CN101401550B - Method for inducing eggplant sporidiolum to form embryoid and special culture medium thereof - Google Patents
Method for inducing eggplant sporidiolum to form embryoid and special culture medium thereof Download PDFInfo
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- CN101401550B CN101401550B CN2008102270141A CN200810227014A CN101401550B CN 101401550 B CN101401550 B CN 101401550B CN 2008102270141 A CN2008102270141 A CN 2008102270141A CN 200810227014 A CN200810227014 A CN 200810227014A CN 101401550 B CN101401550 B CN 101401550B
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Abstract
The invention relates to a method for inducing microspore of eggplant to form embryoid and a special culture medium thereof, and belongs to the field of plant cell engineering. The method for inducing the microspore of the eggplant to form embryoid is to inoculate the microspore of the eggplant to the culture medium and perform dark culture at a temperature of between 25 and 28 DEG C to obtain the embryoid. The culture medium has low cost and good culture effect; the method for inducing the microspore of the eggplant by utilizing the culture medium has the advantages of high embryoid yield and short culture cycle; and the culture medium and the microspore inducing method applied to genetic breeding of the eggplant greatly enrich excellent genotype individuals of the eggplant, obviously shorten the breeding cycle, not only improve the approach for breeding haploidy of the eggplant but also provide technical and theoretical foundations for isolated culture of the microspore of potato family crops, and provide reliable technical support for genetic breeding and genetic research on solanaceous fruit vegetable crops.
Description
Technical field
The present invention relates to method and special culture media thereof that a kind of inducing eggplant microspore in plant cell engineering field forms embryoid.
Background technology
Monoploid has important meaning in genetic breeding.Obtaining haploid a kind of main means at present is that the cultured in vitro microspore obtains haplobiont.Microspore quantity is big, and the little and form homogeneous of single status volume is convenient to research its growth, differentiation and genetic transformation process under the manual control condition, and like induced mutations pollen, chromosome Function Identification etc., thereby microspore is the excellent research material.The in-vitro inducing microspore shows the plant recessive character easily, and then has enriched plant type, and the monoploid and the amphiploid that can also obtain isozygotying simultaneously provide multiple genetic analysis material, have quickened breeding process.Stripped microspore is unicellular, has natural dispersiveness, and enormous amount is easy to obtain, and adds 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.The monoploid that obtains through this method doubles again, obtains doubled haploid colony (DH colony, doubled-haploid progenies).DH colony is the genome that isozygotys in heredity; So it is 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.
The plant Isolated microspore has haploid cell division to form embryoid or callus, and developing into by embryoid that seedling or evoked callus grow then is two kinds of development pathways of plant.And embryoid just has bipolarity in the earliest stages of its generation, i.e. butt (radicle) and stem end (plumule), and do not have directly with the vascular bundle of mother cell or explant and to link, this takes place different with organ.Explain that thus embryoid is the blank of a complete plant at the very start, can from explant or callus, obtain nutrition through butt or similar suspensor structure.Also be easy to separate, grow up to a strain plant under optimum conditions from the surface of callus.
In Plant Tissue Breeding, inducing embryoid body is compared with induced bud, has significant advantage, and the one, quantity is many, and the 2nd, speed is fast, and the 3rd, planting percent is high.Because embryoid has these advantages, so in breeding work and horticulture, available embryoid also has very important significance for theories simultaneously as the individual vegetative propagation means of specific excellent genes type in the research embryonic development.
Summary of the invention
An object of the present invention is to provide a kind of plant tissue culture media.
Plant tissue culture media provided by the present invention is in the KM medium, to add KT and glucose, and to make the final concentration of KT in plant tissue culture media be 0.1-0.5mg/L, make the final concentration of glucose in plant tissue culture media is 50-70g/L; The solvent of said plant tissue culture media is a water; Do not contain glucose in the said KM medium; The pH of said plant tissue culture media is 5.0-6.5.
The final concentration of said KT in plant tissue culture media is preferably 0.5mg/L; The final concentration of said glucose in plant tissue culture media is preferably 65g/L.
Another object of the present invention provides the method that a kind of inducing eggplant microspore forms embryoid.
Inducing eggplant microspore provided by the present invention forms the method for embryoid, is that the eggplant microspore is inoculated into above-mentioned arbitrary said plant tissue culture media, under 25-28 ℃ of condition, secretly cultivates and obtains embryoid.
