CN101781661A - Agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation method - Google Patents
Agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation method Download PDFInfo
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Abstract
The invention relates to an agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation method; in the method, a single bacterial colony of phenol-resistance agrobacterium rhizogene strain which is selected to be bred by self is cultivated in YEB culture solution containing kanamycin to obtain bacterial liquid which is activated; organs of tea tree varieties containing below 25 percent of polyphenol content are selected as explants, and microtrauma pretreatment is carried out to the explants; the explants after carrying out microtrauma pretreatment are dip-dyed in the activated bacterial liquid; and then the dip-dyed explants are co-cultured and carries out bacteria-elimination culture to obtain tea tree rooting; the rooting frequency of the agrobacterium rhizogene mediated tea tree for genetic transformation is improved to 60 percent from 30 percent in research at home and abroad, thereby providing a new effective way for tea tree breed improvement by genetic transformation of the agrobacterium rhizogene mediate and biologically synthesizing secondary metabolism products such as catechin by utilizing tea tree rooting culture.
Description
Technical field the present invention relates to the method for a kind of agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation, belongs to the plant gene engineering technology field.
Background technology Agrobacterium rhizogenes (Agrobacterium rhizogenes) is a kind of important tool of plant genetic conversion and transgenic research, and the root of hair cultivation is the important channel of biosynthesizing Secondary Metabolism of Plant product.Tea tree (Camelliasinensis) is a kind of perennial woody plant of being rich in polyphenols such as catechin, studies show that in a large number: this class polyphenols in the tea tree has obvious sterilization or bacteriostatic action [Mao Qingli, Shi Zhaopeng, Li Ling, etc. tea catechin health care and pharmacological function research new development [J]. Food science .2007 (08).; Wang Jing, Qi Xiangyang, Zhu Xueliang, Deng. the bacteriostatic activity research [J] of NVP-XAA 723 and different oxidation fractions thereof. research and development of natural products .2008: Du Xiao, Li Ning, Zhou Rujuan, Deng. the bacteriostatic action of isolating catechin and polymkeric substance thereof [J] in the plant. the journal .2005 of Sichuan Agricultural University, 23 (3): 374-378], the lignifying structure of this bacteriostatic action and tea tree cell will directly or indirectly hinder the genetic transformation of Agrobacterium rhizogenes to tea tree, it is extremely low that this just utilizes Agrobacterium rhizogenes mediation that tea tree is carried out the genetic transformation rate both at home and abroad, does the Agrobacterium rhizogenes mediated tea tree carry out the only (difference of the root of hair frequency of genetic transformation rate and genetic transformation about 30% of root of hair frequency of genetic transformation?) [Zhang Guanghui, Liang Yuerong, Lu Jianliang. the tea tree root of hair high-frequency induction and the genetic transformation [J] of Agrobacterium rhizogenes mediation. tea science .2006,26 (1): 1-10; Xi Biao, Liu Zusheng, Liang Yuerong, etc. the tea tree genetic transformation [J] of Agrobacterium rhizogenes mediation. tea science .1997,17 (supplementary issue): 155-156.4,5; Zebra M, Banerjee S, Mathur A K, et al.Induction of hairy roots in tea (Camellia sinensisL.) [J] .Current Science.1996,70 (1): 84-86.] and the tea tree transgenic breeding major cause that is difficult to success.
Summary of the invention the purpose of this invention is to provide the method for a kind of agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation, and it is higher that this method makes the Agrobacterium rhizogenes mediated tea tree carry out the root of hair frequency of genetic transformation.For the genetic transformation of Agrobacterium rhizogenes mediation is applied to the tea tree breed improvement and utilizes the tea tree root of hair to cultivate secondary meta-bolites such as biosynthesizing catechin an effective way is provided.
Technical scheme provided by the invention is: the method for a kind of agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation, with single bacterium colony of the phenol-resistance agrobacterium rhizogene strain of seed selection voluntarily, the bacterium liquid of in containing the YEB nutrient solution of kantlex, cultivating activatedly; Select for use contain Polyphenols content below 25% the organ of tea tree breed as explant, explant is carried out the microtrauma pre-treatment; The pretreated explant of microtrauma is contaminated in activated bacterium liquid; The explant of contaminating is obtained the tea tree root of hair through cultivating altogether and taking off the bacterium cultivation.
