CN102860262B - Propagation method of cyrtomium fortunei - Google Patents
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Abstract
The invention discloses a propagation method of cyrtomium fortune. The propagation method comprises the following processing steps of: 1, preparing a culture medium; 2, sterilizing cyrtomium fortune spores; 3, inoculating and cultivating; and 4, reculturing seedling and transplanting cyrtomium fortune sporophyte seedling. The propagation method has the following advantages that: deficiencies of long culture period, high cost and low efficiency of the conventional cyrtomium fortune propagation technology are made up, and a new way for artificial propagation of the cyrtomium fortune is developed. Few spore materials are utilized, so that a large amount of sporophytes can be quickly propagated, the seedling is formed in 65 days, and the planting percent is about 97%, so that large-scale industrial production of the cyrtomium fortune becomes possible, thus requirements of the market on the cyrtomium fortune are satisfied, and meanwhile, damage on the wild resources and the environment can be reduced. Furthermore, the invention further provides a good testing system for developing researches about genetic breeding of species and conservation biology, and wide application prospect is provided.
Description
Technical field
What the present invention relates to is the propagation method of the propagation method of plant, particularly a kind of rhizome of cyrtomium.
Background technology
Rhizome of cyrtomium (Cyrtomium fortunei) is Dryopteridaceae (Dryopteridaceae) perennial evergreen draft, be used as medicine with root-like stock and petiole residue, have the effect of clearing heat and detoxicating, hemostasis, desinsection, be usually used in treating diseases such as influenza, hepatitis B, viral pneumonia, functional uterine bleeding and abdominal pain due to enterositosis; Also be used for the treatment of diseases such as hypertension, dizziness, headache, chronic lead poisoning (Liu Hanzhen, Yu Hao, Xu Wen, what the triumph. the propagation method of rhizome of cyrtomium and culture technique. Chinese wild plant resource, 2005,24(3): 60-63).Simultaneously, rhizome of cyrtomium be again a kind of ornamental plants of grace and good water and soil conservation plant (stone mine. view and admire fern. Beijing: China Forest publishing house, 2002,151-152; Liu Hanzhen, Yu Hao, Xu Wen, what triumph. the propagation method of rhizome of cyrtomium and culture technique. Chinese wild plant resource, 2005,24(3): 60-63).
The source of Chinese medicine rhizome of cyrtomium is extremely complicated, and the rhizome of the multiple pteridophyte that locality or neighborhood produce is all used in various places.At present commercially available rhizome of cyrtomium medicinal material is except C. fortunei, also have surplus dryopteris crassirhizoma (Dryopteris Crassirhixoma), lunathyrium acrostichoidesChing (Lunathyrium acrostichoides), simple bud rhizoma cibotii (Wood wardia unigemmata), the osmund (Osmunda Japonica) etc. 30 more than the kind, belong to 6 sections.By herbal textual, the medicinal source of Chinese medicine rhizome of cyrtomium should be C. fortunei(summer will person of outstanding talent. this grassland plant textual criticism of Chinese medicine rhizome of cyrtomium. and traditional Chinese medicine, 1988,5:23-25; Zhang Lijun, Tian Shaoku, Wu Jianhua. the progress of rhizome of cyrtomium. the Shaanxi traditional Chinese medical science, 2002,23 (8): 748-749).By 10 flavor Chinese herbal medicines (rhizome of cyrtomium, the coptis, Radix Isatidis, the Radix Astragali, kuh-seng, the capsule of weeping forsythia, the bighead atractylodes rhizome, the root of kudzu vine etc.) the external bacteriostatic experiment of salmonella is found, rhizome of cyrtomium, the coptis are to the strongest (Li Guowang of bacteriostasis of salmonella, Miao Zhiguo, Zhao Hengzhang. 10 kinds of Chinese herbal medicines such as Radix Isatidis are to the extracorporeal bacteria inhibitor test of salmonella. the Guizhou agricultural science, 2010,38(2): 142-144).The phloroglucin compounds that contains in the rhizome of cyrtomium, seldom age, the collection season with plant changes, and has powerful anthelmintic action, and rhizomatic powder and crude extract are continued to use as anthelmintic drug since ancient times.Its significantly active and taxonomy be worth be since 19th century people to its carry out a large amount of chemical research reason (Zhourong's Chinese, section golden storehouse for grain, etc. plant chemotaxonomy. Shanghai: Shanghai science tech publishing house, 2005,337-338).