Said microspore can obtain as follows: flower pesticide is inoculated in the pre-culture medium, and dark the cultivation 5-6 days taken out microspore then from flower pesticide under 34-36 ℃ of condition;
The composition of said pre-culture medium can for: in the MS medium, add 2,4-D, KT, vitamin C, sucrose and agar make its final concentration in said pre-culture medium be respectively 0.05-0.5mg/L, 0.5-2mg/L, 6-10mg/L, 2-4%, 5-10g/L; The pH value of said pre-culture medium is 5.5-7; Said MS medium does not contain sucrose and agar; Said percentage composition is the quality percentage composition.
Said flower pesticide can be taken from corolla and be lower than calyx 1-2mm and be higher than the calyx 1-2mm bud in period to corolla.
Culture medium cost of the present invention is low, culture effect good; Utilize medium of the present invention to carry out the method that the eggplant microspore induces and have into the advantage that the embryo rate is high, cultivation cycle is short; Medium of the present invention and microspore abductive approach are applied in the eggplant genetic breeding; The excellent genes type that has greatly enriched eggplant is individual, also can obviously shorten breeding cycle, not only perfect eggplant haploid breeding approach; And technology and theoretical foundation are provided for solanaceous crops microspore cultured in vitro, for accelerating China's solanaceous vegetables Crop Genetic Breeding and genetic research the reliable technique support is provided.
Description of drawings
Fig. 1 a is the microspore of expanding.
Fig. 1 b is the daughter cell that the 1st cell division of microspore forms two equalizations.
Fig. 1 c is that the 1st cell division of microspore forms two unequal daughter cells.
Fig. 1 d is four cell masses that the 2nd division of microspore forms symmetry.
Fig. 1 e is that the 1st cell division of microspore forms two unequal daughter cells.
Fig. 1 f is that cell is proceeded symmetry division formation many cells group.
Fig. 1 g spheroidal embryo.
Fig. 1 h is a heart type embryo.
Fig. 1 i torpedo embryo.
Fig. 1 j cotyledon type embryo.
Fig. 1 k is the cotyledon type embryo.
Figure 11 is the cotyledon type embryo.
Embodiment
Employed experimental technique is conventional method like no specified otherwise in the present embodiment.
The composition of used MS medium is as shown in table 1 among the embodiment:
Table 1, MS medium are formed (do not contain agar and sucrose in this medium, add during use again)
The composition of used KM medium is as shown in table 2 among the embodiment:
The composition of table 2, KM medium (do not contain glucose in this medium, add glucose during use again)
Inducing of embodiment 1, green anti-eggplant microspore
One, green anti-eggplant microspore induces
(1) separation of microspore
1, the selection of bud and collection
Gather that growth on the robust growth plant under field conditions (factors) is normal, the bud of free from insect pests, seamless green anti-eggplant, generally select the bud of full-bloom stage.Observe the developmental stage of microspore through 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, and sepal is about to before and after the cracking, and flower pesticide is generally yellow green.
2, the preparatory cultivation of flower pesticide
(1) 1. the sterilization of bud uses the alcohol of 75% (volumn concentration) to carry out the bud surface sterilization, and disinfecting time is 30sec; 2. use the liquor natrii hypochloritis of 6.5% (quality percentage composition) to soak 15min; 3. with sterile water washing 3 times, each 5min blots subsequent use then with aseptic paper.
(2) be seeded on the superclean bench, from the bud of the poison that disappeared, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium is housed, every culture dish connects the flower pesticide of 2 buds, and every culture dish is equipped with the 8-10mL medium.
Said pre-culture medium I consists of: in every liter of MS medium, add 2,4-D, KT, vitamin C, sucrose and agar make its final concentration be respectively 0.2mgL
-1, 1mgL
-1, 8mgL
-1, 3% (quality percentage composition), 7gL
-1The pH value of said pre-culture medium is 5.8; Said MS medium does not contain sucrose and agar.Adopt autoclaving: 121 ℃ of 15-20min that sterilize down.
(3) heat shock is handled and will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, and heat-shock temperature is 36 ℃, and the processing time is 6 days.
The microspore dedifferentiation is measured: will carry out microspore through the pretreated flower pesticide of excess temperature and dissociate; Per 12 flower pesticide are free in the 5mL sterile water; Under inverted microscope, observe (400X); Every ware is chosen 3 visuals field, write down in each visual field microspore sum with expand the number of microspore, dedifferentiation microspore incidence (%)=expand microspore number/total microspore number * 100%.
