Transformation Protocol for Arabidopsis - Revision history

Torsten Waldminghaus at 16:57, 23 February 2009

2009-02-23T16:57:35Z

←Older revision		Revision as of 16:57, 23 February 2009
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		+	{{back to protocols}}
		+
	Transformation Protocol for Arabidopsis – Abbreviated		Transformation Protocol for Arabidopsis – Abbreviated

Mathias Klode: Added Arabidopsis Category

2008-04-24T16:38:11Z

Added Arabidopsis Category

←Older revision		Revision as of 16:38, 24 April 2008
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	Spring Harbor Press: 55-77.		Spring Harbor Press: 55-77.

-	[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]]	+	[[Category:Protocol]] [[Category:In vitro]] [[Category:Plant]] [[Category:Arabidopsis thaliana]]

Reshma P. Shetty at 17:48, 9 May 2007

2007-05-09T17:48:45Z

←Older revision		Revision as of 17:48, 9 May 2007
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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]]

Harris at 04:21, 20 April 2007

2007-04-20T04:21:30Z

←Older revision		Revision as of 04:21, 20 April 2007
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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]]

Harris at 04:08, 20 April 2007

2007-04-20T04:08:48Z

←Older revision		Revision as of 04:08, 20 April 2007
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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

Harris at 04:08, 20 April 2007

2007-04-20T04:08:30Z

←Older revision		Revision as of 04:08, 20 April 2007
Line 63:		Line 63:

	References:		References:
		+
	Clough SJ and Bent AF, 1998. Floral dip: a simplified method for		Clough SJ and Bent AF, 1998. Floral dip: a simplified method for
	Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J		Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
	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

Harris at 04:08, 20 April 2007

2007-04-20T04:08:09Z

←Older revision		Revision as of 04:08, 20 April 2007
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	pods that have formed prior to transformation.		pods that have formed prior to transformation.

		+
	Prepare bacteria:		Prepare bacteria:
	• Streak YM plus antibiotic plates with bacteria, or Minimal A plus		• Streak YM plus antibiotic plates with bacteria, or Minimal A plus
	antibiotic plates for Agrobacterium.		antibiotic plates for Agrobacterium.
	• Incubate plates for 2-3 days at 28°C.		• Incubate plates for 2-3 days at 28°C.
		+

	Transformation:		Transformation:
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	• Collect seed, store at 4°C		• Collect seed, store at 4°C

		+
	Selection:		Selection:
	• Weigh seed to estimate number (100 seed ≈ 21.8mg)		• Weigh seed to estimate number (100 seed ≈ 21.8mg)

Harris at 04:07, 20 April 2007

2007-04-20T04:07:10Z

←Older revision		Revision as of 04:07, 20 April 2007
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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:

Harris at 04:06, 20 April 2007

2007-04-20T04:06:46Z

←Older revision		Revision as of 04:06, 20 April 2007
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-
-
	Transformation Protocol for Arabidopsis – Abbreviated		Transformation Protocol for Arabidopsis – Abbreviated
-		+
-		+	''Germinate seed in pots
-	Germinate seed in pots	+

	↓ 4 weeks		↓ 4 weeks
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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.
		+

		+	References:
		+	Clough SJ and Bent AF, 1998. Floral dip: a simplified method for
		+	Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
		+	16:735-43.


-	~~Media and solutions for Arabidopsis transformation~~
-
-
-	~~YM Media~~
-	1L
-	~~Mannitol 10g~~
-	~~Yeast extract 0.4g~~
-	~~K2HPO4 (10% w/v stock) 1 ml~~
-	~~KH2PO4 (10% w/v stock) 4 ml~~
-	~~NaCl (10% w/v stock) 1 ml~~
-	~~MgSO4.7H2O (10% w/v stock) 2 ml~~
-
-	~~• pH 6.8~~
-	~~• Agar 15g/L~~
-	~~• Autoclave~~
-	~~• When ready to pour add:~~
-	~~• Antibiotic selection if required~~
-
-	~~Keep poured plates for 2 days at room temperature to visualise any~~
-	~~contamination, then store at 4°C.~~
-
-	~~Minimal A Medium (Svab, et al, 1975)~~
-
-	~~Liquid 1L~~
-	~~Stock solution A 490mL~~
-	~~Stock solution C 11mL~~
-	~~Milli-Q H2O 499mL~~
-
-	~~Solid 1L~~
-	~~Stock solution A 490mL~~
-	~~Stock solution B 499mL~~
-	~~Stock solution C 11mL~~
-
-	~~• Store 4°C~~
-
-
-	~~Minimal A Stock Solution A~~
-	~~Final concentration~~
-	~~g/490mL 1 x mM~~
-	~~K2HPO4 10.5 60~~
-	~~KH2PO4 4.5 33~~
-	~~(NH4)2SO4 1.0 7.6~~
-	~~tri-Sodium citrate.2H20 0.5 1.7~~
-
-	~~• Autoclave in 490mL aliquots~~
-	~~• Store 4°C~~
-	~~• Substituting chemicals~~
-	~~• K2HPO4.3H2O 13.8g/490mL~~
-
-
-
-
-	~~Minimal A Stock Solution B~~
-
-	~~Dissolve 15g agar in Milli-Q H2O, make up to 500mL~~
-
-	~~• Autoclave~~
-	~~• Store room temperature~~
-
-
-	~~Minimal A Stock Solution C~~
-
-	~~Add 1mL 1M MgSO4 to 10mL 40% sucrose~~
-
-	~~• Store room temperature~~

-	~~Infiltration media~~	+	Svab, Z., P. Hajdukiewicz and P. Maliga. 1975. Transgenic tobacco plants by
-	~~200ml Final conc~~.	+	co-cultivation of leaf disks with pPZP Agrobacterium binary vectors. In
-	~~10X MS salts 20ml 1xMS salts~~	+	“Methods in Plant Molecular Biology-A Laboratory Manual”, P. Maliga,
-	~~Sucrose 10g 5%~~	+	D. Klessig, A.. Cashmore, W. Gruissem and J. Varner, eds. Cold
-	~~MES 0~~.~~2g 0~~.1%	+	Spring Harbor Press: 55-77.
-	~~Silwet~~-~~L77 200~~	+

Harris at 04:04, 20 April 2007

2007-04-20T04:04:40Z

←Older revision		Revision as of 04:04, 20 April 2007
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-	Transformation Protocol for Arabidopsis – ~~Summary~~	+	Transformation Protocol for Arabidopsis – Abbreviated


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	Transfer transformed plants to the glasshouse		Transfer transformed plants to the glasshouse
		+