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

Abstract

The invention belongs to the technical field of genetic engineering breeding, in particular to a breeding method based on protocorms germinated from Phalaenopsis seeds as receptors. The method comprises the steps of obtaining protocorms, multiplying and culturing, precultivating, impregnating with Agrobacterium, cocultivating, washing out Agrobacterium, screening and culturing, forming sprouts, rooting and the like. The present invention constructs a set of Phalaenopsis transgenic acceptor system with the protocorm germinated from Phalaenopsis seeds as the acceptor material, and simultaneously establishes a set of genetic transformation system and corresponding detection system based on Agrobacterium dipping, which has the advantages of fast and fast acquisition of acceptor materials. , stable performance, wide range of sources, etc., especially good sensitivity to selective antibiotics, which can effectively avoid the problem of Phalaenopsis insensitivity to antibiotics in the process of inducing protocorms, and is conducive to the screening of resistant transgenic materials, obtaining better , a relatively pure new Phalaenopsis system, which has good promotion and application value for promoting the development of new Phalaenopsis varieties.

Description

A kind of breeding method based on the protocorm of phalaenopsis seed germination as receptor

technical field

The invention belongs to the technical field of genetic engineering breeding, in particular to a breeding method based on protocorms germinated from Phalaenopsis seeds as receptors.

Background technique

Phalaenopsis aphrodite belongs to the Phalaenopsis genus Phalaenopsis. It is an important potted flower with high ornamental value and economic value. However, due to the lack of blue or scented varieties of Phalaenopsis in nature, the traditional Cross-breeding means can not realize the diversification demand to Phalaenopsis flower color, flower fragrance. Therefore, the new transgenic breeding technology has been applied to a certain extent in the cultivation of new varieties of Phalaenopsis. Existing commonly used transgenic breeding techniques include: Agrobacterium-mediated method for indirect transformation of exogenous genes, biolistic method for direct transformation of exogenous genes, microinjection method and PEG-mediated transformation method, etc., and pollen for multi-ovule plants Pipeline method; Among them, the Agrobacterium-mediated method is a gene transformation method that has been studied more, the theoretical mechanism is clearer, and the technical method is relatively mature.

Although the transgenic breeding method has the advantages of clear breeding purpose and short breeding time, in the process of plant transgenic, the selection of recipient material is the basis and key factor for the success of breeding. Appropriate receptor materials help to speed up and improve the smooth progress of genetic transformation and the successful cultivation of transgenic plants. For the transgenic breeding of Phalaenopsis, theoretically, somatic embryos, protocorms and protocorms can be used as ideal recipient materials for genetic transformation of Phalaenopsis, but the current way to obtain somatic embryos and protocorms is mainly through the shoot tip , stems, leaves, root tips, root segments, pedicel axillary buds and other explants, but in the induction process, there are often problems such as browning and low induction rate. Therefore, in the practice of Phalaenopsis transgenic breeding, somatic cells Embryos or protocorm-like receptor materials often have certain limitations. In addition, when somatic embryos or protocorms are used as recipient materials for transgenic breeding, when gene guns or Agrobacterium dipping are used for transgenic operations, there are also defects of low transfection efficiency, and in the process of Agrobacterium dipping, There are also disadvantages such as low sensitivity to antibiotics, so further improvement is urgently needed.

Contents of the invention

The object of the present invention is to provide a kind of breeding method based on the protocorm of Phalaenopsis seed germination as the receptor, the method uses the protocorm of Phalaenopsis seed germination as the transgenic breeding recipient material, which can better overcome the existing explant The browning, low conversion rate, quality difference, long induction time, slow growth rate, and difficulty in succession of the receptor material exist in the induced receptor material; meanwhile, the present invention takes Phalaenopsis "red dragon" and "radiant" varieties as examples , a new Phalaenopsis variety was bred. During the cultivation process of this variety, the transgenic operation was carried out by means of Agrobacterium dipping, and two gene components of flower color and fragrance were introduced respectively, which provided a new reference for the cultivation of new Phalaenopsis germplasm resources. .

The technical solution of the present invention is introduced in detail as follows.

A kind of protocorm based on Phalaenopsis seed germination is the breeding method of acceptor, comprises the following steps:

(1) Under sterile conditions, phalaenopsis seeds are germinated and grown in a seed-induced germination medium to obtain protocorms;

The Phalaenopsis species is preferably "Red Dragon" or "Raining Radiance";

The seed induction germination medium is: 3g/L Huabao No. 1+2g/L activated carbon+2g/L hydrolyzed casein+15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+6.5g/ L agar, pH=5.5~5.6;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000 Lx, sunlight 16h, culture 60~90d;

(2) Cutting the Zhongyuan corm in step (1) into pieces and using it as a receptor material directly or after proliferating and culturing;

Proliferation medium: 3g/L Huabao No. 1+2g/L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6 mg/L2,4-D+ 6.5g/L agar, pH=5.5~5.6;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, sunlight 16h, multiplication culture for 60-90 days, protocorms multiplied to a diameter of about 0.5cm can be used as the recipient material for transgenes;

Step (1) The minimum diameter of the cut piece of Zhongyuan corm should not be less than 0.3cm;

(3) pre-cultivating the receptor material cultured in step (2) for 3 days, and performing Agrobacterium infiltration;

The culture medium and growth conditions used in the pre-cultivation are the same as in step (2);

The Agrobacterium dipping solution is a resuspension liquid, and the resuspension liquid adopts a liquid proliferation medium, and the formula is: 3g/L Huabao No. 1+15g/L sucrose+3mg/L 6-BA+0.6 mg/L 2,4 -D, pH=5.5~5.6;

When dipping with Agrobacterium, resuspension OD ₆₀₀ =0.4~0.6, dipping for 15min;

Bacterial strain in described Agrobacterium dipping liquid is recombinant Agrobacterium strain GV3101, and this bacterial strain comprises plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H ;

The plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H is obtained by transforming the pCAMBIA1301, pCAMBIA1303 vectors, and the specific steps are: first respectively add the CAMV35S promoter ( pCAMV35S) and CAMV35S terminator (T-CAMV35S); then recombine and integrate the rose flower gene RcOOMT2 and the pansy blue gene VwF3'5'H in the genome of the vector; finally, the transformed plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H can be transformed into Agrobacterium strain GV3101;

(4) Place the receptor material after dipping in step (3) on the co-cultivation medium, and culture in the dark at 25°C for 3 days;

The components of the co-cultivation medium are the same as the proliferation medium in step (2);

(5) The acceptor material co-cultured in step (4) is eluted with Agrobacterium, and the elution is carried out by soaking in sterile water containing 300mg/LCef for 20-30min;

(6) Place the receptor material eluted in step (5) on the screening medium. During the screening culture process, perform sieving every 15 days. Before each sieving, the method in step (5) should be followed. Elution of Agrobacterium;

The screening medium is: proliferation medium in step (2)+8 mg/L Hyg+50 mg/L Cef, pH=5.5~5.6;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, daylight 16h;

After four rounds of screening culture, insert into the proliferation medium of step (2) + 50 mg/L Cef medium, and continue to cultivate until it differentiates into buds and grows into seedlings; screening and identification;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, daylight 16h;

(7) Further amplify the correct Phalaenopsis plants screened and identified in step (6) or insert them into the rooting medium to induce rooting;

The rooting medium is: 3g/L Huabao 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 Phalaenopsis transgenic acceptor system with the protocorm germinated from Phalaenopsis seeds as the acceptor material, and establishes a set of genetic transformation system and corresponding detection system based on Agrobacterium dipping at the same time, compared with the existing The obvious technical advantages of the Phalaenopsis transformation system are reflected in the following aspects: First, the receptor system constructed by using seed-germinated protocorms is faster than other methods of obtaining receptor materials through explants; The present invention also provides a protocorm amplification system, so generally speaking, compared with the prior art, the acceptor material of the present invention has stable performance and wide sources; The selective antibiotic sensitivity is better, which can effectively avoid the problem of Phalaenopsis insensitivity to antibiotics in the process of inducing protocorm-like, and is beneficial to the screening of resistant transgenic materials. In general, the present invention has successfully constructed a set of relatively complete moth orchid with good transformation and screening effects through the screening and optimization of the existing moth orchid protocorm breeding system and the improvement and application of the existing moth orchid transgenic breeding system. The Phalaenopsis transgenic breeding system has relatively mature technical means and a good guarantee for breeding effects, and has good promotion and application value for promoting the development of new varieties of Phalaenopsis.

