CN103081721A - Method for fast screening low temperature resistant volvaria volvacea microorganism - Google Patents
Method for fast screening low temperature resistant volvaria volvacea microorganism Download PDFInfo
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- CN103081721A CN103081721A CN2013100280652A CN201310028065A CN103081721A CN 103081721 A CN103081721 A CN 103081721A CN 2013100280652 A CN2013100280652 A CN 2013100280652A CN 201310028065 A CN201310028065 A CN 201310028065A CN 103081721 A CN103081721 A CN 103081721A
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Abstract
The invention relates to a method for fast screening low temperature resistant volvaria volvacea microorganism. The method comprises a first step of adding solanum tuberosum (taking out of juice), glucose and agal-agal into distilled water according to proportion of 180-220g/L:18-22g/L:15-20g/L, mixing the solanum tuberosum, the glucose and the agal-agal with the distilled water evenly, sterilizing mixed solution for 20-23 minutes under the condition of 121DEG C, cooling the mixed solution to 45-50DEG C, pouring the mixed solution to a sterilized petri dish and freezing the mixed solution to obtain a solid medium, and a second step of placing the volvaria volvacea microorganism into the solid medium, carrying out refrigerated process on the volvaria volvacea microorganism for 2-10h at the temperature of 0-4DEG C after the volvaria volvacea microorganism is cultivated for 18-24h under the conditions of darkness and at the temperature of 32-35DEG C, recovering cultivation for 1-5d at the temperature of 32-35 DEG C, scribing a line every 6-8 h during the recovery of the cultivation and then selecting a bacterial strain which is fast in recovery speed and strong in growing vigor to obtain the low temperature resistant volvaria volvacea microorganism. The method is short in needed time, high in efficiency and suitable for volvaria volvacea low temperature resistant introduction or large-batch detection and identification in breeding. The method is easy to operate and very low in cost.
Description
Technical field
The invention belongs to screening bacterial classification field, particularly the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening.
Background technology
Straw mushroom (Volvariella volvacea (Bull ex Fr.) Sing) belongs to Basidiomycetes, Agaricales, light handle mushroom section, Volvariella.Its delicious flavour, nutritious, be the high-quality mushroom kind in edible mushroom.According to research reports, bright straw mushroom is moisture 92.39%, protein 2.66%, fat 2.24%, reducing sugar 1.66%, invert sugar 0.95%, ash content 0.91%; Contain 18 seed amino acids in straw mushroom protein, comprise 8 kinds of essential amino acids that human body can not synthesize.Except edibility, straw mushroom also has the effects such as the body immunity of raising, inhibition cancer cell growth, detoxifcation.Therefore, straw mushroom is very popular, is the export-oriented commodity of China always, finds a good sale in to countries and regions such as the U.S., Canada, Britain, Japan, Singapore, Malaysia.
Straw mushroom originates in the torrid zone and subtropical zone, the optimum growth temperature of mycelium is 32～35 ℃, the optimal temperature of fruit body differentiation and development is 27～31 ℃, the environmental requirement of this high temperature has limited the development of straw mushroom greatly, makes the low temperature resistant straw mushroom bacterial classification of choosing/cultivation become the focus of current research.Usually the low temperature tolerance characteristics of check breeding material, need to could determine through repeatedly repeating fruiting experiment, and each fruiting experiment all will experience female kind-original seed-a series of processes such as cultivated species, could finally obtain fruit body.The shortcomings such as if the extensive fruiting that has no to screen in advance exists workload large, and energy resource consumption is high, and the cycle is long, and is unstable.Therefore, the invention provides a kind of before fruiting experiment the short-cut method of the low temperature resistant bacterial classification of first Screening and Identification straw mushroom, can greatly improve the Breeding Efficiency of low temperature resistant straw mushroom bacterial classification.
Summary of the invention
Technical problem to be solved by this invention is to provide the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening, the method required time is short, efficient is high, only need just to obtain in 1-5 days result, be suitable for large batch of detection evaluation in the low temperature resistant breeding of straw mushroom, and the detection of conventional method needs the 40d left and right, and its operation is also comparatively loaded down with trivial details; The method is simple, and equipment needed thereby and medicine are conventional, and cost is low; This identifies that detecting reliability is high, workable, and detection technique can be completed whole process of the test through simple training.
