International Journal of Plant Genomics / 2009 / Article / Tab 1

Research Article

Agrobacterium-Mediated Gene Transfer to Cereal Crop Plants: Current Protocols for Barley, Wheat, Triticale, and Maize

Table 1

Details on the transformation procedures and the materials needed in barley, wheat, triticale and maize. MS (Murashige and Skoog, for example, Duchefa no. M0221), K4N [11], B5 (Gamborg B5 Vitamin Mixture, e.g., Duchefa no. G0415), Hygromycin (Hygromycin B, e.g., Roche no. 10843555001), IEs—immature embryos. In cases where it is necessary to distinguish different medium compositions, the generic abbreviations of media (PCM, CCM, CIM and RM) are preceeded by a capital letter (B for barley, W for wheat, T for triticale and M for maize) representing the species for which a particular medium has been initially developed.

Treatment/StepBarleyWheatTriticaleMaize

Embryo precultivationScutellum directed up, 5 d on WPCM (4.3 gL−1 MS minerals, 5 𝜇 M CuSO4, 103.1 mgL−1 MS vitamins, 0.5 gL−1 Glutamine, 8 mgL−1 Dicamba, 40 gL−1 Maltose H2O, 0.1 gL−1 Casein hydrolysate, pH = 5.8, 2.5 gL−1 Phytagel), 2 4 C , dark. Incubate 50 IEs per well for 2–4 hours in 6-well plate with 2.5 mL PTM (4.3 gL−1 MS minerals, 5 𝜇 M CuSO4, 103.1 mgL−1 MS vitamins, 0.5 gL−1 Glutamine, 2 mgL−1 2,4-D, 63.75 gL−1 Mannitol-D, 40 gL−1 Maltose H2O, 0.1 gL−1 Casein hydrolysate, pH = 5.8), RT, darkScutellum directed up, 5 d on TPCM (4.3 gL−1 MS minerals, 103.1 mgL−1 MS vitamins, 0.5 gL−1 Glutamine, 6.6 mgL−1 Dicamba, 15 gL−1 Glucose, 15 gL−1 Sucrose, 200 𝜇 M Acetosyringone, 0.1gL-1 Casein hydrolysate, pH = 5.2, 2.5 gL−1 Phytagel), 2 4 C , dark

Inoculation30–50 IEs in a 6-well plate with 2.5 mL BCCM (4.3 gL−1 MS minerals, 1 mgL−1 Thiamine HCl, 0.8 gL−1 L-Cysteine, 0.69 gL−1 L-Proline, 2.5 mgL−1 Dicamba, 30 gL−1 Maltose H2O, 500 𝜇 M Acetosyringone, 1 gL−1 Casein hydrolysate, 0.25 gL−1 Myo-inositol, pH = 5.8) each. Remove BCCM and add 600 𝜇 L Agrobacterium OD600 = 2–2.5, 1 minute 500 mbar, 10 minutes resting at RT, wash for 15 minutes, BCCM Remove PTM and add 400 𝜇 L Agrobacterium, OD600 = 2–2.5, 30 minutes resting at RT, wash 2 x for 5 minutes, WCCM (4.3 gL−1 MS minerals, 103.1 mgL−1 MS vitamins, 0.8 gL−1 L-Cysteine, 0.5 gL−1 Glutamine, 6 mgL−1 2,4-D, 15 gL−1 Glucose, 15 gL−1 Sucrose, 500 𝜇 M Acetosyringone, 0.1 gL−1 Casein hydrolysate, pH = 5.8)Collect 25 precultivated IEs to 2.5 mL BCCM (see barley for media composition). Remove BCCM and add 600  𝜇 L 1 Agrobacterium OD600 = 2.5–3, 1 minute 500 mbar, 10 minutes resting at RT, wash 1-2x for 5 minute, BCCM (see barley for media composition)Collect up to 200 IEs in 1 mL IM (4 gL−1 Chu N6 salt mixture, 4 mgL−1 Chu N6 vitamins, 0.7 gL−1 L-Proline, 1.5 mgL−1 2,4-D, 36 gL−1 Glucose, 68.4 gL−1 Sucrose, 100 𝜇 M Acetosyringone, pH = 5.2), wash 1x, remove IM, add 1ml IM with Agrobacterium OD600 = 0.7, 5 minutes resting at RT, blot IEs dry on 4 filter papers ( 2 1 C  4.5 cm)

