CN110089426A - A method of cultivating Chinese cabbage microspore plant - Google Patents
A method of cultivating Chinese cabbage microspore plant Download PDFInfo
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- CN110089426A CN110089426A CN201810085112.XA CN201810085112A CN110089426A CN 110089426 A CN110089426 A CN 110089426A CN 201810085112 A CN201810085112 A CN 201810085112A CN 110089426 A CN110089426 A CN 110089426A
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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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The present invention discloses a kind of method for cultivating Chinese cabbage microspore plant, specifically comprises the following steps: the selection of (1) bud: choosing petal and anther quantity than the bud for 1:1；(2) it cultivates microspore embryo: the bud of step (1) acquisition is sterilized 10-15 minutes with liquor natrii hypochloritis, aqua sterilisa cleaning, NLN-13 culture medium rinse；Bud after rinse is fully ground, filter, centrifugation, containing 10-30mg/L boron substance NLN-13 culture medium dilution, be dispensed into culture dish and active carbon be added, Heat thermostability, 24-26 DEG C dark culture 14-16 days, formation embryoid；(3) mature embryo germination is at plant: embryoid being carried out shaken cultivation, the green mature embryo of its transfer is transferred in solidified MS media and is cultivated, can directly carry out acclimatization and transplants after cultivating 38-42d.The method of the invention simplifies operating procedure, greatly reduces the operating time, improve plant production efficiency in the case where that can guarantee germ extraction rate.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of to cultivate Chinese cabbage microspore plant
Method.
Technical background
Pakchoi is mainly cross-pollinatd plant, and in traditional breeding method work, needing to obtain purifying parent can only be by normal
Rule selfing, due to inbreeding depression, the breeding of homozygous parent is exactly an extremely difficult problem, and with duration, labor intensive
And financial resources, breeding efficiency are very low.Microspore culture is using cellular omnipotency, by Single cell culture at homozygous plants
Process, can effectively shorten the breeding time limit, a large amount of homozygous plants are achieved in the very short time, are greatly improved
Breeding efficiency.Lichter1982 has found on cabbage type rape for the first time, can obtain embryo shape using microspore culture
Body constantly obtains new progress in terms of this Vegetables in Brassica microspores culture, at present in Brassica genus broccoli, Chinese cabbage, sweet
Success is all achieved in the kinds such as indigo plant, Chinese cabbage.In China, the opposite starting of this technology is slower, is mainly using for reference
In external knowledge, grow rapidly, Cao Mingqing is successfully cultivated using isolated microspore culture technique for the first time within 1993
Embryoid, this later technology all made breakthrough progress in many kinds of Brassica genus in China's fast development,
And sporule regeneration plant is obtained in the broccoli, cabbage mustard, cauliflower of Brassica genus.But the current technology is relatively complicated,
Germ extraction rate is low, and pollution problem is more serious.
Summary of the invention
The problems such as present invention is cumbersome primarily directed to the current technology, and germ extraction rate is low and pollutes improves, thus
Provide a kind of method of the cultivation Chinese cabbage microspore plant of high-efficient simple.
The technical solution that the present invention takes: a method of Chinese cabbage microspore plant is cultivated, is specifically included as follows
Step:
(1) petal and anther length the selection of bud: are chosen than the bud for 1:0.5~2；
(2) it cultivates microspore embryo: the bud of step (1) acquisition being sterilized 10-15 minutes with liquor natrii hypochloritis, sterilizing
Water cleaning, NLN-13 culture medium rinse；Bud after rinse is fully ground, is filtered, centrifugation, yellow-green precipitate is trained with NLN-13
Support base dilution, be dispensed into Heat thermostability in culture dish, 24-26 DEG C dark culture 14-16 days, formation embryoid；
(3) mature embryo germination is at plant: embryoid being carried out shaken cultivation, the green mature embryo of its transfer is transferred to solid
38-42d is cultivated in MS culture medium, directly progress acclimatization and transplants.
Petal and anther length are chosen in step (1) of the present invention than the bud for 1:0.5~2, preferred length is than for 1:1's
Bud, above-mentioned selection petal and anther ratio are in the cell of monokaryotic stage and mid-late uninucleate stage close to the bud in this period of 1:1
At most, the bud in the period is selected, germ extraction rate is high.The preferred materials time is the acquisition of 9 points to 11 points of morning progress samples, this
When ambient temperature it is low, plant moisture content is high, and pollen activity is strong, by the bud of selection, adds distilled water and is put into 4 DEG C of refrigerators
In, pollen activity is kept as far as possible.