Description of drawings

Figure 1 is a schematic diagram of vector transformation of pCAMBIA1301 and pCAMBIA1303;

Figure 2 is a schematic diagram of the construction of pCAMBIA1301 -RcOOMT2, pCAMBIA1303 -VwF3'5'H plasmid expression vectors;

Fig. 3 is the protocorm that seed germination 60d forms in Phalaenopsis tissue culture;

Fig. 4 is the impact of different growth regulator combination treatments on the protocorm proliferation culture;

Figure 5 is the effect of different concentrations of 6-BA and 2,4-D combined treatment on protocorm proliferation and culture;

Fig. 6 is the influence of different activated carbon contents on protocorm growth, wherein A: 0mg/L activated carbon, B: 1mg/L activated carbon, C: 2mg/L activated carbon;

Figure 7 shows the results of antibiotic resistance screening, where 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 the rooting situation of Phalaenopsis;

Figure 9 shows the effects of different concentrations of Cef on the growth of Agrobacterium, from left to right are A-E, where A: 0mg/LCef, B: 50mg/L Cef, C: 100mg/L Cef, D: 200mg/L Cef , E: 300mg/L Cef;

Figure 10 is PCR detection, wherein A is the PCR detection of transforming RcOOMT2 , B is transforming VwF3'5'H , M is Marker, N is Negative, P is Positive; 1#~5# are respectively randomly selected Phalaenopsis transformed plants sample;

Figure 11 is GUS tissue staining detection, wherein A: control, B: GUS positive material.

detailed description

The present invention is further explained below in conjunction with embodiment.

Before introducing the embodiments in detail, a brief introduction to some of the test materials and instruments used in the following embodiments is given below.

Phalaenopsis material: The Phalaenopsis varieties used in the examples include two commercial Phalaenopsis varieties of "Red Dragon" and "Rainbow", and the Phalaenopsis seeds used are obtained from selfing of Phalaenopsis.

Some raw materials of tissue culture medium: Huabao No. 1, 6-BA, 2,4-D, NAA, IBA, hydrolyzed casein, TDZ, activated carbon, sucrose, agar and other materials used in tissue culture medium were purchased from Tongya company.

Some reagents used in transgenic operations: ampicillin (Amp), kanamycin (Kan), rifampin (Rif), gentamicin (Gen), hygromycin (Hyg), cephalosporin (Cef), etc. Antibiotics were purchased from Baosai Company.

Agrobacterium: Competent Agrobacterium strain GV3101 was purchased from Maiqi Company. After recombination, Agrobacterium strain GV3101 contained plasmid expression vectors pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H plasmids. The recombinant strain was constructed by the inventor using genetic engineering technology. The target genes are the RcOOMT2 gene cloned from rose and the VwF3'5'H gene from pansy, respectively . For the specific construction and recombination process, please refer to Figure 1, Figure 2 and Example 1.

Part of the reagents used to construct the vector: The vectors pCAMBIA1301 and pCAMBIA1303 used were from CAMBIA Company. Pst I, Xba I, EcoR I, Sac I, BamH I, Kpn I and other endonucleases used in the construction process, high-fidelity Taq enzyme, pMD18-T vector, cDNA synthesis kit and T4 ligase were purchased from Takara Company , Escherichia coli Top10 competent cells, gel recovery kit, plasmid extraction kit, etc. were purchased from Tiangen Biochemical (Beijing) Co., Ltd.

Relevant LB medium during the cultivation of Escherichia coli and Agrobacterium

LB liquid medium (without antibiotics): 10g/L tryptone+5g/L yeast extract+10g/L sodium chloride;

LB solid medium (without antibiotics): 10g/L tryptone+5g/L yeast extract+10g/L sodium chloride+15g/L agar;

Escherichia coli (containing recombinant plasmid) medium: LB medium + antibiotics, antibiotics: 100 mg/L Kan;

Agrobacterium (containing recombinant plasmid) medium: LB medium + antibiotics, antibiotics: 30mg/L Gen+100mg/L Kan+50 mg/L Rif.

Phalaenopsis tissue culture medium

Seed induction germination medium: 3g/L Huabao No. 1+2g/L activated carbon+2g/L hydrolyzed casein+15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar, pH=5.5~5.6;

Proliferation medium: 3g/L Huabao No. 1+2g/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;

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

Screening medium: proliferation medium+8 mg/L Hyg+50 mg/L Cef, pH=5.5~5.6;

Receptor material pre-culture medium before Agrobacterium infection, and co-cultivation medium after infiltration: the same as the proliferation medium.

It should be noted that after the preparation of the above medium, all the above mediums need to be sterilized by autoclaving at 121°C for 20 minutes. Since antibiotics will lose their effectiveness under high temperature conditions, when preparing LB medium and screening medium containing antibiotics, the medium must first be subjected to high-pressure sterilization. After sterilization, antibiotics such as Gen, Kan, Rif, Hyg, Cef, etc. of corresponding required concentrations are added to the culture medium whose temperature drops below 60°C on the ultra-clean workbench to complete the preparation.

The specific preparation of antibiotic mother solution is as follows:

Kanamycin (Kan), Ampicillin (Amp): Dissolve in distilled water to a final concentration of 100 mg/mL, filter and sterilize, and store at -20°C;

Rifampicin (Rif): dissolved in methanol to a final concentration of 50 mg/mL, sterilized by suction filtration, and stored at -20°C; cephalosporin (Cef): dissolved in distilled water to a final concentration of 100 mg/mL, sterilized by suction filtration, 4 Store at ℃.

The mother solutions of gentamicin (Gen) and hygromycin (Hyg) were purchased from Baosai Company.

Test Reagent GUS Stain

GUS staining solution A (50 mL): 0.2 M phosphate buffer (pH=7.0), 25 mL, 0.1 M potassium ferricyanide, 0.25 mL, 1 M Na2EDTA, 0.5 mL, distilled water, 24.25 mL;

GUS staining solution B: add 50 mg 5-bromo-4-chloro-3-indole-β-glucoside (X-gluc) to 50 mL solution A, and complete the preparation of solution B after fully dissolving.

The main instruments are: ultra-clean workbench, centrifuge, PCR instrument, electrophoresis instrument, ultraviolet gel imager, shaker, pipette, all of which are commonly used equipment in the laboratory.

Example 1

Since the construction of recombinant Agrobacterium strain GV3101, that is, the recombination of plasmid expression vectors pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H into the original Agrobacterium strain GV3101 is a more important link in the present invention, the introduction of the recombinant Agrobacterium strain GV3101 is as follows .

As shown in Fig. 1 and Fig. 2, the obtaining process of recombinant Agrobacterium strain GV3101 is as follows: first, the pCAMBIA1301 and pCAMBIA1303 vectors are transformed, and a CAMV35S promoter and a CAMV35S terminator are respectively added to the multiple cloning sites of the vectors; The rose flower fragrance gene RcOOMT2 and the pansy blue petal key gene VwF3'5'H were respectively recombined and integrated in the genome; finally, the transformed plasmid expression vectors pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H were transformed into the Agrobacterium strain GV3101 That's it. The detailed process is described below.

(1) Transformation of pCAMBIA1301 and pCAMBIA1303 vectors—integration into promoter and terminator

The CAMV35S promoter (pCAMV35S) and CAMV35S terminator (T-CAMV35S) were added to the multiple cloning sites of the pCAMBIA1301 and pCAMBIA1303 vectors, respectively. The sequence of pCAMV35S was cloned in pCAMBIA1301, and the selected enzyme cutting sites were Pst I and Xba I, respectively; the sequence of T-CAMV35S was cloned in pCAMBIA1301, and the selected enzyme cutting sites were EcoR I and Sac I. The specific process is as follows.

