CN102187812B - Method for establishing efficient plant regeneration system by using hevea brasiliensis embryonic cell suspension system - Google Patents
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Abstract
The invention discloses a method for establishing an efficient plant regeneration system by using a hevea brasiliensis embryonic cell suspension system. The method comprises the steps of friable embryonic callus induction with embryonic suspension cells, friable embryonic callus subculture, differentiation of body cell embryoid, plant regeneration and the like. The method is simple in process flow, low in production cost, high in embryoid inductivity and high in plant regeneration rate, and has great application value.
Description
Technical field
The present invention relates to a kind of method of utilizing Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system, belong to plant tissue and cell engineering field.
Background technology
Para rubber tree (Hevea brasiliensis Muell.Arg) is a kind of perennial cross pollination arbor of Euphorbiaceae Hevea; Main source as natural rubber; Extensively plant in the torrid areas, and in development of world economy, occupy an important position always.
The breed breeding of Para rubber tree mainly is to rely on conventional breeding, all derives from the strain of Wei Ke Chinese system and the clone of deriving thereof but breeding cycle length adds the material that is used for conventional breeding, and hereditary basis is narrow.In order to widen Para rubber tree breeding approach, improve its quality, many countries drop into a large amount of manpower, material resources and financial resources and carry out extensive studies and obtained certain progress.At present, still have a lot of difficulties in the Para rubber tree tissue culture, what it was main has: the draw materials restriction of objective condition such as receiving bamboo grows florescence and fruit phase of explant; It is dead that the dedifferentiation callus is prone to brownization, is difficult to long-term subculture and preserves; Germ extraction rate is lower, thereby shoot regeneration frequency is extremely low; Production cost is too high, can't satisfy in the production wilderness demand and the needs of setting up efficient genetic conversion system to tissue cultivating seedling.
For this reason, people hope through setting up Para rubber tree cells,primordial suspension system, and set up efficient plant regeneration system and satisfy and produce and the needs of experiment.Yet, because the restriction of multiple factor utilizes Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system and still faces many difficulties, how to solve these difficulties, finally set up stable, regenerating system becomes the most urgent task at present efficiently.For this reason; The present invention is on the basis of Para rubber tree cells,primordial suspension system; Through correlation technique is studied; Finally set up the efficient plant regeneration system of Para rubber tree, for Para rubber tree self-rooting juvenile-type clone large-scale promotion with further set up the efficient genetic conversion system of Para rubber tree important foundation is provided.
Summary of the invention
The object of the invention provides the Para rubber tree cells,primordial suspension system that utilizes that a kind of production cost is low, the embryoid induction rate high, shoot regeneration frequency is high and sets up the method for efficient plant regeneration system.
The method of utilizing Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system provided by the invention, this method comprises the steps:
A, embryonal suspension cell are induced the friable embryogenic callus: with liquid-transfering gun the culture fluid in the Para rubber tree cells,primordial suspension system is blotted; Choosing small cell cluster is inoculated into the cells,primordial suspension system and induces on the medium of friable embryogenic callus; The dark cultivation 15～30 days; Cultivation temperature is between 25 ℃～28 ℃; It is said that to induce the medium of friable embryogenic callus be MS medium and additional composition; In the MS medium content be potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, supplementary element is 2,4-D 0.5～3.0mg/L, 6-BA 0.1～2.0mg/L, NAA 0.5～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L and Sucus Cocois 30～80ml/L;
B, friable embryogenic callus subculture: the friable embryogenic callus is changed over to successive transfer culture on the medium of inducing the friable embryogenic callus, 15～30 days subcultures are once secretly cultivated, and cultivation temperature is between 25 ℃～28 ℃;
The differentiation of c, somatic cell embryoid: the friable embryogenic callus is inoculated in differential medium; The dark cultivation 20～50 days; To promote differentiation, the maturation of somatic cell embryoid; Cultivation temperature is between 25 ℃～28 ℃; Said differential medium is MS medium and additional composition, and the content in the MS medium is adjusted into potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, and additional composition is 6-BA 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.01～1.0mg/L, GA
30.01～1.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L, activated carbon 1～2g/L and Sucus Cocois 30～80ml/L;
D, plant regeneration: ripe embryoid is inoculated into the medium of emerging; Illumination cultivation; With short its whole plant of regenerating; Cultivation temperature is between 25 ℃～30 ℃; The active ingredient of the said medium of emerging is MS medium and additional composition, and the content in the MS medium is adjusted into potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, and supplementary element is KT 0.5～3.0mg/L, NAA0.01～1.0mg/L, GA
30.1～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L, activated carbon 1～2g/L and Sucus Cocois 30～80ml/L.
