CN100339476C - Hot pepper free spore cell assemblies culture method - Google Patents
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- CN100339476C CN100339476C CNB2004100867018A CN200410086701A CN100339476C CN 100339476 C CN100339476 C CN 100339476C CN B2004100867018 A CNB2004100867018 A CN B2004100867018A CN 200410086701 A CN200410086701 A CN 200410086701A CN 100339476 C CN100339476 C CN 100339476C
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Abstract
The present invention discloses a culture method for free microspore cell masses of hot peppers. In the method, anthers are first cultured in advance, and the free culture of microspore is then carried out. The method develops a path on the basis of a culture system for free microspore of hot peppers, and embryoid and plants thereof are obtained through the free culture of microspore cells and cell masses in the anthers cultured in advance, and microscope examination provides cytology proof of the growth paths of each single cell of the embryoid and haploid. Not only does the establishment of the technical system make a breakthrough in the haploid breeding technology of hot peppers, but also the induction system for embryoid culture through free microspore can possibly become an excellent experiment system for research into the differentiation and the growth of plant cells.
Description
Technical field
The present invention relates to a kind of hot pepper free spore cell assemblies culture method.
Background technology
At present, because the hybrid vigour that plant has stresses in cross-breeding present crop breeding.This can utilize the growth vigor of hybrid to form high yield on the one hand, owing to the complex inheritance background of hybrid, makes its use of should not reserving seed for planting, thereby can protect hybrid development person's interests on the other hand.In cross-breeding, the genetic background of used this material of father and mother must be isozygotied, and just can obtain the hybrid of proterties unanimity after breeding.And to obtain the parent material that genetic background is isozygotied, and in many crops, all must pass through artificial auxiliary pollination self, just can reach relatively through the 5-8 year and isozygoty.Very time-consuming, the effort of this traditional cross-breeding means.The male sex-cell of plant---pollen, owing to only have the karyomit(e) of a cover parent through postmeiotic, it is the haploidy cell, can obtain haplobiont by flower pesticide or unmature pollen (sporule) cultivation, and then become the double haploid material by chromosome doubling, can reach the complete homozygotic state of this plant genetic background like this.When this can overcome heterozygous state on the one hand, dominant gene helped the expression of recessive mutation or gene recombination proterties to the shielding effect of recessive gene, can directly obtain the material that isozygotys on the other hand, thereby be applied to cross-breeding as parent material.Usually only need the 1-2 year by the microspores culture material that obtains to isozygoty, can quicken breeding process greatly, improve breeding efficiency.Therefore in the world the haploid breeding research of crop is paid much attention to, on many crops such as wheat, barley, rape, Chinese cabbage, set up good haploid breeding technical system.
The hybrid vigour of capsicum (Capsicum annuum L.) is obvious, and cross-breeding at present becomes main breeding technique.The anther culture of capsicum has had a lot of researchs, success is in people's such as Wang Yu English in 1973 and George work (Wang Y Y the earliest, Sun C S, Wang C C, et al.The induction of the pollenplantlets of triticale and Capsicum annuum from anther culture.Sci.Sinica, 1973,16:147-151; George L, Narayanswamy S.Haploid Capsicum throughexperimental androgenensis.Protoplasma, 1973,78:467-470).Dumas in 1981 etc. have carried out bigger improvement to the anther culture system afterwards, improved germ extraction rate (Dumas de Vaulx, R., D.Chambonnet, E.Pochard.Culture in vitro d ' antheres de piment (Capsicum annuumL.): Am é lioration des taux d ' obtention de plantes chez diff é rents genotypes par destraitements à+35C.Agronomie, 1981,1:859-864).Many correlation tests (Morrison R A, Koning R E, Evans D A.Anther culture of an interspecifichybrid of Capsicum.J.Plant Physiol., 1986,126:1-9 are arranged again on this basis; Kell K, Andersen S B, Effectsof donor plant temperature, photoperiod, and age on anther culture response ofCapsicum annuum L.Euphytica, 1993,67:105-109; Mityk ó J, Andrasfalvy A, Csillery G, F á ri M, Anther-culture response in different genotypes and F1 hybridsof pepper (Capsicum annuum L) .Plant Breeding, 1995,114:78-80; Wang Lihao, Zhang Baoxi, Guo Jiazhen, etc. the research of some influence factors in the pepper anther culture. gardening journal, 2004,31 (2): 199-204.).Ramon in 1997 etc. have set up the mutually double-deck culture system of solid-liquid, simplified and cultivated operation and further improved germ extraction rate (Ramon Dolcet-sanjuan, Elisabet Claveria, Agustin Huerta.Androgenesis in Capsicum annuum L-Effects of carbohydrate and carbon dioxideenrichment.J.Amer.Soc.Hort.Sci, 1997,122 (4): 468-475).But all these is the achievement in research on anther culture.