It is thimerosal that step (2) of the present invention, which selects liquor natrii hypochloritis, and liquor natrii hypochloritis's mass concentration is 1%,
Gained can be prepared by the following method: 5.6% liquor natrii hypochloritis 2mL is taken, with distilled water constant volume to 11.2mL.With 1%
Liquor natrii hypochloritis is substituted the process that conventionally used alcohol and mercuric chloride carry out disinfection, and method is simple and to reduce pollution several
Furthermore rate also reduces environmental pollution.
Step (2) of the present invention selects NLN-13 culture medium that B5 medium is replaced to carry out rinse and grinding, reduces culture medium
Process for preparation, and with traditional culture solution, instead of lapping liquid.
Step (2) of the present invention is fully ground optional sample grinding machine, such as model are as follows: German LKA control type test tube dispersion machine
S025, filter can be selected cell filter, the diameter of cell filter be preferably 22-26mm, aperture be 38-42 μm, it is more excellent
It is selected as diameter 24mm, aperture is 40 μm.
It is 10 that yellow-green precipitate, which is preferably diluted to cell number, in step (2) of the present invention5A every mL.
Heat thermostability is specially 32-34 DEG C of Heat thermostability 1-2 days in step (2) of the present invention；Heat thermostability used medium
For the NLN-13 culture medium of the active carbon containing 30mg/L containing boron substance and 0.8-1.2mg/mL；Preferably comprise 30mg/L boracic object
The NLN-13 culture medium of the active carbon of matter and 1mg/mL.Inventor, which has found to select, contains boron substance and 0.8-1.2mg/ containing 30mg/L
The NLN-13 culture medium of the active carbon of mL, can increase substantially microspore germ extraction rate, and the presence of active carbon can effectively inhale
The harmful substance being metabolized in attached cell growth process, and boron element can promote the formation of cell wall and cell membrane, to promote
The formation of embryoid.
Culture medium used in dark culture is containing 30mg/L containing boron substance and 0.8-1.2mg/mL in step (2) of the present invention
The NLN-13 culture medium of active carbon；Preferably comprise the NLN-13 culture medium of active carbon of the 30mg/L containing boron substance and 1mg/mL.Hair
Bright people has found the NLN-13 culture medium of active carbon of the selection containing 30mg/L containing boron substance and 0.8-1.2mg/mL, can be significantly
Improve microspore germ extraction rate, the harmful substance that the presence of active carbon can be metabolized in effective adherent cell growth course, and boron
Element can promote the formation of cell wall and cell membrane, to promote the formation of embryoid.
The boron substance of the present invention that contains is boric acid.
Culture medium used in shake culture is to contain boron substance and 0.8-1.2mg/mL containing 30mg/L in step (3) of the present invention
Active carbon NLN-13 culture medium；Preferably comprise the NLN-13 culture medium of active carbon of the 30mg/L containing boron substance and 1mg/mL.
Inventor has found the NLN-13 culture medium of active carbon of the selection containing 30mg/L containing boron substance and 0.8-1.2mg/mL, can be substantially
Degree improves microspore germ extraction rate, the harmful substance that the presence of active carbon can be metabolized in effective adherent cell growth course, and
Boron element can promote the formation of cell wall and cell membrane, to promote the formation of embryoid.
Solidified MS media in step (3) of the present invention is prepared by the following method gained: in regular MS media+
+ 1.2% agar of+3% sucrose of 0.02mg/L NAA+2mg/L 6-BA+1% active carbon, which can be simple and effective, only needs
Once to turn embryo and be achieved with root system stalwartness, the tissue-cultured seedling of robust plant, and vitrifying and browning are less.
