CN102228003B - Culture method for brassica oleracea L. var. acephala microspore regeneration plant - Google Patents
Culture method for brassica oleracea L. var. acephala microspore regeneration plant Download PDFInfo
- Publication number
- CN102228003B CN102228003B CN201110112693XA CN201110112693A CN102228003B CN 102228003 B CN102228003 B CN 102228003B CN 201110112693X A CN201110112693X A CN 201110112693XA CN 201110112693 A CN201110112693 A CN 201110112693A CN 102228003 B CN102228003 B CN 102228003B
- Authority
- CN
- China
- Prior art keywords
- kale
- bud
- culture
- embryoid
- nln
- 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.)
- Expired - Fee Related
Links
- 240000007124 Brassica oleracea Species 0.000 title claims abstract description 85
- 241000196324 Embryophyta Species 0.000 title claims abstract description 59
- 230000008929 regeneration Effects 0.000 title claims abstract description 45
- 238000011069 regeneration method Methods 0.000 title claims abstract description 45
- 241000017163 Acephala Species 0.000 title abstract description 7
- 235000011302 Brassica oleracea Nutrition 0.000 title abstract description 7
- 238000012136 culture method Methods 0.000 title abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 32
- 239000000725 suspension Substances 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000002609 medium Substances 0.000 claims abstract description 21
- 239000006160 differential media Substances 0.000 claims abstract description 17
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 claims description 78
- 235000012905 Brassica oleracea var viridis Nutrition 0.000 claims description 78
- 230000001954 sterilising effect Effects 0.000 claims description 34
- 230000001939 inductive effect Effects 0.000 claims description 29
- 238000000605 extraction Methods 0.000 claims description 21
- 229930006000 Sucrose Natural products 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 210000001161 mammalian embryo Anatomy 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- 239000006870 ms-medium Substances 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 238000005286 illumination Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 230000008021 deposition Effects 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 5
- 206010027336 Menstruation delayed Diseases 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 230000004069 differentiation Effects 0.000 claims description 5
- 239000000575 pesticide Substances 0.000 claims description 5
- 230000002786 root growth Effects 0.000 claims description 5
- 230000035939 shock Effects 0.000 claims description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- 229940064880 inositol 100 mg Drugs 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 229940088594 vitamin Drugs 0.000 claims description 3
- 235000013343 vitamin Nutrition 0.000 claims description 3
- 229930003231 vitamin Natural products 0.000 claims description 3
- 239000011782 vitamin Substances 0.000 claims description 3
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 3
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 claims description 2
- 108010024636 Glutathione Proteins 0.000 claims description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 229930003451 Vitamin B1 Natural products 0.000 claims description 2
- 229930003571 Vitamin B5 Natural products 0.000 claims description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 2
- 229960002079 calcium pantothenate Drugs 0.000 claims description 2
- 230000003203 everyday effect Effects 0.000 claims description 2
- 229940095463 folic acid 0.5 mg Drugs 0.000 claims description 2
- 229960003180 glutathione Drugs 0.000 claims description 2
- 238000007670 refining Methods 0.000 claims description 2
- 125000000185 sucrose group Chemical group 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 235000010374 vitamin B1 Nutrition 0.000 claims description 2
- 239000011691 vitamin B1 Substances 0.000 claims description 2
- 235000009492 vitamin B5 Nutrition 0.000 claims description 2
- 239000011675 vitamin B5 Substances 0.000 claims description 2
- 229940033203 vitamin b6 0.5 mg Drugs 0.000 claims description 2
- 238000001914 filtration Methods 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract description 2
- 210000002257 embryonic structure Anatomy 0.000 abstract 5
- 239000012530 fluid Substances 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 1
- 238000002635 electroconvulsive therapy Methods 0.000 abstract 1
- 239000000706 filtrate Substances 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 abstract 1
- 239000012882 rooting medium Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 238000009395 breeding Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 6
- 239000011521 glass Substances 0.000 description 5
- 239000004033 plastic Substances 0.000 description 5
- 229920003023 plastic Polymers 0.000 description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 239000005708 Sodium hypochlorite Substances 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000011888 foil Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000000306 component Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229960002523 mercuric chloride Drugs 0.000 description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 241000219198 Brassica Species 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241001674939 Caulanthus Species 0.000 description 1
- 241000736199 Paeonia Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 240000005001 Paeonia suffruticosa Species 0.000 description 1
- 235000003889 Paeonia suffruticosa Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a culture method for a brassica oleracea L. var. acephala microspore regeneration plant. The method comprises the following steps: taking inflorescences of brassica oleracea L. var. acephala and rape, directly taking or taking after induction alabastrum of the inflorescences, adding the alabastrum into NLN-13 induced medium after disinfection to form fluid suspension, carrying out filtering, and centrifuging the filtrate to obtain a precipitate; adding NLN-13 induced medium and active carbon mixed liquor in order so as to obtain a microspore fluid suspension; carrying out a heat shock treatment, followed by culturing so as to obtain embryoids in cotyledon stage; inoculating the embryoids to embryoid differential medium until the embryoids are differentiated and regenerates into buds; cutting the regenerated buds to a rooting medium for rooting culture, and hardening and transplanting seedlings to obtain regenerated plants; taking young leaves of the regenerated plants for the detection of ploidy of corresponding regenerated plants and determining regenerated seedlings of rape and brassica oleracea L. var. acephala in the regenerated plants. According to the method provided in the invention, rape which is easy to generate embryos and brassica oleracea L. var. acephala which is difficult to generate embryos are mixedly cultured; the material which is easy to generate embryos is used to spur the material which is difficult to generate embryos; therefore the ratio of embryos of brassica oleracea L. var. acephala is improved.
