Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Abstract

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.

Fig. 1

Various steps in chloroplast genetic engineering. (a) In single-gene (X) chloroplast transformation vectors, coding sequences are flanked by independent 5′ and 3′ regulatory regions, which encompass promoters (P) and terminators (T), respectively. In multiple gene (X, Y, Z) constructs, a single promoter (P) regulates expression of the operon, and individual ribosome binding sites (RBS) are engineered upstream of each open-reading-frame. (b) Gene delivery is usually performed by particle bombardment of chloroplast vectors. Antibiotic selection is performed with spectinomycin or streptomycin, whereas betaine aldehyde is used for plant-derived antibiotic-free selection. (c) The first round of selection generally results in heteroplasmy (left) whereas the second round achieves homoplasmy (right). (d) High-level expression of all the single and multiple gene traits listed here have been achieved, except for PHB and vitamin A operons. This is because of high ploidy of transgenes (5000–10 000 copies per cell). (e) In T₀ reproductive organs, after meiosis, haploid egg and sperm cells are formed (left). The zygote contains only maternal plastids because the paternal plastids disintegrate in the synergid cell (right). Maternal inheritance of transgenes offers biological containment because of lack of gene flow through pollen. Transgenic pollen generally does not contain the foreign gene product. (f) When transgenic seeds are germinated on selectable agents, 100% germination is observed. In contrast with Mendelian segregation of traits, chloroplast transgenes are maternally transmitted without any segregation.