CN102228003A - 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
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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, China was many from ground such as the U.S., Japan, Holland introduction kale, 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 substantially at present, cost an arm and a leg, and the cost height, 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 by introducing external improved seeds, and the cycle is long, need expend lot of manpower and material resources, but the seed selection of uncertain energy is to good kind.Therefore press for the innovation breeding technique, the kind of using on the market is isozygotied 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 sees 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.Relevant rape belongs to studies show that in a large number of Isolated microspore cultivation, 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) gets the donor plant of the inflorescence of the inflorescence of kale and rape as microspores culture; Get bud after inducing directly or with inflorescence, obtain rape and kale and mix 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 filtrate through centrifugal, abandon supernatant and obtain precipitation, 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 of root growth stalwartness through hardening and transplanting, is obtained regeneration plant, identify.
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 sterilizing methods of this area routine 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, standby.
Consisting of of described active carbon mixed liquor: NLN liquid nutrient medium 1L, agarose 2g～5g and 1g active carbon; High-temperature sterilization, standby.
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 precipitation can repeat following operation 1～2 time: in precipitation, add the NLN-13 inducing culture, grind to form suspension, filter gained filtrate through centrifugal, abandon supernatant.Add NLN-13 inducing culture and active carbon mixed liquor after the repetitive operation in the precipitation 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; More 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 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 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, with mass percentage concentration is that 800～1000 times of (multiple of dilution) liquid of 75%～80% tpn (as commercially available tpn wetting powder) 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 cultivates in the greenhouse after 15～20 days and transplants.Come sterilization by 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 the tender leaf of getting regeneration plant detects each regeneration plant ploidy, and identifies rape regrowth and kale regrowth from regeneration plant colony.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, employing easily goes out the rape variety of embryo and the method that difficulty goes out the kale bud Mixed culture of embryo, the material that goes out embryo with the material drive difficulty that easily goes out embryo goes out embryo, improves the germ extraction rate of kale.The inventive method Mixed culture kale germ extraction rate is up to 104/flower bud, and contrast kale germ extraction rate is only up to 0.7/flower bud, 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 that the kale bud is cultivated separately 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; Rape and kale are mixed bud 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, standby again;
3) on superclean bench, (mix the bud sum with 12, wherein the rape bud is 8) sterilization after kale and rape mixing bud place aseptic beaker in the lump, add 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 precipitation 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, carries out culture of rootage under illumination 16 hours every days, 25 ℃; Seedling with the root growth stalwartness after 3 weeks carried out hardening 3 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 800 times of 75% tpn soaked base portion 2 minutes; Plant plant in kale special seedling substrate cave dish the back, and plastic foil covers preserves moisture, and cultivates after 15 days in the greenhouse and transplant, 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) only get in step 1) 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 ", kale in contrast.
The result: the germ extraction rate of kale is 104 a/flower bud in the Mixed culture, and contrast kale germ extraction rate only is 0.7 a/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 13 strain kale microspore seedlings at last, and the contrast strain number that contrast kale bud is cultivated separately 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; Rape and kale are mixed bud 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, standby again;
3) on superclean bench, (mix the bud sum with 15, wherein the rape bud is 5) sterilization after kale and rape mixing bud place aseptic beaker in the lump, add 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 precipitation 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; Embryoid directly is 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, carries out culture of rootage under illumination 16 hours every days, 25 ℃; Seedling with the root growth stalwartness after 3 weeks carried out hardening 5 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 1000 times of 80% tpn soaked base portion 1 minute; Plant plant in kale special seedling substrate cave dish the back, and plastic foil covers preserves moisture, and cultivates after 20 days in the greenhouse and transplant, 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 only is 0.5 a/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 12 strain kale microspore seedlings at last, and the contrast strain number that contrast kale bud is cultivated separately 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; Rape and kale are mixed bud 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, standby again;
3) on superclean bench, kale and rape mixings bud after 14 (mix bud sums, wherein the rape bud is 7) sterilization are placed aseptic beaker in the lump, add the 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 precipitation 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; Embryoid is cut into much the same of 2 block sizes is 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, carries out culture of rootage under illumination 16 hours every days, 25 ℃; Seedling with the root growth stalwartness after 3 weeks carried out hardening 4 days; Clean tissue cultivating seedling root medium with clear water during transplanting, 900 times of 75% tpn soaked base portion 1 minute; Plant plant in kale special seedling substrate cave dish the back, and plastic foil covers preserves moisture, and cultivates after 18 days in the greenhouse and transplant, 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 only is 0.6 a/flower bud; Detect discovery through perusal and flow cytometer, Mixed culture obtains 11 strain kale microspore seedlings at last, and the contrast strain number that contrast kale bud is cultivated separately is 0.
Claims (10)
1. the cultural method of a kale sporule regeneration plant may further comprise the steps:
1) gets the donor plant of the inflorescence of the inflorescence of kale and rape as microspores culture; Get bud after inducing directly or with inflorescence, obtain rape and kale and mix 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 filtrate through centrifugal, abandon supernatant and obtain precipitation, 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 of root growth stalwartness through hardening and transplanting, is obtained regeneration plant, identify.
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 is characterized in that, in the step 1), described condition of inducing is: induced 0.1 hour～48 hours at 3 ℃～5 ℃.
4. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that step 2) in, 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, vitaminB10 .5mg, vitamin B6 0.5mg, vitamin h 0.05mg, folic acid 0.5mg, Na
2EDTA 37.3mg, FeSO
47H
2O27.8mg, inositol 100mg, glycine 2mg, nicotinic acid 5mg, L-glutaminate 800mg, glutathione 30mg, vitamin B5 5mg, the sterile water of serine 100mg and surplus.
5. the cultural method of kale sporule regeneration plant according to claim 1, it is characterized in that step 2) in, described precipitation repeats following operation 1～2 time: add the NLN-13 inducing culture in precipitation, grind to form suspension, filter gained filtrate through centrifugal, abandon supernatant.
6. 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.
7. 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.
8. 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.
9. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that, in the step 4), described embryoid differential medium is: be made of pH5.8～6.1 MS medium 1L, sucrose 20g and agar 10g～12g.
10. the cultural method of kale sporule regeneration plant according to claim 1 is characterized in that, in the step 5), described root media is: be made of pH5.8～6.0 MS medium 1L, sugared 20g～30g and agar 6g～7g; Described sugar is sucrose or white sugar.
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| CN103392600A (en) * | 2013-08-06 | 2013-11-20 | 镇江瑞繁农艺有限公司 | Method for promoting collard stubborn genotype microspore embryogenesis |
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| 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 |
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| 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 |
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