(1) Design primers, PCR clone promoter and terminator sequences

The primer sequences were designed as follows (5'-3'):

pCAMV35S-5': CCTCTAGAAGAGATAGATTTGTAGAGAGAG;

pCAMV35S-3': TGCCTGCAGATGGTGGAGCACGACACTC;

T-CAMV35S-5': CGAATTCAGGTCACTGGATTTTGGTTTTA;

T-CAMV35S-3': TACCGAGCTCCGGCCATGCTAGAGTCCGCAA.

PCR cloning: PCR cloning was performed using the pCAMBIA1301 plasmid as a template,

The 20μL PCR reaction system is as follows:

3' primer, 5' primer, 0.20 μL each;

dNTPs, 0.20 μL;

10×buffer, 2μL;

Plasmid template, 1 μL;

Taq enzyme, 0.15 μL;

_ddH2O , 16.25 μL.

PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min; annealing at 55°C for 1 min; extension at 72°C for 1 min, 31 cycles; extension at 72°C for 10 min; termination at 4°C.

(2) Separation and recovery

The amplified products of PCR clones and DNA molecular markers were separated by 1.0% agarose gel with a little ethidium bromide under the condition of 1×TAE buffer, and then the PCR products were recovered according to the instructions of the gel recovery kit.

(3) Digestion and ligation

The PCR product and the plasmid vector were digested and ligated respectively.

Taking pCAMBIA1301 as an example, the enzyme digestion system is as follows:

Pst I, Xba I, each 1 μL;

10×buffer, 5 μL;

pCAMBIA1301 plasmid, 100ng;

_ddH2O to 50 μL.

Digest overnight at 37°C.

The digested products were separated on 1.0% agarose gel and recovered according to the instructions of the gel recovery kit.

The recovered product was ligated overnight at 16°C with T4 ligase, and the ligation system was as follows:

pCAMBIA1301 plasmid digestion product, 1 μL;

Ligation solution, 5 μL;

pCAMV35S (or T-CAMV35S) PCR clone amplified and the product recovered by enzyme-digestion gel, 4 μL.

(4) Plasmid transformation, amplification, extraction

Transform the ligation product of step (3) into E. coli competent Top10 cells, amplify the plasmid and extract it for later use. The successfully constructed vector plasmid was extracted from Escherichia coli according to the instructions of the plasmid extraction kit (Tiangen Biochemical (Beijing) Co., Ltd.). Only the process of transforming Escherichia coli competent Top10 cells with the ligation product is briefly introduced as follows.

The conversion process is as follows:

A． Place Escherichia coli competent Top10 cells in ice, and after thawing, divide each 50uL into different centrifuge tubes, add 10μL of the ligation product to it, shake gently and place on ice for 30min;

B． After the ice bath is completed, place the centrifuge tube at 42°C for 90 seconds, and then quickly place it on ice for 2 minutes;

C． Add 500 μL LB liquid medium without antibiotics to the centrifuge tube, mix thoroughly, and pre-express at 37°C for 45 minutes;

D． Take 150 μL of pre-expressed bacteria solution and 40 μL of X-gal (20mg/mL) and mix well, use a sterile tip to suck it onto the prepared LB plate containing 100mg/L Amp, and spread the mixture evenly with a spreader Cover the entire flat surface;

E． Incubate the plate upside down at 37°C for 16 hours;

F． Pick 4 white positive clones from the plate cultured in step E on a clean bench, and shake and culture overnight in 5 mL (containing 100 mg/L Amp) LB liquid medium.

G． PCR bacterial liquid identification, the identified correct product is further sequenced and identified, and the sequenced correct bacterial liquid is preserved at -80°C for later use or the extracted plasmid is preserved for later use.

(2) Cloning and vector construction of rose flower fragrance gene RcOOMT2 and pansy blue petal key gene VwF3'5'H

This process is mainly realized through PCR cloning, ligation, enzyme digestion and other technical operations of rose flower fragrance gene RcOOMT2 and pansy blue petal key gene VwF3'5'H . The specific process is briefly introduced as follows.

(5) Cloning of rose RcOOMT2 and pansy VwF3'5'H genes

Based on the full-length sequences of rose flower fragrance gene RcOOMT2 (accession number: AJ439742.1) and pansy VwF3'5'H gene (accession number: AB332097) recorded in the NCBI database, PCR primers were designed according to the obtained gene sequences, The main purpose is to introduce restriction sites for BamH I and Kpn I.

The primer sequences were designed as follows (5'-3'):

RcOOMT2-5 ': CCTTGGATCCATGGAAAGGCTAAACAGCTTT;

RcOOMT2-3 ': CTCATTGAGGTTTATCCTTGAGGTACCAAGG;

VwF3'5'H- 5': CCTTTGGATCCATGGCAATTCTAGTCACCGAC;

VwF3'5'H- 3': CCTTTGGTACCTCAGGTTGCGTACGCGTTTGA.

RNA extraction and cDNA synthesis: The RNA of rose (Double Happiness variety) and pansy were extracted by CTAB method, and cDNA was obtained by reverse transcription according to the instructions of the cDNA synthesis kit.

PCR cloning: Using cDNA as a template, use high-fidelity Taq enzyme to perform PCR cloning of gene sequences respectively. For the PCR reaction system and reaction procedure, refer to step (1) for setting.

(6) Separation and recovery

Separate the amplified products of PCR clones and DNA molecular markers in step (5) with 1.0% agarose gel adding a little ethidium bromide under the condition of 1×TAE buffer, and then perform PCR according to the instructions of the gel recovery kit Product recovery.

(7) connection

The PCR recovered product in step (6) was connected to the pMD18-T vector to obtain the connection products RcOOMT2 -pMD18-T, VwF3'5'H -pMD18-T. The ligation reaction was performed with reference to the pMD18-T kit.

(8) Transformation, amplification, extraction

The ligation products RcOOMT2 -pMD18-T and VwF3'5'H -pMD18-T in step (7) were transformed into E. coli competent cells Top10, and the plasmids were extracted, identified and sequenced. Refer to step (4) for specific operation steps. Store the bacteria solution with correct sequencing at -80°C or extract the plasmid for future use.

(3) Construction of plasmid expression vectors pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H and recombination into the original Agrobacterium strain GV3101

The transformed vectors pCAMBIA1301 and pCAMBIA1303 in step (1) and the plasmids RcOOMT2-pMD18-T and VwF3'5'H -pMD18-T in step (2) were respectively used with BamH I and Kpn Carry out enzyme digestion with I enzyme, digest overnight at 37°C, and refer to step (3) for the enzyme digestion reaction system.

The digested products were separated on 1.0% agarose gel and recovered; then the recovered digested products were ligated at 16°C with T4 ligase, and the ligation reaction system was set according to step (3).

Transform the ligation products, namely the constructed pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H , into Escherichia coli competent cells Top10, positive clones were detected by PCR, and the correct products were further sequenced and identified, and the sequenced bacteria were stored at -80°C Save or extract the plasmid for future use, refer to step (4) for related steps.

The extracted pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H plasmids can be further transformed into Agrobacterium strain GV3101, and the specific steps are as follows:

A． Take the Agrobacterium competent cells and put them on ice to slowly thaw, then add 2 μL of the extracted plasmid DNA to 50 μL of the Agrobacterium competent cells, that is, the extracted pCAMBIA1301 -RcOOMT2 and pCAMBIA1303 -VwF3'5'H plasmids, After mixing, ice bath for 30min;

B． Add liquid nitrogen to freeze for 1 min, and quickly put it in a 37°C water bath for 3-5 min;

C． Add 800 µL of LB liquid medium, shake well, and incubate with shaking at 28°C for 4~5h; then centrifuge at 3000 rpm and 4°C for 3min to collect the bacteria;

D． Spread the bacteria collected in step C evenly on the LB solid medium containing Kan, Rif and Gen, culture at 28°C for 2~3 days, pick 4 single colonies and place them on the LB liquid medium containing the same antibiotic for 28 Cultivate overnight with shaking at 180rpm;

E． The bacteria liquid is detected by PCR, and the positive clones can be used for subsequent transgenic operations (agrobacterium infiltration), or the positive clones can be stored in a -80°C refrigerator for future use.

