CN104770294B

CN104770294B - A kind of protocorm based on the sprouting of iris seed is the breeding method of acceptor

Info

Publication number: CN104770294B
Application number: CN201510153223.6A
Authority: CN
Inventors: 张和臣; 王利民; 孟月娥; 董晓宇; 符真珠; 张晶; 李艳敏; 王慧娟; 蒋卉
Original assignee: Henan Academy of Agricultural Sciences
Current assignee: Henan Academy of Agricultural Sciences
Priority date: 2015-04-02
Filing date: 2015-04-02
Publication date: 2017-07-11
Anticipated expiration: 2035-04-02
Also published as: CN104770294A

Abstract

The invention belongs to genetic engineering breeding technical field, and in particular to a kind of based on the breeding method that the protocorm that iris seed is sprouted is acceptor.This method include obtaining protocorm, Multiplying culture, preculture, During Agrobacterium, co-cultivation, Agrobacterium elution, screening and culturing, into bud seedling, the step such as take root.The present invention constructs a set of iris transgene receptor system using the protocorm of iris seed sprouting as acceptor material, establish a set of genetic conversion system based on During Agrobacterium and corresponding detection architecture simultaneously, with obtaining, acceptor material is quick, performance is stable, the advantages of wide material sources, it is especially preferable to selective antibiotic sensitiveness, can effectively avoid iris induce protocorms during it is insensitive to antibiotic the problem of, beneficial to the screening of resistant transgenic material, obtain preferable, purer new iris system, there is preferable application value for promoting iris new varieties.

Description

A kind of protocorm based on the sprouting of iris seed is the breeding method of acceptor

Technical field

The invention belongs to genetic engineering breeding technical field, and in particular to a kind of protocorm sprouted based on iris seed For the breeding method of acceptor.

Background technology

Iris（Phalaenopsis aphrodite）Belong to orchid family phalaenopsis plant, be a kind of important potted plant flower Grass, with very high ornamental value and economic worth, but is due to that iris lacks with blueness or fragrance in nature Kind, traditional crossbreeding means can not realize the diversified demand to butterfly orchid tint, the fragrance of a flower.Thus new transgenosis is educated The technology of kind has obtained a certain degree of application in terms of iris new varieties are cultivated.Existing conventional transgenic breeding technology bag Include：Particle bombardment, microinjection and PEG that the agrobacterium-mediated transformation of foreign gene indirect reformer, foreign gene are directly converted Mediated transformation method etc., and suitable for the pollen tube passage method of many ovule plants；Wherein agrobacterium-mediated transformation be current research compared with Many, theoretical mechanism is clearer, technical method also more ripe gene transformation method.

Although transgenic breeding method have the advantages that breeding objective clearly, breeding time it is shorter, turn base in plant Because during, the selection of acceptor material is then basis and the key factor of breeding success or not.Suitable acceptor material contributes to Accelerate and improve genetic transformation be smoothed out and genetically modified plants successful incubation.For the transgenic breeding of iris, reason For, somatic embryo, protocorms and protocorm can serve as the preferable genetic transformation acceptor material of iris, but work as Preceding acquisition somatic embryo, the mode of protocorms mainly pass through the explants such as stem apex, stem section, blade, the tip of a root, root segment, pedicel axillary buds The induction of body is obtained, and in Induction Process, is commonly present brown stain, the low problem of inductivity, thus iris transgenic breeding real In trampling, often there is certain limitation as acceptor material using somatic embryo or protocorms.In addition, with somatic embryo or class protocorm When stem carries out transgenic breeding as acceptor material, when carrying out transgeneic procedure using particle gun or During Agrobacterium, there is also The low defect of transfection efficiency, and in During Agrobacterium operating process, also there is the low drawback of Antibiotic Sensitivity, thus it is anxious Need to further it improve.

The content of the invention

It is a kind of based on the breeding method that the protocorm that iris seed is sprouted is acceptor, the party present invention aims at providing The protocorm that method is sprouted using iris seed can preferably be overcome and existing be lured with explant as transgenic breeding acceptor material Lead the browning for obtaining acceptor material and existing, low conversion ratio, quality discrepancy, induction time length, the disadvantage such as the speed of growth is slow, subculture is difficult End；The present invention has cultivated new butterfly orchid variety by taking iris " red dragon " and " shining " kind as an example simultaneously, kind training Transgeneic procedure is carried out in During Agrobacterium mode during educating, pattern, two kinds of gene elements of the fragrance of a flower are directed respectively into, obtained to cultivate The iris germ plasm resource obtained newly provides new reference and reference.

Technical scheme is described in detail as follows.

A kind of protocorm sprouted based on iris seed is the breeding method of acceptor, is comprised the following steps：

（1）Under aseptic condition, by iris seed in Seed inducement germination medium germination and growth, obtain protocorm；

The butterfly orchid variety is preferably " red dragon " or " shining "；

The Seed inducement germination medium is：3g/L spend precious No. 1+2g/L activated carbon+2g/L caseinhydrolysate+ 15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar, pH=5.5 ~ 5.6；

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, the Lx of light intensity 3000, day illumination 16h cultivate 60 ~ 90d；

（2）By step（1）Directly or after Multiplying culture acceptor material is used as after middle protocorm stripping and slicing；

Proliferated culture medium is：3g/L spends No. 1+2g/L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6 mg/L of treasured 2,4-D+ 6.5g/L agar, pH=5.5 ~ 5.6；

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h, 60 ~ 90d of Multiplying culture, propagation Cultivate to diameter be about 0.5cm or so protocorm can as transgenosis acceptor material；

Step（1）Middle protocorm stripping and slicing diameter minimum is no less than 0.3cm；

（3）By step（2）The acceptor material preculture 3d of middle Multiplying culture, carries out During Agrobacterium；

The same step of the preculture used medium and growth conditions（2）；

The During Agrobacterium liquid is re-suspension liquid, and re-suspension liquid uses liquid proliferated culture medium, is formulated and is：3g/L spends precious No. 1 + 15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2,4-D, pH=5.5 ~ 5.6；

During During Agrobacterium, re-suspension liquid OD₆₀₀=0.4 ~ 0.6, contaminate 15min；

Bacterial strain is restructuring agrobacterium strains GV3101 in the During Agrobacterium liquid, and the bacterial strain includes plasmid expression vector pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’H；

The plasmid expression vector pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’HIt is by right PCAMBIA1301, pCAMBIA1303 carrier carry out transformation acquisition, concretely comprise the following steps：First respectively pCAMBIA1301, CAMV35S promoters are added at the multiple cloning sites of pCAMBIA1303 carriers（pCAMV35S）With CAMV35S terminators（T- CAMV35S）；Then in the genome of carrier distinguish recombination and integration Chinese rose floral base becauseRcOOMT2, heartsease Blue GeneVwF3’5’H；To finally the plasmid expression vector pCAMBIA1301 completed be transformed-RcOOMT2、pCAMBIA1303-VwF3’5’H Convert to agrobacterium strains GV3101；

（4）By step（3）Acceptor material is placed on co-cultivation culture medium after dip-dye, light culture 3d under the conditions of 25 DEG C；

The co-cultivation same step of nutrient media components（2）Middle proliferated culture medium；

（5）By step（4）Acceptor material after middle co-cultivation carries out Agrobacterium elution, and elution, which is used, contains 300mg/L Cef sterilized waters, which soak 20 ~ 30min, to be carried out；

（6）By step（5）Acceptor material after middle elution is placed on screening and culturing medium, during screening and culturing, is entered per 15d Row once turns sieve, is required to before turning sieve every time according to step（5）Middle method carries out the elution of Agrobacterium；

The screening and culturing medium is：Step（2）The middle mg/L of proliferated culture medium+8 Hyg+50 mg/L Cef, pH=5.5 ~ 5.6；

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h；

After screening and culturing four-wheel, step is accessed（2）The mg/L of proliferated culture medium+50 Cef culture medium in, continue cultivate Until differentiation and bud formation and growing up to seedling；Screening and Identification；

（7）By step（6）Further amplification or access root media are lured the middle correct Phalaenopsis plants of Screening and Identification Lead and take root；

The root media is：3g/L spend precious No. 1+1.5mg/L IBA+20g/L sucrose+1g/L activated carbon+ 6.5g/L agar, pH=5.5 ~ 5.6.

The present invention constructs a set of iris transgene receptor body using the protocorm of iris seed sprouting as acceptor material System, while a set of genetic conversion system based on During Agrobacterium and corresponding detection architecture are established, compared to existing butterfly Phalaenopsis transformation system, more obvious technical advantage is embodied in following aspects：One is, the protocorm institute sprouted using seed The acceptor systems of structure, it is more quick by explant acquisition acceptor material mode compared to other；The present invention is also provided simultaneously The amplification system of protocorm, thus in general, acceptor material of the invention is compared to prior art, acceptor material performance is steady It is fixed, wide material sources；Two be genetic conversion system constructed by the present invention to selective antibiotic sensitiveness preferably, can effectively avoid Iris induce protocorms during it is insensitive to antibiotic the problem of, beneficial to the screening of resistant transgenic material.It is overall For, the present invention is by breeding the screening of system to existing iris protocorm, optimizing, to existing iris transgenic breeding The improved application of system, successfully constructs a set of more complete and conversion, the preferable iris transgenic breeding of screening effect System, technological means more ripe, breeding effect has preferable guarantee, preferable for promoting iris new varieties to have Application value.

