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Agrobacterium tumefaciens-mediated Transformation of Walnut (Juglans regia)
农杆菌介导的核桃转化   

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Abstract

Like many woody plant species, walnut (Juglans regia) can be difficult to genetically transform and regenerate. However, somatic embryos have been used successfully for over two decades as a target tissue for transformation and regeneration of transgenic walnut plants. Walnut somatic embryos, initiated originally from developing zygotic embryos or anther tissue, will proliferate numerous secondary embryos from single cells in the epidermal layer. These single cells in intact somatic embryos can be efficiently transformed by Agrobacterium tumefaciens (A. tumefaciens). This gene transfer system is most efficient when Agrobacterium binary vector plasmids contain a scorable maker gene (e.g. uidA) and a selectable marker gene (e.g. nptII). This system should be applicable to any crop that undergoes repetitive embryogenesis from single Agrobacterium-susceptible cells. Here we describe the method of transforming somatic embryos in detail so that this technique can be applied to walnut and other woody plant species.

Materials and Reagents

  1. Biological materials
    1. Juglans regia somatic embryo cultures
    2. Disarmed A. tumefaciens strain (e.g. EHA101) harboring binary vector of interest
      For this protocol it is assumed that the vector harbors the nptII selectable marker gene and the uidA scorable marker gene (e.g. pDE00.0201 from Escobar et al., 2002).

  2. Chemicals
    1. Driver Kuniyuki walnut (DKW) medium with vitamins (PhytoTechnology Laboratories®, catalog number: D2470 )
    2. Kanamycin sulfate (PhytoTechnology Laboratories®, catalog number: K378 )
    3. Timentin® (PhytoTechnology Laboratories®, catalog number: T869 )
    4. 4-Hydroxy-3',5'-dimethoxyacetophenone (Acetosyringone) (PhytoTechnology Laboratories®, catalog number: A104 )
    5. GelzanTM CM (Sigma-Aldrich, catalog number: G1910 )
    6. SeaKem® LE agarose (Lonza, catalog number: 50004 )
    7. Bacto® Agar/SuperPureTM agar (BTS, catalog number: A01PD )
    8. Proline (PhytoTechnology Laboratories®, catalog number: D689 )
    9. 5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-gluc) (Sigma-Aldrich, catalog number: B5285 )
    10. 1 M sodium phosphate buffer (pH 7.0)
    11. 0.5 M EDTA (pH 8.0) (PhytoTechnology Laboratories®, catalog number: E 582 )
    12. 0.005 M potassium ferricyanide (Sigma-Aldrich, catalog number: 702587 )
    13. 0.005 M potassium ferrocyanide (Sigma-Aldrich, catalog number: P3289 )
    14. Triton X-100 (Sigma-Aldrich, catalog number: 234729 )
    15. Indole-3-butyric acid (IBA) (Sigma-Aldrich, catalog number: B-5386 )
    16. 6-Benzylaminopurine (BAP) (Sigma-Aldrich, catalog number: B-3408 )
    17. Tryptone (BTS, catalog number: T01PD )
    18. Yeast extract (BTS, catalog number: Y02MG )
    19. NaCl (Thermo Fisher Scientific, catalog number: S271-3 )
    20. Saturated ZnSO4 or NH4NO3 solution
    21. 10x PCR buffer (Applied Biosystems)
    22. PCR primers
    23. dNTPs (Applied Biosystems)
    24. Taq DNA polymerase (Applied Biosystems)
    25. DNeasy Plant Mini Kit (QIAGEN)

  3. Media and supplements
    Microbiological media
    1. Agrobacterium liquid growth medium (LB liquid medium) (see Recipes)
    2. Agrobacterium growth plates (LB solid medium) (see Recipes)  

    Plant media
    1. Driver Kuniyuki walnut (DKW) basal medium (see Recipes)
    2. Virulence induction medium (IM) (see Recipes)
    3. Acetosyringone medium (AS) plates (see Recipes)
    4. KAN/Timentin® selection medium (see Recipes)
    5. KAN only selection medium (see Recipes)
    6. DKW shoot medium (DKW basal medium with 1 mg/L BAP and 0.01 mg/L IBA) (see Recipes)

  4. Glass and Plasticware
    1. Sterile empty 100 x 15 mm Petri plates (VWR International, catalog number: 25384-342 )
    2. Sterile empty 35 x 10 mm Petri plates (BD, FalconTM, catalog number: 25373-041 )
    3. Sterile disposable 50 ml screw-cap centrifuge tubes (BD, FalconTM, catalog number: 352070 )
    4. Sterile disposable cotton-plugged 10 ml pipettes
    5. Plastic pipette tips (20, 200, and 1,000 µl) (Rainin, model: LTS )
    6. Sterile disposable 6-well multiwell plates (BD Biosciences, Falcon®, catalog number: 08-772-1G )
    7. Sterile disposable 96-well multiwell plates (BD Biosciences, Falcon®, catalog number: 08-772-2C )
    8. Filter paper cut to fit in 100 x 15 mm Petri plates and autoclaved
    9. Filter paper discs cut to the diameter of the wells of a 6-well multiwall plate and autoclaved
    10. 150 mm diameter desiccator (e.g. Nalgene, catalog number: 5313-0150 )
    11. Magenta GA-7 vessels (Magenta Corp.)

