CN109136113A - A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method - Google Patents
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method Download PDFInfo
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- CN109136113A CN109136113A CN201811345409.1A CN201811345409A CN109136113A CN 109136113 A CN109136113 A CN 109136113A CN 201811345409 A CN201811345409 A CN 201811345409A CN 109136113 A CN109136113 A CN 109136113A
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Abstract
The invention discloses a kind of sugarcane top rot pathogenic bacteria protoplast preparation and regeneration method, this method specific steps are as follows: (1) sugarcane top rot pathogen spore suspension preparation；(2) preparation of fresh mycelia；(3) configuration of lywallzyme；(4) enzymatic hydrolysis of hyphal cell wall；(5) protoplast is collected；(6) protoplast regeneration；(7) investigation of situation is regenerated.The present invention is at low cost, it is easy to operate, it is low for equipment requirements, enzymolysis time and regeneration period are short simultaneously, substantially increase the preparation efficiency of sugarcane top rot pathogenic bacteria protoplast, protoplast activity is preferably maintained, there is stronger power of regeneration, be conducive to the screening of the genetic transformation and transformant of subsequent pathogen.
Description
Technical field
The present invention relates to the preparation of fungal protoplasts in molecule plant pathology and technical field of regeneration, and in particular to one
The preparation of kind sugarcane top rot pathogenic bacteria protoplast and regeneration method.
Background technique
Sugarcane top rot (Pokkah Boeng) is one as caused by sickle-like bacteria (Gibberella fujikuroi Saw.)
Kind fungal disease.Cause the sickle-like bacteria of sugarcane top rot many kinds of, including Fusarium moniliforme,
The dominant bacteria of Fusarium proliferatum, Fusarium verticillioides etc., different regions sickle-like bacteria are different,
The sugarcane top rot pathogen of China's discovery is mainly based on colyliform sickle mycete (Fusarium verticillioides).It is sweet
Sugarcane top rot pathogen, which mainly passes through air-flow and travels to sugarcane lobus cardiacus and infect, causes harm, and causes blade dead, plant strain growth is obstructed, sternly
Weight can cause plant dead, lead to sugarcane underproduction 5%-20%, and cane sugar reduces by 3%, the shadow caused by susceptible sugar cane breed
Sound is more significant.Each sugarcane production area of China, which has, occurs top rot harm, and in recent years, sugarcane top rot is in the vast sugarcane district in China
Present the trend gradually aggravated.
Currently, carrying out the important set that molecular genetic conversion has become fungal transformation and pathogenesis using protoplast
At part, molecule conversion, which is necessarily dependent upon, prepares efficient, activity height, reproducible protoplast.For sugarcane top rot cause of disease
There has been no relevant reports for the preparation of bacterium protoplast, in order to screen sugarcane top rot pathogen Variation of Virulence bacterial strain, reinforcement pair
Pathogen pathogenic mechanism is studied, and is provided fundamental basis, is needed primary to sugarcane top rot pathogen for sugarcane breeding for disease resistance
The preparation of plastid is studied with regeneration.
Summary of the invention
In order to overcome the deficiencies in the prior art described above, it is former that it is an object of that present invention to provide a kind of sugarcane top rot pathogens
Raw plastid preparation and regeneration method.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
It is activated in base, then the mycelium inoculation at picking colony edge is trained in constant-temperature table in potato glucose water PDW culture medium
It supports, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is cultivated in constant-temperature table, sterilized lens wiping paper filtering obtains fresh mycelia, with pre-cooling 0.8mol/L NaCl solution
Sufficiently washing；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, cultivates in constant-temperature table,
It is primary that period per half an hour carries out microexamination；Protoplast production is observed, avoiding excessively digesting leads to protoplast
Rupture.
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, abandons supernatant, dissolved and precipitated with sorbitol solution STC,
And count under an optical microscope, adjusting protoplast concentration is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, in -80
It is saved backup in DEG C refrigerator；
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in constant incubator and train
It supports；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws 200 μ l protoplast dilutions and be coated on Ma Ling
In potato agar glucose PDA culture medium, as control；
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
As a preferred option of the technical scheme, the potato dextrose agar PDA culture medium, potato glucose water PDW training
It supports base, regeneration culture medium and sucrose solution to use after 121 DEG C of high pressure sterilization 20min, the potato dextrose agar PDA
Culture medium, potato glucose water PDW culture medium contain 100 μ g/ml ampicillins in regeneration culture medium.