The phenol-resistance agrobacterium rhizogene strain of described seed selection voluntarily obtains by laxative remedy: agrobacterium rhizogene strain (Agrobacterium rhizogenes) is transferred to the YEB liquid nutrient medium that contains kantlex carries out actication of culture, then this activation bacterium liquid 100 μ l are coated on that to contain tea-polyphenol concentration be to carry out antibacterial cultivation 24-48 hour on 25-800 μ g/ml and the AB solid medium that contains kantlex, single bacterium colony that picking still can be grown under 200 μ g/ml tea-polyphenol concentration, be transferred to the AB liquid nutrient medium that contains kantlex that contains 200 μ g/ml concentration tea-polyphenol again and carry out actication of culture, then with activatory bacterium liquid 100 μ l separate application to containing 200,400, the flat board of the AB liquid nutrient medium that contains kantlex of the tea-polyphenol of 800 and 1000 μ g/ml concentration carries out once more screening of anti-phenol and activation culture, carries out 2-3 anti-phenol screening according to this until obtaining at still can the grow strong agrobacterium rhizogene strain of anti-phenol of formation bacterium colony of 800-1000 μ g/ml concentration tea-polyphenol substratum.
The described YEB liquid nutrient medium that contains kantlex is obtained by laxative remedy: with the 1g yeast extract, and 5g beef extract, 5g peptone, 0.5gMgSO
47H
2O, the 5g sucrose dissolved is regulated pH value to 7.2 with 1NnaOH again in the distilled water of 900ml, be settled to 1000ml with distilled water then, steam sterilizing treats that the substratum temperature reduces to below 60 ℃ the concentration 100mg/ml kantlex 1ml that adds through the sterilization of 0.22 μ filtering with microporous membrane.
The described kanamycin A B solid medium that contains is obtained by laxative remedy: with 3gK
2HPO
4, 1gNaH
2PO
4, 0.15gKCl, 0.5gMgSO
47H
2O, 1gNH
4Cl, 0.01CaCl
2, 2.5mg FeSO
47H
2O, 5g sucrose and 15g agar are dissolved in the distilled water of 900ml, regulate pH value to 7.2 with 1N NaOH, and steam sterilizing is treated that the substratum temperature is reduced to and added the concentration 100mg/ml kantlex 1ml that sterilizes through 0.22 μ filtering with microporous membrane below 60 ℃; The described kanamycin A B liquid nutrient medium that contains is obtained by laxative remedy: with 3gK
2HPO
4, 1gNaH
2PO
4, 0.15gKCl, 0.5gMgSO
47H
2O, 1gNH
4Cl, 0.01CaCl
2, 2.5mg FeSO
47H
2O and 5g sucrose dissolved are in the distilled water of 900ml, regulate pH value to 7.2 with 1N NaOH, be settled to 1000ml with distilled water then, steam sterilizing treats that the substratum temperature reduces to below 60 ℃ the concentration 100mg/ml kantlex 1ml that adds through the sterilization of 0.22 μ filtering with microporous membrane.
The content of catechin 〉=80% in the described tea-polyphenol.
Described tea-polyphenol contains: caffeine 0.26%, epigallocatechin 2.66%, dl-catechin 0.71%, l-Epicatechol 2.15%, epigallocatechin Nutgalls ester 62.83%, l-Epigallocatechol Nutgalls ester 3.03%, l-Epicatechol Nutgalls ester 16.79%.
Described explant is obtained by laxative remedy: get the tea tree seed and clean with clear water. and strip out exosper, in 75% ethanol, soak 1min, wash down ethanol, use 0.1wt%HgCl again with sterilized water
2Sterilization 10min, aseptic water washing 5 times places 1 week back sprouting on the MS substratum, continues to cultivate at the MS substratum 4-6 week to obtain test-tube plantlet, and the organ (as root or stem) of getting test-tube plantlet is as explant.
