CN105010146A - Rapid propagation method for dysosma versipellis - Google Patents
Rapid propagation method for dysosma versipellis Download PDFInfo
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- CN105010146A CN105010146A CN201510500146.7A CN201510500146A CN105010146A CN 105010146 A CN105010146 A CN 105010146A CN 201510500146 A CN201510500146 A CN 201510500146A CN 105010146 A CN105010146 A CN 105010146A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a rapid propagation method for dysosma versipellis. The rapid propagation method includes the following steps that 1, sterile dysosma versipellis explants are manufactured; 2, the sterile explants are cultured through an induction medium to obtain embryonic callus; 3, the embryonic callus is cultured through a first multiplication medium, and the embryonic callus is subcultured for 2-3 generations to obtain subcultured embryonic callus; 4, the subcultured embryonic callus is cultured through a differentiation medium to obtain somatic embryos; 5, the somatic embryos are cultured through a second multiplication medium to obtain subcultured somatic embryos; 6, the subcultured somatic embryos are cultured through a plant regeneration medium to obtain dysosma versipellis plants. According to the rapid propagation method, the obtained dysosma versipellis embryogenic cell can induce regeneration of the dysosma versipellis plants, and the dysosma versipellis plants have the advantages of being high in growth rate and plant regeneration frequency, stable in character and the like.
Description
Technical field
The present invention relates to biological technical field, refer in particular to the method for quickly breeding of a kind of Dysosma versipellis.
Background technology
Podophyllotoxin (Podophyllotoxin, PTOX) be cyclolignolide in Lignanoids compounds, be from Podophyllum emodi var chinense class plant extraction and isolation to a kind of natural products with antitumor activity, Prof. Du Yucang Etoposide (etoposide, and the precursor of the medicine such as Teniposide (teniposide, VM-26) VP-16).Research shows, podophyllotoxin has special efficacy for chronic disease such as treatment cancer of the esophagus, the cancer of the uterus etc., and the market demand is very huge.Along with continually developing and the increase year by year of mankind's cancer rate in recent years of podophyllotoxin New function, its market demand also will strengthen rapidly.Podophyllotoxin mainly from Berberidaceae Berberidaceae herbaceos perennial as Dysosma, Sinopodophyllum and Diphylleia and sufficient leaf grass to belong to etc. extraction, but limits throughput.Wherein, Dysosma versipellis Dysosma versipellis (Hance) M.Cheng is the main traditional medicine source plant resource producing podophyllotoxin.
Dysosma versipellis is one of China's tradition conventional Chinese medicine, and tool is clearing heat and detoxicating, the function of reducing phlegm and resolving masses, eliminating phlegm and diminishing swelling.For a long time due to excessively excavate and natural propagation rate low, cause wild resource to wane, species are in imminent danger, have been listed in national Precious, Rare, Endangered three-level protective plant at present.In artificial cultivation, because Dysosma versipellis percentage of fertile fruit is low, grain weight is few, and the seedling of seed germination could be gathered in the crops through 5-6, thus limits the production of Dysosma versipellis.
To the research of Dysosma versipellis, the past mainly concentrates on taxonomic identification, analysis of effective component, the aspect such as pharmacology and cultivation of Dysosma versipellis.In recent years, Huang Jiayun etc. (2007) induce Dysosma versipellis callus to produce Multiple Buds, and the formation seedling that takes root.Huang Jiayun etc. (2008) agitated submerged culture Dysosma versipellis callus, and carried out the detection of podophyllotoxin.Tang Fengluan etc. (2008) induce Dysosma versipellis seed seedling to form Multiple Buds, and then callus induction and root, directly form regeneration plant.Wei Ying etc. (2011) report the Induction and differentiation of Dysosma versipellis callus, and successful regeneration goes out plant.Although the tissue cultures of Dysosma versipellis makes some progress, easy brownization of callus, the differentiation rate of induction are low, and easily make a variation, and have had a strong impact on the production practices by quick breeding by group culture Dysosma versipellis seedling.
Somatic embryo be plant callus on solid culture medium after repeatedly subculture, produce meristematic cell, form embryo callus; This in-house cell mass further develops and becomes embryoid.Utilize plant energy high-frequency to produce the experimental system of embryoid, be combined on shaking table and carry out liquid culture, Synchronous Screening is with the consistent high-quality embryoid of acquired character.By plant embryos regeneration plant, there is the advantages such as fast growth, plant regeneration frequency is high, proterties is stable, and can choiceness be screened, the screening of binding activities composition, obtain the high and plant of inheritance stability of active component content, this has become the good receptor material carrying out Secondary Metabolism of Plant and regulatory mechanism.Some medicinal plants such as the coptis (Gui Yaolin etc., 1989), santal (Shekhawat et al.2008), Gynura bicolor (Guo Yangdong etc., 2010) etc. successfully set up cells,primordial suspension culture system.But, at present except the achievement in research in this laboratory, there is no the report about Dysosma versipellis plant somatocyte embryo occurs.
The content of invention
An object of the present invention is to solve at least the problems referred to above and/or defect, and the advantage will illustrated at least is below provided.
A further object of the invention is the technological gap in order to overcome Dysosma versipellis cells,primordial suspension training system, the method of the Fast-propagation of a kind of Dysosma versipellis is provided, in the present invention, the Dysosma versipellis cells,primordial obtained can induce the regeneration of Dysosma versipellis plant, has the advantages such as fast growth, plant regeneration frequency is high, proterties is stable.
