CN101857875B - Method for carrying out gene transformation on manioca by using Agrobacterium tumefaciens mediated transformation - Google Patents
Method for carrying out gene transformation on manioca by using Agrobacterium tumefaciens mediated transformation Download PDFInfo
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Abstract
The invention relates to a method for carrying out gene transformation on manioca by using Agrobacterium tumefaciens mediated transformation, which comprises the following steps: preparing a manioca explant and agrobacterium liquid, and coincubating the mainoca explant after being soaked by the agrobacterium; inserting the coincubated explant into a screening culture medium C13PC for culture to obtain a resistance callus, wherein the screening culture medium C13PC contains 1-3mg/L of Glufosinate ammonium or 1-10mg/L of hygromycin; transferring the obtained resistance callus into a differential medium, and differentiating adventitious buds; and obtaining a transgenosis plant through rooting culture. The method can successively lead in functional genes to carry out genetic improvement on a wild manioca. The conversion efficiency can be about 10% or even more than 10%; the transgenosis inheritance is stable, and the cost is low; a manioca in vitro has good regenerative system stability; and the method has simple operation and high regeneration efficiency, and the time from the induction of the callus to the obtain of the regeneration plant is only 80-90 days.
Description
Technical field
The present invention relates to tissue culture technique field and the genetically engineered field of manioca, refer in particular to the agriculture bacillus mediated method that manioca is carried out gene transformation, thereby obtain transfer-gen plant.
Background technology
Manioca (Jatopha curcas) has another name called the little seeds of a tung oil tree, diesel oil tree, is the Euphorbiaceae Jatropha, and perennial machaka or dungarunga are distributed in the torrid zone and subtropical zone.It is happiness light sun plant, and the sturdy prosperity of root system has stronger drought-resistant barren ability, can be planted on the soil of the difficult growth of other plant, and be dry ideal reproducting tree species.Leprosy seeds benevolence oleaginousness is up to 50%～60%, can extract sulfur-bearing not, pollution-free, the biofuel that meets the Ou Si emission standard, is universally acknowledged bioenergy tree; Also can do medicinal, be usually used in clearing heat and detoxicating, the detumescence stasis of blood etc. of loosing; Discovering in recent years, this plant also has significant anti-cancer activity.Therefore manioca has value of exploiting and utilizing widely.
At present, China just just begins in the work aspect the manioca breeding.Because mostly manioca is wild state, is again xylophyta, adopt conventional breeding to change very difficulty of its inherited character.Therefore, adopt the modern biotechnology means, carry out assistant breeding like transgenic technology and become first-selection.The prerequisite of plant transgenic technology is a Regeneration in Vitro system efficiently.In the last few years, the Cortex jatrophae tissue culture technique was also succeedd, and the explant of employing has embryo, cotyledon, epicotyl, hypocotyl, petiole, blade; Endosperm; Flower pesticide, even the tender stem segments that grows of aged tree (more than 20 years), however regeneration frequency remains further to be improved.
Plant transgenic technology is because of initiatively importing foreign gene, and the directional transformation plant trait receives people's attention day by day.The plant genetic transformation technology can be divided into two big types: one type is the direct gene transfer techniques, comprises that particle bombardment, protoplasm body, liposome method, pollen tube passage method, electricity swash conversion method, PEG mediated transformation method etc., and wherein the particle gun conversion method is representative; Another kind of is the method for transformation of biological mediation, mainly contains agriculture bacillus mediated and virus-mediated two kinds of method for transformation.Transforming with Agrobacterium tumefaciens mediated plant genetic is one of at present valid approach.Yet, because the Study on Genetic Transformation of manioca is started late, therefore, manioca is carried out genetic improvement, through importing foreign gene, it is carried out the canalized character transformation, have crucial economic worth.
Summary of the invention
Technical problem to be solved by this invention is, a kind of agriculture bacillus mediated method that manioca is carried out gene transformation is provided, and solves the problem that existing breeding technique can't genetic improvement.
