CN105052745A - Method for promoting growth of echinacea purpurea regeneration buds - Google Patents
Method for promoting growth of echinacea purpurea regeneration buds Download PDFInfo
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Abstract
The invention belongs to the field of plant biological technologies, and particularly discloses a method for promoting growth of echinacea purpurea regeneration buds. According to the method, according to the biomass of the regeneration buds, the regeneration buds are placed in a growth medium of different DA-6 concentrations to be cultivated, adventitious buds of different biomasses can root well, and when the echinacea purpurea regeneration buds are diploid, the content of DA-6 in the growth medium is 0.08-0.16 mg/L, wherein the adhesion leaf height is 1.5 cm-2.5 cm; when the echinacea purpurea regeneration buds are diploid or triploid, the content of DA-6 in the growth medium is 0.32 mg/L-0.64 mg/L, wherein the adhesion leaf height is 4 cm; when the echinacea purpurea regeneration buds are tetraploid, the content of DA-6 in the growth medium is 0.64 mg/L-1.28 mg/L, wherein the adhesion leaf height of the echinacea purpurea regeneration buds is 4 cm. The height and the root number of the regeneration buds can be increased obviously, and development of related scientific research in echinacea biotechnology seed breeding can be promoted.
Description
Technical field
The invention belongs to plant biotechnology field, particularly, relate to a kind of method that Echinacea regeneration bud grows that promotes.
Background technology
Echinacea (
echinaceapurpureal.) be composite family herbaceos perennial, have good immunoregulation effect, its extract is a kind of immunopotentiating agent and the immunomodulator that are subject to most attention in recent years in the world.Due to the efficacy effect that it is definite, Echinacea has become a kind of world-renowned medicinal plant.People, in order to obtain the Echinacea of multi items, adopt tissue culture technique to carry out regenerating or regenerate clone usually.Utilize tissue culture technique regenerate plant or regenerate clone, usually comprise two steps.The first step produces indefinite bud from explant induction.Second step is that regeneration bud grows into whole plant, in order to transplanting.For Echinacea, in prior art, existing a lot of its explant induction of report research produces indefinite bud; And as how impelling these regeneration bud well-growns, also need more research.
The present inventor is after successfully obtaining some special Echinacea germ plasm resources, the efficiency of taking root of the regeneration bud of the Special germplasm resource found that there is is lower, cause its transplanting survival rate usually lower, reason is the negligible amounts of the plant tip of a root that conventional method is restored.
Diethylamino ethanol caproate (DA-6) is a plant growth regulators, is generally used for field cultivation, is rarely used in tissue cultures, and inventor studies the growth finding that it can promote Echinacea regeneration bud in the early time.But this facilitation of the same display of research is not completely relevant to gene dosage, also has certain lability.For same germplasm materials, be applicable on the long medium of other blastogenesis, the growth of the regeneration bud that part biological amount is less is but suppressed.For addressing this problem, the relation between the effect that inventors have investigated DA-6 and the initial biomass of regeneration bud cultivated.
Summary of the invention
The present invention is in order to overcome in prior art, and the problem that the Echinacea regeneration bud that biomass differs growing way in containing the medium of same concentrations DA-6 differs, provides a kind of method that Echinacea regeneration bud grows that promotes.
Above-mentioned purpose of the present invention is achieved by the following technical programs.
Promote the method that Echinacea regeneration bud grows, the growth medium that the indefinite bud of Echinacea is placed in containing DA-6 is cultivated; Described growth medium is that the Echinacea regeneration bud for different biomass also added the DA-6 of following corresponding content in MS formula except adding 15 ~ 60g/L sucrose, 3 ~ 9g/L agar and 0.01 ~ 0.2mg/LNAA:
When Echinacea regeneration bud is the dliploid connecting the high 1.5 ~ 2.5cm of leaf, in described medium, the content of DA-6 is 0.08 ~ 0.16mg/L;
When Echinacea regeneration bud is dliploid or the triploid connecting leaf height 4cm, in described medium, the content of DA-6 is 0.32 ~ 0.64mg/L;
When Echinacea regeneration bud is the tetraploid connecting leaf height 4cm, in described medium, the content of DA-6 is 0.64 ~ 1.28mg/L.
