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Apr 2019
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Agrobacterium-mediated Transformation of Japonica Rice Using Mature Embryos and Regenerated Transgenic Plants
农杆菌介导粳稻成熟胚和再生转基因植株的转化    

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Abstract

Identification of novel genes and their functions in rice is a critical step to improve economic traits. Agrobacterium tumefaciens-mediated transformation is a proven method in many laboratories and widely adopted for genetic engineering in rice. However, the efficiency of gene transfer by Agrobacterium in rice is low, particularly among japonica and indica varieties. In this protocol, we elucidate a rapid and highly efficient protocol to transform and regenerate transgenic rice plants through important key features of Agrobacterium transformation and standard regeneration media, especially enhancing culture conditions, timing, and growth hormones. With this protocol, transformed plantlets from the embryogenetic callus of the japonica cultivar ‘Taichung 65’ may be obtained within 90 days. This protocol may be used with other japonica rice varieties.

Keywords: Electroporation (电穿孔), Novel genes (新基因), Callus induction (愈伤组织诱导), Binary vector (二进制向量), Oryza Sativa L. (栽培稻)

Background

Genetic transformation and expression of recombinant proteins in plant cells are major factors for plant genetic engineering. This is also a powerful tool for discovering novel genes and exploring genetically controlled traits. Agrobacterium tumefaciens-mediated transformation has been known for its unique ability to transfer a DNA segment from a specialized plasmid into a host plant cell (Gelvin, 2010). This feature allows efficient insertion of stable, unrearranged, single-copy DNA into plant genomes, which may lead to more stable expression than multiple gene copies or scrambled inserts (Iglesias et al., 1997). Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, efforts were made to improve the tool in plant transformation. In most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of host or bacterial genes. The first effective method for Agrobacterium-mediated transformation of japonica rice is now a common protocol in many laboratories (Chan et al., 1993; Hiei et al., 1994; Park et al., 1996). Many functional analyses, for example, PCR, GUS assay, and southern blot analysis, have confirmed the integration of foreign genes into transgenic rice plants obtained by Agrobacterium-mediated transformation (Dai et al., 2001); moreover, Mendelian inheritance of the transgenes was also reported (Pawlowski and Somers, 1996; Hiei et al., 1997).


The transfer of T-DNA and its integration into the rice genome is influenced by numerous factors, such as genotype, explants, Agrobacterium strains, plasmid vectors, addition of vir-gene inducing synthetic phenolic compounds, selection marker, and various conditions of tissue culture and regeneration. In tissue culture systems, selection of actively growing regenerable calli is a principal factor for efficient plant transformation. Likewise, optimization of culture conditions for co-cultivation of rice calli with Agrobacterium (Ozawa and Takaiwa, 2010) and suppression of Agrobacterium overgrowth may be applied to improve the transformation efficiency. Scientists have attempted to improve the plasmid constructions and rice transformation by Agrobacterium-mediated transformation to investigate the function of seed storage protein gene factors as well as cloning genomic candidate regions of more than 10 kb, which may have low restriction enzymes sites required for cloning. In particular, previous limitations of efficient promoter expression were overcome (Gupta et al., 2001; Furtado et al., 2008), especially for endosperm-specific expression (Zhou et al., 2013).


Several transformation methods have been established by many laboratories using mature japonica and indica seeds. Taichung 65 is a japonica rice variety, selected from a cross between the two Japanese varieties, Shinriki and Kameji, in 1923 (Iso, 1957). Taichung 65 is considered an important cultivar for studying rice genetics and breeding. Several point mutations were induced to the genetic stock of Taichung 65 using the methylnitrosourea (MNU) mutagen and have been maintained at the institute of plant genetic resources of Kyushu university. Taichung 65 has been extensively used as a model cultivar for rice biology, breeding research, and genomic studies initiated in many laboratories. However, transformation efficiency is still low in most indica and many japonica varieties due to the difficult regeneration; consequently, the procedure to obtain transgenic plants takes an average of 5 months (Nishimura et al., 2006). Thus, additional improvements in the transformation potentiality are still possible.


Here, we describe a protocol for Agrobacterium-mediated transformation in rice using mature embryos. This protocol is based on the method described by Toki (Toki, 1997), with several modifications; in particular, meropenem is used for plant regeneration (Ogawa and Mii, 2007). Meropenem exhibits the highest antibacterial activity and also achieves a high shoot formation rate in rice and tomato (Ogawa and Mii, 2007). Using this protocol, we succeeded to produce hundreds of independent transgenic lines and transformed several novel genes over the past decade, including Endosperm Storage Protein (ESP1), encoding a eukaryotic chain release factor1 (eRF1) (Elakhdar et al., 2019); Esp2, encoding the protein disulfate isomerase 1-1 (PDI 1-1) (Satoh-Cruz et al., 2010); Glup3, encoding a vacuole processing enzyme (VPE) (Kumamaru et al., 2010); Glup4, encoding the small GTPase Rab5a (Fukuda et al., 2011); and Glup6, encoding the guanine nucleotide exchange factor (GEF) (Fukuda et al., 2013).


In summary, this protocol is a step-by-step approach to obtain stably transformed plants from mature rice embryos by optimizing several stages of Agrobacterium transformation and standard regeneration medium components, including culture conditions, timing, and growth hormones. In this method, we use a 13.8 kb construct carrying the hygromycin phosphotransferase gene (hpt), a firefly luciferase reporter gene (LUC), spectinomycin (Sp), an attR1 site, the chloramphenicol resistance gene (CmR), the ccdB gene, the attR2 site, and the ZmUbi1 promoter (Figure 1 and Figure 2).



Figure 1. Flow chart for Agrobacterium-mediated transformation and regeneration of rice calli using mature embryogenic seed (cv; Taichung 65). (a) Seed embryos. (b) Calli pieces with granular structure, yellowish-white. (c) Growth of transformed Agrobacterium harboring the pSMAH638OX/Ubilp binary vector. (d) Co-cultivation of infected calli. (e) De-colonization of Agrobacterium tumefaciens. (f) Shoot regeneration. (g) Rooting in regenerated shoots. (h) Hardening of rooted plants. (i) Transplanted hardened plants.



Figure 2. Schematic showing embryo calli infection by Agrobacterium. (a) Growth of Agrobacterium-mediated harboring pSMAH638OX/Ubilp vector on LB medium. (b) Mix a small portion of the positive colonies. (c) Collect healthy calli for infection. (d) Calli infection. (e) Remove excess Agrobacterium suspension.

Materials and Reagents

Plasticware and Glass

  1. Petri dish, 90 × 20 mm

  2. Petri dish, 90 × 15 mm

  3. 24-well multi-well plates

  4. Sterile plastic falcon tubes, 50 ml

  5. Filter paper, 70 mm

  6. Parafilm tape (Parafilm Pechiney PM996; 125' L X 4"; www.parafilm.com)

  7. Sterile syringe filters 0.22 μm

  8. Sterile syringe, 30 ml

  9. Stainless-steel sieve

  10. Microspatula

  11. Plant culture pots for rooting (6.5 × 6.5 cm)

  12. Plant soil pots (23 × 23 × 19 cm)


Biological materials

  1. Mature dry seeds of japonica cv. Taichung 65

    The target cultivar of the current transformation method.

