The genome of eukaryotic cells is unevenly distributed and kept in different subcellular compartments. While the vast majority of genetic information is sheltered in the nucleus, small portions of DNA reside in organelles, namely the mitochondria and - in the case of plants - in the plastids. These unique organelles which in their most prominent manifestation are called chloroplasts, developed from cyanobacterial ancestors in a process described by the well accepted endo-symbiosis theory (Gould et al. 2008). In brief, a pre-eukaryotic cell must have engulfed and taken up an ancestor of today's cyanobacteria and subsequently formed a close endosymbiotic relationship with the newly developing organelle. Residues of this evolutionary ancestry are still apparent today in certain prokaryotic characteristics retained by the plastids. There is for example the genome organization in operons, as well as the transcription and translation machinery with their 70S ribosomes, to name only a few features which resemble those of today's bacteria. However, during the adaption process which lasted several billion years the plastids lost their autonomy in that they transferred the majority of their genetic information and the capacity for its regulation. Genes were either lost or transferred to the nucleus, accompanied with the assembly of a regulatory network which operates most of the metabolic processes in plastids. What is present in contemporary plastids is a highly reduced genome retaining some integral features like DNA replication and protein biosynthesis. Furthermore, plastids and especially chloro-plasts have a unique role in that they provide the primary energy source for the plants via photosynthesis and synthesize important compounds like aromatic amino acids. It has only recently become evident that plastids also have crucial roles in

H. Warzecha and A. Hennig

Darmstadt University of Technology, Institute for Botany, Schnittspahnstrasse 3-5, 64287

Darmstadt, Germany e-mail: [email protected]

F. Kempken and C. Jung (eds.), Genetic Modification of Plants, 23

Biotechnology in Agriculture and Forestry 64,

DOI 10.1007/978-3-642-02391-0_2, © Springer-Verlag Berlin Heidelberg 2010

plant development and therefore additionally regulate processes in the cellular metabolism. Several reports provide evidence that plastid genes encode functions which reach beyond its borders, for example, chloroplast protein synthesis is mandatory for regular leaf development and its knock-down will result in aberrant phenotype (Ahlert et al. 2003).

Consequently, the genetic manipulation of chloroplasts became a major focus since it provides the option to study the function of this unique organelle in great detail. Also, the application of chloroplast transformation in biotechnology has advanced due to characteristics which make plastids a promising vehicle for the high-level production of recombinant proteins. The most prominent difference to nuclear genes is the mere number of transgenes which could be introduced into a single cell by transformation of the chloroplast genome. Usually, in green tissue every cell contains up to 100 chloroplasts. Every chloroplast itself contains up to 100 identical copies of the circular plastid DNA, organized in nucleoid structures of about ten aggregated copies (Thomas and Rose 1983). In total this makes up to a 10 000 copies of any gene, outnumbering every nuclear gene by far. This is one reason why chloroplast transformation often results in extraordinarily high levels of recombinant protein accumulation.

However, chloroplast transformation makes high demands on vector design, transformation method as well as plant regeneration. This is exemplified by the so far not solved problem to generate fertile transplastomic lines of the model plant Arabidopsis thaliana (Sikdar et al. 1998), an example depicting the obstacles of this technique which need to be overcome to gain broad applicability. On a routine basis, so far only tobacco chloroplasts are transformed, and therefore most examples given in this chapter refer to tobacco chloroplast transformation. Nevertheless, great progress has been made in expanding the range of this technique to other plant species. Today, there are reports of successful plastid transformation in about 16 species (Table 2.1) and studies with transplastomics give valuable insight into genetics and biochemistry of this unique organelle.

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