CN102119629A - Mutagenesis method for new low-temperature type bacterial strain of straw mushroom and application thereof - Google Patents
Mutagenesis method for new low-temperature type bacterial strain of straw mushroom and application thereof Download PDFInfo
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- CN102119629A CN102119629A CN2010105234286A CN201010523428A CN102119629A CN 102119629 A CN102119629 A CN 102119629A CN 2010105234286 A CN2010105234286 A CN 2010105234286A CN 201010523428 A CN201010523428 A CN 201010523428A CN 102119629 A CN102119629 A CN 102119629A
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Abstract
The invention provides a mutagenesis method for a new low-temperature type bacterial strain of straw mushroom and an application thereof. The method comprises the following steps: inoculating the straw mushroom mycelium on a solid medium containing lithium chloride; irradiating ultraviolet rays; repeatedly treating at 0 DEG C and screening; acquiring a mutagenesis strain; and performing a mushroom-outgoing test, thereby acquiring the low-temperature type bacterial strain of straw mushroom. By using the method provided by the invention, the straw mushroom mycelium can be efficiently induced, thereby acquiring the new low-temperature type bacterial strain of straw mushroom. The invention provides a new breeding method for the inheritance breeding of straw mushroom, namely performing compound mutagenesis by using lithium chloride and ultraviolet rays, and also provides a technical reference for mutagenesis of other edible mushrooms.
Description
Technical field
The invention belongs to edible mushroom mutation breeding technical field, be specifically related to a kind of special method of mutagenesis and the application of the new bacterial strain of a kind of low form straw mushroom.
Background technology
Straw mushroom is a kind of edible mushroom, and is nutritious, and delicious flavour is liked by consumers in general deeply.
Straw mushroom is a typical tropical high temperature modification edible mushroom, mycelia is poor to the resistance of low temperature, especially variation of temperature is very sensitive to the variation of environmental condition: when mean temperature of air below 27 ℃ the time, the straw mushroom fruit body is difficult to form, the utmost point is unfavorable for growing of straw mushroom, greatly affect the yield and quality of straw mushroom, sometimes even cause total crop failure.So to the north of China the Changjiang river the area laundering period shorter, greatly restricted the cultivation scope of straw mushroom.Simultaneously, straw mushroom exists damages to plants caused by sudden drop in temperature, and promptly preserves under 4 ℃ cryogenic conditions, and mycelia is understood autolyze and causes bacterial classification death and fruit body to feel like jelly, liquefy until rotting.This preserves for bacterial classification of straw mushroom and fresh-keeping brought great difficulty postpartum.In addition, the biology efficient of straw mushroom is low.Therefore, the straw mushroom new varieties of cultivation high-yield of low-temperature just seem particularly important.Yet straw mushroom has the very complicated history of life for binding to fungal of the same clan, and mycelia does not have clamp connection, and hybrid selects to lack mark, and this brings great difficulty for the crossbreeding of straw mushroom, and the strain excellent of straw mushroom industry is very poor for a long time.
Prior art has only the mutagenesis of adopting single method about the domestic mutagenesis for straw mushroom of straw mushroom mutagenesis, as ultraviolet mutagenesis or chemical reagent mutagenesis, mutagenesis unstable result.
Summary of the invention
The objective of the invention is to overcome the deficiency of existing straw mushroom induced-mutation technique, a kind of new composite mutagenesis method is provided, make the straw mushroom gene produce variation, in conjunction with 0 ℃ of suitable low temperature screening, thus the new bacterial strain of low form straw mushroom of acquisition genetic stability.