Co-cultivation48–72 hours in 2.5 mL BCCM (see inoculation for composition), 𝜇 , dark 48–72 hours, 25 IEs as stack on filter paper (ø 4.5 cm) soaked with 400 2 1 C L WCCM (see inoculation for composition) + 100 mgL−1 Larcoll, in petri dish (ø 5.5 cm), 𝜇 , dark48–72 hours, 25 IEs as stack on filter paper ( 2 1 C  4.5 cm) soaked with 300 L BCCM (see barley for composition), in petri dish ( 𝜇  5.5 cm), 2 1 C , dark48–72 hours, 40 IEs on MCCM (2 gL−1 Chu N6 salt mixture, 2 mM CaCl2, 112 mgL−1 B5 vitamins, 0.4 gL−1 L-Cysteine, 2.9 gL−1 L-Proline, 4.4 mgL−1 Dicamba, 37.6 gL−1 Maltose 𝜇 H2O, 100 M Acetosyringone, 1 mM DTT, 0.5 gL−1 MES, pH = 5.8, 4 gL−1 Phytagel), 2 4 C , dark
Callus induction10 IEs each for 2x 14 d on BCIM (4.3 gL−1 MS minerals, 5 𝜇 M CuSO4, 1 mgL−1 Thiamine HCl, 0.69 gL−1 L-Proline, 2.5 mgL−1 Dicamba, 30 gL−1 Maltose 2 4 C H2O, 1 gL−1 Casein hydrolysate, 0.25 gL−1 Myo-inositol, pH = 5.8, 3 gL−1 Phytagel, 150 mgL−1 Timentin) + 50 mgL−1 Hygromycin, 2 4 C , dark25 IEs each for 10 d on WCIM (4.3 gL−1 MS minerals, 5 2 4 C M CuSO4, 103.1 mgL−1 MS vitamins, 0.5 gL−1 Glutamine, 2 mgL−1 2,4-D, 40 gL−1 Maltose ·H2O, 0.1 gL−1 Casein hydrolysate, pH = 5.8, 3 gL−1 Phytagel, 150 mgL−1 Timentin), 2 4 C , dark, 25 IEs each for 7 d on WCIM + 20 mgL−1 Hygromycin, 𝜇 , dark10 IEs each for 14 d on BCIM (see barley for composition) without Hygromycin, 2 4 C , dark, 14 d on BCIM + 25 mgL−1 Hygromycin, 3 x , dark40 IEs each for 7 d on MCIM (4 gL−1 Chu N6 salt mixture, 2 mM CaCl2, 5 M silver nitrate, 112 mgL−1 B5 vitamins, 2.9 gL−1 L-Proline, 4.4 mgL−1 Dicamba, 34.2 gL−1 Sucrose, 0.1 gL−1 Casein hydrolysate, 0.5 gL−1 MES, pH = 5.8, 4 gL−1 Phytagel, 150 mgL−1 Timentin), 20 IEs each for 14 d on MCIM + 1.5 mgL−1 Bialaphos, 4–7x 14 d on MCIM + 3 mgL−1 Bialaphos, 2 4 C , dark

Shoot formation 1 3 6 𝜇 14 d on BRM (K4N minerals, 112 mgL−1 B5 vitamins, 146 mgL−1 L-Glutamine, 0.225 mgL−1 6-BAP, 36gL-1 Maltose s 1 m 2 H2O, pH = 5.8, 3 gL−1 Phytagel, 150 mgL−1 Timentin) + 25 mgL−1 Hygromycin, 2 4 C , 16 hours light ( 2 x mol  1 0 0 × 2 0 )see barleysee barley6–10 calluses for 7 d on MRM (4.3 gL−1 MS minerals, 2 mM CaCl2, 103.1 mgL−1 MS vitamins, 60 gL−1 Sucrose, 0.1 gL−1 Myo-inositol, pH = 5.8, 3 gL−1 Phytagel, 75 mgL−1 Timentin) + 1.5 mgL−1 Bialaphos, 2 4 C , dark, 1 7 0 𝜇 14 d on MRM + 1.5 mgL−1 Bialaphos, in high petri dishes ( s 1 m 2  mm), 2 4 C , 16 hours light ( ( 1 3 6 𝜇 m o l s 1 m 2 ) mol  1 0 7 × 9 4 × 9 6 )

Plantlet formationEach plant for 14–28 d on BRM + 25 mgL−1 Hygromycin, in culture vessels (see maize), 2 4 C , 16 hours light 1 7 0 𝜇 see barleysee barley6 plants for 7–14 d on MRM (half strength sucrose compared to shoot formation), in culture vessels ( s 1 m 2  mm), 1 4 / 1 2 C , 16  hours light ( 1 3 6 𝜇 mol  s 1 m 2 )

Plant establishment in soil5-6 weeks in substrate mix (Spezialmischung Petuniensubstrat, Klasmann, Germany), 40g fertiliser “Osmocote” (Scotts, Netherlands) per 7.5 L pot, 2 2 / 2 0 C day/night, 12 hours light ( 1 7 0 𝜇 mol  s 1 m 2 )see barleysee barley2–4 weeks in “Substrat 2” (Klasmann, Germany), 5 . 5 day/night, 16 hours light ( 𝜇 mol  𝜇 )

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