Complete flower pesticide carries out vitro culture together with the unmature pollen grain (sporule) of its inside, is called anther culture.Because the anther wall cell is a diploid cell, therefore the regeneration plant that obtains by anther culture might be from the pollen granule of haploidy, also may can cause mixing of regeneration plant like this, influence the Breeding Application of this method greatly from the diploid cell of medicine wall.Unmature pollen grain (sporule) is separated in flower pesticide, and single culture is called Isolated microspore and cultivates.Because unicellular property, haploidy and the large group characteristic of Isolated microspore, make it on arrenotokous research and using, have than the more advantage of anther culture: as avoiding the interference of anther wall diploid cell fully, obtain a large amount of fully real monoploid or double haploid, help Breeding Application; In addition, the single cell culture system can be used for gene and import, genetic expression, the study on regulation of androgenesis, the division of cell, differentiation and the research of growth scheduling theory.People pay close attention to the foundation of this technology always very much, but the Isolated microspore culture systems of capsicum is but never successfully reported.Illustrating may be because the specificity of chilli microspore needs a unconventional culture technique system.
Summary of the invention
The purpose of this invention is to provide a kind of capsicum (Capsicum annuum L.) free spore cell assemblies culture method.
For achieving the above object, the present invention takes following scheme: earlier flower pesticide is cultivated in advance, carried out the free cultivation of sporule then.
Specifically, this method adopts following steps:
(1) flower pesticide is cultivated in advance: win the bud that sporule is in the monokaryon later stage, peel off the calyx part.After soaking 10 seconds with 70% ethanol, 0.1% mercuric chloride vibration sterilization 10 minutes.Sterile water wash 4 times strips flower pesticide and is inoculated on the PAC substratum, gives 35 ℃ or 32 ℃ of high temperature dark places reason afterwards 8 days, changes 26 ℃ of dark cultivations over to.
(2) Isolated microspore and cell mass thereof are cultivated: will cultivate aseptic flower pesticide after 15 days in advance and transfer in the culture dish (ф 6cm) that fills 2ml PMC liquid nutrient medium on solid medium, 12 flower pesticide of every ware, gently press from both sides flower pesticide with tweezers, discharge sporule and cell mass thereof, then fragments such as anther wall are pressed from both sides out.Clean microspore suspension can obtain the sporule embryoid in 26 ℃ of dark cultivations after 4 weeks.
Available following method is identified:
(1) plant induction: the culture dish of tool embryoid is moved in the darkroom under the illumination condition, after two weeks, will torpedo stage and the embryoid of cotyledon period transfer among the plant induction substratum PPI, 2000Lux, illumination in 16 hours is cultivated down for 25 ℃, induces plant to form.
