An episomal vector system for plastid transformation in higher plants

Mini‐synplastomes for plastid genetic engineering

“…It should be noted that the sequence for the ~7.4 kb homology arms flanking the trnI/trnA integration site present in Gen1 remained in Gen2, although the transgene cassettes were no longer inserted into the integration site, as described above (Figure 3a). In shuttle vectors previously engineered using NICE1 (Staub and Maliga, 1994) and multiple dinoflagellate ori (Min et al ., 2015a), transgene integration with the endogenous plastome was observed; thus, it was necessary to eliminate these recombination events to meet the requirement of the mini‐synplastome engineering platform. After assembly, Gen2 was transformed into potato leaf discs by biolistics and selected against spectinomycin.…”

“…Although autonomously replicating chloroplast vectors were maintained in cultured cells (Daniell et al ., 1990), episomal plastid engineering failed to achieve persistence of the episomal construct in transplastomic plant lines without selection. In fact, even with selection, an episomal plastid engineering construct designed with a dinoflagellate ori was lost after multiple generations in tissue culture (Min et al ., 2015a). The authors hypothesized that the inability to sustain the episome was the result of decreasing copy number throughout the multiple generations.…”

“…Early work showed increase in expression of foreign genes with autonomously replicating chloroplast vectors when compared to vectors without chloroplast ORI in cultured cells (Daniell et al ., 1990), but this was not demonstrated in transplastomic plant lines. Further work demonstrated that episomal constructs were able to express marker genes without integration (Min et al ., 2015a; Staub and Maliga, 1995); however, no attempts were made to quantitate gene expression from these episomes for the purposes of heterologous protein production. In the current work, we demonstrated that some e Gen2 lines were capable of achieving equivalent expression of heterologous genes to the current state‐of‐the‐art HR‐based plastid engineering.…”

“…Using a dinoflagellate ori , it was possible to engineer transplastomic tobacco with extrachromosomal circular DNA, but after relaxing the selective pressure, the plants rapidly lost the episome. Additionally, the recovery of GFP‐positive plants without the extrachromosomal DNA indicated that integration into the native plastome was occurring, preventing achievement of the ultimate goal (Min et al ., 2015a).…”

“…Other chloroplast transformation constructs, with promoter and terminator sequences homologous to host plastid DNA, have also been shown to generate episomal plasmids that persist at least to the T3 generation (Min et al, 2015b). As an alternative approach, a previous attempt was made to develop an episomal plastid transformation system in tobacco that was bioinspired by the dinoflagellate plastome organization (Min et al, 2015a). While the angiosperm plastome is composed of a single circular molecule ranging in size from 107 to 218 kb (Daniell et al, 2016;Shinozaki et al, 1986), in some marine dinoflagellates the plastome is significantly reduced and composed of multiple small minicircles of 2-3 kb (Howe et al, 2003(Howe et al, , 2008Koumandou and Howe, 2007).…”