Chloroplast transformation has several unique advantages. The highest levels of expression in the published literature for engineering agronomic traits (Bacillus thuringiensis insecticidal protein up to 46% of total leaf protein—De Cosa et al., 2001) or human therapeutic proteins (proinsulin up to 72% of total leaf protein—Ruhlman et al., 2010) were achieved using this concept and transplastomic plants maintain normal growth and reproduction despite hyper-expression of foreign proteins. In addition, integration of foreign genes into the chloroplast genome offers transgene containment from pollen transmission because of maternal inheritance of the chloroplast genome in most crops (Daniell, 2007; Hageman, 2010). Furthermore, harvesting therapeutic proteins or industrial products (e.g., biomass-degrading enzymes) from leaves before appearance of any reproductive structures offers effective transgene containment via pollen or seeds. Chloroplast genetic engineering offers a number of other unique advantages including multi-gene engineering in a single transformation event, lack of gene silencing or position effects caused by site-specific transgene integration and minimal or lack of pleiotropic effects caused by subcellular compartmentalization of toxic transgene products (Verma and Daniell, 2007).
Chloroplast transformation was first demonstrated by complementation of deletion mutants in Chlamydomonas, followed by transient foreign gene expression in cultured tobacco cells. After demonstration of the aadA gene as selectable marker in Chlamydomonas chloroplasts, tobacco chloroplast genome was stably transformed (Day and Goldschmidt-Clermont, 2011). Tobacco served as the model system to confer herbicide, insect (lepidopteran, aphids, white flies) or disease resistance (against viral, fungal or bacterial pathogens), drought or salt tolerance or phytoremediation (Clarke and Daniell, 2011). More recently, chloroplast genomes of major crops including cotton and soybean, vegetables (carrot, lettuce, cabbage, eggplant, sugar beet), fruits (tomato), trees (poplar), etc were transformed (Verma and Daniell, 2007; Clarke and Daniell, 2011). Significant progress has been made in expressing vaccine antigens against human bacterial, viral and protozoan pathogens in chloroplasts, and animal studies have demonstrated their efficacy against pathogen or toxin challenge (Daniell et al., 2009). Most importantly, oral delivery of vaccine antigens bioencapsulated in plant cells was shown to be more efficacious than injectable vaccines or in developing oral tolerance against autoimmune disorders (Arlen et al., 2008; Ruhlman et al., 2007; Verma et al., 2010).
The Plant Biotechnology Journal is a leader in publication of articles in chloroplast biotechnology and has published 17.87% of articles in this field (Plant Biotech. J. 17.87%; Plant Physiol. 8.93%; Transgenic Res. 12.84%; Plant Mol. Biol. 8.37%). In this special issue on chloroplast biotechnology, three articles review the history and progress made in chloroplast-selectable markers and removal of marker genes (Day and Goldschmidt-Clermont, 2011), expression of vaccine antigens and biopharmaceuticals (Loessl and Waheed, 2011) or the potential of chloroplast genetic engineering for the manipulation of plastid fatty acid biosynthetic pathways to improve food quality and biofuel production (Rogalski and Carrer, 2011). The Plant Biotechnology Journal is also a leader in the publication of articles in molecular farming engineered via the nuclear (Faye and Gomord, 2010) or chloroplast genome (Rigano et al., 2009; Davoodi-Semiromi et al., 2010; Lee et al., 2011). To further advance this field, this special issue includes several original articles reporting expression of new human therapeutic proteins including oral and injectable insulin (Boyhan and Daniell, 2011), HIV inhibitor cyanovirin (Elghabi et al., 2011), HIV-1 capsid protein p24 (Gonzalez-Rabade et al., 2011), human papillomavirus L1 antigen (Waheed et al., 2011), TGFβ3 (Gisby et al., 2011) and plastidal thioredoxins as modulators of recombinant therapeutic protein production (Sanz-Barrio et al., 2011). The Plant Biotechnology Journal also routinely publishes articles on improving plants to confer useful traits from academic or industry laboratories (Dufourmantel et al., 2007). In this issue, chloroplast transformation has been used to express metallothionein to confer mercury phytoremediation (Ruiz et al., 2011) or modify enzymes in the antioxidant pathway within chloroplasts to confer enhanced salt and cold tolerance (Le Martret et al., 2011). Cultured algal or plant cells offer yet another alternative to crop plants as bioreactors (Rasala et al., 2010; Sattarzadeh et al., 2010). Therefore, in this special issue, the chloroplast system has been used as bioreactor to express higher levels of foreign proteins using a nuclear mutant (Michelet et al., 2011) or by optimization of regulatory sequences in Chlamydomonas (Rasala et al., 2011) or in shoots developed from cultured tobacco cell suspensions in an immersion bioreactor (Michoux et al., 2011).
These diverse applications of chloroplast biotechnology promise to advance this field towards commercial development. Transplastomic plants expressing human therapeutic proteins were first tested in the field in 2007 (Arlen et al., 2007). Recent funding to advance this concept by major pharmaceuticals (Bayer Pharma) or foundations including the Bill and Melinda Gates Foundation or Juvenile Diabetes Research Foundation augurs well for advancing inventions from laboratories to the clinic. Agronomic traits engineered via the chloroplast genome in soybean by Bayer Crop Science (Dufourmantel et al., 2007) are advancing towards field studies. Biomass-degrading enzymes have been engineered via the chloroplast genome (Ziegelhoffer et al., 2009); expression of several such enzymes enabled preparation of cocktails for degradation of pine wood or citrus peel (Verma et al., 2010). Production of low-cost enzymes in non-food/feed crops is ideal to produce biofuel from biomass. It is anticipated that this special issue on chloroplast biotechnology and various other publications appearing in the Plant Biotechnology Journal will be useful for the academic and industry researchers and regulatory agencies.