  2. Binary vector containing gene of interest

    In the following protocol, the binary vector pSMAH638OX/Ubilp (13.8 Kb) construct was derived by the Gateway recombination cloning technology (InvitrogenTM, Thermo Fisher Scientific, Inc.), harboring firefly luciferase (LUC) reporter gene, the hygromycin phosphotransferase (hpt) selectable gene marker, spectinomycin (Sp), an attR1 site, the chloramphenicol resistance gene (CmR), the ccdB gene, and the attR2 site. We used the ZmUbi1 promoter for LUC expression (Hakata et al., 2010; Elakhdar et al., 2019)

  3. Agrobacterium tumefaciens strains EHA101 or EH105 (Hood et al., 1993).


Reagents and chemicals

  1. Ethanol CH3CH2OH (Sigma-Aldrich, catalog number: N0640), 70% (v/v) in distilled water, store at room temperature (RT)

  2. Sodium hypochlorite 10% NaClO (Sigma-Aldrich, catalog number: 105614), store at RT

  3. Sodium chloride, NaCl (Sigma-Aldrich, catalog number: 7647-14-5), store at RT

  4. Sodium hydroxide, NaOH (Sigma-Aldrich, catalog number: 1310-73-2), store at RT

  5. Tween® 20 EMD, (CALBIOCHEM, catalog number: 655205), store at RT

  6. Sucrose C12H22O11 (Wako, catalog number: 196-00015), store at RT

  7. D-Glucose C6H12O6 (Sigma-Aldrich, catalog number: 07-0680-5), store at RT

  8. D-Sorbitol C16H14O6 (Sigma-Aldrich, catalog number: 28-4770-5), store at RT

  9. Casamino acid (Sigma-Alorich, catalog number: 65072-00-6), store at RT in dry condition

  10. L-Proline (PEPTIOE, catalog number: 2718), store at RT

  11. CHU (N6) Basal salt mixture (Sigma-Aldrich, catalog number: C1416-10L), store at 2-8°C

  12. Murashige & Skoog Basal Medium (Sigma-Aldrich, catalog number: M552450L), store at 2-8°C

  13. CHU N6-Vitamin solution X1000 (Phyto, catalog number: PHT:C149), store at 2-6°C

  14. LB Broth, Miller (Luria-Bertani; BD Difco, catalog number: 244620), store at RT

  15. Bacto Peptone (BD Difco, catalog number: 211677), store at RT

  16. Bacteriological agar (Bacto Agar; BD Difco, catalog number: 214010), store at RT

  17. 2,4-Dichlorophenoxyacetic acid (Wako, catalog number: 040-18532), store at RT

  18. 1-Naphthaleneacetic acid NAA C10H7CH2COOH (Wako, catalog number: 86-87-3), store at RT

  19. Kinetin C10H9N5O (Wako, catalog number: 110-00331), store at 2-10°C

  20. Gelrite (0.4%) (Wako, catalog number: 067-04035), store at RT

  21. Carbenicillin sodium salt (Sigma-Aldrich, catalog number: C1389-5G), store at 2-8°C

  22. Hygromycin B (Wako, catalog number: 085-06153), store at -20°C

  23. Acetosyringone (Sigma-Aldrich, catalog number: D134406-5G), store at -20°C

  24. Meropenem (Dainippon Sumitomo Pharma, Osaka, Japan), store at -20°C

  25. Dimethyl sulfoxide DMSO (CH3)2SO (Wako, catalog number: 046-21981), store at RT

  26. KOD-FX (TOYOBO, catalog number: KFX-101), store at -20°C

  27. Sterilizing solution (see Recipes)

    Solution A

    Solution B

  28. Antibiotics (see Recipes)

    Carbenicillin (500 mg/ml)

    Hygromycin (50 mg/ml)

    Kanamycin (50 mg/ml)

    Acetosyringone (100 mg/ml)

    Acetosyringone (10 mg/ml)

    Meropenem (12.5 mg/ml)

    2,4-D (0.2 mg/ml)

    NAA (0.2 mg/ml)

    Kinetin (1 mg/ml)

  29. Cultivation medium (see Recipes; Table 1)

    LB sold medium

    LB liquid medium

    YEP medium

Notes:

  1. All liquids and equipment containing Agrobacterium must be appropriately sterilized.

  2. Essential elements that may influence the effectiveness of transformation are reagents and chemicals. Therefore, we strongly suggest following the methodology, particularly when starting a new protocol. The usage and storage conditions of chemicals are different; therefore, please make sure to follow the recommendations of each company, with particular attention to hazard information on the substances.

Equipment

  1. -86°C Ultra-Low temperature freezer (SANYO, catalog number: MDF-U538)

  2. -30°C Ultra-Low temperature freezer (SANYO, catalog number: MDF-792AT)

  3. MicroPluserTM (Bio-Rad, Hercules)

  4. Spectrophotometer (Thermo ScientificTM, model: NanoDrop 2000)

  5. Incubator/ shaker 28°C for Agrobacterium

  6. Plant growth chamber (SANYO)

  7. Shaker for seed sterilization

  8. Laminar flow hood (SANYO)

  9. Autoclave

  10. pH meter

  11. Spinbar® magnetic stir bar (Sigma-Aldrich, catalog number: Z127116)

  12. Polycarbonate vacuum desiccator (SANSYO, catalog number: SPD-WVGT240)

  13. Thermal cycler (Bio-Rad, model: Tetrad 2 Thermal Cycler (4 × 96-well)

  14. Locking system greenhouse

  15. Rice seedling raising soil (JA Kumiai King Soil; Agr. Japan Co., Ltd.)

Procedure

  1. Agrobacterium culture and transformation

    1. Transformation of competent Agrobacterium cells (EHA101 or EHA105) with binary vector pSMAH638OX/Ubilp (13.8 Kb) constructs (Figure 3).



      Figure 3. Binary vector pSMAH638OX/Ubilp (13.8 Kb) construct carrying the hygromycin phosphotransferase gene (hpt), firefly luciferase reporter gene (luc), spectinomycin (Sp), an attR1 site, the chloramphenicol resistance gene (CmR), the ccdB gene, the attR2 site, and the ZmUbi1 promoter.


