CN102763591B - Atrac tylodes macrocephala koidz conservation method and re-cultivating method - Google Patents
Atrac tylodes macrocephala koidz conservation method and re-cultivating method Download PDFInfo
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Abstract
The invention discloses an Atrac tylodes macrocephala koidz conservation method and re-cultivating method. The conservation method comprises the following steps: (1) taking stem tips of Atrac tylodes macrocephala koidz tissue culture seedlings, and placing the stem tips in a pre-culture solution which is an MS (Murashige and Skoog) fluid nutrient medium containing 0.3 mol/L cane sugar for pre-culture; (2) taking the stem passing through from the step (1) and pre-processing the stem tips in a pre-processing solution which is an MS fluid mutrient medium containing both 2.0 mol/L glycerol and 0.4 mol/L cane sugar; (3) taking the stem tips passing through the step (2) and processing the stem tips in a cryoprotective agent which is prepared in the way that 3.26 mol glycerol, 2.42 mol ethylene glycol, 1.9 mol dimethyl sulfoxide, 0.4 mol cane sugar and the MS fluid nutrient medium are uniformly mixed to 1 L; and (4) putting the stem tips passing through the step (3) into liquid nitrogen for conservation. The Atrac tylodes macrocephala koidz conservation method is low in cost, simple and convenient in operation and high in plant regeneration rate, and is an effective way in vitro conservation of Atrac tylodes macrocephala koidz germplasm resources.
Description
Technical field
The present invention relates to a kind of store method of the bighead atractylodes rhizome and cultural method again.
Background technology
The bighead atractylodes rhizome (Atractylodes macrocephala Koidz.) is composite family [WTBX plant, and its rhizome is used as medicine, and is important medicinal plant.Because the market demand is high, the mankind excavate and environmental disruption for a long time, cause bighead atractylodes rhizome wild resource less, at present mainly by the demand of artificial cultivation with assurance resource satisfying the market.All there is introducing and planting in the most of place of China, practises take Zhejiang in diving, Southern Part of Anhui Province mountain area etc. is as genuine.
At present, often utilize Atractylodes macrocephala seed or piece rhizome to preserve and breed by the field planting mode in preserving garden or breeding garden.Utilize seminal propagation, easily morph, the phenomenon that deterioration of variety, germplasm mix may occur; Utilize the piece root propagation, be difficult to short-term and obtain in a large number germ plasm resource.And the preservation of carrying out in the field planting mode not only occupies cultivated land, also need expend a large amount of manpower and materials and carry out cultivation management, the natural calamities such as arid, flood, low temperature, heat evil, damage by disease and insect also may cause germ plasm resource to be lost, and therefore adopt the field mode should not carry out long preservation.For preserving germ plasm resource, the domestic in vitro tissue of having carried out the bighead atractylodes rhizome is cultivated, fast numerous research, has obtained suitable germination, has expanded tissue culture medium (TCM) and condition of culture numerous, that take root etc. and to cultivate, can obtain in a short time a large amount of group training seedlings.But organize subculture to cultivate, repeatedly subculture also may cause genetic variation, and regularly subculture needs labor intensive, material resources simultaneously.Therefore, still need find the suitableeest long-term preservation method at present.
Summary of the invention
The purpose of this invention is to provide a kind of store method of the bighead atractylodes rhizome and cultural method again.
The invention provides a kind of store method of the bighead atractylodes rhizome, in turn include the following steps:
(1) get the stem apex of bighead atractylodes rhizome group training seedling, carry out preculture in preculture liquid; Described preculture liquid is the MS liquid nutrient medium that contains 0.3mol/L sucrose;
(2) take into the stem apex of step (1), carry out pretreatment in pretreatment fluid; Described pretreatment fluid is the MS liquid nutrient medium that contains 2.0mol/L glycerine and 0.4mol/L sucrose;
(3) take into the stem apex of step (2), process in the vitrifying protectant; The protectant preparation method of described vitrifying is: 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and MS liquid nutrient medium are mixed to 1L;
(4) stem apex of completing steps (3) is dropped in liquid nitrogen and preserves.
In step (1), described stem apex can be the stem apex of 1.5-2mm.
Described bighead atractylodes rhizome group training seedling specifically can be the bighead atractylodes rhizome group training seedling of 2 months seedling ages.
In step (1), described pre-incubated condition can be: 1-4 days (specifically can be 3 days) of 25 ℃ of dark cultivations.
