CN101297635B - Method for breeding spore of Dryopteris varia - Google Patents
Method for breeding spore of Dryopteris varia Download PDFInfo
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- CN101297635B CN101297635B CN2008100291092A CN200810029109A CN101297635B CN 101297635 B CN101297635 B CN 101297635B CN 2008100291092 A CN2008100291092 A CN 2008100291092A CN 200810029109 A CN200810029109 A CN 200810029109A CN 101297635 B CN101297635 B CN 101297635B
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Abstract
The invention relates to a spore reproducing method for Dryopteris varia (L.) Ktunze which can be reproduced fast and is characterized in that the disinfected Dryopteris varia (L.) Ktunze spore is cultured in the culture medium of spore germination and grows into laminar prothallium which is then cultured in multiplication medium for 30 days, in sporophyte inductive medium for 40 days and in sporophyte subculture medium for 20 days in sequence; after forming juvenile sporophyte and grows two to three pieces of juvenile sporophyte leaf, the prothallium is cultured in sporophyte rooting mediumfor 30 days, is transplanted out of bottle after the base part grows brown fine roots with scales and is transplanted to the soil matrix for culturing after seedling is trained. The spore reproducingmethod of the invention has little investment and simple equipment, only needs 110 days from the spore germination to the forming of the juvenile sporophyte, obtains large quantities of Dryopteris varia (L.) Ktunze seedlings in short time with the survival rate of the juvenile sporophyte of 90 percent, and leads the Dryopteris varia (L.) Ktunze to become an excellent foliage plant and perform themedical value thereof.
Description
Technical field
The present invention relates to the sporogenesis method of pteridophyte, specifically relate to a kind of sporogenesis method that can breed Dryopteris varig (L.) O. Ktze (Dryopteris varia (L.) Ktunze) fast.
Background technology
Dryopteris varig (L.) O. Ktze is medium-sized herbaceous plant, in China torrid zone and the subtropical zone extensive distribution is arranged, its sylvan life is autochthonal, common high 40～80cm, and strain shape is attractive in appearance, the leaf look dark green, evergreen all the year round, not only can be developed into to having the ornamental foliage plant of ornamental value, and its rhizome has the drug effect of clearing away heat to and alleviating pain, can be used as medicine, therefore this pteridophyte has good market development value.But it is few about the research of breeding Dryopteris varig (L.) O. Ktze at present.
Summary of the invention
The objective of the invention is to develop a kind of method that can breed Dryopteris varig (L.) O. Ktze fast.
We cultivate in the spore germination medium by the Dryopteris varig (L.) O. Ktze spore that will sterilize and obtain the sheet prothallium, then prothallium is cultivated in proliferated culture medium, sporophyte inducing culture, sporophyte subculture medium successively, wait to grow to change in the sporophyte root media behind the sporophyll and cultivate, treat that base portion grows radicula and just can transplant by bottle outlet, overall process only needs about half a year, thereby has realized purpose of the present invention.
