CN103598096B - One-step inducing method of callus of stem of excellent-quality taxus cuspidata - Google Patents

One-step inducing method of callus of stem of excellent-quality taxus cuspidata Download PDF

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Publication number
CN103598096B
CN103598096B CN201310566846.7A CN201310566846A CN103598096B CN 103598096 B CN103598096 B CN 103598096B CN 201310566846 A CN201310566846 A CN 201310566846A CN 103598096 B CN103598096 B CN 103598096B
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China
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callus
northeast
stem
taxus chinensis
quality
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CN201310566846.7A
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CN103598096A (en
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李春斌
岳玉莲
王剑锋
马洪丰
王德萍
于鹤
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DALIAN JINGUIYUAN TECHNOLOGY DEVELOPMENT Co Ltd
Dalian Minzu University
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DALIAN JINGUIYUAN TECHNOLOGY DEVELOPMENT Co Ltd
Dalian Nationalities University
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Abstract

The invention discloses a one-step inducing method of callus of a stem of excellent-quality taxus cuspidate, namely the stem of an annual taxus cuspidata is cultured on an induction medium prepared by an inventor as an explant, and finally the callus of the stem of the loose, light green or white taxus cuspidate is obtained. The one-step inducing method has the characteristics of high induction rate, good induction state, short time required by induction of the callus, sustainable growth of the callus, and the like. According to the one-step inducing method, the callus of the stem of the taxus cuspidate can be simply and conveniently and simply obtained, and the aim that the callus of the taxus cuspidate is induced in large scale by adopting a plant cell culture technology is radically achieved.

