CN102640659A - Cordyceps militaris fertility mother seed resource breeding method - Google Patents
Cordyceps militaris fertility mother seed resource breeding method Download PDFInfo
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- CN102640659A CN102640659A CN2011100437359A CN201110043735A CN102640659A CN 102640659 A CN102640659 A CN 102640659A CN 2011100437359 A CN2011100437359 A CN 2011100437359A CN 201110043735 A CN201110043735 A CN 201110043735A CN 102640659 A CN102640659 A CN 102640659A
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Abstract
The invention discloses a cordyceps militaris fertility mother seed resource breeding method which is designed for solving the problem that cordyceps militaris cannot be cultured steadily in the mother seed source cultivation caused by a culture degeneration problem in the currently cordyceps militaris labour cultivation, thereby restricting cordyceps militaris technical problems existing in cordyceps militaris large-scale production. According to the method, special fresh cordyceps militaris are used as a component material, an innocuousness surface sterilization agent treatment material is adopted, colony is obtained after spore separation, and the cordyceps militaris fertility seed source with hereditary stability is obtained through technology assessment program such as fertility hypha evaluation screening. The seed source has higher fertility, thereby being applied to cordyceps militaris large-scale factorization cultivation; and the method has importance value on a manual production function, and has wide application prospects.
Description
Technical field
The mother who the present invention relates to a kind of Cordyceps sinensis plants selection, and relating in particular to a kind of northern Chinese caterpillar Fungus can solid sexupara kind resource selection.Belong to edible mushroom, medicinal fungus genetic breeding field.
Background technology
Cordyceps sinensis [Cordyceps sinensis] is the traditional rare medicinal herbs of China, and special pharmacological effect that it had and therapeutic efficiency have obtained people's approval.Owing to the expansion of mankind's activity scope, Ecological environment worsening causes the wild cordyceps continuous decrease in recent years, and price also continues to rise violently.160,000 yuan/kilogram (doing) have been sold to high-quality Chinese caterpillar fungus in 2010.For solving Cordyceps sinensis resource scarcity problem, people begin to pay close attention to Cordyceps militaris very early, and from 1986, people returned the wild cordyceps militaris collection, obtained bacterial classification through separate tissue, and in more than 20 year, the Cordyceps militaris cultivation obtains major technological breakthrough.It has become the best substitute of Cordyceps sinensis at present.Kind surplus the known Chinese caterpillar fungus class in the whole world has 350, China has 62 kinds at present, but the most expensive has only Cordyceps sinensis and Cordyceps militaris.Grass but can not stablize after the production that causes because of the spawn degeneration problem in the northern Chinese caterpillar Fungus artificial cultivation is at present cultivated with female kind provenance, thereby restricted northern Chinese caterpillar Fungus large-scale production.
Summary of the invention:
The present invention grass in order to solve can not stablize after the production that causes because of the spawn degeneration problem in the present northern Chinese caterpillar Fungus artificial cultivation is cultivated with female kind provenance; Thereby restricted the provenance technical problem that northern Chinese caterpillar Fungus large-scale production is brought; The invention provides a kind of northern Chinese caterpillar Fungus can solid sexupara kind resource selection; Be to plant the initial provenance of seed selection as mother with the bright Cordyceps militaris fruiting body of silkworm chrysalis of wild bright Cordyceps militaris or artificial culture; Cordyceps militaris fruiting body is being carried out adopt hydrogen peroxide solution in the separate tissue as the living materials disinfectant; And do not use ethanol and give birth to mercury as disinfectant; Adopt the conidium rubbing method to carry out mother and plant successive transfer culture, successively used unique mother culture media and a whole set of imitative Wild ecological environment breeding method in the screening but estimate bacterium colony and fecundity mycelia, and through the mycelia micromorphology observe and selection bacterium colony fruit body primordium formation time as the seed selection parameter;
The concrete performing step of this method is:
1) the bright Cordyceps militaris fruiting body of silkworm chrysalis with wild bright Cordyceps militaris or artificial culture is that material passes through the female initial provenance of seed selection of planting of separate tissue means acquisition;
2) do not use ethanol and living mercury to handle living materials as disinfectant in the separate tissue, the hydrogen peroxide solution of employing 5% can carry out strain separating work in 20 minutes as the living materials disinfectant behind the spraying disinfection;