Result of the test shows that dedifferentiation frequency takes place green anti-eggplant kind microspore is 6.39%.
3, the free and collection of microspore
With the flower pesticide that aseptic tweezers are chosen pollution-free, no brownization and expanded from the preparatory flower pesticide of cultivating, be put in the culture dish aseptic, that the washing medium is housed.With aseptic scalpel flower pesticide is cut into two sections, pushes flower pesticide more gently, so that microspore is free in the washing medium from flower pesticide.
The above-mentioned washing medium that contains microspore is filtered through 200 order nylon screens, collect filtrating, will filtrate in the centrifugal 1-10min of 500rpm; Remove supernatant, deposition is centrifugal with the washing of washing medium again, repeats 3 times; Collecting precipitation obtains green anti-microspore, is used to induce experiment.
Wherein, consisting of of washing medium: in the MS medium, adding final concentration is the sucrose of 30g/L; The pH value of washing medium is 5.5-7.Adopt autoclaving: 115-121 ℃ of sterilization 15-20min down.
(2) carrying out microspore with liquid nutrient medium i induces
1, microspore is induced the formation embryoid
The liquid nutrient medium i that uses in this experiment is: in the KM medium, add KT and glucose, obtain liquid nutrient medium i, making the final concentration among the KT liquid medium within i is 0.5mg/L, and the final concentration that makes the glucose among the liquid nutrient medium i is 65mg/L; The pH of said liquid nutrient medium i is 5.5; The composition of said KM medium is as shown in table 2.
Liquid nutrient medium i is with 0.22 μ m disposable filter filtration sterilization.
The microspore that separation is obtained is diluted to 4 * 10 with liquid nutrient medium i
5Individual/mL (counting with blood counting chamber) divides to install in the sterile petri dish (Φ 60mm), and each culture dish dress 5mL (flower pesticide of about 2 buds of every ware) seals with the Parafilm film.
Culture dish put into cultivate in the box, under 28 ℃, dark condition, carry out static shallow-layer and secretly cultivate, occur to embryoid, wherein whenever changed once fresh liquid nutrient medium i at a distance from 15-20 days.
When Isolated microspore carries out static cultivation, observe the stripped morphogenetic process of eggplant microspore through inverted microscope, and the number of statistics embryoid, be calculated to be the embryo rate.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have used about 240 buds altogether, and the result shows, repeat to have obtained altogether 12 embryos three times, and on average becoming the embryo rate is 5.0% ± 0.5 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: the generating process of eggplant microspore embryoid is mainly following: After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell.The cell of these two equalizations is similar with typical trophozyte on chromosome, and they continue division, forms embryoid.Can observe the microspore (Fig. 1: a) the 1st cell division takes place, form daughter cell (Fig. 1: b), then observe the four cell mass (Fig. 1: d) that form symmetry through the 2nd division of two equalizations that expands through inverted microscope; Afterwards cell proceed symmetry division form many cells group (Fig. 1: f), until macroscopic spheroidal embryo, heart type embryo, torpedo embryo and cotyledon type embryo (Fig. 1: g, h; I; J, k, l).
The embryo that indivedual microspores are also arranged simultaneously be through the unequal division generation of microspore (plate 1:c, e).
Experimental result also shows; There is asynchronism in the stripped form of microspore embryoid; The time that is embryoid formation is inconsistent; The microspore that under inverted microscope, can observe has just started four cells of formation or the many cells that division forms two cells, has, and the division that but also do not start that has is still a great circle glomus cell; Naked eyes can be observed spheroidal embryo, heart type embryo, torpedo embryo and cotyledon type embryo in same culture dish, and the asynchronism that embryoid is grown also has been described.
Two, green anti-eggplant microspore induces
(1) separation of microspore
1, the selection of bud and collection
Method is with identical described in the experiment one.
2, the preparatory cultivation of flower pesticide
(1) 1. the sterilization of bud uses the alcohol of 70% (volumn concentration) to carry out the bud surface sterilization, and disinfecting time is 1min; 2. use the liquor natrii hypochloritis of 5.0% (quality percentage composition) to soak 5min; 3. with sterile water washing 3 times, each 1min blots subsequent use then with aseptic paper.
(2) be seeded on the superclean bench, from the bud of the poison that disappeared, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium II is housed, every culture dish connects the flower pesticide of 2 buds, and every culture dish is equipped with the 8-10mL medium.
