CN104170653A - Method for cultivating sclerotia of hybrid fungi of morchella esculenta and application of method to manufacturing strains - Google Patents
Method for cultivating sclerotia of hybrid fungi of morchella esculenta and application of method to manufacturing strains Download PDFInfo
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- 241000576755 Sclerotia Species 0.000 title abstract 5
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for cultivating sclerotia of hybrid fungi of morchella esculenta and application of the method to manufacturing strains. The method includes performing cross inoculation on hypha blocks from different strain sources at four corners of each plate cultivation medium by the aid of a four-corner process; transiting fused hyphae to alkalescent cultivation media when hyphae are subjected to hybridization to obtain the fused hyphae; cultivating the fused hyphae under low-temperature and dark conditions to obtain large quantities of the sclerotia of the morchella esculenta. The sclerotia can be used for being stored or manufacturing mother strains or cultivated species of the morchella esculenta. The method and the application have the advantages that efficient heterocaryosis hypha production can be guaranteed by a four-corner inoculation process for performing hybridization fusion on each four corresponding hypha blocks, and high and stable yields of the sclerotia of the morchella esculenta can be guaranteed by the aid of the hyphae and the low-temperature and alkalescent cultivation conditions.
Description
Technical field
The present invention relates to a kind of hickory chick bacterial classification and make and sclerotium culture technique, particularly a kind of mycelia of optimization hybridization mixing operation technology, the high-efficient culture method of sclerotium and the application of preparing at bacterial classification thereof.
Background technology
Hickory chick Morchella esculenta (L.) Pers is under the jurisdiction of Morchellaceae (Morchellaceae) morchella (Morchella), it is internationally famous Precious Edible Fungi, being in great demand in the world, is that Chinese main exit is to the edible fungus of the European countries such as France.And along with the countries such as moral, method, meaning, U.S. increase the demand of hickory chick, its price is constantly soaring.
Hickory chick is recorded in < < Compendium of Materia Medica > > the earliest, and that in China, utilizes is with a long history.Modern science is found to be rich in protein, fatty acid and carbohydrate in hickory chick, particularly multiple rare amino acid is (as cis-3-amino-L-PROLINE, 2-aminoisobutyric acid, 2,4-triamido isobutyric acids etc.), the composition such as mineral element (potassium, phosphorus, magnesium, calcium, iron, zinc, copper, manganese etc.) and multivitamin (thiamine, vitamin b3, niconacid, pantothenic acid, folic acid, pyridoxine, vitamin h, ascorbic acid, VB12) given local flavor and the nutritive value of hickory chick uniqueness, becomes the high-grade food materials in international food and drink.And, hickory chick food and medicine consangunity, modern medicine study shows, hickory chick can kidney tonifying, mend brain, establishing-Yang, refresh oneself, reducing blood lipid, the effect such as antitumor.In addition, hickory chick can be used for the raw material of steeping in wine, and its liquid fermentation and culture thing can be used for again the production of flavouring etc., therefore it is multi-field having broad application prospects such as food, health products, medication chemistries.
At present, the hickory chick of Chinese exports is adopted mostly in naturally wild, and it can not meet domestic and demand international market far away, and the excessive collection of wild resource causes its output sharply to reduce, and has greatly destroyed the bio-diversity of hickory chick.Therefore, the artificial or semi-artificial culture technique of hickory chick is the focus of international edible mushroom research always, and experiment in cultivation is even to be had successfully, but its poor repeatability, fruiting is unstable, can not carry out so far ripe commercialization cultivation.
Scientific research for many years shows, in the history of life of hickory chick, the formation of sclerotium is the only stage which must be passed by of growth fruit body (being fruiting), and the sclerotium that uses hybridization cultural hypha to go out carries out large Tanaka's cultivation experiments and the most easily grows fruit body.Practice is found in producing, during artificial culture hickory chick bacterial classification, lack unified and standard and scientific and efficient bacterial classification cross method, and the inadequacy of condition of culture often causes bacterial classification not form sclerotium or sclerotium disappearance in bacterial classification manufacturing process, cause the hickory chick underproduction even to have no harvest, this is that current hickory chick (partly) artificial cultivation is tested unsettled two major reasons.
Sclerotium fecund, stable yields cultural method and hybridization bacterial classification manufacturing technology that a kind of optimization, the bacterium of hickory chick hybridization are efficiently provided are the problems that the present invention tries hard to solution.
