CN103444542A - Culture method for directly obtaining plant by pepper anther and culture medium - Google Patents

Culture method for directly obtaining plant by pepper anther and culture medium Download PDF

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CN103444542A
CN103444542A CN2013104146898A CN201310414689A CN103444542A CN 103444542 A CN103444542 A CN 103444542A CN 2013104146898 A CN2013104146898 A CN 2013104146898A CN 201310414689 A CN201310414689 A CN 201310414689A CN 103444542 A CN103444542 A CN 103444542A
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plant
culture
cultural method
liquid nutrient
vitamin
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CN103444542B (en
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邓晓梅
李春玲
张树根
张军民
张秦
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BEIJING HAIDIAN PLANT ORGANISM CULTURE TECHNIQUE LABAROTORY
BEIJING HAIHUA BIO-TECH Co Ltd
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BEIJING HAIDIAN PLANT ORGANISM CULTURE TECHNIQUE LABAROTORY
BEIJING HAIHUA BIO-TECH Co Ltd
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Abstract

The invention relates to a culture method for directly obtaining a plant by a pepper anther and a culture medium, and belongs to the field of plant cell engineering. The culture method comprises the steps: (1), selecting a flower bud with microspores in a mid-late uninucleate stage; (2), soaking the flower bud by alcohol with the concentration being 70 percent for 0.5-1min and mercuric chloride with the concentration being1.2 percent for 8-15min, washing for 3-5 times by using sterile water, sucking water to be dry by using sterile filter paper to obtain a sterile anther in the mid-late uninucleate stage; (3), floating the sterile anther in the mid-late uninucleate stage in the liquid culture medium, carrying out dark shake culture for 20-25 days, and then carrying out illumination shake culture; (4), after carrying out illumination shake culture for 10-20 days, transferring a formed cytoledon-stage embryo to an MSO solid culture medium for being cultured, and growing into a normal plant. The culture method disclosed by the invention has the advantages that (1), the induction frequency of a normal embryo is greatly increased, the regeneration frequency of the normal plant is remarkably increased; (2), the operation process is simple, and the plant can be directly obtained from microspores freely dispersing in the liquid culture medium; (3), the culture method is suitable for multiple types of peppers such as cow-horn peppers, capsicum, line peppers and sweetbell redpeppers.

