CN101371650A - Method for inducing dissociate microspore callus of eggplant and regenerating plant strain - Google Patents
Method for inducing dissociate microspore callus of eggplant and regenerating plant strain Download PDFInfo
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- CN101371650A CN101371650A CNA2007101207432A CN200710120743A CN101371650A CN 101371650 A CN101371650 A CN 101371650A CN A2007101207432 A CNA2007101207432 A CN A2007101207432A CN 200710120743 A CN200710120743 A CN 200710120743A CN 101371650 A CN101371650 A CN 101371650A
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Abstract
The invention is a method of inducement of isolated microspore callus of eggplant and plant regeneration. The major procedures are as follows. Appropriate anther is selected for pre-cultivation. Then dissociation and collection of microspore are carried out. And then static shallow pan dark culture is carried out in fluid nutrient medium. After 20 to 30 days, callus emerges successively. Callus is taken for cultivation under sunlight after emergence. When the callus grows to the size with the diameter of 2 to 6 millimeters, callus is transferred to solid regeneration culture medium for further cultivation. After 30 to 45 days, callus begins to differentiate to form buds. When bud grows to size of 1 to 3 centimeters, callus is transferred to rooting culture medium. After 10 to 15 days, strong root is developed. By adopting the method, the probability of obtaining haploid is high and frequency of microspore callus genesis is high. The invention is not only the technological breakthrough of eggplant haploid breeding, the inducing system of isolated microspore for cultivation of embryoid can also be a good experimental system for carrying out research on plant cell differentiation and development, molecular marker and gene map.
Description
Technical field
The present invention relates to a kind of plant cultivation method, particularly relate to a kind of method for breeding haploidy of eggplant.
Background technology
At present, in the eggplant breeding work, the seed selection of inbred line is very important work, but often has problems such as the regeneration period is long.Haploid breeding is one of important breeding method that addresses this problem, and can improve the efficient that breeding is selected greatly, the scale of dwindling breeding population, and shortening the breeding cycle.Monoploid is in the heredity and the application and the meaning of breeding: one quickens inheritance purifying, shortening the breeding cycle; Two can differentiate hereditary type of separation from the gamete level, improve efficiency of selection; Three can effectively utilize the microorganism hereditary technology in higher plant; Four utilize the ploidyization of pollen callus or plant tissue, induce to produce the ploidy plant; Five improve the utilization of distant hybridization in breeding, overcome the incompatibility of distant hybridization, overcome the infertile property of distant hybrids; The 6th, the good experiment material of genetic research; Seven mutant choice; The acceptor material of eight genetic transformations etc.
From the molecular biology angle, obtaining haploid significance also is to obtain the highly purified double haploid that isozygotys by utilizing flower pesticide (powder) to cultivate after the dyed body of monoploid of inducing doubles, set up double haploid colony (DH) by it, it in heredity the genome that isozygotys, DH colony is one of more satisfactory molecular labeling mapping population, be RFLP, AFLP, RAPD, the ideal material of SSR equimolecular mark and genome, can avoid dliploid from two chromosomes of parents nuance, thereby improve the accuracy that gene location is marked on a map greatly at the DNA base sequence.
Anther culture and microspores culture are to obtain haploid effective method.Need to solve but still exist a lot of problems at present in anther culture, because the interference of flower pesticide each several part tissue, cultivating product may cause the somatic cell that produces Different Ploidy to mix body not by pollen.
Summary of the invention
Technical problem solved by the invention provides a kind of eggplant Isolated microspore callus induction and plant regeneration method, this method obtains haploid probability height, microspore callus occurrence frequency height, be not only the technical breakthrough of eggplant haploid breeding, and this Isolated microspore is cultivated the embryoid system of inducing and also may be become and carry out the plant cell differentiation, grow research, the good experimental system of molecular labeling and genome.
