CN106636196B - A kind of peanut method of optimization, efficient mediated by agriculture bacillus - Google Patents
A kind of peanut method of optimization, efficient mediated by agriculture bacillus Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Abstract
The present invention relates to a kind of peanut methods of optimization, efficient mediated by agriculture bacillus.This method optimizes the constructive ways of peanut regeneration bud, and the preinfective preculture process of traditional Agrobacterium is omitted, and substantially reduces the period for obtaining regenerated transformed plants.The differentiation rate and conversion ratio of peanut are improved by the addition of tobacco extract and to the ultrasonication of Peanut in infection processs.The cell division and resistant buds differentiation of Peanut are effectively facilitated by adding zeatin ZT and free radical scavenger in the medium, reduce influence of the exogenous hormone to plant cell, reduce the number of squamous subculture, simultaneously effective alleviates the browning of Peanut.The present invention only needs conventional strain culturing, the experiment consumptive material without the valuableness such as particle gun, bronze.Easy to operation, experimental period is short, high conversion rate, and changing effect is stablized between different genotype peanut varieties.
Description
Technical field
Technical field belonging to the present invention is agricultural biological technical field and plant genetic engineering field.
Background technique
Peanut is important oil plant and industrial crops, is rich in grease and protein.Silk channel injection underlying finite, Resistant germplasm
Lack, produce always by the harm of pest and disease damage and extreme climate, its yield and quality is caused to decline.Therefore, peanut something lost is carried out
Improvement is passed, cultivates new pest-resistant disease-resistant variety with very real meaning.Very using traditional crossbreeding, mutation breeding
Difficulty has the innovation of purpose character, and can artificially select the mode kind in production to carry out external source using technique for gene engineering breeding
The importing of gene obtains purpose character gene transfer, so as to achieve the purpose that Genetic improvement with clearly defined objectively.Currently, plant
More commonly used method is agrobacterium-mediated transformation in genetic transformation.Agrobacterium-mediated transformation can shift biggish DNA fragmentation, easily produce
Raw unit point integration, is not easy to cause molecular rearrangement, while also having many advantages, such as that technically simple and expense is lower.
Peanut belongs to dicotyledonous crops, is the natural host of Agrobacterium, but there is also the foreign genes that legume shares
Import the big disadvantage of difficulty.This is subject to certain restrictions the transgenic breeding work of peanut, is in progress slower.Brazil
In 1991, China Laiyang Agricultural College researcher reported Agrobacterium in nineteen ninety respectively and invades peanut Lacorte et al.
Contaminate power.India Eapen and George was reported for the first time in 1994 obtains transgenic peanuts by Agrobacterium-mediated Transformation.U.S. Cheng
M et al. obtains 5 independent transformation systems by Agrobacterium-mediated Transformation in 1996 and 1997 reports, has bred 52 plants of transgenosis altogether
Plant, but conversion ratio is only 0.2%~0.3%.The LiZhijian in same laboratory etc. improves experimental method, by optimizing agriculture
Time needed for the factors such as bacillus bacterial concentration and antibiotic usage concentration shorten acquisition branch, transformation efficiency are increased to
1.5%.The small equality in country side in 1999 spends No. 4 age seedling tender leafs to make explant with peanut varieties Hubei Province, co-cultures with A GLI bacterial strain,
Transgenic peanuts are obtained through kanamycin screening, transformation efficiency is 2% or so.2000, India Rohini etc. used LBA404 bacterium
Strain obtains transgenic peanuts using identical inoculation technique, and transformation efficiency is increased to 3.3%.
In recent years, the genetic transformation of peanut achieves certain progress with the progress of Somatic Plant Regeneration technology,
Transgenic peanuts regeneration strain can be obtained by way of mediated by agriculture bacillus.But these peanut systems are still generally deposited
It is long in the transformation period, the problem of screening difficulty is big, and transformation efficiency is low and poor repeatability.Therefore, either from the heredity of peanut
Improve angle, or from its angle as gene function analysis carrier crop, establish a kind of short period, high efficiency, it is easy to operate,
The peanut system of low cost is all real and necessary.
Summary of the invention
The purpose of the present invention is to provide a kind of period with the mediated by agriculture bacillus that peanut cotylcdon is conversion explant is short, high
Efficiency, easy to operate, inexpensive genetic transforming method.The present invention with regard to Agrobacterium bacterial concentration involved in genetic transformation, infect
Time co-cultures and newly adds in time, ultrasonic treatment time, tobacco extract concentration, antibiotic usage concentration and culture medium
Plant hormone and the influence conditions such as free radical scavenger concentration improvement is optimized.This method only needs conventional bacterial strain and culture
Base, without expensive devices such as particle guns.Easy to operation, experimental period is short, high conversion efficiency.
The purpose of the present invention is realized by following technical proposals:
(1) it activates, recombinational agrobacterium bacterium solution to be transformed is resuspended, adjust OD600Value is 0.5~0.8；With alcohol, mercuric chloride according to
Secondary processing peanut cotylcdon explant；2.5h is completely impregnated afterwards with aseptic water washing；It will be cut off at cotyledon plumule node after longitudinal sectional
To explant to be transformed；
(2) 5~15min of explant is infected with the Agrobacterium bacterium solution added with tobacco extract, infect while carries out ultrasonic wave
2~8min is handled, after removing extra bacterium solution, explant is placed in and is co-cultured in base, 2~4d of dark culture under the conditions of 26 DEG C；
(3) after co-culturing, explant is taken out with sterile containing 100~300mg/L Cef and 300~500mg/L Carb
After water rinses 4~5 times, is blotted with sterilizing filter paper, be connected to the resistance containing 100~300mg/L Cef and 300~500mg/L Carb
On bud inducement cultivation base, the selection culture 14d under 26 DEG C, 16h illumination condition；After thering is green bud point to be formed, cotyledon, In are removed
Subculture is primary every two weeks in induced medium；
(4) it after green bud point grows into 1~2cm, is inoculated on differential medium and promotes bud differentiation and elongation, with bud
The continued growth of clump, is cut into fritter, and subculture is primary every two weeks, continues differentiation culture；
(5) when luring bud long to 3~4cm high and there are 3~4 leaflets, bud will be lured to cut, is transferred to root media induction
It takes root, completely to seedlings root development, cleans the culture medium on root, be transplanted in the flowerpot of the sandy soil containing sterilizing；Every month is used
Hoagland nutrient solution pours once, while being poured with tap water within every ten days primary.
For techniques described above scheme, step (1) recombinational agrobacterium to be transformed can be special selected from any carrying
Determine the recombinational agrobacterium of target gene；Specifically, having selected two kinds of agricultures for carrying different target gene in embodiments of the present invention
Bacillus respectively converts peanut according to the method for the invention, and has carried out data analysis to changing effect respectively, thus
The method of the invention is demonstrated, validity is not only restricted to the type of recombinational agrobacterium；Specifically, making in the embodiment of the present invention
Two kinds of recombinational agrobacteriums, one are that the area Agrobacterium GV3101, T-DNA with expression vector pCAMBIA1301 is contained
Gus gene is marked by CaMV35S as promoter using hygromycin (HB) as resistance screening.The document of preparation method: agriculture
The nuclear agricultural science reports 2012,26 (9) such as the optimization Qiao Li celestial being of peanut genetic conversion system that bacillus mediates: 1244~1248；It is another
A is that DREB2A gene is contained in the area Agrobacterium EHA105, T-DNA with expression vector pBI121, by rd29A as starting
Son is marked using Geneticin (G418) as resistance screening.The document of preparation method: The Northern Imperial Tomb iris (Iris typhifolia
Kitag.) clone of DREB transcription factor and its genetic transformation [J] Li Xue Northeast Forestry University .2015 (01) to tobacco
67；
For techniques described above scheme, the kind of step (1) described peanut seed is that mound spends 12,17, No. 18；It is rich to spend 1
Number；Spend 14, No. 16 in Shandong.In fact, the method for the invention, is suitable for all types of peanut varieties.
In preferred situation, for techniques described above scheme, alcohol used in step (1), mercuric chloride processing method are to use
70%~75% ethanol postincubation peanut cotylcdon 1~2min of explant handles cotyledon explant with 0.1%~0.2% mercuric chloride later
10~20min.
In preferred situation, for techniques described above scheme, step (2) the Agrobacterium bacterium added with tobacco extract
Liquid is that 1~3ml of tobacco extract is added in every 100ml Agrobacterium bacterium solution.
In preferred situation, for techniques described above scheme, the power of step (2) described ultrasonication is 50w, work
Working frequency is 40KHz.
It is described to co-culture the MS culture medium that base is improvement for techniques described above scheme in preferred situation, wherein adding
Add 1~2mg/L ZT, 4~8mg/L 6-BA, 0.1~0.5g/L casein hydrolysate, 0.05~0.15g/L inositol.PH value is
5.2。
In preferred situation, for techniques described above scheme, the resistant buds induced medium is the MS culture of improvement
Base, wherein addition 1~2mg/L ZT, 4~8mg/L6-BA, 0.1~0.5g/L casein hydrolysate, 2~4g/L L-PROLINE,
0.05~0.15g/L inositol, 0.1~0.3mmol/L vitamin C, 0.1~0.4mmol/L GSH, 0.02~0.1mmol/L dimension
Raw element E.PH value is 5.8.
In preferred situation, for techniques described above scheme, the differential medium is the MS culture medium of improvement, wherein
Add 100~300mg/L Cef, 300~500mg/L Carb, 1~2mg/L ZT, 1~2mg/L NAA, 1~3mg/L KT, 1
~4g/L casein hydrolysate, 0.05~0.15g/L inositol, 20~40g/L sorbierite, 0.1~0.3mmol/L vitamin C,
0.1~0.4mmol/L GSH, 0.02~0.1mmol/L vitamin E.PH value is 5.8.
In preferred situation, for techniques described above scheme, the root media is the 1/2MS culture medium of improvement,
Wherein add 1~2mg/L NAA, 0.05~0.15g/L inositol.PH value is 5.8.
In preferred situation, for techniques described above scheme, the sterilizing sandy soil weight ratio of constituents is husky: soil: vermiculite:
Perlite is 10:10:1:1.
The present invention has the advantages that following and effect compared with the existing technology:
(1) present invention optimizes the constructive ways of peanut regeneration bud, utilize cellular omnipotency and the height of peanut cotylcdon node
Degree differentiation capability reduces variation.The preculture process before traditional Agrobacterium infection processs is eliminated simultaneously, substantially reduces and obtains
Obtain the period of regenerated transformed plants.Because the peanut cotylcdon node of different genotype has active meristematic capacity and de- point mostly
Change effect, therefore the present invention is not limited during the genetic transformation of peanut by genotype.
(2) present invention passes through the addition of tobacco extract and safely and effectively improves to the ultrasonication of Peanut
The differentiation rate of peanut.Conventional method mostly adds acetosyringone, but such materialization in infection processs in agrobacterium liquid
Strong toxicity is learned, it is at high cost, the influence of browning is also easy to produce to induced bud growth.And tobacco extract itself can generate phenolic substances,
To the not additional injury of Peanut.Supercritical ultrasonics technology is to change Peanut cell by cavitation and micromechanics damaging action
The permeability of film and cell wall creates minimally invasive mouth, promotes the exchange of intraor extracellular substance, consequently facilitating the importing of foreign gene.
(3) present invention is appropriate by adding in the co-cultivation base of peanut, induced medium and differential medium
Zeatin ZT is effectively facilitated the cell division of Peanut, accelerates resistant buds differentiation, reduces the number of squamous subculture,
The transformation period is shortened, while playing the role of inhibiting aging to blade.Conventional reformat mode usually adds 2,4-D in the medium
Induce explant, but there are certain chemical toxicities by 2 artificial synthesized, 4-D, and the methylation of 2,4-D induce
Embryos ratio it is higher.And ZT is the n cell mitogen extracting from corn, has to Peanut and breaks
Suspend mode promotes to sprout, the effect of Delaying Leaf-Senescence and yellow.
(4) present invention in the induced medium of peanut and differential medium by adding radicals scavenging appropriate
Agent effectively mitigates the browning of Peanut.The activity of intracellular antioxidase can be improved in free radical scavenger,
The lipid peroxidation degree for reducing cell, to protect cells from damage.By adding vitamin C, dimension life in the medium
The division frequency of protoplast can be improved in plain E, GSH substance, improves the culture situation of protoplast, checks and cause browning
Phenolic substances generation and accumulation.
(5) present invention only needs conventional strain culturing, the experiment consumptive material without the valuableness such as particle gun, bronze.Present invention behaviour
Make simple and easy to do, experimental period is short, high conversion rate, and changing effect is stablized between different genotype peanut varieties, can satisfy flower
The requirement of raw scale transgenosis, has great importance for the analysis of peanut transgenic research and functional gene.
Detailed description of the invention
Fig. 1 is the dyeing qualification figure that Peanut turns gus gene, and A is blade GUS dyeing identification in figure；B is more in figure
Injured tissue GUS dyeing identification (T: transgenosis group, CK: control group)；
Fig. 2 is that addition ZT promotes resistant buds cell division figure, and A is that 2,4-D of addition induces explant growing state in figure；Figure
Middle B is that addition ZT induces explant growing state；
Fig. 3 is that free radical scavenger inhibits explant aging and browning figure, and A is to be not added with free radical scavenger in figure
Plant strain growth situation；B is addition free radical scavenger plant strain growth situation in figure；
Fig. 4 is the PCR qualification figure that peanut turns DREB2A gene, and A is to turn the mound of DREB2A gene to spend No. 17 plant PCR in figure
Qualification figure (M:DL2000；1: negative control；2: water control；3: positive control；4~13: conversion peanut strain) B is to turn in figure
The mound of DREB2A gene spends No. 18 plant PCR qualification figure (M:DL2000；1: negative control；2: water control；3: positive control；4~
13: conversion peanut strain).
Specific embodiment
Technical solution of the present invention is described in further detail with reference to the accompanying drawings and detailed description.The present invention
Including but not limited to following implementation.Changing for substantive content of the present invention is not departed to what the present invention was carried out according to the prior art
Become, still falls within protection scope of the present invention.
1 mediated by agriculture bacillus gus reporter gene of embodiment imports peanut
(1) the Agrobacterium GV3101 with expression vector pCAMBIA1301 (containing hygromycin resistance and gus gene) is lived
Change and (optimization Qiao Lixian Yu Xinling Sui Jiongming grandson's Fan Gancheng generation of the peanut genetic conversion system of mediated by agriculture bacillus is resuspended
Meng Wangjing coral nuclear agricultural science report 2012,26 (9): 1244~1248), OD is adjusted600It is 0.6.Mature and plump, surface are chosen without splitting
The peanut seed (mound spends No. 12) of trace, with 70% ethanol postincubation 1.5min, 0.1% mercuric chloride (W/V) handles 10min, uses sterile water
2.5h is impregnated in sterile water after rinsing 10 times.It is placed on aseptic filter paper and sucks moisture, remove kind of a skin, cotyledon is separated along longitudinal direction,
Plumule is cut off along cotyledon node with scalpel, obtains explant to be transformed.
(2) tobacco seedling tender leaf 2g is taken, smashs taking juice to pieces in sterile centrifugation tube, 2ml sterile water is then added, oscillation is mixed
5min is stood after even, obtains tobacco extract.1~3ml of tobacco extract is wherein added in every 100ml Agrobacterium bacterium solution, then is used
Added with the Agrobacterium bacterium solution infecting peanut explant 10min of tobacco extract, infects while the triangular flask equipped with explant is set
Ultrasonication is carried out in ultrasonic cleaner (power 50w, working frequency 40KHz) center.To tobacco extract concentration and surpass
Two factors of sonication times are arranged different gradients and optimize screening, show that the optium concentration of tobacco extract is 2ml/
100ml, ultrasonication optimum time are 6min (as shown in table 1).Then it is washed with aseptic filter paper and does extra bacterium solution, then will
In explant merging co-cultivation base (MS+2mg/L ZT+5mg/L 6-BA+0.3g/L casein hydrolysate+0.1g/L inositol+
30g/L sucrose+4g/L plant gel), dark culture 3d under the conditions of 26 DEG C.
(3) after co-culturing 3d, it is sterile water that 200mg/L, Carb are 350mg/L that Peanut, which is taken out with containing Cef,
It after rinsing 5 times, is blotted with sterilizing filter paper, being connected to is 20mg/L containing hygromycin, Cef 200mg/L, Carb are the anti-of 350mg/L
On property bud inducement cultivation base (MS+2mg/L ZT+8mg/L 6-BA+0.3g/L casein hydrolysate+2.8g/L L-PROLINE+
0.1g/L inositol+0.15mmol/L vitamin C+0.2mmol/L GSH+0.05mmol/L vitamin E+30g/L sucrose+4g/L plants
Object gel), the selection culture 14d under 26 DEG C, 16h illumination condition；After having green bud point to be formed, cotyledon is removed, is trained in induction
Subculture is primary every two weeks on feeding base, continues culture and promotes bud elongation.
(4) after green bud point grows into 1~2cm, it is inoculated into that hygromycin is 20mg/L, Cef 200mg/L, Carb are
(MS+1mg/L ZT+1mg/L NAA+2mg/L KT+2g/L casein hydrolysate+0.1g/L on the differential medium of 350mg/L
Inositol+30g/L sorbierite+0.15mmol/L vitamin C+0.2mmol/L GSH+0.05mmol/L vitamin E+30g/L sucrose+
4g/L plant gel) promote bud differentiation and elongation, with the continued growth of bud clump, it is cut into fritter, every two weeks subculture one
It is secondary, continue elongation culture.
(5) when resistance lures bud long to 3~4cm high and there are 3~4 leaflets, bud will be lured to cut, be transferred to root media
(1/2MS+1mg/L NAA+0.1g/L inositol+30g/L sucrose+4g/L plant gel) root induction, root starts shape after about 2 weeks
At.Completely to seedlings root development, the culture medium on root is cleaned, the plantlet of root system stalwartness is transplanted to the flower of the sandy soil containing sterilizing
In basin.Wherein sandy soil ingredient is weight ratio sand: soil: vermiculite: perlite is the mixture of 10:10:1:1.Every month is used
Hoagland nutrient solution pours once, while being poured with tap water within every ten days primary.
(6) clip peanut leaf and callus are in X-Gluc solution (10mM EDTA, 100mM phosphate, 0.5mM iron cyanogen
Sour potassium, 0.5mM ferrous potassium cyanate and 0.1%X-Gluc) in carry out GUS dyeing identification, at 37 DEG C overnight, in 70% ethyl alcohol
Dechlorophyllization.There is macroscopic blue spot in GUS positive transgenic peanut leaf and callus, and control group blade
And significant change (as shown in Figure 1) does not occur for callus.It is 68.6% that mound, which spends No. 12 conversion ratios,.With traditional transform mode
It compares, conversion ratio is significantly improved (as shown in table 2).
The influence of 1 tobacco extract concentration of table and ultrasonic treatment time to peanut (mound spends No. 12) differentiation rate
Influence of the 2 two kinds of transform modes of table to peanut (mound spends No. 12) changing effect
2 mediated by agriculture bacillus DREB2A channel genes peanut of embodiment
(1) simultaneously by the Agrobacterium EHA105 activation with expression vector pBI121 (containing G418 resistance and DREB2A gene)
(clone of The Northern Imperial Tomb iris (Iris typhifolia Kitag.) DREB transcription factor and its genetic transformation to tobacco is resuspended
[J] Li Xue Northeast Forestry University .2015 (01) 67), adjust OD600It is 0.7.Choose mature and plump, the crackless peanut in surface
(mound spend No. 17, mound spend No. 18) seed, with 75% ethanol postincubation 1min, 0.1% mercuric chloride (W/V) handles 15min, is rushed with sterile water
2.5h is impregnated in sterile water after washing 10 times.It is placed on aseptic filter paper and sucks moisture, remove kind of a skin, cotyledon is separated along longitudinal direction, is used
Scalpel cuts off plumule along cotyledon node, obtains explant to be transformed.
(2) tobacco seedling tender leaf 2g is taken, smashs taking juice to pieces in sterile centrifugation tube, 2ml sterile water is then added, oscillation is mixed
5min is stood after even, obtains tobacco extract.After optimal screening, with the Agrobacterium bacterium solution added with 3ml/100ml tobacco extract
Infecting peanut explant 8min infects while the triangular flask equipped with explant is placed in ultrasonic cleaner (power 50w, work
Frequency 40KHz) center progress ultrasonication 8min, is then washed with aseptic filter paper and does extra bacterium solution, then set explant
Enter to co-culture in base (MS+1.5mg/L ZT+6mg/L 6-BA+0.5g/L casein hydrolysate+0.1g/L inositol+30g/L sucrose
+ 4g/L plant gel), while (MS+1.5mg/L 2,4-D+6mg/L 6-BA+0.5g/L casein is placed in control medium
Hydrolysate+0.1g/L inositol+30g/L sucrose+4g/L plant gel), dark culture 4d under the conditions of 26 DEG C.
(3) after co-culturing 4d, it is sterile water that 250mg/L, Carb are 400mg/L that Peanut, which is taken out with containing Cef,
After rinsing 5 times, is blotted, be connected to containing the resistance that G418 is 30mg/L, Cef 250mg/L, Carb are 400mg/L with sterilizing filter paper
On bud inducement cultivation base (MS+1.5mg/L ZT+8mg/L6-BA+0.4g/L casein hydrolysate+3.5g/L L-PROLINE+
0.1g/L inositol+0.2mmol/L vitamin C+0.3mmol/L GSH+0.08mmol/L vitamin E+30g/L sucrose+4g/L plants
Object gel), while being placed in control medium (MS+1.5mg/L2,4-D+8mg/L6-BA+0.4g/L casein hydrolysate+
3.5g/L L-PROLINE+0.1g/L inositol+0.2mmol/L vitamin C+0.3mmol/L GSH+0.08mmol/L vitamin E+
30g/L sucrose+4g/L plant gel), selection culture 14d, its upgrowth situation of paired observation under 26 DEG C, 16h illumination condition.It is right
The ratio of the Embryos induced according to culture medium is higher and explant differentiation is sluggish, and the culture medium optimized accelerates cell point
It splits, promotes the differentiation of resistant buds, reduce the number of squamous subculture, shorten transformation period (as shown in Figure 2).When there is green
After bud point is formed, cotyledon is removed, subculture is primary every two weeks on the induction medium, continues culture and promotes bud elongation.
(4) after green bud point grows into 1~2cm, it is inoculated into that G418 is 30mg/L, Cef 250mg/L, Carb are
Differential medium (the MS+1.5mg/L ZT+2mg/L NAA+2.5mg/L KT+3g/L casein hydrolysate+0.1g/ of 400mg/L
L inositol+30g/L sorbierite+0.2mmol/L vitamin C+0.3mmol/L GSH+0.08mmol/L vitamin E+30g/L sucrose+
4g/L plant gel) and control medium (MS+1.5mg/L ZT+2mg/L NAA+2.5mg/L KT+3g/L casein hydrolysate
+ 0.1g/L inositol+30g/L sorbierite+30g/L sucrose+4g/L plant gel) on promote bud differentiation and elongation, paired observation its
Upgrowth situation.There is browning in the partial resistance explant that control medium differentiates, and blade is in yellowish-brown or sepia,
Embryoid is few, and growing way is slow, and partial medium has filiform or dregs to generate.And outside the resistance that the culture medium optimized differentiates
Implant blade is in emerald green, embryoid increase, well-grown (as shown in Figure 3).With the continued growth of bud clump, it is cut into
Fritter, subculture is primary every two weeks, continues elongation culture.
(5) when resistance lures bud long to 3~4cm high and there are 3~4 leaflets, bud will be lured to cut, be transferred to root media
(1/2MS+2mg/L NAA+0.1g/L inositol+30g/L sucrose+4g/L plant gel) root induction, root starts shape after about 2 weeks
At.Completely to seedlings root development, the culture medium on root is cleaned, the plantlet of root system stalwartness is transplanted to the flower of the sandy soil containing sterilizing
In basin.Wherein sandy soil ingredient is weight ratio sand: soil: vermiculite: perlite is the mixture of 10:10:1:1.Every month is used
Hoagland nutrient solution pours once, while being poured with tap water within every ten days primary.
(6) genomic DNA that transgenic peanuts are extracted using CTAB method carries out PCR inspection by positive control of Plasmid DNA
It surveys.Upstream primer: 5'GCTCCTACAAATGCCATCATTGC3', downstream primer: 5'GATAGTGGGATTGTGCGTCATCCC
3'.25 μ l of PCR reaction system, reaction condition: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C extend
1min, 30 circulations；72 DEG C of extension 7min.Reaction product is detected with 1.5% agarose gel electrophoresis, and obtaining size is 205bp
Specific PCR products (as shown in Figure 4).It is 39.4% that mound, which spends No. 17 conversion ratios, and it is 55.8% that mound, which spends No. 18 conversion ratios,.With biography
The transform mode of system is compared, and conversion ratio is significantly improved (as shown in table 3).
Influence of the 3 two kinds of transform modes of table to peanut effect
Examples detailed above is the optimal embodiment of the present invention, but embodiments of the present invention are not by the limit of above-described embodiment
System, those skilled in the art is in made change, modification, substitution, optimization without departing from the spirit and principles of the present invention
Etc. equivalent substitute mode is accordingly to be regarded as, it is included within the scope of the present invention.
Claims (6)
1. a kind of peanut method of mediated by agriculture bacillus, it is characterised in that: including following operating procedure:
(1) it activates, recombinational agrobacterium bacterium solution to be transformed is resuspended, adjust OD600Value is 0.5~0.8；Successively located with alcohol, mercuric chloride
Manage peanut cotylcdon explant；2.5h is completely impregnated afterwards with aseptic water washing；To cut off to obtain at cotyledon plumule node after longitudinal sectional to
Convert explant；
(2) with the Agrobacterium bacterium solution added with tobacco extract infect step (1) acquisition 5~15min of explant, infect at the same into
Row 2~8min of ultrasonication after removing extra bacterium solution, is put into and co-cultures in base, and dark culture 2 under the conditions of 26 DEG C~
4d；
(3) after co-culturing, explant is taken out and is rushed with the sterile water containing 100~300mg/L Cef and 300~500mg/L Carb
It after washing 4~5 times, is blotted with sterilizing filter paper, is connected to the resistant buds containing 100~300mg/L Cef and 300~500mg/L Carb and lures
It leads on culture medium, the selection culture 14d under 26 DEG C, 16h illumination condition；After thering is green bud point to be formed, cotyledon is removed, is being induced
Subculture is primary every two weeks on culture medium；
(4) it after green bud point grows into 1~2cm, is inoculated on differential medium and promotes bud differentiation and elongation, with bud clump
Continued growth is cut into fritter, and subculture is primary every two weeks, continues differentiation culture；
(5) when luring bud long to 3~4cm high and there are 3~4 leaflets, bud will be lured to cut, is transferred to root media induction life
Root completely to seedlings root development cleans the culture medium on root, is transplanted in the flowerpot of the sandy soil containing sterilizing；Every month is used
Hoagland nutrient solution pours once, while being poured with tap water within every ten days primary；
It is described to co-culture the MS culture medium that base is improvement, wherein addition 1~2mg/L ZT, 4~8mg/L 6-BA, 0.1~0.5g/
L casein hydrolysate, 0.05~0.15g/L inositol, pH value 5.2；
The resistant buds induced medium be improvement MS culture medium, wherein addition 1~2mg/L ZT, 4~8mg/L 6-BA,
0.1~0.5g/L casein hydrolysate, 2~4g/L L-PROLINE, 0.05~0.15g/L inositol, 0.1~0.3mmol/L dimension life
Plain C, 0.1~0.4mmol/L GSH, 0.02~0.1mmol/L vitamin E, pH value 5.8；
The differential medium is the MS culture medium of improvement, wherein addition 100~300mg/L Cef, 300~500mg/L
Carb, 1~2mg/L ZT, 1~2mg/L NAA, 1~3mg/L KT, 1~4g/L casein hydrolysate, 0.05~0.15g/L flesh
Alcohol, 20~40g/L sorbierite, 0.1~0.3mmol/L vitamin C, 0.1~0.4mmol/L GSH, 0.02~0.1mmol/L dimension
Raw element E, pH value 5.8.
2. the peanut method of mediated by agriculture bacillus according to claim 1, it is characterised in that: step (2) described ultrasound
The power of wave processing is 50w, working frequency 40KHz.
3. the peanut method of mediated by agriculture bacillus according to claim 1, it is characterised in that: the root media is
The 1/2MS culture medium of improvement, wherein addition 1~2mg/L NAA, 0.05~0.15g/L inositol, pH value 5.8.
4. the peanut method of mediated by agriculture bacillus according to claim 1, it is characterised in that: the sterilizing sandy soil ingredient
Weight ratio is husky: soil: vermiculite: perlite 10:10:1:1.
5. the peanut method of mediated by agriculture bacillus according to claim 1, it is characterised in that: wine used in step (1)
Essence, mercuric chloride processing method are, with 70%~75% ethanol postincubation peanut cotylcdon 1~2min of explant, later with 0.1%~
0.2% mercuric chloride handles 10~20min of cotyledon explant.
6. the peanut method of mediated by agriculture bacillus according to claim 1, it is characterised in that: step (2) it is described added with
The Agrobacterium bacterium solution of tobacco extract is that 1~3ml of tobacco extract is added in every 100ml Agrobacterium bacterium solution.
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