      1. Thaw 50 μl Agrobacterium competent cells (EHA101 or EH105) on ice (Figure 4A).

      2. In 1.5 ml tube, mix 2 µl plasmid DNA (100 picogram/µl) with competent cells.

      3. Incubate the cell mixture on ice for 10 min.

      4. Perform transformation using the MicroPluserTM (Bio-Rad) electroporation method with the “Agr” mode (Figure 4B).

      5. After 10 min, move the cell mixture to the cuvette tube and then the cuvette to chamber slide.

      6. Press the Pluse button until a tone sounds indicating that the pulse has been given.

      7. Immediately add 250 µl YEP medium to cells.

      8. Transfer cells to 25 ml tube containing 3.75 ml YEP medium (Figure 4C).

      9. Warp the tube with foil and incubate for 2 h in the dark at 28°C.

      10. Centrifuge the mixture at 1,500-3,000 × g for 5 min at RT.

      11. Discard around 3.5 ml of the supernatant.

      12. Mix the remaining cells gently.

      13. Plate 20-100 µl of cells on LB agar amended with 50 mg/L hygromycin and 50 mg/L kanamycin.

      14. Make sure that cells are completely dry, then wrap the Petri dish with parafilm and incubate 28°C for 2-3 days.

      15. Pick four single colonies and verify the transformation by PCR (Figure 4D).

      16. Streak out the positive colonies of Agrobacterium strain EHA101 that harbor the gene of interest in a 1 × 4 grid pattern on a single LB agar containing 50 mg/L hygromycin and 50 mg/L kanamycin (Figure 4E).

      17. Incubate the culture for 2-3 days at 28°C.



        Figure 4. Procedure of Agrobacterium-mediated transformation. (A) Agrobacterium competent cells. (B) MicroPluserTM (Bio-Rad). (C) YEP medium. (D-F) Positive transformed Agrobacterium verified by PCR. (G) Mature embryos of Taichung 65. (H) Callus induction after 23 days on N6D medium. (I) Callus infection. (J) Drying calli on sterilized filter paper. (K) Co-cultivated calli on 2N6AS solid medium.


  2. Preparing mature embryos

    1. Seed surface disinfection

      1. Dehusk healthy mature seeds, then hand-cut with a razor. Save the embryo seed halves and discard the endosperm seed halves (Figure 4G).

      2. Disinfect the surface of embryogenic seed parts after soaking and stirring once in 70% ethanol for 10 s in a 50 ml tube (up to 150 seeds).

      3. Rinse the embryo seeds halves in distilled water for 30 s.

      4. Shake the tube vigorously (45 rpm) for 15-20 min in bleach Solution A and then rinse 3-5 times using distilled water.

      5. Wash the seed parts with Solution B under vigorous shaking for 15-20 min, followed by five rinses in distilled water up to 30 s each time.

      6. Place the sterilized embryo seed halves on sterile filter paper over Petri dish (see Note 3).

    2. Callus induction (3-4 weeks)

      1. Partially submerge 8-12 disinfected embryo seed halves per dish within N6D callus induction medium.

      2. Seal the dishes with surgical tape, then wrap every five dishes with aluminum foil and incubate at 28°C in the dark for 3-4 weeks (see Note 4).

    3. Preparation of calli, Agrobacterium suspension, and infection (48 to 72 h)

      1. Mix a small portion of four colonies of Agrobacterium strain EH101 on a micro-spatula and resuspend in 40 ml 2N6D-AS liquid medium containing 16 μl acetosyringone (100 mg/ml stock solution) and mix gently (Agrobacterium colonies must be analyzed by PCR using specific primers to verify the integration of the construct with the bacterial gDNA) (Figure 4F).

      2. Discard brown calli and seed coleoptile from the N6D medium (at the beginning of week 4). Collect the active embryogenic calli (yellowish-white, relatively dry, 1-3 mm in diameter (Nishimura et al., 2006) and globular (Hiei and Komari, 2008) into a 50-ml tube. At this stage, the calli can be used directly for Agrobacterium infection (Figure 4H).

      3. Pour the bacterial suspension prepared in Step B3a into the 50-ml tube.

      4. Soak the calli in the Agrobacterium suspension and gently mix for 90 s (Figure 4I).

      5. Decant the bacterial suspension into a sterilized stainless-steel sieve (12 mesh) and hold in 500-ml sterile beaker.

      6. Blot the sieve on sterilized filter paper placed in a 90 mm × 20 mm Petri dish to remove excess Agrobacterium suspension (Figure 4J).

      7. Place a sterilized filter paper on solid 2N6D-AS medium. Saturate the filter paper by dripping 0.5 ml 2N6D-AS liquid medium and then co-cultivate an appropriate amount of calli (20-25 calli) on the filter paper (Figure 4K).

      8. Seal plates with parafilm tape and wrap with aluminum foil and incubate at 25°C in the dark for 3 days (see Note 5).

    4. Wash calli and select transformed cells (3-4 weeks)

      1. Collect infected calli in a 50 ml sterile tube and wash with sterile water 3-5 times to remove Agrobacterium (until water is clear). Gently shake the tube and pour out the water.

      2. Rinse calli with 40-ml sterile water containing 40 μl carbenicillin 500 mg/ml stock solution to kill Agrobacterium cells.

      3. Pour the water suspension and blot the calli on sterile filter paper held over Petri dish. Dry the calli well (see Note 6).

      4. Transfer calli onto N6D-HC selection medium; 15 to 20 calli can be placed on a single plate.

      5. Seal the plate with surgical tape, and incubate the calli at 28°C in the dark [according to the Toki method; 33°C (light for 10 h)/30°C (dark for 14 h); Saika and Toki (2010)].

      6. Check the culture regularly for contamination. In case of contamination, transfer uncontaminated calli to fresh medium immediately.

      7. Subculture the yellowish-white calli to new fresh N2D-HC medium after 10 to 12 days, seal plates with surgical tape, and incubate at 28°C in the dark. Resistant calli (yellowish-white) can be observed after 20-25 days (Figure 5A).



        Figure 5. Calli selection and regeneration. (A) Hygromycin-resistant calli on N6D-HC selection medium. Arrows indicate resistant calli after 25 days from the infection. (B) Proliferated calli with greening spots. (C) Subculture green pots on the same fresh medium. (D) Rooting. (E) Regenerated plantlets on MS HF medium. (F) Transgenic plants transplanted to soil in the growth chamber. (G-E) Transgenic plants transferred to locking system greenhouse until harvesting. Scale bars: 5 cm in (E), 30 cm in (F), 30 cm in (G), 30 cm in (H).


    5. Regeneration of rice transgenic plants (3-4 weeks)

      1. Transfer resistant calli to REIII regeneration medium containing suitable antibiotics. Using sterilized forceps, subculture a single callus to each well of 24-multi-well plate. Seal the plate with surgical tape and incubate at 30°C under continuous illumination (Figure 5B). Re-transfer transgenic calli to fresh REIII medium every 10 days with the same conditions. The transformed shoot begins to differentiate after 3-4 weeks (Figure 5C).

      2. Transfer 3-4 cm shoots to plantlet pots containing HF medium with appropriate antibiotics at 28°C under 10 h days light (Figure 5D). Several days later, remove the surgical tape from the top of the pots and then remove the pot cover (Figure 5E) (see Note 7).

      3. Transplant transformant rice plants to 6.5 cm pots containing rice seedling raising soil, with one plantlet per pot, in a growth chamber at 28°C (Figure 5F).

      4. When the transformants are 15 cm tall, transplant them to 23-cm pots containing paddy field soil, with one plantlet per pot, in a greenhouse at 28°C until harvest (Figure 5G-5H).