The Para rubber tree cells,primordial suspension system of step a can adopt the routine techniques preparation, also can adopt following method to obtain:
The preparation of a, explant: choose the Para rubber tree monokaryon and keep to the side the bud of phase, carry out disinfection as explant;
B, embryonic callus induction: the stamen that strips out in the bud that disinfects is inoculated in the embryonic callus induction medium, secretly cultivates 30～50 days, and cultivation temperature is between 24 ℃～30 ℃; Said embryonic callus induction medium is MS medium and additional composition; Content in the MS medium is potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L; Supplementary element is 2,4-D 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.5～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L and Sucus Cocois 30～80ml/L;
The subculture of c, embryo callus: change embryo callus over to fresh embryonic callus induction medium successive transfer culture 3～5 times, 7～10 days subcultures are once secretly cultivated, and cultivation temperature is between 25 ℃～28 ℃;
D, embryo callus suspension culture: embryo callus in good condition behind the subculture is broken into 0.1～0.5cm particle; Getting 2～5g embryo callus particle is inoculated in the triangular flask that contains 20～50mL suspending nutrient solution; Place on 80～120r/ minute the shaking table, the dark cultivation, cultivation temperature is between 25 ℃～28 ℃; 7～9 days subcultures once; When changing culture fluid the small cell cluster of the new dispersion that forms of 1～2g changed in the new triangular flask at every turn and cultivates, behind the subculture 4～6 times, promptly obtain Para rubber tree cells,primordial suspension system; Said suspending nutrient solution is MS medium and additional composition; Content in the MS medium is potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L; Supplementary element is 2,4-D 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.5～3.0mg/L, sucrose 30～60g/L and Sucus Cocois 30～80ml/L;
Above-mentioned in step a, earlier with 75～80% alcohol surface sterilizations 30～60 seconds, with 1g/L HgCl sterilization 10～15 minutes, use aseptic water washing at last 3～6 times, each 3～5 minutes again.
Beneficial effect of the present invention is:
The present invention is bamboo grows haploid breeding, polyploid breeding, makes full use of hybrid vigour, cultivates that output is high, resistance and the fast improved seeds of growing, and excellent material is provided for the genetic development of the various economic characters of research.Can obtain self-rooting juvenile-type clone, get rid of the influence of bad stock, improve output; Can obtain the strong and consistent good stock of cold resistance, reduce cold damage; Also can obtain the material of homogeneous, supply scientific research to use, and a considerable approach is provided for the stock breeding of Para rubber tree.
Technological process of the present invention is simple, and production cost is low, and the embryoid induction rate is high, and shoot regeneration frequency is high, has very big using value.
Embodiment
Embodiment one:
Embryonal suspension cell is induced the friable embryogenic callus: with liquid-transfering gun just the culture fluid in the Para rubber tree cells,primordial suspension system blot; Choosing small cell cluster is inoculated into the cells,primordial suspension system and induces on the medium of friable embryogenic callus; The dark cultivation 20 days, cultivation temperature is 28 ℃.The medium of inducing the friable embryogenic callus is MS medium and additional composition; The MS medium content be epsom salt 500mg/L, potassium dihydrogen phosphate 400mg/L, anhydrous calcium chloride 250mg/L and four water manganese sulphate 35mg/L (down with); Supplementary element is 2,4-D 1.5mg/L, 6-BA 0.5mg/L, NAA 1.0mg/L, Phytagel 2.0g/L, sucrose 70g/L and Sucus Cocois 50ml/L.
Friable embryogenic callus subculture: the friable embryogenic callus is changed over to successive transfer culture on the medium of inducing the friable embryogenic callus, 20 days subcultures are once secretly cultivated, and cultivation temperature is 28 ℃.
The differentiation of somatic cell embryoid: callus is inoculated in the dark cultivation of differential medium 40 days, and to promote differentiation, the maturation of somatic cell embryoid, cultivation temperature is 28 ℃.After 40 days, the friable embryogenic callus differentiates the vigorous embryoid of growing way (wherein most of is cotyledon shape embryo, and few part is lopsided embryo), and one little friable embryogenic callus can differentiate the dozens of embryoid.Differential medium is MS medium and additional composition, and additional composition is 6-BA 0.5mg/L, KT 3.0mg/L, NAA0.02mg/L, GA
30.5mg/L, Phytagel 2.0g/L, sucrose 70g/L, activated carbon 1.0g/L and Sucus Cocois 50ml/L.
