CN103820428B - Mental retardation N+inject method and the bacterial strain of institute's selection-breeding of mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain - Google Patents
Mental retardation N+inject method and the bacterial strain of institute's selection-breeding of mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain Download PDFInfo
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Abstract
The invention discloses a kind of mental retardation N+Injecting method and the bacterial strain of institute's selection-breeding of mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain, bacterial strain deposit number is CGMCC No.8651；Selection includes that screening is wanted the Coriolous Dersicolor (Fr.) Quel starting strain of mutation, the acquisition of fusant, selected the steps such as the protoplast material as Low energy N+ ions mutation of fusant bacterial strain, N~+ implantation mutation, the screening of mutagenic strain, stabilization characteristics of genetics checking.The present invention screens the manyzoned polypore sporophore disease-resistant high yield bacterial strain of a strain stabilization characteristics of genetics, and its Coriolous Dersicolor (Fr.) Quel yield is higher than high yield starting strain 10.3%, and disease resistance is apparently higher than disease-resistant starting strain 5%.
Description
Technical field
The present invention relates to biological engineering and edible and medical fungi technical field, be specifically related to use mental retardation N+Ion implantation mutagenesis selects
The method educating manyzoned polypore sporophore disease-resistant high yield bacterial strain, and the mutation Coriolous Dersicolor (Fr.) Quel fungus of the strain disease-resistant high yield obtained by said method
Strain.
Background technology
Coriolous Dersicolor (Fr.) Quel belongs to Basidiomycetes Polyporaceae Coriolous Dersicolor (Fr.) Quel and belongs to fungus, Classification systemCoriolus versicolor(L)F, Coriolous Dersicolor (Fr.) Quel
Being one of the fungus of great medical value, research finds that Coriolous Dersicolor (Fr.) Quel contains polysaccharide, glycopeptide class, steroid, triterpenes, organic acid
Class, alkaloids, glucose alcohols, 29 compounds such as protein-based, press down possibly together with the outer emplastic (ECMM) of born of the same parents, pepsin
Preparation, Fungal elicitor, ferrum bonding chelating agen, cyclic peptide, the aminoacid of 18 kinds of needed by human body and vitamin B1, B2, B6 and copper,
The active trace element of more than the 10 kind of needed by human body such as ferrum, potassium, zinc.Krestin, triterpene in Coriolous Dersicolor (Fr.) Quel become with aminoacid isoreactivity
Point, there is antitumor, immunomodulating, anti-radiation, anti-chemotherapy, calmness, heart tonifying, resist myocardial ischemia, regulate blood fat, blood sugar lowering, flat
Breathe heavily, protect the liver, anti-hypoxia and the anti-ageing effect of waiting for a long time.Clinical research then proves that Coriolous Dersicolor (Fr.) Quel preparation can be used for preventing and treating chronic bronchitis, heavy breathing
Breathe heavily, coronary heart disease, hyperlipidemia, neurasthenia, insomnia, hepatitis, tumor and the auxiliary treatment etc. of chemicotherapy.Prepared from coriolus versicolor mycelium, fermentation
The polysaccharide extracted in liquid is respectively provided with and strong presses down carcinous, through pharmacological research test, Chinese scholars proves that krestin is to little Bai
The suppression ratio of Mus sarcoma 180 and ehrlich carcinoma is respectively 80% and 100%.The kind of Coriolous Dersicolor (Fr.) Quel is chatted the most denumerable the most both at home and abroad, the most scarce
The Coriolous Dersicolor (Fr.) Quel strain that weary disease-resistant high yield, biological character are excellent, its bottleneck having become the production of developmental limitation Coriolous Dersicolor (Fr.) Quel and Coriolous Dersicolor (Fr.) Quel industry is asked
Topic.The most yield of Coriolous Dersicolor (Fr.) Quel strain generally separated from field is the highest, needs through domestication and continuous selection-breeding, to improve cloud
The yield of sesame and the production traits, therefore, the research in terms of the artificially breeding of strain is just particularly important.The effect of spontaneons screening
Rate is relatively low, and takes the method effect of artificially breeding preferably, puts into practice through years of researches, with fruiting body yield and economical character
