CN105230483A - Method for establishing in-vitro regeneration system of Osmunda vachellii - Google Patents
Method for establishing in-vitro regeneration system of Osmunda vachellii Download PDFInfo
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Abstract
The invention discloses a method for establishing an in-vitro regeneration system of Osmunda vachellii, which belongs to the field of agricultural biotechnology. According to the method, sporangiorus is used as explant; after surface disinfection, the explant is inoculated into a variety of mediums containing an improved Beneck 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 (a sapling); and the sapling is transplanted into a matrix after rooting culture, and the seedling of Osmunda vachellii is formed eventually. The method provided by the invention can realize rapid and continuous obtainment of a great number of high-quality seedlings.
Description
Technical field
The invention belongs to agricultural biological technical field, the method that the Osmunda Vachellii Hook vitro Regeneration System of to be a kind of with sporangiorus be explant is set up.
Background technology
Osmunda Vachellii Hook (
osmundavachelliihook.) be osmund Pteridiaceae Osmunda plant, originate in subtropical asia humid region.Be distributed in China Hainan Island, Guangdong and Guangxi Provinces, Fujian, Jiangxi, Zhejiang, Guizhou, south of Yunnan and Hong Kong, also originate in Vietnam, Burma and India.Chang Yesheng in moistening mountain region, thick grass or small stream limit, be the indicator plant of south China acid ground.
The ornamental value that Osmunda Vachellii Hook tool is higher.Plant is up to 1m, and root-like stock is upright, and cylindric, leafage is born in top, and like sago cycas, plant type is attractive in appearance, and leaf attitude is graceful, can for plant in flower garden or indoor pot is viewed and admired.The rhizome of Osmunda Vachellii Hook and the marrow of petiole can be used as medicine, and have clearing heat and detoxicating, hemostasia and promoting granulation effect.Be mainly used in the diseases such as diseases caused by external factors, wind-heat, headache, also can treat traumatism and bleeding, burn and scald, carbuncle furuncle and parotitis.At the end of the nineties in last century, the people such as He Tianshi are formulated as medicinal liquor and medicinal herb tea is drunk for people, have positive effect to body-care, and can separate various hot toxication, pre-anti-cancer and other difficult and complicated illness, nature and flavor are gentle, have no side effect.Relevant research product obtains 4 patents.
The current resource of Osmunda Vachellii Hook mainly relies on wild, but under natural conditions, reproductive efficiency is lower, and artificial propagation generally adopts plant division mode to carry out, and is difficult to extensive cultivation fast.View and admire and medical value because Osmunda Vachellii Hook integrates, its market potential demand is very large.Utilize tissue culture technique to set up pteridophyte regenerating system, fast obtain a large amount of seedling, gardens, health care market can be met to the demand of Osmunda Vachellii Hook, simultaneously the also wild Osmunda Vachellii Hook resource of indirect protection.
Summary of the invention
The object of the present invention is to provide a kind of method that Osmunda Vachellii Hook vitro Regeneration System is set up, namely with the ripe spore of Osmunda Vachellii Hook for explant, through steps such as explant surface sterilization, spore axenic germination, prothallium propagation, sporophyte induction and bottle transplantation of seedlings, establish Osmunda Vachellii Hook vitro Regeneration System, thus obtain a large amount of seedling sustainably fast.
The present invention adopts following technical proposals to realize.The method that Osmunda Vachellii Hook vitro Regeneration System is set up, comprises the following steps:
(1) explant collection and surface sterilization: clip Osmunda Vachellii Hook sporophyll, carries out surface sterilization.
(2) spore axenic germination: be separated the sporangiorus on sporophyll, be inoculated on spore germination medium and cultivate.Spore germination culture medium prescription is: improvement Beneck minimal medium+0 ~ 15g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L
(3) prothallium Multiplying culture: the prothallium that step (2) spore germination is formed is trimmed in proliferated culture medium and carries out prothallium Multiplying culture.Every 50d changes one-step growth medium.Proliferation culture medium formula is: improvement Beneck minimal medium+15g/L sucrose+6-BA0 ~ 0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.
(4) induction of sporophyte: prothallium step (3) formed is pulverized and is forwarded to sporophyte inducing culture induction generation juvenile sporophyte.Sporophyte Fiber differentiation based formulas is: improvement Beneck 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, is forwarded to root media inducing spore body and forms adventive root.Prescription of rooting medium is: improvement Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+2, after 4-D0.1mg/L (or NAA0.1mg/L)+agar 7g/L finds that the adventive root tip of a root is outstanding, unclamp in culturing room immediately, be left unlocked or unlatched bottle cap 10d, culturing gene moisture comparatively fast consumed and shrank period, but unlikely dry, sporophyte structure differentiation is more perfect.
