CN104170653B - 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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- CN104170653B CN104170653B CN201410432139.3A CN201410432139A CN104170653B CN 104170653 B CN104170653 B CN 104170653B CN 201410432139 A CN201410432139 A CN 201410432139A CN 104170653 B CN104170653 B CN 104170653B
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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 Morchella esculenta (L.) Perss strain makes and sclerotium culture technique, particularly to a kind of mycelia hybridization of optimization
Mixing operation technology, the high-efficient culture method of sclerotium and its application in strain preparation.
Background technology
Morchella esculenta (L.) Perss morchella esculenta (l.) pers is under the jurisdiction of Morchellaceae (morchellaceae) sheep
Tripe Pseudomonas (morchella), is internationally famous Precious Edible Fungi, and being in great demand in the world, is Chinese main exit to Europe such as France
The edible fungus of country.And with the country such as moral, method, meaning, U.S., the demand of Morchella esculenta (L.) Perss is increased, its price is constantly climbed
Rise.
Morchella esculenta (L.) Perss are recorded in Compendium of Material Medica earliest, with a long history in China's utilization.Modern science finds in Morchella esculenta (L.) Perss
Rich in proteins, fatty acid and carbohydrate, particularly multiple rare amino acids are (as cis- 3- amino-l- proline, 2- ammonia
Base isopropylformic acid., 2,4- triamido isopropylformic acid. etc.), mineral element (potassium, phosphorus, magnesium, calcium, ferrum, zinc, copper, manganese etc.) and multiple dimension life
Compositions such as plain (thiamine, riboflavin, niconacid, pantothenic acid, Folic Acid, pyridoxol, biotin, ascorbic acid, vb12) imparts sheep
The unique local flavor of tripe bacterium and nutritive value, become the high-grade food materials in international food and drink.And, Morchella esculenta (L.) Perss Medicine Food Homology, modern doctor
Learn research to show, Morchella esculenta (L.) Perss can the kidney invigorating, supplementing the brain, tonifying YANG, refresh oneself, blood fat reducing, the effect such as antitumor.In addition, Morchella esculenta (L.) Perss can use
In raw material of steeping in wine, its liquid fermentation and culture thing can be used for production of flavoring agent etc. again, therefore it is in food, health product, medication chemistry
Have broad application prospects etc. multi-field.
At present, the Morchella esculenta (L.) Perss of Chinese exports are adopted in naturally wild mostly, its far can not meet domestic with international market
Demand, and the excessive collection of wild resource causes its yield drastically to reduce, and greatly destroys the bio-diversity of Morchella esculenta (L.) Perss.Cause
This, the artificial or Semi-artificial cultivation technology of Morchella esculenta (L.) Perss is always the focus of international edible fungi research, and experiment in cultivation occasionally has successfully, but
Its poor repeatability, fruiting is unstable, can not carry out the commercialization cultivation of maturation so far.
Scientific investigations showed that for many years, in the life cycle of Morchella esculenta (L.) Perss, the formation of sclerotium is growth sporophore (i.e. fruiting)
Only stage which must be passed by, and carry out big Tanaka's cultivation experiments using the hybridization sclerotium that goes out of mycelia culture and the most easily grow sporophore.Practice
Find in production, during artificial culture's Morchella esculenta (L.) Perss strain, lack unified and standard and scientific and efficient strain hybridizing method, and in bacterium
In kind manufacturing process, the inadequacy of condition of culture frequently results in strain and does not form sclerotium or sclerotium disappearance, causes Morchella esculenta (L.) Perss to subtract
Produce and even have no harvest, this is two unstable major reasons of current Morchella esculenta (L.) Perss (partly) artificial culture test.
Sclerotium fecund, stable yields cultural method and the hybridization strain system that a kind of optimization, efficient Morchella esculenta (L.) Perss hybridize bacterium is provided
Make technology, be the problem that the present invention tries hard to solve.
Content of the invention
One object of the present invention be aiming at Gaster caprae seu Ovis fungus preparing seeds during hybridization efficiency is high, sclerotium in experiment in cultivation
Form unstable deficiency, provide a kind of Morchella esculenta (L.) Perss to hybridize the sclerotium cultural method of bacterium, the method is easy and simple to handle, intensive, quick,
Mycelia hybridization efficiency is high, has ensured that Morchella esculenta (L.) Perss merge the acquisition of mycelia；Growth promoter habit according to Morchella esculenta (L.) Perss further, design
Low temperature, subalkaline culture environment, can go out sclerotium of morchella esculenta by mass propgation, ensured that Morchella esculenta (L.) Perss land for growing field crops plants to a certain extent
The successful fruiting of training.
