CN102763591A - 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 (Atrac tylodes macrocephala Koidz.) is a composite family [WTBX plant, and its rhizome is used as medicine, and is important medicinal plant.Because the market demand is high, human excavates and environmental disruption for a long time, causes bighead atractylodes rhizome wild resource less, mainly satisfies the demand in market through artificial cultivation to guarantee resource at present.All there is introducing and planting in the most of place of China, and practising with Zhejiang is genuine in dive, Anhui Wannan mountainous area etc.
At present, often utilize bighead atractylodes rhizome seed or piece rhizome in preserving garden or numerous kind of garden, to preserve and breed through the field planting mode.Utilize seminal propagation, be prone to morph, the phenomenon that deterioration of variety, germplasm mix may occur; Utilize the piece root propagation, be difficult to short-term and obtain germ plasm resource in a large number.And the preservation of carrying out with the field planting mode not only occupies cultivated land; Also need the labor manpower and materials to carry out cultivation management; Natural calamities such as arid, flood, low temperature, heat evil, damage by disease and insect also possibly cause germ plasm resource to be lost, and therefore adopt the field mode should not carry out long preservation.Be to preserve 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 a large amount of tissue cultivating seedling in a short time.But organize successive transfer culture, repeatedly subculture also may cause genetic variation, and regularly subculture needs labor intensive, material resources simultaneously.Therefore, still need seek the righttest 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) gets the stem apex of bighead atractylodes rhizome tissue cultivating seedling, cultivating in advance in the culture fluid in advance; Said preparatory culture fluid is the MS liquid nutrient medium that contains 0.3mol/L sucrose;
(2) get the stem apex of completing steps (1), in pretreatment fluid, carry out preliminary treatment; Said pretreatment fluid is the MS liquid nutrient medium that contains 2.0mol/L glycerine and 0.4mol/L sucrose;
(3) get the stem apex of completing steps (2), in the vitrifying protectant, handle; The protectant preparation method of said vitrifying is: with 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and MS liquid nutrient medium mixing to 1L;
(4) preserve in the stem apex input liquid nitrogen with completing steps (3).
In the step (1), said stem apex can be the stem apex of 1.5-2mm.
Said bighead atractylodes rhizome tissue cultivating seedling specifically can be the bighead atractylodes rhizome tissue cultivating seedling of 2 months seedling ages.
In the step (1), said pre-incubated condition can be: 1-4 days (specifically can be 3 days) of 25 ℃ of dark cultivations.
In the step (2), said preliminary treatment can be: with stem apex soaking at room temperature 30 minutes (preferred oscillation treatment) in said pretreatment fluid of completing steps (1).Said room temperature specifically can be 20-30 ℃ (as 25 ℃).
In the step (3), said processing can be: with the stem apex of completing steps (2) in said vitrifying protectant 0 ℃ soaked 60 minutes.
After said step (4) comprised the steps: to accomplish said step (3), the vitrifying protectant that will contain stem apex dropped on the aluminium foil, then aluminium foil was transferred in the frozen pipe that is full of liquid nitrogen, said frozen pipe is dropped in the liquid nitrogen preserve then.Every vitrifying protectant that contains said stem apex specifically can be 16 microlitres.Every vitrifying protectant that contains said stem apex specifically can contain 5 said stem apexs.
The present invention also protect a kind of will more than the bighead atractylodes rhizome of arbitrary said method after preserving carry out cultured method again, the bighead atractylodes rhizome after comprising the steps: above arbitrary said method preserved is cultivated again, obtains bighead atractylodes rhizome plant.
The said cultivation again comprises the steps: the bighead atractylodes rhizome stem apex after thawing was secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose earlier, containing 0.3mg/L6-BA, 0.02mg/L NAA and 0.1mg/L GA then
3The MS solid culture medium in dark cultivate 5-7 days (stem apex returns green and the sign of sprouting is arranged), be transferred in the MS solid culture medium normal illumination more then and cultivate.
Before said cultural method more also is included in and cultivates will more than the bighead atractylodes rhizome of arbitrary said method after preserving with the step of the MS liquid nutrient medium washing that contains 1.2mol/L sucrose.
Said pre-incubated effect: can reduce stem-tip tissue cell free water content, increase protective substance content such as soluble sugar, thereby improve stem apex freezing tolerance, reduce or avoid freezing injury, improve the plant survival rate.
Said 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 said vitrifying agent: add vitrifying agent, make the stem-tip tissue cell in temperature-fall period, reach the vitrifying state, avoid forming in the cell ice crystal damaging cells film.
