**1. Introduction**

The plastids of algae and land plants are semi-autonomous organelles with their own genomes, complete with transcription and translation machinery [1] that arose from endosymbiosis between a eukaryotic ancestor and a photosynthetic cyanobacterium [2]. The plastid genome is a circular doublestranded DNA molecule that encodes 100–250 genes and is maternally inherited in angiosperms [3]. The size of chloroplast genomes varies between species, ranging from 107 kbp (Cathay silver fir, *Cathaya argyrophylla*) to 218 kbp (Geranium, *Pelargonium* spp.). The plastid genome is present in many copies inside the organelle and is protected from gene silencing pathways that typically lower transgene expression, thereby allowing high levels of foreign protein to accumulate, achieving 5–40% total soluble protein (TSP) [4] and up to 70% of total soluble protein in tobacco (*Nicotiana tabacum*) [5–7]. An even higher protein yield (>75% TSP) was recently achieved in tobacco transplastomic plants expressing a hyper-thermostable form of β-glucosidase cloned from the hyperthermophilic bacterium *Pyrococcus furiosus* [8]. Compared to nuclear transformation, plastid transformation has many advantages. First, the maternal inheritance of the chloroplast genome prevents genes from escaping through pollen grains in most plants and thus reduces the spread of transgenes in the environment and avoids issues with Genetically Modified Organisms (GMOs) [9]. Second, the multiple copies of the plastid genome contained within the organelle results in the high expression of foreign genes and the accumulation of the encoded proteins [10]. Third, since plastid transformation involves homologous recombination (HR) of the transgene into a so-called neutral site, the method largely avoids gene silencing caused by position effects [11]. Fourth, the chloroplast employs a prokaryotic gene expression system and allows the easy and simultaneous expression of polycistronic genes [12]. Last, the stacking of multiple transgenes can be performed in a single and highly efficient transformation event [13]. Plastid transformation is, therefore, gaining traction [14].

The main crop species used for plastid transformation are in the Solanaceae, including tobacco, potato (*Solanum tuberosum*), tomato (*S. lycopersicum*), eggplant (*S. melongena*), and pepper (*Capsicum annuum*). Plastid transformation has also been reported in other important crop species, including soybean (*Glycine max*, Fabaceae), lettuce (*Lactuca sativa*, Asteraceae), cauliflower (*Brassica oleracea* var. *botrytis*, Brassicaceae), cotton (*Gossypium* spp., Malvaceae), carrot (*Daucus carota*, Umbelliferae), bitter squash (*Momordica charantia*, Cucurbitaceae) and rice (*Oryza sativa*, Poaceae) (Table 1). To date, the plastids of over 20 flowering plants have been transformed [15] (Table 1). In addition to the crops mentioned above, recent successes in plastid transformation have been reported in the plant species bitter melon [16], and the medicinal plant sweet wormwood (*Artemisia annua*) [17] and licorice weed (*Scoparia dulcis*) [18,19] (Table 1).


**Table 1.** Species in which plastid transformation has been demonstrated.

<sup>1</sup> Kanamycin; <sup>2</sup> Spectinomycin; <sup>3</sup> Streptomycin; <sup>4</sup> PEG-mediated transformations.

Based on these successful cases, plastid transformation should be applicable to many plant families, whether they are monocots or dicots. However, plastid transformation remains much more challenging than nuclear transformation and is not as widespread in plant research. Furthermore, Bock [7] raised the issue of reproducibility of plastid transformation, as the plastids of fewer than ten species have been demonstrably transformed in at least two independent reports since 1988, the year the first example of plastid transformation was published in the unicellular green alga Chlamydomonas (*Chlamydomonas reinhardtii*) [7,40]. Major critical points limiting current plastid transformation attempts

are 1) the method of DNA delivery, 2) homologous recombination efficiency and 3) methods for efficient selection and robust regeneration of transformants. In this review, we will address each of these issues and will highlight recent innovative technologies and strategies in plastid transformation. We hope to give readers a new perspective on the potential of plastid transformation with concrete examples, setting the stage for plastid transformation in more plant species in the future.
