Transgene Expression in Regenerated Roots

This protocol was contributed by Jocelyn Malamy (University of Chicago).

This procedure (Malamy and Benfey 1997), which uses a root transformation protocol based on Marton and Browse (1991), provides a rapid method for assessing gene expression in Arabidopsis roots. It is useful for testing promoter:reporter gene constructs, for expressing genes the overexpression of which is lethal in whole plants, and for transforming the roots of plants that are recalcitrant to conventional transformation techniques. The protocol has been used successfully with Ws, No-0, and RLD ecotypes.

Materials

Germination medium (1 liter) lx MS salts (GIBCO) 0.5 g of MES 45 g of sucrose

Adjust pH to 5.7 with 1 n KOH, and then add 0.7% agar. Surface-sterilized seeds Callus-inducing medium (CIM) (1 liter) lx MS salts (GIBCO) 30 g of sucrose

Adjust pH to 5.9 with 1 n KOH, and then add: 3 ml of 6% KHPO,

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