Description

A kind of pepper anther directly obtains cultural method and the medium of plant
Technical field
The invention belongs to field of plant cell engineering technology, be specifically related to cultural method and medium that a kind of pepper anther directly obtains plant.
Background technology
Although by conventional anther culture, many capsicum varieties have obtained the DH plant, and induction frequency is also very low on the whole.So if the method that can cultivate by Isolated microspore obtains a large amount of embryoids, and then induced development becomes normal regeneration plant, then obtains the DH plant by the method for artificial doubling, and this will improve breeding efficiency greatly.But, make current isolated microspore culture technique become the part of conventional cross-breeding means, still there are a lot of problems, hinder it and be applied to extensive breeding; Two problems that wherein highlight most are:
(1), the microspore root tips of capsicum is still in reduced levels.
How to improve the microspore root tips of capsicum, genotype is generally considered one of most important influence factor.The embryoid induction frequency of microspore is different widely different because of genotype.Kim (2008) carries out the hungry cultivation of carbon source by Isolated microspore, although obtained higher embryoid induction frequency, is only limited to a capsicum variety " Milyang-jare ".Koleva-Gudeva (2009) etc. carry out anther culture to the capsicum of 21 different genotype, only have 12 genotype to induce embryoid.Nowaczyk etc. (2009) only have 6 genotype to obtain embryoid in 19 capsicum varieties, and induction frequency is all lower.In order to improve the induction frequency of embryoid, (2009) such as Ge'mes Juha'sz have attempted capsicum and the pimento of different genotype, and at the microspore Induction period, add maltose and with the Ovary co culture of wheat, inducing in quantity and facilitation arranged qualitatively for embryo.Lantos etc. (2009) are that first hunger is cultivated flower pesticide, then Isolated microspore, and improve the embryoid induction frequency with wheat or capsicum Ovary co culture.Although above these methods slightly are improved microspore embryoid induction frequency to a certain extent, the ratio of Embryos does not reduce, and method is loaded down with trivial details, and impracticable.Therefore set up the chilli microspore cultivating system that an abductive approach has simply again higher embryoid induction frequency and become the task of top priority.
(2), the chilli microspore embryo can not normal development, in regeneration plant, the ratio of normal plant is lower.
The ratio that how to improve normal chick embryo and regular regeneration plant is another major issue during pepper anther and pollen are cultivated.During chilli microspore is cultivated, the incidence of Embryos is higher, and has globular embryo arrest of development phenomenon.In the growth course of embryo, while from globular embryo, being transitioned into heart-shape embryo, cotyledonary embryos, all deformity in various degree can appear on anatomy and function, this is distinct issues very in the androgenesis process.At present, using in the world method more widely is the solid-liquid body Double layer culture method of Supena at report in 2006, but in the embryoid that utilizes the method to obtain, only have 20% to be normal chick embryo, 30% does not have cotyledon and stem apex, grow young root for 50%, an average bud only obtains 0.1 plant (a capsicum bud generally has 6 flower pesticide).(2010) such as Kim etc. (2008), Li Chunling etc. (2008) and Parra-Vega are in the hot pepper free spore incubation, all find to exist spherical embryo and the heart-shape embryo that can not further grow seedling, and a large amount of acotyledons or without the phenomenon of the Embryos of growing point, and various Embryos ratio is higher than normal chick embryo.Therefore, the ratio that how to reduce Embryos in the embryoid induction process becomes an important topic in current capsicum haploid breeding.
Summary of the invention
The object of the invention is to disclose the cultural method that a kind of pepper anther directly obtains plant.
Another object of the present invention has been to disclose the medium of cultivating aseptic flower pesticide in above-mentioned cultural method.
