CN104186310B - A kind of method of the pale reddish brown dish haplobiont of quick acquisition - Google Patents
A kind of method of the pale reddish brown dish haplobiont of quick acquisition Download PDFInfo
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Abstract
The method that the invention discloses the pale reddish brown dish haplobiont of a kind of quick acquisition, including: take pale reddish brown cauliflower flower bud, add B5 after sterilizing and wash culture medium, make suspension, filter gained filtrate and precipitate obtained by centrifugation；It is sequentially added into NLN 13 inducing culture and activated carbon mixed liquor, obtains microspore suspension；Adding aseptic Caulis et Folium Brassicae capitatae flower pesticide after being dispensed in sterile petri dish, after Parafilm sealing, thermal shock processes；After cellar culture, obtain cotyledon type embryoid；It is seeded in embryoid division culture medium until breaking up, regeneration bud；Cut regeneration bud and be seeded on root media root culture, through seedling exercising and transplanting, obtain regeneration plant.The Caulis et Folium Brassicae capitatae flower pesticide and difficulty that easily go out embryo are gone out the pale reddish brown dish Isolated microspore Mixed culture by a certain percentage of embryo by the method, thus promoting that difficulty goes out the pale reddish brown dish fetal development of embryo, it is thus achieved that a large amount of regeneration plants, improve culture efficiency.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly relate to the cultural method of pale reddish brown dish haplobiont.
Background technology
Pale reddish brown dish (Brassicaoleraceavar.botrytis) belongs to Cruciferae Brassica genus brassica specie, originates in the coastal Italian band in West Europe, is the mutation of Brassica oleracea L. var. botrytis L., and bouquet aubergine, best in quality, local flavor is splendid.Other character are similar to Brassica oleracea L. var. botrytis L..Pale reddish brown dish is nutritious, rich in proteins, vitamin C, and crude fibre is few, additionally, possibly together with vitamin A, B1, B2 and sucrose, fructose, selenium etc., local flavor is delicious, can improve immune function of human body, promote liver detoxification, strengthens body constitution and the resistance against diseases of people.The selenium contained can the growth of anticancer.
At present, the pale reddish brown vegetable kind that production uses, it mostly is foreign country's import, seed price is expensive, and domestic germ plasm resource is deficient, under one's control.Pale reddish brown dish hybrid vigor is obvious, selfing separates and obtains the stable selfing line that isozygotys it is generally required to time of 5～6 years, and isolated microspore culture technique can obtain haplobiont in 1～2 year, become homozygous inbred lines after doubling, therefore in the initiative of pale reddish brown dish Elite inbred and raising breeding of new variety efficiency, there is significant application value.Although crucifer microspore culture should be used as a large amount of deep research by Chinese scholars, but the application of relevant pale reddish brown dish microspore culture is not reported.Herein on the basis of early-stage Study, adopt pale reddish brown dish Isolated microspore and Caulis et Folium Brassicae capitatae easily to go out embryo genotype flower pesticide and co-culture, remarkably promote pale reddish brown dish sporidiole fetal development, it is thus achieved that a large amount of regeneration plants, required time is shorter, easy and simple to handle.
Summary of the invention
For current present situation, the method that the invention provides the pale reddish brown dish haplobiont of a kind of quick acquisition, pale reddish brown dish sporidiole and the Caulis et Folium Brassicae capitatae flower pesticide easily going out embryo is adopted to co-culture by some proportionings, remarkably promote pale reddish brown dish sporidiole fetal development, promote that pale reddish brown dish sporidiole goes out embryo, improve the germ extraction rate of microspores culture, it is thus achieved that a large amount of regeneration plants, required time is shorter, easy and simple to handle.
For achieving the above object, the technical scheme that the present invention takes is:
The method of the pale reddish brown dish haplobiont of a kind of quick acquisition, comprises the following steps:
1) pale reddish brown dish and Caulis et Folium Brassicae capitatae alabastrum, sterilizing are taken；
2) being placed in culture fluid by the pale reddish brown dish Isolated microspore after mixing alabastrum sterilizing with Caulis et Folium Brassicae capitatae flower pesticide, Mixed culture after thermal shock, induction obtains cotyledon type embryoid；
3) cotyledon type embryoid is seeded in solid division culture medium, cultivates and obtain regeneration plant；
4) cut regeneration bud from regeneration plant, access root media, it is thus achieved that whole plant；
Described pale reddish brown dish microspore embryoid incidence rate is relatively low, and Caulis et Folium Brassicae capitatae is easier to out embryo.