A kind of eggplant Isolated microspore callus induction and plant regeneration method, key step is to choose suitable flower pesticide to cultivate in advance, carry out the free and collection of microspore then, carrying out static shallow-layer then in the liquid medium within secretly cultivates, callus appears behind 20～30d successively, callus the back occurs and cultivates down in illumination, when callus lines path length to 2～6mm, forward callus on solid regenerated medium continuation cultivation, beginning differentiation on 30～45d callus sprouts a little, forward root media when bud point is long to during to 1～3cm, grow healthy and strong root behind 10～15d.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the wherein said flower pesticide step of choosing is chosen full-bloom stage on the robust growth plant under field conditions (factors) promptly to eggplant, the bud in four door socket periods.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the pre-cultivation of wherein said flower pesticide comprises the steps:
D) sterilization bud;
E) inoculation: on superclean bench, strip flower pesticide gently and be inoculated in the culture dish that pre-culture medium i is housed from the bud of the bacterium of having gone out, every culture dish connects the flower pesticide of 2～3 buds, and every culture dish is equipped with 8～10mL medium;
F) heat shock is handled: will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, heat-shock temperature is 30～36 ℃, and the processing time is 4～8d;
Wherein said pre-culture medium i is as follows: MS medium and 0.05～0.5mgL
-12,4-D and 0.5～2mgL
-1KT and 6～10mgL
-1Vc and 2～4% sucrose and 5～10gL
-1Agar, medium pH value are 5.5～7, adopt autoclaving: 121 ℃ of 15～20min that sterilize down.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the free and collection of wherein said microspore comprises the steps:
D) from the pre-flower pesticide of cultivating, choose pollution-free, no brownization with aseptic tweezers and the flower pesticide that expands is put in the aseptic culture dish;
E) will wash medium ii and add in the top culture dish, cut flower pesticide and extruding, so that microspore is free to washing among the medium ii from flower pesticide with aseptic scalpel;
F) the above-mentioned washing medium ii that contains microspore is filtered through 200 order nylon screens, collect filtrate, remove supernatant, precipitate centrifugally again, repeat 3 times in the centrifugal 1～10min of 500rpm;
Wherein said washing medium ii is as follows: MS medium and 2～3% sucrose, pH value are 5.5～7, adopt autoclaving: 115～121 ℃ of 15～20min that sterilize down.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the cultivation of wherein said Isolated microspore comprises the steps:
C) microspore after centrifugal is diluted to microspore density 2～4 * 10 with liquid nutrient medium iii
5Individual mL
-1., dividing to install in the sterile petri dish, every ware 3～5mL seals with Parafilm;
D) culture dish is put in the cultivation box, under 25～28 ℃, carry out static shallow-layer and secretly be cultured to the callus appearance, change once fresh liquid nutrient medium iii behind 15～20d, occur callus behind 20～30d successively, callus is taken intensity of illumination 1500～2000Lx, illumination 12～16hd after occurring
-1The following cultivation;
Wherein said liquid nutrient medium iii can be any in the following medium: a.KM medium and 0.05～0.5mgL
-12,4～D and 0.1～0.5mgL
-1KT and 0.5～2mgL
-1NAA and 100～300mgL
-1PEG and 5～7% glucose; B.KM medium and 0.5～2mgL
-1NAA and 1～3mgL
-1KT and 150～300mgL
-1PEG and 5～10% glucose;
Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the regeneration of wherein said callus comprises the steps: when callus lines path length to 2～6mm, callus is forwarded among the solid regenerated medium iv, 15～20d subculture once begins differentiation and sprouts a little on 30～45d callus;
Wherein said regeneration culture medium iv is as follows: MS medium and 0～0.2mgL
-1NAA and 0.5～5mgL
-16-BA and 1～5% sucrose and 5～10gL
-1Agar.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the taking root of wherein said regeneration plant comprise the steps: to forward root media v when bud point is long to during to 1～3cm, grow healthy and strong root behind 10～15d; Wherein said root media v is as follows: 1/2MS medium and 0.05～0.5mgL
-1IBA and 1～5% sucrose and 5～10gL
-1Agar.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, wherein said flower pesticide choose preferably the developmental stage of observing microspore by microscopy, choose the microspore of the monokaryon mid-term and the phase of keeping to the side, corresponding bud surface is that corolla is lower than calyx 1～2mm and is higher than calyx 1～2mm to corolla, sepal is about to before and after the cracking, and flower pesticide is yellow green.
