CN106818480A - A kind of method by soybean leaves Callus Regeneration - Google Patents
A kind of method by soybean leaves Callus Regeneration Download PDFInfo
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- CN106818480A CN106818480A CN201710042225.7A CN201710042225A CN106818480A CN 106818480 A CN106818480 A CN 106818480A CN 201710042225 A CN201710042225 A CN 201710042225A CN 106818480 A CN106818480 A CN 106818480A
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- soybean leaves
- callus
- protoplast
- regeneration
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- 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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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Abstract
The invention discloses a kind of method by soybean leaves Callus Regeneration, including：1) acquisition and sterilization of soybean leaves；Wherein, the soybean leaves are first pair of trifoliolate leaf；2) separation and collection of protoplast are carried out from the soybean leaves；3) from the protoplast regeneration callus.It is of the invention simple efficient, substantial amounts of protoplast can be produced the short time and activity is high.Protoplast regeneration callus regeneration rate is high simultaneously.The present invention will be soybean callus further with offer technical support.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of method by soybean leaves Callus Regeneration.
Background technology
Callus plays an important roll in Plant Tissue Breeding, can not only rapid regeneration acquisition using callus
A large amount of whole plants with specific trait, more can for a long time be preserved to the plant resources of specific trait.Conventional callus
Tissue acquisition methods, usually obtain plant explant, such as hypocotyl, cotyledon etc., sterilized rear induction produce callus.
It is a kind of effective side that callus is obtained to obtain callus by protoplast regeneration using plant leaf blade
Formula.But, because plant genotype has a very big impact for the separation of protoplast and regeneration, same plant different genes
The protoplast dedifferentiation of type causes different cultivars Regenerated energy under the same conditions with to break up required condition again different
Power also can be different.
Therefore, those skilled in the art is devoted to developing a kind of soybean protoplast separation in regeneration method.In addition,
Due to current main cultivation soybean varieties protoplast electrofusion and renovation process, there is not been reported, accordingly, it would be desirable to develop suitable for main cultivation
The protoplast electrofusion and regeneration method of soybean varieties.
The content of the invention
The invention provides a kind of method by soybean leaves Callus Regeneration, its protoplast yield is improved, and have
Imitate method of the ground by protoplast regeneration callus.
In better embodiment of the invention, included by the method for soybean leaves Callus Regeneration：
1) acquisition and sterilization of soybean leaves；Wherein, the soybean leaves are first pair of trifoliolate leaf；
2) separation and collection of protoplast are carried out from the soybean leaves；
3) from the protoplast regeneration callus.
Further, step 2) in, using the enzymolysis liquid containing cellulase, pectase and macerozyme to segmentation after it is big
Beans blade carries out lucifuge treatment, and treatment temperature is 28 ± 1 DEG C, to produce protoplast.
Preferably, soybean leaves are divided into 0.5-1mm small bars wide, and lucifuge is processed 4-6 hours, and period gently rocks number
It is secondary.
Further, enzymolysis liquid is to be added with cellulase, 0.2% pectase, 1.0% that mass volume ratio is 2.0%
The CPW-9M solution of macerozyme.
Preferably, per 1g soybean leaves addition 15ml enzymolysis liquids.
Further, the solution obtained after being processed with enzymolysis liquid mixes with isometric KP8 culture mediums, is sieved through with cell
Filter, filtered fluid is centrifuged, and precipitation is washed with KP8 culture mediums, is centrifuged again, obtains protoplast.
Preferably, it is sieved through filter with 200 aim cells, 200 × g centrifugations 10min becomes spherical, and precipitates.It
Suck supernatant as far as possible afterwards, precipitation is resuspended with KP8 culture mediums and rinses once, 200 × g centrifugation 10min collect precipitation, and use KP8
Culture medium is resuspended, obtains protoplast.
Further, step 3) in, by step 2) protoplast that obtains with not solidified containing low melting-point agarose
KP8 culture mediums are resuspended, and drop in culture dish with drops, after after culture medium solidifying, KP8 fluid nutrient mediums added, in 26
Cultivated under the conditions of ± 2 DEG C and lucifuge.
Preferably, the w/v of low melting-point agarose is 1.2%, is first cultivated with the KP8 containing low melting-point agarose
Base adjusts to 2 × 10 protoplast concentration5Individual/mL, 25 μ L/ drops are cultivated in dropping in culture dish.
Further, at the 7th, 14,21 and 28 days of culture, KP8 fluid nutrient mediums are replaced with into KP8:K8 mixed proportions
Respectively 2:1、1:1、0:1、0:1 fluid nutrient medium.
Further, at the 35th day of culture, fluid nutrient medium is replaced with into growth medium, growth medium is addition
There are benzyl aminoadenine, the MSB Liquid Cultures of the Kn and 500mg/L CH of 0.5mg/L of 2,4-D, 0.5mg/L of 0.5mg/L
Base.
