CN102783419A - Efficient regeneration system method for mint internode stems - Google Patents
Efficient regeneration system method for mint internode stems Download PDFInfo
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- CN102783419A CN102783419A CN201210301821XA CN201210301821A CN102783419A CN 102783419 A CN102783419 A CN 102783419A CN 201210301821X A CN201210301821X A CN 201210301821XA CN 201210301821 A CN201210301821 A CN 201210301821A CN 102783419 A CN102783419 A CN 102783419A
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Abstract
An efficient regeneration system method for mint internode stems is established by using morphological second, third and fourth internode stems of an aseptic mint seedling as material, inoculating the material to adventitious bud induction medium, and researching by key technologies such as induction, elongation growth, subculture, seedling strengthening, rooting and the like. The efficient regeneration system method for mint internode stems has the advantages that regeneration rate of induced adventitious buds is up to 95%, stability is high, operation is simple, regeneration cycle is short, and the regeneration system can serve as a receptor system for genetic transformation of Agrobacterium Tumefaciens.
Description
Technical field
The method that the present invention relates to the highly efficient regeneration of a kind of peppermint internode stem section is set up
Technical background
Be rich in volatile oil in the stem of peppermint (Mentha haplocalyx Briq.), the leaf, be widely used in industry such as food, medicine, cosmetics, spices, tobacco.The peppermint all herbal medicine is used to treat anemopyretic cold, wind-warm syndrome from the beginning of, headache, hot eyes, larynx numbness, abscess of throat, aphthae, toothache, nettle rash, rubella etc.Peppermint is one of the important spices of China and medicinal plant, in Jiangsu, ground such as Anhui, Jiangxi widely cultivate, and have become local important agriculture extraordinary economic crops.But single, the deterioration of variety phenomenon of kind that in peppermint produces, occurs makes the breeding work of peppermint more and more noticeable.Utilize transgenic technology to cultivate the good new varieties of peppermint, have the genes of interest source extensively, the advantage of orientable change objective trait.Set up a kind of regenerative system of tissue culture efficiently and it is created as the transgene receptor system and obtains the successful key of transgenic breeding the most at last.At present the quick propagating system to domestic peppermint kind mainly is regrown material with the stem apex, but the short extensive expansion that is suitable for the peppermint kind of this method stem apex breeding time is numerous and be not suitable for the foundation of genetic transforming method.Therefore, for the ease of better carrying out the Study on Genetic Transformation of domestic Chinese medicine peppermint kind, must set up a kind of genetic transformation system of perfect Chinese medicine peppermint main breed.
Summary of the invention
Goal of the invention: the highly efficient regeneration genetic transformation system that the purpose of this invention is to provide a kind of Chinese medicine peppermint kind.The present invention is an object with domestic peppermint cultivar, has set up the genetic transformation system of high regeneration rate, stable, good reproducibility, lays the foundation for utilizing technique for gene engineering to carry out genetic breeding.Aspects such as China's peppermint germ plasm resource innovation and improvement had positive role and realistic meaning
Particular content:
1, the present invention provides a kind of peppermint (Mentha haplocalyx Briq.) stem section tissue culture quick breeding new method, is undertaken by the several steps of following order:
(1) foundation of explant collection and sterile system
Gather the peppermint terminal bud 3～June, after washing powder solution soaks 15min, flowing water flushing 30min.With 75% (V/V) alcohol disinfecting 30s, use 0.2% (m/V) HgCl2 solution soaking 15min again, aseptic water washing 3 times before the inoculation.After blotting surperficial moisture content, peel off outer blade, be cut into the long stem apex of 0.5cm, be seeded in the following medium
25 ± 3 ℃ of temperature, illuminance 1500～3000lx, light application time 10～12h/d cultivates 25～45d follow-up generation behind 30d on the same medium, to obtain the aseptic seedling material.
(2), internode stem section adventitious bud induction culture: under aseptic condition, with sterilizable material the 2nd, 3,4 internode stem sections are inoculated in the following inducing culture.
The MS minimal medium
25 ± 3 ℃ of temperature, secretly cultivate 40～50d, internode stem section adventitious bud induction frequency can reach more than 90%.
