CN109511550A - Seaweed protoplast is separately cultured the method with regeneration plant - Google Patents
Seaweed protoplast is separately cultured the method with regeneration plant Download PDFInfo
- Publication number
- CN109511550A CN109511550A CN201811494026.0A CN201811494026A CN109511550A CN 109511550 A CN109511550 A CN 109511550A CN 201811494026 A CN201811494026 A CN 201811494026A CN 109511550 A CN109511550 A CN 109511550A
- Authority
- CN
- China
- Prior art keywords
- seaweed
- protoplast
- cell
- regeneration plant
- separately cultured
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention provides seaweed protoplast and is separately cultured the method with regeneration plant, belong to field of algae culture, method particularly includes: selection color is normal, uses HgCl with hairbrush cleaning frond surface sludge, miscellaneous algae and other attached impurity without rotten, eugonic frond2Solution disinfection, then it is rinsed well using disinfected sea water, cut fresh frond top young tender part, surface moisture is blotted with filter paper, tiny tissue block or small branch are sheared or be ground into, is first impregnated in sodium potassium tartrate tetrahydrate and polyvinylpyrrolidone mixed solution, seaweed tool enzyme solutions is recycled to carry out digestion enzymatic hydrolysis, then through centrifugation, seaweed somatoplasm is obtained；Protoplast cuhnre is obtained into the cell of regenerative cell's wall, further regeneration plant can be obtained in culture.The problem of present invention provides a kind of cultural method for the seaweed that growing-seedling period is short, seed breeding is high-efficient, it is able to solve asexual reproduction class seaweed seed conservation and breeding, can be used for productive receivers.
Description
Technical field
The invention belongs to Algaculture method fields, and in particular to seaweed protoplast is separately cultured the side with regeneration plant
Method.
Background technique
Though algae produces offspring without constructions such as flower, fruit, seeds, there is miscellaneous mode of reproduction to adapt to environment.
In terms of asexual reproduction, some cells can directly be divided into two, and such as water silk floss, can be broken into several sections, every section is respectively grown into again
Independent individual；Some fronds can produce many spores amphitrichous, can freely move about, and respectively be grown to after each spore is mature
One new individual；When environment is bad, some algae can produce the resting spore of heavy wall, when waiting the environment to be suitable for, then sprout growth
The individual of Cheng Xin.In terms of zoogamy, some algae can produce female, andro gamete, and new individual is just grown up to via post-coitum.
The Chinese invention patent of existing 102144544 A of Publication No. CN, discloses a kind of Eucheuma seaweed plasm
Body is separately cultured the method with regeneration plant, mainly according to the totipotency of seaweed somatocyte development, utilizes seaweed toolenzyme
Eucheuma tissue is decomposed, dissociate somatoplasm body, obtains the body cell and cell line of regenerative cell's wall, is then trained
It supports and obtains regeneration plant；However, the invention enzymolysis efficiency is low, and protoplast cultural condition is rough, and it is numerous to be unfavorable for raising seed
Educate efficiency.
Summary of the invention
The purpose of the present invention is to provide seaweed protoplasts to be separately cultured the method with regeneration plant, provides a kind of nursery
The cultural method for the seaweed that period is short, seed breeding is high-efficient, it is able to solve asexual reproduction class seaweed seed conservation and breeding
The problem of, it can be used for productive receivers.
The technical solution that the present invention is taken to achieve the above object are as follows:
Seaweed protoplast is separately cultured the method with regeneration plant, mainly according to the all-round of seaweed somatocyte development
Property, seaweed is decomposed using seaweed toolenzyme and is organized, and dissociate somatoplasm body, obtains the body cell of regenerative cell's wall and thin
Born of the same parents system, is then cultivated to obtain regeneration plant.Specific step is as follows:
Step 1: selection color is normal, nothing is rotted, eugonic frond, frond surface sludge, miscellaneous algae are cleared up with hairbrush
With other attached impurity, with the HgCl of 0.005-0.008%2Solution disinfection 5-10s, is then rinsed well using disinfected sea water,
Fresh frond top young tender part is cut, blots surface moisture with filter paper, is sheared or be ground into tiny tissue block or small
Branch, first in sodium potassium tartrate tetrahydrate and polyvinylpyrrolidone (PVP) mixed solution impregnate 2-3h after, recycle seaweed tool
Enzyme solutions carry out digestion enzymatic hydrolysis, then through centrifugation, obtain seaweed somatoplasm；
Step 2: Protoplast cuhnre is obtained the cell of regenerative cell's wall, further regeneration plant is can be obtained in culture.