Eggplant Isolated microspore callus induction of the present invention and plant regeneration method, the alcohol of wherein said bud sterilisation step employing 70～75% carries out the bud surface sterilization, disinfecting time 1～3min, soak 5～40min with 5.0～8.0% clorox then, at last with sterile water washing 3 times, each 1～10min blots standby with aseptic paper.
Microspores culture in eggplant Isolated microspore callus induction of the present invention and the plant regeneration method can overcome anther cultural defective, microspores culture is compared the following points advantage with anther culture: one, can get rid of anther wall, flower pesticide every etc. the influence of somatic tissue, eliminate medicine wall and the out of contior influence of other linked groups, and can regulate the various factors of domination androgenesis preferably; Two, there is not the pollen competition problem in cultivating, can improves and obtain haploid probability; Three, microspores culture can induce the microspore callus to take place with higher frequency in bigger genotype scope; Four can observe the overall process that individual cells begins androgenesis; Five since microspore can contact chemistry equably with the mutagen of physics, so microspore also is the ideal material of research absorption, conversion and mutagenesis; In a word, on haploid induction and genetic breeding research efficient, demonstrate the potentiality more much higher than anther culture.
Along with science and technology development, the microspores culture range of application is extensive day by day: (1) is passed through in basic science microspore dedifferentiation in incubation, the research that changes of differentiation and intracellular corresponding Physiology and biochemistry more all has very great help to the research of gene expression and Regulation Mechanism in experimental embryology, physiology and the molecular biology.(2) because microspore quantity is big, the little and form homogeneous of single status volume, thereby be convenient to research its growth, differentiation and genetic transformation process, for example induced mutations pollen, chromosome Function Identification etc. under the manual control condition.(3) the microspore plant recessive character shows easily, thereby plant type is various, and monoploid, double haploid and alien addition line and the substitution line that can obtain isozygotying simultaneously provide multiple genetic analysis material, have quickened breeding process.(4) microspore is unicellular, has natural dispersiveness, enormous amount, be easy to obtain, so having the institute similar to the unicellular microorganism system, microspores culture has superiority, add that the microspore embryo is taken place and the plant regeneration ability is strong, thereby can be used for embryo's clone and a large amount of breedings fast of new genotype or mutant.(5) from the molecular biology angle, haploid significance also is the double haploid colony (doubled～haploid progenies) that sets up by this method, because DH colony is the genome that isozygotys in heredity, so be the ideal material of AFLP, RAPD, SSR equimolecular mark and genome, can avoid dliploid because from two chromosomes of parents nuance, thereby improve the accuracy that gene location is marked on a map greatly at DNA base molecule basic group sequence.
Embodiment
Embodiment 1:
A kind of eggplant Isolated microspore callus induction and plant regeneration method, comprise the steps: that (1) choose flower pesticide: choose full-bloom stage on the robust growth plant under field conditions (factors) promptly to the bud of eggplant, four door sockets, observe the developmental stage of microspore by microscopy, choose the microspore of the monokaryon mid-term and the phase of keeping to the side, corresponding bud surface is that corolla is lower than calyx 1～2mm and is higher than calyx 1～2mm to corolla, sepal is about to before and after the cracking, and flower pesticide is yellow green.
