Transformation protocols

Figure 2: Agrobacterium Plant transformation

Competent Agrobacterium GV3101::pMP90::pSOUP cells

• Grow 8ml overnight culture of Agrobacterium in LB with 5 µg/ml tetracycline + 50 µg/ml rifampicin + 25 µg/ml gentamycin
  (the LB we use for Agrobacterium at all stages has only 5 g/l NaCl)
• Inoculate 8 ml overnight culture in 192 ml LB without antibiotics
• Shake at 28°C until an OD of about 0.5 (generally 3-6 hrs, very strain-dependent)
• Centrifuge at 4000 rpm for 15' at 4°C
• Resuspend pellets in 10 ml of ice-cold 10 mM Tris-HCl (pH 7.5)
• Centrifuge at 4000 rpm for 15' at 4°C
• Resuspend pellets in 20 ml cold LB and freeze in liquid N₂; these cells stay competent for 6 months if kept at -80°C

Agrobacterium transformation in 96-well plates

• 200 µl competent cells + 1 µg plasmid mixed in pre-chilled tubes or plates
• 5 min on ice
• 5 min in liquid N₂
• 5 min at 37°C
• Add 800 µl LB
• 2 h at 28°C with gentle shaking
• Liquid selection of 100 µl in 1.3ml LB + 5 µg/ml tetracycline + 50 µg/ml rifampicin + 50 µg/ml kanamycin + 25 µg/ml gentamycin
• Shake 2 days at 28°C on a Titramax 1000 at 600 rpm
• make a 25% glycerol stock and conserve the plate at -80°C

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PCR 4

using primers Agri51/Agri56/Agri64/Agri69

Agri51 100 µM	0.02 µl
Agri56 100 µM	0.02 µl
Agri64 100 µM	0.02 µl
Agri69 100 µM	0.02 µl
10 x Red Cresol (Aldrich cat no. 11447-2)	1 µl
10x PCR buffer	1 µl
dNTP stock (25 mM of each dNTP)	0.08 µl
Taq enzyme (home made...)	0.2 µl
water	7.14 µl
liquid selection bacteria	0.5 µl

PCR conditions

	94°C	5 min
35 X	94°C	30 sec
	55°C	30 sec
	72°C	2 min
	72°C	5 min

Selection and validation of individual Agrobacterium clones

• Make a 4.10⁵ dilution of the glycerol stock
• Plate 150 µl of the dilution on Q-tray with 200 ml of LB + 5 µg/ml tetracycline + 50 µg/ml rifampicin + 50 µg/ml kanamycin + 25 µg/ml gentamycin
• Grow 2 days at 29°C
• Inoculate four individual colonies each into 1 ml selection medium (LB + Kan, Tet, Rif, Genta)
• Shake 2 days at 29°C
• PCR 5 on culture with primers 51/56/64/69 (same as PCR 4 above)
• Make 25% glycerol stock (add 75 µl of 50% glycerol to 75 µl of the culture)
• Based on PCR 5 results, select correct clones for plant transformation

Plant transformation (infiltration)

• Day 1: sowing; cultivation @ 16 h 145 µE, 20°C, 75% rel. hum. / 8 h 6°C, 75%rel. hum.
• Day 7: transfer to cultivation @ 8 h 145 µE, 20°C, 75% rel. hum. / 16 h 16°C, 75% rel. hum.
• Day 14: transfer into pots: 9 seedlings per pot; continue cultivation @ 8 h 145 µE, 20°C, 75% rel. hum. /16 h 16°C, 75% rel. hum.
• Day 28: transfer to glass house, cultivation @ 16 h 250 µE, 20°C, 80% rel. hum. / 8 h 18°C, 50% rel. hum.
• Day 47: clip primary inflorescence
• Day 54: dip coflorescences into Agrobacterium suspension
• Day 75: cover inflorescences with bags
• Day 89: stop watering
• Day 103: collect bags, store @ 15°C 15% rel. hum. for ca. 2 weeks
• Day 119: harvest seeds into glass tubes, store @ 15°C 15% rel. hum. (for thrips control: 48 h -20°C)

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Selection of transformants

• Day 1: sowing (GA-treatment); cultivation in glass house @ 16 h 250 µE, 20°C, 80% rel. hum. / 8 h 18°C, 50% rel. hum.
• Day 7: BASTA treatment (40 mg/l)
• Day 9: BASTA treatment
• Day 12: BASTA treatment
• Day 14: BASTA treatment
• Day 16: BASTA treatment
• Day 21: transfer resistant seedlings into pots; cultivation in glass house @ 16 h 250 µE, 20°C, 80% rel. hum. / 8 h 18°C, 50% rel. hum.
• Day 75: cover inflorescences with bags
• Day 89: stop watering
• Day 103: collect bags, store @ 15°C 15% rel. hum. for about 2 weeks
• Day 119: harvest seeds into glass tubes, store @ 15°C 15% rel. hum. (for thrips control: 48 h -20°C)

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