The technical purpose of the present invention is realized by following scheme:
A kind of Morchella esculenta (L.) Perss hybridize the sclerotium cultural method of bacterium, comprise the following steps:
By the mycelia block cross inoculation of different Morchella esculenta (L.) Perss bacterium sources in the periphery of plating medium, treat that mycelia hybridization is formed
When merging mycelia, mycelia will be merged and transfer in alkalescence culture medium, and cultivate under low temperature dark condition, obtain Morchella esculenta (L.) Perss hybridization
The sclerotium of bacterium.
Preferably, in the present invention, the ph value of alkalescence culture medium is 7.5-8.5, the culture of culture under the conditions of low temperature is black dull
Temperature is 10-15 DEG C.The too high or too low quantity of formation that all can reduce mycelial growth rate and reduce sclerotium of culture medium ph.Temperature
Spend low impact mycelial growth rate, temperature is too high to be unfavorable for forming sclerotium.
Using culture medium ph7.5-8.5, the environment of cultivation temperature 10-15 DEG C, Morchella esculenta (L.) Perss are conducive to be formed more stable
Sclerote, decreases the sclerotium in conventional Morchella esculenta (L.) Perss incubation and is not formed or sclerotium disappearance degradation phenomena, and then reduce
Agricultural losses.
Described mycelia block can obtain by the following method, and (1) uses the spore of the ripe wild or cultivation mushroom of Morchella esculenta (L.) Perss
Son or piece of tissue 20-25 DEG C in the culture medium under the conditions of carry out sprouting and mycelia purification；Culture medium is pda culture medium herein,
Formula is: 200 grams of Rhizoma Solani tuber osi, 20 grams of glucose, 20 grams of agar, 3 grams of potassium dihydrogen phosphate, 1 gram of magnesium sulfate, vb150 is micro-
Gram, 1000 milliliters of water, ph is natural；Its manufacture method is: peeling potatoes weighed, is cut into small pieces, put in beaker, 800
Milliliter water heated and boiled 30 minutes about, boils thoroughly well cooked but not mushy to potato ball.Then filtered with double gauze, take its filtrate, plus
Enter agar, then heated and boiled makes agar dissolve, and stirs when boiling, and prevents liquid from overflowing.After agar dissolves completely, add
Glucose, heated and stirred allows glucose to be completely dissolved.Finally supply moisture so as to reach 1000 milliliters.It is distributed into test tube while hot
In triangular flask, prevent from solidifying.Test tube adds continuous plug, and triangular flask seals up membrana oralis.The good culture medium of subpackage, goes out in time in pressure cooker
Bacterium, exits three times to exclude cold air in pot, 0.147 MPa about sterilizing 30-40 minute, stops heating, treat pot inner pressure
During pointer zero and after temperature reaches less than 90 DEG C, open pot cover taking-up culture medium.Test tube tiltedly lets cool but making slant medium.
In triangular flask, culture is based on making plating medium in aseptic working platform.
The sprouting of spore with mycelia purification step is: dips in the distilled water suspension inoculating loop of spore in aseptic working platform
Take streak inoculation on plating medium；Then in 20-25 DEG C of dark culturing, whne mycelia grow to 2-5 cm about when, cut
Take Tip Splitting culture in another culture medium of transferring, after continuous switching at least twice, obtain the Morchella esculenta (L.) Pers. Mycelium block of purification.
The sprouting of mushroom piece of tissue with mycelia purification step is: will remove the firm maturation of stem base portion in aseptic working platform
Morchella esculenta (L.) Pers sporophore, cross section above Alcohol Flame, cut, in sporophore surface of internal cavity, the tissue taking Semen Glyciness size, be placed on flat
In 20-25 DEG C of dark culturing in plate culture medium, whne mycelia grow to 2-5 cm about when, cut Tip Splitting and transfer another
Cultivate in one culture medium, after continuous switching at least twice, obtain the Morchella esculenta (L.) Pers. Mycelium block of purification.
Preferably, described Gaster caprae seu Ovis bacteria strain is two kinds it is particularly possible to be wild species bacterial strain and cultigen bacterial strain, every kind of bacterial strain
Mycelia block be respectively two pieces, vaccination ways are by two class mycelia blocks, cross inoculation in the corner location of same plating medium,
It is placed in culture under 20-25 DEG C of dark condition, the secondary hyphae treating the germinating of mycelia block is in flat board centre position overlap, fusion
Afterwards, cut fusion mycelia block and transfer and sclerotium culture or preservation are carried out on alkalescence slant medium.
