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.