      5. Conduct PCR with DNA from the transformed rice plants to confirm T-DNA integration into rice plants (Figure 6).



        Figure 6. Agarose gel profile for verifying the integration of T-DNA insertion into the plant genome. A 509bp LUC gene amplicon size. M, marker; +, construct DNA; -, Taichung 65 gDNA.

Notes

  1. Bacto agar dissolves well with autoclaving. Add the hygromycin after cooling to 50-45°C because hot culture media will degrade the antibiotic. For A. tumefaciens strain EHA105, add 0.2 ml of 50 mg/ml hygromycin per 200 ml LB medium. For strain EHA 101, add 0.2 ml of 50 mg/ml hygromycin and 0.2 ml 500 mg/ml carbenicillin per 200 ml LB medium.

  2. Meropenem (25 mg/L) in the selection medium completely suppresses the overgrowth of Agrobacterium, which results in high transformation efficiency (Ogawa and Mii, 2007).

  3. Seeds one year after harvesting – rather than seeds taken immediately after harvesting – are preferable for callus induction. Healthy mature seeds are critical as starting material for transformation and high-frequency callus formation. Remove lemma and palea (outer coat) from 40-50 seeds (enough for one transformation). Remove starchy endosperm from the seed to reduce contamination.

  4. Callus selection is a key point for efficient transformation. We often check the culture and remove any contaminated seed parts immediately with a sterilized spoon. In such cases, uncontaminated seed parts are transferred to new plates.

  5. Select four single Agrobacterium colonies. Perform PCR using marker-specific primers for the reporter gene to confirm its incorporation into the plant genome. Bacterial suspension density should be OD600 = 0.2 (Ozawa, 2012) or lower (Nishimura et al., 2006), and OD600 = 0.05-0.1 is recommended. This process is important to prevent excess Agrobacterium growth, which can result in damage to calli. Approximately 150 calli can be placed on a single plate (Hiei and Komari, 2008). Seal plates with Parafilm to prevent evaporation.

  6. Wash calli after co-cultivation. Resistant calli contain small nodular embryos on the surface. A critical step is to remove Agrobacterium from calli by washing without causing much physical damage to the nodular embryo cells, as this would prevent the regeneration of the transgene. Prior the co-cultivation, it is very important to ensure that the selectable-antibiotic is removed appropriately so that it does not affect calli growth. Place the calli on the filter paper to allow the extra water to evaporate.

  7. Hardening: The hardening of transgenic plantlets is a crucial step prior to transplanting them into soil. The hardening can be done slowly from low to high light intensity conditions as well as from high to low humidity. The agar medium can be gently removed from the root by rinsing with water.

Recipes

  1. Sterilizing solution

    1. Solution A

      Add 50 ml sodium hypochlorite 10% into 50 ml dH2O

      Store at RT

    2. Solution B

      Add 50 ml Sodium hypochlorite, 50 ml dH2O, and 50 μl Tween® 20

      Store at RT


  2. Antibiotics

    1. Carbenicillin (500 mg/ml)

      Dissolve 5 g carbenicillin powder completely in 10 ml dH2O.

      Sterilize the solution through 0.22 μm syringe filter.

      Store at -20°C in 1 ml aliquots.

    2. Hygromycin (50 mg/ml)

      Dissolve 500 mg hygromycin B in 10 ml dH2O.

      Sterilize the solution through 0.22-μm syringe filter.

      Store at -20°C in 1.5 ml portions.

    3. Kanamycin (50 mg/ml)

      Dissolve 500 mg Kanamycin to 10 ml.

      Sterilize the solution through 0.22-μm syringe filter.

      Store at -20°C in 1.5 ml portions.

    4. Acetosyringone (100 mg/ml)

      Dissolve 1 g acetosyringon to 1 ml DMSO.

      Dilute with 10 ml dH2O.

      Sterilize the solution through 0.22-μm syringe filter.

      Store at -20°C in 1.5-2 ml aliquots.

    5. Acetosyringone (10 mg/ml)

      Dissolve 100 mg acetosyringon to 1 ml DMSO.

      Dilute with 10 ml dH2O.

      Sterilize the solution through 0.22-μm syringe filter.

      Store at -20°C in 1.5-2 ml aliquots.

    6. Meropenem (12.5 mg/ml)

      Dissolved 12.5 g meropenem in 10 ml dH2O.

      Sterilize the solution through 0.22-μm syringe filter.

      Aliquot in 2 ml portions and store at -20°C.

    7. 2,4-D (0.2 mg/ml)

      Dissolve 20 mg of 2,4-D powder in 0.5 ml DMSO.

      Adjust to 100 ml dH2O.

      Store at -20°C.

    8. NAA (0.2 mg/ml)

      Dissolve 20 mg of NAA completely in 1 ml 0.1 N sodium hydroxide solution.

      Adjust volume to 100 ml dH2O.

      Store at -20°C.

    9. Kinetin (1 mg/ml)

      Dissolve 10 mg kinetin in 0.2 ml 1 M sodium hydroxide.

      Adjust the volume to 10 ml with dH2O.

      Store at -20°C.


  3. Prepare cultivation medium (Tanle 1)

    Culture nutritional components: All culture media are prepared fresh in 1,000 ml dH2O, autoclaved at 120°C for 20 min and cooled to 50-40°C; finally, it is asepticaly distributed to approximately 12 (90 mm × 20 mm) Petri dishes in a laminar flow hood. The poured medium plates containing antibiotics can be stored at 4°C for up to one month.


    Table 1. List of callus induction and regeneration medium components

    Medium
    Components N6D 2N6-AS 2N6D-AS solution N6D-HC REIII HF
    Sucrose
    Glucose
    30 g
    -
    30 g
    10 g
    30 g
    10 g
    30 g
    -
    30 g
    -
    30 g
    -
    Sorbitol - - - 30 g
    Casamino acids 0.30 g 0.30 g 0.30 g 0.30 g 1g -
    Proline 2.878 g - - 2.878 g - -
    Murashige and Skoog - - - - 4.4 g 4.4 g
    CHU (N6) 3.981 g 3.981g 3.981g 3.981 g - -
    CHU N6- Vitamin solution 1 ml 1 ml 1 ml 1 ml - -
    2,4-D (0.2 mg/ml) 10 ml 10 ml 10 ml 10 ml -
    Acetosyringon (10 mg/ml) - 1 ml 1 ml - - -
    NAA (x 5000) - - - 10 μl
    Kinetin (x 500) - - - 1 ml
    Gelrite (0.4%) 4 g 4 g - 4 g 4 g 4 g
    Carbenicillin 500 (mg/ml) - - - 1 ml -
    Hygromycin 500 (mg/ml) - - - 1 ml 1 ml
    Meropenem 12.5 (mg/ml) - - - 2 ml

    1. LB solid medium (200 ml)

      LB BROTH 5 g

      Bacto Agar 1.5 g

      Autoclave (120°C for 20 min)

    2. LB liquid medium (200 ml)

      LB BROTH 5 g

      Autoclave (120°C for 20 min)

    3. YEP medium (200 ml)

      Yeast Extract 2 g

      Bacto Peptone 2 g

      NaCl 1 g

      Autoclave (120°C for 20 min)

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (Grant number: 19P19394). We are grateful to professor Calvin O. Qualset, University of California Davis, for reading the manuscript and providing comments and suggestions. This protocol is derived from previous publications (Satoh-Cruz et al., 2010; Fukuda et al., 2011 and 2013; Elakhdar et al., 2019).