For main target strain improvement oneself from initial selection and use, cross-breeding develops into induced mutations breeding, protoplast melts
Close breeding and use Protocols in Molecular Biology to carry out breeding etc., the Breeding of Edible Mushroom method being weeded out the old and bring forth the new, updates,
Breeding efficiency improves constantly.At present, applying more in edible fungus species selection-breeding is ultraviolet mutagenesis selection-breeding, and it has without special
Equipment, to human body non-hazardous effect, be prone to features such as preventing, Mutagenic Effect is the prettyst good, be applied to Pleurotus ostreatus, Agaricus blazei Murrill, Lentinus Edodes
Deng edible fungus variety, and succeed.What the strain improvement research in terms of Coriolous Dersicolor (Fr.) Quel was carried out at present is few, and the present invention uses Low Energy Nitrogen
Ion implantation mutagenesis Coriolous Dersicolor (Fr.) Quel disease-resistant high yield bacterial strain is different from other method for mutation breeding, improves the edible fungus species selection-breeding such as Coriolous Dersicolor (Fr.) Quel
Efficiency.
Ion implanting is a kind of newer method of mutagenesis, injects ions into organism, makes atom be shifted over and resets, making
Element in atom, the ion of injection and the organism of displacement is combined again, forms new molecular structure and gene.External from 20
The intermediary and later stages sixties in century are applied to ion implantation technique the research of field of biology in succession, but progress is the slowest.
Hefei Inst. of Plasma Physics, Chinese Academy of Sciences Yu Zengliang researcher took the lead in a Low Energy Heavy Ion Beam injection technique in 1986 should
For Rice Mutation Breeding, show the statistical result that physiological damage is little, mutational spectrum is wide, mutation frequency is high, and have certain weight
Renaturation and the mutagenic effect of directivity, open the new research direction that low energy ion and organism interact, establish matter
Amount, energy, the machine-processed system of electric charge three factor effect ..., found " ion biotechnology " this cross discipline, and quickly
This new mechanism is applied to mutagenic breeding, cell processing and gene transfer, opens the new way of genetic improvement, thus
Cause extensive concern both domestic and external.Through the development of more than 20 years, the range of application of ion beam was gradually widened, in life sciences
Every field the most progressively shows wide application prospect.So far Mutation technique induced by ion beam, ion beam DNA macromole transfer skill
Art, Assisted by Ion Beam Agrobacterium genetic transfoumation, ion-beam mediated super remote edge molecular hybridization and the processing of ion beam cell
Technology etc. have become the orientation new method of genetic improvement, the new way with China's independent intellectual property right.Ion beam have quality,
The feature of energy duplex mutagenesis effect, x radiation x different from the past, the biological effect that ion implanting causes, existing energy sinks
Long-pending, momentum transmission, has again element quality deposition, first has energy deposition, i.e. injects ion and occurs a series of with biomacromolecule
Collision, when biomacromolecule obtains energy, biological gene bond fission, DNA molecular hits and was originally, leaves scission of link or defect, injects
The omission that unit have a homogenic scission of link of certain probability or DNA is got combines, and with the biochemical reaction of various elements, produces base
Because of sudden change and chromosome disorder；Inject ion and the charge-exchange of target cell, may result in cell surface and be etched, cause cell membrane
Property and the improvement of transmembrane potential thoroughly；Ion beam is squeezed into organism and is produced Braag peak, has stronger ionization, moreover it is possible to produces and lives
The free radical indirect injury effect that property is higher, therefore it may result in higher mutation rate to the effect of organism, additionally, due to note
Enter the different charge numbers of ion, mass number, energy, dosage combination, it is provided that numerous mutagenic conditions, by this electricity, energy, matter
Synergy, affects the physio-biochemical characteristics of biological cell strongly, to cause gene mutation, so variation amplitude is big, has higher
Mutation rate, wider mutational spectrum, the heritability of mutant is more stable, and reverse mutation rate is low.