Example 2

This example focuses on the construction of genetic material based on protocorms germinated from Phalaenopsis seeds as receptors before transgenic breeding in Agrobacterium dipping, and a brief introduction is as follows.

(1) Under sterile conditions, induce the germination and growth of Phalaenopsis seeds in the medium to obtain protocorms;

Specifically: Cut the mature Phalaenopsis fruit from the mother plant, wash it with soapy water, and rinse it under clean water for 30 minutes; put the rinsed fruit into a large sterile beaker on an ultra-clean workbench, add 70% Sterilize with ethanol for 15 minutes, and then sterilize with 10% sodium hypochlorite for 15 minutes. During this period, shake the beaker to make it fully contact; after the disinfection is completed, rinse it with sterile water for 3 times; Cut the orchid fruit, take out the pulp with seeds with sterile tweezers, wash it in a petri dish filled with a small amount of sterile water (the purpose is to separate the seeds attached to it from the pulp), and then use a pipette gun to The sterilized water containing the seeds is evenly sprayed on the sterilized Phalaenopsis seed induction germination medium.

The medium required for the seeds to induce germination is: 3g/L Huabao No. 1+2g/L activated carbon+2g/L hydrolyzed casein+15g/L sucrose+2mg/L 6-BA+0.2mg/L NAA+ 6.5g/L agar, pH=5.5~5.6.

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, sunlight 16h, culture for 60-90d, and grow into protocorms.

As shown in Figure 3, after 60 days of seed culture, larger protocorms (about 0.4-0.5 cm) have been formed, which are used for protocorm proliferation and cultivation.

(2) Cutting the Zhongyuan corm in step (1) directly or after proliferating and culturing it as a receptor material;

Since the number of protocorms after dicing still has a certain limit, it is often necessary to further proliferate and amplify protocorms after dicing to obtain enough receptor materials. After repeated experiments and discussions, the inventor believes that the best tissue culture The conditions are as follows:

Proliferation medium: 3g/L Huabao No. 1+2g/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;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, daylight 16h, multiplication culture for 60-90 days, protocorm with a diameter of about 0.5cm can be used as a transgenic acceptor material after multiplication and culture;

The minimum diameter of protocorm cut pieces used for proliferation culture should not be less than 0.3cm.

The following is a brief introduction to the process of obtaining the optimal culture conditions in the process of protocorm proliferation and culture.

Preliminary screening of different hormone types and concentrations

The combination of plant growth regulators is an important factor in the process of plant tissue culture. A suitable combination of growth regulators can induce the proliferation of tissue culture materials, or change the growth state of tissue culture materials (such as: differentiation of tissue culture materials, rooting, etc. ).

In the following examples, Phalaenopsis protocorms were used as materials. First, a screening test was carried out on the growth regulator combinations, with the aim of selecting a growth regulator combination suitable for protocorm proliferation. The protocorms in the Phalaenopsis multiplication and cultivation process come from the protocorms whose seeds have germinated for 60-90 days, have a diameter of about 0.5 cm, and have not differentiated into buds.

Phalaenopsis protocorm proliferation medium composition: 3g/L Huabao + 2g/L activated carbon + 15g/L sucrose + 6.5g/L agar, pH=5.5~5.6.

Three kinds of hormones were selected: 2,4-D, 6-BA and TDZ, and 12 treatments were set up. The specific treatment combinations are shown in the table below.

When inoculating, inoculate 10 Phalaenopsis protocorms in each bottle of culture medium, and each treatment repeats five bottles.

The day temperature is 25°C, the night temperature is 17°C, the light intensity is 3000Lx, and the daylight is 16h in the culture room.

The statistical items and calculation formula are as follows:

Differentiation and proliferation coefficient (%) = number of cuts that can induce differentiation and proliferation/total number of inoculations × 100%.

The culture results are shown in the table below and Figure 4.

It can be seen from the above table that among the 12 kinds of proliferation medium combinations, treatment 1, treatment 2, treatment 9, treatment 10, treatment 11 and treatment 12 all showed differentiation and proliferation of protocorms after 30 days of culture. Among them, the differentiation and proliferation rates of treatment 9 and treatment 10 reached 90% at 30 days, while the differentiation and proliferation rates of treatments 1 and 2 and treatments 11 and 12 also reached 40% and 25% at 30 days of culture. In treatment 5, 6, 7, and 8, not only did no protocorm differentiation and proliferation occur after 30 days of culture, but even material death occurred at 60 days. The above results show that: the excessive concentration of 2,4-D (greater than 1 mg/L) in the process of protocorm differentiation and proliferation is not only unfavorable for the differentiation and proliferation of protocorms, but even causes the death of protocorm cut pieces; the concentration of 2,4-D is When the concentration of 2,4-D is 0.5 mg/L, although the phenomenon of differentiation and proliferation also occurs in the culture process of 30 days, it is not as good as the differentiation and proliferation efficiency and growth status when the concentration of 2,4-D is 0.5 mg/L. Therefore, the preliminary judgment of the concentration of 2,4-D When it is less than 1mg/L, it is most beneficial to the differentiation and proliferation of Phalaenopsis protocorms.

The combination of growth regulators in treatments 9 and 10 was 2,4-D combined with 6-BA, and in treatments 11 and 12 it was a combination of 2,4-D and TDZ. Treatment 9 and treatment 10 differentiated and proliferated after 60 days of culture The coefficient reached more than 95%, and although treatment 11 and treatment 12 also had a higher differentiation and proliferation coefficient (up to 80%), compared with the first two treatments, the effect was slightly worse. The test results showed that the effect of 6-BA on differentiation and proliferation was better than that of TDZ, but the effect on differentiation and proliferation was not much different when its concentration was 1mg/L and 2mg/L.

In summary, through the comparison of the results of each treatment combination, it is found that the combination of 2,4-D and 6-BA is better than the combination of 2,4-D and TDZ, so it is preliminarily determined that the medium group suitable for protocorm proliferation culture Divided into: 3g/L Huabao+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 Different Concentrations of 2,4-D on Protocorm Proliferation

According to the results of screening tests on hormone types in the proliferating culture of protocorms, although the hormone combination of 2,4-D and 6-BA is beneficial to the proliferation of protocorms, its concentration still needs to be further optimized to meet the actual conditions. Production needs. Since 2,4-D, as an auxin, plays a greater role in protocorm proliferation and culture, the following work first optimized the dosage of 2,4-D.

According to the above results, it is known that when the concentration of 2,4-D is greater than 1 mg/L, it is not conducive to the proliferation and cultivation of protocorms. Therefore, we set the concentration range of 2,4-D between 0 and 1 mg/L. Five different treatments were carried out, namely 0mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L.

The materials used come from the protocorms of the Phalaenopsis "Red Dragon" and "Radiance" seeds that have germinated for 60-90 days, have a diameter of about 0.5 cm, and have not differentiated into buds. 14 Phalaenopsis protocorms were inoculated in each bottle of culture medium, and each treatment was repeated 5 bottles. They were cultured in a culture room with a daytime temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h. After 60 days of multiplication culture, the growth status of the protocorm pieces of the two varieties was counted.

The statistical items and calculation formula are as follows:

Induced proliferation rate of protocorms (%) = number of cut pieces of protocorms formed by proliferation/total number of inoculations × 100%;

Protocorm induction differentiation rate (%) = number of cut pieces differentiated into seedlings/total number of inoculations × 100%;

Protocorm-induced callus rate (%) = number of callus-forming cuts/total number of inoculations × 100%.

The specific processing conditions and statistical results are shown in the table below.