The method of the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening of the present invention comprises:
(1) by proportioning 180-220g/L:18-22g/L:15-20g/L, with potato (getting juice), glucose and agar, add in distilled water, mix, under 121 ℃ of conditions, sterilization 20-30min, then be cooled to 45-50 ℃, pour in sterilized culture dish, solidify, get solid culture medium;
(2) under aseptic condition, in the above-mentioned solid culture medium of straw mushroom bacterial classification access, under dark condition, temperature is 32-35 ℃, after cultivating 18-24h, process 2-10h 0-4 ℃ of refrigeration, then at 32-35 ℃, renewal cultivation 1-5d, during renewal cultivation, every the 6-8h setting-out once, select the bacterial strain that resume speed is fast, mycelial growth is energetic, be low temperature resistant straw mushroom bacterial strain.
In described step (1), the culture dish diameter is 9-12cm.
In described step (1), the pouring volume of medium is the 30%-50% of culture dish volume.
The mycelia of described step (2) medium-height grass mushroom strains is pure white, healthy and strong.
In described step (2), refrigerated storage temperature is preferably 0 ℃.
In described step (2), cold preservation time is preferably 6-10h.
In described step (2), the temperature of renewal cultivation is preferably 32 ℃.
In described step (2), the renewal cultivation time is preferably 3d.
The concrete operations that potato is got juice are: select potato, surface clean is clean, and peeling takes.With potato cutting, adding distil water well-done (generally boil 20-30min, can be poked by glass bar get final product) is with four layers of filtered through gauze.
Beneficial effect
(1) the inventive method required time is short, and efficient is high, only needs 1-5d just can obtain result, be suitable for large batch of detection evaluation in the low temperature resistant breeding of straw mushroom, and conventional method detection straw mushroom resistance to low temperature needs the 40d left and right, and its operation is also comparatively loaded down with trivial details;
(2) the inventive method is simple, and equipment needed thereby and medicine are conventional, and cost is very low;
(3) the present invention identifies that detecting reliability is high, and is workable, and detection technique can be completed whole process of the test through the green hand of simple training.
Description of drawings
Fig. 1 is that " ten " word line is drawn at the culture dish back side;
Fig. 2 be V23 and VH3 mycelia after processing 10h under 0 ℃, recover the situation of growth 72h under 32 ℃;
Fig. 3 is that bacterial strain V23 is at 30 ℃ of lower conventional cultivation fruiting photos;
Fig. 4 is that bacterial strain VH3 is at 28 ℃ of lower conventional cultivation fruiting photos.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
In this test, related straw mushroom bacterial strain has 2, and title is respectively V23 and VH3, is kept at Edible Fungus Inst., Shanghai Academy of Agriculture's edible mushroom preservation center; Wherein the V23 bacterial strain be conventional produce that use bacterial strain, VH3 bacterial strain be the inventor with the V23 bacterial strain through ultraviolet (UV),
60Co-gamma-rays, dithyl sulfate (DES) carry out complex mutation to protoplast, and have obtained Shanghai City kind identification.
Embodiment 1
The Screening and Identification of straw mushroom bacterial strain mycelia resistance to low temperature
(1) sterilization of culture dish: get 10 of the glass culture dishs that clean up, wrap with newspaper, then the 20min that sterilizes under 121 ℃ puts into 60 ℃ of baking oven dry for standby.
(2) PDA medium preparation: potato (getting juice) 200g/L, glucose 20g/L, agar strip 20g/L adds in distilled water, and in the triangular flask of packing into after mixing, 20min sterilizes under 121 ℃; Be cooled to 45-50 ℃, pour in the culture dish of sterilized diameter 9cm, pouring volume is 35% of culture dish volume, solidifies standby.
Wherein potato is got the concrete operations of juice and is: select potato, surface clean is clean, and peeling takes 200g.With potato cutting, adding distil water well-done (generally boil 20-30min, can be poked by glass bar get final product) is with four layers of filtered through gauze.
(3) inoculation and cultivate: after opening ultraviolet lamp sterilization, draw " ten " word line (see figure 1) at the plate back side that sterilization is good, be used for follow-up setting-out and measure and recover growing state.Get the first calcination on flame of card punch of sterilization in advance, then evenly punching on the flat board that covers with the straw mushroom mycelia; Respectively straw mushroom bacterial strain V23 and VH3 are inoculated in solid culture medium central authorities under aseptic condition, mark strain name and date, are placed in and cultivate the 24h left and right in 32 ℃ of incubators.
