CN104186309A - Method for improving embryogenesis rate of Raphanus sativus L. sinoruber makino - Google Patents

Method for improving embryogenesis rate of Raphanus sativus L. sinoruber makino Download PDF

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CN104186309A
CN104186309A CN201410371104.3A CN201410371104A CN104186309A CN 104186309 A CN104186309 A CN 104186309A CN 201410371104 A CN201410371104 A CN 201410371104A CN 104186309 A CN104186309 A CN 104186309A
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rouge radish
culture
medium
broccoli
improves
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CN104186309B (en
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张振超
毛忠良
潘跃平
吴国平
王建华
戴忠良
秦文斌
姚悦梅
潘永飞
肖燕
孙春青
马志虎
孙国胜
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Nanjing Lihua Agricultural Technology Co.,Ltd.
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Zhenjiang Ruifan Agricultural Gardening Co Ltd
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Abstract

The invention discloses a method for improving the embryogenesis rate of Raphanus sativus L. sinoruber makino. The method comprises the following steps: sterilizing Raphanus sativus L. sinoruber makino flower buds, then adding B5 wash medium to prepare a suspension liquid, filtering to obtain a filtrate, and centrifuging to obtain precipitate; sequentially adding NLN-13 induction medium and an activated carbon mixed solution to obtain microspore suspension liquid; subpackaging into a sterile culture dish, then adding sterile broccoli anther, performing Parafilm sealing and then conducting heat-shock treatment; performing routine culture to obtain cotyledonous embryoid; inoculating to an embryoid differentiation medium until differentiation is performed, and regenerating buds; cutting and inoculating regenerated buds to a rooting medium for rooting culture, and hardening seedling and transplanting, to obtain regenerated plants. According to the method, the broccoli anther easy to form embryos and the Raphanus sativus L. sinoruber makino isolated microspore difficult to form embryos are mixed for culture according to certain proportion, so that the embryonic development of the Raphanus sativus L. sinoruber makino can be promoted to obtain a great amount of regenerated plants, and the culture efficiency can be improved.

Description

A kind of method that improves rouge radish embryo incidence
Technical field
The present invention relates to field of plant tissue culture technique, relate in particular to the embryogenetic cultural method of a kind of promotion rouge radish microspore.
Background technology
Rouge radish (Raphanus Sativus L.Sinoruber Makino) is landrace under Cruciferae Rhaphanus radish kind, its fleshy root is nutritious, quality is finer and close, the various dried radish of suitable processing, simultaneously fleshy root skin, the heart are entirely red, high and the pigment quality of radish red pigment content and dissolubility are all better than other carrot, so turnip with red inside is the desirable feedstock crop of extracting natural edible red pigment, in the existing larger area under cultivation in Southwestern China area.From rouge radish, extract the radish red pigment obtain and have that chemical stability is good, storage-stable, the excellent feature of warm tolerance, in addition, it also has certain non-oxidizability, being has the colouring agent of health care to human body.
At present, in production, the rouge radish kind germ plasm resource of use is less, proterties is uneven, and leaf look, blade profile, root type differ in a jumble; False red heart, color of the leather is neat, and yellowish pink be take fickle in love as main, generally mixes the white heart, and partial mass is without typical red heart, and the complete red characteristic of carpel is not remarkable; Pigment content is low, and fresh output is not high.Need extensive collection badly, arrange domestic and international rouge radish germ plasm resource, utilize GENERALIZATION OF MODERN BREEDING TECHNIQUE means to purificate and rejuvenate, select and there is the full rouge radish inbred line breeding material red remarkable characteristic, dissimilar of carpel, and preparing hybrid kind, the level of resources utilization improved.
