CN103444542B - A kind of pepper anther directly obtains cultural method and the medium of plant - Google Patents
A kind of pepper anther directly obtains cultural method and the medium of plant Download PDFInfo
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- CN103444542B CN103444542B CN201310414689.8A CN201310414689A CN103444542B CN 103444542 B CN103444542 B CN 103444542B CN 201310414689 A CN201310414689 A CN 201310414689A CN 103444542 B CN103444542 B CN 103444542B
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Abstract
A kind of pepper anther of the present invention directly obtains cultural method and the medium of plant, belongs to field of plant cell engineering technology.Described cultural method comprises: (1), choose the bud that microspore is in mid-late uninucleate stage; (2), bud with 70% alcohol-pickled 0.5 ~ 1 minute, the mercury chloride of 12 ‰ 8 ~ 15 minutes, aseptic water washing 3 ~ 5 times, aseptic filter paper suck dry moisture, obtains the aseptic flower pesticide of mid-late uninucleate stage; (3), float in liquid nutrient medium by the aseptic flower pesticide of mid-late uninucleate stage, dark concussion cultivation transfers illumination concussion to and cultivates after 20 ~ 25 days; (4), illumination concussion cultivation after 10 ~ 20 days, the cotyledon type embryo of formation is gone in MSO solid culture medium and cultivates, grow up to normal plant.Cultural method of the present invention has the following advantages: (1), greatly improve the induction frequency of normal embryoid, significantly improves the regeneration frequency of normal plant; (2), operating process is simple, can directly by the microspore acquisition plant freely becoming scattered about liquid nutrient medium; (3) polytype capsicums such as long red pepper, goat's horn green pepper, line green pepper, bell pepper, are applicable to.
Description
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 DH plant, and induction frequency is also very low on the whole.If so obtain a large amount of embryoid by the method for microspore-isolated culture, and then induced development becomes normal regeneration plant, then obtain DH plant by the method for artificial doubling, this will improve breeding efficiency greatly.But, current isolated microspore culture technique be made to become a part for conventional cross-breeding means, still there is a lot of problem, hinder it and be applied to extensive breeding; The two problems wherein highlighted most is:
(1), the microspore embryoid induction frequency of capsicum is still in reduced levels.
How to improve the microspore embryoid induction frequency of capsicum, genotype is generally considered one of most important influence factor.The embryoid induction frequency of microspore is widely different because genotype is different.Isolated microspore is carried out carbon source hunger and cultivates by Kim (2008), although obtain higher embryoid induction frequency, is only limited to a capsicum variety " Milyang-jare ".Koleva-Gudeva (2009) etc. carries out anther culture to the capsicum of 21 different genotype, only has 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, Ge'mes Juha'sz etc. (2009) have attempted capsicum and the pimento of different genotype, and at microspore Induction period, add maltose and the Ovary co culture with wheat, for the induction of embryo in quantity and have facilitation qualitatively.Lantos etc. (2009) are then that first hunger cultivates flower pesticide, then Isolated microspore, and improve embryoid induction frequency with wheat or capsicum Ovary co culture.Although these methods slightly improve microspore embryoid induction frequency to a certain extent above, 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 a higher embryoid induction frequency and become the task of top priority.
(2), chilli microspore embryo can not normal development, and in regeneration plant, the ratio of normal plant is lower.
The ratio how improving normal chick embryo and regular regeneration plant is another major issue in pepper anther and pollen cultures.During chilli microspore is cultivated, the incidence of Embryos is higher, and there is globular embryo arrest of development phenomenon.In the growth course of embryo, when being transitioned into heart-shape embryo, cotyledonary embryos from globular embryo, in anatomy and functionally all can occur in various degree deformity, this is very distinct issues in androgenesis process.At present, method is more widely used to be the solid-liquid body Double layer culture method of Supena at report in 2006 in the world, but in the embryoid utilizing the method to obtain, 20% is only had 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).(2008) and the Parra-Vega etc. (2010) such as Kim etc. (2008), Li Chunling are in hot pepper free spore incubation, all find to there is spherical embryo and the heart-shape embryo that can not grow seedling further, and the phenomenon of a large amount of acotyledon or the Embryos without growing point, and various Embryos ratio is higher than normal chick embryo.Therefore, the ratio how reducing Embryos in 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 is to disclose the medium cultivating aseptic flower pesticide in above-mentioned cultural method.