For solving the problems of the technologies described above, the present invention adopts the agriculture bacillus mediated method that manioca is carried out gene transformation of following technical scheme, may further comprise the steps:
(1) preparation manioca explant and agrobacterium liquid, explant carries out common cultivation after During Agrobacterium;
(2) the explant access screening culture medium C13PC that is total to after cultivating cultivates, thereby obtains resistant calli, and said screening culture medium C13PC contains 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin;
(3) differentiate indefinite bud; And
(4) root culture, thus transfer-gen plant obtained.
In said (1) step, cultivate the preferred C13A of employing substratum altogether and contain 50-200 μ M Syringylethanone.
In said (1) step, cultivate altogether and adopt the preferred C13A of employing solid medium, it contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, and the MS substratum of 50-200 μ M Syringylethanone; The preparation of manioca explant is via following technology: select the manioca seed after sterilization, takes out complete embryo and cotyledon, in the MS culture medium culturing, the acquisition cotyledon cuts into explant; Agrobacterium bacterium liquid is preferably to adopt the Agrobacterium body of the centrifugal collection of C13A liquid nutrient medium resuspension to make.
In said (1) step, obtain in the manioca explant technology, be inoculated in the MS substratum and sprout; Be that illumination cultivation 10-12 days then, the culture condition of seed germination was with the dark earlier cultivation of the material that connects 2-4 days; Temperature 23-26 ℃, illumination 12-16 hour/day, intensity of illumination 1800-2000lx.
In said (1) step, the preparation of Agrobacterium bacterium liquid, dip-dye and cultivation altogether further comprise following process step:
(a) mono-clonal of picking Agrobacterium is in containing antibiotic YEP liquid nutrient medium, and 28 ℃, the 200rpm concussion was cultivated 20-24 hour, then according to switching in 1: 100; Concussion is cultured to logarithmic phase, and OD600=0.8-1.0 changes bacterium liquid over to the 50ml centrifuge tube; Normal temperature, centrifugal 20 minutes of 4000rpm collects thalline, with the C13A liquid nutrient medium Agrobacterium that suspends again; Transfer to OD600=0.3-0.6,28 ℃ of concussions are cultivated 1-2 hour, and are subsequent use;
(b) blade that cuts is put into container, add ready bacterium liquid, place shaking table to shake at a slow speed 5-15 minute; And
(c) take out the material of contaminating, outwell bacterium liquid, blade is placed on the filter paper, inhale and remove unnecessary bacterium liquid, insert the C13A solid medium, place 23-26 ℃ of dark to cultivate altogether 2-4 days.
The screening culture medium C13PC of said (2) step is that preferred the employing contained 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin, the MS substratum of 250-500mg/L CEFOTAXIME SODIUM STERILE.
Said (2) step further comprises following process step:
(a) take out explant after cultivating altogether, put into container, add aseptic washing after, with the aseptic washing that contains the 250-500mg/L cephamycin;
(b) callus of washing is placed on the filter paper, inhales and removes unnecessary water, inserts screening culture medium C13PC; The preferable employing of said screening culture medium C13PC MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin; The 250-500mg/L CEFOTAXIME SODIUM STERILE; 2-3% sucrose, 0.7% agar, and pH5.8-6.0); And
(c) place 23-26 ℃ of dark the cultivation 14-21 days, obtain resistant calli.
Said (3) step is with the resistant calli that obtains, and changes division culture medium SR13PC over to and cultivates, and this division culture medium SR13PC preferably contains 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin.
The preferable employing of the division culture medium SR13PC MS substratum of said (3) step contains the 0.25-2.0mg/L6-benzyl purine; 0.1-1.0mg/L the 3-indolebutyric acid, 0.01-0.2mg/L Plant hormones regulators,gibberellins, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin; The 250-500mg/L cephamycin; 20-30g/L sucrose, 7g/L agar, and pH5.8-6.0; The resistant calli that connects can obtain indefinite bud in illumination cultivation 10-60 days at 23-26 ℃, and culture condition does, and illumination 12-16 hour/day, intensity of illumination 1800-2000lx.
