, be left unlocked or unlatched bottle cap 10d, period culturing gene moisture comparatively fast consume and shrink, but unlikely dry, sporophyte structure differentiation is more perfect.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
(6) transplant: the sporophyte seedling of taking out step (5), wash away the gel entrapment culture base of root attachment, entered by seedling replanting in the matrix of high-temperature sterilization, matrix is Cao Tan ︰ vermiculite=3 ︰ 1.Regularly water, cover film, keeps relative moisture more than 90%.
Below in conjunction with embodiment, detailed, complete description is further done to the present invention:
embodiment 1
Explant collection, surface sterilization and inoculation: select the fresh halogen fern sporophyll that sporangiorus is deeply brown, take back in clip sporophyll valve bag, process fresh material in time.Sporophyll surface sterilization: it is the ribbon fragment of 1 × 3 ~ 4cm that sporophyll is cut out, and puts in 100ml wide-mouth bottle, and saturated washing powder solution soaks, and add 1 Tween-80, lid bottle cap, embathes 20min, shaking by swirling bottle is for several times gently for period every 3 ~ 5min; Wide-mouth bottleneck is pricked with gauze, slowly rinses sporophyll 10min with running water, liquid clarification to bottle; On superclean bench, abandon running water in bottle, add the alcohol-pickled 20s of 70%, abandon alcohol, add 0.1%HgCl
2sterilization 2.5min, then with aseptic water washing 5 times, for subsequent use.With the sporangiorus of the scalpel scraping sporophyll outside of belly, be connected to spore axenic germination media surface, added a little sterile water, slight oscillatory, make sporangium disperse to be laid on medium to cultivate.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
Spore axenic germination culture medium prescription is:
A) 1/4MS minimal medium+0/L sucrose+6-BA0.2mg/L+ agar 7g/L.
B) 1/4MS minimal medium+5g/L sucrose+6-BA0.2mg/L+ agar 7g/L.
C) 1/4MS minimal medium+10g/L sucrose+6-BA0.2mg/L+ agar 7g/L.
D) 1/4MS minimal medium+15g/L sucrose+6-BA0.2mg/L+ agar 7g/L.
Cultivate after 50d, result is as follows: in all medium, all has a large amount of spore germination to give prominence to spore cyst wall, forms heart-shaped prothallium, the thickness that difference is heaped only for the color of prothallium, transparency and prothallium.
A) prothallium oyster, more transparent, without heaping.
B) prothallium yellow green, slightly transparent, without heaping.
C) prothallium is green, opaque, has the phenomenon of heap, but easily disperses during switching.
D) prothallium bottle green, opaque, heaps sense obviously, and prothallium caking not easily disperses.
Can be learnt by above-mentioned cultivation results: during the formula of 1/4MS minimal medium forms, the growth conditions of sucrose on spore germination, prothallium has to be affected more significantly.Spore axenic germination culture medium prescription is preferably: 1/4MS minimal medium+10g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+ agar 7g/L.Sucrose (0 or 5g/L), although to cultivate prothallium quantity maximum, grows barren, next step cultivation unfavorable.
embodiment 2
Prothallium inoculation prothallium embodiment 1 obtained is trimmed in prothallium proliferated culture medium and carries out prothallium Multiplying culture.Every 50d changes a subculture.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
Prothallium proliferation culture medium formula is:
A) 1/4MS minimal medium+15g/L sucrose+6-BA0mg/L+2,4-D0.1mg/L+ agar 7g/L.
B) 1/4MS minimal medium+15g/L sucrose+6-BA0.2mg/L+2,4-D0.1mg/L+ agar 7g/L.
C) 1/4MS minimal medium+15g/L sucrose+6-BA0.4mg/L+2,4-D0.1mg/L+ agar 7g/L.
D) 1/4MS minimal medium+15g/L sucrose+6-BA0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.
After cultivating 50d, result is as follows:
A) prothallium fragment can form new prothallium, but limited amount.Prothallium outside of belly rhizoid is flourishing.
B), c), d) prothallium fragment can form new prothallium, as shown in Figure 1, variant in the density of prothallium, with d) intensive, form the lumpiness of likeness in form callus, c) also comparatively dense, but easily disperse during follow-up cultivation operation, better effects if.
Can be learnt by above-mentioned cultivation results: during the formula of 1/4MS minimal medium forms, the propagation of 6-BA on prothallium has to be affected more significantly.Prothallium proliferation culture medium formula is preferably: 1/4MS minimal medium+15g/L sucrose+6-BA0.4mg/L+2,4-D0.1mg/L+ agar 7g/L.
embodiment 3
Prothallium embodiment 2 obtained is pulverized and is inoculated into the formation of inducing seedling in sporophyte inducing culture.Every 50d changes a subculture.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
Sporophyte Fiber differentiation based formulas is:
A) 1/4MS minimal medium+GA
30mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
B) 1/4MS minimal medium+GA
30.4mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
C) 1/4MS minimal medium+GA
30.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
D) 1/4MS minimal medium+GA
31.2mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
After cultivating 50d, result is as follows:
A) sporophyte seedling is had to be formed, average 20 seedlings/bottle, young sporophyll height≤0.6cm.Spire is relatively abundant, some visible vein, as indicated with 2.
B) sporophyte seedling is had to be formed, average 36 seedlings/bottle, young sporophyll height≤1.2cm.Spire slightly extends, more abundant.
C) sporophyte seedling is had to be formed, average 51 seedlings/bottle, young sporophyll height≤1.5cm.Spire extends, still abundant.
D) sporophyte seedling is had to be formed, average 60 seedlings/bottle, young sporophyll height≤2.0cm.Spire is more thin and weak, elongated.