Heterosis In Radish is extremely obvious, the method of traditional separated parental inbred line of inbreeding of more generation generally needs the time of 5～6 years, and Isolated microspore cultural method can obtain haplobiont in 1～2 year, after doubling, become the inbred line of isozygotying, therefore, radish microspore culture has important function in the initiative of good inbred line and the aspects such as efficiency of raising breeding of new variety.Isolated microspore culture technique has very large application potential in radish genetic research and breeding.Through making great efforts for many years, although Chinese scholars has been done a large amount of deep research to improving the research of radish microspore embryo's occurrence frequency aspect, but compare with other crop in cruciferae, its embryoid induction rate and regeneration rate are lower, have seriously hindered the application of microspore culture.And in rouge radish breeding, microspore culture is not applied.On the basis of early-stage Study, adopt rouge radish Isolated microspore and broccoli easily to go out embryo genotype flower pesticide and cultivate altogether herein, significantly promote the embryonic development of rouge radish microspore, obtained a large amount of regeneration plants, required time is shorter, easy and simple to handle.
Summary of the invention
The invention provides the embryogenetic cultural method of a kind of raising rouge radish, adopt rouge radish and the broccoli flower pesticide that easily goes out embryo to cultivate altogether by some proportionings, thereby promote rouge radish microspore to go out embryo, improve the germ extraction rate of microspores culture.
For achieving the above object, the technical scheme that the present invention takes is:
A method that improves rouge radish embryo incidence, comprises the following steps:
1) get rouge radish and broccoli bud, sterilizing;
2) the rouge radish Isolated microspore and the broccoli flower pesticide that mix after bud sterilizing are placed in to culture fluid, Mixed culture after thermal shock, induction obtains cotyledon type embryoid;
3) cotyledon type embryoid is seeded in solid differential medium, cultivates and obtain regeneration plant;
4) from regeneration plant, cut regeneration bud, access root media, obtains whole plant;
Described rouge radish microspore germ extraction rate is poor, and broccoli easily goes out embryo.
The bud of described rouge radish and broccoli is all early stage to double-core late period in monokaryon.
The bud of described rouge radish and broccoli is 1:5～8 with the quantity of flower pesticide ratio.
Described rouge radish microspore and broccoli flower pesticide mixed liquor adopt 32～33 ℃ of thermal shocks to process 1～3 day.
Described culture fluid is the mixture of NLN-13 liquid nutrient medium and sterile active charcoal mixed liquor, wherein consisting of of active carbon mixed liquor: 1L NLN liquid nutrient medium, 2～5g agarose 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 2o8.6mg, 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, Glu 800mg, glutathione 30mg, vitamin B5 5mg, the sterile water of serine 100mg and surplus.
Described microspore suspension is with after flower pesticide mixing thermal shock, after first constant temperature culture occurs to naked eyes visible cell group for 10～15 days under 25 ℃, dark condition, moves on under 25 ℃, dark condition 50～60rpm shaken cultivation 10～15 days, obtains blade profile embryoid.
Described solid differential medium is B5 medium+sucrose 30g/L, agar 10～12g/L, and pH 5.9～6.1.
Described B5 liquid nutrient medium, in 1L, consists of: NaH 2pO 42H 2o 169.5mg, KNO 32500mg, (NH 4) 2sO 4134mg, MgSO 47H 2o 500mg, MnSO 44H 2o 10mg, H 3bO 33mg, ZnSO 47H 2o 2mg, KI 0.75mg, Na 2moO 42H 2o 0.25mg, CuSO 45H 2o 0.025mg, CoCl 26H 2o 0.025mg, Na 2-EDTA 37.3mg, FeSO 47H 2o 27.8mg, CaCl 2.2H 2o 150mg, VB1 10mg, VB6 1mg, VPP 1mg, the distilled water of inositol 100mg and surplus.
Described root media is comprised of 1/2MS medium 1L+0.1～0.2mg/L NAA+0.1～0.2mg/L IBA, sucrose or white granulated sugar 20～30g/L and agar 7～8g/L, and pH 5.8～6.0.
Described MS medium, in 1L, consists of: NH 4hO 31650mg, KNO 31900mg, CaCl 22H 2o440mg, KH 2pO 4170mg, MgSO 47H 2o 370mg, FeSO 47H 2o 27.8mg, 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, vitamin B1 0.1mg, the sterile water of vitamin B6 0.5mg and surplus.