Purpose of the present invention is achieved by the following technical programs:
The method of mutagenesis of the new bacterial strain of a kind of low form straw mushroom is provided, may further comprise the steps:
(1) with the straw mushroom inoculated by hypha block to the solid PDSA flat board that contains 0.6% (W/V) lithium chloride;
Preferred 0.1 * 0.1cm the size of described straw mushroom mycelia piece; Specifically be that the straw mushroom surface mycelia that 32 ℃ of constant incubators can be cultivated 3d scrapes off, the thin inoculated by hypha block that cuts middle and upper part 0.1 * 0.1cm size is to the solid PDSA flat board that contains 0.6% (W/V) lithium chloride.
(2) the solid PDSA flat board that contains lithium chloride that step (1) is connected to the mycelia piece places under the ultraviolet light after the treatment with irradiation lucifuge to cultivate;
The solid PDSA flat board that contains lithium chloride that preferably will be connected to the mycelia piece places the 30cm place irradiation 50min apart from the 15W ultraviolet lamp tube, each level do three groups parallel, wrap with masking foil, lucifuge is cultivated 3d for 30 ℃.Open the about 20min of preheating earlier before the described ultra violet lamp, make light wave stable.
(3) the straw mushroom mycelium that will recover to grow after step (2) is handled cuts into thin piece, is transferred to new PDSA flat board, and places 0 ℃ of low temperature treatment 36～48h, and 28～33 ℃ of normal cultivations are screened.
Preferably, the straw mushroom mycelium that recovers growth is cut into the thin piece of 0.1 * 0.1cm size.
(4) the straw mushroom mycelia piece with survival in the step (3) continues to cut into thin small bacteria block, be forwarded on the fresh PDSA flat board, and (3) middle method screening set by step.The mycelia of survival is repeated screening with identical method, and the mycelia that can survive at last is mutagenic fungi; Preferably repeat to screen 3～6 times.
In the methods of the invention, used medium is conventional straw mushroom cultural hypha base, and filling a prescription is:
PDSA: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, agar 20g, 1 liter in water.
Containing 0.6% (W/V) lithium chloride solid culture based formulas is: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, lithium chloride 6g, agar 20g, 1 liter in water.
Method of the present invention can make the straw mushroom mycelium morph effectively, therefore also is applicable to the mutagenesis of other mycelium of edible fungus bodies, as Asparagus, Xingbao mushroom, flat mushroom etc.
Ultraviolet ray is a kind of Non-ionizing radiation, can cause in the DNA chain break, dna molecular and intermolecular crosslinked, nucleic acid and protein crosslinked, particularly form pyrimidine dimer, hinder the unwinding of dna double chain, duplicate the normal pairing with base, thereby cause gene mutation.Owing to have photoreactivation during ultraviolet mutagenesis, therefore independent ultraviolet mutagenesis DeGrain.Lithium chloride is a kind of alkali halide, can cause the conversion of AT-GC base-pair or cause the disappearance of base, does not also have tangible mutagenesis effect when using separately.Usually adopt the combination of two kinds of methods, the mutagenic strain that promptly carries out obtaining behind the ultraviolet mutagenesis earlier re-uses and is carrying out ultraviolet mutagenesis after LiCl carries out mutagenesis or uses the mutagenic obtained mutagenic strain of LiCl earlier, yet not only the combinatorial mutagenesis that adopts this mode does not make the cold resistance of straw mushroom mutant strain strengthen, and also makes the mutant strain low temperature tolerance ability forfeiture that has obtained sometimes.
The present invention obtains new suitable mutagenesis scheme through creationary experimental summary, has produced following beneficial effect:
The inventive method adopts individual event mutagenesis and two kinds of different mutagenesis experiments of conventional combination of ultraviolet, LiCl and other mutagen, fails the new bacterial strain of cold-resistant straw mushroom that obtains to expect.Through long-term a large amount of experimental study and analysis and summary, the present invention proposes technical solution of the present invention and successfully obtains good low temperature resistant new bacterial strain, by the inventive method starting strain directly is seeded in and contains on the 0.6%LiCl medium, adopt ultraviolet irradiation 50min simultaneously, through 3～6 0 ℃ of low temperature treatment repeatedly, obtain the low temperature resistant straw mushroom mutant strain of energy genetic stability again.The present invention fills up the blank of the effective mutation breeding of edible mushroom, for effective mutation breeding of edible mushroom provides the scientific theory foundation.