For a regeneration bud, make it to grow up to a whole plant, key is to induce adventive root and makes these adventive root well-growns.Inventor in the early time result of study display adds DA-6 and contributes to promoting the growth conditions of regeneration bud, promotes the quantity of its adventive root, thus better reaches and to preserve special planting material and to breed object.The object of the invention is to, according to the regeneration bud of different biomass to the difference of the susceptibility of DA-6, correspondingly add the DA-6 of variable concentrations, inventor finds through great many of experiments, according to the biomass of regeneration bud, the DA-6 adding appropriate concentration in the growth medium of regeneration bud can make the sprouting quantity of the growth conditions of regeneration bud, adventive root be promoted significantly.
In addition, as long as dliploid source, no matter be petiole, the indefinite bud that grows of blade or root explant, the same to the concentration requirement of 6-DA.
For promoting to produce flourishing root system, preferably, when Echinacea regeneration bud is the dliploid of 1.5cm, in described medium, the content of DA-6 is 0.08mg/L.
For promoting to produce flourishing root system, preferably, when Echinacea regeneration bud is the dliploid of 2.5cm, in described medium, the content of DA-6 is 0.16mg/L.
For promoting growing tall and producing flourishing root system of plant, preferably, when Echinacea regeneration bud is the tetraploid of blade 6-8, even leaf height 4cm, in described medium, the content of DA-6 is 0.64mg/L.
For promoting growing tall of plant, preferably, when Echinacea regeneration bud is dliploid or the triploid of blade 6-8, even leaf height 4cm, in described medium, the content of DA-6 is 0.32mg/L.
For promoting to produce flourishing root system, preferably, when Echinacea regeneration bud is dliploid or the triploid of blade 6-8, even leaf height 4cm, in described medium, the content of DA-6 is 0.64mg/L.
Preferably, being seeded in corresponding medium by Echinacea petiole explant regeneration bud, is 25 ± 4 DEG C in cultivation temperature, and intensity of illumination is 1000 ~ 2500lx, and light application time is cultivate 10 ~ 60d under the condition of 10 ~ 16h/d.
Compared with prior art, beneficial effect of the present invention is:
Under the invention solves original technology, the regeneration bud that in the Echinacea regeneration bud grown cultures of different biomass, part biological amount is less grow the outstanding problem be suppressed, for the biotechnology research of Echinacea, particularly for producing seed but the seedling with the Special germplasm material that higher plantation is worth is cloned and had important can being direct appliedly worth.
Accompanying drawing explanation
Fig. 1 is the impact of different DA-6 concentration on the growth of the dliploid regeneration bud of less and medium biomass; Wherein, 1 ~ 5 be respectively with 0,0.01,0.08,0.16, dliploid regeneration bud that the DA-6 process biomass of 0.32mg/L is less; 6 ~ 10 be respectively with 0,0.01,0.08,0.16, dliploid regeneration bud that the DA-6 process biomass of 0.32mg/L is larger.
Fig. 2 is that different DA-6 concentration is on the impact grown compared with mcroorganism amount dliploid, triploid and tetraploid regeneration bud; Wherein, 1 ~ 5 be respectively with 0,0.16,0.32,0.64, the dliploid regeneration bud of the DA-6 process of 1.28mg/L; 6 ~ 10 be respectively with 0,0.16,0.32,0.64, the triploid regeneration bud of the DA-6 process of 1.28mg/L; 11 ~ 15 be respectively with 0,0.16,0.32,0.64, the tetraploid regeneration bud of the DA-6 process of 1.28mg/L.
