CN102144559B - Establishing method for high frequency regeneration system of hemarthria compressa - Google Patents
Establishing method for high frequency regeneration system of hemarthria compressa Download PDFInfo
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- CN102144559B CN102144559B CN201110041447A CN201110041447A CN102144559B CN 102144559 B CN102144559 B CN 102144559B CN 201110041447 A CN201110041447 A CN 201110041447A CN 201110041447 A CN201110041447 A CN 201110041447A CN 102144559 B CN102144559 B CN 102144559B
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Abstract
The invention discloses an establishing method for a high frequency regeneration system of hemarthria compressa. The technology scheme of the organization culture system for the hemarthria compressa in the prior art is not complete or clear, or even the method has not necessary of the defect of repeat reproducibility. The invention provides the method for establishing the high frequency regeneration system by taking the hemarthria compressa young stem or young leaves as the explant. The method comprises the following steps of: taking the young stem or young leaves of the plant as the explant for establishing the high frequency regeneration system of the hemarthria compressa according to the operating processes of callus inducing, callus subculture, callus differentiation training, regrowth propagation culture, regrowth root training and regrowth seedlings transplanting, and optimizing the selection of the explant and the medium components of each cultivating stage for raising the throughput and product quality of each stage of the regeneration system. The method has reliable principle; and the established high frequency regeneration system of the hemarthria compressa has the characteristics of fast growth, high repeatability, simple operation, easy control on condition, and the like.
Description
Technical field
The present invention relates to a kind of Hemarthria compressa high frequency regenerating system method for building up, belong to the plant regeneration field of passing through tissue culture technique in the agricultural cultivation.
Background technology
Hemarthria compressa (Hemarthria compressa (Linn.f.) R.Br) is that the grass family Hemarthria compressa belongs to herbaceos perennial, and stem is soft, the forage grass quality better, and livestock happiness food both can be herded utilization, but also hay curing.In addition, the Hemarthria compressa well developed root system, thick grass is thick close, and wide coverage also is good water and soil conservation plant.The plantation of Hemarthria compressa both can have been satisfied the needs of improvement of the ecological environment, promoted the important measures of sustainable development of animal husbandry especially.
Be published in " Chinese meadow journal " (2007; 29 (2): " foundation of Hemarthria compressa tissue culturing system " literary composition 50～53) discloses the Hemarthria compressa tissue culture technique; This technology is an explant with Hemarthria compressa stem section; Selecting the initial culture optimum formula for use is MS medium+6-BA1.5mg/L+NAA0.2mg/L, and the shoot proliferation culture medium prescription of regrowth is MS medium+6-BA1.5mg/L+NAA0.2/L, and the culture of rootage based formulas is 1/2MS medium+BA0.2mg/L.The disclosed operating process first step of the document starts cultivation and obtains callus; Second step promptly cultivated the shoot proliferation of regrowth; Lacked between two steps necessity that obtains the regrowth incubation from callus has been described, therefore disclosed technical scheme and imperfect.And the disclosed Hemarthria compressa tissue culture technique of the document can not repetition, and just there is technical difficulty in the operation that the first step starts the cultivation stage evoked callus, can't effectively accomplish successor operation.
Summary of the invention
The object of the invention is exactly the deficiency to prior art, and a kind of Hemarthria compressa high frequency regenerating system method for building up is provided, this method good reproducibility, regeneration rate height.For realizing above-mentioned purpose, technical scheme of the present invention is following:
A kind of Hemarthria compressa high frequency regenerating system method for building up carries out according to the operating process of step S1 callus induction, step S2 callus successive transfer culture, step S3 callus differentiation culture, step S4 regrowth enrichment culture, step S5 regrowth culture of rootage, step S6 regrowth acclimatization and transplants; In step S1 callus induction, use and start medium; In step S2 callus successive transfer culture, use subculture medium; In step S3 callus differentiation culture, use the callus differential medium; In step S4 regrowth enrichment culture, use the regrowth proliferated culture medium, in step S5 regrowth culture of rootage, use root media; It is characterized in that: young stem or spire with Hemarthria compressa are explant.
The high frequency regenerating system of plant is to be main means with plant tissue culture technique, and each stage that on whole system, both need guarantee regenerating system cultivates can full implementation, need embody the advantage of regrowth quantity and growth coefficient again.Reach such technique effect, major technology means one are to select appropriate plant explants material, the 2nd, and to the selection and optimization of each stage nutrient media components.
