Chapter 17 Genetic Transformation of Wheat via Particle Bombardment Caroline A. Sparks and Huw D. Jones Abstract Since its first invention in the late 1980s the particle gun has evolved from a basic gunpowder driven machine firing tungsten particles to one more refined which uses helium gas as the propellant to launch alternative heavy metal particles such as gold and silver. The simple principle is that DNA-coated microscopic particles (microcarriers) are accelerated at high speed by helium gas within a vacuum and travel at such a velocity as to penetrate target cells. However, the process itself involves a range of parameters which are open to variation: microparticle type and size, gun settings (rupture pressure, target distance, vacuum drawn, etc.), preparation of components (e.g., gold coating), and preparation of plant tissues. Here is presented a method optimized for transformation of wheat immature embryos using the BioRad PDS-1000/He particle gun to deliver gold particles coated with a gene of interest and the selectable marker gene bar at 650 psi rupture pressure. Following bombardment, various tissue culture phases are used to encourage embryogenic callus formation and regeneration of plantlets and subsequent selection using glufosinate ammonium causes suppression of non-transformed tissues, thus assisting the detection of transformed plants. This protocol has been used successfully to generate transgenic plants for a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.). Key words Wheat, Particle gun, Biolistics, Transformation, Immature embryo, Transgenic plants
1
Introduction The ability to alter the genome of a plant in a precise and predictable manner is a powerful research tool for functional genomics and is fast becoming a key element in the process of commercial varietal improvement. A key component of the genetic modification of crop plants is a robust transformation protocol. Wheat was among the last of the major crops to be transformed with the first fertile transgenic plants being reported using particle bombardment nearly 20 years ago and Agrobacterium transformations becoming routine a few years later [1–6]. However, there remains considerable debate about the respective advantages and disadvantages of these alternative
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_17, © Springer Science+Business Media New York 2014
201
202
Caroline A. Sparks and Huw D. Jones
DNA-delivery methods. There are numerous reports that claim transformation using Agrobacterium tumefaciens tends to give events with single or low transgene copy number with simple insertion patterns. However, it is also well accepted that unwanted “plasmid backbone” (i.e., non T-DNA) sequences are also often integrated into the host genome. In addition, there is a report of Agrobacterium chromosomal DNA also being inserted along with the intended T-DNA [7]. In contrast, it is reported that transformation via biolistics results in multi-copy events with more complex insertion patterns. However, this conclusion has been disputed [8] and can be reduced by using shorter linearized fragments of DNA rather than circular plasmids [9] which simultaneously removes the possibility of plasmid backbone being incorporated into the insertion [10]. These issues become particularly important when the events in question are intended for commercialization and in this case, regardless of the method of transformation, it is common to generate several tens of independent events and screen these for simple, single copy insertions that do not interrupt a native gene or generate other unintended effects. For wheat, regardless of the method of transformation, the procedure can be conveniently divided into two parts: those steps that ensure effective DNA transfer and integration into the plant nuclear genome and those that allow the regeneration and selection of viable adult plants from the transformed cells. We present here two linked chapters. This chapter provides a detailed protocol for the transformation of wheat using the Bio-Rad PDS1000/He particle bombardment device to deliver DNA-coated gold powder to freshly isolated immature embryos. The induction of embryogenic callus, the regeneration and selection steps are described to produce young, fertile wheat plants in 12–16 weeks. Seeds that have inherited the introduced DNA from the primary transgenic plants can be collected after a further 4–5 months. The adjoining linked chapter describes a protocol for Agrobacterium transformation.
2
Materials
2.1 Donor Plants and Surface Sterilization of Immature Seeds
1. Wheat seeds (Triticum aestivum L.) var. Cadenza (see Note 1) are sown on a fortnightly basis to provide a regular supply of good quality donor material which is essential for reliable transformation efficiencies. Plants are grown 5 per 21 cm pot [Nursery Trades (Lea Valley) Ltd., UK] in Rothamsted Prescription mix soil (see Note 2) in controlled environment rooms with 18°C day/15°C night temperatures and a 16 h photoperiod provided by banks of 400 W hydrargyrum quartz iodide (HQI) lamps (Osram Ltd., UK) generating a light intensity of ~700 μmol/m2/s photosynthetically active radiation (PAR) at the pot surface. Relative humidity is maintained
Wheat Transformation by Particle Bombardment
203
at 50–70 %. Pests and disease are kept to a minimum by good housekeeping practices but Amblyseius caliginosus [Nursery Trades (Lea Valley) Ltd., UK] is used routinely to control thrips and the fungicide Talius (DuPont (U.K.) Ltd, UK) is sprayed as a mildew preventative when plants are approximately 4 weeks old. Pots are initially top watered by hand then watered via a flood-bench automated watering system. Plants are stripped to leave the 5 strongest tillers when the plants are 5–6 weeks old. 2. Sterilizing agents: 70 % (v/v) aqueous ethanol, 10 % (v/v) aqueous bleach (see Note 3) and sterile, distilled water. 2.2
Media
All chemicals are from Sigma-Aldrich unless otherwise stated and tissue culture tested or analytical grade chemicals are used. For all stock solutions and media use polished water with a purity of 18.2 MΩ/cm. Sterilization is carried out by autoclaving at 121 °C for 15 min or filter sterilization using a 0.22 μm syringe filter (Sartorius Stedim UK Ltd., UK) or, for larger volumes, MediaKap filters (Medicell International Ltd., UK). Stock solutions are stored at 4 °C for 1–2 months or frozen at −20 °C for up to a year provided no freezing–thawing has occurred (see Note 4). The following stock solutions of the basal salt mixtures, amino acids, and vitamins are required to make the stock plant culture media (see Subheading 2.2, items 7–8). 1. MS macrosalts (×10): 16.5 g/L NH4NO3, 19.0 g/L KNO3, 3.7 g/L MgSO4 ⋅ 7H2O, 1.7 g/L KH2PO4, 4.4 g/L CaCl2 ⋅ 2H2O (see Note 5). Sterilize by autoclaving and store at 4 °C. 2. L7 macrosalts (×10): 2.5 g/L NH4NO3, 15.0 g/L KNO3, 3.5 g/L MgSO4⋅7H2O, 2.0 g/L KH2PO4, 4.5 g/L CaCl2⋅2H2O (see Note 5). Sterilize by autoclaving and store at 4 °C. 3. L microsalts (×1,000): 17.05 g/L MnSO4⋅H2O (see Note 6), 7.5 g/L ZnSO4⋅7H2O, 5.0 g/L H3BO3, 0.75 g/L KI, 0.25 g/L Na2MoO4⋅2H2O, 0.025 g/L CuSO4⋅5H2O, 0.025 g/L CoCl2⋅6H2O. Sterilize by autoclaving and store at 4 °C. 4. 3AA amino acids (×25): 18.75 g/L L-glutamine (see Note 7), 3.75 g/L L-proline, 2.5 g/L L-asparagine. Store at −20 °C in 40 mL aliquots. 5. MS vitamins (– glycine) (×1,000): 500 mg/L pyridoxine HCl, 500 mg/L nicotinic acid, 100 mg/L thiamine HCl. Prepare 100 mL volume, filter-sterilize, and store at 4 °C. 6. L vitamins/inositol (×200): 40.0 g/L myo-inositol, 2.0 g/L thiamine HCl, 0.2 g/L nicotinic acid, 0.2 g/L pyridoxine HCl, 0.2 g/L pantothenic acid (hemi-calcium salt), 0.2 g/L
204
Caroline A. Sparks and Huw D. Jones
ascorbic acid. Prepare 100 mL volume, filter-sterilize, and store at −20 °C in 40 mL aliquots. The following plant culture stock media are prepared from stock solutions (see Subheading 2.2, items 1–6) at 2× concentration to allow mixing 1:1 with double strength Agargel for preparation of final solidified media plates. 7. MSS 3AA/2 9%S (×2): 200 mL/L MS macrosalts, 2 mL/L L microsalts, 2 mL/L MS vitamins (– glycine), 40 mL 3AA solution, 20 mL/L ferrous sulfate chelate solution (×100), 200 mg/L myo-inositol, 180 g/L sucrose. Adjust pH to 5.7 with 5 M NaOH or KOH, filter-sterilize, and store at 4 °C (see Notes 8 and 9). 8. R (×2): 200 mL/L L7 macrosalts, 2 mL/L L microsalts, 10 mL/L L vitamins/inositol, 20 mL/L ferrous sulfate chelate solution (×100), 60 g/L maltose. Adjust pH to 5.7 with 5 M NaOH or KOH, filter-sterilize, and store at 4 °C (see Note 9). 9. Agargel (×2): 10 g/L in water. Prepare in 400 mL volumes and sterilize by autoclaving. Store at room temperature and melt in a microwave prior to use (see Note 10). Supplements are prepared and aliquotted into appropriate amounts for storage. These additions are introduced to media just prior to pouring plates. 10. 2,4-Dichlorophenoxyacetic acid (2,4-D): Prepare at 1 mg/ mL, initially dissolving in ethanol then adding distilled water to volume. Filter-sterilize and store at 4 °C. 11. Silver nitrate (AgNO3): Prepare at 20 mg/mL in distilled water. Filter-sterilize and store at −20 °C in the dark (see Note 11). 12. Copper sulfate (CuSO4 ⋅ 5H2O): Prepare at 25 mg/mL (100 mM) in distilled water. Filter-sterilize and store at 4 °C. 13. Zeatin (mixed isomers): Prepare at 10 mg/mL, initially dissolving in 1 M HCl and adding distilled water to volume. Filter-sterilize and store at −20 °C. 14. Glufosinate ammonium (PPT) (Greyhound Chromatography and Allied Chemicals, UK): Prepare at 10 mg/mL in distilled water. Filter-sterilize and store at −20 °C. To prepare the final culture media for plates, stock media (see Subheading 2.2 items 7–8) are mixed 1:1 with double strength Agargel (see Subheading 2.2, item 9) for solidification, adding supplements (see Subheading 2.2 items 10–14) before pouring (see Note 12). 15. Induction medium: Mix an equal quantity of MSS 3AA/2 9%S (×2) with melted Agargel (×2). Add 0.5 mg/L 2,4-D and 10 mg/L AgNO3 (see Note 11). Pour into 9 cm Petri dishes (~28 mL/dish).
