Novel Approach in Plastid Transformation Aart JE Van Bel*†, Julian Hibberd‡, Dirk Prüfer§ and Michael Knoblauch*
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144 Novel approach in plastid transformation Aart JE van Bel*†, Julian Hibberd‡, Dirk Prüfer§ and Michael Knoblauch* Engineering the nuclear genome of plants is perceived to be dispersal of crop plants engineered for herbicide resistance associated with problems regarding biosafety and the stability may render their weedy relatives insensitive to certain her- of expression of the transgene. Alternative transformation bicides [2]; transgenic pollen may be toxic to nontarget strategies using the genomic outfit of the plastid promise to insects such as butterflies [3••]. be more successful in this respect. Over the past few years progress has been made in screening procedures, and plastid The introduction of genes by engineering the chloroplast transformation technology has allowed function to be assigned has been proposed to remedy the problems associated with to open reading frames, massive expression of insecticidal transgene dispersal into the wild plant population. As there agents and proteins involved in herbicide resistance, and the are no plastids, and hence plastid DNA, in the pollen of accumulation of biopolymers. Recently, the design of a novel most crops (exceptions being alfalfa and possibly rice and femtoinjection technique that allows injection into chloroplasts pea; see [4–6]), any gene introduced into chloroplast-engi- has provided the opportunity to further manipulate and under- neered plants is unlikely to be transferred via the pollen to stand chloroplastic gene expression. the next generation. Introgression of genes from wild rela- tives, however, could eventually allow chloroplast trans- Addresses genes to invade the nontransformed population. The *Institute for General Botany and Plant Physiology, Justus Liebig extent to which introgression of genes between wild rela- University, Senckenbergstrasse 17, D-35390 Giessen, Germany tives occurs is at present uncertain, although in general † e-mail: [email protected] introgression is unusual [7••]. Despite the fact that rates ‡Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom are thought to be low, it has been pointed out by Stewart §Section of Molecular Biotechnology, Fraunhofer Institute IUCT, and Prakash [4] that introgression of the common weed Auf dem Aberg 1, D-57392 Schmallenberg, Germany Raphanus raphanistrum into Brassica napus (oilseed rape) Current Opinion in Biotechnology 2001, 12:144–149 occurred at higher rates [8] than the reciprocal cross of Brassica napus pollen into Raphanus raphanistrum. 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Plastid transformation has several additional advantages Abbreviations compared with nuclear transformation. First of all, the high Bt Bacillus thuringiensis ploidy level of the plastome — 5–80 (Chlamydomonas) or GEF galinstan expansion femtosyringe 500–10,000 (Nicotiana) DNA copies per cell — enables a GFP green fluorescent protein •• •• GM genetically modified high degree of expression [2,9,10 ,11 ]. The high num- ORF open reading frame ber of copies are calculated from the number of DNA copies per chloroplast multiplied by the number of chloro- Introduction plasts per cell. Chloroplast transformation has the potential The public debate on the acceptability of genetically therefore to be used to enhance the yield of transgenic modified (GM) plants revolves around a mixture of issues. products. Highly attractive for future applications is the Besides the respectable emotional and ethical concerns on possibility of polycistronic operon expression, that is, the the introduction of GM plants, several rational arguments stacking of multiple expressed genes in a single transfor- plead for the careful application of transgenic techniques mation event [12]. On top of that, it appears that gene before the release of plants into the environment. The silencing is absent in plastids and expression is therefore insertion of foreign DNA sequences into the nuclear plant more likely to be stable. genome is associated with several potential risks. Agrobacterium-mediated transformation and direct DNA Plastidic production of various protein classes transfer into plant cells often result in the insertion of Current research is exploring the advantages of plastomics vector sequences associated with the gene of interest. for the expression of genes coding for insecticidal proteins or These additional sequences may affect the endogenous allowing for herbicide resistance. Crops expressing the and transgenic expression in an unpredictable fashion. Bacillus thuringiensis (Bt) toxin from the nucleus may only Furthermore, cotransferred plasmid DNA, bacterial produce suboptimal amounts