comai@U.WASHINGTON.EDU (Luca Comai) (06/28/91)
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@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
RINNES@IuBio.Bio.Indiana.Edu (Roger Innes) (07/01/91)
Luca Comai mentioned that I have made some minor modifications in the Valvekens root transformation procedure. Those of you interested in seeing these changes should contact me directly at RINNES@IuBio.Bio.Indiana.Edu. I have recently moved from Berkeley, thus the address Luca gave is no longer correct. Roger Innes Biology dept., Indiana University, Bloomington, IN 47405