Embodiment
Below in conjunction with Figure of description and specific embodiment, the present invention is described in further details, but embodiment does not limit in any form the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Vegetable material: dliploid Echinacea of the present invention is obtained by seed; The preparation method of tetraploid Echinacea can with reference to NilanthiD, ChenXL, ZhaoFC,
etal.Inductionoftetraploidsfrompetioleexplantsthroughcolchic inetreatmentsin
echinaceapurpureal [J] .BioMedResearchInternational, 2009.Triploid Echinacea is obtained Diploid and Tetraploid hybridization.The ploidy of these plant is all identified by root tip chromosomes counting technology.
embodiment 1
The present embodiment have studied the impact of different DA-6 concentration on the growth of the dliploid regeneration bud of less and medium biomass:
S1. the preparation of regeneration bud: first obtain Echinacea petiole explant, petiole explant is from the Echinacea dliploid aseptic seedling preparation of the explant (can with reference to this area convenient technical process) under conditions of tissue culture, then petiole explant to be seeded on regeneration culture medium that (regeneration culture medium is for containing 30g/L sucrose, 0.3mg/LBA, 0.01mg/LNAA, agar solidified with 4.5g/L, at 1.4kgcm
-2the MS medium of lower sterilizing 20min).When regeneration bud grows to Lian Yegaoyue 1.5,2.5cm, bud is scaled off from explantation tissue, just can as the present invention's material---regeneration bud used.
S2. the cultivation of regeneration bud: the bud of the Lian Yegaoyue 1.5 obtained from dliploid Echinacea petiole explant described in step S1,2.5cm is cut out, be inoculated into the addition of variable concentrations DA-6(0,0.08,0.16,0.32,0.64mg/L) growth medium on (growth medium is for containing 30g/L sucrose, 0.01mg/LNAA, agar solidified with 4.5g/L, at 1.4kgcm
-2the MS medium of lower sterilizing 20min), check the situation of regeneration bud growth.
From table 1 and Fig. 1, when root induction, add appropriate DA-6 and help lend some impetus to the root system producing prosperity, when Echinacea regeneration bud is the dliploid of 1.5 ~ 2.5cm, medium containing 0.08 ~ 0.16mg/LDA-6 more can improve tip of a root sum compared to other concentration, and optium concentration is relevant with the initial biomass of regeneration bud.The bud less to biomass, namely dliploid regeneration bud is 1.5cm, and the DA-6 concentration that obtain required for optimum growh effect is lower, is 0.08mg/L, when the addition of DA-6 exceedes optium concentration, is just easy to delay even to suppress it to take root.The bud larger to biomass, namely dliploid regeneration bud is 2.5cm, and the DA-6 concentration reached required for best rooting efficiency is also higher, is 0.16mg/L, and the DA-6 concentration that can tolerate is also higher.
Table 1DA-6 affects the effect of the regeneration bud sprouting adventive root of low, middle biomass
* in table after every columns value different letter representation in Duncan's multiple comparison significant difference (
p<0.05).
embodiment 2
The present embodiment have studied different DA-6 concentration to the impact compared with the dliploid of mcroorganism amount, triploid and the growth of tetraploid regeneration bud:
S1. the preparation of regeneration bud: first obtain Echinacea petiole explant, petiole explant is from the Echinacea dliploid under conditions of tissue culture, triploid, the tetraploid aseptic seedling preparation of the explant (can with reference to this area convenient technical process), then petiole explant to be seeded on regeneration culture medium that (regeneration culture medium is for containing 30g/L sucrose, 0.3mg/LBA, 0.01mg/LNAA, agar solidified with 4.5g/L, at 1.4kgcm
-2the MS medium of lower sterilizing 20min).When regeneration bud grows to Lian Yegaoyue 4cm, bud is scaled off from explantation tissue, just can as the present invention's material---regeneration bud used.
S2. the cultivation of regeneration bud: the bud of the Lian Yegaoyue 4cm obtained from dliploid, triploid, tetraploid Echinacea petiole explant described in step S1 is cut out, be inoculated into the addition of variable concentrations DA-6(0,0.16,0.32,0.64,1.28mg/L) growth medium on (growth medium is for containing 30g/L sucrose, 0.01mg/LNAA, agar solidified with 4.5g/L, at 1.4kgcm
-2the MS medium of lower sterilizing 20min), check the situation of regeneration bud growth.
The condition of culture of adventitious bud inducing regeneration is identical with embodiment 1.Data processing is identical with embodiment 1.