3) female successive transfer culture inoculation method of planting adopts conidium switching method;
4) inoculation back tube is cultivated under following condition: the preceding 2 days lucifuges in 20 ℃ ± 2 ℃ humidity of cultivation temperature, 60% ± 5% inoculation back are cultivated; Whole process was seen the light cultivation from the 3rd day to the 10th day; Light source adopts fluorescent lamp illumination low early and high after, from 1500 luxs to 6000 luxs;
5) tube of selecting proterties preponderant strains silk is as follow-up seed selection starting strain;
6) simulation northern Chinese caterpillar Fungus self-sow environment carries out fruit body primordium and induces; Simulated conditions are: was duration according to the day and night samsara with 12 hours; Give 20 ℃ ± 2 ℃ 6000 lux illumination daytime; Evening, 18 ℃ ± 2 ℃ lucifuges were cultivated, and will under aseptic condition, give mycelia ventilation in the tube before this is handled, and handled continuously 3 days;
7) cultivate from the 4th day with 20 ℃ ± 2 ℃ 6000 lux illumination condition, form manyly with fruit body primordium to observe through anatomical lens or magnifying glass in 7-10 days, evenly, the dark brown tube of color and luster is can solid sexupara kind resource;
8) to seed selection can solid sexupara kind resource carry out concave slide microchamber aseptic culture, under light microscope, carry out morphologic observation, according to conidium on mycelium the state of giving birth to judge that finally obtaining northern Chinese caterpillar Fungus can solid sexupara kind resource.
Characteristics of the present invention and beneficial effect: the bright Cordyceps militaris fruiting body of silkworm chrysalis through with wild bright Cordyceps militaris or artificial culture is a material; Obtain female initial provenance of seed selection of planting through the separate tissue means; Adopt this material as the separate tissue material; Can guarantee the wild proterties of bacterial classification to greatest extent, reduce the degeneration frequency; Adopt hydrogen peroxide solution as the living materials disinfectant; And do not use ethanol and give birth to mercury as disinfectant; Can avoid ethanol as disinfectant to organization material extremely strong soaking kill property, become not enoughly active thereby reduce living materials, also can avoid high toxicity, the medicine of mercuric chloride to be difficult to buy and deficiency that using dosage and processing time should not be grasped; Obtain the initial provenance of female kind through said method and have stable inheritance, its yield traits is all very strong with generation cordycepin ability.This achievement is produced functional Cordyceps militaris for manual work and is had important value, has a extensive future.
Embodiment:
Northern Chinese caterpillar Fungus can solid sexupara kind resource selection; Be to plant the initial provenance of seed selection as mother with the bright Cordyceps militaris fruiting body of silkworm chrysalis of wild bright Cordyceps militaris or artificial culture; Cordyceps militaris fruiting body is being carried out adopt hydrogen peroxide solution in the separate tissue as the living materials disinfectant; And do not use ethanol and give birth to mercury as disinfectant; Adopt the conidium rubbing method to carry out mother and plant successive transfer culture, successively used unique mother culture media and a whole set of imitative Wild ecological environment breeding method in the screening but estimate bacterium colony and fecundity mycelia, and through the mycelia micromorphology observe and selection bacterium colony fruit body primordium formation time as the seed selection parameter;
The concrete performing step of this method is:
The bright Cordyceps militaris fruiting body of silkworm chrysalis with wild bright Cordyceps militaris or artificial culture is that material passes through the female initial provenance of seed selection of planting of separate tissue means acquisition.
Do not use ethanol and living mercury to handle living materials as disinfectant in the separate tissue, the hydrogen peroxide solution of employing 5% can carry out strain separating work in 20 minutes as the living materials disinfectant behind the spraying disinfection.
Being used for the employed culture medium prescription of separate tissue and successive transfer culture is: glucose 30g/L; Peptone 16g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate 2g/L; Manganese sulphate 0.5 gram calcium chloride 0.3 gram water 1000ml, 1000 milliliters of 100 milligrams of agar powders of B B-complex (21 Jin Weita), 18 gram distilled water.
Female successive transfer culture inoculation method of planting adopts conidium switching method.
Inoculation back tube is cultivated under following condition: the preceding 2 days lucifuges in 20 ℃ ± 2 ℃ humidity of cultivation temperature, 60% ± 5% inoculation back are cultivated, and whole process saw that light cultivates from the 3rd day to the 10th day, and light source adopts fluorescent lamp illumination low early and high after, from 1500 luxs to 6000 luxs.