The sporogenesis method of Dryopteris varig (L.) O. Ktze of the present invention, its feature comprises the steps:
(1) collects the Dryopteris varig (L.) O. Ktze spore, in alcohol, soak 25～30s earlier, and then in mercuric chloride solution, soak 7～8min, sterilization back rinsed with sterile water;
(2) the Dryopteris varig (L.) O. Ktze spore with above-mentioned sterilization is made into suspension with sterile water, add in the spore germination medium, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx cultivates under light application time 11～13h/d condition, sprout green filamentous earlier, develop into the sheet prothallium again, described spore germination medium is every liter and contains 6～8g agar, 18～22g sucrose, all the other are MS or 1/2MS, the medium of pH 5.7～5.8;
(3) above-mentioned sheet prothallium is transferred in the proliferated culture medium, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx, under light application time 11～13h/d condition, after cultivating 25～35d, change in the sporophyte inducing culture, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx is under light application time 11～13h/d condition, be cultured to and change the sporophyte subculture medium over to after prothallium sprouts, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx is under light application time 11～13h/d condition, be cultured to the formation juvenile sporophyte, grow 2～3 young sporophylls; Contain 6-benzyl purine 0.15～0.25mg in every liter of the described proliferated culture medium, methyl 0.15～0.25mg, agar 6～8g, sucrose 28～32g, all the other are MS, pH 5.7～5.8; Contain kinetin 6-furfuryl group aminopurine or N6-furfuryladenine 0.4～0.6mg in every liter of the described sporophyte inducing culture, methyl 0.15～0.25mg, agar 6～8g, sucrose 28～32g, all the other are MS, pH 5.7～5.8; Contain methyl 0.05～0.15mg in every liter of the described sporophyte subculture medium, agar 6～8g, sucrose 28～32g, all the other are MS, pH 5.7～5.8;
(4) above-mentioned juvenile sporophyte plant division is transferred on the sporophyte root media, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx, under light application time 11～13h/d condition, when being cultured to the juvenile sporophyte base portion and growing the radicula of brown band scale, but bottle outlet is transplanted, transplanting after the hardening to the pH value is that 6.3～6.4 soil matrix is cultivated, contain methyl 0.25～0.35mg in every liter of the described sporophyte root media, activated carbon 4～6g, agar 6～8g, sucrose 28～32g, all the other are the 1/2MS medium, and pH 5.7～5.8.
The alcohol and the mercuric chloride that adopt in the step (1) are the general disinfectants of vegetable material, and general working concentration alcohol is volume fraction 75%, and mercuric chloride is 1g/L, preferably sterilization rinsed with sterile water 5 times.
The light source of the described illumination of step (2) can be a fluorescent lamp.
The described sheet prothallium of step (3) 35～45d after changing the sporophyte inducing culture over to forms the prothallium that sprouts, described prothallium is changing sporophyte subculture medium 15～25d formation juvenile sporophyte over to, grow 2～3 young sporophylls, described proliferated culture medium, the light source of the illumination of sporophyte inducing culture and sporophyte subculture medium can be a fluorescent lamp.
The light source of the described illumination of step (4) can be a fluorescent lamp, the described hardening time is 3～4d, described soil matrix can be by flower cultivating soil, and the mixing in 2: 1: 1 by volume of vegetable garden mud and plain sand obtains, and also can be obtained by mixing such as pond sludge, wood fragments, humus soil and fine sands.
MS described in step (2), (3) is international medium, its composition and compound method referring to document (Tan Wencheng, Dai Cegang chief editor. the ornamental plants tissue culture technique. Beijing: China Forest publishing house, 1991.); 1/2MS described in step (2), (4) reduces by half the macroelement concentration among the MS, and the constant and medium that forms of other constituent concentrations.
Less investment of the present invention, equipment is simple, and shortened breeding cycle of Dryopteris varig (L.) O. Ktze spore effectively, forming from the spore germination to the juvenile sporophyte only needs about 110d, obtain a large amount of seedlings, children's spore shoot survival percent can reach 90%, makes Dryopteris varig (L.) O. Ktze become good ornamental foliage plant and brings into play its medical value.
Embodiment
Following examples are to further specify of the present invention, but the invention is not restricted to following examples.
Embodiment 1:
The sporophyll of clip maturation, with spore from careful the scraping of leaf margin, the spore of collecting (about 0.1g) is wrapped with filter paper, on superclean bench, use the alcohol-pickled 25s of 10mL volume fraction 75%, the mercuric chloride solution of the putting into 10mL 1g/L again 7min that sterilizes, rinsed with sterile water 5 times is cleaned mercuric chloride, is placed in the aseptic culture dish stand-by at last.
MS medium, 6g agar and 18g sucrose are made the MS spore germination medium 1L of pH 5.7～5.8, and the medium branch of making is installed in 150mL or the 200mL blake bottle, every bottle adds the about 15mL of medium approximately, and room temperature cooling back is stand-by.
