CN104686354A - Tissue culture technology for dryopteris championii - Google Patents
Tissue culture technology for dryopteris championii Download PDFInfo
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- CN104686354A CN104686354A CN201510092707.4A CN201510092707A CN104686354A CN 104686354 A CN104686354 A CN 104686354A CN 201510092707 A CN201510092707 A CN 201510092707A CN 104686354 A CN104686354 A CN 104686354A
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Abstract
The invention discloses a tissue culture technology for dryopteris championii, and relates to a method for quickly breeding seedlings through plant tissue culture technology of the dryopteris championii. According to the tissue culture technology disclosed by the invention, the dryopteris championii mature spore is taken as an explant, a dryopteris championii tissue culture technical system is established through steps of spore germination, prothallus propagation, callus induction, multi-shoot differentiation, rooting, transplanting and the like, and the protection of plant is facilitated; the tissue culture technology can be applied to large-scale production to promote extensive application of the dryopteris championii in gardens.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in Plant Biotechnology, specifically, relate to a kind of wealthy squama shield-fern tissue culture technique.
Background technology
Pteridophyte also claims pteridophyte, is between the most original higher plant---a monoid between bryophyte and spermatophyte.Pteridophyte and bryophyte equally have the alternation of generations phenomenon between gametophyte and sporophyte.The laudatory title that pteridophyte is enjoyed " U.S. without flower ", Ornamental Ferns is liking deeply by the people of various countries with the beauty of line of its gracefulness and graceful beauty of posture.Pteridophyte is because sexual reproduction is to the dependence effect of water, and comparatively just carve the requirement in habitat, growth district is limited.In recent years, due to climate warming, the external causes such as natural calamity and the excessive exploitation of the mankind to pteridophyte cause a lot of fern Wu Zhong Endangered to face extinction.Pteridophyte is as the excellent Ornamental Foliage House Plants of a class and wet-proof plants plant, depend on the introduction of external kind at present, and China is as one of the abundantest area of pteridophytes resources in the world, many outstanding fern kinds are not applied and promote.Therefore, it is very urgent for promoting the investigation and application of Propagtion of Pteridophyta Plants technology.
Wealthy squama shield-fern (
dryopteris championii) be Dryopteridaceae Dryopteris plant, be distributed in the national most area such as Shandong, Henan.Leaf look bud green is glossy, ear pinnule, can adapt to habitats.Drought-enduring happiness light, also resistance to shade can be done sylvan life sheet and plants or plant with mountain stone, is a kind of excellent ground cover plant.The at present existing research to its potted plant introduction and Experiment, but its tissue culture propagation technology there is not been reported.Therefore, set up the protection that wealthy squama shield-fern tissue culture technique system will contribute to this Plants, more can be applied to large-scale production with the extensive use promoting them in gardens.
Summary of the invention
The object of the present invention is to provide out a kind of wealthy squama shield-fern tissue culture technique, the present invention with the ripe spore of wealthy squama shield-fern for explant, by spore germination, prothallium propagation, callus induction, differentiation, take root, transplanting and other steps establishes wealthy squama shield-fern tissue culture technique system, thus achieves object of the present invention.
One of the present invention wealthy squama shield-fern tissue culture technique, is characterized in that comprising the following steps:
(1) spore process and sprouting is cultivated: will have ripe sporangium and the leaf harvest that spore not yet comes off gets off, dust and the impurity on blade face is rinsed gently with clear water, natural air drying in paper bag is put in after drying gently, the spore that collection comes off is in sulfuric acid paper bag, and it is for subsequent use to be positioned over 4 DEG C of Refrigerator stores.Appropriate spore be placed in 1.5mL centrifuge tube and add the Gibberellins solution of 50 ~ 100mg/mL, soaking 5 ~ 10min, with 4000r/min speed with centrifugal 3 ~ 6min, remove supernatant.And then add 2% ~ 5% liquor natrii hypochloritis and to sterilize 5 ~ 15min, with 4000r/min speed with centrifugal 3 ~ 6min, remove supernatant, proceed in 10mL centrifuge tube with after aseptic water washing 4 ~ 5 times, add sterile water and be diluted to density and be about 3 × 10
3the spore suspension of individual mg/L is also inoculated on germination medium and carries out spore germination cultivation.Inoculation is placed on illumination every day 12 ~ 15 hours, and intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate until spore germination under the condition of 25 ~ 28 DEG C.
(2) prothallium Multiplying culture: the prothallium after step (1) spore germination is trimmed in proliferated culture medium and carries out prothallium Multiplying culture.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within every two weeks, changes a subculture.
(3) callus induction: step (1) or (2) acquisition prothallium are inoculated on inducing culture and carry out callus induction.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until form callus.