Wheat Transformation by Particle Bombardment
205
16. Regeneration medium: Mix an equal quantity of R (×2) with melted Agargel (×2). Add 5 mg/L zeatin, 0.1 mg/L 2,4-D, and 25 mg/L CuSO4 (see Note 13). Pour into 9 cm Petri dishes (~28 mL/dish). 17. Selection medium (1): Mix an equal quantity of R (×2) with melted Agargel (×2). Add 5 mg/L zeatin and 4 mg/L PPT. Pour into 9 cm Petri dishes (~28 mL/dish). 18. Selection medium (2): Mix an equal quantity of R (×2) with melted Agargel (×2). Add 4 mg/L PPT. Pour into GA-7 Magenta vessels (Sigma-Aldrich) (~60 mL/vessel). 2.3 Materials for Particle Bombardment
1. Gold particles: 0.6 μm (submicron) gold powder (Bio-Rad Laboratories, UK) (see Note 14). 2. Spermidine (free base): Prepare a 1 M solution by dissolving 1 g spermidine powder in 6.89 mL sterile distilled water. Aliquot into 50 μl volumes and store immediately at −80 °C. For the working stock, dilute an aliquot of 1 M spermidine with sterile distilled water to give 0.1 M, aliquot into 25 μl volumes and store immediately at −20 °C (see Note 15). 3. Calcium chloride (CaCl2⋅2H2O): Prepare at 2.5 M by dissolving 3.67 g in 10 mL distilled water. Filter-sterilize, aliquot into 55 μl volumes, and store at −20 °C. 4. Plasmid DNA: 1 mg/mL in sterile distilled water or sterile TE buffer (pH 8.0) prepared using Qiagen Maxi-prep kit (Qiagen Ltd, UK) or similar. Store in 20 μl aliquots at −20 °C (see Notes 16–18). 5. Sterilizing agents: 100 % ethanol and 10 % (v/v) aqueous Savlon® (Novartis Consumer Healthcare UK Ltd, UK). 6. 650 psi rupture discs (see Note 19), macrocarriers, macrocarrier holders, stopping screens (all Bio-Rad Laboratories, UK).
3
Methods The following method has been optimized for transformation of immature embryos which are the most responsive of the limited range of explants with regeneration potential in wheat (see Note 20).
3.1 Collection and Surface Sterilization of Immature Seeds
Wheat ears are collected from plants in controlled environment donor rooms (see Subheading 2.1) at 12–16 days post anthesis (see Note 21). Separate the immature seeds from the surrounding panicles (see Note 22, Fig. 1a) and surface-sterilize by rinsing in 70 % (v/v) aqueous ethanol for 3–5 min, followed by 10 min in 10 % (v/v) aqueous bleach with occasional gentle shaking. Rinse liberally with sterile distilled water several times then drain to leave the surface-sterilized seeds moist but not immersed in water (see Note 23).
206
Caroline A. Sparks and Huw D. Jones
Fig. 1 Isolation of immature embryos for bombardment. (a) Immature seeds are separated from the panicles of the two outer spikelets. (b) An isolated wheat immature embryo. (c) The embryo axis is removed and the embryos plated scutellum-side uppermost in the central target area of a Petri dish of Induction medium ready for bombardment. Scale bar = 1 mm approximately 3.2 Isolation of Immature Embryos
Isolate the immature embryo from the seed, removing the embryo axis to prevent precocious germination (see Note 24). The embryos should be 0.5–1.5 mm in length and translucent in appearance (see Note 25, Fig. 1b). Once the axis has been removed, place it scutellum-side uppermost onto Induction medium in a 9 cm Petri dish plating 30 embryos per plate in the central 2 cm diameter target area (see Note 26, Fig. 1c). Incubate at 22–23 °C in the dark for 1–2 days’ pre-culture prior to bombardment (see Note 27).
3.3 Preparation of Gold Particles
Gold powder is initially washed, resuspended fully in water, and aliquotted into small volumes for storage. Individual aliquots are then coated with DNA just prior to bombardment. Prepare gold particle stocks as follows. see below: 1. Weigh 20 mg 0.6 μm gold particles (see Note 28) into a sterile Eppendorf tube. Add 1 mL 100 % ethanol and sonicate for 2 min. Centrifuge in a microfuge at top speed for 3 s and remove the supernatant. Repeat the ethanol wash twice more. 2. After removing the final ethanol supernatant, add 1 mL sterile distilled water, sonicate for 2 min and centrifuge in a microfuge at top speed for 3 s. Remove the supernatant and repeat the sterile distilled water wash once more. 3. Resuspend the gold particles in 1 mL sterile distilled water by vortexing. Aliquot 50 μl amounts into sterile 1.5 mL Eppendorf tubes, vortexing between taking each aliquot to ensure an equal distribution of particles. Store at −20 °C. The following steps for coating of gold particles with plasmid DNA should be carried out on ice in a sterile environment immediately prior to bombardment: 4. Defrost a 50 μl aliquot of prepared gold particles (see Subheading 3.3, steps 1–3, Note 29). Sonicate for 1 min to resuspend the particles fully (see Note 30).
Wheat Transformation by Particle Bombardment
207
5. Add up to 5 μl plasmid DNA (1 mg/mL) (see Notes 17, 18, 31 and 32). Vortex for a few seconds to fully mix the DNA with the particles. 6. In the cap of the Eppendorf tube, mix 50 μl 2.5 M Calcium chloride and 20 μl 0.1 M spermidine. Close the lid carefully to avoid displacing the mixture then tap the tube down and immediately vortex to mix all the components together (see Note 33). 7. Centrifuge in a microfuge at full speed for 3–5 s. Remove the supernatant promptly and discard. 8. Resuspend the gold pellet gradually in 150 μl 100 % ethanol, scraping the sides of the tube with the pipette tip and working the pellet back into solution (see Note 34). Vortex briefly then centrifuge in a microfuge at top speed for 3–5 s to pellet the gold. Remove the supernatant promptly. 9. Resuspend in a final volume of 85 μl 100 % ethanol (see Note 35). If bombardment is not to be carried out immediately, seal the tube with Parafilm® (Fisher Scientific, UK) to prevent evaporation of ethanol and store the particles on ice (see Note 36). 3.4 Operation of PDS-1000/He Particle gun
The method describes DNA delivery using the Bio-Rad PDS1000/He particle gun according to manufacturer’s instructions. This is a high pressure device using compressed helium gas which accelerates particles to high velocity: appropriate safety precautions should be taken when operating this equipment. Carry out the following steps to prepare the particle gun components: 1. The following settings are used as standard for this procedure (see Note 37): 2.5 cm gap (distance between rupture disc and macrocarrier), 5.5 cm target distance (distance between stopping screen and target plate), 0.8 cm stopping plate aperture (distance between macrocarrier and stopping screen), 28–29″ Hg vacuum, 5.0 vacuum flow rate, 4.5 vacuum vent rate. 2. Prior to operation of the gun, surface sterilize all equipment and component parts using 90 % (v/v) aqueous ethanol. Spray out the gun chamber, microcarrier launch assembly, rupture disc retaining cap, target shelf, and red plastic seating tool and allow the ethanol to evaporate completely. 3. Dip the macrocarriers, macrocarrier holders, stopping screens, and rupture discs in 100 % ethanol and place on a mesh rack to allow the ethanol to evaporate completely (see Note 38). Once dried, place the macrocarrier holders in sterile Petri dishes and mount a macrocarrier into each one using the seating tool to fix it under the rim (see Note 39). 4. Briefly vortex the DNA-coated gold particles (see Subheading 3.3, steps 4–9), take a 5 μl aliquot and drop centrally onto a
208
Caroline A. Sparks and Huw D. Jones
Fig. 2 PDS/1000-He particle gun component parts. (a) Inverting the macrocarrier holder containing a macrocarrier with DNA-coated gold particles over a stopping screen within the fixed nest of the microcarrier launch assembly. (b) Indented mesh pattern on a macrocarrier left by the stopping screen after a successful shot. (c) Burst rupture disc being removed from the rupture disc retaining cap following a shot
macrocarrier in its holder (see Note 40). Leave on a non-vibrating surface and allow the ethanol to evaporate slowly until a fine film of dried gold remains (see Note 41). Use the prepared macrocarriers as soon as possible after drying (see Note 42). 5. Open the valve on the helium cylinder and turn the regulator adjusting screw to give ~200 psi higher delivery pressure than the rupture disc to be used (see Note 43). Carry out the following steps for the assembly of particle gun components. 6. Load a rupture disc into the rupture disc retaining cap making sure that the disc is seated securely in the recess at the base. Screw the retaining cap onto the gas acceleration tube and tighten firmly using the mini torque wrench (see Note 44, Fig. 3). 7. Place a stopping screen into the fixed nest onto the stopping screen support (see Note 45). Invert a macrocarrier holder with macrocarrier loaded with DNA-coated gold and place above the stopping screen on the top rim of the fixed nest; the macrocarrier should be positioned such that the gold particles are facing down towards the stopping screen (see Fig. 2a). Keep the macrocarrier holder in place using the retaining ring. Mount the whole microcarrier launch assembly into the gun chamber using the second shelf from the top for a gap of 2.5 cm (see Subheading 3.4, step 1, Fig. 3). 8. Place a Petri dish containing a sample on the target shelf and remove the lid. Mount the target shelf on the fourth shelf from the top to give a target distance of 5.5 cm (see Subheading 3.4, step 1, Fig. 3). Close the chamber door. Carry out the following steps to fire the particle gun. 9. Draw a vacuum of 28–29″ Hg in the gun chamber (see Note 46). Once reached, switch the vacuum control switch to the “Hold” position (see Note 47).