Described activated bacterium liquid is obtained by laxative remedy: the phenol-resistance agrobacterium rhizogene strain list bacterium colony that picking is free, and to containing 100mgL
-1The YEB nutrient solution of kantlex, in dark, 25 ℃, the 120rpm shaking table is cultivated 24～36h.
Describedly explant is carried out the microtrauma pre-treatment be: put it under aseptic condition in the MS0 nutrient solution, use 120W, the ultrasonic cleaner of 40KHz carries out the ultrasonication of 15min.
The described dip-dye in activated bacterium liquid is: under aseptic condition, in the bacterium liquid that the aseptic immersion of the pretreated explant somatocyte of microtrauma is activated, soak 10-20min and take out, blot bacterium liquid with aseptic filter paper.
The described explant of cultivating to contaminating altogether places on the MS solid medium that contains IBA5.0 or NAA0.1mg/L, and 25 ℃, the dark 24-48h that cultivates altogether down.
Describedly take off bacterium and cultivate and to be: the explant that will cultivate altogether is through aseptic water washing 4-5 time, moves to behind the aseptic filter paper absorption table water to contain 500mgL
-1Cultivate on the 1/2MS0 solid medium of cephamycin, 25 ℃, under dark condition, take off bacterium and cultivate, 2-5d transfers once, till no bacterial plaque.
The present invention cultivates in advance earlier the pretreated explant of microtrauma again and contaminates in activated bacterium liquid; Described pre-cultivation is: the stem section or (4～6) * (4～6) mm that explant are cut into 8～12mm
2Leaf dish explant is at MS
0The pre-1-3d that cultivates on the substratum.
The present invention is directed to tea tree belongs to the perennial woody plant and is rich in feature and the agriculture bacillus mediated extremely low problem of genetic transformation rate such as Polyphenols, use the phenol-resistance agrobacterium rhizogene strain (as R1000AP) of seed selection voluntarily, select for use and contain the lower kind of Polyphenols (as the green tea kind) and organ (root or stem) is made explant, cooperation is carried out microtrauma pre-treatment (as wither or ultrasonic) to explant somatocyte, the efficient genetic conversion system of tea tree of Agrobacterium rhizogenes Ri mediation has successfully been set up in measures such as dip-dye and common cultivation.Utilize this transformation system, the root of hair frequency that makes the Agrobacterium rhizogenes mediated tea tree carry out genetic transformation brings up to nearly 60% from about 30% of domestic and international research.This genetic transformation for Agrobacterium rhizogenes mediation is applied to the tea tree breed improvement and utilizes the tea tree root of hair to cultivate secondary meta-bolites such as biosynthesizing catechin an effective way is provided.
Embodiment
The seed selection phenol-resistance agrobacterium rhizogene strain: the original agrobacterium rhizogene strain R1000 that stores with the transfering loop picking is transferred to the YEB liquid nutrient medium that contains Km and carries out actication of culture, being applied to this activation bacterium liquid 100 μ l containing tea-polyphenol (TC80) concentration then is to carry out antibacterial cultivation on 100 μ g/ml and the AB solid medium that contains Km, when treating that most of culture dish has bacterium colony to grow (cultivating approximately 24-48 hour), single bacterium colony that picking still can be grown under 200 μ g/ml tea-polyphenol concentration, be transferred to the liquid nutrient medium that contains respective concentration TC80 and carry out actication of culture, then activatory bacterium liquid 100 μ l are applied to and contain 200 respectively, 400, the flat board of the AB liquid nutrient medium that contains kantlex of the tea-polyphenol of 800 and 1000 μ g/ml concentration carries out once more anti-phenol screening and activation culture, carry out twice anti-phenol screening according to this, obtain the strong Agrobacterium rhizogenes of anti-phenol that still can grow at the substratum of 800～1000 μ g/ml tea-polyphenol concentration.