(4) inducing clumping bud: the callus that step (3) obtains is cut into agglomerate of the same size (3mm × 3mm × 3mm) and be transferred in differential medium and carry out inducing clumping bud.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until form Multiple Buds.
(5) culture of rootage: step (4) is cultivated the Multiple Buds obtained and be transferred in root media and carry out root induction.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate until take root under the condition of 25 ~ 28 DEG C.
(6) acclimatization and transplants: when the stem of the young spore seedling of step (5) grows to about 3 ~ 4cm, when root grows to about 2 ~ 3cm, sealed membrane is opened, in culturing room, hardening took out test-tube plantlet after 5 ~ 7 days, wash away the medium of root attachment, by young spore transplantation of seedlings to the peat composed of rotten mosses through high-temperature sterilization: in the mixed-matrix of vermiculite=1:1.After transplanting, regularly water to seedling, and keep humidity more than 90%.
Germination medium described in above-mentioned steps (2) is: 1/2MS+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, and pH value is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, and pH value is 5.4 ~ 5.8.
Inducing culture described in above-mentioned steps (4) is: MS+0.1 ~ 1.0mg/LKT+1 ~ 3mg/L 2,4-D+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (5) is: MS+0.01 ~ 0.3mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Root media described in above-mentioned steps (5) is: MS+0.1 ~ 1.0mg/L NAA+0.1 ~ 0.5mg/L IAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Advantage of the present invention is: relate to wealthy squama shield-fern (
dryopteris championii) by the method for plant tissue culture technique Fast-propagation seedling.The present invention with the ripe spore of wealthy squama shield-fern for explant; by spore germination, prothallium propagation, callus induction, differentiation, take root, transplanting and other steps establishes wealthy squama shield-fern tissue culture technique system; contribute to the protection to this Plants, more can be applied to large-scale production with the extensive use promoting them in gardens.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) spore process and sprouting is cultivated: will have ripe sporangium and the leaf harvest that spore not yet comes off gets off, dust and the impurity on blade face is rinsed gently with clear water, natural air drying in paper bag is put in after drying gently, the spore that collection comes off is in sulfuric acid paper bag, and it is for subsequent use to be positioned over 4 DEG C of Refrigerator stores.Appropriate spore be placed in 1.5mL centrifuge tube and add the Gibberellins solution of 50mg/mL, soaking 6min, with 4000r/min speed with centrifugal 4min, remove supernatant.And then add 2% liquor natrii hypochloritis and to sterilize 5min, with 4000r/min speed with centrifugal 5min, remove supernatant, proceed in 10mL centrifuge tube with after aseptic water washing 4 times, add sterile water and be diluted to density and be about 3 × 10
3the spore suspension of individual mg/L is also inoculated on germination medium and carries out spore germination cultivation.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and cultivation temperature is cultivate 17 days spores under the condition of 25 DEG C namely to sprout, and germination rate is 83.7%.Described germination medium is: 1/2MS+3.5% sucrose+0.5% agar, and pH value is 5.6.
(2) prothallium Multiplying culture: the prothallium after step (1) spore germination is trimmed in proliferated culture medium and carries out prothallium Multiplying culture.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 1500lx, and cultivation temperature is that to cultivate growth coefficient after 45 days under the condition of 25 DEG C be 4.5.The prothallium color of propagation is emerald green, and growing way is vigorous, and both wings are extended into lobate.Described proliferated culture medium is: MS+3.5% sucrose+0.35% agar, and pH value is 5.6.
(3) callus induction: step (1) or (2) acquisition prothallium are inoculated on inducing culture and carry out callus induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and cultivation temperature is cultivate under the condition of 25 DEG C after 28 days namely to form callus, and inductivity is 71.5%.Described inducing culture is: MS+0.1mg/LKT+1mg/L 2,4-D+2.0% sucrose+0.35% agar, pH value is 5.6.
(4) inducing clumping bud: the callus that step (3) obtains is cut into agglomerate of the same size (3mm × 3mm × 3mm) and be transferred in differential medium and carry out inducing clumping bud.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and cultivation temperature is cultivate under the condition of 25 DEG C after 38 days namely to form Multiple Buds, and inductivity reaches 79.4%.Described differential medium is: MS+0.01mg/L NAA+2.0% sucrose+0.35% agar, pH value is 5.6.
(5) culture of rootage: step (4) is cultivated the Multiple Buds obtained and be transferred in root media and carry out root induction.Inoculation is placed on illumination every day 12 hours, and intensity of illumination is 2000lx, and cultivation temperature is cultivate under the condition of 25 DEG C after 26 days namely to take root, and rooting rate reaches 97.5%, and average number of taking root is 6.2.Described root media is: MS+0.5mg/L NAA+0.1mg/L IAA+3.5% sucrose+0.5% agar, pH value is 5.6.