Wheat Transformation by Particle Bombardment
209
Fig. 3 Interior of the PDS/1000-He particle gun chamber showing assembled component parts prior to a shot
10. Press and hold the Fire switch which allows helium to enter the gas acceleration tube. Observe the helium pressure gauge and note at what pressure the rupture disc bursts; it should burst within 10 % of the rupture pressure specified, i.e., 650 psi (see Note 48). 11. Release the Fire switch immediately after the shot and set the vacuum control switch to “Vent” (middle position) to release the vacuum from the chamber (see Note 49). Carry out the following steps to disassemble the particle gun components: 12. Remove the sample, replace the lid, and set aside. 13. Remove the microcarrier launch assembly. Unscrew the retaining ring, remove the macrocarrier holder, and place into 100 % ethanol to re-sterilize. Remove the macrocarrier which has been released from the holder and discard (see Note 50, Fig. 2b). Remove the stopping screen and place in 100 % ethanol to re-sterilize. 14. Using the mini torque wrench, loosen the rupture disc retaining cap then unscrew fully by hand. Remove the burst rupture disc and discard (see Note 51, Fig. 2c). 15. Repeat the assembly/disassembly process for further shots (see Subheading 3.4, steps 6–15) including one or more control shots with gold not coated with DNA (see Subheading 3.3 steps 4–9, Notes 32 and 52). Carry out the following steps to complete the bombardment: 16. Once all experimental plates have been bombarded, close the main valve on the helium cylinder and release the regulator
210
Caroline A. Sparks and Huw D. Jones
adjusting screw. Draw a vacuum in the chamber of ~10″ Hg such that the Fire switch illuminates. Press and release the Fire switch several times until the pressure is drained from the regulator as shown by reducing pressure on the regulator gauge. Turn off the vacuum pump and gun. 17. Drain 100 % ethanol from the macrocarrier holders and stopping screens, rinse in water, and immerse in 10 % (v/v) aqueous Savlon®. Sonicate for 5–10 min to shear any remaining DNA and so prevent any carry over to subsequent experiments. 18. Spray out the gun chamber with 90 % ethanol and clean all components, paying particular attention to wiping round the rupture disc retaining cap and gas acceleration tube connections to remove any residual plastic left from the rupture discs. 3.5 Tissue Culture and Selection of Immature Embryos Following Bombardment
Once bombarded, the embryos need to pass through various tissue culture phases to induce embryogenic callus from which plantlets can be regenerated. In order to preferentially select transformed tissues, the appropriate herbicide or antibiotic for the selectable marker gene is included in the media during the later stages and only plantlets surviving selection are potted to soil for analysis. Steps for the induction of embryogenic callus: 1. Following bombardment, spread the embryos more evenly across the medium dividing between three plates of Induction medium, i.e., 10 per plate (see Note 53). 2. Incubate in the dark at 22–23 °C for 3–4 weeks (see Notes 54–56). Steps for the regeneration of plantlets. 3. After 3–4 weeks of dark culture the embryos should have formed embryogenic calli. Transfer responsive calli to Regeneration medium to initiate regeneration from the somatic embryos. Calli should be transferred whole and not broken up at this stage. 4. Incubate the cultures again at 22–23 °C but now in the light for 3–4 weeks (see Notes 54 and 55, Fig. 4b). Steps for the Selection of transformants. 5. First selection: Once good shoots are developing from the calli, transfer them to Selection medium (1) (see Notes 57– 59). If the regenerating calli are large they should be distributed between a number of dishes to prevent subsequent over-crowding. Calli should still be maintained as one piece at this stage unless they fall apart naturally in which case sections from the same initial callus should be kept together in order to track possible clonal material (see Note 60). Incubate for 3–4 weeks at 22–23 °C in the light (see Notes 54 and 55).
Wheat Transformation by Particle Bombardment
211
Fig. 4 Growth and development of transformed wheat plants. (a) Transient expression of the DsRed reporter gene driven by the maize Ubiquitin promoter + intron [11] in an immature embryo 4 days after bombardment. (b) Embryogenic callus after 2 week’s culture on Regeneration medium in the light. (c) Plantlets surviving or succumbing to selection on Selection medium (2) in a Magenta vessel. (d) Confirmed transgenic plants in GM containment glasshouse showing normal development and fertility. Scale bar = 1 mm approximately
6. Second selection: After 3–4 weeks the effect of selection on the cultures should be apparent; some bleaching of leaves will have occurred and roots may be stunted (see Note 59). Any plantlets which remain green with reasonable root structures should be transferred to Selection medium (2) in MagentaTM vessels for expansion of leaves and roots, placing four to six plantlets per MagentaTM (see Note 61). At this stage it should be possible to divide calli, separating out individual plantlets, but these should be labelled as possible clones (see Note 60). Incubate for a further 3–4 weeks at 22–23 °C in the light. Steps for transferring putative transgenic plants to soil and analysis. 7. Following the second round of selection, transformed plantlets should be fairly obvious in the MagentaTM vessels as those which are green and strong with developed root systems (see Fig. 4c). Remove the plantlets from the MagentaTM vessel, rinsing the roots with water to remove Agargel if necessary, and pot into 8 cm square pots containing moistened Rothamsted prescription mix soil (see Notes 2 and 61). 8. Place the pots in a propagator for 1–2 weeks to create a humid environment for the plantlets to become established (see Note 62) and grow in a suitable GM containment glasshouse (see Note 63). 9. After 2–3 weeks plants should be sufficiently strong to take a leaf sample for genomic DNA extraction and analysis by PCR. 10. Re-pot confirmed positive transgenic plants to soil in 13 cm diameter pots (see Note 2) and grow to maturity in a GM containment glasshouse (see Note 63). Transformed plants usually have no morphological differences to control plants apart from the occasional effects of somaclonal variation from tissue culture: they are generally normal, fertile plants with good seed set (see Fig. 4d).
212
4
Caroline A. Sparks and Huw D. Jones
Notes 1. Although this protocol is typically used for the bread wheat variety Cadenza it can be applied to other wheat varieties but efficiencies may be affected. Over 30 different elite wheat genotypes have been transformed at Rothamsted with some measure of success. 2. Rothamsted prescription mix soil contains: 75 % fine grade peat, 12 % screened sterilized loam, 10 % 6 mm screened limefree grit, 3 % medium vermiculite, 2 kg Osmocote Plus/m3 (slow-release fertilizer, 15 N/11 P/13 K plus micronutrients), 0.5 kg PG mix/m3 (14 N/16 P/18 K granular fertilizer plus micronutrients) from Petersfield Products, UK. 3. Ordinary commercial thin bleach can be used which generally has a sodium hypochlorite content of 4–6 %. 4. Some salts may come out of solution in cold storage so check the solutions and shake before use to resuspend fully if necessary. 5. CaCl2 ⋅ 2H2O should be dissolved in water prior to addition to the other components. 6. MnSO4⋅H2O is the most commonly available form of this salt but if a different hydrated state is used, the amount to add must be calculated to reflect this. 7. L-glutamine can be difficult to dissolve. If this is the case, dissolve separately at pH 9.0 prior to mixing with the other components. 8. Although this transformation protocol describes methods most suitable for bread wheat, durum wheat (Triticum turgidum L.) can also be transformed using this system but with the notable variation that sucrose is adjusted to 4.5 % in the MSS 3AA/2 9%S induction medium for embryogenic callus formation. 9. The osmolarity of stock media should be within the ranges: MSS 3AA/2 9%S (×2) 800–1,100 mOsM and R (×2) 270–300 mOsM. 10. When preparing Agargel it should be shaken before and after autoclaving to ensure even setting which makes subsequent remelting in a microwave easier. Following microwaving, always cool the gelling agent to approximately 50 °C prior to combining with the media solutions. 11. Silver nitrate acts as stress inducing agent in the initial induction phase to promote embryogenesis. It is light sensitive so stocks and any media containing it should be stored in the dark. 12. Ideally plates should be poured and used as fresh as possible yet should be allowed to incubate for a few days at room tem-
Wheat Transformation by Particle Bombardment
213
perature to check for contamination. If stored at 4 °C they can be kept for 2–3 weeks. 13. Copper sulfate acts as a stress-inducing agent in the regeneration phase to promote shooting and can be used in the range 12.5–25 mg/L (50–100 μM). If regeneration is good, 12.5 mg/L may be sufficient as the cultures can become too leafy at 25 mg/L. 14. Although tungsten powder was commonly used with original biolistics equipment, the inert metal gold is now the preferred microcarrier. Different particle sizes are available but small, uniform submicron gold particles (average 0.6 μm diameter) have been found to be best for the small cell size of wheat. 15. Spermidine solutions are hygroscopic, oxidizable, and deaminate with time and consequently should be maintained below −20 °C (preferably at −80 °C) and fresh stocks made regularly. Once thawed any unused aliquots should be discarded. 16. The plasmid can carry one or more gene cassettes. Although pUC-based vectors are commonly used, binary vectors used for Agrobacterium-mediated transformation can also be introduced successfully using particle bombardment. Another alternative is to bombard the gene cassette(s) as an isolated DNA fragment and so remove the plasmid backbone which generally carries an antibiotic resistance gene for selection in E. coli, often regarded as undesirable. 17. A selectable marker gene must also be incorporated either on the same plasmid as the gene(s) of interest or as a separate plasmid which is co-precipitated onto the gold. The selectable marker gene allows selection of transformed tissues, examples being the bar gene (as described in this chapter) which confers resistance to glufosinate ammonium-based broad spectrum herbicides such as Basta®, Challenge®, Liberty®, Harvest®, etc., or nptII which confers resistance to the antibiotics geneticin disulfate (G418), kanamycin, neomycin etc. 18. A reporter gene can also be included, e.g., UidA, GFP, or DsRed to monitor transient and/or stable transformation. As is the case for the selectable marker gene, this can either be on the same plasmid as the gene of interest or on a different plasmid which is co-bombarded. 19. Rupture discs are available from Bio-Rad Laboratories as 450, 650, 900, 1,100, 1,350, 1,550, 1,800, 2,000, and 2,200 psi. A range of rupture pressures should be assessed when optimizing any system; for wheat 650 psi generally gives the best, most consistent results but 450 or 900 psi could also be used. 20. Immature inflorescences are an alternative target tissue which are also responsive in tissue culture and can therefore be transformed. However modifications may need to be made to the media for best results and efficiencies are generally lower.
