*2.2. Harnessing Homologous Recombination in Chloroplasts*

Homologous recombination is a crucial step after DNA delivery into the chloroplast that determines the subsequent success of the transformation. The frequency of HR events is highest when the foreign DNA carries a sequence of at least 121 bp that is identical to the target integration site [48]. Careful consideration should be given to the choice of promoter and regulatory elements (5 and 3 untranslated regions), as well as the insertion site in the plastid genome, to maximize transformation efficiency, as shown in Table 2. The *psbA* promoter, from the plastid-encoded photosystem II protein D1 precursor, was first used over 30 years ago in Chlamydomonas and still appears to be the best position in which to insert a target gene, as the *psbA* gene product is the most highly translated plastid protein [49]. Boynton, Gillham, and colleagues first achieved plastid transformation of Chlamydomonas with this plastid promoter in 1988 [40]; Svab, Hajukiewicz and Maliga followed, in 1990, with the first report of plastid transformation in tobacco, although they used a different fragment of chloroplast DNA in their target plasmid [50]. Plastid transformation has since been reported in many flowering plants, such as tobacco [50–52], Arabidopsis [53,54], potato [55], rice [56], rapeseed [57], and tomato [58]. A protocol for plastid transformation of an elite rapeseed cultivar (*B. napus* L.) has been developed [27]. The highest protein yields were accomplished by using the *rrn* promoter from the plastid rRNA operon, as reported in several reviews [7,8,59,60].

**Table 2.** Promoters, untranslated regions, and insertion sites commonly used for plastid transformation.

