CN103773799A

CN103773799A - Agrobacterium infection method in callus conversion process of Taxus chinensis

Info

Publication number: CN103773799A
Application number: CN201410039375.9A
Authority: CN
Inventors: 皮妍; 蒋科技; 赵东利; 廖志华; 孙小芬; 唐克轩
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2014-01-27
Filing date: 2014-01-27
Publication date: 2014-05-07
Anticipated expiration: 2034-01-27
Also published as: CN103773799B

Abstract

The invention belongs to the technical field of bioengineering, and specifically relates to an agrobacterium infection method in the callus conversion process of Taxus chinensis. The method comprises the following steps: by using hypocotyledonary axis of Taxus chinensis as an explant, inducing a callus tissue, and culturing to obtain an embryo callus; mediating by the agrobacterium tumefaciens, cutting the callus tissue of Taxus chinensis by an agrobacterium-infiltrated blade, guiding a pCAMBIA1304 with a GUS (Glucuronidase) gene plasmid into the callus tissue of Taxus chinensis, detecting the integrating condition of an exogenous hygromycin phosphotransferase (HPT) by PCR (Polymerase Chain Reaction), and detecting expression of the GUS gene in the tissue by a histochemical method to obtain a transgenetic callus tissue of Taxus chinensis, wherein a certain bacterial liquor gradient is formed on the section of the callus tissue by blade contact so as to cover and convert the required optical bacterial liquor concentration to ensure that cells are successfully converted, thereby improving the success rate of agrobacterium-mediated transformation. The method lays a foundation for conducting efficient genetic transformation engineering of the callus tissue of Taxus chinensis.

Description

A kind of method that in Ramulus et folium taxi cuspidatae callus conversion process, Agrobacterium is infected

Technical field

The invention belongs to technical field of bioengineering, be specifically related to a kind of Agrobacterium and infect the method for taxus callus.

Background technology

Ramulus et folium taxi cuspidatae ( taxussp.) claim again Japanese yew, for taxaceae (Taxaceae) Taxus ( taxus) gymnosperm.The Chinese yew genus plants whole world has 11 kinds, is mainly distributed in temperate zone, the Northern Hemisphere to subtropical zone.There are 4 kinds and 1 mutation in China, respectively taxus chinensis in northeast (T.cuspidata), taxusyunnanensis (T.yuennanensis), Chinese Ramulus et folium taxi cuspidatae (T.chinensis), southerm yew (T.chinensis var.mairei), Xizang Taxus chinensis (T.wallichiana) (Chinese Plants will editorial board of the Chinese Academy of Sciences, 1978), be mainly distributed in each provinces and regions in northeast, northwest, North China, southwest, Central-South, south China.Also having introduced a fine variety afterwards Taxus x media (T.x media), is the cross-fertilize seed of taxus chinensis in northeast and european yew (T.baccata), and its female parent is taxus chinensis in northeast, and male parent is european yew.

Taxol (taxol) is a kind of active skull cap components extracting from bark of Ramulus et folium taxi cuspidatae, there is unique anticancer effect, the kinds cancers such as human ovarian cancer, mammary cancer, malignant melanoma are had to outstanding curative effect, leukemia, lung cancer, the cancer of the brain and other some solid tumors etc. are also had to good curative effect, toxic side effect is very little, is one of best natural anti-cancer drugs.According to estimates, the annual requirement of whole world taxol is at least more than 1 000 kg.At present, taxol is only present in 11 kinds of (subspecies) plants of Taxus of gymnosperm taxaceae, and mainly from the stem skin of Chinese yew, obtains.

But the speed of growth of Ramulus et folium taxi cuspidatae is slow, and regenerative power is poor, becomes a useful person and need the 50-250 year.Its stock number is very limited, and particularly content of taxol is low, only accounts for 0.01%～0.02% of bark dry weight.Taxus resource and development contradiction are outstanding especially.In recent years, be subject to ordering about of interests, the wild resource of Ramulus et folium taxi cuspidatae has suffered serious destruction.The medicine source that how to expand taxol has become the task of top priority.The artificial culture of Ramulus et folium taxi cuspidatae has been obtained certain progress, but plantation need account for a large amount of soils, and the cycle is longer.The nearly 30 step reactions of the complete synthesis need of chemistry of taxol, there is no business development and are worth.Culture plant cell, due to the not restriction of climate and soil, is as the effective ways of producing the useful secondary metabolite of plant.In cultured cells of taxus species, the output of taxol is generally not high at present, also all exist the unsettled problem of Taxol Yield even if obtain once in a while higher output, so utilize engineered method to regulate and control related gene in taxol biosynthesizing and branch's approach, become current study hotspot to improve in cultured cells of taxus species Taxol Yield and stability thereof.Inducing culture Ramulus et folium taxi cuspidatae embryo callus subculture, take agrobacterium tumefaciens as mediation, take to infect efficiently method, obtain Ramulus et folium taxi cuspidatae transgenic calli, carry out the research of taxol metabolic engineering to improve content of taxol the final rare effective ways of paclitaxel prodrugs that solve by being.The research of this respect is being carried out in many laboratories both at home and abroad, our laboratory is through groping for many years, successfully set up taxus callus method for transformation and (seen " a kind of genetic transforming method that obtains Ramulus et folium taxi cuspidatae transgenic calli ", Chinese Patent Application No.: 201210121529.0, the applying date: 2012.6.19).But apply conventional Agrobacterium infestation method, the efficiency of conversion and the surviving rate of callus are also not high.