214
Caroline A. Sparks and Huw D. Jones
21. Immature seeds are at about the right stage when they have a whitish bloom on the seed surface and the endosperm is just solidified but not too hard. A few spikelets can be opened at the time of ear collection to determine whether the seeds are at the correct stage. 22. Generally only the outer caryopses of each spikelet are used and none are taken from the tip or base of the ear due to asynchronous seed development. 23. Ideally surface-sterilized seeds should be used the same day. If material needs to be collected in advance it is better to store whole ears at 4 °C with the stems in water prior to removing the seeds; however, it is not recommended to store these longer than overnight. 24. The immature scutellum is the callus-forming tissue; if the embryo axis is left intact it will be prone to germinate on the culture medium thereby preventing callus formation. A practised hand can cut off the embryo axis through the seed coat, leaving a hole through which the scutellum can be removed. 25. Embryos in the size range 0.5–1.5 mm are ideal for transformation via particle bombardment which is slightly smaller than the size advised for Agrobacterium-mediated transformation. Larger embryos may respond if they are still translucent. The size of the embryos is not quite as critical if being used to monitor transient transformation only. 26. Using the gun parameters described, the gold particles typically target a central circular area of ~2 cm diameter, and therefore to maximize transformation the isolated scutella are located in this region. 27. Embryos are incubated prior to bombardment to allow them to recover from the isolation procedure and also because preplasmolysis of cells on the high sucrose medium may increase their ability to withstand bombardment. Experiments to assess the effect of pre-culture have shown that the optimum time for incubation is 1–2 days; transformation is possible after longer periods of pre-incubation (e.g., 4 days) but efficiencies will be reduced. 28. Gold can be prepared at a final concentration of 40 mg/mL if desired (as recommended in the PDS-1000/He manual) but 20 mg/mL has been found to be just as effective for DNA delivery. If prepared at 40 mg/mL, it could give the choice of being used at this concentration or each aliquot diluted by half with sterile distilled water prior to gold coating. 29. 50 μl starting volume of gold will be sufficient for approximately 10–12 shots. The volume of gold and other components can be scaled up or down according to the number of shots required.
Wheat Transformation by Particle Bombardment
215
30. Sonication should resuspend the gold and disrupt any clumps but it should not be sonicated for too long as this may lead to particle agglomeration. 31. The amount of plasmid DNA added should not exceed 5 μg per 50 μl gold as excess DNA can cause clumping of particles. If co-precipitating plasmids, calculate equimolar amounts and use a total of 5 μg DNA. Commonly for co-bombardment of a plasmid of interest and a selectable marker plasmid, a 1.5:1 ratio is used to skew for the gene of interest; plants which survive selection will then have a higher probability of having the gene of interest if containing the marker gene. 32. A gold preparation without DNA should also be prepared at the same time to act as a control. 33. The process of precipitation of DNA onto the gold is very rapid. The calcium chloride and spermidine help to stabilize, precipitate, and bind the DNA to the gold but are mixed first to ensure an even coating, otherwise clumping of particles may occur. 34. The DNA-coated gold suspension needs to be as smooth as possible so it is essential that the particles are resuspended fully at this stage without clumps. Scraping with the pipette tip and repeatedly drawing up and expelling the solution is more effective than vortexing. If the gold appears to be very clumped it is preferable to discard the preparation and start again, checking carefully the amount and concentration of DNA added. 35. The solution should not be aspirated too much during this resuspension step as the ethanol will evaporate and increase the final concentration of particles and may also lead to insufficient volume for the shots required. 36. Ideally the coated particles should be prepared and used as soon as possible but if a number of different treatments are prepared, seal and store the tubes on ice prior to loading onto the macrocarriers. 37. These settings have been optimized for bombardment of wheat immature embryos. Adjustments may need to be made for alternative species or explants. 38. Rupture discs are composed of laminate layers of plastic which may separate if soaked in ethanol for extended periods; as a consequence they should only be dipped briefly in ethanol to sterilize. 39. The macrocarrier must fit completely under the rim of the holder as any gaps will allow the escape of helium. If not secured correctly, the macrocarrier will not be released from the holder and hit the stopping screen with the required pressure to release the gold particles with expected velocity.
216
Caroline A. Sparks and Huw D. Jones
40. In order to ensure that the particles are fully resuspended and equal amounts are placed on each macrocarrier it is important to vortex the gold preparation between taking each sample. 41. Vibration may cause gold particles to clump so the macrocarriers within their Petri dishes should be placed outside of the flow hood on a non-vibrating surface. The macrocarriers can be examined microscopically prior to use to monitor the spread and quality of particles, discarding any on which the gold is unsatisfactory. 42. Macrocarriers should be used quite soon after the ethanol has evaporated so only prepare a few macrocarriers at a time. 43. The regulator must allow sufficient helium through the system to build up and burst the rupture disc: setting the regulator to ~200 psi higher than the rupture disc to be used should be suitable. 44. If the retaining cap is not firmly tightened the rupture disc may become dislodged which means the gun may fail to fire or fires at a lower than expected pressure. 45. If the stopping screen is omitted the released macrocarrier will pass through the hole in the fixed nest straight onto the target tissue, creating rather a mess and potentially contaminating the plate. 46. By creating a vacuum in the chamber there is less air resistance which allows the gold particles to maintain high speed following release from the macrocarrier. 26–30″ Hg is the vacuum range recommended for plants by the manufacturer; in this protocol 28–29″ Hg is routinely drawn. 47. The “Fire” switch should illuminate once the vacuum reaches ~5″ Hg. 48. A metering valve is present on the solenoid valve assembly to regulate the rate of fill of the gas acceleration tube. It should take ~12–15 s to build to bursting pressure. When the rupture disc bursts the macrocarrier is released from its holder onto the stopping screen thus dispersing the particles onto the target tissue. The actual pressure at which the rupture disc bursts should be monitored to confirm a successful shot has occurred. 49. If the Fire switch is released too early no further helium will be discharged and the rupture disc will not burst. To abort a shot prior to the rupture disc bursting, release the Fire switch and set the vacuum switch to Vent. 50. A good mesh indentation should be apparent on the macrocarrier from its contact with the stopping screen if the shot has worked correctly. 51. If the rupture disc is not in the retaining cap it may still be attached to the gas acceleration tube inside the gun. The disc must be removed otherwise it will interfere with further shots.
Wheat Transformation by Particle Bombardment
217
52. Control plates should be included in experiments to allow monitoring of callus formation and regeneration following bombardment. One or more plates of embryos should be bombarded with gold but without DNA such that the embryos have identical treatment but for the presence of DNA. 53. The embryos are spread across different media plates to reduce competition for nutrients and allow for growth. 54. Cultures are incubated in a controlled environment room at 22–23 °C. Cool white fluorescent tubes generate lighting levels of ~250 μmol/m2/s photosynthetically active radiation (PAR) for a 12 h photoperiod. Where dark culture is required, solid trays are covered with foil. 55. Each tissue culture phase can take 3–5 weeks; cultures should be monitored and with experience it will be possible to determine when it is most appropriate to move to the next medium. 56. In order to aid optimization of transformation protocols a reporter gene can be included to act as a visual marker of transformation success (see Note 18). pAHC25 [11] is a convenient plasmid to use having both the selectable marker cassette (bar) but also a cassette containing the β-Glucuronidase gene (UidA). Some embryos can be sacrificed several days postbombardment and assayed for transient gus activity [12] to determine the effectiveness of the bombardment process. Alternative reporter genes also exist which do not require a destructive assay and therefore have the advantage that they can be used to monitor viable tissues in culture over a longer period, e.g., GFP or DsRed (see Fig. 4a and Acknowledgements) 57. Calli from control plates (no DNA) should be transferred to Selection media without PPT but some calli should also be put on Selection media including PPT to demonstrate the effectiveness of the selection. 58. “High lid” Petri dishes can be used at this stage by using upturned Petri dish bases as lids; this creates more space for shoots to develop. 59. Glufosinate ammonium (PPT) is used for selection of plants transformed with the bar gene. 4 mg/L PPT used in the Selection media described should effectively terminate growth of non-transformed tissues although this may not be fully obvious until after the second round of selection. The control plates and Magentas will give a good indication of how well the selection is working. If too many non-transformed escapes are identified, 6 mg/L PPT could be used subsequently to provide more stringent selection pressure. 60. Any plantlets originating from the same callus piece should be regarded as clonal until demonstrated otherwise.
218
Caroline A. Sparks and Huw D. Jones
61. A plant generated from a control plate bombarded with gold but without DNA and subsequently grown on media without plant selection should also be transferred and potted up to act as a negative control which has been through the same experimental conditions. 62. Plantlets derived from tissue culture have little or no waxy cuticle so require a humid environment initially to protect them from desiccation whilst the cuticle forms. 63. GM glasshouse conditions are maintained at 18–20 °C day and 14–16 °C night temperatures with a 16-h photoperiod provided by natural light supplemented with banks of SonT 400 W sodium lamps (Osram Ltd., UK) generating a light intensity of 400–1,000 μmol/m2/s photosynthetically active radiation (PAR).
Acknowledgements Rothamsted Research receives strategic funding from the Biotechnological and Biological Sciences Research Council (BBSRC). Thanks go to Ann Blechl (USDA-ARS, USA) and Jorge Dubcovsky (UC Davis, USA) for providing the DsRed reporter gene construct for our wheat transformation studies. References 1. Blechl AE, Jones HD (2009) Transgenic Applications in Wheat Improvement. In: Carver BF (ed) Wheat: Science and Trade. Wiley-Blackwell, Iowa, pp 397–435 2. Jones HD, Doherty A, Wu H (2005) Review of Methodologies and a Protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1:5 3. Jones HD (2005) Wheat transformation: Current technology and applications to grain development and composition. J Cereal Sci 41:137–147 4. Harwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63:1791–1798 5. Shrawat AK, Loerz H (2006) Agrobacteriummediated transformation of cereals: a promising approach crossing barriers. Plant Biotech J 4:575–603 6. Vasil IK (2007) Molecular genetic improvement of cereals: transgenic wheat (Triticum aesitvum L.). Plant Cell Rep 26:1133–1154 7. Ulker B, Li Y, Rosso MG et al (2008) T-DNA– mediated transfer of Agrobacterium tumefa-
8.
9.
10.
11.