Description
Technical field
The present invention relates to field of plant tissue culture technique, relate in particular to a kind of cultural method of kale sporule regeneration plant.
Background technology
Kale (Brassica oleracea L.var.acephala) has another name called the leaf tree peony; Being the mutation that the Cruciferae rape belongs to brassica specie, is 2 years these plants of sward, and its leaf morphology is attractive in appearance, color is changeable; Whole plant shape such as peony, ornamental value is high.In recent years, how China introduced kale from the U.S., Japan, Holland and other places, because of kale contains a large amount of vitamin As, C, B
2And several mineral materials, particularly calcium, iron, potassium content are very high, and its nutritive value is done the special vegetable cultivation far above common wild cabbage more.Because some kale kind leaf look bright-coloured beauty, red, yellow, green alternate, shrinkage is special; Come in every shape, can be used to decorate the dining table of dinner party, arrange Urban Parks and large-scale flower bed; Beautify central plaza and commercial frontage, therefore, be widely used in seeing the leaf cultivation again.
Kale originates in Mediterranean and little Ya Xiya one band, and Britain, Holland, Germany and U.S.'s plantation are more.China also began introducing and planting and developmental research in recent years, and seed relies on import basically at present, cost an arm and a leg, and cost is high, and tracing it to its cause is because our kale breeding resource is few, can not educate good kind.Traditional breeding method is to carry out the selfing separation through introducing external improved seeds, and the cycle is long, needs the manpower and materials of labor, but the seed selection of uncertain ability is to good kind.Therefore press for the innovation breeding technique, isozygoty the kind of using on the market fast, obtain the breeding resource.The microspores culture method can obtain the double haploid pure lines fast in 1~2 year, shortened the time in 3~4 years than traditional method, thereby shortened breeding process greatly, had improved the efficient of breeding.
A kind of method and special culture media thereof that obtains the kale sporule regeneration plant disclosed among the Chinese patent ZL200610112997.5.This cultural method may further comprise the steps: 1) microspore is inoculated in the embryoid induction medium, to final concentration be 5 * 10
4~5 * 10
5Individual/ML, after then high temperature stress is handled 24~72 hours under 30~35 ℃, dark condition, change inducing embryoid body under 24~26 ℃, dark condition again over to; 2) treat that torpedo stage to cotyledon period embryoid forms after, embryoid is earlier continued to cultivate 5~10 days under 24~26 ℃, 1500~2500LUX, illumination in 14~18 hours/sky condition, again embryoid is inoculated in the differential medium, under the same conditions cultivation; 3) grow stem, leaf after, plant is transferred in the root media, under 24~26 ℃, 1500~2500LUX, illumination in 14~18 hours/sky condition, carry out culture of rootage, obtain the kale regeneration plant.
Since nineteen eighty-two Lichter reported first rape Isolated microspore cultivate to obtain regeneration plant, rape belong to crop at germ extraction rate, go out and all be successful aspect embryo amount, embryo culture and the regeneration plant.The report head that the kale Isolated microspore is cultivated is shown in 1989 (Lichter and Takahata); (2000), Jiang Fengying and Feng Hui people such as (2005) such as people (1992), Tang Qinglin such as Duijs have also done many researchs subsequently, but the not high problem of kale germ extraction rate does not still solve.The big quantity research that relevant rape belongs to the Isolated microspore cultivation shows; The genotype of donor plant is very important to the embryogenetic influence of microspore, and it not only influences produces the embryo rate, and influences quality (the Chuong et al. of embryo; 1988), suppressed the application of microspores culture breeding technique.Therefore, seeking a kind of method that improves the kale germ extraction rate has great importance.
Summary of the invention
The invention provides a kind of cultural method of kale sporule regeneration plant; Adopt the rape variety of high germ extraction rate and the kale kind Mixed culture that difficulty goes out embryo; Obtain the kale microspore seedling that difficulty goes out embryo, improve kale microspores culture germ extraction rate.