Description

High-quality taxus chinensis in northeast callus from stem segment one one-step inducing method
Technical field
The present invention relates to high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method.
Background technology
Taxol (Paclitaxel, trade name Taxol) be the 20th century six a kind of natural tetracyclic diterpene Alkaloids separated from taxaceae (Taxaceae) Taxus (Taxus L.) plant bark of the seventies, extensively paying close attention to by people because of its effect unique in oncotherapy, is best wide spectrum recognized in the world, strong active natural anticancer medical.Due to Chinese yew genus plants poor growth, content of taxol is lower, only accounts for the 0.01%-0.02% of bark dry mass, 100,000/left and right of branches and leaves dry mass.Therefore along with the whole world is to the continuous reinforcement of wild Chinese yew protection of resources measure, utilize wild resource to produce taxol without possible, and present stage artificial planting taxus resource amount also can not meet the demand of plant cell culture far away.
Therefore the exploitation of other approach of taxol just seems very important; Taxus chinensis cell cultures technology is wherein utilized to be exactly an important approach; the protection to Chinese yew Wild ornamental resources and ecotope thereof can be strengthened on the one hand; the gap between supply and demand can be met on the other hand; and culturing and propagating rate is high; condition of culture is easy to optimize and control, and product is more easily separated, is the optimal path solving taxol imbalance between supply and demand in the long run.
Summary of the invention
In order to solve the problem using plant cell culture technology taxus chinensis in northeast callus to be carried out to large-scale culture difficulty, inventor provide high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method.
The technical solution used in the present invention is:
High-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, namely the inducing culture prepared inventor for explant with the stem section of raw taxus chinensis in northeast is then cultivated, is finally loosened, the taxus chinensis in northeast callus from stem segment of light green or white.
Described high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, specifically comprises the following steps:
(1) sterilization of explant
Get the stem section of raw taxus chinensis in northeast then, carry out asepticize process, obtain aseptic taxus chinensis in northeast stem explants for subsequent use;
(2) induction of callus
Be inoculated on inducing culture by the Chinese yew explant of the sterilization of step (1) gained, control temperature is light culture under 18-22 DEG C of condition, obtains the callus after Fiber differentiation after 15 days;
The formula of described inducing culture, is specially according to often liter of calculating:
3792mg KNO 3, 15mg MnSO 4h 2o, 225mg CaCl 22H 2o, 56mgNa 2eDTA2H 2o, 0.37mg CoCl 2.6H 2o, 0.39mg CuSO 45H 2o, 201mg (NH4) 2sO 4, 3mgZnSO 47H 2o, 3.0mg NAA, 41.8mg FeSO 47H 2o, 15mg VB1,0.38mg NaMoO 4.2H 2o, 3.5mg IAA, 4.5mg H 3bO 3, 1.5mg nicotinic acid, 220mgNaH 2pO 4.H 2o, 1.5mgKI, 300mg Vc, 370mg MgSO 47H 2o, 1.5mg VB 6, 268.56mg inositol, 0.5-1mg2,4-D, 2-3mg IBA, 0.3-0.5mg6-BA, 20-30g sucrose, 7-9g agar, 2-3g active carbon and surplus water composition.
Described asepticize process can adopt known any asepticize processing mode, but preferred following asepticize processing mode: the stem section of giving birth to taxus chinensis in northeast then spent the night through running water, then successively with alcohol, mercury chloride sterilization, distilled water is cleaned.The concrete mode more optimized gets raw taxus chinensis in northeast stem section then, running water 10 hours, and then successively with alcohol disinfecting 80-120s, the 0.1% mercury chloride sterilization 10-15min of 70-75%, aseptic water washing for several times, uses aseptic filter paper to be blotted by moisture.
The invention has the beneficial effects as follows:
The present invention with Chinese yew stem section for explant, experiment proves, adopt inducing culture specific to the present invention, one one-step inducing can reach callus induction rate 100%, growth rate 1.0g/L.d, 15d just can grow light green in good condition or white callus and the effect that can constantly grow; Further, the method for the invention is adopted farthest can to suppress outer planting
Body brownization, when operating without error, the generation avoiding brownization of energy 100%.Solve those skilled in the art's problem demanding prompt solution.There is the features such as inductivity is high, induction state good, induction required time is short, step is few, the sustainable growth of callus.
Accompanying drawing explanation
The present invention has accompanying drawing 4 width, wherein:
Fig. 1 is Chinese yew stem section figure;
Fig. 2 is the callus from stem segment figure I obtained according to method provided by the present invention;
Fig. 3 is the callus from stem segment figure II obtained according to method provided by the present invention;
Fig. 4 is the Chinese yew brownization tissue that conventional method that this area is commonly used obtains.
As can be seen from accompanying drawing: adopt the present invention one one-step inducing can reach callus induction rate 100%, growth rate 1.0g/L.d, 15d just can grow light green in good condition or white callus and the effect that can constantly grow; The generation avoiding brownization of energy 100%.
Embodiment
Below by specific embodiment, the present invention is set forth further, but do not limit the present invention.
Embodiment 1
High-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, specifically comprises the steps:
(1) sterilization of explant
Pluck raw taxus chinensis in northeast stem section then, running water 10 hours, then successively with 70% alcohol disinfecting 120s, 0.1% mercury chloride sterilization 10min, for several times, moisture blots aseptic water washing by use aseptic filter paper.
(2) induction of callus
Be inoculated on inducing culture by the Chinese yew explant of the sterilization of step (1) gained, control temperature is light culture under 18 DEG C of conditions, obtains the callus after Fiber differentiation after 15 days;
The formula of described inducing culture, is specially according to often liter of calculating:
3792mg KNO 3, 15mg MnSO 4h 2o, 225mg CaCl 22H 2o, 56mgNa 2eDTA2H 2o, 0.37mg CoCl 2.6H 2o, 0.39mg CuSO 45H 2o, 201mg (NH4) 2sO 4, 3mgZnSO 47H 2o, 3.0mg NAA, 41.8mg FeSO 47H 2o, 15mg VB1,0.38mg NaMoO 4.2H 2o, 3.5mg IAA, 4.5mg H 3bO 3, 1.5mg nicotinic acid, 220mgNaH 2pO 4.H 2o, 1.5mgKI, 300mg Vc, 370mg MgSO 47H 2o, 1.5mg VB 6, 268.56mg inositol, 0.5mg2,4-D, 3mg IBA, 0.3mg6-BA, 30g sucrose, 7g agar, 3g active carbon and surplus water composition.
Embodiment 2
High-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, specifically comprises the steps:
(1) sterilization of explant
Pluck raw taxus chinensis in northeast stem section then, running water 10 hours, then successively with 75% alcohol disinfecting 80s, 0.1% mercury chloride sterilization 15min, for several times, moisture blots aseptic water washing by use aseptic filter paper.
(2) induction of callus
Be inoculated on inducing culture by the Chinese yew explant of the sterilization of step (1) gained, control temperature is light culture under 18-22 DEG C of condition, obtains the callus after Fiber differentiation after 15 days;
The formula of described inducing culture, is specially according to often liter of calculating:
3792mg KNO 3, 15mg MnSO 4h 2o, 225mg CaCl 22H 2o, 56mgNa 2eDTA2H 2o, 0.37mg CoCl 2.6H 2o, 0.39mg CuSO 45H 2o, 201mg (NH4) 2sO 4, 3mgZnSO 47H 2o, 3.0mg NAA, 41.8mg FeSO 47H 2o, 15mg VB1,0.38mg NaMoO 4.2H 2o, 3.5mg IAA, 4.5mg H 3bO 3, 1.5mg nicotinic acid, 220mgNaH 2pO 4.H 2o, 1.5mgKI, 300mg Vc, 370mg MgSO 47H 2o, 1.5mg VB 6, 268.56mg inositol, 1mg2,4-D, 2mg IBA, 0.5mg6-BA, 20g sucrose, 9g agar, 2g active carbon and surplus water composition.
Embodiment 3
High-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, specifically comprises the steps:
(1) sterilization of explant
Pluck raw taxus chinensis in northeast stem section then, running water 10 hours, then successively with 65% alcohol disinfecting 90s, 0.1% mercury chloride sterilization 12min, for several times, moisture blots aseptic water washing by use aseptic filter paper.
(2) induction of callus
Be inoculated on inducing culture by the Chinese yew explant of the sterilization of step (1) gained, control temperature is light culture under 18-22 DEG C of condition, obtains the callus after Fiber differentiation after 15 days;
The formula of described inducing culture, is specially according to often liter of calculating:
3792mg KNO 3, 15mg MnSO 4h 2o, 225mg CaCl 22H 2o, 56mgNa 2eDTA2H 2o, 0.37mg CoCl 2.6H 2o, 0.39mg CuSO 45H 2o, 201mg (NH4) 2sO 4, 3mgZnSO 47H 2o, 3.0mg NAA, 41.8mg FeSO 47H 2o, 15mg VB1,0.38mg NaMoO 4.2H 2o, 3.5mg IAA, 4.5mg H 3bO 3, 1.5mg nicotinic acid, 220mgNaH 2pO 4.H 2o, 1.5mgKI, 300mg Vc, 370mg MgSO 47H 2o, 1.5mg VB 6, 268.56mg inositol, 0.8mg2,4-D, 2.5mg IBA, 0.4mg6-BA, 25g sucrose, 7.5g agar, 2.6g active carbon and surplus water composition.

