Transplastomic plant

Plant gene cassette schematic for plant transplastomics

The first requirement for transplastomic plant generation is to have a suitable gene construct that can be introduced into a plastid like a chloroplast in the form of an E. coli plasmid vector.[8] There are several key features of a suitable gene cassette including but not limited to (1) selectable marker (2) flanking sequences (3) gene of interest (4) promoter sequences (5) 5' UTR (6) 3' UTR (7) intercistronic elements.[9] The selectable marker typically tends to be an antibiotic resistant gene, which would give the plant cell the ability to tolerate being grown on antibiotic containing agar plates.[5] Flanking sequences are crucial for introduction of the gene construct at precise predetermined points of the plastid genome through homologous recombination.[4] The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production.[4] Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an operon.[4] Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively.[4]

The most common method for plastid transformations is biolistics: Small gold or tungsten particles are coated with the plasmid vector and shot into young plant cells or plant embryos, penetrating multiple cell layers and into the plastid.[8] There will then be a homologous recombination event between the shot plasmid vector and the plastid's genome, hopefully resulting in a stable insertion of the gene cassette into the plastid.[8] Whilst the transformation efficiency is lower than in agrobacterial mediated transformation, which is also common in plant genetic engineering, particle bombardment is especially suitable for chloroplast transformation. Other transformation methods include the use of polyethylene glycol (PEG)- mediated transformation, which involves the removal of the plant cell wall in order to expose the "naked" plant cell to the foreign genetic material for transformation in the presence of PEG.[8] PEG- mediated transformation however, is notoriously time consuming, very technical and labor intensive as it requires the removal of the cell wall which is a key protective structural component of the plant cell.[10] Interestingly, a paper released in 2018 has described a successful plastid transformation of the chloroplast from the microalgae species N. oceanica and C. reinhardtii through electroporation.[10] Whilst no study has been attempted yet for plastid transformation of higher plants using electroporation, this could be an interesting area of study for the future.

In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. When foreign DNA is first introduced to the plant tissue, not all chloroplasts will have successfully integrated the introduced genetic material.[5] There will be a mixture of normal and transformed chloroplast within the plant cells. This mix of normal and transformed chloroplasts are defined to be "heteroplasmic" chloroplast population.[5] Stable gene expression of the introduced gene requires a "homoplasmic" population of transformed chloroplasts in the plant cells, where all the chloroplasts in the plant cell has successfully integrated the foreign genetic material.[5] Typically, homoplasmicity can be achieved and identified through multiple rounds of selection on antibiotics.[5] This is where the transformed plant tissue are grown repeatedly on agar plates that contain antibiotics like spectinomycin.[5] Only plant cells that have successfully integrated the gene cassette as shown above will be able to express the antibiotic resistance selectable marker and therefore grow normally on agar plates containing antibiotics.[5] Plant tissue that do not grow normally will have a bleached appearance as the spectinomycin antibiotic inhibits the ribosomes in the plastids of the plant cell, thereby preventing maintenance of the chloroplast[5] However, as heteroplasmic population of chloroplasts may still be able to grow on agar plates effectively, many rounds of antibiotic selection and regrowth are required to cultivate a plant tissue that is homoplasmic and stable.[5] Generation of homoplasmic plant tissue is considered to be a major difficulty in transplastomics and incredibly time consuming.[8]

Example of a tomato plant grafting for agricultural purposes

Some plant species such as Nicotiana tabacum are more receptive to transplastomics compared to members of the same genus such as Nicotiana glauca and Nicotiana benthamiana.[11] An experiment conducted in 2012 highlighted the possibility of facilitating transplastomics for difficult plant species using grafting. Grafting occurs when two different plants are joined together and continue to grow, this technique has been widely employed in agricultural applications and can even occur naturally in the wild.[12] A transplastomic N. tabacum plant was engineered to have spectinomycin resistance and GFP fluorescence.[11] Whilst the nuclear transgenic plants N. benthamiana and N. glauca were engineered to have kanamycin antibiotic resistance and YFP fluorescence.[11] The transplastomic plant and the nuclear transgenic plants were then grafted unto each other and the grafted tissues were then analysed.[11] Fluorescence microscopy and antibiotic selection on agar plates with both kanamycin and spectinomycin revealed that the grafted plant tissue had both transplastomics and nuclear transgene DNA material.[11] This was further confirmed through PCR analysis.[11] This study highlighted that plastids like the chloroplast are able to pass between cells across graft junctions and result in the transfer of genetic material between two different plant cell lines.[11] This finding is significant as it provides an alternative pathway for generation of transplastomic plants for species that are not as easily transformed using our current experimental methodology as seen above.[11]

Inducible expression systems such as theoriboswitches and pentatricopeptide repeat proteins have been widely studied in an effort to control and modulate expression of transgene products in transplastomic plants.[13] One big advantage in using inducible expression systems is to optimize concentration of transgene protein production.[13] For example, young plants need to devote energy and resources into growth and development to become mature plants.[13] Constitutive expression of the transgene would therefore be detrimental for plant growth and development, as it takes away valuable energy and resources to express the foreign gene construct instead.[13] This would result in a poorly developed transplastomic plant with low product yield.[13] Inducible expression expression of the transgene would overcome this limitation and allow the plant to mature fully like a normal wildtype plant before it is induced chemically to begin production of the transgene which can then be harvested.[13]