CN102258010A - Slow refrigerating preservation method for paris polyphylla rootstock - Google Patents

Slow refrigerating preservation method for paris polyphylla rootstock Download PDF

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Publication number
CN102258010A
CN102258010A CN2011102591816A CN201110259181A CN102258010A CN 102258010 A CN102258010 A CN 102258010A CN 2011102591816 A CN2011102591816 A CN 2011102591816A CN 201110259181 A CN201110259181 A CN 201110259181A CN 102258010 A CN102258010 A CN 102258010A
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rhizome
paris rhizome
magnificent
rhizoma paris
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CN102258010B (en
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王跃华
田梦良
林抗雪
刘益丽
张珏
李文光
马良良
张红玉
陈燕
汤有举
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Chengdu University
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Chengdu University
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Abstract

The invention discloses a slow refrigerating preservation method for a paris polyphylla rootstock. In the method, skimmed milk powder with no toxic or side effect is taken as a protectant, a normal refrigerator can be used for finishing cooling operation in the refrigerating preservation process instead of an expensive procedural cooling instrument, the preservation cost and instrument dependence of the material are greatly lowered, and the operation process of the slow refrigerating preservation method is effectively simplified. Due to the adoption of the method, the survival rate of a preserved paris polyphylla rootstock can be about 75-85 percent, and environmentally-friendly and efficient preservation of the paris polyphylla rootstock material is realized.