Preferably, during culture Morchella esculenta (L.) Perss hybridization bacterium sclerotium, mycelia block used is four pieces.When mycelia block used is less than four pieces
When, hybrid fusion efficiency is low；During higher than four pieces, hybrid fusion efficiency is not obviously improved, and complex operation, easy microbiological contamination.
Another object of the present invention is that the sclerotium providing Morchella esculenta (L.) Perss to hybridize bacterium is preparing morchella mother culture and cultigen
On application, its method is with reference to prior art.
Beneficial effects of the present invention:
The method of the present invention is easy and simple to handle, intensive, quick, and effective guarantee Morchella esculenta (L.) Perss merge the acquisition of mycelia；Further
According to the growth promoter habit of Morchella esculenta (L.) Perss, devise low temperature, subalkaline culture environment, Morchella esculenta (L.) Perss bacterium can be gone out by mass propgation
Core, has ensured the successful fruiting of Morchella esculenta (L.) Perss field production to a certain extent.
Figure of description
Fig. 1: make the plating medium corner inocalation method schematic diagram merging mycelia, in figure, 1 expression plating medium；2
Represent the Morchella esculenta (L.) Pers. Mycelium block from different strains with 3.
Specific embodiment
Embodiment one:
1st, in aseptic operating platform, the wild mushroom maturation adopted in the Morchella esculenta (L.) Perss of Sichuan Province Mianyang City Beichuan County is dried spore
Son is inoculated on plating medium (90mm diameter Petri dishes, pda culture medium, ph value nature；Similarly hereinafter), in 25 DEG C of dark trainings
Support.
2nd, Morchella esculenta (L.) Perss spore light culture cut after 2 days 2 cm about mycelia most advanced and sophisticated, switching is trained in another culture medium
Support, continuous 3 switchings obtain the Morchella esculenta (L.) Perss strain of purification.Label and preserve after 4 DEG C.
3rd, on new plating medium, equably inoculate 1-8 truffles silk block (adjacent hyphae respectively around central point is starlike
Block is the different bacterial strain in source), in 25 DEG C of dark culturing.Each mycelia block number mesh does the flat board culture of 20 repetitions.
4th, when mycelia covers with flat board and culture medium all assumes brown, in the annulus ring of plate center, mycelia block composition
Random 50 mycelia of picking in 1/2 radius, merge the quantity of mycelia, and calculate it and account for mycelia quantity in basis of microscopic observation record
Ratio, noted down result with 20 meansigma methodss repeating to test.
5th, statistical result (see Table 1) shows: when inoculating 1-3 truffles silk block on same culture medium flat plate, obtains Morchella esculenta (L.) Perss
The ratio merging mycelia is less than 33%.Inoculating Hyphal anastomosis efficiency during unnecessary 4-8 truffles silk block is 63.09%～66.00%,
Being not significantly different from property, but it is all substantially better than the syncretizing effect of 1～3 truffles kind block；Inoculation block compared to more than 5 pieces,
Plating medium corner inoculate respectively Morchella esculenta (L.) Perss " inocalation method of corner while obtaining high expression and merge mycelia, Neng Goujie
About strain consumption, minimizing microbiological contamination risk, popularization and application in more worth practice.
Embodiment two:
1st, in aseptic operating platform, the fusion mycelia block of homology is inoculated in respectively the pda slant culture of ph4.0～11.0
On base, (18mm × 20cm test tube, containing about 10ml culture medium；Similarly hereinafter), mycelia block size is close, the culture medium inoculated of identical ph value
20 pipes.
2nd, slant medium is placed under 25 DEG C of dark conditions, the Sclerotia forming quantity of observed and recorded Morchella esculenta (L.) Perss, and in
The diameter of sclerotium is measured under microscope.
3rd, statistical result showed (being shown in Table 2): the ph value of pda culture medium is to the quantity of formation of sclerotium of morchella esculenta and diameter
There is obvious influence.Morchella esculenta (L.) Perss can hardly be grown in the acid condition of ph4.0, and ph is more than 10.0 culture medium
Also it is unfavorable for the growth of Morchella esculenta (L.) Perss.Compared to other Acidity of Aikalinity values, can be in inoculation the in the pda culture medium of ph7.5～8.5
Grow more sclerotium of morchella esculentas within two weeks and the 3rd week, will more conducively fruiting in the practice cultivation of land for growing field crops.
The impact that table 2 culture medium ph value is formed to sclerotium of morchella esculenta
Note: a, sclerotium diameter are less than 3mm；The a diameter of 3-5mm of b, sclerotium；C, sclerotium are with diameter greater than 5mm；Sclerotium size distribution
For percentage ratio, meansigma methodss unit is mm.