(6) transplant: the sporophyte 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 improvement Beneck minimal medium formula is: NH
4nO
3200mg/L, KH
2pO
4100mg/L, MgSO
47H
2o100mg/L, CaCl
2100mg/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 DEG C.
In above-mentioned steps (1), surface sterilization condition is preferably as follows: first use the alcohol-pickled 15s of 70%, abandon alcohol, add 0.1% mercuric chloride (HgCl
2) sterilization 2min.
Above-mentioned steps (2) spore germination culture medium prescription is preferably: improvement Beneck minimal medium+5g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L
In above-mentioned steps (3), proliferation culture medium formula is preferably: improvement Beneck minimal medium+15g/L sucrose+6-BA0.6mg/L+2,4-D0.1mg/L+ agar 7g/L.
In above-mentioned steps (4), sporophyte Fiber differentiation based formulas is preferably: improvement Beneck minimal medium+gibberellin (GA
3) 0.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.Vaccination ways is preferably comminuted inoculation.
In above-mentioned steps (5), prescription of rooting medium is preferably: improvement Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+2,4-D0.1mg/L+ agar 7g/L.
The present invention establishes the method for Osmunda Vachellii Hook vitro Regeneration System first, has filled up prior art blank; Use the method for Fast-propagation of the present invention, can realize highly intensive and high density factorial praluction, can obtain the Osmunda Vachellii Hook seedling of a large amount of high-quality within short-term, be the reliable technical guarantee that horticultural production and Chinese medicine cultivation provide.
Accompanying drawing explanation
Fig. 1 Osmunda Vachellii Hook prothallium cultivation effect figure.
Fig. 2 Osmunda Vachellii Hook sporophyte inducing effect figure.
Fig. 3 Osmunda Vachellii Hook transplants design sketch.
Embodiment
Preparation: by formula preparation improvement Beneck minimal medium, its formula is: NH
4nO
3200mg/L, KH
2pO
4100mg/L, MgSO
47H
2o100mg/L, CaCl
2100mg/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 Osmunda Vachellii Hook culture in vitro system provided by the invention is set up, comprises following sequential steps:
(1) explant collection and surface sterilization process: yellowish-brown at Osmunda Vachellii Hook sporangiorus color and luster, when spore rachis is still in yellowish green fresh state, takes back in clip sporophyll valve bag, processes fresh material in time, or storage 4 DEG C of preservations, processes in 2d.Sporophyll surface sterilization: it is the segment of 3 ~ 4cm that sporophyll is cut out, and puts in 50ml 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; Running water slowly rinses sporophyll 10min; On superclean bench, abandon running water in bottle, add the alcohol-pickled 15s of 70%, abandon alcohol, add 0.1%HgCl
2sterilization 2min, then with aseptic water washing 5 times, for subsequent use.
(2) spore axenic germination: be separated the sporangiorus on sporophyll, be inoculated on spore germination medium and cultivate.Spore germination culture medium prescription is: improvement Beneck minimal medium+0 ~ 15g/L sucrose+6-BA0.2mg/L+2,4-D0.1mg/L+ agar 7g/L.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 DEG C.
(3) prothallium Multiplying culture: the prothallium that step (2) spore germination is formed is trimmed in proliferated culture medium and carries out prothallium Multiplying culture.Every 50d changes one-step growth medium.Proliferation culture medium formula is: improvement Beneck 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 DEG C.
(4) induction of sporophyte: prothallium step (3) formed is pulverized and is forwarded to sporophyte inducing culture induction generation juvenile sporophyte.Sporophyte Fiber differentiation basigamy formula is: improvement Beneck 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 DEG 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.Prescription of rooting medium is: improvement Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+2,4-D0.1mg/L (or NAA0.1mg/L)+agar 7g/L.10-15d unclamps immediately, is left unlocked or unlatched bottle cap 10d after finding that each juvenile sporophyte adventive root tip of a root is outstanding successively in culturing room, 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 DEG 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 gathers and surface sterilization process: yellowish-brown at Osmunda Vachellii Hook sporangiorus color and luster, when spore rachis is still in yellowish green fresh state, takes back, process fresh material in time in clip sporophyll valve bag, or storage 4 DEG C of preservations, processes in 2d.Sporophyll surface sterilization: it is the segment of 3 ~ 4cm that sporophyll is cut out, and containing 10-12 sporangiorus, puts in 50ml 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 15s of 70%, abandon alcohol, add 0.1%HgCl
2sterilization 2min, then with aseptic water washing 5 times, for subsequent use.Be separated the sporangiorus on sporophyll, be inoculated on spore germination medium and cultivate, every bottle graft kind 10 sporangioruses.Condition of culture is: 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 DEG C.