The utility model has the advantages that compared with present microspore culture, advantages of the present invention:
This method is mainly simplified in former incubation step, and a kind of efficiently succinct method, this method master are invented
Will be: 1) generation 2 of microspore embryo) at plant, the two aspects are improved and have been simplified for mature embryo germination.1, with 1%
Liquor natrii hypochloritis the process that conventionally used alcohol and mercuric chloride carry out disinfection is substituted, reduce the pollution of environment.2, it uses
NLN-13 cultivates the process that liquid culture medium replaces traditional B5 grinding culture medium to carry out grind away rinse.3, it takes the lead in during grind away
It has used sample grinding machine and cell to cross rate device, the operating time is greatly reduced.4, boron element influences microspore germ extraction rate higher, adds
Boron element is added to can be improved one times of germ extraction rate.And active carbon is mainly after microspore heat shock, 0-4 days to microspore germ extraction rate
Influence it is higher, and have 3-4 times of raising.5, embryo culture at+NAA+6-BA+ active carbon+sucrose in the MS culture medium of plant+
Agar.In the case where that can guarantee germ extraction rate, simplify operating procedure, greatly reduce the operating time, once turn embryo directly at
Seedling improves the time of plant production.
Detailed description of the invention
Fig. 1 is that microspore goes out embryo situation；
Fig. 2 is to cultivate 3 days on MS solid medium；
Fig. 3 is to cultivate 7 days on MS solid medium；
Fig. 4 is to cultivate 30 days on MS solid medium；
Fig. 5 is to cultivate 40 days on MS solid medium.
Fig. 6 is the influence of boron element and active carbon processing to microspore germ extraction rate；
Fig. 7 is the influence of boron element and active carbon processing to ' fast dish ' kind microspore germ extraction rate.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.
Embodiment 1
1, the selection of bud: carrying out the acquisition of sample in 9 points to 11 points of morning, chooses petal and anther ratio close to 1:1
Bud, add a small amount of distilled water and be stored in 4 DEG C of refrigerators.
2, it cultivates microspore embryo: the sample being collected is put into the centrifuge tube of 2ml, it is direct with 1% sodium hypochlorite
Addition carries out disinfection 10-15 minutes, is cleaned 2 times with aqua sterilisa, carries out rinse with NLN-13 for the last time.Cleaned sample
It is transferred in the test tube of sample grinding machine (German LKA control type test tube dispersion machine S025), a small amount of NLN-13 is added, carry out grind away, make
Sample is fully ground.The centrifuge tube of 50mL is got out, places 40 μm of cell filtration plug filtering above it, centrifugation is gone
Clear liquid adds NLN-13 concussion and shakes up, then is centrifuged, and outwells supernatant, leaves yellow-green precipitate.It is so a small amount of to being added in rear test tube
NLN-13, be diluted to cell number be 105A every mL.It is dispensed into the culture dish that diameter is 6mm, the activity of 1mg/mL is added
Charcoal is sealed with sealed membrane.33 DEG C of 1 day Heat thermostabilities are carried out, is put into 25 DEG C of environment and carries out dark culture 15 days after taking out.
3, mature embryo germination is at plant: after dark culture 15 days, embryoid being placed on 2000x, 25 DEG C, the shaking table of 60r/min
The green mature embryo of its transfer, is transferred to solidified MS media and (adds 0.02mg/ in regular MS media by upper carry out shaken cultivation
L NAA, addition 2mg/L 6-BA, addition 1% active carbon, addition 3% sucrose, addition 1.2% agar) in cultivated, cultivate
After 40d, plant grows up to root system stalwartness, and when 3-5 piece true leaf can directly carry out acclimatization and transplants.
In the present embodiment, culture medium used in Heat thermostability, dark culture and shaken cultivation is containing 10-30mg/L boracic
The NLN-13 culture medium of the active carbon of substance and 1mg/mL.
The present embodiment method therefor and the comparison of traditional operation time are as shown in table 1:
1 the method operating time of 1 embodiment of table and traditional operation time compare
Therefore the method for the invention substantially reduces the operating time, once turns the direct seedling of embryo, improves plant
The time of production.
Embodiment 2: the culture of different cultivars Chinese cabbage microspore
Choosing 9 different cultivars, (fast dish arrow shaft is white, ultramarine, two white, green stalks white, Chinese little greens, heart of a cabbage, Wuta-tsai, May
Slowly microspores culture test is carried out using 1 the method for embodiment), their germ extraction rate is counted, compares the invention to microspore
The influence of germ extraction rate.Wherein arrow shaft is white, ultramarine, two Bai Weiyi go out embryo kind, and blueness stalk is white, Chinese little greens, heart of a cabbage are that can go out embryo product
Kind, heart of a cabbage, Wuta-tsai, May are to be not easy out embryo kind slowly.Such as Fig. 1, such as embryo is contrasted as a result, in 9 kinds of selection, out
Embryo rate has reached 100%, wherein 3 be not easy out embryo kind all go out embryo, and easily go out the fast dish of embryo kind reached 13.62 embryos/
Flower bud.