The invention provides a kind of cryopreservation method of the bighead atractylodes rhizome and on the gene that ultralow temperature is preserved, cultivate again, finally obtain 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 to preserve and be subject to the natural calamity influence and cause blastation or destruction; And tissue culture preserve in because of subculture repeatedly causes the danger of genetic character variation or pollution, be an effective way of bighead atractylodes rhizome germ plasm resource {in vitro} conservation.
Description of drawings
Fig. 1 is the photo of tissue cultivating 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 among the following embodiment like no specified otherwise, is conventional method.Used test material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent company and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
6-BA is 6-(benzyl is an amino) purine, and structural formula is seen formula (I).
NAA, i.e. 1-methyl, structural formula is seen formula (II).
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-1,4a-lactone, structural formula are 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 prescription: KNO
319.00g, NH
4NO
316.5g, MgSO
47H
2O3.7g, KH
2PO
41.7g, CaCl
22H
2O4.4g adds water and is settled to 1L), 100 * micro-10ml (100 * trace element formula: MnSO
4H
2O1.69g, ZnSO
47H
2O0.86g, H
3BO
30.62g, KI0.085g, NaMoO
42H
2O0.025g, CuSO
45H
2O0.0025g, CoCl
26H
2O 0.0025g, adding distil water is settled to 1L), 100 * molysite 10ml (100 * molysite prescription: FeSO
47H
2O1.39g, Na
2-EDTA1.86g, adding distil water is settled to 500ml), 200 * organic matter 5ml (200 * organic matter prescription: 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 prescription: inositol 5g, adding distil water is settled to 500ml), pH5.8 is settled to 1L with distilled water.
The MS solid culture medium: it is identical with the MS liquid nutrient medium to fill a prescription, and is settled to 1L, pH5.8 behind the interpolation agar 8.5g.
The MS semisolid culturemedium: it is identical with the MS liquid nutrient medium to fill a prescription, and is settled to 1L, pH5.8 behind the interpolation agar 5-7g.
Bighead atractylodes rhizome tissue cultivating seedling: the public can obtain from crop science research institute of the Chinese Academy of Agricultural Sciences; List of references: Liang Xiaomin, Zhu Chaohui, Hu fine, soft fur, the rapid propagation in vitro research of bighead atractylodes rhizome stem apex, Anhui agricultural science, 2009,37 (35): 17356-17357.
The ultralow temperature of embodiment 1, the bighead atractylodes rhizome is preserved
One, the prescription of related reagent
Preparatory culture fluid: 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.
The vitrifying protectant: with 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and the abundant mixing of MS liquid nutrient medium to 1L.
Two, the ultralow temperature of the bighead atractylodes rhizome is preserved
Get the bighead atractylodes rhizome tissue cultivating 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 100 stem apexs altogether) of 1.5-2mm; Be immersed in the preparatory culture fluid 25 ℃ of dark cultivations 3 days earlier, 30min (normal illumination) are soaked in 25 ℃ of vibrations in the pretreatment fluid then, more then in the vitrifying protectant 0 ℃ soak 60min (normal illumination); On the aseptic aluminum foil strip of every 5mm * 20mm, drip 4 PVS2 vitrifying protectant (every 16 microlitres that contain stem apex more then; Wherein contain 5 stem apexs), at last aluminum foil strip is transferred in the frozen pipe that is full of liquid nitrogen, drop into liquid nitrogen and preserve.
The photo of the bighead atractylodes rhizome tissue cultivating 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 frozen pipe in the taking-up liquid nitrogen takes out the aluminum foil strip in the frozen pipe then, washs (10min at every turn) 2 times with the MS liquid nutrient medium that contains 1.2mol/L sucrose down at 25 ℃, gets stem apex.
2, the stem apex of step 1 was secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose earlier; Containing 0.3mg/L BA, 0.02mg/L NAA and 0.1mg/L GA then
3The MS solid culture medium in dark cultivate 5-7 days (can observe stem apex return green and the sign of sprouting is arranged); Change in the MS solid culture medium normal illumination then 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 normal illumination was cultivated after 7 days in the MS solid culture medium 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 promptly the mean value of plant survival rate is 75.5%; The shoot regeneration frequency of each test is respectively 60%, 67% and 60%, and promptly the mean value of shoot regeneration frequency is 62.2%.
The ultralow temperature of embodiment 2, the bighead atractylodes rhizome is preserved
One, the prescription 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 preparatory culture fluid, 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 preparatory culture fluid, 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 preparatory culture fluid, 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 preparatory culture fluid, 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 preparatory culture fluid, 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 preparatory culture fluid, the average plant survival rate of three tests was 34.4%.The average shoot regeneration frequency of three tests is 23.3%.