12.
ciens chromosomal DNA into plants. Nat Biotech 26:1015–1017 Altpeter F, Baisakh N, Beachy R et al (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327 Fu X, Duc LT, Fontana S et al (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgen Res 9:11–19 Gadaleta A, Giancaspro A, Blechl AE et al (2008) A transgenic durum wheat line that is free of marker genes and expresses 1Dy10. J Cereal Sci 48:439–445 Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen Res 5:213–218 Jefferson RA, Kavanagh TA, Bevan MW (1987) Beta-glucuronidase (Gus) as a sensitive and versatile gene fusion marker in plants. J Cell Biochem 13:3901–3907
1
Introduction The ability to alter the genome of a plant in a precise and predictable manner is a powerful research tool for functional genomics and is fast becoming a key element in the process of commercial varietal improvement. A key component of the genetic modification of crop plants is a robust transformation protocol. Wheat was among the last of the major crops to be transformed with the first fertile transgenic plants being reported using particle bombardment nearly 20 years ago and Agrobacterium transformations becoming routine a few years later [1–6]. However, there remains considerable debate about the respective advantages and disadvantages of these alternative
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_17, © Springer Science+Business Media New York 2014
201
202
Caroline A. Sparks and Huw D. Jones
DNA-delivery methods. There are numerous reports that claim transformation using Agrobacterium tumefaciens tends to give events with single or low transgene copy number with simple insertion patterns. However, it is also well accepted that unwanted “plasmid backbone” (i.e., non T-DNA) sequences are also often integrated into the host genome. In addition, there is a report of Agrobacterium chromosomal DNA also being inserted along with the intended T-DNA [7]. In contrast, it is reported that transformation via biolistics results in multi-copy events with more complex insertion patterns. However, this conclusion has been disputed [8] and can be reduced by using shorter linearized fragments of DNA rather than circular plasmids [9] which simultaneously removes the possibility of plasmid backbone being incorporated into the insertion [10]. These issues become particularly important when the events in question are intended for commercialization and in this case, regardless of the method of transformation, it is common to generate several tens of independent events and screen these for simple, single copy insertions that do not interrupt a native gene or generate other unintended effects. For wheat, regardless of the method of transformation, the procedure can be conveniently divided into two parts: those steps that ensure effective DNA transfer and integration into the plant nuclear genome and those that allow the regeneration and selection of viable adult plants from the transformed cells. We present here two linked chapters. This chapter provides a detailed protocol for the transformation of wheat using the Bio-Rad PDS1000/He particle bombardment device to deliver DNA-coated gold powder to freshly isolated immature embryos. The induction of embryogenic callus, the regeneration and selection steps are described to produce young, fertile wheat plants in 12–16 weeks. Seeds that have inherited the introduced DNA from the primary transgenic plants can be collected after a further 4–5 months. The adjoining linked chapter describes a protocol for Agrobacterium transformation.
2
Materials
2.1 Donor Plants and Surface Sterilization of Immature Seeds
1. Wheat seeds (Triticum aestivum L.) var. Cadenza (see Note 1) are sown on a fortnightly basis to provide a regular supply of good quality donor material which is essential for reliable transformation efficiencies. Plants are grown 5 per 21 cm pot [Nursery Trades (Lea Valley) Ltd., UK] in Rothamsted Prescription mix soil (see Note 2) in controlled environment rooms with 18°C day/15°C night temperatures and a 16 h photoperiod provided by banks of 400 W hydrargyrum quartz iodide (HQI) lamps (Osram Ltd., UK) generating a light intensity of ~700 μmol/m2/s photosynthetically active radiation (PAR) at the pot surface. Relative humidity is maintained
Wheat Transformation by Particle Bombardment
203
at 50–70 %. Pests and disease are kept to a minimum by good housekeeping practices but Amblyseius caliginosus [Nursery Trades (Lea Valley) Ltd., UK] is used routinely to control thrips and the fungicide Talius (DuPont (U.K.) Ltd, UK) is sprayed as a mildew preventative when plants are approximately 4 weeks old. Pots are initially top watered by hand then watered via a flood-bench automated watering system. Plants are stripped to leave the 5 strongest tillers when the plants are 5–6 weeks old. 2. Sterilizing agents: 70 % (v/v) aqueous ethanol, 10 % (v/v) aqueous bleach (see Note 3) and sterile, distilled water. 2.2
Media
All chemicals are from Sigma-Aldrich unless otherwise stated and tissue culture tested or analytical grade chemicals are used. For all stock solutions and media use polished water with a purity of 18.2 MΩ/cm. Sterilization is carried out by autoclaving at 121 °C for 15 min or filter sterilization using a 0.22 μm syringe filter (Sartorius Stedim UK Ltd., UK) or, for larger volumes, MediaKap filters (Medicell International Ltd., UK). Stock solutions are stored at 4 °C for 1–2 months or frozen at −20 °C for up to a year provided no freezing–thawing has occurred (see Note 4). The following stock solutions of the basal salt mixtures, amino acids, and vitamins are required to make the stock plant culture media (see Subheading 2.2, items 7–8). 1. MS macrosalts (×10): 16.5 g/L NH4NO3, 19.0 g/L KNO3, 3.7 g/L MgSO4 ⋅ 7H2O, 1.7 g/L KH2PO4, 4.4 g/L CaCl2 ⋅ 2H2O (see Note 5). Sterilize by autoclaving and store at 4 °C. 2. L7 macrosalts (×10): 2.5 g/L NH4NO3, 15.0 g/L KNO3, 3.5 g/L MgSO4⋅7H2O, 2.0 g/L KH2PO4, 4.5 g/L CaCl2⋅2H2O (see Note 5). Sterilize by autoclaving and store at 4 °C. 3. L microsalts (×1,000): 17.05 g/L MnSO4⋅H2O (see Note 6), 7.5 g/L ZnSO4⋅7H2O, 5.0 g/L H3BO3, 0.75 g/L KI, 0.25 g/L Na2MoO4⋅2H2O, 0.025 g/L CuSO4⋅5H2O, 0.025 g/L CoCl2⋅6H2O. Sterilize by autoclaving and store at 4 °C. 4. 3AA amino acids (×25): 18.75 g/L L-glutamine (see Note 7), 3.75 g/L L-proline, 2.5 g/L L-asparagine. Store at −20 °C in 40 mL aliquots. 5. MS vitamins (– glycine) (×1,000): 500 mg/L pyridoxine HCl, 500 mg/L nicotinic acid, 100 mg/L thiamine HCl. Prepare 100 mL volume, filter-sterilize, and store at 4 °C. 6. L vitamins/inositol (×200): 40.0 g/L myo-inositol, 2.0 g/L thiamine HCl, 0.2 g/L nicotinic acid, 0.2 g/L pyridoxine HCl, 0.2 g/L pantothenic acid (hemi-calcium salt), 0.2 g/L
204
Caroline A. Sparks and Huw D. Jones
ascorbic acid. Prepare 100 mL volume, filter-sterilize, and store at −20 °C in 40 mL aliquots. The following plant culture stock media are prepared from stock solutions (see Subheading 2.2, items 1–6) at 2× concentration to allow mixing 1:1 with double strength Agargel for preparation of final solidified media plates. 7. MSS 3AA/2 9%S (×2): 200 mL/L MS macrosalts, 2 mL/L L microsalts, 2 mL/L MS vitamins (– glycine), 40 mL 3AA solution, 20 mL/L ferrous sulfate chelate solution (×100), 200 mg/L myo-inositol, 180 g/L sucrose. Adjust pH to 5.7 with 5 M NaOH or KOH, filter-sterilize, and store at 4 °C (see Notes 8 and 9). 8. R (×2): 200 mL/L L7 macrosalts, 2 mL/L L microsalts, 10 mL/L L vitamins/inositol, 20 mL/L ferrous sulfate chelate solution (×100), 60 g/L maltose. Adjust pH to 5.7 with 5 M NaOH or KOH, filter-sterilize, and store at 4 °C (see Note 9). 9. Agargel (×2): 10 g/L in water. Prepare in 400 mL volumes and sterilize by autoclaving. Store at room temperature and melt in a microwave prior to use (see Note 10). Supplements are prepared and aliquotted into appropriate amounts for storage. These additions are introduced to media just prior to pouring plates. 10. 2,4-Dichlorophenoxyacetic acid (2,4-D): Prepare at 1 mg/ mL, initially dissolving in ethanol then adding distilled water to volume. Filter-sterilize and store at 4 °C. 11. Silver nitrate (AgNO3): Prepare at 20 mg/mL in distilled water. Filter-sterilize and store at −20 °C in the dark (see Note 11). 12. Copper sulfate (CuSO4 ⋅ 5H2O): Prepare at 25 mg/mL (100 mM) in distilled water. Filter-sterilize and store at 4 °C. 13. Zeatin (mixed isomers): Prepare at 10 mg/mL, initially dissolving in 1 M HCl and adding distilled water to volume. Filter-sterilize and store at −20 °C. 14. Glufosinate ammonium (PPT) (Greyhound Chromatography and Allied Chemicals, UK): Prepare at 10 mg/mL in distilled water. Filter-sterilize and store at −20 °C. To prepare the final culture media for plates, stock media (see Subheading 2.2 items 7–8) are mixed 1:1 with double strength Agargel (see Subheading 2.2, item 9) for solidification, adding supplements (see Subheading 2.2 items 10–14) before pouring (see Note 12). 15. Induction medium: Mix an equal quantity of MSS 3AA/2 9%S (×2) with melted Agargel (×2). Add 0.5 mg/L 2,4-D and 10 mg/L AgNO3 (see Note 11). Pour into 9 cm Petri dishes (~28 mL/dish).
