**3. Can Plastid Transformation Work in Crops? CRISPR-Cas, Morphogenic Regulators, and Protoplast Regeneration Can Help**

Although tobacco is an excellent species for plastid transformation, its leaves are not edible, so any recombinant protein produced in the chloroplast needs to be purified before use. By contrast, plastid transformation of vegetable and fruit plants would offer significant advantages for the production of edible vaccines. Fruits are easier to store and transport relative to leafy vegetables like lettuce. The establishment of plastid transformation in species that bear fruit suitable for human consumption is a hot research topic.

For plastid transformation, the formation of double-strand DNA breaks (DSBs) in the chloroplast genome is a critical factor for HR [56], which might be stimulated at the chosen editing site by the use of the CRISPR nuclease Cas9 or transcription activator-like effector nucleases (TALENs). The CRISPR/Cas9 system allows for precise genome editing by guide RNAs (gRNAs) that direct the Cas9 nuclease to a target site. To increase plastid transformation efficiency, Yoo et al. [68] applied genome editing in Chlamydomonas chloroplasts by introducing two plasmids: one plasmid carried both a gRNA and a Cas9 expression cassette, while the other plasmid bore the donor DNA fragment for integration at the DSB site created by the action of Cas9 and the gRNA. Both plasmids were transformed into the alga by biolistics; cell lysates from individual transformants were screened by PCR 28 days after bombardment. When Cas9/gRNA was placed under the control of the strong chloroplast *psaA* promoter, two transformants out of 20 had the donor DNA at the intended integration site. By contrast, no HR events were observed when Cas9/gRNA were expressed from the weaker plastid *psbD* promoter, or when the first plasmid carried the gRNA but not Cas9. These results indicate that DNA breaks indeed promote donor DNA integration. Although the CRISPR-Cas approach has not yet been applied to plastid transformation in plants, a sequential transformation method was used to generate nuclear HR transgenic lines in Arabidopsis [69]. Two transgenic lines expressing Cas9 were used as parental lines for stable transformation with a second construct carrying the sgRNA and donor DNA. Using this sequential transformation approach, transgenic plants with HR in the target site of the nuclear genome were successfully identified [69]. Similarly, incorporating Cas9 and gRNAs into plastid transformation protocols should accelerate DSB formation and raise the frequency of HR, as high expression of Cas9 would enhance the likelihood of HR in transgenic plants.

TALENs constitute another potential strategy for site-specific gene modification and have been widely used in algae and flowering plants to generate transformants with desired traits [70–72]. For example, Li et al. [73] co-bombarded a plasmid carrying a TALEN construct and a plasmid carrying an HR DNA fragment in rice using biolistics. The transformation efficiency of the TALEN-assisted group was twice as high as that of a group transformed only with the HR fragment-containing plasmid. Together, these recent results provide a convincing foundation for the application of genome editing by Cas9 or TALENs in plastid transformation.

Another limiting factor for any plastid transformation effort is the regeneration efficiency during tissue culture. Harnessing growth-related genes to promote plant growth is not a new concept: in fact, the idea of affecting plant morphogenesis to recover transformants can be traced back to the 1980s [74]. Previous studies have shown that overexpressing morphogenic genes can increase nuclear transformation rates and enhance regeneration ability [75,76]. To date, several morphogenic genes (*Baby boom* (*BBM*) and *Wuschel2* (*WUS2*)) have been successfully used in nuclear transformation [76–78]. This strategy has not yet been applied to plastid transformation and is worth exploring as an approach to support plastid transformation in more species, especially those plants that are recalcitrant to transformation or are only marginally transformable.

Thanks to the advances in transformation technology, research facilities and purity of chemicals, protoplasts might be an option worth (re)-considering for plants that are not amenable to transformation by biolistics or when morphogenic regulators are ineffective. Protoplast regeneration was reported as early as the 1970s [79]. Unfortunately, protoplast regeneration is perceived by many as being hard to establish and labor-intensive and is always considered as a last resort, when it is even considered. This impression might stem from the poor quality of chemicals and laboratory conditions in the early days. With the development of more effective tissue culture protocols and the evolution of laboratory facilities, protoplast regeneration may be established in any plant tissue culture laboratory with minimal effort [44,80]. This technique, in fact, offers many advantages: there is no need for expensive instruments and consumables cost is low. Protoplast transformation may even be considered as being more efficient than other transformation techniques for flowering plants. Multiple rounds of particle bombardment are typically required on hundreds of leaves to obtain transformants, while protoplasts derived from only two leaves and transformed with PEG vesicles loaded with target DNA can produce positive clones in a single experiment [42]. Numerous articles have reported success in PEG-mediated protoplast transformation and regeneration [42,80–82]; some studies also demonstrated that protoplast transformation supported the transformation of non-transformable plants and increased the expression of a foreign (target) gene. In cauliflower, no transformants were obtained by biolistics, but DNA uptake was achieved by protoplasts in the presence of PEG, although the transformation efficiency was not high [82]. In potato, high activity levels were detected for a reporter carrying the *beta-Glucuronidase* (*GUS*) gene transformed into protoplasts using the PEG method. By contrast, transgenic plants

obtained by biolistics showed variable GUS activity levels, and truncated RNA species were detected in plants with low GUS activity [81]. This phenomenon of transgene silencing in lines generated by biolistics is frequently reported, in contrast to Agrobacterium- or PEG-mediated transformation in rice and barley [83,84].

To date, our laboratory has successfully transformed protoplasts with the PEG-mediated method and regenerated whole plants for tobacco and *N. benthamiana*, tomato, wild tomato (*S. peruvianum*), rapid-cycling Brassica and Arabidopsis. Taking tobacco as an example, it only took our laboratory about two years to establish the entire procedure, from protoplast isolation and CRISPR/Cas9 transformation by PEG-mediated transfection to protoplast regeneration [80]; by no means a short time frame, but definitely manageable and attainable. With the incorporation of the latest tools such as SWNTs delivery or morphogenic regulators, we believe that our platform can attain a wider application by reaching more crops or when applied to plastid transformation.