The alabastrum time of drawing materials of described pale reddish brown dish and Caulis et Folium Brassicae capitatae is in monokaryon late period to double-core early stage.
Described pale reddish brown dish and the alabastrum of Caulis et Folium Brassicae capitatae and the quantity of flower pesticide are than for 1:5～8.
Described pale reddish brown dish sporidiole and Caulis et Folium Brassicae capitatae flower pesticide mixed liquor adopt 32～33 DEG C of thermal shocks to process 1～3 day.
Described culture fluid is NLN 13 fluid medium and the mixture of sterile active charcoal mixed liquor, wherein the consisting of of activated carbon mixed liquor: 1LNLN fluid medium, 2～5g agarose and 1g activated carbon.
Described NLN fluid medium, in 1L, consists of: KNO3125mg, Ca (NO3)2·4H2O500mg, MgSO4·7H2O125mg, KH2PO4125mg, H3BO36.2mg, MnSO4·H2O18.95mg, ZnSO4·7H2O8.6mg, Na2MoO4·2H2O0.25mg, CuSO4·5H2O0.025mg, CoCl2·6H2O0.025mg, vitaminB10 .5mg, vitamin B6 0.5mg, biotin 0.05mg, folic acid 0.5mg, Na2EDTA37.3mg, FeSO4·7H2O27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L glutamine 800mg, glutathion 30mg, vitamin B5 5mg, the sterilized water of serine 100mg and surplus.
Described microspore suspension with after flower pesticide mixing thermal shock, first 25 DEG C, after constant temperature culture occurs for 10～15 days to naked eyes visible cell group under dark condition, move on to 25 DEG C, 50～60rpm shaken cultivation 10～15 days under dark condition, obtain blade profile embryoid.
Described solid division culture medium is B5 medium+sucrose 30g/L, agar 10～12g/L, pH5.9～6.1.
Described B5 fluid medium, in 1L, consists of: NaH2PO4·2H2O169.5mg,KNO32500mg, (NH4)2SO4134mg, MgSO4·7H2O500mg, MnSO4·4H2O10mg,H3BO33mg,ZnSO4·7H2O2mg,KI0.75mg,Na2MoO4·2H2O0.25mg,CuSO4·5H2O0.025mg,CoCl2·6H2O0.025mg,Na2‐EDTA37.3mg,FeSO4·7H2O27.8mg,CaCl2.2H2O150mg, VB110mg, VB61mg, VPP1mg, the distilled water of inositol 100mg and surplus.
Described root media is made up of 1/2MS culture medium 1L+0.1～0.2mg/LNAA+0.1～0.2mg/LIBA, sucrose or white sugar 20～30g/L and agar 7～8g/L, pH5.8～6.0.
Described MS culture medium, in 1L, consists of: NH4HO31650mg, KNO31900mg, CaCl2·2H2O440mg, KH2PO4170mg, MgSO4·7H2O370mg, FeSO4·7H2O27.8mg, Na2EDTA37.3mg, H3BO36.2mg, MnSO4·H2O16.9mg, ZnSO4·7H2O8.6mg, Na2MoO4·2H2O0.25mg, CuSO4·5H2O0.025mg, CoCl2·6H2O0.025mg, KI0.83mg, inositol 100mg, glycine 2mg, nicotinic acid 0.5mg, vitaminB10 .1mg, the sterilized water of vitamin B6 0.5mg and surplus.
The invention have the benefit that
(1) the present invention is directed to the problem that pale reddish brown dish microspores culture difficulty goes out embryo, it is provided that the short-cut method of a kind of quick acquisition haplobiont.Mix by a certain percentage with pale reddish brown dish Isolated microspore and co-culture easily going out the Caulis et Folium Brassicae capitatae flower pesticide of embryo, the Caulis et Folium Brassicae capitatae flower pesticide easily to go out embryo drive difficult go out embryo material go out embryo, improve germ extraction rate.The inventive method co-cultures pale reddish brown dish germ extraction rate and is up to 15.5/flower bud, and compare germ extraction rate only up to 1.4/flower bud, and the quality of embryoid is also greatly improved, solves the problem that pale reddish brown dish microspores culture embryo's incidence rate is low, improve the application efficiency of microspore culture.