Because the artificial propagation culture technique of rhizome of cyrtomium relatively lags behind, cause present commercially available rhizome of cyrtomium medicinal material to be mainly derived from the Dryopteris crassirhizoma Nakai resource in northeast, North China and area, the middle and lower reach of Yangtze River.And because market demand is bigger, make these regional rhizome of cyrtomium resources by exhaustive exploitation, the Dryopteris crassirhizoma Nakai resource is seriously damaged.
The artificial fecundation method of rhizome of cyrtomium has division propagation and sporogenesis.Wherein, the growth cycle of division propagation is long and efficient is low, is unfavorable for the large-scale production of medicinal rhizome of cyrtomium.Rhizome of cyrtomium sporogenesis technology generally be the rhizome of cyrtomium spore be seeded on the mixed soil medium cultivate (Liu Hanzhen, Yu Hao, Xu Wen, what triumph. the propagation method of rhizome of cyrtomium and culture technique. Chinese wild plant resource, 2005,24(3): 60-63; Wu Qin. several propagation technique researchs [D] of viewing and admiring pteridophyte. Nanjing: Nanjing Forestry University, 2007), the rhizome of cyrtomium juvenile sporophyte that this method is turned out approximately occurred from after planting 110-190 days, obtain 45-125 days evening time of juvenile sporophyte than the present invention, the reproduction speed of rhizome of cyrtomium is quite slow.Xu Yan uses the method for rhizome of cyrtomium spore aseptic culture, earlier with 5% NaClO solution (containing the 0.1ml Tween-80 in per this solution of 100 ml) with spore 4 min that sterilize, press 4300 spore/cm then
2Inoculum concentration, the rhizome of cyrtomium spore inoculating is cultivated on the 1/2MS solid culture medium, behind spore inoculating, can see in 80～90 days juvenile sporophyte produce (Xu Yan. view and admire pteridophyte germplasm ultralow temperature and preserve research [D]. Beijing: Beijing Forestry University, 2006).
Summary of the invention
What the present invention proposed is a kind of propagation method of rhizome of cyrtomium, and its purpose is intended to remedy deficiencies such as the cultivation period that existing rhizome of cyrtomium propagation technique has is long, cost is high and efficient is low.The spore of rhizome of cyrtomium after sterilization, is inoculated on the MS solid culture medium and cultivates, obtain the sporophyte seedling, obtain rhizome of cyrtomium through transplanting again.
Technical solution of the present invention: a kind of propagation method of rhizome of cyrtomium comprises following processing step:
One, medium preparation;
Two, the sterilization of rhizome of cyrtomium spore;
Three, inoculation and cultivation;
Four, the transplanting of rhizome of cyrtomium sporophyte seedling and management.
Advantage of the present invention: the deficiency that the present invention has remedied that existing rhizome of cyrtomium propagation technique cultivation period is long, cost is high and efficient is low etc., opened up the new way of an artificial propagation rhizome of cyrtomium.With a spot of spore material, can go out a large amount of sporophytes by fast breeding, can become seedling in 65 days; planting percent can reach about 97%; make the large-scale industrial production of rhizome of cyrtomium become possibility, thereby satisfy market to the demand of rhizome of cyrtomium, can reduce its wild resource and environment damage simultaneously.In addition, the present invention also provides good test system for the research of carrying out aspects such as this species genetic breeding and conservation biology, is with a wide range of applications.
