CN103858773B - Take anthocaulus as Ornithogalum caudatum's rapid propagation in vitro method of explant - Google Patents
Take anthocaulus as Ornithogalum caudatum's rapid propagation in vitro method of explant Download PDFInfo
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- CN103858773B CN103858773B CN201410143767.XA CN201410143767A CN103858773B CN 103858773 B CN103858773 B CN 103858773B CN 201410143767 A CN201410143767 A CN 201410143767A CN 103858773 B CN103858773 B CN 103858773B
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Abstract
The invention belongs to flowers propagation method, be specifically related to the asexual rapid propagation method of flowers.With the young tender anthocaulus of Ornithogalum caudatum for explant, dedifferentiation frequency and differentiation rate all can reach more than 70% ~ 75%.The Multiple Buds of strain more than 300 can be differentiated through subculture repeatedly by the callus of one piece of explant induction.Multiple Buds rooting rate can reach more than 90%, transplants survival rate more than 95% under control environment.Make Ornithogalum caudatum's large-scale breeding within the relatively short time go out the neat plant of outward appearance, Variations of Regenerated Plants is low; Regenerating system is very efficient; The cost of average individual plant regrowth is extremely low, is applicable to the in enormous quantities efficient Fast-propagation of Ornithogalum caudatum, is highly suitable for factorial praluction.
Description
Technical field
The invention belongs to flowers propagation method, be specifically related to the asexual rapid propagation method of flowers.
Background technology
Ornithogalum caudatum, another name Urginea maritima, bird breast flower.Be a kind of herbaceos perennial of Liliaceae Rohdea, originate in south, Africa.Bulb ovoid, green, diameter can reach 10 centimetres.Leaf 5-6 piece, banded or strip lanceolar, long 30-60 centimetre, wide 2.5-5 centimetre, also have very high ornamental value as a kind of ornamental flower.Containing OSW-1 (being called for short OSW-1) in Ornithogalum caudatum, in physiologically active screening experiment, find the cancer therapy drug strong ten times even hundred times such as taxol, adriamycin, camptothecine of its Anticancer Activity in vitro than Clinical practice through forefathers.And toxicity is not had to normal cell.From Ornithogalum caudatum, isolated saponin OSW-1 has obvious active anticancer to lung cancer, breast cancer, be common cancer therapy drug camptothecine, adriamycin, 10 ~ 100 times of taxol active anticancer.This surprising discovery will mean that Ornithogalum caudatum likely replaces the medicines such as taxol, adriamycin, camptothecine, and economic worth is very huge.The propagation method of current Ornithogalum caudatum is mainly based on bulb separation and seed propagation, although more easily survive, a maturation plants ball, annual bulb separation is less than 10, and reproductive efficiency is very low, and seed propagation emergence rate is not high, breeding cycle is long, about about 3 years, greatly have impact on production efficiency.Plant Tissue Breeding and Vitro Plant culture technique, the organ (as root, stem, leaf, stem apex, flower, fruit etc.) utilizing plant corpus in vitro, tissue (as formed layer, epidermis, cortex, endosperm etc.) or cell (as megaspore, microspore, somatic cell etc.) and protoplast, under aseptic and suitable synthetic medium and the artificial condition such as illumination, temperature, the technology that the complete plant of callus, indefinite bud, adventive root, formation or generation have the other products of economic worth can be induced.The regeneration plant produced by Plant Tissue Breeding substantially can keep the merit of parent's (explant source plants) except extremely low variation.Make great progress since plant tissue culture technique invention.Utilize plant tissue culture technique Vitro Quick Reproduction Ornithogalum caudatum can produce the unified plant of plant type on a large scale, Fast-propagation Ornithogalum caudatum, meet the demand of domestic and international market further.Although Ornithogalum caudatum's tissue culture experiments that forefathers do tentatively succeeds, usually adopt bulb to be that explant carries out callus induction, due to use bulb for explant leave is taken root comparatively fast, be unfavorable for squamous subculture, reproduction coefficient is lower.Which limits the application of Ornithogalum caudatum's tissue culture technique in actual production.And the present invention can make Ornithogalum caudatum realize Fast-propagation within 1 year, be very applicable to factorial praluction.