Have not yet to see relevant employing mental retardation N+Any report of ion implantation technique mutation Coriolous Dersicolor (Fr.) Quel disease-resistant high yield bacterial strain.
Summary of the invention:
It is an object of the invention to provide a kind of mental retardation N selecting superior strain+Inject mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain
Method and the bacterial strain of institute's selection-breeding.
The technical solution of the present invention is:
A kind of mental retardation N+The method injecting mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain, is characterized in that: comprise the following steps: successively
(1) the Coriolous Dersicolor (Fr.) Quel starting strain of mutation is wanted in screening: selects multiple different Coriolous Dersicolor (Fr.) Quel to produce bacterial strain, carries out the cultivation of Coriolous Dersicolor (Fr.) Quel
Training, selects the highest Coriolous Dersicolor (Fr.) Quel bacterial strain of manyzoned polypore sporophore yield " high yield starting strain " as mutation；Select multiple different cloud
Sesame bacterial strain, carries out the screening of disease-resistant starting strain, filters out a strain " disease-resistant starting strain "；
(2) acquisition of fusant: utilize protoplast fusion method, by " high yield starting strain " and " disease-resistant starting strain "
Carry out the fusion of protoplast, obtain a strain fusant bacterial strain；
(3) protoplast material as Low energy N+ ions mutation of fusant bacterial strain is selected: prepare Coriolous Dersicolor (Fr.) Quel fusant
Protoplast, and make cloud protoplast suspension concentration 105Individual/ml level, is coated in culture dish, carry out Low Energy Nitrogen from
Son injects mutation；
(4) N~+ implantation mutation: step (3) is coated the material in culture dish and carries out N~+ implantation mutation, so
Afterwards with 0.6mol/L sucrose homeo-osmosis agent eluting, coat and cultivate in Coriolous Dersicolor (Fr.) Quel protoplast regeneration culture medium；
(5) screening of mutagenic strain: after bacterium colony grows up to, select mycelia sturdy dense, grow mycelia faster and carry out liquid
Body fermentation test, then carries out the mensuration of hypha biomass, selects Biomass to be stored in higher than the bacterial strain of starting strain more than 5%
As the mutagenic strain of primary dcreening operation in test tube；Then the bacterial strain after primary dcreening operation is carried out Coriolous Dersicolor (Fr.) Quel cultivation with the superior strain that sets out simultaneously
Test, selects the sporophore biological transformation ratio bacterial strain higher than control strain more than 5% as the superior strain of mutation, is simultaneous for
The new strains that above-mentioned screening obtains, then carry out disease-resistant variety screening, selection disease resistance again can be apparently higher than disease-resistant starting strain 5%
Coriolous Dersicolor (Fr.) Quel mutagenic strain as the new strains obtained after multiple sieve；
(6) stabilization characteristics of genetics checking: new strains continuous passage 20 generation that will obtain after above-mentioned mutagenesis screening, so
After carry out experiment in cultivation, select that fruiting body yield is stable, that the unconverted strain of character is ultimately determined on producing is new
Coriolous Dersicolor (Fr.) Quel disease-resistant high yield new strains.
Described N~+ implantation condition be Implantation Energy be 20KeV, implantation dosage is 9 × 1014ions/cm2., target chamber
Vacuum is 10-3Pa, injects with 5s pulsed, is spaced apart l5s.
Regeneration culture medium is: maltose 5 grams, glucose 10 grams, yeast powder 4 grams, and 20 grams of agar, 0.6mol/L mannitol is molten
Liquid l000mL.
