CN106718825A - A kind of black dish microspore-isolated culture method - Google Patents
A kind of black dish microspore-isolated culture method Download PDFInfo
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- CN106718825A CN106718825A CN201611031964.8A CN201611031964A CN106718825A CN 106718825 A CN106718825 A CN 106718825A CN 201611031964 A CN201611031964 A CN 201611031964A CN 106718825 A CN106718825 A CN 106718825A
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- microspore
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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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Abstract
The present invention discloses a kind of black dish microspore-isolated culture method, and the method comprises the steps：Selection microspore-isolated culture embryoid induction rate Chinese cabbage material higher, is hybridized with black dish material to be cultivated, and obtains hybrid seed F1；Hole tray will be seeded in after the hybrid seed F1 low temperature vernalization sprouted, when 34 true leaves, transplanted to big Tanaka, just spent to full-bloom stage, the bud for taking suitable size carries out microspore-isolated culture, obtains regeneration plant.The present invention is hybridized the black dish Chinese cabbage material higher with microspore-isolated culture embryoid induction rate, first-filial generation F1 is the inhereditary material that black dish introduces Chinese cabbage, effectively improve black dish donor plant genotype, improve black dish microspore-isolated culture embryoid induction rate, black dish breeding hereditary basis is widened, black dish breeding efficiency is improved.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of black dish microspore-isolated culture method.
Background technology
Microspore-isolated culture is the emerging technology grown up on Anther Culture basis, is plant cell engineering
One of most active research topic in field.Monoploid, dihaploid can be obtained by isolated microspore culture technique, monoploid and
Dihaploid is extremely precious on crop breeding.The self-mating system that conventional breeding obtains an inheritance stability generally requires 5-8's
Time, and 1-2 is only needed to using microspore culture, therefore carried out the research work of Isolated microspore in many crops
Make.
Black dish is a mutation of Cruciferae Brassica genus Vegetable Crops of Brassica Chinese cabbage subspecies, and its microspore-isolated culture is lured in the presence of difficulty
Embryoid and the low-down problem of embryoid induction rate are derived, and the Chinese cabbage nearer with black dish affiliation is in Isolated microspore
Embryoid is easily induced in culture and inductivity is universal higher.In research at present to have been reported that, genotype is to determine the small spore that dissociates more
Can son culture induce the key factor of embryo and inductivity height, to improve black dish microspore-isolated culture embryoid induction
Rate, now provides a kind of method.
The content of the invention
It is an object of the invention to provide a kind of black dish microspore-isolated culture method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of black dish microspore-isolated culture method, the method comprises the steps：
(2) hole tray will be seeded in after the hybrid seed F1 low temperature vernalization sprouted, when 3-4 piece true leaves, be transplanted to big Tanaka,
Just to full-bloom stage, the bud for taking suitable size carries out microspore-isolated culture, obtains regeneration plant flower.
Microspore-isolated culture step is in the step (2)：
Materials：Using F1 as material, in just flower to full-bloom stage, in the fine day morning 8:00-10:00 chooses healthy and strong no disease and pests harm
Bud on plant, is placed on slide, and drop 2 drips haematine dye liquor, with tweezers and the broken bud of dissecting needle, removal sepal, flower
Medicine etc. is organized, covered, optical microphotograph Microscopic observation mid-late uninucleate stage sporidiole, determines that sporidiole is in mid-late uninucleate stage
The size of bud when ratio is higher, is drawn materials by standard of this size.
Sterilization：The bud of selection is put into the aseptic small beakers of 25mL, first with 70% alcohol 7-10mL sterilize 30s,
Secondly with 2.5% NaClO solution about 10mL sterilization 5min, then with aseptic water washing 3 times；
Sporidiole purifies：After sterilization, it is the B5 sterile liquids of 130g/L that sucrose mass concentration is added in above-mentioned small beaker
Culture medium (pH5.8,116 DEG C of autoclave sterilization 30min) about 8mL, gently being extruded with glass bar makes sporidiole separate out,
350 mesh filter screens are filtered, and collect filtrate in 15mL spiral cover centrifuge tubes, and rotating speed 1000rpm centrifugation 3min abandon supernatant, are repeated 2 times,
Be eventually adding NLN aseptic liquid nutrient mediums that sucrose mass concentration is 130g/L (NLN-13, pH5.8,0.22 μm of aperture it is aseptic
Membrane filtration sterilizes), sporidiole density is reached 1 × 105-2×105Individual/mL (is counted) using blood counting chamber, that is, obtain small
Spore suspension.