Beneficial effect of the present invention is:
(1) the present invention is directed to the problem that rouge radish microspores culture difficulty goes out embryo, a kind of short-cut method that improves germ extraction rate is provided.Easily to go out the broccoli flower pesticide of embryo, mix by a certain percentage common cultivation with rouge radish Isolated microspore, the material that goes out embryo easily to go out the broccoli flower pesticide drive difficulty of embryo goes out embryo, improves germ extraction rate.The inventive method is cultivated altogether rouge radish germ extraction rate and is up to 16.2/flower bud, and contrast radish germ extraction rate is only up to 0.8/flower bud, has solved the low problem of rouge radish microspores culture embryo occurrence frequency, has improved the application efficiency of microspore culture.
(2) finally under same culture conditions, Mixed culture has finally obtained 59 strain rouge radish sporule regeneration plants, and the contrast strain number of cultivating is separately 1 strain.
(3) adopt that method is simple, cultivating the sporule regeneration plant colony obtaining is rouge radish, does not need plant type to identify, thereby has reduced workload, has improved culture efficiency, has operability.
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:
1) B5 washing medium: B5 liquid nutrient medium 1L+ sucrose 30g/L, pH 6.0, HTHP;
Described B5 liquid nutrient medium, in 1L, consists of: NaH 2pO 42H 2o 169.5mg, KNO 32500mg, (NH 4) 2sO 4134mg, MgSO 47H 2o 500mg, MnSO 44H 2o 10mg, H 3bO 33mg, ZnSO 47H2O 2mg, KI 0.75mg, Na 2moO 42H 2o 0.25mg, CuSO 45H 2o 0.025mg, CoCl 26H 2o 0.025mg, Na 2-EDTA 37.3mg, FeSO 47H 2o 27.8mg, CaCl 2.2H 2o 150mg, VB1 10mg, VB6 1mg, VPP 1mg, the distilled water of inositol 100mg and surplus.
2) embryoid differential medium: B5 medium+sucrose 20g/L, agar 10g/L, pH 6.0, autoclave sterilization;
3) NLN-13 inducing culture, NLN liquid nutrient medium 1L+ sucrose 130g/L, pH 6.0, filtration sterilization;
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 2o8.6mg, 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, Glu 800mg, glutathione 30mg, vitamin B5 5mg, the sterile water of serine 100mg and surplus.
4) root media: MS medium+sucrose 30g/L, agar 6g/L, pH 5.8, autoclave sterilization;
Described MS medium, in 1L, consists of: NH 4hO 31650mg, KNO 31900mg, CaCl 22H 2o440mg, KH 2pO 4170mg, MgSO 47H 2o 370mg, FeSO 47H 2o 27.8mg, 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, vitamin B1 0.1mg, the sterile water of vitamin B6 0.5mg and surplus.