Description

A kind of slow freezing store method of magnificent rhizoma paris rhizome
Technical field
The present invention relates to a kind of cryopreservation method of plant germplasm resource, particularly relate to a kind of slow freezing store method of magnificent rhizoma paris rhizome.
Background technology
The Chinese medicine Paris polyphylla of Chinese Pharmacopoeia regulation is the subterranean stem of Yunnan Rhizoma Paridis (Paris polyphylla var.yunnanensis) and magnificent Paris polyphylla (Paris polyphylla var.chinensis), and the former main product is in Yunnan, and latter's main product is in Sichuan.The flea that Shennong's Herbal is put down in writing the earliest not is magnificent Paris polyphylla.That Paris polyphylla has is clearing heat and detoxicating, anti-inflammatory analgetic, activate blood circulation and disperse blood clots, the effect of cool liver arresting convulsion, be important magistral medicines.
Paris polyphylla heightens because of it has antiviral, antitumor action demand in recent years, and price raises up fast.The Paris polyphylla traditional Chinese medicine always provides medicinal with wild resource, because wild resource ground is excessively excavated, causes wild Paris polyphylla resource to suffer destructive destruction in recent years, and is now endangered.For better protection wild Paris chinensis Franch matter resource, the present invention has carried out the rhizomatous ultralow temperature of Paris polyphylla to China and has preserved Study on Technology.
The slow freezing method is long, preservation effect of an a kind of application time cryopreservation method preferably, and it is to be the freezing and storing method that theoretical foundation is set up with the protectiveness dehydration; This method is by finishing in procedural cooling instrument the progressively cooling of freezing preservation material to be handled, the intracellular moisture of material is fully deviate from, in refrigerating process, caused preserving the harm of material because of large quantity of moisture in the cell produces ice crystal thereby avoid preserving material effectively.Yet the cooling step by step of preserving material is handled because this method must use expensive procedural cooling instrument just can finish in actual mechanical process, concrete operations need be finished in procedural cooling instrument and will preserve material with per minute 1-2 ℃ cooling rate and drop to gradually about-100 ℃ from 0 ℃, material are dropped in the liquid nitrogen at last again and preserve.Therefore, conventional slow freezing store method must use expensive procedural cooling instrument on the one hand, and whole freezing preservation process need spend the long time on the other hand; And the conventional employed cryoprotector of slow freezing method mostly is organic reagent greatly, also is unfavorable for the preservation of traditional Chinese medicine germplasm materials.
Summary of the invention
The present invention is directed to the problem that has the store method existence now and the slow freezing store method that a kind of magnificent rhizoma paris rhizome is provided; this method adopts skim milk powder without any side effects as protectant, and has replaced the expensive program cooling instrument that must use originally with general refrigerator.
For achieving the above object, the solution that the present invention adopts comprises the following steps:
(1) preliminary treatment: get magnificent rhizoma paris rhizome, under 4~8 ℃ of conditions, placed 15~20 days earlier, be cut into the bulk of 5~20mm3 then, place pre-culture fluid again: sucrose 4~7%+ proline 0.2~0.5% pre-cultivation 20~24 hours;
(2) freezing processing: the magnificent rhizoma paris rhizome after will cultivating is in advance put into protectant cryovial is housed, place 2 ℃ of refrigerators to place 30~40min earlier, and then dislocation is in-16 ℃ refrigerator and begin to lower the temperature gradually, treat to place 30~60min after temperature is reduced to-24 ℃, drop in the liquid nitrogen rapidly immediately and preserve, described protectant is: skim milk powder 30~40%+ proline 1.0~1.5%;
(3) thaw and wash: magnificent rhizoma paris rhizome preserved at least 1 week in liquid nitrogen after, when needs use rhizome, cryovial is taken out, put into constant incubator rapidly and carry out quick-thawing, thaw finish after, sucrose solution with 0.4~0.5mol/L carries out washing by soaking 3~4 times, and each 10~15min is clean with distilled water flushing at last.
The temperature of constant incubator is 40~60 ℃ in the above-mentioned steps (3).
The survival rate of the rhizoma paris rhizome cell after thawing can detect by the following method: the magnificent rhizoma paris rhizome pH after will washing is 7.0 0.4%TTC (2,3, the 5-triphenyltetrazolium chloride) solution is in 25 ℃ of dark place dyeing 18h, remove TTC solution then, adding absolute ethyl alcohol, to carry out the extraction of TTF in 40 ℃ of thermostat water baths colourless until material, get supernatant and measure light absorption value in the 485nm place, and back material cell relative survival rate is frozen in calculating, cell relative survival rate %=handles the light absorption value X 100% of the light absorption value/material that is untreated of back material, the TTC solution that is adopted is to be formulated in 7.0 the phosphate buffer solution with the TTC material at pH, and compound concentration is 0.4%.