The object of the invention is to be achieved through the following technical solutions:
Pepper anther directly obtains a cultural method for plant, comprises the steps:
(1), float in liquid nutrient medium by the aseptic flower pesticide of mid-late uninucleate stage, dark concussion is cultivated, and transfers illumination concussion to and cultivate after 20 ~ 25 days, 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/LIAA, 0.01mg/L NAA, (namely liquid nutrient medium is by the macroelement of Rm medium for 0.1% active carbon and 6% sucrose, the trace element of Cm medium and molysite, the organic principle of NLN medium, 0.1mg/L BA, 0.1mg/L IAA, 0.01mg/LNAA, 0.1% active carbon and 6% sucrose composition, wherein 0.1% active carbon and 6% sucrose refer in 1000ml medium and add 1g active carbon and 60g sucrose),
(2), illumination concussion cultivation after 10 ~ 20 days, the cotyledon type embryo of formation is gone in MSO solid culture medium and cultivates, grow up to normal plant.
A kind of pepper anther described in technique scheme directly obtains the cultural method of plant, wherein, also comprises the steps: before described method
(i), choose the bud that microspore is in mid-late uninucleate stage;
(ii), bud with 70% alcohol-pickled 0.5 ~ 1 minute, the mercury chloride of 1 ‰ ~ 2 ‰ 8 ~ 15 minutes, aseptic water washing 3 ~ 5 times, aseptic filter paper suck dry moisture, obtains the aseptic flower pesticide of mid-late uninucleate stage.
A kind of pepper anther described in technique scheme directly obtains the cultural method of plant, wherein, is inoculate 15 ~ 25 flower pesticide in 5ml liquid nutrient medium by the inoculum density that the aseptic flower pesticide of mid-late uninucleate stage floats in liquid nutrient medium.
A kind of pepper anther described in technique scheme directly obtains the cultural method of plant, and wherein, the bud being in mid-late uninucleate stage is determined by DAPI dyeing.
A kind of pepper anther described in technique scheme directly obtains the cultural method of plant, and wherein, shaking speed when dark concussion is cultivated and illumination concussion is cultivated is 40 revs/min.
In order to avoid liquid nutrient medium changes osmotic pressure because of moisture evaporation in incubation, therefore on the basis of technique scheme, dark concussion cultivation and illumination concussion supplied liquid nutrient medium to primary quantity every 7 ~ 10 days in cultivating.
The medium of aseptic flower pesticide is cultivated in cultural method described in technique scheme, wherein, 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/LH
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, 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, (namely liquid nutrient medium is by the macroelement of Rm medium for 0.1% active carbon and 6% sucrose, 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 composition, wherein 0.1% active carbon and 6% sucrose refer in 1000ml medium and add 1g active carbon and 60g sucrose).
The present invention has following beneficial effect:
1, cultural method of the present invention have employed dark concussion cultivation in early stage, dark environmental benefits is in the formation of normal germule, the dissolved oxygen rate that the release rate being not only conducive to improving microspore can also improve liquid nutrient medium is cultivated in concussion simultaneously, the microspore discharged can also be allowed to be evenly distributed in liquid nutrient medium simultaneously, these factors make the induction frequency of normal embryoid greatly improve, and thus the regeneration frequency of normal plant is also significantly increased;
2, cultural method of the present invention is simple to operate, directly can obtain plant by the microspore freely becoming scattered about liquid nutrient medium;
3, the embryoid adopting cultural method of the present invention to obtain and normal plant ratio higher.
Embodiment:
Being convenient to for making technical scheme of the present invention understand, below in conjunction with concrete test example, the cultural method that a kind of pepper anther of the present invention directly obtains plant being further described.
embodiment 1:goat's horn type chilli microspore is cultivated:
In June, 2012 fetches bud from planting greenhouse, through DAPI (4,6-diamidine-2-phenylindone) dyeing, pollen confirms that developmental stage is mid-late uninucleate stage, 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 pincet is taken out, is placed in liquid nutrient medium and carries out 25 DEG C of dark concussion cultivations and transfer illumination concussion after 20 ~ 25 days to and cultivate, dark concussion cultivate and illumination concussion cultivation time shaking speed be 40 revs/min, consisting of of this liquid nutrient medium: 2500mg/L KNO
3, 1025mg/L NH
4nO
3, 295mg/L CaCl
22H
2o, 325mg/LMgSO
47H
2o, 145mg/L KH
2pO
4, 67mg/L (NH
4)
2sO
4, 75mg/L NaH
2pO
4h
2o, 100mg/LCa (NO
3)
24H
2o, 13mg/L KCl, 13.9mg/L FeSO
47H
2o, 18.65mg/L Na
2eDTA, 0.695mg/LKI, 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, 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, (namely liquid nutrient medium is by the macroelement of Rm medium for 0.1% active carbon and 6% sucrose, 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 composition, wherein 0.1% active carbon and 6% sucrose refer in 1000ml medium and add 1g active carbon and 60g sucrose, liquid nutrient medium is conventionally prepared),
Dark concussion cultivation 20 ~ 25 days, the visible Embryogenic cell masses of naked eyes; Transfer illumination concussion cultivation 10 ~ 20 days to, existing embryoid produces.Because the induction 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 differentiating the little cotyledon of two panels and go to MSO medium, within 15 ~ 20 days, be grown to normal plantlet, then the normal whole plant of well developed root system is grown up to, on average every 20 flower pesticide produce 15 embryoids, wherein 10 is normal embryoid, normal embryoid ratio is 67%, be far longer than Supena 2006 report solid-liquid Double layer culture method in 20% ratio.In the dark concussion of the present embodiment is cultivated and illumination concussion is cultivated, supplied liquid nutrient medium to primary quantity every 7 ~ 10 days.