Description of drawings
After Fig. 1 is V138 straw mushroom mycelia process ultraviolet and lithium chloride complex mutation, the result through under 32 ℃ of temperature, growing behind 0 ℃ of low temperature treatment 48h;
Fig. 2 mutagenic fungi R6 of the present invention can grow under 18 ℃ of temperature on (a figure left side, among the figure for cultivating the bacterium colony of 3d), and contrast strain V138 under this temperature, can not grow (figure is right)
The new bacterial strain subobject graph that Fig. 3 mutagenesis obtains
Fig. 4 mutagenic strain R6 fruiting lab diagram
Fig. 5 control strain V138 is at 16 ℃ of low-temperature test figure
Fig. 6 mutagenic strain R6 fruit body is at 16 ℃ of low-temperature test figure
After Fig. 7 is straw mushroom mycelia process ultraviolet and lithium chloride complex mutation, the result through under 28 ℃ of temperature, growing behind 0 ℃ of low temperature treatment 36h;
Fig. 8 mutagenic fungi R5 can grow under 18 ℃ of temperature on (a figure left side, among the figure for cultivating the bacterium colony of 3d), and contrast strain V138 under this temperature, can not grow (figure is right)
The new bacterial strain R5 subobject graph that Fig. 9 mutagenesis obtains
Figure 10 mutagenic fungi R5 fruiting lab diagram
Figure 11 control strain V138 is at 16 ℃ of low-temperature test figure
Figure 12 mutagenic strain R5 fruit body at 16 ℃ of low-temperature test figure
Figure 13 adopts new bacterial strain R5 that the AFLP technology obtains mutagenesis and R6 and the contrast V138 difference qualification result at dna level
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.Employed experimental technique is the existing conventional method in present technique field if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are the reagent and the material that can obtain from commercial channels.V138 is the Guangzhou main breed, is provided by agricultural science experimental center, Baiyun District, Guangzhou City, and the visible edible mushroom magazine of relevant information inset is introduced.
Embodiment 1
(1) according to conventional method with the V138 bacterial classification inoculation on the PDSA flat board, cultivate 3d at 32 ℃ of constant incubators, scrape off the straw mushroom surface mycelia on the flat board, the thin inoculated by hypha block that cuts middle and upper part 0.1 * 0.1cm size is to the solid PDSA flat board that contains 0.6% (W/V) lithium chloride;
Described 0.6% (W/V) lithium chloride solid culture based formulas that contains is: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, lithium chloride 6g, agar 20g, 1 liter in water.
(2) open the about 20min of uviol lamp preheating earlier, make light wave stable.The solid PDSA flat board that contains lithium chloride that is connected to the mycelia piece is placed apart from the 30cm place of 15W ultraviolet lamp tube irradiation 50min, each level do three groups parallel, wrap with masking foil, lucifuge is cultivated 3d for 30 ℃.
(3) the straw mushroom mycelium that will recover to grow after step (2) is handled cuts into the thin piece of 0.1 * 0.1cm size, is transferred to new PDSA flat board, and normal the cultivation screened after placing 0 ℃ of low temperature treatment 48h; The present embodiment straw mushroom place under 32 ℃ of temperature, grow behind 0 ℃ of low temperature treatment 48h the results are shown in accompanying drawing 1;
As shown in Figure 1, the inventive method uses ultraviolet ray and LiCl that straw mushroom V138 is carried out complex mutation simultaneously, through 0 ℃ of low temperature repeated treatments and screening, mycelia sudden change or negative sudden change can not survive, thereby filters out the low temperature resistant straw mushroom mutagenic fungi of inheritance stability.Accompanying drawing 1 shows, can survive behind 0 ℃ of low temperature treatment 48h after mycelium morphs (arrow indication among the figure), and not have the mycelia of variation dead after 0 ℃ of low temperature treatment.