(2) final under same culture conditions, Mixed culture has finally obtained the 68 pale reddish brown dish sporule regeneration plants of strain, and the comparison strain number of single culture is 2 strains.
(3) adopt that method is simple, cultivate the sporule regeneration plant colony obtained and be pale reddish brown dish, it is not necessary to plant type is identified, thus decreasing workload, improves culture efficiency, there is operability.
Detailed description of the invention
Embodiment 1
Cultural method carries out as follows.
(1) culture medium preparation: include the culture medium of sporidiole difference cultivation stage, its component and each component weight contained by every liter of culture medium is:
1) B5 washs culture medium: B5 fluid medium 1L+ sucrose 30g/L, pH6.0, High Temperature High Pressure；
Described B5 fluid medium, in 1L, consists of: NaH2PO4·2H2O169.5mg,KNO32500mg, (NH4)2SO4134mg, MgSO4·7H2O500mg, MnSO4·4H2O10mg,H3BO33mg,ZnSO4·7H2O2mg,KI0.75mg,Na2MoO4·2H2O0.25mg,CuSO4·5H2O0.025mg,CoCl2·6H2O0.025mg,Na2‐EDTA37.3mg,FeSO4·7H2O27.8mg,CaCl2.2H2O150mg, VB110mg, VB61mg, VPP1mg, the distilled water of inositol 100mg and surplus.
2) embryoid division culture medium: B5 medium+sucrose 20g/L, agar 10g/L, pH6.0, autoclave sterilization；
3) NLN 13 inducing culture, NLN fluid medium 1L+ sucrose 130g/L, pH6.0, filtration sterilization；
Described NLN fluid medium, in 1L, consists of: KNO3125mg, Ca (NO3)2·4H2O500mg, MgSO4·7H2O125mg, KH2PO4125mg, H3BO36.2mg, MnSO4·H2O18.95mg, ZnSO4·7H2O8.6mg, Na2MoO4·2H2O0.25mg, CuSO4·5H2O0.025mg, CoCl2·6H2O0.025mg, vitaminB10 .5mg, vitamin B6 0.5mg, biotin 0.05mg, folic acid 0.5mg, Na2EDTA37.3mg, FeSO4·7H2O27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L glutamine 800mg, glutathion 30mg, vitamin B5 5mg, the sterilized water of serine 100mg and surplus.
4) root media: MS culture medium+sucrose 30g/L, agar 6g/L, pH5.8, autoclave sterilization；
Described MS culture medium, in 1L, consists of: NH4HO31650mg, KNO31900mg, CaCl2·2H2O440mg, KH2PO4170mg, MgSO4·7H2O370mg, FeSO4·7H2O27.8mg, Na2EDTA37.3mg, H3BO36.2mg, MnSO4·H2O16.9mg, ZnSO4·7H2O8.6mg, Na2MoO4·2H2O0.25mg, CuSO4·5H2O0.025mg, CoCl2·6H2O0.025mg, KI0.83mg, inositol 100mg, glycine 2mg, nicotinic acid 0.5mg, vitaminB10 .1mg, the sterilized water of vitamin B6 0.5mg and surplus.