Can be learnt by above-mentioned cultivation results: during the formula of 1/4MS minimal medium forms, GA
3obvious facilitation is had to the induction of sporophyte.Sporophyte Fiber differentiation based formulas is preferably: 1/4MS minimal medium+GA
30.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.Although d) item sporophyte quantity is maximum, quality is not good, affects the survival rate of follow-up experienced transplantation of seedlings.
embodiment 4
Culture of rootage, hardening and transplanting: separated by the juvenile sporophyte that embodiment 3 is induced, be forwarded to root media inducing spore body and form adventive root.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
Cultivating basigamy formula is:
A) 1/4MS minimal medium+15g/L sucrose+2,4-D0.05mg/L+ agar 7g/L
B) 1/4MS minimal medium+15g/L sucrose+NAA0.05mg/L+ agar 7g/L
After 10-15d finds that each juvenile sporophyte adventive root tip of a root is outstanding successively, unclamp in culturing room immediately, be left unlocked or unlatched bottle cap hardening 10d, sporophyte structure differentiation is more perfect.Before transplanting, when seedling is cleaned, observe and find: a) process the young root formed and relatively process tubbiness, extend again after transplanting, produce more hairs, and b), root look shallow, the root hair that formed of process is comparatively obvious, root hair easy damaged during transplanting, but the later stage also can form new root substitutes original root system.Integrated comparative, with a) medium short-term better processing effect.
Claims (10)
1. the method for halogen fern vitro Regeneration System foundation, is characterized in that comprising the following steps:
(1) explant collection and surface sterilization: clip halogen fern sporophyll, carries out surface sterilization;
(2) spore axenic germination: be separated the sporangiorus on sporophyll, be inoculated on spore axenic germination medium and cultivate;
(3) prothallium Multiplying culture: prothallium montage step (2) obtained carries out prothallium Multiplying culture in prothallium proliferated culture medium;
(4) induction of sporophyte: prothallium step (3) formed pulverizes switching, induction produces juvenile sporophyte;
(5) culture of rootage and hardening: the juvenile sporophyte that step (4) is induced is separated, switching induction juvenile sporophyte is taken root;
(6) transplant: the seedling of taking out step (5), enters seedling replanting in matrix, final formation halogen fern seedling.
2. the method for halogen fern vitro Regeneration System foundation according to claim 1, it is characterized in that: in step (2), spore germination aseptic culture based formulas is: 1/4MS minimal medium+0 ~ 20g/L sucrose+6-benzyl aminopurine 6-BA0.2mg/L+ agar 7g/L.
3. the method for halogen fern vitro Regeneration System foundation according to claim 1, it is characterized in that: prothallium proliferation culture medium formula is: 1/4MS minimal medium+15g/L sucrose+6-BA0 ~ 0.6mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L.
4. the method for halogen fern vitro Regeneration System foundation according to claim 1, is characterized in that: sporophyte Fiber differentiation based formulas is: 1/4MS minimal medium+gibberellin (GA
3) 0 ~ 1.2mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
5. the method for halogen fern vitro Regeneration System foundation according to claim 1, it is characterized in that: in step (5), culture medium prescription is: 1/4MS minimal medium+15g/L sucrose+2,4-D0.05mg/L or naa (NAA) 0.05mg/L+ agar 7g/L.
6. the method that the halogen fern vitro Regeneration System according to claim 2,3,4 or 5 is set up, is characterized in that: above-mentioned 1/4MS minimal medium formula is: NH
4nO
3412.5mg/L, KNO
3475mg/L, KH
2pO
4100mg/L, MgSO
47H
2o92.5mg/L, CaCl
22H
2o110mg/L, KI0.83mg/L, H
3bO
36.2mg/L, MnSO44H
2o22.3mg/L, ZnSO
47H
2o8.6mg/L, Na
2moO
42H
2o0.25mg/L, CuSO
45H
2o0.025mg/L, CoCl
26H
2o0.025mg/L, FeSO
47H
2o6.95mg/L, Na
2-EDTA2H
2o9.325mg/L.
7. the method that the halogen fern vitro Regeneration System according to claim 1,2,3,4 or 5 is set up, is characterized in that: in each step, condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 ° of C.
8. the method that the halogen fern vitro Regeneration System according to claim 1,2,3,4 or 5 is set up, is characterized in that: in step (1), surface sterilization condition is as follows: first use the alcohol-pickled 20s of 70%, abandon alcohol, add 0.1% mercuric chloride (HgCl
2) sterilization 2.5min.
9. the method that the halogen fern vitro Regeneration System according to claim 1,2,3,4 or 5 is set up, is characterized in that: the matrix that seedling replanting adopts is Cao Tan ︰ vermiculite=3 ︰ 1, uses after high-temperature sterilization.
10. the method for halogen fern vitro Regeneration System foundation according to claim 3, is characterized in that: prothallium proliferation culture medium formula is preferably: 1/4MS minimal medium+15g/L sucrose+6-BA0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109331050A (en) * | 2018-12-07 | 2019-02-15 | 广东省林业科学研究院 | Application of the halogen fern water extract in preparation treatment acute gastric ulcer drug |
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| CN109997532B (en) * | 2019-04-08 | 2021-04-23 | 深圳中绿环境集团有限公司 | Method for separately propagating mangrove halopteris |
| CN111616002A (en) * | 2020-07-01 | 2020-09-04 | 中国林业科学研究院热带林业研究所 | Simple and rapid cultivation method for mangrove plant haloperidium aquilinum seedlings |
| CN111616002B (en) * | 2020-07-01 | 2022-03-25 | 中国林业科学研究院热带林业研究所 | Simple and rapid cultivation method for mangrove plant haloperidium aquilinum seedlings |
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