CN110301358A - A method of obtaining pagoda cauliflower microspore DH regeneration plant - Google Patents
A method of obtaining pagoda cauliflower microspore DH regeneration plant Download PDFInfo
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- CN110301358A CN110301358A CN201910740673.3A CN201910740673A CN110301358A CN 110301358 A CN110301358 A CN 110301358A CN 201910740673 A CN201910740673 A CN 201910740673A CN 110301358 A CN110301358 A CN 110301358A
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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
- 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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- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of methods for obtaining pagoda cauliflower microspore DH regeneration plant, donor pagoda cauliflower plant manages with delicacy by cutting ball sparse branching, bud is promoted well to develop, select suitable bud, separate microspore, culture Isolated microspore to cotyledon shape embryoid is formed, cotyledon shape embryoid is inserted into embryoid differential medium, it is expanded to embryoid and regenerates new plant, regeneration plant is inserted in root media and is cultivated, regeneration plant after taking root moves into matrix, sporule regeneration plant Ploidy detection is carried out after surviving, obtain pagoda cauliflower microspore dihaploid DH (doubled haploid) regeneration plant.The method of the present invention obtains homozygous pagoda cauliflower microspore DH regeneration plant, and microspores culture germ extraction rate reaches as high as 20 embryo/flower buds, the germination rate average out to 50% of embryoid.
Description
Technical field
The invention belongs to field of plant tissue culture technique, are related to a kind of acquisition pagoda cauliflower microspore DH regeneration plant
Method.
Background technique
Bouquet pagoda type cauliflower, i.e. pagoda cauliflower (Brassica oleracea L.var.botrytis
Cv.romanesco), it is a kind of cultivated form of cauliflower, comes across the Rome, ITA in 16th century, therefore named Rome flower earliest
Cabbage.This kind of cauliflower type last century end starts to introduce cultivation in China, and cultivation history is shorter, and grower and consumer are to it
It is also more strange.Pagoda cauliflower is rich in vitamin C, K, and eating mouth feel is the same as generally tight bouquet type cauliflower, but its bouquet shape
Especially, big bouquet is sent out with by the similar small bouquet of shape shaped like Buddha's hair worn in a bun or coil, is arranged in Pei Bonaqie ordered series of numbers point shape, especially attraction eye
Ball.
Light green pagoda cauliflower seed is external import in the market, and the country lacks with independent intellectual property rights excellent
Pagoda Varieties of Cauliflower.This is mainly a lack of, another aspect cauliflower hybridization choosing few to the breeding research of cauliflower pagoda, resource
Educate needs 7~8 years, the time is long, and it is time-consuming, block up pagoda Varieties of Cauliflower breeding process.
In conjunction with the requirement of Chinese artichoke crossbreeding and germplasm innovation, can be obtained in the present age using microspores culture method homozygous
Breeding material, next year is obtained with cross combination, shortens breeding cycle to 3~4 years.Microspore culture is cauliflower
One of the important means of high-efficient breeding is of great significance to raising pagoda cauliflower breeding efficiency and level.
The microspore culture of crop in cruciferae is more mature, and cauliflower microspore culture also has more research, but
There is also many difficulties needs to capture, mainly the internal factors such as cultivated form, genotype limitation, donor flower bud development situation,
And the external factors such as culture medium, culture control condition.The pagoda cauliflower of personality type, which is that one kind is more difficult, carries out embryo
One of genotype of induction, mainly pagoda cauliflower resource are few, pagoda cauliflower bolting florescence and common cauliflower difference
The problems such as larger, bennet is thick and inflorescence is few, while pagoda cauliflower flower bud development is irregular, and bud is big but pollen is few, so far state
The inside and outside report that there are no pagoda cauliflower microspore culture and successfully obtain regeneration plant.The application passes through to pagoda character inheritance
Rule is studied, and by directly cultivating or hybridize with the cauliflower for being higher by embryo, or is hybridized with the cauliflower of different colours, to it
Offspring's pagoda cauliflower Isolated microspore is cultivated, and successfully obtains a certain number of dihaploid pagoda regeneration plants, therefrom
The preparation of cross combination can be carried out directly as breeding parent by filtering out the excellent single plant of character, to cultivate there is independent intellectual to produce
The pagoda Varieties of Cauliflower of power provides technology and materials for support.
Summary of the invention
The object of the present invention is to provide a kind of methods that pagoda type cauliflower microspore culture obtains DH regeneration plant, build
Vertical pagoda cauliflower microspore culture regenerating system, to accelerate the pagoda cauliflower breeding process and efficiency of multiple color.