Summary of the invention
The object of the invention is the Traditional breeding processes for overcoming Ornithogalum caudatum, relying on the breeding of nature bulb separation, the shortcoming that reproduction speed is slow; Overcome in forefathers Ornithogalum caudatum tissue culture experiments that differentiation rate is low, gain factor is little, be not suitable for the shortcoming of factorial praluction.A kind of Vitro Quick Reproduction technology being explant with Ornithogalum caudatum anthocaulus is provided.
The technical solution adopted in the present invention is:
1. explant inoculation and callus induction: Ornithogalum caudatum's titbit lower end tender anthocaulus of children is cut off and takes off, anthocaulus tender for children is put into aseptic super-clean bench, by 75% (volume/volume) alcohol-pickled 1 ~ 2 minute, soak 5 ~ 10 minutes with the mercuric chloride solution of 0.1 ~ 0.2% (mass/volume), aseptic water washing 3 ~ 5 times.Anthocaulus tender for children is cut to the fritter of length 3 ~ 5mm, be seeded in containing 6-BA2 ~ 2.5mg/L, IBA0.1 ~ 0.2mg/L, sucrose 3.0 ~ 4.5% (mass/volume), pH value is on the MS medium of 5.5 ~ 6.5,20 DEG C ~ 30 DEG C every day fluorescent lamp irradiate 4 ~ 6 hours, intensity of illumination 1500 ~ 2000Lux.25 ~ 30 days subcultures once, even if can be differentiation-inducing in time growing yellow green callus.
2. the induction of indefinite bud: yellowish green callus is transferred to the MS inductive differentiation medium containing NAA (0.1 ~ 0.5mg/L)+KT (1.5 ~ 2.5mg/L)+agar (5 ~ 7g/L)+3.5 ~ 4.5% sucrose (mass/volume), pH value is 5.5 ~ 6.5,40 ~ 45 days subcultures once, 20 DEG C ~ 30 DEG C fluorescent lamp illumination every day 8 ~ 10 hours, intensity of illumination 1500 ~ 2000Lux.Sustainable more than 1 year of this callus differentiation capability, can differentiate the regrowth of strain more than 300 from one piece of callus through subculture repeatedly.
3. induction piece: aftergrowth forwards 1/2MS+NAA (1 ~ 2mg/L)+agar (5 ~ 7g/L)+sucrose 2% (mass/volume) to, pH value is on the root media of 5.5 ~ 6.5,20 DEG C ~ 30 DEG C fluorescent lamp illumination 12 every day hours, intensity of illumination 1500 ~ 2000Lux.
4. Transplantation of Regenerated Plantlets: can transplant after plant takes root, transplant to the peat composed of rotten mosses: vermiculite: fine sand=3: in the planting matrix of 1: 1, before transplanting, the foreign material such as the agar on plant are rinsed well, planting matrix 121 DEG C of sterilizings 30 ~ 45 minutes, within 8 ~ 12 days, water once and permeablely just can to survive.
The invention has the beneficial effects as follows: with the young tender anthocaulus of Ornithogalum caudatum for explant, dedifferentiation frequency and differentiation rate all can reach more than 70% ~ 75%.The Multiple Buds of strain more than 300 can be differentiated through subculture repeatedly by the callus of one piece of explant induction.Multiple Buds rooting rate can reach more than 90%, transplants survival rate more than 95% under control environment.Make Ornithogalum caudatum's large-scale breeding within the relatively short time go out the neat plant of outward appearance, Variations of Regenerated Plants is low; Regenerating system is very efficient; The cost of average individual plant regrowth is extremely low, is applicable to the in enormous quantities efficient Fast-propagation of Ornithogalum caudatum, is highly suitable for factorial praluction.