A kind of cultural method of kale sporule regeneration plant may further comprise the steps:
1) inflorescence of inflorescence and rape of getting kale is as the donor plant of microspores culture; Get bud after inducing directly or with inflorescence, obtain rape and kale mixing bud;
2) rape after will sterilizing and kale mix and add the NLN-13 inducing culture in the bud; Grind to form suspension; Filter gained filtrating through centrifugal, abandon supernatant and obtain deposition, add NLN-13 inducing culture and active carbon mixed liquor successively, obtain the microspore mixing suspension;
3) microspore suspension was handled 1 day~3 days in 32 ℃~33 ℃ constant temperature heat shocks under dark condition, the back taking-up in 25 ± 2 ℃ of cultivations 20 days~30 days, obtains the cotyledon period embryoid under dark condition;
4) the cotyledon period embryoid is seeded to is cultured to the formation embryoid in the embryoid differential medium; Embryoid is inoculated into continuation cultivation in the embryoid differential medium, until differentiation, the regeneration bud;
5) cut regeneration bud and be seeded on the root media and cultivate, the seedling that root growth is healthy and strong obtains regeneration plant through refining seedling and transplanting, identifies.
In order to reach better effect, preferably:
In the step 1), described bud select on the inflorescence petal and flower pesticide length for use than be 0.6~1.1, monokaryon is more conducive to the cultivation of microspore to the early stage bud of double-core late period.
Bud after inducing also is more conducive to the cultivation of microspore, and described condition of inducing is: induced 0.1 hour~48 hours at 3 ℃~5 ℃.
The consumption of rape bud and kale bud is not generally done special qualification in described rape and the kale mixing bud, and in order to reach better effect, further the quantity of preferred oil cauliflower flower bud is slightly larger than the quantity of kale bud.
Step 2) in, the conventional sterilizing methods in this area is adopted in described sterilization, can adopt sterilized solution that rape and kale are mixed bud sterilization 15~20 minutes, and rape after obtaining sterilizing after cleaning up and kale mix bud.The sterilized solution that generally is used for explant sterilization has mercuric chloride solution, liquor natrii hypochloritis etc., because mercuric chloride solution has very big toxicity, and contaminated environment, harmful; And the effect of clorox sterilization is better, does not poison the preferred liquor natrii hypochloritis of described sterilized solution.In the 1L liquor natrii hypochloritis, consist of: mass percentage concentration is 5.6% 8~10 of aqueous sodium hypochlorite solution 56ml, absolute ethyl alcohol 100ml, liquid detergents and the sterile water of surplus.
Described NLN-13 inducing culture is: be made up of NLN liquid nutrient medium 1L and sucrose 130g, pH 6.0~6.2; Hot and humid sterilization, subsequent use.
Consisting of of described active carbon mixed liquor: NLN liquid nutrient medium 1L, agarose 2g~5g and 1g active carbon; High-temperature sterilization, subsequent use.
Described NLN liquid nutrient medium in 1L, consists of: KNO
3125mg, Ca (NO
3)
24H
2O 500mg, MgSO
47H
2O 125mg, KH
2PO
4125mg, H
3BO
36.2mg, MnSO
4H
2O 18.95mg, ZnSO
47H
2O 8.6mg, Na
2MoO
42H
2O 0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, vitamin B1 (VB
1) 0.5mg, vitamin B6 (VB
6) 0.5mg, vitamin h 0.05mg, folic acid 0.5mg, Na
2EDTA 37.3mg, FeSO
47H
2O 27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, glutathione 30mg, vitamin B5 5mg, the sterile water of serine 100mg and surplus.
Described deposition can repeat following operation 1~2 time: in deposition, add the NLN-13 inducing culture, grind to form suspension, filter gained filtrating through centrifugal, abandon supernatant.Add NLN-13 inducing culture and active carbon mixed liquor after the repetitive operation in the deposition of gained successively, obtain the microspore mixing suspension.
The concentration of microspore is 0.8 * 10 in the described microspore mixing suspension
5Individual/mL~1.2 * 10
5Individual/mL.
Described filtration can be adopted the aseptic filter screen of 45 μ m.
Described centrifugal condition is generally: the centrifugal 3min~5min of 600rpm/min~900rpm/min.
In the step 3), under dark condition be in 25 ± 2 ℃ of concrete steps of cultivating 20 days~30 days: under the dark condition when being cultured to the visible embryoid of naked eyes for 25 ± 2 ℃, again under dark condition under 25 ± 2 ℃ of conditions wave and culture; Wave and culture is healthy and strong with respect to the microspore embryonic development of wave and culture not.
In the step 4), described condition of culture is: illumination 12 hours~18 hours every day and 20 ℃~28 ℃ cultivations down; Further be preferably illumination 16 hours every days and 25 ℃ of cultivations down.General incubation time was 2 week~3 weeks.