Aseptic spore is made into suspension with the 3mL sterile water, gets 0.2mL with aseptic pipette and be added dropwise in the ready blake bottle, slight wave and culture bottle is evenly distributed on the spore germination medium spore.Cultivation temperature is 23～25 ℃, and intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d.The spore germination medium shows green when 25～30d, and spore sprouts green filamentous in a large number.
With 6-benzyl purine 0.15mg, methyl 0.15mg, agar 6g, sucrose 28g and MS are mixed with the proliferated culture medium 1L of pH 5.7～5.8; With kinetin 0.4mg, methyl 0.15mg, agar 6g, sucrose 28g and MS are made into pH 5.7～5.8 sporophyte inducing culture 1L; With methyl 0.05mg, agar 6g, sucrose 28g and MS are made into the sporophyte subculture medium 1L of pH 5.7～5.8.Three kinds of medium making are filled into respectively in 150mL or the 200mL blake bottle, and every bottle adds the about 15mL of medium approximately, and room temperature cooling back is stand-by.
After the spore germination, prothallium is equal well-grown on the spore germination medium, develops into laminated structure behind about 30d, the sheet prothallium is transferred on the prothallium proliferated culture medium, and cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source is a fluorescent lamp, light application time 11～13h/d.Behind the 30d, the prothallium propagation in the prothallium proliferated culture medium is obviously not only widened elongation significantly, and in the middle of the prothallium and top end obviously thicken, at this moment, prothallium is transferred in the sporophyte inducing culture, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d, the prothallium top constantly thickeies, behind about 40d, the prothallium top is induced and is sprouted, and 1 prothallium top has 3～4 buds usually.Change the prothallium that has sprouted in sporophyte subculture medium successive transfer culture, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source is a fluorescent lamp, and light application time 11～13h/d, prothallium no longer breed thickening, bud on the prothallium is constantly grown, behind about 20d, bud grows young sporophyll successively, and a large amount of juvenile sporophytes form.Forming from the spore germination to the juvenile sporophyte only needs about 110d.
With methyl 0.25mg, activated carbon 4g, agar 6g, sucrose 28g, 1/2MS medium make the sporophyte root media 1L of pH 5.7～5.8, and the medium branch of making is installed in 150mL or the 200mL blake bottle, every bottle adds the about 15ml of medium approximately, and room temperature cooling back is stand-by.
When treating that juvenile sporophyte grows 2～3 young sporophylls, its plant division is transferred on the sporophyte root media every bottle of about 10 strain juvenile sporophytes of culture medium inoculated, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d.Through the growth of 30d, the juvenile sporophyte blade is grown up gradually, and the juvenile sporophyte base portion begins to grow very thin root, and root is many and close.Treat that juvenile sporophyte grows 3～4 young sporophylls, when base portion grows the radicula of more brown band scale, but bottle outlet is transplanted.Blake bottle is transplanted in the greenhouse in culturing room, placed spacious bottle hardening 3d under the natural daylight.During transplanting, juvenile sporophyte is taken out from bottle, carefully clean the medium that root adheres to, transplant mixed soil matrix (soil matrix is by flower cultivating soil, and the mixing in 2: 1: 1 by volume of vegetable garden mud and plain sand obtains), after the transplanting, note spraying and moisturizing to the high temperature sterilization.Behind 25～35d, young spore shoot survival percent reaches 90%.
Embodiment 2:
The sporophyll of clip maturation, with spore from careful the scraping of leaf margin, the spore of collecting (about 0.1g) is wrapped with filter paper, on superclean bench, use the alcohol-pickled 30s of 10mL volume fraction 75%, the mercuric chloride solution of the putting into 10mL 1g/L again 8min that sterilizes, rinsed with sterile water 5 times is cleaned mercuric chloride, is placed in the aseptic culture dish stand-by at last.
1/2MS and 8g agar and 22g sucrose are made the 1/2MS spore germination medium 1L of pH 5.7～5.8, the medium branch of making is installed in 150mL or the 200mL blake bottle, every bottle adds medium 15mL approximately, and room temperature cooling back is stand-by.