It can be seen from the above table that the protocorms of different varieties of Phalaenopsis have different proliferation rates under the same 2,4-D concentration treatment; but the same thing is that when the 2,4-D concentration is lower than 0.2mg/L , the proliferation rate of protocorms was low, at this time, most of the cut pieces no longer proliferated to form protocorm clumps, but directly formed buds and further grew into seedlings after growing into a single protocorm; at the concentration of 2,4-D When the concentration of 2,4-D is 0.4-0.6mg/L, the protocorm proliferation rate of the two varieties is high, the highest reaches 70.0%; and when the concentration of 2,4-D is 0.8mg/L, although both varieties have yellow Callus formation, but compared with low concentration (0~0.2mg/L) treatment, the protocorm proliferation rate is relatively high. Therefore, when the concentration of 2,4-D is too low or too high, it is not conducive to the proliferation of Phalaenopsis protocorms. If it is too low, it will easily promote the differentiation and growth of protocorms in a short period of time, which will not only affect the proliferation but also easily cause the volume of protocorms. smaller.

Combination Optimization Screening of Plant Growth Regulator Concentration

According to the optimization screening results of the aforementioned auxin 2,4-D and the preliminary screening results of different types and concentrations of growth regulators, we further designed several concentration combinations of 6-BA and 2,4-D (other components: 3g /L Huabao+2g/L activated carbon+15g/L sucrose+6.5g/L agar, pH=5.5~5.6), through further tissue culture experiments, the protocorm induced proliferation rate was counted (protocorm induced proliferation rate (%) = the number of cut pieces that proliferate to form protocorms/total number of inoculations × 100%), so as to screen out the most suitable combination of medium hormone concentration for protocorm proliferation.

The materials used come from the protocorms of the Phalaenopsis "Red Dragon" and "Radiance" seeds that have germinated for 60-90 days, have a diameter of about 0.5 cm, and have not differentiated into buds. During the tissue culture process, 14 cut protocorms of Phalaenopsis were inoculated in each bottle of culture medium, and each treatment was repeated in 5 bottles. They were cultured in a culture room with a daytime temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h.

The specific ratio and combination of growth regulators are set as follows.

The specific tissue culture results are shown in the table below and Figure 5.

As can be seen from the above table and Figure 5, under the same 2,4-D concentration conditions, the protocorm proliferation rate of the two varieties also increased with the increase of the 6-BA concentration; when the 6-BA concentration At the highest concentration of 5mg/L, high concentration of 2,4-D (0.8mg/L) can inhibit the formation of protocorm clumps, while low concentration (0.4mg/L) of 2,4-D shows bud differentiation state, and when the concentration of 2,4-D was 0.6mg/L, the proliferation of protocorms was dominated by lumps. By comparing the results of three treatments with 2,4-D concentration of 0.6mg/L, it can be seen that the treatment with 6-BA of 1mg/L has weaker proliferation ability than the treatment with high concentration; when the concentration of 6-BA is 5mg/L Although the proliferative ability is strong, there are more buds differentiated in the later stage; and when the concentration of 6-BA is 3mg/L, the proliferation rate of protocorms is not only higher but also the buds are less in the later stage, and the protocorms obtained by proliferating mostly show lumpy. Therefore, according to the analysis of test results, it is found that the hormone ratio of 0.6mg/L 2,4-D+3mg/L 6-BA is suitable for the proliferation and culture of protocorms.

Based on the above screening results of growth regulators, it can be seen that in the process of Phalaenopsis tissue culture, the composition of the better medium, that is, the proliferation medium is: 3g/L Huabao 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.

Influence of Protocorm Cutting Diameter on Proliferation Culture

In the previous work, it was found that the albinism and death of cut pieces often appeared in the process of protocorm proliferation and cultivation. Preliminary research believed that the albino phenomenon was caused by too small cut pieces. In order to grasp the optimal protocorm cut size in practical production and application, the inventor has further discussed the cut size of the protocorm.

Cut the protocorm into three pieces with diameters of A (less than 0.2cm), B (0.2~0.3cm), and C (above 0.3cm), and insert them into the proliferation medium (ie: 3g/L Huabao 1 No.+2g/L activated carbon+15g/L sucrose+3mg/L 6-BA+0.6mg/L 2,4-D+6.5g/L agar, pH=5.5~5.6) After 60 days of culture, the growth status statistics.

14 cut protocorms of Phalaenopsis were inoculated in each bottle of culture medium, and each treatment was repeated for 5 bottles. They were cultured in a culture room with a daytime temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h.

The culture results are shown in the table below.

It can be seen from the above table that the diameter of the protocorm cuts has a great influence on the albinism during the proliferation culture; when the cut diameter is A (less than 0.2cm), 30% of the cuts will appear after 14 days of cultivation Albinism occurs, and the number of albino cuts increases as the number of days of culture increases. After 60 days of culture, the albino rate of the cuts reaches 73%, and the volume of the proliferated protocorms is also small, which cannot be used for subsequent experiments. carried out. When the diameter of the cut piece is B (0.2~0.3cm), although its albino rate is less than that of A within the same culture period, the volume of a single protocorm on the protocorm mass formed by its proliferation is not large enough. Subsequent tests will still produce more obvious whitening phenomenon, which will increase the test error and reduce the test accuracy. When the diameter is C (above 0.3cm), although there are some albino phenomena, 87% of the cut pieces proliferate and form large protocorm clumps, and the individual volume of the protocorm contained in them is also relatively large, while the albino cut pieces Among them, most of them were cut pieces with more cut surfaces, which indicated that severe protocorm damage was not conducive to its proliferation. Therefore, the cutting process of protocorms in actual production should reduce the damage of the cutting and ensure that the diameter of the cutting is large enough.

Effects of Different Contents of Activated Carbon on Growth of Protocorm Diced Proliferation Culture

Browning is a common lethal phenomenon of culture materials in the process of plant tissue culture. Therefore, in the process of tissue culture of plants, substances such as adsorbents or antioxidants are often selected to prevent and control browning, and activated carbon is the most commonly used adsorbent. Due to differences in plant species, the amount of activated carbon added is also different in different plant tissue culture processes, so the inventor further conducted a screening test on the amount of activated carbon added.

The content of activated carbon in the proliferation medium was set to 0g/L, 1g/L, and 2g/L respectively. The materials used were from the seeds of Phalaenopsis "Red Dragon" and "Radiance" after 60-90 days of germination, with a diameter of about 0.5 cm, protocorms undifferentiated into buds. 14 protocorm cuts were inoculated in each bottle of culture medium, and 5 bottles were replicated for each treatment. They were cultured in a culture room with a day temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h.

Observe and count the growth and differentiation state of protocorm cut pieces under different activated carbon contents and count their survival rate and browning rate. The calculation formula is as follows:

Survival rate of protocorm cuts (%) = number of protocorm cuts/total number of inoculations × 100%;

Browning rate of protocorm cuts (%) = number of dead browned cuts/total number of inoculations × 100%.

The specific test results are shown in the table below, and the morphology observation results are shown in Figure 6.

It can be seen from the above table that the higher the content of activated carbon, the fewer the number of cut pieces that will be browned with the increase of the number of days of cultivation; in the treatment without adding activated carbon, a small number of browning phenomena have occurred after 14 days of cultivation, and at 60 days The brown substance released by the material makes the culture medium also turn black; in the latter two treatments, although activated carbon with a content of 1g/L can also reduce the occurrence of browning well, its effect is slightly worse than that of the treatment with a content of 2g/L . In addition, in the course of the test, it was also found that with the increase of the activated carbon content, the value-added effect and growth of the cut pieces were improved. Therefore, on the whole, it is better to choose 2g/L activated carbon to improve the browning condition of protocorm proliferation and culture.

Effects of Antibiotics (Kan and Hyg) on Protocorm Proliferation of Phalaenopsis

Screening agents are used in the preliminary screening of transformed materials in plant genetic transformation experiments, mainly referring to those antibiotic substances that cause toxicity to plants and inhibit their growth. Kan and Hyg are two commonly used screening agents for plant genetic transformation, and are also widely used in Phalaenopsis transgenic research. Since different receptor materials have different sensitivities to different antibiotics, the selection of screening agents usually needs to be determined by conducting multiple antibiotic resistance tests on the receptor materials.

Existing studies have shown that Phalaenopsis protocorms are not sensitive to the toxicity of Kan, and Kan mainly has a greater impact on the formation of Phalaenopsis roots. When the recipient material reaches the rooting stage, it will be screened and cultivated. Hyg is also a kind of screening agent that is widely used in the genetic transformation experiment of Phalaenopsis, and its main performance is that it has a strong inhibitory effect on the growth of Phalaenopsis.

