CN102687667B - Method for inducing haploid endosperm callus tissues by using ripe endosperms of taxales chinensis var.mairei - Google Patents
Method for inducing haploid endosperm callus tissues by using ripe endosperms of taxales chinensis var.mairei Download PDFInfo
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Abstract
The invention discloses a method for inducing haploid endosperm callus tissues by using ripe taxales chinensis var.mairei endosperms, and the method comprises the following steps of: (1) seed exotesta peeling: mechanically breaking a shell, and manually peeling exotesta; (2) explants sterilization: sequentially sterilizing by using 70% alcohol for 90 seconds and 0.1% mercuric chloride for 12 minutes, and washing five times with sterile water for later use; (3) seed pretreatment: sealing with the sterile water, and soaking seeds for 1-5 days at normal temperature; (4) separation of endosperm tissues; and (5) in vitro induction of endosperm callus tissues: inoculating seed endosperms to a callus tissue-inducing culture medium, so as to obtain the 97.92% induction rate after 30 days. By utilizing the method, ripe taxales chinensis var.mairei endosperm callus tissues can be obtained conveniently and quickly, and the ripe taxales chinensis var.mairei endosperm callus tissues lay a foundation for simplifying genetic analysis and chromosome operation and are used as transgenic materials and genomics research materials.
Description
Technical field
The invention belongs to the Plant Tissue Breeding field, be specifically related to the method that the ripe endosperm of a kind of Chinese yew is induced monoploid endosperm callus.
Background technology
Taxol (Paclitaxel, trade name Taxol) be the 20th century six a kind of natural tetracyclic diterpene Alkaloids separated of the seventies from taxaceae (Taxaceae) Taxus (Taxus L.) plant bark, it has significant curative effect at aspects such as treatment cervical carcinoma, breast cancer, small-cell carcinoma of the lung, melanoma and colon cancers.Taxol at first extracts with forming in the layer at the bark of Pacific Ocean yewtree.According to present purification technique, the taxol of producing 1kg approximately needs 10 tons of bark of Ramulus et folium taxi cuspidatae.This approximately need could obtain the Chinese yew of from 2000 to 4000 60-70 lifes.According to the related data introduction, the annual whole world approximately needs to consume the 250kg pure product of paclitaxel.This need extract from 250 kilograms barks or 750,000 Chinese yew could satisfy the needs in this market.Along with increasing progressively day by day the taxol demand, the output of only extracting isolation of taxol from natural Chinese yew woods tree body can not satisfy the medical market needs at present, in addition exploitation without restraint make the Chinese yew natural resources day by day exhausted, the natural updating ability of taxus resource is poor, poor growth, extinction in imminent danger, national governments have expressly provided and have forbidden cutting down wild taxus resource.
Since nineteen twenty-one, Italy botanist Blakesly found the haplobiont of datura at occurring in nature, the monoploid material is because of its higher value, for example control that the hybrid generation separates, improves efficiency of selection, simplifies genetic analysis, the chromosome operation, come into one's own as transgenic line and genomics research material, continue mutually at the existing report of plant.But the acquisition of the monoploid material of report at present is except the monoploid that spontaneous mutation produces, its main path is to obtain by pollen cultivation, ovule cultured in vitro and microspores culture, and the Taxus gymnosperm, because its endosperm of material its own particularity is haploid tissue (Zhang Zongqin etc., Chinese yew, publishing house of Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology, 2010), so just can obtain the monoploid callus by the cultured in vitro of endosperm.The material that contains more inhibition seed germination in the Chinese yew seed, as containing germination inhicbitor matter such as positive enanthic acid, n-nonanoic acid, acetic acid in the southern enqlish yew seed, the soak of its kind skin, secundine and endosperm all has inhibitory action to Chinese cabbage seed germination rate and growth of seedlings, root growth, and with the proportional (Zhang Yanjie etc. of leaching liquor concentration, the research of southern enqlish yew seed kind germination inhicbitor, Nanjing Forestry University's journal (natural science edition), 2007,4(31): 51-58).
Also successfully do not report the report that obtains the monoploid callus by Chinese yew endosperm callus induction according to present research report.
Summary of the invention
The object of the present invention is to provide the ripe endosperm of a kind of Chinese yew to induce the method for monoploid endosperm callus.
The objective of the invention is to realize in the following manner.