In order to realize that above-mentioned technical effect, the present invention are realized especially by following technical scheme:
A method of obtaining pagoda cauliflower microspore DH regeneration plant, comprising the following steps:
1) selection of donor plant and bud
With fertile pagoda cauliflower or its F hybridized with high germ extraction rate cauliflower1In generation, is used as microspores culture donor, plants
Strain bouquet carried out cutting ball in nearly harvest time, carried out sparse branching to bennet, carries out dredging flower bud to inflorescence, keeps plant health-nutrition, promotes
Bud is well developed；
2) separation of bud microspore
After bud sterilization treatment, embryoid induction culture medium is added, suspension is made, filtrate is collected by filtration, centrifugation is abandoned
Supernatant is added active carbon mixed liquor and is compounded into microspore suspension；
3) culture of microspore embryoid
Microspore suspension is placed in constant incubator after dark culture to embryoid appearance, shaken cultivation to cotyledon shape embryo
Shape body is formed；
4) differentiation and sprouting of regeneration plant
Cotyledon shape embryoid is inserted into embryoid differential medium, until embryoid expands and regenerates new plant；
5) culture of rootage and transplanting of regeneration plant
Cutting sprouting has the regeneration plant of normal growth point to insert in root media, culture of rootage is carried out, after taking root
Regeneration plant move into matrix in, cover thin film moisturizing culture；
6) Ploidy detection of regeneration plant
The tender leaf for taking transplant survival regeneration plant detects the DNA ploidy of the regeneration plant with the ploidy analyser, is such as detected as
Dihaploid (doubled haploid, DH), as DH regeneration plant.
Further, with fertile pagoda cauliflower or its F hybridized with high germ extraction rate cauliflower1In generation, trains as microspore
Donor is supported, petal and the anther length ratio of the bud are between 1.0~1.4, in monokaryon advanced stage to the flower of double-core early stage
Flower bud.
Further, the embryoid induction culture medium are as follows: NLN-13 fluid nutrient medium+sucrose 130g/L, pH5.6~
6.0, filtration sterilization.
Further, the active carbon mixed liquor are as follows: NLN-13 fluid nutrient medium+1g/L active carbon, high-temperature sterilization two
It is secondary.
Further, active carbon mixed liquor is added according to the ratio of 0.1mL/10mL embryoid induction culture medium.
Further, the culture of microspore embryoid specifically: the culture dish dispensed is placed in 31-33 DEG C of constant temperature incubation
In case, dark culture 24-48 hours；It is placed on 25 DEG C of constant incubators, dark culture 15-20 days to the visible embryoid of naked eyes occurs；
50rpm shaken cultivation 7-10 days under 25 DEG C of dark again, until cotyledonary embryos shape body is formed.
Further, the embryoid differential medium are as follows: MS culture medium+NAA0.01~0.06mg/L+BAP0.1~
0.5mg/L+ sucrose 30g/L+ agar 10g/L, pH5.6~6.0, high-temperature sterilization.
Further, the root media are as follows: MS culture medium+NAA0.05~0.1mg/L+ sucrose 30g/L+ agar
9g/L, pH5.8, high-temperature sterilization.
The invention has the benefit that
The present invention establishes the microspores culture method of green, orange purple pagoda cauliflower DH regeneration plant production for the first time,
The germ extraction rate of pagoda cauliflower microspore culture reaches as high as 20 embryo/flower buds, the germination rate of embryoid, average out to 50%, regeneration
Plant diploid rate reaches 40%, provides technology and material branch to cultivate pagoda Varieties of Cauliflower with independent intellectual property rights
Support has practical significance to the breeding process and efficiency of accelerating pagoda cauliflower.
Specific embodiment
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
The green pagoda cauliflower microspore culture regeneration plant of embodiment 1
1. the preparation of culture medium:
1.1 embryoid induction culture mediums: NLN-13 fluid nutrient medium+sucrose 130g/L, pH6.0, filtration sterilization, wherein
NLN-13 culture medium prescription is shown in Table 1.
1.2 embryoid differential mediums: MS culture medium+NAA0.02mg/L+BAP0.3mg/L+ sucrose 30g/L+ agar 7g/
L, pH6.0, high-temperature sterilization, wherein MS culture medium prescription is shown in Table 1.
It 1.3 sprouts, root media: MS culture medium+NAA0.05mg/L+ sucrose 30g/L+ agar 7g/L, pH5.8, high temperature
Sterilizing.