The young stem of present technique Scheme Choice Hemarthria compressa or spire are explant, and the sterilization back is inoculated into component under gnotobasis be MS medium+2, carry out callus induction on the startup medium of 4-D and cultivate; The callus that cultivation obtains changes over to behind successive transfer culture in the callus differential medium that component is MS medium+6-BA+NAA and carries out differentiation culture; The regrowth that differentiation culture obtains changes over to behind enrichment culture in the root media that component is the 1/2MS+NAA medium and carries out the regrowth culture of rootage, and the seedling that obtains is finally refined seedling, transplanting.
Under optimum condition, it is explant that said method can be selected the spire of Hemarthria compressa.The Hemarthria compressa high frequency regenerating system of being set up has more good technical effect.
Under optimum condition, said method can be through to the optimization and improvement Hemarthria compressa high frequency regenerating system of nutrient media components in each cultivation stage output capacity and the product quality in each stage.Test data shows, at MS+2, on the 4-D1.0mg/L medium; Spire graininess callus healing rate can reach 100%, and on the differential medium of MS+6-BA 1.0mg/L+NAA 0.2mg/L, the green seedling differentiation rate of spire can reach 100%;, milky callus dry, fine and close by each appearance can produce 5～21 regeneration buds that do not wait, and behind the cultivation 30d, can form the bud clump; Continue enrichment culture, can produce the healthy regrowth of 93～392 strains.Regrowth rooting rate on the 1/2MS root media can reach 100%, and transplanting survival rate also can reach 100%.
Compared with prior art, the invention has the beneficial effects as follows: Hemarthria compressa high frequency regenerating system method for building up provided by the invention has that growth and breeding is fast, repeatability is high, simple to operate, condition is easy to characteristics such as control.Particularly can obtain appearance drying, densification, milky Hemarthria compressa embryo callus through the inventive method; Graininess embryo callus wherein also is applicable to the screening of somatic mutants and the genetic transformation of agrobacterium-mediated transformation, can open up new breeding approach for Hemarthria compressa condition is provided.
Description of drawings
Fig. 1 is that the Hemarthria compressa high frequency regenerating system is set up the techniqueflow sketch map.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are done further to describe.
Embodiment one
As shown in Figure 1, set up the Hemarthria compressa high frequency regenerating system with the inventive method.
1, material
(1) plant explants material: the spire with Hemarthria compressa is an explant material.
(2) main agents:
2,4-D (2,4 dichlorophenoxyacetic acid) mother liquor: 2,4-D with a small amount of anhydrous alcohol solution after, adding distil water is made into the mother liquor of 1mg/L, preserves down for use in 4 ℃.
6-BA (6-benzylaminopurine) mother liquor: 6-BA with a small amount of 10% dissolution of sodium hydroxide after, adding distil water is made into the mother liquor of 1mg/L, preserves down for use in 4 ℃.
NAA (methyl) mother liquor: NAA with a small amount of 10% dissolution of sodium hydroxide after, adding distil water is made into the mother liquor of 1mg/L, preserves down for use in 4 ℃.
(3) basal medium: MS medium, MS medium are adjusted pH to 5.8 before autoclaving.
2, operating procedure
(1) step S1 callus induction
Start medium: MS medium+2,4-D 0.5～2.5mg/L
Explant material is placed 75% the alcohol 30s that sterilizes, takes out the back, be dipped in the about 8min of sterilization in 0.1% the mercury chloride again with aseptic water washing 3 times, after the taking-up with aseptic water washing 4～6 times; Under gnotobasis, explant material is inoculated into to start carries out callus induction on the medium and cultivate.Condition of culture is 23+2 ℃, dark culturing, and every separated 15d changes a subculture.Callus induction is cultivated 60d, and cultivation results is as shown in table 1.
Table 1 callus induction result
(2) step S2 callus successive transfer culture
Subculture medium: MS medium+2,4-D 1.0mg/L
The callus that obtains among the step S1 transferred carry out the callus successive transfer culture in the subculture medium that filters out.The screening of subculture medium is carried out according to tissue culture routine operation method.The callus successive transfer culture is every changes a subculture at a distance from 15d, cultivates 60d, and cultivation results is as shown in table 2.
Table 2 callus successive transfer culture result
(3) step S3 callus differentiation culture
Callus differential medium: MS medium+6-BA1.0～1.4mg/L+NAA 0.1～0.3mg/L
The callus that the growth quality that step S2 is obtained is good changes over to and carries out differentiation culture in the callus differential medium.Condition of culture is 25 ± 2 ℃, 2000～3000lx, 16hr/d.The callus differentiation culture is every changes a subculture at a distance from 10d, cultivates 30d, and cultivation results is as shown in table 3.
Table 3 callus differentiation culture result
(4) step S4 regrowth enrichment culture
Regrowth proliferated culture medium: MS medium+6-BA 1.0～1.4mg/L+NAA0.1～0.3mg/L.