As a preferred option of the technical scheme, pathogen is inoculated in potato dextrose agar PDA culture medium by the step (1)
Middle activation 3-4d, then the mycelium inoculation at picking colony edge is in 100ml potato glucose water PDW culture medium, in revolving speed
For 220rpm, 3~4d is cultivated in the constant-temperature table that temperature is 28 DEG C.
As a preferred option of the technical scheme, the potato glucose water PDW culture medium in the step (2) after inoculation spore suspension
It is 220rpm in revolving speed, the time cultivated in the constant-temperature table that temperature is 28 DEG C is 18-20h.
As a preferred option of the technical scheme, the 0.8mol/L NaCl solution is slow with the phosphoric acid of 0.1mol/L PH 5.5-6.0
Fliud flushing dissolution NaCl be formulated, after 121 DEG C of high pressure sterilization 20min, be placed in 4 DEG C of refrigerators carry out pre-cooling save backup.
As a preferred option of the technical scheme, when the sterilizing lens wiping paper filters, the sterilizing lens wiping paper number of plies is 4-6 layers.
As a preferred option of the technical scheme, the aperture of miillpore filter is 0.22 μm in the step (3).
As a preferred option of the technical scheme, the time being centrifugated in the step (5) is 10min, revolving speed 5000rpm.
As a preferred option of the technical scheme, in the step (5) sorbitol solution STC preparation method are as follows: first configured pH
It is 7.5, the Tris-HCl for the 10mmol/L that concentration is, then add sorbierite and CaCl2, make sorbitol concentration 1.2mol/L,
CaCl2Concentration is 10mmol/L；Configured sorbitol solution STC is placed in 4 DEG C of refrigerators after 121 DEG C of high pressure sterilization 20min
In save backup.
As a preferred option of the technical scheme, regeneration culture medium is to be added to 0.8mol/L sucrose solution in the step (6)
Potato dextrose agar.
As a preferred option of the technical scheme, the regeneration culture medium in step (6) after protoplast is coated with is placed in 28 DEG C of perseverances
3-5d is cultivated in warm incubator.
Potato dextrose agar PDA culture medium in the present invention, potato glucose water PDW culture medium are using existing
There is disclosed configured.
The invention has the following advantages:
(1) present invention is at low cost, easy to operate, low for equipment requirements, while enzymolysis time and regeneration period are short, mention significantly
The high preparation efficiency of sugarcane top rot pathogenic bacteria protoplast, preferably maintains protoplast activity, have it is stronger again
Raw ability, is conducive to the screening of the genetic transformation and transformant of subsequent pathogen.
(2) the protoplast quantity using the method for the invention preparation is more, and quantity can reach 2.7 × 107A/ml is living
Property keep preferably, power of regeneration is strong, and regeneration rate reaches as high as 24.8%.
(3) time of the potato glucose water PDW culture medium constant temp culture of inoculation spore suspension of the invention is 18h progress
Mycelial culture selects the time to be because mycelium at this time, which is just sprouted from spore, to be come out, is in fast growing period,
Very young tender, the protoplast quantity of release is more, is not easy to crack, and activity is high；And culture medium is just totally consumed at this time, is filled
Divide and utilizes resource.
(4) present invention carry out bacterium silk cell wall enzymatic hydrolysis lywallzyme concentration be 10mg/ml, the concentration lywallzyme it is dense
Spend it is low, it is at low cost using the enzymatic hydrolysis that cell wall can be realized on a small quantity, it is high-efficient, it is easy to operate.
(5) the method for the invention selects 0.8mol/L NaCl solution as homeo-osmosis agent, to maintain plasm
The osmotic balance of inside and outside, at the same under the concentration lywallzyme the available further raising of activity, improve enzymatic hydrolysis
Rate.
Detailed description of the invention
Fig. 1 is the observation figure of protoplast of the present invention under the microscope.
Specific embodiment
In order to make those skilled in the art better understand the technical solution in the application, come below in conjunction with embodiment
Technical solution of the present invention is clearly and completely described, it is clear that described embodiment is only a part of the application
Embodiment, based on the embodiment in the application, those of ordinary skill in the art are obtained without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present application.