(6) acclimatization and transplants: when the stem of the young spore seedling of step (5) grows to about 3 ~ 4cm, when root grows to about 2 ~ 3cm, sealed membrane is opened, in culturing room, hardening took out test-tube plantlet after 5 days, wash away the medium of root attachment, by young spore transplantation of seedlings to the peat composed of rotten mosses through high-temperature sterilization: in the mixed-matrix of vermiculite=1:1.After transplanting, regularly water to seedling, and keep humidity more than 90%, transplanting survival rate is 87.1%.
Embodiment 2:
(1) spore process and sprouting is cultivated: will have ripe sporangium and the leaf harvest that spore not yet comes off gets off, dust and the impurity on blade face is rinsed gently with clear water, natural air drying in paper bag is put in after drying gently, the spore that collection comes off is in sulfuric acid paper bag, and it is for subsequent use to be positioned over 4 DEG C of Refrigerator stores.Appropriate spore be placed in 1.5mL centrifuge tube and add the Gibberellins solution of 70mg/mL, soaking 8min, with 4000r/min speed with centrifugal 6min, remove supernatant.And then add 3% liquor natrii hypochloritis and to sterilize 6min, with 4000r/min speed with centrifugal 5min, remove supernatant, proceed in 10mL centrifuge tube with after aseptic water washing 5 times, add sterile water and be diluted to density and be about 3 × 10
3the spore suspension of individual mg/L is also inoculated on germination medium and carries out spore germination cultivation.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate 15 days spores under the condition of 27 DEG C namely to sprout, and germination rate is 88%.Described germination medium is: 1/2MS+3.0% sucrose+0.45% agar, and pH value is 5.8.
(2) prothallium Multiplying culture: the prothallium after step (1) spore germination is trimmed in proliferated culture medium and carries out prothallium Multiplying culture.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2500lx, and cultivation temperature is that to cultivate growth coefficient after 43 days under the condition of 27 DEG C be 4.9.The prothallium color of propagation is emerald green, and growing way is vigorous, and both wings are extended into lobate.Described proliferated culture medium is: MS+3.2% sucrose+0.45% agar, and pH value is 5.8.
(3) callus induction: step (1) or (2) acquisition prothallium are inoculated on inducing culture and carry out callus induction.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate under the condition of 27 DEG C after 26 days namely to form callus, and inductivity is 76%.Described inducing culture is: MS+0.3mg/LKT+1.8mg/L 2,4-D+2.5% sucrose+0.35% agar, pH value is 5.8.
(4) inducing clumping bud: the callus that step (3) obtains is cut into agglomerate of the same size (3mm × 3mm × 3mm) and be transferred in differential medium and carry out inducing clumping bud.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate under the condition of 27 DEG C after 34 days namely to form Multiple Buds, and inductivity reaches 85%.Described differential medium is: MS+0.1mg/L NAA+2.8% sucrose+0.40% agar, pH value is 5.8.
(5) culture of rootage: step (4) is cultivated the Multiple Buds obtained and be transferred in root media and carry out root induction.Inoculation is placed on illumination every day 13 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate under the condition of 27 DEG C after 29 days namely to take root, and rooting rate reaches 100%, and average number of taking root is 5.7.Described root media is: MS+0.8mg/L NAA+0.2mg/L IAA+3.8% sucrose+0.43% agar, pH value is 5.8.
(6) acclimatization and transplants: when the stem of the young spore seedling of step (5) grows to about 3 ~ 4cm, when root grows to about 2 ~ 3cm, sealed membrane is opened, in culturing room, hardening took out test-tube plantlet after 7 days, wash away the medium of root attachment, by young spore transplantation of seedlings to the peat composed of rotten mosses through high-temperature sterilization: in the mixed-matrix of vermiculite=1:1.After transplanting, regularly water to seedling, and keep humidity more than 90%, transplanting survival rate is 90.1%.
