Genetic transformation entails introduction of foreign DNA into a cell (Gietz and Woods 2001) (Fig. 9.1). Genetic transformation has been applied to several algal strains, with C. reinhardtii obtaining the highest rates of transformation (Kindle 2004). Nuclear transformation of various microalgal species such as C. reinhardti is now routine (Walker et al. 2005).

Chloroplast transformation has plastid-specific challenges as compared to nuclear transformation. Nevertheless, chloroplast transformation has been achieved in green (C. reinhardti), red (Porphyridium sp.), and euglenoid algae (E. gracilis) (Wang et al. 2009a). Compared to nuclear transformation, chloroplast transfor­mation has some advantages: primarily, production of high protein levels; the feasibility of expressing multiple proteins from polycistronic mRNAs; and gene containment through the lack of pollen transmission (Wang et al. 2009a). On a final note, attempts in specifically targeting the chloroplast genome of C. reinhardtti and achieving a multiple loci modification in vivo have been performed (O’Neill et al. 2012). The assembly of an ex vivo chloroplast genome using cloning in yeast cells was done targeting a set of genes involved in the photosynthesis pathway (O’Neill et al. 2012). Subsequently, chloroplast transformation was done to achieve the incorporation of genes altering the photosynthesis pathway, more precisely, photosystem II (Nelson and Ben-Shem 2004; Specht et al. 2010).

C. reinhardtii remains the only algal species in which mitochondrial transfor­mation has been reported (Larosa and Remacle 2013; Remacle and Matagne 2004).

Transform into

competent E. coli cells and clone cells

Plasmids are isolated from clones and transformed as in В

Microalgae containing gene of interest

Fig. 9.1 a Transformation of microalgae starts with bacterial cloning to replicate the plasmid that is to be transformed into microalgae. The plasmids are then isolated from the cloning organism via DNA isolation techniques. b Transformation of microalgae can be performed by either vortexing glass beads in the presence of algal cells and DNA plasmids, or electroporating algal cells in a plasmid containing solution

Mitochondrial transformation is still not as common as nuclear or chloroplast transformations due to the small size of the mitochondria. This small size makes it difficult to deliver DNA into the organelles by methods that are used in other transformations. Another challenge that mitochondrial transformation faces is the absence of a relevant gene reporter. The presence of numerous mitochondria in each cell is also an obstacle for manifesting the transformed genotype at the level of the whole cell (Koulintchenko et al. 2012). Co-transformation with chloroplast or nuclear genes and initial selection for these markers is a possible work-around that facilitates the recovery of mitochondrial transformants (Remacle and Matagne 2004).

As for transformation methods, nuclear gene transfer can be achieved using var­ious methods, including electroporation, agitation with glass beads or silicon carbide whiskers, particle bombardment, and agrobacterium vector infection (Table 9.1) (Guo et al. 2013). Lack of a cell wall in the recipient cells (e. g., Dunaliella salina)

U

40

or cell wall deficiency is sometimes necessary to achieve the highest rate of trans­formation. One way to weaken the cell wall in Chlamydomonas is pretreating them with the lytic enzyme autolysin. Autolysin is produced by Chlamydomonas itself through pre-incubation of the cells in a nitrogen-free medium to induce autolysin production, followed by collection of the produced enzyme. An alternative to using autolysin is using cell-wall-deficient mutant cells, such as cw15, for transformations (Walker et al. 2005).