(2) rouge radish microspore and broccoli flower pesticide are cultivated altogether and are promoted embryogenetic cultural method:
1) bud is chosen: get rouge radish petal and anther length than being 1.1, broccoli is 1.1, monokaryon late period is early stage, healthy to double-core, without the bud of damage by disease and insect, as the donor of microspores culture;
2) sterilizing of bud: be mixed with sterilized solution with 1g mercuric chloride+1L sterile water; Rouge radish and broccoli mixing bud are put into sterile petri dish, add sterilized solution, be put on shaking table and shake and carry out surface sterilization 10 minutes, then with aseptic pond, wash after 3 times on superclean bench, standby;
3) on superclean bench, 12 aseptic buds are placed in to aseptic beaker, add 10ml B5 washing medium, crowded broken with tack glass bar, grind to form suspension; This suspension, covers tightly with the aseptic strainer filtering in 45 μ m apertures in 50ml centrifuge tube, and the centrifugal 3min of 600rpm/min outwells supernatant after centrifugal, in precipitation, adds 10ml B5 washing medium, more centrifugal 2 times as stated above, abandons supernatant; Add NLN-13 inducing culture 40ml, then 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, the concentration that obtains microspore is 1 * 10 5the microspore mixing suspension of individual/mL;
4) this microspore suspension is divided and install in 60mm plastics sterile petri dish, every 60mm culture dish adds 4ml microspore mixing suspension and 5 aseptic flower pesticide of broccoli, add a cover the sealing of the rear parafilm of using film, be placed on heat shock in 32.5 ℃ of constant temperature biochemical cultivation cases, under dark condition and process 2 days; Then take out to be placed under 25 ℃ of constant incubators, dark condition and continue to cultivate; During to the visible embryoid of naked eyes, be placed on 25 ℃ of shaking tables, under dark condition, shake and cultivate 20 days, obtain cotyledon type embryoid and reach maturity;
5) cotyledon type embryoid is seeded in embryoid differential medium, at illumination 16 hours every days, 25 ℃, cultivates 3 thoughtful formation embryoids; According to a certain size embryoid, be cut into much the same of 3 block sizes, be inoculated in embryoid differential medium and continue under the same conditions to cultivate, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on root media, at illumination 16 hours every days, 25 ℃, carries out culture of rootage; After 3 weeks, the seedling of root growth stalwartness is carried out to hardening 3 days; During transplanting, with clear water, clean group training shoot root portion medium, rear plant is planted in grow seedlings of vegetable dedicated substrate cave dish, plastic foil covers moisturizing, cultivates and transplant afterwards for 10 days in greenhouse, obtains regeneration plant;
(3) except step 1) in only get rouge radish healthy growth, the donor plant without the inflorescence of damage by disease and insect as microspores culture, other operation is with " (2) rouge radish microspore and broccoli flower pesticide are cultivated altogether and promoted embryogenetic cultural method ", in contrast.
Result: Mixed culture radish germ extraction rate is 13.5/flower bud, and contrast radish germ extraction rate is only 1.1/flower bud.Finally, Mixed culture obtains the pale reddish brown dish sporule regeneration plant of 57 strain, and contrast obtains 1 strain.
Embodiment 2
In step (1): 1) B5 washing medium: B5 liquid nutrient medium 1L+ sucrose 30g/L, pH6.0, HTHP; 2) NLN-13 inducing culture: NLN-13 liquid nutrient medium 1L+ sucrose 130g/L, pH 6.1, filtration sterilization; 3) embryoid differential medium: B5 medium+sucrose 20g/L, agar 11g/L, pH 6.0, autoclave sterilization; 4) root media: MS medium+white sugar 20g/L, agar 7g/L, pH 5.9, hot and humid sterilizing;
(2) rouge radish microspore and broccoli flower pesticide are cultivated altogether and are promoted embryogenetic cultural method:
1) bud is chosen: get rouge radish petal and anther length than being 1.0, broccoli is 1.0, monokaryon late period is early stage, healthy to double-core, without the bud of damage by disease and insect, as the donor of microspores culture;
2) sterilizing of bud: the sterilized solution being mixed with 1g mercuric chloride+1L sterile water; Rouge radish and broccoli mixing bud are put into sterile petri dish, add sterilized solution, be put on shaking table and shake and carry out surface sterilization 12 minutes, then with aseptic pond, wash after 4 times on superclean bench, standby;
3) on superclean bench, 13 aseptic rouge radish buds are placed in to aseptic beaker, add 10ml B5 washing medium, crowded broken with tack glass bar, grind to form suspension; This suspension, covers tightly with the aseptic strainer filtering of 45 μ m in 50ml centrifuge tube, and the centrifugal 5min of 900rpm/min outwells supernatant after centrifugal, in precipitation, adds 10ml B5 inducing culture, more centrifugal 1 time as stated above, abandons supernatant; Add NLN-13 inducing culture 40ml, then 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, the concentration that obtains microspore is 0.8 * 10 5the microspore suspension of individual/mL;
4) this microspore suspension is divided and install in 90mm glass sterile petri dish, every 90mm culture dish adds 10ml microspore mixing suspension and 8 broccoli flower pesticide, add a cover the sealing of the rear parafilm of using film, be placed on heat shock in 33 ℃ of constant temperature biochemical cultivation cases, under dark condition and process 3 days; Rear taking-up is placed under 25 ℃ of constant incubators, dark condition and continues to cultivate; During to the visible embryoid of naked eyes, be placed on 25 ℃ of shaking tables, under dark condition, shake and cultivate 25 days, obtain cotyledon type embryoid and reach maturity;
5) cotyledon type embryoid is seeded in embryoid differential medium, at illumination 16 hours every days, 25 ℃, cultivates 2 thoughtful formation embryoids; Embryoid is directly inoculated in embryoid differential medium and continues under the same conditions to cultivate, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on root media, at illumination 16 hours every days, 25 ℃, carries out culture of rootage; After 3 weeks, the seedling of root growth stalwartness is carried out to hardening 5 days; During transplanting, with clear water, clean group training shoot root portion medium, rear plant is planted in vegetable seedling substrate cave dish, plastic foil covers moisturizing, cultivates and transplant afterwards for 8 days in greenhouse, obtains regeneration plant;
All the other operations are embodiment 1 simultaneously.