Preferably, sodium potassium tartrate tetrahydrate and PVP are configured to the solution that concentration is 0.1-0.5M and 3-6M respectively, body is then pressed
Product is mixed than 1:3-5；Sodium potassium tartrate tetrahydrate and PVP are mutually cooperateed with, and cell wall cellulose can be made to expand, thus
Accelerate cell wall lysis or rupture to cause cell plasmolysis, while can be improved the toughness of cell membrane, finally can be improved
The integrality of frond enzymolysis efficiency and the protoplast for ensuring to obtain.
Preferably, the operation of protoplast electrofusion are as follows: use 250-400 mesh thin,tough silk filtration cell mixed liquor, removing does not disappear
Cell, cell mass, the fragment of change；It takes supernatant 500-850rpm to be centrifuged 8-10min, so that unicellular (protoplast) is sunk, carefully
Born of the same parents' fragment stays in supernatant, discards supernatant liquid, is rinsed and is centrifuged 2-3 with the disinfected sea water containing 1.0-1.2M glucose
Secondary, collection precipitates up to seaweed protoplast.
Preferably, seaweed toolenzyme is the combination of sea snail enzymes or cellulase or both；It is highly preferred that the system of sea snail enzymes
Preparation Method are as follows: conch is cleaned and temporarily supports starvation 2-3 days in disinfected sea water, decladding takes its glandula digestive, is ground to homogenate, 10000-
15000rpm is centrifuged 5-10min, discards precipitating, the acetone of -10~-20 DEG C of pre-coolings is added in supernatant, and centrifugation discards precipitating, then
Repetition addition acetone, which is collected by centrifugation, precipitates to obtain original enzyme liquid, saves backup in -5~-20 DEG C；Original enzyme liquid using when be diluted to seawater
15-25%；The preparation method of cellulase are as follows: molten with the citric acid or acetate buffer solution (pH4.5-5.0) of 40-60mmol/L
Solution, concentration 10-15%, wherein acetate buffer solution is prepared with disinfected sea water.
Preferably, seaweed toolenzyme is in use, be added homeo-osmosis agent, homeo-osmosis agent is selected from mannitol, mountain
One of pears alcohol, glucose, sucrose, KCl, NaCl；Wherein, the suitable concentration of alcohols and carbohydrate be 1.2-2.6M, KCl,
The suitable concentration of NaCl is 0.5-0.8M.
Preferably, the condition of enzymatic hydrolysis are as follows: pH 5.5-7.5 digests 2-4.5h at 15-40 DEG C.
Preferably, Protoplast cuhnre mode are as follows: first by sodium alginate and protoplast mixed in equal amounts drop in calcium chloride
It is formed and is fixed in solution, inoculated in fluid nutrient medium and cultivate；It first keeps at 22-26 DEG C of temperature dark culture 5-7 days, then turns
For illumination cultivation, light application time 8-10h/d, light intensity 2500-3000Lx, every 7 days replacement half culture mediums；Wherein, liquid
Culture medium is to keep culture medium infiltration containing methyl α-naphthyl acetate and 2, the PES culture medium of 4-chlorophenoxyacetic acid, and with sweet dew alcohol and glucose
Pressure；It chelates to form calcium alginate gel not soluble in water using calcium chloride and seaweed acid ion, cell is fixed, can be had
Effect prevents protoplast from sticking together, and can carry out fixed point location observation to protoplast, tracks its growth course, is conducive to mention
High cell splitting rate.
Preferably, Protoplast cuhnre process adds tryptophan after cell carries out first division in the medium
And sodium ascorbate, and adjusting medium pH is 5.5-5.8, continues to cultivate；The additive amount of tryptophan and sodium ascorbate is training
Support the 1-5% and 0.5-2% of base weight amount；The special presence of tryptophan and sodium ascorbate, first is that being conducive to improve protoplast
Division frequency, accelerate cell division to form filamentous, thallus or callus, and then break up and generate plant；Second is that can
It effectively prevent issuable phenolic substances etc. during Protoplast cuhnre to aoxidize, keeps active material in dividing cell
Stability, improve the vigor of cell, promote protoplast differentiation and development, keep higher seed while shortening growing-seedling period
Breed efficiency.