Plant regeneration: the cotyledon shape embryo of maturation is inoculated into the medium of emerging, illumination every day 16 hours, with short its whole plant of regenerating, cultivation temperature is 30 ℃.Cultivate after 10 days, leaf embryo begins to sprout, and after 20 days, grows up to the plantlet of complete stalwartness.The active ingredient of medium of emerging is MS medium and additional composition, and additional composition is KT 1.5mg/L, IAA 0.02mg/L, GA
32.0mg/L, Phytagel 2.0g/L, sucrose 50g/L, activated carbon 1.0g/L and Sucus Cocois 50ml/L.
Para rubber tree cells,primordial suspension system in above-mentioned utilizes the conventional method preparation.
Embodiment two:
The preparation of explant: choose heat and grind the 7-33-97 monokaryon and keep to the side the bud of phase,, use 1g/L HgCl again with 75% alcohol surface sterilization 60 seconds as explant
2Sterilized 10 minutes, and used aseptic water washing at last 3 times, each 5 minutes.
Embryonic callus induction: the stamen that strips out in the bud that disinfects is inoculated on the embryonic callus induction medium in 28 ℃ of dark cultivations 40 days.The effective ingredient of said embryonic callus induction medium is the MS medium and the additional composition of improvement; The composition that the MS medium is revised is: epsom salt 500mg/L; Potassium dihydrogen phosphate 400mg/L, anhydrous calcium chloride 250mg/L, four water manganese sulphate 35mg/L (following steps are together); Additional composition is 2,4-D 2.0mg/L, KT1.0mg/L, NAA 1.0mg/L, Phytagel 2.0g/L, sucrose 70g/L and Sucus Cocois 50ml/L.
The subculture of embryo callus: change the embryo callus of inducing over to fresh embryonic callus induction medium successive transfer culture 4 times, 7 days subcultures once.
Embryo callus suspension culture: embryo callus in good condition behind the subculture is broken into the 0.3cm particle; Getting 3g embryo callus particle is inoculated in the triangular flask that contains the 20mL suspending nutrient solution; Place on 100r/ minute the shaking table, the dark cultivation, cultivation temperature is 28 ℃.7 days subcultures once change the small cell cluster of the 2g new dispersion that form in new triangular flask when changing culture fluid at every turn and cultivate.Behind the subculture 5 times, promptly obtain the cells,primordial suspension system.Said suspending nutrient solution is the MS medium and the additional composition of improvement, and additional composition is 2,4-D 2.0mg/L, KT 1.0mg/L, NAA 1.0mg/L, sucrose 40g/L and Sucus Cocois 50ml/L.
All the other are identical with embodiment one.
Claims (3)
1. a method of utilizing Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system is characterized in that it comprises the steps:
A, embryonal suspension cell are induced the friable embryogenic callus: with liquid-transfering gun the culture fluid in the Para rubber tree cells,primordial suspension system is blotted; Choosing small cell cluster is inoculated into the cells,primordial suspension system and induces on the medium of friable embryogenic callus; The dark cultivation 15～30 days; Cultivation temperature is between 25 ℃～28 ℃; It is said that to induce the medium of friable embryogenic callus be MS medium and additional composition; Content in the MS medium is potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, and supplementary element is 2,4-D 0.5～3.0mg/L, 6-BA 0.1～2.0mg/L, NAA 0.5～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L and Sucus Cocois 30～80ml/L;
B, friable embryogenic callus subculture: the friable embryogenic callus is changed over to successive transfer culture on the medium of inducing the friable embryogenic callus, 15～30 days subcultures are once secretly cultivated, and cultivation temperature is between 25 ℃～28 ℃;
The differentiation of c, somatic cell embryoid: the friable embryogenic callus is inoculated in differential medium; The dark cultivation 20～50 days; To promote differentiation, the maturation of somatic cell embryoid; Cultivation temperature is between 25 ℃～28 ℃; Said differential medium is MS medium and additional composition, and the content in the MS medium is adjusted into potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, and additional composition is 6-BA 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.01～1.0mg/L, GA
30.01～1.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L, activated carbon 1～2g/L and Sucus Cocois 30～80ml/L;
D, plant regeneration: ripe embryoid is inoculated into the medium of emerging; Illumination cultivation; With short its whole plant of regenerating; Cultivation temperature is between 25 ℃～30 ℃; The active ingredient of the said medium of emerging is MS medium and additional composition, and the content in the MS medium is adjusted into potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L, and supplementary element is KT 0.5～3.0mg/L, NAA 0.01～1.0mg/L, GA
30.1～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L, activated carbon 1～2g/L and Sucus Cocois 30～80ml/L.