Embodiment 1
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
3d is activated in base, then the mycelium inoculation at picking colony edge is in revolving speed in potato glucose water PDW culture medium
220rpm, temperature is cultivates 3d in 28 DEG C of constant-temperature table, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is 220rpm in revolving speed, 20h is cultivated in the constant-temperature table that temperature is 28 DEG C, sterilized lens wiping paper filtering obtains fresh
Mycelium is sufficiently washed with pre-cooling 0.8mol/L NaCl solution；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；The phosphate buffer of 0.8mol/L NaCl solution 0.1mol/L PH 5.5
Dissolution NaCl is formulated, and after 121 DEG C of high pressure sterilization 20min, is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, in revolving speed be 80rpm, temperature
For degree to cultivate 3h in 28 DEG C of constant-temperature table, during which progress of per half an hour microexamination is primary, can be under an optical microscope
Observe protoplast production (as shown in Figure 1), from figure 1 it appears that prepared protoplast quantity is more, activity
Keep preferable；
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, and the time of centrifuge separation is 10min, and revolving speed is
5000rpm abandons supernatant, is dissolved and is precipitated with sorbitol solution STC, and counted under an optical microscope, and it is dense to adjust protoplast
Degree is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, is saved backup in -80 DEG C of refrigerators；The preparation of sorbitol solution STC
Method are as follows: first having configured pH is 7.5, the Tris-HCl for the 10mmol/L that concentration is, then adds sorbierite and CaCl2, make sorb
Determining alcohol is 1.2mol/L, CaCl2Concentration is 10mmol/L；Configured sorbitol solution STC is in 121 DEG C of high pressure sterilizations
After 20min, it is placed in 4 DEG C of refrigerators and saves backup
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in 28 DEG C of constant incubator
Middle culture 3d；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws the coating of 200 μ l protoplast dilutions
In in potato dextrose agar PDA culture medium, as control；Regeneration culture medium is the horse for being added to 0.8mol/L sucrose solution
The solid medium of bell potato agar glucose PDA.
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
Used potato dextrose agar PDA culture medium in the present embodiment, potato glucose water PDW culture medium,
Regeneration culture medium and sucrose solution need to use after 121 DEG C of high pressure sterilization 20min；Potato dextrose agar PDA culture medium,
Contain 100 μ g/ml ampicillins in potato glucose water PDW culture medium, regeneration culture medium.
Using the method for the present embodiment, the protoplast quantity finally obtained reaches 0.7 × 107A/ml, regeneration rate reach
20.2%.
Embodiment 2
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
3d is activated in base, then the mycelium inoculation at picking colony edge is in revolving speed in potato glucose water PDW culture medium
220rpm, temperature is cultivates 3d in 28 DEG C of constant-temperature table, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is 220rpm in revolving speed, 18h is cultivated in the constant-temperature table that temperature is 28 DEG C, sterilized lens wiping paper filtering obtains fresh
Mycelium is sufficiently washed with pre-cooling 0.8mol/L NaCl solution；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；The phosphate buffer of 0.8mol/L NaCl solution 0.1mol/L PH 5.5
Dissolution NaCl is formulated, and after 121 DEG C of high pressure sterilization 20min, is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, in revolving speed be 80rpm, temperature
Degree is cultivates 3h in 28 DEG C of constant-temperature table, and during which per half an hour, it is primary to carry out microexamination；
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, and the time of centrifuge separation is 10min, and revolving speed is
5000rpm abandons supernatant, is dissolved and is precipitated with sorbitol solution STC, and counted under an optical microscope, and it is dense to adjust protoplast
Degree is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, is saved backup in -80 DEG C of refrigerators；The preparation of sorbitol solution STC
Method are as follows: first having configured pH is 7.5, the Tris-HCl for the 10mmol/L that concentration is, then adds sorbierite and CaCl2, make sorb
Determining alcohol is 1.2mol/L, CaCl2Concentration is 10mmol/L；Configured sorbitol solution STC is in 121 DEG C of high pressure sterilizations
After 20min, it is placed in 4 DEG C of refrigerators and saves backup
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in 28 DEG C of constant incubator
Middle culture 3d；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws the coating of 200 μ l protoplast dilutions
In in potato dextrose agar PDA culture medium, as control；Regeneration culture medium is the horse for being added to 0.8mol/L sucrose solution
The solid medium of bell potato agar glucose PDA
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
Used potato dextrose agar PDA culture medium in the present embodiment, potato glucose water PDW culture medium,
Regeneration culture medium and sucrose solution need to use after 121 DEG C of high pressure sterilization 20min；Potato dextrose agar PDA culture medium,
Contain 100 μ g/ml ampicillins in potato glucose water PDW culture medium, regeneration culture medium.