of toxins giving rise to an antibiotic genes and plant genome DNA may spread enhanced risk of pests developing Bt resistance [13]. In between and within species coexisting in the environment. attempts to reduce the development of Bt toxin resistance, the gene (cry1A) coding for the Bt toxin Cry1A(c) was insert- Advantages of transplastomic plants ed into the plastome. The high amplification rate resulted in Of 60 major crop plants, only 11 have no wild relatives (i.e., an accumulation of the strongly insecticidal Cry1A(c) pro- representatives of the same genus) [1]. Gene escape lead- toxin [14]. Recently, overexpression of the cry2Aa2 Bt gene ing to the genetic pollution of wild relatives has the poten- using chloroplast engineering in tobacco caused 100% mor- tial to pose a problem. For example, pollen or seed tality of insects that are largely resistant to other Bt proteins Novel approach in plastid transformation van Bel et al. 145 [15••]. In the transformants, the level of protoxin was 20–30- [25•,26•]. Another study showed that chloroplast structure fold higher than in nuclear transgenic plants. and physiology only partly suffered from knocking out plastid-encoded RNA polymerase [28]. In view of the required containment of the herbicide-resis- tant genes, a successful strategy was designed to engineer Targets for chloroplast transformation also include the pro- the plastome for glyphosate resistance [2]. Glyphosate is a teins involved in the metabolic pathways of plastids [29]. potent herbicide that is a competitive inhibitor of an essen- Amongst these are enzymes engaged in carbon fixation, tial step in the aromatic amino acid biosynthetic pathway. like Rubisco [30,31•,32•], and proteins present in the pho- Integration of the petunia EPSPS (5-enol-pyruvyl shikimate- tosynthetic reaction centers [23•,24•]. Chloroplast transfor- 3-phosphate synthase) gene into the tobacco plastome result- mation strategies surmounted previous obstacles to ed in an overproduction of EPSPS leading to glyphosate mutagenise the gene encoding the large subunit of resistance. Likewise, the effect of oxyfluorfen — a diphenyl Rubisco (rbcL) in higher plants. Tobacco plants mutated ether herbicide — was counteracted by plastomic insertion for rbcL showed reduced Michaelis constants for CO2, O2 of the Bacillus subtilis gene encoding protoporphyrinogen oxi- and ribulose bisphosphate, and an increased oxygenase •• • dase (protox) [16 ]. Protoporphyrinogen oxidase catalyzes a activity at limiting O2 levels [32 ]. Additionally, tobacco major step in the production of chlorophyll and heme groups, has also been transformed with the rbcL gene from sun- which is the action site of the diphenyl ether herbicides. flower and that of the cyanobacterium Synechococcus [31•]. Transplastomic lines for the B. subtilis protox exhibited a The latter line produced rbcL mRNA but no large subunit higher degree of oxyfluorfen resistance than the nuclear protein or enzyme activity. In contrast, the hybrid protein transgenic lines. composed of sunflower and tobacco subunits produced a catalytically active hybrid form of the enzyme. In a third The abundant expression capacity of the plastome has the line, a chimeric sunflower–tobacco large subunit arising potential for massive production of foreign molecules in from homologous recombination within the rbcL gene had various plants. Transformation of plastids has already been properties similar to the hybrid enzyme. achieved for tobacco [9,17••], Arabidopsis [18] and potato [19••], looks encouraging for rice [20••] and will presum- Conventional methods of plastome ably be extended to cover several more species in the near transformation future. It is hoped that plastid-mediated molecular farming The genetic transformation of plastids follows the princi- will lead to the biofabrication of a range of biopolymers ples of homologous recombination. For successful transfor- and pharmaceutical proteins. The production of a human mation, the transgene has to be flanked by plastomic somatropin in a soluble biologically active form [10••] and sequences ensuring the integration of the transgene at its biodegradable protein-based polymers in tobacco [11••] defined position in the plastome. The principal methods may be initial steps in this direction. to introduce DNA into chloroplasts are biolistic bombard- ment [12,33] and the use of polyethylene glycol [34,35]. In Dissection of the plastome and functional biolistic bombardment, a particle gun propels gold or tung- analysis of plastome-encoded proteins by sten particles coated with the appropriate DNA at target plastid engineering leaves. The chance that a chloroplast is hit by a particle in Many efforts in plastid engineering have so far dealt with a nondestructive manner is small; nevertheless, the enor-