Summary of the invention

The object of the invention is to overcome deficiency of the prior art, the method that provides the Agrobacterium that can improve transformation efficiency in a kind of Ramulus et folium taxi cuspidatae callus genetic transformation process to infiltrate.

The invention provides the method for the Agrobacterium infiltration that can improve transformation efficiency in Ramulus et folium taxi cuspidatae callus genetic transformation process, in Ramulus et folium taxi cuspidatae callus genetic transformation process, the blade cuts taxus callus piece that adopts Agrobacterium to infiltrate, make to form certain bacterium liquid gradient on the tangent plane of callus of knife face contact, transform required best bacterial concentration thereby cover, guarantee the cell conversion of succeeding, improve the success ratio of Agrobacterium-mediated Transformation, and avoid the toxic action of a large amount of bacterium liquid of traditional infusion method to callus, guarantee the smooth growth of the callus transforming, carry out genetic transformation engineering efficiently for taxus callus and established solid basis.

Specifically, Ramulus et folium taxi cuspidatae callus genetic transformation process comprises: using Ramulus et folium taxi cuspidatae hypocotyl as sugarcane explants through callus induction, and obtain embryo callus subculture through cultivating; With Agrobacterium tumefaciens mediated, the blade cuts taxus callus that adopts Agrobacterium to infiltrate, pCAMBIA1304 plasmid with gus gene is imported to taxus callus, PCR detects external source hygromycin phosphotransferase gene (Hygromycin B phosphotransferase gene, HPT) integration, the expression of gus gene in tissue detects in histochemical method, obtains the transgenic calli of Ramulus et folium taxi cuspidatae.

the inventive method concrete operation step is as follows:

(1) inducing culture of taxus callus: selecting hypocotyl is callus induction explant, secretly cultivates on callus inducing medium, produces gradually white callus, and constantly expand at hypocotyl two ends; Above-mentioned callus inducing medium is take B5 medium as basis, then adds hormone NAA, TDZ and 2,4-D and form;

(2) inducing culture of Ramulus et folium taxi cuspidatae embryo callus: the white callus that hypocotyl is induced is transferred on embryo callus subculture inducing culture, prevention water sample and brownization, and obtain embryo callus fine and close, that hardness increases; This embryo callus subculture inducing culture is take B5 medium as basis, then adds 5 seed amino acids and hormone 2,4-D, NAA and TDZ and form;

(3) genetic transformation: utilize Agrobacterium tumefaciens mediated, take Ramulus et folium taxi cuspidatae embryo callus as acceptor, the blade cuts callus that plant expression vector pCAMBIA1304 agrobacterium tumefaciens containing gus gene is infiltrated, in callus lines, form bacterial concentration gradient, carry out genetic transformation;

(4) de-bacterium, screening and the multiplication culture of resistant embryogenic calli: through genetic transformation, then take off bacterium and cultivate on recovery media, cultivate at the enterprising row filter of screening culture medium, obtain the callus with hygromycin resistance; Resistant calli is accessed to embryo callus subculture medium and carry out multiplication culture, increase the quantity of resistant calli;

(5) PCR detects and GUS histochemical stain method detects transgenosis callus: on PCR detection pCAMBIA1304 plasmid with hygromycin phosphotransferase gene; GUS histochemical stain, immerses callus in the GUS staining fluid preparing, and 37 ℃ of dyeing are spent the night, and the blueness in callus is reporter gene GUS histochemical stain, shows that foreign gene has been incorporated in the karyomit(e) of recipient cell and has expressed.