Described embryoid is inoculated after can being cut into polylith when big.
Described embryoid differential medium is: be made up of pH5.8~6.1 MS medium 1L, sucrose 20g and agar 10g~12g; Hot and humid sterilization.
In the step 5), described root media is: be made up of pH5.8~6.0 MS medium 1L, sugared 20g~30g and agar 6g~7g; Described sugar is sucrose or white sugar; Hot and humid sterilization.
Described MS medium in 1L, consists of: NH
4HO
31650mg, KNO
31900mg, CaCl
22H
2O 440mg, KH
2PO
4170mg, MgSO
47H
2O 370mg, FeSO
47H
2O27.8mg, Na
2EDTA 37.3mg, H
3BO
36.2mg, MnSO
4H
2O 16.9mg, ZnSO
47H
2O 8.6mg, Na
2MoO
42H
2O 0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, KI 0.83mg, inositol 100mg, glycine 2mg, nicotinic acid 0.5mg, vitaminB10 .1mg, the sterile water of vitamin B6 0.5mg and surplus.
Clean tissue cultivating seedling root medium with clear water during transplanting; Using mass percentage concentration is that 800~1000 times of (multiple of dilution) liquid of tpn (like commercially available tpn wetting powder) of 75%~80% soak the tissue cultivating seedling root and plant after 1~2 minute in kale special seedling substrate cave and coil; Plastic foil covers preserves moisture, and in the greenhouse, cultivates after 15~20 days and transplants.Come sterilization through tpn, and give certain buffer environment, adapt to harsher natural environment better to make the tissue cultivating seedling of growing in the laboratory preferably at growing environment.
Described evaluation can be adopted this area methods for ploidy determination commonly used, as gets each regeneration plant ploidy of tender leaf detection of regeneration plant, and from regeneration plant colony, identifies rape regrowth and kale regrowth.The BD FACSCalibur flow cytometer of available U.S. company BD carries out the evaluation of ploidy analysis and regeneration plant kind.
Beneficial effect of the present invention is:
1, it is low to the present invention is directed to existing kale microspores culture germ extraction rate; Especially be very difficult to out the kind of embryo; A kind of method that improves germ extraction rate is provided; Rape variety that employing is prone to embryo and the difficult method that goes out the kale bud Mixed culture of embryo drive the difficult material that goes out embryo with the material that is prone to embryo and go out embryo, improve the germ extraction rate of kale.The inventive method Mixed culture kale germ extraction rate is up to 104/flower bud; Be merely 0.7/flower bud and contrast kale germ extraction rate is the highest; Solve the lower problem of kale microspores culture embryo occurrence frequency, improved the utilization ratio of microspores culture technology.
2, detect discovery through perusal and flow cytometer, Mixed culture can obtain 13 strain kale microspore seedlings at last under identical condition, and the contrast strain number of kale bud alone culture is 0.
Embodiment
Embodiment 1
Cultural method carries out as follows.
(1) medium preparation: comprise the medium of the different cultivation stages of microspore, its component and each component contained weight in every liter of medium is:
Table 1NLN and MS medium component table
1) NLN-13 inducing culture: NLN liquid nutrient medium 1L+ sucrose 130g/L, pH6.0, filtration sterilization;
2) embryoid differential medium: MS medium+sucrose 20g/L, agar 10g/L, pH6.0, hot and humid sterilization;
3) root media: MS medium+sucrose 30g/L, agar 6g/L, pH5.8, hot and humid sterilization;
(2) cultivation of the high germ extraction rate sporule regeneration plant of kale:
1) selection of donor plant bud: get the donor plant of the inflorescence of kale and growth of rape health, no damage by disease and insect as microspores culture; After inducing 24 hours under 4 ℃ of cryogenic conditions, get on the inflorescence petal and flower pesticide length than 0.6, monokaryon late period is to the early stage bud of double-core;
2) sterilization of bud: the sterilized solution that is mixed with 10+sterile water of 5.6% (mass percentage concentration) aqueous sodium hypochlorite solution 56ml/L+ absolute ethyl alcohol 100ml/L+ liquid detergent; Mix bud to rape and kale and put into sterile petri dish, add sterilized solution, be put into to shake on the shaking table and carried out surface sterilization 20 minutes, after washing 3 times with aseptic pond on the superclean bench, subsequent use again;
3) on superclean bench, kale and rape mixings bud after 12 (mix bud sum, wherein the rape bud is 8) sterilization are placed aseptic beaker in the lump, adding 10ml NLN-13 inducing culture, crowded broken with the tack glass bar, grind to form suspension; In the 50ml centrifuge tube, lid is tight with the aseptic strainer filtering of 45 μ m for this suspension, and the centrifugal 3min of 900rpm/min outwells supernatant after centrifugal, and adding 10ml NLN-13 inducing culture in the deposition centrifugal more as stated above 2 times, is abandoned supernatant; Add NLN-13 inducing culture 40ml, add 10 of the active carbon mixed liquors that formed by NLN-13 liquid nutrient medium+agarose 5g/L+1g/L active carbon preparation, high-temperature sterilization again, the concentration that obtains microspore is 1 * 10
5The microspore mixing suspension of individual/mL;
4) this microspore suspension branch is installed in the 60mm glass sterile petri dish, every 60mm culture dish adds 4ml microspore mixing suspension, adds a cover the back and seals with the parafilm film, is placed in the biochemical incubators of 32.5 ℃ of constant temperature, under the dark condition heat shock processing 1 day; The back is taken out and is placed 25 ℃ of constant incubators, dark condition to continue to cultivate down; During to the visible embryoid of naked eyes, placed on 25 ℃ of shaking tables, under the dark condition wave and culture 20 days, obtain the cotyledon period embryoid and reach maturity;