Said (4) step further comprises following process step: the indefinite bud that differentiates is preferably soaked 2-10 minute in the 3-of 0.1-1.0mg/L indolebutyric acid solution after; Be inoculated into the 1/2MS substratum and carry out root culture, can take root in 10-60 days, the root culture condition does; 23-26 ℃; Illumination 12-16 hour/day, intensity of illumination 1800-2000lx, thus obtain transfer-gen plant.
The above-mentioned agriculture bacillus mediated method that manioca is carried out gene transformation, Agrobacterium carries goal gene.
Beneficial effect of the present invention is following: the present invention utilizes During Agrobacterium and common cultivation; On effective resistant calli screening and differentiated system basis; Manioca is carried out transgenic; Make and adopt transgenic method import feature gene pairs manioca to carry out genetic improvement to become possibility, transformation efficiency can reach about 10% even more than 10%.Mostly the gene that transforms because of agriculture bacillus mediated method for transformation tool is single copy, and clear and definite border sequence, inheritance stability are arranged; Majority meets mendelian inheritance; With low cost, and manioca Regeneration in Vitro system stability is good, easy and simple to handle; Regeneration efficiency is high, only needs 80-90 days from the acquisition that is induced to regeneration plant of callus.
The present invention has selected suitable selective agent concentration, has set up effective resistant calli screening system, has both avoided a large amount of escapes of non-conversion callus, has obtained resistant calli again.
Compare with other positions, not only callus draws rate to the cotyledon after utilizing the mature seed of manioca to sprout and the callus differentiation rate is higher as explant, and does not receive the restriction in season, haves laid a good foundation for carrying out Agrobacterium-mediated Transformation in enormous quantities.
The present invention has adopted suitable selective agent concentration in resistant calli differential period, both non-transformed plant is suppressed, and has avoided a large amount of Molecular Detection work of later stage, has guaranteed the normal growth and the differentiation of resistant plant again.
The present invention selects the indolebutyric acid solution soaking for use in the resistant plant stage of taking root, and has obtained higher rooting rate.
Description of drawings
Fig. 1 is the PCR detection figure of transfer-gen plant of the present invention.
Embodiment
The agriculture bacillus mediated method that manioca is carried out gene transformation of the present invention relates generally to next step:
Step 1: the sterilization of seed and sprouting;
Step 2: Agrobacterium-mediated Transformation and common cultivation;
Step 3: the inducing of removal of Agrobacterium and resistant calli;
Step 4: differentiate indefinite bud;
Step 5: root culture;
Step 6: the transplanting of regeneration plant; And
Step 7: the PCR of transfer-gen plant detects.
Further describe in the face of each step down.
Wherein step 1 is gathered China domestic wild manioca seed.The seed that picking is full is peelled off shell, peels off endosperm after sterile-processed, complete embryo and cotyledon is seeded on the MS substratum sprouts, and obtains aseptic seedling.
The MS substratum contains 20-30g/L sucrose, 7g/L agar, and pH5.8-6.0.
The sterilization method of seed comprises following process step:
(1) 75% (volume ratio) alcohol immersion 30 seconds;
(2) aseptic washing is 2-3 time;
(3) 0.1%g/mL mercuric chloride soaked 3-5 minute;
(4) pour out mercuric chloride, with aseptic washing 4-6 time, each 5 minutes;
(5) add sterilized water, soaked 2-4 hour;
(6) peel off endosperm, get complete embryo and cotyledon, be inoculated in the MS substratum (20-30g/L sucrose, 7g/L agar, pH5.8-6.0) in, every flask culture base connects 2-3;
(7) material that connects was cultivated illumination cultivation 10-12 days then 2-4 days dark earlier.
The culture condition of seed germination is, temperature 23-26 ℃, and illumination 12-16 hour/day, intensity of illumination 1800-2000lx.