Agrobacterium tumefaciens of the present invention, there is the biomaterial of public sale in market, can be from many companies as Australian CAMBIA company (CAMBIA, GPO Box 3200, Canberra, ACT 2601, Australia.Trade name: agrobacterium tumefaciens bacterial strain is as EHA105, goods number: Gambar 1) buy.

Compared with the method that infects taxus callus with existing Agrobacterium, the method of the blade cuts taxus callus that the Agrobacterium that the present invention adopts infiltrates, be total in culturing process at taxus callus and Agrobacterium, an Agrobacterium bacterial concentration gradient environment is provided, thereby the concentration that makes taxus callus and Agrobacterium reaches optimal conversion ratio, agriculture bacillus mediated lower to pCAMBIA1304 plasmid importing taxus callus, and avoid the toxic action of a large amount of bacterium liquid of traditional infusion method to callus, improve the survival rate of taxus callus in conversion process, and obtain higher transgene efficiency.This carries out taxus brevifolia alcohol route of synthesis metabolic engineering to the later stage, finally solves medicine source of Taxol scarcity, to meet the large-scale industrialized need of production of medicine industry significant.

The inventive method comprises that in Ramulus et folium taxi cuspidatae biotechnology association area the aspects such as the foundation of detection, Ramulus et folium taxi cuspidatae genetically engineered, cell engineering, metabolic engineering and the Ramulus et folium taxi cuspidatae genetic conversion system of location, the promoter activity of functional protein are with a wide range of applications.

Accompanying drawing explanation

Fig. 1 is cutter blanking method schematic diagram in taxus callus Agrobacterium infection processs.The blade that the Ramulus et folium taxi cuspidatae callus of 1 cubic centimetre of left and right was soaked with Agrobacterium cuts in half lentamente, then the callus tangent plane of two and half sheets is placed in common culture medium down.

Number in the figure: the taxus callus piece before (1) cutting; (2) blade soaking with agrobacterium suspension; (3) the Agrobacterium bacterium liquid on blade; (4) the taxus callus piece after incision; (5) in cutting process, transfer to the Agrobacterium bacterium liquid on callus tangent plane from blade, the bacterial concentration on tangent plane forms a gradient from top to bottom; (6) be total to culture medium; (7) tangent plane is positioned over the taxus callus piece in common culture medium down.

Embodiment

Below embodiments of the invention are elaborated: the present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The induction of Ramulus et folium taxi cuspidatae embryo callus subculture and propagation

1. the breeding of Ramulus et folium taxi cuspidatae aseptic seedling

Peel the crust of Ramulus et folium taxi cuspidatae seed off, the complete endosperm of sterilizing, program is as follows: 75% ethanol disinfection 2 min, 0.1% mercuric chloride, 10 min that sterilize, aseptic water washing 3 times, strip afterwards embryo and be inoculated in the mg/l+2 by B5 medium+6-BA1.0, in the substratum of 4-D 0.1 mg/l+ caseinhydrolysate 1.0 g/l+ gac 1 g/l+ sucrose 20 g/l+ plant gel 2.6 g/l compositions (wherein, the add-on (g) of this component of numeral after component in B5 medium unit volume (l)), first under dark surrounds, make seed germination, then strengthen gradually illumination, the weak slow fire light of application is cultivated, can obtain the aseptic seedling of Ramulus et folium taxi cuspidatae, after seedling grows to 2-3 cm, cut the induction of hypocotyl for callus.

2. the induction of Ramulus et folium taxi cuspidatae embryo callus

Hypocotyl is inoculated on callus inducing medium, this callus inducing medium consists of: B5 medium+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+sucrose 25 g/l+ plant gel 5 g/l, wherein, numeral after component is the add-on (g) of this component in B5 medium unit volume (l), lower same, do not remake explanation one by one; Produce gradually white callus at hypocotyl two ends, and constantly expand.By callus subinoculation on embryo callus subculture inducing culture, this embryo callus subculture inducing culture consists of: B5+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l, screen growing way good and fast, do not have brownization to present white embryo callus.