The ripe endosperm of a kind of Chinese yew is induced the method for monoploid endosperm callus, may further comprise the steps:
(1) peeling off of seed embryo: the southern enqlish yew seed is peeled off out from exosper;
(2) explant sterilization: get the seed that step (1) obtains, sterile water wash is used in the alcohol with 70% and 0.1% mercury chloride surface sterilization again;
(3) preliminary treatment of seed: the seed of step (2) sterilization was soaked 1-5 days with sterile water;
(4) separation of endosperm tissue: the endosperm tissue that under aseptic condition, strips seed;
(5) in-vitro inducing of endosperm callus: the endosperm tissue of peeling off is inoculated into evoked callus on the inducing culture; Described inducing culture is: B
5Medium+Picloram(picloram) 2-5mg/L+6-BA0.5mg/L+GA
33mg/L+ sucrose 20g/L+ agar 7g/L, condition of culture is: secretly cultivate under 24 ± 1 ℃ of conditions.
Chinese yew seed in the described step (1) is life then or 4 ℃ of southern enqlish yew seeds of preserving 1-2.
Described step (2) cleaning and sterilizing process is specific as follows: the seed that will remove exosper is earlier put into aseptic container, and is successively through 70% alcohol disinfecting 90s and 0.1% mercury chloride sterilization 12min, standby behind the aseptic water washing 5 times in superclean bench.
Described step (3) seed preliminary treatment detailed process is as follows: after the seed that will remove exosper is handled through the surface sterilization of step (2), with sterile sealing immersion 1-5 days at ambient temperature of aseptic distilled water.
Step (5) obtains can carrying out the subculture cultivation behind the endosperm callus, and the medium that subculture is cultivated is: B
5Medium+Picloram2-3mg/L+6-BA0.5mg/L+GA
31mg/L+ sucrose 20g/L+ agar 7g/L; Condition of culture is secretly to cultivate under 24 ± 1 ℃ of conditions.
This invention has the following advantages: (1) explant pollution rate is extremely low; (2) the seed preliminary treatment is simple, handles conserve water resource in a large number with hydrostatic; (3) can obtain Chinese yew monoploid callus by step cultivation; Inductivity reaches 97.92%; (4) for simplifying genetic analysis, chromosome operation, laying the first stone as transgenic line and genomics research material, (4) lay the first stone for acquisition Chinese yew monoploid callus carries out the Chinese yew genome sequencing.
Description of drawings
Fig. 1 adopts the inventive method to utilize the ripe endosperm of Chinese yew to induce the process of monoploid endosperm callus,
A. Chinese yew seed and remove the seed of kind of skin wherein;
B. endosperm tissue is cultivated 12d at callus inducing medium;
C. endosperm tissue is cultivated 20d at callus inducing medium;
D. endosperm tissue is cultivated 30d at callus inducing medium;
E. the endosperm callus is cultivated 10d at subculture medium;
Fig. 2 is the flow cytometer cell detection results of the Chinese yew endosperm monoploid callus that obtains among the present invention,
The liploid plant ploidy testing result in F. embryo source wherein;
G. southern enqlish yew monoploid callus ploidy testing result.
Embodiment
Following examples are intended to further specify the present invention, and can not limit the present invention.
Embodiment 1
(1) peeling off of seed embryo: collect the southern enqlish yew seed of giving birth to then, use the broken shell of mechanical bond manual method, seed is peeled off out from exosper;
(2) explant sterilization: the seed that will remove exosper is earlier put into aseptic container, and is through 70% alcohol disinfecting 90s and 0.1% mercury chloride sterilization 12min, standby behind the aseptic water washing 5 times in superclean bench, soaks seed about 5 minutes during cleaning at every turn.
(3) preliminary treatment of seed: after will removing the surface sterilization processing of seed through step (2) of exosper, seal immersion 1-3 days at ambient temperature with aseptic distilled water.
(4) separation of endosperm tissue: under aseptic condition, strip the endosperm tissue of seed, other part rejects;
(5) inducing of endosperm callus: endosperm tissue is inoculated on the inducing culture under 24 ± 1 ℃ of conditions secretly cultivates, can obtain Chinese yew endosperm callus through 30 days cultivations, inductivity reaches 97.92%; Inducing culture is B
5Medium+Picloram3mg/L+6-BA0.5mg/L+GA
33mg/L+ sucrose 20g/L+ agar 7g/L; Constant when other conditions, and when the content of Picloram changed in the 2-5mg/L scope in the inducing culture, the equal well-grown of callus, inductivity all can reach more than 97%.