Wheat Transformation by Particle Bombardment
205
16. Regeneration medium: Mix an equal quantity of R (×2) with melted Agargel (×2). Add 5 mg/L zeatin, 0.1 mg/L 2,4-D, and 25 mg/L CuSO4 (see Note 13). Pour into 9 cm Petri dishes (~28 mL/dish). 17. Selection medium (1): Mix an equal quantity of R (×2) with melted Agargel (×2). Add 5 mg/L zeatin and 4 mg/L PPT. Pour into 9 cm Petri dishes (~28 mL/dish). 18. Selection medium (2): Mix an equal quantity of R (×2) with melted Agargel (×2). Add 4 mg/L PPT. Pour into GA-7 Magenta vessels (Sigma-Aldrich) (~60 mL/vessel). 2.3 Materials for Particle Bombardment
1. Gold particles: 0.6 μm (submicron) gold powder (Bio-Rad Laboratories, UK) (see Note 14). 2. Spermidine (free base): Prepare a 1 M solution by dissolving 1 g spermidine powder in 6.89 mL sterile distilled water. Aliquot into 50 μl volumes and store immediately at −80 °C. For the working stock, dilute an aliquot of 1 M spermidine with sterile distilled water to give 0.1 M, aliquot into 25 μl volumes and store immediately at −20 °C (see Note 15). 3. Calcium chloride (CaCl2⋅2H2O): Prepare at 2.5 M by dissolving 3.67 g in 10 mL distilled water. Filter-sterilize, aliquot into 55 μl volumes, and store at −20 °C. 4. Plasmid DNA: 1 mg/mL in sterile distilled water or sterile TE buffer (pH 8.0) prepared using Qiagen Maxi-prep kit (Qiagen Ltd, UK) or similar. Store in 20 μl aliquots at −20 °C (see Notes 16–18). 5. Sterilizing agents: 100 % ethanol and 10 % (v/v) aqueous Savlon® (Novartis Consumer Healthcare UK Ltd, UK). 6. 650 psi rupture discs (see Note 19), macrocarriers, macrocarrier holders, stopping screens (all Bio-Rad Laboratories, UK).
3
Methods The following method has been optimized for transformation of immature embryos which are the most responsive of the limited range of explants with regeneration potential in wheat (see Note 20).
3.1 Collection and Surface Sterilization of Immature Seeds
Wheat ears are collected from plants in controlled environment donor rooms (see Subheading 2.1) at 12–16 days post anthesis (see Note 21). Separate the immature seeds from the surrounding panicles (see Note 22, Fig. 1a) and surface-sterilize by rinsing in 70 % (v/v) aqueous ethanol for 3–5 min, followed by 10 min in 10 % (v/v) aqueous bleach with occasional gentle shaking. Rinse liberally with sterile distilled water several times then drain to leave the surface-sterilized seeds moist but not immersed in water (see Note 23).
206
Caroline A. Sparks and Huw D. Jones
Fig. 1 Isolation of immature embryos for bombardment. (a) Immature seeds are separated from the panicles of the two outer spikelets. (b) An isolated wheat immature embryo. (c) The embryo axis is removed and the embryos plated scutellum-side uppermost in the central target area of a Petri dish of Induction medium ready for bombardment. Scale bar = 1 mm approximately 3.2 Isolation of Immature Embryos
Isolate the immature embryo from the seed, removing the embryo axis to prevent precocious germination (see Note 24). The embryos should be 0.5–1.5 mm in length and translucent in appearance (see Note 25, Fig. 1b). Once the axis has been removed, place it scutellum-side uppermost onto Induction medium in a 9 cm Petri dish plating 30 embryos per plate in the central 2 cm diameter target area (see Note 26, Fig. 1c). Incubate at 22–23 °C in the dark for 1–2 days’ pre-culture prior to bombardment (see Note 27).
3.3 Preparation of Gold Particles
Gold powder is initially washed, resuspended fully in water, and aliquotted into small volumes for storage. Individual aliquots are then coated with DNA just prior to bombardment. Prepare gold particle stocks as follows. see below: 1. Weigh 20 mg 0.6 μm gold particles (see Note 28) into a sterile Eppendorf tube. Add 1 mL 100 % ethanol and sonicate for 2 min. Centrifuge in a microfuge at top speed for 3 s and remove the supernatant. Repeat the ethanol wash twice more. 2. After removing the final ethanol supernatant, add 1 mL sterile distilled water, sonicate for 2 min and centrifuge in a microfuge at top speed for 3 s. Remove the supernatant and repeat the sterile distilled water wash once more. 3. Resuspend the gold particles in 1 mL sterile distilled water by vortexing. Aliquot 50 μl amounts into sterile 1.5 mL Eppendorf tubes, vortexing between taking each aliquot to ensure an equal distribution of particles. Store at −20 °C. The following steps for coating of gold particles with plasmid DNA should be carried out on ice in a sterile environment immediately prior to bombardment: 4. Defrost a 50 μl aliquot of prepared gold particles (see Subheading 3.3, steps 1–3, Note 29). Sonicate for 1 min to resuspend the particles fully (see Note 30).
Wheat Transformation by Particle Bombardment
207
5. Add up to 5 μl plasmid DNA (1 mg/mL) (see Notes 17, 18, 31 and 32). Vortex for a few seconds to fully mix the DNA with the particles. 6. In the cap of the Eppendorf tube, mix 50 μl 2.5 M Calcium chloride and 20 μl 0.1 M spermidine. Close the lid carefully to avoid displacing the mixture then tap the tube down and immediately vortex to mix all the components together (see Note 33). 7. Centrifuge in a microfuge at full speed for 3–5 s. Remove the supernatant promptly and discard. 8. Resuspend the gold pellet gradually in 150 μl 100 % ethanol, scraping the sides of the tube with the pipette tip and working the pellet back into solution (see Note 34). Vortex briefly then centrifuge in a microfuge at top speed for 3–5 s to pellet the gold. Remove the supernatant promptly. 9. Resuspend in a final volume of 85 μl 100 % ethanol (see Note 35). If bombardment is not to be carried out immediately, seal the tube with Parafilm® (Fisher Scientific, UK) to prevent evaporation of ethanol and store the particles on ice (see Note 36). 3.4 Operation of PDS-1000/He Particle gun
The method describes DNA delivery using the Bio-Rad PDS1000/He particle gun according to manufacturer’s instructions. This is a high pressure device using compressed helium gas which accelerates particles to high velocity: appropriate safety precautions should be taken when operating this equipment. Carry out the following steps to prepare the particle gun components: 1. The following settings are used as standard for this procedure (see Note 37): 2.5 cm gap (distance between rupture disc and macrocarrier), 5.5 cm target distance (distance between stopping screen and target plate), 0.8 cm stopping plate aperture (distance between macrocarrier and stopping screen), 28–29″ Hg vacuum, 5.0 vacuum flow rate, 4.5 vacuum vent rate. 2. Prior to operation of the gun, surface sterilize all equipment and component parts using 90 % (v/v) aqueous ethanol. Spray out the gun chamber, microcarrier launch assembly, rupture disc retaining cap, target shelf, and red plastic seating tool and allow the ethanol to evaporate completely. 3. Dip the macrocarriers, macrocarrier holders, stopping screens, and rupture discs in 100 % ethanol and place on a mesh rack to allow the ethanol to evaporate completely (see Note 38). Once dried, place the macrocarrier holders in sterile Petri dishes and mount a macrocarrier into each one using the seating tool to fix it under the rim (see Note 39). 4. Briefly vortex the DNA-coated gold particles (see Subheading 3.3, steps 4–9), take a 5 μl aliquot and drop centrally onto a
208
Caroline A. Sparks and Huw D. Jones
Fig. 2 PDS/1000-He particle gun component parts. (a) Inverting the macrocarrier holder containing a macrocarrier with DNA-coated gold particles over a stopping screen within the fixed nest of the microcarrier launch assembly. (b) Indented mesh pattern on a macrocarrier left by the stopping screen after a successful shot. (c) Burst rupture disc being removed from the rupture disc retaining cap following a shot
macrocarrier in its holder (see Note 40). Leave on a non-vibrating surface and allow the ethanol to evaporate slowly until a fine film of dried gold remains (see Note 41). Use the prepared macrocarriers as soon as possible after drying (see Note 42). 5. Open the valve on the helium cylinder and turn the regulator adjusting screw to give ~200 psi higher delivery pressure than the rupture disc to be used (see Note 43). Carry out the following steps for the assembly of particle gun components. 6. Load a rupture disc into the rupture disc retaining cap making sure that the disc is seated securely in the recess at the base. Screw the retaining cap onto the gas acceleration tube and tighten firmly using the mini torque wrench (see Note 44, Fig. 3). 7. Place a stopping screen into the fixed nest onto the stopping screen support (see Note 45). Invert a macrocarrier holder with macrocarrier loaded with DNA-coated gold and place above the stopping screen on the top rim of the fixed nest; the macrocarrier should be positioned such that the gold particles are facing down towards the stopping screen (see Fig. 2a). Keep the macrocarrier holder in place using the retaining ring. Mount the whole microcarrier launch assembly into the gun chamber using the second shelf from the top for a gap of 2.5 cm (see Subheading 3.4, step 1, Fig. 3). 8. Place a Petri dish containing a sample on the target shelf and remove the lid. Mount the target shelf on the fourth shelf from the top to give a target distance of 5.5 cm (see Subheading 3.4, step 1, Fig. 3). Close the chamber door. Carry out the following steps to fire the particle gun. 9. Draw a vacuum of 28–29″ Hg in the gun chamber (see Note 46). Once reached, switch the vacuum control switch to the “Hold” position (see Note 47).
