CN101824396B - Induction medium and method using same for isolated culture of tomato microspore to obtain calli - Google Patents

Induction medium and method using same for isolated culture of tomato microspore to obtain calli Download PDF

Info

Publication number
CN101824396B
CN101824396B CN 201010168471 CN201010168471A CN101824396B CN 101824396 B CN101824396 B CN 101824396B CN 201010168471 CN201010168471 CN 201010168471 CN 201010168471 A CN201010168471 A CN 201010168471A CN 101824396 B CN101824396 B CN 101824396B
Authority
CN
China
Prior art keywords
tomato
plant
annexation
carbon source
glutamine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN 201010168471
Other languages
Chinese (zh)
Other versions
CN101824396A (en
Inventor
梁燕
王晓静
李继纲
孙亚东
徐加新
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northwest A&F University
Original Assignee
Northwest A&F University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Northwest A&F University filed Critical Northwest A&F University
Priority to CN 201010168471 priority Critical patent/CN101824396B/en
Publication of CN101824396A publication Critical patent/CN101824396A/en
Application granted granted Critical
Publication of CN101824396B publication Critical patent/CN101824396B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention relates to an induction medium and a method using the same for the isolated culture of tomato microspore to obtain calli. The induction medium comprises the following components: a minimal medium (MS, PM or HN), a carbon source (cane sugar or malt sugar), additives (glutamine and/or AgNO3) and plant growth regulator (NAA+6-BA). By taking various influencing factors in the culture of the tomato micropore into overall consideration and trying various combinations of media and plant growth regulators, the invention acquires the formula of the medium suitable for the growth of microspore; and the callus induction rate is increased by using the pre-culturing method of high-temperature and low-temperature alternating treatment and further increased by the liquid isolated culture, thus laying a solid foundation for the development of the follow-up research.