The invention has the benefit that
1) before the present invention carries out digestion enzymatic hydrolysis to frond using seaweed toolenzyme, first frond is pre-processed: using wine
Stone acid potassium sodium and PVP concentration are respectively the mixed solution immersion frond that 0.1-0.5M and 3-6M are made into；Sodium potassium tartrate tetrahydrate and PVP phase
Mutually collaboration, can be such that cell wall cellulose expands, so that cell wall lysis or rupture be accelerated to cause cytoplasm wall point
From, while can be improved the toughness of cell membrane, finally can be improved frond enzymolysis efficiency and ensuring the protoplast of acquisition
Integrality；
2) training method of protoplast of the present invention uses: first dripping sodium alginate and protoplast mixed in equal amounts in chlorination
It is formed and is fixed in calcium solution, inoculated in fluid nutrient medium and cultivate；It chelates to be formed not using calcium chloride and seaweed acid ion
It is dissolved in the calcium alginate gel of water, cell is fixed, can effectively prevent protoplast and stick together, and can to protoplast
Fixed point location observation is carried out, its growth course is tracked, is conducive to improve cell splitting rate；
3) present invention adds color ammonia after cell carries out first division in Protoplast cuhnre process in the medium
Acid and sodium ascorbate, the special presence of tryptophan and sodium ascorbate, first is that be conducive to improve the division frequency of protoplast,
Accelerate cell division to form filamentous, thallus or callus, and then breaks up and generate plant；Second is that can effectively prevent primary
Issuable phenolic substances etc. aoxidizes in plastid incubation, keeps the stability of active material in dividing cell, mentions
The vigor of high cell promotes protoplast differentiation and development, keeps higher seed breeding efficiency while shortening growing-seedling period.
Present invention employs above-mentioned technical proposals to provide model essay, compensates for the deficiencies in the prior art, design is reasonable, operation side
Just.
Specific embodiment
Present invention is further described in detail with reference to embodiments:
Embodiment 1:
Seaweed algae selects Eucheuma.
Seaweed protoplast is separately cultured the method with regeneration plant, mainly according to the all-round of seaweed somatocyte development
Property, seaweed is decomposed using seaweed toolenzyme and is organized, and dissociate somatoplasm body, obtains the body cell of regenerative cell's wall and thin
Born of the same parents system, is then cultivated to obtain regeneration plant.Specific step is as follows:
(1) selection color it is normal, without rot, eugonic frond, with hairbrush cleaning frond surface sludge, miscellaneous algae and its
Its attached impurity, with 0.005% HgCl2Solution disinfection 5s, is then rinsed well using disinfected sea water, and fresh frond top is cut
Young tender part is held, blots surface moisture with filter paper, tiny tissue block or small branch are sheared or be ground into, first in winestone
After impregnating 2h in sour potassium sodium and PVP mixed solution, seaweed tool enzyme solutions is recycled to carry out digestion enzymatic hydrolysis, it is heavy to be then centrifuged
It forms sediment, obtains seaweed somatoplasm；
Here, sodium potassium tartrate tetrahydrate and PVP are configured to the solution that concentration is 0.1M and 3M respectively, and then 1:3 is mixed by volume
It closes；Sodium potassium tartrate tetrahydrate and PVP are mutually cooperateed with, and cell wall cellulose can be made to expand, to accelerate cell wall
Dissolution or rupture cause cell plasmolysis, while can be improved the toughness of cell membrane, finally can be improved frond enzymatic hydrolysis effect
The integrality of rate and the protoplast for ensuring to obtain；
Here, seaweed toolenzyme selects sea snail enzymes, and sea snail enzymes are temporarily supported in disinfected sea water the preparation method comprises the following steps: conch is cleaned
Middle hungry 2 days, decladding took its glandula digestive, was ground to homogenate, and 10000rpm is centrifuged 10min, discarded precipitating, be added in supernatant-
The acetone of 10 DEG C of pre-coolings, centrifugation discard precipitating, repeat addition acetone and are collected by centrifugation and precipitate to obtain original enzyme liquid, standby in -5 DEG C of preservations
With；Original enzyme liquid using when with seawater be diluted to 15%；
Enzymatic hydrolysis condition are as follows: every gram of frond digests solution using 15ml；The sorbierite of 2.2M is added in enzymolysis liquid as infiltration
Press stabilizer；Keeping enzymolysis liquid pH is to digest 3.5h at 6,30 DEG C；
The operation of protoplast electrofusion are as follows: with 250 mesh thin,tough silk filtration cell mixed liquors, remove indigested cell, cell
Group, fragment；It taking supernatant 500rpm to be centrifuged 10min, unicellular (protoplast) is made to sink, cell fragment stays in supernatant,