In step (2), described pretreatment can be: with the stem apex of completing steps (1) soaking at room temperature 30 minutes (preferred oscillation treatment) in described pretreatment fluid.Described room temperature specifically can be 20-30 ℃ (as 25 ℃).
In step (3), described processing can be: with the stem apex of completing steps (2) in described vitrifying protectant 0 ℃ soaked 60 minutes.
After described step (4) had comprised the steps: described step (3), the vitrifying protectant that will contain stem apex dropped on aluminium foil, then aluminium foil was transferred in the cryopreservation tube that is full of liquid nitrogen, then described cryopreservation tube was dropped in liquid nitrogen and preserved.Every vitrifying protectant that contains described stem apex specifically can be 16 microlitres.Every vitrifying protectant that contains described stem apex specifically can contain 5 described stem apexs.
The present invention also protects a kind of method that the bighead atractylodes rhizome after above arbitrary described method preservation is cultivated again, and the bighead atractylodes rhizome after comprising the steps: above arbitrary described method is preserved is cultivated again, obtains bighead atractylodes rhizome plant.
The described cultivation again comprises the steps: the bighead atractylodes rhizome stem apex after thawing was first secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose, then containing 0.3mg/L 6-BA, 0.02mg/L NAA and 0.1mg/L GA
3The MS solid culture medium in dark cultivate 5-7 days (stem apex returns green and the sign of sprouting is arranged), more then be transferred to normal illumination in the MS solid culture medium and cultivate.
The step that the bighead atractylodes rhizome after before described cultural method more also is included in and cultivates, above arbitrary described method being preserved washs with the MS liquid nutrient medium that contains 1.2mol/L sucrose.
Described pre-incubated effect: can reduce stem-tip tissue cell free water content, increase the protective substance content such as soluble sugar, thereby improve stem apex freezing tolerance, reduce or avoid freezing injury, improve the plant survival rate.
Described pretreated effect: further reduce the stem-tip tissue cell water content, with fast dewatering in the vitrifying protectant of avoiding changing over to higher concentration.
The effect of described vitrifying agent: add vitrifying agent, make the stem-tip tissue cell reach the vitrifying state in temperature-fall period, avoid in cell forming ice crystal damaging cells film.
The invention provides a kind of cryopreservation method of the bighead atractylodes rhizome and cultivate again on the gene that ultralow temperature is preserved, finally obtaining the method for bighead atractylodes rhizome plant.Adopt method provided by the invention to preserve and cultivate, shoot regeneration frequency can reach more than 60%.Method provided by the invention is with low cost, easy and simple to handle, shoot regeneration frequency, avoided the field preservation to be subject to Effect of Natural Disaster and to cause blastation or destruction, and tissue cultivate to preserve in because subculture repeatedly causes genetic character variation or the danger of polluting, be an exsomatize effective way of preservation of Atractylodes macrocephala Germplasm resource.
Description of drawings
Fig. 1 is the photo of group training seedling.
Fig. 2 is the photo of stem apex.
Fig. 3 is for dripping the aluminum foil strip after the PVS2 vitrifying protectant contain stem apex.
Fig. 4 changes the photo of normal illumination cultivation after 7 days in the MS solid culture medium over to.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be conventional method.Test material used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent company.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.
6-BA is that the 6-(benzyl is amino) purine, structural formula is seen formula (I).
NAA, i.e. 1-methyl α-naphthyl acetate, structural formula is seen formula (II).
GA
3, i.e. gibberellin A
3, chemical name is 2,4a, 7-trihydroxy-1-methyl-8-methylene red mould-3-alkene-1,10-dicarboxylic acids-Isosorbide-5-Nitrae a-lactone, structural formula is seen formula (III).
IBA, i.e. 3-indoles-butyric acid, structural formula is seen formula (IV).
MS liquid nutrient medium: sucrose 30g, 10 * macroelement 100ml(10 * macroelement formula: KNO
319.00g, NH
4NO
316.5g, MgSO
47H
2O 3.7g, KH
2PO
41.7g, CaCl
22H
2O 4.4g, add water and be settled to 1L), 100 * micro-10ml(100 * trace element formula: MnSO
4H
2O 1.69g, ZnSO
47H
2O 0.86g, H
3BO
30.62g, KI 0.085g, NaMoO
42H
2O 0.025g, CuSO
45H
2O 0.0025g, CoCl
26H
2O0.0025g, adding distil water is settled to 1L), 100 * molysite 10ml(100 * molysite formula: FeSO
47H
2O 1.39g, Na
2-EDTA 1.86g, adding distil water is settled to 500ml), 200 * organic matter 5ml(200 * organic matter formula: glycine 0.2g, B
10.04g, B
60.05g, nicotinic acid 0.05g, adding distil water is settled to 500ml), 100 * inositol 10ml(100 * inositol formula: inositol 5g, adding distil water is settled to 500ml), pH5.8, be settled to 1L with distilled water.