(6) subculture of endosperm callus is cultivated: the endosperm callus is inoculated on the subculture medium secretly cultivates under 24 ± 1 ℃ of conditions, subculture medium is B
5Medium+Picloram2mg/L+6-BA0.5mg/L+GA
31mg/L+ sucrose 20g/L+ agar 7g/L; Chinese yew endosperm callus can normally be bred cultivation; Constant when other conditions, and when the content of Picloram changed in the 2-3mg/L scope in the subculture medium, Chinese yew endosperm callus also can normally be bred cultivation.The result asks for an interview accompanying drawing.
Claims (4)
1. the ripe endosperm of a Chinese yew is induced the method for monoploid endosperm callus, it is characterized in that, may further comprise the steps:
(1) peeling off of seed embryo: southern enqlish yew (Taxales chinensis var.mairei) seed is peeled off out from exosper;
(2) explant sterilization: get the seed that step (1) obtains, with 70% alcohol surface sterilization 90s and 0.1% mercury chloride surface sterilization 12min, standby behind the aseptic water washing 5 times;
(3) preliminary treatment of seed: the seed of step (2) sterilization was soaked 1-5 days with sterile water;
(4) separation of endosperm tissue: the endosperm tissue that under aseptic condition, strips seed;
(5) in-vitro inducing of endosperm callus: the endosperm tissue of peeling off is inoculated into evoked callus on the inducing culture; Described inducing culture is: B
5Medium+Picloram2-5mg/L+6-BA0.5mg/L+GA
33mg/L+ sucrose 20g/L+ agar 7g/L, condition of culture is: secretly cultivate under 24 ± 1 ℃ of conditions.
2. method according to claim 1 is characterized in that,
Chinese yew seed in the described step (1) is life then or 4 ℃ of southern enqlish yew seeds of preserving 1-2.
3. method according to claim 1 is characterized in that,
Described step (3) seed preliminary treatment detailed process is as follows: after the seed that will remove exosper is handled through the surface sterilization of step (2), with sterile sealing immersion 1-5 days at ambient temperature of aseptic distilled water.
4. method according to claim 1 is characterized in that,
Step (5) obtains carrying out the subculture cultivation behind the endosperm callus, and the medium that subculture is cultivated is: B
5Medium+Picloram2-3mg/L+6-BA0.5mg/L+GA
31mg/L+ sucrose 20g/L+ agar 7g/L; Condition of culture is secretly to cultivate under 24 ± 1 ℃ of conditions.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201210203083 CN102687667B (en) | 2012-06-19 | 2012-06-19 | Method for inducing haploid endosperm callus tissues by using ripe endosperms of taxales chinensis var.mairei |
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| CN 201210203083 CN102687667B (en) | 2012-06-19 | 2012-06-19 | Method for inducing haploid endosperm callus tissues by using ripe endosperms of taxales chinensis var.mairei |
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| CN103598096B (en) * | 2013-11-12 | 2015-07-08 | 大连民族学院 | One-step inducing method of callus of stem of excellent-quality taxus cuspidata |
| CN103563750B (en) * | 2013-11-12 | 2015-03-18 | 大连民族学院 | De-browning method of browned calluses of taxus cuspidata |
| CN111134123A (en) * | 2019-12-19 | 2020-05-12 | 东莞市东阳光冬虫夏草中药有限公司 | Seed germination treatment agent and application |
| CN113475401A (en) * | 2021-08-05 | 2021-10-08 | 云南农业大学 | Rapid propagation method of aconitum vilmorinianum kom |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ATE146527T1 (en) * | 1991-02-12 | 1997-01-15 | Nippon Steel Corp | METHOD FOR PRODUCING TAXOL USING TAXUS SPECIES CELL CULTURE |
| JPH06296493A (en) * | 1993-04-14 | 1994-10-25 | Nippon Steel Corp | Method for producing taxol |
| US5547866A (en) * | 1994-07-20 | 1996-08-20 | The Regents Of The University Of California | Taxane production in haploid-derived cell cultures |
| US6365407B1 (en) * | 2001-03-05 | 2002-04-02 | Council Of Scientific & Industrial Research | Culture medium composition useful for induction and proliferation of Taxus calli |
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