*2.1. Robust Methods for DNA Delivery into the Chloroplast*

The two most common methods for introducing foreign DNA into chloroplasts are biolistic transformation [9] and polyethylene glycol (PEG)-mediated transfection [42]. Biolistics delivers particles coated with DNA into plant cells by high-speed bombardment through a gene gun or a particle delivery system. This method can be applied to various plants by adjusting bombardment parameters such as distance to the target tissue, chamber vacuum pressure, particle size and DNA: particle ratios, to accommodate variations in leaf texture. The PEG-mediated plastid transformation method works on plant cells from which the cell wall has been removed (protoplasts). The co-culture of protoplasts in the presence of PEG vesicles loaded with plasmid DNA allows DNA uptake by protoplasts, leading to the integration of foreign DNA into the plastid genome [42]. Although the PEG method requires the enzymatic digestion of tissues to release protoplasts, it is a more economical procedure because it does not rely on a specialized and expensive delivery system.

A new strategy for plastid transformation via nanoparticles was recently introduced [43]. This method allows DNA to be delivered to chloroplasts simply through single-walled carbon nanotubes (SWNTs) without the need for additional instruments or protoplast isolation, or the aid of chemical reagents. Nanocarriers for transformation consisted of chitosan-complexed single-walled carbon nanotubes (CS–SWNTs). These nanotubes are positively charged and can, therefore, carry negatively charged plasmid DNA via electrostatic interactions; the resulting DNA-SWNT conjugate may easily enter leaf mesophyll cells by infiltration using a syringe from stomatal pores. Once the DNA-SWNT conjugate passes through the leaf surface and enters the mesophyll, it will eventually be trafficked to the chloroplast bilayers by way of lipid exchange envelope penetration [43].

The power of this delivery strategy lies in the fact that the DNA can be selectively released in the chloroplast due to differences in pH within the cell. The acidic cytosol (around pH 5.5) leaves the DNA tightly bound to chitosan; by contrast, the carriers tend to unload the DNA inside the chloroplast due to its weakly alkaline environment (~pH 8.0). This preferential release accomplishes the selective release of DNA at its intended target site. A similar strategy may also be applied to protoplasts by effectively replacing PEG with nanotubes [43]. Indeed, DNA alone does not have the ability to penetrate plant cells in the absence of the SWNT carrier, as demonstrated with protoplasts co-cultured with DNA only. Notably, the efficiency of DNA entrance depends on the zeta potential and hydrodynamic radius of SWNTs rather than the concentration of plasmid DNA. Using a reporter construct carrying the *Yellow Fluorescent Protein* gene (*YFP*), YFP fluorescence was detected after 24 hr incubation of protoplasts with a DNA:SWNT mixture in a 1:6 ratio, and the YFP signal largely coincided with chloroplasts. However, no fluorescence was detected when the DNA:SWNT ratio was increased to 1:1. Excess DNA, bound to SWNTs, will neutralize their surface charge, which will not only reduce the entry of DNA-SWNT conjugates but also increase their instability, resulting in a partial or complete loss of membrane crossing [43].

Using the same YFP reporter, the method was further tested on living plants, in the form here of four-week old arugula (*Eruca sativa*) plants. The authors tested different DNA:SWNT ratios (1:1, 1:3 and 1:6); not surprisingly, based on the reasoning provided above, only ratios of 1:3 and 1:6 expressed YFP. Transient YFP expression reached its peak 48 h after the initial infiltration. An impressive aspect of the study was the low amounts of DNA (~20 ng) required for successful delivery and expression of plasmid DNA to chloroplasts, far less than for PEG-mediated transformation (20–50 μg) or biolistics (5 μg) [44–46].

This article demonstrated the delivery and expression of a transgene to chloroplasts in five different plant species, including four mature living plants and isolated protoplasts, which indicates the potential of this nanoparticle delivery method for widespread application [43]. Recently, another method by Santana et al. used a chloroplast signal peptide as a guide to delivering nanomaterials loaded with chemicals into Arabidopsis chloroplasts [47]. Tagged with a 14-amino acid guide peptide, the cargo was sent to the translocons at the outer and inner membrane of chloroplasts (TOC/TIC) and was transported into the chloroplast stroma. This, therefore, provides a targeted way to deliver materials into plastids via a biorecognition motif [47]. In addition to biolistics and PEG-mediated delivery methods, carbon nanotubes and biorecognition peptides now join the team of plastid transformation tools and combine the advantages of low DNA amounts and high delivery accuracy.

However, all SWNT tests were based on transient expression using a single plasmid carrying flanking regions from switchgrass (*Panicum virgatum*) [43]. Although the YFP signal indeed originated from the chloroplast and thus demonstrated the trafficking function of SWNTs to the organelle, homologous recombination within the chloroplast genome remains to be confirmed. In the case of the signal peptide delivery technique [47], the chemical cargo was the subject of the test and not DNA. Therefore, the ability and the efficiency of stable transformation through these two methods remain to be determined.