In order to obtain the best antibiotic type and screening concentration, the inventors used Phalaenopsis protocorms as test materials to carry out Kan and Hyg resistance screening tests in order to select the most suitable antibiotic type and concentration.

The materials used come from the protocorms of Phalaenopsis "Red Dragon" and "Radiance" that have germinated for 60-90 days and have a diameter of about 0.5 cm. They have not differentiated into buds, and the resistance screening tests of Kan and Hyg were carried out respectively.

Kan resistance screening: Protocorm cuttings were inserted into the proliferation medium with Kan concentrations of 0 mg/L, 10 mg/L, 50 mg/L, and 100 mg/L for Kan resistance screening.

Hyg resistance screening: Protocorm cuts were inserted into the proliferation medium containing 0 mg/L, 1 mg/L, 3 mg/L, 5 mg/L, 8 mg/L, 10 mg/L Hyg for resistance sexual screening.

14 Phalaenopsis protocorms were inoculated in each bottle of culture medium, and each treatment was repeated 5 bottles. They were cultured in a culture room with a daytime temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h. After culturing for one month, the survival rate of each treatment was observed and counted.

The specific test results are shown in the table below, and the morphological observation results are shown in Figure 7.

As for the Kan resistance screening results, it can be seen from the results in the above table that there are differences in the resistance of the two Phalaenopsis varieties to Kan, but the same thing is that the resistance of the two to Kan is not strong, when the concentration of Kan When it reached 100 mg/L, the proliferation of cut pieces of both varieties was severely inhibited, and the survival rates were as low as 7.1% and 18.6%, respectively. Therefore, Kan can be used as a screening agent in transgenic research using protocorms as recipient materials for screening of transformed materials.

As for the results of Hyg resistance screening, it can be seen from the results in the table above that when Hyg is added to the medium, the survival of the cut pieces of the two varieties is inhibited, and with the increase of Hyg content, the survival rate is greatly reduced. When the Hyg content was 3 mg/L, the survival rate was as low as 30% or less; when the Hyg content was 5-8 mg/L, the cuttings showed severe growth inhibition, and when the Hyg content was 10 mg/L, there was almost no cutting. block survives.

According to the screening results of the above two antibiotics, both Kan and Hyg have inhibitory effects on the proliferation of protocorms, but in terms of inhibitory effect, Hyg is better than Kan, because Hyg can be very effective at a low concentration (5mg/L). Good inhibition of protocorm proliferation, and Kan needs a higher concentration (100mg/L) to achieve this effect, therefore, in the genetic transformation of Phalaenopsis is more suitable to use Hyg as a screening agent, and in the concentration selection, In order to reduce the probability of false positives, it is more appropriate to choose Hyg with a concentration of 8 mg/L in the selection of the screening agent concentration.

Effects of Antibacterial Agents on Protocorm Proliferation of Phalaenopsis

Because in the genetic transformation test mediated by Agrobacterium, it is necessary to add an appropriate amount of antibacterial agent to inhibit the growth of Agrobacterium, so as to avoid the negative impact on the acceptor material due to the growth of bacterial plaque.

In order to obtain the optimal dosage of bacteriostatic agent (Cef, cephalosporin), the inventor further carried out the protocorm with a diameter of about 0.5cm based on the germination of Phalaenopsis "red dragon" and "radiant" seeds. Proliferation culture test, brief introduction is as follows.

After the protocorm was cut into pieces, it was inserted into the proliferation medium with Cef contents of 0 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L respectively for cultivation.

14 Phalaenopsis protocorms were inoculated in each bottle of culture medium, and each treatment was repeated 5 bottles. They were cultured in a culture room with a daytime temperature of 25°C, a night temperature of 17°C, a light intensity of 3000Lx, and a daylight of 16h.

After 30 days, the survival rate of cut pieces in each treatment was counted.

The test results are shown in the table below.

It can be seen from the above table that when the content of Cef reaches a certain concentration (200mg/L), it also has a slight inhibitory effect on the proliferation of cut pieces, and the survival rate of cut pieces shows a downward trend; when the content of Cef is 300mg /L, the degree of reduction in the survival rate of cut pieces was more obvious. Due to the additive effect of antibiotics, in order to minimize the effect of Cef on the proliferation of cuttings, it is recommended to use 50 mg/L of Cef to screen for antibacterial agents that inhibit plaque formation during culture.

rooting culture

Because the ultimate purpose of tissue culture needs the plant body to grow root system, thereby finally can transplant successfully, thus inventor has done further experiment for the rooting culture of Phalaenopsis, brief introduction is as follows.

Protocorms continue to be cultured in the proliferation medium, and after more than 90 days, they will gradually differentiate into buds and grow leaves. Transfer the robust buds cultivated by proliferation (with 2-3 leaves on display at the top, the bud height is 2cm, and the leaf length is about 1cm) into the rooting medium for rooting culture. The medium type is: 3g/L Huabao No. 1 +1.5mg/L IBA+20g/L sucrose+1g/L activated carbon+6.5g/L agar, pH=5.5~5.6.

The culture results are shown in Figure 8.

The results showed that with this medium, small roots could be grown at the bud base after 20 days of culture, and the roots of test-tube plantlets reached more than 4 and were relatively strong after 40 days of culture, and the rooting effect was better.

In the genetic transformation of Phalaenopsis, the establishment and optimization of a good receptor system is the key to success. Because the single capsule of Phalaenopsis has a large number of seeds and is easy to germinate, the present invention uses protocorms induced by germination of Phalaenopsis seeds as acceptor materials, and can further satisfy actual production requirements through proliferating and culturing the protocorms.

In the combinatorial screening process of the growth regulators used in the Phalaenopsis proliferation culture, the inventors have explored through multiple experiments and combinations, and believe that the combination of 2,4-D and 6-BA hormones can effectively promote the proliferation of protocorms without the need for Higher concentration ratio, especially 2,4-D, is more beneficial to the proliferation of protocorms as the concentration decreases. Too high concentration not only inhibits the proliferation but also causes the death of the material. Similarly, low concentration of 6-BA is beneficial to its proliferation, but too low concentration will easily cause the protocorms obtained from the proliferation to transition to the budding state once formed, and no longer increase the volume of a single protocorm. Simultaneously, the present invention also further discusses the size of the protocorm cut piece or the amount of activated carbon in the process of multiplication and cultivation. In short, the final result is that the best Phalaenopsis proliferation medium formula is: 3g/L Huabao 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; the minimum diameter of protocorm cut pieces should not be less than 0.3cm.

The results of rooting culture show that when the robust shoots (with 2-3 leaves exhibited at the top, the buds are 2cm higher and the leaf length is about 1cm) are transplanted, the optimal rooting medium formula is: 3g/L Huabao No. 1+1.5mg/L IBA+20g/L sucrose+1g/L activated carbon+6.5g/L agar, pH=5.5~5.6.

One of the conditions that a plant gene transformation receptor system should have is that the receptor system is sensitive to antibiotic selection. When the concentration of screening antibiotics added to the screening medium reaches a certain level, it can inhibit the growth, development and differentiation of non-transformed plant cells, while the transformed plant cells can grow and differentiate normally because they carry corresponding resistance genes, and finally obtain a complete Transform plants. The present invention has carried out the resistance test of Kan and Hyg respectively, and the result shows that the sensitivity of Phalaenopsis protocorm to Kan is poorer than Hyg, but when Kan reaches 100mg/L, the growth of protocorm cut piece also shows serious suppression Phenomena, a large number of cuts appear lethal, this result shows that Kan can also be used for transgenic screening of some Phalaenopsis varieties, which is different from related research reports (Chai M L et al., Stable transformation of protocorm-likebodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Scientia Horticulturae. 2002, 96: 213-224). The low concentration of Hyg (5mg/L) has a good inhibitory effect on the survival of protocorms, which can be applied to the direct screening of receptor materials after dipping. Considering comprehensively, using Hyg with a concentration of 8 mg/L as the screening agent has a better effect, and can reduce the occurrence of false positives during the screening process and increase the conversion rate. In the process of genetic transformation mediated by Agrobacterium, 50 mg/L Cef was selected as the bacteriostatic agent for inhibiting plaque formation in the screening culture process.