5) the cotyledon period embryoid is seeded in the embryoid differential medium, at illumination 16 hours every days, 25 ℃ of following 3 thoughtful formation embryoids of cultivating; Be cut into much the same of 3 block sizes according to a certain size embryoid, be inoculated into and continue under the same conditions in the embryoid differential medium to cultivate, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on the root media, under illumination 16 hours every days, 25 ℃, carries out culture of rootage; Seedling after 3 weeks that root growth is healthy and strong refined seedling 3 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 800 times of 75% tpn soaked base portion 2 minutes; The back is planted the dish in kale special seedling substrate cave to plant, and plastic foil covers preserves moisture, and in the greenhouse, cultivates after 15 days and transplants, and obtains regeneration plant;
7) get the tender leaf of regeneration plant, carry out ploidy analysis, detect each plant ploidy, and from regeneration plant colony, identify rape regrowth and kale regrowth with the BD FACSCalibur flow cytometer of U.S. company BD.
(3) in step 1), only get the donor plant of inflorescence as microspores culture of kale healthy growth, no damage by disease and insect, other operation is with " cultivation of the high germ extraction rate sporule regeneration plant of (2) kale ", as the contrast kale.
The result: the germ extraction rate of kale is 104 a/flower bud in the Mixed culture, and contrast kale germ extraction rate is merely 0.7/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 13 strain kale microspore seedlings at last, and the contrast strain number of contrast kale bud alone culture is 0.
Embodiment 2
Except in the step (1) 1) the NLN-13 inducing culture: NLN-13 liquid nutrient medium 1L+ sucrose 130g/L, pH6.1, filtration sterilization; 2) embryoid differential medium: MS medium+sucrose 20g/L, agar 11g/L, pH6.1, hot and humid sterilization; 3) root media: MS medium+white sugar 20g/L, agar 7g/L, pH5.9, hot and humid sterilization;
(2) cultivation of the high germ extraction rate sporule regeneration plant of kale:
1) selection of donor plant bud: get the donor plant of the inflorescence of kale and growth of rape health, no damage by disease and insect as microspores culture; After inducing 48 hours under 3 ℃ of cryogenic conditions, get on the inflorescence petal and flower pesticide length than 1.1, monokaryon late period is to the early stage bud of double-core;
2) sterilization of bud: the sterilized solution that is mixed with 8+sterile water of 5.6% (mass percentage concentration) aqueous sodium hypochlorite solution 56ml/L+ absolute ethyl alcohol 100ml/L+ liquid detergent; Mix bud to rape and kale and put into sterile petri dish, add sterilized solution, be put into to shake on the shaking table and carried out surface sterilization 15 minutes, after washing 5 times with aseptic pond on the superclean bench, subsequent use again;
3) on superclean bench, kale and rape mixings bud after 15 (mix bud sum, wherein the rape bud is 5) sterilization are placed aseptic beaker in the lump, adding 10ml NLN-13 inducing culture, crowded broken with the tack glass bar, grind to form suspension; In the 50ml centrifuge tube, lid is tight with the aseptic strainer filtering of 45 μ m for this suspension, and the centrifugal 5min of 900rpm/min outwells supernatant after centrifugal, and adding 10ml NLN-13 inducing culture in the deposition centrifugal more as stated above 1 time, is abandoned supernatant; Add NLN-13 inducing culture 40ml, add 10 of the active carbon mixed liquors that formed by NLN-13 liquid nutrient medium+agarose 2g/L+1g/L active carbon preparation, high-temperature sterilization again, the concentration that obtains microspore is 0.8 * 10
5The microspore mixing suspension of individual/mL;
4) this microspore suspension branch is installed in the 90mm plastics sterile petri dish, every 90mm culture dish adds 10ml microspore mixing suspension, adds a cover the back and seals with the parafilm film, is placed in the biochemical incubators of 33 ℃ of constant temperature, under the dark condition heat shock processing 2 days; The back is taken out and is placed 25 ℃ of constant incubators, dark condition to continue to cultivate down; During to the visible embryoid of naked eyes, placed on 25 ℃ of shaking tables, under the dark condition wave and culture 25 days, obtain the cotyledon period embryoid and reach maturity;
5) the cotyledon period embryoid is seeded in the embryoid differential medium, at illumination 16 hours every days, 25 ℃ of following 2 thoughtful formation embryoids of cultivating; Directly be inoculated into continuation cultivation under the same conditions in the embryoid differential medium to embryoid, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on the root media, under illumination 16 hours every days, 25 ℃, carries out culture of rootage; Seedling after 3 weeks that root growth is healthy and strong refined seedling 5 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 1000 times of 80% tpn soaked base portion 1 minute; The back is planted the dish in kale special seedling substrate cave to plant, and plastic foil covers preserves moisture, and in the greenhouse, cultivates after 20 days and transplants, and obtains regeneration plant;
7) get the tender leaf of regeneration plant, carry out ploidy analysis, detect each plant ploidy, and from regeneration plant colony, identify rape regrowth and kale regrowth with the BD FACSCalibur flow cytometer of U.S. company BD.