(2) pale reddish brown dish sporidiole and Caulis et Folium Brassicae capitatae flower pesticide co-culture the embryogenetic cultural method of promotion:
1) alabastrum is chosen: take pale reddish brown cauliflower lobe and anther length than be 0.85, Caulis et Folium Brassicae capitatae be 0.9, monokaryon late period to double-core early stage, healthy, alabastrum without pest and disease damage, as the donor of microspores culture；
2) sterilizing of alabastrum: be configured to sterilized solution with 1g mercuric chloride+1L sterilized water；Pale reddish brown dish and Caulis et Folium Brassicae capitatae mixing alabastrum are put in sterile petri dish, adds sterilized solution, be put on shaking table shake and carry out surface sterilization 10 minutes, then after washing 3 times with aseptic pond on superclean bench, standby；
3) on superclean bench, 12 aseptic alabastrums are placed in sterile beaker, add 10mlB5 wash culture medium, with tack Glass rod squeeze broken, grind to form suspension；This suspension, with in the aseptic strainer filtering in 45 μm of apertures to 50ml centrifuge tube, covers tightly, and 600rpm/min is centrifuged 3min, outwells supernatant after centrifugal, adds 10mlB5 and washs culture medium, be centrifuged 2 times more as stated above, abandon supernatant in precipitation；Adding NLN 13 inducing culture 40ml, add by the activated carbon mixed liquor 10 of NLN 13 fluid mediums+agarose 5g/L+1g/L activated carbon preparation, high temperature sterilize, the concentration obtaining sporidiole is 1 × 105The sporidiole mixing suspension of individual/mL；
4) this microspore suspension is dispensed in 60mm plastics sterile petri dish, every 60mm culture dish adds 4ml sporidiole mixing suspension and 5 aseptic flower pesticide of Caulis et Folium Brassicae capitatae, with the sealing of parafilm film after adding a cover, it is placed on Heat thermostability 2 days in 32.5 DEG C of constant temperature biochemical cultivation cases, under dark condition；Then take out and be placed in 25 DEG C of constant incubators, under dark condition and continue cultivation；During embryoid visible to naked eyes, it is placed in wave and culture 20 days on 25 DEG C of shaking tables, under dark condition, obtains cotyledon type embryoid and reach maturity；
5) cotyledon type embryoid is seeded in embryoid division culture medium, every day 16 h light, cultivate 3 thoughtful formation embryoids at 25 DEG C；It is cut into much the same piece of 3 block sizes according to a certain size embryoid, is inoculated in embryoid division culture medium and continues under the same conditions to cultivate, until differentiation, regeneration bud；
6) regeneration bud cutting healthy growth is seeded on root media, every day 16 h light, carry out root culture at 25 DEG C；After 3 weeks, the Seedling that root growth is healthy and strong is carried out seedling exercising 3 days；Clean tissue cultured seedling root culture medium with clear water during transplanting, after plant is planted in vegetable special seedling substrate hole tray, plastic foil covers moisturizing, cultivates and transplants after 10 days, obtain regeneration plant in greenhouse；
(3) except step 1) in only take pale reddish brown dish healthy growth, without the inflorescence of pest and disease damage as except the donor plant of microspores culture, other operation is with " (2) pale reddish brown dish sporidiole and Caulis et Folium Brassicae capitatae flower pesticide co-culture the embryogenetic cultural method of promotion ", as comparison.
Result: the pale reddish brown dish germ extraction rate of Mixed culture is 9.5/flower bud, and compares germ extraction rate and be only 0.8/flower bud.Finally, Mixed culture obtains the 67 pale reddish brown dish sporule regeneration plants of strain, and compares acquisition 0 strain.
Embodiment 2
Except in step (1) 1) B5 washs culture medium: B5 fluid medium 1L+ sucrose 30g/L, pH6.0, High Temperature High Pressure；2) NLN 13 inducing culture: NLN 13 fluid medium 1L+ sucrose 130g/L, pH6.1, filtration sterilization；3) embryoid division culture medium: B5 medium+sucrose 20g/L, agar 11g/L, pH6.0, autoclave sterilization；4) root media: MS culture medium+white sugar 20g/L, agar 7g/L, pH5.9, hot and humid sterilizing；
(2) pale reddish brown dish sporidiole and Caulis et Folium Brassicae capitatae flower pesticide co-culture the embryogenetic cultural method of promotion:
1) alabastrum is chosen: take pale reddish brown cauliflower lobe and anther length than be 1.0, Caulis et Folium Brassicae capitatae be 1.0, monokaryon late period to double-core early stage, healthy, alabastrum without pest and disease damage, as the donor of microspores culture；
2) sterilizing of alabastrum: the sterilized solution being configured to 1g mercuric chloride+1L sterilized water；Pale reddish brown dish and Caulis et Folium Brassicae capitatae mixing alabastrum are put in sterile petri dish, adds sterilized solution, be put on shaking table shake and carry out surface sterilization 12 minutes, then after washing 4 times with aseptic pond on superclean bench, standby；
3) on superclean bench, 13 aseptic pale reddish brown cauliflower flower buds are placed in sterile beaker, add 10mlB5 wash culture medium, with tack Glass rod squeeze broken, grind to form suspension；This suspension, with in 45 μm of aseptic strainer filterings to 50ml centrifuge tube, covers tightly, and 900rpm/min is centrifuged 5min, outwells supernatant after centrifugal, adds 10mlB5 inducing culture, be centrifuged 1 time more as stated above, abandon supernatant in precipitation；Adding NLN 13 inducing culture 40ml, add by the activated carbon mixed liquor 10 of NLN 13 fluid mediums+agarose 2g/L+1g/L activated carbon preparation, high temperature sterilize, the concentration obtaining sporidiole is 0.8 × 105The microspore suspension of individual/mL；