(2) the pre-cultivation of flower pesticide: comprise the steps:
A) sterilization bud: adopt 70～75% alcohol to carry out the bud surface sterilization, disinfecting time 2min soaks 10～15min with 6.5% clorox then, and with sterile water washing 3 times, 5min blots standby with aseptic paper at every turn at last;
B) inoculation: on superclean bench, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium i is housed from the bud of the bacterium that disappeared, every culture dish connects the flower pesticide of 2 buds, and every culture dish is equipped with 8～10mL medium;
C) heat shock is handled: will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, heat-shock temperature is 32 ℃, the processing time be 8d (my god);
(3) the free and collection of microspore: comprise the steps:
A) from the pre-flower pesticide of cultivating, choose pollution-free, no brownization with aseptic tweezers and the flower pesticide that expands is put in the aseptic culture dish;
B) will wash medium ii and add in the top culture dish, cut flower pesticide and extruding, so that microspore is free to washing among the medium ii from flower pesticide with aseptic scalpel;
C) the above-mentioned washing medium ii that contains microspore is filtered through 200 order nylon screens, collect filtrate, remove supernatant, precipitate centrifugally again, repeat 3 times in the centrifugal 5min of 500rpm;
(4) cultivation of Isolated microspore: comprise the steps:
A) microspore after centrifugal is diluted to desired concn (counting with blood counting chamber), microspore density 3 * 10 with liquid nutrient medium iii
5Individual mL
-1., dividing to install in the sterile petri dish (Φ 60mm), every ware 5mL seals with Parafilm;
B) culture dish is put in the cultivation box, under 25～28 ℃, carry out static shallow-layer and secretly be cultured to the callus appearance, change once fresh liquid nutrient medium iii about every 15d, occur callus behind the 30d successively, callus is taken intensity of illumination 2000Lx, illumination 16hd after occurring
～1The following cultivation;
(5) regeneration of callus: when callus lines path length to 3～5mm, callus is forwarded among the solid regenerated medium iv, the 20d subculture once begins differentiation and sprouts a little on the 45d callus;
(6) taking root of regeneration plant: forward root media v when bud point is long to during to 2～3cm, grow healthy and strong root behind the 10d; Wherein said medium is as follows:
Pre-culture medium i:MS+0.5mgL
-12,4-D+2mgL～1KT+10mgL
-1Vc+4% sucrose+10gL
～1Agar, medium pH value are 6.4, adopt autoclaving: 121 ℃ of 15～20min that sterilize down;
Washing medium ii:MS+2% sucrose, the pH value is 6.4, adopts autoclaving: 115～121 ℃ of 15～20min that sterilize down;
Liquid nutrient medium iii:
A.KM+0.5mgL
-12,4-D+0.5mgL
-1KT+2mgL
-1NAA+300mgL
-1PEG+7% glucose;
B.KM+2mgL
-1NAA+3mgL
-1KT+300mgL
-1PEG+10% glucose;
Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization;
Regeneration culture medium iv:MS+0.2mgL
-1NAA+5mgL
-16-BA+5% sucrose+10gL
-1Agar;
Root media v:1/2MS+0.5mgL
-1IBA+5% sucrose+10gL
-1Agar.
Embodiment 2:
A kind of eggplant Isolated microspore callus induction and plant regeneration method comprise the steps:
(1) chooses flower pesticide: choose full-bloom stage on the robust growth plant under field conditions (factors) promptly to the bud of eggplant, four door sockets, observe the developmental stage of microspore by microscopy, choose the microspore of the monokaryon mid-term and the phase of keeping to the side, corresponding bud surface is that corolla is lower than calyx 1～2mm and is higher than calyx 1～2mm to corolla, sepal is about to before and after the cracking, and flower pesticide is yellow green.