Said pre-culture medium II consists of: in every liter of MS medium, add 2,4-D, KT, vitamin C, sucrose and agar make its final concentration be respectively 0.05mg/L, 0.5mg/L, 6mg/L, 2%, 5g/L; The pH value of said pre-culture medium is 5.5; Said MS medium does not contain sucrose and agar; Said percentage composition is the quality percentage composition.Adopt autoclaving: 121 ℃ of 15-20min that sterilize down.
(3) heat shock is handled and will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, and heat-shock temperature is 34 ℃, and the processing time is 5 days.
The microspore dedifferentiation is measured: will carry out microspore through the pretreated flower pesticide of excess temperature and dissociate; Per 12 flower pesticide are free in the 5mL sterile water; Under inverted microscope, observe (400X); Every ware choose in the record visual field, 3 visuals field the microspore sum with expand the number of microspore, dedifferentiation microspore incidence (%)=expand microspore number/total microspore number * 100%.
Result of the test shows that dedifferentiation frequency takes place the green anti-microspore of eggplant kind is 6.0%.
3, the free and collection of microspore
Method is identical with method described in the experiment one.
(2) carrying out microspore with liquid nutrient medium ii induces
1, microspore is induced the formation embryoid
Carrying out microspore with liquid nutrient medium ii induces
The liquid nutrient medium ii that uses in this experiment is: in the KM medium, add KT and glucose, make its final concentration be respectively 0.1mg/L and 50g/L; The pH of said liquid nutrient medium i is 5.0; The composition of said KM medium is as shown in table 2.
Liquid nutrient medium ii is with 0.22 μ m disposable filter filtration sterilization.
The microspore that separation is obtained is diluted to 4 * 10 with liquid nutrient medium ii
5Individual/mL (counting with blood counting chamber) divides to install in the sterile petri dish (Φ 60mm), and each culture dish dress 5mL (flower pesticide of about 2 buds of every ware) seals with the Parafilm film.
Culture dish put into cultivate in the box, under 25 ℃, dark condition, carry out static shallow-layer and secretly cultivate, occur to embryoid, wherein whenever changed once fresh liquid nutrient medium i at a distance from 15-20 days.
When Isolated microspore carries out static cultivation, observe the stripped morphogenetic process of eggplant microspore through inverted microscope, the number of statistics embryoid, and add up into the embryo rate.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have used about 240 buds altogether, and the result shows, repeat to have obtained altogether 10 embryos three times, and on average becoming the embryo rate is 4.2% ± 0.5 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: the generating process of eggplant microspore embryoid is with identical described in the experiment one; Be that After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell; The cell of these two equalizations is similar with typical trophozyte on chromosome; They continue division, form embryoid.The embryo that indivedual microspores are also arranged simultaneously is through the unequal division generation of microspore.Asynchronism takes place also to exist in the stripped form of microspore embryoid
Three, green anti-eggplant microspore induces
(1) separation of microspore
1, the free and method of collecting of the preparatory cultivation of the selection of bud and collection, flower pesticide, microspore is all with identical described in the experiment one.
Method is with identical described in the experiment one.
2, the preparatory cultivation of flower pesticide
(1) 1. the sterilization of bud uses the alcohol of 75% (volumn concentration) to carry out the bud surface sterilization, and disinfecting time is 3min; 2. use the liquor natrii hypochloritis of 8.0% (quality percentage composition) to soak 40min; 3. with sterile water washing 3 times, each 10min blots subsequent use then with aseptic paper.
(2) be seeded on the superclean bench, from the bud of the poison that disappeared, strip flower pesticide gently and be inoculated in the culture dish (Φ 60mm) that pre-culture medium III is housed, every culture dish connects the flower pesticide of 2 buds, and every culture dish is equipped with the 8-10mL medium.
Said pre-culture medium III consists of: in every liter of MS medium, add 2,4-D, KT, vitamin C, sucrose and agar make its final concentration be respectively 0.5mg/L, 2mg/L, 10mg/L, 4%, 10g/L; The pH value of said pre-culture medium is 7; Said MS medium does not contain sucrose and agar; Said percentage composition is the quality percentage composition.Adopt autoclaving: 121 ℃ of 15-20min that sterilize down.
(3) heat shock is handled and will inoculate good flower pesticide and be put into and carry out the heat shock processing in the dark incubator, and heat-shock temperature is 35 ℃, and the processing time is 6 days.