Liquid is discarded supernatant, is rinsed and is centrifuged with the disinfected sea water containing 1.0M glucose 3 times, collection precipitates up to seaweed plasm
Body；
(2) Protoplast cuhnre is obtained into the cell of regenerative cell's wall, further regeneration plant can be obtained in culture；
Protoplast cuhnre mode are as follows: first form sodium alginate and protoplast mixed in equal amounts drop in calcium chloride solution
It is fixed, it inoculates in fluid nutrient medium and cultivates, the initial plant plate density of protoplast is 1 × 104A/mL；First keep temperature
Dark culture 5 days at 22 DEG C then turn to illumination cultivation, light application time 8h/d, light intensity 2500Lx, replacement half training in every 7 days
Support base；Wherein, fluid nutrient medium is to cultivate first 10 days containing methyl α-naphthyl acetate and 2, the PES culture medium of 4-chlorophenoxyacetic acid, make methyl α-naphthyl acetate
With 2, the concentration of 4-chlorophenoxyacetic acid is respectively 0.03mg/L and 1mg/L, and then the concentration of both adjustment is 1mg/L and 0.1mg/
L, and culture medium osmotic pressure is kept with 1.5M mannitol and 1.2M glucose；It chelates to be formed with seaweed acid ion using calcium chloride
Calcium alginate gel not soluble in water, is fixed cell, can effectively prevent protoplast and sticks together, and to protoplast
Fixed point location observation can be carried out, its growth course is tracked, is conducive to improve cell splitting rate；
Protoplast cuhnre process adds tryptophan and Vitamin C after cell carries out first division in the medium
Sour sodium, and adjusting medium pH is 5.5, continues to cultivate；The additive amount of tryptophan and sodium ascorbate is the 1% of culture medium weight
With 0.5%；The special presence of tryptophan and sodium ascorbate accelerates thin first is that being conducive to improve the division frequency of protoplast
Born of the same parents divide to form filamentous, thallus or callus, and then break up and generate plant；Second is that protoplast can be effectively prevent to train
Issuable phenolic substances etc. aoxidizes during supporting, and keeps the stability of active material in dividing cell, improves cell
Vigor, promote protoplast differentiation and development, shorten growing-seedling period while keep higher seed breeding efficiency.
Embodiment 2:
Seaweed algae selects solieria.
Seaweed protoplast is separately cultured the method with regeneration plant, mainly according to the all-round of seaweed somatocyte development
Property, seaweed is decomposed using seaweed toolenzyme and is organized, and dissociate somatoplasm body, obtains the body cell of regenerative cell's wall and thin
Born of the same parents system, is then cultivated to obtain regeneration plant.Specific step is as follows:
(1) selection color it is normal, without rot, eugonic frond, with hairbrush cleaning frond surface sludge, miscellaneous algae and its
Its attached impurity, with 0.006% HgCl2Solution disinfection 6s, is then rinsed well using disinfected sea water, and fresh frond top is cut
Young tender part is held, blots surface moisture with filter paper, tiny tissue block or small branch are sheared or be ground into, first in winestone
After impregnating 2h in sour potassium sodium and PVP mixed solution, seaweed tool enzyme solutions is recycled to carry out digestion enzymatic hydrolysis, it is heavy to be then centrifuged
It forms sediment, obtains seaweed somatoplasm；
Here, sodium potassium tartrate tetrahydrate and PVP are configured to the solution that concentration is 0.3M and 4.5M respectively, then 1:4 by volume
It mixes；
Here, seaweed toolenzyme selects sea snail enzymes and cellulase mixed in equal amounts, sea snail enzymes the preparation method comprises the following steps: by conch
It cleans and temporarily supports starvation 2 days in disinfected sea water, decladding takes its glandula digestive, is ground to homogenate, and 12000rpm is centrifuged 6min, and it is heavy to discard
It forms sediment, the acetone of -15 DEG C of pre-coolings is added in supernatant, centrifugation discards precipitating, repeats addition acetone and is collected by centrifugation and precipitates to obtain protoenzyme
Liquid is saved backup in -10 DEG C；Original enzyme liquid using when with seawater be diluted to 20%；The preparation method of cellulase are as follows: use
The citric acid or acetate buffer solution (pH 4.8) of 50mmol/L dissolves, concentration 12%, wherein acetate buffer solution disinfected sea water
It prepares；
Enzymatic hydrolysis condition are as follows: every gram of frond digests solution using 15ml；The NaCl of 0.7M is added in enzymolysis liquid as osmotic pressure
Stabilizer；Keeping enzymolysis liquid pH is to digest 3.5h at 6,30 DEG C；
The operation of protoplast electrofusion are as follows: with 300 mesh thin,tough silk filtration cell mixed liquors, remove indigested cell, cell
Group, fragment；It takes supernatant 700rpm to be centrifuged 9min, unicellular (protoplast) is made to sink, cell fragment stays in supernatant, abandons
