**4. What Needs Can Plastid Transformation Fill?**

Plastid transformation was developed over three decades ago, and numerous transplastomic algae and flowering plants have been created successfully. What can these transplastomic plants do? Here, we review the plastid transformation literature to give our readers an understanding of the potential applications.

Engineering chloroplasts with desired agronomic traits has garnered interest in recent years. For instance, expressing a bacterial *4-Hydroxyphenylpyruvate Dioxygenase* (*HPPD*) gene in tobacco or soybean chloroplasts conferred enhanced herbicide resistance [30], while expression of *Betaine Aldehyde Dehydrogenase* (*BADH*) in carrot chloroplasts provided strong salt tolerance [39]. As mentioned earlier, tobacco remains by far the most suitable species for plastid transformation, although the technique has also been successfully applied to other species such as tomato [85,86], potato [37], maize [87], sugar beet [24], cotton [31] and wheat [88]. For example, the simultaneous expression of protease inhibitors and chitinase in transplastomic tobacco plants conferred resistance to multiple biotic and abiotic stresses [89]. Multiple economic and agronomic traits of interest have been engineered into chloroplasts, including resistance to cold, drought, insects or herbicides as well as salt tolerance [30,90–92]. Herbicide resistance is perhaps one of the most notable traits in plastid transformation. Plants resistant to the herbicide glyphosate (commercialized as Roundup) were generated by introducing the *5-Enolpyruvylshikimate-3-Phosphate Synthase* (*EPSPS*) gene into the tobacco plastid genome, which encodes an enzyme that detoxifies glyphosate [4]. Agronomic characters obtained by the engineering of the chloroplast genome are listed in Table 3; most transformations relied on biolistics, although these largely predate the publication of the SWNTs tool, which may provide a new opportunity for scientists thanks to its low cost and ease of use.

**Table 3.** Agronomic traits engineered into crops by plastid transformation.



**Table 3.** *Cont.*

DW: dry weight; FW: fresh weight; NR: not recorded; SD: Shine-Dalgarno sequence; TLP: total leaf protein; TSCP: total soluble cellular protein; TSP: total soluble protein.

#### *4.1. Antigen Vaccines and Protein-based Drugs*

Globally, the number of individuals suffering from diabetes is expected to rise from 170 million in 2000 to a projected 366 million by 2030 [114]. More than 90% of the global population cannot afford the cost of insulin [3,115]. Protein-based drugs such as insulin are expensive because they are produced in yeast fermentation systems and later kept in cold storage, but the final pure product still has a short shelf-life [116]. However, protein-based drugs produced in transplastomic plants may solve many of the associated issues without raising costs or compromising drug efficacy [3]. Many vaccine antigens and biopharmaceuticals have been successfully produced from the chloroplasts of flowering plants.

The induction of insulin production in human subjects is an attractive alternative to daily insulin injections. Exendin-4 (EX4), an analog of the peptide hormone Glucagon-like peptide, was expressed in tobacco chloroplasts, fused to the Cholera toxin B (CTB) subunit to facilitate delivery by crossing the intestinal epithelium. Lyophilized tobacco leaf extracts increased insulin production levels in mice without inducing hypoglycemia, even when a 5000-fold excess dose of CTB-EX4 was delivered orally [117]. In addition, the accumulation of human interferon-gamma in tobacco chloroplasts reached 0.42% of total soluble protein [118]. Unlike microorganisms, plant chloroplasts can perform post-translational modifications of protein-based drugs and promote their proper folding: phosphorylation, amidation, and disulfide bond formation [119].

Human papillomavirus (HPV) is a cause of cervical cancer, which kills over 250,000 women each year. Protein E7 from HPV type 16 (HPV-16 E7) is an attractive anti-cancer vaccine antigen that has been expressed in tobacco via plastid transformation or transient expression [120–122]. The plant-produced proteins successfully induced an immune response and mediated tumor regression in the murine model. Using the transient transfection system with Agrobacterium LBA4404 and the pBIN-NSs vector containing the TSWV NSs silencing suppressor gene, the E7 protein fused with Zera®was expressed only at levels ranging from 0.1–6 g/kg [121]. Via plastid transformation, E7 could reach 0.1% TSP in transplastomic plants [122]. Notably, E7-potato virus X coat protein fusion proteins accumulated to levels around five times higher than the unfused E7 [120,122].

Human coagulation factors made from plants have also been shown to improve immune tolerance in hemophilia murine and canine models [23,123]. In addition, high-level expression of vaccine antigens and therapeutic proteins has been achieved in plant chloroplasts (leaves and roots) or chromoplasts (fruits) for antigens associated with the plague, tetanus, human immunodeficiency virus (HIV), cholera, malaria, Alzheimer's disease and hemophilia [123–130]. Table 4 provides a partial list of vaccine antigens and drug proteins expressed in the chloroplast. Although high levels of protein expression are desirable for chloroplast production of protein-based drugs, excessive expression of foreign proteins may poison host plants. However, the chloroplast of the unicellular green alga Chlamydomonas largely possesses the same machinery necessary for folding and assembling complex eukaryotic proteins, as that of flowering plants and tolerates the accumulation of eukaryotic toxins [20]. Protein-based drugs and vaccine antigens produced in Chlamydomonas chloroplasts are shown in Table 5 [20,131–137].


**Table 4.** Vaccine antigens and protein-based drugs produced by chloroplasts.

DW: dry weight; FW: fresh weight; NR: not recorded; TLP: total leaf protein; TSP: total soluble protein.



DW: dry weight; FW: fresh weight; NR: not recorded; TCP: total cellular protein; TSP: total soluble protein.