The microspore dedifferentiation is measured: will carry out microspore through the pretreated flower pesticide of excess temperature and dissociate; Per 12 flower pesticide are free in the 5mL sterile water; Under inverted microscope, observe (400X); Every ware choose in the record visual field, 3 visuals field the microspore sum with expand the number of microspore, dedifferentiation microspore incidence (%)=expand microspore number/total microspore number * 100%.
Result of the test shows that dedifferentiation frequency takes place the green anti-microspore of eggplant kind is 5.9%.
3, the free and collection of microspore
Method is identical with method described in the experiment one.
(2) carrying out microspore with liquid nutrient medium iii induces
1, microspore is induced the formation embryoid
Carrying out microspore with liquid nutrient medium iii induces
The liquid nutrient medium iii that uses in this experiment is: in the KM medium, add KT and glucose, make its final concentration be respectively 0.5mg/L and 70g/L; The pH of said liquid nutrient medium i is 6.5; The composition of said KM medium is as shown in table 2.
Liquid nutrient medium iii is with 0.22 μ m disposable filter filtration sterilization.
The microspore that separation is obtained is diluted to 4 * 10 with liquid nutrient medium iii
5Individual/mL (counting with blood counting chamber) divides to install in the sterile petri dish (Φ 60mm), and each culture dish dress 5mL (flower pesticide of about 2 buds of every ware) seals with the Parafilm film.
Culture dish put into cultivate in the box, under 27 ℃, dark condition, carry out static shallow-layer and secretly cultivate, occur to embryoid, wherein whenever changed once fresh liquid nutrient medium iii at a distance from 15-20 days.
When Isolated microspore carries out static cultivation, observe the stripped morphogenetic process of eggplant microspore through inverted microscope, the number of statistics embryoid, and add up into the embryo rate.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have used about 240 buds altogether, and the result shows, repeat to have obtained altogether 9 embryos three times, and on average becoming the embryo rate is 3.8% ± 0.2 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: the generating process of eggplant microspore embryoid is with identical described in the experiment one; Be that After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell; The cell of these two equalizations is similar with typical trophozyte on chromosome; They continue division, form embryoid.The embryo that indivedual microspores are also arranged simultaneously is through the unequal division generation of microspore.Asynchronism takes place also to exist in the stripped form of microspore embryoid
Inducing of embodiment 2, Europe long arrow eggplant microspore
One, Europe long arrow eggplant microspore induces
(1) separation of microspore
1, the selection of bud and collection
Gather that growth on the robust growth plant under field conditions (factors) is normal, the bud of free from insect pests, the long arrow eggplant in seamless Europe, generally select the bud of full-bloom stage.Observe the developmental stage of microspore through 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, and sepal is about to before and after the cracking, and flower pesticide is generally yellow green.
2, the preparatory cultivation of flower pesticide
Consistent described in the experiment one among method and the embodiment 1.
Dedifferentiation frequency takes place Europe long arrow eggplant kind microspore is 6.42%.
3, the free and collection of microspore
Consistent described in the experiment one among method and the embodiment 1.
(2) carrying out microspore with liquid nutrient medium i induces
1, microspore is induced the formation embryoid
Consistent described in the experiment one among method and the embodiment 1.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have used about 240 buds altogether, and the result shows, repeat to have obtained altogether 21 embryos three times, and on average becoming the embryo rate is 8.8% ± 0.5 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: consistent described in the experiment one among the generating process of Europe long arrow eggplant microspore embryoid and the embodiment 1; Be that After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell; The cell of these two equalizations is similar with typical trophozyte on chromosome; They continue division, form embryoid.The embryo that indivedual microspores are also arranged simultaneously is through the unequal division generation of microspore.Asynchronism takes place also to exist in the stripped form of microspore embryoid.
Two, Europe long arrow eggplant microspore induces
(1) separation of microspore
1, the selection of bud and collection
Method is with identical described in the experiment one.
2, the preparatory cultivation of flower pesticide
Identical described in the experiment two among method and the embodiment 1.
Result of the test shows that dedifferentiation frequency takes place the long arrow microspore in eggplant kind Europe is 6.1%.
3, the free and collection of microspore
Method is identical with method described in the experiment one.