(2) ploidy is identified: get the about 150mg of blade in age in each bottle seedling, at 2ml LB01 (Dolezel et al.Analysis of nuclear DNA content in plant cells by flow cytometry.Biol.Plant., 1989.31:113-120) in the cell pyrolysis liquid with after the sharp cutter chopping, filter through 50 μ m nylon membranes, filtrate collection is on the BD flow cytometer in the sample pipe.Behind centrifugal 5 minutes of the 1000rpm, remove supernatant liquor, adding 200 μ l concentration in the precipitation is PI (propidium iodide) the nuclear staining agent of 50 μ g/ml, and lucifuge refrigeration was adopted the ploidy level of cells were tested by flow cytometry plant after 20 minutes.Excitation wavelength 488nm.Control material is diplontic donor maternal plant blade.
Advantage of the present invention is: the present invention looks for another way on the Isolated microspore culture system of capsicum, by sporule cell and the cell mass in the free flower pesticide of cultivating after pre-the cultivation, obtained embryoid and plant thereof, microscope inspection provides the cytological evidence of unicellular, the monoploid development pathway of embryoid.The technical breakthrough of capsicum haploid breeding is not only in the foundation of this technical system, and this Isolated microspore cultivation embryoid system of inducing also may become the good experimental system that carries out the vegetable cell differentiation, grows research.
Description of drawings
Fig. 1 is 35 ℃ and cultivates the sporule in the flower pesticide after 5-8 days down in advance: the DAPI fluorescent dye discloses grows the division of medium and small archespore nuclear generation symmetry, does not grow the sporule hypochromatosis.
Fig. 2 is the free spore cell assemblies in the suspension culture.
Fig. 3 differentiates cotyledon end and radiculodium for cell mass.
Fig. 4 is for forming the TTC vigor dyeing of embryoid.
Fig. 5 cultivates the embryoid that is in different development stage that forms for free spore cell assemblies.
Fig. 6 is the whole plant that forms from embryoid.
Embodiment
1 materials and methods
Capsicum (comprising pimento, capsicum variety, Capsicum annuum L) donor maternal plant grows in the plastic greenhouse of tool fly net, and test was carried out between May to late September.
Anther culture: win the bud that sporule is in the monokaryon later stage, peel off the calyx part.After soaking 10 seconds with 70% ethanol, 0.1% mercuric chloride vibration sterilization 10 minutes.Sterile water wash 4 times strips flower pesticide and is inoculated on the solid medium PAC (table 1 with reference to CP substratum (Dumas, 1981), has change), gives 35 ℃ or 32 ℃ of high temperature dark places reason afterwards 8 days, changes 26 ℃ of dark cultivations over to.
Isolated microspore and cell mass thereof are cultivated: adopt direct extrusion Isolated microspore and cell mass thereof in the flower pesticide of pre-cultivation after 15 days, be incubated at liquid nutrient medium PMC (table 1, with reference to the CP substratum, change is arranged) in, every ware (ф 6cm) 2ml, 12 flower pesticide, 26 ℃ of dark cultivations, 4 all " Invest, Then Investigate " embryoids form situation.
In anther culture and free cell culture process, adopt FDA to check vigor (the Heslop HJ.Heslop H Y.Evalution of pollen viability by enzymatically-induced fluorescence:Intracellular hydrolysis of fluorescein diacetate.Stain Technol of cell, 1970,45:115-120).DAPI dyeing, division situation (the Colemen AW of observation of cell nuclear under fluorescent microscope, Goff L J.Application of fluorochrome to pollen biology I.Mithramycin and4 ', 6-diamidino-2-phenylindole (DAPI) as vital stain and for quantification ofnuclear DNA.Stain Technol, 1985,60:145-154).To the embryoid that forms adopt 0.5% triphenyltetrazolium chloride (TTC) dyeing observe embryoid vigor situation (face opens Fu Dengyi for international Seed Inspection association, international Seed Inspection rules, technological standard press, 1976, P24-26,31-32).
Plant induction: the culture dish of tool embryoid is moved in the darkroom under the illumination condition, after two weeks, will torpedo stage and the embryoid of cotyledon period transfer into plant induction substratum PPI (table 1, with reference to the NT substratum) in, 2000Lux, illumination in 16 hours, cultivate down for 25 ℃, induce plant to form.