Embodiment
Below in conjunction with example, the present invention is described further, but content of the present invention is not limited only to following 2 embodiments:
Embodiment one:
1. explant inoculation and callus induction: Ornithogalum caudatum's titbit lower end tender anthocaulus of children is cut off and takes off, anthocaulus tender for children is put into aseptic super-clean bench, by 75% (volume/volume) alcohol-pickled 1 minute, 5 minutes are soaked, aseptic water washing 5 times with the mercuric chloride solution of 0.2% (mass/volume).Anthocaulus tender for children is cut to the fritter of length 3 ~ 5mm, be seeded in containing 6-BA2mg/L, IBA0.15mg/L, sucrose 3.5% (mass/volume), pH value is on the MS medium of 6.2,20 DEG C ~ 30 DEG C every day fluorescent lamp irradiate 4 ~ 6 hours, intensity of illumination 1500 ~ 2000Lux.25 days subcultures once, even if can be differentiation-inducing in time growing yellow green callus.
2. the induction of indefinite bud: yellowish green callus is transferred to the MS inductive differentiation medium containing NAA (0.15mg/L)+KT (2.0mg/L)+agar (7g/L)+4.0% sucrose (mass/volume), pH value is 6.1,40 days subcultures once, 20 DEG C ~ 30 DEG C fluorescent lamp illumination every day 8 ~ 10 hours, intensity of illumination 1500 ~ 2000Lux.Sustainable more than 1 year of this callus differentiation capability, can differentiate the regrowth of 315 strains from one piece of callus through subculture repeatedly.
3. induction piece: aftergrowth forwards 1/2MS+NAA (2mg/L)+agar (6g/L)+sucrose 2% (mass/volume) to, pH value is on the root media of 6.0,20 DEG C ~ 30 DEG C fluorescent lamp illumination 12 every day hours, intensity of illumination 1500 ~ 2000Lux.
4. Transplantation of Regenerated Plantlets: can transplant after plant takes root, transplant to the peat composed of rotten mosses: vermiculite: fine sand=3: in the planting matrix of 1: 1, before transplanting, the foreign material such as the agar on plant are rinsed well, planting matrix 121 DEG C of sterilizings 40 minutes, within 12 days, water once and permeablely just can to survive.
Embodiment two:
1. explant inoculation and callus induction: Ornithogalum caudatum's titbit lower end tender anthocaulus of children is cut off and takes off, anthocaulus tender for children is put into aseptic super-clean bench, by 75% (volume/volume) alcohol-pickled 2 minutes, 10 minutes are soaked, aseptic water washing 5 times with the mercuric chloride solution of 0.1% (mass/volume).Anthocaulus tender for children is cut to the fritter of length 3 ~ 5mm, be seeded in containing 6-BA2.2mg/L, IBA0.1mg/L, sucrose 3.8% (mass/volume), pH value is on the MS medium of 5.8,20 DEG C ~ 30 DEG C every day fluorescent lamp irradiate 4 ~ 6 hours, intensity of illumination 1500 ~ 2000Lux.28 days subcultures once, even if can be differentiation-inducing in time growing yellow green callus.
2. the induction of indefinite bud: yellowish green callus is transferred to the MS inductive differentiation medium containing NAA (0.4mg/L)+KT (2.0mg/L)+agar (5g/L)+3.5% sucrose (mass/volume), pH value is 5.5,45 days subcultures once, 20 DEG C ~ 30 DEG C fluorescent lamp illumination every day 8 ~ 10 hours, intensity of illumination 1500 ~ 2000Lux.Sustainable more than 1 year of this callus differentiation capability, can differentiate the regrowth of 358 strains from one piece of callus through subculture repeatedly.
3. induction piece: aftergrowth forwards 1/2MS+NAA (1.5mg/L)+agar (5g/L)+sucrose 2% (mass/volume) to, pH value is on the root media of 5.6,20 DEG C ~ 30 DEG C fluorescent lamp illumination 12 every day hours, intensity of illumination 1500 ~ 2000Lux.
