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Problems with regeneration of transformed Arabidopsis

Tue Jan 26 12:57:32 EST 1993

We are experiencing difficulties in regenerating transformed
Arabidopsis shoots.  The protocol we are using is the one from
Valvekens et al. (PNAS 85,5536-5540, 1988).
 It involves incubating intact roots on B5 agar supplemented with
 0.5mg/L 2,4-D and 0.05mg/L Kinetin followed by inoculation of sectioned
 roots with Agrobacterium strain AGLO (donated from Bob Ludwig, UCSC) harboring
 various modified Ti binary vectors.  The root explants are then incubated
 on B5 agar containing 0.15mg/L IAA and 5mg/L 2.ipAde.
 50mg/L Kan is used to select for transformed calli and we used 200mg/L
 Timentin to kill the bacteria.
As described by Valvekens et al. green calli developped after 3 weeks and
shoots formed on the control calli (untransformed, no Kan), but the green
transgenic calli on root explant did not regenerate and turned brown
after 6 weeks.
If you have any suggestions regarding this procedure, or if you use a more
effective protocol for Agrobacterium mediated transformation of
Arabidopsis, please, let us know.
        Peter Gogarten, Richard Mercier

We will post a summary on the Arabidopsis net. - Many thanks in advance.

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