Description

Inducing culture and cultivate the method that tomato sporule obtains callus with it
Technical field
The present invention relates to a kind of inducing culture and utilize the free method of cultivating tomato sporule acquisition callus of this inducing culture.
Background technology
In tomato breeding, utilize the monoploid culture technique to have great advantage and potentiality: 1. accelerate the speed of isozygotying of breeding material, accelerate breeding process.Carry out the purifying of breeding material with the conventional breeding means, need to just can bring out stable strain through the selfing in 6～8 generations, also have simultaneously the inconsistent problem of plant phenotype and genotype.Utilize flower pesticide or microspores culture to obtain monoploid (haploid), amphiploid (double haploid obtains isozygotying after dyed body doubles, DH), thus can from hybrid, (comprise the xenogenesis species hybrid) quickly and isolate in a large number isozygoty, stable self-mating system.Genotype and the phenotype of such self-mating system plant are in full accord, have greatly improved efficiency of selection, so the breeding time limit shortens greatly.2. monoploid is cultivated and can accelerate the stable of remote hybrid.Strong proterties can occur and separate in the remote hybrid offspring, a large amount of aneuploid hybrids also often occurs, is difficult to preserve its hereditary property by selfing or natural hybrization.Tomato flower pesticide, the sporule of the first generation of hybrid are carried out isolated culture, again haplobiont is carried out the karyomit(e) artificial doubling, can overcome proterties and separate, obtain rapidly stable novel type.3.DH being conducive to structure, the assignment of genes gene mapping of genetic map, colony reaches according to the Atlas Method clone gene.Tomato flower pesticide with the first generation of hybrid, sporule carries out isolated culture, carry out again the karyomit(e) artificial doubling and just can obtain DH colony, some recessive characters that can not show in hybrid can show in double haploid (DH), there is genotypic difference between the different strains of DH colony, but the genotype in the strain is identical, selfing does not separate, by the self propagated offspring, the genetic composition of each strain can not change, and can eliminate to a great extent the impact of environmental factors, this not only be conducive to the structure of tomato genetic map and quality-base because of the location, what is more important can be carried out accurate location and the clone of quantitative trait locus QTL.The Main Agronomic Characters such as the output of tomato, quality, resistance are quantitative character, relation between the Phenotype and genotype is indefinite, be subject to the interference of ambient conditions, select usually can not reflect truly genotype according to phenotype, utilize DH colony just can overcome to utilize ordinary group to carry out QTL and study existing shortcoming, obtain and the closely linked molecule marker of QTL, the tomato breeding level is improved.4. monoploid is cultivated and to be combined the effect that can improve selection by mutation with selection by mutation.In selection by mutation, because the probability of transgenation is very low, particularly when the recessive gene sudden change occured, mutant character can not directly show on plant, and this just needs a large amount of plant of plantation.Make mutant materials with haplobiont, the sudden change recessive gene just can show, and the speed that obtains mutant can be accelerated greatly, and the breeding time limit is shortened.
Based on above advantage, the monoploid of tomato is cultivated and is subject to great attention, and early stage research focuses mostly on aspect the tomato anther culture.1971 years, Sharp at first cultivated the flower pesticide of cultivation tomato, the callus that has obtained.1978, the hybrid tomato flower pesticide of the cultivation tomatoes (L.peruvianum) such as Cappadocia was therefrom observed spherical embryo.
Early 1980s, Krueget-Lebus etc. cultivate the sporule of tomato variety Nadja and Piccolo, have obtained globular embryo.Khoang etc. (1986) report the haplobiont of regenerating from the flower pesticide of tomato variety Roma, also obtain simultaneously diploid and mixoploid plant.Evans and Morrison (1989) also report and utilize the tomato anther culture to produce haplobiont.