The selection mycelial growth is neat, and bacterium colony woollen blanket shape is smooth, not excessive growth of aerial hyphae, and mycelia annesl fast (changing into yellow then to bronzing) by white, pigment secretion tube how is as follow-up seed selection starting strain.
Starting strain with seed selection is a material; Simulation northern Chinese caterpillar Fungus self-sow environment carries out fruit body primordium and induces program; Institute's simulated conditions give 20 ℃ ± 2 ℃ 6000 lux illumination daytime for being duration according to the day and night samsara with 12 hours, and evening, 18 ℃ ± 2 ℃ lucifuges were cultivated; To under aseptic condition, give mycelia ventilation in the tube before this is handled, handle continuously 3 days.
Cultivate from the 4th day with 20 ℃ ± 2 ℃ 6000 lux illumination condition, form manyly with fruit body primordium (careless bud) to observe through anatomical lens or magnifying glass in 7-10 days, evenly, the dark brown tube of color and luster is can solid sexupara kind resource.
To seed selection can solid sexupara kind resource carry out concave slide microchamber aseptic culture, under light microscope, carry out morphologic observation, according to conidium on mycelium the state of giving birth to judge that finally obtaining northern Chinese caterpillar Fungus can solid sexupara kind resource.
" but the fecundity " that among the present invention the Cordyceps militaris provenance is limited can normally go out grass after meaning the cultivation of used provenance, forms promptly that yield traits is stable, the tangible cordyceps militaris fruit body of morphological indexes.
Embodiment
This method realizes through following step:
The bright Cordyceps militaris fruiting body of silkworm chrysalis with wild bright Cordyceps militaris or artificial culture obtains female initial provenance of seed selection of planting through the separate tissue means;
The employed culture medium prescription of separate tissue is: glucose 30g/L, and peptone 16g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate 2g/L, manganese sulphate 0.5 gram calcium chloride 0.3 gram, water 1000ml, 21 gold medals tie up him 100 milligrams, 1000 milliliters of agar powder 18 grams and distilled water;
Do not use ethanol in the separate tissue and give birth to mercury and handle living materials, but adopt 5% hydrogen peroxide solution, can carry out strain separating work in 20 minutes behind the spraying disinfection as the living materials disinfectant as disinfectant;
Adopt conidium switching method to carry out mother and plant the successive transfer culture inoculation.Be used for the employed culture medium prescription of successive transfer culture with the employed culture medium prescription of above-mentioned separate tissue;
Inoculation back tube is cultivated under following condition: 20 ℃ ± 2 ℃ of cultivation temperature, humidity 60% ± 5%;
Postvaccinal preceding 2 days lucifuges are cultivated, and whole process saw that light cultivates from the 3rd day to the 10th day, and light source adopts fluorescent lamp illumination low early and high after, promptly from 1500 luxs to 6000 luxs.
Select starting strain, select mycelial growth neat, bacterium colony woollen blanket shape is smooth, not excessive growth of aerial hyphae, and mycelia annesl fast (changing into yellow then to bronzing) by white, pigment secretion tube how is as follow-up seed selection starting strain;
To the starting strain that selects; Simulation northern Chinese caterpillar Fungus self-sow environment carries out fruit body primordium induces, and simulated conditions are: was duration according to the day and night samsara with 12 hours, gave 20 ℃ ± 2 ℃ 6000 lux illumination daytime; Evening, 18 ℃ ± 2 ℃ lucifuges were cultivated; Before carrying out this processing, under aseptic condition, give mycelia ventilation in the tube, handled continuously 3 days;
From the 4th day with 20 ℃ ± 2 ℃, 6000 lux illumination conditions are cultivated, and form manyly with fruit body primordium (careless bud) to observe through anatomical lens or magnifying glass in 7-10 days, evenly, the dark brown tube of color and luster is can solid sexupara kind resource;
To seed selection can solid sexupara kind resource carry out concave slide microchamber aseptic culture, under light microscope, carry out morphologic observation, according to conidium on mycelium the state of giving birth to judge that finally obtaining northern Chinese caterpillar Fungus can solid sexupara kind resource.