Claims (9)
1. the store method of a bighead atractylodes rhizome in turn includes the following steps:
(1) gets the stem apex of bighead atractylodes rhizome tissue cultivating seedling, cultivating in advance in the culture fluid in advance; Said preparatory culture fluid is the MS liquid nutrient medium that contains 0.3mol/L sucrose;
(2) get the stem apex of completing steps (1), in pretreatment fluid, carry out preliminary treatment; Said pretreatment fluid is the MS liquid nutrient medium that contains 2.0mol/L glycerine and 0.4mol/L sucrose;
(3) get the stem apex of completing steps (2), in the vitrifying protectant, handle; The protectant preparation method of said vitrifying is: with 3.26mol glycerine, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide (DMSO) and 0.4mol sucrose and MS liquid nutrient medium mixing to 1L;
(4) preserve in the stem apex input liquid nitrogen with completing steps (3).
2. the method for claim 1, it is characterized in that: in the step (1), said stem apex is the stem apex of 1.5-2mm.
3. according to claim 1 or claim 2 method, it is characterized in that: in the step (1), said pre-incubated condition is: 25 ℃ of dark cultivations 1-4 days.
4. like arbitrary described method in the claim 1 to 3, it is characterized in that: in the step (2), said preliminary treatment is: with the stem apex of said completing steps (1) soaking at room temperature 30 minutes in said pretreatment fluid.
5. like arbitrary described method in the claim 1 to 4, it is characterized in that: in the step (3), said being treated to: with the stem apex of said completing steps (2) in said vitrifying protectant 0 ℃ soaked 60 minutes.
6. like arbitrary described method in the claim 1 to 5; It is characterized in that: after said step (4) comprises the steps: to accomplish said step (3); The vitrifying protectant that will contain stem apex drops on the aluminium foil; Then aluminium foil is transferred in the frozen pipe that is full of liquid nitrogen, said frozen pipe is dropped in the liquid nitrogen preserve then.
7. the bighead atractylodes rhizome after arbitrary said method is preserved in the claim 1 to 6 is carried out cultured method again, comprise the steps: the bighead atractylodes rhizome after arbitrary said method is preserved in the claim 1 to 6 is cultivated again, obtain bighead atractylodes rhizome plant.
8. method as claimed in claim 7; It is characterized in that: the said cultivation again comprises the steps: the bighead atractylodes rhizome stem apex after thawing was secretly cultivated 1 day in containing the MS semisolid culturemedium of 0.3mol/L sucrose earlier, containing 0.3mg/L6-BA, 0.02mg/L NAA and 0.1mg/L GA then
3The MS solid culture medium in dark the cultivation 5-7 days, be transferred to illumination cultivation to the MS solid culture medium more then.
9. like claim 7 or 8 described methods, it is characterized in that: the step of before said method also is included in and cultivates the bighead atractylodes rhizome after arbitrary said method is preserved in the claim 1 to 5 being washed with the MS liquid nutrient medium that contains 1.2mol/L sucrose.
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Cited By (6)
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| CN103749309A (en) * | 2014-01-28 | 2014-04-30 | 贵州师范大学 | Method for producing virus-free atractylodes macrocephala koidz seedlings |
| CN104255705A (en) * | 2014-09-04 | 2015-01-07 | 中国农业科学院作物科学研究所 | Method for preventing vitrification of regenerated seedlings of jerusalem artichoke stem tips subjected to ultralow-temperature storage |
| CN105210869A (en) * | 2015-09-30 | 2016-01-06 | 耿跃 | A kind of method of bighead atractylodes rhizome detoxifying fast breeding |
| CN105230493A (en) * | 2015-11-04 | 2016-01-13 | 恩施土家族苗族自治州农业科学院 | Propagation method of bighead atractylodes rhizome seedlings and application thereof |
| 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 |
| CN106718934A (en) * | 2017-02-09 | 2017-05-31 | 河北大学 | A kind of utilization plumular axis and radicle directly break up the bighead atractylodes rhizome regenerating system of adventitious bud |
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| CN103749309A (en) * | 2014-01-28 | 2014-04-30 | 贵州师范大学 | Method for producing virus-free atractylodes macrocephala koidz seedlings |
| CN104255705A (en) * | 2014-09-04 | 2015-01-07 | 中国农业科学院作物科学研究所 | Method for preventing vitrification of regenerated seedlings of jerusalem artichoke stem tips subjected to ultralow-temperature storage |
| 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 |
| CN105230493A (en) * | 2015-11-04 | 2016-01-13 | 恩施土家族苗族自治州农业科学院 | Propagation method of bighead atractylodes rhizome seedlings and application thereof |
| 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 |
| CN106718934A (en) * | 2017-02-09 | 2017-05-31 | 河北大学 | A kind of utilization plumular axis and radicle directly break up the bighead atractylodes rhizome regenerating system of adventitious bud |
| 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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