Described PDSA consists of: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, agar 20g, 1 liter in water.
Mutagenic fungi R6 of the present invention (left side) shown in the accompanying drawing 2 can grow mycelia behind 18 ℃ of cultivation 3d, and contrast strain V138 under this temperature, can not grow (figure is right), illustrate that mutagenic fungi can be in comparison according to growing under the low temperature, control strain can only be grown in the temperature more than 25 ℃.
The new bacterial strain R6 sporophore shape figure that accompanying drawing 3 obtains for mutagenesis.
The district test result figure of the new bacterial strain R6 that accompanying drawing 4 obtains for mutagenesis of the present invention.Show through continuous 3 district's examination experimental results: this bacterial strain fruiting early, fruiting is neat, output is high.Production cycle, comparison was according to short 2～3 days.
Shown in the accompanying drawing 5 result of control strain V138 preservation 48h under 16 ℃ of low temperature.Accompanying drawing 5 shows that fruit body cell rupture, the outflow Heisui River behind preservation 48h under 16 ℃ of low temperature of contrast V138 have lost commodity value.
Accompanying drawing 6 is results of mutagenic strain R6 fruit body preservation 48h under 16 ℃ of low temperature.Fruit body cell behind preservation 48h under 16 ℃ of low temperature of accompanying drawing 6 demonstration mutagenic strain R6 is kept perfectly and does not break, outward appearance is normal, have commodity value, proved absolutely that the inventive method can carry out mutagenesis to the straw mushroom mycelia effectively, obtain the low temperature resistant straw mushroom strain excellent of expection.
New strain morphology feature of R6 and growth characteristics are as follows:
Morphological feature: mycelium milky, fruit body light gray, the high 28.0～37.7mm of egg type phase fruit body, wide 20.7～26.0mm, bacteria cover diameter 15.52mm, mycoderm thickness 3.56mm, the long 15.52mm of stem.
Growth characteristics: mycelia in the growth rate under 30 ℃ of temperature is: 1.51cm/d on 1.93cm/d, the cotton seed hull medium on the PDSA flat board, 27 ℃ of fruit body development temperature, original hase forms (fruiting) time average 7.7d, production cycle 10d, and biological transformation ratio is high by 35～40%.
Embodiment 2
(1) according to conventional method with the V138 bacterial classification inoculation on the PDSA flat board, cultivate 3d at 32 ℃ of constant incubators, scrape off the straw mushroom surface mycelia on the flat board, the thin inoculated by hypha block that cuts middle and upper part 0.1 * 0.1cm size is to the solid PDSA flat board that contains 0.6% (W/V) lithium chloride;
Described 0.6% (W/V) lithium chloride solid culture based formulas that contains is: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, lithium chloride 6g, agar 20g, 1 liter in water.
(2) open the about 20min of uviol lamp preheating earlier, make light wave stable.The solid PDSA flat board that contains lithium chloride that is connected to the mycelia piece is placed apart from the 30cm place of 15W ultraviolet lamp tube irradiation 50min, each level do three groups parallel, wrap with masking foil, lucifuge is cultivated 3d for 28 ℃.
(3) the straw mushroom mycelium that will recover to grow after step (2) is handled cuts into the thin piece of 0.1 * 0.1cm size, is transferred to new PDSA flat board, and normal the cultivation screened after placing 0 ℃ of low temperature treatment 36h; The present embodiment straw mushroom place under 28 ℃ of temperature, grow behind 0 ℃ of low temperature treatment 36h the results are shown in accompanying drawing 7;
As shown in Figure 7, the inventive method uses ultraviolet ray and LiCl that straw mushroom V138 is carried out complex mutation simultaneously, through 0 ℃ of low temperature repeated treatments and screening, mycelia sudden change or negative sudden change can not survive, thereby filters out the low temperature resistant straw mushroom mutagenic fungi R5 of inheritance stability.Accompanying drawing 7 shows, can survive behind 0 ℃ of low temperature treatment 36h after the R5 mycelium morphs (arrow indication among the figure), and not have the mycelia of variation dead after 0 ℃ of low temperature treatment.