Wheat Transformation by Particle Bombardment
209
Fig. 3 Interior of the PDS/1000-He particle gun chamber showing assembled component parts prior to a shot
10. Press and hold the Fire switch which allows helium to enter the gas acceleration tube. Observe the helium pressure gauge and note at what pressure the rupture disc bursts; it should burst within 10 % of the rupture pressure specified, i.e., 650 psi (see Note 48). 11. Release the Fire switch immediately after the shot and set the vacuum control switch to “Vent” (middle position) to release the vacuum from the chamber (see Note 49). Carry out the following steps to disassemble the particle gun components: 12. Remove the sample, replace the lid, and set aside. 13. Remove the microcarrier launch assembly. Unscrew the retaining ring, remove the macrocarrier holder, and place into 100 % ethanol to re-sterilize. Remove the macrocarrier which has been released from the holder and discard (see Note 50, Fig. 2b). Remove the stopping screen and place in 100 % ethanol to re-sterilize. 14. Using the mini torque wrench, loosen the rupture disc retaining cap then unscrew fully by hand. Remove the burst rupture disc and discard (see Note 51, Fig. 2c). 15. Repeat the assembly/disassembly process for further shots (see Subheading 3.4, steps 6–15) including one or more control shots with gold not coated with DNA (see Subheading 3.3 steps 4–9, Notes 32 and 52). Carry out the following steps to complete the bombardment: 16. Once all experimental plates have been bombarded, close the main valve on the helium cylinder and release the regulator
210
Caroline A. Sparks and Huw D. Jones
adjusting screw. Draw a vacuum in the chamber of ~10″ Hg such that the Fire switch illuminates. Press and release the Fire switch several times until the pressure is drained from the regulator as shown by reducing pressure on the regulator gauge. Turn off the vacuum pump and gun. 17. Drain 100 % ethanol from the macrocarrier holders and stopping screens, rinse in water, and immerse in 10 % (v/v) aqueous Savlon®. Sonicate for 5–10 min to shear any remaining DNA and so prevent any carry over to subsequent experiments. 18. Spray out the gun chamber with 90 % ethanol and clean all components, paying particular attention to wiping round the rupture disc retaining cap and gas acceleration tube connections to remove any residual plastic left from the rupture discs. 3.5 Tissue Culture and Selection of Immature Embryos Following Bombardment
Once bombarded, the embryos need to pass through various tissue culture phases to induce embryogenic callus from which plantlets can be regenerated. In order to preferentially select transformed tissues, the appropriate herbicide or antibiotic for the selectable marker gene is included in the media during the later stages and only plantlets surviving selection are potted to soil for analysis. Steps for the induction of embryogenic callus: 1. Following bombardment, spread the embryos more evenly across the medium dividing between three plates of Induction medium, i.e., 10 per plate (see Note 53). 2. Incubate in the dark at 22–23 °C for 3–4 weeks (see Notes 54–56). Steps for the regeneration of plantlets. 3. After 3–4 weeks of dark culture the embryos should have formed embryogenic calli. Transfer responsive calli to Regeneration medium to initiate regeneration from the somatic embryos. Calli should be transferred whole and not broken up at this stage. 4. Incubate the cultures again at 22–23 °C but now in the light for 3–4 weeks (see Notes 54 and 55, Fig. 4b). Steps for the Selection of transformants. 5. First selection: Once good shoots are developing from the calli, transfer them to Selection medium (1) (see Notes 57– 59). If the regenerating calli are large they should be distributed between a number of dishes to prevent subsequent over-crowding. Calli should still be maintained as one piece at this stage unless they fall apart naturally in which case sections from the same initial callus should be kept together in order to track possible clonal material (see Note 60). Incubate for 3–4 weeks at 22–23 °C in the light (see Notes 54 and 55).
Wheat Transformation by Particle Bombardment
211
Fig. 4 Growth and development of transformed wheat plants. (a) Transient expression of the DsRed reporter gene driven by the maize Ubiquitin promoter + intron [11] in an immature embryo 4 days after bombardment. (b) Embryogenic callus after 2 week’s culture on Regeneration medium in the light. (c) Plantlets surviving or succumbing to selection on Selection medium (2) in a Magenta vessel. (d) Confirmed transgenic plants in GM containment glasshouse showing normal development and fertility. Scale bar = 1 mm approximately
6. Second selection: After 3–4 weeks the effect of selection on the cultures should be apparent; some bleaching of leaves will have occurred and roots may be stunted (see Note 59). Any plantlets which remain green with reasonable root structures should be transferred to Selection medium (2) in MagentaTM vessels for expansion of leaves and roots, placing four to six plantlets per MagentaTM (see Note 61). At this stage it should be possible to divide calli, separating out individual plantlets, but these should be labelled as possible clones (see Note 60). Incubate for a further 3–4 weeks at 22–23 °C in the light. Steps for transferring putative transgenic plants to soil and analysis. 7. Following the second round of selection, transformed plantlets should be fairly obvious in the MagentaTM vessels as those which are green and strong with developed root systems (see Fig. 4c). Remove the plantlets from the MagentaTM vessel, rinsing the roots with water to remove Agargel if necessary, and pot into 8 cm square pots containing moistened Rothamsted prescription mix soil (see Notes 2 and 61). 8. Place the pots in a propagator for 1–2 weeks to create a humid environment for the plantlets to become established (see Note 62) and grow in a suitable GM containment glasshouse (see Note 63). 9. After 2–3 weeks plants should be sufficiently strong to take a leaf sample for genomic DNA extraction and analysis by PCR. 10. Re-pot confirmed positive transgenic plants to soil in 13 cm diameter pots (see Note 2) and grow to maturity in a GM containment glasshouse (see Note 63). Transformed plants usually have no morphological differences to control plants apart from the occasional effects of somaclonal variation from tissue culture: they are generally normal, fertile plants with good seed set (see Fig. 4d).
212
4
Caroline A. Sparks and Huw D. Jones
Notes 1. Although this protocol is typically used for the bread wheat variety Cadenza it can be applied to other wheat varieties but efficiencies may be affected. Over 30 different elite wheat genotypes have been transformed at Rothamsted with some measure of success. 2. Rothamsted prescription mix soil contains: 75 % fine grade peat, 12 % screened sterilized loam, 10 % 6 mm screened limefree grit, 3 % medium vermiculite, 2 kg Osmocote Plus/m3 (slow-release fertilizer, 15 N/11 P/13 K plus micronutrients), 0.5 kg PG mix/m3 (14 N/16 P/18 K granular fertilizer plus micronutrients) from Petersfield Products, UK. 3. Ordinary commercial thin bleach can be used which generally has a sodium hypochlorite content of 4–6 %. 4. Some salts may come out of solution in cold storage so check the solutions and shake before use to resuspend fully if necessary. 5. CaCl2 ⋅ 2H2O should be dissolved in water prior to addition to the other components. 6. MnSO4⋅H2O is the most commonly available form of this salt but if a different hydrated state is used, the amount to add must be calculated to reflect this. 7. L-glutamine can be difficult to dissolve. If this is the case, dissolve separately at pH 9.0 prior to mixing with the other components. 8. Although this transformation protocol describes methods most suitable for bread wheat, durum wheat (Triticum turgidum L.) can also be transformed using this system but with the notable variation that sucrose is adjusted to 4.5 % in the MSS 3AA/2 9%S induction medium for embryogenic callus formation. 9. The osmolarity of stock media should be within the ranges: MSS 3AA/2 9%S (×2) 800–1,100 mOsM and R (×2) 270–300 mOsM. 10. When preparing Agargel it should be shaken before and after autoclaving to ensure even setting which makes subsequent remelting in a microwave easier. Following microwaving, always cool the gelling agent to approximately 50 °C prior to combining with the media solutions. 11. Silver nitrate acts as stress inducing agent in the initial induction phase to promote embryogenesis. It is light sensitive so stocks and any media containing it should be stored in the dark. 12. Ideally plates should be poured and used as fresh as possible yet should be allowed to incubate for a few days at room tem-
Wheat Transformation by Particle Bombardment
213
perature to check for contamination. If stored at 4 °C they can be kept for 2–3 weeks. 13. Copper sulfate acts as a stress-inducing agent in the regeneration phase to promote shooting and can be used in the range 12.5–25 mg/L (50–100 μM). If regeneration is good, 12.5 mg/L may be sufficient as the cultures can become too leafy at 25 mg/L. 14. Although tungsten powder was commonly used with original biolistics equipment, the inert metal gold is now the preferred microcarrier. Different particle sizes are available but small, uniform submicron gold particles (average 0.6 μm diameter) have been found to be best for the small cell size of wheat. 15. Spermidine solutions are hygroscopic, oxidizable, and deaminate with time and consequently should be maintained below −20 °C (preferably at −80 °C) and fresh stocks made regularly. Once thawed any unused aliquots should be discarded. 16. The plasmid can carry one or more gene cassettes. Although pUC-based vectors are commonly used, binary vectors used for Agrobacterium-mediated transformation can also be introduced successfully using particle bombardment. Another alternative is to bombard the gene cassette(s) as an isolated DNA fragment and so remove the plasmid backbone which generally carries an antibiotic resistance gene for selection in E. coli, often regarded as undesirable. 17. A selectable marker gene must also be incorporated either on the same plasmid as the gene(s) of interest or as a separate plasmid which is co-precipitated onto the gold. The selectable marker gene allows selection of transformed tissues, examples being the bar gene (as described in this chapter) which confers resistance to glufosinate ammonium-based broad spectrum herbicides such as Basta®, Challenge®, Liberty®, Harvest®, etc., or nptII which confers resistance to the antibiotics geneticin disulfate (G418), kanamycin, neomycin etc. 18. A reporter gene can also be included, e.g., UidA, GFP, or DsRed to monitor transient and/or stable transformation. As is the case for the selectable marker gene, this can either be on the same plasmid as the gene of interest or on a different plasmid which is co-bombarded. 19. Rupture discs are available from Bio-Rad Laboratories as 450, 650, 900, 1,100, 1,350, 1,550, 1,800, 2,000, and 2,200 psi. A range of rupture pressures should be assessed when optimizing any system; for wheat 650 psi generally gives the best, most consistent results but 450 or 900 psi could also be used. 20. Immature inflorescences are an alternative target tissue which are also responsive in tissue culture and can therefore be transformed. However modifications may need to be made to the media for best results and efficiencies are generally lower.
214
Caroline A. Sparks and Huw D. Jones
21. Immature seeds are at about the right stage when they have a whitish bloom on the seed surface and the endosperm is just solidified but not too hard. A few spikelets can be opened at the time of ear collection to determine whether the seeds are at the correct stage. 22. Generally only the outer caryopses of each spikelet are used and none are taken from the tip or base of the ear due to asynchronous seed development. 23. Ideally surface-sterilized seeds should be used the same day. If material needs to be collected in advance it is better to store whole ears at 4 °C with the stems in water prior to removing the seeds; however, it is not recommended to store these longer than overnight. 24. The immature scutellum is the callus-forming tissue; if the embryo axis is left intact it will be prone to germinate on the culture medium thereby preventing callus formation. A practised hand can cut off the embryo axis through the seed coat, leaving a hole through which the scutellum can be removed. 25. Embryos in the size range 0.5–1.5 mm are ideal for transformation via particle bombardment which is slightly smaller than the size advised for Agrobacterium-mediated transformation. Larger embryos may respond if they are still translucent. The size of the embryos is not quite as critical if being used to monitor transient transformation only. 26. Using the gun parameters described, the gold particles typically target a central circular area of ~2 cm diameter, and therefore to maximize transformation the isolated scutella are located in this region. 27. Embryos are incubated prior to bombardment to allow them to recover from the isolation procedure and also because preplasmolysis of cells on the high sucrose medium may increase their ability to withstand bombardment. Experiments to assess the effect of pre-culture have shown that the optimum time for incubation is 1–2 days; transformation is possible after longer periods of pre-incubation (e.g., 4 days) but efficiencies will be reduced. 28. Gold can be prepared at a final concentration of 40 mg/mL if desired (as recommended in the PDS-1000/He manual) but 20 mg/mL has been found to be just as effective for DNA delivery. If prepared at 40 mg/mL, it could give the choice of being used at this concentration or each aliquot diluted by half with sterile distilled water prior to gold coating. 29. 50 μl starting volume of gold will be sufficient for approximately 10–12 shots. The volume of gold and other components can be scaled up or down according to the number of shots required.
Wheat Transformation by Particle Bombardment
215
30. Sonication should resuspend the gold and disrupt any clumps but it should not be sonicated for too long as this may lead to particle agglomeration. 31. The amount of plasmid DNA added should not exceed 5 μg per 50 μl gold as excess DNA can cause clumping of particles. If co-precipitating plasmids, calculate equimolar amounts and use a total of 5 μg DNA. Commonly for co-bombardment of a plasmid of interest and a selectable marker plasmid, a 1.5:1 ratio is used to skew for the gene of interest; plants which survive selection will then have a higher probability of having the gene of interest if containing the marker gene. 32. A gold preparation without DNA should also be prepared at the same time to act as a control. 33. The process of precipitation of DNA onto the gold is very rapid. The calcium chloride and spermidine help to stabilize, precipitate, and bind the DNA to the gold but are mixed first to ensure an even coating, otherwise clumping of particles may occur. 34. The DNA-coated gold suspension needs to be as smooth as possible so it is essential that the particles are resuspended fully at this stage without clumps. Scraping with the pipette tip and repeatedly drawing up and expelling the solution is more effective than vortexing. If the gold appears to be very clumped it is preferable to discard the preparation and start again, checking carefully the amount and concentration of DNA added. 35. The solution should not be aspirated too much during this resuspension step as the ethanol will evaporate and increase the final concentration of particles and may also lead to insufficient volume for the shots required. 36. Ideally the coated particles should be prepared and used as soon as possible but if a number of different treatments are prepared, seal and store the tubes on ice prior to loading onto the macrocarriers. 37. These settings have been optimized for bombardment of wheat immature embryos. Adjustments may need to be made for alternative species or explants. 38. Rupture discs are composed of laminate layers of plastic which may separate if soaked in ethanol for extended periods; as a consequence they should only be dipped briefly in ethanol to sterilize. 39. The macrocarrier must fit completely under the rim of the holder as any gaps will allow the escape of helium. If not secured correctly, the macrocarrier will not be released from the holder and hit the stopping screen with the required pressure to release the gold particles with expected velocity.
216
Caroline A. Sparks and Huw D. Jones
40. In order to ensure that the particles are fully resuspended and equal amounts are placed on each macrocarrier it is important to vortex the gold preparation between taking each sample. 41. Vibration may cause gold particles to clump so the macrocarriers within their Petri dishes should be placed outside of the flow hood on a non-vibrating surface. The macrocarriers can be examined microscopically prior to use to monitor the spread and quality of particles, discarding any on which the gold is unsatisfactory. 42. Macrocarriers should be used quite soon after the ethanol has evaporated so only prepare a few macrocarriers at a time. 43. The regulator must allow sufficient helium through the system to build up and burst the rupture disc: setting the regulator to ~200 psi higher than the rupture disc to be used should be suitable. 44. If the retaining cap is not firmly tightened the rupture disc may become dislodged which means the gun may fail to fire or fires at a lower than expected pressure. 45. If the stopping screen is omitted the released macrocarrier will pass through the hole in the fixed nest straight onto the target tissue, creating rather a mess and potentially contaminating the plate. 46. By creating a vacuum in the chamber there is less air resistance which allows the gold particles to maintain high speed following release from the macrocarrier. 26–30″ Hg is the vacuum range recommended for plants by the manufacturer; in this protocol 28–29″ Hg is routinely drawn. 47. The “Fire” switch should illuminate once the vacuum reaches ~5″ Hg. 48. A metering valve is present on the solenoid valve assembly to regulate the rate of fill of the gas acceleration tube. It should take ~12–15 s to build to bursting pressure. When the rupture disc bursts the macrocarrier is released from its holder onto the stopping screen thus dispersing the particles onto the target tissue. The actual pressure at which the rupture disc bursts should be monitored to confirm a successful shot has occurred. 49. If the Fire switch is released too early no further helium will be discharged and the rupture disc will not burst. To abort a shot prior to the rupture disc bursting, release the Fire switch and set the vacuum switch to Vent. 50. A good mesh indentation should be apparent on the macrocarrier from its contact with the stopping screen if the shot has worked correctly. 51. If the rupture disc is not in the retaining cap it may still be attached to the gas acceleration tube inside the gun. The disc must be removed otherwise it will interfere with further shots.
Wheat Transformation by Particle Bombardment
217
52. Control plates should be included in experiments to allow monitoring of callus formation and regeneration following bombardment. One or more plates of embryos should be bombarded with gold but without DNA such that the embryos have identical treatment but for the presence of DNA. 53. The embryos are spread across different media plates to reduce competition for nutrients and allow for growth. 54. Cultures are incubated in a controlled environment room at 22–23 °C. Cool white fluorescent tubes generate lighting levels of ~250 μmol/m2/s photosynthetically active radiation (PAR) for a 12 h photoperiod. Where dark culture is required, solid trays are covered with foil. 55. Each tissue culture phase can take 3–5 weeks; cultures should be monitored and with experience it will be possible to determine when it is most appropriate to move to the next medium. 56. In order to aid optimization of transformation protocols a reporter gene can be included to act as a visual marker of transformation success (see Note 18). pAHC25 [11] is a convenient plasmid to use having both the selectable marker cassette (bar) but also a cassette containing the β-Glucuronidase gene (UidA). Some embryos can be sacrificed several days postbombardment and assayed for transient gus activity [12] to determine the effectiveness of the bombardment process. Alternative reporter genes also exist which do not require a destructive assay and therefore have the advantage that they can be used to monitor viable tissues in culture over a longer period, e.g., GFP or DsRed (see Fig. 4a and Acknowledgements) 57. Calli from control plates (no DNA) should be transferred to Selection media without PPT but some calli should also be put on Selection media including PPT to demonstrate the effectiveness of the selection. 58. “High lid” Petri dishes can be used at this stage by using upturned Petri dish bases as lids; this creates more space for shoots to develop. 59. Glufosinate ammonium (PPT) is used for selection of plants transformed with the bar gene. 4 mg/L PPT used in the Selection media described should effectively terminate growth of non-transformed tissues although this may not be fully obvious until after the second round of selection. The control plates and Magentas will give a good indication of how well the selection is working. If too many non-transformed escapes are identified, 6 mg/L PPT could be used subsequently to provide more stringent selection pressure. 60. Any plantlets originating from the same callus piece should be regarded as clonal until demonstrated otherwise.
218
Caroline A. Sparks and Huw D. Jones
61. A plant generated from a control plate bombarded with gold but without DNA and subsequently grown on media without plant selection should also be transferred and potted up to act as a negative control which has been through the same experimental conditions. 62. Plantlets derived from tissue culture have little or no waxy cuticle so require a humid environment initially to protect them from desiccation whilst the cuticle forms. 63. GM glasshouse conditions are maintained at 18–20 °C day and 14–16 °C night temperatures with a 16-h photoperiod provided by natural light supplemented with banks of SonT 400 W sodium lamps (Osram Ltd., UK) generating a light intensity of 400–1,000 μmol/m2/s photosynthetically active radiation (PAR).
Acknowledgements Rothamsted Research receives strategic funding from the Biotechnological and Biological Sciences Research Council (BBSRC). Thanks go to Ann Blechl (USDA-ARS, USA) and Jorge Dubcovsky (UC Davis, USA) for providing the DsRed reporter gene construct for our wheat transformation studies. References 1. Blechl AE, Jones HD (2009) Transgenic Applications in Wheat Improvement. In: Carver BF (ed) Wheat: Science and Trade. Wiley-Blackwell, Iowa, pp 397–435 2. Jones HD, Doherty A, Wu H (2005) Review of Methodologies and a Protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1:5 3. Jones HD (2005) Wheat transformation: Current technology and applications to grain development and composition. J Cereal Sci 41:137–147 4. Harwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63:1791–1798 5. Shrawat AK, Loerz H (2006) Agrobacteriummediated transformation of cereals: a promising approach crossing barriers. Plant Biotech J 4:575–603 6. Vasil IK (2007) Molecular genetic improvement of cereals: transgenic wheat (Triticum aesitvum L.). Plant Cell Rep 26:1133–1154 7. Ulker B, Li Y, Rosso MG et al (2008) T-DNA– mediated transfer of Agrobacterium tumefa-
8.
9.
10.
11.
12.
ciens chromosomal DNA into plants. Nat Biotech 26:1015–1017 Altpeter F, Baisakh N, Beachy R et al (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Breeding 15:305–327 Fu X, Duc LT, Fontana S et al (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgen Res 9:11–19 Gadaleta A, Giancaspro A, Blechl AE et al (2008) A transgenic durum wheat line that is free of marker genes and expresses 1Dy10. J Cereal Sci 48:439–445 Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen Res 5:213–218 Jefferson RA, Kavanagh TA, Bevan MW (1987) Beta-glucuronidase (Gus) as a sensitive and versatile gene fusion marker in plants. J Cell Biochem 13:3901–3907