Summary of the invention
One object of the present invention be exactly in, experiment in cultivation not high for the hybridization efficiency in the hickory chick production of hybrid seeds process sclerotium form unsettled deficiency, the sclerotium cultural method of a kind of hickory chick hybridization bacterium is provided, the method is easy and simple to handle, intensive, quick, mycelia hybridization efficiency is high, has ensured the acquisition of hickory chick fusion mycelia; Further, according to the habit of growing of hickory chick, design low temperature, subalkaline culture environment, can turn out in a large number sclerotium of morchella esculenta, ensured to a certain extent the successful fruiting of hickory chick field production.
Technical purpose of the present invention realizes by following scheme:
A sclerotium cultural method for hickory chick hybridization bacterium, comprises the following steps:
The mycelia piece cross inoculation of different hickory chick bacterium sources, in the periphery of plating medium, when mycelia hybridization forms fusion mycelia, will be merged to mycelia and transferred in alkalescence medium, and cultivate under low temperature dark condition, obtain the sclerotium of hickory chick hybridization bacterium.
Preferably, in the present invention, the pH value of alkalescence medium is 7.5-8.5, and the cultivation temperature of cultivating under the black dull condition of low temperature is 10-15 ℃.The too high or too low quantity of formation that all can reduce mycelial growth rate and reduce sclerotium of medium pH.The too low mycelial growth rate that affects of temperature, excess Temperature is unfavorable for forming sclerotium.
Adopt medium pH 7.5-8.5, the environment of cultivation temperature 10-15 ℃, is conducive to hickory chick and forms more multistable fixed sclerote, and the sclerotium having reduced in hickory chick incubation in the past does not form or sclerotium disappearance degradation phenomena, and then has reduced agricultural losses.
Described mycelia piece can obtain by the following method, and (1) uses the spore of the wild of hickory chick maturation or cultivation bacterium mushroom or tissue block is sprouted under 20-25 ℃ of condition on medium and mycelia purifying; Medium is PDA medium herein, fills a prescription to be: 200 grams of potatos, 20 grams of glucose, 20 grams, agar, 3 grams of potassium dihydrogen phosphates, 1 gram, magnesium sulfate, VB
150 micrograms, 1000 milliliters, water, pH nature; Its preparation method is: by peeling potatoes, weigh, be cut into small pieces, put into beaker, 800 ml water heating were boiled about 30 minutes, boiled to potato ball thoroughly well cooked but not mushy.Then with double gauze, filter, get its filtrate, add agar, then heating boils agar is dissolved, while boiling, stir, prevent overflow.After agar dissolves completely, add glucose, add thermal agitation and allow glucose dissolve completely.Finally supply moisture, make it reach 1000 milliliters.Be distributed into while hot in test tube and triangular flask, prevent from solidifying.Test tube adds continuous plug, and triangular flask adds sealed membrane.The medium minute installing, sterilizing in pressure cooker in time, exit three times to get rid of a cold air in pot, and 0.147 MPa left and right sterilizing 30-40 minute, stops heating, when pot inner pressure pointer makes zero and after temperature reaches below 90 ℃, opens pot cover taking-up medium.Test tube tiltedly lets cool and but makes slant medium.In triangular flask, cultivate based on making plating medium in aseptic working platform.
The sprouting of spore and mycelia purification step are: streak inoculation on plating medium after in aseptic working platform, the distilled water suspension of spore being dipped with oese; Then in 20-25 ℃ of dark culturing, when mycelia grows to 2-5 cm left and right, cut most advanced and sophisticated mycelia and transfer and cultivate on another medium, after switching at least twice, obtaining the Morciiella Esculeuta Mycelia piece of purifying continuously.
Sprouting and the mycelia purification step of bacterium mushroom tissue block are: in aseptic working platform, will remove the Morchella esculenta (L.) Pers sporophore of the firm maturation of stem base portion, cross section above Alcohol Flame, in fruit body surface of internal cavity, cut the tissue of getting soya bean size, be placed on plating medium in 20-25 ℃ of dark culturing, when mycelia grows to 2-5 cm left and right, cut most advanced and sophisticated mycelia and transfer and cultivate on another medium, after switching at least twice, obtaining the Morciiella Esculeuta Mycelia piece of purifying continuously.
Preferably, described hickory chick bacterial strain is two kinds, especially can be wild species bacterial strain and cultivated species bacterial strain, the mycelia piece of every kind of bacterial strain is done for oneself two, vaccination ways is by two class mycelia pieces, and cross inoculation is in the corner location of same plating medium, and is placed under the dark condition of 20-25 ℃ and cultivates, secondary hyphae until mycelia piece germinating is overlapping in dull and stereotyped centre position, merge after, cut and merge mycelia piece and transfer and on alkalescence slant medium, carry out sclerotium cultivation or preservation.