Supernatant is removed, is rinsed and is centrifuged with the disinfected sea water containing 1.0M glucose 3 times, collection precipitates up to seaweed plasm
Body；
(2) Protoplast cuhnre is obtained into the cell of regenerative cell's wall, further regeneration plant can be obtained in culture；
Protoplast cuhnre mode are as follows: first form sodium alginate and protoplast mixed in equal amounts drop in calcium chloride solution
It is fixed, it inoculates in fluid nutrient medium and cultivates, the initial plant plate density of protoplast is 5 × 105A/mL；First keep temperature
Dark culture 6 days at 24 DEG C then turn to illumination cultivation, light application time 10h/d, light intensity 2600Lx, replacement half training in every 7 days
Support base；Wherein, fluid nutrient medium is to cultivate first 15 days containing methyl α-naphthyl acetate and 2, the PES culture medium of 4-chlorophenoxyacetic acid, make methyl α-naphthyl acetate
With 2, the concentration of 4-chlorophenoxyacetic acid is respectively 0.03mg/L and 1mg/L, and then the concentration of both adjustment is 1mg/L and 0.1mg/
L, and culture medium osmotic pressure is kept with 1.5M mannitol and 1.2M glucose；
Protoplast cuhnre process adds tryptophan and Vitamin C after cell carries out first division in the medium
Sour sodium, and adjusting medium pH is 5.6, continues to cultivate；The additive amount of tryptophan and sodium ascorbate is culture medium weight
3.2% and 1.1%.
Embodiment 3:
Seaweed algae selects seaweed.
Seaweed protoplast is separately cultured the method with regeneration plant, mainly according to the all-round of seaweed somatocyte development
Property, seaweed is decomposed using seaweed toolenzyme and is organized, and dissociate somatoplasm body, obtains the body cell of regenerative cell's wall and thin
Born of the same parents system, is then cultivated to obtain regeneration plant.Specific step is as follows:
(1) selection color it is normal, without rot, eugonic frond, with hairbrush cleaning frond surface sludge, miscellaneous algae and its
Its attached impurity, with 0.008% HgCl2Solution disinfection 10s, is then rinsed well using disinfected sea water, and fresh frond is cut
Top young tender part blots surface moisture with filter paper, tiny tissue block or small branch is sheared or be ground into, first in wine
After impregnating 3h in stone acid potassium sodium and PVP mixed solution, seaweed tool enzyme solutions is recycled to carry out digestion enzymatic hydrolysis, it is heavy to be then centrifuged
It forms sediment, obtains seaweed somatoplasm；
Here, sodium potassium tartrate tetrahydrate and PVP are configured to the solution that concentration is 0.5M and 6M respectively, and then 1:5 is mixed by volume
It closes；
Here, seaweed toolenzyme selects cellulase, the preparation method of cellulase are as follows: with the citric acid of 60mmol/L or
Acetate buffer solution (pH 5.0) dissolution, concentration 15%, wherein acetate buffer solution is prepared with disinfected sea water；
Enzymatic hydrolysis condition are as follows: every gram of frond digests solution using 15ml；The NaCl of 0.7M is added in enzymolysis liquid as osmotic pressure
Stabilizer；Keeping enzymolysis liquid pH is to digest 3.5h at 6,30 DEG C；
The operation of protoplast electrofusion are as follows: with 400 mesh thin,tough silk filtration cell mixed liquors, remove indigested cell, cell
Group, fragment；It taking supernatant 850rpm to be centrifuged 10min, unicellular (protoplast) is made to sink, cell fragment stays in supernatant,
Liquid is discarded supernatant, is rinsed and is centrifuged with the disinfected sea water containing 1.2M glucose 3 times, collection precipitates up to seaweed plasm
Body；
(2) Protoplast cuhnre is obtained into the cell of regenerative cell's wall, further regeneration plant can be obtained in culture；
Protoplast cuhnre mode are as follows: first form sodium alginate and protoplast mixed in equal amounts drop in calcium chloride solution
It is fixed, it inoculates in fluid nutrient medium and cultivates, the initial plant plate density of protoplast is 1 × 106A/mL；First keep temperature
Dark culture 7 days at 26 DEG C then turn to illumination cultivation, light application time 10h/d, light intensity 3000Lx, replacement half training in every 7 days
Support base；Wherein, fluid nutrient medium is to cultivate first 15 days containing methyl α-naphthyl acetate and 2, the PES culture medium of 4-chlorophenoxyacetic acid, make methyl α-naphthyl acetate
With 2, the concentration of 4-chlorophenoxyacetic acid is respectively 0.03mg/L and 1mg/L, and then the concentration of both adjustment is 1mg/L and 0.1mg/
L, and culture medium osmotic pressure is kept with 1.5M mannitol and 1.2M glucose；
Protoplast cuhnre process adds tryptophan and Vitamin C after cell carries out first division in the medium
Sour sodium, and adjusting medium pH is 5.8, continues to cultivate；The additive amount of tryptophan and sodium ascorbate is the 5% of culture medium weight
With 2%.