The objective of the invention is to be achieved through the following technical solutions:
A kind of pepper anther directly obtains the cultural method of plant, comprises the steps:
(1), the keep to the side aseptic flower pesticide of phase of monokaryon is floated in liquid nutrient medium, dark concussion is cultivated, and transfers the illumination concussion after 20～25 days to and cultivates, consisting of of described liquid nutrient medium: 2500mg/L KNO 3, 1025mg/L NH 4nO 3, 295mg/L CaCl 22H 2o, 325mg/L MgSO 47H 2o, 145mg/L KH 2pO 4, 67mg/L (NH 4) 2sO 4, 75mg/L NaH 2pO 4h 2o, 100mg/L Ca (NO 3) 24H 2o, 13mg/L KCl, 13.9mg/L FeSO 47H 2o, 18.65mg/L Na 2eDTA, 0.695mg/L KI, 3.15mg/L H 3bO 3, 22.13mg/L MnSO 4h 2o, 3.625mg/L ZnSO 47H 2o, 0.188mg/L Na 2moO 42H 2o, 0.016mg/L CuSO 45H 2o, 0.016mg/L CoCl 26H 2o, the 100mg/L inositol, 5mg/L nicotinic acid, the 2mg/L glycine, 0.5mg/L aneurin (vitamin B1), 0.5mg/L puridoxine hydrochloride (vitamin B6), 0.5mg/L folic acid, 0.05mg/L vitamin h, the 800mg/L glutamine, the 100mg/L serine, 30mg/L glutathione (GSH), 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose (are that liquid nutrient medium is the macroelement by the Rm medium, the trace element of Cm medium and molysite, the organic principle of NLN medium, 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose form, wherein 0.1% active carbon and 6% sucrose refer in the 1000ml medium and add 1g active carbon and 60g sucrose),
(2), illumination concussion cultivates after 10～20 days, and the cotyledon type embryo of formation is gone in the MSO solid culture medium and cultivates, and grows up to normal plant.
The described a kind of pepper anther of technique scheme directly obtains the cultural method of plant, wherein, before described method, also comprises the steps:
(i), choose microspore in the keep to the side bud of phase of monokaryon;
(ii), bud is with 70% alcohol-pickled 0.5～1 minute, 1 ‰～2 ‰ mercury chloride 8～15 minutes, aseptic water washing 3～5 times, the aseptic filter paper suck dry moisture, obtain the keep to the side aseptic flower pesticide of phase of monokaryon.
The described a kind of pepper anther of technique scheme directly obtains the cultural method of plant, wherein, by the monokaryon inoculum density that the aseptic flower pesticide of phase floats in liquid nutrient medium that keeps to the side, is to inoculate 15～25 flower pesticide in the 5ml liquid nutrient medium.
The described a kind of pepper anther of technique scheme directly obtains the cultural method of plant, wherein, in the keep to the side bud of phase of monokaryon, is to dye and determine by DAPI.
The described a kind of pepper anther of technique scheme directly obtains the cultural method of plant, and wherein, the shaking speed that dark concussion is cultivated and illumination is shaken while cultivating is 40 rev/mins.
Change osmotic pressure because moisture evaporates for fear of liquid nutrient medium in incubation, therefore, on the basis of technique scheme, dark concussion cultivation and illumination were supplied liquid nutrient medium to primary quantity every 7～10 days in shaking and cultivating.
Cultivate the medium of aseptic flower pesticide in the described cultural method of technique scheme, wherein, the consisting of of described liquid nutrient medium: 2500mg/L KNO 3, 1025mg/L NH 4nO 3, 295mg/L CaCl 22H 2o, 325mg/L MgSO 47H 2o, 145mg/L KH 2pO 4, 67mg/L (NH 4) 2sO 4, 75mg/L NaH 2pO 4h 2o, 100mg/L Ca (NO 3) 24H 2o, 13mg/L KCl, 13.9mg/L FeSO 47H 2o, 18.65mg/L Na 2eDTA, 0.695mg/L KI, 3.15mg/L H 3bO 3, 22.13mg/L MnSO 4h 2o, 3.625mg/L ZnSO 47H 2o, 0.188mg/LNa 2moO 42H 2o, 0.016mg/L CuSO 45H 2o, 0.016mg/L CoCl 26H 2o, the 100mg/L inositol, 5mg/L nicotinic acid, the 2mg/L glycine, 0.5mg/L aneurin (vitamin B1), 0.5mg/L puridoxine hydrochloride (vitamin B6), 0.5mg/L folic acid, 0.05mg/L vitamin h, the 800mg/L glutamine, the 100mg/L serine, 30mg/L glutathione (GSH), 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose (are that liquid nutrient medium is the macroelement by the Rm medium, the trace element of Cm medium and molysite, the organic principle of NLN medium, 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose form, wherein 0.1% active carbon and 6% sucrose refer in the 1000ml medium and add 1g active carbon and 60g sucrose).
The present invention has following beneficial effect:
1, cultural method of the present invention has adopted dark concussion to cultivate in early stage, dark environment is conducive to the formation of normal germule, the dissolved oxygen rate that the release rate not only be conducive to improve microspore can also improve liquid nutrient medium is cultivated in concussion simultaneously, can also allow the microspore discharged be evenly distributed in liquid nutrient medium simultaneously, these factors improve the induction frequency of normal embryoid greatly, thereby the regeneration frequency of normal plant also is significantly increased;
2, cultural method of the present invention is simple to operate, can directly by the microspore that freely becomes scattered about liquid nutrient medium, obtain plant;
3, the embryoid and the normal plant ratio that adopt cultural method of the present invention to obtain are higher.
Embodiment:
For making technical scheme of the present invention be convenient to understand, the cultural method that a kind of pepper anther of the present invention is directly obtained to plant below in conjunction with concrete test example is further described.
embodiment 1:goat's horn type chilli microspore is cultivated:
Fetch bud from planting greenhouse in June, 2012, and pollen confirms that through DAPI (4,6-diamidine-2-phenylindone) dyeing developmental stage is that monokaryon keeps to the side the phase, and the surface sterilization of bud refers to through 75% alcohol 60 seconds, 2 ‰ mercuric chloride 10 minutes, aseptic water washing 5 times.
Carefully flower pesticide is taken out with pincet, be placed in liquid nutrient medium and carry out 25 ℃ of dark concussions and cultivate after 20～25 days and transfer the illumination concussion to and cultivate, dark concussion is cultivated and shaking speed during illumination concussion cultivation is 40 rev/mins, consisting of of this liquid nutrient medium: 2500mg/L KNO 3, 1025mg/L NH 4nO 3, 295mg/L CaCl 22H 2o, 325mg/L MgSO 47H 2o, 145mg/L KH 2pO 4, 67mg/L (NH 4) 2sO 4, 75mg/L NaH 2pO 4h 2o, 100mg/L Ca (NO 3) 24H 2o, 13mg/L KCl, 13.9mg/L FeSO 47H 2o, 18.65mg/L Na 2eDTA, 0.695mg/L KI, 3.15mg/L H 3bO 3, 22.13mg/L MnSO 4h 2o, 3.625mg/L ZnSO 47H 2o, 0.188mg/L Na 2moO 42H 2o, 0.016mg/L CuSO 45H 2o, 0.016mg/L CoCl 26H 2o, the 100mg/L inositol, 5mg/L nicotinic acid, the 2mg/L glycine, 0.5mg/L aneurin (vitamin B1), 0.5mg/L puridoxine hydrochloride (vitamin B6), 0.5mg/L folic acid, 0.05mg/L vitamin h, the 800mg/L glutamine, the 100mg/L serine, 30mg/L glutathione (GSH), 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose (are that liquid nutrient medium is the macroelement by the Rm medium, the trace element of Cm medium and molysite, the organic principle of NLN medium, 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose form, wherein 0.1% active carbon and 6% sucrose refer in the 1000ml medium and add 1g active carbon and 60g sucrose, liquid nutrient medium is prepared according to conventional method),
Dark concussion is cultivated 20～25 days, the visible cells,primordial of naked eyes group; Transfer the illumination concussion to and cultivate 10～20 days, existing embryoid produces.Because inducing of microspore is asynchronous, some embryoid is to torpedo stage or cotyledon period, and also some just has the cell mass of embryo structure.Choose the embryoid that differentiates two little cotyledons and go to the MSO medium, within 15～20 days, be grown to normal plantlet, then the normal whole plant that grows up to well developed root system, average every 20 flower pesticide produce 15 embryoids, wherein 10 is normal embryoid, normal embryoid ratio is 67%, is far longer than Supena ratio of 20% in the solid-liquid Double layer culture method of report in 2006.In the dark concussion cultivation and illumination concussion cultivation of the present embodiment, every 7～10 days, supply liquid nutrient medium to primary quantity.
The above, it is only preferred embodiment of the present invention, not the present invention is done to any formal and substantial restriction, all those skilled in the art, within not breaking away from the technical solution of the present invention scope, when utilizing the disclosed above technology contents, and the equivalent variations of a little change of making, modification and differentiation is equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.