Using the method for the present embodiment, the protoplast quantity finally obtained reaches 2.7 × 107A/ml, regeneration rate reach
24.8%.
Embodiment 3
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
4d is activated in base, then the mycelium inoculation at picking colony edge is in revolving speed in potato glucose water PDW culture medium
220rpm, temperature is cultivates 4d in 28 DEG C of constant-temperature table, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is 220rpm in revolving speed, 18h is cultivated in the constant-temperature table that temperature is 28 DEG C, sterilized lens wiping paper filtering obtains fresh
Mycelium is sufficiently washed with pre-cooling 0.8mol/L NaCl solution；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；The phosphate buffer of 0.8mol/L NaCl solution 0.1mol/L PH 6.0
Dissolution NaCl is formulated, and after 121 DEG C of high pressure sterilization 20min, is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, in revolving speed be 80rpm, temperature
Degree is cultivates 4h in 28 DEG C of constant-temperature table, and during which per half an hour, it is primary to carry out microexamination；
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, and the time of centrifuge separation is 10min, and revolving speed is
5000rpm abandons supernatant, is dissolved and is precipitated with sorbitol solution STC, and counted under an optical microscope, and it is dense to adjust protoplast
Degree is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, is saved backup in -80 DEG C of refrigerators；The preparation of sorbitol solution STC
Method are as follows: first having configured pH is 7.5, the Tris-HCl for the 10mmol/L that concentration is, then adds sorbierite and CaCl2, make sorb
Determining alcohol is 1.2mol/L, CaCl2Concentration is 10mmol/L；Configured sorbitol solution STC is in 121 DEG C of high pressure sterilizations
After 20min, it is placed in 4 DEG C of refrigerators and saves backup
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in 28 DEG C of constant incubator
Middle culture 5d；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws the coating of 200 μ l protoplast dilutions
In in potato dextrose agar PDA culture medium, as control；Regeneration culture medium is the horse for being added to 0.8mol/L sucrose solution
The solid medium of bell potato agar glucose PDA
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
Used potato dextrose agar PDA culture medium in the present embodiment, potato glucose water PDW culture medium,
Regeneration culture medium and sucrose solution need to use after 121 DEG C of high pressure sterilization 20min；Potato dextrose agar PDA culture medium,
Contain 100 μ g/ml ampicillins in potato glucose water PDW culture medium, regeneration culture medium.
The protoplast quantity finally obtained reaches 2.2 × 107A/ml, regeneration rate reach 23.6%.
Embodiment 4
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
4d is activated in base, then the mycelium inoculation at picking colony edge is in revolving speed in potato glucose water PDW culture medium
220rpm, temperature is cultivates 4d in 28 DEG C of constant-temperature table, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is 220rpm in revolving speed, 20h is cultivated in the constant-temperature table that temperature is 28 DEG C, sterilized lens wiping paper filtering obtains fresh
Mycelium is sufficiently washed with pre-cooling 0.8mol/L NaCl solution；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；The phosphate buffer of 0.8mol/L NaCl solution 0.1mol/L PH 5.8
Dissolution NaCl is formulated, and after 121 DEG C of high pressure sterilization 20min, is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, in revolving speed be 80rpm, temperature
Degree is cultivates 3h in 28 DEG C of constant-temperature table, and during which per half an hour, it is primary to carry out microexamination；
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, it is dissolved and is precipitated with sorbitol solution STC, and in optics
It is counted under microscope, adjusting protoplast concentration is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, in -80 DEG C of refrigerators
It saves backup；The preparation method of sorbitol solution STC are as follows: first having configured pH is 7.5, the Tris- for the 10mmol/L that concentration is
HCl, then add sorbierite and CaCl2, make sorbitol concentration 1.2mol/L, CaCl2Concentration is 10mmol/L；Configured mountain
Pears alcoholic solution STC is placed in 4 DEG C of refrigerators and saves backup after 121 DEG C of high pressure sterilization 20min
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in 28 DEG C of constant incubator
Middle culture 5d；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws the coating of 200 μ l protoplast dilutions
In in potato dextrose agar PDA culture medium, as control；Regeneration culture medium is the horse for being added to 0.8mol/L sucrose solution
The solid medium of bell potato agar glucose PDA
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
Used potato dextrose agar PDA culture medium in the present embodiment, potato glucose water PDW culture medium,
Regeneration culture medium and sucrose solution need to use after 121 DEG C of high pressure sterilization 20min；Potato dextrose agar PDA culture medium,
Contain 100 μ g/ml ampicillins in potato glucose water PDW culture medium, regeneration culture medium.