Competing interests

The authors declare that they have no conflict of interests.

References

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简介

[摘要]鉴定水稻新基因及其功能是改善经济性状的关键步骤。根癌农杆菌介导的转化在许多实验室中是一种经过验证的方法,并广泛用于水稻的基因工程。然而,通过基因转移效率的土壤杆菌在水稻是低的,特别是其中粳稻和我ndica品种。在该协议中,我们阐明了一种快速高效的协议,通过农杆菌的重要关键特征转化和再生转基因水稻植物转化和标准再生培养基,特别是增强培养条件、时间和生长激素。使用该方案,可以在 90 天内获得来自粳稻品种“台中 65”的胚胎发生愈伤组织的转化苗。Ť他的协议可以用于与邻疗法粳稻水稻品种。


[背景]植物细胞中重组蛋白的遗传转化和表达是植物基因工程的主要因素。这是也为发现新基因,探索基因控制性状的有力工具。根癌农杆菌介导的转化因其将 DNA 片段从特化质粒转移到宿主植物细胞中的独特能力而闻名(Gelvin , 20 10 )。此功能允许稳定,未重排的单拷贝DNA的高效插入植物基因组中,这可乐一个d更stabl È明示离子比多基因拷贝或加扰的刀片(Iglesias的等人,1997) 。自从 1980 年代初期使用农杆菌产生转基因植物的初步报告以来,人们一直在努力改进植物转化中的工具。在大多数情况下,主要改进涉及植物组织培养转化和再生条件的改变,而不是操纵宿主或细菌基因。农杆菌介导的粳稻转化的第一种有效方法现在是许多实验室的通用方案(Chan等,1993;Hiei等,1994;Park等,1996)。许多功能分析,例如,PCR,GUS测定法,和Southern印迹分析,已证实外源基因整合到由获得的转基因稻植物的农杆菌介导的转化(戴等人,2001); 此外,转基因的孟德尔遗传中也报道(Pawlowski的和Somers的,1996;比睿等人,1997) 。
T-DNA和一体化的转移到水稻基因组是由许多因素,如基因型,外植体,影响土壤杆菌菌株,质粒载体,另外的vir-基因诱导合成的酚类化合物,选择标记,和组织培养的各种条件和再生。在组织培养系统中,选择活跃生长的可再生愈伤组织是有效转化植物的主要因素。同样地,对水稻愈伤组织的有共培养的培养条件优化农杆菌(小泽和高岩,2010)和抑制的离子农杆菌过度生长可被应用,以提高转化效率。科学家们试图以提高质粒构建和水稻遗传转化的农杆菌介导的转化来调查的种子贮藏蛋白基因的因素功能以及克隆的基因组候选区域的超过10 kb的,其可以具有用于克隆所需的低的限制性酶位点。特别地,高效率的启动子表达的先前限制为overcom È (古普塔等人,2001;朵等人,2008) ,特别是对于胚乳特异性表达(周等人,2013。) 。
几种转化方法已使用许多实验室建立了成熟的粳稻和我ndica种子。台中65是Ĵ aponica水稻品种,从之间的交叉选择的两个日本品种,Shinriki和Kameji ,在1923年(ISO ,1957)。台中65被认为是研究的一个重要品种ING [R冰遗传育种。几个点突变诱导的基因库的台中65使用的甲基亚(MNU)诱变和一直保持在植物遗传资源研究所九州大学。台中65是连接广泛用作水稻生物学模型品种,育种研究,并在许多实验室发起的基因组学研究。然而,由于再生困难,大多数籼稻和许多粳稻品种的转化效率仍然较低;因此,为了获得转基因植物的过程需要的平均5个月(西村等人。,200 6 )。因此,在转型潜力其他改进的仍然是可能的。
在这里,我们描述了一个协议,用于农杆菌-介导转化利用成熟胚水稻。该协议基于该方法通过描述岐(岐,1997),与若干修改小号; 在特定的,美罗培南是我们编的植物再生(小川和MII,2007) 。美罗培南展览š最高的抗菌活性,并且还实现了一个在水稻和番茄高芽形成率(小川和MII,2007) 。使用该协议,我们成功编以产生数百独立转基因系和转化的几个新基因在所述过去的十年中,包括胚乳贮藏蛋白(ESP1) ,ENCOD荷兰国际集团一个真核链释放因子1(ERF1)(Elakhdar等人。,2019); ESP2 ,ENCOD荷兰国际集团的蛋白质二硫异构酶1-1(PDI 1-1) (佐藤-克鲁兹等人,2010。); Glup3 ,ENCOD荷兰国际集团液泡加工酶(VPE) (熊丸等人,2010。); Glup4 ,ENCOD荷兰国际集团的小GTP酶RAB5A (福田等人,2011。); 和Glup6 ,ENCOD荷兰国际集团的鸟嘌呤核苷酸交换因子(GEF) (福田等人。,2013) 。
总之,该协议是通过优化农杆菌转化的几个阶段和标准再生培养基成分,包括培养条件、时间和生长激素,从成熟水稻胚胎中获得稳定转化植物的分步方法。在这种方法中,我们使用了一个 13.8 kb 的构建体,它携带潮霉素磷酸转移酶基因 (hpt) 、萤火虫荧光素酶报告基因 (LUC) 、壮观霉素 (Sp) 、attR1 位点、氯霉素抗性基因 (Cm R )、ccdB基因、 attR2位点,并且所述ZmUbi1启动子(图1和图2) 。


图 1.农杆菌介导的水稻愈伤组织转化和再生流程图见d ( cv; Taichung 65 )。(a) 种子胚胎。(b)中愈伤组织片与粒状结构,淡黄-白色。(c)中变换后的生长的农杆菌携带的pSMAH638OX / Ubilp二元载体。(d) 感染愈伤组织的共同培养。(五)非殖民化的根癌农杆菌。(f) 芽再生。(g)在再生枝条中生根。(h) 有根植物的硬化。(i) 移植的硬化植物。


图2.小号电气原理舒克胚胎愈伤组织感染农杆菌。( a )农杆菌介导的携带pSMAH638OX/Ubilp 载体在 LB 培养基上的生长。(b) 混合一小部分阳性菌落。(三)收集健康Ÿ愈伤组织感染。(d)愈伤组织感染。(e) 去除多余的农杆菌悬浮液。

关键字:电穿孔, 新基因, 愈伤组织诱导, 二进制向量, 栽培稻

材料和试剂
 
塑料制品和玻璃
培养皿,90 × 20 毫米
培养皿,90 × 15 毫米
24孔多孔板
无菌塑料猎鹰管,50 毫升
滤纸,70 毫米
Parafilm 胶带(Parafilm Pechiney PM996;125' LX 4";www.parafilm.com)
无菌注射器过滤器 0.22 μm
无菌注射器,30 毫升
不锈钢筛
微铲
用于生根的植物培养盆(6.5 × 6.5 cm)
植物土盆(23 × 23 × 19厘米)
 