Also mainly comprise following process step in the step 2:
1, the preparation of Agrobacterium bacterium liquid is specially:
The mono-clonal of picking Agrobacterium, like EHA105, LBA4404, GV3101 etc.; Contain expression vector such as PEV-GUS respectively, pBI-GUS-hyg, pBI-GUS-bar etc., or other carries the carrier of goal gene; In containing corresponding antibiotic YEP liquid nutrient medium, 28 ℃, the 200rpm concussion was cultivated 20-24 hour; Be that concussion is cultured to logarithmic phase in 1: 100 switching YEP liquid nutrient medium according to volume ratio then, the concentration of Agrobacterium is OD600=0.8-1.0.Change bacterium liquid over to the 50mL centrifuge tube, normal temperature, centrifugal 20 minutes of 4000rpm collects thalline, with the C13A liquid nutrient medium Agrobacterium that suspends again, transfers to OD600=0.3-0.6, cultivates 1-2 individual hour for 28 ℃, and is subsequent use.
Wherein, used YEP liquid culture based formulas and pH value are: 10g/L peptone, 5g/L yeast extract, 10g/L sodium-chlor, pH7.0.
The C13A liquid nutrient medium is meant and contains 1.0-3.0mg/L 6-benzyl purine in the MS substratum, 0.25-1.0mg/L3-indolebutyric acid, 50-200 μ M Syringylethanone, 20-30g/L sucrose, pH5.8-6.0.
2, Agrobacterium-mediated Transformation and common cultivation further comprise following technology:
(1) get the dish of sterilization, middle pad is gone up two layers of filter paper;
(2) take out aseptic seedling, cut growing way cotyledon preferably, put into dish with scissors;
(3) add less water then, be cut into 0.5 * 0.5cm size with cutter;
(4) blade that cuts is put into 50 or the 100mL triangular flask, add ready bacterium liquid, place shaking table to shake at a slow speed 5-15 minute;
(5) take out the material of contaminating, outwell bacterium liquid, blade is placed on the filter paper, inhale and remove unnecessary bacterium liquid, insert the C13A solid medium; Wherein the C13A solid medium is meant that the MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 50-200 μ M Syringylethanone, 20-30g/L sucrose, 7g/L agar, pH5.8-6.0;
(6) material that connects is 23-26 ℃ of dark the cultivation 2-4 days.
Said step 3 further comprises following technology:
(1) takes out material after cultivating altogether, put into 50 or the 100mL triangular flask;
(2) add aseptic washing 2-3 time;
(3) add aseptic washing 2-3 time contain the 500mg/L cephamycin, each 5 minutes;
(4) material of washing is placed on the filter paper, inhales and removes unnecessary water, inserts screening culture medium C13PC; This screening culture medium C13PC is meant that the MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin; The 250-500mg/L CEFOTAXIME SODIUM STERILE; 20-30g/L sucrose, 7g/L agar, pH5.8-6.0;
(5) material that connects obtains resistant calli 23-26 ℃ of dark the cultivation 14-21 days.
(1) with the resistant calli that obtains, changes division culture medium SR13PC over to; This division culture medium SR13PC is meant that the MS substratum contains 0.25-2.0mg/L 6-benzyl purine; 0.1-1.0mg/L the 3-indolebutyric acid, 0.01-0.2mg/L Plant hormones regulators,gibberellins, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin; The 250-500mg/L cephamycin; 20-30g/L sucrose, 7g/L agar, and pH5.8-6.0;
(2) material that connects can obtain indefinite bud in illumination cultivation 10-60 days at 23-26 ℃.Culture condition does, illumination 12-16 hour/day, and intensity of illumination 1800-2000lx.
In the step 5, the indefinite bud that differentiates soaked 2-10 minute in the 3-of 0.1-1.0mg/L indolebutyric acid solution after, be inoculated on the root media and can take root in 10-60 days.
Root media is the 1/2MS substratum.
The root culture condition does, 23-26 ℃, and illumination 12-16 hour/day, intensity of illumination 1800-2000lx.
In the step 6, carry out acclimatization and transplants during the shoot root length to 2 of waiting to regenerate～3cm.
In the step 7,,, carry out pcr amplification, detect and obtain transfer-gen plant with target gene sequences design primer with the genomic dna of CTAB method extraction regeneration plant.