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
Embodiment 1
The propagation method of rhizome of cyrtomium comprises following processing step:
Processing step one, medium preparation,
1) the MS solid culture medium is used: 0.7%(W/V) agar, and pH 5.8;
2) boil the back branch and be filled in the 100ml triangular flask, 121 ℃ then, high pressure (0.1Mpa) steam sterilizing 20 min.
The sterilization of processing step two, rhizome of cyrtomium spore,
1) wrap the ripe spore of rhizome of cyrtomium with filter paper packet, tie paper bag with line, drop into the 4%NaClO aqueous solution, shake frequently, 4 min sterilize;
2) use aseptic water washing 4 times;
3) open paper bag, draw sterile water with dropper, and the rhizome of cyrtomium spore is washed to 10 ml centrifuge tubes, mix making into suspension with vortex mixer, obtain aseptic rhizome of cyrtomium spore suspension.
Processing step three, inoculation and cultivation,
1) under aseptic condition, earlier with the surface wettability of sterile water with the MS medium of above-mentioned preparation, make the MS media surface form the very thin moisture film of one deck, the water that collects in the bottom during apparent dip triangular flask can not allow dropper suct continuous water droplet and be advisable;
2) with dropper the aseptic rhizome of cyrtomium spore suspension that step 2 obtains evenly is seeded on the medium, inoculum density is 2000 spore/cm
2
3) after the inoculation, under 23 ℃, secretly cultivate 24h earlier,
4) go to continuation cultivation under the illumination, intensity of illumination is 2000lx, and light source is fluorescent lamp, and light application time is 10h every day, cultivates 7 days.
Processing step four, rhizome of cyrtomium sporophyte seedling divide seedling, transplanting,
1) cultivate after 7 days, spore began to sprout, and formed gametophyte through 30 days, formed the sporophyte seedling in 65 days, and with height 2cm, blade is that aseptic sporophyte seedling placed the natural lighting lower refining seedling 6 days more than 2; Place air identical illumination condition lower refining seedling 6 days again;
2) open the bottle cap hardening 7 days, press from both sides out the individual plant test-tube plantlet gently with tweezers then;
3) behind the clean medium, the individual plant test-tube seedling transplanting to the cave dish that composite soil is housed, is placed on the slow seedling in shady and cool lucifuge place 4 days, moves to hot-house culture then, cultivation temperature is 23 ℃, and intensity of illumination is 2000lx, and light application time is 10h/ days; The percent by volume of the each component that described composite soil comprises is turfy soil: vermiculite: perlite=2:1:1;
4) seedling grows to 9cm, and it is transplanted in the plastic basin that culture matrix is housed, and puts hot-house culture, and Xia Qiu shades, and keeps 21 ℃ of day temperatures winter in spring, 10 ℃ of nocturnal temperatures, and air humidity 70%, slow seedling 25 days, every first quarter moon waters the MS nutrient solution; Described nutrient solution is except not containing the agar other compositions and MS(Murashige and Skoog) solid culture medium is identical.
The percent by volume of the each component that described culture matrix comprises is: turfy soil: vermiculite: perlite: fertilizer=4:2:2:1.
Embodiment 2
The propagation method of rhizome of cyrtomium comprises following processing step:
Processing step one, medium preparation,
1) the MS solid culture medium is used: 0.7%(W/V) agar, and pH 5.8;
2) boil the back branch and be filled in the 100ml triangular flask, 121 ℃ then, high pressure (0.1Mpa) steam sterilizing 20 min.
The sterilization of processing step two, rhizome of cyrtomium spore,
1) wrap the ripe spore of rhizome of cyrtomium with filter paper packet, tie paper bag with line, drop into the 6%NaClO aqueous solution, shake frequently, 4 min sterilize;
2) use aseptic water washing 5 times;
3) open paper bag, draw sterile water with dropper, and the rhizome of cyrtomium spore is washed to 10 ml centrifuge tubes, mix making into suspension with vortex mixer, obtain aseptic rhizome of cyrtomium spore suspension.
