improved root harvest for transformation

Luca Comai comai at U.WASHINGTON.EDU
Thu Jun 27 13:19:23 EST 1991


Dear Network:
My laboratory follows the Arabidopsis transformation protocol of
Valvekens et al. (1988, PNAS 85,55360) as adapted by Roger Innes (for 
RI address contact "liam1 at violet.berkeley.edu". The protocol is very 
efficient. However, harvesting of root explants is time consuming. We
have found a rapid way to harvest roots. We grow Arabidopsis ecotype
No-0 on large plates (15 cm in diameter, 2.5 cm high -- Falcon 3025) in
the following medium:
5X Murashige and Skoog salt base pH5.7, 1X Gamborg's B5 vitamins, 1%
sucrose, 1% Phytagar. Using an L-shaped glass rod, we form a line of
about 300 to 400 seeds dividing the plate asymmetrically in one third
and two third sections (two parallel lines can also be used). We
incubate the plate vertically with the seed lines parallel to the shelf
on which the plate rests. The root system of the seedling will grow on
the surface of the agar and in about two to three weeks the root tips
will approach the edge of the plate. We harvest the roots by cutting
them below the crown with a sharp blade. Since the roots are growing on
the surface of the agar they can all be removed in a few seconds. We
incubate them on CIM as in Valvekens et al and cut them into sections
just before incubating with Agrobacterium. 
Good Luck,
Luca Comai
University of Washington



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