(2) the pre-cultivation of flower pesticide: comprise the steps:
D) sterilization bud: adopt 70～75% alcohol to carry out the bud surface sterilization, disinfecting time 2min soaks 10～15min with 6.5% clorox then, and with sterile water washing 3 times, 5min blots standby with aseptic paper at every turn at last;
E) inoculation: on superclean bench, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium i is housed from the bud of the bacterium that disappeared, every culture dish connects the flower pesticide of 3 buds, and every culture dish is equipped with 8～10mL medium;
F) heat shock is handled: will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, heat-shock temperature is 35 ℃, the processing time be 8d (my god) about;
(3) the free and collection of microspore: comprise the steps:
D) from the pre-flower pesticide of cultivating, choose pollution-free, no brownization with aseptic tweezers and the flower pesticide that expands is put in the aseptic culture dish;
E) will wash medium ii and add in the top culture dish, cut flower pesticide and extruding, so that microspore is free to washing among the medium ii from flower pesticide with aseptic scalpel;
F) the above-mentioned washing medium ii that contains microspore is filtered through 200 order nylon screens, collect filtrate, remove supernatant, precipitate centrifugally again, repeat 3 times in the centrifugal 5min of 500rpm;
(4) cultivation of Isolated microspore: comprise the steps:
C) microspore after centrifugal is diluted to desired concn (counting with blood counting chamber), microspore density 2 * 10 with liquid nutrient medium iii
5Individual mL
-1., dividing to install in the sterile petri dish (Φ 60mm), every ware 5mL seals with Parafilm;
D) culture dish is put in the cultivation box, under 25～28 ℃, carry out static shallow-layer and secretly be cultured to the callus appearance, change once fresh liquid nutrient medium iii about every 15d, occur callus about 30d successively, callus is taken intensity of illumination 1500Lx, illumination 12hd after occurring
～1The following cultivation;
(5) regeneration of callus: when callus lines path length to 3～5mm, callus is forwarded among the solid regenerated medium iv, 20d left and right sides subculture once begins differentiation and sprouts a little on the callus of the 45d left and right sides;
(6) taking root of regeneration plant: forward root media v when bud point is long to during to 2～3cm, grow healthy and strong root about 10d; Wherein said medium is as follows:
Pre-culture medium i:MS+0.05mgL
-12,4-D+0.5mgL
～1KT+6mgL
-1Vc+2% sucrose+5gL
～1Agar, medium pH value are 5.5, adopt autoclaving: 121 ℃ of 15～20min that sterilize down;
Washing medium ii:MS+2% sucrose, the pH value is 5.5, adopts autoclaving: 115～121 ℃ of 15～20min that sterilize down;
Liquid nutrient medium iii:
A.KM+0.05mgL
-12,4-D+0.1mgL
-1KT+0.5mgL
-1NAA+100mgL
-1PEG+5% glucose;
B.KM+0.5mgL
-1NAA+1mgL
-1KT+150mgL
-1PEG+5% glucose; Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization;
Regeneration culture medium iv:MS+0.5mgL
-16-BA+1% sucrose+5gL
-1Agar;
Root media v:1/2MS+0.05mgL
-1IBA+1% sucrose+5gL
-1Agar.
Embodiment 3:
A kind of eggplant Isolated microspore callus induction and plant regeneration method comprise the steps:
(1) chooses flower pesticide: choose full-bloom stage on the robust growth plant under field conditions (factors) promptly to the bud of eggplant, four door sockets, observe the developmental stage of microspore by microscopy, choose the microspore of the monokaryon mid-term and the phase of keeping to the side, corresponding bud surface is that corolla is lower than calyx 1～2mm and is higher than calyx 1～2mm to corolla, sepal is about to before and after the cracking, and flower pesticide is yellow green.
