Chloroplast transformation is discussed briefly in Chapter 12 of Principles of Gene Manipulation (pp 240-241) but the vast majority of the chapter is devoted to the nuclear transformation of plants. Recently, however, chloroplast transformation has emerged as a serious alternative to nuclear transformation in the biotechnology industry. This is because transplastomic plants have a number of advantages over (nuclear) transgenic plants in terms of protein expression and environmental safety. These advantages can be summarized as follows:

• The plastid genome is highly polyploid. Each plastid may contain multiple copies of the genome and there may be thousands of plastids in a cell. The copy number of any introduced transgene can reach 10,000. This means that transplastomic plants produce extremely high levels of foreign proteins, in the best cases approaching 50% of the total soluble protein in the cell.
• Plastids are not found in mature pollen (they show maternal inheritance only), thus the chances of spreading transgenes by outcrossing are minimal.
• DNA transfer to chloroplasts is achieved by gene targeting, i.e. homologous recombination. Therefore, the transformation construct integrates at a precise and predictable location in the chloroplast genome, eliminating the position effects (influence of position of integration on transgene expression) seen in nuclear transgenics.
• Transgene silencing, a thorn in the side of nuclear transgenics, has not been documented in the plastid genome. This probably reflects both the absence of position effects and the predictability of transgene locus structure (i.e. the absence of inverted and direct repeats, and partial transgene copies).
• Recombinant proteins, in particular those of human origin, appear to fold normally in the chloroplast and to form disulphide bonds as expected. In nuclear transgenics, foreign proteins must be targeted to the secretory pathway to be processed correctly.
• Recombinant proteins that are toxic when expressed at high levels in the cytosol of the plant cell are often non-toxic when they accumulate in the chloroplast.
• Chloroplast genes, like those of bacteria, are arranged in operons. Therefore, multiple transgenes can be expressed in chloroplasts from the same polycistronic transcript.

The first publication reporting the expression of a useful gene (i.e. not a marker gene) in chloroplasts was that of McBride et al. (1995). These investigators showed that a chimeric cry1Ac gene from Bacillus thuringiensis could be expressed at very high levels in chloroplasts and that the resulting transplastomic plants were resistant to a range of insect pests. Further applied studies of chloroplast engineering were not published until the turn of the century (see Table 1). These included reports of chloroplasts expressing transgenes conferring useful agronomic traits (insect resistance, herbicide resistance, drought tolerance), and their use for the production of therapeutic human proteins. One example in the latter category is somatotropin (growth hormone), which is used to treat pituitary dwarfism. This was expressed in tobacco chloroplasts and in the best cases reached 7% of total soluble protein. The protein was folded correctly, appropriate disulphide bonds were formed and it was shown to be biologically active (Staub et al. 2000). Another example is the cholera toxin β subunit (CTB), which can be used as a subunit vaccine. Daniel et al. (2001) succeeded in expressing the CTB in tobacco chloroplasts, and in some plants the recombinant protein accounted for up to 4% of total soluble protein. This is compared to 0.1% for the best nuclear transgenics. In the future, it would be desirable to replicate these expression levels in edible crop plants, so that vaccines can be administered orally without prior purification. Preliminary studies indicate that it should be possible to express recombinant proteins in potato and tomato chloroplasts. Transplastomic tomato plants expressing the aad marker gene contained the recombinant protein in fruits and leaves. The amount of protein in the fruit was about half that in the leaves (Ruf et al. 2001). In potato, high levels of green fluorescent protein were expressed in the leaves (5% total soluble protein) although the amount detected in tubers was 100-fold less (Sidorov et al. 1999).

One unique aspect of chloroplast engineering is the possibility of using operons to express multiple transgenes. In the plant's nuclear genome this is not possible, because each gene produces a separate mRNA. Therefore, multiple transgene expression relies on either the crossing of plants containing single transgenes, or the concurrent transfer of transgenes arranged either on the same vector (cointegrate strategy) or on different vectors (cotransformation strategy) during the initial transformation process. Both these approaches have disadvantages (reviewed by Daniell & Dhingra 2002). In the chloroplast, most genes are transcribed as polycistronic messages. For this reason, the targeted integration of a transgene into the chloroplast genome normally results in its expression as part of a polycistron, unless it is deliberately introduced into a non-transcribed spacer region. However, the deliberate expression of multiple transgenes as part of an operon was achieved for the first time last year. De Cosa et al. (2001) showed that tobacco chloroplasts could express the three genes of the Bacillus thuringiensis cry2Aa2 operon. The cry2Aa2 gene itself is the third gene of the operon. The first two genes encode helper proteins, one of which is a chaperon that folds the toxin protein into a stable conformation and allows it to form crystals (Ge et al. 1998). This approach allowed the recombinant CryAa2 protein to accumulate to the highest levels ever recorded in genetically manipulated plants - over 45% total soluble protein - with potent effects on some of the most difficult-to-control insect pests yet no transfer of the gene or its lethal product into pollen, thus eliminating any danger of harming non-target and beneficial insects. Chloroplast engineering therefore appears to be a safe and environmentally-friendly alternative to nuclear gene transfer in the biotechnology industry. Further information about current applications of chloroplast engineering and the design of vectors and expression cassettes can be found in two recent reviews (Maliga 2002, Daniel et al. 2002).