(2) carrying out microspore with liquid nutrient medium ii induces
1, microspore is induced the formation embryoid
Identical described in the experiment two among method and the embodiment 1.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have inoculated altogether and have used about 240 buds, result to show, repeat to have obtained altogether 19 embryos three times, and on average becoming the embryo rate is 7.9% ± 0.5 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: identical described in the experiment two among the generating process of eggplant microspore embryoid and the embodiment 1; Be that After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell; The cell of these two equalizations is similar with typical trophozyte on chromosome; They continue division, form embryoid.The embryo that indivedual microspores are also arranged simultaneously is through the unequal division generation of microspore.Asynchronism takes place also to exist in the stripped form of microspore embryoid.
Three, Europe long arrow eggplant microspore induces
(1) separation of microspore
1, the selection of bud and collection
Identical described in the experiment three among method and the embodiment 1.
2, the preparatory cultivation of flower pesticide
Identical described in the experiment three among method and the embodiment 1.
Result of the test shows that dedifferentiation frequency takes place the long arrow microspore in eggplant kind Europe is 6.0%.
3, the free and collection of microspore
Identical described in the experiment three among method and the embodiment 1.
(2) carrying out microspore with liquid nutrient medium iii induces
Identical described in the experiment three among method and the embodiment 1.
3 repetition are established in experiment, inoculate 40 culture dishes at every turn, have used about 240 buds altogether, and the result shows, repeat to have obtained altogether 17 embryos three times, and on average becoming the embryo rate is 7.1% ± 0.2 (mean+SD) (become embryo rate=one-tenth embryo number/always number of flowers).
The result shows: identical described in the experiment three among the generating process of eggplant microspore embryoid and the embodiment 1; Be that After microspore mitosis forms the cell of two equalizations and is not divided into trophozyte and reproductive cell; The cell of these two equalizations is similar with typical trophozyte on chromosome; They continue division, form embryoid.The embryo that indivedual microspores are also arranged simultaneously is through the unequal division generation of microspore.Asynchronism takes place also to exist in the stripped form of microspore embryoid.
Claims (3)
1. the method for an inducing eggplant microspore formation embryoid is that the eggplant microspore is inoculated into plant tissue culture media, under 25-28 ℃ of condition, secretly cultivates and obtains embryoid; Said plant tissue culture media is in the KM medium, to add KT and glucose, and to make the final concentration of KT in plant tissue culture media be 0.1-0.5mg/L, make the final concentration of glucose in plant tissue culture media is 50-70g/L; The solvent of said plant tissue culture media is a water; Do not contain glucose in the said KM medium; The pH of said plant tissue culture media is 5.0-6.5.
2. method according to claim 1 is characterized in that: said microspore obtains as follows: flower pesticide is inoculated in the pre-culture medium, and dark the cultivation 5-6 days taken out microspore then from flower pesticide under 34-36 ℃ of condition;
Consisting of of said pre-culture medium: in the MS medium, add 2,4-D, KT, vitamin C, sucrose and agar make its final concentration in said pre-culture medium be respectively 0.05-0.5mg/L, 0.5-2mg/L, 6-10mg/L, 2-4%, 5-10g/L; The pH value of said pre-culture medium is 5.5-7; Said MS medium does not contain sucrose and agar; Said percentage composition is the quality percentage composition.
3. method according to claim 1 and 2 is characterized in that: said flower pesticide is taken from corolla and is lower than calyx 1-2mm and is higher than the calyx 1-2mm bud in period to corolla.
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| CN104542274B (en) * | 2014-07-03 | 2016-09-07 | 郑州市蔬菜研究所 | The culture medium of a kind of Fructus Solani melongenae Anther Culture induction haplobiont and method thereof |
| CN105660406B (en) * | 2016-02-16 | 2017-11-28 | 嵊州北航投星空众创科技有限公司 | A kind of eggplant flower pesticide inducing culture and compound method |
| CN106937594A (en) * | 2017-03-07 | 2017-07-11 | 重庆市农业科学院 | A kind of method for promoting eggplant pollen embryo to breed |
| 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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2008
- 2008-11-18 CN CN2008102270141A patent/CN101401550B/en not_active Expired - Fee Related
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| 李华等.茄子小孢子热激效应和发育潜能的细胞学分析.《种子》.2008,(第10期), * |
| 金丹丹等.茄子组织培养与基因工程研究进展.《分子植物育种》.2004,(第06期), * |
| 顾淑荣.茄子(SolanummelongenaL.)花粉粒离体培养获得植株.《Journal of Integrative Plant Biology》.1979,(第01期), * |
| 马欣等.茄子花药和游离小孢子培养技术研究进展.《长江蔬菜》.2006,(第07期), * |
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