Processing step three, inoculation and cultivation,
1) under aseptic condition, earlier with the surface wettability of sterile water with the MS medium of above-mentioned preparation, make the MS media surface form the very thin moisture film of one deck, the water that collects in the bottom during apparent dip triangular flask can not allow dropper suct continuous water droplet and be advisable;
2) with dropper the aseptic rhizome of cyrtomium spore suspension that step 2 obtains evenly is seeded on the medium, inoculum density is 2500 spore/cm
2
3) after the inoculation, under 27 ℃, secretly cultivate 36 h earlier,
4) go to continuation cultivation under the illumination, intensity of illumination is 2300 lx, and light source is fluorescent lamp, and light application time is 14h every day, cultivates 7 days.
Processing step four, rhizome of cyrtomium sporophyte seedling divide seedling, transplanting,
1) cultivate after 7 days, spore began to sprout, and formed gametophyte through 32 days, formed the sporophyte seedling in 65 days, and with height 4 cm, blade is that aseptic sporophyte seedling placed the natural lighting lower refining seedling 8 days more than 2; Place air identical illumination condition lower refining seedling 8 days again;
2) open the bottle cap hardening 7 days, press from both sides out the individual plant test-tube plantlet gently with tweezers then;
3) behind the clean medium, the individual plant test-tube seedling transplanting to the cave dish that composite soil is housed, is placed on the slow seedling in shady and cool lucifuge place 5 days, moves to hot-house culture then, cultivation temperature is 27 ℃, and intensity of illumination is 2300lx, and light application time is 14h/ days; The percent by volume of the each component that described composite soil comprises is turfy soil: vermiculite: perlite=2:1:1;
4) seedling grows to 11 cm, and it is transplanted in the plastic basin that culture matrix is housed, and puts hot-house culture, and Xia Qiu shades, and keeps 26 ℃ of day temperatures winter in spring, 116 ℃ of nocturnal temperatures, and air humidity 90%, slow seedling 35 days, every first quarter moon waters the MS nutrient solution; Described nutrient solution is except not containing the agar other compositions and MS(Murashige and Skoog) solid culture medium is identical.
The percent by volume of the each component that described culture matrix comprises is: turfy soil: vermiculite: perlite: fertilizer=4:2:2:1.
Claims (1)
1. the propagation method of a rhizome of cyrtomium is characterized in that this method comprises following processing step:
One, medium preparation; Two, the sterilization of rhizome of cyrtomium spore; Three, inoculation and cultivation; Four, branch seedling, the transplanting of rhizome of cyrtomium sporophyte seedling; Described processing step one, medium preparation divide
1) solid culture medium MS(Murashige and Skoog): 0.7%(W/V) agar, pH 5.8;
2) boil the back branch and be filled in the 100ml triangular flask, 121 ℃ then, high pressure 0.1Mpa steam sterilizing 20 min;
The sterilization of described processing step two, rhizome of cyrtomium spore divides
1) wrap the ripe spore of rhizome of cyrtomium with filter paper packet, tie paper bag with line, drop into the 4-6%NaClO aqueous solution, shake frequently, 4 min sterilize;
2) use aseptic water washing 4-5 time;
3) open paper bag, draw sterile water with dropper, and the rhizome of cyrtomium spore is washed to 10 ml centrifuge tubes, mix making into suspension with vortex mixer, obtain aseptic rhizome of cyrtomium spore suspension;
Described processing step three, inoculation and cultivation divide
1) under aseptic condition, earlier with the surface wettability of sterile water with the MS medium of above-mentioned preparation, make the MS media surface form the very thin moisture film of one deck, during with the inclination triangular flask, look the water that collects in the bottom and can not allow dropper suct continuous water droplet to be advisable;
2) with the aseptic rhizome of cyrtomium spore suspension of dropper with processing step two acquisitions, evenly be seeded on the media surface, inoculum density is 2500 spore/cm of 2000-
2