If Fig. 4 to Fig. 7 is easily to go out growing state figure of the fast dish of embryo kind on the MS culture medium after improvement respectively.
Embodiment 3: the influence of boron element and active carbon to different cultivars Chinese cabbage microspore germ extraction rate
2 high kinds of germ extraction rate are had chosen, the research of boron element and active carbon to microspore germ extraction rate has been carried out, are such as schemed
6, it is divided into four groups, control (NLN-13), only addition active carbon only add boron element and add boron element and active carbon simultaneously,
For comparing result it can be found that when what is not all added, the germ extraction rate of two kinds is all very low, adds active carbon and boron element respectively
Afterwards, all have different degrees of raising to germ extraction rate, and after adding active carbon and boron element simultaneously, compare individually addition boron element and
Active carbon, two kinds are all significantly improved, and the 4-5 times of Fig. 7 that be higher by than control is kind ' fast dish ' control (NLN-13) and product
After kind ' fast dish ' adds active carbon and boron element simultaneously, embryo situation map is contrasted.
Claims (10)
1. a kind of method for cultivating Chinese cabbage microspore plant, it is characterised in that specifically comprise the following steps
(1) petal and anther length the selection of bud: are chosen than the bud for 1:0.5~2；
(2) it cultivates microspore embryo: the bud of step (1) acquisition being sterilized 10-15 minutes with liquor natrii hypochloritis, aqua sterilisa is clear
It washes, NLN-13 culture medium rinse；Bud after rinse is fully ground, is filtered, centrifugation, yellow-green precipitate NLN-13 culture medium
Dilution, is dispensed into Heat thermostability in culture dish, 24-26 DEG C dark culture 14-16 days, formation embryoid；
(3) mature embryo germination is at plant: embryoid being carried out shaken cultivation, the green mature embryo of its transfer is transferred to solid MS training
It supports in base and cultivates 38-42d, directly progress acclimatization and transplants.
2. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that choosing in step (1)
Take petal and anther length than the bud for 1:1；The time for choosing bud is 9 points to 11 points of morning.
3. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that step (2) is selected
Liquor natrii hypochloritis's mass concentration is 1%.
4. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that step (2) is abundant
Sample grinding machine is selected in grinding；Cell filter is selected in filtering；The diameter of the cell filter is preferably 22-26mm, aperture 38-
42μm；More preferably diameter 24mm, aperture are 40 μm.
5. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that yellow in step (2)
It is 10 that green precipitate, which is diluted to cell number,5A every mL.
6. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that hot in step (2)
Swash processing be specially 32-34 DEG C Heat thermostability 1-2 days.
7. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that hot in step (2)
Swash the NLN-13 that culture medium used in processing and dark culture is the active carbon containing 30mg/L containing boron substance and 0.8-1.2mg/mL
Culture medium.
8. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that shake in step (3)
Swing the NLN-13 culture medium that culture culture medium used is the active carbon containing 30mg/L containing boron substance and 0.8-1.2mg/mL.
9. according to the described in any item methods for cultivating Chinese cabbage microspore plant of claim 6-8, it is characterised in that institute
Stating containing boron substance is boric acid.
10. the method according to claim 1 for cultivating Chinese cabbage microspore plant, it is characterised in that in step (3)
The proportion of solidified MS media are as follows: MS culture medium, 0.02mg/L NAA, 2mg/L 6-BA, 1% active carbon, 3% sucrose and
1.2% agar.
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Cited By (4)
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| CN110547193A (en) * | 2019-09-12 | 2019-12-10 | 南京农业大学 | Method for cultivating non-heading Chinese cabbage microspore plant |
| CN113080065A (en) * | 2021-05-19 | 2021-07-09 | 南京农业大学 | Method for cultivating non-heading Chinese cabbage microspore plant |
| CN113711920A (en) * | 2021-09-26 | 2021-11-30 | 安徽农业大学 | Method for improving one-time seedling rate of microspore embryoid of non-heading Chinese cabbage |
| CN117121809A (en) * | 2022-05-19 | 2023-11-28 | 南京农业大学 | Method for cultivating microspore plant of non-heading Chinese cabbage |
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| CN117121809A (en) * | 2022-05-19 | 2023-11-28 | 南京农业大学 | Method for cultivating microspore plant of non-heading Chinese cabbage |
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