Claims (7)

1. a pepper anther directly obtains the cultural method of plant, comprises the steps:
(1), the keep to the side aseptic flower pesticide of phase of monokaryon is floated in liquid nutrient medium, dark concussion is cultivated, and transfers the illumination concussion after 20～25 days to and cultivates; Consisting of of described liquid nutrient medium: 2500mg/L KNO 3, 1025mg/L NH 4nO 3, 295mg/L CaCl 22H 2o, 325mg/L MgSO 47H 2o, 145mg/L KH 2pO 4, 67mg/L (NH 4) 2sO 4, 75mg/L NaH 2pO 4h 2o, 100mg/L Ca (NO 3) 24H 2o, 13mg/L KCl, 13.9mg/L FeSO 47H 2o, 18.65mg/L Na 2eDTA, 0.695mg/L KI, 3.15mg/L H 3bO 3, 22.13mg/L MnSO 4h 2o, 3.625mg/L ZnSO 47H 2o, 0.188mg/L Na 2moO 42H 2o, 0.016mg/L CuSO 45H 2o, 0.016mg/L CoCl 26H 2o, 100mg/L inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L aneurin (vitamin B1), 0.5mg/L puridoxine hydrochloride (vitamin B6), 0.5mg/L folic acid, 0.05mg/L vitamin h, 800mg/L glutamine, 100mg/L serine, 30mg/L glutathione (GSH), 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/L NAA, 0.1% active carbon and 6% sucrose;
(2), illumination concussion cultivates after 10～20 days, and the cotyledon type embryo of formation is gone in the MSO solid culture medium and cultivates, and grows up to normal plant.
2. a kind of pepper anther according to claim 1 directly obtains the cultural method of plant, it is characterized in that, before described method, also comprises the steps:
(i), choose microspore in the keep to the side bud of phase of monokaryon;
(ii), bud is with 70% alcohol-pickled 0.5～1 minute, 1 ‰～2 ‰ mercury chloride 8～15 minutes, aseptic water washing 3～5 times, the aseptic filter paper suck dry moisture, obtain the keep to the side aseptic flower pesticide of phase of monokaryon.
3. a kind of pepper anther according to claim 1 and 2 directly obtains the cultural method of plant, it is characterized in that: by the monokaryon inoculum density that the aseptic flower pesticide of phase floats in liquid nutrient medium that keeps to the side, be to inoculate 15～25 flower pesticide in the 5ml liquid nutrient medium.
4. a kind of pepper anther according to claim 1 and 2 directly obtains the cultural method of plant, it is characterized in that: in the keep to the side bud of phase of monokaryon, be to dye and determine by DAPI.
5. a kind of pepper anther according to claim 1 and 2 directly obtains the cultural method of plant, it is characterized in that: the shaking speed that dark concussion is cultivated and illumination is shaken while cultivating is 40 rev/mins.
6. a kind of pepper anther according to claim 1 and 2 directly obtains the cultural method of plant, it is characterized in that: dark concussion is cultivated and illumination is shaken in cultivation, every 7～10 days, supplies liquid nutrient medium to primary quantity.
7. cultivate the medium of aseptic flower pesticide described in claim 1～6 in cultural method, it is characterized in that: the consisting of of described liquid nutrient medium: 2500mg/L KNO 3, 1025mg/L NH 4nO 3, 295mg/L CaCl 22H 2o, 325mg/L MgSO 47H 2o, 145mg/L KH 2pO 4, 67mg/L (NH 4) 2sO 4, 75mg/L NaH 2pO 4h 2o, 100mg/L Ca (NO 3) 24H 2o, 13mg/L KCl, 13.9mg/L FeSO 47H 2o, 18.65mg/L Na 2eDTA, 0.695mg/L KI, 3.15mg/L H 3bO 3, 22.13mg/L MnSO 4h 2o, 3.625mg/L ZnSO 47H 2o, 0.188mg/L Na 2moO 42H 2o, 0.016mg/L CuSO 45H 2o, 0.016mg/L CoCl 26H 2o, 100mg/L inositol, 5mg/L nicotinic acid, 2mg/L glycine, 0.5mg/L aneurin (vitamin B1), 0.5mg/L puridoxine hydrochloride (vitamin B6), 0.5mg/L folic acid, 0.05mg/L vitamin h, 800mg/L glutamine, 100mg/L serine, 30mg/L glutathione (GSH), 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/LNAA, 0.1% active carbon and 6% sucrose.
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CN105028205A (en) * 2015-08-03 2015-11-11 河南科技大学 Method for directly cultivating capsicum annuum L. anthers into seedlings
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CN111374048A (en) * 2020-04-28 2020-07-07 北京市海淀区植物组织培养技术实验室 Chromosome doubling method of pepper or eggplant haploid plant

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CN105638480A (en) * 2016-02-16 2016-06-08 江苏强农农业技术服务有限公司 Formula of anther induction medium for cultivation of capsicum annuum variety
CN111374048A (en) * 2020-04-28 2020-07-07 北京市海淀区植物组织培养技术实验室 Chromosome doubling method of pepper or eggplant haploid plant
CN111374048B (en) * 2020-04-28 2022-03-08 北京市海淀区植物组织培养技术实验室 Chromosome doubling method of pepper or eggplant haploid plant

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