(2) the pre-cultivation of flower pesticide: comprise the steps:
G) sterilization bud: adopt 70～75% alcohol to carry out the bud surface sterilization, disinfecting time 2min soaks 10～15min with 6.5% clorox then, and with sterile water washing 3 times, 5min blots standby with aseptic paper at every turn at last;
H) inoculation: on superclean bench, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium i is housed from the bud of the bacterium that disappeared, every culture dish connects the flower pesticide of 2 buds, and every culture dish is equipped with 8～10mL medium;
I) heat shock is handled: will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, heat-shock temperature is 36 ℃, the processing time be 6d (my god);
(3) the free and collection of microspore: comprise the steps:
G) from the pre-flower pesticide of cultivating, choose pollution-free, no brownization with aseptic tweezers and the flower pesticide that expands is put in the aseptic culture dish;
H) will wash medium ii and add in the top culture dish, cut flower pesticide and extruding, so that microspore is free to washing among the medium ii from flower pesticide with aseptic scalpel;
I) the above-mentioned washing medium ii that contains microspore is filtered through 200 order nylon screens, collect filtrate, remove supernatant, precipitate centrifugally again, repeat 3 times in the centrifugal 5min of 500rpm;
(4) cultivation of Isolated microspore: comprise the steps:
E) microspore after centrifugal is diluted to desired concn (counting with blood counting chamber), microspore density 4 * 10 with liquid nutrient medium iii
5Individual mL
-1., dividing to install in the sterile petri dish (Φ 60mm), every ware 5mL seals with Parafilm;
F) culture dish is put in the cultivation box, under 25～28 ℃, carry out static shallow-layer and secretly be cultured to the callus appearance, change once fresh liquid nutrient medium iii about every 20d, occur callus behind the 30d successively, callus is taken intensity of illumination 2000Lx, illumination 16hd after occurring
～1The following cultivation;
(5) regeneration of callus: when callus lines path length to 3～5mm, callus is forwarded among the solid regenerated medium iv, 20d left and right sides subculture once begins differentiation and sprouts a little on the callus of the 45d left and right sides;
(6) taking root of regeneration plant: forward root media v when bud point is long to during to 2～3cm, grow healthy and strong root behind the 10d; Wherein said medium is as follows:
Pre-culture medium i:MS+0.2mgL
-12,4-D+1mgL～1KT+8mgL
-1Vc+3% sucrose+7gL
～1Agar, medium pH value are 5.8, adopt autoclaving: 121 ℃ of 15～20min that sterilize down;
Washing medium ii:MS+3% sucrose, the pH value is 5.8, adopts autoclaving: 115～121 ℃ of 15～20min that sterilize down;
Liquid nutrient medium iii:
A.KM+0.2mgL
-12,4-D+0.5mgL
-1KT+1mgL
-1NAA+250mgL
-1PEG+6.5% glucose;
B.KM+1mgL
-1NAA+2mgL
-1KT+250mgL
-1PEG+6.5% glucose;
Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization;
Regeneration culture medium iv:MS+0.01mgL
-1NAA+2mgL
-16-BA+2% sucrose+7gL
-1Agar;
Root media v:1/2MS+0.2mgL
-1IBA+2% sucrose+7gL
-1Agar.
Above-described embodiment is described preferred implementation of the present invention; be not that design of the present invention and scope are limited; the present invention relates under the scheme prerequisite not breaking away from; various conspicuous modification and improvement that those skilled in the art make technical scheme of the present invention; all should fall into protection scope of the present invention; the technology contents that the present invention asks for protection all is documented in claims.
Claims (9)
1. eggplant Isolated microspore callus induction and plant regeneration method, it is characterized in that: choose suitable flower pesticide and cultivate in advance, carry out the free and collection of microspore then, carrying out static shallow-layer then in the liquid medium within secretly cultivates, callus appears behind 20～30d successively, callus the back occurs and cultivates down in illumination, when callus lines path length to 2～6mm, forward callus on solid regenerated medium continuation cultivation, beginning differentiation on 30～45d callus sprouts a little, forward root media when bud point is long to during to 1～3cm, grow healthy and strong root behind 10～15d.
2. eggplant Isolated microspore callus induction according to claim 1 and plant regeneration method is characterized in that: the described flower pesticide step of choosing is chosen full-bloom stage on the robust growth plant under field conditions (factors) promptly to eggplant, the bud in four door socket periods.