Liquid fermentation medium is: Rhizoma Solani tuber osi 200g liquor, glucose 20g, KH2P04 10g, MgS ()45g, H20
1000m1。
A kind of mental retardation N+Inject the bacterial strain of the method institute selection-breeding of mutagenic and breeding Coriolous Dersicolor (Fr.) Quel bacterial strain, it is characterized in that: deposit number is
CGMCC No.8651；Classification And Nomenclature: Coriolous Dersicolor (Fr.) Quel, Latin name:Coriolus versicolor.Depositary institution: Chinese micro-life
Thing culture presevation administration committee's common micro-organisms center, address: BeiChen West Road, Chaoyang District, BeiJing City l institute 3, preservation date
On December 19th, 2013.
Advantages of the present invention and good effect be:
The breeding of the edible fungus species such as l, conventional Coriolous Dersicolor (Fr.) Quel generally uses the means such as cross-breeding and UV mutation to carry out,
Have that efficiency of inducing mutation is low, be easily caused negative sudden change and the shortcoming such as hereditary character is unstable, and by using ion implantation technique mutation cloud
The edible fungi protoplasts such as sesame carry out the mutagenic and breeding of bacterial strain, improve the efficiency of mutation and the probability of direct mutation, and mutation
After Coriolous Dersicolor (Fr.) Quel bacterial strain be not susceptible to degenerate.Use mutation of the present invention to go out a strain fruiting body yield to lure higher than the Coriolous Dersicolor (Fr.) Quel of former starting strain
Become bacterial strain, improve yield and the disease resistance of Coriolous Dersicolor (Fr.) Quel, good economic benefit can be created.
2, the present invention utilizes N~+ implantation technology that Coriolous Dersicolor (Fr.) Quel fusant bacterial strain protoplast is carried out mutation, and through various dose
N~+ implantation after, at dose,optimum 9 × 1014ions/cm2.Under, screen the manyzoned polypore sporophore of a strain stabilization characteristics of genetics
Disease-resistant high yield bacterial strain, its Coriolous Dersicolor (Fr.) Quel yield is higher than high yield starting strain 10.3%, and disease resistance is apparently higher than disease-resistant starting strain 5%.
3, result shows, ion implantation technique can be applied in the mutagenic and breeding of Coriolous Dersicolor (Fr.) Quel disease-resistant high yield bacterial strain, and has
Having higher mutation rate and wider mutational spectrum, Mutagenic Effect is good, is a kind of more satisfactory edible fungus species selection.
Below in conjunction with embodiment, the invention will be further described.
The method of Low energy N+ ions mutagenic and breeding disease-resistant high yield Coriolous Dersicolor (Fr.) Quel bacterial strain and the bacterial strain of institute's selection-breeding, deposit number is
CGMCC No.8651；Classification And Nomenclature: Coriolous Dersicolor (Fr.) Quel, Latin name:Coriolus versicolor, depositary institution: Chinese micro-life
Thing culture presevation administration committee's common micro-organisms center, address: BeiChen West Road, Chaoyang District, BeiJing City l institute 3, preservation date
20l3 December 19 days.
Detailed description of the invention:
A kind of Low Energy Nitrogen injects the method for mutagenic and breeding disease-resistant high yield Coriolous Dersicolor (Fr.) Quel bacterial strain, the steps include:
L, the screening of starting strain
1.1 high yields set out bacterial strain screening
From Coriolous Dersicolor (Fr.) Quel strain SYZ1, SYZ2, SYZ3, SYZ4, SYZ5, SYZ6, SYZ7, SYZ8 of domestic introduction different cultivars.