Result: cultivating altogether radish germ extraction rate is 14.5/flower bud, and contrast radish germ extraction rate is only 1.2/flower bud.Finally, Mixed culture obtains 59 strain radish sporule regeneration plants, and contrast obtains 2 strains.
Embodiment 3
In step (1): 1) B5 washing medium: B5 liquid nutrient medium 1L+ sucrose 30g/L, pH6.0, HTHP; 2) NLN-13 inducing culture: NLN-13 liquid nutrient medium 1L+ sucrose 130g/L, pH 6.1, filtration sterilization; 3) embryoid differential medium: B5 medium+sucrose 20g/L, agar 11g/L, pH 6.0, autoclave sterilization; 4) root media: MS medium+white sugar 20g/L, agar 7g/L, pH 5.9, hot and humid sterilizing;
(2) rouge radish microspore and broccoli flower pesticide are cultivated altogether and are promoted embryogenetic cultural method:
1) bud is chosen: get rouge radish petal and anther length than being 0.9, broccoli is 0.9, monokaryon late period is early stage, healthy to double-core, without the bud of damage by disease and insect, as the donor of microspores culture;
2) sterilizing of bud: the sterilized solution being mixed with 1g mercuric chloride+1L sterile water; Rouge radish and broccoli mixing bud are put into sterile petri dish, add sterilized solution, be put on shaking table and shake and carry out surface sterilization 11 minutes, then with aseptic pond, wash after 5 times on superclean bench, standby;
3) on superclean bench, 11 aseptic rouge radish buds are placed in to aseptic beaker, add 10ml B5 inducing culture, with tack glass bar squeeze broken, grind to form suspension; This suspension, covers tightly with the aseptic strainer filtering of 45 μ m in 50ml centrifuge tube, and the centrifugal 5min of 700rpm/min outwells supernatant after centrifugal, in precipitation, adds 10ml B5 inducing culture, more centrifugal 2 times as stated above, abandons supernatant; Add NLN-13 inducing culture 40ml, then 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, the concentration that obtains microspore is 1.2 * 10 5the microspore mixing suspension of individual/mL;
4) this microspore suspension is divided and install in 60mm glass sterile petri dish, every 60mm culture dish adds 4ml microspore mixing suspension and 6 aseptic broccoli flower pesticide, add a cover the sealing of the rear parafilm of using film, be placed on heat shock in 33 ℃ of constant temperature biochemical cultivation cases, under dark condition and process 2 days; Rear taking-up is placed under 25 ℃ of constant incubators, dark condition and continues to cultivate; During to the visible embryoid of naked eyes, be placed on 25 ℃ of shaking tables, under dark condition, shake and cultivate 22 days, obtain cotyledon period embryoid and reach maturity;
5) cotyledon period embryoid is seeded in embryoid differential medium, at illumination 16 hours every days, 25 ℃, cultivates 2.5 thoughtful formation embryoids; Embryoid is cut into much the same of 2 block sizes and is inoculated in embryoid differential medium and continues under the same conditions to cultivate, until differentiation, regeneration bud;
6) regeneration bud that cuts healthy growth is seeded on root media, at illumination 16 hours every days, 25 ℃, carries out culture of rootage; After 3 weeks, hardening is 4 days; During transplanting, with clear water, clean group training shoot root portion medium, then plant is planted in special seedling substrate cave dish, plastic foil covers moisturizing, cultivates and transplant afterwards for 9 days in greenhouse, obtains regeneration plant;
All the other operations are with embodiment 1.