3. eggplant Isolated microspore callus induction according to claim 2 and plant regeneration method is characterized in that: the pre-cultivation of described flower pesticide comprises the steps:
A) sterilization bud;
B) inoculation: on superclean bench, strip flower pesticide gently and be inoculated in the culture dish that pre-culture medium i is housed from the bud of the bacterium of having gone out, every culture dish connects the flower pesticide of 2～3 buds, and every culture dish is equipped with 8～10mL medium;
C) heat shock is handled: will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, heat-shock temperature is 30～36 ℃, and the processing time is 4～8d;
Wherein said pre-culture medium i is as follows: MS medium and 0.05～0.5mgL
-12,4-D and 0.5～2mgL
-1KT and 6～10mgL
-1Vc and 2～4% sucrose and 5～10gL
-1Agar, medium pH value are 5.5～7, adopt autoclaving: 121 ℃ of 15～20min that sterilize down.
4. eggplant Isolated microspore callus induction according to claim 3 and plant regeneration method is characterized in that: the free and collection of described microspore comprises the steps:
A) from the pre-flower pesticide of cultivating, choose pollution-free, no brownization with aseptic tweezers and the flower pesticide that expands is put in the aseptic culture dish;
B) will wash medium ii and add in the top culture dish, cut flower pesticide and extruding, so that microspore is free to washing among the medium ii from flower pesticide with aseptic scalpel;
C) the above-mentioned washing medium ii that contains microspore is filtered through 200 order nylon screens, collect filtrate, remove supernatant, precipitate centrifugally again, repeat 3 times in the centrifugal 1～10min of 500rpm;
Wherein said washing medium ii is as follows: MS medium and 2～3% sucrose, pH value are 5.5～7, adopt autoclaving: 115～121 ℃ of 15～20min that sterilize down.
5. eggplant Isolated microspore callus induction according to claim 4 and plant regeneration method is characterized in that: the cultivation of described Isolated microspore comprises the steps:
A) microspore after centrifugal is diluted to microspore density 2～4 * 10 with liquid nutrient medium iii
5Individual mL
-1, dividing to install in the sterile petri dish, every ware 3～5mL seals with Parafilm;
B) culture dish is put in the cultivation box, under 25～28 ℃, carry out static shallow-layer and secretly be cultured to the callus appearance, per 15～20d changes once fresh liquid nutrient medium iii, occur callus behind 20～30d successively, callus is taken intensity of illumination 1500～2000Lx, illumination 12～16hd after occurring
-1The following cultivation;
Wherein said liquid nutrient medium iii is any in the following medium:
A.KM medium and 0.05～0.5mgL
-12,4～D and 0.1～0.5mgL
-1KT and 0.5～2mgL
-1NAA and 100～300mgL
-1PEG and 5～7% glucose;
B.KM medium and 0.5～2mgL
-1NAA and 1～3mgL
-1KT and 150～300mgL
-1PEG and 5～10% glucose;
Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization.
6. eggplant Isolated microspore callus induction according to claim 5 and plant regeneration method, it is characterized in that: the regeneration of described callus comprises the steps: when callus lines path length to 2～6mm, callus is forwarded among the solid regenerated medium iv, 15～20d subculture once begins differentiation and sprouts a little on 30～45d callus;
Wherein said regeneration culture medium iv is as follows: MS medium and 0～0.2mgL
-1NAA and 0.5～5mgL
-16-BA and 1～5% sucrose and 5～10gL
-1Agar.
7. eggplant Isolated microspore callus induction according to claim 6 and plant regeneration method, it is characterized in that: the taking root of described regeneration plant comprises the steps: to forward root media v when bud point is long to during to 1～3cm, grows healthy and strong root behind 10～15d; Wherein said root media v is as follows: 1/2MS medium and 0.05～0.5mgL
-1IBA and 1～5% sucrose and 5～10gL
-1Agar.
8. eggplant Isolated microspore callus induction according to claim 2 and plant regeneration method, it is characterized in that: described flower pesticide choose the developmental stage of observing microspore by microscopy, choose the microspore of the monokaryon mid-term and the phase of keeping to the side, corresponding bud surface is that corolla is lower than calyx 1～2mm and is higher than calyx 1～2mm to corolla, sepal is about to before and after the cracking, and flower pesticide is yellow green.