The above, be only preferred embodiment of the present invention, not any formal and substantial restriction is done to the present invention, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and a little change made, modify with differentiation equivalent variations, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (5)
1. pepper anther directly obtains a cultural method for plant, comprises the steps:
(1), float in liquid nutrient medium by the aseptic flower pesticide of mid-late uninucleate stage, dark concussion is cultivated, and transfers illumination concussion to and cultivate after 20 ~ 25 days; Shaking speed when described dark concussion is cultivated and illumination concussion is cultivated is 40 revs/min; Dark concussion is cultivated and in illumination concussion cultivation, was supplied liquid nutrient medium to primary quantity every 7 ~ 10 days;
Consisting of of described liquid nutrient medium: 2500mg/L KNO
3, 1025mg/L NH
4nO
3, 295mg/LCaCl
22H
2o, 325mg/L MgSO
47H
2o, 145mg/L KH
2pO
4, 67mg/L (NH
4)
2sO
4, 75mg/LNaH
2pO
4h
2o, 100mg/L Ca (NO
3)
24H
2o, 13mg/L KCl, 13.9mg/LFeSO
47H
2o, 18.65mg/LNa
2eDTA, 0.695mg/L KI, 3.15mg/L H
3bO
3, 22.13mg/L MnSO
4h
2o, 3.625mg/LZnSO
47H
2o, 0.188mg/LNa
2moO
42H
2o, 0.016mg/L CuSO
45H
2o, 0.016mg/LCoCl
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;
(2), illumination concussion cultivation after 10 ~ 20 days, the cotyledon type embryo of formation is gone in MSO solid culture medium and cultivates, grow 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, also comprises the steps: before described method
(i), choose the bud that microspore is in mid-late uninucleate stage;
(ii), bud with 70% alcohol-pickled 0.5 ~ 1 minute, the mercury chloride of 1 ‰ ~ 2 ‰ 8 ~ 15 minutes, aseptic water washing 3 ~ 5 times, aseptic filter paper suck dry moisture, obtains the aseptic flower pesticide of mid-late uninucleate stage.
3. a kind of pepper anther according to claim 1 and 2 directly obtains the cultural method of plant, it is characterized in that: be inoculate 15 ~ 25 flower pesticide in 5ml liquid nutrient medium by the inoculum density that the aseptic flower pesticide of mid-late uninucleate stage floats in 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: the bud being in mid-late uninucleate stage is determined by DAPI dyeing.
5. cultivate the medium of aseptic flower pesticide in cultural method described in Claims 1 to 4, it is characterized in that: consisting of of described liquid nutrient medium: 2500mg/L KNO
3, 1025mg/L NH
4nO
3, 295mg/L CaCl
22H
2o, 325mg/LMgSO
47H
2o, 145mg/L KH
2pO
4, 67mg/L (NH
4)
2sO
4, 75mg/L NaH
2pO
4h
2o, 100mg/LCa (NO
3)
24H
2o, 13mg/L KCl, 13.9mg/L FeSO
47H
2o, 18.65mg/L Na
2eDTA, 0.695mg/LKI, 3.15mg/L H
3bO
3, 22.13mg/L MnSO
4h
2o, 3.625mg/L ZnSO
47H
2o, 0.188mg/LNa
2moO
4.2H
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.
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Families Citing this family (3)
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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 |
| CN105638480B (en) * | 2016-02-16 | 2017-09-01 | 新沂市芭缇雅商贸有限公司 | A kind of capsicum variety, which is cultivated, uses flower pesticide Fiber differentiation based formulas |
| 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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| CN101622960A (en) * | 2009-08-14 | 2010-01-13 | 江苏省农业科学院 | Method for fast obtaining regenerated plant for cayenne pepper anther culture |
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| KR100526161B1 (en) * | 2004-03-04 | 2005-11-08 | 김문자 | A method for culturing isolated microspore of pepper |
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