Example 3

In this example, the Phalaenopsis protocorm cultivated in Example 2 was dipped by the dipping method of Agrobacterium to realize transgenic operation. The dipping solution was the recombinant Agrobacterium strain GV3101 prepared in Example 1, which contained a plasmid expression vector pCAMBIA1301 -RcOOMT2 , pCAMBIA1303 -VwF3'5'H , the bacterial selection marker gene of the plasmid expression vector is Kan, the plant selection marker gene is Hyg, and the reporter gene is GUS. A brief introduction is as follows.

The transgenic operation of Agrobacterium infection includes the following steps.

(1) Pre-cultivation of receptor material

The excellent protocorm obtained in Example 2 with a size of about 0.5 cm and consistent growth was cut into pieces, transferred to the pre-cultivation medium, and pre-cultivated for 3 days to keep the recipient material vigorous vitality. The state is transformed, and the damage caused by the impregnating liquid is reduced at the same time.

The pre-culture medium and culture conditions are the same as the proliferation medium in Example 2.

(2) Agrobacterium infection

The recombinant Agrobacterium strain GV3101 prepared in Example 1 was used to prepare the dipping solution. During the dipping, the OD ₆₀₀ of the recombinant Agrobacterium suspension was 0.4-0.6, and the dipping time was 15 minutes.

Before dipping, put the pre-cultured 3d protocorm cut into a sterile Erlenmeyer flask, try not to bring the culture medium. Pour in the prepared impregnation solution until the receptor material is completely submerged. Gently shake the Erlenmeyer flask continuously, so that the receptor material is in full contact with the liquid, until the required time for dipping is reached.

The preparation steps of the liquid for the recombinant Agrobacterium strain GV3101 are as follows.

A． Take out the recombinant Agrobacterium strain from -80°C, use an inoculation needle to streak on the LB solid medium containing Kan and Rif antibiotics, put it in an incubator, and cultivate it at 28°C for 2-3 days until a single colony appears;

B． Pick a single colony from the plate with an inoculation needle, inoculate it in 5 mL of LB liquid medium containing Kan and Rif antibiotics, and cultivate overnight at 28°C with shaking at 180 rpm;

C． Use a pipette to add the above-mentioned bacterial solution to the LB liquid medium without antibiotics at a ratio of 1:50 and incubate for about 3-5 hours. When the OD ₆₀₀ of the bacterial solution reaches the required value, it can be used for transformation.

When preparing the Agrobacterium resuspension, prepare it according to the following steps: First, use a centrifuge to centrifuge the bacterial solution (that is, the Agrobacterium culture solution prepared in the above step C to be transformed) at 2500rpm for 10min at 4°C, and then collect Bacterial cells; then, when the collected thalline cells were resuspended to the required OD ₆₀₀ value with a liquid proliferation medium (ie, the liquid proliferation medium described in Example 2), they could be used for the next step of transformation.

(3) After dipping, the receptor material was placed on the co-cultivation medium, and cultured in the dark at 25°C for 3 days;

The receptor material has been impregnated. In theory, the exogenous gene has been transformed into the Phalaenopsis genome. After the impregnation, the receptor material was moved to sterile filter paper to dry, and then transferred to a co-cultivation medium (the same as the proliferation medium in Example 2), and cultured in the dark at 25°C for 3 days.

(4) Elution of Agrobacterium

After co-cultivation, the impregnated receptor material needs to be eluted with Agrobacterium before it can be transferred to the screening medium for screening culture. However, since different concentrations of bacteriostatic agents have different effects on the growth of Agrobacterium, it is necessary to screen out the appropriate concentration of bacteriostatic agents for elution before the transfer of the receptor material, so as to ensure the smooth progress of the subsequent screening work.

The specific elution process is as follows: pick out the receptor material after the dark culture in step (3) and place it in a sterile Erlenmeyer flask. Rinse off the silk, and then rinse it with Cef water at an appropriate concentration (finally determined to be 300mg/L) for 2 to 3 times. After soaking in water for 20-30 minutes, the receptor material was picked out and dried on sterile filter paper, and then the receptor material was screened and cultured.

(5) Screening and culturing of receptor materials after dipping

Dry the receptor material eluted in step (4) and transfer it to the screening medium. During the screening culture process, perform a transfer screen every 15 days to maintain the effectiveness of the antibiotic. Before each transfer, you need to follow the steps (4) method for the elution of Agrobacterium.

The screening medium is: proliferation medium+8mg/L Hyg+50mg/L Cef in Example 2, pH=5.5～5.6;

The culture conditions are: day temperature 25°C, night temperature 17°C, light intensity 3000Lx, daylight 16h.

After four rounds of screening culture, the screened protocorms were transferred to the proliferation medium containing only bacteriostatic agent (50mg/L Cef) and continued to culture until they differentiated into buds and grew into seedlings, and then screened and identified.

(6) Further amplify the correct Phalaenopsis plants screened and identified in step (5) or insert them into the rooting medium to induce rooting;

The rooting medium is: 3g/L Huabao No. 1+1.5mg/L IBA+20g/L sucrose+1g/L activated carbon+6.5g/L agar, pH=5.5~5.6.

Discussion on Influencing Factors of Agrobacterium Dip Solution

The above description only gives the final optimization results, but there is no necessary introduction to the optimization process. The following is a brief introduction to the most important influencing factors in the transgenic breeding of Phalaenopsis-the influencing factors related to the Agrobacterium infusion solution.

Since there are many factors affecting the conversion rate of the receptor material in the Agrobacterium-mediated genetic transformation test, the inventors conducted a brief test analysis on the more important factors such as the combination of the receptor material and the liquid, the time of the dipping, and the concentration of the dipping. The results of the study were mainly carried out by the method of transient expression of GUS in the recipient material that had been sterilized after transformation, while considering the degree of damage of the protocorm cuts.

Influence of combination of receptor material and liquid agent on conversion rate

Protocorms obtained from seed induction and protocorms obtained from multiplication culture were respectively selected as recipient materials, and the two materials after pre-cultivation were dipped in two dipping solutions with OD ₆₀₀ of 0.5 (15min), and the difference was counted after 3 days. Results of the conversion rate and damage rate of the receptor material in the combination of the receptor material and the liquid agent.

Combinations A, B, C, and D are set to represent the combination of seed-induced protocorms and bacterial liquid, the combination of proliferating and cultured protocorms and bacterial liquid, the combination of seed-induced protocorms and resuspension, and the proliferating and cultivating protocorms and resuspension.

Transient expression rate of GUS = the number of cut blocks that turned blue/the number of detected cut blocks × 100%;

Damage rate = number of melanized dead pieces/number of detected pieces × 100%.

It can be seen from the above table that for protocorms of the same origin, the transient expression rate of GUS after inoculation in the bacterial solution is higher than that in the heavy suspension (A is higher than C, B is higher than D), but the bacterial solution The damage to the cut protocorm is more serious than that of the heavy suspension (A is higher than C, B is higher than D), and it can cause a large number of cuts to blacken and die in a short period of time; and under the same conditions of the liquid, it is found that the proliferating culture The GUS transient expression rate of the obtained protocorms was higher than that of the directly induced protocorms (B was higher than A, D was higher than C), and the degree of damage to the cut pieces was relatively lower than that of the other type (B was higher than A low, D is lower than C). Therefore, through the comprehensive consideration of the transient expression rate and damage rate of GUS, the protocorm obtained from proliferation culture is used as the receptor material, and the resuspension liquid is selected as the dispensing liquid, which can not only ensure the conversion rate but also reduce the damage degree of the material.

Effect of Dyeing Time on Conversion Rate

Two dipping solutions (both OD ₆₀₀ of 0.5) were selected, namely the bacterial liquid and the resuspension liquid, and two dipping times of 10 min and 15 min were designed respectively, and the above four steps were carried out on the pre-cultured receptor material (protocorm obtained from proliferation culture). Dip dyeing in different ways, after 3 days, the conversion rate and damage rate of the cut pieces are counted.