Bacterial strain, respectively from the Chinese Academy of Agricultural Sciences, northeast and the bacterial strain of this institute preservation, is inoculated on PDA slant medium, keeps away for 28 DEG C
Light is cultivated and is activated for 7 days.By the inoculation of activation in the central authorities equipped with PDA culture medium culturing ware, Coriolous Dersicolor (Fr.) Quel mycelia sprouts record
The time sent out, measure its speed of growth, observe the form (mycelia thickness, dense) of mycelia.Then Coriolous Dersicolor (Fr.) Quel liquid fermentation examination is carried out
Testing, slant strains is seeded in fluid medium (culture medium prescription sees below), and 28 DEG C of lucifuges of 140 revs/min of rotary shaker cultivate 7
My god, then to transfer in fermentation medium, 28 DEG C of lucifuges of 140 revs/min of rotary shaker are cultivated 3 days, and 4000 revs/min are centrifuged 5 points
Clock, collects mycelium, dries to constant weight, measure its quality and obtain Coriolous Dersicolor (Fr.) Quel liquid fermentation Biomass for 65 DEG C.Carry out the cultivation of Coriolous Dersicolor (Fr.) Quel simultaneously
Training, its concrete process is: the slant strains of Coriolous Dersicolor (Fr.) Quel be inoculated in Coriolous Dersicolor (Fr.) Quel fluid medium (liquid culture based formulas sees below),
L40 rev/min of rotary shaker, 28 DEG C of lucifuges are cultivated 7 days, are inoculated in the Coriolous Dersicolor (Fr.) Quel solid medium after sterilizing with the inoculum concentration of 2%,
26 DEG C of lucifuges are cultivated about about 40 days, after mycelia covers with bacterium bag, remove to mushroom room and carry out management of producing mushroom, relative air humidity
85%～95%, temperature 26 DEG C～28 DEG C, scattered light processes, frequent ventilation, and mycelia forms former base, is differentiated to form son real
Body, gathers when waiting manyzoned polypore sporophore full maturity, the yield of statistics Coriolous Dersicolor (Fr.) Quel, calculates the biological transformation ratio of Coriolous Dersicolor (Fr.) Quel, real with Coriolous Dersicolor (Fr.) Quel
Body yield is the highest, and biological transformation ratio soprano is as Coriolous Dersicolor (Fr.) Quel high yield starting strain.Found by statistics simultaneously, manyzoned polypore sporophore
The size of form on PDA plating medium of yield and Coriolous Dersicolor (Fr.) Quel mycelia and liquid fermentation Biomass has positive correlation.At PDA
In plane, mycelia is sturdy dense and fast growth, its manyzoned polypore sporophore yield of the biggest person of liquid fermentation Biomass are high.Test
The rear strain Coriolous Dersicolor (Fr.) Quel bacterial strain determined using SYZ1 from the Chinese Academy of Agricultural Sciences is as the high yield starting strain of mutation.
1.2, the screening of disease-resistant starting strain
1.2.1 Coriolous Dersicolor (Fr.) Quel represents pathogen and screens, and determines two kinds of the main pathogenic fungi (bacillus cereus, penicillium).
1.2.2 resistance against diseases mensuration is carried out by 8 kinds of different Coriolous Dersicolor (Fr.) Quel bacterial strains of introduction.
8 kinds of different Coriolous Dersicolor (Fr.) Quel bacterial strains are at the bacillus cereus (10 determined9 Cfu/mL), penicillium (107Cfu/mL) fermentation
Product filtrate (being concentrated into 25%, the bacteria-free filtrate after being filtered by bacteria filters) and the flat board coating weight (0.5ml/ determined
Flat board) under conditions of, utilizing punching inoculation, cultivated by PDA plate, the size utilizing bacterium circle to grow (is not coated with compareing
The treadmill test of tunning filtrate) bacterial strain bacterium circle growth size compare, draw (resistance level=the test of average resistance
Bacterium loop diameter comparison bacterium loop diameter × 100).The speed of growth of 8 kinds of different Coriolous Dersicolor (Fr.) Quel is measured simultaneously.Life according to different Coriolous Dersicolor (Fr.) Quel
Long speed, the average resistance to antibacterial, the average resistance of penicillium, set up score-system, to determine the disease-resistant energy of different strains
Power, filters out optimal disease-resistant Coriolous Dersicolor (Fr.) Quel bacterial strain.Finally determine that the northeast wild Coriolous Dersicolor (Fr.) Quel bacterial strain of SYZ8 is as disease-resistant starting strain.