(3), indefinite bud subculture grown cultures: the indefinite bud that step (2) obtains is cut, be inoculated in the following medium.
The MS basic culture solution
25 ± 3 ℃ of temperature, illuminance 1500～3000lx, light application time 10～12h/d cultivates 30d;
(4), strong seedling culture: before taking root, need carry out strong seedling culture for what further improve transplanting survival rate.The indefinite bud that step (3) is obtained changes in the following strong seedling culture base.
The MS basic culture solution
At 25 ± 3 ℃ of temperature, illuminance 1000～2000lx, light application time 10～12h/d.Cultivate 20～35d.
(5), culture of rootage: the aseptic seedling with step (4) acquisition plant height 5.0～8.0cm changes in the following root media.
The 1/2MS minimal medium
At 25 ± 3 ℃ of temperature, illuminance 1000～2000lx, light application time 10～12h/d.After cultivating 15～20d, rooting rate can reach more than 90%, and the leaf look dark green, robust growth.
Beneficial effect:
The successful structure of the present invention through internode stem section adventitious shoot regeneration system accomplished explant selection, induced differentiation, strong sprout, key technology such as take root.This regenerating system adventitious bud induction frequency can reach 95%, efficient stable, good reproducibility, and the adventitious shoot regeneration cycle is short, is applicable to the receptor system as genetic transformation.
Embodiment
Below in conjunction with embodiment the present invention is done further explain, but embodiment only is exemplary, and the present invention is not constituted any limitation.What it should be appreciated by those skilled in the art is under the situation that does not depart from spirit and scope of the invention, can make amendment or replace the details of technical scheme of the present invention and form, and these modifications and replacement all falls in protection scope of the present invention.
Embodiment one
Peppermint kind ' 738 ' internode stem section adventitious shoot regeneration, undertaken by the several steps of following order:
1, the foundation of explant collection and sterile system
Gather the peppermint terminal bud 3～June, after washing powder solution soaks 15min, flowing water flushing 30min.With 75% (V/V) alcohol disinfecting 30s, use 0.2% (m/V) HgCl2 solution soaking 15min again, aseptic water washing 3 times before the inoculation.After blotting surperficial moisture content, peel off outer blade, be cut into the long stem apex of 0.5cm, be seeded in the MS medium that contains 1.0mgL-1BA and 0.2mgL-1NAA, contain 5.6gL-1 agar, 30gL-1 sucrose in the medium, pH5.5～5.8,121 ℃ sterilization 20min.Cultivation temperature is 25 ℃, light application time 8～10h, intensity of illumination 1500～2000lx.The healthy aseptic plant of three week of cultivation back choosing is inoculated in and carries out successive transfer culture 30d in the same medium.
2, internode stem section adventitious bud induction culture: select through aseptic peppermint seedling morphology the 2nd, 3,4 positions of initial culture not stem-segment with node as the material of evoking adventive bud.The scale bottom is inoculated into callus inducing medium, and (MS+CW25%+TDZ3.0mgL-1+IAA0.2mgL-1+ sucrose 25g/L+ agar 5.6g/L advance to cultivate in pH5.8).25 ± 3 ℃ of cultivation temperature are secretly cultivated 40d.The differentiation adventitious buds rate can reach more than 95%.
3, indefinite bud elongation is cultivated: the indefinite bud that differentiates is seeded in contains 1.0mgL
-1BA and 0.2mgL
-1In the MS medium of NAA, contain 5.6gL in the medium
-1Agar, 30gL
-1Sucrose, pH5.5～5.8,121 ℃ sterilization 20min.Cultivation temperature is 25 ± 3 ℃, light application time 8～10h, intensity of illumination 1500～2000lx.
4, strong seedling culture: step 4 is obtained the plant of exsomatizing, change strong seedling culture base (MS+6-BA1.0mgL over to
-1+ NAA0.5mgL
-1+ sucrose 20g/L+ agar 5.8g/L cultivates in pH5.8).25 ± 3 ℃ of cultivation temperature, illuminance 1500lx, light application time 12h/d.After cultivating 30d, regeneration plant leaf look dark green, robust growth.