生物材料
1.粳稻成熟干燥种子。台中65      
当前转化方法的目标栽培品种。
2.含有目的基因的二元载体      
在下面的协议中,二元载体pSMAH638OX / Ubilp(13.8 KB)构建体是派生d由Gateway重组克隆技术(Invitrogen公司TM ,赛默飞世尔科技公司),携带萤火虫萤光素酶(LUC )报告基因,所述潮霉素磷酸(hpt ) 选择基因标记、壮观霉素 (Sp)、attR1 位点、氯霉素抗性基因 (CmR)、 ccdB 基因和attR2 位点。我们使用d ZmUbi1 启动子进行 LUC 表达(Hakata等人,2010 年;Elakhdar等人,2019 年)
3.根癌农杆菌菌株 EHA101 或 EH10 5 (Hood et al ., 1993 )。      
 
试剂和化学品
乙醇 CH 3 CH 2 OH(Sigma-Aldrich,目录号:N0640),70%(vl/v)在蒸馏水中,在室温下储存(RT )
次氯酸钠的10%NAC升O(Sigma-Aldrich公司,目录号:105614),保存于室温
氯化钠,NaCl(Sigma-Aldrich,目录号:7647-14-5),储存于室温
氢氧化钠,NaOH(Sigma-Aldrich,目录号:1310-73-2),储存在室温下
Tween ® 20 EMD,(CALBIOCHEM,目录号:655205),在 RT 商店存储
蔗糖 C 12 H 22 O 11 (Wako,目录号:196-00015),储存在 RT
D-葡萄糖 C 6 H 12 O 6 (Sigma-Aldrich,目录号:07-0680-5),储存在 RT
D-山梨糖醇 C 16 H 14 O 6 (Sigma-Aldrich,目录号:28-4770-5),储存在 RT
酪蛋白氨基酸(Sigma-Alorich,目录号:65072-00-6),在室温干燥条件下储存
L-脯氨酸(PEPTIOE,目录号:2718),储存在 RT
CHU(N6)基础盐混合物(Sigma-Aldrich,目录号:C1416-10L),储存在2-8 °C
Murashige & Skoog 基础培养基(Sigma-Aldrich,目录号:M552450L),储存于 2-8 °C
CHU N6-Vitamin solution X1000(Phyto,目录号:PHT:C149),储存在2-6 °C
LB Broth,Miller(Luria-Bertani;BD Difco,目录号:244620),在 RT 商店
Bacto Peptone(BD Difco,目录号:211677),储存在 RT
细菌琼脂(Bacto Agar;BD Difco,目录号:214010),储存在 RT
2,4-二氯苯氧乙酸(Wako,目录号:040-18532),储存于室温
1-萘乙酸NAA C 10 H 7 CH 2 COOH(Wako,目录号:86-87-3),储存于室温
激动素 C 10 H 9 N 5 O(Wako,目录号:110-00331),储存在 2-10 °C
Gelrite(0.4%)(Wako,目录号:067-04035),储存在RT
羧苄青霉素钠盐(Sigma-Aldrich,目录号:C1389-5G),储存于2-8 °C
潮霉素 B(Wako,目录号:085-06153),储存在 -20 °C
乙酰丁香酮(Sigma-Aldrich,目录号:D134406-5G),储存在-20 °C
美罗培南(Dainippon Sumitomo Pharma,大阪,日本),- 20 °C 保存
二甲基亚砜DMSO(CH 3 )2 SO(Wako,目录号:046-21981),室温储存
KOD-FX(TOYOBO,目录号:KFX-101),储存在 -20 °C
消毒液(见配方)
方案一
方案B
抗生素(见食谱)
羧苄青霉素(500 毫克/毫升)
潮霉素(50 毫克/毫升)
卡那霉素(50 毫克/毫升)
乙酰丁香酮 (100 毫克/毫升)
乙酰丁香酮 (10 毫克/毫升)
美罗培南 (12.5 毫克/毫升)
2,4-滴 (0.2 毫克/毫升)
NAA (0.2 毫克/毫升)
激动素 (1 毫克/毫升)
培养基(见配方;表1 )
LB 出售的培养基
LB液体培养基
YEP培养基
笔记:
所有含有农杆菌的液体和设备都必须经过适当消毒。
可能影响转化效率的基本元素是试剂和化学品。因此,我们强烈建议遵循该方法,尤其是在开始新协议时。的使用和储存条件下的C hemicals是不同的; 因此,请务必遵循的建议,第各家公司,尤其要注意对在物质危害信息。
 
设备
 
- 86 °C超低温冰箱(SANYO,目录号:MDF-U538)
-30 °C超低温冰箱(SANYO,目录号:MDF-792AT)
MicroPluser TM (Bio-Rad, Hercules)
分光光度计(Thermo Scientific TM ,型号:NanoDrop 2000)
农杆菌培养箱/摇床 28 °C
植物生长室(SANYO)
种子杀菌摇床
层流罩(SANYO)
高压釜
pH计
Spinbar ®磁力搅拌棒(Sigma-Aldrich,目录号:Z127116)
聚碳酸酯真空干燥器(SANSYO,目录号:SPD-WVGT240)
热循环仪(Bio-Rad,型号:Tetrad 2 热循环仪(4 × 96 孔)
锁定系统温室
水稻育苗土(JA王Kumiai的土壤。AGR日本有限公司。)
 
程序
 
农杆菌培养和转化
用二元载体 pSMAH638OX/Ubilp (13.8 Kb) 构建体转化感受态农杆菌细胞(EHA101 或 EHA105)(图 3)。
 
 
图3.乙inary矢量pSMAH638OX / Ubilp(13.8 KB)构建携带荷兰国际集团的潮霉素磷酸转移酶基因(HPT),萤火虫荧光素酶报告基因(LUC),大观霉素(SP),一个attR1位点,氯霉素抗性基因(CMR),则ccdB基因attR2位,并在ZmUbi1启动子。
 
      在冰上解冻 50 μl农杆菌感受态细胞(EHA101 或 EH105)(图 4A)。
      在 1.5 ml 试管中,将 2 µl 质粒 DNA(100 微微克/µl)与感受态细胞混合。
将细胞混合物在冰上孵育 10分钟。
      使用执行变换的MicroPluser TM (Bio-Rad公司)电穿孔法用“AGR”模式(图4B) 。
      10 分钟后,将细胞混合物移至比色皿管,然后将比色皿移至腔室载玻片。
  按下 Pluse 按钮,直到发出提示音,表示已发出脉冲。
 立即向细胞中加入 250 µl YEP 培养基。
 将细胞转移到含有 3.75 ml YEP 培养基的 25 ml 管中(图 4C)。
   翘曲管与箔和孵育2 h中的黑暗中28℃。
   离心该混合物在1 ,500 - 3 ,000 ×克在5分钟RT 。
 丢弃大约 3.5 ml 的上清液。
   轻轻混合剩余的细胞。
    用50 mg/L 潮霉素和 50 mg/L 卡那霉素修正的 LB 琼脂平板上 20-100 µl 细胞。
      确保该细胞被完全干燥,然后包裹在培养皿用封口膜,并培育28℃下进行2-3天。
      选择四个单菌落并通过 PCR 验证转化(图 4D)。
      条纹出的阳性菌落农杆菌窝藏的兴趣以1基因菌株EHA101 ×在单个LB 4网格图案琼脂含有50mg / L潮霉素和50mg / L卡那霉素(图4E) 。
      在 28°C 下孵育培养物 2-3 天。
 