(4) refrigeration is processed: culture dish that will (3) refrigerates processing 10h under 0 ℃, dark condition.
(5) renewal cultivation and growing state are measured: be placed under 32 ℃, dark condition through culture dish of refrigeration processing in will (4) and cultivate, every 8h setting-out 1 time, measure the situation that V23 and VH3 bacterial strain recover to grow (see Table 1 and Fig. 2), result shows that the low temperature resistant ability of VH3 bacterial strain is better than the V23 bacterial strain.
Table 1V23 and VH3 bacterial strain recover the situation of growth 72h under 32 ℃ after processing 10h under 0 ℃
| Process title | 32 ℃ are recovered growth 72h situation (mm) |
| Process 10h for V23-0 ℃ | 0.28 |
| Process 10h for VH3-0 ℃ | 13.25 |
Embodiment 2
The experiment in cultivation of straw mushroom bacterial strain fruiting temperature
(1) female cultivation of planting: take PDA as medium, each bacterial classification of V23 and VH3 all arranges three groups.First group is that bacterial strain is cultivated 5d at 32 ℃, and second group, the 3rd group is that bacterial strain is cultivated 5d at 30 ℃.
(2) cultivation of original seed: (1) is seeded in Primary spawn material after high-temperature sterilization.First group of bacterial strain of V23 and each bacterial classification of VH3 all cultivated 20d at 32 ℃, all cultivates 20d at 30 ℃ for second group, the 3rd group.
(3) fermentation of composts or fertilisers of cultivating: with cotton seed hulls (95%), lime (5%) water is mixed thoroughly, heap fermentation then, above cover film, and in bottom and overpunch ventilation.3rd, fermentation is proceeded in turning in 4 days, namely can be used for cultivating in the 5th day.
(4) bacterium of cultivated species: (2) are inoculated on cultivation composts or fertilisers of cultivating (3), all cultivate 3d at 32 ℃, cultivate 3d at 30 ℃ for second group, the 3rd group for first group of V23 and each bacterial classification of VH3.
(5) fruiting: after sending out full bacterium on composts or fertilisers of cultivating, first group of bacterial strain of V23 and each bacterial classification of VH3 be at 30 ℃ of lower fruitings, and second group of bacterial strain be at 28 ℃ of lower fruitings, and the 3rd group of bacterial strain be at 26 ℃ of lower fruitings, 4 repetitions of every kind of each fruiting temperature setting of bacterial strain, fruiting two tides.Add up respectively former basic formation time, fruit-body formation time and the biological efficiency thereof of each bacterial strain at each fruiting temperature.
Budding, gather and biological efficiency at table 2V23 fruiting temperature different from the VH3 bacterial strain
As can be seen from Table 2: under three temperature condition, the cultivation result of (30 ℃, 28 ℃, 26 ℃) shows, the squaring period of VH3 is all short than V23 with picking time.In the time of 30 ℃, biological efficiency and the V23 of VH3 are close, have reached respectively 25.4% and 24.8%; In the time of 28 ℃, the biological efficiency of VH3 is significantly higher than V23, nearly exceeds 14% than V23; In the time of 26 ℃, the biological efficiency of VH3 is respectively 16.7% and 10.90% also higher than V23.Biological efficiency at comprehensive three temperature can find that VH3 adaptability to temperature in cultivation is relatively wide, and especially under the higher prerequisite of biological efficiency, the fruiting temperature of VH3 (28 ℃) has reduced at least 2 ℃ than V23 (30 ℃).
These results suggest that the present invention is reliable to the detection method of straw mushroom resistance to low temperature, can be used for the rapid screening of a large amount of bacterial strain resistance to low temperatures of straw mushroom in breeding.The inventive method is easy, and cost is low, has reduced energy consumption, has avoided waste.
Claims (8)
1. the method for the low temperature resistant straw mushroom bacterial classification of rapid screening comprises:
(1) by proportioning 180-220g/L:18-22g/L:15-20g/L, potato (getting juice), glucose and agar are added in distilled water, mix, under 121 ℃ of conditions, then sterilization 20-30min is cooled to 45-50 ℃, pour in sterilized culture dish, solidify, get solid culture medium;
(2) under aseptic condition, in the above-mentioned solid culture medium of straw mushroom bacterial classification access, under dark condition, temperature is 32-35 ℃, after cultivating 18-24h, process 2-10h 0-4 ℃ of refrigeration, then at 32-35 ℃, renewal cultivation 1-5d, during renewal cultivation, every the 6-8h setting-out once, select the bacterial strain that resume speed is fast, mycelial growth is energetic, be low temperature resistant straw mushroom bacterial strain.
2. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (1), the culture dish diameter is 9-12cm.
3. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (1), the pouring volume of medium is the 30%-50% of culture dish volume.
4. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1, it is characterized in that: the mycelia of described step (2) medium-height grass mushroom strains is pure white, healthy and strong.
5. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (2), refrigerated storage temperature is 0 ℃.
6. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (2), cold preservation time is 6-10h.
7. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (2), the temperature of renewal cultivation is 32 ℃.
8. the method for the low temperature resistant straw mushroom bacterial classification of a kind of rapid screening according to claim 1 is characterized in that: in described step (2), the renewal cultivation time is 3d.
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Cited By (7)
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| CN103430855A (en) * | 2013-06-20 | 2013-12-11 | 上海市农业科学院 | Low temperature resistance straw mushroom bacterial and breeding method thereof |
| CN104521712A (en) * | 2014-12-31 | 2015-04-22 | 天津泰达盐碱地绿化研究中心有限公司 | Introduction and screening method for ornamental grass in coastal salt and alkali area |
| CN105145114A (en) * | 2015-08-19 | 2015-12-16 | 黑龙江省科学院微生物研究所 | Method evaluating black fungus mycelium high temperature resistance feature |
| CN114223463A (en) * | 2022-01-04 | 2022-03-25 | 广东省科学院生物与医学工程研究所 | Method for rapidly detecting fruiting performance of ganoderma lucidum |
| CN115807018A (en) * | 2022-08-26 | 2023-03-17 | 上海市农业科学院 | Straw mushroom low temperature resistant gene, screening method and application |
| CN116334111A (en) * | 2023-03-03 | 2023-06-27 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
| CN116640673A (en) * | 2023-07-07 | 2023-08-25 | 上海市农业科学院 | Low-temperature-resistant straw mushroom strain and preparation method thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103430855A (en) * | 2013-06-20 | 2013-12-11 | 上海市农业科学院 | Low temperature resistance straw mushroom bacterial and breeding method thereof |
| CN103430855B (en) * | 2013-06-20 | 2015-08-19 | 上海市农业科学院 | A kind of low temperature resistance straw mushroom bacterial and selection thereof |
| CN104521712A (en) * | 2014-12-31 | 2015-04-22 | 天津泰达盐碱地绿化研究中心有限公司 | Introduction and screening method for ornamental grass in coastal salt and alkali area |
| CN104521712B (en) * | 2014-12-31 | 2017-05-24 | 天津泰达盐碱地绿化研究中心有限公司 | Introduction and screening method for ornamental grass in coastal salt and alkali area |
| CN105145114A (en) * | 2015-08-19 | 2015-12-16 | 黑龙江省科学院微生物研究所 | Method evaluating black fungus mycelium high temperature resistance feature |
| CN114223463A (en) * | 2022-01-04 | 2022-03-25 | 广东省科学院生物与医学工程研究所 | Method for rapidly detecting fruiting performance of ganoderma lucidum |
| CN115807018A (en) * | 2022-08-26 | 2023-03-17 | 上海市农业科学院 | Straw mushroom low temperature resistant gene, screening method and application |
| CN115807018B (en) * | 2022-08-26 | 2024-02-09 | 上海市农业科学院 | Low-temperature-resistant gene of straw mushrooms, screening method and application |
| CN116334111A (en) * | 2023-03-03 | 2023-06-27 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
| CN116334111B (en) * | 2023-03-03 | 2024-02-09 | 上海市农业科学院 | Straw mushroom cellobiose hydrolase and application thereof |
| CN116640673A (en) * | 2023-07-07 | 2023-08-25 | 上海市农业科学院 | Low-temperature-resistant straw mushroom strain and preparation method thereof |
| CN116640673B (en) * | 2023-07-07 | 2024-03-26 | 上海市农业科学院 | Low-temperature-resistant straw mushroom strain and preparation method thereof |
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Application publication date: 20130508 |