5, culture of rootage: step 4 is obtained 5～8cm seedling be inoculated into root media (MS+6-BA0.2mgL
-1+ NAA0.5mgL
-1+ sucrose 20g/L+ agar 5.8g/L carries out root induction in pH5.8).Rooting rate reaches more than 95% several 5-10 bars of taking root after cultivating 30d.
Used reagent in all above-mentioned experimentations all can be given birth to worker's biotechnology Co., Ltd available from Sigma and Shanghai.
Claims (5)
1. the method for peppermint internode stem section highly efficient regeneration is characterized in that may further comprise the steps:
(1) explant selection and sterilization: get the peppermint stem apex as explant, after rinsing well, carry out disinfection;
(2) inducing of internode stem section indefinite bud: get the aseptic seedling internode that obtains in the step (1) as explant, be inoculated in the adventitious bud induction culture base, 25 ± 3 ℃ of cultivation temperature were secretly cultivated 40 days; Said inducing culture is the MS minimal medium and adds hormone TDZ, IAA; TDZ concentration is 2.0-4.0mgL-1 in the said inducing culture, and IAA concentration is 0.2-0.5mgL-1;
(3) the indefinite bud subculture is grown: the indefinite bud that induces in the step (2) is cut to insert in the indefinite bud subculture growth medium cultivated 30 days, said subculture growth medium is the MS medium and adds hormone 6-BA and NAA; 6-BA concentration is 0.5-1.0mgL in the said subculture growth medium
-1, NAA concentration is 0.1-0.5mgL
-1Said normal cultured condition is 25 ± 3 ℃ of cultivation temperature, light application time 8-10h, intensity of illumination 1500-2000lx;
(4) strong seedling culture of aseptic seedling: with in the step (3) to aseptic seedling be inoculated in the strong seedling culture base and cultivated 20-35 days, said subculture growth medium is the MS medium and adds hormone 6-BA and NAA; 6-BA concentration is 0.5-1.0mgL in the said subculture growth medium
-1, NAA concentration is 0.3-0.5mgL
-1Said normal cultured condition is 25 ± 3 ℃ of cultivation temperature, light application time 8-10h, intensity of illumination 1500-2000lx;
(5) culture of rootage: the aseptic seedling that step (4) is obtained is inoculated in the root media cultivated 15-20 days, and said root media is the 1/2MS medium and adds hormone 6-BA and NAA; 6-BA concentration is 0.1-0.5mgL in the said subculture growth medium
-1, NAA concentration is 0.5-1.0mgL
-1Said normal cultured condition is 25 ± 3 ℃ of cultivation temperature, light application time 8-10h, intensity of illumination 1500-2000lx;
All contain agar 5.5-7.0g/L, sucrose 25-35g/L in above-mentioned inducing culture, subculture medium, strong seedling culture base, the root media, and the pH value is 5.6-5.8.
2. according to the said peppermint internode of claim 1 stem section adventitious bud inducing method, it is characterized in that: the internode explant in the said step (2) for (1) step gained aseptic seedling the 2nd, 3,4 successively between the stem section.
3. according to the said peppermint internode of claim 1 stem section adventitious bud inducing method, it is characterized in that: said step also contains Coconut Juice in (2), and Coconut Juice concentration is 20%-25% in the said adventitious bud induction culture base.
4. run into method according to the said peppermint internode of claim 1 stem section indefinite bud, it is characterized in that: the normal cultured time in the step of telling (2) be 40 days; The subculture grown cultures time is 30 days in the said step (3); Growth time in strong sprout in the said step (4) is 20-35 days; The culture of rootage time is 15-20 days in the said step (5).
5. according to the arbitrary said peppermint of claim 1 to 3 internode stem section adventitious bud inducing method not, it is characterized in that: the stem apex explant has drawn from for 3～June in the said step (1); The process that carries out disinfection in the said step (1) is: after explant is soaked 15 minutes with washing powder solution, and flowing water flushing 20-30min.75% ethanol disinfection is 30 seconds on the sterile working platform, uses 0.2%HgCl again
2Solution soaking 15 minutes, aseptic water washing 3 times.
Said internode stem section material is aseptic seedling morphology the 2nd, 3,4 internode stem sections.
Said adventitious bud induction culture base is the MS minimal medium.