Annotate: the YEB liquid nutrient medium that contains Km is obtained by laxative remedy: with the 1g yeast extract, and 5g beef extract, 5g peptone, 0.5gMgSO
47H
2O, the 5g sucrose dissolved is regulated pH value to 7.2 with 1NnaOH again in the distilled water of 900ml, be settled to 1000ml with distilled water then, high pressure steam sterilization (15bf/in
2, 20min), treat that the substratum temperature reduces to below 60 ℃ (feel is not scalded) and add kantlex (Kanamycin sulfate, Km) 100mg/L through 0.22 μ filtration sterilization.
Containing Km AB substratum is obtained by laxative remedy: with 3gK
2HPO
4, 1gNaH
2PO
4, 0.15gKCl, 0.5gMgSO
47H
2O, 1gNH
4Cl, 0.01CaCl
2, 2.5mg FeSO
47H
2O, 5g sucrose is regulated pH value to 7.2 (adding 15g agar when joining solid medium) with 1N NaOH, is settled to 1000ml with distilled water then, high pressure steam sterilization (15bf/in
2, 20min) treat that the substratum temperature reduces to below 60 ℃ (feel is not scalded) and add kantlex (Kanamycinsulfate, Km) 100mg/L through 0.22 μ filtration sterilization.
Tea-polyphenol goods (providing) by Changsha Jinnong Natural Plant Product Industries Co., Ltd, wherein catechin consists of: caffeine 0.26%, epigallocatechin 2.66%, dl-catechin 0.71%, l-Epicatechol 2.15%, epigallocatechin Nutgalls ester 62.83%, l-Epigallocatechol Nutgalls ester 3.03%, l-Epicatechol Nutgalls ester 16.79%, all the other are impurity; Catechin total amount 88.17%.
Agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation
Test-tube plantlet is cultivated: get tea tree (green tea kind, as Fuding Da Bai, East Lake morning and green perfume are early) seed and clean with clear water. and strip out kind of a skin, in 75% ethanol, soak 1min, wash down ethanol, use 0.1%HgCl again with sterilized water
2Sterilization 10min, aseptic water washing 5 times places on the MS substratum (can add BA/IBA=2/1,1mgL
-1/ 0.5mgL
-1) 1 week back sprouts, continue to cultivate to the height of seedling 6cm (about 4-6 week) at the MS substratum and can be used as explant material for conversion.
The activation of bacterial classification:, go to and contain 100mgL from the phenol-resistance agrobacterium rhizogene strain list bacterium colony of the above-mentioned seed selection of YEB slant medium picking
-1The YEB nutrient solution of kantlex (25-30ml/100ml triangular flask), in dark, 25 ℃, 120rpm shake-flask culture 24～36h (bacterium liquid OD
600nm=0.8-1.0 is equivalent to 8*10
6-10
7Individual bacterium/ml bacterium liquid) is used for contaminating.
Ultrasonication: explant puts it under aseptic condition in the MS0 nutrient solution before dip-dye, uses 120W, the ultrasonic cleaner of 40KHz, the ultrasonication of about 15min.
The pre-cultivation: in order to improve the susceptibility of explant, the test tube aseptic seedling is cut into stem section and 5*5mm about 10mm to Agrobacterium
2Leaf dish explant, the pre-1-3d that cultivates on the MS0 substratum.
Contaminate: under aseptic condition, the aseptic explant immersion is in the activated bacterium liquid, soaks 10-20min and take out.Blot unnecessary bacterium liquid with aseptic filter paper.
Cultivate altogether: the explant that will contaminate places on the MS solid medium that contains IBA5.0 or NAA0.1mg/L, and 25 ℃, the dark 24-48h that cultivates altogether down.
Take off bacterium and cultivate to obtain root of hair: the explant of cultivating is through aseptic water washing 4-5 time altogether, moves to behind the aseptic filter paper absorption table water to contain 500mgL
-1(cefotaxime sodium is cultivated 25 ℃ to cephamycin on 1/2MS0 solid medium Cefotaxime), taking off bacterium under dark condition cultivates, the 2-5d switching once generally can be finished after 4-5 switching and take off bacterium (up to no bacterial plaque), and the general back of contaminating just can obtain the tea tree root of hair in 5-6 week.Its induction frequency reaches 60%.