Culture：Microspore suspension is sub-packed in the culture dish of diameter 6cm, per ware about 3.5mL, is sealed with Parafilm
Film sealing after, be immediately placed on 33 DEG C it is black under the conditions of light culture 24h；Then, culture under 25 DEG C of dark conditions is transferred to, there are naked eyes visible
Embryo when occurring, culture dish is transferred on the shaking table of rotating speed 60rpm and continues light culture to forming cotyledon type embryoid；It is average every
Individual bud induces 12.53 embryos；
Plantlet regenerating step is in the step (2)：
Plant regeneration：Light culture 20d or so forms cotyledon type embryoid, selects cotyledon type embryo and transfers into addition 30g/L sugarcanes
Sugar, 8g/L agar, in pH5.8, the MS sterile solid culture mediums of 116 DEG C of autoclave sterilization 30min, in 25 DEG C, illumination 14h/d
Under the conditions of cultivate, formed regeneration plant；
Regeneration plant is taken root：Embryoid is sprouted the seedling inoculation to be formed to addition 30g/L sucrose, 6g/L agar, 1mg/
L6-BA, 0.1mg/LNAA, in pH5.8, the MS sterile solid culture mediums of 116 DEG C of autoclave sterilization 30min, in 25 DEG C, illumination
Cultivated under the conditions of 14h/d, obtain whole plant.Expand numerous, rooting culture to field.
Beneficial effects of the present invention：The present invention is by black dish and microspore-isolated culture embryoid induction rate Chinese cabbage higher
Material is hybridized, and is the inhereditary material that black dish introduces Chinese cabbage, effectively improves black dish donor plant genotype, improves black dish
Microspore-isolated culture embryoid induction rate, widens black dish breeding hereditary basis, improves black dish breeding efficiency.
Brief description of the drawings
For the ease of it will be appreciated by those skilled in the art that the present invention is further illustrated below in conjunction with the accompanying drawings.
Fig. 1 expands schematic diagram for sporidiole of the present invention；
Fig. 2 is cotyledon type embryo shape (A, B) schematic diagram of the present invention；
Fig. 3 is somatic embryogenesis plant schematic diagram of the present invention；
Fig. 4 is that regeneration plant of the present invention expands numerous schematic diagram；
Fig. 5 is regeneration plant field transplanting schematic diagram of the present invention.
Specific embodiment
The present invention is further illustrated with example below in conjunction with the accompanying drawings, but the present invention limited never in any form
System, based on present invention teach that any change or improvement done, belong to protection scope of the present invention.
A kind of black dish microspore-isolated culture method, the method comprises the steps：
(1) selection microspore-isolated culture embryoid induction rate Chinese cabbage material higher, with black dish material to be cultivated
Hybridized, obtained hybrid seed F1, be the inhereditary material that black dish introduces Chinese cabbage, widened black dish hereditary basis；
(2) the hybrid seed F1 sprouted is seeded in hole tray, when 3-4 piece true leaves, is transplanted in big Tanaka, just spent to full blossom
Phase, the bud for taking suitable size carries out microspore-isolated culture according to a conventional method, obtains regeneration plant；
Isolated microspore culture technique step：
(1) draw materials：Using the first-filial generation F1 of black dish and Chinese cabbage as material, in just flower to full-bloom stage, in the fine day morning
8:00-10:Bud on 00 plant for choosing healthy and strong no disease and pests harm, is placed on slide, and drop 2 drips haematine dye liquor, uses tweezers
The tissues such as bud, removal sepal, flower pesticide are crushed with dissecting needle, covered, optical microphotograph Microscopic observation mid-late uninucleate stage is small
Spore, determines size of the sporidiole in mid-late uninucleate stage ratio bud when more, is drawn materials by standard of this size.