In order to realize these objects of the present invention and other advantage, providing the method for quickly breeding of a kind of Dysosma versipellis, comprising the following steps:
Step one: the aseptic explant making Dysosma versipellis;
Step 2: utilize inducing culture to cultivate described aseptic explant, obtain embryo callus, described inducing culture comprises the agar of MS, 2, the 4-D growth hormone of 0.5 ~ 2.0mg/L, the pyroligneous liquor of 1.0 ~ 1.5mg/L, the plants essential oil of 0.2 ~ 0.5mg/L and 6g/L;
Step 3: utilize the first proliferated culture medium to cultivate described embryo callus, squamous subculture 2 ~ 3 generation, obtain subcultured callus, described first proliferated culture medium comprises MS, 2, the 4-D growth hormone of 0.5 ~ 2.0mg/L, the stachyose of 20 ~ 25mg/L and the agar of 6g/L;
Step 4: utilize differential medium to cultivate described subcultured callus, obtain body embryo, described differential medium comprises MS, the 6-benzyl aminoadenine mitogen of 0 ~ 1.0mg/L, the methyl α-naphthyl acetate of 0.5 ~ 2.0mg/L and the agar of 6g/L;
Step 5: utilize the second proliferated culture medium to cultivate described body embryo, obtain subculture body embryo, described second proliferated culture medium comprises MS, the 6-benzyl aminoadenine mitogen of 0 ~ 1.0mg/L, the methyl α-naphthyl acetate of 0.5 ~ 2.0mg/L and 2, the 4-D growth hormone of 0 ~ 2.0mg/L;
Step 6: utilize subculture body embryo described in plant regeneration medium culture, obtain Dysosma versipellis plant, described plant regeneration medium comprises MS, the traditional Chinese medicine powder of 1.0 ~ 1.5mg/L, the indolebutyric acid of the gibberellin 3,0.2 ~ 0.6mg/L of 1.0 ~ 1.4mg/L, the sepiolite powder of 0.3 ~ 0.7mg/L and the agar of 6g/L;
The preparation method of described traditional Chinese medicine powder is: be the Dysosma versipellis of 30 ~ 40 parts by parts by weight, the distinguished and admirable fruit of 18 ~ 25 parts, the Lysimachia foenum-graecum of 23 ~ 28 parts, the Rong'an cumquat of 15 ~ 17 parts and 10 ~ 15 parts larva of a silkworm with batrytis mixing after decoct 30 ~ 40min, filter to obtain Chinese medicine slag, through autoclaving post-drying, then pulverize 200 eye mesh screens, obtain described traditional Chinese medicine powder.
Preferably, the method for quickly breeding of described Dysosma versipellis, also comprises:
Step 7, described Dysosma versipellis plant moved in strong seedling culture base and cultivate 15 ~ 20 days, obtain Dysosma versipellis seedling, described strong seedling culture base comprises short root medium and short stem medium, described strong seedling culture base is positioned in incubator, described short stem medium is positioned at the top of described short root medium, from the below of Dysosma versipellis plant, be immersed in described short root medium by 1/5 of Dysosma versipellis plant, be placed in described short stem medium by 1/3 of Dysosma versipellis plant;
Described short stem medium comprises MS, the described traditional Chinese medicine powder of 1.0 ~ 1.5mg/L, the indolebutyric acid of the gibberellin 3,0.2 ~ 0.6mg/L of 1.0 ~ 1.4mg/L, the sepiolite powder of 0.3 ~ 0.7mg/L and the agar of 6g/L;
Described short root medium comprises MS, the pomelo peel extract of 10 ~ 15mg/L, the indolebutyric acid of 0.8 ~ 1.2mg/L and the pyroligneous liquor of 1.0 ~ 1.5mg/L; The preparation method of the extract of described pomelo peel is: shredded by pomelo peel, be exposed to the sun 1 ~ 2 day, then put into boiling water and boil 30 ~ 40min, filter while hot, and in the filtrate of thermotropism, adding the boric acid of pomelo peel quality 0.1 times, described filter vacuum is evaporated to 6mg/L, and arranging vacuum is 0.05 ~ 0.06MPa, during reduced pressure concentration, control temperature is 55 DEG C, obtains described pomelo peel extract;
Step 8, to cultivate in described Dysosma versipellis seedling replanting to matrix.
Preferably, the method for quickly breeding of described Dysosma versipellis, described incubator comprises upper box and lower box, described upper box and described lower box removably connect, described upper box is the cylindrical shell of up/down perforation, it is the polylactic acid film of 0.01 ~ 0.03 millimeter that the lower plane of described upper box is provided with the thickness of its lower plane consistent size, and described polylactic acid film has multiple through hole, and Dysosma versipellis plant described in a strain is through a described through hole;
Described lower box is the cylindrical shell that bottom closed upper portion is opened wide, and described lower box outer cover is provided with dismountable muff, and described short stem medium is placed on described polylactic acid film, and described short root medium is placed in described lower box.
Preferably, the method for quickly breeding of described Dysosma versipellis, described aseptic explant, described embryo callus, described subcultured callus and the condition of culture of described body embryo in respective medium are: temperature 20 DEG C ~ 24 DEG C, pH value 5.6 ~ 6.0, light culture 15 ~ 20 days, the condition of culture of described subculture body embryo in plant regeneration medium is 24 ~ 26 DEG C, is cultured to Plantlet formation under 1000 ~ 2000Lux illumination condition, and wherein light application time is 10h every day.
Preferably, the method for quickly breeding of described Dysosma versipellis, in described step 8, when cultivating in described Dysosma versipellis seedling replanting to matrix, related for described Dysosma versipellis seedling described short stem medium is transplanted in matrix together with described polylactic acid film, the lower surface of described polylactic acid film is made to be close to described substrate upper surface, the described short stem medium of upper surface carrying of described polylactic acid film, after described Dysosma versipellis seedling cultivates 8 ~ 10 days in matrix, described short stem medium is smashed to pieces, adjust the spacing of described Dysosma versipellis seedling in matrix, two often adjacent described Dysosma versipellis seedling spacing are made to be 30 ~ 35cm, continue to be cultured to Dysosma versipellis maturation,
Described matrix is from top to bottom by grassland ash, and loam, perlite, wood chip, pine needle is 1: 8: 2: 4: 3 formations by weight.
Preferably, the method for quickly breeding of described Dysosma versipellis, before described aseptic explant accesses described inducing culture, also comprise the step obtaining aseptic explant, the step of described acquisition aseptic explant is:
Choose the blade of Dysosma versipellis or root or stem as explant, described explant is placed in beaker, soak with amphoteric surfactant, and be placed in ultrasonic concussion 10 ~ 20min, with deionized water rinsing 40 ~ 50min, in flushing process, scrub the dirt on described explant surface with banister brush gently, then blot the moisture of described explant outside with aseptic filter paper after, be cut into 5mm
2fritter, obtain described aseptic explant;
Described amphoteric surfactant is: the N-long-chain alkoxy carboxylic acid type betain of 20 ~ 25 weight portions, α-cetyl trimethyl the betain of 15 ~ 18 weight portions, N-alkyl-β-the dipropionic acid of 10 ~ 13 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 5 ~ 10 weight portions and the sterile water of 60 ~ 70 weight portions.
Preferably, the method for quickly breeding of described Dysosma versipellis, described body embryo is in described second proliferated culture medium in incubation, and described second proliferated culture medium is arranged in the shaking table that rotating speed is 120r/min, and described second proliferated culture medium changed a subculture every 3 ~ 5 days.