Spore germination culture medium prescription is:
A) Beneck minimal medium+0/L sucrose+6-BA0.2mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
B) Beneck minimal medium+5g/L sucrose+6-BA0.2mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
C) Beneck minimal medium+10g/L sucrose+6-BA0.2mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
D) Beneck minimal medium+15g/L sucrose+6-BA0.2mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
After cultivating 50d, result is as follows:
A) prothallium is obtained by the sporangiorus of 85%, prothallium oyster, more transparent.
B) prothallium is obtained by the sporangiorus of 78%, prothallium yellow green, slightly transparent.
C) obtain prothallium by the sporangiorus of 64%, prothallium is green, opaque.
D) prothallium is obtained by the sporangiorus of 45%, prothallium bottle green, opaque, a small amount of material deuterogenesis brown stain.
Can be learnt by above-mentioned cultivation results: during the formula of improvement Beneck minimal medium forms, the growth conditions of sucrose on spore germination, prothallium has to be affected more significantly.Spore germination culture medium prescription is preferably: improvement Beneck minimal medium+5g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L.Although it is maximum to cultivate prothallium quantity without sucrose, grow barren, next step cultivation unfavorable.
embodiment 2
Prothallium inoculation prothallium embodiment 1 obtained is trimmed in 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 DEG C.
Proliferation culture medium formula is:
A) Beneck minimal medium+15g/L sucrose+6-BA0mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
B) Beneck minimal medium+15g/L sucrose+6-BA0.2mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
C) Beneck minimal medium+15g/L sucrose+6-BA0.4mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
D) Beneck minimal medium+15g/L sucrose+6-BA0.6mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L.
After cultivating 50d, result is as follows:
A) prothallium fragment can form new prothallium, but limited amount, on average 22 every bottle.Prothallium outside of belly rhizoid is flourishing.
B) prothallium fragment can form new prothallium, but limited amount, on average 31 every bottle.The prothallium outside of belly has rhizoid.
C) prothallium fragment can form new prothallium, but quantity is more, arrangement comparatively dense, on average 55 every bottle.The rare rhizoid of the prothallium outside of belly.
D) prothallium fragment can form new prothallium, but quantity is a lot, dense arrangement, and wraps up in mutually layer by layer, and the form of similar romaine lettuce, not easily counts, as shown in Figure 1.The prothallium outside of belly does not have a rhizoid.
Can be learnt by above-mentioned cultivation results: during the formula of improvement Beneck minimal medium forms, the propagation of 6-BA on prothallium has to be affected more significantly.Prothallium proliferation culture medium formula is preferably: improvement Beneck minimal medium+15g/L sucrose+6-BA0.6mg/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 DEG C.
Sporophyte Fiber differentiation basigamy formula is:
A) Beneck minimal medium+GA is improved
30mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
B) Beneck minimal medium+GA is improved
30.4mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
C) Beneck minimal medium+GA is improved
30.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
D) Beneck minimal medium+GA is improved
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 15 seedlings/bottle, young sporophyll height≤1cm.Spire is relatively abundant, some visible vein.
B) sporophyte seedling is had to be formed, average 31 seedlings/bottle, young sporophyll height≤1.5cm.Spire slightly extends, more abundant.
C) sporophyte seedling is had to be formed, average 42 seedlings/bottle, young sporophyll height≤3cm.Spire extends, still abundant, as shown in Figure 2.
D) sporophyte seedling is had to be formed, average 48 seedlings/bottle, young sporophyll height≤4.5cm.Spire is more thin and weak, elongated.
Can be learnt by above-mentioned cultivation results: during the formula of improvement Beneck minimal medium forms, GA
3obvious facilitation is had to the induction of sporophyte.Sporophyte Fiber differentiation based formulas is preferably: improvement Beneck 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 DEG C.
Culture of rootage basigamy formula is:
A) Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+2 is improved, 4-D0.1mg/L+ agar 7g/L
B) Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+NAA0.1mg/L+ agar 7g/L is improved
10-15d unclamps immediately, is left unlocked or unlatched bottle cap 10d after finding that each juvenile sporophyte adventive root tip of a root is outstanding successively in culturing room, period culturing gene moisture comparatively fast consume and shrink, but unlikely dry, 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, as shown in Figure 3, and b) root look shallow, the many hairs that formed of process, 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 Osmunda Vachellii Hook vitro Regeneration System foundation, is characterized in that comprising the following steps:
(1) explant collection and surface sterilization: clip Osmunda Vachellii Hook sporophyll, carries out surface sterilization;
(2) spore axenic germination: be separated the sporangiorus on sporophyll, be inoculated on spore germination medium and cultivate;
(3) prothallium Multiplying culture: the prothallium that step (2) spore germination is formed is trimmed in proliferated culture medium and carries out prothallium Multiplying culture;
(4) induction of sporophyte: prothallium step (3) formed is pulverized and is forwarded to sporophyte inducing culture induction generation juvenile sporophyte;
(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;
(6) transplant: the sporophyte 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.