Embodiment 2

Contain the acquisition of the plant expression vector pCAMBIA1304 agrobacterium tumefaciens engineering bacteria of gus gene

Plant expression vector pCAMBIA1304 is proceeded to agrobacterium tumefaciens (as EHA105, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar 1), the performing PCR of going forward side by side checking.Particularly, first prepare competence agrobacterium tumefaciens (as EHA105): cultivate agrobacterium tumefaciens to bacterium liquid O.D ₆₀₀=0.5, by after bacterium liquid ice bath 30 min, get bacterium in 1.5 ml Eppendorf pipes, in 4 ℃ of 5000 centrifugal 5 min of rpm, remove supernatant; With the 0.1M CaCl of suction filtration sterilizing ₂after (precooling) 400 μ l suspension thalline (beating gently with liquid-transfering gun), place 30 min on ice, centrifugal 5 min of 5000 rpm, remove supernatant, with the 0.1M CaCl of 100 μ l precoolings ₂after suspension thalline, preserve 10 h in 4 ℃ for subsequent use.Then, adopt following methods to obtain the agrobacterium tumefaciens engineering strain containing plant expression vector pCAMBIA1304: the competence of getting pipe 100 μ l, add 2 μ l plasmid pCAMBIA1304, after mixing gently, in placing 5 min and liquid nitrogen on ice, place after 8 min, put into 37 ℃ of water-bath heat shock 5 min; The YEB liquid nutrient medium that adds 800 μ l antibiotic-frees, mixes gently; After shaking culture activation (28 ℃, 200 rpm) 4 h, in room temperature centrifugal (4000 rpm) 10 min, remove supernatant, the resuspended thalline of remaining 200 μ l bacterium liquid mixes; Resuspended the 200 μ l that mix activation bacterium liquid is evenly applied on the YEB solid medium flat board that adds corresponding microbiotic [kantlex (Kan) 100 μ g/ml+ Streptomycin sulphate (Str) 25 μ g/ml+ Rifampin (Rif) 40 μ g/ml], in 28 ℃ of incubators, be inverted and cultivate after 2 days, select resistance list bacterium colony cultivating containing in corresponding antibiotic YEB liquid nutrient medium.Bacterium liquid reaches the PCR that does hygromycin phosphotransferase gene after finite concentration and detects.Result shows, plant expression vector pCAMBIA1304 is successfully building up in agrobacterium tumefaciens bacterial strain.

Embodiment 3

1. Agrobacterium tumefaciens mediated conversion

1.1. the cultivation of agrobacterium tumefaciens

Picking contains the agrobacterium tumefaciens engineering bacteria list bacterium colony of described plant expression vector in substratum (it consists of: YEB liquid nutrient medium+Streptomycin sulphate (Str) 25 μ g/ml+ kantlex (Kan) 100 μ g/ml+ Rifampin (Rif) 40 μ g/ml), 180 rpm shaken overnight on 28 ℃ of shaking tables, work as bacterial concentration in second day and reach OD ₆₀₀=0.5 o'clock, 5000 rpm outwelled upper strata nutrient solution after centrifugal 10 minutes, and the resuspended Agrobacterium that is deposited in bottom, adds 100 μ M Syringylethanones, the upper activation of 28 ℃ of shaking tables (180 rpm) after 2 hours for transforming Ramulus et folium taxi cuspidatae embryo callus subculture.

1.2. the common cultivation of agrobacterium tumefaciens and explant

Moisten aseptic blade by agrobacterium tumefaciens bacterium immersion, with Agrobacterium infiltrate blade longitudinally cut from top to bottom approximately 1 cubic centimetre of big or small embryo callus piece, be divided into two, the disconnected tangent plane (stick and have Agrobacterium bacterium liquid level) of callus that blade cuts is crossed is placed in common substratum down, and (its component is: B5+TDZ (0.5-2.0 mg/l)+2, 4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l) upper in 25 ℃ of dark cultivations 2 days, along with Agrobacterium infiltration up dilution growth gradually, make Agrobacterium concentration reach a best effect that infects.

1.3. the screening of resistant calli and succeeding transfer culture

After cultivating altogether, callus is moved to recovery media, and (recovery media component is: B5+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l+ carboxylic benzyl mycin 250 mg/l) on take off bacterium cultivate, (screening culture medium component is: B5+TDZ (0.5-2.0 mg/l)+2 after 25 days, to be transferred to screening culture medium in 25 ℃ of dark cultivations, 4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l+ carboxylic benzyl mycin 250 mg/l+ Totomycin 5 mg/l) on carry out the screening and culturing of resistant calli, using the each callus that originates from different cells as a clone, (its component is: B5+TDZ (0.5-2.0 mg/l)+2 to be inoculated in subculture medium, 4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l+5 mg/l Totomycin) upper subculture screening, every 20-25 days succeeding transfer culture once.