Comparative example 1:
Algae tissue (is impregnated without the mixed solution of sodium potassium tartrate tetrahydrate and PVP) before being digested without pretreatment；
Remaining operating process is consistent with embodiment 2.
Comparative example 2:
Protoplast cuhnre process does not add tryptophan and sodium ascorbate in culture medium；Remaining operating process and implementation
Example 2 is consistent.
Embodiment 4:
The identification of protoplast:
What fluorescence colour was selected, which is that VBL type is glimmering, is identified to enzymatic hydrolysis gained cell using fluorescent whitening agent decoration method
Optical brightener is made into 0.1% dyeing liquor, carries out dyeing 5min to gained sample, under the microscope in fluorescence microscopy, excitation
The a length of 370nm ultraviolet light of light wave；Lepocyte has green fluorescence and shows that cellulose exists, and protoplast redgreen fluorescence is
Due to chloroplaset presence and send out red fluorescence, show the presence of cell-free wall；
The density measurement of protoplast is counted using blood counting chamber；The measurement of protoplast survival rate uses: primary
Plastid is put into lower percolating solution, and volume energy swells, and being put into can shrink compared with its volume in high-permeation solution, this is living thin
Born of the same parents, and changer is not dead cell to volume；
The protoplast number of (enzymatic hydrolysis 3h) in the embodiment of the present invention or comparative example enzymolysis process is counted, is obtained primary
The density data and survival rate data of plastid, are shown in Table 1；
As shown in Table 1, algae tissue is first pre-processed before being digested: being soaked using sodium potassium tartrate tetrahydrate and PVP mixed solution
Bubble is conducive to improve partition density of the cytoplasm wall separative efficiency to increase protoplast in identical enzymolysis time, and energy
The toughness for enough improving cell membrane, keeps the integrality of protoplast to improve its survival rate.
The partition density and survival rate data of the protoplast of the present invention of table 1
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (9)
1. seaweed protoplast is separately cultured the method with regeneration plant, it is characterised in that: decompose seaweed using seaweed toolenzyme
Tissue, obtains somatoplasm body, via cultivating to obtain regeneration plant；The seaweed toolenzyme digests seaweed tissue
Before, first frond is impregnated with the mixed solution of sodium potassium tartrate tetrahydrate and polyvinylpyrrolidone.
2. seaweed protoplast according to claim 1 is separately cultured the method with regeneration plant, it is characterised in that: described
The concentration of sodium potassium tartrate tetrahydrate and polyvinylpyrrolidone is respectively 0.1-0.5M and 3-6M, and 1:3-5 is mixed by volume.
3. seaweed protoplast according to claim 1 is separately cultured the method with regeneration plant, it is characterised in that: including
Following steps:
Step 1: selection color it is normal, without rot, eugonic frond, with hairbrush cleaning frond surface sludge, miscellaneous algae and its
Its attached impurity, with the HgCl of 0.005-0.008%2Solution disinfection 5-10s, is then rinsed well using disinfected sea water, is cut new
Fresh frond top young tender part, blots surface moisture with filter paper, is sheared or be ground into tiny tissue block or small branch,
After first impregnating 2-3h in sodium potassium tartrate tetrahydrate and polyvinylpyrrolidone mixed solution, seaweed tool enzyme solutions is recycled to disappear
Change enzymatic hydrolysis, then through centrifugation, obtains seaweed somatoplasm；
Step 2: Protoplast cuhnre is obtained the cell of regenerative cell's wall, further regeneration plant is can be obtained in culture.
4. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
The operation of protoplast electrofusion are as follows: use 250-400 mesh thin,tough silk filtration cell mixed liquor, remove indigested cell, cell mass, broken
Piece；It takes supernatant 500-850rpm to be centrifuged 8-10min, so that protoplast is sunk, cell fragment stays in supernatant, discards supernatant
Liquid is rinsed and is centrifuged 2-3 times with the disinfected sea water containing 1.0-1.2M glucose, and collection precipitates up to seaweed plasm
Body.
5. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
Seaweed toolenzyme is the combination of sea snail enzymes or cellulase or both；Wherein, sea snail enzymes the preparation method comprises the following steps: conch is cleaned temporary
Starvation 2-3 days in disinfected sea water are supported, decladding takes its glandula digestive, is ground to homogenate, and 10000-15000rpm is centrifuged 5-10min,
Precipitating is discarded, the acetone of -10~-20 DEG C of pre-coolings is added in supernatant, centrifugation discards precipitating, repeats addition acetone and is collected by centrifugation
Original enzyme liquid is precipitated to obtain, is saved backup in -5~-20 DEG C；Original enzyme liquid using when with seawater be diluted to 15-25%；Cellulase is matched
Method processed are as follows: dissolved with the citric acid or acetate buffer solution of 40-60mmol/L, concentration 10-15%, wherein acetate buffer solution is used
Disinfected sea water is prepared.
6. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
Seaweed toolenzyme in use, be added homeo-osmosis agent, homeo-osmosis agent be selected from mannitol, sorbierite, glucose, sucrose,
One of KCl, NaCl；Wherein, the suitable concentration of alcohols and carbohydrate is 1.2-2.6M, and the suitable concentration of KCl, NaCl are 0.5-
0.8M。
7. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
Enzymatic hydrolysis condition are as follows: pH 5.5-7.5 digests 2-4.5h at 15-40 DEG C.
8. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
Protoplast cuhnre mode are as follows: first sodium alginate and protoplast mixed in equal amounts drop are formed in calcium chloride solution and fixed, then
It is inoculated into fluid nutrient medium and cultivates；It first keeps at 22-26 DEG C of temperature dark culture 5-7 days, then turns to illumination cultivation, when illumination
Between be 8-10h/d, light intensity 2500-3000Lx, every 7 days replacement half culture mediums；Wherein, fluid nutrient medium be containing methyl α-naphthyl acetate and
2, the PES culture medium of 4-chlorophenoxyacetic acid, and culture medium osmotic pressure is kept with sweet dew alcohol and glucose.
9. seaweed protoplast according to claim 2 is separately cultured the method with regeneration plant, it is characterised in that: described
Protoplast cuhnre process adds tryptophan and sodium ascorbate, and adjust after cell carries out first division in the medium
Section medium pH is 5.5-5.8, continues to cultivate；The additive amount of tryptophan and sodium ascorbate be culture medium weight 1-5% and
0.5-2%。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811494026.0A CN109511550B (en) | 2018-12-07 | 2018-12-07 | Method for separating and culturing seaweed protoplast and regenerating plant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811494026.0A CN109511550B (en) | 2018-12-07 | 2018-12-07 | Method for separating and culturing seaweed protoplast and regenerating plant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109511550A true CN109511550A (en) | 2019-03-26 |
| CN109511550B CN109511550B (en) | 2021-11-23 |
Family
ID=65795174
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811494026.0A Active CN109511550B (en) | 2018-12-07 | 2018-12-07 | Method for separating and culturing seaweed protoplast and regenerating plant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109511550B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0568442A (en) * | 1991-09-14 | 1993-03-23 | Kaiyo Bio Technol Kenkyusho:Kk | Simple method for sterile culture of algae |
| CN1631119A (en) * | 2004-12-24 | 2005-06-29 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding raising technology |
| CN1754426A (en) * | 2004-09-30 | 2006-04-05 | 中国科学院海洋研究所 | The method of the separation and purification of asparagus protoplast and regeneration plant |
| CN1806526A (en) * | 2004-12-24 | 2006-07-26 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding breeding technology |
| CN101422128A (en) * | 2007-10-29 | 2009-05-06 | 中国水产科学研究院黄海水产研究所 | Separation and regeneration method of gulfweed protoplast |
| CN102144544A (en) * | 2011-01-10 | 2011-08-10 | 中国海洋大学 | Method for separating Eucheuma seaweed protoplast and culturing regenerated plant |
| CN102160524A (en) * | 2011-01-10 | 2011-08-24 | 中国海洋大学 | Separation culture and plant regeneration method for kappaphycus seaweed protoplast |
| CN105754927A (en) * | 2016-03-22 | 2016-07-13 | 中国海洋大学 | Preparation method of gracilaria salicornia protoplast |