Said certain density plant hormone is TDZ2.0～4.0mgL
-1And IAA0.2～0.5mgL
-1
Said additive is 20%～25% Coconut Juice.
With inducing the indefinite bud that differentiates after 40 days to be inoculated on the bud elongation growth medium.Said strong seedling culture base is MS minimal medium+0.5～1.0mgL
-16-BA+0.3～1.0mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH5.5～5.8; Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx; Cultivate 20d, switching propagation 2 times;
Said root media is 1/2MS+0.1～0.5mgL
-16-BA+0.3～1.0mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH5.5～5.8; Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx; Cultivate 20d.
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| CN201210301821XA CN102783419A (en) | 2012-08-23 | 2012-08-23 | Efficient regeneration system method for mint internode stems |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563743A (en) * | 2012-08-07 | 2014-02-12 | 高山林 | Commercial crop meristem culture virus-free novel technology |
| CN105393920A (en) * | 2015-12-18 | 2016-03-16 | 江苏省中国科学院植物研究所 | Method for establishing mint leaf high-efficiency regeneration system |
| CN105794639A (en) * | 2016-03-28 | 2016-07-27 | 安徽科技学院 | Mint leaf adventitious bud induction and plant regeneration method |
| CN106755063A (en) * | 2016-11-28 | 2017-05-31 | 山东省科学院生态研究所 | Agriculture bacillus mediated salt tolerant American mint stem section conversion system |
| CN107197673A (en) * | 2017-06-19 | 2017-09-26 | 贵州山乡原生态农业开发有限公司 | The cultural method and nutrient solution of a kind of peppermint |
| CN109234312A (en) * | 2017-07-11 | 2019-01-18 | 江苏省中国科学院植物研究所 | It is a kind of using peppermint internode stem section as the genetic transforming method of explant |
| CN115777533A (en) * | 2022-11-08 | 2023-03-14 | 中国林业科学研究院热带林业研究所 | Regeneration method taking betula alnoides internode stem section as explant |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
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| CN1181419A (en) * | 1996-10-29 | 1998-05-13 | 科学工业研究委员会 | Tissue culture processfor producing large number of viable mint plants in vitro |
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| XIA LI等: "EFFICIENT PLANT REGENERATION OF NATIVE SPEARMINT (MENTHA SPICATA L.)", 《IN VITRO CELL. DEV. BIOL.-PLAN》 * |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563743A (en) * | 2012-08-07 | 2014-02-12 | 高山林 | Commercial crop meristem culture virus-free novel technology |
| CN105393920A (en) * | 2015-12-18 | 2016-03-16 | 江苏省中国科学院植物研究所 | Method for establishing mint leaf high-efficiency regeneration system |
| CN105794639A (en) * | 2016-03-28 | 2016-07-27 | 安徽科技学院 | Mint leaf adventitious bud induction and plant regeneration method |
| CN106755063A (en) * | 2016-11-28 | 2017-05-31 | 山东省科学院生态研究所 | Agriculture bacillus mediated salt tolerant American mint stem section conversion system |
| CN106755063B (en) * | 2016-11-28 | 2020-05-19 | 山东省科学院生态研究所 | Agrobacterium-mediated salt-tolerant peppermint stem segment transformation system |
| CN107197673A (en) * | 2017-06-19 | 2017-09-26 | 贵州山乡原生态农业开发有限公司 | The cultural method and nutrient solution of a kind of peppermint |
| CN109234312A (en) * | 2017-07-11 | 2019-01-18 | 江苏省中国科学院植物研究所 | It is a kind of using peppermint internode stem section as the genetic transforming method of explant |
| CN109234312B (en) * | 2017-07-11 | 2021-11-30 | 江苏省中国科学院植物研究所 | Genetic transformation method taking mint internode stems as explants |
| CN115777533A (en) * | 2022-11-08 | 2023-03-14 | 中国林业科学研究院热带林业研究所 | Regeneration method taking betula alnoides internode stem section as explant |
| CN115777533B (en) * | 2022-11-08 | 2023-08-29 | 中国林业科学研究院热带林业研究所 | Regeneration method using betula alnoides internode stem as explant |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
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Application publication date: 20121121 |