China has also carried out research in this respect subsequently.Gao Xiuyun, Wang Jifang etc. (1979,1980) carry out tomato tomato anther culture, find to have haplobiont.Yuan Yinan (1999) cultivates sporule to spherical embryo and class heart-shape embryo stage to cultivate tomato and wild-type tomato as material.
Compare with the tomato anther culture, sporule has been haploid cell, inducing behind the sporule plantlet that develops into through callus or embryoid all is monoploid, when can eliminating anther culture, this disturbs the mixed times phenomenon that occurs because of somatic tissues such as anther wall, filigree, connectives, fixing and purification efficiency to material is higher, therefore, be a focus of current tomato breeding technical study.Cultivating tomato Microsporogenesis callus is key technique.Also there is not at present the general tomato sporule of a cover to obtain the cultivation program of callus, the comprehensive microspore development of the present invention period of living in, donor plant genotype, pretreatment process, particularly minimum medium and a kind of methods of cultivating tomato Microsporogenesis callus of many factors proposition such as plant-growth regulator and additive composition and ratio.
Summary of the invention
The purpose of this invention is to provide a kind of inducing culture and utilize the free method of cultivating tomato sporule acquisition callus of this inducing culture.
For achieving the above object, the present invention at first provides a kind of inducing culture, and this inducing culture includes minimum medium, carbon source, annexation and plant-growth regulator, and described minimum medium is MS, PM or HN; Described plant-growth regulator is NAA0.1mg.L -1+ 6-BA0.2mg.L -1, NAA0.01mg.L -1+ 6-BA0.1mg.L -1, NAA0.01mg.L -1+ 6-BA0.1mg.L -1, 6-BA0.01mg.L -1Or NAA0.01mg.L -1+ 6-BA0.2mg.L -1Described carbon source is 0.3% sucrose and/or 0.3% maltose; Described annexation is 500mg.L -1Glutamine and/or 3mg.L -1AgNO 3
The present invention also provides the optimal technical scheme of above-mentioned inducing culture:
Described minimum medium is MS, and plant-growth regulator is NAA0.1mg.L -1+ 6-BA0.2mg.L -1, NAA0.01mg.L -1+ 6-BA0.1mg.L -1Or NAA0.01mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose.
Described minimum medium is HN, and annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose, plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.1mg.L -1Perhaps minimum medium is PM, and annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose, plant-growth regulator is 6-BA0.01mg.L -1
The present invention also further provides and has utilized above-mentioned inducing culture to cultivate the method that the tomato sporule obtains callus, and the method may further comprise the steps:
(1) the tomato monokaryon sterilization of phase bud of keeping to the side;
(2) the tomato sporule is free;
(3) pre-treatment of tomato sporule;
(4) cultivation of tomato sporule is cultivated on above-mentioned inducing culture, until obtain callus.
The present invention also provides the above-mentioned inducing culture that utilizes to cultivate the optimal technical scheme that the tomato sporule obtains the callus method:
In the step (1), obtain the keep to the side method of phase bud of described tomato monokaryon and be: the plant beginning of buddingging, take bud from plant, aceto-camine dyeing compressing tablet microscopy obtains the tomato monokaryon phase bud that keeps to the side.
In the step (1), the method for described sterilization with the flowing water flushing tomato monokaryon phase bud 10～20min that keeps to the side, with 70% alcohol disinfecting, 20～40s and the 5% clorox 15～20min that sterilizes, is used aseptic water washing for first more at last.
In the step (2), described free method is that the tomato monokaryon phase bud that keeps to the side is put in the sterile test tube, then adds 0.3mol/L mannitol solution 6-8mL, pulverize, gained suspension is crossed 300 purpose cells sieve, the centrifugal 2～5min of 600～1200rpm, remove supernatant liquor, repeat 2～4 times.At last, outwell supernatant liquor, add each described inducing culture suspension tomato sporule of claim 1～3, and dilution gained tomato sporule is 1.5～2 * 10 to concentration 5Individual/mL.
In the step (3), the pre-treatment of described tomato sporule is that step (2) gained tomato sporule is divided in the culture dish, and sealing then in the dark, after leaving standstill 1～3 day under 3～5 ℃, was left standstill under 30～40 ℃ 1～3 day again.