The protoplast quantity finally obtained reaches 0.5 × 107A/ml, regeneration rate reach 22.3%.
Embodiment 5
A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method, include the following steps,
(1) pathogen the preparation of sugarcane top rot pathogen spore suspension: is inoculated in potato dextrose agar PDA culture
3d is activated in base, then the mycelium inoculation at picking colony edge is in revolving speed in potato glucose water PDW culture medium
220rpm, temperature is cultivates 4d in 28 DEG C of constant-temperature table, centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW training
It supports in base, is 220rpm in revolving speed, 19h is cultivated in the constant-temperature table that temperature is 28 DEG C, sterilized lens wiping paper filtering obtains fresh
Mycelium is sufficiently washed with pre-cooling 0.8mol/L NaCl solution；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml with pre-cooling 0.8mol/L NaCl solution
Enzyme solution, it is spare after filtering with microporous membrane degerming；The phosphate buffer of 0.8mol/L NaCl solution 0.1mol/L PH 5.6
Dissolution NaCl is formulated, and after 121 DEG C of high pressure sterilization 20min, is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, in revolving speed be 80rpm, temperature
Degree is cultivates 4h in 28 DEG C of constant-temperature table, and during which per half an hour, it is primary to carry out microexamination；
(5) protoplast is collected: the enzymolysis liquid with sterilizing lens wiping paper filtration step (4) is residual to remove the mycelia not digested
Body washs filtrate with pre-cooling 0.8mol/L NaCl solution, is centrifugated, and the time of centrifuge separation is 10min, and revolving speed is
5000rpm abandons supernatant, is dissolved and is precipitated with sorbitol solution STC, and counted under an optical microscope, and it is dense to adjust protoplast
Degree is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, is saved backup in -80 DEG C of refrigerators；The preparation of sorbitol solution STC
Method are as follows: first having configured pH is 7.5, the Tris-HCl for the 10mmol/L that concentration is, then adds sorbierite and CaCl2, make sorb
Determining alcohol is 1.2mol/L, CaCl2Concentration is 10mmol/L；Configured sorbitol solution STC is in 121 DEG C of high pressure sterilizations
After 20min, it is placed in 4 DEG C of refrigerators and saves backup
(6) protoplast regeneration: taking the Protoplast suspension prepared to dilute 100 times with 0.8mol/L sucrose solution,
With direct rubbing method, draws 200 μ l protoplast dilutions and be coated on regeneration culture medium, be placed in 28 DEG C of constant incubator
Middle culture 4d；It takes isometric Protoplast suspension to be diluted with sterile water simultaneously, draws the coating of 200 μ l protoplast dilutions
In in potato dextrose agar PDA culture medium, as control；Regeneration culture medium is the horse for being added to 0.8mol/L sucrose solution
The solid medium of bell potato agar glucose PDA
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B is then primary
Plastid regeneration rate=(A-B) × 100/ protoplast sum × 100%.
Used potato dextrose agar PDA culture medium in the present embodiment, potato glucose water PDW culture medium,
Regeneration culture medium and sucrose solution need to use after 121 DEG C of high pressure sterilization 20min；Potato dextrose agar PDA culture medium,
Contain 100 μ g/ml ampicillins in potato glucose water PDW culture medium, regeneration culture medium.
The protoplast quantity finally obtained reaches 1.6 × 107A/ml, regeneration rate reach 22.1%.
To sum up, the time for being inoculated with the potato glucose water PDW culture medium constant temp culture of spore suspension is that 18h carries out mycelium
Culture, more protoplasts can be generated, obtain higher regeneration rate.