Preferably, while cultivating hickory chick hybridization bacterium sclerotium, mycelia piece used is four.When mycelia piece used is during lower than four, hybridization fusion efficiencies is low; During higher than four, hybridization fusion efficiencies does not obviously promote, and complex operation, easily microbiological contamination.
Another object of the present invention is to provide the sclerotium of hickory chick hybridization bacterium in the application of preparing on morchella mother culture and cultivated species, and its method is with reference to prior art.
beneficial effect of the present invention:
Method of the present invention is easy and simple to handle, intensive, quick, effective guarantee hickory chick merge the acquisition of mycelia; Further, according to the habit of growing of hickory chick, design low temperature, subalkaline culture environment, can turn out in a large number sclerotium of morchella esculenta, ensured to a certain extent the successful fruiting of hickory chick field production.
figure of description
Fig. 1: make the four jiaos of inoculation method schematic diagrames of plating medium that merge mycelia, in figure, 1 represents plating medium; 2 and 3 represent to derive from the Morciiella Esculeuta Mycelia piece of different strains.
Embodiment
embodiment mono-:
1, in aseptic operating platform, the wild mushroom mushroom of the hickory chick of Mianyang City Beichuan County, Jiang Caiyu Sichuan Province ripe dry spore inoculating (90mm diameter culture dish, PDA medium, pH value nature on plating medium; Lower same), in 25 ℃ of dark culturing.
2, the dark mycelia tip that cuts 2 cm left and right after 2 days of cultivating of hickory chick spore, switching is cultivated on another medium, and continuous 3 switchings obtain the hickory chick bacterial classification of purifying.After labelling in 4 ℃ of preservations.
3, on new plating medium, around the starlike 1-8 ferfas silk piece (adjacent mycelia piece is the different bacterial strain in source) of inoculating respectively equably of central point, in 25 ℃ of dark culturing.Each mycelia piece number is made the flat board of 20 repetitions and is cultivated.
4, when mycelia is covered with flat board and medium and all presents brown, random 50 mycelia of picking in annulus ring 1/2 radius forming in dull and stereotyped center, mycelia piece, under microscope, observed and recorded merges the quantity of mycelia, and calculate the ratio that it accounts for mycelia quantity, with 20 mean values that repeat experiment result of noting down.
5, statistics (in Table 1) shows: while inoculating 1-3 ferfas silk piece on same culture medium flat plate, the ratio that obtains hickory chick fusion mycelia is less than 33%.Mycelia fusion efficiencies while inoculating unnecessary 4-8 ferfas silk piece is 63.09%～66.00%, there is no the significance difference opposite sex, but it is all obviously better than the syncretizing effect of 1～3 ferfas kind piece; Than more than 5 inoculation pieces, four jiaos of plating mediums, inoculate respectively " when four jiaos of inoculation methods are obtaining high expressed amount and merge mycelia, can saving bacterial classification consumption, reduce microbiological contamination risk, apply in more worth practice of hickory chick.
embodiment bis-:
1,, in aseptic operating platform, the fusion mycelia piece of homology is inoculated in respectively on the PDA slant medium of pH4.0～11.0, (18mm * 20cm test tube, containing about 10ml medium; Lower same), mycelia block size is close, culture medium inoculated 20 pipes of identical pH value.
2, slant medium is placed under 25 ℃ of dark conditions, the sclerotium quantity of formation of observed and recorded hickory chick, and under microscope, measure the diameter of sclerotium.
3, statistical result showed (in Table 2): the pH value of PDA medium has obvious influence to the quantity of formation of sclerotium of morchella esculenta and diameter.Almost can not growth sheep tripe bacterium in the acid condition of pH4.0, and pH is greater than the growth that 10.0 medium is also unfavorable for hickory chick.Compared to other Acidity of Aikalinity values, on the PDA medium of pH7.5～8.5, can and within the 3rd week, grow more sclerotium of morchella esculenta at inoculation second week, in the practice cultivation of land for growing field crops, will be more conducive to fruiting.
The impact that table 2 Medium's PH Value forms sclerotium of morchella esculenta
Note: A, sclerotium diameter are less than 3mm; B, sclerotium diameter are 3-5mm; C, sclerotium diameter are greater than 5mm; Sclerotium size distribution is percentage, and mean value unit is mm.
embodiment tri-:
1,, in aseptic operating platform, by the fusion inoculated by hypha block of homology, on the PDA of pH8.0 slant medium, (18mm * 20cm test tube, containing about 10ml medium; Lower same), mycelia block size is close.
2, slant medium is placed in respectively under the dark condition of 4～30 ℃, the bacterial classification under uniform temp condition is respectively cultivated 20 pipes.The sclerotium quantity of formation of hickory chick after observed and recorded 3 weeks, and under microscope, measure the diameter of sclerotium.