Brief description of the drawings

Fig. 1 is pCAMBIA1301, pCAMBIA1303 vector modification schematic diagram；

Fig. 2 is pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’HPlasmid expression vector builds schematic diagram；

Fig. 3 sprouts the protocorm that 60d is formed by seed in iris tissue cultures；

Fig. 4 is influence of the different combination of regulators processing to Protocorm Multiplication culture；

Fig. 5 is influence of various concentrations 6-BA, 2,4-D combined treatment to Protocorm Multiplication culture；

Fig. 6 is influence of the different activities carbon content to protocorm Stem nematode, wherein A：0mg/L activated carbons, B：1mg/L activity Charcoal, C：2mg/L activated carbons；

Fig. 7 is antibiotic resistance the selection result, wherein A：Control（CK）, B：1mg/L Hyg, C：3mg/L Hyg, D：5mg/ L Hyg, E：8mg/L Hyg, F：10mg/L Hyg, G：10mg/L Kan, H：50mg/L Kan, I：100mg/L Kan；

Fig. 8 is taken root situation for iris；

Fig. 9 is the influence that different concentration of Ce f grows to Agrobacterium, and A-E, wherein A are followed successively by from left to right：0mg/L Cef, B：50mg/L Cef, C：100mg/L Cef, D：200mg/L Cef, E：300mg/L Cef；

Figure 10 detects that wherein A is conversion for PCRRcOOMT2, B for conversionVwF3’5’HPCR detection, M is Marker, N For Negative, P is Positive；1# ~ 5# is the iris transformed plant sample randomly selected respectively；

Figure 11 is the detection of GUS tissue stainings, wherein A：Control, B：GUS detects positive material.

Embodiment

With reference to embodiment the present invention will be further explained explanation.

Before specifically embodiment is introduced, part test material and instrument used in following embodiments are briefly situated between first Continue as follows.

Iris material：Butterfly orchid variety used includes " red dragon ", " shining " two business irises in embodiment Kind, iris seed used is obtained by iris selfing.

Tissue culture culture medium part material：It is used in tissue culture culture medium to spend precious No. 1,6-BA, 2,4-D, NAA, IBA, hydrolysis junket The materials such as albumen, TDZ, activated carbon, sucrose, agar are purchased from Tong Ya companies.

Portion of reagent used in transgeneic procedure：Ampicillin（Amp）, kanamycins（Kan）, rifampin（Rif）, celebrating Big mycin（Gen）, hygromycin（Hyg）, cephalosporin（Cef）Bao Sai companies are purchased from Deng antibiotic.

Agrobacterium：Agrobacterium strains GV3101 competence is purchased from its company advanced in years, contains after restructuring in agrobacterium strains GV3101 There is plasmid expression vector pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’HPlasmid, recombinant bacterial strain is adopted by inventor Built-up with technique for gene engineering, wherein target gene is respectively to clone from Chinese roseRcOOMT2Gene, heartseaseVwF3’5’HGene, the specific regrouping process that builds can be found in Fig. 1, Fig. 2 and embodiment 1.

Portion of reagent used by carrier construction：Used carrier pCAMBIA1301, pCAMBIA1303 derive from CAMBIA companies. Used by building processPstI、XbaI、EcoRI、Sac I、BamH I、KpnThe restriction endonucleases such as I, high-fidelity Taq enzyme, pMD18-T Carrier, cDNA synthetic agent box and T4 ligases etc. are purchased from Takara companies, Escherichia coli Top10 competent cells, glue reclaim Small extraction reagent kit of kit, plasmid etc. is biochemical purchased from Tiangeng（Beijing）Co., Ltd.

Correlation LB culture mediums in Escherichia coli, Agrobacterium incubation

LB fluid nutrient mediums（Antibiotic-free）：10g/L tryptone+5g/L yeast extract+10g/L sodium chloride；

LB solid mediums（Antibiotic-free）：10g/L tryptone+5g/L yeast extract+10g/L sodium chloride+ 15g/L agar；

Escherichia coli（Containing recombinant plasmid）Culture medium：LB culture mediums+antibiotic, antibiotic is：100 mg/L Kan；

Agrobacterium（Containing recombinant plasmid）Culture medium：LB culture mediums+added with antibiotic, antibiotic is：30mg/L Gen+100 mg/L Kan+50 mg/L Rif。

Iris culture medium for tissue culture

Seed inducement germination medium：3g/L spends precious No. 1+2g/L activated carbon+2g/L caseinhydrolysate+15g/L sugarcane Sugar+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar, pH=5.5 ~ 5.6；

Proliferated culture medium：3g/L spends No. 1+2g/L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2 of treasured, 4-D+ 6.5g/L agar, pH=5.5 ~ 5.6；

Liquid proliferated culture medium：3g/L spends treasured No. 1+15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2,4-D, pH= 5.5~5.6；

Screening and culturing medium：The mg/L Hyg+50 mg/L Cef of proliferated culture medium+8, pH=5.5 ~ 5.6；

Before During Agrobacterium culture medium is co-cultured after acceptor material precultivation medium, dip-dye：Same proliferated culture medium.

It should be noted that above culture medium is both needed to 121 DEG C of autoclaving 20min after the completion of preparing, because antibiotic is high It can be failed under the conditions of temperature, therefore when preparing the LB culture mediums containing antibiotic, screening and culturing medium, need that first culture medium goes out through high pressure After bacterium, be down on superclean bench in less than 60 DEG C of culture medium to temperature add it is corresponding needed for the Gen of concentration, Kan, The antibiotic such as Rif, Hyg, Cef are to prepare completion.

Antibiotic mother liquor is specifically formulated as follows：

Kanamycins（Kan）, ampicillin（Amp）：With distillation water dissolves, final concentration 100mg/mL, suction filtration sterilizes ,- 20 DEG C of preservations；

Rifampin（Rif）：Dissolved with methanol, final concentration 50mg/mL, suction filtration sterilizing, -20 DEG C of preservations；Cephalosporin （Cef）：With distillation water dissolves, final concentration 100mg/mL, suction filtration sterilizing, 4 DEG C of preservations.

Gentamicin（Gen）, hygromycin（Hyg）Mother liquor is purchased from Bao Sai companies.

Testing reagent GUS coloring agents

GUS dyes A liquid（50 mL）：0.2 M phosphate buffers（pH=7.0）, 25 mL, the 0.1 M potassium ferricyanides, 0.25 ML, 1 M Na2EDTA, 0.5 mL, distilled water, 24.25mL；

GUS dyes B liquid：Add the chloro- 3- indoles-beta-glucosidases of the 50 bromo- 4- of mg 5-（X-gluc）In 50 mL A liquid In, the preparation of B liquid is fully completed after dissolving.

Key instrument has：Superclean bench, centrifuge, PCR instrument, electrophoresis apparatus, ultraviolet gel imaging instrument, shaking table, pipettor, It is laboratory common equipment.

Embodiment 1

Due to building recombinational agrobacterium bacterial strain GV3101, i.e., by plasmid expression vector pCAMBIA1301-RcOOMT2、 pCAMBIA1303-VwF3’5’HLink more important in being the present invention into Agrobacterium original strain GV3101 is recombinated, thus To being described below for restructuring agrobacterium strains GV3101.

As shown in Figure 1 and Figure 2, recombinational agrobacterium bacterial strain GV3101 acquisition process is：First to pCAMBIA1301, PCAMBIA1303 carriers are transformed, whole plus CAMV35S promoters and CAMV35S at the multiple cloning sites of carrier respectively It is only sub；Then in the genome of carrier distinguish recombination and integration Chinese rose floral base becauseRcOOMT2, the crucial base of the blue petal of heartsease CauseVwF3’5’H；To finally the plasmid expression vector pCAMBIA1301 completed be transformed-RcOOMT2、pCAMBIA1303-VwF3’ 5’HConvert to agrobacterium strains GV3101.Detailed process is described below.

（One）PCAMBIA1301, pCAMBIA1303 vector modification-be integrated into promoter and terminator

CAMV35S promoters are added at the multiple cloning sites of pCAMBIA1301, pCAMBIA1303 carrier respectively （pCAMV35S）With CAMV35S terminators（T-CAMV35S）.Wherein pCAMV35S sequence is cloned in pCAMBIA1301, chooses Restriction enzyme site be respectivelyPstI andXbaI；T-CAMV35S sequences are cloned in pCAMBIA1301, and the restriction enzyme site of selection isEcoRI andSac I.Detailed process is as follows.

（1）Design primer, PCR cloning promoters and terminator sequence

Primer sequence design is following (5'-3')：

pCAMV35S-5’：CCTCTAGAAGAGATAGATTTGTAGAGAGAG；

pCAMV35S-3’：TGCCTGCAGATGGTGGAGCACGACACTC；

T-CAMV35S-5’：CGAATTCAGGTCACTGGATTTTGGTTTTA；

T-CAMV35S-3’：TACCGAGCTCCGGCCATGCTAGAGTCCGCAA.

PCR is cloned：Enter performing PCR clone by template of pCAMBIA1301 plasmids,

20 μ L PCR reaction systems are as follows：

3 ' primers, 5 ' primers, each 0.20 μ L；

DNTPs, 0.20 μ L；

10 × buffer, 2 μ L；

Plasmid template, 1 μ L；

Taq enzyme, 0.15 μ L；

ddH₂O, 16.25 μ L.

PCR response procedures：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C extend 1min, 31 Individual circulation；72 DEG C re-extend 10min；4 DEG C of terminations.

（2）Separation, recovery

By PCR clonal expansions product and DNA molecular Marker under 1 × TAE buffer conditions, with a little bromination second of addition The 1.0% Ago-Gel separation of ingot, then carries out PCR primer recovery according to glue reclaim kit specification.

（3）Digestion, connection

Digestion, connection are carried out to PCR primer and plasmid vector respectively.

By taking pCAMBIA1301 as an example, digestion system is as follows：

Pst I、XbaI, each 1 μ L；

10 × buffer, 5 μ L；

PCAMBIA1301 plasmids, 100ng；

ddH₂O to 50 μ L.

37 DEG C of digestions are stayed overnight.

Digestion products are carried out with 1.0% Ago-Gel separation and is reclaimed according to glue reclaim kit specification.

Recovery product is stayed overnight with the 16 DEG C of connections of T4 ligases, and linked system is as follows：

PCAMBIA1301 plasmid enzyme restriction recovery products, 1 μ L；

Ligation solution, 5 μ L；

pCAMV35S（Or T-CAMV35S）Digestion glue reclaim product after PCR clonal expansions, 4 μ L.

（4）Plasmid conversion, amplification, extraction

By step（3）Connection product convert to E. coli competent Top10 cells, amplification plasmid simultaneously extracts standby. The vector plasmid successfully constructed is extracted from Escherichia coli, by plasmid extraction kit（Tiangeng is biochemical（Beijing）Co., Ltd）Explanation Book is carried out.Only the process that connection product converts E. coli competent Top10 cells is briefly discussed below.

Conversion process is as follows：

A. E. coli competent Top10 cells are placed in ice, after melting, different centrifuge tubes are divided in per 50uL, 10 μ L connection product is added thereto, is gently shaken up and is placed in 30min on ice；

B. centrifuge tube is placed in 42 DEG C of heat shock 90s after the completion of ice bath, then is immediately placed in 2min on ice；

C. the LB fluid nutrient mediums that 500 μ L are free of antibiotic are added into centrifuge tube, are fully mixed, 37 DEG C of pre- expression 45min；

D. 150 μ L are taken to express the X-gal of bacterium solution and 40 μ L in advance（20mg/mL）Fully mix, be drawn to and prepared with sterile pipette tip The LB flat boards containing 100mg/L Amp, mixed liquor is uniformly coated with to whole planar surface with spreader；

E. flat board is inverted 37 DEG C of culture 16h；

F. by cultured flat board in step E on super-clean bench 4 white positive colonies of picking, in 5mL（Contain 100mg/L Amp）LB fluid nutrient mediums in concussion and cultivate stay overnight.

G.PCR bacterium solutions are identified, identify the correct further sequencing identification of product, and correct -80 DEG C of guarantor bacterium of bacterium solution will be sequenced Standby or extraction plasmid is saved backup.

（Two）Chinese rose floral base becauseRcOOMT2, the blue petal key gene of heartseaseVwF3’5’HClone and vector construction

The process mainly by Chinese rose floral base becauseRcOOMT2, the blue petal key gene of heartseaseVwF3’5’H The technical operation such as PCR clones, connection, digestion realize that detailed process is briefly discussed below.

（5）Chinese roseRcOOMT2And heartseaseVwF3’5’HGene cloning

With the Chinese rose floral base that is recorded in ncbi database becauseRcOOMT2（Indexed number：AJ439742.1）And heartseaseVwF3’5’HGene（Indexed number：AB332097）Full length sequence be defined, PCR primer is designed according to the gene order of acquisition, it is main Syllabus is to introduceBamHI andKpnI restriction enzyme site.

Primer sequence design is following (5'-3')：

RcOOMT2-5’：CCTTGGATCCATGGAAAGGCTAAACAGCTTT；

RcOOMT2-3’：CTCATTGAGGTTTATCCTTGAGGTACCAAGG；

VwF3'5'H-5’：CCTTTGGATCCATGGCAATTCTAGTCACCGAC；

VwF3'5'H-3’：CCTTTGGTACCTCAGGTTGCGTACGCGTTTGA.

RNA is extracted, cDNA synthesis：Chinese rose is extracted respectively using CTAB methods（Double Happiness kind）, heartsease RNA, press Reverse transcription, which is carried out, according to cDNA synthetic agent box specification obtains cDNA.

PCR is cloned：Using cDNA as template, the PCR clones of gene order are carried out respectively with high-fidelity Taq enzyme.PCR reactants System and response procedures refer to step（1）It is middle to set.

（6）Separation, recovery

By step（5）In PCR clonal expansions product and DNA molecular Marker under 1 × TAE buffer conditions, with adding Enter the 1.0% Ago-Gel separation of a little ethidium bromide, then carrying out PCR primer according to glue reclaim kit specification returns Receive.

（7）Connection

By step（6）Middle PCR recovery products are connected with pMD18-T carriers, obtain connection productRcOOMT2- pMD18-T,VwF3’5’H-pMD18-T.Coupled reaction is carried out with reference to pMD18-T kits.

（8）Conversion, amplification, extraction

By step（7）In connection productRcOOMT2- pMD18-T,VwF3’5’H- pMD18-T is converted to Escherichia coli sense By state cell Top10, extract plasmid and identify, be sequenced.Concrete operation step refers to step（4）.Correct bacterium solution -80 will be sequenced DEG C protect bacterium it is standby or extract plasmid save backup.

（Three）Plasmid expression vector pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’HStructure and restructuring Into Agrobacterium original strain GV3101

By step（One）After middle transformation（Add promoter and terminator）Carrier pCAMBIA1301, pCAMBIA1303 With step（Two）InRcOOMT2- pMD18-T,VwF3’5’H- pMD18-T plasmids are used respectivelyBamHI andKpnI enzymes carry out digestion, 37 DEG C of digestions are stayed overnight, and endonuclease reaction system refers to step（3）Set.

Digestion products carry out 1.0% Ago-Gel separation and glue reclaim；The digestion products of recovery are connected using T4 afterwards Connect enzyme accordingly to be connected at 16 DEG C, coupled reaction system sets and refers to step（3）.

It is constructed pCAMBIA1301- by connection productRcOOMT2、pCAMBIA1303-VwF3’5’HConvert to big Enterobacteria competent cell Top10, PCR detection positive colony, identifies the correct further sequencing identification of product, and sequencing is correct - 80 DEG C of bacterium solution protect that bacterium are standby or extract plasmid and save backup, correlation step refers to step（4）.

To the pCAMBIA1301- extractedRcOOMT2、pCAMBIA1303-VwF3’5’HPlasmid is further converted to agriculture Bacillus strain GV3101, is comprised the following steps that：

A. take Agrobacterium competent cell to be placed on ice slowly to melt, then add into 50 μ L Agrobacterium competent cells Enter the DNA that 2 μ L are extracted, that is, the pCAMBIA1301- extractedRcOOMT2、pCAMBIA1303-VwF3’5’HPlasmid, Ice bath 30min after mixing；

B. liquid feeding nitrogen frost 1min, is put into rapidly 37 DEG C of water-baths and is incubated 3 ~ 5min；

C. 800 μ L LB fluid nutrient mediums are added, are shaken up, 28 DEG C of 4 ~ 5h of concussion and cultivate；Then 3000 rpm, 4 DEG C of centrifugations 3min, collects thalline；

D. the step C thalline collected are uniformly coated on the LB solid mediums containing Kan, Rif and Gen, 28 DEG C of cultures 2 ~ 3d, respectively 4 single bacterium colonies of picking be placed on the LB fluid nutrient mediums containing same antibiotic 28 DEG C, 180rpm concussion and cultivates Overnight；

E.PCR detects bacterium solution, and positive colony can be used to follow-up transgeneic procedure（During Agrobacterium）, or by the positive It is standby that -80 DEG C of refrigerators of clone protect bacterium.

Embodiment 2

The present embodiment highlights the protocorm sprouted in During Agrobacterium before transgenic breeding based on iris seed The structure of the genetic stocks of acceptor, is briefly discussed below.

（1）Under aseptic condition, iris seed is induced into germination and growth in the medium, protocorm is obtained；

Specially：Ripe iris fruit is cut from maternal plant, after being cleaned with suds, is positioned under clear water and rinses 30min；Fruit after flushing is put into sterile large beaker on superclean bench, 70% ethanol disinfection 15min is added, 10% is used afterwards Hypochlorite disinfectant 15min, during which not stopping to rock beaker makes it fully contact；Aseptic water washing is used after the completion of sterilization 3 times；With The tissue culture knife of sterilizing cuts the iris fruit being placed on aseptic paper, will be taken out with seed-bearing pulp with aseptic nipper, Rinsed in the culture dish for filling a small amount of sterilized water（Purpose is to separate the seed adhered to thereon with pulp）, after with liquid-transfering gun will Uniformly sprayed in the iris Seed inducement germination medium after sterilizing containing seed-bearing sterilized water.

The Seed inducement sprouts required culture medium：3g/L spends precious No. 1+2g/L activated carbons+2g/L hydrolysis junket egg + 15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar, pH=5.5 ~ 5.6 in vain.

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h cultivate 60 ~ 90d, grow into original Bulb.

As shown in figure 3, after seed culture 60d, having formed larger protocorm（0.4 ~ 0.5cm or so）, for protocorm Multiplying culture.

Because quantity still has certain limitation after protocorm stripping and slicing, therefore often needed further for protocorm after stripping and slicing Propagation amplification to obtain enough acceptor materials, by experiment discussion repeatedly, it has been recognised by the inventors that optimal condition of tissue culture is such as Under：

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h, 60 ~ 90d of Multiplying culture, propagation Culture be about 0.5cm or so to diameter protocorm after can as transgenosis acceptor material；

Protocorm stripping and slicing diameter minimum for Multiplying culture is no less than 0.3cm.

The acquisition process to optimal culture condition in Protocorm Multiplication incubation is briefly discussed below below.

The preliminary screening of hormon species and concentration

Plant growth regulator combination is the important factor in order in plant tissue culture course, suitable growth regulator Combination can induce the Multiplying culture of group training material, or change the growth conditions of group training material（Such as：It is the breaking up of group training material, raw Root etc.）.

Following examples carry out screening test, purpose to combination of regulators first using iris protocorm as material It is the combination of regulators for selecting suitable Protocorm Multiplication.Protocorm during iris Multiplying culture is sprouted from seed 60 ~ 90d is sent out, diameter is about 0.5cm, the undifferentiated protocorm into bud.

Iris Protocorm Multiplication medium component is：3g/L spends treasured+2g/L activated carbon+15g/L sucrose+6.5g/L Agar, pH=5.5 ~ 5.6.

Hormone selects 2, tri- kinds of 4-D, 6-BA and TDZ, sets 12 kinds of processing, and specific processing combination is as shown in the table.

During inoculation, 10 iris protocorms of every bottle of inoculation of medium, each processing repeats five bottles.

Cultivated in 25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h culturing room.

Count item and computing formula is as follows：

Differentiation and growth coefficient（%）=it can induce differentiation, stripping and slicing number/inoculation sum × 100% of propagation.

Cultivation results are as shown in following table and Fig. 4.

As can be seen from the above table, in 12 kinds of proliferated culture medium combinations, processing 1, processing 2, processing 9, processing 10, processing 11 The differentiation and proliferation of protocorm is occurred as soon as when cultivating 30d with processing 12.The differentiation and proliferation rate of wherein processing 9 and processing 10 exists 90% has been reached during 30d, and the differentiation and proliferation rates of processing 1,2 and processing 11,12 when cultivating 30d also respectively reach 40% and 25%. And not only breed in processing 5,6,7,8 after culture 30d without protocorm differentiation and occur, to even occurring during 60d, material death is existing As.The above results show：2,4-D concentration is excessive in protocorm differentiation breeding（More than 1mg/L）Not only bad for protocorm Differentiation and proliferation culture, or even cause the death of protocorm stripping and slicing；When 2,4-D concentration are 1mg/L, although in 30d culture Also occur differentiation and proliferation phenomenon in journey, but differentiation and proliferation efficiency and upgrowth situation when being 0.5mg/L not as good as 2,4-D concentration, because This, it is most favourable to the differentiation and proliferation culture of iris protocorm when tentatively judging 2,4-D concentration less than 1mg/L.

Processing 9,10 in growth regulator be combined as 2,4-D collocation 6-BA, processing 11,12 in be then 2,4-D and TDZ Combination, processing 9 and processing 10 culture 60d after differentiation and proliferation coefficient reach more than 95%, though and processing 11 and processing 12 in So also there is higher differentiation and proliferation coefficient（Reach 80%）, but then effect is slightly worse for contrast the first two processing.Result of the test table It is bright：The effect of 6-BA differentiation and proliferation is better than TDZ, but the difference that its concentration influences in 1mg/L and 2mg/L on differentiation and proliferation is not Greatly.

To sum up, found by the Comparative result to each processing combination：2,4-D and 6-BA combination is better than 2,4-D and TDZ Combination, thus preliminary judgement be applied to Protocorm Multiplication culture nutrient media components be：3g/L spends treasured+0.5mg/L 2,4-D + 1.0mg/L 6-BA+15g/L sucrose+2g/L activated carbon+6.5g/L agar, pH=5.5 ~ 5.6.

Effects of the various concentrations 2,4-D to Protocorm Multiplication

It can be seen from the Multiplying culture of foregoing protocorm on hormone kind screening test result, 2,4-D and 6-BA Though hormone combinations be conducive to the propagation of protocorm, still need to further optimize its concentration, to meet actual production need Will.Because 2,4-D is acted on more greatly as auxin in Protocorm Multiplication incubation, thus following work are firstly for 2,4- Screening is optimized in D consumption.

It can be seen from aforementioned result, it is unfavorable for the Multiplying culture of protocorm when being more than 1mg/L due to 2,4-D concentration, therefore, 2,4-D concentration range is set between 0 ~ 1mg/L by we, and 5 different disposals are devised altogether, is 0mg/L, 0.2mg/ respectively L、0.4mg/L、0.6mg/L、0.8mg/L。

Material therefor sprouts 60 ~ 90d from iris " red dragon " and " shining " seed, and diameter is about 0.5cm, the undifferentiated protocorm into bud.14 iris protocorms of every bottle of inoculation of medium, each processing repeats 5 bottles. Cultivated in 25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h culturing room.To two kinds after Multiplying culture 60d The upgrowth situation of protocorm stripping and slicing is counted.

Count item and computing formula is as follows：

Protocorm proliferative induction rate（%）=propagation forms stripping and slicing number/inoculation sum × 100% of protocorm；

Protocorm induces differentiation rate（%）=it is divided into the stripping and slicing number of seedling/inoculation sum × 100%；

Protocorm callus induction rate（%）The stripping and slicing number of=formation callus/inoculation sum × 100%.

Specific disposition and statistics are as shown in the table.

As can be known from the above table, different cultivars iris protocorm is under same 2,4-D concentration processing, the proliferation rate of protocorm Have differences；But something in common is that the proliferation rate of protocorm is relatively low, now most when 2,4-D concentration is less than 0.2mg/L Stripping and slicing no longer breeds to form protocorm agglomerate, but directly into bud and further growth seedling after single protocorm is grown up to； When 2,4-D concentration are 0.4 ~ 0.6mg/L, the Protocorm Multiplication rate of two kinds is higher, and highest reaches 70.0%；And when 2, When 4-D concentration is 0.8mg/L, although have the formation of yellow callus in two kinds, but compared to low concentration（0~0.2mg/ L）Processing for Protocorm Multiplication rate it is of a relatively high.Therefore, it is unfavorable for iris protocorm when 2,4-D concentration is too low or too high The propagation of stem, the differential growth of too low easy promotion protocorm in a short time, not only influence is bred and easily causes protocorm agglomerate Small volume.

The Combinatorial Optimization screening of plant growth regulator concentration

According to aforementioned growth element 2,4-D optimal screening result and the preliminary screening of different growth regulator species and concentration As a result, we further devise 6-BA and 2,4-D several groups of concentration combinations（Other components：3g/L spends treasured+2g/L activated carbons + 15g/L sucrose+6.5g/L agar, pH=5.5 ~ 5.6）, tested by further tissue culture, count protocorm proliferative induction rate （Protocorm proliferative induction rate（%）=propagation forms stripping and slicing number/inoculation sum × 100% of protocorm）, so as to filter out optimum In the hormone in medium concentration combination of Protocorm Multiplication.

Material therefor sprouts 60 ~ 90d from iris " red dragon " and " shining " seed, and diameter is about 0.5cm, the undifferentiated protocorm into bud.During tissue culture, the 14 iris protocorm strippings and slicings of every bottle of inoculation of medium, each Processing repeats 5 bottles.In 25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx is cultivated in day illumination 16h culturing room.

Specific growth regulator proportioning combination sets as follows.

Specific tissue culture result is as shown in following table and Fig. 5.

The protocorms of two kinds is can be seen that under identical 2,4-D concentration conditions from upper table and Fig. 5, with 6-BA The raising Protocorm Multiplication rate of concentration is also improved therewith；When 6-BA concentration reaches maximum concentration 5mg/L, the 2,4-D of high concentration （0.8mg/L）The formation of protocorm agglomerate can be suppressed, and concentration is low（0.4mg/L）2,4-D are then presented budding differentiation state, and When 2,4-D concentration is 0.6mg/L, the propagation ability of protocorm is based on lumps.By being 0.6mg/L's to 2,4-D concentration Three results relatively find out that 6-BA is weaker than the multiplication capacity of high concentration for 1mg/L processing；6-BA concentration is 5mg/ Though multiplication capacity is relatively strong during L, late-stage differentiation is into the more of bud；And when 6-BA concentration is 3mg/L, the proliferation rate of protocorm is not Lumps are presented the protocorm that the only higher and later stage obtains into the less of bud, propagation more.Therefore, sent out according to the analysis of result of the test Existing 0.6mg/L 2,4-D+3mg/L 6-BA hormone combination is applied to the Multiplying culture of protocorm.

Summary can be seen that in iris tissue culture procedures on the selection result of growth regulator, compared with Excellent culture medium is that proliferated culture medium composition is：3g/L spends precious No. 1+3mg/L 6-BA+0.6mg/L 2,4-D+2g/L activity Charcoal+15g/L sucrose+6.5g/L agar, pH=5.5 ~ 5.6.

Influence of the protocorm stripping and slicing diameter to Multiplying culture

Found in the conventional course of work, often occur that the albefaction death of stripping and slicing is existing during the Multiplying culture of protocorm As Primary Study is considered to cause the generation of albinism because stripping and slicing is too small.For the ease of being held in production application Optimal protocorm stripping and slicing size, inventor has carried out further discussion for the stripping and slicing size of protocorm.

A diameter of A is respectively cut into protocorm（Less than 0.2cm）、B（0.2~0.3cm）、C（More than 0.3cm）It is three kinds big Small stripping and slicing, accesses proliferated culture medium（I.e.：3g/L spends No. 1+2g/L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6 of treasured Mg/L 2,4-D+6.5g/L agar, pH=5.5 ~ 5.6）Its growth conditions is counted after middle culture 60d.

14 iris protocorm strippings and slicings of every bottle of inoculation of medium, each processing repeats 5 bottles.In 25 DEG C of day temperature, night temperature 17 DEG C, light intensity 3000Lx is cultivated in day illumination 16h culturing room.

Cultivation results are as shown in the table.

As can be seen from the above table, the diameter of protocorm stripping and slicing has a larger shadow to albefaction during Multiplying culture Ring；As a diameter of A of stripping and slicing（Less than 0.2cm）When, there is albinism in cultivating has 30% stripping and slicing after 14d, and with culture day The quantity of several increase albefaction strippings and slicings is consequently increased, and stripping and slicing albefaction rate has reached 73% after 60d is cultivated, and breeds the original Bulb volume is also smaller, it is impossible to the progress for follow-up test.In a diameter of B of stripping and slicing（0.2~0.3cm）When, during identical culture Though in its albefaction rate lacking compared with A, the volume that it breeds single protocorm on the protocorm agglomerate formed is also not big enough, if with Obvious albinism can be still produced in follow-up test, increases test error, and reduce experiment accuracy.As a diameter of C （More than 0.3cm）When, occur albinism individually though also having, 87% stripping and slicing breeds to form big protocorm agglomerate and include The single volume of protocorm it is also larger, and be mostly the more stripping and slicing of tangent plane in the stripping and slicing of albefaction, illustrate that protocorm damage is tighter Also it is unfavorable for its propagation during weight.Therefore, stripping and slicing damage and guarantee stripping and slicing should be reduced to the stripping and slicing processing of protocorm in actual production Diameter is sufficiently large.

The influence that different content activated carbon grows to protocorm stripping and slicing Multiplying culture

Brown stain is common a kind of culture materials lethality in plant tissue culture course.Therefore, in the tissue of plant In incubation, often selection adds the material such as adsorbent or antioxidant to carry out the preventing and treating of brown stain, and wherein activated carbon is The most commonly used adsorbent.Due to floristics difference, in different plant tissue culture courses, activated carbon addition also not phase Together, thus inventor has further carried out screening test to the addition of activated carbon.

The content of activated carbon in proliferated culture medium is respectively set to 0g/L, 1g/L, 2g/L, material therefor derives from butterfly Blue " red dragon " and " shining " seed sprout 60 ~ 90d, and diameter is about 0.5cm, the undifferentiated protocorm into bud.Every bottle 14 protocorm strippings and slicings of inoculation of medium, each processing sets 5 bottles of repetitions, in 25 DEG C of day temperature, 17 DEG C of night temperature, light intensity Cultivated in 3000Lx, day illumination 16h culturing room.

Observation statistics different activities carbon content under protocorm stripping and slicing Growth and Differentiation state and count its survival rate and browning Rate, computing formula is as follows：

Protocorm stripping and slicing survival rate（%）=produce protocorm stripping and slicing number/inoculation sum × 100%；

Protocorm stripping and slicing melting brown rate（%）=browning death stripping and slicing number/inoculation sum × 100%.

Specific test result is as shown in the table, and morphologic observation result is as shown in Figure 6.

As can be seen from the above table, activated carbon content is higher, as the stripping and slicing number that browning occurs for the increase of cultivated days is fewer； In the processing that inactive charcoal is added, there is browning in existing minority after culture 14d, the brown discharged to material during 60d Material make it that culture medium is also changed into black；In latter two processing, although the activated carbon of 1g/L contents can also reduce brown stain very well Generation, but its effect is more slightly worse than what 2g/L content was handled.In addition, it has also been found that with activated carbon content in process of the test Raising, the value-added effect and growing way of stripping and slicing increase.Therefore, in general, the activated carbon from 2g/L is used to improve Brown stain situation in Protocorm Multiplication incubation is preferable.

Antibiotic（Kan and Hyg）Influence to iris Protocorm Multiplication

Selective agent is used for the preliminary screening of converting material in Genetic Transformation in Higher Plants experiment, is primarily referred to as those and plant is made Into murder by poisoning, so as to suppress the antibiotic substance of its growth.Kan and Hyg is two kinds of conventional selective agents of Genetic Transformation in Higher Plants, in butterfly It is also widely used in phalaenopsis transgenic research.Because different acceptor materials has differences to the sensitiveness of different antibiotic, Therefore, the selection of selective agent generally need to carry out the resistant proof of Multiple Classes of Antibiotics to determine to acceptor material.

Now there are some researches show iris protocorm is insensitive to Kan toxicity, and the lifes of Kan mainly to iris root system Into there is considerable influence, therefore the Kan of larger dose is often selected in iris Transgenic studies as selective agent, or in acceptor Material to proceed to and carry out screening and culturing to it again when taking root the stage.Hyg is also using more in iris genetic transformation test A kind of selective agent, its mainly show be to iris growth have stronger inhibitory action.

To obtain optimal Antibiotics and screening concentration, inventor is carried out using iris protocorm as test material Kan and Hyg resistance screening experiment, to selecting most suitable class of antibiotic and concentration.

Material therefor sprouts 60 ~ 90d from iris " red dragon " and " shining " seed, and diameter is about 0.5cm, the undifferentiated protocorm into bud carries out Kan, Hyg resistance screening experiment respectively.

Kan resistance screening：Protocorm stripping and slicing is respectively connected to Kan concentration for 0 mg/L, 10 mg/L, 50 mg/L, 100 Kan resistance screening is carried out in mg/L proliferated culture medium.

Hyg resistance screening：Protocorm stripping and slicing is respectively connected to containing 0 mg/L, 1 mg/L, 3 mg/L, 5 mg/L, 8 Resistance screening is carried out in mg/L, 10mg/LHyg proliferated culture medium.

14 iris protocorms of every bottle of inoculation of medium, each processing repeats 5 bottles.In 25 DEG C of day temperature, 17 DEG C of night temperature, Cultivated in light intensity 3000Lx, day illumination 16h culturing room.After culture one month, the survival rate of each processing of observation statistics.

Specific result of the test is as shown in the table, and morphologic observation result is as shown in Figure 7.

For Kan resistance screening result, it can be seen that two butterfly orchid varieties from upper table result and Kan resisted Property has differences, but something in common is that the two resistance to Kan is not strong, when Kan concentration reaches 100 mg/L, two product The propagation for planting stripping and slicing is heavily suppressed, survival rate difference as little as 7.1% and 18.6%.Therefore, using protocorm as acceptor material Can be from Kan as selective agent in the transgenic research of material, the screening for converting material.

For Hyg resistance screening result, it be can be seen that from upper table result when adding Hyg in culture medium, two The survival of kind stripping and slicing is suppressed, and with the raising of Hyg contents, survival rate is substantially reduced.When Hyg contents are 3mg/L When, survival rate as little as less than 30%；When Hyg contents are 5 ~ 8mg/L, stripping and slicing shows the suppressed phenomenon of serious growth, arrives Survived when content is 10mg/L almost without stripping and slicing.

According to the selection result of two above antibiotic, Kan and Hyg have inhibitory action to the propagation of protocorm, but just For inhibition, Hyg is then better than Kan, because Hyg is in low concentration（5mg/L）When can suppress the increasing of protocorm well Grow, and Kan then needs higher concentration（100mg/L）This effect is can be only achieved, therefore, on the genetic transformation of iris more It is adapted to using Hyg as selective agent, and in concentration selection, in order to reduce the probability of false positive appearance, in the choosing of screening agent concentration The Hyg for selecting selection content 8mg/L concentration is more suitable.

Influence of the bacteriostatic agent to iris Protocorm Multiplication

Due in being tested with Agrobacterium-mediated genetic transformation, it is necessary to which adding appropriate bacteriostatic agent suppresses Agrobacterium Growth, to avoid because bacterial plaque grows the negative effect produced to acceptor material.

To obtain bacteriostatic agent（Cef, cephalosporin）Optimum amount, inventor is further with iris " red dragon " and " light Awns four is penetrated " seed sprouting, Multiplying culture experiment has been carried out based on the protocorm that diameter is about 0.5cm, has been described as follows.

After protocorm stripping and slicing, be linked into Cef contents be respectively 0 mg/L, 50mg/L, 100mg/L, 200mg/L, Cultivated in 300mg/L proliferated culture medium.

14 iris protocorms of every bottle of inoculation of medium, each processing repeats 5 bottles.In 25 DEG C of day temperature, 17 DEG C of night temperature, Cultivated in light intensity 3000Lx, day illumination 16h culturing room.

The survival rate of stripping and slicing in each processing is counted after 30d.

Result of the test is as shown in the table.

As can be seen from the above table, when Cef content up to finite concentration（200mg/L）When, the propagation to stripping and slicing is also produced Fraction of inhibition, the survival rate of stripping and slicing shows downward trend；When Cef contents are 300mg/L, stripping and slicing survival rate drop Low degree is more obvious.Due to the effect of antibiotic have it is additive, therefore, in order to reduce as far as possible Cef to stripping and slicing increase The influence grown, it is proposed that be used for during screening and culturing the bacteriostatic agent for suppressing bacterial plaque formation from 50mg/L Cef.

Culture of rootage

Because the final purpose of tissue cultures needs plant to grow root system, so that success can be finally transplanted, thus hair A person of good sense has done further experiment for the culture of rootage of iris, is described as follows.

Protocorm continues to cultivate in proliferated culture medium, after 90d, can be eventually differentiated into bud, and grow blade.Will propagation The healthy and strong bud turned out（2 ~ 3 blade exhibitions are arranged at top, and bud is higher than 2cm, length of blade 1cm or so）It is transferred in root media Culture of rootage is carried out, type of culture medium is：3g/L spend precious No. 1+1.5mg/L IBA+20g/L sucrose+1g/L activated carbon+ 6.5g/L agar, pH=5.5 ~ 5.6.

Cultivation results are as shown in Figure 8.

As a result show, using this culture medium, rootlet, culture to test tube seedling after 40d can be gone out after culture 20d in bastem minister Root reach more than 4 and more sturdy, rooting efficiency is preferable.

In the genetic transformation application of iris, the foundation and optimization of good receptor system are successful keys.Due to The single capsule seed amount of iris is more and is easy to sprout, thus the present invention sprouts the formed protocorm of induction with iris seed Stem is acceptor material, and further can meet needs of production by the Multiplying culture of protocorm.

During to the combined sorting of growth regulator used by iris Multiplying culture, inventor passes through test of many times group Close and inquire into, it is believed that 2,4-D and 6-BA hormone combinations can be effectively promoted Protocorm Multiplication, and be not required to higher concentration and match somebody with somebody Than, become apparent especially with 2,4-D, as the reduction of concentration is more conducive to Protocorm Multiplication, too high not only Inhibit proliferaton but also Material can be caused dead.Similarly, the 6-BA of low concentration is conducive to it to breed, but concentration is too low easily causes the obtained protocorm of propagation Stem no longer carries out the growth of single protocorm volume once being formed just into bud status transition.The present invention is trained for propagation simultaneously Protocorm stripping and slicing size or activated carbon dosage are also further explored during supporting.In a word, final result thinks optimal Iris proliferation culture medium formula is constituted：3g/L spend precious No. 1+3mg/L 6-BA+0.6mg/L 2,4-D+2g/L activated carbon+ 15g/L sucrose+6.5g/L agar, pH=5.5 ~ 5.6；Protocorm stripping and slicing diameter minimum is no less than 0.3cm.

And culture of rootage result shows, the healthy and strong bud that Multiplying culture is gone out（2 ~ 3 blade exhibitions are arranged at top, and bud is higher than 2cm, length of blade 1cm or so）During switching, preferably prescription of rooting medium is：3g/L spends No. 1+1.5mg/L IBA+ of treasured 20g/L sucrose+1g/L activated carbon+6.5g/L agar, pH=5.5 ~ 5.6.

One of condition that genetic plant transformations receptor system should possess is exactly receptor system to screening antibiotic sensitive.Work as sieve When selecting the screening antibiotic concentration added in culture medium to reach to a certain degree, growth, the development of non-transformed plant cell can be suppressed And differentiation, and convert plant cell due to carry corresponding resistant gene can normal growth, differentiation, and finally obtain complete Transformed plant.The present invention has carried out Kan and Hyg resistant proof respectively, as a result shows sensitiveness of the iris protocorm to Kan It is poor compared with Hyg, but when Kan reaches 100mg/L, the growth of protocorm stripping and slicing also shows serious suppressed phenomenon, largely cuts There is lethal situation in block, the result shows Kan can be used for the transgenosis screening of some butterfly orchid varieties, this to it is related Research report has differences（Chai M L et al., Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Scientia Horticulturae. 2002,96：213-224）.Low concentration Hyg（5mg/L）The good inhibitory action survived to protocorm, can Suitable for the direct screening of acceptor material after dip-dye.Consider, the Hyg using concentration as 8mg/L is that selective agent effect is preferable, together When can reduce the generation of false positive in screening process, improve conversion ratio.And during with Agrobacterium-mediated genetic transformation, It is preferable as the bacteriostatic agent effect for suppressing bacterial plaque formation during screening and culturing from 50mg/L Cef.

Embodiment 3

The present embodiment use During Agrobacterium mode to the iris protocorm cultivated in embodiment 2 carried out contaminate with Transgeneic procedure is realized, dip dyeing liquid for shell is the recombinational agrobacterium bacterial strain GV3101 prepared by embodiment 1, and the bacterial strain includes plasmid table Up to carrier pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’H, the bacteria screening marker gene of plasmid expression vector For Kan, plant screening mark gene is Hyg, and reporter gene is GUS.It is briefly discussed below.

During Agrobacterium transgeneic procedure, comprises the following steps.

（1）The preculture of acceptor material

It is about that the consistent excellent protocorm of 0.5cm, growing way is cut into block by the size obtained in embodiment 2, transfers into pre- In culture medium, preculture 3d is made acceptor material keep vigorous vitality, converted with preferably state, dropped simultaneously The injury that low dip dyeing liquid for shell is caused to it.

Proliferated culture medium in the precultivation medium, condition of culture be the same as Example 2.

（2）During Agrobacterium

Dip dyeing liquid for shell is prepared with the recombinational agrobacterium bacterial strain GV3101 prepared by embodiment 1, during dip-dye, recombinational agrobacterium is resuspended Liquid OD₆₀₀=0.4 ~ 0.6, contaminate 15min.

Before dip-dye, preculture 3d protocorm stripping and slicing is clamped into sterile triangular flask, band culture medium of trying not. Enter the dip dyeing liquid for shell that is ready for being totally submerged acceptor material.Triangular flask constantly is gently shaken, acceptor material is filled with dip dyeing liquid for shell Tap is touched, until reaching required immerged time requirement.

Recombinational agrobacterium bacterial strain GV3101 dip dyeing liquid for shell preparation process, with reference to as described below.

A. from -80 DEG C of taking-up recombinational agrobacterium bacterial strains, with transfer needle on the LB solid mediums containing Kan, Rif antibiotic Rule, after be placed in incubator, under the conditions of 28 DEG C cultivate 2 ~ 3d to single bacterium colony occur；

B. transfer needle picking single bacterium colony from flat board is used, LB fluid nutrient mediums of the 5mL containing Kan, Rif antibiotic is inoculated in In, 28 DEG C, 180rpm shaken overnight cultures；

C. above-mentioned bacterium solution is pressed 1 with pipettor:50 ratio be added in the LB fluid nutrient mediums of antibiotic-free culture 3 ~ 5h or so, to bacterium solution OD₆₀₀Used during for desirable value for conversion.

When preparing Agrobacterium re-suspension liquid, prepare according to the following steps：First with centrifuge by bacterium solution（That is institute in above-mentioned steps C The Agrobacterium nutrient solution to be transformed used prepared）Under the conditions of 4 DEG C, 2500rpm centrifugation 10min collect thalline afterwards；Then use Liquid proliferated culture medium（Liquid proliferated culture medium i.e. described in embodiment 2）Required for the thalline being collected into is suspended into again OD₆₀₀During value, you can the conversion for next step.

（3）Acceptor material is placed on co-cultivation culture medium after dip-dye, light culture 3d under the conditions of 25 DEG C；

Acceptor material is by contaminating, for theory, and foreign gene has been converted into iris genome.After the completion of dip-dye, Acceptor material is moved on on aseptic filter paper and dried, is transferred afterwards into co-cultivation culture medium（Proliferated culture medium in be the same as Example 2）On, Light culture 3d under the conditions of being placed in 25 DEG C.

（4）The elution of Agrobacterium

The acceptor material contaminated is after co-cultivation, it is necessary to which carrying out the elution of Agrobacterium can be transferred in screening and culturing medium again Carry out screening and culturing.But it is due to that the influence that various concentrations bacteriostatic agent grows to Agrobacterium is different, therefore need to filters out suitable dense The bacteriostatic agent of degree carries out the washing steps before acceptor material switching, just can ensure that being smoothed out for follow-up screening operation.

Specifically elution process is：By step（3）Acceptor material after middle light culture terminates is chosen and is placed in sterile conical flask, First the culture medium of acceptor material surface attachment and macroscopic mycelium are rinsed out with sterilized water, then, then with suitable dense Degree（It is ultimately determined to 300mg/L）Cef water rinse 2 ~ 3 times, during which constantly shake conical flask, finally use same suitable concentration （It is ultimately determined to 300mg/L）Cef water soak 20 ~ 30min after acceptor material chosen dried to aseptic filter paper, then carry out The screening and culturing of acceptor material.

（5）The screening and culturing of acceptor material after dip-dye

By step（4）It is middle to be gone to after the acceptor material of elution dries on screening and culturing medium, during screening and culturing, often 15d is once turned sieve, to keep the effectiveness of antibiotic, is required to before turning sieve every time according to step（4）Middle method carries out agriculture bar The elution of bacterium.

The screening and culturing medium is：Proliferated culture medium+8mg/L Hyg+50mg/L Cef, pH=5.5 ~ 5.6 in embodiment 2；

Condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, light intensity 3000Lx, day illumination 16h.

After screening and culturing four-wheel, the protocorm filtered out is transferred into comprising only bacteriostatic agent（50mg/L Cef）Propagation training To support continue in base and cultivate, until differentiation and bud formation and grow up to seedling, Screening and Identification.

（6）By step（5）Further amplification or access root media are lured the middle correct Phalaenopsis plants of Screening and Identification Lead and take root；

During Agrobacterium liquid influence factor is inquired into

Final optimization pass result is only gived in foregoing description, but lacks necessary introduction for optimization process, below it is right The most important influence factor in iris transgenic breeding --- the influence factor closed with During Agrobacterium liquid phase is briefly introduced It is as follows.

The factor of influence acceptor material conversion ratio has multiple in being tested due to Agrobacterium-mediated genetic transformation, inventor's pin Wherein more important acceptor material is combined with dip dyeing liquid for shell, immerged time and contaminates the factors such as concentration and carried out brief experiment point Analysis.Result of study is mainly carried out by the method to the GUS transient expressions through degerming acceptor material after conversion, while considering former The degree of injury of bulb stripping and slicing.

Acceptor material combines the influence for conversion ratio with dip dyeing liquid for shell

The protocorm that the protocorm and Multiplying culture that selection Seed inducement is obtained respectively are obtained is acceptor material, by preculture Two kinds of materials afterwards are respectively in OD₆₀₀To be contaminated in 0.5 two kinds of dip dyeing liquid for shell（15min）, different acceptor materials are counted after 3d The damage ratio result of conversion ratio and acceptor material in being combined with dip dyeing liquid for shell.

Combination A, B, C, D are set, represent respectively Seed inducement protocorm combined with bacterium solution, Multiplying culture protocorm and bacterium solution Combination, Seed inducement protocorm and re-suspension liquid, Multiplying culture protocorm and re-suspension liquid.

Stripping and slicing number × 100% of the blue stripping and slicing number of GUS transient expressions rate=change/detected；

Stripping and slicing number × 100% of the dead stripping and slicing number of damage ratio=melanism/detected.

As can be seen from the above table, for the protocorm of same source approach, the GUS transient expressions after being contaminated in bacterium solution Rate is compared with the height in re-suspension liquid（A is high compared with C, and B is high compared with D）, but bacterium solution is serious compared with re-suspension liquid to the injury of protocorm stripping and slicing（A is high compared with C, B It is high compared with D）, a large amount of stripping and slicing melanism can be caused dead in a short time；And, find Multiplying culture in dip dyeing liquid for shell under the same conditions The GUS transient expressions rate of obtained protocorm is than directly inducing the expression rate of obtained protocorm high（B is high compared with A, and D is high compared with C）, And the degree of injury of stripping and slicing is more another kind of relatively low（B is low compared with A, and D is low compared with C）.Therefore, by GUS transient expressions rate and damage Hinder and consider of both rate, the protocorm obtained with Multiplying culture makees acceptor material, when selection re-suspension liquid is dip dyeing liquid for shell not It only can guarantee that conversion ratio and the degree of injury of material can be reduced.

Influence of the immerged time for conversion ratio

Two kinds of dip dyeing liquid for shell of bacterium solution and re-suspension liquid are selected respectively（OD₆₀₀It is 0.5）, each two leachings of design 10min and 15min The dye time, to the acceptor material after preculture（The protocorm that Multiplying culture is obtained）The dip-dye of aforementioned four different modes is carried out, The conversion ratio and damage ratio of stripping and slicing are counted after 3d.

Statistics is as shown in the table.

As can be seen from the above table, no matter in which kind of dip dyeing liquid for shell, immerged time is advisable for 15min, has reached 53.6%.

Dip dyeing liquid for shell concentration influences for transformation efficiency

By the acceptor material after preculture（The protocorm that Multiplying culture is obtained）, respectively at OD₆₀₀For 0.2,0.4,0.6, Contaminated in 0.8 re-suspension liquid（15min）, count the conversion ratio and damage ratio of acceptor material.

Statistics is as shown in the table after 3d.

OD is can be seen that from upper table result₆₀₀When=0.2, the degree of injury of stripping and slicing is minimum, but now conversion ratio is 0%；With OD₆₀₀The increase of value, GUS transient expression rates are also gradually stepped up, but work as OD₆₀₀When=0.8, decline is but presented in GUS transient expressions rate State, while the damage ratio of stripping and slicing reaches highest.OD₆₀₀When=0.4 ~ 0.6, GUS transient expression rates are of a relatively high, are respectively 39.3% and 46.4%, now, the damage ratio and OD of stripping and slicing₆₀₀Comparing when=0.8 is relatively low.Therefore, dip dyeing liquid for shell concentration 0.4 ~ The dip-dye of acceptor material is may be incorporated between 0.6.

Eluent-cephalosporin of Agrobacterium（Cef）The screening of concentration

To determine the suitable concentration of Cef water, first in LB solid mediums（Containing antibiotic）Middle addition bacteriostatic agent Cef, contains Amount is respectively set to 0 mg/L, 50mg/L, 100mg/L, 200mg/L, 300mg/L, then carries out the culture of recombinational agrobacterium, sees Examine in identical incubation time（2~3d）The upgrowth situation of Agrobacterium, so that it is determined that most appropriate Cef concentration.

Specific cultivation results are as shown in following table and Fig. 9.

Note：+++, ++ ,+,-represent that normal proliferation, propagation are suppressed, it is heavily suppressed to breed respectively, do not have Have.

As can be seen from the above table, when Cef concentration is 300mg/L, the growth of Agrobacterium is completely suppressed, therefore, optional The Cef for being 300mg/L with concentration carries out the elution of Agrobacterium.

Inspection example

Tissue-cultured seedling after the genetic transformation of embodiment 3 needs to carry out every detection, to ensure that the gene for intending conversion is correctly integrated Enter iris genome.Choose resistance screening and reach that 2cm or so tissue-cultured seedling carries out items and referred to for positive and length of blade Mark is to be checked.Detection method includes PCR detections and Histological stain method detects two classes, is briefly discussed below.

Detection

During screening and culturing, some are unconverted, and successful acceptor material can also show the resistance to selective agent and progress Growth and Differentiation formation " false positive " plant.Therefore need to enter performing PCR detection whether to determine target gene by antagonism material Successfully it is incorporated into the genome of resistant material.The PCR of resistant material is detected with matter included in recombinational agrobacterium in the present invention Grain is positive control（P）, the Phalaenopsis leaves without dip-dye are negative control（N）.Comprise the following steps that：

（1）The DNA of the iris resistant material filtered out is extracted

Iris material DNA is extracted using modified CTAB method, comprised the following steps that：

Take 0.1g fresh iris tissue to be put into 2mL centrifuge tubes, pulverized after adding liquid nitrogen, then in centrifuge tube Add 1000 μ L CTAB, after be put into water-bath 90min in 65 DEG C of thermostat water baths, during which centrifuge tube need to be taken out turn upside down 2 ~ 3 times.