Ploidy is identified: get the about 150mg of blade in age in each bottle seedling, adopt flow cytometer (FACSCalibur, U.S. company BD) to measure the ploidy level of plant.The nuclear staining agent is propidium iodide (propidium iodide), concentration 50 μ g/ml, excitation wavelength 488nm.Control material is diplontic donor maternal plant blade.It is CellQuest that the DNA data are obtained software, and ploidy analysis software is ModFit.
The used substratum of table 1 chilli microspore plant induction
| Form | Composition | Flower pesticide is cultivated (PAC) in advance | Isolated microspore is cultivated (PMC) | Plant induction (PPI) | |
| A large amount of (mg/l) molysite (mg/l) trace (mg/l) organic (mg/l) hormone (mg/l) sugar (g/l) active carbon (g/l) agar (g/l) pH | (NH 4) 2SO 4 NH 4NO 3 KNO 3 Ca(NO 3) 2.4H 2O CaCl 2.2H 2O MgSO 4.7H 2O KH 2PO 4 NaH 2PO 4.H 2O KCl FeSO 4.7H 2O EDTA.Na 2 KI CoCl 2.6H 2O H 3BO 3 Na 2MoO 4.2H 2O MnSO 4.H 2O MnSO 4.4H 2O CuSO 4.5H 2O ZnSO 4.7H 2The plain cobalamin glutamine of O inositol nicotinic acid pyridoxol thiamine calcium pantothenate folic acid biological glycine kinetine 2,4-D | 34 1238 2150 50 313 412 142 38 7 13.9 18.65 0.695 0.016 3.15 0.188 22.13 0.016 3.625 50.3 0.7 5.5 0.6 0.5 0.005 0.03 0.1 0.01 0.01 30 1.5 8 5.9 | 34 1238 2150 50 313 412 142 38 7 13.9 18.65 0.695 0.016 3.15 0.188 22.13 0.016 3.625 50.3 0.7 5.5 0.6 0.5 0.005 0.03 0.1 0.1 30 5.8 | 825 950 220 1233 680 27.8 37.3 0.83 0.03 6.2 0.25 22.3 0.025 8.6 100 5 0.5 0.5 0.5 0.05 2 0.1 30 10 5.9 | |
2 results and analysis
2.1 the cytological evidence that embryoid is grown
Tested microscopy and cultivated the cultivation that begins to free cell in advance, whole growth courses that embryoid forms from flower pesticide.
35 ℃ of pyroprocessing are after 5 days, and sporule dissociates in flower pesticide.FDA checks demonstration, and about 25% sporule still has vigor preferably, and these sporule cells expand full and circular, and other cell does not send green fluorescence, loses vigor, the state of the obvious germinal furrow of tool when cell still keeps just free.DAPI checks demonstration, and in the cystite, homoeokinesis (Fig. 1) has taken place nucleus, and the control material sporule nucleus of always cultivating under 26 ℃ is asymmetric division substantially, is formed on the dyeing depth, discrepant vegetative cell and sexual cell nuclear on the size.
35 ℃ of cultivations are in the time of 10 days, and many divisions of 2-have taken place the sporule nucleus, and polus animalium is asynchronous; Cell walls forms or does not form, thereby has formed syncyte or many cells group.Being formed with of cell walls produces immediately after a nuclear fission and causes fissionally, also has just to form cell walls after nuclear fission repeatedly, produces fissionally, even in a cell nucleus about 12 arranged and still do not take place fissional.Observe also as seen, the splitted cell taken place deviate from pollen wall, although and do not have the splitted cell that multinuclear has been arranged, still still be in the pollen wall, the deuterogenesis cell fission, just can deviate from pollen wall after forming cell mass.