In the step (4), the cultivation of described tomato sporule refers under 24～30 ℃, secretly cultivates in each described inducing culture of claim 1～3, until there is callus to occur, forwards 24～30 ℃ of lower illumination 14～18h to again and carries out the light cultivation.
The culture temperature of described dark cultivation is 27 ℃, when having callus to occur, forwards 27 ℃ of lower illumination 16h to again and carries out the light cultivation.
The present invention has considered the many factors that affects the tomato microspores culture, comprises that donor plant genotype, microspore development period of living in, minimum medium kind, carbon source kind, plant-growth adjust kind combination and concentration proportioning, pretreatment process etc.By attempting the combination of multiple basic medium and plant-growth regulator etc., obtained the culture medium prescription of suitable sporule growth, and utilize the high and low temperature alternate treatment to carry out pre-incubated method and improved healing rate, and cultivate sporule and further improved healing rate by liquid is free, for carrying out smoothly of follow-up study work established solid basis.
Description of drawings
Fig. 1 is in the keep to the side tomato sporule of phase of monokaryon.
The callus that Fig. 2 tomato sporule suspension culture forms.
Embodiment
The invention will be further described with specific embodiment for the below, can be implemented so that those skilled in the art can better understand the present invention also, but illustrated embodiment is not as a limitation of the invention.
The preparation of inducing culture:
1) minimum medium: 1. MS; 2. PM; 3. HN.
2) carbon source: 1. sucrose 3%; 2. maltose 3%
3) annexation: 500mg.L -1Glutamine; 3mg.L -1AgNO 3
4) plant-growth regulator:
①NAA0.01mg.L -1；
②NAA0.01mg.L -1+6-BA0.01mg.L -1；
③NAA0.01mg.L -1+6-BA0.1mg.L -1；
④6-BA0.01mg.L -1；
⑤NAA0.01mg.L -1+6-BA0.2mg.L -1；
⑥NAA0.1mg.L -1+6-BA0.2mg.L -1。
5) minimum medium prescription (mg.L -1)
(1) MS:NH 4NO 31650, KNO 31900, CaCl 22H 2O 440, MgSO 47H 2O 370, KH 2PO 4170, H 3BO 36.2, ZnSO 44H 2O 8.6, MnSO 44H 2O 22.3, and KI 0.83, Na 2MoO 42H 2O 0.25, CuSO 45H 2O 0.025, CoCl 26H 2O 0.025, FeSO 47H 2O27.8, Na 2EDTA 37.3, pantothenic acid 1.0, inositol 100, nicotinic acid 1.0, Vb 61.0, Vb 1Vb 61.0 Vb 0.01, pH 5.8.
(2) PM:MgSO 47H 2O 185, CaCl 2166, KH 2PO 468, KNO 3950, NH 4NO 3200, Fe-EDTA 40, inositol 450, and pH 5.8.
(3) HN:MgSO 47H 2O 185, CaCl 2166, KH 2PO 468, MnSO 4H 2O 25, H 3BO 310, ZnSO 47H 2O 10, NaMoO 42H 2O 0.25, and Fe-EDTA 40, nicotinic acid 5, and vitamin 0.5, pyridoxine hydrochloride 0.5, inositol 100, glycine 2, pH 5.6.
Utilize the free tomato sporule key step of cultivating of inducing culture provided by the invention as follows:
1) the tomato monokaryon sterilization of phase bud of keeping to the side: common tomato plant enters the florescence, in time removes the flower of having opened, and makes tomato plant always be in the florescence state.From can observing bud, get the buds of different sizes with tweezers from plant, conventional aceto-camine dyeing compressing tablet microscopy is determined microspore development period.Select microspore development to be in the keep to the side bud of phase of monokaryon, the flowing water flushing, aseptic water washing is used in then alcohol disinfecting and clorox sterilization at last.
2) the tomato sporule is free: get the tomato bud with tweezers and be put in the sterile test tube, add mannitol solution, pulverize; It is centrifugal to put into centrifuge tube after suspension sieves by cell; Remove supernatant liquor, add again mannitol solution, cross cell sieve, centrifugal, repeat 2～4 times.Then, outwell supernatant liquor, in centrifuge tube, add inducing culture suspension sporule again, blood counting chamber counting, dilution sporule concentration to 1.5～2 * 10 5Individual .mL -1
3) pre-treatment of tomato sporule: the tomato sporule of gained is divided in the culture dish, and sealing then in the dark, after leaving standstill 1～3 day under 3～5 ℃, was left standstill 1～3 day in 30～40 ℃ again.
4) cultivation of tomato sporule: under 24～30 ℃, in inducing culture, secretly cultivate, until there is callus to occur, forwards again 24～30 ℃ of lower illumination 14～18h to and in inducing culture, carry out the light cultivation
Embodiment 1
Test materials: common tomato, cherry tomato, wild-type tomato and first generation of hybrid tomato are at the tomato material of 8 kinds of interior different genotype.
The tomato variety of table 1.8 kind of different genotype
Figure GSA00000096468300051