Claims (6)
1. a wealthy squama shield-fern tissue culture technique, is characterized in that comprising the following steps:
(1) spore process and sprouting is cultivated: will have ripe sporangium and the leaf harvest that spore not yet comes off gets off, dust and the impurity on blade face is rinsed gently with clear water, natural air drying in paper bag is put in after drying gently, the spore that collection comes off is in sulfuric acid paper bag, and it is for subsequent use to be positioned over 4 DEG C of Refrigerator stores, appropriate spore is placed in 1.5mL centrifuge tube and adds the Gibberellins solution of 50 ~ 100mg/mL, soak 5 ~ 10min, with 4000r/min speed with centrifugal 3 ~ 6min, remove supernatant, and then add 2% ~ 5% liquor natrii hypochloritis and to sterilize 5 ~ 15min, with 4000r/min speed with centrifugal 3 ~ 6min, remove supernatant, proceed in 10mL centrifuge tube with after aseptic water washing 4 ~ 5 times, add sterile water to be diluted to density and to be about 3 × 10
3the spore suspension of individual mg/L is also inoculated on germination medium and carries out spore germination cultivation, and inoculation is placed on illumination every day 12 ~ 15 hours, and intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is cultivate until spore germination under the condition of 25 ~ 28 DEG C,
(2) prothallium Multiplying culture: the prothallium after step (1) spore germination is trimmed in proliferated culture medium and carries out prothallium Multiplying culture, inoculation is placed on illumination every day 12 ~ 16 hours, intensity of illumination is 1500 ~ 2500lx, cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within every two weeks, changes a subculture;
(3) callus induction: step (1) or (2) acquisition prothallium are inoculated on inducing culture and carry out callus induction, inoculation is placed on illumination every day 12 ~ 16 hours, intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate until form callus under the condition of 25 ~ 28 DEG C;
(4) inducing clumping bud: the callus that step (3) obtains is cut into agglomerate of the same size (3mm × 3mm × 3mm) and be transferred in differential medium and carry out inducing clumping bud, inoculation is placed on illumination every day 12 ~ 16 hours, intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate until form Multiple Buds under the condition of 25 ~ 28 DEG C;
(5) culture of rootage: step (4) is cultivated the Multiple Buds obtained and be transferred in root media and carry out root induction, inoculation is placed on illumination every day 12 ~ 16 hours, intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate until take root under the condition of 25 ~ 28 DEG C;
(6) acclimatization and transplants: when the stem of the young spore seedling of step (5) grows to about 3 ~ 4cm, when root grows to about 2 ~ 3cm, sealed membrane is opened, in culturing room, hardening took out test-tube plantlet after 5 ~ 7 days, wash away the medium of root attachment, by young spore transplantation of seedlings to the peat composed of rotten mosses through high-temperature sterilization: in the mixed-matrix of vermiculite=1:1, after transplanting, regularly water to seedling, and keep humidity more than 90%.
2. one according to claim 1 wealthy squama shield-fern tissue culture technique, it is characterized in that the germination medium described in step (2) is: 1/2MS+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
3. one according to claim 1 wealthy squama shield-fern tissue culture technique, it is characterized in that the proliferated culture medium described in step (3) is: MS+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
4. one according to claim 1 wealthy squama shield-fern tissue culture technique, it is characterized in that the inducing culture described in step (4) is: MS+0.1 ~ 1.0mg/LKT+1 ~ 3mg/L 2,4-D+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
5. one according to claim 1 wealthy squama shield-fern tissue culture technique, is characterized in that the differential medium described in step (5) is: MS+0.01 ~ 0.3mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
6. one according to claim 1 wealthy squama shield-fern tissue culture technique, it is characterized in that the root media described in step (5) is: MS+0.1 ~ 1.0mg/L NAA+0.1 ~ 0.5mg/L IAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106538393A (en) * | 2016-12-08 | 2017-03-29 | 大连民族大学 | A kind of method of mountain region high-yield culturing Herba Fimbristylis dichotomae |
| CN106613977A (en) * | 2016-12-22 | 2017-05-10 | 中国科学院昆明植物研究所 | Sterilization and inoculation method of pteridophyte spores |
| CN106942052A (en) * | 2017-03-02 | 2017-07-14 | 东北农业大学 | The industrial seedling rearing and artificial cultivation method of a kind of dryopteris fragrans |
-
2015
- 2015-03-02 CN CN201510092707.4A patent/CN104686354A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 王晓倩等: "阔鳞麟毛蕨孢子的无菌繁殖", 《植物生理学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106538393A (en) * | 2016-12-08 | 2017-03-29 | 大连民族大学 | A kind of method of mountain region high-yield culturing Herba Fimbristylis dichotomae |
| CN106613977A (en) * | 2016-12-22 | 2017-05-10 | 中国科学院昆明植物研究所 | Sterilization and inoculation method of pteridophyte spores |
| CN106942052A (en) * | 2017-03-02 | 2017-07-14 | 东北农业大学 | The industrial seedling rearing and artificial cultivation method of a kind of dryopteris fragrans |
| CN106942052B (en) * | 2017-03-02 | 2019-01-15 | 东北农业大学 | A kind of industrial seedling rearing and artificial cultivation method of dryopteris fragrans |
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Application publication date: 20150610 |