3) after the inoculation, at temperature 23-27 ℃, cultivate 24-36 h under the unglazed photograph;
4) go to continuation cultivation under the illumination, its intensity of illumination is 2000-2300 lx, and light source is fluorescent lamp, and light application time is 10-14h every day, cultivates 7 days;
Described processing step four, rhizome of cyrtomium sporophyte seedling divide seedling, transplanting, divide
1) cultivate after 7 days, spore began to sprout, and formed gametophyte through 30-32 days, formed the sporophyte seedling, and as blade height 2-4 cm, was that aseptic sporophyte seedling placed the natural lighting lower refining seedling 6-8 days more than 2 with blade in 65 days; Place air natural lighting lower refining seedling 6-8 days again;
2) open the bottle cap hardening 7 days, press from both sides out the individual plant test-tube plantlet gently with tweezers then;
3) clean medium after, the individual plant test-tube seedling transplanting to the cave dish that composite soil is housed, and was placed on shady and cool lucifuge place slow seedling 4-5 days, move to hot-house culture then, the temperature of hot-house culture is 23-27 ℃, and intensity of illumination is 2000-2300 lx, and light application time is 10-14h/ days; The percent by volume of the each component that described composite soil comprises is turfy soil: vermiculite: perlite=2:1:1;
4) seedling grows to 9-11 cm, it is transplanted in the plastic basin that culture matrix is housed, put hot-house culture, Xia Qiu shades, keep day temperature 21-26 ℃ winter in spring, nocturnal temperature 10-16 ℃, air humidity 70-90%, slow seedling 25-35 days, every first quarter moon watered MS(Murashige and Skoog) nutrient solution; Described nutrient solution is except not containing the agar other compositions and MS(Murashige and Skoog) solid culture medium is identical;
Described culture matrix, the percent by volume of the each component that it comprises is: turfy soil: vermiculite: perlite: fertilizer=4:2:2:1.
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Families Citing this family (9)
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| CN104604493A (en) * | 2015-01-26 | 2015-05-13 | 河南科技学院 | Cyrtomium fortunei spore reproduction method |
| CN104782484B (en) * | 2015-04-13 | 2017-02-01 | 云南省农业科学院生物技术与种质资源研究所 | Method for obtaining tissue culture plantlets fast by utilizing homogenate of stems and leaves of hydrilla verticillata |
| CN105230492B (en) * | 2015-11-04 | 2017-11-24 | 蒋向军 | A kind of scythian lamb rhizome spore tissue culture mating system |
| CN107667859A (en) * | 2017-10-18 | 2018-02-09 | 陈培党 | A kind of rhizome of cyrtomium tissue culture and rapid propagation method |
| CN107593462A (en) * | 2017-11-14 | 2018-01-19 | 陈培党 | A kind of rhizoma woodwardiae tissue culture technique |
| CN108496960B (en) * | 2018-05-17 | 2021-03-30 | 吉林农业科技学院 | Ultralow-temperature preservation method for rhizoma dryopteris crassirhizomae |
| CN109197398B (en) * | 2018-11-09 | 2021-07-09 | 贵州大学 | Method for collecting clean basidiospores of agaricus fungus |
| 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 |
| CN112931212A (en) * | 2021-03-29 | 2021-06-11 | 江苏蕨美植物科技有限公司 | Method for breeding adiantum ladder |
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Application publication date: 20130109 Assignee: Beijing Huamei Wanxiang Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019980000688 Denomination of invention: Propagation method of cyrtomium fortunei Granted publication date: 20130710 License type: Common License Record date: 20191122 Application publication date: 20130109 Assignee: Yangzhou little apple gardening Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019980000696 Denomination of invention: Propagation method of cyrtomium fortunei Granted publication date: 20130710 License type: Common License Record date: 20191125 Application publication date: 20130109 Assignee: Jiangsu Bigen Fruit Industry Development Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019980000706 Denomination of invention: Propagation method of cyrtomium fortunei Granted publication date: 20130710 License type: Common License Record date: 20191125 |
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