Described PDSA consists of: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, agar 20g, 1 liter in water.
Mutagenic fungi R5 of the present invention (left side) shown in the accompanying drawing 8 can grow mycelia behind 18 ℃ of cultivation 3d, and contrast strain V138 under this temperature, can not grow (figure is right), illustrate that mutagenic fungi can be in comparison according to growing under the low temperature, control strain can only be grown in the temperature more than 25 ℃.
The sporophore shape figure of the new bacterial strain R5 that accompanying drawing 9 obtains for mutagenesis.Accompanying drawing 10 is tested the diagram egg type phase for the scale district examination of the new bacterial strain R5 that mutagenesis of the present invention obtains.Through continuous 3 district examination experimental results show this bacterial strain fruiting early, grow thickly, the output height.Production cycle, comparison was according to short 2～3 days.
Shown in the accompanying drawing 11 result of control strain V138 preservation 48h under 16 ℃ of low temperature.Accompanying drawing 11 shows that fruit body cell rupture, the outflow Heisui River behind preservation 48h under 16 ℃ of low temperature of contrast V138 have lost commodity value.
Accompanying drawing 12 is results of preservation 48h under 16 ℃ of low temperature of the new bacterial strain fruit body that obtains of mutagenesis.Fruit body cell behind preservation 48h under 16 ℃ of low temperature of the new bacterial strain that accompanying drawing 12 demonstration mutagenesis obtain is kept perfectly and does not break, outward appearance is normal, have commodity value, proved absolutely that the inventive method can carry out mutagenesis to the straw mushroom mycelia effectively, obtain the low temperature resistant straw mushroom strain excellent of expection.Morphological feature and the growth characteristics of new bacterial strain R5 are as follows:
Morphological feature: mycelium milky, fruit body grey, the high 26.0～42.3mm of egg type phase fruit body, wide 20.3～30.0mm, bacteria cover diameter 17.67mm, mycoderm thickness 3.67mm, the long 16.56mm of stem.
Growth characteristics: mycelia in the growth rate under 30 ℃ of temperature is: 1.49cm/d on 1.84cm/d, the cotton seed hull medium on the PDSA flat board, 27 ℃ of fruit body development temperature, original hase forms (fruiting) time average 7.7d, production cycle 10d, and biological transformation ratio is high by 35～40%.
Accompanying drawing 13 is the new bacterial strains and the difference qualification result of contrast V138 at dna level that adopt the AFLP technology that mutagenesis is obtained.Accompanying drawing 13 has shown that new bacterial strain R5 (swimming lane 2) and R6 (swimming lane 3) that mutagenesis obtains have a tangible band difference with control strain V138 (swimming lane 1), illustrate that effective sudden change has taken place the new bacterial strain of two strains that uses the inventive method to obtain, and is new bacterial strain on dna level.M is DNA MarkerIII (size is respectively 4500bp, 3000bp, 2000bp, 1200bp, 600bp, 500bp, 200bp from top to down) among the figure.Simultaneously, experiment showed, that two new bacterial strains all are low temperature resistant, illustrate that the inventive method is stable on the mutagenesis effect of straw mushroom.