The present invention at least comprises following beneficial effect:
The first, in the present invention, the Dysosma versipellis cells,primordial obtained can induce the regeneration of Dysosma versipellis plant, there is the advantages such as fast growth, plant regeneration frequency is high, proterties is stable, and can choiceness be screened, the screening of binding activities composition podophyllotoxin, obtain the high and plant of inheritance stability of active component podophyllotoxin content, this is the good receptor material carrying out Secondary Metabolism of Plant and regulatory mechanism, simultaneously also for the production of podophyllotoxin provides the raw material sources of a kind of high-quality and sustainable use;
The second, first the explant sterilization of Dysosma versipellis adopts amphoteric surfactant to soak, the amphoteric surfactant of beet alkalescence has good antibiotic property, the growth and breeding of part bacterium and fungi can be suppressed, the betain of the alkyl dimethyl simultaneously in amphoteric surfactant has good sequestering power to cupric, by cupric, from polyphenol oxidase, chelating is out, make polyphenol oxidase lose oxidability, reduce the brown stain of Dysosma versipellis explant;
Three, various trace elements required in Dysosma versipellis Plantlet formation process and benefit materials is contained in traditional Chinese medicine powder, traditional Chinese medicine powder is added in plant regeneration medium, improve the resistance against diseases of Dysosma versipellis plant, the survival rate more than 90% that later stage is transplanted, add simultaneously sepiolite powder with the use of, absorb the harmful substance in medium, contribute to the Dysosma versipellis plant cultivating high-quality; Add agar formation in short stem medium and solidify shape medium, traditional Chinese medicine powder and sepiolite powder is added in this medium, to Dysosma versipellis seedling supply nutrition and to medium sterilization while, the permeability of short stem medium can be improved, make the stem of the Dysosma versipellis seedling cultivated strongr;
Four, larger by obtaining rhizome to the further strong seedling culture of Dysosma versipellis plant, the Dysosma versipellis seedling that immunity is stronger, again Dysosma versipellis seedling is transplanted, make the Dysosma versipellis seedling of transplanting strongr, the survival rate that further guarantee Dysosma versipellis is transplanted, environment in strong seedling culture process needed for Dysosma versipellis root growth and the difference of nutrition, adopt the collocation of short root medium and short stem medium, accelerate the growth rate of Dysosma versipellis plant, ensure the nutrition of the Dysosma versipellis seedling fast-ripenin formed;
Five, upper and lower dismountable incubator project organization is simple, easy and simple to handle, for Dysosma versipellis seedling provides two kinds of different culture medias simultaneously, muff can be sleeved on lower box outside when temperature is lower, ensures the environment needed for Dysosma versipellis growth of seedling;
6th, related for Dysosma versipellis seedling short stem medium is transplanted in matrix together with polylactic acid film, certain laundering period is provided to Dysosma versipellis seedling, environmental catastrophe is avoided to stimulate the excessive and death that causes to Dysosma versipellis seedling, improve the survival rate that Dysosma versipellis is transplanted, after Dysosma versipellis accommodate substrate environment, short stem medium is smashed to pieces, short stem medium still can provide nutriment for it in the late growing stage process of Dysosma versipellis, polylactic acid film is degradable film, cover the effect that matrix top has insulation warming to matrix, polylactic acid film is finally degraded to the nutriment needed for Dysosma versipellis, improve the economic use value of short stem medium and polylactic acid film.
Accompanying drawing explanation
Fig. 1 is the structural representation of incubator described in embodiment in the present invention.
Embodiment:
< embodiment 1>
A method for quickly breeding for Dysosma versipellis, comprises the following steps:
Step one: the aseptic explant making Dysosma versipellis, choose the blade of Dysosma versipellis or root or stem as explant, described explant is placed in beaker, soak with amphoteric surfactant, and be placed in ultrasonic and shake 10min, use deionized water rinsing 40min, in flushing process, scrub the dirt on described explant surface with banister brush gently, then blot the moisture of described explant outside with aseptic filter paper after, be cut into 5mm
2fritter, obtain described aseptic explant, first the explant sterilization of Dysosma versipellis adopts amphoteric surfactant to soak, the amphoteric surfactant of beet alkalescence has good antibiotic property, can suppress the growth and breeding of part bacterium and fungi, the betain of the alkyl dimethyl simultaneously in amphoteric surfactant has good sequestering power to cupric, and by cupric, from polyphenol oxidase, chelating is out, make polyphenol oxidase lose oxidability, reduce the brown stain of Dysosma versipellis explant;
Described amphoteric surfactant is: the N-long-chain alkoxy carboxylic acid type betain of 20 weight portions, α-cetyl trimethyl the betain of 15 weight portions, N-alkyl-β-the dipropionic acid of 10 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 5 weight portions and the sterile water of 60 weight portions;
Step 2: utilize inducing culture to cultivate described aseptic explant, condition of culture is temperature 20 DEG C, pH value 5.6, light culture obtained embryo callus after 15 days, described inducing culture comprises the agar of 2, the 4-D growth hormone of MS, 0.5mg/L, the pyroligneous liquor of 1.0mg/L, the plants essential oil of 0.2mg/L and 6g/L;
Step 3: utilize the first proliferated culture medium to cultivate described embryo callus, squamous subculture 2 generation, condition of culture is temperature 20 DEG C, pH value 5.6, light culture obtained subcultured callus after 15 days, described first proliferated culture medium comprises 2, the 4-D growth hormone of MS, 0.5mg/L, the stachyose of 20mg/L and the agar of 6g/L;
Step 4: utilize differential medium to cultivate described subcultured callus, condition of culture is temperature 20 DEG C, pH value 5.6, and light culture obtained body embryo after 15 days, and described differential medium comprises the methyl α-naphthyl acetate of MS, 0.5mg/L and the agar of 6g/L;
Step 5: utilize the second proliferated culture medium to cultivate described body embryo, condition of culture is temperature 20 DEG C, pH value 5.6, and light culture obtains subculture body embryo after 15 days, described second proliferated culture medium comprises the methyl α-naphthyl acetate of MS, 0.5mg/L; Described second proliferated culture medium is arranged in the shaking table that rotating speed is 120r/min, and described second proliferated culture medium changed a subculture every 3 ~ 5 days;
Step 6: utilize subculture body embryo described in plant regeneration medium culture, condition of culture is 24 DEG C, Plantlet formation is cultured under 1000Lux illumination condition, light application time is 10h every day, described plant regeneration medium comprises the agar of the traditional Chinese medicine powder of MS, 1.0mg/L, the indolebutyric acid of gibberellin 3,0.2mg/L of 1.0mg/L, the sepiolite powder of 0.3mg/L and 6g/L;
The preparation method of described traditional Chinese medicine powder is: the Dysosma versipellis by parts by weight being 30 parts, the distinguished and admirable fruit of 18 parts, the Lysimachia foenum-graecum of 23 parts, 30min is decocted after the larva of a silkworm with batrytis mixing of the Rong'an cumquat of 15 parts and 10 parts, filter to obtain Chinese medicine slag, through autoclaving post-drying, then 200 eye mesh screens were pulverized, obtain described traditional Chinese medicine powder, containing various trace elements required in Dysosma versipellis Plantlet formation process and benefit materials in traditional Chinese medicine powder, traditional Chinese medicine powder is added in plant regeneration medium, improve the resistance against diseases of Dysosma versipellis plant, the survival rate more than 90% that later stage is transplanted, add simultaneously sepiolite powder with the use of, absorb the harmful substance in medium, contribute to the Dysosma versipellis plant cultivating high-quality,
Step 7, described Dysosma versipellis plant moved in strong seedling culture base and cultivate 15 days, obtain Dysosma versipellis seedling, described strong seedling culture base comprises short root medium and short stem medium, described strong seedling culture base is positioned in incubator, described short stem medium is positioned at the top of described short root medium, from the below of Dysosma versipellis plant, be immersed in described short root medium by 1/5 of Dysosma versipellis plant, be placed in described short stem medium by 1/3 of Dysosma versipellis plant;
Described short stem medium comprises the agar of the described traditional Chinese medicine powder of MS, 1.0mg/L, the indolebutyric acid of gibberellin 3,0.2mg/L of 1.0mg/L, the sepiolite powder of 0.3mg/L and 6g/L; Add agar formation in short stem medium and solidify shape medium, traditional Chinese medicine powder and sepiolite powder is added in this medium, to Dysosma versipellis seedling supply nutrition and to medium sterilization while, the permeability of short stem medium can be improved, make the stem of the Dysosma versipellis seedling cultivated strongr;
Described short root medium comprises the pomelo peel extract of MS, 10mg/L, the indolebutyric acid of 0.8mg/L and the pyroligneous liquor of 1.0mg/L; The preparation method of the extract of described pomelo peel is: shredded by pomelo peel, be exposed to the sun 1 day, then put into boiling water and boil 30min, filter while hot, and in the filtrate of thermotropism, adding the boric acid of pomelo peel quality 0.1 times, described filter vacuum is evaporated to 6mg/L, and arranging vacuum is 0.05MPa, during reduced pressure concentration, control temperature is 55 DEG C, obtains described pomelo peel extract;
Step 8, cultivate in described Dysosma versipellis seedling replanting to matrix, related for described Dysosma versipellis seedling described short stem medium is transplanted in matrix together with described polylactic acid film 3, the lower surface of described polylactic acid film 3 is made to be close to described substrate upper surface, the described short stem medium of upper surface carrying of described polylactic acid film 3, related for Dysosma versipellis seedling short stem medium is transplanted in matrix together with polylactic acid film 3, certain laundering period is provided to Dysosma versipellis seedling, environmental catastrophe is avoided to stimulate the excessive and death that causes to Dysosma versipellis seedling, improve the survival rate that Dysosma versipellis is transplanted, after described Dysosma versipellis seedling cultivates 8 days in matrix, described short stem medium is smashed to pieces, after Dysosma versipellis accommodate substrate environment, short stem medium is smashed to pieces, short stem medium still can provide nutriment for it in the late growing stage process of Dysosma versipellis, adjust the spacing of described Dysosma versipellis seedling in matrix, two often adjacent described Dysosma versipellis seedling spacing are made to be 30cm, continue to be cultured to Dysosma versipellis maturation, polylactic acid film 3 is degradable film, and cover the effect that matrix top has insulation warming to matrix, polylactic acid film 3 is finally degraded to the nutriment needed for Dysosma versipellis, improves the economic use value of short stem medium and polylactic acid film 3,
Described matrix is from top to bottom by grassland ash, and loam, perlite, wood chip, pine needle is 1: 8: 2: 4: 3 formations by weight.
As shown in Figure 1, wherein, described incubator comprises upper box 1 and lower box 2, described upper box 1 removably connects with described lower box 2, described upper box 1 is the cylindrical shell of up/down perforation, it is the polylactic acid film 3 of 0.01 millimeter that the lower plane of described upper box 1 is provided with the thickness of its lower plane consistent size, described polylactic acid film 3 has Dysosma versipellis plant described in the strain of multiple through hole 4, through a described through hole 4;
The cylindrical shell that described lower box 2 opens wide for bottom closed upper portion, described lower box 2 outer cover is provided with dismountable muff 5, and described short stem medium is placed on described polylactic acid film 3, and described short root medium is placed in described lower box 2;
Upper and lower dismountable incubator project organization is simple, easy and simple to handle, for Dysosma versipellis seedling provides two kinds of different culture medias simultaneously, muff 5 can be sleeved on lower box 2 outside when temperature is lower, ensure the environment needed for Dysosma versipellis growth of seedling.
< embodiment 2>
A method for quickly breeding for Dysosma versipellis, comprises the following steps:
Step one: the aseptic explant making Dysosma versipellis, choose the blade of Dysosma versipellis or root or stem as explant, described explant is placed in beaker, soak with amphoteric surfactant, and be placed in ultrasonic and shake 20min, use deionized water rinsing 50min, in flushing process, scrub the dirt on described explant surface with banister brush gently, then blot the moisture of described explant outside with aseptic filter paper after, be cut into 5mm
2fritter, obtain described aseptic explant, first the explant sterilization of Dysosma versipellis adopts amphoteric surfactant to soak, the amphoteric surfactant of beet alkalescence has good antibiotic property, can suppress the growth and breeding of part bacterium and fungi, the betain of the alkyl dimethyl simultaneously in amphoteric surfactant has good sequestering power to cupric, and by cupric, from polyphenol oxidase, chelating is out, make polyphenol oxidase lose oxidability, reduce the brown stain of Dysosma versipellis explant;
Described amphoteric surfactant is: the N-long-chain alkoxy carboxylic acid type betain of 25 weight portions, α-cetyl trimethyl the betain of 18 weight portions, N-alkyl-β-the dipropionic acid of 13 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 10 weight portions and the sterile water of 70 weight portions;
Step 2: utilize inducing culture to cultivate described aseptic explant, condition of culture is temperature 24 DEG C, pH value 6.0, light culture obtained embryo callus after 20 days, described inducing culture comprises the agar of 2, the 4-D growth hormone of MS, 2.0mg/L, the pyroligneous liquor of 1.5mg/L, the plants essential oil of 0.5mg/L and 6g/L;
Step 3: utilize the first proliferated culture medium to cultivate described embryo callus, squamous subculture 3 generation, condition of culture is temperature 24 DEG C, pH value 6.0, light culture obtained subcultured callus after 20 days, described first proliferated culture medium comprises 2, the 4-D growth hormone of MS, 2.0mg/L, the stachyose of 25mg/L and the agar of 6g/L;
Step 4: utilize differential medium to cultivate described subcultured callus, condition of culture is temperature 24 DEG C, pH value 6.0, light culture obtained body embryo after 20 days, and described differential medium comprises the 6-benzyl aminoadenine mitogen of MS, 1.0mg/L, the methyl α-naphthyl acetate of 2.0mg/L and the agar of 6g/L;
Step 5: utilize the second proliferated culture medium to cultivate described body embryo, condition of culture is temperature 24 DEG C, pH value 6.0, light culture obtains subculture body embryo after 20 days, described second proliferated culture medium comprises the 6-benzyl aminoadenine mitogen of MS, 1.0mg/L, the methyl α-naphthyl acetate of 2.0mg/L and 2, the 4-D growth hormone of 2.0mg/L; Described second proliferated culture medium is arranged in the shaking table that rotating speed is 120r/min, and described second proliferated culture medium changed a subculture every 3 ~ 5 days;
Step 6: utilize subculture body embryo described in plant regeneration medium culture, condition of culture is 26 DEG C, Plantlet formation is cultured under 2000Lux illumination condition, light application time is 10h every day, and described plant regeneration medium comprises the agar of the traditional Chinese medicine powder of MS, 1.5mg/L, the gibberellin 3 of 1.4mg/L, the indolebutyric acid of 0.6mg/L, the sepiolite powder of 0.7mg/L and 6g/L;
The preparation method of described traditional Chinese medicine powder is: the Dysosma versipellis by parts by weight being 40 parts, the distinguished and admirable fruit of 25 parts, the Lysimachia foenum-graecum of 28 parts, 40min is decocted after the larva of a silkworm with batrytis mixing of the Rong'an cumquat of 17 parts and 15 parts, filter to obtain Chinese medicine slag, through autoclaving post-drying, then 200 eye mesh screens were pulverized, obtain described traditional Chinese medicine powder, containing various trace elements required in Dysosma versipellis Plantlet formation process and benefit materials in traditional Chinese medicine powder, traditional Chinese medicine powder is added in plant regeneration medium, improve the resistance against diseases of Dysosma versipellis plant, the survival rate more than 90% that later stage is transplanted, add simultaneously sepiolite powder with the use of, absorb the harmful substance in medium, contribute to the Dysosma versipellis plant cultivating high-quality,
Step 7, described Dysosma versipellis plant moved in strong seedling culture base and cultivate 20 days, obtain Dysosma versipellis seedling, described strong seedling culture base comprises short root medium and short stem medium, described strong seedling culture base is positioned in incubator, described short stem medium is positioned at the top of described short root medium, from the below of Dysosma versipellis plant, be immersed in described short root medium by 1/5 of Dysosma versipellis plant, be placed in described short stem medium by 1/3 of Dysosma versipellis plant;
Described short stem medium comprises the agar of the described traditional Chinese medicine powder of MS, 1.5mg/L, the gibberellin 3 of 1.4mg/L, the indolebutyric acid of 0.6mg/L, the sepiolite powder of 0.7mg/L and 6g/L; Add agar formation in short stem medium and solidify shape medium, traditional Chinese medicine powder and sepiolite powder is added in this medium, to Dysosma versipellis seedling supply nutrition and to medium sterilization while, the permeability of short stem medium can be improved, make the stem of the Dysosma versipellis seedling cultivated strongr;
Described short root medium comprises the pomelo peel extract of MS, 15mg/L, the indolebutyric acid of 1.2mg/L and the pyroligneous liquor of 1.5mg/L; The preparation method of the extract of described pomelo peel is: shredded by pomelo peel, be exposed to the sun 2 days, then put into boiling water and boil 40min, filter while hot, and in the filtrate of thermotropism, adding the boric acid of pomelo peel quality 0.1 times, described filter vacuum is evaporated to 6mg/L, and arranging vacuum is 0.06MPa, during reduced pressure concentration, control temperature is 55 DEG C, obtains described pomelo peel extract;
Step 8, cultivate in described Dysosma versipellis seedling replanting to matrix, related for described Dysosma versipellis seedling described short stem medium is transplanted in matrix together with described polylactic acid film 3, the lower surface of described polylactic acid film 3 is made to be close to described substrate upper surface, the described short stem medium of upper surface carrying of described polylactic acid film 3, related for Dysosma versipellis seedling short stem medium is transplanted in matrix together with polylactic acid film 3, certain laundering period is provided to Dysosma versipellis seedling, environmental catastrophe is avoided to stimulate the excessive and death that causes to Dysosma versipellis seedling, improve the survival rate that Dysosma versipellis is transplanted, after described Dysosma versipellis seedling cultivates 10 days in matrix, described short stem medium is smashed to pieces, after Dysosma versipellis accommodate substrate environment, short stem medium is smashed to pieces, short stem medium still can provide nutriment for it in the late growing stage process of Dysosma versipellis, adjust the spacing of described Dysosma versipellis seedling in matrix, two often adjacent described Dysosma versipellis seedling spacing are made to be 35cm, continue to be cultured to Dysosma versipellis maturation, polylactic acid film 3 is degradable film, and cover the effect that matrix top has insulation warming to matrix, polylactic acid film 3 is finally degraded to the nutriment needed for Dysosma versipellis, improves the economic use value of short stem medium and polylactic acid film 3,
Described matrix is from top to bottom by grassland ash, and loam, perlite, wood chip, pine needle is 1: 8: 2: 4: 3 formations by weight.
As shown in Figure 1, wherein, described incubator comprises upper box 1 and lower box 2, described upper box 1 removably connects with described lower box 2, described upper box 1 is the cylindrical shell of up/down perforation, it is the polylactic acid film 3 of 0.03 millimeter that the lower plane of described upper box 1 is provided with the thickness of its lower plane consistent size, described polylactic acid film 3 has Dysosma versipellis plant described in the strain of multiple through hole 4, through a described through hole 4;
The cylindrical shell that described lower box 2 opens wide for bottom closed upper portion, described lower box 2 outer cover is provided with dismountable muff 5, and described short stem medium is placed on described polylactic acid film 3, and described short root medium is placed in described lower box 2;
Upper and lower dismountable incubator project organization is simple, easy and simple to handle, for Dysosma versipellis seedling provides two kinds of different culture medias simultaneously, muff 5 can be sleeved on lower box 2 outside when temperature is lower, ensure the environment needed for Dysosma versipellis growth of seedling.
< embodiment 3>
A method for quickly breeding for Dysosma versipellis, comprises the following steps:
Step one: the aseptic explant making Dysosma versipellis, choose the blade of Dysosma versipellis or root or stem as explant, described explant is placed in beaker, soak with amphoteric surfactant, and be placed in ultrasonic and shake 15min, use deionized water rinsing 45min, in flushing process, scrub the dirt on described explant surface with banister brush gently, then blot the moisture of described explant outside with aseptic filter paper after, be cut into 5mm
2fritter, obtain described aseptic explant, first the explant sterilization of Dysosma versipellis adopts amphoteric surfactant to soak, the amphoteric surfactant of beet alkalescence has good antibiotic property, can suppress the growth and breeding of part bacterium and fungi, the betain of the alkyl dimethyl simultaneously in amphoteric surfactant has good sequestering power to cupric, and by cupric, from polyphenol oxidase, chelating is out, make polyphenol oxidase lose oxidability, reduce the brown stain of Dysosma versipellis explant;
Described amphoteric surfactant is: the N-long-chain alkoxy carboxylic acid type betain of 23 weight portions, α-cetyl trimethyl the betain of 17 weight portions, N-alkyl-β-the dipropionic acid of 12 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 8 weight portions and the sterile water of 65 weight portions;
Step 2: utilize inducing culture to cultivate described aseptic explant, condition of culture is temperature 22 DEG C, pH value 5.8, light culture obtained embryo callus after 18 days, described inducing culture comprises the agar of 2, the 4-D growth hormone of MS, 1.3mg/L, the pyroligneous liquor of 1.3mg/L, the plants essential oil of 0.4mg/L and 6g/L;
Step 3: utilize the first proliferated culture medium to cultivate described embryo callus, squamous subculture 3 generation, condition of culture is temperature 22 DEG C, pH value 5.8, light culture obtained subcultured callus after 18 days, described first proliferated culture medium comprises 2, the 4-D growth hormone of MS, 1.3mg/L, the stachyose of 23mg/L and the agar of 6g/L;
Step 4: utilize differential medium to cultivate described subcultured callus, condition of culture is temperature 22 DEG C, pH value 5.8, light culture obtained body embryo after 18 days, and described differential medium comprises the 6-benzyl aminoadenine mitogen of MS, 0.5mg/L, the methyl α-naphthyl acetate of 1.3mg/L and the agar of 6g/L;
Step 5: utilize the second proliferated culture medium to cultivate described body embryo, condition of culture is temperature 22 DEG C, pH value 5.8, light culture obtains subculture body embryo after 18 days, described second proliferated culture medium comprises the 6-benzyl aminoadenine mitogen of MS, 0.5mg/L, the methyl α-naphthyl acetate of 1.3mg/L and 2, the 4-D growth hormone of 1.0mg/L; Described second proliferated culture medium is arranged in the shaking table that rotating speed is 120r/min, and described second proliferated culture medium changed a subculture every 4 days;
Step 6: utilize subculture body embryo described in plant regeneration medium culture, condition of culture is 25 DEG C, Plantlet formation is cultured under 1500Lux illumination condition, light application time is 10h every day, and described plant regeneration medium comprises the agar of the traditional Chinese medicine powder of MS, 1.3mg/L, the gibberellin 3 of 1.2mg/L, the indolebutyric acid of 0.4mg/L, the sepiolite powder of 0.5mg/L and 6g/L;
The preparation method of described traditional Chinese medicine powder is: the Dysosma versipellis by parts by weight being 35 parts, the distinguished and admirable fruit of 22 parts, the Lysimachia foenum-graecum of 26 parts, 35min is decocted after the larva of a silkworm with batrytis mixing of the Rong'an cumquat of 16 parts and 13 parts, filter to obtain Chinese medicine slag, through autoclaving post-drying, then 200 eye mesh screens were pulverized, obtain described traditional Chinese medicine powder, containing various trace elements required in Dysosma versipellis Plantlet formation process and benefit materials in traditional Chinese medicine powder, traditional Chinese medicine powder is added in plant regeneration medium, improve the resistance against diseases of Dysosma versipellis plant, the survival rate more than 90% that later stage is transplanted, add simultaneously sepiolite powder with the use of, absorb the harmful substance in medium, contribute to the Dysosma versipellis plant cultivating high-quality,
Step 7, described Dysosma versipellis plant moved in strong seedling culture base and cultivate 18 days, obtain Dysosma versipellis seedling, described strong seedling culture base comprises short root medium and short stem medium, described strong seedling culture base is positioned in incubator, described short stem medium is positioned at the top of described short root medium, from the below of Dysosma versipellis plant, be immersed in described short root medium by 1/5 of Dysosma versipellis plant, be placed in described short stem medium by 1/3 of Dysosma versipellis plant;
Described short stem medium comprises the described traditional Chinese medicine powder of MS, 1.3mg/L, the gibberellin of 1.2mg/L, the agar of the indolebutyric acid of 0.4mg/L, the sepiolite powder of 0.5mg/L and 6g/L; Add agar formation in short stem medium and solidify shape medium, traditional Chinese medicine powder and sepiolite powder is added in this medium, to Dysosma versipellis seedling supply nutrition and to medium sterilization while, the permeability of short stem medium can be improved, make the stem of the Dysosma versipellis seedling cultivated strongr;
Described short root medium comprises the pomelo peel extract of MS, 13mg/L, the indolebutyric acid of 1.0mg/L and the pyroligneous liquor of 1.3mg/L; The preparation method of the extract of described pomelo peel is: shredded by pomelo peel, be exposed to the sun 1.5 days, then put into boiling water and boil 35min, filter while hot, and in the filtrate of thermotropism, adding the boric acid of pomelo peel quality 0.1 times, described filter vacuum is evaporated to 6mg/L, and arranging vacuum is 0.05MPa, during reduced pressure concentration, control temperature is 55 DEG C, obtains described pomelo peel extract;
Step 8, cultivate in described Dysosma versipellis seedling replanting to matrix, related for described Dysosma versipellis seedling described short stem medium is transplanted in matrix together with described polylactic acid film 3, the lower surface of described polylactic acid film 3 is made to be close to described substrate upper surface, the described short stem medium of upper surface carrying of described polylactic acid film 3, related for Dysosma versipellis seedling short stem medium is transplanted in matrix together with polylactic acid film 3, certain laundering period is provided to Dysosma versipellis seedling, environmental catastrophe is avoided to stimulate the excessive and death that causes to Dysosma versipellis seedling, improve the survival rate that Dysosma versipellis is transplanted, after Dysosma versipellis accommodate substrate environment, short stem medium is smashed to pieces, short stem medium still can provide nutriment for it in the late growing stage process of Dysosma versipellis, after described Dysosma versipellis seedling cultivates 9 days in matrix, described short stem medium is smashed to pieces, adjust the spacing of described Dysosma versipellis seedling in matrix, two often adjacent described Dysosma versipellis seedling spacing are made to be 33cm, continue to be cultured to Dysosma versipellis maturation, polylactic acid film 3 is degradable film, and cover the effect that matrix top has insulation warming to matrix, polylactic acid film 3 is finally degraded to the nutriment needed for Dysosma versipellis, improves the economic use value of short stem medium and polylactic acid film 3,
Described matrix is from top to bottom by grassland ash, and loam, perlite, wood chip, pine needle is 1: 8: 2: 4: 3 formations by weight.
As shown in Figure 1, wherein, described incubator comprises upper box 1 and lower box 2, described upper box 1 removably connects with described lower box 2, described upper box 1 is the cylindrical shell of up/down perforation, it is the polylactic acid film 3 of 0.02 millimeter that the lower plane of described upper box 1 is provided with the thickness of its lower plane consistent size, described polylactic acid film 3 has Dysosma versipellis plant described in the strain of multiple through hole 4, through a described through hole 4;
The cylindrical shell that described lower box 2 opens wide for bottom closed upper portion, described lower box 2 outer cover is provided with dismountable muff 5, and described short stem medium is placed on described polylactic acid film 3, and described short root medium is placed in described lower box 2;
Upper and lower dismountable incubator project organization is simple, easy and simple to handle, for Dysosma versipellis seedling provides two kinds of different culture medias simultaneously, muff 5 can be sleeved on lower box 2 outside when temperature is lower, ensure the environment needed for Dysosma versipellis growth of seedling.
Experimental comparison
< comparative example 1>
After following three kinds of Disinfection Methods are adopted to the blade of Dysosma versipellis or the explant of root or stem, utilize cultivation method of the present invention, cultivation regulates identical, often kind of sterilization method processes the explant of 40 Dysosma versipelliss respectively, its survival rate is in table 1, wherein liquid detergent sterilization adopt to be mass fraction be 0.1% liquid detergent aqueous solution soaking 8min, scrub described explant surface smut gently with writing brush in immersion process, after taking out described explant, use running water 18min; What ethanol disinfection adopted is first with the alcohol solution dipping 10s that mass fraction is 70%, is the hypochlorite disinfectant 20min of 1.0%, uses aseptic water washing 30min afterwards with mass fraction; What amphoteric surfactant sterilization adopted is the N-long-chain alkoxy carboxylic acid type betain of 23 weight portions, α-cetyl trimethyl the betain of 16 weight portions, N-alkyl-β-the dipropionic acid of 12 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 8 weight portions and the sterile water of 65 weight portions soaks, and be placed in ultrasonic and shake 15min, use deionized water rinsing 45min, in flushing process, scrub the dirt on described explant surface with banister brush gently.
Table 1 adopts three kinds of sterilization methods on the impact of Dysosma versipellis explant sterilization on its browning rate
< comparative example 2>
Two kinds of following methods are adopted to transplant to Dysosma versipellis plant, method one, Dysosma versipellis plant is directly transplanted in matrix, method two, by Dysosma versipellis plant by transplanting method in embodiment 3, first cultivate in incubator after 18 days and obtain Dysosma versipellis seedling, then by Dysosma versipellis seedling replanting in matrix, the matrix in two kinds of methods is identical with the matrix in embodiment 3.Often kind of method transplants cultivation 50 strain Dysosma versipellis plant.
Table 2 method one and method two are on the impact of the transplanting survival rate of Dysosma versipellis plant
| Transplanting method | Transplant survival number/strain | Survival rate/% |
| Method one | 42 | 84 |
| Method two | 48 | 96 |
< comparative example 3>
Dysosma versipellis method for quickly breeding A, does not add traditional Chinese medicine powder in the plant regeneration medium of subculture body embryo; Method B, the plant regeneration medium of subculture body embryo with the plant regeneration medium in embodiment 3, two kinds of other incubation steps of method all with other incubation steps in embodiment 3, with 50 Dysosma versipellis explants in often kind of method.
Table 3 method A and way B Fast-propagation Dysosma versipellis are on the impact of Dysosma versipellis survival rate
| Propagation method | Dysosma versipellis survives number/strain | Survival rate/% |
| Method A | 45 | 80 |
| Method B | 49 | 98 |
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (7)
1. a method for quickly breeding for Dysosma versipellis, is characterized in that, comprises the following steps:
Step one: the aseptic explant making Dysosma versipellis;
Step 2: utilize inducing culture to cultivate described aseptic explant, obtain embryo callus, described inducing culture comprises the agar of MS, 2, the 4-D growth hormone of 0.5 ~ 2.0mg/L, the pyroligneous liquor of 1.0 ~ 1.5mg/L, the plants essential oil of 0.2 ~ 0.5mg/L and 6g/L;
Step 3: utilize the first proliferated culture medium to cultivate described embryo callus, squamous subculture 2 ~ 3 generation, obtain subcultured callus, described first proliferated culture medium comprises MS, 2, the 4-D growth hormone of 0.5 ~ 2.0mg/L, the stachyose of 20 ~ 25mg/L and the agar of 6g/L;
Step 4: utilize differential medium to cultivate described subcultured callus, obtain body embryo, described differential medium comprises MS, the 6-benzyl aminoadenine mitogen of 0 ~ 1.0mg/L, the methyl α-naphthyl acetate of 0.5 ~ 2.0mg/L and the agar of 6g/L;
Step 5: utilize the second proliferated culture medium to cultivate described body embryo, obtain subculture body embryo, described second proliferated culture medium comprises MS, the 6-benzyl aminoadenine mitogen of 0 ~ 1.0mg/L, the methyl α-naphthyl acetate of 0.5 ~ 2.0mg/L and 2, the 4-D growth hormone of 0 ~ 2.0mg/L;
Step 6: utilize subculture body embryo described in plant regeneration medium culture, obtain Dysosma versipellis plant, described plant regeneration medium comprises MS, the traditional Chinese medicine powder of 1.0 ~ 1.5mg/L, the indolebutyric acid of the gibberellin 3,0.2 ~ 0.6mg/L of 1.0 ~ 1.4mg/L, the sepiolite powder of 0.3 ~ 0.7mg/L and the agar of 6g/L;
The preparation method of described traditional Chinese medicine powder is: be the Dysosma versipellis of 30 ~ 40 parts by parts by weight, the distinguished and admirable fruit of 18 ~ 25 parts, the Lysimachia foenum-graecum of 23 ~ 28 parts, the Rong'an cumquat of 15 ~ 17 parts and 10 ~ 15 parts larva of a silkworm with batrytis mixing after decoct 30 ~ 40min, filter to obtain Chinese medicine slag, through autoclaving post-drying, then pulverize 200 eye mesh screens, obtain described traditional Chinese medicine powder.
