*4.5. Everything Looks Great, Right?*

There are still many challenges facing the widespread adoption of plastid transformation technologies. These include: low transformation efficiency; lack of efficient screening methods for homoplasmy in transgenic plants outside of tobacco, especially in important crops such as rice, corn and other monocotyledonous plants, due to the lack of suitable selection markers and regulatory elements [167]; lack of appropriate tissue-specific regulatory sequences [168]; degradation of foreign proteins [169]; and foreign protein expression sometimes causing male sterility, yellow leaves and stunting. Inducible systems such as the ethanol-induced T7 promoter system, IPTG-lac system, and theophylline-inducible riboswitch system were developed because of the damage caused by constitutive foreign protein expression in host plants, but even these systems suffer from drawbacks: they are complex, toxic and costly. Although plastid transformation has been applied to various fields, it will also be critical to raise awareness among the general public of the usefulness of marker-free transplastomic plants.

#### **5. Conclusions and Prospects**

There is no doubt that plastid transformation has afforded a new direction for plant genetic engineering and constitutes a research hotspot because of its many advantages over nuclear transformation. Indeed, high transgene expression and engineering of polygenic traits are not amenable to classical nuclear transformation. However, plastid transformation can meet human needs, it is cost-effective, environmentally friendly, safe and efficient. It can be used to modify agronomic traits, and for phytoremediation and biofuels production. More importantly for human health, antigen vaccines and protein-based drugs can be produced in chloroplasts. Although plastid transformation has been achieved in many crops, many others still remain recalcitrant to plastid transformation [137]. The protocols of plastid transformation in crops would offer significant advantages for the production of edible vaccines and medical proteins, biofuels and industrial enzymes, as well as enhanced agronomic traits. In this review, we have illustrated the potential advantages from new studies and technologies like CRISPR-Cas9 for introducing double-strand DNA breaks for HR and create new varieties/mutants (*acc2*) to increase selection efficiency during plastid transformation. No longer limited to biolistic methods, nanotubes can be an alternative material for DNA delivery to increase the donor DNA into chloroplasts. Although they have not yet been applied to plastid transformation, the morphogenic regulators (*BBM*, *WUS2*, and cytokinin biosynthesis genes) and the new chemicals/technology for tissue culture and regeneration can increase the regeneration of transformed cells. These new approaches should also entice researchers to reconsider protoplast-based strategies for plastid transformation. Combined with nuclear transformation and other methods, plastid transformation may allow the production of important proteins.

**Author Contributions:** C.S.L. conceived and designed the review. C.-S.L., P.-C.Y. and W.-J.C. prepared the plastid transformation section. Y.Y., P.-C.Y., W.-J.C., and K.Y. prepared the plastid transformation application section. P.-C.Y., W.-J.C. and C.-S.L. wrote the manuscript with input from all co-authors. All authors read and approved the final manuscript.

**Funding:** This research was supported by Academia Sinica, Innovative Translational Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10, AS-KPQ-108-ITAR-10, AS-KPQ-109-ITAR-10), and the Ministry of Science and Technology (105-2313-B-001-007 -MY3; 108-2313-B-001 -011 -), Taiwan.

**Acknowledgments:** We thank Plant Editors for English editing.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**