Can realize that through above method manioca carries out gene transformation, it produces chemotactic response with agrobacterium tumefaciens to the chemical substance that plant discharges, and concentrates to the plant wounded tissue.After cultivating altogether, the cytolemma that the chemical inducer of injury sees through Agrobacterium makes the Vir gene activation on the Ti-plasmids.The Vir gene product makes the T-DNA on the Ti-plasmids get into vegetable cell, and is incorporated in the plant nucleus gene group.And goal gene is inserted in T-DNA right sides within battery limit; Therefore the goal gene that is inserted in T-DNA right sides within battery limit also is incorporated on the plant chromosome thereupon, thereby makes goal gene (like degeneration-resistant relevant CN gene of plant disease-resistant and ERFs gene) in vegetable cell, obtain expressing.Carry the Agrobacterium of various various objectives genes, all can realize mediation as stated above, different according to the goal gene that inserts like this; Just can realize manioca is carried out directional transformation, thereby realize genetic improvement, to improve its various characteristics manioca; As improve oil-contg, output; Cold-resistant, disease-resistant, drought resisting etc.
Below in conjunction with instance description embodiment of the present invention are detailed.Need to prove that following instance is illustrative, is not determinate, can not limit protection scope of the present invention with following instance.
Instance 1
This instance changes gus gene over to Yuanmou, Yunnan wild manioca with agriculture bacillus mediated, specifically sets forth the method for the invention, and the total quantity of sample and the survival quantity after various processes please refer to table 1.Each process step is specific as follows:
Step 1: gather the wild manioca seed in Yuanmou, Yunnan.The seed that picking is full is peelled off shell, soaks 30 seconds in 75% ethanol, and aseptic water washing 2-3 time was sterilized 3-5 minute in 0.1% mercury chloride then, and aseptic water washing 5-7 time is peelled off endosperm with the sterilized water immersion after 2-4 hour then.Get complete embryo and cotyledon, be inoculated in the MS substratum, every bottle graft 2-3,23-26 ℃ of dark the cultivation after 2-4 days changes illumination cultivation 10-12 days over to, obtains testing the aseptic seedling of usefulness.
Step 2: the mono-clonal of picking Agrobacterium EHA105 (containing expression vector PEV-GUS) is in the YEP substratum that contains rifamycin antibiotic flat (20mg/L) and kantlex (50mg/L); 28 ℃; The 200rpm concussion was cultivated 20-24 hour; Be forwarded in the 300mLYEP substratum according to 1: 100 volume ratio then, logarithmic phase is cultivated in concussion, and Agrobacterium concentration is OD600=0.8-1.0.Change bacterium liquid over to the 50mL centrifuge tube, normal temperature, 4000rpm collected thalline in centrifugal 20 minutes; (the MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 50-200 μ M Syringylethanone with the C13A liquid nutrient medium; 20-30g/L sucrose, and pH5.8-6.0) Agrobacterium that suspends again, transfer to OD600=0.3-0.6; Cultivate 1-2 hour for 28 ℃, subsequent use.
Get the dish of sterilization, middle pad is gone up two layers of filter paper.Choose robust growth, the smooth uniform aseptic seedling cotyledon in blade face adds less water, is cut into 0.5 * 0.5cm (but being not limited to) size.The blade that cuts is put into 50 or the 100mL triangular flask, add ready bacterium liquid, place shaking table to shake at a slow speed 10 minutes.Take out the material of contaminating then, outwell bacterium liquid, blade is placed on the filter paper; Unnecessary bacterium liquid is removed in suction, and (the MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid to insert the C13A solid medium; 50-200 μ M Syringylethanone, 20-30g/L sucrose, 7g/L agar; And pH5.8-6.0), place 23-26 ℃ of dark the cultivation 2-4 days.