Gene Product Application Reference

*5-enolpyruvoylshikimate-3-phosphate synthetase

References:

Daniell H, Dhingra A (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Curr Opin Biotechnol 13, 136-41.

Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7, 84-91.

Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311, 1001-9.

De Cosa B, Moar W, Lee SB, Miller M, Daniell H. (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nature Biotechnol 19, 71-4.

DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol 127, 852-62.

Ge B, Bideshi D, Moar WJ, Federici BA (1998) Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis. FEMS Microbiol Lett 165, 35-41.

Guda C, Lee S-B, Daniell, H (2000) Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Reps 19, 257-62.

Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ (1999) Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc Natl Acad Sci USA 96, 1840-5.

Lutz KA, Knapp JE, Maliga P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol. 125, 1585-90.

Maliga P (2002) Engineering the plastid genome of higher plants. Curr Opin Plant Biol 5, 164-72.

McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Bio/Technology 13, 362-5.

Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19, 870-5.

Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, Nehra NS. Technical Advance. Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 19, 209-16.

Staub JM, Garcia B, Graves J, Hajdukiewicz PT et al. (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nature Biotechnol 18, 333-8.

Ye GN, Hajdukiewicz PT, Broyles D, Rodriguez D et al. (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25, 261-70.

The next generation of selectable marker genes for plants?

In Chapter 12 of Principles of Gene Manipulation , we discuss selectable marker genes used for the transformation of plants (Box 12.2, pp 231-232). Traditionally, these have been antibiotic or herbicide resistance markers that allow transformed plant cells to survive in the presence of otherwise toxic concentrations of the selective agent. One problem with such markers is that the selective agent can adversely affect plant growth and regeneration. Furthermore, there are concerns that such markers could spread in the environment or pose a threat to human health by encouraging the evolution of resistant strains of pathogenic bacteria. These issues have driven research in the direction of safer and friendlier marker genes. The next generation of selectable markers may well be based on genes that control plant growth and development.

The first such marker to be used was the Agrobacterium tumefaciens ipt gene, encoding isopentyl transferase, an enzyme catalysing one of the early steps in cytokinin synthesis. Cytokinins are required for shoot development, but constant high levels of the phytohormone cause abnormal growth. Therefore, the ipt gene cannot be constitutively expressed in the same way as traditional markers. Two approaches have been used to avoid this. In one approach, the marker has been excised from the transgenic plant by inducible site-specific recombination. Marker-removal strategies are also used to get rid of traditional antibiotic and herbicide resistance markers (reviewed by Hohm et al. 2001, Ow 2001). The other approach has been to place the ipt gene under an inducible promoter, so that cytokinins are produced only when needed. Thus, regeneration is carried out in cytokinin-free medium in the presence of the inducing agent, so that only transformed cells produce the enzyme required to stimulate cytokinin synthesis. Transgenic shoots are then transferred to unsupplemented medium to allow normal growth (Kunkel et al. 1999). Plant ipt genes have also been investigated as possible selectable markers (Takei et al. 2001, Kakimoto 2001).

One disadvantage of the ipt gene as a marker is that the cytokinins produced by transformed cells can diffuse away from their source and provide cross-protection to non-transformed cells, resulting in a high proportion of so-called escapes. An alternative system has been developed based on the Arabidopsis CKI1 (CYTOKININ INDEPENDENT 1) gene, which encodes a component of the cytokinin signal transduction pathway. This gene provides the same benefits as ipt (i.e. inducible expression promotes shoot growth on a cytokinin-free medium) but the product does not diffuse, so the number of escapes is reduced (see Zuo et al. 2002b for details).

A variety of recently characterized plant regulatory genes that could potentially be used as markers is discussed in a recent review (Zuo et al. 2002b). These genes generally fall into two classes: those that promote organogenesis and those that promote somatic embryogenesis. The ipt gene is useful for plants that regenerate via organogenesis since it promotes shoot growth, but it cannot be used for plants that regenerate via the alternative somatic embryogenesis route. However, the leafy cotyledon genes are promising candidate markers for somatic embryogenesis, and the Arabidopsis WUS (WUSCHEL) gene has also been identified as a key regulator of the vegetative-to-embryogenic transition (Zuo et al. 2002a).

References:
Hohn B, Levy AA, Puchta H (2001) Elimination of selectable markers from transgenic plants. Curr Opin Biotechnol 12, 139-43.

Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentyltransferases. Plant Cell Physiol 42, 677-85.

Kunkel T, Niu Q-W, Chan YS, Chua N-H (1999) Inducible isopentyl transferase as a high-efficiency marker for plant transformation. Nature Biotechnol 17, 916-9.

Ow D (2001) The right chemistry for marker gene removal. Nature Biotechnol 19, 115-6.

Takei K, Sakakibara H, Sugiyama T (2001) Identification of genes encoding adenylate isopentyltranaferase, a cytokine biosynthesis enzyme, in Arabidopsis thaliana. J Biol Chem 276, 26405-10.

Zuo J, Niu Q-W, Frugis G, Chua N-H (2002a) The WUSCHEL gene promotes vegetative-to embryonic transition in Arabidopsis. Plant J 30, 349-59.

Zuo J, Niu Q-W, Ikeda Y, Chua N-H (2002b) Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes. Curr Opin Biotechnol 13, 173-80.