Claims (10)
1. a kind of sugarcane top rot pathogenic bacteria protoplast preparation and regeneration method, it is characterised in that: include the following steps,
(1) preparation of sugarcane top rot pathogen spore suspension: pathogen is inoculated in potato dextrose agar PDA culture medium
Activation, then the mycelium inoculation at picking colony edge is cultivated in constant-temperature table in potato glucose water PDW culture medium,
Centrifugal process collects thallus, and sterile water is configured to spore suspension；
(2) preparation of fresh mycelia: prepared spore suspension 1ml is taken to be inoculated in 100ml potato glucose water PDW culture medium
In, it is cultivated in constant-temperature table, sterilized lens wiping paper filtering obtains fresh mycelia, abundant with pre-cooling 0.8mol/L NaCl solution
Washing；
(3) configuration of lywallzyme: weighing 100mg lywallzyme, is configured to 10mg/ml enzyme solution with pre-cooling 0.8mol/L NaCl solution,
It is spare after filtering with microporous membrane degerming；
(4) enzymatic hydrolysis of hyphal cell wall: taking 0.5g fresh mycelia, and 10ml lywallzyme is added, cultivates in constant-temperature table, during which
It is primary that per half an hour carries out microexamination；
(5) protoplast is collected: with the enzymolysis liquid of sterilizing lens wiping paper filtration step (4) to remove the mycelia residuum not digested,
Filtrate is washed with pre-cooling 0.8mol/L NaCl solution, is centrifugated, abandons supernatant, dissolved and precipitated with sorbitol solution STC, and
It counts under an optical microscope, adjusting protoplast concentration is 1 × 107A/ml is sub-packed in 1.5ml centrifuge tube, in -80 DEG C
It is saved backup in refrigerator；
(6) protoplast regeneration: taking the Protoplast suspension for preparing to dilute 100 times with 0.8mol/L sucrose solution, with straight
Rubbing method is connect, 200 μ l protoplast dilutions is drawn and is coated on regeneration culture medium, be placed in constant incubator and cultivate；Together
When take isometric Protoplast suspension to be diluted with sterile water, draw 200 μ l protoplast dilutions be coated on potato Portugal
In grape sugar agar PDA culture medium, as control；
(7) it regenerates the investigation of situation: setting the clump count grown on regeneration culture medium as A, control clump count B, then protoplast
Regeneration rate=(A-B) × 100/ protoplast sum × 100%.
2. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
The potato dextrose agar PDA culture medium, potato glucose water PDW culture medium, regeneration culture medium and sucrose solution warp
It is used after crossing 121 DEG C of high pressure sterilization 20min；The potato dextrose agar PDA culture medium, potato glucose water PDW training
Contain 100 μ g/ml ampicillins in feeding base, regeneration culture medium.
3. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
Pathogen is inoculated in potato dextrose agar PDA culture medium by the step (1) activates 3-4d, then picking colony edge
Mycelium inoculation in 100ml potato glucose water PDW culture medium, be 220rpm in revolving speed, temperature is that 28 DEG C of constant temperature shakes
3~4d is cultivated in bed.
4. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
Potato glucose water PDW culture medium in the step (2) after inoculation spore suspension is 220rpm in revolving speed, and temperature is 28 DEG C
The time cultivated in constant-temperature table is 18-20h.
5. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
The 0.8mol/L NaCl solution is formulated with the phosphate buffer of 0.1mol/L PH 5.5-6.0 dissolution NaCl, in 121
After DEG C high pressure sterilization 20min, it is placed in 4 DEG C of refrigerators and is pre-chilled, saved backup.
6. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
It is 80rpm that fresh mycelia, which is added after lywallzyme in revolving speed, in the step (4), is cultivated in the constant-temperature table that temperature is 28 DEG C
Time is 3-4h.
7. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
The time being centrifugated in the step (5) is 10min, revolving speed 5000rpm.
8. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
The preparation method of sorbitol solution STC in the step (5) are as follows: first having configured pH is 7.5, the 10mmol/L that concentration is
Tris-HCl, then add sorbierite and CaCl2, make sorbitol concentration 1.2mol/L, CaCl2Concentration is 10mmol/L；It configures
Sorbitol solution STC after 121 DEG C of high pressure sterilization 20min, be placed in 4 DEG C of refrigerators and save backup.
9. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, it is characterised in that:
Regeneration culture medium is the solid training for being added to the potato dextrose agar PDA of 0.8mol/L sucrose solution in the step (6)
Support base.
10. sugarcane top rot pathogenic bacteria protoplast preparation according to claim 1 and regeneration method, feature exist
In: the regeneration culture medium in step (6) after protoplast is coated with is placed in 28 DEG C of constant incubators and cultivates 3-5d.
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| CN113462633A (en) * | 2021-08-16 | 2021-10-01 | 广西大学 | Method for separating and extracting protoplast of sugarcane young leaf in different development stages |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113462633A (en) * | 2021-08-16 | 2021-10-01 | 广西大学 | Method for separating and extracting protoplast of sugarcane young leaf in different development stages |
| CN113462633B (en) * | 2021-08-16 | 2023-05-05 | 广西大学 | Method for separating and extracting protoplast of sugarcane young leaf in different development periods |
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