All the other operations are embodiment 1 simultaneously.
The result: the germ extraction rate of Mixed culture kale is 100 a/flower bud, and contrast kale germ extraction rate is merely 0.5/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 12 strain kale microspore seedlings at last, and the contrast strain number of contrast kale bud alone culture is 0.
Embodiment 3
Except in the step (1) 1) the NLN-13 inducing culture: NLN-13 liquid nutrient medium 1L+ sucrose 130g/L, pH6.2, filtration sterilization; 2) embryoid differential medium: MS medium+sucrose 20g/L, agar 12g/L, pH6.0, hot and humid sterilization; 3) root media: MS medium+sucrose 25g/L, agar 6.5g/L, pH6.0, hot and humid sterilization;
(2) cultivation of the high germ extraction rate sporule regeneration plant of kale:
1) selection of donor plant bud: get the donor plant of the inflorescence of kale and growth of rape health, no damage by disease and insect as microspores culture; Get on the inflorescence petal and flower pesticide length than 0.8, monokaryon late period is to the early stage bud of double-core;
2) sterilization of bud: the sterilized solution that is mixed with 9+sterile water of 5.6% (mass percentage concentration) aqueous sodium hypochlorite solution 56ml/L+ absolute ethyl alcohol 100ml/L+ liquid detergent; Mix bud to rape and kale and put into sterile petri dish, add sterilized solution, be put into to shake on the shaking table and carried out surface sterilization 20 minutes, after washing 4 times with aseptic pond on the superclean bench, subsequent use again;
3) on superclean bench, kale and rape mixings bud after 14 (mix bud sum, wherein the rape bud is 7) sterilization are placed aseptic beaker in the lump, adding 10ml inducing culture, crowded broken with the tack glass bar, grind to form suspension; In the 50ml centrifuge tube, lid is tight with the aseptic strainer filtering of 45 μ m for this suspension, and the centrifugal 4min of 700rpm/min outwells supernatant after centrifugal, and adding 10ml NLN-13 inducing culture in the deposition centrifugal more as stated above 2 times, is abandoned supernatant; Add NLN-13 inducing culture 40ml, add 10 of the active carbon mixed liquors that formed by NLN-13 liquid nutrient medium+agarose 3.5g/L+1g/L active carbon preparation, high-temperature sterilization again, the concentration that obtains microspore is 1.2 * 10
5The microspore mixing suspension of individual/mL;
4) this microspore suspension branch is installed in the 60mm glass sterile petri dish, every 60mm culture dish adds 4ml microspore mixing suspension, adds a cover the back and seals with the parafilm film, is placed in the biochemical incubators of 32.5 ℃ of constant temperature, under the dark condition heat shock processing 2 days; The back is taken out and is placed 25 constant incubators, dark condition to continue to cultivate down; During to the visible embryoid of naked eyes, placed on 25 ℃ of shaking tables, under the dark condition wave and culture 25 days, obtain the cotyledon period embryoid and reach maturity;
5) the cotyledon period embryoid is seeded in the embryoid differential medium, at illumination 16 hours every days, 25 ℃ of following 2.5 thoughtful formation embryoids of cultivating; Be cut into much the same of 2 block sizes to embryoid and be inoculated into continuation cultivation under the same conditions in the embryoid differential medium, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on the root media, under illumination 16 hours every days, 25 ℃, carries out culture of rootage; Seedling after 3 weeks that root growth is healthy and strong refined seedling 4 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 900 times of 75% tpn soaked base portion 1 minute; The back is planted the dish in kale special seedling substrate cave to plant, and plastic foil covers preserves moisture, and in the greenhouse, cultivates after 18 days and transplants, and obtains regeneration plant;
7) get the tender leaf of regeneration plant, carry out ploidy analysis, detect each plant ploidy, and from regeneration plant colony, identify rape regrowth and kale regrowth with the BD FACSCalibur flow cytometer of U.S. company BD.