9. eggplant Isolated microspore callus induction according to claim 3 and plant regeneration method, it is characterized in that: the alcohol of described bud sterilisation step employing 70～75% carries out the bud surface sterilization, disinfecting time 1～3min, soak 5～40min with 5.0～8.0% clorox then, at last with sterile water washing 3 times, each 1～10min blots standby with aseptic paper.
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| CNA2007101207432A CN101371650A (en) | 2007-08-24 | 2007-08-24 | Method for inducing dissociate microspore callus of eggplant and regenerating plant strain |
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| CNA2007101207432A CN101371650A (en) | 2007-08-24 | 2007-08-24 | Method for inducing dissociate microspore callus of eggplant and regenerating plant strain |
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| CN103444552A (en) * | 2013-09-28 | 2013-12-18 | 武汉市蔬菜科学研究所 | Method for inducing eggplant anther to regenerate haplobiont |
| CN104041415A (en) * | 2014-06-18 | 2014-09-17 | 南京市蔬菜科学研究所 | Method for obtaining haploid regenerated plant by eggplant anther culturing |
| CN104542274A (en) * | 2014-07-03 | 2015-04-29 | 郑州市蔬菜研究所 | Culture medium of induced haplobiont for culturing eggplant anther and method of culture medium |
| CN105165620A (en) * | 2015-10-09 | 2015-12-23 | 金陵科技学院 | Eggplant flower bud surface disinfection technology |
| CN105766652A (en) * | 2016-04-22 | 2016-07-20 | 福州市蔬菜科学研究所 | Method for inducing formation and plant regeneration of calluses of isolated microspores of long eggplant |
| CN106962187A (en) * | 2017-03-24 | 2017-07-21 | 金陵科技学院 | A kind of eggplant haplobiont method for doubling |
| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
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| CN103444552A (en) * | 2013-09-28 | 2013-12-18 | 武汉市蔬菜科学研究所 | Method for inducing eggplant anther to regenerate haplobiont |
| CN103444552B (en) * | 2013-09-28 | 2015-08-19 | 武汉市蔬菜科学研究所 | A kind of method of inducing eggplant flower pesticide regeneration haplobiont |
| CN104041415A (en) * | 2014-06-18 | 2014-09-17 | 南京市蔬菜科学研究所 | Method for obtaining haploid regenerated plant by eggplant anther culturing |
| CN104041415B (en) * | 2014-06-18 | 2015-09-23 | 南京市蔬菜科学研究所 | A kind of eggplant anther culture obtains the method for monoploid regeneration plant |
| CN104542274A (en) * | 2014-07-03 | 2015-04-29 | 郑州市蔬菜研究所 | Culture medium of induced haplobiont for culturing eggplant anther and method of culture medium |
| CN105165620A (en) * | 2015-10-09 | 2015-12-23 | 金陵科技学院 | Eggplant flower bud surface disinfection technology |
| CN105766652A (en) * | 2016-04-22 | 2016-07-20 | 福州市蔬菜科学研究所 | Method for inducing formation and plant regeneration of calluses of isolated microspores of long eggplant |
| CN105766652B (en) * | 2016-04-22 | 2018-03-20 | 福州市蔬菜科学研究所 | One kind induces long eggplant Isolated microspore calli induction and plant regeneration method |
| CN106962187A (en) * | 2017-03-24 | 2017-07-21 | 金陵科技学院 | A kind of eggplant haplobiont method for doubling |
| CN110845266A (en) * | 2019-11-22 | 2020-02-28 | 顾霆 | Embryonic germ cell dedifferentiation culture medium and preparation method thereof |
| CN116998405A (en) * | 2023-08-23 | 2023-11-07 | 金陵科技学院 | Method for improving embryoid seedling rate of eggplant anther culture |
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