The present invention has adopted skim milk powder without any side effects as protectant, has realized that the green of rare traditional Chinese medicine is preserved; Though skim milk powder can not enter in the cell as the impermeability protectant; but it is dissolvable in water in the tissue juice between the cell to increase the concentration of tissue juice; thereby make and form permeable pressure head between intracellular fluid and tissue juice; can impel deviating from of ICW effectively; with the content of free water in the minimizing cell, thereby can avoid material in process of cryopreservation, to cause the injury of pair cell owing to the formation of ice crystal in the cell.Simultaneously, the present invention has realized finishing smoothly the cooling operation of process of cryopreservation in general refrigerator, really broken away from the dependence of conventional slow freezing store method to the expensive program cooling instrument, has reduced the time and the cost of the freezing preservation of material widely.
Below in conjunction with embodiment the present invention is described in further details, but content of the present invention is not limited in this.
Embodiment
Embodiment 1
(1) preliminary treatment: choose and grow more than 5 years and the magnificent rhizoma paris rhizome of healthy growth, under 4 ℃ of conditions, placed 20 days earlier, be cut into 15mm then 3Bulk, place pre-culture fluid again: sucrose 7%+ proline 0.5% is pre-to be cultivated 24 hours;
(2) freezing processing: the magnificent rhizoma paris rhizome after will cultivating is in advance put into protectant cryovial is housed, place 2 ℃ of refrigerators to place 30min earlier, and then dislocation is in-16 ℃ refrigerator and begin to lower the temperature gradually, treat to place 40min after temperature is reduced to-24 ℃, drop in the liquid nitrogen rapidly immediately and preserve, described protectant is: skim milk powder 40%+ proline 1.5%;
(3) thaw and wash: magnificent rhizoma paris rhizome preserved for 2 weeks in liquid nitrogen after, when needs use rhizome, cryovial is taken out, put into 55 ℃ of constant incubators rapidly and carry out quick-thawing, thaw finish after, sucrose solution with 0.5mol/L carries out washing by soaking 3 times, and each 10min is clean with distilled water flushing at last;
(4) freeze the detection of material cell survival rate afterwards: the magnificent rhizoma paris rhizome after will washing is put into beaker, add that an amount of to regulate pH with phosphate buffer solution be that 7.0 0.4%TTC solution is in 25 ℃ of constant temperature dark places dyeing 18h, inhale then and remove TTC solution, add absolute ethyl alcohol and in 40 ℃ of thermostat water baths, carry out the extraction of TF until colourless, get supernatant and measure light absorption value at the 485nm place on spectrophotometer, it is 85.4% that back material cell relative survival rate is frozen in calculating.
Embodiment 2
(1) preliminary treatment: choose and grow more than 5 years and the magnificent rhizoma paris rhizome of healthy growth, under 4 ℃ of conditions, placed 20 days earlier, be cut into 15mm then 3Bulk, place pre-culture fluid again: sucrose 4%+ proline 0.2% is pre-to be cultivated 24 hours;
(2) freezing processing: the magnificent rhizoma paris rhizome after will cultivating is in advance put into protectant cryovial is housed, place 2 ℃ of refrigerators to place 30min earlier, and then dislocation is in-16 ℃ refrigerator and begin to lower the temperature gradually, treat to place 40min after temperature is reduced to-24 ℃, drop in the liquid nitrogen rapidly immediately and preserve, described protectant is: skim milk powder 30%+ proline 1%;
(3) thaw and wash: magnificent rhizoma paris rhizome preserved for 2 weeks in liquid nitrogen after, when needs use rhizome, cryovial is taken out, put into 40 ℃ of constant incubators rapidly and carry out quick-thawing, thaw finish after, sucrose solution with 0.5mol/L carries out washing by soaking 3 times, and each 10min is clean with distilled water flushing at last;
(4) freeze the detection of material cell survival rate afterwards: the magnificent rhizoma paris rhizome after will washing is put into beaker, add that an amount of to regulate pH with phosphate buffer solution be that 7.0 0.4%TTC solution is in 25 ℃ of constant temperature dark places dyeing 18h, inhale then and remove TTC solution, add absolute ethyl alcohol and in 40 ℃ of thermostat water baths, carry out the extraction of TF until colourless, get supernatant and measure light absorption value at the 485nm place on spectrophotometer, it is 77.4% that back material cell relative survival rate is frozen in calculating.
Embodiment 3
(1) preliminary treatment: choose and grow more than 5 years and the magnificent rhizoma paris rhizome of healthy growth, under 4 ℃ of conditions, placed 15 days earlier, be cut into 5mm then 3Bulk, place pre-culture fluid again: sucrose 7%+ proline 0.5% is pre-to be cultivated 20 hours;
(2) freezing processing: the magnificent rhizoma paris rhizome after will cultivating is in advance put into protectant cryovial is housed, place 2 ℃ of refrigerators to place 30min earlier, and then dislocation is in-16 ℃ refrigerator and begin to lower the temperature gradually, treat to place 40min after temperature is reduced to-24 ℃, drop in the liquid nitrogen rapidly immediately and preserve, described protectant is: skim milk powder 40%+ proline 1.5%;
(3) thaw and wash: magnificent rhizoma paris rhizome preserved for 2 weeks in liquid nitrogen after, when needs use rhizome, cryovial is taken out, put into 55 ℃ of constant incubators rapidly and carry out quick-thawing, thaw finish after, sucrose solution with 0.4mol/L carries out washing by soaking 3 times, and each 10min is clean with distilled water flushing at last;
(4) freeze the detection of material cell survival rate afterwards: the magnificent rhizoma paris rhizome after will washing is put into beaker, add that an amount of to regulate pH with phosphate buffer solution be that 7.0 0.4%TTC solution is in 25 ℃ of constant temperature dark places dyeing 18h, inhale then and remove TTC solution, add absolute ethyl alcohol and in 40 ℃ of thermostat water baths, carry out the extraction of TF until colourless, get supernatant and measure light absorption value at the 485nm place on spectrophotometer, it is 80.4% that back material cell relative survival rate is frozen in calculating.