The statistical results are shown in the table below.

It can be seen from the above table that no matter what kind of liquid is used, the best time for dipping is 15 minutes, reaching 53.6%.

Effect of concentration of liquor on transformation efficiency

The pre-cultured receptor materials (protocorms obtained from proliferation culture) were soaked (15 min) in the suspensions with OD ₆₀₀ of 0.2, 0.4, 0.6, and 0.8 respectively, and the transformation rate and damage rate of the receptor materials were counted.

Transient expression rate of GUS = the number of cut blocks that turned blue/the number of detected cut blocks × 100%;

Damage rate = number of melanized dead pieces/number of detected pieces × 100%.

The statistical results after 3d are shown in the table below.

It can be seen from the results in the above table that when OD ₆₀₀ =0.2, the degree of damage to the cut piece is the smallest, but the conversion rate is 0% at this time; with the increase of OD ₆₀₀ value, the transient expression rate of GUS also gradually increases, but when OD 600 When ₆₀₀ =0.8, the transient expression rate of GUS showed a decline, and the damage rate of cut pieces reached the highest. When OD ₆₀₀ =0.4~0.6, the transient expression rate of GUS was relatively high, 39.3% and 46.4%, respectively. At this time, the damage rate of cut pieces was also lower compared with OD ₆₀₀ =0.8. Therefore, the impregnation solution concentration between 0.4 and 0.6 can be used for the impregnation of the receptor material.

Agrobacterium eluate - screening of cephalosporin (Cef) concentration

In order to determine the appropriate concentration of Cef water, the bacteriostatic agent Cef was first added to LB solid medium (containing antibiotics), and the content was set to 0 mg/L, 50mg/L, 100mg/L, 200mg/L, 300mg/L respectively, Then culture the recombinant Agrobacterium, observe the growth of Agrobacterium within the same culture period (2~3d), so as to determine the most suitable concentration of Cef.

The specific culture results are shown in the following table and Figure 9.

Note: ++++, ++, +, - indicate normal proliferation, inhibited proliferation, severely inhibited proliferation, and no, respectively.

It can be seen from the above table that when the concentration of Cef is 300mg/L, the growth of Agrobacterium is completely inhibited. Therefore, Cef with a concentration of 300mg/L can be selected for the elution of Agrobacterium.

Test case

The tissue-cultured seedlings after gene transformation in Example 3 need to undergo various tests to ensure that the gene to be transformed is correctly integrated into the Phalaenopsis genome. The tissue-cultured seedlings whose resistance screening was positive and whose leaves reached about 2 cm in length were selected for various indicators to be tested. The detection methods include PCR detection and tissue staining detection, which are briefly introduced as follows.

detection

During the screening culture process, some recipient materials that have not been successfully transformed can also show resistance to the screening agent and undergo growth and differentiation to form "false positive" plants. Therefore, it is necessary to perform PCR detection on resistant materials to determine whether the target gene is successfully integrated into the genome of resistant materials. In the PCR detection of the resistant material in the present invention, the plasmid contained in the recombinant Agrobacterium is used as the positive control (P), and the uninfected Phalaenopsis leaves are used as the negative control (N). Specific steps are as follows:

(1) DNA extraction of the screened Phalaenopsis resistant materials

The modified CTAB method was used to extract the DNA of the Phalaenopsis material, and the specific steps were as follows:

Take 0.1g of fresh Phalaenopsis tissue into a 2mL centrifuge tube, add liquid nitrogen and grind it into powder, then add 1000 μL of CTAB to the centrifuge tube, and then put it in a constant temperature water bath at 65°C for 90 minutes. Take it out and turn it upside down 2-3 times.

After the water bath, take out the test tube and place it at room temperature, then add 1/2 volume (CTAB volume) of chloroform/isoamyl alcohol (24:1) to the centrifuge tube, and gently mix it up and down for more than 5 minutes. Pay attention to this operation process Perform in a fume hood. After mixing, centrifuge at 12000 rpm for 10 min. Aspirate 600 μL of the supernatant into a pre-marked 1.5ml centrifuge tube, be careful not to absorb impurities. Add 1/2 volume (supernatant volume) of isopropanol to the centrifuge tube containing the supernatant, invert and mix well, and centrifuge at 10,000 rpm for 30s. Discard the supernatant and dry the pellet in the centrifuge tube.

Add 500uL of 75% ethanol to fully wash the DNA; centrifuge at 12000rpm for 2min; discard the supernatant, and dry the DNA at room temperature; add 100uL ddH ₂ O to shake to fully dissolve the DNA for later use.

(2) PCR detection

According to the sequence information of RcOOMT2 and VwF3'5'H genes, primers were designed for PCR detection, and the primers were designed as follows (5'-3'):

RcOOMT2-5 ':TACGGGAACCATCAGCCAA,

RcOOMT2-3 ': GAAACCAGCATCAGTGAAGAG;

VwF3'5'H- 5': ACCTCAACTTCTCCAACCGC;

VwF3'5'H- 3': GCTCCTTCACCATCTTCGTC.

PCR template: the Phalaenopsis DNA of the screened resistant material is used as a template, the corresponding recombinant plasmid is used as a positive control, and the untransformed Phalaenopsis DNA is used as a negative control.

The PCR reaction system (20 μL) is as follows:

3' primer, 5' primer, 0.20 μL each;

dNTPs, 0.20 μL;

Template, 2 μL;

Taq enzyme, 0.15 μL;

10×buffer, 2μL;

_ddH2O , 15.25 μL.

PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min; annealing at 58°C for 1 min; extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 5 min; termination at 4°C.

The PCR amplification product and DNA molecular marker were separated by 1.0% agarose gel with a little ethidium bromide under the condition of 1×TAE buffer solution, and the detection results were observed in a UV gel imager. The detection results are shown in the figure 10.

(3) Detection by tissue staining

Since the reporter gene of the plasmid expression vector selected in this method contains GUS. Therefore, in addition to PCR detection of resistant materials, GUS tissue staining can also be used to detect whether the reporter gene is normally expressed to indirectly indicate whether the target gene is normally expressed in the resistant materials. Specific steps are as follows:

First, collect young leaves or other young organs from the transgenic plants screened out for resistance. The material should not be too large (about 0.2 cm), and the larger material should be sliced with a tissue culture knife. Put the slices into a centrifuge tube and add solution A, the volume of solution A is appropriate to cover the material. Place the centrifuge tube in a constant temperature incubator at 37°C for 1 h.

Pour out liquid A, then add the same volume of liquid B to the centrifuge tube. Incubate in a constant temperature incubator at 37°C for several hours until a blue color is observed on the material (in order to fully react, the material can also be treated overnight). Note that after adding solution B, the centrifuge tube should be wrapped with tinfoil to prevent light from affecting the effect of solution B.

Rinse the material with water, and if there is no interference from other pigments, the processing result of the material can be directly observed. This process can be performed under a dissecting microscope or directly observed with the naked eye. In order to avoid the interference of other pigments contained in the leaves (such as chlorophyll, etc.), you can add 95% ethanol to the centrifuge tube to elute the pigment after pouring out the B solution. The decolorization is 30min each time, and it can be eluted several times. , until no other interfering pigments exist. The GUS test results are shown in Figure 11.

In summary, the present invention has proved through different combination tests that the protocorm obtained by proliferation culture is used as the acceptor material, and when the resuspension liquid is selected as the liquid for dyeing, the transformation rate can be guaranteed and the degree of damage of the material can be reduced; During the process, the appropriate time for dipping is 15 minutes; the concentration of Agrobacterium in the dipping solution is OD ₆₀₀ =0.4~0.6, and the transformation efficiency is higher. In a word, the present invention, on the basis of exploring and setting up the optimum receptor material of Phalaenopsis and optimum tissue culture conditions, is the receptor material with the propagation culture tissue of the protocorm of Phalaenopsis, has carried out actual agrobacterium dipping test, successfully will The exogenous gene has been integrated into the Phalaenopsis genome, which provides a new way and direction for the cultivation of new varieties of Phalaenopsis, so it has good promotion and application value.

Claims (5)

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：

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

（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.