Transformation Protocol for Arabidopsis: Difference between revisions
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Transformation Protocol for Arabidopsis – Abbreviated | Transformation Protocol for Arabidopsis – Abbreviated | ||
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Transfer transformed plants to the glasshouse'' | Transfer transformed plants to the glasshouse'' | ||
'''Transformation Protocol for Arabidopsis''' Method modified from Clough and Bent (1988) | |||
'''Transformation Protocol for Arabidopsis''' Method modified from Clough and Bent (1988) | |||
Plant material: | Plant material: | ||
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• Incubate overnight at 4°C then in light at 20°C. After 10 days select | • Incubate overnight at 4°C then in light at 20°C. After 10 days select | ||
transformants, transplant to soil. | transformants, transplant to soil. | ||
* [[Media and solution preparation for this protocol]] | |||
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16:735-43. | 16:735-43. | ||
Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by | Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by | ||
co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In | co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In | ||
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D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold | D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold | ||
Spring Harbor Press: 55-77. | Spring Harbor Press: 55-77. | ||
[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]] [[Category:Arabidopsis thaliana]] |
Latest revision as of 09:57, 23 February 2009
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Transformation Protocol for Arabidopsis – Abbreviated
Germinate seed in pots
↓ 4 weeks
Streak bacteria onto YM/MinA
↓ 2-3 days 28°C
Spray/dip bacterial suspension onto plants
↓ 1 day in box
Let plants set seed
↓ 3-4 weeks
Plate seed to GM + selection
↓
Transfer transformed plants to the glasshouse
Transformation Protocol for Arabidopsis Method modified from Clough and Bent (1988)
Plant material: • Thinly sow Arabidopsis thaliana cv C24 seed into soil. Grow at 20°C under 16h light until plants are beginning to flower. Cut off any seed pods that have formed prior to transformation.
Prepare bacteria:
• Streak YM plus antibiotic plates with bacteria, or Minimal A plus
antibiotic plates for Agrobacterium.
• Incubate plates for 2-3 days at 28°C.
Transformation:
• Measure about 20mL Infiltration Medium for each bacterial strain.
• Scrape or wash bacteria from plate with sterile loop and suspend in
20mL of Infiltration media.
• Adjust density to an OD600 0.9-1.0.
• Dip flowers and unopened buds of Arabidopsis into the bacterial
suspension, or alternatively the plants can be sprayed with bacterial
suspension using a plant mister.
• Cover plants to maintain humidity overnight. Grow at 20°C in light.
• Repeat dipping/spraying after 7 days.
• Collect seed, store at 4°C
Selection:
• Weigh seed to estimate number (100 seed ≈ 21.8mg)
• Sterilise for 2 mins in 70% (v/v) ethanol followed by 20 mins in 20%
(v/v) bleach (Zixo brand) + 0.02% Triton X-100. Keep the seeds
moving in the bleach solution by putting tubes on a rotator wheel.
• Wash seeds 5 times with sterile distilled water (in laminar flow hood)
• Plate to Germination Medium + selection plates, seal with Micropore
tape.
• Incubate overnight at 4°C then in light at 20°C. After 10 days select
transformants, transplant to soil.
References:
Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.
Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by
co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In
“Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga,
D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold
Spring Harbor Press: 55-77.