(2) sterilize：The bud of selection is put into the aseptic small beakers of 25ml, is sterilized with 70% alcohol 7-10ml first
30s, secondly with 2.5% NaClO solution about 10ml sterilization 5min, then with aseptic water washing 3 times；
(3) sporidiole purifying：After sterilization, it is the B5 sterile liquids of 130g/L that sucrose mass concentration is added in above-mentioned small beaker
Body culture medium about 8ml, gently being extruded with glass bar makes sporidiole separate out, the filtering of 350 mesh filter screens, collects filtrate and is revolved in 15mL
In lid centrifuge tube, rotating speed 1000rpm centrifugation 3min abandon supernatant, are repeated 2 times, and it is 130g/L's to be eventually adding sucrose mass concentration
NLN (NLN-13,0.22 μm of sterilised membrane filter filtration sterilization in aperture, pH5.8) liquid asepsis culture medium, reaches sporidiole density
1×105-2×105Individual/mL (is counted) using blood counting chamber, that is, obtain microspore suspension.
(4) cultivate：Microspore suspension is sub-packed in the culture dish of diameter 6cm, per ware 3.5ml, is sealed with Parafilm
Membrana oralis is sealed, and is immediately placed on 33 DEG C of dark culturing 24h；Then, culture (Fig. 1) under 25 DEG C of dark conditions is transferred to, 12d has visually can
When the embryo seen occurs, it is transferred to and continues light culture to formation cotyledon type embryoid (Fig. 2) on the shaking table of rotating speed 60rpm；It is average every
Individual bud induces 6-8 embryo, up to 12.53；
(5) plant regeneration：Embryoid growth 20d or so, selects cotyledon type embryo and transfers into addition 30g/L sucrose, 8g/L fine jades
During fat, pH are 5.8,116 DEG C of MS culture mediums of autoclave sterilization 30min, cultivated under the conditions of 25 DEG C of illumination 14h/d, formed
Regeneration plant；Not direct seedling and formed callus embryoid be transferred to addition 1mg/L6-BA above-mentioned culture medium on, make
It continues to develop seedling (Fig. 3).
(6) regeneration plant is taken root：Embryoid is sprouted the seedling to be formed be inoculated into addition 30g/L sucrose, 6g/L agar,
During 1mg/L6-BA, 0.1mg/LNAA, pH are 5.8,116 DEG C of MS root medias of autoclave sterilization 30min, in 25 DEG C, light
Cultivated according under the conditions of 14h/d, obtain whole plant, expand numerous, rooting culture to field (Fig. 4, Fig. 5).
Material selection of the present invention crow dish carries out microspore-isolated culture with the first-filial generation F1 of Chinese cabbage, compared to directly entering
Row crow dish microspore-isolated culture, F1 introduces the inhereditary material of Chinese cabbage, improves black dish microspore-isolated culture embryoid and lures
Conductance, solves the low technical problem of root tips, widens black dish breeding hereditary basis, improves black dish breeding efficiency.
Above content is only citing made for the present invention and explanation, and affiliated those skilled in the art are to being retouched
The specific embodiment stated is made various modifications or supplement or is substituted using similar mode, without departing from invention or super
More scope defined in the claims, all should belong to protection scope of the present invention.
Claims (3)
1. a kind of black dish microspore-isolated culture method, it is characterised in that the method comprises the steps：
(1) selection microspore-isolated culture embryoid induction rate Chinese cabbage material higher, is carried out with black dish material to be cultivated
Hybridization, obtains hybrid seed F1, is the inhereditary material that black dish introduces Chinese cabbage；
(2) hole tray will be seeded in after the hybrid seed F1 low temperature vernalization sprouted, when 3-4 piece true leaves, be transplanted to big Tanaka, just spent
To full-bloom stage, the bud for taking suitable size carries out microspore-isolated culture, obtains regeneration plant.