1) the tomato monokaryon sterilization of phase bud of keeping to the side: the aobvious flower bud of plant begins, and in time removes the flower of having opened, and makes tomato plant always be in the florescence state.From can observing bud, get the buds of different sizes with tweezers from plant, aceto-camine dyeing compressing tablet microscopy obtains the keep to the side tomato bud of phase of monokaryon.Select microspore development to be in the keep to the side tomato bud of phase of monokaryon, flowing water flushing 10～20min, 70% alcohol disinfecting, 20～40s, clorox sterilization 15～20min of 5%, deionization aseptic water washing 2～5 times, each 3～5min.
2) the tomato sporule is free: get the tomato bud with tweezers and be put in the sterile test tube, add 0.3mol.L -1Mannitol solution 6-8ml, then with glass stick the tomato bud is pulverized, suspension is put into the centrifuge tube with scale, in the centrifugal 2～5min of 600～1200rpm by behind the 300 purpose cells sieves.Abandon supernatant liquor, add again 0.3mol.L -1Mannitol solution 6-8ml, the centrifugal 2～5min of 600～1200rpm.Then, outwell supernatant liquor, in centrifuge tube, add inducing culture suspension sporule again, blood counting chamber counting, dilution sporule concentration to 1.5～2 * 10 5Individual .mL -1
3) pre-treatment of tomato sporule: the amount with 3mL is divided in the culture dish of 60 * 15mm at last, with the Paraflim sealing, then in the dark, after leaving standstill 2 days under 4 ℃, leaves standstill 2 days in 36 ℃ again.
4) cultivation of tomato sporule: under 27 ℃, secretly cultivate at inducing culture, until there is callus to occur, forwards again 27 ℃ of lower illumination 16h to and in inducing culture, carry out the light cultivation
The composition of inducing culture:
Minimum medium is MS, and plant-growth regulator is NAA0.1mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose.
Embodiment 2
Test materials: with embodiment 1.
1) the tomato monokaryon sterilization of phase bud of keeping to the side: the aobvious flower bud of plant begins, and every morning 8:00-10:00 gets the buds of different sizes with tweezers from plant, takes back the laboratory with ice chest, and aceto-camine dyeing compressing tablet microscopy is determined microspore development period.Select microspore development to be in the keep to the side bud of phase of monokaryon, flowing water flushing 15min, 70% alcohol disinfecting 30s, clorox sterilization 15～20min of 5%, deionization aseptic water washing 3 times, each 3～5min.
2) the tomato sporule is free: get the tomato bud with tweezers and be put in the sterile test tube, add 0.3mol.L -1Mannitol solution 6～8mL; With glass stick the tomato bud is pulverized, gained suspension is put into the centrifuge tube with scale, the centrifugal 5min of 600rpm by 300 purpose cells sieve; Abandon supernatant liquor, add 6～8mL mannitol solution, the centrifugal 5min of 600rpm repeats 3 times.At last, abandon supernatant liquor, add inducing culture suspension sporule, blood counting chamber counting, dilution sporule concentration to 1.5～2 * 10 5Individual .mL -1
3) pre-treatment of tomato sporule: the amount of microspore suspension with 3mL is divided in the culture dish of 60 * 15mm, seals with Paraflim.Under the dark condition, under 4 ℃, left standstill 2 days, then under 36 ℃, left standstill 2 days.
4) cultivation of tomato sporule: under 27 ℃, secretly cultivate, until there is callus to occur, forwards again 27 ℃ of illumination 16h to and carry out the light cultivation.
The composition of inducing culture: with embodiment 1.
Experimental result:
The sporule of the tomato variety of different genotype has all obtained a large amount of callus in embodiment 1 and embodiment 2.
Embodiment 3
Each step of experiment is with embodiment 2, and experiment material is common tomato, and each is tested used inducing culture and sees Table 2.
Table 2. is respectively tested used inducing culture
Figure GSA00000096468300071
Experimental result:
The sporule of the tomato variety of different genotype has obtained a large amount of callus in following inducing culture, see Table 3.
Table 3. has obtained the list of experiments of a large amount of callus
The sporule of the tomato variety of different genotype has obtained a small amount of callus in following inducing culture, see Table 4.
Table 4. has obtained the list of experiments of a small amount of callus
Figure GSA00000096468300082
Other inducing culture does not obtain callus.
The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited to this.Being equal to that those skilled in the art do on basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.