Further, the callus after being cultivated in above-mentioned growth medium after 7 days is transferred to and contains 0.6% agar
MSB media surfaces, make callus continued growth, every 2 weeks using the MSB culture mediums containing 0.6% agar carry out once after
It is commissioned to train foster.
Preferably, in step 1) before, selection is full to show that smooth and without spot ripe soybean kernel is seeded in little Hua
(vermiculite/perlite=3 after sterilizing in basin：1) appropriate Hoagland nutrient solutions, are added, until the first couple of seedling three goes out
Compound leaf is fully deployed.
Further, step 1) in, the sterilization is the soybean leaves to be rinsed 3~4 times using flowing water, Ran Houyong
20% NaClO vibrates in 180r/min, and sterilize 20min under the conditions of 23 ± 1 DEG C, afterwards with sterile water wash 3-5 times.
Further, the soybean varieties that the above method is used are grand No. 1 of day.
It is of the invention simple efficient, substantial amounts of protoplast can be produced the short time and activity is high.While protoplast regeneration
Callus regeneration rate is high.The present invention will be soybean callus further with offer technical support.
The technique effect of design of the invention, specific steps and generation is described further below with reference to accompanying drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Brief description of the drawings
Fig. 1 is that the soybean leaves of a preferred embodiment of the invention produce protoplast schematic diagram.
Fig. 2 is the trypan blue staining detection protoplast activity schematic diagram of a preferred embodiment of the invention.
Fig. 3 is the protoplast regeneration callus schematic diagram of a preferred embodiment of the invention.
Specific embodiment
The material used in specific embodiment, if without specified otherwise, can directly buy acquisition.
CPW solution formulas are：
| 27.2mg/L | |
| 101.0mg/L | |
| 1480.0mg/L | |
| 246.0mg/L | |
| 0.025Mg/L |
pH 5.8；
CPW-9M is the CPW solution containing 9% mannitol；
KP8 culture medium prescriptions are：
K8 culture medium prescriptions are：
MSB Liquid Culture based formulas are：MSB 4.44g/L, sucrose 20g/L, pH 5.8.
In the specific embodiment of the present invention, by the method for soybean leaves Callus Regeneration, including：1) Soybean Leaves
The acquisition and sterilization of piece；Wherein, the soybean leaves are first pair of trifoliolate leaf；2) protoplast is carried out from the soybean leaves
Separation and collection；3) from the protoplast regeneration callus.
Step 1) in, soybean kernel is sowed and is cultivated, until first pair of trifoliolate leaf of seedling is fully deployed.Clip this three
Go out compound leaf to carry out disinfection；Young leaflet tablet cytoactive is most strong, and first pair of trifoliolate leaf is the most strong blade of cytoactive, therefore choosing
Selecting the blade carries out subsequent operation.
Step 2) in, the trifoliolate leaf after sterilization is split, small bar or small bulk are divided into, addition contains cellulose
Enzyme, pectase, the enzymolysis liquid of macerozyme, lucifuge processes to produce protoplast at 28 ± 1 DEG C.By above-mentioned enzymolysis liquid and culture
Base mixes, sieving, leaves the solution containing protoplast, and centrifugation obtains protoplast, then is washed with culture medium one time, is centrifuged again
And be resuspended in culture medium.Counted and Trypan Blue, checked protoplast yield and survival rate；
Step 3) in, protoplast is diluted with the culture medium containing low melting-point agarose, make its concentration be 2 × 105Individual/
ML, in then dropping in culture dish with drops, add appropriate nutrient solution carries out initial stage training under the conditions of 26 ± 2 DEG C of lucifuge
Support.Fluid nutrient medium is replaced weekly within three weeks afterwards, replace with the culture medium of KP8 contents decline.Band callus increases and changes
After for yellow green, fluid nutrient medium is replaced with and is added with the 2 of 0.5mg/L, the benzyl aminoadenine (BA) of 4-D, 0.5mg/L,
The MSB fluid nutrient mediums of the Kn and 500mg/L CH of 0.5mg/L.The callus for growing up to can be in the MSB cultures containing agar
Primary surface growth stimulator is passed on, to be enlarged culture.
Embodiment
1. the acquisition and sterilization of soybean young leaflet tablet
Choose and full show that smooth and without spot ripe grand No. 1 soybean kernel in day is seeded in small flower (after sterilizing
Vermiculite/perlite=3：1) appropriate Hoagland nutrient solutions, are added.Until first pair of trifoliolate leaf of seedling is opened up completely
Open.The young leaflet tablet that clip is fully deployed, 3~4 times are rinsed using flowing water, then with 20% NaClO sterilizations 20min (180r/
Min vibrates, 23 ± 1 DEG C), afterwards with sterile water wash 3~5 times until yellow disappears, will NaClO clean up.