2. the method for utilizing Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system according to claim 1 is characterized in that, the Para rubber tree cells,primordial suspension system of step a adopts following method to obtain:
The preparation of a, explant: choose the Para rubber tree monokaryon and keep to the side the bud of phase, carry out disinfection as explant;
B, embryonic callus induction: the stamen that strips out in the bud that disinfects is inoculated in the embryonic callus induction medium, secretly cultivates 30～50 days, and cultivation temperature is between 24 ℃～30 ℃; Said embryonic callus induction medium is MS medium and additional composition; Content in the MS medium is potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L; Supplementary element is 2,4-D 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.5～3.0mg/L, Phytagel 2～3g/L, sucrose 50～90g/L and Sucus Cocois 30～80ml/L;
The subculture of c, embryo callus: change embryo callus over to fresh embryonic callus induction medium successive transfer culture 3～5 times, 7～10 days subcultures are once secretly cultivated, and cultivation temperature is between 25 ℃～28 ℃;
D, embryo callus suspension culture: embryo callus in good condition behind the subculture is broken into 0.1～0.5 cm particle; Getting 2～5 g embryo callus particles is inoculated in the triangular flask that contains 20～50 mL suspending nutrient solutions; Place on 80～120r/ minute the shaking table, the dark cultivation, cultivation temperature is between 25 ℃～28 ℃; 7～9 days subcultures once; When changing culture fluid the small cell cluster of the new dispersion that forms of 1～2 g changed in the new triangular flask at every turn and cultivates, behind the subculture 4～6 times, promptly obtain Para rubber tree cells,primordial suspension system; Said suspending nutrient solution is MS medium and additional composition; Content in the MS medium is potassium dihydrogen phosphate 400～550mg/L, anhydrous calcium chloride 200～450mg/L, four water manganese sulphate 20～45mg/L and epsom salt 450～650mg/L; Supplementary element is 2,4-D 0.5～3.0mg/L, KT 0.5～3.0mg/L, NAA 0.5～3.0mg/L, sucrose 30～60g/L and Sucus Cocois 30～80ml/L.
3. the method for utilizing Para rubber tree cells,primordial suspension system to set up efficient plant regeneration system according to claim 2 is characterized in that, in step a, earlier with 75～80% alcohol surface sterilizations 30～60 seconds, uses 1g/L HgCl again
2Sterilized 10～15 minutes, and used aseptic water washing at last 3～6 times, each 3～5 minutes.
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| CN102676574A (en) * | 2012-04-19 | 2012-09-19 | 中国热带农业科学院橡胶研究所 | Agrobacterium mediated method for obtaining transgenic plant of hevea brasiliensis |
| WO2014017513A1 (en) * | 2012-07-24 | 2014-01-30 | 日産化学工業株式会社 | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN104031936A (en) * | 2014-06-16 | 2014-09-10 | 中国热带农业科学院橡胶研究所 | Method of promoting generation of lateral buds of Heveabrasiliensis by trans-AtWUS (Arabidopsisthaliana WUSCHEL) gene |
| CN108243960B (en) * | 2018-02-06 | 2021-03-19 | 江苏农林职业技术学院 | High-frequency somatic embryo regeneration culture medium without germplasm genotype limitation and application thereof |
| CN112841037B (en) * | 2021-03-31 | 2022-04-15 | 中国热带农业科学院橡胶研究所 | Embryogenic callus induction culture medium for rubber tree, method for inducing somatic embryogenesis of rubber tree and application |
| CN114717175B (en) * | 2022-04-11 | 2024-07-16 | 中国热带农业科学院橡胶研究所 | Method for establishing embryogenic cell line of rubber tree with high embryogenic capacity and maintaining embryogenic morphology of embryogenic cell line |
| CN116439137B (en) * | 2023-06-02 | 2024-03-12 | 中国热带农业科学院橡胶研究所 | Method for improving self-rooting young clone propagation efficiency of Brazilian rubber tree through ultrasonic treatment |
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| CN101485290A (en) * | 2008-01-17 | 2009-07-22 | 中国热带农业科学院橡胶研究所 | Method for establishing rubber tree internal integument regeneration system |
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| CN101180948A (en) * | 2007-12-14 | 2008-05-21 | 中山大学 | Method for establishing high-efficiency somatic cell embryogenesis regeneration plant of plantain banana and Brazilian banana |
| CN101485290A (en) * | 2008-01-17 | 2009-07-22 | 中国热带农业科学院橡胶研究所 | Method for establishing rubber tree internal integument regeneration system |
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