Claims (2)

1. the slow freezing store method of a magnificent rhizoma paris rhizome is characterized in that comprising the steps:
(1) preliminary treatment: get magnificent rhizoma paris rhizome, under 4~8 ℃ of conditions, placed 15~20 days earlier, be cut into 5~20mm then 3Bulk, place pre-culture fluid again: sucrose 4~7%+ proline 0.2~0.5% is pre-to be cultivated 20~24 hours;
(2) freezing processing: the magnificent rhizoma paris rhizome after will cultivating is in advance put into protectant cryovial is housed, place 2 ℃ of refrigerators to place 30~40min earlier, and then dislocation is in-16 ℃ refrigerator and begin to lower the temperature gradually, treat to place 30~60min after temperature is reduced to-24 ℃, drop in the liquid nitrogen rapidly immediately and preserve, described protectant is: skim milk powder 30~40%+ proline 1.0~1.5%;
(3) thaw and wash: magnificent rhizoma paris rhizome preserved at least 1 week in liquid nitrogen after, when needs use rhizome, cryovial is taken out, put into constant incubator rapidly and carry out quick-thawing, thaw finish after, sucrose solution with 0.4~0.5mol/L carries out washing by soaking 3~4 times, and each 10~15min is clean with distilled water flushing at last.
2. a kind of magnificent rhizoma paris rhizome slow freezing store method according to claim 1 is characterized in that: the temperature of constant incubator is 40~60 ℃ in the step (3).
CN 201110259181 2011-09-02 2011-09-02 Slow refrigerating preservation method for paris polyphylla rootstock Expired - Fee Related CN102258010B (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105123253A (en) * 2015-10-15 2015-12-09 王婧 Seedling method for paris polyphylla
CN108684657A (en) * 2018-03-27 2018-10-23 中国医学科学院药用植物研究所海南分所 A kind of dalbergia wood seed cryopreservation method
CN108719280A (en) * 2018-07-14 2018-11-02 沿河后花园农业观光旅游综合开发有限公司 A kind of store method of lily ball tissue
CN111693475A (en) * 2020-07-28 2020-09-22 扬州大学 Improved method for measuring activity of plant root system in sections

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JPH1056821A (en) * 1996-08-22 1998-03-03 Koichi Naito Greening foundation member
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CN101884321A (en) * 2010-07-13 2010-11-17 大连普瑞康生物技术有限公司 Cryopreservation method for Sinkiang saussurea involucrata cell line

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JPH1056821A (en) * 1996-08-22 1998-03-03 Koichi Naito Greening foundation member
CN1532279A (en) * 2003-03-26 2004-09-29 ����������Ʒ�о��� Freeze-drying and cryopreservation methods of ginseng callus cells
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105123253A (en) * 2015-10-15 2015-12-09 王婧 Seedling method for paris polyphylla
CN105123253B (en) * 2015-10-15 2017-10-27 向君民 A kind of method for culturing seedlings of paris polyphylla
CN108684657A (en) * 2018-03-27 2018-10-23 中国医学科学院药用植物研究所海南分所 A kind of dalbergia wood seed cryopreservation method
CN108684657B (en) * 2018-03-27 2021-02-12 中国医学科学院药用植物研究所海南分所 Ultralow-temperature preservation method for dalbergia wood seeds
CN108719280A (en) * 2018-07-14 2018-11-02 沿河后花园农业观光旅游综合开发有限公司 A kind of store method of lily ball tissue
CN111693475A (en) * 2020-07-28 2020-09-22 扬州大学 Improved method for measuring activity of plant root system in sections

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