Step 3: take out the material (outer planting body number is 580) after cultivating altogether, put into 50 or the 100mL triangular flask, adds aseptic washing 2-3 time, usefulness contains aseptic washing 2-3 time of 500mg/L cephamycin, each 5 minutes again.The material of washing is placed on the filter paper, inhales and removes unnecessary water, and (the MS substratum contains 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid to insert screening culture medium C13PC; The 1-3mg/L Glufosinate ammonium, 250-500mg/L CEFOTAXIME SODIUM STERILE, 20-30g/L sucrose; 7g/L agar, and pH5.8-6.0), 23-26 ℃ of dark the cultivation 14-21 days; Obtain 320 of resistant callis, the resistant calli rate is about 55.2%, sees table 1.
Step 4: with the resistant calli that obtains, (the MS substratum contains 0.25-2.0mg/L 6-benzyl purine, 0.1-1.0mg/L 3-indolebutyric acid to change division culture medium SR13PC over to; 0.01-0.2mg/L Plant hormones regulators,gibberellins, 1-3mg/L Glufosinate ammonium, 250-500mg/L cephamycin; 20-30g/L sucrose, 7g/L agar, and pH5.8-6.0); 23-26 ℃, illumination cultivation can obtain indefinite bud in 30 days.Culture condition does, illumination 12-16 hour/day, and intensity of illumination 1800-2000lx.70 of differentiation callus survivals, the resistant calli differentiation rate is 18%.
Step 5: the indefinite bud that differentiates soaked 5 minutes in the 3-of 0.5mg/L indolebutyric acid solution after, be inoculated into root media 1/2MS and go up illumination and cultivate and to take root in 30 days, carry out acclimatization and transplants to the greenhouse during shoot root length to 2 of waiting to regenerate～3cm.Obtaining the regeneration plant number is 55 strains.
At last, extract the genome DNA of regeneration plant with the CTAB method, with a pair of primer of gus gene sequences Design:
P1：5′GTGAGCGTCGCAGAAC?ATTAC?3′
P4：5′ACGCCATTTGAAGCCGATGT?3′
Carry out pcr amplification, detect and obtain transfer-gen plant, with reference to Fig. 1; Wherein 7 are negative contrast (unconverted plant), and 1-5 is transgenic sample (other sample omits from view because of no signal), and 8 are negative contrast (water); 6 is that 9 is dna molecular scalar (DNA Ladder) over against photograph (plasmid).Show through the pcr amplification result, have 5 strain samples to expand with plasmid over against according to band of the same size (about 1000bp), be positive, be transfer-gen plant.Its transformation efficiency is 9.1%.
Resistant calli in table 1 instance of the present invention draws rate and differentiation rate
Claims (7)
1. the agriculture bacillus mediated method that manioca is carried out gene transformation may further comprise the steps:
(1) preparation manioca explant and agrobacterium liquid, explant carries out common cultivation after During Agrobacterium, and said the cultivation altogether adopted the C13A solid medium; Wherein the C13A solid medium is meant that the MS substratum contains 1.0-3.0mg/L 6-benzyl purine; 0.25-1.0mg/L the 3-indolebutyric acid, 50-200 μ M Syringylethanone, 20-30g/L sucrose; 7g/L agar, pH5.8-6.0;
(2) the explant access screening culture medium that is total to after cultivating is cultivated, thereby obtains resistant calli, and said screening culture medium is the C13PC substratum; It is for containing 1.0-3.0mg/L 6-benzyl purine, 0.25-1.0mg/L 3-indolebutyric acid, 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin; The 250-500mg/L CEFOTAXIME SODIUM STERILE; 2-3% sucrose, 0.7% agar, and the MS substratum of pH5.8-6.0;
(3) differentiate indefinite bud: be with the resistant calli that obtains, change division culture medium SR13PC over to and cultivate, this SR13PC substratum; Be that the MS substratum contains 0.25-2.0mg/L 6-benzyl purine, 0.1-1.0mg/L 3-indolebutyric acid, 0.01-0.2mg/L Plant hormones regulators,gibberellins; 1-3mg/L Glufosinate ammonium or 1-10mg/L Totomycin, 250-500mg/L cephamycin, 20-30g/L sucrose; 7g/L agar, and pH5.8-6.0; And
(4) root culture is the indefinite bud that differentiates is soaked 2-10 minute in the 3-of 0.1-1.0mg/L indolebutyric acid solution after, to be inoculated into the 1/2MS substratum and to carry out root culture, thereby obtains transfer-gen plant.
2. agriculture bacillus mediated method of manioca being carried out gene transformation as claimed in claim 1; It is characterized in that: in said (1) step; The preparation of manioca explant is via following technology: select the manioca seed after sterilization, take out complete embryo and cotyledon, sprout in the MS substratum; Obtain cotyledon, cut into explant; Wherein, the said MS substratum that is inoculated in is sprouted, and is to cultivate 2-4 days the material that connects is dark earlier, and illumination cultivation 10-12 days then, the culture condition of seed germination was, and temperature 23-26 ℃, illumination 12-16 hour/day, intensity of illumination 1800-2000lx; Agrobacterium bacterium liquid is that the Agrobacterium body with the centrifugal collection of C13A liquid nutrient medium resuspension makes.
3. the agriculture bacillus mediated method that manioca is carried out gene transformation as claimed in claim 2 is characterized in that: in said (1) step, the preparation of Agrobacterium bacterium liquid, dip-dye and cultivation altogether further comprise following process step:
(a) mono-clonal of picking Agrobacterium is in containing antibiotic YEP liquid nutrient medium, and 28 ℃, the 200rpm concussion was cultivated 20-24 hour, then according to switching in 1: 100; Concussion is cultured to logarithmic phase, and OD600=0.8-1.0 changes bacterium liquid over to the 50ml centrifuge tube; Normal temperature, centrifugal 20 minutes of 4000rpm collects thalline, with the C13A liquid nutrient medium Agrobacterium that suspends again; Transfer to OD600=0.3-0.6,28 ℃ of concussions are cultivated 1-2 hour, and are subsequent use; Wherein, said YEP liquid culture based formulas and pH value are: 10g/L peptone, 5g/L yeast extract, 10g/L sodium-chlor, pH7.0; Said C13A liquid nutrient medium is meant and contains 1.0-3.0mg/L 6-benzyl purine in the MS substratum, 0.25-1.0mg/L 3-indolebutyric acid, 50-200 μ M Syringylethanone, 20-30g/L sucrose, pH5.8-6.0;
(b) blade that cuts is put into container, add ready bacterium liquid, place shaking table to shake at a slow speed 5-15 minute; And
(c) take out the material of contaminating, outwell bacterium liquid, blade is placed on the filter paper, inhale and remove unnecessary bacterium liquid, insert the C13A solid medium, place 23-26 ℃ of dark to cultivate altogether 2-4 days.
4. the agriculture bacillus mediated method that manioca is carried out gene transformation as claimed in claim 1, it is characterized in that: said (2) step further comprises following process step:
(a) take out explant after cultivating altogether, put into container, add aseptic washing after, with the aseptic washing that contains the 250-500mg/L cephamycin;
(b) explant of washing is placed on the filter paper, inhales and removes unnecessary water, inserts screening culture medium C13PC; And
(c) place 23-26 ℃ of dark the cultivation 14-21 days, obtain resistant calli.
5. agriculture bacillus mediated method of manioca being carried out gene transformation as claimed in claim 1; It is characterized in that: the resistant calli that said (3) step connects is at 23-26 ℃; Can obtain indefinite bud in illumination cultivation 10-60 days; Culture condition does, illumination 12-16 hour/day, and intensity of illumination 1800-2000lx.
6. agriculture bacillus mediated method of manioca being carried out gene transformation as claimed in claim 1; It is characterized in that: said (4) step further comprises following process step: the root culture condition does; 23-26 ℃; Illumination 12-16 hour/day, intensity of illumination 1800-2000lx, thus obtain transfer-gen plant.
7. like each described agriculture bacillus mediated method that manioca is carried out gene transformation among the claim 1-6, it is characterized in that: said Agrobacterium carries goal gene.
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| Soham Trivedi et al.Establishment of Agrobacterium-mediated genetic transformation in Jatropha curcas L.《international Journal of Agriculture Sciences》.2009,第1卷(第2期),全文. * |
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