4. Transplantation of Regenerated Plantlets: can transplant after plant takes root, transplant to the peat composed of rotten mosses: vermiculite: fine sand=3: in the planting matrix of 1: 1, before transplanting, the foreign material such as the agar on plant are rinsed well, planting matrix 121 DEG C of sterilizings 35 minutes, within 10 days, water once and permeablely just can to survive.
Claims (1)
1. be Ornithogalum caudatum's rapid propagation in vitro method of explant with anthocaulus, it is characterized in that comprising the following steps:
1) explant inoculation and callus induction: Ornithogalum caudatum's inflorescence lower end tender anthocaulus of children is cut off and takes off, anthocaulus tender for children is put into aseptic super-clean bench, by 75% (volume/volume) alcohol-pickled 1 ~ 2 minute, soak 5 ~ 10 minutes with the mercuric chloride solution of 0.1 ~ 0.2% (mass/volume), aseptic water washing 3 ~ 5 times; Anthocaulus tender for children is cut to the fritter of length 3 ~ 5mm, be seeded in containing 6-BA2 ~ 2.5mg/L, IBA0.1 ~ 0.2mg/L, sucrose 3.0 ~ 4.5% (mass/volume), pH value is on the MS medium of 5.5 ~ 6.5, cultivation temperature 20 DEG C ~ 30 DEG C, every day, fluorescent lamp irradiated 4 ~ 6 hours, intensity of illumination 1500 ~ 2000Lux; 25 ~ 30 days subcultures once, can be differentiation-inducing in time growing yellow green callus;
2) induction of indefinite bud: yellowish green callus is transferred to the NAA containing 0.1 ~ 0.5mg/L, the KT of 1.5 ~ 2.5mg/L, the agar of 5 ~ 7g/L, the MS inductive differentiation medium of 3.5 ~ 4.5% (mass/volume) sucrose, pH value is 5.5 ~ 6.5,40 ~ 45 days subcultures once, cultivation temperature 20 DEG C ~ 30 DEG C, fluorescent lamp illumination 8 ~ 10 hours every day, intensity of illumination 1500 ~ 2000Lux, sustainable more than 1 year of this callus differentiation capability, can differentiate the regrowth of strain more than 300 from one piece of callus through subculture repeatedly;
3) induction of root: aftergrowth forwards the sucrose of the agar+2% (mass/volume) of the NAA+5 ~ 7g/L of 1/2MS+1 ~ 2mg/L to, pH value is on the root media of 5.5 ~ 6.5, cultivation temperature 20 DEG C ~ 30 DEG C, fluorescent lamp illumination 12 every day hours, intensity of illumination 1500 ~ 2000Lux;
4) Transplantation of Regenerated Plantlets: can transplant after plant takes root, transplant to the peat composed of rotten mosses: vermiculite: fine sand=3: in the planting matrix of 1: 1, before transplanting, the foreign material such as the agar on plant are rinsed well, planting matrix 121 DEG C of sterilizings 30 ~ 45 minutes, within 8 ~ 12 days, water once and permeablely just can to survive.
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| CN105815214B (en) * | 2016-03-21 | 2018-01-02 | 云南省农业科学院农业环境资源研究所 | A kind of blade seedling rapid propagation method of Ornithogalum caudatum |
| CN106069756B (en) * | 2016-06-16 | 2018-04-06 | 四川天艺优境环境科技有限公司 | A kind of quick breeding method for tissue culture of white flower Ornithogalum caudatum |
| CN106508684B (en) * | 2016-12-08 | 2018-08-03 | 云南省热带作物科学研究所 | A kind of South Africa Star of Bethlehem in-vitro culture method |
| CN109430059A (en) * | 2018-12-25 | 2019-03-08 | 云南省农业科学院花卉研究所 | A kind of cut-flower Ornithogalum caudatum in vitro culture quick-breeding method |
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