Recent achievements obtained by chloroplast transformation

Abstract

Chloroplasts play a great role for sustained wellbeing of life on the planet. They have the power and raw materials that can be used as sophisticated biological factories. They are rich in energy as they have lots of pigment-protein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in minerals imported from the plant cell. Chloroplast genome transformation offers multiple advantages over nuclear genome which among others, include: integration of the transgene via homologus recombination that enables to eliminate gene silencing and position effect, higher level of transgene expression resulting into higher accumulations of foreign proteins, and significant reduction in environmental dispersion of the transgene due to maternal inheritance which helps to minimize the major critic of plant genetic engineering. Chloroplast genetic engineering has made fruit full progresses in the development of plants resistance to various stresses, phytoremediation of toxic metals, and production of vaccine antigens, biopharmaceuticals, biofuels, biomaterials and industrial enzymes. Although successful results have been achieved, there are still difficulties impeding full potential exploitation and expansion of chloroplast transformation technology to economical plants. These include, lack of species specific regulatory sequences, problem of selection and shoot regeneration, and massive expression of foreign genes resulting in phenotypic alterations of transplastomic plants. The aim of this review is to critically recapitulate the latest development of chloroplast transformation with special focus on the different traits of economic interest.

Keywords: Chloroplast transformation; Homologus recombination; Novel traits; Regulatory sequences; Transgene.

Fig. 1

Diagrammatic representation of the processes for chloroplast genome transformation. a Basic design of a typical vector for transforming the plastid genome. Both the expression cassette and the selection cassette are placed between the two plastid regions. These flanking regions are taken from the wild-type plastid genome of a plant species whose plastome is to be manipulated, to allow a crossover event take place to integrate DNA sequences between them. Green arrows in the chloroplast expression vector represent promoters (P) and the direction of transcription, whereas terminators (T) are indicated by red rectangles. The untranslated regions are represented by white circles. The thin dotted lines with arrows indicate homologous recombination. b Delivery of transforming plasmids into chloroplasts in leaf cells using a particle delivery system. The plasmid DNA is coated on the surface of the microparticles of either gold or tungsten and then shot on to the abaxial surface of 4- to 6-week-old sterile leaves using a gene gun. The bombarded leaves are incubated for 48 h in the dark, cut into small discs and placed on regeneration medium supplemented with the appropriate antibiotic and hormones. Primary shoots generally arise within 2–3 months. c The process of recovering a stable homoplasmic transplastomic plant line. Initially, only a few copies of the plastome are transformed, and therefore the explant contains a mixture of both transformed as well as untransformed copies, a state known as heteroplasmy. The wild-type copies (indicated by light-coloured ovals) are sorted out gradually by repeating two or three regeneration cycles under selection to reach homoplasmy, a state where all copies of the plastome are transformed (indicated by dark grey ovals). Adopted from Ref. Ahmad et al. [113]