2. the PCR of transgenosis taxus callus detects

First adopt conventional CTAB (Cetyltrimethyl Ammonium bromide, hexadecyl trimethyl ammonium bromide) method extracts the DNA of transgenosis Ramulus et folium taxi cuspidatae hygromycin resistance callus, then, by PCR method, the hygromycin phosphotransferase gene in transgenosis Ramulus et folium taxi cuspidatae resistant calli is detected.PCR reaction conditions is: 94 ℃ of sex change 5 min → 30 circulation (94 ℃ of 50 sec → 58 ℃ 50 sec → 72 ℃ 1 min) → 72 ℃ of 6 min.Result shows, utilize designed PCR special primer amplification transgenosis taxus callus DNA, can amplify the special object fragment (0.8 kb) consistent with expected results, and during as template, not amplify any fragment take non-transformed Ramulus et folium taxi cuspidatae genomic dna.Illustrate that hygromycin phosphotransferase gene has been incorporated in camplotheca acuminata genome.

3. the histological chemistry of transgenosis taxus callus is detected

The histochemical stain of gus reporter gene, immerses callus the staining fluid (100 mM phosphoric acid buffers, 5 mM high-potassium ferricyanides, 5 mM yellow prussiate of potash, the 10 mM Na that prepare ₂eDTA, 50 mg/ml X-Gluc) in, 37 ℃, dyeing time is 16 hours, the blueness in callus is reporter gene GUS histochemical stain, shows that foreign gene has been incorporated in the karyomit(e) of recipient cell and has expressed.

The genetic transforming method that the Agrobacterium that the present embodiment adopts infiltrates blade cuts taxus callus, has improved survival rate and the transformation efficiency of callus in conversion process, has improved 4-6 doubly than the method for former pure immersion.For the later stage is carried out Ramulus et folium taxi cuspidatae metabolic engineering and large-scale production taxol and the final alkaloid scarcity that solves provides a kind of effective solution.

Claims

1. the method that in a Ramulus et folium taxi cuspidatae callus conversion process, Agrobacterium is infected, it is characterized in that: be in Ramulus et folium taxi cuspidatae callus genetic transformation process, the blade cuts taxus callus piece that adopts Agrobacterium to infiltrate, make to form certain bacterium liquid gradient on the tangent plane of callus of knife face contact, transform required best bacterial concentration thereby cover, guarantee the cell conversion of succeeding.

2. method according to claim 1, is characterized in that described Ramulus et folium taxi cuspidatae callus genetic transformation process comprises: using Ramulus et folium taxi cuspidatae hypocotyl as sugarcane explants through callus induction, and obtain embryo callus subculture through cultivating; With Agrobacterium tumefaciens mediated, the blade cuts taxus callus that adopts Agrobacterium to infiltrate, imports taxus callus by the pCAMBIA1304 plasmid with gus gene; PCR detects the integration of external source hygromycin phosphotransferase gene (HPT); The expression of gus gene in tissue detects in histochemical method, obtains the transgenic calli of Ramulus et folium taxi cuspidatae.

3. method according to claim 2, is characterized in that concrete operation step is as follows:

4. method according to claim 3, it is characterized in that, the described taxus callus inducing culture component of step (1) is: B5 medium+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+sucrose 25 g/l+ plant gel 5 g/l; Wherein, the numeral after component is the add-on (g) of this component in B5 medium unit volume (l), lower same, does not remake explanation one by one.

5. method according to claim 3, it is characterized in that, the described embryo callus subculture inducing culture component of step (2) is: B5 medium+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l.

6. method according to claim 3, it is characterized in that, the callus lines volume size of the blade cuts that the described Agrobacterium of step (3) infiltrates is about 1 cubic centimetre, be cut into and be divided into two, and the disconnected tangent plane of the callus that blade cuts is crossed sticks and have Agrobacterium bacterium liquid level to be placed on common substratum down to cultivate.

7. method according to claim 3, it is characterized in that, the component of the recovery media described in step (4) is: B5+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l+ carboxylic benzyl mycin 250 mg/l.

8. method according to claim 3, it is characterized in that, the described callus screening culture medium component of step (4) is: B5 medium+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/L+250 mg/L carboxylic benzyl mycin+5 mg/L Totomycin.

9. method according to claim 3, it is characterized in that, the component of the subculture medium described in step (4) is: B5 medium+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l+5 mg/l Totomycin.

10. method according to claim 6, it is characterized in that, described common nutrient media components is: B5+TDZ (0.5-2.0 mg/l)+2,4-D (1.0-2.0 mg/l)+NAA (1.0-2.0 mg/l)+tyrosine 0.8 g/l+ L-Ala 0.4 g/l+ proline(Pro) 0.4 g/l+ Methionin 0.4 g/l+ L-glutamic acid 0.4 g/l+ sucrose 25 g/l+ plant gel 5 g/l.