2. the preparation and separation of protoplast
Blade after sterilization is divided into 0.5-1mm small bars wide.Per 1g tissue using 15ml enzymolysis liquids two 60 ×
Lucifuge processes 4~6hr at 28 ± 1 DEG C in the plastic culture dish of 15mm, and period gently rocks for several times, is able to observe that plasm
Body is discharged, and is sunken to bottom, as shown in Figure 1.
Wherein, enzymolysis liquid is that CPW-9M (pH 5.8) adds 2.0%w/v cellulases (Cellulase Onozuka
R10), 0.2%w/v pectases (Pectolyase Y23), 1.0%w/v macerozymes (macerozyme R10), 55 DEG C of water-baths
10min, 1500rpm is centrifuged 15 minutes after being cooled to room temperature, takes supernatant, uses 0.22um filtering with microporous membrane, and 4 DEG C of lucifuges are protected
Deposit.
Will enzymolysis solution mix with isometric KP8 culture mediums, with 200 aim cells sieve filtering solution after, 200 × g from
Heart 10min becomes spherical, and precipitates.Supernatant is sucked as far as possible afterwards, protoplast is resuspended with KP8 culture mediums and rinses
Once, 200 × g centrifugations 10min, collects precipitation, and be resuspended in centrifuge tube with KP8 culture mediums.
Plasmic yield is counted using blood counting chamber and light microscope, the yield for finding protoplast is
1.5×107Individual/mL (when start vane is organized as 1g), while protoplast activity is detected using trypan blue staining,
What is be not colored in the visual field is active protoplasm somatocyte, and dye blueness is the cell for losing vigor, such as Fig. 2 institutes
Show.Statistics finds that the survival rate of protoplast is 89.8%.
3. callus regeneration
1.2% low melting-point agarose solution is prepared using KP8 culture mediums, and when agarose is cooled down but do not solidified, will
The protoplast concentration of preparation is adjusted to 2 × 105Individual/mL, and culture in the culture dish of 60mm is placed in the form of 25 μ L drops
(10 drops/ware), after after droplet solidification, add the KP8 culture mediums of 5.0mL in culture dish.Culture dish is placed in into 26 ± 2 DEG C to keep away
Cultivated under optical condition 7 days, culture dish is placed under illumination condition cultivates afterwards.
At the 7th, 14,21 and 28 days of culture, replacement fluid nutrient medium was KP8:K8 mixed proportions are respectively 2:1,1:1,
0:1,0:1 fluid nutrient medium.Such replacement frequency and culture medium are configured, and are in order that histocyte acclimatizing culture medium
Osmotic pressure.A subculture was changed every 7 days, the osmotic pressure of culture medium is gradually reduced, until reaching normal tissue growth infiltration
Pressure.It can be found that occurring the callus of protoplast electrofusion regeneration among drop in incubation, small white spots shape is presented,
And gradually increase and be changed into yellow green.
When waiting callus to increase and be changed into yellow green, fluid nutrient medium is replaced with into growth medium, usually
Carried out at the 35th day or so.Growth medium be 2,4-D, 0.5mg/L for being added with 0.5mg/L benzyl aminoadenine (BA),
The kinetin (KN) of 0.5mg/L and the MSB fluid nutrient mediums of 500mg/L caseinhydrolysates (CH).
Be transferred to for Agarose plug or droplet and contain by (i.e. after Callus Regeneration Adaptable growth culture medium) within the 7th day afterwards
The MSB media surfaces of 0.6% agar, make the callus continued growth for having regenerated.And every 2 weeks using containing 0.6% agar
MSB culture mediums carry out a squamous subculture.Callus from protoplast regeneration is as shown in Figure 3.
Preferred embodiment of the invention described in detail above.It should be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations with design of the invention.Therefore, all technical staff in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a kind of method by soybean leaves Callus Regeneration, it is characterised in that methods described includes：
1) acquisition and sterilization of soybean leaves；Wherein, the soybean leaves are first pair of trifoliolate leaf；
2) separation and collection of protoplast are carried out from the soybean leaves；
3) from the protoplast regeneration callus.
2. the method for soybean leaves Callus Regeneration as claimed in claim 1, it is characterised in that step 2) in, using containing
The enzymolysis liquid for having cellulase, pectase and macerozyme carries out lucifuge treatment to the soybean leaves after segmentation, and treatment temperature is 28
± 1 DEG C, to produce protoplast.
3. the method for soybean leaves Callus Regeneration as claimed in claim 2, it is characterised in that the enzymolysis liquid is addition
It is 1.0% macerozyme for 0.2% pectase, mass volume ratio to have cellulase, mass volume ratio that mass volume ratio is 2.0%
CPW-9M solution.