Equipment

  1. Spectrophotometer (e.g. Bio-Rad Laboratories, model: SmartSpec 3000 )
  2. Laboratory centrifuge (e.g. Sigma-Aldrich, Laborzentrifugen Laboratory Centrifuge 3K 10 )
  3. Micro-centrifuge (e.g. Denville, model: 260D )
  4. Continuous power supply units for electrophoresis (e.g. Thermo EC, model: EC 105 and Hoefer, model: HE 33 Mini Submarine unit)
  5. Electrophoresis units for agarose gels (e.g. Fisher Scientific, models: 03-500-124 , 03-500-132 , and 03-500-134 )
  6. Laminar flow hood (e.g. EdgeGard, Baker, model: EG3252 )
  7. Pipetman® 20, 200, and 1,000 µl (Rainin, model: LTS )
  8. Analytical balance (e.g. Mettler Toledo, model: AT261 DeltaRange® )
  9. Top loading electronic balance (e.g. Mettler, model: PM 2000 )
  10. pH meter (e.g. Corning Pinnacle, model: 540 )
  11. Constant temperature incubator (e.g. Napco, model: 301 )
  12. Water baths (e.g.Thermo Scientific, model: 2870 )
  13. Vortex mixer (e.g.: Scientific Industries, model: G 560 )
  14. Freezer (- 80 °C) (e.g. New Brunswick, model: U 700 Premium)

Procedure

  1. Obtain actively multiplying walnut somatic embryo cultures or initiate new cultures from zygotic embryos or anther tissue (Dandekar et al., 1989; Mendum and McGranahan, 1995; Leslie et al., 2006).
    Several days before initiation of transformation:
    1. Streak Agrobacterium strains out on a LB plates with appropriate antibiotics. Incubate at 28 °C for 48 h.
    2. Culture sufficient numbers of embryos of the desired genotypes.
    3. Prepare appropriate antibiotics.
    4. Make LB liquid medium.
    5. Make co-cultivation liquid medium.
    6. Make DKW basal, AS, and selection media.
    The day before initiation of transformation:
    Select embryos: Pick out 60-70 or more rapidly growing, small, white, uniform embryos (Figure 1A) and place them onto fresh DKW basal medium.
    Day 1 (prepare liquid cultures)
    1. Inoculate each strain of Agrobacterium to be used into a 50 ml conical centrifuge tube containing 20 ml of LB medium.
    2. Place the capped tubes on a rotary shaker at ~200 rpm at 25 °C.
    3. After 2 h add the appropriate selective antibiotics for the vector used and return to shaker.
    Day 2
    1. After shaking overnight, the bacterial cultures should be turbid. Determine the Abs600nm of a 10-1 dilution of the culture.
    2. For 60-70 embryos, approximately 25 ml of co-cultivation suspension will be utilized. The desired Abs600nm of the co-cultivation suspension is ~0.5 (an Abs600nm reading of 0.5 is approximately equivalent to 2.5 x 108 bacteria/ml). To calculate the needed volume of overnight culture, use the following equation:

      (Co-cultivation volume needed) x (desired Abs600nm of the co-cultivation suspension)
      (Abs600nm of the Agrobacterium culture) x (dilution factor)

      For example: If you need 25 ml of co-cultivation suspension at a concentration of 2.5 x 108 bacteria/ml and an aliquot of the Agrobacterium culture diluted 1: 10 gives an A600 reading of 0.371, how much of the Agrobacterium culture do you need to dilute for use?

      (25) x (0.5)     =   3.36 ml
      (0.371) x (10)

    3. Using a sterile pipette, place the calculated volume of Agrobacterium culture into a sterile plastic 50 ml centrifuge tube and centrifuge for 10 min at 4,000 x g and ambient temperature to pellet the bacteria.
    4. Pour or pipette the supernatant into a waste container and resuspend the pellet in the co-cultivation medium. The pellet is easier to resuspend in a small volume (0.5 ml first) by carefully pipetting up and down, followed by bringing the solution to required final volume.
    5. Return the tubes to the rotary shaker at ~200 rpm at 25 °C for 1-2 h.

  2. Co-cultivation
    1. Place desired number of embryos into a well of a sterile six-well multiwell plate. The well should be no more than half filled with embryos (about 20-23 embryos). Use more than one well for each transformation in case of contamination problems. Transformations of multiple genotypes can be performed in different wells of the same plate but if using multiple bacterial vectors it is advisable to use separate plates to avoid cross-contamination.   
    2. Dispense the appropriate volume of Agrobacterium co-cultivation suspension (about 8 ml, or enough to cover the embryos) into each well using sterile 10 ml pipettes with cotton-plugged ends.
    3. Incubate at ambient temperature for at least 10-15 min (Figure 1B and Note 1).
    4. Place a sterile filter paper into a number of empty sterile Petri plates equal to the number of wells used.
    5. Pipet as much excess co-cultivation liquid as possible from each well into a waste container.  
    6. Transfer the embryos from each well onto the filter paper in the Petri plates using sterile forceps. This will wick excess co-cultivation liquid from the embryos.
    7. Transfer the embryos to labeled plates of AS medium (about 10 per plate) and place the plates in the dark for 48 h at 20-22 °C (see Video 1).