1.3 utilize Protoplast Fusion Technique, and high yield starting strain and disease-resistant starting strain are carried out protoplast fusion,
Finally give fusant bacterial strain YRH1.
It is 5 days in mycelia cell age, prepares high yield under the lywallzyme of 2% 30 DEG C of enzymolysis Parameter Conditions of 5 hours respectively and set out bacterium
Strain and the protoplast of disease-resistant starting strain, and be 10 with homeo-osmosis dilution agent to concentration5Individual/ml.By examination before merging
Test investigation strain mycelial growth rate, mode of appearance and mycelia colouring matter secretion situation, protoplast regeneration situation etc., select bacterial strain
SYZ1 inactivates 2 hours at 55 DEG C, takes each 1 milli of the SYZ1 protoplast suspension after SYZ8 protoplast suspension and inactivation the most respectively
Rising, vortex mixes, and is added dropwise over PEG6000, merges at 30 DEG C.Then sterilized water washing is centrifugal is diluted to 1ml, coats
Cultivate in protoplast regeneration culture medium, pick out and all have with starting strain the bacterial strain of antagonism to obtain fusant YRH1.
2, Low energy N+ ions mutation
(1) sample pre-treatments: fusant bacterial strain YRH1 cultivates the mycelia of 5 days, with 2% lywallzyme enzymolysis 5 hours at 30 DEG C,
Filter enzymolysis solution with 8 layers of lens paper, with the homeo-osmosis agent washing and filtering by several times of 8ml altogether, take filtrate.4000r/min is centrifuged
10 minutes, remove supernatant, with homeo-osmosis agent 8ml centrifuge washing, obtain the protoplast of purification.By protoplast osmotic pressure
Stabilizer 10ml dilutes use, makes protoplast suspension, counts with blood counting chamber.Refined protoplast suspension is diluted to
105Individual/ml.Taking 0.1ml and coat in culture dish, sterile wind air-dries, and carries out Low energy N+ ions immediately.
(2) ion implanting conditions: Implantation Energy is 20KeV, implantation dosage is 0,300,600,900,1200, l500,
l800×1012ions/cm2., target chamber vacuum is l0-3Pa, injects with 5s pulsed, is spaced apart 15s.Calculate it at each dosage
Under Survival curves.Its result shows, along with the increase of implantation dosage, survival volume presents first reduction, rear rising and reduces
" saddle-shape " variation tendency.The range being likely due to low dose ion injection cell is shorter, and cell surface is only carried out by ion
Damage and etching, thus survival rate is higher；Along with the increase of dosage, the etching of cell surface is serious, and ion undermines cell interior
And produce substantial amounts of free radical and soft rays etc., cause survival rate drastically to decline；But when dropping to certain value, saturated phenomenon occurs,
When dose accumulation to certain value, certain repair mechanism of cell is activated, and survival rate has been gone up；When dosage continues to increase,
Cell injury will be unable to repair.
(3) post processing of sample: by through the culture dish of N~+ implantation and the culture dish 1m1 0.6mol/L of comparison
Sucrose homeo-osmosis agent eluting, takes 0.1m1 and coats on regeneration culture medium flat board, and 28 DEG C of lucifuges count after cultivating 96h, by terms of
Calculate survival volume.Survival volume is through N+The surviving colonies number that ion implanting processes.Then with implantation dosage as abscissa, survival volume is
Vertical coordinate, draws the relation curve of survival volume and implantation dosage.
