International Journal of Plant Genomics

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/Step	Barley	Wheat	Triticale	Maize

Embryo precultivation	—	Scutellum directed up, 5 d on WPCM (4.3 gL⁻¹ MS minerals, 5M CuSO₄, 103.1 mgL⁻¹MS vitamins, 0.5 gL⁻¹ Glutamine, 8 mgL⁻¹Dicamba, 40 gL⁻¹ MaltoseH₂O, 0.1 gL⁻¹ Casein hydrolysate, pH = 5.8, 2.5 gL⁻¹ Phytagel), , 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, 5M 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 MaltoseH₂O, 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⁻¹MS vitamins, 0.5 gL⁻¹ Glutamine, 6.6 mgL⁻¹ Dicamba, 15 gL⁻¹ Glucose, 15 gL⁻¹ Sucrose, 200M Acetosyringone, 0.1gL^-1 Casein hydrolysate, pH = 5.2, 2.5 gL⁻¹ Phytagel), , 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 MaltoseH₂O, 500M Acetosyringone, 1 gL⁻¹ Casein hydrolysate, 0.25 gL⁻¹ Myo-inositol, pH = 5.8) each. Remove BCCM and add 600L Agrobacterium OD₆₀₀ = 2–2.5, 1 minute 500 mbar, 10 minutes resting at RT, wash for 15 minutes, BCCM	Remove PTM and add 400L Agrobacterium, OD₆₀₀ = 2–2.5, 30 minutes resting at RT, wash 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, 500M 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 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, 100M 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 ( 4.5 cm)

Co-cultivation	48–72 hours in 2.5 mL BCCM (see inoculation for composition), , dark	48–72 hours, 25 IEs as stack on filter paper (ø 4.5 cm) soaked with 400L WCCM (see inoculation for composition) + 100 mgL⁻¹Larcoll, in petri dish (ø 5.5 cm), , dark	48–72 hours, 25 IEs as stack on filter paper ( 4.5 cm) soaked with 300L BCCM (see barley for composition), in petri dish ( 5.5 cm), , 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 MaltoseH₂O, 100M Acetosyringone, 1 mM DTT, 0.5 gL⁻¹ MES, pH = 5.8, 4 gL⁻¹ Phytagel), , dark
Callus induction	10 IEs each for 2x 14 d on BCIM (4.3 gL⁻¹ MS minerals, 5M CuSO₄, 1 mgL⁻¹ Thiamine HCl, 0.69 gL⁻¹ L-Proline, 2.5 mgL⁻¹ Dicamba, 30 gL⁻¹ MaltoseH₂O, 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, , dark	25 IEs each for 10 d on WCIM (4.3 gL⁻¹ MS minerals, 5M 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), , dark, 25 IEs each for 7 d on WCIM + 20 mgL⁻¹ Hygromycin, , dark	10 IEs each for 14 d on BCIM (see barley for composition) without Hygromycin, , dark, 14 d on BCIM + 25 mgL⁻¹ Hygromycin, , dark	40 IEs each for 7 d on MCIM (4 gL⁻¹ Chu N6 salt mixture, 2 mM CaCl₂, 5M 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, , dark

Shoot formation	14 d on BRM (K4N minerals, 112 mgL⁻¹ B5 vitamins, 146 mgL⁻¹L-Glutamine, 0.225 mgL⁻¹6-BAP, 36gL^-1MaltoseH₂O, pH = 5.8, 3 gL⁻¹Phytagel, 150 mgL⁻¹ Timentin) + 25 mgL⁻¹ Hygromycin, , 16 hours light (mol )	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, , dark, 14 d on MRM + 1.5 mgL⁻¹Bialaphos, in high petri dishes ( mm), , 16 hours light (mol )

Plantlet formation	Each plant for 14–28 d on BRM + 25 mgL⁻¹ Hygromycin, in culture vessels (see maize), , 16 hours light	see barley	see barley	6 plants for 7–14 d on MRM (half strength sucrose compared to shoot formation), in culture vessels ( mm), , 16 hours light (mol )

Plant establishment in soil	5-6 weeks in substrate mix (Spezialmischung Petuniensubstrat, Klasmann, Germany), 40g fertiliser “Osmocote” (Scotts, Netherlands) per 7.5 L pot, day/night, 12 hours light (mol )	see barley	see barley	2–4 weeks in “Substrat 2” (Klasmann, Germany), day/night, 16 hours light (mol )