      Video 1. Walnut transformation


    Day 4: Selection
    After co-cultivating for 48 h, transfer the embryos to plates of KAN/Timentin medium containing 200 mg/L kanamycin and 200 mg/L Timentin (see Note 2). Other selection medium may be utilized depending on the selectable marker gene (see Note 3). Incubate the culture plates in the dark at ambient temperature (20-24 °C).
    Day 6 and onward:
    1. Transfer embryos to fresh KAN/Timentin medium after two days and again after an additional five days. This helps to reduce bacterial overgrowth. Thereafter transfer the embryos to fresh KAN/Timentin medium weekly for 8-12 weeks.
    2. As new somatic embryos begin to develop from the surface of the original (E0) embryos, separate them from the parent embryos.  Label these new embryos as E1 embryos.  Repeat this process for one more generation (E2 embryos; Figure 1C).
    3. After 8-12 weeks of selection, embryos can be moved to selection medium containing only kanamycin. Observe the embryo cultures carefully for the next several weeks to ensure that no residual Agrobacterium has survived.

  3. Scoring for GUS expression
    1. As E2 embryos emerge, test them for GUS (uidA) activity (Jefferson, 1987).
    2. Pipette 40 µl of X-gluc staining solution (see recipes) into wells of a sterile 96-well multiwell plate.
    3. Using a fine point scalpel remove a small piece of tissue (cotyledon tips work well) from each well-formed and healthy E2 embryo of interest. Put the tissue piece in the X-gluc solution and label and mark the location of the embryo from which it was excised.
    4. Incubate at 37 °C and monitor the development of blue color. Color change should be apparent in 10 min to 2 h (Figure 1D).
    5. If the tissue piece developed the distinctive blue color, the E2 embryo from which it was cut should be separated and multiplied on selection medium until E3 embryos are available for DNA analysis.


      Figure 1. Walnut transformation. A. Embryos with well-develped cotyledens ready for transformation. B. Embyos in co-cultivation suspension. C. Primary embryo (E0) developing secondary embryos (E1 and E2). D. GUS test, transformed embryos turn blue in X-Gluc solution.

  4. PCR based verification of transformation
    1. Select actively proliferating E3 embryos from different embryo lines and use for DNA isolation.
    2. Isolate total DNA using a DNeasy Plant Mini Kit according to the manufacturer’s protocols.
    3. Perform PCRs using appropriate primers to confirm the presence of the nptII gene (or other gene of interest). Set up the PCR reactions using 250 ng of genomic DNA in each 25 μl PCR reaction.
    4. After PCR is completed, 5 μl of PCR product is electrophoresed to verify amplification of an appropriately-sized band. DNA from untransformed embryos can be used as a negative control (Figure 2).


      Figure 2. Confirmation of transgenic nature. Agarose gel of PCR products showing 790 bp bands in transformed lines 8A and 1A for APh3/Aph4 primers. Negative controls or non-transformed lines does not show any bands.

  5. Somatic embryo germination and plant production
    1. After verification of transformation, some E3 embryos from the desired embryo lines can be desiccated to initiate germination. Choose well-formed somatic embryos and place them in 35 x 10 mm sterile Petri plates with no medium. Cover the plates but leave them unsealed (do not wrap with Parafilm®) and place them in the dark at ambient temperature (20-24 °C) on the rack of a well-sealed desiccator containing 10-15 ml of saturated ZnSO4 or NH4NO3 in the bottom (Figure 3A).
    2. After the embryos become an opaque white (typically 2-7 days) remove the embryos from the desiccator and place them on DKW shoot medium in Magenta GA-7 vessels or Petri plates.
    3. Culture at ambient temperature under cool white fluorescent lights (16 h day length, ~100 µE) for 2 to 8 weeks.
    4. Most embryos will produce roots, but typically fewer than 10% of embryos develop shoots.  Roots will usually emerge from embryos in 7-10 days (Figure 3B).  
    5. Fully germinated embryos possessing both roots and shoots should be removed from the medium as soon as possible and planted in potting soil (for example UC Mix, 25%: 42%: 33% sand: fir bark: peat moss). Alternatively, epicotyls can be excised, micropropagated on DKW shoot medium, and then rooted to generate multiple plants.
    6. To acclimatize, keep plants on soil at 100% humidity for 2 weeks and then gradually reduce the humidity.
    7. Established plants can be repotted to larger containers as needed and maintained in a greenhouse or lath house.


      Figure 3. Plant production using trnasgenic somatic embryos. A. Drying embryos in dessicator B. New shoots emerging from  transformed walnut somatic embryos.

Notes

  1. Physical wounding is not necessary when somatic embryos are used for transformation.
  2. Transfer embryos in a well-spaced pattern on each plate so that if Timentin-resistant bacteria begin to multiply they are not moved to all the embryos on the plate.
  3. Hygromycin B (25 mg/L) can serve as an alternative selectable marker in place of kanamycin or as a second selectable marker if performing co-transformation to insert two genes simultaneously.
  4. Kanamycin sulfate in solution has a very high pH. If used at a concentration greater than 100 mg/L for selection adjust the pH of the kanamycin stock solution to 5.5 with dilute HCl prior to filter sterilizing.

Recipes

  1. Kanamycin sulfate (50 mg/ml stock)
    5 g dissolved in 100 ml dH2O
    Filter-sterilize
    Stored at -20 °C in 10-15 ml aliquots
  2. Timentin (100 mg/ml stock)
    6.2 g dissolved in 62 ml dH2O
    Filter sterilize
    Stored at -20 °C in 10-15 ml aliquots
  3. 100 mM Acetosyringone
    19.6 mg dissolved in 1 ml 95% EtOH
    Use capped centrifuge tube and vortex
    Parafilm
    Stored at room temperature
  4. X-Gluc staining solution
    Dissolve X-Gluc to a 0.3% w/v solution in dimethylformamide
    Dilute with 100 mM sodium phosphate buffer (pH 7.0) containing 0.006% Triton X-100 and 0.5 mM K+Fe cyanide to make a 1 mM X-gluc working solution
    Filter-sterilize
    Stored refrigerated
  5. Indole-3- butyric acid (IBA) (0.1 mg/ml stock)
    10 mg dissolved in 100 ml
    Dissolve IBA powder in a few drops of 1 N KOH
    Diluted with dH2O
  6. 6-Benzylaminopurine (BAP) (1 mg/ml stock)
    25 mg dissolved in 25 ml
    Dissolve BAP powder in a few drops of 1 N KOH
    Diluted with dH2O
  7. Media
    Driver Kuniyuki walnut (DKW) basal medium
    To make 1 L:
    5.32 g DKW basal medium with vitamin powder dissolved in dH2O
    30 g sucrose
    pH to 5.5 using 1 N KOH
    2.2 g/L GelzanTM
    Autoclave and pour into 40 100 x 15 mm Petri plates
  8. LB liquid medium
    To make 100 ml
    1 g Tryptone
    0.5 g yeast extract
    1 g NaCl
    pH 6.8-7.2
    Autoclave
  9. LB growth plates
    To make 100 ml
    1 g Tryptone
    0.5 g yeast extract
    1 g NaCl
    0.8 g Bacto® agar
    pH 6.8-7.2
    Autoclave and pour in 100 x 15 mm Petri plates
  10. Virulence induction medium (IM)
    To make 500 ml
    2.66 g DKW basal medium with vitamin powder
    15 g sucrose
    0.5 ml 100 µM acetosyringone (to make 1 µM final concentration)
    575 mg proline (to make 1mM final concentration)
    pH 5.2 using 1 N KOH
    Filter sterilize
    Stored refrigerated (4 °C) in 50 ml aliquots
  11. Acetosyringone medium (AS) plates
    To make 1 L
    5.32 g DKW basal medium with vitamin powder
    30 g sucrose
    1 ml 100 µM acetosyringone (add this before autoclaving)
    pH to 5.5 using 1 N KOH
    2.2 g/L GelzanTM
    Autoclave and pour in 100 x 15 mm Petri plates
  12. KAN/Timentin selection medium
    To make 1 L
    5.32 g DKW basal medium with vitamin powder
    30 g sucrose
    pH 5.5 using 1 N KOH
    Dispense into 1 L screw-cap bottles (500 ml per bottle)
    1.1 g GelzanTM to each bottle
    Autoclave, and cool to 60 °C in a water bath
    Then add 200 mg/L pH adjusted filter sterilized kanamycin and 200 mg/L filter sterilized Timentin (see Note 4)
    Mix thoroughly and pour into sterile 100 x 15 mm Petri plates.
    When solidified, store refrigerated in the original plastic sleeves until ready for use
  13. KAN only selection medium
    The same procedure as KAN/Timentin selection medium but without the Timentin
  14. DKW shoot medium
    To make 1 L
    5.32 g of DKW basal medium with vitamin powder
    30 g sucrose
    1 mg/L BAP and 0.01 mg/L IBA
    pH to 5.5
    2.1 g/L Gelzan®
    Microwave until the medium boils
    Mix thoroughly on a stir plate
    Dispense into Magenta Corporation GA7 vessels (approximately 30 ml of medium each)
    Autoclave

Acknowledgments

Note that a similar protocol has been described by Leslie et al. (2006) and Dandekar et al. (1989).

References

  1. Dandekar, A. M., McGranahan, G. H., Leslie, C. A. and Uratsu, S. L. (1989). Agrobacterium-mediated transformation of somatic embryos as a method for the production of transgenic plants. J Tissue Culture Methods 12(4): 145-150.
  2. Escobar, M. A., Leslie, C. A., McGranahan, G. H. and Dandekar, A. M. (2002). Silencing crown gall disease in walnut (Juglans regia L.). Plant Science 163(3): 591-597.
  3. Jefferson, R. A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5(4): 387-405.
  4. Leslie, C. A., Uratsu, S. L., McGranahan, A. M., and Dandekar, G. H. (2006). Methods Mol Biol. In: Wang K. (ed). Agrobacterium protocols 2nd ed, Vol. 2. Humana Press, Vol. 344: 297 -312.
  5. Mendum, M. L., and McGranahan, G. (1995). Somatic embryogenesis of clonal chandler. Walnut Res Rep, California Walnut Board 25-27.

简介

像许多木本植物物种一样,核桃( Juglans regia )可能难以进行遗传转化和再生。然而,体细胞胚已经成功使用超过二十年作为转基因核桃植物的转化和再生的靶组织。核桃体细胞胚,最初从发育的合子胚或花药组织开始,将从表皮层中的单个细胞增殖许多次级胚。在完整的体细胞胚中的这些单细胞可以通过根癌土壤杆菌(根瘤土壤杆菌)有效地转化( tumefaciens )。当农杆菌二元载体质粒含有可打分的基因(例如uidA)和选择性标记基因(例如nptII)时,该基因转移系统是最有效的。该系统应适用于从单个农杆菌感受细胞经历重复胚胎发生的任何作物。在这里我们详细描述转化体细胞胚胎的方法,使这种技术可以应用于核桃和其他木本植物物种。

材料和试剂

  1. 生物材料
    1. 体细胞胚胎培养
    2. 已撤防。 含有感兴趣的二元载体的tumefaciens 菌株(例如 EHA101) 对于   该协议假定载体含有 nptII 可选择标记基因和 uidA 可评分标记基因(例如 pDE00.0201,来自Escobar 等人,2002)。

  2. 化学品
    1. 具有维生素( Phyto Technology Laboratories ,目录号:D2470)的驱动剂Kuniyuki核桃(DKW)培养基
    2. 卡那霉素硫酸盐( Phyto Technology Laboratories ,目录号:K378)
    3. Timentin ®( Phyto Technology Laboratories ®,目录号:T869)
    4. 4-羟基-3',5'-二甲氧基苯乙酮(Acetosyringone)( Phyto Technology Technology Laboratories ,目录号:A104)
    5. Gelzan TM CM(Sigma-Aldrich,目录号:G1910)
    6. (Lonza,目录号:50004)
    7. Bacto Agar/SuperPure TM 琼脂(BTS,目录号:A01PD)
    8. Proline( Phyto 技术实验室®,目录号:D689)
    9. 5-溴-4-氯-3-吲哚基-β-D-葡糖苷酸(X-gluc)(Sigma-Aldrich,目录号:B5285)
    10. 1M磷酸钠缓冲液(pH 7.0)
    11. 0.5M EDTA(pH 8.0)( Technology Laboratories ,目录号:E 582)
    12. 0.005M铁氰化钾(Sigma-Aldrich,目录号:702587)
    13. 0.005M氰亚铁酸钾(Sigma-Aldrich,目录号:P3289)
    14. Triton X-100(Sigma-Aldrich,目录号:234729)
    15. 吲哚-3-丁酸(IBA)(Sigma-Aldrich,目录号:B-5386)
    16. 6-苄氨基嘌呤(BAP)(Sigma-Aldrich,目录号:B-3408)
    17. 胰蛋白胨(BTS,目录号:T01PD)
    18. 酵母提取物(BTS,目录号:Y02MG)
    19. NaCl(Thermo Fisher Scientific,目录号:S271-3)
    20. 饱和的ZnSO 4或NH 4 NO 3溶液
    21. 10x PCR缓冲液(Applied Biosystems)
    22. PCR引物
    23. dNTPs(Applied Biosystems)
    24. Taq DNA聚合酶(Applied Biosystems)
    25. DNeasy植物迷你包(QIAGEN)

  3. 媒体和补充
    微生物培养基
    1. 土壤杆菌液体生长培养基(LB液体培养基)(参见Recipes)
    2. 土壤杆菌生长板(LB固体培养基)(参见Recipes)

    植物培养基
    1. 司机Kuniyuki核桃(DKW)基础培养基(参见食谱)
    2. 毒性诱导培养基(IM)(见配方)
    3. Acetosyringone介质(AS)板(见配方)
    4. KAN/Timentin ®选择培养基(参见配方)
    5. KAN只选择介质(见配方)
    6. DKW苗培养基(具有1mg/L BAP和0.01mg/L IBA的DKW基础培养基)(参见Recipes)

  4. 玻璃和塑料制品
    1. 无菌空100×15mm培养皿(VWR International,目录号:25384-342)
    2. 无菌空35×10mm培养皿(BD,Falcon ,目录号:25373-041)
    3. 无菌一次性50毫升螺旋盖离心管(BD,Falcon ,目录号:352070)
    4. 无菌一次性棉塞10 ml移液器
    5. 塑料移液管吸头(20,200和1000μl)(Rainin,型号:LTS)
    6. 无菌一次性6孔多孔板(BD Biosciences,Falcon ,目录号:08-772-1G)
    7. 无菌一次性96孔多孔板(BD Biosciences,Falcon ,目录号:08-772-2C)
    8. 将滤纸切成适合100×15mm培养皿并高压灭菌
    9. 将滤纸圆盘切成6孔多壁板的孔的直径并高压灭菌
    10. 直径150mm的干燥器(例如Nalgene,目录号:5313-0150)
    11. 洋红GA-7容器(Magenta Corp.)

设备

  1. 分光光度计(例如Bio-Rad Laboratories,型号:SmartSpec 3000)
  2. 实验室离心机(例如Sigma-Aldrich,Laborzentrifugen Laboratory Centrifuge 3K 10)
  3. 微离心机(例如,Denville,型号:260D)
  4. 用于电泳的连续电源装置(例如Thermo EC,型号:EC 105和Hoefer,型号:HE 33 Mini Submarine unit)
  5. 用于琼脂糖凝胶的电泳单元(例如Fisher Scientific,型号:03-500-124,03-500-132和03-500-134)
  6. 层流罩(例如 EdgeGard,Baker,型号:EG3252)
  7. Pipetman 20,200和1,000μl(Rainin,型号:LTS)
  8. 分析余额(例如 Mettler Toledo,型号:AT261 DeltaRange
  9. 顶部载入电子天平(例如 Mettler,型号:PM 2000)
  10. pH计(例如 Corning Pinnacle,型号:540)
  11. 恒温培养箱(如 Napco,型号:301)
  12. 水浴(例如Thermo Scientific,型号:2870)
  13. 涡旋混合器(例如:Scientific Industries,型号:G 560)
  14. 冷冻( - 80°C)(如 New Brunswick,型号:U 700 Premium)

程序

  1. 获得主动繁殖核桃体细胞胚胎培养物或从合子胚或套组织启动新的培养物(Dandekar等人,1989; Mendum和McGranahan,1995; Leslie等人, 2006)。
    开始转化前几天:
    1. Streak农杆菌在具有适当抗生素的LB平板上产生。 在28℃孵育48小时
    2. 培养足够数量的所需基因型的胚胎
    3. 准备适当的抗生素。
    4. 使LB液体介质。
    5. 共培养液体培养基。
    6. 制作DKW basal,AS和选择介质。
    变革开始前一天:
    选择胚胎:选择60-70或更快速生长,小,白色,均匀的胚胎(图1A),并将其放置在新鲜的DKW基础培养基上。
    第1天(制备液体培养物)
    1. 将每个土壤杆菌菌株接种到含有20ml LB培养基的50ml锥形离心管中。
    2. 将加盖的管放在旋转振荡器上,在约200rpm,25℃
    3. 2小时后,向所用载体中加入合适的选择性抗生素,并返回振荡器。
    第2天
    1. 振荡过夜后,细菌培养物应是混浊的。 确定培养物的10μL-1稀释液的Abs 600nm。
    2. 对于60-70胚胎,约25ml共培养悬浮液 将被利用。所述共培养悬浮液的所需Abs 600nm 是〜0.5(0.5的Abs 600nm读数大约相当于2.5x  10 8个细菌/ml)。为了计算所需的过夜培养体积, 使用以下公式:

      (共培养悬浮液的需要的Abs <600> <600> > (农杆菌培养物的Abs <600nm)×(稀释因子)

      对于  例如:如果你需要25毫升的共培养悬浮液 浓度为2.5×10 8个细菌/ml,以1:10稀释的土壤杆菌培养物的等分试样得到的A 600读数为0.371,如何 大多数土壤杆菌文化你需要稀释才能使用?

      (25)x(0.5)      =   3.36 ml
      (0.371)×(10)

    3. 使用无菌移液管,放置计算体积的农杆菌 培养成无菌塑料50ml离心管并离心 10分钟(4,000转/分钟)和环境温度下使细菌沉淀
    4. 将上清液倒入或吸取到废物容器中并重悬 所述沉淀在共培养培养基中。 丸更容易 通过小心吸取重悬于小体积(0.5ml第一)   ,然后使溶液达到所需的最终体积
    5. 将管在约200rpm下在25℃下返回旋转振荡器1-2小时。

  2. 共培养
    1. 将所需数量的胚胎放入无菌六孔的孔中 多孔板。 井应该不超过一半 胚胎(约20-23胚胎)。 每个使用多个孔 在污染问题的转变。 变换 多个基因型可以在同一板的不同孔中进行   但如果使用多个细菌载体,建议使用单独   以避免交叉污染。  
    2. 分配 适当体积的农杆菌共培养悬浮液(约8 ml,或足以覆盖胚胎) 移液器带棉塞端。
    3. 在环境温度下孵育至少10-15分钟(图1B和注释1)。
    4. 将无菌滤纸置于与所用孔数相同的空无菌培养皿中
    5. 将尽可能多的过量共培养液体从每个孔中取出进入废物容器。  
    6. 将胚胎从每个井转移到Petri的滤纸上   板使用无菌镊子。 这将灯芯过度共同培养 液体从胚胎。
    7. 转移胚胎标记板   的AS培养基(每板约10个),并将板置于黑暗中 在20-22℃下48小时(参见视频1)。

      视频1.核桃转化
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    第4天:选择
    共培养48小时后,将胚转移至含有200mg/L卡那霉素和200mg/L Timentin的KAN/Timentin培养基平板(见注2)。 根据选择标记基因,可以使用其它选择培养基(参见注释3)。 在环境温度(20-24℃)下在黑暗中孵育培养板。
    第6天及以后:
    1. 转移胚胎到新鲜KAN/Timentin培养基,两天后 后额外五天。 这有助于减少细菌 过度生长。 然后将胚转移到新鲜的KAN/Timentin培养基中   每周8-12周。
    2. 随着新的体细胞胚开始发育   从原始(E )胚胎的表面,将它们与胚胎分离 亲代胚胎。 将这些新胚胎标记为E 胚胎。 重复此操作 过程再生一代(E 胚胎;图1C)。
    3. 后 8-12周的选择,胚胎可以移动到选择培养基 仅含卡那霉素。 仔细观察胚胎培养物   接下来几周以确保没有残留的土壤杆菌 存活。

  3. GUS表达的评分
    1. 当E sub 2胚胎出现时,测试它们用于GUS( uidA )活性(Jefferson,1987)。
    2. 吸取40μlX-gluc染色溶液(见配方)到无菌96孔多孔板的孔中。
    3. 使用细点解剖刀去除一小块组织(子叶 尖端工作良好)从每个良好形成并且健康的感兴趣的E 2/2胚胎。  将组织片放在X-gluc溶液中,并标记和标记 切除胚胎的位置。
    4. 在37℃孵育并监测蓝色的发展。颜色变化应在10分钟至2小时内显现(图1D)。
    5. 如果组织片显现出独特的蓝色,则从其切割的E 2子胚应当分离并增殖 选择培养基,直到E 胚胎可用于DNA分析。


      图  1.核桃转化。 A.具有良好发育的子叶的胚胎 准备转型。 B.共生培养悬浮液中的Embyos。 C。 发育二次胚胎(E 1和E 2)的原代胚胎(E )。 D. GUS 测试,转化的胚胎在X-Gluc溶液中变成蓝色。

  4. 基于PCR的转化验证
    1. 选择来自不同胚胎系的活跃增殖的E 胚胎,并用于DNA分离。
    2. 使用DNeasy Plant Mini Kit根据制造商的说明书分离总DNA
    3. 使用适当的引物进行PCR以确认存在 nptII基因(或其他感兴趣的基因)。 设置PCR反应使用 在每25μlPCR反应中含有250ng基因组DNA。
    4. PCR后   完成后,对5μlPCR产物进行电泳以验证 放大适当大小的带。 来自未转化的DNA 胚胎可用作阴性对照(图2)

      图2。 转基因本性的确认。 PCR产物的琼脂糖凝胶显示 在APh3/Aph4引物的转化株系8A和1A中的790bp条带。 阴性对照或未转化的细胞系不显示任何条带。

  5. 体细胞胚发芽和植物生产
    1. 在转化的验证后,从所需的胚胎中选择一些E 3 胚胎系可以干燥以引发萌发。 选择 良好形成的体细胞胚,并将其放置在35×10mm无菌Petri 平板无培养基。 覆盖板,但让它们开封(不要 用Parafilm ®包装),并将其在环境温度下置于黑暗中 (20-24℃)在包含10-15ml的良好密封的干燥器的架子上 的饱和ZnSO 4或NH 4 NO 3在底部(图3A)。
    2. 后 胚胎变成不透明的白色(通常2-7天)去除 胚胎从干燥器和将它们放在DKW射击介质上 洋红GA-7容器或培养皿。
    3. 在冷白色荧光灯(16小时天长度,〜100μE)下在环境温度下培养2至8周
    4. 大多数胚胎会产生根,但通常少于10% 胚胎发育芽。 根在7-10通常会从胚胎出现 天(图3B)。  
    5. 完全发芽的胚胎拥有两者 根和芽应尽快从培养基中除去 并种植在盆栽土壤(例如UC Mix,25%:42%:33%沙:冷杉)中   树皮:泥炭藓)。 或者,可以切除上胚轴, 微量繁殖在DKW苗培养基上,然后根生生根 多个植物。
    6. 为了适应环境,将植物在100%湿度的土壤中保持2周,然后逐渐降低湿度。
    7. 建立的植物可以根据需要转移到较大的容器中,并保存在温室或板条房中

      图   3.使用致畸体细胞胚的植物生产。 A。 干燥胚胎   在干燥剂B中。 转化核桃体细胞 胚胎。

笔记

  1. 当体细胞胚用于转化时,物理伤害不是必需的
  2. 在每个板上以良好间隔的模式转移胚胎,使得如果Timentin抗性细菌开始增殖,它们不会移动到板上的所有胚胎。
  3. 如果进行共转化以同时插入两个基因,潮霉素B(25mg/L)可以作为替代卡那霉素的备选选择标记或作为第二选择标记。
  4. 硫酸卡那霉素在溶液中具有非常高的pH。 如果以大于100mg/L的浓度使用以进行选择,则在过滤灭菌之前用稀HCl调节卡那霉素储备溶液的pH至5.5。

食谱

  1. 硫酸卡那霉素(50mg/ml母液)
    5g溶解在100ml dH 2 O中 过滤灭菌
    储存于-20°C,以10-15 ml等份
  2. 特美汀(100mg/ml母液)
    6.2g溶解在62ml dH 2 O中 过滤灭菌
    储存于-20°C,以10-15 ml等份
  3. 100 mM乙酰丁香酮 19.6mg溶解在1ml 95%EtOH中 使用有盖离心管和涡流
    parafilm
    在室温下贮存
  4. X-Gluc染色溶液 将X-Gluc溶解于0.3%w/v的二甲基甲酰胺溶液中
    用含有0.006%Triton X-100和0.5mM K + Fe氰化物的100mM磷酸钠缓冲液(pH 7.0)稀释,制成1mM X-gluc工作溶液。
    过滤灭菌
    储存冷藏
  5. 吲哚-3-丁酸(IBA)(0.1mg/ml母液) 10mg溶解在100ml中
    将IBA粉末溶解在几滴1N KOH中 用dH 2 O稀释
  6. 6-苄氨基嘌呤(BAP)(1mg/ml母液)
    25mg溶于25ml
    将BAP粉末溶解在几滴1N KOH中 用dH 2 O稀释
  7. 媒体
    司机Kuniyuki核桃(DKW)基础培养基
    使1 L:
    5.32g DKW基础培养基,其中维生素粉末溶于dH 2 O中 30克蔗糖 pH至5.5,使用1N KOH
    2.2 g/L Gelzan TM
    高压灭菌并倒入40×100×15mm培养板中
  8. LB液体培养基
    要做到100毫升
    1克胰蛋白胨
    0.5g酵母提取物
    1克NaCl
    pH 6.8-7.2
    高压灭菌器
  9. LB生长板
    要做到100毫升
    1克胰蛋白胨
    0.5g酵母提取物
    1克NaCl
    0.8g Bacto 琼脂
    pH 6.8-7.2
    高压灭菌并倒入100 x 15 mm培养板中
  10. 毒性诱导培养基(IM)
    使500毫升
    2.66克DKW基础培养基和维生素粉末
    15克蔗糖 0.5ml100μM乙酰丁香酮(使1μM终浓度)
    575mg脯氨酸(使1mM终浓度)
    pH 5.2,使用1N KOH
    过滤灭菌
    储存冷藏(4℃),以50ml等分
  11. Acetosyringone培养基(AS)培养板
    使1 L
    5.32克DKW基础培养基与维生素粉末
    30克蔗糖 1 ml 100μM乙酰丁香酮(在高压灭菌前加入) pH至5.5,使用1N KOH
    2.2 g/L Gelzan TM
    高压灭菌并倒入100 x 15 mm培养板中
  12. KAN /特门汀选择培养基
    使1 L
    5.32克DKW基础培养基与维生素粉末
    30克蔗糖 pH 5.5,使用1N KOH
    分配到1L螺旋盖瓶(每瓶500ml)
    1.1 g Gelzan TM 到每个瓶子
    高压灭菌,并在水浴中冷却至60℃。
    然后加入200mg/L pH调节过滤灭菌的卡那霉素和200mg/L过滤灭菌的特美汀(见注4)。
    充分混合并倒入无菌的100×15mm培养皿中。
    固化后,将其储存在原始塑料套筒中,直到准备使用
  13. KAN只选择介质
    与KAN/Timentin选择培养基相同,但没有Timentin
  14. DKW拍摄媒体
    使1 L
    5.32g含维生素粉的DKW基础培养基
    30克蔗糖 1mg/L BAP和0.01mg/L IBA
    pH至5.5 2.1 g/L Gelzan ®
    微波直到介质沸腾
    在搅拌板上彻底混合
    分配到Magenta Corporation GA7容器(每个约30ml培养基) 高压灭菌器

致谢

注意,类似的方案已由Leslie等人(2006)和Dandekar等人(1989)描述。

参考文献

  1. Dandekar,A.M.,McGranahan,G.H.,Leslie,C.A。和Uratsu,S.L。(1989)。 土壤杆菌 - 介导的体细胞胚转化作为转基因植物的生产方法。组织培养方法 12(4):145-150
  2. Escobar,M.A.,Leslie,C.A.,McGranahan,G.H.and Dandekar,A.M。(2002)。 在核桃中沉默冠状病( Juglans regia L。)。 Plant Science 163(3):591-597。
  3. Jefferson,R.A。(1987)。 在植物中分析嵌合基因:GUS基因融合系统。 Plant Mol Biol Rep 5(4):387-405。
  4. Leslie,C.A.,Uratsu,S.L.,McGranahan,A.M。,和Dandekar,G.H。(2006)。 Methods Mol Biol 。 In:Wang K.(ed)。 A grobacterium protocols 2nd ed, 2。 Humana Press,Vol。 344:297 -312。
  5. Mendum,M.L。,和McGranahan,G。(1995)。 克隆钱包的体细胞胚胎发育 核桃研究会,加利福尼亚核桃委员会 25- 27.
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Walawage, S. L., Leslie, C. A., Escobar, M. A. and Dandekar, A. M. (2014). Agrobacterium tumefaciens-mediated Transformation of Walnut (Juglans regia). Bio-protocol 4(19): e1258. DOI: 10.21769/BioProtoc.1258.
  2. Araji, S., Grammer, T. A., Gertzen, R., Anderson, S. D., Mikulic-Petkovsek, M., Veberic, R., Phu, M. L., Solar, A., Leslie, C. A., Dandekar, A. M. and Escobar, M. A. (2014). Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut. Plant Physiol 164(3): 1191-1203.
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