Embodiment three:
1st, in aseptic operating platform, the mycelia block that merges of homology is inoculated in (18mm on the pda slant medium of ph8.0
× 20cm test tube, containing about 10ml culture medium；Similarly hereinafter), mycelia block size is close.
2nd, slant medium is respectively placed under 4～30 DEG C of dark condition, the strain under identical temperature conditionss is respectively trained
Support 20 pipes.The Sclerotia forming quantity of observed and recorded Morchella esculenta (L.) Perss after 3 weeks, and measure the diameter of sclerotium under microscope.
3rd, statistical result showed (being shown in Table 3): cultivation temperature has bright to the quantity of formation of sclerotium of morchella esculenta and diameter
Aobvious influence, the too low speed of growth that have impact on mycelia of temperature, and the too high formation being also unfavorable for sclerotium of temperature.By Gaster caprae seu Ovis
When bacterium is placed in culture in the range of 10～15 DEG C, its sclerotium increment is maximum, averagely reaches 18.5～21.5/pipe.
The impact that table 3 cultivation temperature is formed to sclerotium of morchella esculenta
Example IV:
1st, in aseptic operating platform, respectively will be from Sichuan Province Beichuan County, Qingchuan County, Xuanhan County and Lijiang County In Yunnan Province city
Morchella esculenta (L.) Perss merge mycelia block and be inoculated on the pda slant medium of ph8.0 that (18mm × 20cm test tube, containing about 10ml culture medium；
Similarly hereinafter), mycelia block size is close.
2nd, slant medium is respectively placed under 12 DEG C of dark condition, the sclerotium of Morchella esculenta (L.) Perss in Continuous Observation record 3 weeks
Quantity of formation, and measure the diameter of sclerotium under microscope.The strain in each region source does 20 pipe cultures to be repeated to test.
3rd, statistical result showed (being shown in Table 4): ph8.0 pda culture medium with 12 DEG C, under dark condition, different geographical is come
The Morchella esculenta (L.) Perss life sclerotigenic time in source is different, shows that Xuanhan County and Lijiang City start from first week, and Beichuan, Qinghai and Sichuan
Sclerotium of morchella esculenta come across second week；After cultivating the second time-of-week, the Morchella esculenta (L.) Perss in above-mentioned source all show higher bacterium
Core volume of production, reaches 15-17 sclerotium/pipe, illustrates that, under ph8.0,12 DEG C and dark condition, Morchella esculenta (L.) Perss can stably produce bacterium
Core.
Table 4 sclerotium production stability is tested
Note: a, sclerotium diameter are less than 3mm；The a diameter of 3-5mm of b, sclerotium；C, sclerotium are with diameter greater than 5mm；Sclerotium size distribution
For percentage ratio, meansigma methodss unit is mm.
Claims (4)
1. a kind of Morchella esculenta (L.) Perss hybridize the sclerotium cultural method of bacterium, and its feature exists: comprises the following steps: different Gaster caprae seu Ovis bacteria strains are come
The mycelia block cross inoculation in source, in the periphery of plating medium, when mycelia hybridization forms and merges mycelia, will merge mycelia switching
In alkalescence culture medium, and cultivate under low temperature dark condition, obtain the sclerotium that Morchella esculenta (L.) Perss hybridize bacterium；Described Gaster caprae seu Ovis bacteria strain is
Two kinds, the mycelia block of every kind of bacterial strain is respectively two pieces, and vaccination ways are to cultivate two class mycelia block cross inoculations in same flat board
The corner location of base, is placed in culture under 20-25 DEG C of dark condition, and the secondary hyphae treating the germinating of mycelia block is in flat board interposition
After putting overlap, merging, cut fusion mycelia block and transfer sclerotium culture or preservation are carried out on alkalescence slant medium.
2. Morchella esculenta (L.) Perss according to claim 1 hybridize bacterium sclerotium cultural method it is characterised in that: described alkalescence inclined-plane
The ph value of culture medium is 7.5-8.5.
3. Morchella esculenta (L.) Perss according to claim 1 hybridize bacterium sclerotium cultural method it is characterised in that: described low temperature dark bar
The temperature of part is 10-15 DEG C.
4. morchella mother culture and cultivation are being prepared by the sclerotium that the Morchella esculenta (L.) Perss that the method culture described in claim 1 obtains hybridize bacterium
Application in kind.
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| CN106416751B (en) * | 2016-09-27 | 2019-11-22 | 四川省农业科学院土壤肥料研究所 | The method that hickory chick single-ascospore strain is cultivated using " artificial pollination " |
| 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 |
| CN110122174B (en) * | 2019-06-11 | 2021-05-28 | 北京农学院 | Method for obtaining hybrid strain from strain obtained by monospore cross breeding of morchella esculenta |
| 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 |
| 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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