Based on the frustrating efforts to produce viable protoplasts from microalgae discussed earlier, efforts were initated to develop other methods for introducing DNA into microalgal cells through the intact algal cell walls. At the time this research was going on, the only microalga for which there was a reproducible transformation system was C. reinhardtii. Early efforts to transform this organism were facilitated by the availability of wall-less cells, either genetic mutants (cw — 15), or cells whose walls were degraded using autolysin, a species-specific cell wall-degrading enzyme produced during mating by C. reinhardtii gametes. High-frequency nuclear transformation was accomplished by agitating these wall-less cells in the presence of plasmid DNA, glass beads, and polyethylene glycol (Kindle 1990). This method was reported to work for walled cells, but at a very low frequency. DNA could also be introduced into walled cells of Chlamydomonas and into higher plant cells using microprojectile bombardment, or biolistics; however, this technique requires very expensive, specialized equipment. (This technique will be described in detail “Development of a Genetic Transformation System for the Diatoms Cyclotella and Navicula.”)

During the early 1990s, several reports demonstrated the feasibility of using silicon carbide whiskers (SiC) to mediate the entry of DNA into intact plant cells (Kaeppler et al. 1990; Asano et al. 1991). NREL researcher Terri Dunahay decided to try this approach to introduce DNA into intact algal cells. As reliable selectable markers were not yet available for any oleaginous microalgal strain, she decided to use Chlamydomonas as a model system. A strain of C reinhardtii that contains a defect in the gene for nitrate reductase (CC2453 nit1-305 mt-) was obtained from the Chlamydomonas Genetics Center at Duke University, Durham, North Carolina. These cells cannot use nitrate as a N source, but grow well in the presence of ammonia or urea. Kindle (1990) had shown previously that NR-deficient cells could be transformed with the Chlamydomonas wild-type gene for NR; transformed cells expressing the added DNA could be detected by their ability to grow on nitrate as the sole N source. A plasmid containing the wild-type NR gene from Chlamydomonas was obtained from Dr. P. Lefebvre at the University of Minnesota, St. Paul, Minnesota. A protocol for SiC-mediated transformation was developed based on the glass bead transformation protocol of Kindle (1990). Exponentially growing cells were washed once in NH4+-free medium, then suspended in the same medium with plasmid DNA, sterilzed SiC whiskers, and polyethylene glycol (mw 8,000) to a final concentration of 4.5%-5.0% w/v. The samples were agitated using a vortex mixer for periods from 30 seconds to 10 minutes, then diluted into NH4+-free medium containing 0.6% agar (top agar) and plated onto agar plates that contained the same medium. Transformed colonies (containing a funtional NR gene) appeared in 1-2 weeks.

Attempts to transform walled cells of Chlamydomonas using SiC were made in parallel with glass bead-mediated transformation to compare the two procedures. The results of a typical experiment are shown in Figure II. B.7. The number of transformants obtained using SiC varied

between experiments, but generally were in the range of 10-100 per 107 cells, comparable to transformation efficiency obtained with glass beads. Probably the most significant finding was the difference in cell viability after being agitated with either glass beads or SiC fibers. The viability of the cultures was greater than 80% even after agitation with SiC fibers for 10 minutes; only 10% of the cells survived agitation with glass beads for 60 seconds. The fact that SiC — mediated transformation appears to be a more “gentle” protocol than glass bead treatment may be important when adapting the transformation procedure to other species that may have different wall properties. This work resulted in two publications (Dunahay 1993; Dunahay et al. 1997). The second paper was a collaboration with Dr. Jonathan Jarvik at Carnegie Mellon University. Dr. Jarvik’s laboratory adopted and refined the SiC protocol and now uses it routinely to generate transformants in Chlamydomonas strains with intact walls. After the initial development of the SiC protocol, there was some work at NREL to adapt this procedure for other algal strains of interest to the biodiesel project. Initially, no genetic markers for these strains were available; however, the viability of Monoraphidium and Cyclotella were tested following agitation with SiC; both strains showed high survival rates after extended agitation with SiC. However, the successful development of a transformation system for Cyclotella using biolistics (discussed later) precluded further work on SiC-mediated algal transformation. A few attempts were made to generate transformants of Cyclotella or Navicula using SiC once a selectable marker system was developed. Only one transformant was generated in one experiment. The silica frustule of the diatoms likely acts as a significant barrier to penetration by SiC fibers. SiC would probably work better for introducting DNA into non-diatom cells such as Monoraphidium; these cells are very small and may not be a good target for biolistics, but might be readily pierced by SiC fibers.

. Cell survival and transformation efficiency of intact C. reinhardtii following vortex mixing with SiC fibers or glass beads. (Source: Dunahay 1993.)