Claims (10)
1. the method for agrobacterium rhizogene mediated tea tree rooting high-frequency induction and genetic transformation is with single bacterium colony of the phenol-resistance agrobacterium rhizogene strain of seed selection voluntarily, the bacterium liquid of cultivating activatedly in containing the YEB nutrient solution of kantlex; Select for use contain Polyphenols content below 25% the organ of tea tree breed as explant, explant is carried out the microtrauma pre-treatment; The pretreated explant of microtrauma is contaminated in activated bacterium liquid; The explant of contaminating is obtained the tea tree root of hair through cultivating altogether and taking off the bacterium cultivation;
The phenol-resistance agrobacterium rhizogene strain of described seed selection voluntarily obtains by laxative remedy: agrobacterium rhizogene strain (Agrobacterium rhizogenes) is transferred to the YEB liquid nutrient medium that contains kantlex carries out actication of culture, then this activation bacterium liquid 100 μ l are coated on that to contain tea-polyphenol concentration be to carry out antibacterial cultivation 24-48 hour on 25-800 μ g/ml and the AB solid medium that contains kantlex, single bacterium colony that picking still can be grown under 200 μ g/ml tea-polyphenol concentration, be transferred to the AB liquid nutrient medium that contains kantlex that contains 200 μ g/ml concentration tea-polyphenol again and carry out actication of culture, then with activatory bacterium liquid 100 μ l separate application to containing 200,400, the flat board of the AB liquid nutrient medium that contains kantlex of the tea-polyphenol of 800 and 1000 μ g/ml concentration carries out once more screening of anti-phenol and activation culture, carries out 2-3 anti-phenol screening according to this until obtaining at still can the grow strong agrobacterium rhizogene strain of anti-phenol of formation bacterium colony of 800-1000 μ g/ml concentration tea-polyphenol substratum.
2. method according to claim 1 is characterized in that: described explant is obtained by laxative remedy: get the tea tree seed and clean with clear water. and strip out exosper, in 75% ethanol, soak 1min, wash down ethanol, use 0.1wt%HgCl again with sterilized water
2Sterilization 10min, aseptic water washing 5 times places 1 week back sprouting on the MS substratum, continues to cultivate at the MS substratum 4-6 week to obtain test-tube plantlet, and the organ of getting test-tube plantlet is as explant.
3. method according to claim 1 and 2 is characterized in that: activated bacterium liquid is obtained by laxative remedy: the phenol-resistance agrobacterium rhizogene strain list bacterium colony that picking is free, and to containing 100mgL
-1The YEB nutrient solution of kantlex, in dark, 25 ℃, the 120rpm shaking table is cultivated 24～36h.
4. method according to claim 1 and 2 is characterized in that: describedly explant is carried out the microtrauma pre-treatment be: put it under aseptic condition in the MS0 nutrient solution, use 120W, the ultrasonic cleaner of 40KHz carries out the ultrasonication of 15min.
5. method according to claim 1 and 2, it is characterized in that: dip-dye is in activated bacterium liquid: under aseptic condition, in the bacterium liquid that the aseptic immersion of the pretreated explant somatocyte of microtrauma is activated, soak 10-20min and take out, blot bacterium liquid with aseptic filter paper.
6. method according to claim 1 and 2 is characterized in that: the described explant of cultivating to contaminating altogether places on the MS solid medium that contains IBA5.0 or NAA0.1mg/L, and 25 ℃, the dark 24-48h that cultivates altogether down.
7. method according to claim 1 and 2, it is characterized in that: describedly take off bacterium and cultivate and to be: the explant that will cultivate altogether is through aseptic water washing 4-5 time, move on the 1/2MS0 solid medium that contains the 500mgL cephamycin behind the aseptic filter paper absorption table water and cultivate, 25 ℃, taking off bacterium under dark condition cultivates, 2-5d transfers once, till no bacterial plaque.