Claims (8)

1. an inducing culture includes minimum medium, carbon source, annexation and plant-growth regulator, it is characterized in that: described inducing culture is MS for (1) minimum medium, and plant-growth regulator is
NAA0.1mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (2) minimum medium is MS, and plant-growth regulator is NAA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (3) minimum medium is MS, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.1mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (4) minimum medium is MS, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (5) minimum medium is MS, and plant-growth regulator is NAA0.1mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (6) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (7) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (8) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.1mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (9) minimum medium is HN, and plant-growth regulator is 6-BA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (10) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (11) minimum medium is HN, and plant-growth regulator is NAA0.1mg.L -1+ 6-BA0.2mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% sucrose; (12) minimum medium is MS, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.1mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose; (13) minimum medium is MS, and plant-growth regulator is 6-BA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose; (14) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose; (15) minimum medium is HN, and plant-growth regulator is NAA0.01mg.L -1+ 6-BA0.1mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose; Perhaps, (16) minimum medium is PM, and plant-growth regulator is 6-BA0.01mg.L -1, annexation is 500mg.L -1Glutamine+3mg.L -1AgNO 3, carbon source is 0.3% maltose.
2. utilize inducing culture claimed in claim 1 to cultivate the method that the tomato sporule obtains callus, it is characterized in that may further comprise the steps:
(1) the tomato monokaryon sterilization of phase bud of keeping to the side;
(2) the tomato sporule is free;
(3) pre-treatment of tomato sporule;
(4) cultivation of tomato sporule is cultivated on inducing culture claimed in claim 1, until obtain callus.
3. the method for acquisition callus according to claim 2, it is characterized in that: in the step (1), obtain the keep to the side method of phase bud of described tomato monokaryon and be: the plant beginning of buddingging, take bud from plant, aceto-camine dyeing compressing tablet microscopy obtains the tomato monokaryon phase bud that keeps to the side.
4. the method for acquisition callus according to claim 2, it is characterized in that: in the step (1), the method of described sterilization is for first with the flowing water flushing tomato monokaryon phase bud 10～20min that keeps to the side, with 70% alcohol disinfecting, 20～40s and 5% clorox sterilization, 15～20min, use at last aseptic water washing again.
5. the method for acquisition callus according to claim 2, it is characterized in that: in the step (2), described free method is that the tomato monokaryon phase bud that keeps to the side is put in the sterile test tube, then add 0.3mol/L mannitol solution 6～8mL, pulverize, gained suspension is crossed 300 purpose cells sieve, the centrifugal 2～5min of 600～1200rpm, remove supernatant liquor, repeat 2～4 times; At last, outwell supernatant liquor, add inducing culture suspension tomato sporule claimed in claim 1, and dilution gained tomato sporule is 1.5～2 * 10 to concentration 5Individual/mL.
6. the method for acquisition callus according to claim 2, it is characterized in that: in the step (3), the pre-treatment of described tomato sporule is that step (2) gained tomato sporule is divided in the culture dish, sealing, then in the dark, after leaving standstill 1～3 day under 3～5 ℃, under 30～40 ℃, left standstill 1～3 day again.
7. the method for each described acquisition callus according to claim 2～6, it is characterized in that: in the step (4), the cultivation of described tomato sporule refers under 24～30 ℃, in inducing culture claimed in claim 1, secretly cultivate, until there is callus to occur, forwards again 24～30 ℃ of lower illumination 14～18h to and carry out the light cultivation.
8. the method for acquisition callus according to claim 7, it is characterized in that: the culture temperature of described dark cultivation is 27 ℃, when having callus to occur, forwards 27 ℃ of lower illumination 16h to again and carries out light and cultivate.
CN 201010168471 2010-05-11 2010-05-11 Induction medium and method using same for isolated culture of tomato microspore to obtain calli Expired - Fee Related CN101824396B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201010168471 CN101824396B (en) 2010-05-11 2010-05-11 Induction medium and method using same for isolated culture of tomato microspore to obtain calli