4. the method for soybean leaves Callus Regeneration as claimed in claim 2, it is characterised in that after being processed with enzymolysis liquid
The solution of acquisition mixes with isometric KP8 culture mediums, is filtered with cell sieve, and filtered fluid is centrifuged, and precipitation is washed with KP8 culture mediums
Wash, be centrifuged again, obtain protoplast.
5. the method for soybean leaves Callus Regeneration as claimed in claim 1, it is characterised in that step 3) in, by step
2) protoplast for obtaining is resuspended with the not solidified KP8 culture mediums containing low melting-point agarose, and is dripped with drops
In culture dish, after after culture medium solidifying, KP8 fluid nutrient mediums are added, cultivated under the conditions of 26 ± 2 DEG C and lucifuge.
6. the method for soybean leaves Callus Regeneration as claimed in claim 5, it is characterised in that in culture the 7th, 14,
21 and 28 days, KP8 fluid nutrient mediums are replaced with into KP8:K8 mixed proportions are respectively 2:1、1:1、0:1、0:1 Liquid Culture
Base.
7. the method for soybean leaves Callus Regeneration as claimed in claim 6, it is characterised in that at the 35th day of culture,
Fluid nutrient medium is replaced with into growth medium, the growth medium is be added with 0.5mg/L 2, the benzyl of 4-D, 0.5mg/L
The MSB fluid nutrient mediums of aminoadenine, Kn the and 500mg/L CH of 0.5mg/L.
8. the method for soybean leaves Callus Regeneration as claimed in claim 7, it is characterised in that will be in the grown cultures
Callus after being cultivated in base after 7 days is transferred to the MSB media surfaces containing 0.6% agar, makes callus continue to give birth to
It is long, carry out a squamous subculture using the MSB culture mediums containing 0.6% agar within every 2 weeks.
9. the method for soybean leaves Callus Regeneration as claimed in claim 1, it is characterised in that step 1) in, it is described to disappear
Poison is that the soybean leaves are rinsed 3~4 times using flowing water, is then vibrated in 180r/min with 20% NaClO, 23 ± 1 DEG C
Under the conditions of sterilize 20min, afterwards with sterile water wash 3-5 times.
10. the method for soybean leaves Callus Regeneration as claimed in any one of claims 1-9 wherein, it is characterised in that use
Soybean varieties be grand No. 1 of day.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710042225.7A CN106818480B (en) | 2017-01-20 | 2017-01-20 | A method of by soybean leaves Callus Regeneration |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710042225.7A CN106818480B (en) | 2017-01-20 | 2017-01-20 | A method of by soybean leaves Callus Regeneration |
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| CN106818480A true CN106818480A (en) | 2017-06-13 |
| CN106818480B CN106818480B (en) | 2019-03-12 |
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| CN201710042225.7A Expired - Fee Related CN106818480B (en) | 2017-01-20 | 2017-01-20 | A method of by soybean leaves Callus Regeneration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109370976A (en) * | 2018-12-20 | 2019-02-22 | 江苏省农业科学院 | Chinese small iris mesophyll protoplast and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63313579A (en) * | 1987-06-16 | 1988-12-21 | Kikkoman Corp | Preparation of callus originated from protoplast of leguminous plant |
| CN1083525A (en) * | 1992-09-03 | 1994-03-09 | 东北农学院 | A kind of new (ZSP) soybean protoplast culture medium |
| CN105219693A (en) * | 2015-09-19 | 2016-01-06 | 安徽科技学院 | A kind of soybean leaves protoplast electrofusion method for Subcellular Localization |
-
2017
- 2017-01-20 CN CN201710042225.7A patent/CN106818480B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63313579A (en) * | 1987-06-16 | 1988-12-21 | Kikkoman Corp | Preparation of callus originated from protoplast of leguminous plant |
| CN1083525A (en) * | 1992-09-03 | 1994-03-09 | 东北农学院 | A kind of new (ZSP) soybean protoplast culture medium |
| CN105219693A (en) * | 2015-09-19 | 2016-01-06 | 安徽科技学院 | A kind of soybean leaves protoplast electrofusion method for Subcellular Localization |
Non-Patent Citations (5)
| Title |
|---|
| F.W. SCHWENK等: ""Soybean mesophyll protoplasts"", 《PLANT SCIENCE LETTERS》 * |
| O. L. GAMBORG等: ""Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean"", 《PLANT CELL REPORTS》 * |
| 孙文丽等: ""大豆再生体系的研究进展"", 《安徽农业科学》 * |
| 罗希明等: ""大豆原生质体的植株再生"", 《植物学报》 * |
| 罗希明等: ""大豆叶肉细胞原生质体的游离和培养"", 《吉林农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109370976A (en) * | 2018-12-20 | 2019-02-22 | 江苏省农业科学院 | Chinese small iris mesophyll protoplast and preparation method thereof |
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