2. the method for quickly breeding of Dysosma versipellis as claimed in claim 1, is characterized in that, also comprise:
Step 7, described Dysosma versipellis plant moved in strong seedling culture base and cultivate 15 ~ 20 days, obtain Dysosma versipellis seedling, described strong seedling culture base comprises short root medium and short stem medium, described strong seedling culture base is positioned in incubator, described short stem medium is positioned at the top of described short root medium, from the below of Dysosma versipellis plant, be immersed in described short root medium by 1/5 of Dysosma versipellis plant, be placed in described short stem medium by 1/3 of Dysosma versipellis plant;
Described short stem medium comprises MS, the described traditional Chinese medicine powder of 1.0 ~ 1.5mg/L, the indolebutyric acid of the gibberellin 3,0.2 ~ 0.6mg/L of 1.0 ~ 1.4mg/L, the sepiolite powder of 0.3 ~ 0.7mg/L and the agar of 6g/L;
Described short root medium comprises MS, the pomelo peel extract of 10 ~ 15mg/L, the indolebutyric acid of 0.8 ~ 1.2mg/L and the pyroligneous liquor of 1.0 ~ 1.5mg/L; The preparation method of the extract of described pomelo peel is: shredded by pomelo peel, be exposed to the sun 1 ~ 2 day, then put into boiling water and boil 30 ~ 40min, filter while hot, and in the filtrate of thermotropism, adding the boric acid of pomelo peel quality 0.1 times, described filter vacuum is evaporated to 6mg/L, and arranging vacuum is 0.05 ~ 0.06MPa, during reduced pressure concentration, control temperature is 55 DEG C, obtains described pomelo peel extract;
Step 8, to cultivate in described Dysosma versipellis seedling replanting to matrix.
3. the method for quickly breeding of Dysosma versipellis as claimed in claim 2, it is characterized in that, described incubator comprises upper box and lower box, described upper box and described lower box removably connect, described upper box is the cylindrical shell of up/down perforation, it is the polylactic acid film of 0.01 ~ 0.03 millimeter that the lower plane of described upper box is provided with the thickness of its lower plane consistent size, and described polylactic acid film has multiple through hole, and Dysosma versipellis plant described in a strain is through a described through hole;
Described lower box is the cylindrical shell that bottom closed upper portion is opened wide, and described lower box outer cover is provided with dismountable muff, and described short stem medium is placed on described polylactic acid film, and described short root medium is placed in described lower box.
4. the method for quickly breeding of Dysosma versipellis as claimed in claim 3, it is characterized in that, described aseptic explant, described embryo callus, described subcultured callus and the condition of culture of described body embryo in respective medium are: temperature 20 DEG C ~ 24 DEG C, pH value 5.6 ~ 6.0, light culture 15 ~ 20 days, the condition of culture of described subculture body embryo in plant regeneration medium is 24 ~ 26 DEG C, is cultured to Plantlet formation under 1000 ~ 2000Lux illumination condition, and wherein light application time is 10h every day.
5. the method for quickly breeding of Dysosma versipellis as claimed in claim 4, it is characterized in that, in described step 8, when cultivating in described Dysosma versipellis seedling replanting to matrix, related for described Dysosma versipellis seedling described short stem medium is transplanted in matrix together with described polylactic acid film, the lower surface of described polylactic acid film is made to be close to described substrate upper surface, the described short stem medium of upper surface carrying of described polylactic acid film, after described Dysosma versipellis seedling cultivates 8 ~ 10 days in matrix, described short stem medium is smashed to pieces, adjust the spacing of described Dysosma versipellis seedling in matrix, two often adjacent described Dysosma versipellis seedling spacing are made to be 30 ~ 35cm, continue to be cultured to Dysosma versipellis maturation,
Described matrix is from top to bottom by grassland ash, and loam, perlite, wood chip, pine needle is 1: 8: 2: 4: 3 formations by weight.
6. the method for quickly breeding of Dysosma versipellis as claimed in claim 5, is characterized in that, before described aseptic explant accesses described inducing culture, also comprise the step obtaining aseptic explant, the step of described acquisition aseptic explant is:
Choose the blade of Dysosma versipellis or root or stem as explant, described explant is placed in beaker, soak with amphoteric surfactant, and be placed in ultrasonic concussion 10 ~ 20min, with deionized water rinsing 40 ~ 50min, in flushing process, scrub the dirt on described explant surface with banister brush gently, then blot the moisture of described explant outside with aseptic filter paper after, be cut into 5mm
2fritter, obtain described aseptic explant;
Described amphoteric surfactant is: the N-long-chain alkoxy carboxylic acid type betain of 20 ~ 25 weight portions, α-cetyl trimethyl the betain of 15 ~ 18 weight portions, N-alkyl-β-the dipropionic acid of 10 ~ 13 weight portions, the mixed solution of the dodecyldimethylammonium hydroxide inner salt of 5 ~ 10 weight portions and the sterile water of 60 ~ 70 weight portions.
7. the method for quickly breeding of Dysosma versipellis as claimed in claim 6, it is characterized in that, described body embryo is in described second proliferated culture medium in incubation, described second proliferated culture medium is arranged in the shaking table that rotating speed is 120r/min, and described second proliferated culture medium changed a subculture every 3 ~ 5 days.
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| CN117305217A (en) * | 2023-10-24 | 2023-12-29 | 临沂市农业科学院 | Optimized stem tip tissue culture medium, culture method and application |
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