Claims (2)

1. high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, namely with the stem section of raw taxus chinensis in northeast then for explant is cultivated on inducing culture, finally to be loosened, the taxus chinensis in northeast callus from stem segment of light green or white;
Described high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method, specifically comprises the following steps:
(1) sterilization of explant
Get the stem section of raw taxus chinensis in northeast then, carry out asepticize process, obtain aseptic taxus chinensis in northeast stem explants for subsequent use;
(2) induction of callus
Be inoculated on inducing culture by the Chinese yew explant of the sterilization of step (1) gained, control temperature is light culture under 18-22 DEG C of condition, obtains the callus after Fiber differentiation after 15 days;
The formula of described inducing culture, is specially according to often liter of calculating:
3792mg KNO 3, 15mg MnSO 4h 2o, 225mg CaCl 22H 2o, 56mgNa 2eDTA2H 2o, 0.37mg CoCl 2.6H 2o, 0.39mg CuSO 45H 2o, 201mg (NH4) 2sO 4, 3mgZnSO 47H 2o, 3.0mg NAA, 41.8mg FeSO 47H 2o, 15mg VB1,0.38mg NaMoO 4.2H 2o, 3.5mg IAA, 4.5mg H 3bO 3, 1.5mg nicotinic acid, 220mgNaH 2pO 4.H 2o, 1.5mgKI, 300mg Vc, 370mg MgSO 47H 2o, 1.5mg VB 6, 268.56mg inositol, 0.5-1mg2,4-D, 2-3mg IBA, 0.3-0.5mg6-BA, 20-30g sucrose, 7-9g agar, 2-3g active carbon and surplus water composition.
2. high-quality taxus chinensis in northeast callus from stem segment one one-step inducing method according to claim 1, it is characterized in that: raw taxus chinensis in northeast stem section then is specifically got in the asepticize process described in step (1), running water 10 hours, then successively with alcohol disinfecting 80-120s, the 0.1% mercury chloride sterilization 10-15min of 70-75%, aseptic water washing for several times, uses aseptic filter paper to be blotted by moisture.
CN201310566846.7A 2013-11-12 2013-11-12 One-step inducing method of callus of stem of excellent-quality taxus cuspidata Expired - Fee Related CN103598096B (en)

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CN104620985A (en) * 2015-02-03 2015-05-20 北华大学 Tissue culture and quick propagation method of taxus cuspidata
CN106718914A (en) * 2016-12-23 2017-05-31 叶宗耀 A kind of Chinese yew culture medium

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CN102687667A (en) * 2012-06-19 2012-09-26 湖南农业大学 Method for inducing haploid endosperm callus tissues by using ripe taxales chinensis var.mairei endosperms
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JPH1042888A (en) * 1996-07-30 1998-02-17 Jumoku Seiri Kinousei Butsushitsu Gijutsu Kenkyu Kumiai Production of taxol by culturing
US6365407B1 (en) * 2001-03-05 2002-04-02 Council Of Scientific & Industrial Research Culture medium composition useful for induction and proliferation of Taxus calli
CN1388245A (en) * 2002-06-21 2003-01-01 中山大学 Method of raising taxol yield of south taxad
CN101904303A (en) * 2010-07-13 2010-12-08 大连普瑞康生物技术有限公司 Chinese yew cell culture and method for large-scale subculture of same
CN102640710A (en) * 2012-05-21 2012-08-22 上海应用技术学院 Method for induction and subculture of taxus chinensis leaf callus and culture medium used by method
CN102687667A (en) * 2012-06-19 2012-09-26 湖南农业大学 Method for inducing haploid endosperm callus tissues by using ripe taxales chinensis var.mairei endosperms
CN102715086A (en) * 2012-06-19 2012-10-10 湖南农业大学 Method for obtaining callus and suspension cells under induction of Taxus chinensis test tube plantlet
CN102715085A (en) * 2012-06-19 2012-10-10 湖南农业大学 Method for inducing seed embryos of Chinese yew to obtain calluses and suspension cells

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