2. a kind of black dish microspore-isolated culture method according to claim 1, it is characterised in that in the step (2)
Microspore-isolated culture step is：
Materials：Using F1 as material, in just flower to full-bloom stage, in the fine day morning 8:00-10:00 chooses healthy and strong no disease and pests harm plant
On bud, be placed on slide, drop 2 drips haematine dye liquors, with the broken bud of tweezers and dissecting needle, removal sepal, flower pesticide group
Knit, covered, optical microphotograph Microscopic observation mid-late uninucleate stage sporidiole, determine sporidiole be in mid-late uninucleate stage ratio compared with
The size of bud when high, is drawn materials by standard of this size；
Sterilization：The bud of selection is put into the aseptic small beakers of 25mL, first with 70% alcohol 7-10mL sterilize 30s, secondly
Sterilized 5min with 2.5% NaClO solution about 10mL, then with aseptic water washing 3 times；
Sporidiole purifies：After sterilization, it is the B5 sterile liquid cultures of 130g/L that sucrose mass concentration is added in above-mentioned small beaker
Base about 8mL, gently being extruded with glass bar makes sporidiole separate out, the filtering of 350 mesh filter screens, collects filtrate and is centrifuged in 15mL spiral covers
Guan Zhong, rotating speed 1000rpm are centrifuged 3min, abandon supernatant, are repeated 2 times, and are eventually adding the NLN that sucrose mass concentration is 130g/L aseptic
Fluid nutrient medium, makes sporidiole density reach 1 × 105-2×105Individual/mL, that is, obtain microspore suspension；
Culture：Microspore suspension is sub-packed in the culture dish of diameter 6cm, it is close with Parafilm sealed membranes per ware about 3.5mL
It is honored as a queen, is immediately placed under 33 DEG C of dark conditions and cultivates 24h；Then, culture under 25 DEG C of dark conditions is transferred to, there is macroscopic embryo
During appearance, culture dish is transferred on the shaking table of rotating speed 60rpm and continues light culture to formation cotyledon type embryoid；Average each flower
Flower bud induces 12.53 embryos.
3. a kind of black dish microspore-isolated culture method according to claim 1, it is characterised in that in the step (2)
Plantlet regenerating step is：
Light culture 20d formed cotyledon type embryoid, select cotyledon type embryoid transfer into addition 30g/L sucrose, 8g/L agar,
In pH5.8, the MS culture mediums of 116 DEG C of autoclave sterilization 30min, cultivated under the conditions of 25 DEG C, illumination 14h/d, form regeneration
Plant；
Embryoid is sprouted the seedling inoculation to be formed to addition 30g/L sucrose, 6g/L agar, 1mg/L6-BA, 0.1mg/LNAA,
In pH5.8, the MS root medias of 116 DEG C of autoclave sterilization 30min, cultivated under the conditions of 25 DEG C, illumination 14h/d, obtained
Whole plant, expands numerous, rooting culture to field.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109328717A (en) * | 2018-10-26 | 2019-02-15 | 安徽农业大学 | A method of promote B. campestris L.ssp. Chinensis to bloom ahead of time |
| CN113973715A (en) * | 2021-11-26 | 2022-01-28 | 安徽农业大学 | Method for improving sporogenous rate of microspores of black-bone vegetables |
| CN114916441A (en) * | 2022-05-24 | 2022-08-19 | 安徽农业大学 | Method for improving germ production rate of free microspores of lindera aggregate by using melatonin |
| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
| CN115024218A (en) * | 2022-07-06 | 2022-09-09 | 安徽农业大学 | Method for improving autotetraploid inductivity of black-bone cabbage and identifying ploidy of black-bone cabbage |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109328717A (en) * | 2018-10-26 | 2019-02-15 | 安徽农业大学 | A method of promote B. campestris L.ssp. Chinensis to bloom ahead of time |
| CN113973715A (en) * | 2021-11-26 | 2022-01-28 | 安徽农业大学 | Method for improving sporogenous rate of microspores of black-bone vegetables |
| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
| CN114916441A (en) * | 2022-05-24 | 2022-08-19 | 安徽农业大学 | Method for improving germ production rate of free microspores of lindera aggregate by using melatonin |
| CN115024218A (en) * | 2022-07-06 | 2022-09-09 | 安徽农业大学 | Method for improving autotetraploid inductivity of black-bone cabbage and identifying ploidy of black-bone cabbage |
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Application publication date: 20170531 |