To cultivate in the regrowth obtain the long regrowth that 3 intact leafs are arranged through step S3 changes over to and carries out the regrowth enrichment culture in the regrowth proliferated culture medium.Condition of culture is 23 ± 2 ℃, 1500～2000lx, 12hr/d; The regrowth enrichment culture is every changes a subculture at a distance from 10d, cultivates 60d, and cultivation results is as shown in table 4.
Table 4 regrowth enrichment culture result
Annotate: 1. growth coefficient=number/inoculation strain number sprouts; 2. " ++ " plant strain growth gesture a little less than, the blade light green color, growth coefficient is lower; 3. " +++" the plant strain growth stalwartness, cluster, dark green leaf, growth coefficient is high.
(5) step S5 regrowth culture of rootage
Root media: 1/2MS medium+NAA0～0.4mg/L
The regrowth that the upgrowth situation that step S4 is obtained is good changes the culture of rootage of carrying out regrowth in the regrowth root media over to.Condition of culture is 28 ± 2 ℃, 2000～3000lx, 16hr/d.Culture of rootage 30d, culture of rootage result is as shown in table 5.
Table 5 regrowth culture of rootage result
(6) step S6 regrowth acclimatization and transplants
The seedling of growth 20～30d is refined seedling, be specially: at first blake bottle is shifted out culturing room, at room temperature refine seedling 2～3d, open and seal film, add running water, be advisable, continue refining seedling 3d to flood medium.Seedling through the refining seedling is transplanted to little basin behind the careful clean residual agar of base portion, and transplanting medium is sandy soil.Transplant and begin to occur tillering after 15 days, transplanting survival rate is 100%.
Embodiment two
Set up the Hemarthria compressa high frequency regenerating system with the inventive method, itself and embodiment one something in common no longer repeat, and its difference is:
(1) step S1 callus induction
Start medium: MS medium+2,4-D 0.5～2.5mg/L+6-BA 0～0.4mg/L
Callus induction is cultivated 60d, and cultivation results is as shown in table 6.
Table 6 callus induction result
Claims (6)
1. a Hemarthria compressa high frequency regenerating system method for building up carries out according to the operating process of step S1 callus induction, step S2 callus successive transfer culture, step S3 callus differentiation culture, step S4 regrowth enrichment culture, step S5 regrowth culture of rootage, step S6 regrowth acclimatization and transplants; In step S1 callus induction, use and start medium; In step S2 callus successive transfer culture, use subculture medium; In step S3 callus differentiation culture, use the callus differential medium; In step S4 regrowth enrichment culture, use the regrowth proliferated culture medium, in step S5 regrowth culture of rootage, use root media; It is characterized in that: the startup nutrient media components that said step S1 uses is MS medium+2; 4-D 0.5～2.5mg/L; The subculture medium component that said step S2 uses is MS medium+2; 4-D1.0mg/L; The callus differential medium component that said step S3 uses is MS medium+6-BA1.0～1.4mg/L+NAA0.1～0.3mg/L, and the regrowth proliferated culture medium component that said step S4 uses is MS medium+6-BA 1.0～1.4mg/L+NAA0.1～0.3mg/L, and the root media component that said step S5 uses is 1/2MS+NAA 0 ~ 0.4mg/L; High frequency regenerating system is an explant with the young stem or the spire of Hemarthria compressa.
2. method according to claim 1 is characterized in that: the startup nutrient media components that said step S1 uses is MS medium+2,4-D0.6～1.4mg/L.
3. method according to claim 1 is characterized in that: the startup nutrient media components that said step S1 uses is MS medium+2,4-D 1.0mg/L.
4. according to the arbitrary described method of claim 1～3, it is characterized in that: the startup nutrient media components that said step S1 uses also contains 6-BA 0～0.4mg/L.
5. method according to claim 4 is characterized in that: the startup nutrient media components that said step S1 uses contains 6-BA 0～0.1mg/L.
6. according to claim 1 or 2 or 3 or 5 described methods, it is characterized in that: the regrowth proliferated culture medium component that callus differential medium that said step S3 uses or said step S4 use is MS medium+6-BA1.0mg/L+NAA 0.2mg/L.
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Non-Patent Citations (3)
| Title |
|---|
| 刘金平等."扁穗牛鞭草水培无性苗构件组成特点的研究".《福建林业科技》.2005,第32卷(第4期),第111-113,117页. |
| 刘金平等."扁穗牛鞭草组织培养体系建立".《中国草地学报》.2007,第29卷(第2期),第50-53页. |
| 刘金平等."西南地区扁穗牛鞭草有性生殖多样性研究".《种子》.2006,第25卷(第8期),第17-21页. |
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