4) this microspore suspension is dispensed in 90mm glass serum culture dish, every 90mm culture dish adds 10ml sporidiole mixing suspension and 8 Caulis et Folium Brassicae capitatae flower pesticide, with the sealing of parafilm film after adding a cover, it is placed on Heat thermostability 3 days in 33 DEG C of constant temperature biochemical cultivation cases, under dark condition；Rear taking-up is placed in 25 DEG C of constant incubators, under dark condition and continues cultivation；During embryoid visible to naked eyes, it is placed in wave and culture 25 days on 25 DEG C of shaking tables, under dark condition, obtains cotyledon type embryoid and reach maturity；
5) cotyledon type embryoid is seeded in embryoid division culture medium, every day 16 h light, cultivate 2 thoughtful formation embryoids at 25 DEG C；Embryoid direct inoculation to embryoid division culture medium continuing under the same conditions cultivate, until differentiation, regeneration bud；
6) regeneration bud cutting healthy growth is seeded on root media, every day 16 h light, carry out root culture at 25 DEG C；After 3 weeks, the Seedling that root growth is healthy and strong is carried out seedling exercising 5 days；Clean tissue cultured seedling root culture medium with clear water during transplanting, after plant is planted in vegetable seedling substrate hole tray, plastic foil covers moisturizing, cultivates and transplants after 8 days, obtain regeneration plant in greenhouse；
All the other operations are with embodiment 1.
Result: co-culturing pale reddish brown dish germ extraction rate is 14.2/flower bud, and compares germ extraction rate and be only 1.5/flower bud.Finally, Mixed culture obtains the 66 pale reddish brown dish sporule regeneration plants of strain, and compares acquisition 1 strain.
Embodiment 3
Except in step (1) 1) B5 washs culture medium: B5 fluid medium 1L+ sucrose 30g/L, pH6.0, High Temperature High Pressure；2) NLN 13 inducing culture: NLN 13 fluid medium 1L+ sucrose 130g/L, pH6.1, filtration sterilization；3) embryoid division culture medium: B5 medium+sucrose 20g/L, agar 11g/L, pH6.0, autoclave sterilization；4) root media: MS culture medium+white sugar 20g/L, agar 7g/L, pH5.9, hot and humid sterilizing；
(2) pale reddish brown dish sporidiole and Caulis et Folium Brassicae capitatae flower pesticide co-culture the embryogenetic cultural method of promotion:
1) alabastrum is chosen: take pale reddish brown cauliflower lobe and anther length than be 1.1, Caulis et Folium Brassicae capitatae be 1.1, monokaryon late period to double-core early stage, healthy, alabastrum without pest and disease damage, as the donor of microspores culture；
2) sterilizing of alabastrum: the sterilized solution being configured to 1g mercuric chloride+1L sterilized water；Pale reddish brown dish and Caulis et Folium Brassicae capitatae mixing alabastrum are put in sterile petri dish, adds sterilized solution, be put on shaking table shake and carry out surface sterilization 11 minutes, then after washing 5 times with aseptic pond on superclean bench, standby；
3) on superclean bench, 11 aseptic pale reddish brown cauliflower flower buds are placed in sterile beaker, add 10mlB5 inducing culture, with tack Glass rod squeeze broken, grind to form suspension；This suspension, with in 45 μm of aseptic strainer filterings to 50ml centrifuge tube, covers tightly, and 700rpm/min is centrifuged 5min, outwells supernatant after centrifugal, adds 10mlB5 inducing culture, be centrifuged 2 times more as stated above, abandon supernatant in precipitation；Adding NLN 13 inducing culture 40ml, add by the activated carbon mixed liquor 10 of NLN 13 fluid mediums+agarose 3.5g/L+1g/L activated carbon preparation, high temperature sterilize, the concentration obtaining sporidiole is 1.2 × 105The sporidiole mixing suspension of individual/mL；
4) this microspore suspension is dispensed in 60mm glass serum culture dish, every 60mm culture dish adds 4ml sporidiole mixing suspension and 6 aseptic Caulis et Folium Brassicae capitatae flower pesticide, with the sealing of parafilm film after adding a cover, it is placed on Heat thermostability 2 days in 33 DEG C of constant temperature biochemical cultivation cases, under dark condition；Rear taking-up is placed in 25 DEG C of constant incubators, under dark condition and continues cultivation；During embryoid visible to naked eyes, it is placed in wave and culture 22 days on 25 DEG C of shaking tables, under dark condition, obtains cotyledon period embryoid and reach maturity；
5) cotyledon period embryoid is seeded in embryoid division culture medium, every day 16 h light, cultivate 2.5 thoughtful formation embryoids at 25 DEG C；Embryoid is cut into much the same piece of 2 block sizes be inoculated in embryoid division culture medium and continue cultivation under the same conditions, until differentiation, regeneration bud；
6) regeneration bud cutting healthy growth is seeded on root media, every day 16 h light, carry out root culture at 25 DEG C；Seedling exercising 4 days after 3 weeks；Cleaning tissue cultured seedling root culture medium with clear water during transplanting, then plant is planted in special seedling substrate hole tray, plastic foil covers moisturizing, transplants, obtain regeneration plant in greenhouse after cultivating 9 days；
All the other operations are with embodiment 1.