3, statistical result showed (in Table 3): cultivation temperature has obvious influence to the quantity of formation of sclerotium of morchella esculenta and diameter, the too low growth rate that affects mycelia of temperature, and excess Temperature is also unfavorable for the formation of sclerotium.Hickory chick is placed in while cultivating within the scope of 10～15 ℃, and its sclerotium amount of growth is maximum, on average reaches 18.5～21.5/pipe.
The impact that table 3 cultivation temperature forms sclerotium of morchella esculenta
embodiment tetra-:
1,, in aseptic operating platform, (18mm * 20cm test tube, containing about 10ml medium on the PDA of pH8.0 slant medium respectively the hickory chick that derives from Beichuan County, Sichuan Province, Qingchuan County, Xuanhan County and Lijiang County In Yunnan Province city to be merged to inoculated by hypha block; Lower same), mycelia block size is close.
2, slant medium is placed in respectively under the dark condition of 12 ℃, the sclerotium quantity of formation of 3 weeks interior hickory chicks of Continuous Observation record, and under microscope, measure the diameter of sclerotium.The bacterial classification in each source, region is done 20 pipes cultivations and is repeated experiment.
3, statistical result showed (in Table 4): under the PDA of pH8.0 medium and 12 ℃, dark condition, the raw sclerotigenic asynchronism(-nization) of hickory chick in different geographical source, show that Xuanhan County and Lijiang City start from first week, and the sclerotium of morchella esculenta of Beichuan, Qinghai and Sichuan comes across second week; Cultivate second week after the time, the hickory chick in above-mentioned source has all shown higher sclerotium output, reaches 15-17 sclerotium/pipe, illustrates that under pH8.0,12 ℃ and dark condition, hickory chick can stably produce sclerotium.
The experiment of table 4 sclerotium production stability
Note: A, sclerotium diameter are less than 3mm; B, sclerotium diameter are 3-5mm; C, sclerotium diameter are greater than 5mm; Sclerotium size distribution is percentage, and mean value unit is mm.
Claims (5)
1. the sclerotium cultural method of hickory chick hybridization bacterium, it is characterized in that: comprise the following steps: by the mycelia piece cross inoculation of different hickory chick bacterium sources in the periphery of plating medium, when mycelia hybridization forms fusion mycelia, to merge mycelia transfers in alkalescence medium, and cultivate under low temperature dark condition, obtain the sclerotium of hickory chick hybridization bacterium.
2. according to the sclerotium cultural method of the hickory chick hybridization bacterium described in claim 1, it is characterized in that: described hickory chick bacterial strain is two kinds, the mycelia piece of every kind of bacterial strain is done for oneself two, vaccination ways is in the corner location of same plating medium by two class mycelia piece cross inoculations, and be placed under the dark condition of 20-25 ℃ and cultivate, secondary hyphae until mycelia piece germinating is overlapping in dull and stereotyped centre position, merge after, cut merge mycelia piece transfer in alkalescence slant medium on carry out sclerotium cultivation or preservation.
3. the sclerotium cultural method of hickory chick hybridization bacterium according to claim 2, is characterized in that: the pH value of described alkalescence slant medium is 7.5-8.5.
4. the sclerotium cultural method of hickory chick hybridization bacterium according to claim 1, is characterized in that: the temperature of described low temperature dark condition is 10-15 ℃.
5. the sclerotium of a hickory chick hybridization bacterium is in the application of preparing on morchella mother culture and cultivated species.
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| CN108410744B (en) * | 2018-03-21 | 2021-06-18 | 福建师范大学 | Fusion strain for producing polysaccharide, adenosine and cordycepin |
| CN109247192A (en) * | 2018-10-25 | 2019-01-22 | 天全县营生食用菌种植农民专业合作社 | A kind of cultural method of hickory chick |
| CN110122174A (en) * | 2019-06-11 | 2019-08-16 | 北京农学院 | The method of hybrid strain is obtained from hickory chick monospore crossbreeding obtained strains |
| CN113455287A (en) * | 2021-07-07 | 2021-10-01 | 重庆市酉阳县琦睿峰食用菌有限责任公司 | Method for directly separating morchella tissue by PDA (personal digital Assistant) plate to obtain pure strain |
| CN116508646A (en) * | 2023-04-28 | 2023-08-01 | 十堰市农业科学院 | Lentinus edodes double-single hybridization device and method |
| CN116508646B (en) * | 2023-04-28 | 2024-03-26 | 十堰市农业科学院 | Lentinus edodes double-single hybridization device and method |
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