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201010168471 CN101824396B (en) 2010-05-11 2010-05-11 Induction medium and method using same for isolated culture of tomato microspore to obtain calli

Publications (2)

Publication Number Publication Date
CN101824396A CN101824396A (en) 2010-09-08
CN101824396B true CN101824396B (en) 2013-04-10

Family

ID=42688560

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201010168471 Expired - Fee Related CN101824396B (en) 2010-05-11 2010-05-11 Induction medium and method using same for isolated culture of tomato microspore to obtain calli

Country Status (1)

Country Link
CN (1) CN101824396B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164303B (en) * 2017-07-19 2020-11-20 泗县微腾知识产权运营有限公司 Pumpkin isolated microspore induction culture medium and induction culture method
CN113728923B (en) * 2021-09-30 2022-07-22 云南省农业科学院热区生态农业研究所 Method for inducing tomato anther callus to rapidly proliferate

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1915002A (en) * 2006-09-04 2007-02-21 中国科学院植物研究所 Method for inducing embryo callus of larch, and dedicated culture medium

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1915002A (en) * 2006-09-04 2007-02-21 中国科学院植物研究所 Method for inducing embryo callus of larch, and dedicated culture medium

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
周敏，等.谷氨酰胺和硝酸银对花生幼叶芽再生的促进作用.《植物生理学通讯》.2002,第38卷(第3期),240－241. *
彭新红,等.芦笋花药培养胚状体诱导条件优化的初探.《江西农业大学学报》.2006,第28卷(第1期),39-43. *
谷佳南，等.黄瓜花药培养愈伤组织诱导及再生的研究.《江苏农业科学》.2009,(第3期),37－39，46. *

Also Published As

Publication number Publication date
CN101824396A (en) 2010-09-08

Similar Documents

Publication Publication Date Title
CN104871964B (en) A kind of method improving wild rice and cultivated rice distant hybridization embryo crash breeding efficiency
Pauk et al. Protocol for wheat (Triticum aestivum L.) anther culture
JP2018535672A (en) Polyploid rice light and temperature sensitive gene male sterility system and its breeding method
WO2013097343A1 (en) Method for breeding poplar haploid
Herath et al. Effect of culture media for anther culture of indica rice varieties and hybrids of indica and japonica
CN104041414A (en) Method for obtaining haploid regenerated plant by chilli anther culturing
Barnabás Protocol for producing doubled haploid plants from anther culture of wheat (Triticum aestivum L.)
CN101011028B (en) Breeding method of chrysanthemum haploid
Li et al. Callus induction, suspension culture and protoplast isolation in Camellia oleifera
CN101824396B (en) Induction medium and method using same for isolated culture of tomato microspore to obtain calli
CN108464242B (en) Lily tenuifolius somatic embryo direct generation method capable of remarkably shortening induction time
CN104041415A (en) Method for obtaining haploid regenerated plant by eggplant anther culturing
CN103782908A (en) Anther culture method for indica japonica hybrid rice
CN107155874B (en) A kind of method and its culture medium obtaining Dihaploid Potato plant using Anther Culture
CN107012162B (en) Agrobacterium-mediated cotton embryo tip rapid transformation method
CN106801065A (en) It is a kind of to improve cotton regenerated and transformation efficiency method and application
CN101401550B (en) Method for inducing eggplant sporidiolum to form embryoid and special culture medium thereof
CN104871965B (en) A kind of method utilizing wild rice simultaneously to cultivate japonica rice and long-grained nonglutinous rice
CN103314849B (en) Base eggplant body embryo generation abductive approach in a kind of wild-type tomato
CN101622960A (en) Method for fast obtaining regenerated plant for cayenne pepper anther culture
CN108739391A (en) A kind of method of monoploid onion Regeneration in Vitro and its special explant
CN105850737A (en) Rhododendron Delavayi 'Cosmopolitan' chromosome doubling method
CN105123495A (en) Application of partially indica-type paddy rice dominant dwarf mutant
CN109984030A (en) A kind of Cymbidium lianpan unisexuality kind vitro Regeneration System is established and rapid propagation method
Gniech-Karasawa Gametic embryogenesis, somatic embryogenesis, plant cell cultures, and protoplast fusion: Progress and Opportunities in Biofuel Production

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20130410

Termination date: 20140511