 
图 4.农杆菌介导的转化过程。( A )农杆菌感受态细胞。( B ) MicroPluser TM (Bio-Rad)。( C ) YEP 培养基。( DF )通过 PCR 验证的阳性转化农杆菌。( G )台中65成熟胚。( H )在 N6D 培养基上培养 23 天后的愈伤组织诱导。(一)愈伤组织感染。( J )在无菌滤纸上干燥愈伤组织。( K ) C在 2N6AS 固体培养基上培养的愈伤组织。
 
 
准备成熟胚胎
1.种子表面消毒离子      
去壳健康成熟的种子,然后用剃刀手工切割。保存胚胎种子HAL VES和丢弃的胚乳种子HAL VES (图4G)。
在 50 毫升管(最多 150 个种子)中在 70% 乙醇中浸泡和搅拌一次10 秒后,对胚发生种子部分的表面进行消毒。
冲洗胚胎种子HAL VES在蒸馏水中30秒。
摇晃剧烈管(45转),用于漂白15-20分钟小号olution A,然后用蒸馏水冲洗3-5次。
洗种子与零件小号下olution乙剧烈SHAK ING 15-20分钟,接着在蒸馏水中五个漂洗至30每个时间s。
放置灭菌胚胎种子半部在无菌滤纸上在陪替氏培养皿(小号EE注3)。
2.愈伤组织诱导(3-4 周)      
P artially š ubmerge 8-12消毒胚胎种子半部每个培养皿N6D愈伤组织诱导培养基中。
密封用外科胶带菜,然后包裹每五个菜用铝箔,并培育在28℃下在所述暗3-4周(小号EE注4)。
3.制备愈伤组织,土壤杆菌悬浮液,和感染(48至72小时)      
一种。混合一小部分的四个菌落农杆菌在含有1个40毫升2N6D-AS液体介质中的微抹刀,重悬应变EH101 6μl的乙酰丁香酮(100毫克/毫升储液),并轻轻混合(农杆菌菌落必须被ANALY捷思通过PCR使用特异性引物š验证与细菌的gDNA的构建体)的整合(图4 ˚F )。    
湾 从 N6D 培养基中丢弃棕色愈伤组织和种子胚芽鞘(在第4周开始时)。收集活性胚性愈伤组织(微黄色-白色的,相对干燥,1-3毫米直径(西村等人,200。6 )和球状(比睿和Komari,2008)到一个50毫升的管中,在这个阶段。,将愈伤组织可以使用ð直接用于农杆菌感染(图4H)。    
C。倾中制备的细菌悬浮液小号TEP B3一个在到50-ml管中。    
d. 将愈伤组织浸泡在农杆菌悬浮液中,轻轻混合 90 秒(图 4I)。    
e. 倒出细菌悬浮液至灭菌污点少钢筛(12目)和豪在500米LD升无菌烧杯中。    
F。在放置在90 mm × 20 mm培养皿中的灭菌滤纸上吸干筛子,以去除多余的农杆菌悬浮液(图 4J)。     
G。将无菌滤纸放在固体 2N6D-AS 介质上。通过滴入 0.5 ml 2N6D-AS 液体培养基使滤纸饱和,然后在滤纸上共同培养适量的愈伤组织(20-25 个愈伤组织)(图 4K)。    
H。密封板用石蜡膜带和包装用铝箔,并培育在25 ℃下在黑暗中3天(小号EE注5)。   
4.洗涤愈伤组织并选择转化细胞(3-4 周)      
将感染的愈伤组织收集在 50 ml 无菌管中,并用无菌水冲洗 3-5 次以去除农杆菌(直至水清澈)。ģ ently š无须鳕管和倒出水。
用含有 40 μl 羧苄青霉素 500 mg/ml 原液的 40 ml 无菌水冲洗愈伤组织,以杀死农杆菌细胞。
倒入水悬浮液,将愈伤组织放在培养皿上的无菌滤纸上。ð RY愈伤组织以及(小号EE注6)。
将愈伤组织转移到 N6D-HC 选择培养基上;15 到 20 个愈伤组织可以放在一个盘子上。
密封用外科胶带的板,孵育在28愈伤组织℃下在黑暗中[根据该岐方法; 33°C(亮10小时)/30°C(暗14小时);Saika 和 Toki ( 2010) ] 。
定期检查培养物是否受到污染。在污染的情况下,立即将未受污染的愈伤组织转移到新鲜培养基中。
传代培养淡黄-白色的愈伤组织到新鲜N2D-HC介质后10〜12天S,密封高原é s的医用胶带,并孵育在28 ℃下在所述暗。ř esistant愈伤组织(微黄色-白色)可以后20-25天(可以观察到图5A) 。
 
 
图 5.愈伤组织选择和再生。( A ) N6D-HC 选择培养基上的耐潮霉素愈伤组织。一个rrow小号表明resistan牛逼后,从感染25天愈伤组织。( B )增殖的愈伤组织,带有绿点。( C )在相同的新鲜培养基上继代培养绿色盆栽。( D )生根。(E )在 MS HF 培养基上再生的小植株。( F )移植到生长室土壤中的转基因植物。( GE )转基因植物转移到锁定系统温室直至收获。比例尺:5厘米(E )、30厘米(F )、30厘米(G)、30厘米(H)。
 
5 、水稻转基因植株的再生(3-4周)      
一种。将抗性愈伤组织转移到含有合适抗生素的 REIII 再生培养基中。使用消毒镊子,将单个愈伤组织传代到 24 孔板的每个孔中。用手术胶带密封板,并在 30 °C下连续照明(图 5B)孵育。在相同条件下每10 天将转基因愈伤组织重新转移到新鲜的REIII 培养基中。转化后的枝条在3-4 周后开始分化(图 5C)。    
湾 将 3-4 厘米的芽转移到含有 HF 培养基和适当抗生素的植物盆中,在 28 °C下在 10 小时光照下(图 5D)。几天后,从罐的顶部取出手术胶带,然后祛瘀Ë锅凹圆R(图5E) (小号EE注7)。    
C。移植转化体稻类植物到6.5厘米锅小号containin克水稻育苗土壤,以每盆植株一个,在28在生长室℃下(图5 ˚F )。    
d. 当TR ansformants 15厘米高,移植米到23 -厘米含有稻田土壤的盆中,每盆植株一个,在温室中在28 ℃下未直到收获(图5G- 5 H)。    
e. 行为PCR用DNA FR ö M中的转化的水稻植物,以证实T-DNA整合到水稻植物小号(图6)。    
 
 
图6.琼脂糖凝胶轮廓为验证荷兰国际集团T-DNA插入物的整合到植物基因组中。509bp LUC基因扩增子大小。M ,标记;+ ,构建 DNA ; - ,台中65 gDNA。
 
笔记
 
              乙雅图琼脂溶解小号以及用高压灭菌。冷却至 50-45°C 后加入潮霉素,因为热培养基会降解抗生素。为A.癌农杆菌菌株EHA105 ,添加0.2毫升50mg / ml的潮霉素每200毫升LB培养基。对于菌株 EHA 101 ,每 200 毫升 LB 培养基添加 0.2 毫升 50毫克/毫升潮霉素和 0.2 毫升 500 毫克/毫升羧苄青霉素。
              美罗培南(25毫克/升)在选择培养基中完全suppresse š的过度生长的土壤杆菌,这导致高的转化效率(小川和MII,2007) 。
              收获一年后的种子 -而不是在收获后立即采取种子-更可取的愈伤组织诱导。健康成熟的种子作为转化和高频愈伤组织形成的起始材料至关重要。从 40-50 粒种子(足以进行一次转化)中去除外稃和外稃(外涂层)。从种子中去除含淀粉的胚乳以减少污染。
              C allus 选择是高效转化的关键点。我们经常检查文化和移除任何污染的种子立即零件与一消毒勺。在这种情况下,未污染的种子部分是传递红色至新板中。
              选择四个单一的农杆菌菌落。执行PCR用标记-特异性引物小号报导基因,以确认其掺入到植物基因组。细菌悬浮液的密度应该是OD 600 = 0.2 (小泽,2012)或降低(西村等人,200。6 ),和OD 600 = 0.05 - 0.1是建议编辑。这个过程对于防止土壤杆菌过度生长很重要,这会导致愈伤组织受损。大约 150 个愈伤组织可以放在一个板上(Hiei 和 Komari,2008 年)。用封口膜密封板以防止蒸发。
              共培养后清洗愈伤组织。抗性愈伤组织在表面含有小的结节状胚。一个关键的步骤是通过洗涤从愈伤组织中去除农杆菌,而不会对结节状胚胎细胞造成太大的物理损伤,因为这会阻止转基因的再生。P rior共培养,这是非常重要的,以确保选择的抗生素被删除适当所以这不会影响愈伤组织的生长。将愈伤组织放在滤纸上,让多余的水蒸发。
              淬火:该转基因植株的硬化是一个关键的一步之前,他们移植的土壤。硬化可以从低到高光强度条件以及从高到低湿度缓慢进行。琼脂培养基可以通过用水冲洗轻轻地从根部去除。
 
食谱
 
消毒液
方案一
将 50 ml 10% 次氯酸钠加入50 ml dH 2 O
存储在 RT
方案B
加入50mL次氯酸钠,将50ml卫生署2 ö ,和50μl的Tween ® 20
存储在 RT
 
抗生素
羧苄青霉素(500 毫克/毫升)
将 5 g 羧苄青霉素粉末完全溶解在 10 ml dH 2 O 中。
通过 0.22 μm 注射器过滤器对溶液进行消毒。
以 1 ml 等分试样在 -20°C 下储存。
潮霉素(50 毫克/毫升)
将 500 毫克潮霉素 B 溶解在10 毫升 dH 2 O 中。
通过 0.22 - μm 注射器过滤器对溶液进行消毒。
以 1.5 毫升的份量在 -20°C 下储存。
卡那霉素(50 毫克/毫升)
将 500 mg 卡那霉素溶解至 10 ml。
通过 0.22 - μm 注射器过滤器对溶液进行消毒。
以 1.5 毫升的份量在 -20°C 下储存。
乙酰丁香酮 (100 毫克/毫升)
将 1 g 乙酰丁香酮溶解到 1 ml DMSO 中。
用 10 ml dH 2 O稀释。
通过 0.22 - μm 注射器过滤器对溶液进行消毒。
以 1.5-2 ml 等分试样在 -20°C 下储存。
乙酰丁香酮 (10 毫克/毫升)
将 100 mg 乙酰丁香酮溶解到 1 ml DMSO 中。
用 10 ml dH 2 O稀释。
通过 0.22 - μm 注射器过滤器对溶液进行消毒。
以 1.5-2 ml 等分试样在 -20°C 下储存。
美罗培南 (12.5 毫克/毫升)
将 12.5 g 美罗培南溶解在10 ml dH 2 O 中。
通过 0.22 - μm 注射器过滤器对溶液进行消毒。
分装 2 毫升,并在 -20°C 下储存。
2,4-滴 (0.2 毫克/毫升)
将 20 mg 2,4-D 粉末溶解在 0.5 ml DMSO 中。
调整到 100 ml dH 2 O。
储存在-20°C。
NAA (0.2 毫克/毫升)
将 20 mg NAA 完全溶解在1 ml 0.1 N 氢氧化钠溶液中。
将音量调整到 100 ml dH 2 O。
储存在-20°C。
激动素 (1 毫克/毫升)
将 10 mg 激动素溶解在0.2 ml 1 M 氢氧化钠中。
用 dH 2 O将体积调节至 10 ml 。
储存在-20 °C 。
 
准备培养基(Tanle 1)
培养营养成分:所有培养MEDI一个准备新鲜在1 ,000毫升卫生署2 ö ,在120高压灭菌℃下20分钟和冷却至50 - 40℃ ; 最后,它是无菌ALY distribut编到大约12 (90毫米× 20毫米)在层流罩培养皿中。倾倒的含有抗生素的培养基板可以在 4°C 下储存长达 1 个月。


表 1. 愈伤组织诱导和再生培养基成分列表
 
                       LB溶胶我d培养基(200ml)中
磅肉汤 5 克
细菌琼脂 1.5 克
高压釜(120℃为20分钟)
                       LB液体培养基(200毫升)
磅肉汤 5 克
高压釜(120℃为20分钟)
                       YEP 培养基(200 毫升)
酵母提取物 2 克
Bacto 蛋白胨 2 克
氯化钠 1 克
高压釜(120℃为20分钟)
 
致谢
 
这个工作是由格兰特在急救来自日本学术振兴科学研究(KAKENHI)(:19P19394授权号)的部分资助。我们感谢教授卡尔文O. Qualset,美国加州大学戴维斯分校,阅读手稿并提供意见和建议。该协议源自之前的出版物(Satoh-Cruz et al ., 2010 ; Fukuda et al ., 2011 and 2013; Elakhdar et al ., 2019 )。              
 
 
利益争夺
 
该作者宣称,他们没有利益冲突小号。
 
参考
 
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引用:Elakhdar, A., Fukuda, M. and Kubo, T. (2021). Agrobacterium-mediated Transformation of Japonica Rice Using Mature Embryos and Regenerated Transgenic Plants. Bio-protocol 11(18): e4143. DOI: 10.21769/BioProtoc.4143.
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