MS solid culture medium: fill a prescription identical with the MS liquid nutrient medium, be settled to 1L, pH5.8 after interpolation agar 8.5g.
MS semisolid culturemedium: fill a prescription identical with the MS liquid nutrient medium, be settled to 1L, pH5.8 after interpolation agar 5-7g.
Bighead atractylodes rhizome group training seedling: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; List of references: Liang Xiaomin, Zhu Chaohui, the Hu fine, soft fur, the rapid propagation in vitro research of bighead atractylodes rhizome stem apex, the Agriculture of Anhui science, 2009,37(35): 17356-17357.
The ultralow temperature of embodiment 1, the bighead atractylodes rhizome is preserved
One, the formula of related reagent
Preculture liquid: the MS liquid nutrient medium that contains 0.3mol/L sucrose.
Pretreatment fluid: the MS liquid nutrient medium that contains 2.0mol/L glycerine and 0.4mol/L sucrose.
Vitrifying protectant: 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and MS liquid nutrient medium are fully mixed to 1L.
Two, the ultralow temperature of the bighead atractylodes rhizome is preserved
get the bighead atractylodes rhizome group training seedling of 2 months seedling ages, successively peel off outer blade, surround shoot apical meristem to remaining 1-2 sheet leaf primordium, cut the stem apex (obtaining altogether 100 stem apexs) of 1.5-2mm, first be immersed in 25 ℃ of dark cultivations 3 days in preculture liquid, then in pretreatment fluid, the 30min(normal illumination is soaked in 25 ℃ of vibrations), then 0 ℃ of immersion 60min(normal illumination in the vitrifying protectant again), then drip again 4 PVS2 vitrifying protectant (every 16 microlitres that contain stem apex on the aseptic aluminum foil strip of every 5mm * 20mm, wherein contain 5 stem apexs), aluminum foil strip is transferred in the cryopreservation tube that is full of liquid nitrogen finally, the input liquid nitrogen is preserved.
The photo of the bighead atractylodes rhizome group training seedling of 2 months seedling ages is seen Fig. 1.The photo of the stem apex that cuts is seen Fig. 2.The aluminum foil strip that dropping contains after the PVS2 vitrifying protectant of stem apex is seen Fig. 3.
Three, cultivation and survival rate, regeneration rate are added up again
1, the cryopreservation tube in the taking-up liquid nitrogen, then take out the aluminum foil strip in cryopreservation tube,, with the MS liquid nutrient medium washing that contains 1.2mol/L sucrose 2 times (each 10min), gets stem apex under 25 ℃.
2, the stem apex of step 1 was first secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose; Then containing 0.3mg/L BA, 0.02mg/L NAA and 0.1mg/L GA
3The MS solid culture medium in dark cultivate 5-7 days (can observe stem apex returns green and the sign of sprouting is arranged); Then change in the MS solid culture medium normal illumination over to and cultivate, that observes when normal illumination is cultivated 15 days that normal leaf grows is the survival plant, and normal illumination is cultivated the plant that 30 days durations go out leaf and root and is regeneration plant.
Change the photo that in the MS solid culture medium, normal illumination was cultivated after 7 days over to and see Fig. 4.
Plant survival rate=survival plant quantity ÷ stem apex quantity * 100%.Stem apex quantity=100 wherein.
Shoot regeneration frequency=regeneration plant quantity ÷ stem apex quantity * 100%.Stem apex quantity=100 wherein.
Carry out repeated test three times, the plant survival rate of each test is respectively 66%, 93%, 67%, and namely the mean value of plant survival rate is 75.5%; The shoot regeneration frequency of each test is respectively 60%, 67% and 60%, and namely the mean value of shoot regeneration frequency is 62.2%.
The ultralow temperature of embodiment 2, the bighead atractylodes rhizome is preserved
One, the formula of related reagent
All with the step 1 of embodiment 1.
Two, the ultralow temperature of the bighead atractylodes rhizome is preserved
25 ℃ of dark times of cultivating adopted respectively 1 day, 2 days, 3 days, 4 days or 5 days in preculture liquid, and other is all with the step 2 of embodiment 1.
