Poster: Cytoskeleton structure & dynamics
Abs #
690: Adapting Agrobacterium transformation to a marine alga, Acetabularia acetabulum
Transformation with vectors expressing GFP fusion proteins has greatly facilitated studies of morphogenesis and development. Acetabularia acetabulum, a giant (2-4 cm) unicellular alga, makes a complex thallus (“body plan”) with morphologically and physiologically distinct regions. However, it is a powerful model because it is simple – it is unicellular and uninucleate for most of its lifecycle. To understand how A. acetabulum can execute its remarkable morphogenesis with just one nucleus we would like to visualize subcellular locations of specific cell components such as the cytoskeleton. Agrobacterium is widely used to transform organisms from vascular plants to humans. In this case, Agrobacterium-mediated transformation entails transferring foreign DNA from the bacteria into the nucleus of a host alga and requires co-incubation of the alga (a marine organism) with Agrobacterium (a fresh water soil microbe) in a common medium so that the transfer of DNA can occur. The haplophase is ideal for transformation because gametangia are mitotically active (required for transformation by Agrobacterium) and millions of different transformants can be obtained from one individual alga. To maximize percent transformation, we optimized survival of gametangia during each step prior to co-incubation. For mature gametangia, 93-98% survived removal of microbes with a protease, 95% survived mechanical isolation from the cap, and >85% of the gametangia survived the required 2 day incubation in distilled water. Data on selection of transformants with herbicides and screening for expression of GFP constructs will also be presented. These data will be correlated with morphological and cell biological changes in the gametangia as they mature, e.g. changes in wall thickness.