Water-bath is taken out test tube and placed in room temperature, backward centrifuge tube plus 1/2 volume after terminating（CTAB volumes）Chloroform/ Isoamyl alcohol（24:1）, gently turn upside down mixing more than 5min, notices that this operating process is carried out in fume hood.Mix after 12000 rpm centrifuge 10min.Clear 600 μ L are sucted in having marked in advance in the 1.5ml centrifuge tubes of order, are careful not to be drawn onto impurity. Add 1/2 volume into the centrifuge tube equipped with supernatant（Supernatant volume）Isopropanol, overturn mix after, 10000 rpm centrifugation 30s.Supernatant is abandoned, makes the precipitation drying in centrifuge tube.

75% ethanol 500uL is separately added into, DNA is fully washed；2min is centrifuged in 12000rpm；Abandon supernatant, room The lower dry DNA of temperature；Add 100uL ddH₂It is standby that O concussions make DNA fully dissolve.

（2）PCR is detected

According toRcOOMT2、VwF3’5’HThe sequence information design primer of gene detects that design of primers is as follows for PCR (5'-3')：

RcOOMT2-5'：TACGGGAACCATCAGCCAA,

RcOOMT2-3'：GAAACCAGCATCAGTGAAGAG；

VwF3'5'H-5'：ACCTCAACTTCTCCAACCGC；

VwF3'5'H-3'：GCTCCTTCACCATCTTCGTC.

Pcr template：Sun is made as template, and with corresponding recombinant plasmid using the iris DNA of resistant material that is filtered out Property control, negative control is used as using unconverted iris DNA.

PCR reaction systems（20μL）It is as follows：

3 ' primers, 5 ' primers, each 0.20 μ L；

DNTPs, 0.20 μ L；

Template, 2 μ L；

Taq enzyme, 0.15 μ L；

10 × buffer, 2 μ L；

ddH₂O, 15.25 μ L.

PCR response procedures：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 1min；58 DEG C of annealing 1min；72 DEG C extend 30sec, 30 Individual circulation；72 DEG C re-extend 5min；4 DEG C of terminations.

By pcr amplification product and DNA molecular Marker under 1 × TAE buffer conditions, with a little ethidium bromide of addition 1.0% Ago-Gel is separated, and observes in ultraviolet gel imaging instrument testing result, and testing result is shown in Figure 10.

（3）Histological stain method is detected

Because the reporter gene of the plasmid expression vector selected in this method contains GUS.Therefore, except antagonism material is carried out PCR detections are outer, GUS Histological stain method detections can also be carried out to it, whether normal expression is come side light by reporter gene Target gene whether in resistant material normal expression.Comprise the following steps that：

Young leaflet tablet or other young tender organs are collected first from the transfer-gen plant that resistance screening goes out, material is unsuitable excessive （About 0.2cm）, slicing treatment is carried out to larger material with tissue culture knife.Section is put into centrifuge tube and A liquid is added, A liquid Volume with by material covering be advisable.Centrifuge tube is put into 37 DEG C of constant incubator and incubates 1h.

A liquid is poured out, the B liquid of same volume is then added in centrifuge tube.Number is incubated in 37 DEG C of constant incubator small When, untill observing that blueness is presented on material（To react fully, also material can be carried out to handle overnight）.Note adding Entering need to be in centrifuge tube outer wrapping masking foil after B liquid, it is to avoid the action effect of illumination effect B liquid.

Material is rinsed well with water, if can directly observe the result of material without the interference of other pigments.The process can Carry out, also can directly visually observe under disecting microscope.To avoid the interference of other pigments contained in blade（As leaf is green Element etc.）, can after B liquid is poured out in centrifuge tube add 95% ethanol carry out pigment elution, decolourize 30min every time, can be through many Secondary elution, until existing for stopping without other interference pigments.GUS testing results are shown in Figure 11.

In summary, the present invention is demonstrated by different composite tests, and acceptor is made with the protocorm that Multiplying culture is obtained Material, selection re-suspension liquid not only can guarantee that conversion ratio and can reduce the degree of injury of material when being dip dyeing liquid for shell；In the process of dip-dye In, immerged time is advisable with 15min；The concentration of Agrobacterium is with OD in dip dyeing liquid for shell₆₀₀Transformation efficiency is higher when=0.4 ~ 0.6.In a word, The present invention is on the basis of the optimal acceptor material of iris and optimal conditions of tissue culture are set up in discussion, with iris protocorm Multiplying culture is organized as acceptor material, has carried out the During Agrobacterium experiment of reality, has successfully entered exogenous origin gene integrator Iris genome, new approach and direction are provided for iris rearing new variety, thus with preferable popularization and application valency Value.

Claims

1. it is a kind of based on iris seed sprout protocorm be acceptor breeding method, it is characterised in that this method include with Lower step：

（2）By step（1）Directly or after Multiplying culture acceptor material is used as after gained protocorm stripping and slicing；

（3）By step（2）The acceptor material preculture of gained, carries out During Agrobacterium；

（4）By step（3）The acceptor material of dip-dye is placed on co-cultivation culture medium, light culture 3d under the conditions of 25 DEG C；

（5）By step（4）Acceptor material after middle co-cultivation carries out Agrobacterium elution, elution using containing 300mg/L Cef without Bacterium water, which soaks 20 ~ 30min, to be carried out；

（6）By step（5）Acceptor material after elution, which is placed on screening and culturing medium, carries out screening and culturing, during screening and culturing, Once turned sieve per 15d, be required to before turning sieve every time according to step（5）Middle method carries out the elution of Agrobacterium；Screening and culturing After four-wheel, continue to cultivate up to differentiation and bud formation and grow up to seedling；Screening and Identification；

（7）By step（6）Further amplification or the access root media induction life of the middle correct Phalaenopsis plants of Screening and Identification Root；

Step（1）Described in butterfly orchid variety be " red dragon " or " shining "；

The Seed inducement germination medium is constituted：3g/L spend precious No. 1+2g/L activated carbon+2g/L caseinhydrolysate+ 15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar, pH=5.5 ~ 5.6；

Step（1）Middle protocorm stripping and slicing diameter is not less than 0.3cm；

Step（2）Described in Multiplying culture use for proliferated culture medium, proliferated culture medium composition is：3g/L spends No. 1+2g/ of treasured L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2,4-D+ 6.5g/L agar, pH=5.5 ~ 5.6；

Step（4）Described in co-culture culture medium the same proliferated culture medium of composition.

2. as claimed in claim 1 based on the breeding method that the protocorm that iris seed is sprouted is acceptor, it is characterised in that step Suddenly（1）Middle condition of culture is：25 DEG C of day temperature, 17 DEG C of night temperature, the Lx of light intensity 3000, day illumination 16h cultivate 60 ~ 90d.

3. as claimed in claim 1 based on the breeding method that the protocorm that iris seed is sprouted is acceptor, it is characterised in that step Suddenly（2）Described in Multiplying culture condition be：25 DEG C of day temperature, 17 DEG C of night temperature, the Lx of light intensity 3000, day illumination 16h, Multiplying culture 60 ~ 90d, the protocorm that Multiplying culture to diameter is 0.5cm is used as the acceptor material of transgenosis；

Step（6）Described in screening and culturing medium be：The mg/L Hyg+50 mg/L of proliferated culture medium+8 Cef；

Step（6）Continuing culture used medium after middle screening and culturing four-wheel is：The mg/L of proliferated culture medium+50 Cef.

4. as claimed in claim 1 based on the breeding method that the protocorm that iris seed is sprouted is acceptor, it is characterised in that step Suddenly（3）Described in preculture incubation time be 3d；

During Agrobacterium liquid used is re-suspension liquid during the During Agrobacterium, and re-suspension liquid uses liquid proliferated culture medium, is formulated and is： 3g/L spends treasured No. 1+15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2,4-D, pH=5.5 ~ 5.6；

During During Agrobacterium, 15min is contaminated in re-suspension liquid OD600=0.4 ~ 0.6；

Bacterial strain is restructuring agrobacterium strains GV3101 in the During Agrobacterium liquid, and the bacterial strain includes plasmid expression vector pCAMBIA1301-RcOOMT2、pCAMBIA1303-VwF3’5’H。

5. as claimed in claim 1 based on the breeding method that the protocorm that iris seed is sprouted is acceptor, it is characterised in that step Suddenly（7）Described in take root the root media of use,

The root media is constituted：3g/L spends No. 1+1.5mg/L IBA+20g/L sucrose+1g/L activated carbons+6.5g/ of treasured L agar, pH=5.5 ~ 5.6.