3, the screening of high-yield disease resisting bacterial strain
(1) primary dcreening operation: by the flat board 0.6mol/L sucrose homeo-osmosis agent eluting after nitrogen ion beam injects, take
0.1ml coats on regeneration culture medium flat board, after bacterium colony grows up to, select mycelia sturdy dense, grow mycelia faster and carry out
Liquid fermentation is tested, and then carries out the mensuration of hypha biomass, and the bacterial strain selecting Biomass to be higher than starting strain more than 5% is protected
It is stored in test tube the mutagenic species as primary dcreening operation
(2) multiple sieve: then the bacterial strain after primary dcreening operation is carried out Coriolous Dersicolor (Fr.) Quel experiment in cultivation with the superior strain that sets out simultaneously, select
Biological transformation ratio as the superior strain of mutation, is simultaneous for the bacterial strain of above-mentioned screening higher than the bacterial strain of control strain more than 5%,
Using the lab screening disease-resistant variety new method set up to carry out Resistance screening, selection disease resistance again can be apparently higher than the disease-resistant bacterium that sets out
The Coriolous Dersicolor (Fr.) Quel mutagenic strain of strain 5%, finally gives the Coriolous Dersicolor (Fr.) Quel new strains of a strain disease-resistant high yield.
4, the N~+ implantation impact on bacterial strain mutation rate
Using starting strain as comparison, the bacterium colony that random picking is formed after accepting ion implantation mutagenesis carries out liquid respectively and sends out
Ferment is tested, 20 bacterial strains of each dosage picking, investigates the Biomass of bacterial strain.Higher than comparison more than 5% for direct mutation, less than right
According to more than 5% for negative sudden change, it is positive mutation rate that the clump count of direct mutation accounts for the ratio of taken total plate count, the bacterium colony of negative sudden change
It is negative mutation rate that number accounts for the ratio of taken total plate count, does not has definite proportionate relationship, but note between mutation rate and implantation dosage
Enter dosage 6 × 1014 ions·cm-2Time, bacterial strain mutation rate is the highest.The positive mutation rate of Coriolous Dersicolor (Fr.) Quel bacterial strain is along with N+Ion implanting agent
Amount increase and present the trend of rising, dosage reaches 9.00 × 1014 ions·cm-2When positive mutation rate reach maximum
18.75%, the most on a declining curve, therefore select this dosage and the results are shown in Table 1 as the optimal dose of mutation.
Table l N+The ion implantation dosage impact on Coriolous Dersicolor (Fr.) Quel fungus plant mutant rate
5, hereditary stability checking
The bacterial strain obtained after mutagenesis screening is carried out 20 times pass on, carry out Coriolous Dersicolor (Fr.) Quel experiment in cultivation the most again, with first generation ratio
Relatively, select fruiting body yield stable (there is not significant difference, statistical analysis, variance p between the yield of the most each generation bacterial strain >
0.05), character is unconverted as final Coriolous Dersicolor (Fr.) Quel mutagenicity high-yield bacterial strain.Finishing screen chooses a strain disease-resistant high yield Coriolous Dersicolor (Fr.) Quel mutagenic bacteria
Strain, higher by 10.3% than the former high yield producing strain that sets out, disease resistance, apparently higher than disease-resistant starting strain 5%, stabilization characteristics of genetics, is protected
Hide numbered CGMCC No.8651.
Culture medium prescription:
Protoplast regeneration culture medium is: maltose 5 grams, glucose 10 grams, yeast powder 4 grams, 20 grams of agar, 0.6mol/L
Mannitol solution l000mL, PH are natural,.
PDA culture medium: Rhizoma Solani tuber osi 200g liquor, glucose 20g, KH2P04 10g, MgS ()45g, 20 grams of agar, H20
1000m1。
Fluid medium: Rhizoma Solani tuber osi 200g liquor, glucose 20g, KH2P04 10g, MgS ()45g, H20 1000m1。
Fermentation medium: glucose 20g, Semen Maydis powder 20g, soybean cake powder 20g, KH2P0410g, MgS045g, VBl100mg,
H20 1000ml。
Solid state cultivation culture medium: cotton seed hulls 83%, wheat bran 15%, sucrose l%, Gypsum Fibrosum powder l%, moisture are 60%.
Claims (1)
1. a Coriolous Dersicolor (Fr.) Quel (Coriolus versicolor) bacterial strain, it is characterized in that: deposit number is CGMCC No.8651.
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