Claims (9)
1. the method for mutagenesis of the new bacterial strain of low form straw mushroom is characterized in that may further comprise the steps:
(1) with the straw mushroom inoculated by hypha block to the solid PDSA flat board that contains 0.6% (W/V) lithium chloride; Described 0.6% (W/V) lithium chloride solid culture based formulas that contains is: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, lithium chloride 6g, agar 20g, 1 liter in water;
(2) the solid PDSA flat board that contains lithium chloride that step (1) is connected to the mycelia piece places under the ultraviolet light after the treatment with irradiation lucifuge to cultivate;
(3) will handle the straw mushroom mycelium that recovers growth in the back through step (2) and cut into thin piece, be transferred to new PDSA flat board, place 0 ℃ of low temperature treatment after normal cultivation screen; Described PDSA plate pack becomes: peeling potato 200g, glucose 20g, KH
2PO
43g, MgSO
41.5g, pangamic acid～10mg, agar 20g, 1 liter in water;
(4) the straw mushroom mycelia piece with survival in the step (3) continues to cut into thin small bacteria block, be forwarded on the fresh PDSA flat board, and (3) middle method repeats screening set by step, and the mycelia of survival is mutagenic fungi.
2. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that the described straw mushroom mycelia of step (1) piece is 0.1 * 0.1cm size.
3. according to the method for mutagenesis of claim 1 or the new bacterial strain of 2 described low form straw mushrooms, it is characterized in that the described straw mushroom mycelia of step (1) is for cultivating 3d at 32 ℃ of constant incubators.
4. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that the described ultraviolet light of step (2) adopts the 15W ultraviolet lamp tube, described ultraviolet lamp tube is apart from straw mushroom mycelia piece 30cm, and irradiation time is 50min.
5. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that the thin block size of step (3) or the described straw mushroom mycelium of step (4) is 0.1 * 0.1cm.
6. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that described 0 ℃ of low temperature treatment time of step (3) is 36～48h.
7. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that the described normal cultivation temperature of step (3) is 28～33 ℃.
8. according to the method for mutagenesis of the new bacterial strain of the described low form straw mushroom of claim 1, it is characterized in that described to repeat to screen number of times be 3～6 times to step (4).
9. the application of the method for mutagenesis of the new bacterial strain of each described low form straw mushroom of claim 1～8 is characterized in that being applied to the mutation breeding aspect of Asparagus, Xingbao mushroom or flat mushroom.
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| CN103098649A (en) * | 2013-01-31 | 2013-05-15 | 上海市农业科学院 | Method capable of rapidly evaluating extent of low temperature injury of straw mushroom |
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| CN103081721A (en) * | 2013-01-25 | 2013-05-08 | 上海市农业科学院 | Method for fast screening low temperature resistant volvaria volvacea microorganism |
| CN103098649B (en) * | 2013-01-31 | 2014-02-19 | 上海市农业科学院 | Method capable of rapidly evaluating extent of low temperature injury of straw mushroom |
| CN103098649A (en) * | 2013-01-31 | 2013-05-15 | 上海市农业科学院 | Method capable of rapidly evaluating extent of low temperature injury of straw mushroom |
| CN103210791A (en) * | 2013-05-09 | 2013-07-24 | 河北大学 | Method for cultivating pleurotus citrinopileatus by using persimmon sawdust |
| CN103283484A (en) * | 2013-05-09 | 2013-09-11 | 河北大学 | Method for culturing hericium erinaceus by persimmon sawdust |
| CN103210791B (en) * | 2013-05-09 | 2015-04-22 | 河北大学 | Method for cultivating pleurotus citrinopileatus by using persimmon sawdust |
| CN103283484B (en) * | 2013-05-09 | 2015-04-22 | 河北大学 | Method for culturing hericium erinaceus by persimmon sawdust |
| 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 |
| CN103937683A (en) * | 2014-04-18 | 2014-07-23 | 丁荣兴 | Preparation method of straw mushroom mycelium containing organic iodine |
| CN103937683B (en) * | 2014-04-18 | 2016-09-28 | 丁荣兴 | A kind of Mycelia of Straw Mushroom, Volvariel volvacea preparation method containing organic iodine |
| 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: 20110713 |