1 NLN-13 and MS culture medium prescription of table
2. the culture of green pagoda cauliflower microspore regeneration plant
2.1 donor plant management and bud select: using the green fertile germ plasm resource of pagoda cauliflower as donor material,
Or its F for hybridizing with the tight bouquet type white cauliflower material that microspores culture embryo has occurred1In generation, is used as donor material.For
Body plant strain growth selects the balanced sufficient, plant of NPK nutrition and flower bud growth health, the plant without the obvious state of an illness in plastic greenhouse
Donor is made in strain, selects just open 1-2 colored inflorescence, and therefrom winning petal with anther length ratio is 1.2, is kept to the side in monokaryon
10, the bud of phase to double-core early stage.
The sterilizing of 2.2 buds: it is first cleaned bud 3 times with pure water, then puts it into sterilized solution (0.1%HgCl2+ 0.1%
Polysorbas20) in, shaking table slight wobble (50rpm) carries out surface sterilization 12 minutes, then uses sterile water wash 4 times on the super-clean bench
Afterwards, spare.
Separation, mixture and the packing of 2.3 bud microspores: the bud after sterilizing is placed in sterile beaker on the super-clean bench
In, 1mL embryoid induction culture medium is added, is gently embossed flower bud with tack glass rod, then adds 5mL same medium, stir into outstanding
Supernatant liquid；By the suspension with 40 μm of sterile nylon strainer filterings in centrifuge tube, 1000rpm is centrifuged 4 minutes, abandons supernatant；Again
5mL embryoid induction culture medium is added to be centrifuged in the same way and abandon supernatant；Embryoid induction culture medium is added, is adjusted to small spore
Sub- density is 2.0 × 106A microspore/ml.Active carbon mixed liquor is added in 0.1mL/10mL culture medium ratio again, and (formula is shown in
On), it is compounded into microspore suspension；The microspore suspension is sub-packed in the sterile petri dish that diameter is 6cm, is used after capping
The sealing of parafilm film, it is spare.
The culture of 2.4 microspore embryoids: the culture dish dispensed is placed in 33 DEG C of constant incubators, dark culture 24 is small
When；It is placed on 25 DEG C of constant incubators, dark culture 10-15 days to embryoid occurs；The 50rpm shaken cultivation under 25 DEG C of dark again
7-10 days, until cotyledonary embryos shape body is formed.
The differentiation and sprouting of 2.5 regeneration plants are cultivated: by cotyledon type embryoid in super-clean bench, lying against embryoid differentiation
On culture medium, is cultivated at daily illumination 16 hours, 25 DEG C and expanded to embryoid within 15 days and regenerate new plant.
The culture of rootage and transplanting of 2.6 regeneration plants: cutting sprouting has the regeneration plant of normal growth point to insert in training of taking root
It supports in base, carries out culture of rootage at daily illumination 16 hours, 25 DEG C；Regeneration plant after taking root, which moves into, contains peat and pearl
In matrix of the rock by 2 ︰ 1 of volume preparation, sprinkles profoundly water, cultivated 1 week at 25 DEG C after plastic covering film.
The Ploidy detection of 2.7 regeneration plants: taking the tender leaf of transplant survival regeneration plant, is examined with partecPA the ploidy analyser
Survey the DNA ploidy of the regeneration plant.
Totally 18 plants of green pagoda cauliflower microspore regeneration plant obtained with the example method culture, through Ploidy detection,
The regeneration plant of middle diploid shares 8 plants, and diploid rate accounts for the 44.4% of total strain number.
2 purple pagoda cauliflower microspore culture regeneration plant of embodiment
In the present embodiment, by the green fertile germ plasm resource of pagoda cauliflower its with the tight bouquet type cauliflower material of purple
The F of hybridization1In generation, is used as donor material.It chooses flower bud development and be in 10 buds based on mid-late uninucleate stage and double-core early stage, it is colored
Valve and anther length ratio are 1.3.Microspore separation is carried out after bud sterilizing, embryoid induction culture medium is then added, is adjusted to
Microspore density is 4.0 × 106A microspore/ml.Active carbon mixed liquor (formula is added in 0.1mL/10mL culture medium ratio again
On seeing).It is compounded into microspore suspension, which is sub-packed in the aseptic plastic that diameter is 6cm by 4mL/ ware and is trained
It supports in ware, is sealed after capping with parafilm film, cultivated 24 hours under the conditions of culture dish is placed in 32.5 DEG C；The embryoid of acquisition
It is placed in embryoid differential medium MS culture medium+NAA0.05mg/L+BAP0.4mg/L+ sucrose 30g/L+ agar 8g/L, pH5.8
In, it is cultivated under the conditions of daily 25 DEG C of illumination in 16 hours 18 days and goes out plant to somatic embryogenesis；NLN-13, MS culture medium prescription are same
Table 1；Remaining process is same as embodiment 1.