2. the method for Osmunda Vachellii Hook vitro Regeneration System foundation according to claim 1, is characterized in that: yellowish-brown at Osmunda Vachellii Hook sporangiorus color and luster, and when spore rachis is still in yellow green fresh state, clip sporophyll, processes fresh material in time.
3. the method for Osmunda Vachellii Hook vitro Regeneration System foundation according to claim 1, it is characterized in that: spore germination culture medium prescription is: improvement Beneck minimal medium+0 ~ 15g/L sucrose+6-benzyl aminopurine (6-BA) 0.2mg/L+2,4 dichlorphenoxyacetic acids (2,4-D) 0.1mg/L+ agar 7g/L.
4. the method for Osmunda Vachellii Hook vitro Regeneration System foundation according to claim 1, it is characterized in that: in step (3), proliferation culture medium formula is: improvement Beneck minimal medium+15g/L sucrose+6-BA0 ~ 0.5mg/L+2,4-D0.1mg/L+ agar 7g/L.
5. the method for Osmunda Vachellii Hook vitro Regeneration System foundation according to claim 1, it is characterized in that: in step (4), sporophyte Fiber differentiation based formulas is: improvement Beneck minimal medium+gibberellin (GA
3) 0 ~ 1.2mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
6. the method for Osmunda Vachellii Hook vitro Regeneration System foundation according to claim 1, it is characterized in that: in step (5), culture of rootage basigamy formula is: improvement Beneck minimal medium+10g/L sucrose+6-BA0.05mg/L+2,4-D0.1mg/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 10d, period culture of rootage gene moisture comparatively fast consume and shrink, but unlikely dry.
7. the method that the Osmunda Vachellii Hook vitro Regeneration System according to claim 3,4,5 or 6 is set up, is characterized in that: improvement Beneck minimal medium formula is: NH
4nO
3200mg, KH
2pO
4100mg, MgSO
47H
2o100mg, CaCl
2100mg, KI0.83mg, H
3bO
36.2mg, MnSO44H
2o22.3mg, ZnSO
47H
2o8.6mg, Na
2moO
42H
2o0.25mg, CuSO
45H
2o0.025mg, CoCl
26H
2o0.025mg, FeSO
47H
2o27.8mg, Na
2-EDTA2H
2o37.3mg.
8. the method that the Osmunda Vachellii Hook vitro Regeneration System according to claim 1,2,3,4,5 or 6 is set up, is characterized in that: the condition of culture that culture in vitro system is set up is that 12h illumination+12h is dark, intensity of illumination 2500Lx, 25 ± 2 DEG C.
9. the method that the Osmunda Vachellii Hook vitro Regeneration System according to claim 1,2,3,4,5 or 6 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 that the Osmunda Vachellii Hook vitro Regeneration System according to claim 1,2,3,4,5 or 6 is set up, is characterized in that: sporophyte Fiber differentiation based formulas is: improvement Beneck minimal medium+GA
30.8mg/L+5g/L sucrose+agar 7g/L+ caseinhydrolysate 100mg/L.
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| CN106171999A (en) * | 2016-07-21 | 2016-12-07 | 深圳市仙湖植物园管理处 | Guangdong osmund can educate Spore cultivation and propagation method thereof |
| CN106718899A (en) * | 2016-12-08 | 2017-05-31 | 大连民族大学 | A kind of method of mountain region high-yield culturing common vetch dish |
| CN106718878A (en) * | 2016-11-22 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of fan fern quick breeding by group culture method with young sporangiorus as explant |
| CN109601387A (en) * | 2019-01-25 | 2019-04-12 | 徐州生物工程职业技术学院 | With the Osmunda Vachellii Hook tissue culture propagation method of the GGB approach of young sporangiorus induction |
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| CN106718878A (en) * | 2016-11-22 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of fan fern quick breeding by group culture method with young sporangiorus as explant |
| CN106718899A (en) * | 2016-12-08 | 2017-05-31 | 大连民族大学 | A kind of method of mountain region high-yield culturing common vetch dish |
| CN109601387A (en) * | 2019-01-25 | 2019-04-12 | 徐州生物工程职业技术学院 | With the Osmunda Vachellii Hook tissue culture propagation method of the GGB approach of young sporangiorus induction |
| CN109601387B (en) * | 2019-01-25 | 2021-08-24 | 徐州生物工程职业技术学院 | Tissue culture propagation method of osmunda vachellii with GGB route induced by juvenile sporocyst group |
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