Result: cultivating altogether rouge radish germ extraction rate is 10.6/flower bud, and contrast germ extraction rate is only 0.7/flower bud.Finally, Mixed culture obtains 60 strain radish sporule regeneration plants, and contrast obtains 3 strains.

Claims (9)

1. a method that improves rouge radish embryo incidence, comprises the following steps:
1) get the bud of rouge radish and broccoli, sterilizing;
2) the rouge radish Isolated microspore and the broccoli flower pesticide that mix after bud sterilizing are placed in to culture fluid, Mixed culture after thermal shock, induction obtains cotyledon type embryoid;
3) cotyledon type embryoid is seeded in solid differential medium, cultivates and obtain regeneration plant;
4) from regeneration plant, cut regeneration bud, access root media, obtains whole plant.
2. a kind of method that improves rouge radish embryo incidence according to claim 1, is characterized in that, described rouge radish microspore germ extraction rate is poor, and broccoli easily goes out embryo.
3. according to a kind of method that improves rouge radish embryo incidence described in claim 1 or 2, it is characterized in that, the bud of described rouge radish and broccoli is all early stage to double-core late period in monokaryon.
4. a kind of method that improves rouge radish embryo incidence according to claim 3, is characterized in that, described rouge radish bud with the quantity of broccoli flower pesticide than being 1:5～8.
5. according to a kind of method that improves rouge radish embryo incidence described in claim 1 or 2 or 4, it is characterized in that, rouge radish microspore and broccoli flower pesticide mixed liquor adopt 32～33 ℃ of thermal shocks to process 1～3 day.
6. according to a kind of method that improves rouge radish embryo incidence described in claim 1 or 2, it is characterized in that, step 2) culture fluid described in is the mixture of NLN-13 liquid nutrient medium and sterile active charcoal mixed liquor, wherein consisting of of active carbon mixed liquor: 1L NLN liquid nutrient medium, 2～5g agarose and 1g active carbon.
7. a kind of method that improves rouge radish embryo incidence according to claim 5, it is characterized in that, described microspore suspension is with after flower pesticide mixing thermal shock, after first under 25 ℃, dark condition, constant temperature culture occurs to naked eyes visible cell group for 10～15 days, move on under 25 ℃, dark condition 50～60rpm shaken cultivation 10～15 days, obtain cotyledon type embryoid.
8. according to a kind of method that improves rouge radish embryo incidence described in claim 1 or 2 or 4 or 6, it is characterized in that step 3) described in solid differential medium be B5 medium+sucrose 30g/L, agar 10～12g/L, pH 5.9～6.1.
9. a kind of method that improves rouge radish embryo incidence according to claim 8, it is characterized in that, described root media is comprised of 1/2MS medium 1L+0.1～0.2mg/L NAA+0.1～0.2mg/L IBA, sucrose or white granulated sugar 20～30g/L and agar 7～8g/L, and pH 5.8～6.0.
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CN114027181A (en) * 2021-09-23 2022-02-11 江苏省农业科学院 Radish microspore culture method
CN114946661A (en) * 2022-06-21 2022-08-30 华盛农业集团股份有限公司 Culture medium and culture method for tissue culture seedling of immature radish seeds
CN115316269A (en) * 2021-09-23 2022-11-11 江苏省农业科学院 Method for improving embryogenic rate of radish microspore

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