Claims (5)
1. northern Chinese caterpillar Fungus can solid sexupara kind resource selection; Be to plant the initial provenance of seed selection as mother with the bright Cordyceps militaris fruiting body of silkworm chrysalis of wild bright Cordyceps militaris or artificial culture; It is characterized in that: Cordyceps militaris fruiting body is being carried out adopt hydrogen peroxide solution in the separate tissue as the living materials disinfectant; And do not use ethanol and give birth to mercury as disinfectant; Adopt the conidium rubbing method to carry out mother and plant successive transfer culture, successively used unique mother culture media and a whole set of imitative Wild ecological environment breeding method in the screening but estimate bacterium colony and fecundity mycelia, and through the mycelia micromorphology observe and selection bacterium colony fruit body primordium formation time as the seed selection parameter;
The concrete performing step of this method is:
1) the bright Cordyceps militaris fruiting body of silkworm chrysalis with wild bright Cordyceps militaris or artificial culture is that material passes through the female initial provenance of seed selection of planting of separate tissue means acquisition;
2) do not use ethanol and living mercury to handle living materials as disinfectant in the separate tissue, the hydrogen peroxide solution of employing 5% can carry out strain separating work in 20 minutes as the living materials disinfectant behind the spraying disinfection;
3) female successive transfer culture inoculation method of planting adopts conidium switching method;
4) inoculation back tube is cultivated under following condition: the preceding 2 days lucifuges in 20 ℃ ± 2 ℃ humidity of cultivation temperature, 60% ± 5% inoculation back are cultivated; Whole process was seen the light cultivation from the 3rd day to the 10th day; Light source adopts fluorescent lamp illumination low early and high after, from 1500 luxs to 6000 luxs;
5) tube of selecting proterties preponderant strains silk is as follow-up seed selection starting strain;
6) simulation northern Chinese caterpillar Fungus self-sow environment carries out fruit body primordium and induces; Simulated conditions are: was duration according to the day and night samsara with 12 hours; Give 20 ℃ ± 2 ℃ 6000 lux illumination daytime; Evening, 18 ℃ ± 2 ℃ lucifuges were cultivated, and will under aseptic condition, give mycelia ventilation in the tube before this is handled, and handled continuously 3 days;
7) cultivate from the 4th day with 20 ℃ ± 2 ℃ 6000 lux illumination condition, form manyly with fruit body primordium to observe through anatomical lens or magnifying glass in 7-10 days, evenly, the dark brown tube of color and luster is can solid sexupara kind resource;
8) to seed selection can solid sexupara kind resource carry out concave slide microchamber aseptic culture, under light microscope, carry out morphologic observation, according to conidium on mycelium the state of giving birth to judge that finally obtaining northern Chinese caterpillar Fungus can solid sexupara kind resource.
2. northern Chinese caterpillar Fungus according to claim 1 can solid sexupara kind resource selection; It is characterized in that: being used for the employed culture medium prescription of separate tissue and successive transfer culture is: glucose 30g/L; Peptone 16g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate 2g/L; Manganese sulphate 0.5 gram calcium chloride 0.3 gram water 1000ml, 1000 milliliters of 100 milligrams of agar powders of B B-complex, 18 gram distilled water.
3. northern Chinese caterpillar Fungus according to claim 1 can solid sexupara kind resource selection, it is characterized in that: the tube of the good mycelia of said proterties, for mycelial growth neat; Bacterium colony woollen blanket shape is smooth; Not excessive growth of aerial hyphae, the mycelia annesl is fast, the many tubes of pigment secretion.
4. northern Chinese caterpillar Fungus according to claim 2 can solid sexupara kind resource selection, and it is characterized in that: said B B-complex adopts 21 Jin Weita.
5. northern Chinese caterpillar Fungus according to claim 1 can solid sexupara kind resource selection, and it is characterized in that: the bacterium colony cultivation temperature is 18-20 ℃ ± 2 ℃, intensity of illumination in 1500 luxs to 6000 luxs.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104054513A (en) * | 2014-07-17 | 2014-09-24 | 宋显和 | Cordyceps militaris strain cultivation method |
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Patent Citations (6)
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| CN1995321A (en) * | 2006-12-29 | 2007-07-11 | 上海市农业科学院 | Northern aweto strain and its selection breeding method |
| CN101353628A (en) * | 2008-09-12 | 2009-01-28 | 江苏省农业科学院 | Rapid identification method of Chinese caterpillar fungus fertile bacterial strain |
| KR20100104408A (en) * | 2009-03-17 | 2010-09-29 | 주식회사머쉬텍 | Cordyceps militaris mycellia cultured by using rice and poultry soup for health using the smae |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104054513A (en) * | 2014-07-17 | 2014-09-24 | 宋显和 | Cordyceps militaris strain cultivation method |
| CN104054513B (en) * | 2014-07-17 | 2015-10-28 | 宋显和 | Coragyceps militaris bacterium culture method |
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