. 2009 Jun 21;2009:835608. doi: 10.1155/2009/835608

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/Step	Barley	Wheat	Triticale	Maize
Embryo precultivation	—	Scutellum directed up, 5 d on WPCM (4.3 gL⁻¹ MS minerals, 5 μM CuSO₄, 103.1 mgL⁻¹MS vitamins, 0.5 gL⁻¹ Glutamine, 8 mgL⁻¹Dicamba, 40 gL⁻¹ Maltose·H₂O, 0.1 gL⁻¹ Casein hydrolysate, pH = 5.8, 2.5 gL⁻¹ Phytagel), 24°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 μM 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, dark	Scutellum 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 μM Acetosyringone, 0.1 gL⁻¹ Casein hydrolysate, pH = 5.2, 2.5 gL⁻¹ Phytagel), 24°C, dark	—

Inoculation	30–50 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 μM Acetosyringone, 1 gL⁻¹ Casein hydrolysate, 0.25 gL⁻¹ Myo-inositol, pH = 5.8) each. Remove BCCM and add 600 μL Agrobacterium OD₆₀₀ = 2–2.5, 1 minute 500 mbar, 10 minutes resting at RT, wash for 15 minutes, BCCM	Remove PTM and add 400 μL Agrobacterium, OD₆₀₀ = 2–2.5, 30 minutes resting at RT, wash 2x 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⁻¹2,4-D, 15 gL⁻¹ Glucose, 15 gL⁻¹ Sucrose, 500 μM 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 μL⁻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⁻¹Chu 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 μM 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 (ø 4.5 cm)

Co-cultivation	48–72 hours in 2.5 mL BCCM (see inoculation for composition), 21°C, dark	48–72 hours, 25 IEs as stack on filter paper (ø 4.5 cm) soaked with 400 μL WCCM (see inoculation for composition) + 100 mgL⁻¹Larcoll, in petri dish (ø 5.5 cm), 21°C, dark	48–72 hours, 25 IEs as stack on filter paper (ø 4.5 cm) soaked with 300 μL BCCM (see barley for composition), in petri dish (ø 5.5 cm), 21°C, dark	48–72 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·H₂O, 100 μM Acetosyringone, 1 mM DTT, 0.5 gL⁻¹ MES, pH = 5.8, 4 gL⁻¹ Phytagel), 21°C, dark

Callus induction	10 IEs each for 2x 14 d on BCIM (4.3 gL⁻¹ MS minerals, 5 μM CuSO₄, 1 mgL⁻¹ Thiamine HCl, 0.69 gL⁻¹ L-Proline, 2.5 mgL⁻¹ Dicamba, 30 gL⁻¹ Maltose·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, 24°C, dark	25 IEs each for 10 d on WCIM (4.3 gL⁻¹ MS minerals, 5 μM CuSO₄, 103.1 mgL⁻¹ MS vitamins, 0.5 gL⁻¹ Glutamine, 2 mgL⁻¹2,4-D, 40 gL⁻¹ Maltose ·H₂O, 0.1 gL⁻¹ Casein hydrolysate, pH = 5.8, 3 gL⁻¹ Phytagel, 150 mgL⁻¹ Timentin), 24°C, dark, 25 IEs each for 7 d on WCIM + 20 mgL⁻¹ Hygromycin, 24°C, dark	10 IEs each for 14 d on BCIM (see barley for composition) without Hygromycin, 24°C, dark, 14 d on BCIM + 25 mgL⁻¹ Hygromycin, 24°C, dark	40 IEs each for 7 d on MCIM (4 gL⁻¹ Chu N6 salt mixture, 2 mM CaCl₂, 5 μM 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, 24°C, dark

Shoot formation	3x 14 d on BRM (K4N minerals, 112 mgL⁻¹ B5 vitamins, 146 mgL⁻¹L-Glutamine, 0.225 mgL⁻¹6-BAP, 36gL⁻¹Maltose·H₂O, pH = 5.8, 3 gL⁻¹Phytagel, 150 mgL⁻¹ Timentin) + 25 mgL⁻¹ Hygromycin, 24°C, 16 hours light (136 μmol s⁻¹ m⁻²)	see barley	see barley	6–10 calluses for 7 d on MRM (4.3 gL⁻¹ MS minerals, 2 mM CaCl₂, 103.1 mgL⁻¹ MS vitamins, 60 gL⁻¹Sucrose, 0.1 gL⁻¹Myo-inositol, pH = 5.8, 3 gL⁻¹Phytagel, 75 mgL⁻¹Timentin) + 1.5 mgL⁻¹ Bialaphos, 24°C, dark, 2x 14 d on MRM + 1.5 mgL⁻¹Bialaphos, in high petri dishes (100 × 20 mm), 24°C, 16 hours light (170 μmol s⁻¹ m⁻²)

Plantlet formation	Each plant for 14–28 d on BRM + 25 mgL⁻¹ Hygromycin, in culture vessels (see maize), 24°C, 16 hours light (136 μmol s⁻¹ m⁻²)	see barley	see barley	6 plants for 7–14 d on MRM (half strength sucrose compared to shoot formation), in culture vessels (107 × 94 × 96 mm), 24°C, 16 hours light (170 μmol s⁻¹ m⁻²)

Plant establishment in soil	5-6 weeks in substrate mix (Spezialmischung Petuniensubstrat, Klasmann, Germany), 40g fertiliser “Osmocote” (Scotts, Netherlands) per 7.5 L pot, 14/12°C day/night, 12 hours light (136 μmol s⁻¹ m⁻²)	see barley	see barley	2–4 weeks in “Substrat 2” (Klasmann, Germany), 22/20°C day/night, 16 hours light (170 μmol s⁻¹ m⁻²)