| CN106893690A (en) * | 2016-12-27 | 2017-06-27 | 中国海洋大学 | The preparation method of Chilean fragrant plant mentioned in ancient texts protoplast |
-
2018
- 2018-12-07 CN CN201811494026.0A patent/CN109511550B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0568442A (en) * | 1991-09-14 | 1993-03-23 | Kaiyo Bio Technol Kenkyusho:Kk | Simple method for sterile culture of algae |
| CN1754426A (en) * | 2004-09-30 | 2006-04-05 | 中国科学院海洋研究所 | The method of the separation and purification of asparagus protoplast and regeneration plant |
| CN1631119A (en) * | 2004-12-24 | 2005-06-29 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding raising technology |
| CN1806526A (en) * | 2004-12-24 | 2006-07-26 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding breeding technology |
| CN101422128A (en) * | 2007-10-29 | 2009-05-06 | 中国水产科学研究院黄海水产研究所 | Separation and regeneration method of gulfweed protoplast |
| CN102144544A (en) * | 2011-01-10 | 2011-08-10 | 中国海洋大学 | Method for separating Eucheuma seaweed protoplast and culturing regenerated plant |
| CN102160524A (en) * | 2011-01-10 | 2011-08-24 | 中国海洋大学 | Separation culture and plant regeneration method for kappaphycus seaweed protoplast |
| CN105754927A (en) * | 2016-03-22 | 2016-07-13 | 中国海洋大学 | Preparation method of gracilaria salicornia protoplast |
| CN106893690A (en) * | 2016-12-27 | 2017-06-27 | 中国海洋大学 | The preparation method of Chilean fragrant plant mentioned in ancient texts protoplast |
Non-Patent Citations (10)
| Title |
|---|
| M.POLNE-FULLER等: "Digestion of seaweeds by the marine amoeba Trichosphaerium", 《HYDROBIOLOGIA》 * |
| SHENGHAO LIU等: "Optimization of parameters for isolation of protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L", 《J APPL PHYCOL》 * |
| 崔翠菊等: "大叶藻原生质体的分离和培养", 《水产科学》 * |
| 张思: "红翎菜科三种海藻原生质体分离培养及外植体发育模式研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 戴继勋: "紫菜原生质体的纯系培育、诱变处理和种间细胞融合的研究", 《海洋与湖沼》 * |
| 戴继勋等: "坛紫菜原生质体的发育研究", 《遗传学报》 * |
| 李春艳等: "植物原生质体培养与融合的研究进展 ", 《海南师范学院学报(自然科学版)》 * |
| 王素娟等: "坛紫菜营养细胞和原生质体培养的研究I", 《海洋与湖沼》 * |
| 王钟霞: "龙须菜原生质体制备、融合和再生及孢子融合的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 许星鸿等: "制备条斑紫菜原生质体的工具酶研究", 《淮海工学院学报(自然科学版)》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109511550B (en) | 2021-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102150624B (en) | Tissue culture and rapid propagation method for pinellia tuber plant | |
| CN102217539B (en) | Isolated rooting culture method for fir clone | |
| CN104920201B (en) | A kind of kelp seedling breeding method | |
| CN102144547A (en) | Method for quickly breeding and transplanting grape stock unit | |
| CN108849498B (en) | Method for promoting accumulation of flavonoid substances in leaf tissues of ornamental crabapple | |
| CN105993909A (en) | Pyropia haitanensis pure line seedling cultivation method | |
| CN103583357B (en) | Method for sterile seeding of lithops and establishing regeneration system | |
| CN104686361A (en) | Induction and culture method of embryonic callus of grape | |
| CN105132354A (en) | Extraction method of gracilaria blodgettii protoplast | |
| CN106434817A (en) | Method for improving Haematococcus pluvialis to produce astaxanthin by using alkali pretreatment technology | |
| CN107129962B (en) | A kind of primary culture method of Hirudo japonica salivary gland cell | |
| CN116814525B (en) | Culture medium for culturing Babylonia embryos and method for culturing Babylonia embryos outside capsules | |
| CN105123512B (en) | A kind of breeding method of roxburgh anoectochilus terminal bud | |
| CN109511550A (en) | Seaweed protoplast is separately cultured the method with regeneration plant | |
| CN115340955A (en) | Method for efficiently culturing arbuscular mycorrhizal fungi | |
| Rusig et al. | Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture | |
| KR100898416B1 (en) | Method of cultivation for Ecklonia stolonifera | |
| KR100334629B1 (en) | Method for manufacturing high quality young seedling of phalaenopsis in bioreactor by using tissue of flower stalk before blooming | |
| CN102144544B (en) | Method for separating Eucheuma seaweed protoplast and culturing regenerated plant | |
| Takebe et al. | Isolation and culture of protoplasts: tobacco | |
| CN111296286A (en) | Moringa oleifera tetraploid induction and propagation method | |
| David | Conifer protoplasts | |
| Steiner | Protoplast isolation and transformation in sweetpotato | |
| CN110839532A (en) | Asexual propagation method of umbrellas roses | |
| CN109479727A (en) | A method of inducing cells,primordial using Afriocan agapanthus blade as explant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |