CN107529551A - A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair - Google Patents
A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair Download PDFInfo
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Abstract
The invention discloses a kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair, belong to the technical field of Crop Genetic Breeding, this method passes through needle point method, the hypocotyl of seedling is infected with Agrobacterium rhizogenesA4 bacterial strain, obtain 90% rooting rate, the tea tree for making to infect has flourishing hair root lateral root, establish the tea tree transgenosis hair root system of efficient stable, it is transgenosis that this method, which obtains root system, the non-transgenic mixed type tea tree of overground part branches and leaves, it can be applied not only to the scientific research of tea root developmental regulation biology, it can be used for the high-yield culturing of degeneration-resistant infertile mountainous area in tea production.
Description
Technical field
The present invention relates to the technical field of Crop Genetic Breeding, more particularly to it is a kind of with transgenosis root of hair
The highly effective revulsion induction method of mixed type tea tree.
Background technology
Tea is the first big non-alcoholic beverage for being only second to water, has significant healthy functions, very popular.But plantation is extensive
China seed tea tree, overloading planted in the hills of soil depletion and mountain region, regularly by a variety of biologies and abiotic stress
Stress.The tea germplasm for establishing resistance of wide spectrum is always the target of industry.Mixed type tea tree with transgenosis root of hair should be
Realize an approach of this target.Tea tree is difficult to realize genetic transformation by the mediation of Agrobacterium.Though occasionally have agriculture bacillus mediated
Tea tree transgenosis reported success, but be difficult to repeat.The oxidation of polyphenol oxides and its right during this may be organized with tea tree
The depression effect of Agrobacterium is relevant.
Tea is the first big non-alcoholic beverage for being only second to water, has significant healthy functions, very popular.The tealeaves of China
Yield (1467467 tons) occupies the first in the world, followed by India's (991180 tons), Kenya, Sri Lanka and Turkey.Entirely
The tea yield in the world has reached 4520000 tons, and the average daily Tea Consumption amount of Britain is 644.1 tons.Tea tree root is rich in by root
Unique L-thiamine of portion's synthesis, but accumulated in the blade of tea tree.L-thiamine is due to reducing anxiety, suppression in recent years
Aspect is loosened in hypertension, raising memory capability and promotion processed beneficial effect, so his importance gradually steps up.It is rich in tealeaves
Containing polyphenol substance, this is a kind of material that there is anticancer, antiallergy, antiviral, anti-inflammatory, antibacterial, strengthen immunity to act on.Naturally
Activity of the polyphenol due to interior free yl can be eliminated by secondary metabolic pathways, so the antioxygen that it may be beneficial to human body
Change acts on.The content of the most monomer flavonols catechin of content is in 30-40% (DW), wherein epigallocatechin in green tea
Gallate (EGCG) content highest.It has been reported that the galloyl in ECG and EGCG can be by being directly incorporated in bacterium
Peptidoglycan layer and interfere it biosynthesis produce antibacterial action.
Tea tree is to be grown under different country and different weather situation to determine optimal growth state by climate change
Rainwater is irrigated, a kind of crops of single crop system.However, climate change constrains the production of tealeaves on influence caused by economy
Amount.In view of the increase of drought environment and soil salinity, it is necessary to pass through the tolerance to being related to tea tree salinity and arid
The principle of molecule and physiology course is analyzed, to improve the agronomic traits of tea tree.Recent years, use agrobacterium mediation converted
The gene transfer that method is carried out is used on production transfer-gen plant by a large amount of, to improve the agricultural of plant and nutritional character, but it is right
It is an obstacle in terms of the biotechnology exploitation that the genomics of the same Main Agronomic Characters crop of tea is applied.
Although having had been reported that the example of some tea tree genetic transformations was appeared in the clone of Assam kind, but tea
Set and tea tree biology is hampered to the recalcitrant of Agrobacterium_mediated method and develops the research in terms of famous-brand and high-quality tea tree breed.Will gram
Take this bottleneck, it is necessary to the method for establishing a tea tree genetic transformation.Under normal circumstances, the antibacterial polyphenol of high concentration is to plant
Tissue is poisonous, and the oxidation generation quinones with tannic acid in the case of adverse circumstance and injury also has very high reaction
Activity.Although less compared to the Polyphenols in Callus of Leaf, the accumulation of polyphenol substance can be in inoculation and adverse environmental factor
Such as explant excision, the injured and co-cultivation of Agrobacterium, the selection of antibiotic, conditions of exposure and using on explant surface
Appropriate increase under the conditions of disinfectant.In vitro during agrobacterium mediation converted, most of crops are generally required for two to arrive
The co-cultivation time of three days.But tea tree then needs time of five to six days to improve transformation efficiency.The growth of time may
Cause the brown stain that explant is excessive.In order to optimize tea tree agrobacterium mediation converted efficiency and control the phenols oxygen of explantation tissue
Change, many scholars have used different condition of culture and culture medium to overcome the challenge of the outer hair root of inductor.Although culture medium
It is middle to have used different adsorbents and antioxidant to slow down Tissue Browning (necrosis), but these materials may be situated between to Agrobacterium
Conversion is led to produce important negative effect and prevent the expression of gene.
Expanding genetic analysis and the research of gene function needs effective transformation technology.The different explants of tea tree are for example tender
Branch, hypocotyl, cotyledon, cotyledonary node etc. have all been planted with biolistic injection method or Agrobacterium_mediated method to produce transgenosis
Strain.However, the big multiaction of these methods is limited and labour intensive.The gene related to root biology is being done and nutrition suction
Research in terms of receipts, pathogen interaction, symbiosis, hormone transport.These problems can be mediated with agrobacterium rhizogenes
Be converted solution.Its oncogene is shifted to Plant Genome from the extrachromosomal replicon Ri plasmids of agrobacterium rhizogenes
In DNA and then start a large amount of formation of independent hairy root, and can be analyzed in a short period of time.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of mixed type tea with transgenosis root of hair
The highly effective revulsion induction method of tree, to solve the low technical problem of agriculture bacillus mediated tea tree transgene efficiency.
The present invention is achieved by the following technical solutions：
The invention provides a kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair, including following step
Suddenly：
(1) full tea tree seed is screened, kind is germinateed two in the Nutrition Soil of sterilizing under the conditions of 26 ± 2 DEG C after sterilization
Month, tea tree seedling is obtained, the tea tree seedling for screening health is standby；
(2) by gene transformation into Agrobacterium rhizogenesA4 bacterial strain, restructuring agrobacterium rhizogenes is obtained, root of hair agriculture will be recombinated
Bacillus is inoculated in A4 improvement solid mediums and cultivated, and obtains and collects agrobacterium rhizogenes bacterium colony；Wherein, the restructuring root of hair agriculture
The preparation method and incubation of bacillus for details, reference can be made to document using conventional gene engineering method：Methods in
Molecular Biology, vol.343：Agrobacterium Protocols, 2/e, volume 1；
(3) the agrobacterium rhizogenes bacterium colony of syringe needle picking step (2) is used, stabs the hypocotyl of the tea tree seedling into step (1), pin
Head will pass through hypocotyl middle part；Then the agrobacterium rhizogenes bacterium colony of some steps (2) is dipped with triangular glass rod, is applied
It is put on the acupuncture region of hypocotyl；Obtain the seedling contaminated；
(4) seedling of dip-dye is put into humidity cabinet, starts to be hanged with the A4 bacterial strains containing 10% (volume ratio) fortnight
Supernatant liquid waters to seedling, and the later stage irrigates seedling 3 months with the MS culture mediums and water of 1% (volume ratio), that is, obtaining has transgenosis
The mixed type tea tree of root of hair, the tea tree have flourishing hair root lateral root, and root system is transgenosis, and overground part branches and leaves turn base to be non-
Cause.
Further, in the step (1), the method for screening full tea tree seed is：By fresh tea tree seed water
After washing, soaked overnight in water, seed hull and the seed of floating are screened out, selects the seed sunk to the bottom, as full tea tree kind
Son.
Further, in the step (1), the formula of Nutrition Soil is：Pin Shi matrix peat soil and vermiculite in mass ratio 3:1
Mixing.
Further, in the step (2), the preparation method of A4 improvement solid mediums is：By 10g sucrose, 0.2g
MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO4, 0.1gNaCl, 1% (wt/vol) 1.0ml NaMoO4, 1% (wt/vol)
1ml C6H5FeO4, 1% (wt/vol) 1ml boric acid, 1.0g yeast extracts be dissolved into 900ml sterile distilled water, Ran Houding
Hold 1000ml, adjust pH to 6.8-7.0, add the agar of 1% mass ratio, after being sterilized at 121 DEG C, saved backup at 4 DEG C.
Further, in the step (4), the preparation method of the A4 bacterial strain suspension containing 10% (volume ratio) is：Will
Restructuring agrobacterium rhizogenes is activated with A4 improvement fluid nutrient mediums and is enlarged culture, is obtained stoste, 100ml stostes is taken, with nothing
Bacterium distilled water is settled to 1000ml, and the preparation method of the A4 improvement fluid nutrient medium is：By 10g sucrose, 0.2g
MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO4, 0.1gNaCl, 1% (wt/vol) 1.0ml NaMoO4, 1% (wt/vol)
1ml C6H5FeO4, 1% (wt/vol) 1ml boric acid, 1.0g yeast extracts be dissolved into 900ml sterile distilled water, Ran Houding
Hold 1000ml, adjust pH to 6.8-7.0, after being sterilized at 121 DEG C, saved backup at 4 DEG C.
Further, in the step (4), the preparation method of the MS culture mediums of 1% (volume ratio) is：Respectively plus 25ml
10 × MS major salt, 5ml 100 × MS minor salt and 5ml 100 × Fe EDTA to 500ml sterile distilled waters
In, then constant volume to 1000ml adjusts pH to 5.8,121 DEG C of high-temperature sterilizations 15 minutes, obtains MS culture mediums, treats culture medium temperature
When dropping to 45 DEG C, 10ml MS culture mediums are taken into 990ml sterile distilled waters, are preserved under 25 DEG C of no light conditions.
The present invention has advantages below compared with prior art：The invention provides a kind of mixed type with transgenosis root of hair
The highly effective revulsion induction method of tea tree, this method infect the hypocotyl of seedling with Agrobacterium rhizogenesA4 bacterial strain, obtains 90% and takes root
Rate, there is flourishing hair root lateral root, establish the tea tree transgenosis hair root system of efficient stable, this method obtains root system
For transgenosis, the non-transgenic mixed type tea tree of overground part branches and leaves, tea root developmental regulation biology can be applied not only to
Scientific research, in tea production can be used for degeneration-resistant infertile mountainous area high-yield culturing.
Brief description of the drawings
Fig. 1 is tea shoot root of hair situation comparison diagram, wherein, A (1,2) is the control being uninfected by, and A (3,4) is the tea of non-acupuncture
Seedling compares, and B is the tea shoot of acupuncture infection；
Fig. 2 is the agarose gel electrophoresis result of Molecular Identification.
Embodiment
Embodiment 1
1st, material
1.1 reagent
1) agrobacterium rhizogenes strain A4 [Agricultural Culture Collection ofChina (ACCC)]
2)10×MS major salts：Chemical Reagent Co., Ltd., Sinopharm Group is purchased from, and is prepared by product description,
Preserved at 4 DEG C.
3)100×MS majorsalts：Chemical Reagent Co., Ltd., Sinopharm Group is purchased from, and is prepared by product description,
Preserved at 4 DEG C.
4)100×Fe EDTA：Chemical Reagent Co., Ltd., Sinopharm Group is purchased from, and is prepared by product description, at 4 DEG C
Preserve.
5) 100pmol rolC primers：
Sense primer：5’-CCAAGCTTGTCAGAAAACTTCAGGG-3’
Anti-sense primer：5’-CCGGATCCAATACCCAGCGCTTT-3’
6) 100pmol aux1 primers：
Sense primer：5'-CCAAGCTTGTCAGAAAACTTCAGGG-3'
Anti-sense primer：5'-CCGGATCCAATACCCAGCGCTTT-3'.
7) 4% carbendazim：4g carbendazim is dissolved into 900ml sterile distilled water, then constant volume to 1000ml,
Preserve at room temperature.
8) 0.1%HgCl2：By 1g HgCl2It is dissolved into 900ml sterile distilled water, then constant volume to 1000ml,
Preserve at room temperature.
9) A4 improves fluid nutrient medium (growth agrobacterium rhizogenes)：By 10g sucrose, 0.2g MgSO4·7H2O、0.5g
K2HPO4、0.2g CaSO4, 0.1g NaCl, 1% (wt/vol) 1.0ml NaMoO4, 1% (wt/vol) 1ml C6H5FeO4, 1%
(wt/vol) 1ml boric acid, 1.0g yeast extracts are dissolved into 900ml sterile distilled water, then constant volume to 1000ml.Regulation
PH to 6.8-7.0, preserved at 4 DEG C.
10) A4 improves solid medium：The agar that 1% mass ratio is added in fluid nutrient medium is improved for A4.
11) 1% (wt/vol) agar plate：2g agar is added in 200ml distilled water.High-temperature sterilization 15 divides at 121 DEG C
Clock, temperature fall 10ml into sterile petri dish (90 × 15mm) after dropping to 40 DEG C.
12) the A4 bacterial strain suspension of 10% (volume ratio) is contained：Restructuring agrobacterium rhizogenes is improved into fluid nutrient medium with A4
Activate and be enlarged culture, obtain stoste, take 100ml stostes, 1000ml is settled to sterile distilled water
13) the MS culture mediums of 1% (volume ratio) (plant is infected in watering)：Respectively plus 25ml 10 × MS major salt,
In 5ml 100 × MS minor salt and 5ml 100 × Fe EDTA to 500ml sterile distilled waters, then constant volume arrives
1000ml, pH to 5.8 is adjusted, 121 DEG C of high-temperature sterilizations 15 minutes, obtains MS culture mediums.When temperature drops to 45 DEG C, 10ml is taken to arrive
100ml is fallen in 990ml sterile distilled waters, after mixing into bottle, 25 DEG C are stored under no light condition.
14) cultivation tray is filled：The Pin Shi matrix peat soil and vermiculite that high-temperature sterilization (121 DEG C of 15min) will be passed through press 3:1
Ratio is added in basin, is cooled to 20 DEG C.
1.2 equipment
1) glass culture dish (90*15mm) that can be sterilized
2) vinyl disc (700*500*50mm)
3) syringe needle 21G*1.25Prime (0.8*32mm)
4) sealed membrane (4IN.*125FT)
5) band handle triangular glass rod
6) humidity cabinet
7) electrophoretic apparatus
8) Artificial Growth case
9) shaking table (the sincere 100H of intelligence)
1.3 device configuration
1) humidity cabinet：In order to keep humidity, mini plastic film cover should be put into vinyl disc back cover above in cultivation tray.
Pallet should be carried out disinfection with 70% ethanol in advance.
2) Artificial Growth case：The photoperiod of growth case should be adjusted to 16h illumination periods and 8h dark phase, and temperature is 26 ± 2
℃.Light source uses cold light (40-50mmol m-2s-1).
3) bacteriological incubator：The temperature of culture agrobacterium rhizogenes, which is done something in disregard of obstacles, is maintained at 28 DEG C.
4) shaking table：Agrobacterium rhizogenes mixes with 10%A4 fluid nutrient mediums and is maintained at 150rpm to obtain uniform bacterium
Suspension.
5) PCR cycle program and electrophoresis：Prepare Easy-Taq polymerases (the full formula gold biotechnology in Beijing containing 1Unit
Co., Ltd), 2.5 μ L 10x buffer, 2 μ L dNTP (2.5mmolL-1), each 1 μ L (10mmolL- of front and rear primer
1), the totally 25 μ L of 10ng gDNA and 17.5 μ L water reaction system enters performing PCR.The program of PCR instrument (Bio-Rad S1000) is
First 95 DEG C of 3min of denaturation temperature, the amplifications (95 DEG C of 30s, 58 DEG C of 30s and 72 DEG C of 1min) of 30 circulations, then 72 DEG C of 10min.
PCR primer carries out electrophoresis in 1.5% agaropectin, then is observed with ethidium bromide staining.
2 methods
The collection (autumn) of 2.1 seeds
The mature seed collected from the anti-early kind of agriculture of 7 years age of trees in the tealeaves Experimental Base of Agricultural University Of Anhui；
The screening of 2.2 seeds
In order to allow tea tree seedling hair tonic hairy root, fresh seed will pass through the washing and selection of running water；Seed hull
It need to be removed by the immersion at a night.Then filter, the seed for floating and sinking to the bottom is separated, and broadcast from the seed sunk to the bottom
Kind, remove the seed of floating.
2.3 thes surface of the seed sterilize
(1) 100 seeds are placed into 25ml Conical flasks, are washed twice with distilled water.
(2) it is immersed in again in the sterilization ling solution of 4% (volume ratio) overnight, is then made a return journey with distillation water washing and remove fungi.
(3) with the HgCl of 0.1% (volume ratio)2Solution carries out the sterilization of 4 minutes to the surface of the seed, then clear with distilled water
Wash three times.
(4) add (volume ratio) ethanol of 100ml 70% and then gentle agitation is filtered for two minutes.
(5) remove the ethanol of residual with substantial amounts of water and clean seed.
The germination of 2.4 seeds
Seed kind is being filled with the basin of sterilized Pin Shi matrix peat soil and vermiculite and in growth case (26 ± 2 DEG C)
Bimestrial germination process is carried out, obtains tea tree seedling.
The preparation (agrobacterium rhizogenes) of 2.5 inoculation bacterium
The agrobacterium rhizogenes for the preservation containing 50% glycerine taken out from -80 DEG C of refrigerators is drawn on A4 improvement solid mediums
The long single bacterium colony of plank.Plank with Agrobacterium rhizogenesA4 strain is cultivated two days in 28 DEG C of incubator, then picking one
Individual single bacterium falls in a new A4 plate and cultivated 4 days.
2.6 hairy root inductions
Healthy life by the screening tea tree seedling big two months in case is grown is used for transgenosis reality
Test, the bacterium colony grown in step 2.5 is collected into a culture dish.With one small Agrobacterium of syringe needle picking, then stab into children
The hypocotyl of seedling, syringe needle will pass through hypocotyl middle part.Then some Agrobacteriums are dipped with triangular glass rod, is applied to
The acupuncture region of hypocotyl.The seedling infected is put into humidity cabinet, and used in the fortnight of beginning containing 10% A4 bacterium
Strain suspension waters to seedling, and the later stage irrigates seedling 3 months with the MS culture mediums and water of 1% (volume ratio).
2.7 collect root tissue
Collection is infected infects root with non-, first rinses 1 hour of root with running water, is then rinsed again with sterile distilled water,
Last root surface is carried out disinfection processing with 70% alcohol again, the material as Molecular Identification.
2.8 Molecular Identification
Root genomic DNA is extracted, and using root genomic DNA as template, clone agrobacterium rhizogenes T-DNA regions
RolC and auxI genes, to confirm whether the T-DNA on Agrobacterium plasmid has been incorporated into the genome sequence of plant.rolC
It is as shown in Figure 2 with auxI gene PCR results.
2.9 results count
The tea shoot root of hair Efficiency Statistics table of table 1
Table 1 understands that very big probability (90%) obtains hairy root by this method.This technology can be used for controlling root
Growth improve the yield of L-thiamine.Moreover, the transgenosis A4 bacterial strains for carrying target gene can be used for promoting root system
Unite to make plant adapt to biological and abiotic stress.This method is in all tea tree breeds or other woody and medicinal plants
Similar changing effect can be obtained.
3rd, conclusion
The present invention utilizes agriculture bacillus mediated live body method for transformation, has successfully infected recalcitrant Chinese Tea Varieties agriculture and has resisted
It is early, it is grown hairy root, the Molecular Identification of the hairy root to growing finds that the T-DNA on Agrobacterium plasmid is successfully integrated
Into the genome of the hairy root newly grown, it was demonstrated that can be imported any foreign gene with the method that agrobacterium rhizogenes mediates
In tea tree, transgenic hairy root lateral root is obtained, the limitation that in vitro method is applied in tea tree is overcome, establishes the tea of efficient stable
Transgenosis hair root system is set, can be as the important tool of the research in terms of root system biology and cometabolism.
Be above a kind of detailed embodiment of the invention and specific operating process, be using technical solution of the present invention before
Put and implemented, but protection scope of the present invention is not limited to the above embodiments.
Claims (6)
1. a kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair, it is characterised in that comprise the following steps：
(1) full tea tree seed is screened, kind is germinateed two months under the conditions of 26 ± 2 DEG C, obtained in the Nutrition Soil of sterilizing after sterilization
Tea tree seedling is obtained, the tea tree seedling for screening health is standby；
(2) by gene transformation into Agrobacterium rhizogenesA4 bacterial strain, restructuring agrobacterium rhizogenes is obtained, agrobacterium rhizogenes will be recombinated
It is inoculated in A4 improvement solid mediums and cultivates, obtains and collect agrobacterium rhizogenes bacterium colony；
(3) the agrobacterium rhizogenes bacterium colony of syringe needle picking step (2) is used, stabs the hypocotyl of the tea tree seedling into step (1), syringe needle will
Through hypocotyl middle part；Then the agrobacterium rhizogenes bacterium colony of some steps (2) is dipped with triangular glass rod, is applied to
Hypocotyl acupuncture region；Obtain the seedling contaminated；
(4) seedling of dip-dye is put into humidity cabinet, starts fortnight to use the A4 bacterial strain suspension containing 10% (volume ratio)
Watered to seedling, the later stage irrigates seedling 3 months with the MS culture mediums and water of 1% (volume ratio), that is, obtaining has transgenosis root of hair
Mixed type tea tree.
2. a kind of highly effective revulsion induction method of mixed type tea tree with transgenosis root of hair according to claim 1, its feature
It is, in the step (1), the method for screening full tea tree seed is：After fresh tea tree seed is washed with water, in water
Soaked overnight, seed hull and the seed of floating are screened out, selects the seed sunk to the bottom, as full tea tree seed.
3. a kind of highly effective revulsion induction method of mixed type tea tree with transgenosis root of hair according to claim 1, its feature
It is, in the step (1), the formula of Nutrition Soil is：Pin Shi matrix peat soil and vermiculite in mass ratio 3:1 mixing.
4. a kind of highly effective revulsion induction method of mixed type tea tree with transgenosis root of hair according to claim 1, its feature
It is, in the step (2), the preparation method of A4 improvement solid mediums is：By 10g sucrose, 0.2g MgSO4·7H2O、
0.5g K2HPO4、0.2g CaSO4, 0.1g NaCl, 1% (wt/vol) 1.0ml NaMoO4, 1% (wt/vol) 1ml
C6H5FeO4, 1% (wt/vol) 1ml boric acid, 1.0g yeast extracts be dissolved into 900ml sterile distilled water, then constant volume arrives
1000ml, pH to 6.8-7.0 is adjusted, add the agar of 1% mass ratio, after being sterilized at 121 DEG C, saved backup at 4 DEG C.
5. a kind of highly effective revulsion induction method of mixed type tea tree with transgenosis root of hair according to claim 1, its feature
It is, in the step (4), the preparation method of the A4 bacterial strain suspension containing 10% (volume ratio) is：Root of hair agriculture bar will be recombinated
Bacterium A4 bacterial strains are activated with A4 improvement fluid nutrient mediums and are enlarged culture, are obtained stoste, 100ml stostes are taken, with sterile distillation
Water is settled to 1000ml, and the preparation method of the A4 improvement fluid nutrient medium is：By 10g sucrose, 0.2g MgSO4·
7H2O、0.5g K2HPO4、0.2g CaSO4, 0.1gNaCl, 1% (wt/vol) 1.0ml NaMoO4, 1% (wt/vol) 1ml
C6H5FeO4, 1% (wt/vol) 1ml boric acid, 1.0g yeast extracts be dissolved into 900ml sterile distilled water, then constant volume arrives
1000ml, pH to 6.8-7.0 is adjusted, after being sterilized at 121 DEG C, is saved backup at 4 DEG C.
6. a kind of highly effective revulsion induction method of mixed type tea tree with transgenosis root of hair according to claim 1, its feature
It is, in the step (4), the preparation method of the MS culture mediums of 1% (volume ratio) is：Respectively plus 25ml 10 × MS major
In salt, 5ml 100 × MS minor salt and 5ml 100 × Fe EDTA to 500ml sterile distilled waters, then constant volume arrives
1000ml, pH to 5.8 is adjusted, 121 DEG C of high-temperature sterilizations 15 minutes, MS culture mediums is obtained, when culture medium temperature drops to 45 DEG C, takes
100ml MS culture mediums preserve into 900ml sterile distilled waters under 25 DEG C of no light conditions.
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| CN113141965A (en) * | 2021-05-30 | 2021-07-23 | 浙江农林大学 | Construction and optimization of simple and efficient apocarya agrobacterium transformation system |
| CN113331059A (en) * | 2021-07-21 | 2021-09-03 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
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| 奚彪等: "发根农杆菌介导的茶树遗传转化", 《茶叶科学》 * |
| 高秀清: "茶氨酸生物合成研究", 《中国优秀博硕士学位论文全文数据库（硕士）工程科技Ⅰ辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113141965A (en) * | 2021-05-30 | 2021-07-23 | 浙江农林大学 | Construction and optimization of simple and efficient apocarya agrobacterium transformation system |
| CN113331059A (en) * | 2021-07-21 | 2021-09-03 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
| CN113331059B (en) * | 2021-07-21 | 2022-09-09 | 贵州大学 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
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