Sanjeet Kumar
Chloroplasts are the site of photosynthesis in plant mostly seen in leaves which provides the primary source of the world’s food productivity. Plastids of higher plants are generally semiautonomous. The plastid genome is a circular molecule of double stranded DNA. Plastids are plant cellular organelles with a ~120–150 kb genome present in 1,000–10,000 copies per cell (Bendich, 1987), and maternally inherited in most angiosperm plant species (Hagemann, 2004). Chloroplast transformation has become an attractive alternative to nuclear gene transformation due to its advantages: high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission (Maliga, 2002; Bock, 2007; Kittiwongwattana et al., 2007).
Introduction
The concept of chloroplast genetic engineering was developed in the 1980s (Daniell and Mc Fadden, 1987). and Boynton et al. (1988) was reported successfully chloroplast transformation in Chlamydomonas through gene gun. In 1989, stable chloroplast transformation in higher plants was achieved in Pal Malinga’s Laboratory (Svab et al., 1990). Chloroplast transformation generally results from homologous recombination, with a fragment of transforming DNA replacing the corresponding chloroplast DNA. Two decades ago, it has become a most attractive alternate to nuclear gene transformation due to great potential: high protein levels, the feasibility of expressing multiple proteins. Keeping the importance of this technique authors try to highlight the recent trends in Chloroplast engineering.
It was generally achieved by the biolistic process, with which the Escherichia coli plasmids containing a marker gene and the gene of interest were introduced into chloroplasts or plastids. The foreign genes were inserted into plasmid DNA by homologous recombination via the flanking sequences at the insertion site.
Plastid expression vectors possessed left and right flanking sequences each with 1–2 kb in size from the host plastid genome, which are used for foreign gene insertion into plastid DNA via homologous recombination. The site of insertion in the plastid genome is determined by the choice of ptDNA segment flanking the marker gene and the gene of interest. Insertion of foreign DNA in intergenic regions of the plastid genome had been accomplished at 16 sites (Maliga, 2004). Three of the insertion sites, trnV-3'rps12，trnI-trnA and trnfM-trnG, were most commonly used (Maliga, 2004).
Regulation sequences
The gene expression level in plastids is predominately determined by promoter and 5′-UTR elements (Gruissem and Tonkyn, 1993). Therefore, suitable 5′-untranslated regions (5′-UTRs) including a ribosomal binding site (RBS) are important elements of plastid expression vectors (Eibl et al., 1999). In order to obtain high-level protein accumulation from expression of the transgene, the first requirement is a strong promoter to ensure high levels of mRNA. Most laboratories used the strong plastid rRNA operon (rrn) promoter (Prrn). Stability of the transgenic mRNA is ensured by the 5′-UTR and 3′-UTR sequences flanking the transgenes.
Other applications
· Production of biopharmaceuticals
· In metabolic pathway engineering
· In research on RNA editing
· Production of therapeutic proteins
Several environmental stress such as disease, drought, insect pests, salinity and freezing, can severely limit plant growth and development. In order to improve the plant traits, many researchers had done a series attempts. Many important agronomic traits have already been engineered via the plastid genome, such as herbicide resistance, insect resistance, and tolerance to drought and salt.
Biotic stresses
The insect resistance genes were investigated for high-level expression from the chloroplast genome. Cry genes could be expressed extremely well in the plastid genome and there was no requirement to adjust the codon usage nor any need for other sequence manipulation (Kota et al., 1999; Cosa et al., 2001).
Abiotic stresses
Chloroplast engineering had been successfully applied for the development of plants with tolerance to salt, drought and low temperature. Previous research has shown that over expression of enzymes for Glycine betaine （GlyBet）biosynthesis in transgenic plants improved tolerance to various abiotic stresses (Rhodes and Hanson, 1993).
Conclusions
The chloroplast engineering provide a good platform of foreign gene expression and holds great potential for the introduction of agronomic traits as well as the production of therapeutic proteins or vaccines in plants indigenous to developing countries such as India where people do not have access to these medicinal compounds. It has become a powerful Biotechnological tool for the study of biogenesis. Therefore rapid research is needed to obtain the commercial application of chloroplast engineering.
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