Aseptic spore is made into suspension with the 3mL sterile water, getting 0.2mL with aseptic pipette is added dropwise in the ready spore germination blake bottle, slight wave and culture bottle, spore is evenly distributed on the medium, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d.The spore germination medium shows green when 25～30d, and spore sprouts green filamentous in a large number.
With 6-benzyl purine 0.25mg, methyl 0.25mg, agar 8g, sucrose 32g and MS are made into the proliferated culture medium 1L of pH 5.7～5.8; With kinetin N6-furfuryladenine 0.6mg, methyl 0.25mg, agar 8g, sucrose 32g and MS are made into pH 5.7～5.8 sporophyte inducing culture 1L; With methyl 0.15mg, agar 8g, sucrose 32g and MS are made into the sporophyte subculture medium 1L of pH 5.7～5.8.Three kinds of medium making are divided respectively install in 150mL or the 200mL blake bottle, every bottle adds the about 15mL of medium approximately, and room temperature cooling back is stand-by.
After the spore germination, prothallium is equal well-grown on 1/2MS spore germination medium, develop into laminated structure behind about 30d, the sheet prothallium is transferred on the prothallium proliferated culture medium, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d.Prothallium propagation behind about 30d in the prothallium proliferated culture medium obviously, not only widen elongation significantly, and prothallium is middle and top end is obviously thickeied, at this moment, prothallium is transferred in the sporophyte inducing culture, cultivation temperature is 23～25 ℃, and intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d, the prothallium top constantly thickeies, and the primary liquid top is induced and sprouted behind about 40d, and 1 prothallium top has 3～4 buds usually.Change the prothallium that has sprouted in sporophyte subculture medium successive transfer culture, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source is a fluorescent lamp, light application time 11～13h/d, prothallium no longer breed thickening, and the bud on the prothallium is constantly grown, bud grows young sporophyll successively behind about 20d, and a large amount of juvenile sporophytes form.Forming from the spore germination to the juvenile sporophyte only needs about 110d.
With methyl 0.35mg, activated carbon 6g, agar 8g, sucrose 32g, 1/2MS medium make the sporophyte root media 1L of pH 5.7～5.8, and the medium of making is filled in 150mL or the 200mL blake bottle, every bottle adds medium 15mL approximately, and room temperature cooling back is stand-by.
When treating that juvenile sporophyte grows 2～3 young sporophylls, its plant division is transferred on the sporophyte root media every bottle of about 10 strain juvenile sporophytes of culture medium inoculated, cultivation temperature is 23～25 ℃, intensity of illumination 2000～2500lx, light source are fluorescent lamp, light application time 11～13h/d.Through the growth of about 30d, the juvenile sporophyte blade is grown up gradually, and the juvenile sporophyte base portion begins to grow very thin root, and root is many and close.Treat that juvenile sporophyte grows 3～4 young sporophylls, when base portion grows the radicula of more brown band scale, but bottle outlet is transplanted.Blake bottle is transplanted in the greenhouse in culturing room, placed spacious bottle hardening 4d under the natural daylight.During transplanting, juvenile sporophyte is taken out from bottle, carefully clean the medium that root adheres to, transplant, after the transplanting, note spraying and moisturizing to the mixed soil matrix of high temperature sterilization.Behind about 25～35d, young spore shoot survival percent reaches 90%.