Cultivate after 15 days, sporule and cell mass thereof are dissociated out in flower pesticide carry out liquid culture.It is still very asynchronous to observe the division of finding cell, and many cells group is arranged, and the cell (Fig. 2) that only is in secondary split is also arranged.Can continued growth break up in the cell mass liquid medium within, after 10 days, can observe some cell masses has had polarity to form, and produces the differentiation (Fig. 3) of cotyledon and radicle in free cultivation.The free cultivation after 25 days, from globular stage embryo progressively form to the embryoids at different levels of cotyledon period embryo, the development degree of embryoid shows extremely asynchronous characteristic (Fig. 5), this and early stage fissional asynchronism are corresponding to.
Under dark culture condition, the embryoid color that forms in the liquid nutrient medium is white or canescence.Adopt the TTC staining to carry out the vigor inspection to it, the visible better vigor of most of embryoid tool (Fig. 4).
2.2 plant regeneration from embryoid
The sporule embryoid that suspension culture obtains through secondary succeeding transfer culture (about 40 days), has obtained the normal plant of plant type (Fig. 6) after the plant induction substratum is gone in switching.Table 2 has been listed the relevant work and the result thereof of capsicum haploid induction technology in the world.The isolated microspore culture technique of our foundation no matter be on germ extraction rate or the quality (closely related with the regeneration plant ability) at embryo, all is enhanced than the work of report in the past as can be seen.
Table 2 capsicum (Capsicum annuum L) haploid breeding different technologies and effect thereof
*
| Material type | Embryoid formation time (week) | Tool embryo flower pesticide number/100 flower pesticide | Embryo number/100 flower pesticide | Regeneration plant number/100 flower pesticide | The author |
| The floating flower pesticide flower pesticide of flower pesticide flower pesticide flower pesticide flower pesticide flower pesticide flower pesticide flower pesticide Isolated microspore | 4-8 6 9 6 | 18.7 34.0 16.5 4.0 | 13 0.23 125 84.4 3.5 6.0 38.3 183 | 4.8 0.01 8.0 28.9 43 | Gyulai, 2000 Hwang, 1998 Ramon, 1997 Mityko, 1995 Kristiansen, 1993 Dumas, 1981 Wang Li are great, the 2004 Li Chun tinkling of pieces of jade, 2001 sub-monarchs, 2000 the present invention |
*Listed numerical value all is the numerical value of best result in this report in the table
All the time it is very asynchronous to exist an embryoid to grow in the anther culture of capsicum, and it is undesired, of poor quality to grow, the problem that seedling rate is low.Its normal chick embryo only accounts for the 1/20-1/10 of whole embryoids, and last plant seedling rate only is the 1/10-1/100 of embryoid sum
[8]Therefore the limiting factor of pepper anther culture is not on the number of somatoblast frequency and embryoid, but in the further normal development of embryoid, thereby particularly the normal development of proembryo improves the tool cotyledon, on the ratio of the normal embryoid of vegetative point.This research is free sporule cell and cell mass in the flower pesticide of pre-cultivation after 15 days, on the one hand, the cultivation that has proved the cell mass that these development degrees are not waited can obtain the sporule embryoid, on the other hand may since cell mass in suspension culture nutrition supply and the growing space than good in the flower pesticide, obtained to be up to the result that 12 flower pesticide produce 22 embryoids in this research, and the cotyledon period embryo ratio be about 23%, with conventional anther culture relatively, performance is better.
Undoubtedly, free spore cell assemblies is cultivated route among the present invention, for studying the influence of substratum factor or other factors for sporule cell fission, differentiation, the growth and the morphogenesis of research vegetable cell, optimize the androgenesis technology of capsicum, improving normal chick embryo shape body frequency is an excellent research system.
2.3 the ploidy of regeneration plant is identified
Get well-grown blade, adopt flow cytometer to carry out the ploidy analysis.The result shows, in regeneration strain, exists monoploid, diploid and mixoploid plant.Because the early stage microscopy that embryoid is grown shows that somatoblast group is from sporule, therefore the latter two should be in the division atomization of spore cell assemblies, the double haploid material that spontaneously doubled haploid forms, and in the same cell mass of possibility, the part cell chromosome has taken place to double, and the mixoploid material that chromosome doubling produces does not take place the part cell.