From table 2 and Fig. 2, in root induction and promotion plant strain growth, when Echinacea regeneration bud is dliploid or the triploid of 4cm, the medium containing 0.32 ~ 0.64mg/LDA-6 more comprehensively can improve plant height and tip of a root sum compared to other concentration; When Echinacea regeneration bud is the tetraploid of 4cm, the medium containing 0.64 ~ 1.28mg/LDA-6 more comprehensively can improve plant height and tip of a root sum compared to other concentration.Regeneration bud for above-mentioned initial biomass comparatively large (6 ~ 8, blade, even leaf height 4cm) will obtain maximum tip of a root number, and the optium concentration of adding DA-6 in dliploid, triploid or tetraploid medium is 0.64mg/L; To obtain the effect of best raising plant height, the optium concentration of adding DA-6 in dliploid or triploid medium is 0.32mg/L, and the optium concentration of adding DA-6 in tetraploid medium is 0.64mg/L.
Table 2DA-6 affects the effect grown compared with the regeneration bud of high-biomass
* in table after every columns value different letter representation in Duncan's multiple comparison significant difference (
p<0.05).
Claims (6)
1. promote to it is characterized in that the method that Echinacea regeneration bud grows, the growth medium that Echinacea indefinite bud is placed in containing DA-6 is cultivated; Described growth medium is that the Echinacea regeneration bud for different biomass also added the DA-6 of following corresponding content in MS formula except adding 15 ~ 60g/L sucrose, 3 ~ 9g/L agar and 0.01 ~ 0.2mg/LNAA:
When Echinacea regeneration bud is the dliploid connecting the high 1.5 ~ 2.5cm of leaf, in described growth medium, the content of DA-6 is 0.08 ~ 0.16mg/L;
When Echinacea regeneration bud is dliploid or the triploid connecting leaf height 4cm, in described growth medium, the content of DA-6 is 0.32 ~ 0.64mg/L;
When Echinacea regeneration bud is the tetraploid connecting leaf height 4cm, in described growth medium, the content of DA-6 is 0.64 ~ 1.28mg/L.
2. method according to claim 1, is characterized in that, when Echinacea regeneration bud is the dliploid of 1.5cm, in described medium, the content of DA-6 is 0.08mg/L.
3. method according to claim 1, is characterized in that, when Echinacea regeneration bud is the dliploid of 2.5cm, in described medium, the content of DA-6 is 0.16mg/L.
4. method according to claim 1, is characterized in that, when Echinacea regeneration bud is the tetraploid of blade 6-8, even leaf height 4cm, in described medium, the content of DA-6 is 0.64mg/L.
5. method according to claim 1, is characterized in that, when Echinacea regeneration bud is dliploid or the triploid of blade 6-8, even leaf height 4cm, in described medium, the content of DA-6 is 0.32mg/L.
6. method according to claim 1, it is characterized in that, be seeded in corresponding medium by Echinacea petiole explant regeneration bud, is 25 ± 4 DEG C in cultivation temperature, intensity of illumination is 1000 ~ 2500lx, and light application time is cultivate 10 ~ 60d under the condition of 10 ~ 16h/d.
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| CN106258978A (en) * | 2016-08-30 | 2017-01-04 | 华南农业大学 | One step obtains culture medium and the tissue culture method thereof of complete Echinacea regrowth |
| EP3808172A1 (en) | 2019-10-14 | 2021-04-21 | University of Science and Technology | Method of obtaining sprouts by way of somatic embryogenesis from fragments of leaf blades with echinacea purpurea l. (moench) |
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| CN106106186A (en) * | 2016-08-17 | 2016-11-16 | 桂林伯林生物技术有限公司 | The root media of a kind of Cyclocarya paliurus Iljinskaja test tube Seedling and the tissue culture and rapid propagation method of Cyclocarya paliurus Iljinskaja |
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| EP3808172A1 (en) | 2019-10-14 | 2021-04-21 | University of Science and Technology | Method of obtaining sprouts by way of somatic embryogenesis from fragments of leaf blades with echinacea purpurea l. (moench) |
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Application publication date: 20151118 |