Result: co-culturing pale reddish brown dish germ extraction rate is 13.6/flower bud, and compares germ extraction rate and be only 1.5/flower bud.Finally, Mixed culture obtains the 70 pale reddish brown dish sporule regeneration plants of strain, and compares acquisition 3 strains.
Claims (7)
1. a method for the pale reddish brown dish haplobiont of quick acquisition, comprises the following steps:
1) peek amount than for the pale reddish brown dish of 1:5～8 with easily go out the alabastrum of Caulis et Folium Brassicae capitatae of embryo, sterilizing；
2) being placed in culture fluid by the pale reddish brown dish Isolated microspore after mixing alabastrum sterilizing with the Caulis et Folium Brassicae capitatae flower pesticide easily going out embryo, Mixed culture after thermal shock, induction obtains cotyledon type embryoid；
3) cotyledon type embryoid is seeded in solid division culture medium, cultivates and obtain regeneration plant；
4) cut regeneration bud from regeneration plant, access root media, it is thus achieved that whole plant.
2. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to claim 1, it is characterised in that the alabastrum of described pale reddish brown dish and the Caulis et Folium Brassicae capitatae that easily goes out embryo is monokaryon late period to double-core early stage in period of drawing materials.
3. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to claim 1, it is characterised in that step 2) described in pale reddish brown dish sporidiole adopt 32～33 DEG C of thermal shocks to process 1～3 day with the Caulis et Folium Brassicae capitatae flower pesticide mixed liquor easily going out embryo.
4. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to any one of claims 1 to 3, it is characterized in that, step 2) described in culture fluid be NLN-13 fluid medium and the mixture of sterile active charcoal mixed liquor, consisting of of wherein said activated carbon mixed liquor: 1LNLN fluid medium, 2～5g agarose and 1g activated carbon.
5. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to any one of claims 1 to 3, it is characterized in that, described microspore suspension with after flower pesticide mixing thermal shock, first 25 DEG C, after constant temperature culture occurs for 10～15 days to naked eyes visible cell group under dark condition, move on to 25 DEG C, 50～60rpm shaken cultivation 10～15 days under dark condition, obtain cotyledon type embryoid.
6. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to any one of claims 1 to 3, it is characterised in that step 3) described in solid division culture medium be B5 medium+sucrose 30g/L, agar 10～12g/L, pH5.9～6.1.
7. the method for the pale reddish brown dish haplobiont of a kind of quick acquisition according to any one of claims 1 to 3, it is characterized in that, described root media is made up of 1/2MS culture medium 1L+0.1～0.2mg/LNAA+0.1～0.2mg/LIBA, sucrose or white sugar 20～30g/L and agar 7～8g/L, pH5.8～6.0.
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| The ploidy of plants obtained from anther culture of cauliflowers(Brassica oleracea var.botrytis);D.J.OCKENDON, ET AL.;《Ann.appl.Biol》;19881231;第113卷;第319-325页。 * |
| 小孢子培养获得松花型花椰菜DH再生植株;顾宏辉等;《农业生物技术学报》;20071231;第15卷(第2期);第301-305页，尤其是摘要以及第302页第1.1-1.5节，第2.2节 * |
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