Claims (1)
1. hot pepper free spore cell assemblies culture method is characterized in that: earlier flower pesticide is cultivated in advance, carried out the free cultivation of sporule then, concrete steps are as follows:
(1) anther culture: win the bud that sporule is in the monokaryon later stage, peel off the calyx part; After soaking 10 seconds with 70% ethanol, 0.1% mercuric chloride vibration sterilization 10 minutes; Sterile water wash 4 times strips flower pesticide and is inoculated on the PAC substratum, gives 35 ℃ or 32 ℃ of high temperature dark places reason afterwards 8 days, changes 26 ℃ of dark cultivations over to;
(2) Isolated microspore and cell mass thereof are cultivated: in the flower pesticide of pre-cultivation after 15 days, adopt direct extrusion Isolated microspore and cell mass thereof, be incubated in the PMC substratum, and every ware 2ml, 12 flower pesticide, 26 ℃ of dark cultivations can obtain the sporule embryoid after 4 weeks;
The concrete component and the concentration of PAC substratum and PMC substratum are as follows:
Form Composition Flower pesticide is cultivated (PAC) in advance Isolated microspore is cultivated (PMC)
A large amount of (mg/l) molysite (mg/l) trace (mg/l) organic (mg/l) (NH
4)
2SO
4 NH
4NO
3 KNO
3 Ca(NO
3)
2.4H
2O CaCl
2.2H
2O MgSO
4.7H
2O KH
2PO
4 NaH
2PO
4.H
2O KCl FeSO
4.7H
2O EDTA.Na
2 Kl CoCl
2.6H
2O H
3BO
3 Na
2MoO
4.2H
2O MnSO
4.H
2O MnSO
4.4H
2O CuSO
4.5H
2O ZnSO
4，7H
2The plain cobalamin of O inositol nicotinic acid pyridoxol thiamine calcium pantothenate folic acid biological 34 1238 2150 50 313 412 142 38 13.9 18.65 0.695 0.016 3.15 0.188 22.13 0.016 3.625 50.3 0.7 5.5 0.6 0.5 0.005 0.03 34 1238 2150 50 313 412 142 38 13.9 18.65 0.695 0.016 3.15 0.188 22.13 0.016 3.625 50.3 0.7 5.5 0.6 0.5 0.005 0.03
Hormone (mg/l) sugar (g/l) active carbon (g/l) agar (g/l) pH Glutamine glycine kinetine 2.4-D 0.1 0.01 0.01 30 1.5 8 5.9 0.1 0.1 30 5.8
(3) ploidy is identified: get the about 150mg of blade in age in each bottle seedling, with after the sharp cutter chopping, through the filtration of 50 μ m nylon membranes, filtrate collection is on flow cytometer in the sample pipe in 2ml LB01 cell pyrolysis liquid; Behind centrifugal 5 minutes of the 1000rpm, remove supernatant liquor, adding 200 μ l concentration in the precipitation is the propidium iodide nuclear staining agent of 50 μ g/ml, lucifuge refrigeration is after 20 minutes, adopt the ploidy level of cells were tested by flow cytometry plant, excitation wavelength 488nm, control material are diplontic donor maternal plant blade.
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| CN1418537A (en) * | 2002-12-26 | 2003-05-21 | 北京市海淀区植物组织培养技术实验室 | Method for breeding allied hybrid species utilizing anther of Capsicum hybrid species |
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| Title |
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| 国内外辣椒(C.annuum)单倍体育种技术研究进展 龙洪进，西南农业学报，第17卷 2004 * |
| 辣椒花药培养和小孢子培养研究 刘独臣，中国优秀博硕士学位论文全文数据库 2003 * |
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