8. method according to claim 1 and 2 is characterized in that: the described YEB liquid nutrient medium that contains kantlex is obtained by laxative remedy: with the 1g yeast extract, and 5g beef extract, 5g peptone, 0.5gMgSO
47H
2O, the 5g sucrose dissolved is regulated pH value to 7.2 with 1NnaOH again in the distilled water of 900ml, be settled to 1000ml with distilled water then, steam sterilizing treats that the substratum temperature reduces to below 60 ℃ the concentration 100mg/ml kantlex 1ml that adds through the sterilization of 0.22 μ filtering with microporous membrane; The described AB solid medium that contains kantlex is obtained by laxative remedy: with 3gK
2HPO
4, 1gNaH
2PO
4, 0.15gKCl, 0.5gMgSO
47H
2O, 1gNH
4Cl, 0.01CaCl
2, 2.5mgFeSO
47H
2O, 5g sucrose and 15g agar are dissolved in the distilled water of 900ml, regulate pH value to 7.2 with 1N NaOH, and steam sterilizing is treated that the substratum temperature is reduced to and added the concentration 100mg/ml kantlex 1ml that sterilizes through 0.22 μ filtering with microporous membrane below 60 ℃; The described AB liquid nutrient medium that contains kantlex is obtained by laxative remedy: with 3gK
2HPO
4, 1gNaH
2PO
4, 0.15gKCl, 0.5gMgSO
47H
2O, 1gNH
4Cl, 0.01CaCl
2, 2.5mg FeSO
47H
2O and 5g sucrose dissolved are in the distilled water of 900ml, regulate pH value to 7.2 with 1N NaOH, be settled to 1000ml with distilled water then, steam sterilizing treats that the substratum temperature reduces to below 60 ℃ the concentration 100mg/ml kantlex 1ml that adds through the sterilization of 0.22 μ filtering with microporous membrane; The content of catechin 〉=80% in the described tea-polyphenol.
9. method according to claim 8, it is characterized in that: described tea-polyphenol contains: caffeine 0.26%, epigallocatechin 2.66%, dl-catechin 0.71%, l-Epicatechol 2.15%, epigallocatechin Nutgalls ester 62.83%, l-Epigallocatechol Nutgalls ester 3.03%, l-Epicatechol Nutgalls ester 16.79%.
10. method according to claim 1 and 2 is characterized in that: the pretreated explant of microtrauma is cultivated earlier more in advance contaminated in activated bacterium liquid; Described pre-cultivation is: the stem section or (4～6) * (4～6) mm that explant are cut into 8～12mm
2Leaf dish explant, the pre-1-3d that cultivates on the MS substratum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107114131A (en) * | 2017-06-09 | 2017-09-01 | 中国林业科学研究院亚热带林业研究所 | The quick microbial inoculum inoculation method in forest root |
| CN107529551A (en) * | 2017-09-06 | 2018-01-02 | 安徽农业大学 | A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair |
| CN108834900A (en) * | 2018-07-31 | 2018-11-20 | 安徽农业大学 | Inhibit the cultural method of explant oxidizing brown stain and endophytic bacterial contamination in tea-tree tissue culture |
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| CN101063149A (en) * | 2007-04-25 | 2007-10-31 | 东北师范大学 | Agriculture bacillus mediated alfalfa genetic conversion method |
| CN101280319A (en) * | 2008-03-27 | 2008-10-08 | 成都大学 | Agrobacterium-mediated transgenic method for woad |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107114131A (en) * | 2017-06-09 | 2017-09-01 | 中国林业科学研究院亚热带林业研究所 | The quick microbial inoculum inoculation method in forest root |
| CN107529551A (en) * | 2017-09-06 | 2018-01-02 | 安徽农业大学 | A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair |
| CN107529551B (en) * | 2017-09-06 | 2020-06-19 | 安徽农业大学 | Efficient induction method of mixed tea trees with transgenic hairy roots |
| CN108834900A (en) * | 2018-07-31 | 2018-11-20 | 安徽农业大学 | Inhibit the cultural method of explant oxidizing brown stain and endophytic bacterial contamination in tea-tree tissue culture |
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Application publication date: 20100721 |