Claims (2)
1. the sporogenesis method of a Dryopteris varig (L.) O. Ktze, its feature comprises the steps:
(1) collects Dryopteris varig (L.) O. Ktze (Dryopteris varia (L.) Ktunze) spore, in alcohol, soak 25～30s earlier, and then in mercuric chloride solution, soak 7～8min, sterilization back rinsed with sterile water;
(2) the Dryopteris varig (L.) O. Ktze spore with above-mentioned sterilization is made into suspension with sterile water, add in the spore germination medium, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx, cultivate under light application time 11～13h/d condition, sprout green filamentous earlier, develop into the sheet prothallium again, described spore germination medium is every liter and contains 6～8g agar, 18～22g sucrose, all the other are MS or 1/2MS, the medium of pH5.7～5.8, described 1/2MS reduces by half the macroelement concentration among the MS, and the constant and medium that forms of other constituent concentrations;
(3) above-mentioned sheet prothallium is transferred in the proliferated culture medium, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx, under light application time 11～13h/d condition, after cultivating 25～35d, change in the sporophyte inducing culture, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx is under light application time 11～13h/d condition, be cultured to and change the sporophyte subculture medium over to after prothallium sprouts, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx is under light application time 11～13h/d condition, be cultured to the formation juvenile sporophyte, grow 2～3 young sporophylls; Contain 6-benzyl purine 0.15～0.25mg in every liter of the described proliferated culture medium, methyl 0.15～0.25mg, agar 6～8g, sucrose 28～32g, all the other are MS, pH5.7～5.8; Contain kinetin 0.4～0.6mg in every liter of the described sporophyte inducing culture, methyl 0.15～0.25mg, agar 6～8g, sucrose 28～32g, all the other are MS, pH5.7～5.8; Contain methyl 0.05～0.15mg in every liter of the described sporophyte subculture medium, agar 6～8g, sucrose 28～32g, all the other are MS, pH5.7～5.8;
(4) above-mentioned juvenile sporophyte plant division is transferred on the sporophyte root media, 23～25 ℃ of temperature, intensity of illumination 2000～2500lx, under light application time 11～13h/d condition, when being cultured to the juvenile sporophyte base portion and growing the radicula of brown band scale, but bottle outlet is transplanted, transplanting after the hardening to the pH value is that 6.3～6.4 soil matrix is cultivated, and contains methyl 0.25～0.35mg, activated carbon 4～6g in every liter of the described sporophyte root media, agar 6～8g, sucrose 28～32g, all the other are the 1/2MS medium, pH5.7～5.8, described 1/2MS reduces by half the macroelement concentration among the MS, and the constant and medium that forms of other constituent concentrations.
2. the sporogenesis method of a kind of Dryopteris varig (L.) O. Ktze according to claim 1, it is characterized in that the alcohol described in the step (1) is volume fraction 75%, mercuric chloride is 1g/L, rinsed with sterile water 5 times of sterilization back, the described hardening time of step (4) is 3～4 days, described soil matrix is by flower cultivating soil, and the mixing in 2: 1: 1 by volume of vegetable garden mud and plain sand obtains or obtained by pond sludge, wood fragments, humus soil and fine sand mixing.
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| CN101790962B (en) * | 2010-04-12 | 2012-09-05 | 东莞市生物技术研究所 | Production method of adiantum |
| CN102283090A (en) * | 2011-06-27 | 2011-12-21 | 江苏省中国科学院植物研究所 | Dryopteris erythrosora spore propagation method |
| CN102860262B (en) * | 2012-10-25 | 2013-07-10 | 南京林业大学 | Propagation method of cyrtomium fortunei |
| CN105724246A (en) * | 2014-12-08 | 2016-07-06 | 张秀国 | Method for rapid propagation of floating fern seedling |
| CN106613160A (en) * | 2016-11-23 | 2017-05-10 | 云南云投生态环境科技股份有限公司 | Spore breeding method for arsenic hyperaccumulator Pteris cretica |
| 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 |
| CN112021179B (en) * | 2020-09-03 | 2022-07-01 | 西南林业大学 | Tissue culture method of Dryopteris fragrans |
| CN112293253B (en) * | 2020-10-28 | 2022-02-18 | 中国科学院昆明植物研究所 | Isolated preservation culture medium and isolated preservation method for Adiantum furiosaeanum |
| CN116138165B (en) * | 2022-11-20 | 2024-01-30 | 江苏萤火虫环境科技有限公司 | Spore propagation method of Botrytis cinerea of lead-cadmium repair plant |
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| 安晓云等.绵马鳞毛蕨的组织培养与植株再生.植物生理学通讯43 4.2007,43(4),739-740. |
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