All the other operations are embodiment 1 simultaneously.
The result: the germ extraction rate of Mixed culture kale is 102 a/flower bud, and contrast kale germ extraction rate is merely 0.6/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 11 strain kale microspore seedlings at last, and the contrast strain number of contrast kale bud alone culture is 0.
Claims (6)
1. the cultural method of a kale sporule regeneration plant may further comprise the steps:
1) gets the inflorescence of rape variety of inflorescence and high germ extraction rate of kale kind that difficulty goes out embryo as the donor plant of microspores culture; Get bud after inducing directly or with inflorescence, obtain rape and kale mixing bud; Described condition of inducing is: induced 0.1 hour~48 hours at 3 ℃~5 ℃;
2) rape after sterilization and kale mix and add the NLN-13 inducing culture in the bud; Grind to form suspension; Filter gained filtrating through centrifugal, abandon supernatant and obtain deposition, add NLN-13 inducing culture and active carbon mixed liquor successively, obtain the microspore mixing suspension; Described NLN-13 inducing culture is: be made up of NLN liquid nutrient medium 1L and sucrose 130g, pH 6.0~6.2;
Consisting of of described active carbon mixed liquor: NLN liquid nutrient medium 1L, agarose 2g-5g and 1g active carbon;
Described NLN liquid nutrient medium in 1L, consists of: KNO
3125mg, Ca (NO
3)
24H
2O 500mg, MgSO
47H
2O 125mg, KH
2PO
4125mg, H
3BO
36.2mg, MnSO
4H
2O 18.95mg, ZnSO
47H
2O 8.6mg, Na
2MoO
42H
2O 0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, vitamin B1 0.5mg, vitamin B6 0.5mg, vitamin h 0.05mg, folic acid 0.5mg, Na
2EDTA 37.3mg, FeSO
47H
2O 27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, glutathione 30mg, vitamin B5 5mg, the sterile water of serine 100mg and surplus;
3) microspore suspension was handled 1 day~3 days in 32 ℃~33 ℃ constant temperature heat shocks under dark condition, the back taking-up in 25 ± 2 ℃ of cultivations 20 days~30 days, obtains the cotyledon period embryoid under dark condition;
4) the cotyledon period embryoid is seeded to is cultured to the formation embryoid in the embryoid differential medium; Embryoid is inoculated into continuation cultivation in the embryoid differential medium, until differentiation, the regeneration bud; Described embryoid differential medium is: be made up of pH5.8~6.1 MS medium 1L, sucrose 20g and agar 10g~12g;
5) cut regeneration bud and be seeded on the root media and cultivate, the seedling that root growth is healthy and strong obtains regeneration plant through refining seedling and transplanting, identifies; Described root media is: be made up of pH5.8~6.0 MS medium 1L, sugared 20g~30g and agar 6g~7g; Described sugar is sucrose or white sugar.
2. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that, in the step 1), described bud be on the inflorescence petal and flower pesticide length than be 0.6~1.1, monokaryon late period is to the early stage bud of double-core.
3. the cultural method of kale sporule regeneration plant according to claim 1; It is characterized in that step 2) in, operation below the described deposition repetition 1~2 time: in deposition, add the NLN-13 inducing culture; Grind to form suspension, filter gained filtrating through centrifugal, abandon supernatant.
4. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that step 2) in, the concentration of microspore is 0.8 * 10 in the described microspore mixing suspension
5Individual/mL~1.2 * 10
5Individual/mL.
5. the cultural method of kale sporule regeneration plant according to claim 1; It is characterized in that; In the step 3); Under dark condition be in 25 ± 2 ℃ of concrete steps of cultivating 20 days~30 days: under the dark condition when being cultured to the visible embryoid of naked eyes for 25 ± 2 ℃, again under dark condition under 25 ± 2 ℃ of conditions wave and culture.
6. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that, in the step 4), described condition of culture is: illumination 12 hours~18 hours every day and 20 ℃~28 ℃ cultivations down.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201110112693XA CN102228003B (en) | 2011-05-03 | 2011-05-03 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201110112693XA CN102228003B (en) | 2011-05-03 | 2011-05-03 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102228003A CN102228003A (en) | 2011-11-02 |
CN102228003B true CN102228003B (en) | 2012-11-07 |
Family
ID=44840640
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201110112693XA Expired - Fee Related CN102228003B (en) | 2011-05-03 | 2011-05-03 | Culture method for brassica oleracea L. var. acephala microspore regeneration plant |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102228003B (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102577962A (en) * | 2012-02-28 | 2012-07-18 | 江苏丘陵地区镇江农业科学研究所 | Culture method for improving embryonic birth rate of cabbage stalk |
CN103392600B (en) * | 2013-08-06 | 2015-06-10 | 镇江瑞繁农艺有限公司 | Method for promoting collard stubborn genotype microspore embryogenesis |
CN110089426B (en) * | 2018-01-29 | 2022-03-08 | 南京农业大学 | Method for cultivating non-heading Chinese cabbage microspore plant |
CN109937876A (en) * | 2019-03-22 | 2019-06-28 | 南京农业大学 | A method of improving Chinese cabbage microspore embryo's inductivity |
CN111903520A (en) * | 2020-08-01 | 2020-11-10 | 梁江 | Method for regenerating plant by using isolated microspore embryoid of ginseng |
CN113229148A (en) * | 2021-06-10 | 2021-08-10 | 南京新创蔬菜分子育种研究院有限公司 | Method for obtaining regenerated plants by culturing collard free microspores |
WO2023193475A1 (en) * | 2022-12-20 | 2023-10-12 | 唐山师范学院 | Method for improving cabbage microspore doubling and plant regeneration |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1922986A (en) * | 2006-09-14 | 2007-03-07 | 北京市农林科学院 | Method for obtaining Brassica oleracea var. acephala DC. small spore regenerated plants and special culture medium |
CN101822156A (en) * | 2010-03-16 | 2010-09-08 | 北京市农林科学院 | Method for quickly culturing homozygous transgenic ornamental collard with insect resistance and herbicide resistance |
-
2011
- 2011-05-03 CN CN201110112693XA patent/CN102228003B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1922986A (en) * | 2006-09-14 | 2007-03-07 | 北京市农林科学院 | Method for obtaining Brassica oleracea var. acephala DC. small spore regenerated plants and special culture medium |
CN101822156A (en) * | 2010-03-16 | 2010-09-08 | 北京市农林科学院 | Method for quickly culturing homozygous transgenic ornamental collard with insect resistance and herbicide resistance |
Non-Patent Citations (1)
Title |
---|
林顺权.培养基及其配制.《植物细胞工程》.厦门大学出版社,2000,(第1版),第33页倒数第6-9行. * |
Also Published As
Publication number | Publication date |
---|---|
CN102228003A (en) | 2011-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102228003B (en) | Culture method for brassica oleracea L. var. acephala microspore regeneration plant | |
CN102893870B (en) | Rapid propagation and seedling raising method for beautiful millettia root seedling tissue culture | |
CN101617631B (en) | Culture method of high diplont rate sporule regeneration plant of broccoli | |
CN103190347B (en) | Teapot dates tissue culturing method | |
CN104885773B (en) | A kind of method of quickly breeding blue berry early stage sizing tissue culture commercial seedling | |
CN106561456B (en) | A kind of Helen's pocket orchid aseptic seeding rapid propagation method | |
CN103416294A (en) | Concolor paphiopedilum crossbreeding method and seedling breeding method thereof | |
CN102613076A (en) | Vegetative propagation method for butterfly orchid | |
CN102239803B (en) | Method for culturing regeneration plants of Brassica oleracea microspores | |
CN109258468A (en) | A kind of potato saline-alkali tolerant improvement breeding method | |
CN101595824B (en) | Rapid in-vitro seedling raising method by utilizing sandalwood seed embryo | |
CN102577962A (en) | Culture method for improving embryonic birth rate of cabbage stalk | |
CN102428872B (en) | Culture medium and culture method for cultivating immature embryo of double-petal paeonia lactiflora | |
CN105875414A (en) | Method for cultivating butterfly orchid by promoting rapid proliferation of butterfly orchid protocorm-like body | |
CN103392600B (en) | Method for promoting collard stubborn genotype microspore embryogenesis | |
CN106305427A (en) | Tissue culture and rapid propagation method for Acer ginnala | |
CN103392604A (en) | Tissue culture method for broccoli | |
CN104186309B (en) | Method for improving embryogenesis rate of Raphanus sativus L. sinoruber makino | |
CN104082145A (en) | Method for rapidly propagating adiantum soboliferum | |
CN105010123B (en) | The method and culture medium of strawberry distant hybrid are obtained by rescue isolated culture | |
CN100391333C (en) | Aseptic seedling tissue culturing and test tube seedling hardening off and transplating technology for anthurium andraeanum | |
CN102362579B (en) | Method for cultivating artificial hybridization hexaploid rape microspore regeneration plants | |
CN110495393A (en) | A method of obtaining purple cauliflower microspore DH regeneration plant | |
CN105875410A (en) | Rapid propagation method for hybrid cymbidium seedlings of cymbidium sinense and cymbidium hookerianum | |
CN103598093B (en) | A kind of abductive approach of blueberry embryoid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121107 Termination date: 20200503 |
|
CF01 | Termination of patent right due to non-payment of annual fee |