Three, cultivation and survival rate, regeneration rate are added up again
Step 3 with embodiment 1.
Carry out repeated test three times, results averaged.
When 25 ℃ of dark times of cultivating were 1 day in preculture liquid, the average plant survival rate of three tests was 69.8%.The average shoot regeneration frequency of three tests is 54.9%.
When 25 ℃ of dark times of cultivating were 2 days in preculture liquid, the average plant survival rate of three tests was 66.0%.The average shoot regeneration frequency of three tests is 52.6%.
When 25 ℃ of dark times of cultivating were 3 days in preculture liquid, the average plant survival rate of three tests was 75.6%.The average shoot regeneration frequency of three tests is 62.2%.
When 25 ℃ of dark times of cultivating were 4 days in preculture liquid, the average plant survival rate of three tests was 57.1%.The average shoot regeneration frequency of three tests is 46.5%.
When 25 ℃ of dark times of cultivating were 5 days in preculture liquid, the average plant survival rate of three tests was 34.4%.The average shoot regeneration frequency of three tests is 23.3%.
Claims (3)
1. the store method of a bighead atractylodes rhizome in turn includes the following steps:
(1) get the stem apex of bighead atractylodes rhizome group training seedling, carry out preculture in preculture liquid; Described preculture liquid is the MS liquid nutrient medium that contains 0.3mol/L sucrose; Described pre-incubated condition is: 25 ℃ of dark 1-4 days that cultivate; Described stem apex is the stem apex of 1.5-2mm;
(2) take into the stem apex of step (1), carry out pretreatment in pretreatment fluid; Described pretreatment fluid is the MS liquid nutrient medium that contains 2.0mol/L glycerine and 0.4mol/L sucrose; Described pretreatment is: with the stem apex of described completing steps (1) soaking at room temperature 30 minutes in described pretreatment fluid;
(3) take into the stem apex of step (2), process in the vitrifying protectant; The protectant preparation method of described vitrifying is: 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and MS liquid nutrient medium are mixed to 1L; Described being treated to: with the stem apex of described completing steps (2) in described vitrifying protectant 0 ℃ soaked 60 minutes;
(4) stem apex of completing steps (3) is dropped in liquid nitrogen and preserves.
2. the method for claim 1; it is characterized in that: after described step (4) has comprised the steps: described step (3); the vitrifying protectant that will contain stem apex drops on aluminium foil; then aluminium foil is transferred in the cryopreservation tube that is full of liquid nitrogen, then described cryopreservation tube is dropped in liquid nitrogen and preserves.
3. the method that the bighead atractylodes rhizome after the described method of claim 1 or 2 being preserved is cultivated again, the bighead atractylodes rhizome after comprising the steps: the described method of claim 1 or 2 is preserved is cultivated again, obtains bighead atractylodes rhizome plant;
The described cultivation again comprises the steps: the bighead atractylodes rhizome stem apex after thawing was first secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose, then containing 0.3mg/L6-BA, 0.02mg/L NAA and 0.1mg/L GA
3The MS solid culture medium in dark cultivate 5-7 days, more then be transferred to illumination cultivation in the MS solid culture medium;
The step that the bighead atractylodes rhizome after before described method also is included in and cultivates, the described method of claim 1 or 2 being preserved washs with the MS liquid nutrient medium that contains 1.2mol/L sucrose.
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| CN103749309B (en) * | 2014-01-28 | 2015-01-14 | 贵州师范大学 | Method for producing virus-free atractylodes macrocephala koidz seedlings |
| CN104255705B (en) * | 2014-09-04 | 2015-11-04 | 中国农业科学院作物科学研究所 | One overcomes the vitrified method of regrowth after jerusalem artichoke stem apex Excised Embryos |
| CN105210869A (en) * | 2015-09-30 | 2016-01-06 | 耿跃 | A kind of method of bighead atractylodes rhizome detoxifying fast breeding |
| CN105230493B (en) * | 2015-11-04 | 2018-03-13 | 恩施土家族苗族自治州农业科学院 | A kind of propagation method of bighead atractylodes rhizome seedling and its application |
| CN106561637A (en) * | 2016-10-28 | 2017-04-19 | 上海市农业生物基因中心 | Cryopreservation and recovery culture methods for dioscorea alata protocrom-like bodies |
| CN106718934B (en) * | 2017-02-09 | 2019-01-18 | 河北大学 | A kind of Rhizoma Atractylodis Macrocephalae regenerating system directly breaking up adventitious bud using plumular axis and radicle |
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