The orange pagoda cauliflower microspore culture regeneration plant of embodiment 3
In the present embodiment, by the green fertile germ plasm resource of pagoda cauliflower, it hybridizes with orange type cauliflower material
F1In generation, is used as donor material.It chooses flower bud development and is in 10 buds based on mid-late uninucleate stage and double-core early stage, petal and flower
Medicine length ratio is 1.2.Microspore separation is carried out after bud sterilizing, embryoid induction culture medium is then added, is adjusted to microspore
Density is 5.0 × 106A microspore/ml.Active carbon mixed liquor (formula see on) is added in 0.1mL/10mL culture medium ratio again.
It is compounded into microspore suspension, which is sub-packed in the aseptic plastic culture dish that diameter is 6cm by 4mL/ ware,
It is sealed after capping with parafilm film, is cultivated 48 hours under the conditions of culture dish is placed in 32 DEG C；The embryoid of acquisition is placed in embryo shape
In body differential medium MS culture medium+NAA0.05mg/L+BAP0.2mg/L+ sucrose 30g/L+ agar 8g/L, pH6.0, daily
It is cultivated under the conditions of 25 DEG C of illumination in 16 hours 18 days and goes out plant to somatic embryogenesis；NLN-13, MS culture medium prescription are the same as table 1；Remaining
Process is same as embodiment 1.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Claims (8)
1. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant, which comprises the following steps:
1) selection of donor material and bud
With fertile pagoda cauliflower or its F hybridized with high germ extraction rate cauliflower1In generation, is used as microspores culture donor, plant
Ball carried out cutting ball in nearly harvest time, carried out sparse branching to bennet, carries out dredging flower bud to inflorescence, keeps plant health-nutrition, promotes bud
Good development；
2) separation of bud microspore
After bud sterilization treatment, embryoid induction culture medium is added, suspension is made, filtrate is collected by filtration, supernatant is abandoned in centrifugation
Liquid is added active carbon mixed liquor and is compounded into microspore suspension；
3) culture of microspore embryoid
Microspore suspension is placed in constant incubator after dark culture to embryoid appearance, shaken cultivation to cotyledon shape embryoid
It is formed；
4) differentiation and sprouting of regeneration plant
Cotyledon shape embryoid is inserted into embryoid differential medium, until embryoid expands and regenerates new plant；
5) culture of rootage and transplanting of regeneration plant
Cutting sprouting has the regeneration plant of normal growth point to insert in root media, carries out culture of rootage, after taking root again
Raw plant moves into matrix, covers thin film moisturizing culture；
6) Ploidy detection of regeneration plant
The tender leaf for taking transplant survival regeneration plant detects the brilliance DNA ploidy of the regeneration plant with the ploidy analyser, is such as detected as
Dihaploid, as DH regeneration plant.
2. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
With fertile pagoda cauliflower or its F hybridized with high germ extraction rate cauliflower1In generation, is used as microspores culture donor, the bud
Petal and anther length ratio between 1.0~1.4, in monokaryon advanced stage to the bud of double-core early stage.
3. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
The embryoid induction culture medium are as follows: NLN-13 fluid nutrient medium+sucrose 130g/L, pH 5.6~6.0, filtration sterilization.
4. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
The active carbon mixed liquor are as follows: NLN-13 fluid nutrient medium+1g/L active carbon, high-temperature sterilization is twice.
5. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
Active carbon mixed liquor is added according to the ratio of 0.1mL/10mL embryoid induction culture medium.
6. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
The culture of microspore embryoid specifically: the culture dish dispensed is placed in 31-33 DEG C of constant incubator, dark culture 24-48
Hour；It is placed on 25 DEG C of constant incubators, dark culture 15-20 days to the visible embryoid of naked eyes occurs；Again under 25 DEG C of dark
50rpm shaken cultivation 7-10 days, until cotyledon shape embryoid is formed.
7. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
The embryoid differential medium are as follows: MS culture medium+NAA 0.01~0.06mg/L+BAP, 0.1~0.5mg/L+ sucrose
30g/L+ agar 10g/L, pH 5.6~6.0, high-temperature sterilization.
8. a kind of method for obtaining pagoda cauliflower microspore DH regeneration plant according to claim 1, which is characterized in that
The root media are as follows: MS culture medium+NAA 0.05~0.1mg/L+ sucrose 30g/L+ agar 9g/L, pH 5.8, high temperature
Sterilizing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114831023A (en) * | 2022-03-28 | 2022-08-02 | 浙江省农业科学院 | Efficient genetic transformation method for cauliflower by taking flower stalks as explants |
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